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

通过催化残基突变获得新功能的糖基化酶的研究。

• 批准号: 14360043
• 负责人: KIMURA Atsuo
• 金额: $ 9.41万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14360043/
• 关键词: transglycosidation; glycosylase; synthase

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.

糖基化酶是水解糖苷键的酶。这些酶还催化转糖苷作用，其中糖基残基转移到受体底物。转糖苷作用是生产具有食品价值的低聚糖和合成具有生物活性的糖链的重要反应。转糖苷和水解同时进行，这意味着即使在转糖苷的条件下，用于转糖苷的底物及其产物也通过水解裂解。研究了糖基化酶的反应，发现了只催化转糖苷反应的现象。在这项研究中，我们分析了有价值现象的机制并进行了它们的应用。结果总结如下。1）用硅化物底物法测定了带负电荷的催化残基。突变酶（合成酶），其中催化残基被取代，生产和纯化。该酶不发生水解反应，只催化氟底物和受体合成寡糖。芳基糖苷的受体是一个很好的底物，这意味着芳基基团和亚位点+2之间的疏水相互作用是重要的。2)构建了识别反应中间体模拟物平面底物的突变酶，并对其合成寡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。向反应混合物中加入乙醇也是有效的，但是高浓度的乙醇降低了寡糖的产生。我们发现了一种对酒精有抗性的糖苷酶。目前，将醇稳定酶转化为同类型突变酶的研究正在尝试中。

项目成果

Barley proteome analysis, starch degrading enzymes and proteinaceous inhibitors

大麦蛋白质组分析、淀粉降解酶和蛋白质抑制剂

• 发表时间: 2003
• 期刊: J.Appl.Glycosci. 50・2
• 作者: C.Finnie

J.Wongchawalit: "Purification and Properties of α-Glucosidase II from Japanese Honeybee, Apis cerna japonica"J.Appl.Glycosci.. 50・2. 297-298 (2003)

J.Wongchawalit：“日本蜜蜂 (Apis cerna japonica) 的 α-葡萄糖苷酶 II 的纯化和性质”J.Appl.Glycosci.. 50・298 (2003)。

Characterization, and Sequence Analysis of Two α-Amylase Isoforms from Azuki Bean, Vigna angularis, Showing Different Affinity towards β-Cyclodextrin

红豆、绿豆中两种对 β-环糊精表现出不同亲和力的 α-淀粉酶异构体的表征和序列分析

• 发表时间: 2003
• 期刊: Biosci.Biotechnol.Biochem. 67・5
• 作者: S.S.Mar

Involvement of individual subsites and secondary substrate binding sites in multiple attack on amylose by barley α-amylase.

单个亚位点和次级底物结合位点参与大麦 α-淀粉酶对直链淀粉的多重攻击。

• 发表时间: 2005
• 期刊: Biochemistry 44(6)
• 作者: Kramhoft B;Bak-Jensen KS;Mori H;Juge N;Nohr J;Svensson B
• 通讯作者: Svensson B

Purification, characterization, and sequence analysis of two α-amylase isoforms from azuki bean, Vigna angularis, showing different affinity towards β-cyclodextrin.

红豆、绿豆中两种 α-淀粉酶亚型的纯化、表征和序列分析，显示出对 β-环糊精的不同亲和力。

• 发表时间: 2003
• 期刊: Biosci Biotechnol Biochem 67(5)
• 作者: Mar SS;Mori H;Fukuhara A;Okuyama M;Saburi W;Fukuda K;Lee JH;Chiba S;Kimura A
• 通讯作者: Kimura A