Bioavailability of organic compounds solubilized in nonionic surfactant micelles

Whether direct availability of organic compound solubilized in nonionic surfactant micelles (bioavailability) in a bioremediation or biotransformation process is uncertain to some extent, which is partially attributed to the difficulty by direct experimental determination. In another point of view, it should be ascribed to the fuzzy concept about the solubilization of organic compound in a nonionic surfactant micelle aqueous solution. In this mini-review, the solubilization of organic compound in surfactant micelles aqueous solution is fully discussed; especially saturated solubilization and unsaturated solubilization have been emphasized. Then the current methods for estimation of bioavailability of organic compounds solubilized in micelles are introduced, in which the possible drawbacks of each method are stressed. Finally, the conclusion that organic compound solubilized in micelles is unavailable directly by microbes has been drawn and the intensification of bioremediation or biotransformation by nonionic surfactant micelle aqueous solution is contributed to enhancement of the hydrophobic organic compounds dissolution.     

Assessing bioavailability of the solubilization of organic compound in nonionic surfactant micelles by dose–response analysis

It is uncertain in some extent that organic compounds solubilized in micelles of a nonionic surfactant aqueous solution are bioavailable directly by the microbes in an extractive microbial transformation or biodegradation process. In this work, a dose–response method, where a bioequivalence concept is introduced to evaluate the synergic toxicity of the nonionic surfactants and the organic compounds, was applied to analyze the inhibition effect of organic compounds (naphthalene, phenyl ether, 2-phenylethanol, and 1-butanol) in nonionic surfactant Triton X-100 micelle aqueous solutions and Triton X-114 in aqueous solutions forming cloud point systems. Based on the result, a mole solubilization ratio of organic compounds in micelle was also determined, which consisted very well with those of classic semi-equilibrium dialysis experiments. The results exhibit that bioavailability of organic compounds solubilized in micelles to microbial cells is negligible, which provides a guideline for application of nonionic surfactant micelle aqueous solutions or cloud point systems as novel media for microbial transformations or biodegradations.     

Formulation and Characterization of Phytophenol-Carrying Antimicrobial Microemulsions

Phytophenols were solubilized in nonionic surfactant micelles to form antimicrobially active and thermodynamically stable microemulsions. Formulation of phytophenols in microemulsions has previously been shown to improve their antimicrobial activity in model microbiological and food systems. Carvacrol and eugenol were incorporated in micellar solutions of two nonionic surfactants (Surfynol® 485W and Surfynol® 465) by mixing at room temperature. Particle size of formed microemulsions was determined by dynamic light scattering, and structural information about the mixed micellar system was obtained by nuclear magnetic resonance spectroscopy (NMR). Uptake of carvacrol and eugenol in surfactant micelles as determined by ultrasonic velocity measurements was very rapid, e.g., below the maximum additive concentration, the phytophenols were completely solubilized in the micelles in less than 30 min. Depending on the surfactant–phytophenol combination, the self-assembled surfactant–phytophenol aggregates had mean particle diameters between 3 and 17 nm. Elucidation of the structure of aggregates by 1H NMR studies indicated that micelles had a “bracket-like” structure with phytophenols being located inside the palisade layer of the micelle in direct contact with adjacent surfactant monomers. Encapsulation of phytophenols in surfactant micelles enables the incorporation of large amounts of hydrophobic antimicrobials in aqueous phases. Formulation of antimicrobial microemulsions may thus offer a means to deliver high concentrations of phytophenols to the bacterial surfaces of foodborne pathogens to affect kill.     

Affinity precipitation of proteins by surfactant-solubilized, ligand-modified phospholipids.

