A kinetic model for enzymatic reactions in reverse micellar systems involving water‐insoluble substrates and enzyme activators

The activity of Chromobacterium viscosum lipase (glycerol‐ester hydrolase, EC 3.1.1.3) entrapped in AOT/isooctane reverse micelles was significantly increased by the addition of short chain polyethylene glycols (PEGs) or methoxypolyethylene glycols (MPEGs) for the hydrolysis of olive oil. To understand enzyme activity in the presence of PEG 400 or MPEG 550 molecules, a kinetic model was proposed. The validity of this model was verified by experimental data on the lipase‐catalyzed hydrolysis of olive oil in AOT/isooctane reverse micellar systems, in which PEG 400 or MPEG 550 had been added. The large value of the equilibrium constant (k_D) for enzyme activation indicated that the affinity between C viscosum lipase and PEG 400 or MPEG 550 molecules was very strong. The Michaelis constant (K_m) predicted by the proposed model explained enzymatic reactions more exactly than that by the previously published model. Copyright © 2003 Society of Chemical Industry     

Enzymatic activity of Chromobacterium viscosum lipase in an AOT/Tween 85 mixed reverse micellar system

To enhance the Chromobacterium viscosum lipase (glycerol‐ester hydrolase; EC 3.1.1.3) activity for the reaction of water‐insoluble substrates, the AOT/isooctane reverse micellar interface was modified by co‐adsorption of a non‐ionic surfactant. Polyoxyethylene sorbitan trioleate (Tween 85) was used as the non‐ionic surfactant and olive oil as a water‐insoluble substrate. An appreciable increase of lipase activity was observed and at higher W_o values (where W_o = molar ratio of water to total surfactants of the micellar system) there was no sharp fall of the enzyme activity such as a typical bell‐shaped profile. The kinetic model for the lipase‐catalysed hydrolysis of olive oil in AOT/isooctane reverse micellar system was applied to the enzymatic reaction in this mixed reverse micellar system. It was found that the predictions of the model agree well with the experimental kinetic results and that the adsorption equilibrium constant of olive oil molecules between the micellar phase and the bulk phase of the organic solvent is smaller in AOT/Tween 85 mixed reverse micellar systems than in simple AOT reverse micellar systems. © 1999 Society of Chemical Industry     

A kinetic model for enzymatic reactions in a reverse micellar system,involving water-insoluble substrate

A kinetic model was proposed for enzymatic reactions in a reverse micellar system, involving a water-insoluble substrate. Though surfactant is one of the main structural components of reverse micelles, an increase in the surfactant concentrations affects the enzyme activity remarkably. A relationship between the enzyme activity and the surfactant concentration is discussed. In this study it was assumed that free substrate in the organic phase was in adsorption equilibrium with the surface of the micellar surfactant, and that the adsorption coefficient and the true K_m value (Michaelis constant) were independent of the surfactant concentration. The validity of this model was verified by data on the hydrolysis of olive oil, catalyzed by Chromobacterium viscosum lipase (Glycerol-ester hydrolase; EC 3.1.1.3) in an AOT/isooctane reverse micellar system. The activity value predicted by the model equation agreed well with the experimental data.     

Increased activity and stability of Candida rugosa lipase in reverse micelles formed by chemically modified AOT in isooctane

Sodium bis (2‐ethylhexyl polyoxyethylene) sulfosuccinates, which can be structurally viewed as chemically modified AOT (MAOT), were prepared and successfully used to form reverse micelles in isooctane. The activity and stability of Candida rugosa lipase (glycerol‐ester hydrolase, EC 3.1.1.3) appreciably increased in the MAOT/isooctane reverse micellar system compared with the AOT/isooctane system taking the hydrolysis of olive oil as a model reaction. The best polymerization number of oxyethylene group was found to be 2.0, which corresponded to a maximum enzyme activity of twice that in the AOT/isooctane system. The half‐life of lipase in the MAOT micellar system was twice that in the AOT micellar system. The optimal operational conditions remained unchanged. The Michaelis kinetics showed that the maximum reaction rate obviously increased in the MAOT micellar system compared with that in the AOT micellar system, while the change in the Michaelis constant was insignificant. © 2001 Society of Chemical Industry     

Pretreatment of Chromobacterium viscosum lipase with acetone increases its activity in sodium bis‐(2‐ethylhexyl) sulfosuccinate (AOT) reverse micelles

