Resíduos do processamento de peixes comerciais como fonte de

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UNIVERSIDADE FEDERAL DE PERNAMBUCO
CENTRO DE CIÊNCIAS BIOLÓGICAS
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS BIOLÓGICAS
NÍVEL DOUTORADO
RESÍDUO DO PROCESSAMENTO DE PEIXES COMERCIAIS
COMO FONTE DE PROTEASES ALCALINAS E SEU POTENCIAL
USO BIOTECNOLÓGICO
TALITA DA SILVA ESPÓSITO
RECIFE - PE
2009
UNIVERSIDADE FEDERAL DE PERNAMBUCO
CENTRO DE CIÊNCIAS BIOLÓGICAS
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS BIOLÓGICAS
RESÍDUO DO PROCESSAMENTO DE PEIXES COMERCIAIS
COMO FONTE DE PROTEASES ALCALINAS E SEU POTENCIAL
USO BIOTECNOLÓGICO
TALITA DA SILVA ESPÓSITO
Tese apresentada ao Programa de Pós-Graduação em
Ciências Biológicas da Universidade Federal de
Pernambuco, como parte dos requisitos para obtenção
do título de Doutor em Ciências Biológicas, área de
concentração Biotecnologia.
Prof. Dr. Ranilson de Souza Bezerra
Orientador
Prof. Dr. Luiz Bezerra de Carvalho Júnior
Co-orientador
RECIFE – PE
Outubro de 2009
Espósito, Talita da Silva
Resíduos do processamento de peixes comerciais como fonte de
proteases alcalinas e seu potencial uso biotecnológico/ Talita da Silva
Espósito – Recife: O Autor, 2009
182 folhas: il., fig., tab.
Tese (doutorado) – Universidade Federal de Pernambuco.
CCB. Ciências Biológicas. Biotecnologia, 2009.
Inclui bibliografia e anexos
1. Peixes- aplicação biotecnológica. 2. Proteases. 3. Brasilaquicultura. I Título.
572.76
CDD (22.ed.)
UFPE/CCB – 2010- 013
utubr
Talita da Silva Espósito
Sumário
SUMÁRIO
DEDICATÓRIA......................................................................................................................i
AGRADECIMENTOS...........................................................................................................ii
LISTA DE TABELAS..........................................................................................................iv
LISTA DE FIGURAS...........................................................................................................vi
RESUMO..............................................................................................................................xi
ABSTRACT.........................................................................................................................xii
1. INTRODUÇÃO..................................................................................................................1
1.1. Produtos pesqueiros brasileiros...........................................................................2
1.1.1. Aquicultura continental........................................................................4
1.1.1.1. Tambaqui...........................................................................................5
1.1.1.2. Carpa..................................................................................................6
1.1.2. Pesca extrativa marinha........................................................................8
1.1.2.1. Carapeba..........................................................................................10
1.1.2.2. Ariocó..............................................................................................10
1.2. Proteases............................................................................................................13
1.2.1. Proteases alcalinas de peixes..............................................................14
1.2.2. Purificação e avaliação de proteases alcalinas de peixes....................17
1.3. Aplicações tecnológicas de proteases alcalinas.................................................23
1.3.1. Aplicação de proteases alcalinas na indústria de detergentes.............26
Sumário
2.
OBJETIVOS.............................................................................................................28
3.
REFERÊNCIAS BIBLIOGRÁFICAS.....................................................................29
4.
ARTIGOS CIENTÍFICOS…………………………………………………….......50
4.1.
Artigo 1: Surfactants- and oxidants-resistant alkaline proteases from
common carp (Cyprinus carpio L) processing waste…………………………...…51
4.2.
Artigo 2: Fish processing waste as a source of alkaline proteases for laundry
detergent…………………………………………………………….......................66
4.3.
Artigo 3: Trypsin-like enzyme from tambaqui (Colossoma macropomum):
Purification and characterization of an alkaline enzyme stable to commercial
detergents and oxidizing agents…………………………………………………....73
4.4.
Artigo 4: Purificação e caracterização de uma protease alcalina das vísceras
da carapeba prateada (Diapterus rhombeus)..........................................................104
4.5.
Artigo 5: Alkaline protease from the processing waste of the lutjanid and its
compatibility with oxidant, surfactants and commercial detergent………………131
5. CONCLUSÕES..............................................................................................................164
6. ANEXOS........................................................................................................................165
6.1.
Normas do periódico Bioresource Technology..........................................166
6.2.
Normas do periódico Brazilian Journal of Food Technology....................171
6.3.
Normas do periódico Journal of Industrial Microbiology &
Biotechnology.........................................................................................................176
Dedicatória
DEDICATÓRIA
À minha querida avó Maria Espósito (in memorian), com
muitas saudades.
Aos meus pais, irmãos, sobrinhos e a Igor da Mata.
i
Agradecimentos
AGRADECIMENTOS
A Deus, pelo dom da vida e por todas as bênçãos.
Após longos anos de estudos e aprendizagens, hoje e sempre serei grata pelos esforços
realizados pelos meus pais, em quem pude me espelhar e fazer minha pequena parte
perante todos os estímulos e apoio que sempre me foram dados. Às vezes distantes, porém
sempre ao meu lado, foram o motivo para o qual me dediquei nesses anos.
Aos meus irmãos, Samuel e Ramiro Espósito por todo o apoio e aos meus sobrinhos André
Filipe, Luis Eduardo e João da Mata pelas divertidas horas de brincadeiras.
Ao companheiro de todos os momentos: Igor da Mata, pelo amor, paciência e
compreensão.
Ao meu orientador e amigo professor Ranilson Bezerra, o pai da família LABENZ.
Ao professor Luiz Bezerra Jr., pelas sempre sábias contribuições.
À FACEPE e à CAPES pelo auxílio concedido em forma de bolsa.
Aos membros da banca, os professores Dr.Carlos Prentice, Dra Tereza Correia, Dra Graça
Cunha e Dra Patrícia Fernandes, por disporem do seu tempo para prestar-me valiosas
sugestões.
À família LABENZ: Augusto Vasconcelos, Caio Dias, Dárlio Teixeira, Diego Buarque,
Fábio Marcel, Helane Costa, Ian Porto, Janilson Felix, Juliana Santos, Juliett Xavier,
Karina Ribeiro, Marina Marcuschi, Mirela Assunção Patrícia Castro, Renata França,
Robson Liberal (in memorian), Suzan Diniz, Thiago Cahu, Welayne Mendes,
companheiros, neste trabalho e na vida.
ii
Agradecimentos
Aos alunos de iniciação científica: Fernanda, Karoll, Amanda, Gilmar e Robson.
Aos colegas da turma do doutorado, pelos bons momentos do primeiro ano de doutorado.
À Adenilda e aos professores e funcionários do Departamento de Bioquímica da UFPE, em
especial a Albérico, João, Neidinha e Miron pelos imprescindíveis favores prestados nesses
anos.
iii
Lista de Tabelas
LISTA DE TABELAS
Página
Tabela 1:
Produção pesqueira brasileira por modalidade. (Fonte: IBAMA,
2008)......................................................................................
Tabela 2:
Propriedades de proteases alcalinas extraídas das vísceras de
peixes tropicais........................................................................
Tabela 3:
17
Algumas das diversas fontes de proteases alcalinas com potencial
para aplicação em diferentes indústrias.......................................
Artigo 1:
4
25
Surfactants- and oxidants-resistant alkaline proteases from common
carp (Cyprinus carpio L) processing waste
Tabela 1:
Partial purification of proteases from Cyprinus carpio intestine….
Tabela 2:
Effect of surfactants on proteolytic activity of Cyprinus carpio
intestine purified by ethanol precipitation…………………………
Tabela 3:
57
61
Comparation of pH, temperature and bleach stability properties of
commercial detergent proteases with alkaline protease from
Cyprinus carpio.......................................................................
Artigo 2:
61
Fish processing waste as a source of alkaline proteases for laundry
detergent
Tabela 1
Effect of surfactants on proteases of C. macropomum pyloric
caeca and intestine purified by ethanol precipitation……………
Artigo 3:
70
Trypsin-like enzyme from tambaqui (Colossoma macropomum):
Purification and characterization of an alkaline enzyme stable to
commercial detergents and oxidizing agents
Tabela 1
Effect of metal ions on the second order kinetic parameters
(Kcat/Km) of the trypsin-like from tambaqui………………………
Tabela 2
98
Kinetic parameters from the hydrolysis of two series of synthetic
iv
Lista de Tabelas
fluorogenic peptides substrates by trypsin-like from tambaqui.
Abz-RXFK-Eddnp (X represents P1’) and Abz-XRFK-Eddnp (X
represents P2)……………………………………………………...
Tabela 3
Stability of Alcalase
®
from Novozymes , Commercial Porcine
®
and Trypsin-like from tambaqui in the
Trypsin from Sigma
99
®
presence of commercial laundry detergents, surfactants and H2O2
for 60 min at 25 °C………………………………………………...
Artigo 4
100
Purificação e caracterização de uma protease alcalina das vísceras da
carapeba prateada (Diapterus rhombeus)
Tabela 1
Efeito de íons e inibidores de protease sobre a atividade da
tripsina purificada da carapeba prateada (D. rhombeus)................
Tabela 2
Parâmetros cinéticos para a tripsina da carapeba prateada (D.
rhombeus), utilizando o substrato BAPNA (1,2 mM)...................
Artigo 5
126
127
Alkaline protease from the processing waste of the lutjanid and its
compatibility with oxidant, surfactants and commercial detergent
Tabela 1
Purification of trypsin-like enzyme from the pyloric caeca of the
lane snapper………………………………………………………..
Tabela 2
Ion effect on the trypsin-like enzyme from the pyloric caeca of the
lane snapper………………………………………………………..
Tabela 3
154
155
Values are expressed in ± standard deviation; n = 3; the specific
enzyme activity of the control sample (100%) was 50,000 U/mg
using azocasein as substrate……………………………………….
156
v
Lista de Figuras
LISTA DE FIGURAS
Página
Figura 1:
Tambaqui, Colossoma macropomum. (Foto: Marcuschi, M.) .............
6
Figura 2:
Carpa comum, Cyprinus carpio. (Fonte: fao.org).................................
7
Figura 3:
Carapeba prateada, Diapterus rhombeus. (Foto: Silva, J.F.)................
10
Figura 4:
Ariocó, Lutjanus synagris. (Foto: Espósito, T.S.).................................
11
Figura 5:
Representação da (A) tripsina clivando o lado carboxil da arginina e
resíduos de lisina, enquanto (B) a trombina cliva ligações Arg-Gly
em sequências especificamente particulares. (Fonte: BERG et al.,
2004)....................................................................................................
Artigo 1:
14
Surfactants- and oxidants-resistant alkaline proteases from common carp
(Cyprinus carpio L) processing waste
Figura 1:
Sodium dodecyl sulfate polyacrylamide gel electrophoresis of
alkaline protease from Cyprinus carpio viscera. Lane 1: crude
extract; Lane 2: heated crude extract; Lane 3: precipitate with 30–
70% ethanol. The molecular weights standard protein makers used
were: bovine serum albumin (66 kDa), ovoalbumin (45 kDa),
glyceraldehydes 3-phosphate dehydrogenase (36 kDa), carbonic
anhydrase (29 KDa), trypsinogen (24 kDa)…………………………..
Figura 2:
58
Effect of pH on proteolytic activity (a) and on stability (b) of
protease. Buffer solutions 0.1 M phosphate (); Tris–HCL (z) and
NaOH/glycine (Ÿ) of Cyprinus carpio viscera 30–70% ethanol
fraction………………………………………………………………...
Figura 3:
Temperature profile (a) and thermal stability (b) of Cyprinus carpio
viscera 30–70% ethanol fraction………………………………….......
Figura 4:
59
60
Effect of peroxide on activity of proteases from Cyprinus carpio
alimentary canal 30–70% ethanol fraction at 40C……………………
62
vi
Artigo 2:
Lista de Figuras
Fish processing waste as a source of alkaline proteases for laundry
detergent
Figura 1:
SDS-PAGE of alkaline protease from the viscera of C. macropomum.
Lane 1: molecular weights of standard protein markers (bovine
serum albumin 66 KDa, ovoalbumin 45 KDa, glyceraldehydes 3phosphate dehydrogenase 36 KDa, carbonic anhydrase 29 KDa,
trypsinogen 24 KDa, and a-lactoalbumin 14,2 KDa); lane 2: crude
extract; lane 3: precipitate with 30–70% ethanol; lane 4: zymogram
of the crude extract and lane 5: zymogram of the precipitate with 30–
70% ethanol…………………………………………………………..
Figura 2:
69
Effect of pH on the activity (a) and alkaline stability (b) of proteases
from C. macropomum pyloric caeca and intestine, precipitated with
30–70% ethanol. The enzyme activity on azocasein was established
at different pH levels provided by the following buffer solutions: 0.1
M phosphate (), Tris–HCl (z) and NaOH/glycine (Ÿ). The
specific enzyme activity of control sample (100%) was 142.0 U/mg
using azocasein as substrate…………………………………………..
Figura 3:
70
Temperature profile (a) and thermal stability (b) of proteases from C.
macropomum pyloric caeca and intestine precipitated by 30–70%
ethanol. (a) The protease activity was assayed at indicated
temperatures, 0.1 M NaOH/glycine, pH 11.0, and (b) the enzyme
preparation was incubated for 30 min. at the indicated temperatures,
and after the preparation had reached 25°C their proteolytic activities
were assayed. The specific enzyme activity of the control sample
(100%) was 142.0 U/mg using azocasein as substrate……………….
Figura 4:
70
The inactivation curve of the H2O2 of proteases from the C.
macropomum pyloric caeca and intestine precipitated by 30–70%
ethanol. Enzyme preparations were incubated at pH 11.0 and 40°C
with H2O2 at the concentrations of 5% (z), 10% (Ÿ), 15% (ź).
Samples were withdrawn at time intervals, their activities
(duplicates) were established using azocasein as substrate and
compared to the non-treated sample (). The specific enzyme
vii
Lista de Figuras
activity of the control sample (100%) was 146.0 U/ mg using
azocasein as substrate…………………………………………………
Figura 5:
71
The stability of protease in commercially available detergents.
Protease (0.2 mg mL-1) was incubated at 40°C in the presence of
detergents at 7 mg mL-1. Activity of the control sample devoid of any
detergent incubated under similar conditions (z), Surf® (Ƒ), Ala®
ǻ), Bem-te-vi® (ź), Omo Multi-Ação® (¸). The specific enzyme
activity of the control sample (100%) was 146.0 U/mg using
71
azocasein as substrate…………………………………………………
Trypsin-like enzyme from tambaqui (Colossoma macropomum):
Artigo 3:
Purification and characterization of an alkaline enzyme stable to
commercial detergents and oxidizing agents
Figura 1:
Molecular mass of the purified trypsin-like from tambaqui. A. SDSPAGE of the purified trypsin-like from tambaqui. Line 1 - Pattern of
standard proteins bands; Line 2 - Final purification step (affinity
chromatography), showing a single band of 27.5 kDa. B. Mass
spectrum from the purified enzyme, comprising of two main peaks:
one with 24 kDa and other with half this value (12 kDa)……………
Figura 2:
101
A. Effect of pH on the second order kinetic parameters (Kcat/Km) of
the trypsin-like from tambaqui using z-FR-mca as substrate. B.
Effect of temperature on the second order kinetic parameters
(Kcat/Km) of the trypsin-like from tambaqui using z-FR-mca as
substrate. C. Thermal stability of trypsin-like from tambaqui.
Aliquots were incubated at 40 °C (
(
), 70 °C (
), 55 °C (
),60 °C (
), 65 °C
) and samples were taken at various time (X axis).
Residual activity (Y axis), relative to the initial activity (0 hours),
was
measured
at
25.5
°
C,
using
z-FR-mca
as
substrate……………………………...………………………………..
102
viii
Figura 3:
Lista de Figuras
Comparison of the amino acid N-terminal sequences from tambaqui
trypsins-like with other trypsin from the literature. The dots
represents residues identical to the tambaqui trypsin whereas the
letters indicate the different ones……………………………………...
Artigo 4:
103
Purificação e caracterização de uma protease alcalina das vísceras da
Figura 1:
Eletroforese em gel de poliacrilamida – SDS-PAGE da tripsina
purificada da carapeba prateada (D. rhombeus). Na linha 1 está o
padrão de peso molecular e na linha 2 o fração obtida da coluna de
afinidade....................................................................................
Figura 2:
128
Efeito do pH sobre a atividade da tripsina da carapeba prateada (D.
rhombeus). Os tampões utilizados no ensaio foram fosfato (ŶS+
a 7,5), Tris- HCl (żS+D*OLFLQD-NaOH (ŸS+D
(A), Efeito da temperatura sobre a atividade da tripsina da carapeba
prateada (D. rhombeus). O valor mais alto de atividade enzimática
específica obtida a 55°C, foi estipulada como o 100% (B), Efeito da
temperatura sobre a estabilidade da tripsina da carapeba prateada (D.
rhombeus) (C)............................................................................
Figura 3:
129
Alinhamento da sequência N-terminal da tripsina símile da carapeba
prateada (Diapterus rhombeus) com outras de tripsina de peixes e
uma tripsina bovina. Os pontos representam resíduos de aminoácido
iguais à sequência principal (presente trabalho) e as letras indicam os
aminoácidos que são diferentes.......................................................
Artigo 5:
130
Alkaline protease from the processing waste of the lutjanid and its
compatibility with oxidant, surfactants and commercial detergent
Figura 1:
SDS-PAGE of intestine and pyloric caeca purified trypsin from the
lane snapper; Lane 1: Standard proteins; Lane 2: Pool collected by pAminobenzamidine Sepharose 6B; molecular weight was estimated
ix
Lista de Figuras
using the protein standards galctosidase (116 kDa), phosphorylase b
(97.4 kDa), bovine serum albumin (66 kDa), alcoholdehydrogenase
(37.6 kDa), carbonic anhydrase (28.5 kDa), myoglobin (18.4 kDa)
and lysozyme (14 kDa)………………………………………………..
Figura 2:
Michaelis–Menten plot for trypsin kinetics; BApNA concentrations
(1.8–0.01875 mM); R2=0.99………………………………………….
Figura 3:
159
160
Effect of temperature (A), thermal stability (B) and pH (C) on
trypsin-like enzyme from lane snapper intestine and pyloric caeca;
The purified enzyme collected from p-aminobenzamidine sepharose
6B was incubated with BApNA (8mM) at the temperatures and pH
indicated for 30 min. The products were measured at 405 nm.
Thermal stability was determined by assaying activity (25-75ºC)
after pre-incubation for 30 min at the temperatures indicated. All the
experiments were carried out in triplicate. Values (mean ± SD) are
expressed as percentage of highest activity…………………………
Figura 4:
161
Inactivation curve of H2O2 on protease from the pyloric caeca and
intestine of L. synagris precipitated with 40-80% ethanol. Enzyme
preparations were incubated at pH 9.0 and 40 ºC, with H2O2 at
concentrations of 5% (z) and 10% (S). Samples were withdrawn at
time intervals; their activities (duplicates) were established using
azocasein as substrate and compared to the non-treated sample ()...
Figura 5:
162
Stability of protease in commercially available detergents. Protease
(0.2 mg mL-1) was incubated at 25 ºC and 40 ºC in presence of
detergents at 7mg mL-1. Activity of control sample devoid of any
detergent incubated under similar conditions (Ŷ 6XUI Ɣ $OD
(Ÿ%HP-te-vi® (ź2PR0XOWL-Ação® (i)………………………
163
x
Resumo
RESUMO
Neste trabalho testou-se a aplicabilidade das proteases de vísceras de peixes como aditivo
de detergentes em pó comerciais. Para extração das enzimas foram utilizadas vísceras de
Colossoma macropomum (tambaqui) e de Cyprinus carpio (carpa), principal peixe nativo e
segundo peixe exótico da aquicultura continental nacional, respectivamente, e de Diapterus
rhombeus (carapeba prateada) e Lutjanus synagris (ariocó), peixes de grande importância
para a pesca extrativa estuarina e marinha no nordeste brasileiro, respectivamente. A partir
deste material obteve-se o extrato bruto. Em um primeiro estudo dos peixes dulcícolas, o
extrato bruto passou por uma semi-purificação fracional com etanol. O extrato bruto obtido
das vísceras da carapeba prateada, do ariocó e em um segundo estudo o de tambaqui foi
inicialmente fracionado com sulfato de amônio e posteriormente purificado em colunas de
gel-filtração e de afinidade. As frações obtidas da precipitação com etanol ou sulfato de
amônio tiveram sua atividade enzimática e quantidade de proteínas determinadas para
escolha da fração a ser trabalhada. A fração saturada com 30-70% de etanol apresentou
maior atividade específica tanto no tambaqui quanto na carpa. A fração de sulfato de
amônio com saturações de 30-60% para o tambaqui, 60-90% para a carapeba prateada e
40-80% para ariocó apresentou maior rendimento e foi a escolhida para os processos
posteriores de purificação da enzima símile a tripsina dos peixes. Verificou-se em que
temperatura e pH as proteases da fração com 30-70% de etanol e da enzima pura
apresentavam maior atividade, além da sua estabilidade em relação a estes parâmetros.
Para testar a compatibilidade com detergentes comerciais, foram utilizados quatro
detergentes comerciais, cinco agentes surfactantes e peróxido de hidrogênio em diferentes
concentrações. Os resultados obtidos sugerem que as proteases alcalinas encontradas nas
vísceras dos peixes estudados apresentam características ideais para utilização na indústria
de detergentes em pó, como: retenção de mais de 50% da sua atividade na presença de
Surf® e na presença de 5% de H2O2 após 1 hora de incubação a 40ºC. Além disso, a
atividade da enzima foi estimulada na presença de surfactantes não-iônicos (tween 20 e
tween 80) e iônicos (saponin e colato de sódio).
xi
Abstract
ABSTRACT
The objective of this research was to test alkaline proteases from fish viscera as an additive
in commercially available detergent formulations. Viscera from Colossoma macropomum
(Amazonian tambaqui) and Cyprinus carpio (carp), the most important native fish and the
second exotic fish in importance for Brazilian aquaculture, and from Diapterus rhombeus
(silver mojarra) and Lutjanus synagris (lane snapper) important components of commercial
fishery in Brazilian northeast waters were extracted and used as a source of enzyme for this
research. In a first study the crude extracts from freshwater fishes were submitted to a
partial purification with ethanol. The crude extract from silver mojarra and lane snapper
and in a second study the crude extract from Amazonian tambaqui were initially
fractionated with ammonium sulfate and further purified by gel filtration and affinity
chromatography. The protein content and the proteolytic activity of the fractions were
assessed. The fraction presenting the highest proteolytic activity was further studied. The
fraction with 30-70% of ethanol was the selected for the freshwater fishes. The fraction
with 30-60% (from Amazonian tambaqui), 60-90% (from silver mojarra) and 40-80%
(from lane snapper) of ammonium sulfate were chosen to purify the trypsin-like enzyme.
