As mentioned before, the decrease in specific activity typically means either that you are losing your protein along the way (unpurifying it, you might say), it is unstable and is losing catalytic activity during the purification, or it is being degraded by proteases. You should consider changing the buffer conditions to help maintain stability. Examples include adding sugars (glycerol, sucrose, or trehalose) as stabilizers and adjusting the pH. Make sure all purification steps are done in the cold and all fractions are stored on ice. Include protease inhibitor cocktail. Work through the purification as quickly as possible. Check for proteolytic degradation by Western blot, if you have a suitable antibody.

Also consider the possibility that the cellulase activity is actually due to a mixture of different enzymes, so that as you purify one of them you lose the activity of the others.

Dear,

Decreasing sp activity simply implies that your protein is being lost at every stage of purification. Pls take all necessary precautions in each purification step.

It depends on your purification conditions including:

1. The % of protein recovery based on the method (gel filtration, ion exchange, RP-HPLC). 

2. pH value during purification

3. Avoiding denaturing condition

4. Temperature during purification.

5. Protease may work during purification and so the activity may be reduced.

Good luck

Hi,

       In each purification steps, the total amount of proteins are decreasing and just your enzyme is keeping on your samples. Please note that you must use specific substrate to enzymatic assay or specific affinity chromatography. Finally, you will find the lowest Unit activity of your purified molecule but when you calculate specific activity you will see the highest value. for guidance, please see a purification paper on XX enzyme.

Cheers,  A.

hi,

apart from the above comments, 1) u need to be very sure about the enzyme stability. 2) in my opinion i recommend ion exchange chromatography before going for SEC.

good luck

you need to determine the quantity of protein after each step to be able to determine the specific activity.

Hi there,

During efficient purification steps, Specific Activity (activity expressed by mg of protein) is increasing because the protein of interest is getting more abundant. If it's not the case, it's either because you lose your protein during purification or because the activity becomes unstable during purification and/or assay...

Given that it is the  specific activity that gets lower after each purification step, this indicates that your enzyme becomes more and more inactive. Besides the reasons mentioned by Kamran, one reason might be the loss of a cofactor. Some cellulases need Ca2+, Mg2+ or other divalent metal ions for optimal activity. Those might get leached out during your purification steps, depending on the buffers you use.

Thanks everyone. I used phosphate buffer, pH 7.0.

As mentioned before, the decrease in specific activity typically means either that you are losing your protein along the way (unpurifying it, you might say), it is unstable and is losing catalytic activity during the purification, or it is being degraded by proteases. You should consider changing the buffer conditions to help maintain stability. Examples include adding sugars (glycerol, sucrose, or trehalose) as stabilizers and adjusting the pH. Make sure all purification steps are done in the cold and all fractions are stored on ice. Include protease inhibitor cocktail. Work through the purification as quickly as possible. Check for proteolytic degradation by Western blot, if you have a suitable antibody.

Also consider the possibility that the cellulase activity is actually due to a mixture of different enzymes, so that as you purify one of them you lose the activity of the others.

Thanks all.

• The most common reason for loss of activity is enzyme instability.you should therefore spend some time testing conditions to render the enzyme stable during purification.First of all eliminate the obviouS factors like proteolytic activity that could destroy the enzyme  as

The reason may be because of repeated purification the lytic enzyme dominate in the bacteria and slows or block the other activity of enzymes 

Could the choice of substrate influence this loss of activity? For example, could the use of lignocellulosic biomass rather than a pure cellulosic substrate such as CMC, Avicel, etc, cause this problem?

no the substrate does not influence this loss of activity, rather it is your purification system e.g. the matrix etc that are actually inhibiting.

The reason I mentioned substrate choice as a possible cause of loss of activity is because lignocellulosic biomass might contain some amounts of protein which could induce the production of proteases along with the desired enzyme in the culture supernatant. Many have suggested the action of proteases here as a possible reason for loss of activity. So, if one uses CMC or avicel  (which do not contain protein) for example, the chances of having proteases in the culture supernatant might be reduced. This is just my thinking.

Yes, agree with above comments. In my experience, it happens when your protein of interest get co-purified with some inactive form of enzyme, which leads to great loss of activity. I reccommend you may take a look and re-confirm at the gene encoding protein sequence, that it did not have cause any undesirable point mutation at crucial residue that might alters overall protein conformation and stability. Besides, i also suggest that you need to optimize your purification buffer conditions to get fully active and stable protein.

Best luck!

Regards,

Irshad Baig

1. Optimization of your conditions should be done where you have to spend some time doing it.

2. Try to do the purification in a cold room.

3. Check after each purification process with gel electrophoresis to rule out any dimer or multimer formation.

How you tried to use different kind of buffers (phosphate, tris-HCl, etc.). 

