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Purification and partial amino acid sequence ofpropionicin PLG-1, a bacteriocin produced by

Propionibacterium thoenii P127Hd Paik, Ba Glatz, Non Renseigné

To cite this version:Hd Paik, Ba Glatz, Non Renseigné. Purification and partial amino acid sequence of propionicin PLG-1, a bacteriocin produced by Propionibacterium thoenii P127. Le Lait, INRA Editions, 1995, 75(4_5), pp.367-377. �hal-00929443�

Lait (1995) 75, 367-377© Elsevier/INRA

367

Original article

Purification and· partial amino acid sequenceof propionicin PLG-1, a bacteriocin produced

by Propionibacterium thoenii P127 1

HD Paik, BA Glatz*

Department of Food Science and Human Nutrition, Iowa State University, Ames, Iowa, USA

Summary - Propionicin PLG-1 , a bacteriocin produced by Propionibacterium thoenii P127, was puri-fied to homogeneity by ammonium sulfate precipitation foliowed by ion exchange column chromatog-raphy and reversed-phase high-performance Iiquid chromatography. According to amino acid compositionanalysis, propionicin PLG-1 has a calculated molecular weight of 9328 and eontains 99 ami no acidresidues, of whieh 42% are hydrophobie (Ala, Ile, Leu, Val and Pro). A 10-amino acid sequence fromthe N-terminal endwas identified: NH2-1Asn-2VaI-3AspAAla(Thr)_5Arg-6Thr(Cys)-7 Ala(Thr)-8Arg-9Thr(Ala)_10pro. No homology of this sequence to sequences of other bacteriocins from lactic acid bac-teria was seen in a search of the SWISS-PROT data bank.

baeterioein 1 propionibacteria 1 propionicin 1composition 1 purification

Résumé - Purification et séquence partielle des acides aminés de la propionicine PLG-1, unebactériocine produite par Propionibacterium thoenii P127. La propionicine PLG-1, une bactério-cine produite par Propionibacterium thoenii P127, a été purifiée jusqu'à homogénéité par précipitationau sulfate d'ammonium suivie d'une chromatographie sur colonne d'échange d'ions et d'une chroma-tographie liquide haute-performance en phase inverse. D'après l'analyse de la composition en acidesaminés, la propionicine PLG-1 a un poids moléculaire calculé de 9328 et contient 99 résidus d'acidesaminés, dont 42% sont hydrophobes (Ala, Ile, Leu, Val et Pro). Une séquence de 10 acides aminés dela partie N-terminale a été identifiée: NH2-1Asn2Vaf-3AspAAla(Thr)-SArg-6Thr(Cysj-lAla(Thr).8Arg-9Thr(Ala)-10Pro. Aucune homologie de séquence avec d'autres bactériocines de bactéries lactiques n'aété trouvée en consultant la base de données SWISS-PROT.

bactériocine / bactérie propionique / propionicine / séquence d'acides aminés / purification

1 Journal paper no J-16306 of the Iowa Agriculture and Home Economies Experiment Station,Ames, Iowa, project no 3129 .• Correspondence and reprints: 2312 Food Sciences Sldg, Iowa State University, Ames, lA 50011,USA

368 HO Paik, BA Glatz

INTRODUCTION

Bacteriocins are defined as bactericidal pro-teins with a narrow spectrum of activity tar-geted toward species related to the pro-ducer culture (Tagg et al, 1976). Becausebacteriocins are natural products of manymicroorganisms associated with foods, thereis currently much interest in their use as nat-ural food preservatives.

Numerous bacteriocins from gram-posi-tive bacteria, particularly from lactic acid bac-teria, have been identified (Klaenhammer,1988), but only a few bacteriocins have beenfound in propionibacteria. Among the dairypropionibacteria, 2 bacteriocins have beenreported: propionicin PLG-1 from P thoeniiP127 (Lyon and Glatz, 1991,1993) andjenseniin G from P jensenii P126 (Grinsteadand Barefoot, 1992). Propionicin PLG-1 isactive against a variety of microorganisms(Lyon and Glatz, 1991) and has been shownto have a molecular weight of 10 000 afterpurification by ion exchange chromatogra-phy and isoelectric focusing (Lyon and Glatz,1993).

