Simple method of purification and sequencing of a bacteriocin produced by Pediococcus acidilactici UL5.
A bacteriocin produced by a strain of Pediococcus acidilactici was successfully purified sequentially by acid extraction (at pH 2) and reverse-phase high-performance liquid chromatography (HPLC). Cell extracts of derivative strains deficient in bacteriocin production exhibited a similar HPLC elution profile to the active extracts except for the two peaks containing bacteriocin activity. The sequence of the antibacterial peptide consisted of 44 amino acid residues of which 42 were identified, and its molecular weight was 4624 Da, as determined by mass spectrometry. Moreover, according to the molecular weight of the peptide, the unidentified residues in the bacteriocin sequence must correspond to two tryptophan residues, confirming that the peptide isolated from Ped. acidilactici UL5 is pediocin PA-1. However, oxidized forms of the bacteriocin produced during storage also showed bacteriocin activity and resulted in a second peak with activity in the chromatograms. HPLC chromatograms of cell surface preparations from the active and from the deficient strains were confirmed by capillary electrophoresis. The purification method used is simple and effective in comparison with traditional methods, permitting a selective recovery of cell-associated bacteriocin at low pH, and its isolation in pure form for sequencing.
Daba
H
H
Centre de recherche STELA, Université LAVAL, Ste-Foy (Québec), Canada.
Lacroix
C
C
Huang
J
J
Simard
R E
RE
Lemieux
L
L
D000595:Amino Acid Sequence; D001430:Bacteriocins / Q000737:chemistry* / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid; D004586:Electrophoresis; D013058:Mass Spectrometry; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010376:Pediococcus / Q000737:chemistry*; D017421:Sequence Analysis
D001430:Bacteriocins