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 PY2 ) was evolved by synergistically engineering the substrate-binding pocket and active-site lids. The resulting mutant Pul PY2 -M2 exhibited 5-fold improvement in catalytic efficiency ( k cat / K m ) compared to that of Pul PY2 . Pul PY2 -M2 was utilized to develop a one-pot reaction system for efficient production of maltooligosaccharides. The maltooligosaccharides conversion rate of Pul PY2 -M2 reached 96.1%, which was increased by 5.4% compared to that of Pul PY2 . Furthermore, when employed for glucose production, the glucose productivity of Pul PY2 -M2 was 25.4% and 43.5% higher than that of Pul PY2 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 PY2 -M2 in starch sugar industry.