Purification and characterization of solvent-tolerant, thermostable, alkaline metalloprotease from alkalophilic Pseudomonas aeruginosa MTCC 7926

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. KM and Vmax, 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) >95% in metal ions (Mn2+, Ca2+, Mg2+, Fe2+) for 1 h, (c) >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

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