D-扁桃酸脱氢酶能催化D-扁桃酸及D-卤代扁桃酸的氧化，在氯吡格雷中间体生物合成中起关键作用。现有D-扁桃酸脱氢酶对D-邻氯扁桃酸催化活性较低，且其影响机制尚不明确，限制了它的应用范围。本项目拟以前期获得的高活性D-扁桃酸脱氢酶LhDMDH为亲本：（1）开展半理性设计辅助的定向进化、结构解析、分子对接和分子动力学模拟，系统研究底物中氯的取代位置对D-扁桃酸脱氢酶活性的影响，并阐明其分子机制；（2）通过理性设计及多点定点突变等技术，对酶的底物选择性进行理性改造，获得更高D-邻氯扁桃酸活性的突变体。本项目有望揭示D-氯代扁桃酸氯的取代位置影响D-扁桃酸脱氢酶活性的分子机制，拓宽D-扁桃酸脱氢酶的应用范围，为其它酶底物选择性的改造提供新思路，对丰富和完善酶工程基础理论及方法具有重要学术意义。

D-mandelate dehydrogenase (DMDH) can catalyze the oxidation of D-mandelic acid and D- halogenated mandelic acid, and plays key role in biosynthesis of clopidogrel intermediate. However, the existing DMDHs have very low activity against D-o-chloromandelic acid, and its mechanism is still unclear. Therefore, its application is limited. To overcome the bottleneck, some strategies will be adopted to explore the influence mechanisms and improve its catalytic properties. The LhDMDH obtained by our preliminary study has high catalytic activity, and will be used as parent in this project. Firstly, molecular mechanism of chloride substitution position affecting catalytic activity of D-mandelate dehydrogenase will be systemically explored by some advanced strategies, including semi-rational design aided directed evolution, crystal structure determination, molecular docking and molecular dynamics simulation. Secondly, the further rational modification for improving catalytic activity on D-o-chloromandelic acid will be implemented by rational design and multi site-directed mutagenesis. This project is expected to reveal the molecular mechanism of chloride substitution position affecting catalytic activity of D-mandelate dehydrogenase, and broaden the application of DMDH, which may provide a new strategy to modify the substrate selection of enzyme. In addition, this project has prominent academic significance to develop the basic theories and methods of biochemical engineering and enzyme engineering.