A new (S)-ketoprofen specific esterase (EST-Y29) was identified from a metagenome library from environmental samples, which showed homologies with class C-beta lactamase, penicillin binding protein, and other lipases/esterases. In order to investigate the biochemical and biophysical properties, the recombinant protein was overexpressed, purified to homogeneity, and characterized. This EST-Y29 has high catalytic activity against p-nitrophenyl esters of short fatty acids (C(2) and C(4)) and alpha-naphthyl acetate with activation energy of 30.4 kJ/mol. We have further characterized EST-Y29 using high performance liquid chromatography (HPLC), circular dichroism (CD), dynamic light scattering (DLS) and size exclusion chromatography (SEC).