粘质沙雷氏菌H3010发酵型D-乳酸脱氢酶基因的克隆表达、纯化及酶学性质研究  

Molecular cloning,gene expression,purification and characterization of fermentative D-lactate dehydrogenase from S.marcescens H3010

作　　者：邱昱[1] 张燎原[2] 魏东芝[1] 周文瑜[1] 沈亚领[1] 储炬[1] 朱家文[3] 

机构地区：[1]华东理工大学生物反应器工程国家重点实验室,上海200237 [2]福建农林大学生命科学学院,福州350002 [3]华东理工大学化工分离研究所,上海200237

出　　处：《工业微生物》2012年第4期30-37,共8页Industrial Microbiology

基　　金：863专题项目资助(2006AA02Z243);国家重点实验室专项经费资助(2060204);上海市重点学科建设项目资助(B505)

摘　　要：通过PCR技术从粘质沙雷氏菌H3010基因组DNA中扩增出该D-乳酸脱氢酶基因,连接至pET-28a(+)表达载体,转入大肠杆菌BL21(DE3)中进行了重组表达,优化了酶纯化的条件,并对其酶学性质进行初步研究。结果表明,获得的该酶编码基因全长993 bp,编码330个氨基酸,大小为37 kDa。经优化表达及纯化条件后重组酶纯度可达90%。酶学性质研究发现,该重组酶最适反应温度为60℃,最适酶促反应pH为7.5(0.2 mol/L磷酸盐缓冲液),37℃下测得对底物丙酮酸的动力学参数Km=3.39 mmol/L,Vmax=6.87 mmol/(mg·min),对辅酶NADH的动力学参数Km=1.43mmol/L,Vmax=1.61mmol/(mg·min)。为酶法生产D-乳酸及利用代谢工程构建产D-乳酸的基因工程菌打下基础。The d-ldh gene coding for d-lactate dehydrogenase from Serratia marcescens H3010 was amplified by PCR, and was ligated with the expression plasmid of pET-28a（ purification conditions for the enzyme were optimized ＋ ） and transformed into E. colt BL21 （DE3） and the enzyme characteristics were investigated for expression. The as well. The results showed that the ORF length of d-ldh gene was 993 bp and encoded protein of 330 residues, and its molecular weight was 37 kDa. The purity of the enzyme up to 90% could be obtained through expression and purification optimization. Studies on the enzyme characteristics revealed that the optimal reaction temperature was 60℃, and the optimal reaction pH was 7.5 （0.2 mol/L phosphate buffer）. Further more, the Km and Vmax of the enzyme for pyruvic acid were 3.39 mmol/L and 6.87 mmol/（ mg rain）, meanwhile these two kinetic parameters for NADH were 1.43 mmol/L and Vmax = 1.61 mmoV（ mg rain） , respectively. These studies laid a good foundation for production of D-lactate by enzymic method and construction of D-lactate producing genetic engineering strain.

关 键 词：D-乳酸脱氢酶 粘质沙雷氏菌 表达 纯化 酶学性质 

分 类 号：Q78[生物学—分子生物学]