Improving the enantioselectivity of halohydrin dehalogenase for the synthesis of (R)-benzyl glycidyl ether via biocatalytic azidolysis.

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.