Thermostability improvement of Orpinomyces sp. xylanase by directed evolution
Autor(a) principal: | |
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Data de Publicação: | 2012 |
Outros Autores: | , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | LOCUS Repositório Institucional da UFV |
Texto Completo: | https://doi.org/10.1016/j.molcatb.2012.04.021 http://www.locus.ufv.br/handle/123456789/21788 |
Resumo: | The methodology of directed evolution, using the mutagenic technique of error-prone PCR has been used to improve the thermostability of enzymes. This method was applied to the endo-β-1,4-xylanase from Orpinomyces strain PC-2. The constructed library of xylanase (xynA) mutants was subjected to several screening cycles in plates with azo-xylan-agarose as substrate and four thermostable mutants (M1–M4) were selected. Homology models for these thermostable mutants were constructed to identify the location of the residues changed by error-prone PCR and to investigate the effect of these mutations on the xylanase properties. Xylanase activities of the mutants and wild type were maximal at 60 °C and in the pH range of 5–7. The mutants displayed higher thermostability than the wild type XynA, where the wild type showed a half-life at 60 °C of 7.92 min, while half-life values for M1, M2, M3 and M4 were 209, 33.2, 401 and 15.3 min, respectively. Additionally, M3 and M4 presented a good performance in more extreme pH conditions. The mutants retained their ability to hydrolyze birchwood and oat spelt xylans, which are substrates presenting different degrees of branching. |
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Trevizano, Larissa MattosVentorim, Rafaela ZandonadeRezende, Sebastião Tavares deSilva Junior, Floriano PaesGuimarães, Valéria Monteze2018-09-12T19:08:21Z2018-09-12T19:08:21Z2012-0913811177https://doi.org/10.1016/j.molcatb.2012.04.021http://www.locus.ufv.br/handle/123456789/21788The methodology of directed evolution, using the mutagenic technique of error-prone PCR has been used to improve the thermostability of enzymes. This method was applied to the endo-β-1,4-xylanase from Orpinomyces strain PC-2. The constructed library of xylanase (xynA) mutants was subjected to several screening cycles in plates with azo-xylan-agarose as substrate and four thermostable mutants (M1–M4) were selected. Homology models for these thermostable mutants were constructed to identify the location of the residues changed by error-prone PCR and to investigate the effect of these mutations on the xylanase properties. Xylanase activities of the mutants and wild type were maximal at 60 °C and in the pH range of 5–7. The mutants displayed higher thermostability than the wild type XynA, where the wild type showed a half-life at 60 °C of 7.92 min, while half-life values for M1, M2, M3 and M4 were 209, 33.2, 401 and 15.3 min, respectively. Additionally, M3 and M4 presented a good performance in more extreme pH conditions. The mutants retained their ability to hydrolyze birchwood and oat spelt xylans, which are substrates presenting different degrees of branching.engJournal of Molecular Catalysis B: Enzymaticv. 81, p. 12- 18, set. 2012Elsevier B.V.info:eu-repo/semantics/openAccessError-prone PCRXylanaseThermostabilityOrpinomycesThermostability improvement of Orpinomyces sp. xylanase by directed evolutioninfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfreponame:LOCUS Repositório Institucional da UFVinstname:Universidade Federal de Viçosa (UFV)instacron:UFVORIGINALartigo.pdfartigo.pdftexto completoapplication/pdf606983https://locus.ufv.br//bitstream/123456789/21788/1/artigo.pdfaa18fa57be4d6f1cd1f8254bcfeafc89MD51LICENSElicense.txtlicense.txttext/plain; charset=utf-81748https://locus.ufv.br//bitstream/123456789/21788/2/license.txt8a4605be74aa9ea9d79846c1fba20a33MD52THUMBNAILartigo.pdf.jpgartigo.pdf.jpgIM Thumbnailimage/jpeg5169https://locus.ufv.br//bitstream/123456789/21788/3/artigo.pdf.jpg8dba1927d2ce648832af2ebaa9875977MD53123456789/217882018-09-12 23:00:32.648oai:locus.ufv.br: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Repositório InstitucionalPUBhttps://www.locus.ufv.br/oai/requestfabiojreis@ufv.bropendoar:21452018-09-13T02:00:32LOCUS Repositório Institucional da UFV - Universidade Federal de Viçosa (UFV)false |
