Purification and characterization of enterocin 4, a bacteriocin produced by Enterococcus faecalis INIA 4.
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ABSTRACT:
SUBMITTER: Joosten HM
PROVIDER: S-EPMC168244 | biostudies-other | 1996 Nov
REPOSITORIES: biostudies-other
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Project description:Lactic acid bacteria exhibiting activity against the gram-positive bacterium Bacillus subtilis were isolated from rice bran. One of the isolates, identified as Enterococcus faecalis RJ-11, exhibited a wide spectrum of growth inhibition with various gram-positive bacteria. A bacteriocin purified from culture fluid, designated enterocin RJ-11, was heat stable and was not sensitive to acid and alkaline conditions, but it was sensitive to several proteolytic enzymes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that enterocin RJ-11 had a molecular weight of 5,000 in its monomeric form. The amino acid sequence determined for purified enterocin RJ-11 exhibited high levels of similarity to the sequences of enterocins produced by Enterococcus faecium.
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Project description:The structural genes for the two-peptide bacteriocin enterocin 1071 (Ent1071) in Enterococcus faecalis FAIR-E 309 were cloned. DNA sequence analysis showed that the enterocin 1071A (Ent1071A) peptide of strain FAIR-E 309 differed by two amino acids from the Ent1071A reported for E. faecalis BFE 1071 (E. Balla, L. M. T. Dicks, M. Du Toit, M. J. van der Merwe, and W. H. Holzapfel, Appl. Environ. Microbiol. 66:1298-1304, 2000), while the Ent1071B gene encoded identical peptides in these strains. However, resequencing of ent1071A from E. faecalis BFE 1071 showed that the Ent1071A peptide sequence reported previously was incorrect in two amino acids. Also, ent1071B in E. faecalis FAIR-E 309 encoded a prepeptide that was three amino acids shorter than that previously reported for E. faecalis BFE 1071 Ent1071B. A presumptive immunity gene (eni1071) was located downstream of the bacteriocin structural genes. This gene was cloned into the heterologous host E. faecalis ATCC 19433 and was shown to confer immunity. A truncated ABC transporter gene was located upstream of the Ent1071 structural genes.
| S-EPMC127528 | biostudies-literature
Project description:Use of bacteriocins in food preservation has received great attention in recent years. The goal of this study is to characterize enterocin RM6 from Enterococcus faecalis OSY-RM6 and investigate its efficacy against Listeria monocytogenes in cottage cheese. Enterocin RM6 was purified from E. faecalis culture supernatant using ion exchange column, multiple C18-silica cartridges, followed by reverse-phase high-performance liquid chromatography. The molecular weight of enterocin RM6 is 7145.0823 as determined by mass spectrometry (MS). Tandem mass spectrometry (MS/MS) analysis revealed that enterocin RM6 is a 70-residue cyclic peptide with a head-to-tail linkage between methionine and tryptophan residues. The peptide sequence of enterocin RM6 was further confirmed by sequencing the structural gene of the peptide. Enterocin RM6 is active against Gram-positive bacteria, including L. monocytogenes, Bacillus cereus, and methicillin-resistant Staphylococcus aureus (MRSA). Enterocin RM6 (final concentration in cottage cheese, 80 AU/mL) caused a 4-log reduction in population of L. monocytogenes inoculated in cottage cheese within 30 min of treatment. Therefore, enterocin RM6 has potential applications as a potent antimicrobial peptide against foodborne pathogens in food.
| S-EPMC3697273 | biostudies-literature
Project description:Enterococcus faecalis WHE 96, a strain isolated from soft cheese based on its anti-Listeria activity, produced a 5,494-Da bacteriocin that was purified to homogeneity by ultrafiltration and cation-exchange and reversed-phase chromatographies. The amino acid sequence of this bacteriocin, named enterocin 96, was determined by Edman degradation, and its structural gene was sequenced, revealing a double-glycine leader peptide. After a comparison with other bacteriocins, it was shown that enterocin 96 was a new class II bacteriocin that showed very little similarity with known structures. Enterocin 96 was indeed a new bacteriocin belonging to class II bacteriocins. The activity spectrum of enterocin 96 covered a wide range of bacteria, with strong activity against most gram-positive strains but very little or no activity against gram-negative strains.
