Bounded water kinetic model of β-galactosidase in reverse micelles

In the present investigation, -galactosidase was solubilized into Aerosol OT (AOT)/isooctane reverse micelles. Kinetic data for the hydrolysis of o-nitrophenyl--D-galactopyranoside (ONPG) at different pH values and molar ratios of water to AOT (W_o) were collected. It was observed that the usual kinetic model used for -galactosidase catalysis in aqueous systems failed to represent the experimental data. A bounded water model, however, showed a better correlation between enzymatic activity and W_o. In contrast to the aqueous system, controlling the water concentration in the reverse micelles allows the rate constants for the reaction between water molecules and glycosyl-enzyme complexes to be evaluated.     

Kinetics of lipase-catalyzed hydrolysis of palm oil in lecithin/izooctane reversed micelles

Candida rugosa lipase has been used to investigate the hydrolysis of palm oil in a lecithin/isooctane reversed micellar system. The reaction obeys Michaelis-Menten kinetics for the initial conditions. Kinetic parameters such as maximum rate and Michaelis constant (K _m) were determined for lipase-catalyzed hydrolysis in n-hexane and isooctane. According to the K _m values, the enzyme affinity towards the substrate was increased in isooctane. The maximum degree of hydrolysis was generally decreased as the initial substrate concentration was increased. This may suggest that the hydrolysis in lecithin reversed micelles should be regarded as a one-substrate first-order reversible reaction. It is shown in this study that the proposed one-substrate first-order kinetic model can serve for the precise prediction of the degree of hydrolysis for a known reaction time or vice versa, when the initial substrate concentration is less than 0.325 mol/dm³. A disagreement with this model was found when the initial substrate concentration was higher than approximately 0.3 mol/dm³. This may be due to the effects of the products on lipase activity or even to the conversion of the reversed micellar system to other systems. Received: 16 May 1997 / Received revision: 22 October 1997 / Accepted: 24 October 1997     

Lipase-catalyzed hydrolysis of olive oil in chemically-modified AOT/isooctane reverse micelles in a hollow fiber membrane reactor

The hydrolysis of olive oil catalyzed by Candida rugosa lipase in sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/isooctane and the synthetic sodium bis(2-ethylhexyl polyoxyethylene)sulfosuccinate (MAOT)/isooctane reverse micellar systems was investigated in a polysulfone hollow fiber membrane reactor with recycle of the reaction mixture. Lipase was completely retained by the membrane while olive oil and oleic acid freely passed through. The retention of reverse micelles depended on W ₀ (molar ratio of water to surfactant). At an olive oil concentration of 0.23 mol l^–1 the final substrate conversion in the MAOT micellar system was about 1.4 times of that in the AOT micellar system.     

Reactivity of trypsin in reverse micelles: pH-effects on the W0 versus enzyme activity profiles

pH-Dependence of hydrolytic activity of trypsin has been studied in cationic reverse micellar system of cetyltrimethylammonium bromide (CTAB) in (50% v/v) chloroform/isooctane using a positively charged substrate N_α-benzoyl-L-arginine ethyl ester (BAEE). The pH of the medium was varied from 4.0 to 8.5 with addition of 0.025 M citrate-phosphate buffer containing 1 mM CaCl₂. Optimum pH for maximum enzyme activity, pH_opt in reverse micelles is found to be similar to that observed in bulk aqueous solution (8.0–8.5). However, changes in activity of trypsin (k_cat) as a function of water content W₀ (W₀ = [H₂O]/[CTAB]) in reverse micelles are found to be pH dependent. At low pH (4.0) and low water content (W₀ = 5) the enzyme is more active in reverse micelles than in bulk aqueous solution by a factor of 2. This ‘superactivity’ is lost at higher W₀ values and the k_cat in reverse micelles is found to be similar to that observed in aqueous bulk. At pH 5, the enzyme activity is found to be independent of W₀ while at pH 6.0–6.5 the enzyme activity is low at W₀ 5 and increases with water content to a constant value which is still 50% lower than that in aqueous buffer. Above pH 7, the W_o-activity profile becomes distinctly bell shaped with W₀ optimum around 10–15. The enzyme activity at optimum W₀ is close to that observed in aqueous bulk.     

油包水微乳液中抗体酶催化布洛芬酯选择性水解的酶学特性

根据过渡态理论设计和合成了能诱导产生催化选择性水解布洛芬甲酯的催化抗体的四面体硫酸盐半抗原,并与牛血清白蛋白(BSA)偶联制备成免疫源,通过免疫手段成功筛选出具有加速选择性水解生成S-布洛芬的特异性催化抗体.其Kcat,app/Kuncat,app达1.6x104.进一步地将催化抗体运用到W/O微乳体系(反胶束)中进行布洛芬酯的选择性水解研究,其动力学研究证明其催化过程同样遵循Michaelis.Menten方程.考察了pH值和温度对催化初速度影响,Wo(体系中水和琥珀酸二辛酯磺酸钠(AOT)的摩尔比)对催化初速度影响呈现为钟罩型,最适的Wo.为21.     

