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Aim We aim to explore the non-structural sugars from white wine grape pomace (WWGP) as the input carbon source for the co-production of multiple high-value products by the non-fastidious yeast Rhodotorula babjevae to create a sustainable and economically appealing process. Methods and Results Water extraction of unfermented, soluble sugars from WWGP yielded extracts with similar amounts of glucose and fructose, which were used to prepare a growth medium. Rhodorotula babjevae multiplied as fast on WWGP-based medium as on a reference medium but achieved higher cell dry weight (CDW) and lower intracellular triacylglycerol accumulation (22.5% vs. 28.6%) in WWGP-based medium. In addition, R. babjevae produced mannitol and arabitol and carotenoids and secreted polyol esters of fatty acids, a rare type of glycolipid as confirmed by Fourier transform-infrared, nuclear magnetic resonance and high-performance liquid chromatography analyses. Remarkably, R. babjevae consumed simultaneously both fructose and glucose when on WWGP-based medium and left glucose practically untouched in the reference medium, evidencing a fructophilic character. Conclusions Rhodorotula babjevae, a metabolic versatile yeast, proliferated on a minimally processed extract and successfully converted glucose and fructose into high-value products. Significance and Impact of Study Different chemicals with market potential can be produced through the valorization of abundant waste feedstocks generated by the wine industry to which R. babjevae can contribute.

Spent coffee grounds (SCG) were treated with subcritical water (SBW) in a semi-continuous reactor at different temperatures, pressures, and water flowrates. Yields of carbohydrates and phenolics reached 33.7 g and 4 g per 100 g-dry SCG, respectively, at 200 oC, 70 bar and 10 mL/min. Liquors collected at the beginning of assays were richer in phenolics and poorer in carbohydrates, whose yield increased sharply when temperature reached 150 oC. Most carbohydrates were recovered as oligosaccharides, and practically all cellulose remained in the SCG recovered at the end of assays. Extracts exhibited DPPH antioxidant capacity and antibacterial activity against both Gram-positive and Gram-negative bacteria.

UID/AMB/04085/2019. LAQV UID/QUI/50006/2019. PD\BD\135170\2017. IF/01146/2015. Tungsten is a critical raw material for European and U.S. economies. Tungsten mine residues, usually considered an environmental burden due to e.g. arsenic content, are also secondary tungsten resources. The electrodialytic (ED) process and deep eutectic solvents (DES) have been successfully and independently applied for the extraction of metals from different complex environmental matrices. In this study a proof of concept demonstrates that coupling DES in a two-compartment ED set-up enhances the removal and separation of arsenic and tungsten from Panasqueira mine secondary resources. Choline chloride with malonic acid (1:2), and choline chloride with oxalic acid (1:1) were the DES that in batch extracted the average maximum contents of arsenic (16%) and tungsten (9%) from the residues. However, when ED was operated at a current intensity of 100 mA for 4 days, the extraction yields increased 22% for arsenic and 11% for tungsten, comparing to the tests with no current. From the total arsenic and tungsten extracted, 82% and 77% respectively were successfully removed from the matrix compartment, as they electromigrated to the anolyte compartment, from where these elements can be further separated. This achievement potentiates circular economy, as the final treated residue could be incorporated in construction materials production, mitigating current environmental problems in both mining and construction sectors. proof published

The aim of this study was to valorize spent coffee grounds (SCG) into bioactive extracts for improving skin health. These extracts were obtained by subcritical water extraction (SWE) at 100 bar and temperatures up to 220 °C, in a semicontinuous mode. They were analyzed for phenolic acids, carbohydrates, antioxidant activity (AA) measured by the DPPH assay, ROS-scavenging activity in keratinocyte cells, and elastase and tyrosinase inhibitory activity. SCG extracts collected up to 140 °C had higher phenolic acids content (19.9 mgGA/gdry SCG), higher AA (EC50 of 20.6 μg mL–1), and lower carbohydrate content (38 mgsugars/gdry SCG) than the fraction collected from 140 to 220 °C (5.7 mgGA/gdry SCG, EC50 of 132.2 μg mL–1, and 286 mgsugars/gdry SCG). The extracts were proven to have antiaging and skin lightening effects by inhibiting elastase (99% and 97.9%) and tyrosinase (78.6% and 92.1%) activity. The extracts were incorporated in shear thinning and acidic hydrogels for topical application. Release (70%) and p...

Deep Eutectic Systems (DESs) are an emerging class of green solvents with a myriad of applications, from biotechnology to material science. The high viscosity of most DESs is an obstacle that can be circumvented by the addition of water. But how much water is too much water? There is a need to understand the effect of water in the organizational dynamics of DESs, and to clarify the role of water on the physical properties of the DES, and as a functional additive. In this work, NMR spectroscopy was used to study choline chloride:glycerol (at a 1:2 molar ratio) systems with added water, at water contents ranging from 1 wt% (xw = 0.06) to 70 wt% (xw = 0.94). We identified three distinct water behaviour domains. Up to a water content of 11 wt%, water does not disrupt the DES structure; in fact, water has a glue-like effect, acting as a bridge between the DES components. Between water contents of 11 wt% and 35 wt%, the solvation of the DES components starts to occur, but the DES nanostructure is still present. At 35 wt% of water, the DES structure is disrupted, and the system transitions to a DES-in-water solution.

