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

Abstract

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 Km and decreased Vmax 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. The study revealed the pH-specific effects of Ca2+ on the mineralization of phytic acid