Purification and characterization of solvent‐tolerant, thermostable, alkaline metalloprotease from alkalophilic <i>Pseudomonas aeruginosa</i> MTCC 7926

Abstract

Abstract BACKGROUND: Microbial proteases are becoming imperative for commercial applications. The protease secreted by Pseudomonas aeruginosa MTCC 7926, isolated from solvent‐contaminated habitat was purified and characterized for activity at various edaphic conditions. The purified alkaline protease was investigated for dehairing of animal skin, anti‐staphylococcal activity and processing of X‐ray film. RESULTS: The protease was 24‐fold purified by ammonium sulfate fractionation, sephadex G‐100 gel filtration and DEAE‐cellulose, with 36% recovery. K M and V max , using casein were 2.94 mg mL −1 and 1.27 µmole min −1 , respectively. The apparent molecular mass by SDS‐PAGE was 35 kDa. Alkaline protease was active at pH 6–11 and temperature 25–65 °C. Its activity was (a) 86.8% in 100 mmol L −1 NaCl, (b) &gt;95% in metal ions (Mn 2+ , Ca 2+ , Mg 2+ , Fe 2+ ) for 1 h, (c) &gt;90% in bleaching agents and chemical surfactants, (d) 135.4 ± 2.0% and 119.9 ± 6.2% with rhamnolipid and cyclodextrin, respectively, (e) stable in solvents for 5–30 days at 27 °C, and (f) inhibited by EDTA, indicating metalloprotein. CONCLUSION: This work showed that purified protease retained its activity in surfactants, solvents, metals, and bleaching agents. The enzyme is an alternative for detergent formulations, dehairing of animal skin, X‐ray film processing, treatment of staphylococcal infections and possibly non‐aqueous enzymatic peptide synthesis. Copyright © 2009 Society of Chemical Industry