The activity of Chromobacterium viscosum lipase for hydrolysis of olive oil in sodium bis‐(2‐ethylhexyl) sulfosuccinate (AOT) reverse micelles was increased by pretreatment with acetone. In contrast to the untreated lipase, no sharp fall in the activity of the treated lipase at higher W₀ (water to AOT molar ratio) values was observed. The fluorescence emission intensity of the treated lipase in reverse micelles was higher than that of the untreated lipase but the maximal emission wavelength (λ_max) was the same for both lipases. A kinetic model that considers the free substrate in equilibrium with the substrate adsorbed on the micellar surface was successfully used to better understand the activity enhancement. The Michaelis constant (K_m) and substrate adsorption equilibrium constant (K_ad) were reduced by lipase pretreatment with acetone whereas the maximum reaction rate (v_max) remained unaltered. Copyright © 2005 Society of Chemical Industry     

AOT逆胶束体系脂肪酶催化合成油酸乙酯

在二-(2-乙基己基)琥珀酸酯磺酸钠和异辛烷构建的逆胶束体系中,以Lipex脂肪酶为催化剂,合成了油酸乙酯,考察了各影响因素对其产率的影响,并进行了脂肪酶紫外荧光检测和体系粒度分析. 结果表明,Lipex脂肪酶具有良好的催化活性,反应条件优化选用异辛烷为溶剂,在反应温度25℃、缓冲液pH 6.5、水/表面活性剂(摩尔比)10、乙醇/油酸(摩尔比)20及Lipex脂肪酶浓度0.035 g/L、油酸浓度0.005 mol/L、摇床转速150 r/min、反应36 h的条件下,油酸乙酯产率达到71.25%. 逆胶束粒度和酶构象直接影响酶活性,最适逆胶束粒度约为80 nm.     

Extraction of Candida rugosa lipase from aqueous solutions into organic solvents by forming an ion‐paired complex with AOT

Candida rugosa lipase was extracted from aqueous solutions into organic solvents by forming an ion‐paired complex with sodium bis(2‐ethylhexyl)sulfosuccinate (AOT). The optimal aqueous pH for lipase recovery was 4.5 and the optimal CaCl₂ concentration was 10 mmol dm^?3. The lipase recovery decreased with increasing aqueous enzyme concentration but increased with increasing AOT concentration in the organic phase. The presence of polar co‐solvents in the aqueous phase did not obviously improve the lipase recovery, which was also little influenced by the type of hydrophobic organic solvent used for solubilising AOT. Surprisingly, no detectable activity of the ion‐paired C. rugosa lipase was observed for both the esterification of lauric acid with 1‐propanol in isooctane and the hydrolysis of olive oil in isooctane containing an appropriate amount of water. The ion‐paired C. rugosa lipase mediated the enantioselective crystallisation of racemic ketoprofen in isooctane, indicating the feasibility of using it as a chiral mediator for the enantioseparation of hydrophobic racemic compounds in organic systems. Copyright © 2006 Society of Chemical Industry     

A colorimetric determination of fatty acids as a new assay of lipases in reverse micelles

A new simple spectrophotometric method is described for the determination of the free fatty acid content in triglyceride oils or other lipophilic samples. The method utilizes phenol red as fatty acid indicator, which is solubilized phenol red as fatty acid indicator, which is solubilized in reverse micelles formed by AOT [sodiumbis(2-ethylhexyl) sulfosuccinate] in isooctane. Fatty acid determinations in vegetable oils can be carried out rapidly with oil samples of less than 100 mg. The acid value of three different oils tested agreed quite well with the acid value obtained for the same samples with another colorimetric determination using cupric acetate. The method can be extended to a continuous determination of fatty acids which are released during the initial stage of a lipase catalyzed hydrolysis of triglyceride substrates in reverse micelles. This new sensitive lipase assay has been applied for a lipase ofPseudomonas bacteria with lipase concentrations as low as 0.1 μg/ml. Using trioctanoylglycerol as substrate in 50 mM AOT/isooctane with w_o=[H₂O]/[AOT]=11.1 (pH 9.0), the apparent overall Michaelis-Menten constant (K_m′app,ov) is 27 mM and the turnover number (k_cat) 44 sec⁻¹.     

