universidade federal de pernambuco - RI UFPE

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

Talita da Silva Espósito

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

Talita da Silva Espósito

Dedicatória

DEDICATÓRIA

À minha querida avó Maria Espósito (in memorian), com muitas saudades. Aos meus pais, irmãos, sobrinhos e a Igor da Mata.

i

Talita da Silva Espósito

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.

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Talita da Silva Espósito

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.

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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

Talita da Silva Espósito

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

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Talita da Silva Espósito

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

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Talita da Silva Espósito

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

Talita da Silva Espósito

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

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Talita da Silva Espósito

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 carapeba prateada (Diapterus rhombeus)

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

Talita da Silva Espósito

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  6XUIŠ Ɣ  $ODŠ (Ÿ %HP-te-vi® (ź 2PR0XOWL-Ação® (i)………………………

163

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Talita da Silva Espósito

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).

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Talita da Silva Espósito

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).

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Talita da Silva Espósito

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

Talita da Silva Espósito

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

Talita da Silva Espósito

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).

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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

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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.

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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

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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

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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

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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).

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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

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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

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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

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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

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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

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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

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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).

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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

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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.

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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.

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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).

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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

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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

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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

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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).

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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 Aditivo de detergentes

Bactéria

-

Aditivo de detergentes

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)

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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

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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).

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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.

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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.

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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, 1999.

BANIK, R.M.; PRAKASH, M. Laundry detergent compatibility of the alkaline protease from Bacillus cereus. Microbiological Research, v. 159, p. 135-140, 2004.

BARRETT, A. J. Classification of peptidases. In: BARRETT, A. J. (ed). Methods in Enzymology. New York: Academic Press, 1994. v. 244, p. 1-59.

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Referências Bibliográficas

BERG, J. M; STRYER, L.; TYMOCZKO, J. L. Bioquímica. 5 ed. Rio de Janeiro: Guanabara Koogan, 2004. 1104 p.

BEG, Q.K.; GUPTA, R. Purification and characterization of an oxidation-stable, thioldependent serine alkaline protease from Bacillus mojavensis. Enzyme and Microbial Technology, v. 32, p. 294-304, 2003.

BERNARDINO,

G.

Desenvolvimento

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BEZERRA, R. S. et al. Characterization of stomach and piloric caeca proteinases of tambaqui (Colossoma macropomum). Journal of Food Biochemistry, v. 24, p.198-199, 2000.

BEZERRA, R. S.; VIEIRA, V. L. A; CARVALHO Jr., L. B. Proteases no trato digestivo de peixes: tripsina do tambaqui (Colossoma macropomum), modelo alternativo para o aproveitamento de sub-produtos da indústria pesqueira. Biotecnologia Ciência & Desenvolvimento, n. 22, p. 46-49, setembro/outubro, 2001a.

BEZERRA, R. S. et al. Partial purification and characterization of a thermostable trypsin from pyloric caeca of tambaqui (Colossoma macropomum). Journal of Food Biochemistry, v. 25, p. 199-210, 2001b.

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Referências Bibliográficas

BEZERRA, R. S. et al. Alkaline proteinase from intestine of Nile tilapia (Oreochromis niloticus). Process Biochemistry, v. 40, p. 1829-1834, 2005.

BJARNASSON, J. B.; MANTYLA, E. O.; ASGEIRSSON, B. Purification and characterization of proteolytic digestive enzymes from the pyloric caeca of Atlatic cod. In: WALTHER, B. T.; FYHN, H. J. (eds). Physiological and Biochemical Aspects of Fish Development. Norway: University of Bergen, 1993.

BOSSI, A.; RIGHETTI, P. G.; VECCHIO, G.; SERVINSEN, S. Focusing of alkaline protease (Subtilisins) in pH 10-12 immobilized gradients. Electrophoresis, 12, 1535-1540, 1994.

BOUGATEF, A., et al. Purification and characterization of trypsin from the viscera of sardine (Sardina pilchardus). Food Chemistry, v.102, p.343-350, 2007.

BRACHT, A.; ISHII-IWAMOTO, E. L. Métodos de Laboratório em Bioquímica. Barueri: Manole, 2002. p. 77-192.