These fractions were assayed at different temperatures and pH aiming to estimate the
conditions for optimum proteolysis. Temperarture and pH stability experiments were also
carried out. Different commercially available detergents, surfactants and hydrogen
peroxide were used to test the compatibility of these proteases with detergent formulations.
The results reveal that these alkaline proteases show desirable characteristics for its use in
laundry industry such as: retention of more than 50% of its initial activity in the presence
of Surf® and 5% H20 2, after 1 hour of incubation at 40ºC. In addition to that it was
observed a slight increase of the proteolytic activity in the presence of non-ionic (tween 20
and tween 80) and ionic surfactants (saponin and sodium cholate).
xii
Introdução
1. INTRODUÇÃO
Diferentemente do comportamento verificado na segunda metade dos anos 90, onde
a balança comercial brasileira de produtos pesqueiros apresentou déficits continuados,
nesta década, pelo terceiro ano consecutivo, registraram-se superávits crescentes,
chegando, inclusive, em 2003, a superar as importações totais de pescado (IBAMA, 2008).
O aumento na produção pesqueira é proporcional aos resíduos deixados por essa
indústria. De modo geral, os resíduos provenientes do processamento de pescados no
Brasil não são tratados, sendo considerados produtos sem valor comercial. Doode (1996) e
Castillo-Yáñez et al. (2005) registraram que o descarte do lixo proveniente da industria
pesqueira está causando um grave problema de poluição no México, enfatizando a
necessidade do uso comercial desses sub-produtos em caráter de urgência, mostrando que a
biotecnologia proporciona um meio de extrair desse resíduo produtos valiosos, como as
enzimas.
Essa crescente poluição ambiental e o reconhecimento de que o uso dos recursos
biológicos é limitado têm enfatizado a necessidade de utilização de sub-produtos da
indústria pesqueira. O trato digestório, que é usualmente desperdiçado, constitui cerca de
5% do peso do peixe (GILDBERG, 1992). A biotecnologia promove um meio de
transformar esse material em valiosos produtos, como as enzimas, trazendo uma fonte
alternativa para indústrias que utilizam catalisadores em seus processos (CASTILLOYÁÑEZ et al., 2005).
Segundo Bezerra et al. (2001a), a grande quantidade de vísceras eliminada pelo
setor pesqueiro torna as proteases de teleósteos viáveis para processamentos industriais
1
Introdução
específicos, principalmente na indústria de alimentos e detergentes. Dessa forma, otimiza o
aproveitamento do pescado e, consequentemente, reduz o desperdício.
1.1. Produtos pesqueiros brasileiros
O Brasil possui 12% da água doce do planeta, 3,5 milhões de km2 de ZEE (Zona
Econômica Exclusiva), 8.400 km de costa, além de clima, diversidade de espécies
aquáticas, mercados com demanda insatisfeita interna e externamente, disponibilidade de
infra-estrutura de apoio e outras condições extremamente favoráveis (BERNARDINO,
2001). Possui, portanto, um grande potencial de mercado tanto para a produção pesqueira
quanto para os advindos da aquicultura. Apesar deste potencial, historicamente, o país tem
apresentado pequena participação no cenário mundial da atividade (GEO BRASIL, 2002).
A pesca no Brasil concentra seus esforços pesqueiros sobre poucas espécies. Em
geral, aquelas que oferecem condições, em termos de concentração e potencial, de suportar
uma atividade econômica sustentada e mais rentável. Este fato pode ser explicado devido
às características predominantemente tropicais e subtropicais, que contribuem para
determinar a inexistência de estoques densos (GEO BRASIL, 2002).
Dentre os recursos estuarinos e marinhos que suportam as principais pescarias
brasileiras destacam-se: o camarão-rosa da costa Norte (Farfantepenaeus subtilis e
Farfantepenaeus brasiliensis), que é responsável pela principal pescaria da Costa Norte do
Brasil; as lagostas (Panulirus argus e Panulirus laevicauda) - os mais importantes recursos
pesqueiros da região Nordeste; o pargo (Lutjanos purpureus) e outros lutjanídeos que são,
historicamente, um importante recurso para a pesca do Nordeste e, mais recentemente, para
o Norte; o caranguejo-uçá (Ucides cordatus), que é considerado um dos componentes mais
importantes da fauna dos manguezais, sendo encontrado ao longo do litoral brasileiro, mas
2
Introdução
sua pesca mais significativa se dá nos estados do Maranhão e Pará (COSTA, 1979; MELO,
1996); a sardinha-verdadeira (Sardinella brasiliensis), espécie que suporta a principal
pescaria industrial na região Sudeste e Sul do Brasil; peixes demersais do Sudeste e Sul;
camarões rosa (Farfantepenaeus brasiliensis e F. paulensis), branco (Litopenaeus
schmitti), sete-barbas (Xiphopenaeus kroyeri), barba-ruça (Artemesia longinaris) e Santana
(Pleoticus muelleri) nas regiões Sudeste e Sul pela pesca comercial e os atuns e afins:
bonito-listrado (Katsuwonus pelamis), as albacoras (Thunnus albacares, T. alalunga, T.
atlanticus), o espadarte (Xiphias gladius), o dourado (Coryphaena hyppurus), a cavala
(Scomberomorus cavalla), a serra (Scomberomorus brasiliensis), os agulhões (Istiophorus
albicans, Makaira nigricans e Tetrapterus albidus) e várias espécies de tubarões, dentre
outras, sendo a pescas detes uma das mais complexas, seja pela variedade de métodos de
captura que utiliza, seja pela quantidade de espécies envolvidas, além de ser praticada ao
longo de toda a costa (DIAS-NETO; DORNELLES, 1996).
A aquicultura brasileira é uma atividade que envolve 98.557 produtores, instalados
numa área de 78.552 hectares, o que perfaz uma área média de 0,80 hectares/propriedade.
A produção média é de 1,46 toneladas/ha. A heterogeneidade dos sistemas de produção
torna a média global um dos indicadores de desempenho com pouco poder de explicação.
Entretanto, esses dados indicam que a aquicultura no Brasil, com exceção do setor da
carcinicultura, é sustentada principalmente por pequenos produtores. Quanto ao número de
espécies cultivadas, ao contrário do que ocorre nos principais países produtores, onde é
cultivado um reduzido número de espécies, pelo menos 62 espécies vêm sendo utilizadas
comercialmente ou experimentalmente na aquicultura brasileira, sendo peixes (51),
crustáceos (5), moluscos (4), anfíbios (1) e algas (1) (BERNARDINO, 2001).
3
Introdução
Segundo o IBAMA (2008) a produção de pescado estimada em 2006 no Brasil foi
de 1.050.808 toneladas. A produção pesqueira por modalidade pode ser observada na
Tabela 1.
Tabela 1: Produção pesqueira brasileira por modalidade. (Fonte: IBAMA, 2008).
Ano: 2006
Toneladas
Valores (R$)
Pesca extrativa marinha
527.871,5
1.690.364.770,00
Pesca extrativa continental
251.241,0
586.397.460,05
Maricultura
80.512,0
302.614.500,00
Aquicultura continental
191.183,5
715.227.400,00
1.050.808,0
3.294.604.130,05
Total
1.1.1. Aquicultura continental
Em 2006, a aquicultura continental, com uma produção de 191.183,5 t, representou
18,2% da produção de pescado total do Brasil. O valor estimado foi de R$ 715.227.400,00,
um crescimento de 6,4% em relação ao ano de 2005. Esta modalidade apresentou
crescimento
nas regiões Norte de 12,1%, na Nordeste de 2,1%, na Sudeste de 13,2%, na Sul de 6,1% e
no Centro-Oeste de 1,3% em 2006. As principais espécies de peixes utilizadas na
aquicultura dessas regiões são: tilápia, carpa, tambaqui, tambacu e curimatã (IBAMA,
2008).
Quanto à participação das regiões na produção total da aquicultura do País, em
2006, a região Sul continua ocupando o primeiro lugar, com 32,9% do total, sendo seguida
pela Sudeste, com 19%, a Nordeste com 18,9%, a Centro-Oeste com 17,7% e a Norte com
4
Introdução
11,6%. Entre os estados do Sul e Sudeste, a produção de pescado está concentrada no Rio
Grande do Sul, com a maior produção, Santa Catarina, Paraná e São Paulo. A principal
explicação para o fato de a produção aquícola ser mais significativa nas regiões Sudeste e
Sul, é, certamente, o uso de tecnologias apropriadas, a disponibilidade de insumos e a
mobilização das associações de produtores (IBAMA, 2008).
O cultivo intensivo de peixes é realizado em viveiros projetados especialmente para
este fim, possuindo sistemas de abastecimento e escoamento controlados e povoamento
com peixes de valor comercial. As taxas de estocagem são programadas para uma criação
comercial de alta produtividade e, com o intuito de aumentar o crescimento dos peixes usase, além da fertilização, a ração balanceada. Para a criação ser economicamente viável, a
ração deve proporcionar elevada conversão alimentar capaz de promover um crescimento
rápido, e o peixe, por sua vez, deve alcançar alto valor de mercado (VINATEA, 1997).
1.1.1.1. Tambaqui
Dentre as principais espécies de peixes cultivados no Brasil está o tambaqui,
Colossoma macropomum, Cuvier, 1818, (Figura 1) pertence à família Characidae, subfamília Serrasalminae (NELSON, 1984). Esta é uma espécie bentopelágica dulcícola nativa
do Brasil.
A característica mais relevante das espécies do gênero Colossoma é a presença de
um grande número de cecos pilóricos, que variam de 30 a 40, mas podendo chegar até a 75
(HONDA, 1974 apud MACHADO-ALLISON, 1982). Zedzian; Barnard (1967) sugerem
que este órgão tem função similar ao pâncreas de outros vertebrados, responsáveis pela
produção de proteases alcalinas.
5
Introdução
A alimentação principal do tambaqui é constituída por microcrustáceos
planctônicos e frutas, ingerindo também algas filamentosas, plantas aquáticas frescas e em
decomposição, insetos aquáticos e terrestres que caem na água, caracóis, caramujos, frutas
secas e carnosas e sementes duras e moles (LOVSHIN, 1995).
Nos viveiros os tambaquis podem ser alimentados com frutas, tubérculos, sementes
e rações peletizadas e extrusadas (VINATEA, 1997). O tambaqui alimenta-se rápido e
agressivamente, não dando tempo para outros peixes comerem, no entanto, em sistema de
policultivo pode ser cultivado junto com curimatã, carpas e tilápia. Atinge peso médio de
1,5 kg em um ano de cultivo (HANCZ, 1993; TEICHERT-CODDINGTON, 1996).
Figura 1: Tambaqui, Colossoma macropomum. (Foto: Marcuschi, M.)
1.1.1.2. Carpa
A segunda espécie de peixe exótico mais importante para a aquicultura continental
do Brasil é a carpa comum, Cyprinus carpio, Linnaeus, 1758 (Figura 2). Este peixe
pertence à Classe Actinopterygii, Ordem Cypriniformes, família Cyprinidae, na qual são
encontrados peixes de água doce ou salobra. Atualmente é a espécie de peixe doméstica
mais importante do mundo e é cultivada há aproximadamente 4.000 anos (WOHLFARTH,
1993). É uma das quatro espécies sobre as quais existe um maior conhecimento científico e
6
Introdução
tecnológico de cultivo (CARVALHO et al., 2004; FRANCIS et al., 2002; HIDALGO et
al., 1999; NANDEESHA et al., 2002; RITVO et al., 2004; RUANE et al., 2002; SALAM
et al., 2005; WANG et al., 2006; YAMAMOTO et al., 2003).
No Brasil, é a espécie mais cultivada na principal região piscicultora, responsável
por mais de 54% da produção do Sul (IBAMA, 2008). Suas características mais positivas
são: a rusticidade, a capacidade de reprodução natural em cativeiro, o crescimento rápido, a
aceitação de um amplo espectro de alimentos e o tamanho que atinge (PROENÇA;
BITTENCOURT, 1994).
Embora considerada onívora, a carpa apresenta preferência por pequenos
organismos animais. O primeiro alimento das larvas são rotíferos, seguidos de cladóceros.
À medida que crescem, as carpas demonstram nítida preferência por organismos
bentônicos, como larvas de quironomídeos, poliquetas e pequenos moluscos. Dependendo
da disponibilidade destes organismos, a carpa pode ingerir detritos (nos quais bactérias e
protozoários constituem-se nas principais fontes de nutrientes), sementes de plantas
aquáticas e organismos zooplanctônicos (PROENÇA; BITTENCOURT, 1994).
Em um ano de cultivo atinge peso médio de 1,0 Kg (SCOTT; CROSSMAN, 1973).
No sistema de policultivo, se adapta bem com o tambaqui, a carpa capim, a carpa prateada
e a tilápia (SALAM et al., 2005).
Figura 2: Carpa comum, Cyprinus carpio. (Fonte: fao.org)
7
Introdução
1.1.2. Pesca extrativa marinha
Com uma produção de 527.871,5 t em 2006, a pesca extrativa marinha representou
50,2% da produção total de pescado do Brasil, apresentando um crescimento de 3,9% em
relação ao ano anteiror. A região Nordeste, com uma produção de 155.162 toneladas é a
segunda maior região produtora de pescado do Brasil, por meio desta modalidade. O valor
total estimado da produção foi de R$ 723.561.235,00 (IBAMA, 2008).
A pesca no Brasil situa-se entre as quatro maiores fontes de proteína animal para o
consumo humano no país. Além de ser responsável pela geração de 800 mil empregos
diretos, com um parque industrial composto por cerca de 300 empresas relacionadas à
captura e ao processamento (GEOBRASIL, 2002). Ao se considerar o aspecto da geração
de empregos e fonte de alimentos para um contingente de brasileiros que vivem no litoral
do país e áreas ribeirinhas (a pesca nacional é uma das poucas atividades que absorve mãode-obra de pouca ou nenhuma qualificação, quer seja de origem urbana ou rural, sendo em
alguns casos a única oportunidade de emprego para certos grupos de indivíduos,
principalmente para a população excluída), pode-se verificar a real importância dessa
atividade. Esses fatos demonstram que a pesca brasileira é um componente fundamental
para a socioeconomia brasileira.
No que diz respeito à pesca marítima no Brasil pode-se classificá-la, segundo sua
finalidade ou categoria econômica em: pesca amadora, pesca de subsistência, pesca
artesanal ou de pequena escala e pesca empresarial/industrial. A pesca amadora é
praticada ao longo de todo o litoral brasileiro, com a finalidade de turismo, lazer ou
desporto, e o produto da atividade não pode ser comercializado ou industrializado. A pesca
de subsistência é exercida com o objetivo de obtenção do alimento, não tendo finalidade
8
Introdução
comercial e é praticada com técnicas rudimentares. A pesca artesanal (ou de pequena
escala) contempla tanto as capturas com o objetivo comercial, associado à obtenção de
alimento para as famílias dos participantes, como o da pesca com o objetivo
essencialmente comercial. Pode, inclusive, ser alternativa sazonal ao praticante, que se
dedica durante parte do ano à agricultura - pescador/agricultor. Tem como fundamento o
fato de que os produtores são proprietários de seus meios de produção (redes, anzóis etc.).
A pesca empresarial/industrial pode ser em dividida duas subcategorias: a desenvolvida
por armadores de pesca e a empresarial ou industrial. A pesca empresarial desenvolvida
por armadores de pesca caracteriza-se pelo fato de os proprietários das embarcações e dos
petrechos de pesca (os armadores) não participarem de modo direto do processo produtivo,
função delegada ao mestre da embarcação. Estas são de maior porte e raio de ação que
aquelas utilizadas pela pequena escala e exigem uma certa divisão de trabalho entre os
tripulantes (mestre, cozinheiro, gelador, maquinista, pescador, etc). Além dos seus motores
propulsores, dispõem ainda de certos equipamentos auxiliares à pesca, exigindo algum
treinamento formal para determinadas funções Na pesca industrial, a empresa é
proprietária tanto das embarcações, como dos apetrechos de pesca. É organizada em
diversos setores e, em alguns casos, integra verticalmente a captura, o beneficiamento e a
comercialização. As embarcações dispõem de mecanização não só para deslocamento, mas
também para o desenvolvimento das fainas de pesca, como o lançamento e recolhimento
de redes e, em alguns casos, beneficiamento do pescado a bordo, o que não acontece com
as artesanais (DIAS-NETO; DORNELLES, 1996).
9
Introdução
1.1.2.1. Carapeba
Dentre as espécies de grande representatividade para a pesca artesanal no litoral da
região Nordeste do Brasil está a carapeba prateada (Diapterus rhombeus, Cuvier, 1829)
(IBAMA, 2008), espécie costeira, predominantemente estuarina presente nas águas
tropicais do Oceano Atlântico (AUSTIN, 1973) pertencente à família Gerreidae. De acordo
com IBAMA (2008), em 2006 foram capturados 2.080t de Gerrídeos oriundos da pesca
artesanal.
A carapeba prateada possui corpo comprimido e alto (Fig. 3), atingindo um
comprimento máximo de 40 cm, possue boca protáctil, que se estende em forma de tubo
durante a alimentação, que é constituida em geral de pequenos organismos encontrados na
areia e lodo (GILMORE; GREENFIELD, 2002).
Fig. 3: Carapeba prateada. (Foto: Silva, J.F.)
1.1.2.2. Ariocó
O ariocó, Lutjanus synagris (Perciformes, Percoidei, Lutjanidae, Lutjaninae),
Linnaeus, 1758, (Figura 4) possui como características morfológicas principais o corpo
alongado, coberto por escamas ctenóides e cabeça caracteristicamente triangular em vista
10
Introdução
lateral, com o perfil superior mais fortemente inclinado que o inferior. Essa espécie possui
10 espinhos e 12 raios na nadadeira dorsal (raramente 11 ou 13 raios) e apresenta uma
mancha negra bem evidente, acima da linha lateral, logo abaixo dos primeiros raios da
nadadeira dorsal (MENEZES; FIGUEIREDO, 1980). A boca contém dentes caninos
moderados, similares em ambas as mandíbulas, com placas de dentes no céu da boca em
forma de âncora e o maxilar chega à margem anterior do olho. O corpo possui uma
coloração cor de rosa na lateral inferior e avermelhado na superior, com 8-10 faixas
horizontais amarelo-dourado e 3-4 faixas irregulares e delgadas na cabeça (CARVALHOFILHO, 1999; CERVIGÓN, et al. 1992).
Figura 4: Ariocó, Lutjanus synagris. (Foto: Espósito, T.S.)
O L. synagris é encontrado em todo o oceano Atlântico oeste, desde a Carolina do
Norte até o sudeste do Brasil (MANOOCH; MASON, 1984), sendo muito abundante nas
Antilhas, Panamá e na costa norte da América do Sul.
Nos últimos anos, a biologia e ecologia dos Lutjanídeos têm sido estudadas e
revisadas intensamente, sendo considerados como importantes recursos pesqueiros em toda
a sua área de ocorrência. O gênero Lutjanus é o mais diversificado da família Lutjanidae e
o mais importante sob o ponto de vista econômico, pois há muitas espécies distribuídas por
todos os mares tropicais do mundo. A maior parte das espécies alcança tamanho comercial,
sendo grandemente apreciadas pela excelente qualidade de sua carne (ACERO; GARZÓN,
1985).
11
Introdução
A pesca de lutjanídeos na costa brasileira é feita de forma artesanal e responde por
6% da captura extrativa marinha no Nordeste, um total de 7.151,5 toneladas nesse ano. São
na maioria vendidos eviscerados e inteiros para grandes países importadores como os
Estados Unidos. A exportação do pargo rendeu ao Brasil mais de US$ 13 milhões em
2006. Das 22 empresas que exportam igual ou acima de 4 milhões de dólares, quase a
totalidade (15) esta localizadas no Nordeste (IBAMA, 2008).
Além de ser uma espécie considerada importante nas comunidades de peixes
demersais costeiros (RIVERA-ARRIAGA et al, 1996), o ariocó, Lutjanus synagris, é alvo
de relevantes pescarias do Caribe (LUCKHURST et al, 2000; ACOSTA; APPELDOORN,
1992) ao Brasil (ALEGRÍA; MENEZES, 1970). Porém, quase tudo aquilo que se sabe da
espécie é derivado de estudos sobre adultos. Em particular, o crescimento na fase juvenil é
deduzido a partir daqueles indivíduos que sobrevivem até a fase adulta. A única exceção
são trabalhos de A.W. David, ainda em andamento, que se referem a variação latitudinal do
crescimento e das características de vida do juvenil ao longo da plataforma oeste da
Flórida. No entanto, visto a variabilidade existente no crescimento da espécie, é pouco
provável que resultados obtidos no hemisfério norte sejam diretamente aplicáveis no
hemisfério sul. Essa falta de conhecimento básico da biologia da espécie impossibilita a
elaboração de medidas que possam favorecer o seu manejo através da proteção da fase
juvenil.
Os juvenis do L. synagris utilizam os estuários para alimentar-se, enquanto que
adultos migram para profundidades que podem ultrapassar 40 m de profundidade e
distâncias maiores que 70 km da linha de costa (RIVERA-ARRIAGA et al., 1996).
Quando adulto L. synagris é carnívoro generalista e oportunista com atividade
alimentar diurna e crepuscular (DUARTE; GARCIA, 1999), sendo que estes hábitos
12
Introdução
podem variar de acordo com a disponibilidade do alimento, a sazonalidade e tamanho
(RIVERA-ARRIAGA et al., 1996).
1.2. Proteases
As proteases são enzimas que catalisam, in vivo, proteólises, a hidrólise das
ligações peptídicas entre as proteínas. De acordo com a IUBMB as proteases estão
inseridas no subgrupo 4 do grupo 3 (Hidrolases), pois por uma reação de hidrólise, elas
clivam a proteína adicionando uma molécula de água à ligação peptídica (BERG et al.,
2004). No entanto, as proteases não se adaptam tão bem a esse sistema geral de
nomenclatura de enzima, pois apresentam uma grande variedade de estruturas e de ações.
