Mutagenesis And Breeding Of Alkaline Protease High-yield Strain

Alkaline proteases belong to proteases that can hydrolyze protein-peptide bonds under alkaline conditions,with a wide range of p H values between 9 and 11.Most of its active centers contain serine,also known as serine protease.It is widely used in detergent,food,medical,silk,leather and other industries.At present,large foreign companies monopolize protease preparations,while domestic enterprises have production problems such as low enzyme output,high price,and unsatisfactory enzyme properties,which make the enterprises low profit.Therefore,how to improve the enzyme production capacity of strains and optimize fermentation conditions were still hot topic.In this experiment,2709 alkaline protease strain of Henan Yangshao Biochemical Company,was selected as the research object,and a variety of mutation methods were used to select a high-yield strain,and the enzymatic properties were studied,then the fermentation process was optimized.A stable strain Y9(15582 U/m L)was isolated from 2709 alkaline protease strain.The mutation breeding of strain Y9 was carried out.The results showed that when the UV was 30 W and the distance was 20 cm,the mutation was 60 s and the positive mutation rate was higher.The compound mutagenesis of the strain was carried out by using diethyl sulfate and ultraviolet light.After treatment with 1.5%concentration of diethyl sulfate for 30minutes,ultraviolet mutagenesis was carried out.The strain was also mutated by atmospheric pressure room temperature plasma.The gas source was argon,and the power was 100 W.When the mutation time was 70 s,the positive mutation rate was higher.After repeated mutagenesis with the above mutagenic parameters,a strain A59with alkaline protease high-yield was obtained.And its enzyme activity was 23485U/m L.Through genetic stability analysis,it was found that the genetic stability was good.Compared with the original strain,the enzyme activity of the strain increased by50.72%.The properties of alkaline protease produced by strain A59 were studied.The results were as follows:the optimum temperature of the enzyme was 55?to 60?,the optimum p H of the enzyme was 9 to 11,the enzyme had good thermal stability when the temperature was below 45?,the enzyme had good p H stability and alkali resistance when the p H was between 8 to 10.5,the enzyme had strong antioxidant activity to 1 mol/L H₂O₂ when the enzyme was used,Ca²⁺and Mn²⁺could slightly increase the enzyme activity,Fe²⁺and Zn²⁺could almost completely inhibit the enzyme reaction,Mg²⁺,Na⁺,K⁺had no significant effect on enzyme activity.Through the study of enzymatic properties,it was found that the enzymatic properties of the strain were almost the same as that of the original strain,indicating that the enzymatic properties of the strain had not changed.Taking strain A59 as the research object,nine factors affecting the strain were studied.The results showed that:soybean meal 3.6%,corn meal 5.2%,disodium hydrogen phosphate concentration 0.8%,Mg²⁺concentration 0.15%,Tween-80concentration 0.12%,and inoculation amount 5%,the enzyme activity was 36829U/m L.Further study showed that the most important factors affecting enzyme production were Na₂HPO₄ concentration,Mg²⁺concentration and Tween-80concentration.Three factors and five levels of response surface tests were conducted on these factors.The results showed that:Na₂HPO₄ concentration 0.9115%,Mg²⁺concentration 0.1593%,and Tween-80 concentration 0.09%,the predicted enzyme activity of the strain reached 38758.626 U/m L.Under this condition,through further verification,the enzyme activity was 38650 U/m L,increased by 64.57%.According to the fermentation kinetics analysis of strain A59,the enzyme production of strain A59reached the highest value of 38650 U/m L at 42 h,about 4 h ahead of the original strain.Based on the optimized process of shake flask,the A59 was scaled up.After optimization,the fermentation process parameters of the 1 m³ fermentation tank were as follows:charging coefficient 0.7,inoculation volume 5%,temperature 38?,dissolved oxygen maintained at 20%to 30%,at this time,the speed was 200 r/min,and the ventilation volume was 1:0.5 to 1:2.5.The p H of the fermentation liquid was7.0.At 32 h,the enzyme activity was the highest.After 8 batches of tests,the average enzyme activity was 52000 U/m L.According to the same principle of the volume oxygen transfer coefficient k_L·a in liquid phase,the dissolved oxygen was controlled at20%to 30%in 5 m³ fermentation tank.The fermentation time was about 30h,and the enzyme production performance was stable.Through five batches of tests,the average enzyme activity was 52764 U/ml,which can be used in industrial production.Through the scale-up test of the 35 m³ fermentation tank,the dissolved oxygen was controlled at 20%to 30%.At this time,the stirring speed was 160 r/min,and the ventilation volume was 1:0.5 to 1:2.5.The fermentation time was about 29 h.The average enzyme activity reached 52784 U/ml through 8 batches of experiments,and the enzyme production performance was stable.Compared with the fermentation kinetic curve of the small-scale experiment,the maximum enzyme production was achieved 3 h in advance,which shortened the fermentation time.