菜豆环氧水解酶归一性水解间硝基环氧苯乙烷的研究

doi:10.13560/j.cnki.biotech.bull.1985.2019-0624

摘要/Abstract

摘要： 旨在研究菜豆环氧水解酶PvEH2对映归一性水解外消旋（rac-）间硝基环氧苯乙烷（mNSO）的特性,为制备光学纯（R）-间硝基苯乙二醇（mNPED）提供优良的生物催化剂。构建表达有重组PvEH2的工程菌E. coli/pveh2。以rac-mNSO为底物,借助手性色谱法分析E. coli/pveh2全细胞的EH活性和区域选择性。基于有机助溶剂种类和剂量对酶活性的影响,筛选并优化缓冲液/助溶剂反应体系。结果显示,在菜豆植株中,PvEH2基因的转录水平可能受体外环境诱导因素的影响。E. coli/pveh2的EH活性为15.4 U/g_干细胞,水解反应的区域选择性系数α_S和β_R分别为90.3%和96.4%。在经过筛选优化的含有5%（V/V）丙三醇的磷酸盐缓冲液中,最高底物浓度为40 mmol/L,是原缓冲液体系的4倍。当反应至12 h时,所得（R）-mNPED的光学纯度ee_p及产率分别为84.9%和90.2%。E. coli/pveh2或PvEH2在水解rac-mNSO中表现出优秀的区域选择性,在制备光学纯（R）-mNPED中具有较好的应用潜力。

关键词: 环氧水解酶, 对映归一性水解, 间硝基环氧苯乙烷, 间硝基苯乙二醇

Abstract: This work aims to provide a superior biocatalyst for producing optically pure（R）-m-nitrophenyl-1,2-ethanediol（mNPED）via studying the catalytic properties of PvEH2,a Phaseolus vulgaris epoxide hydrolase,in the enantioconvergent hydrolysis of racemic（rac-）m-nitrostyrene oxide（mNSO）. The engineered Escherichia coli strain expressing the recombinant PvEH2,designated E. coli/pveh2,was constructed. Using rac-mNSO as substrate,the EH activity and regioselectivity of E. coli/pveh2 were assayed by chiral chromatography. Then,according to the effects of several organic solvents and their dosages on enzymatic activity,the buffer/co-solvent reaction system was screened and optimized. Results showed that the transcription level of PvEH2 gene in P. vulgaris might be affected by the induction factors in vitro. The EH activity of E. coli/pveh2 was 15.4 U/g_{dry cell},and its regioselectivity coefficients,α_S and β_R,were 90.3% and 96.4%,respectively. In the optimized phosphate buffer containing 5%（V/V）glycerol,the maximum allowable substrate concentration（40 mmol/L）was 4 times higher than that in sole buffer.（R）-mNPED with 84.9% ee_p and 90.2% yield was obtained at 12 h reaction. E. coli/pveh2 or PvEH2 demonstrates an excellent regioselectivity in the hydrolysis of rac-mNSO,thus making it a promising biocatalyst to prepare optically pure（R）-mNPED.

Key words: epoxide hydrolase, enantioconvergent hydrolysis, m-nitrostyrene oxide, m-nitrophenyl-1, 2-ethanediol

李闯, 文正, 刘畅, 邬敏辰. 菜豆环氧水解酶归一性水解间硝基环氧苯乙烷的研究[J]. 生物技术通报, 2020, 36(1): 73-80.

LI Chuang, WEN Zheng, LIU Chang, WU Min-chen. Enantioconvergent Hydrolysis of m-Nitrostyrene Oxide by Phaseolus vulgaris Epoxide Hydrolase[J]. Biotechnology Bulletin, 2020, 36(1): 73-80.

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