Hello. I am determining the enzyme activity of a cellulase, expressed as umol glucose/mL enzyme/hour. In vials I am adding 1 mL of citrate buffer and 0.5 mL of the diluted enzyme to 50 mg of filter paper. After 1 hour incubation at 50 °C, I take 0.5 mL of the mixture and read it with DNS (in this case I add 3 mL of DNS, boil for 5 min, add 20 mL of water and read at 540 nm).
In order to calculate the amount of glucose generated, I need to compare it with a standard curve. Should I prepare my standards (I usually use 1.0 g/L, 2.0 g/L, 3.0 g/L, 5.0 g/L and 6.7 g/L) and dilute them in 1 mL of buffer as well? The enzyme I am working with is C2730 from Trichoderma ressei, has anyone determined IU for it, and if so, what value did you get?
You need to make the standard graph with glucose (10 mg/ml). Make serial dilutions from the same of 2 mg-10 mg/ml. Add 0.5 ml of these serially diluted samples just as you add 0.5 ml of enzyme during the assay and perform all the activities as mentioned in IUPAC protocol
1 ml citrate buffer + 0.5 ml of serially diluted glucose std + 3 ml of DNS- Boil for 5 min. Add 200 ul of sample and dilute with 2.5 ml water. Read absorbance at 540 nm
Thank you for your answer. Shouldn't I take 0.5 mL of the mixture (1 mL buffer + 0.5 mL std) and mix that with the 3 mL of DNS? And after boiling, take the 200 uL and dilute with 2.5 mL water?
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