Effects of water activity and aqueous solvent ordering on thermal stability of lysozyme, alpha-chymotrypsinogen A, and alcohol dehydrogenase

Effects of water activity (aW) and solvent ordering were separately analyzed on the thermal unfolding of lysozyme and alpha-chymotrypsinogen A, and also on the thermal deactivation of yeast alcohol dehydrogenase (YADH) in aqueous solutions with various additives. With the coexistence of additives, water activity was the determinant of the extent of the change in the thermal stability of proteins while solvent ordering was the determinant of the direction of the change. The parameter alpha, determined from the activity coefficient of water, representing the deviation of aW from that of the ideal solution, was useful as a quantitative index of the solvent ordering showing good correlations with the unfolding temperature and enthalpy of lysozyme and alpha-chymotrypsinogen A and also with the thermal deactivation rate constant of YADH at a constant aW. Solvent ordering seemed to affect the thermal stability of proteins mainly through its effect on the intramolecular hydrophobic interaction among amino acid residues in a protein molecule but the contribution of the electrostatic interaction including hydrogen bonding through the change in permittivity of solution was also suggested.     

Effects of water activity and aqueous solvent ordering on thermal stability of lysozyme, α-chymotrypsinogen A, and alcohol dehydrogenase

Effects of water activity (a_W) and solvent ordering were separately analyzed on the thermal unfolding of lysozyme and α-chymotrypsinogen A, and also on the thermal deactivation of yeast alcohol dehydrogenase (YADH) in aqueous solutions with various additives. With the coexistence of additives, water activity was the determinant of the extent of the change in the thermal stability of proteins while solvent ordering was the determinant of the direction of the change. The parameter α, determined from the activity coefficient of water, representing the deviation of a_W from that of the ideal solution, was useful as a quantitative index of the solvent ordering showing good correlations with the unfolding temperature and enthalpy of lysozyme and α-chymotrypsinogen A and also with the thermal deactivation rate constant of YADH at a constant a_W. Solvent ordering seemed to affect the thermal stability of proteins mainly through its effect on the intramolecular hydrophobic interaction among amino acid residues in a protein molecule but the contribution of the electrostatic interaction including hydrogen bonding through the change in permittivity of solution was also suggested.     

Relationship between preferential interaction of a protein in an aqueous mixed solvent and its solubility

The present paper is devoted to the derivation of a relation between the preferential solvation of a protein in a binary aqueous solution and its solubility. The preferential binding parameter, which is a measure of the preferential solvation (or preferential hydration) is expressed in terms of the derivative of the protein activity coefficient with respect to the water mole fraction, the partial molar volume of protein at infinite dilution and some characteristics of the protein-free mixed solvent. This expression is used as the starting point in the derivation of a relationship between the preferential binding parameter and the solubility of a protein in a binary aqueous solution. The obtained expression is used in two different ways: (1) to produce a simple criterion for the salting-in or salting-out by various cosolvents on the protein solubility in water, (2) to derive equations which predict the solubility of a protein in a binary aqueous solution in terms of the preferential binding parameter. The solubilities of lysozyme in aqueous sodium chloride solutions (pH=4.5 and 7.0), in aqueous sodium acetate (pH=8.3) and in aqueous magnesium chloride (pH=4.1) solutions are predicted in terms of the preferential binding parameter without any adjustable parameter. The results are compared with experiment, and for aqueous sodium chloride mixtures the agreement is excellent, for aqueous sodium acetate and magnesium chloride mixtures the agreement is only satisfactory.     