Stability of a Fusarium solani pisi recombinant cutinase in phosphatidylcholine reversed micelles

Summary A Fusarium solani pisi recombinant cutinase solubilized in phosphatidylcholine/isooctane reversed micelles was used to catalyse the esterification reaction of butyric acid with 2-butanol at pH 10.7. The influence of temperature, Wo and substrates on lipase stability was evaluated. The enzyme displays a better stability, with a half-life over 125 days, at a temperature of 22°C and for a low water content (W_O= 6.5). Butyric acid increased the cutinase deactivation (t_1/2=0.56h), while 2-butanol led to a similar half-life (t_1/2=14h) as without substrate.     

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.     

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.     

Catalytic activity of lignin peroxidase and partition of veratryl alcohol in AOT/isooctane/toluene/water reverse micelles

The activity of lignin peroxidase (LiP) and the partition of its optimum substrate veratryl alcohol (VA) in sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/isooctane/toluene/water reverse micelles were studied in this paper to understand the microheterogeneous effect of the medium on the catalytic properties of LiP hosted in the reverse micelle. Results showed that LiP from Phanerochaete chrysosporium could express its activity in the reverse micelles, but its activity depended, to a great extent, on the composition of the reverse micelles. Optimum activity occurred at a molar ratio of water to AOT (ω₀) of 11, a pH value of 3.6, and a volume ratio of isooctane to toluene of 7–9. Under optimum conditions, the half-life of LiP was circa 12 h. The dependence of LiP activity on the volume fraction of water in the medium (θ), at a constant ω₀ value of 11, indicated that VA was mainly solubilized in the pseudophase of the reverse micelle. Based on the pseudobiphasic model and the corresponding kinetic method, a linear line can be obtained in a plot of apparent Michaelis constant of VA vs θ, and the partition coefficient of VA between the pseudophase and the organic solvent phase was determined to be 35.8, which was higher than that (22.3) between bulk water and the corresponding mixed organic solvent. H₂O₂ inhibited LiP at concentrations higher than 80 μM; this concentration value seems to be different from that in aqueous solution (about 3 mM). The differences mentioned above should be ascribed to the microheterogeneity and the interface of the AOT reverse micelle.     

Characteristics of lipase-catalyzed hydrolysis of olive oil in AOT-isooctane reversed micelles

Candida rugosa lipase solubilized in organic solvents in the presence of both surfactant and water could catalyze the hydrolysis of triglycerides, and kinetic analysis of the lipase-catalyzed reaction was found to be possible in this system. Among eight organic solvents tested, isooctane was most effective for the hydrolysis of olive oil in reversed micelles. Temperature effect, pH profile, K(m,app) and V(max,app) were determined. Among various chemical compounds, Cu(2+), Hg(2+), and Fe(3+) inhibited lipase severely. But the enzyme activity was restorable partially by adding histidine or glycine to the system containing these metal ions. The enzyme activity was dependent on R (molar ratio of water to surfactant) and maximum activity was obtained at R = 10.5. Upon addition of glycerol to the reversed micelles, lipase activity was affected in a different fashion depending on the R values. Stability of the lipase in reversed micelles was also dependent on R, and it was most stable at R = 5.5.     

The behavior of proteases in lecithin reverse micelles

Reverse micelles, formed in isooctane/alcohol by phosphatidylcholines of variable chain length (i.e. 6, 7 or 8 C atoms in the fatty acid moiety) have been studied, mostly in relation to their capability of solubilizing trypsin and alpha-chymotrypsin. It has been found that the capability of the lecithin reverse micellar systems to solubilize water is strongly affected by the chain length of the alkyl group and by the alcohol used as co-surfactant. The C8-lecithin system, i.e. 1,2-dioctanoyl-sn-glycero-3-phosphocholine, in isooctane/hexanol is the system which affords the maximal solubilization of water (up to wo 60, where wo = [H2O]/[lecithin]) and of the enzymes. The water of the water pool of lecithin reverse micelles has been investigated by 1H-NMR; the proton chemical shift as a function of wo was found to be similar to the case of reverse micelles formed by the well known negatively charged surfactant sodium bis(2-ethylhexyl sulfosuccinate). 31P-NMR studies show that the ionization behavior of phosphate groups is similar to that in bulk water, suggesting no anomaly in the pH behavior of this water pool. The stability of trypsin and alpha-chymotrypsin in the various lecithin reverse micellar system is similar and occasionally better than that in aqueous solution. The same holds for the kinetic behavior (kcat and Km have been determined for a few systems). The bell-shaped curve of the pH/activity profile in lecithin reverse micelles is, for both enzymes, shifted towards more alkaline values with respect to water. Bell-shaped curves are also obtained when studying the influence of wo on the enzyme activity, with an optimal wo which is in the range 7-10, a surprisingly small value considering that we are dealing with hydrolases. Circular dichroic studies have been carried out in order to correlate the activity with the protein conformation: for both enzymes, generally no marked perturbations appear as a consequence of the solubilization in the lecithin reverse micelles, but conditions can be found under which significant alterations are present. Certain properties of the two enzymes, which in water solution are very similar, become sharply different in reverse micelles, showing that occasionally the micellization is able to enhance the relatively small structural differences between the two proteins.     

