Dynamic transition of α-helix to β-sheet structure in linear surfactin correlating to critical micelle concentration

Summary Linear heptapeptide surfactin was prepared by alkaline cleavage of the lactone ring of cyclic surfactin. The structure of linear surfactin was characterised and confirmed by FAB-mass-spectroscopy, FT-IR and HPLC analysis. It was found that linear surfactin easily forms micelles in aqueous solutions by coordinating -sheet formation from -helical monomolecules, and the cmc value found to be 1.28×10^–5 M. The CD spectra indicates conformational change of linear surfactin from -helical below cmc to -sheet above cmc.     

Tuning micelles of a bioactive heptapeptide biosurfactant via extrinsically induced conformational transition of surfactin assembly

We have studied the effects of extrinsic environmental conditions on the conformation of surfactin, a heptapeptide biosurfactant from Bacillus subtilis, in aqueous solutions. It has been made clear that temperature, pH, Ca²⁺ ions and the synthetic nonionic surfactant hepta-ethylene glycol (C₁₂E₇) affect the conformation of surfactin in aqueous solutions. The β-sheet formation reached a maximum at 40°C both in presence and absence of (C₁₂E₇) and the nonionic surfactant enhances the β-sheet formation even at 25°C. Ca²⁺ induced the formation of a-helices and caused this transition at 0.3 mm with surfactin monomers or at 0.5 mm with surfactin micelles, but above these transition concentrations of Ca²⁺ β-sheets were observed. In micellar solution the β-sheet structure was stabilized at pH values below 7 or upon addition of Ca²⁺ in concentrations above 0.5 mm . Our results indicated that the bioactive conformation of surfactin is most likely the β-sheets when the molecules are assembled in micelles. The β-sheet structure in micelles could be retained by tuning the micelles. Surfactin micelles could be tuned in the bioactive conformation by manipulating pH, temperature, Ca²⁺ or (C₁₂E₇) concentrations in surfactin solutions. Our results strongly indicated that Ca²⁺ and other molecules (such as C₁₂E₇) may function as directing templates in the assembly and conformation of surfactin in micelles. Thus, we suggest environmental manipulation and template-aided micellation (TAM) as a new approach for preparing predesigned micelles, microemulsions or micro-spheres for specific application purposes. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Interactions of bioactive lipopeptides, iturin A and surfactin from Bacillus subtilis.

The antifungal activity of iturin A and its interaction with erythrocyte membranes were enhanced in the presence of surfactin. The modification of the properties of iturin A was explained by the formation of mixed iturin A-surfactin micelles. Such mixed micelles were easily generated when both lipopeptides were in aqueous solutions in the absence of mineral salts but the formation of these micelles did not occur when the solutions contained a high molarity of mineral cations.     

Physicochemical studies of the interaction of the lipoheptapeptide surfactin with lipid bilayers of L-alpha-dimyristoyl phosphatidylcholine

To understand the biological action of surfactin from Bacillus subtilis we investigated its effects on the phase transition of L-alpha-dimyristoyl phosphatidylcholine (DMPC)-vesicles from the crystalline to the fluid state using differential scanning calorimetry; light scattering; small angle neutron scattering and cryo-electron microscopy. DSC-thermograms revealed two phase transition peaks. Light scattering profiles showed two branches with characteristic hysteresis phenomena. With both techniques the same values of the phase transition temperatures T(m1) and T(m2) of 23.5 and 23 degrees C were obtained indicating two forms of DMPC-surfactin aggregates which could be visualized by cryo-electron microscopy. Until 4 mol% surfactin the vesicular form predominated, but was accompanied by bilayered membrane fragments by increasing the biosurfactant concentrations. At surfactin concentrations higher than 15 mol% smaller DMPC-surfactin micelles of ellipsoidal conformation were formed, as demonstrated by small angle neutron scattering. In addition, by "Poor Man's" temperature-jump-relaxation spectroscopy slow transients in the phase transition of vesicular DMPC-surfactin aggregates with relaxation times of 20-30 s were detected which presumably indicate the slow dissipation of intermediate lipid-and surfactin domains formed after the main phase transition on the way to the fluid state. This process is accelerated by surfactin.     

