Modulating the pH profile of the pullulanase from Pyrococcus yayanosii CH1 by synergistically engineering the active center and surface

A preferable pullulanase with high thermostability and catalytic activity at pH 4.5-5 is desired to match with glucoamylase in the starch-saccharification process. However, most of them exhibit low activity under such low pH conditions. Here, the optimal pH of the hyperthermostable pullulanase from Pyrococcus yayanosii (PulPY2) was successfully shifted from 6.4 to 5 with a 2-fold increase in the specific activity based on synergistic engineering of the active center and surface. Synergistic engineering was performed by introducing histidine within 6 Å of the active sites, and by enhancing negative charges on the enzymatic surface. Two single-site mutants of PulPY2-Q13H and PulPY2-I25E with higher hydrolytic activity were obtained, the optimal pH of which was shifted to pH 5 and 5.4, respectively; the combined mutant PulPY2-Q13H/I25E exhibited the optimal pH of 5, 3.2-fold increasing catalytic efficiency at pH 5, and high thermostability compared to PulPY2. These results not only obtained an applicable pullulanase for industrial application, but also provided a strategy for shifting the optimal pH of the enzyme based on synergistic engineering of the active center and surface.