Synthesis of Glucosyltrehaloses by a Thermostable α-Glucosidase with Broad Substrate Specificity and High Transglucosylation Activity

Assessment Using Wild-type Enzyme and Mutants with Amino Acid Substitution near the Active Site

Abstract

Glucosyltrehaloses, such as 6-O-α-glucosyltrehalose, have both anti-dental caries activities and growth-promoting effects on the genus Bifidobacterium, which plays important roles in improving human fecal flora. Here we show the enzymatic formation of glucosyltrehaloses from trehalose as a sole substrate (glucosyl donor and acceptor) using a thermostable α-glucosidase from Bacillus sp. SAM1606. This enzyme has high transglucosylation activity and broad substrate specificity, and can efficiently act on trehalose. Reaction of the enzyme with 1.32M trehalose at 60°C and pH 6.0 yielded a significant amount of tri- and tetrasaccharides, which were found to be 6-O-α-glucosyltrehalose and 6-O-α-isomaltosyltrehalose, respectively, by MS and NMR analyses. We also analyzed the effect of mutations at position 273 of the enzyme on the reactivity and specificity of transglucosylation. All mutant enzymes showed reduced glucosyl transfer activity; in particular, mutants substituted with Lys, Arg, His, Phe, Trp, or Tyr showed virtually no glucosyl transfer activity to trehalose.