Catalysis by Laccase (From Coriolus uersicolor) in Microheterogeneous Media of the Water/Organic Solvent/Surfactant Type

Catalysis by laccase from Coriolus uersicolor solubilized in the ternary systems of surfactant/water/organic solvent type, namely, Aerosol OT/water/octane, Brij 56/water/cyclohexane and egg lecithin/water/octane + pentanol + methanol mixture, has been studied. The laccase activity is found to depend, in principle, not only on the water/surfactant molar ratio, but on the surfactant concentration (with its hydration degree being constant) as well. The following inferences should be emphasized. Firstly, in all the systems under study, the catalytic activity (k_cat) of laccase entrapped into surfactant reversed micelles increases more than 50 times (when the surfactant concentration is extrapolated to zero) compared with the k_cat value in aqueous solution. Secondly, the catalytic activity (k_cat) of laccase entrapped in hydrated Aerosol OT aggregates, having lamellar, reversed cylindrical (hexagonal) and reversed micellar structure, depends greatly on the aggregate type. In other words, the phase transitions, i.e. an alteration in the packing of hydrated Aerosol OT molecules, evokes a sharp reversible change in the enzymatic activity. Thirdly, in the same phase, the catalytic activity of the solubilized enzyme depends on the linear dimensions of water cavities inside the surfactant aggregates (i.e. on the water content in the system under study). All these effects, regulating enzymatic activity, are probably caused by an alteration of the conformational mobility of laccase molecules incorporated into the inner polar cavities inside the surfactant aggregates.     

Influence of nonionic surfactant on aggregation state of scleroglucan in aqueous solution

The interactions between nonyl phenol polyethylene oxide and scleroglucan are investigated by turbidimetry, viscosimetry filterability tests and measurements of elastic modulus and adsorption. The phase diagrams of this ternary system have been established as a function of temperature and composition. It is shown that the surfactant molecules are adsorbed by the polymer at a low surfactant concentration, c_s; the adsorption induces a breaking down of the polymer aggregates and the filterability properties of the solutions are greatly improved. An excess of surfactant phase separation is observed by heating at a temperature that is a decreasing function of c_s. This is explained by the formation of a complex polymer-surfactant which has the same thermodynamic properties (lower critical solution temperature) as polyethylene oxide and the derived nonionic 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.     

Viscometric and spectroscopic studies on the solution behaviour of hydrophobically modified cellulosic polymers

The solution properties of hydroxyethyl cellulose (HEC) and hydrophobically modified hydroxyethyl cellulose (HM-HEC) have been investigated by means of viscometric and spectroscopic techniques involving free radical and fluorescent probes. The greater viscosity of HM-HEC solutions above a critical polymer concentration (C_p) of approximately 0·2% has been interpreted in terms of the formation of a three-dimensional network structure in which the polymer chains are effectively crosslinked by the intermolecular association of neighbouring hydrophobic side chains. C_p is considerably less than the predicted polymer coil overlap concentration (C*) of approximately 1%.

The interaction of the polymers with an anionic surfactant, sodium dodecyl sulphate (SDS) has also been investigated. A mechanism involving the interaction of free surfactant with the regions of intermolecular hydrophobic association is suggested to account for the considerable differences in the rheological behaviour of the polymers in the presence of SDS.     

Enzymatic activity of an extremely halophilic phosphatase from the Archaea Halobacterium salinarum in reversed micelles

Alkaline p-nitrophenylphosphate phosphatase (pNPPase) from the halophilic archaeon Halobacterium salinarum (previously halobium) was solubilized in reversed micelles of cetyltrimethylammonium bromide (CTAB) in cyclohexane with 1-butanol as cosurfactant. The hydrolysis reaction appears to follow Michaelis–Menten kinetics. The dependency of the maximum reaction rate (V_max) on the water content θ (% v/v) (or ω₀ value: molar ratio of water to surfactant concentrations) showed a bell-shaped curve for 0.3 M CTAB, but not for 0.2 M CTAB. The enzyme activity increased with the surfactant concentration at a constant ω₀ value (10.27). When the surfactant concentration was increased at a constant θ, the enzyme activity decreased. The enzyme was more stable in reversed micelles than in aqueous media.     