As enzimas proteolíticas diferem marcadamente em relação ao seu grau de
especificidade pelo substrato. A subtilisina, por exemplo, que é encontrada em certas
bactérias, praticamente não discrimina seu substrato: ela cliva qualquer ligação peptídica
levando pouco em conta a identidade das cadeias do lado adjacente. Já a tripsina, uma
enzima digestiva secretada pelo pâncreas, é mais especifica e catalisa a quebra das ligações
peptídicas só no lado carboxil da lisina e resíduos de arginina. A trombina, uma enzima
que participa da coagulação sanguínea, é ainda mais especifica que a tripsina. Ela só
catalisa a hidrolise de ligações arginina-glicina só em sequências particulares de peptídeos
(Figura 6) (BERG et al., 2004).
As proteases podem ser divididas em dois principais grupos: exopeptidases e as
endopeptidases. As do primeiro grupo atuam próximo das extremidades das cadeias e as
endopeptidases atuam preferencialmente nas regiões internas das cadeias polipeptídicas
(BARRETT, 1994; RAO et al., 1998). Levando-se em conta o valor do pH no qual
apresentam atividade máxima, estas enzimas podem ser classificadas em: proteases ácidas,
13
Introdução
neutras ou alcalinas (RAO et al., 1998). Neste segundo grupo encontram-se as principais
proteases industriais.
HIDRÓLISE
HIDRÓLISE
Figura 5: Representação da (A) tripsina clivando o lado carboxil da arginina e resíduos de
lisina, enquanto (B) a trombina cliva ligações Arg-Gly em sequências especificamente
particulares. (Fonte: BERG et al., 2004).
1.2.1. Proteases alcalinas de peixes
Com o auxilio das proteases, as proteínas adquiridas na dieta são degradadas até
que seus peptídeos e aminoácidos constituintes possam ser utilizados para a síntese de
novas proteínas (BERG et al., 2004).
Em teleósteos, as proteases digestivas são amplamente encontradas nas suas
vísceras, um dos principais resíduos deixados pela indústria pesqueira. A produção e
excreção das proteases digestivas destes peixes ocorrem de forma muita parecida ao
observado nos mamíferos (KOLODZIEJSKA; SIKORSKI, 1996).
O estômago secreta HCl e contém pepsina, uma protease que é produzida no
epitélio sob a forma de pepsinogênio. Esse zimogênio é ativado por autocatálise, liberando
cerca de 40 a 50 resíduos de aminoácidos da sua região N-terminal. As proteases digestivas
do pâncreas são produzidas sob a forma de zimogênios, como o tripsinogênio, e são
ativadas no lúmen do intestino pela ação da enteroquinase (enteropeptidase), uma protease
14
Introdução
do intestino delgado, que hidrolisa uma ligação peptídica específica no tripsinogênio,
transformando-o em tripsina ativa. A partir disto, as moléculas de enteroquinase
juntamente com as de tripsina (recém ativadas) promovem um efeito cascata, responsável
pela ativação de novos tripsinogênios e outros zimogênios como o quimiotripsinogênio,
procarboxipeptidase, proelastase e profosforilase (BRODY, 1994). Segundo Glass et al.
(1989), em algumas espécies, como entre os teleósteos, o pâncreas não é individualizado,
encontrando-se difuso em outros órgãos, como nos cecos pilóricos.
O primeiro registro de estudo sobre proteases digestivas de peixes data da década
de 40, quando uma pepsina de salmão foi cristalizada (NORRIS; ELAM, 1940). Desde
então, proteases digestivas de peixes de águas temperadas vêm sendo comumente
estudadas, incluindo não só as pepsinas, mas também as tripsinas, quimotripsinas,
gastricsinas e elastases.
Em muitas espécies de peixes, tripsinogênios e zimogênios são secretados pelos
cecos pilóricos. A atividade da tripsina (EC 3.4.21.4) em diferentes peixes de clima
temperado vem sendo estudada: Salmo gairdneri (KITAMIKADO; TACHINO, 1960),
Gadus morhua (ASGEIRSSON et al., 1989; BJARNASSON et al., 1993; OVERNELL,
1973),
Sardinos
melanostia
(MURAKAMI;
NODA,
1981),
Mallotus
villosus
(HJELMELAND; RAA, 1982); Protoptera aethiopicus (DE HAEN et al., 1977),
Parasilurus asotus (YOSHINAKA et al., 1984), Gadus ogac (SIMPSON; HAARD, 1984),
Tautogolabrus adspersus (SIMPSON; HAARD, 1985), Salmo solar (STOCKNES;
RUSTAD, 1995), Siganus canaliculatus (SABAPATHY; TEO, 1995), Engraulis japônica
(KISHIMURA et al., 2005), Sardinops melanosticts e Pleuroprammus azonus
(KISHIMURA et al., 2006), Sardina pilchardus (BOUGATEF et al., 2007), Sebastes
schlegelii e Alcichthys alcicornis (KISHIMURA et al., 2007). O pH ótimo das tripsinas
15
Introdução
desses peixes é alcalino, similarmente ao encontrado em tripsinas de invertebrados e de
outros vertebrados.
De acordo com Ritskes (1971) e Orejana; Liston (1981), a tripsina é um
componente importante na preparação de arenques. Simpson; Haard (1987) comprovaram
que quando adicionada no processo de aceleração da fermentação do arenque a tripsina do
bacalhau do Atlântico provocou maior solubilização das proteínas quando comparado com
a tripsina bovina. Outro uso de tripsina de bacalhau é na extração de carotenoproteínas de
resíduos do camarão, que também demonstrou ser mais eficiente que a tripsina bovina
(CANO-LOPEZ et al., 1987).
Recentemente, a caracterização e purificação de proteases alcalinas extraídas das
vísceras de peixes tropicais vêm sendo realizadas. Os resultados destas pesquisas mostram
proteases com características peculiares para aplicações biotecnológicas, principalmente na
indústria de detergente, que requer proteases com pH ótimo elevado e termoestabilidade
em temperaturas altas (Tabela 2).
Peixes de clima temperado, mas muito bem adaptados ao clima tropical do Brasil,
como as carpas, também apresentaram características interessantes, como foi mostrado por
Aranishi et al. (1998) quando da purificação de dipeptidades de Cyprinus carpio, onde
estas tiveram maior atividade no pH 9,0 e a 60ºC. Apesar desse potencial já ter sido
relatado desde a década de 80 por Cohen et al. (1981a,b) e Jónás et al. (1983).
A diversidade biológica dos peixes permite uma variedade de proteases com
propriedades únicas (DE VECCHI; COPPES, 1996), fato que, aliado à grande quantidade
de vísceras disponíveis no mercado, tornam as proteases desses teleósteos potencialmente
viáveis para processos industriais específicos, principalmente nas indústrias de alimentos e
detergentes (BEZERRA et al., 2001a).
16
Introdução
Tabela 2: Propriedades de proteases alcalinas extraídas das vísceras de peixes tropicais.
Ambiente
Marinho
Dulcícola
Espécie
Propriedades
Autor/ano
pH
ótimo
Temperatura
ótima (ºC)
7,0-9,0
55
Alencar et al. (2003)
9,0
55
Klomklao et al. (2004)
7,0-8,0
50
Castillo-Yánez et al. (2005)
9,0
8,5
55
60
Souza et al. (2007)
Klomklao et al. (2007a)
9,0
Klomklao et al. (2009)
8,0
9,0
55
60
50
40
Bezerra et al. (2005)
El-Shemy; Levin (1997)
7,0-9,0
65
Bezerra et al. (2000)
Brycon orbignyanus
9,5
10
60
60
Bezerra et al. (2001b)
Garcia-Carreño et al. (2002)
Hoplias malabaricus
7,0-9,0
55
Alencar et al. (2003)
Pseudupeneus maculatus
Caranx hippos
Sparisoma sp.
Katsuwonus pelami
Thunnus albacores
Thunnus tonggol
Sardinops sagax
caerulea
Pseudupeneus maculatus
Katsuwonus pelami
(do baço)
Katsuwonus pelami
(do intestino)
Oreochromis niloticus
Tilapia nlotica/aurea
Colossoma macropomum
1.2.2. Purificação e avaliação de proteases alcalinas de peixes
O processo de purificação de uma proteína requer primeiramente a separação desta
dos componentes celulares. Os tecidos e células são rompidos em solução tampão,
obedecendo a certos critérios que evitam a desnaturação da proteína de escolha, de modo
que se forma uma mistura denominada extrato bruto (BRACHT; ISHII-IWAMOTO,
2002). Estas biomoléculas podem ser purificadas de acordo com diferentes métodos que se
17
Introdução
baseiam em diferenças físicas como, tamanho da molécula protéica, carga elétrica e
afinidade com outras moléculas (NELSON; COX, 2004).
Não existe uma sequência exata dos métodos de purificação a serem usados em
todas as proteínas. Devlin (1998) e Voet; Voet (2005) relatam que deve-se escolher uma
sequência de técnicas de purificação que resulte em um elevado grau de purificação e alto
rendimento. A obtenção de métodos sensíveis e específicos para distinguir e medir
quantitativamente a proteína que se pretende isolar é também indispensável.
Como pode ser observado na Tabela 2, há um grande interesse no estudo das
proteases alcalinas das vísceras de peixes, seja com o objetivo de conhecer a fisiologia
digestiva do peixe ou para demonstrar o potencial biotecnológico destas moléculas. Para
estudar estas proteases é necessário primeiramente separá-las dos outros componentes
celulares ou possíveis contaminantes encontrados nas vísceras destes animais. Vários
trabalhos têm sido publicados com diferentes técnicas para esta finalidade (BEZERRA et
al., 2001b; GARCIA-CARREÑO et al. 2002; KOMKLAO et al., 2004; BEZERRA et al.,
2005; CASTILLO-YÁNEZ et al. 2005; KISHIMURA et al., 2005, 2006, 2007;
BOUGATEF et al., 2007; SOUZA et al., 2007). As técnicas mais comuns utilizadas (e que
tem apresentado melhores resultados quando utilizadas combinadas) por estes autores para
purificar proteases alcalinas de vísceras de peixes são: Centrifugação, Tratamento térmico,
Fracionamento por “Salting-out”, Gel filtração, Cromatografia de afinidade.
18
Introdução
Centrifugação
A primeira etapa em um típico protocolo de purificação de proteína é a
centrifugação. O princípio deste método é que diferentes partículas em suspensão (células,
organelas ou moléculas), tendo diferentes massas ou densidades, estabelecer-se-ão no
fundo de diferentes índices (DEVLIN, 1998). A centrifugação diferencial separa proteínas
solúveis de materiais insolúveis; a força centrífuga e a duração da centrifugação são
ajustadas para assegurar que os materiais insolúveis sedimentem, formando precipitados,
de forma que as proteínas solúveis permaneçam no líquido sobrenadante. As proteínas aí
contidas podem ser então separadas por outros métodos de purificação (NELSON; COX,
2004).
Tratamento térmico
O tratamento térmico do extrato bruto têm sido uma ferrramenta inicial ou
intermediária eficiente no processo de purificação de proteases alcalinas de peixes. Esta
técnica foi primeiramente testada para enzimas de peixes por Bezerra et al. (2001b), com o
propósito de diminuir a quantidade de proteases, eliminando aquelas termolábeis.
Fracionamento salino e com etanol: Purificações fundamentadas nas diferenças de
solubilidade
Uma vez o extrato bruto aquecido pronto, vários métodos têm sido usado para
purificar as enzimas de peixes. Um método comum que tem sido adotado por vários
autores é submeter o extrato a tratamentos que separem a proteína em diferentes frações,
baseados em propriedades como tamanho ou carga.
19
Introdução
Muitos solventes orgânicos miscíveis em água são capazes de precipitar enzimas.
Devido a sua baixa constante dielétrica (quando comparado com a água), solventes
orgânicos aumentam a atração entre as moléculas de proteínas, formando agregados, até
que as partículas assumam proporções macroscópicas e precipitem. Este fenômeno
consiste na remoção da água de solvatação da proteína, permitindo que forças eletrostáticas
induzam regiões de cargas opostas da proteína a se atraírem. Neste caso, a água é removida
tanto pelo solvente orgânico, como pela estruturação ao redor da molécula orgânica. Como
consequência, a constante dielétrica é diminuída (SCOPES, 1988; WANG et al., 1979;
HARRISON, 1993).
Os álcoois - metanol, etanol e isopropanol - são os mais importantes precipitantes
industriais. O etanol, no entanto, apresenta o balanço ideal entre o efeito na solubilidade e
características hidrofóbicas adequadas para reduzir a desnaturação. A precipitação com
etanol é uma técnica promissora que pode ser aplicada para muitos tipos de proteínas em
escala industrial. O etanol é, depois da água, o mais importante dos solventes, por possuir
boas características físico-químicas, como uma completa miscibilidade com a água, baixo
ponto de fusão, ausência de risco de misturas explosivas, alta volatilidade, inércia química,
baixa toxicidade e baixo custo, especialmente no Brasil (CORTEZ; PESSOA Jr., 1999).
Os sais neutros têm efeito pronunciado sobre a solubilidade de proteínas. Para
Nelson; Cox (2004) os sais de íons divalentes, tais como MgCl2 e (NH4)2SO4, são muito
mais eficientes na solubilização do que os sais de íons monovalentes como o NaCl, NH4Cl
e KCl. Com o uso dos sais ocorre o aumento de solubilidade (salting in) ou perda de
solubilidade (salting out) das proteínas. O sulfato de amônio é o sal mais usado para a
precipitação, pois tem solubilidade acentuada e produz força iônica elevada (BRACHT;
ISHII-IWAMOTO, 2002).
20
Introdução
Cromatografia em Gel
Os métodos mais eficientes de fracionamento de proteases de peixes fazem uso de
colunas de cromatografia, que podem estar baseada nas diferenças de carga das proteínas,
no tamanho, na afinidade com o ligante e outras propriedades. Um material poroso sólido
com propriedades químicas apropriadas (a fase estacionária) é colocado na coluna e uma
solução tampão (fase móvel) percorre através dele. A solução contendo a proteína,
colocada no da coluna percorre através da matriz sólida (NELSON; COX, 2004).
A cromatografia de exclusão molecular, também conhecida como cromatografia em
gel ou ainda gel filtração, separa as proteínas em função do seu peso molecular. Neste
método proteínas maiores atravessam a coluna mais rapidamente que as proteínas de
tamanho menor. A fase sólida consiste de esferas com poros projetados ou cavidades de
tamanhos particulares. As proteínas grandes não conseguem entrar nos poros, portanto tem
uma curta (e rápida) passagem pela coluna em volta das esferas. As proteíns pequenas
entram nas cavidades e, como resultado, migram através da coluna mais lentamente
(NELSON; COX, 2004).
As colunas de gel filtração mais comumente utilizadas para purificar enzimas de
peixes são: Sefadex G-50, G-75 e G-100 (KISHIMURA et al., 2007; BEZERRA et al.,
2005; SOUZA et al., 2007; BOUGATEF et al., 2007).
Cromatografia de afinidade: Purificações fundamentadas na separação por adsorção
seletiva
A cromatografia de afinidade baseia-se no principio de que as proteínas podem ser
separadas de acordo com a sua capacidade de se ligar de forma não-covalente a outra
molécula. Esse tipo de cromatografia apresenta ligantes que podem ser substratos
enzimáticos ou outras moléculas, como anticorpos. A coluna de afinidade reage quando a
21
Introdução
proteína de interesse se liga ao ligante, sendo que as outras proteínas passam livremente
pela coluna. As proteínas ligadas à coluna são, então, decantadas pela adição de excesso do
ligante ou pela mudança na concentração de sal ou pH (BRACHT; ISHII-IWAMOTO,
2002; DEVLIN, 1998).
Os parâmetros de solubilidade para separar proteínas são usados, sobretudo nas
fases iniciais de purificação protéica, mas eles não fornecem a resolução elevada dos
métodos cromatográficos e eletroforéticos, bem mais precisos em relação às impurezas
remanescentes (DEVLIN, 1998).
Determinação da sequência de aminoácidos N- terminal
Frequentemente, após a purificação e o cálculo da massa molecular da proteína de
interesse, a análise realizada é o sequenciamento dos seus aminoácidos. A partir da
determinação da estrutura primária da proteína, várias informações podem ser obtidas
sobre a sua função e a história evolutiva.
Pehr Edman estabeleceu um método de marcação e clivagem do aminoácido
amino-terminal sem romper as ligações peptídicas entre os outros aminoácidos. Esta
técnica, denominada degradação de Edman, remove sequencialmente um aminoácido de
cada vez da extremidade amino-terminal do peptídeo. Para tanto, o fenilisotiocinato
(reagente de Edman) reage primeiramente com o grupo amino-terminal do peptídeo.
Posteriormente, esse peptídeo é submetido a tratamento com ácido diluído em baixas
temperaturas para que ocorra a remoção do resíduo n-terminal como um derivado
fenilhidantoínico, o qual pode ser identificado por cromatografia. O restante da cadeia é
submetido à outra série destas reações, permitindo a identificação de novos resíduos nterminal, determinando assim a sequência do peptídeo (VOET e VOET, 2005; NELSON e
COX, 2004).
22
Introdução
A informação da estrutura desses resíduos é de fundamental importância para o
conhecimento do mecanismo molecular de ação do peptídeo, para a comparação de
sequências entre proteínas análogas de um mesmo indivíduo, de membros da mesma
espécie e de espécies relacionadas.
Com base nestas informações muitas enzimas digestivas, principalmente as serino
proteases como a tripsina de peixes marinhos, já foram submetidas aos métodos de
identificação e tiveram seus resíduos de aminoácidos n-terminal sequenciados: salmão do
Atlântico (Salmo salar) (MALE et al., 1995); anchovas (Engraulis japonicus) (AHSAN;
WATANABE, 2001); Pomatomus saltatrix (KLOMKLAO et al., 2007b); sardinha
(Sardina pilchardus) (BOUGATEF et al., 2007); Sebastes schlegelii e Alcichthys alcicorns
(KISHIMURA et al., 2007).
1.3. Aplicações tecnológicas de proteases alcalinas
À parte de sua importância biológica, como ativação de zimogênios, transporte e
composição do sangue, entre outras funções, as proteases são altamente relevantes no
contexto biotecnológico (MAURER, 2004). A utilização de proteases na indústria é
responsável por aproximadamente 60% do mercado total de enzimas, entre estas, as
alcalinas são as mais aplicadas (ANWAR; SALEEMUDDIN, 1998; GUPTA et al., 2002).
As proteases são muito usadas na indústria de alimentos em processos de
fermentação, na produção de gelatina hidrolisada, produção de leite de soja, fabricação de
pães, produção de queijo, tenderização e amaciamento de carnes (DE VECCHI; COPPES,
1996). São também empregadas na formulação de detergentes (BANERJEE, 1999), na
produção de papel (STEELE; STOWERS, 1991), na indústria do couro (GEORGE et al.,
1995), na produção de cerveja (DONAGHY; MACKAY, 1993), na recuperação de prata
23
Introdução
nos filmes de raios X (KUMAR; TAKAGI, 1999), na indústria farmacêutica, elaboração
de laticínios (ANWAR; SALEEMUDDIN, 1998) e no processamento industrial de
resíduos (PASTOR et al., 2001). Por seu envolvimento no ciclo de vida de microrganismos
patogênicos, tem se tornado um alvo no desenvolvimento de agentes terapêuticos contra
doenças fatais como câncer e AIDS (RAO; DESHPANDE, 1997). Entre todas as
aplicações, o uso de proteases nos setores alimentício e na indústria de detergentes são os
mais descritos na literatura e os que mais cresceram (RAO et al., 1998) (Tabela 3).
A vasta diversidade de proteases contrasta com a sua especificidade e tem atraído
cientistas do mundo todo para esforçar-se em utilizá-las com finalidades fisiológicas e
biotecnologias (FOX et al., 1991; POLDERMANS, 1990). As proteases para uso industrial
podem ser obtidas de qualquer ser vivo: microrganismos, vegetais, e animais (ELBELTAGY et al, 2004; DE VECCHI; COPPES, 1996), embora o estudo de proteases
extraídas de animais aquáticos marinhos e dulciaquícolas não seja extenso (EL-BELTAGY
et al., 2004).
24
Introdução
Tabela 3: Algumas das diversas fontes de proteases alcalinas com potencial para aplicação
em diferentes indústrias.
Espécie
Streptococcus sp.
Fonte
Bactéria
pH ótimo
8,0
Aplicação industrial
Indústria
de
laticineos/produção de
queijo
Detergente e sabão em
pó
Formulação
de
detergente
para
lavanderia
Formulação
de
detergente
para
lavanderia
Dissolução de misturas
racemicas de D,Lfenilalanina e glicina
Formulação
de
detergente
Industria
de
couro/depilação
Detergentes
comerciais
Detergente industrial
Bacillus stearothermophilus
Bactéria
9,5
Tritirachium album
(proteinase T)
Fungo
9,0-12,0
Tritirachium album
(proteinase R)
Fungo
7,0-10,0
Conidiobolus coronatus
(proteinase alcalina B)
Fungo
9,7
Bacillus sp. Y (BYA)
Bactéria
10,0-12,5
Bacillus sp. (AH-101)
Bactéria
12,0-13,0
Conidiobolus coronatus
(NCL 86.8.20)
Bacillus firmus
Fungo
8,5
Bactéria
8,0
Bacillus sp.
Bactéria
8,5
Bacillus sp.
(Savinase/Durazym)
Bacillus licheniformis
(Alcalase)
Bacillus subtilis
Bacillus brevis
Bactéria
9,0-11,0
Bactéria
8,2
Bactéria
Bactéria
8,5
10,5
Bacillus sp. JB-99
Bactéria
11,0
Nocardiopsis SP
Fungo
10,5
Bacillus mojavensis
Bacillus cereus
Bactéria
Bactéria
10,5
10,5-11
Vibrio metschnikovii DL 3351
Bacillus sp.
Bactéria
12,0
Industria
couro/depilação
Formulação
de
detergentes
Sintese de peptídeos
biologicamente ativos
Industria de couro
Detergente
de
lavanderia
Formulação
de
detergentes
Aditivo de detergente
para lavanderia
Aditivo de detergentes
Aditivo de detergente
para lavanderia
Bactéria
-
Vibrio fluvialis
Bactéria
8,0
Ȗ-Proteobacterium
Bactéria
9,0
Referência
Van
Boven
al.(1988)
et
Sato et al.(1990)
Samal et al. (1990)
Samal et al. (1990)
Sutar et al. (1991)
Shimogaki
et
al.
