Purification, amino acid sequence and characterization of the class IIa bacteriocin weissellin A, produced by Weissella paramesenteroides DX

Bioresource Technology 102 (2011) 6730–6734

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Purification, amino acid sequence and characterization of the class IIa bacteriocin weissellin A, produced by Weissella paramesenteroides DX Maria Papagianni a,⇑, Emmanuel M. Papamichael b a b

Department of Hygiene and Technology of Food of Animal Origin, School of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki 54006, Greece Department of Chemistry, University of Ioannina, Ioannina 45-110, Greece

a r t i c l e

i n f o

Article history: Received 31 January 2011 Received in revised form 30 March 2011 Accepted 30 March 2011 Available online 3 April 2011 Keywords: Weissela paramesenteroides Weissellin A Bacteriocin Amino acid sequence Biopreservative

a b s t r a c t Weissella paramesenteroides DX has been shown to produce a 4450-Da class IIa bacteriocin, weissellin A, composed of 43 amino acids with the sequence KNYGNGVYCNKHKCSVDWATFSANIANNSVAMAGLTGGNAGN. The bacteriocin shares 68% similarity with leucocin C from Leuconostoc mesenteroides. Computational analyses predict that the bacteriocin is a hydrophobic molecule with a beta-sheet type conformation. Weissellin A exhibited various levels of activity against all gram-positive bacteria tested, but was not active against Salmonella enterica Enteritidis. The antimicrobial activity was not associated with target-cell lysis. The bacteriocin retained activity after exposure to 121 °C for 60 min or to 20 °C for 6 months, and to pH 2.0–10.0. It was not sensitive to trypsin, a-chymotrypsin, pepsin and papain, but was inactivated by proteinase K. At a dissolved oxygen concentration of 50%, weissellin A was produced with growth-associated kinetics. The properties of weissellin A make this bacteriocin a potentially suitable agent for food and feed preservation. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Many bacteria produce ribosomally synthesized antimicrobial peptides (bacteriocins) with diverse structures, molecular masses, biochemical characteristics, spectra of activities and sequence homologies (Papagianni, 2003). These are usually membrane-permeabilizing cationic peptides with less than 50 amino acid residues (Papagianni, 2003). Depending on the presence of modified amino acid residues, the bacteriocins are classified as class I (modification) or class II (no modifications). One group of class II bacteriocins, the class IIa of ‘‘pediocin-like’’ bacteriocins, are gaining increasing interest because of their strong antilisterial activity, and heat and pH stability (Fimland et al., 2005). A considerable number of pediocin-like bacteriocins have been reported in the literature but only a few of them have been isolated and thoroughly characterized (Hastings et al., 1991; Héchard et al., 1992; Henderson et al., 1992; Holck et al., 1992; Metivier et al., 1998; Tichaczek et al., 1992). Elucidation of their amino acid sequences and the genetic determinants have provided valuable insights into the common characteristics of the subclass, e.g. the -YGNGV- motif in the N-terminus, the structure/function relationships and the modes of action of these compounds. In this study we report the complete amino acid sequence and describe the characteristics of a class IIa bacteriocin, designated ⇑ Corresponding author. Tel.: +30 2310 999804; fax: +30 2310 999829. E-mail address: [email protected] (M. Papagianni). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.03.106

as weissellin A, which is produced by Weissella paramesenteroides DX isolated from a traditional Greek sausage. 2. Methods 2.1. Bacterial strains and growth conditions The sausage isolate DX used in this study, was identified as W. paramesenteroides phylogenetically by comparison of a 708base pair 16SrDNA sequence using BLAST (results not shown here). Listeria inocua ATCC BAA-680D was used as indicator microorganism for bacteriocin activity, and the strains listed in Table 1 were used to delineate the antimicrobial spectrum of the bacteriocin. 2.2. Bacteriocin production in batch fermentation The isolated strain of W. paramesenteroides DX was maintained in M17 + 2% glucose agar (prepared with addition of 1.5% granulated agar to broth media) and grown statically in M17 + 2% glucose broth at 30 °C for 60 h (M17 was from Sharlau Microbiology, Spain). Mid-logarithmic phase cultures (OD600 = 1.2) were used as inoculums (2% v/v) for cultivation in a BIOFLO 110 New Brunswick Scientific stirred tank reactor with a working volume of 2L. The agitation system consisted of two 6-bladed Rushton-type impellers (52 mm), operated at the stirrer speed of 150 rpm. The temperature was maintained at 30 °C. The culture pH at inoculation time was 6.0 and no pH control was applied during cultivation. The

