Modulating the pH profile of the pullulanase from Pyrococcus yayanosii CH1 by synergistically engineering the active...

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 (Pul_PY2) 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 Pul_PY2-Q13H and Pul_PY2-I25E with higher hydrolytic activity were obtained, the optimal pH of which was shifted to pH 5 and 5.4, respectively; the combined mutant Pul_PY2-Q13H/I25E exhibited the optimal pH of 5, 3.2-fold increasing catalytic efficiency at pH 5, and high thermostability compared to Pul_PY2. 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.