The influence of several parameters on the fermentative production of nisin Z by Lactococcus lactis IO-1 was studied. Considerable attention has been focused on the relationship between the primary metabolite production of bacteriocin and lactate and cell growth, which has so far not been clarified in detail. Production of nisin Z was optimal at 30 degrees C and in the pH range 5.0-5.5. The addition of Ca2+ to the medium showed a stimulating effect on the production of nisin Z. A maximum activity of 3150 IU/ml was obtained during pH-controlled batch fermentation in the medium supplemented with 0.1 M CaCl2. It was about three times higher than that obtained under the optimal conditions for cell growth and lactic acid production.

Staphylococcus warneri ISK-1, which we had previously reported as Pediococcus sp. ISK-1, produces a novel bacteriocin, nukacin ISK-1. Edman degradation of the chemically reduced nukacin ISK-1 produced a sequence of 27 amino acids, 7 of which were unidentified. Using single-specific-primer-PCR product as a probe, a 3.6-kb HindIII fragment containing the nukacin ISK-1 structural gene (nukA) was cloned and sequenced. The deduced amino acid sequence of nukacin ISK-1 had 57 amino acids, including a 30-amino acid leader region. The propeptide sequence showed significant similarity to those of lacticin-481 type lantibiotics. In the region upstream of nukA, a part of a long open reading frame (ORF), designated as nukM, encoding a putative modification enzyme was oriented in the opposite direction. In the region downstream of nukA, ORF1 was found in which the sequence of the putative translational product was similar to various response regulatory proteins.

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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%.