PURIFICATION AND CHARACTERIZATION OF AN ENANTIOSELECTIVE AMIDASE FROMPSEUDOMONAS-CHLORORAPHIS B23

An amidase produced by Pseudomonas chlororaphis B23 was purified and c haracterized. The purification procedure used included ammonium sulfat e precipitation and hydrophobic, anion-exchange, gel filtration, and c eramic hydroxyapatite chromatography steps. This amidase has a native molecular mass of about 105 kDa and is a homodimer whose subunits have a molecular mass of 54 kDa. The enzyme exhibited maximal activity at 50 degrees C and at pH values ranging from 7.0 to 8.6. We found no evi dence that metal ions were required, and the enzyme was inhibited by s everal thiol reagents. This amidase exhibited activity against a broad range of aliphatic and aromatic amides and exhibited enantioselectivi ty for several aromatic amides, including 2-phenylpropionamide (enanti omeric excess [ee] = 100%), phenylalaninamide (ee = 55%), and 2-(4-chl orophenyl)-3-methylbutyramide (ee = 96%), but not 2-(6-methoxy-2-napht hyl) propionamide (the amide form of naproxen) (ee = 0%). The characte ristics of the P. chlororaphis B23 amidase are the same as the charact eristics of enantioselective amidases described by Mayaux et al. (J. F . Mayaux, E. Cerbelaud, F. Soubrier, D. Faucher, and D. Petre, J. Bact eriol. 172:6764-6773, 1990; J. F. Mayaux, E. Cerbelaud, F. Soubrier, P . Yeh, F. Blanche, and D. Petre, J. Bacteriol. 173:6694-6704, 1991) an d Kobayashi et at (M. Kobayashi, H. Komeda, T. Nagasawa, M. Nishiyama, S. Horinouchi, T. Beppu, H. Yamada, and S. Shimizu, fur. J. Biochem. 217:327-336, 1993).