Caracterización de proteínas con actividad antifúngica producidas
Transcription
Caracterización de proteínas con actividad antifúngica producidas
UNIVERSIDAD DE EXTREMADURA MENÚ SALIR MENÚ SALIR Edita: Universidad de Extremadura Servicio de Publicaciones Caldereros 2. Planta 3ª Cáceres 10071 Correo e.: [email protected] http://www.unex.es/publicaciones MENÚ SALIR ! " #$ % # & '( ! ) + * ,- . / , % ! % # & ", # % , 1 , 0 / 0 - ) 4556 * ) & 23 MENÚ SALIR ( 78. " 9 . ) ! #$ % # & '( ! ) + * , - . / , % ! % # & ", # % , 1 , 0 / 0 - ) 4556 * (78. 9 . ) & 23 MENÚ SALIR 0 : " ) #$ % #& '( ) + ! ! * , - . / , % ! % # & ", # % , 1 , 0 / 0 - ) ) 4556 * 0 : & 23 MENÚ SALIR !"#$% % / # , 8, 0 / ,- ' 0 # ) ) % , # ) 0 ( 78. / ,- : 0 . ' # 0 # 0 ) # / /# 0 / # ! , $ ; , 0 ! 8 / =< 8 # ' < ' / % / =< 9 # # ' ? # 0 ! - 0 0 / ,; 0 0 / # , ! 0 , # 0 / ' , 9 # ) ! 1, @ # ) , 0 : , > 8 ! . # 0 , # , : &% 0 # " < 8 # 9 ( ! 0 - $, 0 > .? " / , # # 0 # . /, /, 0 , ' ) ! A /B # # & : < '! # / , . 0 ! , , , , % # , 0 , A' / -B % , # 0 , MENÚ SALIR 0 $ / 0 @ 8 7 0 , 0 / / 0 , / < < 9 < # / !! ! #< ! , - 0 C 0 < # -- D @E F / F$ / E F / / GE F # / F/ E F ;/ / / @ / / 0 / / / # > / ! / 9 E/ @ ! ? / -- # ! ! , ! . , , E / ) / 1, # , ' 0 @ / /! E # ' / # # E E / / : / ) 0 ) 0 / - / 0 - # / #0 / #0 ) , / # - 7 0 ! 0 ' 0 / $ 0 ! 0 # / ) 0 # ! $@' ' . # & : 4553G5HI3HG @# , ,- # / % #; % 0 ) ) @! # 0 / MENÚ SALIR MENÚ SALIR MENÚ SALIR !"# $%&%$!'('!)*! +!,%(-"$(-.(+',!"#$%.!#$/-+!.-'-' 0 '%!!"# $%!)&%12! !"# $%&%$!'('3(+( .!)'(%)!"# $%&%$!'('"-,(-$,%$( '%!!"#$%!)&%12! -,!%,!,# /+( "%%42! -,!,# /+( "- 5 !"2!)"( -,!,# /+( "-%!# (12!#-,!,# /+("- 6 ! " 5 5 7 # $ 8 "9 : ; < MENÚ SALIR " # = 5 % # & % # " ' ( ) * ) + ' ' ) ! ) * ) + ' * ) + , ! ) " * ) + - # . / , $ , ) * ) + # ) , * ) + ' # ?284569 ) 0 +12134526714689 8: ) ; 1 98<5= 1946:>9)1= ?284569 :28@ 0 595463 3+12134526714689 8: 4+5 )595 5938A69) 4+5 1946:>9)1= :28@ 0 25= 6@6912B 5<1=>14689 8:4+5 48C6364B 19A D59D646<64B 48 A6::52594 42514@594D 8: 4+5 1946:>9)1= ?284569 ) :28@ MENÚ SALIR +12134526714689 8: ) + ; 1 98<5=3+648D191D5 E64+ 1946:>9)1= 0 1346<64B:28@ ,' 0 595463 3+12134526714689 8: 4+5 )595 59389A69) 4+5 98<5= 3+648D191D5 ) + :28@ 0 ::5348:A6 ::5259442514@594D 89 4+5 3+648D191D5 19A 1946:>9)1= 1346<64B8: ) + :28@ # # ! # ( " # ) * ) + , # ) # * ) + * ) * ) + # " 5 * 4 > * 5 * 4 & < ? " # "" # '' MENÚ SALIR MENÚ SALIR INTRODUCCIÓN I. INTRODUCCIÓN I.1. POBLACIÓN FÚNGICA EN ALIMENTOS Las condiciones ambientales en que se procesan o se almacenan diversos tipos de alimentos favorecen la proliferación en su superficie de mohos que tienen la capacidad de adaptarse y desarrollarse en sustratos muy diversos. Las especies de mohos aisladas con mayor frecuencia en alimentos pertenecen a los géneros Penicillium, Aspergillus, Fusarium y Eurotium, aunque las características intrínsecas de cada tipo de producto condicionan el desarrollo de unas especies u otras (Filtenborg y col., 1996). Así, frutas y verduras frescas suelen alterarse durante su almacenamiento por P. expansum, P. crustosum, P. solitum, P. digitatum o P. italicum (Pitt y Hocking, 1997; Moss, 2008), y los cereales en cambio pueden ser colonizados por P. polonicum, P. verrucosum, P. aurantiogriseum, P. viridicatum, A. flavus, A. niger o A. versicolor (Filtenborg y col., 1996). En productos cárnicos madurados y quesos es frecuente el desarrollo una población fúngica superficial durante la maduración que puede tener efectos deseables, ya que contribuye en gran medida a la formación del sabor, aroma o textura del producto acabado (Kinsella y Hwang, 1976; Martín y col., 2004, 2006), e incluso puede ejercer un efecto protector frente a otros microorganismos no deseados (Geisen y col., 1992; Leistner, 1994; Singh y Dincho, 1994; Berwal y Dincho, 1995; Molimard y col., 1995; Decker y Nielsen, 2005). Por ello, en quesos es frecuente el uso de determinadas cepas de mohos como P. roqueforti o P. camemberti como cultivos iniciadores (Nielsen y col., 1998), y se ha propuesto la elaboración de este tipo de cultivos para su utilización en productos cárnicos madurados a partir de cepas no toxigénicas de P. chrysogenum (Benito y col., 2005; Martín y col., 2006), Penicillium olsonii (López-Díaz y col., 2001) o Penicillium camemberti (Bruna y col., 2003). Pero el desarrollo de mohos no siempre es deseable en alimentos. En quesos se han descrito cepas alterantes de P. commune y P. nalgiovense que pueden conducir a un deterioro en las características sensoriales (Filtenborg y col., 1996), debido normalmente a la liberación de exoenzimas durante su desarrollo. Estos enzimas pueden continuar su actividad independientemente de que se produzca la eliminación del micelio, causando desde olores o coloraciones desagradables hasta la completa desintegración de la estructura del alimento. No obstante, el aspecto más preocupante respecto del desarrollo de una 1 MENÚ SALIR INTRODUCCIÓN población fúngica incontrolada en alimentos es su capacidad para producir micotoxinas. El desarrollo de mohos en quesos puede conllevar la producción de micotoxinas (Marth y Yousef, 1991), muchas de las cuales han sido detectadas en estos productos (Lafont y col., 1979; Le Bars, 1979; Northolt y col., 1980; Jarvis, 1983; Siemens y Zawistowski, 1993). De manera similar, en productos cárnicos madurados la población microbiana superficial está constituida fundamentalmente por mohos, muchos de los cuales son toxigénicos (Bullerman y col., 1969; Escher y col., 1973; Halls y Ayres, 1973; Wu y col., 1974; Leistner, 1984; Núñez y col., 1996; López-Díaz y col., 2001). I.2. CONSECUENCIAS DEL DESARROLLO DE MOHOS EN PRODUCTOS CÁRNICOS MADURADOS Con respecto a la población fúngica que puede colonizar la superficie de productos cárnicos, existen tanto mohos que contribuyen en gran medida a la formación del sabor, aroma o textura del producto acabado, como especies que pueden deteriorar las características sensoriales o producir micotoxinas. I.2.1. EFECTOS BENEFICIOSOS La población fúngica que coloniza los productos cárnicos madurados presenta diversas actividades metabólicas que pueden mejorar las características sensoriales del producto. Así, la presencia de mohos puede ofrecer actividad antioxidante, lipolítica, proteolítica, contribuir al aroma y sabor del producto e incluso ejercer un efecto protector por su actividad antimicrobiana (Figura I1). El efecto antioxidante de los mohos se atribuye a que la mayoría de las cepas aisladas de jamón poseen una actividad catalasa moderada (Núñez, 1995), destruyendo peróxidos y reduciendo las reacciones oxidativas (Burgos, 1981), por lo que disminuye el enranciamiento y contribuyen a la estabilización del color del producto (Liepe, 1982; Smith y Palumbo, 1983; Geisen, 1993). Además, muchos mohos producen compuestos con acción antioxidante (Ludemann y col., 2004). Por otra parte, numerosas especies de mohos pertenecientes al género Penicillium poseen exoenzimas con actividad lipolítica que pueden incrementar la concentración de ácidos grasos libres y compuestos volátiles en jamón (Huerta y col., 1987; Molina y col., 1991) y embutidos (Toledo y col., 1996), contribuyendo así al desarrollo del sabor y aroma del producto. Se ha observado actividad lipolítica en mohos del género Penicillium 2 MENÚ SALIR INTRODUCCIÓN aislados de jamón ibérico (Núñez, 1995; Alonso, 2004) y de embutidos (Selgas y col., 1999; Ludemann y col., 2004). Figura I1. Efectos beneficiosos por el desarrollo de mohos durante la maduración del jamón (Núñez, 1995). Adicionalmente, aunque la proteolisis en los productos cárnicos se ha asociado generalmente a la actividad de los enzimas presentes en el tejido muscular (Toldrá y col., 1992; Sárraga y col., 1993), algunas cepas aisladas de jamón curado producen una intensa hidrólisis de proteínas miofibrilares y un gran incremento de aminoácidos libres cuando se inoculan en carne de cerdo estéril y productos cárnicos madurados (Rodríguez y col., 1998; Martín y col., 2001, 2002, 2004). En este sentido, se ha demostrado una intensa actividad proteolítica de una cepa de P. chrysogenum (Rodríguez y col., 1998; Martín y col., 2001, 2002; Benito y col., 2003; Alonso, 2004), a partir de la cual se ha purificado una proteasa muy activa en las condiciones de maduración de diferentes productos cárnicos (Benito y col., 2002). También se han aislado de embutidos mohos muy proteolíticos de los géneros Mucor y Penicillium (Trigueros y col., 1995), capaces de incrementar la concentración de aminoácidos libres en estos productos (Bruna y col., 2001). Los mohos pueden favorecer la formación del aroma característico de los productos curados por diversas vías. En primer lugar, los aminoácidos liberados, además de potenciar el sabor de los alimentos (Suyama y Shimuzu, 1982; Nishimura y Kato, 1988), pueden 3 MENÚ SALIR INTRODUCCIÓN intervenir en reacciones de condensación de Maillard (Shahidi y col., 1986; Mottram y Whitfield, 1995) y de degradación de Strecker (Barbieri y col., 1992), en las que se pueden formar compuestos responsables del aroma del jamón curado (García y col., 1991; Ruiz y col., 1998; Whitfield, 1992). Finalmente, los mohos pueden tener un efecto protector frente a microorganismos patógenos o alterantes (Geisen y col., 1992; Leistner, 1994; Singh y Dincho, 1994; Berwal y Dincho, 1995). Así, el 75% de los mohos aislados en jamón ibérico inhiben el crecimiento de Staphylococcus aureus (Núñez y col., 1996), y diversas especies no toxigénicas de mohos tienen la capacidad de inhibir la producción de micotoxinas e incluso de destruir las formadas por otras especies toxigénicas (Mislivec y col. 1988). No obstante, este efecto podría deberse a actividades tan poco deseables como la producción de toxinas o de antibióticos, o bien a mecanismos más deseables como a interferencias metabólicas o a la liberación de proteínas antimicrobianas. I.2.2. EFECTOS PERJUDICIALES El desarrollo de ciertos mohos, como los géneros Mucor y Rhizopus, puede provocar un aspecto no deseable al jamón o embutidos (Figura I2). Por otro lado, los mohos con gran capacidad lipolítica pueden ocasionar olores anómalos (Sanz, 1986). De hecho, se ha identificado a P. commune como el responsable del “defecto del ácido fénico”, caracterizado por conferir al jamón un fuerte olor a fenol (Spotti y col., 1988). Figura I2. Contaminación fúngica superficial en jamón curado. 4 MENÚ SALIR INTRODUCCIÓN No obstante, el aspecto más preocupante respecto al desarrollo de una población fúngica superficial en estos productos es su capacidad para producir micotoxinas. Las micotoxinas son metabolitos secundarios tóxicos que al ser ingeridos en pequeñas cantidades, pueden ocasionar diferentes patologías relacionadas con inmunosupresión, carcinogénesis, neurotoxicidad, etc. (Samson y col., 1995; Richard, 2007). Existen micotoxinas que sólo son producidas por un grupo reducido de mohos, y otras generadas por especies muy diversas incluso de géneros diferentes (Frisvad y Thrane, 1995). En este sentido, se han descrito especies como Penicillium griseofulvum capaces de producir gran variedad de micotoxinas (Frisvad y Filtenborg, 1990). No obstante, el perfil y la concentración de micotoxinas que puede producir un moho, así como la posterior difusión al interior del alimento, no sólo dependen de la naturaleza del microorganismo sino también de la composición del alimento y del procesado al que esté sometido. Además, estos metabolitos son muy resistentes a los tratamientos físicos y químicos, por lo que una vez que las micotoxinas están en el alimento pueden permanecer en él incluso después del procesado (Osborne y col., 1996; Castellá y col., 1998; Katta y col., 1999). Muchos mohos colonizadores de alimentos son capaces además de producir antibióticos como la penicilina (Andersen y Frisvad, 1994), lo que supone un riesgo para los consumidores alérgicos a este antibiótico (Bremmelgaard, 1998). El potencial toxigénico de los mohos aislados de productos cárnicos se ha establecido en distintos trabajos. Se ha comprobado que el 80% de 1481 aislados del género Penicillium a partir de embutidos son potencialmente toxigénicos (Eckardt y col., 1979). Más del 75% de los mohos aislados en jamón ibérico muestran toxicidad o mutagenicidad en ensayos biológicos (Núñez y col., 1996). Entre estos mohos, el más frecuente es P. commune, productor de ácido ciclopiazónico (Núñez y col., 1996; Sosa y col., 2002), destacando también P. restrictum, A. flavus y A. niger. Además, todos los aislados de P. commune obtenidos de chorizo de Cantimpalos mostraron una alta toxicidad para larvas de Artemia salina (López-Díaz y col., 2001). Estas especies pueden producir micotoxinas en los productos cárnicos sobre los que se desarrollan (Núñez y col., 2007), habiéndose comprobado en embutidos y jamones curados la presencia de citrinina, patulina, rugulosina, ocratoxina A, ácido ciclopiazónico, citreoviridina, breviamida A, fumitremorgina B, griseofulvina y verruculógeno TR1 (Leistner, 1984). Además, las micotoxinas pueden difundir al interior de los productos, donde pueden mantenerse incluso cuando el micelio se ha eliminado (Leistner, 1984). 5 MENÚ SALIR INTRODUCCIÓN Por todo esto, resulta preocupante el crecimiento incontrolado de mohos potencialmente toxigénicos en la superficie de los productos cárnicos madurados (Núñez y col., 1996; Sabater-Vilar y col., 2003) y sería recomendable controlar su desarrollo en este tipo de productos. I.3. CONTROL DE MOHOS EN PRODUCTOS CÁRNICOS MADURADOS Existen numerosos mecanismos para controlar el desarrollo microbiano en distintos tipos de alimentos. Entre los métodos físicos de control se encuentra la utilización de tratamientos térmicos, radiaciones ionizantes o ultravioleta, altas presiones, pulsos eléctricos o de luz, o campos magnéticos. También pueden emplearse agentes químicos, incluyendo ácidos orgánicos, aceites esenciales, peróxido de hidrógeno, etc., así como el envasado en atmósferas controladas o modificadas. Estos tratamientos resultan eficaces para evitar el desarrollo de mohos en determinados alimentos, como frutas, verduras, pan, etc. No obstante, en los productos cárnicos madurados está limitada la aplicación directa de fungicidas. Además, las medidas higiénicas para prevenir la contaminación durante la elaboración se ven desbordadas en los secaderos y bodegas, donde resulta muy difícil eliminar totalmente la contaminación ambiental. Por otra parte, la inespecificidad de estos tratamientos evitaría la posible contribución beneficiosa de los mohos a la maduración de estos productos. Por todo ello, en productos cárnicos madurados es necesario adoptar estrategias alternativas que garanticen el desarrollo de los microorganismos deseables y la seguridad de estos alimentos. I.3.1. CULTIVOS PROTECTORES Para controlar la población fúngica que se desarrolla en la superficie de los productos cárnicos madurados podrían utilizarse cultivos protectores formados por mohos capaces de inhibir por competición a los mohos no deseables, siempre teniendo en cuenta que deben ser no toxigénicos y mostrar una actividad metabólica adecuada. No obstante, esta opción se ve limitada por la evolución de las condiciones ecológicas durante la maduración, resultando muy poco probable que los mohos seleccionados superen a todas las especies habituales en la amplísima gama de temperatura y actividad de agua que tiene lugar durante la maduración (Rodríguez y col., 1994). Una posibilidad radicaría en el empleo de mohos que dispusieran de algún elemento adicional que favoreciese su 6 MENÚ SALIR INTRODUCCIÓN desarrollo, como la producción de antibióticos. Lógicamente, esta opción se ha descartado por las posibles reacciones alérgicas en el consumidor o la inducción de resistencia en otros microorganismos como consecuencia de su empleo continuado. Otra posible estrategia consiste en el empleo de proteínas antibacterianas como las bacteriocinas, que están siendo ampliamente utilizadas en otro tipo de alimentos, o de los microorganismos que las producen. Así, la nisina es una bacteriocina autorizada como conservante alimentario (Gálvez y col., 2007). Pero este tipo de proteínas, a pesar de tener un espectro de inhibición muy amplio, no inhibe a los mohos. Por ello, ni las bacteriocinas ni los microorganismos que las producen pueden ser empleados para evitar el crecimiento fúngico en alimentos. Sin embargo, la reciente descripción de proteínas antifúngicas naturales elaboradas por mohos abre nuevas perspectivas en este sentido. La utilización de cepas de mohos productoras de proteínas antifúngicas como cultivos protectores en productos cárnicos madurados, permitiría limitar el desarrollo de mohos toxigénicos sin afectar a los deseables, contribuyendo así a estandarizar las características del producto. I.3.2. PROTEÍNAS ANTIFÚNGICAS La investigación de nuevos agentes antifúngicos ha experimentado un notable avance en los últimos años como consecuencia del incremento de presentación de micosis en pacientes inmunodeprimidos por el SIDA o por tratamientos anticancerígenos, especialmente infecciones por distintas especies de Candida o en aspergilosis invasivas (Anaissie, 2008; de la Torre y col., 2008; Pemán y Almirante, 2008). Esta búsqueda de nuevos compuestos antifúngicos se ha extendido también a otros campos como la agricultura, donde la proliferación de mohos patógenos en los cultivos ocasiona grandes pérdidas económicas. Así, se han caracterizado nuevas proteínas con actividad antifúngica (Upadhyay y Srivastava., 2008; Yan y col., 2008) y se han producido plantas transgénicas que las expresen (Girlanda y col., 2008; Swathi-Anyradha y col., 2008). En los últimos años se han purificado y caracterizado proteínas con efecto antifúngico a partir de plantas (Chu y col., 2003; Ng y col., 2003), bacterias (Yang y col., 2002), mohos (Marx y col., 2007; Meyer, 2008), artrópodos (Tomie y col., 2003), o reptiles (Gomes y col., 2005), así como también se han diseñado péptidos sintéticos (Patrzykaty col., 2003). 7 MENÚ SALIR INTRODUCCIÓN El mecanismo de acción de las proteínas antifúngicas es tan variado como los organismos que las producen (Selitrennikoff, 2001; Theis y Stahl, 2004). Algunos atraviesan la membrana e interfieren en la síntesis de ADN, ARN o de proteínas, inhiben la cadena respiratoria celular (Cociancich y col., 1993), disminuyen el ATP (Cociancich y col., 1993), inactivan los ribosomas fúngicos (Hwu y col., 2000) o provocan la desporalización de la membrana (Thevissen y col., 1997). Otros en cambio forman canales y poros en la membrana alterando su permeabilidad y provocando la pérdida de iones y otros solutos (Thevissen y col., 1999; Lee y col., 2001) o pueden interferir en la síntesis de componentes esenciales de la pared celular como el -1,3-glucano (Odds y col., 2003), la quitina (Yang y Gong, 2002; Von der Weid y col., 2003) o el quitosán (Shimosaka y col., 2005), pudiendo incluso hidrolizarlos (Sela-Buurlage y col., 1993; Graham y Sticklen, 1994). Esto debilita la pared y origina la lisis de la célula. No obstante, las proteínas antifúngicas de mayor utilidad son las que presentan mecanismos de acción específicos para la cubierta externa de los hongos (Groll y col., 1998), ya que suelen ser poco tóxicas para las células de mamíferos. En la interacción de la proteína con la membrana desempeñan un papel destacado la carga (Hoover y col., 2003) y el carácter catiónico o hidrofóbico del compuesto (Friedrich y col., 1999; Hancock y Chapple, 1999). Así, la hidrofobicidad y la estructura en -hélice, se correlacionan con la toxicidad para las células de mamíferos, mientras que la carga positiva está más relacionada con la actividad antimicrobiana (Hong y col., 2001). Esto se debe a que las membranas celulares de bacterias y mohos se componen principalmente de fosfolípidos aniónicos que favorecen la unión de la proteína antifúngica y su actividad, mientras que estos fosfolípidos no están presentes en muchos eucariotas superiores o están confinados en la cara interna de la membrana plasmática, no estando en contacto con el medio extracelular (Rivas y Andreu, 2003; Theis y Stahl, 2004). Por otra parte, existe una gran variabilidad en cuanto a la composición de aminoácidos y estructura secundaria y terciaria de las proteínas antifúngicas, aunque la mayoría se caracterizan por ser catiónicas y por formar estructuras anfipáticas (Hwang y Vogel, 1998). Se han descrito cuatro proteínas antifúngicas catiónicas y de bajo peso molecular producidas por mohos: AFP de Aspergillus giganteus (Nakaya y col., 1990; Lacadena y col., 1995), PAF de Penicillium chrysogenum (Marx y col., 1995), Anafp de Aspergillus niger (Lee y col., 1999) y AcAFP de A. clavatus (Skouri-Gargouri y Gargouri, 2008). Todas ellas se caracterizan por su pequeño tamaño (51-58 aminoácidos), su alto 8 MENÚ SALIR INTRODUCCIÓN contenido en residuos de cisteína y su fuerte carácter básico. Además, sus estructuras terciarias están estabilizadas por tres o cuatro puentes disulfuro y forman estructuras con un marcado carácter anfipático (Nakaya y col., 1990; Lacadena y col., 1995). Sin embargo, la aplicación de los mohos productores como cultivos protectores en alimentos madurados en general y en productos cárnicos en particular exige que dichos mohos estén adaptados a las condiciones ecológicas de estos alimentos, que no los alteren y que no sean productores de micotoxinas. Ninguna de las cepas a partir de las que se han purificado proteínas antifúngicas ha sido obtenida a partir de productos madurados. Por ello, es esperable que no se adapten a las condiciones ecológicas particulares de este tipo de productos de manera que puedan competir con los mohos que crecen de forma espontánea. Además, algunos de estos mohos pertenecen al género Aspergillus, que sólo es predominante en estos alimentos en las últimas fases de su elaboración/maduración, cuando la aw es muy baja. Por lo tanto, la utilización de mohos requiere encontrar cepas adaptadas a este tipo de alimentos y que sean capaces de elaborar las proteínas antifúngicas en estos productos. En este caso, las obtenidas de especies del género Penicillium, empleadas como cultivos iniciadores, plantearían menos objeciones que las de Aspergillus, debido a que este último género se relaciona más frecuentemente con la alteración de alimentos y la producción de micotoxinas (Perrone y col., 2007). I.3.3. PROTEÍNAS ANTIFÚNGICAS DE Penicillium chrysogenum En estudios previos se seleccionaron los aislados no toxigénicos de P. chrysogenum RP42C y AS51D, procedentes de jamón curado, por su capacidad para inhibir el desarrollo de mohos toxigénicos frecuentes en alimentos. Adicionalmente, se purificaron dos proteínas con actividad antifúngica, PgAFP y PgChP, a partir de RP42C y AS51D, respectivamente (Acosta y col., 2009a). PgAFP y PgChP se obtuvieron como consecuencia de una búsqueda exhaustiva de proteínas antifúngicas con carga neta positiva, debido a que el carácter catiónico de este tipo de proteínas se asocia con una menor toxicidad para las células de mamíferos. Así, PgAFP y PgChP fueron obtenidas mediante cromatografía líquida para la purificación rápida de proteínas (FPLC) empleando inicialmente columnas de intercambio catiónico y posteriormente de filtración en gel (Acosta y col., 2009a). La proteína PgAFP mostró un amplio espectro de inhibición frente a mohos toxigénicos aislados de jamón curado, tales como A. flavus, A. niger, A. parasiticus, P. 9 MENÚ SALIR INTRODUCCIÓN griseolfulvum, P. solitum o P. restrictum. En cambio, la proteína PgChP sólo afectó a P. commune, aunque no se descarta que tenga actividad antifúngica frente a otros mohos no estudiados (Acosta y col., 2009a, b). Las proteínas PgAFP y PgChP mostraron un peso molecular muy diferente por SDS-PAGE, siendo de aproximadamente 9 y 37 kDa, respectivamente. El punto isoeléctrico de PgAFP fue de 9,2, por lo que la proteína resultó ser muy catiónica. Por otra parte, el hecho de que PgAFP perdiera actividad frente a algunas cepas del género Aspergillus tras el tratamiento con α-amilasa, sugirió la posibilidad de algún tipo de glicosilación en la proteína, hecho que se confirmó mediante una tinción de ácido periódico-Schiff (PAS; Zacharius y col., 1969) siguiendo el método descrito por Deepak y col. (2003). Además, la secuencia amino terminal parecía indicar que se trataba de una proteína antifúngica no descrita hasta el momento. No obstante, el análisis mediante MALDI-TOF de los fragmentos obtenidos de PgAFP tras la digestión con tripsina no ofreció demasiada información sobre su composición aminoacídica, por lo que apenas se obtuvieron datos relativos a la estructura primaria de esta proteína (Acosta y col., 2009b). También se confirmó la producción de PgAFP en condiciones similares a las de las etapas iniciales de la maduración. Esto podría suponer para P. chrysogenum RP42C una ventaja adicional para competir frente al resto de mohos que se desarrollan en la superficie de los productos cárnicos madurados, lo que sería de gran valor para su utilización como cultivo protector. Además, PgAFP mostró sensibilidad al tratamiento con enzimas como tripsina, pepsina y lisozima, lo cual permite ser optimistas respecto a su uso en alimentos, ya que puede ser degradada en el tracto gastrointestinal (Acosta, 2006). Por otra parte, aunque la información disponible de PgChP se limita a su peso molecular y a su capacidad para inhibir a P. commune, dadas las diferencias en la masa y en el espectro de inhibición entre PgAFP y PgChP, en este trabajo se aborda la caracterización de ambas proteínas. I.4. CARACTERIZACIÓN DE PROTEÍNAS Para el estudio de las proteínas se pueden determinar distintas características, entre las que destacan el peso molecular, el punto isoeléctrico, la secuencia de aminoácidos y la del gen que la codifica, así como la sensibilidad a distintos tratamientos (térmicos, pHs, iones, etc.). 10 MENÚ SALIR INTRODUCCIÓN I.4.1. DETERMINACIÓN DEL PESO MOLECULAR I.4.1.1. Electroforesis en gel de poliacrilamida Uno de los métodos más habituales para estimar el peso molecular de una proteína es la electroforesis en gel de poliacrilamida, que puede utilizarse para separar proteínas en función de su carga y tamaño en condiciones no desnaturalizantes o nativas (Bonnerjea y col., 1986), o bien sólo en función del tamaño de sus subunidades, en condiciones desnaturalizantes (Weber y Osborn, 1969). Muchos autores han utilizado la electroforesis desnaturalizante tanto para comprobar la pureza como para calcular el peso molecular de proteínas antifúngicas (Lacadena y col., 1995; Marx y col., 1995; Martínez-Ruiz y col., 1997; Lee y col., 1999). La electroforesis en gel de poliacrilamida tiene la ventaja de que puede llevarse a cabo en cualquier laboratorio ya que la inversión en material e instrumentos es mínima. Sin embargo, este método puede llevar a errores en el peso molecular estimado. De hecho, proteínas con modificaciones post-traduccionales presentan una movilidad anómala ya que estas modificaciones provocan que no se una adecuadamente el SDS en electroforesis desnaturalizantes (Russ y Poláková, 1973; Bagger y col., 2007). Igualmente, proteínas con puntos isoeléctricos extremos pueden repeler las cargas negativas del SDS y no migrar como lo deberían hacer teóricamente en función de su peso molecular (Mondstadt y Holldorf, 1990). I.4.1.2. Espectrometría de masas En los últimos años se ha aplicado una técnica muy conocida en otros campos, aunque novedosa en el estudio de proteínas: la espectrometría de masas. La mayor parte de los análisis bioquímicos por espectrometría de masas utilizan la ionización por electrospray (ESI) o por desorción con láser asistida por matriz (MALDI), junto a un analizador de tiempo de vuelo (TOF) (Ashcroft, 2005). ESI y MALDI son métodos de ionización “suave” que producen moléculas cargadas con poca fragmentación, aún tratándose de muestras de muy alta masa molecular. Tanto en ESI como en MALDI se protona la muestra a analizar y como resultado se genera un espectro masa/carga (m/z) que tiene una amplia distribución de múltiples iones cargados. Este espectro m/z puede ser convertido a un espectro con las masas reales (Figura I3). Manualmente, la masa real puede ser calculada con la fórmula m/z= (M + nH+)/n, donde M es la masa molecular de la proteína y n es el número de protones 11 MENÚ SALIR INTRODUCCIÓN asociados a ella. El error asociado a esta técnica suele rondar el 0,01% de la masa (Ashcroft, 2005). Figura I3. Determinación del peso molecular mediante espectrometría de masas con ionización por ESI y MALDI. Tanto MALDI como ESI son técnicas ampliamente utilizadas para la determinación del peso molecular de las proteínas, aunque requieren una fuerte inversión en instrumentos, así como tener amplios conocimientos en su manejo y en la interpretación de los resultados. A efectos prácticos, la ionización MALDI suele ser más atractiva porque tiene mayor tolerancia a sustancias químicas o sales presentes en la muestra, reduciendo así la necesidad de purificación que requiere la ionización ESI (Standing, 2003). Por lo demás, ambas son técnicas muy valiosas y precisas para determinar el peso molecular de una proteína. I.4.2. DETERMINACIÓN DEL PUNTO ISOELÉCTRICO Las técnicas más frecuentes para el cálculo del punto isoeléctrico de una proteína suelen basarse en el isoelectroenfoque, que puede llevarse a cabo mediante electroforesis en gel en una o dos dimensiones, así como mediante electroforesis capilar. 12 MENÚ SALIR INTRODUCCIÓN I.4.2.1. Isoelectroenfoque El isoelectroenfoque es un tipo de electroforesis que permite separar los componentes de una muestra en función de su punto isoeléctrico. Se basa en la capacidad de las moléculas de variar su carga neta en función del pH del medio. La electroforesis se lleva a cabo añadiendo una mezcla de anfolitos con diferentes puntos isoeléctricos en el medio, para crear un gradiente de pH entre dos electrodos con carga eléctrica opuesta. Al aplicar una diferencia de potencial cada anfolito se mueve hacia uno u otro electrodo en función de su propia carga y se detiene cuando el pH del medio que le rodea es igual a su punto isoeléctrico. Esta técnica se caracteriza por una gran repetibilidad y resolución (Fuentes Arderiu y col., 1998). I.4.2.2. Electroforesis bidimensional Este tipo de electroforesis consiste en realizar dos separaciones electroforéticas consecutivas de una misma muestra en función de propiedades diferentes en dos direcciones distintas. Lo más frecuente es realizar la primera separación por isoelectroenfoque según el punto isoeléctrico, y la segunda en gel de poliacrilamida con SDS (PAGE-SDS) según la masa molar (Figura I4). Esta técnica se suele emplear para la separación de muestras complejas de proteínas, pudiendo calcular puntos isoeléctricos, masas molares, cuantificar proteínas, etc. ) * + ! & ! " # $% ' ($ Figura I4. Electroforesis bidimensional. 13 ! MENÚ SALIR INTRODUCCIÓN I.4.2.3. Electroforesis capilar por isoelectroenfoque La electroforesis capilar es una técnica desarrollada en los últimos años que consiste en realizar técnicas electroforéticas convencionales (electroforesis en gel, isoelectroenfoque, etc.) en capilares de sílice fundida con un diámetro interno de entre 25 y 75 µm y una longitud que puede oscilar entre 7 y 100 cm. Tanto uno de los extremos del capilar como los electrodos se encuentran en una disolución tampón. La muestra se añade cerca del extremo anódico y se aplica una elevada diferencia de potencial al sistema, que hace que los componentes de la muestra se muevan a lo largo del capilar y pasen a través de un detector colocado cerca del cátodo (Holland y Sepaniak, 1993; Berg y col., 2008). Estudios comparativos entre isoelectroenfoque, electroforesis bidimensional y electroforesis capilar revelan que las tres técnicas proporcionan una información fiable acerca del punto isoeléctrico (Valenzuela y col., 1999). Sin embargo, la electroforesis capilar aporta ventajas como la necesidad de una mínima cantidad de muestra, alta reproducibilidad y rapidez en los análisis comparativos. A pesar de esto, la electroforesis bidimensional es una de las técnicas más utilizadas en laboratorios de proteómica para la separación de mezclas de proteínas y la determinación del punto isoeléctrico. I.4.3. IDENTIFICACIÓN DE PROTEÍNAS En la actualidad, en la identificación de proteínas se utilizan diversos procedimientos, entre los que se incluyen la secuenciación del extremo N-terminal, detección con anticuerpos específicos, composición de aminoácidos, etc. Todos estos métodos generalmente son lentos, laboriosos o caros (Gil, 2004). Hasta hace poco tiempo, para obtener la secuencia aminoacídica de una proteína la técnica más utilizada era la degradación de Edman. Pero la espectrometría de masas, debido a su rapidez y elevada sensibilidad, se ha convertido en el método de elección para la identificación de proteínas a gran escala, que es el primer paso para el estudio de proteínas de distintos organismos (Russeth y col., 2006; Samyn y col., 2006). Además, esta técnica también permite la caracterización de modificaciones post-traduccionales que presentan relevancia fisiológica, tales como la glicosilación y la fosforilación (Claverol y col., 2003; Morelle y col., 2006). El análisis de las proteínas mediante espectrometría de masas ha sido posible gracias al desarrollo de varios métodos de ionización suave como ESI o MALDI para convertir biomoléculas grandes, polares y no volátiles, en iones en fase gaseosa. 14 MENÚ SALIR INTRODUCCIÓN Esta técnica implica varios pasos: la conversión de los péptidos en fase gaseosa mediante técnicas de ionización suave como MALDI o ESI; la separación de los iones según su m/z en un analizador de masas (por ejemplo un analizador de tiempo de vuelo); la fragmentación opcional de los iones seleccionados, por ejemplo mediante una disociación inducida por colisión (CID) en un espectrómetro de masas en tándem, combinando dos analizadores diferentes; y por último la medida de las masas en un detector obteniendo un espectro de masas que refleja la abundancia de los iones frente a su valor m/z. Para la identificación de proteínas se han desarrollado dos estrategias que se encuentran representadas esquemáticamente en la Figura I5: identificación mediante mapeo peptídico o huella peptídica (PMF: peptide mass fingerprinting) e identificación mediante etiqueta de secuencia o peptídica, que conlleva la fragmentación de péptidos y la obtención de la secuencia aminoacídica total o parcial utilizando un espectrómetro de masas en tándem. I.4.3.1. Mapeo peptídico o huella peptídica. El mapeo peptídico o huella peptídica es una técnica utilizada rutinariamente para identificar proteínas de forma rápida, normalmente a partir de geles de SDS-PAGE o 2DPAGE, que se realiza en un espectrómetro de masas tipo MALDI-TOF o ESI-TOF. La huella peptídica de una determinada proteína es un conjunto de péptidos generados mediante la digestión con una proteasa específica. Estas masas peptídicas experimentales son comparadas con las masas peptídicas teóricas de proteínas presentes en bases de datos. Para ello se han desarrollado diversos algoritmos (PEPSEA, PROFOUND, MS-FIT, PEPTIDENT, etc) disponibles en Internet. Para la identificación correcta de la proteína se requiere que las masas de un gran número de péptidos coincidan con sus masas teóricas, y que cubran parte de la secuencia de la proteína de la base de datos. Además, si la cantidad de proteína es pequeña, el número de péptidos observados puede ser reducido y por tanto la proteína no se puede identificar con fiabilidad. 15 MENÚ SALIR INTRODUCCIÓN * , * !- , ! - ./ # 0 1 ! ! 0* ! 2 3 Figura I5. Identificación de proteínas mediante espectrometría de masas. I.4.3.2. Secuencia peptídica o etiqueta peptídica. La secuencia peptídica es una estrategia para la identificación de proteínas no incluidas en las bases de datos o que presentan identificaciones ambiguas mediante MALDI/ESI-TOF. Los espectrómetros de masas en tándem MS/MS permiten también la determinación de la secuencia de aminoácidos. Para ello se selecciona un ión por la masa en un primer espectrómetro y se fragmenta por colisión con un gas. Posteriormente los fragmentos obtenidos se analizan en un segundo espectrómetro. Puede utilizarse con una fuente de ionización tipo MALDI o ESI. A partir de la protonación de los péptidos, estos iones pueden ser seleccionados y sometidos a una disociación inducida por colisión (CID), en la 16 MENÚ SALIR INTRODUCCIÓN cual interaccionan con el gas de colisión (normalmente nitrógeno o argón), y tiene lugar la fragmentación a lo largo del esqueleto peptídico. Los espectros de fragmentación MS/MS proporcionan información sobre la identidad y posición del aminoácido en el péptido. Por tanto es posible adquirir la secuencia completa del péptido si se parte de una fragmentación de buena calidad. No obstante a veces sólo se obtiene una secuenciación parcial del péptido. La información sobre la secuencia parcial, la masa del péptido, y la localización exacta de los aminoácidos, pueden permitir la identificación de la proteína en las bases de datos. A esta aproximación se le denomina identificación por etiqueta peptídica. Existen diversas herramientas bioinformáticas que se pueden utilizar para la interpretación de la fragmentación de un péptido (PEPSEA, SEQUEST, PEPFRAG, MSTAG y MASCOT). El método de la secuenciación de novo es la estrategia de elección para aquellas proteínas de las que se desconozca la secuencia del gen que las codifica. El objetivo de esta aproximación es obtener secuencias completas de aminoácidos que se deduzcan de novo de los espectros MS/MS (bien mediante interpretación manual o con ayuda de programas informáticos) y buscar homologías con proteínas presentes en bases de datos. Esta estrategia permite encontrar proteínas homólogas en otras especies utilizando programas de búsqueda como BLAST o FASTA. Finalmente, aunque la proteína de interés pueda no ser identificada o sea una proteína desconocida, la información sobre las secuencias obtenidas se puede utilizar para diseñar cebadores que permitan la obtención y clonación del gen que la codifica. I.5. CARACTERIZACIÓN DEL GEN QUE CODIFICA UNA PROTEÍNA Caracterizar el gen que codifica una proteína es de gran utilidad porque permite en muchos casos completar la secuencia de aminoácidos. Adicionalmente, el conocimiento de la secuencia genética que codifica una proteína permite su clonación en un vector de expresión y una síntesis más eficiente de la proteína en un organismo distinto del que normalmente la produce. I.5.1. BIBLIOTECAS DE ADN COMPLEMENTARIO La obtención de bibliotecas de ADN complementario o ADNc es una de las estrategias que se han utilizado con proteínas antifúngicas (Marx y col., 1995). Para ello es necesario elegir un vector o plásmido que se utilizará como herramienta para la 17 MENÚ SALIR INTRODUCCIÓN construcción de la biblioteca. Los vectores plasmídicos son capaces de clonar fragmentos de ADNc desde cientos hasta unas 9000 pares de bases, ya que tamaños mayores necesitan otros vectores como los cósmidos. La elección del plásmido depende de los sitios de clonación, de restricción y de los lugares de resistencia a antibióticos. Una vez elegido el vector se une al ADNc sintetizado. Posteriormente se introducen en el microorganismo competente elegido, formándose los transformantes que constituirán la biblioteca. Los clones relacionados con la síntesis de la proteína se detectan mediante una sonda de ADN (Redkar y col., 1996) que puede elaborarse a partir de la secuencia del extremo amino terminal de la proteína purificada. Otras estrategias encaminadas a encontrar los genes relacionados con la síntesis de la proteína están basadas en la realización de PCRs directamente sobre el ADN genómico (Geisen, 2000) o el ARN mensajero (RT-PCR) del microorganismo productor de la proteína. Para ello se pueden utilizar cebadores diseñados a partir de las secuencias amino y carboxilo terminal de la proteína purificada. I.5.2. AMPLIFICACIÓN RÁPIDA DE EXTREMOS DE ADNc (RACE-PCR) Existen técnicas que permiten la amplificación de un gen partiendo de un fragmento conocido de la proteína sin que ésta corresponda necesariamente a los extremos amino o carboxilo terminal. Una de estas técnicas se basa en la amplificación de extremos de un gen y es denominada amplificación rápida de extremos de ADNc mediante PCR o RACEPCR (“ Rapid Amplification of cDNA Ends - Polymerase Chain Reaction” ). Para llevar a cabo esta técnica se requiere conocer una parte del gen que queremos caracterizar. Esta región se puede obtener mediante el diseño de cebadores a partir de secuencias de aminoácidos obtenidas previamente y la posterior amplificación por PCR utilizando ADN genómico del microorganismo. Existen dos tipos de RACE-PCR en función del extremo que se vaya a amplificar: 5’-RACE y 3’-RACE (Scotto-Lavino y col., 2006a y b). En la Figura I6 se representa un esquema general de la amplificación de un gen completo mediante RACE-PCR. En primer lugar es necesaria la extracción de ARN a partir de un cultivo del microorganismo en el cual se ha inducido la transcripción del gen que codifica la proteína. Seguidamente, se realiza una transcripción reversa empleando unos adaptadores o secuencias conocidas que hibridan con los extremos 5’ y 3’ del ARN inicial (Figura I7). Por lo tanto, el ADNc resultante llevará consigo estas secuencias conocidas, generando dos poblaciones de ADNc 18 MENÚ SALIR INTRODUCCIÓN 5’ y 3’ para las amplificaciones de los extremos 5’ y 3’, respectivamente. Para la amplificación final de los extremos del gen a caracterizar se llevan a cabo dos reacciones de PCR, una para el extremo 5’ y otra para el extremo 3’, que emplearán respectivamente las dos poblaciones de ADNc 5’ y 3’ obtenidas anteriormente por retrotranscripción. Se utilizan además dos cebadores por cada reacción: uno complementario a la secuencia conocida del adaptador que se unió a los extremos 5’ o 3’ (LUP o SUP), y otro diseñado a partir de la parte conocida del gen que codifica la proteína (5’-RACE GSP o 3’-RACE GSP). Para su confirmación, los fragmentos son clonados y secuenciados. Una vez conocidos los extremos 5’ y 3’, la obtención del gen completo es una simple reacción de PCR empleando dos cebadores diseñados a partir de los extremos 5’ y 3’ conocidos del gen. La determinación de la secuencia de ADN que codifica la proteína antifúngica puede ser de gran interés para la transformación de otros microorganismos, con el fin de expresar la proteína con una alta eficacia para su producción a nivel industrial. Esto facilitaría su utilización como aditivo en alimentos en los que la presencia de mohos no sea deseable. Por otra parte, también podría servir para expresar la proteína en otros microorganismos aislados de productos cárnicos madurados que presenten unas características deseables, pero que sean incapaces de competir con el resto de la población fúngica. 19 MENÚ SALIR INTRODUCCIÓN ' 12 ! ! " 41 ' ) $ ' 2 5 4 1 ' )$ ' 2 $% & ' & '% & ' !" $6 # !" $6 4 & 54 )! 6 4+ 5 4 7 0 ! 6 4+ 5 4 & ' 2 ! !* Figura I6. Amplificación de un gen mediante RACE-PCR. 20 MENÚ SALIR INTRODUCCIÓN $% & '% & & ! " ' 12 # ' 12 2 .' ' ' ' ' =2 8 9 9 9 9 9 4 & 2 . ' ' ' ' '5 4 =2 8 9 9 9 9 9 4 54 ! " # 4 ((( 5 4 ) )) 2 .' ' ' ' ' 4 54 ((( ))) 2 .' ' ' ' ' 2 899999 2 899999 2 .' ' ' ' ' 4 54 54 4 () 54 4 4 ((( 54 )) ) " '% 4 2 89 9999 54 2 .' ' ' ' ' 4 () 4 * 541 ' )$ ( 54 ;: 54 2 899999 2 .' ' ' ' ' 54 2 899999 4 4 2 899999 ;: " $% * 4 2 .' ' ' ' ' 54 2 .' ' ' ' ' 2 .' ' ' ' ' 54 2 899999 54 54 54 4 $6 2 .' ' ' ' ' 54 4 41 ' ) $ ( : 54 ' < 4 ((( =2 8 9 9 9 9 9 ! ! 54 4 $6 54 =2 8 9 9 9 9 9 ) 0 4 54 4 4 54 4 4 54 541 ' ) $ ( 41 ' ) $ ( 4 54 4 4 ;: 4 1 ' )$ ( <) 0 " * 41 ' )$ 5 4 1 ' )$ ( <) 0 " * 5 41 ' )$ Figura I7. Amplificación Rápida de Extremos de ADNc (RACE-PCR). 21 : < MENÚ SALIR INTRODUCCIÓN I.6. EVALUACIÓN DE TOXICIDAD EN PROTEÍNAS ANTIFÚNGICAS Un importante prerrequisito para cualquier aplicación de proteínas antifúngicas es confirmar su inocuidad mediante ensayos de toxicidad. En este tipo de ensayos se usan células, tejidos vivos, líneas celulares u organismos, para evaluar los efectos de cualquier sustancia. Básicamente consisten en su exposición a determinadas concentraciones de la proteína a evaluar por un tiempo determinado. El material biológico empleado para los ensayos, ya sean células u organismos, debe tener alta sensibilidad a los tóxicos, ya que al establecer las concentraciones seguras para este tipo de material, se espera proteger a los organismos superiores sobre los que se pretenden emplear las proteínas antifúngicas. Uno de los métodos comúnmente empleados para demostrar la seguridad y selectividad de una proteína es el ensayo de actividad hemolítica (Hickey y col., 2003; Jin y col., 2009). En este método, la evaluación de la posible toxicidad de la proteína objeto de estudio se realiza empleando glóbulos rojos procedentes de mamíferos y comprobando la hemólisis producida. Si se produce hemólisis, la proteína tiene efectos citotóxicos y no es apta para su utilización; si por el contrario la proteína no ocasiona la lisis de los glóbulos rojos, merecerá seguir siendo estudiada. La ausencia de actividad hemolítica generalmente se acepta como prueba de que la proteína es segura (Van’t Hof y col., 2001). Algunas ventajas de la evaluación de la actividad hemolítica con respecto a otros métodos son que se evita el sacrificio de animales de experimentación y es un método que no requiere equipos o instrumentos específicos y puede ser realizado en cualquier laboratorio de investigación. Sin embargo, los ensayos in vitro a menudo no reproducen las condiciones fisiológicas en los consumidores, donde la proteína puede sufrir cambios que afecten a su toxicidad (Theis y Stahl, 2004). Por lo tanto, conviene complementar esta prueba con otro tipo de evaluaciones. Un método adicional para la comprobación de efectos citotóxicos en proteínas son los ensayos con líneas celulares, procedentes de células humanas que han perdido la capacidad de controlar su división celular. La ventaja de las líneas celulares es que tienen características similares a las células del mismo tipo en el organismo vivo, y a su vez evitan el sacrificio de animales de experimentación para la obtención de cultivos primarios. No obstante, ya que las líneas celulares han sufrido ciertas modificaciones en factores como los del control de la división celular, los cultivos primarios normalmente procedentes 22 MENÚ SALIR INTRODUCCIÓN de ratas o ratones de laboratorio siguen siendo ampliamente utilizados para la evaluación de la toxicidad (Szappanos y col., 2005, 2006). Entre los ensayos que emplean organismos vivos para la evaluación de la toxicidad, uno de los más útiles y sencillos de desarrollar es el empleo de larvas de Artemia salina. En este ensayo, las larvas son expuestas a la sustancia a evaluar y se calcula el porcentaje de mortalidad de las mismas tras un tiempo determinado. La principal ventaja de este método es que las larvas son especialmente susceptibles a sustancias tóxicas, junto con la fácil disponibilidad de este organismo. Además, las larvas de Artemia salina no requieren condiciones especiales de cultivo, simplemente necesitan ser incubadas en un medio que simula al agua de mar, donde normalmente se desarrolla este organismo. Por todo ello, este método ha sido ampliamente empleado en múltiples estudios, incluyendo la evaluación de la presencia de micotoxinas en alimentos (Núñez y col., 1996, 2007; López-Díaz y col., 2001; González y col., 2007), producción de micotoxinas de hongos patógenos de plantas (Moretti y col., 2007), en hierbas medicinales (González y col., 2007) o la evaluación de sustancias empleadas en odontología (Milhem y col., 2008) e incluso de nueva síntesis (Chohan, 2009). I.7. EFECTO DE DIFERENTES COMPUESTOS SOBRE LA ACTIVIDAD DE PROTEÍNAS ANTIFÚNGICAS. El efecto de diversos tratamientos o sustancias sobre la actividad antifúngica tiene gran interés para la caracterización de estas proteínas. En primer lugar permitirá formular hipótesis con respecto a los posibles mecanismos de acción implicados en la actividad que muestran frente a distintos mohos sensibles. Por otra parte, la influencia de determinados compuestos o procesos que puedan estar presentes o incorporarse en los alimentos condicionará en gran medida sus posibles aplicaciones. Por lo tanto, para su caracterización, se puede estudiar el efecto que ejercen en las proteínas diversos factores como tratamientos térmicos (Munimbazi y Bulleman, 1998; von der Weid y col., 2003; Skouri-Gargouri y Gargouri, 2008), diferentes valores de pH (Harighi y col., 2007), o distintas concentraciones de cationes (Almeida y col., 2000; Kaiserer y col., 2003), quelantes (Edlind y col., 2002), detergentes (Naeem y col., 2006) o fosfato (Liu y col., 2002). 