gdh和ldh基因共表达重组大肠杆菌合成D-乳酸研究  

Synthesis of D-Lactate by Stereospecific Reduction of Pyruvic Acid Using Recombinant E.coli Co-Expressing Lactate Dehydrogenase and Glucose Dehydrogenase

作　　者：胡媛[1] 周文瑜[1] 魏东芝[1] 沈亚领[1] 

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鲁华生物技术研究所,上海200237

出　　处：《药物生物技术》2014年第1期13-17,共5页Pharmaceutical Biotechnology

基　　金：上海市重点学科建设资助项目(No.B505);生物反应器工程国家重点实验室重点资助项目(No.2060204)

摘　　要：D-聚乳酸是一种生物可降解材料,具有高热稳定性,生物合成其单体D-乳酸将具有广阔的前景。以枯草芽孢杆菌基因组DNA为模板,PCR扩增葡萄糖脱氢酶基因gdh,与表达载体pETduet-1连接获得pETduetGDH;再以粘质沙雷氏菌基因组DNA为模板,PCR扩增乳酸脱氢酶基因ldh,与已构建的pETduet-GDH连接获得pETduet-LG,转化E.Coli BL21共表达。过表达GDH和LDH大肠杆菌可不对称还原丙酮酸合成D-乳酸。经全细胞转化条件优化发现,添加NAD+能提高起始反应速度,在最佳温度37℃、0.2 mol/L磷酸缓冲液、pH7.0、细胞浓度OD值为40和丙酮酸20 g/L时,底物转化率可达86.5%,D-乳酸产量为17.3 g/L。D-lactic of highly optical purity has a wide perspective and practicability in biopolymer material,agriculture,chemical industry,etc.Bio-catalysis has become the most promising method in asymmetric synthesis because of its high conversion ratio,good stereo specificity and the mild reaction condition.The poly lactic acid（PLA） which has high thermostability is a kind of biodegradable material.D-lactic acid biosynthesis possesses a broad prospect.The glucose dehydrogenase gene gdh was amplified from Bacillus subtilis by PCR technique,and inserted into the plasmid pETduet-1 to construct the recombinant plasmid pETduet-GDH.The lactate dehydrogenase gene ldh was amplified from Serratia marcescens H30,and then inserted into pETduet-GDH to construct the recombinant plasmid pETduet-LG.The positive plasmid was transformed into E.coli.BL21,and a recombinant strain E.coli pETduet-LG was obtained.Adding NAD＋helped in increasing the initial reaction velocity.And the best catalysis conditions were：temperature：37 ℃;buffer solution：0.2 mol/L phosphate buffer;pH7.0;OD：40;substrate concentration：20 g/L.The conversion rate was 86.5% and the yield of D-lactic acid was 17.3 g/L.

关 键 词：D-乳酸 乳酸脱氢酶 葡萄糖脱氢酶 共表达 辅酶再生 全细胞转化 

分 类 号：Q784[生物学—分子生物学]