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 µmol of tyrosine/min per mg 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 55 °C. The enzyme activity was inhibited by PMSF, suggesting the presence of serine residues at the active site. The enzyme had a Km of 1.66 mg/ml and a Vmax of 526 units/min per mg of protein with casein as the substrate.