Protein partitioning in weakly charged polymer-surfactant aqueous two-phase systems

The study includes partitioning of proteins in aqueous two-phase systems consisting of the polymer dextran and the non-ionic surfactant C₁₂E₅ (pentaethylene glycol mono-n-dodecyl ether). In this system a micelle-enriched phase is in equilibrium with a polymer-enriched phase. Charges can be introduced into the micelles by the addition of charged surfactants. The charge of the mixed micelles is easily varied in sign and magnitude independently of pH, by the addition of different amounts of negatively charged surfactant, sodium dodecyl sulphate (SDS), or positively charged surfactant dodecyl trimethyl ammonium chloride (DoTAC). A series of water-soluble model proteins (BSA, β-lactoglobulin, myoglobin, cytochrome c and lysozyme), with different net charges at pH 7.1, have been partitioned in non-charged systems and in systems with charged mixed micelles or charged polymer (dextran sulphate). It is shown that partition coefficients for charged proteins in dextran-C₁₂E₅ systems can be strongly affected by addition of charged surfactants (SDS, DoTAC) or polymer (dextran sulphate) and that the effects are directly correlated to protein net charge.     

Effect of aprotic solvents on the enzymatic activity of lipase in AOT reverse micelles

The modification of reverse micellar systems composed of AOT, isooctane, water by the addition of aprotic solvents has been performed. The impact of this change on the activity, stability and kinetics of solubilized Chromobacterium viscosum lipase (glycerol-ester hydrolase, EC 3.1.1.3) was investigated. Of seven aprotic solvents tested, dimethyl sulfoxide (DMSO) was found to be most effective. It was found that lipase activity was enhanced by optimizing some relevant parameters, such as water–AOT molar ratio (W₀), buffer pH and surfactant concentration. A kinetic model that considers the free substrate in equilibrium with the substrate adsorbed on the micellar surface was successfully used to deduce some kinetic parameters (V_max, K_m and K_ad), and the values of K_m and K_ad were significantly reduced by the presence of DMSO. Higher lipase stability was found in AOT reverse micelles with DMSO compared with that in simple AOT systems with half-life of 125 and 33 days, respectively. Fluorescence spectroscopy and Fourier transform infrared spectroscopy (FT-IR) were used to elucidate the effects of DMSO on the properties of AOT reverse micelles.     

Differential scanning calorimetric studies on bovine serum albumin IV. Effect of anionic surfactants with various lengths of hydrocarbon chain

Using defatted and SH-blocked bovine serum albumin (BSA), measurements of differential scanning calorimetry (DSC) have been made at pH 7 on the complexes of BSA and a series of sodium alkyl sulfates. Alkyl sulfates used were sodium decyl sulfate (SDeS), sodium octyl sulfate (SOS), sodium hexyl sulfate (SHS) and sodium ethyl sulfate (SES). Results obtained were compared with those on the system BSA-sodium dodecyl sulfate (SDS) studied previously. Two peaks P 1 and P2 existed in the DSC curve of BSA. These peaks originate in the heat-induced transition of BSA. The pattern of DSC curve changed with the amount of the ligand added, i.e. with the molar mixing ratio ligand/BSA (1). The change for systems BSA-SDeS, BSA-SOS and BSA-SHS was qualitatively the same as that for the system BSA-SDS (2). Interestingly, SES, which is not a surfactant, interacts with BSA. The change for the system BSA-SES was qualitatively the same as that for the system BSA-Na₂SO₄. All alkyl sulfates suppressed the heat-induced transition at lower concentrations. A linear relationship was obtained for the plots of log(D/A)₁ versus log CMC, where (D/A)₁ is the molar mixing ratio of anionic surfactant (D) to BSA (A) at which the most heat-stable complex is formed. This suggests that the hydrophobic force has a serious effect on the formation of heat-stable complexes.     

Dependence of Lipase Activity on Water Content and Enzyme Concentration in Reverse Micelles

The activity of Candida rugosa lipase (EC 3.1.1.3) in reverse micelles has been measured at various concentrations of water and enzyme with the aim of answering the question, why is the enzyme activity affected by the molar ratio of water to surfactant (w₀ = [H₂O]/[Surfactant])? In the low range of water content (below w₀ ≈ 6), the activity increases with increasing water content, indicating the requirement of a minimum amount of water for the full expression of enzymatic activity. The minimal w₀-value for obtaining maximal activity depends on the enzyme concentration: The higher the enzyme concentration, the higher w_{0, max}. In addition, it was found that, at least for the case of Candida rugosa lipase, the measured dependence of enzyme activity on w₀ does not represent a true chemical equilibrium. Changing the w₀-value during the reaction does not change the activity as expected on the basis of the w₀-activity profile obtained for single w₀ point measurements. All these observations, however, cannot be directly generalized to all enzymes in reverse micelles, due to the peculiarity of lipase. In particular, the enzyme seems to inactivate irreversibly during the solubilization process.     

