不对称还原苯丙酮酸的L-乳酸脱氢酶L-LcLDH2的表达及生物信息学分析  

Expression and Bioinformatic Analysis of an L-Lactate Dehydrogenase(L-LcLDH2)for the Asymmetric Reduction of Phenylpyruvic Acid

作　　者：李雪晴[1] 袁风娇[1] 刘艳 李剑芳[1] 邬敏辰 LI Xueqing;YUAN Fengjiao;LIU Yan;LI Jianfang;WU Minchen(School of Food Science and Technology,Jiangnan University,Wuxi 214122,China;Wuxi School of Medicine,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学食品学院,江苏无锡214122 [2]江南大学无锡医学院,江苏无锡214122

出　　处：《食品与生物技术学报》2019年第12期25-30,共6页Journal of Food Science and Biotechnology

基　　金：国家自然科学基金项目(21676117)。

摘　　要：以干酪乳杆菌(Lactobacillus casei)基因组DNA为模板,采用PCR技术扩增一种L-乳酸脱氢酶(L-LcLDH2)的编码基因Lcldh2;借助表达质粒pET-28a(+)将Lcldh2在大肠杆菌BL21(DE3)中实施异源表达。实验结果表明,细胞超声破碎液中L-LcLDH2催化苯丙酮酸的酶活性为47 U/mg总蛋白质;采用重组E.coli/Lcldh2全细胞催化苯丙酮酸还原,所获产物L-苯乳酸的对映体过量(eep)值>99%。生物信息学分析表明,Lcldh2开放阅读框长906 bp,编码含301个氨基酸的L-LcLDH2,预测其相对分子质量和等电点分别为32585和5.5;L-LcLDH2含有典型的NAD+结合位点序列(GXGXXG),属于NAD依赖型L-乳酸脱氢酶,且是一种非分泌、非跨膜、无信号肽和定位于细胞质的酶蛋白。L-LcLDH2的获得为生物法制备高光学纯L-苯乳酸提供了新的酶源。Using the genomic DNA from Lactobacillus casei CICIM B1192 as the templet,we amplified the coding gene Lcldh2 by PCR technique,which encodes an L.casei L-lactate dehydrogenase(L-LcLDH2).Then,Lcldh2 was successfully expressed in E.coli BL21(DE3)mediated by an expression vector pET-28a(+).Using PPA as the substrate,L-LcLDH2 displayed the enzymeactivity of 47 U/mg,by which the asymmetric reduction of the substrate PPA afforded L-PLA with enantiomeric excess(eep)of more than 99%.Bioinformatic prediction revealed that Lcldh2 is 906 bp in length,encoding 301 amino acids.Theoretical relative molecular mass and isoelectric point of L-LcLDH2 is 32585 and 5.5,respectively.L-LcLDH2 is a stable cytoplasmic protein without signal peptide and has a highly conserved sequence GXGXXG.The acquisition of L-LcLDH2 has provided a new biocatalyst for the preparation ofhighly optically pureL-phenyllactic acid.

关 键 词：LACTOBACILLUS CASEI L-乳酸脱氢酶 苯丙酮酸 不对称还原 表达 生物信息学 

分 类 号：Q786[生物学—分子生物学]