Screening Keratinase Production Strain And Optimizing Fermentation Process And Studying On Characterization And Application

Keratin is a kind of insoluble structural protein,that can not be degraded by dilute acids,diluted alkali and salt solution.The poultry feathers contains more than 80%keratin.A large number of abandoned feather caused environmental pollution,but at the same time,it lead a serious waste of resources,and its comprehensive utilization,not only can solve the shortage of feed protein,but also can reduce environmental pollution In this study,a kind of keratinase producing strain was isolated with feather as the sole carbon source and nitrogen source,and the optimization of fermentation condition,separation and purification,characterization of the enzyme and application in feather degradation.The main research content is as follows:(1)Rotten feather and soil samples were obtained from the chicken farm,and a strain of high yield keratinase was screened with feathers as the sole carbon and nitrogen source: CJPE209,which was identified as Bacillus sp.And the preservation number was CCTCC M 2015734.(2)The fermentation conditions were as follows: fermentation time 48 h,fermentation temperature 37 ?,inoculation voulme 2%,shaking speed 220r/min,loading volume 25 mL/250 mL,initial pH7.The culture components were further optimized by PB design and CCD design,the best medium was sucrose 13.6 g/L,feather meal 5.6 g/L,urea 4 g/L,potassium dihydrogen phosphate 0.4 g/L,magnesium sulfate 1.44 g/L,calcium chloride 1.1 g/L and sodium chloride 5 g/L.Under the conditions of optimum culture conditions and optimum culture medium,the enzyme activity of fermentation broth reached 503.5 U/mL,which was 1.88 times before optimization.(3)The keratinase produced by Bacillus sp.CJPE209 was purified by a series of processes: ammonium sulfate precipitation,Sephadex G-25 gel filtration desalting,DEAE cellulose DE-52 chromatography and Sephadex G-75 gel filtration.It specific activity was 8527.19 U/mL,the purification fold was 4.59 times and the yield was12.35%.The molecular weight of the keratin was determined by sodium dodecyl sulfate electrophoresis at 43 kDa.The optimum reaction temperature of the keratin was 60 ?,the optimum reaction pH was 8,and the stability was maintained in neutral and alkaline environment.And to studied the effects of metal ions and chemical reagents on keratinase activity and the kinetics parameters of keratinase.(4)The degradation ability of keratinase from Bacillus sp.CJPE209 was determined by the feather degradation test.The crude enzyme solution could reduce the feathers by half during 48 h,and produce soluble amino nitrogen.The degradation products can be used as feed additives for animal feed.