I work on cellulase enzyme and express it in E.coli(BL21). In SDS-PAGE I have good bands, but after purification with Ni-NTA column and dialysis, it's activity is very low (according to enzymatic assay). My enzyme didn't have good activity before purification so although the protein band is very sharp on gel electrophoresis, after purification it's activity drops further. My dialysis buffer is 20mM Tris.
What are the units of your activity, ie is it a specific activity? And why do you think the activity is low? We need more details. Is the low activity based on literature values?
Do you actually know this enzyme is a cellulase? Are you assaying this with cellulose and measuring the released glucose? Is it an endo or exo cellulase? Or are you using PNP-glycosides? Are you using cellobioside or glucoside?Might it work on other sugars such as xylose or B-1,3-linked glucose?
Generally I would expect a nice band on a gel to produce an active enzyme, but you may not have the correct assay conditions. For instance many cellulases like low pH and some require Mg2+. We need the details of your assay to be able to help.
Which organism's Cellulase have you expressed in E.Coli is the primary question? Because in Eucaryotes that is fungi and yeast, the cellulase are highly glycosylated proteins whereas in Bacteria the protein cannot be glycosylated. Moreover most of the bacteria produce cellulase as cellulosomes. Cellulosomes are multi-enzyme complexes which are associated with the cell surface and mediate cell attachment to the insoluble substrate and degrade it to soluble products which are then absorbed. You may get a good band in SDS PAGE but if it does not reflect the functionality of the enzyme. you need to furnish more details so that i can help you
it's an endoglucanase, from thermophile acidophilic bacteria.according to litrature optimum temperature is 65 in 3 minute. for enzymatic assay my substrate is CMC,and measure released glucose units with DNS reagent- as diffirenec color and especophotometric absorbance in540nm,it's activity is low-although protein band is very sharp
Parastou, then play a bit with different pH and temperature range. May be the recombinant protein show a little shift in its optimum pH and temperature... Hope it works for you...
Deepti@yes i should define pH and temperature profile for it,is there another factor important for protein activity?how i can retain protein activity for along time?is there an agent that don't interfere with enzymic activity and stabilize it's structure?thanks
The pH and temperature do play a very important role..... Normally CMC'ase activity is carried out at pH 4.5. When I was working with Paenibacillus cellulase at this pH I was getting very low acitvities. Then I used Citrate phosphate buffer which working range from 4.0 to 7.0 pH. and prepared substrates in 4,5,6,and 7 pH buffers. You will be amazed to know that my enzyme activity increased by 4 folds...Try to lyophilize your protein sample so as to preserve your enzyme activity if possible for you.
3 minutes is avery short assay time - it is easy to miss the time by 10% and thus ruin your assay. Instead you should dilute your enzyme by 10 fold and run the assay for 10 times as long.
CMC is not necessarily a good substrate - some enzymes are known to cleave CMC but not cellulose and vice versa. If this is a new enzyme you might like to try another substrate, such as Avicel.
Also your protein may not be properly folded - you can check by circular dichroism or ITC. If you only have a small percent correctly folded you wont know by SDS-PAGE.
Deepti@sure,I will examine pH range for it.thanks for your help.
Ellis@yes i assay activity until 10 min,and different concentration of enzyme and substrate unfortunately don't see significant difference between them!!
another thing!I have good express after 24 hours in 22 degree, can this long time effect on protein folding or activity?
If you induce your protein for too long then you can start to get degradation, which you obviously do not have with a single band on PAGE. How bad is your activity? Is it 0.1% of what you expect, or just 50%? Is it possible there is a cofactor missing, such as a metal ion?
Cellulase can be endocellulase or exocellulase or Beta glycosidase If you used CMC and measured the product with DNS then you were measuring the exocelulase activity Have you tried zymogram adsay with cellulose as the substrate added to the acrylamide gel ? I agree with others that the reaction condition for your enzyme assay has to be optimised in term of pHs temperature ionic strength and possible activator cations for cellulase
Mr Ellis,sorry for this delay for responding!!Ok,after purification,activity drop 50%.i want bring back it's activity with diyalysis and changing buffer,i extract enzyme with 200mM imidazole from Ni-NTA column,and my dialysis buffer is tris 20mM ,pH 6.5(according to pH profile).but activity drop alot,about80%.somebody says adding DTT(dithitritol)ii dialysis buffer,I didn't do that until now and another thing Ca ion,i hope it works!
Maggy@thanks, i have a question about zymogram test!how can i do that?i boil Enz-sub cocktail with DNS for in 5 min for enzymatic assay.i don't know?!i should add CMC in acrylamide gel and after running, boil gel with DNS!!!!
You cannot measure absolute activity before purification because you do not know the exact amount of enzyme you have in the total protein mix. Unless you are looking for an industrial enzyme supply, 10% total of extremely pure enzyme is still good for assays and even some biotransformations. You can easily get 10 fold variation between the same enzyme from different species, particularly if you have not optimised the assay conditions.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Proteins