Bacillus Subtilis The Antagonism Strain B29 Active Substances To The Formation Of The Conditions Of Its Antibacterial Protein Isolated And Purified

Bacillus subtilis strain B29 showed a strong activity to inhibit the growth of someplant pathogenic fungi. The optimum fermentation conditions of antifungal substanceproduction of strain B29 were fermentation temperature 30℃, original pH7.5, amountof load 75ml(in 250ml flask), and fermentation cycle 120h. Under these conditions theinhibition efficiency of the fermentation supernatant reached 42.43％.The antifungal protein from the fermentation supernatant of strain B29 wasprecipitated by (NH₄)₂SO₄. The optimal saturation of (NH₄)₂SO₄ was from 30％to 70％.The rough antifungal protein of strain B29 was stable to high temperature at someextent, partially tolerant to proteinase K, and more sensitive to trypsinase and pepsin.One antagonistic peak was obtained from the rough antifungal protein afterDEAE-52 and Bio-Gel^?P-100 column chromatography, and its purity was 97.81％. Thispeak showed only one single band(B29I) with 42.3KD and pI 5.69 in SDS-PAGE andIEF-PAGE, respectively. B291 was inhibitory to spore germination of pathogens andsuppressive to germ-tube elongation.The antifungal protein B29I separated by SDS-PAGE was analysed after in-geldigestion by NCBA(national center of biomedical analysis). The PMF (peptide-massfingerprinting) result showed that B29I may be a new protein because the PMF result ofB29I was not similar to any other proteins in Mascot. Three peptide fragment sequenceswere obtained: KTHVLEDEFK, KGYQTGDFGAYLH and RTYEVAEESPVLGL.