Recycled paper mill sludge (RPMS) was hydrothermally treated by subcritical water (SBW) in two modes of operation, namely semi-continuous and batch, at temperatures in the range 150–250 °C, and 100 bar. The performance of both systems was analyzed in terms of degree of hydrolysis, product yield and formation of degradation products. The highest degree of RMPS hydrolysis was achieved using the batch reactor configuration, while in the semi-continuous system higher yields of water soluble compounds (WSC) and monosaccharides were achieved. By using the semi-continuous system at 100 bar, 250 °C and a water flow rate of 5 mL/min, 40% of the organic carbon content of RPMS was recovered in the form of soluble compounds. Additionally 20% of total carbohydrates and 75% of total lignin of RPMS were recovered as soluble material. Detailed mass balances performed for both operational modes revealed that under batch mode, the naturally occurring compounds found in RPMS underwent significant transformation, generating derivatives referred to as degradation products. An analysis of the severity factor was performed for both operational modes. It was concluded that significant values of WSC were only obtained for severity factors higher than four.

In this work we report a new way of producing wrinkled soft Janus spheres by using scCO 2 . The wrinkles appear in one of the half-hemispheres by the asymmetric UV irradiation of the particles followed by an anisotropic scCO 2 swelling. This treatment allows the fabrication of banded as well as sinusoidal packed wrinkles in the irradiated spheres surface. The particles were prepared from an elastomeric network consisting of two soft segments, polypropylene oxide and polybutadiene. The spheres to be treated by scCO 2 were selected taking into account the chemical composition as well as the elastic and wettability characteristics of the materials.

Liquid water at temperatures above the boiling point and high pressures, also known as pressurized hot water, or subcritical water (SBCW), is an effective solvent for both polar and non-polar organic solutes. This is often associated to the decrease of water's dielectric constant at high temperatures, apparently allowing water to behave like an organic solvent. The decrease of the solubility at high pressures, in turn, is explained by a mild increase of the dielectric constant of water. Nevertheless, the relationship between the dielectric constant of water, hydration, and the solubility of polar and non-polar molecules in SBCW, remains poorly understood. Here, we study through molecular dynamics, the hydration thermodynamic parameters and the solubility of non-polar and polar aromatic model systems, for which a solubility increase in SBCW is observed. We show that the temperature dependence of the hydration free energy of the model non-polar solutes is nonmonotonic, exhibiting a solute size independent maximum at ~475 K, above which hydration becomes entropically favorable and enthalpically unfavorable. The monotonic increase of the solubility, separated here in hydration and vaporization or sublimation components of the pure liquid or solid solute, respectively, is, in turn, related to the temperature increase of the latter, and only to a minor extent with the decrease of the hydration free energy above ~475 K, via the hydration entropy. A solubility increase or decrease is also found at high pressures for different solutes, explained by the relative magnitude of the hydration and the vaporization or sublimation components of the solubility. For the model solid polar system studied, the hydration free energy increases monotonically with the temperature, instead, and the solubility increase is caused by the decrease of the sublimation component of the solubility. Thus, despite of the observed increase of the hydration free energy with pressure, related to the entropic component decrease, our results indicate that the dielectric constant plays no significant role on the solubility increase of non-polar and polar solutes in SBCW, opposite to the dielectric constant picture. The structure of water next to the solutes is also investigated and a structural enhancement at room temperature is observed, resulting in significantly stronger pair interactions between a water molecule and its third and fourth nearest water neighbors. This structural and energetic enhancement nearly vanishes, however, at high temperatures, contributing to a positive hydration entropy.

Enzymatic esterification is an effective route that can contribute for the separation of racemic (rac) mixtures and is thus of great importance for the pharmaceutical, food, and cosmetic industries...

We applied Pulsed Gradient Spin Echo diffusion with high-resolution magic angle spinning NMR to study sol-gel matrices used to encapsulate enzymes for biocatalysis (TMOS/MTMS and TMOS/BTMS) to gain insight into the local chemical microenvironment. Transport properties of solvents with different polarities (1-pentanol, acetonitrile and n-hexane) were studied through their apparent self-diffusion coefficients. The spin echo attenuation of the solvents shows two distinct diffusion domains, one with fast diffusion (Dfast ) associated with interparticle diffusion and another with slow diffusion (Dslow ) corresponding to the displacement inside the pores within the sol-gel particles. The analysis of the root mean square displacements at different diffusion times showed that the Dfast domain has a free diffusion regime in both matrices (the root mean square displacement is linearly dependent of the diffusion time), while the Dslow domain shows a different regime that depends on the matrix. We investigated the exchange regime between the two diffusion sites. In both matrices, n-hexane was in intermediate exchange between diffusion domains, while the polar solvents were in slow exchange in TMOS/BTMS and in intermediate exchange in TMOS/MTMS. Data were fitted for TMOS/BTMS with the Karger model, and the physical parameters were obtained. The results add to the evidence that the pores are a hydrophobic environment but that the presence of some free hydrophilic groups inside the pore, as observed in the TMOS/BTMS, has a key role in slowing down the exchange of polar solvents and that this is relevant to explain previously reported enzyme activity in these materials. Copyright © 2016 John Wiley & Sons, Ltd.

