Influence of homogenization treatment on physicochemical properties and enzymatic hydrolysis rate of pure cellulose fibers.

The aim of this study is to compare the effect of different homogenization treatments on the physicochemical properties and the hydrolysis rate of a pure bleached cellulose. Results obtained show that homogenization treatments improve the enzymatic hydrolysis rate of the cellulose fibers by 25 to 100 %, depending of the homogenization treatment applied. Characterization of the samples showed also that homogenization had an impact on some physicochemical properties of the cellulose. For moderate treatment intensities (pressure below 500 b and degree of homogenization below 25), an increase of water retention values (WRV) that correlated to the increase of the hydrolysis rate was highlighted. Result also showed that the overall crystallinity of the cellulose properties appeared not to be impacted by the homogenization treatment. For higher treatment intensities, homogenized cellulose samples developed a stable tridimentional network that contributes to decrease cellulase mobility and slowdown the hydrolysis process.

Influence of steam explosion on physicochemical properties and hydrolysis rate of pure cellulose fibers.

The aim of this study is to compare the effect of different steam explosion treatments on the physicochemical properties and the hydrolysis rate of a pure bleached cellulose. The results showed that moderate steam explosion treatments (severity factor below 5.2) did not appear to improve the enzymatic hydrolysis rate of the cellulose fibers. However, characterization of the samples showed a modification of the physicochemical properties of the cellulose, resulting in an increase of the water retention values (WRV) coupled to an increase of the overall crystallinity. For higher treatment intensities, an important thermal degradation of the cellulose was highlighted. This thermal degradation caused an important modification of the cellulose composition which leads to a decrease of the hydrolysis rate.

Interaction of hexadecylbetainate chloride with biological relevant lipids.

The present work investigates the interaction of hexadecylbetainate chloride (C(16)BC), a glycine betaine-based ester with palmitoyl-oleoyl-phosphatidylcholine (POPC), sphingomyelin (SM), and cholesterol (CHOL), three biological relevant lipids present in the outer leaflet of the mammalian plasma membrane. The binding affinity and the mixing behavior between the lipids and C(16)BC are discussed based on experimental (isothermal titration calorimetry (ITC) and Langmuir film balance) and molecular modeling studies. The results show that the interaction between C(16)BC and each lipid is thermodynamically favorable and does not affect the integrity of the lipid vesicles. The primary adsorption of C(16)BC into the lipid film is mainly governed by a hydrophobic effect. Once C(16)BC is inserted in the lipid film, the polar component of the interaction energy between C(16)BC and the lipid becomes predominant. Presence of CHOL increases the affinity of C(16)BC for membrane. This result can be explained by the optimal matching between C(16)BC and CHOL within the film rather by a change of membrane fluidity due to the presence of CHOL. The interaction between C(16)BC and SM is also favorable and gives rise to highly stable monolayers probably due to hydrogen bonds between their hydrophilic groups. The interaction of C(16)BC with POPC is less favorable but does not destabilize the mixed monolayer from a thermodynamic point of view. Interestingly, for all the monolayers investigated, the exclusion surface pressures are above the presumed lateral pressure of the plasma membranes suggesting that C(16)BC would be able to penetrate into mammalian plasma membranes in vivo. These results may serve as a useful basis in understanding the interaction of C(16)BC with real membranes.

Penetration behaviour of alkylbetainate chlorides into lipid monolayers.

In this paper, the penetration behaviour of the alkylbetainate chloride surfactants (C(n)BC, n=10-16) into lipid monolayers of dipalmitoylphosphatidylserine (DPPS), dipalmitoylphosphatidic acid (DPPA), dipalmitoylphosphatidylethanolamine (DPPE), palmitoyoleoylphosphatidylcholine (POPC) and cholesterol (CHOL) is investigated using the Langmuir trough technique. The penetration of C(n)BC is followed by measurement of the surface pressure increase (Δπ) at a constant surface area after the injection of C(n)BC into the aqueous phase, underneath the lipid monolayer previously spread at the air-water interface at 25°C and at different initial surface pressures (π(i)). The influence of both the lipid head group and the surfactant hydrocarbon chain length on the effectiveness of C(n)BC penetration into these monolayers is discussed. The results have shown that C(n)BC adsorb at the air-water interface giving evidence of their surface-active properties. The adsorption kinetics of C16BC into different lipid monolayers are lipid head charge and lipid head volume-dependent. The magnitude of the surface pressure increase (Δπ) arises in the following order: DPPA>DPPS≫CHOL≈DPPE>POPC. C(n)BC penetration into negatively-charged (DPPS and DPPA) monolayers does not seem to depend on surfactant alkyl-chain length compared to uncharged (CHOL) and zwitterionic (DPPE and POPC) monolayers for which Δπ increases with a larger alkyl-chain length. Electrostatic interactions are mainly involved in the affinity of C(n)BC with monolayers but the hydrophobic effect plays also a role.

