Hello. I am studying about bacteriocin.
My cell-free supernatant(CFS) from bacillus subtilis can inhibit indicator strain. I want to know, inhibition factor in CFS is protein or peptide. So i treated proteinase K(final concentration is 1mg/ml, 37degree, 2hr). But my case, i saw inhibition zone. Is not my inhibition factor bacteriocin? Did bacteriocin have resistant to proteinase K?
A CFS is too crude to perform such an assay. Usually CFS contains multiple active components.
Besides that, the bacteriocin might be resistant to proteinase K digestion or the digestion is insufficient. Fractionate the CFS further and use multiple strains both gram negative and positive for activity analysis of all the fractions with each strain, with and without proteinase k digestion.
Dear Mangal
To show wheather the inhibition of indicator bacterium is due to bacteriocin or something else.CFS should be treated with different proteases as you made.When you make the assay you can find inhibition after treatment by certain protease (proteinaseK in yr case).This is possible since proteases act at specific sites in the plypeptide chain.This site is a peptide linkage betwen 2 amino acids.Those 2 amino acids cold not be available in a linkage and another reason that this bacteriocin may contain lipid or glucidic moieties and can affect on protease action.Yr result is correct and is not important all proteases degrade the bacteriocin peptide.
Hi Mangal. Just as it has been pointed out, CFS may contain other substances which may interfere in bacteriocin assay and such should be eliminated to avoid interference. But firstly I suggest that you confirm through PCR if the organism from which the CFS was obtained actually encodes genes for bacteriocin production. It is actually after this confirmation that you may proceed with bacteriocin production and assay from producer organisms
Thanks for your answer. If i will do more testing(papain, trypsin), is this enough?
what if after trypsin and proteinase K digestion there is still activity. Will you conclude that the bacteriocin is absent.
Mangal Singh
Hello, more than answer your question I want to do one, I am also working with Bacillus but we have a problem when filtering to obtain cell-free supernatants we have a loss of activity, when we test the supernatant just by centrifuging we have a good inhibition and after filtering disappears
Did not this happen to you?
Adolfo Hernández-Barboza We have been trying several treatments to get the cells to release the active compounds, such as sonication or tween but we did not achieve positive results, apparently for it to have activity the cell must be present, you who have tried?
Noemi Aguila I have performed the Agar Well Diffusion Assay with Whole Bacterial Culture of our LABs in question and L. plantarum against the reference microorganisms, by which we obtained results, but when applying the same Agar Well Diffusion Assay with the Cell-Free Supernatant, no result was obtained.
Therefore I am investigating, whether the bacteriocins or the antibacterial molecules are subject to the membrane of filtration or the LABs and L. plantarum are stimulated or some kind of upregulation in the production of the antibacterial molecules in the presence of the target microorganisms (thus increasing their concentration) or some metabolic product that is same secretes.
On the other hand, in my search I have found that for example pyocin ( a class of bacteriocins)produced by Pseudomonas aeruginosa is upregulating by hydrogen peroxide, a metabolic product of LABs. Therefore this is an approach that I should investigate, if my LABs are responding to a metabolite of target microorganisms to produce more antibacterial agent.
Here the link...Article Microarray analysis of Pseudomonas aeruginosa reveals induct...
Adolfo Hernández-Barboza In our case, the problem is that this occurs with different strains of several Bacillus species and when faced with different pathogenic strains, it will be difficult to know which is the inducer of the activity.
Me podrías proporcionar algun correo electronico para poder estar en contacto? creo seria mas facil
Noemi Aguila My Insitutional email: [email protected]
My Personal email: [email protected]
Muchas gracias por el interes. Saludos
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Peptides