Isolation And Identification Of Bacillus Subtilis A_3-2 Strain And Separation And Purification Of The Antifungal Protein Secreted By A_3-2 Strain

A bacterium named A3.2 is screened from soil sample of Mountain Tai. It secretes large amount of antifugal proteins with strong inhibiting activity against several plant deseases.The determination shows that it is a strain of Bacillus subtilis. The cultural conditions such as temperature, fermentation peried and the compound of medium are studied . The results of tests show that suitable factors for both bacterium growth and antimicrobial substance production are 30C, 72 hours. Further study shows concentration of ammonium sulfate between 50% and 70% is the most fitting saturation for antagonistic protein precipit ability.Crude proteins are sbtained from preciptation of the cellfree culture of Aj.2 strain with ammonium sulphate at 70% saturation,then purified by column chromatography on Sephades G-75 and DEAE-Sepharose Fast Flow. The purified protein was demonstrated to be one single protein band by SDS-polyacrylamide gel electrophoresis with a molecular weight of 349ID. The purified protein is insensitive to heat, resistant to trypsin , proteinase K and chloroform.Its active pH range is wide .from 4.0 to more than 12.0,but partially sensitive to high pH.To treat the pathogenetic fungi hyphas with purified protein and then observe at regular intervals under normal microscope.lt is shown that some cells of hyphas began to dilate and become to deformed spherical structure when cultured for a short penal ,and more and more of the deformed cell and larger and larger of dilate cell with the increase of culture time .So that the hyphas seem to form some strings of beads. Furher treatments make the deformedspherical cells separated to each other and hyphas breaken into many small segments ,and the cytoplasm oozed out of the cells so as to most of cells become empty.Then the cell wall of these separated empty cells disintegrate and hyphas collapse.