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Environment

Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays

Published: October 1st, 2013

DOI:

10.3791/50399

1Department of Biology, The University of Mississippi

Microplate based procedures are described for the colorimetric or fluorometric analysis of extracellular enzyme activity. These procedures allow for the rapid assay of such activity in large numbers of environmental samples within a manageable time frame.

Much of the nutrient cycling and carbon processing in natural environments occurs through the activity of extracellular enzymes released by microorganisms. Thus, measurement of the activity of these extracellular enzymes can give insights into the rates of ecosystem level processes, such as organic matter decomposition or nitrogen and phosphorus mineralization. Assays of extracellular enzyme activity in environmental samples typically involve exposing the samples to artificial colorimetric or fluorometric substrates and tracking the rate of substrate hydrolysis. Here we describe microplate based methods for these procedures that allow the analysis of large numbers of samples within a short time frame. Samples are allowed to react with artificial substrates within 96-well microplates or deep well microplate blocks, and enzyme activity is subsequently determined by absorption or fluorescence of the resulting end product using a typical microplate reader or fluorometer. Such high throughput procedures not only facilitate comparisons between spatially separate sites or ecosystems, but also substantially reduce the cost of such assays by reducing overall reagent volumes needed per sample.

Microorganisms such as bacteria and fungi obtain nutrients and carbon from complex organic compounds through the production of extracellular enzymes. These enzymes typically hydrolyze polymers into smaller subunits that can be taken into the cell. Therefore, at an ecological level, these microbial extracellular enzymes are responsible for much of the nutrient mineralization and organic matter decomposition that occurs in natural environments. Enzymes such as cellobiohydrolase (CBH) and β-glucosidase are important for cellulose degradation and work in unison to catalyze the hydrolysis of cellulose to glucose1,2, which provides a utilizable carbon substr....

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Colorimetric Analysis of Extracellular Enzyme Activity in Soils and Sediments

1. Preparation of Substrate and Buffer Solutions for Colorimetric Analyses of Enzyme Activity

  1. Prepare 50 mM acetate buffer (pH 5.0-5.5) by mixing 50 ml 0.1 M acetic acid (2.87 ml glacial acetic acid in 500 ml water), 150 ml 0.1 M sodium acetate, and 200 ml distilled H2O. Adjust pH to 5.0-5.5 with 0.1 M acetic acid if necessary.
  2. Prepare a solution of 1 M sodium hydroxide (NaOH) in distill.......

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Soils and aquatic sediments typically have appreciable levels of extracellular enzyme activity as a result of attached microbial communities (biofilms) growing on the surface of particles. Figure 3 shows how this activity changes depending on the size of particles obtained from the surface sediment of a third order stream in northern Mississippi, USA. A previous study has shown that the bacterial communities on sediment particles from this stream can be separated into three distinct groups based on molec.......

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Determining the activity of a variety of microbial extracellular enzymes in soils and sediments can provide useful insights into rates of nutrient mineralization and organic matter processing17. However, soils can vary in their moisture levels, so it is important to standardize activity to soil dry weight. This requires an additional drying step (typically of two days) beyond simply measuring enzyme activity. Thus, in contrast to assays of enzyme activity in water samples that provide near instantaneous result.......

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Funding for aspects of this work was provided by various sources including the United States Department of Agriculture Specific Cooperative Agreement 58-6408-1-595 and the National Science Foundation (award 1049911).

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Name Company Catalog Number Comments
REAGENTS AND MATERIALS
Glacial acetic acid Various suppliers
Sodium acetate Various suppliers
Sodium hydroxide Various suppliers
p-Nitrophenol Fisher BP612-1 Alternates available
p-Nitrophenyl (pNP)-phosphate Sigma N3234 pNP-substrate
pNP-β-glucopyranoside Sigma N7006 pNP-substrate
pNP-β-N-acetylglucosaminide Sigma N9376 pNP-substrate
Clear 96-well microplates Fisher 12-563-301 Alternates available
96-well deep well blocks Costar 3958 Alternates available
Aluminum weigh pans Various suppliers
Sterile 15 ml centrifuge tubes Various suppliers
Sterile 50 ml centrifuge tubes Various suppliers
4-Methylumbelliferone Sigma M1381
4-Methylumbelliferyl (MUB)-phosphate Sigma M8883 MUB-substrate
4-MUB-glucopyranoside Sigma M3633 MUB-substrate
4-MUB-N-acetylglucosaminide Sigma M2133 MUB-substrate
Sodium bicarbonate Various suppliers
Black 96-well microplate Costar 3792
Pipette reservoir Various suppliers
EQUIPMENT
Centrifuge Eppendorf 5810R
Centrifuge rotor Eppendorf A-4-81 For microplates/deep-well blocks
Microplate reader BioTek Synergy HT Alternates available
Microplate fluorometer BioTek FLx 800 Alternates available
8-channel pipettor Various suppliers

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