Enzyme assays under dried conditions

Enzymatic assays (Fig. 1) were performed by adding a buffered solution containing the corresponding fluorogenic substrate (0.1 mM, final concentration; AMC, MUG and MUP) (Gonzalez, Portillo & Piñeiro-Vidal, 2015) to natural soil (2 mg aliquots) or the sterilized soil supplemented with bacterial species. Until this point samples and solutions were maintained on ice and once the fluorogenic substrates were added, the soil mixtures were frozen at −80 °C to minimize substrate degradation and modification of sample conditions during handling. Samples aimed to perform enzyme activity measurements at reduced water activities aliquots were freeze dried to reduce the water content in the soil mixture and reach the required water activity. The final water activity was determined as detailed above using a Rotronic water activity probe. Samples were only freeze-dried a single period and those at a water activity close to the required water content were used for the assay. If the resulting water activity at a given soil subsample was distant from the expected value that subsample was not processed further. Reactions at different temperatures and water activities were carried out in triplicate. Once the required water activity is obtained, the aliquots were incubated in a sealed container at the required temperatures (20 °C or 60 °C) for different time periods. Different aliquots were placed in different tubes so that for each time point three replicates were extracted from incubation. Incubation times were below 10 min because around this time the kinetic curve leveled-off. That time period was sufficient to estimate the linear slope of fluorescence vs incubation time in the studied cases. The enzyme assays for natural samples were incubated at 20 °C and 60 °C. Enzyme assays for bacterial cultures were incubated at the strain optimum growth temperature. Time zero was considered when the reaction reached the desired incubation temperature. After the incubation period, the reactions were stopped by adding ethanol (Stemmer, 2004) and the pH was adjusted with ice-cold glycine-NaOH (0.1 M; pH 11) to maximize the fluorescence signal while preserving the undigested fluorogenic substrate and the fluorescent product. The stopped reaction mixture was vortexed and the solution was cleared from soil particles by centrifugation at 5,000×g for 5 min (4 °C). Fluorescent measurements were carried out in an Omega fluorometer (BMG LabTech GmbH, Ortenberg, Germany) using the filter sets recommended by the manufacturer (excitation 355 nm; emission 460 nm). The rate of enzyme activity was estimated by linear regression (Model I, only Y variable is subject to error) (Sokal & Rohlf, 2012) as the slope of fluorescence vs incubation time during the linear portion of the curve. Correlation and regression analysis (Model II, both variables are subject to error) between the results from assays following the above protocol and those performed by the classic, in-solution method (see below) where performed according to Sokal & Rohlf (2012).