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目的检测螠蛏共附枯草芽孢杆菌110次级代谢产物的抑菌活性组分,并探讨抑菌活性组分的抗肿瘤作用。方法用0.22μm滤膜过滤将螠蛏共附枯草芽孢杆菌110的菌体和发酵液分开,用硫酸铵沉淀发酵液的蛋白组分并用SDS-PAGE检测,用滤纸片法分别检测菌株发酵液、菌体及胞外蛋白的抑菌活性,琼脂块法检测抑菌活性组分的抑真菌活性,用海虾毒性法和MTT法检测抑菌活性组分的抗肿瘤活性。结果螠蛏共附枯草芽孢杆菌110的发酵液抑菌活性远远高于菌体的抑菌活性,发酵液的硫酸铵沉淀物具有很强的抑菌活性,硫酸铵沉淀的110活性粗蛋白主要由分子量约为60、45和25kDa的3条带组成。110活性粗蛋白对8种真菌指示菌具有抑制作用,其中对辣椒青枯、番茄灰霉和瓜类枯萎的抑菌圈直径超过20mm。该活性粗蛋白还对白血病细胞K562、肝癌细胞HepG-2和人乳腺癌细胞MDA-MB-231具有抑制作用,其中200μg/mL的蛋白对人肝癌细胞HepG-2抑制率达74.01%。结论螠蛏共附枯草芽孢杆菌110胞外蛋白有抗肿瘤活性和广谱抑菌活性,具有潜在的开发价值。
Objective To detect the antibacterial activity components of secondary metabolites of Bacillus subtilis 110 and to study the anti-tumor effect of antibacterial activity components. Methods The bacterial cells and fermentation broth of Bacillus subtilis 110 were separated by filtration with 0.22 μm membrane filter. The protein components of the fermentation broth were precipitated by ammonium sulfate and detected by SDS-PAGE. The fermentation broth of the strains was detected by filter paper method. The antibacterial activity of the bacterium and the extracellular protein was tested. The fungistatic activity of the antibacterial activity component was detected by the agar block method. The antitumor activity of the antibacterial activity component was tested by shrimp toxicity method and MTT method. Results The antibacterial activity of the fermentation broth of Bacillus subtilis 110 was much higher than that of the bacterium. The ammonium sulfate precipitate of the fermentation broth had strong antibacterial activity. The crude protein precipitated by ammonium sulfate was mainly Consists of three bands with molecular weights of about 60, 45 and 25 kDa. 110 active crude protein had inhibitory effect on eight fungi. Among them, the inhibition zones of withered pepper, tomato gray mold and melon were more than 20mm in diameter. The active crude protein also has inhibitory effects on K562 leukemia cells, HepG-2 hepatoma cells and MDA-MB-231 human breast cancer cells. The inhibitory rate of 200μg / mL protein on HepG-2 cells was 74.01%. Conclusion The total protein of Bacillus subtilis 110 extracellular protein has anti-tumor activity and broad-spectrum antibacterial activity, which has potential value for development.