Method for Rapid Purification of Class IIa Bacteriocins and Comparison of Their Activities

Abstract

A three-step method was developed for the purification of mesentericin Y105 (60% yield) from the culture supernatant of Leuconostoc mesenteroides Y105. The same procedure was successfully applied to the purification of five other anti-Listeria bacteriocins identified by mass spectrometry. Specific activities of the purified bacteriocins were compared.The increasing numbers of food contaminations by Listeria spp. have greatly stimulated research on class IIa bacteriocins produced by lactic acid bacteria. The number of such peptidic compounds may be limited since many of the newly detected strains express previously described bacteriocins. Consequently, the detection and characterization of still-unknown antagonistic peptides is becoming difficult. One of the more time-consuming steps in such studies is the purification of antagonistic compounds. Because bacteriocins are secreted into the culture medium, most strategies start with a step to concentrate bacteriocins from the culture supernatant, such as pH-dependent adsorption of bacteriocins on heat-killed producer bacteria (7), diatomite calcium silicate (Micro-Cel) (9), or rice hull ash or precipitation with silicic acid (19), ammonium sulfate (7), or ethanol (31). Although these procedures are used principally to reduce the working volume, they typically do not provide a high degree of purity. Therefore, subsequent steps of preparative isoelectric focusing (31) and/or multiple chromatographic separations, including cation exchange (7, 10, 22), gel filtration (12, 22), hydrophobic interaction (7,10,22), and reverse-phase liquid chromatography (6,10,22,24), are necessary to achieve significant purification of the bacteriocins. Usually, but not always, the protein yields obtained are low (for a review, see reference 7). This is probably due to the high number of steps in the protocol, leading to time-consuming processes and subsequently low yields.The primary structures of anti-Listeria bacteriocins are presented in Fig. 1. All of these peptides are characterized by the consensus sequence YGNGV(n)C(n) 4 C(n)V(n) 4 A (where n is any amino acid). More generally, the first 20 residues are well conserved, while a hydrophobic C-terminal half moiety is characterized by a large structural diversity, suggesting its implication in host specificity (11). We hypothesize that these variable C-terminal parts of the peptides result in differences in their specific anti-Listeria activities. Indeed, the sensitivity of a particular target strain depends on the bacteriocin concentration of the solution or culture supernatant used for the activity assay. Therefore, the host specificity described for a bacteriocin could depend on host sensitivity. Moreover, comparison of the bacteriocin specific activities described in the literature remains difficult because the methods and the target strains used are heterogeneous.Mesentericin Y105, a 37-amino-acid-long peptide produced by Leuconostoc mesenteroides Y105 was first characterized by Héchard et al. in 1992 (17). The method used to puri...

Expression of mptC of Listeria monocytogenes induces sensitivity to class IIa bacteriocins in Lactococcus lactis

“…lactis is known to be insensitive to class IIa bacteriocins (Eijsink et al, 1998;Guyonnet et al, 2000) and, in our study, Lc. lactis MG-Con was unaffected by a leucocin A concentration of 400 mg ml 21 .…”

“…Leucocin A was synthesized as described previously (Ramnath et al, 2000) and resuspended in 50 % acetonitrile to a final concentration of 2 mg ml 21 . Pediocin PA-1 (Henderson et al, 1992) and enterocin A (Aymerich et al, 1996) were produced by Pediococcus acidilactici NRRL B5627 and Enterococcus faecium 336, respectively, and purified as described by Guyonnet et al (2000). All bacteriocin stocks were stored at 220 uC until used.…”

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

“…The major problem is that the centrifugation and precipitation steps become cumbersome when one is working with large volumes. Moreover, ammonium sulfate precipitation of peptides from culture medium results in variable and often low yields (about 50% but in some instances down to 10%), in part because much of the precipitate floats even after centrifugation and is consequently difficult to collect (8,15,26,27). It is our experience that ammonium sulfate precipitation of the pediocin-like bacteriocin pediocin PA-1 has a yield of 40% Ϯ 20% (average and standard deviation of 22 experiments; unpublished results).…”

“…It is our experience that ammonium sulfate precipitation of the pediocin-like bacteriocin pediocin PA-1 has a yield of 40% Ϯ 20% (average and standard deviation of 22 experiments; unpublished results). In a recently published procedure for purification of pediocin-like bacteriocins, concentration by ammonium sulfate precipitation was replaced with cation-exchange chromatography (15). Although this replacement was reported to increase the yield of purified bacteriocins (overall yields of between 10 and 66% were obtained), purification from a 100-ml culture took 6 to 8 h (15).…”

“…In a recently published procedure for purification of pediocin-like bacteriocins, concentration by ammonium sulfate precipitation was replaced with cation-exchange chromatography (15). Although this replacement was reported to increase the yield of purified bacteriocins (overall yields of between 10 and 66% were obtained), purification from a 100-ml culture took 6 to 8 h (15). In this study we present a rapid and simple two-step procedure suitable for both small-and large-scale purification of pediocin-like bacteriocins and other cationic peptides.…”