Effect of citrate on Aspergillus niger phytase adsorption and catalytic activity in soil

Abstract

Phytase enzymes from bacteria, fungus and plant root exudates are known to hydrolyse organic phosphorus (Po) to bioavailable inorganic orthophosphate in soil. Exploiting such biochemical functions in agricultural systems, offers the potential for alternative sustainable phosphorus sources. Phytase adsorption to soil particles and phytate metal complexation has been shown to inhibit phytate (InsP6) dephosphorylation. Organic acid anions such as citrate increase phytase catalytic efficiency towards complexed forms of InsP6, but the mechanisms are poorly understood. The aim of this work was to evaluate Aspergillus niger phytase inactivation and changes in its catalytic properties upon addition to soil, as well as the effect of citrate on phytase adsorption and activity towards free, precipitated and adsorbed InsP6.We performed a series of enzyme hydrolysis activity assays when the enzymes were free, in soil solution and adsorbed to soil under varying chemical conditions, with and without citrate. A. niger phytase showed a relatively low absorption affinity for the Cambisol test soil. Phytase activity reduced by 37.3% due to adsorption. Citrate had no effect on the rate or total amount of phytase adsorption or soil adsorbed phytase activity thereafter. Free phytases and phytases in soil solution showed optimum activity (≥80%) at pH4.5–5.5. Activity decreased slightly for soil adsorbed enzymes compared to enzymes which were free or in soil solution>pH5 and <4 in the pH dependency curve. A decrease in activity was seen only at ionic strengths (NaCl)>0.6M in adsorbed and free enzymes, while activity from enzymes in soil solution reduced in all tested ionic strengths. Citrate significantly increased phytase activity towards InsP6 adsorbed to soil when the phytases were free (p≤0.003) but not in other treatments (Na, Al and Ca-phytate). These results suggest that the effect of citrate on soil InsP6 dephosphorylation is associated with the availability of the substrate (InsP6) rather than its effect on the enzyme per se. The ionic strength and pH of soil solution has been shown to impact on phytase activity, suggesting that salinity, quality of irrigation water, wetting/drying cycles and fertilisation will have discrete impacts on the activity of phytases once released in soil and thus the ability to make Po available for uptake by plants and microbes. The optimum acidic pH of A. niger also brings into question its suitability for application in many agricultural soils.