Isolation And Identification Of A Keratin-Degrading Strain,Fermentation And Enzymatic Properties Of Keratinase

Feathers, as by-products of poultry slaughtering industry, have a huge annual production. Feathers are mainly composed of keratin, as much as90%, which is rich in necessary amino acids for animal growth, and also contains macroelement, microelement, as well as some unknown growth factors. Because the chemical structure of keratin is very stable, it is insoluble under general conditions, and it is difficult to hydrolyze by general proteinase. By using physical and chemical treatment could produce a low quality, severely damage of amino acids, high energy consumption, high pollution problem, however, the use of biological degradation of keratin is a good alternative, not only improve the nutritional value of feather keratin, but also make use of environmental waste. At the same time, the use of keratin and keratinase in fertilizer, medicine, cosmetics and other areas have valuable use and broad prospects for further development.A strain of degrading feather keratin bacterium with high efficiency was isolated from environment by adopting the culture medium taking feather meal as unique carbon and nitrogen source. This strain was identified as Bacillus pumilus by morphology, physiological and biochemical experiments and16Sr RNA gene analysis, and named as Bacillus pumilus WHK4. The degradation rate of feather keratin reached to85.76%after48h fermentation with the strain. Using Kunming white mouse as the experimental animals, using the number of viable bacteria in up to8.7×1013CFU/mL Bacillus pumilus WHK4that had been cultivated for24h, and supernatant medium of WHK4feathers that had been fermented for60h fed mice. The acute toxicity tests showed that mouse group did not die, they were alive and well. Execution of the test group and control group mouse, anatomy observed that liver, kidney, lungs, intestines and other organs of experimental group mouse had no change. Above results showed that Bacillus pumilus WHK4and its fermentation are non-toxic products on mouse. The fermentation mediums of Bacillus pumilus WHK4were optimized. The optimum fermentation medium was determined:feathers100.0g/L, MgSO4·7H2O0.15g/L, NaCl0.15g/L, CaCl20.005g/L, K2HPO40.09g/L, KH2PO40.54g/L. The optimum fermentation conditions were initial pH8.0,6.0%inoculum, bacteria age18h, temperature37℃, rotation speed180r/min. The results showed that the degradation rate of feather was52.0%at60h under the optimized conditions. If feather was taken the place of equivalence father power, the degradation rate of feather power was74.4%at60h.To research feather fermentation process under the best medium composition and culture conditions. The results showed that dark muddy brown liquid was appeared in the cone-shaped bottle after36h fermentation with the strain. Diversification of feather scanned by electron microscopy and feather degradation rate determinate, the results showed that feather sprig and feather terrier were degradated simultaneously. Feather structure was damaged obviously after24h fermentation and the degradation rate of feather was21.5%. All of the feather sprigs were off almost and deep depression was appeared in the father terrier surface after60h fermentation and the degradation rate of feather was52.0%. Feather terrier was fractured and separated after72h fermentation and the degradation rate of feather was54.2%. Fermentation supernatant of SDS-PAGE electrophoresis showed that soluble small molecule protein in the fermentation supernatant was increasing although the extension of fermentation time. The content of soluble protein in the fermentation supernatant was the largest and the concentration was achieved to1.84mg/mL after84h fermentation.Fermentation processes which taking Bacillus pumilus WHK4with a high capability of degrading feather keratin as material were carried out to produce enzymes. Crude enzyme was prepared through low-temperature centrifugalization, and then its some properties were determined:the optimal temperature is60℃; it can maintain a good thermal stability in40℃-50℃; the optimal pH value8.0; DTT could improve its enzyme activity but PMSF intensively inhibits its activity.This study indicated that Bacillus pumilus WHK4is an appropriate strain for exploring microorganic fermentation system to degrade feather keratin, and had a great applied potential in producing high quality protein feed.