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Comparison of Two Methods for Purification of Enterocin B, A Bacteriocin Produced by Enterococcus Faecium W3 a
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Halil Dündar , Mehmet Atakay , Ömür Çelikbiçak , Bekir Salih & Faruk Bozoğlu a
Department of Biotechnology, Middle East Technical University, Ankara, Turkey
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Department of Chemistry, Hacettepe University, Beytepe, Ankara, Turkey
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Department of Food Engineering, Middle East Technical University, Ankara, Turkey Accepted author version posted online: 02 Sep 2014.
To cite this article: Halil Dündar, Mehmet Atakay, Ömür Çelikbiçak, Bekir Salih & Faruk Bozoğlu (2014): Comparison of Two Methods for Purification of Enterocin B, A Bacteriocin Produced by Enterococcus Faecium W3, Preparative Biochemistry and Biotechnology, DOI: 10.1080/10826068.2014.958165 To link to this article: http://dx.doi.org/10.1080/10826068.2014.958165
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COMPARISON OF TWO METHODS FOR PURIFICATION OF ENTEROCIN B, A BACTERIOCIN PRODUCED By Enterococcus faecium W3 Halil Dündar1, Mehmet Atakay2, Ömür Çelikbiçak2, Bekir Salih2, Faruk Bozoğlu3 1
Department of Biotechnology, Middle East Technical University, Ankara, Turkey, Department of Chemistry, Hacettepe University, Beytepe, Ankara, Turkey, 3Department of Food Engineering, Middle East Technical University, Ankara, Turkey
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Abstract
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This study aimed to compare two different approaches for the purification of enterocin B from Enterococcus faecium strain W3 based on the observation that the bacteriocin was found both in cell associated form and in culture supernatant. The first approach employed ammonium sulfate precipitation, cation-exchange chromatography and
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sequential reverse-phase high performance liquid chromatography. The latter approach exploited pH mediated cell adsorption-desorption method to extract cell bound
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bacteriocin, and one run of reverse-phase chromatography. The first method resulted in purification of enterocin B with a recovery of 4% of the initial bacteriocin activity found in culture supernatant. MALDI-TOF MS analysis and de novo peptide sequencing of the purified bacteriocin confirmed that the active peptide was enterocin B. The second method achieved the purification of enterocin B with a higher recovery (16%) and
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Correspondence Addressed to Halil Dündar: E-mail: [email protected]
enabled us to achieve pure bacteriocin within a shorter period of time by avoiding time consuming purification protocols. The purity and identity of the active peptide was confirmed again by MALDI-TOF MS analysis. Although both approaches were satisfactory to obtain sufficient amount of enterocin B for use in MS and amino acid
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sequence analysis, the latter was proved to be applicable in large-scale and rapid purification of enterocin B.
INTRODUCTION
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Bacteriocins from lactic acid bacteria (LAB) are of great importance because of their use in food preservation.[1] Bacteriocins are ribosomally synthesized antimicrobial peptides displaying antagonistic effect against other bacteria.[2] They exhibit bactericidal activity on the sensitive cells through depolarization of cell membrane or through the inhibition
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of cell wall synthesis. The bacteriocins from LAB have received considerable attention regarding food safety because of the generally recognized as safe (GRAS) status of these
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bacteria.[3] Bacteriocins produced from Enterococcus spp. are the most widely produced and characterized among LAB bacteriocins.[4] Among them, enterocin B shows important properties such as stability and solubility over a wide pH range, heat stability and inhibition spectrum, making it an attractive candidate for food preservation. Enterocin B was shown to inhibit the growth of Listeria monocytogenes, Enterococcus faecalis,
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chromatography, MALDI-TOF, peptide sequencing
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KEYWORDS: bacteriocin, enterocin B, bacteriocin purification, reverse-phase
Clostridium sporogenes, Clostridium tyrobutyricum, Staphylococcus aureus.[5] It showed significant antilisterial effect in minced pork meat, pork liver patè cooked ham and debondened chicken breasts[6] as well as inhibiting the production of slime by L. sakei CTC746 in vacuum packaged sliced cooked pork ham.[7] The synergistic action of enterocin B with high hydrostatic pressure (HHP) was shown in cooked ham model with
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reduction in the intensity of HHP treatment.[8] The first attempts to purify enterocin B employed its cationic and hydrophobic nature and hence, cation-exchange and hydrophobic interaction chromatography with repeated reverse-phase chromatography steps were applied on the concentrated culture supernatant.[5] Various methods were
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reported for the purification of enterocins: enterocin A purification; ammonium sulfate
exchange column), hydrophobic interaction chromatography (Octyl-Sepharose CL-4B
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column), followed by C 2/C18 reverse-phase chromatography,[9] enterocin B purification;
extraction with Amberlite XAD-16, cation-exchange chromatography (SP-Sepharose Fast Flow cation-exchange column), hydrophobic interaction chromatography (OctylSepharose CL-4B column), followed by C2/C18 reverse-phase chromatography,[5]
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enterocins L50A and L50B purification; extraction with Amberlite XAD-16, cationexchange chromatography (SP-Sepharose Fast Flow cation-exchange column),
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hydrophobic interaction chromatography (Octyl-Sepharose CL-4B column), followed by C2/C18 reverse-phase chromatography,[10] enterocin I purification; ammonium sulfate precipitation, cation-exchange chromatography (SP-Sepharose Fast-Flow column), hydrophobic interaction chromatography (Phenyl-Sepharose CL-4B column), followed by C2/C18 reverse-phase chromatography,[11] enterocin P purification; ammonium sulfate
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precipitation, cation-exchange chromatography (SP-Sepharose Fast Flow cation-
precipitation, gel filtration (G25 PD10 columns), cation exchange chromatography (SPSepharose Fast Flow column), hydrophobic-interaction chromatography (OctylSepharose CL-4B column), followed by C2/C18 reverse-phase chromatography.[12] Although these methods worked to achieve purification to homogeneity for elucidation of primary structure, the yields were variable and low, the protocols being time consuming
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and not suitable for large-scale applications. Therefore, methods for rapid and large-scale purification of bacteriocins need to be devised for use in controlling food spoilage and in clinical applications.
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Micro-Cel,[13] Rice hull ash and silicic acid[14] were exploited for extraction of
was studied also.[15,16] Bacteriocins of lactic acid bacteria are adsorbed on the cells of
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producer strain depending of pH of the environment, adsorption occurs at around pH 6.0 and desorption occurs around pH 2.0. Based on this property of LAB bacteriocins,
pediocin AcH, leuconocin Lcm1 and sakacin A, and the lantibiotic nisin were purified on
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a large-scale.[17]
In this study we tested the utility of pH mediated cell adsorption-desorption method with
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the subsequent reverse-phase high performance liquid chromatography to purify enterocin B. The method was compared with the usual purification protocol consisting of ammonium sulfate precipitation, cation-exchange chromatography and sequential reverse-phase high performance liquid chromatography.
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bacteriocins from culture supernatant. Extraction with aqueous two-phase partitioning
MATERIALS AND METHODS
Microorganisms
Bacteriocin producer Enterococcus faecium W3 was a food strain previously isolated in our laboratory Middle East Technical University, Food Microbiology Labotatory) and
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identified as E. faecium by gram staining, endospore staining, catalase reaction, heterofermentative/homofermentative distinguishing and 16S rDNA analysis.
Bacteriocin Activity Assay
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Bacteriocin activity was evaluated by a microtiter plate assay. Two-fold serial dilutions
μL de Man Rogasa Sharp (MRS) broth. 150 μL of indicator strain Lactobacillus
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delbrueckii subsp. delbrueckii RSSK 498 (100-fold diluted) was added to the bacteriocin containing wells and the plate was incubated at 30°C for 6 h. Bacteriocin activity was evaluated by measuring the growth at 620 nm by a microtiter plate reader reader (Multiscan Ascent; Labsystems, Helsinki, Finland). One bacteriocin unit (BU) was
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defined as the amount of bacteriocin inhibiting the growth of the indicator strain by 50%.
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Temperature And Ph Optimization For Bacteriocin Production MRS broth adjusted to an initial pH of 6.5 was inoculated (1%, v/v) with an overnight culture of strain W3 and incubated at 10, 15, 20, 25, 30 and 37°C without pH control for 16 h. For determination of the effect of initial pH on bacteriocin production, MRS broth adjusted to pH 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 was inoculated (1%, v/v) with an overnight
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of preparations containing bacteriocin were added to microtiter plate wells containing 50
culture of strain W3 and incubated at 30°C without pH regulation for 16 h. Bacteriocin activity was measured as described above.