反胶团酶催化研究新进展

评述了近年来反胶团酶催化研究的新进展。在AOT/异辛烷反胶团中加入非离子型表面活性剂如Tween 85、小相对分子质量聚乙二醇等可有效降低酶与表面活性剂间的静电和疏水作用 ,显著提高酶的活性。对AOT进行化学修饰及合成结构与磷脂类似的新表面活性剂以用其构建新的反胶团体系 ,酶的活性较常用的AOT/异辛烷反胶团体系有显著提高。在反胶团酶反应动力学研究中考虑水含量或底物在反胶团表面吸附的影响等 ,提出了进一步研究的设想 ,包括开发新型表面活性剂以进一步提高酶的活性和稳定性及有利于产物分离     

Activity of horseradish peroxidase in aqueous and reverse micelles and back‐extraction from reverse‐micellar phases

Studies on the activity of the enzyme horseradish peroxidase (HRP) have been carried out in micellar as well as reverse‐micellar media. The activity of the enzyme was studied in the presence of different classes of surfactants – ionic as well as non‐ionic. In aqueous media, the activity of the enzyme varied depending on whether the concentration of the surfactant used was above or below the critical micellar concentration (CMC). The enzyme was also studied in reverse‐micellar systems. HRP was introduced into the reverse micellar phase by the injection method and its activity within the reverse micelles was determined. The effect of water to surfactant ratio (W_o) on activity within reverse micelles was studied, and an almost two‐fold increase in activity was seen when the enzyme was encapsulated within reverse micelles of aqueous phase fractional hold‐up (?) of 0.0072 (v/v) consisting of sodium bis‐(2‐ethylhexyl) sulfosuccinate (AOT) in isooctane at a W_o of 20. The activity of HRP was measured over a wide range of AOT concentrations having different W_o values. Back‐extraction of HRP from these reverse micelles was carried out at varying ionic strengths of phosphate buffer. Back extraction was found to be highest at pH 7.0 in 40 mol m^?3 phosphate buffer and 100 mol m^?3 sodium chloride. © 2001 Society of Chemical Industry     

Kinetic behavior of yeast alcohol dehydrogenase in AOT/isooctane reverse micelles

The kinetic behavior of yeast alcohol dehydrogenase (YADH) in sodium di-2-ethylhexylsulfosuccinate (AOT) isooctane reverse micelles has been studied using methyl ethyl ketone (MEK), NADH and Tris as the substrate, coenzyme and buffer, respectively. The solubility diagrams of aqueous buffer in the isooctane solution of AOT were established as a function of temperature and molar ratio of water of surfactant (ω₀) at various Tris and AOT concentrations. The dependence of enzyme activity on enzyme concentration, pH, ω₀ and Tris concentration was determined. The optimal ω₀ was at 10–15, increasing slightly with an increase in Tris concentration. The YADH entrapped in reverse micelles exhibited minimum activity at a Tris concentration of 0·1 mol dm^?3, while in aqueous buffer enzyme activity was not significantly affected by Tris concentration. Comparing the rate equation of the reduction of MEK by YADH in reverse micelles with that in aqueous buffer, the association of YADH and NADH could apparently have proceeded with an irreversible reaction before the substrate was bound, when performed in a reverse micellar system. Although the YADH entrapped in reverse micelles was less stable than when dissolved in aqueous buffer, the enzyme retained at least 20% activity after 21 h at 25°C and ω₀ = 20. This result was an improvement over previously reported data.     

Kinetics and stability of a chromobacterium viscosum lipase in reversed micellar and aqueous media

The lipolytic activity of Chromobacterium viscosum lipase B (EC 3.1.1.3.; triacylglycerol hydrolase) solubilized both in water and AOT/isooctane reversed micelles has been investigated using triolein as a substrate. The influence of relevant parameters in the catalytic activity such as temperature, pH, surfactant and substrate concentrations, and water content was tested and compared in both media. A study of stability of the lipase was carried out, with particular reference to the influence of pH. Three major effects of the encapsulation of the lipase in the micelles were observed: increased activity (up to 7 times higher than in water), greater stability, specially at pH 7, and higher resistance to thermal deactivation.     

SEPES/异辛烷反胶团中脂肪酶的特性

<正>反胶团酶反应兼有水相与有机相酶反应的共同优点,但至今尚无工业应用的重要原因是反胶团中酶的活性和稳定性不高.常用的离子型表面活性剂,如二-(2-乙基己基)琥珀酸双酯磺酸钠(AOT)在有机溶剂中形成的反胶团,因相界面与酶分子间的静电作用将造成酶在反胶团中迅速失活.而非离子型表面活性剂形成的反胶团中,酶一般具有较高的活性和稳定性,但这种体系需加入助表面活性剂,给下游加工过程带来麻烦.作者制备了具有不同氧乙烯聚合数(n)的二-(2-乙基己基聚氧乙烯)琥珀酸双酯磺酸钠(简称SEPES),其分子结构与AOT相似,但其极性头与疏水尾间连有聚氧乙烯链非离子性亲水基团.本文报道脂肪酶在SEPES/异辛烷反胶团体系中的特性.     

