Testing for proteic nature of a putative bacteriocin:
I used 1mg/ml Proteinase K to digest the putative bacteriocine in a cell free supernatent (with unknown concentration, but the supernatant was harvested from bacterial culture with McF3) , then incubated for 3 hours at 37°C , then deactivated it at 100°C for 10min.
I then performed a deep well diffusion agar assay and find out that the putative bacteriocine treated with proteinase K was partially digested with proteinase K: In comparison with the untreated putative bacteriocine , the halo is norrower but still have an inhibition zone.
Can you please help me understand why is the inhibiting activity remaining after proteinase K treatement?
If it is not a protein , why is there a difference between the untreated and treated sample?
any suggestions please?
In what cases does proteinase K not affect proteins?
Thanks in advance.
I suppose that you do have a negative control with proteinase K and no bacteriocin to show that proteinase K doesn't inhibit the growth of your indicator bacteria ?
Dear Lyna M'Rabet,
Have you considered the possibility that there is more than one antimicrobial metabolite in your cell-free supernatant? For example lipopeptides, are usually resistant to enzymatic digestion.
If you only expect bacteriocins, my recommendation is that you increase the enzyme concentration (there are papers that use up to 10 mg / ml) and that you use other enzymes such as pronase E, in my particular case with this enzyme I achieved a total inactivation.
I wish you good luck.
Regards
Dear Mariana Emilia Cozzolino,
Thank you for your answer and suggestions.
I checked for lipids whith Lipase 1mg/ml and I had no difference between the untreated and treated samples. I will try to apply what you recommended and see if it works.
Bests,
Lyna M'Rabet
Dear Lyna M'Rabet,
Proteinase K is a serine protease. Although it has broad-spectrum substrate specificity, it might not digest all types of proteins.
My recommendation is that you can set some concentration gradients of protease K and compare the inhibiting activities or try to use other stronger proteinases, and it seems that you can consider whether there are any substances that can antagonize proteinase K in your supernatant.
Bests.
Dear Jing Yu,
Thank you for your answer and recommendation.
I will take that into account.
Bests,
Lyna M'Rabet
Hi
First, you may consider the optimal temperature of protinase k between 50-65 degree celcius although it has activity at over 20 degre. You may try at 55 degree for even prolonged time more than 3 hours.
Second, you can thik about whether any inhibitor of protinase k might present in your sample/buffer may contain PMSF or something else? Also if possible you can check pH that should have 7.5 to 12 for optimal activity of protinase k.
It is possible that the new generated bacteriocin fragments upon proteinase K treatment also induce some inhibitory activities against very susceptible targets.
Try to perform the experiment against different bacterial targets that require higher titer of bacteriocin to be inhibited.
In this case although the untreated bacteriocin would show small zone of inhibition, however, the digested fragments might loss the antibacterial effects due to unstable structure of the peptide/protein.
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