23 MENÚ SALIR INTRODUCCIÓN I.7.1. TEMPERATURA En diversos alimentos es frecuente la aplicación de tratamientos térmicos, por lo que la estabilidad de la proteína antifúngica al calor será imprescindible para su posible aplicación en este tipo de productos. La termoestabilidad de una proteína está directamente relacionada con su estructura. De hecho las proteínas resistentes a altas temperaturas poseen gran cantidad de aminoácidos cargados y residuos aromáticos en su estructura primaria, son frecuentes las interacciones iónicas o modificaciones post-traduccionales en las mismas, y además son también muy estables al tratamiento con proteasas (Trivedi y col., 2006; Zhou y col., 2007). Además, las proteínas plegadas o con gran cantidad de puentes disulfuro son más resistentes a altas temperaturas. Las proteínas antifúngicas para uso en alimentos no requieren una alta termoestabilidad, ya que no irán destinadas a productos sometidos a un tratamiento de esterilización en los que generalmente no van a sobrevivir microorganismos. Por el contrario, sí puede ser de interés que resistan tratamientos equivalentes a la pasterización que se pueden aplicar en productos lácteos o cárnicos. I.7.2. pH La evaluación de la estabilidad de las proteínas antifúngicas ante distintos valores de pH permitiria evaluar su aplicación en alimentos ácidos o fermentados como embutidos o quesos madurados y encurtidos, siendo el pH de estos últimos incluso menor de 3,7. Del mismo modo que la temperatura, la resistencia de una proteína a diferentes pHs puede indicar el grado de estabilidad de su estructura. I.7.3. CATIONES El estudio de la influencia de distintos cationes sobre las proteínas es de gran valor para su caracterización, ya que muchas proteínas antimicrobianas, especialmente las que actúan sobre membranas, son sensibles a distintas concentraciones de los mismos (Theis y Stahl, 2004). No obstante existen proteínas antifúngicas, como algunas defensinas de plantas, que mantienen su actividad incluso con altas concentraciones de iones en el medio (Thevissen y col., 1999). La presencia de cationes puede además modificar la estructura o conformación de la proteína (Parker y col., 1996; Sissi y Palumbo, 2009) e incluso interferir en la unión con las membranas sobre las que actúan. En este tipo de proteínas los 24 MENÚ SALIR INTRODUCCIÓN cationes pueden interferir en la unión tanto con los fosfolípidos (Osborn y col., 1995; Thevissen y col., 1997, 1999), como con los receptores específicos sobre los que actúan en las membranas de los microorganismos sensibles (Almeida y col., 2000; Kaiserer y col., 2003; Theis y col., 2003). Además en algunos casos, el efecto sobre la actividad de la proteína es diferente si están presentes cationes monovalentes o divalentes (Broekaert y col., 1995). Por ello, la exposición de proteínas a diferentes cationes como Ca2+, Mg2+, Na+ o K+, puede ofrecer una valiosa información sobre el tipo de mecanismo de acción implicado en la actividad antimicrobiana. Por otro lado, la resistencia a cationes es esencial para la aplicación de proteínas antimicrobianas en alimentos con alta concentración de sales, como es el caso de algunos productos cárnicos madurados, ya que estas sales podrían provocar una disminución de la actividad de la proteína e incluso llegar a inactivarla (Theis y Stahl, 2004). De manera similar, una baja concentración de iones en el medio también puede modificar la actividad de una proteína, tanto potenciándola como inhibiéndola (Edlind y col., 2002). Por ello es necesario evaluar también el efecto sobre la proteína de secuestradores de iones como EDTA y EGTA. Algunos como EDTA y fosfato suelen ser empleados como quelantes de metales en alimentos no ácidos que requieren aditivos sin sabor (Calvo, 1991), por lo que es necesario estudiar la influencia de estos compuestos en la actividad antimicrobiana si se pretende aplicar la proteína en este tipo de productos. I.7.4. DETERGENTES Los detergentes, dependiendo de su carácter iónico, pueden influir en las proteínas de distinta manera, modificando su estructura e incluso desnaturalizándolas, o potenciando su actividad al favorecer la solubilidad de sus sustratos. Los detergentes no aniónicos, como tween 20, tween 80 y Triton X-100, se utilizan como suplementos en medios de cultivo para favorecer la dispersión de los microorganismos inoculados. Este tipo de detergentes solubilizan los sustratos sin alterar la estructura de las proteínas, por lo que no suelen ser muy agresivos contra microorganismos o proteínas. Por el contrario los detergentes iónicos, como docecil sulfato sódico (SDS) o ácido deoxicólico, tienen gran actividad antimicrobiana, ya que ocasionan la disrupción de las membranas y tienen efectos líticos, por lo que son conocidos como potentes 25 MENÚ SALIR INTRODUCCIÓN desnaturalizantes de proteínas. Usualmente, estos detergentes son utilizados para el estudio de proteínas ya que pueden alterar la conformación de las mismas. Así, la adición de ciertos detergentes puede llevar a la inducción de una estructura en α–hélice en ciertas proteínas (Naeem y col., 2006). El uso de detergentes no iónicos como emulsionantes también está extendido en la industria alimentaria. De hecho en España se emplean tween 20 y tween 80 como emulsionantes en confitería, repostería y para la elaboración de galletas (Calvo, 1991). La resistencia de la actividad antimicrobiana de las proteínas a estos detergentes haría que fuesen unas buenas candidatas para su uso en este tipo de alimentos. 26 MENÚ SALIR MENÚ SALIR ! $ "# % & " ! ' + '& , "# % $- ' ()' * % & . ( & " . / , % " " * % % ! $ ' " ! . $ %0 % "# %0 ! ! 3 ♦ 4 " $' 1 + ♦ . * % "! 2, ! ' ()' * ' + ' * $ , " % ! $ . ! * ♦ , ! ' ()' * ' + ' ♦ 0 & %0 ! $ % $ " * " % ' ()' * ' + '$ % MENÚ SALIR " " 6 ' ()' " , " " ' + '6 *2 7 6 &" 8 & $ " % " '+ ' " $9 6 . ' ()' *$ ) & " ' + '$ "" 6 : 6 . " 6 " " 1 , "' ()' * '+ ' $ ♦ , " ♦ % * %*% " *$ "" " " , 6 :6 : 6 $ %" *" % " ♦ 6 , %0 ♦ " " " "" " " % . "" & 6 6 * "' ()' 6 " $ * " " * "' ()' 5 ' + '$ $ * "% $ MENÚ SALIR MENÚ SALIR ! '( )* "! # ! $%! & & )+, - % & . / & ! "! # ! $%! & 01 )2 & 3 . 4 3 / 5 !$( 3) & / , 1 ), 1 ), 3$ & 6 ), ! ! "! # ! $%! & & & 0"! $2 / & &/ 7 . 3$ & 3$ & 5 , / , " % 3 & 0) )3! . / & 2 # # 0 )13 )2 3 3 & 7 - / , 8& # 7 , MENÚ SALIR & &# . 3 #8& 3 7 - , & #0 )13 ); )2, 3 6< 8& % 6 ), # $ # & 3 = , 3 / /< ! . !$( 8& / , % $ & # & &# . 3 1 & (4 / 8& , 3( 4 3 3 / 7 5 5 , / !$( # ' ( 4 ! 5 3 4 , ) & 3 !$( 0( $ 3!$(2, = 3 , . % , & ! , 9: "! MENÚ SALIR * ( + ! ) % 3 # & 8& - /< 7? # 8& # . 8& # / # & , 5 & & . # 8& - 8& 3 & & 8& # & @ / & , 3 & ? 8& ? # / / , , - ! & >9, :? % & )!)) 7 04 A - &B #, 9> & 2#& MENÚ SALIR MENÚ SALIR MENÚ SALIR RESULTADOS Characterization of PgAFP, a novel antifungal protein from Penicillium chrysogenum Andrea Rodríguez-Martín, Susan Liddella, Raquel Acosta, Félix Núñez, Miguel A. Asensio* Higiene y Seguridad Alimentaria, Facultad de Veterinaria. Universidad de Extremadura, Avda. de la Universidad, s/n. 10071- Cáceres, Spain. a Division of Animal Sciences, University of Nottingham. Sutton Bonington Campus. Loughborough, Leicestershire. LE12 5RD. United Kingdom. * Corresponding author: Telephone (+34) 927 257 125; fax: (+34) 927 257 110. E-mail: [email protected] ABSTRACT A new antifungal protein PgAFP from Penicillium chrysogenum is described. The protein showed a molecular weight of 6.5 kDa by electrospray ionization mass spectrometry (ESIMS). Peptide sequences were obtained by digesting the protein with trypsin and chymotrypsin and analyzing the resulting peptides by ESI-MS/MS. The obtained sequences showed high similarity with the antifungal protein Anafp from Aspergillus niger. No evidence for N- or O- deglycosylations was observed upon chemical and enzymatic treatments of PgAFP. PgAFP is a new protein that belongs to a group of small, cysteine-rich, and basic proteins with antifungal activity. However, the characterization of PgAFP should be exhaustive to demonstrate that this protein is harmless to humans before any potential use as preservative in foods. KEY WORDS: antifungal protein, dry-fermented food, growth inhibition, posttranslational modifications. INTRODUCTION The antifungal protein PgAFP was purified in previous studies by fast protein liquid chromatography (Acosta et al., 2009a) from the strain RP42C, which was isolated from dry-cured ham and characterized as P. chrysogenum based on classical taxonomical methods (Pitt and Hocking, 1997). PgAFP showed a strong antifungal activity against selected toxigenic moulds (Acosta et al., 2009a). 33 MENÚ SALIR MANUSCRITO 1 Many fungal proteins have been reported including ribosome inactivating proteins, antifungal proteins, ribonucleases, ubiquitin-like proteins, lectins, cellulases, xylanases, laccases, invertases, and trehalose phosphorylases (Ng, 2004). There is only scant information available about antifungal proteins of fungal origin. PgAFP showed some similarity (Acosta et al., 2009b) to a group of antifungal peptides secreted filamentous fungi of the group Ascomycetes, composed of proteins with common characteristics including being cysteine rich, highly basic, and of low molecular weight. Only four proteins have been reported in this group: PAF from Penicillium chrysogenum (Marx et al., 1995), AFP from Aspergillus giganteus (Nakaya et al., 1990; Lacadena et al., 1995), Anafp from Aspergillus niger (Lee et al., 1999) and AcAFP from Aspergillus clavatus (Skouri-Gargouri and Gargouri, 2008). The amino acid sequence for all these proteins is known, except for AcAFP. In previous studies (Acosta et al., 2009b) PgAFP was positive for glycosylation by Periodic Acid Schiff reaction (Deepak et al., 2003). No glycosylations have been described for PAF, AFP, Anafp, and AcAFP, but this kind of post-translational modification has been described in the antifungal protein from Urginea indica (Deepak et al., 2003) or chitosanases from Mycobacter AL-1 and P. islandicum (Hedges and Wolfe, 1974; Fenton and Eveleigh, 1981). The aim of this work was to determine the amino acid sequence and molecular weight of PgAFP. In addition, PgAFP was exposed to enzymatic and chemical deglycosylation treatments, and analyzed for possible changes in the protein by SDS-PAGE and electrospray ionization mass spectrometry. The genetic identity of the producer mould was also confirmed. MATERIALS AND METHODS Microbial strain The strain RP42C was isolated from dry-cured ham, and it was identified as Penicillium chrysogenum according to classical morphological and biochemical studies (Acosta et al., 2009a). This mould produces the antifungal protein PgAFP (Acosta et al., 2009a). 34 MENÚ SALIR RESULTADOS Genetic identification of the producer mould Genetic characterization of RP42C was carried out by amplifying the fragments 18S-28S rRNA intergenic spacer (ITS) by polymerase chain reaction (White et al., 1990) and determining the DNA sequence. DNA isolation. The mould RP42C was grown in malt extract broth made of glucose 2% (w/v), malt extract 2% (w/v), and peptone 0.1% (w/v), pH 4.5, at 25ºC for 5 days. After freezing by adding liquid nitrogen, two grams of mycelium were ground up using a mortar and pestle. Lysis was performed by homogenizing the obtained powder in 4 ml 0.05 M Tris-HCl, pH 8, 0.005 M ethylenediamine tetraacetic acid (EDTA), 0.05 M NaCl (TES) buffer and 1% (w/v) sodium dodecyl sulfate (SDS). Afterwards, 200 µl proteinase K (20 µg/µl) were added, and the mixture was incubated at 60ºC for 35 min, then cooled on ice and extracted with phenol-chloroform-isoamyl alcohol (25:24:1). The suspension was centrifuged at 3,000 ×g for 2 min at 4ºC. The upper phase was transferred to a fresh tube and DNA was precipitated by the addition of 3 M sodium acetate, pH 5.2 to a final concentration of 10% (w/v) and two volumes of cold ethanol. After centrifugation, the pellet was cleaned with ethanol at 70%, centrifuged again, resuspended in sterile water and treated with 50 µl RNase (10 µg/µl) at 37ºC for 60 min. Finally, DNA was extracted with phenol-chloroform-isoamyl alcohol and precipitated again as indicated above, resuspended in water and stored at -70ºC. The quantity and quality of the purified DNA was determined using a Biophotometer (Eppendorf AG, Hamburg, Germany). ITS amplification. DNA fragments for sequence analysis were obtained by PCR using primers ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4 (TCCTCCGCTTATTGATAT GC). Amplified products comprised the internal transcribed spacer 1 (ITS1), 5.8S, and ITS2 regions of the ribosomal DNA. For each 50 µl reaction, a mixture was prepared containing 50 ng of genomic DNA, 0.1 vol. of 10× PCR buffer (10 mM Tris-HCl, pH 8.8, 50 mM KCl, 0.1% Triton X-100), 4 mM MgCl2, 1 mM of deoxynucleoside triphosphates (Roche, Mannheim, Germany), 0.4 µM of each primer, and 2 U of DyNAzyme I DNA Polymerase (Finnzymes, Espoo, Finland). Reactions were run on a programmable thermal cycler Mastercycler epgradient (Eppendorf AG) with an initial denaturation of 3 min at 96°C, followed by 30 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 40 s, with a final extension at 72°C for 10 min. Aliquots of the PCR were electrophoresed on 1% (w/v) agarose gels in 1× TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8) using a Sub-Cell GT apparatus (Bio-rad Laboratories, Hercules, USA). Products were visualized using the 35 MENÚ SALIR MANUSCRITO 1 UV transillumination G-Box (Syngene, Fredericks, USA) and photographed with a GeneSnap camera (Syngene). DNA fragments were gel-purified and sequenced at the Institute of Biomedicine (CSIC, Valencia, Spain). The sequences obtained were searched against the public databases using the Basic Local Alignment Search Tool (BLAST). PgAFP purification P. chrysogenum RP42C was grown in malt extract broth at 25ºC for 15 days. The medium was filtered through a 0.45 µm diameter pore size cellulose acetate membrane and PgAFP was purified using an ÄKTA fast protein liquid chromatography system (Amersham Pharmacia Biotech AB, Uppsala, Sweden) according to Acosta et al. (2009a). For this, filtered medium was loaded on a HiTrap SP HP 5 ml ion exchange column (Amersham Pharmacia Biotech AB) equilibrated with 0.02 M sodium acetate buffer, pH 4.5. Bound proteins were eluted with a gradient of NaCl to 1 M (0 to 25% in 15 column volumes (CV), increasing to 100% in 2 CV, followed by 15 CV at 100%) in 0.02 M sodium acetate buffer. Eluted proteins were detected at 214 nm. The fraction that showed antifungal activity in previous studies (Acosta et al., 2009a) was further purified using a HiLoad 26/60 Superdex 75 prep grade gel filtration column (Amersham Pharmacia Biotech AB) equilibrated with 50 mM sodium phosphate, 0.15 M NaCl buffer, pH 7, at a constant flow rate of 2.5 ml/min. Data collection and processing were performed using the UNICORN software 4.12 (Amersham Pharmacia Biotech AB). Finally, the fraction containing PgAFP was desalted and concentrated with YM-3 Microcon Centrifugal Filter Units (Millipore Iberica S.A.U., Madrid, Spain), before storage at -20ºC. Protein characterization Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE was performed as described Laemmli (1970). The protein PgAFP was separated on 4% (v/v) polyacrylamide stacking and 12% (v/v) polyacrylamide resolving gels. Gels were stained with Imperial Protein Stain (Pierce Biotechnology, Rockford, USA). Two molecular weight marker sets were used: Precision Plus Protein Standards with ten proteins between 10 and 250 kDa (Bio-Rad) and Low Molecular Weight Electrophoresis calibration kit with proteins between 14.4 and 97 kDa (Pharmacia, Uppsala, Sweden). Molecular weight determination by ESI-MS. The molecular weight of PgAFP was determined by electrospray mass spectrometry in a Q-TOF2 mass spectrometer (Waters 36 MENÚ SALIR RESULTADOS Ltd, Manchester, UK). The protein was concentrated and dissolved in ammonium acetate. PgAFP (0.1 µg/µl) with 1% (v/v) formic acid was desalted using C18 reverse phase pipette Zip-Tips (Millipore) according to the manufacturer’s instructions. Samples were eluted into 50% (v/v) methanol, 0.1% (v/v) formic acid, and were loaded into nanospray needles (Waters) for analysis. The mass spectrometer was operated with a capillary voltage of 9001200 V and the sampling cone at 45-50 V. Data were acquired between 400 and 3000 m/z with a cycle time of 1 second. Mass spectra resulted from combining typically 5 min of data. Spectra were deconvoluted using the MaxEnt1 maximum entropy software (Waters) with an output range of 4,000 and 11,000 Da, at a resolution of 1 Da and a peak width of 0.75 Da. Reduction and alkylation of PgAFP. Reduction and alkylation of the protein prior to deglycosylation or enzymatic digestion were performed with the protein both in acrylamide gel pieces and in solution. After SDS-PAGE, PgAFP (25 µg per well) was stained using Imperial Protein Stain. The band was excised from the gel and washed three times by incubation in 100 µl of de-stain solution made of 50 mM ammonium bicarbonate and 50% acetonitrile for 15 min at 40ºC with intermittent vortexing. Gel pieces were incubated at 56ºC in 100 µl of reducing solution (10 mM dithiothreitol in 50 mM ammonium bicarbonate) that was removed after 45 min. One hundred µl of alkylation solution (55 mM iodoacetamide in 50 mM ammonium bicarbonate) was added and incubation continued at room temperature in the dark for 45 min. The gel slice was then washed in 100 µl of 50 mM ammonium bicarbonate for 5 min at 40ºC and the supernatant was discarded. Gel pieces were washed twice in 100 µl of 50 mM ammonium bicarbonate in 50% (v/v) acetonitrile for 1 min, and centrifuged. Sample was dehydrated in 100 µl of acetonitrile and incubated for 1-2 min. Finally, the supernatant was discarded and the gel pieces were dried at 40ºC for a few min. After addition of the digestive enzyme, the tube containing the gel pieces was cooled at 4°C for 20 min and finally incubated at the optimal temperature of each enzyme. To digest PgAFP in solution, different quantities of the protein (12.5, 25 and 62.5 µg) were added to tubes with 50 mM ammonium bicarbonate up to a volume of 5 µl. Dithiothreitol 5 mM (dissolved in 50 mM ammonium bicarbonate) was added (5 µl) and the final solution was incubated at 50ºC for 30 min. After adding 10 µl of 20 mM iodoacetamide (dissolved in 50 mM ammonium bicarbonate), sample was incubated at room temperature for 30 min in the dark before the digestion with each deglycosidase or protease. 37 MENÚ SALIR MANUSCRITO 1 Analysis for glucidic residues. Chemical deglycosylation was carried out with PgAFP in solution using two different chemical methods: acid hydrolysis with HCl (Krapf et al., 2006; Leis et al., 1997) and -elimination with NaOH (Geert and Thomas-Oates, 1998; Tarelli, 2007). For acid hydrolysis with HCl, 1 µg of PgAFP was incubated in 20 mM HCl at 50ºC for 3 h. -elimination with NaOH was performed incubating 1µg of PgAFP with 0.2 M NaOH at 45ºC for 16 h. Finally, both samples were analyzed by ESI-MS. Enzymatic deglycosylation was carried out using endoglycosidase F1 (Northstar BioProducts, Liverpool, UK), PNGase F (New England Biolabs, Ipswich, UK), and the EDEGLY commercial kit (Sigma, Madrid, Spain). PgAFP protein (200 µg) was treated with 4 µl of endoglycosidase F1 in 250 mM NaH2PO4 at pH 4.5, the sample was incubated at 37ºC for 1 h, and analyzed by ESI-MS. PNGase F was used following the manufacturer’s instructions. Two additional assays were performed without detergent with PgAFP in-gel and in-solution, one with a prior reduction and alkylation as above and another one heating PgAFP at 100ºC for 5 minutes before adding the enzyme. All samples were analyzed by SDS-PAGE and ESI-MS. The E-DEGLY deglycosylation kit (Sigma), containing PNGase F, -2(3,6,8,9) neuraminidase, O-glycosidase, (1-4)-galactosidase, and −N- acetylglucosaminidase, was also used. Reactions were carried out under native and denaturing conditions according to the manufacturer’s instructions and analysed by SDSPAGE. Amino acid sequencing Digestion with enzymes Trypsin digestion. PgAFP was digested with trypsin gold (Promega, Hampshire, UK). Different treatments with and without reduction and alkylation steps were performed prior to digestion. Gel piece samples were incubated with trypsin gold (10 ng/µl) in 50 mM ammonium bicarbonate at 40ºC or 60ºC for 1.5, 3, and 16 h. PgAFP in-solution was incubated at 37ºC for 16 h in 50 mM ammonium bicarbonate, 5% (v/v) acetonitrile and 10 ng/µl of trypsin gold. Chymotrypsin digestion. Digestions with the reduced and alkylated protein were performed with PgAFP in-gel and in-solution. Gel pieces containing PgAFP were digested at different concentrations (1, 5, 10, and 15 ng/µl) of chymotrypsin (Roche, Welwyn Garden City, UK) in buffer (100 mM Tris-HCl, 10 mM CaCl2, pH 7.8) at 25ºC and 30ºC for 16 h. 38 MENÚ SALIR RESULTADOS PgAFP in-solution was digested by chymotrypsin at 0.1, 1, 5 or 10 ng/µl with 5% acetonitrile and the same buffers detailed above. Temperatures and incubation time were identical to the in-gel samples. Electrospray ionization mass spectrometry (ESI-MSMS) analysis for peptide sequence determination Peptide samples were made to 0.1% (v/v) formic acid, then desalted using Zip-Tip C18 reverse phase pipette tips (Millipore) according to manufacturer’s instructions. Peptides were eluted with 50% (v/v) acetonitrile, 0.1% (v/v) formic acid. Samples were loaded into individual borosilicate nanospray needles (Waters) using 1-10 µl GELoader pipette tips (Eppendorf) and analyzed in a Q-TOF2 mass spectrometer (Waters), a high resolution hybrid orthogonal angle-Time of Flight tandem LC mass spectrometer, fitted with a nanoflow ESI (electrospray ionization) source (Waters). The mass spectrometer was operated with a capillary voltage of 900–1,200 V in positive ion mode, using argon as the collision gas. Survey scans of peptide mass spectra were performed with the sampling cone set at 45–50 V and data were acquired from 400–1,600 m/z with a cycle time of 2.4 seconds. Peptide mass spectra presented resulted from combining typically 5 min of data acquisition. Individual peptide ions were selected manually for tandem MS analysis. Collision voltages were optimised manually for individual peptides. Tandem MS fragmentation spectra were collected typically from 50 to 1,600 m/z. Tandem MS spectra were deconvoluted into singly charged, mono-isotopic masses using the MaxENT3 maximum entropy software, and peptide sequences were determined by manual interpretation using the PepSeq software within the MassLynx V 4.0 package (Waters). De novo peptide sequences were searched against the public database BLASTP using parameter settings for “short and nearly exact matches”. RESULTS Molecular genetic characterisation of P. chrysogenum RP42C Amplification of the 18S-28S rRNA intergenic spacer (ITS) sequence produced a band with 394 bp. The DNA sequence of the amplified was determined and compared against other sequences using BLAST. The sequence from RP42C mould shared 100% of identity and showed a maximum score with the 18S-28S rRNA intergenic spacer of Penicillium 39 MENÚ SALIR MANUSCRITO 1 chrysogenum strain L1 (Accession number in GenBank: FJ467371). Thus, the producer mould RP42C was confirmed as Penicillium chrysogenum. Molecular weight determination The molecular mass of purified PgAFP was estimated from its migration on SDS-PAGE. The protein migrated as a single band of approximately 10 kDa (Figure 1). Figure 1. SDS-PAGE of PgAFP (5 µg). M: Pharmacia Low Molecular Weight Markers. In ESI-MS, PgAFP resolved as a short series of multiply charged peaks, with the dominant peak set of around 1,083 Da corresponding to the +6 charge state (Figure 2). The m/z spectrum was deconvoluted and converted to the molecular mass profile using Maximum Entropy processing (Figure 3). The mass profile shows a dominant peak of 6,494 Da, corresponding to the mass of mature PgAFP. 40 MENÚ SALIR RESULTADOS Figure 2. Electrospray mass spectra of PgAFP. A) Spectrum showing the major multiply charged peaks. B) Expanded spectrum showing the +6 charge state peak set. 41 MENÚ SALIR MANUSCRITO 1 6494.29 6592.22 mass Figure 3. Molecular mass profile from Maximum Entropy processing. PgAFP resolves as a single major peak with a mass of 6,494 Da. Analysis for glucidic residues Analysis of PgAFP for glycosylations did not show any evidence of N- or O-linkedoligosaccharides. Chemical deglycosylation with acid hydrolysis or -elimination revealed no changes in the mass spectrum profiles obtained in ESI-MS compared with untreated controls. No differences on gel-mobility of PgAFP were observed on SDS-PAGE after attempting deglycosylation by enzymatic means using endoglycosidase F1 (data not shown), PNGase F (Figure 4), or the E-DEGLY deglycosylation kit (Figure 4). ESI-MS analysis showed no change in the mass spectrum profiles of PgAFP treated with endoglycosidase F1 and PNGase F (data not shown). 42 MENÚ SALIR RESULTADOS Figure 4. SDS-PAGE analysis of attempted enzymatic deglycosylation of PgAFP. A) PNGase F-treated PgAFP (1) untreated PgAFP (2). B) E-DEGLY deglycosylation kit under denaturing conditions (1, 2) or native conditions (3, 4). 1 and 3 are untreated controls. M: Protein molecular weight standards. Peptide sequences The peptides obtained from PgAFP after digestion by trypsin and chymotrypsin were analyzed by electrospray ionization tandem mass spectrometry and the amino acid sequences deduced. Mass spectrum profiles of peptides obtained from PgAFP digestions are shown in Figure 5. There were no major differences in the peptide profiles for in-gel compared to in-solution digestions. Six de novo peptide sequences were obtained by treatment with trypsin and chymotrypsin (Figure 5, Table 1). The survey spectrum and the processed tandem MS spectrum of a representative peptide are shown in Figure 6. 43 MENÚ SALIR MANUSCRITO 1 Figure 5. Mass spectra of PgAFP peptides after trypsin (A) and chymotrypsin (B) digestions. Table 1. De novo obtained peptide sequences. Lower case letters indicate amino acids which were tentative assignments. Dashes indicate the presence of additional amino acids which were unassigned. Note that L and I are interchangeable here since these two residues are isobaric. Sequence Number de novo sequence Peptide Charge Digestive Mass state enzyme 1 LSKFGGECSL 549.26 +2 Chymotrypsin 2 FGGECSLK 449.24 +2 Trypsin 3 HNTCTYLK 490.26 +2 Trypsin 4a NHVVNCGSAANKK 466.88 +3 Trypsin 607.308 +2 Trypsin 4b NHVVNCGSAANK 5 -yDEHHK 468.19 +2 Trypsin 6 -cqTPV 540.92 +3 Chymotrypsin 44 MENÚ SALIR RESULTADOS Figure 6. ESI MS (A) and MS/MS spectrum (B) of a PgAFP tryptic peptide with a charge state of +3 and precursor mass of 607.308 (peptide 4b NHVVNCGSAANK in Table 1). 45 MENÚ SALIR MANUSCRITO 1 DISCUSSION To confirm the previous identification of PgAFP based on morphology and biochemical characteristics (Acosta et al., 2009a), the 18S-28S rRNA intergenic spacer was analyzed. The obtained sequence confirmed that the producer mould RP42C belongs to P. chrysogenum. The molecular mass of PgAFP obtained by SDS-PAGE was estimated to be over 10 kDa (Figure 1), whereas ESI-MS revealed a lower mass of 6.5 kDa. For large molecules, such as intact proteins, ESI-MS is capable of determining the molecular weight to within 0.01% of the total molecular weight. For a mass of 6,494 Da, this is 0.65 Da. Therefore, it seems likely that PgAFP migrates anomalously in SDS-PAGE. Many characteristics of proteins can cause them to migrate atypically on SDS-PAGE, in particular, lipoproteins and glycoproteins migrate anomalously because SDS cannot bind with lipids or oligosaccharides (Grefrath and Reynolds, 1974). In previous studies (Acosta et al., 2009b) PgAFP showed evidences of glycosylations by Periodic Acid Schiff reaction (Deepak et al., 2003). Of all protein postranslational modifications currently known, such as acetylation, phosphorylation, formylation, or glycosylation, the last is by far the most common. Moreover, glycosylated proteins are not uncommon among antifungal proteins, such as an antifungal glycoprotein from Urginea indica (Deepak et al., 2003) or chitosanases from Mycobacter AL-1 and P. islandicum (Hedges and Wolfe, 1974; Fenton and Eveleigh, 1981). We therefore used a range of enzymatic and chemical deglycosylation methods to assess possible N-linked and O-linked glycosylation of PgAFP. We found no evidence of N- or O-linked oligosaccharides. Although in some cases the assay products were assessed using only SDS-PAGE, in which the resolution may have been insufficient to reveal relatively small mass changes, no evidence of mass changes were found using highly resolving MS analysis. Atypical migration on SDSPAGE can be due to other causes that do not involve post-translational modifications. For example, proteins with extreme isoelectric points can show a higher apparent molecular weight (around a 25% higher) due to electrostatic repulsions causing less SDS to bind and a consequent decrease in their migration velocity (Mondstadt and Holldorf, 1990). Notably, the 42 residues obtained using de novo sequencing that together constitute around 72% of PgAFP (a calculated molecular weight of 4.7 kDa, compared with the 6.5 kDa mass observed by ESI-MS), have a predicted pI of pH 8.8. In addition, in previous studies 46 MENÚ SALIR RESULTADOS this protein showed an experimental isoelectric point of pH 9.2 by capillary isoelectric focusing electrophoresis (Acosta et al. 2009b). Amino acid sequences of PgAFP peptides were obtained using tandem MS and de novo sequencing (Table 1). The sequences were searched against the public sequence databases to find any similar proteins present. A multiple alignment was performed with the most similar proteins (Figure 7). PgAFP Anafp A.niger PAF AFP A.clavatus LSKFGGECSLKHNTCTYL ------------------------------------------LSKYGGECSLEHNTCTYR ---MQLTSIAIILFAAMGAIANPIAAE---ADNLVARE--AELSKYGGECSVEHNTCTYL ---MQITTVALFLFAAMGGVATPIES---VSNDLDARAEAGVLAKYTGKCTKSKNECKYK MKFVSLASLGFALVAALGAVATPVEADSLTAGGLDARDESAVLATYNGKCYKKDNICKYK MKVVSLASLGFALVAALGVVASPVDADSLAAGGLDARDESAVQATYDGKCYKKDNICKYK PgAFP Anafp A.niger PAF AFP A.clavatus -K HVVNCGSAANKK yDEHHK cqTPV -KDGKNHVVSCPSAANLRCKTDRHHCRYDDHHKTVDCQTPV -KGGKDHIVSCPSAANLRCKTERHHCEYDEHHKTVDCQTPV NDAGKDTFIKCPKFDNKKCTKDNNKCTVDTYNNAVDCD--AQSGKTAICKCYV---KKCPRDGAKCEFDSYKGKCYC---AQSGKTAICKCYV---KVCPRDGAKCEFDSYKGKCYC---- Figure 7. Alignment of PgAFP amino acid sequences with the most similar proteins: Anafp (Lee et al., 1999), XP_001391221 (hypothetical protein from Aspergillus niger CBS 513.88), AAA92718 (PAF from P. chrysogenum), P17737 (AFP from A. giganteus), and XP_001267787 (hypothetical protein from A. clavatus). The PgAFP amino acid sequences had the highest homology with Anafp (81%), and a hypothetical protein (79 %), both from Aspergillus niger. Three other proteins had lower levels of similarity with PgAFP; PAF (33%), AFP (26%), and a hypothetical protein from Aspergillus clavatus (21%). Both PgAFP and PAF are produced by P. chrysogenum, but they are clearly different proteins. The aligned proteins and PgAFP have common characteristics: they are small (5.8-6.6 kDa), cysteine-rich, and basic, due to the presence of a high amount of lysine residues (Marx, 2004). The mature proteins PAF and AFP are cleavage products of precursor forms which carry a pre-pro-sequence, with a signal peptide and a prosequence that keep the proteins in an inactive form until exported to the extracellular medium (Marx, 2004). Since PgAFP may belong to this group of proteins, it could also be produced as a precursor form. In contrast with other antifungal proteins, such as some chitosanases, no glycosylations have been described in this group. The apparent lack of glycosylation of PgAFP is consistent with it being a member of this group. 47 MENÚ SALIR MANUSCRITO 1 Recently, the genome of P. chrysogenum Wisconsin 54-1255 has been sequenced (Van den Berg et al., 2008). A putative protein sequence is present (accession number CAP80456) which shows homology with the amino acid sequences obtained from PgAFP in this work. However, the production of the protein by this mould has not been reported. PgAFP is a new antifungal protein obtained from P. chrysogenum. Since PgAFP was produced by a species generally recognized as safe by the United States Food and Drug Administration (Marx, 2004) it may be permissible for use as preservative in foods. However, the characterization of PgAFP should be exhaustive to demonstrate that this protein is harmless to humans. For this, future objectives will include obtaining the coding sequence of this novel and potent antifungal protein. ACKNOWLEDGEMENTS This work was supported by the Spanish Ministry of Education and Science (AGL200406546-ALI) and FEDER. Andrea Rodríguez and Raquel Acosta were recipient of FPI grants from the Spanish Ministry of Education and Science. REFERENCES Acosta, R., Rodríguez-Martín, A., Martín, A., Núñez, F., and Asensio, M.A. Selection of antifungal protein-producing molds from dry-cured meat products. Int. J. Food Microbiol. Submitted (2009a). Acosta, R., Rodríguez-Martín, A., Bermúdez, E., Núñez, F., and Asensio, M.A. Partial characterization and antifungal activity of a protein from Penicillium chrysogenum. Food Microbiol. Submitted (2009b). Deepak, A.V., Thippeswamy, G., Shivakameshwari, M.N., and Salimath, B.P. Isolation and characterization of a 29-kDa glycoprotein with antifungal activity from bulbs of Urginea indica. Biochem. Biophys. Res. Comm. 311: 735-742 (2003). Fenton, D. M. and Eveleigh, D. E. Purification and mode of action of a chitosanase from Penicillium islandicum. J. Gen. Microbiol. 126: 151-165 (1981). Geert, J. D. and Thomas-Oates, J. Analysis of glycoproteins and glycopeptides using fast-atom bombardment. Methods Mol. Biol. 61: 231-241 (1998). Grefrath, S.P. and Reynolds, J.A. The molecular weight of the major glycoprotein from the human erythrocyte membrane. Proc. Natl. Acad. Sci. U S A. 71: 3913-3916 (1974). 48 MENÚ SALIR RESULTADOS Hedges, A. and Wolfe, R. S. Extracellular enzyme from Myxobacter Al-1 that exhibits both -1,4 glucanase and chitosanase activities. J. Bact. 120: 844-853 (1974). Krapf, D., Vidal, M., Arranz, S. E., and Cabada, M. O. Characterization and biological properties of L-HGP, a glycoprotein from the amphibian oviduct with acrosome-stabilizing effects. Biol. Cell. 98: 403–413 (2006). Lacadena, J., Martínez del Pozo, A., Gasset, M., Patiño, B., Campos-Olivas, R., Vázquez, C., Martínez-Ruiz, A., Mancheño, J.M., Oñaderra, M., and Gavilanes, J.G. Characterization of the antifungal protein secreted by the mould Aspergillus giganteus. Arch. Biochem. Biophys. 20: 273-281 (1995). Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685 (1970). Lee, D.G., Shin, S.Y., Maeng, C-Y., Jin, Z.Z., Kim, K.L., and Hahm, K-S. Isolation and characterization of a novel antifungal peptide from Aspergillus niger. Biochem. Biophy. Res. Commu. 263: 646-651 (1999). Leis, O., Madrid, J.F., Ballesta, J., and Hernández, F. N- and O-linked oligosaccharides in the secretory granules of rat paneth cells: an ultrastructural cytochemical study. J. Histoch. Cytoch. 45: 285-294 (1997). Marx, F., Haas, H., Reindl, M., Stöffler, G., Lottspeich, F., and Redl, B. Cloning, structural organization and regulation of expression of the Penicillium chrysogenum paf gene encoding an abundantly secreted protein with antifungal activity. Gene. 167: 167-171 (1995). Marx, F. Small, basic antifungal proteins secreted from filamentous ascomycetes: a comparative study regarding expression, structure, function and potential application. Appl. Microbiol. Biotechnol. 65: 133-142 (2004). Mondstadt, G.M. and Holldorf, A.W. Arginine deaminase from Halobacterium. salinarum: purification and properties. Biochem. J. 273: 739-745 (1990). Nakaya, K., Omata, K., Okahashi, I., Nakamura, Y., Kolkenbrock, H., and Ulbrich, N. Amino acid sequence and disulfide bridges of an antifungal protein isolated from Aspergillus giganteus. Eur. J. Biochem. 193: 31-38 (1990). Ng, T.B. Peptides and proteins from fungi. Peptides. 25: 1055-1073 (2004). 49 MENÚ SALIR MANUSCRITO 1 Pitt, J.I. and Hocking, A.D. Fungi and food spoilage. Blackie Academic and Professional. London, United Kingdom (1997). Skouri-Gargouri, H. and Gargouri, A. First isolation of a novel thermostable antifungal peptide secreted by Aspergillus clavatus. Peptides. 29: 1871-1877 (2008). Tarelli, E. Resistance to deglycosylation by ammonia of IgAl O-glycopeptides: implications for the -elimination of O-glycans linked to serine and threonine. Carboh. Res. 342: 2322-2325 (2007). Van den Berg, M.A., Albang, R., Albermann, K., Badger, J.H., Daran, J.M., Driessen, A.J., Garcia-Estrada, C., Fedorova, N.D., Harris, D.M., Heijne, W.H., Joardar, V., Kiel, J.A., Kovalchuk, A., Martin, J.F., Nierman, W.C., Nijland, J.G., Pronk, J.T., Roubos, J.A., van der Klei, I.J., van Peij, N.N., Veenhuis, M., von Dohren, H., Wagner, C., Wortman, J., and Bovenberg, R.A. Genome sequencing and analysis of the filamentous fungus Penicillium chrysogenum. Nat. Biotechnol. 26: 1161-1168 (2008). White, T.J., Bruns, T., Lee, S., and Taylor, J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Editors: M. A. Innis, D. H. Gelfand, J. J. Sninsky, J.S. and T. J. White. PCR Protocols: A Guide to Methods and Applications. Academic Press, Inc., San Diego. Pages 315-322 (1990). 50 MENÚ SALIR Manuscrito 2: Genetic characterization of the gene encoding the novel antifungal protein PgAFP from Penicillium chrysogenum MENÚ SALIR RESULTADOS Genetic characterization of the gene encoding the novel antifungal protein PgAFP from Penicillium chrysogenum Andrea Rodríguez-Martín, Raquel Acosta, Félix Núñez, Mª José Benitoa, Miguel A. Asensio* Higiene y Seguridad Alimentaria, Facultad de Veterinaria. Universidad de Extremadura, Avda. de la Universidad, s/n. 10071- Cáceres, Spain. a Nutrición y Bromatología, Escuela de Ingenierías Agrarias, Universidad de Extremadura, Carretera de Cáceres s/n, 06071- Badajoz, Spain * Corresponding author: Telephone (+34) 927 257 125; fax: (+34) 927 257 110. E-mail: [email protected] ABSTRACT The strain RP42C from Penicillium chrysogenum produces a small protein PgAFP that inhibits the growth of some toxigenic moulds. The gene encoding the precursor of this antifungal protein has been characterized. cDNA was prepared from isolated total RNA by RT-PCR. The cDNA fragment encoding the pgafp gene was obtained by PCR using degenerate primers based on two known amino acid sequences of the purified PgAFP. Full-length cDNA of pgafp was obtained by RACE-PCR. Comparison of nucleotide sequence from genomic fragment and cDNA of the gene, revealed the presence of a 279 bp coding region which is interrupted by two introns of 63 and 62 bp in length. The precursor of the antifungal protein consists of 92 amino acids and appears to be processed to the mature 58 amino acids PgAFP by two-step process. The deduced amino acid sequence of the preproprotein revealed 75 % identity to an antifungal protein predicted from the Aspergillus niger CBS 513.88 genome. KEY WORDS: preproprotein, protective cultures, dry fermented foods. INTRODUCTION The strain RP42C of the mould Penicillium chrysogenum secretes a small protein PgAFP that has been purified for its ability to inhibit toxigenic moulds isolated from dry-cured meat products (Acosta et al., 2009a,b). Several amino acid sequences of this protein were obtained by electrospray ionization mass spectrometry (Rodríguez-Martín et al., 2009) revealing a high homology with the antifungal protein Anafp (Lee et al., 1999). Anafp protein belongs to a new group of antimicrobial proteins named small, basic and cysteine51 MENÚ SALIR MANUSCRITO 2 rich antifungal proteins. The recent sequencing of the genome of different moulds has shown several genes that could encode proteins with common characteristics to this group. However, only four proteins have been reported: Anafp from Aspergillus niger (Lee et al., 1999), PAF from Penicillium chrysogenum (Marx et al., 1995), AFP from Aspergillus giganteus (Nakaya et al., 1990; Lacadena et al., 1995), and AcAFP from Aspergillus clavatus (Skouri-Gargouri and Gargouri, 2008). Only AFP and PAF have been genetically characterizated. In addition a gene homologous to paf gene has been described from Penicillium nalgiovense (Geisen, 2000). The genes encoding PAF and AFP have open reading frames interrupted by two introns with conserved splice sites. They encode products of 92 and 94 amino acids, respectively, and these proteins are synthesized as a long precursor with a peptide signal and a presequence that are removed to obtain mature proteins. Genetic characterization of the gene encoding PgAFP will allow to clon it in other microorganisms, to increase PgAFP production and to obtain protective cultures for future applications in foods, environment or medicine. In addition, the genetic sequence will give valuable information for a better characterization of the protein. MATERIALS AND METHODS Microbial strain P. chrysogenum RP42C was isolated from dry-cured ham, and belongs to the fungal collection of Food Hygiene, University of Extremadura. DNA isolation P. chrysogenum RP42C was grown in malt extract broth made with 2% (w/v) glucose, 2% (w/v) malt extract, and 0.1% (w/v) peptone, pH 4.5 at 25ºC under continuous shaking for 5 days. The mycelium was obtained by filtering the culture through Whatman paper nº2. Two grams of mycelium were broken with mortar and pestle after freezing by adding liquid nitrogen. Fungal lysis was performed by homogenizing the obtained powder in 4 ml 0.05 M Tris-HCl, pH 8; 0.005 M ethylenediamine tetraacetic acid (EDTA); 0.05 M NaCl (TES) buffer and 1% (w/v) sodium dodecyl sulfate (SDS). Then, 200 µl proteinase K (20 µg/µl) were added, the mix was incubated at 60ºC for 35 min, cooled on ice, and extracted with phenol-chloroform-isoamyl alcohol (25:24:1). The suspension was centrifuged at 52 MENÚ SALIR RESULTADOS 3000×g for 2 min at 4 ºC. The upper phase containing DNA was transferred to a fresh tube and precipitated by adding 3 M sodium acetate pH 5.2 to a final concentration of 10% (v/v) and two volumes of cold ethanol. After centrifugation, the pellet was cleaned with 70% (v/v) ethanol, centrifuged again in the conditions above mentioned, resuspended in sterile water, and treated with 50 µl RNase (10 µg/µl) at 37ºC for 60 min. Finally, DNA was extrated with phenol-chloroform-isoamyl alcohol, precipitated again as indicated above, resuspended in water, and stored at -70ºC. Quantity and quality of purified DNA was determined spectrophotometrically in a Biophotometer (Eppendorf AG, Hamburg, Germany). Genomic DNA amplification Polymerase chain reactions were performed with genomic DNA (100 ng) as template in 50 µl reaction mixtures containing 50 pmol each of forward and reverse primers, 0.5 mM each of the deoxyribonucleotide triphosphates (dNTPs), 0.1 vol of 10× PCR buffer (22.5 mM MgCl2, 500 mM Tris-HCl pH 9.2, 140 mM ammonium sulfate), 1.8 mM MgCl2 and 2.5 units pfu turbo polymerase (Stratagene, La Jolla, USA). The degenerated primers, pgafpDPF1 and pgafp-DPR1A (Table 1), were designed from amino acid sequences previously obtained by mass spectrometry (Rodríguez-Martín et al., 2009). Three reactions were done: one with both forward and reverse primers, another only with forward, and another only with reverse primer. Table 1. Degenerated primers used to amplify the pgafp gene. Primer names Amino acid sequences and designed primers* L pgafp-DPF1 H N T C T 5’-TN AAR CAY AAY ACN TGN AC-3’ C pgafp-DPR1A K G S A A N 3’-CR CCN AGN CGN CGN TT-5’ * R for purine, Y for pirimidine, N for A, C, T, or G. DNA amplification was carried out in a Px2 thermocycler (Thermo Scientific, Cramlington, UK). The PCR program consisted of initial denaturation (94ºC for 5 min), 35 53 MENÚ SALIR MANUSCRITO 2 cycles of denaturation (94ºC for 5 s), annealing (45-57ºC range for 5 s, gradient 12), and extension (72ºC for 1 s) steps, followed by a final extension at 72 ºC for 4 min. PCR products were electrophoresed on 2% (w/v) agarose gels with MetaPhor agarose (BMA, Rockland, ME, USA). Two different markers were used for agarose gels: DNA marker from Gibco (0.15-12kb bands) (Gibco, GrandIsland, NY, USA) and DNA size standard from Bio-Rad (0.05-2kb) (Bio-rad Laboratories, Madrid, Spain). PCR products detected only in the reactions with both reverse and forward primers were gel-purified and cloned into the pCR2.1-TOPO vector (Invitrogen, Barcelona, Spain). One Shot TOP10 Chemically Competent E. coli (Invitrogen) were transformed with that vector. To confirm the insert in the vector, digestions with restriction enzyme EcoRI or PCR with M13 forward and reverse primers (Invitrogen) were carried out. Finally, vectors were sequenced in the Institute of Biomedicine (CSIC, Valencia, Spain). RNA isolation P. chrysogenum RP42C was grown in malt extract broth as indicated for DNA isolation. Subsequently, mycelium was quickly frozen with liquid nitrogen and stored at -80ºC until RNA extraction. Mycelium was broken with mortar and pestle after adding liquid nitrogen. RNA extraction was performed with RNeasy Plant Mini Kit (Qiagen) following the manufacturer’s instructions. The final extracted RNA was resuspended in 40 µl of RNasefree deionized water. Rapid amplification of cDNA ends-Polymerase Chain Reaction (RACE-PCR) Amplification of 5’ and 3’ ends of pgafp gene was carried out using the SMART RACE cDNA Amplification Kit (Clontech Laboratories, Saint Germain en Laye, France). cDNA synthesis by reverse transcriptase-polymerase chain reaction (RT-PCR). Two separate populations of cDNA were synthesized according to the amplification kit instructions: 5’-RACE-Ready cDNA and 3’-RACE-Ready cDNA. After incorporating the SMART sequence into both the 5’- and 3’-RACE-Ready cDNA populations, RACE PCR reactions were performed using the Universal Primer A Mix (UPM), in conjunction with distinct gene-specific primers. Gene-specific primers design. A gene-specific primer (GSP) was designed for amplifying the 3’- end (3’-RACE GSP: 5’-GGGTGGAAAGAACCATGTAGTCAATTGCG-3’) from 54 MENÚ SALIR RESULTADOS the sequence previously obtained by PCR with degenerate primers pgafp-DPF1 and pgafpDPR1A. Amplification of the 5’- end was carried out designing a gene-specific primer (5’RACE GSP: 5’-AACTGGGGTCTGGCAGTCAACCCTC-3’) from the 3’-end also previously obtained. Polymerase chain reactions were performed with 5’- or 3’-RACE-Ready cDNAs (50 ng) as template in 50 µl reaction mixtures containing 1 mM of each of the dNTPs (Roche, Madrid, Spain), 0.5 µM of the 5’- or 3’-RACE GSP primers, 5 µl of Universal Primer A Mix (10×), 0.1 vol of 10× PCR buffer (10 mM Tris-HCl, pH 8.8, 50 mM KCl, 0.1 % Triton X-100), 4 mM MgCl2 and 2.5 units pfu turbo polymerase (Stratagene). The PCR program for obtaining the 3’-end consisted of 35 cycles of denaturation (94ºC for 30s), annealing (68ºC for 30s), and extension (72ºC for 3 min). The PCR program for 5’-end amplification was: 5 cycles of 2 steps (94ºC for 30 s and 72ºC for 3 min), 5 cycles of 3 steps (94ºC for 30 s, 70ºC for 30 s and 72ºC for 3 min) and 27 cycles of 3 steps (94ºC for 30 s, 68ºC for 30 s and 72ºC for 3 min). Finally, 5’- and 3’RACE-PCR products were gel-purified and cloned into pCR2.1-TOPO vectors, which were used to transform One Shot TOP10 Chemically Competent E. coli (Invitrogen) before purifying and sequencing the plasmid. Digestions with restriction enzyme Eco RI (Roche) or PCR with M13 forward primer (Invitrogen) were performed to confirm the insert in the vector. Full-length cDNA amplification. The full cDNA fragment of the gene pgafp was obtained by PCR with specific primers (Fwd-PrePro pgafp: 5’-ATGCAGATCACCAGCATTGCC3’ and Rev-PrePro/Mature pgafp: 5’-TCAAACTGGGGTCTGGCAGTC-3’) designed from 5’ and 3’ ends. PCR reaction mixtures at a final volume of 50 µl contained 5µl of 5’RACE-Ready cDNA, 0.5 µM each of forward and reverse primers, 0.5 mM each of the dNTPs, 0.1 vol of 10× PCR buffer (10 mM Tris-HCl, pH 8.8, 50 mM KCl, 0.1% Triton X100), 4 mM MgCl2 and 2.5 units pfu turbo polymerase (Stratagene). The PCR program consisted of 35 cycles of denaturation (94ºC for 30 s), annealing (65ºC for 30 s), and extension (72ºC for 3 min). PCR products were gel-purified and cloned into pCR2.1TOPO vectors, which were used to transform One Shot TOP10 Chemically Competent E. coli (Invitrogen) before purifying and sequencing the plasmid. Digestions with restriction enzyme Eco RI (Roche) or PCR with M13 forward primer (Invitrogen) were performed to confirm if the insert was in the vector. 