Purification and identification of a phytase from fruity bodies of the winter mushroom, Flammulina velutipes
A phytase, with a molecular mass of 14.8 kDa as determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis was isolated from fresh fruiting bodies of the mushroom Flammulina velutipes. The chromatographic procedure used for isolation of the phytase included ion exchange chromatography on DEAE-cellulose, CM-cellulose, Q-Sepharose, affinity chromatography on Affi-gel blue gel and fast protein liquid chromatography-gel filtration on Superdex 75. The enzyme was adsorbed on DEAE-cellulose, unadsorbed on CM-cellulose and Affi-gel blue gel, and adsorbed on QSepharose. It presented an N-terminal sequence different from those of reported fungal phytases. It exhibited maximal activity at about 45°C and it did not manifest drastic fluctuations in activity over the pH of 3 to 9. The phytase presented activity towards a variety of phosphorylated compounds with the following ranking of activity: ATP > fructose-6-phosphate ≈ glucose-6-phosphate > AMP > ADP> β- glycerophosphate > sodium phytate. It did not present antifungal activity.
Key words: Phytase, Flammulina velutipes, Mushroom, Isolation.