Study of Thermomyces lanuginosa lipase in the presence of tributyrylglycerol and water.

The Thermomyces lanuginosa lipase has been extensively studied in industrial and biotechnological research because of its potential for triacylglycerol transformation. This protein is known to catalyze both hydrolysis at high water contents and transesterification in quasi-anhydrous conditions. Here, we investigated the Thermomyces lanuginosa lipase structure in solution in the presence of a tributyrin aggregate using 30 ns molecular-dynamics simulations. The water content of the active-site groove was modified between the runs to focus on the protein-water molecule interactions and their implications for protein structure and protein-lipid interactions. The simulations confirmed the high plasticity of the lid fragment and showed that lipid molecules also bind to a secondary pocket beside the lid. Together, these results strongly suggest that the lid plays a role in the anchoring of the protein to the aggregate. The simulations also revealed the existence of a polar channel that connects the active-site groove to the outside solvent. At the inner extremity of this channel, a tyrosine makes hydrogen bonds with residues interacting with the catalytic triad. This system could function as a pipe (polar channel) controlled by a valve (the tyrosine) that could regulate the water content of the active site.

Effect of cholesterol and fatty acids on the molecular interactions of fengycin with Stratum corneum mimicking lipid monolayers.

The combination of atomic force microscopy (AFM) and the Langmuir trough technique was used in this work to investigate the molecular interactions of fengycin with lipid monolayers constituted of the major lipid classes found in human stratum corneum (SC). AFM imaging o f spread SC lipids/fengycin monolayers showed that fengycin preferentially partitions into cholesterol-rich phases surrounding 2D domains mainly constituted of ceramide and fatty acid molecules. Penetration experiments of fengycin from the subphase into SC-mimicking monolayers clearly indicated that the lipopeptide insertion at the lipid interface is enhanced in the presence of cholesterol. AFM analysis of mixed SC lipids/fengycin monolayers obtained after lipopeptide penetration revealed that cholesterol strongly interacts with fengycin and undergoes specific molecular interactions with more disordered, loosely packed ceramide molecules. These results highlight the capacity of fengycin to interact with the lipid constituents of the extracellular matrix of SC and, in particular, with cholesterol.

Enzymatically prepared n-alkyl esters of glucuronic acid: the effect of freeze-drying conditions and hydrophobic chain length on thermal behavior.

In this work, some of the physicochemical properties of enzymatically prepared n-alkyl esters of glucuronic acid are presented. Two questions are addressed. The first concerns the influence of post-purification freeze-drying conditions on octyl glucuronate thermotropic behavior. Depending on the amount of water added before freeze-drying, the alpha/beta anomeric ratio determined by (1)H NMR is affected and differences are observed in DSC thermograms probably due to polymorphism. The second question concerns the effect of hydrophobic chain length on the thermal behavior. An increase of both transition temperature and transition enthalpy is observed by increasing the number of carbon atoms in the alkyl chain (C8

Molecular organization of surfactin-phospholipid monolayers: effect of phospholipid chain length and polar head.

Mixed monolayers of the surface-active lipopeptide surfactin-C(15) and various lipids differing by their chain length (DMPC, DPPC, DSPC) and polar headgroup (DPPC, DPPE, DPPS) were investigated by atomic force microscopy (AFM) in combination with molecular modeling (Hypermatrix procedure) and surface pressure-area isotherms. In the presence of surfactin, AFM topographic images showed phase separation for each surfactin-phospholipid system except for surfactin-DMPC, which was in good agreement with compression isotherms. On the basis of domain shape and line tension theory, we conclude that the miscibility between surfactin and phospholipids is higher for shorter chain lengths (DMPC>DPPC>DSPC) and that the polar headgroup of phospholipids influences the miscibility of surfactin in the order DPPC>DPPE>DPPS. Molecular modeling data show that mixing surfactin and DPPC has a destabilizing effect on DPPC monolayer while it has a stabilizing effect towards DPPE and DPPS molecular interactions. Our results provide valuable information on the activity mechanism of surfactin and may be useful for the design of surfactin delivery systems.

Penetration of surfactin into phospholipid monolayers: nanoscale interfacial organization.

