D-扁桃酸脱氢酶底物选择性的分子机制及其理性改造

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.

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-扁桃酸脱氢酶在L-邻氯苯甘氨酸的生物合成中起关键作用，但是现有的D-扁桃酸脱氢酶对D-邻氯扁桃酸的催化活性极低，这在很大程度上制约了L-邻氯苯甘氨酸生物合成的发展。因此，为了消除邻氯扁桃酸盐的邻位氯取代对酶活性的抑制作用，提高对邻氯扁桃酸盐的生物催化性能，本项目以D-扁桃酸脱氢酶为主要研究对象，开展了以下四方面的研究工作：.1. 将短乳杆菌来源的D-扁桃酸脱氢酶LbDMDH进行了定向进化，成功获得了对D-邻氯扁桃酸具有更高催化活性的D-扁桃酸脱氢酶突变体LbDMDHN253S，其催化活性约为LbDMDH的29倍。.2. 以LbDMDH为探针，从哈尔滨乳杆菌中挖掘出1个新型的D-扁桃酸脱氢酶LhDMDH，其对D-扁桃酸的活性约为探针的4倍；对LhDMDH进行3轮实验室进化，获得了1个对D-邻氯扁桃酸活性显著提高的突变体LhDMDHA59G/N252G。.3. 借助多模板同源建模、分子对接和分子动力学模拟等技术，从氯原子的空间位阻、氢键作用及电子分布等方面探索了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。.4. 优化了全细胞催化D,L-扁桃酸制备L-苯甘氨酸的条件，在优化的最佳条件下，使300 mM D,L-扁桃酸，反应12 h，L-苯甘氨酸的得率和时空产率分别达到87.89%和79.70 g·L-1·d-1，明显高于优化前的产量。.本项目的实施，为加速实现L-邻氯苯甘氨酸高效的生物合成奠定坚实的基础，对丰富和完善酶工程基础理论及方法具有重要学术意义，而且对优化提升工程酶在生物化工先进制造中的效率和拓宽酶的工业应用范围具有实际应用前景。