The fractionation of red wine grape pomace (RWGP) by subcritical water (SBW) extraction/hydrolysis was studied in a semi-continuous reactor. Different temperature programs were used, comprising ramps and plateaus at different temperatures, with different time lengths. Pressure was kept constant at 100 bar. A program applied to previously defatted RWGP (def-RWGP) featuring plateaus at 50, 100 and 190 °C allowed the recovery of virtually all of the hemicellulose, while leaving in the residue essentially all of the lignin and the cellulose. The latter was readily hydrolyzed enzymatically. When using whole RWGP, it was shown that over 85 % of the lipids remained in the residue. The RWGP extracts had contents of phenolic compounds up to 29 g/100 gextract, and were rich in hemicellulose-derived carbohydrates, mostly in the form of oligomers. The extracts exhibited an antioxidant capacity that compared favorably with that of gallic acid.

The continuous production of biodiesel (fatty acid methyl esters; FAME) from chicken feather meal (CFM) was carried out in supercritical carbon dioxide (sc-CO 2 ), in a process combining extraction of oil from CFM followed by transesterification of the CFM oil with methanol. First, extraction experiments were carried out at different pressure, temperature and solvent flow rate conditions to assess the influence of these process parameters on the extraction rate and composition of extracted oil. Over 90% of the oil available in CFM was extracted by sc-CO 2 in 60 min. Oleic acid (C18:1), palmitic acid (C16:0) and linoleic acid (C18:2) were the major components of the oil extracted, accounting for ca. 84% w/w. The integrated extraction and enzymatic transesterification of CFM oil was then carried out at 40 °C and 250 bar, at solvent flow rates in the range 30–150 g/min, for oil:methanol molar ratios of 1:6–1:24, using Lipozyme RM IM® as biocatalyst, in a pilot plant unit. The lowest FAME yield obtained was 96.7%. Both the extraction and reaction steps were modeled, based on a broken and intact cells approach and the consideration of three consecutive steps, respectively, and a good agreement with experimental data was obtained.

The amorphization of the readily crystallizable therapeutic ingredient and food additive, menthol, was successfully achieved by inclusion of neat menthol in mesoporous silica matrixes of 3.2 and 5.9 nm size pores. Menthol amorphization was confirmed by the calorimetric detection of a glass transition. The respective glass transition temperature, Tg = −54.3 °C, is in good agreement with the one predicted by the composition dependence of the Tg values determined for menthol:flurbiprofen therapeutic deep eutectic solvents (THEDESs). Nonisothermal crystallization was never observed for neat menthol loaded into silica hosts, which can indicate that menthol rests as a full amorphous/supercooled material inside the pores of the silica matrixes. Menthol mobility was probed by dielectric relaxation spectroscopy, which allowed to identify two relaxation processes in both pore sizes: a faster one associated with mobility of neat-like menthol molecules (α-process), and a slower, dominant one due to the hindered mobil...

White wine grape pomace (WWGP) was treated with subcritical water in a semi-continuous reactor, at 170, 190 and 210 °C. Assays at 210 °C led to highest recovery of carbohydrates (85% of total amount of carbohydrates of WWGP) and phenolic compounds (2.6 g/100 gWWGP). Moreover, 48% of initial lignin was solubilized. The amount of carbohydrates recovered increased sharply in the initial stages of the process, due to the extraction of soluble carbohydrates, and then less pronouncedly, as hydrolysis of WWGP took place. The difference in the progress curves for the yields of carbohydrates and of phenolics allowed the separate recovery of carbohydrate-rich, and phenolics-enriched, GP extracts. The latter exhibited 10 times higher antioxidant activity (AA) than a GP extract obtained by hydro-alcoholic extraction, and 8 times lower AA than pure ascorbic acid. WWGP extracts were also found to have 4 times higher antimicrobial activity against Gram-positive than Gram-negative bacteria. info:eu-repo/semantics/publishedVersion

