摘要
人工合成经密码子优化的羰基还原酶基因Sys1,并与葡萄糖脱氢酶基因Sygdh共克隆至双启动子表达质粒p ETDuet-1中,获重组质粒p ETDuet-Sygdh-Sys1。将其转化大肠杆菌(Escherichia coli)BL21(DE3),构建了共表达羰基还原酶和葡萄糖脱氢酶的重组工程菌E.coli BL21/p ETDuet-Sygdh-Sys1。以经IPTG诱导的重组菌为生物催化剂,不对称还原间-氯苯甲酰甲基氯(m-CPC),制备手性药物中间体(R)-2-氯-1-(3-氯苯基)乙醇((R)-CCE)。在m-CPC 30 mmol/L、重组湿菌体70 mg/m L、葡萄糖40 mmol/L、辅酶NADP+0.2 mmol/L,以及p H 7.0、反应温度40℃和反应时间3 h等条件下,所获手性化合物(R)-CCE的摩尔产率高达99.0%,对映体过量值(e.e.值)为100%。
A codon-optimized gene Sys1,which encodes a carbonyl reductase,was synthesized,and cloned into an expression plasmid p ETDuet-1 with double promoters together with a glucose dehydrogenase-encoding gene Sygdh. The recombinant E. coli BL21/p ETDuet-Sygdh-Sys1,expressing the carbonyl reductase and the glucose dehydrogenase together,was constructed by transforming the recombinant plasmid,p ETDuet-Sygdh-Sys1,into E. coli BL21（DE3）. Using recombinant E. coli cells induced by IPTG as biocatalysts,（R）-2-chloro-1-（3-chlorophenyl）ethanol（（R）-CCE）,a chiral drug intermediate,was synthesized by asymmetrically reducing mchlorophenacyl chloride（m-CPC）. Under the reaction conditions of 30 mmol/L of m-CPC,70 mg/m L of wet recombinant E. coli cells,40 mmol/L of glucose,0.2 mmol/L of NADP＋at p H 7.0 and 40 ℃ for 3 h,the molar yield of（R）-CCE reached 99.0% with an enantiomeric excess（e.e.） value of 100%.
出处
《食品与生物技术学报》
CAS
CSCD
北大核心
2016年第3期278-283,共6页
Journal of Food Science and Biotechnology
基金
国家自然科学基金项目(31271811)
