一种来源于运动替斯崔纳菌KA081020-065的新型卤醇脱卤酶的表达纯化及性质分析

Expression and characterization of a novel halohydrin dehalogenase from Tistrella mobilis KA081020-065

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HheTM，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。HheTM是一种同源四聚体；最适温度为50 ℃；不同的 pH 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，HheTM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。

Abstract:

Halohydrin dehalogenase is of great significance for biodegradation of the chlorinated pollutants, and also serves as an important biocatalyst in the synthesis of chiral pharmaceutical intermediates. A putative halohydrin dehalogenase (HheTM) gene from Tistrella mobilis 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 °C. 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 °C. 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.