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

DOI：10.3969/j.issn.1673-1689.2019.12.004

中文关键词: Lactobacillus casei L-乳酸脱氢酶 苯丙酮酸 不对称还原 表达 生物信息学

英文关键词: Lactobacillus casei,L-lactate dehydrogenase,phenylpyruvic acid,asymmetricreduction,expression,bioinformatics

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中文摘要:

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32 585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。

英文摘要:

Using the genomic DNA fromLactobacillus caseiCICIM 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 32 585 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.

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