et al. [208] studied the influence of Na+ ions on surfactin‐C16 by fluorescence using pyrene as the fluorescent probe. From the plot of I 1/I 3 vs log S t a value of 2.47 × 10−5 M (0.05 M Tris buffer, pH 8.5–8.6, 293 K) was obtained. These authors also observed that the micropolarity surrounding the pyrene molecules decreases with the addition of enough Na+. The authors observed a decrease of the aggregation number (determined by the steady‐state fluorescence quenching method) with increasing Na+, which is contrary to what it should be expected. A similar study was conducted by these authors [209] to analyze the effect of other ions as Li+, K+, Mg2+, and Ca2+. As previously, monovalent ions reduced the micropolarity, and tend to originate small spherical micelle particles. The effect of Mg2+ concentration on micropolarity (expressed by the I 1/I 3 ratio) is less obvious than for monovalent ions while the behavior in the presence of Ca2+ is different as the ratio strongly increased to reach a maximum and then “was almost unchanged at other concentrations.” The morphology of surfactin‐C16 micelles with different counterions was observed by Freeze‐Fracture Transmission Electron Microscopy (FFTEM). The smallest micelles were observed in the Li+ solution. In the presence of divalent cations, large aggregates about 200 nm wide and more than 500 nm length were observed.
Surfactin was studied by ITC in phosphate buffer of pH 7.4 at 30 °C [210]. Values of 11.09 kJ/mol and 3.8 × 10−5 M were obtained for the enthalpy of micellization and the cmc, respectively. As the process is endothermic at this temperature, the process has to be entropy‐driven. The distribution of hydrodynamic radius (dynamic light scattering experiments), in terms of the relative number, shows one peak at 4–6 nm at both 0.1 and 0.3 mM surfactin concentrations. In terms of intensity, large aggregates were also observed (~85 nm at 0.1 mM surfactin and ~108 nm at 0.3 mM surfactin). TEM images at 0.3 mM also show the coexistence of small micelles and large aggregates. CD spectra are concentration dependent, showing that the secondary structure of surfactin adopts a β‐turn at low concentrations (0.1–0.3 mM) and begins to adopt a β‐sheet conformation at a relatively high concentration (0.5 mM).
At pH 8.7 (0.1 M NaHCO3) Ishigami et al. [211] measured a value of 9.4 × 10−6 M for the cmc of surfactin (3‐hydroxymyristic acid) at which the surface tension was 30 mN/m (25 °C). In these conditions, CD measurements confirmed that the secondary structure of surfactin was a ‐sheet conformation, the molar ellipticity being stable, while the lactone ring was confirmed by FTIR. The micellar aggregation number was 173 and, assuming a cylindrical shape, from static light scattering measurements, the dimensions would be 231 nm (length) and 5.8 nm (diameter). The authors concluded thati“surfactin formed a large elongated rod‐shaped micelle in spite of its bulky molecular structure.”
The surface pressure increases from areas A o = 1.84, 1.82, and 2.02 nm2 and reaches breaking points at areas of A t = 0.89, 0.81, and 0.79 nm2, the values being obtained at pH values of 4.2, 4.8, and 5.4, respectively (20 °C, pKa = 5.8). The A t values are close to the molecular area (0.75 nm2) estimated for a surfactin model with alkyl chains and the peptide ring vertically and supine oriented, respectively, to the plane surface. It was concluded that the compression from A o to A t leads to a reorientation of the alkyl chains (from flat on the surface to vertical with respect to the surface plane) but the peptide rings remain with a supine orientation. Different values were obtained by Maget‐Dana and Ptak [212].
Knoblich et al. [213] studied the aggregates of surfactin by cryo‐TEM in water at pH 7, 9.5, and 12. Spherical, ellipsoidal, and/or cylindrical micelles were observed as a function of pH. At pH 7, spherical (diameters between 5 and 9 nm) and ellipsoidal micelles (length 19 nm and width 11 nm) were measured. At pH 9.5 the dimension of cylindrical micelles had a length of 40–160 nm and a width of 10–14 nm. At this pH, the addition of 0.1 M NaC1 and 0.2 M CaC12 transforms the cylindrical micelles into spherical and/or ellipsoidal micelles of small size. However, at pH 12 the micrographs show mostly spherical (average diameter ~ 8 nm) and ellipsoidal micelles (size ~9 and 6 nm). At this pH the characteristic FTIR band for the lactone group disappeared, meaning that this group is opened. Consequently, the authors suggest that the formation of the surfactin micelles at pH 12 is different from that at low pH values.
