Computer-aided real-time estimation of reaction conversion for lipase-catalyzed esterification in solvent-free systems

Real-time conversion estimation through macroscopic balancing was investigated for enzymatic esterification reactions in a solvent-free system. In principle, the conversion of ester synthesis can be determined from the amount of water produced by the reaction because water is formed as a by-product in the same molar ratio as the product. In this study, we show that the water production rate, and thereby the reaction conversion, can be estimated on-line from measurements of the relative humidity of the inlet and outlet air and the material balances of water in the system. In order to test the performance of the real-time conversion estimation method, the lipase-catalyzed esterification reaction of n-capric acid and n-decyl alcohol in solvent-free media was conducted while controlling the water activity at various values. When the reaction conversions estimated on-line were compared with those analyzed off-line by gas chromatography, good agreement was obtained: the average mean absolute error was +/- 2.4% of the reaction conversion despite the simplicity of the method. The on-line estimation method presented here requires no expensive or complicated analytical instruments and no sampling of reaction medium. It can be used for monitoring nonaqueous enzymatic reactions where water is produced or consumed during reaction.     

Computer-aided control of water activity for lipase-catalyzed esterification in solvent-free systems

A computer system for on-line monitoring and control of the water activity (a(w)) in solvent-free media has been developed. The performance of this system was investigated by carrying out the lipase-catalyzed esterification of n-capric acid with n-decyl alcohol. A humidity sensor measured the relative humidity in the reactor headspace, which was then transmitted electrically to a digital computer that was used as a feedback controller. The water activity control was achieved by sparging either humidified air or dried air through the reaction medium at a flow rate determined by the digital feedback controller. The use of humid air and dry air for a(w) control made it possible to induce a larger a(w) gradient and thereby higher water transfer rate. As a result, the water activity quickly reached the desired a(w) values. We tested whether water activity in the reaction medium can be monitored by measuring relative humidity in the headspace. When the water activity in the liquid phase was determined from measurements of water content in the medium and compared to that measured directly with the humidity sensor, the a(w) in the reaction medium did not differ significantly from that in the headspace. This indicates that there is a near-equilibrium between the liquid medium and the exit air stream. Water activity was also successfully maintained close to the set point despite the massive production of water during the esterification process. Thus, the control system developed in this study is particularly useful for systems where large amounts of water are produced and where conventional methods make it difficult to control water activity as a result of a low water transfer rate. The effects that computer control of the water activity had on the reaction rate and yield were also examined. The reaction yield was significantly improved with water activity control. The conversions obtained at 28 h without and those with water activity control were 70% and 96%, respectively. In addition, from the fact that the final yields increased with decreasing a(w), computer-aided water activity control was performed with a set-point change. By controlling a(w) at 0.55 during initial reaction phase, followed by a step change of a(w) from 0.55 to 0 after 11 h of reaction, it was possible to enhance the final conversion to 100%.     

Enzymatic production of enantiopure ketoprofen in a solvent-free two-phase system

Enzymatic hydrolysis conducted in a medium composed of solely substrate is considered to resolve racemic ketoprofen esters. In a system composed of two components, the pure liquid substrate (organic phase) and water (aqueous phase), hydrolysis products can be efficiently removed from the reaction mixtures. Accordingly, in this study we designed a solvent-free two-phase system for the enantioselective enzymatic hydrolysis of ketoprofen esters. In order to further optimize this system, the influences of various factors, such as the pH of the aqueous phase, temperature, enzyme content, and the alcohol chain length of esters, were examined on conversion and enantiomeric excess. 1N NaHCO₃ was identified as the most efficient aqueous phase for the extraction of ketoprofen. Changes in the amount of enzyme did not significantly affect the maximum conversion or the enantiomeric excess. On the other hand, ketoprofen esters with shorter alcohol chains displayed higher initial reaction rates and conversions in solventless media. In the case of ketoprofen propyl ester, for example, the productivity of the solvent-free two-phase system was about 10–100 times higher than that obtained to date for ketoprofen esterification with alcohols in organic solvents. The enantioselectivities obtained in solvent-free media were similar to those obtained for the enantioselective esterification of ketoprofen in organic solvents.     

