![]() ![]() The supernatant collected was assayed for bacteriocin activity via the agar well diffusion method. The samples were centrifuged at 10,000 rpm for 15 min. casseliflavus MI001 and incubated at different temperatures (25 ☌, 30 ☌, 35 ☌, 40 ☌, and 45 ☌) and pH values ranging from 5 to 9 for 48 h. Optimum conditions were determined by growing the test organism at a wide range of temperatures and pH values. 2.2 Optimization of culture conditions for bacteriocin production 2.2.1 Effect of incubation temperature and pH on bacteriocin production Finally, the Muller–Hinton agar (Hi-Media Chemicals, India) medium was used for bacteriocin activity. The de Man, Rogosa, and Sharpe (MRS) broth medium (Hi-Media Chemicals, India) was used for production of the bacteriocin. A nutrient broth medium (Hi-Media Chemicals, India) was used for maintenance and growing the indicator organisms for bacteriocin activity. The cultures were revived and stored in refrigerated conditions for further work. The indicator organisms were procured from the MTCC at the Institute of Microbial Technology, Chandigarh. An attempt has been made to characterize the bacteriocin ABC transporter ATP-binding protein from the Enterococcus casseliflavus MI001 strain. However, an improved understanding of the structure, function, and action mechanism of these transporters have enabled us to develop new approaches for investigating lead molecules in terms of treating various diseases caused by problematic organisms. ABC transporters were found to play a key role in virulence and also identified as suitable targets for the development of antibacterial vaccines. The importance of these transporters in the multiple cellular functions and biosynthetic pathways of bacteria represents a novel strategy for the secretion of bacterial proteins and points out potential drug targets. The transporter acts as an efflux system, which helps the secretion of bacteriocins, proteins, polysaccharides, toxic compounds, and enzymes. Due to the presence of a glycine leader on the N-terminal side, these transporters remove the N-terminal leader peptide from its bacteriocin precursor by cleavage at a Gly-Gly bond and transport the mature bacteriocin across the cytoplasmic membrane. Bacteriocins are secreted by either a double glycine leader in the N-terminal part of the pre-bacteriocin or secreted by a sec-system. Bacteriocins synthesized as propeptides are processed through this ABC transporter system and exported from cells as mature peptides. The proteolytic domain resides in the N-terminal region, the ABC transporter domain is in the C-terminal and central multi-pass transmembrane region. They export bacteriocin across the cell membrane via a proteolytic function. One such substrate is bacteriocin, and the transporter proteins exporting bacteriocin are called bacteriocin ABC transporters. Based on the type of substrate exported, they are categorized into various types. Importers mediate the uptake of essential nutrients, vitamins, and trace metals from the surrounding environment, whereas exporters export substrates to the surrounding environment. These transporters are the primary transporters functioning as both importers and exporters. They play diverse roles in both prokaryotes and eukaryotes. ATP-binding cassette (ABC) transporters are one of the largest super families of transport proteins present in all forms of life. Living organisms depend on various means of transport for the uptake of external nutrients and sequestration of waste products into the surrounding environment. The ABC transporter ATP-binding protein could be used as a potential alternative for food preservation, and it may be considered as a bio-preservative agent in food processing industries. Further, the bacteriocin ABC transporter showed antimicrobial activity against food spoilage microorganisms. The NMR spectrum of purified bacteriocin revealed the presence of amino acids, namely lysine, methionine, cysteine, proline, threonine, tryptophan, and histidine. The bacteriocin was purified with an eightfold purification scheme resulting with a specific activity of 15,000 AU/mg. Purification was performed using ammonium sulphate precipitation, gel filtration, and DEAE ion exchange chromatography. The optimal conditions for the production of bacteriocin were found to be at 35 ☌, a pH 5.5, and an incubation time of 24 h. In the present study, bacteriocin isolated from the Enterococcus casseliflavus MI001 strain was identified as an ABC transporter ATP-binding protein. ![]() ATP-binding cassette (ABC) transporters constitute one of the largest transporter protein families and play a role in diverse biological processes. ![]()
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