Purification, biochemical characterization and performance evaluation of an alkaline serine protease from Aspergillus flavus MTCC 9952 mutant

A ~38 kDa alkaline protease of mutant Aspergillus flavus MTCC 9952 was purified upto homogeneity by 18.5-fold with 21.0% recovery. The protease was optimally active at pH 11.0 and temperature 40 °C. It revealed significant stability in pH range 8.0–11.0 and temperature range 35–50 °C. Casein was the most preferred substrate which displayed Km=1.95±0.03 mg/ml and Vmax=476.2±9.4 µg tyrosine/ml/min. The catalytic activity was enhanced by Fe++, Ca++ and Ni++ while completely inhibited by Cu++ and Hg++. Phenyl Methyl Sulphonyl Fluoride caused complete inhibition whereas, activity was trivially inhibited by Ethylene Diamine Tetra Acetic Acid and activated by reducing agents beta-mercaptoethanol and di-thiothreitol. The enzyme exhibited excellent stability towards H2O2, surfactants and laundry detergents. The purified protease was activated in the presence of benzene (120.1%) and chloroform (122.3%), nevertheless, retained significant stability with Acetone (90.2%), Diethyl ether (93.0%), n-Hexane (92.4%), Octane (99.8%), and Dodecane (95.0%). In application studies, the protease completely removed blood stains from cotton cloths when used with detergents and hydrolyzed gelatin from waste X-ray films. Presumably, this is the first report on alkaline protease from A. flavus, exhibiting organic solvent stability and gelatin hydrolysis from used X-ray films. Conclusively, this protease could potentially be used in detergent formulation, silver recovery from waste X-ray films and peptide synthesis.