A lot of the answers so far suggest instability, proteolysis etc.   If it really is SPECIFIC activity that is going down and not just total activity, this makes it more likely that it is due to removal of a cofactor or an accessory protein. Proteolysis, for example, would produce fragments or nicked molecules that would almost certainly be separated. In that case you would see a big drop in yield (i.e. total activity) but still an increase in the specific activity of the remaining active fractions. To see if you are removing an essential piece of the machinery, such as a cofactor, metal ion etc., you could try adding back fractions just as in the heroic early days of enzymology!  Happy New Year!

Paul

Mushafau, your observation is in order. When you go through various steps in enzyme purification, the crude enzyme would have higher enzyme activity to the enzymes resulting from subsequent purification. This is because on ammonium sulfate precipitation, the enzyme concentration that is precipitated decreases. In other words, not all the enzyme are precipitated out and that is the essence of the process. At a given %ammonium sulphate, a range of enzyme mol. weight is precipitated out- and not all of the mol.weighted enzymes that can carryout the catalytic activity. A good textbook on enzyme purification will explain all these better and the accompanying calculations for purification yield.

I completely agree with Paul C. Engel suggestions.

Paul C Engel,  As per your answer can we add these cofactors, and even DTT  after each purification step.  How to determine the appropriate amount which should be added.

In addition to DTT you can add a stabilising agent like 30% w/v glycerol to the buffers.

You mentioned ammonium sulphate. Similar case happened to my enzyme. You can try using your crude, add some ammonium sulphate at low concentration, and do assay. If the activity drop, maybe your protein do not like the salt. You can consider using other precipitation approaches or use cross flow to concentrate. 

I had a similar experience after using acetone.

you need to study if the enzyme is dependent on certain co-factors.

Decreasing specific activity during purificarion is either due to inactivation of your enzyme (proteolysis being most likely) or loss of an activating co-factor. The website BRENDA contains a wealth of information on activators and inhibitors of cellulases,as well as structural and mechanistic information, which is well worth consulting.

http://www.brenda-enzymes.org/enzyme.php?ecno=3.2.1.4#METALS%20and%20IONS

Given your problem with enzyme stability, before embarking on too many different purification steps, I would suggest measuring the stability of your enzyme in a crude extract over time under different conditions: temperature (4 deg and 25 deg); buffer pH; addition of various potential stabilising agents alone and in combination, e.g. protease inhibitor cocktail; EDTA (0.1 mM - heavy metals); DTT (1 mM - thiols); BSA (1 mg/ml). If an activator is essential, then dialyse your enzyme an measure specific activity before and after treatment. The dialysate can be added back after concentration to see if it restores activity - if not then it is loss of a cofactor. Ammonium sulfate should be of the highest purity as this reagent can contain heavy metals.

An excellent book for further information is Protein Purification; Principles and Practice by Robert Scopes.

getting It is possible that your enzyme is stabilised by interaction with other proteins. Protection from protease action has already been mentioned. The addition of sugar can often protect an enzyme from denaturation. Loss of activity/denaturation can be particularly likely when the total protein concentration is getting very low as the enzyme approaches purity.

Peter

I endorse Alan's answer. It covers most of the likely causes of the loss of enzyme activity during purification and suggests ways of preventing it.

Hi, There are two possible reasons:

1- The enzyme is unstable and it is being lost.

2- Check the amount of protein then calculate specific activity. If it is higher than specific activity of previous step so you are going to purify your sample. but please consider recovery and purification folds value.

I do agree with Adam Shapiro.  As cellulase is a mixture of enzymes, you might have purified only one among them. check the activity using specific substrates in each step of purification.

Though late, I suppose adding a voice to available suggestions will be very helpful in future researches into specific activities of complex enzymes involved in complex hydrolytic pathways.

Summarily, I wish to state that your observation is in order and my humble suggestion has been captured in an earlier submission by Dr. Adam B Shapiro  in his last paragraph: "Also consider the possibility that the cellulase activity is actually due to a mixture of different enzymes, so that as you purify one of them you lose the activity of the others". 

Recall that hydrolysis of lignocellulosics requires several enzymes which act in synergy  but  sequentially to produce a glucose and/or xylose sugars. Recall also that all types of  cellulases and hemicellulases (since hemicellulose also contain some amount of glucose) are produced in an inductive medium with ligno-cellulosics as carbon source.

All these enzymes could have contributed to the higher specific activity of the crude extract. However, at each purification stage, you definitely would have lost some with more restrictive activities (for instance, beta glucosidase and arabinofuranosidase-which are dependent on the hydrolytic products of endoglucanases and xylanases respectively- might have been loss). This will definitely will affect your specific activity since your "standard" is glucose sugar not "glycerin". So, the more you purify, the more you loose other important enzymes with either lower or higher molecular weight. 

 Hope this helps!

Specific activity of an enzyme decrease:i) loss of active enzymes, ii) denaturation of enzymes.

To avoid, maintain buffer condition, check you  working pH  and temperature and presence of inhibiting metal salts.