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

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.