Folding of outer membrane protein A in the anionic biosurfactant rhamnolipid

Folding and stability of bacterial outer membrane proteins (OMPs) are typically studied in vitro using model systems such as phospholipid vesicles or surfactant. OMP folding requires surfactant concentrations above the critical micelle concentration (cmc) and usually only occurs in neutral or zwitterionic surfactants, but not in anionic or cationic surfactants. Various Gram-negative bacteria produce the anionic biosurfactant rhamnolipid. Here we show that the OMP OmpA can be folded in rhamnolipid at concentrations above the cmc, though the thermal stability is reduced compared to the non-ionic surfactant dodecyl maltoside. We discuss implications for possible interactions between OMPs and biosurfactants in vivo.     

Effect of surface-active substances on bioluminescence intensity of bacteria

The study of sensitivity of luminous bacteria isolated from the Black and Azov seas to surfactants from various classes was carried out. It was shown that cationic surfactants had a strong inhibition effect on bacterial luminescence in contrast to anionic and in particular nonionic surfactants. To increase the luminous bacteria sensitivity to the action of OP-10 (nonionic surfactant) and ABS (anionic surfactant), which are widely used in industry, several approaches have been developed. They include modulation of bacterial sensitivity by the additives of cationic substances, use of luminous bacteria at a logarithmic stage of growth, realization of biotesting at low pH = 5.5. The use of these approaches allows to lower effective concentrations of OP-10 and ABS, which caused a decrease of bioluminescence by 50%, 3-200 times and opens perspectives for the use of the bioluminescent method to study these surfactants toxicity on the principle of biosensorics.     

Role of surfactant on the proteolysis of aqueous bovine serum albumin

Nonionic and ionic surfactants diminish the initial rate of proteolysis of aqueous bovine serum albumin (BSA) by subtilisin Carlsberg. Surfactants studied include: nonionic tetraethylene glycol monododecyl ether (C12E4); anionic sodium dodecyl sulfate (SDS), anionic sodium dodecylbenzenesulfonate (SDBS), and cationic dodecyltrimethylamonium bromide (DTAB). Kinetic data are obtained using fluorescence emission. Special attention is given to enzyme kinetic specificity determined by fitting initial-rate data to the Michaelis-Menten model. All surfactants reduce the rate of proteolysis, most strongly at concentrations near and above the critical micelle concentration (CMC). Circular dichroism (CD), tryptophan/tyrosine fluorescence spectra, and tryptophan fluorescence thermograms indicate that BSA partially unfolds at ionic surfactant concentrations near and above the CMC. Changes in BSA conformation are less apparent at ionic surfactant concentrations below the CMC and for the nonionic surfactant C12E4. Subtilisin Carlsberg activity against the polypeptide, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, decreased due to enzyme-surfactant interaction. At the concentrations and time frames studied, there was no enzyme autolysis. Importantly, aqueous proteolysis rates are significantly reduced at high surfactant concentrations where protein-micellar-surfactant aggregates occur. To explain the negative effect of surfactant on subtilisin Carlsberg proteolytic activity against BSA, we propose that micelle/protein complexes hinder enzyme access.     

In Situ emulsification and mobilization of gasoline range hydrocarbons using surfactants

In this article the conditions that govern surfactant‐enhanced emulsification and mobilization of petroleum hydrocarbons in soil are reviewed. The effect of soil properties, groundwater constituents, and differing surfactant solutions on the emulsification process is discussed. A constant head soil flushing apparatus used to characterize surfactant‐enhanced mobilization of m‐xylene is described. Data showing the effect of surfactant‐enhanced mobilization on m‐xylene removal efficiency in washed sand is presented. Flushing solutions were used at concentrations from below to well above the critical micelle concentration (CMC) of the surfactants used. Removal efficiencies are shown to vary with surfactant concentration and with surfactant type. Flushing solutions of anionic, nonionic, and anionic/nonionic surfactant mixtures were evaluated.     

