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

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993bp，编码330个氨基酸，大小为37kDa。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（O．2mol／L磷酸盐缓冲液），37℃下测得对底物丙酮酸的动力学参数Km=3．39mmol／L，Vmax=6．87mmol／（mg·min），对辅酶NADH的动力学参数Km=1．43mmol／L，Vmax=1．61mmo]／（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。

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

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.