School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, NO 1, Wenyuan Road, Nanjing 210023, People's Republic of China.
School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, NO 1, Wenyuan Road, Nanjing 210023, People's Republic of China.
Int J Biol Macromol. 2021 Feb 15;170:123-128. doi: 10.1016/j.ijbiomac.2020.12.104. Epub 2020 Dec 19.
Halohydrin dehalogenases (HHDHs) are valuable biocatalysts for the synthesis of enantiopure benzyl glycidyl ether (BGE) and its derivatives, which are important synthetic intermediates for anti-cancer and anti-obesity drugs. However, all the reported HHDHs exhibit low enantioselectivity. In this study, we screened site-saturation mutagenesis libraries of AbHHDH at positions R89, A136, V137, P178, N179, F180, I181, Y186 and F187 for mutants with enhanced enantioselectivity toward BGE. The four improved variant R89V, R89Y, R89K and V137I were identified, and the double mutant R89Y/V137I showed 2.9-fold higher enantioselectivity than the wild type. The regions of HHDH containing the identified mutations were analyzed by homology modeling to explain the changes of enantioselectivity. Kinetic resolution of 20 to 100 mM BGE using whole cells of Escherichia coli expressing the mutant R89Y/V137I resulted in (R)-BGE yields of 42 to 32.5%, with ee >99%. This study improves our understanding of the enantioselectivity of HHDHs and contributes improved biocatalysts for the kinetic resolution of BGE.
卤代醇脱卤酶（HHDHs）是合成对映体纯苄基缩水甘油醚（BGE）及其衍生物的有价值的生物催化剂，BGE 及其衍生物是抗癌和抗肥胖药物的重要合成中间体。然而，所有报道的 HHDHs 的对映选择性都较低。在这项研究中，我们筛选了 AbHHDH 在位置 R89、A136、V137、P178、N179、F180、I181、Y186 和 F187 的饱和突变文库，以寻找对 BGE 具有增强对映选择性的突变体。鉴定出四个改进的变体 R89V、R89Y、R89K 和 V137I，双突变体 R89Y/V137I 的对映选择性比野生型高 2.9 倍。通过同源建模分析了包含鉴定出的突变的 HHDH 区域，以解释对映选择性的变化。使用表达突变体 R89Y/V137I 的大肠杆菌全细胞对 20 至 100mM BGE 进行动力学拆分，得到（R）-BGE 的产率为 42%至 32.5%，ee 值>99%。这项研究提高了我们对 HHDHs 对映选择性的理解，并为 BGE 的动力学拆分提供了改进的生物催化剂。
