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Enzymatic transesterification is becoming a commercially competitive route to biodiesel. Supercritical CO2 (scCO2) is well established as a solvent for extraction processes. Here we analyze the economy of a scCO2-based, enzymatic process, for the production of biodiesel from waste cooking sunflower oil (WCO). The conversion of WCO to fatty acid ethyl esters (FAEE) catalyzed by immobilized lipase from Thermomyces lanuginosus (Lipozyme TL IM), and downstream separation to recover biodiesel conforming to the EN14214 norm, were studied in a pilot plant unit. The data generated was used to design an envisaged industrial plant, for which an energy balance was carried out. Investment and production costs were estimated for the conversion of 8000 tonWCO/year. This led to biodiesel costs of 1.64€/L and 0.75€/L (for a WCO price of 0.25€/kg; enzyme prices of 800€/kg and 8€/kg, respectively), which already reflect the ca. 10% impact of glycerol sales.

The continuous kinetic resolution of (R, S)-1-phenylethanol via enzymatic transesterification was chosen as a benchmark reaction for the study of a complete reaction/separation process using supercritical carbon dioxide. Phase equilibrium data for binary and ternary systems was acquired as a starting point to determine the best conditions of operation. Total conversion of the (R)-isomer of the alcohol was achieved using a 10% molar excess of vinyl laurate. Three separators operating at given temperatures and pressures allowed the recovery of (S)-1-phenylethanol with a purity of 86%. The phase equilibrium data obtained indicates that an additional separation step should allow the recovery of both (S)-1-phenylethanol and (R)-1-phenylethylaurate with over 95% purity. Due to the low solubilities of the target compounds, CO2 from the outlet gas stream of the last separator must be recycled to ensure the technical viability of the integrated reaction/separation of (R, S)-1-phenylethanol.

This paper presents vapor-liquid equilibrium (VLE) data for systems containing high-pressure CO2, an ionic liquid, and products of an enzymatic reaction between succinic anhydride and (R,S)-2-octanol in the presence of a lipase. VLE experiments were used to calculate partition coefficients of reaction products between the ionic liquid-rich phase and the CO2-rich phase. Experiments of extraction with CO2 from an ionic liquid of (S)-2-octanol in conditions similar to those of the postreaction mixture were also carried out. The extraction at 11 MPa and 35 degrees C allows one to recover completely the unreacted enantiomer of 2-octanol with very high enantiomeric excess (98.42%).

Deep eutectic solvents (DES) can be formed by bioactive compounds or pharmaceutical ingredients. A therapeutic DES (THEDES) based on ibuprofen, a non-steroidal anti-inflammatory drug (NSAID), and menthol was synthesized and its thermal behavior was analyzed by differential scanning calorimetry (DSC). A controlled drug delivery system was developed by impregnating a starch:poly-ϵ-caprolactone polymeric blend (SPCL 30:70) with the menthol:ibuprofen THEDES in different ratios (10 and 20 wt%), after supercritical fluid sintering at 20 MPa and 50 °C. The morphological characterization of SPCL matrices impregnated with THEDES was performed by scanning electron microscopy (SEM) and micro-computed tomography (micro-CT). Drug release studies were carried out in a phosphate buffered saline. The results obtained provide important clues for the development of carriers for the sustainable delivery of bioactive compounds., Projects ENIGMA - PTDC/EQU-EPR/121491/2010, PTDC/QUI-QUI/119210/2010, PTDC/EQU-EQU/122106/2010, PEst-C/EQB/LA0006/2013 from the European Union’s Seventh Framework Programme (FP7/2007-2013) under grant agreement n° REGPOT - CT2012-316331-POLARIS and from Project “Novel smart and biomimetic materials for innovative regenerative medicine approaches (Ref.: RL1 - ABMR - NORTE-01-0124-FEDER-000016)” co-financed by North Portugal Regional Operational Programme (ON.2–O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF).

