White wine grape pomace as a suitable carbon source for lipid and carotenoid production by fructophilic Rhodorotula babjevae.

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.

Valorization of Cork Using Subcritical Water.

Granulated cork was submitted to subcritical water extraction/hydrolysis in a semi-continuous reactor at temperatures in the range of 120-200 °C and with a constant pressure of 100 bar. The influence of temperature on the composition of the cork extracts obtained was assessed-namely, their content of carbohydrates and phenolics. The extraction yield increased with the temperature, and this was associated with the decrease in the dielectric constant of water and the increase in its ionic product. Extracts composed of up to 36% phenolics were obtained at temperatures of up to 120 °C, with an antioxidant activity only two times lower than that of pure gallic acid, but in low amounts. Assays at higher temperatures generated extracts richer in carbohydrates and with a phenolics content of ca. 20 wt.% in comparatively far higher amounts. Neither the amount of suberin nor its structure were affected by the subcritical water treatment.

Stabilizing Unstable Amorphous Menthol through Inclusion in Mesoporous Silica Hosts.

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 mobility of menthol molecules adsorbed at the inner pore walls (S-process). The fraction of molecular population governing the α-process is greater in the higher (5.9 nm) pore size matrix, although in both cases the S-process is more intense than the α-process. A dielectric glass transition temperature was estimated for each α (Tg,dielc(α)) and S (Tg,dielc(S)) molecular population from the temperature dependence of the relaxation times to 100 s. While Tg,dielc(α) agrees better with the value obtained from the linearization of the Fox equation assuming ideal behavior of the menthol:flurbiprofen THEDES, Tg,dielc(S) is close to the value determined by calorimetry for the silica composites due to a dominance of the adsorbed population inside the pores. Nevertheless, the greater fraction of more mobile bulk-like molecules in the 5.9 nm pore size matrix seems to determine the faster drug release at initial times relative to the 3.2 nm composite. However, the latter inhibits crystallization inside pores since its dimensions are inferior to menthol critical size for nucleation. This points to a suitability of these composites as drug delivery systems in which the drug release profile can be controlled by tuning the host pore size.

Probing sol-gel matrices microenvironments by PGSE HR-MAS NMR.

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 Kärger 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.

Production of Electrospun Fast-Dissolving Drug Delivery Systems with Therapeutic Eutectic Systems Encapsulated in Gelatin.

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.

Subcritical Water as a Pre-Treatment of Mixed Microbial Biomass for the Extraction of Polyhydroxyalkanoates.

Polyhydroxyalkanoate (PHA) recovery from microbial cells relies on either solvent extraction (usually using halogenated solvents) and/or digestion of the non-PHA cell mass (NPCM) by the action of chemicals (e.g., hypochlorite) that raise environmental and health hazards. A greener alternative for PHA recovery, subcritical water (SBW), was evaluated as a method for the dissolution of the NPCM of a mixed microbial culture (MMC) biomass. A temperature of 150 °C was found as a compromise to reach NPCM solubilization while mostly preventing the degradation of the biopolymer during the procedure. Such conditions yielded a polymer with a purity of 77%. PHA purity was further improved by combining the SBW treatment with hypochlorite digestion, in which a significantly lower hypochlorite concentration (0.1%, v/v) was sufficient to achieve an overall polymer purity of 80%. During the procedure, the biopolymer suffered some depolymerization, as evidenced by the lower molecular weight (Mw) and higher polydispersity of the extracted samples. Although such changes in the biopolymer's molecular mass distribution impact its mechanical properties, impairing its utilization in most conventional plastic uses, the obtained PHA can find use in several applications, for example as additives or for the preparation of graft or block co-polymers, in which low-Mw oligomers are sought.

Solubility of Polar and Nonpolar Aromatic Molecules in Subcritical Water: The Role of the Dielectric Constant.

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 nonpolar organic solutes. This is often associated with 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 among the dielectric constant of water, hydration, and the solubilities of polar and nonpolar molecules in SBCW remains poorly understood. Here, we study, through molecular dynamics, the hydration thermodynamic parameters and the solubility of nonpolar 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 nonpolar aromatic 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 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 nonpolar 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.

