Study on keratinase from highly efficient feather hydrolyzing microorganisms

Isolation of microbes producing keratinolytic enzymes from various sources in Thailand was performed. 9 isolates possessing feather degradation of 36.58-91.83 percent by weight were found. According to secondary screening of optimum pH, optimum temperature, pH stability and temperature stability of keratinase, the isolate K6, K82 and K103 were selected. The keratinase of K6, K82 and K103 showed optimum pH at 5-7, 7-10 and 6-9 and pH stability of 6-7, 5-10 and 5-10 respectively. They all played optimum temperature and temperature stability for 1 hour at 60 and 40-50 deg C, respectively. when synergistic reaction was studied, enzymatic activity co-operation of isolate K6 and K82 was higher than alone for 1.2 times whereas either of them cooperating to K103 resulted in reducing enzymatic activity. Therefore K6 and K82 were selected for further studies. By taxonomic study, isolate K6 and K82 belonged to Bacillus licheniformis and B. pumilus respectively. Lyophilization, Biogel-P100 gel filtration and strong anion exchange chromatography (Econo-Pac Q Cartridge (-N+(CH3)3) were used to purity enzymes. Sodium dodesyl sulfate electrophoresis indicated that the purified keratinase from K6 and K82 were dimeric designated SUK A as well as SUK B and monomeric as SUK C, and have molecular weight of 70, 70 and 45 kDa, respectively. Enzymatic kinetic directing to amino acid production was studied. The value of Km and Vmax from SUK A, SUK B and SUK C were 5.32*10*[-4), 5.21*10*[-4), 2.02*10*[-4) mol/l and 0.406, 0.412, 1.120 micromol/min respectively resulting in higher activity of SUK C. This result also support higher amino acid production of 1.5-30.8 percent due to the cooperation of SUK C to SUK A, SUK B or SUK A and SUK B. Study on the enzymatic activity of SUK A and SUK B, showing higher feather degradation than SUK C for 1.7 and 1.9 times respectively. The cooperation of SUK A and SUK B produced the hydrolyzed feather with highest increased pepsin digestibility of 27.26 percent comparing to action alone when the ratio of 5:1 was used.