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

Abstract

A rapid and simple two-step procedure suitable for both small-and large-scale purification of pediocin-like bacteriocins and other cationic peptides has been developed. In the first step, the bacterial culture was applied directly on a cation-exchange column (1-ml cation exchanger per 100-ml cell culture). Bacteria and anionic compounds passed through the column, and cationic bacteriocins were subsequently eluted with 1 M NaCl. In the second step, the bacteriocin fraction was applied on a low-pressure, reverse-phase column and the bacteriocins were detected as major optical density peaks upon elution with propanol. More than 80% of the activity that was initially in the culture supernatant was recovered in both purification steps, and the final bacteriocin preparation was more than 90% pure as judged by analytical reverse-phase chromatography and capillary electrophoresis.Gene-encoded, ribosomally synthesized antimicrobial peptides are 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 amphiphilic or hydrophobic, and many of them kill bacteria by permeabilizing the target cell membrane. The peptides may be developed into new and useful antimicrobial additives and drugs. An example of this is the antimicrobial peptide nisin, which is produced by lactic acid bacteria (LAB). This peptide is used as a food preservative (9) and has been considered for use for treatment of gastric Heliobacter infections and/or ulcers (16).There has especially been considerable interest in antimicrobial peptides (bacteriocins) produced by LAB because of the "food-grade quality" and industrial importance of these bacteria. LAB are used in food production, are part of the natural microbial flora in food that humans have consumed for centuries, and constitute a significant part of the indigenous flora of mammals, including humans. Thus, LAB and the bacteriocins that they produce may be considered safe agents for preventing growth of pathogenic and/or undesirable microorganisms.Many of the LAB bacteriocins belong to the pediocin-like family; these bacteriocins are of special interest because of their antilisterial activity. The family contains at least 15 different bacteriocins, of which pediocin PA-1 (3,19,23,30), leucocin A UAL-187 (17), mesentericin Y105 (18), sakacin P (35) and curvacin A (identical to sakacin A [20,35]) were the first to be identified. All pediocin-like bacteriocins are cationic, contain between 35 and 50 amino acid residues, permeabilize target cell membranes, and have very similar primary structures but differ markedly with respect to their target cell specificity (5-7, 10-14, 22, 24, 29, 32, 37).The use of pediocin-like bacteriocins and other antimicrobial peptides as additives or drugs requires a simple and rapid method by which large quantities may be purified to homogeneity. Present methods for purification of pediocin-like bacteriocins and other cationic bacteriocins generally include a centrifugati...

Structure-Function Analysis of Immunity Proteins of Pediocin-Like Bacteriocins: C-Terminal Parts of Immunity Proteins Are Involved in Specific Recognition of Cognate Bacteriocins

“…Enterocin A, leucocin A, leucocin C, pediocin PA-1, curvacin A, and sakacin P were purified to homogeneity from 0.5-or 1.0-liter cultures of their producer strains by cationexchange chromatography and reverse-phase chromatography, as described pre- viously (32). The purity of the bacteriocins was analyzed by analytical chromatography on a RPC SC 2.1/10 C 2 C 18 column (Amersham Biosciences) using the SMART chromatography system (Amersham Biosciences) and water-2-propanol containing 0.1% (vol/vol) trifluoroacetic acid for the mobile phase.…”

Comparison of two methods for purification of plantaricin ST31, a bacteriocin produced by Lactobacillus plantarum ST31

“…A rapid and two-step procedure suitable for both small-and large-scale purification of pediocin-like bacteriocins and other cationic peptides have been reported (15). Bacterial cells and anionic compounds passed through the column, with cationic bacteriocins being eluted subsequently with 1 M NaCl (15).…”

Structure analysis of the two-peptide bacteriocin lactococcin G by introducing d-amino acid residues

“…When the indicator strains L. lactis MG1363 and Lactococcus sp. LMGT-2077 contained the unmodified pMG36e plasmid (van de Guchte et al, 1989) or the pLcnGim expression plasmid (Oppegård et al, 2010), erythromycin was added to the growth media to a final concentration of 2 mg ml 21 to ensure that the plasmids remained in the cells.…”

“…The wild-type lactococcin G peptides were produced by a heterologous expression system (Axelsson & Holck, 1995;Axelsson et al, 1998;Oppegård et al, 2007) and purified using a two-step purification method as previously described (Oppegård et al, 2007;Uteng et al, 2002). The six peptides containing D-amino acid residues were synthesized and purified to more than 90 % purity by GenScript (NJ, USA).…”