Atomic force microscopy (AFM) combined with surface pressure-area isotherms were used to probe the interfacial behavior of phospholipid monolayers following penetration of surfactin, a cyclic lipopeptide produced by Bacillus subtilis strains. Prior to penetration experiments, interfacial behavior of different surfactin molecules (cyclic surfactins with three different aliphatic chain lengths--S13, S14, and S15--and a linear surfactin obtained by chemical cleavage of the cycle of the surfactin S15) has been investigated. A more hydrophobic aliphatic chain induces greater surface-active properties of the lipopeptide. The opening of the peptide ring reduces the surface activity. The effect of phospholipid acyl chain length (dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine- (DPPC), and distearoylphosphatidylcholine) and phospholipid polar head (DPPC, dipalmitoylphosphatidylethanolamine and dipalmitoylphosphatidylserine) on monolayer penetration properties of the surfactin S15 has been explored. Results showed that while the lipid monolayer thickness and the presence of electrostatic repulsions from the interfacial film do not significantly influence surfactin insertion, these parameters strongly modulate the ability of the surfactin to alter the nanoscale organization of the lipid films. We also probed the effect of surfactin structure (influence of the aliphatic chain length and of the cyclic structure of the peptide ring) on the behavior of DPPC monolayers. AFM images and isotherms showed that surfactin penetration is promoted by longer lipopeptide chain length and a cyclic polar head. This indicates that hydrophobic interactions are of main importance for the penetration power of surfactin molecules.

Nanoscale properties of mixed fengycin/ceramide monolayers explored using atomic force microscopy.

To gain insight into the interactions between fengycin and skin membrane lipids, mixed fengycin/ceramide monolayers were investigated using atomic force microscopy (AFM) (monolayers supported on mica) and surface pressure-area isotherms (monolayers at the air-water interface). AFM topographic images revealed phase separation in mixed monolayers prepared at 20 degrees C/pH 2 and composed of 0.25 and 0.5 fengycin molar ratios, in the form of two-dimensional (2-D) hexagonal crystalline domains of ceramide surrounded by a fengycin-enriched fluid phase. Surface pressure-area isotherms as well as friction and adhesion AFM images confirmed that the two phases had different molecular orientations: while ceramide formed a highly ordered phase with crystalline chain packing, fengycin exhibited a disordered fluid phase with the peptide ring lying horizontally on the substrate. Increasing the temperature and pH to values corresponding to the skin parameters, i.e., 37 degrees C/pH 5, was found to dramatically affect the film organization. At low fengycin molar ratio (0.25), the hexagonal ceramide domains transformed into round domains, while at higher ratio (0.5) these were shown to melt into a continuous fengycin/ceramide fluid phase. These observations were directly supported by the thermodynamic analysis (deviation from the additivity rule, excess of free energy) of the monolayer properties at the air-water interface. Accordingly, this study demonstrates that both the environmental conditions (temperature, pH) and fengycin concentration influence the molecular organization of mixed fengycin/ceramide monolayers. We believe that the ability to modulate the formation of 2-D domains in the skin membrane may be an important biological function of fengycin, which should be increasingly investigated in future pharmacological research.

Combined enzymatic hydrolysis and HPAEC method for simultaneous analysis of galacturonic acid and neutral sugars of pectin.

Pectic substances are heteropolysaccharides from plant cell walls, which mainly consist of a homogalacturonan backbone of predominantly alpha-(1-->4) linked galacturonic acid (GalA) residues. This chain is interrupted by ramified rhamnogalacturonan regions with a certain amount of neutral sugars (rhamnose, arabinose, galactose, glucose, xylose and mannose) present as side-chains. The GalA residues can be methyl-esterified at the carboxyl group. Usually uronic acids of pectin are determined as anhydrogalacturonic acid by spectrophotometry, using the metahydrodiphenyl method (Thibault, 1979), while the monosaccharides are determined by High-Performance Liquid Chromatography (HPLC) after a methanolysis (Quemener and Thibault, 1990) or by Gaz Liquid Chromatography (GLC) as alditol acetates according to Blakeney et al. (1983). In this paper, a combined enzymatic hydrolysis and HPLC method is validated for simultaneous analysis of galacturonic acid and neutral sugars without any derivatisation.

Insecticide activity of surfactins and iturins from a biopesticide Bacillus subtilis Cohn (S499 strain).

Surfactin C14, surfactin C15, and iturin C15 are lipopeptides purified from Bacillus subtilis (S499 strain). They were incorporated to artificial diet of the fruit fly Drosophila melanogaster (Meigen) (Diptera, Drosophilidae) to assess their potential insecticide activity. Surfactins with long fatty acid chain (C14 and C15) showed insecticide effect on the fruit fly, D. melanogaster. On the contrary, iturin was not toxic to fruit fly D. melanogaster. At 100 ppm, surfactin C14 and C15 showed respectively 85.4 and 92.6% adults mortality after one-day exposure. F1 progeny fly emergence inhibition by C14 and C15 were respectively 79.8% and 91.3%. To check whether the biocide activity of lipopeptides was due to their surface-active properties, detergent Triton X100, SDS, CTAB and Tween 80 were tested. No adult mortality was recorded with the detergents but Triton X100 and SDS showed F1 progeny emergence inhibition similar to that of surfactins. We showed that there was a dose-response activity with surfactin C15.