Lipase catalyzed esterification of glycidol in organic solvents

We studied the resolution of racemic glycidol through esterification with butyric acid catalyzed by porcine pancreatic lipase in organic media. A screening of seven solvents (log P values between 0.49 and 3.0, P being the n-octanol-water partition coefficient of the solvent) showed that neither log P nor the logarithm of the molar solubility of water in the solvent provides good correlations between enantioselectivity and the properties of the organic media. Chloroform was one of the best solvents as regards the enantiomeric purity (e. p.) of the ester produced. In this solvent, the optimum temperature for the reaction was determined to be 35 degrees C. The enzyme exhibited maximum activity at a water content of 13 +/- 2% (w/w). The enantiomeric purity obtained was 83 +/- 2% of (S)-glycidyl butyrate and did not depend on the alcohol concentration or the enzyme water content for values of these parameters up to 200 mM and 25% (w/w), respectively. The reaction was found to follow a BiBi mechanism. (c) 1993 John Wiley & Sons, Inc.     

Predicting the Activity Coefficients of Free-Solvent for Concentrated Globular Protein Solutions Using Independently Determined Physical Parameters

The activity coefficient is largely considered an empirical parameter that was traditionally introduced to correct the non-ideality observed in thermodynamic systems such as osmotic pressure. Here, the activity coefficient of free-solvent is related to physically realistic parameters and a mathematical expression is developed to directly predict the activity coefficients of free-solvent, for aqueous protein solutions up to near-saturation concentrations. The model is based on the free-solvent model, which has previously been shown to provide excellent prediction of the osmotic pressure of concentrated and crowded globular proteins in aqueous solutions up to near-saturation concentrations. Thus, this model uses only the independently determined, physically realizable quantities: mole fraction, solvent accessible surface area, and ion binding, in its prediction. Predictions are presented for the activity coefficients of free-solvent for near-saturated protein solutions containing either bovine serum albumin or hemoglobin. As a verification step, the predictability of the model for the activity coefficient of sucrose solutions was evaluated. The predicted activity coefficients of free-solvent are compared to the calculated activity coefficients of free-solvent based on osmotic pressure data. It is observed that the predicted activity coefficients are increasingly dependent on the solute-solvent parameters as the protein concentration increases to near-saturation concentrations.     

Enzymatic esterification in organic media: role of water and organic solvent in kinetics and yield of butyl butyrate synthesis

Summary The respective roles of organic solvent and of water in butyl butyrate synthesis from n-butanol and n-butyric acid in n-hexane by Mucor miehei lipase have been investigated by analysis of the kinetics and the reaction balances. Esterificaton was found to take place in both low water systems containing solid enzyme in hexane and in biphasic aqueous enzyme solution/hexane systems. In the solid enzyme system, the enzyme adsorbed the water produced, thus delaying the appearance of a discrete aqueous phase. As expected, the presence of some water was indispensable for this system, as its removal or exclusion by various means (adsorption, distillation) affected enzyme activity. However, water removal had little effect on the final yield of esterification. Reaction velocities were quite similar for the solid enzyme/hexane system and for the biphasic aqueous enzyme solution/hexane system. In the latter case, the butyl butyrate formed was almost exclusively found in the organic phase. Ethyl butyrate, a more polar compound, was synthesized with a lower yield. These results allow the conclusion that the reaction took place in a phase consisting of either solid hydrated enzyme with no discrete aqueous phase or of an aqueous enzyme solution by basically similar mechanisms according to the amount of water available to the system, the esterification being driven to completion by transfer of the ester into the organic phase because of a favourable partition coefficient. Offprint requests to: F. Monot     

Role of organic solvents on Pa-hydroxynitrile lyase interfacial activity and stability

