定点饱和突变提高Lactobacillus casei L-乳酸脱氢酶对苯丙酮酸的催化效率

Improving catalytic efficiency of an Lactobacillus casei L-lactate dehydrogenase for phenylpyruvic acid production by site-saturated mutagenesis

摘要:

【背景】光学纯L-苯乳酸是一种天然防腐剂，也是一种高附加值的手性分子，在食品、制药和材料等领域有广阔的应用前景。本实验室已发现来源于Lactobacillus casei CICIM B1192的NADH依赖型L-乳酸脱氢酶(L-LcLDH)可不对称还原苯丙酮酸制备L-苯乳酸，但其活性较低。为提高L-LcLDH催化苯丙酮酸的催化效率，构建了一个单突变体L-LcLDHQ88R，其催化效率kcat/Km是L-LcLDH的4.9倍。【目的】为进一步提高L-LcLDHQ88R催化苯丙酮酸的催化效率，采用饱和突变技术将位于L-LcLDHQ88R底物结合口袋附近的氨基酸残基Ile229随机替换为其他氨基酸，以获得活性更高的优良突变体。【方法】以重组表达质粒pET-22b-LcldhQ88R为模板，采用全质粒PCR技术对L-LcLDHQ88R基因(LcldhQ88R)中编码Ile229的密码子实施饱和突变，构建突变转化子文库。以催化苯丙酮酸的活性为指标，从文库中筛选出优良的突变转化子。【结果】突变转化子(Escherichia coli/LcldhQ88R/I229Q)表达出一种由Arg和Gln分别替换了Gln88和Ile229的双突变体L-LcLDHQ88R/I229Q。重组表达产物L-LcLDHQ88R/I229Q的酶学性质分析表明：L-LcLDHQ88R/I229Q的比活性是L-LcLDH的18.5倍，是L-LcLDHQ88R的2.3倍；其催化效率分别为后两者的6.8倍和1.4倍。L-LcLDH突变前后的温度和pH特性改变不大。根据分子对接结果推测出，双突变Q88R/I229Q导致L-LcLDH的底物结合口袋的入口变大和构型的变化可能对其催化活性的提高发挥了重要作用。【结论】双突变Q88R/I229Q显著提高了L-LcLDH的活性和催化效率，使得L-LcLDHQ88R/I229Q在不对称还原苯丙酮酸制备L-苯乳酸中成为有潜力的工具酶。

Abstract:

[Background] Optically pure L-phenyllactic acid (L-PLA) is a natural antimicrobial agent and also a highly value-added chiral compound with potential applications in food, pharmaceutical and biomaterial areas. Although L-PLA can be produced by asymmetric reduction of phenylpyruvic acid (PPA) by L-LcLDH, an L-lactate dehydrogenase from Lactobacillus casei CICIM B1192, it displays low activity. To improve the activity of a wild-type L-LcLDH towards PPA, an L-LcLDH mutant, L-LcLDHQ88R, was successfully constructed by our lab. Its catalytic efficiency (kcat/Km) was 4.9-fold higher than that of L-LcLDH. [Objective] To further improve the catalytic efficiency of L-LcLDHQ88R towards PPA, the amino acid Ile229 located in the substrate-binding pocket of L-LcLDHQ88R or L-LcLDH was randomly substituted with any one of 20 amino acids. [Methods] Using a recombinant plasmid pET-22b-LcldhQ88R as the template, the Ile229-encoding codon in LcldhQ88R was subjected to site-saturated mutagenesis by whole-plasmid PCR technique. Then, the mutant library of L-LcLDHQ88R was constructed by transforming pET-22b-LcldhQ88R variants into E. coli BL21(DE3), respectively. Finally, an E. coli transformant expressing the highest activity towards PPA was screened from this library. [Results] The selected transformant, E. coli/LcldhQ88R/I229Q, contains a double-mutant gene in which the Gln88- and Ile229-encoding codons in Lcldh was substituted with Arg- and Gln-encoding ones, respectively. The specific activity of purified L-LcLDHQ88R/I229Q towards PPA was 18.5- and 2.3-fold those of L-LcLDH and L-LcLDHQ88R, while its catalytic efficiency was 6.8- and 1.4-folds those of the latter two, respectively. Additionally, the pH and temperature properties of L-LcLDHQ88R/I229Q did not obviously changes compared to L-LcLDH. The analysis of catalytic mechanism by molecular docking (MD) simulation showed that the double-mutation of Q88R/I229Q enlarges the inlet of substrate-binding pocket and alters the steric structure of active centre, which may contribute to the increase of activity. [Conclusion] The double-mutation of Q88R/I229Q in L-LcLDH primary structure greatly enhanced its specific activity and catalytic efficiency, making L-LcLDHQ88R/I229Q a promising candidate for asymmetric reduction of PPA.