IMOBILIZAÇÃO DE LACASE A PARTÍCULAS MAGNÉTICAS DE

Transcription

UNIVERSIDADE FEDERAL DE PERNAMBUCO
CENTRO DE CIÊNCIAS BIOLÓGICAS
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS BIOLÓGICAS
IMOBILIZAÇÃO DE LACASE A PARTÍCULAS
MAGNÉTICAS DE POLISILOXANO-ÁLCOOL
POLIVINÍLICO
ROZIANA CUNHA CAVALCANTI JORDÃO
RECIFE, 2010
UNIVERSIDADE FEDERAL DE PERNAMBUCO
CENTRO DE CIÊNCIAS BIOLÓGICAS
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS BIOLÓGICAS
IMOBILIZAÇÃO DE LACASE A PARTÍCULAS
MAGNÉTICAS DE POLISILOXANO-ÁLCOOL
POLIVINÍLICO
Tese apresentada ao Programa de Pós-Graduação em
Ciências Biológicas, nível Doutorado, como parte dos
requisitos para obtenção do grau de Doutor em Ciências
Biológicas, na área de Biotecnologia pela Universidade
Federal de Pernambuco
Doutoranda: Roziana Cunha Cavalcanti Jordão
Orientador: Prof. Dr. Luiz Bezerra de Carvalho Júnior
RECIFE, 2010
Jordão, Roziana Cunha Cavalcanti
Imobilização de lacase a partículas magnéticas de polisiloxano-álcool
polivínilico/ Roziana Cunha Cavalcanti Jordão. – Recife: O Autor, 2010.
95 folhas : il., fig., tab.
Orientador: Luiz Bezerra de Carvalho Júnior.
Tese (doutorado) – Universidade Federal de Pernambuco.
Centro de Ciências Biológicas. Biotecnologia, 2010.
Inclui bibliografia e anexos.
1. Lacase (enzima) 2. Proteína 3. Fenóis 4. Álcool I. Título.
572.7
CDD (22.ed.)
UFPE/CCB-2010-106
Dedico este trabalho a minha família, aos meus professores, amigos e a todos que de alguma forma contribuíram para meu crescimento pessoal e profissional durante o doutorado. v
SUMÁRIO
AGRADECIMENTOS .................................................................................................... vi
LISTA DE FIGURAS ................................................................................................... viii
LISTA DE TABELAS ..................................................................................................... x
LISTA DE ABREVIAÇÕES .......................................................................................... xi
RESUMO ....................................................................................................................... xii
ABSTRACT .................................................................................................................. xiii
1 INTRODUÇÃO ........................................................................................................... 15
2 REVISÃO DA LITERATURA ................................................................................... 16
2.1 Lacases .................................................................................................................. 16
2.1.1 Origem e distribuição ..................................................................................... 16
2.1.2 Modo de ação das lacases .............................................................................. 17
2.1.3 Propriedades gerais das lacases ...................................................................... 19
2.1.4 Aplicações ...................................................................................................... 20
2.2 Imobilização.......................................................................................................... 21
2.2.1 Considerações gerais ...................................................................................... 21
2.2.2 Imobilização de lacases .................................................................................. 22
2.3. Suporte ................................................................................................................. 24
2.3.1 Considerações gerais ...................................................................................... 24
2.3.2 Partículas magnéticas de polisiloxano álcool polivinílico (mPOS-PVA) ...... 25
3 REFERÊNCIAS BIBLIOGRÁFICAS ........................................................................ 28
4 OBJETIVOS ................................................................................................................ 35
4.1. Objetivo geral ...................................................................................................... 35
4.2. Objetivos específicos ........................................................................................... 35
5 ARTIGOS CIENTÍFICOS .......................................................................................... 37
Artigo I – Laccase from Agaricus bisporus immobilized on magnetic polysiloxanepolyvinyl alcohol composite for the oxidation of phenolic mixtures ......................... 37
Artigo II - Immobilization of Trametes versicolor laccase on magnetic polysiloxanepolyvinyl alcohol composite and application in phenolic mixtures oxidation ........... 58
6. CONCLUSÕES .......................................................................................................... 78
7. ANEXOS .................................................................................................................... 79
vi
AGRADECIMENTOS
Primeiramente agradeço a Deus pela vida!
A minha amada família pelo apoio, carinho, ensinamentos e sábias lições de vida.
Ao Prof. Dr. Luiz Bezerra de Carvalho Junior pela orientação prestada durante a
elaboração deste estudo e confiança durante todos os anos de pesquisa no grupo de
Imobilização de Biomoléculas.
A Luiza Rayanna (Ray) pelo carinho e grande colaboração durante todos os
experimentos e redação da tese, especialmente nas companhias, caronas e lanches com
sua família, da qual fiz parte temporariamente.
Meus sinceros agradecimentos aos amigos e professores Alexandra Salgueiro, Maria
Helena, Leonie, Lúcia Fernanda, Sérgio Paiva e Valdemir pelos ensinamentos
repassados relacionados à pesquisa bem como relacionados às experiências de vida.
A Universidade de Pernambuco e Universidade Católica de Pernambuco pela liberação
parcial das atividades.
Ao Prof. Dr. José Luiz de Lima Filho, diretor do LIKA, por ceder as instalações para
realização de parte da pesquisa;
Agradeço a todos que fazem parte do Laboratório de Bioquímica do LIKA-UFPE pela
agradável convivência, especialmente a Mariana Cabrera e Vanessa Brustein por toda
amizade e apoio constantes;
As grandes amigas Lúcia, Janeide e Valéria pelos conselhos e boas risadas. Aos amigos
Ucêmicos pela amizade que espero que perdure por muito tempo.
Agradeço a Adenilda Eugênia de Lima, secretária do PPGCB pela ajuda constante;
vii
A todos os funcionários do LIKA pelo apoio técnico;
Agradeço ao CNPq pelo suporte financeiro durante o curso deste trabalho;
A todos que, de forma direta ou indireta, contribuíram para a realização deste trabalho.
viii
LISTA DE FIGURAS
REVISÃO DA LITERATURA
Figura 1
Estrutura tridimensional da lacase de Trametes versicolor.
15
Figura 2
Sítio ativo das lacases.
16
Figura 3
Ciclo catalítico das lacases.
17
Figura 4
Estrutura da matriz híbrida POS-PVA.
25
Figura 5
Reações químicas para preparação da matriz híbrida de POS-PVA
26
ARTIGO I Laccase from Agaricus bisporus immobilized on magnetic
polysiloxane-polyvinyl alcohol composite for the oxidation of
phenolic mixtures
Figure 1 Best immobilization conditions for the laccase concentration (A), time
(B) and pH (C) reaction coupling.
45
Figure 2 Optimum pH (A) and pH stability profiles (B) of the soluble and
immobilized laccase.
46 Figure 3
Optimum temperature (A) and temperature stability profiles (B) of the
soluble and immobilized laccase.
47 Figure 4
Reusability of laccase immobilized on mPOS-PVA.
48
ix
ARTIGO II
Immobilization of Trametes versicolor laccase on magnetic polysiloxanepolyvinyl alcohol composite and application in phenolic mixtures
oxidation
Figure 1
Pareto chart for the laccase immobilization.
65
Figure 2
Response surface of the effect of laccase concentration, immobilization pH
66
and their mutual interaction on protein loading.
Figure 3
Response surface of the effect of laccase concentration, immobilization pH
67
and their mutual interaction on specific activity.
Figure 4
Pareto chart for phenol biotransformation.
Figure 5
Response surface plots for transformation of phenolic mixture as a function
of: (a) phenol concentration and pH for 32 h; (b) reaction time and phenol
71
concentration at pH 6.0; (c) pH and time of reaction at 1.0 mM of phenol
concentration.
70
x
LISTA DE TABELAS
REVISÃO DA LITERATURA
Tabela 1.
ARTIGO II
Propriedades de lacases de diferentes origens imobilizadas por ligação
covalente em suportes insolúveis em água 22
Immobilization of Trametes versicolor laccase on magnetic polysiloxanepolyvinyl alcohol composite and application in phenolic mixtures
oxidation
Table 1.
Experimental design and results according to the CCRD 22.
Table 2.
ANOVA of protein loading of mPOS-PVA including laccase concentration,
immobilization as well as their interactions.
65
Table 3.
Experimental design and results according to the CCRD 23.
69
Table 4.
ANOVA of phenol transformation using immobilized laccase on mPOS-PVA
including pH, phenol concentration and time reaction.
70
64
xi
LISTA DE ABREVIAÇÕES
ABTS - ácido 2,2´-azino-bis-(3-etilbenzotiazol-6-sulfônico)
DEI – Derivado Enzimático Imobilizado
EC - Enzyme Commission
kDa - quilo Daltons
mPOS-PVA - Polissiloxano Álcool Polivinílico Magnetizado
POS - Polissiloxano
PVA - Álcool Polivinílico
SGZ – Siringaldazina
xii RESUMO
Imobilização de lacase de Trametes versicolor a partículas magnéticas polissiloxano
álcool polivinílico (mPOS-PVA) e sua aplicação para a remoção de compostos
fenólicos de uma mistura modelo de fenol foram estudados. As partículas de mPOSPVA
foram preparadas usando o processo sol-gel e magnetizadas por co-precipitação de
íons Fe2+ e Fe3+. As condições de imobilização e de oxidação de fenóis foram
investigados. Delineamento composto central rotacional e metodologia de superfície de
resposta foram utilizados para avaliar os efeitos de parâmetros de imobilização, como
concentração de enzima, pH e tempo de imobilização. A quantidade de lacase
imobilizada foi 3,0 mg/g de suporte sob condições otimizadas (50 μg ml -1 de lacase, pH
4,5, 180 min e temperatura de 25◦C). Excesso de proteína imobilizada ao suporte
resultou em baixa eficiência do biocatalisador. A lacase imobilizada foi utilizada para a
oxidação de uma mistura de cinco compostos fenólicos (fenol, guaiacol, pirogalol,
resorcinol e ácido tânico) comumente presentes em efluentes da indústria papeleira. Os
compostos fenólicos foram oxidadas pela lacase formando produtos insolúveis, os quais
foram removidos do meio de reação por filtração em membrana. Para obter as melhores
condições para oxidação de fenol, um delineamento composto central rotacional com
diferentes combinações de pH, concentração de fenol e tempo de reação foi realizada. O
derivado imobilizado reduziu 65,1% de teor de fenóis totais da solução modelo sob
condições ótimas (concentração de fenóis de 1mM, pH 6,0 durante 32h). Os resultados
destes experimentos indicam que a metodologia de superfície de resposta foi um
método promissor para a otimização de imobilização de proteínas e que lacase
imobilizada em mPOS-PVA é eficaz na transformação de misturas de compostos
fenólicos.
Palvras chave: Imobilização, Álcool polivinílico, Polissiloxano, Lacase, Agaricus
bisporus, partículas magnéticas.
xiii ABSTRACT
Laccase from Trametes versicolor immobilization on magnetic polysiloxane-polyvinyl
alcohol particles (mPOS-PVA) and its application for removing phenolic compounds
from a phenol model mixture were studied. The mPOS-PVA particles were prepared by
using sol-gel process and magnetized by Fe2+ and Fe3+ co-precipitation. The
immobilization conditions and of the application phenol oxidation were investigated.
Central composite rotatable design and response surface methodology were employed
to evaluate the effects of immobilization parameters, such as enzyme concentration,
immobilization pH and immobilization time. The amount of immobilized laccase on the
mPOS-PVA particles was 3.0 mg/g particles under optimized working conditions (50
μg ml-1 laccase, pH 4.5, 180 min and 25 ◦C), whereas higher loadings gave rise to a
less-efficient biocatalyst. The immobilized laccase was used for the oxidation of
phenolic compounds (phenol, guaiacol, pyrogallol, resorcinol and tannic acid) chosen
among those present in paper-mill industry. Phenol compounds were oxidized by
laccase mostly in insoluble products, which were simultaneously removed from reaction
medium by filtration through the membrane. To obtain the optimum conditions for the
phenol oxidation a central composite rotatable design, with different combinations of
pH, phenol concentration and time reaction was performed. Under optimum conditions
(phenol concentration 1mM, pH 6.0 during 32h) the immobilized derivative reduced
65.1% of the original phenol content from the model solution. Results of these
experiments indicated that response surface methodology is a promising method for
optimization of protein immobilization and that immobilized laccase on magnetic
polysiloxane-polyvinyl alcohol particles is effective in the transformation of phenolic
mixtures.
Keywords Immobilization, Polyvinyl alcohol, Polysiloxane, Laccase, Agaricus bisporus,
Magnetic particles.
Jordão, R.C.C - Imobilização de lacases a partículas magnéticas de Polisiloxano- Álcool Polivinílico
1 INTRODUÇÃO
As lacases (benzenediol: oxigênio oxidoredutase, E.C. 1.10.3.2) são membros da
família de proteínas multi-cobres, que incluem ascorbato oxidase, ceruloplasmina e
bilirrubina oxidases. Estas enzimas catalisam a oxidação de polifenóis e substâncias
aromáticas com concomitante redução de oxigênio dissolvido à água (BALDRIAN,
2006).
Lacases tem baixa especificidade, podendo agir sobre um amplo espectro de
substratos e quando se adiciona ao meio de reação uma molécula mediadora são capazes
de degradar compostos aromáticos recalcitrantes com potencial de óxido-redução
elevado. Tais características fornecem a esta enzima um grande valor para
desenvolvimento de tecnologias ambientalmente seguras nos processos de polpa e papel
e na biorremediação de efluentes industriais contendo compostos aromáticos
(NILADEVI e PREMA, 2008).