The use of ligand-modified phospholipids solubilized in aqueous solution by nonionic surfactant for affinity precipitation of proteins is described. Avidin was precipitated by contact with solutions in which dimyristoylphosphatidylethanolamine (DMPE) functionalized with biotin (DMPE-B) was solubilized in octaethylene glycol mono-n-dodecyl ether (C12E8) solutions. The nonionic surfactant solubilizes the phospholipid in micelles above its critical micelle concentration (CMC) and in small submicellar aggregates below this concentration. At C12E8 concentrations significantly exceeding its CMC, determined to be about 100 microM, precipitation of avidin by solubilized DMPE-B is not observed. In this regime, binding of protein by DMPE-B was monitored by a hyperchroic shift in the protein's UV-visible spectrum at 231.5 nm. The data were analyzed using a model that considers the four binding sites on the protein to be independent and identical in binding strength for DMPE-B. Below the CMC of C12E8, precipitation is observed and is monitored by increasing turbidity of the solution. The kinetics of precipitation and the aggregate size measured by quasielastic light scattering were analyzed using Smoluchowski kinetics and the Mie scattering theory. These results help establish more completely the factors that influence the precipitation of proteins by ligand-modified phospholipids, and they are helpful in specifying conditions for the precipitation of other proteins.     

Effect of a commercial alcohol ethoxylate surfactant (C<Subscript>11-15</Subscript>E<Subscript>7</Subscript>) on

Biodegradation of poorly soluble polycyclic aromatic hydrocarbons (PAHs) has been a challenge in bioremediation. In recent years, surfactant-enhanced bioremediation of PAH contaminants has attracted great attention in research. In this study, biodegradation of phenanthrene as a model PAHs solubilized in saline micellar solutions of a biodegradable commercial alcohol ethoxylate nonionic surfactant was investigated. The critical micelle concentration (CMC) of the surfactant and its solubilization capacity for phenanthrene were examined in an artificial saline water medium, and a type of marine bacteria, Neptunomonas naphthovorans, was studied for the biodegradation of phenanthrene solubilized in the surfactant micellar solutions of the saline medium. It is found that the solubility of phenanthrene in the surfactant micellar solutions increased linearly with the surfactant concentrations, but, at a fixed phenanthrene concentration, the biodegradability of phenanthrene in the micellar solutions decreased with the increase of the surfactant concentrations. This was attributed to the reduced bioavailability of phenanthrene, due to its increased solubilization extent in the micellar phase and possibly lowered mass transfer rate from the micellar phase into the aqueous phase or into the bacterial cells. In addition, an inhibitory effect of the surfactant on the bacterial growth at high surfactant concentrations may also play a role. It is concluded that the surfactant largely enhanced the solubilization of phenanthrene in the saline water medium, but excess existence of the surfactant in the medium should be minimized or avoided for the biodegradation of phenanthrene by Neptunomonas naphthovorans.     

Determination of molecular weight of membrane proteins by the use of low-angle laser light scattering combined with high-performance gel chromatography in the presence of a non-ionic surfactant

An assessment study was carried out to evaluate the performance of the low-angle laser light scattering technique combined with high-performance gel chromatography in the presence of a nonionic surfactant, octaethyleneglycol n-dodecyl ether, precision differential refractometry and ultraviolet photometry. It was found that the combined technique is highly promising as a method for the determination of the molecular weight of a membrane protein solubilized by the surfactant. For trial, molecular weights of the following membrane proteins of Escherichia coli, both solubilized in oligomeric forms, were measured; porin that forms the transmembrane diffusion pore in the outer membrane, and lambda-receptor protein that facilitates the diffusion of maltose-maltodextrins across the outer membrane. The result obtained indicates that both porin and lambda-receptor protein exist as trimers in the surfactant solution.     

Degradation of polycyclic aromatic hydrocarbons in the presence of synthetic surfactants.

The biodegradation of polycyclic aromatic hydrocarbons (PAH) often is limited by low water solubility and dissolution rate. Nonionic surfactants and sodium dodecyl sulfate increased the concentration of PAH in the water phase because of solubilization. The degradation of PAH was inhibited by sodium dodecyl sulfate because this surfactant was preferred as a growth substrate. Growth of mixed cultures with phenanthrene and fluoranthene solubilized by a nonionic surfactant prior to inoculation was exponential, indicating a high bioavailability of the solubilized hydrocarbons. Nonionic surfactants of the alkylethoxylate type and the alkylphenolethoxylate type with an average ethoxylate chain length of 9 to 12 monomers were toxic to a PAH-degrading Mycobacterium sp. and to several PAH-degrading mixed cultures. Toxicity of the surfactants decreased with increasing hydrophilicity, i.e., with increasing ethoxylate chain length. Nontoxic surfactants enhanced the degradation of fluorene, phenanthrene, anthracene, fluoranthene, and pyrene.     