Bacteriocin Purification
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Purification By Ammonium Sulfate Precipitation, Cation Exchange Chromatography On SP-Sepharose Fast Flow Column And Reverse-Phase High Performance Liquid Chromatography On Akta Purifier. 1000 mL of MRS broth was inoculated with an overnight culture of E. faecium W3 (1%
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v/v) and incubated for 16 h with an initial pH of 6.5 at 30°C. Bacteriocin containing
4°C. Ammonium sulfate (40 g per 100 mL) was added to 500 mL of culture supernatant
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in centrifuge tubes and then shaked at 4°C for 40 min. The mixture was centrifugated
(13000 rpm for 30 min at 4°C) to precipitate the bacteriocin from supernatant. The pellet was dissolved in 25 mL of sterile distilled water and diluted to 100 mL with sterile distilled water. This sample was applied to 5 mL of SP-Sepharose Fast Flow (GE
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Healthcare Biosciences) column, which was equilibrated with 10 mM acetic acid. The column was washed with 2 column volumes (CV) of 10 mM sodium phosphate buffer
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(pH 6.0) and 2 CV of sodium phosphate buffer (10 mM, pH 6.0) containing 100 mM NaCI. Subsequently, the column was eluted with a step-wise gradient of 2 CV of 300 mM NaCI and 2 CV of 1.0 M NaCI. Fractions of 2.5 mL at a flow rate of 1 mL were collected and assayed for bacteriocin activity. Fractions with the highest bacteriocin activity from SP Sepharose Fast flow column, the last 7.5 mL (300 mM NaCI) and the first 5 mL (1.0
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supernatant was obtained after centrifugation of the culture at 13000 rpm for 30 min at
M NaCI), were combined and applied to Resource 15 RPC 3 mL reverse-phase column (100 mm length, 6.4 mm i.d., and with 15 µm pore size) equilibrated with 0.1% (v/v) (TFA) in distilled water. Elution was carried out with a 5 CV linear gradient of 15 to 55% 2-propanol with 0.1% TFA at a flow rate of 1 mL/min using Äkta Purifier fast protein liquid chromatography system. Fractions of 1 mL were collected and assayed for
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bacteriocin activity. Active fractions from first reverse-phase chromatography were applied to reverse-phase chromatography under the same conditions and bacteriocin activity was assayed as described above. Purified bacteriocin was subjected to MALDI-
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TOF mass spectrometry and amino acid sequencing analysis.
Phase High Performance Liquid Chromatography
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The second purification protocol employed cell-associated form of the bacteriocin as an initial sample to extract relatively pure bacteriocin based on our observation that the bacteriocin produced by E. faecium W3 was detected in cell associated form (bound to
producer cell surface) as well as in culture supernatant fluid. The pelleted cells of the 1 L
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of culture was mixed with 500 mL of culture supernatant left from the same fermentation flask. The pH of the heat treated culture was adjusted to 6.0 by addition of 1.0 M NaOH
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solution. The culture was stirred for 1 h at 4°C to allow the bacteriocin to adsorb onto bacterial cells. The cells were collected by centrifugation and washed with 200 mL of 5.0 mM sodium phosphate buffer (pH 6.0) to remove impurities. After centrifugation for 30 min at 4°C (13000 rpm), the pelleted cells were resuspended in 100 mL of 5.0 mM sodium phosphate buffer (pH 6.0) containing 100 mM NaCI, and the pH was adjusted to
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Purification By Ph Mediated Cell Adsorption-Desorption Method And Reverse-
2.0 by gradual addition of HCI. The cell suspension was stirred in cold room (4°C) overnight, centrifuged and filtered. The extract was concentrated with boiling to a volume of 12 mL and assayed for bacteriocin activity. After freeze-drying of this fraction, it was resuspended in 1 mL of 0.1% TFA and applied to reverse-phase high performance liquid chromatography by injecting 100 µL aliquots into Vydac C18 column (150 mm length,
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4.6 mm i.d., and with 5 µm pore size) incorporated in Agilent 1200 Series Quaternary HPLC system. Detection was carried out at wavelength range between 190-700 nm. Mobile phase was composed using varying amounts of water with 0.1% TFA (v/v) (A) and acetonitrile (ACN) with 0.085% TFA (v/v) (B). Mobile phase was varied in the
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following gradients: 0 to 5 min, 96% to 4% (v/v); 5 to 80 min, 60% to 40% (v/v); 80 to
collected in separate vials for 1 min intervals and assayed for bacteriocin activity. Protein
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concentrations were measured spectrophotometrically at 280 nm. Purified bacteriocin
was subjected to MALDI-TOF mass spectrometry and amino acid sequencing analysis.
MALDI-TOF Analysis
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Active fractions were dried in water bath at 70°C, and finally in vacuum oven at 30°C. Mass spectrometric analyses were performed by dissolving the dried peptide in 200 μL
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ACN:H2O mixture in 50:50 (v/v) containing 0.1% TFA (v/v). Mass spectra of purified bacteriocin were acquired on a Voyager-DETM PRO MALDI-TOF mass spectrometer (Applied Biosystems, USA) as described.[18]
Amino Acid Sequencing
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90 min, 10% to 90% (v/v); and 90 to 100 min, 10% to 90% (v/v). Peptide fractions were
Mass spectrometry data were acquired using a Q-STAR Elite Q-TOF mass spectrometer (Applied Biosystems) with a nano-ESI source. MS/MS data were obtained via Information Dependent Acquisition (IDA) method, where doubly and triply charged parent ions were selected for fragmentation by collision induced dissociation (CID) using nitrogen as collision gas. Protein identification was performed by searching the NCBI
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database using the Mascot search engine with the following search criteria: monoisotopic mass, peptide charge +2, 50 ppm mass accuracy, trypsin as digesting enzyme with 1 missed cleavage allowed, carbamidomethylation of cysteine as a fixed modification, oxidation of methionine as allowable variable modification. For all de novo algorithms,
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methionine oxidation was selected as variable modification and carbamidomethylation of
tolerance and MS/MS tolerance of 0.3 Da. A maximum of one missed-cleavage was
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allowed. All data were searched for tryptic peptides.
Mode Of Action Of Ph Mediated Cell Surface Adsorption-Desorption Fraction Cell suspension of Listeria monocytogenes RSSK 475 and Enterococcus faecalis was
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grown to exponential growth phase. Monitoring of bacterial growth was carried out by determining the absorbance at 600 nm (A600). Exponentially growing culture of the
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organism was divided into two equal parts and pH mediated cell surface adsorptiondesorption fraction from E. faecium W3 was added to each part with a final concentration of 300 AU mL. Control culture and bacteriocin treated culture were incubated and at selected intervals samples were taken from both cultures for total viable colony counting and A600 measurement. Total viable counts (CFU/mL) were determined by pour plating
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cysteines as fixed modification. QSTAR data were de novo sequenced using peptide
technique using the serial dilutions of indicator organism. A600 values were measured by a spectrophotometer (path length, 1 cm; UV-160 UV-visible spectrophotometer; Shimadsu). All assays were performed in duplicate.
RESULTS AND DISCUSSION
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Effect Of Temperature And Ph On Bacteriocin Production Growth and bacteriocin production occurred at temperatures between 20 and 37 oC with an initial pH of 6.5-7.0 under uncontrolled conditions. Production of bacteriocin by the producer organism resulted in 320 AU/mL bacteriocin activity at 30 and 37 oC in CSF and
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160 AU/mL at 20 and 25 oC). The producer organism produced the same amount of
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Nevertheless, no bacterial growth and bacteriocin production were detected under pH 5.0.
Purification By Ammonium Sulfate Precipitation, Cation-Exchange
Chromatography On SP-Sepharose Fast Flow, And Three Consecutive ReversePhase HPLC On Resource RPC 3 Ml Column Using Äkta Purifier.
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The culture supernatant had an activity of 320 BU/mL and ammonium sulfate precipitation resulted in 80% recovery of the initial bacteriocin activity. The precipitate
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was subjected to cation-exchange chromatography on SP Sepharose Fast Flow column. The recovery by cation-exchange chromatography was low (14%) due to activity loss in flow-through. The results of the protocol is shown in Table 1. The highest bacteriocin activity from SP Sepharose Fast flow column, the last 7.5 mL (300 mM NaCl) and the first 5 mL (1.0 M NaCl), were pooled (19200 BU/mL) and applied to Resource 15 RPC 3
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bacteriocin whent the initial pH was between 5.5 and 7.0 (320 AU/mL) at 30 oC.
mL reverse-phase column equilibrated with 0.1% (v/v) (TFA) in distilled water. Bacteriocin activity was detected in fractions 11, 12, and 13 (Figure 1). Fraction 12 with the highest bacteriocin activity (6400 BU/mL) was diluted ten times with 0.1% (v/v) TFA and run on Resource 15 RPC 3 mL column with Akta purifier under the same conditions. The second run of reverse-phase HPLC resulted in one symmetrical peak with bacteriocin
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activity in fraction 18 (6400 BU/mL) (Figure 2), which was concentrated by SpeedVac, applied for MALDI-TOF and de novo peptide sequencing. MALDI-TOF analysis of this active peak resulted in a mass of 5462 Da (Figure 3) and its de novo sequencing produced the following amino acid sequence from position 8 to 21, position 23 to 35 and position
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37 to 50: NH2--------NEI/LNRPNNI/LS-----CGAAI/LAGGI/LFGI/LP-
Thus, the sequence and MALDI-TOF mass spectrometry data confirmed that active peak
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is identical to enterocin B with the following sequence:
ENDHRMPNELNRPNNLSKGGAKCGAAIAGGLFGIPKGPLAWAAGLANVYSKCN.
