Biosynthesis of Glucosyl Glycerol, a Compatible Solute, Using Intermolecular Transglycosylation Activity of Amylosucrase from Methylobacillus flagellatus KT

A putative α-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 α-amylase. The optimal reaction temperature and pH for the sucrose hydrolysis activity of MFAS were determined to be 45 °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 °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 1 H and 13 C NMR, their chemical structures were determined to be (2S)-1- O -α- d -glucosyl-glycerol or (2R)-1- O -α- d -glucosyl-glycerol and 2- O -α- d -glucosyl-glycerol, in which a glucose molecule is linked to glycerol via an α-glycosidic linkage.