重组菌转化4-氯乙酰乙酸乙酯为R-(+)-4-氯-3-羟基丁酸乙酯的研究  

Asymmetric Reduction of Ethyl 4-Chloroacetoacetate to R-(+)-4-Chlor-3-Hydroxybutyrate by Recombinant Escherichia Coli Strains

作　　者：李静[1] 林建平[1] 徐志南[1] 谭天伟[2] 岑沛霖[1] 

机构地区：[1]浙江大学材料与化学工程学院,浙江杭州310027 [2]北京化工大学生命科学与技术学院,北京100029

出　　处：《化学反应工程与工艺》2006年第4期350-355,共6页Chemical Reaction Engineering and Technology

基　　金：北京市生物加工过程重点实验室开放资助项目(STS100100421)

摘　　要：通过醛基还原酶和6-磷酸葡萄糖脱氢酶的基因构建重组大肠杆菌,并考察加入诱导剂的时间、浓度和诱导时间对酶活的影响,优化重组大肠杆菌的表达。实验结果表明,重组大肠杆菌在对数生长中期加入0.4 mmol/L的诱导剂,在32℃下培养8 h,酶活可达到5.27 U/mg-protein。以重组大肠杆菌为催化剂,研究4-氯乙酰乙酸乙酯转化为-R(+)-4-氯-3-羟基丁酸乙酯的反应,发现利用基因重组技术构建的大肠杆菌,可使醛基还原酶和葡萄糖脱氢酶共同表达,实现辅酶再生,提高产物对映体过量值。在转化反应过程中,随着温度升高,产物收率先增加;当转化温度在32℃以上,收率反而会下降。底物对反应有抑制作用,采用分批加入底物的方式可以减少抑制作用,提高产物收率。NADP-dependent aldehyde reductase gene ALR and glucose-6-phosphate dehydrogenase gene gdh223 were cloned from Sporobolomyces salmonicolor and Bacillus megaterium ZJU0310 respectively, then the recombinant E. coil M15 （pQE30-ALRgdh223） strain was constructed and optimized by investigating the effects of the time of adding isopropylthiogalactoside （IPTG）, IPTG concentration and temperature on enzyme activity. Experiment results showed that optimized conditions were IPTG concentration 0.4 mmol/L, 32 ℃, 8 h. Under optimized conditions, 5.27 U/mg-protein of enzyme activity was obtained after 8 h cultivation. The asymmetric reduction of ethyl 4-chloroacetoacetate to R- （＋） -4-chlor-3-hydroxybutyrate was carried out and it was found that recombinant Escherichia Coli Strains could make coenzyme regenerate and increase ee. When reaction temperature was increased, product yield was increased, but over 32 ℃, product yield was decreased. The reactant had an inhabiting effect on the reaction. The inhabiting could be weaken and product yield could be increased by batch feed way.

关 键 词：不对称还原 4-氯乙酰乙酸乙醇 R-(+)-4-氯-3-羟基丁酸乙酯 

分 类 号：Q781[生物学—分子生物学] Q554