RESUMEN
The peptide antibiotic mutacin 1140 belongs to the group of antibiotics called lantibiotics. They are ribosomally synthesized and undergo extensive enzymatic modifications before being excreted into the culture medium. By using reverse-phase-high-performance liquid chromatography (RP-HPLC) and a semiquantitative bacteriocin bioassay to track mutacin 1140, an efficient ammonium sulfate (AS) precipitation method has been developed for removing mutacin 1140 from a complex medium containing 5% yeast extract. This method minimizes the amounts of fermentation by-products and media components that make downstream purification processes more difficult and economically infeasible. The method may be adaptable for the initial purification step of other lantibiotics. A threefold decrease in the precipitation of the medium components found in yeast extract, at pH 8.0 vs. pH 2.0, may have broad utility for the isolation of secondary metabolites produced in this complex medium. The average yield of mutacin 1140 from the fermentation medium was determined as 66%.
Asunto(s)
Antibacterianos/aislamiento & purificación , Bacteriocinas/aislamiento & purificación , Fermentación , Microbiología Industrial/métodos , Péptidos/aislamiento & purificación , Levaduras/química , Secuencia de Aminoácidos , Antibacterianos/química , Bacteriocinas/química , Cromatografía Líquida de Alta Presión , Mezclas Complejas/química , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/aislamiento & purificación , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Péptidos/química , Alineación de SecuenciaRESUMEN
The peptide antibiotic nisin A belongs to the group of antibiotics called lantibiotics. They are classified as lantibiotics because they contain the structural group lanthionine. Lanthionines are composed of a single sulfur atom that is linked to the beta-carbons of two alanine moieties. These sulfur atoms are vulnerable to environmental oxidation. A mild oxidation reaction was performed on nisin A to determine the relative effects it would have on bioactivity. High-mass-accuracy Fourier transform ion cyclotron resonance mass spectrometry data revealed the addition of seven, eight, and nine oxygens. These additions correspond to the five lanthionines, two methionines, and two histidines that would be susceptible to oxidation. Subsequent bioassays revealed that the oxidized form of nisin A had a complete loss of bactericidal activity. In a competition study, the oxidized nisin did not appear to have an antagonistic affect on the bioactivity of nisin A, since the addition of an equal molar concentration of the oxidized variant did not have an influence on the bactericidal activity of the native antibiotic. Electron microscopy data revealed that the oxidized forms were still capable of assembling into large circular complexes, demonstrating that oxidation does not disrupt the lateral assembly mechanism of the antibiotic. Affinity thin-layer chromatography and fluorescence microscopy experiments suggested that the loss of activity is due to the inability of the oxidized form of nisin to bind to the cell wall precursor lipid II. Given the loss of bioactivity following oxidation, oxidation should be an important factor to consider in future production, purification, pharmacokinetic, and pharmacodynamic studies.