Propriedades bioquímicas e cinético-enzimáticas de cisteíno- proteases do intestino médio da lagarta da soja

Detalhes bibliográficos
Autor(a) principal: Mendonça, Eduardo Gomes de
Data de Publicação: 2008
Tipo de documento: Dissertação
Idioma: por
Título da fonte: LOCUS Repositório Institucional da UFV
Texto Completo: http://locus.ufv.br/handle/123456789/2394
Resumo: Protease inhibitors that react with insect specific proteases are candidates to have their genetic code inserted in genetically modified plants. The first step to achieve this is the characterization of proteolytic enzymes of these insects intestines. Cysteine-protease of the soluble and insoluble fractions from the midgut of Anticarsia gemmatalis were characterized using the substrate LBApNA. The Soluble Fraction, called Fraction I, was obtained from the suspension medium after nine cycles of freezing and thawing of the intestine of A. gemmatalis, and the Insoluble Fraction, called Fraction II, was obtained from the maceration with detergent Brij 35 of the resulting pellet of the Fraction I. Two pH values were found with high activity in both Fractions they were pH 3.6 and 8.0 for the Fraction I and 4.6 and 8.0 for the Fraction II. And the peak of activity when tested the influence of temperature was at 35oC for the Fraction I and 60°C for the Fraction II. The KM app found for the Fractions I and II were 2.28 mM and 0.44 mM, respectively, and the Vmax app for the respective Fractions were 297.68 nM.s-1 and 122.95 nM.s-1. Four protease inhibitors were tested, which one from one class of protease: TLCK (inhibitor of serine-protease), E-64 (inhibitor for cysteine-protease), EDTA (inhibitor of metallo-protease) and Pepstatin A (inhibitor of aspartyl-protease). The higher inhibition was observed when E-64 was added to the medium for both Fractions, once that all concentrations tested decreased significantly the activity of cysteine- protease. An increase of TLCK into the medium made a gradual decrease in the activity of cysteine-protease, due to the reaction with the amino acid histidine residue in the catalytic triad, which is also part of the triad of cisteine-protease. The effect of EDTA in the activity of cysteine-protease of A. gemmatalis on L-BApNA shows the difference between the two Fractions analyzed. The Fraction I do not depend on the divalent ions as Ca2 + for its activity. However, the Fraction II is a calcium- dependent, since EDTA reduced significantly its activity. The Pespstatin A did not influenced the cysteine-proteases of the Fraction I, exerting little influence on the Fraction II.
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spelling Mendonça, Eduardo Gomes dehttp://lattes.cnpq.br/8989382342757236Oliveira, Joel Antônio dehttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4707224A0Oliveira, Maria Goreti de Almeidahttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4790894D6Guedes, Raul Narciso Carvalhohttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4721108T2Moreira, Maurílio Alveshttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4796105P2Oliveira, Tânia Toledo dehttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4787758J2Brumano, Maria Helena Nasserhttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4791952U32015-03-26T13:07:23Z2009-02-122015-03-26T13:07:23Z2008-02-27MENDONÇA, Eduardo Gomes de. Biochemistry and kinectics-enzymatics properties of cysteine-proteases of the velvetbean caterpillar midgut. 2008. 