Endoxylanase catalyzes the hydrolysis of xylan, a plant cell wall heteropolysaccharide that contains a backbone of 1,4-linked xylose residues. Xylanases have been found to be effective in reducing chlorine dosage requirements in the pulp-bleaching process. It is desirable that xylanases used for industries are stable and active under alkaline conditions at high temperatures.
In this study, the activity and stability of xylanases from Bacillus sp. were improved by directed evolution. Three evolved mutants were isolated after sequential rounds of error-prone PCR to introduce random mutations, and membrane-based screening of the resultant mutant library. They were identified as having one(mutant 3-5), two(mutant 6-8), and three(mutant 10-61) amino acid substitutions, respectively. All the substituted amino acids of mutant 10-61 were located on the protein surface, and had polar residues with hydroxyl group. Compared to the wild type enzyme, the stability of three mutants in alkaline condition were enhanced without the loss of specific activity. Mutant 10-61 was 2.5-fold more active than wild type enzyme after 30h incubation in Tris-CI buffer(pH 9.0). In addition to alkaline stability, thermostability was also improved in the three mutants.