Although the protocol worked well enough to be able to identify the bacteriocin, it caused
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substantial loss of bacteriocin activity with a final recovery of 4%. The remarkable activity loss was observed during cation-exchange chromatography where much
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bacteriocin activity was lost in flow through (Table 1). Reverse-phase chromatography also contributed to the loss of bacteriocin activity during the first run.
Cell-associated form of the bacteriocin was used as initial extract in the second purification method where the bacteriocin was eluted from the cell surface by low pH
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GPI/LAWAAGI/LANVYS where I/L denotes that the residue is isoleucine or leucine.
(pH 2.0) and 150 mM NaCI. This extraction method recovered 48% of the bacteriocin activity found in culture supernatant. The pH 2 extract was purified to homogeneity with the application of reverse-phase HPLC on Vydac C18 column incorporated in Agilent 1200 Series Quaternary HPLC system. The pH dependent adsorption-desorption method resulted in purification with 21.5 fold increase but the largest increase in specific activity
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was provided by reverse-phase chromatography with a 4408 fold increase (Table 2). RPHPLC resulted in six peaks, the fourth peak showed bacteriocin activity (Figure 4). The retention time of the bacteriocin on Vydac C18 column was about 80 min, corresponding to 90% acetonitrile. MALDI-TOF MS analysis confirmed the purity and molecular mass
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of enterocin B (Figure 5). MALDI mass spectrum of active peptide was obtained using
mass spectrum of the sample was obtained in α-cyano-4-hydroxycinnamic acid matrix
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and is given in Figure 5. Three mass value for the active peptide were observed by MALDI-TOF analysis including 5444, 5466 and 5482 Da as a result of the second
purification protocol (Figure 5). The main peak with a molecular mass of 5465 Da is the same as the calculated molecular mass (5465.2 Da). The peak with a molecular mass of
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5482 Da probably represents Enterocin B with an oxidized methionine residue as reported previously.[5,19] The related molecular mass values of enterocin B including 5466
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and 5482 Da were observed both in pH mediated cell surface adsorption-desorption extract and in the active fraction from reverse-phase chromatography. MALDI-MS spectra of this active fraction were also carried out at low and at very high mass ranges up to 200000 Da. No other peptide and proteins were observed in the same active peptide fraction (Figure 5). Amino acid sequence analysis of the active fraction showed that it
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positive ion and linear mode in various novel MALDI matrices but the suitable MALDI
was identical to the sequence obtained from the first purification protocol. The pH mediated cell adsorption-desorption method was carried out with some modifications as described previously.[17] It is known that bacteriocin producing strains of lactic acid bacteria adsorb their own bacteriocins around pH 6.0 and release around pH 2.0.[20] Dextranicin 24 produced by Leuconostoc sp.,[21] pediocin AcM produced by
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Pediococcus acidilactici,[22] leucocins A-, B- and C-TA33a produced by Leuconostoc mesenteroides TA33a[23] and plantaricin C19 produced by Lactobacillus plantarum C19[24] were purified by pH mediated cell adsorption-desorption method and reversephase chromatography. Bovicin 255 produced by Streptococcus sp. was purified by pH
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mediated cell adsorption-desorption method, ammonium sulphate precipitation, reverse-
by Pediococcus pentosaceus was purified by pH mediated cell adsorption-desorption
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method and gel filtration chromatography on FPLC system.[26] In this study, pH mediated cell adsorption-desorption method provided relatively pure enterocin B as seen after running of the sample by reverse-phase chromatography on Vydac column. Enterocin B obtained by pH mediated cell adsorption-desorption method can be used in ex situ
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applications taken into account its purity and higher yield without performing expensive purification protocols. Most purification schemes devised for purification of enterocins
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and other bacteriocins from lactic acid bacteria take advantage of the cationic and relatively hydrophobic natures of these molecules, and hence involve applications of cation-exchange chromatography, hydrophobic interaction chromatography and reversephase high performance liquid chromatography. Although these methods has been successful for the purification of many bacteriocins for identification, they are time
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phase and gel filtration chromatography on FPLC system.[25] Pediocin ACCEL produced
consuming, costly and not suitable for large-scale production.
Inhibition Spectrum Of Cell Surface Adsorbed-Desorbed Enterocin B Both partially purified bacteriocin (pH mediated cell surface adsorption desorption fraction) and its processed product through reverse-phase chromatography was tested
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against a set of Gram-positive and Gram-negative bacteria. Pure enterocin B and cell surface adsorption-desorption fraction showed the same inhibitory spectrum and inhibited the growth of Lc. mesenteroides, L. delbrueckii, L. cremoris, L. monocytogenes, L. innocua, E. faecalis, B. cereus and some lactic acid bacteria. However, no Gram-negative
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bacteria tested were inhibited by pure enterocin B and the cell surface adsorption-
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Mode Of Action
Turbidity of the bacteriocin treated culture of L. monocytogenes declined rapidly after the first 20 min (Figure 6). The decrease in A600 continued throughout the incubation as opposed the control culture. L. monocytogenes RSSK 475 lost its viability by 99.99 %
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within the first 20 min; viable colony count dropped rapidly from 8x10 8 to 104 (a 5.9 log reduction). After 4 hours, the viable colony count of the bacteriocin treated culture was
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400 CFU/mL, that is a 6.3 log reduction in viability (Figure 6).
CONCLUSION
Comparison of the two purification approach indicated that pH mediated cell surface adsorption-desorption method together with RP-HPLC can be used successfully for rapid
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desorption fraction (Table 3).
purification of enterocin B, which is a non-pediocin like one peptide bacteriocin (class IIc), as performed for many pediocin-like bacteriocins. The application of pH mediated cell surface adsorption-desorption method avoided time consuming ammonium sulfate precipitation, cation-exchange chromatography and hydrophobic interaction chromatography steps which might cause activity losses during the purification. Although
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both methods achieved purification of enterocin B with a similar specific activity, a significant improvement in the yield of enterocin B was achieved (16%) by pH mediated cell surface adsorption with a following reverse-phase chromatography compared with the yield obtained by ammonium sulfate precipitation, cation-exchange chromatography
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and final sequential reverse-phase chromatography (4%). Both purification approaches is
high purity and yield of the bacteriocin preparation obtained from pH mediated cell
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surface adsorption-desorption method make it possible its use in a concentrated form as a
food preservative. The cell of the producer strain functioned as a specific ligand to adsorb the enterocin B from culture supernatant, and provided both purification and concentation of the molecule. It is known that some stains of E. faecium produces enterocin A together
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with enterocin B which shows ynergistic action towards pathogenic and food-spoilage bacteria when used in combination. The rapid purification method mentioned here can be
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exploited for the large-scale purification of enterocin A and enterocin B together as well as for the bacteriocins of multiple bacteriocin producer strains. Combined and partial purification of different bacteriocins might be an alternative approach to avoid development of bacteriocin resistant strains. The feasibility of pH mediated cell surface adsorption and desorption method can be analysed for the multiple bacteriocins from
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successful to the extent that primary structure of the bacteriocin was obtained. However,
different LAB bacteria by mixing relavant bacteriocin supernatants and optimization of adsorption onto a suitable producer cell, which can provide large-scale and concomitant purification of a bacteriocin to achieve a strong bacteriocin cocktail targeting the resistant cells.
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characterization of enterocin I from Enterococcus faecium 6T1a, a novel antilisterial plasmid-encoded bacteriocin which does not belong to the pediocin family of bacteriocins. Appl. Environ. Microbiol. 1998, 64, 4883–4890.
Herranz, C.; Mukhopadhyay, S.; Casaus, P.; Martínez, J.M.; Rodríguez, J.M.;
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Nes, I.F.; Cintas, L.M.; Hernández, P.E. Biochemical and genetic evidence of enterocin P
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production by two Enterococcus faecium-like strains isolated from fermented sausages. Curr. Microbiol. 1999, 39, 282–290. 13.
Coventry, M.J.; Gordon, J.B.; Alexander, M.; Hickey, M.W.; Wan, J. A food-
grade process for isolation and partial purification of bacteriocins of lactic acid bacteria that uses diatomite calcium silicate. Appl. Environ. Microbiol. 1996, 62, 1764–1769.
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Enterocins L50A and L50B, two novel bacteriocins from Enterococcus faecium L50, are
14.
Janes, M.E.; Nannapaneni, R.; Proctor, A.; Johnson, M.G. Rice hull ash and
silicic acid as adsorbents for concentration of bacteriocins. Appl. Environ. Microbiol. 1998, 64, 4403–4409.
17
15.
Métivier, A.; Boyaval, P.; Duffes, F.; Dousset, X.; Compoint, J.P.; Marion, D.
Triton X-114 phase partitioning for the isolation of a pediocin-like bacteriocin from Carnobacterium divergens. Lett. Appl. Microbiol. 2000, 30, 42–46. 16.
Li, C.; Bai, J.; Li, W.; Cai, Z.; Ouyang, F. Optimization of conditions for
17.
Yang, R.; Johnson, M.C.; Ray, B. Novel method to extract large amounts of
18.