Water activity control: a way to improve the efficiency of continuous lipase esterification

During continuous lipase-catalyzed oleic acid esterification by ethanol in n-hexane, the oleic acid conversion, initially at 95%, decreases to 20% after 2 h. This decrease is caused by the accumulation of the water produced in the course of the reaction in the packed-bed reactor (PBR). In order to improve the PBR efficiency, it is necessary to evacuate the water produced. In this study, different approaches have been tested to control the water content in the PBR during continuous esterification. The first approach consisted in improving the water solubility by increasing the reaction medium polarity. The addition of polar additives to n-hexane, the use of more polar solvents, and the use of solvent-free reaction medium were tested as a means to favor the water evacuation from the PBR. First of all, the use ofn-hexane supplemented with acetone (3 M) or 2-methyl-2-propanol (1 M) enabled the conversion to be maintained at higher values than those obtained in pure n-hexane. The replacement of n-hexane by a more polar solvent, like the 5-methyl-2-hexanone, resulted in the same effect. In all cases, conversions at steady-state were always less than 95%, as obtained in pure n-hexane. This is explained by a decrease in the enzyme activity due to the increase in the medium polarity. Nevertheless, an increase in enzyme quantity allowed 90% conversion to be maintained during 1 week using 3 M acetone amended n-hexane. Good results (a steady-state conversion of about 80%) were obtained when esterification was carried out in a solvent-free reaction medium containing 2 M 2-methyl-2-propanol as a polar additive. The second approach consisted in the evaporation of the accumulated water by use of an intermittent airflow. Although this process did not enable constant esterification rate to be maintained, it did enable the initial conversion (95%) to be restored intermittently.     

Lipase-catalyzed esterification of conjugated linoleic acid with <Emphasis Type="SmallCaps">l</Emphasis>-carnitine in solvent-free system and acetonitrile

Lipase-catalyzed esterification of conjugated linoleic acid (CLA) with l-carnitine in solvent-free system and acetonitrile was studied. Three lipases (Novzym 435, Amamo AY30 and Amano AYS) have been assayed as suitable biocatalysts in the reaction. It was found that Amano AY30 was the most effective biocatalyst in both solvent-free system and acetonitrile. The conversion rate varied from 8.05 to 60.9% in terms of reaction conditions such as the amount of lipase, the presence of water, the amount of molecular sieves and reaction time. The conversions of substrate in solvent-free system were higher than that in acetonitrile. When the substrates were 1 mmol CLA and 1 mmol l-carnitine, the maximum conversion (60.9%) was obtained in solvent-free system with 150 mg lipase AY30, 50% water content and 150 mg molecular sieves at the reaction time of 24 h. A novel CLA ester product was successfully isolated and characterized by ESI-MS and ¹H NMR.     

Chemometric analysis of lipase-catalyzed synthesis of xylitol esters in a solvent-free system

Immobilized Candida antarctica lipase B-catalyzed esterification of xylitol and two fatty acids (capric and caproic acid) were studied in a solvent-free system. The Taguchi orthogonal array method based on three-level-four-variables with nine experiments was applied for the analysis and optimization of the reaction parameters including time, substrate molar ratio, amount of enzyme, and amount of molecular sieve. The obtained conversion was higher in the esterification of xylitol and capric acid with longer chain length. The optimum conditions derived via the Taguchi approach for the synthesis of xylitol caprate and xylitol caproate were reaction time, 29 and 18 h; substrate molar ratio, 0.3 and 1.0; enzyme amount, 0.20 and 0.05 g, and molecular sieve amount of 0.03 g, respectively. The good correlation between the predicted conversions (74.18% and 61.23%) and the actual values (74.05% and 60.5%) shows that the model derived from the Taguchi orthogonal array can be used for optimization and better understanding of the effect of reaction parameters on the enzymatic synthesis of xylitol esters in a solvent-free system.     

