title: [Purification and characterization of esterase from Morganella morganii ZJB-09203].
abstract: Enantioselective hydrolysis of 2-carboxyethyl-3-cyano-5-methylhexanoic acid (CNDE) is the key step in chemoenzymatic synthesis of pregabalin. We purified an intracellular carboxyl esterase from Morganella morganii ZJB-09203, which exhibited high enantioselectivity and activity towards CNDE. The carboxyl esterase was purified to electrophoretic homogeneity by ammonium sulfate fraction precipitation, Phenyl Sepharose 6 FF hydrophobic interaction chromatography, anion exchange with DEAE Sephadex A-50 and Bio-Scale CHT column. The purified enzyme was a monomer with molecular mass of 68 kDa determined by SDS-PAGE and gel chromatography. Substrate specificity of the enzyme towards p-nitrophenyl esters suggested that the purified enzyme was an esterase. The optimal reaction pH for CNDE hydrolysis was 9.0, and optimal temperature was 45 degrees C. The esterase was stable between pH 7.0 and 9.0, and at 40 degrees C. The enzyme activity was enhanced by Ca2+, Cu2+ and Mn2+, whereas strongly inhibited by Co2+, Fe3+, Ni2+ and EDTA. Meanwhile, we investigated the kinetic parameters of the esterase towards p-nitrophenyl esters and effect of CNDE concentration on conversion. The present study reported the esterase capable of stereospecific hydrolysis of CNDE for the first time. Our research will provide foundations for industrial production of Pregabalin using the new biocatalyst.
authors.forename: Renchao
authors.initials: R
authors.lastname: Zheng
authors.forename: Tianzhen
authors.initials: T
authors.lastname: Wang
authors.forename: Xiaojun
authors.initials: X
authors.lastname: Li
authors.forename: Yuguo
authors.initials: Y
authors.lastname: Zheng
mesh_terms: D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004950:Esterases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D020613:Morganella morganii / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D004950:Esterases