PURIFICATION AND CHARACTERIZATION OF PHYTASE FROM ASPERGILLUS FUMIGATUS ISOLATED FROM AFRICAN GIANT SNAIL (ACHATINA FULICA)

Abstract

Phytase, an enzyme that catalyzes the stepwise hydrolysis of myo-inositol-1,2,3,4,5,6-hexakisphosphate (phytate) into phosphorous and organophosphate compound and capable of reducing environmental pollution and metal chelating effect of phytate, was purified from Aspergillus fumigatus isolated from African Giant Snail (Achatina fulica); a member of class gastropoda and a macrophytophagus herbivore; that feeds on a wide variety of plants and decaying materials. Phytase isolated from Aspergillus fumigatus was subjected to ammonium sulphate precipitation, followed by ion exchange chromatography (DEAE Sephacel) and then gel filtration (Sephacryl S-200). Effect of pH and Temperature on the activity and stability of the purified phytase were determined, while effects of metal ions, chemical inhibitors, molecular weight and kinetic parameters were also investigated. Approximately 45%- fold purification was achieved with an overall recovery of 15%. The physicochemical properties revealed that the purified phytase has an optimum temperature activity at 40oC and it retained 80 % of its initial activity after 1 hour incubation period at 50oC and maintained about 100% stability at pH 5.0 after 6 hours with over 80% remaining activity between pH 4-7. The kinetic parameters of the purified enzyme, Vmax and Km were determined to be 35.7 μmol/min and 7.2 mM respectively. The phytase activity was enhanced in the presence of Ca2+, Cu2+ and Fe2+, while EDTA also enhanced enzymatic activity at low concentration but was greatly inhibited by Zn2+, Hg2+ and Al3+, Sodium Dodecyl Sulphate (SDS) and urea. The result showed that phytase produced from A. fumugatus may contribute significantly to the phytate degrading enzyme system in African giant snail and may serve a useful commercial purpose.