SI No.: Scope of The Document

SI No.
CONTENTS
1 Scope of the Document
2 Principle
3 Conditions
4 Method
5 Reagents
6 Sample Preparation
7 Procedure
8 Calculation
9 Enzyme Definition
10 Reference
1. Scope of the Document:
This document is applicable for determination of Cellulase enzyme in products containing Cellulase as an active
component.
2. Principle: This assay is based on measurement, by the DNS reagent, of glucose equivalents (GE) liberated from
whatman No.1 filter paper as a result of enzyme action for over 15 minutes. GE measured in a control is deducted
from those found in the test before conversion of GE units to enzyme activity units.
Cellulase
Cellulose + H2O Reducing Sugar (measured as Glucose)
3.Conditions: T = 60°C, pH = 4.8, A540 nm, Light path = 1 cm
4. Method: Spectrophotometric Stop Rate Determination
5. Reagents:
5.1. Citrate Buffer, pH 4.8, Concentration 0.05 Molar:
5.2. 3,5-Di-Nitro Salicylic acid (Sigma Aldrich - 128848 ALDRICH) Reagent (DNS Reagent)
For 1000ml of DNS reagent

a) DNS - 10 gm
b) Potassium Sodium Tartarate - 300 gm
c) Sodium hydroxide (Pellets) - 16 gm
First, weigh the required chemicals in separate beakers. 10gm of DNS is weighed and dissolved in 50ml of DW and
poured into 1litre conical flask and kept for stirring until it is completely dissolved. Sodium hydroxide is dissolved in
about 50ml of DW. Now, add Potassium Sodium Tartarate which was previously dissolved in minimum amount of
water (about 700ml of water), to the DNS mixture and then finally add Sodium hydroxide solution. White droplets
which may form will not interfere. Add another 200-250ml of DW and keep for stirring until all the white droplets
get dissolved. Then, make up the volume to 1000ml using DW, filter it if necessary and pour into 1ltr brown bottle
and label it as DNS.
5.3. Substrate
Filter Paper Strips: Whatmann Filter Paper No.1 (1 x 6 cm strip)
5.4. Standard Glucose.

Prepared by adding 100 mg of glucose per 100 ml of Citrate Buffer pH 4.8 0.05M
6. Sample Preparation
500mg accurately weighed sample was transferred to a test tube containing 15ml of Citrate Buffer pH 4.8 0.05M, mix
by shaking, sonicate for 1 cycle (8 minutes), filter and the filtrate obtained is used for assay. In case the absorbance is
above 0.7 or below 0.1, prepare a new analytical sample to get the absorbance in the range of 0.1-0.7 of standard.
7. Procedure
Pre-incubate buffer, whatmann filter paper stripes and enzyme preparations separately for 5 minutes in water bath set
at 600C temperature. Prepare tests, controls and standards in duplicate.
7.1. Standards:
Tube Standard Standard Citrate Buffer Absorbance at
Name Volume Concentration(in pH 4.8 Add 1ml DNS reagent and keep 540nm
(in µl) µg) (0.05M) in boiling water bath for 5min
S1 200 200 To make up and makeup the volume to 10ml
S2 400 400 to
S3 600 600 1ml
S4 800 800
S5 1000 1000
Blank 0 0 1
7.2. Tests:
For tests add 1 ml of Citrate Buffer pH 4.8 0.05M, 1 strip of substrate, 0.5 ml of enzyme preparation in a test tube,
place in a water bath at 600C for 15 minutes. After incubation immediately add 1 ml of DNS reagent, mix, transfer
test tubes and place in a boiling water bath for 5 min and make up the volume to 10 ml with water.
Controls:
For control add 1 ml of Citrate Buffer pH 4.8 0.05M, immediately add 1 ml of DNS reagent, mix, transfer test tubes
and place in a boiling water bath for 5 min. After boiling, make up the volume to 10 ml.
NOTE: Tests, controls and standards should be treated with DNS and boiled at the same time.
Absorbance is read at 540 nm in a UV-Vis Spectrophotometer of tests, controls and standards. For all readings use
cuvettes.If cloudy, centrifuge first before reading. Determine the mean absorbance for each pair of duplicates.
Construct a standard curve and determine the reducing sugar concentration of the tests and controls.
8.Calculation
Cellulase activity per gram of sample = AT - AC

AS X CS X DU X 19
MWS X VT X I
Definitions:
AT = Absorbance of test
AC = Absorbance of control
AS = Absorbance of standard
CS = Concentration of standard
DU = Dilution factor of Unknown
19 = Factor for activity definition
MWS= Molecular weight of standard
VT = Volume of Test
I = Incubation time for activity
9. Enzyme Definition:
One Unit of enzyme is defined as the amount of Cellulase activity that liberates 0.05 µmol of glucose equivalents per
minute from the substrate.
10. Reference
Measurement Of Hemicellulase Activities, Part 1: Cellulase., Pure &Appi. Chem., Vol. 59, No.12, Pp. 1739—1752,
1987. International Union Of Pure And Applied Chemistry Applied Chemistry Division Commission On
Biotechnology.

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SI No.

1 Scope of the Document

3.Conditions: T = 60°C, pH = 4.8, A540 nm, Light path = 1 cm

4. Method: Spectrophotometric Stop Rate Determination

5.1. Citrate Buffer, pH 4.8, Concentration 0.05 Molar:

For 1000ml of DNS reagent

5.4. Standard Glucose.

Cellulase activity per gram of sample = AT - AC

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