Thermobarostability of alpha-chymotrypsin in reversed micelles of aerosol OT in octane solvated by water-glycerol mixtures

Thermostability of alpha-chymotrypsin at normal pressure in reversed micelles depends on both an effective surfactant solvation degree and glycerol content in the system. The difference in alpha-chymotrypsin stability in reversed micelles at various glycerol concentrations [up to 60% (v/v)] was more pronounced at high surfactant degrees of solvation, R >/= 16. After a 1-h incubation at 40 degrees C in "aqueous" reversed micelles (in the absence of glycerol), alpha-chymotrypsin retained only 1% of initial catalytic activity and 10, 22, 59, and 48% residual activity in glycerol-solvated micelles with 20, 30, 50, and 60% (v/v) glycerol, respectively. The explanation of the observed effects is given in the frames of micellar matrix structural order increasing in the presence of glycerol as a water-miscible cosolvent that leads to the decreasing mobility of the alpha-chymotrypsin molecule and, thus the increase of its stability. It was found that glycerol or hydrostatic pressure could be used to stabilize alpha-chymotrypsin in reversed micelles; a lower pressure is necessary to reach a given level of enzyme stability in the presence of glycerol.     

Enhancement of plasminogen activation by surfactin C: augmentation of fibrinolysis in vitro and in vivo

The reciprocal activation of plasminogen and prourokinase (pro-u-PA) is an important mechanism in the initiation and propagation of local fibrinolytic activity. We have found that a bacterial lipopeptide compound, surfactin C (3-20 microM), enhances the activation of pro-u-PA in the presence of plasminogen. This effect accompanied increased conversions of both pro-u-PA and plasminogen to their two-chain forms. Surfactin C also elevated the rate of plasminogen activation by two-chain urokinase (tcu-PA) while not affecting plasmin-catalyzed pro-u-PA activation and amidolytic activities of tcu-PA and plasmin. The intrinsic fluorescence of plasminogen was increased, and molecular elution time of plasminogen in size-exclusion chromatography was shortened in the presence of surfactin C. These results suggested that surfactin C induced a relaxation of plasminogen conformation, thus leading to enhancement of u-PA-catalyzed plasminogen activation, which in turn caused feedback pro-u-PA activation. Surfactin C was active in enhancing [125I]fibrin degradation both by pro-u-PA/plasminogen and tcu-PA/plasminogen systems. In a rat pulmonary embolism model, surfactin C (1 mg/kg, i.v.) elevated 125I plasma clot lysis when injected in combination with pro-u-PA. The present results provide first evidence that pharmacological relaxation of plasminogen conformation leads to enhanced fibrinolysis in vivo.     

Enhanced production of surfactin from Bacillus subtilis by addition of solid carriers

Addition of a small quantity of solid porous carriers (e.g., activated carbon or expanded clay) into fermentation broth significantly increased surfactin production with Bacillus subtilis ATCC 21332. Culture medium containing 25 g L(-1) of activated carbon gave an optimal surfactin yield of 3600 mg L(-1), which was approximately 36-fold higher than that obtained from carrier-free liquid culture. The marked increase in surfactin production was primarily attributed to stimulation of cell growth due to the presence of activated carbon carriers. Concentration of limiting carbon substrate (glucose) is also an important factor affecting the production of surfactin, as an initial glucose concentration of 40 g L(-1) resulted in optimal surfactin production. An appropriate agitation rate also benefited surfactin production, as the best yield appeared at an agitation rate of 200 rpm. Surfactin was purified from fermentation broth via a series of acidic precipitation and solvent extraction. The resulting product was nearly 90% pure with a recovery efficiency of ca. 72%. The purified surfactin reduced the surface tension of water from 72 to 27 mN m(-1) with a critical micelle concentration of ca. 10 mg L(-1). The surfactin product also attained an emulsion index of 70% for kerosene and diesel at a low concentration of 100 and 600 mg L(-1), respectively.     

