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.     

Structure and activity of trypsin in reverse micelles

The kinetic properties of trypsin have been studied in reverse micelles formed by two surfactant systems, namely bis(2-ethylhexyl) sodium sulfosuccinate (AOT) in isooctane, and hexadecyltrimethyl ammonium bromide (CTAB) in chloroform/isooctane (1:1, by vol.). Three substrates have been used, namely N alpha-benzoyl-L-Arg ethyl ester, N alpha-benzoyl-L-Phe-L-Val-L-Arg p-nitroanilide (BzPheValArg-NH-Np) in AOT and N alpha-benzyloxycarbonyl-L-Lys p-nitrophenyl ester (ZLysO-Np) in CTAB. One of the main aims of the work was to compare the behaviour of trypsin in reverse micelles with that of alpha-chymotrypsin, for which an enhancement of kcat had been observed with respect to aqueous solutions. The pH profile is not significantly altered in reverse micelles with respect to water, however the kinetic parameters (kcat and Km) differ widely from one another, and are markedly affected by the micellar conditions, in particular by the water content wo (wo = [H2O]/[AOT]). Whereas in the case of BzPheValArg-NH-Np kcat is much smaller than in water, in the case of ZLysO-Np at pH 3.2 (but not at pH 6.0) a slight enhancement with respect to water is observed. On the basis of rapid kinetic spectrophotometry (stopped-flow) and solvent isotope effect studies, this enhancement is ascribed to a change in the rate-limiting step (acylation rather than hydrolysis). As in the case of alpha-chymotrypsin, the maximal activity is found for all substrates at rather small wo values (below 12), which is taken to suggest that the enzyme works better when is surrounded by only a few layers of tightly bound water. Spectroscopic studies [ultraviolet absorption, circular dichroism (CD) and fluorescence] have been carried out as a function of wo. Whereas the absorption properties are practically unchanged, the CD spectrum in AOT micelles has a lower intensity than in water, which is interpreted as a partial unfolding. The intensity is partly restored when Ca2+ ions are added, indicating that the micellar environment may cause a partial denaturation by depleting it of calcium ions. Fluorescence data show that the emission properties of the protein in reverse micelles match those in aqueous solution at around wo = 13 approx., whereas lambda max shifts towards the red by increasing wo, indicating an exposure of the tryptophan residues and probably an unfolding of the whole protein, at wo values above 15. Finally the reaction between trypsin and its specific macromolecular Kunitz inhibitor from soybeans is studied.(ABSTRACT TRUNCATED AT 400 WORDS)     

Activity and stability of horse-liver alcohol dehydrogenase in sodium dioctylsulfosuccinate/cyclohexane reverse micelles

Horse liver alcohol dehydrogenase (EC 1.1.1.1) solubilized in sodium dioctylsulfosuccinate (AOT)/cyclohexane reverse micelles was used for the oxidation of ethanol and reduction of cyclohexanone in a coupled substrate/coenzyme recycling system. The activity of the enzyme was studied as a function of pH and water content. The enzyme was optimally active in microemulsions prepared with buffer of pH around 8. An increase in enzymatic activity was observed as a function of increasing water content. The Km values for the substrates were calculated based on the total reaction volume. The apparent Km for ethanol in reverse micelles was about eight times lower as compared to that in buffer solution, whereas the Km for cyclohexanone was almost unaltered. Storage and operational stability were investigated. It was found that the specific activity of the alcohol dehydrogenase operating in reverse micellar solution was good for at least two weeks. The steroid eticholan-3 beta-ol-17-one was also used as a substrate. In this case the reaction rate was approximately five times higher in a reverse micellar solution than in buffer.     

Substrate effects on the enzymatic activity of alpha-chymotrypsin in reverse micelles

Six different substrates have been used for measuring the activity of alpha-chymotrypsin in reverse micelles formed by sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in isooctane. The substrates were glutaryl-Phe p-nitroanilide, succinyl-Phe p-nitroanilide, acetyl-Phe p-nitroanilide, succinyl-Ala-Ala-Phe p-nitroanilide, succinyl-Ala-Ala-Pro-Phe p-nitroanilide and acetyl-Trp methyl ester. It has been shown that the dependence of the kinetic constants (kcat and Km) on the water content of the system, on wo (= [H2O]/[AOT]), is different for the different substrates. This indicates that activity-wo profiles for alpha-chymotrypsin in reverse micelles not only reflect an intrinsic feature of the enzyme alone. For the p-nitroanilides it was found that the lower kcat (and the higher Km) in aqueous solution, the higher kcat as well as Km in reverse micelles. "Superactivity" of alpha-chymotrypsin could only be found with the ester substrate and with relatively "poor" p-nitroanilides. The presence of a negative charge in the substrate molecule is not a prerequisite for alpha-chymotrypsin to show "superactivity".     

