This protocol is reported in a journal written by Paolucci-Jeanjean et al. (2000).
I think it is not necessary, I just boil my enzyme for a few minutes to inactivate it. I work with alkaline proteases. I think its also depends on the type of enzyme you work on...but I think that just boiling it you will inactivate it
I agree with Ivana. Enzymes are very sensitive to heat, change of optimum pH etc. If you don't work with the Taq Polymerase, which can work up to like 72°C, and you bring your solution to boiling temperature...then your enzyme should be inactivated. I would probably look up references about your enzyme and inactivation by heat, if there are any specifics-how hot for how long...just in case...
I also agree with both the answers. Until and unless your enzyme is thermophilic, you can inactivate your enzyme only by boiling.
If you use the protocol of Paolucci-Jeanjean et al. (Biotechnol Bioeng., 68, 71, 2000) it is perhaps required to lower the pH since the enzyme is active at 80 C. Did you check the paper by Brooks and Griffin in J Food Sci 52, 712, 1987?
There are many ways to inactivate enzymes besides boiling. Adding acid or base, a denaturant such as urea or SDS, chelating an essential metal ion with EDTA, and adding a specific inhibitor are all possibilities.
which kind of enzyme do you have? if your enzyme is immobilized one do not use any denaturation process, otherwise you can not use your enzyme repeatedly. after your exact time to measure activity just centrifuge your immobilized preparation at 4°C then take liquid phase with any pipette to measure its activity. finally wash your preparation with appropriate buffer several times and keep it in the buffer at 4°C till next measurement.
You should also consider and check the effects of your inactivation method on the substrate/products if you are using this to stop an assay.
In general, boiling alone will be sufficient. For many enzymes adding acid or alkali will also do the trick. For inactivation during an enzyme assay system, boiling may not be fast enough for accurate following of kinetics and so addition of ice cold acid, alkali or some of the other reagents mentioned by others in their comments. For kinetic work the reactions needs to be stopped instantly and we have found that this is almost impossible to achieve by plunging a reagent tube into boiling water.
Peter Butterworth
If the substrate is present, it may stabilize the enzyme towards inactivation. Empirical testing is best to determine the conditions. And I agree with Peter too. For instant inactivation, other denaturant techniques would work. The technique you use may depend on what you are trying to accomplish.
Exactly, quenching rapidly can be done by SDS/Proteinase K. The SDS unfolds the enzyme allowing the proteinase to degrade it. Proteinase K has very high activity.
I am doing enzyme kinetics of starch hydrolysis by a-amylase and amyloglucosidase. Instant deactivation of enzyme activity is essential. From all the methods suggested, I believe SDS quebching is easilycarried out in my lab. Does any one has experience on the concentration of SDS and volume to be used (I need a value to begin with for my studies). Thanks =)
Sometimes with SDS, one has to boil the sample to completely denature. I would recommend hIgh concentrations of acid or base for instant quenching. Or add proteinase K to the SDS as Jovencio mentioned.
Now I have Proteinase K and SDS in my lab. The Proteinase K was bought from Merck, and it has activity of 600 Manson-U/ml. How much do I need to put in in order to inactivate the enzymes (a-amylase and amyloglucosidase)? Kindly recommend.
I would make a stock 1 mg/ml solution of proteinase K in your buffer system, and then add 2-5ul per about 100ul reaction. This should be overkill. You can add more if you test it and find that the activity is increasing or not fully quenched. I don't know what your assay is, and how you detect activity, so I don't know if the proteinase K concentration is an issue. I would try to measure things in mg/ml rather than arbitrary units.
There is no need to adjust the pH for deactivation of enzyme because all enzymes are heat sensitive some are thermophilic but they also deactivate or denature after boiling.
I pH deactivate my enzymes as my product will ppt out of solution when heated, also dropping the pH is more rapid than heating . So it depends on your end product on what you choice to do.
No, absolutely. There is no any need of pH adjust for heat inactivation of enzymes; in fact, all enzymes are heat sensitive and even thermophilic ones are inactive (by denaturalization) after boiling
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