| S-EPMC2704840 | biostudies-literature
Project description:BackgroundBecause Candida albicans is resistant to several antifungal antibiotics, there is a need to identify other less toxic natural products, particularly antimicrobial proteins, peptides or bacteriocin like inhibitory substances. An attempt has been made to purify and characterise an anti-Candida compound produced by Enterococcus faecalis.ResultsAn anti-Candida protein (ACP) produced by E. faecalis active against 8 C. albicans strains was characterised and partially purified. The ACP showed a broad-spectrum activity against multidrug resistant C. albicans MTCC 183, MTCC 7315, MTCC 3958, NCIM 3557, NCIM 3471 and DI. It was completely inactivated by treatment with proteinase K and partially by pronase E.The ACP retained biological stability after heat-treatment at 90°C for 20 min, maintained activity over a pH range 6-10, and remained active after treatment with α-amylase, lipase, organic solvents, and detergents. The antimicrobial activity of the E. faecalis strain was found exclusively in the extracellular filtrate produced in the late logarithmic growth phase. The highest activity (1600 AU mL-1) against C. albicans MTCC 183 was recorded at 48 h of incubation, and activity decreased thereafter. The peptide showed very low haemagglutination and haemolytic activities against human red blood cells. The antimicrobial substance was purified by salt-fractionation and chromatography.Partially purified ACP had a molecular weight of approximately 43 KDa in Tricine-PAGE analysis. The 12 amino acid N terminal sequence was obtained by Edman degradation. The peptide was de novo sequenced by ESI-MS, and the deduced combined sequence when compared to other bacteriocins and antimicrobial peptide had no significant sequence similarity.ConclusionsThe inhibitory activity of the test strain is due to the synthesis of an antimicrobial protein. To our knowledge, this is the first report on the isolation of a promising non-haemolytic anti-Candida protein from E. faecalis that might be used to treat candidiasis especially in immunocompromised patients.
| S-EPMC3585888 | biostudies-literature
Project description:The Enterococcus faecalis class IIa bacteriocin MC4-1 encoded by the sex pheromone-responding, multiple-antibiotic resistance plasmid pAMS1 exhibits "siblicidal" (sibling-killing) activity under certain conditions. Stabs of plasmid-containing cells on solid medium containing lawns of bacteria of the same (plasmid-containing) strain give rise to zones of inhibition. If the plasmid-containing host also produces gelatinase, bacteriocin cannot be detected.
| S-EPMC2681816 | biostudies-literature
Project description:In this study, the antimicrobial activity of a bacteriocin produced by Enterococcus faecalis KT11, isolated from traditional Kargı Tulum cheese, was determined, and bacteriocin KT11 was partially characterized. The results showed that bacteriocin KT11 was antagonistically effective against various Gram-positive and Gram-negative test bacteria, including vancomycin- and/or methicillin-resistant bacteria. The activity of bacteriocin KT11 was completely abolished after treatment with proteolytic enzymes (proteinase K, α-chymotrypsin, protease and trypsin), which demonstrates the proteinaceous nature of this bacteriocin. Additionally, bacteriocin KT11 remained stable at pH values ranging from 2 to 11 and after autoclaving at 121℃ for 30 min. In addition, the activity of bacteriocin KT11 was stable after treatment with several surfactants (EDTA, SDS, Triton X-100, Tween 80 and urea) and organic solvents (chloroform, propanol, methanol, ethyl alcohol, acetone, hexane and ethyl ether). Cell-free supernatant of E. faecalis KT11 was subjected to ammonium sulfate precipitation and then desalted by using a 3.5-kDa cut-off dialysis membrane. The bacteriocin activity was determined to be 711 AU/mL in the dialysate. After tricine-SDS-PAGE analysis, one peptide band, which had a molecular weight of ~3.5 kDa, exhibited antimicrobial activity. Because the bacteriocin KT11, isolated from E. faecalis KT11, exhibits a broad antimicrobial spectrum, heat stability and stability over a wide pH range, this bacteriocin can be used as a potential bio-preservative in foods. Additionally, bacteriocin KT11 alone or in combination with conventional antibiotics may provide a therapeutic option for the treatment of multidrug-resistant clinical pathogens after further in vivo studies.
