Producing peroxidase enzyme—a key oxidoreductase that uses hydrogen peroxide (H₂O₂) to oxidize phenolic compounds—is valuable for enhancing resin polymerization and pigmentation in Aquilaria trees. It can be integrated into inoculants like BarIno™ LaccaResin™ or ResinRush™ to simulate oxidative stress during agarwood formation.
Peroxidase Enzyme Production (Lab-Scale SOP)
What is Peroxidase?
- Enzyme Class: EC 1.11.1.x
- Function: Catalyzes oxidation of phenolic substrates (e.g., guaiacol, lignin) using H₂O₂
- Biological Role: Lignin biosynthesis, plant defense, and oxidative signaling
Step-by-Step Production Protocol
1. Select a Peroxidase-Producing Microorganism or Source
| Source | Type | Characteristics |
|---|---|---|
| Horseradish (Armoracia rusticana) | Plant root | Standard industrial source (HRP) |
| Aspergillus niger | Fungus | Easy to culture, moderate yield |
| Bacillus subtilis | Bacteria | Rapid growth, lower output |
| Pleurotus ostreatus | Fungi | Also produces laccase |
For field-friendly agarwood use: Aspergillus niger or plant-based HRP
2. Plant-Based Peroxidase Extraction (Horseradish Root)
Materials:
- Fresh horseradish root (100–200g)
- 0.1 M phosphate buffer, pH 6.5
- Cheesecloth, blender, centrifuge
Procedure:
- Peel and slice horseradish root
- Blend 100g root in 300 mL cold phosphate buffer
- Filter through cheesecloth
- Centrifuge filtrate at 10,000 rpm for 15 min
- Collect clear supernatant = crude HRP extract
- Optional: Add 10% glycerol for stability
Store at 4°C or freeze at −20°C
3. Microbial Peroxidase Fermentation (Fungi/Bacteria)
Medium Example (for Aspergillus niger):
- Glucose: 1%
- Yeast Extract: 0.2%
- KH₂PO₄: 0.1%
- MgSO₄·7H₂O: 0.05%
- MnSO₄: 0.001% (enhancer)
- Guaiacol (0.01%) or Tannic Acid – Inducer
- pH: 5.5–6.0
Procedure:
- Inoculate sterilized medium with spores (5–10%)
- Incubate at 28–30°C, 120 rpm for 5–7 days
- Filter and centrifuge culture broth
- Supernatant = crude fungal peroxidase
4. Peroxidase Activity Assay (Guaiacol Method)
Reagents:
- 0.05 M phosphate buffer, pH 6.0
- 20 mM guaiacol
- 12 mM H₂O₂
Procedure:
- Mix 2.5 mL buffer + 0.1 mL guaiacol + 0.1 mL enzyme
- Add 0.1 mL H₂O₂
- Measure absorbance increase at 470 nm for 3–5 mins
Activity Unit:
1 U = amount of enzyme that oxidizes 1 µmol of guaiacol/min at 25°C
Storage Tips
| Condition | Duration |
|---|---|
| 4°C with glycerol (10%) | 1 month |
| −20°C (aliquoted) | 6 months |
| Freeze-dried | >12 months (ideal for field kits) |
Boosting Production
- Add manganese (Mn²⁺) or inducers like guaiacol or lignin
- Solid substrates (wheat bran, sawdust) work well in SSF
- Maintain acidic pH for fungal peroxidase (pH 4.5–6.0)
Application in Agarwood Induction
- Combine with laccase or FeCl₃ for enhanced oxidative polymerization
- Use in mature trees to darken resin and enhance chromogenic reactions
- Suggested dose: 5–10 U/mL, 2–5 mL per injection site