Melhoramento por evolução dirigida da termoestabilidade da xilanase xynA de Orpinomyces sp. PC-2

Detalhes bibliográficos
Autor(a) principal: Trevizano, Larissa Mattos
Data de Publicação: 2009
Tipo de documento: Dissertação
Idioma: por
Título da fonte: LOCUS Repositório Institucional da UFV
Texto Completo: http://locus.ufv.br/handle/123456789/2408
Resumo: Xylan is the most common hemicellulosic polysaccharide in cell walls of land plants. This polysaccharide is composed of a main-chain polymer made up exclusively of β-1,4 xylose residues. This backbone possesses branches containing arabinofuranosyl, acetyl, and glucuronosyl residues. Enzymatic hydrolysis of xylans is brought about by a variety of enzyme activities that are grouped under the generic term hemicellulases. Endoxylanases (EC 3.2.1.8) hydrolyze internal β-1,4 bonds in the main-chain and generate soluble xylooligosaccharides. A variety of microorganisms, including bacteria, yeast and filamentous fungi, have been reported to produce xylanase, in which the most potent producers are fungi. Researchers are especially interested in fungal xylanases because they are secreted extracellularly and their activity is much higher than the xylanases from yeasts and bacteria. Among the biotechnological applications of xylanases, it can be outstanding the bioconversion of lignocellulosic materials into fermentative products, improvement of digestibility of animal feedstock, clarification of juices, facilitating the release of lignin from the pulp and reducing the amount of chlorine required for bleaching in pulp in the paper industry. Over the last few decades, there has been a growing interest in lignocellulose bioconversion as a renewable energy source and xylanases can be effectively used with cellulases to hydrolyze the lignocellulosic biomass and bioethanol production. Most of the fungal xylanases show optimal activities at neutral or acidic pH and at temperatures below 45 °C. Xylanase (XynA), originally isolated from the anaerobic fungus Orpinomyces PC-2, has been tested for several industrial applications, in pilot scale. This enzyme presented extremely high activity with the xylan substrate of animal ration, cellulose pulp and other biomass types. Studies demonstrated that the use of the native enzyme has been more efficient in optimal conditions as pH 5.0-5.5 and temperature of 50-55 °C. However, the industrial process is usually accomplished at higher temperature and alkaline pH, what justifies the interest for xylanases that are active in these conditions. Directed evolution has emerged as a successful alternative in the genetic engineering of enzymes and it has been used to improve the functional properties of those molecules. In this study, error-prone PCR was used to improve the thermostability of endo-β-1,4-xylanase from Orpinomyces PC-2. The constructed library of xylanase (xynA) mutants was submitted to several screening cycles. The transformants were first exposed to 60 ºC during one hour and then the thermostable mutants were selected with the azo-xylan-agarose 0.2% pH 6.5 as substrate. Six mutants selected were sequenced and these amino acid sequences were analyzed to identify the mutations. Two mutants displayed higher thermal stability than the xylanase without mutations (wild-type). Whereas the wild-type lost 60% of its activity after 10 min at 60 ºC, mutants M4 and M6 showed enhanced thermostability and retained approximately 50% of its activities after treatment at 60 ºC for 60 min. In addition, M4 retained about of 40% of its initial activity after incubation at 75 ºC for 60 min. In order to evaluate the mutation effects in the enzyme properties, these ones were characterized for the optimum temperature, optimum pH and substrate specificity. The mutants and the wild-type showed an optimal temperature and pH for xylanase activity at 60 ºC and pH range of 5.0-7.0. The enzymes showed activity against the two tested substrates. An initial structural modeling study was accomplished with the wild-type and two mutants, which presented higher thermostability, seeking the best understanding of the relationship between structure and function of those xylanases.
