Alkalophilic adaptation of XynB endoxylanase from Aspergillus niger via rational design of pKa of catalytic residues.

Based on the strategy of changing pH-stability profiles by altering pKa values of catalytic residues, rational protein engineering was applied to improve alkalophilic adaptation of Aspergillus niger endoxylanase XynB. Computational predictions and molecular modeling were carried out using PROPKA server and SWISS-MODEL server, respectively. Three endoxylanase mutant of S108V, N151E, and Q178R, in which the pKa values of either catalytic glutamate residues shifted, were generated. In agreement with expectation, the variant of Q178R improved alkalophilic performances. The mutant Q178R raised the optimum pH of XynB from 5.5 to 6.0 and retained 37% of the maximum activity at pH 8.0. Interestingly, the pKa values of Glu84 and Glu175 in Q178R are 7.91 and 6.32, respectively. The pKa of Glu175 is lower than that of Glu84, as opposed to the fact that the pKa of Glu84 is lower than that of Glu175 in other GH11 xylanases. It indicated that Glu175 may convert into a nucleophile residue and Glu84 into an acid/base residue.