(1991)
Takami et al. (1992)
Phadatare et al. (1993)
Moon;
Parulekar
(1993)
de Loperena et al. (1994)
Bossi et al. (1994)
Chen et al. (1995)
Hameed et al. (1996)
Banerjee et al. (1999)
Johnvesly;
Naik
(2001)
Moreira et al. (2002)
Beg; Gupta (2003)
Banik; Prakash (2004)
Mei; Jiang (2005)
Nascimento; Martins
(2006)
Aditivo de detergente Venugopal; Saramma
para lavanderia
(2006)
Aditivo de detergentes Sana et al. (2006)
25
Introdução
1.3.1. Aplicação de proteases alcalinas na indústria de detergentes
A indústria de detergente está, cada vez mais, emergindo como o principal
consumidor de diversas enzimas hidrolíticas que agem em pH altamente alcalino.
Atualmente, as proteases são um dos principais ingredientes de uma grande variedade de
detergentes, desde aqueles usados para limpezas domésticas, àqueles usados para limpeza
de lentes de contato ou dentaduras. O principal uso das proteases compatíveis com
detergentes é na formulação de detergentes em pó (aproximadamente 25% do mercado
total de enzimas) (RAO et al., 1998).
Um pré-requisito para que enzimas proteolíticas possam ser usadas na formulação
de detergentes é que elas sejam alcalinas e termostáveis, com um pH ótimo alto. Essas
características são importantes devido ao pH do sabão em pó, que é geralmente entre 9-12
e a temperatura de lavagem que varia de 50 a 60ºC (TAKAMI et al., 1989; MANACHINI;
FORTINA, 1998).
No entanto, existem outros fatores envolvidos na seleção de proteases para
detergentes, como sua compatibilidade com o sabão em pó e os componentes presentes na
sua fórmula, tais como agentes surfactantes, oxidantes, perfumes e alvejantes (KUMAR et
al., 1998).
A preparação do primeiro detergente contendo enzimas data de antes de 1913,
consistia de carbonato de sódio e um extrato pancreático bruto. O primeiro detergente
contento enzimas bacterianas foi introduzido em 1956 (RAO et al., 1998). No entanto, a
importância econômica das proteases alcalinas só surgiu quando proteases alcalinas de
bactérias do gênero Bacillus foram introduzidas nos anos 60 para facilitar a liberação de
26
Introdução
material de origem protéica em manchas como aquelas de sangue, molhos, ovos e leite
(KUMAR; TAKAGI, 1999; GUPTA et al., 2002).
A adição de enzimas proteolíticas alcalinas na formulação de detergentes aumenta
consideravelmente o potencial de limpeza entre 30 e 40% (MOREIRA et al., 2002). O
benefício do uso destas enzimas envolve também a conservação das fibras dos tecidos e
seu caráter biodegradável.
Atualmente, a enzima utilizada em todos os detergentes em pó é a subtilisina, uma
serinoprotease (US patente nº 1240058, 374971, 370482, e 4266031, e UK patente nº
13155937). Em 2002, a União Européia produziu e usou cerca de 900 toneladas dessas
proteases (MAURER, 2004). Apesar de ser adicionada até os dias atuais, ela não é uma
protease ideal para este fim, devido a sua baixa estabilidade térmica na presença de
detergente e curta vida de prateleira (SAMAL et al., 1990), além de requerer metodologias
de filtração de custo intensivo para obter preparação de enzimas livre de microrganismos.
A utilização de proteases de fungos tem sido proposta por alguns autores (SAMAL
et al., 1990; PHADATARE et al., 1993; MOREIRA et al., 2002). Apesar de oferecerem a
vantagem de seu micélio ser facilmente removido por filtração, para que haja uma
produção economicamente viável é necessário otimizar o meio de fermentação
(PHADATARE et al., 1993). Por esta razão, é essencial buscar novas fontes de protease
(BANERJEE et al., 1999).
27
Objetivos
2. OBJETIVOS
2.1. Objetivo geral
Aplicar proteases que podem ser obtidas a partir de resíduos da indústria pesqueira
brasileira.
2.2. Objetivos específicos
Semi-purificar proteases alcalinas de vísceras de Colossoma macropomum e
Cyprinus carpio utilizando etanol como agente precipitante;
Purificar e sequenciar a porção N- terminal de uma protease símile a tripsina das
vísceras de C. macropomum e Diapterus rhombeus;
Purificar proteases alcalinas extraídas de vísceras de Lutjanus synagris;
Determinar o pH e temperatura ótima da(s) protease(s) obtidas;
Investigar a estabilidade da(s) protease(s) em relação ao pH e a temperatura;
Verificar a compatibilidade dessas proteases com detergentes comerciais, agentes
oxidantes e surfactantes.
28
Referências Bibliográficas
3. REFERÊNCIAS BIBLIOGRÁFICAS
ACERO, P.; GARZÓN, F., J. Los pargos (Pisces: Perciformes: Lutjanidae) del Caribe
Colombiano. Actualités biologiques, v. 14, n. 53, p. 89-99, 1985.
ACOSTA, A., APPELDOORN, R., S. Estimation of growth, mortality and yield per recruit
for Lutjanus synagris (Linnaeus) in Puerto Rico. Bulletin of Marine Science, v. 50, n. 2,
p. 282-291, 1992.
AHSAN, M. N., WATANABE, S. Kinetic and structural properties of two isoforms of
trypsin isolated from the viscera of japanese anchovy, Engraulis japonicus. Journal of
Protein Chemistry, v. 20, n. 1, p. 49-58, 2005.
ALEGRIA, J. R. C., MENEZES, M. F. Edad y crecimento del ariacó, Lutjanus synagris
(Linnaeus), en el nordeste del Brasil. Arquivos de Ciências do Mar, v. 10, n. 1, p. 65-68,
1970.
ALENCAR, R. B. et al. Alkaline proteases from digestive tract of four tropical fishes.
Brazilian Journal of Food Technology, v. 6, p. 279-284, 2003.
ANWAR, A.; SALEEMUDDIN, M. Alkaline pH acting digestive enzymes of the
polyphagous insect pest Spilosoma obliqua: stability and potential as detergent additives.
Biotechnology and Applied Biochemistry, v. 25, p. 43-46, 1997.
29
ANWAR, A.; SALEEMUDDIN, M. Alkaline proteases: a review. Bioresource
Technology, v. 64, p. 175-183, 1998.
ARANISHI, F. et al. Purification and characterization of thermostable dipeptidase from
carp intestine. Journal of Marine Biotecnology, v. 6, p. 116-123,1998.
ASGEIRSSON, B.; FOX, J. W.; BJARNASON, J. Purification and characterization of
trypsin from the poikilotherm Gadus morhua. European Journal of Biochemistry, v.
180, p. 85-94, 1989.
AUSTIN, H. M. Northern range extension of the rhomboid mojarra, Diapterus rhombeus
Cuvier and Valencienes (Gerreidae). Chesapeaker Science, v. 3, p. 222, 1973.
BANERJEE, U. C. et al. Thermostable alkaline protease from Bacillus brevis and its
characterization as a laundry detergent additive. Process Biochemistry, v. 35, p. 213-219,
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49
Artigos Científicos
4. ARTIGOS CIENTÍFICOS
50
4.1.
Artigo 1
Artigo 1: Surfactants- and oxidants-resistant alkaline proteases from common
carp (Cyprinus carpio L) processing waste
Publicado no peródico Journal of Food Biochemistry
51
Artigo 1
52
Artigo 1
53
Artigo 1
54
Artigo 1
55
Artigo 1
56
Artigo 1
57
Artigo 1
58
Artigo 1
59
Artigo 1
60
Artigo 1
61
Artigo 1
62
Artigo 1
63
Artigo 1
64
Artigo 1
65
4.2.
Artigo 2
Artigo 2: Fish processing waste as a source of alkaline proteases for laundry
detergent
Publicado no periódico Food Chemistry
66
Artigo 2
67
Artigo 2
68
Artigo 2
69
Artigo 2
70
Artigo 2
71
Artigo 2
72
4.3.
Artigo 3
Artigo 3: Trypsin-like enzyme from tambaqui (Colossoma macropomum):
Purification and characterization of an alkaline enzyme stable to commercial
detergents and oxidizing agents
A ser submetido ao periódico Bioresource Technology
73
Artigo 3
TRYPSIN-LIKE ENZYME FROM TAMBAQUI (Colossoma macropomum):
PURIFICATION AND CHARACTERIZATION OF AN ALKALINE ENZYME
STABLE TO COMMERCIAL DETERGENTS AND OXIDIZING AGENTS
Marina Marcuschia, Talita S. Espósito a, Maurício F. M. Machadob, Marcelo F. M.
Machado b, Márcia V. Silva a, Luiz B. Carvalho Jra, Vitor Oliveirab, Ranilson S.
Bezerraa1
a
Laboratório de Enzimologia (LABENZ), Departamento de Bioquímica (CCB) and
Laboratório de Imunopatologia Keizo Asami (LIKA), Universidade Federal de
Pernambuco, Av. Prof. Moraes Rego, s/n, Cidade Universitária, 50670-910 Recife,
Pernambuco, Brazil
b
Departamento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São
Paulo, Rua Três de Maio, 100, São Paulo 04044-020, Brazil
1
Corresponding author. Tel.: +55 81 2126 8540; fax: +55 81 2126 84 85 / 85 76.
E-mail addresses: [email protected] (R.S.Bezerra).
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Abstract
A detergent stable trypsin-like of 27.5 kDa was purified from the pyloric caeca of
tambaqui (Colossoma macropomum). The enzyme presented the N-terminal amino acid
sequence IVGGYECKAHSQPHVSLNI, optimum pH and temperature of 9.0 and 50°C,
respectively, and stability to temperatures up to 60°C. Using two series of fluorescence
peptide substrate, tambaqui trypsin-like showed higher efficiency to hydrolyze substrates
with leucine and lysine at P2 and serine and arginine at P1’, being also able to hidrolyse
substrates with proline at P1’. The tambaqui trypsin-like was only significantly inhibited
by TLCK and PMSF, by the ions Cu +2, Zn+2 and Ni+2 and by the surfactant SDS. The
enzyme was very stable in the presence of various commercial laundry detergents and
oxidizing agents. These results are evidence of the versatility from the tambaqui trypsinlike, which is an alternative alkaline protease for laundry detergent additive.
Keywords: Tropical fish, tambaqui (Colossoma macropomum), trypsin, fluorescent
substrate, laundry detergent.
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1. Introduction
The term trypsin was coined by W. F. Küne in 1876 to describe the proteolytic
activity found in animal pancreas. Nowadays, trypsin (EC 3.4.21.4) is known as the serine
endoproteases that hydrolyzes peptides bonds in the carboxylic end of the amino acid
residues Arginine (R) and Lysine (K) (Norioka and Sakiyama, 2004). This enzyme can be
found in several organisms such as animals, bacterias and viruses, playing a pivotal role in
their digestive physiology (Hedstrom, 2002). In most teleost fishes, trypsin is synthesized
in the pyloric caeca cells as an inactive precursor (trypsinogen), being then secreted into
the intestine lumen and activated by enteroproteases (Kapoor et al., 1975).
Proteinases, like trypsin, present several biotechnological applications, among which
the most common are in the industry of laudry detergent, food, leather and waste
degradation processes (Anwar and Saleemuddin; 1998, Maurer, 2004). The utmost targeted
protease for detergent formulations is the microbial enzyme subtilisin (Wolfgang, 2004;
Rawlings et al., 2007). However, its use still bares many important questions regarding to
storage, shelf time and production costs (Anwar and Saleemuddin, 1998). Thus, there is a
demand for alternative sources of protease that are stable in mediums with alkaline pH,
chelant and oxidant agents and are able to hydrolyze proteins bind to insoluble substrates,
such as cloth, denture and contact lenses (Wolfgang, 2004).
A possible enzyme source for biotechnological application is the fish digestive
orgains, a common subproduct from the fishery industry. Proteases from tropical fishes are
usually thermostable, have long shelf life and are highly activite over a wide range of pH
(Bezerra et al., 2005; Espósito et al., 2009). In the Northern Brazil, one of the most
important continental fishes is tambaqui (Colossoma macropomum), a Characidae fish
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found mainly in rivers, lakes and flooded regions from the Amazon (Almeida et al., 2006).
This tropical fish has an omnivorous habit, feeding mostly from seeds, fruits (during the
flood seasons) and zooplancton (during the dry season) (Almeida et al., 2006). Moreover,
this fish is well adapted to acid pH water (Aride et al., 2007) and is tolerant to Cd +2 and
Cu +2 at moderate concentrations (Matsuo et al., 2005).
The acid and alkaline proteases from tambaqui were first characterized by Bezerra et
al. (2000). Afterward, Bezerra et al. (2001) purified and characterized a thermostable
trypsin-like of 38.5 kDa from the pyloric caeca of this same fish. In a more recent work,
Espósito et al. (2009) partially purified alkaline proteases from tambaqui viscera and
investigated its potential application as detergent additive. Therefore, the present work
aims to purify a trypsin-like isoform from tambaqui pyloric caeca, compare its N-terminal
sequence with those from other animals, characterize it with fluorescent substrates, test its
stability against detergent components and compare these results with commercial alkaline
proteases.
2. Materials and Methods
2.1. Enzyme extraction
Juvenile specimens of tambaqui (Colossoma macropomum), with an average weight
of 316.7 g (±73.2) and total length of 24.9 cm (±2.2) were kindly provided by the rearing
units from Universidade Federal Rural de Pernambuco. The animals were sacrificed in ice
bath, the pyloric caeca tissues (mean of 0.7 g per fish) were surgically removed and then
homogenized in 10 mM Tris-HCl 15 mM NaCl (0,2 g of tissue per buffer mL). The
resulting preparation was centrifuged at 10,000 x g for 15 min at 4 oC to remove cell debris
and nuclei. The supernatant (crude extract) was used in the purification steps (Bezerra et
al., 2005).
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2.2. Enzyme purification
In the purification steps, first the crude extract was incubated for 30 min at 45 oC
(heat treatment) and centrifuged at 10,000 xg for 15 min at 4 oC as described by Bezerra et
al., (2001). The obtained supernatant was fractionated with ammonium sulphate to the
saturation of 0–30% (fraction F1) and 30–60% (fraction F2). The fractions were centrifuged
at 10,000 xg for 15 min at 4 oC. The pellets (fractions F1 and F2) were resuspended with 2
mL of 100 mM Tris–HCl pH 8.0 at 4 oC and dialysed against this same buffer for 24h at 4
o
C. Afterward, the dialysed fraction F2 was applied into a gel filtration column (120 cm3
with 9g Sephadex® G-75, Sigma®) at a flow rate of 0.4 ml/min of elution buffer (100 mM
Tris–HCl pH 8.0). Fractions with tryptic activity were pooled (30 mL) and applied into an
affinity column (2 cm3 with 1 mL of benzamidine-agarose, Sigma®) at a flow of 0.5
mL/min of binding buffer (100 mM Tris–HCl pH 8.0). When all non-ligand molecules
were washed out, a 500 mM KCl-HCl pH 2.0 buffer was used to elute the trypsin. To each
P/IUDFWLRQFROOHFWHGLWZDVDGGHG/RI07ULV–HCl buffer pH 8.5 to recover the
enzymes from the denatured state. These fractions were pooled, dialysed against 100 mM
Tris–HCl pH 8.0 buffer for 24h at 4 oC and used in the following assays.
2.3. Enzymatic activity and protein determination
For the purification steps monitoring, the tryptic activity was assayed with N-Įbenzoyl-L-arginine 4-nitroanilide hydrochloride (BApNA) prepared in Dimethylsulfoxide
(DMSO). The reaction mixture was composed of 4 mM BAPNA (30 µL), 100 mM Tris–
HCl pH 8.0 (140 µL) and sample (30 µL). The release of p-nitroaniline (product) was
followed by the increase in absorbance against a blank after 10 min at 25 oC (triplicate) in
a microplate reader at Ȝ 405 nm (BioRad Model 680). One unit of enzymatic activity was
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defined as the amount of enzyme capable to hydrolyze one µmol of BApNA per min under
the established conditions, using the molar extinction coefficient of 9,100 mM -1cm -1.
For the characterization steps, the trypsin-like activity was assayed using the
fluorescence substrate carbobenzoxy-Phe-Arg-7-amido-4-methylcoumarin (z-FR-MCA)
prepared in DMSO. The reaction mixture used to assay the effect of pH, temperature and
metal ions was composed of 100 µM z-FR-MCA (0.2 µL), 100 mM Tris–HCl pH 8.0 (1
mL) and sample (10 µL). The mixture was kept under agitation in a Hitachi F-2500
(Tokyo, Japan) fluorimeter for 25 min and the release of free MCA (fluorophore) was
continuously measured at ȜEX 380 nm and ȜEM 460 nm. The absorbance values were used
to calculate the apparent second-order rate constant (kcat/Km) assayed under pseudo-firstorder conditions, where [S] << Km, using the software Grafit 5.0.0 (Leatherbarrow, 2001).
These values were later converted to relative percentage of Kcat/Km.
The thermal stability and effect of inhibitors were assayed with the same
fluorescence substrate (z-FR-MCA), but there were only obtained the initial velocity
values. The reaction mixture was composed of 1 mM z-FR-MCA (1 µL), 100 mM Tris–
HCl pH 8.0 (1 mL) and sample (1-2 µL). The release of free MCA was followed at ȜEX 380
nm and ȜEM 460 nm in a Hitachi F-2500 (Tokyo, Japan) fluorimeter for 90 seconds at 25
°C. One unit of enzymatic activity was defined as the amount of enzyme capable to
hydrolyze one µmol of z-FR-MCA per second under the established conditions. The results
were reported as the activity relative to the non-treated samples.
In the experiments of compatibility with hydrogen peroxide, surfactant and detergent
the total proteolytic activity was assayed according to Bezerra et al. (2005). The reaction
mixture was composed of 1% Azocasein (50 µL), prepared in 0.1 M Tris-HCl, pH 8.0 and
enzyme sample (50 µL). After 60 min of incubation at 25 oC, the reaction was stopped by
addition of 10% (w/v) trichloroacetic acid (240 µL) and the mixture was centrifuged for 5
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min at 8,000xg. The supernatant (70 µL) was mixed with 1 M NaOH (130 µL) and the
released azo-dye (product) was measured against a blank at Ȝ 450 nm using a microplate
reader (Bio-Rad Model 680). One unit (U) of enzymatic activity was defined as the amount
of enzyme required to hydrolyze azocasein and cause an increase in the absorbance of
0.001 per min. The results were reported as the activity relative to the non-treated samples.
The total protein content of the samples was estimated according to Bradford (1976),
using bovine serum albumin (BSA) as standard protein.
2.4. Effect of pH and temperature
The effect of pH on the tryptic activity was evaluated at 25 °C in a range of 5 to 11.
(100mM Citrate-phosphate pH 5.0, 6.0 and 7.0; 100 mM Tris-HCl pH 7.5, 8.0, 8.5 and 9.0;
100 mM Glicine-NaOH pH 10 and 11). The effect of temperature was evaluated in a range
of 22 °C to 70 °C, using 100 mM Tris-HCl pH 8.0 as buffer.
2.5. Thermal stability
The thermal stability of the trypsin-like was evaluated at the temperatures 40, 55, 60,
65 and 70 °C. Samples were incubated under each temperature and at every hour an aliquot
/ ZDV FROOHFWHG DQG PL[HG ZLWK P0 7ULV-HCl pH 8.0 (1mL) and 1 mM z-FR0&$/WRDVVD\WKHUHVLGXDODFWLYLWLHV
2.6. Effect of metal ions
To assess the effect of metal ions on the tryptic activity, the following salts were
used CaCl2; CuSO4; MgCl2; NiCl2; NaCl; KCl; ZnCl2 in a final concentration range of 1 to
P06DPSOHV/ZHUHLQFXEDWHGZLWKWKHVDOWVROXWLRQLQP07ULV-HCl pH 8.0
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(1mL) and mixed with 1 mM z-FR-0&$/WRDVVD\WKHUHVLGXDODFWLYLWLHVDW&
The results were compared to a control without ions.
2.7 Effect of synthetic inhibitors
7KH HIIHFW RI WKH IROORZLQJ V\QWHWLF LQKLELWRUV RQ WU\SVLQ ZDV HYDOXDWHG 0
tosyl lysine chloromethyl ketone (TLCK), 1mM phenylmethylsulfonylfluoride (PMSF),
0WRV\OSKHQ\ODODQLQHFKORURPHWK\ONHWRQH73&.02-Fenantrolina, 10 mM
HWK\OHQHGLDPLQH WHWUDFHWLF DFLG ('7$ 0 (-64. The sample was incubated for 15
minutes with the inhibitors, at a ratio of 1:1 (v/v). Then an aliquot was withdraw from the
LQFXEDWLRQ/DQGPL[WXUHZLWKP07ULV-HCl pH 8.0 (1mL) and 1 mM Z-FR-MCA
/WRDVVD\WKHUHVLGXDODFWLYLWLHV
2.8 Determination of cleavage specificity
To determine the substrate specificity of the trypsin-like from tambaqui, there were
used two series of fluorescence resonance energy transfer peptides containing orthoaminobenzoyl (Abz) and 2,4-dinitrophenyl (Dnp), synthesized by Barros et al. (2007).
Their general sequences were Abz-XRFK(Dnp)-OH and Abz-RXFK(Dnp)-OH, in which
X denotes the position of the altered amino acid. The assay mixture comprised of sample
(/P07ULV-HCl pH 8.0 (1mL) and substrate in an increasing final concentration
UDQJLQJ IURP WR 0 7KH DFWLYLWLHV IRU HDFK VXVEWUDWH FRQFHQWUDWLRQ ZHUH
assayed for 120 seconds at 25 °C (ȜEX 320 nm and ȜEM 420 nm). The kinetic parameters Km
and Vmax were calculated by non-linear regression data analysis using the program Grafit
5.0.0 (Leatherbarrow, 2001). The turnouver value (Kcat) was calculated by dividing the
Vmax value by the final concentration of trypsin in the reaction.
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The specificity of the peptide bond cleavage was monitored through HPLC analysis,
using a C-FROXPQP[PP7ZRHOXWLRQVROYHQWV\VWHPVZHUHXVHG$
trifluoroacetic acid and water (1:1000, v/v); (B) trifluoroacetic acid, methanol and water
(1:900:100, v/v/v.), at a flow rate of 1.0 ml/min with a 0–90% gradient of solvent B over
60 min. The hydrolysis product were detercted by UV Ȝ 365 nm (to detect the Dnp portion)
and fluorimeter ȜEX 320 nm and ȜEM 420 nm (to detect the Abz portion).
