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65 strains of proteinase producer bacteria were separated from the fresh milk among which 3 strains, produce more Proteinase, were selected to do some physiology and biochemistry experiments and optimize their condition about producing proteinase. Finally ,the strains which can produce most Proteinase were selected to study enzymology and do mutagenize in order to elevate their ability about producing proteinase .Bs4、Bs15 and Bs2917 their ability higher and stationary about producing proteinase were identitied by physiology and biochemistry experiments . According to 16S rDNA homology analysis,strain of Bs4 belong to Bacillus subtilis primarily.The best condition of culture of Bs4 were fermentation temperature37℃,PH6.5, culturing duration36h,amount of inoculation3% by optimization,and the activity of enzyme reached 1888.73U/mL. The best condition of culture of Bs15 were fermentation temperature32℃, PH7.5 , culturing duration36h , amount of inoculation3% by optimization,and the activity of enzyme reached 712.18U/mL.The best condition of culture of Bs2917 were fermentation temperature32℃,PH7.0, culturing duration48h,amount of inoculation3% by optimization , and the activity of enzyme reached 367.57U/mL.Liquid state medium was optimized through orthogonal experiment tand one-factor experiments .The suitable medium contained of Bs4 (g/L): corn powder 33.4g/L, bran33.4g/L, bean cake powder30.8g/L, (NH4)2HPO4 10.75g/L ,KH2PO4 1.34g/L.The activity of enzyme reached 2129.31U/mL after culturing at fermentation temperature37℃,culturing duration36h.The suitable medium contained of Bs15 (g/L): corn powder29.95g/L, bran29.95g/L, bean cake powder 31.8g/L, (NH4)2HPO4 /NaH2PO4 5.9g/L. The activity of enzyme reached 1390.79U/mL after culturing at fermentation temperature32℃,culturing duration 36h.Study of enzyme characterizations about strain Bs4 found out that the optimum temperature and pH value for enzyme producing are 57℃and PH7.0 respectively. Mg2+、Ca2+、K+ can activate enzyme-producing ,and then Zn2、+Fe3+have a inhibition to enzyme. In order to improve the strain to get a high proteinase producing variety,different physical and chemic mutagens were used to mutagenize the initial strain Bs4.UB-12 high yield strain was obtained after mutagenized by UV,Nitrite and Microwave and the enzyme activity was about3014U/mL which was 40% higher than the initial strain and shows fine stability.
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