HPLC purification and re-evaluation of chemical identity of two circular bacteriocins, gassericin A and reutericin 6

Aim: The study aimed for the complete purification and recharacterization of the highly hydrophobic circular bacteriocins, gassericin A and reutericin 6. Methods and Results: Gassericin A and reutericin 6 were purified to homogeneity using previously described method and reverse-phase HPLC with an octyl column and eluents of aqueous acetonitrile and 2-propanol. Mass analysis, N-terminal sequencing and bacteriocin assay of the HPLC-purified bacteriocins showed the two bacteriocins had identical seamless circular structures with the same m/z value (5651) of [M + H]+ and both had the same specific activity. d/l-amino acid composition analysis using two distinct methods with the chiral fluorescent derivatization reagents (+)-1-(9-fluorenyl)ethyl chloroformate and o-phthalaldehyde/N-acetyl-l-cystein revealed neither gassericin A nor reutericin 6 contained d-alanine residues contrary to our previous results. Conclusion: Purified gassericin A and reutericin 6 are chemically identical circular molecules containing no d-alanine residues. Significance and Impact of the Study: The HPLC conditions developed in this study will facilitate advanced purification and correct characterization of other highly hydrophobic bacteriocins.