I am trying to precipitate a bacterial cellulase produced on a lignocellulosic hydrolysate as the first step in the purification of the enzyme.
I did step-wise ammonium sulphate fractionation at 0-40% and 40-90% saturation. Initially I did not see any precipitate in the two fractions but after repeated centrifugation at high speeds (up to 20000g for 20 minutes at 4degC) I was able to get some little pellets which I resuspended in little quantity of buffer. These two fractions have very little specific activity (less than 1 unit/mg) but the crude culture supernatant and the 90% supernatant have high specific activities (16-18 units/mg).
What could be the reason why I am not getting active fraction at up to 90% saturation? I am afraid that adding the salt up to 95% might also not give me the desired protein? Please kindly advise.
Some proteins are soluble even at high salt concentration. Example is the S-100 proteins, soluble at 100% saturation of ammonium sulfate. The reasons are not totally clear, but they used to be small, have affinity to calcium and contain some saccharide moiety. If you want purified the protein, perhaps you can precipitate all other proteins and they submit the superantant to dialysis and concentration.
Thank you Francisco Solano. Do you suggest that I should further try 100% saturation for the precipitation?
Did you try to change the pH. Proteins are less soluble close to their pI.
Dear Jose,
Several month ago I had similar problem on proteases of Beauveria bassiana. I had high activity in crude but no precipitation on Ammonium sulphate. When I used inhibitor and negative control I found the enzyme is so little in the sample. I changed te media and increased incubation time. In your case, I suggest to use negative control or inhibitor (more recommended) to find whether you have the enzyme in crude or not. As you know, spectrophotometry just detect OD without caring to real activity.
Good Luck,
Arash
There is nothing wrong in your experiment. I would suggest to go for 90 to 100% Ammonium sulphate cut and see the activity in both the precipitate and the supernatant. Most of the unwanted proteins have been removed using 0 to 90% cut and now you have a more purer enzyme....If you have activity in supernatant (90 to 100% saturation) then remove the Ammonium sulphate from the supernatant and I am very much sure you will get much more specific activity. Wish you good luck.
The precipitation can sometimes be very slow. Have you tried leaving the preparation overnight in the cold with 95% ammonium sulphate.
Peter
Peter is right, u should try this. Better to add ammonium sulphate slowely at 4 degree on magnetic stirrer with low speed and then leave o/n in cold room (4 degree C) before centrifugation.
I agree with former comments. You can leave the solution saturated in ammonium sulphate at 4ºC for very slow stirring overnight and see what you get. Good luck
Do you still see activity in the supernatant after treatment with 90% ammonium sulphate? The protein might simply have been denatured. You may want to try ion exchange fractionation of your crude as your first enrichment step.
If most of the activity is in the supernatant with high recovery you might proceed with your purification by using the supernatant may the enzyme have very low Mw and not ppt by ammonium sulfate you can use gel filtration our and ion exchange chromatography
Then your enzyme was purified by discarding almost the other proteins in the precipitate. You will verify this on SDS-PAGE.
You can also try precipitation with acetone.
Saturated ammonium sulfate (SAS) fractionation is almost proportional to molecular weight of protein and is influenced by concentration. Impurities were already removed by SAS frac. What about the ion-exchange column chromatography to concentrate you r target? Good luck.
Change the pH of the solution, in general for negatively charge proteins, lowering pH to acid conditions (pH 4-5), may help in precipitation of proteins in ammonium sulfate saturation process. All proteins close to isoelectric point are less soluble. Your proteins must be stable at that the pH you choose, and after recovering activity at optimum pH.
HN
Did you allow time for the precipitation? The salting out can sometimes be quite slow and so leaving the 95% fraction overnight (in the cold) may prove to be effective.
Did you test for activity in the fraction that is soluble in 90% salt? Dialyze that fraction into an appropriate buffer to ensure that the activity is in fact still there.
If so - attempt any of the above suggestions.
However, if you don't detect specific activity in the highly soluble fraction, and it isn't in the earlier fractions either, then your activity might possibly require two separate proteins, each precipitating at different ammonium sulfate concentrations. Their separation leads to highly reduced activity in all fractions.
1. .Add ammonium sulphate to 75% saturation, pH of solution should be 4.5. Precipitate day-night at 5oC.
2. Try to isolate enzyme with the cold ethanol.
The possible reasons why u r unable to get active fractions may be all procedure u followed for amm. sul. ppt. is not done in right way. I mean there are some precautions for doing amm. sul. ppt. like temp. should be low during the addition of amm sul. to the crude extract and mixing of salts in medium should be slow n gentle to avoid formation of air bubble as air bubble is an enemy for the protein and there will be a chance of protein degradation also. At last after addition of salts it should be kept at low temp. without any disturbances. And u should try to collect more protein (mg) in crude step (more specific activity grtr dan 20 U/mg is better. So that u will finally got a good result. All d best.
Can you suggest some paper , as I am also facing the same problem.
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