Interesterification and synthesis by Candida cylindracea lipase in microemulsions

Unusual reactions of interesterification and synthesis catalyzed by Candida cylindracea lipase have been tested in reverse microemulsions. The microemulsions used are made of fatty acids or triglycerides, the enzyme dissolved in a very low water quantity, Brij 35 used as surfactant and an alcoholic cosurfactant. In such a system, fats and alcohols are both the substrates of the enzyme and the microemulsion components. Incidentally, non specific Candida cylindracea lipase does not catalyze interesterification of short chain triglycerides, revealing a specificity for the chain length. Interesterification reactions tested in the presence of a given water quantity but with varying water activities show that it is the water activity and not the water quantity which is a fundamental parameter of the system. The effect of the surfactant (Brij 35) on the interesterification reaction is studied. Heptyl-oleate synthesis catalyzed by non-specific lipase is obtained in microemulsions at a 98% yield. Synthesis of glycerol esters is also tested in monophasic medium and mono and diglycerides are obtained.     

Kinetic resolution of ibuprofen catalyzed by Candida rugosa lipase in ionic liquids

Candida rugosa lipase-catalyzed esterification of ibuprofen with 1-propanol was conducted in seven ionic liquids and the results were compared with those in isooctane. Although the enzyme showed comparable or higher activity in some ionic liquids compared to that in isooctane, only in the case of [BMIM]PF6 was the enantioselectivity (E = 24.1) almost twice that (E = 13.0) of isooctane. In another six ionic liquids the enzyme enantioselectivity was much poorer (E = 1.1-6.4). At the same conversion of 30%, E of [BMIM]PF6 is more than triple that of isooctane. The lipase stability in [BMIM]PF6 was improved by 25% of that in isooctane. It was concluded that [BMIM]PF6 could be applied to substitute the conventional organic solvent (isooctane) in the esterification of ibuprofen.     

Action of Candida rugosa lipase in carvone isomers as solvents

The activity and enantioselectivity of Candida rugosa lipase were investigated in chiral solvents, (–)-, (+)- and racemic carvone, for the resolution of 2-chloro-propionic acid with n-butanol via esterification. The activity of the enzyme studied was about 50% higher in (–)-carvone than in (+)-carvone, however the enantioselectivity was similar.     

Cyclic resolution of racemic ibuprofen via coupled efficient lipase and acid-base catalysis

Extracellular lipase LIP prepared in our lab from the yeast Yarrowia lipolytica was used for the resolution of racemic ibuprofen. The (S)-enantiomer was preferred by lipase LIP, and the unreacted (R)-enantiomer was extracted and racemized in basic solvent-water medium to be re-resolved. Solvent, content of solvent, base concentration, and temperature have a strong effect on racemization. The (S)-ester was separated and hydrolyzed to (S)-ibuprofen in acidic dimethyl sulfoxide-water mixture containing 70% dimethyl sulfoxide. The high purity (S)-ibuprofen (ee = 0.98) was obtained using lipase LIP to catalyze hydrolysis of (S)-ester in 0.1 M phosphate buffer (pH = 8).     

Solubilization and growth of Candida pseudotropicalis in water-in-oil microemulsions

Some new aspects of microbiology in water-in-oil microemulsions are investigated using Candida pseudotropicalis in a hexadecane solution containing Tween85/Span80 (each 5% wt:wt) as surfactant, and limited amount of water (up to 3%, vol:vol), Microemulsion solutions containing cells up to 10 mg fresh weight per milliliter can be prepared, which display a greater time stability and a much smaller light scattering than aqueous suspensions having the same cell concentration. This is ascribed to a lower aggregation tendency of the cells in the microemulsion environment. It is also shown that C. pseudotropicalis cells are able to grow (up to a factor of approximately 6-7 within a few days) in the microemulsion system containing nutrient medium in the aqueous microphase; but they are also able to grow at the expense of the hexadecane. This is proved with radioactive-labeled hexadecane by measuring the increase of radioactivity in the cells as well as the emission of (14)CO(2). The growth rate of the cells is then compared with the growth rate of cellular proteins during cell reproduction in the microemulsion system. Two regimes are observed: a first one, in which cells growth rate and protein growth rate proceed parallel to each other; and a second one (established after 0.5-1 day) characterized by depletion of proteins in the microemulsion system. The implications of these findings for cell metabolism in microemulsion and for possible biotechnological applications are discussed.     

Effect of solvent on enantioselective esterification of naproxen by lipase with trimethylsilyl methanol

Improvement of stereoselective resolution of racemic Naproxen, 2-(6-methoxy-2-naphthyl)propionic acid, was attempted with esterifcation reaction by Candida cylindracea lipase. By carefully selecting the organic medium, a 72-time enhancement of yield of the desired S-ester was achieved. The optimal reaction temperature was approximately 53 degrees C, and an alcohol concentration between 20 mM and 40 mM in an 80% (v/v) isooctane and 20% (v/v) toluene mixture was found. (c) 1994 John Wiley & Sons, 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.     

