Enterococcus faecium isolated from Lombo, a Portuguese traditional meat product: characterisation of antibacterial compounds and factors affecting bacteriocin production

2012 ◽  
Vol 3 (4) ◽  
pp. 319-330 ◽  
Author(s):  
S.D. Todorov ◽  
L. Favaro ◽  
P. Gibbs ◽  
M. Vaz-Velho
Keyword(s):  
Mode Of Action ◽  
Enterococcus Faecium ◽  
Proteolytic Enzymes ◽  
Sodium Dodecyl ◽  
Meat Product ◽  
Maximum Activity ◽  
Tween 20 ◽  
Potential Candidate ◽  
Spoilage Bacteria ◽  
Faecium Strain

Strain ST211CH, identified as a strain of Enterococcus faecium, isolated from Lombo produced a bacteriocin that inhibited the growth of Enterococcus spp., Listeria spp., Klebsiella spp., Lactobacillus spp., Pseudomonas spp., Staphylococcus spp. and Streptococcus spp. The mode of action of the bacteriocin named as bacteriocin ST211Ch was bactericidal against Enterococcus faecalis ATCC19443. As determined by Tricine-SDS-PAGE, the approximate molecular mass of the bacteriocin was 8.0 kDa. Loss in antimicrobial activity was recorded after treatment with proteolytic enzymes. Maximum activity of bacteriocin ST211Ch was measured in broth cultures of E. faecium strain ST211Ch after 24 h; thereafter, the activity was reduced. Bacteriocin ST211Ch remained active after exposure to various temperatures and pHs, as well as to Triton X-100, Tween-80, Tween-20, sodium dodecyl sulfate, NaCl, urea and EDTA. Effect of media components on production of bacteriocin ST211Ch was also studied. On the basis of PCR reactions targeting different bacteriocin genes, i.e. enterocins, curvacins and sakacins, no evidences for the presence of these genes in the total DNA of E. faecium strain ST211Ch was obtained. The bacterium most probably produced a bacteriocin different from those mentioned above. Based on the antimicrobial spectrum, stability and mode of action of bacteriocin ST211CH, E. faecium strain ST211Ch might be considered as a potential candidate with beneficial properties for use in biopreservation to control food spoilage bacteria.

Characterization and Antimicrobial Spectrum of Bifidocin B, a Bacteriocin Produced by Bifidobacterium bifidum NCFB 1454†‡

1998 ◽  
Vol 61 (1) ◽  
pp. 47-51 ◽  
Author(s):  
ZELIHA YILDIRIM ◽  
MICHAEL G. JOHNSON
Keyword(s):  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Bifidobacterium Bifidum ◽  
Maximum Activity ◽  
Proteinase K ◽  
Food Borne ◽  
Spoilage Bacteria ◽  
Catalase Peroxidase ◽  
Protease Iv

Five strains of Bifidobacterium bifidum (ATCC 11863 and 29591, and NCFB 1453, 1454, and 1455) were examined for production of bacteriocins in MRS broth with 0.05% cysteine. Only strain NCFB 1454 excreted a bacteriocin into the broth; it was designated bifidocin B. Bifidocin B was sensitive to several proteolytic enzymes (protease IV, pronase E, protease XVII, proteinase K, trypsin, α-chymotrypsin, papain, and pepsin), but was resistant to catalase, peroxidase, lipase, lysozyme, cellulase, ribonuclease A, and amylases. It was also resistant to organic solvents such as ethyl alcohol, acetone, hexane, chloroform, methanol, and ether, and to heating at 90°C for 15, 30, and 60 min or at 121°C for 15 min. Bifidocin B remained active after storage at −20 or −70°C for 3 months and retained biological activity after exposure to pH values of 2 to 10. Bifidocin B was active against some food-borne pathogens and food spoilage bacteria such as Listeria, Enterococcus, Bacillus, Lactobacillus, Leuconostoc, and Pediococcus species but was not active against the other gram-positive and gram-negative bacteria tested. Bifidocin B was produced during exponential phase, reaching a maximum activity of 3,200 AU/ml at early stationary phase. Bifidocin B had a molecular mass of about 3.3 kDa as analyzed by Tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis.