Um obstáculo que ainda deve ser superado para aplicação industrial das lacases
é o alto custo de produção da enzima, que pode ser minimizado com a pesquisa de
novas fontes de lacases, como também a obtenção de derivados enzimáticos
imobilizados com boas propriedades operacionais e capazes de preservar sua atividade
catalítica em vários ciclos oxidativos consecutivos (DURAN et al. 2002).
A imobilização da lacase em suportes sólidos geralmente melhora sua
desempenho, pois reduz a susceptibilidade à desnaturação e aumenta sua estabilidade
sob condições adversas no ambiente de reação, potencializando sua aplicação em
processos industriais.
O compósito Polisiloxano-Álcool Polivinílico (POS-PVA) foi descrito para a
imobilização de enzimas com obtenção de derivados com boas características catalíticas
e estabilidade operacional (NERI et al. 2009). O POS-PVA apresenta grande área de
superfície, alta porosidade, estabilidade térmica, óptica e química, configurando boa
alternativa para a imobilização de lacases para aplicação na área ambiental (SANTOS et
al., 2008; LIMA-BARROS et al., 2002).
15
2 REVISÃO DA LITERATURA
2.1 Lacases
2.1.1 Origem e distribuição
As lacases (benzenodiol: oxigênio oxidoredutases, E.C. 1.10.3.2) foram descritas
inicialmente em 1883, quando extraída da planta Rhus vernicifera e aproximadamente
10 anos depois foram isoladas de fungos (THURSTON, 1994).
Lacases são proteínas globulares, com peso molecular de 60 a 100 kDa,
apresentando de 10-25 % de carboidrato N-ligado. Estas enzimas são produzidas por
fungos (Ascomicetos, Deuteromicetos e Basidiomicetos), bactérias (Azospirillum
lipoferum e Bacillus subtilis), insetos (Drosophila melanogaster) e plantas
(SUGUMARAN et al., 1992). A grande maioria das lacases descritas na literatura
apresenta estrutura monomérica, como a lacase produzida pelo fungo Trametes
versicolor (PIONTEK et al., 2002) e outros ocorrem como dímeros que é o caso da
lacase de (Agaricus bisporus), conforme sugerido por PERRY et al. (1993).
Figura 1. Estrutura de lacase de Trametes versicolor. Os átomos de cobre são
mostrados como esferas azuis
Fonte: PIONTEK, ANTORINIS e CHOINOWSKI, 2002.
16
2.1.2 Modo de ação das lacases
As lacases são caracterizadas por conter quatro átomos de cobre no seu sítio
catalítico (Figura 2). Os átomos de cobre são classificados em três tipos: cobre Tipo 1,
responsável pela oxidação do substrato e pela cor azul-esverdeada típica de proteínas
multi-cobres (GIARDINA et al., 2010); cobre Tipo 2 e Tipo 3, sendo este último
constituído por dois átomos de cobre antiferromagneticamente acoplados (SUNDARAN
et al., 1997). Os cobres Tipo 2 e 3 se combinam para formar uma estrutura trinuclear
envolvida na ligação com o oxigênio molecular, a qual está coordenada por 8 resíduos
de histidina, (COLE, CLARK, e SOLOMON, 1990).
Figura 2. Sítio ativo das lacases
Fonte: RIVA, 2006.
A figura 3 ilustra o ciclo catalítico das lacases, o qual compreende três passos
principais: redução do cobre Tipo 1 pelo substrato; transferência eletrônica interna do
cobre Tipo 1 para os cobres Tipo 2 e 3; transferência de elétrons do cobre para o O2,
17
reduzindo-o a H2O. Os quatro átomos de cobre da lacase nativa apresentam estado de
oxidação 2+, e à medida que a enzima promove a oxidação de seus substratos, os átomos
de cobre são reduzidos e transferem seus elétrons, através dos três resíduos de
aminoácidos His-Cis-His. Desta forma, o sítio 1 poderia promover a oxidação de um
substrato, até a completa redução de todos os sítios e sua reoxidação formando água
para retomar novamente o ciclo (VILLELA, 2006; WESENBERG, KYRIAKIDES e
AGATHOS, 2003).
Figura 3. Ciclo catalítico das lacases
Fonte: BALDRIAN, 2006
A oxidação de substratos fenólicos pela lacase resulta na produção de quinonas
ou radicais livres instáveis, os quais são estabilizados pela polimerização espontânea.
Os produtos insolúveis formados apresentam alto peso molecular e podem ser isolados
por sedimentação ou filtração (BRYJAK et al., 2007).
18
19
Por muitos anos, a participação de lacase na degradação da lignina foi
considerada limitada à oxidação de unidades fenólicas, que compreendem unicamente
de 10 a 20% do polímero. Entretanto, nos anos noventa foi demonstrado que lacases
também pode oxidar unidades não-fenólicas de lignina na presença de compostos
oxidáveis de baixa massa molar, conhecidos como mediadores, que incluem substratos
artificiais e metabólitos fúngicos (BALDRIAN, 2006).
Mediadores são moléculas de baixa massa molar que atuam como carreadores de
elétrons. Após serem oxidadas por lacases (no cento T1), dinfundem-se para fora do
sítio ativo da enzima e ao encontrar o substrato retira seus elétrons, que devido ao seu
tamanho, não poderiam alcançar de forma direta o sítio ativo forma, este processo pode
levar a um mecanismo de oxidação que não seria possível para a enzima sozinha,
estendendo, assim, a atividade oxidativa desta enzima (BALDRIAN, 2006).
Diversos compostos sintéticos têm sido descritos como mediadores eficientes de
lacases, entretanto, o mais comumente usado é o 1-Hidroxibenzotriazol (HBT). Lacases
na presença de mediador oxidam substâncias recalcitrantes como: anilinas, estruturas
relacionadas a compostos organofosforados, compostos modelos não-fenólicos de
lignina,
fenóis,
clorofenóis,
corantes
aromáticos
e
outras
(WESENBERG,
KYRIAKIDES e AGATHOS, 2003).
2.1.3 Propriedades gerais das lacases
Muitos fungos secretam diferentes isoformas de lacases. O número de
isoenzimas pode variar entre espécies ou dentro da mesma espécie, dependendo se a
enzima é induzida ou não. Elas podem apresentar diferenças quanto à estabilidade, pH
ótimo, temperatura ótima e afinidade por diferentes substratos.
O pH ótimo de lacases depende do tipo de substrato utilizado. Quando o
substrato é ABTS, o pH ótimo é mais ácido, na faixa de 3,0 a 5,0. A diferença no
potencial de óxido-redução entre o substrato fenólico e o cobre do sítio T1 pode
aumentar a oxidação do substrato em valores altos de pH, contudo a ligação do ânion
hidróxido aos cobres do sítio T2/T3 pode resultar em inibição da atividade lacase
devido à diminuição da transferência de elétrons entre centro T1 e T2/T3. Estes efeitos
opostos devem ser considerados na determinação do pH ótimo de lacases (SALIS,
2009).
A temperatura ótima das lacases pode variar, dependendo da origem da enzima.
Essas enzimas têm suas atividades inibidas por azida, cianeto (Agaricus bisporus,
Trametes gallica, Trametes sanguinea) e fluoreto, os quais se ligam aos átomos de
cobre impedindo a transferência interna de elétrons, e os íons de Hg2+ (Chaetomium
termophilum, Daedalea quercina, Lentinula edodes, Lentinula edodes) que podem
induzir mudanças conformacionais na proteína (BALDRIAN, 2006; GIANFREDA, XU
e BOLLAG, 1999).
Atividades de lacases foram detectadas em meios de cultura de ampla variedade
de fungos degradadores de lignina, especialmente fungos da degradação branca da
madeira, como Trametes (Coriolus) versicolor and Pycnoporus sanguineus. Umas das
limitações para aplicação de lacases fúngicas em larga escala é a baixa velocidade de
produção, a qual pode ser melhorada pela adição de indutores como xilidina e ácido
ferúlico. Lacases têm sido purificadas e caracterizadas no ccogumelo Agaricus bisporus,
uma fonte de baixo custo. A. bisporus é a espécie mais conhecida e consumida de
cogumelos comestíveis, gerando grande quantidade de resíduos, os quais são utilizados
para produção de extratos enzimáticos. As lacases obtidas desta espécie fúngica são
glicoproteínas diméricas com 15% de carboidrato e com peso molecular de 100 kDa.
(WOOD, 1980; TREJO- HERNANDEZ, LOPES-MANGUIA, RAMIREZ, 2001).
2.1.4 Aplicações
Devido à capacidade de oxidar compostos aromáticos, particularmente fenóis,
lacases estão recebendo grande atenção em várias aplicações industriais diretas e
indiretas como clareamento de corantes, processamento de papel, deslignificação,
produção de etanol, detoxificação de poluentes ambientais, prevenção da descoloração
de vinhos, nanotecnologia, biosensores, etc. (DURAN et al. 2002).
20
As lacases têm sido utilizadas na indústria têxtil, particularmente na
descoloração de efluentes têxteis, devido ao seu alto potencial de degradação de
corantes de diferentes estruturas químicas (ABADULLA et al., 2000; BLÁNQUEZ et
al., 2004; HOU et al., 2004), incluindo corantes sintéticos (COUTO et al., 2004, 2005).
Em 1996, a Novozyme (Novo Nordisk, Dinamarca) lançou a lacase comercializada
como DeniLite® para aplicação na indústria têxtil, precisamente em acabamento de
brim (COUTO e HERRERA, 2006).
Lacases também foram aplicadas na remoção de compostos aromáticos,
particularmente fenólicos, presentes em efluentes das indústrias papeleiras e de
produção de óleo de oliva, entre outras (WANG, THIELE e BOLLAG, 2002;
AGGELIS et al., 2003; ZHANG et al., 2009).
O estudo destas enzimas nas indústrias de papel tem sido realizado a fim de
diminuir o impacto ambiental desses processos. A preparação industrial do papel requer
a separação e a degradação da lignina na polpa da madeira, e este processo pode ser
realizado com a utilização de lacases (CAMARERO et al., 2004).
As lacases podem ser aplicadas para eliminação de compostos fenólicos
indesejáveis, que conferem cor e gosto desagradáveis aos sucos, cervejas e vinhos
(SERVILLI et al., 2000; MINUSSI, PASTORE e DURÁN, 2002).
Na área analítica, lacase tem sido aplicada na detecção de fenóis e derivados, no
monitoramento ambiental (ROSATTO et al., 2001), em análise de fármacos (BAUER,
et al., 1999; FERRY E LEECH, 2005) e para eletroimunoensaios (KUZNETSOV et al.,
2001).
2.2 Imobilização
2.2.1 Considerações gerais
As enzimas apresentam muitas características que tornam sua aplicação
tecnológica e ambiental vantajosa comparada aos catalisadores químicos convencionais.
21
Contudo, sua aplicação ainda é limitada devido a sua alta sensibilidade a agentes
desnaturantes, altos custos de produção e impossibilidade de reuso. Tais desvantagens
podem ser contornadas por meio da utilização de enzimas imobilizadas. A imobilização
é a fixação da enzima sobre ou dentro de suportes sólidos, resultando em sistemas
heterogêneos. A enzima imobilizada é mais resistente a mudanças no ambiente de
reação, permitindo recuperação e reuso. Comparado com a enzima livre, o sistema
imobilizado usualmente tem sua atividade diminuída e a constante de Michaelis
aumentada. Estas alterações estruturais introduzidas na enzima pelo procedimento de
imobilização resultam da criação de um micro ambiente diferente do meio de reação
(CASTRO et al., 2008).
A aplicação em escala industrial de tecnologias usando biocatalisadores
imobilizados é ainda limitada (GERBSCH e BUCHHOLZ, 1995). Dentre estas
aplicações destacam-se exemplos da utilização de enzimas imobilizadas na produção de
xarope de milho rico em frutose, através da isomerização contínua de glicose com a
enzima glicose isomerase pela Clinton Corn Producing, Estados Unidos; a produção
contínua de L-aminoácidos com a enzima aminoacilase pela Tanabe Seiyacu, Japão
(CARVALHO et al., 2006) e síntese do ácido 6-aminoppenicilânico (penicilina semisintética) com a enzima penicilina acilase pela Bristol-Myer Squibb, Inglaterra. Quanto
à utilização de células imobilizadas destaca-se a produção de vinagre por células viáveis
de Acetobacter e a produção de acrilamida a partir de acrilonitrila empregando células
imobilizadas não viáveis de Rhodococcus rhodochrous pela Nitto Chemical Industries,
Japão; síntese do ácido 6-aminopenicilínico (penicilina semi-sintética) com a enzima
penicilina-G ou L-acilase pela Bristol-Myer Squibb, Inglaterra, cuja produção mundial é
da ordem de 5.000 toneladas/ano; produção de aspartame com a termolisina
imobilizada; e a hidrólise da lactose presente no soro de queijo (CASTRO et al., 2008).
2.2.2 Imobilização de lacases
A imobilização de lacases de origens diferentes tem sido estudada
extensivamente por vários métodos (adsorção, enclausuramento, ligações cruzadas e
ligação covalente) baseados principalmente em mecanismos químicos e ou físicos.
