Identification of proteases produced by entomopathogenic fungi Beauveria bassiana (Bals) Vuill. strain CG432 previously activated in coffee berry borer alive (Hypothenemus hampei)
Autor(a) principal: | |
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Data de Publicação: | 2013 |
Outros Autores: | , , , , , , , |
Tipo de documento: | Artigo |
Idioma: | por |
Título da fonte: | Semina. Ciências Agrárias (Online) |
Texto Completo: | https://ojs.uel.br/revistas/uel/index.php/semagrarias/article/view/9652 |
Resumo: | Conidia of entomopathogenic fungi can penetrate the insect exoskeleton both by mechanic action of the germinative tube and by production of multiple proteases, chitinases, and lipases in response to the composition of the insect cuticle. Therefore the purpose of this work was to produce, to purify, and to characterize the structure of the proteases produced by the fungus Beauveria bassiana CG432, previously activated in adults of coffee berry borer (Hypothenemus hampei) grown under submerged culture conditions. A suspension containing 106 activated conidia/mL was inoculated to a culture medium at 28ºC and 150 rpm for 3 days. Protease extracts, were obtained by centrifugation at 8000g for 20 minutes, were fractionated and were concentrated by ultrafiltration using controlled porosity membranes of 100 kDa and 3 kDa, respectively. Gel filtration chromatography on Sephadex G-100 isolated one protein peak (peak II), which contained 56% of residues of the aminoacid aspartic acid, characterized by HPLC using an ODS-C18 reverse phase column. The peak protein showed specific activity 43 times superior when compared to the free-cell extract in bovine serum albumin, subtilisin-like protease activity, and a single protein band reveled by Brilliant Coomassie Blue on a gelatin zimogram PAGE electrophoresis, by native conditions. The homogeneity of peak II was confirmed by revelation of a single band during determination of the isoelectric pH 4.5, but molecular mass determination by PAGE-2D electrophoresis showed 2 bands of 23 and 26 kDa. Proteases were characterized as serine proteases with cysteine residues important to activity, since they had been inhibited by phenylmethylsulphonyl fluoride and p-chlormercuricbenzoic acid. Peak II proteases showed Km of 4x10-4 on subtilisin-like substrate. |
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Semina. Ciências Agrárias (Online) |
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Identification of proteases produced by entomopathogenic fungi Beauveria bassiana (Bals) Vuill. strain CG432 previously activated in coffee berry borer alive (Hypothenemus hampei)Identificação de proteases produzidas pelo fungo entomopatogênico Beauveria bassiana (Bals) Vuill. Cepa CG432 previamente ativada em insetos vivos de broca do café ((Hypothenemus hampei))Beauveria bassianaProteasesPurificationStructural characterization.Beauveria bassianaProteasesPurificaçãoCaracterização estrutural.Bioquímica de MicrorganismosEnzimologiaProteômicaConidia of entomopathogenic fungi can penetrate the insect exoskeleton both by mechanic action of the germinative tube and by production of multiple proteases, chitinases, and lipases in response to the composition of the insect cuticle. Therefore the purpose of this work was to produce, to purify, and to characterize the structure of the proteases produced by the fungus Beauveria bassiana CG432, previously activated in adults of coffee berry borer (Hypothenemus hampei) grown under submerged culture conditions. A suspension containing 106 activated conidia/mL was inoculated to a culture medium at 28ºC and 150 rpm for 3 days. Protease extracts, were obtained by centrifugation at 8000g for 20 minutes, were fractionated and were concentrated by ultrafiltration using controlled porosity membranes of 100 kDa and 3 kDa, respectively. Gel filtration chromatography on Sephadex G-100 isolated one protein peak (peak II), which contained 56% of residues of the aminoacid aspartic acid, characterized by HPLC using an ODS-C18 reverse phase column. The peak protein showed specific activity 43 times superior when compared to the free-cell extract in bovine serum albumin, subtilisin-like protease activity, and a single protein band reveled by Brilliant Coomassie Blue on a gelatin zimogram PAGE electrophoresis, by native conditions. The homogeneity of peak II was confirmed by revelation of a single band during determination of the isoelectric pH 4.5, but molecular mass determination by PAGE-2D electrophoresis showed 2 bands of 23 and 26 kDa. Proteases were characterized as serine proteases with cysteine residues important to activity, since they had been inhibited by phenylmethylsulphonyl fluoride and p-chlormercuricbenzoic acid. Peak II proteases showed Km of 4x10-4 