Use of immobilized 3-phytase and flow injection for the determination of phosphorus species in natural waters

A proportion of dissolved organic phosphorus (DOP) compounds found in natural waters may be utilized by algae and bacteria through hydrolysis by extracellular phosphomonoesterases such as alkaline phosphatase. The fraction of DOP remaining unhydrolysed may consist of organic phosphorus species such as myo-inositol hexakisphosphate (phytic acid), and comprise part of the pool of biologically unavailable or even refractory DOP. Of this bio-unavailable DOP fraction, a major portion is amenable to hydrolysis by 3-phytase, a phosphohydrolytic enzyme which is not found widely in aquatic systems. This paper describes the development and application of a flow injection system which utilises immobilized 3-phytase for the determination of phytase hydrolysable phosphorus (PHP) present in natural waters. The product of enzymatic hydrolysis is orthophosphate, and a conventional phosphomolybdenum blue detection method was used. The use of a suitable flow injection manifold and a multiple linear regression calibration procedure for quasi-simultaneous determination of phytase-hydrolysable and dissolved reactive phosphorus (DRP) coexisting in natural waters is outlined. The proposed technique is rapid (ca. 40 injections h−1), sensitive and reproducible, and the immobilized phytase exhibited good operational and storage stability over a period of several months.