Expression of sfp gene and hydrocarbon degradation by Bacillus subtilis

Bacillus subtilis C9 produces a lipopeptide-type biosurfactant, surfactin, and rapidly degrades alkanes up to a chain length of C₁₉. The nucleotide sequence of the sfp gene cloned from B. subtilis C9 was determined and its deduced amino acid sequence showed 100% homology with the sfp gene reported before [Nakano et al. (1992) Mol. Gen. Genet. 232: 313–321]. To transform a non-surfactin producer, B. subtilis 168, to a surfactin producer, the sfp gene cloned from B. subtilis C9 was expressed in B. subtilis 168. The transformed B. subtilis SB103 derivative of the strain 168 was shown to produce surfactin measured by its decrease in surface tension, emulsification activity, and TLC analysis of the surface active compound isolated from the culture broth. Like B. subtilis C9, B. subtilis SB103 containing sfp gene readily degraded aliphatic hydrocarbons (C_10–19), though its original strain did not. The addition of surfactin (0.5%, w/v) to the culture of B. subtilis 168 significantly stimulated the biodegradation of hydrocarbons of the chain lengths of 10–19; over 98% of the hydrocarbons tested were degraded within 24 h of incubation. These results indicate that the lipopeptide-type biosurfactant, surfactin produced from B. subtilis enhances the bioavailability of hydrophobic hydrocarbons.     

Influence of Water on the Primary Photosynthetic Activity of Rhodospirillum Rubrum in Reverse Micelles

The effect of water on the primary photosynthetic activity of purple bacterium Rhodospirillum rubrum was studied in Hexadecane-Tween-Spane (HTS)- and phospholipid (PLC)-reverse micelles. Reverse micelles offer the possibility of modulating the amount of water to which enzymes and multienzymatic complexes are exposed. Fast bacteriochlorophyll (BChl) fluorescence induction kinetics and reaction centre absorption changes at 820 nm were used as an assay for the functional transfer of bacterial cells into HTS-reverse micelles and bacterial photosynthetic complexes (BPC) into PLC-reverse micelles. Both the bacterial cells and BPC showed an increase in the rate of primary photosynthetic activity by increasing the concentration of water in the reverse micelles. The bacterial cells could be kept viable for many hours in HTS-reverse micelles in presence of 6% (v/v) water. NMR studies indicated that the photosynthetic activity was affected by the availability of water in reverse micelles. The bacterial cells in HTS or BPC in PLC reverse micelles could be used to further understand the influence of water on the organisation and function of photosynthetic complexes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Production of a lipopeptide antibiotic, surfactin, by recombinant Bacillus subtilis in solid state fermentation

Production of a lipopeptide antibiotic, surfactin, in solid state fermentation (SSF) on soybean curd residue, Okara, as a solid substrate was carried out using Bacillus subtilis MI113 with a recombinant plasmid pC112, which contains lpa-14, a gene related to surfactin production cloned at our laboratory from a wild-type surfactin producer, B. subtilis RB14. The optimal moisture content and temperature for the production of surfactin were 82% and 37 degrees C, respectively. The amount of surfactin produced by MI113 (pC112) was as high as 2.0 g/kg wet weight, which was eight times as high as that of the original B. subtilis RB14 at the optimal temperature for surfactin production, 30 degrees C. Although the stability of the plasmid showed a similar pattern in both SSF and submerged fermentation (SMF), production of surfactin in SSF was 4-5 times more efficient than in SMF. (c) 1995 John Wiley & Sons, Inc.     