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bacteriocins from lactic acid bacteria. Appl. Environ. Microbiol. 1992, 58, 3355–3359. Ceyhan, T.; Yüksek, M.; Yağlıoğlu, H.G.; Salih, B.; Erbil, M.K.; Elmalı, A.;
Bekaroğlu, Ö. Synthesis, characterization and nonlinear absorption of novel octakisPOSS substituted metallophthalocyanines and strong optical limiting property of CuPc.
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Dalton Trans. 2008, 2407–2413.
Franz, C.M.A.P.; Worobo, R.W.; Quadri, L.E.N.; Schillinger, U.; Holzapfel,
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W.H.; Vederas, J.C.; Stiles, M.E. Atypical genetic locus associated with constitutive production of enterocin B by Enterococcus faecium BFE 900. Appl. Environ. Microbiol. 1999, 65, 2170–2178. 20.
Klaenhammer, T.R. Genetics of bacteriocins produced by lactic acid bacteria.
FEMS Microbiol. Rev. 1993, 12, 39–85.
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experimental designs). Biotechnol. Prog. 2001, 17, 366–368.
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bacteriocin extraction in PEG (Salt aqueous two-phase systems using statistical
21.
Revol-Junelles, A.M.; Lefebvre, G. Purification and N-terminal amino acid
sequence of dextranicin 24, a bacteriocin of Leuconostoc sp. Curr. Microbiol. 1996, 33, 136–137.
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22.
Elegado, F.B.; Kim, W.J.; Kwon, D.Y. Rapid purification, partial characterization,
and antimicrobial spectrum of the bacteriocin, Pediocin AcM, from Pediococcus acidilactici M. Int. J. Food Microbiol. 1997, 37, 1–11. 23.
Papathanasopoulos, M.A.; Dykes, G.A.; Revol-Junelles, A.M.; Delfour, A.; Von
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Holy, A.; Hastings, J.W. Sequence and structural relationships of leucocins A-, B- and C-
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Atrih, A.; Rekhif, N.; Moir, A.J.G.; Lebrihi, A.; Lefebvre, G. Mode of action,
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purification and amino acid sequence of plantaricin C19, an anti-Listeria bacteriocin
produced by Lactobacillus plantarum C19. Int. J. Food Microbiol. 2001, 68, 93-104. 25.
Whitford, M.F.; McPherson, M.A.; Forster, R.J.; Teather, R.M. Identification of
bacteriocin-like inhibitors from rumen Streptococcus spp. and isolation and
26.
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characterization of bovicin 255. Appl. Environ. Microbiol. 2001, 67, 569–574. Wu, C.W.; Yın, L.J.; Jiang, S.T. Purification and characterization of bacteriocin
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from Pediococcus pentosaceus ACCEL. J. Agric. Food Chem. 2004, 52, 1146–1151.
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TA33a from Leuconostoc mesenteroides TA33a. Microbiol. 1998, 144, 1343–1348.
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TABLE 1. First purification protocol of enterocin B
Stage Culture
Total act
Protein
Sp acta
Yield
(BU)
(mg/mL)
(BU/mg)
(%)
500
160000
30.83
10.37
25
128000
6.22
823.15
12.5
22400
0.42
4266
First run
3
12800
Second run
1
6400
supernatant
SPSepharose
a
14
411
4740
8
457
0.12
53333
4
5143
ep te d
Specific activity is total BU divided by the total protein.
b
79
0.9
M
RP-FPLC b
1
80
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(NH4)2SO4
RP-FPLC, fast-performance liquid chromatography.
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Precipit.
100
Fold
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Purification Vol (mL)
20
TABLE 2 Second purification protocol of enterocin B Purification
Vol
Total
Stage
(mL)
act
Protein
Sp acta
(mg/mL) (BU/mg)
Yield
fold
(%)
500
160000
30.83
10.37
100
1
pH 2 extract
12
76800
28.66
223.3
48
21.5
RP-HPLC b
1
25600
0.56
45714
16
4408
a
an us
Specific activity is total BU divided by the total protein.
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Culture sup.
b
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M
RP-FPLC, fast-performance liquid chromatography.
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(BU)
21
TABLE 3. Inhibition spectrum of enterocin B and enterocin pool from E. faecium W3. pH 2 extract
Purified entB
Medium b
a
a
temp(oC)
L. fermentum
-
-
L. casei
-
-
L. acidophilus
-
-
L. plantarum LP73
-
-
L. plantarum Z11L
-
-
L. plantarum HU
-
L. plantarum RSSK 675
+
L. plantarum RSSK 10
+
L. plantarum 80B
+
L. delbrueckii RSSK 498
+
sensitivity
MRS
30
MRS
37
MRS
30
MRS
30
-
MRS
30
+
MRS
30
+
MRS
30
+
MRS
30
+
MRS
30
an us
MRS
M
ep te d
30
L. cremoris RSSK 708
+
+
MRS
30
L. salivarious TNO M7
-
-
MRS
30
L. plantarum LMG 2003
+
+
MRS
30
L. sakei LMG 2313
+
+
MRS
30
+
+
MRS
30
E. faecalis LMG 2602
+
+
GM17
30
L. innocua LMG 2813
+
+
GM17
30
L. monocytogenes RSSK
+
+
GM17
30
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sensitivity
Growth
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Indicator organism
L. mesenteroides RSSK 923
22
471 L. monocytogenes RSSK
+
+
GM17
30
+
+
GM17
30
+
+
B. cereus LMG 2732
+
+
S. aureus
+
P. pentosaceous LMG
-
L. monocytogenes RSSK 475
30
+
GM17
30
-
TSB
30
-
TSB
30
-
-
TSB
30
-
-
TSB
30
-
-
TSB
30
-
-
TSB
30
-
-
TSB
30
-
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E. coli LMG 3083
M
30
-
S. paratyphi GATA
GM17 GM17
P. fluorescens LMG 3020
3085
30
+
2001
S. enterica typh LMG
GM17
an us
478
A2961
S. paratyphi GATA
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L. monocytogenes RSSK
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472
B33653
S. paratyphi GATA B33656 S. paratyphi GATA B34323
23
S. paratyphi GATA
-
-
TSB
30
-
-
TSB
30
-
-
TSB
30
-
-
TSB
30
B38359 S. paratyphi GATA
S. paratyphi GATA
S. paratyphi GATA
-
-
TSB
30
-
TSB
30
-
TSB
30
-
-
TSB
30
-
-
TSB
30
38383 S. enteritidis GATA
-
38055 -
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S. enteritidis GATA
M
S. enteritidis GATA
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B4620
33280
S. cholerasuis GATA 34088
S. cholerasuis GATA 21 a
+, presence of inhibition; -, absence of inhibition; -+,presence of weak inhibition;
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B38518
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B38470
b
MRS: de Man-Rogosa-Sharpe broth (Merck); GM17: M17+0.5% glucose; TSB:
Tryptone soy broth (Oxoid); NB: Nutrient Broth (Oxoid). ATCC: American type Culture Collection, Rockville, Maryland, USA; DSM: Deutsche Sammlung von Mikroorganismen, Braunschweig, Germany; TNO: Netherlands Organisation for Applied Scientific Research; LMG: Laboratory of Microbial Gene
24
Technology, Department of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences, As, Norway; RSSK: Refik Saydam National Type Culture Collection, Ankara, Turkey; GATA: Gülhane Military Medical Academy, Ankara,
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Turkey.
25
Figure 1. First run of RP-HPLC profile of the first purification protocol. Bacteriocin
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activity was highest in fractions 12 (6400 BU/mL).
26
Figure 2. Second run of RP-HPLC profile of the first purification protocol. Bacteriocin
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activity was highest in fraction 18 (3200 BU/mL).
27
Figure 3. MALDI-TOF/MS analysis of the active fraction (fraction 18) obtained from the second run RP-HPLC of the first purification protocol. The m/z value of 5462 Da
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corresponds to [M + H] + indicating a molecular mass of 5461 Da.
28
Figure 4. Reverse-phase HPLC profile of pH dependent cell adsorption-desorption extract (pH 2 extract) with Vydac C18 column incorporated in Agilent 1200 Series Quaternary HPLC system. Bacteriocin activity was detected in the fraction indicated with
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arrow.
29
Figure 5. MALDI-TOF/MS analysis of the active fraction from reverse-phase HPLC of the second purification protocol. The m/z value of 5466 Da corresponds to [M + H] +
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indicating a molecular mass of 5465 Da.
30
Figure 6. Effect of pH mediated cell surface adsorption-desorption fraction containing enterocin B against growing cells of L. monocytogenes RSSK 475 with 300 BU/mL final concentration. Symbols: ● and ○, cell viability (log10 CFU/mL); ▲ and Δ, Turbidity (A600 nm) (appropriate broth); ○ and Δ, culture with bacteriocin; ● and ▲, culture
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without bacteriocin.
31
Preparative Biochemistry and Biotechnology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb20
Comparison of Two Methods for Purification of Enterocin B, A Bacteriocin Produced by Enterococcus Faecium W3 a
b
b
b
c
Halil Dündar , Mehmet Atakay , Ömür Çelikbiçak , Bekir Salih & Faruk Bozoğlu a
Department of Biotechnology, Middle East Technical University, Ankara, Turkey
b
Department of Chemistry, Hacettepe University, Beytepe, Ankara, Turkey
c
Department of Food Engineering, Middle East Technical University, Ankara, Turkey Accepted author version posted online: 02 Sep 2014.