Purification and characterization of enterocin MC13 produced by a potential aquaculture probiontEnterococcus faeciumMC13 isolated from the gut ofMugil cephalus

2011 ◽  
Vol 57 (12) ◽  
pp. 993-1001 ◽  
Author(s):  
R. Satish Kumar ◽  
P. Kanmani ◽  
N. Yuvaraj ◽  
K.A. Paari ◽  
V. Pattukumar ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Enterococcus Faecium ◽  
Vibrio Vulnificus ◽  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Probiotic Bacterium ◽  
Mass Spectrometry Analysis ◽  
Native Page ◽  
Spectrometry Analysis

A bacteriocin producer strain MC13 was isolated from the gut of Mugil cephalus (grey mullet) and identified as Enterococcus faecium . The bacteriocin of E. faecium MC13 was purified to homogeneity, as confirmed by Tricine sodium dodecyl sulphate – polyacrylamide gel electrophoresis (SDS–PAGE). Reverse-phase high-performance liquid chromatography (HPLC) analysis showed a single active fraction eluted at 26 min, and matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry analysis showed the molecular mass to be 2.148 kDa. The clear zone in native PAGE corresponding to enterocin MC13 band further substantiated its molecular mass. A dialyzed sample (semicrude preparation) of enterocin MC13 was broad spectrum in its action and inhibited important seafood-borne pathogens: Listeria monocytogenes , Vibrio parahaemolyticus , and Vibrio vulnificus . This antibacterial substance was sensitive to proteolytic enzymes: trypsin, protease, and chymotrypsin but insensitive to catalase and lipase, confirming that inhibition was due to the proteinaceous molecule, i.e., bacteriocin, and not due to hydrogen peroxide. Enterocin MC13 tolerated heat treatment (up to 90 °C for 20 min). Enterococcus faecium MC13 was effective in bile salt tolerance, acid tolerance, and adhesion to the HT-29 cell line. These properties reveal the potential of E. faecium MC13 to be a probiotic bacterium. Enterococcus faecium MC13 could be used as potential fish probiotic against pathogens such as V. parahaemolyticus, Vibrio harveyi , and Aeromonas hydrophila in fisheries. Also, this could be a valuable seafood biopreservative against L. monocytogenes.

scholarly journals Purification and Characterization of an Extracellular Protease from Penicillium chrysogenum Pg222 Active against Meat Proteins

2002 ◽  
Vol 68 (7) ◽  
pp. 3532-3536 ◽  
Author(s):  
María J. Benito ◽  
Mar Rodríguez ◽  
Félix Núñez ◽  
Miguel A. Asensio ◽  
María E. Bermúdez ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Penicillium Chrysogenum ◽  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Maximum Activity ◽  
Extracellular Protease ◽  
Collagenolytic Activity ◽  
Sds Page

ABSTRACT An extracellular protease from Penicillium chrysogenum (Pg222) isolated from dry-cured ham has been purified. The purification procedure involved several steps: ammonium sulfate precipitation, ion-exchange chromatography, filtration, and separation by high-performance liquid chromatography. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and gel filtration, the purified fraction showed a molecular mass of about 35 kDa. The hydrolytic properties of the purified enzyme (EPg222) on extracted pork myofibrillar proteins under several conditions were evaluated by SDS-PAGE. EPg222 showed activity in the range of 10 to 60°C in temperature, 0 to 3 M NaCl, and pH 5 to 7, with maximum activity at pH 6, 45°C, and 0.25 M NaCl. Under these conditions the enzyme was most active against tropomyosin, actin, and myosin. EPg222 showed collagenolytic activity but did not hydrolyze myoglobin. EPg222 showed higher activity than other proteolytic enzymes like papain, trypsin, and Aspergillus oryzae protease. The N-terminal amino acid sequence was determined and was found to be Glu-Asn-Pro-Leu-Gln-Pro-Asn-Ala-Pro-Ser-Trp. This partial amino acid sequence revealed a 55% homology with serine proteases from Penicillium citrinum. The activity of this novel protease may be of interest in ripening and generating the flavor of dry-cured meat products.

