title: Cloning and Characterization of a Novel Esterase from Rhodococcus sp. for Highly Enantioselective Synthesis of a Chiral Cilastatin Precursor.
abstract: A novel nonheme chloroperoxidase (RhEst1), with promiscuous esterase activity for enantioselective hydrolysis of ethyl (S)-2,2-dimethylcyclopropanecarboxylate, was identified from a shotgun library of Rhodococcus sp. strain ECU1013. RhEst1 was overexpressed in Escherichia coli BL21(DE3), purified to homogeneity, and functionally characterized. Fingerprinting analysis revealed that RhEst1 prefers para-nitrophenyl (pNP) esters of short-chain acyl groups. pNP esters with a cyclic acyl moiety, especially that with a cyclobutanyl group, were also substrates for RhEst1. The Km values for methyl 2,2-dimethylcyclopropanecarboxylate (DmCpCm) and ethyl 2,2-dimethylcyclopropane carboxylate (DmCpCe) were 0.25 and 0.43 mM, respectively. RhEst1 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.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.
authors.forename: Jiang
authors.initials: J
authors.lastname: Pan
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.
authors.forename: Zheng-Jiao
authors.initials: ZJ
authors.lastname: Luan
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China guochaoxu@163.com jianhexu@ecust.edu.cn.
authors.forename: Guo-Chao
authors.initials: GC
authors.lastname: Xu
authors.affiliation: Department of Chemical and Biomolecular Engineering, Pusan National University, Busan, South Korea.
authors.forename: Sunghoon
authors.initials: S
authors.lastname: Park
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China guochaoxu@163.com jianhexu@ecust.edu.cn.
authors.forename: Jian-He
authors.initials: JH
authors.lastname: Xu
mesh_terms: D000595:Amino Acid Sequence; D002711:Chloride Peroxidase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D015377:Cilastatin / Q000378:metabolism*; D003001:Cloning, Molecular; D016000:Cluster Analysis; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D011480:Protease Inhibitors / Q000378:metabolism*; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D004269:DNA, Bacterial; D011480:Protease Inhibitors; D015377:Cilastatin; D002711:Chloride Peroxidase