Isolation and characterization of a halotolerant <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">subtilis</Emphasis> BBK-1 which produces three kinds of lipopeptides: bacillomycin L,plipastatin, and surfactin

Twenty-three halotolerant and biosurfactant producing strains were collected from salty conditions in central Thailand. One of the strains designated BBK-1 produced the biosurfactants with the highest activity. BBK-1 was isolated from fermented foods and was identified as B. subtilis based on its physiological characteristics and 16S rRNA gene sequence. We show that the strain grows in media containing NaCl up to 16% (w/v) and produces biosurfactants in NaCl up to 8%. We found that B. subtilis BBK-1 produces three kinds of surface-active lipopeptides simultaneously. By their respective molecular weights and amino acid compositions, it is indicated that these lipopeptides are bacillomycin L, plipastatin, and surfactin. In order to analyze the production mechanism of lipopeptides further in the strain, a generally important biosynthetic gene encoding 4'-phosphopantetheinyl transferase was cloned and sequenced. The gene existed in a single copy in the genome and the deduced amino acid sequence was almost identical to that of Lpa-14 from B. subtilis strain RB14, which co-produces iturin A and surfactin.     

Surfactin isoforms from Bacillus subtilis HSO121: separation and characterization

Natural surfactin is a mixture of cyclic lipopeptides built from variants of a heptapeptide and a beta-hydroxy fatty acid. A biosurfactant-producing strain, Bacillus subtilis HSO121, was isolated from the production water of an oil field. The strain was able to produce eight surfactin isoforms which have been isolated by acid-precipitation followed by extraction into methanol. A novel procedure for the purification of surfactin was achieved. It consists of a solid-phase extraction on C(18) gel followed by reversed-phase high performance liquid chromatography using Prep. HiQ sil C18W, column. The surfactin isoforms were eluted by linear acetonitrile gradient from 80-100%. The peaks were analyzed by TLC on silica gel, and after acid hydrolysis their amino acid compositions were determined by HPLC analysis. Eight isoforms of surfactin had nearly the same amino acid composition and appeared a single spot in TLC. According to the R(f) values with the amino acid composition, these peaks belong to the surfactin group of lipopeptides. Infrared analysis of the purified samples also revealed a pattern similar to that of surfactin. This is a very effective method for isolating and fractionating lipopeptides, of the same or different nature.     

Comparison of tyrosinase activity and stability in aqueous and nearly nonaqueous environments

The activity and stability of tyrosinase were compared in aqueous and two nearly nonaqueous environments (a low-water solvent system and reversed micelles). Initial rates of oxidation of methyl- and butyl-catechols in aerosol OT, sodium di-2-ethylhexylsulfosuccinate, (AOT)/isooctane micelles were higher than in aqueous solution, showing superactivity, whereas lower rates were obtained in cetyltri-methylammonium bromide (CTAB)/hexane/chloroform micelles and in chloroform containing celite-supported enzyme. The enzyme was most stable in chloroform, whereas half-lives in aqueous buffer and in both AOT and CTAB micelles were lower. The optimal reaction temperatures were higher in both micelles than in water but lower in chloroform. Thus, tyrosinase was active in ≤3.5% v/v water with apparent K_m, V_max, and activation energies reasonably similar to those in aqueous solution.     

Epidermal growth factor-conjugated poly(ethylene glycol)-block- poly(delta-valerolactone) copolymer micelles for targeted delivery of chemotherapeutics

