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Cellulase - Assay

Source:
Trichoderma reesei ATCC#26921
CAS:
9012-54-8
EC:
3.2.1.4
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Cellulase Assay

Method
Cellulase activity is determined by its effect on microcrystalline cellulose with respect to glucose formation. Released glucose is determined in a hexokinase/glucose-6-phosphate dehydrogenase system at 340 nm. One unit of activity releases 0.01 mg glucose per hour from micro-crystalline cellulose at 37°C and pH 5.0 under the specified conditions.
Reagents
  • 0.05 M Acetic acid, pH 5.0
  • Microcrystalline cellulose (Avicel, F.M.C. or equivalent))
  • Glucose determination reagent*

ATP 0.77 μmol/ml
Hexokinase 1.5 units/ml
NAD 0.91 μmol/ml
Glucose-6-phosphate dehydrogenase ≥1.9 units/ml
Tris⋅HCl buffer pH 7.6 ±0.2 0.1 M

*Can be replaced with a glucose assay utilizing a standard curve.

Enzyme

Dissolve enzyme in reagent grade water at a concentration of 1-0.1 mg/ml.

Procedure

Measure into clean dry test tubes as follows:

  Test Blank
Microcrystalline cellulose 200 mg 200 mg
0.05 M Acetic acid 4.0 ml 4.0 ml
Reagent grade water ----- 1.0 ml
Enzyme dilution 1.0 ml -----

Incubate with stirring or shaking for 2 hours at 37°C. Remove tubes to an ice bath and allow sediment to settle. Clarify by centrifugation. Store in an ice bath.

Place 3.0 ml glucose reagent in a cuvette and incubate in spectrophotometer set at 340 nm and 25°C to achieve temperature equilibration. Record the A340 of the glucose reagent in the cuvette. Add 0.1 ml of the supernatant from each reaction tube and record increase in A340 until no further change occurs in 3-5 minutes. Record final A340.

Calculation

Worthington purified cellulases contain less than 0.02 units per mg dry weight lipase activity. One unit of lipase activity releases one micromole of fatty acid per minute from emulsified olive oil at 25°C and pH 8.0.

Nuclease activity is evaluated by incubating 1 µg and 10 µg cellulase with 1 µg phi-X174DNA in 0.5 ml at 37°C for 16 hours under optimum conditions. Degradation of the DNA (evidence of exonuclease) and conversion of the DNA to the Rf2 or linear form (evidence of endonuclease) are monitored by agarose electrophoresis. CEL cellulase exhibits no evidence of nuclease contamination at the 1 µg level, and only a trace of endonuclease contamination at a 10 µg level. CELF grade shows slight endonuclease contamination at the 1 µg level, and partial exonuclease contamination at a 10 µg level.

One mg of purified cellulase exhibits less tryptic-like proteolytic activity on a casein-agar radial diffusion plate than 0.01µg purified trypsin after 16 hours at 25°C.

Cellulase Products

Description
Activity
Code
Cat. #
Size
Price
Description
Cellulase
Source:
Trichoderma reesei ATCC#26921
Purified complex containing exo- and endoglucanase activities. A diafiltered, lyophilized powder. Tested for lipase, protease, and nuclease.
Store at 2-8°C.
…
Activity
≥45 units per mg dry weight
Code
CEL
LS002598
250 mg
$40.00
LS002601
1 gm
$96.00
LS002603
10 gm
$815.00
LS002600
Bulk
---
The minimum amount for bulk packaging/pricing for this product is 200000 x 1 mg. Please contact custservice@worthington-biochem.com to request a quote for smaller amounts.
Description
Cellulase
Source:
Trichoderma reesei ATCC#26921
A partially purified, lyophilized powder.
Store at 2-8°C.
…
Activity
≥25 units per mg dry weight
Code
CELF
LS002610
1 gm
$53.00
LS002611
10 gm
$376.00
LS002609
Bulk
---
The minimum amount for bulk packaging/pricing for this product is 200 x 1 gm. Please contact custservice@worthington-biochem.com to request a quote for smaller amounts.
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