BRODY, T. Nutritional Biochemistry. USA: Academic Press, 1994. 657p.

CANO-LOPEZ, A.; SIMPSON, B. K.; HAARD, N. F. Extraction of carotenoprotein from shrimp process wastes with the aid of trypsin from Atlantic cod. Journal of Food Science, v. 52, p. 503-506, 1987.

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Referências Bibliográficas

CARVALHO, A. P. et al. Solubility and peptide profile affect the utilization of dietary protein by common carp (Cyprinus carpio) during early larval stages. Aquaculture, v. 234, p. 319-333, 2004.

CARVALHO-FILHO, A. Peixes: Costa brasileira. 3 ed. São Paulo: Melro, 1999.

CASTILLO-YÁÑES, F. J.; PACHECO-AGUIAR, R.; GARCÍA-CARREÑO, F.L.; TORO, M.A.N. Isolation and characterization of trypsin from pyloric caeca of Monterrey sardine Sardinops sagax caerulea. Comparative Biochemistry and Physiology (Part B), v. 140, p.91-98, 2005.

CERVIGÓN, F. et al. Fichas FAO de Identificación de Especies para los Fines de La Pesca. Guía de campo de las especies comerciales marinas y de aguas salobres de la costa septentrional de sur America. Comisión de Comunidades Europeas y de NORAD. Roma, FAO, 1992. 513p.

CHEN, S. T.; KAO, C. L.; WANG, K. T. Alkaline protease catalysis of a secondary amine to form a peptide bond. International Journal of Peptide and Protein Research, v. 46, p. 314-319, 1995.

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YOSHINAKA, R.; SATO, M.; SUZUKI, T.; IKEDA, S. Enzymatic characterization of anionic trypsin of the catfish Parasilurus asotus. Comparative Biochemistry and Physiology (Part B), v. 77, p. 1-6, 1984.

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Artigos Científicos

4. ARTIGOS CIENTÍFICOS

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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

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Artigo 2

Artigo 2: Fish processing waste as a source of alkaline proteases for laundry detergent

Publicado no periódico Food Chemistry

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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

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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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Artigo 3

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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Artigo 3

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 76

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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). 77

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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 78

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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 79

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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\WKHUHVLGXDODFWLYLWLHVDWƒ& 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-FROXPQ—P[PP 7ZRHOXWLRQVROYHQWV\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 82

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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 83

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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 84

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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 85

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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). 88

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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 89

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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. 90

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Acknowledgements This work was financially supported by SEAP, CNPq, FINEP, UFPE/FACEPE and PETROBRAS.

References Alencar, R.B., Biondi, M.M., Paiva, P.M.G., Vieira, V.L.A., Carvalho Junior, L.B., Bezerra, R.S., 2003. Alkaline proteases from the digestive tract of four tropical fishes. Brazilian Journal of Food Technology. 6 (2), 279-284. Almeida. L.C., Lundstedt, L.M., Moraes, G., 2006. Digestive enzyme responses of tambaqui (Colossoma macropomum) fed on different levels of protein and lipid. Aquaculture Nutrition. 12, 443–450. Anwar, A., Saleemuddin, M., 1998. Alkaline peptidases: a review. Bioresource Technology. 64, 175-183. Aride, P.H.R., Roubach, R., Val, A.L., 2007.Tolerance response of tambaqui Colossoma macropomum (Cuvier) to water pH. Aquaculture Research. 38, 588-594. Baird, T., Wang, B, Lodder, M., Hecht, S.M., Craik, C.S., 2000. Generation of active trypsin by chemical cleavage. Tetrahedron. 56, 9477-9485. Barros, N.M.T., Campos, M., Bersanetti, P.A., Oliveira, V., Juliano, M.A., Boileau, G., Juliano, L., Carmona, A.K., 2007. Neprilysin carboxydipeptidase specificity studies and improvement in its detection with fluorescence energy transfer peptides. Biological Chemistry. 388, 447-455. Bezerra, R.S., Santos, J.F, Carvalho Júnior., L.B., Lino, M.A.S., Vieira, V.L.A., 2000. Characterization of stomach and pyloric caeca proteinases of tambaqui (Colossoma macropomum). Journal of Food Biochemistry. 24, 189-199. 91