Epidermal growth factor (EGF)-conjugated copolymer micelles were prepared from a mixture of diblock copolymers of methoxy poly(ethylene glycol)-block-poly(delta-valerolactone) (MePEG-b-PVL) and EGF-PEG-b-PVL for targeted delivery to EGF receptor (EGFR)-overexpressing cancers. The block copolymers and functionalized block copolymers were synthesized using PEG as the macroinitiator and HCl-diethyl ether as the catalyst. The MePEG-b-PVL and the carboxyl-terminated PEG-b-PVL (HOOC-PEG-b-PVL) copolymers were found to have molecular weights of 5940 and 5900, respectively, as determined by gel permeation chromatography (GPC) analyses. The HOOC-PEG-b-PVL copolymers were then activated by N-hydroxysuccinimide and subsequently reacted with EGF to form the EGF-PEG-b-PVL copolymers. The efficiency for the conjugation of EGF to the copolymer was found to be 60.9%. A hydrophobic fluorescent probe, CM-DiI, was loaded into both the nontargeted MePEG-b-PVL micelles and the targeted EGF-conjugated PEG-b-PVL micelles. The effective mean diameters of the CMDiI-loaded nontargeted and the CMDiI-loaded targeted micelles were found to be 32 +/- 1 nm and 45 +/- 2 nm, respectively, as determined by dynamic light scattering (DLS). The zeta potentials for the nontargeted micelles (no CM-DiI-loaded), CM-DiI-loaded nontargeted micelles, and CM-DiI-loaded targeted micelles were found to be -6.5, -8.7, and - 13.5 mV, respectively. Evaluation of the in vitro release of CM-DiI from the MePEG-b-PVL micelles in phosphate buffer saline (0.01 M, pH = 7.4) containing 10% (v/v) fetal bovine serum at 37 degrees C revealed that approximately 20% of the probe was released within the first 2 h. Confocal laser scanning microscopy (CLSM) analysis revealed that the targeted micelles containing CM-DiI accumulated intracellularly in EGFR-overexpressing MDA-MB-468 breast cancer cells following a 2 h incubation period, while no detectable cell uptake was observed for the nontargeted micelles. Results obtained from the confocal images were confirmed in an independent study by measuring the intracellular CM-DiI fluorescence in cell lysate. In addition, the presence of free EGF was found to decrease the extent of uptake of the targeted micelles. Nuclear staining of the cells with Hoechst 33258 indicated that the targeted micelles mainly localized in the perinuclear region and some of the micelles were localized in the nucleus. These results demonstrate that the EGF-conjugated copolymer micelles developed in this study have potential as vehicles for targeting hydrophobic drugs to EGFR-overexpressing cancers.     

Optimizing iron supplement strategies for enhanced surfactin production with Bacillus subtilis

Supplement of Fe(2+) into fermentation medium was utilized as a tool to optimize the iron-mediated enhancement of surfactin production from Bacillus subtilis ATCC 21332. Up to 3000 mg L(-)(1) of surfactin was produced using an iron-enriched minimal salt (MS) medium amended with an optimal Fe(2+) dosage of 4.0 mM, leading to 8-fold and 10-fold increase in cell concentration and surfactin yield, respectively, as compared to those without Fe(2+) supplement. In addition to resulting in an optimal production yield of surfactin, a supplement of 4.0 mM of Fe(2+) also propelled maximum overall surfactin production rate to a highest value of 24 mg L(-)(1) h(-)(1). Our results also show that production of surfactin followed a growth-associated kinetic model. The best yield coefficient estimated from the model was ca. 162 mg surfactin (g dry cell)(-)(1). The supernatant of the iron-enriched culture of B. subtilis ATCC 21332 exhibited the ability to emulsify kerosene and achieved a maximum emulsion index (E(24)) of 80% for culture supplemented with 4.0 mM of Fe(2+). Comparison of emulsion index and the corresponding surfactin production indicates that the emulsification activity was essentially contributed by surfactin.     

A micellar model system for the role of zeaxanthin in the non-photochemical quenching process of photosynthesis—chlorophyll fluorescence quenching by the xanthophylls

