It is hard to quantify the substrate cellulose, thus cellulase activity is determined by measuring the product, glucose. Thus, the activity is measured as mol glucose produced in one second (mol/s = katal), or a suitable fraction thereof, say, nkat or fkat.

To convert the colour formed in your glucose assay into a concentration, you need to perform the same assay with a glucose solution of known concentration. The molecular mass of glucose is 180, so a 180 g/L solution would be 1 M. Again, depending on the sensitivity of your assay, you use appropriate dilutions, say, mM. Plot the absorbance vs glucose concentration and you should get a straight line, the slope is the molar extinction coefficient of glucose in your assay, you can then use Lambert-Beer's law to calculate the concentration of glucose in your samples.

In your enzyme assay, measure the glucose concentration at various time points and plot it vs time, the data should form a straight line, the slope of which is the enzymatic activity. Never, ever be tempted to use only one time point, as then you cannot know whether the reaction is linear. This mistake has finished the career of many an enzymologist!

If you measure activity vs enzyme concentration, you should again get a straight line, the slope of which is the specific activity (kat/mg), which is proportional to the purity of your enzyme. Provided your enzyme is pure, you can use the molecular mass of the enzyme to calculate its turnover number (molecules of glucose produced by one molecule of enzyme in one second).

Segel's Biochemical Calculations is an old book, but very useful to get your head around all the maths used in our trade. Scanned copies float around the internet, if your library doesn't have it. Biswanger's Enzyme Kinetics is a modern intro to the field.

Engelbert's remarks are true for the general case of enzymes acting on soluble, homogeneous substrates. However, in the case of cellulases, the time course of the reaction is NEVER linear. This is because the substrate itself is hetereogeneous and displays a whole range of sites defferring in their degree of accessibility or crystallinity. This is why activity units are usually defined empirically, by specifying the substrate and the release of a defined amount of reducing sugar expressed as glucose equivalents. For example, "filter paper units" being defined as the activity actually releasing a specified amount of glucose equivalents per unit of time using filter paper (not cotton, not Avicel, not acid-swollen cellulose etc.). Thus, calculations based on linear extrapolation of other values of the assay are not legitimate.

As per my knowledge only FPU assay considers the release of 2 mg glucose from enzyme dilution when the same is incubated with 50 mg Whatman Filter Paper 1 at 50 Degree Celsius and pH 4.8 for one hour. If your enzyme is unable to use release 2 mg glucose within one hour then shift your enzyme assay from FPU activity to CMC'ase activity. Sodium Salt of Carboxy methyl cellulose is a much easier substrate as compared Filter paper for hydrolysis.

Dear Deepti,

Instead of using the FPU unit for measuring the cellulase activity, you can calculate its activity base on U/ml. In this way it is not necessary to measure the enzyme activity base on releasing of 2 mg/0.5 ml glucose in the enzyme solution.

Enzyme Activity(μmol/min ml) or (U/ml) = (Consumed Substrate) ×Total Reaction Volume / (Reaction time (min)) × (Enzyme volume(ml))

Dear Mina Karimi Avargani ,

Can you some more detail? what means by substrate consumed? if I want to find cellulase activity U/mL then how to do it?@ Mina Karimi Avargani

How to represent the correlation between enzyme production and enzyme activity?