Solvent Effects on Equilibrium Position and Initial Rate of Lipase-catalyzed Esterification Reactions in Organic Solvents: Experimental Results and Prediction Capabilities

The solvent effect on the equilibrium position and the initial rate of esterification of 1-hexanol with acetic acid catalyzed by a lipase has been experimentally investigated. A variety of non-polar and polar solvents have been considered and the results obtained indicate that the solvent effect on the equilibrium conversion is very important compared to that for transesterification reactions. A theoretically sound methodology using the group-contribution UNIFAC model for the prediction of solvent effects on the equilibrium position of enzymatic reactions is presented and it is applied to the reaction of 1-hexanol with acetic acid as well as to a similar reaction from the literature. The results obtained are better than those from empirical methods proposed in the literature such as correlations with the octanol-water partition coefficient of the solvent, as well as the solubility of water in the solvent. Moreover, the proposed methodology can be used for the determination of the equilibrium constant of the reaction. For the prediction of the solvent effect on the initial rate of enzymatic reactions it is found that it is more accurately determined using the product of the activities of the reactants, which can be predicted by the UNIFAC model, than the octanol-water partition coefficient of the solvent or the solubility of water in the solvent.     

Solvent Effects on Equilibrium Position and Initial Rate of Lipase-catalyzed Esterification Reactions in Organic Solvents: Experimental Results and Prediction Capabilities

The solvent effect on the equilibrium position and the initial rate of esterification of 1-hexanol with acetic acid catalyzed by a lipase has been experimentally investigated. A variety of non-polar and polar solvents have been considered and the results obtained indicate that the solvent effect on the equilibrium conversion is very important compared to that for transesterification reactions. A theoretically sound methodology using the group-contribution UNIFAC model for the prediction of solvent effects on the equilibrium position of enzymatic reactions is presented and it is applied to the reaction of 1-hexanol with acetic acid as well as to a similar reaction from the literature. The results obtained are better than those from empirical methods proposed in the literature such as correlations with the octanol-water partition coefficient of the solvent, as well as the solubility of water in the solvent. Moreover, the proposed methodology can be used for the determination of the equilibrium constant of the reaction. For the prediction of the solvent effect on the initial rate of enzymatic reactions it is found that it is more accurately determined using the product of the activities of the reactants, which can be predicted by the UNIFAC model, than the octanol-water partition coefficient of the solvent or the solubility of water in the solvent.     

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

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

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

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

Integrated bioproduction and extraction of 3-methylcatechol

Pseudomonas putida MC2 is a solvent-tolerant strain that accumulates 3-methylcatechol. In aqueous media, 10 mM of 3-methylcatechol was produced and production was limited by 3-methylcatechol toxicity to the biocatalyst. Production levels increased by introduction of a second, organic phase that provides the substrate toluene and extracts the product from the culture medium. Octanol was shown to be an appropriate second phase with respect to tolerance of the strain for this solvent and with respect to partitioning of both substrate and product. Per unit of overall reactor volume (octanol and water), best results were obtained with 50% (v/v) of octanol: an overall 3-methylcatechol concentration of 25 mM was reached with 96% of the product present in the octanol phase. These product concentrations are much higher than in aqueous media without organic solvent, indicating that biocatalysis in an organic/aqueous two-phase system is an improved set-up for high production levels of 3-methylcatechol.     

Sol-gel immobilisation of Saccharomyces cerevisiae enhances viability in organic media

The polyhydroxylated silane network of a sol-gel protected immobilised Saccharomyces cerevisiae against the effects of five organic solvents. The viability of immobilised yeast directly correlated with the logarithm of the partition coefficient of the solvent in an octanol/water two phase system increasing the decimal reduction time (D) and reaching the maximum with octanol, the most hydrophobic solvent assayed. The D value increased from 0.16 min for free yeast to 1.9 and to 22 min for immobilised yeast exposed to ethanol and 1-octanol respectively.     

Application of a computational model of natural deep eutectic solvents utilizing the COSMO-RS approach for screening of solvents with high solubility of rutin

The screening of natural deep eutectic solvents (NADES) to identify those with the ability to strongly solvate rutin was conducted using the COSMO-RS methodology. A NADES model was constructed that took into account the possible ionic and neutral forms of its constituents. The distributions of all forms were computed based on the equilibrium constants of neutralization reactions between amino and carboxylic acids. The proposed model was validated against the experimental solubilities of 15 NADES. A linear relationship between these data and the estimated activity coefficient values was found. The screening encompassed 126 different NADES. It was found that ten of them outperformed the best reference system. The most effective two-component solvent comprised proline combined with 2,3-diaminosuccinic acid, and the solubility of rutin in this solvent was found to be 130% greater than its solubility in the best reference system. The amino acids associated with the highest rutin solubilities were all cyclic, and the use of carboxylic acids with two carboxyl groups and a main chain consisting of two methylene groups with two amino substituents was observed to yield the best rutin solubilities. Because of the acidic properties of rutin, the presence of basic sites on the components of the NADES generally leads to enhanced solubility.     

