Enzymatic flow-injection determination of phytase-hydrolysable phosphorus (PHP) in natural waters using immobilized 3-phytase

Phytic acid, also known as myo-inositol hexakisphosphate, is a naturally occurring, non-toxic phosphoric ester that is abundant in seeds and plants as well as aquatic sediments and in soils. It is hydrolysed to a less phosphorylated form by phytases, which are naturally occurring phosphohydrolase enzymes. Because of their non-specificity, phytases also hydrolyse other phosphoester compounds, liberating bioavailable orthophosphate. A flow-injection (FI) method for the determination of phytase hydrolysable phosphorus (PHP) using immobilized phytase is proposed. The method is suitable for the determination of PHP in natural waters, a parameter that may provide a measure of potential long-term bioavailability of organic phosphorus in aquatic systems. Sample volume, temperature, and buffer composition for the immobilized phytase hydrolysis were all optimized. The effects of the potentially interfering ions Al3+, Fe2+, Fe3+, Mn2+, Cu2+, Pb2+, NO2-, NO3-, I-, SO42, AsO43-, and EDTA and urea were also investigated. The method has a limit of detection of 5 mu g P L-1, and the optimized RSD was typically < 4% (n = 4). The spike recoveries of phytic acid as P were >= 95%. The optimized method was applied to the determination of phytase hydrolysable phosphorus in the Tamar Estuary, and the results obtained (14-25 mu g P L-1) show that this fraction constitutes a significant portion of the total dissolved P pool in the estuary.