Study On Breeding And Fermentation Technology Of Highly Mutated Strains Of Alkaline Protease

Alkaline protease is a kind of protease which is suitable for pH 9~11,which belongs to serine protease.It not only hydrolyzes peptide bond,but also has the function of hydrolyzing ester bond,amide bond and transesterification and transpeptide.Alkaline protease in the detergent,food,medical,brewing,silk,leather and other industries have applied.Although protease research has been for many years,the protease preparations is mainly monopoly by foreign companies at the moment.Domestic enterprises also have production,but low yield,expensive and other issues lead to less corporate profits.Therefore,it is still a hot research to improve the ability of producing strains and the optimization of culture technology.The starting strain of this experiment is Henan Yangshao Biochemical Company of alkaline protease production strains,2709 alkaline protease strains.The strain was first resuscitated and the F12 strain was obtained.The activity of the strain was 9796.8 U / mL,which was 9% higher than that of the original strain.In order to obtain high alkaline protease producing strain,low energy ion beam gene modification technique was used to mutagenize the high yield strain.The mutagenesis parameters of the F12 strain were optimized by ion beam implantation,The results showed that the mutagenic effect is better under the energy was 30 KeV and the dose was 1×1015 ions cm-2,screening to obtain alkaline protease high yield strain A3,and the average activity of the enzyme was 11892.8 U/mL,which was 32.4% higher than that of the original strain.The A3 strain was subjected to UV mutagenesis.The results showed that the lethal rate of the strain was 81.82% and 90.91% at 120 s and 150 s,respectively.The Z26 strain was screened at the same time.The average enzyme activity was 13517.7 U/mL,which was 13.7% higher than that of ion beam mutant strain A3,which was 50.4% higher than that of the original strain.The change of enzyme activity of Z26 strain was between 97% and 103%,which showed good genetic stability and can be produced as an industrial strain.To reduce costs and increase production.The Z26 high yield mutagenic strains as the starting strains,the single factor and orthogonal experiments were carried out on the main fermentation conditions affecting their enzyme production.The experimental results show that the best fermentation conditions of the strain: The addition of soybean meal amount was 4%,the corn flour was added at 7%,the fermentation temperature was 37%,the initial pH was 7,the inoculation amount was 7%,and the fermentation time was 36 h.Under the condition of the experiment,the activity of Z26 strain was 13929.7 U/mL after fermentation,3% higher than the enzyme activity of before optimization.Finally,the preliminary enzymatic study of the alkaline protease was carried out.The results show that the optimum temperature is 37 ?,the optimum pH is 11,the temperature is not high and the temperature is good at 50 ?,and the p H is stable at 7-11.There is a certain degree of oxidation resistance for 1mol/L H202,Cu2+ almost completely inhibited the enzyme reaction;Ca2+,Mn2+ can improve the enzyme activity.So the alkaline protease performance is good to be widely used in industry.