Fast-dissolving delivery systems (FDDS) have received increasing attention in the last years. Oral drug delivery is still the preferred route for the administration of pharmaceutical ingredients. Nevertheless, some patients, e.g. children or elderly people, have difficulties in swallowing solid tablets. In this work, gelatin membranes were produced by electrospinning, containing an encapsulated therapeutic deep-eutectic solvent (THEDES) composed by choline chloride/mandelic acid, in a 1:2 molar ratio. A gelatin solution (30% w/ v) with 2% (v/v) of THEDES was used to produce electrospun fibers and the experimental parameters were optimized. Due to the high surface area of polymer fibers, this type of construct has wide applicability. With no cytotoxicity effect, and showing a fast-dissolving release profile in PBS, the gelatin fibers with encapsulated THEDES seem to have promising applications in the development of new drug delivery systems., The research leading to these results has received funding from Fundação para a Ciência e a Tecnologia (FCT) through the projects ENIGMA - PTDC/EQU-EPR/ 121491/2010 and UID/CTM/50025/2013, LAQVREQUIMTE: UID/QUI/50006/2013, UCIBIO-REQUIMTE: UID/Multi/04378/2013 (co-financed by the ERDF under the PT2020 Partnership Agreement [POCI-01-0145-FEDER- 007728]) and by FEDER through the COMPETE 2020 Programme. Marta Martins is grateful for financial support from FCT through the grant BIM/PTDC/EQUEPR/121491/ 2010/ENIGMA. This research has also received funding from the European Union Seventh Framework Programme (FP7/ 2007-2013) under grant agreement number REGPOTCT2012-316331-POLARIS and from the project BNovel smart and biomimetic materials for innovative regenerative medicine approaches^ 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)., info:eu-repo/semantics/publishedVersion

The phase equilibrium of the binary system (CO2 + propionic anhydride) was determined experimentally at temperatures of 308, 313, and 323 K and pressures up to 10 MPa. Measurements were carried out in a high-pressure visual cell with variable volume. The experimental data were modeled using the Peng–Robinson equation of state and the Mathias–Klotz–Prausnitz mixing rule. A good correlation was achieved with this model, with a total average absolute deviation of 0.21%.

Introducción: Se ha descrito una incidencia de hipofosfatemia en pacientes con soporte nutricional especializado (SNE) de hasta el 30-40%. La hipofosfatemia leve y la moderada son generalmente asintomáticas, mientras que la severa es el hecho fundamental del síndrome de realimentación. Objetivo: Evaluar la incidencia y gravedad de la hipofosfatemia en pacientes hospitalizados no críticos con nutrición enteral (NE). Material y métodos:Se diseñó un estudio observacional y prospectivo en condiciones de práctica clínica habitual. Se recogieron datos clínicos, antropométricos y analíticos de 181 pacientes a los que se les inició nutrición enteral. El seguimiento fue de 7 días. Resultados: El 51.9% de los pacientes estaban en riesgo de desarrollar síndrome de realimentación según las guías del United Kingdom National Institute For Health and Clinical Excellence (NICE). La incidencia de hipofosfatemia fue del 31.5%, la de la hipofosfatemia severa del 1.1%. De todos los parámetros clínicos, antropométricos y analíticos analizados sólo la edad y unas proteínas séricas más bajas se correlacionaron de forma estadísticamente significativa con el aumento en la incidencia de hipofosfatemia. Conclusiones: La incidencia de hipofosfatemia grave en nuestra serie es muy baja, lo que hace imposible extraer conclusiones específicas para este grupo de pacientes.

In this work the phase behavior of the ternary system ethanol/(±)-menthol/CO2 was studied at the temperatures of 313 and 323 K, in the pressure range 8–10 MPa. Experiments were performed using a high pressure phase equilibrium apparatus with a visual cell. CO2 is more selective toward ethanol, separation factors ranging from 1.6 to 7.6 at the conditions tested. The pTxy data obtained were correlated with the Peng–Robinson equation of state combined with the Mathias–Klotz–Prausnitz mixing rule. The model gave a good fitting to the data, with a total average absolute deviation of 3.7%, and was able to predict the occurrence of a three-phase region observed experimentally at 313 K and 9 MPa.

The thermal behavior and transport properties of several ion jellys (IJs), a composite that results from the combination of gelatin with an ionic liquid (IL), were investigated by dielectric relaxation spectroscopy (DRS), differential scanning calorimetry (DSC), and pulsed field gradient nuclear magnetic resonance spectroscopy (PFG NMR). Four different ILs containing the dicyanamide anion were used: 1-butyl-3-methylimidazolium dicyanamide (BMIMDCA), 1-ethyl-3-methylimidazolium dicyanamide (EMIMDCA), 1-butyl-1-methylpyrrolidinium dicyanamide (BMPyrDCA), and 1-butylpyridinium dicyanamide (BPyDCA); the bulk ILs were also investigated for comparison. A glass transition was detected by DSC for all materials, ILs and IJs, allowing them to be classified as glass formers. Additionally, an increase in the glass transition temperature upon dehydration was observed with a greater extent for IJs, attributed to a greater hindrance imposed by the gelatin matrix after water removal, rendering the IL less mobile. While crystallization is observed for some ILs with negligible water content, it was never detected for any IJ upon thermal cycling, which persist always as fully amorphous materials. From DRS measurements, conductivity and diffusion coefficients for both cations (D+) and anions (D-) were extracted. D+ values obtained by DRS reveal excellent agreement with those obtained from PFG NMR direct measurements, obeying the same VFTH equation over a large temperature range (ΔT ≈ 150 K) within which D+ varies around 10 decades. At temperatures close to room temperature, the IJs exhibit D values comparable to the most hydrated (9%) ILs. The IJ derived from EMIMDCA possesses the highest conductivity and diffusion coefficient, respectively, ∼10(-2) S·cm(-1) and ∼10(-10) m(2)·s(-1). For BMPyrDCA the relaxational behavior was analyzed through the complex permittivity and modulus formalism allowing the assignment of the detected secondary relaxation to a Johari-Goldstein process. Besides the relevant information on the more fundamental nature providing physicochemical details on ILs behavior, new doorways are opened for practical applications by using IJ as a strategy to produce novel and stable electrolytes for different electrochemical devices.

