Enhancement of the thermostability of halohydrin dehalogenase from Agrobacterium radiobacter AD1 by constructing a combinatorial smart library

International Journal of Biological Macromolecules
Xiong WangYanxia Sun

Abstract

Halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) is a key enzyme in preparing β-substituted alcohols and optically pure epoxides. However, the weak thermostability of HheC greatly limits its industrial application and needs to be improved. In this study, a combinatorial smart library was constructed and screened for enhancing the thermostability of HheC. Mutation sites Lys203 and Lys204 located at the protein surface were selected and simultaneously randomized with charged amino acids (Arg, Asp, Glu, Lys), resulting in a combinatorial smart library. After screening a total of only 48 clones, three positive variants CSL1 (Lys203Arg), CSL2 (Lys204Arg) and CSL3 (Lys203Arg/Lys204Arg) were obtained. Compared to the wild type HheC, variant CSL2 exhibited an increase in thermal stability with a nearly 6.9-fold improvement of half-life (τ1/2, 55°C) at 55 °C without compromising catalytic activity. In addition, the double mutant CSL3 displayed approximately 3.4-fold higher τ1/2, 55°C value and 1.8-fold higher kcat value toward 1,3-dicholoro-2-propanol (1,3-DCP) than that of the wild-type HheC. Homology modeling analysis highlights that the newly gained hydrogen bonds and optimizing charge-charge interactions at the prot...Continue Reading

Citations

Nov 20, 2019·Biochimica Et Biophysica Acta. Proteins and Proteomics·Victor SayousCarlos G Acevedo-Rocha

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