Breeding Of Thermotolerant Alkaline Protease Producing Strain, Study On Unhairing Process And Property Of Its Enzyme

Based on the preparation of Bacillus sp. SD-142, we studied all factors effecting on formation and regeneration of protoplasts. Under the optimum conditions, the rates of formation and regeneration of protoplasts of stain SD-142 had reached to 86.8% and 31.3% 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 primitive strain Bacillus sp. SD-142 were prepared. After many times of complex mutagenetic treatment of protoplasts of SD-142 and selection from a lot of mutants obtained, a mutant CH-17-9 was finally obtained with properties of high-output, stable and Thermotolerant Alkaline Protease. The enzyme activity of mutant rose up to 2759.4U/mL as compared with 516U/mL of the primitive stain.By studying on the supernatant of the culture fluids, the enzymatic characteristics were concluded. The optimum temperature and pH for protease activity was 60℃ and pH9.5. It was stable in the pH range 6.5-11.0 and had good thermal stability. The enzyme retains 100% of activity after 24h of heat treatment at 40℃ and 53.4% of activity after 30min at 60℃. Addition of CaCl2(10mM) andglycine (1M), individually and in combination improve the thermostability of enzyme. The enzyme retained 60.8% of its maximum activity after 1h at 60℃ in the presence of glycine.The enzyme retained about 58.8% of its maximum activity after 10min at 40℃ in the presence of SDS. The enzyme retained main activity after lh at 40℃ in washing powder(14mg/mL). The extracellular production of the enzyme, its thermostable nature and compatibility with most commercial detergents are features that suggest its application in detergent industry. We have also studied the ability of the enzyme in unhairing, which is the foundation of the further research.