Optimization of protease production by Bacillus sp. EBTA6 and investigation of industrial application potentials of the enzyme

In this study, 32 Bacillus isolates, that were previously isolated from soil and food samples, were screened for protease production and one of the best producers, Bacillus sp. EBTA6, was selected for the optimization of protease production and the determination of the properties of the enzyme as well as the industrial applications. Central Composite Design (CCD) of Response Surface Methodology was used for the optimization. For that purpose, three important independent variables (pH, temperature and yeast extract) and two dependent variables (protease activity and growth) have been determined. The results showed that all of the independent factors have significant effect on the enzyme activity and yeast extract concentration and temperature had significant effect on the growth. Optimum conditions for protease production were determined as 30°C, pH 9, and 15 g/L yeast extract concentration with the activity of 3207,01 U/mL. A 2,6 times increment in the activity have been achieved after the optimization. The optimum pH and temperature of the enzyme was determined as 8 and 50-60°C, respectively. The enzyme was stable at a broad pH range between 7 and 10 (82-93 %). It retained 55% of its activity at 50°C in 150 min. Amongst the metal ions, K+, Cu2+, Sn2+ and Zn2+ slightly inhibited the activity while Ca2+ and Mg2+ increased it. EDTA, PMSF and SDS have decreased the the activity, on the other hand Tween 20 and Tween 80 did not effect it. Km and Vmax values of the enzyme were deterimed as 2,762 mg/mL and 3125 U/mL, respectiely. It possessed considerably high milk clotting activity in the presence of CaCl2. Washing performance of the enzyme which were tested on blood and ketchup stains showed that it was quite effective at 50°C. The studies have revealed that the protease from Bacillus sp. EBTA6 was serin alkaline and had industrial potential.