The binding of anionic and nonionic surfactants to collagen through the hydrophobic effect

The adsorption of nonionic surfactants on hide powder previously treated with anionic surfactants has been studied. The adsorption of nonionic surfactants takes place through hydrophobic interactions. A mechanism has been proposed for this interaction, assuming that the nonionic surfactant has been fixed by means of secondary adsorption (hydrophobic interaction) after the primary adsorption of the anionic surfactant (ionic and hydrophobic interaction) which makes it possible.     

Biodegradation of naphthalene in aqueous nonionic surfactant systems.

The principal objective of this study was to quantify the bioavailability of micelle-solubilized naphthalene to naphthalene-degrading microorganisms comprising a mixed population isolated from contaminated waste and soils. Two nonionic surfactants were used, an alkylethoxylate, Brij 30 (C12E4), and an alkylphenol ethoxylate, Triton X-100 (C8PE9.5). Batch experiments were used to evaluate the effects of aqueous, micellized nonionic surfactants on the microbial mineralization of naphthalene and salicylic acid, an intermediate compound formed in the pathway of microbial degradation of naphthalene. The extent of solubilization and biodegradation under aerobic conditions was monitored by radiotracer and spectrophotometric techniques. Experimental results showed that surfactant concentrations above the critical micelle concentration were not toxic to the naphthalene-degrading bacteria and that the presence of surfactant micelles did not inhibit mineralization of naphthalene. Naphthalene solubilized by micelles of Brij 30 or Triton X-100 in liquid media was bioavailable and degradable by the mixed culture of bacteria.     

Behavior of fragmented calcium (II) adenosine triphosphatase from sarcoplasmic reticulum in detergent solution.

The behavior of Ca2+-ATPase from sarcoplasmic reticulum in detergent solution was compared with that of Ca2+-ATPase which had been cleaved in half by limited trypsin digestion. Attempts to dissociate the fragments (I and II) with an excess of detergent micelles demonstrated that fragments I and II are structurally dependent upon each other, and that they must be denatured in order to be dissociated. Partial dissociation of the fragmented ATPase was found to occur in the bile salt detergents, deoxycholate and cholate, and optical data showed that there was an accompanying change in conformation. No dissociation of the fragmented ATPase was observed in nonionic detergents. The fragmented ATPase retained the same specific activity and stability as the intact ATPase under a variety of conditions when solubilized in Tween 80 or dodecyl octaoxyethylene glycol monoether. The data demonstrate that the noncovalent interactions that maintain the native conformation of the ATPase are not affected by either trypsin cleavage or solubilization in nonionic detergent solution.     

Interactions between charged polypeptides and nonionic surfactants

The influence of molecular characteristics on the mutual interaction between peptides and nonionic surfactants has been investigated by studying the effects of surfactants on amphiphilic, random copolymers of alpha-L-amino acids containing lysine residues as the hydrophilic parts. The hydrophobic residues were either phenylalanine or tyrosine. The peptide-surfactant interactions were studied by means of circular dichroism spectroscopy and binding isotherms, as well as by 1D and 2D NMR. The binding of surfactant to the peptides was found to be a cooperative process, appearing at surfactant concentrations just below the critical micellar concentration. However, a certain degree of peptide hydrophobicity is necessary to obtain an interaction with nonionic surfactant. When this prerequisite is fulfilled, the peptide mainly interacts with self-assembled, micelle-like surfactant aggregates formed onto the peptide chain. Therefore, the peptide-surfactant complex is best described in terms of a necklace model, with the peptide interacting primarily with the palisade region of the micelles via its hydrophobic side chains. The interaction yields an increased amount of alpha-helix conformation in the peptide. Surfactants that combine small headgroups with a propensity to form small, nearly spherical micelles were shown to give the largest increase in alpha-helix content.     

