Isolation of keratinase producing bacteria and increasing of enzyme production by molecular methods

In the present study, three keratinolytic Bacillus strains were isolated from the feather containing soil samples collected from the poultry enterprise of Çukurova University. The isolates were entitled as Bacillus sp. MK1, MK2, and MK3, respectively. The optimum enzyme activities were observed at 40°C for MK1 and MK3 keratinases whereas 50 ºC for MK2 keratinase. Similarly, optimum pH value for MK1 and MK3 keratinase was 9.0, whereas 8.0 for MK2 keratinase. The specific activities of MK1, MK2, and MK3 keratinases were 2.76, 0.77 and 5.48 U/mg protein at 40°C, respectively. Maximum enzyme productions of isolates were observed after 36 hours for MK1, and 24-36 hours for MK2, and MK3. Over-expressing mutant varieties MK1-M3, MK1-M4 and MK1-M5 from MK1, MK2-M3 and MK2-M4 from MK2, and MK3-M1, MK3-M3, MK3-M4 and MK3-M5 from MK3 were obtained after EtBr treatment. MK1-M3, MK1-M4, MK1-M5, MK2-M3, MK2-M4, MK3-M1, MK3-M3, MK3-M4 and MK3-M5 have produced 186, 117, 133, 144, 171, 122, 116, 214 and %187 keratinase according to their own wild type strains, respectively. PMSF, urea, CaCl2 and Tween 80 were determined common activators for all enzymes. However, EDTA has also inhibited all these three enzymes. According to BLAST analysis, Bacillus sp. MK1 rDNA sequence was similar to B. subtilis NCDO 1769 rDNA sequence at the rate of 96%. On the other hand, Bacillus sp. MK2 and MK3 rDNA sequences were similar to B. subtilis NRRL B-4219 and B. tequilensis 10b rDNA sequences at the rate of 99%, and B. subtilis SBMP4 rDNA sequence at the rate of 98%.