Recently, many bacteriocins have beenpurified to homogeneity, and the amino acidsequences of man y of these purified bac-teriocins have been determined. Becausebacteriocins are usually extracellular prod-ucts, the first purification step concentratesthe bacteriocin from the culture super-natants, usually by ammonium sulfate pre-cipitation. Several chromatographie meth-ods, such as gel filtration, ion exchangeand/or hydrophobie interaction chromatog-raphy, have been recommended to achievesignificant further purification. Reversed-phase chromatography was used as thefinal purification step for several bacteri-ocins, including pediocin PA-1 (Lozano etal, 1992), curvacin A (Tichaczek et al, 1992),sakacin A (Holck et al, 1992), plantaricin A(Nissen-Meyer et al, 1993), bavaricin A(Larsen et al, 1993) and piscicolin 61 (Holcketal,1994).

Separation on reversed-phase supportsin high-performance liquid chromatography(HPLC) has also been used to obtain highlypurified preparations of leucocin A-UAL 187(Hastings et al, 1991), lactacin F (Murianaand Klaenhammer, 1991), mesentericinY105 (Hechard et al, 1992), lacticin 481(Piard et al, 1992), salivaricin A (Ross etal, 1993), curvaticin FS47 (Garver andMuriana, 1994) and staphylococcin 1580(Sahl, 1994). The hydrophobie nature ofthese bacteriocins allows their purificationby reversed-phase HPLC.

The goal of this study was the determi-nation of amino acid composition and partialsequence of propionicin PLG-1. For such astudy, highly purified bacteriocin wasneeded. This article reports an improvedprocedure for purification of propionicin PLG-1, as weil as its amino acid composition andN-terminal amino acid sequence.

MATERIALS AND METHODS

Bacterial cultures and media

Producer strain Propionibacterium thoenii P127was maintained as described previously by Lyonand Glatz (1991). Working cultures were propa-gated in sodium lactate broth (NLB) without shak-ing at 32°C. Lactobacil/us delbrueckii ATCC 4797was obtained from Dr S Barefoot (Clemson Uni-versity, Clemson, SC). Stock cultures were main-tained at -60°C in Lactobacilli MRS broth (Difco,Detroit, MI) containing 20% glycerol. Working cul-tures were prepared from stock cultures andgrown in Lactobacilli MRS broth without shakingat37"C.

Production of propionicin PLG-1

Strain P127 was grown in 14 1 of NLB under con-trolled conditions in a 19 1 fermenter (modelNLF22, Bioengineering AG, Wald, Switzerland) inthe Iowa State University Fermentation Facility.The fermentation was started with a 1% (v/v)

Characterization of propionicin PLG-1

inoculum of an 18 h culture in NLB, and was incu-bated for 14 d at 32°C. Agitation was at 100 rpmwithout aeration. The pH was controlled at 7.0 ±0.1 by the addition of 3 molli HCI or 3 molli NaOH.

Ammonium sulfate precipitation

The procedure reported by Lyon and Glatz (1993)was modified as follows: Ammonium sulfate wasadded slowly, with constant stirring, to culturesupernatants (- 1 150 ml) to 75% saturation at4°C over approximately a 10 h period. Slow stir-ring was continued for an additional 3 h. Precip-itated proteins were pelleted by centrifugation at24 000 x 9 for 30 min at 4°C, resuspended in20 mmol/l 2-N-morpholino-ethane-sulfonic acid(MES; Sigma Chemical Co, St Louis, MO) butter,pH 6.5, + 0.1% Tween 80, and dialyzed against 31of 10 mmol/l MES butter, pH 6.5, + 0.1% Tween80, for 12-18 h in Spectra-Por no 3 dialysis tub-ing (molecular mass cutott, 3 500; Spectrum Med-icallndustries, Los Angeles, CA).