55 MENÚ SALIR MANUSCRITO 2 Amplification of the whole pgafp gene The gene sequence was amplified by PCR using Fwd-PrePro pgafp and RevPrePro/Mature pgafp primers. PCR mixtures were composed by 100 ng of genomic DNA, 0.5 µM each of forward and reverse primers, 0.5 mM each of the dNTPs, 0.1 vol of 10× PCR buffer (10 mM Tris-HCl, pH 8.8, 50 mM KCl, 0.1% Triton X-100), 4 mM MgCl2, and 2.5 units pfu turbo polymerase (Stratagene) and deionized water until a final volume of 50µl. The PCR program consisted of 30 cycles of denaturation (94ºC for 30 s), annealing (65ºC for 30 s), and extension (72ºC for 3 min). PCR products were cloned in pCR2.1TOPO vector that later was used to transform cells of One Shot TOP10 Chemically Competent E. coli. The plasmid was purified and sequenced. Introns sequences were confirmed by GenScan software. RESULTS Partial amplification of pgafp gene using degenerated primers The pgafp gene partial amplification was done using the two degenerate primers, pgafpDPF1 and pgafp-DPR2A (Table 1). After different reactions, the optimal annealing temperature was 45.1ºC. PCR products run in agarose gel showed one small band of around 75 bp that was amplified only in reactions with both the forward and the reverse primers (Figure 1). This band was selected for cloning and sequencing. Figure 1. Products obtained by PCR using DPF1 primer (F), DPF1+DPR2A primers (F+R), and DPR2A (R) primer. M: DNA standards. The arrow indicates the selected band for cloning and sequencing. 56 MENÚ SALIR RESULTADOS After sequencing, the fragment found was 70 bp length (5’-tgaagcataatacgtgcacatacctaaag ggtggaaagaaccatgtagtcaattgcggttctgccgctaa-3’), and the translated sequence (KHNTCTYL KGGKNHVVNCGSAA) was compared in Basic Local Alignment Search Tool (BLAST) showing common sequences with Anafp from Aspergillus niger. Sequence of pgafp gene by RACE-PCR Both 3’ and 5’ -ends of the gene pgafp were obtained by amplification from cDNA. mRNA was extracted from a 5 days-old culture of P. chrysogenum RP42C and was used to obtain the two different populations of 5’- and 3’-RACE-Ready cDNA by RT-PCR. The gene specific primer 3’-RACE GSP (5’-GGGTGGAAAGAACCATGTAGTCAATTGCG-3’), designed from the known pgafp sequence of genomic DNA, allowed to amplify the 3’-end by RACE-PCR. The resulting product of about 450 bp included 121 bp of the 3’-end of pgafp (Figure 2). Amplification of the 5’-end was carried out with a new gene specific primer, 5’-RACE GSP (5’-AACTGGGGTCTGGCAGTCAACCCTC-3’), designed from 3’-end sequence. The obtained band, of approximately 440 bp, included the full pgafp cDNA gene. A) B) Figure 2. cDNA fragments of 5’- and 3’-RACE PCR (A and B, respectively). M: DNA standards. Amplifications of complete gene in genomic DNA and cDNA were performed by designing two new primers Fwd-full pgafp (5’-ATGCAGATCACCAGCATTGCC-3’) and Rev-full pgafp (5’-TCAAACTGGGGTCTGGCAGTC-3’). The sequence of genomic DNA was 404 bp, whereas the amplified cDNA was 279 bp (Figure 3). The difference between 57 MENÚ SALIR MANUSCRITO 2 the sequences revealed the existence of two introns of 63 and 62 bp (Figure 4), absent in the cDNA, which were confirmed by GenScan software. Figure 3. pgafp gene amplification of the with genomic DNA (1) and cDNA (2). M: DNA standards. 5’-atgcagatcaccagcattgccattgtcttcttcgccgcaatgggtgcggttgctaacccc M Q I T S I A I V F F A A M G A V A N P atcgcgagggagtcggacgatcttgatgcccgagacgtacagcttagtaaattcggagga I A R E S D D L D A R D V Q L S K F G G gtaagttcttcttacaagacgtctatatagaaatagcactaacctttctgaaccacttta << Intron 1 caggaatgcagcttgaaacacaacacgtgcacatacctaaagggtggaaagaaccatgta >> E C S L K H N T C T Y L K G G K N H V gtcaattgcggttcggccgccaacaagaaggtaggttccgattcgattcggggccaattg V N C G S A A N K K << Intron 2 atttgttcttatcatttaatcttcatctacagtgcaagtctgatcgccaccactgtgaat >> C K S D R H H C E acgatgagcaccacaagagggttgactgccagaccccagtttga-3’ Y D E H H K R V D C Q T P V - Figure 4. Full-length DNA sequence of pgafp gene. The open reading frame has been translated to amino acids. Consensus splice sequences and internal marker of fungal introns are underlined. The open reading frame was translated to protein by ExPASy proteomics server at the Swiss Institute of Bioinformatics (http://www.expasy.org). Analysis of the signal peptidase 58 MENÚ SALIR RESULTADOS cleavage site predicted by the program SignalP (Nielsen et al., 1997) suggested that the pre-sequence consisted of the first 18 amino acids (MQITSIAIVFFAAMGAVA). DISCUSSION P. chrysogenum RP42C produces a high amount of the PgAFP in the first week of incubation (Acosta, 2006). Thus, to obtain appropriate cDNA populations, a 5 days-old culture was used to make sure that the mRNA from gene encoding PgAFP was actively being produced. The RACE-PCR was effective to obtain pgafp gene. Thus, the designed primers and obtained cDNA populations were adequate to amplify the 3’ and 5’ –ends of pgafp. When the complete sequence was obtained, a difference between the pgafp gene from genomic DNA (404 bp) and from cDNA (279 bp) could be observed (Figure 3). Thus, the existence of two introns of 63 and 62 bp in the gene, which are absent in the cDNA, was confirmed by GenScan software. The small size of these introns compared to those of mammalian introns is a typical feature of fungal genes (Gurr et al., 1987). The 5’and 3’-ends of the two introns are quite similar (Figure 4) and coincide with the consensus splice sequences for fungal introns: 5’-splice donor site GTNNGT (N= A, G, T or C) and the consensus 3’-splice acceptor site DYAG (D= A, G or T; Y= C or T). In addition, the first intron has the internal CTAAC sequence, very common in fungal introns (Wiesner et al., 1988). The pgafp open reading frame encodes a 92 amino acid-long precursor of the PgAFP. The first 18 amino acids correspond with a secretion signal sequence according to the SignalP program (Nielsen et al., 1997). The next residues (19-34) were not found in the amino acid sequence obtained from the purified protein (Rodríguez-Martín et al., 2009). These 16 amino acids constitute a prosequence that would be removed before or during the release of mature PgAFP. Prosequences play an important role by preventing protein activity before secretion. Only after the prosequence is cleaved off, the mature protein adopts its correct tertiary structure and gains its final activity. Antifungal proteins produced as preproproteins, such as PAF and AFP (Nakaya et al., 1990; Marx et al., 1995), have been described from other moulds. The deduced mature PgAFP has 58 amino acid residues. It shows a high percentage (27.6 %) of basic amino acids. The isoelectric point was estimated to be 8.8 using the Compute pI tool from Expasy (Gasteiger et al., 2005). The isoelectric point of PgAFP estimated by isoelectrofocusing in previous studies (Acosta et al., 2009b) was 9.2, higher than the 59 MENÚ SALIR MANUSCRITO 2 predicted value with Compute pI tool. However, the prediction can be not exact for highly basic and small proteins (Gasteiger et al., 2005). A pI of 9.2 results in a net positive charge under physiological conditions, which is a common characteristic among antifungal proteins from moulds. The accessibility of the whole genome sequence of an increasing number of microorganisms facilitates the search for genes encoding potential antifungal proteins. Three of these genes, those from Aspergillus niger, Neosartorya fischeri, and Gibberella zeae, show partial homology with PgAFP protein. The sequences of the hypothetical proteins from these genes as well as other reported antifungal proteins are aligned in Figure 5. The protein showing the highest homology (75%) with the precursor of PgAFP was the hypothetical protein from A. niger. Despite both PAF and PgAFP precursors are produced by Penicillium chrysogenum, they only share a similarity of 43%. There are six cysteines conserved in all the similar proteins (Figure 5). Those residues would play an important role on stabilizing of the tertiary structure of PgAFP by formation of disulfide bridges (Nakaya et al. 1990; Marx et al., 1995; Lee et al., 1999). PgAFP Anafp A.niger N.fischeri G.zeae PAF AFP A.clavatus ---MQITSIAIVFFAAMGAVANPIARE---SDDLDARD--VQLSKFGGECSLKHNTCTYL ------------------------------------------LSKYGGECSLEHNTCTYR ---MQLTSIAIILFAAMGAIANPIAAE---ADNLVARE--AELSKYGGECSVEHNTCTYL ---MQITKISLFLFVGIGVVASPIHAE---SDGLNARAVNAADLEYKGECFTKDNTCKYK ---MQFSTIIPLFVAAMGVVATPVNSP---AQELDARGNLFPRLEYWGKCTKAENRCKYK ---MQITTVALFLFAAMGGVATPIESV---SNDLDARAEAGVLAKYTGKCTKSKNECKYK MKFVSLASLGFALVAALGAVATPVEADSLTAGGLDARDESAVLATYNGKCYKKDNICKYK MKVVSLASLGFALVAALGVVASPVDADSLAAGGLDARDESAVQATYDGKCYKKDNICKYK PgAFP Anafp A.niger N.fischeri G.zeae PAF AFP A.clavatus K-GGKNHVVNCGSAANKKCKSDRHHCEYDEHHKRVDCQTPV K-DGKNHVVSCPSAANLRCKTDRHHCRYDDHHKTVDCQTPV K-GGKDHIVSCPSAANLRCKTERHHCEYDEHHKTVDCQTPV ID-GKTYLAKCPSAANTKCEKDGNKCTYDSYNRKVKCDFRH NDKGKDVLQNCPKFDNKKCTKDGNSCKWDSASKALTCY--NDAGKDTFIKCPKFDNKKCTKDNNKCTVDTYNNAVDCD--AQSGKTAICKCYV---KKCPRDGAKCEFDSYKGKCYC---AQSGKTAICKCYV---KVCPRDGAKCEFDSYKGKCYC---- Figure 5. Alignment of PgAFP amino acid sequences (Rodríguez-Martín et al., 2009) with other antifungal proteins. Accession numbers in Genbank are XP_001391221 (hypothetical protein from Aspergillus niger CBS 513.88), XP_001262586 (hypothetical protein from Neosartorya fischeri NRRL 181), XP_384921 (hypothetical protein from Gibberella zeae PH1), AAA92718 (PAF from P. chrysogenum), P17737 (AFP from A. giganteus) and XP_001267787 (hypothetical protein from Aspergillus clavatus). Common amino acids are denoted by shaded background letters. 60 MENÚ SALIR RESULTADOS In previous studies PgAFP showed a molecular weight of 6,494 Da by mass spectrometry (Rodríguez-Martín et al., 2009). Translating the genetic sequence according to Gasteiger et al. (2005) the predicted mass of the mature protein was 6,500 Da, very close to the weight determined by mass spectrometry. In previous works, PgAFP protein was tested for having glycosylations by treatments with deglycosilases and chemical compounds, and no linkedoligosaccharides were found. The minimal difference found between predicted and experimental molecular weights confirms that this protein is not glycosylated. Recently, the genome of P. chrysogenum Wisconsin 54-1255 has been sequenced (Van den Berg et al., 2008). A putative protein can be found in BLAST (accession number CAP80456) that differs from the precursor of PgAFP in only three amino acids. However, the production of the protein by this mould has not been reported. In conclusion, the gene enconding the antifungal protein PgAFP has been sequenced. Such as similar proteins, PgAFP is synthesized as a long precursor with a peptide signal and a presequence that are removed to obtain mature proteins. The characterization of pgafp gene will permit its cloning for overproduction or heterologous expression of the protein in other microorganisms. ACKNOWLEDGEMENTS This work was supported by the Spanish Ministry of Education and Science (AGL200406546-ALI) and FEDER. Andrea Rodríguez and Raquel Acosta were the recipients of FPI grants from the Spanish Ministry of Education and Science. Authors would like to thank Dr. Susan Liddell (Division of Animal Sciences) and Prof. Ian Connerton (Division of Food Sciences) for their support during Andrea Rodriguez stay’s at Nottingham University. REFERENCES Acosta, R. Doctoral Thesis: “Selección de Penicillium productores de péptidos antifúngicos para su utilización en productos cárnicos madurados”. University of Extremadura. Spain. 2006. Acosta, R., Rodríguez-Martín, A., Martín, A., Núñez, F., and Asensio, M.A. Selection of antifungal protein-producing molds from dry-cured meat products. Int. J. Food Microbiol. Submitted (2009a). 61 MENÚ SALIR MANUSCRITO 2 Acosta, R., Rodríguez-Martín, A., Bermúdez, E., Núñez, F., and Asensio, M.A. Partial characterization and antifungal activity of a protein from Penicillium chrysogenum. Food Microbiol. Submitted (2009b). Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M.R., Appel, R.D., and Bairoch, A. Protein Identification and Analysis Tools on the ExPASy Server; (In) John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press. Full text - Copyright Humana Press (2005). Geisen, R. P. nalgiovense carries a gene which is homologous to the paf gene of P. chrysogenum which codes for an antifungal peptide. Int. J. Food Microbiol. 62: 95-101 (2000). Gurr, S.J., Uncles, S.E., and Kinghorn, J.R. The structure and organization of nuclear genes of filamentous fungi. In: Kinghorn, J.R. (Ed.). Gene structure in Eukaryotic Microbes. IRL Press, Oxford, pp. 93-193 (1987). Lacadena, J., Martínez del Pozo, A., Gasset, M., Patiño, B., Campos-Olivas, R., Vázquez, C., Martínez-Ruiz, A., Mancheño, J.M., Oñaderra, M., Gavilanes, J.G. Characterization of the antifungal protein secreted by the mould Aspergillus giganteus. Arch. Biochem. Biophys. 20: 273-281 (1995). Lee, D.G., Shin, S.Y., Maeng, C-Y., Jin, Z.Z., Kim, K.L. and Hahm, K-S. Isolation and characterization of a novel antifungal peptide from Aspergillus niger. Biochem. Biophy. Res. Commu. 263: 646-651 (1999). Marx, F., Haas, H., Reindl, M., Stöffler, G., Lottspeich, F. and Redl, B. Cloning, structural organization and regulation of expression of the Penicillium chrysogenum paf gene encoding an abundantly secreted protein with antifungal activity. Gene. 167: 167-171 (1995). Nakaya, K., Omata, K., Okahashi, I., Nakamura, Y., Kolkenbrock, H., and Ulbrich, N. Amino acid sequence and disulfide bridges of an antifungal protein isolated from Aspergillus giganteus. Eur. J. Biochemistry. 193: 31-38 (1990). Nielsen, H., Engelbrecht, J., Brunak, S., and Von Heijne, G. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10: 1-6. (1997). 62 MENÚ SALIR RESULTADOS Rodríguez-Martín, A., Liddell, S., Acosta, R., Nuñez, F., and Asensio, M.A. Characterization of PgAFP, a novel antifungal protein from Penicillium chrysogenum. Manuscrito 1 (2009). Skouri-Gargouri, H. and Gargouri, A. First isolation of a novel thermostable antifungal peptide secreted by Aspergillus clavatus. Peptides. 29: 1871-1877 (2008). Van den Berg, M.A., Albang, R., Albermann, K., Badger, J.H., Daran, J.M., Driessen, A.J., Garcia-Estrada, C., Fedorova, N.D., Harris, D.M., Heijne, W.H., Joardar, V., Kiel, J.A., Kovalchuk, A., Martin, J.F., Nierman, W.C., Nijland, J.G., Pronk, J.T., Roubos, J.A., van der Klei, I.J., van Peij,N.N., Veenhuis,M., von Dohren, H., Wagner, C., Wortman, J. and Bovenberg, R.A. Genome sequencing and analysis of the filamentous fungus Penicillium chrysogenum. Nat. Biotechnol. 26: 1161-1168 (2008). Wiesner, P., Beck, J., Beck, K.F., Ripka, S., Müller, G., Lücke, S. and Schweizer, E. Isolation and sequence analysis of the fatty acid synthetase FAS2 gene from Penicillium patulum. Eur. J. Biochem. 177: 69-79 (1988). 63 MENÚ SALIR Manuscrito 3: Preliminary evaluation of the toxicity and sensitivity to different treatments of the antifungal protein PgAFP from Penicillium chrysogenum MENÚ SALIR RESULTADOS Preliminary evaluation of the toxicity and sensitivity to different treatments of the antifungal protein PgAFP from Penicillium chrysogenum Andrea Rodríguez-Martín, Raquel Acosta, Félix Núñez, Miguel A. Asensio* Higiene y Seguridad Alimentaria, Facultad de Veterinaria. Universidad de Extremadura, Avda. de la Universidad, s/n. 10071- Cáceres, Spain. * Corresponding author: Telephone (+34) 927 257 125; fax: (+34) 927 257 110. E-mail: [email protected] ABSTRACT The protein PgAFP obtained from Penicillium chrysogenum RP42C inhibits toxigenic moulds found in foods. The aim of this work was to evaluate the potential toxicity of PgAFP and to study basic characteristics of its antifungal activity, including the effect of treatments and compounds common in foods. No haemolytic activity or toxicity on Artemia salina larvae was detected. PgAFP showed neither chitinase nor chitosanase activities. The protein showed a high stability to heat treatments up to 100ºC for 15 min, and at pH values from 1 to 12. Some detergents potentially present in food as indirect additives interfered with the antifungal activity, but tween 20 had no impact on the protein. Only high concentration of other compounds, such as EDTA and sodium phosphate, affected PgAFP activity when low levels of the protein were present. Monovalent ions showed very little impact, whereas Ca2+ blocked the antifungal activity even at low concentrations. These results indicate that PgAFP could be used to prevent mould growth in foods with high salt levels or where thermal or acid stability are essential. KEY WORDS: antifungal activity, food preservative, protective culture. INTRODUCTION Penicillium chrysogenum has been proposed as starter culture for mould fermented foods (Martín et al., 2002; Benito et al., 2005), mainly due to its ability to produce volatile compounds that contribute to flavour development (Ramírez et al., 1995; Jacobsen and Hinrichsen, 1997; Martín et al., 2006). However, the continuous evolution of the physicochemical characteristics along ripening can favour the development of undesirable moulds, which would not only lead to a negative flavour of the product (Lund et al., 1995), but also 65 MENÚ SALIR MANUSCRITO 3 to mycotoxin production (Núñez et al., 1996, 2000; Nielsen et al., 1998; López-Díaz et al., 2001; Sosa et al., 2002; Sabater-Vilar et al., 2003). Some mould can produce different antifungal compounds, including proteins. The use of natural antifungal proteins could be attractive to satisfy consumer's demand. Therefore, fungal cultures with antagonistic activity against spoilage and toxigenic moulds could be of interest to improve the microbial safety of the product. In previous studies, P. chrysogenum RP42C isolated from dry-cured ham showed a broadspectrum inhibition against toxigenic moulds, including Aspergillus niger, A. flavus, and P. commune due to the protein PgAFP (Acosta et al., 2009a, b). Various mechanisms of action have been described for antifungal proteins, including interference in synthesis of DNA, RNA, or proteins, damage to cellular ribosome (Hwu et al., 2000), and membrane channel and pore formation (Thevissen et al., 1999). Nevertheless, the most useful antifungal proteins are those specific against fungal membrane and cell wall, because of their lesser toxic effects on mammalian cells (Groll et al., 1998). Cationic antifungal proteins, such as AFP from A. giganteus and PAF from P. chrysogenum, have proved to be specific for fungal membranes (Marx et al., 2008; Meyer et al., 2008) and not for mammalian cells (Szappanos et al., 2005 and 2006). Interestingly, PgAFP shows similar characteristics to AFP and PAF, both in the amino acid sequence and the isoelectric point (Marx, 2004; Acosta et al., 2009b; Rodríguez-Martín et al., 2009a, b). A prerequisite for any application of antifungal proteins or the producer mould on foods is the lack of toxicity for consumers. The use of antifungal proteins from ascomycetes can be limited by chemical compounds present and physical treatments applied to foods. Many antifungal proteins, especially membrane-acting peptides, are sensitive to cations, which can be a crucial point in the application to food products with high salt concentrations (Theis and Stahl, 2004). Given that PgAFP is produced by a mould isolated from a substrate with high salt concentration, its antifungal activity could withstand high-cation levels. Moreover, antifungal proteins should also be active under the singular conditions of foods where moulds can grow, particularly in a wide range of pH values. Other compounds that can be present on foods, such as detergents and chelators, may also interfere with antifungal proteins. In addition, stability to heat treatments can be of utmost interest for the potential use of antifungal proteins as food preservative. The aim of this work was to carry out a preliminary evaluation of the potential toxicity of 66 MENÚ SALIR RESULTADOS PgAFP, as well as the impact of salts, heat treatments, different pH values, and detergents on its antifungal activity. MATERIALS AND METHODS Microbial strains The PgAFP producing mould, P. chrysogenum RP42C, and the toxigenic moulds Penicillium restrictum Pr341 and Aspergillus niger An261, were isolated from dry-cured ham and belong to the fungal collection of Food Hygiene (University of Extremadura, Spain). PgAFP purification P. chrysogenum RP42C was grown at 25ºC for 15 days in malt extract broth made of glucose 2% (w/v), malt extract 2% (w/v), and peptone 0.1% (w/v), pH 4.5. Then, the medium was filtered through a 0.45 µm diameter pore size cellulose acetate membrane and PgAFP was purified using an ÄKTA fast protein liquid chromatography system (Amersham Pharmacia Biotech AB, Uppsala, Sweden) according to Acosta et al. (2009a). For this, filtered medium was loaded on a HiTrap SP HP 5 ml ion exchange column (Amersham Pharmacia Biotech AB) equilibrated with 0.02 M sodium acetate buffer, pH 4.5. Bound proteins were eluted with the following gradient of 0.02 M sodium acetate, 1 M NaCl buffer, pH 4.5: 0% to 25% in 15 column volumes (CV), increase to 100% in 2 CV, and finally keeping 100% for 15 CV. Eluted proteins were detected at 214 nm. The fraction that showed antifungal activity in previous studies (Acosta et al., 2009a) was purified using a HiLoad 26/60 Superdex 75 prep grade gel filtration column (Amersham Pharmacia Biotech AB) equilibrated with 50 mM sodium phosphate, 0.15 M NaCl buffer, pH 7, at a constant flow rate of 2.5 ml/min. Data collection and processing were done using the UNICORN software 4.12 (Amersham Pharmacia Biotech AB). Finally, the fraction containing PgAFP was desalted and concentrated with YM-3 microcon centrifugal filter units (Millipore Iberica S.A.U., Madrid, Spain) and kept at -20ºC. Protein was quantified by Johnson method according to Córdoba (1990). An aliquot of the purified protein (100 µl) was dried at 100 ºC. The sample was digested by adding 200 µl of sulfuric acid at 120 ºC. Then, 4.8 ml of distilled water, 3 ml of 4 N NaOH, and 2 ml of Nessler reactive made of KI 0.4% (w/v), 0.4% HgI2 (w/v), and 0.175% (w/v) arabic gum 67 MENÚ SALIR MANUSCRITO 3 were added. After 10 min at room temperature, the absorbance was measured at 490 nm in a spectrophotometer. A calibration curve was obtained with different concentrations of ammonium sulfate. Sodium dodecyl sulphate-polyacrylamide gel eletrophoresis (SDS-PAGE) SDS-PAGE was performed as described Laemmli (1970). The protein PgAFP was separated on a 4% (v/v) polyacrylamide stacking and 8% (w/v) polyacrylamide resolving gels. Gels were stained with Imperial Protein Stain (Pierce Biotechnology, Inc., Rockford, U.S.A.). The molecular weight was determined by comparison of its electrophoretic mobility with proteins of the molecular mass marker (6.5-66 kDa) from Sigma (SigmaAldrich Química S.A., Madrid, Spain). Haemolytic activity assay The haemolytic activity of the protein was determined using sheep defibrinated blood (Oxoid). The erythrocytes were isolated by centrifugation (1,000 ×g for 6 min at room temperature) and washed three times with 50 mM sodium phosphate, 0.15 M NaCl buffer, pH 7. The cell concentration in the stock suspension was adjusted to 1×109 cells/ml. A volume of 50 µl of cells suspension was added to 200 µl of sterile concentrated protein (300 µg/ml) and incubated at 37ºC. After 0, 8, and 24 h, the samples were placed on ice, immediately centrifuged at 4ºC, and the supernatant was carefully collected. The haemolysis was determined by measuring the absorbance of the supernatant at 570 nm (Moerman et al., 2002) and 541 nm (Lai et al., 2005). Controls for 0% and 100% haemolysis were obtained suspending sheep red blood cells in the buffer or distilled water, respectively. The haemolysis percentage was calculated using the following equation: Percentage haemolysis of supernatants = [(Abs in the peptide solution − Abs in buffer)/Abs in water solution] × 100. Three independent experiments were conducted with triplicate readings for each one. Toxicity assay using brine shrimp larvae (Artemia salina) The toxicity of PgAFP was evaluated using brine shrimp larvae, following the Harwig and Scott method (1971) with modifications. Brine shrimp medium (BSM) was made of 0.3% (w/v) dipotassium glycerophosphate, 3% (w/v) NaCl, 0.03% (w/v) CaCl2 • 2H2O, 0.05% 68 MENÚ SALIR RESULTADOS (w/v) MgSO4 • 7H2O, 0.15% (w/v) MgCl2 • 6H2O, 0.08% (w/v) KCl, 0.01% (w/v) MgBr2 • 6H2O, and 0.6% (w/v) glycine. The medium was adjusted to pH 6.5. Brine shrimp larvae (100-200 mg) were grown in 100 ml BSM under continuous shaking (140 rpm) for 30 h at 30ºC. The test was carried out in Erlenmeyer flasks containing 20 to 40 larvae in 10 ml BSM. PgAFP was added at final concentrations of 3, 16, 33, 57, and 75 µg/ml. Buffer without PgAFP was used in negative controls. Dead larvae were counted just before incubation and after 24 h at 30ºC in a shaker at 80 rpm. Finally, the mortality percentage was calculated. PgAFP was indicated as nontoxic when death rate of larvae was not significantly different (p <0.05) from spontaneous mortality in controls. Evaluation of chitinase and chitosanase activities Chitinase and chitosanase activities were determined with a modification of the 3,5dinitrosalicylic acid method (Su et al., 2006) by measuring the reducing sugars liberated during the hydrolysis of chitin or chitosan. One ml of the 1% (w/v) chitin or chitosan (Sigma-Aldrich) in 1% (v/v) acetic acid was added to 1 ml of PgAFP (300 µg/ml) and incubated for 2-3 h at 37ºC. Then, to detect the hydrolisis reaction, 2 ml of dinitrosalicylic acid reagent, made of 1% (w/v) 3,5-dinitrosalicylic acid, 0.05% (w/v) sodium sulphite and 1% (w/v) phenol dissolved, were added and the sample was incubated at 100ºC for 45 min. Finally, 1 ml of 40% (w/v) potassium sodium tartrate was added and the solution was centrifuged. The supernatant was measured at 520 and 575 nm. To quantify the reducing sugars liberated a calibration curve was obtained with different concentrations of D-(+)Glucosamine hydrochloride (Sigma-Aldrich). A chitinase from Streptomyces griseus (Sigma-Aldrich) with both chitinase and chitosanase activities was used as positive control. Preparation of colloidal chitin: Colloidal chitin was obtained following the method of Roberts and Selitrennikott with minor modifications (Harighi et al., 2007). Ten grams of chitin powder were added slowly into 100 ml of H3PO4 (85%) at 25ºC under vigorous shaking for 2 h. This solution was carefully added over 1 liter of absolute ethanol at -20ºC. The mix was shaken for 30 min and stored until use. Ten ml of suspension were centrifuged, the pellet was washed three times with 50 mM sodium phosphate buffer pH 7, and finally resuspended in distilled water. The final concentration of colloidal chitin was around 1%. 69 MENÚ SALIR MANUSCRITO 3 Chitinase activity after gel electrophoresis The chitinase activity assay was carried out as described by Trudel and Asselin (1989). PAGE-SDS was performed in 12% (v/v) polyacrylamide gradient gel containing 0.01% (w/v) glycol chitin and 0.1% (w/v) SDS. A stacking gel with 4% (v/v) polyacrylamide containing 0.1% (w/v) SDS was used. Samples were boiled for 5 min with 20% (v/v) glycerol, 0.025% (w/v) bromophenol blue, 2% (w/v) SDS and 5% (v/v) -mercaptoethanol in 62.5 mM Tris-HCl (pH 6.8). The gel was run at 100 V for 1 h. After electrophoresis, the gel was incubated for 2 h at 37 ºC under continuous shaking in 100 mM sodium acetate buffer (pH 5.0) containing 1% (v/v) Triton X-100. Then, the gel was stained with a 0.01% (w/v) calcofluor white M2R (Sigma-Aldrich) in 500 mM TrisHCl (pH 8.9). After 5 min, this solution was removed and the gel was incubated in distilled water at room temperature for about 1 h. Lytic zones were visualized in the UV transilluminator Syngene G-Box (Synoptics, Fredericks, USA). The gel was photographed and analyzed with the GeneSnap software (Synoptics). An additional gel was stained only with Imperial Protein Stain (Pierce Biotechnology, Rockford, U.S.A.). The chitinase from S. griseus (Sigma) was used as positive control. The molecular mass marker used was 666.5 kDa (Sigma). Glycol chitin synthesis: 0.2 g glycol chitosan was dissolved in 4 ml of 10 % (v/v) acetic acid and the mixture was shaken overnight at 22ºC. Methanol (18 ml) was slowly added and the solution was filtered through 0.45 µm pore size cellulose acetate membrane filters. Acetic anhydride (0.3 ml) was added and kept for 30 min. The resulting gel was cut into small pieces and after adding 1 vol of methanol was shaken for 4 min. This suspension was centrifuged at 27000 ×g at 4ºC for 15 min and the pellet was resuspended in 20 ml of 0.02% (w/v) sodium azide. The final concentration of the glycol chitin was 1% (w/v). Evaluation of antifungal activity Quantitative assay for fungal growth inhibition was performed following the protocol developed by Broekaert et al. (1990) with some modifications (Acosta et al., 2009a). The toxigenic strains P. restrictum Pr341 and A. niger An261 (Núñez et al., 1996; Díaz, 1999) were used as reference strains. To perform the inhibition test in microtitter plates, 100 µl of the fraction obtained by gel filtration chromatography were dispensed in triplicate wells. Each well was loaded with 100 µl of concentrated malt extract broth (4% malt extract, 4% 70 MENÚ SALIR RESULTADOS glucose and 0.2% peptone, pH 4.5) inoculated with the reference strain to c.a. 106 spores/ml. The plates were incubated for up to 96 h at 25ºC. Fungal growth was determined by measuring the optical density variation at 595 nm after 96 h. Effect of heat-treatments The study of the protein stability after heat treatment was carried out according to Okkers et al. (1999). Aliquots of PgAFP protein (300 µg/ml) were exposed to different combinations of temperatures (60, 80, and 100ºC) and times (10, 20, and 30 min), as well as to sterilization at 121ºC for 15 min. After the heat-treatment, the samples were cooled on ice and tested for antifungal activity in microtiter plates. PgAFP antifungal activity after exposure at different pH values PgAFP (300 µg/ml) was retained in microcon centrifugal filter units YM-3 (Millipore) and then dissolved in different buffers at the following values: pH 1, 2 (HCl/KCl); pH 3 (glycine/HCl); pH 4, 5, 6 (citric acid/sodium phosphate); pH 7 (sodium phosphate/NaCl); pH 8 (Tris/HCl); pH 9, 10 (Tris/NaOH); and pH 12 (KCl/NaOH). Samples were incubated at 25ºC for 2 h and the pH adjusted to 4.5 before testing the antifungal activity. Effect of different chemicals on PgAFP antifungal activity To study the influence of some chemicals on the antifungal activity, several assays were carried out with 3, 30, and 300 µg PgAFP/ml in presence of the following compounds: NaCl, CaCl2, KCl, or MgCl2 (0.1, 1, 10, and 100 mM); EDTA or EGTA at 0.01, 0.1, and 1 mM; 1% of Triton X-100, tween-20, tween-80, SDS, or deoxycholic acid; NaH2PO4 at 0, 0.1, 1, 10, and 100 mM. Statistical analysis Data were statistically analyzed by SPSS software package (version 13.0. SPSS Inc., Chicago, USA) for a variance analysis (one way ANOVA) and the means were separated by the Tukey honest significant difference test. 71 MENÚ SALIR MANUSCRITO 3 RESULTS Evaluation of PgAFP toxicity None of the readings at either 570 or 541 nm during the incubation time revealed haemolytic activity. The concentration of PgAFP tested (240 µg/ml) showed 0% haemolysis after 24 h. In an additional assay with Artemia salina, the larvae mortality was 0% even at the highest concentration of the protein 75 µg/ml after 24 h incubation. Therefore, PgAFP showed no toxicity at any concentration tested. Chitosanase and chitinase activities No chitosanase or chitinase activity was observed by the 3,5-dinitrosalicylic acid method, as no glucosamine was liberated by PgAFP in the conditions assayed. To further evaluate the chitinase activity, chitin hydrolysis was tested by electrophoresis in a glycol chitin gel. No activity was observed loading the polyacrylamide gel with 50 µg of PgAFP (Figure 1). Lytic zones by chitinase from S. griseus used as positive control were observed as black bands after staining with calcofluor white M2R. Figure 1. Chitinase activity by gel electrophoresis. SDS-PAGE with glycol chitin was stained with Imperial Protein Stain (A) and Calcofluor White M2R (B). M: protein marker, Q: chitinase from S. griseus (100 µg). 72 MENÚ SALIR RESULTADOS Effect of heat-treatments and different pH values PgAFP protein showed a remarkable stability at high temperatures. PgAFP kept antifungal activity against P. restrictum and A. niger even after being heat-treated at 80ºC for 30 min "# $ % & ! ' or 100ºC for 15 min (Figure 2). 10 20 30 10 20 30 3 10 15 20 30 15 15a 60ºC 80ºC 100ºC 121ºC 10 20 30 10 20 30 3 10 15 20 30 15 15a 60ºC 80ºC 100ºC 121ºC Figure 2. Antifungal activity of heat-treated PgAFP expressed as mean and standard deviation of reference moulds growth after 96 h. Asterisks indicate statistically significant differences to positive control with non heat-treated PgAFP (***p<0.001). C+: positive control with non heattreated PgAFP; C-: negative control without PgAFP. a: Autoclave cycle. Incubation in buffers at pH values from 1 to 12 had no effect on the stability of PgAFP and the protein remained active against the reference moulds (Figure 3). 73 MENÚ SALIR MANUSCRITO 3 "# $ % & ! ' * ( ) $+ Figure 3. Antifungal activity of PgAFP after treatment at different pH values expressed as mean and standard deviation of reference moulds growth after 96 h. C-: negative control without PgAFP. Effect of cations The impact of different monovalent and divalent cations at 0.1, 1, 10, and 100 mM on the inhibitory activity of PgAFP (3, 30, and 300 µg/ml) over P. restrictum and A. niger was tested (Figure 4). Monovalent cations did not seem to exert a strong impact on the antifungal activity. The antifungal activity on A niger decreased only with at the highest concentration of K+ or Na+ (100 mM) and the lowest level of PgAFP. The remaining conditions tested did not alter the activity of PgAFP against P. restrictum and A. niger. Divalent cations showed a deeper impact on PgAFP activity against both sensitive moulds. The highest level (100 mM) of Ca2+ decreased PgAFP activity, irrespectively of the protein concentration tested. Even 10 mM Ca2+ reduced the inhibition with 3 µg/ml of PgAFP. Mg2+ showed a lower effect than Ca2+, given that only 100 mM decreased the activity with low PgAFP concentrations. 74 MENÚ SALIR RESULTADOS ' µ . * µ . * µ . * µ . "# $ % & ! ' * / 0% / ,% % 0% " -' 1' µ . * µ . * µ . * * "# $ % & ! ' µ . - % - ,% % % " -' Figure 4. Effect of monovalent (A) and divalent (B) cations on antifungal activity of PgAFP expressed as mean and standard deviation of reference moulds growth after 96 h. Asterisks indicate statistically significant differences to the negative control without PgAFP (*p<0.05; **p<0.01; ***p<0.001). Effect of calcium chelators EDTA and EGTA showed a different effect on PgAFP activity. EGTA had no influence on PgAFP antifungal activity, even at the highest concentration tested (1 mM). EDTA at 75 MENÚ SALIR MANUSCRITO 3 levels up to 0.01 mM had no impact on PgAFP antifungal activity against either sensitive mould, whereas 0.1 mM limited the activity of lower levels of PgAFP just on P. restrictum (Figure 5). The highest concentration of EDTA (1 mM) prevented growth of P. restrictum and A. niger (data not shown), and no further effect could be established. µ . * µ . * µ . * * "# $ % & ! ' µ . - - - 23 4 % - " -' Figure 5. Effect of EDTA on the antifungal activity of PgAFP expressed as mean and standard deviation of reference moulds growth after 96 h. Asterisks indicate statistically significant differences to the negative control without PgAFP (***p<0.001). Effect of detergents Even though non-ionic detergents limited the growth of the reference moulds, it was possible to evaluate their effect on PgAFP activity. However, SDS totally prevented growth of reference moulds, and no effect on PgAFP could be established. For both sensitive strains, tween 20 had no effect, tween 80 only reduced the activity with 3 µg/ml of PgAFP, and Triton X-100 decreased the activity at all PgAFP concentrations (Figure 6). With deoxycholic acid PgAFP did not show antifungal activity against P. restrictum, whereas the effect of PgAFP on A. niger could not be observed due to the extremely poor growth of this mould. 76 MENÚ SALIR RESULTADOS µ . * µ . * µ . * * "# $ % & ! ' µ . 4 4 " 5' 4) 3 4 3 "5' 4 4) 3 " 5' Figure 6. Effect of Triton X-100 (T100), tween 20 (T20), tween 80 (T80), and deoxycholic acid (DAc) on the antifungal activity of PgAFP expressed as mean and standard deviation of reference moulds growth after 96 h. Asterisks indicate statistically significant differences to the negative control without PgAFP (**p<0.01; ***p<0.001). Effect of sodium phosphate Sodium phosphate influenced the antifungal activity against both reference moulds, particularly on A. niger (Figure 7). With P. restrictum, the effect was limited to high levels of phosphate (10-100 mM) with the lowest PgAFP concentration. However, the lowest phosphate concentration (0.1 mM) produced a significant effect on the activity against A. niger with 3 µg/ml of PgAFP. This effect increased gradually with the phosphate concentration. 77 MENÚ SALIR MANUSCRITO 3 µ . * µ . * µ . * * "# $ % & ! ' µ . ! $!% % " -' Figure 7. Effect of phosphate on the antifungal activity of PgAFP expressed as mean and standard deviation of reference moulds growth after 96 h. Asterisks indicate statistically significant differences to the negative control without PgAFP (**p<0.01; ***p<0.001). DISCUSSION P. chrysogenum RP42C has been proposed as protective culture on mould fermented foods (Acosta et al., 2009b), and the use of PgAFP as additive could also be of interest to prevent unwanted moulds. Common methods to evaluate safety are the haemolytic assay (Theis et al, 2004) and the brine shrimp larvae (Artemia salina) test (Harwig and Scott, 1971; Milhem et al., 2008; Chohan, 2009). The absence of cytolytic activity to red blood cells is generally accepted as proof that the protein can be regarded as safe (Van’t et al., 2001). Brine shrimp larvae (Artemia salina) test has been also used to screen fungal toxins (Harwig and Scott, 1971) or synthetic compounds (Milhem et al., 2008; Chohan, 2009) for toxicity. The results here presented revealed neither haemolytic activity against red blood cells nor lethal effects on brine shrimp larvae when PgAFP was tested up to 240 and 75 µg/ml, respectively. The information available on other antifungal proteins from moulds reveals that neither PAF nor AFP was toxic up to 100 µg/ml to human umbilical vein endothelial cells (Szappanos et al., 2005, 2006). There are also bacterial antifungal proteins non-toxic to blood red cells, such as PPEBL21 from Escherichia coli at 1,250 µg/ml (Yadav et al., 2007). On the other hand, some antifungal proteins from bacteria have shown toxic effects. 78 MENÚ SALIR RESULTADOS SAP protein isolated from Streptomyces sp. strain AP77 was non-toxic up to 250 µg/ml to human dermal fibroblasts but toxic at all higher doses (Woo et al., 2002). A more potent toxicity was found for syringomycin, a protein that has shown 100% haemolysis at 20 µg/ml (Sorensen et al., 1996). Antifungal peptides purified from Bacillus cereus were also reported to be highly toxic to human erythrocytes (Latoud et al., 1986). The toxicity of most reported antifungal proteins to mammalian cells has become a major limitation for their use to develop new antifungal drugs or preparing improved antifungal formulations. Therefore, the lack of toxicity showed by PgAFP in these preliminary tests supports its potential for food use, although a more complete evaluation is required. It is known that most chitin containing organisms produce chitinases, which are presumably required for morphogenesis of cell walls and exoskeleton (Gooday, 1971). Apart from these functions, these enzymes have also been found to be involved in the host defence against pathogens (Somashekar and Joseph, 1996). Chitinases from moulds have been described to have antifungal activity, such as from Thermomyces lanuginosus (Guo et al., 2005) and from Fusarium chlamydosporum (Mathivanan et al., 1998). In addition, in a parallel work an antifungal protein from P. chrysogenum AS51D that hydrolyses deacetylated chitin has been described (Rodríguez-Martín et al., 2009c). However, PgAFP did not show either chitinase or chitosanase activities when tested in solution or in gel (Figure 1). Therefore, the antifungal activity of this protein cannot be based on the degradation of these compounds commonly found in fungal cell wall. Some detergents can be present on various foods as a consequence of their use as direct or indirect additives. Detergents are unique among protein denaturants, and some of them can alter protein conformation at low concentrations. The addition of detergents can either increase or decrease the helical conformation, and even convert forms into helices (Naeem et al., 2006). The effect of ionic and non-ionic detergents on PgAFP varied according to the sensitive mould and the protein concentration tested (Figure 6). Triton X100 and deoxycholic acid inhibited the antifungal activity, irrespectively of PgAFP concentration. However, the inhibitory effect of both tweens was limited to tween 80 at the lowest PgAFP concentration tested. Ionic detergents tend to denature proteins by altering native three dimensional structures. Some ionic detergents, such as deoxycholic acid, can also act as open-channel blockers, as it has been shown for porins (Duret and Delcour, 2006). No further information is available to suggest whether the effect of deoxycholic acid is mainly due to changes in the tertiary structure of PgAFP or in the fungal membrane. 79 MENÚ SALIR MANUSCRITO 3 In this study, the non-ionic detergent Triton X-100 seems to counteract the antifungal activity of PgAFP. However, the partial inhibition on growth of reference moulds made it difficult to evaluate the particular impact of Triton X-100 on PgAFP activity. Finally, the activity of PgAFP decreased as sodium phosphate concentration increased, although the protein remained active up to 100 mM of phosphate. Among the main factors that can interfere with the antifungal activity in foods are salts, pH and heat treatments. PgAFP was highly resistant to a wide range of temperature and pH values. As it is shown in figure 2, PgAFP displayed antifungal activity after treatment at 80ºC for 30 min, and even at 100ºC for 15 min. Similarly, incubation in buffers at pH values from 1 to 12 had no effect on PgAFP antifungal activity. Additionally, PgAFP showed a high stability to proteases, such as pepsin and papain (Acosta, 2006). These characteristics are similar to those of other antifungal proteins from moulds. AFP from A. giganteus is stable at high temperature, protease degradation, and a broad pH range due to a specific folding pattern (Lacadena et al., 1995). The mature form of AFP folds into five antiparallel strands which define a small and compact -barrel, stabilized by internal disulfide bridges formed by eight cysteine residues (Campos-Olivas et al., 1995). It has been assumed that Anafp, NAF, and PAF have the same tertiary structure, although only three disulfide bridges can be formed by the six cysteine residues present in these proteins (Marx, 2004). PgAFP has common characteristics to these antifungal proteins, including a high stability, six cysteine residues, and a quite high similarity in the amino acid sequence (Rodríguez-Martín et al., 2009a, b). Thus, PgAFP could also have a tertiary structure based on a compact -barrel. On the other hand, cation sensitivity has been described for basic antimicrobial proteins, including AFP and PAF. In fact, AFP activity decreases in the presence of Ca2+, K+, and Na+ ions (Theis et al., 2003), and PAF activity is sensitive to Mg2+, K+ and Na+ (Kaiserer et al., 2003). Cations seem to disturb the interaction of antifungal proteins with negatively charged membrane components (Barkai-Golan et al., 1978) or specific receptors (Broekaert et al., 1992). The latter has been suggested for AFP (Theis et al., 2003) and PAF (Kaiserer et al., 2003). The influence of high cation levels on PgAFP depended on the ion charge, protein concentration and the sensitive mould. The highest effect was obtained with the divalent cations (Figure 4). Defensins are also more sensitive to divalent than to monovalent cations 80 MENÚ SALIR RESULTADOS (Terras et al., 1992), particularly to Ca2+ (Lehrer et al., 1989; Cociancich et al., 1993; Osborn et al., 1995). This indicates that the effect of monovalent cations depended on the reference mould, with no effect on the activity against P. restrictum. The antagonistic effect of ions over plant defensins is dependent on the fungus and thus on the conformation of the putative target site (Terras et al., 1992; Osborn et al., 1995). Thus, the distinct effect of cations on PgAFP activity against each sensitive mould tested can be explained by differences on receptor conformation. In addition, the results obtained for PgAFP suggest that the ratio of protein to ion concentration plays a decisive role on the interaction with protein activity. EDTA can also be present in foods due to its use as metal sequestrant additive. The ability of EDTA and EGTA for chelating ions is well known, being EDTA the most potent of them (Choucair et al., 2006). However, EDTA chelates both Ca2+ and Mg2+, whereas EGTA is more specific for Ca2+ ions. EGTA did not influence PgAFP activity, whereas EDTA inhibited this activity only against P. restrictum at the lower concentrations of the protein tested (Figure 5). Thus, an excess of divalent cations has a stronger effect on PgAFP activity than the lack of those ions. In summary, PgAFP protein is stable over a wide range of pH values and active in presence of high concentrations of ions and at high temperatures. All this information revealed that PgAFP has potential to prevent mould growth in a wide range of foods with high salt levels or where thermal or acid stability are essential. However, the use of this protein as additive requires an in-depth toxicity evaluation. ACKNOWLEDGEMENTS This work was supported by the Spanish Ministry of Education and Science (AGL200406546-ALI) and FEDER. Andrea Rodríguez and Raquel Acosta were the recipients of a FPI grant from the Spanish Ministry of Education and Science. REFERENCES Acosta, R. Doctoral Thesis: “Selección de Penicillium productores de péptidos antifúngicos para su utilización en productos cárnicos madurados”. University of Extremadura (2006). 