Screening Of Novel Ester Hydrolyases And Application For Resolution Of Racemic Flurbiprofen

More than 60 strains with selectivity to (S) or (R)- flurbiprofen ethyl ester were isolated from soil samples by enrichment culture in a medium containing flurbiprofen ethyl ester as a sole carbon source. The strains NH33 and C71 were the best strains in term of their activity and enantioselectivity, and were identified as Pesudomonas putida NH33 and Bacillus cereus C71 respectively. The two strains produced enantiomerically pure (S)-flurbiprofen and (R)-flurbiprofen from their corresponding racemic ethyl ester respectively. After optimizing the hydrolysis reaction conditions including pH, temperature, surfactants and solvents, a small-scale conversion containing lOmmol/L flurbiprofen ethyl ester was carried out with the two strains. The total yield about 60% with a 98%ee of (S)-isomer and a 93%ee of (R)-isomer was achieved. DNA library of P. putida NH33 was constructed, and a positive clone, pNHIO, expressing a single ester hydrolyase was selected. The pNHIO showed a higher stereospecificity compared with the wild type strain, and produced (S)-flurbiprofen (>99%ee) from its ethyl ester.