22
Lacases imobilizadas sobre diversos suportes e suas aplicações foram revisadas por
Duran et al. (2002), os quais reportaram a imobilização de lacases sobre vidro de
porosidade controlada, quitosana, eupergite®, poliuretano, caolinita, sílica gel, alumina
G, sefarose, entre outros. Pesquisas recentes foram publicadas descrevendo a
imobilização da lacase em suportes como quitosana e sílica com modificações,
derivados de celulose, copolímeros de acrilato e terra de diatomácea (Tabela 1). A
literatura não descreve a imobilização de lacases a partículas magnéticas de POS-PVA e
apenas uma publicação foi encontrada descrevendo a imobilização da lacase de
Agaricus bisporus em cerâmica-quitosana (SHANG, LIU e WANG, 2009).
Tabela 1: Propriedades de lacases de diferentes origens imobilizadas por ligação
covalente em suportes insolúveis em água.
Origem
Suporte
pH ótimo
Temperatura ótima (°C)
Referência
Agaricus bisporus
Compósito cerâmica-quitosana
3,0
Shang, Liu,
Wang, 2009
50
Cerrena unicolor
Granocel-4000, a base de celulose
6,5 SGZ
5,9 ABTS
55
Rekuc et al.,
2009
Cerrena unicolor
Copolímero butil acrilato e etileno
glicol dimetacrilato
6,0
30
Bryjak et al.,
2007
23
Coriolopsis polyzona
Terra de diatomácea comercial
-
Cabana et al.,
2009
-
Coriolus versicolor
Quitosana
Zhang et al.,
2009
4,5
Myceliophthora thermophila
Esferas EC-EP3 e NK (polímeros a base de polimetacrilato)
3,0
Kunamneni et
al., 2008
60
Pleurotus sajor-caju
Sílica mesoporosa funcionalizada e poliamida
-
-
Salis et al., 2009
e Rasera et al.
2009
55
Jiang et al., 2005
45
Yang et al., 2006
Pycnoporus sanguineus
Microesferas de quitosana magnéticas
3,0
Rhus vernicifera
Quitosana
8,0
Rhus vernificera
Poli(GMA/EGDMA)
poli(glicidil
metacrilato/
etilenoglicol
dimetacrilato)
6,0
50
Arica et al.,
2008
24
Trametes versicolor
Esferas de sílica mesoporosa magnéticas
3,6
30
Zhu et al, 2007
Trametes versicolor
Nanopartículas e caolinita
6,0
45
Hu et al., 2007
Trametes versicolor
Polímero de rede semi-interpenetreda
5,5
40
Yamak et al.,
2009
Trametes versicolor and P. cinnabarinus
Esferas de celulose macroporosa magnéticas
Rotkova et al.,
2009
SGZ – siringaldazina; ABTS - ácido 2,2´-azino-bis-(3-etilbenzotiazol-6-sulfônico)
2.3. Suporte
2.3.1 Considerações gerais
As recentes tecnologias para a imobilização de enzimas requerem materiais com
propriedades especiais, as quais aliadas à melhoria na estratégia do processo de
imobilização configuram fatores essenciais para o sucesso da imobilização. Para ser
efetivo na imobilização o suporte deve deixar a enzima acessível aos substratos, manter
sua atividade por longo período e permitir que o sistema (suporte/enzima) seja
regenerado ao final do processo, sem que ocorram perdas na atividade enzimática.
25
2.3.2 Partículas magnéticas de polisiloxano álcool polivinílico (mPOS-PVA)
O Polisiloxano-Álcool Polivinílico (POS-PVA), compósito entre polisiloxano
(POS) e álcool polivinílico (PVA), apresenta boas características para aplicação em
imobilização de enzimas, devido a sua grande área de superfície, alta porosidade,
estabilidade térmica, óptica e química (LIMA-BARROS et al., 2002).
Figura 4. Estrutura da matriz híbrida POS-PVA (R = grupos etil)
Fonte: SANTOS, et al. 2008
De acordo com Ingersoll e Bright (1997) a síntese do suporte começa com
hidrólise do alcóxido de silício formando um produto hidroxilado e o álcool
correspondente. O segundo passo é a condensação entre um grupo alcóxido não
hidrolisado e uma hidroxila, ou entre duas hidroxilas apenas, formando uma mistura
coloidal (sol). O último passo envolve policondensação entre os componentes dessa
mistura coloidal e uma rede adicional (PVA) resultando em matriz híbrida porosa
(Figura 5).
26
Figura 5. Preparação da matriz híbrida de POS-PVA
Fonte: SANTOS et al., 2008
A matriz híbrida de POS-PVA pode ser conjugada a magnetita (Fe3O4) através
da co-precipitação de Fe2+ e Fe3+, formando um compósito com partículas magnéticas,
possibilitando uma rápida separação quando o mesmo é submetido a um campo
magnético, reduzindo, deste modo, os custos operacionais do processo. Robinson,
Dunnill e Lilly, 1973, foram os primeiros pesquisadores a investigar as propriedades de
um suporte magnético para a imobilização de enzimas.
Materiais magnéticos têm recebido grande atenção devido a sua aplicação em
áreas como biologia, medicina e meio ambiente. Estes materiais são compostos de um
núcleo de óxido de ferro que revestem moléculas orgânicas ou inorgânicas. As
partículas magnéticas respondem a um campo magnético externo, porém não interagem
entre elas em ausência do mesmo.
Diversas aplicações incluem o uso de partículas magnéticas, tais como:
imobilização de enzimas (DEKKER et al. 1989), isolamento de células (HAIK, PAI e
CHEN, 1999; HANCOCK e KEMSHEAD, 1993; MOLDAY e MOLDAY, 1984),
imunoensaio (RICHARDSON et al. 2001), adsorção e purificação de proteínas,
separação de ácidos nucléicos (LEVISON et al. 1998; UHLEN et al. 1989) e liberação
de drogas (RUUGE e RUSETSKI, 1993).
27
Após magnetização, o mPOS-PVA pode ser ativado com adição de
glutaraldeído, que age como braço químico para ligação de biomoléculas, tornando o
suporte uma superfície mais compatível para a imobilização covalente.
O POS-PVA tem sido utilizado em diversas aplicações, como por exemplo,
imobilização de anticorpos (COÊLHO et al., 2003; MELO et al., 2008), de enzimas
(NERI et al., 2009) e como fase sólida para ensaios quimiluminescentes (COÊLHO et
al., 2002).
28
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35
4 OBJETIVOS
4.1. Objetivo geral
Investigar a imobilização de lacases a partículas magnéticas de rede semi
interpenetrada de Polisiloxano-Álcool Polivínilico e aplicar o biocatalisador obtido na
transformação de misturas contendo compostos fenólicos.
4.2. Objetivos específicos
Imobilizar lacases em partículas magnéticas de rede semi interpenetrada de
polisiloxano e álcool polivínilico;
Investigar propriedades do derivado sintetizado, tais como: retenção de
atividade específica; pH ótimo; temperatura ótima; estabilidade térmica; tempo
de meia-vida;
Comparar as propriedades do derivado imobilizado com aquelas descritas para
a enzima solúvel;
Realizar o estudo das condições ótimas para a oxidação de misturas de fenóis
constituintes de efluentes industriais.
36
5 ARTIGOS CIENTÍFICOS
Artigo I - Manuscrito submetido para publicação no periódico Journal of Industrial
Microbiology & Biotechnology. Fator de Impacto: 1.9
Laccase from Agaricus bisporus immobilized on magnetic polysiloxane-polyvinyl
alcohol composite for the oxidation of phenolic mixtures
Roziana Cunha Cavalcanti Jordão, Luiza Rayanna Amorim de Lima, Luiz Bezerra de
Carvalho Junior
Departamento de Bioquímica and Laboratório de Imunopatologia Keizo Asami (LIKA),
Universidade Federal de Pernambuco, Av. Morais Rego, Campus Universitário,
50670-910, Recife, Pernambuco, Brazil.
Abstract Polysiloxane-polyvinyl alcohol particles were prepared by using sol-gel
process and magnetized by Fe2+ and Fe3+ co-precipitation. Laccase from Agaricus
bisporus was immobilized onto these magnetic particles via glutaraldehyde. The best
immobilization conditions were found to be: protein/particles ratio equal to 100 µg per
10 mg, pH 3.0 and one hour of coupling reaction. The protein and enzyme specific
activity retained under these conditions were 6.6 µg mg-1 and 70% of that found for the
free enzyme (2 U mg-1). The immobilized biocatalyst exhibited the maximal activity at
pH 3.0 and 50 ºC. Also, it presented higher thermal stability compared to free enzyme.
The apparent Km (145 ± 9 µM) was higher than that estimated for the free enzyme (85 ±
37
7 µM) using ABTS as substrate. After five consecutive oxidative cycles 70% of the
initial activity was retained. The immobilized laccase was used for the oxidation of a
mixture of three compounds (phenol, guaiacol and tannic acid) chosen among those
generally present in paper-mill effluents. After five days 60 % of the total phenols were
removed from the system. The results suggest that immobilized laccase on magnetic
polysiloxane-polyvinyl alcohol particles is effective in the transformation of phenolic
mixtures.
Keywords Immobilization, Polyvinyl alcohol, Polysiloxane, Laccase, Agaricus
bisporus, Magnetic particles.
Introduction
Laccases (benzenediol:oxygen oxidoreductase E.C. 1.10.3.2) are multicopper
oxidases widely distributed in higher plants, fungi and bacteria. These enzymes catalyze
the oxidation of various aromatic and inorganic substrates whilst simultaneously
reducing molecular oxygen to water. The substrates oxidized by laccase include ortho-,
para-, diphenol, and aromatic compounds containing hydroxyl and amine groups and
the range of substrate may be extended by the inclusion of a redox mediator in reaction
mixtures [2]. The catalytic ability of laccases combined with the fact that they require
only molecular oxygen (unlike peroxidase) for the transformation of aromatic
compounds, led to diverse biotechnological applications. Phenols and derivatives
compounds are introduced into surface water from industrial effluents such as those
from the pulp-and-paper plants, textile industry, petroleum refining and domestic
38
wastewaters. The presence of these compounds in drinking and irrigation water
represents a health and environmental hazard because of their toxicity and possible
accumulation in the environment.
Phenol, guaiacol and tannic acid are phenolic
compounds usually found in paper-mill effluents [19]. Laccase of A. bisporus have been
isolated from different sources, purified and characterized. They are dimeric proteins
with molecular mass around 65 kDa, acidic isoeletric pH, between 3 and 4 [4, 20, 28].
The immobilization of enzymes offers several advantages (reuse, increase of
stability, ease of recovery of enzyme and products exempt of contamination) and those
onto magnetic support can be easily separated by a magnetic field from the reaction
mixture and can be easily implemented as continuous enzyme-catalyzed process.
Immobilized laccases on solid supports and their application have been
extensively reviewed by Duran et al. [10]. Recent research work described laccase
immobilization onto copolymer of butyl acrylate and ethylene glycol dimethacrylate [2],
nanoparticle and kaolinite [12], magnetically separable mesoporous silica spheres [30],
Poly(GMA/EGDMA) beads [1], diatomaceous earth support Celite ® R-633 [6], epoxyactivated carriers [14], cellulose based Granocel 4000 [21], magnetic bead cellulose
[22], functionalized SBA-15 mesoporous silica [24] and magnetic chitosan
nanoparticles [11].
A previous study demonstrated that a semi-interpenetrated network of polyvinyl
alcohol (PVA) and polysiloxane (POS) can be easily magnetized by Fe2+ and Fe3+ coprecipitating to form a composite with magnetite particles of Fe3O4 (mPOS–PVA).
These particles presented good characteristic for immobilization of β-galactosidase [17]
and the biocatalyst obtained was used for galacto-oligosaccharides production and
lactose hydrolysis [18]. From the best of our knowledge, the studies about
39
immobilization of laccase on magnetic POS-PVA have not being reported in the
literature up the present time.
The objective of this study was to propose a magnetically separable immobilized
laccase onto mPOS-PVA particles, to investigate some properties and compare with
those found for the free enzyme.
Materials and methods
Chemicals
Laccase from A. bisporus; 2,2, Azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)
(ABTS) and bovine serum albumine (BSA) were purchased from Sigma Chemical Co.
(St. Louis, MO, USA). Ethanol (minimum 99%) and polyvinyl alcohol (MW 72,000)
were purchased from Reagen Chemical Co. (São Paulo, Brazil). Glutaraldehyde (25%)
and tetraethoxysilane (TEOS) were from Aldrich Chemical Co. (Milwaukee, WI, USA).
Phenol, guaiacol, tannic acid and all other reagents were of analytical grade.
Preparation of magnetic POS-PVA particles
Beads of POS-PVA hybrid composite were synthesized by sol-gel process according to
[3]. The POS-PVA discs were mechanically ground and submitted to magnetization
according to Carneiro Leão et al. [7]. The resulting magnetic POS-PVA particles
40
(mPOS-PVA) were thoroughly washed with distilled water until pH 7.0 and dried at 50
ºC overnight and finally sieved (<100µm).
Laccase immobilization onto magnetic POS-PVA particles
Magnetic POS-PVA particles (mPOS-PVA) were chemically activated by adding 1 ml
of 2.5 % glutaraldehyde solution in 0.1 M H2SO4.
