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

淀粉去支化酶催化支链淀粉、糖原和普鲁兰等α-葡聚糖的α-1，6-葡萄糖苷键的水解，用于工业淀粉糖化过程。根据底物的特性，这些酶分为普鲁兰酶和异淀粉酶两类。在本项目中，我们旨在阐明底物专一性的机理，并设计具有工业价值的酶。我们首先解决了枯草杆菌普鲁兰聚糖酶的晶体结构，并比较了几种淀粉脱膜酶的晶体结构和氨基酸序列。在此基础上，我们将突变引入Klebsiella Pullulanase的活性部位，发现活性部位附近柔性环上的甘氨酸残基被Ala或Pro取代的突变株对支链淀粉的活性比野生型酶更高，热稳定性也更好。

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.