Tetrachlorofluorescein TInsP5 As a Substrate Analog Probe for Measuring Phytase Activity in Environmental Samples.

An innovative approach for determining phytase activity in environmental samples is presented. A fluorogenic substrate analog of phytic acid, 1D-myo-5-O-(1-oxo-1-(2',4,7,7'-tetrachloro-3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-6-yl)-5,8,11-trioxa-2-azatridecan-13-yl)-inositol 1,2,3,4,6-pentakis-O-(dihydrogen) phosphate, referred to as tetrachlorofluorescein (TET) tethered (T)InsP₅, has been developed allowing direct measurement of the phosphate ester bond-cleavage reaction. Test enzymes, wheat (4-) and Aspergillus niger (3-) phytase, hydrolyze TET TInsP₅ producing flurogenic dephosphorylated probe species, which are readily separated by reversed-phase high-performance liquid chromatography (RP HPLC). Because dephosphorylated probe species retain the TET group, highly sensitive quantification can be achieved using florescence detection (Excitation/Emission λ = 245/540 nm). Calibration curves for TET TInsP₅ which may be used as a standard for quantifying all probe species, were linear (R² > 0.999) over the range of concentrations tested. Phytase-generated dephosphorylated probe species (e.g., TET TInsP₄ and TET TInsP₃) were characterized/identified with RP HPLC-mass spectrometry. The TET TInsP₅ molecular probe was successfully used to detect phytase activity in pond water. Phytase activity associated with unfiltered pond water was greater than that observed for the filterable fraction (<0.22 μm). Moreover, it appears that 3- and 4-phytase were both active in pond water based on an analysis of the chromatographic profile of the dephosphorylated probe species produced. The TET TInsP₅ molecular probe should prove useful when it comes to assessing the nutritive status of P in surface water ecosystems. The advent of a fluorogenic substrate analog of phytic acid affords environmental scientists the means to unambiguously quantify an extremely small amount of phytase-generated product(s), enabling the measurement of phytase activity, over a reasonably short time duration, in an environmental sample containing low concentrations of enzyme.