The activated support was
recuperated by magnetic field and washed with distilled water. Attempts were made to
optimize the conditions for laccase immobilization conditions on mPOS-PVA. The
crucial parameters like concentration of enzyme, contact time and pH were varied and
subjected to study. Laccase solutions (1.0 ml), containing a variable protein content in
the 50 to 1200 µg range in acetate buffer (pH 3.0-9.0) was mixed under rotary stirring
(60 rev min-1) with activated mPOS-PVA (10 mg) at 4 ºC for 10 min, 30 min, 1, 5, 12,
24 e 48 h. Subsequently, the laccase immobilized on mPOS-PVA was recovered by
magnetic field. Then the magnetic particles were washed with 0.15 M NaCl solution
followed by 0.1 M acetate buffer solution (pH 4.5) stirring to remove the unbound
protein. The washing procedure was repeated ten times. Finally, the immobilized
enzyme was directly used for the activity measurement and kept at 4 ºC until further
use.
Determination of laccase activity and protein estimation
41
The activity of the free and immobilized laccase was spectrophotometrically determined
in a reaction mixture containing 0.5 mM ABTS (0.5 ml) as the substrate in 0.1 mM
citrate buffer pH 3.0 (0.45 ml), containing a suitable amount of laccase (0.1 IU), at 25o
C [13, 23]. Three minutes later, the absorbance of the solution or supernatant at 420 nm
was spectrophotometrically determined (Ultrospec® 3000 Pro of Amersham Pharmacia
Biotech – UV/Visible spectrophotometer). The molar extinction coefficient of ABTS is
36 x 10-3 M-1 cm-1 and one unit of activity was defined as the amount of enzyme
required to oxidize 1 µmol of substrate per minute under the experimental conditions.
The protein content was estimated according to Lowy´s method [15]. The quantity of
protein bound to the support was calculated by subtracting the protein recovered in the
washing of the support-enzyme complex from the protein used for immobilization.
Optimum pH and pH stability
To investigate the optimum pH, the activities of the free and immobilized laccase were
determined by measuring the activities in different buffer solutions pH 3.0-9.0 (3.0
using 0.1 M sodium citrate buffer, 4.0-5.0 using 0.1 M sodium acetate buffer, 6.0-7.0
using 0.1 M sodium phosphate buffer and 9.0 using 0.1 M glycine-NaOH buffer). The
pH stability was examined after pre-incubating the enzyme sample at 25 ºC for 6 h in
the pH range 3.0-5.0. The residual activity was assayed as above described.
Optimum temperature and thermal stability
42
The optimum temperature of the free and immobilized laccase were determined at
temperature range of 30-80 ºC in sodium citrate buffer pH 3,0. To investigate thermal
stability of the laccase, the free and immobilized enzyme were incubated in the absence
of substrate at the temperatures ranging from 30 ºC to 80 ºC in buffer solution of pH
3.0 for one hour and their activities were immediately measured as above described.
The enzyme activity of the not incubated laccase was taken as 100%.
Kinetics studies
The apparent Michaelis constants (Km) of the free and immobilized laccase were
determined by examining the enzyme activity on increasing concentrations of ABTS
(0.05 – 1.5 mM).
Reusability
The reusability of the immobilized enzyme derivative (1.4 U mg-1) was evaluated by
performing eight consecutive oxidative cycles using ABTS 0.5 mM as substrate (1ml).
After each oxidation cycle, the biocatalyst was washed three times with buffer solution
and the procedure repeated with fresh substrate solutions.
43
Half-life
For evaluation of the half-life, the immobilized enzyme was stored at 4 ºC for several
days in 0.2 M sodium acetate buffer (pH 3.0). The remaining activity of enzymes was
determined at 25o C in as previously described. The half-life was calculated according
Zille et al. [31].
Treatment of phenolic compounds by the soluble and immobilized laccase
The soluble and immobilized laccase containing about 2 UI were incubated at 25º C for
5 days with 1 ml of phenol solution (1mM) composed of phenol, guaiacol and tannic
acid [27]. Afterwards, samples were withdrawn at 0 and 5 days of incubation, filtered
through Millipore membrane (0.45 µM) and the total phenol was estimated according to
the Singleton and Rossi method [26]. The total phenol content of the samples was
expressed as milligram of gallic acid equivalents (GAE) per milliliter. Phenol
concentration of the untreated sample was taken as 100%. Usually two controls assays
were performed: one without the enzyme to evaluate spontaneous transformation of
phenol mixture and other with heat-denatured laccase.
Statistical analysis
All laccase immobilization process, enzymatic activity, protein estimation, pH and
temperature profiles, kinetics studies, reuse, half-life and oxidation of phenolic
44
45
compounds were performed at least three times. Means and standard errors were
calculated using Microsoft Origin (Version 7.0).
Results
The mPOS-PVA particles synthesis was carried out in two steps: firstly, beads of
a
network
of
polysiloxane-polyvinyl
alcohol
molecules
was
formed
using
glutaraldehyde as an arm under acid catalysis [3] and afterwards smashed and
magnetized [7]. Several aspects, including (i) laccase concentration, (ii) immobilization
time and (iii) immobilization pH, were studied in order to find the best immobilization
conditions. Figure 1A shows the amount of immobilized laccase on the support as a
direct function of enzyme concentration. A linear correlation can be seen between
enzyme concentration and protein retention up to a certain enzyme concentration, when
a saturation value is reached and no more laccase can be immobilized. Figure 1B shows
the immobilized amount and the activity of laccase as a function of time when the
laccase concentration was 100 μg ml-1. The immobilization of laccase on POS-PVA was
achieved in only 1 h, and after that time there was a small decline of specific activity of
immobilized enzyme. Figure 1C shows the immobilized amount and activity of laccase
on the support as a function of pH when the laccase concentration was 100 µg m-1 and
immobilization time was one hour. The higher specific activities of the immobilized
preparation were obtained in a pH range from 3.0 to 4.0. A pH value of 3.0 was used in
the successive steps, as this has been reported in the literature as the optimum pH value
for the enzymatic activity of free laccase [2]. The mPOS-PVA showed to be capable to
fix 6.6 µg protein per mg of magnetic particles. At the same time the higher laccase
specific activity was approximately 70% comparing with free laccase.
A
20
Protein Loading (mg/g mPOs-PVA)
-1
Specific Actvity * 10 (U/mg)
18
18
16
16
14
14
12
12
10
10
8
8
6
6
4
4
2
2
0
0
200
400
600
800
Specific Activity * 10-1 (U/mg)
Protein Loading (mg/g mPOS-PVA)
20
0
1200
1000
Laccase concentration (μg/mL)
B
12.0
Specific Activity (U/mg)
11.5
11.5
11.0
11.0
10.5
10.5
10.0
10.0
9.5
9.5
9.0
9.0
8.5
8.5
8.0
8.0
7.5
7.5
7.0
7.0
6.5
6.5
6.0
6.0
5.5
5.5
Protein Loading (mg/ g mPOS-PVA)
12.0
5.0
5.0
0
10
20
30
40
50
Time (h)
C
8
7
7
6
6
5
5
4
4
3
3
2
2
1
1
8
0
0
2
3
4
5
6
7
8
9
10
pH
Fig. 1 Best immobilization conditions for the laccase concentration (A), time (B) and
pH (C) reaction coupling. The retention of protein and specific activity were correlated
with different amounts of enzyme (50-1200 µg ml-1), incubation time (0.16-48 h) and
pH (3.0-9.0).
46
47
The activities of the free and immobilized laccase on ABTS at different pH
values are shown in Figure 2A. Both preparations presented maximal activity at pH 3.0
and negligible activity above pH 5.0. The pH stability in terms of the residual activities
is demonstrated in Fig. 2B. The immobilized laccase fully retained its activity after 6 h
in pH range 3.0-5.0 while free laccase lost 20% of its original activity in the pH range
4.0-5.0.
The effect of temperature on the relative activity of the free and immobilized
laccase is shown in Fig. 3A. At the optimum pH value, free and immobilized laccase
had maximal activity in the temperature range 50-60 ºC. The activities of both free and
immobilized enzymes decreased gradually above their optimum temperature. The free
laccase retained 60% of its maximal activity even at 70ºC, while its immobilized
counterpart demonstrated more than 90% of its maximal activity. Furthermore,
immobilized laccase showed a broadening of the temperature range of enzyme activity.
A 130
B Soluble laccase
Immobilized laccase
120
120
110
100
105
90
100
Relative Activity (%)
Relative activity (%)
Soluble laccase
Immobilized laccase
115
110
80
70
60
50
40
30
95
90
85
80
75
70
65
20
60
10
55
0
3
4
5
6
pH
7
8
9
50
3.0
3.5
4.0
4.5
5.0
pH
Fig. 2 Optimum pH (A) and pH stability profiles (B) of the soluble and immobilized
laccase. The enzyme activity for the soluble and immobilized enzyme was established
at pH range (3.0-9.0) whereas for the pH stability the activity was measured after preincubating the enzyme at 25 ºC for 6 h at pH 3.0; 4.0 and 5.0. The soluble laccase (2U
mg-1) was taken as 100%.
48
Figure 3B shows the activity of the free and immobilized laccases treated at 3070 ºC for one hour. The activity of the immobilized laccase decreased more slowly than
for the free enzyme. The residual activities of free and immobilized laccases after one
hour at 50 ºC were 50 and 80%, respectively.
A B
120
Soluble laccase
Immobilized laccase
120
100
100
Soluble laccase
Immobilized laccase
80
60
40
80
60
40
20
20
0
0
30
40
50
60
70
80
30
40
Temperature (ºC)
50
60
70
Temperature (ºC)
Fig. 3 Optimum temperature (A) and temperature stability profiles (B) of the soluble
and immobilized laccase. The enzyme activity for the soluble and immobilized enzyme
was established at temperature range (30-80°C) whereas for the temperature stability
the activity was measured after pre-incubating the enzyme for 1 h at temperature range
(30-70°C).
The apparent Km value of immobilized laccase (145 ± 8.7 µM) was found to be
1.7 times higher than that for the free laccase (85 ± 6.8 µM).
One of the most important advantages of enzyme immobilized is the possibility
of its reuse for several reaction cycles, since it can be easily separated by the reaction
medium and added to a fresh substrate solution. The reusability of laccase immobilized
on mPOS-PVA is shown in Fig. 4. After five consecutive operations, the retained
relative activity for immobilized laccase was 70 % and after eight cycles the
immobilized derivative retained around 30 % of original activity, which indicates that
biocatalyst had a regular performance in the mentioned conditions. The half-life of the
immobilized laccase was 38.7 days.
49
120
Residual activity (%)
100
80
60
40
20
0
0
1
2
3
4
5
6
7
8
9
Cicles number
Fig. 4 Reusability of laccase immobilized on mPOS-PVA. The reuse was established by
incubating the particles with 0.5 mM ABTS at 25°C pH 3.0. After each oxidation cycle,
the biocatalyst was washed three times with buffer solution and the procedure repeated
with other aliquot as substrate.
The soluble laccase completely oxidized a mixture containing phenol, guaiacol
and tannic acid (1mM) after five days of incubation whereas a percent of about 60%
was estimated for the immobilized preparation. The products from both reactions
polymerized yielding a purple precipitates that it can be removed from the reaction
medium by filtration [9]. This property has raised interest in the treatment of
wastewaters containing toxic phenolic compounds.
Discussion
In this paper, immobilization of the laccase from A. bisporus on magnetic POSPVA particles was evaluated. Immobilization of great amount of protein resulted in a
decrease of laccase activity showing that there is a limit after which immobilized
enzyme molecules are inactive, due to probably the steric hindrance effect. This
phenomenon was previously observed for other immobilized enzymes at high loading
[25, 30]. This result also can be explained in terms of limitations due the diffusion of
the substrates inside the support before reaching the enzyme active site [10]. To avoid
the overloading phenomenon all experiments used to characterize the immobilized
enzyme were carried out using a preparation synthesized with 100 μg ml-1 of the soluble
laccase. The immobilization of laccase on mPOS-PVA was fast, it was achieved in only
1 h, and after that time there was a small decline of specific activity of the immobilized
enzyme. The immobilization of laccase from Trametes versicolor on different supports
was reported by Salis et al. [24]. The laccase immobilization process was reached after
100 min for SBA-15 mesoporous silica, after 30 min for amberlite IRA-400 and after
120 min for montmorillonite. The different immobilization times for the different
supports were explained taking into account that the immobilization of a protein
macromolecule depends of the support characteristics, such as extension and chemical
nature the surface of the support. The pH-value of the mixture in the immobilization
step is one of the most important factors affecting laccase immobilization as it was
demonstrated in this work. The value chosen was pH 3.0 which coincided with the
optimum pH described in the literature for laccase activity on ABTS as substrate and
also with the pI value for laccase from A. bisporus [3].
Laccase activity after immobilization on mPOS-PVA was maximized when
using optimum conditions of immobilization, such as protein concentration 100 μg ml-1
50
after one hour and pH 3.0. The mPOS-PVA showed to be capable to fix 6.6 mg protein
per g of magnetic particles. At the same time the highest laccase activity was
approximately 70% comparing with free laccase.
Several publications describe the investigation of new supports for laccase
immobilization via covalent immobilization. For example, laccase was immobilized on
non-porous polyglycidil metacrylate beads by covalent attachment and the amount of
immobilized laccase was about 5.6 mg g-1 support and the activity of immobilized
laccase was 53 % of free counterpart [1]. Laccase was immobilized on Al2O3 pellets
after glutaraldehyde activation and the amount of immobilized enzyme was also about
5.6 mg g-1 support and the retained activity varied from 14 % to 66 % of the free
counterpart [8].
