Analyses of molecular structures and functional relationships of starch debranching enzymes aiming to improve industrial starch saccharification processes

淀粉脱支酶催化α-葡聚糖如支链淀粉、糖原和支链淀粉的α-1，6-糖苷键的水解，并用于工业淀粉糖化过程。这些酶根据底物特异性分为两类，普鲁兰酶和异淀粉酶。本项目旨在阐明底物特异性的机制，设计具有工业价值的酶。本文首先解析了枯草芽孢杆菌普鲁兰酶的晶体结构，并比较了几种淀粉脱支链酶的晶体结构和氨基酸序列。在此基础上，我们对克雷伯氏菌普鲁兰酶的活性位点进行了突变，发现突变体中活性位点附近的Gly残基被Ala或Pro取代，从而提高了酶的活性，并改善了酶的热稳定性。

Starch-debranching enzymes catalyze the hydrolysis of the α-1,6-glucosidic linkage of α-glucans such as amylopectin, glycogen and pullulan, and are used in an industrial starch saccharification process. These enzymes classified into two groups, pullulanase and isoamylase based on the substrate specificities. In this project, we aimed to clarify the mechanism of substrate specificity and design industrially valuable enzyme. We first solved the crystal structure of Bacillus subtilis pullulanase, and compared the crystal structures and amino acid sequences of several starch-debraniching enzymes. Then we introduced mutations into active sites of Klebsiella pullulanase and found some mutants in which Gly residues located on flexible loop in the vicinity of active site are replaced by Ala or Pro showed higher activity toward amylopectin and improved thermostability than wild type enzyme.