Enhanced and suppressed phosphorus mineralization by Ca complexation: NMR and CD spectroscopy investigation

With the increasing demand for P fertilizer for world food production, the use of soil organic P fraction via mineralization could become an important P resource in agricultural soils. However, the predominant organic P species, phytic acid, has been considered rather recalcitrant to mineralization due to its active interaction with dissolved metals like Ca²⁺ in soil pore water. Calcium ions can be an inhibitor to many phytases, yet the mechanism was not clear. The objective of this study was to understand the effects of Ca²⁺(aq) on the phytase activity and inhibitory mechanisms using batch degradation kinetic experiments, Nuclear Magnetic Resonance (NMR) spectroscopy, Saturation Transfer Difference (STD) NMR, and Circular dichroism (CD) spectroscopy. The phytase activity followed Michaelis-Menten kinetics and increased Michaelis constant K_m and decreased V_max with Ca²⁺ addition were observed at pH 6. Therefore, mixed inhibition was the inhibition mechanism which was likely a result of the allosteric effect of Ca²⁺. The near-UV CD spectra supported phytase secondary conformational change upon the interaction between Ca²⁺ and the enzyme. It was found that phytase initially reacted with the D/L-3 phosphate of phytic acid at pH 6. At pH 8, the overall phytase activity decreased, yet the effect of Ca²⁺ on phytase activity was the opposite of that of pH 6. Enhanced phytase activity with Ca²⁺ addition was attributed to the structural change of phytic acid upon the Ca²⁺ complexation, which was confirmed by NOE spectra. The Ca²⁺-phytic acid complex might be a more favorable substrate than the free phytic acid. Unlike the findings from pH 6, Ca²⁺ didn't induce significant changes in either the near- or far-UV region of the CD spectra at pH 8. Furthermore, P5 was found to be the target of phytase at pH 8.