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.