To get an insight to the mechanism of the zeaxanthin-dependent non-photochemical quenching in photosystem II of photosynthesis, we probed the interaction of some xanthophylls with excited chlorophyll-a by trapping both pigments in micelles of triton X-100. Optimal distribution of pigments among micelles was obtained by proper control of the micelle concentration, using formamide in the reaction mixture, which varies the micellar aggregation number over three orders of magnitude. The optimal reaction mixture was obtained around 40% (v/v) formamide in 0.2-0.4% (v/v) triton X-100 in water. Zeaxanthin in the micellar solution exhibited initially absorption and circular dichroism spectral features corresponding to a J-type aggregate. The spectrum was transformed over time (half-time values vary—an average characteristic figure is roughly 20 min) to give features representing an H-type aggregate. The isosbestic point in the series of spectral curves favors the supposition of a rather simple reaction between two pure J and H-types dimeric species. Violaxanthin exhibited immediately stable spectral features corresponding to a mixture of J-type and more predominately H-type dimers. Lutein, neoxanthin and β-carotene did not show any aggregated spectral forms in micelles. The spectral features in micelles were compared to spectra in aqueous acetone, where the assignment to various aggregated types was established previously. The specific tendency of zeaxanthin to form the J-type dimer (or aggregate) could be important for its function in photosynthesis. The abilities of five carotenoids (zeaxanthin, violaxanthin, lutein, neoxanthin and β-carotene) to quench chlorophyll-a fluorescence were compared. Zeaxanthin, in its two micellar dimeric forms, and β-carotene were comparable good quenchers of chlorophyll-a fluorescence. Violaxanthin was a much weaker quencher, if at all. Lutein and neoxanthin rather enhanced the fluorescence. The implications to non-photochemical quenching process in photosynthesis are discussed.     

Cell-free biosynthesis of surfactin, a cyclic lipopeptide produced by Bacillus subtilis

The lipopeptide antibiotic surfactin is a potent extracellular biosurfactant produced by various Bacillus subtilis strains. Biosynthesis of surfactin was studied in a cell-free system prepared from B. subtilis ATCC 21332 and OKB 105, which is a transformant producing surfactin in high yield [Nakano, M. M., Marahiel, M. A., & Zuber, P. (1988) J. Bacteriol. 170, 5662-5668]. Cell material was disintegrated by treatment with lysozyme and French press. A cell-free extract was prepared by ammonium sulfate fractionation, which catalyzed the formation of surfactin at the expense of ATP. Lipopeptide biosynthesis required the L-amino acid components of surfactin and D-3-hydroxytetradecanoyl-coenzyme A thioester. D-Leucine which is present in surfactin was not utilized but inhibited the biosynthetic process. The structure of surfactin, synthesized enzymatically in vitro, was confirmed by chromatographic comparison with the authentic compound and by amino acid analyses. An enzyme fraction was prepared by gel permeation chromatography which catalyzed ATP/pyrophosphate exchange reactions dependent on the component amino acids of surfactin. This enzyme fraction was capable of binding substrate amino acids covalently, probably via thioester linkages. The formation of these intermediates was inhibited by various thiol blocking reagents and phenylmethanesulfonyl fluoride. De novo synthesis of the lipopeptide was not observed with this partially purified enzyme fraction most likely due to the lack of an acyltransferase activity required for linking the beta-hydroxy fatty acid to the peptide moiety.     

A micellar model system for the role of zeaxanthin in the non-photochemical quenching process of photosynthesis--chlorophyll fluorescence quenching by the xanthophylls

