一种来源于红螺菌科细菌新型卤醇脱卤酶的克隆表达及其酶学性质鉴定

Expression and characterization of a novel halohydrin dehalogenase from Rhodospirillaceae bacterium

摘要:

作为一类多功能生物催化剂，卤醇脱卤酶在手性β-取代醇和环氧化合物合成应用方面备受关注。目前催化功能较为清楚的卤醇脱卤酶不足40种，且绝大部分催化性能并不能满足科学研究和实际应用的要求，因此挖掘并鉴定更多的卤醇脱卤酶具有重要意义。本文克隆表达了来源于红螺菌科细菌Rhodospirillaceae bacterium中 一个假定卤醇脱卤酶 (HHDH-Ra) 并对其催化特性以及酶学性质进行研究。将HHDH-Ra基因克隆到表达宿主大肠杆菌Escherichia coli BL21(DE3)，结果显示目的蛋白为可溶性表达。底物特异性研究显示HHDH-Ra对1,3-二氯-2-丙醇 (1,3-DCP) 和4-氯-3-羟基丁酸乙酯 (CHBE) 具有良好的特异性。以1,3-DCP为反应底物获得HHDH-Ra的最适pH和最适温度分别为8.0和30 ℃。pH 稳定性结果显示HHDH-Ra在pH 6.0–8.0具有较好的稳定性且经过100 h处理以后仍能保持70%左右的酶活。温度稳定性结果显示HHDH-Ra在30 ℃、40 ℃条件下的半衰期为60 h，且当温度提高到50 ℃时，该酶的半衰期仍有20 h，远高于已报道的酶。因此，来源于Rhodospirillaceae bacterium新型卤醇脱卤酶具有较好的温度、pH稳定性以及催化活性，在合成关键化学、医药中间体中具有一定的应用潜力。

Abstract:

As a class of multifunctional biocatalysts, halohydrin dehalogenases are of great interest for the synthesis of chiral β-substituted alcohols and epoxides. There are less than 40 halohydrin dehalogenases with relatively clear catalytic functions, and most of them do not meet the requirements of scientific research and practical applications. Therefore, it is of great significance to excavate and identify more halohydrin dehalogenases. In the present study, a putative halohydrin dehalogenase (HHDH-Ra) from Rhodospirillaceae bacterium was expressed and its enzymatic properties were investigated. The HHDH-Ra gene was cloned into the expression host Escherichia coli BL21(DE3) and the target protein was shown to be soluble. Substrate specificity studies showed that HHDH-Ra possesses excellent specificity for 1,3-dichloro-2-propanol (1,3-DCP) and ethyl-4-chloro-3-hydroxybutyrate (CHBE). The optimum pH and temperature for HHDH-Ra with 1,3-DCP as the reaction substrate were 8.0 and 30 °C, respectively. HHDH-Ra was stable at pH 6.0–8.0 and maintained about 70% of its original activity after 100 h of treatment. The thermal stability results revealed that HHDH-Ra has a half-life of 60 h at 30 °C and 40 °C. When the temperature is increased to 50 °C, the enzyme still has a half-life of 20 h, which is much higher than that of the reported enzymes. To sum up, the novel halohydrin dehalogenase from Rhodospirillaceae bacterium possesses good temperature and pH stability as well as catalytic activity, and shows the potential to be used in the synthesis of chemical and pharmaceutical intermediates.