For surfactin‐C15, Zou et al. [214] obtained a cmc value of 1.54 × 10−5 M and γ cmc = 27.7 mN/m (0.01 M phosphate buffer at pH 7.4; 25 °C). From the Gibbs isotherm, a value of 107.8 Å2 was calculated for the molecular area at the interface. The experimental SANS data, fitting the curves for sphere‐like aggregates, show that the radius of gyration of surfactin aggregates increases from 16 ± 0.4 Å to 20.1 ± 0.6 Å when the concentration increases from 4.0 × 10−5 M to 2.4 × 10−4 M. The pressure‐area isotherm at the air–water interface shows that the pressure starts to increase at 231 Å2/molecule and reaches a breaking point at 123 Å2/molecule (pH 7.4, 25 °C). These values are higher than those published by Ishigami et al. [211] at lower pH values.
Osman et al. [215] have studied the effects of pH, Ca2+ ions, and the nonionic surfactant C12E7 on the conformation of surfactin in aqueous solutions using CD. Gradual alterations in the CD spectra of surfactin were observed that were related to the aggregational behavior of surfactin. The aggregation number (static light scattering measurements) raised to 144 at the surfactin/C12E7 molar ratio of 25 : 75, indicating enhancement of micellization. The molar ellipticity suggests that C12E7 enhanced the formation of micelles by promoting the assembly of surfactin molecules in sheets, even at very low surfactin concentrations. This could be due to an intercalation of C12E7 surfactin molecules in the micelle. The formation of sheets is enhanced by a temperature increase. Below the cmc, at pH > 8.5, CD spectra suggest an unordered conformation, but at neutral pH the conformation changed to sheets and the surfactin monomers have a helical conformation. Above the cmc, the pH effect was different. At pH 9, helices were formed, and below this pH, until a value of 6 occurred, sheets were formed. At pH > 9, the cyclic lactone ring of surfactin may be cleaved to form linear surfactin in solutions stored for long periods of time, and consequently above this pH value the observations could be related to the linear surfactin derivative, in concordance with Knoblich et al. [213]. The transitions induced in the monomers were dependent on the concentration of Ca2+ (α‐helices–unordered structure–β‐sheet conformation), a phenomenon that could be due to the binding of surfactin to Ca2+ and formation of surfactin clusters. These transitions were also observed above the cmc. Thus Ca2+ affects the surfactin conformation and also induces concentration‐dependent transitions. These results suggest that β‐sheets is the preferred bioactive conformation of surfactin.
Shen et al. [216] have produced from the B. subtilis strain three different deuterated surfactins (one perdeuterated, one with the four leucines perdeuterated, and one with everything except the four leucines perdeuterated) and used them in NR and SANS studies at pH 7.5. As expected, the largest signal in the reflectivity profiles is from the perdeuterated sample. Fitting the layer as a Gaussian distribution normal to the surface, in all three cases the area per molecule at the surface is 147 ± 10 Å2 and the overall thickness of the layer is 14 ± 2 Å. The results also suggest an unusually close‐packed surface layer and that the alkyl chain must be folded back into the leucines of the heptapeptide ring in order to give the observed compactness and the low extent of immersion in the aqueous subphase. Therefore, surfactin adopts a compact globular structure at the surface. The best fit of data was obtained by a core‐shell model in which the core contains the alkyl chain and the four leucines, and the remaining head group, water, and the counterions are at the shell. The aggregation number was 20 ± 4, the overall micelle diameter 50 ± 5 Å, and the radius of the hydrophobic core 22 ± 2 Å.
The structure of other cyclolipopeptides different from surfactin have been reviewed by Kaspar et al. [20]. Among them families of iturins (heptapeptides), fengycin (decapeptides), or