EMERGE 2016 Autochamber Sites Enzyme Assays


METHODS:
The activity of five hydrolytic enzymes β-d-glucosidase, β-d-xylosidase, N-acetyl-β-d-glucosaminidase, arylsulphatase, and phosphatase was assessed using a fluorometric enzyme assay following methods adapted from Saiya-Cork et al. 2002 and DeForest, 2009. Briefly, a soil slurry was prepared for each sample by blending 1 g of soil with 125 mL of sodium acetate buffer (50 mM; pH 6.2). The soil slurry was then transferred to a 96 well flat-bottom black microplate. Following,  4-methylumbelliferyl (MUB) standard solution and fluorescently linked enzyme substrates were added to the respective wells and the plates were incubated at 25°C for 45 min for β-d-glucosidase, N-acetyl-β-d-glucosaminidase, arylsulphatase, and phosphatase, and 30 min for β-d-xylosidase. Incubation times and substrate concentrations were chosen based on a V-max test performed to capture peak enzyme activity. A buffer only control column and control columns containing only soil slurry and standard were included so background fluorescence could be extracted from assay wells. Fluorescence was read in a BioTek Synergy HT microplate reader at a wavelength of 460 nm emission and  360 nm excitation. Final enzyme activity was reported as µmol activity g-1 dry soil h-1.

The oxidative enzyme activity of phenol oxidase was assessed with a colorimetric enzyme assay (DeForest, 2009). A soil slurry was prepared for each sample by blending 1 g of soil with 125 mL of sodium acetate buffer (50 mM; pH 6.2), and slurries were transferred to a 96 deep-well plate. A blank column containing only buffer was included in the plate, as well as controls containing only buffer and substrate. L-3,4,-dihydroxy phenylalanine (L-DOPA) was chosen as the substrate for measuring phenol oxidase activity. L-DOPA was added in a non- limiting quantity at 25mM concentration and plates were incubated at 25°C for 24 hours. Following incubation, the supernatant was transferred to a 96 well flat-bottom clear microplate, and absorbance was read in a BioTek Synergy HT microplate reader at 460 nm. Final activity was reported as µmol activity g-1 dry soil h-1.

DeForest, JL. 2009. The influence of time, storage temperature, and substrate age on potential soil enzyme activity in acidic forest soils using MUB-linked substrates and L DOPA. Soil Biol. Biochem.  41:1180-1186.
Saiya-Cork, KR, RL Sinsabaugh, DR Zak. 2002. The effects of long term nitrogen deposition  on extracellular enzyme activity in an Acer saccharum forest soil. Soil Biol and Biochem  34: 1309-1315.

COLUMN DEFINITIONS:
ARYL = Arylsulfatase
BG = beta-glucosidase
NAG = N-acetylglucosidase
XYLO = Xylosidase
PHOS = Phosphatase
PhenOx= Phenol Oxidase

FUNDING:
This research is a contribution of the EMERGE Biology Integration Institute, funded by the National Science Foundation, Biology Integration Institutes Program, Award # 2022070.
We thank the Swedish Polar Research Secretariat and SITES for the support of the work done at the Abisko Scientific Research Station. SITES is supported by the Swedish Research Council’s grant 4.3-2021-00164.
This study was also funded by the Genomic Science Program of the United States Department of Energy Office of Biological and Environmental Research, grant #s DE-SC0010580 and DE-SC0016440.