Fluorescence anisotropy as a means to determine extracellular polysaccharide hydrolase activity in environmental samples

Abstract

Current approaches to measure the activities of microbial extracellular enzymes in aquatic environments are hampered by slow throughput or by differences between the structure of simple substrate proxies and macromolecules. Here we show that measurements of fluorescence anisotropy can be used to deter- mine the hydrolysis rate of two fluorescently labeled polysaccharides, laminarin and xylan, in environ- mental samples. A simple analysis shows that the anisotropy of these fluorescently labeled polysaccharides can be approximated using a modification of the Perrin equation. /C2112008 Elsevier Inc. All rights reserved. The rates and structural specificities of microbial extracellular enzymes help to control the bioavailability and turnover of high- molecular-weight dissolved organic matter (DOM) in natural waters [1]. The most widely used method to assess these enzyme activities is based on small substrate proxies (e.g., 4-methylumbel- liferyl-b-d-glucopyranoside [2], l-leucine-7-amido-4-methylcouma- rin [3]) that consist of a monomer bound to a fluorophore that fluoresces when the fluorophore–monomer bond is cleaved. This method is inexpensive, precise, and rapid, but the structural differ- ences between macromolecules and small substrate proxies mean that many aspects of enzyme structural selectivity cannot be ex- plored, and the hydrolysis rates obtained with these substrate prox- ies may differ from those measured with the corresponding polymer [4]. Furthermore, small substrate proxies can diffuse into cellular periplasm, so they may record a combination of extracellular and periplasmic enzymatic activity [5]. An alternative approach is to measure directly, either by chromatographic analysis [6–8] or by electron paramagnetic resonance (EPR) spectroscopy [9], changes…