Studies on membrane fusion. 1. Interactions of pure phospholipid membranes and the effect of myristic acid,lysolecithin, proteins and dimethylsulfoxide

The interaction and mixing of membrane components in sonicated unilamellar vesicles and also non-sonicated multilamellar vesicles prepared from highly purified phospholipids suspended in NaCl solutions has been examined. Electron microscopy and differential scanning calorimetry were used to characterize the extent and kinetics of mixing of membrane components between different vesicle populations. No appreciable fusion was detected between populations of non-sonicated phospholipid vesicles incubated in aqueous salt (NaCl) solutions. Mixing of vesicle membrane components via diffusion of phospholipid molecules between vesicles was observed in populations of negatively charged phosphatidylglycerol vesicles but similar exchange diffusion was not detected in populations of neutral phosphatidylcholine vesicles. Incubation of sonicated vesicle populations at temperatures close to or above the phospholipid transition temperature resulted in an increase in vesicle size and mixing of vesicle membrane components as determined by a gradual change in the thermotropic properties of the mixed vesicle population. The interaction of purified phospholipid vesicles was also examined in the presence of myristic acid and lysolecithin. Our results indicate that while these agents enhance mixing of vesicle membrane components, in most cases mixing probably proceeds via diffusion of phospholipid molecules rather than by fusion of entire vesicles. Increased mixing of vesicle membrane components was also produced when vesicles were prepared containing a purified hydrophobic protein (myelin proteolipid apoprotein) or were incubated in the presence of dimethylsulfoxide. In these two systems, however, the evidence suggests that mixing of membrane components results from the fusion of entire vesicles.     

Inhibition of Aflatoxin Production by Surfactants

The effect of 12 surfactants on aflatoxin production, growth, and conidial germination by the fungus Aspergillus flavus is reported. Five nonionic surfactants, Triton X-100, Tergitol NP-7, Tergitol NP-10, polyoxyethylene (POE) 10 lauryl ether, and Latron AG-98, reduced aflatoxin production by 96 to 99% at 1% (wt/vol). Colony growth was restricted by the five nonionic surfactants at this concentration. Aflatoxin production was inhibited 31 to 53% by lower concentrations of Triton X-100 (0.001 to 0.0001%) at which colony growth was not affected. Triton X-301, a POE-derived anionic surfactant, had an effect on colony growth and aflatoxin production similar to that of the five POE-derived nonionic surfactants. Sodium dodecyl sulfate (SDS), an anionic surfactant, and dodecyltrimethylammonium bromide, a cationic surfactant, suppressed conidial germination at 1% (wt/vol). SDS had no effect on aflatoxin production or colony growth at 0.001%. The degree of aflatoxin inhibition by a surfactant appears to be a function of the length of the hydrophobic and hydrophilic chains of POE-derived surfactants.     

Comparative Studies of Lipase from Rhizopus Delemar in Various Microemulsion Systems

Hydrolysis of triglycerides by lipase from Rhizopus delemar has been studied in three different types of microemulsion systems. Microemulsions were prepared by using anionic (AOT), cationic (CTAB) and nonionic (C₁₂E₄) surfactants. Various parameters affecting the reaction, such as temperature, pH optimum, water content (R = [H₂O]/[surfactant]), as well as K_m.app and V_app, were determined using triolein and tributyrin as substrates. Maximum enzyme activity was obtained at R = 9, T = 30°C and pH = 6.5 in anionic surfactant systems, while in cationic, it was found at R = 7, T = 22.5°C and pH = 5.8. The stability of the enzyme was also studied in anionic and cationic systems under various conditions. The enzymatic reaction was also found to be very slow when it was studied in the C₁₂E₄ systems.     

Enhancement of enzymatic hydrolysis of cellulose by surfactant

Effects of surfactants on enzymatic saccharification of cellulose have been studied. Nonionic, amphoteric, and cationic surfactants enhanced the saccharification, while anionic surfactant did not. Cationic and anionic surfactants denatured cellulase in their relatively low concentrations, namely, more than 0.008 and 0.001%, respectively. Using nonionic surfactant Tween 20, which is most effective to the enhancement (e.g., the fractional conversion attained by 72 h saccharification of 5 wt % Avicel in the presence of 0.05 wt % Tween 20 is increased by 35%), actions of surfactant have been examined. As the results, it was suggested that Tween 20 plays an important role in the hydrolysis of crystalline cellulose and that Tween 20 disturbs the adsorption of endoglucanase on cellulose, i.e., varies the adsorption balance of endo- and exoglucanase, resulting in enhancing the reaction. The influence of Tween 20 to the saccharification was found to remain in simultaneous saccharification and fermentation of Avicel.     