Enzymatic glycerolysis of a triglyceride in aqueous and nonaqueous microemulsions

Enzymatic solvolysis of a model triglyceride, palm oil, was performed in microemulsions containing isooctane, sodiumbis(2-ethylhexyl)sulfosuccinate (AOT), palm oil and a combination of water and glycerol as the polar component. Using a 1,3-specific lipase both hydrolysis, leading to the formation of fatty acid and one mole of monoglyceride, and glycerolysis, giving three moles of monoglyceride, occur. The reaction was very slow in a completely nonaqueous system. Addition of a small amount of water led to an increased rate of glycerolysis, in addition to hydrolysis. It was found that by using³H labelled material reaction products originating from the two reactions were formed in equimolar amounts. The products probably emanate from a common intermediate. The molar ratio, R, of water and glycerol to surfactant turned out to be critical, optimum R-value being 3.4. Four different lipases, one from porcine pancreas and three of fungal origin, were tested. No marked differences in ratio of monoglyceride to fatty acid formed were obtained, indicating that the ratio between glycerolysis and hydrolysis is constant regardless of the lipase used.     

Acquisition of Water Solubility Diagrams in Ternary Systems (AOT/Organic Solvent/Alcohol) and Extraction of α-Lactalbumin Using Reverse Micellar Systems

In this study water solubility curves were constructed and calorimetric measurements obtained for reverse micellar systems consisting of an alcohol (isopropanol or butanol), surfactant (AOT) and organic solvent (isooctane or hexane). Also evaluated were the effects of alcohol and solvent type and surfactant concentration on the extraction of the α-lactalbumin (α–la). From the obtained solubility diagrams for ternary systems, it was concluded that isooctane presented the highest water solubility capacity in the center of the micelle systems with hexane, since isooctane has greater molecular volume and greater effect of the surfactant aggregation number. With respect to the alcohols, it was observed that isopropanol and butanol act in the system as a co-surfactant, since they prefer to adsorb at the water/solvent interface. It was also verified that butanol improved water solubility inside the reverse micellar due to its contribution to increase the critical packing parameter. The amount of α-la extracted increased proportionally with the AOT concentration for systems with isooctane and hexane. However, for systems with the latter solvent, the concentration of extracted protein first increases and then decreases. The extraction power of reverse micellar systems with isooctane was influenced by the type of alcohol with butanol showing better results. For systems containing hexane there was no effect of the alcohol added to the system on extraction power of α-lactalbumin.     

Invert emulsion as a medium for fungal lipase activity

An extracellular lipase from the fungusPythium ultimum was active in an invert [water-in-oil] emulsion consisting of 4% water emulsified into edible oils with taurocholic acid as the surfactant. The pH range for optimum lipolytic activity was 7.5–8.5, and the optimum temperature for activity was 45°C. Specific activity of the purified lipase was 919.5 μmol/min/mg protein in the invert emulsion. Water content of the invert emulsion influenced activity of the lipase differently, depending on the substrate. The rate of olive oil hydrolysis with thePythium lipase decreased with time, possibly due to inactivation of the enzyme and inhibition by free fatty acid products of the reaction. Total hydrolysis of olive oil by thePythium lipase was compared with that by lipases fromCandida rugosa andRhizopus arrhizus in the invert emulsion. Hydrolysis essentially ceased within 24 h or less for the lipases from each source. However, the addition of aqueous solution at 8 h from the beginning of incubation stimulated hydrolysis byC. rugosa andR. arrhizus lipases by 1.8-fold and 2.5-fold, respectively, but not by theP. ultimum lipase, over corresponding controls after 48 h.     

