The lipopeptide lichenysin (cyclo-[L-Gln1-->D-Leu2-->L-Leu3-->L-Val4--> L-Asp5-->D-Leu6-->L-Ile7-beta-OH fatty acid]) produced by Bacillus licheniformis structurally resembles surfactin from Bacillus subtilis. The main difference is the presence of a glutaminyl residue in position 1 of the peptide sequence in place of glutamic acid in surfactin. This local variation causes significant changes in the properties of the molecule compared to surfactin. Lichenysin has a higher surfactant power, the critical micellar concentration (c.m.c.) being strongly reduced from 220 to 22 microM and a much higher hemolytic activity because 100% hemolysis was observed with only 15 microM instead of 200 microM. Lichenysin is also a better chelating agent because its association constants with Ca2+ and Mg2+ are increased by a factor of 4 and 16, respectively. This effect is assigned to an increase in the accessibility of the carboxyl group to cations owing to a change in the side chain topology induced by the Glu/Gln exchange. Additionally, the propensity of the lipopeptide for extensive hydrophobic interactions, as illustrated by its low c.m.c., contributes to further stabilization of the cation and an increase in the partitioning of lichenysin into the erythrocyte membrane. Our data support the formation of a lichensyin-Ca2+ complex in a molar ratio of 2:1 instead of 1:1 with surfactin, suggesting an intermolecular salt bridge between two lichenysin molecules. Therefore, when Ca2+ ions are present in the solution, micellization occurs via a dimer assembly, with a possible long-range effect on the spatial arrangement of the micelles or other supramolecular structures. Finally, among all the surfactin peptidic variants so far known, lichenysin is the one for which the three tested activities are the most substantially improved.
A series of 9 lactonic lipopeptide biosurfactants was isolated from Bacillus licheniformis IM 1307 as representatives of the lichenysin group and we propose to name them lichenysins G. They were recovered from the culture mediumas complex mixtures of molecules having different peptide sequences and different structures of /?-hydroxy fatty acids. Their separation was achieved by a reversed-phase HPLCmethod leading to eight well-separated compounds. The complete structure of individual isoforms was proposed following the results of amino acid and fatty acid analysis, LSI-MS and 2D NMRspectroscopies. Compared to surfactin, lichenysins G are at least 10 fold more efficient biosurfactants.
When Bacillus subtilis S 499 was grown on a culture medium containing L-alanine as nitrogen source, a mixture of surfactins was obtained. Suitable chromatographic conditions allowed the separation of isoforms. Among these compounds, a new variant of surfactin was isolated and its structure was established by chemical and spectrometric methods, especially by NMR spectrometry. It contains a peptide sequence which differs from that of standard surfactin by the replacement of the Lvaline residue by L-alanine residue in position 4. The folding mode of [Ala4]surfactin as deduced from NMR results was compared with that of standard surfactin and the structure/properties relationship issuing from the study of this new isoform is discussed.Several strains of Bacillus subtilis have been reported to be producers of a powerful biosurfactant, surfactin, and the strain S 499 was found as one of the best producers [1, 21. In addition to its biosurfactant activity, surfactin possesses moderate antibiotic properties, it lyses erythrocytes [3] and it is an inhibitor of fibrin clotting [l]. The most attractive property is its surfactant activity which may have potential industrial applications [4].The primary structure of surfactin has been determined by Kakinuma et al. [5]. It is a macrolide containing a heptapeptide sequence LGlu-LLeu-DLeu-LVal-LAsp-DLeu-LLeu linked to a P-hydroxy fatty acid with 13, 14 or 15 carbon atoms.Recently an isoform of surfactin has been described in which the seventh amino acid of the peptide chain of surfactin, namely L-leucine, is replaced by L-valine and it was named [Val7]surfactin. The nomenclature of this isoform is based on the symbol and the position of the unusual amino acid present in the peptide chain, as suggested by IUPAC-IUB [5a]. The initially described parent compound in this series [5] Abbreviations.[Ala4]Surfactin, surfactin in which Val4 has been replaced by Ala; LSIMS, liquid secondary ion mass spectrometry; DQF-COSY, double-quantum-filter correlation spectroscopy, TOCSY, total correlation spectroscopy ; NOESY, nuclear Overhauser enhancement spectroscopy; ycMc, surface tension close to the critical micellar concentration.they have characterized an [Ile7]surfactin by NMR spectromThe production of [Val7]surfactin was found highly dependent on the culture medium and it was enhanced by supplementation of L-valine or L-isoleucine [8]. Moreover, some differences in the surface-active properties of surfactins could result from modifications in the peptide sequence. With the object of obtaining new isoforms of surfactin, suitable experimental conditions were established for the separation of isoforms by chromatographic methods. Thus a new isoform was isolated and its structure was determined by chemical and spectrometric methods. etry ~71. MATERIALS AND METHODS Culture conditionsSurfactins were produced by Bacillus subtilis S 499. A slant culture of the strain was inoculated in 50 ml brain heart infusion (bioMkrieux, France). After 24 h at 32°C the culture broth was transferred into a groov...
The biosynthesis of bacterial isoleucyl-rich surfactins was controlled by supplementation of L-isoleucine to the culture medium. Two new variants, the [Ile4,7]- and [Ile2,4,7]surfactins, were thus produced by Bacillus subtilis and their separation was achieved by reverse-phase HPLC. Amino acids of the heptapeptide moiety were analysed by chemical methods, and the lipid moiety was identified by beta-hydroxy anteiso pentadecanoic acid by combined GC/MS. Sequences were established on the basis of two-dimensional NMR data. Because conformational parameters issuing from NMR spectra suggested that the cyclic backbone fold was globally conserved in the new variants, structure-activity relationships were discussed in details on the basis of the three-dimensional model of surfactin in solution. Indeed, both variants have increased surface properties compared with that of surfactin, and this improvement is assigned to an increase of the hydrophobicity of the apolar domain favouring micellization. Furthermore, the additional Leu-to-Ile substitution at position 2 in the [Ile2,4,7]surfactin leads to a substantial increase of its affinity for calcium, when compared with that of [Ile4,7]surfactin or surfactin. This effect is assigned, from the model, to an increase in the accessibility of the acidic side chains constituting the calcium binding site. Thus, the propensities of such active lipopeptides for both hydrophobic and electrostatic interactions were improved, further substantiating that they can be rationally designed.
Bacillus subtilis coproduces several surfactin variants that are powerful biosurfactants and have potential applications in biology and industry. A single amino acid substitution in the heptapeptide moiety of surfaetins strongly modifies their properties. To better establish structure-activity relationships and to search new variants with enhanced properties, Bacillus subtilis was grown into two modified culture media. Two new variants were isolated by chromatographic methods and studied by NMR spectroscopy. As planned, modifications consisted in the substitution of the L-valine residue at the fourth position by a more hydrophobic residue, i.e., leucine or isoleucine. These [Leu4]-and [Ile4]surfactins have a higher affinity for hydrophobic solvents and a twice improved surfactant power. Structure-property correlations were confirmed by analysis of the hydrophobic residue distribution in the three-dimensional model of the structure of surfactin in solution.
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