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M. Papagianni, E.M. Papamichael / Bioresource Technology 102 (2011) 6730–6734 Table 1 Inhibition spectrum of culture supernatant of W. paramesenteroides DX containing weissellin A.

a

Indicator organism

Medium

Incubation temperature

Aeration

Sensitivity to weissellin Aa

Bacillus cereus LMG13569 Clostridium sporogenes NCTC533 Clostridium thiaminolyticum ATCC15579 Enterococcus faecalis NCTC8176 Lactobacillus brevis ATCC8287 Lactobacillus bulgaricus LMG13551 Lactobacillus casei ATCC344 Lactobacillus curvatus ATCC51436 Lactobacillus jensenii ATCC25258 Lactobacillus plantarum CECT220 Lactobacillus sakei CECT906T Lactococcus lactis LM0230 L. lactis ATCC11454 L. lactis IL1403 L. lactis subsp. cremoris MC1363 Leuconostoc mesenteroides ATCC19254 Listeria inocua ATCC BAA-680D Listeria monocytogenes ATCC19111 Micrococcus luteus CECT241 Pediococcus acidilactici ATCC25740 Pediococcus pentosaceus ATCC 33316 P. pentosaceus LMG13560 Salmonella enteritidis ATCC13076 Staphylococcus carnosus LMG13564

BHI RCM RCM MRS MRS MRS MRS MRS MRS MRS MRS MRS MRS MRS MRS MRS BHI BHI NB MRS MRS MRS SS BHI

37 37 37 37 37 37 37 30 37 30 30 30 30 30 30 25 30 30 30 30 30 30 25 37

Aerobic Anaerobic Anaerobic Microaerophilic Microaerophilic Microaerophilic Microaerophilic Microaerophilic Microaerophilic Microaerophilic Microaerophilic Microaerophilic Microaerophilic Microaerophilic Microaerophilic Microaerophilic Microaerophilic Microaerophilic Aerobic Microaerophilic Microaerophilic Microaerophilic Microaerophilic Microaerophilic

85 100 78 75 65 65 75 10 70 70 20 75 20 72 15 20 92 100 100 55 50 50 0 92

Mean values of at least three experiments, expressed as a percentage of the inhibition zone diameter of the standard test strain Micrococcus flavus ATTC 400.

dissolved oxygen tension (DOT) was maintained at 50% by sparging the reactor with a mixture of N2 and atmospheric air, adjusted by using two mass flow controllers, and the DOT was kept constant by feedback regulation. Samples were taken every 2 h. Runs were carried out in triplicate and repeated if experimental variation exceeded 10%. Fermentation kinetic parameters were calculated by numerical differentiation in MS Excel. 2.3. Determination of biomass, lactate, and glucose concentrations Cell dry weight was determined by filtering 5 ml of broth through nitrocellulose filters (pore size, 0.45 lm, dried in a microwave oven at 150 W for 15 min), washing twice with 10 ml of distilled water and dried in a microwave oven (150 W, 15 min). Lactic acid concentration was determined with the EnzyPlus D/L Lactic Acid kit by Diffchamb AB (Diffchamb, Sweden). Glucose was determined using a glucose oxidase/peroxidase assay kit by Sigma Aldrich (Sigma Aldrich Co., UK). 2.4. Determination of bacteriocin activity Antimicrobial compound attached to cell walls was removed by using the cell adsorption–desorption method for bacteriocin extraction as was described by Yang et al. (1992) with minor modifications. The pH of samples of W. paramesenteroides DX cultures was adjusted to 6.5 by addition of 1 N NaOH. The cultures were stirred at 150 rpm for 3 h at room temperature, the cells were collected by centrifugation at 10,000g (4 °C) for 30 min, washed twice with 100 ml of 5 mM sodium phosphate buffer (pH 6.5), collected again by centrifugation at 10,000g (4 °C) for 30 min and finally resuspended in 50 ml of 100 mM NaCl and HCl solution (pH 2.0). The cell suspension was stirred for 12 h at 4 °C, then subjected to centrifugation at 10,000g (4 °C) for 15 min. The supernatant was filtered through 0.22 lm pore-size filters, concentrated to 0.1 volume by polyethylene glycol dialysis (PEG, Sigma, Mr 20,000) and again filter sterilized. This material was designated as ‘‘crude bacteriocin preparation’’ and was frozen at 20 °C when not used immediately. For activity determinations, serial twofold