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Bezerra, R.S., Santos, J.F., Paiva, P.M.G., Correia, M.T.S., Coelho, L.C.B.B., Vieira, V.L.A., Carvalho Júnior, L.B., 2001. Partial purification and characterization of a thermostable trypsin from pyloric caeca of tambaqui (Colossoma macropomum). Journal of Food Biochemistry. 25(3), 199-210. Bezerra, R.S., Lins, E.J.F., Alencar, R.B., Paiva, P.M.G., Chaves, M.E.C., Coelho, L.C.B.B., Carvalho Júnior, L.B., 2005. Alkaline proteinase from intestine of Nile tilapia (Oreochromis niloticus). Process Biochemistry. 40, 1829-1834. Bougatef, A., Souissi, N, Fakhfakh, N., Ellouz-Triki,Y., Nasri, M., 2007. Purification and characterization of trypsin from the viscera of sardine (Sardina pilchardus). Food Chemistry. 102, 343-350. Bradford, M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye-binding. Analytical Biochemistry. 72, 248. Castillo-Yañez, F.J., Pacheco-Aguilar, R, García-Carreño, F.L., Navarrete-Del Toro, M.A., 2005. Isolation and characterization of trypsin from pyloric caeca of Monterey sardine Sardinops sagax caerulea. Comparative Biochemistry and Physiology Part B. 140, 9198. Espósito, T.S., Amaral, I.P.G., Buarque, D.S., Oliveira, G.B., Carvalho Jr., L.B., Bezerra, R.S., 2009a. Fish processing waste as a source of alkaline proteases for laundry detergent. Food Chemistry. 112, 125-130. Espósito, T.S.; Amaral, I.P.G.; Marcuschi, M.; Carvalho Jr, l.b.; Bezerra, R.S. 2009b. Surfactants- and oxidants-resistant alkaline proteases from common carp (cyprinus carpio l) processing waste. Journal of Food Biochemistry, In press. Fuchise, T., Kishimura, H., Sekizaki, H., Nonami, Y.,Kanno, G., Klomklao, S., Benjakul, S., Chun, B.S., 2009. Purification and characteristics of trypsins from cold-zone fish, 92

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pacific cod (Gadus macrocephalus) and saffron cod (Eleginus gracilis). Food Chemistry. 116, 611-616. Gudmundsdóttir, A., Pálsdóttir, H.M., 2005. Atlantic cod trypsins: from basic research to practical applications. Marine biotechnology. 7, 77-88. Hedstrom, L., 2002. Serine protease mechanism and specificity. Chemical Reviews. 102, 4501-4523. Jellouli, K., Bougatef, A., Daassi, D., Balti, R., Barkia, A., Nasri, M., 2009. New alkaline trypsin from the intestine of grey triggerfish (Balistes capriscus) with high activity at low temperature: purification and characterisation. Food Chemistry. 116, 644-650. Kasserra, H.P., Laidler, K.J., 1969. Mechanisms of action of trypsin and chymotrypsin. Canadian Journal of Chemistry. 47, 4031-4039. Kapoor, B.G.; Smit, H.; Verighina, I.A., 1975. The alimentary canal and digestion in teleosts. Advancens in marine biology. 13, 109-239. Kishimura, H., Hayashi, K., Miyashita,Y., Nonami, Y., 2006. Characteristics of trypsins from the viscera of true sardine (Sardinops melanostictus) and the pyloric ceca of arabesque greenling (Pleuroprammus azonus). Food Chemistry, 97, 65-70. Kishimura, H, Tokuda, Y., Yabe, M., Klomklao, S., Benjakul, S., Ando, S., 2007. Trypsins from the pyloric ceca of jacopever (Sebastes schlegelii) and elkhorn sculpin (Alcichthys alcicornis): Isolation and characterization. Food Chemistry. 100, 1490-1495. Kishimura, H., Klomklao, S., Benjakul, S.,Chun, B.S., 2008. Characteristics of trypsin from the pyloric ceca of walleye pollock (Theragra chalcogramma). Food Chemistry. 106, 194-199. Klomklao, S, Benjakul, S., Visessanguan, W., Kishimura, H., Simpson B.K., Saeki, H., 2006. Trypsins from yellowfin tuna (Thunnus albacores) spleen: purification and characterization. Comparative Biochemistry and Physiology Part B. 144, 47-56. 93