Evaluation of Drug Load and Polymer by Using a 96-Well Plate Vacuum Dry System for Amorphous Solid Dispersion Drug Delivery

It is well recognized that poor dissolution rate and solubility of drug candidates are key limiting factors for oral bioavailability. While numerous technologies have been developed to enhance solubility of the drug candidates, poor water solubility continuously remains a challenge for drug delivery. Among those technologies, amorphous solid dispersions (SD) have been successfully employed to enhance both dissolution rate and solubility of poorly water-soluble drugs. This research reports a high-throughput screening technology developed by utilizing a 96-well plate system to identify optimal drug load and polymer using a solvent casting approach. A minimal amount of drug was required to evaluate optimal drug load in three different polymers with respect to solubility improvement and solid-state stability of the amorphous drug-polymer system. Validation of this method was demonstrated with three marketed drugs as well as with one internal compound. Scale up of the internal compound SD by spray drying further confirmed the validity of this method, and its quality was comparable to a larger scale process. Here, we demonstrate that our system is highly efficient, cost-effective, and robust to evaluate the feasibility of spray drying technology to produce amorphous solid dispersions.     

Rapid screening for improved solubility of small human proteins produced as fusion proteins in Escherichia coli

A prerequisite for structural genomics and related projects is to standardize the process of gene overexpression and protein solubility screening to enable automation for higher throughput. We have tested a methodology to rapidly subclone a large number of human genes and screen these for expression and protein solubility in Escherichia coli. The methodology, which can be partly automated, was used to compare the effect of six different N-terminal fusion proteins and an N-terminal 6*His tag. As a realistic test set we selected 32 potentially interesting human proteins with unknown structures and sizes suitable for NMR studies. The genes were transferred from cDNA to expression vectors using subcloning by recombination. The subcloning yield was 100% for 27 (of 32) genes for which a PCR fragment of correct size could be obtained. Of these, 26 genes (96%) could be overexpressed at detectable levels and 23 (85%) are detected in the soluble fraction with at least one fusion tag. We find large differences in the effects of fusion protein or tag on expression and solubility. In short, four of seven fusions perform very well, and much better than the 6*His tag, but individual differences motivate the inclusion of several fusions in expression and solubility screening. We also conclude that our methodology and expression vectors can be used for screening of genes for structural studies, and that it should be possible to obtain a large fraction of all NMR-sized and nonmembrane human proteins as soluble fusion proteins in E. coli.     

Toward advanced ionic liquids. Polar,enzyme-friendly solvents for biocatalysis

Ionic liquids, also called molten salts, are mixtures of cations and anions that melt below 100°C. Typical ionic liquids are dialkylimidazolium cations with weakly coordinating anions such as (MeOSO₃) or (PF₆). Advanced ionic liquids such as choline citrate have biodegradable, less expensive, and less toxic anions and cations. Deep eutectic solvents are also included in the advanced ionic liquids. Deep eutectic solvents are mixtures of salts such as choline chloride and uncharged hydrogen bond donors such as urea, oxalic acid, or glycerol. For example, a mixture of choline chloride and urea in 1:2 molar ratio liquefies to form a deep eutectic solvent. Their properties are similar to those of ionic liquids. Water-miscible ionic liquids as cosolvents with water enhance the solubility of substrates or products. Although traditional water-miscible organic solvents also enhance solubility, they often inactivate enzymes, while ionic liquids do not. The enhanced solubility of substrates can increase the rate of reaction and often increases the regioor enantioselectivity. Ionic liquids can also be solvents for non-aqueous reactions. In these cases, they are especially suited to dissolve polar substrates. Polar organic solvent alternatives inactivate enzymes, but ionic liquids do not even when they have similar polarities. Besides their solubility properties, ionic liquids and deep eutectic solvents may be greener than organic solvents because ionic liquids are nonvolatile, and can be made from nontoxic components. This review covers selected examples of enzyme catalyzed reaction in ionic liquids that demonstrate their advantages and unique properties, and point out opportunities for new applications. Most examples involve hydrolases, but oxidoreductases and even whole cell reactions have been reported in ionic liquids.     

