Endo-1,4-β-D-xylanase Assay Using Azo-Xylan and Variants Thereof

Materials and reagents

Biological materials

1. Cell-free supernatants (CFSs), mixture of extracellularly secreted enzymes obtained from filtration of microbial culture broth at 0.22 μm

Reagents

1. Azo-Xylan from Birchwood (Megazyme, catalog number: S-AXBP)

3. Ethanol (Honeywell, catalog number: 02870)

4. Citric acid (Merck, catalog number: 1.00244.1000)

5. Sodium dihydrogen phosphate (NaH₂PO₄) (Chemlab, catalog number: CL00.1497.1000)

6. Disodium hydrogen phosphate (Na₂HPO₄) (Chemlab, catalog number: CL00.1463.1000)

7. Potassium dihydrogen phosphate (KH₂PO₄) (Sigma-Aldrich, catalog number: P5655)

8. Tris (Neofroxx, catalog number: 1125KG001)

9. CAPS (Sigma-Aldrich, catalog number: C2632)

10. Hydrochloric acid (HCl) (Honeywell, catalog number: 30721)

11. Sodium hydroxide (NaOH) (Honeywell, catalog number: 30620)

Solutions

1. Azo-Xylan substrate (see Recipes)

2. Citrate buffer (see Recipes)

3. Sodium phosphate buffer pH 6.0 (see Recipes)

4. Sodium phosphate buffer pH 7.0 (see Recipes)

5. Sodium phosphate buffer pH 8.0 (see Recipes)

6. Potassium phosphate buffer (see Recipes)

7. Tris-HCl buffer (see Recipes)

8. CAPS buffer (see Recipes)

9. Precipitant solution (see Recipes)

Recipes

1. Azo-Xylan substrate (100 mL)

Reagent	Final concentration	Quantity or Volume
Azo-Xylan powder	1% w/v	1 g
Distilled water (dH2O)	n/a	to 100 mL, see note
Total	1% w/v	100 mL

Note: The Azo-Xylan powder should be added to 80 mL of boiling distilled water under vigorous stirring on a hot plate stirrer. Turn off the heat and continue stirring until the suspension has mixed completely. Add water to 100 mL.

2. Citrate buffer (100 mL)

Reagent	Final concentration	Quantity or Volume
Citric acid	2.63 M	9.60 g
HCl (6 N)	n/a	to pH 3.0, 4.0, or 5.0
dH2O	n/a	to 100 mL, see note
Total	n/a	100 mL

Note: Dissolve the citric acid in 80 mL of dH₂O. Adjust the pH to the desired value. Add water to 100 mL.

3. Sodium phosphate buffer pH 6.0 (100 mL)

Reagent	Final concentration	Quantity or Volume
NaH2PO4 solution (1 M)	438 mM	43.80 mL
Na2HPO4 solution (1 M)	62 mM	6.20 mL
dH2O	n/a	to 100 mL
Total	n/a	100 mL

4. Sodium phosphate buffer pH 7.0 (100 mL)

Reagent	Final concentration	Quantity or Volume
NaH2PO4 solution (1 M)	195 mM	19.50 mL
Na2HPO4 solution (1 M)	305 mM	30.50 mL
dH2O	n/a	to 100 mL
Total	n/a	100 mL

5. Sodium phosphate buffer pH 8.0 (100 mL)

Reagent	Final concentration	Quantity or Volume
NaH2PO4 solution (1 M)	34 mM	3.40 mL
Na2HPO4 solution (1 M)	466 mM	46.60 mL
dH2O	n/a	to 100 mL
Total	n/a	100 mL

6. Potassium phosphate buffer (100 mL)

Reagent	Final concentration	Quantity or Volume
KH2PO4	0.50 M	6.80 g
HCl (6 N)	n/a	to pH 6.0 or 7.0
dH2O	n/a	to 100 mL, see note
Total	n/a	100 mL

Note: Dissolve KH₂PO₄ in 80 mL of dH₂O. Adjust the pH to the desired value. Add water to 100 mL.

7. Tris-HCl buffer (100 mL)

Reagent	Final concentration	Quantity or Volume
Tris-base powder	0.50 M	6.06 g
HCl (6 N)	n/a	to pH 7.0, 8.0 or 9.0
dH2O	n/a	to 100 mL, see note
Total	n/a	100 mL

Note: Dissolve the Tris-base powder in 80 mL of dH₂O. Adjust the pH to the desired value. Add water to 100 mL.

8. CAPS buffer (100 mL)

Reagent	Final concentration	Quantity or Volume
CAPS powder	0.50 M	11.06 g
NaOH (5 M)	n/a	to pH 10.0, 11.0
dH2O	n/a	to 100 mL, see note 5
Total	n/a	100 mL

Note: Dissolve the CAPS powder in 80 mL of dH₂O. Adjust the pH to the desired value. Add water to 100 mL.

9. Precipitant solution (100 mL)

Reagent	Final concentration	Quantity or Volume
Ethanol 95%	95% v/v	100 mL
Total	95% v/v	100 mL

Laboratory supplies

1. 1.5 mL plastic tubes (Sarstedt, catalog number: 72.690.001)

2. 200 μL pipette tips (Manufacturer, catalog number: 70.3030.020)

3. 1000 μL pipette tips (Manufacturer, catalog number: 70.3050)

4. 96-well plates (Sarstedt, catalog number: 83.3924.500)

Equipment

1. LLG-uniTHERMIX Pro thermo shaker (Lab Logistics Group GmbH, catalog number: 6.263 484)

2. Centrifuge (Eppendorf, model: 5430 R)

3. BioTek Synergy Neo2 hybrid multi-mode reader (Agilent Technologies, model: BTNEO2)

Software and datasets

1. Gen5^TM Microplate Reader and Imager Software (BioTek Instruments, Inc., version 3.10)

2. GraphPad Prism 9

3. Excel

Procedure

A. Standard procedure

1. Pre-incubation

a. Place 50 μL of 1% w/v Azo-Xylan substrate and 200 µL of each cell-free supernatant (CFS) in separate 1.5 mL plastic tubes.

b. Prewarm samples in a thermo shaker for 5 min at 50 °C under static conditions.

