The Key Laboratory of Industrial Biotechnology of Ministry of Education , Jiangnan University , 1800 Lihu Avenue , Wuxi 214122 , China.
State Key Laboratory of Bioreactor Engineering, Biomedical Nanotechnology Center, School of Biotechnology , East China University of Science and Technology , Shanghai 200237 , P.R. China.
J Agric Food Chem. 2019 Aug 28;67(34):9611-9617. doi: 10.1021/acs.jafc.9b03376. Epub 2019 Aug 15.
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 () was cloned from CH1, isolated from a deep-sea hydrothermal site. Pul 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 Pul 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 Pul. Pul and the truncation mutant enzyme could realize the combined use of pullulanase with α-amylase during the starch liquefaction process to improve hydrolysis efficiency.
普鲁兰酶是淀粉加工工业中常用的支链淀粉酶。由于淀粉液化过程需要高温，因此需要一种热稳定的普鲁兰酶。在这里，从深海热液区分离到的 CH1 中克隆了一种新型超耐热 II 型普鲁兰酶基因()。Pul 在 pH6.6 和 95°C 下具有最佳活性，在 95°C 孵育 10 小时后保留超过 50%的活性。其热稳定性明显高于以前报道的大多数普鲁兰酶。为了进一步提高其活性和热稳定性，截短了 Pul 的 N 端和 C 端结构域。组合截短突变体 Δ28N + Δ791C 的最适温度提高到 100°C，比野生型 Pul 的比活性提高到 32.18 U/mg，提高了 6 倍。Pul 和截短突变体酶可以在淀粉液化过程中实现普鲁兰酶与α-淀粉酶的联合使用，以提高水解效率。
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