Isolation and Purification of Enterocin EL1, a Bacteriocin Produced by a Strain of Enterococcus faecium†

1995 ◽  
Vol 58 (8) ◽  
pp. 890-898 ◽  
Author(s):  
WANDA J. LYON ◽  
DENNIS G. OLSON ◽  
ELSA A. MURANO
Keyword(s):  
Enterococcus Faecium ◽  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Producer Strain ◽  
Isolation And Purification ◽  
Heat Stable ◽  
Listeria Ivanovii ◽  
Extraction Buffer ◽  
Approximate Molecular Weight

A meat isolate, identified as Enterococcus faecium L1, was found to produce a bacteriocin designated enterocin EL1 Enterocin EL1 was active against a narrow spectrum of microorganisms, inhibiting all tested strains of Listeria. Identification of the producer strain was determined phenotypically by biochemical and morphological tests. Enterocin EL1 was heat stable, sensitive to several proteolytic enzymes, and stable from pH 2 to 11. Adsorption of the bacteriocin to producer cells was dependent on ionic interaction of the bacteriocin and the cell surface at various pHs. By changing the pH of the extraction buffer, enterocin EL1 was extracted from E. faecium L1 cells in a concentrated form. Enterocin EL1 isolated by cell extraction was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a protein with an approximate molecular weight of 2,300. Partially purified enterocin EL1 added to sensitive cells of Listeria ivanovii was bactericidal; however, the bacteriocin did not inhibit the producer strain L1.

scholarly journals Actividad enzimática de proteasas de Cyprinus carpio (Cypriniformes: Cyprinidae) extraídas de una laguna contaminada en México

2017 ◽  
Vol 65 (2) ◽  
Author(s):  
Arisaí Hernández-Sámano ◽  
Xochitl Guzmán-García ◽  
Raquel García-Barrientos ◽  
Isabel Guerrero-Legarreta
Keyword(s):  
Proteolytic Activity ◽  
Cyprinus Carpio ◽  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Waste Water Treatment ◽  
Sodium Dodecyl ◽  
Maximum Activity ◽  
Protease Production ◽  
Human Consumption ◽  
Alkaline Ph

Common carp (Cyprinus carpio) is an aquatic organism of commercial value able to survive in polluted environments; carps contain proteolytic enzymes of physiological importance and potential industrial application. The objective of this work was partially purify and study the proteolytic activity at different pH of carp proteases living in a polluted environment. Three carps were captured in different zones of Zumpango polluted lagoon (Mexico) at 1 m of maximum deep. Protease crude extracts were obtained from dorsal muscle by aqueous extraction and fractionated by 20 %, 50 %, 80 %-saturated (NH4)2SO4. Fractions extracted with 50 % and 80 %-saturated (NH4)2SO4 were selected for their high proteolytic activity and concentrated by ultrafiltration through 100 kDa molecular weight cutoff membranes and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The crude proteolytic extract had significantly higher activity (19.7 - 20.3 U / mg) at pH 2, 5, and 7 (P < 0.001). Fractions obtained with 20 %, 50 % and 80 % - saturated (NH4)2SO4 showed peak activity at pH 5 (2.8 U / mg) and pH 6 (2.2 U / mg); pH 6 (4.3 U / mg) and pH 3 - 4 (3.6 - 3.7 U / mg); pH 3 (10.8 U / mg) and pH 10 (10.6 U / mg); respectively. Subfractions of < 100 kDa, obtained with 50 % and 80 %-saturated (NH4)2SO4, had peak proteolytic activity at alkaline pH. A < 100 kDa fraction, obtained with 80 %-saturated (NH4)2SO4, had the highest proteolytic activity (37.3 - 43.7 U / mg) at pH 8 - 10, purification factor of 3 and 19.1 % recovery. Thirteen proteins between 9.8 to 104.8 kDa were identified in the crude extract. Peak protein concentration was observed for 31 - 33 and 39 - 41 kDa, suggesting the possibility predominance of serine- and aspartyl- proteases, respectively. We suggest this protease with maximum activity at alkaline pH is related to the adaptation of C. carpio to polluted waters with high pH. Although unsuitable for human consumption, these organisms can be a source of protease production aimed to several uses as in the industry and waste water treatment among others.

scholarly journals Production and characterization of bacteriocin produced by lactic acid bacteria isolated from rusip