dilutions of the crude bacteriocin extract were made in sterile distilled water, the pH of samples was adjusted to 6.5 with 1 N NaOH and 30 ll were delivered into wells in M17 + 2% glucose agar plates previously seeded with 10 ll of a fresh overnight culture of Micrococcus flavus ATCC 400 (106 cells/ml) and incubated at 30 °C. The sample titer was defined as the reciprocal of the highest dilution at which activity was still obtained and was expressed in activity units (AU)/ml (Papagianni et al., 2006). Bacterial strains and growth conditions for the activity spectrum determination of the crude bacteriocin preparation are listed in Table 1. For each indicator organism the appropriate solid medium was used. Anaerobic and microaerophilic growth was insured by growing the cultures in an incubator with controlled CO2 partial pressure. 30 ll aliquots of cell-free culture supernatant fluid (pH adjusted at 6.5) were spotted on the appropriate solid media (1.5% w/v agar) seeded with a fresh culture of test cells, at logarithmic phase of growth. Plates were incubated at the optimum conditions for each test microorganism (Table 1) and examined for the presence of clear zones of inhibition. Strain sensitivities were expressed as a percentage (based on zone diameter measurements) of M. flavus ATCC 400 sensitivity to 200 AU of bacteriocin. 2.5. Crude bacteriocin preparation Following extraction from producer cells, the bacteriocin-containing solution was concentrated using a vacuum concentrator centrifuge and subjected to a series of blue native polyacrylamide gel electrophoresis (BN PAGE) and native PAGE runs according to Wittig et al. (2006). Isolation of the protein of interest in a single band was achieved by carrying out native electrophoresis at pH 6.1 in the absence of urea, using gels prepared with a mixture of 15% (w/v) acrylamide and 0.4% (w/v) bisacrylamide. The exact position of the antimicrobial protein was determined after covering the native electrophoresis gel with solid medium (nutrient agar) containing cells of the indicator microorganism (M. flavus). The Rainbow protein molecular weight marker (2.35–46 kDa) (Amersham International, Amersham UK) was used for molecular weight comparisons.

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2.6. Molecular mass determination and amino acid sequencing of the bacteriocin The recovered peptide from native-PAGE gels was subjected to tricine-SDS–PAGE, carried out according to Schägger (2003), for a first estimation of its molecular weight. For mass determination by mass spectrometry, a gel slice containing the peptide of interest was excised from 15% polyacrylamide tricine-SDS–PAGE gels containing 0.1% bromophenol blue and the peptide was with formic acid/water/2-propanol (1:3:2 v/ v/v). The eluate was treated as described by Cohen and Chait (1997) and the protein was finally eluted into the electrospray source with a solution consisting of 70% acetonitrile/2.5% acetic acid at 6 ml/min. Electrospray ionization mass spectrometric analysis (ESI-MS) was performed on a TSQ-700 triple-quadrupole mass spectrometer (Finnigan MAT Corp., San Jose, CA) according to Mirza et al. (1995). For determination of the amino acid sequence, the protein sample was transferred from SDS–PAGE gel onto a polyvinylidene (PVDF) membrane and the protein was subjected to N-terminal sequencing (Komatsu, 2009). The protein was cleaved with AspN protease (P3303 Sigma; 0.1 lg enzyme to 20 lg peptide, incubated for 2 h at 30 °C) into two fragments. The fragments were subjected to native-PAGE and electroblotting onto PVDF membranes and the amino acid sequence of the peptide fragments was determined by Edman degradation sequencing of the protein using an Applied Biosystems Procise Sequenser (ABI 494 protein sequencer). 2.7. Sequence-based analysis and bioinformatics tools The software Clone Manager 7 (version 7.11, Sci Ed Central) was used for sequence-based computation analyses. The following analyses were performed: Molecular weight, isoelectric point, amino acid composition profile, three-line graphs plots for the predicted values for alpha-helix, beta-sheet and beta-turn configurations according to Garnier et al. (1978), hydrophilicity analysis and plots according to Kyte and Doolittle (1982) and Hopp and Woods (1982), and surface exposure (SE) analysis according to Janin et al. (1978). Homology searches were performed with BLAST (Basic Local Alignment Search Tool, NCBI-algorithm blastp) and the Cobalt Multiple Alignment Tool (NCBI, 0000).

