KERATINOLYTIC PROTEASE PRODUCTION AND CHARACTERIZATION FROM BACILLUS SP. ISOLATED FROM POULTRY WASTES

摘要

Keratinases originating from microorganisms are used in many industrial fields such as the recycle of keratinous wastes, leather, textile, the 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 on the basal feather-meal medium. Strain H62 with the highest keratinase activity was determined as the keratinase producer and identified as Bacillus licheniformis with microscopic, biochemical (VITEK-2, 90%) and molecular analysis (16S rRNA, 99%, B. licheniformis 9945A). 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-meal 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. 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 enzyme was 100% stable whereas activity was decreased at the other temperatures. At room temperature, a loss of activity was determined after the 24th hour. EDTA, SDS and Urea increased the enzyme activity; however, Tween-20 was decreased. The enzyme was seen to be a single band with a molecular weight of 26 kDa. As a result, keratinase B. Licheniformis H62 is an enzyme that can be used in mesophyll and alkaline conditions, particularly in medical applications and as feed supplements.