A Hyperthermostable Type II Pullulanase from a Deep-Sea Microorganism Pyrococcus yayanosii CH1

Pullulanase is a commonly used debranching enzyme in the starch processing industry. Because the starch liquefaction process requires high temperature, a thermostable pullulanase is desired. Here, a novel hyperthermostable type II pullulanase gene (pul(PY)) was cloned from Pyrococcus yayanosii CH1, isolated from a deep-sea hydrothermal site. Pul(PY) was optimally active at pH 6.6 and 95 degrees C, retaining more than 50% activity after incubation at 95 degrees C for 10 h. The thermostability was significantly higher than those of most pullulanases reported previously. To further improve its activity and thermostability, the N-terminal and C-terminal domains of Pul(PY) were truncated. The optimum temperature of the combined truncation mutant Delta 28N + Delta 791C increased to 100 degrees C with a specific activity of 32.18 U/mg, which was six times higher than that of wild-type Pul(PY). Pul(PY) and the truncation mutant enzyme could realize the combined use of pullulanase with a-amylase during the starch liquefaction process to improve hydrolysis efficiency.