81 MENÚ SALIR MANUSCRITO 3 Acosta, R., Rodríguez-Martín, A., Martín, A., Núñez, F., and Asensio, M.A. Selection of antifungal protein-producing molds from dry-cured meat products. Int. J. Food Microbiol. Submitted (2009a). Acosta, R., Rodríguez-Martín, A., Bermúdez, E., Núñez, F., and Asensio, M.A. Partial characterization and antifungal activity of a protein from Penicillium chrysogenum. Food Microbiol. Submitted (2009b). Barkai-Golan R., Mirelman D., and Sharon, N. Studies on growth inhibition by lectins of Pencillia and Aspergilli. Arch. Microbiol. 116: 119–124 (1978). Benito, M.J., Núñez, F., Córdoba, M.G, Martín, A., and Córdoba, J.J. Generation of non-protein nitrogen and volatile compounds by Penicillium chrysogenum Pg222 activity on pork myofibrillar proteins. Food Microbiol. 22 (6): 513-519 (2005). Broekaert, W.F., Terras, F.R.G., Cammue, B.P.A., and Vanderleyden, J. An automated quantitative assay for fungal growth inhibition. FEMS Microbiol. 69: 55-60 (1990). Broekaert, W.F., Mariën, W., Terras, F.R.G., De Bolle, M.F.C., Proost, P., Van Damme, J., Dillen, L., Claeys, M., Rees, S.B., Vanderleyden, J., and Cammue, B.P.A. Antimicrobial peptides from Amaranthus caudatus seeds with sequence homology to the cysteine/glycine-rich domain of chitin-binding proteins. Biochemistry. 31: 4308-4314 (1992). Campos-Olivas, R., Bruix, M., Santoro, J., Lacadena, L., Martínez del Pozo, A., Gavilanes, J.G., and Rico, M. NMR solution structure of the antifungal protein from Aspergillus giganteus: evidence for cysteine pairing isomerism. Biochemistry. 34: 30093021 (1995). Chohan, Z.H. Synthesis of organometallic-based biologically active compounds: In vitro antibacterial, antifungal and cytotoxic properties of some sulfonamide incorporated ferrocences. J. Enzyme Inhib. Med. Chem. 24: 169-75 (2009). Choucair N., Laporte V., Levy R., Tranchant C., Gies J. P., Poindron P., and Lombard, Y. The role of calcium and magnesium ions in uptake of beta-amyloid peptides by microglial cells. Int. J. Immunopathol. Pharmacol. 19: 683-96 (2006). 82 MENÚ SALIR RESULTADOS Cociancich, S., Ghazi, A., Hetru, A., Hoffman, J.A., and Letellier, L. Insect defensin, an inducible antibacterial peptide, forms voltage-dependent channels in Micrococcus luteus. J. Biol. Chem. 268: 19239-19245 (1993). Córdoba, J.J. Doctoral Thesis: Transformaciones de los componentes nitrogenados durante la maduración del jamón de cerdo ibérico. Universidad de Extremadura (1990). Díaz, M.C. Doctoral Thesis: “Caracterización de mohos toxigénicos aislados de jamón curado mediante técnicas de ácidos nucleicos, cromatográficas y electroforéticas”. University of Extremadura. Spain. (1999). Duret, G. and Delcour, A.H. Deoxycholic acid blocks Vibrio cholerae OmpT but not OmpU Porin. Jour. biol. chem. 281(29): 19899-19905 (2006). Gooday, G.W. An audioradiographic study of hyphal growth of some fungi. J. Gen. Microbiol. 67: 125-133 (1971). Groll, A.H., De Lucca, A.J., and Walsh, T.J. Emerging targets for the development of novel antifungal therapeutics. Trends Microbiol. 6: 117-124 (1998). Guo, R.F., Li, D.C., and Wang, R. Purification and properties of a thermostable chitinase from thermophilic fungus Thermomyces lanuginosus. Wei Sheng Wu Xue Bao. 45(2): 2704 (2005). Harighi, M. J., Zamani, M. R., and Motallebi, M. Evaluation of antifungal activity of purified chitinase 42 from Trichoderma atroviride PTCC5220. Biotech. 6: 28-33 (2007). Harwig, J. and Scott, P.M. Brine shrimp (Artemia salina L.) larvae as a screening system for fungal toxins. Appl. Microbiol. 21(6): 1011–1016 (1971). Hong, S.Y., Park, T.G., and Lee, K.H. The effect of charge increase on the specificity and activity of a short antimicrobial peptide. Peptides. 22: 1669-1674 (2001). Hwu, L., Huang, C.C., Chen, D.T., and Lin, A. The action mode of the ribosomeinactivating protein -sarcin. J. Biomed. Sci. 7: 420-428 (2000). Jacobsen, T. and Hinrichsen, L. Bioformation of flavour by Penicillium candidum on glucose, peptone, maize oil and meat extract. Food Chem. 3: 409-416 (1997). Kaiserer, L., Oberparleiter, C., Weiler-Görz, R., Burgstaller, W., Leiter, E., and Marx, F. Characterization of the Penicillium chrysogenum antifungal protein PAF. Arch Microbiol. 180: 204-210 (2003). 83 MENÚ SALIR MANUSCRITO 3 Lacadena, J., Martínez del Pozo, A., Gasset, M., Patiño, B., Campos-Olivas, R., Vázquez, C., Martínez-Ruiz, A., Mancheño, J.M., Oñaderra, M., and Gavilanes, J.G. Characterization of the antifungal protein secreted by the mould Aspergillus giganteus. Arch. Biochem. Biophys. 20: 273-281 (1995). Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685 (1970). Lai, Y-P., Peng, Y-F., Zuo, Y., Li, J., Huang, J., Wang, L-F., and Wu, Z.R. Functional and structural characterization of recombinant dermcidin-1L, a human antimicrobial peptide. Biochem. Bioph. Research. Commun. 328: 243–250 (2005). Latoud, C., Peypoux, F., Michel, G., Genet, R., and Morgat, J. Interactions of antibiotics of the iturin group with human erythrocytes. Biochim. Biophys. Acta. 856: 526– 535 (1986). Lehrer, R.I., Barton, A., Daher, K.D., Harwig, S.S., Ganz, T., and Selsted, M.E. Interaction of human defensins with Escherichia coli. Mechanism of bactericidal activity. J. Clin. Investig. 84: 553-561 (1989). López-Díaz, T.M., Santos, J.A., García-López, M.L., and Otero, A. Surface mycoflora of a Spanish fermented meat sausage and toxigenicity of Penicillium isolates. Int. J. Food Microbiol. 68: 69-74 (2001). Lund, F., Filtenborg, O., and Frisvad, J.C. Associated mycoflora of cheese. Food Microbiol. 12: 173-180 (1995). Martín, A., Asensio, M.A., Bermúdez, M.E., Córdoba, M.G., Aranda, E., and Córdoba, J.J. Proteolytic activity of Penicillium chrysogenum and Debaryomyces hansenii during controlled ripening of pork loins. Meat Sci. 62: 129-137 (2002). Martín, A., Córdoba, J.J., Aranda, E., Córdoba, M.G., and Asensio, M.A. Contribution of a selected fungal population to the volatile compounds on dry-cured ham. Int. J. Food Microbiol. 110(1): 8-18 (2006). Marx, F. Small, basic antifungal proteins secreted from filamentous ascomycetes: a comparative study regarding expression, structure, function and potential application. Appl. Microbiol. Biotechnol. 65: 133-142 (2004). 84 MENÚ SALIR RESULTADOS Marx, F., Binder, U., Leiter, É. y Pócsi, I. The Penicillium chrysogenum antifungal protein PAF, a promising tool for the development of new antifungal therapies and fungal cell biology studies. Cell. Mol. Life Sci. 65: 445-454 (2008). Mathivanan, N., Kabilan, V., and Murugesan, K. Purification, characterization, and antifungal activity of chitinase from Fusarium chlamydosporum, a mycoparasite to groundnut rust, Puccinia arachidis. Can. J. Microbiol. 44(7): 646-51 (1998). Milhem, M. M., Al-hiyasat, A.S. and Darmani, H. Toxicity testing of restorative dental materials using brine shrimp larvae (Artemia Salina). J. Appl. Oral Sci. 16: 297-301 (2008). Moerman, L., Bosteels, S., Noppe, W., Willems, J., Clonen, E., Schoofs, L., Thevissen, K., Tytgat, J., Van Eldere, J., and Verdonck, J. W. F. Antibacterial and antifungal properties of alpha-helical, cationic peptides in the venom of scorpions from southern Africa European. J. Biochem. 269: 4799-4805 (2002). Naeem, A., Fatima, S., and Khan R.H. Characterization of partially folded intermediates of papain in presence of cationic, anionic, and nonionic detergents at low pH. Biopolymers. 83: 1-10 (2006). Nielsen, M.S., Frisvad, J.C., and Nielsen P.V. Colony interaction and secondary metabolite production of cheese-related fungi in dual culture. J. Food Prot. 61(8): 10231029 (1998). Núñez, F., Rodríguez, M.M., Bermúdez, M.E., Córboba, J.J., and Asensio, M.A. Composition and toxigenic potential of the mould population on dry-cured ham. Int. J.Food Microbiol. 32: 185-197 (1996). Núñez, F., Díaz, M.C., Rodríguez, M.M., Aranda, E., Martín, A., and Asensio, M.A. Effects of substrate, water activity, and temperature on grown and verrucosidin production by Penicillium polonicum isolated from dry-cured ham. J. Food Prot. 63: 231-236 (2000). Okkers, D.J., Dicks, L.M.T., Silvester, M., Joubert, J.J. and Odendaal, H.J. Characterization of pentocin TV35b, a bacteriocin-like peptide isolated from Lactobacillus pentosus with a fungistatic effect on Candida albicans. J. Appl. Microbiol. 87: 726-734 (1999). Osborn, R.W., De Samblanx, G.W., Thevissen, K., Goderis, I., Torrekens, S., Van Leuven, F., Attenborough, S., Rees, S.B., and Broekaert, W.F. Isolation and 85 MENÚ SALIR MANUSCRITO 3 characterization of plant defensins from seeds of Asteraceae, Fabaceae, Hippocastanaceae and Saxifragaceae. FEBS Lett. 368: 257-262 (1995). Ramírez, J., Guerrero, I., Ponce, E., and Prado, A. Changes in flavour attributes during ripening of fermented sausages. J. Muscle Foods. 6: 257-269 (1995). Rodríguez-Martín, A., Liddell, S., Acosta, R., Nuñez, F., and Asensio, M.A. Characterization of PgAFP, a novel antifungal protein from Penicillium chrysogenum. Manuscrito 1 (2009a). Rodríguez-Martín, A., Acosta, R., Nuñez, F., Benito, M.J., and Asensio, M.A. Genetic characterization of the gene encoding the novel antifungal protein PgAFP from Penicillium chrysogenum. Manuscrito 2 (2009b). Rodríguez-Martín, A., Liddell, S., Acosta, R., Nuñez, F., and Asensio, M.A. Characterization of PgChP, a novel chitosanase with antifungal activity from Penicillium chrysogenum. Manuscrito 4 (2009c). Sabater-Vilar, M., Nijmeijer, S., and Fink-Gremmels, J. Genotoxicity assessment of five tremorgenic mycotoxins (Fumitremorgen B, Paxilline, Penitrem A, Verruculogen, and Verrucosidin) produced by molds isolated from fermented meats. J. Food Prot. 66: 21232129 (2003). Somashekar, D. and Joseph, R. Chitosanases-Properties and applications: a review. Biores. Tech. 55: 35-45 (1996). Sorensen, K.N., Kim, K.H., and Takemoto, J.Y. In vitro antifungal and fungicidal activities and erythrocytes toxicities of Pseudomonas syringae pv. syringae. Antimicrob. Agents Chemother. 40: 2710–2713 (1996). Sosa, M.J., Córdoba, J.J., Díaz, M., Rodríguez, M.M., Bermúdez, E., Asensio, M.A., and Núñez, F. Production of cyclopiazonic acid by Penicillium commune isolated from dry-cured ham on a meat-extract based substrate. J. Food Prot. 65: 988-992 (2002). Su, C., Wang, D., Yao, L., and Yu, Z. Purification, characterization, and gene cloning of a chitosanase from Bacillus species strain S65. Agric. Food Chem. 54: 4208-4214 (2006). Szappanos, H., Szigeti, G.P., Pál, B., Rusznák, Z., Szucs, G., Rajnavölgyi, E., Balla, J., Balla, G., Nagy, E., Leiter, E., Pócsi, I., Marx, F., and Csernoch, L. The Penicillium chrysogenum-derived antifungal peptide shows no toxic effects on mammalian cells in the 86 MENÚ SALIR RESULTADOS intended therapeutic concentration. Naunyn Schmiedebergs Arch Pharmacol. 371(2):122132 (2005). Szappanos, H., Szigeti, G.P., Pál, B., Rusznák, Z., Szucs, G., Rajnavölgyi, E., Balla, J., Balla, G., Nagy, E., Leiter, E., Pócsi, I., Hagen, S., Meyer, V., and Csernoch, L. The antifungal protein AFP secreted by Aspergillus giganteus does not cause detrimental effects on certain mammalian cells. Peptides. 27(7):1717-1725 (2006). Terras, F.R., Schoofs, H.M., De Bolle, M.F., Van Leuven, F., Rees, S.B., Vanderleyden, J., Cammue, B.P., and Broekaert, W.F. Analysis of two novel classes of plant antifungal proteins from radish (Raphanus sativus L.) seeds. J. Biol. Chem. 267(22): 15301–15309 (1992). Theis, T., Wedde, M., Meyer, V., and Stahl, U. The antifungal protein from Aspergillus giganteus causes membrane permeabilization. Antimicrob. Agents Chemother. 47: 588-593 (2003). Theis, T. and Stahl, U. Antifungal proteins: targets, mechanisms and prospective applications. Cell Mol. Life Sci. 61(4): 437-55 (2004). Thevissen, K., Terras, F.R.G., and Broekaert, W.F. Permeabilization of fungal membranes by plant defensins inhibits fungal growth. Appl. Env. Microbiol. 65: 5451-5458 (1999). Trudel, J. and Asselin, A. Detection of chitinase activity after polyacrvlamide gel electrophoresis. Anal. Biochem. 178: 362-366 (1989). Van’t, H., W., Veerman, E.C.I., Helmerhorst, E.J., and Amerongen, A.V.N. Antimicrobial peptides: properties and applicability. Biol. Chem. 382: 597-619 (2001). Woo, J. H., Kitamura, E., Myuoga, H., and Yuto, K. An antifungal protein from the marine bacterium Streptomyces sp. strain AP77 is specific for Pythium porphyrae, a causative agent of red rot disease in Porphyra spp. Appl. Environ. Microbiol. 68: 2666– 2675 (2002). Yadav, V., Mandhan, R., Pasha, Q., Pasha, S., Katyal, A., Chhillar, A.K., Gupta, J., Dabur, R., and Sharma, G. L. An antifungal protein from Escherichia coli. J. Medical Microbiol. 56: 637–644 (2007). 87 MENÚ SALIR Manuscrito 4: Characterization of PgChP, a novel chitosanase with antifungal activity from Penicillium chrysogenum MENÚ SALIR RESULTADOS Characterization of PgChP, a novel chitosanase with antifungal activity from Penicillium chrysogenum Andrea Rodríguez-Martín, Susan Liddella, Raquel Acosta, Félix Núñez, Miguel A. Asensio* Higiene y Seguridad Alimentaria, Facultad de Veterinaria. Universidad de Extremadura, Avda. de la Universidad, s/n. 10071- Cáceres, Spain. a Division of Animal Sciences, University of Nottingham. Sutton Bonington Campus. Loughborough, Leicestershire. LE12 5RD. United Kingdom. * Corresponding author: Telephone (+34) 927 257 125; fax: (+34) 927 257 110. E-mail: [email protected] ABSTRACT A new fungal chitosanase with antifungal activity, PgChP from Penicillium chrysogenum, is described. Two isoforms were found by SDS-PAGE in the purified extract of PgChP. After enzymatic deglycosylation only the smaller isoform was observed by SDS-PAGE. Identical amino acid sequences were obtained from the two proteins and these showed a high similarity with fungal chitosanases. Analysis of the molecular mass by electrospray ionization mass spectrometry (ESI-MS) revealed the existence of six peaks from 30 to 31 kDa differentiated by a mass multiple of 162 Da, the typical mass of galactose or mannose residues. This could be due to different levels of glycosylation of the same protein PgChP. The isoelectric point was aproximately pH 5 estimated by two-dimensional polyacrylamide gel electrophoresis. PgChP is the first chitosanase to be described from P. chrysogenum. KEY WORDS: antifungal protein, food preservative, glycosylation, N-linked oligosaccharides, protective culture, toxigenic mould. INTRODUCTION The antifungal protein PgChP was selected in previous studies (Acosta et al., 2009a) from the strain AS51D isolated from dry-cured ham and characterized as Penicillium chrysogenum according to classical taxonomical methods (Pitt and Hocking, 1997). PgChP was purified by fast protein liquid chomatography and showed antifungal activity against selected toxigenic moulds (Acosta et al., 2009a). 89 MENÚ SALIR MANUSCRITO 4 Antifungal proteins have been described from diverse organisms including plants and animals, but only scant information is available about antifungal proteins of fungal origin. Many antifungal proteins have interesting applications because they attack compounds of fungal walls that are not present in mammalian cells, such as glucanases, chitinases and chitosanases (Adams, 2004). Fungal infections and contaminations have led to an increasing demand for antifungal drugs in diverse fields including agriculture, medicine and food protection. Many antifungal drugs have low efficacy rates, show severe side effects and cause resistance on the microorganisms, as antibiotics (Meyer, 2008). This makes it necessary to study new compounds, including fungal proteins such as PgChP. However, before any application of PgChP, an exhaustive characterization of the protein is required. In previous studies, another antifungal protein from P. chrysogenum, named PgAFP, showed evidence of glycosylation by periodic acid-Schiff staining (Acosta et al., 2009b). Glycan chains in glycoproteins participate in many biological processes, such as protein interactions, or confer stability on the protein against proteases or heat treatments (Morelle et al., 2006). These modifications introduce variations in the molecular mass and net charge of the protein. Enzymatic and chemical deglycosylation treatments followed by SDS-PAGE and electrospray ionization mass spectrometry are commonly used to evaluate this sort of post-translational modification (Claverol et al., 2003). Additional information on the molecular mass and amino acid sequences is typically obtained by mass spectrometry after digestion in-gel or in-solution with proteolytic enzymes (Habermann et al., 2004). This paper describes the amino acid sequencing, molecular mass determination and isoelectric point estimation of the antifungal protein PgChP. MATERIALS AND METHODS Microbial strain P. chrysogenum AS51D was isolated from dry-cured ham, being identified according to classical morphological and biochemical studies (Acosta et al., 2009a). 90 MENÚ SALIR RESULTADOS Genetic identification of the mould Genetic characterization of AS51D was carried out by amplifying the fragments 18S-28S rRNA intergenic spacer (ITS) by polymerase chain reaction (White et al., 1990). DNA isolation. The mould AS51D was grown in malt extract broth, made of glucose 2% (w/v), malt extract 2% (w/v), and peptone 0.1% (w/v), pH 4.5, at 25ºC for 5 days. Two grams of mycelium were broken with morter and pestle after freezing by adding liquid nitrogen. Lysis was performed by homogenizing the powder in 4 ml 0.05 M Tris-HCl, pH 8, 0.005 M ethylenediamine tetraacetic acid (EDTA), 0.05 M NaCl (TES) buffer and 1% (w/v) sodium dodecyl sulfate (SDS). Afterwards, 200 µl proteinase K (20 µg/µl) was added, and the mixture was incubated at 60ºC for 35 min, cooled on ice and extracted with phenol-chloroform-isoamyl alcohol (25:24:1). The suspension was centrifuged at 3,000 ×g for 2 min at 4ºC. The upper phase containing DNA was transferred to a fresh tube and precipitated by the addition of 3 M sodium acetate pH 5.2 to a final concentration of 10% (w/v) and two volumes of cold ethanol. After centrifugation, the pellet was washed with 70% (v/v) ethanol, centrifuged again in the conditions mentioned above, resuspended in sterile water and treated with 50 µl RNase (10 µg/µl) at 37ºC for 60 min. Finally, DNA was extracted with phenol-chloroform-isoamyl alcohol and precipitated again as indicated above, resuspended in water and stored at -70ºC. The quantity and quality of purified DNA was determined spectrophotometrically in a Biophotometer (Eppendorf AG, Hamburg, Germany). ITS amplification. PCR fragments for sequence analysis were obtained using primers ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4 (TCCTCCGCTTATTGATATGC) covering the internal transcribed spacer 1 (ITS1) region, 5.8S, and ITS2 region of the ribosomal DNA. For each 50 µl reaction, a mixture was prepared containing 50 ng of genomic DNA, 0.1 vol. of 10× PCR buffer (10 mM Tris-HCl, pH 8.8, 50 mM KCl, 0.1% Triton X-100), 4 mM MgCl2, 1 mM of deoxynucleoside triphosphates (Roche, Mannheim, Germany), 0.4 µM of each primer, and 2 U of DyNAzyme I DNA Polymerase (Finnzymes, Espoo, Finland). Reactions were run on a programmable thermal cycler Mastercycler epgradient (Eppendorf AG) with an initial denaturation of 3 min at 96°C, followed by 30 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 40 s, with a final extension at 72°C for 10 min. Aliquots of the PCR were electrophoresed on 2% (w/v) agarose gels in 1× TAE buffer (40 mM Tris-acetate, 1 mM EDTA) pH 8, using a Sub-Cell GT apparatus (Bio-rad Laboratories, Hercules, USA). Products were visualized using the UV transillumination G91 MENÚ SALIR MANUSCRITO 4 Box (Syngene, Fredericks, USA) and photographed with a GeneSnap camera (Syngene). DNA fragments were gel-purified and sequenced at the Institute of Biomedicine (CSIC, Valencia, Spain). The sequences obtained were searched against the public databases using the Basic Local Alignment Search Tool (BLAST). PgChP purification P. chrysogenum AS51D was grown in malt extract broth at 25ºC for 15 days. The medium was filtered through a 0.45 µm diameter pore size cellulose acetate membrane and PgChP was purified using an ÄKTA fast protein liquid chromatography system (Amersham Bioscience AB, Uppsala, Sweden) according to Acosta et al. (2009a). For this, filtered medium was loaded on a HiTrap SP HP 5 ml ion exchange column (Amersham Pharmacia Biotech AB) equilibrated with 0.02 M sodium acetate buffer, pH 4.5. Bound proteins were eluted with a gradient of NaCl to 1 M (0 to 25% in 15 column volumes (CV), increasing to 100% in 2 CV, followed by 15 CV at 100%) in 0.02 M sodium acetate buffer. Eluted proteins were detected at 214 nm. The fraction that showed antifungal activity in previous studies (Acosta et al., 2009a) was purified using a HiLoad 26/60 Superdex 75 prep grade gel filtration column (Amersham Pharmacia Biotech AB) equilibrated with 50 mM sodium phosphate, 0.15 M NaCl buffer pH 7, at a constant flow rate of 2.5 ml/min. Data collection and processing were done using the UNICORN software 4.12 (Amersham Pharmacia Biotech AB). Finally, the fraction containing PgChP was desalted and concentrated with YM-10 Microcon Centrifugal Filter Units (Millipore Iberica, Madrid, Spain) and stored at 20ºC. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE was performed as described Laemmli (1970). The protein PgChP was separated on a 4% (v/v) polyacrylamide stacking and 8% (v/v) polyacrylamide resolving gel. Gels were stained with Imperial Protein Stain (Pierce Biotechnology, Rockford, USA). Two preparations of molecular weight markers were used: Precision Plus Protein Standards with ten proteins between 10 and 250 kDa (Bio-Rad) and the Low Molecular Weight Electrophoresis calibration kit with proteins between 14.4 and 97 kDa (Pharmacia, Uppsala, Sweden). 92 MENÚ SALIR RESULTADOS Reduction and alkylation of PgChP Reduction and alkylation of the protein prior to deglycosylation or enzymatic digestion were performed with the protein in SDS-PAGE gel pieces or in solution. After SDSPAGE, PgChP (25 µg per well) was stained using Imperial Protein Stain. The band was excised from the gel and washed three times by incubation in 100 µl of de-stain solution (50 mM ammonium bicarbonate, 50% acetonitrile) for 15 min at 40ºC with intermittent vortexing. Gel pieces were incubated at 56ºC in 100 µl of reducing solution (10 mM dithiothreitol in 50 mM ammonium bicarbonate) which was discarded after 45 min and replaced with 100 µl of alkylation solution (55 mM iodoacetamide in 50 mM ammonium bicarbonate) and incubation continued at room temperature in the dark for 45 min. The gel pieces were then washed in 100 µl of 50 mM ammonium bicarbonate for 5 min at 40ºC and the supernatant was discarded. The gel was washed twice in 100 µl of 50 mM ammonium bicarbonate in 50% acetonitrile for 1 min and centrifuged. Sample was dehydrated in 100 µl of acetonitrile and incubated for 1-2 min. Finally, the supernatant was discarded and the gel pieces were dried at 40ºC for a few min. After addition of the digestive enzyme, the tube containing the gel pieces was cooled at 4°C for 20 min and finally incubated at the optimal temperature of each enzyme. For digestion of PgChP in-solution, different quantities of the protein (1.25, 2.5, and 6.25 µg) were added into tubes containing 50mM ammonium bicarbonate up to a final volume of 5 µl. Dithiothreitol 5 mM dissolved in 50 mM ammonium bicarbonate was added (5 µl) and the final solution was incubated at 50ºC for 30 min. After adding 10 µl of 20 mM iodoacetamide dissolved in 50 mM ammonium bicarbonate, sample was incubated at room temperature for 30 min in the dark before digestion with each enzyme. Digestion with enzymes for amino acid sequencing Trypsin digestion. Digestion with the reduced and alkylated protein was performed with PgChP in-gel and in-solution. Gel pieces containing PgChP were incubated with 10 ng/µl trypsin gold (Promega, Hampshire, United Kingdom) in 50 mM ammonium bicarbonate at 40 or 60ºC for 1.5, 3, or 16 h. Additionally, further automated in-gel digestions were carried out. For these, samples of PgChP in-gel pieces were de-stained, dehydrated, reduced, alkylated, washed, and digested using the ProteomeWorks MassPREP robotic liquid handling station (Waters, Manchester, UK), essentially as described above. For the digestion step, the microtiter plate containing the gel pieces was cooled to 6°C for 10 min 93 MENÚ SALIR MANUSCRITO 4 before addition of 25 µl per well of trypsin gold at 10 ng/µl. The plate was incubated at 6°C for 20 min and incubated at 37°C for 4 h. PgChP was digested in-solution by incubating at 37ºC for 16 h after adding 50 mM ammonium bicarbonate, containing 5% acetonitrile and 10 ng/µl of trypsin gold. All digested protein samples were stored at 4°C until ESI-MS analysis. Chymotrypsin digestion. Digestion with the reduced and alkylated protein was performed with PgChP in-gel and in-solution. Gel pieces were digested at different concentrations (1, 5, 10, and 15 ng/µl) of chymotrypsin (Roche, Welwyn Garden City, UK) in buffer (100 mM Tris-HCl, 10 mM CaCl2 buffer pH 7.8), at 25ºC and 30ºC for 16 h. PgChP in-solution was digested by chymotrypsin at 0.1, 1, 5, or 10 ng/µl with 5% (v/v) acetonitrile and the same buffer mentioned above. Temperatures and incubation time were identical to those used for in-gel sample digestions. Analysis for glucidic residues Chemical deglycosylation was carried out with PgChP in solution and using two different chemical methods: acid hydrolysis with HCl (Leis et al., 1997; Krapf et al., 2006) and elimination with NaOH (Geert and Thomas-Oates, 1998; Tarelli, 2007). For acid hydrolysis with HCl, 1 µg of PgChP was incubated in 20 mM HCl at 50ºC for 3 h. elimination with NaOH was performed by incubating 1µg of PgChP in 0.2 M NaOH at 45ºC for 16 h. Finally, both samples were analyzed by ESI-MS. Enzymatic deglycosylation PNGase F (New England Biolabs, Ipswich, UK) was used following the manufacturer’s instructions and samples were analyzed by SDS-PAGE. E-DEGLY deglycosylation kit (Sigma, Madrid, Spain) contained the following deglycosylases: PNGase F, -2(3,6,8,9) neuraminidase, O-glycosidase, (1-4)-galactosidase, and −N-acetylglucosaminidase. Reactions were carried out under native and denaturing conditions according to manufacturer’s instructions and products were subjected to SDSPAGE analysis. 94 MENÚ SALIR RESULTADOS Electrospray ionization mass spectrometry (ESI-MS) analysis for peptide sequence determination After deglycosylation and enzymatic digestion, formic acid was added to a final concentration of 0.1% (v/v); then the sample was desalted using Zip-Tip C18 reverse phase pipette tips (Millipore) according to manufacturer’s instructions. Peptides were eluted with an equal volume of 50% (v/v) acetonitrile and 0.1% (v/v) formic acid. Samples were loaded into individual borosilicate nanospray needles (Waters) using 1-10 µl GELoader pipette tips (Eppendorf) and analyzed in a Q-TOF2 mass spectrometer (Waters). ESI-MS was carried out with a capillary voltage of 900–1200 V in positive ion mode, using argon as the collision gas. Survey scans of peptide mass spectra were performed with the sampling cone set at 45–50 V and data were acquired from 400–1500 m/z with a cycle time of 2.4 seconds. Peptide mass spectra presented resulted from combining typically 5 – 15 min of data acquisition. Individual peptide ions were selected manually for tandem MS analysis. Collision voltages were optimised manually for individual peptides. Tandem MS fragmentation spectra were collected typically from 50 to 1600 m/z. Tandem MS spectra were deconvoluted into singly charged, mono-isotopic masses using the MaxENT3 maximum entropy software and peptide sequences were determined by manual interpretation using the PepSeq software within the MassLynx V 4.0 package (Waters, UK). De novo peptide sequences were searched against the public databases using BLASTP with parameter settings for “short and nearly exact matches". A multiple sequence alignment of fungal chitosanases was produced using the program T-Coffee at www.ebi.ac.uk. Molecular weight determination by ESI-MS The molecular weight of PgChP was determined by electrospray mass spectrometry in a QTOF2 mass spectrometer (Waters, UK). PgChP (0.125 µg/µl) in 1% (v/v) formic acid was desalted using C18 reverse phase pipette Zip-Tips (Millipore) according to the manufacturer’s instructions. Protein was eluted into 50% (v/v) methanol, 1% (v/v) formic acid, and loaded into nanospray needles for analysis. The mass spectrometer was operated with a capillary voltage of 900-1200 V and the sampling cone at 45-50 V. Data were acquired between 400 and 3000 m/z with a cycle time of 1 second. Mass spectra resulted from combining typically 5 min of data. Spectra were deconvoluted using the MaxEnt1 95 MENÚ SALIR MANUSCRITO 4 maximum entropy software with an output range of 20,000 and 40,000 Da, at a resolution of 1 Da and a peak width of 0.75 Da. Isoelectric point estimation by 2D-PAGE Isoelectric focusing was performed on 7cm IPG ReadyStrips pH 3–10NL (Bio-Rad). Concentrated protein (20 µg) was dissolved in a sample buffer containing 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 0.005% (w/v) bromophenol blue, 0.5% (v/v) destreak reagent (GE Healthcare, Uppsala, Sweden) and 0.5% ampholyte and incubated at room temperature for 60 min. The first-dimension separation was performed on a Protean IEF Cell System (Bio-Rad). The IPG strip was loaded using active in-gel rehydration. Electrofocusing was performed as follows: 250 V for 15 min, then at a linear gradient from 250-4,000 V for 2 h, followed by 4,000 V until a total of 18,000 V/hr was reached. The voltage decreased to a holding voltage of 500 V. After IEF, the strip was equilibrated with a sample equilibration buffer (SEB) containing 6 M urea, 375 mM Tris-HCl (pH 8.8), 2% (w/v) Sodium Dodecyl Sulphate (SDS), 30% (v/v) glycerol, and 0.0125% (w/v) bromophenol blue. In the first incubation, SEB contained 1% (w/v) dithiothreitol, after 8 min this solution was replaced with SEB containing 25% (w/v) iodoacetamide and the strip was incubated for a further 8 min. The IEF strip was placed onto 8% (v/v) polyacrylamide gels and sealed in with agarose. SDS-PAGE was then carried out at 125 V for 50 min. Proteins were visualized by staining with Imperial Protein Stain (Pierce Biotechnology) and gel images were scanned on a GS800 scanner (BioRad). Precision Plus Protein Standards (Bio-Rad) were used as molecular weight markers. A separate 2D-gel was performed with 2-D SDS-PAGE Standards (15 µl) (Bio-Rad) to provide calibrated pI points to allow estimation of the pI of PgChP. RESULTS Genetic characterisation of P. chrysogenum AS51D Amplification of the 18S-28S rRNA intergenic spacer (ITS) sequence revealed a band of 585 bp, which was sequenced and compared against other sequences in BLAST. The strain AS51D displayed 100% of identity and the maximum score with the 18S-28S rRNA intergenic spacer of Penicillium chrysogenum strain FRR 807, accession number in GenBank: AY373839. Thus, AS51D was confirmed as P. chrysogenum. 96 MENÚ SALIR RESULTADOS Analysis of PgChP protein by SDS-PAGE SDS-PAGE of purified PgChP to estimate the molecular mass was revealed two discrete bands (Figure 1): an intensely stained band showing a molecular mass of 37 kDa and a fainter one of around 40 kDa. Figure 1. SDS-PAGE of PgChP protein, showing two bands of 40 kDa (1) and 37 kDa (2). M: Pharmacia low molecular weight standard. Mass spectrometric analysis of PgChP digestions Figure 2 shows typical survey spectrum peptide profiles obtained by mass spectometry for the 37 kDa protein band digested with trypsin and chymotrypsin. The 40 kDa band was also analysed in a similar manner. Overall, the peptide mass spectrum profiles obtained for the upper and lower bands showed a high degree of similarity (Figure 3). 97 MENÚ SALIR MANUSCRITO 4 Figure 2. Mass spectra showing typical peptide profiles obtained from trypsin (A) and chymotrypsin (B) digestions of PgChP (37 kDa band). 98 MENÚ SALIR RESULTADOS Figure 3. Mass spectra showing the highly similar peptide profiles obtained from trypsin digestion of the 37 kDa (A) and 40 kDa (B) bands from SDS-PAGE of PgChP. De novo amino acid sequences Tandem MS was also performed on peptides from PgChP trypsin and chymotrypsin digestions to obtain amino acid sequences. All of the 40 kDa band peptide sequences obtained were also present in the 37 kDa band. Together with the mass spectrum profiles, the data indicated that the two bands observed on SDS-PAGE analysis were likely to represent the same protein or two very similar proteins. The 37 kDa band was subjected to extensive tandem MS analysis to provide increased amino acid sequence coverage of the protein. A total of twelve de novo peptide sequences were obtained from trypsin and chymotrypsin digestions from the 37 kDa band and four from the 40 kDa band (Table 1). 99 MENÚ SALIR MANUSCRITO 4 Table 1. PgChP-de novo peptide sequences obtained from trypsin and chymotrypsin digestions from 37 kDa and 40 kDa. L and I are interchangeable. Italic letters indicate amino acids which were tentative assignments. Sequence Number Peptide de novo sequence mass Charge Digestive enzyme 1 NKPDGGPPGSYF 618.29 +2 Chymotrypsin 2a -FAAGSSLPVAALQSAAAK 959.81 +3 Trypsin 2b* YFAAGSSLPVAALQSAAAK 911.48 +2 Trypsin 2c* PVAALQSAAAK 1026.61 +1 Trypsin 3 -PDATYPLDGDNGA- 866.40 +2 Trypsin 4 -TIHSDWAK 528.79 +2 Trypsin 5 -WLADMDVDCDGLD- 1139.03 +2 Trypsin 6 GNPDGQHQTNFGAL- 728.33 +2 Trypsin 7 -AAYEVPFFVIPDR 988.21 +3 Trypsin 8a GALPGNNVGAVICDGK 771.40 +2 Trypsin 8b GALPGNNVGAVICDGK 720.89 +2 Chymotrypsin 9a -PQVLGEASWLMAR 1053.50 +2 Trypsin 9b* MFYGIYGDSDGDTPQVLGEASRFFAR 972.43 +3 Trypsin 10a FTGDDSVLPSSALNK 775.87 +2 Trypsin 10b* -TYILFTGDDSVLPSSALNK 1352.95 +3 Trypsin 11 NYVTNFTTLR 614.82 +2 Trypsin 12 RSMGDKLM 469.22 +2 Chymotrypsin *: Indicates sequences from the 40 kDa band. -: indicates the presence of additional unassigned amino acids. 100 MENÚ SALIR RESULTADOS Analysis for glucidic residues After enzymatic deglycosylation of PgChP with the PNGase F or the E-DEGLY deglycosylation kit, only the band of 37 kDa was detected (Figure 4). However, chemical deglycosylation with HCl and NaOH revealed no changes in spectra with untreated and treated PgChP by ESI-MS. gChP Figure 4. SDS-PAGE of purified PgChP untreated (lane 1), and treated with PNGase F (lane 2). The arrow indicates the 37 kDa band which remains after deglycosylation. M: Precision Plus Protein Standard. Molecular mass determination of PgChP protein by electrospray ionization mass spectrometry ESI-MS was used to establish the accurate mass of intact PgChP. The m/z spectrum was deconvoluted and converted to the molecular mass profile using Maximum Entropy processing (Figure 5A). The mass profile shows a dominant peak of 30,771.9 Da with a series of surrounding peaks differing from each other by approximately 162 Da (Figure 5B). 101 MENÚ SALIR MANUSCRITO 4 Figure 5. Molecular mass profile from Maximum Entropy processing of PgChP. The most abundant peak has a mass of 30771.9 Da (A). Detailed view revealing a series of peaks separated by approximately 162 Da (B). Additional peaks surrounding the major peaks represent sodium adducted forms (+/- 22 Da). 102 MENÚ SALIR RESULTADOS Isoelectric point estimation 2D-PAGE analysis showed that PgChP migrates on a broad pH range gel in a discrete region of the gel at approximately pH 5 (Figure 6). A) B) Figure 6. Isoelectric point (pI) determination of PgChP by 2D SDS-PAGE. A: PgChP is indicated with an arrow. B: 2D SDS-PAGE standards. Numbers within figure B indicate the pIs of 4 standard proteins that migrate as a series of spots. DISCUSSION In previous studies the strain AS51D was characterized as P. chrysogenum using methods based on morphology and biochemical characteristics, including the production of secondary metabolites by high performance liquid chromatography (Acosta et al., 2009a). In the present study, the sequence of the 18S-28S rRNA intergenic spacer confirmed the previous characterization. P. chrysogenum is regarded as a safe mould of starter cultures (Núñez et al., 1996; Benito et al., 2003) together with other close species, such as Penicillium nalgiovense (Geisen, 2000). To fulfill this role, AS51D could be used either as a source for antifungal proteins or as protective culture. 103 MENÚ SALIR MANUSCRITO 4 Two bands were found in the concentrated PgChP fraction by SDS-PAGE analysis with estimated molecular weights of 37 and 40 kDa. The comparison between the amino acid sequences obtained from both bands showed a high degree of similarity (Table 1). All sequences obtained from the 40 kDa band were derived from three different regions of the 37 kDa protein: sequences 2a, 2b, and 2c in one region; 9a and 9b in a second; and 10a and 10b in a third region. Where they overlapped, peptide sequences from the 40 kDa band were identical to those from the 37 kDa band, with the only exception being for peptide 9b for which 3 of the 5 C-terminal residues could be only tentatively assigned from the tandem MS spectrum. The higher number of sequences obtained from the 37 kDa band result from the more in-depth analysis carried out with this protein, including digestion with chymotrypsin, and in-gel and in-solution digestions. After exposing the PgChP protein to N-Glycosidase F (PNGase F) only the protein of 37 kDa was observed (Figure 4). This deglycosylation enzyme cleaves between the innermost GlcNAc and asparagine residues of high mannose hybrid and complex oligosaccharides from N-linked glycoproteins (Maley et al., 1989; Plummer and Tarentino, 1991). Thus, the protein of 40 kDa is glycosylated, and its peptidic core migrates at the same molecular mass as the 37 kDa protein. Chemical deglycosylation with HCl and NaOH produced no changes in mass spectra when treated and untreated PgChP were compared by ESI-MS. Thus, neither sialic acid residues nor O-linked oligosaccharides are present (Leis et al., 1997; Geert and Thomas-Oates, 1998; Krapf et al., 2006; Tarelli, 2007). The analysis of the molecular mass of the protein by mass spectrometry revealed the existence of six major peaks centring a dominant peak of around 30.8 kDa, differentiated by a mass multiple of 162 Da, the mass of a galactose or mannose residue. It is therefore possible that the purified fraction of PgChP has multiple forms of the protein each with a different number of mannose or galactose residues. The glycosylation of the protein likely explains the difference in the mass estimated by SDS-PAGE and ESI-MS. It is known that glycoproteins can migrate atypically by SDS-PAGE because SDS does not bind oligosaccharides (Nelson, 1971; Russ and Poláková, 1973; Bagger et al., 2007). Given that the ESI-MS provides an exact molecular mass to within 0.01%, it can be deduced that the protein has an atypical migration by SDS-PAGE. Glycosylated proteins are not uncommon among antifungal enzymes, such as an antifungal glycoprotein from Urginea indica (Deepak et al., 2003) or chitosanases from Mycobacter AL-1 and P. islandicum (Hedges and Wolfe, 1974; Fenton and Eveleigh, 1981). 104 MENÚ SALIR RESULTADOS Glycosylation represents the most common form of protein post-translational modifications. Carbohydrates can be found in both eukaryotes and prokaryotes and they participate in many biological processes including inter- and extra-cellular signalling, protein regulations and interactions or molecular recognition. Glycan chains are added to proteins as a set of variations on a core structure, and can vastly increase the complexity of protein molecules. Glycans can determine the localization, activity and function of proteins and can influence physicochemical characteristics of proteins such as folding, solubility, resistance to degradation by proteases or increase in thermal stability (Hildegard and Geyer, 2006; Morelle et al., 2006). In N-glycosylation, glycans are attached to the protein via N-acetylglucosamine (GlcNAc) to the amide group of asparagine within an Asn-X-Ser/Thr (sometimes also Cys) motif, where X is any amino acid apart from proline (Morelle et al., 2006). Although the complete amino acid sequence of PgChP is unknown, at least one consensus site for Nglycosylation is present in the protein (Table 1, peptide 11 -NFT-). This potential site for glycosylation in PgChP supports the interpretation that the 37 kDa band represents the deglycosylated core of the 40 kDa band. The de novo amino acid sequences obtained from PgChP (Table 1) showed the highest similarity with sequences of seven described or predicted fungal chitosanases from A. fumigatus (accession number: XP_754126), N. fischeri (XP_001262949), A. niger (XP_001390407), A. oryzae-1 (BAD08218), A. oryzae-2 (XP_001824257), A. terreus (XP_001209034), and A. clavatus (XP_001274664). The twelve peptide sequences from PgChP were placed onto a multiple sequence alignment of these protein sequences (Figure 7). Three highly conserved amino acid sequences have been described in all of the fungal chitosanases. These three regions are located in the vicinity of the centre of the open reading frames, and are considered to be important for catalytic function (Shimosaka et al., 2005). In each of these three regions there is one conserved carboxylic amino acid (Glu or Asp) considered to be essential or important for the catalytic function as a proton donor in a large number of glycosyl hydrolases (Monzingo et al., 1996; Robertus et al., 1998). At least one of these conserved regions corresponds with the de novo peptide sequence 9 of PgChP, which has the conserved glutamic acid (underlined): PQVLGEASWLMAR. 105 MENÚ SALIR MANUSCRITO 4 PgChP A.fumigatus N.fischeri A.niger A.oryzae-1 A.oryzae-2 A.terreus A.clavatus * *** * * ***** * * NKPDGGPPGSYFAAGSSLPVAALQSAAAK MPSTTIIRQLAISLALCN-SALGQVVNGADYNKPNGGPPASFFAAASTMPVAALQAAAAK MPSKTIIRQLAISVALCN-SALGQVVNGADYNKPNGGPPASFFAAASTMPVAALQAAAAK MAFKTTAG--LAFLALAG-SVKAQSVDGSKYNSPSNGPPASYFAAATTLPVAALQSAAAK MPIKSFASRLALSLAICG-TAMGQKVNGADYNKPDGGPPAKFFQASSSIPVAAIQAAAAK MPIKSFASRLALSLAICG-TAMGQKVNGADYNKPDGGPPAKFFQASSSIPVAAIQAAAAK MVFKKAAIGLTLPFALFSSVALGQTVDGSDYDSPNGGPPGSYFAAASTMPVAALQAAAAK MIFKSSLSHAAASLLLLT-PALAQKVQGPEYNKPSAGPPASFFAAAPTMPVAALKSAVAR PgChP A.fumigatus N.fischeri A.niger A.oryzae-1 A.oryzae-2 A.terreus A.clavatus * ** *** ** ********** PDATYPLDGDNGA TIHSDWAK WLADMDVDCDGLD ATKVPSLATYPVSQDKGAAKSTIHTDWASFSEGASISWVADMDVDCDGLNSGC------ASKVPSLATYPVSQDSGAAKSTIHTDWASFSEGASISWVADMDVDCDGLNSGC------ASSVPSKATYPVNTDDDSPKSTIHSDWVKFNQGAALSWVADMDVDCDGIDYKC------ASKVPSHATYPIGQ--GSTKSTIHSDWAGFSEGAAFSFIADMDVDCDGLNHGC------ASKVPSHATYPIGQ--GSTKSTIHSDWAGFSEGAAFSFIADMDVDCDGLNHGC------ATKVPSYATYPVSQDDNAKKSTIHSDWASFSQGAAISWVADMDVDCDGIDSGCEHEIKTS ASVVPKNAAYPVNQD-GGPTATIHADWASLPTAAAYVYTADMDVDCDGLDHNC------- PgChP A.fumigatus N.fischeri A.niger A.oryzae-1 A.oryzae-2 A.terreus A.clavatus ** ** ** *** ***** *** * ** **** ** GNPDGQHQTNFGALAAYEVPFFVIPDR AGALPGNNVGAVIC----DGK QGNPDGQPQTNWGALSAYEVPFIVIPDKYLSANSGALPGNNIAAVIC----NGKMFYGIL QGNPDGQPQTNWGALSAYEVPFIVIPDKYLSANTGALPGNNIAAVIC----NGKMFYGIL KGNGDGLPETNWGALSAYEVPWIVIPDQFLTANEDLLPGNNVAAVIC----NGKMYYGIL KGNPDGQKETNWGALSAYEVPFIVIPQEFLDANKGTLKGNAVAAVIC----NGKMFYGIF KGNPDGQKETNWGALSAYEVPFIVIPQEFLDANKGTLKGNAVAAVICATSSNGKMFYGIF QGNPDGQDATNWGALAAYEVPFIVIPQKYLDHNGNALKGNNIAAVIC----NGKMFYGIL KGNPDGQPDTNFGALAAYEVPFVVIPDRFATTYASALPGNNIVAVIC----DGKMFYGIF PgChP A.fumigatus N.fischeri A.niger A.oryzae-1 A.oryzae-2 A.terreus A.clavatus * * ********* * ** ** PQVLGEASWLMAR LLFTGDDSVLPSSALNKNY GDSNGDSPQVTGEASWLMARTCFPNEGLNGNNGHTGVDVTYIVFTGKNAVLPSSALTKNY GDSNGDSPQVTGEASWLMARTCFPNEGLNGNNGHTGVDVTYIVFTGKDAVLPSSALTKNY GDSNGDDPEVTGEASWLMARTCFPDDDLNGAEGHAEADVTCKPYT-------RSALNKNY GDSNGDSPQVTGEASWLMARTCFPKEDLNGNKGHTAADVTYIVFTGDKAVLPSSALNKNY GDSNGDSPQVTGEASWLMARTCFPEEDLNGNKGHTAADVTYIVFTGDKAVLPSSALNKNY GDANGDEPQVTGEASWLMARTCFPNEGLNGNKGHTAADVTYILFTGDESVLPSSALNENY GDTDGDHPQVIGEASWLMARTCFPNDNLNGDSGHVPADVTYIFFTGKDSVLPSSAVNKNY PgChP A.fumigatus N.fischeri A.niger A.oryzae-1 A.oryzae-2 A.terreus A.clavatus * * ****** * VTNFTTLRSMGDKLM ITNFTTLRSMGDKLVNALVSNLGLSGTPPTK---------TTTRVTTTTTKPT-SAASCS ITNFTTLRSMGDKLVNALASKLGLSGTPPTK---------TTTLVTTTTTKPT-STASCS ITNFSTLRSMGDKLVGALASNLGLTSSA------------------------SGSTATCS ITNFDTLRSMGDSLVGALAKNLNLGGGGGNP---------PTTLTTTSIPEPTGGSGSCS ITNFDTLRSMGDSLVGALAKNLNLGGGGGNP---------PTTLTTTSIPEPTGGSGSCS ITNFSTLRSMGDKLVNALASNLGLSGGSGGT---------PTT--TTTATQPT-STGTCS ITDFTKLRSMGDSLVNAFASQLGISSGGGNGGGDGGHTTLHTTTATRTATAPT-STATCD PgChP A.fumigatus N.fischeri A.niger A.oryzae-1 A.oryzae-2 A.terreus A.clavatus WAGHCL-----GASCSSDDDCADALVCTAGKCSVDGAA-TCSWEGHCEGASCSSDDDCSD WAGHCL-----GASCSSNDDCADALVCAAGKCSVDGAA-TCSWEGHCEG----------WQGHCE-----GAICSTEDDCSDDLVCDSGKCSSPDED---------------------WPGHCA-----GATCSSNDDCSDDLTCQNGKCASDGSAETCSWEGHCKGATCSSNDDCSD WPGHCAGFKNKGATCSSNDDCSDDLACQNGKCASDGSAETCSWEGHCKGATCSSNDDCSD WAGHCE-----GASCSTNDDCSDQLACKNGVCSTDGEV-VCSWEGHCEGATCSSNDDCSD WEGHCL-----GTACSNNGECSDPFGCINGFCNYDPTL-TCQWAGHCVGASCSSNDQCSD PgChP A.fumigatus N.fischeri A.niger A.oryzae-1 A.oryzae-2 A.terreus A.clavatus DLYCKSGSCTA------------------------------------P----------------------------VYS-------------------------------------DGDDEDDEDEEENDEDDEDDDDEDDEDDE-----ELACISGICSVDNGVETCEWEGHCEGASCSSHDDCDGNLACKNGKCSA--ELACISGICSVDNGVETCEWEGHCEGASCSSHDDCDGNLACKNGKCSA--ELYCAGGACTSA--------------------------------------PFACIDGACAVDT-SLDCSRKGHCAGTTCLSDSDCSRPLSCILGVCANQSG Figure 7. Alignment of PgChP amino acid sequences with the most similar proteins in the public sequence databases. Shaded-background letters show similarities of the proteins with the obtained sequences of PgChP. Accession numbers in Genbank are XP_754126 (A. fumigatus), XP_001262949 (N. fischeri), XP_001390407 (A. niger), BAD08218 (A. oryzae-1), XP_001824257 (A. oryzae-2), XP_001209034 (A. terreus), and XP_001274664 (A. clavatus). Asterisks indicate amino acid residues completely conserved in all sequences. Numbered segments correspond to de novo obtained peptide sequences for PgChP (see Table 1). 