Solubilization and insertion into reverse micelles of the major myelin transmembrane proteolipid

The Folch-Pi proteolipid has been isolated from bovine white matter and characterized with respect to phospholipid and glycolipid composition. The protein-lipid complex has been solubilized in aqueous reverse micelles of di(2-ethylhexyl) sodium sulfosuccinate and isooctane. Solubilization of this otherwise water-insoluble proteolipid requires small amounts of water, the percent of solubility being maximum for a low molar ratio of water to surfactant (Wo = 5.6). Unlike hydrophilic proteins, the extent of incorporation into the micellar system is negligible at 50 mM surfactant and reaches 90Vo only at 300 mM. However, the conformation of the proteolipid in reverse micelles as studied by fluorescence emission spectroscopy and circular dichroism was not affected by variations of the surfactant concentration. These results are consistent with the peculiar properties of the aqueous environment of the proteolipid within the reverse micelles and may reflect the membrane-like character of these bio-assemblies.     

3-hydroxy-3-methylglutaryl-CoA reductase: solubilization in the presence of proteolytic inhibitors, partial purification, and reversible phosphorylation-dephosphorylation

A growing body of evidence indicates that 3-hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34, reductase) is degraded by proteolytic enzymes during solubilization by traditional freeze-thaw techniques. We have solubilized reductase in an active, stable form with nonionic detergents [Lubrol WX or poly(oxyethylene) ether type W-1]. Solubilization proceeded in high (greater than 70%) yield in the presence of the proteolytic inhibitors leupeptin, phenylmethanesulfonyl fluoride, and ethylene glycol bis(beta-aminoethyl ether)-N,N,-N',N'-tetraacetic acid and was independent of prior freeze-thawing of the microsomes. We have purified detergent-solubilized reductase 40-fold in high yield by means of sucrose density gradient centrifugation and dye-ligand chromatography. Detergent-solubilized reductase is heat labile, unlike reductase solubilized by the freeze-thaw method. Detergent-solubilized reductase can be inactivated up to 90% by use of reductase kinase. This inactivation requires both adenosine 5'-triphosphate and adenosine 5'-diphosphate, as has been previously observed for both microsomal and freeze-thaw solubilized reductase. Inactivation is reversed by subsequent treatment with a phosphoprotein phosphatase.     

Characteristics of halorhodopsin-bacterioruberin complex from Natronomonas pharaonis membrane in the solubilized system

Halorhodopsin is a retinal protein with a seven-transmembrane helix and acts as an inward light-driven Cl(-) pump. In this study, structural state of the solubilized halorhodopsin (NpHR) from the biomembrane of mutant strain KM-1 of Natronomonas pharaonis in nonionic detergent was investigated. A gel filtration chromatography monitored absorbances at 280 and 504 nm corresponding to the protein and a lipid soluble pigment of bacterioruberin (BR), respectively, has clearly detected an oligomer formation of the NpHRs and a complex formation between the NpHR and BR in the solubilized system. A molar ratio of NpHR:BR in the solubilized complex was close to 1:1. Further SDS-PAGE analysis of the solubilized NpHR cross-linked by 1% glutaraldehyde has revealed that the NpHR forms homotrimer in detergent system. Although this trimeric structure was stable in the presence of NaCl, it was dissociated to the monomer by the heat treatment at 45 °C in the desalted condition. The same tendency has been reported in the case of trimeric NpHR expressed heterologously on the E. coli membrane, leading to a conclusion that the change of strength of the trimeric association dependent on the ion binding is a universal feature of the NpHR. Interestingly, the trimer dissociation on the NpHR was accompanied by the complete dissociation of the BR molecule from the protein, indicated that the cavity formed by the NpHR protomers in the trimeric conformation is important for tight binding of the BR. Because the binding affinity for Cl(-) and the resistance to hydroxylamine under light illumination showed only minor differences between the NpHR in the solubilized state and that on the biomembrane, the influences of solubilization to the tertiary structure and function of the protein are thought to be minor. This NpHR-BR complex in the solubilized system has a potential to be a good model system to investigate the intermolecular interaction between the membrane protein and lipid.     

