Rapid Two-Step Procedure for Large-Scale Purification of Pediocin-Like Bacteriocins and Other Cationic Antimicrobial Peptides from Complex Culture Medium

A rapid and simple two-step procedure suitable for both small-and large-scale purification of pediocin-like bacteriocins andother cationic peptides has been developed. In the first step,the bacterial culture was applied directly on a cation-exchangecolumn (1-ml cation exchanger per 100-ml cell culture). Bacteriaand anionic compounds passed through the column, and cationicbacteriocins were subsequently eluted with 1 M NaCl. In thesecond step, the bacteriocin fraction was applied on a low-pressure,reverse-phase column and the bacteriocins were detected as majoroptical density peaks upon elution with propanol. More than80% of the activity that was initially in the culture supernatantwas recovered in both purification steps, and the final bacteriocinpreparation was more than 90% pure as judged by analytical reverse-phasechromatography and capillary electrophoresis.

INTRODUCTION

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Introduction

Gene-encoded, ribosomally synthesized antimicrobial peptidesare widely distributed in nature, being produced by bacteria,plants, and a wide variety of animals, including humans (28,29, 32, 34). The peptides are often cationic and amphiphilicor hydrophobic, and many of them kill bacteria by permeabilizingthe target cell membrane. The peptides may be developed intonew and useful antimicrobial additives and drugs. An exampleof this is the antimicrobial peptide nisin, which is producedby lactic acid bacteria (LAB). This peptide is used as a foodpreservative (9) and has been considered for use for treatmentof gastric Heliobacter infections and/or ulcers (16).

There has especially been considerable interest in antimicrobialpeptides (bacteriocins) produced by LAB because of the "food-gradequality" and industrial importance of these bacteria. LAB areused in food production, are part of the natural microbial florain food that humans have consumed for centuries, and constitutea significant part of the indigenous flora of mammals, includinghumans. Thus, LAB and the bacteriocins that they produce maybe considered safe agents for preventing growth of pathogenicand/or undesirable microorganisms.

Many of the LAB bacteriocins belong to the pediocin-like family;these bacteriocins are of special interest because of theirantilisterial activity. The family contains at least 15 differentbacteriocins, of which pediocin PA-1 (3, 19, 23, 30), leucocinA UAL-187 (17), mesentericin Y105 (18), sakacin P (35) and curvacinA (identical to sakacin A [20, 35]) were the first to be identified.All pediocin-like bacteriocins are cationic, contain between35 and 50 amino acid residues, permeabilize target cell membranes,and have very similar primary structures but differ markedlywith respect to their target cell specificity (5-7, 10-14, 22,24, 29, 32, 37).

The use of pediocin-like bacteriocins and other antimicrobialpeptides as additives or drugs requires a simple and rapid methodby which large quantities may be purified to homogeneity. Presentmethods for purification of pediocin-like bacteriocins and othercationic bacteriocins generally include a centrifugation stepfor the removal of cells from the bacterial culture medium,followed by a step in which the peptides may be concentrated.This latter step often involves precipitation of peptides withammonium sulfate and collecting the precipitate by centrifugation.The peptides are subsequently purified to homogeneity by sequentialchromatography on cation exchange, hydrophobic interaction,and/or reverse-phase columns. Although these procedures functionreasonably well when one is purifying smaller quantities ofpeptides from a <1-liter bacterial culture, they tend tobecome unmanageable when one is working with the large volumesthat are needed for large-scale industrial production and forproduction of peptides for structural and functional studies.The major problem is that the centrifugation and precipitationsteps become cumbersome when one is working with large volumes.Moreover, ammonium sulfate precipitation of peptides from culturemedium results in variable and often low yields (about 50% butin some instances down to 10%), in part because much of theprecipitate floats even after centrifugation and is consequentlydifficult to collect (8, 15, 26, 27). It is our experience thatammonium sulfate precipitation of the pediocin-like bacteriocinpediocin PA-1 has a yield of 40% ± 20% (average and standarddeviation of 22 experiments; unpublished results). In a recentlypublished procedure for purification of pediocin-like bacteriocins,concentration by ammonium sulfate precipitation was replacedwith cation-exchange chromatography (15). Although this replacementwas reported to increase the yield of purified bacteriocins(overall yields of between 10 and 66% were obtained), purificationfrom a 100-ml culture took 6 to 8 h (15). In this study we presenta rapid and simple two-step procedure suitable for both small-and large-scale purification of pediocin-like bacteriocins andother cationic peptides.

