I have done FPU activity for cellulase. The enzyme is in solid form. At 0.2 mg of quantity in 1.5 ml buffer+50 mg whatman filter paper-1 it gives 2 mg glucose in 60 min. (as per NREL protocol), std DNS protocol. How can i calculate FPU activity for that enzyme.
Respected Madam
I am using solid enzyme. My reaction protocol is as follows.
1 ml buffer+0.5 ml buffer containing 0.4 mg enzyme (So, enzyme concentration is 0.8 mg/ml) +50 mg What man filter paper-1-------> this mixture was heated at 50°C for 60 min. Then 3 ml DNS added and kept for 5 min. in boiling water. Cool and then add 20 ml water and OD checked at 540 nm. How can i calculate FPU for that.
please guide
Hi!
In most DNS protocols, the enzyme unit is taken into account, which is represented as the amount of reducing sugar released per mL and per minute of reaction. If you have 2mg of glucose released in 60 min of reaction, just do the calculations to determine the amount of glucose released per minute in the reaction, then calculate per ml of enzyme that was added in the reaction
1 U = glucose / min / mL
Hi,
FPU is used to calculate the exoglucanase activity of the cellulase complex. The IUPAC method stated by Ghose in the late 1980s, which is also approved by the NREL could be used. It is available online. The substrate used would be Whatmann No.1 filter paper (cut to specific size: 1cm x 5cm). 1 FPU/mL/ 1 IU/mL of exoglucanase would refer to 1micro.mole of the reducing sugar released per minute per mL of the enzyme complex used.
Dear Jayesh,
I understand that you have a powder sample of the cellulase complex, which is later diluted. I would recommend you to prepare a stock and dilute it to required concentrations (1mg/mL, 3mg/mL etc.,) Later, considering this solution as your enzyme, go for the IUPAC Ghose assay. They have a threshold reducing sugar release of 2 mg to further calculate the FPU from it. I hope it helps.
You can find calculation example in this protocol: https://www.google.com/url?sa=t&source=web&rct=j&url=https://www.nrel.gov/docs/gen/fy08/42628.pdf&ved=2ahUKEwi4m-uY2KjoAhUz6KYKHU4lBVUQFjACegQIAxAB&usg=AOvVaw3mLeLtUyHe_eIPWzSk6RzW&cshid=1584694817074
I suppose the main problem that you using mass (mg/mL) concentration.
The term "concentration" in this assay is used to represent the proportion of the original enzyme solution present in the dilution added to the assay mixture. For example a 1:10 dilution of the 1:20 working stock of enzyme will have a "concentration" of 0.005.
Dear All
There is lot of methods of DNSA reagent preparation available in literature. Which is suitable method. Becaus my reagent reduced with time. The initial OD of reagent against water is 0.22-0.24 and after storage at 4-10°C in amber colour bottle, it get reduce and showing yellow to dark brown colour and OD against water is 4-5. The method of DNSA preparation is as follows: Weigh 1 g DNSA and dissolve in 50 ml DW with the help of a magnetic stirrer for approximately 30 min. Weigh 30 g of sodium potassium tartarate and add slowely in 50 ml soln stirrer for 30 min. Then add 20 ml 2 N NaOH solution in the DNSA and make the volume up to 100 ml. This complete preparation conducted at 25-30°C (RT)
At what temperature DNSA reagent should be stored? Please suggest
Dear @jayesh
I prepare the DNS reagent in two separate solutions and both with heating. the first is 1% DNS in 30ml and the second solution is 0.4M NaOH plus 1M sodium tartrate and potassium in 30ml. submit the 2 solutions at 50-55 ° C with agitation until all is diluted. let it cool a little, mix the 2 solutions and make up to 100ml with water. the color of the dns reagent is orange. store in an amber bottle at room temperature. if when you use the reagent you notice that there are visible lumps, heat it up a little until you dilute them.
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