Study On The Interactions Of Microbial Lipopeptide With Phospholipid

Microbial lipopeptides basically produced by microorganisms is generally composed of a hydrophobic fatty acid chain and a hydrophilic peptide moiety, and is an important type of biosurfactants. Surfactin is reported as one of the most surface-active microbial lipopeptides. Except for its exceptional surface activity, surfactin also possesses peculiar biological properties such as anti-microbial, antiviral and hemolytic activities which were proposed to be determined by the interactions of surfactin with biological membranes. In the present study, phospholipid monolayer and vesicle were used as biomimicking membrane models and the interaction of surfactin with phospholipid at the air-water interface and in solution were investigated.The lipopeptide sample, which was centrifuged from the cell free broth of Bacillus subtilis HSO 121, were separated by column chromatography and further isolated and collected by semipreparative reversed-phase high performance liquid chromatography. Among them, four surfactin analogues have the same peptide moiety and the structural difference lies in the fatty acid chain length, and they are denoted as surfactin-C12, surfactin-C13, surfactin-C14 and surfactin-C15 in the context. Another surfactin analogue has different amino acid sequence in the peptide moiety (N-Asp-Leu-Leu-Val-Glu-Leu-Leu), and it was denoted as [Asp1, Glu5]surfactin-C16 in the context.Binary system of surfactin-C14 and DMPC (L-α-dimyristylphosphatidylcholine) in the mixed monolayers at the air-water interface was studied by Langmuir film balance. Based on the surface pressure-molecular area (π-A) isotherms, the excess area of mixing (Aex) was calculated, and the mixed monolayers were characterized by atomic force microscopy. When molar fraction of surfactin-C14 (XS) in the mixed monoayer is 0.10, the miscibility between surfactin and DMPC are non-ideal. When XS=0.75, the Aex is positive, it can be the sign of the formation of bidimensional clusters. Based on the AFM characterization and thermodynamic analysis, it was suggested that, at surface pressure of 25 mN·m-1, the presence of DMPC in the mixed monolayer promoted the self-aggregation of surfactin-C14 at the air-water interface. Above results indicate that the interaction between surfactin-C14 and DMPC in the mixed monolayer is greatly influenced by the molar fraction of surfactin as well as surface pressure. Furthermore, the consecutive compression-expansion cycles of surfactin-C14 monolayer were studied. It was found that a larger hysteresis loop was observed when the compression isotherm underwent a "plateau region". With a decrease in the subphase pH the hysteresis loop became smaller. The surface morphologies of the surfactin-C14 monolayer in the "plateau region" were characterized by scanning electron microscopy (SEM). From the SEM images surface aggregates with diameters ranging from tens to hundreds of nanometers were observed. It is suggested that, at the "plateau region", surfactin-C14 molecules tend to aggregate into three-dimensional ordered structures.The effects of fatty acid chain length in surfactin molecule, the subphase pH, surface pressure as well as surfactin interfacial concentration on the molecular interaction between surfactin and DMPC were examined. It is suggested the subphase pH exerts impact on the interaction, which is mainly resulted from the change of ionization state in peptide moiety in the surfactin molecule as well as inter-and intra-molecular hydrogen bonding. Furthermore, the interaction between two components is dominated by repulsive force when the mixed monolayer is under liquid expanded state, while the interaction became less repulsive when the mixed monolayer is under liquid condensed state.The mixed behavior of a novel [Asp1, Glu5]surfactin-C16 and DMPC was also studies by Langmuir film balance. From the Aex-XS plots, it was found that the miscibility between [Aspi, Glu5]surfactin-C16 and DMPC on pure water depends on the surfactin molecular fraction and surface pressures. It is proposed that, on acidic subphase the two carboxyl residues in surfactin molecules can be hardly protonated, as a result of steric hindrance, the DMPC molecules can hardly interact with surfactin in mixed monolayer. On alkaline subphase, the fully ionized carboxyl residues in surfactin would enhance the hydrophilicity of the surfactin molecules, the DMPC molecules can be easily interact with surfactin by hydrophobic interactions.Small angle X-ray scattering (SAXS), pyrene fluorescence probe spectrometry and freeze-fracture transmission electron microscopy (FF-TEM) were applied to investigate the interaction between [Asp1,Glu5]surfactin-C16 and phospholipid vesicles. SAXS results indicated that the addition of surfactin changed the structure of phospholipid vesicles. When higher concentration of surfactin was added, the destruction of DMPC vesicles was observed. The tendency of pyrene fluorescence intensity I1/I3 suggests the interaction between surfactin and DMPC makes the pyrene moving towards more hydrophobic micro-environment. The leakage of DMPC vesicles with the addition of surfactin was recorded with Freeze-fracture transmission electron microscopy (FF-TEM), and a destruction process of DMPC vesicles by surfactin was proposed.