A putative alpha-amylase gene (accession number, CP000284) of Methylobacillus flagellatus KT ATCC51484 was cloned in Escherichia coli, and its gene product was expressed and characterized. The purified recombinant enzyme (MFAS) displayed a typical amylosucrase (ASase) activity by the demonstration of multiple activities of hydrolysis, isomerization, and polymerization although it was designated as an alpha-amylase. The optimal reaction temperature and pH for the sucrose hydrolysis activity of MFAS were determined to be 45 A degrees C and pH 8.5, respectively. MFAS has relatively high thermostable characteristics compared with other ASases, as demonstrated by a half-life of 19.3 min at 50 A degrees C. MFAS also showed polymerization activity using sucrose as a sole substrate. Glycerol was transglycosylated by the intermolecular transglycosylation activity of MFAS. Two major products were observed by thin-layer chromatography and isolated by paper chromatography and recycling HPLC. Using H-1 and C-13 NMR, their chemical structures were determined to be (2S)-1-O-alpha-d-glucosyl-glycerol or (2R)-1-O-alpha-d-glucosyl-glycerol and 2-O-alpha-d-glucosyl-glycerol, in which a glucose molecule is linked to glycerol via an alpha-glycosidic linkage.