摘要
卤醇脱卤酶(Halohydrin dehalogenase)不仅对于含氯污染物的生物降解、净化环境具有重要意义,而且在手性医药中间体合成中也是一种重要的生物催化剂,但其数量较少。采用基因挖掘在基因组数据库中获得一种来自运动替斯崔纳菌Tistrella mobilis KA081020-065的新型卤醇脱卤酶基因Hhe TM,将该基因克隆、表达在大肠杆菌BL21(DE3)中,利用镍柱亲和层析将该酶进行纯化并研究了其酶学性质。Hhe TM是一种同源四聚体;最适温度为50℃;不同的p H缓冲液对其活性有较大影响;在碱性、30℃以下的条件下稳定性高。在氰根离子存在的条件下,Hhe TM能够催化(S)-4-氯-3-羟基丁酸乙酯合成(R)-4-氰基-3-羟基丁酸乙酯,为酶法合成阿托伐他汀关键中间体提供了一种新的卤醇脱卤酶。
Halohydrin dehalogenase is Ot great slgnltlcance for Olouegraaanon oi me cnlorlnateu pollutants, and also serves as an important biocatatyst in the synthesis of chiral pharmaceutical intermediates. A putative halohydrin dehalogenase （HheTM） gene from TistreIla rnobilis KA081020-065 was cloned and over-expressed in Escherichia coli BL21 （DE3）. The recombinant enzyme was purified by Ni-NTA column and characterized. Gel filtration and SDS-PAGE analysis showed that the native form of HheTM was a tetramer. It exhibited the highest activity at 50 ℃. The nature and pH of the buffer had a great effect on its activity. The enzyme maintained high stability under the alkaline conditions and below 30 ℃. HheTM catalyzed the transformation of ethyl（S）-4-chloro-3-hydroxybutyrate in the presence of cyanide, to give ethyl （R）-4-cyano-3- hydroxybutyrate, a key intermediate for the synthesis of atorvastatin.
出处
《生物工程学报》
CAS
CSCD
北大核心
2015年第5期659-669,共11页
Chinese Journal of Biotechnology
基金
中国科学院重点部署项目(No.KSED-EW-Z-015)资助~~