37 °C for 1 h. All enzymes were from Sigma and used at the final concentration of 2 mg/ml. Purified bacteriocin in buffers without enzyme, enzyme-buffer solutions, and buffers were used as controls. Following incubation, enzymes were inactivated by heating for 3 min at 100 °C, and samples were examined with tricineSDS–PAGE on 15% gels (w/v) and also for antimicrobial activity.

2.11. Mode of action of the bacteriocin Purified bacteriocin, at a final concentration of 200 AU/ml, was added to M. flavus ATCC400 cultures at mid-logarithmic phase of growth, at 30 °C. Changes in turbidity were recorded at 600 nm and the number of colony-forming units (cfu) was determined for samples and controls (bacteriocin-free cultures of M. flavus). All samples in which bacteriocin was added, were then centrifuged at 10,000g (4 °C) for 15 min and the supernatant was examined for the presence of proteins and DNA by measuring the OD at 280 and 260 nm, respectively.

3. Results and discussion 3.1. Bacteriocin production, activity spectrum and properties Weissella paramesenteroides DX produces a bacteriocin, named weissellin A, active against a range of Gram-positive bacteria, but not the Gram-negative bacterium, Salmonella enterica servora enteritidis (Table 1). Among the bacteria tested, Listeria monocytoges, L. inocua and Clostridium sporogenes were the most sensitive, pediococci showed intermediate sensitivity and Lactobacillus curvatus, Lactobacillus sakei, Lactococcus lactis subs. cremoris and L. lactis subs. lactis ATCC11454, the producer of nisin, were the least sensitive. Weissellin A thus shows an antimicrobial spectrum similar to that of class IIa bacteriocins (Fimland et al., 2005). With M. flavus ATCC400 as indicator organism, weissellin A exhibited a non-lytic mode of activity since no decrease in turbidity of exposed cultures nor an increase in OD at 260 and 280 nm of the culture supernatant were observed (data not shown). Possibly, this bacteriocin acts on the cytoplasmic membrane of target cells by forming hydrophilic pores which cause an efflux of important cellular metabolites and subsequent cell death as observed for other class IIa bacteriocins (Fimland et al., 2005).

2.8. Effect of temperature on bacteriocin activity 1200

Crude bacteriocin was dissolved in distilled water at a concentration of 200 AU/ml. The pH of the samples was adjusted to the range of pH values of 2.0–14.0, using 1 N HCl or 1 N NaOH solutions. Following incubation at 37 °C for 30 min, the samples were neutralized to pH 6.0, and tested for antimicrobial activity. 2.10. Sensitivity of the bacteriocin to proteases

1000

20

800 15 Glucose

10

Biomass Weissellin A

400

5

200

0

0 0

Resistance to proteolytic enzymes was determined by incubating samples of the purified bacteriocin in the presence of proteinase K, trypsin, a-chymotrypsin (0.05 M sodium phosphate buffer, pH 7.0), pepsin and papain (0.2 M citric acid buffer, pH 2.0), at

600

pH Lactic acid

Weissellin A (AU/ml)

2.9. Effect of pH on bacteriocin activity

Biomass, Glucose, LA (g/L), pH

25

Crude bacteriocin samples (pH 6.0, 200 AU/ml) were exposed to heat treatments (using a heat block) of 40, 60, 80, and 100 °C for 10, 30, and 60 min and 121 °C for 10 and 20 min. The samples were afterwards tested for antimicrobial activity. Samples were also stored for 6 months at 20, 4, and 30 °C and were assayed for antimicrobial activity at 1-week intervals.

10

20

30

40

50

60

Time (h) Fig. 1. Growth of W. paramesenteroides DX in a stirred tank bioreactor at 150 rpm and 50% DOT. Time-courses for biomass, weissellin A, lactic acid, glucose concentrations, pH.