Dialysis against polyethylene glycol

When reduction of sample volume was required,the sam pie in dialysis tubing was placed in aweighboat containing about 50 g of polyethyleneglycol (PEG; Mr 15 000-20 000; Sigma ChemicalCo, St Louis, MO) and incubated for 1-3 h at4°C.

Ion exchange chromatography

The procedure reported by Lyon and Glatz (1993)was modified as follows: The column dimensionswere 1.6 x 23 cm and the bed volume of the car-boxymethyl Sepharose (Sigma) was 39 ml. Thecolumn was equilibrated with 20 mmol/l MESbutter (pH 6.5) + 0.1 % Tween 80 and concen-trated partially purified bacteriocin was appliedin a descending mode at 4°C. The column waswashed with several volumes of the same loadingbutter to separate unadsorbed proteins and thenadsorbed proteins were eluted from the column bymeans of a Iinear salt gradient (0 to 1.0 molliNaCI, 500 ml) in MES butter (pH 6.5) + 0.1 %Tween 80. Fractions (4.2 ml) were monitored for

369

protein content by absorbance at 280 nm andassayed for bacteriocin activity.

Reversed-phase high~performanceliquid chromatography

Samples (20 Ill) were applied to a 30 cm IlBon-dapak C18 column (Supelcosil LC-18; Supelco,Inc, Bellefonte, PA), which was equilibrated with0.1% (vlv) trifluoroacetic acid (TFA; Sigma), in aShimadzu HPLC (model LC-600, Shimadzu Cor-poration, Kyoto, Japan). Elution was with a 90 minlinear gradient from 100% butter A to 100% butterB, th en 20 min at 100% buffer B. Butter A was0.1 % (v/v) TFA in water; butter B was 0.1 % TFAin 100% 2-propanol (Fisher Scientific, Fair Lawn,NJ). The flow rate was maintained at 0.4 ml/minand the eluate was monitored at 220 nm bymeans of a Shimadzu UV-Vis Spectrophotometer(model SPD-6AV, Shimadzu Corporation). Frac-tions of 2 ml volume were collected and assayedfor bacteriocin activity.

SDS-PAGE

Aliquots (3-5 Ill) of fractions obtained from HPLCwere subjected to SDS-PAGE. Approximately50-75 ng protein were loaded into each lane.Electrophoresis was performed using 10-20%gradient polyacrylamide gels (Mini-PROTEAN IlReady Gels; Bio-Rad Laboratories, Hercules,CA), with the butter system described by Laemmli(1970) at constant voltage (100 V) for 100 min.Gels were fixed in 30% ethanol-1 0% glacial ace ticacid solution for 1 h and silver stained accordingto the manufacturer's instructions (Bio-Rad).

Protein determination

Protein content of samples at the ditterent purifi-cation steps was determined by the bicinchoninicacid (BCA) assay (Stoscheck, 1990) accordingto the manufacturer's specifications (PierceChemical Co, Rockford, IL). Bovine serum albu-min (BSA; Sigma) was used as the protein stan-dard. Enhanced protocol, which involved colordevelopment at 60°C for 30 min, was used.

370 HO Paik, BA Glatz

Amino acid compositionand sequence analysis

Amino acid composition and sequence analy-sis of purified prapionicin PLG-1 were performedin the Iowa State University Protein Facility.Amino acid analysis was performed with anami no acid analyzer model 420A (Perkin-Elmer,Applied Biosystems Div, Foster City, CA)equipped with an integrated hydrolysis system.The amino acid sequence was determined byEdman degradation (Edman and Begg, 1967)using an Applied Biosystems 477A proteinsequencer (Perkin-Elmer, Applied BiosystemsDiv) with an on-Iine 120A phenylthiohydantoinamino acid analyzer.

The sequence was compared to those in theSWISS-PROT data base, by using the SequenceAnalysis Software Package, licensed fram theGenetics Computer Group (University of Wis-consin, Madison, WI) (Devereux et al, 1984).