The activities of both preparations free and immobilized laccase presented
maximal activity at pH 3.0 and minimum activity above pH 5.0 using ABTS as
substrate. Shang et al. [25] reported identical optimum pH value for the laccase from A.
bisporus immobilized on ceramic-chitosan composite support using ABTS as substrate.
The optimum reaction pH for the laccase activity varies depending on the type of
substrate. Rotkova et al. [22] studied the effect of pH on laccase activity from Trametes
versicolor with two substrates and concluded that the optimal pH for the reaction
environment was 5.0 and 3.5 for the substrates syringaldazine and ABTS, respectively.
The immobilized laccase presented higher pH-stability comparing with the free laccase
in the pH range 3.0-5.0. These results indicated that the immobilization improved the
stability of the laccase in the acidic region.
At the optimum pH value, free and immobilized laccase had maximal activity in
the temperature range 50-60 ºC. For free laccases, optimum temperatures have been
51
reported in the same range by other researchers [21, 25]. However, the immobilized
laccase presented higher relative activity comparing with the free laccase for
temperatures above 60ºC, which agree with other investigators studding immobilized
laccase from A. bisporus [25] and laccase from other fungi [1, 14, 29]. The activity of
the immobilized laccase decreased more slowly than for the free enzyme after one hour
at 50 ºC. The thermal stability is one of the most important features for biotechnological
application of the biocatalyst and the enhanced thermal stability of laccase arising from
immobilization would be an advantage for its industrial application.
Values of the Michaelis constants (Km) of different laccases widely vary for the
same substrate. Establishing comparison between characteristics of laccase isolated
from fungal sources is very delicate due to the variation in experimental conditions [16].
The apparent Km value of immobilized laccase (145 µM) was found to be
approximately 1.7 times higher than that for the free laccase (85 µM). The enzyme has
usually its activity lowered and the apparent Km increased after immobilization. These
alterations result from structural changes introduced to the enzyme by the applied
immobilization procedure and from the creation of a microenvironment in which the
enzyme works, different from the bulk solution [10]. The apparent Km value was 66.64
µM reported to immobilized laccase from A. bisporus on ceramic composite by using
glutaradehyde using with ABTS as substrate was in pH 3.0 buffer [25]. Cabana et al. [6]
reported an increasing of approximately six times in apparent Km value (31.0 μM) to
free laccase from Trametes versicolor on ABTS at pH 3.0 after immobilization
procedure laccase on the diatomaceous earth support (192.0 μM) and Rekuc et al. [21]
reported an increasing of five times apparent Km value (39.4 μM) to free laccase from
Cerrena unicolor after immobilization on cellulose based carrier Granocel (214.9 μM).
52
The result of this work demonstrated a regular performance of the biocatalyst
which showed 70 % of its original activity even after five cycles of use. Furthermore,
the half-life of the immobilized laccase was 38.7 days.
In conclusion, this work demonstrated that immobilization of laccase from A.
bisporus by covalent binding on mPOS-PVA retained 70% of its specific activity, with
improved stability at pH 3.0-5.0 and temperature at 50 ºC. The immobilized enzyme
offered the advantage of quick separation under a magnetic field and retained 70% of its
original activity after five consecutive operations. Furthermore, this immobilized
preparation was able to oxidize phenolic compounds (phenol, guaiacol and tannic) at
lower performance (60%) than the soluble enzyme but its reusability overcomes this
limitation.
Acknowledgements This work has been supported by grants of CNPq (Brazilian
research Agency).
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57
Artigo II – submetido para publicação no periódico Biotechnology Letters Fator de
Impacto: 1.6.
Immobilization of Trametes versicolor laccase on magnetic polysiloxane-polyvinyl alcohol composite
and application in phenolic mixtures oxidation
Roziana Cunha Cavalcanti Jordãoa, Luiza Rayanna Amorim de Limab, Valdemir Alexandre dos Santosa,
Luiz Bezerra de Carvalho Juniorb
b
L. B. Carvalho Junior () and L. R. A. Lima
Laboratório de Imunopatologia Keizo Asami (LIKA) and Departamento de Bioquímica,
Universidade Federal de Pernambuco,
Av. Morais Rego, Campus Universitário, 50670-910,
Recife, Pernambuco, Brazil.
E-mail: [email protected]
Tel: 55 81 21268484, Fax: 55 81 32283242.
a
Roziana C. C. Jordão and Valdemir Alexandre dos Santos
Centro de Ciências e Tecnologia,
Universidade Católica de Pernambuco,
Rua do Príncipe, 526, Boa Vista, CEP: 50050-900
Recife, Pernambuco, Brazil.
e-mail: [email protected]
Abstract – Abstract – Laccase from Trametes versicolor immobilization on magnetic polysiloxanepolyvinyl alcohol particles (mPOS-PVA) and its application for removing phenolic compounds from a
phenol model mixture were studied. The mPOS-PVA particles were prepared by using sol-gel process
and magnetized by Fe2+ and Fe3+ co-precipitation. Immobilization conditions and application of the
immobilized enzyme in the phenol oxidation were investigated. Central composite rotatable design and
response surface methods were employed to evaluate the effects of immobilization parameters, such as
58
enzyme concentration, immobilization pH and immobilization time. The amount of immobilized laccase
on the mPOS-PVA particles was determined as 3.0 mg/g particles under optimized working conditions
(50 µg ml-1 laccase, pH 4.5, 180 min and 25 ◦C). The recovered activity of the immobilized laccase on the
mPOS-PVA particles was about 50% compared to free enzyme, whereas higher loadings gave rise to a
less-efficient biocatalyst. The immobilized laccase was used for the oxidation of a mixture of five
phenolic compounds (phenol, guaiacol, pyrogallol, resorcinol and tannic acid) chosen among those
present in paper-mill industry. Phenol compounds were transformed via oxidation reaction catalyzed by
laccase mostly in an insoluble product which was simultaneously separated by filtration through the
membrane. To obtain the optimum conditions for the phenol oxidation a central composite rotatable
design with different combinations of pH, phenol concentration and reaction time was performed. Under
optimum conditions, the immobilized derivative on mPOS–PVA reduced 65.1% of the original phenol
content from the model solution. Results of these experiments indicated that response surface
methodology was a promising method for optimization of protein immobilization and that immobilized
laccase on magnetic polysiloxane-polyvinyl alcohol particles is effective in the transformation of phenolic
mixtures.
Keywords Polysiloxane - Polyvinyl alcohol - Laccase - Immobilization - Magnetic particles-Trametes
versicolor
Introduction
Laccases or benzenediol: oxygen oxidoreductase (E.C. 1.10.3.2) are glycosylated polyphenol
oxidases which contain four copper ions per molecule and catalyze the one-electron oxidation of four
reducing-substrate molecules concomitant with the four-electron reduction of molecular oxygen to water.
These enzymes oxidize a broad range of substrates, such as polyphenols, methoxy-substituted phenols,
diamines, and some inorganic compounds. In the presence of mediators, fungal laccases exhibit an
enlarged substrate range and are then able to oxidize compounds with a redox potential exceeding their
own (Baldrian 2006). Generally, the oxidation of a subtrate by laccases leads to polymerization of the
products through C-O and C-C oxidative coupling reactions. This process leads to detoxification of water
59
polluted by precipitation of phenolic contaminats (Dec and Bollag 1990; Ko and Chen 2008). Laccase
genes from a number of ligninolytic fungi, including Trametes versicolor, have previously been cloned
and characterized (Roy-Arcand and Archibald 1991; Jonsson et al. 1995; Collins and Dobson 1997;
Piontek et al. 2002; Ha et al. 2005; Lorenzo et al. 2006; Matijosyte et al. 2008).
Laccases are used in various industrial processes such as pulp delignification, wood fiber
modification, dye decolorisation, wine clarification (Minussi et al. 2007) and biosensor development
(Odaci et al. 2007), as well as in environmental processes like bioremediation of soils and water (Ryan et
al. 2007, Moldes and Sanroman 2006). This research area is the subject of intense biotechnology activity
in the biodegradation of effluents containing aromatic compounds, especially phenolic substances
(Canfora et al. 2008; Ceylan et al. 2008; Kurniawati and Nicell 2008). Phenols and derivatives
compounds are introduced into surface waters from industrial effluents such as those from the pulp-andpaper plants, textile industry, petroleum refining and domestic wastewaters.
Enzyme immobilization has gained increasing interest in recent years, owing to its many
advantages in comparison with their soluble form, such as the reutilization of the biocatalyst, increase of
stability, ease of recovery of enzyme and products exempt of contamination. Furthermore, the enzymes
immobilized onto magnetic support can be easily separated from the reaction mixture by a magnetic field
and can be implemented as continuous enzyme-catalyzed process (Brady and Jordaan 2009).
Laccases have been immobilized on a variety of support materials, by different methods (Duran
et al. 2002). Recent research work described laccase immobilization onto copolymer of butyl acrylate and
ethylene glycol dimethacrylate (Bryjak et al. 2007), nanoparticles and kaolinite [Hu et al. 2007],
magnetically separable mesoporous silica spheres (Zhu et al. 2007), epoxy-activated carriers (Kunamneni
et al. 2008), Poly(GMA/EGDMA) beads (Arica et al. 2009), diatomaceous earth support Celite® R-633
(Cabana et al. 2009), cellulose based Granocel 4000 (Rekuc et al. 2009), magnetic bead cellulose
(Rotkova et al. 2009), functionalized SBA-15 mesoporous silica (Salis et al. 2009) and magnetic chitosan
nanoparticles (Fang et al. 2009).
Magnetized Polysiloxane-Polyvinyl alcohol (mPOS–PVA) presented good characteristic for
immobilization of proteins (Barros 2002, Coêlho 2002) and was used for galactooligosaccharides
production from lactose (Neri et al. 2009).
60
The application of experimental design in enzyme immobilization process can result in reduction
in the number of experiments and improved statistical interpretation possibilities. Additionally, the factors
that influence the experiments are identified, optimized and the interaction that may exist between these
factors can be evaluated (Rodrigues and Iemma 2005).
There have been several studies to evaluate the laccase immobilization. These studies require a
large number of experiments to describe the effect of individual factors and are time consuming.
Experimental design and optimization of protein immobilization processes have been reported for
different enzymes such as pectinase (Li et al. 2007), invertase (Marquez et al. 2008), lipase (Chang et al.
2008) and protease (Ortega et al. 2009). However, only few recent studies aiming to the optimization of
laccase immobilization are available (Silva et al. 2007).
Therefore, the present work reports the optimum reaction conditions for immobilization of
laccase from Tramtes vesricolor on mPOS-PVA particles, using a biocatalyst immobilized in optimized
conditions, in the transformation of phenolic mixtures.
Materials and Methods
Chemicals
Laccase from T. versicolor 2,2, Azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) and bovine
serum albumine (BSA) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Ethanol
(minimum 99%) and polyvinyl alcohol (MW 72,000) were purchased from Reagen Chemical Co. (São
Paulo, Brazil). Glutaraldehyde (25%) and tetraethoxysilane (TEOS) were acquired from Aldrich
Chemical Co. (Milwaukee, WI, USA).
other reagents were of analytical grade.
Phenol, guaiacol, tannic acid, pyrogallol and resorcinol and all
61
Preparation of mPOS-PVA particles
Beads of POS-PVA hybrid composite were synthesized by sol-gel process according to Barros et al.
(2002). The POS-PVA beads were mechanically ground and submitted to magnetization according to
Carneiro Leão et al. (1991). The resulting mPOS-PVA particles were thoroughly washed with distilled
water until pH 7.0 and dried at 50 ºC overnight and finally sieved (<100µm).
Laccase immobilization onto mPOS-PVA particles (mPOS-PVA-laccase)
The mPOS-PVA particles were previously activated with 1 m l-1 2.5 % glutaraldehyde solution in 0.1 M
H2SO4 and washed with distilled water. The immobilization process was carried out by incubating mPOSPVA (10 mg) with 1.0 ml-1 of laccase solutions containing from 16 to 44 µg ml-1 of enzyme at different
pHs (acetate buffer 0.2 M to pH 4.58 and 5.0; phosphate buffer 0.2 M to pH 6.0, 7.0 and 7.4). The mPOSPVA was maintained in laccase solutions at 4° C for 3 h under rotary stirring (60 rpm). After that, the
mPOS-PVA-laccase particles were recovered by magnetic field and washed with 0.15 M NaCl solution
followed by adequate buffer solution and the supernatant was kept for protein measurements.
Determination of laccase activity and protein estimation
The activity of the free and immobilized laccase was spectrophotometrically determined in a reaction
mixture containing 0.05 mM ABTS as the substrate in 0.2 mM acetate buffer to pH 4.5 (free laccase) and
pH 3.0 (mPOS-PVA-laccase), containing a suitable amount of laccase, at 25o C (Hublik and Schinner
2000, Roy-Arcand and Archibald 1991). The absorbance of the solution (free) or supernatant
(immobilized) at 420 nm was spectrophotometrically determined (Ultrospec® 3000 Pro of Amersham
Pharmacia Biotech – UV/Visible spectrophotometer). The molar extinction coefficient of ABTS is 36 x
62
10-3 M-1 cm-1 and one unit of activity was defined as the amount of enzyme required to oxidize 1 µmol of
substrate per minute under the experimental conditions. The protein content was estimated according to
Lowry et al. (1951). The quantity of protein bound to the support was calculated by subtracting the
protein recovered in the washing of the support-enzyme complex from the protein used for
immobilization.
