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Assessing the antimicrobial activities of Ocins.

Choyam S, Lokesh D, Kempaiah BB, Kammara R - Front Microbiol (2015)

Bottom Line: The generation of a zone of inhibition on a solid substrate indicates the bioactivity of antimicrobial peptides such as bacteriocin and enterocin.However, universal well diffusion assays for antimicrobials, irrespective of their ability to diffuse (bacteriocin and enterocin), do not exist.The ability of different zymography components to generate non-specific activities have rarely been explored in the literature.

View Article: PubMed Central - PubMed

Affiliation: Department of Protein Chemistry and Technology, Central Food Technological Research Institute (CFTRI) Mysore, India.

ABSTRACT
The generation of a zone of inhibition on a solid substrate indicates the bioactivity of antimicrobial peptides such as bacteriocin and enterocin. The indicator strain plays a significant role in bacteriocin assays. Other characteristics of bacteriocins, such as their dispersal ability and the different zymogram components, also affect bacteriocin assays. However, universal well diffusion assays for antimicrobials, irrespective of their ability to diffuse (bacteriocin and enterocin), do not exist. The ability of different zymography components to generate non-specific activities have rarely been explored in the literature. The purpose of the present work was to evaluate the impact of major factors (diffusion and rate of diffusion) in a solid substrate bioassay, and to document the adverse effects of sodium dodecyl sulfate in zymograms used to estimate the approximate molecular weight of bacteriocins.

No MeSH data available.


Related in: MedlinePlus

(A) Zymography to show the formation and disappearance of the non-specific zone of inhibition (ZOI). The procedure in brief, After the successful separation of proteins by SDS-PAGE, and native PAGE the gel was rinsed twice with phosphate buffer. Later, it was overlaid on indicator strain containing plate. Left overnight at 37°C. The sequence shown on the gel are: lane 1, molecular weight marker; lane 2, enterocin; lanes 3–5, overlaid gel showing the ZOI, and decreased ZOI; lane 6, Reappearance of bacterial colonies; and lanes 7–9, existing ZOI after prolonged incubation. (B) Structure of zerumbone oxime and its oxime-esters. Star represents for antimicrobial peptide.
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Figure 2: (A) Zymography to show the formation and disappearance of the non-specific zone of inhibition (ZOI). The procedure in brief, After the successful separation of proteins by SDS-PAGE, and native PAGE the gel was rinsed twice with phosphate buffer. Later, it was overlaid on indicator strain containing plate. Left overnight at 37°C. The sequence shown on the gel are: lane 1, molecular weight marker; lane 2, enterocin; lanes 3–5, overlaid gel showing the ZOI, and decreased ZOI; lane 6, Reappearance of bacterial colonies; and lanes 7–9, existing ZOI after prolonged incubation. (B) Structure of zerumbone oxime and its oxime-esters. Star represents for antimicrobial peptide.

Mentions: In a 100-ml two-neck round-bottom flask, zerumbone oxime (1 mM) was added to 20 ml of CH2Cl2, 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI, 2.5 mM), and 4-(Dimethylamino)pyridine (DMAP, 0.2 mM). Magnetic stirring was performed under a nitrogen atmosphere for 5 min to mix them efficiently. Subsequently, carboxylic acid (1 mM) was added, and the resultant reaction mixture was stirred at room temperature until the reaction was completed. The progress of the reaction was monitored by thin-layer chromatography using ethyl acetate and the hexane mixture as an eluting solvent for the disappearance of zerumbone oxime. After the reaction was completed, it was diluted with 40 ml water. The organic layer was separated, dried over anhydrous Na2SO4, and filtered. The clear filtrate was concentrated to yield a crude product, which was purified by triturating with petroleum ether. Pure compounds (2–4) were characterized by nuclear magnetic resonance, infrared, and high-resolution mass spectroscopy. The structures of zerumbone oxime and its oxime esters have been shown in the Figure 2B.


