Screening Of Methyl Phenylglycidate Hydrolase Producing Microorganisms And Characterization Of The Corresponding Esterase

Methyl (2S,3R)-phenylglycidate [(2S,3R)-MPG] is an important intermediate for production of Taxol and (-)-Clausenamide. In this paper, a new strain, Enterobacter sp. ECU1107, was isolated from over200soil samples through a two-step screening strategy by using racemic MPG as the sole carbon source. Whole cells of Enterobacter sp. ECU1107were employed for the enantioselective hydrolysis of racemic MPG. The optimum temperature and pH of the whole-cell mediated bioreaction were40°and6.0, respectively. Organic-aqueous biphasic system was employed and isooctane was selected as the best organic solvent. A decagram scale preparation of (2S,3R)-MPG was performed with a substrate loading of600mM, affording (2S,3R)-MPG in>99%ee and43.5%isolated yield.An esterase, nEst01, was cloned from Enterobacter sp. ECU1107and expressed in Escherichia coli. The recombinant E. coli whole cells expressing nEst01were used in enzymatic hydrolysis of100mM racemic MPG, the enantiomeric excess of the remaining (2S,3R)-MPG reached>99%at about50%conversion after2h reaction. The optimal reaction pH was10.5. The recombinant EnEst01showed high activity toward alkyl carboxylates with short chains and the activities decreased sharply with the increase of the carbon chain length. Thermostability of the esterase EnEst01was relatively poor, with a half-life of only4.6h at40℃and deactivated completely within several minutes at50℃. The inactivation energy was determined to be216kJ/mol. A molecular structure modulation of EnEst01was constructed by homogenous modeling and the low thermostability was confirmed through B-factor modulation.