Studies on the key amino acid residues responsible for the alkali-tolerance of the xylanase by site-directed or random mutagenesis

Asparagine (Asn)-71 of the xylanase (XYN) from Bacillus pumilus A-30 was found highly conserved in alkaline xylanases of family G/11. The mutated gene fragments containing different substitutions of Asn-71 was obtained by site-directed mutagenesis to study its role in the alkali-tolerant mechanism of xylanase. The xylanase activity was completely lost if Asn-71 residue was replaced by alkaline arginine (Arg) or lysine (Lys) residues, but obviously depressed with a shift in the pH optimum of the enzyme from 6.7 to 6.3 if substituted by serine (Ser) or aspartate (Asp) residues. No mutant with a shift of the pH optimum to a more basic value was found. Furthermore, N71D lost its activity in the alkaline pH range completely, while N71S did not lose as much as that of N71D. Except for Asn-71, the random mutagenesis to other residues of the xylanase was also studied. The alkali-tolerant mechanism of the xylanase was analyzed by their charged character, ionized state, and the hydrogen bond network of the residues surrounding the two catalytic residues on the basis of homology modeling of the mutated xylanases.