2.9 SDS-PAGE and mass spectrometer
The polyacrylamide gel electrophoresis (SDS–PAGE) was carried out according to
Laemmli (1970), using a 4% (w/v) stacking gel and a 12.5% (w/v) separation gel. The gel
was stained for protein with 0.01% (w/v) Coomassie Brilliant Blue R-250, dissolved in
10% (v/v) acetic acid with 25% (v/v) ethyl alcohol. The background of the gel was
destained by washing with the same solution, without Coomassie. The molecular mass and
purity of the purified trypsin was also checked by MALDI-TOF (matrix-assisted laserdesorption ionization–time-of-flight) mass spectrometry (TofSpec-E, Micromass).
2.10. Determination of N-terminal amino acid sequence
The N-terminal sequence was determined by the method of Edman degradation using
a protein sequencer PPSQ-23 (Shimadzu Tokyo, Japan) and an isocratic HPLC system.
2.11 Effect of oxidizing agent, surfactants and compatibility with commercial
detergents
The stability of three proteases (trypsin-like from tambaqui, Alcalase® from
Novozymes® and porcine trypsin from Sigma®) was investigated in the presence of
hydrogen peroxide (H2O2), ionic (Chaps) and non-ionic surfactants (SDS, Tween 20, triton
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x-100). For the compatibility with laundry detergents, the proteases were incubated at 25
o
C under agitation with commercially available detergents: Omo Multiação; Minerva -
Unilever do Brasil; Ala - Lever Igarassu and Bem-te-vi - ASA to a final concentration
of 7 mg of detergent mL-1$OLTXRWV/ZHUHZLWKGUDZDIWHUDPLQRILQFXEDWLRQ
and the residual proteolytic activity in each sample was determined at 25 oC with azocasein
and compared to a control without additive, according to Moreira et al. (2002). The results
were reported as the activity relative to the control.
2.12 Statistical analysis
The statistical analysis was done using the software Sigmastat 3.5, using on-wayAnova followed by Tukey, with p<0.05.
3. Results and discussion
3.1 Enzyme purification
The purification of the trypsin-like from tambaqui was carried out in four steps. The
first purification step (heat treatment) has been previously used in the purification of
tambaqui protease (Bezerra et al., 2001; Espósito et al., 2009). Although the heat treatment
only increased the specific enzymatic activity by 1.6 folds, it is an important step, since it
induces the denaturation and hydrolysis of thermolabile undesired contaminants (Bezerra
et al., 2001, Bougatef et al., 2007). In the second purification step (ammonium sulphate
fractioning) there were obtained three fractions, among which the second one (30-60%)
showed the highest specific activity (5.06 U · min-1 · mg-1), hence being further used in the
purification steps. In the third step (size exclusion chromatography) the specific activity of
the sample significantly increased (8.19 folds). In the present work, this inclusion of this
step was crutial to clear the sample from contaminants, such as pigments and lipids. The
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last step (affinity chromatography) provided an enzyme pool with low purification yield
(3%), but high specific activity (62.19 U · min-1 · mg-1), which was 40.28 folds higher than
that from the crude extract. The high purification folds values and low yields seen in this
work were within the average found for the other fish trypsin purification (Castillo-Yáñez
et al., 2005; Bougatref et al., 2007; Souza et al, 2007).
3.2 Trypsin Characterization
The purified enzyme showed a single band of 27.5 kDa on the SDS-PAGE (Fig. 1a)
and a main peak of 24kDa on the mass spectrum (Fig. 1b). Other trypsin from various fish
species showed similar molecular mass results, such as Gadus macrocephalus, Eleginus
gracilis (Fuchise et al., 2009), Balistes capriscus (Jellouli et al., 2009), Theragra
chalcogramma (Kishimura et al., 2008), Sebastes schlegelii, Alcichthys alcicornis
(Kishimura et al., 2007) and Pseudupeneus maculatus (Souza et al., 2007).
The purified trypsin-like from tambaqui was highly active at a pH range from 8.0 to
9.5, with optimum pH at 9.0 (Fig. 2a). Similar results have been found for other tropical
fishes such as Colossoma macropomum (trypsin-like isoform of 38.5 kDa described by
Bezerra et al., 2001), Oreochromis niloticus (Bezerra et al., 2005) and Pseudupeneus
maculatus (Souza et al., 2007). A possible reason for the poor catalytic efficiency of
trypsin-like enzymes at lower pH values is the protonation of the histidine residue in the
catalytic under this conditions. This event impairs the nucleophilic attack to the scissile
peptide bond, performed by the serine residue. On the other hand, a high pH value also
reduces catalytic activity by promoting the deprotonation of the N-terminal isoleucine, thus
disrupting the active center conformation (Kasserra and Laidle, 1969).
The trypsin-like from tambaqui was highly active at temperatures from 30 to 65 °C,
with optimum temperature around 50 °C (Fig. 2b). The optimum temperature of trypsin
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from tambaqui was similar to those from some temperate fishes, like Sardinops sagax
caerulea (Castillo-Yañez et al., 2005). Alcichthys alcicornis (Kishimura et al., 2007)
Gadus macrocephalus (Fuchise et al., 2009) and lower than those from the subtropical
fishes Pomatomus saltatrix (55 °C) (Klomklao et al., 2007a), Sardina pilchardus (60 °C)
(Bougatef et al., 2007), and the tropical fish Katsuwonus pelamis (60 °C) (Klomklao et al.,
2007b).
The thermal stability of the tambaqui trypsin-like is showed in Figure 2c. The
enzyme maintained 32% of its initial activity after 6 hours at 60 °C. However, the activity
was reduced to 50% after 1h at 65 °C and was completely lost after 1h at 70 °C, possibly
due to enzyme denaturation. Likewise, trypsin from other tropical fishes, such as Thunnus
albacores (Klomklao et al., 2006) and Katsuwonus pelamis (Klomklao et al., 2007b;
Klomklao et al., 2009) are very stable at temperatures up to 60 °C, but rather unstable at
temperatures higher than 70 °C. On the other hand, trypsin from subtropical fishes, like S.
Pilchardus (Bougatef et al., 2007), S. caerulea (Castillo-Yañez et al., 2005), B. capriscus
(Jellouli et al., 2009) and temperate fishes, like S. schlegelii, A. alcicornis (Kishimura et
al., 2007), G. macrocephalus (Fuchise et al., 2009) are stable at temperatures below 40 °C,
but lose more than 80% of their activity at temperatures higher than 60 °C.
Kishimura et al. (2008) showed that there is a positive correlation between the
temperature of fish habitat and thermal stability from their trypsin. According to
Gudmundsdóttir and Pálsdóttir (2005), trypsin from cold adapted fish has higher catalytic
efficiency and lower thermal stability due to their molecular flexibility. On the other hand,
tropical fish trypsins are more stable due to their stronger interactions, lower surface
hydrophilicity and stronger hydrophobic interactions in the protein center (Klomklao et al.,
2007b; Klomklao et al., 2009). Furthermore, the stability at higher temperatures of the
tambaqui trypsin-like can be related to the thermal selection performed by the heat
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treatment step carried out during the purification process. This is an interesting
characteristic for the detergent industry, since that to be used for this purpose the enzymes
should be stable to a wide range of temperatures for a great period of time (Moreira et al.,
2002; Espósito et al, 2009).
The effect of metal ions on the catalytic efficiency of tambaqui trypsin-like was
analyzed at pH 8.0 using z-FR-mca (Table 1). The ions Cu +2, Zn+2 and Ni+2, in
concentrations until 50 mM, reduced the tambaqui trypsin-like catalytic efficiency to less
then 50% in comparisson to the control without ions. Similar results for inhibition of fish
trypsin activity were found for the ions Cu+2 and Zn+2 (Souza et al., 2007; Lu et al., 2008;
Bougatef et al., 2007). According to Matsuo et al. (2005), soft water fishes are very
susceptible to metal poisoning due to the low availability of other cations in this
environment. However, these authors showed that the tambaqui Na+ influx was not
significantly affected by concentrations up to 6.5 mM of Cu+2 ions.
The ions Mg+2, Ca+2, K+ and Na+ showed a milder inhibitory effect of tambaqui
trypsin-like catalytic efficiency at 50 mM. In fact, a slightly increase on the enzyme
catalytic efficiency was observed in the presence of K+ ions at concentrations up to 5 mM
and a significant increase was seen in the presence of 5 and 10 mM Ca+2. The Ca+2 ions
have been known to enhance trypsin activity and protect it from autolysis, mainly in
mammalian (Lu et al., 2008). Many authors have shown that fish trypsin activity can
slightly increase (Lu et al., 2008, Bougatef et al., 2007) or remain the same in the presence
of Ca+2 (Souza et al., 2007). A slight inhibition in the presence of K+ has been reported for
other fish trypsin (Souza et al., 2007). In the presence of Na+ a gradual decrease of tryptic
activity was also observed for other fishes, which can be related to an induced salting-out
precipitation (Klomklao et al., 2009; Klomklao et al., 2007a).
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The effect of various proteases inhibitors on the activity of the trypsin-like from
tambaqui was also determined. The enzyme was strongly inhibited by TLCK (trypsin
inhibitor) and PMSF (serine protease inhibitor), maintaining 10.04% ± 0.09 and 27.20% ±
3.41 of residual activity respectively. Similar results were found for other fish trypsin
(Bougatef et al., 2007; Kishimura et al., 2007; Kishimura et al., 2008; Lu et al., 2008;
Klomklao et al., 2009).
The chelating reagent, EDTA, slightly inhibited the enzyme activity (82.97% ± 4.31
residual activity) as seen for other fish trypsin (Castillo-Yanez et al., 2005; Klomklao et al.,
2006; Klomklao et al., 2007a; Kishimura et al., 2008). The inhibitors TPCK (quimotrypsin
inhibitor), O-Fenantrolina (metallo-proteases inhibitor) and E-64 (cystein protease
inhibitor) had no significant effect on the trypsin-like (113.58% ± 5.31, 110.69% ± 9.18
and 102.99% ± 2.85 residual activity respectively), as found in the literature for other
fishes (Castillo-Yanez et al., 2005; Klomklao et al., 2006; Kishimura et al., 2007;
Klomklao et al., 2007; Lu et al., 2008; Klomklao et al., 2009).
Table 2 presents the kinetic parameters, Michaelis constant (Km), turnover number
(Kcat) and catalytic efficiency (Kcat/KM), for the hydrolysis of two series of synthetic
fluorogenic tetrapeptide. These substrates were used to determine the tambaqui trypsin-like
cleavage preferences for positions P1’ and P2. The nomenclature system used to indicate
the position of the amino acids residue in the protein substrate was formulated by
Schechter and Berger (1967). In this system, the scissile bond resides between positions P1
and P1’. From the scissile bond to the N-terminal side of substrate, the amino acid residues
are numbered from P1 to Pn. Toward the C-terminal side the amino acids are numbered
from P1’ to Pn’. The substrates hydrolised by trypsins preferably presents a lysine (K) or
an arginine (R) at P1.
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In the present study, HPLC analysis (data not shown) confirmed that the tambaqui
trypsin-like cleaved only peptide bonds on the carboxyl site of arginine (R) and eventually
lysine (K), when it was available. The preference for each substrate was compared through
the catalytic efficiency values (Kcat/KM) shown in Table 2.
The tambaqui trypsin-like showed higher efficiency to hydrolyze substrates with
serine (S) and arginine (R) at P1’. Low affinity was seen for glicine (G), valine (V),
glutamine (Q), aspartic acid (D), glutamic acid (E) and proline (P). One of the most
interesting result was that the trypsin-like was able to hydrolyze the substrate with proline
(P) at P1’. The turnover value for proline (P) at this position was low, but also was the Km,
which indicates that this enzyme is able to bind to this substrate, but it does not cleaves it
in a rapid or efficient fashion. Although it is well reported that trypsin does not hydrolyze
substrate with proline (P) at P1’, recent studies have shown that it is possible to occur even
for commercial mammalian trypsin (Rodriguez et al., 2008).
As for the P2 amino acid composition, the tambaqui trypsin-like showed preference
for leucine (L) and lysine (K), whereas trypsin from rat (Baird et al., 2000) and cockroach
(Marana et al., 2002) presented very low affinity for these residues at this same positions.
Trypsin-like from tambaqui showed low affinity for the residues glicine (G), glutamine
(Q), triptophan (W) and glutamic acid (E) at position P2.
The following 20 amino acid residues IVGGYECKAHSQPHVSLNI were identified
from the NH2-terminal region of tambaqui trypsin-like. In comparison to other vertebrates
(Fig. 3), tambaqui presented higher NH2-terminal homology to the tropical marine fishes
Thunnus albacores (Klomklao et al., 2006) and Katsuwonus pelamis (Klomklao et al.,
2007b; Klomklao et al., 2009), as well as to the temperate fish Sebastes schlegelii
(Kishimura et al., 2007) and the subtropical fish Sardinops melanostictus (Kishimura et al.,
2006).
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The alignment from Figure 3 indicates that the first seven NH2-terminal amino acid
residues (IVGGYEC) and the residues between positions 15 and 19 (QVSLN) are
conserved in vertebrates trypsin. However, in mammals, the glutamic acid (E) in position 6
is replaced by a threonine (T). The preservation of the NH2-terminal amino acid residues
(isoleucine) is very important to trypsin activity, since it forms a saline bridge with the
amino acid Asp-179, that promotes a molecular rearrangement, enabling the catalytic
activity (Hedstrom, 2002). Another important structural feature for proteins is the
disulphide bonds. In vertebrates trypsin, there can be found up to six bonds, one of which
occurs between Cys-7 and Cys-142 (Roach et al., 1997). The conservation of a cysteine
residue in position 7, is an indicator for the possible existence of a similar bond in the
trypsin-like from tambaqui.
Significant differences were also found between tambaqui trypsin-like NH2-terminal
sequence and other fish. In position 8, instead of the usual neutral side chains amino acid
residues, like glutamine (Q) or threonine (T), tambaqui trypsin-like exhibit a positively
charged lysine (K). In the position 20, while most trypsin have a serine (S) residue,
tambaqui presented the apolar amino acid isoleucine (I). For the numbering applied here it
was considered the N-terminal Isoleucine the number one amino acid residue.
3.3 Effect of surfactants, oxidizing agent and commercial detergents
As seen in previous works, tambaqui proteases are potential sources of addictive for
the laundry industry (Espósito et al., 2009). In the present work the stability of the
tambaqui trypsin-like was compared to two commercial enzymes (Alcase and porcine
trypsin) in the presence of laundry detergents, surfactants and oxidants (Table 3).
Alcalase® relative activity was significantly higher than those from the other enzymes,
when incubated for 1h with all of the studied surfactants. In the presence of high
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concentration of SDS (1% w/v), the trypsin tambaqui retained 24.72% of initial activity,
while enzymes from Bacillus sp. were completely inhibited in concentrations of only 0.3%
SDS (Nascimento and Martins, 2006).
The hydrogen peroxide in concentrations ranging from 5 to 15% significantly
increased the activity from the three enzymes tested. The same was not observed for
Colossoma macropoum (Espósito et al. 2009a) and Cyprinus carpio (Espósito et al.
2009b).
In the presence of laundry detergents (Table 3), the enzymes Alcalase ® and the
trypsin-like from tambaqui have shown great stability, whereas the porcine trypsin was
completely denatured in this conditions. These special properties can make tambaqui
trypsin biotecnologically attractive and can be proposed it use in the detergent and food
industry.
According to Mei & Jiang (2005), extensive study have been done with alkaline
proteases microorganisms, however, very few published reports are available on the
compatibility of the alkaline proteases with detergent ingredients. Works regarding the use
of fish enzymes as possible detergent are still very new, but they have already been
showing some promising results results (Espósito 2009a; Espósito 2009b).
4. Conclusions
The N-terminal sequences alignment allied with the inhibition and molecular weight
results indicates that the enzyme purified from tambaqui in this work is, most likely, a
trypsin. Moreover, the results regarding the stability of this trypsin-like in the presence
surfactants, oxidizing agent and commercial detergents, combined with those of
thermostability and optimum pH reassures the possibility for application of this enzyme as
a detergent additive.
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Acknowledgements
This work was financially supported by SEAP, CNPq, FINEP, UFPE/FACEPE and
PETROBRAS.
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Figure Captions
Figure 1. Molecular mass of the purified trypsin-like from tambaqui. A. SDS-PAGE of the
purified trypsin-like from tambaqui. Line 1 - Pattern of standard proteins bands; Line 2 Final purification step (affinity chromatography), showing a single band of 27.5 kDa. B.
Mass spectrum from the purified enzyme, comprising of two main peaks: one with 24 kDa
and other with half this value (12 kDa).
Figure 2.A. Effect of pH on the second order kinetic parameters (Kcat/Km) of the trypsinlike from tambaqui using z-FR-mca as substrate. B. Effect of temperature on the second
order kinetic parameters (Kcat/Km) of the trypsin-like from tambaqui using z-FR-mca as
substrate. C. Thermal stability of trypsin-like from tambaqui. Aliquots were incubated at
40 °C (
), 55 °C (
),60 °C (
), 65 °C (
), 70 °C (
) and samples were taken at
various time (X axis). Residual activity (Y axis), relative to the initial activity (0 hours),
was measured at 25.5 °C, using z-FR-mca as substrate.
Figure 3. Comparison of the amino acid N-terminal sequences from tambaqui trypsins-like
with other trypsin from the literature. The dots represents residues identical to the tambaqui
trypsin whereas the letters indicate the different ones.
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Tables
Table 1. Effect of metal ions on the second order kinetic parameters (Kcat/Km) of the
trypsin-like from tambaqui.
Residual Kcat/Km (%)
Ions
1 mM
5 mM
10 mM
88.57 ±2.76
162.86 ±0.34
140.00 ±0.32
CaCl2
75.71 ±0.15
59.43 ±0.28
32.00 ±0.06
CuSO4
MgCl2
95.24 ±0.13
82.29 ±0.60
92.00 ±2.86
71.43 ±0.21
35.43 ±0.07
29.71 ±0.57
ZnCl2
74.29 ±0.62
45.71 ±0.09
34.29 ±0.23
NiCl2
102.86 ±1.43
91.43 ±0.58
78.57±1.31
NaCl
110.86 ±3.43
110.86 ±3.43
62.86 ±1.14
KCl
*
NT – Not tested. In this concentration the salt became insoluble.
50 mM
62.86 ±0.09
5.26 ±1.26
76.00 ±0.57
NT*
10.51 ±0.07
73.14 ±3.43
56.00 ±2.29
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Table 2. Kinetic parameters from the hydrolysis of two series of synthetic fluorogenic
peptides substrates by trypsin-like from tambaqui. Abz-RXFK-Eddnp (X represents P1’)
and Abz-XRFK-Eddnp (X represents P2).
Abz-RĻ;).-Eddnp
Abz-XRĻ).-Eddnp
Kcat/K m
Substrat
Kcat (s-1) Km 0 0-1 se
1
)
Substrat
Kcat (s-1)
e
Km
0
Kcat/Km
0-1 s1
)
RGFK
9.15
15.94
0.57
GRFK
31.94
16.30
1.960
RVFK
4.59
18.31
0.25
VRFK
46.57
2.87
16.22
RPFK
0.10
3.10
0.03
FRFK
8.87
0.53
16.68
RSFK
16.63
1.03
16.30
LRFK
53.23
1.01
52.60
RYFK
10.65
1.66
6.40
YRFK
27.72
5.57
4.98
RNFK
16.63
4.93
3.38
NRFK
30.61
7.44
4.12
RQFK
13.31
7.38
1.80
QRFK
12.64
8.93
1.42
RDFK
2.33
8.08
0.29
WRFK
6.32
3.26
1.94
REFK
7.49
12.89
0.58
ERFK
17.30
6.86
2.52
RRFK
26.61
1.69
16.46
KRFK
53.23
0.58
91.58
RHFK
8.32
1.85
4.49
HRFK
21.07
6.76
3.11
RTFK
18.30
3.25
5.63
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Table 3. Stability of Alcalase® from Novozymes®, Commercial Porcine Trypsin from
Sigma® and Trypsin-like from tambaqui in the presence of commercial laundry detergents,
surfactants and H2O2 for 60 min at 25 °C.
(%)
Alcalase®
Porcine trypsin
Tambaqui trypsin
Omo multi ação ®
80.41 ± 3.50
0.00 ± 4.08
76.80 ± 0.73
Bem-te-vi®
92.27 ± 1.31
13.07 ± 5.55
89.86 ± 0.73
66.19 ± 2.70
0.00 ± 6.30
102.58 ± 3.64
88.87 ± 2.23
0.00 ± 1.96
93.47 ± 4.37
Triton X-100
101.85 ± 3.55
71.06 ± 2.59
93,66
SDS
109.60 ± 3.14
82.09 ± 2.17
24,72
Tween 20
111.27 ± 5.61
101.73 ± 3.42
94,15 ±0,69
Tween 80
113.12 ± 4.28
102.08 ± 2.82
92,2 ±6,9
Chaps
69.10 ± 4.01
99.43 ± 1.59
*NT
5%
134.63 ± 2.00
111.03 ± 3.47
121.67 ± 0.20
10%
138.11 ± 2.35
129.58 ± 3.34
130.8 ± 4.73
177.46 ± 6.22
144.61 ± 3.20
138.3 ± 4.78
Effectors
Commercial Detergent
(7mg/mL)
Minerva
Ala
®
®
Surfactants
(1%)
Oxidant Agent (H2O2)
15%
*NT – Not tested
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Artigo 3
Figures
1
2
A
B
Figure 1a and 1b
100
Artigo 3
A
B
C
Figure 2a, 2b and 2c
101
Artigo 3
Figure 3
102
4.4.
Artigo 4
Artigo 4: Purificação e caracterização de uma protease alcalina das vísceras da
A ser submetido ao periódico Brazilian Journal of Food Technology
103
Artigo 4
PURIFICAÇÃO E CARACTERIZAÇÃO DE UMA PROTEASE ALCALINA DAS
VÍSCERAS DA CARAPEBA PRATEADA (Diapterus rhombeus)
Janilson F. Silvaa, Talita S. Espósitoa, Marina Marcuschia, Karina Ribeiro a, Ronaldo
O. Cavallib, Ranilson S. Bezerraa,*.
a
Laboratório de Enzimologia (LABENZ), Departamento de Bioquímica e Laboratório de
Imunopatologia Keizo Asami (LIKA), Universidade Federal de Pernambuco, Cidade
Universitária, 50670-420, Recife-PE, Brasil.
b
Departamento de Pesca e Aquicultura, Universidade Federal Rural de Pernambuco,
Recife-PE, Brasil.
