Cloning and Characterization of a Novel Esterase from Rhodococcus sp. for Highly Enantioselective Synthesis of a Chiral Cilastatin Precursor

ABSTRACT A novel nonheme chloroperoxidase ( Rh Est1), with promiscuous esterase activity for enantioselective hydrolysis of ethyl ( S )-2,2-dimethylcyclopropanecarboxylate, was identified from a shotgun library of Rhodococcus sp. strain ECU1013. Rh Est1 was overexpressed in Escherichia coli BL21(DE3), purified to homogeneity, and functionally characterized. Fingerprinting analysis revealed that Rh Est1 prefers para -nitrophenyl ( p NP) esters of short-chain acyl groups. p NP esters with a cyclic acyl moiety, especially that with a cyclobutanyl group, were also substrates for Rh Est1. The K m values for methyl 2,2-dimethylcyclopropanecarboxylate (DmCpCm) and ethyl 2,2-dimethylcyclopropane carboxylate (DmCpCe) were 0.25 and 0.43 mM, respectively. Rh Est1 could serve as an efficient hydrolase for the bioproduction of optically pure ( S )-2,2-dimethyl cyclopropane carboxylic acid (DmCpCa), which is an important chiral building block for cilastatin. As much as 0.5 M DmCpCe was enantioselectively hydrolyzed into ( S )-DmCpCa, with a molar yield of 47.8% and an enantiomeric excess ( ee ) of 97.5%, indicating an extremely high enantioselectivity ( E = 240) of this novel and unique biocatalyst for green manufacturing of highly valuable chiral chemicals.