Xu Wenjing, Chen Zhi, Chen Lei, Lin Jinping, Wei Dongzhi
School of Biotechnology, East China University of Science and Technology, Shanghai 200237, China.
Sheng Wu Gong Cheng Xue Bao. 2021 Apr 25;37(4):1298-1311. doi: 10.13345/j.cjb.200719.
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.
作为一类多功能生物催化剂，卤代醇脱卤酶在合成手性β-取代醇和环氧化物方面具有重要意义。目前具有相对明确催化功能的卤代醇脱卤酶不足40种，且大多数不符合科研和实际应用的要求。因此，挖掘和鉴定更多的卤代醇脱卤酶具有重要意义。在本研究中，表达了一种来自红螺菌科细菌的假定卤代醇脱卤酶（HHDH-Ra）并对其酶学性质进行了研究。将HHDH-Ra基因克隆到表达宿主大肠杆菌BL21(DE3)中，结果表明目标蛋白是可溶的。底物特异性研究表明，HHDH-Ra对1,3-二氯-2-丙醇（1,3-DCP）和4-氯-3-羟基丁酸乙酯（CHBE）具有优异的特异性。以1,3-DCP为反应底物时，HHDH-Ra的最适pH和温度分别为8.0和30℃。HHDH-Ra在pH 6.0-8.0范围内稳定，处理100小时后仍保持约70%的原始活性。热稳定性结果表明，HHDH-Ra在30℃和40℃下的半衰期为60小时。当温度升至50℃时，该酶仍有20小时的半衰期，远高于已报道的酶。综上所述，这种来自红螺菌科细菌的新型卤代醇脱卤酶具有良好的温度和pH稳定性以及催化活性，显示出在化学和药物中间体合成中应用的潜力。