Bacteriocin assay

The weil diffusion assay as described by Lyonand Glatz (1993) was modified as follows: Thebasal layer of NLA contained 2.5% agar and 0.1%Tween 80 and was 5 mm deep. Alter pouring theagar layer, plates were incubated 24 h at roomtemperature before wells were eut. Alter 7 mmdiameter wells were eut, plates were incubated at3rC for 2 h or at room temperature for 2 d todry the plates and to facilitate sam pie diffusioninto the agar. Indicator strain was L delbrueckiiATCC 4797, which was added to 5 ml soit agar(0.7% agar) overlays of MRS medium at about107 cells per overlay. Serially diluted samples(200 Ill) were added to wells, allowed to diffuse at4°C, and the base agar was flipped into the Petridish lid before the overlay was applied. Plateswere incubated anaerobically in the BBL Gas-Pak system (Becton Dickinson, Cockeysville, MD)for 12 h at 3rC before diameters of zones ofinhibition were measured. Minimum detectablezone diameter was 9 mm (1 mm beyond weildiameter). Activity units (AU) per ml of originalculture were calculated from the reciprocal of thehighest dilution that praduced a detectable zoneof inhibition. If the inhibition zone at this dilutionwas large (> 11 mm diameter), additional incre-mental dilutions were assayed to define the titermore precisely.

RESULTS

Purification of bacteriocin

Propionicin PLG-1 was previously purified tohomogeneity by ammonium sulfate precip-itation, ion exchange chromatography andisoelectric focusing (Lyon and Glatz, 1993).To obtain highly purified preparations foramino acid composition and sequence anal-ysis, the previously reported purificationscheme was followed but was modified asrequired. Sorne changes in ammonium sul-fate precipitation conditions and ionexchange chromatography were made.Reversed-phase HPLC replaced isoelectricfocusing as the final step.

The purification steps and their associ-ated recoveries of propionicin PLG-1 aregiven in table 1. Propionicin PLG-1 was puri-fied from the supernatant fraction of culturesgrown in semidefined medium, sodium lac-tate broth, to minimize the presence of con-taminating proteins. In preliminary studies,sorne bacteriocin activity was detected inthe proteins precipitated at 50% saturationof ammonium sulfate. Therefore, to avoidloss of bacteriocin, a single precipitation at75% saturation of ammonium sulfate wasused.

Upon consideration of the amount of pro-tein to be applied to the ion exchange Car-boxymethyl Sepharose column and the bedvolume that could accomodate this amount,a smaller column (1.6 x 23 cm, 39 ml bedvolume) was used than in previous work(Lyon and Glatz, 1993). After application ofthe bacteriocin preparation, the column waswashed with MES buffer. A large proteinpeak was eluted within the first 63 ml (15fractions) of buffer (fig 1). This peak of unad-sorbed proteins was completely separatedfrom a protein peak eluted at about 0.04 to0.16 molli NaCI. A small portion at the tail ofthis peak contained 60% of the bacteriocinactivity originally applied to the column.

Table 1. Purification of propionicin PLG-1.Purification de la propionicine PLG-t.

Sample after given step Volume(ml)

Propionicinectivity(AU/ml)

Total propionicinectivlty (AU) a

Protein Totalconcentration protein(mg/ml) b (mg)

Spactivity(AU/mg)

Activityrecovered(%)

Foldpurification

Culture supernatant 1 150 2.5 2875 3.36 3864 0.74 100

Ammonium sulfate 2.7 2560 6912 10.77 29.08 237.69 240.4 321 oprecipitation and PEG zr

Il>concentration P3oIon exchange 21 217 4557 0.2114 4.44 1 026.4 158.5 1387 ~

N'el.