Bacterial strains and bacteriocin assay.

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Bacterial strains and...

Pediocin PA-1 (3, 19, 23, 30) was produced by Pediococcus acidilacticiLMG 2351, which was isolated from commercial starter culturesobtained from Christian Hansen Laboratories, Copenhagen, Denmark.Sakacin P (35) and a sakacin P mutant were produced by bacteriocinexpression systems developed recently (2, 11, 22). CurvacinA (35), leucocin A (17), lactococcin A (21), lactococcin G (33),and nisin Z (25) were produced by Lactobacillus curvatus LTH1174(35), Leuconostoc mesenteroides 6 (36), Lactococcus lactis subsp.cremoris LMG 2130 (21), L. lactis LMG 2081 (33), and L. lactisLMG 2077, respectively. Both enterocins A and B were producedby Enterococcus faecium CTC492 (31). The lactococcal strainswere grown at 30°C in M17 (Biokar Diagnostica) supplementedwith glucose and Tween 80 to final concentrations of 0.4% (wt/vol)and 0.1% (vol/vol), respectively. All the other strains weregrown at 30°C in MRS broth (Oxoid).

Bacteriocin activity was measured by using a microtiter plateassay system, essentially as described previously (11, 21).Lactobacillus sake NCDO 2714 (type strain) was used as the indicatorstrain during the assaying of pediocin PA-1, sakacin P, a sakacinP mutant, curvacin A, leucocin A, nisin Z, and enterocins Aand B. L. lactis subsp. lactis IL 1403 was used as the indicatorstrain during the assaying of lactococcins A and G. One bacteriocinunit (BU) was defined as the amount of bacteriocin that inhibitedthe growth of the indicator strain by 50%.

Purification of pediocin-like bacteriocins by passage of bacterial cultures through cation exchanger followed by reverse-phase chromatography of peptides.

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Purification of pediocin-like...

The rapid two-step purification procedure that was developedavoids centrifugation and precipitation steps and is thus easilyscaled up. In the first step, the bacterial culture was applieddirectly on a SP Sepharose Fast Flow cation-exchange column(Amersham Pharmacia Biotech). This approach is similar to thatreported by Callewaert and De Vuyst for purification and concentrationof the bacteriocin amylovorin L471 on an expanded bed cation-exchangecolumn to which crude fermentation medium had been applied (4).Bacteria and anionic compounds passed through the SP SepharoseFast Flow cation-exchange column, whereas cationic bacteriocinswere eluted with 1 M NaCl after the column had first been washedwith a phosphate buffer containing between 0.1 and 0.2 M (dependingon the bacteriocin which is being purified) NaCl. In the secondstep, the 1 M NaCl fraction from the cation exchanger was appliedat a high flow rate on a low-pressure Resource reverse-phasecolumn (Amersham Pharmacia Biotech). Bacteriocins bind to thecolumn and were detected as major peaks on the optical densityprofile upon elution with propanol. This second step could berepeated if necessary for obtaining peptides with a purity ofmore than 90%.

The procedure may be illustrated with the purification of pediocinPA-1. More than 80% of the activity that was initially in theculture supernatant was recovered in both purification steps(Table 1), and the bacteriocin was detected as a major opticaldensity peak upon reverse-phase chromatography in the secondstep (Fig. 1). The identity of the purified peptide obtainedafter reverse-phase chromatography was verified by mass spectrometry;it was more than 90% pure as judged by analytical reverse-phasechromatography and capillary electrophoresis (results not shown).

