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. 1995 Aug;61(8):2873–2878. doi: 10.1128/aem.61.8.2873-2878.1995

Improvement of solubility and stability of the antimicrobial peptide nisin by protein engineering.

H S Rollema 1, O P Kuipers 1, P Both 1, W M de Vos 1, R J Siezen 1
PMCID: PMC167563  PMID: 7487019

Abstract

Nisin is a 3.4-kDa antimicrobial peptide that, as a result of posttranslational modifications, contains unsaturated amino acids and lanthionine residues. It is applied as a preservative in various food products. The solubility and stability of nisin and nisin mutants have been studied. It is demonstrated that nisin mutants can be produced with improved functional properties. The solubility of nisin A is highest at low pH values and gradually decreases by almost 2 orders of magnitude when the pH of the solution exceeds a value of 7. At low pH, nisin Z exhibits a decreased solubility relative to that of nisin A; at neutral and higher pH values, the solubilities of both variants are comparable. Two mutants of nisin Z, which contain lysyl residues at positions 27 and 31, respectively, instead of Asn-27 and His-31, were produced with the aim of reaching higher solubility at neutral pH. Both mutants were purified to homogeneity, and their structures were confirmed by one- and two-dimensional 1H nuclear magnetic resonance. Their antimicrobial activities were found to be similar to that of nisin Z, whereas their solubilities at pH 7 increased by factors of 4 and 7, respectively. The chemical stability of nisin A was studied in the pH range of 2 to 8 and at a 20, 37, and 75 degrees C. Optimal stability was observed at pH 3.0. Nisin Z showed a behavior similar to that of nisin A. A mutant containing dehydrobutyrine at position 5 instead of dehydroalanine had lower activity but was significantly more resistant to acid-catalyzed chemical degradation than wild-type nisin Z.

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Selected References

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  1. Casadaban M. J., Cohen S. N. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol. 1980 Apr;138(2):179–207. doi: 10.1016/0022-2836(80)90283-1. [DOI] [PubMed] [Google Scholar]
  2. De Vuyst L., Vandamme E. J. Influence of the carbon source on nisin production in Lactococcus lactis subsp. lactis batch fermentations. J Gen Microbiol. 1992 Mar;138(3):571–578. doi: 10.1099/00221287-138-3-571. [DOI] [PubMed] [Google Scholar]
  3. Gasson M. J. Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-induced curing. J Bacteriol. 1983 Apr;154(1):1–9. doi: 10.1128/jb.154.1.1-9.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Gross E., Morell J. L. The structure of nisin. J Am Chem Soc. 1971 Sep 8;93(18):4634–4635. doi: 10.1021/ja00747a073. [DOI] [PubMed] [Google Scholar]
  5. Gross E. alpha, beta-Unsaturated and related amino acids in peptides and proteins. Adv Exp Med Biol. 1977;86B:131–153. doi: 10.1007/978-1-4757-9113-6_9. [DOI] [PubMed] [Google Scholar]
  6. Kuipers O. P., Beerthuyzen M. M., Siezen R. J., De Vos W. M. Characterization of the nisin gene cluster nisABTCIPR of Lactococcus lactis. Requirement of expression of the nisA and nisI genes for development of immunity. Eur J Biochem. 1993 Aug 15;216(1):281–291. doi: 10.1111/j.1432-1033.1993.tb18143.x. [DOI] [PubMed] [Google Scholar]
  7. Kuipers O. P., Boot H. J., de Vos W. M. Improved site-directed mutagenesis method using PCR. Nucleic Acids Res. 1991 Aug 25;19(16):4558–4558. doi: 10.1093/nar/19.16.4558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kuipers O. P., Rollema H. S., Yap W. M., Boot H. J., Siezen R. J., de Vos W. M. Engineering dehydrated amino acid residues in the antimicrobial peptide nisin. J Biol Chem. 1992 Dec 5;267(34):24340–24346. [PubMed] [Google Scholar]
  9. Lian L. Y., Chan W. C., Morley S. D., Roberts G. C., Bycroft B. W., Jackson D. Solution structures of nisin A and its two major degradation products determined by n.m.r. Biochem J. 1992 Apr 15;283(Pt 2):413–420. doi: 10.1042/bj2830413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Liu W., Hansen J. N. Some chemical and physical properties of nisin, a small-protein antibiotic produced by Lactococcus lactis. Appl Environ Microbiol. 1990 Aug;56(8):2551–2558. doi: 10.1128/aem.56.8.2551-2558.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Mulders J. W., Boerrigter I. J., Rollema H. S., Siezen R. J., de Vos W. M. Identification and characterization of the lantibiotic nisin Z, a natural nisin variant. Eur J Biochem. 1991 Nov 1;201(3):581–584. doi: 10.1111/j.1432-1033.1991.tb16317.x. [DOI] [PubMed] [Google Scholar]
  12. Rauch P. J., De Vos W. M. Characterization of the novel nisin-sucrose conjugative transposon Tn5276 and its insertion in Lactococcus lactis. J Bacteriol. 1992 Feb;174(4):1280–1287. doi: 10.1128/jb.174.4.1280-1287.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Van de Ven F. J., Van den Hooven H. W., Konings R. N., Hilbers C. W. NMR studies of lantibiotics. The structure of nisin in aqueous solution. Eur J Biochem. 1991 Dec 18;202(3):1181–1188. doi: 10.1111/j.1432-1033.1991.tb16488.x. [DOI] [PubMed] [Google Scholar]
  15. Vos P., Simons G., Siezen R. J., de Vos W. M. Primary structure and organization of the gene for a procaryotic, cell envelope-located serine proteinase. J Biol Chem. 1989 Aug 15;264(23):13579–13585. [PubMed] [Google Scholar]
  16. de Vos W. M., Mulders J. W., Siezen R. J., Hugenholtz J., Kuipers O. P. Properties of nisin Z and distribution of its gene, nisZ, in Lactococcus lactis. Appl Environ Microbiol. 1993 Jan;59(1):213–218. doi: 10.1128/aem.59.1.213-218.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

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