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spelling Trevizano, Larissa Mattoshttp://lattes.cnpq.br/3860787717001357Rezende, Sebastião Tavares dehttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4787599A3Santoro, Marcelo Matoshttp://buscatextual.cnpq.br/buscatextual/index.jspGuimarães, Valéria Montezehttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4798758T3Fietto, Luciano Gomeshttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4763824H8Passos, Flávia Maria Lopeshttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4781817D32015-03-26T13:07:27Z2013-12-122015-03-26T13:07:27Z2009-04-29TREVIZANO, Larissa Mattos. Improvement of the thermostability of Orpinomyces sp. PC-2 xylanase by directed evolution. 2009. 80 f. Dissertação (Mestrado em Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal) - Universidade Federal de Viçosa, Viçosa, 2009.http://locus.ufv.br/handle/123456789/2408Xylan is the most common hemicellulosic polysaccharide in cell walls of land plants. This polysaccharide is composed of a main-chain polymer made up exclusively of β-1,4 xylose residues. This backbone possesses branches containing arabinofuranosyl, acetyl, and glucuronosyl residues. Enzymatic hydrolysis of xylans is brought about by a variety of enzyme activities that are grouped under the generic term hemicellulases. Endoxylanases (EC 3.2.1.8) hydrolyze internal β-1,4 bonds in the main-chain and generate soluble xylooligosaccharides. A variety of microorganisms, including bacteria, yeast and filamentous fungi, have been reported to produce xylanase, in which the most potent producers are fungi. Researchers are especially interested in fungal xylanases because they are secreted extracellularly and their activity is much higher than the xylanases from yeasts and bacteria. Among the biotechnological applications of xylanases, it can be outstanding the bioconversion of lignocellulosic materials into fermentative products, improvement of digestibility of animal feedstock, clarification of juices, facilitating the release of lignin from the pulp and reducing the amount of chlorine required for bleaching in pulp in the paper industry. Over the last few decades, there has been a growing interest in lignocellulose bioconversion as a renewable energy source and xylanases can be effectively used with cellulases to hydrolyze the lignocellulosic biomass and bioethanol production. Most of the fungal xylanases show optimal activities at neutral or acidic pH and at temperatures below 45 °C. Xylanase (XynA), originally isolated from the anaerobic fungus Orpinomyces PC-2, has been tested for several industrial applications, in pilot scale. This enzyme presented extremely high activity with the xylan substrate of animal ration, cellulose pulp and other biomass types. Studies demonstrated that the use of the native enzyme has been more efficient in optimal conditions as pH 5.0-5.5 and temperature of 50-55 °C. However, the industrial process is usually accomplished at higher temperature and alkaline pH, what justifies the interest for xylanases that are active in these conditions. Directed evolution has emerged as a successful alternative in the genetic engineering of enzymes and it has been used to improve the functional properties of those molecules. In this study, error-prone PCR was used to improve the thermostability of endo-β-1,4-xylanase from Orpinomyces PC-2. The constructed library of xylanase (xynA) mutants was submitted to several screening cycles. The transformants were first exposed to 60 ºC during one hour and then the thermostable mutants were selected with the azo-xylan-agarose 0.2% pH 6.5 as substrate. Six mutants selected were sequenced and these amino acid sequences were analyzed to identify the mutations. Two mutants displayed higher thermal stability than the xylanase without mutations (wild-type). Whereas the wild-type lost 60% of its