Pulsed field gradient spin echo high resolution magic angle spinning nuclear magnetic resonance spectroscopy is a powerful technique to characterize confined biosystems. We used this approach to assess the diffusion of solvent and reaction species within sol–gel matrices differing in enzyme loading.

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.

In this work for the first time Ion-Jelly® membranes were developed and characterized. Ion-Jelly® is a new green and sustainable gel material, obtained by addition of an ionic liquid to gelatine. Three different ionic liquids ([EMIM][DCA], [BMIM][DCA] and [BMPyr][DCA]), were used to study the difference in the membrane properties introduced by changes in the ionic liquid cation. The physical properties of the membranes were characterized by traction, contact angles, and SEM. These membranes were further tested in separation processes using supercritical carbon dioxide for the fractionation of a model mixture made of oleic acid and squalene and for the separation of biodiesel products and reagents. The permeability to pure gases (H2, N2, O2, CO2 and CH4) was also determined and ideal selectivities calculated.

Faculty of Pharmacy Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL) University of Lisbon

Up to 30-40% of the patients starting artificial nutritional support develop hypophosphatemia. In general, patients with mild and moderate hypophosphatemia do not have symptoms, but severe hypophosphatemia is the hallmark of refeeding syndrome.To determine the incidence of hypophosphatemia in not critically ill patients receiving enteral feeding.Prospective study. We assessed during seven days 181 not critically ill patients started on enteral artificial nutrition support during seven days.51.9% of the patients were considered to be at risk of developing refeeding syndrome (United Kingdom National Institute for Health and Clinical Excellence criteria). The incidence of hypophosphatemia was 31.5%, but only 1.1% of the patients developed severe hypophosphatemia. Older age and lower plasma proteins were significantly associated with hypophosphatemia.The incidence of severe hypophosphatemia in our study is low, so we can't offer robust conclusions about the risk of hypophosphatemia in the type of patients receiving enteral nutrition.INTRODUCCIÓN: se ha descrito una incidencia de hipofosfatemia en pacientes con soporte nutricional especializado (SNE) de hasta el 30-40%. La hipofosfatemia leve y la moderada son generalmente asintomáticas, mientras que la severa es el hecho fundamental del síndrome de realimentación. OBJETIVO: evaluar la incidencia y gravedad de la hipofosfatemia en pacientes hospitalizados no críticos con nutrición enteral (NE).MATERIAL Y MÉTODOS: se diseñó un estudio observacional y prospectivo en condiciones de práctica clínica habitual. Se recogieron datos clínicos, antropométricos y analíticos de 181 pacientes a los que se les inició nutrición enteral. El seguimiento fue de siete días.RESULTADOS: el 51,9% de los pacientes estaban en riesgo de desarrollar síndrome de realimentación según las guías del United Kingdom National Institute for Health and Clinical Excellence (NICE). La incidencia de hipofosfatemia fue del 31,5% y la de la hipofosfatemia severa, del 1,1%. De todos los parámetros clínicos, antropométricos y analíticos analizados, solo la edad y unas proteínas séricas más bajas se correlacionaron de forma estadísticamente significativa con el aumento en la incidencia de hipofosfatemia. CONCLUSIÓN: la incidencia de hipofosfatemia grave en nuestra serie es muy baja, lo que hace imposible extraer conclusiones específicas para este grupo de pacientes.

