Yes, you have to dialyze the enzyme solution before use. 

After ammonium sulphate precipitation you have to necessarily remove the excess by extensive dialysis in order to refold your enzyme. 

Maybe the sulfate concentration was not enough and you did not manage to recover the enzyme. I usually use 80%.

Try to determine activity in crude extract

Increases the sulfate concentration

Dialyzed until the Ba(OH)2 test is negative

How easily did your protein go back into solution after precipitation? Have you checked the structure retention to determine that it is still folded? I would recommend dialysis  and then if you still don't see any activity,  run circular dichroism to make sure you don't have random coils instead of the 3D structure you need for an active protein. Also, double check that your protein concentration in solution is high enough to measure activity. 

After dialysis also , not getting activity. I have cross checked whther activity was present in fermented broth or not ? But found activity in the broth but not in the fration obtained after amm. Sulphate precipitation (40-80 percent). Should I start with 10-80 percent ammm. Ppt or with acetone? Please suggest me.

Hi Vijay,

I suggest at first check that the enzyme is precipated by the amount of amm. Sul. that you use. And since amm. sul. almost has no adverse effect on protein structure, I think that amm sul inhibit your enzyme activity, you test it by addition of amm sul to the active crude extract and checking the enzyme activity. 

Acetone is not a good precipitant in your case because it would denature your protein

Yes, you should always dialyze the enzyme solution after amm. sulfate precipitation, if you want to check the activity. Amm. sulfate often inteferes with activity- by interacting with the enzyme or with the components of the assay.

Have you verified (by SDS-PAGE) that your enzyme is present in your sample that you are using for enzyme activity? Is your protein solution too dilute after reconstituting the  ppt? I would also check all fractions (ppt and supernatent), after the amm. sulfate pptn. step, for the presence of your protein first, to locate which fraction it is present in. It can be possible that the protein did not get precipitated at 70% ?

2) Acetone precipitation is not recommended for an enzyme. You may use it simply to concentrate a fraction for the purpose of loading onto a gel, but it will generally destroy activity, so be cautious using it.  

Ammonium sulphate precipitation needs to be done on ice (not in the cold room, which - as Fleischer wrote in his book on protein purification - chill the operator, not the sample), with constant stirring and slow addition of AS (literally crystal by crystal) to avoid local high concentration. Even then, some proteins do not survive this procedure. Proteins are like people: every fool is different.

After AS precipitation the excess salt needs to be removed from the redissolved protein to recover full activity (e.g., by dialysis or desalting). Salts lower the effective water concentration and enzymes need water as "grease" for conformational changes during their cycle. Indeed, this is how AS precipitation works in the first place.

Acetone (also ethanol and methanol) can be used to precipitate some enzymes in the active form, but you need to be even more careful than with AS to avoid irreversible denaturation. Because mixing water and org. solvents generates heat, the solvent needs to be cooled to -10 °C and added VERY slowly. After centrifugation of the precipitate at -5 °C the pellet is lyophilised to remove the solvent. The resulting "acetone-powder" can be very stable if stored dry and cold (-80 °C). Again, success depends on your enzyme and is not really predictable.

Thank you all for your valuable suggestions