Tetrachlorofluorescein TInsP^sub 5^ as a Substrate Analog Probe for Measuring Phytase Activity in Surface Water: Proof of Concept

• Published in: Journal of environmental quality 2013-01, Vol.42 (1), p.56
• Main Authors: Berry, Duane F, Harich, Kim
• Format: Article
• Language: English
• Subjects: Acids; Aquatic ecosystems; Chromatography; Liquid chromatography; Mass spectrometry; Microorganisms; Phosphate esters; Ponds; Potatoes; Surface water
• ISSN: 0047-2425; 1537-2537

An innovative approach for measuring phytase activity (PA) in surface water is presented. A substrate analog of myo-inositol hexakis(dihydrogen) phosphate (InsP^sub 6^), commonly referred to as 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)InsP^sub 5^, has been developed that can be used to monitor the (phytase-catalyzed) phosphate ester bond- cleavage reaction. Test phytases, (wheat [4-] and Aspergillus niger [3-] phytase) sequentially remove phosphate groups from TET TInsP^sub 5^, producing dephosphorylated probe species that were readily separated by reversed-phase high-performance liquid chromatography (RP-HPLC). Because dephosphorylated probe species retain the TET group, highly sensitive quantification could be achieved using fluorescence detection (excitation/emission λ = 245/540 nm). Calibration curves for TET TInsP^sub 5^, which could be used as a standard for quantifying all probe species, were linear (R^sup 2^ > 0.999) over the range of concentrations tested. Phytase-generated dephosphorylated probe species were characterized or identified using RP-HPLC with mass spectrometry. Results of mass spectrometry analysis show that the RP-HPLC system was capable of distinguishing between dephosphorylated probe species at the regioisomeric level. The TET TInsP^sub 5^ molecular probe was used to successfully measure PA in pond water. We found that the PA associated with the particulate plus water-soluble fraction was greater than that observed for the water-soluble fraction alone. Moreover, it appeared that 4- and 3-phytase were active in pond water based on an analysis of the chromatographic profile (i.e., elution sequence) of dephosphorylated probe species produced. The advent of a fluorescent substrate analog of InsP^sub 6^ affords environmental scientists with the means to unambiguously quantify an extremely small amount of phytase-generated dephosphorylated product(s), enabling the measurement of PA over a reasonably short time duration, in an environmental sample containing low concentrations of enzyme. [PUBLICATION ABSTRACT]