Specificity analysis of a glycosyl hydrolase

Bacillus sp.SAM1606 α-葡萄糖基化酶催化剂的转化sucrose to produce三个regioisomers of the glucoses， with theanderose （6-O-D1 G-D1-葡萄糖基化酶催化剂）作为最基本的转移产品。要找到活性位点氨基酸替代方案，其中可以影响葡萄糖基转移的反应性和地理性，在活性位点附近发生1.75 M蔗糖反应时， 16个突变体的氨基酸替代物在60 ° C和pH值为6.0的情况下，并采用转化糖基化的途径作为分析的产品特性良好。选定的氨基酸含量位点是由比较所需的氨基酸序列位置附近的SAM 1606酶活性位点与那些芽孢杆菌寡核苷酸-1，6-葡萄糖苷（O 16 G）的活性位点，在活性位点附近具有非常高的氨基酸序列相似性，但有一个不同的物质特性。结果显示，在测试了突变的SAM 1606酶时，只有带有Gly 273的替代品的突变体和专业展示了转换糖基化的改变反应能力和特性;这些突变体显示了葡萄糖基转移的显著增强的初始速度，生长异构体（6-O-D1 F-D1-葡萄糖基化）作为主要转移产品。这些结果表明，Gly 273的替代品以正确的方式由过渡糖基化的增强反应能力和改变的特性管理。在这一位置上，氨基酸居留的运动是对葡萄糖基转移特性的决定性的进一步确认，即B.。Cereus O 16 G，其中有一个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.