Residue mutations of xylanase in Aspergillus kawachii alter its optimum pH

Abstract

Aspergillus kawachii and Aspergillus niger have been traditionally used as molds for commercial microbial fermentation because of their capability to grow in extremely acidic environments and produce acid-stable enzymes. Endo-1,4-β-xylanase cleaves the glycosidic bonds in the xylan backbone, consequently reducing the degree of polymerization of the substrate. The amino acid sequences of xylanases from A. kawachii and A. niger only differ in one amino acid residue. However, the xylanases from A. kawachii and A. niger show different optimum pH values of 2.0 and 3.0, respectively. In this study, we synthesized the A. kawachii xylanase gene (XynC) on the basis of the bias codon of yeast and mutated the gene in the dominating region related to optimum pH shifting during gene synthesis. After the overexpression of this gene in Pichia pastoris G115, the mutant (Thr64Ser) enzyme (XynC-C) showed an optimum pH of 3.8, which indicated partial alkalinity compared with the original xylanase from A. kawachii. Similar to that of the enzyme with one residue mutation (Asp48Asn), the optimum pH of the enzyme with two residue mutations (Thr64Ser and Asp48Asn) shifted to 5.0. The result indicated that mutation Asp48 was more important than mutation Thr64 in optimum pH shifting. We proposed a model that explains the lower optimum pH of XynC-C than other members of the xylanase family G. XynC-C showed similar proteolytic resistance and Km and Vmax values for beechwood xylan to other xylanases.