The Selection Of High-yield Alkaline Protease Strains And Analysis Of Enzymetic Properties

Alkaline proteases are a physiologically and commercially important group of enzymes. It has been applied universally to industrial of detergents, leather processing and silk. Besides, it has a wonderful prospect of application potential in medical purpose, food processing, silver recovery and environment protection, as well as basic research. Therefore, it is important to improve the enzyme activity of the strains of producing alkaline protease continuously. The complex mutagenetic treatments on protoplasts are a kind of effective method to get high-output strains.Taking Bacillus licheniformis S23 as the initial strain, its enzyme activity is 20387μ/mL. The complex mutagenetic treatments on protoplasts were used in this study. The fermentation conditions, the method of purification and properties of Alkaline proteases were studied. The main contents of the study were as follows:1 .All factors effecting on formation and regeneration of protoplasts were studied. These factors include penicillin, lysozyme, aminoacetic acid and stabilizers. Under the optimum conditions of protoplasts formation and regeneration, the protoplasts from the original strain S23 were prepared. The optimum conditions were confirmed: aminoacetic acid 1mg/mL, penicillin 0.4μ/mL; lysozyme 2mg/mL, temperature 37℃, time 6 hours. The rates of formation and regeneration of protoplasts of strain S23 had reached to 93.8% and 37.5% respectively.2. The complex mutagenetic treatments on protoplasts were studied. The optimum conditions were as follows: The dosage of ultraviolet is 3 minutes (simple treament); The dosage of ultraviolet is 1min and the concentration of EMS is 30mg/mL (complex treatments). The spore were heated at 80℃ for 20 minutes. After several complex mutagenetic treatments on protoplasts of strain S23 and a large amount of the regenerative mutants, a stable mutant SH-59 producing high-output alkaline protease is obtained. The enzyme activity is 32356μ/mL.3.The fermentation medium and the conditions of a high yield strain producing high alkaline protease were studied. The main components of the medium were substituted. The optimum medium were consist of: 6.5% corn flour, 4.0% soybean flour, 0.04% KH₂PO₄,0.3% Na₂HPO₄, 0.4%CaCl₂, pH9.0. The optimum fermentation conditions were confirmed: seed age 12h, inoculum volume 2%, a 250ml flask containing 25ml medium, 210r/min, 34℃ .And the highest enzyme activity rose up to 38925μ/ml as compared with 20387μ/ml of the originalstrain after cultivated 44h.4.The alkaline protease was purified from the culture of Bacillus lichenifonnis SH-59 by means of ammonium sulfate precipitation followed by DEAE-Sephadex-A-50 anion-exchange column chromatography,CM-Sephadex-C-50 hydroxyaptite column chromatography.The purified proteinase was demonstrated to be electrophoretic homogeneity by SDS-PAGE,with a molecular weight of 28kDa.5.The properties of the alkaline protease were studied. The optimum pH and temperature toward the hydrolysis of casein were pH 10.0 and 55"C.The alkaline protease was stable up to 50°C,in the pH range from 7-11. hi addition, it can resist on 1M H2O2.