Producing cellulase enzyme involves cultivating cellulase-producing microorganisms, especially fungi or bacteria, under conditions that stimulate them to secrete cellulolytic enzymes. Here’s a complete guide to lab-scale cellulase production, particularly useful for agarwood inoculant formulations like BarIno™ BioFusion™.
Cellulase Production Guide
What is Cellulase?
A group of enzymes that break down cellulose into glucose. It includes:
- Endoglucanases (CMCase) – breaks internal bonds
- Exoglucanases (cellobiohydrolase) – cleaves chain ends
- β-glucosidases – converts cellobiose to glucose
Step-by-Step Cellulase Production (Lab-Scale)
1. Select a Microorganism
| Organism | Type | Notable Feature |
|---|---|---|
| Trichoderma reesei | Fungus | High cellulase yield |
| Aspergillus niger | Fungus | Also produces pectinase/xylanase |
| Bacillus subtilis | Bacteria | Fast-growing, safer handling |
| Penicillium spp. | Fungus | Broad enzyme activity |
For agarwood use, Trichoderma reesei or Aspergillus niger is recommended.
2. Prepare the Culture Medium
Use a cellulose-rich medium to induce cellulase production.
Example Medium (CMC Broth):
- Carboxymethyl cellulose (CMC): 1.0%
- Yeast extract: 0.2%
- K₂HPO₄: 0.1%
- MgSO₄·7H₂O: 0.05%
- NH₄NO₃ or Peptone: 0.5%
- Distilled water: to 1000 mL
- pH: Adjust to 5.5–6.0
Sterilize at 121°C for 15 minutes.
3. Inoculation & Incubation
- Inoculate medium with fungal or bacterial spores (5% inoculum).
- Incubate at:
- Fungi: 28–30°C for 5–7 days
- Bacteria: 35–37°C for 2–3 days
- Agitation: 120–150 rpm (for submerged culture)
- Solid-state fermentation: Can be done on wheat bran, rice straw, or sugarcane bagasse (moistened)
4. Harvest the Enzyme
- After incubation, filter the culture through cheesecloth or muslin to remove biomass.
- Centrifuge at 10,000 rpm for 15 min to clarify.
- The supernatant is crude cellulase extract.
5. Optional: Activity Assay
- Use DNS method or CMC assay to determine cellulase activity in U/mL.
- One unit (U) = amount of enzyme releasing 1 µmol of glucose per minute.
Storage
- Use enzyme fresh or store at:
- 4°C (for short term)
- Freeze-dried or glycerol-stabilized at −20°C (for longer storage)
Scale-Up Tips
| Scale | Substrate | Notes |
|---|---|---|
| Lab | CMC broth | Quick testing |
| Pilot | Wheat bran or sugarcane bagasse | Cost-effective for scale-up |
| Industrial | Steam-exploded biomass | Requires bioreactor & downstream purification |
Application in Agarwood Inoculants
- Combine cellulase (10 mg/mL) with Fusarium oxysporum in injection cocktails
- Helps soften wood tissues and enhance fungal penetration in BarIno™ BioFusion™.