title: Improving the thermostability and catalytic efficiency of Bacillus deramificans pullulanase by site-directed mutagenesis.
abstract: Pullulanase (EC 3.2.1.41) is a well-known starch-debranching enzyme. Its instability and low catalytic efficiency are the major factors preventing its widespread application. To address these issues, Asp437 and Asp503 of the pullulanase from Bacillus deramificans were selected in this study as targets for site-directed mutagenesis based on a structure-guided consensus approach. Four mutants (carrying the mutations D503F, D437H, D503Y, and D437H/D503Y) were generated and characterized in detail. The results showed that the D503F, D437H, and D503Y mutants had an optimum temperature of 55°C and a pH optimum of 4.5, similar to that of the wild-type enzyme. However, the half-lives of the mutants at 60°C were twice as long as that of the wild-type enzyme. In addition, the D437H/D503Y double mutant displayed a larger shift in thermostability, with an optimal temperature of 60°C and a half-life at 60°C of more than 4.3-fold that of the wild-type enzyme. Kinetic studies showed that the Km values for the D503F, D437H, D503Y, and D437H/D503Y mutants decreased by 7.1%, 11.4%, 41.4%, and 45.7% and the Kcat/Km values increased by 10%, 20%, 140%, and 100%, respectively, compared to those of the wild-type enzyme. Mechanisms that could account for these enhancements were explored. Moreover, in conjunction with the enzyme glucoamylase, the D503Y and D437H/D503Y mutants exhibited an improved reaction rate and glucose yield during starch hydrolysis compared to those of the wild-type enzyme, confirming the enhanced properties of the mutants. The mutants generated in this study have potential applications in the starch industry.
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, China.
authors.forename: Xuguo
authors.initials: X
authors.lastname: Duan
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
authors.forename: Jing
authors.initials: J
authors.lastname: Wu
mesh_terms: D000064:Acclimatization / Q000502:physiology*; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis; D004926:Escherichia coli; D005087:Glucan 1,4-alpha-Glucosidase; D006026:Glycoside Hydrolases / Q000378:metabolism* / Q000502:physiology; D007700:Kinetics; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D013213:Starch / Q000235:genetics; D013696:Temperature
chemical_list: D013213:Starch; D006026:Glycoside Hydrolases; D005087:Glucan 1,4-alpha-Glucosidase; C100181:pullulanase