To cite this article: Halil Dündar, Mehmet Atakay, Ömür Çelikbiçak, Bekir Salih & Faruk Bozoğlu (2014): Comparison of Two Methods for Purification of Enterocin B, A Bacteriocin Produced by Enterococcus Faecium W3, Preparative Biochemistry and Biotechnology, DOI: 10.1080/10826068.2014.958165 To link to this article: http://dx.doi.org/10.1080/10826068.2014.958165
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COMPARISON OF TWO METHODS FOR PURIFICATION OF ENTEROCIN B, A BACTERIOCIN PRODUCED By Enterococcus faecium W3 Halil Dündar1, Mehmet Atakay2, Ömür Çelikbiçak2, Bekir Salih2, Faruk Bozoğlu3 1
Department of Biotechnology, Middle East Technical University, Ankara, Turkey, Department of Chemistry, Hacettepe University, Beytepe, Ankara, Turkey, 3Department of Food Engineering, Middle East Technical University, Ankara, Turkey
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2
Abstract
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This study aimed to compare two different approaches for the purification of enterocin B from Enterococcus faecium strain W3 based on the observation that the bacteriocin was found both in cell associated form and in culture supernatant. The first approach employed ammonium sulfate precipitation, cation-exchange chromatography and
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sequential reverse-phase high performance liquid chromatography. The latter approach exploited pH mediated cell adsorption-desorption method to extract cell bound
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bacteriocin, and one run of reverse-phase chromatography. The first method resulted in purification of enterocin B with a recovery of 4% of the initial bacteriocin activity found in culture supernatant. MALDI-TOF MS analysis and de novo peptide sequencing of the purified bacteriocin confirmed that the active peptide was enterocin B. The second method achieved the purification of enterocin B with a higher recovery (16%) and
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Correspondence Addressed to Halil Dündar: E-mail: [email protected]
enabled us to achieve pure bacteriocin within a shorter period of time by avoiding time consuming purification protocols. The purity and identity of the active peptide was confirmed again by MALDI-TOF MS analysis. Although both approaches were satisfactory to obtain sufficient amount of enterocin B for use in MS and amino acid
1
sequence analysis, the latter was proved to be applicable in large-scale and rapid purification of enterocin B.
INTRODUCTION
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Bacteriocins from lactic acid bacteria (LAB) are of great importance because of their use in food preservation.[1] Bacteriocins are ribosomally synthesized antimicrobial peptides displaying antagonistic effect against other bacteria.[2] They exhibit bactericidal activity on the sensitive cells through depolarization of cell membrane or through the inhibition
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of cell wall synthesis. The bacteriocins from LAB have received considerable attention regarding food safety because of the generally recognized as safe (GRAS) status of these
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bacteria.[3] Bacteriocins produced from Enterococcus spp. are the most widely produced and characterized among LAB bacteriocins.[4] Among them, enterocin B shows important properties such as stability and solubility over a wide pH range, heat stability and inhibition spectrum, making it an attractive candidate for food preservation. Enterocin B was shown to inhibit the growth of Listeria monocytogenes, Enterococcus faecalis,
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chromatography, MALDI-TOF, peptide sequencing
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KEYWORDS: bacteriocin, enterocin B, bacteriocin purification, reverse-phase
Clostridium sporogenes, Clostridium tyrobutyricum, Staphylococcus aureus.[5] It showed significant antilisterial effect in minced pork meat, pork liver patè cooked ham and debondened chicken breasts[6] as well as inhibiting the production of slime by L. sakei CTC746 in vacuum packaged sliced cooked pork ham.[7] The synergistic action of enterocin B with high hydrostatic pressure (HHP) was shown in cooked ham model with
2
reduction in the intensity of HHP treatment.[8] The first attempts to purify enterocin B employed its cationic and hydrophobic nature and hence, cation-exchange and hydrophobic interaction chromatography with repeated reverse-phase chromatography steps were applied on the concentrated culture supernatant.[5] Various methods were
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reported for the purification of enterocins: enterocin A purification; ammonium sulfate
exchange column), hydrophobic interaction chromatography (Octyl-Sepharose CL-4B
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column), followed by C 2/C18 reverse-phase chromatography,[9] enterocin B purification;
extraction with Amberlite XAD-16, cation-exchange chromatography (SP-Sepharose Fast Flow cation-exchange column), hydrophobic interaction chromatography (OctylSepharose CL-4B column), followed by C2/C18 reverse-phase chromatography,[5]
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enterocins L50A and L50B purification; extraction with Amberlite XAD-16, cationexchange chromatography (SP-Sepharose Fast Flow cation-exchange column),
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hydrophobic interaction chromatography (Octyl-Sepharose CL-4B column), followed by C2/C18 reverse-phase chromatography,[10] enterocin I purification; ammonium sulfate precipitation, cation-exchange chromatography (SP-Sepharose Fast-Flow column), hydrophobic interaction chromatography (Phenyl-Sepharose CL-4B column), followed by C2/C18 reverse-phase chromatography,[11] enterocin P purification; ammonium sulfate
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precipitation, cation-exchange chromatography (SP-Sepharose Fast Flow cation-
precipitation, gel filtration (G25 PD10 columns), cation exchange chromatography (SPSepharose Fast Flow column), hydrophobic-interaction chromatography (OctylSepharose CL-4B column), followed by C2/C18 reverse-phase chromatography.[12] Although these methods worked to achieve purification to homogeneity for elucidation of primary structure, the yields were variable and low, the protocols being time consuming
3
and not suitable for large-scale applications. Therefore, methods for rapid and large-scale purification of bacteriocins need to be devised for use in controlling food spoilage and in clinical applications.
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Micro-Cel,[13] Rice hull ash and silicic acid[14] were exploited for extraction of
was studied also.[15,16] Bacteriocins of lactic acid bacteria are adsorbed on the cells of
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producer strain depending of pH of the environment, adsorption occurs at around pH 6.0 and desorption occurs around pH 2.0. Based on this property of LAB bacteriocins,
pediocin AcH, leuconocin Lcm1 and sakacin A, and the lantibiotic nisin were purified on
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a large-scale.[17]
In this study we tested the utility of pH mediated cell adsorption-desorption method with
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the subsequent reverse-phase high performance liquid chromatography to purify enterocin B. The method was compared with the usual purification protocol consisting of ammonium sulfate precipitation, cation-exchange chromatography and sequential reverse-phase high performance liquid chromatography.
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bacteriocins from culture supernatant. Extraction with aqueous two-phase partitioning
MATERIALS AND METHODS
Microorganisms
Bacteriocin producer Enterococcus faecium W3 was a food strain previously isolated in our laboratory Middle East Technical University, Food Microbiology Labotatory) and
4
identified as E. faecium by gram staining, endospore staining, catalase reaction, heterofermentative/homofermentative distinguishing and 16S rDNA analysis.
Bacteriocin Activity Assay
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Bacteriocin activity was evaluated by a microtiter plate assay. Two-fold serial dilutions
μL de Man Rogasa Sharp (MRS) broth. 150 μL of indicator strain Lactobacillus
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delbrueckii subsp. delbrueckii RSSK 498 (100-fold diluted) was added to the bacteriocin containing wells and the plate was incubated at 30°C for 6 h. Bacteriocin activity was evaluated by measuring the growth at 620 nm by a microtiter plate reader reader (Multiscan Ascent; Labsystems, Helsinki, Finland). One bacteriocin unit (BU) was
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defined as the amount of bacteriocin inhibiting the growth of the indicator strain by 50%.
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Temperature And Ph Optimization For Bacteriocin Production MRS broth adjusted to an initial pH of 6.5 was inoculated (1%, v/v) with an overnight culture of strain W3 and incubated at 10, 15, 20, 25, 30 and 37°C without pH control for 16 h. For determination of the effect of initial pH on bacteriocin production, MRS broth adjusted to pH 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 was inoculated (1%, v/v) with an overnight
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of preparations containing bacteriocin were added to microtiter plate wells containing 50
culture of strain W3 and incubated at 30°C without pH regulation for 16 h. Bacteriocin activity was measured as described above.