Catalytic behavior of Pseudomonas cepacia lipase in w/o microemulsions

The activity of purified Pseudomonas cepacia lipase has been investigated in esterification reactions of various aliphatic alcohols with natural fatty acids. The reactions were carried out in microemulsions formed in isooctane by bis-(2-ethylhexyl)sulfosuccinate sodium salt (AOT). Kinetic studies showed that the reaction follows a ping-pong bi-bi mechanism with inhibition by both substrates. The apparent kinetic parameters of the reaction were found to be K(m octanol) = 310 mM, K(m lauric acid) = 78 mM, and V(max) = 250 mumol min(-1) mg(-1). The same system was used for the synthesis of mono- and diglycerides from glycerol and lauric acid, which was successful at very low w(o) values. The catalytic behavior of P. cepacia lipase was also studied in esterification reactions performed in a nonionic microemulsion system formulated by tetraethyleneglycoldodecylether (C(12)E(4)). The optimum activity was found at about w(o) = 8. The apparent values of V(max app) and K(m app) for octanol were calculated and found to be 100 mumol min(-1) mg(-1) and 76 mM, respectively. (c) 1995 John Wiley & Sons, Inc.     

Enzymatic interesterification of triglyceride with surfactant-coated lipase in organic media

Several surfactant-coated enzymes have been prepared by coating lipases of various origins with a nonionic surfactant, glutamic acid dioleylester ribitol (2C(18)Delta(9)GE). Enzymatic interesterification of tripalmitin with oleic acid using the surfactant-coated lipase was carried out in organic media. The surfactant-coated lipases could effectively catalyze the interesterification of glycerides better than did the powder lipases. A suitable organic solvent was an aliphatic hydrocarbon such as isooctane. The enzymatic activity for the interesterification strongly depended on the origin of the lipase. The surfactant-coated lipase prepared by Mucor javanicus showed the highest enzymatic activity for the interesterification of glycerides, although its powder lipase did not show enzymatic activity. Selective interesterification of glycerides could be performed by adjusting the concentration ratio of oleic acid to tripalmitin in isooctane. Di-substituted glyceride could be selectively produced when the concentration ratio of carboxylic acid to glycerides was 7. (c) 1995 John Wiley & Sons, Inc.     

Kinetics of lipase deactivation in AOT/isooctane reversed micelles

The stability of lipase in AOT/isooctane reversed micellar solution was investigated. It was found that the lipase deactivated to a stable state that was not completely inactivated. The lipase residual activity after achieving the stable state in AOT/isooctane reversed micelles at 30 °C, pH 7.0, W₀=8.0 was found to be 0.15, and the first-order deactivation rate coefficient of lipase at the same conditions was regressed to be 0.75 h⁻¹. The stability of lipase was increased while oleic acid was added. Assuming the protection of oleic acid to lipase stability is due to the lipase–oleic acid complex does not decay, the kinetic model of lipase deactivation in AOT/isooctane reversed micellar solution including the influence of oleic acid was established. It was shown with the model equation that the increase in stability of the enzyme by oleic acid could be quantitatively estimated by the dissociation constant of lipase–oleic acid complex which was determined by product inhibition experiments. The model equation fit the experimental data well with an average relative deviation of 3.40%.     

Kinetic study of lipase catalyzed esterification reactions in water-in-oil microemulsions

The kinetics of the esterification of lauric acid by (-)menthol, catalyzed by Penicillium simplicissimum lipase, was studied in water/bis-(2-ethylhexyl)sulfosuccinate sodium salt (AOT)/isooctane microemulsions. Due to their low water content, microemulsions assist in reversing the direction of lipase activity, favoring synthetic reactions. The kinetics of this synthesis follows a Ping-Pong Bi--Bi mechanism. The values of all apparent kinetic parameters were determined. The theoretical model for the expression of enzymic activity in reverse micelles, proposed by Verhaert et al. (Verhaert, R., Hilhorst, R., Vermüe, M., Schaafsma, T. J., Veeger, C. 1990. Eur. J. Biochem. 187: 59-72) was extended to express the lipase activity in an esterification reaction involving two hydrophobic substrates in microemulsion systems. The model takes into account the partitioning of the substrates between the various phases and allows the calculation of the intrinsic kinetic constants. The experimental results showing the dependence of the initial velocity on the hydration ratio, W(o) = [H(2)O]/[AOT], of the reverse micelles, were in accordance with the theoretically predicted pattern. (c) 1993 John Wiley & Sons, Inc.     

Ultrasonication enhanced hydrolytic activity of lipase in water/isooctane two-phase systems

The hydrolysis of olive oil catalyzed by Chromobacterium viscosum lipase (EC 3.1.1.3) in a water/isooctane two-phase system was carried out both under ultrasound and conventional stirring. The maximum activity of lipase in the ultrasonicated system was 1.75 times higher than that in the stirred system. The lipase activity was dependent on ultrasonic power and volume ratio of isooctane to water. The optimum reaction temperature in both systems was around 25°C. The stability of lipase at 25°C in the ultrasonicated system decreased more rapidly than that in the stirred system. In the presence of exogenous oleic acid, however the half-life of lipase in the ultrasonicated system was improved to a value, which was respectively half and twice of that in stirred systems with and without oleic acid. The maximum reaction rate (V_max) was increased by ultrasonication whereas the Michaelis constant (K_m) remained unaltered.     

