Obtenção de linhagens recombinantes de Penicillium griseoroseum para produção de poligalacturonase
Autor(a) principal: | |
---|---|
Data de Publicação: | 2005 |
Tipo de documento: | Tese |
Idioma: | por |
Título da fonte: | LOCUS Repositório Institucional da UFV |
Texto Completo: | http://locus.ufv.br/handle/123456789/1530 |
Resumo: | Analysis of a 170 bp DNA fragment of the promoter region of the pgg2 gene containing a putative CAAT box at - 270 bp from ATG code by electrophoretic mobility shift assay with nuclear extracts prepared from mycelia grown under pectin- containing medium revealed a high mobility complex that was subsequently confirmed when analysis was conducted employing a double-stranded oligonucleotide spanning the CCAAT motif. A substitution in the core sequence for GTAGG partially abolished formation of specific complexes, showing the involvement of the CCAAT box in the regulation of the polygalacturonase gene studied. A 23 bp sinthetic double- stranded oligonucleotide containing a cis-element resembling the cAMP response element (CTACAGTG) observed in yeasts and mammals promoters was also used as probe in EMSA assays. Band shifts were not seen when nuclear extracts prepared from mycelia cultured on pectin and glucose- containing medium supplemented with yeast extract were used in the reactions. This result suggests that the CTACAGTG element tested is not a target site for regulatory activators. In order to inactivate the P. griseoroseum pgg2 gene, a disruption vector, named pPG15pgg2Δniafo was constructed by inserting a 4 Kb HindIII DNA fragment, containing the nia D gene from Fusarium oxysporum in the coding region of pgg2. The disruption of pgg2 resulted in transformant strains producing at most 12 % of PG activity of the host strain (PG63), indicating that this gene responds for almost 90% of PG total activity in P. griseoroseum. To overexpress extracellular polygalacturonase in P. griseoroseum, an expression cassete was constructed, designated pAN52-pgg2 that contained the pgg2 gene under the control of the strong constitutive promoter gpdA from Aspergillus nidulans. The transformation of the PG63 mutant strain using this vector and the selective plasmid (pNPG1) resulted in the isolation of a recombinant strain (146) that, when cultivated in glucose, sucrose or sugar cane juice as sole carbon source, produced a PG activity 12 times higher than that produced by the mutant strain cultivated in pectin and yeast extract. The PG produced by the recombinant strain possesses an optimum pH range from 3,5 to 5,0 and is inactivated at the extrem pHs 2,5 and 8,0. It was demonstrated that this enzyme acts efficiently hydrolising polygalacturonic acid in a broad range of temperature (30 to 60 oC) with the optimal temperature ranging from 30 to 45 oC. In addition, storage of the recombinant strain culture supernatants at temperatures of - 20, 4, and 30 oC for five weeks did not affect enzyme activity. On the other hand, the enzyme exhibited low thermostability, being inactivated when pre-incubated for 1 hour at temperatures of 40, 50, and 60oC. |
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Ribeiro, João Batistahttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4707778D1Araujo, Elza Fernandes dehttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4783675E2Queiroz, Marisa Vieira dehttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4785812Z5Passos, Flávia Maria Lopeshttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4781817D3Dias, Eustáquio Souzahttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4769445U0Schwan, Rosane Freitashttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4721861H82015-03-26T12:50:52Z2008-05-212015-03-26T12:50:52Z2005-12-09RIBEIRO, João Batista. Obtention of recombinant strains from Penicillium griseoroseum for polygalacturonase production. 2005. 108 f. Tese (Doutorado em Associações micorrízicas; Bactérias láticas e probióticos; Biologia molecular de fungos de interesse) - Universidade Federal de Viçosa, Viçosa, 2005.http://locus.ufv.br/handle/123456789/1530Analysis of a 170 bp DNA fragment of the promoter region of the pgg2 gene containing a putative CAAT box at - 270 bp from ATG code by electrophoretic mobility shift assay with nuclear extracts prepared from mycelia grown under pectin- containing medium revealed a high mobility complex that was subsequently confirmed when analysis was conducted employing a double-stranded oligonucleotide spanning the CCAAT motif. A substitution in the core sequence for GTAGG partially abolished formation of specific complexes, showing the involvement of the CCAAT box in the regulation of the polygalacturonase gene studied. A 23 bp sinthetic double- stranded oligonucleotide containing a cis-element resembling the cAMP response