Natural deep eutectic solvents (NADES) have shown to be promising sustainable media for a wide range of applications. Nonetheless, very limited data is available on the properties of these solvents. A more comprehensive body of data on NADES is required for a deeper understanding of these solvents at molecular level, which will undoubtedly foster the development of new applications. NADES based on choline chloride, organic acids, amino acids and sugars were prepared, and their density, thermal behavior, conductivity and polarity were assessed, for different NADES compositions. The NADES studied can be stable up to 170 °C, depending on their composition. The thermal characterization revealed that all the NADES are glass formers and some, after water removal, exhibit crystallinity. The morphological characterization of the crystallizable materials was performed using polarized optical microscopy which also provided evidence of homogeneity/phase separation. The conductivity of the NADES was also assessed from 0 to 40 °C. The more polar, organic acid-based NADES presented the highest conductivities. The conductivity dependence on temperature was well described by the Vogelâ Fulcherâ Tammann equation for some of the NADES studied., Fundação para a Ciência e a Tecnologia (FCT/MEC) through the grants PTDC/EQUEPR/12191/2010/ENIGMA, SFRH/BD/47088/2008 and SFRH / BPD / 44946 / 2008. We further acknowledge the financial support of FCT/MEC through the project ENIGMA — PTDC/EQU-EPR/121491/2010, and the project PEst-C/ EQB/LA0006/2013 and FCOMP-01-0124-FEDER-020646.We also acknowledge the funding from the European Union Seventh Framework Programme (FP7/2007–2013) under grant agreement no. REGPOT-CT2012-316331-POLARIS, and from the Project “Novel smart and biomimetic materials for innovative regenerative medicine approaches (Ref.: RL1 — ABMR — NORTE-01-0124-FEDER- 000016)” co-financed by the North Portugal Regional Operational Programme (ON.2 — O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF).

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., The research leading to these results has received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no 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). Funding was also provided by Fundacao para a Ciencia e a Tecnologia, through contracts LAQV, REQUIMTE: UID/QUI/50006/2013 and UCIBIO, REQUIMTE: UID/Multi/04378/2013.

Ion Jelly materials combine the chemical versatility and conductivity of an ionic liquid (IL) with the morphological versatility of a biopolymer (gelatin). They exhibit very interesting properties, such as conductivities up to 10 − 4 S cm − 1 , and high thermostability up to 180 °C, and have been used successfully to design electrochromic windows. In this work we report on the preparation of Ion Jelly fibers through electrospinning in order to obtain high surface area conductive materials. We have used the IL 1-(2-hydroxyethyl)-3-methyl-imidazolium tetrafluoroborate ([C 2 OHmim]BF 4 ), which exhibits conveniently high ionic conductivity (over 10 − 3 S cm − 1 ) and electrochemical stability (electrochemical window over 6.0 V). The morphology of the obtained fibers was quantified using Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). We found that on average the effect of the IL on fiber diameter differs for lower and higher IL concentrations and that this effect was correlated with the initial conductivity and viscosity of Ion Jelly electrospinning solution. Moreover we also found that conductivities of Ion Jelly fibers are of the same order of magnitude as the conductivities of Ion Jelly dense films (~ 10 − 4 S cm − 1 ). To the best of our knowledge, this is the first report on the incorporation of an IL into gelatin fibers using electrospinning. This opens up new opportunities for the application of gelatin fibers in electrochemical and biomedical devices.

The kinetics of glucose oxidase (GOD) and horseradish peroxidase (HRP) on a transparent gelatin–ionic liquid functional polymer has been investigated using a colorimetric assay of H 2 O 2 with phenol-4-sulfonic acid and 4-aminoantipyrine, as color-generating precursors. The effect of different ionic liquids 1-ethyl-3-methyl-imidazolium ethyl sulfate ([emim][EtSO 4 ]), 1-butyl-3-methyl-imidazolium dycianamide ([bmim][N(CN) 2 ]), and 1-butyl-3-methyl-imidazolium chloride ([bmim][Cl]), 1-butyl-3-methyl-imidazolium tetrafluoroborate ([bmim][BF 4 ]), gelatin type A and water on the activity of the two free enzymes was studied. The selection of the ionic liquid [emim][EtSO 4 ] and gelatin type A with subsequent maturation of the functional polymer at water activity of 0.76 have been found as the most suitable conditions for the entrapment of both enzymes. The activity of GOD and HRP on the gelatin–[emim][EtSO 4 ] solid disk (6.2 × 1.5 mm of diameter and thickness) formed in the bottom of the well of ELISA plate showed to be respectively 16 times and 13 times lower than in free sodium phosphate buffer. The storage stability at 4 °C of the both immobilized enzymes showed that GOD can retain up to 70% of the initial activity after 2 weeks, where HRP retained 91% of its initial activity. The drop cast deposition of the transparent gelatin–[emim][EtSO 4 ] functional polymer containing the two enzymes (GOD, HRP) and the color-generating precursors on filter paper allowed to demonstrate their applicability on colorimetric glucose detection.

A continuous process for biodiesel production in supercritical carbon dioxide was implemented. In the transesterification of virgin sunflower oil with methanol, Lipozyme TL IM led to fatty acid methyl esters yields (FAME) that exceeded 98% at 20 MPa and 40 °C, for a residence time of 20 s and an oil to methanol molar ratio of 1:24. Even for moderate reaction conversions, a fractionation stage based on two separators afforded FAME with >96% purity. Lipozyme TL IM was less efficient with waste cooking sunflower oil. In this case, a combination of Lipozyme TL IM and Novozym 435 afforded FAME yields nearing 99%.

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%).