Thermodynamically based solvent design for enzymatic saccharide acylation with hydroxycinnamic acids in non-conventional media

Enzyme-catalyzed synthesis has been widely studied with lipases (EC 3.1.1.3), but feruloyl esterases (FAEs; EC 3.1.1.73) may provide advantages such as higher substrate affinity and regioselectivity in the synthesis of hydroxycinnamate saccharide esters. These compounds are interesting because of their amphiphilicity and antioxidative potential. Synthetic reactions using mono- or disaccharides as one of the substrates may moreover direct new routes for biomass upgrading in the biorefinery. The paper reviews the available data for enzymatic hydroxycinnamate saccharide ester synthesis in organic solvent systems as well as other enzymatic hydroxycinnamate acylations in ionic liquid systems. The choice of solvent system is highly decisive for enzyme stability, selectivity, and reaction yields in these synthesis reactions. To increase the understanding of the reaction environment and to facilitate solvent screening as a crucial part of the reaction design, the review explores the use of activity coefficient models for describing these systems and - more importantly - the use of group contribution model UNIFAC and quantum chemistry based COSMO-RS for thermodynamic predictions and preliminary solvent screening. Surfactant-free microemulsions of a hydrocarbon, a polar alcohol, and water are interesting solvent systems because they accommodate different substrate and product solubilities and maintain enzyme stability. Ionic liquids may provide advantages as solvents in terms of increased substrate and product solubility, higher reactivity and selectivity, as well as tunable physicochemical properties, but their design should be carefully considered in relation to enzyme stability. The treatise shows that thermodynamic modeling tools for solvent design provide a new toolbox to design enzyme-catalyzed synthetic reactions from biomass sources.     

Reproductive and developmental effects and physical and chemical properties of pentaerythritol tetranitrate (PETN) in the rat

Pentaerythritol tetranitrate (PETN) is an explosive chemical that has been detected in environmental media. Although previous toxicology studies have shown PETN to be relatively benign, a lack of available information concerning developmental and reproductive effects from oral PETN exposure was needed. Sprague‐Dawley rats were exposed to oral daily adjusted volumetric doses of 0, 100, 500, or 1,000 mg PETN/kg body mass in a corn oil vehicle for up to 56 days. Mating, duration of gestation, body weight, feed consumption, overall condition of adults, and the number, sex, and condition of pups were recorded. Histological examinations were also performed on the ovaries, testes, and epididymides of animals from the control and the highest dose groups. Other environmental criteria, water solubility, octanol/water partition coefficient, and biodegradation rates of neat PETN were also determined. Only body weights and feed consumption were affected by treatment; however, these differences may be attributed more to volumetric adjustments of vehicle in the control and high‐dose groups than to PETN toxicity. No adverse effects on development or reproduction from PETN exposure were observed. Water solubility, octanol water partition coefficient, and water suspension and biodegradation rates suggest PETN is unlikely to transport or bioaccumulate in the environment to any appreciable extent. Additionally, biotic processes are most likely faster in breaking down PETN than the abiotic processes involved in dissolving PETN in water. Birth Defects Res (Part B) 86:65‐71, 2009. © 2009 Wiley‐Liss, Inc.     

Extracting hydrophobic free energies from experimental data: relationship to protein folding and theoretical models

Solubility and vapor pressure measurements of hydrocarbons in water are generally thought to provide estimates of the strength of the hydrophobic effect in the range 20-30 cal/(mol.A2). Our reassessment of the solubility data on the basis of new developments in solution thermodynamics suggests that the hydrophobic surface free energy for hydrocarbon solutes is 46-47 cal/(mol.A2), although the actual value depends strongly on curvature effects [Nicholls et al. (1991) Proteins (in press); Sharp et al. (1991) Science 252, 106-109]. The arguments to support such a significant increase in the estimate of the hydrophobic effect stem partly from theoretical considerations and partly from the experimental results of De Young and Dill [(1990) J. Phys. Chem. 94, 801-809] on benzene partition between water and alkane solvents. Previous estimates of the hydrophobic effect derive from an analysis of solute partition data, which does not fully account for changes in volume entropy. We show here how the ideal gas equations, combined with experimental molar volumes, can account for such changes. Revised solubility scales for the 20 amino acids, based on cyclohexane to water and octanol to water transfer energies, are derived. The agreement between these scales, particularly the octanol scale, and mutant protein stability measurements from Kellis et al. [(1989) Biochemistry 28, 4914-4922] and Shortle et al. [(1990) Biochemistry 29, 8033-8041] is good. The increased strength of the hydrophobic interaction has implications for the energetics of protein folding, substrate binding, and nucleic acid base stacking and the interpretation of computer simulations.     

