Screening Of Alkaline Protease Producing Bacteria From Soil And Optimization Of Enzyme Producing Conditions

Protease is a kind of enzymes that hydrolyze protein peptide chain to amino acids.According to different adaptive p H reaction system,there are acid protease in acid system,neutral protease in neutral system and alkaline protease in alkaline system.Among them,alkaline protease in alkaline environment can hydrolyze protein peptide bond,and the optimum p H range is 9-11.Alkaline protease in the system of its action is efficient hydrolysis,site-specific shear,hydrolysis controllable.It is widely used in leather,unhairing,protein screening,brewing,food processing and other industries.The protease produced by "production factory" with microorganism as carrier can meet the needs of industrial production because of its low cost,easy access and considerable yield.In this paper,a strain with high enzyme activity and yield was screened from the soil with more humus,and the alkaline protease in the strain was studied.Firstly,the strain was identified as Bacillus by morphology and molecular biology,and then it was identified as Bacillus amyloliquefaciens by molecular biology,and named as Bacillus amyloliquefaciens t-18.The conditions of ARTP were as follows: power 120 W,gas volume 10 SLM,treatment distance2 mm,set temperature of cooling water circulator 20 ?.After mutation breeding,a Bacillus amyloliquefaciens strain t-18-2 with alkaline protease activity of 337 U / ml was obtained.The enzyme activity was 29.1% higher than that of the original strain.After five successive generations of culture,the alkaline protease activity of the fermentation broth of the strain t-18-2 remained 97%compared with that of the first generation strain,which had good genetic stability.Taking Bacillus amyloliquefaciens t-18-2 as the starting strain,through single factor test and response surface methodology(RSM)to optimize medium composition,the results showed that:soluble starch 4.9%,peptone 2.7%,Mg SO4 0.192%,Na Cl 1%;The optimal fermentation conditions were as follows: culture temperature 37 ?,initial p H 7.0,liquid volume 100 ml,inoculum volume3%,fermentation time 88 H.Compared with before optimization,the enzyme activity increased from337 U / ml to 1299 U / ml.The alkaline protease produced by Bacillus amyloliquefaciens t-18-2 was analyzed.The optimum temperature of alkaline protease was 50 ?,the stability of alkaline protease was good in the range of 40-50 ?,and it could retain more than 50% of the enzyme activity after 90 min;The optimum p H of alkaline protease produced by Bacillus amyloliquefaciens t-18-2 was 10.5.After 24 h storage in buffer solution with p H of 10.5,it still had more than 85% enzyme activity and good alkali resistance.