Engineering hyperthermophilic pullulanase to efficiently utilize corn starch for production of maltooligosaccharides and glucose.

Pullulanase is a starch-debranching enzyme that hydrolyzes side chain of starch, oligosaccharides and pullulan. Nevertheless, the limited activities of pullulanases constrain their practical application. Herein, the hyperthermophilic type II pullulanase from Pyrococcus yayanosii CH1 (Pul) was evolved by synergistically engineering the substrate-binding pocket and active-site lids. The resulting mutant Pul-M2 exhibited 5-fold improvement in catalytic efficiency (k/K) compared to that of Pul. Pul-M2 was utilized to develop a one-pot reaction system for efficient production of maltooligosaccharides. The maltooligosaccharides conversion rate of Pul-M2 reached 96.1%, which was increased by 5.4% compared to that of Pul. Furthermore, when employed for glucose production, the glucose productivity of Pul-M2 was 25.4% and 43.5% higher than that of Pul and the traditional method, respectively. These significant improvements in maltooligosaccharides and glucose production and the efficient utilization of corn starch demonstrated the potential of the engineered Pul-M2 in starch sugar industry.