Autor para correspondência
Ranilson S. Bezerra
Laboratório de Enzimologia (LABENZ), Departamento de Bioquímica e Laboratório de
Imunopatologia Keizo Asami (LIKA), Universidade Federal de Pernambuco, Cidade
Universitária, 50670-420, Recife-PE, Brasil.
Tel, +55 81 21268540; Fax, +55 81 21268576
email: [email protected]
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Artigo 4
RESUMO
Uma protease alcalina foi encontrada nas vísceras da carapeba prateada (Diapterus
rhombeus) obtendo-se um incremento de 86,80 vezes na atividade específica e um
rendimento de 22,34%. O processo de purificação foi realizado em três etapas: tratamento
térmico (45oC por 30min), precipitação com sulfato de amônio e cromatografia de
exclusão molecular (Sephadex G-75). Uma alíquota do extrato purificado foi aplicada em
gel de poliacrilamida (SDS-PAGE) e o seu peso foi estimado em 24,5 kDa. O pH ótimo e a
temperatura ótima para a atividade enzimática foram 8,5 e 55 °C, respectivamente. A
enzima demonstrou ser sensível a temperaturas superiores a 45 ºC, após incubação por 30
min, perdendo 100% de sua atividade. Os valores de K m e do Kcat da protease foram 0,266
mM e 0,116 s-1 0 -1, respectivamente, usando benzoil-DL-arginina-p-nitroanilida
(BAPNA) como substrato. Sua atividade foi aumentada na presença dos íons K +, Li+ e
Ca2+ e inibidas pelos íons Fe2+, Cd 2+, Cu2+, Al3+, Hg2+, Zn2+ e Pb 2+. Testes com inibidores
de proteases mostraram que a enzima foi fortemente inibida por TLCK e benzamidina,
inibidores clássicos de tripsina. A sequência dos 15 primeiros aminoácidos do N-terminal
da protease foi IVGGYECTMHSEAHE e mostrou alta homologia com tripsinas de várias
espécies de peixes.
Palavras-chaves: protease, tripsina, peixe, carapeba prateada, Diapterus rhombeus
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Artigo 4
INTRODUÇÃO
A piscicultura é um ramo da aquicultura que cresce em todo o mundo, tornando-se
necessária para compensar a demanda por produtos pesqueiros, uma vez que os recursos
naturais estão cada vez mais escassos. O aumento da produção de pescado invariavelmente
resulta na produção de resíduos ligados a esta atividade agroindustrial. As vísceras estão
entre as partes do peixe que não são consumidas e correspondem por 5% do peso total do
animal (Simpson & Haard, 1987). Ao serem eliminados sem qualquer tratamento, estes
resíduos representam um grave problema ambiental. De acordo com Bezerra et al. (2001a),
as vísceras dos peixes são conhecidas por serem ricas em enzimas digestivas viáveis para
utilização em determinados processos biotecnológicos, o que as tornam uma importante
fonte de enzimas de interesse industrial.
No trato digestório dos peixes, uma das principais enzimas é a tripsina, uma
endopeptidase da classe das serinoprotease, que cliva ligações peptídicas na extremidade
carboxi-terminal dos resíduos de aminoácido arginina e lisina (Kishimura et al., 2007).
Esta enzima desempenha uma função chave na digestão de proteínas advindas da dieta,
sendo responsável também pela ativação do tripsinogênio e de outros zimogênios
(Klomklao et al., 2007a). Devido à sua ampla aplicabilidade em processos biotecnológicos,
grande atenção tem sido dada para o estudo desta enzima (SHI, et al., 2007).
Várias isoformas de tripsina de diversas espécies de peixes, têm sido purificadas e
caracterizadas. Trabalhos com Cyprinus carpio (Cao et al., 2000), Colossoma
macropomum (Bezerra et al., 2001b), Oreochromis niloticus (Bezerra et al., 2005),
Sardinops sagax caerulea (Castillo-Yañez et al., 2005), Scomber australasicus (Kishimura
et al., 2006), Sardinops melanostictus e Pleuroprammus azonus (Kishimura et al., 2006),
Oncorhynchus tshawytscha (Kurtovic et al., 2006), Pricanthus macracanthus (Hau &
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Artigo 4
Benjakul, 2006), Macruronus novaezealandiae (Shi et al., 2007), Pomatomus saltatrix
(Klomklao et al., 2007a), Sarda sarda (Klomklao et al., 2007b), Sebastes schlegelii e
Alcichthys alcicornis (Kishimura et al., 2007c), Ctenopharyngodon idellus (Liu et al.,
2007), Sardina pilchardus (Bougatef et al., 2007) e Theragra chalcogramma (Kishimura et
al., 2008) ressaltam características nestas enzimas que as tornam passíveis de utilização em
processos industriais relevantes, tais como aditivo para sabão em pó (Espósito et al 2009) e
produção de alimentos (Shahidi et al 2001).
Uma espécie muito importante para a pesca no litoral da região Nordeste do Brasil
é a carapeba prateada (Diapterus rhombeus) (Beltrão, 1988). Pertencente à família
Gerreidae, ela pode ser encontrada em regiões costeiras estuarinas nas águas tropicais do
Oceano Atlântico (Austin, 1973). Esta espécie, em conjunto com representantes dos
gêneros Eucinostomus, Eugerres e Gerres, apresenta um papel relevante tanto na
ictiofauna estuarina quanto na pesca de subsistência de algumas localidades (Chen et al.,
2007). Apesar de registros de cultivos de carapebas em sistemas extensivos nas zonas
estuarinas nesta região do país (Cerqueira, 2004), a pesca artesanal é mais representativa.
De acordo com os dados do IBAMA (2008), em 2006, esta região capturou 2.080t de
Gerrídeos oriundos da pesca extrativa marinha artesanal. Toda esta produção gera um
descarte anual estimado de aproximadamente 100t de vísceras. A investigação de enzimas
presentes nestes resíduos pode otimizar o aproveitamento do pescado e, consequentemente,
reduzir o desperdício de produtos da aquicultura ou da pesca e agregar valor ao pescado.
Neste sentido, o presente estudo teve como objetivo purificar uma tripsina do trato
digestório da carapeba prateada e caracterizá-la quanto as suas propriedades físicas e
químicas, como efeito de temperatura, pH, íons, inibidores, concentração de substrato e
sequências de aminoácidos N-terminal.
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Artigo 4
MATERIAL E MÉTODOS
Obtenção dos exemplares
Espécimes de D. rhombeus foram obtidos de uma comunidade pesqueira localizada
no município de Itapissuma-PE, Brasil. Os peixes foram acondicionados em gelo e
transportados para o Laboratório de Enzimologia da Universidade Federal de Pernambuco
(LABENZ - UFPE). No laboratório os peixes foram pesados e medidos e apresentaram
peso e comprimento médio de 350±20g e 28±2cm, respectivamente. Os exemplares foram
dissecados para retirada do intestino e cecos, obtendo-se um total de 30g de vísceras. Estas
foram armazenadas em freezer a -25 °C até o momento das análises.
Extrato enzimático
O material foi descongelado e homogeneizado na concentração de 40mg/mL
(peso/volume) de tecido em solução de Tris-HCl 0,01M, pH 8,0 com 0,9% NaCl. Para
tanto, utilizou-se um homogeneizador de tecidos (Bodine Electric Company – Chicago,
EUA). O homogeneizado foi então centrifugado a 9.000xg por 25 minutos a 4ºC para
remoção das partículas insolúveis. O sobrenadante obtido (extrato bruto) foi coletado e
armazenado em freezer a -25ºC para ser utilizado nos processos de purificação.
Ensaio enzimático e dosagem protéica
As atividades enzimáticas foram realizDGDVXWLOL]DQGRR%$31$1Į-benzoil-DLarginina-p-nitroanilida) como substrato específico para tripsina. O ensaio foi realizado em
triplicata, utilizando-se 30µL de BAPNA 8mM dissolvido em DMSO (Dimetilsulfóxido),
140µL de Tris–HCl 0,1M pH 8,0 e 30µL da amostra. A liberação do produto (pnitroanilina) foi acompanhada em espectrofotômetro de microplaca (xMarktm BIORAD) a
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Artigo 4
Ȝ 405nm por 10 minutos a 25°C. Uma unidade (U) de atividade enzimática foi definida
como a quantidade de enzima capaz de hidrolisar o BAPNA, produzindo 0,001 de
mudança na absorbância por minuto sob as condições estabelecidas, utilizando-se o
coeficiente de extinção molar de 9,1 mM -1cm
-1
padrão para este substrato. O conteúdo
protéico foi obtido a partir da mensuração da absorbância das amostras em 260 e 280nm,
usando a equação:
>proteína@ mg/mL = A280nm x 1,5 – A260nm x 0,75 (Warburg & Christian, 1941).
Processos da purificação enzimática
O extrato bruto foi acondicionado por 30min em banho maria a 45ºC e,
posteriormente, colocado no gelo para resfriamento rápido. Esse material foi centrifugado
a 9.000xg durante 25 min a 4ºC, o precipitado foi descartado e o sobrenadante (146mL)
coletado e utilizado como extrato bruto aquecido (EBA). Posteriormente, o EBA foi
submetido à precipitação com sulfato de amônio obtendo-se as frações 0-30%, 30-60% e
60-90% de saturação salina. Para tanto, o sal foi lentamente adicionado ao extrato sob
agitação. Após a solubilização total do sal, o extrato permaneceu em repouso por 4h a 4ºC.
Posteriormente o material foi centrifugado a 9.000xg durante 25 min a 4ºC e o precipitado,
ressuspendido com 38,5mL de solução tampão Tris-HCl 0,1M, pH 8,0. Em seguida
realizou-se a atividade específica para tripsina das frações para definição do material a ser
utilizado nas demais etapas de purificação. O material obtido foi dialisado em solução
tampão Tris-HCl 0,01M, pH 8,0 por um período de 24h. Após a diálise, o material foi
OLRILOL]DGR SDUD REWHQomR GH PJ GH SURWHtQD H UHVVXVSHQGLGRV HP / GH 7ULV-HCl
0,1M pH 8,0 para aplicação em coluna de gel filtração Sephadex G-75 pré-equilibrada com
solução tampão Tris-HCl 0,1M pH 8,0. Mantendo-se um fluxo de 20mL/h coletaram-se
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Artigo 4
alíquotas de 2mL, as quais foram então analisadas quanto ao conteúdo protéico e atividade
enzimática específica.
Para a atividade enzimática específica das frações calculou-se a unidade da
atividade enzimática (U), utilizando-se o substrato BAPNA 8mM, dividido pela
concentração de proteína (mg) encontrada na amostra. O resultado foi demonstrado em
U/mg de proteína.
Eletroforese
Uma alíquota do “pool” com maior atividade específica da coluna de SephadexG75 foi liofilizada e utilizada para eletroforese em gel de poliacrilamida (SDS-PAGE),
seguindo metodologia descrita por Laemmli (1970), usando gel de concentração a 4% e gel
de separação a 15%. O gel foi corado com uma solução composta de 0,01% de Azul
brilhante de Coomassie, 25%, metanol e 10% ácido acético e foi descorado em uma
solução com a mesma composição, mas desprovida do corante.
O peso molecular da banda da proteína purificada foi estimado por comparação
com um padrão de peso molecular (Amersham Biosciences – Reino Unido) composto por
miosina (205 kDa), B-galactosidase (116 kDa), fosforilase b (97 kDa), transferrina (80
kDa), BSA (66 kDa), glutamato dihidrogenase (55 kDa), ovalbumina (45 kDa), anidrase
carbônica (30 kDa) e inibidor de tripsina (21 kDa).
Efeitos de pH
A atividade da tripsina frente a variações de pH, na faixa de 4,0 a 11,0, foi
mensurada utilizando BApNA a 8 mM (30µL) como substrato específico. Para tanto, 30µL
da amostra foi adicionado a 140µL de solução tampão Citrato-Fosfato 0,1M para a faixa de
pH de 4,0 a 7,5; Tris-HCl 0,1M com pH variando de 7,2 a 8,5 e Glicina-NaOH 0,1M com
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Artigo 4
pH variando de 8,6 a 11,0. Após 10min, foi mensurada a absorbância em
espectrofotômetro de microplaca (xMarktm BIORAD) a Ȝ 405nm a 25°C. No resultados,
foi estipulada como sendo o 100%, o valor mais alto de atividade enzimática específica
obtida no experimento.
Efeitos de temperatura
A temperatura ótima e a estabilidade térmica da enzima purificada foram avaliadas
perante diferentes temperaturas que variaram de 25º a 80ºC, com intervalos de 5ºC. O
perfil da atividade proteolítica frente à variação de temperatura foi avaliado incubando-se a
amostra (30µL) com o tampão Tris–HCl 0,1M pH 8,0 (140µL) e BAPNA 8mM (30µL) em
banho maria por 10min. No ensaio de estabilidade térmica, para cada temperatura, a
enzima foi incubada por 30min em banho maria. Em seguida, a atividade residual da
enzima foi aferida por 10min a 25ºC. Para tanto, foi adicionado 30µL da enzima incubada
a 140µL de Tris–HCl 0,1M pH 8,0 e 30µL de BAPNA 8mM. Todos os ensaios foram
realizados em triplicata e acompanhados em espectrofotômetro de microplaca (xMarktm
BIORAD) a Ȝ 405nm.
Efeitos de inibidores
Os testes de inibição foram realizados segundo metodologia adaptada por Alencar
et al. (2003) e Bezerra et al. (2005). Para tanto, 30µL de enzima purificada foram
incubados em microplacas durante 30min com 30µL de diferentes inibidores de protease
mantendo uma concentração final de 4mM. Assim, para o referido ensaio foi empregado o
ácido etilenodiamino tetra-acético – EDTA (inibidor de metaloproteases), o Emercaptoetanol (redutor de grupos S-S), o fluoreto de fenilmetilsulfonil – PMSF (inibidor
de serino-proteases), a benzamidina (inibidor de tripsina), o tosil lisina clorometil cetona –
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Artigo 4
TLCK (inibidor de tripsina) e o tosil fenilalanil clorometil cetona – TPCK (inibidor de
quimotripsina). Posteriormente, adicionou-se 110µL de solução tampão Tris-HCl 0,1M e
30uL de BAPNA. Após 10min, a leitura das absorbâncias foi realizada em leitor de
microplacas (xMarktm BIORAD), em um comprimento de onda de 405nm.
Efeitos dos íons metálicos
Alíquotas de 30/GDHQ]LPa purificada foram incubadoVFRP/GHGLIHUHQWHV
sais metálicos (AlCl3, BaCl2, CaCl2, CdCl2, CuCl2, FeCl2, HgCl2, KCl, LiCl, MnCl2,
PbCl2, ZnCl2), por 30min em microplacas com concentração final de 1mM. Em seguida,
IRLDGLFLRQDGR/GH7ULV-HCl 0,1M pH 8,0 e 30/GRVXEVWUDWR%$3NA. Após 10min
de reação, foi dosada a atividade enzimática em um leitor de microplaca a 405nm.
Parâmetros cinéticos
O substrato utilizado no ensaio cinético foi BAPNA (concentração final de 0 a
4,8mM), preparado com DMSO (Dimetilsulfóxido). A reação foi realizada em triplicata
em microplaca e consistiu da mistura de 30µL de solução da enzima purificada (109µg
proteína/mL), com 140µL de Tris-HCl 0,1M em pH 8,0 e 30µL de substrato. A liberação
do produto (p-nitroanilina) foi acompanhada por intermédio de um leitor de microplacas a
405nm. Os valores de atividade (U s-1) obtidos para cada concentração de substrato foram
plotados num gráfico e os parâmetros assintóticos da cinética de Michaelis-Mente (Vmax e
Km) foram calculados empregando o programa MicrocalTM OriginTM versão 6.0 (Software,
Inc, EUA). A constante catalítica da taxa da enzima (Kcat) foi calculada dividindo-se o
valor de Vmax (s-1) pela concentração final de enzima na reação (mM). Adicionalmente foi
calculado o valor de Kcat/Km, o qual representa a eficiência catalítica da reação.
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Artigo 4
Obtenção da sequência amino-terminal
A tripsina purificada foi sequenciada no Laboratório de Bioquímica da Escola
Paulista de Medicina – UNIFESP. A sequência amino-terminal foi obtida através da
degradação de Edman utilizando um sequenciador modelo PPSQ-23 (Shimadzu, Tóquio,
Japão).
RESULTADOS E DISCUSSÃO
Uma tripsina dos cecos pilóricos e intestino da carapeba prateada (D. rhombeus) foi
isolada utilizando diferentes etapas de purificação. A primeira etapa da purificação foi o
aquecimento do extrato bruto, que resultou em um aumento discreto do rendimento da
purificação. Na segunda etapa (fracionamento com sulfato de amônio) a fração com maior
atividade específica foi a de 30% a 60%. Após a passagem pela coluna de gel filtração
(Sephadex-G75), o pool obtido apresentou um grau de purificação 86,80 vezes maior em
relação ao extrato bruto. A fração recuperada desta cromatografia, quando aplicada ao gel
SDS – PAGE, mostrou a migração de uma única banda com peso molecular estimado em
26,54 kDa (Figura 1). Resultados similares foram observados em outras espécies isoladas
de peixes como a enguia (Anguilla japonica 26 kDa) (Yoshinaka et al., 1985), truta arcoíris (Oncorhynchus mykiss 26 kDa) (Kristjansson, 1991), hoki fish (Macruronus
novaezeaalandiae 26 kDA) (Shi et al., 2007) e carpa (Ctenopharyngodon idellus 26,4 kDa)
(Liu et al., 2007), nos quais também foram avaliadas outras tripsinas isoladas.
O protocolo aqui empregado tem sido eficiente na purificação de tripsina de peixes
tropicais (Bezerra et al. 2001b; 2005; Souza et al. 2007). Bezerra et al. (2001b) reportam a
importância da etapa de aquecimento na purificação de uma tripsina do tambaqui
(Colossoma macropomum). Apesar do baixo fator de purificação obtido nesta etapa, o
aquecimento elimina as proteínas termolábeis e também promove uma hidrólise das
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Artigo 4
proteínas contaminantes, tornado-as peptídeos que são mais hidrofílicos. Esta propriedade
melhora o desempenho das etapas posteriores de precipitação por sulfato de amônio e
cromatografia em gel de filtração (Sephadex-G75).
Após a purificação avaliaram-se as características físico-químicas da tripsina
isolada do trato digestório de D. rhombeus. Os testes para definição do pH ótimo revelaram
maior atividade enzimática na faixa de pH alcalino (7,5-11,0), mostrando um pico de
atividade em 8,5 (Fig. 2A). Este resultado é comum para a atividade de enzimas digestivas
de peixes (Castillo-Yáñez et al., 2005) como reportado em Walleye pollock (Theragra
chalcogramma) (Kishimura et al., 2008), bluefish (Pomatomus saltatrix) (Klomklao et al.,
2007a),
hoki fish (Macruronus novaezealandiae) (Shi et
al.,
2007),
salmão
(Onchorhynchus tshawytscha) (Kurtovic et al., 2006) e tilápia do Nilo (Oreochromis
niloticus) (Bezerra et al., 2005) que apresentaram atividade ótima na faixa de pH de 8,0 a
9,0.
A temperatura ótima da enzima purificada (Fig. 2B) foi 55 °C, sendo idêntica à
encontrada para a tainha (Mugil cephalus) (Guizani et al., 1991) e semelhantes as tripsinas
de outros peixes tropicais como tambaqui (Colossoma macropomum) (Bezerra et al.,
2000), tilápia do Nilo (Oreochromis niloticus) (Bezerra et al., 2005) e saramunete
(Pseudopeneus maculatus) que apresentaram maior atividade proteolítica a 60 ºC, 50 ºC e
52 ºC, respectivamente. Quanto à termoestabilidade, a tripsina destes peixes também
apresentou-se sensível em temperaturas acima de 45 ºC, o que se assemelha ao resultado
encontrado no presente estudo (Fig. 2C).
O efeito de íons metálicos (1mM) sobre a atividade da tripsina de D. rhombeus está
apresentado na Tabela 2. A atividade da enzima foi aumentada em relação ao controle
(100%) quando a mesma foi incubada na presença dos íons K+ (34%), Li+ (46%) e Ca2+
(83%). O cálcio é um ativador clássico para tripsina de mamíferos (Souza et al., 2007). No
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Artigo 4
entanto, Bezerra et al. (2005) e Souza et al. (2007) encontraram que a tripsina da tilápia do
Nilo e saramunete sofreram inibição pelo cálcio. Este fato sugere diferenças no sítio que
liga o cálcio à enzima dos referidos peixes. A atividade da tripsina da tilápia do Nilo e do
saramunete também foi inibida na presença dos íons Mn2+ e Ba2+. Entretanto, a tripsina
isolada da espécie estudada não demonstrou traços de inibição enzimática para esses íons.
Os íons Fe2+, Cd2+, Cu 2+, Al3+ diminuíram em torno de 20 a 35% a atividade da enzima
analisada, já o Hg2+ e o Zn2+ inibiram a atividade da tripsina em 53,11% e 71,23%,
respectivamente. Não obstante, estes valores de inibição são menos expressivos do que os
descritos para o saramunete. Na presença do Pb 2+, constatou-se a inativação total da
enzima purificada do D. rhombeus.
A influência de diversos inibidores específicos sobre a atividade da enzima
purificada do D. rhombeus está apresentada na Tabela 1. A enzima foi completamente
inibida por TLCK. Estudos com Tilápia do Nilo (Bezerra et al., 2005), bluefish (Klomklao
et al., 2007a), atum (Thunnus albacores) (Klomklao et al., 2006) e bonito do Atlântico
(Sarda sarda) (Klomklao et al., 2007b) também demonstraram inibição pelo TLCK. Estes
resultados estão relacionados com o fato do TLCK ser um inibidor específico de tripsina,
além de inativar enzimas com atividade similares. Essa inibição ocorre a partir da ligação
covalente com o resíduo de histidina na porção catalítica da molécula, bloqueando, assim,
a ligação do substrato ao centro ativo da enzima (Jeong et al., 2000).
A bezamidina (inibidor de tripsina) inibiu 75% da atividade enzimática. Quando
LQFXEDGD FRP ȕ-mercaptoetanol, ocorreu uma redução de 36% da atividade residual da
enzima. Na presença de PMSF em concentrações de 2 mM e 4 mM, a tripsina foi inibida
em 22,8% e 71,36% respectivamente. O EDTA inibiu apenas 21,5% da atividade
enzimática da tripsina. A enzima não sofreu nenhum efeito na sua atividade proteolítica
quando exposta ao TPCK.
