Characterization of Halomonas sp Strain H11 α-Glucosidase Activated by Monovalent Cations and Its Application for Efficient Synthesis of α-D-Glucosylglycerol

An alpha-glucosidase (HaG) with the following unique properties was isolated from Halomonas sp. strain H11: (i) high transglucosylation activity, (ii) activation by monovalent cations, and (iii) very narrow substrate specificity. The molecular mass of the purified HaG was estimated to be 58 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). HaG showed high hydrolytic activities toward maltose, sucrose, and p-nitrophenyl alpha-D-glucoside (pNPG) but to almost no other disaccharides or malto-oligosaccharides higher than trisaccharides. HaG showed optimum activity to maltose at 30 degrees C and pH 6.5. Monovalent cations such as K+, Rb+, Cs+, and NH4+ increased the enzymatic activity to 2- to 9-fold of the original activity. These ions shifted the activity-pH profile to the alkaline side. The optimum temperature rose to 40 degrees C in the presence of 10 mM NH4+, although temperature stability was not affected. The apparent K-m and k(cat) values for maltose and pNPG were significantly improved by monovalent cations. Surprisingly, k(cat)/K-m for pNPG increased 372- to 969-fold in their presence. HaG used some alcohols as acceptor substrates in transglucosylation and was useful for efficient synthesis of alpha-D-glucosylglycerol. The efficiency of the production level was superior to that of the previously reported enzyme Aspergillus niger alpha-glucosidase in terms of small amounts of by-products. Sequence analysis of HaG revealed that it was classified in glycoside hydrolase family 13. Its amino acid sequence showed high identities, 60%, 58%, 57%, and 56%, to Xanthomonas campestris WU-9701 alpha-glucosidase, Xanthomonas campestris pv. raphani 756C oligo-1,6-glucosidase, Pseudomonas stutzeri DSM 4166 oligo-1,6-glucosidase, and Agrobacterium tumefaciens F2 alpha-glucosidase, respectively.