A microplate fluorimetric assay for the study of enzyme diversity in soils |
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| Institution: | 1. Department of Soil Science, The University of Reading, Whiteknights. PO Box 233, Reading RG6 6DW, UK;2. Institute of Grassland and Environmental Research, North Wyke, Okehampton, Devon EX20 2SB, UK;1. School of Forest Resources and Environmental Science, Michigan Technological University, Houghton, MI 49931, USA;2. Department of Biological Sciences, University of Cincinnati, 312 College Drive, Cincinnati, OH 45221, USA;3. US Forest Service, Northern Research Station, 410 MacInnes Dr., Houghton, MI 49931, USA;1. Dept. of Soil Science of Temperate Ecosystems, University of Göttingen, Germany;2. Institute of Physicochemical and Biological Problems in Soil Science, Russian Academy of Sciences, Pushchino, Russia;3. Dept. of Agricultural Soil Science, University of Göttingen, Germany;4. Institute of Environmental Science, Kazan Federal University, 420049, Kazan, Russia;1. Max Planck Institute for Biogeochemistry, Hans-Knoell-Str. 10, 07745 Jena, Germany;2. Institute of Soil Science and Land Evaluation, Soil Biology Section, University of Hohenheim, Emil-Wolff-Str. 27, 70599 Stuttgart, Germany;3. Institute of Geography, Friedrich Schiller University Jena, Löbdergraben 32, 07743 Jena, Germany;1. Department of Agricultural and Environmental Sciences, Tennessee State University, Nashville, TN 37209, USA;2. State Key Laboratory of Loess and Quaternary Geology, Key Laboratory of Aerosol Chemistry and Physics, Institute of Earth Environment, Chinese Academy of Sciences, Xi’an 710061, China;3. Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK 73019, USA;4. Climate Change Science Institute and Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA;5. Department of Biological Sciences, Tennessee State University, Nashville, TN 37209, USA |
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| Abstract: | 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. |
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