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

Summary

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.

Abstract

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.

Introduction

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 glucose^1,2, which provides a utilizable carbon substr....

Protocol

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 distilled H₂O.
3. Prepare pNP-linked substrate solutions in 50 mM acetate buffer. To assay phosphatase prepare 5 mM pNP-p....

Discussion

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.......

Materials

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