Three Systems Used for Biocatalysis in Organic Solvents a Comparative Study

The activity and operational stability of horse liver alcohol dehydrogenase (HLADH) and α-chymotrypsin were investigated in three systems commonly used for biocatalysis in organic solvents:

1. enzyme adsorbed on a solid support (celite) and added to the organic solvent (isooctane)

2. enzyme powder directly added to the organic solvent (isooctane).

3. enzyme dissolved in a microemulsion (AOT/isooctane).

The activity and the operational stability in all systems were strongly dependent on the water content. The initial reaction rate was high in both the microemulsion and the celite system, but was much lower when adding the enzymes directly to the organic solvent. HLADH was observed to be more stable when added directly to the organic solvent or dissolved in the microemulsion than when adsorbed on celite, whereas for α-chymotrypsin stability was higher when adsorbed on celite or added directly to the organic solvent. For a hydrolytic reaction, a microemulsion was preferred due to the high water content. When adding the enzymes directly to the organic solvent both HLADH and chymotrypsin were adsorbed strongly to the glass walls of the reaction vessel. None of the systems were superior in all respects for the two enzymes studied.     

Two-Step biocatalytic conversion of an ester to an aldehyde in reverse micelles

Lipases from Candida cyclindracea (L-1754) and wheat germ (L-3001) have been used to hydrolyze esters to their corresponding alcohols and acids in reverse micelles. Alcohol dehydrogenase from baker's yeast (YADH) was subsequently used to reduce the alcohol products to aldehydes. Cofactor recycling in the redox reaction was achieved using a sacrificial cosubstrate, as described previously. Four surfactants (sodium dioctylsulfosuccinate, Nonidet P-40 with Triton X-35, polyoxyethylene, 10-cetyl-ether, polyoxyethylene sorbitan trioleate) were employed to determine the effect of amphiphile on ester hydrolysis and redox reaction rates separately. The effect of type of organic solvent, W(0) [(water]/[surfactant)], and substrate concentration on separte enzyme activity were also investigated. A brief investigation of a single phase, two-step reaction catalyzed by the combination of lipase and YADH in reverse micelles is also reported. The activities of the enzymes are significantly different when used together instead of independently. (c) 1994 John Wiley & Sons, Inc.     

Immobilized horse liver alcohol dehydrogenase as an on‐line high‐performance liquid chromatographic enzyme reactor for stereoselective synthesis

Horse liver alcohol dehydrogenase (HLADH) has been non‐covalently immobilized on an immobilized artificial membrane (IAM) high‐performance liquid chromatography (HPLC) stationary phase. The resulting IAM‐HLADH retained the reductive activity of native HLADH as well as the enzyme's enantioselectivity and enantiospecificity. HLADH was also immobilized in an IAM HPLC stationary phase prepacked in a 13 × 4.1 mm ID column to create an immobilized enzyme reactor (HLADH‐IMER). The reactor was connected through a switching valve to a column containing a chiral stationary phase (CSP) based upon p‐methylphenylcarbamate derivatized cellulose (Chiralcel OJR‐CSP). The results from the combined HLADH‐IMER/CSP and chromatographic system demonstrate that the enzyme retained its activity and stereoselectivity after immobilization in the column and that the substrate and products from the enzymatic reduction could be transferred to a second column for analytical or preparative separation. The combined HLADH‐IMER/CSP system is a prototype for the preparative on‐line use of cofactor‐dependent enzymes in large‐scale chiral syntheses. Chirality 11:39–45, 1999. © 1999 Wiley‐Liss, Inc.     

