Purification and Identification of Fungistatic Protein from Bacillus subtilis CF-3
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Abstract:
Ammonium sulfate precipitation, Sephadex G-75 gel column chromatography, and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) were used to purify and identify the primary fungistatic proteins in Cephalothecium fungi that act on the biocontrol agent Bacillus subtilis CF-3. The results demonstrated that the active protein could be optimally precipitated with 60~70% ammonium sulfate with a content of 149.03 μg/mL, which was significantly higher than the yield obtained from other ammonium sulfate concentrations (p≤0.01). Crude proteins separated by ammonium sulfate at a saturation of 60~70% were further purified by Sephadex G-75 gel column chromatography, yielding two absorption peaks and eight fractions. The second fraction had a significantly greater fungistatic effect than the other fractions (p≤0.05) and was further purified. Band 2.3 of the flowthrough had the highest fungistatic activity and was cut from the gel for collection. Biological mass spectrometry was used to identify the bands as ?-glutamyl transpeptidase and intracellular serine protease, with molecular masses of 42.5 ku and 33.9 ku, respectively. These results laid the foundation for the application of this bacterial strain and these fungistatic proteins.
