Optimization Of Fermentation Conditions, Partial Characterization And Purification Of Antifungal Substance From Bacillus Subtilis G8 Strain

Bacillus subtilis G8 strain showed a strong activity to inhibit the growth of some plant pathogenic fungi. The inhibition mechanism is producing antifungal protein. In this paper, fermentation medium and culture conditions of the strain were optimized. Subsequently, partial character and purification of antifungal protein were studied. All this lay a foundation for further study of characteristics and structure of antifungal protein, antifungal mechanism and genetic modification of strain.To increase protein yield, the medial components and fermentation conditions for antifungal activity of G8 strain were optimized by single factor and orthogonal design test in shake-flask. The result showed that the optimal medium were composed of 5g corn starch,5g soybean tryptone, 20g ammonium acetate, 0.5g calcium carbonate; The optimal culture conditions were at temperature 35℃with initial pH 7, inoculum volume 30 ml/L, medium volume 70-100 mL in 500 mL flask and rotation speed 180 r/ min; the optimal seed age was 12 h, and the fermentation time was 48 h. Inhibitory rate against Sclerotinia.sclerotiorum of the optimal broth was 2.1 times higher than the broth of LB medium when precipitated by ammonium sulfate,protein concentration reached 103.9mg/L,2.3 times higher than LB media.To gain physical and chemical properties of the antifungal protein, the effects of heat, pH, metal ions, surfactants and proteases were studied, and the capacity of degrading specific substrates was detected also.The result showed that the antifungal protein were thermal stable, about complete activity remained after heating at 100°C for 1h, the antifungal protein still retains 90% of initial activity when autoclaved at 121°C for 30min; they were stable to acid(pH 2) and metal ions,could interact with surfactants and tolerant to trypsinase and pepsin,partial sensitive to protease K. The degrading test showed that the target protein was not chitinase, cellulase orβ-1,3– glucanase.Crude protein precipitated with ammonium sulphate were purified by column chromatography on Sephadex G-100, Cellulose DE-52, CM Sepharose CL-6B, Pheny-Sepharose 6 FF, then analyse with SDS-PAGE, Tricine-SDS-PAGE and activity test. The results show that the antifungal protein may be peptide with a molecular weight about 4000 Da. Crude protein precipitated with hydrochloric acid were extracted with methanol, the crude extract was separated on thin layer and analysed on infrared spectrometer. The results showed that the antifungal protein may be a linear lipodecapeptide.To estimate the antagonistic effect of the antifungal protein to disease pathgen, the inhibition activities on mycelium growth of the antifungal protein against S. sclerotiorum(Lib.)de Bary were determined by mycelium growth rate method in lab. The results showed that its EC50 value was 12.4μg/mL, which was significantly lower than fungicide diethofencarb and pyrimethanil. In addition, osmotic potential testing showed that the antifungal protein could make effect on cellular membrane of S. sclerotiorum, as a result, intracellular substances extravasated and mycelium became thinner than the control.