State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China.
Wuxi School of Medicine, Jiangnan University, Wuxi, 214122, China.
J Biotechnol. 2018 Sep 10;281:193-198. doi: 10.1016/j.jbiotec.2018.05.011. Epub 2018 May 22.
To improve the specific activity and catalytic efficiency of l-LcLDH1, 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, Lcldh1 and Lcldh1, were constructed by whole-plasmid PCR as designed theoretically, and expressed in E. coli BL21(DE3), respectively. The purified mutant, l-LcLDH1 or l-LcLDH1, 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) more than 99%. Their catalytic efficiencies (k/K) without d-fructose-1,6-diphosphate (d-FDP) were 4.8- and 5.2-fold that of l-LcLDH1. Additionally, the k/K values of l-LcLDH1 and l-LcLDH1 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-LcLDH1 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-LcLDH1.
为提高来源于干酪乳杆菌（Lactobacillus casei）的 NADH 依赖型变构 l-乳酸脱氢酶（l-LcLDH1）对苯丙酮酸（PPA）的比活性和催化效率，基于半理性设计对其进行定向改造。采用全质粒 PCR 技术分别构建了理论设计的三个突变基因 Lcldh1、Lcldh1 和 Lcldh1，并在大肠杆菌 BL21（DE3）中表达。纯化后的突变体 l-LcLDH1 和 l-LcLDH1 对 PPA 的比活性分别达到 451.5 和 512.4 U/mg，由此不对称还原 PPA 得到的 l-苯乳酸（PLA）的对映体过量值（ee）大于 99%。在无 d-果糖-1,6-二磷酸（d-FDP）时，其催化效率（k/K）分别是 l-LcLDH1 的 4.8 倍和 5.2 倍。此外，当有 d-FDP 存在时，l-LcLDH1 和 l-LcLDH1 的 k/K 值分别比无 d-FDP 时高 168.4 倍和 8.5 倍。通过分子对接（MD）模拟分析表明，用丙氨酸替代 l-LcLDH1 中的 I229 扩大了底物结合口袋的空间，而用精氨酸替代 Q88 使口袋入口比 l-LcLDH1 更大。
