菜豆环氧水解酶归一性水解间硝基环氧苯乙烷的研究  

Enantioconvergent Hydrolysis of m-Nitrostyrene Oxide by Phaseolus vulgaris Epoxide Hydrolase

作　　者：李闯[1] 文正 刘畅 邬敏辰 LI Chuang;WEN Zheng;LIU Chang;WU Min-chen(School of Biotechnology,Jiangnan University,Wuxi 214122;Wuxi School of Medicine,Jiangnan University,Wuxi 214122)

机构地区：[1]江南大学生物工程学院,无锡214122 [2]江南大学无锡医学院,无锡214122

出　　处：《生物技术通报》2020年第1期73-80,共8页Biotechnology Bulletin

基　　金：国家自然科学基金面上项目(21676117)

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,水解反应的区域选择性系数αS和βR分别为90.3%和96.4%。在经过筛选优化的含有5%(V/V)丙三醇的磷酸干细胞盐缓冲液中,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eep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。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,αS andβR,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.

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 

分 类 号：S64[农业科学—蔬菜学]