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 (pulPY) was cloned from Pyrococcus yayanosii CH1, isolated from a deep-sea hydrothermal site. PulPY was optimally active at pH 6.6 and 95 °C, retaining more than 50% activity after incubation at 95 °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 PulPY were truncated. The optimum temperature of the combined truncation mutant Δ28N + Δ791C increased to 100 °C with a specific activity of 32.18 U/mg, which was six times higher than that of wild-type PulPY. PulPY and the truncation mutant enzyme could realize the combined use of pullulanase with α-amylase during the starch liquefaction process to improve hydrolysis efficiency.
Keywords: CH1; domain truncation; hyperthermophilic enzyme; pullulanase; starch debranching enzyme; starch liquefaction.