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-LcLDH^(Q88R),其催化效率kcat/Km是L-LcLDH的4.9倍。【目的】为进一步提高L-LcLDH^(Q88R)催化苯丙酮酸的催化效率,采用饱和突变技术将位于L-LcLDH^(Q88R)底物结合口袋附近的氨基酸残基Ile^(229)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p ET-22b-LcldhQ88R为模板,采用全质粒PCR技术对L-LcLDH^(Q88R)基因(LcldhQ88R)中编码Ile^(229)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/Lcldh^(Q88R/I229Q))表达出一种由Arg和Gln分别替换了Gln88和Ile^(229)的双突变体L-LcLDH^(Q88R/I229Q)。重组表达产物L-LcLDH^(Q88R/I229Q)的酶学性质分析表明:L-LcLDH^(Q88R/I229Q)的比活性是L-LcLDH的18.5倍,是L-LcLDH^(Q88R)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-LcLDH^(Q88R/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。
[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, pharmaceuticaland biomaterial areas. Although L-PLA can be produced by asymmetric reduction of phenylpyruvicacid（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-LcLDH（Q88R）, was successfully constructed by our lab. Its catalytic efficiency（kcat/Km） was4.9-fold higher than that of L-LcLDH. [Objective] To further improve the catalytic efficiency ofL-LcLDH（Q88R） towards PPA, the amino acid Ile（229） located in the substrate-binding pocket ofL-LcLDH（Q88R） or L-LcLDH was randomly substituted with any one of 20 amino acids. [Methods]Using a recombinant plasmid p ET-22 b-LcldhQ88 R as the template, the Ile（229）-encoding codon inLcldhQ88 R was subjected to site-saturated mutagenesis by whole-plasmid PCR technique. Then, themutant library of L-LcLDH（Q88R） was constructed by transforming p ET-22 b-LcldhQ88 R 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/Lcldh（Q88R/I229Q）, contains a double-mutant gene in which the Gln88-and Ile（229）-encoding codons in Lcldh wassubstituted with Arg-and Gln-encoding ones, respectively. The specific activity of purifiedL-LcLDH（Q88R/I229Q） towards PPA was 18.5-and 2.3-fold those of L-LcLDH and L-LcLDH（Q88R）, whileits catalytic efficiency was 6.8-and 1.4-folds those of the latter two, respectively. Additionally, thepH and temperature properties of L-LcLDH（Q88R/I229Q） did not obviously changes compared toL-LcLDH. The analysis of catalytic mechanism by molecular docking（MD） simulation showed thatthe double-mutation of Q88 R/I229 Q enlarges the inlet of substrate-binding pocket
作者
李雪晴
刘艳
袁风娇
李剑芳
邬敏辰
LI Xue-Qing;LIU Yan;YUAN Feng-Jiao;LI Jian-Fang;WU Min-Chen(School of Food Science and Technology,Jiangnan University,Wuxi,Jiangsu 214122,China;Wuxi School of Medicine,Jiangnan University,Wuxi,Jiangsu 214122,China)
出处
《微生物学通报》
CAS
CSCD
北大核心
2018年第7期1401-1407,共7页
Microbiology China
基金
国家自然科学基金(21676117)~~
