Analysis of Functional Domains and Improvement of Alkaliphily of an Alkaline Xylanase on the Basis of Its Three-dimensional Structure

Abstract

Xylanase J (XynJ) from alkaliphilic Bacillus sp. strain 41M-1 is a multi-domain enzyme consisting of a glycoside hydrolase (GH) family 11 catalytic domain and a carbohydrate-binding module family 36 xylan-binding domain (XBD). Structural comparison of the GH family 11 catalytic domains indicated that there were several specific salt bridges in the catalytic cleft of XynJ. Mutant enzymes were prepared by substituting several amino acid residues responsible for the characteristic salt bridges. Elimination of the salt bridges caused an acidophilic shift in optimum pH, suggesting that the characteristic salt bridges contributed to the alkaliphily of XynJ. On the other hand, reinforcing one of the characteristic salt bridges in the catalytic domain shifted the optimum pH of XynJ from 8.5 to 9.0. Furthermore, introducing excess Arg residues on the protein surface was also effective to improve the alkaliphily of XynJ. Xylan-binding properties of various XBD mutants were investigated as fusion proteins with glutathione-S-transferase. Furthermore, the same substitutions were introduced into the XBD region of XynJ and insoluble xylan-hydrolyzing activity was measured. The results indicated that some Asp, Trp and Tyr residues were important for xylan-binding activity of the XBD, and that xylan-hydrolyzing activity of XynJ was closely correlated to xylan-binding activity of the XBD region.