Lid dynamics of porcine pancreatic lipase in non-aqueous solvents

BackgroundUnderstanding the dynamics of enzymes in organic solvents has wider implications on their industrial applications. Pancreatic lipases, which show activity in their lid open-state, demonstrate enhanced activity in organic solvents at higher temperatures. However, the lid dynamics of pancreatic lipases in non-aqueous environment is yet to be clearly understood.MethodsDynamics of porcine pancreatic lipase (PPL) in open and closed conformations was followed in ethanol, toluene, and octanol using molecular simulation methods. In silico double mutant D250V and E254L of PPL (PPL_mut-Cl) was created and its lid opening dynamics in water and in octanol was analyzed.ResultsPPL showed increase in solvent accessible surface area and decrease in packing density as the polarity of the surrounded solvent decreased. Breaking the interactions between D250-Y115, and D250-E254 in PPL_mut-Cl directed the lid to attain open-state conformation. Major energy barriers during the lid movement in water and in octanol were identified. Also, the trajectories of lid movement were found to be different in these solvents.ConclusionsOnly the double mutant at higher temperature showed lid opening movement suggesting the essential role of the three residues in holding the lid in closed conformation. The lid opening dynamics was faster in octanol than water suggesting that non-polar solvents favor open conformation of the lid.General significanceThis study identifies important interactions between the lid and the residues in domain 1 which possibly keeps the lid in closed conformation. Also, it explains the rearrangements of residue–residue interactions during lid opening movement in water and in octanol.     

Dynamic interactions between enzyme activity and the microstructured environment

A new approach for the study of an enzyme's relationship with its own reaction medium has been developed. One technique of micellar enzymology is the use of pseudohomogeneous systems composed of surfactant/water/organic solvent. In such systems, the physicochemical properties and textures of the medium depend on the relative ratios of the different components. Enzymes are catalytically active in such systems and up to the present have been studied in different microenvironments, such as micelles, microemulsions and lyotropic liquid crystals. Our purpose was to develop a system in which the enzyme could, by its activity, modify one of the components in such a way that the relative ratios among them changed sufficiently to produce a transition from one phase domain to another. The three components, water (or glucose in water), octanol and octyl-beta-D-glucoside, form a classical ternary water/oil/surfactant system. The relevant phase diagram shows different macroheterogeneous phases and microstructured domains. The enzyme beta-D-glucosidase hydrolyses octyl-beta-D-glucoside to form glucose and octanol. The enzyme was found to change the relative ratios of water (or glucose in water), octanol and octyl-beta-D-glucoside in such a manner that the physicochemical structure of the medium was modified. At the beginning of the reaction beta-D-glucosidase was present in a micellar solution of octyl-beta-D-glucoside in water. As the enzymatic reaction proceeded, the medium became biphasic. One of the two phases was the micellar solution of octyl beta-D-glucoside in water, while the other phase was either a microemulsion or a liquid crystalline phase. In addition the enzyme, through its catalytic activity, was able to modify the physiocochemical properties of the reaction medium.     

Enzymatic Reactions in Non-Conventional Media: Prediction of Solvent Water Content for Optimum Water Activity

Most enzymes provide their optimum performance at a given water activity (aw), which is generally solvent independent. For a given organic liquid solvent at a specific temperature or for a supercritical solvent at a specific temperature and pressure this corresponds to a water concentration in which water has the desired activity. We present here a methodology for predicting this water concentration thus reducing substantially the amount of experimental work needed to find the optimum solvent with respect to equilibrium conversion.

If the enzyme optimum water activity is known, the methodology predicts the required water content in the solvent to achieve this aw value. If, in addition, the enzyme water activity curve is available, this methodology provides the total water that must be added to the system (enzyme plus solvent) so that a specific water activity can be obtained.

The same methodology can also be applied to predict the effect of the total water content of the system (initial or initial plus produced) on the water activity values. It is shown that: (a) for esterification reactions taking place in hydrophobic organic solvents, the produced water can lead to a substantial change in water activity, but not for less hydrophobic solvents; (b) introduction of dry CO₂ into a system, pre-equilibrated to a certain water activity at atmospheric pressure, can lead to a substantial decrease in the water activity especially at temperatures just above the critical one of the solvent and pressures larger than that.     

