| Alkali protease, serine protease is extensive used in detergent,leather,silk industry, pharmaceutical engineering, food engineering, peptide synthesis, wastewater treatment and so on. Relative molecular of alkali protease is about20000-34000, less than that of neutral protease. But isoelectric point of alkali protease is about8to9, higher than that of neutral protease. Most of alkali protease acts well under the condition of pH7-11, but is thermolabile. The purpose in this study is to screen a thermostable strain which produces alkaline protease using corn straw hydrolysate as a carbon source. The thermostable alkaline protease is subsequently obtained. It is not easy to make the thermostable alkaline protease inactive and the growth of harmful microorganisms is prevented under the condition of high temperature. The cost of material culture is reduced and energy is saved when using free corn straw as raw material culture. In this study five strains producing alkali protease were screened from the soil and compost. The strain20101with highest enzyme activity was selected by fermentation product qualitative and quantitative test. The strain20101was analyzed to be bacillus subtilis by16S rDNA analysis. Alkali protease activity from strains20101was improved by the method of ultraviolet mutagenesis and culture medium optimization. The crude enzyme was obtained from fermentation liquor by salting out and dialysis. Main properties of the crude enzyme was then studied. The results is as follows:1. Five strains with large hydrolysis circle of casein, which produce alkali protease is screened from the soil and compost in jilin agricultural university. Enzymatic activity is determinated under the condition of50℃. The data shows that enzyme activity from Strain20101is the highest, about119.8U/mL.2. There is scattering type drape on the surface of Strain20101colonial morphology, which is large, close to round. There is mycoderm on the surface of liquid culture medium when Strain20101is cultured in liquid culture medium. Strain20101is gram-positive with the shape of straight pole.16SrDNA Sequence identification shows that the strain is Bacillus subtilis.3. Strain20101Z1is obtained by three rounds ultraviolet mutagenesis. Alkali protease activity from Strain20101Z1is improved, about299.5U/mL. Enzyme activity is increased150.1%than that from strain20101. Strain20101Z1shows good genetic stability after subculturies8times.4. The optimal hydrolysis conditions of corn straw by orthogonal test is as follows:hydrolysis time is105min, hydrolysis temperature is95℃, Solid liquid ratio is1:15, acid concentration is2.5%. Hydrolysis rate of corn straw is38.6%.5. The flask culture condition of strain20101by the single factor experiment and response surface test is optimize.The optimal culture process is as follows:culture temperature is50℃, sugar density is15.6g/L, inoculum size is6.7mL, yeast powder content is20g/L. Under the optimal culture medium, the enzyme activity is312.5U/mL.6. A few brown alkaline protease is obtained by salting and dialyzing fermentation liquid. The data shows that the optimal temperature of alkaline protease is60℃, the optimal pH is10. Alkaline protease is stable when it is heated under the condition of60℃, residual enzyme activity is96%for30min,91%for60min, still81%for120min. |
