I have also used DNS assay to calculate the activity.
This is the method I used to calculate the activity
If the OD 540 value is 1.04 OD, then it is equal to 0.53mg of D-galacturonic acid( from the standard graph of D-galacturonic acid (plot wit d D-galacturonic acid concentration of 1mg/ml)
Details of my DNS assay are;
Vol of substrate (1% citrus pectin) = 0.5 ml
vol of crude enzyme = 0.5 ml
vol of DNS reagent = 1.5 ml
Dilution factor = 10
Reaction time = 15 min
Then what will be the enzyme activity? Please help me to calculate the enzyme activitu (U/mi)?
You are basically asking us to do your homework which I feel is not right. You need to learn to do it yourself.
International units of enzyme activity are:
µmols (notice this is units of mass, not concentration) of substrate converted into product per minute. That you should calculate by extrapolation from your standard curve and using the assay time span.
U/ml is the volumetric enzyme activity, so basically you must divide the total units of activity by the volume of enzyme added (in mL to match the units you requested).
A formal definition of these units and values is available here:
http://www.iupac.org/publications/pac/2001/pdf/7306x0927.pdf
You should also look at previous threads before asking something:
https://www.researchgate.net/post/How_will_I_calculate_enzyme_activity_Total_and_Specific_activity
https://www.researchgate.net/post/what_should_be_the_right_way_to_calculate_the_enzyme_activity_of_microbial_lipase_using_p-NPP_as_a_substrate?_tpcectx
and search other available resources, such as university pages:
http://cst.ur.ac.rw/library/Food%20Science%20books/batch1/Food%20Science%20and%20Technology%20Published%20articles/Analysis%20Protocols/Calculation%20of%20enzyme%20activity.ppt
and even YouTube:
https://www.youtube.com/watch?v=eXMvcLroUbU
Best wishes
Rogelio
Thank you very much Rogelio Rodríguez-Sotres Sir and John H.T. Luong Sir for your valuable comments...
I would like to ask, why some researcher used glucose as a standar for pectinase activity? We know that pectin monomers is galacturonic acid.
And remember to use the official unit which is the katal, officially adopted in 1999 by the General Conference on Weights and Measures. One katal is the amount of enzyme that converts 1 mole of substrate per second; abbreviated as kat (nanokatal is the usual order of magnitude)
Why glucose: probably because this is a measure of reducing end-groups and the sugars are not extremely different in their response. But GalA would be better.
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