Research on Glycosylases which Obtained New Functions by Mutation on Catalytic Residue.

糖基化酶是水解糖苷键的酶。这些酶也催化转糖苷化，其中糖基残基转移到受体底物上。转糖苷化是一种重要的反应，一是生产有食品价值的低聚糖，二是合成具有生物活性的糖链。转糖苷化和水解是同时进行的，这意味着即使在转糖苷化的条件下，转糖苷化的底物及其产物也会被水解裂解。对糖基化酶的反应进行了研究，发现了只催化转糖苷化的现象。在本研究中，我们分析了有价值现象的机理并进行了应用。结果总结如下：1)采用自杀底物法测定了负电荷催化残渣。制备并纯化了取代催化残基的突变体酶（合酶）。该酶无水解反应，仅催化氟-底物和受体合成低聚糖。芳基糖苷受体是一个很好的底物，这意味着芳基与亚位+2之间的疏水相互作用是重要的。2)构建了能识别平面底物（反应中间体的模拟化合物）的突变酶，并对其合成低聚糖的能力进行了研究。观察到产量低。我们改变了底物浓度，成功地提高了产率。在反应混合物中加入醇也是有效的，但高浓度的醇降低了低聚糖的产量。我们发现了一种抗酒精的糖苷酶。目前，醇稳定型酶向同类型突变型酶的转化还处于尝试阶段。

Glycosylases are enzymes that hydrolyze the glycosidic linkage. These enzymes also catalyze the transglycosidation, in which the glycosyl residue is transferred to the acceptor substrate. The transglycosidation is an important reaction i)to produce oligosaccharides valuable for foods and ii)to synthesize bio-active sugar-chains. Transglycosidation and hydrolysis proceed in the same time, meaning that the substrate for transglycosidation as well as its product(s) is cleaved by hydrolysis even under conditions of transglycosidation. We have studied the reactions of glycosylase, and have found the phenomena that catalyzed the transglycosidation only. In this study, we analyze the mechanism of valuable phenomena and perform their application. The results are summarized as follows. 1)We have determined the catalytic residue of negatively charged by the method using suicide substrate. Mutant enzyme (synthase), of which catalytic residue was replaced, was produce and purified. The enzyme showed no hydrolytic reaction, only catalyzed the synthesis of oligosaccharide(s) from fluoride-substrate and acceptor. Acceptor of aryl glycoside is a good substrate, meaning that the hydrophobic interaction between aryl-group and subsite +2 is important. 2)Mutant enzyme, which recognized the plane-shaped substrate, a mimic compound of reaction intermediate, was constructed, and its ability of oligosaccharide-synthesis was studied. The low production was observed. We changed the substrate concentration, and succeeded in the improvement of yield. Addition of alcohol to reaction mixture was also effective, but the high concentration of alcohol decreased the production of oligosaccharide. We have found a glycosidase resistant for alcohol. Currently, the conversion of alcohol-stable enzyme to mutant enzyme of same type is trying.