Solubilization of soybean mitochondria in AOT/isooctane water-in-oil microemulsions

The water-in-oil microemulsion system bis(2 ethyl-hexyl-sodium-succinate (AOT)/isooctane/water is able to solubilize soybean nodules mitrochrondria. Transparent and thermodynamically stable hydrocarbon solutions are obtained, which can be assayed for mitochondrial activity just as aqueous solutions. Malate dehydrogenase (MDH) activity was measured in vivo and gave in reverse micelles very similar results as in water. However the kinetic behavior of this reaction in AOT/isooctane reverse micelles shows some differences with respect to water. Mitochondria in reverse AOT micelles are able to retain about 70% of their initial MDH activity after three days. Mitochondria can be back-transferred from reverse micelles to water and show respiratory activity almost identical to the native organelles. Electron microscopy studies show that the dimensions of mitochondria back-transferred into water from AOT micelles are comparable to the dimensions of the native organelles.     

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.     

Enzymatic catalysis in microemulsions: enzyme reuse and product recovery

A technique for enzyme reuse and product recovery from enzymatic catalysis in microemulsions is demonstrated. The enzymatic reaction is performed in a homogeneous isotropic microemulsion; AOT (sodium bis-(2-ethyl- hexyl)sulfosuccinate)/isooctane/buffer or C(12)E(5)(penta ethylene glycol dodecyl ether)/heptane/buffer. By small temperature changes the systems are shifted to two phase regions, where an oil-rich phase, containing the product, coexists with a water-rich phase containing surfactant and enzyme. The oil-rich phase may be replaced by an oil solution containing new substrate. Thus, the reaction may be continued and the enzyme reused. This procedure was repeated nine times in the present study. Data on phase behavior in presence and in absence of protein, partitioning of the components and a radioactive-labelled protein between the phases, and the repeated use of horse liver alcohol dehydrogenase (HLADH) in the microemulsions are presented.     

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.     

Biodegradation of pesticides in nonionic water-in-oil microemulsions of tween 85: Relationship between micelle structure and activity

Water-in-oil microemulsion systems have been studied in recent years for a number of applications in protein separation and enzymology. Although it is well established that reversed micelle systems provide an excellent medium for nonaqueous biocatalytic studies, there is still much speculation as to the interaction of the enzyme with the surfactant interface. Polyoxyethylene sorbitan trioleate (Tween 85) is a nonionic surfactant which has some interesting properties for microemulsion formation and protein solubilization. In conjunction with a separate article describing the structural features of Tween 85 reversed micelles in hexane with isopropanol as a cosurfactant, this work describes the activity of an enzyme, organophosphorus hydrolase, for degrading organophosphorus pesticides in this microemulsion system. Ternary phase diagrams were constructed to outline the phase boundaries at different temperatures and isopropanol concentrations, which elucidate the role of the cosurfactant alcohol, as well as some features of micelle structure. Kinetic and stability studies with organophosphorus hydrolase show the effect of enzyme partitioning between the micelle surfactant layer and aqueous core. (c) 1994 John Wiley & Sons, Inc.     

Renaturation and interaction of ribonuclease A with AOT surfactant in reverse micelles

This study extended works on effects of solute on the percolation of reverse micelles to the effects of interactions between protein and surfactants on protein refolding by reverse micelles. The changes in percolation behavior were identified and attributed to the position of solutes in the core aqueous phase and the interaction between the solute and the surfactants. The percolation behavior of reverse micelles with solutes was related to protein renaturation and the reverse micelle. This study aims to highlight the involvement of the interface and the interaction of the protein with the surfactant during protein refolding. Ribonuclease A and AOT reverse micelles together constitute a model system considered here. The systemic parameters of the reverse micelle, water content (W(o)) and pH value, were applied to modify the interaction between the denatured protein molecules and the surfactant interface. The interactions and the locations of the protein molecules were determined from changes in percolation temperature measured by conductivity. The percolation and protein activity show that a stronger interaction of the protein molecules with surfactant corresponds to superior recovery of protein activity. The investigation concludes that the refolding of protein by reverse micelles is not only facilitated by the isolation of reverse micelles but also by the interaction due to the interface of the reverse micelle.     

