Hi dear Romina:

The crystalline structure of cellulose can only be attacked by a type of cellulases, the exoglucanases or cellobiohydrolases, these hydrolyze the terminal glycosidic bonds, releasing cellobiose as the main product. This type of enzymes represent 40-70% of the total cellulase system. They are monomeric enzymes with a molecular mass between 50 and 65 kDa. They have an optimum pH of activity between 4 and 5 and their optimum temperature is 37 to 60 ºC.

I have determined these enzymes in enzymatic crudes from fungi in a simple way from the Test with filter paper according to the method of Adney et al. 2009 This method consists of preparing different test tubes: mixtures of enzymes (enzymatic crude), control of enzymes and target; in the case of the tubes that are going to contain the enzyme mixture, a rolled strip of Whatman 1 filter paper (1.0 x 6.0 cm) equivalent to 50 mg was placed, which serves as a substrate. Then 1 mL of 0.05 mol / L sodium citrate buffer at pH = 4.8 was added to all the test tubes, except the blank, which was added with 1.5 mL of said solution. Then, the test mixture of enzymes and the blank at 50 ° C were placed in the thermostatic bath for one minute to balance the substrate with the buffer. Once the system was stabilized, 0.5 mL of the different enzymes were added to the respective test tubes. Then they were placed in the hot water bath at 50˚C for 60 minutes. To stop the reaction and develop color all add 3 mL of the dinitrosalicylic acid (DNS ) reagent (develop a color intensity proportional to the concentration of reducing sugars) and for 5 minutes in a heating bath with water at 100˚C. After cooling all the tubes in a water bath to let settle any solid and perform the reading of the absorbance at 540 nm in the spectrophotometer for it is added to each tube an amount of water equivalent to 2.5 mL in 0.2 mL of reaction mixture. A standard glucose curve in 0.05 mol / L sodium citrate buffer and pH = 4.8 is constructed from different standard glucose concentrations (10 mg / mL) and the corresponding absorbance values ​​to determine the concentration of glucose. To determine the enzyme concentration, the IUPAC (2008) defines that the formation of 2 mg of glucose / 0.5mL of buffer from 50 mg of Whatman1 paper equals 0.37 IU / mL, therefore by a proportionality relationship (1) Using the above information and determining the concentration of glucose through the standard curve, the enzymatic activity of each sample is found.The determination of the β1-4 enzyme exoglucanase is expressed in international units per milliliter (IU / ml). Which refers to the glucose micro-moles released per minute of reaction under the conditions of the activity assay. enzyme activity (IU / mL) = (0.37 IU / mL * concentration (mg / glucose / (0.5 mL))) / ((2 mg / glucose) / (0.5 mL))

Thank you very much everyone for your contribution. In fact, I measure FPasas, but I need to know the specific contribution of the CBHs (I optimize culture media for this enzymatic activity) and I have the reagents. I wanted to know if there is any standardized protocol for the measurement of enzymatic activity and the curve (iupac, nrel) to know if I am doing well and to be able to compare with other works.

Dear Kou Hayakawa: Unfortunately, I do not have HPLC!