Catalytic activity and adsorption of Pa-hydroxynitrile lyase (Pa-Hnl) was investigated at various organic solvent/water interfaces. We focused on the role of solvent polarity in promoting activity and stability in two-phase systems, specifically for the solvents heptane, dibutyl ether (DBE), diisopropyl ether (DIPE), butylmethyl ether (BME), and methyl tert-butyl ether (MTBE). Enzyme activity towards mandelonitrile cleavage was determined in a recycle reactor with a well-defined interfacial area as described by Hickel, et al. 1999. Here the recycle reactor was modified to permit exchange of the aqueous phase. With this modification, irreversibility of enzyme adsorption was determined as a function of the adsorption time at the interface. Irreversibility of enzyme adsorption was also investigated by measuring the surface pressure of a sessile-drop upon washout. We find that Pa-Hnl exhibits the highest stability but the lowest initial catalytic activity at the aqueous/organic solvent interface with the most polar organic solvents. Thus, DIPE and MTBE display no loss in enzyme activity over a period of several hours. However, the more apolar the solvent is the higher the initial Pa-Hnl activity, but the faster the loss of activity. Dynamic tensiometry reveals that Pa-Hnl adsorbs more strongly at the interface of the more apolar solvents. Surprisingly, Pa-Hnl develops some irreversible adsorption after 30 min at the DIPE/water interface, but does not lose catalytic activity.     

Activity and stability of cross-linked tyrosinase aggregates in aqueous and nonaqueous media

Cross-linked tyrosinase aggregates were prepared by precipitating the enzyme with ammonium sulfate and subsequent cross-linking with glutaraldehyde. Both activity and stability of these cross-linked enzyme aggregates (CLEAs) in aqueous solution, organic solvents, and ionic liquids have been investigated. Immobilization effectively improved the stability of the enzyme in aqueous solution against various deactivating conditions such as pH, temperature, denaturants, inhibitors, and organic solvents. The stability of the CLEAs in various organic solvents such as tert-butanol (t(1/2)=326.7h at 40°C) was significantly enhanced relative to that in aqueous solution (t(1/2)=5.5h). The effect of thermodynamic water activity (a(w)) on the CLEA activity in organic media was examined, demonstrating that the enzyme incorporated into CLEAs required an extensive hydration (with an a(w) approaching 1.0) for optimizing its activity. The impact of ionic liquids on the CLEA activity in aqueous solution was also assessed.     

Studies on synthesis of short chain alkyl esters catalyzed by goat pregastric lipase

Goat pregastric lipase, in the form of a suspended enzyme powder, was found to be active in catalyzing the synthesis of alkyl esters in anhydrous organic solvents. The rate of catalyzed synthesis of esters was very dependent on the solvent medium, and maximum activity was found when a hydrocarbon was used as the solvent. The optimal temperature for the catalyzed synthesis ranged from 30 to 40°C and the maximal temperature was 35°C for the synthesis of butyl caproate in isooctane. The selectivity for the carbon-chain length of the fatty acid by the lipase was similar to that seen in hydrolysis reactions in aqueous solution, and the optimal rate of synthesis of alkyl esters was found for synthesis of the esters which had 8 or 10 carbons in the alkyl moieties from the two individual substrates. The rate of synthesis was also dependent on the water content in the system, with maximum activity occurring at 1% w/w water in isooctane.     

Enzymatic oxidation of ethanol in the gaseous phase

The enzymatic conversion of gaseous substrates represents a novel concept in bioprocessing. A critical parameter in such systems is the water activity, A(w) The present article reports the effect of A(w) on the catalytic performance of alcohol oxidase acting on ethanol vapors. Enzyme activity in the gas-phase reaction increases several orders of magnitude, whereas the thermostability decreases drastically when A(w) is increased from 0.11 to 0.97. The enzyme is active on gaseous substrates even at hydration levels below the monolayer coverage. Enhanced thermostability at lower hydrations results in an increase in the optimum temperature of the gas-phase reaction catalyzed by alcohol oxidase. The apparent activation energy decreases as A(w) increases, approaching the value obtained for the enzyme in aqueous solution. The formation of a pread-sorbed ethanol phase on the surface of the support is not a prerequisite for the reaction, suggesting that the reaction occurs by direct interaction of the gaseous substrate with the enzyme. The gas-phase reaction follows Michaelis-Menten kinetics, with a K(m) value almost 100 times lower than that in aqueous solution. Based on vapor-liquid equilibrium data and observed K(m) values, it is postulated that during the gas-phase reaction the ethanol on the enzyme establishes an equilibrium with the ethanol vapor similar to that between ethanol in water and ethanol in the gas phase.     

