一种不对称水解(R,S)-2,6-二甲基苯基氨基丙酸甲酯的新酯酶基因的克隆表达和酶学性质研究

Cloning and expression of a novel esterase capable of enantioselective hydrolysis of methyl( R,S)-N-(2,6-dimethylphenyl) alaninate and its enzymatic properties

本文将来自反硝化无色杆菌Achromobacterdenitrificans1104的酯酶基因EHest,转化大肠杆菌中,成功表达了具有不对称水解农药甲霜灵的中间体(R,S)-2,6-二甲基苯基氨基丙酸甲酯(MAP)活性的酯酶EHesterase。用重组酯酶EHesterase催化MAP的水解,底物浓度50 g/L,反应1h的转化率29.5%,产物(R-酸)的eep是85.1%。该酶的最适反应pH和温度分别为9.0和50℃,在50℃以下和pH5~9之间具有较好的稳定性。该酶水解MAP的米氏动力学参数Vm、Km分别是0.733 g/(L·min)和7.49 g/L。加入10%DMSO对酶EHesterase的立体选择性和催化速度有一定的促进作用。Cu^(2+)、Fe^(3+)对酶活有明显抑制作用。该酶水解MAP的活性与水解p-对硝基苯乙酸酯的活性数量级相当,是水解橄榄油活性的333倍。

The esterase gene（ EHest） from Achromobacterdenitrificans 1104 was ligated with plasmids p ET28a（ ＋）,transformed into E. coli BL21Gold（ DE3） plys S. The resulting recombinants successfully expressed the esterase（ EHesterase） capable of catalyzing enatioelective hydrolysis of methyl（ R,S）-N-（ 2,6-dimethylphenyl） alaninate（ MAP）,a key intermediate for the synthesis of metalaxyl. Catalyzing the hydrolysis of MAP（ 5% m/v） by EHesterase for1 h at 37 ℃,the substrate conversion was 29. 5% and eepof the product acid（ major in R configuration） was 85. 1%. The optimum reaction pH and temperature of EHesterase were 9. 0 and 50 ℃ respectively. The enzyme was stable at below 50℃ and between pH 5 and pH 9. The Michaelis-Menten kinetics parameters Vmand Kmfor MAP＇s hydrolysis catalyzed by EHesterase were 0. 733 g/（ L·min） and 7. 49 g/L respectively. 10% DMSO somewhat increased the enantioselectivity and activity of the enzyme. 1 mmol/L of Cu^2＋,Fe^3＋significantly inhibited its activity. Using MAP as substrate,the hydrolysis activity of EHesterase was the same order as that using p-nitrophenyl acetate as substrate and was 333 times as high as that using olive oil as substrate.