Sep 20, 2017
Version 2
Extracellular enzyme assays (NAG, BG, and CBH) V.2
- Mia Maltz1,
- Allison SD2,
- German D2,
- Looby C.2
- 1UC Riverside;
- 2UC Irvine
External link: https://doi.org/10.1371/journal.pone.0184991
Protocol Citation: Mia Maltz, Allison SD, German D, Looby C. 2017. Extracellular enzyme assays (NAG, BG, and CBH). protocols.io https://dx.doi.org/10.17504/protocols.io.jg3cjyn
Manuscript citation:
Maltz MR, Treseder KK, McGuire KL (2017) Links between plant and fungal diversity in habitat fragments of coastal shrubland. PLoS ONE 12(9): e0184991. doi: 10.1371/journal.pone.0184991
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: August 19, 2017
Last Modified: March 15, 2018
Protocol Integer ID: 7419
Keywords: hydrolytic, enzymes, litter, potential enzyme activity
Abstract
We conducted fluorimetric assays for each of three hydrolytic enzymes: b-glucosidase and cellobiohydrolase, and N-acetyl-glucosaminidase in black microplates. We measured fluorescence at 365 nm excitation and 450 nm emission. From each sample, we recorded fluorescence values for MUB substrate (substrate control), homogenate (homogenate control), MUB standards in the presence of maleate buffer (standard), and MUB in the presence of homogenate. We calculated potential extracellular enzyme activity of NAG, BG, and CBH from each litter sample.
Attachments
Materials
MATERIALS
NaOH
Sodium Acetate Buffer
MilliQ Water
4-MUB-β-D-cellobioside (in refrigerator) 12.5 mg/50 mL 500 μM
4-Methylumbelliferyl β-D-glucopyranoside 16.9 mg/50 mL 1000 μM
1000 μM 4-Methylumbelliferone 0.89 mg/50 ml 100 μM
4-MUB-N-acetyl--D-glucosaminide
Before start
Reagents:
1.0 M NaOH
4 g NaOH pellets; 100 mL DI water
50 mM sodium acetate buffer, pH 5.0 (can make a 10X stock solution; pH can vary with soil)
4.374 g sodium acetate trihydrate
1.1 ml glacial acetic acid (add more to make pH = 5)
1 L sterile DI water (miliQ water)
Substrate solutions (1000 μM unless otherwise noted):
| Enzyme | Substrate | |
| Cellobiohydrolase (CBH) | 4-MUB-β-D-cellobioside (in refrigerator) 12.5 mg/50 mL 500 μM | |
| β-Glucosidase (BG) | 4-Methylumbelliferyl β-D-glucopyranoside 16.9 mg/50 mL 1000 μM | |
| α-Glucosidase (AG) | 4-Methylumbelliferyl α-D-glucopyranoside 16.9 mg/50 mL 1000 μM | |
| β-xylosidase (BX) | 4-Methylumbelliferyl β-D-xylopyranoside 15.4 mg/50 mL 1000 μM | |
| MUB standard | 1000 μM 4-Methylumbelliferone 0.89 mg/50 ml 100 μM |
Make substrate and fluorescent standard solutions in 125 mL red glass bottles using milliQ water. Store solutions in the 4°C refrigerator. Substrates are in the freezer unless noted. Remake solutions every week, and make them one day BEFORE you want to start working, as this affects the measured activity levels. MUB standard should be diluted to 25 μM before use, depending on expected sample fluorescence. MUB is made by heating the solution to 90°C for a few minutes, with constant stirring (don’t let it boil).
Sample Preparation: Soils
Sample Preparation: Soils
Weigh other part (1-2 g wet weight), record mass, and place in labeled 500 ml container.
Add 125 ml acetate buffer and blend on highest speed for 1 minute to make a homogenate slurry.
125 mL acetate buffer
00:01:00 blending on highest speed
Rinse blender with DI water between samples.
Assay Set-up
Assay Set-up
Using the multi-channel pipette with wide-mouth tips (i.e., trimmed with scissors), add the homogenate to all wells receiving homogenate
All wells should now contain 250 μl. Place plates in the incubator at a proper temperature for soils (e.g., 10°C for Alaskan soils). Incubate for one hour.
01:00:00 incubation
Assay Termination
Assay Termination
After one hour of incubation, add 10 μL of 1 M NaOH to each well, noting time.
Note
This is not done in advance. Add the NaOH to the plates and wait 10 min, then read the plates. This is important. Give the NaOH time to interact with the MUB. MUB will only fluoresce in alkaline conditions.
10 µL 1 M NaOH
Reading Plates
Reading Plates
Read fluorescence plates at 365 nm excitation and 450 nm emission.
Standard Curve Construction
Standard Curve Construction
It is important to calculate the standard curve for the MUB based on the MUB standard wells (wells 9-10).
First, obtain the “homogenate blank” value by averaging the values in column 11.
Then, take the MUB standard values, average them for each MUB concentration, and subtract the homogenate blank from the
fluorescence values for each concentration.
Construct a standard curve using linear regression (MUB concentration on the x-axis, and Fluorescence on the y-axis) to obtain the slope. Make sure this is calculated with the MUB concentrations in μ mols/L (which is synonymous with nmols/mL).
The slope is your Extinction Coefficient (ε ).
To calculate the “quench”, simply construct a second standard curve (in a separate plate) that receives buffer and no
homogenate. This “unquenched” curve gives the true fluorescence of MUB.
The ratio of the slope of the standard curve containing soils to the standard curve containing buffer tells you how much the soil
quenches the fluorescence of the MUB.
Fluorescence Activity
Fluorescence Activity
Activity (nmol h-1g-1) = {NFU x Buffer vol(mL)}/{ε x Homogenate vol(mL) x Time(h) x Soil mass(g)} NFU = net fluorescence units = {(Assay – Homogenate Control)/Quench Coeff} – Substrate control Quench coefficient = (Quench control – Homogenate control)/Standard Fluorescence
ε = extinction coefficient Assay volume = 0.250 mL Homogenate volume = 0.200 mL Buffer vol = volume of buffer used to dilute the soil when making homogenate (likely 125 mL) Soil mass = mass of soil included when making homogenate (g) For soils, the mass of the soil can be wet weight, dry weight, organic matter, or Cmic.