Effect of surfactant solubilization on biodegradation of polychlorinated biphenyl congeners by Pseudomonas LB400

A variety of commercial surfactants were tested to determine their effect on polychlorinated biphenyl (PCB) transformation by Pseudomonas LB400. Initial tests determined that most surfactants were fully or partially able to solubilize the PCB congeners 2,5,2′-chlorobiphenyl (CBP), 2,4,2′,4′-CBP, 2,3,5,2′,5′-CBP and 2,4,5,2′,4′,5′-CBP, at concentrations above the surfactants' critical micelle concentration (CMC). Surfactants were also found to have no negative effect on bacterial survival, as cell numbers were the same or higher after incubation in the presence of surfactants than after incubation without surfactants. A comparison of the extent of biotransformation of single PCB congeners by the bacterium revealed that, at surfactant concentrations above the CMC, the presence of an anionic surfactant promoted while nonionic surfactants inhibited PCB transformation, compared to a control with no surfactant. The rates of transformation of PCB congeners were also higher in the presence of the anionic surfactant compared to the control. The inhibitory effects of a nonionic surfactant, Igepal CO-630 at a concentration above its CMC, on transformation of 2,4,5,2′,5′-CBP could be eliminated by diluting the surfactant/PCB solution to a concentration close to the surfactant CMC. Received: 26 October 1998 / Received revision: 5 March 1999 / Accepted: 14 March 1999     

表面活性剂对分枝杆菌KR2菌株降解菲的影响

采用同位素示踪方法,从表面活性剂的浓度、离子类型和直链长度三方面研究了表面活性剂对分枝杆菌KR2菌株降解菲的影响。结果表明,表面活性剂的存在不能促进KR2菌对菲的降解;高浓度表面活性剂(≥20mg·L^-1)的存在,使菲的降解出现延迟期,非离子表面活性剂Tween80在低浓度时(≤10mg·L^-1)可以优先作为营养基质被分枝杆菌KR2菌株利用,表面活性剂的离子类型对菲降解的抑制作用的顺序为阳离子表面活性剂TDTMA>阴离子表面活性剂LAS>非离子表面活性剂Tween80,表面活性剂的直链长度对菲降解的影响为直链越短,对微生物的毒性越大,菲降解得越不完全。     

Coaggregate of amphiphilic zinc chlorins with synthetic surfactants in an aqueous medium to an artificial supramolecular light-harvesting system

Aqueous assemblies of zinc chlorins possessing a nonionic (oligo)oxyethylene, a cationic quaternary ammonium or an anionic sulfonate group were prepared in the presence of a synthetic surfactant. The nonionic zinc chlorin formed aggregates when admixed with a nonionic surfactant such as Triton X-100 to give a highly ordered oligomeric J-aggregate similarly as natural bacteriochlorophyll-c or d does in a chlorosome. In addition, the coassemblies of the cationic zinc chlorin with an anionic surfactant and of the anionic zinc chlorin with a cationic surfactant gave large oligomers of these chlorophyllous pigments. The structures of hydrophilic groups in both the zinc chlorin and surfactant molecules controlled their aqueous coassemblies.     