Oxidation of phenolic mixtures using the immobilized laccase
Phenolic mixtures composed of phenol, guaiacol, tannic acid, pyrogallol and resorcinol were prepared
and the oxidation was monitored at different conditions of pH, reaction time and phenol concentration.
The immobilized laccase was incubated with 1 ml of the phenolic mixture at 25º C protected from light.
Afterwards, samples were withdrawn and filtered through Millipore membrane (0.45 µm) and the total
phenol content was estimated according to the Singleton and Rossi (1965). The total phenol content of the
samples was expressed as milligram of gallic acid equivalents (GAE) per milliliter of phenolic mixture.
Phenol concentration of the untreated sample was taken as 100%. Control assays were performed using
phenolic mixture incubated without and with only mPOS-PVA support to evaluate non enzymatic
oxidations.
Experimental design and statistical analysis
The immobilization parameters were optimized using response surface methodology (RSM) and central
composite rotatable design (CCRD). For the determination of the factors considered as optimum two
types of CCRD were used: CCRD (22 plus axial and central points) for the laccase immobilization and
CCRD (23 plus axial and central points) for the phenol oxidation (Rodrigues and Iemma, 2005). Results
were analysed using the software Statistica® 7.0. The adjustment of the experimental data for the
independent variables was represented by the second-order polynomial equation:
63
64
y = β 0 + ∑ β j ⋅ x j + ∑ β ij x i x j + ∑ β jj x 2j + e
j
ip j
(1)
j
where y is the dependent variable (response variable) to be modeled; β0, βj, βij and βjj are regression
coefficients and xi and xj are the independent variables (factors) and e is the error. The model was
simplified by dropping terms that were not statistically significant (p > 0.05) by analysis of variance –
ANOVA (Montgomery 1991).
Results and discussion
Immobilization conditions of laccase on m-POS-PVA
Preliminary experiments were carried out to screen the parameters that influence the covalent
immobilization of laccase on mPOS-PVA particles via glutaraldehyde and to determine the experimental
area that could be used for optimization analysis (data not shown). The analysis of the overall data
indicated that the concentration of enzyme had the most pronounced effect on responses, although the
immobilization pH exerted a statistically significant effect. In fact, the immobilization time was not a
significant factor. Therefore, an immobilization time of 180 min was chosen as optimum for rest of
experiments. The most suitable operational condition to be used for further optimization using CCRD
was: pH 5.0-7.0; 20-40 µg ml-1 of laccase concentration and 180 min contact time.
The experimental design and the results obtained from the experiments are shown in Table 1.
Among the various treatments, the higher protein retentions (14.8 mg per g of support) was in the zero
level (pH 6.0 and 30 µg ml-1) and the smallest protein retention (6.5 mg per g of support) was pH 7.0 and
20 µg ml-1 (run 2).
65
Table 1 Experimental design and results according to the CCRD 22.
Variable level
Response
Laccase concentration (µg ml-1)
pH
Protein retention
X1
X2
(mg/g support)
1
-1(20)
-1(5)
12.00
2
-1(20)
+1(7)
6.50
3
+1(40)
-1(5)
13.00
4
+1(40)
+1(7)
12.00
5
-1.42 (16)
0 (6)
7.60
6
+1.42 (44)
0 (6)
12.00
7
0 (30)
-1.42 (4.6)
12.40
8
0 (30)
+1.42 (7.41)
10.30
9
0 (30)
0 (6)
14.40
10
0 (30)
0 (6)
14.80
11
0 (30)
0 (6)
14.60
12
0 (30)
0 (6)
14.70
Run
The ANOVA presented in Table 2 indicated that the model was statistically significant and
adequate to represent the actual relationship between the independent variables (factors) and the
dependent variable or response (protein loading), which can be confirmed by the F-ratio values (ratio for
calculated by established F values). The fitness of the model was expressed by the R2 value, which was
0.977, indicating that 97.7 % of the variability in the response can be explained by the model. This
suggested that the model accurately represented the data in the experimental region. The reduced value
for the pure error demonstrated dominium of the technique.
66
Table 2 ANOVA of protein loading of mPOS-PVA including laccase concentration, immobilization as
well as their interactions a.
Source
SS
df
MS
F-ratio
p
(X1): laccase concentration
20.23288
1
20.23288
68.4934
0.00012
(X2): pH immobilization
11.20975
1
11.20975
37.9478
0.00029
(X1 )(X1)
34.96900
1
34.96900
118.378
0.00005
(X2)(X2)
15.62500
1
15.62500
52.8945
0.00017
(X1)(X2)
5.06250
1
5.06250
17.1378
0.00094
Pure Error
0.08750
3
0.02917
a
SS, sum of squares; df, degrees of freedom, MS, mean square, R2= 0.977
Fig. 1 shows the graphical representation (Pareto chart) of the “size effect” of each of the
parameters investigated upon protein loading. The quadratic term for the laccase concentration (X1)
showed the most pronounced effect on the response and in the second place comes the linear term of this
same factor, together with the quadratic term of pH (X2).
-4.675
X 1(Q)
3.180635
(1)X 1(L)
-3.125
X 2(Q)
-2.36746
(2)X 2(L)
1Lby2L
2.25
2
3
4
Effect Estimate (Absolute Value)
Fig. 1 Pareto chart for the laccase immobilization.
5
67
The protein loading (Y) as a function of the X1 and X2 factors was obtained in the form of the
following quadratic equation:
Y = −40.0811 + 0.8865 ⋅ X1 − 0.0234 ⋅ X12 + 14.1913 ⋅ X 2 − 1,5625 ⋅ X 22 + 0.1125 ⋅ X1 ⋅ X 2
(2)
Equation (2) was used and three dimensional plots were drawn. Fig. 2 shows a well defined
region of optimum factor values for the protein loading. The response surface showed a maximum region
corresponding to pH 6.0 and laccase concentration of 30 µg ml-1 and with protein loading of about 15 mg
per gram of support.
Fig. 2 Response surface of the effect of laccase concentration, immobilization pH and their mutual
interaction on protein loading (immobilization time = 180 min).
The immobilization process can affect the physical and chemical properties of enzymes. The
properties of the immobilized derivative obtained are due to the following factors: diffusional effects or
mass transfer limitations - come from the diffusion resistance of the substrate to the catalytic site of the
enzyme, conformational effects of the enzyme molecule due to the change in the tertiary structure of the
activesite; microenvironmental effects - resulting from the method of immobilization used and nature of
support. As the specific activity is an essential parameter for the application of the biocatalyst in the
oxidation of phenol, this parameter was investigated in the same conditions described before for protein
loading. Fig. 3 shows the response surface obtained, where one can observe an opposite behavior to the
one shown in Fig. 2.
Fig. 3 Response surface of the effect of laccase concentration, immobilization pH and their mutual
interaction on specific activity (time immobilization = 180 min).
The analysis of effect of specific activity as function of laccase concentration and pH showed
that the response variable (specific activity) can reach higher values when the pH is kept around 4.0 and
laccase concentration > 50 µg ml-1. As expected, the higher protein loading in the zero level (30 µg ml-1
and pH 6.0) presented smaller specific activity. This phenomenon previously observed for other
immobilized enzymes at high loading can be explained in terms of limitations due the diffusion of the
substrates inside the support before reaching the enzyme active site (Zhu et al. 2007). Additional tests
using phenol as a substrate showed that 50 µg ml-1 laccase concentration, pH 4.5 and 180 min
immobilization time were the best conditions for the preparation of immobilized biocatalyst. The choice
of values which are not coincident with the ones suggested in Fig. 3 can be explained by a considerable
68
69
interaction between the factors X1 and X2 (projection of the response surface) which makes it difficult to
explain the phenomenon based on only one factor.
Optimization of oxidation of phenolic mixtures using immobilized laccase on mPOS-PVA
The immobilized laccase capacity for oxidation of phenol mixtures was evaluated and the
experimental design and the results obtained from the experiments are shown in Table 3. The conditions
to reach the higher phenol oxidation (65.1 %) were observed in zero level (1.0 mM of phenol
concentration, pH 6.0 and 32 h of reaction time) and not detected oxidation at pH 7.0, 1.5 mM of phenol
concentration and 16 h of reaction time (run 7).
The ANOVA presented in Table 4 indicated that the model was statistically significant and
adequate to represent the actual relationship between the factors and the phenol oxidation, which can be
confirmed by the F-ratio values. The fitness of the model was 0.937, indicating that 93.7 % of the
variability in the response can be explained by the model. The reduced experimental error revealed an
excellent dominium of the operational technique.
Fig. 4 shows the graphical representation (Pareto chart) of the parameters investigated upon
phenol oxidation.
The quadratic term for the pH (X1), phenol concentration (X2) and time reaction (X3) variables
showed pronounced effect on the oxidation and in the second place comes the linear term of X3 variable.
The oxidation of phenol (Y) as a function of the X1, X2 and X3 factors was obtained in the form
of the following quadratic equation:
Y = −429.844 + 138.811 ⋅ X1 − 13.302 ⋅ X12 + 88.258 ⋅ X 2 − 62.402 ⋅ X 22 _ 1.176 ⋅ X 3 −
- 0.051 ⋅ X 32 + 6.900 ⋅ X1 ⋅ X 2 + 0.487 ⋅ X1 ⋅ X 3 − 0.194 ⋅ X 2 ⋅ X 3
(3)
70
Equation (3) was used and three dimensional plots were drawn. Figs. 5a-c shows well defined
regions of optimum factor values for the phenol oxidation. The response surfaces showed maximum
region corresponding to the phenol concentration 1.0 mM, pH 6.0 and 32 h of reaction time obtaining
reduction of 65.1 % original phenol content.
Table 3 Experimental design and results according to the CCRD 23
Response
variable level
pH
Phenol concentration (mM)
Time (h)
run
X1
X2
X3
phenol oxidation (%)
1
-1(5.0)
-1(0.5)
-1(16)
12.0
2
-1(5.0)
-1(0.5)
+1(48)
40.0
3
-1(5.0)
+1(1.5)
-1(16)
14.4
4
-1(5.0)
+1(1.5)
+1(48)
15.0
5
+1(7.0)
-1(0.5)
-1(16)
5.0
6
+1(7.0)
-1(0.5)
+1(48)
43.0
7
+1(7.0)
+1(1.5)
-1(16)
0.0
8
+1(7.0)
+1(1.5)
+1(48)
53.0
9
-1.68(4.3)
0(1.0)
0(32)
26.0
10
+1.68(7.7)
0(1.0)
0(32)
28.0
11
0(6.0)
-1.68(0.16)
0(32)
18.0
12
0(6.0)
+1.68(1.84)
0(32)
23.0
13
0(6.0)
0(1.0)
-1.68(5)
20.0
14
0(6.0)
0(1.0)
+1.68(59)
36.0
15
0(6.0)
0(1.0)
0(32)
65.1
16
0(6.0)
0(1.0)
0(32)
64.5
17
0(6.0)
0(1.0)
0(32)
64.2
18
0(6.0)
0(1.0)
0(32)
65.1
19
0(6.0)
0(1.0)
0(32)
64.8
20
0(6.0)
0(1.0)
0(32)
64.2
71
Table 4 - ANOVA of phenol oxidation using immobilized laccase on mPOS-PVA including pH, phenol
concentration and time reaction.
Source
SS
df
MS
F-ratio
p
(X1): pH
38.613
1
38.613
35.2316
0.00002
(X2): phenol concentration
6.186
1
6.186
5.6439
0.00170
(X3): time
1571.723
1
1571.723
1434.103
0.00000
(X1 )(X1)
2550.153
1
2550.153
2326.861
0.00000
(X2)(X2)
3507.379
1
3507.379
3200.272
0.00000
(X3)(X3)
2416.399
1
2416.399
2204.818
0.00000
(X1)(X2)
95.220
1
95.220
86.88251
0.00000
(X1)(X3)
486.720
1
486.720
444.1027
0.00000
(X2)(X3)
19.220
1
19.220
17.5370
0.00012
Pure Error
0.829
5
0.166
a
SS, sum of squares; df, degrees of freedom, MS, mean square, R2= 0.937.
-31.2011
X 2(Q)
-26.6049
X 1(Q)
-25.8978
X 3(Q)
21.4557
(3)X 3(L)
15.6
1Lby3L
1Lby2L
6.9
3.362939
(1)X 1(L)
-3.1
2Lby3L
-1.346
(2)X 2(L)
0
5
10
15
20
25
Effect Estimate (Absolute Value)
Fig. 4 Pareto chart for phenol oxidation.
30
35
(a)
(b)
(c)
Fig. 5 Response surface plots for oxidation of phenolic mixture as a function of: (a) phenol concentration
and pH for 32 h; (b) reaction time and phenol concentration at pH 6.0; (c) pH and time of reaction at 1.0
mM of phenol concentration.
72
The ability of laccase from T. versicolor to exert catalytic activity on many types of aromatic
compounds has been demonstrated and particularly the oxidation of phenols substrates under various
reaction conditions has been reported (Canfora et al. 2008; Kurniawati and Nicell 2008; Roy-Arcand and
Archibald 1991). At the same time only few studies have been reported about the application of
immobilized laccase in the biodegradation of phenolic mixtures (Bayramoglu and Arica 2009; Salis et al.
2009). Different authors used variables times (10 to 24 h) for degradation, and this parameter is
dependent on the time course of the reaction, initial concentration, and mainly, the complexity of the
substrates utilized (Gianfreda et al. 1999; Giardina et al. 2010). Russo et al. (2007) reported that the
decrease of the phenol oxidation rate after a long time is due to the possible accumulation of the
degradation products, causing inhibitory effect in the enzymatic degradation process. The mixture of
phenolic compounds used in this study contain large molecules such as tannic acid and oxidation process
could be improved with addition of a mediator molecule in the reaction medium.