Phase diagrams of a CTAB/organic solvent/buffer system applied to extraction of enzymes by reverse micelles

A partial pseudo-ternary phase diagram has been studied for the cethyltrimethylammonium bromide/isooctane:hexanol:butanol/potassium phosphate buffer system, where the two-phase diagram consisting of the reverse micelle phase (L2) in equilibrium with the solvent is indicated. Based on these diagrams two-phase systems of reverse micelles were prepared with different compositions of the compounds and used for extraction and recovery of two enzymes, and the percentage of enzyme recovery yield monitored. The enzymes glucose-6-phosphate dehydrogenase (G6PD) and xylose redutase (XR) obtained from Candida guilliermondii yeast were used in the extraction procedures. The recovery yield data indicate that micelles having different composition give selective extraction of enzymes. The method can thus be used to optimize enzyme extraction processes.     

Engineering of functional supramacromolecular complexes of proteins (enzymes) using reversed micelles as matrix microreactors.

The size of the inner water cavity of reversed micelles formed in a triple system 'water-surfactant-organic solvent' can be widely varied by changing the degree of surfactant hydration. This gives grounds to use reversed micelles as matrix microreactors for the design of supramolecular complexes of proteins. Using ultracentrifugation analysis, it has been demonstrated that the oligomeric composition of various enzymes (ketoglutarate dehydrogenase, alkaline phosphatase, lactic dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase) solubilized in reversed micelles of Aerosol OT [sodium bis(2-ethylehexyl)sulfosuccinate] in octane changes upon variation of the degree of hydration. An oligomeric complex forms under conditions when the radius of the micelle inner cavity is big enough to incorporate this complex as a whole. At lower degrees of hydration the micelles 'uncouple' such complexes to their components. The catalytic properties of various oligomeric complexes have been studied. Possibilities of using reversed micelles for the separation of subunits of oligomeric enzymes under non-denaturating conditions have been demonstrated. In particular, the isolated subunits of alkaline phosphatase, lactic dehydrogenase and glyceraldehyde-3-phosphate have been found to be active in Aerosol OT reversed micelles. The dependences of the catalytic activity of oligomeric enzymes represent saw-like curves. The maxima of the catalytic activity observed at these curves relate to the functioning of various oligomeric forms of an enzyme. The radii of the micelle inner cavity under conditions when these maxima are observed correlate with the linear dimensions of the enzyme oligomeric forms. Correlation of the position of a maximum with the shape of an oligomeric complex is discussed.     

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.     

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.     

Structure-Activity Relationship of a U-Type Antimicrobial Microemulsion System

The structure-activity relationship of a U-type antimicrobial microemulsion system containing glycerol monolaurate and ethanol at a 1∶1 mass ratio as oil phase and Tween 20 as surfactant were investigated along a water dilution line at a ratio of 80∶20 mass% surfactant/oil phase, based on a pseudo-ternary phase diagram. The differential scanning calorimetry results showed that in the region of up to 33% water, all water molecules are confined to the hydrophilic core of the reverse micelles, leading to the formation of w/o microemulsion. As the water content increases, the water gains mobility, and transforms into bicontinuous in the region of 33–39% water, and finally the microemulsion become o/w in the region of above 39% water. The microstructure characterization was confirmed by the dynamic light scattering measurements and freeze-fracture transmission electron microscope observation. The antimicrobial activity assay using kinetics of killing analysis demonstrated that the microemulsions in w/o regions exhibited relatively high antimicrobial activity against Escherichia coli and Staphylococcus aureus due to the antimicrobial oil phase as the continuous phase, while the antimicrobial activity started to decrease when the microemulsions entered the bicontinuous region, and decreased rapidly as the water content increased in the o/w region, as a result of the dilution of antimicrobial oil droplets in the aqueous continuous phase.     

