Determination of Cellulase Activity

(Spectrophotometric method)
1 Definition of the Unit
One unit (U) of cellulase activity is defined as the amount of enzyme which liberates 1
μmol reducing sugar per minute from 5mg/mL substrate (carboxymethylcellulose sodium)

solution at 50.0℃ and pH 4.8 under the conditions of the test.

2 Principle of the method

The cellulase is incubated with the carboxymethylcellulose sodium under specified
temperature and pH conditions. During incubation, carboxymethylcellulose sodium is
hydrolyzed by cellulase, remains reducing sugar, which can deoxidize 3,5-dinitrosalicylic

acid（DNS） into orange- brown chemicals. There is linear relation between the amount of
reducing sugar and the color intensity. So the activity of cellulase in reaction solution can be
calculated by determinating the chroma of reaction solution with spectrothotometric method.
3 Reagents and solutions
3.1 Water
Distilled water, or equivalent.
3.2 Acid solution, 0.05mol/L
Dissolve 3.94g citric acid monohydrate and 9.19g trisodium citrate dihydrate into 800ml
water completely, and make the final solution volume to 1000mL. Adjust the solution pH
value to 4.80 with hydrochloric acid solution (3mol/L) or sodium hydroxide solution (3mol/L).
3.3 Sodium hydroxide solution, 200g/L
Dissolve 20.0g sodium hydroxide into 100ml water completely.
3.4 Glucose solution,10mg/mL
Dissolve 1.0000g glucose into 100ml water completely.
3.5 Substrate--carboxymethylcellulose sodium solution, 10mg/mL
Weigh 1.000g carboxymethylcellulose sodium solution (Sigma C5678), slowly add

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80mL acid solution(0.05mol/L), magnetically stir and heat up the solution to make the
carboxymethylcellulose sodium solution dissolved completely. Stop heating, then cool
solution to room temperature. Adjust the solution pH value to 4.80 with hydrochloric acid
solution(3mol/L) or sodium hydroxide solution(3mol/L), and then make up volume to 100mL

with acid solution(0.05mol/L). The solution is effective in 7 days under storing at 4℃.

3.6 DNS reagent

Dissolve 3.15g 3,5-dinitrosalicylic acid with 500mL water, stirring for 3s~5s and

incubating under the temperature of 45℃. Slowly add 100mL 200g/L Sodium Hydroxide

solution and go on stirring to make the solution clear (the temperature do not exceed

+48℃). Gradually add 91.0g potassium sodium tartrate tetrahydrate, 2.50g phenol and 2.50g

anhydrous sodium sulfite into the solution. Incubating the solution (45℃) and then add 300ml

water, stir the solution to dissolve the above substance completely. Stop incubating and cool
the solution to room temperature, fill up to 1000ml with water and filter it. This solution should
be stored in brown container at room temperature and be useable after 7 days. The DNS
regent is effective within 180 days.
4 Apparatus
4.1 General apparatus in analytical laboratory.

4.2 Water bath, capable of being equilibrated at 50.0 ± 0.1℃

4.3 Spectrometer set at 540 nm, with a 10 mm cuvette

4.4 Magnetic Stirrer
4.5 Vortex mixer
4.6 pH meter, being accurate to 0.01
4.7 Centrifuge, capable of 4000r/min
5 Procedure
5.1 Calibration curve
Mix 2mL acid solution with 3mL DNS reagent together, boil the solution for 5min. Cool

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the solution to room temperature and fill up to 10mL to make up the standard blank sample.
Prepare the glucose solution as the table 1.

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 Table 1. Preparation for Glucose Solution
Glucose Acid Solution Final Concentration of
Solution (mL) Standard Glucose Solution
(mL) (mg/mL)

1.00 0.10
2.00 0.20
3.00 0.30
4.00 Make up to the final volume of 0.40
5.00 100mL 0.50
6.00 0.60
7.00 0.70
Each dilution of the two parallel is set aside 1mL into the graduated test tube, and then
add 1mL acid solution and 3.0mL DNS solution. Stir it for 3-5s, boil for 5min. Then cool it to
room temperature, fill up to 10mL. All Absorbance value of each dilution are collected under
the wavelength of 540nm, based on the standard blank sample. Draw the calibration curve:
glucose concentration as Y-coordinate, absorbance value as X-coordinate.
Note: The calibration curve should be drawn again when the DNS solution is prepared
again.
5.2 Preparation of sample solution
Precisely weigh (0.001g accuracy) cellulase sample (in duplicate) in a beaker, and
dissolve with 50ml acid solution in 100ml volumetric flasks respectively, stir strongly for
30min by magnetic stirrer and make up to the final volume to 100mL.The liquid sample is
diluted and filled up to the final volume with acid solution directly.
Centrifuge the two samples for 3-5min at 4000r/min. Transfer different volume
supernatant solution and diluted by acetic acid- sodium acetic acid buffer to ensure the
concentration of cellulase at 0.16U/mL-0.19U/mL.

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5.3 Reaction
Perform the procedure as the follows (Table 2). The time interval of reagent addition
from substance must be consistent.
Table 2. Reaction Procedure

Reaction Procedure Enzyme blank Enzyme sample

1. 10mL carboxymethylcellulose
√ √
sodium substrate

2. Preheat, 5min, 50℃ √ √

3. 5mL Sample/enzyme solution √ √

4. Preheat, 5min, 50℃ √ √

5. Add 1mL enzyme solution in the

√ √
scale cube
6. Add 3mL DNS solution √ ----------
7. Stir for 3-5s for stopping the
√ ----------
hydrolyzing
8. Add 1mL carboxymethylcellulose
√ √
sodium solution
9. Stir for 3-5s √ √

10. Incubate,15min, 50℃ √ √

11. Add 3mL DNS solution ---------- √

12. Stir for 3-5s for stopping the
---------- √
hydrolyzing

13. Boil, 5 min, 100℃ √ √

14. Cool reaction solution to room

√ √
temperature in cold water
15. Fill up the final volume to 10mL
√ √
with water

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 16. Stir for 3-5s √ √
17. Collect the absorbance value (A)
√ √
under wavelength 540nm, based on
(AB) (AE)
the standard blank sample as blank.

6 Calculation and expression

In which:
X – Cellulase activity, U/g or U/mL.
AE – Absorbance value of enzyme sample solution.
AB – Absorbance value of enzyme blank solution.
K– Slope of calibration curve
CO– Intercept of calibration curve.

M – Molecular Weight (C6H12O6)=180.2

t – Reaction time, min.
m – Weight or volume of the sample, g or mL.
n– Dilution multiple.
7 Deviation permitted
The relative deviation of two parallel values from one sample should be less than 8%.

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