Micellar catalysis of polyphenol oxidase in AOT/cyclohexane

The catalytic behaviour of mushroom polyphenol oxidase has been studied in dioctylsulphosuccinate (AOT)/cyclohexane reverse micelles. The steady-state conditions were accomplished up to 20 min and 17 μg protein in the assay towards 4-methylcatechol and no loss of specific activity was observed relative to aqueous medium. The pH activity profile of the enzyme was kept in reverse micelles as in water, showing a plateau between 5 and 6.5. The stability of polyphenol oxidase to pH was also studied and about 20% inactivation was found in reverse micelles relative to aqueous medium at neutral pHs. Moreover there was a decrease of stability at acidic pHs. The optimum W_o obtained was 20 and the enzyme was nearly independent of the surfactant concentration at constant W_o.

Kinetic studies of polyphenol oxidase towards several substrates showed that the substrate inhibition by p-cresol and 4-methylcatechol observed in buffer was not kept in AOT/cyclohexane reverse micelles. Moreover, the K_m increased and the catalytic efficiency (V/K_m) of the enzyme decreased as the hydrophobicity of substrates was increased.     

Microviscosity in dilute aqueous solutions of SDS and non-ionic cellulose derivatives of different hydrophobicity: fluorescence probe investigations

The microviscosity in mixed micelles formed in dilute aqueous solutions of sodium dodecyl sulphate (SDS) and a set of non-ionic cellulose ethers of different hydrophobicity has been determined by means of steady-state fluorescence probe techniques. Two hydrophobic probes have been applied in this investigation: 1,3-di(1-pyrenyl)propane (P3P) and perylene. Reference measurements of microviscosity have also been performed on SDS solutions including the uncharged polymers poly(ethyleneoxide) (PEO) or poly(vinylpyrrolidone) (PVP). All compositions investigated showed qualitatively the same general behaviour with an abrupt increase in microviscosity at the critical surfactant concentration where the polymer-surfactant interaction starts (c₁) followed by a maximum and an asymptotically declining region as the surfactant concentration was increased further. Comparison with a recent investigation of a specific ethyl(hydroxyethyl)cellulose (EHEC fraction CST-103)/ SDS/water system (Evertsson & Nilsson (1997) Macromolecules, 30, 2377) revealed that the maximum in microviscosity generally corresponds to a low degree of SDS adsorption (≈ 0.5 mmol of SDS per gram of polymer) and consequently to a high polymer content of the mixed micelles formed in the type of systems studied herein. The hydrophobicity of the cellulose derivatives was found to correlate to the amplitude of the overall microviscosity pattern for the mixed micelles, i.e. an increased polymer hydrophobicity gave an increased rigidity of the polymersurfactant aggregates. An approximately exponential relation was demonstrated between the maxima in microviscosity of the different mixed micelles and the surface activities of the corresponding cellulose derivatives. All polymer/surfactant combinations investigated gave aggregates with a higher rigidity than ordinary SDS micelles. The microviscosity of the mixed micelles of the cellulose derivatives and SDS formed close to c₁ increased as the temperature rose from 20 to 50 °C. This effect was attributed to an increased hydrophobicity of the cellulose ethers upon temperature elevation, hence giving rise to further close-packing of the aggregate structures.     

Tyrosinase activity in reversed micelles

The hydroxylase and oxidase activities of mushroom tyrosinase were studied in both sodium di-2-ethylhexylsulfosuccinate (AOT)/isooctane and cetyltrimethylammonium bromide (CTAB)/hexane/chloroform reversed micelles. The enzyme presented its highest activity when the water to surfactant molar ratio (W ₀) was 20 for both systems. When entrapped in the AOT reversed micelles, the enzyme activity decreased with the increase in AOT concentration at a constant W ₀, and the enzyme not only presented a higher reaction rate related to its oxidase activity but also a shorter lag period related to its hydroxylase activity. The relation between water activity and W ₀ revealed that enzyme activity in reversed micelles was more related to the size of the micelles which was determined by W ₀ and less to the water activity. Tyrosinase in CTAB reversed micelles showed potential for the analysis of o-diphenols.     

