Measurement of phytase activity in soil using a chromophoric tethered phytic acid probe

The lack of a reliable method for measuring phytase activity (PA) in soil impedes efforts to elucidate the influence of these phosphomonoesterases on phytate degradation and subsequently the P cycling process in soils and sediments. The objective of our research is to develop a convenient method for measuring PA in soil using the novel chromophoric substrate analog of phytate, 5-O-[6-(benzoylamino)hexyl]-d-myo-inositol-1,2,3,4,6-pentakisphosphate i.e., T(tethered)InsP5. This artificial substrate permits direct measurement of the phytase-catalyzed dephosphorylation reaction (hydrolysis of phosphate groups) using reversed-phase high-performance liquid chromatography. TInsP5 can serve as an external standard for quantification of phosphorylated probe intermediate species on a molar basis, as UV detection relies on the same benzamido chromophore (λmax 226 nm), which is unaffected by the number of phosphate groups present on the inositol moiety. Under soil slurry assay conditions we observed dephosphorylation of TInsP5 with subsequent production of phosphorylated probe intermediates and 5-O-[6-(benzoylamino)hexyl]-D-myo-inositol, i.e., TIns. PA was determined by quantifying production of dephosphorylated probe species for a given incubation time period. Results of this investigation support our position that TInsP5 will function as a useful probe for measuring PA in soil.