on subtilisin-like substrate. Conídios de fungos entomopatogênicos atravessam o exoesqueleto do inseto pela ação mecânica do tubo germinativo e produção de múltiplas isoformas de proteases, quitinases e lipases em resposta à composição da cutícula do inseto. Desta forma o objetivo deste trabalho foi extrair, purificar e caracterizar a estrutura de proteases produzidas em cultivo submerso por Beauveria bassiana CG432 previamente ativada em adultos vivos de broca-do-café (Hypothenemus hampei). Uma suspensão contendo 106 conídios ativados/mL foi inoculada em meio de cultura líquido a 28ºC, 150 rpm por 3 dias. O extrato de proteases (EP) foi obtido da centrifugação a 8000 g por 20 minutos, fracionado e concentrado por ultrafiltração em membrana de porosidade controlada 100 kDa e 3 kDa, respectivamente. A cromatografia de gel filtração em Sephadex G-100 separou um pico proteico (Pico II) que apresentou 56% de resíduos do aminoácido ácido aspártico quando analisado por HPLC em coluna de fase reversa ODS-C18; atividade específica 43 vezes superior ao EP sobre soro albumina bovina; atividade de protease tipo-subtilisina e uma única banda proteica revelada por nitrato de prata e Coomassie Brilhant Blue em zimograma sobre gelatina por eletroforese PAGE em condições nativas. A homogeneidade do Pico II foi confirmada pela revelação de uma única banda durante a determinação do pH isoelétrico igual a 4,5, porém a determinação da massa molecular separou 2 bandas de 23 e 26 kDa por eletroforese PAGE-2D. As proteases foram caracterizadas como serino proteases com resíduo cisteína importante para a atividade, pois foram inibidas por fluoreto fenil-metil-sufonil e ácido p-cloromercúriobenzóico. As proteases do Pico II apresentaram Km 4x10-4 sobre substrato tipo-subtilisina.UEL2013-02-28info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionapplication/pdfhttps://ojs.uel.br/revistas/uel/index.php/semagrarias/article/view/965210.5433/1679-0359.2012v33n6Supl2p3055Semina: Ciências Agrárias; Vol. 33 No. 6Supl2 (2012); 3055-3068Semina: Ciências Agrárias; v. 33 n. 6Supl2 (2012); 3055-30681679-03591676-546Xreponame:Semina. Ciências Agrárias (Online)instname:Universidade Estadual de Londrina (UEL)instacron:UELporhttps://ojs.uel.br/revistas/uel/index.php/semagrarias/article/view/9652/pdfCopyright (c) 2013 Semina: Ciências Agráriashttp://creativecommons.org/licenses/by-nc/4.0info:eu-repo/semantics/openAccessVaréa, Geni SilvaOliveira, Jakeliny Akemi YamamotoSugahara, Vanessa HitomiIto, Eliana TiemiPinto, Jurandir PereiraTrevisan, DalvaRamos, Humberto Josué de OliveiraMagalhães, Diogo Maciel dePereira, Luiz Filipe Protásio2023-01-26T15:08:22Zoai:ojs.pkp.sfu.ca:article/9652Revistahttp://www.uel.br/revistas/uel/index.php/semagrariasPUBhttps://ojs.uel.br/revistas/uel/index.php/semagrarias/oaisemina.agrarias@uel.br1679-03591676-546Xopendoar:2023-01-26T15:08:22Semina. Ciências Agrárias (Online) - Universidade Estadual de Londrina (UEL)false |
dc.title.none.fl_str_mv |
Identification of proteases produced by entomopathogenic fungi Beauveria bassiana (Bals) Vuill. strain CG432 previously activated in coffee berry borer alive (Hypothenemus hampei) Identificação de proteases produzidas pelo fungo entomopatogênico Beauveria bassiana (Bals) Vuill. Cepa CG432 previamente ativada em insetos vivos de broca do café ((Hypothenemus hampei)) |
title |
Identification of proteases produced by entomopathogenic fungi Beauveria bassiana (Bals) Vuill. strain CG432 previously activated in coffee berry borer alive (Hypothenemus hampei) |
spellingShingle |
Identification of proteases produced by entomopathogenic fungi Beauveria bassiana (Bals) Vuill. strain CG432 previously activated in coffee berry borer alive (Hypothenemus hampei) Varéa, Geni Silva Beauveria bassiana Proteases Purification Structural characterization. Beauveria bassiana Proteases Purificação Caracterização estrutural. Bioquímica de Microrganismos Enzimologia Proteômica |
title_short |
Identification of proteases produced by entomopathogenic fungi Beauveria bassiana (Bals) Vuill. strain CG432 previously activated in coffee berry borer alive (Hypothenemus hampei) |
title_full |
Identification of proteases produced by entomopathogenic fungi Beauveria bassiana (Bals) Vuill. strain CG432 previously activated in coffee berry borer alive (Hypothenemus hampei) |
title_fullStr |
Identification of proteases produced by entomopathogenic fungi Beauveria bassiana (Bals) Vuill. strain CG432 previously activated in coffee berry borer alive (Hypothenemus hampei) |
title_full_unstemmed |
Identification of proteases produced by entomopathogenic fungi Beauveria bassiana (Bals) Vuill. strain CG432 previously activated in coffee berry borer alive (Hypothenemus hampei) |
title_sort |
