Fluorometric assay for phenol oxidase activity in soils and its controlling variables

Abstract

Phenolic compounds constitute the major component of recalcitrant carbon pools in soils and undergo degradation by enzymes such as phenol oxidases. Despite their significance, a common, appropriate assay to determine its activity is yet to be found. Until recently, only colorimetric assays, using substrates such as L-DOPA, pyrogallol, and ABTS, have been dominantly employed to measure phenol oxidase activity, despite their drawbacks, such as interference of soil background color, unavailability of a commercial product, and low sensitivity. Therefore, this study aimed to identify the optimal conditions for the novel fluorometric Amplex Red assay, compare the effectiveness and reproducibility of this assay with the conventional colorimetric L-DOPA assay, and reveal the key controlling variables of phenol oxidase activity by applying it to various soil types. Results showed that the optimal phenol oxidase activity can be determined at a substrate concentration of 500 μM and when the soil suspension is prepared with sonication only, using deionized water as a solvent. The comparison of the Amplex Red and L-DOPA assay indicated that the fluorometric assay has greater reproducibility, with a significantly smaller coefficient of variation (19.9 % and 107 %, respectively). For the L-DOPA assay, there was substantial variability between replicates and strong dependence on soil suspension preparation methods. Although this study reveals that the Amplex Red assay is more suitable for measuring phenol oxidase activity, additional research on other soil types is important to confirm its applicability, and adjustments to incubation conditions may be necessary for specific soil types. The application of the Amplex Red assay on various soil types uncovered soil pH and organic matter as the two most important factors controlling phenol oxidase activity, which aligned with previous findings. A positive linear correlation between phenol oxidase activity and soil pH (R = 0.734, P ≤ 0.000) but a negative exponential correlation with soil organic matter content (R = −0.547, P ≤ 0.01).