BACKGROUND: Purification and characterization of a novel protease produced by Bacillus sp. RKY3, has been investigated, with special emphasis on the stability of the enzyme in the presence of different oxidizing and reducing agents as well as organic solvents. The enzyme was purified in two steps through concentration of the crude enzyme by ammonium sulfate precipitation, followed by anion exchange chromatography. RESULTS: The purified protease had a molecular mass of approximately 38 kDa, which was highly active over a broad range of pH between 7.0 and 9.0 and was also stable over a wide pH range from 5.0 to 11.0. Although the optimum temperature for enzyme activity was found to be 60 degrees C, it was rapidly deactivated at temperatures above 60 degrees C. It also showed good stability at 50 degrees C, with a 70 min half-life. Ca2+ ions did not greatly enhance the activity or the stability of the enzyme. PMSF (1 mmol L-1) completely inhibited the protease activity, and thus the purified protease was considered to be serine protease. The purified protease was stable with oxidants (H2O2, 2%), reducing agents (sodium dodecyl sulfate, 2%), and organic solvents (25%) such as benzene, hexane, and toluene. CONCLUSION: The purified enzyme, protease, seems to possess potential applications in protease-based detergent and bleaching industries. The enzymatic activity against a wide variety of substrates suggests that the purified enzyme should be investigated for a range of commercial applications, especially for soy protein and gelatin hydrolysis in the food processing industry. (C) 2008 Society of Chemical Industry