A metal‐chelate affinity reverse micellar system for protein extraction

A new nonionic reverse micellar system is developed by blending two nonionic surfactants, Triton X‐45 and Span 80. At total surfactant concentrations lower than 60 mmol/L and molar fractions of Triton X‐45 less than 0.6, thermodynamically stable reverse micelles of water content (W₀) up to 30 are formed. Di(2‐ethylhexyl) phosphoric acid (HDEHP; 1–2 mmol/L) is introduced into the system for chelating transition metal ions that have binding affinity for histidine‐rich proteins. HDEHP exists in a dimeric form in organic solvents and a dimer associated with one transition metal ion, including copper, zinc, and nickel. The copper‐chelate reverse micelles (Cu‐RM) are characterized for their W₀, hydrodynamic radius (R_h), and aggregation number (N_ag). Similar with reverse micelles of bis‐2‐ethylhexyl sodium sulfosuccinate (AOT), R_h of the Cu‐RM is also linearly related to W₀. However, N_ag is determined to be 30–90 at W₀ of 5–30, only quarter to half of the AOT reverse micelles. Then, selective metal‐chelate extraction of histidine‐rich protein (myoglobin) by the Cu‐RM is successfully performed with pure and mixed protein systems (myoglobin and lysozyme). The solubilized protein can be recovered by stripping with imidazole or ethylinediaminetetraacetic acid (EDTA) solution. Because various transition metal ions can be chelated to the reverse micelles, it is convinced that the system would be useful for application in protein purification as well as simultaneous isolation and refolding of recombinant histidine‐tagged proteins expressed as inclusion bodies. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Chirality of reverse micelles

Four chiral analogues of the surfactant Aerosol-OT (AOT) have been synthesized and characterized. All of them form reverse micelles in apolar solvents in the w₀ range 0–30 (w₀ = [water]/[tenside]). Reverse micellar solutions have been investigated by UV absorption and circular dichroism spectroscopies with the aim of clarifying whether the formation of the macromolecular micellar structure induces the appearance of new chromophoric bands or perturbs the existing ones. Methanolic solutions of the surfactants, in which no micellar aggregates are formed, were taken as references. One of the products 1(S),1′(S)-dimethylbisheptylsulphosuccinate sodium salt (MH-AOT) was capable of forming reverse micelles of relatively high water content (w₀ up to 40) and this process was accompanied by a specific increase in the intensity of the circular dichroism band associated with the ester absorbance of the molecule. As no concomitant changes were seen in the UV absorbance spectrum, it was concluded that this observation reflected conformational events occurring within the surfactant rather than chromophoric perturbation. These results are qualitatively similar to those found recently for lecithin reverse micelles which, however, form gels at sufficiently high water contents. The chiroptical properties of these supramolecular aggregates are compared with those of covalent macromolecular systems such as polypeptides.     

Continuous lipolysis in a reversed micellar membrane bioreactor

The enzymatic hydrolysis of olive oil using Chromobacterium viscosum lipase B encapsulated in reversed micelles of AOT in isooctane was carried out in a continous reversed micellar membrane bioreactor. A tubular ceramic membrane installed in an ultrafiltration module was used to retain the lipase and separate the products from the reaction media. Water filled micelles were supplemented to the reactor together with the substrate/solvent solution to compensate for the permeation of reversed micelles. The influence of substrate concentration, residence time and water content in the productivity and conversion of the system were investigated. A linear relationship between productivity and conversion degree was found for each substrate concentration tested. Operational stability of the bioreactor was tested in a long term operation confirming the high stability of this catalytic system.List of Symbols a(S ₀ ), b(S ₀ ) parameters of Eq. (3) - [AOT] mM AOT concentration - C _c mM concentration in the concentrate - C _p mM concentration in the permeate - E _t mg total amount of lipase - [lipase] mg/cm³ overall lipase concentration - [OIL] mM olive oil concentration - [OLEIC] mM oleic acid concentration in the permeate - P mol/(min · mg) oleic acid productivity - Q _in cm³/min inlet flow rate - Q _out cm³/min outlet flow rate (equal to permeate flow rate) - Q _r cm³/min recirculation flow rate - W _o ratio of water to AOT molar concentrations - X steady state conversion degree in the permeate stream - T °C temperature - rejection coefficient     