Mixed micelles of monomeric and dimeric cationic surfactants with phospholipids: effect of hydrophobic interactions

Surface tension (gamma) and time resolved fluorescence quenching (TRFQ) measurements have been performed on the binary mixtures of monomeric as well as dimeric alkylammonium bromides with l-alpha-dimyristoylphosphatidycholine (DMPC) and L-alpha-dipalmitoylphosphatidycholine (DPPC). The critical micelle concentration (cmc) has been evaluated from the gamma measurements. The gamma plots show two breaks in the gamma versus [total surfactant] curves in most of the cases. The first break (C1) has been attributed to the mixed vesicle formation process. The break down of the vesicles leads to the mixed micellization between the surfactant and phospholipid monomers at the second break (C2). The amount of surfactant used in the vesicle breakdown process (DeltaC) increases linearly with the increase in the amount of phospholipid and depends significantly on the hydrophobicities of the cationic components. The surface area per molecule (a) evaluated from the gamma plots indicates compact monolayer formation in the case of monomeric surfactants with lower hydrophobicities and reverse is observed for dimeric surfactants. The pyrene life time (tau) of the solubilized pyrene in the hydrophobic environment of mixed micelles, fully supports the conclusion that derived from a.     

Effect of water-soluble polymers on the state of aggregation, vesicle size, and phase transformations in mixtures of phosphatidylcholine and sodium cholate.

The state of aggregation and the steady-state size of mixed aggregates made of phospholipids and surfactants are both determined by the surfactant/lipid ratio in the mixed aggregates (Re). Water-soluble polymers, such as dextrans and polyethylene glycols (PEGs) of different molecular weights, induce reversible aggregation of phospholipid vesicles, mostly due to dehydration of the vesicle surface and depletion forces, and only at much higher concentrations, PEGs (but not dextran) also induce irreversible size growth of the vesicles. Here we show that the water-soluble polymers dextrans and PEGs do not affect the vesicle-micelle phase boundaries in mixtures of phosphatidylcholine and the anionic surfactant sodium cholate. By contrast, these polymers affect markedly the steady-state size of cholate-containing vesicles. As compared with pure phosphatidylcholine vesicles, the cholate-containing vesicles have a lower tendency to undergo polymer-induced aggregation, probably due to the electrostatic repulsion between the negatively charged vesicles, but a higher tendency to undergo irreversible size growth at relatively low polymer concentrations. Such irreversible size growth was observed not only for PEG but also for dextran, which in the absence of cholate is incapable of inducing vesicle size growth. These findings are consistent with the prevailing concept that the polymer-induced size growth is due to the effect of large structural fluctuations in the bilayers of deformed aggregated vesicles, the surface of which is dehydrated by the polymer. The presence of cholate in the bilayers at sufficiently high concentrations induces such fluctuations, yielding irreversible size growth within the clusters of dehydrated vesicles formed upon mixing with polymers.     

Real-time Observation of Lipoplex Formation and Interaction with Anionic Bilayer Vesicles

A novel development has allowed for the direct observation of single, pairwise interactions of linear DNA with cationic vesicles and of DNA-cationic lipid complexes with anionic vesicles. A new cationic phospholipid derivative, l,2-dioleoyl-sn-glycero-3-ethylphosphocholine, was used to prepare giant bilayer vesicles and to form DNA-cationic lipid complexes (lipoplexes). The cationic vesicles were electrophoretically maneuvered into contact with DNA, and similarly, complexes were brought into contact with anionic phospholipid vesicles composed of dioleoylphosphatidylglycerol (DOPG; 100%), DOPG/dioleoylphosphatidylethanolamine (DOPE; 1:1) or DOPG/dioleoylphosphatidylcholine (DOPC; 1:1). Video fluorescence microscopy revealed that upon contact with phospholipid anionic vesicles, lipoplexes exhibited four different types of behavior: adhesion, vesicle rupture, membrane perforation (manifested as vesicle shrinkage and/or content loss), and expansion of DNA (which was always concomitant with membrane perforation.) In one instance, the lipoplex was injected into the target vesicle just prior to DNA expansion. In all other instances, the DNA expanded over the outer surface of the vesicle, and expansion was faster, the larger the area of vesicle over which it expanded. Given the likelihood of incorporation of cellular anionic lipids into lipoplexes, the expansion of the DNA could be important in DNA release during cell transfection. Upon contact with naked DNA, giant cationic vesicles usually ruptured and condensed the DNA into a small particle. Contact of cationic vesicles that were partially coated with DNA usually caused the DNA to wrap around the vesicle, leading to vesicle rupture, vesicle fusion (with other attached vesicles or lipid aggregates), or simply cessation of movement. These behaviors clearly indicated that both DNA and vesicles could be partly or fully covered by the other, thus modifying surface charges, which, among others, allowed adhesion of DNA-coated vesicles with uncoated vesicles and of lipid-coated DNA with uncoated DNA.     

