Development of Purification Method and Identification of a Peptide Antibiotic Produced by Lactococcus lactis IO-1.

Abstract

The culture supernatant of Lactococcus lactis I0-1, which was isolated in our laboratory, inhibited the cell growth of various Gram-positive bacteria but did not inhibit the nisin A-producing strain, L. Iactis NCDO 497. A nisin-like peptide antibiotic produced by L. Iactis I0-1 was efficiently purified sequentially by acid treatment (at pH 3), ammonium sulfate precipitation, cation-exchange chromatography and reversed-phase high performance liquid chromatography. Dissociation of the peptide aggregates with high molar concentrations of urea resulted in successful purification. The molecular mass of this peptide antibiotic was 3335.67 by fast atom bombardment-mass spectrometry, confirming that the peptide antibiotic from L. Iactis I0-1 is nisin Z, a natural nisin variant. The purification method used is rapid, simple and effective, permitting the specific activity to increase 122-fold, and the recovery was 24%.

Some Characteristics of Nisin Z, a Peptide Antibiotic Produced by Lactococcus lactis IO-1.

“…We also investigated the relationship between the primary metabolite production of nisin Z and lactate and cell growth, which had so far not been clarified in detail. This study resulted in a 3-fold activity of nisin Z in the culture broth under optimal conditions (Matsusaki et al, 1996b). Nisin Z has the possibility of use as a food preservative similarly to nisin A.…”

“…Bacterial strains and culture conditions Lactococcus lactis IO-I isolated in our laboratory was used as the nisin Z-producing strain (Matsusaki et al, 1996a). To compare nisin Z with nisin A, L. Iactis NCDO 497, a nisin Aproducing strain, was also used.…”

“…To compare nisin Z with nisin A, L. Iactis NCDO 497, a nisin Aproducing strain, was also used. The indicator strain used for the assay of antimicrobial activity was Bacillus subtilis C I as described previously (Matsusaki et al, 1996a coagulans JCM 2257T, B. stearothermophilus JCM 2501T Micrococcus luteus IF012708 and Escherichia coli JMI09…”

“…All lactic acid bacteria, bacilli and C. acetobutylicum were grown as described previously (Matsusaki et al, 1996a …”

“…Iactis IO-I isolated in our laboratory produces a peptide antibiotic, which was purified and identified to be nisin Z (Matsusaki et al, 1996a). We also investigated the relationship between the primary metabolite production of nisin Z and lactate and cell growth, which had so far not been clarified in detail.…”