Thermostability improvement of Orpinomyces sp. xylanase by directed evolution

详细信息    查看全文

• 作者：Larissa Mattos Trevizano ; Rafaela Z ; onade Ventorim ; Sebasti?o Tavares de Rezende ; Floriano Paes Silva Junior ; Val¨¦ria Monteze Guimar?es
• 关键词：Error-prone PCR ; Xylanase ; Thermostability ; Orpinomyces
• 刊名：Journal of Molecular Catalysis B: Enzymatic
• 出版年：2012
• 出版时间：September, 2012
• 年：2012
• 卷：81
• 期：Complete
• 页码：12-18
• 全文大小：592 K

文摘

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-¦Â-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 ¡ãC 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 ¡ãC 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.