| Soybean protein is a kind of protein with nutrition and healthcare function and unique processing performance. As an important raw material, it has been widely used in vegetarian and meat-based food products. However, some components of soybean protein such as α-subunit and α’-subunit of β-conglycinin are critical allergen, which has become a serious problem to the allergy sufferers. Allergic reactions can cause the cutaneous, respiratory, and gastrointestinal adverse reactions, and may lead to shock or death. Therefore, it is important to efficiently remove the allergen in soybean protein. Bacillus sp. BBE201108 was screened in our previous work, the protease of which could degrade soybean allergen α-subunit of β-conglycinin. In order to further analyze the property and function of this protease, a series of strategies were conducted in this study. First, the protease from Bacillus sp. BBE201108 was purified, and its property and effect on soybean protein allergen were studied. Then the compound enzymes of Bacillus sp. BBE201108 protease, alcalase and papain were performed to hydrolyze soybean protein allergen. Finally, the method of immobilization of Bacillus sp. BBE201108 was optimized, and the property and effect on soybean allergen were also studied.The results of this study were described as follows:(1) The protease produced by Bacillus sp. BBE201108 was purified. The protease was purified by ammonium sulfate precipitation, dialysis, anion exchange chromatography, and gel filtration chromatography. A purified protease was obtained with 6.48% recovery and an overall purification of 11.55-fold. The specific activity of protease increased from 244.20 U?mg-1 to 2,575.45 U?mg-1. The property of the Bacillus sp. BBE201108 protease was studied. The optimum p H and temperature were 8.0 and 50°C. The protease activity was inhibited by Mn2+, Fe2+, Cu2+, sodium dodecyl sulfate, Tween 80 and dithiothreitol, and the influence of inhibitors showed that the protease was a serine protease. The kinetic parameters Km, Vmax, kcat and kcat/Km of the protease were 4.19 g?L-1, 4.04 g?L-1?s-1, 107.73 s-1 and 25.71 L?g-1?s-1, respectively. The result of SDS-PAGE verification showed that both of the α-subunit and α’-subunit of β-conglycinin could be hydrolyzed by Bacillus sp. BBE201108 protease under the optimum conditions.(2) Bacillus sp. BBE201108 protease, alcalase, papain and their compound enzymes were chosen to figure out their effects on soybean protein isolate. The result showed that a recipe that Bacillus sp. BBE201108 protease: alcalase: papain=2:1:1 was the most efficient compound enzyme, and the hydrolysis degree of soybean protein isolate was obviously improved.(3) Bacillus sp. BBE201108 protease was immobilized using sodium alginate as a carrier. Based on the single-factors tests, the optimum immobilization conditions of Bacillus sp. BBE201108 protease were: sodium alginate of 30 g?L-1, Ca Cl2 of 20 g?L-1, Bacillus sp. BBE201108 protease with sodium alginate ratio of 1:2, and incubation for 1 h. Under this condition, the recovery rate of protease activity was 63.42%. Compared to the free protease, the optimum temperature of the immobilized protease increased by 10°C. Meanwhile, the adaptation scopes of p H and temperature were wider, and the thermal stability also significantly improved. The SDS-PAGE verification of the hydrolyzed soybean protein isolate by the immobilized protease showed that the immobilized protease could also remove the α-subunit and α’-subunit of β-conglycinin. Compared with the free protease, the specificity of immobilized protease for α-subunit and α’-subunit of β-conglycinin further increased. |
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