[ganesh2gig]

Hi All ,

Thank you for taking time to read this topic .

I have been trying to standardize few enzyme assay procedures, I have chosen parameters like buffer concentration  , pH , temperature of incubation , time  of incubation, substrate preparation  Etc...

The problem I am facing is I never get repeatable result .  Could some one let me know why this issue occurs , is somebody facing the same problem ? 

I have tried to estimate Xylananase using Xylan substrate and DNS reagent ,  Starch for amylase etc..

[Arkcon]

Its an interesting problem, but its going to be hard for us to pin down without more specifics.  (You given us some good specific information, but I feel we need more.)

If I follow you correctly, you've selected matrix conditions, and reproducibility is poor.

OK, are the matrix conditions good?  Is pH too close the the enzyme's pI?  It may be changing its structure throughout the reaction.  Are you using reasonable concentration for substrate and enzyme?  I know you should get reproducibility first, then work on linearity, but maybe you should try 2 or 3 points to see if you're at a reasonable concentration.

Furthermore, how far are you from published references on how this reaction is usually run?  It would be worthwhile just coping something like that, to try and pin down if your matrix is OK-- pH is really what you think it is, there aren't contaminants in your water or buffers, that your enzyme isn't bad, etc.

[Babcock_Hall]

Are you working from a published protocol?  If not, have you tried looking for one?  The universe of possibilities is very large here, and we would need much more information about the specifics of what you are trying to do to me of much help.

Here are a couple of random thoughts.  If you have a very large concentration of enzyme, the assay may be nonlinear in time because of substrate depletion or product accumulation, for example.  Occasionally an enzyme sticks to the pipet due to some adsorption process at low enzyme concentration; when this happened to me, my rates were all over the map.  I used a little bit of detergent in the enzyme stock solution, and the problem essentially went away.

[ganesh2gig]

Thanks for the insight .

I did refer a standard protocol from Sigma Aldrich ,  few other publications and texts. The issue is Enzyme behave differently , depending on source . Example - Xylanase from Bacterial source is active at pH 10 , whereas from fungal source it is at pH 5 , this depends entirely on source .

I will definitely try using a detergent in the enzyme extraction procedure , what detergent did you use ? As I know , detergent may denature protein , did you use non ionic detergent .

But determining the rates is one thing , the other arbitrary part is enzyme units , how can there be so much of  arbitrary units , even though the specific activity is defined .

[Babcock_Hall]

I would not include detergent in the prep, unless it is part of the procedure.  The only reason to try detergent in an assay is that when one assays an enzyme, its concentration is often very low, in the nanomolar range.  Therefore, if it has a tendency to adsorb onto surfaces, such as the transfer pipet, one can lose a significant fraction of it.  Whether or not it helps is likely to be very dependent on the enzyme itself.  You could try Tween20, but I would use a fresh bottle of whatever detergent you used, because they sometimes form peroxides.

When I typed xylanase into BRENDA, I received about twenty hits.  It might be worth a look to see if your particular xylanase is represented.
http://www.brenda-enzymes.org/index.php4

A unit of activity is the amount of enzyme that will convert one micromole of substrate to product per minute under defined conditions.  Specific activity is = (activity)/(mass of enzyme in mg).  The conditions chosen might vary from one xylanase to another, based on the properties of each particular xylanase.  The optimal conditions probably depend upon which organism or which isozyme within a single organism is being studied.  Not just pH but also substrate concentration, ionic strength, and the identity of the buffer should be specified IMO.