Enzymatic synthesis of geraniol esters in a solvent-free system by lipases

Geraniol esters were synthesised by direct esterification catalysed by esterases and lipases (five enzymes were tested) in a solvent-free system at 37°C. The best conversions yields, about 85%, on geranyl butyrate and valerate obtained with esterase 30 000 from Mucor miehei. The effect of substrate molar ratio alcohol/acid variation was studied. A study of the water production was made in parallel during the esterification reaction.     

Stability and activity of lipase in subcritical 1,1,1,2-tetrafluoroethane (R134a)

The stability and activity of commercial immobilized lipase from Candida antarctica (Novozym 435) in subcritical 1,1,1,2-tetrafluoroethane (R134a) was investigated. The esterification of oleic acid with glycerol was studied as a model reaction in subcritical R134a and in solvent-free conditions. The results indicated that subcritical R134a treatment led to significant increase of activity of Novozym 435, and a maximum residual activity of 300% was measured at 4 MPa, 30 °C after 7 h incubation. No deactivation of Novozym 435 treated with subcritical R134a under different operation factors (pressure 2–8 MPa, temperature 30–60 °C, incubation time 1–12 h, water content 1:1, 1:2, 1:5 enzyme/water, depressurization rate 4 MPa/1 min, 4 MPa/30 min, 4 MPa/90 min) was observed. While the initial reaction rate was high in subcritical R134a, higher conversion was obtained in solvent-free conditions. Though the apparent conversion of the reaction is lower in subcritical R134a, it is more practicable, especially at low enzyme concentrations desired at commercial scales.     

Enzymatic Production of Alkyl Esters Through Lipase-Catalyzed Transesterification Reactions in Organic Solvents: Solvent Effects and Prediction Capabilities of Equilibrium Conversions

The solvent effect on the equilibrium position of the transesterification reaction of hexanol with ethyl acetate catalyzed by a lipase has been investigated in a variety of non-polar and polar solvents - and binary mixtures. The results obtained indicate that the solvent effect on the equilibrium conversion is very small as compared to that for the direct esterification reactions.

Equilibrium conversions were then predicted using the equilibrium constant for the reaction obtained from Gibbs free energy of formation information for reactants and products in combination with the UNIFAC activity coefficient model. A solvent independent equilibrium conversion was obtained, which was in good agreement with the observed average value for all solvents. This indicates that UNIFAC provides satisfactory estimates of the activity coefficients but its group contribution structure does not allow the prediction of the small differences in conversion among the solvents examined.

Finally plots of these conversions versus the solvent octanol/water partition coefficient or the solubility of water in the solvent, that provide the correct trend in direct esterification reactions, did not achieve the same for transesterification.     

Enzymatic Synthesis of Ethyl-glucoside Monooleate with Lipase in Solvent-free Medium

Ethylglucoside monooleate was synthesized by esterification between ethylglucoside and oleic acid with immobilized lipase from Candida antarctica in a solvent-free system. It was shown that a stirred tank reactor was suitable for the enzymatic reaction process involving substrates with low miscibility, in which the biocatalyst was recycled five times without significant activity loss. Removal of the co-product, water, from the reaction medium by carrying out the reaction under reduced pressure benefited the esterification reaction and increased the monooleate yield up to 97% within 8 hours.     

Optimisation of n-octyl oleate enzymatic synthesis over Rhizomucor miehei lipase

Octyl oleate is a useful organic compound with several applications in cosmetic, lubricant and pharmaceutical industry. At first, the enzymatic synthesis of n-octyl oleate by direct lipase-catalysed esterification of oleic acid and 1-octanol was investigated in a stirred batch reactor in solvent-free system. A systematic screening and optimisation of the reaction parameters were performed to gain insight into the kinetics mechanism. Particularly, enzyme concentration, reaction temperature, stirrer speed, water content, substrates concentration and molar ratio were optimised with respect to the final product concentration and reaction rate. The kinetics mechanism of the reaction was investigated. Finally, a comparison of the experimental results obtained in a solvent free-system with those using two different solvents, supercritical carbon dioxide (SC-CO₂) and n-hexane, was proposed. It resulted that in SC-CO₂ higher concentration of the desired product was attained, requiring lower enzyme concentrations to achieve comparable conversion of free fatty acid into fatty acid ester.     