We performed a comparative study on the activity of Fusarium solani pisi cutinase immobilized on zeolites NaA and NaY, in n-hexane, acetonitrile, supercritical ethane (sc-ethane) and sc-CO2, at two different water activity (aW) values set by salt hydrate pairs in situ and at acid–base conditions fixed with solid-state buffers of aqueous pKa between 4.3 and 10.6. The reaction studied was the transesterification of vinyl butyrate by (R,S)-2-phenyl-1-propanol. The transesterification activity of cutinase was highest and similar in sc-ethane and in n-hexane,about one order of magnitude lower in acetonitrile and even lower in sc-CO2. Activity coefficients (γ) generated for the two substrates indicated that they were better solvated in acetonitrile and thus less available for binding at the active site than in the other three solvents. γ data also suggested higher reaction rates in sc-ethane than in n-hexane, as observed, and provided evidence for a direct negative effect of sc-CO2 on enzyme activity. Manipulation of the acid–base conditions of the media did not afford any improvement of the initial rates of transesterification relative to the blanks (no added acid–base buffer, only salt hydrate pair), except in the case of cutinase immobilized on zeolite NaA in sc-ethane at aW = 0.7. The poor performance of the blank in this case and the great improvement observed in the presence of a basic buffer suggest a deleterious acidic effect in the medium which, an experiment without additives confirmed, was not due to the known acidic character of the salt hydrate pair used to set aW = 0.7. In acetonitrile, increasing aW was accompanied by a decrease in initial rates of transesterification, unlike in the other solvents. There was considerable hydrolysis in acetonitrile, where initial rates of hydrolysis increased about 20-fold from aW = 0.2 to 0.7. Hydrolysis was less pronounced in sc-ethane and in n-hexane, and only at aW = 0.7, and in sc-CO2 butyric acid was detected only at very long reaction times, in agreement with a generally low catalytic activity. Cutinase enantio-selectivity towards the alcohol substrate was low and unaffected by any manipulation of medium conditions. This work has been supported by Fundação para a Ciência e Tecnologia (FCT, Portugal) through the contracts PRAXIS/PBIO/14314/1998 and POCTI/35429/QUI/2000 and the grant PRAXIS XXI/BD/21615/99 (S. Garcia), and by FEDER.We thank Ricardo Baptista for help in the production of cutinase.

The catalytic activities of cutinase immobilized on zeolite NaY and Candida antarctica lipase B immobilized on an acrylic resin (Novozym 453) were measured in a model transesterification reaction in three imidazolium cation-based ionic liquids (RTILs), supercritical ethane (sc-ethane), sc-CO2 and n-hexane, at a water activity (aW) of 0.2 and 0.7. The transesterification activity of cutinase was highest and similar in 1-n-butyl-3-methylimidazolium hexafluorophosphate ([C4mim][PF6]), sc-ethane and n-hexane, more than one order of magnitude lower in sc-CO2, and increased with an increase in aW. Hydrolysis was not detected in sc-fluids and n-hexane, and was observed in RTILs at aW 0.7 only. Both initial rates of transesterification and of hydrolysis of Novozym decreased with an increase in aW. sc-CO2 did not have a deleterious effect on Novozym activity, which was as high as in sc-ethane and n-hexane. The low reaction rates obtained in this case in RTILs suggested the existence of internal diffusion limitations absent in the cutinase preparation where the enzyme is only adsorbed at the surface of the support. sc-CO2 did not adversely affect the catalytic activity of cutinase suspended in [C4mim][PF6], suggesting a protective effect of the RTIL. In the case of Novozym, a marked increase in the rate of transesterification was obtained in the [C4mim][PF6]/sc-CO2 system, compared to the RTIL alone. This may reflect improved mass transfer of solutes to the pores of the immobilization matrix due to a high concentration of dissolved CO2 and a reduction in viscosity of the RTIL.

We have previously demonstrated that a solid-state buffer could be successfully used to control the ionization state of subtilisin Carlsberg cross-linked microcrystals (CLECs) suspended in supercritical ethane (sc-ethane) in the presence of acid-base active species such as salt hydrates and zeolite molecular sieves. Here we studied the effect of six zwitterionic proton/sodium (pH-pNa) solid-state acid-base buffers on the catalytic activity of subtilisin CLECs in sc- ethane at high and low water activity (a(W)). CLECs were strongly activated by increasing a(W). At high a(W), and despite the high hydrolysis rates, transesterification activities were still about one order of magnitude higher than those observed at lower a(W). This is in contradiction with what was previously reported in the absence of acid-base control and supports the hypothesis that the poor catalytic performance of subtilisin CLECs at high a(W) observed in those studies was due to the inhibitory effect of the hydrolytic by- product, rather than to the competition of water with propanol for the acyl-enzyme intermediate. Although the catalytic activity of subtilisin showed a general positive correlation with the aqueous pK(a) of the acid-base buffers tested here, our results also show that as expected, the acid-base behavior of the buffers in nonaqueous media is more complex than what can be predicted from aqueous-based parameters alone. This work further confirms the usefulness of solid-state acid-base buffers in supercritical biocatalysis but highlights the need for further research on the topic.

