摘要
羰基还原酶(CRED)是手性药物合成中的重要工具酶,已在工业生产中广泛应用。该研究利用多种基因挖掘策略获得了96个不同来源的CRED基因,于大肠埃希菌中克隆、表达,从而构建了CRED酶库1.0,并利用8种不同结构化合物考察了酶库的催化活性。结果表明,不同的酶对不同结构底物的催化活性有显著差别;并且,酶库中包含多个具有高催化活性的CRED,如K21、K42、K38和K73等,可分别高效催化还原乙酰乙酸乙酯、4-氯代乙酰乙酸乙酯、苯乙酮和苯甲酰乙酸乙酯。随后,采用K42和K87高效催化制备了光学手性醇(S)-4-氯-3-羟基丁酸酯(ee>99%),采用K94催化合成(R)-4-氯-3-羟基丁酸酯(ee>99%),时空收率高达800g·(L·d)^(–1)。该研究所构建的CRED酶库1.0为手性药物的工业合成提供了新的选择。
Carbonyl reductases(CREDs)are valuable tools in the synthesis of chiral drugs.In this study,96CREDs from different sources were obtained by gene mining strategy,then cloned and expressed in Escherichia coli to construct CRED library 1.0.The catalytic performance of the enzyme library for eight substrates with different structures was investigated.The results showed that the catalytic performance of CREDs on various substrates with distinct structure was significantly different.Several enzymes with high catalytic activity were screened out from the enzyme library,such as K21,K42,K38 and K73,which can efficiently catalyze ethyl acetoacetate,4-chloroacetoacetate ethyl ester,acetophenone and ethyl benzoylacetate,respectively.Later,the process development for the efficient preparation of optically chiral alcohol(S)-4-chloro-3-hydroxybutyrate(ee>99%)by carbonyl reductase K42 and K87,the synthesis of(R)-4-chloro-3-hydroxybutyrate(ee>99%)by K94 were successfully realized with a space-time yield of 800 g·(L·d)^(–1).The carbonyl reductase library 1.0 provided a new tool box for industrial synthesis of chiral drugs.
作者
汤佳伟
陈浩
张露文
张蕊
吴海燕
奕栋
张福利
陈少欣
TANG Jiawei;CHEN Hao;ZHANG Luwen;ZHANG Rui;WU Haiyan;YI Dong;ZHANG Fuli;CHEN Shaoxin(State Key Lab.of New Drug and Pharmaceutical Process,Shanghai Institute of Pharmaceutical Industry,China State Institute of Pharmaceutical Industry,Shanghai 201203)
出处
《中国医药工业杂志》
CAS
CSCD
北大核心
2023年第9期1337-1344,共8页
Chinese Journal of Pharmaceuticals
基金
科技部国家重点研发计划“绿色生物制造”项目(2021YFC2102100)
上海市浦江人才计划项目(21PJ1423200)。
关键词
羰基还原酶
酶库
重组表达
催化活性
手性醇
carbonyl reductase
enzyme library
recombinant expression
catalytic activity
chiral alcohol