A fluorimetric method for the estimation of the critical micelle concentration of surfactants

The present work investigates the possibility of a rapid estimation of critical micelle concentration (cmc) of surfactants by means of soluble fluorescent probes. The effect of nonionic or differently charged surfactants on the fluorescent properties of the anionic 8-anilino-1-naphtalenesulfonic acid magnesium salt (ANS) or cationic rhodamine 6G has been investigated. The possibility of cmc evaluation depends on the appropriate selection of the dye-detergent couple. ANS has to be used with anionic surfactants; on the other hand, rhodamine 6G has to be used with cationic detergents. Both ANS and rhodamine 6G have been proved to be effective with either zwitterionic or nonionic surfactants. Plots of ANS fluorescence increase or rhodamine 6G decrease vs surfactant concentration give two straight lines whose intersection indicates the cmc of the detergent. Under all these conditions the fluorescent probe does not interfere with the micellization process. Excitation of the fluorescent probes at the isosbestic point does not affect the evaluation of the cmc of the detergent. The method applies for linear or steroid surfactants and is independent of the cmc value within a wide range of concentrations.     

Versatile interactions of the antimicrobial peptide novispirin with detergents and lipids

Novispirin G-10 is an 18-residue designed cationic peptide derived from the N-terminal part of an antimicrobial peptide from sheep. This derivative is more specific for bacteria than the parent peptide. We have analyzed Novispirin's interactions with various amphipathic molecules and find that a remarkably wide variety of conditions induce alpha-helical structure. Optimal structure induction by lipids occurs when the vesicles contain 40-80% anionic lipid, while pure anionic lipid vesicles induce aggregation. SDS also forms aggregates with Novispirin at submicellar concentrations but induces alpha-helical structures above the cmc. Both types of aggregates contain significant amounts of beta-sheet structure, highlighting the peptide's structural versatility. The cationic detergent LTAC has a relatively strong affinity for the cationic peptide despite the peptide's net positive charge of +7 at physiological pH and total lack of negatively charged side chains. Zwitterionic and nonionic detergents induce alpha-helical structures at several hundred millimolar detergent. We have solved the peptide structure in SDS and LTAB by NMR and find subtle differences compared to the structure in TFE, which we ascribe to the interaction with an amphiphilic environment. Novispirin is largely buried in the SDS-micelle, whereas it does not enter the LTAC-micelle but merely forms a dynamic equilibrium between surface-bound and nonbound Novispirin. Thus, electrostatic repulsion can be overruled by relatively high-detergent concentrations or by deprotonating a single critical side chain, despite the fact that Novispirin's ability to bind to amphiphiles and form alpha-helical structure is sensitive to the electrostatics of the amphiphilic environment. This emphasizes the versatility of cationic antimicrobial peptides' interactions with amphiphiles.     

Bacterial decolorization of acid orange 7 in the presence of ionic and non-ionic surfactants

The effects of the non-ionic surfactant Triton X-100, the cationic surfactant cetyltri-methylammonium bromide (CTAB) and the anionic surfactant sodium N-lauroyl sarcosinate (SLS) on the decolorization of the reaction medium containing the monoazo dye Acid Orange 7 (AO7) by Alcaligenes faecalis and Rhodococcus erythropolis were studied. It was found that the surfactants influenced in different ways the rate of decolorization. At all concentrations tested the non-ionic surfactant Triton X-100 decreased the decolorization rate of R. erythropolis. At concentrations above the critical micelle concentration (CMC) Triton X-100 upset the usually observed exponential decay of the dye with A. faecalis due probably to the existence of an outer membrane in this organism. In concentrations above the CMC the anionic surfactant SLS inhibited the decolorization and, at prolonged incubation, caused partial release of the bound dye. The cationic surfactant CTAB in concentrations above and below the CMC accelerated drastically the binding of AO7 to the cells causing a rapid staining of the biomass and complete decolorization of the reaction medium. An attempt was made for explanation of the observed differences by the negative electrostatic charge of the living bacterial cell.     