Reverse micelles in protein separation: the use of silica for the back-transfer process

In order to use reverse micellar solutions successfully for the separation of proteins, good methods are needed to recover the biomolecules into an aqueous environment after solubilization into organic micellar media. Usually the recovery is accomplished by equilibrating the protein-loaded reverse micellar solution with a water phase containing an appropriate salt (back-transfer). In this article we describe an alternative "back extraction" procedure which is based on the addition of silica to the protein-containing reverse micellar solution. In this way, the water is stripped from the reverse micellar solution. [i.e., bis(2-ethylhexyl) sodium sulfosuccinate (AOT)/isooctane/water] and the proteins adsorb to the silica particles. The adsorption process is shown to be practically quantitative. The subsequent recovery of the proteins form the silica into an aqueous solution turns out to be most efficient at alkaline pH (pH 8); 60-80 of the total protein (alpha-chymotrypsin or trypsin) could be recovered. The specific enzyme activity at the end of the whole cycle can be as high as 80-100%. The procedure is applied also for the back extraction from micellar solutions in which, instead of AOT, a biocompatible surfactant such as a synthetic short-chain lecithin was used. It is shown that the recovery of a alpha-chymotrypsin and trypsin is also achievable under these conditions in quite good yield and under good maintenance of the enzyme's catalytic activity. (c) 1993 John Wiley & Sons, Inc.     

Enzymatic hydrolysis of microcrystalline cellulose in reverse micelles

The activities of cellulases from Trichoderma reesei entrapped in three types of reverse micelles have been investigated using microcrystalline cellulose as the substrate. The reverse micellar systems are formed by nonionic surfactant Triton X-100, anionic surfactant Aerosol OT (AOT), and cationic surfactant cetyltrimethyl ammonium bromide (CTAB) in organic solvent media, respectively. The influences of the molar ratio of water to surfactant omega0, one of characteristic parameters of reverse micelles, and other environmental conditions including pH and temperature, on the enzymatic activity have been studied in these reverse micellar systems. The results obtained indicate that these three reverse micelles are more effective than aqueous systems for microcrystalline cellulose hydrolysis, and cellulases show "superactivity" in these reverse micelles compared with that in aqueous systems under the same pH and temperature conditions. The enzymatic activity decreases with the increase of omega0 in both AOT and Triton X-100 reverse micellar systems, but reaches a maximum at omega0 of 16.7 for CTAB reverse micelles. Temperature and pH also influence the cellulose hydrolysis process. The structural changes of cellulases in AOT reverse micelles have been measured by intrinsic fluorescence method and a possible explanation for the activity changes of cellulases has been proposed.     

Activity and stability of yeast alcohol dehydrogenase (YADH) entrapped in aerosol OT reverse micelles

The activity and stability of yeast alcohol dehydrogenase (YADH) entrapped in aerosol OT reverse micellar droplets have been investigated spectrophotometrically. Various physical parameters, e.g., water pool size, w(0), pH, and temperature, were optimized for YADH in water/AOT/isooctane reverse micelles. It was found that the enzyme exhibits maximum activity at w(0) = 28 and pH 8.1. It was more active in reverse micelles than in aqueous buffers at a particular temperature and was denatured at about 307deg;C in both the systems. At a particular temperature YADH entrapped in reverse micelles was less stable than when it was dissolved in aqueous buffer.     

Esterification reactions of lipase in reverse micelles

The activities of lipase from Candida cylindracea and Rhizopus delemar have been investigated in water/AOT/iso-octane reverse micellar media through the use of two esterification reactions: fatty acid-alcohol esterification and glyceride synthesis. Such media promotes the occurrence of these two lipase-catalyzed reactions due to its low water content. The effect of various parameters on the activity of lipase from C. cylindracea in reverse micelles was determined and compared to results where alternate media were employed. It was observed that the structure of the media, as dictated by the type and concentration of the substrates and products and by the water/AOT ratio, w(0), had a strong impact on enzyme activity. Strong deactivation of both typase types occurred in reverse micelles, especially in the absence of substrates and for w(0) values greater than 3.0. Glyceride synthesis was realized with lipase from R. delemar, but not with that from C. cylindracea; the temperature and concentration of substrates and water strongly dictated the reaction rate and the percent conversion.     

Reversed micelles of polymeric surfactants in nonpolar organic solvents. A new microheterogeneous medium for enzymatic reactions.