Bacteriocin Purification
5
Purification By Ammonium Sulfate Precipitation, Cation Exchange Chromatography On SP-Sepharose Fast Flow Column And Reverse-Phase High Performance Liquid Chromatography On Akta Purifier. 1000 mL of MRS broth was inoculated with an overnight culture of E. faecium W3 (1%
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v/v) and incubated for 16 h with an initial pH of 6.5 at 30°C. Bacteriocin containing
4°C. Ammonium sulfate (40 g per 100 mL) was added to 500 mL of culture supernatant
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in centrifuge tubes and then shaked at 4°C for 40 min. The mixture was centrifugated
(13000 rpm for 30 min at 4°C) to precipitate the bacteriocin from supernatant. The pellet was dissolved in 25 mL of sterile distilled water and diluted to 100 mL with sterile distilled water. This sample was applied to 5 mL of SP-Sepharose Fast Flow (GE
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Healthcare Biosciences) column, which was equilibrated with 10 mM acetic acid. The column was washed with 2 column volumes (CV) of 10 mM sodium phosphate buffer
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(pH 6.0) and 2 CV of sodium phosphate buffer (10 mM, pH 6.0) containing 100 mM NaCI. Subsequently, the column was eluted with a step-wise gradient of 2 CV of 300 mM NaCI and 2 CV of 1.0 M NaCI. Fractions of 2.5 mL at a flow rate of 1 mL were collected and assayed for bacteriocin activity. Fractions with the highest bacteriocin activity from SP Sepharose Fast flow column, the last 7.5 mL (300 mM NaCI) and the first 5 mL (1.0
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supernatant was obtained after centrifugation of the culture at 13000 rpm for 30 min at
M NaCI), were combined and applied to Resource 15 RPC 3 mL reverse-phase column (100 mm length, 6.4 mm i.d., and with 15 µm pore size) equilibrated with 0.1% (v/v) (TFA) in distilled water. Elution was carried out with a 5 CV linear gradient of 15 to 55% 2-propanol with 0.1% TFA at a flow rate of 1 mL/min using Äkta Purifier fast protein liquid chromatography system. Fractions of 1 mL were collected and assayed for
6
bacteriocin activity. Active fractions from first reverse-phase chromatography were applied to reverse-phase chromatography under the same conditions and bacteriocin activity was assayed as described above. Purified bacteriocin was subjected to MALDI-
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TOF mass spectrometry and amino acid sequencing analysis.
Phase High Performance Liquid Chromatography
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The second purification protocol employed cell-associated form of the bacteriocin as an initial sample to extract relatively pure bacteriocin based on our observation that the bacteriocin produced by E. faecium W3 was detected in cell associated form (bound to
producer cell surface) as well as in culture supernatant fluid. The pelleted cells of the 1 L
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of culture was mixed with 500 mL of culture supernatant left from the same fermentation flask. The pH of the heat treated culture was adjusted to 6.0 by addition of 1.0 M NaOH
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solution. The culture was stirred for 1 h at 4°C to allow the bacteriocin to adsorb onto bacterial cells. The cells were collected by centrifugation and washed with 200 mL of 5.0 mM sodium phosphate buffer (pH 6.0) to remove impurities. After centrifugation for 30 min at 4°C (13000 rpm), the pelleted cells were resuspended in 100 mL of 5.0 mM sodium phosphate buffer (pH 6.0) containing 100 mM NaCI, and the pH was adjusted to
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Purification By Ph Mediated Cell Adsorption-Desorption Method And Reverse-
2.0 by gradual addition of HCI. The cell suspension was stirred in cold room (4°C) overnight, centrifuged and filtered. The extract was concentrated with boiling to a volume of 12 mL and assayed for bacteriocin activity. After freeze-drying of this fraction, it was resuspended in 1 mL of 0.1% TFA and applied to reverse-phase high performance liquid chromatography by injecting 100 µL aliquots into Vydac C18 column (150 mm length,
7
4.6 mm i.d., and with 5 µm pore size) incorporated in Agilent 1200 Series Quaternary HPLC system. Detection was carried out at wavelength range between 190-700 nm. Mobile phase was composed using varying amounts of water with 0.1% TFA (v/v) (A) and acetonitrile (ACN) with 0.085% TFA (v/v) (B). Mobile phase was varied in the
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following gradients: 0 to 5 min, 96% to 4% (v/v); 5 to 80 min, 60% to 40% (v/v); 80 to
collected in separate vials for 1 min intervals and assayed for bacteriocin activity. Protein
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concentrations were measured spectrophotometrically at 280 nm. Purified bacteriocin
was subjected to MALDI-TOF mass spectrometry and amino acid sequencing analysis.
MALDI-TOF Analysis
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Active fractions were dried in water bath at 70°C, and finally in vacuum oven at 30°C. Mass spectrometric analyses were performed by dissolving the dried peptide in 200 μL
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ACN:H2O mixture in 50:50 (v/v) containing 0.1% TFA (v/v). Mass spectra of purified bacteriocin were acquired on a Voyager-DETM PRO MALDI-TOF mass spectrometer (Applied Biosystems, USA) as described.[18]
Amino Acid Sequencing
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90 min, 10% to 90% (v/v); and 90 to 100 min, 10% to 90% (v/v). Peptide fractions were
Mass spectrometry data were acquired using a Q-STAR Elite Q-TOF mass spectrometer (Applied Biosystems) with a nano-ESI source. MS/MS data were obtained via Information Dependent Acquisition (IDA) method, where doubly and triply charged parent ions were selected for fragmentation by collision induced dissociation (CID) using nitrogen as collision gas. Protein identification was performed by searching the NCBI
8
database using the Mascot search engine with the following search criteria: monoisotopic mass, peptide charge +2, 50 ppm mass accuracy, trypsin as digesting enzyme with 1 missed cleavage allowed, carbamidomethylation of cysteine as a fixed modification, oxidation of methionine as allowable variable modification. For all de novo algorithms,
cr ip t
methionine oxidation was selected as variable modification and carbamidomethylation of
tolerance and MS/MS tolerance of 0.3 Da. A maximum of one missed-cleavage was
an us
allowed. All data were searched for tryptic peptides.
Mode Of Action Of Ph Mediated Cell Surface Adsorption-Desorption Fraction Cell suspension of Listeria monocytogenes RSSK 475 and Enterococcus faecalis was
M
grown to exponential growth phase. Monitoring of bacterial growth was carried out by determining the absorbance at 600 nm (A600). Exponentially growing culture of the
ep te d
organism was divided into two equal parts and pH mediated cell surface adsorptiondesorption fraction from E. faecium W3 was added to each part with a final concentration of 300 AU mL. Control culture and bacteriocin treated culture were incubated and at selected intervals samples were taken from both cultures for total viable colony counting and A600 measurement. Total viable counts (CFU/mL) were determined by pour plating
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cysteines as fixed modification. QSTAR data were de novo sequenced using peptide
technique using the serial dilutions of indicator organism. A600 values were measured by a spectrophotometer (path length, 1 cm; UV-160 UV-visible spectrophotometer; Shimadsu). All assays were performed in duplicate.
RESULTS AND DISCUSSION
9
Effect Of Temperature And Ph On Bacteriocin Production Growth and bacteriocin production occurred at temperatures between 20 and 37 oC with an initial pH of 6.5-7.0 under uncontrolled conditions. Production of bacteriocin by the producer organism resulted in 320 AU/mL bacteriocin activity at 30 and 37 oC in CSF and
cr ip t
160 AU/mL at 20 and 25 oC). The producer organism produced the same amount of
an us
Nevertheless, no bacterial growth and bacteriocin production were detected under pH 5.0.
Purification By Ammonium Sulfate Precipitation, Cation-Exchange
Chromatography On SP-Sepharose Fast Flow, And Three Consecutive ReversePhase HPLC On Resource RPC 3 Ml Column Using Äkta Purifier.
M
The culture supernatant had an activity of 320 BU/mL and ammonium sulfate precipitation resulted in 80% recovery of the initial bacteriocin activity. The precipitate
ep te d
was subjected to cation-exchange chromatography on SP Sepharose Fast Flow column. The recovery by cation-exchange chromatography was low (14%) due to activity loss in flow-through. The results of the protocol is shown in Table 1. The highest bacteriocin activity from SP Sepharose Fast flow column, the last 7.5 mL (300 mM NaCl) and the first 5 mL (1.0 M NaCl), were pooled (19200 BU/mL) and applied to Resource 15 RPC 3
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bacteriocin whent the initial pH was between 5.5 and 7.0 (320 AU/mL) at 30 oC.
mL reverse-phase column equilibrated with 0.1% (v/v) (TFA) in distilled water. Bacteriocin activity was detected in fractions 11, 12, and 13 (Figure 1). Fraction 12 with the highest bacteriocin activity (6400 BU/mL) was diluted ten times with 0.1% (v/v) TFA and run on Resource 15 RPC 3 mL column with Akta purifier under the same conditions. The second run of reverse-phase HPLC resulted in one symmetrical peak with bacteriocin
10
activity in fraction 18 (6400 BU/mL) (Figure 2), which was concentrated by SpeedVac, applied for MALDI-TOF and de novo peptide sequencing. MALDI-TOF analysis of this active peak resulted in a mass of 5462 Da (Figure 3) and its de novo sequencing produced the following amino acid sequence from position 8 to 21, position 23 to 35 and position
cr ip t
37 to 50: NH2--------NEI/LNRPNNI/LS-----CGAAI/LAGGI/LFGI/LP-
Thus, the sequence and MALDI-TOF mass spectrometry data confirmed that active peak
an us
is identical to enterocin B with the following sequence:
ENDHRMPNELNRPNNLSKGGAKCGAAIAGGLFGIPKGPLAWAAGLANVYSKCN.
Although the protocol worked well enough to be able to identify the bacteriocin, it caused
M
substantial loss of bacteriocin activity with a final recovery of 4%. The remarkable activity loss was observed during cation-exchange chromatography where much
ep te d
bacteriocin activity was lost in flow through (Table 1). Reverse-phase chromatography also contributed to the loss of bacteriocin activity during the first run.
