Isolation and characterization of alkalophilic Pseudomonas sp. and optimization of culture conditions for alkaline protease production

Proteases are well known enzymes for their wide range application in food industry, detergent industry and pharmaceuticals industry. They are also widely used in leather industry for dehairing and bating of hides as an alternative for toxic chemicals which in turn hamper the environment. In the present investigation a total of 20 bacteria were isolated from the leather industry plant effluent. All isolates were screened for proteolytic activity using alkaline skim milk agar plate, among 20 bacterial isolates only 3 isolates showed alkaline proteolytic activity, one isolate was selected for further study. The effect of carbon sources, nitrogen sources, pH and temperature on production of alkaline protease was investigated by one factor-at-a-time method. The best enzyme activity was observed at pH 8 and temperature 37 °C. Protease activity was enhanced by Cu2+and Mg2+. Hg2+ and Zn2+ slightly activated the protease activity. Fe2+ slightly inhibited the proteolytic activity. Extracellular alkaline protease was purified from Pseudomonas isolate in a two-step procedure involving ammonium sulphate precipitation and Sephadex G-100 column chromatography. The molecular size of the alkaline protease enzyme was determined to be 43 kDa by SDS-PAGE. Isolation and characterization of alkalophilic Pseudomonas sp. and optimization of culture conditions for alkaline protease production