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TABLE 1. Purification of pediocin PA-1 by new procedure

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FIG. 1. Reverse-phase chromatography of the 1 M NaCl fraction obtained in the cation-exchange chromatography step (step 1, Table 1). After addition of propanol and trifluoroacetic acid to final concentrations of 5 and 0.1% (vol/vol), respectively, the 1 M NaCl fraction from step 1 (Table 1) was applied at a flow rate of 5 ml/min on a 3-ml Resource reverse-phase column. Peptides binding to the column were eluted at a flow rate of 1 ml/min with a linear propanol gradient (shown in the figure) in 0.1% trifluoroacetic acid. Only the fractions under the horizontal bar contained bacteriocin activity, which coincided with the absorbance peak under the bar. The first 42 ml represents the flowthrough fraction that contained substances that did not bind to the column. To prevent fouling of the reverse-phase column, samples applied to the column were cleared of gross contaminants by centrifugation at about 4,000 x g for 10 min.

For comparison, pediocin PA-1 was in parallel also isolatedfrom the same culture using our earlier standard purificationprocedure (Table 2). Due to the much-higher yields obtainedwith the new procedure, about 300 µg was purified from400-ml culture using the new procedure, whereas only about 45µg (average and standard deviation of 22 earlier experimentswere 80 ± 30 µg) was purified using our earlierprocedure (Table 1). Moreover, pediocin PA-1 was purified inless than 2 h using the new procedure, whereas our earlier proceduretook more than twice that time when starting with a 400-ml culture.This time difference becomes much larger when one starts withlarger culture volumes, since the new procedure, in contrastto our earlier procedure, may easily be scaled up with relativelylittle increase in purification time.

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TABLE 2. Purification of pediocin PA-1 by earlier standard procedure, essentially as described in reference 35

Other pediocin-like bacteriocins that have been tested—curvacinA, leucocin A, sakacin P, and a sakacin P mutant—alsobound to the cation exchanger column and were recovered withyields of 90% or more after bacterial cultures were passed throughthe column. However, the optimal concentration of NaCl for washingthe column before elution of the bacteriocins with 1 M NaCl(see Table 1, footnote c) differed among these bacteriocins,presumably reflecting their net positive charge. When pediocinPA-1 (net positive charge of about 5 to 6) was being isolated,0.2 M NaCl could be used without loss of the bacteriocin (lessthan 1% loss) from the column. When sakacin P (net positivecharge of about 3 to 4) was being isolated, the concentrationof NaCl had to be reduced to 0.15 M in order to avoid loss ofbacteriocin (between 1 and 20% loss at 0.2 M NaCl) when thecolumn was washed. The bacteriocins appeared as a major opticaldensity peak upon reverse-phase chromatography in the secondpurification step.

Capacity of cation-exchange column: >100-ml cell culture may be applied to 1-ml cation exchanger.

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Capacity of cation-exchange...

To determine the amount of cell culture that could be appliedto the cation exchanger in the first purification step, an overnightstationary bacterial culture producing pediocin PA-1 was appliedon a 3-ml SP Sepharose Fast Flow cation-exchange column, andflowthrough fractions were assayed for bacteriocin activity(Fig. 2). Only about 4 and 7% of the bacteriocin activity werelost in the flowthrough fraction when, respectively, 300 and500 ml of the culture were applied to the column (Fig. 2). Bindingof the bacteriocin was, however, significantly reduced whenmore than 600 ml of culture was applied to the 3-ml column.Of the activity which was applied after 600 ml of culture hadpassed through the column, less than 50% bound to the column,and essentially no further binding was obtained after 1 literhad passed through the column (Fig. 2). In our bacteriocin purificationexperiments, no more than 100 ml of culture was applied per1-ml ion exchanger. After use, the ion exchanger could be regeneratedby washing with several column volumes of 2 M NaOH followedby column equilibration.

