| In order to produce keratinase and elucidate the mechanism of keratin degradation, a strain withhigh keratinolytic activity and well feather-degrading capability named KD-N2 was screened. Theculture medium and conditions were optimized and processes of fermentation in feather and hairsubstrates in 5L bioreactor were discussed; then the purification and characterization of keratinaseproduced in feather substrate were discussed as well as the processes and mechanism of keratindegradation; at last the dehairing capabilities of crude keratinases on goat skin and calf skin weredetermined.The results were shown as below:(1) A strain KD-1 with feather-degrading capability was screened and identified as Bacillus subtilisbased on morphological, physiochemical, and phylogenetic characteristics. A mutant named KD-N2with 1.5 times higher keratinolytic activity was screened with NTG mutagenisis and the mutant hadoptimal growing conditions of 28℃and pH value of 6.6, respectively. The crude keratinase showedoptimum reaction temperature and pH value of 50C and 8.0, respectively. Soluble keratin and keratinazure were used as substrates for keratinase activity determination.(2) Effects of keratin-containing substrates of feathers, silk, wool and hair on keratinase productionwere studied and the optimum substrate was of 10 g/L feathers. Optimized fermentation conditions wereof initial pH value of 7.5 to 8.0, 2％to 5％inoculum size, 23℃and inoculum age of 16h. Orthonaldesign experiments resulted that the optimal culture medium in 10 g/L feather as sole carbon andnitrogen source were (g/L): NaC1 0.5, MgSO4 0.2, KH2PO4/K2HPO4 0.35/0.7. In this medium, thekeratinase activity reached 66.5 U/mL after 24h and maximum in 30h as well as soluble protein. After30h cultivation, essential amino acids of Threonine, Valine, Methionine, Isoleucine, Phenylalanine andLysine were produced and the maximal one was Cysteine in concentration of 0.154 mg/mL, NH3 wasof 0.4424 mg/mL. Further experiments showed that 0.5 g/L beef extract as well as 2 g/L corn flour wereoptimal for keratinase production. And the optimized medium were (g/L): NaC1 0.5, MgSO4 0.2,KH2PO4/K2HPO4 0.35/0.7, CaCl2 0.01, beef extract 0.5 and corn flour 2.0. Adding carbon and nitrogensources at the same time had negative effects for the inducible keratinase production.(3) Optimal conditions for keratinase production in hair substrate were of 16 g/L hair, initial pHvalue 6.5, 20ml culture volume and 10％inoculum size; the optimal production period was 36h in 10g/L hair, 5％inoculum size at 28C and 200 rpm; sucrose, corn flour and yeast extract in 10 g/L wereoptimal for keratinase production. Fractional factorial design experiments showed that MgSO4 andK2HPO4 were the main factors for keratinase production and central composite design experimentsresulted that the optimal culture medium were (g/L): MgSO4 0.832, K2HPO4 2.38, NaCl 1.0, CaCl20.05, KH2PO4 0.4, sucrose 3. Keratinase activity produced in the optimized medium was 124.8 U/mLand was 83％higher than the primary culture medium.(4) During fermentation processes in both feather and hair substrates, the pH value, content ofsoluble amino acids and protein increased gradually, and amino acids production in feather substratecould be described with Peal-Reed model: Y=3.5479/1+6.5259e3.4823x2-0.42916x-0.00105x3The kinetic models of cell growth and product formation in feather substrate could be described asdx/dt=0.1013(1-x/0.3742)xanddp/dt=276.69 dx/dt-3.6x, respectively.The kenetic models of cell growth and product formation in hair substrate could be described asdx/dt=0.0728 (1-x/0.197)xanddp/dt=422.34 dx/dt+5.187x, respectively.(5) The keratinase was purified using 40-80％saturation ammonium sulphate precipitation,Sephadex G-75 and DEAE-Sepharose chromatographic techniques. The purified enzyme had themolecular mass of 30.5 kDa as determined by SDS-PAGE analysis. The optimum pH at 50℃was 8.5and the optimum temperature at pH 8.5 was 55℃. The keratinase was made partially inactive by Ca2+,Mg2+, Zn2+, Al3+, methanol, ethanol, DMSO, isopropanol and serine protease inhibitor PMSE EDTA inconcentration of 2.5 mM, 5 mM and 10 mM showed stimulatory effects on the keratinase activity.Reducing agents of DTT, mercaptoethanol, L-cysteine, sodium sulphite and chemicals of SDS,ammonium sulfatate, DMSO stimulated the enzyme activity towards substrate of feather meal. Besidesfeather keratin, the enzyme showed activity towards soluble proteins of ovalbumin, BSA, casein andinsoluble ones of sheep wool and human hair. Calf hair, silk and collagen could not be hydrolyzed bythe enzyme.(6) With the increasing of feather concentration, the degradation rate of substrate decreasedgradually, and the optimal conditions in 10 g/L feather content were initial pH value of 8.0-8.5, 32℃,2％inoculum size and 20h inoculum age. Scanning electron microscopy showed the degradation offeather, hair and silk to some extent during cultivation, and it was presumed to be the results ofsubstrates swell in culture, cell growth, keratinase hydrolysis and mechanical vibration.(7) Dehairing capabilities of keratinase produced in both feather and hair substrates were carriedout and the results showed that both crude keratianses could remove hair from goat and calf skins. Thedeharing capabilities were better in higher temperature. And the calf skin was more easily dehairedwithin 12-16h by keratinase produced in feather substrate and within 16-20h by keratinase from hairsubstrate.
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