Activity and enantioselectivity of wildtype and lid mutated Candida rugosa lipase isoform 1 in organic solvents

The activity and enantioselectivity of lipase 1 from Candida rugosa and of a chimera enzyme obtained by replacing the lid of isoform 1 with the lid of isoform 3 were compared in organic solvents. The alcoholysis of chloro ethyl 2-hydroxy hexanoate with methanol and of vinyl acetate with 6-methyl-5-hepten-2-ol were used as model reactions in different reaction conditions. The chimera enzyme was less active and enantioselective than the wildtype in all the conditions tested. A rationale for such decreases could be that the chimera lipase has a lower proportion of enzyme molecules in the open form. This might lead to a hindered access to the enzyme active site, thus affecting the catalytic activity.     

Resolution of (R,S)‐ibuprofen catalyzed by immobilized Novozym40086 in organic phase

The enantioselective esterification of ibuprofen catalyzed by Novozym40086 was successfully conducted in organic solvent. Removing‐water reagent was added into the reaction mixture to remove water produced in the esterification. The effects of temperature, n‐hexanol concentration, ibuprofen concentration, and loading of enzymes were investigated. Under the condition of equilibrium, the thermodynamic equilibrium constant (K) of 7.697 and enantioselectivity (E) of 8.512 were obtained. The esterification reaction achieved its equilibrium in approximately 30 hours with conversion of 56% and ee_S of 93.78%. The predicted values of X and ee_S were 67.90% and 95.60%, respectively. The experimental value is approximately equal to the theoretical value, which indicates the feasibility of ideal models.     

Optimization of carbohydrate fatty acid ester synthesis in organic media by a lipase from Candida antarctica

The effect of solvents and solvent mixtures on the synthesis of myristic acid esters of different carbohydrates with an immobilized lipase from C. antarctica was investigated. The rate of myristyl glucose synthesized by the enzyme was increased from 3.7 to 20.2 micromol min(-1) g(-1) by changing the solvent from pure tert-butanol to a mixture of tert-butanol:pyridine (55:45 v/v), by increasing the temperature from 45 degrees C to 60 degrees C, and by optimizing the relative amounts of glucose, myristic acid, and the enzyme preparation. Addition of more than 2% DMSO to the tert-butanol:pyridine system resulted in a reduction of enzyme activity. Lowering the water content of the enzyme preparation below 0.85% (w/w) resulted in significant decreases in enzyme activity, while increasing the water content up to 2.17% (w/w) did not significantly affect the enzyme activity. The highest yields of myristyl glucose were obtained when an excess of unsolubilized glucose was present in the reaction system. In this case, all of the initially solubilized and a significant amount of the initially unsolubilized glucose was converted to the ester within 24 h of incubation, resulting in a myristyl glucose concentration of 34 mg/mL(-1). Myristic acid esters of fructose (22.3 micromol min(-1) g(-1)), alpha-D-methyl-glucopyranoside (26.9 micromol min(-1) g(-1)) and maltose (1.9 micromol min(-1) g(-1)) could also be prepared using the tert-butanol:pyridine solvent system. No synthesis activity was observed with maltotriose, cellobiose, sucrose, and lactose as substrate.     

Kinetics of Enantioselective Esterification of Naproxen by Lipase in Organic Solvents

The kinetics of stereoselective esterification of racemic Naproxen with trimethylsilyl methanol by Candida cylindracea lipase in organic solvents has been investigated. A Ping-Pong Bi Bi mechanism with competitive inhibition by this alcohol for each enantiomer -has been identified. The rate equations were further analyzed in the time-course reaction after considering the effect of enzyme deactivation in the organic mixtures, but not in isooctane. Effects of the hydrophobicity of solvent on the solubility of the racemate, the kinetic parameters and their combinations are also discussed.     

Influence of water activity on the enantioselective esterification of (R,S)-ibuprofen by crosslinked crystals of Candida antarctica lipase B in organic solvent media.

The lipase B from Candida antarctica was purified from a commercial source and crystallized. The microcrystals were crosslinked using glutaraldehyde. The crosslinked crystals were then used to catalyze the esterification of (R,S)-ibuprofen with dodecanol in octane at various water activities. As for the commercial preparation immobilized on acrylic resin, Novozym 435, low water activities foster better enantioselectivity, and the maximum reaction rates are obtained for a(W) = 0.1.     

Lipase-catalyzed esterification of 2-(4-substituted phenoxy)propionic acids in organic solvents: substituent effect controlling enantioselectivity toward racemic acids

The enantioselectivity for lipase-catalyzed esterifications of 2-(4-substituted phenoxy)propionic acids in organic solvents was found to be mainly controlled by both size (steric) and electronic effects of substituents: H, F, Cl, CF₃ and CH₃. For the similar substituents in size, CF₃ and CH₃, however, their electronic effects play an important role in controlling the enantioselectivity. A model for the enantiorecognition is proposed by the discussion based on the value of the Michaelis constant obtained for the enantiomers.