68 f. Dissertação (Mestrado em Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal) - Universidade Federal de Viçosa, Viçosa, 2008.http://locus.ufv.br/handle/123456789/2394Protease inhibitors that react with insect specific proteases are candidates to have their genetic code inserted in genetically modified plants. The first step to achieve this is the characterization of proteolytic enzymes of these insects intestines. Cysteine-protease of the soluble and insoluble fractions from the midgut of Anticarsia gemmatalis were characterized using the substrate LBApNA. The Soluble Fraction, called Fraction I, was obtained from the suspension medium after nine cycles of freezing and thawing of the intestine of A. gemmatalis, and the Insoluble Fraction, called Fraction II, was obtained from the maceration with detergent Brij 35 of the resulting pellet of the Fraction I. Two pH values were found with high activity in both Fractions they were pH 3.6 and 8.0 for the Fraction I and 4.6 and 8.0 for the Fraction II. And the peak of activity when tested the influence of temperature was at 35oC for the Fraction I and 60°C for the Fraction II. The KM app found for the Fractions I and II were 2.28 mM and 0.44 mM, respectively, and the Vmax app for the respective Fractions were 297.68 nM.s-1 and 122.95 nM.s-1. Four protease inhibitors were tested, which one from one class of protease: TLCK (inhibitor of serine-protease), E-64 (inhibitor for cysteine-protease), EDTA (inhibitor of metallo-protease) and Pepstatin A (inhibitor of aspartyl-protease). The higher inhibition was observed when E-64 was added to the medium for both Fractions, once that all concentrations tested decreased significantly the activity of cysteine- protease. An increase of TLCK into the medium made a gradual decrease in the activity of cysteine-protease, due to the reaction with the amino acid histidine residue in the catalytic triad, which is also part of the triad of cisteine-protease. The effect of EDTA in the activity of cysteine-protease of A. gemmatalis on L-BApNA shows the difference between the two Fractions analyzed. The Fraction I do not depend on the divalent ions as Ca2 + for its activity. However, the Fraction II is a calcium- dependent, since EDTA reduced significantly its activity. The Pespstatin A did not influenced the cysteine-proteases of the Fraction I, exerting little influence on the Fraction II.Inibidores de protease que atuam em proteases específicas de insetos são candidatos para terem seus códigos genéticos inseridos em plantas geneticamente modificadas. Um primeiro passo para se alcançar isso é a caracterização das enzimas proteolíticas do intestino desses insetos. Cisteíno-proteases da Fração solúvel e da Fração insolúvel do intestino médio de Anticarsia gemmatalis foram caracterizadas utilizando o substrato L-BApNA. A Fração solúvel, chamada de Fração I, foi obtida do sobrenadante após nove ciclos de congelamento e descongelamento do intestino de A. gemmatalis e a Fração insolúvel, chamada de Fração II, obtida da maceração com o detergente Brij 35 do pellet resultante da Fração I. Verificaram-se dois valores de pH com pronunciada atividade em ambas as Frações, sendo eles pH 3,6 e 8,0 para a Fração I e 4,6 e 8,0 para a Fração II. Já o pico de atividade quando testada a influência da temperatura foi em 35oC para a Fração I e 60°C para a Fração II. O KM app encontrado para as Frações I e II foram de 2,28 mM e 0,44 mM respectivamente. A Vmáx app para as respectivas Frações foram 297,68 nM.s-1 e 122,95 nM.s-1. Quatro inibidores de proteases foram testados, sendo cada um deles de uma classe de protease: TLCK (inibidor de serino-protease), E-64 (inibidor de cisteíno-protease), EDTA (inibidor de metaloprotease) e Pepstatina A (inibidor de aspartil- protease). A inibição mais pronunciada foi verificada quando E-64 foi adicionado ao meio para ambas as Frações, uma vez que todas as concentrações testadas diminuíram significativamente a atividade de cisteíno-protease. Um aumento de TLCK no meio fez com que a atividade de cisteíno-protease caísse gradativamente devido à reação desse com resíduo de aminoácido histidina na tríade catalítica, o qual faz parte de tríade de cisteíno-protease também. O efeito de EDTA na atividade de cisteíno-protease de A. gemmatalis sobre L-BApNA mostra a diferença entre as duas Frações analisadas. A Fração I não depende de íons divalentes como Ca2+ para sua atividade. Já a Fração II é cálcio-dependente, pois EDTA diminuiu significativamente sua