Purification and characterization of a Bacillus sp. DG0303 thermostable α-glucosidase with oligo-1,6-glucosidase activity

Extracellular alpha-glucosidase was purified to homogeneity from moderately thermophilic Bacillus sp. DG0303. The thermostable alpha-glucosidase was purified by ammonium sulfate fractionation, ion-exchange chromatography, preparative polyacrylamide gel electrophoresis (PAGE), and electroelution. The molecular weight of the enzyme was estimated to be 60 kDa by SDS-PAGE. The optimum temperature for the action of the enzyme was at 60 degrees C. It had a half-life of 35 min at 60 degrees C. The enzyme was stable at the pH range of 4.5 similar to 7.0 and had an optimum pH at 5.0. The enzyme preparation did not require any metal ion for activity. The thermostable alpha-glucosidase hydrolyzed the alpha-1,6-linkages in isomaltose, isomaltotriose, and panose, and had little or no activity with maltooligosaccharides and other polysaccharides. The K-m (mM) for p-nitrophenyl-alpha-D-glucopyranoside (pNPG), panose, isomaltose, and isomaltotriose were 4.6, 4.7, 40.8, and 3.7 and the V-max (mu mol . min(-1) mg(-1)) for those substrates were 5629, 1669, 3410, and 1827, respectively. The N-terminal amino acid sequence of the enzyme was MERVWWKKAV. Based on its substrate specificity and catalytic properties, the enzyme has been assigned to be an oligo-1,6-glucosidase.