Production and characterization of Orpinomyces mutant xylanases with improved temperature and pH stabilities

Abstract

The error-prone PCR technique has been widely used in order to obtain thermostable enzymes more suitable for industrial conditions. The Orpinomyces xynA mutant library allowed the selection of four thermostable mutants (M1-M4). Molecular dynamics (MD) predicted an N-terminal tail as being a destabilizing structural region and allowed further enhancing of the mutant xylanases thermostability. Thus, removal of the 27 amino acid residues enabled an increase in the enzyme half-life values (t1/2). However, besides the improved thermostability, the large enzyme production and high catalytic performance are also relevant for the biotechnological application of enzymes. During the mutant enzymes production in E. coli, the IPTG induction protocol allowed high expression levels of soluble and active xylanases. The mutant xylanases without the 27 amino acid residues showed improved thermostability and the shorter versions of M2 and M4 (named as SM2 and SM4) also presented a good performance in more extreme pH conditions.