Response surface methodology (RSM) was employed to enhance the production of organic solvent-stable protease by Bacillus sphaericus DS11. A significant influence of glycerol, MgSO(4)center dot 7H(2)O, and pH on organic solvent-stable protease production was noted with Plackett-Burman design. Then, a three-level Box-Behnken design was employed to optimize the medium composition and culture conditions for the production of the protease in shake-flask. Using this methodology, the quadratic regression model of producing organic solvent-stable protease was built and the optimal combinations of media constituents and culture conditions for maximum protease production were determined as glycerol 12.47 g/L, MgSO(4)-7H(2)O 0.73 g/L, and pH 8.25. Protease production obtained experimentally coincident with the predicted value and the model was proven to be adequate. The enhancement of protease from 465.06 U/mL to 1182.68 U/mL was achieved with the optimization procedure. (c) 2010 Elsevier Ltd. All rights reserved.