element (CTACAGTG) observed in yeasts and mammals promoters was also used as probe in EMSA assays. Band shifts were not seen when nuclear extracts prepared from mycelia cultured on pectin and glucose- containing medium supplemented with yeast extract were used in the reactions. This result suggests that the CTACAGTG element tested is not a target site for regulatory activators. In order to inactivate the P. griseoroseum pgg2 gene, a disruption vector, named pPG15pgg2Δniafo was constructed by inserting a 4 Kb HindIII DNA fragment, containing the nia D gene from Fusarium oxysporum in the coding region of pgg2. The disruption of pgg2 resulted in transformant strains producing at most 12 % of PG activity of the host strain (PG63), indicating that this gene responds for almost 90% of PG total activity in P. griseoroseum. To overexpress extracellular polygalacturonase in P. griseoroseum, an expression cassete was constructed, designated pAN52-pgg2 that contained the pgg2 gene under the control of the strong constitutive promoter gpdA from Aspergillus nidulans. The transformation of the PG63 mutant strain using this vector and the selective plasmid (pNPG1) resulted in the isolation of a recombinant strain (146) that, when cultivated in glucose, sucrose or sugar cane juice as sole carbon source, produced a PG activity 12 times higher than that produced by the mutant strain cultivated in pectin and yeast extract. The PG produced by the recombinant strain possesses an optimum pH range from 3,5 to 5,0 and is inactivated at the extrem pHs 2,5 and 8,0. It was demonstrated that this enzyme acts efficiently hydrolising polygalacturonic acid in a broad range of temperature (30 to 60 oC) with the optimal temperature ranging from 30 to 45 oC. In addition, storage of the recombinant strain culture supernatants at temperatures of - 20, 4, and 30 oC for five weeks did not affect enzyme activity. On the other hand, the enzyme exhibited low thermostability, being inactivated when pre-incubated for 1 hour at temperatures of 40, 50, and 60oC.Análise de um fragmento de DNA de 170 pb da região promotora do gene pgg2 contendo um possível CCAAT-box localizado a -270 pares de bases (pb) do códon de início da tradução, por meio de ensaios de migração retardada em gel, revelou a formação de complexos específicos deste fragmento de DNA com proteínas nucleares obtidas a partir de micélio cultivado em meio contendo pectina. A ligação de proteínas no elemento CCAAT foi confirmada quando a análise foi realizada utilizando um oligonucleotídeo fita dupla contendo essa seqüência. Substituição da seqüência original pela seqüência GTAGG diminuiu a formação de complexos específicos, comprovando o envolvimento do elemento CCAAT na regulação da expressão do gene pgg2. Um oligonucleotídeo de 23 pb contendo a seqüência CTACAGTG, similar à seqüência de um cis-elemento de resposta a AMPc encontrado em promotores de leveduras e mamíferos, também foi usado como sonda em ensaios de migração retardada em gel. Complexos de DNA-proteínas não foram detectados quando se utilizou extratos protéicos nucleares, preparados a partir de micélio cultivado em glicose e extrato de levedura, sugerindo que o elemento CTACAGTG não representa um sítio para a ligação de proteínas ativadoras. Foi construído um vetor para inativação do gene pgg2 de P. griseoroseum, denominado pPG15pgg2Δniafo, o qual contém o gene pgg2 interrompido por um fragmento de DNA de 4,0 Kb, correspondente ao gene nia de Fusarium oxysporum. A inativação do gene pgg2, demonstrou que a poligalacturonase codificada por este gene é responsável por 90 % da atividade total de PG de P. griseoroseum. Foi construído um vetor para expressão do gene pgg2 sob o controle do promotor constitutivo do gene gpdA de Aspergillus nidulans, o qual foi utilizado na transformação da linhagem PG63 de P. griseoroseum. Foram obtidas linhagens transformantes que, quando cultivadas na presença de glicose, sacarose ou caldo de cana, apresentaram aumento na atividade de PG de até 12 vezes em relação à linhagem PG63 cultivada em pectina e extrato de levedura. A linhagem recombinante 146 apresentou aumento na atividade de PG quando cultivada em meio contendo glicose (1%) por 48 a 96 horas, utilizando-se 106 ou 107 conídios/mL do meio de cultura. Embora tenha sido observado menor massa micelial seca, quando as linhagens recombinantes foram cultivadas em sacarose e caldo de cana, a atividade de PG foi de 90 % da determinada quando estas linhagens foram cultivadas em glicose. A poligalacturonase produzida pela linhagem recombinante 146 de P. griseoroseum apresentou alta atividade na faixa de pH de 3,5 a 5,0 e nas temperaturas de 30 a 60oC, sendo estável quando armazenada por pelo menos 5 semanas nas temperaturas de -20, 4 e 30oC. No entanto, apresentou baixa