Influence of electrical properties on the evaluation of the surface hydrophobicity of Bacillus subtilis.

The surface hydrophobicity of nine Bacillus subtilis strains in different states (spores, vegetative cells, and dead cells) was assessed by water contact angle measurements, hydrophobic interaction chromatography (HIC) and bacterial adhesion to hydrocarbon (BATH). Electrokinetic properties of B. subtilis strains were characterized by zeta potential measurements and found to differ appreciably according to the strain. Correlations between HIC data, BATH data and zeta potential showed that HIC and BATH are influenced by electrostatic interactions. Water contact angle measurements thus provide a better estimate of cell surface hydrophobicity. The water contact angle of B. subtilis varied according to the strain and the state, the spores tending to be more hydrophobic than vegetative cells.

Influence of culture conditions on lipopeptide production by Bacillus subtilis.

Bacillus subtilis produces various families of lipopeptides with different homologous compounds. To produce "new molecules" with improved activities and to select strains that produced a reduced number of homologs or isomers, we studied the effects of different media on the nature of the synthesis of fatty acid chains for each lipopeptide family. This study focused on two B. subtilis strains cultivated in flasks. Optimized medium for lipopeptide production and Landy medium modified by replacing glutamic acid with other alpha-amino acids were used. We found that the intensity of production of homologous compounds depends on the strain and the culture medium. Analysis of these lipopeptides by high-performance liquid chromatography showed that the strain B. subtilis NT02 yielded various homologous compounds when cultivated in Landy medium (L-Glu), but primarily one homologous product in high relative amounts when cultivated in the optimized medium. Mass spectrometric analysis and determination of the amino acid composition of this molecule enabled us to identify it as Bacillomycine L c15.

Characterization of two Acacia gums and their fractions using a langmuir film balance.

The mechanical properties of monolayers from two Acacia gums [Acacia senegal (L.) Willd. and Acacia seyal Del.] and their three fractions isolated by hydrophobic interaction chromatography were studied with a Langmuir film balance to obtain a more complete understanding of their action mode. The analysis of compression isotherms revealed that A. senegal gums globally exhibit better interfacial properties than A. seyal ones. The behavior of the whole gums appeared to be strongly influenced by their arabinogalactan-protein complex.

Nanometer scale organization of mixed surfactin/phosphatidylcholine monolayers.

Mixed monolayers of the surface-active lipopeptide surfactin-C(15) and of dipalmitoyl phosphatidylcholine (DPPC) were deposited on mica and their nanometer scale organization was investigated using atomic force microscopy (AFM) and x-ray photoelectron spectroscopy (XPS). AFM topographic images revealed phase separation for mixed monolayers prepared at 0.1, 0.25, and 0.5 surfactin molar ratios. This was in agreement with the monolayer properties at the air-water interface indicating a tendency of the two compounds to form bidimensional domains in the mixed systems. The step height measured between the surfactin and the DPPC domains was 1.2 +/- 0.1 nm, pointing to a difference in molecular orientation: while DPPC had a vertical orientation, the large peptide ring of surfactin was lying on the mica surface. The N/C atom concentration ratios obtained by XPS for pure monolayers were compatible with two distinct geometric models: a random layer for surfactin and for DPPC, a layer of vertically-oriented molecules in which the polar headgroups are in contact with mica. XPS data for mixed systems were accounted for by a combination of the two pure monolayers, considering respective surface coverages that were in excellent agreement with those measured by AFM. These results illustrate the complementarity of AFM and XPS to directly probe the molecular organization of multicomponent monolayers.

Purification of antifungal lipopeptides by reversed-phase high-performance liquid chromatography.

A rapid procedure for the purification of antifungal lipopeptides from Bacillus subtilis, a potential agent for biocontrol of plant diseases, was tested. It consists of a solid-phase extraction on C18 gel followed by reversed-phase chromatography using a biocompatible PepRPC HR 5/5 column with a pharmacia fast protein liquid chromatographic system. This is a very effective method for isolating and fractionating iturin A and surfactin, two lipopeptides of different nature, co-produced by Bacillus subtilis strain S499. The presence of homologous lipopeptides was easily detected.