Cloning and characterization of a novel esterase from Rhodococcus sp.for highly enantioselective synthesis of a chiral cilastatin precursor


Yan Zhang;


A novel non-heme chloroperoxidase(RhEstl)with promiscuous esterase activity in enantioselectively hydrolysis of ethyl(S)-2,2-dimethylcylopropanecarboxylate hydrolase was identified from the shot-gun library of Rhodococcus sp.ECU1013.RhEstl was overexpressed in Escherichia coli BL21(DE3),purified to homogeneity and functionally characterized.Fingerprinting analysis revealed that RhEstl prefers to para-nitrophenyl(pNP)esters with short-length acyl chain.pNP esters with a cyclic acyl chain,especially that with a cyclobutanyl group,might also be potential substrates for RhEstl.The K_m values for methyl2,2-dimethylcyclopropanecarboxylate(DmCpCm)and ethyl 2,2-dimethylcyclopropane carboxylate(DmCpCe)were 0.25 and 0.43 mM,respectively.RhEstl could serve as an efficient hydrolase for the bioproduction of optically pure(S)-2,2-dimethyl cyclopropane carboxylic acid(DmCpCa),an important chiral building block for cilastatin.As much as 0.5 M of DmCpCe was enantioselectively hydrolyzed into(S)-DmCpCa with a molar yield of 47.8%and an enantiomeric excess(ee)of 97.1%,indicating an extremely high enantioselelctivtiy(E=204)and a promising prospective of this novel RhEstl for biomanufacturing valuable chiral chemicals.




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