利用重组大肠杆菌还原4氯乙酰乙酸乙酯不对称合成S4氯3羟基丁酸乙酯研究.pdfVIP

利用重组大肠杆菌还原4-氯乙酰乙酸乙酯 不对称合成(S)-4-氯-3-羟基丁酸乙酯 叶齐 严明 许琳 应汉杰1 1 南京工业大学材料化学工程国家重点实验室，江苏 南京210009） （ 摘要： 从毕赤酵母(Pichia Stipitis) 中克隆NADPH 依赖性的羰酰还原酶PsCR 基因，构建了重组大肠杆菌 E. coli BL21 （pET-22b-PsCR ），该重组菌能够大量可溶地表达羰酰还原酶，通过考察诱导OD、诱导浓度、 诱导时间和温度对酶活的影响，优化了重组大肠杆菌的表达条件，结果表明：在重组菌 OD 为 0.6 时加入 终浓度为0.8mmol/L 的IPTG 在25 ℃诱导10h, 比酶活可达32U/mg 。利用重组菌进行不对称还原4-氯乙酰 乙酸乙酯为(S)-4-氯-3-羟基丁酸乙酯的反应，在添加辅酶NADPH 、葡萄糖脱氢酶及分批加入底物的情况下， 产物浓度可达23.4g/L ，转化率为93.7%，产物的对映体过量值为100%。表明该重组菌对4-氯乙酰乙酸乙 酯有极高的活性及立体选择性。 关键词: 重组大肠杆菌； 4-氯乙酰乙酸乙酯； (S)-4-氯-3-羟基丁酸乙酯；不对称合成 Asymmetric Reduction of Ethyl 4-Chloroacetoacetate to S-(-)-4-Chlor-3-Hydroxybutyrate by Recombinant E. Coli YE Qi YAN Ming XU Lin YING Hanjie State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing University of Technology, Nanjing 210009, China Abstract ：A carbonyl reductase gene was cloned from Pichia stipitis (PsCR)and 0.86-kb DNA fragment including PsCR gene was inserted into pET-22b. Then the recombinant E. coli BL21(DE3) harboring pET-22b-PsCR was constructed and optimized by investigating the effects of the time of adding isopropylthiogalactoside (IPTG), IPTG concentration, temperature and time on enzyme activity. Experiment results showed that optimized conditions were IPTG concentration 0.8 mmol/L, 25℃, 10 h. Under optimized conditions, 32 U/mg protein of enzyme activity was obtained. Reducing COBE to S-CHBE by recombinant E. coli was reacted in an aqueous monophase system for18h, while the molar yield of (S)-CHBE is 93.7% and the optical purity of the product was more than 99% enantiomeric excess (e.e). Key words ：recombinant E. coli; asymmetric reduction; ethyl 4-chloroacetoacetate; S-(-)-4-chloro-3-hydroxybutyrate 联系人：应汉杰. 第一作者：叶齐（1984—），男，