atividade. Pespstatina A não influenciou cisteíno-proteases da Fração I, exercendo pouca influência na Fração II.Coordenação de Aperfeiçoamento de Pessoal de Nível Superiorapplication/pdfporUniversidade Federal de ViçosaMestrado em Bioquímica AgrícolaUFVBRBioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animalSojaLagarta da sojaCisteíno-proteasesSoybeanVelvetbean caterpillarCysteine-proteasesCNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::ENZIMOLOGIAPropriedades bioquímicas e cinético-enzimáticas de cisteíno- proteases do intestino médio da lagarta da sojaBiochemistry and kinectics-enzymatics properties of cysteine-proteases of the velvetbean caterpillar midgutinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisinfo:eu-repo/semantics/openAccessreponame:LOCUS Repositório Institucional da UFVinstname:Universidade Federal de Viçosa (UFV)instacron:UFVORIGINALtexto completo.pdfapplication/pdf610164https://locus.ufv.br//bitstream/123456789/2394/1/texto%20completo.pdfd4acc98ff4458d460a2e468db7ed737dMD51TEXTtexto completo.pdf.txttexto completo.pdf.txtExtracted texttext/plain101692https://locus.ufv.br//bitstream/123456789/2394/2/texto%20completo.pdf.txta5e90e813e49550a4ce4aa07fe84705bMD52THUMBNAILtexto completo.pdf.jpgtexto completo.pdf.jpgIM Thumbnailimage/jpeg3686https://locus.ufv.br//bitstream/123456789/2394/3/texto%20completo.pdf.jpg52eb479035c93a16cf653c6b0fb700c6MD53123456789/23942016-04-07 23:11:36.082oai:locus.ufv.br:123456789/2394Repositório InstitucionalPUBhttps://www.locus.ufv.br/oai/requestfabiojreis@ufv.bropendoar:21452016-04-08T02:11:36LOCUS Repositório Institucional da UFV - Universidade Federal de Viçosa (UFV)false
dc.title.por.fl_str_mv Propriedades bioquímicas e cinético-enzimáticas de cisteíno- proteases do intestino médio da lagarta da soja
dc.title.alternative.eng.fl_str_mv Biochemistry and kinectics-enzymatics properties of cysteine-proteases of the velvetbean caterpillar midgut
title Propriedades bioquímicas e cinético-enzimáticas de cisteíno- proteases do intestino médio da lagarta da soja
spellingShingle Propriedades bioquímicas e cinético-enzimáticas de cisteíno- proteases do intestino médio da lagarta da soja
Mendonça, Eduardo Gomes de
Soja
Lagarta da soja
Cisteíno-proteases
Soybean
Velvetbean caterpillar
Cysteine-proteases
CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::ENZIMOLOGIA
title_short Propriedades bioquímicas e cinético-enzimáticas de cisteíno- proteases do intestino médio da lagarta da soja
title_full Propriedades bioquímicas e cinético-enzimáticas de cisteíno- proteases do intestino médio da lagarta da soja
title_fullStr Propriedades bioquímicas e cinético-enzimáticas de cisteíno- proteases do intestino médio da lagarta da soja
title_full_unstemmed Propriedades bioquímicas e cinético-enzimáticas de cisteíno- proteases do intestino médio da lagarta da soja
title_sort Propriedades bioquímicas e cinético-enzimáticas de cisteíno- proteases do intestino médio da lagarta da soja
author Mendonça, Eduardo Gomes de
author_facet Mendonça, Eduardo Gomes de
author_role author
dc.contributor.authorLattes.por.fl_str_mv http://lattes.cnpq.br/8989382342757236
dc.contributor.author.fl_str_mv Mendonça, Eduardo Gomes de
dc.contributor.advisor-co1.fl_str_mv Oliveira, Joel Antônio de
dc.contributor.advisor-co1Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4707224A0
dc.contributor.advisor-co2.fl_str_mv Oliveira, Maria Goreti de Almeida
dc.contributor.advisor-co2Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4790894D6
dc.contributor.advisor1.fl_str_mv Guedes, Raul Narciso Carvalho
dc.contributor.advisor1Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4721108T2
dc.contributor.referee1.fl_str_mv Moreira, Maurílio Alves
dc.contributor.referee1Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4796105P2
dc.contributor.referee2.fl_str_mv Oliveira, Tânia Toledo de
dc.contributor.referee2Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4787758J2
dc.contributor.referee3.fl_str_mv Brumano, Maria Helena Nasser