106 MENÚ SALIR RESULTADOS A protein sequence homology analysis was performed calculating the percentages of amino acid identity between the sequences obtained from PgChP protein shown in table 1 and the aligned sequences from the seven fungal chitosanases (Table 2). Table 2. Similarity between the amino acid sequences obtained from PgChP and the aligned amino acid sequences of the similar fungal chitosanases described in Figure 7. Percentages higher than 85% are background shaded. Similarity (%) PgChP A. fumigatus N. fischeri A. niger A. oryzae-1 A. oryzae-2 A. terreus A. clavatus 1 75 75 67 75 75 75 67 2a 78 78 83 83 83 78 61 2b* 74 74 84 79 79 79 53 3 61 54 38 46 46 46 38 4 75 75 75 75 75 75 75 5 85 85 92 85 85 92 85 6 86 86 64 79 79 79 86 7 77 77 69 69 69 77 92 8a,b 76 76 71 65 65 65 82 9a 92 92 85 92 92 92 92 9b* 73 73 69 73 73 69 73 10a 72 78 67 83 83 83 78 10b* 76 81 43 86 86 86 81 11 90 90 80 80 80 80 70 12 87 87 87 75 75 87 75 Sequences All peptide sequences from PgChP showed high similarity (from 38 to 92%) with the fungal chitosanases (Table 2). In addition to sequence 9, already discussed as a conserved region in fungal chitosanases, sequence 5 had a similarity higher than 85% in all the compared proteins. The high similarity with fungal chitosanases and the conserved region identified in sequence 9, confirm that PgChP belong to this kind of proteins. Chitosanases (E.C. 3.2.1.132) are the glycoside hydrolases that cleave the glycosidic bonds of chitosan. These enzymes have been reported from viruses, bacteria and fungi (Wang et al., 2008). Chitosanases are classified into five glycoside hydrolase families, GH-5, GH-8, GH-46, GH-75, and GH-80, according to amino acid sequence homology. Fungal chitosanases 107 MENÚ SALIR MANUSCRITO 4 belong to the GH-75 family (Cheng et al., 2005), which is composed mainly by chitosanases from Aspergillus (Cheng and Li, 2000; Zhang et al., 2000) and Fusarium (Shimosaka et al., 1996; Cantarel et al., 2008). Most chitosanases are characterized by a low molecular mass in the range of 10-50 kDa (Fenton and Eveleigh, 1981; Somashekar and Joseph, 1992; Alfonso et al., 1992). The isoelectric point (pI) estimated from 2D-PAGE and isoelectrofocusing was close to pH 5, which is within the range of isoelectric points, from pH 4 to 5, described for fungal chitosanases (Fenton and Eveleigh, 1981; Alfonso et al., 1992; Shimosaka et al., 1993). The range of predicted isoelectric points (pH 3.83-4.97) and molecular masses (33-40 kDa) of the selected fungal chitosanases are quite similar to the experimental values obtained for PgChP (Table 3). Table 3. Isoelectric points (pI) and molecular masses of PgChP and selected fungal chitosanases. Isoelectric Point Molecular Mass (kDa) Protein/producer mould 2D-PAGE/IEF Predicted* ESI-MS Theoretical* PgChP/ P. chrysogenum 5 - 30-31 - XP_001274664/ A. fumigatus - 4.64 - 35.1 XP_001262949/ N. fischeri - 4.97 - 33.1 XP_001390407/ A. niger - 3.83 - 33.4 BAD08218/ A. oryzae-1 - 4.69 - 38.9 XP_001824257/ A. oryzae-2 - 4.71 - 39.8 XP_001209034/ A. terreus - 4.25 - 36.0 XP_001274664/ A. clavatus - 4.64 - 40.1 * Predicted pI values and theoretical molecular masses were calculated using the ProtParam tool in the ExPASy proteomics server at the Swiss Institute of Bioinformatics (http://www.expasy.org). Recently, the genome of P. chrysogenum Wisconsin 54-1255 has been sequenced (Van den Berg et al., 2008). A putative protein can be found in BLAST (accession number CAP80409) that shows high homology with the amino acid sequences obtained from PgChP in this work. However, the production of the protein by this mould has not been reported. Currently, only two chitosanases have been reported from the genus Penicillium, one from P. spinulosum and another from P. islandicum (Fenton et al., 1981; Ak et al., 1998), but none from P. chrysogenum. Given that P. chrysogenum is used to obtain generally 108 MENÚ SALIR RESULTADOS recognized as safe (GRAS) enzymes, PgChP could be used as a food preservative against toxigenic moulds. However, before using PgChP for food protection, an in-depth toxicological evaluation of the antifungal protein is a prerequisite for its application. Thus, a full characterization should be done for this novel chitosanase. ACKNOWLEDGEMENTS This work was supported by the Spanish Ministry of Education and Science (AGL200406546-ALI) and FEDER. Andrea Rodríguez was the recipient of a FPI grant from the Spanish Ministry of Education and Science. Thanks to Alex Rathbone, University of Nottingham, for tuition in 2D gels. REFERENCES Acosta, R., Rodríguez-Martín, A., Martín, A., Núñez, F., and Asensio, M.A. Selection of antifungal protein-producing molds from dry-cured meat products. Int. J. Food Microbiol. Submitted (2009a). Acosta, R., Rodríguez-Martín, A., Bermúdez, E., Núñez, F., and Asensio, M.A. Partial characterization and antifungal activity of a protein from Penicillium chrysogenum. Food Microbiol. Submitted (2009b). Adams, D.J. Fungal cell wall chitinases and glucanases. Microbiology. 150: 2029-35 (2004). Ak, O., Bakir, U., and Guray, T. Production, purification and characterization of chitosanase from Penicillium Spinulosum. Biochem. Arch. 14: 221-225 (1998). Alfonso, C., Martinez, M. J., and Reyes, F. Purification and properties of two endochitosanases from Mucor rouxii implicated in its cell wall degradation. FEMS Microbiol. Letters. 95: 187-194 (1992). Bagger, H.L., Hoffmann, S.V., Fuglsang, C.C., and Westh, P. Glycoprotein-surfactant interactions: A calorimetric and spectroscopic investigation of the phytase-SDS system. Biophys. Chem. 129: 251-258 (2007). Benito, M.J., Córdoba, J.J., Alonso, M., Asensio, M.A., and Núñez, F. Hydrolytic activity of Penicillium chrysogenum Pg222 on pork myofibrillar proteins. Int. J. Food Microbiol. 89: 155-61 (2003). 109 MENÚ SALIR MANUSCRITO 4 Cantarel, B.L., Coutinho, P.M., Rancurel, C., Bernard, T., Lombard, V., and Henrissat, B. The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics. Nucleic Acids Res. 37 (Database issue):D233-8. (2008). Cheng, C. and Li, Y. K. An Aspergillus chitosanase with potential for large-scale preparation of chitosan oligosaccharides. Biotechnol. Appl. Biochem. 32: 197-203 (2000). Cheng, C., Chang, C., Wu, Y., and Li, Y. Exploration of glycosyl hydrolase family 75, a chitosanase from Aspergillus fumigatus. J. Biol. Chem. 281: 3137-3144 (2005). Claverol, S., Burlet-Schiltz, O., Gairin, J.G. and Monsarrat, B. Characterization of protein variants and post-translational modifications: ESI-MS analyses of intact proteins eluted from polyacrylamide gels. Mol. Cell. Proteomics. 2: 483-493 (2003). Deepak, A.V., Thippeswamy, G., Shivakameshwari, M.N. and Salimath, B.P. Isolation and characterization of a 29-kDa glycoprotein with antifungal activity from bulbs of Urginea indica. Biochem. Biophys. Res. Comm. 311: 735-742 (2003). Fenton, D. M. and Eveleigh, D. E. Purification and mode of action of a chitosanase from Penicillium islandicum. J. Gen. Microbiol. 126: 151-165 (1981). Geert, J. D. and Thomas-Oates, J. Analysis of glycoproteins and glycopeptides using fast-atom bombardment. Methods Mol. Biol. 61: 231-241 (1998). Geisen, R. P. nalgiovense carries a gene which is homologous to the paf gene of P. chrysogenum which codes for an antifungal peptide. Intern. J. Food Microbiol. 62: 95-101 (2000). Habermann, B., Oegema, J., Sunyaev, S. and Shevchenko, A. The power and the limitations of cross-species protein identification by mass spectrometry-driven sequence similariy searches. Mol. Cell. Proteomics. 3: 238-249 (2004). Hedges, A. and Wolfe, R. S. Extracellular enzyme from Myxobacter Al-1 that exhibits both -1,4 glucanase and chitosanase activities. J. Bact. 120: 844-853 (1974). Hildegard, G. and Geyer, R. Strategies for analysis of glycoprotein glycosylation. Bioch. Bioph. 1764: 1853-1869 (2006). Krapf, D., Vidal, M., Arranz, S. E., and Cabada, M. O. Characterization and biological properties of L-HGP, a glycoprotein from the amphibian oviduct with acrosome-stabilizing effects. Biology of the Cell. 98: 403-413 (2006). 110 MENÚ SALIR RESULTADOS Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227: 680-685 (1970). Leis, O., Madrid, J. F., Ballesta, J., and Hernández, F. N- and O-linked oligosaccharides in the secretory granules of rat paneth cells: an ultrastructural cytochemical study. J. Histoch. Cytoch. 45: 285-294 (1997). Maley, F., Trimble, R. B., Tarentino, A. L. and Plummer, T. H. Characterization of glycoproteins and their associated oligosaccharides through the use of endoglycosidases. Anal. Biochem. 180: 195-204 (1989). Meyer, V. A small protein that fights fungi: AFP as a new promising antifungal agent of biotechnological value. Appl. Microbiol. Biotechnol. 78: 17-28 (2008). Morelle, W., Canis, K., Chirat, F., Faid, V. and Michalski, J-C. The use of mass spectrometry for the proteomic analysis of glycosylation. Proteomics. 6: 3993-4015 (2006). Mondstadt, G. M. and Holldorf, A. W. Arginine deaminase from Halobacterium. salinarum: purification and properties. Biochem. J. 273: 739-745 (1990). Monzingo, A.F., Marcotte, E.M., Hart, P.J., and Robertus, J.D. Chitinases, chitosanases, and lysozymes can be divided into procaryotic and eucaryotic families sharing a conserved core. Nat. Struc. Biol. 3: 133-140 (1996). Nelson, C.A. Binding of detergents to proteins: maximum amount of dodecyl sulfate bound to proteins and resistance to binding of several proteins. J. Biol. Chem. 246: 38953901 (1971). Núñez, F., Rodriguez, M.M., Bermtidez, M.E., Córdoba, J.J. and Asensio M.A. Composition and toxigenic potential of the mould population on dry-cured Iberian ham. Int. J. Food Microbiol. 32: 185-197 (1996) Pitt, J.I. and Hocking, A.D. Fungi and food spoilage. Blackie Academic and Profesional. London, United Kingdom (1997). Plummer, T.H. and Tarentino, A.L. Purification of the oligosaccharide-cleaving enzymes of Flavobacterium meningosepticum. Glycobiol. 1: 257-263 (1991). 111 MENÚ SALIR MANUSCRITO 4 Robertus, J.D., Monzingo, A.F., Marcotte, E.M., and Hart, P.J. Structural analysis shows five glycohydrolase families diverged from a common ancestor. J. Exp. Zool. 282: 127-132 (1998). Russ, G. and Poláková, K. Molecular-weight determination of proteins and glycoproteins of RNA enveloped viruses by polyacrylamide-gel electrophoresis in SDS. Biochem. Biophys. Res. Commun. 55: 666-672 (1973). Shimosaka, M., Nogawa, M., Ohno, Y., and Okazaki, M. Chitosanase from the plant pathogenic fungus, Fusarium solani f. sp. phaseoli-purification and some properties. Biosci. Biotechnol. Biochem. 57: 231-235 (1993). Shimosaka, M., Kumehara, M., Zhang, X., Nogawa, M., and Okazaki, M. Cloning and characterization of a chitosanase gene from the plant pathogenic fungus Fusarium solani. J. Ferment. Bioeng. 82: 426-431 (1996). Shimosaka, M., Sato, K., Nishiwaki, N., Miyazawa, T. and Okazaki, M. Analysis of essential carboxylic amino acid residues for catalytic activity of fungal chitosanases by site-directed mutagenesis. J. Bios. Bioeng. 5: 545-550 (2005). Somashekar, D. and Joseph, R. Partial purification and properties of a novel chitosanase secreted by Rhodotorula gracilis. Lett. Appl. Microbiol. 14: 1-4 (1992). Tarelli, E. Resistance to deglycosylation by ammonia of IgAl O-glycopeptides: implications for the -elimination of O-glycans linked to serine and threonine. Carboh. Res. 342: 2322-2325 (2007). Van den Berg, M.A., Albang, R., Albermann, K., Badger, J.H., Daran, J.M., Driessen, A.J., Garcia-Estrada, C., Fedorova, N.D., Harris, D.M., Heijne, W.H., Joardar, V., Kiel, J.A., Kovalchuk, A., Martin, J.F., Nierman, W.C., Nijland, J.G., Pronk, J.T., Roubos, J.A., van der Klei, I.J., van Peij, N.N., Veenhuis, M., von Dohren, H., Wagner, C., Wortman, J. and Bovenberg, R.A. Genome sequencing and analysis of the filamentous fungus Penicillium chrysogenum. Nat. Biotechnol. 26: 1161-1168 (2008). Wang, J., Zhou, W., Yuan, H., and Wang, Y. Characterization of a novel fungal chitosanase Csn2 from Gongronella sp. JG. Carboh. Res. 343: 2583-2588 (2008). White, T.J., Bruns, T., Lee, S., and Taylor, J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Editors: M. A. Innis, D. H. Gelfand, J. J. 112 MENÚ SALIR RESULTADOS Sninsky, and T. J. White. PCR Protocols: A Guide to Methods and Applications. Academic Press, Inc., San Diego. Pages 315-322 (1990). Zhang, X., Dai, A., Zhang, X., and Kuroiwa, K. Purification and characterization of chitosanase and exo- -D-Glucosaminidase from a Koji Mold, Aspergillus oryzae IAM2660. Biosci. Biotechnol. Biochem. 64: 1896-1902 (2000). 113 MENÚ SALIR Manuscrito 5: Genetic characterization of the gene enconding the novel chitosanase PgChP from Penicillium chrysogenum MENÚ SALIR RESULTADOS Genetic characterization of the gene encoding the novel chitosanase PgChP from Penicillium chrysogenum Andrea Rodríguez-Martín, Raquel Acosta, Félix Núñez, Mª José Benitoa, Miguel A. Asensio* Higiene y Seguridad Alimentaria, Facultad de Veterinaria. Universidad de Extremadura, Avda. de la Universidad, s/n. 10071- Cáceres, Spain. a Nutrición y Bromatología, Escuela de Ingenierías Agrarias, Universidad de Extremadura, Carretera de Cáceres s/n, 06071- Badajoz, Spain * Corresponding author: Telephone (+34) 927 257 125; fax: (+34) 927 257 110. E-mail: [email protected] ABSTRACT The protein PgChP is a new chitosanase with antifungal activity produced by Penicillium chrysogenum AS51D. The gene encoding the chitosanase PgChP has been sequenced. A DNA fragment of the gene encoding pgchp was obtained by PCR using degenerate primers based on two known amino acid sequences of the purified PgChP. cDNA was prepared for RACE-PCR from total RNA of P. chrysogenum by RT-PCR. Full-length cDNA and complete genomic DNA of pgchp were obtained using primers designed from 5’ and 3’ – ends. Comparing the cDNA and genomic DNA, four introns of 60, 53, 59, and 59 bp were observed. Full-length cDNA has 915 bp, corresponding to an open reading frame encoding a protein of 304 amino acid residues. The first 21 amino acids matched a signal peptide. The predicted mass of the translated nucleotidic sequence was 32 kDa and the predicted isoelectric point was 4.8. The translated sequence of pgchp revealed that the protein had two carboxylic amino acids (Glu and Asp) described as essential for the catalytic function of fungal chitosanases. Two N-glycosylation consensus sequences were observed in the translated pgchp gene. The putative amino acid sequence showed high homology with fungal chitosanases. KEY WORDS: antifungal activity, glycosylation, N-linked oligosaccharides, dryfermented foods. INTRODUCTION P. chrysogenum AS51D was isolated from meat products in previous studies (Acosta et al., 2009a). This mould produces the protein PgChP, selected for its ability of inhibiting the 115 MENÚ SALIR MANUSCRITO 5 growth of some toxigenic moulds. In previous works, different de novo amino acid sequences obtained from PgChP showed significant similarities with the respective sequences of seven fungal chitosanases, belonging to the GH-75 family of fungal chitosanases (Rodríguez-Martín et al., 2009). Chitosanases (E.C. 3.2.1.132) are produced by a large number of microorganisms including bacteria and fungi. Numerous bacterial chitosanases are able to degrade exogenously added chitosan (Shimosaka et al., 2000; Yoon et al., 2002; Lee et al., 2006; Zhang et al., 2007). On the contrary, fungal chitosanases have been reported from only a limited number of strains and their physiological roles are unclear (Fenton and Eveleigh, 1981; Zhang et al., 2000). The synthesis of some chitosanases can be induced by adding different substrates such as glucosamine (Zhang et al., 2001) and chitosan (Zhu et al., 2007). Analysis of the molecular weight of PgChP by ESI-MS revealed six isoforms differing from each other by approximately 162 Da, the mass of one residue of mannose. These evidences indicated that the chitosanase PgChP was a glycoprotein with different degrees of glycosylation. Various deglycosylation treatments revealed the presence of N-linked oligosaccharides in PgChP (Rodríguez-Martín et al., 2009). The aim of this paper was to study the genetic sequence encoding the chitosanase PgChP. This sequence will allow studying the existence of introns, the complete putative amino acid sequence, and glycosylation consensus sequences. MATERIALS AND METHODS Microbial strain P. chrysogenum AS51D was isolated from dry-cured ham and belongs to the fungal collection of Food Hygiene, University of Extremadura. DNA isolation P. chrysogenum AS51D was grown in malt extract broth made with 2% (w/v) glucose, 2% (w/v) malt extract, and 0.1% (w/v) peptone, pH 4.5 at 25ºC under continuous shaking, for 5 days. The mycelium was obtained by filtering the culture through paper Whatman nº2. Two grams of mycelium were broken with mortar and pestle after freezing by adding liquid nitrogen. Fungal lysis was performed by homogenizing the obtained powder in 4 ml 0.05 M Tris-HCl, pH 8; 0.005 M ethylenediamine tetraacetic acid (EDTA); 0.05 M NaCl 116 MENÚ SALIR RESULTADOS (TES) buffer and 1% (w/v) sodium dodecyl sulfate (SDS). Next, 200 µl proteinase K (20 µg/µl) were added, and the mix was incubated at 60ºC for 35 min, cooled on ice and extracted with phenol-chloroform-isoamyl alcohol (25:24:1). The suspension was centrifuged at 3000×g for 2 min at 4ºC. The upper phase containing DNA was transferred to a fresh tube and precipitated by adding 3 M sodium acetate pH 5.2 to a final concentration of 10% (w/v) and two volumes of cold ethanol. After centrifugation, the pellet was cleaned with 70% (v/v) ethanol, centrifuged again, resuspended in sterile water, and treated with 50 µl RNase (10 µg/µl) at 37ºC for 60 min. Finally, DNA was extracted with phenol-chloroform-isoamyl alcohol, and precipitated again as indicated above, resuspended in water, and stored at -70ºC. Quantity and quality of purified DNA was determined spectrophotometrically in a Biophotometer (Eppendorf AG, Hamburg, Germany). Genomic DNA amplification Polymerase chain reactions were performed with genomic DNA (100 ng) as template in 50 µl reaction mixtures containing 50 pmol each of forward and reverse degenerated primers, 0.5 mM each of deoxyribonucleotide triphosphates, (dNTPs, Roche, Madrid, Spain), 0.1 vol of 10× PCR buffer (22.5 mM MgCl2, 500 mM Tris-HCl pH 9.2, 140 mM ammonium sulfate), 1.8 mM MgCl2 and 2.5 units of pfu turbo polymerase (Stratagene, La Jolla, USA). The degenerated primers pgchp-DPF1 and pgchp-DPR2A (Table 1) were designed from amino acid sequences previously obtained by mass spectrometry (Rodríguez-Martín et al., 2009). Additionally, for pgchp-DPR2A the contiguous residues conserved in fungal chitosanases (Shimosaka et al., 2005) were also included. 117 MENÚ SALIR MANUSCRITO 5 Table 1. Degenerated primers used to amplify the pgchp gene. Primer names Amino acid sequences and designed primers* N pgchp-DPF1 P D G G P 5’-AAY AAR CCN GAY GGN GGN CC-3’ M pgchp-DPR2A K A R T C F P 3’-AC CGN GCN TGN ACR AAR GG-5’ * R for purine, Y for pirimidine, N for A, C, T, or G. Conserved amino acids used for designing the primer pgchp-DPR2A are underlined. DNA amplification was carried out in a Px2 thermocycler (Thermo Scientific, Cramlington, UK). Three reactions were done, one with both forward and reverse primers, and two with either forward or reverse primers. The PCR program consisted of initial denaturation (94ºC for 5 min), 35 cycles of denaturation (94ºC for 1min), annealing (5660ºC range for 30 s, gradient 12), and extension (72ºC for 30 s), followed by a final extension at 72ºC for 4 min. PCR products were electrophoresed on 2% (w/v) agarose gels in 1× TAE buffer (40 mM Tris-acetate, 1 mM EDTA), pH 8 ussing a Sub-Cell GT apparatus (Bio-rad Laboratories, Madrid, Spain). Two different markers were used for agarose gels: 1kb DNA ladder (0.25-12 kb bands) (Promega, Hampshire, United Kingdom) and DNA size standard (0.05-2kb) (Bio-Rad). Then, products detected only in the reactions with both reverse and forward primers were gel-purified and cloned into the pCR2.1TOPO vector (Invitrogen, Barcelona, Spain). One Shot TOP10 Chemically Competent E. coli cells (Invitrogen) were transformed with that vector. To confirm the insert in the vector, digestions with restriction enzyme EcoRI (Roche) were carried out. Finally, vectors were sequenced in the Institute of Biomedicine (CSIC, Valencia, Spain). RNA isolation P. chrysogenum AS51D was grown in malt extract broth, pH 4.5 at 25ºC under continuous shaking for 4 days. Chitosanase production was induced in a medium with glucosamine (Zhang et al., 2001) made with 0.3% (w/v) NaNO3, 0.05% (w/v) KCl, 0.05% (w/v) MgSO4•7H2O, 0.01% (w/v) FeSO4•7H2O, and 2% (w/v) D(+)-glucosamine hydrochloride (Sigma, Dorset, United Kingdom), dissolved in 25 mM phosphate buffer pH 5.8. Mould 118 MENÚ SALIR RESULTADOS was cultured in glucosamine medium at 25ºC under continuous shaking for 17 h. Subsequently, mycelium was quickly frozen with liquid nitrogen and stored at -80ºC until RNA extraction. Frozen mycelium was broken with mortar and pestle after adding liquid nitrogen. RNA extraction was performed with RNeasy Plant Mini Kit (Qiagen, Crawley, UK) following the manufacturer’s instructions. Total extracted RNA was resuspended in 40 µl of RNase-free deionized water. Rapid amplification of cDNA ends by Polymerase Chain Reaction (RACE-PCR) Amplification of 5’ and 3’ ends of the pgchp gene was carried out using the SMART RACE cDNA Amplification Kit (Clontech Laboratories, Saint Germain en Laye, France). cDNA synthesis by reverse transcriptase-polymerase chain reaction (RT-PCR). Two separate populations of cDNA were synthesized according to the amplification kit instructions: 5’-RACE-Ready cDNA and 3’-RACE-Ready cDNA. After incorporating the SMART sequence into both the 5’- and 3’-RACE-Ready cDNA populations, RACE PCR reactions were performed using the Universal Primer A Mix (UPM), in conjunction with distinct gene-specific primers. Gene-specific primers design. Two gene-specific primers (GSP) were designed for amplifying the 5’ and 3’- ends (5’-RACE GSP: CCATTGCAGATAACGGCACCAACG and 3’-RACE GSP: GTTCGACAAGGGTGCGGCATATG). Polymerase chain reactions were performed with 5’- or 3’-RACE-Ready cDNAs (50 ng) as template in 50 µl reaction mixtures containing 1 mM of dNTPs (Roche), 0.5 µM of the 5’- or 3’-RACE GSP primers, 5 µl of Universal Primer A Mix (10×), 0.1 vol of 10× PCR buffer (10 mM Tris-HCl, pH 8.8, 50 mM KCl, 0.1% Triton X-100), 4 mM MgCl2, and 2.5 units pfu turbo polymerase (Stratagene). A program for touchdown PCR was carried out for 5’-end amplification: 5 cycles of 2 steps (94ºC for 30 s and 72ºC for 3 min), 5 cycles of 3 steps (94ºC for 30 s, 70ºC for 30 s and 72ºC for 3 min) and 27 cycles of 3 steps (94ºC for 30 s, 68ºC for 30 s and 72ºC for 3 min). The PCR program to obtain the 3’-end consisted of 30 cycles of denaturation (94ºC for 30 s), annealing (68ºC for 30 s), and extension (72ºC for 3 min). Finally, 5’- and 3’-RACE-PCR products were gel-purified and cloned into pCR2.1-TOPO vectors, which were used to transform One Shot TOP10 Chemically Competent E. coli (Invitrogen). Then plasmids were purified and sequenced. Digestions with restriction enzyme EcoRI (Roche) were performed to confirm the insert in the vector. 119 MENÚ SALIR MANUSCRITO 5 Full-length cDNA amplification. The full cDNA fragment of the gene pgchp was obtained by PCR with specific primers Fwd-pgchp (ATGATGACCTACAGCCGCTTAATCCC) and Rev-pgchp (TCAATCACTGGGGCATTTTCCAC) designed from 5’ and 3’ ends. PCR reaction mixtures at a final volume of 50 µl contained 5’-RACE-Ready cDNA (50 ng), 0.5 µM each of forward and reverse primers, 0.5 mM each of the dNTPs, 0.1 vol of 10× PCR buffer (10 mM Tris-HCl, pH 8.8, 50 mM KCl, 0.1% Triton X-100), 4 mM MgCl2 and 2.5 units pfu turbo polymerase (Stratagene). The PCR program consisted of 30 cycles of denaturation (94ºC for 30 s), annealing (65ºC for 30 s), and extension (72ºC for 3 min). PCR products were gel-purified and cloned into pCR2.1-TOPO vectors. These plasmids were used to transform One Shot TOP10 Chemically Competent E. coli cells (Invitrogen). Then vectors were purified and sequenced. Digestions with restriction enzyme EcoRI (Roche) were performed to confirm the wanted insert in the vector. Amplification of the whole genomic gene The genomic gene sequence was amplified by PCR using Fwd-pgchp and Rev-pgchp primers. PCR reactions contained 100 ng of genomic DNA, 0.5 µM each of forward and reverse primers, 0.5 mM each of the dNTPs, 0.1 vol of 10× PCR buffer (10 mM Tris-HCl, pH 8.8, 50 mM KCl, 0.1% Triton X-100), 4 mM MgCl2 and 2.5 units pfu turbo polymerase (Stratagene), and deionized water until a final volume of 50µl. The PCR program consisted of 30 cycles of denaturation (94ºC for 30 s), annealing (65ºC for 30 s), and extension (72ºC for 3 min). PCR products were gel-purified and cloned into pCR2.1-TOPO vectors. These plasmids were used to transform One Shot TOP10 Chemically Competent E. coli cells (Invitrogen). Then vectors were purified and sequenced. Alignment between gene sequences obtained from genomic DNA and cDNA was performed. Introns sequences were confirmed by GenScan software. RESULTS Partial amplification of pgchp gene using degenerated primers The pgchp gene partial amplification from genomic DNA was done using two degenerated primers: pgchp-DPF1 and pgchp-DPR2A (Table 1). After different reactions, the optimal annealing temperature was 57.7ºC. PCR products obtained with these primers were run in 120 MENÚ SALIR RESULTADOS agarose gel. Just one band with high intensity was amplified in reactions with both forward and reverse primers (Figure 1). This band was cloned and sequenced. Figure 1. PCR products using forward DPF1 (F), both forward DPF1 and reverse DPR2A (F+R), and reverse DPR2A (R) primers. M: DNA standards. The arrow indicates the selected band. After sequencing, the selected fragment was found to consist of 657 bp. The obtained sequence was translated and compared in Basic Local Alignment Search Tool (BLAST) with known sequences. A high degree of similarity with several fungal chitosanases was found, as it is discussed later. The common sequences included three introns described for genes encoding fungal chitosanases. Sequence of the pgchp gene by RACE PCR After induction with glucosamine, mRNA was extracted and used to obtain both 5’- and 3’-RACE-Ready cDNA populations by RT-PCR. Two gene specific primers were designed from the obtained genomic DNA sequence without introns to get 5’- and 3’-ends: 5’-RACE GSP (5’-CCATTGCAGATAACGGCACCAACG-3’) and 3’-RACE GSP (5’GTTCGACAAGGGTGCGGCATATG-3’), respectively. After 5’ and 3’-RACE reactions, two bands with the expected size (Figure 2) were gel-purified from 5’- and 3’-RACE 121 MENÚ SALIR MANUSCRITO 5 PCRs, cloned, and sequenced. The cDNA ends of the gene 5’ and 3’ were 559 bp and 796 bp, respectively. Figure 2. cDNA fragments of 5’- and 3’-RACE PCR (A and B, respectively). M: DNA standards. Next, two new primers were designed to obtain the full encoding cDNA (Fwd-pgchp: 5’ATGATGACCTACAGCCGCTTAATCCC-3’ and Rev-pgchp: 5’- TCAATCACTGGGGCATTTTCCAC-3’). The full-length cDNA contained a 915 bp open reading frame encoding a protein of 304 amino acid residues (Figure 3) that included the known PgChP sequences. Genomic DNA of pgchp gene, amplified using the same primers for cDNA, was 1146 bp length. Comparison of cDNA and genomic sequences revealed that pgchp gene had four introns with a length of 60, 53, 59, and 59 bp (Figure 3). These introns were confirmed by GenScan program. 122 MENÚ SALIR RESULTADOS 5’-atgatgacctacagccgcttaatccccttcgctctctttgccttgtttggcacagggctc M M T Y S R L I P F A L F A L F G T G L gcgcaaacagtcgatggctccaaattcaacaagccagacggcggtccaccaggaagctac A Q T V D G S K F N K P D G G P P G S Y ttcgctgctggctcgtctattcccgtcgctgctttgcagagcgctgctgcaaaggctcgc F A A G S S I P V A A L Q S A A A K A R actcccgtgccagatgcaacctatcctattaatggcgataacggtgctaagaaagttacc T P V P D A T Y P I N G D N G A K K V T atccacagcgactgggctaagttcgacaaggtagatactctcactggtcggttaattgca I H S D W A K F D K << Intron 1 aggtccgcatgcttattaaatggatcatagggtgcggcatatgtttggattgcggatatg >> G A A Y V W I A D M gacgtcgactgcgacggcattgactacaagtgcaaggtacggtcactattgttgtctgtc D V D C D G I D Y K C K << Intron 2 tgagatgttattgctgacccgatgttcagggaaacccggatggtcagcaccaaaccaact >> G N P D G Q H Q T N tcggagctttggctgcgtatgaagtgccgttctttgtgattcccgacaggtttggaacca F G A L A A Y E V P F F V I P D R F G T agtacgcgaagcagcttcctggaaacaacgttggtgccgttatctggtatggtttcctac K Y A K Q L P G N N V G A V I C<< ctttgtaagatatctcagcggcgtcttgttctaaccgaaagctagcaatggaaagatgtt Intron 3 >> N G K M F ctacggaatttacggagactccgatggcgatacccctcaggtcatcggcgaggcctcatg Y G I Y G D S D G D T P Q V I G E A S W gttaatggcccggacctgcttccctaatgatgacttgaatggcaatagtggccatggtga L M A R T C F P N D D L N G N S G H G D tgttgatgtgacctgtaagtttgcagccttggcacatctagatccgtattctgtcaacta V D V T << Intron 4 ctaatctaaccagatatcctcttcactggcgacgactcggtgctccccagcagcgctctc >>Y I L F T G D D S V L P S S A L aacaagaattatgtcaccaacttcaccactctgcgctctatgggagacaaactcatgact N K N Y V T N F T T L R S M G D K L M T gctcttgcgaagaacctcaagttggtagatggaggtgatggaggttctccaacaactacg A L A K N L K L V D G G D G G S P T T T gctgggtccaaccctactagcgggtcttgtgagtgggagggacactgtgctggtgcgtct A G S N P T S G S C E W E G H C A G A S tgcaaagatgaaaatgattgctccgatcaactggtctgcaaaagtggaaaatgccccagt C K D E N D C S D Q L V C K S G K C P S gattga-3’ D - Figure 3. Full-length DNA sequence of pgchp gene. The open reading frame has been translated to amino acids. Typical sequences of fungal introns are underlined. The open reading frame was translated to protein by ExPASy proteomics server at the Swiss Institute of Bioinformatics (http://www.expasy.org). Analysis of the signal peptidase cleavage site predicted by the SignalP program (Nielsen et al., 1997) suggested that the pre-sequence consisted of the first 21 amino acids (MMTYSRLIPFALFALFGTGLA). 123 MENÚ SALIR MANUSCRITO 5 DISCUSSION RACE-PCR is a method widely used in the characterization of genes. The successful of this technique is highly depended on the amount of mRNA from the gene enconding the studied protein. Thus, the production of PgChP was induced by glucosamine, as it has been reported for other chitosanases (Zhang et al., 2001). After the mRNA extraction, both populations 5’- and 3’-RACE-Ready cDNAs were satisfactorily obtained by RT-PCR. Using gene specific primers designed from the sequence amplified with degenerated primers, the 5’- and 3’-ends were obtained by RACE-PCR. When the complete sequence was obtained, a difference between the pgchp gene from genomic DNA (1146 bp) and from cDNA (915 bp) could be observed (Figure 3). Thus, the existence of four introns of 60, 53, 59, and 59 bp in the gene, which are absent in the cDNA, was confirmed by GenScan software. The small size of these introns compared to mammalian introns is a typical feature from fungal genes (Gurr et al., 1987). The 5’- and 3’-ends of the four introns are very similar (Figure 3) and coincide with the consensus splice sequences for fungal introns 5’-splice donor site (GT), the 3’-splice acceptor site (AG). In addition, three of them have the internal CTRAC sequence (Figure 3), typical of fungal introns (Wiesner et al., 1988). The translated sequence of the gene pgchp encodes a chitosanase of 304 amino acids of the PgChP protein. Comparing the putative amino acid sequence of pgchp gene with seven fungal chitosanases, the homology was about 60 %. Several common sequences were found among all these proteins (Figure 4). Three conserved regions have been described in fungal chitosanases as important for catalytic function (Shimosaka et al., 2005). In two of these regions there are one aspartic and one glutamic acid residues, essentials for catalytic function of the chitosanase from F. solani. The predicted amino acid sequence of PgChP also shows these two residues (Figure 4). Carboxylic amino acids (Glu or Asp) have been identified as necessary residues for the catalytic function as proton donors in a large number of glycosyl hydrolases (Monzingo et al., 1996; Robertus et al., 1998). PgChP and the other seven fungal chitosanases show eleven common residues of aspartic acid as well as two of glutamic acid (Figure 4). Moreover, 10 cysteines of PgChP are found in all these fungal chitosanases (Figure 4). Cysteine residues are known to play an important role on the stabilization of the tertiary structure by the formation of disulfide bridges in fungal proteins (Nakaya et al., 1990; Lacadena et al., 1995; Marx et al., 1995; Lee et al., 1999). 124 MENÚ SALIR RESULTADOS PgChP A.fumigatus N.fischeri A.niger A.oryzae-1 A.oryzae-2 A.terreus A.clavatus --MMTYSRLIPFALFALFG-TGLAQTVDGSKFNKPDGGPPGSYFAAGSSIPVAALQSAAA MPSTTIIRQLAIS-LALCN-SALGQVVNGADYNKPNGGPPASFFAAASTMPVAALQAAAA MPSKTIIRQLAIS-VALCN-SALGQVVNGADYNKPNGGPPASFFAAASTMPVAALQAAAA MAFKTTAG--LAF-LALAG-SVKAQSVDGSKYNSPSNGPPASYFAAATTLPVAALQSAAA MPIKSFASRLALS-LAICG-TAMGQKVNGADYNKPDGGPPAKFFQASSSIPVAAIQAAAA MPIKSFASRLALS-LAICG-TAMGQKVNGADYNKPDGGPPAKFFQASSSIPVAAIQAAAA MVFKKAAIGLTLP-FALFSSVALGQTVDGSDYDSPNGGPPGSYFAAASTMPVAALQAAAA MIFKSSLSHAAAS-LLLLT-PALAQKVQGPEYNKPSAGPPASFFAAAPTMPVAALKSAVA 57 58 58 56 58 58 59 58 PgChP A.fumigatus N.fischeri A.niger A.oryzae-1 A.oryzae-2 A.terreus A.clavatus KARTPVPDATYPINGDNGAKKVTIHSDWAKFDKGAAYVWIADMDVDCDGIDYKC-----KATKVPSLATYPVSQDKGAAKSTIHTDWASFSEGASISWVADMDVDCDGLNSGC-----KASKVPSLATYPVSQDSGAAKSTIHTDWASFSEGASISWVADMDVDCDGLNSGC-----KASSVPSKATYPVNTDDDSPKSTIHSDWVKFNQGAALSWVADMDVDCDGIDYKC-----KASKVPSHATYPIGQ--GSTKSTIHSDWAGFSEGAAFSFIADMDVDCDGLNHGC-----KASKVPSHATYPIGQ--GSTKSTIHSDWAGFSEGAAFSFIADMDVDCDGLNHGC-----KATKVPSYATYPVSQDDNAKKSTIHSDWASFSQGAAISWVADMDVDCDGIDSGCEHEIKT RASVVPKNAAYPVNQD-GGPTATIHADWASLPTAAAYVYTADMDVDCDGLDHNC------ 111 112 112 110 110 110 119 111 PgChP A.fumigatus N.fischeri A.niger A.oryzae-1 A.oryzae-2 A.terreus A.clavatus -KGNPDGQHQTNFGALAAYEVPFFVIPDRFGTKYAKQLPGNNVGAVIC----NGKMFYGI -QGNPDGQPQTNWGALSAYEVPFIVIPDKYLSANSGALPGNNIAAVIC----NGKMFYGI -QGNPDGQPQTNWGALSAYEVPFIVIPDKYLSANTGALPGNNIAAVIC----NGKMFYGI -KGNGDGLPETNWGALSAYEVPWIVIPDQFLTANEDLLPGNNVAAVIC----NGKMYYGI -KGNPDGQKETNWGALSAYEVPFIVIPQEFLDANKGTLKGNAVAAVIC----NGKMFYGI -KGNPDGQKETNWGALSAYEVPFIVIPQEFLDANKGTLKGNAVAAVICATSSNGKMFYGI SQGNPDGQDATNWGALAAYEVPFIVIPQKYLDHNGNALKGNNIAAVIC----NGKMFYGI -KGNPDGQPDTNFGALAAYEVPFVVIPDRFATTYASALPGNNIVAVIC----DGKMFYGI 166 167 167 165 165 169 175 166 PgChP A.fumigatus N.fischeri A.niger A.oryzae-1 A.oryzae-2 A.terreus A.clavatus 226 227 227 218 225 229 235 226 PgChP A.fumigatus N.fischeri A.niger A.oryzae-1 A.oryzae-2 A.terreus A.clavatus YGDSDGDTPQVIGEASWLMARTCFPNDDLNGNSGHGDVDVTYILFTGDDSVLPSSALNKN LGDSNGDSPQVTGEASWLMARTCFPNEGLNGNNGHTGVDVTYIVFTGKNAVLPSSALTKN LGDSNGDSPQVTGEASWLMARTCFPNEGLNGNNGHTGVDVTYIVFTGKDAVLPSSALTKN LGDSNGDDPEVTGEASWLMARTCFPDDDLNGAEGHAEADVTCKPYT-------RSALNKN FGDSNGDSPQVTGEASWLMARTCFPKEDLNGNKGHTAADVTYIVFTGDKAVLPSSALNKN FGDSNGDSPQVTGEASWLMARTCFPEEDLNGNKGHTAADVTYIVFTGDKAVLPSSALNKN LGDANGDEPQVTGEASWLMARTCFPNEGLNGNKGHTAADVTYILFTGDESVLPSSALNEN FGDTDGDHPQVIGEASWLMARTCFPNDNLNGDSGHVPADVTYIFFTGKDSVLPSSAVNKN * * YVTNFTTLRSMGDKLMTALAKNLKLVDGGDGG---------SPT--TTAGSNPT--SGSC YITNFTTLRSMGDKLVNALVSNLGLSGTPPTK---------TTTRVTTTTTKPT-SAASC YITNFTTLRSMGDKLVNALASKLGLSGTPPTK---------TTTLVTTTTTKPT-STASC YITNFSTLRSMGDKLVGALASNLGLTSSA------------------------SGSTATC YITNFDTLRSMGDSLVGALAKNLNLGGGGGNP---------PTTLTTTSIPEPTGGSGSC YITNFDTLRSMGDSLVGALAKNLNLGGGGGNP---------PTTLTTTSIPEPTGGSGSC YITNFSTLRSMGDKLVNALASNLGLSGGSGGT---------PTT--TTTATQPT-STGTC YITDFTKLRSMGDSLVNAFASQLGISSGGGNGGGDGGHTTLHTTTATRTATAPT-STATC PgChP A.fumigatus N.fischeri A.niger A.oryzae-1 A.oryzae-2 A.terreus A.clavatus EWEGHCA-----GASCKDENDCSDQLVCKSGKCPSD-----------------------SWAGHCL-----GASCSSDDDCADALVCTAGKCSVDGAA-TCSWEGHCEGASCSSDDDCS SWAGHCL-----GASCSSNDDCADALVCAAGKCSVDGAA-TCSWEGHCEGVYS------SWQGHCE-----GAICSTEDDCSDDLVCDSGKCSSPDED--------------------SWPGHCA-----GATCSSNDDCSDDLTCQNGKCASDGSAETCSWEGHCKGATCSSNDDCS SWPGHCAGFKNKGATCSSNDDCSDDLACQNGKCASDGSAETCSWEGHCKGATCSSNDDCS SWAGHCE-----GASCSTNDDCSDQLACKNGVCSTDGEV-VCSWEGHCEGATCSSNDDCS DWEGHCL-----GTACSNNGECSDPFGCINGFCNYDPTL-TCQWAGHCVGASCSSNDQCS 304 331 324 288 331 340 337 339 PgChP A.fumigatus N.fischeri A.niger A.oryzae-1 A.oryzae-2 A.terreus A.clavatus ---------------------------------------------------DDLYCKSGSCTA------------------------------------P----------------------------------------------------------------------DGDDEDDEDEEENDEDDEDDDDEDDEDDE-----DELACISGICSVDNGVETCEWEGHCEGASCSSHDDCDGNLACKNGKCSA--DELACISGICSVDNGVETCEWEGHCEGASCSSHDDCDGNLACKNGKCSA--DELYCAGGACTSA--------------------------------------DPFACIDGACAVDT-SLDCSRKGHCAGTTCLSDSDCSRPLSCILGVCANQSG 273 277 277 254 276 280 283 285 344 317 380 389 350 390 Figure 4. Alignment of PgChP with other fungal chitosanases. Common amino acids are denoted by background shaded letters. Asterisks indicate essential amino acid residues for catalytic activity of fungal chitosanases. Accession numbers in Genbank are XM_754126 (A. fumigatus), XM_001262949 (N. fischeri), XM_001390407 (A. niger), BAD08218 (A. oryzae-1), XP_001824257 (A. oryzae-2), XM_001209034 (A. terreus), XM_001274664 (A. clavatus). 125 MENÚ SALIR MANUSCRITO 5 Six glycosylated isoforms of PgChP with N-linked oligosaccharides have been reported (Rodríguez-Martín et al., 2009). In N-glycosylations, glycans are attached to the protein via N-acetylglucosamine (GlcNAc) to the amide group of asparagine within an Asn-XSer/Thr/Cys motif, where X is any amino acid apart from proline (Morelle et al., 2006). Two N-glycosylation consensus sequences were found in the translated pgchp gene: NFT (residues 230-232) and NDC (residues 288-290). Glycosylation is the most common post-translational modification in proteins. Carbohydrates participate in important biological processes, including inter- and extracellular signalling, protein regulations and interactions, and molecular recognition. Glycans can determine the localization, activity and function of proteins, as well as influence on physicochemical characteristics of proteins such as folding, solubility, resistance to proteases or thermal stability (Geyer et al., 2006; Morelle et al., 2006). The potential role of the N-linked oligosaccharides of PgChP on intra- or extracellular signalling could be essential for the antifungal activity of this protein. The predicted mass of the translated nucleotidic sequence, according to the Compute pI/Mw tool (Gasteiger et al., 2005), was 31,991 Da, which is close to the 30 to 31 kDa determined for the six excreted isoforms of the protein by ESI-MS (Rodríguez-Martín et al, 2009). The difference between the predicted mass and the determined weight can be explained by the lack of the signal peptide (2,300 Da) in the excreted protein and the presence of N-linked oligosaccharides. The predicted isoelectric point (Gasteiger et al., 2005) was 4.8, which is close to the value of 5 determined by 2D-PAGE—IEF (RodríguezMartín et al., 2009). Recently, the genome of P. chrysogenum Wisconsin 54-1255 has been sequenced (Van den Berg et al., 2008). A putative protein can be found in BLAST (accession number CAP80409) that differs from the precursor of PgChP in only four amino acids. However, the production of the protein by this mould has not been reported. In conclusion, the gene encoding the chitosanase PgChP with antifungal activity has been sequenced. The putative amino acid sequence of PgChP shows two consensus sites for glycosylations. The role of N-linked oligosaccharides from PgChP on antifungal activity deserves further investigation. 126 MENÚ SALIR RESULTADOS ACKNOWLEDGEMENTS This work was supported by the Spanish Ministry of Education and Science (AGL200406546-ALI) and FEDER. Andrea Rodríguez and Raquel Acosta were recipients of a FPI grants from the Spanish Ministry of Education and Science. Authors would like to thank Dr. Susan Liddell (Division of Animal Sciences) and Prof. Ian Connerton (Division of Food Sciences) for their support during Andrea Rodriguez stay’s at Nottingham University. REFERENCES Acosta, R., Rodríguez-Martín, A., Martín, A., Núñez, F., and Asensio, M.A. Selection of antifungal protein-producing molds from dry-cured meat products. Int. J. Food Microbiol. Submitted (2009a). Fenton, D. M. and Eveleigh, D. E. Purification and mode of action of a chitosanase from Penicillium islandicum. J. Gen. Microbiol. 126: 151-165 (1981). Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M.R., Appel, R.D., and Bairoch, A. Protein Identification and Analysis Tools on the ExPASy Server; (In) John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press. Full text - Copyright Humana Press (2005). Geyer, H. and Geyer, R. Strategies for analysis of glycoprotein glycosylation. Proteins and Proteomics. 1764: 1853-1869, (2006). Gurr, S.J., Uncles, S.E., and Kinghorn, J.R. The structure and organization of nuclear genes of filamentous fungi. In: Kinghorn, J.R. (Ed.). Gene structure in Eukaryotic Microbes. IRL Press, Oxford, pages 93-193 (1987). Lacadena, J., Martínez del Pozo, A., Gasset, M., Patiño, B., Campos-Olivas, R., Vázquez, C., Martínez-Ruiz, A., Mancheño, J.M., Oñaderra, M., and Gavilanes, J.G. Characterization of the antifungal protein secreted by the mould Aspergillus giganteus. Arch. Biochem. Biophys. 20: 273-281 (1995). Lee, D.G., Shin, S.Y., Maeng, C., Jin, Z.Z., Kim, K.L., and Hahm, K. Isolation and characterization of a novel antifungal peptide from Aspergillus niger. Biochem. Biophys. Res. Commun. 263: 646-651 (1999). 127 MENÚ SALIR MANUSCRITO 5 Lee, Y.S., Yoo, J.S., Chung, S.Y., Lee, Y.C., Cho, Y.S., and Choi, Y.L. Cloning, purification, and characterization of chitosanase from Bacillus sp. DAU101. Appl. Microbiol. Biotechnol. 73: 113-21 (2006). Marx, F., Haas, H., Reindl, M., Stöffler, G., Lottspeich, F., and Redl, B. Cloning, structural organization and regulation of expression of the Penicillium chrysogenum paf gene encoding an abundantly secreted protein with antifungal activity. Gene. 167: 167-171 (1995). Monzingo, A.F., Marcotte, E.M., Hart, P.J., and Robertus, J.D. Chitinases, chitosanases, and lysozymes can be divided into procaryotic and eucaryotic families sharing a conserved core. Nat. Struc. Biol. 3: 133-140 (1996). Morelle, W., Canis, K., Chirat, F., Faid, V., and Michalski, J-C. The use of mass spectrometry for the proteomic analysis of glycosylation. Proteomics. 6: 3993-4015 (2006). Nakaya, N., Omata, K., Okahashi, I., Nakamura, Y., Kolkenbrock, H.J., and Ulbrich, N. Amino-acid sequence and disulphide bridges of an antifungal protein isolated from Aspergillus giganteus. Eur. J. Biochem. 193: 31-38 (1990). Nielsen, H., Engelbrecht, J., Brunak, S., and Von Heijne, G. Analysis identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10: 1-6. (1997). Robertus, J.D., Monzingo, A.F., Marcotte, E.M., and Hart, P.J. Structural analysis shows five glycohydrolase families diverged from a common ancestor. J. Exp. Zool. 282: 127-132 (1998). Rodríguez-Martín, A., Liddell, S., Acosta, R., Nuñez, F., and Asensio, M.A. Characterization of PgChP, a novel chitosanase with antifungal activity from Penicillium chrysogenum. Manuscrito 4 (2009). Shimosaka, M., Fukumori, Y., Zhang, X.Y., He, N.J., Kodaira, R., and Okazaki, M. Molecular cloning and characterization of a chitosanase from the chitosanolytic bacterium Burkholderia gladioli strain CHB101. Appl. Microbiol. Biotechnol. 54: 354-60 (2000). Shimosaka, M., Sato, K., Nishiwaki, N., Miyazawa, T., and Okazaki, M. Analysis of essential carboxylic amino acid residues for catalytic activity of fungal chitosanases by site-directed mutagenesis. J. Bios. Bioeng. 5: 545-550 (2005). 128 MENÚ SALIR RESULTADOS Van den Berg, M.A., Albang, R., Albermann, K., Badger, J.H., Daran, J.M., Driessen, A.J., Garcia-Estrada, C., Fedorova, N.D., Harris, D.M., Heijne, W.H., Joardar, V., Kiel, J.A., Kovalchuk, A., Martin, J.F., Nierman, W.C., Nijland, J.G., Pronk, J.T., Roubos, J.A., van der Klei, I.J., van Peij, N.N., Veenhuis, M., von Dohren, H., Wagner, C., Wortman, J., and Bovenberg, R.A. Genome sequencing and analysis of the filamentous fungus Penicillium chrysogenum. Nat. Biotechnol. 26: 1161-1168 (2008). Wiesner, P., Beck, J., Beck, K.F., Ripka, S., Müller, G., Lücke, S., and Schweizer, E. Isolation and sequence analysis of the fatty acid synthetase FAS2 gene from Penicillium patulum. Eur J Biochem. 177: 69-79 (1988). Yoon, H.G., Lee, K.H., Kim, H.Y., Kim, H.K., Shin, D.H., Hong, B.S., and Cho, H.Y. Gene cloning and biochemical analysis of thermostable chitosanase (TCH-2) from Bacillus coagulans CK108. Biosci. Biotechnol Biochem. 66: 986-95 (2002). Zhang, X., Dai, A., Zhang, X., and Kuroiwa, K. Purification and characterization of chitosanase and exo- -D-Glucosaminidase from a Koji Mold, Aspergillus oryzae IAM2660. Biosci. Biotechnol. Biochem. 64: 1896-1902 (2000). Zhang, X., Dai, A., Zhang, X., Kuroiwa, K., Kodaira, R., Nogawa, M., Shimosaka, M., and Okazaki, M. Cloning and characterizacion of a chitosanase gene from the Koji Mold Aspergillus oryzae Strain IAM 2660. Biosci. Biotechnol. Biochem. 64 (4): 977-981 (2001). Zhang, J. and Sun, Y. Molecular cloning, expression and characterization of a chitosanase from Microbacterium sp. Biotechnol. Lett. 29: 1221-5 (2007). Zhu, X., Zhou, Y., and Feng, J. Analysis of both chitinase and chitosanase produced by Sphingomonas sp. CJ-5. J. Zhejiang. Univ. Sci. B. 8(11): 831-838 (2007). 129 MENÚ SALIR Manuscrito 6: Effect of different treatments on chitosanase and antifungal activity of PgChP from Penicillium chrysogenum MENÚ SALIR RESULTADOS Effect of different treatments on chitosanase and antifungal activity of PgChP from Penicillium chrysogenum Andrea Rodríguez-Martín, Raquel Acosta, Félix Núñez, Miguel A. Asensio* Higiene y Seguridad Alimentaria, Facultad de Veterinaria. Universidad de Extremadura, Avda. de la Universidad, s/n. 10071- Cáceres, Spain. * Corresponding author: Telephone (+34) 927 257 125; fax: (+34) 927 257 110. E-mail: [email protected] ABSTRACT The protein PgChP showed antifungal and chitosanase activities with potential application in food industry as protective additive and production of chitosan oligomers, respectively. Haemolytic assay was negative, essential characteristic for using it for human applications. PgChP was resistant at heat treatments even higher than 60ºC. Different ions influenced on chitosanase and antifungal activities of the protein; thus, Na+ and Mg2+ affected to the chitosanase activity, whereas the antifungal activity was only affected for Mg2+ and Ca2+ and not for monovalent ions. Non-ionic detergents remained or improved the chitosanase action, although decreased the antifungal activity. Ionic detergents inhibited both antifungal and chitosanase activities. EGTA (0.1 mM), Triton X-100 (1%) and phosphate (1 mM) increased the chitosanase activity of PgChP. The antifungal activity does not seem to be related to chitosan degradation in sensitive moulds. KEY WORDS: chitosan oligomer, chitosanase, antifungal activity, food preservative, haemolytic activity. INTRODUCTION Natural antifungal proteins are a new source of clinically useful therapeutics. Antifungal proteins have different action mechanisms. Some of them interfere on DNA, RNA, and protein synthesis, or they can act on fungal walls in diverse ways. Some of them decrease the ATP level (Cociancich et al.., 1993) or depolarize the membrane (Thevissen et al.., 1996). Other antifungal proteins interfere with essential elements of fungal walls, such as -1,3-glucan (Odds et al.., 2003), chitin (Yang and Gong, 2002; Von der Weid et al., 2003), or chitosan (Davis and Eveleigh, 1984). These compounds can be even hydrolized by some antifungal proteins like glucanases, chitinases, and chitosanases. 131 MENÚ SALIR MANUSCRITO 6 The most useful antifungal proteins are those that act specifically against fungal membranes because of their lesser toxic effects on mammalian cells (Groll et al., 1998). However, none of these antifungal proteins is currently utilized because they have shown to be cation-sensitive. Sensitivity to high-ionic-strength conditions may be a crucial point in the application of antifungal proteins under physiological conditions, or in many food products with high salt concentration. The protein PgChP was previously purified from the strain P. chrysogenum AS51D for its ability to inhibit the growth of toxigenic moulds (Acosta et al., 2009). Given that this mould was isolated from dry-cured ham, a substrate with a high salt concentration, PgChP could withstand cations and have adequate characteristics for the use in foods. PgChP showed high homology with fungal chitosanases according to its amino acid sequence (Rodríguez-Martín et al., 2009a and 2009b). Thus, PgChP could have chitosanase as well as antifungal activity. Chitosanases can degrade a variety of chitosans with different degrees of acetylation (Ohtakara et al., 1988; Osswald et al., 1993) to yield chitosan oligomers, which have interesting biological properties, including antifungal and antibacterial activity (Allan and Hadwiger, 1979; Hirano and Nagano, 1989), as food preservatives (Rivas et al., 2000). Chitosan is a polysaccharide found in fungal walls (Zhang et al., 2000; Kumiko et al., 2002; Di Mario, 2008) and its degradation by chitosanases can be responsible for inhibiting target moulds (Saito et al., 2009). Thus, the antifungal activity of PgChP could be related to the chitosanase activity. To design potential applications, the impact of environmental factors on PgChP activity should be known. The aim of the present work was to study the influence of factors such as temperature, different substrates, ions, and detergents on both antifungal and chitosanase activities. MATERIALS AND METHODS Microbial strain The PgChP producing mould, P. chrysogenum AS51D, and the toxigenic mould Penicillium commune Pc332 were isolated from dry-cured ham and belong to the fungal collection of Food Hygiene (University of Extremadura, Spain). 