termoestabilidade, sofrendo desnaturação quando pré- incubada por 1 hora nas temperaturas de 40, 50 e 60oC. Os níveis enzimáticos relatados neste trabalho e as propriedades físico-químicas da PG II produzida pela linhagem recombinante 146 de P. griseoroseum demonstram a eficiência do sistema de expressão desenvolvido e que a PG II é uma enzima adequada para aplicação na indústria de processamento de sucos de frutas.Conselho Nacional de Desenvolvimento Científico e Tecnológicoapplication/pdfporUniversidade Federal de ViçosaDoutorado em Microbiologia AgrícolaUFVBRAssociações micorrízicas; Bactérias láticas e probióticos; Biologia molecular de fungos de interesseLinhagens recombinantesPenicillium griseoroseumPoligalacturonaseRecombinant strainsPenicillium griseoroseumPolygalacturonaseCNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA MOLECULAR E DE MICROORGANISMOSObtenção de linhagens recombinantes de Penicillium griseoroseum para produção de poligalacturonaseObtention of recombinant strains from Penicillium griseoroseum for polygalacturonase productioninfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/openAccessreponame:LOCUS Repositório Institucional da UFVinstname:Universidade Federal de Viçosa (UFV)instacron:UFVORIGINALtexto completo.pdfapplication/pdf474965https://locus.ufv.br//bitstream/123456789/1530/1/texto%20completo.pdf47631e534720bfd9352b5ce8a705f9e9MD51TEXTtexto completo.pdf.txttexto completo.pdf.txtExtracted texttext/plain204507https://locus.ufv.br//bitstream/123456789/1530/2/texto%20completo.pdf.txtc7a5abb081a29531776572d25c0e82cfMD52THUMBNAILtexto completo.pdf.jpgtexto completo.pdf.jpgIM Thumbnailimage/jpeg3505https://locus.ufv.br//bitstream/123456789/1530/3/texto%20completo.pdf.jpg92a74d12f69b9e8de42fc343b99c626aMD53123456789/15302016-04-06 23:10:54.588oai:locus.ufv.br:123456789/1530Repositório InstitucionalPUBhttps://www.locus.ufv.br/oai/requestfabiojreis@ufv.bropendoar:21452016-04-07T02:10:54LOCUS Repositório Institucional da UFV - Universidade Federal de Viçosa (UFV)false |
dc.title.por.fl_str_mv |
Obtenção de linhagens recombinantes de Penicillium griseoroseum para produção de poligalacturonase |
dc.title.alternative.eng.fl_str_mv |
Obtention of recombinant strains from Penicillium griseoroseum for polygalacturonase production |
title |
Obtenção de linhagens recombinantes de Penicillium griseoroseum para produção de poligalacturonase |
spellingShingle |
Obtenção de linhagens recombinantes de Penicillium griseoroseum para produção de poligalacturonase Ribeiro, João Batista Linhagens recombinantes Penicillium griseoroseum Poligalacturonase Recombinant strains Penicillium griseoroseum Polygalacturonase CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA MOLECULAR E DE MICROORGANISMOS |
title_short |
Obtenção de linhagens recombinantes de Penicillium griseoroseum para produção de poligalacturonase |
title_full |
Obtenção de linhagens recombinantes de Penicillium griseoroseum para produção de poligalacturonase |
title_fullStr |
Obtenção de linhagens recombinantes de Penicillium griseoroseum para produção de poligalacturonase |
title_full_unstemmed |
Obtenção de linhagens recombinantes de Penicillium griseoroseum para produção de poligalacturonase |
title_sort |
Obtenção de linhagens recombinantes de Penicillium griseoroseum para produção de poligalacturonase |
author |
Ribeiro, João Batista |
author_facet |
Ribeiro, João Batista |
author_role |
author |
dc.contributor.authorLattes.por.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4707778D1 |
dc.contributor.author.fl_str_mv |
Ribeiro, João Batista |
dc.contributor.advisor1.fl_str_mv |
Araujo, Elza Fernandes de |
dc.contributor.advisor1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4783675E2 |
dc.contributor.referee1.fl_str_mv |
Queiroz, Marisa Vieira de |
dc.contributor.referee1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4785812Z5 |
dc.contributor.referee2.fl_str_mv |
Passos, Flávia Maria Lopes |
dc.contributor.referee2Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4781817D3 |
dc.contributor.referee3.fl_str_mv |
Dias, Eustáquio Souza |
dc.contributor.referee3Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4769445U0 |
dc.contributor.referee4.fl_str_mv |
Schwan, Rosane Freitas |
dc.contributor.referee4Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4721861H8 |
contributor_str_mv |
Araujo, Elza Fernandes de Queiroz, Marisa Vieira de Passos, Flávia Maria Lopes Dias, Eustáquio Souza Schwan, Rosane Freitas |
dc.subject.por.fl_str_mv |
Linhagens recombinantes Penicillium griseoroseum Poligalacturonase |
topic |
Linhagens recombinantes Penicillium griseoroseum Poligalacturonase Recombinant strains Penicillium griseoroseum Polygalacturonase CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA MOLECULAR E DE MICROORGANISMOS |