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Klomklao, S., Benjakul, S., Visessanguan, W., Kishimura H., Simpson, B.K. Trypsin from the pyloric caeca of bluefish (Pomatomus saltatrix)., 2007a. Comparative Biochemistry and Physiology Part B. 148, 382-389. Klomklao, S., Benjakul, S., Visessanguan, W., 2007b. Purification and characterisation of trypsins from the spleen of skipjack tuna (Katsuwonus pelamis). Food Chemistry. 100, 1580-1589. Klomklao, S., Kishimura, H., Nonami, Y., Benjakul, S., 2009. Biochemical properties of two isoforms of trypsin purified from the Intestine of skipjack tuna (Katsuwonus pelamis). Food Chemistry. 115, 155-162. Laemmli U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227, 680–685. Leatherbarrow, R.J., 2001. Grafit. Erithacus Software Limited. Lu, B.J, Zhou, L.G., Cai, Q.F., Hara, K., Maeda, A., Su, W.J., Cao, M.J., 2008. Purification and characterisation of trypsins from the pyloric caeca of mandarin fish (Siniperca chuatsi). Food Chemistry. 110, 352-360. Marana, S.R., Lopes, A.R., Juliano, L., Juliano, M.A., Ferreira, C., Terra, W.R., 2002. Subsites of trypsin active site favor catalysis or substrate binding. Biochemical and Biophysical Research Counications. 290, 494-497. Matsuo, A.Y.O., Wood, C.M., Val, A.L., 2005. Effects of copper and cadmium on ion transport and gill metal binding in the Amazonian teleost tambaqui (Colossoma macropomum) in extremely soft water. Aquatic Toxicology. 74, 351-364. Maurer, K.H., 2004. Detergent proteases. Current Opinion in Biotechnology. 15, 330334. Mei, C.; Jiang, X. 2005. A novel surfactant- and oxidation-stable alkaline protease from Vibrio metschnikovii DL 33–51. Process Biochemistry 40, 2167–2172. 94

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Moreira, K.A., Albuquerque, B.F., Teixeira, M.F.S., Porto, A.L.F., Lima Filho, J.L., 2002. Application of protease from Nocardiopsis sp. as a laundry detergent additive. World Journal of Microbiology & Biotechnology. 18, 307-312. Norioka, S., Sakiyama, F., 2004. Trypsin. In: Barrett, A. J., Rawlings, N. D., Woessner, J.F. (Eds). Handbook of Proteolytic Enzymes. Academic Press, London,. 2nd eddition, pp. 1483-1488. Rawlings, N.D., Morton, F.R., Barrett, A.J., 2007. An Introduction to Peptidases and the Merops Database. In: Polaina, J. and MacCabe, A.P (editores). Industrial Enzymes: Structure, Function and Application. Springer, 1st eddition. pp. 161-179 Roach, J.C., Wang, K., Gan, L., Hood, L., 1997. The molecular evolution of the vertebrate trypsinogens. Journal of Molecular Evolution. 45, 640-652. Rodriguez, J., Gupta, N., Smith, R.D., Pevzner, P.A., 2008. Does trypsin cut before proline? Journal of Proteome Research. 7(1), 300-305. Souza, A.A.G., Amaral, I.P.G., Santo, A.R.E., Carvalho Jr, L.B., Bezerra, R.S., 2007. Trypsin-like enzyme from intestine and pyloric caeca of spotted goatfish (Pseudupeneus maculatus). Food Chemistry. 100, 1429-1434. Wolfgang, A., 2004. Enzymes in Household Detergents. In: Enzymes in Industry. 3rd eddition, Wiley-VCh. p.p. 154-180.