132 MENÚ SALIR RESULTADOS PgChP purification P. chrysogenum AS51D was grown at 25ºC for 15 days in malt extract broth made of glucose 2% (w/v), malt extract 2% (w/v) and peptone 0.1% (w/v), pH 4.5. Then, the medium was filtered though a 0.45 µm diameter pore size cellulose acetate membrane and PgChP was purified using an ÄKTA fast protein liquid chromatography system (Amersham Pharmacia Biotech AB, Uppsala, Sweden) according to Acosta et al. (2009). For this, filtered medium was loaded on a HiTrap SP HP 5 ml ion exchange column (Amersham Pharmacia Biotech AB) equilibrated with 0.02 M sodium acetate buffer, pH 4.5. Bound proteins were eluted with a gradient of 0.02 M sodium acetate, 1 M NaCl buffer, pH 4.5: 0% to 25% in 15 column volumes (CV), increase to 100% in 2 CV, and finally keeping 100% for 15 CV. Eluted proteins were detected at 214 nm. The fraction that showed antifungal activity in previous studies (Acosta et al., 2009) was purified using a HiLoad 26/60 Superdex 75 prep grade gel filtration column (Amersham Pharmacia Biotech AB) equilibrated with 50 mM sodium phosphate, 0.15 M NaCl buffer, pH 7, at a constant flow rate of 2.5 ml/min. Data collection and processing were done using the UNICORN software 4.12 (Amersham Pharmacia Biotech AB). Finally, the fraction containing PgChP was desalted and concentrated with YM-10 microcon centrifugal filter units (Millipore Iberica S.A.U., Madrid, Spain) and kept at -20ºC. Protein was quantified by Johnson method according to Córdoba (1990). An aliquot of the purified protein (100µl) was dried in a heat cabinet at 100 ºC. The sample was digested by adding 200 µl of sulfuric acid in a sand bath at 120 ºC. Then, 4.8 ml of distilled water, 3 ml of 4N NaOH and 2 ml of Nessler reactive made of KI 0.4% (w/v), 0.4% HgI2 (w/v) and 0.175% (w/v) arabic gum were added. After 10 min at room temperature the absorbance was measured at 490 nm in a spectrophotometer. A calibration curve was obtained with different concentrations of ammonium sulfate. Sodium dodecyl sulphate-polyacrylamide gel eletrophoresis (SDS-PAGE) SDS-PAGE was performed as described Laemmli (1970). The protein PgChP was separated on a 4% (v/v) polyacrylamide stacking and 8% (v/v) polyacrylamide resolving gels. Gels were stained with Imperial Protein Stain (Pierce Biotechnology, Inc., Rockford, U.S.A.). The molecular weight was determined by comparison of its electrophoretic mobility with proteins of the molecular mass marker (66-6.5 kDa) from Sigma (SigmaAldrich Química S.A., Madrid, Spain). 133 MENÚ SALIR MANUSCRITO 6 Haemolytic activity assay The haemolytic activity of the protein was determined using sheep defibrinated blood (Oxoid). The erythrocytes were isolated by centrifugation (1000 ×g for 6 min at room temperature) and washed three times with 50 mM sodium phosphate, 0.15 M NaCl buffer, pH 7. The cell concentration in the stock suspension was adjusted to 1×109 cells/ml. A volume of 50 µl of cells suspension was added to 200 µl of sterile concentrated protein (10 µg/ml) and incubated at 37ºC. After 0, 8, and 24 h, the samples were placed on ice, immediately centrifuged at 4ºC, and the supernatant was carefully collected. The haemolysis was determined by measuring absorbance of the supernatant at 570 nm (Moerman et al., 2002) and 541 nm (Lai et al., 2005). Controls for 0% and 100% haemolysis were obtained suspending sheep red blood cells in the buffer or distilled water, respectively. The haemolysis percentage was calculated using the following equation: Percentage haemolysis of supernatants = [(Abs in the peptide solution − Abs in buffer)/Abs in water solution] × 100. Three independent assays were conducted with triplicated readings for each one. Evaluation of chitinase and chitosanase activities Chitinase and chitosanase activities were determined with a modification of the 3,5dinitrosalicylic acid method (Su et al., 2006) by measuring the reducing sugars liberated during the hydrolysis of chitin or chitosan. One ml of the 1% (w/v) chitin or chitosan (Sigma-Aldrich) in 1% (v/v) acetic acid was added to 1 ml of the PgChP solution (10µg/ml) and incubated for 2-4 h at 37ºC. Then, to detect the hydrolisis reaction, 2 ml of dinitrosalicylic acid reagent, made of 1% (w/v) 3,5-dinitrosalicylic acid (Sigma-Aldrich), 0.05% (w/v) sodium sulfite, and 1% (w/v) phenol were added and the sample was incubated at 100ºC for 45 min. Finally, 1 ml of 40% (w/v) potassium sodium tartrate was added and the solution was centrifuged. The supernatant was measured at 520 and 575 nm. To quantify the reducing sugars liberated, a calibration curve was obtained with different concentrations of D-(+)-Glucosamine hydrochloride (Sigma-Aldrich). A chitinase from Streptomyces griseus (Sigma-Aldrich) with both chitinase and chitosanase activities was used as positive control. Preparation of colloidal chitin: The colloidal chitin was obtained following the method of Roberts and Selitrennikott with minor modifications (Harighi et al., 2007). Ten grams of 134 MENÚ SALIR RESULTADOS chitin powder were added slowly into 100 ml of H3PO4 (85%) at 25ºC under vigorous shaking for 2 h. This solution was carefully added over 1 liter of absolute ethanol at -20ºC. The mix was shaken for 30 min and stored until use. Ten ml of suspension were centrifuged, the pellet was washed three times with 50 mM sodium phosphate buffer pH 7, and resuspended in distilled water. The final concentration of colloidal chitin was around 1%. Chitinase activity assay by gel electrophoresis The chitinase activity assay was carried out as described by Trudel and Asselin (1989). PAGE-SDS was performed in 4% (v/v) polyacrylamide stacking gel and 8% (v/v) separating polyacrylamide gradient gel containing 0.01% (w/v) glycol chitin and 0.1% (w/v) SDS. Samples were boiled for 5 min with 20% (v/v) glycerol, 0.025% (w/v) bromophenol blue, 2% (w/v) SDS and 5% (v/v) -mercaptoethanol in 62.5 mM Tris-HCl (pH 6.8). The gel was run at 100 V for 1 h. After electrophoresis, the gel was incubated for 2 h at 37 ºC under continuous shaking in 100 mM sodium acetate buffer (pH 5.0) containing 1% (v/v) Triton X-100. Then, the gel was stained with a 0.01% (w/v) calcofluor white M2R (Sigma-Aldrich) in 500 mM TrisHCl (pH 8.9). After 5 min, this solution was removed and the gel was incubated in distilled water at room temperature for about 1 h. Lytic zones were visualized in the UV transilluminator Syngene G-Box (Synoptics, Fredericks, USA). The gel was photographed and analyzed with the GeneSnap software (Synoptics). An additional gel was stained only with Imperial Protein Stain (Pierce Biotechnology, Inc., Rockford, U.S.A.). The chitinase from S. griseus (Sigma) was used as positive control. The molecular mass marker used was 66-6.5 kDa (Sigma). Glycol chitin synthesis: 0.2 g glycol chitosan was dissolved in 4 ml of 10% acetic acid and the mixture was shaken overnight at 22ºC. Methanol (18 ml) was slowly added and the solution was filtered through 0.45 µm pore size cellulose acetate membrane filters. Acetic anhydride (0.3 ml) was added and kept for 30 min. The resulting gel was cut into small pieces and after adding 1 vol of methanol was shaken for 4 min. This suspension was centrifuged at 27000 ×g at 4ºC for 15 min and the pellet was resuspended in 20 ml of 0.02% (w/v) sodium azide. The final concentration of the glycol chitin was 1% (w/v). 135 MENÚ SALIR MANUSCRITO 6 Evaluation of Antifungal activity Quantitative assay for fungal growth inhibition was performed following the protocol developed by Broekaert et al. (1990) with some modifications (Acosta et al., 2009). P. commune Pc332 (Núñez et al., 1996) was used as reference strain. To perform the inhibition test in microtitter plates, 100 µl of the fraction obtained by gel filtration chromatography were dispensed in triplicate wells. Each well was loaded with 100 µl of concentrated malt extract broth (4% malt extract, 4% glucose and 0.2% peptone, pH 4.5) inoculated with the reference strain to c.a. 106 spores/ml. The plates were incubated for up to 72 h at 25 ºC. Fungal growth was determined by measuring the optical density variation at 595 nm. Effect of heat-treatments The study of the protein stability after heat treatment was carried out according to Okkers et al. (1999). Aliquots of PgChP protein (10 µg/ml) were exposed to different combinations of temperatures (60, 80, and 100 ºC) and times (10, 20, and 30 min), as well as to sterilization at 121 ºC for 15 min. After the heat-treatment, the samples were cooled on ice. Then, antifungal and chitosanase activities were evaluated as described above. Influence of different chemicals on chitosanase and antifungal activity To study the influence of some chemicals on the protein, the activity of PgChP (10 µg /ml) was tested with different compounds: NaCl, CaCl2, KCl, or MgCl2 (0.1, 1, 10, and 100 mM); EDTA or EGTA at 0.01, 0.1, and 1 mM; 0.5% and 1% of Triton X-100, tween-20, tween-80, SDS, or deoxycholic acid; NaH2PO4 at 0, 0.1, 1, 10, and 100 mM. Statistical analysis Data were statistically analyzed using SPSS software package (version 13.0. SPSS Inc., Chicago, USA) by a variance analysis (one way ANOVA) and the means were separated by the Tukey honest significant difference test. 136 MENÚ SALIR RESULTADOS RESULTS Haemolytic activity To study the haemolytic activity of PgChP on mammalian cells, the effect of the protein was examined on sheep red blood cells. The protein PgChP did not show haemolytic activity, being the value obtained of 0%, even after 24 h. Chitosanase and chitinase activities The protein PgChP showed chitosanase activity, but no chitinase activity was found by the 3,5-dinitrosalicylic acid method (Figure 1). Figure 1. Chitinase and chitosanase activities of PgChP expressed as liberated D-glucosamine after 4 h. Asterisks indicate statistically significant differences to the negative control without protein (***p<0.001). Chitinase from S. griseus (50 µg/ml) was used as positive control. To further evaluate the chitinase activity, the hydrolysis of chitin was tested by electrophoresis in a glycol chitin gel. No activity was observed loading the polyacrilamide gel with 18 µg of PgChP (Figure 2). Lytic zones by chitinase from S. griseus, used as positive control, were observed as black bands after staining with calcofluor white M2R. 137 MENÚ SALIR MANUSCRITO 6 &% # &% # "' "& "' "& " " &" &" % % Figure 2. Chitinase activity of PgChP evaluated by gel electrophoresis. SDS-PAGE gels with glycol chitin were stained with Imperial Protein Stain (A) and Calcofluor White M2R (B). M: protein marker, Q: Chitinase from S. griseus (100 µg). Protein stability to different temperatures PgChP protein showed a remarkable stability at high temperatures. The chitosanase activity did not show a significant reduction after heat treatments even at 60ºC for 30 min (Figure 3). " # ! " # % $ ! % " ! # % " ! Figure 3. Effect of heat-treatment on PgChP chitosanase activity expressed as mean and standard deviation of liberated glucosamine after 2 h. Asterisks indicate statistically significant differences (p<0.05) to the positive control without heat-treatment (C+). 138 MENÚ SALIR RESULTADOS The antifungal activity seems to withstand even treatments of 80ºC up to 20 min (Figure 4), whereas higher temperatures inactivated PgChP antifungal activity allowing an %'% + unrestricted growth of the sensitive mould. & ) * ,- " " # ! " $ ! # % % ! " # % ( " ! Figure 4. Antifungal activity of heat-treated PgChP expressed as mean and standard deviation of P. commune Pc332 growth after 48 h. Asterisks indicate statistically significant differences (p<0.05) to the positive control with non heat-treated PgChP. C+: positive control with non heat-treated PgChP; C-: negative control without PgChP. Effect of ions on PgChP chitosanase and antifungal activities PgChP chitosanase activity was not affected by the presence of up to 100 mM CaCl2 or KCl ions whereas it decreased at the highest NaCl or MgCl2 concentration (Figure 5). 139 MENÚ SALIR MANUSCRITO 6 . 0 / 1 " " Figure 5. Effect of monovalent (A) and divalent (B) ions on the chitosanase activity of PgChP expressed as mean and standard deviation of liberated glucosamine after 3 h. Asterisks indicate statistically significant differences to the positive control without ions (closed bars) (**p<0.01; ***p<0.001). On the other hand, the inhibition of the reference mould was unaffected by monovalent ions, K+ and Na+, but significantly decreased with high concentrations of divalent ions (Figure 6). 140 MENÚ SALIR RESULTADOS . ( 0 ,- ) * %'% + / 1 ( " ,- ) * %'% + " Figure 6. Effect of monovalent (A) and divalent (B) ions on antifungal activity of PgChP against P. commune Pc332 expressed as mean and standard deviation of optical density after 72 h. Asterisks indicate statistically significant differences to the negative control (C-) without PgChP (* p<0.05; ** p<0.01; *** p<0.001). Effect of cation chelators Activity of PgChP was also assayed in presence of EDTA and EGTA. The chitosanase activity was strongly affected by high levels of chelators. Thus, 1 mM EDTA inhibited chitosanase activity, whilst even low levels of EGTA increased chitosan hydrolysis by PgChP (Table 1). 141 MENÚ SALIR MANUSCRITO 6 Table 1. Effect of EDTA and EGTA on chitosanase activity of PgChP after 2 h incubation. !" " ! a " ! $% & #%2 ' & 2 $' & #%2 ' ' 2 #! " ! % # $ 2 ! #3 3 2 2 &$ 3'2 '& Results are expressed as mean ± SD of liberated glucosamine. Asterisks indicate statistically significant differences to the positive control without chelators (*p<0.05; ***p<0.001). The antifungal activity of PgChP was observed at the lowest EDTA concentration (0.01 mM) but not at 0.1 mM (Table 2). The highest level of EDTA tested inhibited growth of the reference mould, and no further effect on the antifungal activity could be observed. EGTA did not seem to inhibit growth of the reference mould even at the highest concentration tested. No effect of EGTA was observed on the antifungal activity against P. commune Pc332, irrespectively of the concentration used (Table 2). Table 2. Effect of EDTA and EGTA on the antifungal activity of PgChP against P. commune Pc332 after 48 h incubation. ( ) " ! * #+ , " ! / $% & # / $' & a %" 2 # 2 " % % &$ #3 2 & #" 2 . "! ! %& 2 % 2 - " ! 2 %" 2 " & " "' 2 # ! & 2 2 2 % 2 %% #3 2 "$ &% 2 #& 2 #$ 3 Results are expressed as mean ± SD of optical density. Asterisks indicate statistically significant differences to the negative control without PgChP (C-) (*p<0.05; **p<0.01; ***p<0.001). 142 MENÚ SALIR RESULTADOS Resistance to protein-denaturing detergents Detergents displayed different effects on the chitosanase activity of PgChP (Figure 7). Non-ionic detergents showed no inhibition on the activity. In fact, tween 20, tween 80, and Triton X-100 even increased the chitosanase activity. In contrast, the ionic detergents sodium dodecil sulphate (SDS) and deoxycholic acid partially or completely inhibited the chitosanase activity, respectively, for the 2 h incubation time. 6+ " 6+ $ 6 7( 4 4 000 % % % 4 8 . 000 % % 5 Figure 7. Effect of different detergents on chitosanase activity of PgChP expressed as mean and standard deviation of liberated glucosamine after 2 h. Asterisks indicate statistically significant differences to the positive control without detergents (*p<0.05; ***p<0.001). On the other hand, PgChP showed no antifungal activity on P. commune Pc332 with 0.5 or 1% (w/v) of the tested detergents. 143 MENÚ SALIR MANUSCRITO 6 ( 6+ " 6+ $ 6 4 4 7( 8 . ) * ,- %'% + 1 000 000 % % 000 000 % 000 % 4 % 5 Figure 8. Effect of different detergents on antifungal activity of PgChP against P. commune Pc332 expressed as mean and standard deviation of optical density after 72 h. Asterisks indicate statistically significant differences to the negative control (C-) without PgChP (*** p<0.001). Effect of sodium phosphate on the PgChP activity The chitosanase activity decreased at the highest concentration of sodium phosphate tested, whereas levels below 100 mM favoured this activity (Table 3). Table 3. Influence of phosphate on the chitosanase activity of PgChP after 2 h of incubation. !" , ! #%2 a ! ' &'2 " " #! " ! $" %&2 ! &$ %'2 ! &" &2 Results are expressed as mean ± SD of liberated glucosamine. Asterisks indicate statistically significant differences to the positive control without phosphate (** p<0.01; *** p<0.001). Phosphate also seems to affect the antifungal activity of PgChP, but the uneven growth of the reference mould in negative controls did not allow to obtain more consistent results (Table 4). 144 MENÚ SALIR RESULTADOS Table 4. Influence of phosphate on the antifungal activity of PgChP against P. commune Pc332 after 72 h. ( ) " ! * , ! " / &$ 2 %$ 2 a ! $ 2 " " " - 3 . "! " ! 2 " #+ , ! $% 2 &' %# 2 $3 2 # '% 2 ! " % '$ 2 3$ 2 3 # Results are expressed as mean ± SD of optical density. Asterisks indicate statistically significant differences to the negative control without PgChP (** p<0.01; *** p<0.001). DISCUSSION As it has been shown in a previous work, the protein PgChP showed antifungal activity against P. commune Pc332 (Acosta et al., 2009), a toxigenic mould present in foods (Sosa et al., 2002). PgChP was obtained by ion exchange chromatography looking for cationic proteins (Acosta et al., 2009), because the cationic character in proteins is related to a higher antifungal activity, but to a lower toxicity for mammalian cells (Hong et al., 2001). Given that PgChP shows an isoelectric point close to 5 (Rodríguez-Martín et al., 2009a), the protein is not cationic under physiological conditions and its potential as toxic for humans has to be assessed. The haemolysis assay to test for cytolytic activity is generally accepted as proof that the protein can be regarded as safe (Van’t Hof et al., 2001). PgChP did not show any haemolytic activity even though in depth tests are needed to confirm the safety of this protein. The amino acid sequence of PgChP revealed a high homology to fungal chitosanases (Rodríguez-Martín et al., 2009a, 2009b). Thus, the chitosanase activity of PgChP was evaluated. As it is shown in figure 1, the protein showed a significant chitosanase activity. Some microbial chitosanases, such us the chitosanase II from Acinetobacter sp. strain CHB101, hydrolyze colloidal chitin and glycol chitin besides chitosan (Shimosaka et al., 1995). However, PgChP did not show activity on either of these substrates, which agrees with the fact that most chitosanases do not have chitinase activity (Su et al., 2006; Fukamizo and Brzezinski, 1997). Fungal chitosanases differ in their thermostability. There are chitosanases such as Csn2 from Gongronella sp. JG (Wang et al., 2008) that resist 50 and 55ºC for 30 and 11 min, respectively. Bacterial chitosanases have shown stability within a similar or lower range of 145 MENÚ SALIR MANUSCRITO 6 temperature, such as those from Acinetobacter sp. C-17 (Zhu et al., 2003) and Sphingomonas sp. CJ.5, that are stable at 30-40ºC and 45ºC for one hour, respectively (Zhu et al., 2007). Given that PgChP kept chitosanase activity after heating at 60ºC for 30 min (Figure 3), it can be regarded as more stable than other chitosanases. On the other hand, the antifungal activity of PgChP was more heat-resistant than chitosanase activity. PgChP antifungal activity resisted even at 80 ºC for up to 20 min. The fact that chitosanase and antifungal activities of PgChP differ in thermostability indicates that chitosan-hydrolysis is not essential for the antifungal activity, as it will be discussed later. The presence of K+ and Ca2+ did not affect the chitosanase activity of PgChP at the highest tested concentration, but 100 mM Mg2+ and Na+ inhibited that activity. However, K+ and Ca2+ activate several fungal chitosanases (Alfonso et al., 1992; Fenton and Eveleigh, 1981). As a consequence, metal ions can have different impact on fungal chitosanases. This effect can be related to the influence of different concentrations of particular ions on the structure of chitosanases (Wang et al., 2008). Regarding the antifungal activity of PgChP, over 1 mM Mg2+ or Ca2+ reduced the inhibition on P. commune, whereas monovalent ions Na+ or K+ did not affect the antifungal activity (Figure 6). The fact that divalent ions repressed the antifungal activity could mean that these ions compete with PgChP for potential target sites in the fungal wall, as it has been described for metal ions on other antifungal proteins (Broekaert et al., 1992; Kaiserer et al., 2003; Theis et al., 2003). In addition, the divergent effect of Na+ and Ca2+ on PgChP chitosan hydrolysis and fungal inhibition supports that different mechanisms of action are responsible for these activities. The activity of proteins can also be influenced by low concentrations of metal ions. The addition 1 mM of EDTA, a chelator of Mg2+ and Ca2+ (Choucair et al., 2006), decreased the chitosanase activity. Then, the absence of Mg2+ seems to inhibit the chitosanase activity of PgChP. In contrast, EGTA inhibited neither chitosanase nor antifungal activity of PgChP, and even notably increased the chitosanase activity (Table 1). EGTA is a quite specific chelator of Ca2+ (Choucair et al., 2006). Thus, the chitosanase activity of PgChP was not affected by the absence of this ion. On the contrary, the antifungal activity was not affected by EGTA or low levels of EDTA (Table 2), whereas high levels of EDTA strongly limited the development of the reference 146 MENÚ SALIR RESULTADOS mould. Once more, the differences on chitosanase and antifungal activities of PgChP are evidenced, due to the impact of low levels of metal ions. Ionic and non-ionic detergents had a distinct effect on PgChP activities. Non-ionic detergents (tween 20, tween 80, and Triton X-100) led to an increase of chitosanase activity (Figure 7), probably due to an improved protein-substrate interaction. However, no clear effect of these detergents could be observed on the antifungal activity, due to the low growth of P. commune Pc332 even when added at 0.5%. The ionic detergents SDS and deoxycholic acid caused loss of both chitosanase and antifungal activities. The effect of these detergents can be attributed to protein denaturation that prevents PgChP from being active. Finally, sodium phosphate increased the chitosanase activity of PgChP, except at the highest concentration (100 mM) which caused loss of activity. These results are somehow similar to those obtained with identical levels of NaCl. Thus, the main impact of sodium phosphate on this activity can be attributed to Na+ concentration. The evaluation of phosphate effect on the antifungal activity was hampered by an uneven growth of Pc332 in control wells without PgChP. As it can be observed in table 4, 10 mM phosphate PgChP exerted a significant inhibition (p<0.01) on P. commune, but this was not observed at lower concentrations. The poorer growth reached by P. commune in the negative controls can mask statistically significant differences due to the antifungal activity. As it has been evidenced mainly by the residual activity after heat treatments and under high Na+ or Ca+ concentrations, antifungal and chitosanase activities of PgChP are independent from each other. This is in contrast to the antifungal activity of the protein CtoA from Amycolatopsis sp. that has been attributed to the hydrolysis of chitosan in the cell wall of the sensitive Rhizopus oryzae (Saito et al., 2009). In addition, it is unlike that the antifungal activity of PgChP is related to the degradation of chitosan in the fungal wall because chitosan has not been described in P. commune. Applications of PgChP for chitosan-hydrolysis could take advantage of the thermal stability to 60ºC and the increased activity of the protein by adding EGTA (0.1 mM), Triton X-100 (1%) or phosphate (10 mM). Nontheless, the conditions of pH and temperature for an optimum chitosanase activity should be determined. The antifungal activity of the PgChP protein was not affected by high temperatures and monovalent cations, indicating that PgChP have potential applications as a preservative in 147 MENÚ SALIR MANUSCRITO 6 the food industry, particularly on substrates with a high concentration of salts. Thus, the addition of PgChP could prevent the growth of unwanted moulds in dry-cured meat products, where Na+ and K+ are abundant. Even though a complete study about the safety of PgChP is necessary, the information here reported is promising for a potential use of this protein as food preservative. ACKNOWLEDGEMENTS This work was supported by the Spanish Ministry of Education and Science (AGL200406546-ALI) and FEDER. Andrea Rodríguez and Raquel Acosta were the recipients of a FPI grant from the Spanish Ministry of Education and Science. REFERENCES Acosta, R., Rodríguez-Martín, A., Martín, A., Núñez, F., and Asensio, M.A. Selection of antifungal protein-producing molds from dry-cured meat products. Int. J. Food Microbiol. Submitted (2009). Alfonso, C., Martínez, M. J., and Reyes, F. Purification and properties of two endochitosanases from Mucor rouxii implicated in its cell wall degradation. FEMS Microbiol. Lett. 95: 187-194 (1992). Allan, C.R. and Hadwiger, L.A. The fungicidal effect of chitosan on fungi of varying cell wall composition. Exp. Mycol. 3: 285-287 (1979). Broekaert, W.F., Terras, F.R.G., Cammue, B.P.A., and Vanderleyden, J. An automated quantitative assay for fungal growth inhibition. FEMS Microbiol. 69: 55-60 (1990). Broekaert, W.F., Mariën, W., Terras, F.R.G., De Bolle, M.F.C., Proost, P., Van Damme, J., Dillen, L., Claeys, M., Rees, S.B., Vanderleyden, J., and Cammue, B.P.A. Antimicrobial peptides from Amaranthus caudatus seeds with sequence homology to the cysteine/glycine-rich domain of chitin-binding proteins. Biochemistry. 31: 4308-4314 (1992). Choucair N., Laporte V., Levy R., Tranchant C., Gies J. P., Poindron P., and Lombard, Y. The role of calcium and magnesium ions in uptake of beta-amyloid peptides by microglial cells. Int. J. Immunopathol. Pharmacol. 19: 683-96 (2006). 148 MENÚ SALIR RESULTADOS Cociancich, S., Ghazi, A., Hetru, A., Hoffman, J.A., and Letellier, L. Insect defensin, an inducible antibacterial peptide, forms voltage-dependent channels in Micrococcus luteus. J. Biol. Chem. 268: 19239-19245 (1993). Córdoba, J.J. Doctoral Thesis: “Transformaciones de los componentes nitrogenados durante la maduración del jamón de cerdo ibérico”. University of Extremadura. Spain. (1990). Davis, B. and Eveleigh, D.E. Chitosanases: occurrence, production and immobilization. In Chitin, Chitosan and Related Enzymes, pp. 161-179. J. P. Zikakis. New York: Academic Press (1984). Di Mario, F., Rapana, P., Tomati, U., and Galli., E. Chitin and chitosan from Basidiomycetes. J. Biol. Macromols. 43: 8-12 (2008). Fenton, D.M. and Eveleigh, D.E. Purification and mode of action of a chitosanase from Penicillium islandicum. J. Gen. Microbiol. 126: 151-165 (1981). Fukamizo, T., and Brzezinski, R. Chitosanase from Streptomyces sp. strain N174: a comparative review of its structure and function. Biochem. Cell. Biol. 75: 687-696 (1997). Groll, A.H., De Lucca, A.J., and Walsh, T.J. Emerging targets for the development of novel antifungal therapeutics. Trends Microbiol. 6: 117-124 (1998). Hancock, R.E.W. and Chapple, D.S. Peptide antibiotics. Antimicrob. Agents Chemother. 43: 1317-1323 (1999). Harighi, M. J., Zamani, M. R., and Motallebi, M. Evaluation of antifungal activity of purified chitinase 42 from Trichoderma atroviride PTCC5220. Biotech. 6: 28-33 (2007). Hirano S. and Nagano N. Effects of chitosan, pectic acid, lysozyme and chitinase on the growth of several phytopathogens. Agric. Biol. Chem. 53: 3065–3066 (1989). Hong, S.Y., Park, T.G., and Lee, K.H. The effect of charge increase on the specificity and activity of a short antimicrobial peptide. Peptides. 22: 1669-1674 (2001). Kaiserer, L., Oberparleiter, C., Weiler-Görz, R., Burgstaller, W., Leiter, E., and Marx, F. Characterization of the Penicillium chrysogenum antifungal protein PAF. Arch Microbiol. 180: 204-210 (2003). 149 MENÚ SALIR MANUSCRITO 6 Kumiko, S., Matsuda, H., and Kawamukai, M. Functional expression of chitinase and chitosanase, and their effects on morphologies in the yeast Shizosaccharomyces pombe. Biosci. Biotechnol. Biochem. 66: 1143-1147 (2002). Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685 (1970). Lai, Y-P., Peng, Y-F, Zuo, Y., Li, J., Huang, J., Wang, L-F., and Wu, Z.R. Functional and structural characterization of recombinant dermcidin-1L, a human antimicrobial peptide. Biochem. Bioph. Research. Commun. 328: 243–250 (2005). Moerman, L., Bosteels, S., Noppe, W., Willems, J., Clonen, E., Schoofs, L., Thevissen, K., Tytgat, J., Van Eldere, J., and Verdonck, J. W. F. Antibacterial and antifungal properties of alpha-helical, cationic peptides in the venom of scorpions from southern Africa European. J. Biochem. 269: 4799-4805 (2002). Núñez, F., Rodríguez, M.M., Bermúdez, M.E., Córboba, J.J., and Asensio, M.A. Composition and toxigenic potential of the mould population on dry-cured ham. Int. J. Food Microbiol. 32: 185-197 (1996). Odds, F.C., Brown, A.J.P., and Gow, N.A.R. Antifungal agents: mechanisms of action. Trends Microbiol. 11: 272-279 (2003). Ohtakara, A., Izume, M., and Mitsutomi, M. Action of microbial chitinases on chitosan with different degrees of deacetylation. Agric. Biol. Chem. 52: 3181-3182 (1988). Okkers, D.J., Dicks, L.M.T., Silvester, M., Joubert, J.J., and Odendaal, H.J. Characterization of pentocin TV35b, a bacteriocin-like peptide isolated from Lactobacillus pentosus with a fungistatic effect on Candida albicans. J. Appl. Microbiol. 87: 726-734 (1999). Osswald, W.F., Shapiro J.P., McDonald, R.E., Niedz, R.P., and Mayer, R.T. Some citrus chitinases also possess chitosanase activities. Experientia. 49: 888-892 (1993). Rivas, L.A., Parro, V., Moreno-Paz, M., and Mellado, P. The Bacillus subtilis 168 csn gene encondes a chitosanase with similar properties to a Streptomyces enzyme. Micriobiol. 146: 2929-2936 (2000). Rodríguez-Martín, A., Liddell, S., Acosta, R., Nuñez, F., and Asensio, M.A. Characterization of PgChP, a novel chitosanase with antifungal activity from Penicillium chrysogenum. Manuscrito 4 (2009a). 150 MENÚ SALIR RESULTADOS Rodríguez-Martín, A., Acosta, R., Nuñez, F., Benito, M.J., and Asensio, M.A. Genetic characterization of the gene enconding the novel chitosanase PgChP from Penicillium chrysogenum. Manuscrito 5 (2009b). Saito, A., Ooya, T., Miyatsuchi, D., Fuchigami, H., Terakado, K., Nakayama, S-y., Watanabe, T., Nagata, Y., and Ando, A. Molecular characterization and antifungal activity of a family 46 chitosanase from Amycolatopsis sp. CsO-2. FEMS Microbiol. Lett. 293(1): 79-84 (2009). Shimosaka, M., Nogawa, M., Wang, X., Kumehara, M., and Okazaki, M. Production of two chitosanases from a chitosan-assimilating bacterium, Acinetobacter sp. strain CHB101. Appl. Environ. Microbiol. 61: 438-442 (1995). Sosa, M.J., Córdoba, J.J., Díaz, C., Rodríguez, M., Bermúdez, E., Asensio, M.A., and Núñez, F. Production of cyclopiazonic acid by Penicillium commune isolated from drycured ham on a meat extract-based substrate. J Food Prot. 65: 988-92 (2002). Su, C., Wang, D., Yao, L., and Yu, Z. Purification, characterization, and gene cloning of a chitosanase from Bacillus Species Strain S65. Agric. Food Chem. 54: 4208-4214 (2006). Theis, T., Wedde, M., Meyer, V., and Stahl, U. The antifungal protein from Aspergillus giganteus causes membrane permeabilization. Antimicrob. Agents Chemother. 47: 588-593 (2003). Thevissen, K., Ghazi, A., De Samblanx, G.W., Brownlee, C., Osborn, R.W., and Broekaert, W.F. Fungal membrane responses induced by plant defensins and thionins. J. Biol. Chem. 271: 15018-15025 (1996). Trudel, J. and Asselin, A. Detection of chitinase activity after polyacrvlamide gel electrophoresis. Anal. Biochem. 178: 362-366 (1989). Van’t Hof, W., Veerman, E.C.I., Helmerhorst, E.J., and Amerongen, A.V.N. Antimicrobial peptides: properties and applicability. Biol. Chem. 382: 597-619 (2001). Von der Weid, I., Alviano, D.S., Santos, A.L.S., Soares, R.M.A., Alviano, C.S., and Seldin, L. Antimicrobial activity of Paenibacillus peoriae strain NRRL BD-62 against a broad spectrum of phytopathogenic bacteria and fungi. J. Appl. Microbiol. 95: 1143-1151 (2003). Wang, J., Zhou, W., Yuan, H., and Wang Y. Characterization of a novel fungal chitosanase Csn from Gongronella sp. JG. Carboh. Res. 343: 2583-2588 (2008). 151 MENÚ SALIR MANUSCRITO 6 Yang, Q. and Gong, Z. Purification and characterization of an ethyleneinduced antifungal protein from leaves of guilder rose (Hydrangea macrophylla). Prot. Express. Purif. 24: 7682 (2002). Zhang, X., Dai, A., Zhang, X., and Kuroiwa, K. Purification and characterization of chitosanase and exo- -D-Glucosaminidase from a Koji Mold, Aspergillus oryzae IAM2660. Biosci. Biotechnol. Biochem. 64: 1896-1902 (2000). Zhu, X-F., Wu, X-Y., and Dai, Y. Fermentation conditions and properties of a chitosanase from Acinetobacter sp. C-17. Biosci. Biotechnol. Biochem. 67(2): 284-290 (2003). Zhu, X-F., Zhou, Y., and Feng, J-l. Analysis of both chitinase and chitosanase produced by Sphingomonas sp. CJ-5. J. Zheijang Univ. Sci. B. 8(11): 831-838 (2007). 152 MENÚ SALIR V. DISCUSIÓN MENÚ SALIR DISCUSIÓN V. DISCUSIÓN Las proteínas PgAFP y PgChP, purificadas a partir de dos cepas de Penicillium chrysogenum aisladas de jamón curado, mostraron actividad antifúngica en estudios previos. Debido a la potencialidad que esta actividad presenta para su aplicación en el control de mohos indeseables en alimentos se planteó la caracterización de dichas proteínas. V. 1. CARACTERIZACIÓN BIOQUÍMICA La masa molecular de la proteína PgAFP se había estimado en torno a 9 kDa mediante SDS-PAGE. Sin embargo, el análisis de la proteína purificada mediante espectrometría de masas SELDI-TOF (Acosta y col., 2009b) y ESI-MS (Manuscrito 1) reveló un peso molecular de únicamente 6,5 kDa (Figura V.1). 10000 Figura V.1. Peso molecular de PgAFP en SDS-PAGE (A) y análisis por SELDI-TOF (B; Acosta y col., 2009b). Esta discrepancia entre los resultados de las dos técnicas podría deberse a la existencia de modificaciones post-traduccionales en la proteína (Pitt-Rivers y Ambesi Impiombato, 1968; Tozzini y col., 1994; Murayama y col., 2003). Así se han descrito migraciones anómalas de proteínas glicosiladas en SDS-PAGE atribuidas a una menor capacidad de las mismas para unirse al SDS (Nelson, 1971; Russ y Poláková, 1973; Bagger y col., 2007). PgAFP mostró además una reacción positiva a la tinción con ácido periódico-Schiff (Acosta y col., 2009b), el cual oxida los grupos 1,2-glicol presentes en los polisacáridos a aldehídos, que a su vez reaccionan con la fucsina para formar bandas de color magenta o rosado (Zacharius y col., 1969). Por lo tanto, este resultado es indicativo de la presencia de cadenas de glicano unidas a la proteína. Un posterior análisis de la proteína mediante espectrometría de masas MALDI-TOF reveló la presencia de picos 153 MENÚ SALIR DISCUSIÓN atípicos de 215 Da que no pudieron interpretarse como secuencias de aminoácidos, lo que apoyó la hipótesis de que PgAFP tuviera algún tipo de glicosilación unida a su núcleo proteico (Acosta, 2006). No obstante en el presente estudio, el tratamiento con una amplia variedad de deglicosidasas no reveló ningún indicio de glicosilaciones, hecho que fue confirmado posteriormente con la secuencia deducida a partir del gen que codifica dicha proteína (Manuscritos 1 y 2). En consecuencia, la migración anómala mostrada por PgAFP en los geles de poliacrilamida podría ser explicada por su alto punto isoeléctrico, estimado en 9,2 (Acosta, 2006), que puede originar repulsiones electrostáticas que dificulten la unión con el SDS impidiendo una correcta migración en los geles (Mondstadt y Halldorf., 1990). De manera similar, la proteína PgChP también mostró diferencias en el peso molecular obtenido mediante SDS-PAGE y espectrometría de masas, aunque en esta proteína sí pudo confirmarse la existencia de glicosilaciones (Manuscrito 4). Durante la purificación de la proteína se procedió a su concentración, lo que permitió visualizar dos bandas de 37 y 40 kDa en gel de poliacrilamida (Manuscrito 4, figura 1), la segunda de ellas no detectada anteriormente (Acosta y col., 2009a). Después de tratamientos de deglicosilación con PNGasa F se observó una única banda de 37 kDa en el gel. Estos resultados indican que la cadena proteica posee esta masa molecular y posteriormente es glicosilada, formándose una molécula de 40 kDa. Adicionalmente, la secuenciación aminoacídica de ambas proteínas de 37 y 40 kDa reveló secuencias de aminoácidos idénticas (Tabla 3, manuscrito 4), ratificando que las dos bandas observadas en los geles correspondían a dos isoformas de la misma proteína, una de ellas glicosilada. Posteriormente, un análisis más exhaustivo mediante espectrometría de masas con fuente de ionización por electrospray reveló la existencia de glicosilaciones en PgChP, ya que se detectaron seis isoformas de entre 30 y 31 kDa (Figura V.2) cuyo peso molecular difería en múltiplos de 162 Da, masa que se corresponde con un residuo de galactosa o manosa. 154 MENÚ SALIR DISCUSIÓN Figura V.2. Peso molecular de las seis isoformas de PgChP determinado por ESI-MS. La glicosilación de la proteína se confirmó posteriormente mediante el análisis de la secuencia de aminoácidos que se deduce del gen que codifica PgChP (Manuscrito 5). Así, la diferencia entre el peso molecular de la proteína deducida (29691 Da) y el valor experimental de cada isoforma (30285, 30447, 30610, 30772, 30934 y 31098 Da), corresponde a la parte glicídica de cada una de ellas, lo cual supone un nivel de glicosilación en PgChP que va desde un 1,6 a un 4,5 % dependiendo de la isoforma. Adicionalmente, el análisis tanto de los péptidos obtenidos tras la digestión con tripsina y quimotripsina, como de la secuencia de aminoácidos deducida del gen que codifica PgChP, reveló la presencia de dos secuencias consenso de glicosilación (Morelle y col., 2006) en esta proteína: Asn-Phe-Thr (aminoácidos 230-232) y Asn-Asp-Cys (aminoácidos 288290). La glicosilación representa la forma más común de las modificaciones posttraduccionales en proteínas tanto de eucariotas como de procariotas, donde participan en numerosos procesos biológicos, tales como señalización inter- e intracelular, regulación de proteínas o reconocimiento celular. Los glicanos pueden determinar la localización, actividad y función de las proteínas que los contienen, así como influir en las características fisicoquímicas de las mismas, tales como plegamiento, solubilidad, resistencia a proteasas o aumento de la estabilidad a altas temperaturas (Hildegard y Geyer, 2006; Morelle y col., 2006). De hecho estos grupos glicídicos parecen estar implicados en la actividad antifúngica de algunas proteínas purificadas a partir de plantas (Islam y col., 2002; Deepak y col., 2003, Yamunarani y col., 2005; Kuo y col., 2008). 155 MENÚ SALIR DISCUSIÓN Profundizando en el estudio de las secuencias de aminoácidos deducidas a partir de los genes que codifican la producción de las proteínas PgChP y PgAFP, el análisis con el programa SignalP (Nielsen y col., 1997a) permitió predecir la existencia de péptidos señal o presecuencias en ambas proteínas. Estas secuencias comprenden los primeros 21 (MMTYSRLIPFALFALFGTGLA) y 18 (MQITSIAIVFFAAMGAVA) aminoácidos del extremo N-terminal de PgChP y PgAFP, respectivamente (Manuscritos 2 y 4). En el caso de PgAFP pudo confirmarse además la presencia de una prosecuencia comprendida por los aminoácidos 19 a 34. Este fragmento, que difiere del péptido señal, no está presente en la proteína madura, por lo que no pudo obtenerse mediante la secuenciación aminoacídica de la proteína purificada a partir del medio de cultivo. Se han descrito prosecuencias similares en proteínas con actividad antimicrobiana producidas tanto por mohos (Oka y col., 1990; Wnendt y col., 1994; Marx y col., 1995), como por levaduras (Tao y col., 1990; Schmitt y Breinig, 2002) y bacterias (Bormann y col., 1999), que mantienen inactiva a la proteína en el interior de la célula. De hecho, es necesaria la eliminación de estos fragmentos durante el proceso de secreción al medio para que la proteína adopte una correcta estructura terciaria y adquiera su actividad (Eder y Fersht, 1995). El análisis de las secuencias de aminoácidos deducidas permitió además estimar tanto la masa como el pI de ambas proteínas (Gasteiger y col., 2005). Así, las masas moleculares estimadas de PgChP y PgAFP una vez eliminados los respectivos péptidos señal y la preprosecuencia fueron de 29691 y 6500 Da, respectivamente. Los pIs estimados para PgChP (pH 4,8) y PgAFP (pH 8,8) fueron muy próximos a los de 5,0 y 9,2 respectivamente, determinados por electroforesis en gel de dos dimensiones (Manuscrito 4, figura 5) y mediante electroforesis capilar de isoelectroenfoque (Acosta y col., 2009b). Numerosos autores han relacionado el carácter catiónico con la actividad antifúngica (Broekaert y col., 1992; Nielsen y col., 1997b; Koo y col., 1998; Hong y col., 2001), ya que la presencia de aminoácidos de carga neta positiva sugiere la capacidad de interaccionar con los fosfolípidos aniónicos de membrana de mohos (Sitaram y Nagaraj, 1999) pudiendo provocar alteraciones en la permeabilidad de la misma. Por lo tanto, el punto isoeléctrico de ambas proteínas puede tener una gran relación con la actividad de las mismas, fundamentalmente en el caso de PgChP, ya que presentan una carga neta negativa a pH fisiológico. No obstante, la posible implicación de la carga en la actividad de ambas proteínas se comentará más adelante. 156 MENÚ SALIR DISCUSIÓN V. 2. CARACTERIZACIÓN GENÉTICA Los genes que codifican la producción de PgAFP y de PgChP fueron secuenciados mediante RACE-PCR. Los genes pgafp y pgchp, compuestos por 276 y 912 nucleótidos, codifican los precursores de 92 y 304 aminoácidos, respectivamente (Tabla V.1). En ambos casos el marco de lectura abierto (ORF) está interrumpido por pequeños intrones de 63 y 62 pb en pgafp, y de 60, 53, 59 y 59 pb en pgchp, típicos de genes fúngicos (Gurr y col., 1987). Todos los intrones incluyen en los extremos 5’ y 3’ las secuencias consenso de ensamblamiento o “consensus splice sequences” (Manuscrito 2, figura 5; Manuscrito 5, figura 4), y muchos de ellos además contienen la secuencia interna CTRAC, siendo todas estas secuencias típicas de genes fúngicos (Wiesner y col., 1988). Tabla V.1. Características de los genes pgafp y pgchp. !" * + # $ %&$' # () & '$*$& , - &. / ,/ - , Uno de los factores más importantes para emplear una proteína a nivel industrial es el bajo coste para producirla. Para identificar las condiciones de cultivo ideales y los factores que estimulan su producción es necesario conocer los mecanismos que regulan la expresión de los genes que la codifican. En este sentido, el estudio de la región promotora de los genes proporciona valiosa información sobre las condiciones de expresión de las proteínas que codifican. Así, la caracterización del promotor del gen que codifica para la proteína PAF de P. chrysogenum permitió confirmar la presencia de cuatro sitios de unión a CreA de represión de catabolitos de carbono y dos sitios de unión a AreA de represión de metabolitos de nitrógeno (Marx, 2004). En este tipo de regulación, la transcripción del gen se inhibe en presencia ciertos hidratos de carbono, fundamentalmente glucosa y fructosa (Marx y col., 1995; Vollebregt y col., 1994), y algunos compuestos nitrogenados pueden tanto inhibir como intensificar la producción, como es el caso de glutamina y amonio respectivamente (Marx y col., 1995). Por otra parte, la expresión de la proteína AFP producida por A. giganteus está regulada por otro tipo de factores ambientales como el pH 157 MENÚ SALIR DISCUSIÓN y la presencia de fosfatos (Meyer, 2008). De hecho, el gen afp posee dos secuencias consenso para un factor de regulación por pH (Tilburn y col., 1995) que hacen que la producción de esta proteína se incremente cuando el moho productor se incuba en medios alcalinos (Meyer y Stahl, 2002). La presencia de fosfatos por el contrario provoca una gran disminución de la producción (Meyer y Stahl, 2002). Debido a la influencia que numerosos factores ambientales pueden ejercer sobre la expresión de PgAFP y PgChP, se hace necesario plantear nuevas investigaciones encaminadas a la obtención de la región no codificante de pgafp y pgchp y al estudio de los elementos de regulación presentes en los promotores, lo que permitirá optimizar las condiciones de obtención de ambas proteínas. Por otra parte, ya se están llevando a cabo diferentes ensayos para la expresión de ambos genes en microorganismos con mayor velocidad de crecimiento como Escherichia coli o Pichia pastoris lo que facilitará la obtención rápida de grandes cantidades de proteína, y de esta forma se posibilitará la evaluación para una posible aplicación a nivel industrial. La clonación en E. coli permitirá, en el caso de PgChP, la expresión de la proteína en un microorganismo procariota obteniéndose una proteína recombinante sin glicosilaciones. Esta PgChP recombinante puede resultar de gran interés para evaluar el papel que desempeñan las glicosilaciones en la proteína y si este tipo de modificaciones post-traduccionales son necesarias para su actividad antifúngica. V. 3. COMPARACIÓN DE LA SECUENCIAS AMINOACÍDICAS DE PgAFP Y PgChP CON OTRAS PROTEÍNAS La caracterización a nivel bioquímico de PgChP y PgAFP reveló la existencia de grandes diferencias entre ellas, aún tratándose ambas de proteínas con actividad antifúngica producidas por P. chrysogenum. Así, en el caso de PgChP se trata de una glicoproteína aniónica de unos 30 kDa que en principio muestra escasas características comunes a PgAFP, de sólo 6,5 kDa y marcado carácter catiónico. El análisis de las secuencias tanto aminoacídica como genética y su posterior comparación en bases de datos permitió corroborar la existencia de tales diferencias. De hecho PgChP y PgAFP mostraron semejanzas con diversas proteínas producidas por mohos con actividad quitosanasa o antifúngica respectivamente. PgChP reveló homologías tanto con quitosanasas fúngicas descritas como con secuencias deducidas del genoma de distintos mohos, aunque la mayor similitud se 158 MENÚ SALIR DISCUSIÓN encontró entre PgChP y siete quitosanasas fúngicas con los siguientes números de adquisición en Genbank: XP_754126 (Aspergillus fumigatus), XM_001262949 (N. fischeri), XM_001390407 (Aspergillus niger), BAD08218 (Aspergillus oryzae-1), XP_001824257 (A. oryzae-2), XM_001209034 (Aspergillus terreus) y XP_001274664 (Aspergillus clavatus). PgChP presentó cerca del 60% de homología con las siete proteínas, siendo la más parecida la quitosanasa CSN de A. fumigatus. Las quitosanasas (E.C. 3.2.1.132) son glicosil hidrolasas (glicosidasas) que cortan enlaces glicosídicos del quitosán, y son producidas por virus, bacterias y hongos (Wang y col., 2008a, Wang y col., 2008b). En función de sus secuencias aminoacídicas se han clasificado en cinco familias: GH-5, GH-8, GH-46, GH-75 y GH-80. Las quitosanasas fúngicas están incluidas en la familia GH-75 (Cheng y col., 2005), compuesta principalmente por glicosidasas producidas por mohos de los géneros Aspergillus y Fusarium (Shimosaka y col., 1996; Cantarel y col., 2008). Como era de esperar, la secuencia aminoacídica deducida del gen pgchp presentó numerosas regiones comunes con las siete proteínas que mostraron mayor homología (Manuscrito 5, figura 5), incluyendo tres regiones conservadas en quitosanasas muy relacionadas con su actividad y dos aminoácidos, glutámico y aspártico, esenciales para la función catalítica de este tipo de enzimas, tal como se ha descrito para la quitosanasa de F. solani (Shimosaka y col., 2005). De la misma manera, los diez residuos de cisteína presentes en PgChP están conservados en las siete quitosanasas del alineamiento, y probablemente confieren estabilidad mediante la formación de enlaces disulfuro (Nakaya y col., 1990; Marx y col., 1995; Lee y col., 1999). Por otra parte, en la familia GH-75 existen varias hidrolasas producidas por bacterias del género Streptomyces. PgChP mostró una homología del 25% con quitosanasas deducidas de los genes de S. griseus y S. avermitilis. Dado que se ha descrito que las quitosanasas bacterianas y fúngicas tienen un origen evolutivo diferente (Zhang y col., 2000; Shimosaka y col., 2005), esa similitud se debería a la presencia de una serie de aminoácidos de gran importancia para la estructura y la actividad catalítica del enzima (Monzingo y col., 1996). De hecho los aminoácidos glutámico y aspártico, esenciales para la actividad de las quitosanasas fúngicas (Shimosaka y col., 2005), están presentes también en estas dos proteínas bacterianas (Figura V.3). Por el contrario, las tres secuencias altamente conservadas en quitosanasas fúngicas no parecen tener relación con las homólogas en las secuencias de las proteínas bacterianas (Figura V.3). 