Biochemical properties and substrate recognition mechanism of GH31 α-glucosidase from Bacillus sp. AHU 2001 with broad substrate specificity.

α-Glucosidases are ubiquitous enzymes that hydrolyze the α-glucosidic linkage at the non-reducing end of substrates. In this study, we characterized an α-glucosidase (BspAG31A) belonging to glycoside hydrolase family 31 from Bacillus sp. AHU 2001. Recombinant BspAG31A, produced in Escherichia coli, had high hydrolytic activity toward maltooligosaccharides, kojibiose, nigerose, and neotrehalose. This is the first report of an α-glucosidase with high activity toward neotrehalose. The transglucosylation products, nigerose, kojibiose, isomaltose, and neotrehalose, were generated from 440 mm maltose. Substitution of Tyr268, situated on the β → α loop 1 of BspAG31A, with Trp increased hydrolytic activity toward isomaltose. This mutation reduced the hydrolytic activity toward maltooligosaccharides more than toward kojibiose, nigerose, and neotrehalose. Analysis of the Y173A mutant of BspAG31A showed that Tyr173, situated on the N-terminal domain loop, is associated with the formation of subsite +2. In Y173A, the kcat/Km for maltooligosaccharides slightly decreased with an increasing degree of polymerization compared with wild type. Among the amino acid residues surrounding the substrate binding site, Val543 and Glu545 of BspAG31A were different from the corresponding residues of Bacillus thermoamyloliquefaciens α-glucosidase II, which has higher activity toward isomaltose than BspAG31A. The E545G mutation slightly enhanced isomaltase activity without a large reduction of hydrolytic activities toward other substrates. V543A showed 1.8-3.5-fold higher hydrolytic activities toward all substrates other than neotrehalose compared with wild type, although its preference for isomaltose was unchanged.