dc.contributor.referee3Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4791952U3
contributor_str_mv Oliveira, Joel Antônio de
Oliveira, Maria Goreti de Almeida
Guedes, Raul Narciso Carvalho
Moreira, Maurílio Alves
Oliveira, Tânia Toledo de
Brumano, Maria Helena Nasser
dc.subject.por.fl_str_mv Soja
Lagarta da soja
Cisteíno-proteases
topic Soja
Lagarta da soja
Cisteíno-proteases
Soybean
Velvetbean caterpillar
Cysteine-proteases
CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::ENZIMOLOGIA
dc.subject.eng.fl_str_mv Soybean
Velvetbean caterpillar
Cysteine-proteases
dc.subject.cnpq.fl_str_mv CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::ENZIMOLOGIA
description Protease inhibitors that react with insect specific proteases are candidates to have their genetic code inserted in genetically modified plants. The first step to achieve this is the characterization of proteolytic enzymes of these insects intestines. Cysteine-protease of the soluble and insoluble fractions from the midgut of Anticarsia gemmatalis were characterized using the substrate LBApNA. The Soluble Fraction, called Fraction I, was obtained from the suspension medium after nine cycles of freezing and thawing of the intestine of A. gemmatalis, and the Insoluble Fraction, called Fraction II, was obtained from the maceration with detergent Brij 35 of the resulting pellet of the Fraction I. Two pH values were found with high activity in both Fractions they were pH 3.6 and 8.0 for the Fraction I and 4.6 and 8.0 for the Fraction II. And the peak of activity when tested the influence of temperature was at 35oC for the Fraction I and 60°C for the Fraction II. The KM app found for the Fractions I and II were 2.28 mM and 0.44 mM, respectively, and the Vmax app for the respective Fractions were 297.68 nM.s-1 and 122.95 nM.s-1. Four protease inhibitors were tested, which one from one class of protease: TLCK (inhibitor of serine-protease), E-64 (inhibitor for cysteine-protease), EDTA (inhibitor of metallo-protease) and Pepstatin A (inhibitor of aspartyl-protease). The higher inhibition was observed when E-64 was added to the medium for both Fractions, once that all concentrations tested decreased significantly the activity of cysteine- protease. An increase of TLCK into the medium made a gradual decrease in the activity of cysteine-protease, due to the reaction with the amino acid histidine residue in the catalytic triad, which is also part of the triad of cisteine-protease. The effect of EDTA in the activity of cysteine-protease of A. gemmatalis on L-BApNA shows the difference between the two Fractions analyzed. The Fraction I do not depend on the divalent ions as Ca2 + for its activity. However, the Fraction II is a calcium- dependent, since EDTA reduced significantly its activity. The Pespstatin A did not influenced the cysteine-proteases of the Fraction I, exerting little influence on the Fraction II.
publishDate 2008
dc.date.issued.fl_str_mv 2008-02-27
dc.date.available.fl_str_mv 2009-02-12
2015-03-26T13:07:23Z
dc.date.accessioned.fl_str_mv 2015-03-26T13:07:23Z
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dc.type.driver.fl_str_mv info:eu-repo/semantics/masterThesis
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dc.identifier.citation.fl_str_mv MENDONÇA, Eduardo Gomes de. Biochemistry and kinectics-enzymatics properties of cysteine-proteases of the velvetbean caterpillar midgut. 2008. 68 f. Dissertação (Mestrado em Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal) - Universidade Federal de Viçosa, Viçosa, 2008.
dc.identifier.uri.fl_str_mv http://locus.ufv.br/handle/123456789/2394
identifier_str_mv MENDONÇA, Eduardo Gomes de. Biochemistry and kinectics-enzymatics properties of cysteine-proteases of the velvetbean caterpillar midgut. 2008. 68 f. Dissertação (Mestrado em Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal) - Universidade Federal de Viçosa, Viçosa, 2008.
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