159 MENÚ SALIR DISCUSIÓN PgChP A.niger A.oryzae-1 A.oryzae-2 A.fumigatus S.avermitilis S.griseus --MMTYSRLIPFALFALFGTGLAQTVDGSKFNKPDGGPPGSYFAAGSSIPVAALQSAAAK MAFKTTAGLA---FLALAGSVKAQSVDGSKYNSPSNGPPASYFAAATTLPVAALQSAAAK MPIKSFASRLALS-LAICGTAMGQKVNGADYNKPDGGPPAKFFQASSSIPVAAIQAAAAK MPIKSFASRLALS-LAICGTAMGQKVNGADYNKPDGGPPAKFFQASSSIPVAAIQAAAAK MPSTTIIRQLAIS-LALCNSALGQVVNGADYNKPNGGPPASFFAAASTMPVAALQAAAAK MRTRTLTLAAAAGAALLATAALPATASPSGTKAPASA------QEGS-VSAASLLAKVTS MRTRVLAL----TSAACCAALLGAAALPASATGPGAGQEPLAAEAGAPATAAELLAEVRA PgChP A.niger A.oryzae-1 A.oryzae-2 A.fumigatus S.avermitilis S.griseus ARTPVPDATYPINGDNGAKKVTIHSDWAKFDKGAAYVWIADMDVDCDGIDYK-CKGNPDASSVPSKATYPVNTDDDSPKSTIHSDWVKFNQGAALSWVADMDVDCDGIDYK-CKGNGDASKVPSHATYPIG--QGSTKSTIHSDWAGFSEGAAFSFIADMDVDCDGLNHG-CKGNPDASKVPSHATYPIG--QGSTKSTIHSDWAGFSEGAAFSFIADMDVDCDGLNHG-CKGNPDATKVPSLATYPVSQDKGAAKSTIHTDWASFSEGASISWVADMDVDCDGLNSG-CQGNPDCSQI-SNGKY--KTDDETS-ATI----PVCGKNGAVFWKADMDIDCDGQVTGKCNGTTDP CSRI-SKGAY--RTDSGASRATV----PVCGTGDAVFWKADMDIDCDGRETKACSRKTDP PgChP A.niger A.oryzae-1 A.oryzae-2 A.fumigatus S.avermitilis S.griseus -GQHQTNFGA-----LAAYEVPFFVIPD--RFGTKYAKQLPGNNVGAVIC----NGKMFY -GLPETNWGA-----LSAYEVPWIVIPD--QFLTANEDLLPGNNVAAVIC----NGKMYY -GQKETNWGA-----LSAYEVPFIVIPQ--EFLDANKGTLKGNAVAAVIC----NGKMFY -GQKETNWGA-----LSAYEVPFIVIPQ--EFLDANKGTLKGNAVAAVICATSSNGKMFY -GQPQTNWGA-----LSAYEVPFIVIPD--KYLSANSGALPGNNIAAVIC----NGKMFY WFQDDTAFHQSDGKPLRADSLPYVVVPSSSSIWNYASAGIKGGGVVAVIY----NNKVEY YFLPETAFPSSRGEPLDSAVLPHVVVPGASKVWDYRRSGLTGGSVVAVVY----RDRVRY PgChP A.niger A.oryzae-1 A.oryzae-2 A.fumigatus S.avermitilis S.griseus GIYGDSDGDTPQVIGEASWLMARTCFPNDDLNGNSGHGDVDVTYILFTGDDSVLPSSALN GILGDSNGDDPEVTGEASWLMARTCFPDDDLNGAEGHAEADVTCKPYT-------RSALN GIFGDSNGDSPQVTGEASWLMARTCFPKEDLNGNKGHTAADVTYIVFTGDKAVLPSSALN GIFGDSNGDSPQVTGEASWLMARTCFPEEDLNGNKGHTAADVTYIVFTGDKAVLPSSALN GILGDSNGDSPQVTGEASWLMARTCFPNEGLNGNNGHTGVDVTYIVFTGKNAVLPSSALT AVVGDTG--PTKIIGEASYATAQALGIDPD--PETGGTDSGVTYILFKNS-QVSP----GVIGDTG--PTGIIGEASYAMAKTLGIDPD--PSTGGAESGVTYIAFKDS-RVSP----- PgChP A.niger A.oryzae-1 A.oryzae-2 A.fumigatus S.avermitilis S.griseus KNYVTNFTTLRSMGDKLMTALAKNLKLVDGGDGGSP--TTTAGSNPT--SGSCEWEGHCA KNYITNFSTLRSMGDKLVGALASNLGLTSSA---------------SGSTATCSWQGHCE KNYITNFDTLRSMGDSLVGALAKNLNLGGGGGNPPTTLTTTSIPEPTGGSGSCSWPGHCA KNYITNFDTLRSMGDSLVGALAKNLNLGGGGGNPPTTLTTTSIPEPTGGSGSCSWPGHCA KNYITNFTTLRSMGDKLVNALVSNLGLSGTPPTKTTTRVTTTTTKPT-SAASCSWAGHCL ---IESHSAAVSLGDSLAKQFLQNN-------------------------------------IESRARARSRGAERAREFVGR------------------------------------ PgChP A.niger A.oryzae-1 A.oryzae-2 A.fumigatus S.avermitilis S.griseus -----GASCKDE----------------------------------------------------GAICSTEDDCSDDLVCDSGKCSSPDEDDGDDEDDEDEEENDEDDEDDDDE---------GATCSSNDDCSDDLTCQNGKCASDGSAETCSWEGHCKGATCSSNDDCSDELACIS GFKNKGATCSSNDDCSDDLACQNGKCASDGSAETCSWEGHCKGATCSSNDDCSDELACIS -----GASCSSDDDCADALVCTAGKCSVDGA-ATCSWEGHCEGASCSSD--------------------------------------------------------------------------------------------------------------------------------- PgChP A.niger A.oryzae-1 A.oryzae-2 A.fumigatus S.avermitilis S.griseus --------------------------NDCSDQLVCKSGKCPSD --------------------------DDEDDE----------GICSVDNGVETCEWEGHCEGASCSSHDDCDGNLACKNGKCSAGICSVDNGVETCEWEGHCEGASCSSHDDCDGNLACKNGKCSA--------------------------DDCSDDLYCKSGSCTAP ------------------------------------------------------------------------------------- 00 22 00 $1 22 00 $1 , $1 22 Figura V.3. Alineamiento de PgChP con otras quitosanasas fúngicas y bacterianas. Los aminoácidos sombreados son comunes a todas las quitosanasas del alineamiento. Las tres zonas altamente conservadas en quitosanasas fúngicas están englobadas en cuadros. *: Residuos de ácido aspártico y glutámico esenciales para la función catalítica. Números de adquisición de las proteínas: XP_001390407 (A. niger), BAD08218 (A. oryzae1), XP_001824257 (A. oryzae-2), XP_754126 (A. fumigatus), NP_823026 (S. avermitilis) y YP_001822750 (S. griseus). 160 MENÚ SALIR DISCUSIÓN De las quitosanasas fúngicas conocidas, sólo dos están producidas por mohos del género Penicillium, concretamente P. islandicum y P. spinulosum (Fenton y Eveleigh, 1981; Ak y col., 1998), no habiéndose descrito hasta el momento ninguna quitosanasa producida por P. chrysogenum. Por otra parte, aunque son frecuentes las glicosilaciones en proteínas con actividad antifúngica purificadas a partir de plantas (Islam y col., 2002; Deepak y col., 2003; Yamunarani y col., 2005), hasta el momento sólo se ha descrito este tipo de modificación postraduccional en las quitosanasas de Mycobacter AL-1 (Hedges y Wolfe, 1974) y P. islandicum (Fenton y Eveleigh, 1981). Adicionalmente, tanto el peso molecular en torno a 30 kDa como el pI de 5 determinados para PgChP (Manuscrito 4), se encuentran dentro de los rangos descritos para otras quitosanasas, de entre 10 y 50 kDa y pIs entre 4 y 10 (Somashekar y Joseph, 1996). La quitosanasa glicosilada con 30 kDa y pI 4,2 de P. islandicum (Fenton y Eveleigh, 1981) es la más similar a PgChP desde el punto de vista bioquímico. No obstante, hasta el momento se desconoce su secuencia aminoacídica, por lo que no es posible establecer una homología entre ambas proteínas. Por todo ello se puede afirmar que PgChP es una nueva quitosanasa de 30 kDa producida por P. chrysogenum incluida en la familia GH-75. Además, esta proteína ha mostrado actividad antifúngica frente a cepas de interés en alimentos (Acosta y col., 2009a), lo cual no ha sido comprobado en otras quitosanasas. En relación con PgAFP, el análisis de las secuencias tanto aminoacídica como genética y su posterior comparación en bases de datos, permitió descartar la similitud con PgChP. Así, PgAFP mostró homología con un conjunto de proteínas con actividad antifúngica secretadas por mohos, concretamente: PAF de P. chrysogenum (Marx y col., 1995), AFP de A. giganteus (Nakaya y col., 1990; Lacadena y col., 1995), Anafp de A. niger (Lee y col., 1999) y AcAFP de A. clavatus (Skouri-Gargouri y Gargouri, 2008). Estas proteínas tienen una estructura primaria diferente a la descrita en otras proteínas antifúngicas producidas por plantas, invertebrados o anfibios, pero comparten características comunes con ellas, como su pequeño tamaño (51-60 aminoácidos; 5,8-6,7 kDa), alto contenido en residuos de cisteína y un marcado carácter básico debido a la presencia de lisina y arginina (Theis y Stahl, 2004). Adicionalmente, aunque hasta el momento no se han descrito modificaciones post-traduccionales en este tipo de proteínas, sí se han encontrado discrepancias en el peso molecular determinado mediante distintas técnicas. Concretamente PAF corresponde a una banda de unos 12 kDa en SDS-PAGE 161 MENÚ SALIR DISCUSIÓN (Marx y col., 1995), pero a partir de la secuencia aminoacídica deducida del gen su peso sólo se estima en 6,3 kDa (Marx, 2004); esta diferencia de masa también se aprecia en Anafp y AcAFP (Lee y col., 1999; Skouri-Gargouri y Gargouri, 2008). Estos resultados son similares a los expuestos anteriormente para PgAFP, si bien estas diferencias podrían atribuirse, como se indicó anteriormente, a su marcado carácter catiónico (Mondstadt y Holldorf 1990). Por lo tanto, PgAFP no sólo muestra una gran similitud en sus secuencias genética y aminoacídica con este tipo de proteínas antifúngicas producidas por mohos, sino que también comparte otra serie de características comunes, ya que se trata de una proteína de 58 aminoácidos, con una masa molecular de 6,5 kDa y pI de 9,2. Además, tal como se recoge en la tabla V.2, esta proteína presenta otras características comunes, como un alto contenido de aminoácidos básicos y seis residuos de cisteína. Tabla V.2. Parámetros de las formas maduras de las proteínas PgAFP, Anafp, PAF, AFP, y las deducidas de Penicillium nalgiovense (NAF), A. niger, Neosartorya fischeri y Giberella zeae. " ! 9 78 " # $ %&$' # 78 " : -# 78 " ;-#< " = / =< $ " = / < / * = / <, +/ /, ,< =/ /, ,< =/ < /, /+ < =/= / < / 7 /= 7 *$#&. "$ = C "% !" # ' ')&$' # 3 4 " 5 "$ ! 4 ' ) ! $# & 7 8 " ' '()$#$&$1 = * " &$ > #! !" ? ! 1"$& / < =/= & @ A ; , - & 4/ "5 - & 4/ $# B / '! - & 4/ & @ A & = @ A,= 46&!"$& Los seis residuos de cisteína presentes en PgAFP están conservados en todas las proteínas antifúngicas de mohos (Marx, 2004), aunque AFP que posee dos residuos adicionales (Wnendt y col., 1994). De hecho, la gran estabilidad mostrada por este tipo de 162 MENÚ SALIR DISCUSIÓN proteínas frente a proteasas, altas temperaturas o pHs extremos (Lacadena y col., 1995) se ha atribuido a la presencia de estos residuos, que estabilizan la estructura terciaria mediante la formación de puentes disulfuro (Campos-Olivas y col., 1995). Con respecto a las proteínas antifúngicas producidas por mohos, sólo han sido caracterizados los genes que codifican AFP y PAF (Wnendt y col., 1994; Marx y col., 1995). Asimismo, se ha descrito un gen homólogo al que codifica PAF en P. nalgiovense, denominándose la proteína deducida NAF, aunque ésta no se ha purificado (Geisen, 2000). El análisis de las secuencias de aminoácidos revela homologías del 31 al 42% entre PAF, AFP y Anafp, mientras que PAF es 100 % idéntica a NAF. De las proteínas descritas y purificadas, Anafp mostró la mayor similitud con PgAFP, concretamente un 79%. Tal como ya se ha indicado en el apartado anterior para PgAFP, tanto AFP como PAF son sintetizadas como preproproteínas, es decir contienen una secuencia péptido señal para su secreción y una prosecuencia que es eliminada antes o durante su liberación al medio (Marx, 2004). Con respecto a Anafp, aunque sólo se ha determinado la secuencia aminoacídica de la proteína madura (Lee y col., 1999), se asume por su gran similitud con el resto que también está codificada como preproproteína (Marx, 2004). En relación a la proteína AcAFP sólo se han descrito los 21 primeros aminoácidos, de los cuales 20 y 12 residuos coinciden con la región amino terminal de AFP y PAF respectivamente. En los últimos años, gracias a la secuenciación del genoma de diferentes mohos, se han ido recogiendo en bases de datos proteínas deducidas que muestran similitud con este grupo de proteínas antifúngicas. De hecho PgAFP, al igual que PAF, AFP y Anafp, ha mostrado homologías con proteínas deducidas de N. fischeri y G. zeae (Manuscrito 2, figura 6). Una de estas proteínas deducidas, concretamente del genoma de A. niger, ha mostrado junto a Anafp una alta similitud con PgAFP, con un 75 y 79 % de homología, respectivamente. Por otra parte, a pesar de que tanto PgAFP como PAF son producidas por cepas de P. chrysogenum sólo mostraron un 43 % de homología, por lo que se trata de proteínas diferentes. Por último, las secuencias que codifican las proteínas PgAFP y PgChP se han localizado en el genoma de P. chrysogenum Wisconsin 54-1255 cuya secuencia completa ha sido determinada recientemente (Van den Berg et al., 2008). 163 MENÚ SALIR DISCUSIÓN V. 4. MECANISMO DE ACCIÓN DE LA ACTIVIDAD ANTIFÚNGICA DE PgAFP Y PgChP Conocer el mecanismo de acción de proteínas antifúngicas como PgChP y PgAFP puede ser de gran utilidad para el desarrollo de nuevas estrategias antimicrobianas y para determinar su posible aplicación en determinados productos. Los mecanismos de acción de estas proteínas son tan variados como los organismos que las producen, e incluyen degradación de polímeros de las paredes celulares fúngicas, formación de canales o poros en la membrana, inactivación de ribosomas, alteración de la síntesis de ADN o inhibición del ciclo celular (Selitrennikoff, 2001). No obstante, aún se desconoce el modelo de acción de muchos de estos compuestos. En cualquier caso, las proteínas antifúngicas de mayor utilidad son las que presentan mecanismos de acción específicos para las membranas de los hongos (Groll y col., 1998), ya que suelen ser poco tóxicas para las células de mamíferos. Los factores que desempeñan un papel más destacado en la interacción del péptido con la membrana son la carga (Hoover y col., 2003) y la hidrofobicidad del compuesto (Friedrich y col., 1999; Hancock y Chapple, 1999). De hecho, el carácter catiónico se ha relacionado de manera directa con la actividad antifúngica (Broekaert y col., 1992; Nielsen y col., 1997b; Koo y col., 1998; Hong y col., 2001). La base de la especificidad de este tipo de proteínas catiónicas es la composición lipídica de la membrana, ya que al estar cargadas positivamente pueden interaccionar con los fosfolípidos aniónicos (Sitaram y Nagaraj, 1999) presentes en las membranas de bacterias y hongos. Por el contrario, en el caso de los eucariotas superiores, esta unión es muy débil, ya que sus membranas están compuestas principalmente por fosfolípidos sin cargas negativas (Theis y Stahl, 2004). El hecho de que tanto PgChP como PgAFP muestren actividad antifúngica pero no produzcan hemólisis de glóbulos rojos de mamíferos, puede apuntar a un mecanismo de acción específico para las membranas de mohos. Aunque la composición de la pared celular fúngica varía en función del género y la especie, los polímeros más frecuentes son quitina, 1,3-glucano y 1,6-glucano. Otros polímeros como el quitosán sólo se han descrito en un limitado número de hongos de los géneros Rhizopus, Absidia, Fusarium o Mucor (Price y Storck, 1975; Alfonso y col., 1995; Gao y col., 1995). Se ha atribuido un papel de defensa a las quitosanasas por su capacidad de hidrolizar quitosán desestabilizando la pared celular (Somashekar y Joseph, 1996), y en algunos casos se ha relacionado directamente la actividad antifúngica con la actividad 164 MENÚ SALIR DISCUSIÓN quitosanasa (Saito y col., 2009). Sin embargo, distintos compuestos y tratamientos térmicos provocaron efectos diferentes sobre la actividad quitosanasa y antifúngica de la proteína PgChP, lo que podría significar que la actividad antifúngica no se debe a la degradación del quitosán. Además, PgChP mostró actividad frente a P. commune, especie en la que hasta el momento no se ha descrito la presencia de quitosán. Por otra parte, tanto PgChP como PgAFP poseen carga neta positiva al pH de 4,5 empleado para evaluar la actividad antifúngica. Esta característica, junto a la capacidad de formar estructuras anfipáticas, es común a todas las proteínas que actúan sobre las membranas microbianas, aun presentando éstas estructuras primarias muy diversas y secundarias tanto en hoja- como en α-hélice (Tossi y col., 2000). Se ha descrito un mecanismo de acción para algunas de estas proteínas, el modelo Shai-Mat-suzaki-Huang (SMH), que propone la interacción de éstas con los fosfolípidos de carga neta negativa presentes en la membrana plasmática de bacterias y hongos provocando la formación de poros en la misma (Matsuaki, 1999; Shai, 1999; Yang y col., 2000). Entre estas proteínas se encuentran algunas defensinas, que son un grupo de proteínas y péptidos antimicrobianos producidos por mamíferos (Ganz y col., 1990), insectos (Lamberty y col., 1999) o plantas (Broekaert y col., 1995), muy similares a las proteínas antifúngicas producidas por mohos en cuanto a su pequeño tamaño, carácter catiónico y presencia de puentes disulfuro en su estructura. Sin embargo las defensinas producidas por mamíferos e insectos, de menor tamaño (3 a 5 kDa), forman canales iónicos en la membrana plasmática provocando la pérdida de minerales y metabolitos esenciales (Lehrer y col., 1989; White y col., 1995). Adicionalmente, algunas defensinas de plantas producen una desporalización de la membrana como consecuencia de su unión a los fosfolípidos aniónicos (Thevissen y col., 1999). No obstante, PgAFP no presenta actividad frente a bacterias y levaduras (Acosta y col., 2009b). Estos datos coinciden con lo descrito hasta ahora para las proteínas antifúngicas más similares a PgAFP, como PAF (Kaiserer y col., 2003), AFP (Lacadena y col., 1995; Theis y col., 2003) y AcAFP (Skouri-Gargouri y Gargouri, 2008). Anafp en cambio, es la única proteína de este grupo activa frente a levaduras (Lee y col., 1999), si bien hasta el momento se desconoce su mecanismo de acción. La mayoría de estas proteínas además de no mostrar actividad frente a levaduras y bacterias, manifiestan diferentes espectros de inhibición frente a mohos (Marx, 2004). Por ello, tal como se ha descrito para algunas defensinas de plantas (Thevissen y col., 1997; Thomma y col., 2002), se ha propuesto un mecanismo de acción basado en la existencia de receptores específicos 165 MENÚ SALIR DISCUSIÓN para cada proteína en las membranas plasmáticas de los mohos sensibles (Marx y col., 2007; Meyer, 2008). Esta idea se ve apoyada por el hecho de que la actividad de PAF y AFP es fuertemente inhibida en presencia de Mg2+ y K+ (Theis y col., 2003) y Ca2+, Na+ y K+ (Kaiserer y col., 2003), respectivamente, ya que estos cationes podrían saturar los receptores existentes en las membranas de los mohos sensibles impidiendo la unión de las proteínas antifúngicas y con ello su actuación (Theis y col., 2003). Al igual que ha sido descrito para PAF y AFP, tanto PgChP como PgAFP han mostrado diferente sensibilidad a los cationes presentes en el medio. Así, aunque altas concentraciones de Mg2+ y Ca2+ inhiben la actividad antifúngica de ambas proteínas, los cationes monovalentes Na+ y K+ únicamente afectan a la actividad de PgAFP (Manuscritos 3 y 6). Concretamente, al combinar una pequeña cantidad de proteína (3µg/ml) con una alta concentración de iones (100 mM) se aprecia una pérdida de actividad, aunque sólo frente a A. niger. Por lo tanto, el mecanismo de acción de PgChP y PgAFP podría basarse en la existencia de receptores específicos en las membranas de los mohos sensibles, ausentes en levaduras o bacterias. Estos receptores tendrían mayor afinidad por los cationes divalentes y además diferirían en función del moho sensible, siendo por ejemplo los receptores para PgAFP en A. niger más afines a Na+ y K+ que los presentes en P. restrictum. Por otra parte, aunque el mecanismo de acción de PAF y AFP podría estar mediado por la presencia de receptores específicos en la membrana, ambas proteínas ejercen efectos diferentes sobre los mohos sensibles. Concretamente, la unión de PAF con el receptor específico incrementa la permeabilidad al K+, provocando una hiperpolarización de la membrana que conlleva la entrada de la proteína mediante endocitosis. A partir de ese momento se activan distintos mecanismos, como la producción de especies reactivas de oxígeno o mitoptosis (formación de poros en las membranas mitocondriales), que ocasionan la muerte celular programada del moho sensible (Marx y col., 2007). A pesar de las similitudes con PAF, el efecto de AFP es diferente, ya que sólo los mohos resistentes son capaces de introducirla por endocitosis posiblemente para una degradación con fines nutricionales. La unión de AFP a los receptores específicos en los mohos sensibles, provoca tanto alteraciones en la membrana como la inhibición de la enzima quitín sintasa que interviene en la biosíntesis de la pared celular fúngica (Hagen y col., 2007; Meyer, 2008). La proteína AFP posee además en su extremo amino terminal la secuencia de unión a la quitina CKYKAQ (Hagen y col., 2007), no descrita en PAF, Anafp, PgAFP, ni PgChP. 166 MENÚ SALIR DISCUSIÓN Por todo ello, aunque la información disponible sugiere que el mecanismo de acción de PgAFP y PgChP está basado en la interacción específica con receptores presentes en las membranas fúngicas, sería necesario confirmarlo, y en su caso estudiar tanto los receptores implicados, como el efecto que las proteínas ejercen tras la unión con los mismos. V. 5. APLICACIONES DE LAS CEPAS DE P. chrysogenum Y LAS PROTEÍNAS PgAFP Y PgChP Como ya se ha descrito en esta memoria, las condiciones ecológicas que ofrecen los productos cárnicos madurados favorecen el desarrollo de una población fúngica superficial, que aunque puede contribuir a la maduración (Martín y col., 2004, 2006), también puede originar defectos o alteraciones (Sanz, 1986; Spotti y col., 1988) e incluso producir micotoxinas (Núñez y col., 1996, 2007; López-Díaz y col., 2001; Sabater-Vilar y col., 2003). Para controlar la población fúngica podrían utilizarse cultivos iniciadores que al implantarse en el producto fuesen capaces de inhibir por competición a los mohos no deseados. No obstante, la evolución de las condiciones ecológicas a lo largo de la maduración puede hacer que los cultivos terminen siendo desplazados por otras cepas mejor adaptadas. En cambio para los aislamientos de P. chrysogenum RP42C y AS51D, la producción de las proteínas PgAFP y PgChP con actividad antifúngica, puede suponer una ventaja adicional para competir frente al resto de mohos. De hecho, tanto PgAFP como PgChP se purificaron como resultado de la búsqueda de mohos productores de proteínas antifúngicas para su utilización como cultivos protectores en productos cárnicos madurados (Acosta, 2006). Además, ambas cepas han sido aisladas de jamón curado, por lo que se trata de mohos adaptados a las condiciones de temperatura, actividad de agua y pH que se dan a lo largo del proceso de maduración. Adicionalmente, P. chrysogenum ha sido propuesto como cultivo iniciador para la elaboración de productos madurados por su contribución a la formación de sabor y aroma en el producto acabado (Martín y col., 2004, 2006; Benito y col., 2005). Dado que P. chrysogenum RP42C produce proteína activa en condiciones similares a las de las etapas iniciales de la maduración (Acosta, 2006), será de gran interés confirmar la producción tanto de PgAFP como de PgChP en productos madurados. Además, el amplio espectro de inhibición mostrado fundamentalmente por P. chrysogenum RP42C frente a mohos toxigénicos de interés en estos productos, como A. flavus, A. parasiticus o P. commune, permite ser optimistas respecto a su utilización como cultivo protector para garantizar la seguridad alimentaria en este tipo de alimentos. 167 MENÚ SALIR DISCUSIÓN No obstante, también podrían emplearse ambas proteínas purificadas en productos en los que no sea deseable el desarrollo de mohos. Además PgChP, debido a su actividad quitosanasa, podría también emplearse para la producción de oligómeros de quitosán a nivel industrial. De forma general y como paso previo a la aplicación de una proteína a nivel industrial, resulta de gran importancia garantizar que la actividad antifúngica se mantiene en presencia de determinados compuestos que puedan estar presentes en el producto donde se vaya a aplicar (Theis y Stahl, 2004). Adicionalmente el efecto de ciertos tratamientos a temperaturas o pHs extremos puede condicionar también sus posibles aplicaciones. Por otra parte, la posibilidad de incrementar la actividad de estas proteínas mediante la adición de determinados sustratos es otro factor a tener en cuenta antes de proponer futuras aplicaciones. V. 5. 1. APLICACIÓN DE PgChP Y PgAFP COMO AGENTES ANTIFÚNGICOS Como se ha comentado anteriormente, no sólo resulta de gran interés la posibilidad de utilizar mohos productores de proteínas antifúngicas como cultivos protectores en productos madurados, sino que también puede ser útil el uso de PgAFP y PgChP como aditivo en alimentos en los que no sea deseable el desarrollo del moho productor. El uso de proteínas antimicrobianas como protectores en alimentos no es un concepto nuevo. Concretamente la nisina, bacteriocina producida por varias cepas de Lactococcus lactis, ha sido aprobada como GRAS (“Generally Regarded as Safe”) en ocho países de la Unión Europea. Esta proteína es ampliamente utilizada en productos lácteos, vegetales, carne o pescado (Delves-Broughton y col., 1996) para evitar el desarrollo de bacterias patógenas gram positivas como Bacillus cereus, Listeria monocytogenes o determinadas especies de Enterococcus, Streptococcus y Staphylococcus (Lubelski y col., 2008). Además, la nisina ha sido comercializada para el tratamiento de infecciones gástricas causadas por Helicobacter spp. y algunas de sus variantes están siendo probadas en ensayos clínicos para tratar enfermedades producidas por enterococos resistentes a la vancomicina (Reddy y col., 2004). No obstante, antes de proponer a PgAFP y PgChP para su utilización como compuestos antifúngicos en alimentos se deben evaluar los posibles efectos indeseables que pudieran ocasionar en los consumidores para evitar cualquier peligro de tipo sanitario. Por otra parte, la estabilidad de las mismas tras el tratamiento a diferentes temperaturas y 168 MENÚ SALIR DISCUSIÓN pHs o en presencia de determinados compuestos que se encuentren en los sustratos donde se pretendan utilizar, condicionará en gran medida sus posibles aplicaciones. Como se ha mencionado, tanto PgChP como PgAFP fueron inicialmente purificadas mediante cromatografía de intercambio catiónico (Acosta y col, 2009a) con el objeto de obtener proteínas con carácter básico, ya que esta característica está relacionada con una mayor actividad antifúngica y una menor toxicidad para las células de mamíferos (Hong y col., 2001). No obstante, si bien PgAFP mostró un pI de 9,2 (Acosta y col., 2009b), PgChP mostró un pI de 5 (Manuscrito 4, figura 6), lo que supone que esta proteína no es catiónica a pH fisiológico. La ausencia de actividad hemolítica de PgChP y PgAFP frente a glóbulos rojos (Manuscritos 3 y 6), y en el caso de PgAFP también de toxicidad para larvas de Artemia salina (Manuscrito 3), permite ser optimistas hasta el momento con respecto a la inocuidad de estas proteínas, ya que estas técnicas son comúnmente empleadas para chequear la toxicidad de nuevas sustancias (Van’t Hof y col., 2001; Theis y Stahl, 2004; Milhem y col., 2008; Chohan, 2009). La ausencia de efectos negativos podría permitir la utilización de PgAFP y PgChP en diversos campos de aplicación, si bien el presente estudio se ha enfocado hacia una posible aplicación en alimentos. Por ello, se evaluó el efecto sobre la actividad antifúngica tanto de diferentes tratamientos como de diversos compuestos comúnmente encontrados en alimentos, como sales, detergentes o fosfatos (Tabla V.3; Manuscritos 3 y 6). 169 MENÚ SALIR DISCUSIÓN Tabla V.3. Resistencia de PgAFP y PgChP a tratamientos térmicos, distintos pHs, cationes, fosfato sódico, quelantes y detergentes en las proteínas, expresada como los valores máximos a los que se mantiene actividad antifúngica. D E: / F G $ = E: / $ ' HI 7 I I : G I G > / / > 7 F G J J = "$! @G L L M& ' 5$&14$& G K GGG K GGG GGG' GGG & GGG GGG GGG a : Máxima concentración empleada 100 mM. : Máxima concentración empleada 1 mM. c : Máxima concentración empleada 1 %. d ---: Pérdida de la actividad antifúngica de la proteína. b n.c.: Afectó al crecimiento del moho y su influencia no pudo determinarse. n.d.: No determinado. Muchos productos alimentarios se someten de forma rutinaria a tratamientos térmicos, ya sea como consecuencia del propio proceso de elaboración o con el objeto de garantizar su estabilidad microbiológica, lo que condiciona en gran medida la aplicación de ambas proteínas. En este sentido, tanto PgChP como PgAFP mostraron actividad antifúngica incluso tras tratamientos térmicos a 80 y 100ºC, respectivamente. Dado que concentraciones de Ca2+ de 10 a 100 mM pueden inhibir la actividad de estas proteínas, su uso se vería limitado en alimentos ricos en calcio como el queso. Si embargo, la actividad de PgAFP y PgChP se mantuvo en presencia de altas concentraciones de Na+ y K+, lo que puede ser de gran interés en productos cárnicos madurados, donde abunda el cloruro sódico y puede haber otras sales de sodio y potasio. 170 MENÚ SALIR DISCUSIÓN Además, estas proteínas muestran una mayor resistencia a estos iones que otras como PAF o AFP, cuya actividad ya se reduce en presencia de 40-60 mM de Na+ (Kaiserer y col., 2003) o Na+ y K+ (Theis y col., 2003), respectivamente. Otras sustancias como el EDTA y los fosfatos se utilizan como aditivos, entre otras razones, por su capacidad de secuestrar metales en alimentos no ácidos que requieren aditivos sin sabor (Calvo, 1991). Si bien los quelantes no tuvieron un gran impacto en la actividad antifúngica de PgChP y PgAFP, ésta se redujo en presencia de una alta concentración (100 mM) de fosfato, aunque esta disminución, en el caso de PgAFP, depende de la concentración de la proteína y del moho sensible utilizado. El uso de detergentes no iónicos como emulsionantes también está extendido en la industria alimentaria. De hecho, en España se emplean en confitería, repostería y para la elaboración de galletas (Calvo, 1991), si bien de los detergentes evaluados en este estudio sólo tween 20 y tween 80 están autorizados en alimentos (E-432 y E-433, respectivamente). Los detergentes inhibieron la actividad antifúngica de PgChP, pero PgAFP se mantuvo activa con un 1% de tween 20 o de tween 80. Dado que esta concentración supera los niveles habituales en los alimentos de 0,1-0,3% (R.D. 142/2002), la presencia de estos aditivos no afectaría a la actividad de PgAFP. Además de la utilización de PgAFP y PgChP de forma individual, la combinación de ambas o con algún tratamiento físico o químico podría suponer un efecto sinérgico que incrementara la actividad antifúngica en los alimentos. Adicionalmente, aunque PgAFP haya mostrado un espectro de inhibición muy amplio (Acosta y col, 2009b), el uso combinado con PgChP podría ampliar la actividad a mohos que posean quitosán en la pared celular como Mucor o Rhizopus (Shimosaka y col., 2005; Somashekar y Joseph, 1996), géneros alterantes en productos cárnicos madurados. Por otra parte, la estabilidad mostrada por ambas proteínas al tratamiento con temperaturas y pHs extremos abre un gran abanico de posibilidades respecto de su combinación con diferentes tratamientos físicos o químicos. La combinación de compuestos antimicrobianos con tratamientos térmicos, químicos o físicos como las altas presiones o los campos eléctricos pulsados, está siendo ampliamente estudiada. Así, la nisina ha sido combinada con lisozima y campos eléctricos pulsados (Sobrino-López y Martín-Belloso, 2008), con tratamientos de frío (Cao-Hoang y col., 2008), con calor y acidificación (Naim y col., 2008), o con ácido láctico y monolaurina (Tokarsky y Marshall, 171 MENÚ SALIR DISCUSIÓN 2008) para controlar diversos patógenos en alimentos. Combinaciones similares no sólo pueden suponer un incremento en la actividad de PgAFP y PgChP, sino que también podrían modificar su especificidad. De hecho la proteína antifúngica cecropina B, que por sí sola no tiene efecto frente a E. coli, en combinación con lisozima es eficaz para combatir este microorganismo (Casteels y col., 1993). Por todo lo anterior, PgChP y PgAFP parecen tener características idóneas para ser empleadas como agentes protectores en diversos alimentos que requieran un control frente a mohos alterantes o toxigénicos. No es previsible que la utilización de estas proteínas como aditivo provoque alteraciones en la flora intestinal beneficiosa del consumidor ya que ambas poseen un mecanismo de acción específico frente a mohos. Por otra parte, estudios realizados con PgAFP muestran la sensibilidad de esta proteína a tripsina, pepsina y lisozima (Acosta, 2006), por lo que podría ser degradada en el tracto gastrointestinal. No obstante, la aplicación de PgAFP y PgChP no tiene por qué estar limitada a un uso en alimentos. De hecho se ha propuesto la utilización de proteínas antifúngicas similares en otros campos como la agricultura o en terapéutica humana (Marx y col., 2007; Meyer, 2008). En este sentido, la incorporación de genes que codifican proteínas antifúngicas ha permitido la obtención de cultivos transgénicos resistentes a plagas de hongos (Campo y col., 2008; Swathi-Anyradha y col., 2008) y ciertas proteínas antifúngicas están siendo ya estudiadas para su uso como agentes farmacológicos (Cappelletty y Eiselstein-McHitrick, 2007; Wiederhold y Lewis, 2007; Fortún y col., 2009). V. 5. 2. APLICACIÓN DE PgChP COMO QUITOSANASA Dado que la caracterización aminoacídica de PgChP reveló que pertenecía a la familia G-75 de quitosanasas, fue necesario comprobar su capacidad para hidrolizar quitosán. Aunque algunas quitosanasas pueden hidrolizar polisacáridos distintos al quitosán (Pelletier y Sygusch, 1990; Shimosaka y col., 1995; Kimoto y col., 2002), PgChP no presentó indicios de degradación de la quitina (Manuscrito 6), como suele ser habitual entre las quitosanasas (Fukamizo y Brzezinski, 1997; Su y col., 2006). La quitina es el componente estructural mayoritario del exoesqueleto de invertebrados y de las paredes celulares fúngicas (Lower, 1984; Ornum, 1992). Debido a que la degradación de la quitina es muy lenta, la acumulación de desechos resultantes del procesado de crustáceos es un problema emergente en la industria de productos de la pesca 172 MENÚ SALIR DISCUSIÓN (Shahidi y Synowiecki, 1991). No obstante, estos desechos pueden ser empleados para la producción de sustancias muy beneficiosas a nivel industrial, como son la quitina, el quitosán, o los productos de la degradación de los mismos. De hecho, los oligómeros resultantes de la degradación del quitosán tienen múltiples aplicaciones: como inhibidores de la producción de micotoxinas (Cuero y col., 1991), purificadores de agua (Jeuniaux, 1986; Muzzarelli y col., 1989), neutralizantes de acidez en zumos de fruta y vino (Chen y Li, 1996; Rwan y Wu, 1996; Spagna y col., 1996), e incluso como agentes antitumorales y potenciadores del sistema inmune (Pae y col., 2001). Adicionalmente, y como resultado de la búsqueda de nuevos antimicrobianos naturales, los oligómeros del quitosán han mostrado actividad antimicrobiana frente a bacterias, levaduras y mohos (Yalpani y col., 1992). El quitosán puede ser degradado mediante hidrólisis química o enzimática, obteniéndose oligómeros de menor (monómeros-trímeros) o mayor grado de polimerización (pentámeros-heptámeros), respectivamente. No obstante, los oligómeros resultantes de una hidrólisis enzimática con quitosanasas muestran mejores características funcionales (Kendra y Hadwiger, 1984; Uchida y col., 1988), lo que ha incrementado el interés por este tipo de enzimas. Dado que las quitosanasas difieren en sus propiedades enzimáticas (Wang y col., 2008b), es necesario conocer el efecto sobre PgChP de diversos factores como iones o detergentes para potenciar su actividad. Los iones Ca2+ y K+ no tuvieron efecto alguno sobre la actividad quitosanasa de PgChP a concentraciones de 100 mM, mientras que Mg2+ y Na+ sí influyeron en la actividad por encima de 10 mM. En el caso de los quelantes, aunque la actividad se redujo en presencia de una alta concentración (1 mM) de EDTA, con EGTA se llegó a triplicar la cantidad de glucosamina liberada (Manuscrito 6). La influencia de altas concentraciones de Mg2+ y Na+ sobre PgChP puede deberse a que los iones afecten a la estructura de la proteína impidiendo su actividad, tal y como ha sido descrito para otras quitosanasas de mohos (Wang y col., 2008b). De hecho, el tratamiento con SDS y ácido deoxicólico, detergentes iónicos con conocido efecto desnaturalizante, produjo una inhibición de la actividad de PgChP. Por el contrario los detergentes no iónicos, fundamentalmente el Tritón X-100, favorecieron la actividad de la proteína. De forma similar, en presencia de fosfato (10 mM) se duplicó la cantidad de glucosamina liberada. 173 MENÚ SALIR DISCUSIÓN En conclusión, la producción de oligómeros de quitosán mediante hidrólisis con PgChP puede incrementarse en presencia de EGTA, Tritón X-100 y fosfato, siendo las concentraciones más apropiadas 0,1 mM, 1 % y 10 mM, respectivamente. La proteína podría además ser empleada en presencia de altos niveles Ca2+ y K+. No obstante, la actividad quitosanasa de PgChP debe ser evaluada a diferentes temperaturas y pHs con el fin de encontrar las condiciones óptimas para la degradación del quitosán. 174 MENÚ SALIR VI. CONCLUSIONES MENÚ SALIR CONCLUSIONES VI. CONCLUSIONES 1. PgAFP es una nueva proteína antifúngica de 6,5 kDa, en la que no se han detectado glicosilaciones. De acuerdo con su secuencia primaria, pertenece a un grupo de proteínas antifúngicas de pequeño tamaño, básicas y con alto contenido en cisteína, que son producidas por mohos. 2. La proteína antifúngica PgChP presenta diversas isoformas glicosiladas con pesos moleculares comprendidos entre 30 y 31 kDa y, de acuerdo con su secuencia aminoacídica y su actividad enzimática, es una nueva quitosanasa perteneciente a la familia GH-75, mayoritariamente compuesta por quitosanasas fúngicas. 3. Las secuencias aminoacídicas deducidas de los genes que codifican estas dos proteínas revelan la existencia de un péptido señal en ambas y una prosecuencia en PgAFP que no están presentes en las proteínas purificadas. 4. La ausencia de actividad hemolítica y de toxicidad frente a larvas de Artemia salina, junto con la reconocida inocuidad de P. chrysogenum, permite plantear la aplicación de estas dos proteínas en alimentos. 5. Ambas proteínas antifúngicas muestran una gran estabilidad a tratamientos térmicos, y mantienen su actividad en presencia de quelantes o fosfato sódico a las concentraciones que pueden encontrarse en alimentos. 6. La actividad antifúngica de las dos proteínas sólo se ve limitada en presencia de altas concentraciones de iones divalentes o de detergentes iónicos, si bien los detergentes no iónicos afectan a la proteína PgChP. Por lo tanto, estas proteínas pueden ser de utilidad en un amplio rango de alimentos. 7. La actividad quitosanasa de PgChP es independiente de la actividad antifúngica y no se ve limitada por altas concentraciones de iones de calcio y detergentes no iónicos. 8. Las proteínas PgAFP y PgChP muestran características adecuadas para controlar el crecimiento de mohos indeseables en alimentos. 175 MENÚ SALIR CONCLUSIONES VI. CONCLUSIONS 1. PgAFP is a novel antifungal protein of 6.5 kDa with no glycosylations. According to its primary sequence, it belongs to a group of small, basic, and cystein-rich antifungal proteins produced by moulds. 2. The antifungal protein PgChP has several glycosylated isoforms with molecular weights ranging from 30 to 31 kDa. According to primary sequence and enzymatic activity, it is a novel chitosanase belonging to the GH-75 family, mainly composed by fungal chitosanases. 3. The putative amino acid sequences reveal a signal peptide in both proteins and one prosequence in PgAFP that are not found in the purified proteins. 4. The lack of haemolytic activity and toxicity against Artemia salina larvaes together with the safety granted to P. chrysogenum support the potential application of these two proteins in foods. 5. Both antifungal proteins show a high thermal stability and keep their activity with chelators or sodium phosphate at the concentrations found in foods. 6. The antifungal activity of the two proteins is only restricted by high concentrations of divalent ions or ionic detergents, whereas non-ionic detergents only affect to PgChP. Therefore, these two proteins can be of use in a wide range of foods. 7. The chitosanase activity of PgChP is not related to the antifungal activity, and it is restricted by neither high calcium ion concentrations nor non-ionic detergents. 8. Both proteins show appropriate characteristics to control growth of unwanted moulds in foods. 176 MENÚ SALIR VII. BIBLIOGRAFÍA MENÚ SALIR BIBLIOGRAFÍA VII. BIBLIOGRAFÍA Acosta, R. Tesis Doctoral: “Selección de Penicillium productores de péptidos antifúngicos para su utilización en productos cárnicos madurados”. Universidad de Extremadura (2006). Acosta, R., Rodríguez-Martín, A., Martín, A., Núñez, F. y Asensio, M.A. Selection of antifungal protein-producing molds from dry-cured meat products. Int. J. Food Microbiol. Submitted (2009a). Acosta, R., Rodríguez-Martín, A., Bermúdez, E., Núñez, F. y Asensio, M.A. Partial characterization and antifungal activity of a protein from Penicillium chrysogenum. Food Microbiol. Submitted (2009b). Ak, O., Bakir, U. y Guray, T. Production, purification and characterization of chitosanase from Penicillium Spinulosum. Biochem. Arch. 14: 221-225 (1998). Alfonso, C., Nuero, O.M., Santamaria, F. y Reyes, F. Purification of a heat-stable chitin deacetylase from Aspergillus nidulans and its role in cell wall degradation. Curr. Microbiol. 30: 49-54 (1995). Almeida, M.S., Cabral, K.M.S., Zingali, R.B. y Kurtenbach, E. Characterization of two novel defense peptides from pea (Pisum sativum) seeds. Arch. Biochem. Biophy. 378 (2): 278-286 (2000). Alonso, M. Efecto de la utilización de cultivos iniciadores de Penicillium chrysogenum Pg222, Debaryomyces hansenii Dh345 y Staphylococcus xylosus Sx5EA en productos cárnicos. Tesis Doctoral. Universidad de Extremadura, España (2004). Anaissie, E.J. Diagnosis and therapy of fungal infection in patients with leukemia-new drugs and immunotherapy. Best Pract. Res. Clin. Haematol. 21: 683-90 (2008). Andersen, S.J. y Frisvad, J.C. Penicillin production by Penicillium nalgiovense. Lett. Appl. Microbiol. 19: 486-488 (1994). Ashcroft, A.E. Recent developments in electrospray ionisation mass spectrometry: noncovalently bound protein complexes. Nat. Prod. Rep. 22: 452-464 (2005). Bagger, L., Hoffmann, V., Fuglsang, C. y Westh, P. Glycoprotein-surfactant interactions: a calorimetric and spectroscopic investigation of the phytase-SDS system. Biophys. chem. 129(2-3): 251-8 (2007). Barbieri, G., Bolzoni, L., Parolari, G., Virgili, R., Careri, M. y Mangia, A. Flavor compounds of dry-cured hams. J. Agric. Food Chem. 40: 2389- 2394 (1992). 177 MENÚ SALIR BIBLIOGRAFÍA Benito, M.J., Rodríguez, M., Núñez, F., Asensio, M.A., Bermúdez, M.E. y Córdoba, J.J. Purification and Characterization of an Extracellular Protease from Penicillium chrysogenum Pg222 Active against Meat Proteins. Appl. Microbiol. 68: 3532-3536 (2002). Benito, M.J., Córdoba, J.J., Alonso, M., Asensio, M.A. y Núñez, F. Hydrolytic activity of Penicillium chrysogenum Pg222 on pork myofibrillar proteins. Int. J. Food Microbiol. 89: 155-161 (2003). Benito, M.J., Núñez, F., Córdoba, M.G., Martín, A. y Córdoba, J.J. Generation of non-protein nitrogen and volatile compounds by Penicillium chrysogenum Pg222 activity on pork myofibrillar proteins. Food Microbiol. 22 (6): 513-519 (2005). Berg, J.M., Stryer, L., Tymoczko, J. y Macarulla, J.M. Biochemistry. Editorial Reverté, sexta edición (2008). Berwal, J.S. y Dincho, D. Molds as protective cultures for raw dry sausages. J. Food Prot. 58: 817-819 (1995). Bonnerjea, J., Oh, S., Hoare, M. y Dunnell, P. Protein purification: The right step at the right time. Biotechnol. 4: 954-958 (1986). Bormann, C., Baier,D., Horr, I., Raps, C., Berger, J., Jung,G. y Schwarz, H. Characterization of a novel, antifungal, chitin-binding protein from Streptomyces tendae Tu901 that interferes with growth polarity. J. Bacteriol. 181: 7421–7429 (1999). Bremmelgaard, A. Truslen fra multiresistente mikroorganismer. Ugeskrift for Læger. 160: 6329-6344 (1998). Broekaert, W.F., Mariën, W., Terras, F.R.G., De Bolle, M.F.C., Proost, P., Van Damme, J., Dillen, L., Claeys, M., Rees, S.B., Vanderleyden, J. y Cammue, B.P.A. Antimicrobial peptides from Amaranthus caudatus seeds with sequence homology to the cysteine/glycine-rich domain of chitin-binding proteins. Biochemistry. 31: 4308-4314 (1992). Broekaert, W.F., Terras, F.R., Cammue, B.P. y Osborn, R.W. Plant defensins: novel antimicrobial peptides as components of host defense system. Plant Physiol. 108: 1353-1358 (1995). Bruna, J.M., Ordóñez, J.A., Fernández, M., Herranz, B. y de la Hoz, L. Microbial y physico-chemical changes during the ripening of dry fermented sausages superficially inoculated with or having added an intracellular cell-free extract of Penicillium aurantiogriseum. Meat Sci. 59: 87-96 (2001). 178 MENÚ SALIR BIBLIOGRAFÍA Bruna, J.M., Hierro, E.M., de la Hoz, L., Mottramb, D.S., Fernández, M. y Ordóñez, J.A. Changes in selected biochemical and sensory parameters as affected by the superficial inoculation of Penicillium camemberti on dry fermented sausages. Int. J. Food Microbiol. 85: 111-125 (2003). Bullerman, L.B., Hartman, P.A. y Ayres, J.C. Aflatoxin production in meats. II. Aged dry salamis and aged country cured hams. Appl. Microbiol. 18: 718-722 (1969). Burgos, J. Las decoloraciones en los productos cárnicos. Cárnica 2000. Abril: 49-56 (1981). Calvo, M. Aditivos Alimentarios. Propiedades, aplicaciones y efectos sobre la salud. Mira Editores (1991). Campo, S., Manrique, S., García-Martínez, J. y San Segundo, B. Production of cecropin A in transgenic rice plants has an impact on host gene expression. Plant Biotechnol. J. 6: 585-608 (2008). Campos-Olivas, R., Bruix, M., Santoro, J., Lacadena, L. y Martínez del Pozo, A., Gavilanes, J.G., Rico, M. NMR solution structure of the antifungal protein from Aspergillus giganteus: evidence for cysteine pairing isomerism. Biochemistry. 34: 3009-3021 (1995). Cantarel, B.L., Coutinho, P.M., Rancurel, C., Bernard, T., Lombard, V. y Henrissat, B. The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics. Nucleic Acids Res. 37(Database issue):D233-8. (2008). Cao-Hoang, L., Marechal, P.A., Le-Thanh, M. y Gervais, P. Synergistic action of rapid chilling and nisin on the inactivation of Escherichia coli. Appl. Microbiol. Biotechnol. 79: 105-109 (2008). Cappelletty, D. y Eiselstein-McKitrick, K. The echinocandins. Pharmacotherapy. 27: 369-88 (2007). Casteels, P., Ampe, C., Jacobs, F. y Tempst, P. Functional and chemical characterization of hymenoptaecin, an antimicrobial polypeptide that is infection-inducible in the honeybee (Apis mellifera). J. Biol. Chem. 268: 7044–7054 (1993). Castellá, M.M., Summer, S.S., Bullerman, L.B. Stability of fumonisins in thermally processed corn products. J. Food Prot. 61: 1030-1033 (1998). Chen, Y., Li, C. Studies on the application of chitosan to clarification of grapefruit juice. Food Sci. 23: 617-628 (1996). Cheng, C., Chang, C., Wu, Y. y Li, Y. Exploration of glycosyl hydrolase family 75, a chitosanase from Aspergillus fumigatus. J. Biol. Chem. 281: 3137-3144 (2005). 179 MENÚ SALIR BIBLIOGRAFÍA Chohan, Z.H. Synthesis of organometallic-based biologically active compounds: In vitro antibacterial, antifungal and cytotoxic properties of some sulfonamide incorporated ferrocences. J. Enzyme Inhib. Med. Chem. 24: 169-75 (2009). Chu, K.T., Liu, K.H. y Ng, T.B. Cicerarin, a novel antifungal peptide from the green chickpea. Peptides. 24: 659-663 (2003). Claverol, S., Burlet-Schiltz, O., Gairin, J.E. y Monsarrat, B. Characterization of protein variants and post-translational modifications: ESI-MS analyses of intact proteins eluted from polyacrylamide gels. Mol. Cell. Proteomics. 