Specificity analysis of a glycosyl hydrolase

The Bacillus sp.SAM1606α-glucosidase catalyzes transglucosylation of sucrose to produce three regioisomers of the glucosylsucroses，with theanderose(6-O怡D1G齐埃D1-glucosylsucrose)as the most abundant transfer product。To find the active-site amino acid residues which can affect the reactivity and regiospecificity of the glucosyl transfer，16mutants with amino acid substitutions near the active site were reacted with 1.75M sucrose at 60℃，pH6.0，and the course of transglucosylation as well as the product specificity were analyzed。The sites of the amino acid substitutions were selected by comparing the conserved amino acid sequences located near the active site of the SAM1606enzyme with those of the Bacillus oligo-1，6-glucosidases(O16G)，which have very high amino acid sequence similarities near the active site but have a distinct substrate specificity。The results showed that，among the mutated SAM1606enzymes examined，only the mutants with the substitution of Gly273 with Pro showed an altered reactivity and specificity of transglucosylation；these mutants exhibited a significantly enhanced initial velocity of glucosyl transfer，yielding isomelezitose(6-O I D1-glucosylsucrose)instead of theanderose as the jmaor transfer product。These results indicate that the substitution of Gly273 with Pro critically governs the enhanced reactivity and altered specificity of the transglucosylation。The notion that amino acid residue at this position is the determinant of the glucosyl-transfer specificity was further confirmed by observation that the B.cereus O16G，which has a proline at the corresponding position，produced isomelezitose as the major transfer product during transglucosylation with sucrose.

The Bacillus sp. SAM1606 α-glucosidase catalyzes transglucosylation of sucrose to produce three regioisomers of the glucosylsucroses, with theanderose (6-OィイD1GィエD1-glucosylsucrose) as the most abundant transfer product. To find the active-site amino acid residues which can affect the reactivity and regiospecificity of the glucosyl transfer, 16 mutants with amino acid substitutions near the active site were reacted with 1.75M sucrose at 60℃, pH 6.0, and the course of transglucosylation as well as the product specificity were analyzed. The sites of the amino acid substitutions were selected by comparing the conserved amino acid sequences located near the active site of the SAM1606 enzyme with those of the Bacillus oligo-1,6-glucosidases (O16G), which have very high amino acid sequence similarities near the active site but have a distinct substrate specificity. The results showed that, among the mutated SAM1606 enzymes examined, only the mutants with the substitution of Gly273 with Pro showed an altered reactivity and specificity of transglucosylation; these mutants exhibited a significantly enhanced initial velocity of glucosyl transfer, yielding isomelezitose (6-OィイD1FィエD1-glucosylsucrose) instead of theanderose as the major transfer product. These results indicate that the substitution of Gly273 with Pro critically governs the enhanced reactivity and altered specificity of the transglucosylation. The notion that amino acid residue at this position is the determinant of the glucosyl-transfer specificity was further confirmed by observation that the B. cereus O16G, which has a proline at the corresponding position, produced isomelezitose as the major transfer product during transglucosylation with sucrose.