Directed modification of l-LcLDH1, an l-lactate dehydrogenase from Lactobacillus casei, to improve its specific activity and catalytic efficiency towards phenylpyruvic acid

Abstract To improve the specific activity and catalytic efficiency of l - Lc LDH1, an NADH-dependent allosteric l -lactate dehydrogenase from L. casei , towards phenylpyruvic acid (PPA), its directed modification was conducted based on the semi-rational design. The three variant genes, Lcldh1 Q88R , Lcldh1 I229A and Lcldh1 T235G , were constructed by whole-plasmid PCR as designed theoretically, and expressed in E. coli BL21(DE3), respectively. The purified mutant, l - Lc LDH1 Q88R or l - Lc LDH1 I229A , displayed the specific activity of 451.5 or 512.4 U/mg towards PPA, by which the asymmetric reduction of PPA afforded l -phenyllactic acid (PLA) with an enantiomeric excess ( ee p ) more than 99%. Their catalytic efficiencies ( k cat / K m ) without d -fructose-1,6-diphosphate ( d -FDP) were 4.8- and 5.2-fold that of l - Lc LDH1. Additionally, the k cat / K m values of l - Lc LDH1 Q88R and l - Lc LDH1 I229A with d -FDP were 168.4- and 8.5-fold higher than those of the same enzymes without d -FDP, respectively. The analysis of catalytic mechanisms by molecular docking (MD) simulation indicated that substituting I229 in l - Lc LDH1 with Ala enlarges the space of substrate-binding pocket, and that the replacement of Q88 with Arg makes the inlet of pocket larger than that of l - Lc LDH1.