Remarkable antiagglomeration effect of a yeast biosurfactant, diacylmannosylerythritol, on ice-water slurry for cold thermal storage

Antiagglomeration effects of different surfactants on ice slurry formation were examined to improve the efficiency of an ice-water slurry system to be used for cold thermal storage. Among the chemical surfactants tested, a nonionic surfactant, poly(oxyethylene) sorbitan dioleate, was found to show a greater antiagglomeration effect on the slurry than anionic, cationic, or amphoteric surfactants. More interestingly, diacylmannosylerythritol, a glycolipid biosurfactant produced by a yeast strain of Candida antarctica, exhibited a remarkable effect on the slurry, attaining a high ice packing factor (35%) for 8 h at a biosurfactant concentration of 10 mg/L. These nonionic glycolipid surfactants are likely to effectively adsorb on the ice surface in a highly regulated manner to suppress the agglomeration or growth of the ice particles. This is the first report on the utilization of biosurfactant for thermal energy storage, which may significantly expand the commercial applications of the highly environmentally friendly slurry system.     

Giant extracellular Glossoscolex paulistus Hemoglobin (HbGp) upon interaction with cethyltrimethylammonium chloride (CTAC) and sodium dodecyl sulphate (SDS) surfactants: Dissociation of oligomeric structure and autoxidation

The effects of two ionic surfactants on the oligomeric structure of the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) in the oxy - form have been studied through the use of several spectroscopic techniques such as electronic optical absorption, fluorescence emission, light scattering, and circular dichroism. The use of anionic sodium dodecyl sulphate (SDS) and cationic cethyltrimethyl ammonium chloride (CTAC) has allowed to differentiate the effects of opposite headgroup charges on the oligomeric structure dissociation and hemoglobin autoxidation. At pH 7.0, both surfactants induce the protein dissociation and a significant oxidation. Spectral changes occur at very low CTAC concentrations suggesting a significant electrostatic contribution to the protein–surfactant interaction. At low protein concentration, 0.08 mg/ml, some light scattering within a narrow CTAC concentration range occurs due to protein–surfactant precipitation. Light scattering experiments showed the dissociation of the oligomeric structure by SDS and CTAC, and the effect of precipitation induced by CTAC. At higher protein concentrations, 3.0 mg/ml, a precipitation was observed due to the intense charge neutralization upon formation of ion pair in the protein–surfactant precipitate. The spectral changes are spread over a much wider SDS concentration range, implying a smaller electrostatic contribution to the protein–surfactant interactions. The observed effects are consistent with the acid isoelectric point (pI) of this class of hemoglobins, which favors the intense interaction of HbGp with the cationic surfactant due to the existence of excess acid anionic residues at the protein surface. Protein secondary structure changes are significant for CTAC at low concentrations while they occur at significantly higher concentrations for SDS. In summary, the cationic surfactant seems to interact more strongly with the protein producing more dramatic spectral changes as compared to the anionic one. This is opposite as observed for several other hemoproteins. The surfactants at low concentrations produce the oligomeric dissociation, which facilitates the iron oxidation, an important factor modulating further oligomeric protein dissociation.     

Action of surface-active substances of biological membranes. III. Comparison of hemolytic activity of ionic and nonionic surfactants

The hemolytic action of a number of homologous series of cationic surfactants on human erythrocytes was measured. The hemolytic effects of anionic, nonionic and cationic surface-active agents are compared. The relationship which exists between the key physicochemical properties of surfactants (critical micelle concentration, hydrophile-lipophile balance) and their hemolytic capacities is discussed. The parameters required to compare the actions of various surfactants on different cellular membranes are considered in relation to the study of the correlation between the surfactant lytic effects and the features of the membrane molecular organization.