activity after 10 min at 60 ºC, mutants M4 and M6 showed enhanced thermostability and retained approximately 50% of its activities after treatment at 60 ºC for 60 min. In addition, M4 retained about of 40% of its initial activity after incubation at 75 ºC for 60 min. In order to evaluate the mutation effects in the enzyme properties, these ones were characterized for the optimum temperature, optimum pH and substrate specificity. The mutants and the wild-type showed an optimal temperature and pH for xylanase activity at 60 ºC and pH range of 5.0-7.0. The enzymes showed activity against the two tested substrates. An initial structural modeling study was accomplished with the wild-type and two mutants, which presented higher thermostability, seeking the best understanding of the relationship between structure and function of those xylanases.Xilana é o polissacarídeo hemicelulósico mais comum da parede celular de plantas. Este polissacarídeo é composto por uma cadeia principal constituída exclusivamente por resíduos de xilose unidos por ligação β-1,4. Esta cadeia principal possui ramificações contendo resíduos de arabinofuranosil, acetil e glucuronosil. A hidrólise enzimática de xilanas é realizada por uma variedade de enzimas que são agrupadas pelo termo genérico de hemicelulases. Endoxilanases (EC 3.2.1.8) hidrolisam ligações β-1,4 internas da cadeia principal e geram oligossacarídeos de xilose solúveis. Uma variedade de microrganismos, incluindo bactéria, leveduras e fungos filamentosos têm sido descritos como produtores de xilanase, sendo que os mais potentes produtores são os fungos. Pesquisadores estão especialmente interessados em xilanases fúngicas porque elas são secretadas extracelularmente e sua atividade é muito maior que as xilanases provenientes de leveduras e bactérias. Dentre as aplicações biotecnológicas das xilanases, pode ser enfatizada a bioconversão de materiais lignocelulósicos em produtos fermentáveis, melhoria da digestibilidade de rações animais, clarificação de sucos, auxílio na liberação da lignina da polpa de celulose e redução da quantidade de cloro requerido para o branqueamento da polpa na indústria de papel. Nas últimas décadas, tem surgido um crescente interesse na bioconversão de lignocelulose como uma fonte de energia renovável, e xilanases podem ser efetivamente usadas, em associação com as celulases, para hidrólise de biomassa lignocelulósica e produção de bioetanol. Muitas das xilanases fúngicas mostram atividade ótima em pHs neutros ou ácidos e em temperaturas abaixo de 45 ºC. Xilanase (XynA), originalmente isolada do fungo anaeróbico Orpinomyces PC-2, foi testada para diversas aplicações industriais em escala piloto. Esta enzima apresentou atividade extremamente alta com o substrato xilana de ração animal, polpa de celulose e outros tipos de biomassa. Estudos demonstraram que o uso da enzima nativa foi mais eficiente em condições de pH 5.0-5.5 e temperatura de 50-55 ºC. No entanto, os processos industriais são normalmente realizados em temperaturas mais elevadas e em pH alcalino, o que justifica o interesse por xilanases que sejam ativas nestas condições. A metodologia de evolução dirigida do DNA surgiu como uma alternativa de sucesso na engenharia genética de enzimas e tem sido empregada para melhorar as propriedades funcionais dessas moléculas. Neste trabalho, a técnica de error-prone PCR foi utilizada para aprimorar a termoestabilidade da endo-β-1,4-xylanase de Orpinomyces PC-2. A biblioteca de mutantes (xynA) construída foi submetida a vários ciclos de screening. Os transformantes foram inicialmente expostos a 60 ºC durante uma hora e os mutantes termoestáveis foram selecionados com o substrato azo-xilana- agarose 0,2% pH 6,5. Seis mutantes selecionados foram seqüenciados e suas seqüências de aminoácidos foram analisadas para identificação das mutações. Dois mutantes apresentaram maior estabilidade