工业微生物2016，Vol.46Issue(5)：35-41,7.DOI:10.3969/j.issn.1001-6678.2016.05.006
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
摘要
Abstract
The esterase gene ( EHest ) from Achromobacterdenitrificans 1104 was ligated with plasmids pET28 a (+) , transformed into E. coli BL21Gold ( DE3 ) plysS. 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 for 1 h at 37℃, the substrate conversion was 29. 5% and eep of 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 Vm and Km for 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 Cu2+, Fe3+ 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.关键词
反硝化无色杆菌/酯酶/克隆表达/(R,S)-2,6-二甲基苯基氨基丙酸甲酯/酶学性质Key words
Achromobacterdenitrificans/esterase/gene cloning and expression/methyl(R,S)-N-(2,6-dimethylphenyl) alaninate/enantioselective hydrolysis/enzymatic properties引用本文复制引用
董剑鸣,张利坤,卢亚南,姚星超,杨玉莹,陆跃乐,张朝晖..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[J].工业微生物,2016,46(5):35-41,7.基金项目
国家自然科学基金(21447005)。 （21447005）
