Cloning, expression and characterization of a gene encoding the esterase hydrolyzing R,S-metalaxyl high R-enantioselectively

Abstract:

[Objective] A gene encoding the esterase to hydrolyze R, S-metalaxyl high enantioseclectively to R-metalaxyl acid was cloned and expressed in E. coli. The properties of the recombinant esterase were studied. [Methods] Based on known N-terminal 10 amino acid sequence of the target esterase, a candidate esterase gene matching with it was found in the sequenced Albibacters sp. zjut528 genome. It had a length of 969 bp encoding 322 amino acids and was named RMest. The RMest DNA segment was amplified by PCR, ligated with pET-28a(+), transformed into E. coli BL21Gold(DE3) to construct the recombinant strain. The recombinant esterase was expressed by induction with IPTG and purified with Ni²⁺ resin. [Results] The recombinant RMesterase was expressed successfully in RMest-pET-28a(+)-E. coli BL21 Gold (DE3). Its size was about 46 kDa in SDS-PAGE. Hydrolysis of 10 g/L of R,S-metalaxyl for 6 h catalyzed by intracellular crude enzyme from the recombinant strain, the substrate conversion rate reached 49.8% with product (metalaxyl acid) ee_p of 99.3%. The main product was R-metalaxyl acid. The optimal temperature and pH of RMestrase was 40℃ and 9.0 respectively. The activity of RMesterase was inhibited by methanol, the other product of metalaxyl hydrolysis. After aligning the amino acid sequence of RMest with other proteins in Uniprot KB database by Blast+, the neighboring-joining phylogenetic tree was constructed. It shows that the esterase RMest had a long evolution distance with homologous Lysophospholipases, AB hydrolase-1 domain-containing proteins and other esterases. That indicates that the esterase RMest was a new one that evolved relative independently. [Conclusion] The esterase gene RMest was successfully cloned and expressed in RMest-pET-28a(+)-E. coli BL21 Gold (DE3). The recombinant RMesterase could hydrolyze R, S-metalaxyl high R-enantioselectively to produce R-metalaxyl acid.