I am working in the production of cellulase from fungus (molds) like Aspergillus niger, but I have problems with the precipitation with ammonium sulfate. I don't see any precipitation in the different fractions, neither after centrifugation at 14,000 rpm for 10 min. To produce the enzymes, I use solid state fermentation and for the extraction sodium phosphate buffer 50mM ph=6. I don't see precipitation yet, even at 70% concentration of ammonium sulfate.
I tried precipitation with acetone with good results, but in some cases the protein loses the activity or became insoluble.
I tried different protocols to precipitation with ammonium sulfate but I don't see good results. The concentration of protein in my crude extracts are about 0.05 mg/ml to 0.5 mg/ml (bradford method).
Usually, cellulases of molds have the isoelectric point at pH near to 4. Try to test the effect of extract' pH (at the presence of ammonium sulphate) on enzyme precipitation.
pH plays an important part in Ammonium sulphate precipitation. Try to lower the pH during precipitation (by Ammonium Sulphate) you may get better result.....Try between 5 to 5.5.
We usually precipitated extracellular endocellulases using 80% ammonium sulfate and proteins are recovered by centrifugation at 7500rpm for 45min at 4°C. The pellet is resuspended in 100mM sodium acetate buffer (pH5).
It works very well. May be the quantity of cellulases produced is low, so it will be better to try to optimize the induction to scale up the production of cellulases.
I am certainly agree with augustin, have you ever tried to find out is there any activity in your crude pellet and suparnatant separately, one of the most important point to purify your desired protein with salting out you are supposed to be get enough crude protein at first but i think the concentration of 0.05 mg/ml to 0.5 mg/ml are not enough to exract your enzyme from precipitate, it could be resulted in losing your enzyme during purification. Firstly try to get activity in crude media and then salting it out, if there is no any activity in your media so change the fermentation protocol to get better enzyme concentration.
Tracking the desired enzyme in each saturation is some times forgettable. Because we usually directly cited from other paper. whereas, cellulose has varied characters which is have different optimum protocol to purified it. Hence, the suggestion from Augustin and chenk is precise.
Try to concentrate the broth 10 fold by ultrafiltration membrane 30kDa MWCO before Ammonium sulphate pptn..Monitor Protein content and enzyme activity at each step. Hope this helps
To check a feasibility of above suggestions, first of all, try to increase the protein concentration using small volume of extracting buffer. By the way, dist. water is very appropriate for extraction of cellulases.
You have to use minimal extraction volume to increase the protein concentration and go for 80-90% ammonium sulfate saturation. Let the saturated soln stand for 3-4 hrs at 4°C before centrifugation. Hope it will work.
I have seen very usuful opinions about your problem. However, more info is needed about the SSF process like the type of support you are using; is this a natural (bagasse, wheat bran etc) or an inert support?. This is important cause some natural supports may tightly adsorbe the enzyme thus extraction protocol have to be changed. A useful advise is to concentrate the extract first by ultrafiltration (previously centrifugated and filtrated to avoit fine support particles). Also important is to know the purpose of precipitation.
As suggested my Vijay try concentration using the right cutoff membrane and try solvent such as dimethoxy ethane, I have used it for laccase and it doesn't effect the laccase activity..all the best
For ultrafiltration use 10KDa membranes. Anyway, it is important the protocol you are using to extract the enzymes from the SSF samples.
Thanks all for your answers, are really useful! I will consider it .
One more comment from my own experience. When you salt out your protein, in what tube do you centrifuge it? What shape does the bottom has? If you use flat bottom, the pellet will be dispersed all around so that you will not see it.Us something with conical bottom, like Falcon tube, so that the pellet is concentrated on one small spot.
Good luck with that.
You should use acetone. I can give you full protocol and one most important thing that you have to keep the sample at 4 °C or low temp. for overnight after adding either ammonium sulfate or acetone u will not see any ppt at the same time when you are adding these.
You can extract with citrate buffer (0.05 M, pH 4.8) in a substrate: buffer ratio of 1:10 employing a simple contact method. Then filtered by Whatmann filter paper no. 1 then filtrates were centrifuged at 10,000 rpm for 20 min at 4°C. Now you can get more enzymes (in mg/ml) as crude samples from supernatant. By the which substrates you used for fermentation can also be responsible for lower/higher enzyme production.
A late contribution: ammonium sulfate precipitation works best when protein concentration is >1 mg/ml.
For acetone precipitation you need to work in the cold, acetone is much more denaturing for proteins than ammonium sulphate.
The sample should be in an ice bath, the acetone should be cooled to -20 °C before addition (mixing acetone and water produces heat of hydration!). Add the acetone slowly and drop-wise with constant stirring (as vigourous as possible without foam production). The centrifuge needs to be refrigerated to -5 °C, and remaining acetone removed from the pellet by lyophilisation. The resulting acetone powder is often quite stable when stored dry at -20 °C. Personally, I put the container into a jam jar with patent lid (the kind that made "klick" when you first opened it) containing desiccant. These things seal very well.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Enzymology
- Enzymes