Cell-associated form of the bacteriocin was used as initial extract in the second purification method where the bacteriocin was eluted from the cell surface by low pH
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GPI/LAWAAGI/LANVYS where I/L denotes that the residue is isoleucine or leucine.
(pH 2.0) and 150 mM NaCI. This extraction method recovered 48% of the bacteriocin activity found in culture supernatant. The pH 2 extract was purified to homogeneity with the application of reverse-phase HPLC on Vydac C18 column incorporated in Agilent 1200 Series Quaternary HPLC system. The pH dependent adsorption-desorption method resulted in purification with 21.5 fold increase but the largest increase in specific activity
11
was provided by reverse-phase chromatography with a 4408 fold increase (Table 2). RPHPLC resulted in six peaks, the fourth peak showed bacteriocin activity (Figure 4). The retention time of the bacteriocin on Vydac C18 column was about 80 min, corresponding to 90% acetonitrile. MALDI-TOF MS analysis confirmed the purity and molecular mass
cr ip t
of enterocin B (Figure 5). MALDI mass spectrum of active peptide was obtained using
mass spectrum of the sample was obtained in α-cyano-4-hydroxycinnamic acid matrix
an us
and is given in Figure 5. Three mass value for the active peptide were observed by MALDI-TOF analysis including 5444, 5466 and 5482 Da as a result of the second
purification protocol (Figure 5). The main peak with a molecular mass of 5465 Da is the same as the calculated molecular mass (5465.2 Da). The peak with a molecular mass of
M
5482 Da probably represents Enterocin B with an oxidized methionine residue as reported previously.[5,19] The related molecular mass values of enterocin B including 5466
ep te d
and 5482 Da were observed both in pH mediated cell surface adsorption-desorption extract and in the active fraction from reverse-phase chromatography. MALDI-MS spectra of this active fraction were also carried out at low and at very high mass ranges up to 200000 Da. No other peptide and proteins were observed in the same active peptide fraction (Figure 5). Amino acid sequence analysis of the active fraction showed that it
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positive ion and linear mode in various novel MALDI matrices but the suitable MALDI
was identical to the sequence obtained from the first purification protocol. The pH mediated cell adsorption-desorption method was carried out with some modifications as described previously.[17] It is known that bacteriocin producing strains of lactic acid bacteria adsorb their own bacteriocins around pH 6.0 and release around pH 2.0.[20] Dextranicin 24 produced by Leuconostoc sp.,[21] pediocin AcM produced by
12
Pediococcus acidilactici,[22] leucocins A-, B- and C-TA33a produced by Leuconostoc mesenteroides TA33a[23] and plantaricin C19 produced by Lactobacillus plantarum C19[24] were purified by pH mediated cell adsorption-desorption method and reversephase chromatography. Bovicin 255 produced by Streptococcus sp. was purified by pH
cr ip t
mediated cell adsorption-desorption method, ammonium sulphate precipitation, reverse-
by Pediococcus pentosaceus was purified by pH mediated cell adsorption-desorption
an us
method and gel filtration chromatography on FPLC system.[26] In this study, pH mediated cell adsorption-desorption method provided relatively pure enterocin B as seen after running of the sample by reverse-phase chromatography on Vydac column. Enterocin B obtained by pH mediated cell adsorption-desorption method can be used in ex situ
M
applications taken into account its purity and higher yield without performing expensive purification protocols. Most purification schemes devised for purification of enterocins
ep te d
and other bacteriocins from lactic acid bacteria take advantage of the cationic and relatively hydrophobic natures of these molecules, and hence involve applications of cation-exchange chromatography, hydrophobic interaction chromatography and reversephase high performance liquid chromatography. Although these methods has been successful for the purification of many bacteriocins for identification, they are time
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phase and gel filtration chromatography on FPLC system.[25] Pediocin ACCEL produced
consuming, costly and not suitable for large-scale production.
Inhibition Spectrum Of Cell Surface Adsorbed-Desorbed Enterocin B Both partially purified bacteriocin (pH mediated cell surface adsorption desorption fraction) and its processed product through reverse-phase chromatography was tested
13
against a set of Gram-positive and Gram-negative bacteria. Pure enterocin B and cell surface adsorption-desorption fraction showed the same inhibitory spectrum and inhibited the growth of Lc. mesenteroides, L. delbrueckii, L. cremoris, L. monocytogenes, L. innocua, E. faecalis, B. cereus and some lactic acid bacteria. However, no Gram-negative
cr ip t
bacteria tested were inhibited by pure enterocin B and the cell surface adsorption-
an us
Mode Of Action
Turbidity of the bacteriocin treated culture of L. monocytogenes declined rapidly after the first 20 min (Figure 6). The decrease in A600 continued throughout the incubation as opposed the control culture. L. monocytogenes RSSK 475 lost its viability by 99.99 %
M
within the first 20 min; viable colony count dropped rapidly from 8x10 8 to 104 (a 5.9 log reduction). After 4 hours, the viable colony count of the bacteriocin treated culture was
ep te d
400 CFU/mL, that is a 6.3 log reduction in viability (Figure 6).
CONCLUSION
Comparison of the two purification approach indicated that pH mediated cell surface adsorption-desorption method together with RP-HPLC can be used successfully for rapid
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desorption fraction (Table 3).
purification of enterocin B, which is a non-pediocin like one peptide bacteriocin (class IIc), as performed for many pediocin-like bacteriocins. The application of pH mediated cell surface adsorption-desorption method avoided time consuming ammonium sulfate precipitation, cation-exchange chromatography and hydrophobic interaction chromatography steps which might cause activity losses during the purification. Although
14
both methods achieved purification of enterocin B with a similar specific activity, a significant improvement in the yield of enterocin B was achieved (16%) by pH mediated cell surface adsorption with a following reverse-phase chromatography compared with the yield obtained by ammonium sulfate precipitation, cation-exchange chromatography
cr ip t
and final sequential reverse-phase chromatography (4%). Both purification approaches is
high purity and yield of the bacteriocin preparation obtained from pH mediated cell
an us
surface adsorption-desorption method make it possible its use in a concentrated form as a
food preservative. The cell of the producer strain functioned as a specific ligand to adsorb the enterocin B from culture supernatant, and provided both purification and concentation of the molecule. It is known that some stains of E. faecium produces enterocin A together
M
with enterocin B which shows ynergistic action towards pathogenic and food-spoilage bacteria when used in combination. The rapid purification method mentioned here can be
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exploited for the large-scale purification of enterocin A and enterocin B together as well as for the bacteriocins of multiple bacteriocin producer strains. Combined and partial purification of different bacteriocins might be an alternative approach to avoid development of bacteriocin resistant strains. The feasibility of pH mediated cell surface adsorption and desorption method can be analysed for the multiple bacteriocins from
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successful to the extent that primary structure of the bacteriocin was obtained. However,
different LAB bacteria by mixing relavant bacteriocin supernatants and optimization of adsorption onto a suitable producer cell, which can provide large-scale and concomitant purification of a bacteriocin to achieve a strong bacteriocin cocktail targeting the resistant cells.
15
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Aymerich, M.T.; Garriga, M.; Costa, S.; Monfort, J.M.; Hugas, M. Prevention of
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2.
ropiness in cooked pork by bacteriocinogenic cultures. Int. Dairy J. 2002, 12, 239–246. 8.
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synergism through bacteriocins and high pressure in a meat model system during storage. Food Microbiol. 2002, 19, 509–518.
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9.
Aymerich, T.; Holo, H.; Håvarstein, L.S.; Hugas, M.; Garriga, M.; Nes, I.F.
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cr ip t
10.
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Floriano, B.; Ruiz-Barba, J.L.; Jiménez-Díaz, R. Purification and genetic
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11.
characterization of enterocin I from Enterococcus faecium 6T1a, a novel antilisterial plasmid-encoded bacteriocin which does not belong to the pediocin family of bacteriocins. Appl. Environ. Microbiol. 1998, 64, 4883–4890.
Herranz, C.; Mukhopadhyay, S.; Casaus, P.; Martínez, J.M.; Rodríguez, J.M.;
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Nes, I.F.; Cintas, L.M.; Hernández, P.E. Biochemical and genetic evidence of enterocin P
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production by two Enterococcus faecium-like strains isolated from fermented sausages. Curr. Microbiol. 1999, 39, 282–290. 13.
Coventry, M.J.; Gordon, J.B.; Alexander, M.; Hickey, M.W.; Wan, J. A food-
grade process for isolation and partial purification of bacteriocins of lactic acid bacteria that uses diatomite calcium silicate. Appl. Environ. Microbiol. 1996, 62, 1764–1769.
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Enterocins L50A and L50B, two novel bacteriocins from Enterococcus faecium L50, are
14.
Janes, M.E.; Nannapaneni, R.; Proctor, A.; Johnson, M.G. Rice hull ash and
silicic acid as adsorbents for concentration of bacteriocins. Appl. Environ. Microbiol. 1998, 64, 4403–4409.
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15.
Métivier, A.; Boyaval, P.; Duffes, F.; Dousset, X.; Compoint, J.P.; Marion, D.
