Measurement of phytase activity using tethered phytic acid as an artificial substrate: Methods development [An article from: Soil Biology and Biochemistry]

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Description:
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 ^3^1P 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.