Probing the microenvironment of sol-gel entrapped cutinase: the role of added zeolite NaY.

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.

A comparison between pure active pharmaceutical ingredients and therapeutic deep eutectic solvents: Solubility and permeability studies.

THEDES, so called therapeutic deep eutectic solvents are here defined as a mixture of two components, which at a particular molar composition become liquid at room temperature and in which one of them is an active pharmaceutical ingredient (API). In this work, THEDES based on menthol complexed with three different APIs, ibuprofen (ibu), BA (BA) and phenylacetic acid (PA), were prepared. The interactions between the components that constitute the THEDES were studied by NMR, confirming that the eutectic system is formed by H-bonds between menthol and the API. The mobility of the THEDES components was studied by PFGSE NMR spectroscopy. It was determined that the self-diffusion of the species followed the same behavior as observed previously for ionic liquids, in which the components migrate via jumping between voids in the suprastructure created by punctual thermal fluctuations. The solubility and permeability of the systems in an isotonic solution was evaluated and a comparison with the pure APIs was established through diffusion and permeability studies carried out in a Franz cell. The solubility of the APIs when in the THEDES system can be improved up to 12 fold, namely for the system containing ibu. Furthermore, for this system the permeability was calculated to be 14×10-5cm/s representing a 3 fold increase in comparison with the pure API. With the exception of the systems containing PA an increase in the solubility, coupled with an increase in permeability was observed. In this work, we hence demonstrate the efficiency of THEDES as a new formulation for the enhancement of the bioavailability of APIs by changing the physical state of the molecules from a solid dosage to a liquid system.

Sol-gel encapsulation: an efficient and versatile immobilization technique for cutinase in non-aqueous media.

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.

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.

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.

Enhancement of enzyme activity in supercritical carbon dioxide via changes in acid-base conditions.

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.

Water activity effects on geranyl acetate synthesis catalyzed by novozym in supercritical ethane and in supercritical carbon dioxide.

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)).

Ion jelly conductive properties using dicyanamide-based ionic liquids.

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.

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 Young's Modulus of 1.0GPa. Results show that SCG can be a bioresource for polyhydroxyalkanoates production with interesting properties.

Ion jelly: a tailor-made conducting material for smart electrochemical devices.

We present a new concept for the design of a polymeric conducting material that combines the chemical versatility of an organic salt (ionic liquid) with the morphological versatility of a biopolymer (gelatin); the resulting 'ion jelly' can be applied in electrochemical devices, such as batteries, fuel cells, electrochromic windows or photovoltaic cells.

Salt hydrates for in situ water activity control have acid-base effects on enzymes in nonaqueous media.

Salt hydrates very frequently are utilized as in situ water activity buffers in reaction mixtures of enzymes in nonaqueous media. In addition to buffering water activity, there is evidence that salt hydrates also often affect initial rates in other ways. This has been generally overlooked or thought to be related to water transfer effects. Here we show that salt hydrates can have important acid-base effects on enzymes in nonaqueous media. We performed transesterification reactions in n-hexane and in supercritical ethane catalyzed by cross-linked crystals of subtilisin, differing in the method used to set a(W), and confirmed that the presence of salt hydrate pairs significantly affected the catalytic performance of the enzyme. However, in the presence of a solid-state acid-base buffer, salt hydrates had no effect on enzymatic activity. Direct evidence for the acid-base effects of salt hydrates was obtained by testing their effect on the protonation state of an organo-soluble H(+)/Na(+) indicator. The four salt hydrate pairs tested affected the indicator to very different extents. By promoting the exchange of H(+) for Na(+), salt hydrates will tend to affect the ionization state of acidic residues in the protein and, hence, enzymatic activity. In fact, salt hydrates were able to affect the pH memory of subtilisin lyophilized from different aqueous pHs, bringing about up to 20-fold enhancements and up to 5-fold decreases in catalytic activity. The possibility of such acid-base effects need to be considered in all experiments using salt hydrates to control water activity.

Zeolite molecular sieves have dramatic acid-base effects on enzymes in nonaqueous media.

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.

Effect of immobilization support, water activity, and enzyme ionization state on cutinase activity and enantioselectivity in organic media.

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).