工业微生物2012，Vol.42Issue(4)：30-37,8.DOI:10.3969/j.issn.1001-6678.2012.04.006
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
摘要
Abstract
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( + ) and transformed into E. coli BL21 (DE3) for expression. The purification conditions for the enzyme were optimized and the enzyme characteristics were investigated 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 60t, 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 min) , meanwhile these two kinetic parameters for NADH were 1. 43 mmol/L and Vmax = 1. 61 mmol/( mg min) , 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Key words
D-lactate dehydrogenase/ S. marcescens H3010/ expression/ purification/ characterization of enzyme引用本文复制引用
邱昱,张燎原,魏东芝,周文瑜,沈亚领,储炬,朱家文..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[J].工业微生物,2012,42(4):30-37,8.基金项目
863专题项目资助(2006AA02Z243) （2006AA02Z243）
国家重点实验室专项经费资助(2060204) （2060204）
上海市重点学科建设项目资助(B505) （B505）