To get an insight to the mechanism of the zeaxanthin-dependent non-photochemical quenching in photosystem II of photosynthesis, we probed the interaction of some xanthophylls with excited chlorophyll-a by trapping both pigments in micelles of triton X-100. Optimal distribution of pigments among micelles was obtained by proper control of the micelle concentration, using formamide in the reaction mixture, which varies the micellar aggregation number over three orders of magnitude. The optimal reaction mixture was obtained around 40% (v/v) formamide in 0.2-0.4% (v/v) triton X-100 in water. Zeaxanthin in the micellar solution exhibited initially absorption and circular dichroism spectral features corresponding to a J-type aggregate. The spectrum was transformed over time (half-time values vary-an average characteristic figure is roughly 20 min) to give features representing an H-type aggregate. The isosbestic point in the series of spectral curves favors the supposition of a rather simple reaction between two pure J and H-types dimeric species. Violaxanthin exhibited immediately stable spectral features corresponding to a mixture of J-type and more predominately H-type dimers. Lutein, neoxanthin and beta-carotene did not show any aggregated spectral forms in micelles. The spectral features in micelles were compared to spectra in aqueous acetone, where the assignment to various aggregated types was established previously. The specific tendency of zeaxanthin to form the J-type dimer (or aggregate) could be important for its function in photosynthesis. The abilities of five carotenoids (zeaxanthin, violaxanthin, lutein, neoxanthin and beta-carotene) to quench chlorophyll-a fluorescence were compared. Zeaxanthin, in its two micellar dimeric forms, and beta-carotene were comparable good quenchers of chlorophyll-a fluorescence. Violaxanthin was a much weaker quencher, if at all. Lutein and neoxanthin rather enhanced the fluorescence. The implications to non-photochemical quenching process in photosynthesis are discussed.     

An efficient method for separation of surfactin from Bacillus amyloliquefaciens fmb50 broth by flocculation

An efficient method for removing microbial cells and macromolecular impurities and purification of surfaction from fermentation broth produced by Bacillus amyloliquefaciens fmb50 was carried out in this study. Among three inorganic flocculants and a macromolecular flocculants, the combination use of CaCl₂ and Na₂HPO₄ was the most effective separation process. Addition of 50% ethanol into fermentation broth could not only disrupt the surfactin micells, but also promote the permeating of surfactin in filtration. The flocculation condition was optimized by an L₉ (3⁴) orthogonal design. The light transmittance, surfactin recovery rate, protein removal rate and filtration flux could reach to 96.3%, 95.31%, 56.59% and 3204.41 L m⁻² h⁻¹ respectively, the surfactin purity reached to 79.5% and the residual protein was 8.1% in separated product under the optimal flocculation condition (flocculants dosage 0.5%, pH 5.0, and temperature 35 °C). Validation test also demonstrated stable results under the optimal conditions. Due to higher efficiency, lower cost and scale-up more easily of flocculation and filtration processes, it is feasible to separate surfactin from fermentation broth.     

Molecular mechanism of membrane permeabilization by the peptide antibiotic surfactin

Surfactin, an acidic lipopeptide produced by various strains of Bacillus subtilis, behaves as a very powerful biosurfactant and possesses several other interesting biological activities. This work deals with the molecular mechanism of membrane permeabilization by incorporation of surfactin. The surfactin-induced vesicle contents leakage was monitored by following release of carboxyfluorescein entrapped into unilamellar vesicles made of palmitoyloleoylphosphatidylcholine (POPC). The effect of the addition of cholesterol, dipalmitoylphosphatidylcholine (DPPC) and palmitoyloleoylphosphatidylethanolamine (POPE) was also checked. It was observed that surfactin was able to induce content leakage at concentrations far below the onset surfactin/lipid ratio for membrane solubilization to occur, which in our system was around 0.92. Electron microscopy showed that vesicles were present after addition of surfactin at a ratio below this value, whereas no vesicles could be observed at ratios above it. Cholesterol and POPE attenuated the membrane-perturbing effect of surfactin, whereas the effect of DPPC was to promote surfactin-induced leakage, indicating that bilayer sensitivity to surfactin increases with the lipid tendency to form lamellar phases, which is in agreement with our previous observation that surfactin destabilizes the inverted-hexagonal structure. Fourier-transform infrared spectroscopy (FTIR) was used to specifically follow the effect of surfactin on different parts of the phospholipid bilayer. The effect on the C=O stretching mode of vibration of POPC indicated a strong dehydration induced by surfactin. On the other hand, the C-H stretching bands showed that the lipopeptide interacts with the phospholipid acyl chains, resulting in considerable membrane fluidization. The reported effects could be useful to explain surfactin-induced 'pore' formation underlying the antibiotic and other important biological actions of this bacterial lipopeptide.