dc.subject.eng.fl_str_mv |
Recombinant strains Penicillium griseoroseum Polygalacturonase |
dc.subject.cnpq.fl_str_mv |
CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA MOLECULAR E DE MICROORGANISMOS |
description |
Analysis of a 170 bp DNA fragment of the promoter region of the pgg2 gene containing a putative CAAT box at - 270 bp from ATG code by electrophoretic mobility shift assay with nuclear extracts prepared from mycelia grown under pectin- containing medium revealed a high mobility complex that was subsequently confirmed when analysis was conducted employing a double-stranded oligonucleotide spanning the CCAAT motif. A substitution in the core sequence for GTAGG partially abolished formation of specific complexes, showing the involvement of the CCAAT box in the regulation of the polygalacturonase gene studied. A 23 bp sinthetic double- stranded oligonucleotide containing a cis-element resembling the cAMP response element (CTACAGTG) observed in yeasts and mammals promoters was also used as probe in EMSA assays. Band shifts were not seen when nuclear extracts prepared from mycelia cultured on pectin and glucose- containing medium supplemented with yeast extract were used in the reactions. This result suggests that the CTACAGTG element tested is not a target site for regulatory activators. In order to inactivate the P. griseoroseum pgg2 gene, a disruption vector, named pPG15pgg2Δniafo was constructed by inserting a 4 Kb HindIII DNA fragment, containing the nia D gene from Fusarium oxysporum in the coding region of pgg2. The disruption of pgg2 resulted in transformant strains producing at most 12 % of PG activity of the host strain (PG63), indicating that this gene responds for almost 90% of PG total activity in P. griseoroseum. To overexpress extracellular polygalacturonase in P. griseoroseum, an expression cassete was constructed, designated pAN52-pgg2 that contained the pgg2 gene under the control of the strong constitutive promoter gpdA from Aspergillus nidulans. The transformation of the PG63 mutant strain using this vector and the selective plasmid (pNPG1) resulted in the isolation of a recombinant strain (146) that, when cultivated in glucose, sucrose or sugar cane juice as sole carbon source, produced a PG activity 12 times higher than that produced by the mutant strain cultivated in pectin and yeast extract. The PG produced by the recombinant strain possesses an optimum pH range from 3,5 to 5,0 and is inactivated at the extrem pHs 2,5 and 8,0. It was demonstrated that this enzyme acts efficiently hydrolising polygalacturonic acid in a broad range of temperature (30 to 60 oC) with the optimal temperature ranging from 30 to 45 oC. In addition, storage of the recombinant strain culture supernatants at temperatures of - 20, 4, and 30 oC for five weeks did not affect enzyme activity. On the other hand, the enzyme exhibited low thermostability, being inactivated when pre-incubated for 1 hour at temperatures of 40, 50, and 60oC. |
publishDate |
2005 |
dc.date.issued.fl_str_mv |
2005-12-09 |
dc.date.available.fl_str_mv |
2008-05-21 2015-03-26T12:50:52Z |
dc.date.accessioned.fl_str_mv |
2015-03-26T12:50:52Z |
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info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/doctoralThesis |
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doctoralThesis |
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publishedVersion |
dc.identifier.citation.fl_str_mv |
RIBEIRO, João Batista. Obtention of recombinant strains from Penicillium griseoroseum for polygalacturonase production. 2005. 108 f. Tese (Doutorado em Associações micorrízicas; Bactérias láticas e probióticos; Biologia molecular de fungos de interesse) - Universidade Federal de Viçosa, Viçosa, 2005. |
dc.identifier.uri.fl_str_mv |
http://locus.ufv.br/handle/123456789/1530 |
identifier_str_mv |
RIBEIRO, João Batista. Obtention of recombinant strains from Penicillium griseoroseum for polygalacturonase production. 2005. 108 f. Tese (Doutorado em Associações micorrízicas; Bactérias láticas e probióticos; Biologia molecular de fungos de interesse) - Universidade Federal de Viçosa, Viçosa, 2005. |
url |
http://locus.ufv.br/handle/123456789/1530 |
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Universidade Federal de Viçosa |
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Doutorado em Microbiologia Agrícola |
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UFV |
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BR |
dc.publisher.department.fl_str_mv |
Associações micorrízicas; Bactérias láticas e probióticos; Biologia molecular de fungos de interesse |
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Universidade Federal de Viçosa |
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