M. Papagianni, E.M. Papamichael / Bioresource Technology 102 (2011) 6730–6734

70,00 60,00

-1

Specific growth rate, µ (h )

0,40 qp

Specific weissellin A production rate, q p (kAU/g/h)

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0,35

µ

0,30

50,00

0,25 40,00 0,20 30,00 0,15 20,00

0,10

10,00

0,05

0,00

0,00 0

20

40

60

Time (h) Fig. 2. Growth of W. paramesenteroides DX in a stirred tank bioreactor at 150 rpm and 50% DOT. Time-courses for specific growth rate and specific production rate of weissellin A.

In bioreactor culture, weissellin A production takes place during the exponential phase of growth. The maximum production rate was observed at around 12 h of fermentation which corresponds to late exponential phase. Production reached a maximum of 1120 AU/ml at 18 h and maintained at that levels until 40 h, but declined sharply thereafter possibly because of proteolytic degradation (Fig. 1). The time-courses of specific rates of growth and weissellin A production are shown in Fig. 2. Obviously, weissellin A is produced as a growth-associated fermentation product and as such a product, its levels can be influenced by the growth rate. Bacteriocins of LAB are usually growth-associated metabolites and many studies have shown that their production is strongly influenced by a large number of fermentation parameters that reflect on the growth rate of the producer organism. The identification of conditions which yield high titers of weissellin A in fermentation should be the subject of further studies. Weissellin A showed heat resistance even up to 121 °C for 1 h and maintained its activity over the pH range of 2.0–10.0 (data not shown). This stability is a common feature of class IIa bacteriocins (Papagianni, 2003; Fimland et al., 2005). Storage for 6 months at 20, 4, and 30 °C did not cause any significant changes to antimicrobial activity compared to fresh crude preparation samples (data not shown). Among the proteases tested, only proteinase K caused 100% inactivation of antibacterial activity.

Fig. 3. Coomassie-stained 15% acrylamide SDS–PAGE gel of purified weissellin A in different concentrations. The arrows denote the weissellin A band.

3.2. Bacteriocin purification and molecular characterization Weissellin A was purified by native PAGE and SDS–PAGE indicated that it has a molecular weight of 5 kDa (Fig. 3). The peptide consists of 43 amino acid residues (Fig. 4) and has a predicted molecular weight of 4448.9, which is in good agreement with the experimentally derived value by ESI-MS of 4450 kDa. In the N-terminal region, the sequence contains the motif (pediocin box) YGNGV- and the two cysteine residues which is characteristic for bacteriocins belonging to class IIa (Papagianni, 2003; Fimland et al., 2005). The calculated isoelectric point for the peptide is 9.46 due to its high content (18.6%) of asparagines residues. In this respect, weissellin A resembles leucosin C, produced by Leuconostoc mesenteroides (Fimland et al., 2002). Computer-based structure analysis predict that weissellin A has a hydrophilic N- and a hydrophobic C-terminus, but as a whole, the peptide appears to be strongly hydrophobic, with more that 75% of its amino acid residues being hydrophobic. The hydrophobic nature of weissellin A was evidenced by the successful extraction in the formic acid/water/2propanol solution. The prediction of N-terminal region showed two b-sheets maintained in a hairpin conformation. Also, residues

Fig. 4. Multiple alignment of the amino acid sequence of weissellin A and other class IIa bacteriocins. Similarity format and color areas of high matches at the same position. Dashes represent gaps introduced to optimize the alignment. (See above-mentioned references for further information.)

