Susana Barreiros

A comparative study of biocatalysis in non-conventional solvents: Ionic liquids, supercritical fluids and organic media

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.

Novozym 435 activity in compressed gases. Water activity and temperature effects

Abstract 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.

Lipase-catalyzed enantioselective esterification of glycidol in supercritical carbon dioxide

Abstract 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.

Production of polyhydroxyalkanoates from spent coffee grounds oil obtained by supercritical fluid extraction technology

Spent coffee grounds (SCG) oil was obtained by supercritical carbon dioxide (scCO2) extraction in a pilot plant apparatus, with an oil extraction yield of 90% at a 35kgkg(-1) CO2/SCG ratio. Cupriavidus necator DSM 428 was cultivated in 2L bioreactor using extracted SCG oil as sole carbon source for production of polyhydroxyalkanoates. The culture reached a cell dry weight of 16.7gL(-1) with a polymer content of 78.4% (w/w). The volumetric polymer productivity and oil yield were 4.7gL(-1)day(-1) and 0.77gg(-1), respectively. The polymer produced was a homopolymer of 3-hydroxybutyrate with an average molecular weight of 2.34×10(5) and a polydispersity index of 1.2. The polymer exhibited brittle behaviour, with very low elongation at break (1.3%), tensile strength at break of 16MPa and Youngs Modulus of 1.0GPa. Results show that SCG can be a bioresource for polyhydroxyalkanoates production with interesting properties.

Lipase catalysed mono and di-acylation of secondary alcohols with succinic anhydride in organic media and ionic liquids

The acylation of the model substrate (R,S)-2-octanol with succinic anhydride catalysed by immobilised Candida antarcticalipase B was studied in two water-miscible organic solvents, two water-immiscible ones, nine 1-alkyl-3-methylimidazolium ionic liquids (RTILs) and two quaternary ammonium RTILs. From previous reports, the reaction was expected to yield an acidic half ester (hemiester). However, it was found that the diester was also produced, in many cases at an even higher yield. The major reaction products were the (R)-hemiester and the (R,R)-diester. In some of the solvents, the amount of hemiester formed increased and then remained constant, whereas in others the amount of hemiester first peaked and then decayed. This behaviour could be explained by the coupling of two reaction pathways: the hydrolysis of the hemiester, and the esterification of the hemiester. The solubility limit of succinic acid, produced in the former pathway, was found to be a discriminating parameter, the precipitation of the acid acting as a driving force for the consumption of the hemiester formed. The impact of the choice of solvent on the outcome of the acylation with succinic anhydride was confirmed by conducting the reaction on a preparative scale: in acetonitrile, the reaction produced 32% (w/w) of (R)-hemiester and 68% (w/w) of (R,R)-diester, whereas in tetrahydrofuran 85% (w/w) of (R)-hemiester and 15% (w/w) of (R,R)-diester were obtained.

Effect of pressure on the catalytic activity of subtilisin Carlsberg suspended in compressed gases.

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.

Enzymatic resolution of Indinavir precursor in ionic liquids with reuse of biocatalyst and media by product sublimation

Efficient enzymatic resolution of (±)-cis-benzyl N-(1-hydroxyindan-2-yl)carbamate in [aliq][N(CN)2] by acylation using CALB as a biocatalyst was achieved, with the possibility of catalyst and medium reuse. The reaction proceeds to the formation of the corresponding acetate in moderate yield and high enantiomeric excess for the first and second cycles. Additionally, the biocatalyst can be recovered by filtration and reaction products can be easily removed from the ionic liquid by direct sublimation under high vacuum.

Design of controlled release systems for THEDES—Therapeutic deep eutectic solvents, using supercritical fluid technology

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.

Solvent effects on the catalytic activity of subtilisin suspended in compressed gases.

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.

Dissolution enhancement of active pharmaceutical ingredients by therapeutic deep eutectic systems

A therapeutic deep eutectic system (THEDES) is here defined as a deep eutectic solvent (DES) having an active pharmaceutical ingredient (API) as one of the components. In this work, THEDESs are proposed as enhanced transporters and delivery vehicles for bioactive molecules. THEDESs based on choline chloride (ChCl) or menthol conjugated with three different APIs, namely acetylsalicylic acid (AA), benzoic acid (BA) and phenylacetic acid (PA), were synthesized and characterized for thermal behaviour, structural features, dissolution rate and antibacterial activity. Differential scanning calorimetry and polarized optical microscopy showed that ChCl:PA (1:1), ChCl:AA (1:1), menthol:AA (3:1), menthol:BA (3:1), menthol:PA (2:1) and menthol:PA (3:1) were liquid at room temperature. Dissolution studies in PBS led to increased dissolution rates for the APIs when in the form of THEDES, compared to the API alone. The increase in dissolution rate was particularly noticeable for menthol-based THEDES. Antibacterial activity was assessed using both Gram-positive and Gram-negative model organisms. The results show that all the THEDESs retain the antibacterial activity of the API. Overall, our results highlight the great potential of THEDES as dissolution enhancers in the development of novel and more effective drug delivery systems.