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Artigo 4
Os parâmetros cinéticos da tripsina do D. rhombeus para o substrato BAPNA
(específico para tripsina) estão apresentados na Tabela 2. Dentre os parâmetros analisados,
têm-se a constante de Michaelis (Km) que é um indicador da afinidade da enzima pelo
substrato e a eficiência catalítica (Kcat), que indica o número de moléculas de substrato
convertidas em produto por segundo. Os valores de Km e do Kcat foram 0,266 mM e 0,930
s-1, respectivamente. O valor do Km da tripsina do D. rhombeus foi mais baixo que aqueles
encontrados para bigeye snapper (Priacanthus macrachantus) (Hau e Benjakul, 2006) e
tilápia do Nilo (Oreochromis niloticus) (Bezerra et al., 2005) mostrando maior afinidade
pelo substrato BAPNA. Valores de Km inferiores ao encontrado neste trabalho foram
relatados para sardinha Monterey (Sardinops sagax caerula) (Castillo-Yáñez et al., 2005) e
anchova (Engraulis japonica) (Heu et al., 1995).
Para o sequenciamento do N-terminal da tripsina do D. rhombeus foram
identificados 15 aminoácidos, compondo a seqüência IVGGYECTMHSEAHE, a qual foi
alinhada com outras seqüências de peixes e uma bovina (Fig. 3). De acordo com Cao et al.
(2000), geralmente os sete primeiros resíduos de aminoácidos (IVGGYEC) do N-terminal
de tripsina de peixes demonstram alta homologia. Além disso, todas as tripsinas de peixes
apresentam um resíduo de Glu na posição 6, enquanto em mamíferos, é comum a presença
de Thr, em tripsinas pancreáticas. Como mostrado na Figura 3, a tripsina do D. rhombeus
exibiu esses padrões e apresentou uma maior homologia com os peixes Gadus
macrocephalus (Fuchise et al., 2009), Alcichthys alcicornis (Kishimura et al., 2007),
Theragra chalcograma (Kishimura et al., 2008), Eleginus gracilis (Fuchise et al., 2009) e
Pleuroprammus azonus (Kishimura et al, 2006).
Os dados obtidos sugerem que a protease purificada é uma tripsina símile. Esta
enzima apresentou características compatíveis para sua utilização em processos
industriais (Ex.: indústria alimentícia e detergentes comerciais), demonstrando
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Artigo 4
assim, a viabilidade da utilização dos resíduos do peixe D. rhombeus como fonte de
biomoléculas de interesses biotecnológicos.
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characteristics of trypsin from pyloric ceca of spotted mackerel (Scomber australasicus).
Journal of Food Biochemistry, n. 30, p. 466-477.
Kishimura, H., Hayashi, K., Miyashita, Y., Nonami, Y. (2006b) Characteristic of trypsins
from the viscera of true sadine (Sardinops melanostictus) and the pyloric ceca of arabesque
greeling (Pleuroprammus azonus). Food Chemistry, v. 97, p. 65-70.
Kristjánsson, M. M. (1991) Purification and characterization of trypsin from the pyloric
caeca of rainbow trout (Oncorhynchus mykiss). Journal of Agricultural and Food
Chemistry, v. 39, p. 1738-1742.
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Kurtovic, I., Marshall, S. N., Simpson, B. K. (2006) Isolation and characterization of a
trypsin fraction from the yloric ceca of chinook salmon (Oncorhynchus tshawytscha).
Comparative Biochemistry and Physiology, Part B, v. 143, p. 432-440.
Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature. v. 227. p. 680-685.
Liu, Z. Y., Wang, Z., Xu, S. Y. (2007) Two trypsin from the intestin of grass carp
(Ctenopharyngodon idellus). Comparative Biochemistry Physiology, Part B, v. 177, p.
655-666.
Outzen, H., Berglund, G. I., Smalds, A.0., Willassen, N.P. (1996) Temperature and pH
Sensitivity of Trypsins from Atlantic Salmon (Salmo salar) in Comparison with Bovine
and Porcine Trypsin. Comp. Biochem. Physiol. Vol. 115B, No. 1, pp. 33-45.
Shahidi, F., Kamil, Y.V.A.J. (2001) Enzymes from fish and aquatic invertebrates and their
application in the food industry. Trends in Food Science & Technology 12 435–464.
Shi, C., Marshall, S. N., Simpsom, B. K. (2007) Purification and characterization of trypsin
from the ceca of the New Zealand hoki fish (Macruronus novaezealandiae). Journal of
Food Biochemistry, v. 31, p. 772-796.
Simpson, B.K., Haard, N.F. (1987) Trypsin and trypsin-like enzymes from the stomach
less cunner (Tautogolabrus adspersus): catalytic and other physical characteristics.
Journal of Agricultural and Food Chemistry, v. 35, p. 652-656.
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Souza, A.A.G., Amaral, I.P.G., Santo, A.R.E., Carvalho Jr, L.B.., Bezerra, R.S. (2007)
maculatus). Food Chemistry, v. 100, p. 1429-1434.
Warburg, O., Christian, W. (1941) Isolierung und kristallisation des garungs ferments
enolasc. Biochemical Zeitschrift, v. 310, p. 384-421.
Yoshinaka, R., Sato, M., Suzuki, T., Ikeda, S. (1985) Characterization of an anionic trypsin
from the eel (Anguilla japonica). Comparative Biochemistry Physiology, Part B, v. 80,
p. 11-14.
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Tabela 1 – Efeito de íons e inibidores de protease sobre a atividade da tripsina purificada
da carapeba prateada (D. rhombeus).
Atividade residual (%)
Controle
100,00
Ions (1mM)
Ca2+
183,13 ± 0,75
Li2+
146,18 ± 14,32
K2+
134,46 ± 3,77
Ba2+
108,35 ± 4,52
Mn2+
101,24 ± 4,52
2+
79,57 ± 8,53
Cd 2+
78,69 ± 0,75
Cu 2+
69,27 ± 0,00
Al2+
66,96 ± 0,75
Hg2+
46,89 ± 4,52
Zn2+
28,77 ± 13,41
Pb2+
0,00 ± 0,00
Fe
Inibdores (2mM)
PMSF
77,40 ± 7,37
PMSF (4mM)
32,64 ± 3,03
TPCK
103,64 ± 13,03
TLCK
0,00 ± 0,00
Benzamidina
25,01 ± 0,47
EDTA
78,51 ± 11,09
ȕ-mercaptoetanol
64,61 ± 1,87
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Tabela 2 – Parâmetros cinéticos para a tripsina da carapeba prateada (D. rhombeus),
utilizando o substrato BAPNA (1,2 mM).
Parâmetros
Espécies
Referências
Km
(mM)
Kcat
(s-1)
Kcat/Km
(s-1 mM-1)
Carapeba (D. rhombeus)
Anchova (E. japonica)
0,266
0,049
0,93
1,55
3,48
31,00
Este trabalho
HEU et al., 1995
Sépia (S. officinalis)
0,064
2,32
36,25
BALTI et al., 2009
Bigeye snapper (P.
macracanthus)
Bacalhau (G. morhua)
0,312
1,06
3,40
HAU e BENJAKUL, 2006
0,102
0,70
6,80
ASGEIRSSON et al., 1989
Bovina
0,650
2,00
3,10
ASGEIRSSON et al., 1989
Suína
0,820
1,55
1,89
OUTZEN et al., 1996
Salmão (S. salar)
0,300
0,80
2,67
OUTZEN et al., 1996
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1
2
Figura 1 - Eletroforese em gel de poliacrilamida – SDS-PAGE da tripsina purificada da
carapeba prateada (D. rhombeus). Na linha 1 está o padrão de peso molecular e na linha 2
o fração obtida da coluna de afinidade.
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Artigo 4
A
B
C
Figura 2 – Efeito do pH sobre a atividade da tripsina da carapeba prateada (D. rhombeus).
Os tampões utilizados no ensaio foram fosfato (ŶS+D, Tris- HCl (żS+D
8,5), Glicina-NaOH (ŸS+D) (A), Efeito da temperatura sobre a atividade da
tripsina da carapeba prateada (D. rhombeus). O valor mais alto de atividade enzimática
específica obtida a 55°C, foi estipulada como o 100% (B), Efeito da temperatura sobre a
estabilidade da tripsina da carapeba prateada (D. rhombeus) (C).
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Figura 3 - Alinhamento da sequência N-terminal da tripsina símile da carapeba prateada
(Diapterus rhombeus) com outras de tripsina de peixes e uma tripsina bovina. Os pontos
representam resíduos de aminoácido iguais à sequência principal (presente trabalho) e as
letras indicam os aminoácidos que são diferentes.
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4.5.
Artigo 5
Artigo 5: Alkaline protease from the processing waste of the lane snapper
(Lutjanus synagris) and its compatibility with oxidants, surfactants and
commercial detergents
A ser submetido ao periodico Journal of Industrial Microbiology & Biotechnology
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ALKALINE PROTEASE FROM THE PROCESSING WASTE OF THE LANE
SNAPPER (Lutjanus synagris) AND ITS COMPATIBILITY WITH OXIDANTS,
SURFACTANTS AND COMMERCIAL DETERGENTS
Talita S Espósito, Marina Marcuschi, Ian P G Amaral, Luiz B Carvalho Jr, Ranilson
S Bezerra*
Laboratório de Enzimologia – LABENZ, Departamento de Bioquímica and Laboratório de
Imunopatologia Keizo Asami – LIKA, Universidade Federal de Pernambuco, Brazil
*Corresponding author:
Ranilson S. Bezerra.
Laboratório de Enzimologia – LABENZ, Departamento de Bioquímica, Universidade Federal de
Pernambuco, Cidade Universitária, Recife-PE, Brazil, CEP 50670-420, Tel.: + 55-81-21268540,
Fax: + 55-81-21268576, E-mail address: [email protected]
Running title: Fish protease as a laundry detergent additive
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Abstract
An alkaline protease from the viscera of the lane snapper (Lutjanus synagris) was puri¿HG
by heat treatment, fractionation with ammonium sulfate and affinity chromatography. The
molecular weight of the enzyme was estimated to be 28.4 kDa (SDS-PAGE). The purified
enzyme was capable of hydrolyzing the specific substrate for trypsin benzoyl-arginine-pnitroanilide (BApNA) and was inhibited by benzamidine and tosyl lysine chloromethyl
ketone (TLCK), synthetic trypsin inhibitors and phenylmethylsulphonyl Àuoride (PMSF),
which is a serine-protease inhibitor. The enzyme exhibited maximal activity at pH 9.0 and
45 °C and retained 50% of the activity after incubation at the optimal temperature for 30
min. At a concentration of 10 mM, activity was slightly activated by Ca2+ and inhibited by
the following ions in decreasing order: Cd2+> Hg2+> Cu2+> Zn2+> Al3+. The effects of Ba2+,
K1+ and Li1+ proved to be less intensive. Using 1% (w/v) azocasein as substrate, the
enzyme revealed high resistance (60% residual activity) when incubated with 10% H2O2
for 75 min. The enzyme retained more than 80% activity after 60 min in the presence of
different surfactants (Tween 20, Tween 80 and sodium choleate). The alkaline protease
demonstrated compatibility with commercial detergents (7 mg/mL), such as Bem-te-vi®,
Surf® and Ala®, retaining more than 50% of initial activity after 60 min at 25 ºC and 30
min at 40 ºC. The thermostability and compatibility of this enzyme with commercial
detergents suggests a good potential for application in the detergent industry.
Keywords: lane snapper (Lutjanus synagris), alkaline protease, purification, affinity
chromatography, detergent compatibility
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1. Introduction
The lane snapper (Lutjanus synagris) is a reef-associated lutjanid distributed in the
western Atlantic from the southeast of the USA to southern Brazil [1]. This species is an
important component of commercial fishery in northeastern Brazil and is captured by the
artisanal and commercial fishing fleets [2]. Brazil exports the lane snapper either whole or
without viscera, mainly to the USA [3]. According to the Brazilian environmental agency
IBAMA [2], the Brazilian production of L. synagris in 2006 was of 1863 tons, the majority
of which was captured on the northeastern coast.
Fish processing generates large amounts of solid and liquid waste, such as heads,
tails, skin, bones and viscera. This processing waste is a huge problem for the ¿shery
industry and its disposal has a major economic and environmental impact [4-7]. The use of
fish viscera as a source of biomolecules for biotechnological application is a viable
alternative. This waste is regarded as one of the richest sources of proteolytic enzymes and
it is possible to recover about 1 g of the enzyme per 1 kg of viscera [8-10]. Proteases have
been purified from the processing waste of various fish species, such as tambaqui, Nile
tilapia, Monterey sardine, Japonese anchovy, spotted goatfish, true sardine, arabesque
greenling, jacopever, elkhorn sculpin and sardine [9,11-17]. The studies cited describe the
isolation, puri¿cation and characterization of trypsin (EC 3.4.21.4), which is one of the
main digestive proteases detected in the pyloric caeca and intestine of fish.
Alkaline proteases, mainly trypsin and subtilisin, are the most important group of
industrial enzymes, with applications in the leather, food and pharmaceutical industries as
well as bioremediation processes [18,19]. However, their major application (about 60% of
all protease sold) is in the detergent industry. Biological detergents are commonly used in
domestic laundry soaps because the enzymes provide the additional benefit of low
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temperature washes with improved cleaning performance. The addition of proteases to
detergents considerably increases the cleaning effect by removing protein stains and the
consumption of surface-active substances, thereby decreasing the pollution load [20,21].
Currently, subtilisins are chosen as the enzyme for detergent formulations (US
patent nos. 1240058, 374971, 370482 and 4266031 and UK patent n. 13155937), despite
not being the ideal detergent enzymes due to low thermal stability in presence of detergents
and short shelf-life [22]. Thus, it is desirable the search for new proteases with novel
properties from as many different sources as possible [23].
The lane snapper has a typical carnivorous digestive tract [24] composed of the
stomach followed by the pyloric caeca, which precedes a very short intestine. The
developed pyloric caeca is likely responsible for a higher amount of alkaline proteases. No
information regarding the characteristics of trypsin from the intestine and pyloric caeca of
the lane snapper (Lutjanus synagris) has been reported, despite the importance of this
species to the Brazilian market (mainly the northeastern region) and exportation as an
appreciated marine fish.
The aim of the present study was to purify this enzyme and test the viability of its
biotechnological use in detergent formulations.
2. Materials and Methods
2.1. Preparation of crude extract
Crude extracts from the intestine and pyloric caeca of Lutjanus synagris were
prepared using the method described by Bezerra et al. [11]. Fresh fish were collected in
both the dry and rainy seasons. The intestine and pyloric caeca of these fish were collected
and homogenized (Bodine Electric Company – Chicago, USA) at a proportion of 1 g of
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tissue for each 5 mL of 0.9% NaCl (w/v) prepared in 0.1-M Tris-HCl buffer (pH 8.0). The
homogenate was centrifuged (Sorvall RC-6 Superspeed Centrifuge – North Carolina,
USA) at 10,000 xg for 10 min at 4 ºC and the supernatant (crude extract) was used for the
purification steps.
2.2. Precipitation of enzymes
For the partial purification of the enzymes, the crude extracts were first submitted
to a heat treatment at 45 ºC for 30 min and centrifuged at 10,000 xg for 10 min at 4 ºC
[11]. The supernatant was used in a two-step fractionation with ammonium sulfate (40 and
80% saturation). The precipitate formed at 0-40% and 40-80% saturation of ammonium
sulfate was collected by centrifugation at 10,000 xg at 4 ºC for 15 min, resuspended in 0.1M Tris-HCl buffer (pH 8.0) and dialyzed twice against 4 L of 0.01 M Tris-HCl buffer (pH
8.0) for 12 h. All steps in the enzyme precipitation process were carried out at 4 ºC.
2.3. Purification of trypsin-like enzyme
Aliquots of the fraction with 40-80% ammonium sulfate saturation (5 mg.mL-1
protein) were applied to a column of p-Aminobenzamidine Sepharose 6B (1.5 × 0.2 cm2).
The matrix was balanced with 0.1-M Tris-HCl buffer (pH 8.0). For the elution of trypsin
from the column, 0.2 M of K-Cl buffer (pH 2.0) was used. Fractions of 0.5 mL were
collected at a flow rate of 30 mL.h-1 and 30 PL of 1M Tris-HCl, pH 9.0, were added to
each tube. Fractions containing detectable protein using the Warburg and Christian method
[25] were pooled and dialyzed twice against 2 L of 0.01 M Tris-HCl buffer, pH 8.0, for 12
h. This procedure was repeated five times to obtain 13.6 mg of the purified enzyme.
2.4. Assay for alkaline protease and trypsin activity
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Protease activity was assayed using 1% azocasein (w/v) as substrate, as described
by Bezerra et al. [11], using microplate reader. In triplicate, using microcentrifuge tubes,
1% azocasein (w/v) was incubated with the sample for 60 min at 25 °C. Trichloroacetic
acid (TCA) was added to stop the reaction and after 15 min the tubes were centrifuged for
5 min at 8,000 xg. The supernatant was then added to 1 M NaOH on a microtiter plate and
the absorbance of the mixture was measured in a microtiter plate reader at 450 nm against
a blank in which distilled water was used instead of the tryspsin-like enzyme. One unit (U)
of enzyme activity was defined as the amount of enzyme able to hydrolyze azocasein to
produce a change of 0.001 units of absorbance per minute.
Trypsin activity was determined using the method described by Souza et al. [17]
with adaptations, using 8 mM of benzoyl-arginine-p-nitroanilide (BApNA) as a substrate.
P-nitroaniline release was followed at 405 nm using a microtiter plate reader (Bio-Rad
680). One unit of enzyme activity was defined as the amount of enzyme required to
K\GURO\]HPRORI%$S1$SHUPLQXWH
2.5. Protein determination
The protein content was determined based on the method described by Warburg
and Christian [25], measuring the absorbance of the samples at 280 and 260 nm and using
the following equation: [protein] mg/mL = Abs280 nm × 1.5 - Abs260 nm × 0.75. Porcine
trypsin (Sigma) was used as a standard protein.
2.6. Electrophoresis
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was
carried out using a 4% stacking gel (w/v) and 15% separating gel (w/v) (Vertical
Electrophoresis System Bio-Rad Laboratories, Inc.) based on the method described by
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Artigo 5
Laemmli [26]. The gel was stained with 0.1% Coomassie Brilliant Blue (w/v) and
destained with 10% acetic acid (v/v) and 25% methanol (v/v). The dialyzed trypsin-like
enzyme (50 µg of protein) was concentrated by lyophilization.
2.7. Effect of protease inhibitors
The effect of inhibitors was determined based on the methods described by Alencar
et al. [27] and Bezerra et al. [11], incubating trypsin-like enzyme from the lane snapper
with different specific protease inhibitors (phenylmethylsulfonylfluoride – PMSF; tosyl
lysine chloromethyl ketone – TLCK and benzamidine) at 8 mM. After incubating the
mixture for 30 min, 8 mM of BApNA was added, the residual activity was measured and
the percentage of inhibition was calculated.
2.8. Kinetic studies
The kinetic parameters Vmax and Km were calculated by fitting the reaction rates to
a Michaelis–Menten graph using the Origin Version 6.0 software program (Microcal
Software, Inc). Activity was assayed with different final concentrations of BApNA
prepared in DMSO ranging from 0.01875 to 1.8 mM. The reactions were prepared in
triplicate in a 96-ZHOOPLFURWLWHUSODWHO RIHQ]\PHORI-M Tris–HCl buffer
S+DQGVWDUWHGE\WKHDGGLWLRQRIORI%$S1$%ODQNVZHUHSUHSDUHGZLWKRXWWKH
enzyme.
2.9. Effect of metal ions
The effect of metal ions was assayed using the methods described by Souza et al.
[17]. Samples of the purified enzyme (30 µL) were added to a 96-well microtiter plate with
a 10-mM solution (70 µL) of AlCl3, BaCl2, CdSO4, CuSO4, HgCl2, KCl, LiCl, Pb and
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ZnSO4. After 30 min of incubation, Tris–HCl buffer (70 µL), pH 8.0, and 8 mM of
BAPNA (30 µL) were added. The p-nitroaniline produced was recorded in a microplate
reader (Bio-Rad 680) at 405 nm after 30 min of reaction.
2.10. Effect of pH and temperature
To evaluate the effects of pH and temperature on trypsin, activity was determined
in different 0.1-M buffer solutions (phosphate: pH 6.5-7.5; Tris-HCl: pH 7.2-9.0; and
NaOH-Glycine: pH 8.6-11.0) at 25 ºC. Temperature dependencies of enzyme activity were
determined at pH 9.0 and various temperatures (25-75ºC). Thermal stability was recorded
at 25 ºC after pre-incubating the enzyme for temperatures ranging from 25-60ºC at
intervals of 5 ºC at pH 9.0 for 30 min.
2.11. Effect of oxidizing agents
Hydrogen peroxide stability of the proteases from the lane snapper was investigated
by incubating samplHV / ZLWK +2O2 / DW FRQFHQWUDWLRQV RI DQG
DW & 6DPSOHV / ZHUH ZLWKGUDZQ DW DQG PLQXWH LQWHUYDOV WR
establish their activities (duplicates) on azocasein and to compare them to the non-treated
sample [21].
2.12. Effect of surfactants
Stability with regard to ionic (saponin and sodium choleate) and non-ionic
surfactants (SDS, Tween 20 and Tween 80) was investigated by incubating the purified
enzyme in a 1% concentration of surfactant solution (w/v) for 30 and 60 min at 40 ºC, after
which enzyme activity was assayed [21].
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2.13. Compatibility with commercial detergents
The trypsin-like enzyme from the lane snapper at a concentration of 0.20 mg mL-1
was incubated at 40 ºC with commercial detergents: Ala® (Protec & Gamble); Bem-te-vi®
(Alimonda); Omo Multi Ação® (UniLever) and Surf® (UniLever) in a final concentration
of 7 mg.mL-1. Samples were collected at 10-min intervals for 60 min. The residual
proteolytic activity in each sample was determined at 25 ºC and compared with the control
sample incubated in Tris-HCl, pH 9.0, at 40 ºC. Protease activity was assayed using 1%
azocasein (w/v) as substrate, as described by Bezerra et al. [11], using a microplate reader.
In triplicate, using microcentrifuge tubes, 1% azocasein (w/v) was incubated with the
sample for 60 min at 25 °C. Trichloroacetic acid (TCA) was added to stop the reaction and,
after 15 min, the tubes were centrifuged for 5 min at 8000 xg. The supernatant was then
added to 1 M of NaOH on a microtiter plate and the absorbance of the mixture was
measured in a microtiter plate reader at 450 nm against a blank in which distilled water
was used instead of the tryspsin-like enzyme. One unit (U) of enzyme activity was defined
as the amount of enzyme able to hydrolyze azocasein to produce a change of 0.001 units of
absorbance per minute.