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.     

Comparison of direct esterification and transesterification of fructose by Candida antartica lipase

Fructose oleates synthesis was performed in a batch reactor by trans- or direct esterification. An immobilized lipase from Candida antartica was used. When a solvent was used, 65% and 46% of conversion of fructose were obtained by transesterification and direct esterification, respectively. These two reactions were also compared in a solvent-free melt. Both in molten media and with cosolvent, two isomeric forms of fructose oleates were produced.     

Kinetics of enzymatic synthesis of L-ascorbyl acetate by Lipozyme TLIM and Novozym 435

L-ascorbyl acetate was synthesized through lipase-catalyzed esterification using Lipozyme TLIM and Novozym 435. Four solvents, including methanol, ethanol, acetonitrile, and acetone were investigated for the reaction, and acetone and acetonitrile were found to be suitable reaction media. The influences of several parameters such as water activity (a _w), substrate molar ratio, enzyme loading, and reaction temperature on esterification of L-ascorbic acid were systematically and quantitatively analyzed. Through optimizing the reaction, lipase-catalyzed esterification of L-ascorbic acid gave a maximum conversion of 99%. The results from using Lipozyme TLIM and Novozym 435 as biocatalysts both showed that a _w was an important factor for the conversion of L-ascorbic acid. The effect of pH value on lipase-catalyzed L-ascorbic acid esterification in acetone was also investigated. Furthermore, results from a kinetic characterization of Lipozyme TLIM were compared with those for Novozym 435, and suggested that the maximum reaction rate for Lipozyme TLIM was greater than that for Novozym 435, while the enzyme affinity for substrate was greater for Novozym 436.     

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.     

Lipase of <Emphasis Type="Italic">Aspergillus niger</Emphasis> NCIM 1207: A Potential Biocatalyst for Synthesis of Isoamyl Acetate

Commercial lipase preparations and mycelium bound lipase from Aspergillus niger NCIM 1207 were used for esterification of acetic acid with isoamyl alcohol to obtain isoamyl acetate. The esterification reaction was carried out at 30°C in n-hexane with shaking at 120 rpm. Initial reaction rates, conversion efficiency and isoamyl acetate concentration obtained using Novozyme 435 were the highest. Mycelium bound lipase of A. niger NCIM 1207 produced maximal isoamyl acetate formation at an alcohol/acid ratio of 1.6. Acetic acid at higher concentrations than required for the critical alcohol/acid ratio lower than 1.3 and higher than 1.6 resulted in decreased yields of isoamyl acetate probably owing to lowering of micro-aqueous environmental pH around the enzyme leading to inhibition of enzyme activity. Mycelium bound A. niger lipase produced 80 g/l of isoamyl acetate within 96 h even though extremely less amount of enzyme activity was used for esterification. The presence of sodium sulphate during esterification reaction at higher substrate concentration resulted in increased conversion efficiency when we used mycelium bound enzyme preparations of A. niger NCIM 1207. This could be due to removal of excess water released during esterification reaction by sodium sulphate. High ester concentration (286.5 g/l) and conversion (73.5%) were obtained within 24 h using Novozyme 435 under these conditions.     

