Measuring Substrate Specificity of Extracellular Enzymes in Arctic Soils (Svalbard)

Microbial extracellular enzymes mediate most interactions between soil heterotrophic microbes and soil organic matter, because microbes use these enzymes to initiate the degradation of high molecular weight organic molecules. Activities of these enzymes are commonly determined via fluorescence spectroscopy using fluorogenic substrate proxies such as leucyl 7-amino-4-methylcoumarin or 4-methylumbelliferyl-glucopyranoside. The degradation rate of a given substrate is measured using a fluorescence detector and is commonly interpreted to be proportional to the activity of a specific extracellular enzyme corresponding to the substrate. Metagenomic studies, however, suggest that a diverse set of extracellular enzymes, many of which are functionally similar but not identical, are present in soils. It is therefore difficult to know precisely which enzymes a given assay is sensitive to.

Here we present measurements of the substrate specificities of extracellular hydrolases in permafrost and active-layer soils from Svalbard, in the high Arctic. We assess enzyme substrate specificity by measuring the extent to which related fluorogenic and chromogenic enzyme substrates competitively inhibit one another, using an adaptation of a high performance liquid chromatography (HPLC)-based enzyme assay. We discuss optimization of this method for enzyme specificity measurements and the implications of extracellular enzyme promiscuity for Arctic soil microbial ecology. Understanding the effects of substrate specificity will help build a more representative model of the enzymology and microbial community interactions within this remote ecosystem.