The Separation And Purification Of The Antifungal Substances Produced By Bacillus Lichenjformis

A strain of inhibition to many kinds of plant pathogenic fungi, named BS-3, was isolatedfrom soils. The strain had quite strong fungistatic activities on plant pathogenic fungi such asrice sheath blight, rice blast. We found that it had the percent 100 homologous to Bacilluslicheniformis,Bacillus subtilis and Bacillus amyloliquefaciens by 16S rDNA sequencesBLAST analysis, plus the results of morphological and biochemical parameters, it belongs toBacillus licheniformis.Using the optimum fermentation conditions to produce antifungal substance, selectingrice sheath blight fungus (Thanatephorus cucumeris) which inhibited obviously by the strainas the indicator bacteria, we utilized inhibition zone method to detect antifungal activity offermentation product belong to the strain BS-3. The antifungal protein from the fermentationsupernatant of the strain BS-3 was precipitated by (NH₄)2SO₄. The optimal saturation of(NH4)2SO4 was 50%. Detecting the fermented liquid after the treatment of centrifuging and50% saturated ammonium sulfate, the results showed that the supernatant fluid treated bycentrifuging and the centrifugation after the treatment of 50% saturated ammonium sulfateand centrifuging had quite strong fungistatic activities. The rough antifungal protein of strainBS-3 had quite strong thermal stability, tolerance to proteinase K and trypsinase to a certainextent.After the rough antifungal protein to pass through the chromatography of DEAE-52 andSephadex G-75, the peak which had antifungal activity to rice sheath blight fungus wasobtained. This peak showed only one single band with 31.0kD and pI 5.41 in SDS-PAGE andIEF-PAGE, respectively. After thorough detection made by HPLC, the purity of the antifungalprotein in this peak is 98.25%.