Purification And Characteristics Of Several Antifungal Proteins And Peptides

Antifungal peptides(AFPs), serve to protect organisms against fungal invasion, have been isolated from a variety of plants species.The experimental materials of present investigation was seeds of tartary buckwheat (Fagopyrum tataricum).The purification procedure involved extraction with aqueous buffer, heat treatment, cation exchange chromatography on Resource S column, and size exclusion chromatography (SEC) on Superdex Peptide.The apparent molecular weight of purified peptide was approximately 8.0 kDa by Tricine-SDS-PAGE,and actual molecular weight was 3.909 kDa by surface-enhanced laser desorption ionization-time of flight (SELDI-TOF). This peptide showed strong antifungal activity against Panus conchatus, Trichoderma reesei and Alternaria alternata. Light microscopic examination disclosed antifungal peptide induced swelling of hyphal tips, hyphal distortions and fragmentation in the Trichoderma reesei.A chitinase with antifungal activity was purified from naked oat (Avena chinensis) seeds by extraction with an aqueous buffer, cation exchange and size exclusion chromatography. The molecular weight and isoelectric point of the purified chitinase were estimated at 35 kDa and 8.9, respectively. Peptide mass fingerprint analysis indicated the primary structure of the purified naked oat (Avena chinensis) chitinase is highly homologic to oat (Avena sativa) class I chitinase.When colloidal chitin is used as a substrate, with a specific activity 3.596 U/mg, which is consistent with reported chitinase activity, the purified chitinase is highly active.The chitinase activity is dependent on both pH and temperature, with an optimum pH of 7.0 and optimum temperature of 40℃.Measurement of in vitro antifungal activity demonstrated that the purified chitinase has potent, dose-dependent inhibitory activity against fungi Panus conchatus and Trichoderma reesei. All these results provided evidence that the chitinase purified from naked oat seeds is a class I chitinase with antifungal activity..To isolate and purify an antifungal proteins from seeds of adlay(Coix chinensis Tod.)and analyze its antifungal activity. Methods The procedure involved extraction with aqueous buffer, ion exchange chromatography on Resource S,and size exclusion chromatography (SEC) on Superdex 75.Results The molecule weight of antifungal protein is approximately 28kDa, which was determined by SDS-PAGE.It shows strong fungal growth inhibitory activity against Alternaria alternate, Trichoderma reesei and Panus conchatus, in a dose-dependent manner. Conclusion The method is simple,and the purify of protein is over 95%,it can be used for purification of some novel antifungal protein from plant seeds.