Abstract
Chiral epichlorohydrin (ECH) is an attractive intermediate for chiral pharmaceuticals and chemicals preparation. The asymmetric synthesis of chiral ECH using 1,3-dicholoro-2-propanol (1,3-DCP) catalyzed by a haloalcohol dehalogenase (HHDH) was considered as a feasible approach. However, the reverse ring opening reaction caused low optical purity of chiral ECH, thus severely restricts the industrial application of HHDHs. In the present study, a novel selective conformation adjustment strategy was developed with an engineered HheCPS to regulate the kinetic parameters of the forward and reverse reactions, based on site saturation mutation and molecular simulation analysis. The HheCPS mutant E85P was constructed with a markable change in the conformation of (S)-ECH in the substrate pocket and a slight impact on the interaction between 1,3-DCP and the enzyme, which resulted in the kinetic deceleration of the reverse reactions. Compared with HheCPS, the catalytic efficiency (kcat(S)-ECH/Km(S)-ECH) of the reversed reaction dropped to 0.23-fold (from 0.13 to 0.03 mM−1 s−1), while the catalytic efficiency (kcat(1,3-DCP)/Km(1,3-DCP)) of the forward reaction only reduced from 0.83 to 0.71 mM−1 s−1. With 40 mM 1,3-DCP as substrate, HheCPS E85P catalyzed the synthesis of (S)-ECH with the yield up to 55.35% and the e.e. increased from 92.54 to >99%. Our work provided an effective approach for understanding the stereoselective catalytic mechanism as well as the green manufacturing of chiral epoxides.
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Acknowledgements
This work was financially supported by the National Key Research and Development Program of China (No. 2021YFA1501400 and 2019YFA0905000) and State Key Laboratory of Catalytic Materials and Reaction Engineering (RIPP, SINOPEC).
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National Key Research and Development Program of China,2021YFA1501400,Xiao-Ling Tang,National Key Research and Development Program of China,2019YFA0905000,Xiao-Jian Zhang
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XJZ designed and directed the main experiments, and revised the overall manuscript. MYH designed and carried out the experiments, and wrote the manuscript. HZD assisted in experiments and manuscript revise. MC, XXP, YCG and XLT provided technical support. ZQL provided an overall plan for this paper and reviewed the manuscript. YGZ proposed scientific and in-depth knowledge for this study.
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Zhang, XJ., Huang, MY., Peng, XX. et al. Preparation of (S)-epichlorohydrin using a novel halohydrin dehalogenase by selective conformation adjustment. Biotechnol Lett (2024). https://doi.org/10.1007/s10529-024-03479-y
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DOI: https://doi.org/10.1007/s10529-024-03479-y