Results and discussion
In the present study, a trypsin-like enzyme was purified from the intestine and
pyloric caeca of the lane snapper in three steps: heat treatment, ammonium sulfate
precipitation and affinity chromatography. The enzyme was purified 63.85-fold from the
crude extract (Table 1). Protein purification strategies are generally of high cost and time
consuming. Indeed, this is an important limiting factor for the commercial use of fish
processing waste as a source of proteases. However, the procedures employed in the
present study are of relatively low cost and the raw material (fish viscera) has little or no
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cost at all, often being discarded. The present study also confirms the previously observed
efficiency of heat treatment and ammonium sulfate precipitation as step in purifying
trypsins from tropical fish [9,11,12,17]. P-Aminobenzamidine is a highly effective and
specific ligand for purification of trypsin-like enzymes [28,29] and was used effectively in
the present study as the final purification step. This technique was able to purify a protease
(only one band in SDS-PAGE) with an estimated molecular weight of 28.4 kDa (Fig. 1).
Similar results have been found for trypsin from other fish: Oreochromis niloticus (23.5
kDa) [11], Sardina pilchardus (25 kDa) [12], Engraulis japonica (24 kDa) [14], Sardinops
melanostictus, Pleuroprammus azonus (24 kDa) [15], Sebastes schlegelii, Alcichthys
alcicornis (24 kDa) [16] and Pseudupeneus maculatus (24.5 kDa) [17].
As this protease was obtained from a biological source that lives in an open system
(marine fish), it is subject to seasonal changes that could be reflected in many aspects of its
physiology, including protease synthesis in the digestive tract. In tropical northeastern
Brazil, the seasonal effect is minimized; the water temperature generally ranges from 24 to
28 oC throughout the year. Moreover, fish diet may vary seasonally in tropical costal
environments, changing in both quality and quantity, mainly with oscillation of salinity
generated during the rainy and dry seasons. The lane snapper is known to be a generalist
carnivore and trophic opportunist, preying on a wide range of food sources [30, 31].
Despite the different environmental condictions (e.g., salinity, temperature and food
availability) between the dry and rainy seasons, no differences were observed in trypsin
activity in the lane snapper (54.3 and 54.2 U/mg, respectively).
The trypsin-like protease from the lane snapper was inhibited by the serine
proteinase inhibitor PMSF (45%) and exhibited strong inhibition in the presence of TLCK
(81.22%) and benzamidine (77.75%), which are synthetic trypsin inhibitors. These results
indicate that this enzyme is probably a trypsin. Similar results are reported in a study by
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Bezerra et al. [11], in which an alkaline protease from Nile tilapia intestine was also
inhibited by PMSF, TLCK and benzamidine (approximately 55, 100 and 87.5%,
respectively).
The BApNA hydrolysis rates obeyed the Michaelis-Menten kinetics model
regarding the concentration of substrate examined in the present study (Fig.2). Km and
Vmax values for the trypsin-like enzyme from the lane snapper acting on BApNA were 0.66
± 0.044 mM and 2370.40 ± 65.93 mU, respectively. This Km value is similar to that
reported by Martinez et al. [32] for trypsin from anchovy (Engraulis encrasicolus), which
is a pelagic marine fish that feeds on plankton, unlike the lane snapper, which is a reefassociated marine fish that feeds on small fish, bottom-living crabs, shrimp, worms,
gastropods and cephalopods. In comparison to other marine fish, this value is lower than
trypsin from the spotted goatfish (Pseudupeneus maculates, Km=1.94 and 1.82) and higher
than trypsin from Monterey sardine (Sardinops sagax caerulea, Km= 0.051), mullet (Mugil
cephalus, Km=0.49), other species of anchovy (E. japonica, Km= 0.049) and salmon
(Oncorhynchus keta, Km=0.029) [17, 13, 33-35]. The Km value for the lane snapper was
also different from trypsin found in freshwater fish – lower than Nile tilapia (Oreochromis
niloticus, Km= 0.772) and higher than carp (Cyprinus carpio, Km=0.039) [11, 37].
Table 2 displays the effects of the ions on trypsin-like enzyme activity from the
intestine of the lane snapper. This enzyme proved sensitive to ions, mainly Al3+, Cd2+,
Cu 2+, Hg2+ and Zn2+. It is known that Cd 2+, Co2+ and Hg2+ act on sulphhydryl residues in
proteins [40]. The inhibition caused by these metal ions suggests the relevance of
sulfhydryl residues for the catalytic action of this protease [11]. In other studies on tropical
fish proteases, these ions also inhibited trypsin-like enzyme activity in samples from the
intestine of the Nile tilapia and the pyloric caeca of the spotted goatfish [11,17]. Despite
the effects of Cd2+, Zn2+ and Al3+ (respectively, 6.07%, 56.39% and 57.69%) on enzyme
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activity from the intestine of the lane snapper, its influence is lesser than that recorded for
the Nile tilapia enzyme (respectively, 3.0%, 30.5% and 0%). However, the effects of Cu2+
and Hg2+ (respectively, 13.09% and 11.39%) proved to be stronger than those recorded for
the Nile tilapia enzyme (respectively, 43.9% and 38.0%). The inhibition effects of Li+, K+
and Ba2+ on lane snapper enzyme activity were less intensive than those displayed by the
ions mentioned above. Only Ca2+ increased enzyme activity. This result suggests that this
enzyme possesses the primary calcium-binding site found in mammalian pancreatic trypsin
and trypsin from other fish [41, 42].
Enzymes used in detergent formulations should have high optimal pH and thermal
stability [21], which are characteristics of the protease purified from the lane snapper (Fig.
3) and other reef-associated marine fish, such as the cunner (Tautogolabrus adspersus)
[43]. Compared to bacterial enzymes used as additives in detergent (Alcalase,
Savinase, Esperase - Novozymes, Denmark; Maxatase- Gist-brocades, The
Netherlands), the enzyme from the lane snapper has a lower optimal temperature activity
(Fig. 3a), but retained 50% of its activity after 30 min of incubation at 45 qC, while most of
the bacterial enzymes used have low thermal stability above this temperature (Fig. 3b).
The effect of pH on trypsin activity is illustrated in Fig. 3c. The enzyme hydrolyzed
BApNA effectively at alkaline pH with optimal activity at pH 8.0-10.5, which is similar to
that of other ¿sh trypsins [9, 11, 17, 27, 44-48]. This characteristic likely contributes to its
physiological role in intestinal tissue, where pH is high [40], and is a relevant aspect that
enables its use in detergent formulations, as the pH of laundry detergents is commonly
alkaline [23].
The performance of proteases in detergent is influenced by several factors (e.g., pH,
ionic strength, washing temperature, detergent composition, bleach systems and
mechanical handling). To test the compatibility of this enzyme, its proteolytic activity was
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assayed under different conditions resembling these factors. Fig. 4 displays the oxidant
stability of the lane snapper protease in the presence of hydrogen peroxide. The peroxide
inactivation curve indicates that the lane snapper digestive protease is stable even at high
concentrations of H2O2 (15% v/v). These results are similar to those found for alkaline
proteases from the tambaqui and carp [49,50]. As bleach stability has only been attained by
site-directed mutagenesis [51, 52] or protein engineering [53] of bacterial enzymes, this
characteristic can be considered relevant from the biotechnological standpoint.
Enzyme activity of the trypsin-like enzyme from the lane snapper was analyzed in
the presence of non-ionic (Tween 20 and Tween 80) and ionic (sodium choleate)
surfactants using azocasein as substrate. Table 3 shows the high stability of this enzyme
when incubated with these surfactants. Tween 80 increased protease activity by 33.6%
after 30 min of incubation. After 60 min of incubation with Tween 20 and sodium
choleate, the protease retained 94.5% and 99.1% of its initial activity. Only sodium
dodecyl sulfate (SDS) was capable of strongly inhibiting the enzyme after 60 min.
The alkaline protease from L. synagris demonstrated stability and compatibility
with a wide range of commercial detergents at 25 °C and 40 °C (Fig.5). The enzyme
retained about 50% of its activity after 1 h in the presence of the Surf®, Ala® and Bem-tevi® detergents at 25 °C. After 1h at 40 °C, the enzyme retained more than 60% of its
activity in the Surf® detergent and retained about 50% of its activity in the presence of
Ala® and Bem-te-vi® after 30 min. The Omo® detergent inhibited the activity of the
enzyme, with about 70% loss of activity after 30 min of incubation at 40 °C. Espósito et al.
[49] found that tambaqui proteases retained more than 50% of their activity when
incubated with the Ala®, Bem-te-vi® and Omo® detergents for 1 h at 40 qC. Maximal
stability was observed with the Surf® detergent, as the enzyme retained 73.70% of its
activity. Studies on the compatibility of proteases from the fungi Conidiobolus coronatus
143
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and Nocardiopsis sp in the presence of detergents demonstrate activity retention of 64%
and 90%, respectively [21,54]. Studies on alkaline proteases from species of Bacillus
describe retention of more than 70% of activity after 1 h at 40 ºC [55].
Enzymes from fish viscera contribute toward sustainable development by being
isolated from waste that is usually discarded. In the search for an alkaline-stable protease
for use in the detergent industry and based on the results of the present study, trypsin from
L. synagris viscera was easely purified through affinity chromatography with high
recovery (about 90%). It therefore demonstrates good potential for application in laundry
detergents. Moreover, the economy in production would make this enzyme suitable for
low-cost operations in the industry.
Acknowledgments
We would like to express our thanks to CAPES, PETROBRAS, SEAP/PR, CNPq,
FINEP and FACEPE for providing the necessary financial support to complete this work.
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Table 1:
Purification of trypsin-like enzyme from the pyloric caeca of the lane snapper
Purfication steps
Crude extract
Step 1: Heat treatment
Step 2: Ammonium sulfate
precipitation
F1(NH4)2SO4 (0-40%)
F2 (NH4)2SO4 (40-80%)
Step 3: Affinity
chromatography
Total protein
Specific activity
Total
(mg)
(U/mg protein)
activity
(U)
121,410.99 142.71
850.76
124,747.25 61.29
2035.36
100.0
102.7
1.0
2.4
0.3
664.84
2.85
102.626.37 49.50
109,076.92 2.01
0.5
84.5
89.84
2.4
0.3
63.85
233.58
2073.26
54,317.8
Recovery Purification
(%)
(fold)
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Table 2:
Ion effect on the trypsin-like enzyme from the pyloric caeca of the lane snapper
Ion (10
mM)
Al3+
Ba2+
Ca2+
Cd2+
Cu2+
Hg2+
K+
Li+
Zn2+
Residual Activity±SD (%)
57.69 ± 8.81
72.11±1.14
115.25±1.24
6.07±0.8
13.09±0.76
11.39±1.97
90.52±4.10
96.02±5.10
56.36±3.11
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Table 3:
Values are expressed in ± standard deviation; n = 3; the specific enzyme activity of the
control sample (100%) was 50,000 U/mg using azocasein as substrate
Surfactants
(1% w/v)
Residual activity
(%)
After 30 min
After 60 min
Sodium choleate
SDS
Tween 20
Tween 80
71.8±10.8
61.1±10.6
84.0±5.8
133.6±22.9
99.1±12.3
3.6±3.1
94.5±9.4
81.8±6.9
155
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List of figures:
Fig. 1: SDS-PAGE of intestine and pyloric caeca purified trypsin from the lane snapper;
Lane 1: Standard proteins; Lane 2: Pool collected by p-Aminobenzamidine Sepharose 6B;
molecular weight was estimated using the protein standards galctosidase (116 kDa),
phosphorylase b (97.4 kDa), bovine serum albumin (66 kDa), alcoholdehydrogenase (37.6
kDa), carbonic anhydrase (28.5 kDa), myoglobin (18.4 kDa) and lysozyme (14 kDa)
Fig. 2: Michaelis–Menten plot for trypsin kinetics; BApNA concentrations (1.8–0.01875
mM); R2=0.99
Fig.3: Effect of temperature (A), thermal stability (B) and pH (C) on trypsin-like enzyme
from lane snapper intestine and pyloric caeca; The purified enzyme collected from paminobenzamidine sepharose 6B was incubated with BApNA (8mM) at the temperatures
and pH indicated for 30 min. The products were measured at 405 nm. Thermal stability
was determined by assaying activity (25-75ºC) after pre-incubation for 30 min at the
temperatures indicated. All the experiments were carried out in triplicate. Values (mean ±
SD) are expressed as percentage of highest activity.
Fig. 4: Inactivation curve of H2O2 on protease from the pyloric caeca and intestine of L.
synagris precipitated with 40-80% ethanol. Enzyme preparations were incubated at pH 9.0
and 40 ºC, with H2O2 at concentrations of 5% (z) and 10% (S). Samples were withdrawn
at time intervals; their activities (duplicates) were established using azocasein as substrate
and compared to the non-treated sample ().
156
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Fig. 5: Stability of protease in commercially available detergents. Protease (0.2 mg mL-1)
was incubated at 25 ºC and 40 ºC in presence of detergents at 7mg mL-1. Activity of
control sample devoid of any detergent incubated under similar conditions (Ŷ6XUIƔ
Ala® (Ÿ%HP-te-vi® (ź2PR Multi-Ação® (i)
157
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Fig.1:
Espósito, T.S.
1
2
116 kDa 97.4 kDa 66 kDa 37.6 kDa -
28.5 kDa -
28.4 kDa
18.4 kDa 14 kDa -
158
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Fig. 2:
Espósito, T.S.
2000
1800
1600
Activity (um/mL)
1400
1200
1000
800
Km = 0.66 mM
Vmax = 2,370.40 mU
600
400
200
0
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
mM
159
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Fig. 3:
.
Espósito, T.S.
110
A
100
90
Residual Activity (%)
80
70
60
50
40
30
20
10
0
20
30
40
50
60
70
80
o
Temperature ( C)
110
100
90
B
80
70
60
50
40
30
20
10
0
20
30
40
50
60
70
80
Temperature (ºC)
110
C
100
90
Relative Activity (%)
80
70
60
50
40
30
20
10
0
4
5
6
7
8
9
10
11
pH
160
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Fig. 4:
Espósito, T.S.
Control
5%
10%
15%
110
100
90
80
70
60
50
40
30
20
10
0
0
10
20
30
40
50
60
70
80
Time (min)
161
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Fig. 5:
Espósito, T.S.
o
25 C
110
100
90
Residual activity (%)
80
70
60
50
40
30
20
10
0
0
10
20
30
40
50
60
Time (min)
110
40ºC
100
90
80
70
60
50
40
30
20
10
0
0
10
20
30
40
50
60
Time (min)
162
Conclusões
5 CONCLUSÕES
O presente estudo indicou que:
1. A precipitação com etanol demonstrou ser efetiva na obtenção de proteases
alcalinas (principalmente enzimas símiles a tripsina) das vísceras de Colossoma
macropomum e Cyprinus carpio. Essas proteases agiram efetivamente em pH e
temperatura recomendados para enzimas utilizadas como aditivos de detergentes
em pó. As proteases semi-purificadas também apresentaram estabilidade no pH e
temperatura recomendados, bem como na presença de surfactantes iônicos e nãoiônicos. Além disso, as enzimas permaneceram estáveis em altas concentrações de
H2O2 e na presença de diversos detergentes em pó comerciais;
2. Os resultados de inibição e o calculo do peso molecular indicam que a enzima
purificada das vísceras de C. macropomum, Diapterus rhombeus e Lutjanus
synagris, neste trabalho, é uma enzima símile a tripsina. O alinhamento da
sequência N- terminal da enzima purificada das espécies C macropomum e D.
rhombeus corroboram estes resultados. A estabilidade destas enzimas na presença
de agentes surfactantes e oxidantes e detergentes comerciais combinado com a
termoestabilidade e pH ótimo alto, demonstram a possibilidade aplicação destas
enzimas como aditivos de detergentes em pó.
163
Anexos
6. ANEXOS
164
Anexos
6.1. NORMAS DO PERIÓDICO BIORESOURCE TECHNOLOGY
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justification, i.e., do not use a constant right-hand margin.) Ensure that each new paragraph
is clearly indicated. Present tables and figure legends on separate pages at the end of the
manuscript. If possible, consult a recent issue of the journal to become familiar with layout
and conventions. Number all pages consecutively, use 12 pt font size and standard fonts.
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Page length: Maximum page length should be 12, 35 and 40 pages for Short
Communication, Original article/case study and review paper, including text, references,
tables and figures. Each figure and table must be put separately on a single page.
Corresponding author: Clearly indicate who is responsible for correspondence at all stages
of refereeing and publication, including post-publication. Ensure that telephone and fax
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consult a recent journal paper for style if possible.
Abstract: Each paper should be provided with an Abstract of about 100-150 words,
reporting concisely on the purpose and results of the paper.
Keywords: Immediately after the abstract, provide a maximum of five keywords (avoid, for
example, 'and', 'of'). Be sparing with abbreviations: only abbreviations firmly established in
the field may be eligible.
Symbols: Abbreviations for units should follow the suggestions of the British Standards
publication BS 1991. The full stop should not be included in abbreviations, e.g. m (not m.),
ppm (not p.p.m.), '%' and '/' should be used in preference to 'per cent' and 'per'. Where
abbreviations are likely to cause ambiguity or not be readily understood by an international
readership, units should be put in full.
Units: Follow internationally accepted rules and conventions: use the international system
of units (SI). If, in certain instances, it is necessary to quote other units, these should be
added in parentheses. Temperatures should be given in degrees Celsius. The unit 'billion' is
ambiguous and must not be used.
Maths: Authors should make clear any symbols (e.g. Greek characters, vectors, etc.) which
may be confused with ordinary letters or characters. Duplicated use of symbols should be
avoided where this may be misleading. Symbols should be defined as they arise in the text
and
separate
Nomenclature
should
also
be
supplied.
References: All publications cited in the text should be presented in a list of references
(maximum 20, 35 and 75 references for short communication, original research paper/case
study and review papers, respectively) following the text of the manuscript.
Text: All citations in the text should refer to:
1. Single author: the author's name (without initials, unless there is ambiguity) and the year
of publication;
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3. Three or more authors: first author's name followed by 'et al.' and the year of
publication.
Citations may be made directly (or parenthetically). Groups of references should be listed
first alphabetically, then chronologically. Examples: "as demonstrated (Allan, 1996a,
1996b, 1999; Allan and Jones, 1995). Kramer et al. (2000) have recently shown ...."
List: References should be arranged first alphabetically and then further sorted
chronologically if necessary. More than one reference from the same author(s) in the same
year must be identified by the letters "a", "b", "c", etc., placed after the year of publication.
Examples:
Reference to a journal publication: Van der Geer, J., Hanraads, J.A.J., Lupton, R.A., 2000.
The art of writing a scientific article. J. Sci. Commun. 163, 51-59.
Reference to a book: Strunk Jr., W., White, E.B., 1979. The Elements of Style, third ed.
Macmillan, New York.
Reference to a chapter in an edited book: Mettam, G.R., Adams, L.B., 1999. How to
prepare an electronic version of your article, in: Jones, B.S., Smith , R.Z. (Eds.),
Introduction to the Electronic Age. E-Publishing Inc., New York, pp. 281-304.
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Colour Costs and Queries: For colour illustrations, a colour printing fee is charged to the
author per colour unit. Further information concerning colour illustrations and costs is
available
from
Author
Support
at
[email protected],
and
at
http://authors.elsevier.com/locate/authorartwork.
FREE ONLINE COLOUR If, together with your accepted article, you submit usable colour
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Tables: Tables should be numbered consecutively and given suitable captions and each
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to tables should be typed below the table and should be referred to by superscript
lowercase letters. Note that the maximum number of figures allowed for Original article,
case study, and review papers is 6.
Figures: Please make sure that figure files are in an acceptable format (TIFF, EPS or MS
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submit usable color figures then Elsevier will ensure, at no additional charge, that these
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Note that the maximum number of figures allowed for Original article, case study, and
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etc...), while retaining the maximum limit of 6.
Conclusions: State here the inferences drawn from the results, preferably in running text
form, in maximum 100 words. No results should be given here.
Electronic Annexes: We strongly encourage you to submit electronic annexes, such as
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are submitting on hardcopy, please supply 3 disks/CD ROMs containing the electronic
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article." Production will insert the relevant URL at the typesetting stage after this
statement.
Notification: Authors will be notified of the acceptance of their paper by the editor. The
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Copyright: All authors must sign the Transfer of Copyright agreement before the article
can be published. This transfer agreement enables Elsevier to protect the copyrighted
168
Anexos
material for the authors, but does not relinquish the authors' proprietary rights. The
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For more information on proofreading please go to our proofreading page
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6.2. NORMAS DO PERIÓDICO BRAZILIAN JOURNAL OF FOOD TECHNOLOGY
170
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6.3. NORMAS DO PERIÓDICO JOURNAL OF INDUSTRIAL MICROBIOLOGY
& BIOTECHNOLOGY
Edited by: Allen I. Laskin
Print ISSN: 1367-5435
Online ISSN: 1476-5535
Impact Factor: 1.919 (JCR-2008)
Author Guidelines
The Journal of Industrial Microbiology and Biotechnology is an international journal
which publishes papers describing original research, short communications, and critical
reviews in the fields of biotechnology, fermentation and cell culture, biocatalysis,
environmental microbiology, natural products discovery and biosynthesis, marine natural
products, metabolic engineering, genomics, bioinformatics, and other areas of applied
microbiology.
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-in-Chief for final acceptance.
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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 Editorin-Chief in advance to determine the suitability of the topic.
All manuscripts are subject to copy-editing after acceptance.
ONLINE SUBMISSION
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.
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
176
Anexos
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
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Anexos
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”
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 wellcontrasted 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
178
Anexos
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
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, lowerquality 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.
Scan resolution:
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Anexos
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
e-mail: [email protected]
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:
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.
Proofreading
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Anexos
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.
Offprints
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
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.
Authors must provide a signed Copyright Transfer Statement for their paper (see "After
Acceptance")
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.
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Order Open Choice
After 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 OpenChoice,
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.
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.
Proof reading
The purpose of the proof is to check for typesetting or conversion errors and the
completeness and accuracy of the text, tables and figures. Substantial changes in
content, e.g., new results, corrected values, title and authorship,
are not allowed without the approval of the Editor.
After online publication, further changes can only be made in the form of an Erratum,
which will be hyperlinked to the article.
Online First
The article will be published online after receipt of the corrected proofs. This is the
official first publication citable with the DOI. After release of the printed version, the
paper can also be cited by issue and page numbers.
182

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