| S-EPMC6238024 | biostudies-literature
Project description:A dramatic increase in bacterial resistance towards currently available antibiotics has raised worldwide concerns for public health. Therefore, antimicrobial peptides (AMPs) have emerged as a promisingly new group of therapeutic agents for managing infectious diseases. The present investigation focusses on the isolation and purification of a novel bacteriocin from an indigenous sample of cow milk and it's mode of action. The bacteriocin was isolated from Weissella confusa A3 that was isolated from the sample and was shown to have inhibitory activity towards pathogenic bacteria namely Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa and Micrococcus luteus. The bacteriocin was shown to be heat stable and functioned well at low pH (2 to 6). Reduction of activity was shown after treatment with proteinase K, trypsin and peptidase that confirmed the proteinaceous nature of the compound. MALDI-TOF analysis of the sample gave a mass approximating 2.7 kDa. The membrane of the bacteria was disrupted by the bacteriocin causing SYTOX® green dye to enter the cell and bind to the bacterial DNA giving fluorescence signal. Bacterial cell treated with the bacteriocin also showed significant morphological changes under transmission electron microscope. No virulence and disease related genes can be detected from the genome of the strain.
| S-EPMC4608715 | biostudies-literature
Project description:A bacteriocin produced by a vaginal isolate of Enterococcus faecium strain 62-6, designated enterocin 62-6, was characterized following purification and DNA sequence analysis and compared to previously described bacteriocins. Enterocin 62-6 was isolated from brain heart infusion (BHI) culture supernatants using ammonium sulfate precipitation followed by elution from a Sepharose cation exchange column using a continuous salt gradient (0.1-0.7 M NaCl). SDS-PAGE of an active column fraction resulted in an electrophoretically pure protein, which corresponded to the growth inhibition of the sensitive Lactobacillus indicator strain in the gel overlay assay. Purified enterocin 62-6 was shown to be heat- and pH-stable, and sensitive to the proteolytic enzymes alpha-chymotrypsin and pepsin. Results from mass spectrometry suggested that it comprised two peptides of 5206 and 5219+/-1 Da, which was confirmed by DNA sequence analysis. The characteristics of enterocin 62-6 as a small, heat- and pH-stable, cationic, hydrophobic, two-peptide, plasmid-borne bacteriocin, with an inhibitory spectrum against a broad range of Gram-positive but not Gram-negative bacteria, were consistent with its classification as a class IIc bacteriocin. Furthermore, its wide spectrum of growth inhibitory activity against Gram-positive bacteria of vaginal origin including lactobacilli, and stability under the acidic conditions of the vagina, are consistent with our hypothesis that it could have potential significance in disrupting the ecology of the vaginal tract and pave the way for the establishment of the abnormal microbiota associated with the vaginal syndrome bacterial vaginosis. This is the first class IIc bacteriocin produced by a strain of E. faecium of vaginal origin to be characterized.
| S-EPMC2705065 | biostudies-literature
Project description:New anticancer agents are continually needed because cancerous cells continue to evolve resistance to the currently available chemotherapeutic agents. The aim of the present study was to screen, purify and characterize a hepatotoxic bacteriocin from Enterococcus species. The production of bacteriocin from the Enterococcus isolates was achieved based on their antibacterial activity against indicator reference strains. Enterococcus isolates showed a broad spectrum of antibacterial activity by forming inhibition zones with diameters ranged between 12 and 29 mm. The most potent bacteriocin producing strain was molecularly identified as Enterococcus thailandicus. The crude extracted bacteriocin was purified by cation exchange and size exclusion chromatography that resulted in 83 fractions. Among them, 18 factions were considered as bacteriocins based on their positive antibacterial effects. The anticancer effects of the purified bacteriocins were tested against HepG2 cell line. The most promising enterocin (LNS18) showed the highest anticancer effects against HepG2 cells (with 75.24% cellular inhibition percentages), with IC50 value 15.643 μM and without any significant cytotoxic effects on normal fibroblast cells (BJ ATCC® CRL-2522™). The mode of anticancer action of enterocin LNS18 against HepG2 cells could be explained by its efficacy to induce cellular ROS, decrease HepG2 CD markers and arrest cells in G0 phase. Amino acid sequence of enterocin LNS18 was determined and the deduced peptide of the structural gene showed 86 amino acids that shared 94.7% identity with enterocin NKR-5-3B from E. faecium. Enterocin LNS18 consisted of 6 α-helices; 5 circular and one linear. Model-template alignment constructed between enterocin LNS18 and NKR-5-3B revealed 95.31% identity. The predicted 3D homology model of LNS18, after circularization and release of 22 amino acids, showed the formation of a bond between Leu23 and Trp86 amino acid residues at the site of circularization. Furthermore, areas of positive charges were due to the presence of 6 lysine residues resulting in a net positive charge of +4 on the bacteriocin surface. Based on the above mentioned results, our characterized bacteriocin is a promising agent to target liver cancer without any significant toxic effects on normal cell lines.
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