Hello Mariana,

Your stock solution  will be 5 mg/ml. Look up what is the specific activity  indicated by the provider (Sigma); this will give you an approximation of the range of dilutions that you should test in order to get 2 mg of glucose in 1 h. The problem with cellulase assays is that the kinetics of the reaction is highly non linear, so you cannot extrapolate from data that are either too high or too low to match the definition. But if you have a standanrd curve with enough points (let's say  with a 1.5- fold increase between points) that brackets the 2 mg/h value, you can interpolate between the two experimental points that bracket this value to find out which exact dilution should have given 2 mg/h. From that you can  calculate the specific activity of your enzyme.

Good luck,

Pierre Béguin

Thank you very much for your answer. 

Other information provided by the manufacturer states the following: the product has 5 KU in total (5000 U) and 1008 mg. Therefore, the U concentration is 4.96 U/mg. Considering that the solubility of the product is 5 mg/mL, then I would have 24,8 U/mL of resuspended product, is this correct?

In another type of product (same enzyme, but liquid form: Sigma C2730), it says there are more than 700 U/g of product. The density is between 1.1-1.3 g/mL, therefore the amount of U/mL would be 840 U/mL, right? 

Would this imply that in terms of U, I would have to add 33,8 times more of the lyophilized product?

Thank you. 

Mariana,

If you can tell me which Lot number of C8546 that you have, I can give you some more specific information on how to handle and use this enzyme. I am a Principal Scientist for MilliporeSigma. One thing to note that the Dilution does not include the reconstitution of the enzyme. Are you using an Enzymatic method provided by Sigma Aldrich(Millipore  

Thank you very much for your answer.

The Lot# is SLBS1785V. I am not using the method provided by Sigma Aldrich, but the NREL method using filter paper as the substrate and citrate buffer at 50 °C for 1 hour. 

I am attaching the data I obtained by using this method and trying to calculate FPU/mL of the resuspended product. 

Thank you. 

Mariana,

         Ok, I see that Lot SLBS1785V was released using our assay method, which is based on a Glucose/Hexokinase Cocktail, at 4.96 units/mg solid, So if you have 5 KU  vial that be 1008 mg. You need to establish what your NREL assay result is by weighing up a small amount of the material and then dissolve it, and so now once it is dissolved you will just need to determine the units/ml of Enzyme. This dissolution is not considered a dilution in the assay. Have you considered using our assay for your work, it is probably very similar, the incubation is @ 37C and for 30 minutes with shaking.  Just contact our Tech Service Department and they can send you the assay, as I as a researcher do not have copies that can be shared with customers.

Thank you very much for the information. I believe this is the protocol: http://www.sigmaaldrich.com/technical-documents/protocols/biology/enzymatic-assay-of-cellulase.html 

If I dilute 5 mg/mL of C8546, then I would obtain 24,8 U/mL, right? 

If I wish to use this enzyme instead of the already prepared one, C2730, then I would have to use about 30 times more of the C8546, right?

Yes, at 5 mg/ml of C8546 with 4.96 units/mg, you would have a solution at 24.8 units/ml. 

I wish I could give you specific information for a specific lot of C2730, however, this is not produced in St. Louis, so I can not access QC information unless I know what other site this is manufactured/sold from. However, if you have a 50 ml bottle as it is supplied and you have a concentration of 1.1 mg/ml (minimum) you have a total of 55 grams of Protein, or 55,000 mg. The Specific Activity is at a minimum of 700 units/g, so the total amount of Enzyme is 38,500 units/ml. So the units/ml concentration is 770 units/ml. So yes, you would be able to use less C2730 in comparison to C8546. 

Thank you very much for the clarifications!