Zeolite molecular sieves very commonly are used as in situ drying agents in reaction mixtures of enzymes in nonaqueous media. They often affect enzyme behavior, and this has been interpreted in terms of altered hydration. Here, we show that zeolites can also have dramatic acid-base effects on enzymes in low water media, resulting from their cation-exchange ability. Initial rates of transesterification catalyzed by cross-linked crystals of subtilisin were compared in supercritical ethane, hexane, and acetonitrile with water activity fixed by pre-equilibration. Addition of zeolite NaA (4 A powder) still caused remarkable rate enhancements (up to 20-fold), despite the separate control of hydration. In the presence of excess of an alternative solid-state acid-base buffer, however, zeolite addition had no effect. The more commonly used Merck molecular sieves (type 3 A beads) had similar but somewhat smaller effects. All zeolites have ion-exchange ability and can exchange H+ for cations such as Na+ and K+. These exchanges will tend to affect the protonation state of acidic groups in the protein and, hence, enzymatic activity. Zeolites pre-equilibrated in aqueous suspensions of varying pH-pNa gave very different enzyme activities. Their differing basicities were demonstrated directly by equilibration with an indicator dissolved in toluene. The potential of zeolites as acid-base buffers for low-water media is discussed, and their ability to overcome pH memory is demonstrated.

We report on the performance of cross-linked enzyme microcrystals (CLECs) of subtilisin Carlsberg in supercritical fluids (SC-fluids). The catalytic activity of CLECs in SC- ethane was found to be 2- to 10-fold greater than in hexane under the same conditions, using CLECs dried by propanol washing. Air-dried CLECs and lyophilized powders showed much lower activities, reflecting the same hydration hysteresis effects as in organic solvents. Reaction rates were much lower in SC-CO2, especially at higher water activity, probably as a result of acid-base effects of carbonic acid on the enzyme.

We studied a model transesterification reaction catalyzed by subtilisin Carlsberg suspended in carbon dioxide, propane, and mixtures of these solvents under pressure. To account for solvent effects due to differences in water partitioning between the enzyme and the bulk solvents, we measured water sorption isotherms for the enzyme in each solvent. We measured catalytic activity as a function of enzyme hydration and obtained bell-shaped curves with maxima at the same enzyme hydration (12%) in all the solvents. However, the activity maxima were different in all media, being much higher in propane than in either CO2 or the mixtures with 50 and 10% CO2. Considerations based on the solvation ability of the solvents did not offer an explanation for the differences in catalytic activity observed. Our results suggest that CO2 has a direct adverse effect on the catalytic activity of subtilisin. © 1996 John Wiley & Sons, Inc.

We studied the performance of Fusarium solani pisi cutinase, immobilized on a zeolite, in supercritical fluids. The catalytic activity of the enzyme was strongly dependent on water activity, was unaffected by pressure up to 300 bar, and was higher in supercritical ethylene than in supercritical carbon dioxide. The enzyme was very selective toward one of the isomers of 1-phenylethanol, with an enantiomeric excess of virtually 100%, regardless of water activity, pressure, solvent, and temperature. We used the X-ray crystal structure of the enzyme and did a computer modeling of the structures of the transition states formed by the two enantiomers. The differences between these structures helped elucidate the preference for the (R)-enantiomer.