Assessing the antimicrobial activities of Ocins.

Choyam S, Lokesh D, Kempaiah BB, Kammara R - Front Microbiol (2015)

(A) Zymography to show the formation and disappearance of the non-specific zone of inhibition (ZOI). The procedure in brief, After the successful separation of proteins by SDS-PAGE, and native PAGE the gel was rinsed twice with phosphate buffer. Later, it was overlaid on indicator strain containing plate. Left overnight at 37°C. The sequence shown on the gel are: lane 1, molecular weight marker; lane 2, enterocin; lanes 3–5, overlaid gel showing the ZOI, and decreased ZOI; lane 6, Reappearance of bacterial colonies; and lanes 7–9, existing ZOI after prolonged incubation. (B) Structure of zerumbone oxime and its oxime-esters. Star represents for antimicrobial peptide.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4585010&req=5

Figure 2: (A) Zymography to show the formation and disappearance of the non-specific zone of inhibition (ZOI). The procedure in brief, After the successful separation of proteins by SDS-PAGE, and native PAGE the gel was rinsed twice with phosphate buffer. Later, it was overlaid on indicator strain containing plate. Left overnight at 37°C. The sequence shown on the gel are: lane 1, molecular weight marker; lane 2, enterocin; lanes 3–5, overlaid gel showing the ZOI, and decreased ZOI; lane 6, Reappearance of bacterial colonies; and lanes 7–9, existing ZOI after prolonged incubation. (B) Structure of zerumbone oxime and its oxime-esters. Star represents for antimicrobial peptide.
Mentions: In a 100-ml two-neck round-bottom flask, zerumbone oxime (1 mM) was added to 20 ml of CH2Cl2, 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI, 2.5 mM), and 4-(Dimethylamino)pyridine (DMAP, 0.2 mM). Magnetic stirring was performed under a nitrogen atmosphere for 5 min to mix them efficiently. Subsequently, carboxylic acid (1 mM) was added, and the resultant reaction mixture was stirred at room temperature until the reaction was completed. The progress of the reaction was monitored by thin-layer chromatography using ethyl acetate and the hexane mixture as an eluting solvent for the disappearance of zerumbone oxime. After the reaction was completed, it was diluted with 40 ml water. The organic layer was separated, dried over anhydrous Na2SO4, and filtered. The clear filtrate was concentrated to yield a crude product, which was purified by triturating with petroleum ether. Pure compounds (2–4) were characterized by nuclear magnetic resonance, infrared, and high-resolution mass spectroscopy. The structures of zerumbone oxime and its oxime esters have been shown in the Figure 2B.

Bottom Line: The generation of a zone of inhibition on a solid substrate indicates the bioactivity of antimicrobial peptides such as bacteriocin and enterocin.However, universal well diffusion assays for antimicrobials, irrespective of their ability to diffuse (bacteriocin and enterocin), do not exist.The ability of different zymography components to generate non-specific activities have rarely been explored in the literature.

View Article: PubMed Central - PubMed

Affiliation: Department of Protein Chemistry and Technology, Central Food Technological Research Institute (CFTRI) Mysore, India.

ABSTRACT
The generation of a zone of inhibition on a solid substrate indicates the bioactivity of antimicrobial peptides such as bacteriocin and enterocin. The indicator strain plays a significant role in bacteriocin assays. Other characteristics of bacteriocins, such as their dispersal ability and the different zymogram components, also affect bacteriocin assays. However, universal well diffusion assays for antimicrobials, irrespective of their ability to diffuse (bacteriocin and enterocin), do not exist. The ability of different zymography components to generate non-specific activities have rarely been explored in the literature. The purpose of the present work was to evaluate the impact of major factors (diffusion and rate of diffusion) in a solid substrate bioassay, and to document the adverse effects of sodium dodecyl sulfate in zymograms used to estimate the approximate molecular weight of bacteriocins.

No MeSH data available.


Related in: MedlinePlus