Ion exchange and PEG 0.4 11 393 4557 11.10 4.44 1 026.4 158.5 1387 ëï:::J

concentration C 8-"'C

Injection onto HPLCdël

20 III 11393 227.86 11.10 0.2220 1 026.4 158.5 1387 "'Co':::J

C'8 reversed-phase HPLC 8 25 200 0.0134 0.1072 1 865.7 139.1 2521 o'S'"Ur

a Bacteriocin activity was determined by the weil-diffusion assay. b Protein concentration was determined by BCA method. C Volume ot active samples trom ion exchange col-0~

umn reduced tram 21 to 0.4 ml by PEG concentration. Ali values for ion exchange were recalculated based on this volume change. d Only 20 III of concentrated sam pie appliedto high-performance liquid chromatography (HPLC). Ali values were recalculated based on this volume.a Activité de la bactériocine déterminée par la méthode de diffusion radiale. b Concentration en protéines déterminée par la méthode BGA. C Volume d'échantillon actifprovenant de la colonne d'échange d'ions réduit de 21 ml à 0,4 ml par concentration au PEG. Toutes les valeurs pour l'échange d'ions ont été recalculées en se basant surce changement en volume. d Injection de seulement 20 III d'échantillon concentré. Toutes les valeurs ont été recalculées en se basant sur ce volume.

W-....1....

372 HD Paik, BA Glatz

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II- 3500 c-E0.8 -

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V II- 1000 00.2 "53

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0 en0.0 1 1 1 1

0 20 40 60 80 100 120 140Fraction

The previously reported final step in pro-pionicin purification was by isoelectric focus-ing (Lyon and Glatz, 1993). However,despite several attempts with different com-binations of ampholytes, we were not able toobtain a single protein band after isoelec-tric focusing. Therefore, reversed-phaseHPLC was tried as an alternative method.Reversed-phase HPLC can be used forpurification and/or large-scale preparationof proteins that are stable in organic mobilephases, or proteins that can renature afterunfolding occurs during the elution process(Chicz and Regnier, 1990). A number ofbacteriocins have been purified by thismethod.

Various acids and organic solvents usedin reversed-phase HPLC were first testedfor their effect on propionicin activity. Equalvolumes of partially purified bacteriocin andmethanol, 2-propanol, acetonitrile, ethanol,0.2% (v/v) trifluoroacetic acid (TFA) and 2%(v/v) phosphoric acid were incubated for 2 h,then assayed for bacteriocin activity in theweil diffusion assay. No effect of any ofthese solvents and acids on measured activ-ity was seen, nor did the solvents and acidsinhibit the indicator strain. The TFA and 2-propanol were selected for use because of

Fig 1. Elution profile ofpropionicin PLG-1 inCM-Sepharose columnchromatography. •A28ü' 0 bacteriocin activ-ity.Profil d'élution de la pro-pionicine PLG-1 aucours de la chromato-graphie sur colonne car-boxyméthyl sepharose.• A28o, 0 Activité de labactériocine.

their excellent solubilizing properties (Chiczand Regnier, 1990).

The 5 fractions from the ion exchangecolumn containing bacteriocin activity (21ml total volume) were pooled, reduced to0.4 ml volume by dialysis against PEG toconcentrate the bacteriocin, and 20 III ofthis concentrate were applied to an analyt-ical C18 reversed-phase HPLC column. Apreparative scale column would be preferredwhen large quantities of purified protein aredesired, but the column used in this studywas sufficient to produce enough proteinfor subsequent composition and sequenceanalyses. The elution of the propionicinpreparation from the C18 column was mon-itored at A220. Two independent trials wereperformed, and gave similar results. Onetrial is shown in figure 2. Several small,sharp peaks were seen, but bacteriocinactivity was detected only in 10 fractions,with highest activity seen in 4 fractions con-taining a single peak that eluted with 84% 2-propanol. A total of 56 fractions wereobtained from the column. When the purityof the fractions containing bacteriocin activ-ity was assessed by SDS-PAGE, a singleprotein band with apparent molecular weight9 690 was detected in 4 fractions with high

Fig 2. C'8 reversed-phase HPLC analysis ofpropionicin PLG-1obtained from ionexchange chromatogra-phy .• Bacteriocin activ-ity.Analyse par chro-matographie RP-HPLC(colonne CIS) de la pro-pionicine PLG-t obtenuepar chromatographied'échange d'ions .• Activ-ité de la bactériocine.