The transesterification reaction of n -butyl acetate by 1-hexanol catalyzed by Novozym 435 (immobilized Candida antarctica lipase B from Novo Nordisk) suspended in near-critical carbon dioxide, near-critical ethane, and compressed propane at 35°C and 100 bar was studied. Separate equilibration of the solvents with mixtures of salt hydrates known to give a certain water activity ( a w ) allowed us to convert water concentration into water activity. In all the solvents, the catalytic activity of Novozym was nearly insensitive to a w up to saturation. Reaction rates were higher in propane and ethane than in CO 2 , possibly due to the direct adverse effect of CO 2 already observed with other enzymes. By sieving the enzyme preparation into different size series, it was possible to verify the existence of internal diffusion limitations. These were significantly more severe in propane than in the other two solvents which can be explained by the fact that propane behaves like a conventional liquid in this study whereas CO 2 and ethane are closer to the critical point. An increase in temperature at constant pressure activated the enzyme in the three solvents. This effect was most pronounced in CO 2 and comparable in ethane and propane. In propane, activation by temperature was almost negligible when the whole enzymatic preparation was used which should reflect the existence of more severe mass transfer limitations in this solvent. Experiments at fixed density showed that the activation of Novozym in ethane was solely a temperature effect whereas in CO 2 , this effect was combined with one mediated by changes in solvation.

In this work, blend of starch and poly-e-caprolactone (PCL) doped with different concentrations of 1-butyl-3-methylimidazolium acetate ([BMIM]Ac) or 1-butyl-3-methylimidazolium chloride ([BMIM]Cl) was studied. The blends were characterized by mechanical analysis, infra-red spectroscopy (FTIR), differential scanning calorimetry (DSC) and dielectric relaxation spectroscopy (DRS), evaluating the IL doping effect. The samples were submitted to supercritical carbon dioxide foaming and the morphology of the structures was assessed. DSC shows a single glass transition and melting endotherm for foamed and unfoamed samples, undergoing no effect upon IL doping, and DRS shows increased molecular mobility for blends with higher IL concentrations, and some hindrance for lower ones. Conductivity for SPCL doped with 30% [BMIM]Cl, before and after foaming, is comparable to conductivity of the IL but exhibiting more stable conductivity values, opening doors for applications as self-supported conductive materials., The research leading to these results has received funding from Fundacao da Ciencia e Tecnologia (FCT) through the project ENIGMA - PTDC/EQU-EPR/121491/2010 and from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no. REGPOT-CT2012-316331-POLARIS, PEst-C/EQB/LA0006/2013 and FEDER Marta Martins Rita Craveiro and Alexandre Paiva are grateful for financial support from Fundacao da Ciencia e Tecnologia (FCT) through the grant PTDC/EQUEPR/12191/2010/ENIGMA, BIM/PTDC/EQU-EPR/ 121491/2010/ENIGMA and SFRH/BD/47088/2008.

The enzymatic resolution of racemic glycidol in supercritical CO2 is described. The enzymatic route chosen was the esterification with butyric acid catalyzed by either free or immobilized porcine pancreatic lipase. The solubility of glycidol, the least soluble reactant, was measured in CO2 at 35°C and pressures in the range 70–180 bar for concentrations of butyric acid up to 500 mM. The pressure selected for subsequent experiments was 140 bar. The partitioning of water between CO2 and the enzyme preparations was quantified for various amounts of added water. The experimental sorption isotherms obtained were used to derive the water content of the enzyme preparations assayed in kinetic studies. For the free enzyme, maximum reaction rates were obtained for an enzyme water content of 10 ± 2% w/w, the same as in organic solvents, although the corresponding initial rate of 0.0045 ± 0.0003 m h−1 g−1 was much lower than for these solvents. A series of supports covering a wide range of hydrophilicities were screened for enzyme activity, the best results being for the more hydrophilic ones, Sephadex G-25 and Bio-gel P6. Enzyme immobilized on these supports performed better than the free enzyme, leading to optimized initial rates of 0.0110 ± 0.0007 m h−1 g−1 and purities of 83 ± 2% of (S)-glycidyl butyrate, at 25–30% conversion and 20–25% hydration of the enzyme preparations. The selectivity observed is the same as the highest value obtained in organic solvents. As in those solvents, enantioselectivity in CO2 does not depend on enzyme hydration.

