Selection Of High-yield Alkaline Protease Producing Strain By Complex Mutagenesis Of Protoplasts And Study On Its Enzymetic Characterization

Based on the preparation of Bacillus licheniformis JF-20, we studied all factors effecting on formation and regeneration of protoplasts. Under the optimum conditions, the rates of formation and regeneration of protoplasts of strain JF-20 had reached to 86.9% and 38.4% respectively. The related phenomenon of formation and regeneration of protoplasts were observed by optical microscope.Under the optimal conditions of protoplasts formation and regeneration, the protoplasts from the original strain Bacillus licheniformis JF-20 were prepared. After some complex mutagenetic treatments on protoplasts of strain JF-20 and selections from a lot of mutants obtained ,we obtained a mutant called JF-UN122, which has a high-out alkaline protease. The enzyme activity of JF-UN122 rose up to 8129.4 u/ml as compared with 894.4u/ml of the original strain . The fermentation medium is as follows: 6.0% dextrin , 3.0% ground soybean , 0.3% KH2PO4 , 0.04% Na2HPO4 and 0.08% Na2CO3, pH8.5.An alkaline protease was purified from the culture of Bacillus licheniformis JF-UN122 by means of ammonium sulfate precipitation followed by DEAE-Sephadex-A-50 anion-exchange column chromatography, CM-Sephadex-C-50 hydroxyaptite column chromatography andSephadex-G-75 column chromatography. The purified proteinase was demonstrated to be electrophoretic homogeneity by SDS-PAGE , with a molecular weight of 31.6kDa . The Km for casein was 5.26 ug/mL , Vm , 20.8 ug/min . The optimum pH and temperature for hydrolysis of casein were 9.0 and 55℃, respectively . The enzyme was stable up to 55℃, in the pH range from 5-11. PMSF nearly inhabited its activity while other ions and reagents such as calcium , EDTA , urea and SDS had no notable effects on the activity of the protease . In addition , it can resist on 1M H2O2 and be protected by dithiothreitol(DTT).