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In 1968, Arima et al. [196] reported the isolation and characterization of a potent clotting inhibitor produced in the culture fluids of several strains of Bacillus subtilis. As its surface activity was much stronger than that of sodium lauryl sulfate they named the product surfactin. Figure 1.9 shows the structure of a typical surfactin. Other structures are also known [20].

Figure 1.9 Schematic structure of a surfactin.

According to Arima et al., surfactin is a peptide lipid composed of L‐aspartic acid, L‐glutamic acid, L‐valine, L‐leucine, D‐leucine (1 : 1 : 1 : 2 : 2) and unidentified fatty acids. The complete elucidation of the structure was published by Kakinuma et al. in a series of papers one year later [197–200]. Bonmatin et al. [201] studied surfactin by two‐dimensional ¹H‐NMR in DMSO and observed two conformations characterized by a saddle‐shape topology with polar Glu and Asp side chains oppositely oriented to that of the C11–13 aliphatic chain. The conformation of surfactin was reinvestigated by FTIR spectroscopy by Vass et al. [202]. Circular dichroism (CD) and FTIR spectroscopic data in different solvents with or without Ca²⁺ ions indicate that surfactin has a unique ability of adopting strongly different conformations depending on the conditions. The carboxyl groups of Glu¹ and Asp⁵ are responsible for Ca²⁺ binding at low concentration (ratio of calcium/lipopeptide < 1). The NMR structure of surfactin has been determined in sodium dodecyl sulfate and dodecylphosphocholine micellar solutions [203]. pKa values of Asp and Glu are around 4.3 and 4.5 [204, 203]. When comparing results for surfactins from different authors it is important to notice that different hydrophobic alkyl chains may be involved. Razafindralambo et al. [205] have isolated a homologous series of surfactins containing β‐hydroxy fatty acids having different alkyl chain lengths (13, 14, or 15 carbon atoms). These authors have investigated their dynamic surface properties and found a dependence with both bulk concentration and hydrophobic character of the alkyl chain. The cmc values depend on the length of the alkyl chain and the tendency is the same as that observed for classical alkyl chain surfactants, i.e. the values are lower the larger is the alkyl chain. The surface tension at cmc changes in the same direction. These authors also observed that at low concentrations, the longer the alkyl chain, the faster is the decrease of the surface with time, but at high concentrations the maximum rate was observed for n _c = 14. Surfactin reduces to 27 mN/m at a concentration as low as 2 × 10⁻⁵ M. For surfactin, several authors have found two break points in the plot surface tension vs log (concentration), both being dependent on the experimental conditions [177]. The first one of 1.89 × 10⁻⁶ M (20 mM phosphate buffer) has been ascribed to a premicellization phenomenon, while the second one was assigned to the cmc (Table 1.2).

The effect of monovalent and divalent cations on surfactin was studied by Thimon et al. [206]. The experiments were performed at pH 9.5 (5 mM Tris), and Mg²⁺, Ca²⁺, and Mn²⁺ chloride salts were added at 0.5 mM concentration and monovalent ions (Li⁺, Na⁺, K⁺, and Rb⁺) were added at 0.1 M. Both type of cations, as well their concentrations, had a strong effect on cmc. For instance, values of 2.0 × 10⁻⁵ M (Ca²⁺), 5.7 × 10⁻⁵ M (Mn²⁺), 6.0 × 10⁻⁷ M (Li⁺) and 1.47 × 10⁻⁶ M (Rb⁺) were found. Molecular areas (determined from the analysis of the premicellar surface tension‐log (concentration) plot) are also affected and the authors concluded that the micellization of surfactin is highly favored in the presence of divalent cations. Later Thimon et al. [207] calculated the association constants of some cations, the obtained values being K = 1.5 × 10⁵/M and 1.9 × 10⁴/M for Ca²⁺ and Mg²⁺, respectively. The stoichiometry of complexes is 1 : 1 (Ca²⁺) or 2 : 1 (Rb⁺).

Li 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, pK_a = 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 Ų 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 Ų/molecule and reaches a breaking point at 123 Ų/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 Ų 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 locillomycins (nonapeptides) can be mentioned, as well as linear lipopeptides. In comparison to surfactin, much less is known about their physicochemical properties.