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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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Figures 1

2 A

B

Figure 1a and 1b

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A

B

C

Figure 2a, 2b and 2c

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Figure 3

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Artigo 4

Artigo 4: Purificação e caracterização de uma protease alcalina das vísceras da carapeba prateada (Diapterus rhombeus)

A ser submetido ao periódico Brazilian Journal of Food Technology

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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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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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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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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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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 108

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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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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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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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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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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 113

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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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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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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 116

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assim, a viabilidade da utilização dos resíduos do peixe D. rhombeus como fonte de biomoléculas de interesses biotecnológicos.

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Bezerra, R. S., Vieira, V. L. A., Santos, A. J. G. (2001a) Clico reprodutivo da carapeba prateada Diapterus rhombeus (Cuvier, 1829), no litoral de Pernambuco. Tropical Oceanography, v. 1, p. 67-78.

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Bezerra, R. S., Lins E. J. F., Alencar R. B., Paiva, P. M. G., Chaves, M. E. C., Coelho L. C. B. B., Carvalho Jr L. B. (2005) Alkaline proteinase from intestine of Nile tilapia (Oreochromis niloticus). Process Biochemistry, v. 40, p. 1829-1834.

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Castillo-Yáñes, F.J., Pacheco-Aguiar, R., García-Carreño, F.L., Toro, M.A.N. (2005) Isolation and characterization of trypsin from pyloric caeca of monterrey sardine Sardinops sagax caerulea. Comparative Biochemistry Physiology, Part B, v. 140, p. 91-98.

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Cerqueira, V. R. Cultivo de peixes marinhos. In: Poli, C, R.; Poli, A. T. B.; Andreata, E.; Beltrame, E. Aquicultura. Experiências brasileiras. Ed. Multitarefa , 2004, 445 p.

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Hau, P. V., Benjakul, S. (2006) Purification and characterization of trypsin from caeca of bigeye snapper (Pricanthus macracanthus). Journal of Food Biochemistry, v. 30, p. 478495.

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Klomklao, S., Benjakul, S., Visessanguan, W. Khishimura, H., Simpsom, B. K. (2007a) Trypsina from the pyloric ceca of bluefish (Pomatomus saltatrix). Comparative Biochemistry Physiology, Part B, v. 148, p. 382-389.

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Klomklao, S., Benjakul, S., Visessanguan, W., Kishimura, H., Simpsom, B. K. (2007b) 29 kDa trypsin from the pyloric ceca of Atlantic bonito (Sarda sarda): recovery and chacterization. Journal of Agricultural and Food Chemistry, v. 55, p. 4548-4553.

Kishimura, H., Tokuda, Y., Yabe, M., Klomklao, S., Benjakul, S., Ando, S. (2007) Trypsin from the ceca of jacopever (Sebastes schlegelii) and elkorn sculpin (Alcichthys alcicornis): Isolation and characterization. Food Chemistry, v. 100, p. 1490-1495.

Kishimura, H. Klomklao, S., Benjakul, S, Chun, B. S. (2008) Charateristics of trypsin from ceca of walleye pollock (Teragra chalcogramma). Food Chemistry, v. 106, p. 194-199.

Kishimura, H., Tokuda, Y., Klomklao, S., Benjakul, S., Ando, S. (2006a) Enzymatic 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.

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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.

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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) Trypsin-like enzyme from intestine and pyloric caeca of spotted goatfish (Pseudupeneus maculatus). Food Chemistry, v. 100, p. 1429-1434.

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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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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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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\]H—PRORI%$S1$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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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-ZHOOPLFURWLWHUSODWH—O RIHQ]\PH—ORI-M Tris–HCl buffer S+ DQGVWDUWHGE\WKHDGGLWLRQRI—ORI%$S1$%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  ž& 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 142

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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

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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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21.Moreira KA, Albuquerque BF, Teixeira MFS, Porto ALF, Lima Filho JL (2002) Application of protease from Nocardiopsis sp. as a laundry detergent additive. World J Microbiol Biotechnol 18: 307-312

22. Samal BB, Kara B, Stabinsky Y (1990) Stability of two novel serine proteinases in commercial laundry detergent formulations. Biotechnol Bioeng 35: 650-652

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23. Banerjee UC, Sani RK, Azmi W, Soni R (1999) Thermostable alkaline protease from Bacillus brevis and its characterization as a laundry detergent additive. Process Biochem 35: 213-219. doi:10.1016/S0032-9592(99)00053-9

