Daba H, Lacroix C, Huang J, Simard R E, Lemieux L
Centre de recherche STELA, Université LAVAL, Ste-Foy (Québec), Canada.
J Appl Bacteriol. 1994 Dec;77(6):682-8. doi: 10.1111/j.1365-2672.1994.tb02819.x.
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.
一株嗜酸乳杆菌产生的细菌素通过酸提取（pH 2）和反相高效液相色谱（HPLC）成功地依次纯化。缺乏细菌素产生能力的衍生菌株的细胞提取物，除了含有细菌素活性的两个峰外，其HPLC洗脱图谱与活性提取物相似。抗菌肽序列由44个氨基酸残基组成，其中42个已被鉴定，通过质谱测定其分子量为4624 Da。此外，根据该肽的分子量，细菌素序列中未鉴定的残基必定对应于两个色氨酸残基，这证实了从嗜酸乳杆菌UL5分离出的肽是片球菌素PA - 1。然而，储存过程中产生的细菌素氧化形式也表现出细菌素活性，并在色谱图中产生了第二个有活性的峰。活性菌株和缺陷菌株的细胞表面制剂的HPLC色谱图通过毛细管电泳得到证实。与传统方法相比，所使用的纯化方法简单有效，能够在低pH下选择性回收与细胞相关的细菌素，并将其纯化为测序形式。