2: 483-493 (2003). Cociancich, S., Ghazi, A., Hetru, A., Hoffman, J.A. y Letellier, L. Insect defensin, an inducible antibacterial peptide, forms voltage-dependent channels in Micrococcus luteus. J. Biol. Chem. 268: 19239-19245 (1993). Cuero, R.G., Osuji, G. y Washington, A. N-Carboxymethyl chitosan inhibition of aflatoxin production: role of zinc. Biotech. Lett. 13: 441-444 (1991). Decker, M. y Nielsen, P.V. The inhibitory effect of Penicillium camemberti and Geotruchum candidum on the associated funga of white mould cheese. Int. J. Food Microbiol. 104: 51-60 (2005). Deepak, A.V., Thippeswamy, G., Shivakameshwari, M.N. y Salimath, B.P. Isolation and characterization of a 29-kDa glycoprotein with antifungal activity from bulbs of Urginea indica. Biochem. Biophys. Res. Comm. 311: 735-742 (2003). de la Torre, P., Meyer, D.K. y Reboli, A.C. Anidulafungin: a novel echinocandin for candida infections. Future Microbiol. 3: 593-601 (2008). Delves-Broughton, J., Blackburn, P., Evans, R.J. y Hugenholz, J. Applications of the bacteriocin, nisin. Anton. Van Leeuw. 69: 193-202 (1996). Eckardt, C., Ramming, G., Trapper, D. y Leistner, L. Vorkommen toxinogener Penicillium-Arten bei Lebens- und futtermittein. Jahresber. Bundesanst. Fleischforsch. Kulmbach, C. 24-25 (1979). Eder, J. y Fersht, A. R. Pro-sequence-assisted protein folding. Mol. Microbiol. 16: 609– 614 (1995). Edlind, T., Smith, L., Henry, K., Katiyar, S. y Nickels, J. Antifungal activity in Saccharomyces cerevisiae is modulated by calcium signalling. Mol. Microbiol. 46(1): 257-268 (2002). Escher, F.E., Koehler, P.E. y Ayres, J.C. Production of ochratoxins A and B on country cured ham. Appl. Microbiol. 26: 27-30 (1973). 180 MENÚ SALIR BIBLIOGRAFÍA Fenton, D. M. y Eveleigh, D. E. Purification and mode of action of a chitosanase from Penicillium islandicum. J. Gen. Microbiol. 126: 151-165 (1981). Filtenborg, O., Frisvad, J.C. y Thrane, U. Moulds in food spoilage. Int. J. Food Microbiol. 33: 85-102 (1996). Fortún, J., Martín-Dávila, P., Montejo, M., Muñoz, P., Cisneros, J.M., Ramos, A., Aragón, C., Blanes, M., San Juan, R., Gavaldá, J., Llinares, P. y GESITRA Study Group. Prophylaxis with caspofungin for invasive fungal infections in highrisk liver transplant recipients. Transplantation. 87: 424-35 (2009). Friedrich, C., Scott, M.G., Karunaratne, N., Yan, H. y Hancock, R.E.W. Salt-Resistant alpha-helical cationic antimicrobial peptides. Antimicrob. Agents Chemother. 43: 1542-1548 (1999). Frisvad, J.C. y Filtenborg, O. Secondary metabolites as consistent criteria in Penicillium taxonomy and a synoptic key to Penicillium subgenus Penicillium. En: Modern Concepts in Penicillium y Aspergillus Classification. Eds.: R.A. Samson y J.I. Pitt. Plenum Press. New York. pp.: 373-384 (1990). Frisvad, J.C. y Thrane, U. Mycotoxin production by food-borne fungi. En: Introduction to Food-borne Fungi. 4th ed. Eds.: R.A. Samson, E.S. Hoekstra, J.C. Frisvad y O. Filtenborg. Centraalbureau voor Schimmelcultures. Baarn, Netherlands. Pp.: 251-260 (1995). Fuentes Arderiu, X., Castiñeiras Lacambra, M.J. y Queraltó Compañó, J.M. Bioquímica Clínica y Patología Molecular: Volumen 1. Ediciones Reverté (1998). Fukamizo, T. y Brzezinski, R. Chitosanase from Streptomyces sp. strain N174: a comparative review of its structure and function. Biochem. Cell. Biol. 75: 687-696 (1997). Gálvez, A., Abriouel, H., Lucas López, R. y Omar, N.B. Bacteriocin-based strategies for food biopreservation. Int. J. Food Micriobiol. 120: 51-70 (2007). Ganz, T., Selsted, M.E. y Lehrer, R.I. Defensins. Eur. J. Haematol. 44: 1-8 (1990). Gao, X.D., Katsumoto, T. y Onodera, K. Purification and characterization of chitin deacetylase from Absidia coerulea. J. Biochem. 117: 257-263 (1995). García, C., Berdagué, J.J., Antequera, T., López-Bote, C., Córdoba, J.J. y Ventanas, J. Volatile compounds of dry cured Iberian ham. Food Chem. 41: 23-32 (1991). Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M.R., Appel, R.D. y Bairoch, A. Protein identification and analysis tools on the expasy server; 181 MENÚ SALIR BIBLIOGRAFÍA (In) John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press. Full text - Copyright Humana Press (2005). Geisen, R., Luecke, F.K. y Kroeckel, L. Starter and protective cultures for meat and meat products. Fleischwirtsch. 72: 894-901 (1992). Geisen, R. Fungal starters cultures for fermented food: molecular aspects. Trends Food Sci. Technol. 4: 251-256 (1993). Geisen, R. P. nalgiovense carries a gene which is homologous to the paf gene of P. chrysogenum which codes for an antifungal peptide. Int. J. Food Microbiol. 62: 95101 (2000). Gil, C. La metodología proteómica, una herramienta para la búsqueda de función. Sociedad Española de Microbiología. 35: 12-20 (2004). Girlanda, M., Bianciotto, V., Cappellazzo, G.A., Casieri, L., Bergero, R., Martino, E., Luppi, A.M. y Perotto, S. Interactions between engineered tomato plants expressing antifungal enzymes and nontarget fungi in the rhizosphere and phyllosphere. FEMS Microbiol. Lett. 288: 9-18 (2008). Gomes, V.M., Carvalho, A.O., Da Cunha, M., Keller, M.N., Bloch Jr., C., Deolindo, P. y Alves, E.W. Purification and characterization of a novel peptide with antifungal activity from Bothrops jararaca venom. Toxicon. 45(7): 817-27 (2005). González, A.M., Presa, M., Latorre, M.G. y Lurá, M.C. Detection of fungal metabolites showing toxic activity through Artemia salina bioassay. Rev. Iberoam. Micol. 24(1): 59-61 (2007). Graham, L.S. y Sticklen, M.B. Plant chitinases. Can. J. Bot. 72: 1057-1083 (1994). Groll, A.H., De Lucca, A.J. y Walsh, T.J.. Emerging targets for the development of novel antifungal therapeutics. Trends Microbiol. 6: 117-124 (1998). Guerrero, I., Ponce, E. y Prado, A. Changes in flavour attributes during ripening of fermented sausages. J. Muscle Foods. 6: 257-269 (1995). Gurr, S.J., Uncles, S.E. y Kinghorn, J.R. The structure and organization of nuclear genes of filamentous fungi. In: Kinghorn, J.R. (Ed.). Gene structure in Eukaryotic Microbes. IRL Press, Oxford, pp. 93-193 (1987). Hagen, S., Marx, F., Ram, A.F. y Meyer, V. The antifungal protein AFP from Aspergillus giganteus inhibits chitin synthesis in sensitive fungi. Appl. Environ. Microbiol. 73: 2128-2134 (2007). Halls, N.A. y Ayres, J.C. Potential production of sterigmatocystin on country cured ham. Appl. Microbiol. 26: 636-637 (1973). 182 MENÚ SALIR BIBLIOGRAFÍA Hancock, R.E.W. y Chapple, D.S. Peptide antibiotics. Antimicrob. Agents Chemother. 43: 1317-1323 (1999). Harighi, M. J., Zamani, M. R. y Motallebi, M. Evaluation of antifungal activity of purified chitinase 42 from Trichoderma atroviride PTCC5220. Biotech. 6: 28-33 (2007). Hedges, A. y Wolfe, R. S. Extracellular enzyme from Myxobacter Al-1 that exhibits both -1,4 glucanase and chitosanase activities. J. Bact. 120: 844-853 (1974). Hickey, R.M., Twomey, D.P., Ross, R.P. y Hill, C. Production of enterolysin A by a raw milk enterococcal isolate exhibiting multiple virulence factors. Microbiology. 149: 655-64 (2003). Hildegard, G. y Geyer, R. Strategies for analysis of glycoprotein glycosylation. Bioch. Bioph. 1764: 1853-1869 (2006). Holland, R.D. y Sepaniak, M.J. Qualitative analysis of mycotoxins using micellar electrokinetic capillary chromatography. Anal. Chem. 65: 1140- 1146 (1993). Hong, S.Y., Park, T.G. y Lee, K.H. The effect of charge increase on the specificity and activity of a short antimicrobial peptide. Peptides. 22: 1669- 1674 (2001). Hoover, D.M., Wu, Z., Tucker, K., Lu, W. y Lubkowski, J. Antimicrobial characterization of human -defensin 3 derivatives. Antimicrob. Agents Chemother. 47: 2804-2809 (2003). Huerta, T., Sanchis, V., Hernández, J. y Hernández, E. Mycoflora of dry-salted spanish ham. Proceedings of the ICoMST. 2: 1047-1050 (1987). Hwang, P.M. y Vogel, H.J. Structure-function relationships of antimicrobial peptides. Biochem. Cell. Biol. 76: 235-246 (1998). Hwu, L., Huang, C.C., Chen, D.T. y Lin, A. The action mode of the ribosomeinactivating protein -sarcin. J. Biomed. Sci. 7: 420-428 (2000). Islam, S. Z., Honda, Y., Sawa, Y. y Babadoost, M. Characterization of antifungal glycoprotein in red-light-irradiated broadbean leaflets. Mycoscience. 43: 471-473 (2002). Jacobsen, T. y Hinrichsen, L. Bioformation of flavour by Penicillium candidum on glucose, peptone, maize oil and meat extract. Food Chem. 3: 409-416 (1997). Jarvis, B. Mould and mycotoxins in mouldy cheeses. Microbiol. Alim. Nutr. 1: 187-191 (1983). 183 MENÚ SALIR BIBLIOGRAFÍA Jeuniaux, C. Chitosan as a tool for the purification of waters. Chitin in nature and technology. (Muzzarelli, R.A.A., Jeuniaux, C., Gooday, G.W., eds), Pg. 551-570. Plenum Press, New York, USA (1986). Jin, L.L., Song, S.S., Li, Q., Chen, Y.H., Wang, Q.Y. y Hou, S.T. Identification and characterisation of a novel antimicrobial polypeptide from the skin secretion of a Chinese frog (Rana chensinensis). Int. J. Antimicrob. Agents. (en prensa) (2009). Kaiserer, L., Oberparleiter, C., Weiler-Görz, R., Burgstaller, W., Leiter, E. y Marx, F. Characterization of the Penicillium chrysogenum antifungal protein PAF. Arch. Microbiol. 180: 204-210 (2003). Katta, S.K., Jackson, L.S., Summer, S.S., Hanna, M.A. y Bullerman, L.B. Effect of temperature and screw speed on stability of fumonisin B1 in extrusion cooked corn grits. Cereal Chem. 76: 16-20 (1999). Kendra, D.F. y Hadwiger, L.A. Characterization of the smallest chitosan oligomer that is maximally antifungal to Fusarium solani and elicits pisatin formation in Pisum sativum. Exp. Mycol. 8: 276-281 (1984). Kimoto, H., Kusaoke, H., Yamamoto, I., Fujii, Y., Onodera, T. y Taketo, A. Biochemical and genetic properties of Paenibacillus Glycosyl hydrolase having chitosanase activity and discoidin domain. J. Biol. Chem. 277: 14695-14702 (2002). Kinsella, J.E. y Hwang, D.H. Enzymes of Penicillium roqueforti involved in the biosynthesis of cheese flavor. Crit. Rev. Food Sci. Nutr. 12: 191- 228 (1976). Koo, J.C., Lee, S.Y., Chun, H.J., Cheong, Y.H., Choi, J.S., Kawabata, S.I., Miyagi, M., Tsunasawa, S., Ha, K.S., Bae, D.W., Han, C.D., Lee, B.L. y Cho, M.J. Two hevein homologs isolated from the seed of Pharbitis nil L. exhibit potent antifungal activity. Biochim. Biophys. Acta. 1382: 80-90 (1998). Kuo, C-J., Liao, Y-C., Yang, J-H., Huang, L-C., Chang, C-T. y Sung, H-Y. Cloning and characterization of an antifungal class III chitinase from suspension-cultured bamboo (Bambusa oldhamii) cells. J. Agric. Food Chem 56(23):11507-11514 (2008). Lacadena, J., Martínez del Pozo, A., Gasset, M., Patiño, B., Campos-Olivas, R., Vázquez, C., Martínez-Ruiz, A., Mancheño, J.M., Oñaderra, M. y Gavilanes, J.G. Characterization of the antifungal protein secreted by the mould Aspergillus giganteus. Arch. Biochem. Biophys. 20: 273-281 (1995). Lafont, P., Siriwardana, M.G., Combemale, I. y Lafont, J. Mycophenolic acid in marketed cheeses. Food Cosmet. Toxicol. 17: 147-149 (1979). 184 MENÚ SALIR BIBLIOGRAFÍA Lamberty, M., Ades, S., Uttenweiler-Joseph, S., Brookhart, G., Bushey, D., Hoffmann, J.A. y Bulet, P. Insect inmunity. Isolation from the lepidopteran Heliothis virescens of a novel insect defensin with potent antifungal activity. J. Biol. Chem. 274: 9320-9326 (1999). Le Bars, J. Cyclopiazonic acid production by Penicillium camemberti Thom and natural occurrence of this mycotoxin in cheese. Appl. Env. Microbiol. 38: 1052-1055 (1979). Lee, D.G., Shin, S.Y., Maeng, C-Y., Jin, Z.Z., Kim, K.L. y Hahm, K-S. Isolation and characterization of a novel antifungal peptide from Aspergillus niger. Biochem. Biophy. Res. Commu. 263: 646-651 (1999). Lee, D.G., Kim, D.H., Park, Y., Kim, H.K., Kim, H.N., Shin, Y.K, Choi, C.H. y Hahm, K.S. Fungicidal effect of antimicrobial peptide, PMAP-23, isolated from porcine myeloid against Candida albicans. Biochem. Biophys. Res. Commun. 282: 570-574 (2001). Lehrer, R.I., Barton, A., Daher, K.D., Harwig, S.S., Ganz, T. y Selsted, M.E. Interaction of human defensins with Escherichia coli. Mechanism of bactericidal activity. J. Clin. Investig. 84: 553-561 (1989). Leistner, L. Toxinogenic penicillia occurring in feeds and foods: a review. Food Technol. Aust. 36: 404-406 (1984). Leistner, L. Starter and protective cultures for foods in Europe. Microbes for better living, Micon 94 and 35th AMI Conference. 417-423 (1994). Liepe, H.U. Starter cultures in meat production. Biotechnology. Eds.: H.J. Rehm y G. Reeds. Verlag-Chemie, Weinheim. 5: 399-424 (1982). Liu, Y., Tortora, G., Ryan, M. E., Lee, H-M. y Golub, L. M. Potato dextrose agar antifungal susceptibility testing for yeast and molds: evaluation of phosphate effect on antifungal activity of CMT-3. Antimic. Agents. Chemoth. 46: 1455-1461 (2002). López-Díaz, T. M., Santos, J. A., García-López, M. L. y Otero, A. Surface mycoflora of a Spanish fermented meat sausage and toxigenicity of Penicillium isolates. Int. J. Food Microbiol. 68: 69-74 (2001). Lower, S.E. Polymers from the sea chitin and chitosan. Manufacturing Chemist. 55: 73-75 (1984). Lubelski, J., Rink, R., Khusainov, G.N. y Kulpers, O.P. Biosynthesis, immunity, regulation, mode of action and engineering of the model lantibiotic nisin. Cell. Mol. Life. Sci. 65: 455-476 (2008). 185 MENÚ SALIR BIBLIOGRAFÍA Ludemann, V., Pose, G., Pollio, L.M. y Segure, J. Determination of growth characteristics lipolytic and proteolytic activities of Penicillium strains isolated from Argentinean salami. Int. J. Food Microbiol. 96: 13-18 (2004). Marth, E.H. y Yousef, A.E. Fungi and dairy products. Handbook of Applied Mycology, Foods and Feeds. Eds.: Arora, D.K.; Mukerji, K.G. y Marth, E.H. Marcel Dekker. New York. 3: 375-414 (1991). Martín, A., Córdoba, J.J., Rodríguez, M.M., Núñez, F. y Asensio, M.A. Evaluation of microbial proteolysis in meat products by Capillary Electrophoresis. J. Appl. Microbiol. 89: 163-171 (2001). Martín, A., Asensio, M.A., Bermúdez, M.E., Córdoba, M.G., Aranda, E. y Córdoba, J.J. Proteolytic activity of Penicillium chrysogenum and Debaryomyces hansenii during controlled ripening of pork loins. Meat Sci. 62: 129-137 (2002). Martín, A., Córdoba, J.J., Núñez, F., Benito, M.J. y Asensio, M.A. Contribution of a selected fungal population to proteolysis on dry-cured ham. Int. J. Food Microbiol. 94: 55-66 (2004). Martín, A., Córdoba, J.J., Aranda, E., Córdoba, M.G. y Asensio, M.A. Contribution of a selected fungal population to the volatile compounds on dry-cured ham. Int. J. Food Microbiol. 110(1): 8-18. (2006). Martínez-Ruiz, A., Martínez del Pozo, A., Lacadena, J., Mancheño, J.M., Oñaderra, M. y Gavilanes, J.G. Characterization of a natural larger form of the antifungal protein (AFP) from Aspergillus giganteus. Biochim. Biophys. Act. 1340: 81-87 (1997). Marx, F., Haas, H., Reindl, M., Stöffler, G., Lottspeich, F. y Redl, B. Cloning, structural organization and regulation of expression of the Penicillium chrysogenum paf gene encoding an abundantly secreted protein with antifungal activity. Gene. 167: 167-171 (1995). Marx, F. Small, basic antifungal proteins secreted from filamentous ascomycetes: a comparative study regarding expression, structure, function and potential application. Appl. Microbiol. Biotechnol. 65: 133-142 (2004). Marx, F., Binder, U., Leiter, É. y Pócsi, I. The Penicillium chrysogenum antifungal protein PAF, a promising tool for the development of new antifungal therapies and fungal cell biology studies. Cell. Mol. Life Sci. 65: 445-454 (2008). 186 MENÚ SALIR BIBLIOGRAFÍA Matsuzaki, K. Why and how are peptide-lipid interactions utilized for self defense? magainins and tachyplesins as archetypes. Biochim. Biophys. Acta. 1462: 1-10 (1999). Meyer, V. y Stahl, U. New insights in the regulation of the afp gene encoding the antifungal protein of Aspergillus giganteus. Curr. Genet. 42: 36-42 (2002). Meyer, V. A small protein that fights fungi: AFP as a new promising antifungal agent of biotechnological value. Appl. Microbiol. Biotechnol. 78: 17-28 (2008). Milhem, M. M., Al-hiyasat, A.S. y Darmani, H. Toxicity testing of restorative dental materials using brine shrimp larvae (Artemia Salina). J. Appl. Oral Sci. 16: 297-301 (2008). Mislivec, P.B., Trucksess, M.W. y Stoloff, L. Effect of other toxigenic mold species on aflatoxin production by Aspergillus flavus in sterile broth shake culture. J. Food Prot. 51: 449-451 (1988). Molimard, P., Vassal, L., Bouvier, I. y Spinnler, H.E. Suivi de croissance de Penicillium camemberti et Geotrichum candidum en culture pure et en association au cours de l`affinage de fromages expérimentaux á pâte molle de type camembert. Lait. 75 : 316 (1995). Molina, I., Nieto, P., Flores, J., Silla, H. y Bermell, S. Study of the microbial flora of dry cured ham. 5. Lipolytic activity. Fleischwirtsch. 71: 906- 913 (1991). Mondstadt, G. M. y Holldorf, A. W. Arginine deaminase from Halobacterium. salinarum: purification and properties. Biochem. J. 273: 739-745 (1990). Monzingo, A.F., Marcotte, E.M., Hart, P.J. y Robertus, J.D. Chitinases, chitosanases, and lysozymes can be divided into procaryotic and eucaryotic families sharing a conserved core. Nat. Struc. Biol. 3: 133-140 (1996). Morelle, W., Canis, K., Chirat, F., Faid, V. y Michalski, J-C. The use of mass spectrometry for the proteomic analysis of glycosylation. Proteomics. 6: 3993-4015 (2006). Moretti, A., Mulè, G., Ritieni, A. y Logriego, A. Further data on the production of beauvericin, enniatins and fusaproliferin and toxicity to Artemia salina by Fusarium species of Gibberella fujikuroi species complex. Int. J. Food Microbiol. 118(2):15863 (2007). Moss, M.O. Fungi, quality and safety issues in fresh fruits and vegetables. J. Appl. Microbiol. 104: 1239–1243 (2008). 187 MENÚ SALIR BIBLIOGRAFÍA Mottram, D.S. y Whitfield, F.B. Maillard-lipid interactions in nonaqueous systems: volatiles from the reaction of cysteine and ribose with phosphatidylcholine. J. Agric. Food Chem. 43: 1302-1306 (1995). Munimbazi, C. y Bullerman, L.B. Isolation and partial characterization of antifungal metabolites of Bacillus pumilus. J. Appl. Microbiol. 84: 959-968 (1998). Murayama, N., Saguchi, K., Mentele, R., Assakura, M.T., Ohi, H., Fujita, Y., Camargo, A.C.M., Higuchi, S. y Serrano, S.M.T.. The unusual high molecular mass of Bothrops protease A, a trypsin-like serine peptidase from the venom of Bothrops jararaca, is due to its high carbohydrate content. Biochim. Biophys. Acta. 1652: 1-6 (2003). Muzzarelli, R.A.A., Weckx, M. y Fillipini, O. Removal of trace metal ions from industrial waters, unclear effluents and drinking water, with the aid of cross-linked N-carboxymethyl chitosan. Carbohydr. Poly. 11: 293-296 (1989). Naeem, A., Fatima, S. y Khan R.H. Characterization of partially folded intermediates of papain in presence of cationic, anionic, and nonionic detergents at low pH. Biopolymers. 83: 1-10 (2006). Naim, F., Zareifard, M.R., Zhu, S., Huizing, R.H., Grabowski, S. y Marcotte, M. Combined effects of heat, nisin and acidification on the inactivation of Clostridium sporogenes spores in carrot-alginate particles: from kinetics to process validation. Food Microbiol. 25: 936-941 (2008). Nakaya, K., Omata, K., Okahashi, I., Nakamura, Y., Kolkenbrock, H. y Ulbrich, N. Amino acid sequence and disulfide bridges of an antifungal protein isolated from Aspergillus giganteus. Eur. J. Biochem. 193: 31-38 (1990). Nelson, C.A. Binding of detergents to proteins: maximum amount of dodecyl sulfate bound to proteins and resistance to binding of several proteins. J. Biol. Chem. 246: 3895-3901 (1971). Ng, T.B., Parkash, A. y Tso, W.W. Purification and characterization of-and-benincasins, arginine/glutamate-rich peptides with translation-inhibiting activity from wax gourd seeds. Peptides. 24: 11-16 (2003). Nielsen, H., Engelbrecht, J., Brunak, S. y Von Heijne, G. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10: 1-6. (1997a). 188 MENÚ SALIR BIBLIOGRAFÍA Nielsen, K.K., Nielsen, J.E., Madrid, S.M. y Mikkelsen, J.D. Characterization of a new antifungal chitin-binding peptide from sugar beet leaves. Plant Physiol. 113: 83-91 (1997b). Nielsen, M., Frisvad, J. y Nielsen, P. Protection by fungal starters against growth and secondary metabolite production of fungal spoilers of cheese. Int. J. Food Microbiol. 42: 91-99 (1998). Nishimura, T. y Kato, H. Taste of free amino acids and peptides. Food Rev. Int. 4: 175194 (1988). Northolt, M.D., Van Egmond, H.P., Soentoro, P. y Deijll, E. Fungal growth and the presence of sterigmatocystin in hard cheese. J. Assoc. Off. Anal. Chem. 63: 115-119 (1980). Núñez, F. Flora fúngica en jamón ibérico y su importancia tecnológica y sanitaria. Tesis Doctoral. Facultad de Veterinaria. Universidad de Extremadura (1995). Núñez, F., Rodríguez, M.M., Bermúdez, M.E., Córdoba, J.J. y Asensio, M.A. Composition and toxigenic potential of the mould population on dry-cured ham. Int. J.Food Microbiol. 32: 185-197 (1996). Núñez, F., Westphal, C.D., Bermúdez, E. y Asensio, M.A. Production of secondary metabolites by some terverticillate penicillia on carbohydrate-rich and meat substrates. J. Food Prot. 70(12): 2829-36 (2007). Odds, F.C., Brown, A.J.P. y Gow, N.A.R. Antifungal agents: mechanisms of action. Trends Microbiol. 11: 272-279 (2003). Oka, T., Natori, Y., Tanaka, S., Tsurugi, K. y Endo, Y. Complete nucleotide sequence of cDNA for the cytotoxin alpha sarcin. Nucleic Acids Res. 18: 1897 (1990). Ornum, V.J. Shrimp waste-must it be wasted. Infofish Int. 6: 48-52 (1992). Osborn, R.W., De Samblanx, G.W., Thevissen, K., Goderis, I., Torrekens, S., Van Leuven, F., Attenborough, S., Rees, S.B. y Broekaert, W.F. Isolation and characterization of plant defensins from seeds of Asteraceae, Fabaceae, Hippocastanaceae and Saxifragaceae. FEBS Lett. 368: 257-262 (1995). Osborne, B.G., Ibe, F., Brown, G.L., Petagine, F., Scudamore, K.A., Banks, J.N., Hetmanski, N.T. y Leonard, C.T. The effects of milling and processing on wheat contaminated with ochratoxin A. Food Add. Cont. 13: 141- 153 (1996). Pae, H.O., Seo, W.G., Kim, N.Y., Oh, G.S., Kim, G.E., Kim, Y.H., Kwak, H.J., Yun, Y.G., Jun, C.D. y Chung, H.T. Induction of granulocytic differentiation in acute 189 MENÚ SALIR BIBLIOGRAFÍA promyelocytic leukemia cells (HL-60) by water-soluble chitosan oligomer. Leuk. Res. 25: 339-46 (2001). Parker, M.S., Crowley, W.R. y Parker, S.L. Differences in cation sensitivity of ligand binding to Y1 and Y2 subtype of neuropeptide Y receptor of rat brain. Eur. J. Pharmacol. 318(1): 193-200 (1996). Patrzycat, A., Gallant, J.W., Seo, J.K., Pytyck, J. y Douglas, S.E. Novel antimicrobial peptides derived from flatfish genes. Antimicrob. Agents Chemother. 47: 2464-2470 (2003). Pelletier, A. y Sygusch, J. Purificacion and characterization of three chitosanase activities from Bacillus megaterium P1. Appl. Environ. Microbiol. 56: 844-848 (1990). Pemán, J. y Almirante, B. Advances in the diagnosis and treatment of yeast infections: role of the new antifungal agents. Enferm Infecc Microbiol Clin. 13: 38-46 (2008). Perrone, G., Susca, A., Cozzi, G., Ehrlich, K., Varga, J., Frisvad, J.C., Meijer, M., Noonim, P., Mahakarnchanakul, W. y Samson, R.A. Biodiversity of Aspergillus species in some important agricultural products. Stud. Mycol. 59: 53-66 (2007). Pitt, J.I. y Hocking, A.D. Fungi and Food Spoilage, 2nd edn. London: Blackie Academic & Professional (1997). Pitt-Rivers, R. y Ambesi-Impiombato, F.S. The binding of sodium dodecyl sulphate to various proteins. Biochem. J. 109: 825–830 (1968). Price, J. S. y Storck, R. Production, purification, and characterization of an extracellular chitosanase from Streptomyces. J. Bacteriol. 124: 1574-1585 (1975). Real Decreto 142/2002, de 1 de febrero, por el que se aprueba la lista positiva de aditivos distintos de colorantes y edulcorantes para su uso en la elaboración de productos alimenticios, así como sus condiciones de utilización. Reddy, K.V.R., Yedery, R.D. y Aranha, C. Antimicrobial peptides: premises and promises. Int. J. Antimicrobial Agents. 24: 536-547 (2004). Redkar, R.J., Lemke, P.A. y Singh, N.K. Isolation of differentially expressed cDNA clones from salt adapted Aspergillus nidulans. Curr. Genet. 29: 130-135 (1996). Richard, J.L. Some major mycotoxins and their mycotoxicoses- an overview. Int. J. Food Microbiol. 119: 3-10 (2007). Rivas, L. y Andreu, D. Péptidos antibióticos eucarióticos: ¿una nueva alternativa en clínica? Enf. Infeccio. Microbiol. Clínica. 21: 358 – 365 (2003). Rodríguez, M., Núñez, F., Córdoba, J.J., Sanabria, C., Bermúdez, M.E. y Asensio, M.A. Characterization of Staphylococcus spp. and Micrococcus spp. isolated from 190 MENÚ SALIR BIBLIOGRAFÍA Iberian ham throughout the ripening process. Int. J. Food Microbiol. 24: 329-335 (1994). Rodríguez M.M., Nuñez, F., Córdoba, J.J., Bermúdez, M.E. y Asensio, M.A. Evaluation of proteolytic activity of micro-organisms isolated from dry cured ham. J. Appl. Microbiol. 85: 905-912 (1998). Ruiz, J., Cava, R., Ventanas, J. y Jensen, M.T. Headspace solid phase microextraction for the analisis of volatiles in a meat product: dry cured Iberian ham. J. Agric. Food Chem. 46: 4688-4694 (1998). Russ, G. y Poláková, K. The molecular weight determination of proteins and glycoproteins of RNA enveloped viruses by polyacrylamide gel electrophoresis in SDS. Biochem. Biophys. Res. Commun. 55(3): 666–672 (1973). Russeth, K.P., Higgins, L. y Andrews, M.T. Identificacion of proteins from non-model organism using mass spectrometry: aplication to a hibernating mammal. J. Proteome Research. 5: 829-839 (2006). Rwan, J. y Wu., J. Deacification of grapefruit juice with chitosan. Food Sci. 23: 509-519 (1996). Saito, A., Ooya, T., Miyatsuchi, D., Fuchigami, H., Terakado, K., Nakayama, S.Y., Watanabe, T., Nagata, Y. y Ando, A. Molecular characterization and antifungal activity of a family 46 chitosanase from Amycolatopsis sp. CsO-2. FEMS Microbiol. Lett. 293: 79-84 (2009). Sabater-Vilar, M., Nijmeijer, S. y Fink-Gremmels, J. Genotoxicity assessment of five tremorgenic mycotoxins (Fumitremorgen B, Paxilline, Penitrem A, Verruculogen, and Verrucosidin) produced by molds isolated from fermented meats. J. Food Prot. 66: 2123-2129 (2003). Samson, R.A., Hoekstra, E.S., Frisvad, J.C. y Filtenborg, O. Introduction to Foodborne Fungi. 4th edition. Eds.: Samson, R.A.; Hoekstra, E.S.; Frisvad, J.C. y Filtenborg, O. Centraalbureau voor Schimmelcultures. Baarn, Netherlands. pp.: 308312 (1995). Samyn, B., Sergeant, K., Memmi, S., Debyser, G., Devreese, B. y Beeumen, J.V. MALDI-TOF/TOF de novo sequence analysis of 2D PAGE-separated proteins from Halorhodospira halophila, a bacterium with unsequenced genome. Electrophoresis. 27: 2702-2711(2006). Sanz, B. Factores que influyen en sabores y olores del jamón. Cárnica 2000, 34: 57-61 (1986). 191 MENÚ SALIR BIBLIOGRAFÍA Sárraga, C., Gil, M. y García-Regueiro, J.A. Comparison of calpain and capthesin (B, L and D) activities during dry-cured ham processing from heavy and light large white pigs. J. Sci. Food Agric. 62: 71-75 (1993). Schmitt, M. J. y Breinig, F. The viral killer system in yeast: from molecular biology to application. FEMS Microbiol. Lett. 26: 257–276 (2002). Scotto-Lavino, E., Du, G. y Frohman, M.A. 3' end cDNA amplification using classic RACE. Nat. Protoc. 1: 2742-2745 (2006a). Scotto-Lavino, E., Du, G. y Frohman, M.A. 5' end cDNA amplification using classic RACE. Nat. Protoc. 1: 2555-2562 (2006b). Sela-Buurlage, M.B., Ponstein, A.S., Vloemans, S.A., Melchers, L.S., Van den Elzen, P.J.M. y Cornelissen, B.J.C. Only specific tobacco (Nicotinia tabacum) chitinases and -1,3-glucanases exhibit antifungal activity. Plant Physiol. 101: 857-863 (1993). Selgas, M.D., Casas, C., Toledo, V.M. y García, M.L. Effect of selected mould strains on lipolysis in dry fermented sausages. Europ. Food Res. Technol. 209: 360-365 (1999). Selitrennikoff, C.P. Antifungal proteins. Appl. Env. Microbiol. 67: 2883-2894 (2001). Shahidi, F., Rubin, L.J. y D´Souza, L.A. Meat flavour volatiles: A review of the composition, techniques of analysis, and sensory evaluations. CRC. Crit. Rev. Food Sci. Nutr. 24: 141-243 (1986). Shahidi, F. y Synowiecki, J. Isolation and characterization of nutrients and value-added products from snow crab (Chinoecetes Opilio) and shrimp (Pandalus Borealis) processing discards. J. Agric. Food Chem. 39: 1527-1532 (1991). Shai, Y. Mechanism of binding, insertion and destabilization of phospholipid bilayer membranes by α-helical antimicrobial and cell non-selective membrane-lytic peptides. Biochim. Biophys. Acta. 1462: 55-70 (1999). Shimosaka, M., Nogawa, M., Wang, X., Kumehara, M. y Okazaki, M. Production of two chitosanases from a chitosan-assimilating bacterium, Acinetobacter sp. strain CHB101. Appl. Environ. Microbiol. 61: 438-442 (1995). Shimosaka, M., Kumehara, M., Zhang, X., Nogawa, M. y Okazaki, M. Cloning and characterization of a chitosanase gene from the plant pathogenic fungus Fusarium solani. J. Ferment. Bioeng. 82: 426-431 (1996). Shimosaka, M., Sato, K., Nishiwaki, N., Miyazawa, T. y Okazaki, M. Analysis of essential carboxylic amino acid residues for catalytic activity of fungal chitosanases by site-directed mutagenesis. J. Bios. Bioeng. 5: 545-550 (2005). 192 MENÚ SALIR BIBLIOGRAFÍA Siemens, K. y Zawistowski, J. Occurrence of PR imine, a metabolite of Penicillium roqueforti, in blue cheese. J. Food Prot. 56: 317-319 (1993). Singh, B.J. y Dincho, D. Molds as protective cultures for raw dry sausages. J. Food Prot. 57: 928-930 (1994). Sissi, C. y Palumbo, M. Effects of magnesium and related divalent metal ions in topoisomerase structure and function. Nucleic Acids Res. 37(3): 702-11(2009). Sitaram, N. y Nagaraj, R. Interaction of antimicrobial peptides with biological and model membranes: structural and charge requirements for activity. Biochim. Biophys. Acta. 1462: 29-54 (1999). Skouri-Gargouri, H. y Gargouri, A. First isolation of a novel thermostable antifungal peptide secreted by Aspergillus clavatus. Peptides. 29: 1871-1877 (2008). Smith, J.L. y Palumbo, S.A. Use of starter cultures on meat. J. Food Prot. 46: 997-1006 (1983). Sobrino-López, A. y Martín-Belloso, O. Enhancing the lethal effect of high-intensity pulsed electric field in milk by antimicrobial compounds as combined hurdles. J. Dairy Sci. 91: 1759-1768 (2008). Somashekar, D. y Joseph, R. Chitosanases-Properties and applications: a review. Biores. Tech. 55: 35-45 (1996). Sosa, M.J., Córdoba, J.J., Díaz, M., Rodríguez, M.M., Bermúdez, E., Asensio, M.A. y Núñez, F. Production of cyclopiazonic acid by Penicillium commune isolated from dry-cured ham on a meat-extract based substrate. J. Food Prot. 65: 988-992 (2002). Spagna, G., Pifferi, P.G., Rangoni, C., Mattivi, F., Nicolini, G. y Palmonari, R. The stabilization of white wines by adsorption of phenolic compounds on chitin and chitosan. Food Res. Intern. 29: 241-248 (1996). Spotti, E., Mutti, P. y Campanini, M. Indagine microbiologica sul Diffeto dell'acido fenico del prosciutto durante la stagionatura. Ind. Conserve. 63: 343-346 (1988). Standing, K.G. Peptide and protein de novo sequencing by mass spectrometry. Cur. Opinion Struct. Biol. 13: 595-601 (2003). Su, C., Wang, D., Yao, L. y Yu, Z. Purification, characterization, and gene cloning of a chitosanase from Bacillus species strain S65. Agric. Food Chem. 54: 4208-4214 (2006). Suyama, M. y Shimuzu, T. Buffering capacity and taste of carnosine and its methylated compounds. Bull. Jap. Soc. Sci. 48: 89-94 (1982). 193 MENÚ SALIR BIBLIOGRAFÍA Swathi-Anyradha, T., Divya, K., Jami, S.K. y Kirti, P.B. Transgenic tobacco and peanut plants expressing a mustard defensin show resistance to fungal pathogens. Plant Cell Rep. 27: 1777-86 (2008). Szappanos, H., Szigeti, G.P., Pál, B., Rusznák, Z., Szucs, G., Rajnavölgyi, E., Balla, J., Balla, G., Nagy, E., Leiter, E., Pócsi, I., Marx, F. y Csernoch, L. The Penicillium chrysogenum-derived antifungal peptide shows no toxic effects on mammalian cells in the intended therapeutic concentration. Naunyn Schmiedebergs Arch. Pharmacol. 371(2):122-132 (2005). Szappanos, H., Szigeti, G.P., Pál, B., Rusznák, Z., Szucs, G., Rajnavölgyi, E., Balla, J., Balla, G., Nagy, E., Leiter, E., Pócsi, I., Hagen, S., Meyer, V. y Csernoch, L. The antifungal protein AFP secreted by Aspergillus giganteus does not cause detrimental effects on certain mammalian cells. Peptides. 27(7):1717-1725 (2006). Tao, J., Ginsberg, I., Banerjee, N., Held, W., Koltin, Y. y Bruenn, J. A. Ustilago maydis KP6 killer toxin: structure, expression in Saccharomyces cerevisiae, and relationship to other cellular toxins. Mol. Cell. Biol. 10: 1373–1381 (1990). Theis, T., Wedde, M., Meyer, V. y Stahl, U. The antifungal protein from Aspergillus giganteus causes membrane permeabilization. Antimicrobial agents chemo. 47: 588593 (2003). Theis, T. y Stahl, U. Antifungal proteins: targets, mechanisms and prospective applications. Cell Mol Life Sci. 61: 437-55 (2004). Thevissen, K., Osborn, R.W., Acland, D.P. y Broekaert, W.F. Specific, high affinity binding sites for an antifungal plant defensin on Neurospora crassa hyphae and microsomal membranes. J. Biol. Chem. 272: 32176-32181 (1997). Thevissen, K., Terras, F.R.G. y Broekaert, W.F. Permeabilization of fungal membranes by plant defensins inhibits fungal growth. Appl. Env. Microbiol. 65: 5451-5458 (1999). Thomma, B.P., Cammue, B.P. y Thevissen, K. Plant defensins. Planta. 216: 193-202 (2002). Tilburn, J., Sarkar, S., Widdick, D.A., Espeso, E.A., Orejas, M., Mungroo, J., Penalva, M.A. y Arst, H.N. The Aspergillus PacC zinc finger transcription factor mediates regulation of both acid-and alkaline-expressed genes by ambient pH. EMBO. J. 14: 779–790 (1995). 194 MENÚ SALIR BIBLIOGRAFÍA Tokarskyy, O. y Marshall, D.L. Mechanism of synergistic inhibition of Listeria monocytogenes growth by lactic acid, monolaurin, and nisin. Appl. Environ. Microbiol. 74: 7126-9 (2008). Toldrá, F., Miralles, M.C. y Flores J. Protein extractability in dry-cured ham. Food Chem. 44: 391-394 (1992). Toledo, V.M., Selgas, M.D., Casas, C., Fernández, M. y García, M.L. Cambios en la fracción de ácidos grasos libres en embutidos con mohos. X Congreso Nacional de Microbiología de Alimentos. Valencia. pp. 97 (1996). Tomie, T., Ishibashi, J., Furukawa, S., Kobayashi, S., Sawahata, R., Asaoka, A., Tagawa, M. y Yamakawa, M. Scarabaecin, a novel cysteinecontaining antifungal peptide from the rhinoceros beetle, Oryctes rhinoceros. Biochem. Biophys. Res. Commun. 307: 261-266 (2003). Tossi, A., Sandri., L. y Giangaspero, A. Amphipathic, α-helical antimicrobial peptides. Biopolymers. 55: 4-30 (2000). Tozzini, A.C., Ek, B., Palva, E.T. y Hopp, H.E. Potato virus X coat protein: a glycoprotein. Virology. 202: 651-658 (1994). Trigueros, G., García, M.L., Casas, C., Ordóñez, J.A. y Selgas, M.D. Proteolytic y lipolytic activities of moulds strains isolated from Spanish dry fermented sausage. Z. Lebensm. Unters. Forch. 201: 298-305 (1995). Trivedi, S., Gehlot, H.S. y Rao, S.R. Protein thermostability in Archaea and Eubacteria. Genet. Mol. Res. 5(4): 816-27 (2006). Uchida, Y., Izume, M. y Ohtakara, A. Preparation of chitosan oligomers with purified chitosanase and its application. Proceeding from the 4th International Conference on Chitin and Chitosan, pp. 373-382. Trondheim, Norway (1988). Upadhyay, A. y Srivastava, S. Characterization of a new isolate of Pseudomonas fluorescens strain Psd as a potential biocontrol agent. Lett. Appl. Microbiol. 47: 98105 (2008). Valenzuela, M.A., Gamarra, N., Gómez, L., Kettlun, A.M., Sardón, M., Pérez, L.M., Vinagre, J. y Guzman, N.A. A comparative study of fish species identification by gel isoelectrofocusing two-dimensional gel electrophoresis, and capillary zone electrophoresis. J. Capill. Electrophor. Microchip. Technol. 6(3-4): 85-91 (1999). Van den Berg, M.A., Albang, R., Albermann, K., Badger, J.H., Daran, J.M., Driessen, A.J., Garcia-Estrada, C., Fedorova, N.D., Harris, D.M., Heijne, W.H., Joardar, 195 MENÚ SALIR BIBLIOGRAFÍA V., Kiel, J.A., Kovalchuk, A., Martin, J.F., Nierman, W.C., Nijland, J.G., Pronk, J.T., Roubos, J.A., van der Klei, I.J., van Peij,N.N., Veenhuis,M., von Dohren, H., Wagner, C., Wortman, J. y Bovenberg, R.A. Genome sequencing and analysis of the filamentous fungus Penicillium chrysogenum. Nat. Biotechnol. 26: 1161-1168 (2008). Van’t Hof, W., Veerman, E.C.I., Helmerhorst, E.J. y Amerongen, A.V.N. Antimicrobial peptides: properties and applicability. Biol. Chem. 382: 597-619 (2001). Vollebregt, A.W.H., Van Solingen, P. y Bovernberg, R.A.L. Isolation and characterization of gene Y of Penicillium chrysogenum: close relation of the gene product to the antifungal protein of Aspergillus giganteus. Abstr. Eur. Conf. Fungal Genet. 2. (1994). von der Weid, I., Alviano, D.S., Santos, A.L.S., Soares, R.M.A., Alviano, C.S. y Seldin, L. Antimicrobial activity of Paenibacillus peoriae strain NRRL BD-62 against a broad spectrum of phytopathogenic bacteria and fungi. J. Appl. Microbiol. 95: 11431151 (2003). Wang, S.L., Peng, J.H., Liang, T.W. y Liu, K.C. Purification and characterization of a chitosanase from Serratia marcescens TKU011. Carbohydr. Res. 343: 1316-1323 (2008a). Wang, J., Zhou, W., Yuan, H. y Wang, Y. Characterization of a novel fungal chitosanase Csn2 from Gongronella sp. JG. Carbohydr Res. 343: 2583-2588 (2008b). Weber, K. y Osborn, M. The reliability of molecular weight determinations by dodecyl sulphate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244: 4406-4412 (1969). White, S.H., Wimley, W.C. y Selsted, M.E. Structure, function, and membrane integration of defensins. Curr. Opin. Struct. Biol. 5: 521-527 (1995). Whitfield, F.B. Volatiles from interactions of Maillard reactions and lipids. Crit. Rev. Food Sci. Nutr. 31: 1-58 (1992). Wiederhold, N.P. y Lewis, J.S. The echinocandin micafungin: a review of the pharmacology, spectrum of activity, clinical efficacy and safety. Expert. Opin. Pharmacother. 8: 1155-66 (2007). Wiesner, P., Beck, J., Beck, K.F., Ripka, S., Müller, G., Lücke, S. y Schweizer, E. Isolation and sequence analysis of the fatty acid synthetase FAS2 gene from Penicillium patulum. Eur. J. Biochem. 177: 69-79 (1988). 196 MENÚ SALIR BIBLIOGRAFÍA Wnendt, S., Felske-Zech, H., Henze, P. P. C., Ulbrich, N. y Stahl, U. Characterization of the gene encoding alphasarcin, a ribosome-inactivating protein secreted by Aspergillus giganteus. Gene. 124: 239–244 (1993). Wnendt, S., Ulbrich, N. y Stahl, U. Molecular cloning, sequence analysis and expression of the gene encoding an antifungal-protein from Aspergillus giganteus. Curr. Genet. 25: 519-523 (1994). Wu, M.T., Ayres, J.C. y Koehler, P.E. Production of citrinin by Penicillium viridicatum on country-cured ham. Appl. Microbiol. 27: 427-428 (1974). Yalpani, M., Johnson, F. y Robinson, L.E. Antimicrobial activity of some chitosan derivatives. Advances in chitin and chitosan (Brine, C.J., Sandford, P.A., Zikakis, J.P., eds), pp. 543-555. Elsevier Applied Science, London, UK (1992). Yamunarani, K., Jaganathan, R., Bhaskaran, R., Govindaraju, P. y Velazhahan, R. In vitro antifungal activity of a 29-kDa glycoprotein purified from the galls of Quercus infectoria. Acta Phytopath. Entomol. Hungarica. 40: 43-54 (2005). Yan, R., Hou, J., Ding, D., Guan, W., Wang, C., Wu, Z. y Li, M. In vitro antifungal activity and mechanism of action of chitinase against four plant pathogenic fungi. J. Basic Microbiol. 48: 293-301 (2008). Yang, L., Weiss, T.M., Lehrer, R.I. y Huang, H.W. Crystallization of antimicrobial pores in membranes: magainin and protegrin. Biophys. J. 79: 2002- 2009 (2000). Yang, L., Tan, R.X., Wang, Q., Huang, W. y Yin, Y. Antifungal cyclopeptides from Halobacillus litoralis YS3106 of marine origin. Tetrah. Lett. 43: 6545-6548 (2002). Yang, Q., Gong, Z. Purification and characterization of an ethyleneinduced antifungal protein from leaves of guilder rose (Hydrangea macrophylla). Prot. Express. Purif. 24: 76-82 (2002). Zacharius, R.M., Zell, T.E., Morrison, J.H. y Woodlock, J.J. Glycoprotein staining following electrophoresis on acrylamide gels. Anal. Biochem. 30: 148–52 (1969). Zhang, X., Dai, A., Zhang, X. y Kuroiwa, K. Purification and characterization of chitosanase and exo- -D-Glucosaminidase from a Koji Mold, Aspergillus oryzae IAM2660. Biosci. Biotechnol. Biochem. 64: 1896-1902 (2000). Zhou, X.X., Wang, Y.B., Pan, Y.J. y Li, W.F. Differences in amino acids composition and coupling patterns between mesophilic and thermophilic proteins. Amino Acids. 34(1): 25-33 (2007). 197 MENÚ SALIR VIII. RESUMEN MENÚ SALIR RESUMEN VIII. RESUMEN Las condiciones ambientales en que se procesan o se almacenan diversos tipos de alimentos favorecen el desarrollo en su superficie de mohos que tienen la capacidad de adaptarse y desarrollarse en sustratos muy diversos. En productos cárnicos madurados y quesos es frecuente el desarrollo una población fúngica superficial que puede tener efectos deseables, ya que contribuyen en gran medida a la formación del sabor, aroma o textura del producto acabado. Pero el desarrollo de mohos no siempre es deseable en alimentos. Muchos mohos son alterantes, llevando al deterioro de las características sensoriales de los alimentos. No obstante, el aspecto más preocupante respecto del desarrollo de una población fúngica en alimentos es su capacidad para producir micotoxinas. Existen numerosos mecanismos para controlar el desarrollo microbiano en distintos tipos de alimentos, incluyendo métodos físicos y químicos. Estos tratamientos resultan eficaces para evitar el desarrollo de mohos en determinados alimentos, pero no son adecuados para otros productos como los madurados, donde la presencia de ciertos mohos es beneficiosa y además la aplicación de fungicidas está limitada. Una posible solución en este tipo de alimentos sería el empleo de proteínas antifúngicas que evitaran el desarrollo de mohos indeseables y que a la vez permitieran el crecimiento de mohos beneficiosos en los productos madurados. En estudios previos se seleccionaron dos cepas no toxigénicas de P. chrysogenum, RP42C y AS51D, procedentes de jamón curado, por su capacidad para inhibir el desarrollo de mohos toxigénicos frecuentes en alimentos. Además, se purificaron dos proteínas con actividad antifúngica, PgAFP y PgChP a partir de RP42C y AS51D, respectivamente. El objetivo fundamental de este trabajo ha sido la caracterización de las dos proteínas PgAFP y PgChP. Esta caracterización se ha basado en: la determinación de sus secuencias aminoacídicas, la obtención de las secuencias de los genes que las codifican; la evaluación preliminar de su toxicidad; el estudio de la influencia de diversos tratamientos sobre la estabilidad de las proteínas y el efecto sobre la actividad antifúngica de compuestos comúnmente encontrados en alimentos. Pese a que tanto PgAFP como PgChP son proteínas con efecto antifúngico, han resultado ser muy diferentes en cuanto a sus pesos moleculares, puntos isoeléctricos y secuencias aminoacídicas. PgAFP tiene un peso molecular de 6,5 kDa, un punto isoeléctrico determinado previamente de 9,2, y no posee glicosilaciones pese a que 199 MENÚ SALIR RESUMEN estudios preliminares apuntaban a que presentaba esta modificación. Este hecho se confirmó con la obtención de la secuencia del gen que codifica la proteína y la consiguiente determinación de la secuencia aminoacídica deducida. PgChP sin embargo, mostró tener seis isoformas glicosiladas con pesos moleculares comprendidos entre 30 y 31 kDa, separados por una masa de 162 Da que podría corresponder a un residuo de manosa o galactosa. Además, la secuencia deducida del gen permitió encontrar dos posibles sitios de glicosilación (Asn-Phe-Thr y Asn-Asp-Cys) y la presencia de cadenas glucídicas se demostró mediante deglicosilaciones enzimáticas. PgChP además presentó un punto isoeléctrico cercano a 5, por lo que la proteína no es catiónica. Las secuencias obtenidas de los genes pgafp y pgchp que codifican PgAFP y PgChP tuvieron un tamaño de 276 y 912 pb, que codifican 92 y 304 aminoácidos, respectivamente. Además, estos genes presentaron varios intrones pequeños con marcadores típicos de intrones fúngicos. La obtención de las secuencias aminoacídicas permitió encontrar proteínas con cierta similitud en su estructura primaria. Así, PgAFP presentó un 79% de homología con la proteína Anafp de A. niger y PgChP resultó tener cerca del 60% de similitud con la familia de hidrolasas GH-75 compuesta por quitosanasas principalmente fúngicas. Tanto PgAFP como PgChP presentan en su secuencia aminoacídica deducida una parte que corresponde a un péptido señal necesario para que ambas proteínas sean secretadas al medio extracelular. Además PgAFP, al igual que Anafp y otras proteínas con estructura primaria parecida, presenta una prosecuencia, que parece mantener la proteína inactiva para no causar daño a su moho productor, hasta que este fragmento es eliminado durante su secreción. La actividad antifúngica de PgAFP y de PgChP se inhibe por altas concentraciones (100 mM) de ciertos cationes, especialmente los divalentes Ca2+ y Mg2+. Al igual que lo propuesto para otras proteínas como las defensinas, el mecanismo de acción de PgChP y PgAFP podría basarse en la existencia de receptores específicos en las membranas de los mohos sensibles y ausentes en los mohos resistentes. Los cationes podrían saturar estos receptores específicos e impedir que las proteínas se unieran a ellos y ejercieran su acción antifúngica. PgChP muestra actividad hidrolítica sobre quitosán, lo que junto a la homología de su secuencia aminoacídica con otras quitosanasas fúngicas confirma que pertenece a este 200 MENÚ SALIR RESUMEN grupo de enzimas. Esta capacidad de PgChP para degradar quitosán puede hacer que su espectro de inhibición incluya a hongos con quitosán en su pared celular, como los géneros Rhizopus y Mucor, alterantes habituales en alimentos madurados. Además, las quitosanasas son utilizadas en la industria para la degradación de caparazones de moluscos, así como para la obtención de polímeros de quitosán, de alto valor como protectores frente a patógenos en alimentos, inhibidores de micotoxinas, purificadores de agua, etc. Por otro lado, es necesario demostrar la inocuidad antes de proponer la aplicación de cualquier nueva proteína en alimentos. Aunque será necesario ampliar estos estudios, PgAFP y PgChP no mostraron evidencias de toxicidad sobre glóbulos rojos y larvas de Artemia salina a concentraciones superiores a las que poseen actividad antifúngica. De forma general y como paso previo a la aplicación de una proteína a nivel industrial, resulta de gran importancia garantizar que la actividad antifúngica se mantiene en presencia de determinados compuestos que puedan estar presentes en el producto donde se vaya a aplicar. Adicionalmente el efecto de ciertos tratamientos a temperaturas o pHs extremos puede condicionar también sus posibles aplicaciones. Ambas proteínas PgAFP y PgChP mostraron ser estables a altas temperaturas, permaneciendo activas tras ser sometidas a 100 ºC 15 minutos y 80 ºC 20 minutos, respectivamente. PgAFP además mostró ser estable a valores de pH entre 1 y 12. La actividad antifúngica de PgAFP y PgChP se mantuvo en presencia de altas concentraciones (100 mM) de sodio y potasio, lo que puede ser de gran interés en alimentos madurados, donde abunda el cloruro sódico y puede haber otras sales de sodio y potasio. Los quelantes EDTA y EGTA no tuvieron un gran impacto en la actividad antifúngica de PgChP y PgAFP, mientras que el fosfato sódico a alta concentración (100 mM) sí pudo inhibir esta actividad. Los detergentes también inhibieron la actividad antifúngica de PgChP, pero PgAFP se mantuvo activa a concentraciones de tween 20 o de tween 80 más altas a las empleadas en los alimentos. En definitiva, PgAFP y PgChP han mostrado alta estabilidad, han resultado ser inocuas en los ensayos realizados y resisten componentes comúnmente encontrados en alimentos. Aunque es necesario profundizar en su estudio, estas proteínas y las cepas de P. chrysogenum que las producen muestran características adecuadas para su utilización frente a mohos alterantes y toxigénicos en alimentos crudos madurados. 201