乳酸脱氢酶突变体D-LDHY52L在大肠杆菌中的重组表达及其合成D-苯基乳酸的研究

The recombinant expression of D-lactic dehydrogenase mutant D-LDHY52L in E. coli and its application in D-phenyllactic acid synthesis research

Lactobacillus pentosus D-乳酸脱氢酶的三维构象表明氨基酸序列中第52位的酪氨酸非常接近底物酮酸盐C3位上的基团。该研究利用定点突变技术将第52位的亲水性的酪氨酸替换成疏水性的亮氨酸,有效提高了D-乳酸脱氢酶对底物苯丙酮酸的催化活力。在大肠杆菌中重组表达后,利用重组菌全细胞转化苯丙酮酸合成D-苯基乳酸。结果表明,D-苯基乳酸的产量比未突变菌株提高了46%,产量达18.47 g/L,转化率达65.96%。

The three-dimensional structures of D-lactic dehydrogenase from Lactobacillus pentosus imply that the side chain of 52 Tyr of D-LDH can be located very near the C3 substituent group of 2-ketoacid substrate,thereby,associated with the recognition of substrate side chain. In this study,The replacement of hydrophilic Tyr 52 with hydrophobic Leu was done by the site-directed mutagenesis. The single amino acid replacement of Tyr52 with Leu significantly increased the affinity and activity toward phenylpyruvic acid. The mutant D-LDHY52 L was over-expressed in Eschrichia coli BL21（ DE3）. The whole cell transformation of PPA into D-PLA was researched in this study. The results showed that production of D-PLA was increased 46% by the mutant enzyme,compared to the native enzyme.The highest production of D-PLA was 18. 47 g / L in the fed-batch transformation,and the conversion ratio was65. 96%.