A simple method for preparation of α-sarcin and an antifungal protein (AFP) from mold (Aspergillus giganteus MDH 18894) has been developed. α-Sarcin and AFP were purified simultaneously by chitin affinity column chromatography and gel filtration. By this method, 4.5 mg of pure α-sarcin and 6.9 mg of pure AFP were obtained from 2 liters of culture medium. Compared with other purification methods such as ion-exchange column chromatography, this procedure was very simple and specific. The purified α-sarcin and AFP were homogeneous as characterized by SDS-polyacrylamide gel electrophoresis. Both α-sarcin and AFP exhibited the binding activity to generated chitin. Soluble glycochitin decreased the intensity of fluorescence of α-sarcin and made the λemm shift from 340 to 347 nm. Titration of α-sarcin with N-bromosuccinimide under native conditions revealed that two tryptophans (Trps) were all located in the core part of α-sarcin molecule. This indicated that Trps were not involved in the binding of α-sarcin to chitin. Glycochitin in the culture medium increased the expression of α-sarcin, while it had no effect on the expression of AFP. Unlike other ligands such as Cibacron blue for the affinity purification of α-sarcin and AFP, glycochitin increased the nuclease activity of α-sarcin.
- Antifungal protein (AFP)
- Aspergillus giganteus MDH 18894
- Chitin affinity column chromatography
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