Candida Antarctica Lipase B-Catalysed Synthesis Of Dihydroxyacetone Fatty Acid Esters

The enzymatic syntheses of 1-lauroyl-dihydroxyacetone and 1, 3-dilauroyl-dihydroxyacetone were investigated. Lipase B from Candida Antarctica (SP435) was used to catalyse the acylation of dihydroxyacetone (DHA) with lauric acid in organic solvent media at controlled water activity. High conversions of dihydroxyacetone (< 90%) are achieved when the water activity is 0.11 or below in solvents of various hydrophobicities, such as diethyl ether, methyl-terr-butyl ether (MTBE) and diphenyl ether. The main product in the esterification of DHA with lauric acid is 1-lauroyl-DHA, while the amount of 1, 3-dilauroyl-DHA that is produced can be increased by changing the reaction conditions. Thus, hasing the water activity from 0.75 to 0.06 resulted in an increase in the total yield of 1, 3-dilauroyl-DHA from 3% to 20%. Solvents which have high logP values favoured the acylation of 1-lauroyl-DHA and thereby the formation of 1, 3-dilauroyl-DHA. Thus, when diphenyl ether was used in this reaction, the yield of 1, 3-dilauroyl-DHA was 45%. Complete acylation to 1, 3-dilauroyl-DHA was achieved when a fatty acid vinyl ester was used as acyl donor in a closed reactor.     

Optimized lipase-catalyzed synthesis of adipate ester in a solvent-free system

Immobilized Candida antarctica lipase-catalyzed esterification of adipic acid and oleyl alcohol was investigated in a solvent-free system (SFS). Optimum conditions for adipate ester synthesis in a stirred-tank reactor were determined by the response surface methodology (RSM) approach with respect to important reaction parameters including time, temperature, agitation speed, and amount of enzyme. A high conversion yield was achieved using low enzyme amounts of 2.5% w/w at 60°C, reaction time of 438 min, and agitation speed of 500 rpm. The good correlation between predicted value (96.0%) and actual value (95.5%) implies that the model derived from RSM allows better understanding of the effect of important reaction parameters on the lipase-catalyzed synthesis of adipate ester in an organic solvent-free system. Higher volumetric productivity compared to a solvent-based system was also offered by SFS. The results demonstrate that the solvent-free system is efficient for enzymatic synthesis of adipate ester.     

Kinetic biosynthesis of l-ascorbyl acetate by immobilized Thermomyces lanuginosus lipase (Lipozyme TLIM)

Thermomyces lanuginosus lipase (Lipozyme TLIM)-catalyzed esterification of l-ascorbic acid was studied. It was suggested that Lipozyme TLIM was a suitable biocatalyst for enzymatic esterification of l-ascorbic acid. Three solvents were investigated for the reaction, and acetone was found to be a suitable reaction medium. Furthermore, it was found that water activity could notably affect the conversion. Moreover, pH memory of Lipozyme TLIM lipase for catalyzing l-ascorbic acid esterification in acetone was observed and the effect of pH on the reaction was estimated. In addition, the influences of other parameters such as substrate mole ratio, enzyme loading, and reaction temperature and reusability of lipase on esterification of l-ascorbic acid were also analyzed systematically and quantitatively. Kinetic characterization of Lipozyme TLIM showed that K _m,a and V _max were 80.085 mM and 0.747 mM min⁻¹, respectively. As a result, Lipozyme TLIM-catalyzed esterification of l-ascorbic acid gave a maximum conversion of 99%.     

Effect of water activity on the lipase catalyzed esterification of geraniol in ionic liquid [bmim]PF6

Enzymatic reactions in non-aqueous media have been shown to be effective in carrying out chemical transformation where the reactants are insoluble in water or water is a byproduct limiting conversion. Ionic liquids, liquid organic salts with infinitesimal vapor pressure, are potentially useful alternatives to organic solvents. It is known that the thermodynamic water activity is an important variable affecting the activity of enzymes in non-aqueous solvents. This study investigated the influence of water activity on the esterification of geraniol with acetic acid in ionic liquid [bmim]PF6 catalyzed by immobilized Candida antarctica lipase B. The conversion of geraniol in [bmim]PF6 was significant although the reaction rate was slower than in organic solvents. The profile of initial reaction rate-water activity was determined experimentally, and differed from the data reported for other non-aqueous solvents. A maximum in the initial reaction rate was found at aw = 0.6. The pseudo reaction equilibrium constant, Kx, was measured experimentally for the reaction. The average value of Kx in [bmim]PF6 was 12, 20-fold lower than the value reported for the same system in hexane.