米曲霉果糖基转移酶溶液结构、催化机制及分子改造研究

项目名称： 米曲霉果糖基转移酶溶液结构、催化机制及分子改造研究

项目编号： No.21476216

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 有机化学

项目作者： 毛多斌

作者单位： 郑州轻工业学院

项目金额： 82万元

中文摘要： 酶在溶液中三维结构及其催化机制是当今酶催化研究前沿科学问题。米曲霉果糖基转移酶溶液结构至今尚未得到解析，因此溶液中该酶与反应底物识别及转糖基反应机制并不清楚。本课题以米曲霉果糖基转移酶AoFT催化蔗糖与葡萄糖-6-乙酸酯生成蔗糖-6-乙酸酯为模式反应，采用异核多维液相核磁共振技术解析AoFT溶液结构及其与底物的相互作用，结合生化突变、圆二色谱、荧光色谱和差示扫描量热方法，分析其结构域功能，确定AoFT与底物结合的活性位点精细结构，揭示溶液中AoFT催化底物的识别机制；通过研究AoFT主链动力学性质，深入分析其溶液构象的动态变化与动力学之间关系，阐明溶液中AoFT催化作用机制；在获得AoFT三维结构基础上进行分子设计，拓宽AoFT对底物选择性，进而提高酶催化效率。研究成果将揭示果糖基转移酶溶液结构及催化机制，在糖基转移酶定向改造以及糖基化学品生物合成等方面具有重要理论意义和使用价值。

中文关键词： 溶液结构；动态结构；主链动力学；催化机制；分子改造

英文摘要： Enzymatic three-dimensional structure in solution and catalytic mechanism is the frontier scientific issues in the fields of biological catalysis. Solution structure of the fructosyltransferase from Aspergillus oryzae have not been resolved, so substrate recognition and transglycosylation reaction mechanism of the enzyme in solution are not clear. In this study, the synthesis of sucrose-6-acetate from sucrose and glucose-6-acetate catalyzed by the fructosyltransferase AoFT will be as a model reaction, and detailed studys on the solution structure and the mechanism of substrate recognition will be carried out by liquid-state heteronuclear and multidimensional NMR chromatography to clarify domain functions, active sites and mechanisms of substrates recognition of AoFT, combined with biochemical mutation, circular dichroism, fluorescence spectroscopy and different scanning calorimetry. Through the analysis of the backbone dynamics of AoFT, the relations between dynamics changes of its conformation and reaction kinectics in solution will be established, and catalytic mechanisms of AoFT in solution will be elucidated. Molecular design will be performed based on the 3D structure of AoFT in order to broaden the substrate selectivity and improve the catalytic efficiency of the enzyme. The research results will clarify the catalytic mechanism of fructosyltransferases, and have important theoretical significance and practice value for directed designs of the glycosyltransferase and biosynthesis of glycosyl-based production of chemicals.

英文关键词： solution structure;dynamic structure;backbone dynamics;catalytic mechanism;molecular modification