térmica se comparado a xilanase sem mutações (tipo selvagem). Enquanto o tipo selvagem perdeu 60% de sua atividade depois de 10 min a 60 ºC, os mutantes M4 e M6 mostraram maior termoestabilidade e mantiveram aproximadamente 50% de suas atividades depois do tratamento a 60 ºC por 60 min. Em adição, M4 manteve cerca de 40% de sua atividade inicial depois de incubação a 75 ºC por 60 min. Com o objetivo de avaliar os efeitos das mutações nas propriedades das enzimas, estas foram caracterizadas quanto à temperatura ótima, pH ótimo e especificidade por substratos. Os mutantes e a xilanase tipo selvagem apresentaram temperatura ótima de 60 ºC e pH ótimo na faixa de 5,0-7,0 para atividade de xilanase. As enzimas apresentaram atividade com os dois substratos testados. Um estudo inicial de modelagem estrutural da xilanase tipo selvagem e duas mutantes, que apresentaram maior termoestabilidade, foi realizado, visando o melhor entendimento da relação entre estrutura e função dessas xilanases.Coordenação de Aperfeiçoamento de Pessoal de Nível Superiorapplication/pdfporUniversidade Federal de ViçosaMestrado em Bioquímica AgrícolaUFVBRBioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animalEvolução dirigidaXilanaseOrpinomyces sp.Directed evolutionXylanaseOrpinomyces sp.CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICAMelhoramento por evolução dirigida da termoestabilidade da xilanase xynA de Orpinomyces sp. PC-2Improvement of the thermostability of Orpinomyces sp. 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dc.title.por.fl_str_mv Melhoramento por evolução dirigida da termoestabilidade da xilanase xynA de Orpinomyces sp. PC-2
dc.title.alternative.eng.fl_str_mv Improvement of the thermostability of Orpinomyces sp. PC-2 xylanase by directed evolution
title Melhoramento por evolução dirigida da termoestabilidade da xilanase xynA de Orpinomyces sp. PC-2
spellingShingle Melhoramento por evolução dirigida da termoestabilidade da xilanase xynA de Orpinomyces sp. PC-2
Trevizano, Larissa Mattos
Evolução dirigida
Xilanase
Orpinomyces sp.
Directed evolution
Xylanase
Orpinomyces sp.
CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA
title_short Melhoramento por evolução dirigida da termoestabilidade da xilanase xynA de Orpinomyces sp. PC-2
title_full Melhoramento por evolução dirigida da termoestabilidade da xilanase xynA de Orpinomyces sp. PC-2
title_fullStr Melhoramento por evolução dirigida da termoestabilidade da xilanase xynA de Orpinomyces sp. PC-2
title_full_unstemmed Melhoramento por evolução dirigida da termoestabilidade da xilanase xynA de Orpinomyces sp. PC-2
title_sort Melhoramento por evolução dirigida da termoestabilidade da xilanase xynA de Orpinomyces sp. PC-2
author Trevizano, Larissa Mattos
author_facet Trevizano, Larissa Mattos
author_role author
dc.contributor.authorLattes.por.fl_str_mv http://lattes.cnpq.br/3860787717001357
dc.contributor.author.fl_str_mv Trevizano, Larissa Mattos
dc.contributor.advisor-co1.fl_str_mv Rezende, Sebastião Tavares de
dc.contributor.advisor-co1Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4787599A3
dc.contributor.advisor-co2.fl_str_mv Santoro, Marcelo Matos
dc.contributor.advisor-co2Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/index.jsp
dc.contributor.advisor1.fl_str_mv Guimarães, Valéria Monteze
dc.contributor.advisor1Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4798758T3
dc.contributor.referee1.fl_str_mv Fietto, Luciano Gomes
dc.contributor.referee1Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4763824H8
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
contributor_str_mv Rezende, Sebastião Tavares de
Santoro, Marcelo Matos
Guimarães, Valéria Monteze
Fietto, Luciano Gomes
Passos, Flávia Maria Lopes
dc.subject.por.fl_str_mv Evolução dirigida
Xilanase
Orpinomyces sp.
topic Evolução dirigida
Xilanase
Orpinomyces sp.
Directed evolution
Xylanase
Orpinomyces sp.
CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA
dc.subject.eng.fl_str_mv Directed evolution
Xylanase
Orpinomyces sp.
dc.subject.cnpq.fl_str_mv CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA
description Xylan is the most common hemicellulosic polysaccharide in cell walls of land plants. This polysaccharide is composed of a main-chain polymer made up exclusively of β-1,4 xylose residues. This backbone possesses branches containing arabinofuranosyl, acetyl, and glucuronosyl residues. Enzymatic hydrolysis of xylans is brought about by a variety of enzyme activities that are grouped under the generic term hemicellulases. Endoxylanases (EC 3.2.1.8) hydrolyze internal β-1,4 bonds in the main-chain and generate soluble xylooligosaccharides. A variety of microorganisms, including bacteria, yeast and filamentous fungi, have been reported to produce xylanase, in which the most potent producers are fungi. Researchers are especially interested in fungal xylanases because they are secreted extracellularly and their activity is much higher than the xylanases from yeasts and bacteria. Among the biotechnological applications of xylanases, it can be outstanding the bioconversion of lignocellulosic materials into fermentative products, improvement of digestibility of animal feedstock, clarification of juices, facilitating the release of lignin from the pulp and reducing the amount of chlorine required for bleaching in pulp in the paper industry. Over the last few decades, there has been a growing interest in lignocellulose bioconversion as a renewable energy source and xylanases can be effectively used with cellulases to hydrolyze the lignocellulosic biomass and bioethanol production. Most of the fungal xylanases show optimal activities at neutral or acidic pH and at temperatures below 45 °C. Xylanase (XynA), originally isolated from the anaerobic fungus Orpinomyces PC-2, has been tested for several industrial applications, in pilot scale. This enzyme presented extremely high activity with the xylan substrate of animal ration, cellulose pulp and other biomass types. Studies demonstrated that the use of the native enzyme has been more efficient in optimal conditions as pH 5.0-5.5 and temperature of 50-55 °C. However, the industrial process is usually accomplished at higher temperature and alkaline pH, what justifies the interest for xylanases that are active in these conditions. Directed evolution has emerged as a successful alternative in the genetic engineering of enzymes and it has been used to improve the functional properties of those molecules. In this study, error-prone PCR was used to improve the thermostability of endo-β-1,4-xylanase from Orpinomyces PC-2. The constructed library of xylanase (xynA) mutants was submitted to several screening cycles. The transformants were first exposed to 60 ºC during one hour and then the thermostable mutants were selected with the azo-xylan-agarose 0.2% pH 6.5 as substrate. Six mutants selected were sequenced and these amino acid sequences were analyzed to identify the mutations. Two mutants displayed higher thermal stability than the xylanase without mutations (wild-type). Whereas the wild-type lost 60% of its activity after 10 min at 60 ºC, mutants M4 and M6 showed enhanced thermostability and retained approximately 50% of its activities after treatment at 60 ºC for 60 min. In addition, M4 retained about of 40% of its initial activity after incubation at 75 ºC for 60 min. In order to evaluate the mutation effects in the enzyme properties, these ones were characterized for the optimum temperature, optimum pH and substrate specificity. The mutants and the wild-type showed an optimal temperature and pH for xylanase activity at 60 ºC and pH range of 5.0-7.0. The enzymes showed activity against the two tested substrates. An initial structural modeling study was accomplished with the wild-type and two mutants, which presented higher thermostability, seeking the best understanding of the relationship between structure and function of those xylanases.
publishDate 2009
dc.date.issued.fl_str_mv 2009-04-29
dc.date.available.fl_str_mv 2013-12-12
2015-03-26T13:07:27Z
dc.date.accessioned.fl_str_mv 2015-03-26T13:07:27Z
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dc.identifier.citation.fl_str_mv TREVIZANO, Larissa Mattos. Improvement of the thermostability of Orpinomyces sp. PC-2 xylanase by directed evolution. 2009. 80 f. Dissertação (Mestrado em Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal) - Universidade Federal de Viçosa, Viçosa, 2009.
dc.identifier.uri.fl_str_mv http://locus.ufv.br/handle/123456789/2408
identifier_str_mv TREVIZANO, Larissa Mattos. Improvement of the thermostability of Orpinomyces sp. PC-2 xylanase by directed evolution. 2009. 80 f. Dissertação (Mestrado em Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal) - Universidade Federal de Viçosa, Viçosa, 2009.
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