反胶团萃取蛋白质特性的研究

实验将一种阴离子表面活性剂（AOT）和一种阳离子表面活性剂（Aliquat336）分别溶于异辛烷（isooctane）中，构成了两种不同的反胶团体系．通过用两种不同的反胶团溶液萃取六种蛋白质的实验，研究了水相pH值及离子强度对反胶团体系中水含量Wo和蛋白质萃取率影响的规律．实验发现不论哪种反胶团体系，水相pH值对Wo的影响都不大，但pH值却对蛋白质萃取率有很大的影响，对AOT反胶团体系而言，随着pH值的降低，蛋白质的萃取率升高；对Aliquat336反胶团体系而言，随着水相pH值的降低，蛋白质的萃取率下降．随着离子强度的增大，AOT反胶团体系的水含量大幅度地降低；而对Aliquat336体系水含量的影响很小，但水相离子强度对蛋白质萃取率的影响是相同的，随着离子强度的增大，两种体系对蛋白质的萃取率均下降。     

Hydrolysis of edible oils by lipases immobilized on hydrophobic supports: Effects of internal support structure

The hydrolysis of edible oil by immobilized lipases on novel support materials was investigated. Six hydrophobic polymers were studied with the following techniques: (i) determination of the surface area of each support by BET (Brunauer-Emmett-Teller) analysis of nitrogen adsorption isotherms; (ii) electron photomicrography; and (iii) measuring lipase activity by hydrolysis of olive oil with lipase fromCandida cylindracea immobilized on each support. A detailed structural analysis on one support also was carried out by mercury porosimetry. The composition and porosity of a support are more important than the surface area in determining activity for immobilized lipases. Furthermore, having selected the “most efficient” support, five lipases fromC. cylindracea, Rhizomucor miehei, andPseudomonas cepacia, were immobilized, and their hydrolytic activities were determined at several temperatures and pH values with olive oil and beef tallow as substrates in solvent-free systems. For each parameter, twelve successive 2.5-h hydrolysis reactions were conducted on a laboratory-scale under batch conditions. Lipase AY fromC. cylindracea had the highest hydrolytic activity, in the range of 30–50°C at pH 5.5 with olive oil as the substrate. For beef tallow, lipase PS, fromP. cepacia, displayed the highest activity at 50°C and pH 7.     

Concentrated phases of an ionic surfactant (Aerosol OT) in polar solvents

We have studied concentrated phases of Aerosol OT (AOT) in polar (water, ethylene glycol, formamide, N,N-dimethylformamide) and apolar (isooctane) solvents. We investigated two surfactant volume fractions (φ = 0.2 and φ = 0.6) with small angle x-ray scattering (SAXS), rheology and electrical conductivity experiments. AOT self-assembles differently depending on solvent type and concentration. SAXS experiments show that the AOT/water system displays a lamellar phase. In the other cases, only formamide displays a lamellar phase for φ = 0.6. The other solvents (and formamide at φ = 0.2) promote the self-assembly of AOT in other microstructures. The SAXS spectra display correlation peaks consistent with a disordered array of cylindrical aggregates. The microstructure of the AOT/isooctane system at φ = 0.2 is that of an arrangement of spherical aggregates. The results are explained in terms of surfactant packing models and solvent properties. For instance, the ability of ethylene glycol to form hydrogen bonds with AOT promotes the formation of cylindrical aggregates by increasing the area per surfactant polar head. As N,N-dimethylformamide is slightly miscible in hydrocarbons it increases the volume of the surfactant tail promoting reverse structures.     

Fabrication and Characterization of Gradient Refractive Index Plastic Rods Containing Inorganic Nanoparticles

A novel method was developed for the preparation of GRIN plastic rods containing inorganic nanoparticles. Silver nanoparticles were prepared by using W/O (water in oil) reversed micelle technique in the presence of bis(2-ethylhexyl) sulpho-succinate (AOT) surfactant. The effects of w values (w=[H₂O]/[AOT]), silver nitrate concentration, AOT/isooctane/H₂O ratio, and initiator concentration on the nanoparticle size were investigated. Optical absorption spectra of the micellar samples were recorded on a spectrophotometer at room temperature in the range of 200–900 nm. The nanoparticle size was confirmed by TEM technique. To introduce the nanoparticles into the GRIN plastic rods, methyl methacrylate (MMA) was used instead of isooctane in the organic phase. Nanoparticles were found to increase the refractive index of plastic rods effectively. However, the existence of surfactant may cause the aggregation of nanoparticles leading to the occurrence of light scattering and becoming opaque. We estimated both real image transmission and the three dimensional refractive index distributions of the GRIN plastic rods prepared in this investigation. The results in this investigation suggest that nanoparticles could be used as a dopant to fabricate GRIN plastic rods and increase its refractive index effectively.