Multiple-substrate enzyme assays: a useful approach for profiling enzyme activity in soils?

This study focuses on the applicability of multiple-substrate enzyme assays to simultaneously determine various soil enzyme activities within one assay. Mineral soils from agricultural field sites differing in soil properties and management were used to optimise substrate composition and concentration of 4-methylumbelliferone and 7-amino-4-methylcoumarin derivatives as model substrates. In contrast to conventional assays, enzyme activity was measured at soil pH since optimum pH is not more applicable using a multiple-substrate approach. Furthermore, enzyme activity was not calculated from the product formed but from substrate decrease. After incubation the added substrates were re-extracted, separated by high-performance liquid chromatography and quantified by UV-absorption at 320 nm. This approach allows simultaneous measurement of the activity of β-d-glucosidase, N-acetyl-β-d-glucosaminidase, β-d-glucuronidase, β-d-xylosidase, phosphomonoesterase, sulfoesterase and leucine-aminopeptidase within one assay and with sufficient accuracy. However, incomplete re-extraction due to adsorption of substrates to the soil matrix was observed. In addition, certain competitive inhibition effects due to chemically similar substrates were found. Compared to conventional methods, no distinct differences in enzyme activity profile were detected, with both assays—conventional and multiple-substrate approach—leading to similar differentiation among the investigated soils. In conclusion, the multiple-substrate approach may serve as time-saving alternative to standard enzyme assays in mineral soils. Certainly, since the multiple-enzyme assay is conducted at soil pH, the procedure leads to reduced comparability of soils with contrasting pH values.