Mutant Strain İmprovement From Bacillus subtilis E6-5 By Chemical Mutagenesis (ethyl methane sulfonate, EMS) for Enhanced Production of Protease and Optimization of Growth Medium

In this study, the parent strain Bacillus subtilis E6-5 was exposed to EMS by classical mutation procedure, and 82 mutants were obtained. Among of these mutants, a mutant strain was selected based on its ability to produce highest zone formation (15 mm), and strain was named as Bacillus subtilis KE20. Obtained mutant was tested for enzyme production capacity in the liquid media, the mutant KE20 showed 9.2 fold increase in protease production over the parent strain Bacillus subtilis E6-5. It was found that mutant strain produced protease on maximum level at 48th hour. In addition, the effects of nutrional andphysical factors on enzyme production were examined. For this purpose, the effects of major medium ingredients (different carbon sources, nitrogen sources and metal ions) on the production of the enzyme were used. The best carbon source was found to be corn starch (560 IU/mL). Among the organic nitrogen sources, the highest enzyme production was obtained in the presence of skimmilk (624 IU/mL), CaCl2 (196 U/mL) was detected as effective as metal ion. In the physical parameters, the best results was obtained at 30°C, pH 8.0, 100 rpm asagitation rate, 3% as inoculum size and 3 days as inoculum age. A new medium was obtained by optimizing the incubation conditions (nutrional and physical factors) of protease production from KE20 mutant. In this medium, enzyme yield was enhanced 79% compared to basal medium. The production of protease by KE20 was inreased 16.5 fold compared to the parental strain. The mutant strain produced may have a prominent potential for protease production on an industrial scale.