不对称水解(R,S)-2,6-二甲基苯基氨基丙酸甲酯新菌种的分离鉴定及酯酶基因的克隆、表达

Cloning and expression of an esterase gene from a new strain capable of enantioselective hydrolyzing methyl (R,S)-N-(2,6-dimethylphenyl) alaninate

摘要:

[目的] 筛选鉴定1株可以选择性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）的菌株，并克隆、表达该菌株中的酯酶基因。[方法] 以MAP为唯一碳源，对活性污泥样品中的微生物进行富集培养，采用罗丹明B平板显色法进行初筛，通过摇瓶复筛得到了1株对MAP具有最高对映体选择性和水解活力的新菌株，根据其形态、生理生化特征及16S rRNA序列分析，确立该菌株的系统发育学地位。构建该菌株的基因文库，筛选获得含目的基因的克隆子，通过序列分析和引物扩增得到酯酶基因，将基因与表达载体pET28a（+）连接后，转化大肠杆菌BL21Gold（DE3） plysS，构建重组菌。[结果] 该菌属于革兰氏阴性菌，结合16S rRNA基因、形态特征和生理生化实验结果，鉴定该菌为反硝化无色杆菌。通过基因文库法，找到了该菌中的酯酶基因EHest，并成功构建了重组大肠杆菌EHest-pET28a（+）-BL21Gold（DE3） plysS，表达了来自Achromobacter denitrificans 1104且具有不对称水解MAP活性的酯酶EHesterase，大小约27 kDa，表达酶活是原始菌株的27.1倍。用EHesterase催化MAP水解，底物浓度50 g/L，反应1 h，底物转化率为29.5%，产物（酸）的ee_p为85.1%，对映体选择性为R型。该酶的最适反应pH和温度分别为pH 9.0和50℃。它水解MAP的活性分别是水解橄榄油和乙酸乙酯活性的333倍和667倍。[结论] 筛选到1株具有不对称水解MAP能力的新菌株Achromobacter denitrificans 1104。

Abstract:

[Objective] We screened and identified a strain capable of enantioelectively hydrolyzing methyl (R,S)-N-(2,6-dimethylphenyl) alaninate (MAP), a key intermediate for the synthesis of metalaxyl, followed by cloning and expressing the esterase in E. coli.[Methods] We used MAP as the sole carbon source in the medium inoculated with an active sludge specimen to enrich the target microorganism. The strain with the highest hydrolysis activity and enatioelectivity was identified by 16S rRNA sequence analysis, morphological observation and physiological and biochemical properties. From the gene library of the strain, the DNA sequence fragment containing the target gene was found. By DNA sequence analysis and PCR amplification, the esterase gene was obtained. It was ligated with plasmids pET28a (+), then transformed into E. coli BL21Gold (DE3) plysS.[Results] We isolated a gram-negative bacterial strain capable of enantioelective hydrolyzing MAP. It was identified as Achromobacter denitrificans. From its gene library, the esterase gene named EHest was found. The recombinant EHest-pET28a(+)-BL21Gold (DE3) plysS was constructed. The recombinant expressed esterase (EHesterase) capable of catalyzing enatioelective hydrolysis of methyl (R,S)-N-(2,6-dimethylphenyl) alaninate. Its size was 27 kDa. The expression activity was 27.1 times as high as that in the original strain. Hydrolysis of MAP (5% M/V) by EHesterase for 1 h at 37℃, the substrate conversion was 29.5% and ee p of the product acid (major in R configuration) was 85.1%. The optimum pH was 9.0 and temperature 50℃.[Conclusions] A new isolate Achromobacter denitrificans 1104 capable of enantioelective hydrolyzing MAP was found and identified.