Soil enzyme activities analysis

The enzyme activities involved in α-glucosidase (EC 3.2.1.20) hydrolyzing of soluble saccharides); β-glucosidase (EC 3.2.1.21) hydrolyzing of cellulose; 1,4-β-cellobiosidase (EC 3.2.1.91) hydrolyzing of cellulose; 1,4-β-xylosidase (EC 3.2.1.37) hydrolyzing of hemicellulose, XS), were measured as described by previous studies^32,53. Briefly, 1 g of field-moist, frozen soil was homogenized by magnetic stirrer in 90 ml of Tris buffer (pH = 7.8) which was approximate pH of the soil samples. The extracellular enzymes were determined by a fluorometric microplate assay using a 100 μM solution of 4-methylumbelliferone (MUB). 50 μL aliquots of 200 μM MUB-substrate were dispensed in the soil assay and control. The slurries were then added to 96- well microplates using eight-channel micropipette for each soil sample, and an additional eight replicates of quench controls. Assays were run with one quenched blank column (50 μL buffer, 100 μL slurries and 50 water), one quenched standard column (50 μL buffer, 100 μL slurries and 50 μL MUB), one MUB-substrate assay column (50 μL buffer, 100 μL slurries and 50 μL MUB-substrate), one blank column (200 μL buffer), one blank standard column (150 μL buffer and 50 μl MUB) one MUB-substrate blank column (150 μL buffer and MUB-substrate). The plates were incubated in the dark for 3 h at 30 °C and measured with a M200 pro (TECEN, Austria) multilabel fluorescence reader using excitation 355 nm and emission 460 nm⁵⁴. Specific enzyme activities were calculated by dividing enzyme activities over the SOC⁵⁵.