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V30 through L35 show a propensity for a b-sheet (Supplementary Fig. 1). Weissellin A is therefore a b-sheet-rich peptide consistent with the general characteristic of other known pediocin-like bacteriocins (Fimland et al., 2005). Alignment of the weissellin A sequence with other class IIa bacteriocin sequences shows that weissellin A shares 68% similarity with leucocin C, 61% with sakacin P, and 55% with pediocin PA-1 (Fig. 4). In recent years, there have been a large number of reports dealing with the antimicrobial spectrum, purification and characterization of bacteriocins produced by LAB. However, because of the potential use of bacteriocins of LAB as food preservatives, it is of great importance to gain insight into their chemical structure. For the substantial number of different bacteriocins that have been described so far, their primary structure is lacking and only a few have been described in detail at the molecular level. In-depth characterization of the purified form of a bacteriocin at such a level is absolutely necessary in assessing its food-related applications and in designing an effective downstream processing scheme. To our knowledge, there has been no report of an amino acid sequence or even an amino acid composition of a bacteriocin isolated from a Weissella strain. Structural descriptions such as those presented in this work add to a growing knowledge base for the antimicrobial peptides of LAB and may provide valuable information for further studies on the efficient production of these peptides for industrial applications. 4. Conclusions Weissellin A, a 4450 Da peptide produced by W. paramesenteroides DX, has the characteristics of a class IIa bacteriocin. Its properties make it a promising agent in food and feed preservation. It inhibited growth of a range of Gram-positive bacteria, as well as of Listeria monocytogenes. It is heat resistant and could be used therefore in pasteurized and sterilized foods. Its stability over a wide pH range indicates that it could be used in acidic and nonacidic foods. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biortech.2011.03.106. References Aymerich, T., Holo, H., Håvarstein, L.S., Hugas, M., Garriga, M., Nes, I.F., 1996. Biochemical and genetic characterization of enterocin A from Enterococcus faecium, a new antilisterial bacteriocin in the pediocin family of bacteriocins. Appl. Environ. Microbiol. 62, 1676–1682. Bennik, M.H., Vanloo, B., Brasseur, R., Gorris, L.G., Smid, E.J., 1998. A novel bacteriocin with a YGNGV motif from vegetable-associated Enterococcus mundtii: full characterization and interaction with target organisms. Biochim. Biophys. Acta 1373, 47–58. Bhugaloo-Vial, P., Dousset, X., Metivier, A., Sorokine, O., Anglade, P., Boyaval, P., Marion, D., 1996. Purification and amino acid sequences of piscicolins V1a and V1b, two class IIa bacteriocins secreted by Carnobacterium piscicola V1 that display significantly different levels of specific inhibitory activity. Appl. Environ. Microbiol. 62, 4410–4416. Cintas, L.M., Casaus, P., Havarstein, L.S., Hernandez, P.E., Nes, I.F., 1997. Biochemical and genetic characterization of enterocin P, a novel sec- dependent bacteriocin from Enterococcus faecium P13 with a broad antimicrobial spectrum. Appl. Environ. Microbiol. 63, 4321–4330.

