以1,4-丁二醇为辅底物再生NADPH的醇脱氢酶的构建及催化机制研究

项目名称： 以1,4-丁二醇为辅底物再生NADPH的醇脱氢酶的构建及催化机制研究

项目编号： No.21506073

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 有机化学

项目作者： 许国超

作者单位： 江南大学

项目金额： 21万元

中文摘要： 醇脱氢酶催化的不对称还原是合成手性仲醇最具应用潜力的绿色途径之一，具有底物偶联型辅因子再生系统的醇脱氢酶可便捷地实现底物的还原和辅因子在位再生，而备受工业青睐。相比异丙醇，1,4-丁二醇作为这类酶的“智能”辅底物因含两个羟基可实现辅因子高效、绿色再生。但仅少数酶可将其氧化以再生NADH，且无NADPH再生的报道，辅因子循环的机制也不明确，因此亟待研究和阐明。本项目拟以申请人前期获得的来源于Kluveromyces polyspora的醇脱氢酶KpADH为研究对象，系统研究参与辅底物结合的氨基酸位点；对结合位点进行组合饱和突变和DNA改组，高通量筛选获得兼具氧化1,4-丁二醇再生NADPH和高对映选择性的突变酶；结合晶体结构、反应动力学和光谱学结果，揭示酶与辅底物的识别规律，阐明催化机制。项目将提供一种以1,4-丁二醇为辅底物高效再生NADPH的工具酶，并为辅因子再生系统的改造提供理论指导。

中文关键词： 醇脱氢酶；底物偶联型辅因子再生；1；4-丁二醇；蛋白质工程；催化机制

英文摘要： Biocatalytic asymmetric reduction employing alcohol reductases is a green and promising alternative for the synthesis of chiral secondary alcohol. The alcohol dehydrogenases harboring substrate-coupled cofactor regeneration system are industrially preferable due to its ability in elegant achievement the asymmetric reduction of prochiral ketone and cofactor regeneration in situ. Compared with isopropanol, 1,4-butanediol, having two free hydroxyl groups, could work as a ‘smart’ co-substrate for these alcohol dehydrogenases in the efficient and green regeneration of cofactor. However, only several alcohol dehydrogenases were reported with the ability in regeneration of NADH by oxidation of 1,4-butanediol and no NADPH-dependent dehydrogenases was discovered. And also we have little knowledge yet for the catalytic mechanism of this substrate-coupled cofactor regeneration. The proposed project plans first to investigate the relationship between co-substrate and residues in the binding pocket from newly mined alcohol dehydrogenase, KpADH from Kluveromyces polyspora, to get the hotspots for mutation. Through the high-throughput screening of the combinatorial saturation and DNA shuffling libraries of the hotspots, variants possessed the ability in regeneration of NADPH with 1,4-butanediol and retained the high enantioselectivity in the reduction of substrate are returned The interactions of residues and 1,4-butanediol and the molecular mechanism of NADPH regeneration are uncovered via analysis the structures of enzyme-substrate complex, reaction kinetics and spectroscopy data. The results will provide biocatalyst for efficient NADPH regeneration with 1,4-butanediol as co-substrate and the theoretical basis for the molecular engineering of the cofactor regeneration of the substrate-coupled alcohol dehydrogenase.

英文关键词： Alcohol dehydrogenase;Substrate-coupled cofactor regeneration;1;4-butanediol;Protein engineering;Catalytic mechanism