Cloning And Expression Of A New Protease From Bacillus Subtilis And Its Physicochemical Research

A Bacillus subtilis strain BLYS-1 which could produce protease was screened from thelocal soil. The protease production of Bacillus subtilis strain BLYS-1could ganeratedhydrolyzed food protein with no bittereass, hence, are valuable for use in food industry. Inthis paper, the protease gene was cloned by using Homology-Based Candidate Gene Methodand was expressed in E.coli BL21, in which the protease showed the hydrolysis ability. Thephysicochemical properties of the protease were investigated after extracted from the Bacillusstrain BLYS-1 after cultured under the optimized fermentation conditions. The physiologicalfunction of the soybean protein hydrolyzate produced by strain BLYS-1 was studied byfeeding them to the mice. The results were as follows:Cloning and expression of the protease The gene producing the protease by Bacillussubtilis strain BLYS-1 was cloned successfully based on the homology sequences. Thesequence analysis showed that the protease gene had a high homology with Bacillus subtilisextracellular metalloprotease up to 99.39%. Based on pET28a, the recombination pET28aNPplasmid carrying the protease gene NP was constructed, and then transformed intoEscherichia coli strain BL21. The protease gene could be expressed in BL21 induced byIPTG, the protease molecular weight was about 30kDa according to SDS-PAGE.Optimization of the fermentation conditions In this study, thefermentationconditions were optimized for the Bacillus subtilis strain BLYS-1 to produce the protease,such as pH,temperature,rate of rotation,volume and inoculum size; as well as thecomponent of culture media as carbon source,sugar source and inorganic salts. The resultsshowed that the pH 9.0,temperature 35℃,volume100ml/250ml,inoculum size 8% and therate of rotation150r/min could increase the protease activity. According to the result ofResponse Surface, 1% starch and 1.5% corn were good for increasing the protease activity,Under the optimized conditions, the highest protease activity with 800.650 U/ml wasobtained.Physiochemical property of the protease The protease produced by Bacillus subtilisstrain BLYS-1 was extracted and purified. The effect of pH value, temperature, organicsolutions, metal ions, oxidant and EDTA on the activity of the protease were investigated. Theresults indicated that the protease has higher activity ranging from pH 5 to pH 8, while theoptimum pH was nearly 7 and temperature was 40-60℃, respectively. It was also showed thatthe protease would be precipitated by 70% ammonium sulfate. The activity of the proteasewas inhibited by tween 80, isopropyl alcohol, SDS, SnP²⁺P, CaP²⁺P, NiP+P et al, while it waspromoted by MgP²⁺Pand ZnP²⁺P. The dynamic parameters with the substrate of casein was Km2.53g/L and Vmax6.67μg/s.Pphysiological function of the soybean protein hydrolyzate produced by strain BLYS-1 The six groups of mice of Kunming series were feed with purified water, soybean polypeptide solution, the enzymolysis solution of soybean protein, soybean milk, soybean protein solution and soybean protein hydrolyzate solution produced by strain BLYS-1 for thirty days, respectively. And then the anti-fatigue ability, the functions of spleen, ren and liver were investigated. According to the results, it was demonstrated that the soybean protein hydrolyzate solution had good effects on increasing the weight, prolonging the climbing-time and swimming-time as well as increasing the exponent of the spleen and ren of the mice. And the reserve ability of glycogen in liver was also inhenced.Compared with purified water , other lavaged solutionscould increase the content of lactic acid dehydrogenase in mice blood, while the soybean protein hydrolyzate solution could reduce the nitrogen content of hematuria in mice blood.