To enhance the specific activity and catalytic efficiency (k_cat/K_m) of an NADH-dependent LpPPR, its directed modification was performed based on the computer-aided design using molecular docking simulation and multiple sequence alignment. Firstly, five single-site variants of an LpPPR-encoding gene (lpppr) were amplified and expressed in E. coli BL21 (DE3). The asymmetric reduction of 20 mM phenylpyruvic acid (PPA) was carried out using 50 mg/mL E. coli/lpppr^R53Q or /lpppr^A79V whole wet cells at 37 °C for 20 min, giving d-phenyllactic acid (PLA) with 41.1 or 44.3% yield, being 1.17- or 1.26-fold that by E. coli/lpppr. Secondly, double-site variants were obtained by saturation mutagenesis of Ala79 in LpPPR^R53Q. Among all tested E. coli transformants, E. coli/lpppr^R53Q/A79V exhibited the highest d-PLA yield of 85.3%. The specific activity and k_cat/K_m of the purified LpPPR^R53Q/A79V increased to 67.5 U/mg and 169.8 mM⁻¹ s⁻¹, which were 3.0- and 13.2-fold those of LpPPR, respectively. Finally, the catalytic mechanism analysis of LpPPR^R53Q/A79V by molecular docking simulation indicated that the replacement of Arg53 in LpPPR with Gln expanded its substrate-binding pocket, while that Ala79 with Val formed an additional π-sigma interaction with phenyl group of PPA.

为了提高NADH依赖性Lp PPR的比活性和催化效率（k _cat / K _m ），基于计算机辅助设计，使用分子对接模拟和多序列比对进行定向修饰。首先，在大肠杆菌BL21 (DE3)中扩增并表达了Lp PPR 编码基因 ( lpppr )的五个单位点变体。使用 50 mg/mL大肠杆菌/ lpppr ^R53Q或/ lpppr ^A79V全湿细胞在 37 °C 下对20 mM 苯基丙酮酸 (PPA) 进行不对称还原20 分钟，得到d-苯乳酸 (PLA) 的收率为 41.1 或 44.3%，是大肠杆菌/ lpppr的 1.17 或 1.26 倍。其次，通过对Lp PPR ^R53Q中的Ala79进行饱和诱变获得双位点变体。在所有测试的大肠杆菌转化体中，大肠杆菌/ lpppr ^R53Q/A79V的d -PLA 产率最高，为 85.3%。纯化的Lp PPR ^R53Q/A79V的比活性和k _cat / K _m增加到 67.5 U/mg 和 169.8 mM ^-1  s ^-1，分别是Lp PPR的 3.0 倍和 13.2 倍。最后，通过分子对接模拟对Lp PPR ^R53Q/A79V的催化机理分析表明，Lp PPR 中的 Arg53 被Gln 取代扩大了其底物结合口袋，而 Ala79 与 Val 形成了额外的 π-sigma 相互作用与苯基购电协议。