Hi all,
I have an anaerobic bacteria, which could utilize xylan as the sole carbon source. I tested and found that the xylan-degrading enzyme was cell-associated protein. I harvested the cell mass which growth on the xylan (incompletely soluble in broth culture) by centrifugation. The I did the sonication to break down the cell wall and release the cell-associcated protein. Then I centrifuged to collect the supernatant. I precipitated the supernatant by 40% (NH4)2SO4, centrifuge to harvest the pellet. Did dialysis overnight by 1kDa-cut-off dialysis membrane.
Then I had the crude extract enzyme. I incubate the crude enzyme with substrate were oligosaccharides of xylan, from xylose (X1) to hexose (X6). for 24h. But when I develop the hydrolysis product on TLC, the patern of spots were strange. In the hydrolysis of all sets, presence of spots at X3, X4 and X5, even in incubation of crude extract enzyme with Xylose.
Therefore, I think, the presence of X3, X4, X5 in the product enzyme reaction with xylose, might be the remaining oligosaccharide products from broth culture.
Could you give me some suggestion or recommendation to remove this remaining products from crude extract enzyme?
Thank you in advanced.
11/23/23
Dear Tra Thi,
I will try to answer your questions and provide some suggestions.
After your initial recovery of the enzyme (i.e., ammonium sulfate (AS) precipitation and dialysis), did you first assay the enzyme on xylan oligosaccharides to see if the enzyme actually had any activity? A quick and easy way to do this would be to incubate the enzyme w/ xylan oligosaccharides, then test for an increase in reducing power. Even without enzyme treatment, xylan oligosaccharides are reducing sugars. However, if they are hydrolyzed by the enzyme, lower molecular weight sugars (e.g., xylose, X2) should be released, so the reducing power after enzymolysis should be higher. There are a number of methods for measuring reducing sugar, e.g., methods based on copper reduction, etc. They are quick and easy to do. One is the Nelson method (Nelson, N., J. Biol. Chem. 153:375-380). It's an OLD method, but it works well. There are other methods that will work. These can be found by doing a google search. This would tell you if your enzyme was active when you did your incubations.
In your incubation, you tested the enzyme w/ xylan oligosaccharides (X3, X4, X5). Even if the enzyme was active, it is possible that it did not completely hydrolyze these sugars, so some of them would remain following incubation, and would show up on TLC. Was xylose present in your incubation mixture along w/ X3, X4, X5? Xylose is one of the sugars you would be looking for by TLC of the hydrolysis products. If it was initially present in your reaction mixture, there's no way to tell if it came from hydrolysis of the larger sugars. Setting up an incubation mixture w/ enzyme plus only xylose might tell you if the enzyme contained other enzyme(s) that converted xylose into something else, although that is not very likely.
When you ran your TLC, did you include enzyme alone as one of the spots on the TLC plate, w/o X3, X4, X5? This should have told you if the enzyme contained any remaining oligosaccharide products from the broth culture. This seems unlikely. When you did your initial AS precipitation, these sugars should have been left in the AS supernatant after centrifugation of the enzyme pellet. Even if some of the sugars had remained, they should have been removed by the dialysis step.
If some of the sugars are still in your partially-purified enzyme, there are a couple of things you can do. One thing is to do a second AS precipitation as before to separate the enzyme from any soluble sugars. Another thing is to re-dialyze the enzyme. This is easier than AS precipitation. If I were doing this, I wouldn't use a 1kDa-cut-off dialysis membrane. I would use a dialysis membrane with a higher mol. wt. cut-off, e.g., 3500-5000 daltons.
I hope this information helps you. Good luck w/ your research!
Bill Colonna
Dept. of Food Science & Human Nutrition
Iowa State University, Ames, Iowa, USA
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