Purification and characterization of an alkaline phytase from Lilium longiflorum

Phytases (myo-inositol-hexakisphosphate phosphohydrolases) are the principle enzymes responsible for the hydrolysis of phytic acid. They catalyze the sequential hydrolysis of phosphate monoesters on the myo-inositol ring, generating less phosphorylated myo-inositol derivatives and orthophosphate.; In plant seeds and pollen, phytic acid accounts for 1-5% by dry weight and is the major storage form of phosphorus and essential minerals. In animal cells, phytic acid is often the most abundant inositol phosphate. Phytases have long been of interest from a commercial perspective as an animal feed supplement. Recently, the identification of multiple-inositol polyphosphate phosphatases (phytic acid-degrading enzymes) in mammalian cells has indicated a role for phytases in the inositol phosphate mediated Ca2+-mobilization during signal transduction.; This dissertation describes the purification and characterization of alkaline phytase from a plant source. Purification of alkaline phytase from pollen grains of Lilium longiflorum was achieved by means of selective precipitation by heat and ammonium sulfate, anion exchange and chromatofocusing chromatography and gel electrophoresis. Alkaline phytase was purified ∼3,000-fold with an overall recovery of 4.2%. Estimates of the native molecular mass indicated a MR of 113-149 kD by gel filtration chromatography. The enzyme displayed Michaelis-Menten kinetics against Na-phytate with a K M of 77 muM and a Vmax of 0.272 U/mg. Separation by 2-D gel electrophoresis and MALDI-TOF mass spectrometric analysis suggest the presence of multiple mass and charge isoforms with pI values between 7.3 and 8.3. The purified enzyme was thermally-stable, retaining 100% of original activity after incubation at 55°C for thirty minutes.; Several studies have established a correlation between phytate degradation and an increase in acid phytase activity during pollen tube formation and seed germination. In contrast, during the germination of lily pollen, the activity of alkaline phytase was seen to decrease by 70% from an initial value of 5.7 x 10-4 U/mg pollen to 1.6 x 10-4 U/mg pollen after six hours of germination. The level of the alkaline phytase gene transcript however, did not change as a function of germination time.