Lactococcus lactis strain NIZO 221 86 produces an extracellular, lanthionine-containing 3.5-kDa polypeptide with antimicrobial activity. Its retention time on reversed-phase (RP) HPLC and its amino acid composition showed high similarities but no complete identity to nisin. The gene for this lantibiotic, designated nisZ, has been cloned and its nucleotide sequence was found to be identical to that of the precursor nisin gene apart from a single mutation resulting in the substitution His27Asn in the mature polypeptide. NMR studies of the natural nisin variant, which has been designated nisin Z, confirmed the His27Asn substitution and indicated that it has a similar structure to nisin.Lantibiotics are ribosomally synthesized antimicrobial polypeptides containing the unusual amino acids lanthionine and fl-methyllanthionine [I]. Genetic analysis has shown that the lantibiotics are produced as precursor molecules consisting of an uncommon leader region and a structural region that is post-translationally processed [1 -51. Nisin is a 34-aminoacid lantibiotic produced by Lactococcus lactis that has wide applications as a food preservative 161. It also contains dehydroalanine and dehydrobutyrine (P-methyldehydroalanine) [7]. An extensive characterization of nisin by onedimensional and two-dimensional NMR analysis has recently been described [8, 91. Long range contacts were observed in the NOE spectra of nisin, indicating the presence of a preferred conformation [8]. In order to obtain further insight into the structure/function relationship of nisin, we purified and characterized an antimicrobial peptide produced by L. luctis strain NIZO 22186 that showed high similarity to nisin. Our detailed analysis on the biochemical and genetic level shows that this compound is a natural nisin variant, designated nisin Z, that differs from nisin in a single amino acid. MATERIALS AND METHODS Cultivation and purification L . luctis, subspecies luctis, strains NIZO R5 [lo] and NIZO22186 (NIZO collection) were grown for 16 h at 30°C in a medium containing 1% sucrose, 1% peptone, 1% yeast extract, 0.2% NaCI, 0.002% MgS04 . 7 H 2 0 and 1 YO KH2P04, pH 6.8 (L. de Vuyst, personal communication). After addition of 0.5 M (NH4)2S04, the cell-free supernatant (5 1) was run on a Fractogel TSK butyl650-S (Merck) column (5 x 20 cm), Correspondence to W. M. de Vos, Netherlands Institute for DairyAbbreviations. RP, reversed phase; Ile, isoleucine. Note. The novel nucleotide sequence data published here has been deposited with the EMBL sequence data banks and is available under accession number X61144.The novel amino acid sequence data published here has been deposited with the EMBL sequence data bank.Research (NIZO), P.O. Box 20, NL-6710 BA Ede, The Netherlands which was washed with 1.51 0.5M (NH4)2S04 and subsequently with deionized water until A220 < 0.5 was achieved. The antimicrobial substance was then eluted using 10mM HC1 and concentrated by ultrafiltration (Filtron, Novacell, Omega, molecular mass cut off 1 kDa). Final purification and analy...

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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 1 H 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 20, 37, and 75؇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.

Whereas protein engineering of enzymes and structural proteins nowadays is an established research tool for studying structure-function relationships of polypeptides and for improving their properties, the engineering of posttranslationally modified peptides, such as the lantibiotics, is just coming of age. The engineering of lantibiotics is less straightforward than that of unmodified proteins, since expression systems should be developed not only for the structural genes but also for the genes encoding the biosynthetic enzymes, immunity protein and regulatory proteins. Moreover, correct posttranslational modification of specific residues could in many cases he a prerequisite for production and secretion of the active lantibiotic, which limits the number of successful mutations one can apply. This paper describes the development of expression systems for the structural lantibiotic genes for nisin A, nisin Z, gallidermin, epidermin and Pep5, and gives examples of recently produced site-directed mutants of these lantibiotics. Characterization of the mutants yielded valuable information on biosynthetic requirements for production. Moreover, regions in the lantibioties were identified that are of crucial importance for antimicrobial activity. Eventually, this knowledge will lead to the rational design of lantibiotics optimally suited for fighting specific undesirable microorganisms. The mutants are of additional value for studies directed towards the elucidation of the mode of action of lantibiotics.

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Casein micelles in milk are stable colloidal particles with a stabilizing hairy brush of kappa-casein. During cheese production rennet cleaves kappa-casein into casein macropeptide and para-kappa-casein, thereby destabilizing the casein micelle and resulting in aggregation and gel formation of the micelles. Heat treatment of milk causes impaired clotting properties, which makes heated milk unsuitable for cheese production. In this paper we compared five different techniques, often described in the literature, for their suitability to quantify the enzymatic hydrolysis of kappa-casein. It was found that the technique is crucial for the yield of casein macropeptide and this yield then affects the calculated enzymatic inhibition caused by heat treatment, ranging from 5 to 30%. The technique, which we found to be the most reliable, demonstrates that heat-induced calcium phosphate precipitation does not affect the enzymatic cleavage, while whey protein denaturation causes a very slight reduction of enzyme activity. By using diffusing wave spectroscopy, a very sensitive technique to monitor gelation processes, we demonstrated that heat-induced calcium phosphate precipitation does not affect the clotting. Whey protein denaturation does not affect the start of flocculation but has a clear effect on the clotting process. This work adds to a better understanding of the processes causing the impaired clotting properties of heated milk.

Structural properties of whey protein (WP)/gum arabic (GA) coacervates were investigated by measuring the diffusivity of WP and GA in their coacervate phase as a function of pH by means of three different complementary techniques. The combination of these measurements revealed new insights into the structure of coacervates. Nuclear magnetic resonance (NMR) measured the self-diffusion coefficient of the GA in the coacervate phase prepared at various pH values. Fluorescence recovery after photobleaching (FRAP) was measured using a confocal scanning laser microscope. The WP and GA were covalently labeled with two different dyes. The time of fluorescence recovery, related to the inverse of the diffusion coefficient, was evaluated from the measurements, and the diffusivity of the WP and GA on a long time scale could be individually estimated at each pH value. Diffusing wave spectroscopy (DWS) combined with transmission measurement was carried out in the coacervate phase, and the diffusion coefficient, corresponding to the averaged diffusion of all particles that scattered in the system, was calculated as a function of pH. Independently of the technique used, the results showed that the diffusion of the WP and GA within the coacervate phase was reduced as compared to a diluted biopolymer mixture. NMR, DWS, and FRAP measurements gave similar results, indicating that the biopolymers moved the slowest in the coacervate matrix at pH 4.0-4.2. It is assumed that the diffusion of the WP and GA is reduced because of a higher electrostatic interaction between the biopolymers. Furthermore, FRAP results showed that in the coacervate phase WP molecules diffused 10 times faster than GA molecules. This result is very relevant because it shows that WP and GA move independently in the liquid coacervate phase. Finally, DWS measurements revealed that the coacervate phase rearranged with time, as evidenced by a decrease of the diffusion coefficient and a loss of the turbidity of the sample. A more homogeneous transparent coacervate phase was obtained after a few days/weeks. Faster rearrangement was obtained at pH 3.0 and 3.5 than at higher pH values.