Isolation, Production, Purification, Assay and Characterization of Alkaline Protease Enzyme from Aspergillus niger and its Compatibility with Commercial Detergents

Aspergillus niger is a highly potent fungus used in the production of alkaline protease. Extra cellular alkaline protease was purified from A. niger in a twostep procedure involving ammonium sulphate precipitation and Sephadex G100 column chromatography. The molecular mass of the enzyme was determined to be 60 kDa by SDS-PAGE. The enzyme activity was also analyzed by zymogram with gelatin. The enzyme was more stable over a wide range of pH (6–12) and the temperatures up to 37 °C. It showed optimum enzyme activity at pH 8.0 and a temperature of 30°C. If the enzyme concentration is gradually increased then activity increases and if the enzyme concentration is kept constant, and the substrate concentration is gradually increased then also activity increases. The activity of the enzyme on substrate casein also increases with increase in concentration of the enzyme. The protease enzyme was completely inhibited by the serine protease inhibitor of phenylmethylsulfonyl fluoride (PMSF) and Calcium chloride was the most suitable activator. The crystallization of the purified enzyme was performed by hanging drop vapour diffusion method using PEG 6000 as the precipitant. The micro crystals occurred in 40-50% of PEG 6000. The enzyme was most compatible with commercial detergent Tide in absence of substrate and most compatible with sunlight in presence of substrate.