【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-Lc LDH2)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-Lc LDH2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-Lc LDH2,预测其相对分子质量和等电点分别为32 585和5.5;L-Lc LDH2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-Lc LDH2的获得为生物法制备高光学纯L-苯乳酸提供了新的酶源。更多还原

【Abstract】 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-28 a(+). 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(ee_p) of more than 99%. Bioinformatic prediction revealed that Lcldh2 is906 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-Lc LDH2 has provided a new biocatalyst for the preparation ofhighly optically pureL-phenyllactic acid.更多还原