Producing xylanase enzyme involves culturing xylanase-producing microorganisms—most commonly fungi or bacteria—on substrates rich in xylan (a major hemicellulose component in plant cell walls). Xylanase plays a key role in degrading hemicellulose, making it useful for enhancing fungal penetration during agarwood inoculation (e.g., in BarIno™ XyloPrime™ or BioFusion™ formulations).
How to Produce Xylanase Enzyme (Lab-Scale SOP)
What is Xylanase?
- Enzyme Class: EC 3.2.1.8
- Function: Hydrolyzes β-1,4-xylosidic bonds in xylan (hemicellulose) → produces xylo-oligosaccharides and xylose
- Role: Breaks down plant cell wall structures, aiding resin-inducing fungi in penetrating wood
Step-by-Step Production Protocol
1. Select a Xylanase-Producing Microorganism
| Microbe | Type | Features |
|---|---|---|
| Aspergillus niger | Fungus | High enzyme yield, GRAS status |
| Trichoderma reesei | Fungus | Strong cellulase + xylanase |
| Bacillus subtilis | Bacterium | Fast-growing, moderate xylanase |
| Penicillium chrysogenum | Fungus | Broad-spectrum hemicellulase |
Recommended for agarwood inoculants: Aspergillus niger or Trichoderma reesei
2. Prepare Xylanase Production Medium
Inducing Carbon Source:
- Birchwood xylan (0.5–1%) — best inducer
- Alternatively: corn cobs, wheat bran, sugarcane bagasse (5–10%)
Liquid Fermentation Medium Example:
- Xylan: 0.5–1%
- Peptone or Yeast Extract: 0.2%
- KH₂PO₄: 0.1%
- MgSO₄·7H₂O: 0.05%
- NaCl: 0.1%
- Adjust pH to 5.5
- Sterilize at 121°C for 15 minutes
3. Inoculation & Incubation
- Inoculate sterile medium with spores (5–10% v/v)
- Incubate at:
- 28–30°C (fungi) or 37°C (bacteria)
- 120–150 rpm shaking incubator (for submerged culture)
- Incubation period: 4–7 days
- Solid-State Option: Moist wheat bran or sugarcane bagasse (60–70% moisture) also works for SSF
4. Harvesting Xylanase
- Filter out biomass using cheesecloth or filter paper
- Centrifuge the supernatant at 10,000 rpm for 10–15 minutes
- The clear supernatant contains crude xylanase enzyme
5. Assaying Xylanase Activity (Optional)
Use DNS (Dinitrosalicylic Acid) method to measure reducing sugars released from xylan.
Activity Definition:
1 Unit (U) of xylanase = amount of enzyme that releases 1 µmol of xylose per minute under assay conditions
Storage
- Use fresh or store:
- 4°C for up to 1 week
- –20°C for longer shelf life
- Stabilize with glycerol (10–20%) if needed
Yield Expectations
| Method | Expected Yield |
|---|---|
| Submerged fermentation | 50–200 U/mL |
| Solid-state fermentation | 100–500 U/g dry substrate |
| With birchwood xylan | 2–3× induction vs. glucose |
Tips for Enhanced Production
- Use inducers like xylan, corn cob powder, or wheat bran
- Add trace Cu²⁺ or Mn²⁺ to boost expression in some fungi
- Optimize pH (5–6) and temp (28–30°C) for best fungal yields
- Combine with cellulase fermentation if producing dual enzymes
Application in Agarwood Induction
- Inject 5–10 mg xylanase per tree hole as part of pre-treatment
- Enhances Fusarium colonization and resin canal disruption
- Combine with cellulase, laccase, or FeCl₃ in BarIno™ BioFusion™