Purification And Identification Of An Antifungal Protein From Bacillus Subtilis Strain B-916

Bacillus subtilis Strain Bs-916 was isolated from rice field soil. B-916 was used incontrolling rice shealth blight and rice false smut in field, while showed a high controleffect. The Bs-916 and its extract showed a high antifungal (antibacterial) activityagainst hizoctonia solani, Fusarium oxysporum Schlecht. f. fabae Yu et Fang, Fusariummoniliforme Sheld., Fusarium oxysporum (Schl.) f.sp.nieveum (E.F.Smith)SnyderetHansen, Pyricularia grisea, Ustilaginoidea virens, Xanthomonas oryzae pv. oryzae(Ishiyama) Zoo, Xanthomonas oryzae pv. oryzicola Dowson. Bs-916 and its extractshowed a broad-spectrum antifungal activity. The extract of Bacillus subtilis strain B-916was deposited by acetone, PEG6000, ammonium sulfate and ultra-4 centrifugal filterdevices exhibited a high antifungal activity (detected toward Rhizoctonia solani). Theantifungal activity of the extract treated with protease K was lower than that withoutprotease K treated. The results implied that the extract of Bs-916 secrete an antifungalprotein.The filtrated cultured liquid of Bacillus subtilis strain Bs-916 was precipitated ingrades with the ammonium sulfate to draw the antifungal proteins, and eight kinds crudeproteins were drawn, we studied character of two kinds crude proteins drawn withammonium sulfate at 40%～50% and 50%～60% saturation respectively, which showeda strong antifungal activity. The concentration of crude proteins drawn with ammoniumsulfate at 40%～50% and 50%～60% saturation were 17.261μg/ml and 18.899μg/mlrespectively. Both of them showed high antifungal activity against R solani, F.moniliforme while no antifungal activity against P. infestans. Crude proteins drawn withammonium sulfate at 40%～50% and 50%～60% saturation showed a stable and highantifungal activity against R. solani during 0～40℃, decreased antifungal activity whenthe temperature was more than 40℃, kept only a little antifungal activity at 120℃. Crudeproteins drawn with ammonium sulfate at 40%～50% and 50%～60% saturation showed a stable and high antifungal activity in broad range of pH value, however, lost fullantifungal activity when pH value was 13.6, kept partial antifungal activity after treatedwith protease K, protease E, Pepsin, Papatin and Trypsin. Results of SDS-PAGE showedthat Bacillus subtilis Bs-916 secreted abundance of proteins.Bacillus subtilis strain Bs-916 secreted the antifungal protein, but litter is knownabout the antifungal protein. In this study, the crude proteins were purified by AmershamAKTA Explore system. The crude proteins were eluted through the DEAE SepharoseFast Flow column and three peaks were found; only the second fraction (fraction B) hadantifungal activity. After the fraction B were pooled and eluted in the Phenyl Sepharose 6F.F. column, two peaks were obtained, only the second fraction (Fraction E) hadantifungal activity. When the Fraction E were pooled and eluted in the hydroxyapatitecolumns, there were also two peaks and the last peak (Fraction G) exhibited antifungalactivity, which showed only one protein band in the SDS-PAGE, designated as Bacisubinwith 41.9 kDa molecular weight compared with standard proteins.Bacisubin was measured antifungal activity toward Rhizoctonia solani, Fusariummoniliforme, Magnaporthe grisease, Sclerotinia sclerotiorum, Alternaria oleracea, A.brassicae, C. capsici and Botrytis cinerea. The results exhibited that the antifungalprotein had a broad-spectrum of antifungal activity and high virulence. Especially, theantifungal protein exhibited a stronger antifungal to pathology fungi, such as genericAlternaria. EC₅₀ value of antifungal protein to Rhizoctonia solani, Botrytis cinerea,Alternaria oleracea and A. brassicae were 4.01,2.74,0.08 and 0.055μM, respectively.The mechanism to antifungal activity of Bacisubin caused mycelia alimentation obstacle.Mycelia apex appeared distortion, tumescence, rupture and cell wall thickened. HoweverBacisubin did not showed protease activity, protease inhibitory activity, but exhibited acertain ribonuclease activity and hemagglutinating activity,.Antifungal protein bacisubin was identified with MALDI-TOF-MS and Q-TOFafter digested with trypsin. When Bacisubin (digested with trypsin) was identified withMALDI-TOF-MS, 22 peaks of absorbance were found and identification in MatrixScience Database, where were no observably protein related. Then mixed peptides wereidentified with Q-TOF-MS, five peptide tags (PT) were get. Five PT sequence wasVTIVDEKGR, FSFSDSDVHNR, VYIADSTNFK, ELPISENLASVNMR, and EAEWAYMITGK respectively. These PTs were used to search in Matrix Science Database; we found two proteins were observably related with this antifungal protein. The two proteinswas oxalte decarboxylase. One was an oxalte decarboxylase from Bacillus subtilis strainB-168, another was an oxalte decarboxylase from the Bacillus licheniformis ATCC14580.However, antifungal activity of oxalte decarboxylase was not reported elsewhere.