Conclusion
Immobilized laccase from T. versicolor was prepared on POS-PVA magnetic particles and the
immobilization process was optimized. The laccase immobilized on mPOS-PVA prepared in this study
had the capacity of oxidation of phenolic mixtures by enzymatic biodegradation. The use of experimental
design permitted the rapid screening of a large experimental domain for optimization of the laccase
immobilization. The characteristics of the immobilized laccase from T. versicolor on mPOS–PVA were
influenced by pH and laccase concentration. The fit of the model was confirmed by the value of the
determination coefficient (R2) indicating that 97.7 % of total variations were explained by the model. The
optimized conditions for laccase immobilization (3.3 mg per gram of support) were: initial laccase
concentration of 50 ug ml-1, pH 4.5 and 3 hours contact time.
This research work showed that the phenol content of the mixture could be reduced 65.1% of its
original amount after 32 h oxidation by immobilized laccase. The fit of the model was checked by the
value of the determination coefficient (R2) indicating that 93.7 % of total variations were explained by the
model. Furthermore, the products were removed from the system by filtration.
73
Acknowledgements This work has been supported by grants of CNPq (Brazilian research Agency).
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immobilization by adsorption in ionic exchange resin for sucrose hydrolysis. J Mol Catal B Enzym
51: 86-92.
Matijošyte I, Arends IWCE, Sheldon RA, Vries S (2008) Pre-steady state kinetic studies on the
microsecond time scale of the laccase from Trametes versicolor. Inorg Chim Acta 361: 1202-1206.
Minussi RC, Rossi M, Bologna L, Rotilio D, Pastore GM, Duran N (2007) Phenols removal in musts:
strategy for wine stabilization by laccase. J Mol Cat B: Enz 45: 102–107.
Moldes D, Sanromán MA (2006) Amelioration of the ability to decolorize dyes by laccase: relationship
between redox mediators and laccase isoenzymes in Trametes versicolor. World J Microbiol
Biotechnol 22: 1197-1204.
Montgomery DC (1991) Design and Analysis of Experiments. John Wiley and Sons, London.
76
Neri DFM, Balcão VM, Carneiro-da-Cunha MG, Carvalho Jr. LBC, Teixeira J (2008) Immobilization of
β-galactosidase from Kluyveromyces lactis onto a polysiloxane- polyvinyl alcohol magnetic (mPOSPVA) composite for lactose hydrolysis. Catal Comm 9: 2334-2339.
Neri DFM, Balcão VM, Costa RS, Rocha ICAP, Ferreira EMFC, Torres DPM, Rodrigues LRM,
Carvalho Jr. LBC, Teixeira JA (2009) Galacto-oligosaccharides production during lactose hydrolises
by free Aspergillus oryzae β-galactosidase and immobilized on magnetic polysiloxane-polyvinyl
alcohol. Food Chem 115: 92-99.
Odaci D, Timur S, Pazarlioglu N, Montereali MR, Vastarella W, Pilloton R, Telefoncu A (2007)
Determination of phenolic acids using Trametes versicolor laccase. Talanta 71: 312-317.
Ortega N, Perez-Mateos M, Pilar MC, Busto MD (2009) Neutrase immobilization on alginateglutaraldehyde beads by covalent Attachment. J Agric Food Chem 57: 109-115.
Piontek K, Antorini M, Choinowski T (2002) Crystal structure of a laccase from the fungus Trametes
versicolor at 1.90-Å resolution containing a full complement of coppers. J Biol Chem 277: 3766337669.
Rekuc A, Bryjak J, Szymanska K, Jarzebski AB (2009) Laccase immobilization on mesostructured
cellular foams affords preparations with ultra high activity. Process Biochem 44: 191-198.
Rodrigues MI, Iemma AF (2005) Planejamento de experimentos e otimização de processos. Casa do pão,
São Paulo.
Rotkova J, Sulakova R, Korecka L, Zdrazilova P, Jandova M, Lenfeld J, Horak D, Bilkova Z (2009)
Laccase immobilized on magnetic carriers for biotechnology applications. J Magn Magn Mater 321:
1335–1340.
Roy-Arcand L, Archibald FS (1991) Direct dechlorination of chiorophenolic compounds by laccases from
Trametes (Coriolus) versicolor. Enzyme Microb Technol 13: 194-203.
Ryan D, Leukes W, Burton S (2007) Improving the bioremediation of phenolic wastewaters by Trametes
versicolor. Bioresour Technol 98: 579-587.
77
Salis S, Pisano M, Monduzzi M, Solinas V, Sanjust E (2009) Laccase from Pleorotus sajor-caju on
functionalized SBA-15 mesoporous silica: immobilization and use for the oxidation of phenolic
compounds. J Mol Catal B Enzym 5: 175-180.
Silva C, Silva CJ, Zille A, Guebitz GM, Cavaco-Paulo A (2007) Laccase immobilizaation on
enzymatically functionalized polyamide 6,6 fibres. Enzyme Microb Technol 41 867-875.
Singleton VL, Rossi JA (1965) Colorimetry of totalphenolics with phosphomolybdic-phosphotungstic
acid reagents. Am J Enol Viticul 16:144-158.
Zhu Y, Kaskel S, Shi J, Wage T, Van Pée K-H (2007) Immobilization of Trametes versicolor lacase on
magnetically separable mesoporous silica spheres. Chem Mater 19: 6408-6413.
78
6. CONCLUSÕES
Lacases de Agaricus bisporus e Trametes versicolor foram imobilizadas em
partículas magnéticas de rede semi interpenetrada de polisiloxano e álcool polivínilico
(m-POS-PVA);
O derivado enzimático obtido foi capaz de oxidar misturas contendo fenóis
comumente encontrados na indústria papeleira;
A metodologia de superfície de resposta foi um método eficiente para a
otimização de imobilização de proteínas.
79
80
7. ANEXOS
7.1 ANEXO 1
INSTRUCTIONS
FOR
AUTHORS
-
JOURNAL
OF
INDUSTRIAL
MICROBIOLOGY & BIOTECHNOLOGY
Editor-in-Chief: Allen I. Laskin
ISSN: 1367-5435 (print version)
ISSN: 1476-5535 (electronic version)
Editorial procedure
Authors should submit their articles online to facilitate even quicker and more efficient
processing. Electronic submission substantially reduces the editorial processing and
reviewing time and shortens overall publication time.
Please log directly onto the link below and upload your manuscript following the
instructions given on screen.
All manuscripts are subject to review by at least two members of the journal’s editorial
board or other experts.
Upon receipt, manuscripts will be assigned a manuscript tracking number and
forwarded to a Senior Editor. The corresponding author will be notified via e-mail
when the manuscript is received and informed of the manuscript tracking number and
the Senior Editor who will be handling the review. Queries regarding the review and
revisions of the manuscript should be directed to the Senior Editor. The manuscript
tracking number should be included in all correspondence regarding the submission.
The Senior Editor advises the corresponding author of his/her and the reviewers’
comments. If minor or major revisions are recommended, revised manuscripts should
be returned to the Senior Editor handling the manuscript. Revised manuscripts should
be submitted directly to the Senior Editor as e-mail attachments. At this point in the
review process, higher-quality figures may be requested if necessary. Accepted
manuscripts will not be forwarded to the publisher without an electronic copy of the
final revision. Papers that do not conform to the journal norms will be returned to the
authors for revision before being considered for publication. When the Senior Editor is
satisfied that the manuscript is ready for acceptance, s/he forwards it to the Editor-inChief for final acceptance.
The journal accepts manuscripts for the following sections:
•
Original Papers should normally not exceed 16 printed pages (one printed page
corresponds to about 850 words of text or 3 illustrations with their legends).
•
Short Communications should not exceed 3 printed pages.
•
Letters to the Editor should not exceed 2 printed pages.
•
Review Papers, including mini-reviews, should be critical reviews on subjects
of interest to industrial and applied microbiologists. The length of the article will
depend on the subject. Authors considering preparation of a review should contact the
Editor-in-Chief in advance to determine the suitability of the topic.
All manuscripts are subject to copy-editing after acceptance.
Manuscript preparation
General remarks:
Manuscripts should be typed double spaced, including figure legends, any footnotes to
tables or figures, and references.
All manuscripts must conform to the current edition of the CBE Style Manual for
Biological Editors and are subject to copy-editing.
Title page:
The title page must include the name(s) of the author(s), a concise and informative title,
the affiliation(s) and address(es) of the author(s), and the e-mail address and telephone
and fax numbers of the corresponding author.
Abstract:
Each paper, including Reviews, must be preceded by an abstract of approximately 250
words or less presenting the questions being addressed or the hypothesis being tested,
the general methods used (e.g. liquid chromatography and mass spectroscopy) and the
most important results and conclusions.
81
82
Keywords:
Up to 5 keywords should be supplied after the Abstract for indexing purposes.
Abbreviations:
All abbreviations should be introduced parenthetically in the text when the term first
appears (except for standard physiological and biochemical abbreviations).
Subsequently only the abbreviation should be used.
Footnotes:
Footnotes to the text are numbered consecutively. Those to tables should be indicated
by superscript lower-case letters, beginning with "a" in each table. (Asterisks may be
used for statistical values or data).
Introduction:
The Introduction should state the purpose of the investigation and give a short review
of the pertinent literature. It should conclude with a concise statement of the author’s
objectives.
Materials and Methods:
The Materials and Methods section should follow the Introduction and should provide
enough information to permit repetition of the experimental work. The name of
suppliers or sources of equipment and chemicals should be given parenthetically, with
the city, state/province/county and country. This information need not be given for
common
equipment
found
in
most
laboratories
(balances,
pH
meters,
spectrophotometers) or common chemicals (NaCl, DNA) the source of which is not
crucial to repetition of the work. The grade of chemicals should be stated if it is
important for repetition of the work.
Results:
Present your findings, stating the major trends shown by data in figures or tables, but
do not repeat in the text data that are obvious from the figures or tables. The number of
replicates involved and the number of independent repetitions of the experiment or
measurement should be stated here or as a footnote to a table.
Discussion:
State your conclusions from the data and discuss how they compare with previously
published information on the subject. If appropriate, suggest theoretical implications
and propose future studies. It may be appropriate to combine Results and Discussion,
particularly where the results of one experiment are the basis for the next experiment
reported in this paper.
Acknowledgements:
These should be as brief as possible. Any grant that requires acknowledgement should
be mentioned. The names of funding organizations should be written in full.
References:
The list of references should include only works that are cited in the text and that have
been published or accepted for publication. Abstracts, theses and presentations at
meetings are not acceptable as references. Personal communications should be
mentioned in the text with the affiliation of the individual providing the communication
and not included in the list of references.
Citations in the text should be identified by numbers in square brackets, and the list of
references at the end of the paper should be both alphabetized under the first author’s
name and numbered. References by the same author or team of authors should be listed
in chronological order. Here are a few examples of the style of references:
1. Atlas RM (2005) Handbook of media for environmental microbiology. CRC Press,
Boca Raton, Fla, USA
2. van Ginkel CG, Middlehuis BJ, Spijk F, Abma WR (2005) Cometabolic reduction of
bromate by a mixed culture of microorganisms using hydrogen gas in a gas-lift reactor.
J Ind Microbiol Biotechnol 32:1-6
3. Heeschan W, Hahn G (1982) Quality control of media for Lactobacillus and
Streptococcus. In: Corry JE (ed) Culture media. GIT, Darmstadt, Germany, pp 109-119
If available, the Digital Object Identifier (DOI) of the cited literature should be added at
the end of the reference in question, e.g. “...J Ind Microbiol Biotechnol 30:1-5. DOI
10.10007/s10295-002-0001-5”
83
Illustrations and Tables:
All figures (photographs, graphs or diagrams) and tables should be cited in the text, and
both figures and tables should be numbered separately and consecutively throughout.
Figure parts should be identified by lower-case letters.
Line drawings:
Please submit high-quality images. Inscriptions should be clearly legible. See
"Preparing your manuscript" for preferred file formats.
Halftone illustrations:
Black-and-white and color halftone illustrations should be submitted as well-contrasted
images correctly aligned with the top facing up. Magnification should be indicated by a
scale bar.
Size of figures:
Figures should match the width of either one column (8.6 cm) or two columns (17.6
cm). The maximum length is 23.5 cm, including the legend.
Figure legends:
Legends must be brief, self-sufficient explanations of the illustrations. The legends
should be placed together at the end of the text.
Tables:
Each table should have a title. Abbreviations, except widely used ones (e.g. s, M, or
cm), should be identified in a footnote. Footnotes to tables should be indicated by
superscript lower-case letters, beginning with “a” in each table. (Asterisks may be used
for significance values and other statistical data.)
Color illustrations
Online publication of color illustrations is free of charge. Since JIMB does not have
funds to support color illustrations, authors wishing to have illustrations in color will be
required to pay € 950 (plus 19% VAT), irrespective of the number of color figures in
the article.
Preparing your manuscript
84
Layout guidelines:
1. Use a normal, plain font (e.g., Times Roman) for text.
Other style options:
a) For textual emphasis, use italics.
b) For special purposes, such as for vectors, use boldface.
2. Use the automatic page-numbering function to number the pages.
3. Lines should be numbered consecutively throughout the text.
4. Do not use field functions.
5. For indents use tab stops or other commands, not the space bar.
6. Use the table functions of your word processing program, not spreadsheets,
to make tables.