2013 ◽  
Vol 8 (1) ◽  
pp. 13
Author(s):  
Arifah Kusumarwati ◽  
Ninoek Indriati ◽  
Irma Hermana
Keyword(s):  
Lactic Acid ◽  
High Temperature ◽  
Lactic Acid Bacteria ◽  
Proteolytic Enzymes ◽  
Sodium Dodecyl ◽  
Tween 80 ◽  
Proteinase K ◽  
Tween 20 ◽  
Bacterial Inhibition ◽  
Lauryl Sarcosine

Research was conducted to produce and characterize bacteriocin produced by lactic acid bacteria (LAB) isolated from rusip, a traditional Bangkanese fermented fish product. Experiment was started by isolation of lactic acid bacteria from rusip, followed by screening to obtain the best isolate which has the highest bacterial inhibition activity. The selected isolate was then identified and used to produce crude bacteriocin. The crude bacteriocin was characterized through its stability in high temperature and proteolytic enzymes, inhibitory spectrum, pH sensitivity and effect of surfactants. The result showed that CN1.10a isolate which was identified as Lactococcus lactis subsp lactis has the highest bacterial inhibition activities; therefore it was selected to produce crude bacteriocin. The bacteriocin produced was heat stable, sensitive to proteolytic enzymes i.e. proteinase-K and papain but not to RNase. It inhibited Escherichia coli, Listeria monocytogenes and Lactobacillus plantarum. It stable at pH 2.0 to 6.0. Among surfactans used sodium dodecyl sulphate (SDS), lauryl sarcosine and EDTA were able to stimulate bacteriocin production, while the production were strongly inhibited by Tween 20, Tween 80, Triton X-100 and urea. Based on the above characteristic, the bacteriocin was suitable to be used as a preservative of food which has to be processed at high temperature.

scholarly journals Partial purification and biochemical characterization of a new highly acidic NYSO laccase from Alcaligenes faecalis

2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Soad A. Abdelgalil ◽  
Ahmad R. Attia ◽  
Reyed M. Reyed ◽  
Nadia A. Soliman
Keyword(s):  
Enzyme Activity ◽  
Sodium Dodecyl ◽  
Alcaligenes Faecalis ◽  
Maximum Activity ◽  
Sodium Oxalate ◽  
Partial Purification ◽  
Bromophenol Blue ◽  
Bromocresol Purple ◽  
Laccase Enzyme

Abstract Background Due to the multitude industrial applications of ligninolytic enzymes, their demands are increasing. Partial purification and intensive characterization of contemporary highly acidic laccase enzyme produced by an Egyptian local isolate designated Alcaligenes faecalis NYSO were studied in the present investigation. Results Alcaligenes faecalis NYSO laccase has been partially purified and intensively biochemically characterized. It was noticed that 40–60% ammonium sulfate saturation showed maximum activity. A protein band with an apparent molecular mass of ~ 50 kDa related to NYSO laccase was identified through SDS-PAGE and zymography. The partially purified enzyme exhibited maximum activity at 55 °C and pH suboptimal (2.5–5.0). Remarkable activation for enzyme activity was recognized after 10-min exposure to temperatures (T) 50, 60, and 70 °C; time elongation caused inactivation, where ~ 50% of activity was lost after a 7-h exposure to 60 °C. Some metal ions Cu2+, Zn2+, Co2+, Ni2+, Mn2+, Cd2+, Cr2+, and Mg2+ caused strong stimulation for enzyme activity, but Fe2+ and Hg2+ reduced the activity. One millimolar of chelating agents [ethylenediamine tetraacetic acid (EDTA), sodium citrate, and sodium oxalate] caused strong activation for enzyme activity. Sodium dodecyl sulfate (SDS), cysteine-HCl, dithiothreitol (DTT), β-mercaptoethanol, thioglycolic acid, and sodium azide caused strong inhibition for NYSO laccase activity even at low concentration. One millimolar of urea, imidazole, kojic acid, phenylmethylsulfonyl fluoride (PMSF), H2O2, and Triton X-100 caused activation. The partially purified NYSO laccase had decolorization activity towards different dyes such as congo red, crystal violet, methylene blue, fast green, basic fuchsin, bromophenol blue, malachite green, bromocresol purple eriochrome black T, and Coomassie Brilliant Blue R-250 with various degree of degradation. Also, it had a vast range of substrate specificity including lignin, but with high affinity towards p-anisidine. Conclusion The promising properties of the newly studied laccase enzyme from Alcaligenes faecalis NYSO strain would support several industries such as textile, food, and paper and open the possibility for commercial use in water treatment. It will also open the door to new applications due to its ligninolytic properties in the near future.

