Microbial production, purification, and characterization of (S)-specific N-acetyl-2-amino-1-phenyl-4-pentene amidohydrolase from Rhodococcus globerulus K1/1

Rhodococcus globerulus K1/1 was found to express an inducible (S)-specific N-acetyl-2-amino-1-phenyl-4-pentene amidohydrolase. Optimal bacterial growt h and amidohydrolase expression were both observed at about pH 6.5. Purific ation of the enzyme to a single band in a Coomassie blue-stained SDS-PAGE g el was achieved by nucleic acid and ammonium sulfate precipitation of Rhodo coccus globerulus K1/1 crude extract and column chromatography on TSK Butyl -650(S) Fractogel and Superose 12HR. The amidohydrolase was purified to a h omogeneity leading to a tenfold increase of the specific activity with a re covery rate of 65%. At pH 7.0 and 23 degrees C the enzyme showed no loss of activity after 30 days incubation. The amidohydrolase was stable up to 55 degrees C. The enzyme was inhibited strongly only by 10 mM Zn2+ among the t ested metal cations and was inhibited 100% by 0.01 mM phenylmethanesulfonyl fluoride. The molecular weight of the native enzyme was estimated to be 92 kDa by gel filtration and 55 kDa by SDS-PAGE, suggesting a homodimeric str ucture.