Dear Mushafau Oke

At first dilute your culture samples using 0.05 Molar Citrate buffer (pH 4.8) in different ratios such as 2 folds (times), 5 folds , 10 fold , 15 folds ,…( each sample should be serially diluted separately). Then, you use DNS method ( add 3 ml Dinitorosalicyclic acid Reagent to the diluted samples and mix, followed by placing samples in boiling water for 5 minutes and cooled to room temperature) for measuring optical density (OD =absorbance) of the glucose released by Cellulase (Filter Paper Activity) in the samples at 540 nm using a Spectrophotometer. Note that the OD value (absorbance value) should be between 0 to 2. If the OD value is more than 2, you should dilute more your samples and if the OD value is 0 or less than 0 you decrease the dilution rate OR use samples without diluting.

Please note that when you use spectrophotometer, measure OD of reagent blank and OD of enzyme blank then measure OD value of diluted sample. Then subtract the OD of reagent blank and enzyme blank from the OD of diluted sample.

Then, you prepare a standard graph (curve) for glucose [add different amount s of glucose (mg) to distilled water (ml) to make for example 0.1 mg/ml, 0.2 mg/ml, 0.3 mg.ml ,… then measure OD for each and draw a graph using Excel program from Microsoft Office).

You use the standard graph for the samples and according to their OD, calculate their respective glucose concentration. Then, calculate the concentration of diluted sample by dividing 1 to dilution rate of sample for example: 1 /dilution rate of 5= 1/5. Then, draw a standard curve based on the mg glucose inside the sample and the value of its respective concentration. Then you see the standard curve obtained and see which of two concentrations of diluted samples includes mg glucose very close to 2 mg glucose (above and below the 2 mg) so that one of them contain slightly more than 2 mg glucose and the latter contains slightly less than 2 mg glucose. Note that, use the Excel program from Microsoft Office to draw a standard curve and find the equation of curve (linear curve; Y= aX ) and then use the equation for finding the concentration which contains 2 mg glucose e.i. 2=aX, so X (concentration)= 2/a. Then follow IUPAC method that is 0.37/X= FPU. (Last time, Excel program was not and they used traditional calculation form the curve).

Then the value of FPU calculated is multiplied by dilution rate used for example the FPU is 0.7 and the dilution rate is 15 , therefore 0.7 × 15= 10.5 FPU. Hence, the activity of the Cellulase in the sample is 10.5 FPU

Please note that if the diluted sample gives OD value of 0 or less than it, there is no need to dilute the sample and calculate enzyme activity (FPU) based on the IUPAC method.

Kind regards,

Peyman Abdeshaian

Postdoctoral researcher,

Department of Chemical and Process Engineering, National University of Malaysia (UKM)

Dear Mushafau Oke

At first dilute your culture samples using 0.05 Molar Citrate buffer (pH 4.8) in different ratios such as 2 folds (times), 5 folds , 10 fold , 15 folds ,…( each sample should be serially diluted separately). Then, you use DNS method ( add 3 ml Dinitorosalicyclic acid Reagent to the diluted samples and mix, followed by placing samples in boiling water for 5 minutes and cooled to room temperature) for measuring optical density (OD =absorbance) of the glucose released by Cellulase (Filter Paper Activity) in the samples at 540 nm using a Spectrophotometer. Note that the OD value (absorbance value) should be between 0 to 2. If the OD value is more than 2, you should dilute more your samples and if the OD value is 0 or less than 0 you decrease the dilution rate OR use samples without diluting.

Please note that when you use spectrophotometer, measure OD of reagent blank and OD of enzyme blank then measure OD value of diluted sample. Then subtract the OD of reagent blank and enzyme blank from the OD of diluted sample.

Then, you prepare a standard graph (curve) for glucose [add different amount s of glucose (mg) to distilled water (ml) to make for example 0.1 mg/ml, 0.2 mg/ml, 0.3 mg.ml ,… then measure OD for each and draw a graph using Excel program from Microsoft Office).

You use the standard graph for the samples and according to their OD, calculate their respective glucose concentration. Then, calculate the concentration of diluted sample by dividing 1 to dilution rate of sample for example: 1 /dilution rate of 5= 1/5. Then, draw a standard curve based on the mg glucose inside the sample and the value of its respective concentration. Then you see the standard curve obtained and see which of two concentrations of diluted samples includes mg glucose very close to 2 mg glucose (above and below the 2 mg) so that one of them contain slightly more than 2 mg glucose and the latter contains slightly less than 2 mg glucose. Note that, use the Excel program from Microsoft Office to draw a standard curve and find the equation of curve (linear curve; Y= aX ) and then use the equation for finding the concentration which contains 2 mg glucose e.i. 2=aX, so X (concentration)= 2/a. Then follow IUPAC method that is 0.37/X= FPU. (Last time, Excel program was not and they used traditional calculation form the curve).

Then the value of FPU calculated is multiplied by dilution rate used for example the FPU is 0.7 and the dilution rate is 15 , therefore 0.7 × 15= 10.5 FPU. Hence, the activity of the Cellulase in the sample is 10.5 FPU

Please note that if the diluted sample gives OD value of 0 or less than it, there is no need to dilute the sample and calculate enzyme activity (FPU) based on the IUPAC method.

Kind regards,

Peyman Abdeshaian

Postdoctoral researcher,

Department of Chemical and Process Engineering, National University of Malaysia (UKM)

Thank you very much Dr. Peyman. Your explanation is very explicit and helpful. I later got more detailed explanation from NREL LAP on cellulase measurement and it is just exactly as you explained. However, can one also use micro crystalline cellulose instead of filter paper and would one still need to dilute the enzyme in such situation? Thanks once again.

Dr. Peyman Abdeshaian,

If one decides to use microcrystalline cellulose as the substrate for total cellulase activity, would one still need to dilute the enzyme (bacterial)?

I understand that the IUPAC method stipulated the use of filter paper units and not glucose equivalents because the hydrolysis of filter paper is non-linear due to its non-uniform distribution of amorphous and crystalline regions, but I think microcrystalline cellulose is more uniform and it is therefore okay to use direct glucose equivalents to express the cellulase activity.

Please am i right on these assumptions? The IUPAC method is too complex and tedious, so I am considering using microcrystalline cellulose for my total cellulase activity tests instead. Your kind comments will be appreciated. Thanks.

Dear Dr. Peyman,

I have an issue regarding concentration of my bacterial enzyme. I followed your suggestion that do not dilute the enzyme. But the issue is that my enzyme concentration did not reach 2 mg glucose standard. I need to quantify my enzyme without any other techniques like purification or concentration etc. Kindly help me out in this regard.