Screening,identification And Characterization Of Bacillus Halotolerans.DS5 With Alkaline Protease Production

Alkaline proteases are a class of enzymes that can efficiently hydrolyze proteins in an alkaline environment and have a wide range of uses in the washing,leather,and food industries.With the development of industrialization,the demand for alkaline proteases is increasing.Alkaline protease produced by microbial fermentation has low fermentation cost and mature extraction technology,and has become the main source of enzyme preparations.In this study,a strain of DS5 with high protease production was screened by the primary and secondary screening of microorganisms in the soil.After 16Sr DNA,and physiological and biochemical experiments,the strain was initially identified as Bacillus halotolerans.The strain was positive for Gram staining and was able to use glucose,sucrose,lactose,maltose,soluble starch and many other substances and grow normally at10%salt concentration.The DS5 strain has a short growth cycle,and alkaline protease enzyme activity was detected in the supernatant at 4 h of fermentation in the initial medium（Dextrin 1%,soluble starch 2%,yeast extract 1%,Na Cl 0.5%,p H 7.0）,with maximum activity at 12h of fermentation.The protease from the fermentation broth was purified in two steps by 70%ammonium sulfate precipitation and Superdex-75 gel filtration chromatography column to obtain a purified alkaline protease with a molecular weight of approximately 27.0 k Da,an enzyme activity recovery of 16%,a protein purification multiple of 5.6 times and a specific activity of 1501 U/mg.The protease has an optimum temperature of 50°C and an optimum p H of 9.0,has broad p H stability,and retains 70%of its activity when incubated for 2.5 h in a buffer solution of p H 7.0-12.0,after incubation at 50°C for 2.5 h,40%of the activity was still retained;Ca²⁺,Mg²⁺,and Mn²⁺significantly increased enzyme activity,with 15 m M of Mn²⁺increasing protease activity by 86.24%;The reducing agents DTT andβ-mercaptoethanol at a concentration of 1 m M can increase enzyme activity,and PMSF has an inhibitory effect on the enzyme;it has a high salt tolerance,and the enzyme activity remains unchanged after 1h incubation in a 2.5 M high salt solution;it a broad spectrum of substrate enzymes,capable of hydrolysing a wide range of proteins,the most suitable substrate being casein;JB-02 in GB/T131742008 states that the washing capacity of protein soiled cloths is the main indicator to test the performance of protease on protein washing,compared to enzyme-free detergents,detergents with DS5 alkaline protease have excellent detergency and are more effective in washing protein soiled cloths.The fermentation conditions and media components of DS5 strain were then optimised.The optimum carbon,nitrogen,inorganic salts and metal ions in the medium were determined by single-factor experiments to be soluble starch,sesame powder,KH₂PO₄,K₂SO₄and Mg²⁺respectively;the optimum fermentation conditions are:12%loading volume,initial p H of fermentation broth 5.0.After optimizing the medium with Design-expert 8.0.6 software,the optimized medium components were:soluble starch:4%,sesame powder:1%,KH₂PO₄:1.2%,Mg²⁺:10 m M,K₂SO₄:0.4%,p H:6.0,and fermentation was carried out at 12%loading volume is 4%inoculation rate,200 rpm,37°C for 60h.The fermentation enzyme activity of the optimised medium was 824.60 U/m L,which is a 6.84-fold increase compared to the initial medium fermentation of 120.58U/m L.