Cutinase, an esterase from Fusarium solani pisi, was immobilized in sol-gel matrices of composition 1:5 tetramethoxysilane (TMOS):n-alkyltrimethoxysilane (n-alkylTMS). Fluorescence spectroscopy using the single tryptophan (Trp-69) residue of cutinase as a probe revealed that the polarity of the matrices decreased as their hydrophobicity increased up to the TMOS/n-butylTMS pair, which correlates with an increase in cutinase activity. Fluorescence emission was suppressed (a higher than two orders of magnitude reduction) in the TMOS/n-octylTMS matrix, suggesting a greater proximity of the tryptophan to a nearby disulfide bridge. When sol-gel matrices were prepared with added zeolite NaY, the fluorescence emission intensity maximum (lambda(max)) of the tryptophan did not change. And although the presence of the zeolite led to the recovery of fluorescence emission from the TMOS/n-octylTMS matrix, the corresponding lambda(max) fell in line with the values obtained for the matrices with lower n-alkyl chain lengths, indicating that the tryptophan does not sense the zeolite. On the other hand, the presence of the zeolite led to increases in cutinase activity in all the matrices. This suggests that the zeolite is in a position to affect the active site of the enzyme, located at the opposite pole of the enzyme molecule. Scanning electron microscopy and energy dispersive X-ray spectroscopy revealed that the zeolite particles were segregated to the pores of the matrices. Optical microscopy following the staining of the protein with a fluorescent dye showed that the enzyme was distributed throughout the material, and tended to accumulate around zeolite particles. By promoting the accumulation of the enzyme at the pores of the material, the zeolite should improve the accessibility of the enzyme to the substrates and lead to a higher enzymatic activity. Data obtained for sol-gel matrices with epoxy or SH groups provided further evidence that cutinase responded to changes in the chemical nature of the precursors.

Cutinase from Fusarium solani pisi was encapsulated in sol−gel matrixes prepared using combinations of tetramethoxysilane and monoalkyl-methoxysilane precursors with different chain lengths (from methyltrimethoxysilane to n-octyltrimethoxysilane), in molar proportion of 1 to 5. The specific activity of the encapsulated cutinase in a model transesterification reaction in n-hexane has been correlated with the structure of the organically modified silica (ORMOSIL) support. The matrixes were characterized by diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy and by solid-state 29Si and 1H nuclear magnetic resonance (NMR). The enhancement of the bioactivity with increasing alkyl chain lengths up to C4 was correlated with the nature of the ORMOSIL surface groups, namely, with a decrease in the hydrophilic−lipophilic balance (HLB) of the matrix. Such trend suggests that the kinetics of the catalyzed transesterification in n-hexane is a diffusion-controlled process. For coprecursors with longer a...

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.

We applied Pulsed Gradient Spin Echo diffusion with high-resolution magic angle spinning NMR to study sol-gel matrices used to encapsulate enzymes for biocatalysis (TMOS/MTMS and TMOS/BTMS) to gain insight into the local chemical microenvironment. Transport properties of solvents with different polarities (1-pentanol, acetonitrile and n-hexane) were studied through their apparent self-diffusion coefficients. The spin echo attenuation of the solvents shows two distinct diffusion domains, one with fast diffusion (D

Functionalized electrospun fibers are of great interest for biomedical applications such as in the design of drug delivery systems. Nevertheless, in some cases the molecules of interest have poor solubility in water or have high melting temperatures. These drawbacks can be overcome using deep eutectic solvents. In this work, poly(vinyl alcohol) (PVA), a common biodegradable biopolymer, was used to produce new functionalized fibers with the eutectic mixture choline chloride:citric acid in a molar ratio of (1:1) ChCl:CA (1:1), which was used as a model system. Fibers were produced from an aqueous solution with 7.8% (w/v) and 9.8% (w/v) of 95% hydrolyzed PVA and a 2% (v/v) of ChCl:CA (1:1). Smooth, uniform fibers with an average diameter of 0.4 μm were obtained with a content of 19.8 wt % of ChCl:CA (1:1) encapsulated, FCT/MEC through the project ENIGMA - PTDC/EQU-EPR/121491/2010, and the project PEst-C/EQB/LA0006/2013. European Union’s Seventh Framework Programme (FP7/2007-2013) under grant agreement n°REGPOT-CT2012-316331-POLARIS, and from the Project “Novel smart andbiomimetic materials for innovative regenerative medicine approaches (Ref.: RL1 - ABMR - NORTE-01-0124-FEDER-000016) ”cofinanced by the North Portugal Regional Operational Programme (ON.2 − O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF). This work was also partly funded by FEDER funds through the COMPETE 2020 Programme and National Funds through FCT - Portuguese Foundation for Science and Technology under the project UID/CTM/50025/2013.

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).

