Rational design of a Bacillus circulans xylanase by introducing charged residue to shift the pH optimum

Introduction or disruption of long-range electrostatic interactions can be an effective way to change the pK_as of catalytic residues and the pH optima of enzymes. In particular, shifting the pH optima toward the acidic or basic limb is an important issue for industrial applications of xylanases, e.g., for the paper or food industries. Here, we suggest an effective strategy to shift the pH optimum of an enzyme by introducing charged residue. Our strategy is to alter the titration behavior of the strongly interacting catalytic glutamates in Bacillus circulans xylanase by introducing acidic or basic residue in juxtaposition to the natively present acidic residues surrounding the catalytic site, thereby shifting the pH-activity profile. Mutation sites were chosen to be long distances (>8.5) away from the catalytic sites. The strategy was verified by site-directed mutagenesis experiments. The results show that the pH optimum can be changed (-0.5 to 1.5 unit) by strategically selecting the mutation sites. The strategies developed can effectively be applied to change the pH optima of the families of enzymes harboring acidic residues as catalytic residues.