Producing laccase enzyme involves culturing laccase-producing fungi or bacteria—typically white-rot fungi like Trametes versicolor, Pleurotus ostreatus, or Aspergillus spp.—under inducing conditions that promote enzyme secretion.
Here is a full guide for lab-scale laccase production suitable for integration into agarwood inoculant formulations like BarIno™ LaccaResin™ or BioFusion™.
Laccase Enzyme Production (Lab-Scale SOP)
What is Laccase?
- Enzyme Class: EC 1.10.3.2
- Function: Oxidizes phenols, lignins, and aromatic compounds, essential in lignin degradation and resin polymerization
- Cofactor: Copper-dependent oxidase
Step-by-Step Laccase Production Protocol
1. Select a Laccase-Producing Organism
| Microorganism | Type | Characteristics |
|---|---|---|
| Trametes versicolor | White-rot fungus | High laccase output |
| Pleurotus ostreatus | Oyster mushroom | Easy to cultivate |
| Aspergillus niger | Filamentous fungus | Faster growth, moderate laccase |
| Bacillus subtilis (rare strains) | Bacterium | Low output, robust |
Recommendation for Agarwood: T. versicolor or P. ostreatus
2. Prepare the Laccase Induction Medium
Basic Laccase Production Medium:
- Glucose or Glycerol: 1.0%
- Yeast Extract: 0.2%
- KH₂PO₄: 0.2%
- MgSO₄·7H₂O: 0.05%
- CuSO₄·5H₂O: 0.001–0.01% (critical copper inducer)
- pH: Adjust to 5.5 (fungal laccase works best at pH 4.5–6.0)
- Volume: Make up to 1000 mL with distilled water
Sterilize at 121°C for 15 minutes
3. Inoculation & Incubation
- Inoculate with fungal spore suspension or mycelial plugs (5–10% v/v)
- Incubate at 28–30°C for 5–10 days
- Use shaking incubator at 120–150 rpm for submerged fermentation
- For solid-state fermentation (SSF), use moist wheat bran, sugarcane bagasse, or sawdust
Laccase production peaks between Day 5 and Day 8
4. Harvesting the Crude Enzyme
- Filter out fungal biomass using gauze or cheesecloth
- Centrifuge supernatant at 10,000 rpm for 15 minutes
- Crude laccase extract is now ready for use
Store at 4°C short-term or freeze at −20°C for longer stability
5. Laccase Activity Assay (ABTS Method)
Use ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) as substrate
- Mix 1.5 mL of 2 mM ABTS in acetate buffer (pH 5.0)
- Add 0.5 mL enzyme extract
- Measure absorbance at 420 nm over 3–5 minutes
- Use extinction coefficient: ε = 36,000 M⁻¹cm⁻¹
1 Unit (U) of laccase = amount oxidizing 1 µmol of ABTS/min
Yield Expectations
| Culture Type | Yield (U/mL) |
|---|---|
| Shaking (liquid) | 20–50 U/mL (peak at Day 7) |
| SSF (solid substrate) | 100–300 U/g substrate |
| With Cu²⁺ induction | 2–4× increase in output |
Tips for Boosting Production
- Add 0.01% CuSO₄ after 48–72 hours to induce laccase genes
- Use lignin-rich waste like coconut husk or wood powder in SSF
- Maintain acidic pH (4.5–5.5) during production
- Supplement with veratryl alcohol or ethanol (0.1%) to boost yield
Use in Agarwood Inoculants
- Blend with Fusarium oxysporum or FeCl₃ in BarIno™
- Inject at 5–10 U/mL, 2–5 mL per hole
- Best used in trees older than 6 years for darker resin output