• nisin;
  • protein engineering;
  • charged residue;
  • antimicrobial activity;
  • autoregulation

Three mutants of the lantibiotic nisin Z, in which the Val32 residue was replaced by a Glu, Lys or Trp residue, were produced and characterized for the purpose of establishing the role of charge differences in the C-terminal part of nisin on antimicrobial activity and signaling properties. 1H-NMR analyses showed that all three mutants harbor an unmodified serine residue at position 33, instead of the usual dehydroalanine. Apparently, the nature of the residue preceding the serine to be dehydrated, strongly affects the efficiency of modification. Cleavage of [Glu32,Ser33]nisin Z by endoproteinase Glu-C yielded [Glu32]nisin Z(1–32)-peptide, which has a net charge difference of -2 relative to wild-type nisin Z. The activity of [Lys32, Ser33]nisin Z against Micrococcus flavus was similar to that of wild-type nisin, while [Trp32,Ser33]nisin Z, [Glu32, Ser33]nisin Z and [Glu32]nisin Z(1–32)-peptide exhibited 3–5-fold reduced activity, indicating that negative charges in the C-terminal part of nisin Z are detrimental for activity. All variants showed significant loss of activity against Streptococcus thermophilus. The potency of the nisin variants to act as signaling molecules for auto-induction of biosynthesis was significantly reduced. To obtain mutant production, extracellular addition of (mutant) nisin Z to the lactococcal expression strains was essential.