Materials and Reagents

Liquid nitrogen
BCA Reagent (Tiangen Biotech, catalog number: PA115-01 )
Bovine serum albumin
AZC L-galactomannan (Megazyme, catalog number: I-AZGMA )
Commercial Aspergillus niger endo-β-mannanase (Megazyme, catalog number: E-BMANN )
100 mM phenylmethanesulfonyl fluoride (PMSF) (see Recipes)
0.5 M ethylene diaminete traacetic acid (EDTA) (see Recipes)
Extraction buffer (see Recipes)
0.1 M sodium acetate buffer (pH 5.0) (see Recipes)

Equipment

Mortar and pestle
10,000 Mr cut-off filter (EMD Millipore, catalog number: UFC801096 )
Centrifuge
Incubator shaker
Water bath
Microplate reader or Spectrophotometer

Procedure

Samples (~ 10 g developing xylem or leaves from one-year-old poplar) are ground in liquid nitrogen to a fine powder and homogenized at 4 °C in 1.5-volume fold of extraction buffer for 1 h (strong enzymatic activity can be detected in developing xylem).
The homogenate is centrifuged at 10,000 x g for 30 min at 4 °C.
The supernatant is then passed through a 10,000 Mr cut-off filter and dehydrated to < 500 μl, then the protein is diluted to ~1 μg/μl in 0.1 M sodium acetate buffer (pH 5.0) at 4 °C.
The protein extraction is measured by BCA Reagent using bovine serum albumin as a standard.
200 μl of reaction mixture containing 100 μl of 1% AZC L-galactomannan (w/v, in 0.1 M sodium acetate buffer, pH 5.0) and 20 μg of extracted proteins or heated inactive proteins (100 °C 10 min, as control) is incubated at 40 °C for 2 h with continuous shake.
The reaction mixture is boiled at 100 °C for 5 min and centrifuged at 12,000 x g for 5 min.
The absorbance (A) of the supernatant at 590 nm is determined. The background values (A₀) obtained using heated inactive proteins are subtracted from values (A₁) obtained using active extract (A = A₁ - A₀).
Standardization
1. Enzyme activity of a serial dilutions of a commercial Aspergillus niger endo-β-mannanase (E-BMANN, Megazyme) is determined under the conditions: 200 μl of reaction mixture containing 100 μl of 1% AZC L-galactomannan and E-BMANN is incubated at 40 °C for 2 h.
2. A standard curve correlated with E-BMANN activity is shown in Figure 1. For absorbance values in a range of 0.05~0.9, these values can be calculated by reference to the equation: Y = SX + C. Where:
  Y = endo-β-Mannanase activity (in micro-Units/assay, i.e. per 200 μl)
  S = Slope of the calibration graph
  X = Absorbance of the reaction at 590 nm (A)
  C = Intercept on the Y-axis
3. According to the manufacturer’s instruction, one Unit of activity is defined as the amount of enzyme required to release one micromole of mannose reducing-sugar equivalents per minute under the defined assay conditions (1 micro-Unit = 1 pmol/min).
Calculation of enzyme activity: endo-β-Mannanase activity in the sample is determined by reference to the standard curve to convert absorbance values to micro-Units per assay (Y), then further to micro-Units per μg protein (Y/[20 μg protein], pmol/min/[μg protein]).

Figure 1. Endo-β-mannanase standard curve on the commercial endo-β-mannanase.

Recipes

100 mM Phenylmethanesulfonyl fluoride (PMSF) 10 ml
Mix 0.174 g of PMSF with 10 ml isopropanol
Store in small aliquots at -20 °C
0.5 M Ethylene diamine tetraacetic acid (EDTA) (pH 8.0) (1 L)
Dissolve 186.1 g EDTA-Na^.2H₂O in 800 ml dH₂O
Adjust pH to 8.0 with NaOH (~20 g NaOH particles)
Add dH₂O to 1 L
Autoclave at 121 °C for 20 min
Store at RT
Extraction buffer (1 L)
1 M sodium acetate buffer (pH 5.0)
10 mM EDTA
10 mM sodium azide
3 mM PMSF
Mix 57 ml glacial acetic acid (1.05 g/ml) and 20 ml 0.5 M EDTA with 800 ml dH₂O
Adjust pH to 5.0 with NaOH
Add 0.65 g sodium azide
Add dH₂O to 1 L
Add 30 μl of 100 mM PMSF per ml extraction buffer immediately before use
Note: Do not add the sodium azide until pH is adjusted. Acidification of sodium azide will release a poisonous gas.
0.1 M sodium acetate buffer (pH 5.0)
Mix 5.7 ml glacial acetic acid (1.05 g/ml) with 800 ml dH₂O
Adjust pH to 5.0 with NaOH
Add 0.65 g sodium azide
Add dH₂O to 1 L

Acknowledgments

This protocol was adapted from Zhao et al. (2013).

References

Zhao, Y., Song, D., Sun, J. and Li, L. (2013). Populus endo-beta-mannanase PtrMAN6 plays a role in coordinating cell wall remodeling with suppression of secondary wall thickening through generation of oligosaccharide signals. Plant J 74(3): 473-485.