• KSBB Journal
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• v.25 no.5
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• pp.429-436
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• 2010

Keratinase is widely used in certain industrial applications. The present study sought to improve the culture conditions of Bacillus subtilis SMMJ-2 to facilitate mass production of keratinase. Strain SMMJ-2 was irradiated by ultraviolet light and the resulting isolates were tested for keratinase activity. Isolates displaying elevated keratinase activity were selected and used to determine the optimum temperature (24, 30, 37, 45, $55^{\circ}C$) for bacterial keratinase production during a 4 day incubation period. The highest enzyme activity (55 units/mL/min), from a Bacillus subtilis SMMJ-2 mutant (mutant No. 2) was demonstrated following incubation at $30^{\circ}C$. The effects of carbon and nitrogen sources on keratinase production were confirmed by measuring the enzyme activity from the culture broth of the mutant strain cultured in various media containing different carbon source and nitrogen sources during a 4 day period. The optimal medium composition for producing keratinase consisted of 1% glucose, 0.7% $K_2HPO_4$, 0.2% $K_2HPO_4$, and 1.2% soybean meal. Optimal initial pH and temperature for producing keratinase were 7.0 and $30^{\circ}C$, respectively. Keratinases produced by B. subtilis SMMJ-2 and the mutant No. 2 were purified from the culture broth which used soybean meal as a nitrogen source. Membrane ultrafiltration, DEAE-sephacel ion exchange and Sephadex G-100 gel chromatography were used to purify the enzymes. The purified keratinases from both B. subtilis SMMJ-2 and the mutant No. 2 showed single bands and their molecular weights were estimated as 28 kDa and 42 kDa, respectively on SDS-polyacrylamide gel electrophoresis.