14-25: Improvement of endo-1,4-β-xylanase thermostability from Thermoascus aurantiacus by mutagenic PCR

Hemicelluloses, which include β-glucans and xylans, are major constituents of plant cell walls. The endo-1,4-β-xylanases (EC 3.2.1.8) are responsible for the hydrolysis of β-1,4 connections present in the internal chain of xylan. The xylanase in study is a potential enzyme, with many industrial aplication, as in pulp and paper industry, beside they can help in bioconversion of lignocellulosic biomass into biofuels. Therefore, the aim is study a xylanase present in the thermophilic fungus genome, Termoascus aurantiacus, reporting the production of the recombinant enzyme by cloning and expression in S. cerevisiae and improve of the thermostability of the xylanase using PCR mutagenic (error-prone PCR). For this purpose, at the time, the insert of xylanase was cloned into vector YPGK1, a bifunctional vector for constitutive expression in S. cerevisiae under the control of the PGK1 gene promoter and the LEU2 auxotrophic mark, and expressed in S. cerevisiae. After, it was made the first mutants generation, the material transformed into S. cerevisiae by heat shock and the xylanolytic activity was evaluated in the plate assay in medium with 1% xylan. The next step will be check the increase of the mutant enzymes thermal stability in test plate and as needed will be processed new round of mutations. After selection of mutant xylanase, the two lines of xylanases: native and mutated will be purified and characterized for comparative studies.
Keywords: endo-1,4-β-xylanase; PCR mutagenic; thermophilic fungus; Thermoascus aurantiacus.