Microbial extracellular enzyme activity in marine sediments: extreme pH to terminate reaction and sample storage

The current analytical technique for measuring microbial extracellular enzyme activity within natural samples of water, soil or sediment involves incubation of samples with specific substrates conjugated with a fluorophore such as 7-amino-4-methylcoumarin (AMC) or methylumbelliferone (MUF). In this procedure, the extracellular enzymatic reaction is usually terminated by freezing and the reaction mixture is then stored frozen for later analysis. Unfortunately, the precision of this assay is Limited by the reactivation of extracellular enzymes which can occur when samples are thawed and by the adsorption of Liberated fluorophore onto the sediment which cannot be quantified without proper controls. To overcome these limitations, we describe a simple method using extreme pH (<5.5; >11) to terminate extracellular enzymatic reactions. Studies of aminopeptidase (AMPase) and beta-glucosidase (beta Gase) activity in marine sediments have also shown that addition of HCl and NaOH, respectively, eliminated the reactivation of extracellular enzymes when frozen samples were thawed. Furthermore, acetone and NaOH additions also reduced the interference caused by the adsorption of fluorophores to the sediment. Linearity of standard curves for fluorophores in the presence of these added chemicals were maintained and additional use of acetone increased AMC fluorescence (similar to 20 %). Modifications described in this report significantly reduced the level of variability in the determination of extracellular enzyme activity in marine sediments and allowed the simultaneous treatment of numerous samples.