7. Use the equation editor of your word processing program or MathType for
equations.
8. Place any figure legends or tables at the end of the manuscript.
9. Submit all figures as separate files and do not integrate them into the text.
Data formats:
Save your text file in an MS Word-compatible format.
Illustrations:
The preferred figure formats are EPS for graphics exported from a drawing program
and TIFF for halftone illustrations. Legible, lower-quality formats (JPG, JPEG) may
be used in order to meet file size restrictions on initial submission, but the original,
higher-quality files may be requested during the review process. Each figure should
be in a separate file, and the file name should include the figure number. Figure
legends should be appended to the text after the reference list and not placed in the
figure files.
Color illustrations:
Store color illustrations as RGB (8 bits per channel) in TIFF format. Legible,
lower-quality formats (JPG, JPEG) may be used in order to meet file size
restrictions on initial submission, but the original, higher-quality files may be
requested during the review process.
85
Scan resolution:
Scanned line drawings should be digitized with a resolution that will yield a
resolution of at least 800 dpi in the published figure. For digital halftones, 300
dpi is usually sufficient.
Vector graphics:
Fonts used in the vector graphics must be included. Do not draw with
hairlines. The minimum line width is 0.2 mm (0.567 pt) in the published
figure.
General information on data delivery:
After acceptance of a manuscript, it will be forwarded electronically to the
publisher at the following address:
Journal Production Life Sciences/Chemistry
Springer-Verlag
Tiergartenstr. 17
69121 Heidelberg
Germany
Tel: +49-6221-487-8500
Fax: +49-6221-487-8527
If additional materials (i.e. print-quality figures) are required, the publisher
will contact the corresponding author at the e-mail address listed on the
submission. The publisher will also provide proofs electronically to the
corresponding author for review prior to publication.
Electronic supplementary material
Electronic supplementary material (ESM) for a paper will be published in the electronic
edition of the journal provided the material is:
1. Submitted in electronic form together with the manuscript
2. Accepted after peer review
ESM may consist of:
86
Information that cannot be printed: animations, video clips, sound recordings (use
QuickTime, AVI, MPEG, animated GIFs, or any other common file format)
Information that is more convenient in electronic form: sequences, spectral data, etc.
Large quantities of original data that relate to the paper: additional tables, large numbers
of illustrations (color or black-and-white), etc.
Legends for ESM tables and figures must be brief, self-sufficient explanations. ESM is
to be numbered and referred to as S1, S2, etc. After acceptance for publication, ESM
will be published as received from the author in the online version of the article only. It
is referred to in the printed version
Proof readingProofreading
Authors are informed by e-mail that a temporary URL has been created from which they
can obtain their proofs. Proofreading is the responsibility of the author. Authors should
make their proof corrections (formal corrections only) on a printout of the PDF file
supplied, checking that the text is complete and that all figures and tables are included.
Substantial changes in content, e.g. new results, corrected values, title and authorship,
are not allowed without the approval of the responsible editor. In such a case please
contact the journal’s Editorial Office before returning the proofs to the publisher. After
online publication, corrections can only be made in exceptional cases and in the form of
an erratum, which will be hyperlinked to the article.
OffprintsOffprints
Twenty-five offprints of each contribution are supplied free of charge. Orders for
additional offprints can be placed by filling out the order form that is provided with the
proofs. When ordering additional offprints, an author is entitled to receive, upon
request, a PDF file of the article for personal use.
Online First
Papers will be published online about one week after receipt of the corrected proofs.
Papers published online can be cited by their DOI. After release of the printed version,
the paper can also be cited by issue and page numbers.
Legal requirements
87
The author(s) guarantee(s) that the manuscript will not be or has not been published
elsewhere in any language without the consent of the copyright holder (the Society for
Industrial Microbiology); that the rights of third parties will not be violated; and that the
reviewers, editors, publisher, or SIM will not be held legally responsible should there be
any claims for compensation.
Authors wishing to include figures, tables or text passages that have already been
published elsewhere are required to obtain permission from the copyright holder(s) and
to include evidence that such permission has been granted when submitting their papers.
Any material received without such evidence will be assumed to originate from the
authors. Authors of an article published in JIMB may use figures and tabular material in
their own subsequent publications without permission, as long as the original JIMB
paper is credited
The editors reserve the right to reject manuscripts that do not comply with the above
requirements. The author will be held responsible for false statements or failure to fulfill
these requirements.
Springer Open Choice
In addition to the traditional publication process, Springer now provides an alternative
publishing option: Springer Open Choice (Springer's open access model). A Springer
Open Choice article receives all the benefits of a regular article, but in addition is made
freely available through Springer's online platform SpringerLink. To publish via
Springer Open Choice upon acceptance of your manuscript, please click on the link
below to complete the relevant order form and provide the required payment
information. Payment must be received in full before free access publication.
After AcceptanceAfter Acceptance
Upon acceptance of your article you will receive a link to the special Author Query
Application at Springer’s web page where you can sign the Copyright Transfer
Statement online and indicate whether you wish to order Open Choice,
paper offprints, or printing of figures in color.
Once the Author Query Application has been completed, your article will be processed
and you will receive the proofs.
88
Copyright transfer
Authors will be asked to sign the Copyright Transfer Statement
for their paper. This will
ensure the widest possible protection and dissemination of information under
copyright laws.
Open Choice articles do not require transfer of copyright as the copyright remains with
the author. In opting for open access, they agree to the Springer Open Choice Licence.
Offprints
Additional offprints can be ordered by the corresponding author.
Color illustrations
Online publication of color illustrations is free of charge. For color in the print version,
authors will be expected to make a contribution towards the extra costs.
89
7.2 ANEXO II
INSTRUCTIONS FOR AUTHORS - BIOTECHNOLOGY LETTERS
Executive Editor: C. Ratledge
ISSN: 0141-5492 (print version)
ISSN: 1573-6776 (electronic version)
MANUSCRIPT SUBMISSION
Submission of a manuscript implies: that the work described has not been published
before; that it is not under consideration for publication anywhere else; that its
publication has been approved by all co-authors, if any, as well as by the responsible
authorities – tacitly or explicitly – at the institute where the work has been carried out.
The publisher will not be held legally responsible should there be any claims for
compensation.
Permissions
Authors wishing to include figures, tables, or text passages that have already been
published elsewhere are required to obtain permission from the copyright owner(s) for
both the print and online format and to include evidence that such permission has been
granted when submitting their papers. Any material received without such evidence
will be assumed to originate from the authors.
How to Submit
Manuscripts should preferably be submitted in the original file format. Please follow
the hyperlink “Submit online” on the right to open an e-mail to the editor and attach the
files.
If this is not possible, one printout of the manuscript must be submitted to the editor.
TITLE PAGE
The title page should include:
The name(s) of the author(s)
A concise and informative title
The affiliation(s) and address(es) of the author(s)
90
The e-mail address, telephone and fax numbers of the corresponding author
Abstract
Please provide an abstract of 150 to 250 words. The abstract should not contain any
undefined abbreviations or unspecified references.
Keywords
Please provide 4 to 6 keywords which can be used for indexing purposes.
TEXT
Text Formatting
Manuscripts should be submitted in Word.
Use a normal, plain font (e.g., 10-point Times Roman) for text.
Use italics for emphasis.
Use the automatic page numbering function to number the pages.
Do not use field functions.
Use tab stops or other commands for indents, not the space bar.
Use the table function, not spreadsheets, to make tables.
Use the equation editor or MathType for equations.
Note: If you use Word 2007, do not create the equations with the default equation
editor but use the Microsoft equation editor or MathType instead.
Save your file in doc format. Do not submit docx files.
Word template
Manuscripts with mathematical content can also be submitted in LaTeX.
LaTeX macro package
Headings
Please use no more than three levels of displayed headings.
Abbreviations
Abbreviations should be defined at first mention and used consistently thereafter.
Footnotes
91
Footnotes can be used to give additional information, which may include the citation of
a reference included in the reference list. They should not consist solely of a reference
citation, and they should never include the bibliographic details of a reference. They
should also not contain any figures or tables.
Footnotes to the text are numbered consecutively; those to tables should be indicated by
superscript lower-case letters (or asterisks for significance values and other statistical
data). Footnotes to the title or the authors of the article are not given reference symbols.
Always use footnotes instead of endnotes.
Acknowledgments
Acknowledgments of people, grants, funds, etc. should be placed in a separate section
before the reference list. The names of funding organizations should be written in full.
SCIENTIFIC STYLE
Please always use internationally accepted signs and symbols for units, SI units.
Genus and species names should be in italics.
REFERENCES
Citation
Cite references in the text by name and year in parentheses. Some examples:
Negotiation research spans many disciplines (Thompson 1990).
This result was later contradicted by Becker and Seligman (1996).
This effect has been widely studied (Abbott 1991; Barakat et al. 1995; Kelso and Smith
1998; Medvec et al. 1993).
Reference list
The list of references should only include works that are cited in the text and that have
been published or accepted for publication. Personal communications and unpublished
works should only be mentioned in the text. Do not use footnotes or endnotes as a
substitute for a reference list.
Reference list entries should be alphabetized by the last names of the first author of
each work.
92
Journal article
Gamelin FX, Baquet G, Berthoin S, Thevenet D, Nourry C, Nottin S, Bosquet L (2009)
Effect of high intensity intermittent training on heart rate variability in prepubescent
children. Eur J Appl Physiol 105:731-738. doi: 10.1007/s00421-008-0955-8
Ideally, the names of all authors should be provided, but the usage of “et al” in long
author lists will also be accepted:
Smith J, Jones M Jr, Houghton L et al (1999) Future of health insurance. N Engl J Med
965:325–329
Article by DOI
Slifka MK, Whitton JL (2000) Clinical implications of dysregulated cytokine
production. J Mol Med. doi:10.1007/s001090000086
Book
South J, Blass B (2001) The future of modern genomics. Blackwell, London
Book chapter
Brown B, Aaron M (2001) The politics of nature. In: Smith J (ed) The rise of modern
genomics, 3rd edn. Wiley, New York, pp 230-257
Online document
Cartwright J (2007) Big stars have weather too. IOP Publishing PhysicsWeb.
http://physicsweb.org/articles/news/11/6/16/1. Accessed 26 June 2007
Dissertation
Trent JW (1975) Experimental acute renal failure. Dissertation, University of
California
Always use the standard abbreviation of a journal’s name according to the ISSN List of
Title Word Abbreviations, see
www.issn.org/2-22661-LTWA-online.php
93
TABLES
All tables are to be numbered using Arabic numerals.
Tables should always be cited in text in consecutive numerical order.
For each table, please supply a table caption (title) explaining the components of the
table.
Identify any previously published material by giving the original source in the form of
a reference at the end of the table caption.
Footnotes to tables should be indicated by superscript lower-case letters (or asterisks
for significance values and other statistical data) and included beneath the table body.
ARTWORK
For the best quality final product, it is highly recommended that you submit all of your
artwork – photographs, line drawings, etc. – in an electronic format. Your art will then
be produced to the highest standards with the greatest accuracy to detail. The published
work will directly reflect the quality of the artwork provided.
Electronic Figure Submission
Supply all figures electronically.
Indicate what graphics program was used to create the artwork.
For vector graphics, the preferred format is EPS; for halftones, please use TIFF
format. MS Office
ELECTRONIC SUPPLEMENTARY MATERIAL
Springer accepts electronic multimedia files (animations, movies, audio, etc.) and other
supplementary files to be published online along with an article or a book chapter. This
feature can add dimension to the author's article, as certain information cannot be
printed or is more convenient in electronic form.
Submission
Supply all supplementary material in standard file formats.
94
Please include in each file the following information: article title, journal name,
author names; affiliation and e-mail address of the corresponding author.
To accommodate user downloads, please keep in mind that larger-sized files may
require very long download times and that some users may experience other problems
during downloading
Audio, Video, and Animations
Always use MPEG-1 (.mpg) format.
Text and Presentations
Submit your material in PDF format; .doc or .ppt files are not suitable for long-term
viability.
A collection of figures may also be combined in a PDF file.
Spreadsheets
Spreadsheets should be converted to PDF if no interaction with the data is intended.
If the readers should be encouraged to make their own calculations, spreadsheets
should be submitted as .xls files (MS Excel).
Specialized Formats
Specialized format such as .pdb (chemical), .wrl (VRML), .nb (Mathematica
notebook), and .tex can also be supplied.
Collecting Multiple Files
It is possible to collect multiple files in a .zip or .gz file.
Numbering
If supplying any supplementary material, the text must make specific mention of the
material as a citation, similar to that of figures and tables.
Refer to the supplementary files as “Online Resource”, e.g., "... as shown in the
animation (Online Resource 3)", “... additional data are given in Online Resource 4”.
Name the files consecutively, e.g. “ESM_3.mpg”, “ESM_4.pdf”.
95
Captions
For each supplementary material, please supply a concise caption describing the
content of the file.
Processing of supplementary files
Electronic supplementary material will be published as received from the author
without any conversion, editing, or reformatting.
Accessibility
In order to give people of all abilities and disabilities access to the content of your
supplementary files, please make sure that
The manuscript contains a descriptive caption for each supplementary material
Video files do not contain anything that flashes more than three times per second (so
that users prone to seizures caused by such effects are not.
96

IMOBILIZAÇÃO DE LACASE A PARTÍCULAS MAGNÉTICAS DE

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