Keratinolytic protease production and characterization from bacillus sp. isolated from poultry wastes

Keratinases originated from microorganisms are used in many industrial fields such as recycle of keratinous wastes, leather, textile, detergent industry and medical applications. In this study, 42 Bacillus strain were isolated from Cukurova University Research and Application and Chicken Management Unit. 8 of these isolates showed proteolytic activity on skim-milk and keratinolytic activity with keratin-azure when they were produced on the basal feather medium. Strain H62 with the highest keratinase activity was determined as the keratinase producer and identified as Bacillus licheniformis with microscopic (Gram +, endospor +, bacil), biochemical (VITEK-2, 90%) and molecular analysis (16S rRNA, 99%, 9945A). Conditions for enzyme production was optimized. The highest enzyme production was carried out at 40°C for 45 hours by adding 0.1 g/L mannitol (as carbon source), 0.1 g/L ammonium nitrate (as nitrogen source) and 15 g/L feather meal into the basal feather medium. Although keratinase showed the activity at 20-90°C and pH 5.0-13.0, optimum activity was obtained at 40°C and pH 9.5. Enzyme was treated with different buffers (pH 7.0-13.0) for 30 minutes. 100% of stability was determined at pH 8.0, whereas the loss of activity was observed at pH 7.0-9.0. After a pre-incubation at 20-100°C for 30 minutes enzyme was 100% stable whereas activity was decreased at the other temperatures. When the enzyme was hold on at the room temperature, a loss of activity was determined after 24th hour. EDTA, SDS and Urea increased the enzyme activity however Tween-20 was decreased. Enzyme was seem to be a single band with the molecular weight of 26 kDa. As a result, keratinase B. Licheniformis H62 is an enzyme that can be used in mesophile and alkali conditions, particularly in medical applications and as feed suplements.