Purification and characterization of alkaline protease from Alcaligenes faecalis

Extracellular alkaline protease from the alkalophilic bacterium Alcaligenes faecalis was purified by a combination of ion-exchange and size-exclusion chromatographic methods, and its properties were examined. The purified enzyme had a specific activity of 563.8 mumol of tyrosine/min per ring of protein and gave a single band on native PAGE and SDS/PAGE with a molecular mass of 67 kDa. Gelatin zymogram also revealed one clear zone of proteolytic activity which corresponded to the band obtained with native PAGE and SDS/PAGE. The enzyme had an optimal pH of 9.0 and exhibited its highest activity at SS degreesC. The enzyme activity was inhibited by PMSF, suggesting the presence of serine residues at the active site. The enzyme had a K-m of 1.66 mg/ml and a V-max of 526 units/min per mg of protein with casein as the substrate.