Characterization of propionicin PLG-1 373

;?100 ~

...............0

(5 ...................c: ...................<Il

500- o .....e0- 1.4 60N

1.2 50 E1.0 :3

40 ~0 0.8 ~~ 30 ~« 0.6 <Il

0.4 20 .=00

10.C:

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0 10 20 30 40 50 60

Fraction

bacteriocin activity (fig 3). The other frac-tions contained this band plus possibly anaddition al faint band. We concluded that the9 690 Mr protein purified at this step is pro-pionicin PLG-1.

Amino acid composition and sequence

The amino acid composition of purified pro-pionicin PLG-1 is given in table II. Propi-onicin PLG-1 contains 99 amino acidresidues with a calculated molecular weightof 9 328. This agrees closely with the molec-ular weight of 9 690, determined by the posi-tion of propionicin PLG-1 compared tomolecular weight markers in 10-20% gra-dient gels in SDS-PAGE (fig 3). Neutral (Gly)and hydrophobic (Ala, Ile, Leu, Val and Pro)residues make up a significant portion ofpropionicin PLG-1: 20 and 42%, respec-tively. It should be noted that tryptophancan be destroyed by the acid hydrolysismethod employed. If propionicin PLG-1 con-tained some tryptophan residues, they couldgo undetected.

The sequence of the first 10 N-terminalamino acids was determined as follows:NH2-1Asn-2VaI-3Asp.4Ala(Thr)_5Arg-

6Thr(Cys).7 Ala(Thr)_8Arg-9Thr(Ala)_1OPro.When an amino acid is listed in parenthesesafter another, this indicates that either maybe present, with the first being more likely.This amino acid sequence was compared

Table Il. Amino acid composition of propionicinPLG-1.Composition en acides aminés de la propionicinePLG-t.

Residuesper molecule

Mole %

23.796.276.914.37

20.2611.752.843.851.215.954.113.154.77

Aminoacid

24674

20123416435

Total no of ami no acids 99

Alanine (Ala)Arginine (Arg)Aspartic acid (Asp)Glutamic acid (Glu)Glycine (Gly)Isoleucine (Ile)Leucine (Leu)Lysine (Lys)Proline (Pro)Serine (Ser)Threonine (Thr)Tyrosine (Tyr)Valine (Val)

374

kDa

66.045.0 ........36.0 '---29.024.0 -....... '20.0 -r14.26.5 1

HO Paik, BA Glatz

2 3 4 5 6 7 8 9 10- - '- .- - - ,.......,- --'<.-

Fig 3. SOS-PAGE analysis of fractions recov-ered from reversed-phase HPLC that contain pro-pionicin PLG-1. Lanes 1, 3 and 10: empty; lanes2 and 8: Mr standards (top to bottom, bovineserum albumin [Mr 66 000], chicken egg ovalbu-min [Mr 45 000], rabbit muscle glyceraldehyde-3-phosphate dehydrogenase [Mr 36 000], bovineerythrocytes carbonic anhydrase [Mr 29 000],bovine pancreas trypsinogen [Mr 24 000], soy-bean trypsin inhibitor [Mr 20 000], bovine milk œ-lactalbumin [Mr 14 200], bovine lung aprotinin[Mr 6 500]; lanes 4, 5, 6, 7 and 9: HPLC fractions33,34,35,36 and 37, respectively).Analyse par SOS-PAGE de fractions obtenuespar chromatographie RP-HPLC qui contiennent lapropionicine PLG-1. Puits 1, 3 et 10: vide; puits2 et 8 : standards de poids moléculaire (de hauten bas: sérum albumine bovine [Mr 66 OOOJ,ovalbumine d'œuf de poule [Mr 45 OOOJ,gly-céraldéhyde-3-phosphate déshydrogénase demuscle de lapin [Mr 36 OOOJ,anhydrase car-bonique d'érythrocytes bovins [Mr 29 OOOJ,trypsinogène de pancréas bovin [Mr 24 OOOJ,inhibiteur trypsique de soja [Mr 20 OOOJ,a-lactal-bumine bovine [Mr 14 200J, apoprotine depoumon bovin [Mr 6 500J ; puits 4, 5, 6, 7 et 9 :fractions HPLC 33, 34, 35, 36 et 37, respective-ment.

to others listed in the SWISS-PROT databank. No homology to other listed proteins,including other bacteriocins from lactic acidbacteria, was found. Therefore, propionicinPLG-1 seems to be different from other pre-viously reported bacteriocins from lactic acidbacteria.