A new microheterogeneous non-aqueous medium for enzymatic reactions, based on reversed micelles of a polymeric surfactant, was suggested. The surfactant termed CEPEI, was synthesized by successive alkylation of poly(ethyleneimine) with cetyl bromide and ethyl bromide and was found to be able to solubilize considerable amounts of water in benzene/n-butanol mixtures. The hydrodynamic radius of polymeric-reversed micelles was estimated to be in the range 22-51 nm, depending on the water content of the system, as determined by means of the quasi-elastic laser-light scattering. Polymeric reversed micelles were capable of solubilizing enzymes (alpha-chymotrypsin and laccase) in nonpolar solvents with retention of catalytic activity. Due to the strong buffering properties of CEPEI over a wide pH range, it could maintain any adjusted pH inside hydrated reversed micelles. It was found that catalytic behavior of enzymes entrapped in polymeric reversed micelles was rather insensitive to the pH of the buffer solution introduced into the system as an aqueous component, but determined mostly by acid-base properties of the polymeric surfactant itself. Both catalytic activity and stability of entrapped alpha-chymotrypsin and laccase were found to increase with increasing water content of the system. Under certain conditions, the entrapment of alpha-chymotrypsin into CEPEI reversed micelles resulted in a considerable increase in catalytic activity and stability as compared to aqueous solution. CEPEI reversed micelles were demonstrated to be promising enzyme carriers for use in membrane reactors. Owing to the large dimensions of CEPEI reversed micelles, they are effectively kept back by a semipermeable membrane, thus allowing an easy separation of the reaction product and convenient recovery of the enzyme.     

Relaxation phenomena in systems of protein-containing reverse micelles of surfactants in organic solvents

The research was aimed to establish the equilibrium processes in protein-containing systems of AOT reverse micelles in octane. As chromophore label for tracing the kinetics of the process, the acid-base indicator, p-nitrophenol, was used. The establishing of the equilibrium in the reverse micelle system notably decelerated in the presence of a solubilized protein (native and stearoylated alpha-chymotrypsin). During the establishing of the equilibrium, the solubilized enzyme can be irreversibly inactivated. The level of the residual activity of the enzyme in the equilibrium system depended on the procedure of micellar system preparation. The methods have been offered to set up the equilibrium in the reverse micelle system without inactivation of the solubilized enzyme.     

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.     

Phospholipid-based reverse micelles

Physicochemical investigations on the aggregation of phospholipids (mainly phosphatidylcholines) in organic solvents are reviewed and compared with the aggregation behaviour of phospholipids in aqueous medium. In particular we review the data showing that phosphatidylcholines (lecithins) form reverse micellar structures in certain apolar solvents. In these systems not only low molecular weight compounds but also catalytically active enzymes and entire cells can be solubilized. In addition, highly viscous phosphatidylcholine gels can be obtained in organic solvents upon solubilizing a critical amount of water. Generally, phospholipid-based reverse micelles can be regarded as thermodynamically stable models for inverted micellar lipid structures possibly occurring in biological membranes.     

A comparison of lipase-catalyzed ester hydrolysis in reverse micelles, organic solvents, and biphasic systems

The performance of lipases from Candida rugosa and wheat germ have been investigated in three reaction media using three acetate hydrolyses as model reactions (ethyl acetate, allyl acetate, and prenyl acetate). The effect of substrate properties and water content were studied for each system (organic solvent, biphasic system, and reverse micelles). Not unexpectedly, the effect of water content is distinct for each system, and the optimal water content for enzyme activity is not always the same as that for productivity. A theoretical model has been used to simulate and predict enzyme performance in reverse micelles, and a proposed partitioning model for biphasic systems agrees well with experimental results. While the highest activities observed were in the micellar system, productivity in microemulsions is limited by low enzyme concentrations. Biphasic systems, however, support relatively good activity and productivity. The addition of water to dry organic solvents, combined with the dispersion of lyophilized enzyme powders in the solvent, resulted in significant enzyme aggregation, which not surprisingly limits the applicability of the "anhydrous" enzyme suspension approach. (c) 1995 John Wiley & Sons, Inc.     

