A microplate fluorimetric assay for the study of enzyme diversity in soils

A fluorimetric microplate enzyme assay has been developed to study enzyme diversity in soil as an approach to understanding functional diversity. The microplate assay allows a large number of soil samples and/or enzymes to be analysed in a short time. The substrates used are conjugates of the highly fluorescent compounds 4-methylumbelliferone (MUB) and 7-amino-4-methyl coumarin (AMC). The main advantage of using fluorimetrically-labelled substrates is that product formation can be measured directly in the microplate without previous extraction and purification of the product. A detailed protocol for this new technology is presented and some potential applications are outlined. A comparative study was carried out between the new microplate fluorimetric assay and a standard colorimetric enzyme assay based on p-nitrophenyl substrates. The kinetics of β-glucosidase and phosphatase activities were investigated in soils with different fertilizer backgrounds. Both methods generated similar values for Vmax (maximum rate of activity) whereas the affinity of β-glucosidase and phosphatase for their respective substrates (as indicated by Km values, Michaelis–Menten constant) was up to two orders of magnitude greater for the 4-methylumbelliferyl substrates compared to the p-nitrophenyl substrates.