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

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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.