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FIG. 2. The amount of bacteriocin activity in the flowthrough fraction as a function of the amount of bacterial culture (overnight culture of P. acidilactici LMG 2351; produces pediocin PA-1) that has passed through a 3-ml SP Sepharose Fast Flow cation-exchange column. The horizontal, stippled line indicates the activity in the bacterial culture before it is applied to the column.

Other cationic peptides secreted by bacteria may also be concentratedand purified by simply passing the bacterial cultures througha cation exchanger. After passage of a culture producing thetwo-peptide bacteriocin lactococcin G (33), 33% of the recoveredactivity was found in the flowthrough fraction and 67% was foundin the 1 M NaCl fraction. Although the percentage of lactococcinG which bound to the column was not as high as that obtainedfor the pediocin-like bacteriocins, it was higher than thatobtained (about 35% binding to the column) in the cation-exchangechromatography step of the earlier standard purification procedure(33). Binding of lactococcin G to cationic exchangers is generallynot as efficient as one would expect from the cationic characterof the two peptides that constitute lactococcin G, possiblydue to interactions between lactococcin G and other components(33). A recovery similar to that obtained with lactococcin Gwas also obtained when a culture of E. faecium CTC492 was passedthrough a cation exchanger. This strain produces two bacteriocins:enterocin B and the pediocin-like bacteriocin enterocin A (31).The recovery of the two other bacteriocins that were tested,nisin Z and lactococcin A, was lower, being only about 30 and15%, respectively. However, there was no effort to optimizechromatography conditions, and optimization, perhaps by reducingthe pH of the culture medium before application on the cationexchanger, might possibly improve the recovery.

The influence of the pH on recovery was clearly demonstratedby Callewaert and De Vuyst (4). In their report on the purificationof amylovorin L471, crude fermentation medium containing cellswas applied directly to an expanded bed cation-exchange column.The highest recovery (30%) was obtained upon applying the bacteriocinsto the column at pH 3.6 and eluting it at pH 6.5 (4). The recoverywas reduced by 50% or more if both sample application and elutionwere carried out at the same pH, irrespective of whether thiswas 3.6 or 6.5 (4). The bacteriocins was lost in the flowthroughfraction when both application and elution were carried outat pH 6.5, whereas the loss appeared to be due to poor releasefrom the column when both application and elution were carriedout at pH 3.6 (4). It is our experience that many peptide bacteriocinshave affinity to the matrix of commonly used columns. Some bacteriocins,such as plantaracin JK, may interact strongly with the columnmatrix, and elution requires 3 M guanidine-HCl (1). Other bacteriocinsappear to interact weakly with the matrix and may be releasedunder gentler elution conditions. Even for these bacteriocins,however, recovery may in some cases be improved with the useof guanidine-HCl. We consequently recommend that elution with3 M guanidine-HCl be included as a final elution step upon columnchromatography of bacteriocins in order to ascertain that theelution has been complete. The use of guanidine-HCl has onedrawback, however, in that guanidine-HCl may interfere withthe binding of the bacteriocin to the column in the subsequentchromatography step, thus necessitating dilution or dialysisof the sample.

The new purification procedure has proved to be extremely useful.The short purification time and high yields have enabled usto purify in a few hours milligram quantities of pediocin-likebacteriocins that are needed for our structural studies, whereasthis could take up to a week or more using our previous purificationprotocol.

ACKNOWLEDGMENTS

This work was supported by a grant from the Norwegian ResearchCouncil.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biochemistry, University of Oslo, Post Box 1041, Blindern, 0316 Oslo, Norway. Phone: 47-22 85 66 32. Fax: 47-22 85 44 43. E-mail: gunnar.fimland{at}biokjemi.uio.no.

REFERENCES

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