The esterification reaction of geraniol with acetic acid (100 mM/100 mM) catalyzed by immobilized Candida antarctica lipase B (Novozym 435) was studied in supercritical carbon dioxide (sc-CO2) and in sc-ethane in a packed-bed reactor(PBR). In sc-CO2 it was easy to adjust the water activity (aw) in the reaction mixture to levels leading to good enzyme performance. Enzyme stability was high and steady-state conversions could be achieved that exceeded the largest conversions measured in batch stirred-tank reactors (BSTRs), which is probably due to the lower aw levels achieved in the PBR. In sc-ethane, where the solubility of water is lower, high steady-state conversions could be attained only by preventing the accumulation on the enzyme bed of the water produced during reaction. The kinetic parameters for the reaction in sc-CO2 were determined using previously published data obtained in a BSTR, and a model was developed for the PBR that included those kinetic parameters. This model was able to predict with reasonable accuracy the behavior of the PBR. Slight differences were observed for some operating regions, probably due to the influence of aw in the activity of the enzyme, which is not included in the model. This work was supported by Fundação para a Ciência e Tecnologia (FCT, Portugal) through grants SFRH/BD/13084/2003 (A.S.R.) and SFRH/BPD/41546/2007 (P.V.), FEDER, and by the European Commission in the framework of the Marie Curie Research Training Network “Green Chemistry in Supercritical Fluids: Phase Behaviour, Kinetics and Scale-up” (EC Contract No. MRTN-CT-2004-504005). We thank Novo Nordisk Bioindustrial, Spain, for the gift of Novozym 435.

The kinetics of the lipase-catalyzed synthesis of decyl acetate, by the transesterification reaction of vinyl acetate with decanol, was investigated at 30 °C using n-hexane as the solvent. Novozym 435 was found to be the most active catalyst among the immobilized lipases tested. Given the nonideality of the reaction mixture, only a thermodynamic activity-based kinetics was found to be suitable to represent the experimental data in the entire range of compositions tested (0.1-1.4 M). The reaction follows a ping-pong bi-bi mechanism, in which inhibition only by excess of alcohol was identified. Although intraparticle diffusional limitations were detected, intrinsic kinetic parameters were obtained by crushing the catalyst particles. The results were compared to those obtained with supercritical CO2 as the solvent. For the conditions tested, Candida antarctica lipase B showed higher activity in the organic medium. The authors thank Fundacção para a Ciência e Tecnologia (Portugal) for A.S.R.’s Ph.D. grant (SFRH/BD/13084/2003) and for the financial support of the project POCI/EQU/56732/2004, FEDER for financing LSRE (FEDER/POCI/2010), Professor Romualdo Salcedo from FEUP for the optimization code, and Novozymes and Amano Enzyme Inc. for kindly providing the catalysts.

The esterification reaction of geraniol with acetic acid catalyzed by Novozym was studied in supercritical ethane (sc-ethane) and in supercritical carbon dioxide (sc-CO(2)). Water activity (a(W)) had a very strong effect on enzyme activity, with reaction rates increasing up to a(W) = 0.25 and then decreasing for higher a(W). Salt hydrate pairs could not prevent changes in a(W) during the course of reaction but were able to control a(W) to some extent and had a beneficial effect on both initial rates of esterification and conversion in sc-ethane. The enzyme was more active in sc-ethane than in sc-CO(2), confirming the deleterious effect of the latter already observed with some enzymes. Temperatures between 40 and 60 degrees C did not have a strong effect on initial rates of esterification, although reaction progress declined considerably in that temperature range. For the mixture of 50 mM acetic acid plus 200 mM geraniol, 100% conversion was achieved at a reaction time of 10 h at 40 degrees C, 100 bar, an a(W) of incubation of 0.25, and a Novozym concentration of 0.55 mg cm(-)(3) in sc-ethane. Conversion was below 50% in sc-CO(2) at otherwise identical conditions. With an equimolar mixture of the two substrates (100 mM), 98% conversion was reached at 10 h of reaction in sc-ethane (73% conversion in sc-CO(2)).

We studied the effect of pressure up to 300 bar on the catalytic efficiency of subtilisin Carlsberg suspended in compressed propane, near-critical ethane, near-critical carbon dioxide and tert-amyl alcohol, at constant temperature and fixed enzyme hydration. Increasing pressure lowered the catalytic efficiency of the enzyme in all the solvents, resulting in positive activation volumes, delta V#. The delta V# values in compressed propane and in tert-amyl alcohol were similar and larger in magnitude than the value reported in the literature for the same reaction in an aqueous buffer, although within the range of typical delta V# values in aqueous media. In the near-critical fluids, the delta V# were much larger, e.g., an increase in pressure of only 200 bar causing a sixfold decrease in the catalytic efficiency of subtilisin in carbon dioxide. These data should reflect the proximity of ethane and carbon dioxide to the critical point, and the resulting condensation of solvent molecules about the solutes, yielding negative solute partial molar volumes.