Stability and Enzymatic Studies of Glucose Dehydrogenase from the Archaeon Haloferax mediterranei in reverse micelles

Reverse micelles were used as a cytoplasmic model to study the kinetics of an extreme halophilic enzyme such as the recombinant glucose dehydrogenase from the Archaeon Haloferax mediterranei. This enzyme was solubilized in reverse micelles of hexadecyltrimethylammoniumbromide in cyclohexane, with 1-butanol as co-surfactant. Glucose dehydrogenase retained its catalytic properties in this organic medium, showing good stability at low water content, even at low salt concentration (125 mM NaCl). The dependence of the enzymatic activity on the molar water surfactant ratio (w₀=[H₂O]/[surfactant]) increased with rising water content. Surprisingly, the activity of this extreme halophilic enzyme did not depend on the salt concentration in reverse micelles. The kinetic of the enzymatic oxidation of β-D-glucose to D-glucono-1,5-lactone using NADP⁺ as coenzyme for the glucose dehydrogenase from Haloferax mediterranei was also studied in the reverse micellar system.     

Selective oxidation of glucose on bismuth-promoted Pd-Pt/C catalysts prepared from NOct4Cl-stabilized Pd-Pt colloids

Charcoal-supported Pd-Pt catalysts based on Pd-Pt/NOct₄Cl colloidal alloys have superior activity and selectivity in the oxidation of glucose to gluconic acid compared with industrial heterogeneous Pd-Pt catalysts. According to transmission electron microscopy, X-ray diffraction/Debye function analysis, X-ray photoelectron spectroscopy, X-ray absorption near edge structure, and extended X-ray absorption fine structure analysis the chemical coreduction of PdCl₂ and PtCl₂ in the appropriate ratio with NOct₄BEt₃H yielded the alloyed Pd-Pt colloids in organic solvents. They are screened by the lipophilic NOct₄Cl surfactant layer from coagulation and poisoning. TEM showed colloids of particle sizes in the range from 1.5 to 3 nm.     

Amidase encapsulated in TTAB reversed micelles for the study of transamidation reactions

Amidase, an amide hydrolase enzyme (E.C.3.5.1.4) with acyl transferase activity, was encapsulated in a reversed micellar system composed of the cationic surfactant tetradecyltrimethyl ammonium bromide (TTAB) in heptane/octanol (80/20%) and phosphate buffer at w ₀ 11. The reaction used to study the effect of the reversed micellar system on the enzyme behaviour was a transamidation reaction. The effect of surfactant concentration, buffer molarity and pH on the enzyme kinetics was evaluated. Both initial velocities and product yield were measured. The results indicated that a high initial velocity of hydroxamic acid synthesis and also the highest yield (98%) were obtained using the lowest pH value. The effect of TTAB concentration was dependent on the buffer molarity used. The effect of buffer molarity on reversed micelle dimensions was analysed by light scattering. These results showed that the buffer molarity had a strong influence on the reversed micelle radius that correlated with enzyme activity.     

Effects of Surfactant Protein-A on the Interaction of Pneumocystis murina with its Host at Different Stages of the Infection in Mice

ABSTRACT. We examined the effects of surfactant protein A (SP-A), a collectin, on the interaction of Pneumocystis murina with its host at the beginning, early to middle, and late stages of infection. Pneumocystis murina from SP-A wild-type (WT) mice inoculated intractracheally into WT mice (WT_S-WT_R) adhered well to alveolar macrophages, whereas organisms from SP-A knockout (KO) mice inoculated into KO mice (KO_S-KO_R) did not. Substitution of WT mice as the source of organisms (WT_S-KO_R) or recipient host macrophages (KO_S-WT_R) restored adherence to that found with WT_S-WT_R mice. In contrast, when immunosuppressed KO and WT mice were inoculated with P. murina from a homologous source (KO_S-KO_R, WT_S-WT_R) or heterologous source (WT_S-KO_R, KO_S-WT_R) and followed sequentially, WT_S-KO_R mice had the highest levels of infection at weeks 3 and 4; these mice also had the highest levels of the chemokine macrophage inflammatory protein-2 and neutrophils in lavage fluid at week 3. Surfactant protein-A administered to immunosuppressed KO_S-KO_R mice with Pneumocystis pneumonia for 8 wk as a therapeutic agent failed to lower the organism burden. We conclude that SP-A can correct the host immune defect in the beginning of P. murina infection, but not in the middle or late stages of the infection.     

