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Enzymes stable and active in non-aqueous media are in large demand for their increasing application in organic synthesis. A solvent-stable protease is reported from a solvent tolerant strain (PseA) of Pseudomonas aeruginosa. Production of this novel protease was optimized by varying the culture conditions. Glycerol (0.7%) as carbon source, casein (0.4%) and yeast extract (0.6%) as nitrogen sources, CaCl₂ (0.6mM) as metal ion in culture media (pH 7.0), inoculated with 2.5% of 36h grown mother culture and incubation for 24h at 30°C with 250rpm shaking were found to be the best culture conditions leading to maximum growth and protease production (1601U/ml). The enzymatic properties of the protease revealed, pH and temperature optimum of 9.0 and 55°C, respectively. It retained 100%, 91% and 80% of its initial activity after heating for 30min at 45, 50 and 55°C, respectively. The enzyme exhibited …