Thermostability improvement of Orpinomyces sp. xylanase by directed evolution

• Autor(a) principal: Trevizano, Larissa Mattos
• Data de Publicação: 2012
• Outros Autores: Ventorim, Rafaela Zandonade, Rezende, Sebastião Tavares de, Silva Junior, Floriano Paes, Guimarães, Valéria Monteze
• 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
• topic: Error-prone PCR; Xylanase; Thermostability; Orpinomyces

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.