Filter Paper Activity (FPase) Assay For Cellulases
Descriptions: This protocol is a little modified but it is based on IUPAC procedure.
Basically, this measures the ability of the cellulose to hydrolyze both crystalline and
amorphous regions of cellulose. Therefore, it is generally believed that FPase assay is the
best measure of the activities of both endo- and exo- type in a cellulase with a strip of
filter paper (Whatman #1, 50mg, 1 X 6 cm) for 1hour reaction at the given temperature.
The standard procedure for the filter paper assay is as follows;
1. Add 1 mL buffer to a test tube having a cap.
2. Add 0.5mL enzyme, diluted in the buffer. At least two dilutions must be made of
each enzyme sample investigated. One dilution should release slightly less than 2
mg of glucose in the reaction conditions.
3. Temperature on 50°C or specific temp. and add one filter paper strip into the test
tube.
4. Incubate 50°C for 1hr.
5. Boil the mixture for 20 min.
6. Add 20mL water. Mix it by vigorous shaking.
7. Filter the mixture with a glass filterpaper.
8. The filtrate is measured against reference at 540 nm.
Reference; 1.5 mL buffer, 3 ml DNS, 20min boil, 20ml water
Blank ; 1 mL buffer, 0.5mL enzyme, 3 ml DNS, 20min boil, 20ml water
Unit Calculation:
1. Construct a linear glucose standard using the absolute amounts of glucose
(mg/0.5mL) plotted against 540nm.
2. Using this standard, translate the absorbance values of the sample tubes (after
subtraction of enzyme blank) into glucose.
3. Translate the dilutions used into enzyme concentrations;
Concentration 
1
Volume of enzyme in dilution

Dilution
Total volume of dilution
4. Estimate the concentration of enzyme which would have released exactly 2mg of
glucose by plotting glucose produced against enzyme concentration on
semilogarithmic graph paper.
5. Calulate FPase Unit (FPU):
FPU 
0.37
units  ml-1
Enzyme concentration to release 2mg glucose
The units of FPU is based on the International Unit (IU)
1 IU = 1 micromole min-1 of substrate converted
= 1 micromole min-1 of glucose formed during the hydrolysis reaction
= 0.18mg min-1 when product is glucose