Producing pectinase enzyme—a group of enzymes that degrade pectin, a structural polysaccharide in plant cell walls—is particularly useful for softening woody tissues, aiding fungal penetration, and enhancing resin canal expansion in Aquilaria trees. It can be used in BarIno™ BioFusion™, XyloPrime™, or pre-treatment blends for agarwood induction.
How to Produce Pectinase Enzyme (Lab-Scale SOP)
What is Pectinase?
- Enzyme Class: EC 3.2.1.15 (Polygalacturonase, Pectin lyase, Pectin esterase, etc.)
- Function: Breaks down pectin and pectic acid in plant cell walls
- Use: Softens plant tissues, facilitates fungal colonization in resin-induction protocols
Step-by-Step Pectinase Production Protocol
1. Select a Pectinase-Producing Microorganism
| Microbe | Type | Characteristics |
|---|---|---|
| Aspergillus niger | Fungus | Most commonly used; high pectinase yield |
| Penicillium notatum | Fungus | Strong activity on citrus peel |
| Bacillus subtilis | Bacterium | Fast-growing, moderate yield |
| Saccharomyces cerevisiae | Yeast | Mild pectinolytic activity |
Recommended: Aspergillus niger (widely used in industry)
2. Prepare Pectinase Induction Medium
Liquid Medium (Submerged Fermentation):
- Pectin (citrus or apple): 1.0%
- Yeast Extract or Peptone: 0.2%
- KH₂PO₄: 0.2%
- MgSO₄·7H₂O: 0.05%
- (Optional) NaCl: 0.1%
- Adjust pH to 5.0–5.5
- Sterilize at 121°C for 15 minutes
For solid-state fermentation (SSF), use moistened orange peel, banana peel, or sugarcane bagasse (60–70% moisture).
3. Inoculation & Incubation
- Inoculate medium with 5–10% fungal spore suspension
- Incubate at 28–30°C for 5–7 days
- Shaking speed: 120–150 rpm (for submerged culture)
- For SSF: Maintain moisture and temperature; cover trays to avoid contamination
4. Harvesting Pectinase
- After incubation, filter broth using cheesecloth or muslin
- Centrifuge the filtrate at 10,000 rpm for 10–15 mins
- Collect the clear supernatant = crude pectinase extract
Store at 4°C (short term) or freeze for long-term use
5. Assaying Pectinase Activity (Polygalacturonase Activity)
Reagents:
- 1% pectin solution in acetate buffer (pH 5.0)
- DNS reagent (for reducing sugar detection)
Procedure:
- Mix 0.5 mL enzyme + 1 mL pectin solution
- Incubate at 37°C for 30 minutes
- Add DNS reagent and boil for 5 mins
- Measure absorbance at 540 nm
1 Unit (U) = amount of enzyme that releases 1 µmol of galacturonic acid/min
Storage & Stabilization
| Condition | Shelf Life |
|---|---|
| 4°C | 2 weeks |
| −20°C | 3–6 months |
| Freeze-dried | ≥12 months (ideal for field kits) |
Application in Agarwood Inoculation
- Use in pre-injection treatments to weaken parenchyma and resin canal walls
- Ideal in older trees (6+ years) or compact-wood species
- Combine with:
- Fusarium oxysporum (for biotic colonization)
- Cellulase and xylanase (for complete wall degradation)
- FeCl₃ or MnO₂ (for oxidative stress)
Suggested Dose: 5–10 mg/mL or 10–20 U/mL
Inject 2–5 mL per hole depending on tree girth and spacing
Enhancers
- Induction boosted by adding citrus peel powder (1–2%)
- Keep pH near 5.0 for optimal fungal expression
- Optional: Add trace Ca²⁺ to stabilize pectinase activity