Purified propionicin PLG-1 obtained fromreversed-phase HPLC was stable to stor-age in the Iyophilized state at both 4 and-60°C for 3 months. No significant change

in activity was seen in samples stored overthis period (data not shown).

DISCUSSION

A highly purified bacteriocin was obtainedin this study through a sequence of stepsincluding ammonium sulfate precipitation,ion exchange chromatography andreversed-phase HPLC. Bacteriocin was pre-cipitated from more than 1 1 of culture super-natant. The addition of so much ammoniumsulfate to such large volumes of culturesupernatant took over 10 h; the sampleswere further stirred slowly at 4°C for an addi-tional 3 h. This much longer incubation withammonium sulfate compared to that usedpreviously with smaller (50-100 ml) super-natants (usually 3 h to add salt plus an addi-tional 30 min of stirring after salt addition)seemed to improve bacteriocin recovery.For example, in 1 batch, the measured activ-ity in a 50 ml sample was 2.4-fold lower thanthat measured in a 1 1 sample. Possibly thelonger incubation facilitates interaction ofbacteriocin molecules with each other orwith other proteins that can then precipitate.Other bacteriocins have been reported toprecipitate poorly. For example, lactacin F(Muriana and Klaenhammer, 1991) and lac-tocin S (Mortvedt et al, 1991) have beenreported to be lost as a floating fraction dur-ing ammonium sulfate precipitation, possiblydue to their hydrophobie character. Slowaddition of ammonium sulfate plus continuedstirring for at least 3 h are recommendedfor bacteriocin recovery.

A single-step ammonium sulfate precip-itation at 75% saturation was used, ratherthan taking the proteins precipitated between50 and 75% saturation, because weobserved that some bacteriocin activity waslost in the proteins precipitated at 50% sat-uration. However, this change affected theamount of protein present in the precipitateand the fold purification of bacteriocin

Characterization of propionicin PLG-1

obtained. In the current study, an estimated321-fold purification was obtained afterammonium sulfate precipitation. This com-pares to a 600-fold purification in this stepreported previously (Lyon and Glatz, 1993).

For ion exchange chromatography, asmall column with a correspondingly sm ailbed volume was used. Bacteriocin waseluted from the column in a tight band, andwas completely separated from unadsorbedproteins. However, the elution profile fromthe ion exchange column indicated that pro-pionicin was not completely separated fromother absorbed proteins. Therefore, a finalstep was necessary for complete purifica-tion. Reversed-phase HPLC proved to bean effective final step.

Recently, reversed-phase HPLC hasbeen used to obtain highly purified prepa-rations of a number of other bacteriocins,including leucocin A-UAL 187 (Hastingset al, 1991), lactacin F (Muriana andKlaenhammer, 1991), mesentericin Y105(Hechard et al, 1992), lacticin 481 (Piard etal, 1992), salivaricin A (Ross et al, 1993),curvaticin FS47 (Garver and Muriana, 1994)and staphylococcin 1580 (Sahl, 1994). Thehydrophobic nature of these bacteriocins,and the apparent hydrophobicity of propi-onicin PLG-1, allows their purification byreversed-phase HPLC. Hydrophobicity mayalso contribute to the tendency of many bac-teriocins from lactic acid bacteria to asso-ciate with other substances to form largemacromolecular complexes. For instance,lactacin B (Barefoot and Klaenhammer,1984), helveticin J (Joerger and Klaen-hammer, 1986) and lactacin F (Muriana andKlaenhammer, 1991) have been shown toform associations with lipid and carbohy-drate. Bacteriocins from Lactobacillus sphave been reported to associate with pro-tein-detergent (Tween 80 in MRS broth)micelles (Garver and Muriana, 1994). Asso-ciation of propionicin PLG-1 with other pro-teins or aggregation of several moleculesof propionicin into multimeric forms is the

