L-乳酸脱氢酶在大肠杆菌BL-21(DE3)中的表达  

Expression of L-lactate dehydrogenase in Escherichia coli BL-21(DE3)

作　　者：李倩[1] 王梦[1] 刘珞[1] 谭天伟[1] 

机构地区：[1]北京化工大学生命科学与技术学院北京市生物加工过程重点实验室,北京100029

出　　处：《生物加工过程》2011年第6期21-25,共5页Chinese Journal of Bioprocess Engineering

基　　金：国家自然科学基金资助项目(20876011);北京市自然科学基金资助项目(2071002);国家重点基础研究发展计划(973计划)资助项目(2007CB707804);国家高技术研究发展计划(863计划)资助项目(2006AA020103;2006AA020102;2006AA020201;2007AA100404;2007CB714304)

摘　　要：为了在大肠杆菌中构建利用葡萄糖生产L-乳酸的途径,以鼠李糖乳杆菌(Lactobacillus rhamnosus)LA-04-01基因组为模板,设计引物扩增L-乳酸脱氢酶基因L-ldh。将该基因连接到表达载体pET-28a(+)上,并转化大肠杆菌Top10。通过卡那霉素抗性平板筛选,提取重组质粒pET28a-L-ldh并测序,结果正确。将pET28a-L-ldh转化大肠杆菌BL-21(DE3),通过卡那霉素抗性平板筛选,得到产乳酸的大肠杆菌基因工程菌。经IPTG诱导后,SDS-PAGE电泳,检测到目的蛋白条带,L-乳酸脱氢酶比酶活力达到9.44 U/mL。该基因工程菌通过摇瓶发酵,L-乳酸产量达到3g/L,成功构建出一条在大肠杆菌中生产L-乳酸的新途径。In order to construct a new metabolic pathway of lactic acid in Escherichia coli,the Lactobacillus rhamnosus genome was used as the template to amplify the L-lactate dehydrogenase gene（L-ldh）.The gene was inserted into vector of pET-28a（＋）,then transformed into E.coli Top10 and screened with erythromycin.The results showed that the sequence of the recombinant vector was correct.It was transformed into E.coli BL-21（DE3） which is deficient of L-lactate dehydrogenase.After induction with IPTG,the recombinant protein was analyzed and the enzyme activity was measured as 9.44 U/mL.Both results indicated the L-ldh was expressed in the E.coli BL-21（DE3）.By the flask fermentation,3 g/L L-lactate acid was achieved.

关 键 词：大肠杆菌 L-乳酸脱氢酶基因 鼠李糖乳杆菌 

分 类 号：TQ028.8[化学工程]