Cutinase from Fusarium solani pisi was encapsulated in sol-gel matrices prepared with a combination of alkyl-alkoxysilane precursors of different chain-lengths. The specific activity of cutinase in a model transesterification reaction at fixed water activity in n-hexane was highest for the precursor combination tetramethoxysilane/n-butyltrimetoxysilane (TMOS/BTMS) in a 1:5 ratio, lower and higher chain lengths of the mono-alkylated precursor or decreasing proportions of the latter relative to TMOS leading to lower enzyme activity. Results obtained using combinations of three precursors confirmed the beneficial effect of the presence of BTMS in the preparations. Scanning electron microscopy of the 1:5 TMOS/n-alkylTMS gels showed a direct correlation between the macropore dimensions and the alkyl chain length of the alkylated precursor and revealed that TMOS/n-octylTMS gels suffered extensive pore collapse during the drying process. The specific activity of TMOS/BTMS sol-gel entrapped cutinase was similar to that exhibited by the enzyme immobilized by adsorption on zeolite NaY. However, the incorporation of different additives (zeolites, silica, Biogel, grinded sol-gel, etc.) having in common the capability to react with residual silanol groups of the sol-gel matrix brought about remarkable enhancements of cutinase activity, despite the fact that the global porosity of the gels did not change. The behavior of the gels in supercritical CO 2 (sc-CO 2) paralleled that exhibited in n-hexane, although cutinase activity was ca. one order of magnitude lower (i.e. sol-gel encapsulation did not prevent the deleterious effect of CO 2. The impact that functionalization of some of the additives had on cutinase activity indicates that the enzyme/matrix interactions must play an important role. Some of the best additives from the standpoint of enzyme activity were also the best from the standpoint of its operational stability (ca. 80% retention of enzyme activity at the tenth reutilization cycle). None of the additives that proved effective for cutinase could improve the catalytic activity of sol-gel encapsulated Pseudomonas cepacia lipase.

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.

We report on the effect of salt hydrate pairs and of zeolite NaA on the Novozym catalyzed esterification of geraniol with acetic acid in an organic solvent and in supercritical fluids. The zeolite was pre-equilibrated to the water activity ( a W ) of the experiment to avoid drying effects. Medium conditions were selected to minimize changes in a W during the course of initial rate measurements. Highest enzyme activity was obtained in the presence of zeolite NaA at initial a W = 0.2. An organosoluble chromoionophoric indicator revealed that the salt hydrate pairs used to set initial a W = 0.1 or 0.7 were highly acidic when compared to the salt hydrate pair used to set a W = 0.2, and that zeolite NaA was even more basic than the latter, and able to noticeably make the medium less acidic in the presence of acetic acid. The impact of zeolite NaA is attributed to its ability to promote the formation of a more basic form of enzyme through ion exchange and thus facilitate the establishment of a formal negative charge on the enzyme active site, as required by the catalytic mechanism. This work highlights the dual role that zeolites may play in tuning a W and the ionization state of enzymes in nonaqueous media, also in esterification reactions.

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 studied the reaction between vinyl butyrate and 2-phenyl-1-propanol in acetonitrile catalyzed by Fusarium solani pisi cutinase immobilized on zeolites NaA and NaY and on Accurel PA-6. The choice of 2-phenyl-1-propanol was based on modeling studies that suggested moderate cutinase enantioselectivity towards this substrate. With all the supports, initial rates of transesterification were higher at a water activity (a(w)) of 0.2 than at a(w) = 0.7, and the reverse was true for initial rates of hydrolysis. By providing acid-base control in the medium through the use of solid-state buffers that control the parameter pH-pNa, which we monitored using an organo-soluble chromoionophoric indicator, we were able, in some cases, to completely eliminate dissolved butyric acid. However, none of the buffers used were able to improve the rates of transesterification relative to the blanks (no added buffer) when the enzyme was immobilized at an optimum pH of 8.5. When the enzyme was immobilized at pH 5 and exhibited only marginal activity, however, even a relatively acidic buffer with a pK(a) of 4.3 was able to restore catalytic activity to about 20% of that displayed for a pH of immobilization of 8.5, at otherwise identical conditions. As a(w) was increased from 0.2 to 0.7, rates of transesterification first increased slightly and then decreased. Rates of hydrolysis showed a steady increase in that a(w) range, and so did total initial reaction rates. The presence or absence of the buffers did not impact on the competition between transesterification and hydrolysis, regardless of whether the butyric acid formed remained as such in the reaction medium or was eliminated from the microenvironment of the enzyme through conversion into an insoluble salt. Cutinase enantioselectivity towards 2-phenyl-1-propanol was indeed low and was not affected by differences in immobilization support, enzyme protonation state, or a(w).

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.

Enzyme performance is often impaired in supercritical carbon dioxide. We were able to enhance enzyme activity in this medium via changes in acid-base conditions by using ion-exchange materials (solid H + /Na + buffer pairs and a zeolite), which were selected on the basis of the response of an organosoluble acid-base indicator. The concentration of ion-exchange materials had an important effect on the catalytic activity of subtilisin Carlsberg cross-linked enzyme crystals (CLECs), and this was related to the protonation and hydration states of the enzyme. The buffer Na 2 CO 3 / NaHCO 3 gave the highest enhancement in enzyme activity (by a factor of 54), probably as a result of its high basicity and capacity to counteract the deleterious effect of carbonic acid to a greater extent than the other materials tested.

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.

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.