Demonstration of Pulmonary Surfactant by Tracheal Injection of Tricomplex Salt Mixture: Electron Microscopy

Laboratory animals were perfused with glutaraldehyde through the right ventricle before filling the whole lung intratracheally with a solution of 0.05 N Pb(NO₃)₂ and K₃Fe(CN)₆ and incubating it at 37 C for 30 min. An electron-dense reaction product on the surface of epithelial cells and in certain well-localized regions of the alveolar septa results. Nonspecific, intracellular, precipitates do not occur. Lungs which are sliced before glutaraldehyde fixation and immersed in the tricomplex salt mixture show patchy localization of reaction product. Diffuse, electron-dense deposits measuring 100-2000 mμ in diameter are seen in the cytoplasm of epithelial, endothelial and interstitial cells although occasionally there is localization on the surface of the epithelial cells and in specific sites within cells. Injection of the whole lung with tricomplex salt mixture after fixation with glutaraldehyde by vascular perfusion is a better method for the ultrastructural demonstration of pulmonary surfactant than immersion fixation and staining.     

Surfactants Based on Renewable Raw Materials

Under the European Commission's European Climate Change Programme, a group of experts studied the possibilities of using more renewable raw materials as chemical feedstock and assessed the related potential for greenhouse gas (GHG) emission reduction. Surfactants were among the products studied. Surfactants are currently produced from both petro-chemical feedstocks and renewable resources (oleochemical surfactants). Assuming, in a first step, that total surfactant production in the European Union remains constant until 2010, it was estimated that the amount of oleochemical surfactants could be increased from about 880 kilotons (kt) in 1998 to approximately 1, 100 kt in 2010 (an increase of 24%). This substitution reduces the life-cycle CO₂ emissions from surfactants by 8%; the theoretical maximum potential for total substitution is 37%. Because the surfactant market is expected to grow, the avoided emissions will probably exceed 8% of the current life-cycle CO₂ emissions from surfactants. If compared to the CO₂ emissions from the total industrial sector and, even more so, if compared to the total economy, the relative savings are much lower (0.02% to 0.09%). This leads to the conclusion that the increased production and use of biobased surfactants should be part of an overall GHG emission reduction strategy consisting of a whole range of measures addressing both energy demand and supply. This article also discusses policies and measures designed to increase the use of biobased surfactants.     

Influence of Igepal reverse micellar and micellar systems on activity and stability of heme peroxidases

The activities of horseradish peroxidase (HRP) and lactoperoxidase (LPO) entrapped in reverse micelles of Igepal CO-520 in cyclohexane were studied. When the molar ratio of water to surfactant, w ₀ was ≥13, the activity of HRP encapsulated in the water pool of the reverse micelle was comparable with that measured in buffer. For LPO, however, lower activity was observed after its incorporation into the same system.

The activity of the investigated peroxidases was also measured in an aqueous solution of Igepal CO-720 or after incubation with this surfactant. The enzymes became inactivated in an aqueous micellar solution of Igepal CO-720, although this process was reversible.

The stability of HRP and LPO at 37 or 50°C was lower in the micellar systems than in buffer with the exception for HRP in reverse micelles at 50°C.     

Characteristics of Lipase-Catalyzed Glycerolysis of Triglyceride in AOT-Isooctane Reversed Micelles

Chromobacterium viscosum lipase, solubilized in microemulsion droplets of glycerol containing small amounts of water and stabilized by a surfactant, could catalyze the glycerolysis of triolein. Kinetic analysis of the lipase-catalyzed reaction was possible in the reversed micellar system. Among surfactants and organic solvents tested, bis(2-ethylhexyl)sodiumsulfosuccinate (AOT) and isooctane were respectively most effective, for the glycerolysis of triolein in reversed micelles. Temperature effects, pH profile, K_m,app, and V_max,app were determined. Among various chemical compounds, Fe³⁺, Cu²⁺, and Hg²⁺ inhibited the lipase-catalyzed glycerolysis severely. However, the glycerolysis activity was partially restorable by adding histidine or glycine to the system containing these metal ions. The glycerolysis activity was dependent on water content and maximum activity was obtained at an R value of 1.21. Higher stability of the lipase was obtained in the reversed micellar system.