dc.title.en.fl_str_mv |
Thermostability improvement of Orpinomyces sp. xylanase by directed evolution |
title |
Thermostability improvement of Orpinomyces sp. xylanase by directed evolution |
spellingShingle |
Thermostability improvement of Orpinomyces sp. xylanase by directed evolution Trevizano, Larissa Mattos Error-prone PCR Xylanase Thermostability Orpinomyces |
title_short |
Thermostability improvement of Orpinomyces sp. xylanase by directed evolution |
title_full |
Thermostability improvement of Orpinomyces sp. xylanase by directed evolution |
title_fullStr |
Thermostability improvement of Orpinomyces sp. xylanase by directed evolution |
title_full_unstemmed |
Thermostability improvement of Orpinomyces sp. xylanase by directed evolution |
title_sort |
Thermostability improvement of Orpinomyces sp. xylanase by directed evolution |
author |
Trevizano, Larissa Mattos |
author_facet |
Trevizano, Larissa Mattos Ventorim, Rafaela Zandonade Rezende, Sebastião Tavares de Silva Junior, Floriano Paes Guimarães, Valéria Monteze |
author_role |
author |
author2 |
Ventorim, Rafaela Zandonade Rezende, Sebastião Tavares de Silva Junior, Floriano Paes Guimarães, Valéria Monteze |
author2_role |
author author author author |
dc.contributor.author.fl_str_mv |
Trevizano, Larissa Mattos Ventorim, Rafaela Zandonade Rezende, Sebastião Tavares de Silva Junior, Floriano Paes Guimarães, Valéria Monteze |
dc.subject.pt-BR.fl_str_mv |
Error-prone PCR Xylanase Thermostability Orpinomyces |
topic |
Error-prone PCR Xylanase Thermostability Orpinomyces |
description |
The methodology of directed evolution, using the mutagenic technique of error-prone PCR has been used to improve the thermostability of enzymes. This method was applied to the endo-β-1,4-xylanase from Orpinomyces strain PC-2. The constructed library of xylanase (xynA) mutants was subjected to several screening cycles in plates with azo-xylan-agarose as substrate and four thermostable mutants (M1–M4) were selected. Homology models for these thermostable mutants were constructed to identify the location of the residues changed by error-prone PCR and to investigate the effect of these mutations on the xylanase properties. Xylanase activities of the mutants and wild type were maximal at 60 °C and in the pH range of 5–7. The mutants displayed higher thermostability than the wild type XynA, where the wild type showed a half-life at 60 °C of 7.92 min, while half-life values for M1, M2, M3 and M4 were 209, 33.2, 401 and 15.3 min, respectively. Additionally, M3 and M4 presented a good performance in more extreme pH conditions. The mutants retained their ability to hydrolyze birchwood and oat spelt xylans, which are substrates presenting different degrees of branching. |
publishDate |
2012 |
dc.date.issued.fl_str_mv |
2012-09 |
dc.date.accessioned.fl_str_mv |
2018-09-12T19:08:21Z |
dc.date.available.fl_str_mv |
2018-09-12T19:08:21Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
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publishedVersion |
dc.identifier.uri.fl_str_mv |
https://doi.org/10.1016/j.molcatb.2012.04.021 http://www.locus.ufv.br/handle/123456789/21788 |
dc.identifier.issn.none.fl_str_mv |
13811177 |
identifier_str_mv |
13811177 |
url |
https://doi.org/10.1016/j.molcatb.2012.04.021 http://www.locus.ufv.br/handle/123456789/21788 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.ispartofseries.pt-BR.fl_str_mv |
v. 81, p. 12- 18, set. 2012 |
dc.rights.driver.fl_str_mv |
Elsevier B.V. info:eu-repo/semantics/openAccess |
rights_invalid_str_mv |
Elsevier B.V. |
eu_rights_str_mv |
openAccess |
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application/pdf |
dc.publisher.none.fl_str_mv |
Journal of Molecular Catalysis B: Enzymatic |
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Journal of Molecular Catalysis B: Enzymatic |
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reponame:LOCUS Repositório Institucional da UFV instname:Universidade Federal de Viçosa (UFV) instacron:UFV |
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LOCUS Repositório Institucional da UFV |
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