Activity and stability of lipase in Aerosol-OT/isooctane reverse micelles

The stability of Candida rugosa lipase, which catalyzes the hydrolysis reaction of olive oil in AOT/isooctane reverse micelles, decreased with the increase of ₀ (defined as the molar ratio of water to surfactant) and Aerosol-OT concentration. The addition of a non-ionic cosurfactant, tetraethylene glycol dodecyl ether (C₁₂E₄), preserved enzymatic activity. The residual activity of the lipase was 53% after 24 h, while the enzyme completely lost its activity within 6 h in the absence of C₁₂E₄ addition. The stabilizing effect of C₁₂E₄ resulted in the increase of conversion. The enhancement of the activity and stability of lipase in reverse micelles by the addition of C₁₂E₄ may contribute to increase the rigidity of the micellar matrix stabilizing the enzyme structure.     

Kinetic behaviour of alpha-chymotrypsin in reverse micelles. A stopped-flow study.

At the aim of investigating whether the early rapid phase of enzyme turnover is different in reverse micelles compared with bulk water, the kinetic properties of alpha-chymotrypsin have been studied in reverse micelles formed by sodium bis(2-ethylhexyl)sulfosuccinate in isooctane. Pre-steady state and steady-state kinetic constants, in water and in reverse micelles, have been determined by stopped-flow spectrophotometry for the hydrolysis of two substrates, namely acetyl-L-tryptophan-p-nitrophenyl ester and p-nitrophenyl acetate. It has been shown that, for both substrates, the acylation rate constant (k2) is very much lower in reverse micelles than in water. However, the deacylation rate constant (k3) and the turnover number (kcat) are not significantly changed in reverse micelles with respect to bulk water. Therefore, despite considerable rate changes in the acylation step, deacylation is rate limiting both in water as well as in reverse micelles, under the experimental conditions used.     

Enhanced enzymatic activity in reverse micelles

Summary The bell shaped dependence of the superactivity of enzymes solubilized in ionic reverse micelles (RMs) on the hydration ratio (W₀) is theoretically explained. The superactivity is due to enhanced concentration of the substrate (which has the same kind of charge as that of the surfactant head groups) near the enzyme surface. The opposing effects of the increase in the absolute charge of the surface of the RM and in the water pool width with W₀ cause a maximum in activity.     

A comparative study of lysozyme conformation in various reverse micellar systems

The activity and conformation of lysozyme solubilized in apolar solvents via reverse micelles was investigated. The systems used were sodium di-2-ethylhexylsulfosuccinate (AOT)/isooctane/H₂O, cetyltrioctylammoniumbromide (CTAB)/CHCl₃, isooctane/H₂O; tetraethyleneglycoldodecylether (EO₄C₁₂)/isooctane/H₂O, and bulk water. CD spectra of lysozyme in reverse micellar solutions were investigated as a function of w₀ (= [H₂O]/[AOT]) and were compared to the spectra in aqueous solutions. No marked changes were found in the EO₄C₁₂ or in the CTAB systems with respect to water, which indicates that no sizeable conformational changes of the enzyme occurred upon solubilization in the reverse micellar systems. In agreement with previous studies [C. Grandi, R. E. Smith, and P. L. Luisi (1981) J. Biol. Chem. 256 , 837–843] dramatic conformational changes can be inferred in the AOT system on the basis of CD studies. This is taken as an indication that the enzyme denatures in this micellar system. This is particularly striking because the enzyme is fully active in AOT reverse micelles. The apparent paradox is solved by the observation that the native CD spectrum (and by inference, the native conformation) is maintained when lysozyme is bound to NAG or NAG₃, and by inference, when the substrate is bound, e.g., during enzyme turnover. However, in the absence of added NAG, NAG₃, or substrate, the enzyme in the AOT reverse micellar system rapidly denatures. Together with CD studies, fluorescence and nmr data confirm the hypothesis of an irreversible denaturation of lysozyme in the AOT system, the denaturation being slowed down when the substrate is present. The activity of the enzyme has been studied as a function of pH and w₀ using the chromophoric substrate 3,4-dinitrophenyl-tetra-N-acetyl-β-D -chitotetraoside (3,4-DNP-NAG₄). Generally speaking, the kinetic parameters are comparable to those found in bulk water solution. More detailed, in the CTAB system, k_cat tends to be smaller than in aqueous solution (with quite similar K_M), whereas in the EO₄C₁₂ system (at pH 7.0) the turnover number is larger and K_M is smaller than in water. In the AOT system, the kinetic parameters at pH 7.0 are also quite comparable to those found in water.     

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.