基于理性设计的新型菜豆环氧化物水解酶定向改造

Chiral vicinal diols are widely applied in the synthesis of pharmaceuticals, fine chemicals and so on. The preparation of chiral vicinal diols has attracted much attention by epoxide hydrolases (EHs) via enantioconvergent hydrolysis of racemic epoxides, having characteristics of the high enantiomeric purity and yield of products and an environmentally friendly process. Currently, only few EHs can catalyze the enantioconvergent hydrolysis alone, but usually the catalytic effect was unsatisfactory. How to excavate novel EHs or perform modification towards existing EHs is the key to break the bottleneck of their industrial development. In this project, a novel EH from Phaseolus vulgaris (PvEH1) is used as a research object. Firstly, based on computer-aided rational design, the site-saturation and iterative-saturation mutagenesis of several key amino acids around the substrate binding pocket of PvEH1 are carried out to construct the primary-, secondary- and multi-stage PvEH1 mutant libraries. Secondly, using styrene oxide, 1,2-epoxyhexane and phenyl glycidyl ether as model substrate molecules, 6-9 mutants with high catalytic activity and enantioconvergence are obtained via high-throughput screening. Then, the hydrolytic processes are optimized to prepare chiral vicinal diols by excellent PvEH1 mutants. Finally, the molecular mechanism of the improved catalytic activity and enantioconvergence of mutants is resolved by means of molecular docking and dynamics simulation along with the determination of catalytic properties. This project is expected to promote the industrial development of PvEH1 mutants, and also provides a new idea of directed modification for other EHs or enzymes.

手性邻二醇在合成药物和精细化学品等方面用途广泛。采用环氧化物水解酶(EHs)催化外消旋环氧化物对映归一性水解制备手性邻二醇，具有产物对映体纯度和产率高以及对环境友好等优点而备受关注。目前仅有极少数EHs能单一酶催化底物对映归一性水解，但效果不尽人意，如何挖掘新型EHs或对现有EHs进行改造，是突破该酶产业发展瓶颈的关键。本项目拟以新型菜豆环氧化物水解酶(PvEH1)为研究对象，基于计算机辅助的理性设计，对其底物结合口袋周围关键氨基酸进行定点和迭代饱和突变，构建一级、二级和多级突变酶文库；针对3种模式底物分子进行高通量筛选，获得催化活性和对映归一性高的6-9个突变酶，并优化制备手性邻二醇的水解工艺；借助分子对接和动力学模拟等手段并结合催化特性测定，解析突变酶催化活性和对映归一性提高的分子机制。本研究成果将促进PvEH1突变酶产业化生产和应用的发展，并为其它EHs或酶的定向改造提供新思路。

对映纯环氧化物和邻二醇广泛应用于药物和精细化学品等的合成。采用环氧化物水解酶(EHs, EC 3.3.2.-) 催化外消旋环氧化物不对称水解制备手性环氧化物和/或邻二醇，具有产物光学纯度和产率高以及对环境友好等优点而备受关注。本项目重点围绕菜豆环氧化物水解酶PvEH1，对多种植物来源的8种EHs展开了研究：. 1) 植物EHs的基因克隆、表达和酶学特性。以PvEH1为模板，在GenBank数据库中挖掘了菜豆PvEH2、PvEH3和PvEH4，绿豆VrEH3，番茄SlEH1和SlEH2及大豆GmEH3。采用分子生物学实验技术，将8种植物EHs在大肠杆菌中实现了可溶性表达，并对重组EHs的酶学特性和底物谱进行了系统的研究。. 2) EHs定向改造的理性设计。构建了包含多序列比对、三维结构同源建模、分子对接和分子动力学模拟等的理性设计策略，分析了植物EHs底物结合口袋独特的双底物通道 (tunnel 1和2)，确定了结合口袋中“热点”氨基酸位点。基于该理性设计策略，不仅实现了PvEH1对映归一性或对映选择性的定向改造，还通过对其他植物EHs的定向改造论证了该理性设计的可行性。. 3) 优良突变酶的获得和催化特性。针对芳香族环氧乙烷、缩水甘油醚和脂肪族环氧乙烷这3类底物，采用定点突变、定点或迭代饱和突变及组合突变等手段，完成了一级、二级和多级EH突变体文库的构建和高通量筛选，获得了10个以上针对不同底物的高对映归一性或对映选择性的突变酶。采用动力学拆分和对映归一性水解，高效制备了多种对映纯环氧化物和/或邻二醇。. 4) EHs对映归一性或对映选择性提高的分子机制。采用同源建模构建了野生型酶和突变酶的三维结构，揭示了突变酶的结构改变与其催化特性变化之间的联系；运用AutoDock程序将野生型酶或突变酶分别与(R)-和(S)-环氧化物进行分子对接，再运用Discovery Studio软件对酶与底物的对接复合物在300 K下进行分子动力学模拟，解析突变酶对映归一性或对映选择性提高的分子机制。. 本项目经过适当的调整和拓展，圆满完成了预定的研究内容，所获成果将促进环氧化物水解酶产业化生产和应用的发展。