Micro-Assay Method for Measuring Soil Phytase Activity with the TInsP5 Molecular Probe.

The lack of a convenient method for measuring phytase activity (PA) associated with complex natural environments has constrained efforts to elucidate the impact of phytase on P cycling process in soils and sediments. Our objective is to develop a soil PA assay using the chromophoric substrate analog of phytic acid, 5-O-[6-(benzoylamino)hexyl]-D-myo-inositol-1,2,3,4,6-pentakisphosphate, i.e., T(tethered)InsP5. The TInsP5 molecular probe permits direct measurement of the phytase-catalyzed dephosphorylation reaction by quantifying production of chromophoric probe intermediate species (wavelength maximum 226 nm) with reversed-phase high-performance liquid chromatography. A newly developed micro-assay technique, described in this report, was used to measure PA of Groseclose agricultural soil. The concentration of soil-generated phosphorylated intermediate probe species (e.g., TInsP4, TInsP3) was estimated, on a molar basis, using TInsP5 as the external standard. PA was determined using a mass balance approach by quantifying production of dephosphorylated probe species over a given time period. The micro-assay method for determining PA in soil was straightforward and delivered reproducible results. Also included in this report is a comprehensive approach for identifying previously unsubstantiated soil-produced dephosphorylated intermediate probe species.