"I'm working on characterizing an endolysin derived from an A. baumannii-specific bacteriophage and would like to test its activity against purified peptidoglycan. If anyone has experience isolating peptidoglycan specifically from A. baumannii, your insights would be very helpful. Also, if you can suggest any reliable biochemical assays for evaluating endolysin activity against peptidoglycan (e.g., turbidity reduction, dye-release assays, etc.), I would greatly appreciate that as well."
Thank you in advance for your help!
hi Inam Ullah
If you want to test endolysin activity on peptidoglycan, the gold standard is to use commercially available peptidoglycan from Micrococcus or Bacillus species, which are offered by various suppliers.
For any scientific publication, it's essential to demonstrate that the material you are working with is precisely what you claim it to be. This is especially important for complex molecules, making it preferable to obtain them from reputable commercial sources.
Sigma and many other suppliers offer a variety of peptidoglycan formulations. I recommend using one of these for consistency and reliability.
Best,
Povilas
Povilas Kavaliauskas Thank you very much for your suggestion regarding the use of commercially available purified peptidoglycan. That indeed sounds like a convenient and standardized approach for testing endolysin activity.
I have a follow-up question: if I use purified PG as a substrate, how can I determine which specific bond my endolysin is targeting (e.g., glycosidic, amide, or peptide bonds)? I was considering analytical technique such as HPLC, to characterize the cleavage products.
Apart from this, are there any feasible biochemical assays that could help identify the type of enzymatic activity? Any recommendations or experience with such methods would be greatly appreciated.
Thanks again for your valuable input!
Hi Inam Ullah
This is a somewhat complex question, and I believe the best approach depends on the techniques and time available. While I’m not a deep specialist in peptidoglycan, here are a few thoughts:
If commercially available, you might consider using PG preparations with specific modified bonds. Observing loss of activity under such modifications could provide insights into the mechanism of action, which could be further validated by HPLC or mass spectrometry.
Alternatively, a potentially simpler approach would be to examine the conserved motifs within your endolysin. If there are structural motifs known to be associated with specific activities (e.g., glycosidic, amide, or peptide bond cleavage), you could identify the relevant residues and generate mutants lacking them. You could then validate the resulting cleavage products using HPLC.
Hope this helps, and let me know if you’d like to discuss further.
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