2. Start of reactions: mix 50 µL of each prewarmed CFS with 50 µL of prewarmed Azo-Xylan substrate.

3. Incubation: Incubate for 2 h at 50 °C under static conditions.

4. End of reactions

a. Add 250 µL of precipitant solution to each plastic tube.

b. Vortex the tubes.

c. Place at room temperature for 10 min.

5. Clarification: Centrifuge at 10,000× g for 5 min (Figure 2).

6. Absorbance measurement

a. Transfer 200 µL of each clarified reaction mixture into a well of a 96-well plate.

b. Measure the absorbance at 590 nm.

Figure 2. Reactions in tubes throughout the assay phases. (A) Reactions carried out incubating Azo-Xylan substrate and cell-free supernatants (CFSs). (B) Negative controls prepared by adding precipitant solution to the Azo-Xylan substrate before adding CFSs.

B. Variations on the standard procedure

1. Determination of the optimal temperature (Figure 3A)

a. Carry out sample pre-incubations and reaction incubations at different temperatures.

2. Determination of the optimal pH (Figure 3B)

a. Add 10 µL of each buffer solution to tubes with 50 µL of Azo-Xylan substrate.

b. Pre-incubate substrates and samples at the optimum temperature.

c. Start reactions by adding 40 µL of each prewarmed CFS to the tubes with the buffered substrate.

d. Incubate for 1 h at the optimum temperature.

3. Determination of enzyme thermostability (Figure 3C)

a. Pre-incubate CFS aliquots at their optimum temperature and at 5 or 10 °C below their optimum.

b. Withdraw 40 µL aliquots at different time points.

c. Carry out assays at the optimum pH and temperature with 1 h incubation time.

4. Determination of enzyme storage stability (Figure 3D)

a. Periodically test CFSs stored at 4 °C via an assay with the same pH, temperature, and incubation time as previous assays for comparison.

Critical points: When handling multiple reaction tubes, make sure to stop the reactions in the same order they were started and with the same time interval between one tube and the next one to have the same incubation time for all tubes.

Figure 3. Summary of variations to the standard protocol. (A) Determination of optimum temperature. (B) Determination of optimum pH. (C) Determination of enzymatic thermostability. (D) Determination of enzymatic storage stability.

Data analysis

Calculate the mean and standard deviation of absorbance values between technical triplicates. A template for the determination of temperature and pH optima, thermostability, and shelf life is available as an Excel file in Supplementary information.

Validation of protocol

All experiments were run in technical triplicates of each biological triplicate. Negative controls were prepared by mixing 250 µL of the precipitant solution with 50 µL of Azo-Xylan (with 10 µL of buffer solution in the case of buffered assays) before adding 50 µL of CFS (or 40 µL in the case of buffered assays).

This protocol or parts of it has been used and validated in the following research articles:

1. Bombardi et al. [20]. Lignocellulolytic Potential of Microbial Consortia Isolated from a Local Biogas Plant: The Case of Thermostable Xylanases Secreted by Mesophilic Bacteria. Int J Mol Sci.

2. Bombardi et al. [19]. Thermophilic Hemicellulases Secreted by Microbial Consortia Selected from an Anaerobic Digester. Int J Mol Sci.

General notes and troubleshooting

Troubleshooting

Problem 1: The absorbance reading at 590 nm exceeds the linearity range of the spectrophotometer.

Possible cause: The enzymes in the CFSs are too concentrated.

Solution: Reduce the incubation time of the reactions.

Problem 2: The absorbance reading of negative controls is too high (>0.1).

Possible causes: The Azo-Xylan polymer has degraded over time, perhaps because of contamination with xylanases.

Solution: Prepare a fresh Azo-Xylan suspension every 2–3 months and store it at 4 °C when not in use. Cover the bottle with aluminum foil for protection from light.

Problem 3: The volume of liquid in the vials at the end of the incubation time is lower than expected.

Possible cause: The incubation temperature caused evaporation of some of the liquid.

Solution: At the end of the incubation time, place the vials on ice for a few seconds before opening them to add the precipitant solution.

Supplementary information

The following supporting information can be downloaded here:

1. Excel file: Data analysis template

Acknowledgments

Contributions of each author: Conceptualization, S.F.; Investigation, L.B. and A.C.; Writing—Original Draft, L.B. and A.C.; Writing—Review & Editing, S.F.; Funding acquisition, S.F.; Supervision, S.F. All authors have read and agreed to this version of the manuscript.

Funding sources that supported the work: This research was funded by MUR - Italian Ministry of University and Research, grant number “CUP B31I18000230006” and by Next Generation EU in the framework of National Biodiversity Future Center, grant number “CUP B33C22000660001”.

The protocol was described and validated from Bombardi et al [19,20].

Acknowledgment: All authors are thankful for the support received by Dr. Luca Zuliani, Dr. Andrea Salini, and Dr. Paola Bordoli during protocol optimization.

Competing interests

The authors declare no conflicts of interest.

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