Optimization of hydrolytic and oxidative enzyme methods for ecosystem studies

Microbial digestive enzymes in soil and litter have been studied for over a half century, yet the understanding of microbial enzymes as drivers of ecosystem processes remains hindered by methodological differences among researchers and laboratories. Modern techniques enable the comparison of enzyme activities from different sites and experiments, but most researchers do not optimize enzyme assay methods for their study sites, and thus may not properly assay potential enzyme activity. In this review, we characterize important procedural details of enzyme assays, and define the steps necessary to properly assay potential enzyme activities in environmental samples. We make the following recommendations to investigators measuring soil enzyme activities: 1) run enzyme assays at the environmental pH and temperature; 2) run proper standards, and if using fluorescent substrates with NaOH addition, use a standard time of 1 min between the addition of NaOH and reading in a fluorometer; 3) run enzyme assays under saturating substrate concentrations to ensure Vmax is being measured; 4) confirm that product is produced linearly over the duration of the assay; 5) examine whether mixing during the reaction is necessary to properly measure enzyme activity; 6) find the balance between dilution of soil homogenate and assay variation; and 7) ensure that enzyme activity values are properly calculated. These steps should help develop a unified understanding of enzyme activities in ecosystem ecology.