Cohen, S.L., Chait, B.T., 1997. Mass spectrometry of whole proteins eluted from sodium dodecyl sulphate-polyacrylamide gel electrophoresis gels. Anal. Biochem. 247, 257–267. Fimland, G., Johnsen, L., Dalhus, B., Nissen-Meyer, J., 2005. Pediocin-like antimicrobial peptides (class IIa bacteriocins) and their immunity proteins: biosynthesis, structure, and mode of action. J. Peptide Sci. 11, 688–696. Fimland, G., Sletten, K., Nissen-Meyer, J., 2002. The complete amino acid sequence of the pediocin-like antimicrobial peptide leucocin C. Biochem. Biophys. Res. Com. 295, 826–827. Garnier, J., Osguthorpe, D.J., Robson, B., 1978. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J. Mol. Biol. 120, 97–120. Hastings, J.W., Sailer, M., Johnson, K., Roy, K.L., Vederas, J.C., Stiles, M.E., 1991. Characterization of leucocin A-UAL 187 and cloning of the bacteriocin gene from Leuconostoc gelidum. J. Bacteriol. 173, 7491–7500. Héchard, Y., Derijard, B., Letellier, F., Canatiempo, Y., 1992. Characterization and purification of mesentericin Y105, an anti-Listeria bacteriocin from Leuconostoc mesenteroides. J. Gen. Microbiol. 138, 2725–2731. Henderson, J.T., Chopco, A.L., van Wassenaar, P.D., 1992. Purification and primary structure of pediocin PA-1 produced by Pediococcus acidilactici PAC1 0. Arch. Biochem. Biophys. 295, 5–12. Holck, A., Axelsson, L., Birkeland, S.E., Aukrust, T., Blom, H., 1992. Purification and amino acid sequence of sakacin A, a bacteriocin from Lactobacillus sake. J. Gen. Microbiol. 138, 2715–2720. Hopp, T.P., Woods, K.R., 1982. Prediction of protein antigenic determinants from amino acid sequences. Proc. Natl. Acad. Sci. USA 78, 3824–3828. Jack, R.W., Wan, J., Gordon, J., Harmark, K., Davidson, B.E., Hillier, A.J., Wettenhall, R.E.H., Hickey, M.W., Coventry, M.J., 1996. Characterization of the chemical and antimicrobial properties of piscicolin 126, a bacteriocin produced by Carnobacterium piscicola JG126. Appl. Environ. Microbiol. 62, 2897–2903. Janin, J., Wodak, S., Levitt, M., Maigret, B., 1978. Conformation of amino acid sidechains in proteins. J. Mol. Biol. 125, 357–386. Kaiser, A.L., Montville, T.J., 1996. Purification of bacteriocin bavaricin MN and characterization of its mode of action against Listeria monocytogenes Scott A cells and lipid vesicles. Appl. Environ. Microbiol. 62, 4529–4535. Komatsu, S., 2009. Western blotting/Edman sequencing using PVDF membrane. Methods Mol. Biol. 536, 163–171. Kyte, J., Doolittle, R.F., 1982. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157, 105–132. Le Marrec, C., Hyronimus, B., Bressollier, P., Verneuil, B., Urdaci, M.C., 2000. Biochemical and genetic characterization of coagulin, a new antilisterial bacteriocin in the pediocin family of bacteriocins, produced by Bacillus coagulans I4. Appl. Environ. Microbiol. 66, 5213–5220. McCormick, J.K., Worobo, R.W., Stiles, M.E., 1996. Expression of the antimicrobial peptide carnobacteriocin B2 by a signal peptide-dependent general secretory pathway. Appl. Environ. Microbiol. 62, 4095–4099. Metivier, A., Pilet, M.F., Dousset, X., Sorokine, O., Anglade, P., Zagorec, M., Piard, J.C., Marion, D., Cenatiempo, Y., Fremaux, C., 1998. Divercin V41 a new bacteriocin with disulphide bonds produced by Carnobacterium divergens V41: primary structure and genomic organization. Microbiology 144, 2837–2844. Mirza, U.A., Chait, B.T., Lander, H.M., 1995. Monitoring reactions of nitric-oxide with peptides and proteins by electrospray-ionization mass-spectrometry. J. Biol. Chem. 270, 17185–17188. NCBI. . Papagianni, M., 2003. Ribosomally synthesized peptides with antimicrobial properties: biosynthesis, structure, function, and applications. Biotechnol. Adv. 21, 465–499. Papagianni, M., Avramidis, N., Filioussis, G., Dasiou, D., Ambrosiadis, I., 2006. Determination of bacteriocin activity with bioassays carried out on solid and liquid substrates: assessing the factor ‘‘indicator microorganism’’. Microb. Cell Fact. 5, 30. Schägger, H., 2003. SDS electrophoresis techniques. In: Hunte, C., von Jagow, G., Schägger, H. (Eds.), Membrane Protein Purification and Crystallization. A Practical Guide, 2nd edn. Academic, San Diego, California, USA, pp. 4.85–4.103. Simon, L., Fremaux, C., Cenatiempo, Y., Berjeaud, J.M., 2002. A new type of antilistrial bacteriocin. Appl. Environ. Microbiol. 68, 6416–6420. Tichaczek, P.S., Nissen-Meyer, J., Nes, I.F., Vogel, R.F., Hammes, W.P., 1992. Characterization of the bacteriocins curvacin A from Lactobacillus curvatus LTH1174 and sakacin P from Lactobacillus sake LTH673. Syst. Appl. Microbiol. 15, 460–468. Wittig, I., Braun, H.P., Schagger, H., 2006. Blue native PAGE. Nat. Protocols 1, 418– 428. Yang, R., Johnson, M.C., Ray, B., 1992. Novel method to extract large amounts of bacteriocins from lactic acid bacteria. Appl. Environ. Microbiol. 58, 3353–3359. Yildirim, Z., Winters, D.K., Johnson, M.G., 1999. Purification, amino acid sequence and mode of action of bifidocin B produced by Bifidobacterium bifidum NCFB 1454. J. Appl. Microbiol. 86, 45–54.