Identification Of Alkaline Protease Producing Strain BN-2 And Its Enzymatic Properties

Protease is a hydrolytic enzyme that can degrade proteins into small peptides and amino acids.Alkaline protease?AP?,as a kind of protease with high activity and good stability in alkaline environment,is widely used in detergent,bioremediation,waste management,photography,diagnosis and other industries.Microbial alkaline protease has become an important source of alkaline protease production because of its low production cost and high productivity.Bacteria and fungi are good sources of various alkaline proteases in microorganisms.At present,the main enzymatic strains suitable for industrial production are Bacillus,such as Bacillus subtilis,Bacillus licheniformis,Bacillus alkalophilus and so on.In this paper,a strain with high enzyme activity was screened from natural environment,and the alkaline protease in the strain was studied.Firstly,a strain BN-2 with high protease activity was isolated from naturally fermented soybeans.The strain was identified as Bacillus subtilis subsp.natto BEST195 by morphology and molecular biology.Then the genomic DNA of BN-2 strain was successfully extracted by CTAB method.The gene of alkaline protease?AP?was amplified by PCR,and inserted into pBE2R vector to construct pBE2R-AP and then transfected to Bacillus subtilis WB600 for secretory expression.When dextrin and soluble starch were used as carbon sources,the recombinant protease activity was 1.2 times compared to the original strain.At the same time,p43 promoter in pBE2R vector was replaced by P195 promoter of alkaline protease in BN-2 strain.The activity of alkaline protease regulated by p43 promoter was about 1.4 times higher than that regulated by P195 promoter in every milliliter fermentation broth.The enzymatic properties of the recombinant protease were studied using casein as a substrate.The results showed that the optimum pH of the recombinant protease was 8.0,and the residual enzymatic activity was 94% in pH 8.0 buffer for 24 hours;The optimum temperature of theenzyme was 50?,but the activity of enzyme was decreased to 51% after incubation at 50? for 30min;The enzymatic activity was enhanced by 1 mM Ca²⁺ or Mn²⁺,1 mmol/L Mn²⁺ increased the enzyme activity by 48%,1 mmol/L Ca²⁺ increased the enzyme activity by 8%;1% Triton X-100 inhibited the enzyme activity by 13%,1% Tween20 inhibited the enzyme activity by 8%,and 1% Tween80 inhibited the enzyme activity by 15%.