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 φX174DNA 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 0.01 μg purified trypsin after 16 hours at 25°C.