Hello everyone,
We have isolated bacteriocin or bacteriocin like inhibitory substance from B. clausii having antimicrobial activity against gram positive and negative bacteria.
On checking it's sensitivity to proteolytic enzymes (proteinase k, trypsin and pepsin), it retained 50-60% of its original activity. Will it be considered as Bacteriocin?
We purified it by combination of Ammonium sulfate precipitation, dialysis and column purification and checked for molecular weight using SDS-PAGE. And we could see single intact band at around 12kDa. Is there any reported bacteriocin having molecular weight > 9kDa?
Then we sent it for nano LC-MS/MS analysis in order to identify protein sequence. They did not provide us raw file but instead analyzed the result and gave us protein sequence and results of different peptides having similarity to reported sequence. Molecular mass analysis of resulting protein sequence is similar to SDS results. Here
We found that it shows similarity to probable thiol peroxidase from B.clausii. What could be the possible reason for its bacteriocin like activity? Can we consider this as bacteriocin like inhibitory substance?
Thank You for your valuable time.
With Best
The diverse substances classified as bacteriocins include proteins larger than 9 kDa. An example is 27 kDa lysostaphin. There is a nice article about bacteriocins in Wikipedia:
https://en.wikipedia.org/wiki/Bacteriocin
Thank You very much for your reply..
Sir, can you please tell me what could be the mode of action of identified protein (thiol peroxidase), which accounted for antimicrobial activity?
Have you demonstrated that this protein actually possesses thioredoxin peroxidase catalytic activity, or do you only know that it has sequence similarity to thioredoxin peroxidase?
Sir for now we only know that protein possesses thioredoxin peroxidase activity based on sequence similarity...
There are many bacteriocins larger than 9 kDa, A list of all the bacteriocins and reference papers can be found on this link
http://bagel2.molgenrug.nl/index.php/bacteriocin-database/63-bacteriocins-larger-than-10kd
As Adam is suggesting you can confirm by performing an assay for thioredoxin peroxidase catalytic activity.
Did you also run the gel of proteolytic digests? If the protein is resisting cleavage how MS/MS was performed.
May be peroxidase is a probable match? How many residues (or stretch) of MS/MS sequence actually match with it?
Hello, Sorry for the late reply,
On doing blastp, it is showing 79% query coverage and 37% identity to best match sequence.
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