Purificação parcial das quitinases, Pbcts1 e Pbcts2, do fungo Paracoccidiodes brasiliensis
Autor(a) principal: | |
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Data de Publicação: | 2008 |
Tipo de documento: | Dissertação |
Idioma: | por |
Título da fonte: | Repositório Institucional da UFG |
Texto Completo: | http://repositorio.bc.ufg.br/tede/handle/tde/1280 |
Resumo: | Paracoccidoides brasiliensis is a human pathogenic dimorphic fungus. The recombinant chitinase from P. brasiliensis, Pbcts1r, was overexpressed in Escherichia coli using pET-32a (+) as vector. The enzyme was produced as inclusion bodies and became soluble by Sarkosyl being purified by a single step using a Ni-NTA resin. Pbcts1r showed activity against 4-MU-(GlcNAc)3 and 4-MU-(GlcNAc)2, presenting a endochitinase activity. Immunoblot reaction with anti-Pbcts1r identified two proteins in yeast crude extract. A partial purification of P. brasiliensis yeast crude extract by cationic-exchange chromatography on HPLC revealed two different chitinases, Pbcts1 and Pbcts2, with molecular mass of 45 kDa and 34 kDa, respectively. Pbcts2 has exochitinase activity and Pbcts1 has endochitinase activities. Reactions with anti- Pbcts1r showed the presence of Pbcts1 and Pbcts2 in crude extracts of yeast and transition from mycelium to yeast. On mycelium crude extracts was found only Pbcts1 and on yeast cell wall extract only Pbcts2. Both proteins were found to be secreted by yeast parasitic phase showing their probable importance in the permanence of the fungus in the human host. Phylogenetic relationships between the orthologs Pbcts1 and the putative Pbcts2 indicated the presence of a common ancestral. During evolution, P. brasiliensis could have acquired Pbcts2 and Pbcts1 playing distinct roles in order to growth and survive in diverse environment on saprophytic and parasitic phases |
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PEREIRA, Maristelahttp://lattes.cnpq.br/1345781867765758http://lattes.cnpq.br/4570454631558386SANTANA, Lidiane Aparecida da Penha2014-07-29T15:16:35Z2010-04-222008-04-03SANTANA, Lidiane Aparecida da Penha. Partial purification of chitinases, and Pbcts1 Pbcts2, fungus Paracoccidioides brasiliensis. 2008. 81 f. Dissertação (Mestrado em Ciências Biolóicas) - Universidade Federal de Goiás, Goiânia, 2008.http://repositorio.bc.ufg.br/tede/handle/tde/1280Paracoccidoides brasiliensis is a human pathogenic dimorphic fungus. The recombinant chitinase from P. brasiliensis, Pbcts1r, was overexpressed in Escherichia coli using pET-32a (+) as vector. The enzyme was produced as inclusion bodies and became soluble by Sarkosyl being purified by a single step using a Ni-NTA resin. Pbcts1r showed activity against 4-MU-(GlcNAc)3 and 4-MU-(GlcNAc)2, presenting a endochitinase activity. Immunoblot reaction with anti-Pbcts1r identified two proteins in yeast crude extract. A partial purification of P. brasiliensis yeast crude extract by cationic-exchange chromatography on HPLC revealed two different chitinases, Pbcts1 and Pbcts2, with molecular mass of 45 kDa and 34 kDa, respectively. Pbcts2 has exochitinase activity and Pbcts1 has endochitinase activities. Reactions with anti- Pbcts1r showed the presence of Pbcts1 and Pbcts2 in crude extracts of yeast and transition from mycelium to yeast. On mycelium crude extracts was found only Pbcts1 and on yeast cell wall extract only Pbcts2. Both proteins were found to be secreted by yeast parasitic phase showing their probable importance in the permanence of the fungus in the human host. Phylogenetic relationships between the orthologs Pbcts1 and the putative Pbcts2 indicated the presence of a common ancestral. During evolution, P. brasiliensis could have acquired Pbcts2 and Pbcts1 playing distinct roles in order to growth and survive in diverse environment on saprophytic and parasitic phasesParacoccidioides brasiliensis é um fungo dimórfico patogênico humano. A quitinase recombinante de P. brasiliensis, Pbcts1r, foi superexpressa em Escherichia coli utilizando pET-32(a)+ como vetor. A enzima foi produzida em corpos de inclusão e se tornou