375

probable reason it is seen to elute in gel fil-tration at apparent molecular weights ofmore th an 150 000 and approximately10 000, while under dissociating conditionsit elutes at 10 000 (Lyon and Glatz, 1993).As has been demonstrated most completelyfor the bacteriocins nisin and subtilin(Montville and Kaiser, 1993), the hydropho-bicity of these molecules promotes interac-tion with cell membranes, leading to bacte-ricidal action through the formation of poresin the membranes.

The successful determination of theamine acid composition and the N-terminalsequence of propionicin PLG-1 indicatesthat propionicin does not contain unusualor modified amine acids, as is the case withlantibiotics such as nisin, nor does it have ablocked N-terminus. It does contain a rela-tively high percentage of hydrophobic amineacids; such hydrophobicity is commonamong bacteriocins of lactic acid bacteria(Garver and Muriana, 1994). For example,about 50% of the ami no acids are hydropho-bic in lactococcin A (Holo et al, 1991), lac-tocin S (Mortvedt et al, 1991) and curvaticinFS47 (Garver and Muriana, 1994). How-ever, curvacin P and sakacin P (TichaczeketaI, 1992) contain only about 20-25%hydrophobic residues. The high proportionof glycine residues in propionicin shouldprovide a significant amount of f1exibility tothe molecule (Garver and Muriana, 1994).Glycine occupies very little space and allowsa wide range of conformations in the foldingof polypeptide chains (Stryer, 1988).

Propionicin PLG-1 was not found to sharehomology with other proteins (including otherbacteriocins) Iisted in the SWISS-PROT databank. In contrast to this result, many bacte-riocins of lactic acid bacteria have beenreported to share significant degrees ofhomology. For example, bavaricin A (Larsenet al, 1993) was found to share 66% homol-ogy with pediocin PA-1 produced by Pedio-coccus acidilactici (Marugg et al, 1992)and 39% homology with leucocin A-UAL

376 HD Paik, BA Glatz

(Hastings et al, 1991). Sakacin A (Holck etal, 1992) was reported to share sornehomology, especially in the N-terminalregion, with the newly sequenced bacteri-ocins leucocin A-UAL 187 (Hastings et al,1991), pediocin PA-1 (Lozano et al, 1992)and sakacin P (Tichaczek et al, 1992). Theamino acid sequence of leucocin B-Ta11a(Felix et al, 1994) was reported to be sig-nificantly homologous to the sequence ofleucocin A-UAL 187 (Hastings et al, 1991). Abacteriocin produced by Pediococcus acidi-lactici was shown to have the identical pri-mary amino acid sequence as pediocin PA-1 (Henderson et al, 1992) and was, in tact,the same molecule. Staphylococcin 1580(Sahl, 1994) was shown to be identical toepidermin, a lantibiotic, by amino acid com-position analysis, determination of molecu-lar weight and limited N-terminal sequenc-ing.

ln conclusion, we have obtained a highlypurified preparation of propionicin PLG-1by sequential steps of ammonium sulfateprecipitation, ion exchange chromatogra-phy and reversed-phase HPLC. The aminoacid sequence of propionicin PLG-1 indi-cates that it is different from other previouslyreported bacteriocins from lactic acid bac-te ria. Its hydrophobic nature lends support tothe suggestion that propionicin PLG-1, likeother bacteriocins, interacts with and mayhave as its primary target the cytoplasmicmembrane of sensitive organisms.

ACKNOWLEDGMENTS

We are indebted to Dr E Hammond who provideduse of his HPLC, Dr A Myers who providedaccess to the SWISS-PROT data base throughthe Sequence Analysis Software Package, theIowa State University Protein Facility for aminoacid composition and sequence analysis and theIowa State University Fermentation Facility foruse of fermentation equipment. This work wassupported by the Binational Agricultural Researchand Development Fund (BARD), grant no US-2080-91.

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