Comparative thermostability of mesophilic and thermophilic alcohol dehydrogenases: Stability-determining roles of proline residues and loop conformations

The present study demonstrates the comparative thermal, conformational and kinetic stabilities of the three closely related enzymes; the mesophilic yeast alcohol dehydrogenase (YADH), horse liver alcohol dehydrogenase (HLADH), and the extreme-thermophilic Thermoanaerobacter brockii alcohol dehydrogenase (TBADH). The mid-point unfolding temperatures for TBADH and HLADH were at least 10 °C and 6 °C higher, respectively, than that of YADH. When YADH was completely inactivated by thermal stress, the residual activities of HLADH and TBADH were 70% and 100%, respectively. The optimum temperature (T_opt) activities of HLADH and TBADH were at least 40 °C and 55 °C higher, respectively, than that of YADH. Due to the higher rigidity of HLADH and TBADH, the enzymatic activation energies of HLADH and TBADH were higher than that of YADH. Geometric X-ray analysis indicated a comparatively higher coil (turn and loop) percentage in TBADH and HLADH than in YADH. Pairwise alignment for TBADH/HLADH exhibited a similarity score approximately 2.5-fold greater than that of the TBADH/YADH pair. Multiple alignments made with ClustalW revealed a higher number of conserved proline residues in the two most stable enzymes (HLADH/TBADH). These extra prolines tend to occur in surface loops and are likely to be responsible for the increased stability of TBADH and HLADH, by loop rigidification.     

Candida Rugosa lipase reactions in nonionic w/o-microemulsion with a technical surfactant

In enzyme catalysis there is a great interest in finding suitable organic media for less water soluble substrates in order to increase the substrate concentration and therefore the reaction rates. These requirements are fulfilled by using w/o-microemulsions as reaction media. The influences of pH, temperature, water concentration and the kinetic parameters of Candida Rugosa Lipase in a nonionic w/o-microemulsion with a surfactant of technical grade, Marlipal O13-60, are presented. In an example the enantiospecific esterification of racemic menthol with propionic anhydride using this nonionic microemulsion likely to be affordable in large scale applications is shown. For a continuous process an ultrafiltration unit is attached to a reactor within a loop. In this way, the reverse micelles containing the enzymes can be separated from the oil, containing the product, and reused afterwards.     

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.     

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.     

Efficient Repeated Use of Alcohol Dehydrogenase with NAD + Regeneration in an Aqueous-organic Two-phase System

Bioconversion of cinnamyl alcohol to cinnamaldehyde was carried out in an aqueous-organic two-phase reaction system by the repeated use of horse liver alcohol dehydrogenase (HLADH) and NAD + with coenzyme regeneration. Both HLADH and the coenzyme were efficiently entrapped in the aqueous phase, while the substrate was supplied successively from the organic phase and the product was accumulated in the organic phase. Optimum conditions for cinnamaldehyde production in the aqueous-organic two-phase system were also examined, including substrate concentration, pH, and organic solvent type. Under suitable conditions, both HLADH and NAD + in the aqueous-organic two-phase system could be reused, and NAD + cycling numbers of 3040 were obtained after repeated operation for 40 &#117 h.     

Efficient Repeated Use of Alcohol Dehydrogenase with NAD + Regeneration in an Aqueous-organic Two-phase System

Bioconversion of cinnamyl alcohol to cinnamaldehyde was carried out in an aqueous-organic two-phase reaction system by the repeated use of horse liver alcohol dehydrogenase (HLADH) and NAD + with coenzyme regeneration. Both HLADH and the coenzyme were efficiently entrapped in the aqueous phase, while the substrate was supplied successively from the organic phase and the product was accumulated in the organic phase. Optimum conditions for cinnamaldehyde production in the aqueous-organic two-phase system were also examined, including substrate concentration, pH, and organic solvent type. Under suitable conditions, both HLADH and NAD + in the aqueous-organic two-phase system could be reused, and NAD + cycling numbers of 3040 were obtained after repeated operation for 40 λh.