Polarographic measurement of oxygen uptake using lipoxygenase in reverse micelles

Lipoxygenase-catalyzed linoleic acid peroxidation was chosen as a model system to study the applicability of oxygraphy to monitor the oxygen uptake in organic solvents containing reverse micelles. Care was taken to control the oxygen back transfer from the atmosphere to the sample micellar solution, resulting in a significant improvement of electrode response. Under these conditions, lipoxygenase activity was linear up to 100 mug of enzyme. Given the quality of the calibration curve and the good correlation between lipoxygenase and ascorbate oxidase, the described technique is proposed as an alternative method for determining lipoxygenase activity in reverse micelles. The reliability of this technique was confirmed by the good agreement between polarography and classic spectrophotometry in kinetic studies. Preliminary experiments carried out on soybean cells solubilized in a Tween 85-isopropylpalmitate system demonstrated that a light-dependent oxygen uptake can be measured. The authors propose that the Clark-type electrode be employed to study both the activity of oxidasic enzymes in reverse micelles and cell viability and physiology in organic solvents.     

Kinetic model for enzymatic hydrolysis in reverse micelles

A bound water model is developed for the interpretation of kinetic data of b-galactosidase in reverse micelles. Assessing the kinetic parameters of p-nitrophenyl-b-D-galactopyranoside hydrolysis in aqueous and reverse micellar system reveals that the major effect on hydrolytic rate is owing to the amount of free water in reverse micelles, not the enzyme molecules' structural change.     

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.     

Synthesis of AcPheLeuNH(2) by alpha-chymotrypsin in TTAB reversed micelles: Application of response surface methodology to the optimization of the system

The influence of the long chain alcohols, hexanol, octanol, and decanol, as cosurfactants of the reverse micellar system of tetradecyltrimethylammonium bromide on the alpha-chymotrypsin-mediated AcPheLeuNH(2) synthesis was studied. The effect of temperature, buffer molarity, pH, and substrate concentration was also evaluated. The enzyme was chemically modified and the effect of this modification upon the enzyme activity was also analyzed. Octanol allowed a higher activity for both enzyme forms. The peptide synthesis/substrate hydrolysis ratio is independent of the long chain alcohol used. The chemical modification decreases the alpha-chymotrypsin activity under the system conditions studied, but increases the initial velocity of peptide synthesis relative to the ester substrate hydrolysis. The response surface methodology was applied to optimize the dipeptide synthesis in the system containing octanol as cosurfactant. (c) 1994 John Wiley & Sons, Inc.     

Interconnection between activity and conformational mobility of alpha-chymotrypsin in reverse micelle systems

A comparative study of the catalytic activity of alpha-chymotrypsin and the spin label rotation frequency in the alpha-chymotrypsin active center of reverse micellar systems solvated by H2O-organic mixtures was carried out. It was found that the decrease in the label rotation frequency resulting from the substitution of water in the micellar inner cavity by glycerol, 2.3-butanediol and dimethylsulfoxide (up to 95%) caused a marked increase (20-fold in the case of 2.3-butanediol) of the enzyme catalytic activity. The experimental results are quantitatively interpreted in terms of a simple kinetic scheme postulating the existence of the enzyme in two interconvertible forms differing in the conformational (intramolecular) mobility, i.e., the resting one predominantly existing in aqueous solution, and the tense one whose proportion rises with an increase in the concentration of the water-miscible organic solvent in the reverse micellar system. The value of kcat (2.4 s-1) for the tense form of the enzyme exceeded by more than 25 times the catalytic activity of chymotrypsin in aqueous solution (0.09 s-1) for the resting form.     

Enzymatic production of L-tryptophan in a reverse micelle reactor

The enzymatic production of tryptophan from indole and serine was investigated in a micellar solution of the surfactant Brij 56 in cyclohexane. An anion exchanger was employed to facilitate the transfer of tryptophan and serine between the water pool of the reverse micelle and the bulk organic phase. The influence of potassium ion, water content, pH, and co-surfactant on enzyme activity is reported. Kinetic studies indicate that the enzyme is not inhibited by indole in the micellar system and that the enzyme is more stable in reverse micelles than in bulk water. The design of a continuous reverse micelle reactor, which accommodates both product recovery and enzyme reactivation, is discussed.     

Solubilization of enzymes in apolar solvents via reverse micelles

The solubilizing of enzymes via reverse micelles provides a method for the catalytic bioconversion of water-insoluble material, for example, reduction of steroids and enoates, cholesterol oxidation, and inter- and trans-esterification of lipophilic substances. Proteases in reverse micelles have been used for the synthesis of water-insoluble peptides. Whole cells solubilized by micellar solutions remain viable in organic solvents. The solubilization of proteins in reverse micelles or water-in-oil microemulsions also provides a method for extracting and purifying enzymes from biological sources. All these uses of reverse micelles represent potential but, as yet, unproven, applications of the technology. What is needed is a deeper understanding of the fundamental processes involved and the development of appropriate reactor systems.