Triton X-114 phase partitioning for the isolation of a pediocin-like bacteriocin from Carnobacterium divergens. Lett. Appl. Microbiol. 2000, 30, 42–46. 16.
Li, C.; Bai, J.; Li, W.; Cai, Z.; Ouyang, F. Optimization of conditions for
17.
Yang, R.; Johnson, M.C.; Ray, B. Novel method to extract large amounts of
18.
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bacteriocins from lactic acid bacteria. Appl. Environ. Microbiol. 1992, 58, 3355–3359. Ceyhan, T.; Yüksek, M.; Yağlıoğlu, H.G.; Salih, B.; Erbil, M.K.; Elmalı, A.;
Bekaroğlu, Ö. Synthesis, characterization and nonlinear absorption of novel octakisPOSS substituted metallophthalocyanines and strong optical limiting property of CuPc.
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W.H.; Vederas, J.C.; Stiles, M.E. Atypical genetic locus associated with constitutive production of enterocin B by Enterococcus faecium BFE 900. Appl. Environ. Microbiol. 1999, 65, 2170–2178. 20.
Klaenhammer, T.R. Genetics of bacteriocins produced by lactic acid bacteria.
FEMS Microbiol. Rev. 1993, 12, 39–85.
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experimental designs). Biotechnol. Prog. 2001, 17, 366–368.
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bacteriocin extraction in PEG (Salt aqueous two-phase systems using statistical
21.
Revol-Junelles, A.M.; Lefebvre, G. Purification and N-terminal amino acid
sequence of dextranicin 24, a bacteriocin of Leuconostoc sp. Curr. Microbiol. 1996, 33, 136–137.
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Elegado, F.B.; Kim, W.J.; Kwon, D.Y. Rapid purification, partial characterization,
and antimicrobial spectrum of the bacteriocin, Pediocin AcM, from Pediococcus acidilactici M. Int. J. Food Microbiol. 1997, 37, 1–11. 23.
Papathanasopoulos, M.A.; Dykes, G.A.; Revol-Junelles, A.M.; Delfour, A.; Von
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Holy, A.; Hastings, J.W. Sequence and structural relationships of leucocins A-, B- and C-
24.
Atrih, A.; Rekhif, N.; Moir, A.J.G.; Lebrihi, A.; Lefebvre, G. Mode of action,
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purification and amino acid sequence of plantaricin C19, an anti-Listeria bacteriocin
produced by Lactobacillus plantarum C19. Int. J. Food Microbiol. 2001, 68, 93-104. 25.
Whitford, M.F.; McPherson, M.A.; Forster, R.J.; Teather, R.M. Identification of
bacteriocin-like inhibitors from rumen Streptococcus spp. and isolation and
26.
M
characterization of bovicin 255. Appl. Environ. Microbiol. 2001, 67, 569–574. Wu, C.W.; Yın, L.J.; Jiang, S.T. Purification and characterization of bacteriocin
ep te d
from Pediococcus pentosaceus ACCEL. J. Agric. Food Chem. 2004, 52, 1146–1151.
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TA33a from Leuconostoc mesenteroides TA33a. Microbiol. 1998, 144, 1343–1348.
19
TABLE 1. First purification protocol of enterocin B
Stage Culture
Total act
Protein
Sp acta
Yield
(BU)
(mg/mL)
(BU/mg)
(%)
500
160000
30.83
10.37
25
128000
6.22
823.15
12.5
22400
0.42
4266
First run
3
12800
Second run
1
6400
supernatant
SPSepharose
a
14
411
4740
8
457
0.12
53333
4
5143
ep te d
Specific activity is total BU divided by the total protein.
b
79
0.9
M
RP-FPLC b
1
80
an us
(NH4)2SO4
RP-FPLC, fast-performance liquid chromatography.
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Precipit.
100
Fold
cr ip t
Purification Vol (mL)
20
TABLE 2 Second purification protocol of enterocin B Purification
Vol
Total
Stage
(mL)
act
Protein
Sp acta
(mg/mL) (BU/mg)
Yield
fold
(%)
500
160000
30.83
10.37
100
1
pH 2 extract
12
76800
28.66
223.3
48
21.5
RP-HPLC b
1
25600
0.56
45714
16
4408
a
an us
Specific activity is total BU divided by the total protein.
cr ip t
Culture sup.
b
ep te d
M
RP-FPLC, fast-performance liquid chromatography.
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(BU)
21
TABLE 3. Inhibition spectrum of enterocin B and enterocin pool from E. faecium W3. pH 2 extract
Purified entB
Medium b
a
a
temp(oC)
L. fermentum
-
-
L. casei
-
-
L. acidophilus
-
-
L. plantarum LP73
-
-
L. plantarum Z11L
-
-
L. plantarum HU
-
L. plantarum RSSK 675
+
L. plantarum RSSK 10
+
L. plantarum 80B
+
L. delbrueckii RSSK 498
+
sensitivity
MRS
30
MRS
37
MRS
30
MRS
30
-
MRS
30
+
MRS
30
+
MRS
30
+
MRS
30
+
MRS
30
an us
MRS
M
ep te d
30
L. cremoris RSSK 708
+
+
MRS
30
L. salivarious TNO M7
-
-
MRS
30
L. plantarum LMG 2003
+
+
MRS
30
L. sakei LMG 2313
+
+
MRS
30
+
+
MRS
30
E. faecalis LMG 2602
+
+
GM17
30
L. innocua LMG 2813
+
+
GM17
30
L. monocytogenes RSSK
+
+
GM17
30
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sensitivity
Growth
cr ip t
Indicator organism
L. mesenteroides RSSK 923
22
471 L. monocytogenes RSSK
+
+
GM17
30
+
+
GM17
30
+
+
B. cereus LMG 2732
+
+
S. aureus
+
P. pentosaceous LMG
-
L. monocytogenes RSSK 475
30
+
GM17
30
-
TSB
30
-
TSB
30
-
-
TSB
30
-
-
TSB
30
-
-
TSB
30
-
-
TSB
30
-
-
TSB
30
-
ep te d
E. coli LMG 3083
M
30
-
S. paratyphi GATA
GM17 GM17
P. fluorescens LMG 3020
3085
30
+
2001
S. enterica typh LMG
GM17
an us
478
A2961
S. paratyphi GATA
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L. monocytogenes RSSK
cr ip t
472
B33653
S. paratyphi GATA B33656 S. paratyphi GATA B34323
23
S. paratyphi GATA
-
-
TSB
30
-
-
TSB
30
-
-
TSB
30
-
-
TSB
30
B38359 S. paratyphi GATA
S. paratyphi GATA
S. paratyphi GATA
-
-
TSB
30
-
TSB
30
-
TSB
30
-
-
TSB
30
-
-
TSB
30
38383 S. enteritidis GATA
-
38055 -
ep te d
S. enteritidis GATA
M
S. enteritidis GATA
an us
B4620
33280
S. cholerasuis GATA 34088
S. cholerasuis GATA 21 a
+, presence of inhibition; -, absence of inhibition; -+,presence of weak inhibition;
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B38518
cr ip t
B38470
b
MRS: de Man-Rogosa-Sharpe broth (Merck); GM17: M17+0.5% glucose; TSB:
Tryptone soy broth (Oxoid); NB: Nutrient Broth (Oxoid). ATCC: American type Culture Collection, Rockville, Maryland, USA; DSM: Deutsche Sammlung von Mikroorganismen, Braunschweig, Germany; TNO: Netherlands Organisation for Applied Scientific Research; LMG: Laboratory of Microbial Gene
24
Technology, Department of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences, As, Norway; RSSK: Refik Saydam National Type Culture Collection, Ankara, Turkey; GATA: Gülhane Military Medical Academy, Ankara,
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Turkey.
25
Figure 1. First run of RP-HPLC profile of the first purification protocol. Bacteriocin
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activity was highest in fractions 12 (6400 BU/mL).
26
Figure 2. Second run of RP-HPLC profile of the first purification protocol. Bacteriocin
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activity was highest in fraction 18 (3200 BU/mL).
27
Figure 3. MALDI-TOF/MS analysis of the active fraction (fraction 18) obtained from the second run RP-HPLC of the first purification protocol. The m/z value of 5462 Da
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corresponds to [M + H] + indicating a molecular mass of 5461 Da.
28
Figure 4. Reverse-phase HPLC profile of pH dependent cell adsorption-desorption extract (pH 2 extract) with Vydac C18 column incorporated in Agilent 1200 Series Quaternary HPLC system. Bacteriocin activity was detected in the fraction indicated with
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arrow.
29
Figure 5. MALDI-TOF/MS analysis of the active fraction from reverse-phase HPLC of the second purification protocol. The m/z value of 5466 Da corresponds to [M + H] +
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indicating a molecular mass of 5465 Da.
30
Figure 6. Effect of pH mediated cell surface adsorption-desorption fraction containing enterocin B against growing cells of L. monocytogenes RSSK 475 with 300 BU/mL final concentration. Symbols: ● and ○, cell viability (log10 CFU/mL); ▲ and Δ, Turbidity (A600 nm) (appropriate broth); ○ and Δ, culture with bacteriocin; ● and ▲, culture
cr ip t an us M
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without bacteriocin.
31
