High-throughput fluorometric measurement of potential soil extracellular enzyme activities

Microbes in soils and other environments produce extracellular enzymes to depolymerize and hydrolyze organic macromolecules so that they can be assimilated for energy and nutrients. Measuring soil microbial enzyme activity is crucial in understanding soil ecosystem functional dynamics. The general c...

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Bibliographic Details
Published in:Journal of visualized experiments 2013-11, Vol.81 (81), p.e50961-e50961
Main Authors: Bell, Colin W, Fricks, Barbara E, Rocca, Jennifer D, Steinweg, Jessica M, McMahon, Shawna K, Wallenstein, Matthew D
Format: Article
Language:English
Subjects:
Archaea - enzymology
Bacteria - enzymology
BASIC BIOLOGICAL SCIENCES
Environmental Sciences
Enzymes - analysis
Enzymes - metabolism
Fluorescent Dyes - chemistry
Fluorescent Dyes - metabolism
Fluorometry - methods
Fungi - enzymology
High-Throughput Screening Assays - methods
Science & Technology - Other Topics
Soil - chemistry
Soil Microbiology
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Summary:Microbes in soils and other environments produce extracellular enzymes to depolymerize and hydrolyze organic macromolecules so that they can be assimilated for energy and nutrients. Measuring soil microbial enzyme activity is crucial in understanding soil ecosystem functional dynamics. The general concept of the fluorescence enzyme assay is that synthetic C-, N-, or P-rich substrates bound with a fluorescent dye are added to soil samples. When intact, the labeled substrates do not fluoresce. Enzyme activity is measured as the increase in fluorescence as the fluorescent dyes are cleaved from their substrates, which allows them to fluoresce. Enzyme measurements can be expressed in units of molarity or activity. To perform this assay, soil slurries are prepared by combining soil with a pH buffer. The pH buffer (typically a 50 mM sodium acetate or 50 mM Tris buffer), is chosen for the buffer's particular acid dissociation constant (pKa) to best match the soil sample pH. The soil slurries are inoculated with a nonlimiting amount of fluorescently labeled (i.e. C-, N-, or P-rich) substrate. Using soil slurries in the assay serves to minimize limitations on enzyme and substrate diffusion. Therefore, this assay controls for differences in substrate limitation, diffusion rates, and soil pH conditions; thus detecting potential enzyme activity rates as a function of the difference in enzyme concentrations (per sample). Fluorescence enzyme assays are typically more sensitive than spectrophotometric (i.e. colorimetric) assays, but can suffer from interference caused by impurities and the instability of many fluorescent compounds when exposed to light; so caution is required when handling fluorescent substrates. Likewise, this method only assesses potential enzyme activities under laboratory conditions when substrates are not limiting. Caution should be used when interpreting the data representing cross-site comparisons with differing temperatures or soil types, as in situ soil type and temperature can influence enzyme kinetics.
ISSN:1940-087X
1940-087X
DOI:10.3791/50961