24. Dominici-Arosemena A, Wolff M (2005) Reef fish community structure in Bocas del Toro (Caribbean, Panama) along spatial scales and gradients in habitat complexity. Caribb J Sci 41:613–637. doi: 10.1007/s10152-006-0045-4

25. Warburg O, Christian W (1941) Isolierung und kristallisation des garungs ferments enolase. Biochem Z 310: 384–421

26. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of Bacteriophage T4. Nature 227: 680-685

27. Alencar RB, Biondi MM, Paiva PMG, Vieira VLA, Carvalho Jr LB, Bezerra RS (2003) Alkaline proteases from the digestive tract of four tropical fishes. Brazilian Journal of Food Technology 6: 279-284

28. Osnes KK, Mohr V (1985) On the purification and characterization of three serine-type peptide hydrolases from antarctic krill, Euphausia superba. Comp Biochem Physiol B 82: 607–19. doi:10.1016/0305-0491(85)90497-3

29. Rick W (1974) Trypsin. In: Bergmeyer HU, editor. Methods of Enzymatic Analysis, 2nd edn. New York: Academic Press. pp1013–23

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30. Duarte L, Garcia C (1999). Diet of the mutton snapper Lutjanus analis (Cuvier) from the gulf of Salamanca. Colombia, Caribbean Sea. Bulletin of Marine Science 65(2):453465

31. Sierra LM, Claro R, Popota OA (1994) Alimentación y relaciones tróficas. In: Claro R (ed), Ecología de los peces marinos de Cuba, pp. 263-320. Instituto de Oceanología. Academia de Ciencias de Cuba y Centro de Investigaciones de Quintana Roo, México.

32. Martínez A, Olsen RL, Serra JL (1988) Purification and characterization of two trypsin-like enzymes from the digestive tract of anchovy Engraulis encrasicholus.Comp Biochem Physiol B 91(4):677-84. doi:10.1016/0305-0491(88)90191-5

33. Guizani N, Rolle RS, Marshall MR, Wei CI (1991) Isolation, purification and characterization of a trypsin from the pyloric caeca of mullet (Mugil cephalus). Comp Biochem Physiol B 98: 517-521. doi:10.1016/0305-0491(91)90246-A

34. Heu MS, Kim HR, Pyeun JH (1995) Comparison of trypsin and chymotrypsin from the viscera of anchovy, Engraulis japonica. Comp Biochem Physiol B 112(3):557-67. doi: doi:10.1016/0305-0491(95)00111-5

35. Sekizaki H, Itoh K, Murakami M, Toyota E, Tanizawa K (2000) Anionic trypsin from chum salmon: activity with p-amidinophenyl ester and comparison with bovine and Streptomyces

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39. Aranishi F, Watanabe T, Osatomi K, Cao M, Hara K, Ishihara T (1998) Purification and characterization of thermostable dipeptidase from carp intestine. J Mar Biotechnol 6:116–123

40. Male R, Lorens LB, Smalas AO, Torrissen KR (1995). Molecular cloning and characterization of anionic and cationic variants of trypsin from Atlantic salmon. Eur J Biochem 232: 677–685

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46. Sabapathy U, Teo LH (1995) Some properties of the intestinal proteases of the rabbitfish, Siganus canaliculatus (Park). Fish Physiol Biochem 14: 215-221

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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

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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 ().

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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 (Ŷ 6XUIŠƔ  Ala® (Ÿ %HP-te-vi® (ź 2PR Multi-Ação® (i)

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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 -

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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

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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

Residual Activity (%)

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

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Fig. 4:

Espósito, T.S.

Control 5% 10% 15%

110 100 90 Residual Activity (%)

80 70 60 50 40 30 20 10 0 0

10

20

30

40

50

60

70

80

Time (min)

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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 Residual Activity (%)

80 70 60 50 40 30 20 10 0 0

10

20

30

40

50

60

Time (min)

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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.

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6. ANEXOS

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6.1. NORMAS DO PERIÓDICO BIORESOURCE TECHNOLOGY Edited by: S.C. Ricke

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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

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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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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

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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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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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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.

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