Solubilization capacity of nonionic surfactant micelles exhibiting strong influence on export of intracellular pigments in Monascus fermentation

In this study, perstractive fermentation of intracellular Monascus pigments in nonionic surfactant micelle aqueous solution had been studied. The permeability of cell membrane modified by nonionic surfactant might have influence on the rate of export of intracellular pigments into its extracellular broth while nearly no effect on the final extracellular pigment concentration. However, the solubilization of pigments in nonionic surfactant micelles strongly affected the final extracellular pigment concentration. The solubilization capacity of micelles depended on the kind of nonionic surfactant, the super‐molecule assembly structure of nonionic surfactant in an aqueous solution, and the nonionic surfactant concentration. Elimination of pigment degradation by export of intracellular Monascus pigments and solubilizing them into nonionic surfactant micelles was also confirmed experimentally. Thus, nonionic surfactant micelle aqueous solution is potential for replacement of organic solvent for perstractive fermentation of intracellular product.     

Ester Synthesis by a Recombinant Cutinase in Reversed Micelles of a Natural Phospholipid

Fusarium solani pisi recombinant cutinase solubilized in reversed micelles of a nonionic surfactant (phosphatidylcholine) in isooctane was used to catalyze the esterification of fatty acids with 2-butanol. Various parameters affecting the catalytic activity of the microencapsulated cutinase, such as pH, w_o (molar ratio water/surfactant), temperature and substrate concentration were investigated. Maximal specific activity were obtained with w_o=13, at pH 10.7 and 35d`C. The cutinase showed a higher specific activity for short length fatty acids, namely butyric acid. Calculation of the apparent kinetic parameters (k_m and V_max) for the synthesis of butyl butyrate, showed a low apparent affinity of the cutinase in phosphatidylcholine reversed micelles for both substrates.     

Effect of cationic surfactants on the conformation and aggregation of poly(L-glutamic acid)

The effects of three cationic surfactants, dodecylammonium chloride (DAC), dodecyldimethylammonium chloride (DDAC), and dodecyldimethylammonium chloride (DTDAC), on the conformation of poly(L -glutamic acid) and at neutral pH were examined by CD. The maximum extent of the α-helix induction occurs for each surfactant when the mixing ration is about unity. Different effects specific to each surfactant, as described below, appear in the range of mixing ratios larger than that required for the maximum induction. In the case of DTAC, the α-helices disintegrate into random coils. In the case of DDAC, the aggregation of α-helices takes place eventually leading to precipitation. Solubilization of the precipitates occurs at high mixing ratios. The most complex behavior is seen in the case of DAC; aggregation of α-helices occurs only to a small extent and the formation of a small complex predominates over aggregation takes place again as DAC concentration increases further. Induction of the α-helix is favored by dilution at a constant mixing ratio but is suppressed by the addition of NaCl.     

Dissolution improvement of high drug-loaded solid dispersion

This study focused on an investigation of a high drug-loaded solid dispersion system consisting of drug, carrier, and surfactant. Solid dispersions of a water-insoluble ofloxacin (OFX) with polyethylene glycol (PEG) of different molecular weights, namely binary solid dispersion systems, were prepared at drug to carrier not less than 5∶5. Polysorbate 80, a nonionic surfactant, was incorporated into the binary solid dispersion systems as the third component to obtain the ternary solid dispersion systems. The powder x-ray diffraction and differential scanning calorimetric studies indicated that crystalline OFX existed in the solid dispersions with high drug loading. However, a decreased crystallinity of the solid dispersions obtained revealed that a portion of OFX was in an amorphous state. The results indicated a remarkably improved dissolution of drug from the ternary solid dispersion systems when compared with the binary solid dispersion systems. This was because of polysorbate 80, which improved wettability and solubilized the non-molecularly dispersed or crystalline fraction of OFX.     

Solubilization and reconstitution of vesicular stomatitis virus envelope using octylglucoside.

Reconstituted vesicular stomatitis virus envelopes or virosomes are formed by detergent removal from solubilized intact virus. We have monitored the solubilization process of the intact vesicular stomatitis virus by the nonionic surfactant octylglucoside at various initial virus concentrations by employing turbidity measurements. This allowed us to determine the phase boundaries between the membrane and the mixed micelles domains. We have also characterized the lipid and protein content of the solubilized material and of the reconstituted envelope. Both G and M proteins and all of the lipids of the envelope were extracted by octylglucoside and recovered in the reconstituted envelope. Fusion activity of the virosomes tested either on Vero cells or on liposomes showed kinetics and pH dependence similar to those of the intact virus.     

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.