solúvel pela adição de sarkosyl, sendo purificada em um único passo com a utilização da resina Ni-NTA. Pbcts1r mostrou atividade diante de 4-MU-(GlcNAc)3 e 4- MU-(GlcNAc)2, apresentando atividade de endoquitinase. A reação de imunoblot com anti-Pbcts1r identificou duas proteínas no extrato bruto de levedura. A purificação parcial do extrato bruto de P. brasiliensis por cromatografia de troca-catiônica em HPLC revelou duas quitinases diferentes, Pbcts1 e Pbcts2, com massas moleculares de 45 e 34 kDa, respectivamente. Pbcts2 tem atividade de exoquitinase e Pbcts1 de endoquitinase. Reações com anti-Pbcts1r mostraram a presença de Pbcts1 e Pbcts2 no extrato bruto de levedura e transição de micélio para levedura. No extrato bruto de micélio foi encontrado somente Pbcts1 e no extrato de parede celular de levedura somente Pbcts2. Ambas as proteínas foram encontradas secretadas pela fase parasitária (levedura), mostrando a provável importância dessas proteínas na permanência do fungo no hospedeiro. Relações filogenéticas entre os ortólogos Pbcts1 e a provável Pbcts2 indicam a presença de um ancestral comum. Durante a evolução, P. brasiliensis poderia ter adquirido Pbcts2 e Pbcts1 desempenhando diferentes papéis para o crescimento e sobrevivência do fungo na fase saprofítica e parasitáriaMade available in DSpace on 2014-07-29T15:16:35Z (GMT). No. of bitstreams: 1 dissertacao parte 1 lidiane biologia.pdf: 35041 bytes, checksum: c58f88ce1be4655ee71c7b5ab0bef247 (MD5) Previous issue date: 2008-04-03application/pdfhttp://repositorio.bc.ufg.br/TEDE/retrieve/4039/dissertacao%20parte%201%20lidiane%20biologia.pdf.jpgporUniversidade Federal de GoiásMestrado em BiologiaUFGBRCiências BiolóicasParacoccidiodes brasiliensis, quitinase, purificaçãoParacoccidioides brasiliensis, chitinase, purificationCNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULARPurificação parcial das quitinases, Pbcts1 e Pbcts2, do fungo Paracoccidiodes brasiliensisPartial purification of chitinases, and Pbcts1 Pbcts2, fungus Paracoccidioides brasiliensisinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UFGinstname:Universidade Federal de Goiás (UFG)instacron:UFGORIGINALdissertacao parte 1 lidiane biologia.pdfapplication/pdf35041http://repositorio.bc.ufg.br/tede/bitstreams/471546fb-9ddd-4525-8cb3-9ed4ff6b225b/downloadc58f88ce1be4655ee71c7b5ab0bef247MD51THUMBNAILdissertacao parte 1 lidiane biologia.pdf.jpgdissertacao parte 1 lidiane biologia.pdf.jpgGenerated Thumbnailimage/jpeg2144http://repositorio.bc.ufg.br/tede/bitstreams/59925666-fade-4135-b851-33bc6d9ffc40/download8731166403969277e7c6a2f91d056cbeMD52tde/12802014-07-30 03:10:34.912open.accessoai:repositorio.bc.ufg.br:tde/1280http://repositorio.bc.ufg.br/tedeRepositório InstitucionalPUBhttp://repositorio.bc.ufg.br/oai/requesttasesdissertacoes.bc@ufg.bropendoar:2014-07-30T06:10:34Repositório Institucional da UFG - Universidade Federal de Goiás (UFG)false |
dc.title.por.fl_str_mv |
Purificação parcial das quitinases, Pbcts1 e Pbcts2, do fungo Paracoccidiodes brasiliensis |
dc.title.alternative.eng.fl_str_mv |
Partial purification of chitinases, and Pbcts1 Pbcts2, fungus Paracoccidioides brasiliensis |
title |
Purificação parcial das quitinases, Pbcts1 e Pbcts2, do fungo Paracoccidiodes brasiliensis |
spellingShingle |
Purificação parcial das quitinases, Pbcts1 e Pbcts2, do fungo Paracoccidiodes brasiliensis SANTANA, Lidiane Aparecida da Penha Paracoccidiodes brasiliensis, quitinase, purificação Paracoccidioides brasiliensis, chitinase, purification CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR |
title_short |
Purificação parcial das quitinases, Pbcts1 e Pbcts2, do fungo Paracoccidiodes brasiliensis |
title_full |
Purificação parcial das quitinases, Pbcts1 e Pbcts2, do fungo Paracoccidiodes brasiliensis |
title_fullStr |
Purificação parcial das quitinases, Pbcts1 e Pbcts2, do fungo Paracoccidiodes brasiliensis |
title_full_unstemmed |
Purificação parcial das quitinases, Pbcts1 e Pbcts2, do fungo Paracoccidiodes brasiliensis |
title_sort |
Purificação parcial das quitinases, Pbcts1 e Pbcts2, do fungo Paracoccidiodes brasiliensis |