Imitating Prebiotic Homochirality on Earth

We show how the amino acids needed on prebiotic earth in their homochiral L form can be produced by a reaction of L-alpha-methyl amino acids—that have been identified in the Murchison meteorite—with alpha-keto acids under credible prebiotic conditions. When they are simply heated together they perform a process of decarboxylative transamination but with almost no chiral transfer, and that in the wrong direction, producing D-amino acids from the L-alpha-methyl amino acids. With copper ion a square planar complex with two of the reaction intermediates is formed, and now there is the desired L to L transformation, producing small enantioexcesses of the normal L-amino acids. We also show how these can be amplified, not by making more of the L form but by increasing its concentration in water solution. The process can start with a miniscule excess and in one step generate water solutions with L/D ratios in the over 90% region. Kinetic processes can exceed the results from equilibria. We have also examined such amplifications with ribonucleosides, and have shown that initial modest excesses of the D-nucleosides can be amplified to afford water solutions with D to L ratios in the high 90’s. We have shown that the homochiral compound has two effects on the solubility of the racemate. On one hand it decreases the solubility of the racemate by its role in the solubility product, as a theoretical equation predicts. On the other hand, it increases the solubility of the racemate by changing the nature of the solvent, acting as a cosolvent with the water. This explains why the amplification, while large, is not as large as the simple theoretical equation predicts. Thus when credible examples are produced where small enantioexcesses of D-ribose are created under credible prebiotic conditions, the prerequisites for the RNA world will have been exemplified.     

Chemicobiological interactions and the use of partition coefficients in their correlation

Using octanol/water partition coefficients as an operational definition of hydrophobicity, 70 examples are given in which the hydrophobic interactions of organic compounds with themselves (in micelles) with macromolecules or with biological systems can be quantitatively correlated by the expression: log RBR = a log P + b. In this expression RBR is a binding constant or a relative biological response, P is the octanol/water partition coefficient and a and b are constants obtained via the method of least squares. These results are strong support for the utility of log P in the correlation of hydrophobic interactions. They also illustrate the extremely wide range of processes in which hydrophobic bonding plays a critical role.     

Biotransformation in double-phase systems: physiological responses of Pseudomonas putida DOT-T1E to a double phase made of aliphatic alcohols and biosynthesis of substituted catechols

Pseudomonas putida strain DOT-T1E is highly tolerant to organic solvents, with a logP(ow) (the logarithm of the partition coefficient of a solvent in a two-phase water-octanol system of > or =2.5. Solvent tolerant microorganisms can be exploited to develop double-phase (organic solvent and water) biotransformation systems in which toxic substrates or products are kept in the organic phase. We tested P. putida DOT-T1E tolerance to different aliphatic alcohols with a logP(ow) value between 2 and 4, such as decanol, nonanol, and octanol, which are potentially useful in biotransformations in double-phase systems in which compounds with a logP(ow) around 1.5 are produced. P. putida DOT-T1E responds to aliphatic alcohols as the second phase through cis-to-trans isomerization of unsaturated cis fatty acids and through efflux of these aliphatic alcohols via a series of pumps that also extrude aromatic hydrocarbons. These defense mechanisms allow P. putida DOT-T1E to survive well in the presence of high concentrations of the aliphatic alcohols, and growth with nonanol or decanol occurred at a high rate, whereas in the presence of an octanol double-phase growth was compromised. Our results support that the logP(ow) of aliphatic alcohols correlates with their toxic effects, as octanol (logP(ow) = 2.9) has more negative effects in P. putida cells than 1-nonanol (logP(ow) = 3.4) or 1-decanol (logP(ow) = 4). A P. putida DOT-T1E derivative bearing plasmid pWW0-xylE::Km transforms m-xylene (logP(ow) = 3.2) into 3-methylcatechol (logP(ow) = 1.8). The amount of 3-methylcatechol produced in an aliphatic alcohol/water bioreactor was 10- to 20-fold higher than in an aqueous medium, demonstrating the usefulness of double-phase systems for this particular biotransformation.     

Chelation therapy for methylmercury(II) poisoning. Synthesis and determination of solubility properties of MeHg(II) complexes of thiol and dithiol antidotes

Methylmercury(II) complexes of the most widely studied antidotes for mercury poisoning have been prepared, and both the water solubility and 1-octanol/water partition coefficients determined for these complexes and the L-cysteine complex. New complexes of N-acetyl-D,L-penicillamine, 2-mercaptosuccinic acid, meso-dimercaptosuccinic acid, and Unithiol have been synthesized and characterized, and are found to have the formulations MeHgSCMe2CH(NHCOMe)CO2H, MeHgSCH(CO2H)CH2CO2H, MeHgSCH(CO2H)CH(CO2H)SHgMe, and Na[MeHgSCH2CH-(SHgMe)CH2SO3], respectively. Trends in octanol/water partition coefficients are consistent with reported studies of the effectiveness of antidotes for MeHg(II) poisoning and redistribution of MeHg(II) on administration of antidotes, particularly for British anti-Lewisite, Unithiol, and meso-dimercaptosuccinic acid.