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

Abstract

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)InsP 5. This artificial substrate permits direct measurement of the phytase-catalyzed dephosphorylation reaction (hydrolysis of phosphate groups) using reversed-phase high-performance liquid chromatography. TInsP 5 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 ( λ max 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 TInsP 5 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 TInsP 5 will function as a useful probe for measuring PA in soil.