In this case, the isotopic fractionation caused by phytase from Aspergillus niger will be determined by the equilibrium of the reaction. These observations suggest that the reformation of IP6 from the two products of the reaction (myo-inositol and Pi) is taking place at a rate, which is within the time scale of the experiment. In addition, we observe a change in the oxygen isotope composition of Pi when using myo-inositol and potassium-dihydrogen-phosphate as sole substrates in the enzymatic assays with phytase from Aspergillus niger. In contrast, the results from assays with phytase from Aspergillus niger indicate that the exchange of oxygen occurs at more than one third of the total 24 oxygen which are associated to the phosphates in IP6. This is a value very similar to those reported for acid phosphatases (6).

Provided that, the isotopic fractionation can be calculated with an isotopic mass balance resulting in -8.4â€° (Ã-3.6 SD). This suggests that during the enzymatic hydrolysis, four P-O bonds and two C-O bonds are broken. From this we conclude that the incorporation of one oxygen atom from water occurs only at four phosphate molecules of IP6, while two phosphate molecules do not experience an incorporation of oxygen. In the case of the assays with phytase from wheat germ, our results indicate that one sixth of the total 24 oxygen which are associated to the phosphates in IP6 are exchanged with oxygen from water.

Assays with phytase from wheat germ lead to a turnover of the substrate close to 100%, while assays with phytase from Aspergillus niger lead to a turnover of the substrate close to 80%. Enzymatic assays with phytase from wheat germ and Aspergillus niger were prepared under sterile and temperature controlled conditions in order to determine the effect of phytases on the oxygen isotope composition of phosphate, which has been liberated from IP6 via enzymatic hydrolysis. Phytases catalyze the hydrolysis of myo-inositol-hexakis-phosphate (IP6), which is an important component of organic P in many ecosystems (7). While the effect of phosphomonoesterases and phosphodiesterases on the oxygen isotope composition of phosphate has been examined, there are, so far, no studies dealing with the effect of phytases (4-6). During the incorporation, an isotopic fractionation occurs, which might be used to identify the origin of Pi in the environment (3-6). The enzymatic hydrolysis leads, via a nucleophilic attack, to the incorporation of one oxygen atom from the water into the newly formed Pi molecule. These enzymes catalyze the hydrolysis of phosphoesters leading to a release of Pi. Plants and microorganisms under phosphorus (P) stress release extracellular phosphatases as a strategy to acquire inorganic phosphate (Pi) (1-2). The Effect of Phytase on the Oxygen Isotope Composition of Phosphate

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