scholarly journals Emergence of a daptomycin-non-susceptible Enterococcus faecium strain that encodes mutations in DNA repair genes after high-dose daptomycin therapy

2016 ◽  
Vol 9 (1) ◽  
Author(s):  
Takashi Matono ◽  
Kayoko Hayakawa ◽  
Risen Hirai ◽  
Akira Tanimura ◽  
Kei Yamamoto ◽  
...  
Keyword(s):  
Dna Repair ◽  
Enterococcus Faecium ◽  
High Dose ◽  
Dna Repair Genes ◽  
Repair Genes ◽  
Faecium Strain

scholarly journals Draft Genome Sequence of Probiotic Enterococcus faecium Strain L-3

2016 ◽  
Vol 4 (1) ◽  
Author(s):  
Alena Karaseva ◽  
Anna Tsapieva ◽  
Justin Pachebat ◽  
Alexander Suvorov
Keyword(s):  
Genome Sequence ◽  
Russian Federation ◽  
Enterococcus Faecium ◽  
Draft Genome ◽  
Bacteriocin Production ◽  
Draft Genome Sequence ◽  
Bacteriocin Producer ◽  
Probiotic Preparation ◽  
The Russian Federation ◽  
Faecium Strain

We report here the draft genome sequence of the bacteriocin producer Enterococcus faecium strain L-3, isolated from a probiotic preparation, Laminolact, which is widely used in the Russian Federation. The draft genome sequence is composed of 74 contigs for a total of 2,643,001 bp, with 2,646 coding genes. Five clusters for bacteriocin production were found.

scholarly journals Mucin biosynthesis. Properties of a bovine tracheal mucin β-6-N-acetylglucosaminyltransferase

1985 ◽  
Vol 227 (2) ◽  
pp. 405-412 ◽  
Author(s):  
P W Cheng ◽  
W E Wingert ◽  
M R Little ◽  
R Wei
Keyword(s):  
Enzyme Activity ◽  
Freezing Point ◽  
Cetylpyridinium Chloride ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Gas Chromatography Mass Spectrometry ◽  
Tween 20 ◽  
Group A ◽  
Michaelis Constants ◽  
Triton X

We have characterized a bovine tracheal mucin beta-6-N-acetylglucosaminyltransferase that catalyses the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to the C-6 of the N-acetylgalactosamine residue of galactosyl-β 1→3-N-acetylgalactosamine. Optimal enzyme activity was obtained between pH 7.5-8.5, at 5mM-MnCl2, and at 0.06-0.08% (v/v) Triton X-100 (or Nonidet P-40), or 0.5-5.0% (v/v) Tween 20. Ba2+, Mg2+ and Ca2+ could partially replace Mn2+, but Co2+, Fe2+, Cd2+ and Zn2+ could not. Sodium dodecyl sulphate, cetylpyridinium chloride, sodium deoxycholate, octyl beta-D-glucoside, digitonin and alkyl alcohols were less effective in enhancing enzyme activity, and dimethyl sulphoxide was ineffective. The apparent Michaelis constants were 1.25 mM for UDP-N-acetylglucosamine, 0.94-3.34 mM for freezing-point-depressing glycoprotein and 0.19 mM for periodate-treated blood-group-A porcine submaxillary mucin. Asialo ovine submaxillary mucin could not serve as the glycosyl acceptor. The structure of the 14C-labelled oligosaccharide obtained by alkaline-borohydride treatment of the product was identified as Gal beta 1→3(Glc-NAc beta 1→6)N-acetylgalactosaminitol by beta-hexosaminidase treatment, gas chromatography-mass spectrometry and 1H-n.m.r. (270 MHz) analysis. The enzyme is important in the regulation of mucin oligosaccharide biosynthesis.

Sign in / Sign up

Export Citation Format

Share Document