Yeast Protease Preparations

This research takes Xinjiang natural fermentation yeast as the starting strain, and screen the yeast with high yield protease by pure culture and Mutagenesis techniques.Yield optimization protease enzyme-producing conditions on the basis of single factor,And the separation and purification of protease ammonium sulfate precipitation method.This research takes chitosan as material and focused on the producing technology and properties of chitosan microspheres, protease immobilizing technology, enzymology properties of immobilized protease and influences of ethanol, ethylene glycol, sodium bisulfite, sodium chloride, urea and sucrose reagent agents to it. Studies are included in the thesis:1. Compound mutant screening of UV aluminum chloride form starting strain,the strain relative to the starting strain increased 68.49U?ml-1. The optimal p H value for proteinase producing is 10. Proteinase strain was obtained by compound mutation of UV and Aicl on mutant strain and proteinase activity is100 U?ml-1.The genetic stability of mutant strain is very good.2. Study of single factor(carbon source and nitrogen sources)and orthogonal test discovered that the fermentation medium 2% glycerol, 1.5% peptone, 0.025% calcium sulfate,Ga+ can activate enzyme-producing ability.Study of enzyme characterizations found out that the optimum temperature for enzyme producing are 60℃.3. The extraction and purification of the protease were investigated. The culture broth was centrifuged to separate solids. Solid ammonium sulfate was added to supernate with 50% saturation and the precipitate was removed as the impurity proteins. Solid ammonium sulfate was added to the supernate with 70% saturation. The precipitate was collected by centrifugation, and then dried by vacuum freeze dryer to obtain the solid crude powder of the neutral protease. The specific activity of purification multiple was 3.34, energy recovery for 61.34%.4. Making activity chitosan microspheres uses emulsion-crosslinking, with sole factor assays, the chitosan microspheres through crosslink methods is analyzed at the aspects of chitosan acid sol concentration, NaOH-ethanol, stirring time, glutaraldehyde and ethyl acetate dosage on the preparation. The optimal protease immobilization conditions are determined through orthogonal rotation tassays, which include:chitosan acidic sol concentration was 2.5%; Na OH ethanol is 1:1; stirring time was 80min; glutaraldehyde dosage was 1.5 mL; ethyl acetate amount to7.5mL. With this optimal conditions, the products of chitosan microsphere has good adsorption property, good swelling, forms size and alkaline endurance etal. Applied to the process of high temperature treatment and will not be degraded.5. The immobilized protease through crosslink methods is analyzed at the aspects of enzyme giving amount, glutaral pentanedial eventual concentration, cross-link and adsorption time. The results showed that the optimum conditions for the immobilization of protease were: the concentration of glutaraldehyde was 4%, the cross-linking time was 3h and the adsorption time was 15 h.6. The enzymology properties of immobility protease is studied, and results show that: the optimal p H is 10, with one unit offset towards acidic values compared with free enzyme; the optimal temperature is 70℃, 10℃ higher than free enzymes. The stabilities of immobilized protease in acidic or alkaline solutions and heat endurance are significantly improved compared with free enzyme, and these are advantages in improving its utilization ratio in industrial use.7. Different reagents for different enzymes. The results showed that: ethanol, ethylene glycol solution, sodium bisulfite solution, sodium chloride solution for both enzymes exhibited inhibition greater role for the free enzyme. Urea solution and sucrose are the two enzyme reagents have a greater activation. At the same time, data show that immobilized protease shows an inert change in activation and inhibition compared with free enzyme.