Characterization of an α-glucosidase, HdAgl, from the digestive fluid of Haliotis discus hannai

摘要

Previously, we isolated two α-amylase isozymes, HdAmy58 and HdAmy82, from the digestive fluid of the Pacific abalone Haliotis discus hannai (Kumagai et al., 2013, Comp. Biochem. Physiol., B 164, 80-88). These enzymes degraded starch producing maltooligosaccharides but not glucose. However, the digestive fluid itself could produce glucose from starch, indicating that the digestive fluid contains α-glucosidase-like enzymes together with the α-amylases. Thus, in the present study, we isolated this α-glucosidase-like enzyme from the digestive fluid and characterized it to some extent. Isolation of this enzyme was carried out by ammonium sulfate fractionation followed by conventional column chromatographies and FPLC. The purified enzyme showed an apparent molecular mass of 97. kDa on SDS-PAGE, and optimal temperature and pH of 45°C and 3.8-5.5, respectively. This enzyme could degrade various sizes of maltooligosaccharides into glucose and released glucose from starch producing no appreciable intermediate oligosaccharides. We concluded that this enzyme is an α-glucosidase (EC 3.2.1.20) exolitically acting on polymer substrate and named HdAgl. HdAgl efficiently degraded maltose but hardly degraded p-nitrophenyl α. -. D-glucopyranoside (α-. pNPG) and isomaltose. This enzyme was regarded as a maltase-like α-glucosidase that preferably degrades maltose but scarcely aryl glucosides. When starch was used as a substrate, HdAgl converted approximately 40% (w/w) of the starch to glucose. If an abalone α-amlylase HdAmy58 was added to the reaction mixture, the glucose yield increased to 60% (w/w). These results suggested that both HdAgl and HdAmy58 play important roles for the production of glucose from dietary starch in the digestive fluid. The amino-acid sequence of 887 residues for HdAgl was deduced by the cDNA method. This sequence showed 41-46% amino-acid identities to those of mammalian and avian α-glucosidases belonging to glycoside-hydrolase-family31.