Navegando por Autor "Silva Junior, Floriano Paes"
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Item Thermostability improvement of Orpinomyces sp. xylanase by directed evolution(Journal of Molecular Catalysis B: Enzymatic, 2012-09) Trevizano, Larissa Mattos; Ventorim, Rafaela Zandonade; Rezende, Sebastião Tavares de; Silva Junior, Floriano Paes; Guimarães, Valéria MontezeThe 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.