Cellulase (Filter Paper Unit) Assay
By Jian Shi
Reagents and Materials
1 DNS Reagent: (Store in dark bottle!!!)
1. Mix distilled water 400 mL with 3, 5-Dinitrosalicylic acid 10.0 g and 19.3N (50% w/w ) sodium hydroxide 20.75mL; slightly heat to help dissolving; 2. Add Rochelle salts (sodium potassium tartrate Tetrahydrate) 300 g; make sure all the salts are dissolved; add DI water up to 1L. 3. Keep the solution in dark bottle by cover Al foil, store in dark place. Chemicals 1 1/2 1/10 3, 5-Dinitrosalicylic acid 10.0 g 5.0 g 1.0 g sodium hydroxide 19.3N 20.75 mL 10.4 mL 2.07 mL DI water 400 mL 200 mL 40 mL
Sodium potassium tartrate 3H2O 300 g 150 g 30 g Add water up to 1 L 500 mL 100 mL 2 Citrate Buffer: For Trichoderma reesei, cellulase assays are carried out in 0.05 M citrate buffer pH 4.8. For other cellulase enzymes, the pH and the assay temperature may be different. The assay conditions must be defined when reporting results.
1. Mix citric acid monohydrate 210 g with DI water 750 mL; 2. Dilute to 1 L and check pH; 3. Adjust by adding NaOH until pH equals 4.5; the solution above should be 1M citrate buffer 4. Dilute 1M buffer to 0.05M by adding 1: 19 folders DI water. Chemicals 1 1/2 1/5 citric acid monohydrate 210 g 105 g 42 g DI water a To 1 L To 500mL To 200mL NaOH adjusted adjusted adjusted DI water b 1:19 1:19 1:19
3 Filter Paper: prepare 50 mg Whatman No. 1 filter paper strip (approximately 1.0 x 6.0 cm).
Glucose standards 1. Prepare working stock solution of anhydrous glucose (2mg/mL) by dissolving 0.4g glucose up to 200mL pH 4.8 citrate buffer, use immediately or tightly seal and store frozen(store at 4°C will not last long). 2. Vortex the solution before using it to ensure adequate mixing. 3. Dilute the working stock solution to different concentration by the following manner: 0.5 mL + 0 mL buffer = 1.0 mg/0.5mL, 400 µL + 100 µL buffer = 0.8 mg/0.5mL, 300 µL + 200 µL buffer = 0.6 mg/0.5mL, 200 µL + 300 µL buffer = 0.4 mg/0.5mL, 100 µL + 400 µL buffer = 0.2 mg/0.5mL; 4. Add 0.5 mL of each of the above glucose dilutions to 1.0 mL citrate buffer in test tube with cap, incubate at 50°C for exactly 60 min; 5. Remove each assay tube from the 50°C bath; add 3.0 mL DNS reagent to each tubes and mixing.
Procedure for the Filter Paper Assay
1. Place a rolled filter paper strip into each 13 x 100 test tube; 2. Add 1.0 mL 0.05 M Na-citrate, pH 4.8 to the tube, let the buffer saturate the filter paper strip; 3. Equilibrate tubes with buffer and substrate to 50°C. 4. Add 0.5 mL appropriately diluted enzyme (contain about 0.1 unit FPU activity) in citrate buffer, run additional dilutions if the enzyme activity is not clear. 5. Incubate at 50°C for exactly 60 min; 6. At the end of the incubation period, remove each assay tube from the 50°C bath and stop the enzyme reaction by immediately adding 3.0 mL DNS reagent to each tubes and mixing. 7. Prepare one reagent blank with 1.5 mL citrate buffer and go through the procedures above; 8. Prepare one enzyme control for each of the enzyme samples: 1.0 mL citrate buffer + 0.5 mL enzyme dilution (prepare a separate control for each dilution tested) and go through the procedures above; 9. Prepare one substrate control: 1.5 mL citrate buffer + filter-paper strip and go through the procedures above Color development
1. Boil all samples, controls, blanks, and glucose standards tubes for exactly 5.0 minutes in a vigorously boiling water bath containing sufficient water to cover the portions of the tubes occupied by the reaction mixture plus reagent; 2. After boiling, transfer to a cold ice water bath; 3. Let the tubes sit until all the pulp has settled, or centrifuge briefly; move the sample to spectrophotometer cuvette; 4. Determine color formation by measuring absorbance against the reagent blank at 540 nm. With this dilution the glucose standards described above should give absorbance in the range of 0.1 to 1.0 A.
Calculations
1. Construct a linear glucose standard curve using the absolute amounts of glucose
(mg/0.5 mL) plotted against A540. The data for the standard curve should closely fit a calculated straight line, with the correlation coefficient for this straight line fit being very near to one. 2. Verify the standard curve by running a calibration verification standard, an independently prepared solution of containing a known amount of glucose which falls about midpoint on the standard curve. 3. Use standard curve to determine the amount of glucose released for each sample tube after subtraction of enzyme blank. 4. Calculate FPU by the following formula:
amount of glucose released in0.5mL sample(mg) * dilution rate FPU unit IU mL1 0.18mg min 1*60min
Note: Derivation of the FPU Unit The unit of FPU is based on the International Unit (IU) 1 IU = 1 µmol min-1 of glucose released = 0.18 mg min-1 glucose released Dilution rate = 1000/ #of µL original enzyme solution in the 0.5mL diluted enzyme solution. = 1/ Fraction of original enzyme solution in the 0.5mL diluted enzyme solution.