Measurement of phytase activity using tethered phytic acid as an artificial substrate: Methods development

Abstract The artificial chromophoric substrate analog of phytic acid, 5- O -[6-(benzoylamino)hexyl]- d - myo -inositol-1,2,3,4,6-pentakisphosphate (T-IP 5 ), may prove useful in measuring soil phytase activity. This chemical probe allows for direct measurement of phytase-catalyzed dephosphorylation (i.e., hydrolysis of the phosphoester bond) using high-performance liquid chromatography with UV detection. Before T-IP 5 can be used to measure phytase activity in environmental samples (soil, stream sediment, manure) refinement of the T-IP 5 probe methodology is required. Using 31 P nuclear magnetic resonance (NMR) spectroscopy, we identified 5- O -[6-(benzoylamino)hexyl]- d - myo -inositol-trisphosphate (T-IP 3 ) as the key intermediate that accumulates during phytase-catalyzed dephosphorylation of T-IP 5 . An improved HPLC method for separation of reactants is also presented.