author |
SANTANA, Lidiane Aparecida da Penha |
author_facet |
SANTANA, Lidiane Aparecida da Penha |
author_role |
author |
dc.contributor.advisor1.fl_str_mv |
PEREIRA, Maristela |
dc.contributor.advisor1Lattes.fl_str_mv |
http://lattes.cnpq.br/1345781867765758 |
dc.contributor.authorLattes.fl_str_mv |
http://lattes.cnpq.br/4570454631558386 |
dc.contributor.author.fl_str_mv |
SANTANA, Lidiane Aparecida da Penha |
contributor_str_mv |
PEREIRA, Maristela |
dc.subject.por.fl_str_mv |
Paracoccidiodes brasiliensis, quitinase, purificação |
topic |
Paracoccidiodes brasiliensis, quitinase, purificação Paracoccidioides brasiliensis, chitinase, purification CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR |
dc.subject.eng.fl_str_mv |
Paracoccidioides brasiliensis, chitinase, purification |
dc.subject.cnpq.fl_str_mv |
CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR |
description |
Paracoccidoides brasiliensis is a human pathogenic dimorphic fungus. The recombinant chitinase from P. brasiliensis, Pbcts1r, was overexpressed in Escherichia coli using pET-32a (+) as vector. The enzyme was produced as inclusion bodies and became soluble by Sarkosyl being purified by a single step using a Ni-NTA resin. Pbcts1r showed activity against 4-MU-(GlcNAc)3 and 4-MU-(GlcNAc)2, presenting a endochitinase activity. Immunoblot reaction with anti-Pbcts1r identified two proteins in yeast crude extract. A partial purification of P. brasiliensis yeast crude extract by cationic-exchange chromatography on HPLC revealed two different chitinases, Pbcts1 and Pbcts2, with molecular mass of 45 kDa and 34 kDa, respectively. Pbcts2 has exochitinase activity and Pbcts1 has endochitinase activities. Reactions with anti- Pbcts1r showed the presence of Pbcts1 and Pbcts2 in crude extracts of yeast and transition from mycelium to yeast. On mycelium crude extracts was found only Pbcts1 and on yeast cell wall extract only Pbcts2. Both proteins were found to be secreted by yeast parasitic phase showing their probable importance in the permanence of the fungus in the human host. Phylogenetic relationships between the orthologs Pbcts1 and the putative Pbcts2 indicated the presence of a common ancestral. During evolution, P. brasiliensis could have acquired Pbcts2 and Pbcts1 playing distinct roles in order to growth and survive in diverse environment on saprophytic and parasitic phases |
publishDate |
2008 |
dc.date.issued.fl_str_mv |
2008-04-03 |
dc.date.available.fl_str_mv |
2010-04-22 |
dc.date.accessioned.fl_str_mv |
2014-07-29T15:16:35Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
format |
masterThesis |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
SANTANA, Lidiane Aparecida da Penha. Partial purification of chitinases, and Pbcts1 Pbcts2, fungus Paracoccidioides brasiliensis. 2008. 81 f. Dissertação (Mestrado em Ciências Biolóicas) - Universidade Federal de Goiás, Goiânia, 2008. |
dc.identifier.uri.fl_str_mv |
http://repositorio.bc.ufg.br/tede/handle/tde/1280 |
identifier_str_mv |
SANTANA, Lidiane Aparecida da Penha. Partial purification of chitinases, and Pbcts1 Pbcts2, fungus Paracoccidioides brasiliensis. 2008. 81 f. Dissertação (Mestrado em Ciências Biolóicas) - Universidade Federal de Goiás, Goiânia, 2008. |
url |
http://repositorio.bc.ufg.br/tede/handle/tde/1280 |
dc.language.iso.fl_str_mv |
por |
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por |
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info:eu-repo/semantics/openAccess |
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openAccess |
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application/pdf |
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Universidade Federal de Goiás |
dc.publisher.program.fl_str_mv |
Mestrado em Biologia |
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UFG |
dc.publisher.country.fl_str_mv |
BR |
dc.publisher.department.fl_str_mv |
Ciências Biolóicas |
publisher.none.fl_str_mv |
Universidade Federal de Goiás |
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Universidade Federal de Goiás (UFG) |
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