Identification of proteases produced by entomopathogenic fungi Beauveria bassiana (Bals) Vuill. strain CG432 previously activated in coffee berry borer alive (Hypothenemus hampei) |
author |
Varéa, Geni Silva |
author_facet |
Varéa, Geni Silva Oliveira, Jakeliny Akemi Yamamoto Sugahara, Vanessa Hitomi Ito, Eliana Tiemi Pinto, Jurandir Pereira Trevisan, Dalva Ramos, Humberto Josué de Oliveira Magalhães, Diogo Maciel de Pereira, Luiz Filipe Protásio |
author_role |
author |
author2 |
Oliveira, Jakeliny Akemi Yamamoto Sugahara, Vanessa Hitomi Ito, Eliana Tiemi Pinto, Jurandir Pereira Trevisan, Dalva Ramos, Humberto Josué de Oliveira Magalhães, Diogo Maciel de Pereira, Luiz Filipe Protásio |
author2_role |
author author author author author author author author |
dc.contributor.author.fl_str_mv |
Varéa, Geni Silva Oliveira, Jakeliny Akemi Yamamoto Sugahara, Vanessa Hitomi Ito, Eliana Tiemi Pinto, Jurandir Pereira Trevisan, Dalva Ramos, Humberto Josué de Oliveira Magalhães, Diogo Maciel de Pereira, Luiz Filipe Protásio |
dc.subject.por.fl_str_mv |
Beauveria bassiana Proteases Purification Structural characterization. Beauveria bassiana Proteases Purificação Caracterização estrutural. Bioquímica de Microrganismos Enzimologia Proteômica |
topic |
Beauveria bassiana Proteases Purification Structural characterization. Beauveria bassiana Proteases Purificação Caracterização estrutural. Bioquímica de Microrganismos Enzimologia Proteômica |
description |
Conidia of entomopathogenic fungi can penetrate the insect exoskeleton both by mechanic action of the germinative tube and by production of multiple proteases, chitinases, and lipases in response to the composition of the insect cuticle. Therefore the purpose of this work was to produce, to purify, and to characterize the structure of the proteases produced by the fungus Beauveria bassiana CG432, previously activated in adults of coffee berry borer (Hypothenemus hampei) grown under submerged culture conditions. A suspension containing 106 activated conidia/mL was inoculated to a culture medium at 28ºC and 150 rpm for 3 days. Protease extracts, were obtained by centrifugation at 8000g for 20 minutes, were fractionated and were concentrated by ultrafiltration using controlled porosity membranes of 100 kDa and 3 kDa, respectively. Gel filtration chromatography on Sephadex G-100 isolated one protein peak (peak II), which contained 56% of residues of the aminoacid aspartic acid, characterized by HPLC using an ODS-C18 reverse phase column. The peak protein showed specific activity 43 times superior when compared to the free-cell extract in bovine serum albumin, subtilisin-like protease activity, and a single protein band reveled by Brilliant Coomassie Blue on a gelatin zimogram PAGE electrophoresis, by native conditions. The homogeneity of peak II was confirmed by revelation of a single band during determination of the isoelectric pH 4.5, but molecular mass determination by PAGE-2D electrophoresis showed 2 bands of 23 and 26 kDa. Proteases were characterized as serine proteases with cysteine residues important to activity, since they had been inhibited by phenylmethylsulphonyl fluoride and p-chlormercuricbenzoic acid. Peak II proteases showed Km of 4x10-4 on subtilisin-like substrate. |
publishDate |
2013 |
dc.date.none.fl_str_mv |
2013-02-28 |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
https://ojs.uel.br/revistas/uel/index.php/semagrarias/article/view/9652 10.5433/1679-0359.2012v33n6Supl2p3055 |
url |
https://ojs.uel.br/revistas/uel/index.php/semagrarias/article/view/9652 |
identifier_str_mv |
10.5433/1679-0359.2012v33n6Supl2p3055 |
dc.language.iso.fl_str_mv |
por |
language |
por |
dc.relation.none.fl_str_mv |
https://ojs.uel.br/revistas/uel/index.php/semagrarias/article/view/9652/pdf |
dc.rights.driver.fl_str_mv |
Copyright (c) 2013 Semina: Ciências Agrárias http://creativecommons.org/licenses/by-nc/4.0 info:eu-repo/semantics/openAccess |
rights_invalid_str_mv |
Copyright (c) 2013 Semina: Ciências Agrárias http://creativecommons.org/licenses/by-nc/4.0 |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
UEL |
publisher.none.fl_str_mv |
UEL |
dc.source.none.fl_str_mv |
Semina: Ciências Agrárias; Vol. 33 No. 6Supl2 (2012); 3055-3068 Semina: Ciências Agrárias; v. 33 n. 6Supl2 (2012); 3055-3068 1679-0359 1676-546X reponame:Semina. Ciências Agrárias (Online) instname:Universidade Estadual de Londrina (UEL) instacron:UEL |
instname_str |
Universidade Estadual de Londrina (UEL) |
instacron_str |
UEL |
institution |
UEL |
reponame_str |
Semina. Ciências Agrárias (Online) |
collection |
Semina. Ciências Agrárias (Online) |
repository.name.fl_str_mv |
Semina. Ciências Agrárias (Online) - Universidade Estadual de Londrina (UEL) |
repository.mail.fl_str_mv |
semina.agrarias@uel.br |
_version_ |
1799306065502797824 |