Influence of solvent and water activity on kinetically controlled peptide synthesis.

alpha-Chymotrypsin deposited on Celite was used to catalyse peptide synthesis reactions between N-protected amino acid esters and leucine amide in organic media with low water content. The influence of the solvent and the thermodynamic water activity on the reaction kinetics was studied. The substrate specificity in the reactions was shown to be a combination of the substrate specificity of the enzyme in aqueous media and the influence of the solvents. The magnitude of the solvent effects differed greatly depending on the substrates used. In hydrophobic solvents high reaction rates were observed and the competing hydrolysis of the ester substrate occurred to only a minor extent. Reactions occurred at water activities as low as 0.11, but the rate constants increased with increasing water activity and were about two orders of magnitude higher at the highest water activity tested (0.97).     

Effects of osmolytes on solvent features of water in aqueous solutions

The solvatochromic solvent features of water (dipolarity/polarizability, π*, hydrogen bond donor acidity, α, and hydrogen bond acceptor basicity, β) of water have been determined in aqueous solutions of erythritol, glucose, inositol, sarcosine, xylitol and urea with concentrations from 0 to ~3 M and higher. The concentration effects of the osmolytes on the solvent features of water were characterized and compared with those reported previously for sorbitol, sucrose, trimethylamine N-oxide (TMAO), and trehalose. The solvent features of water in solutions of all osmolytes except TMAO and sarcosine were established to be linearly interrelated. It is shown that the concentration effects of essentially all nonionic osmolytes depend on osmolytes’ lipophilicity, molecular polarizability, and polar surface area. It is demonstrated that solubility of various compounds in aqueous solutions of glucose, sucrose, sorbitol, and urea of varied concentrations may be described in terms of solvent dipolarity/polarizability of water in these solutions. Surface tension of aqueous solutions of sucrose and sorbitol may also be described in the same terms. The relative permittivity of aqueous solutions of glucose and sucrose may be described in terms of the solvent hydrogen bond donor acidity of water. It is suggested that the effects of nonionic osmolytes on behavior of proteins and nucleic acids in aqueous media may be considered in terms of the altered solvent features of water instead of “nano-molecular crowding” effect.     

DMSO enhanced conformational switch of an interfacial enzyme

Interfacial proteins function in unique heterogeneous solvent environments, such as water–oil interfaces. One important example is microbial lipase, which is activated in an oil‐water emulsion phase and has many important enzymatic functions. A unique aprotic dipolar organic solvent, dimethyl sulfoxide (DMSO), has been shown to increase the activity of lipases, but the mechanism behind this enhancement is still unknown. Here, all‐atom molecular dynamics simulations of lipase in a binary solution were performed to examine the effects of DMSO on the dynamics of the gating mechanism. The amphiphilic α5 region of the lipase was a focal point for the analysis, since the structural ordering of α5 has been shown to be important for gating under other perturbations. Compared to the closed‐gorge ensemble in an aqueous environment, the conformational ensemble shifts towards open‐gorge structures in the presence of DMSO solvents. Increased width of the access channel is particularly prevalent in 45% and 60% DMSO concentrations (w/w). As the amount of DMSO increases, the α5 region of the lipase becomes more α‐helical, as we previously observed in studies that address water–oil interfacial and high pressure activation. We believe that the structural ordering of α5 plays an essential role on gating and lipase activity.     

The water-dependence of the catalytic activity of bilirubin oxidase suspensions in low-water systems.

We investigated the enzymic activity of bilirubin oxidase when it is suspended as a lyophilized powder in a low-water system. The enzyme required buffer salts and a source of water to show activity. This study investigated the complete range of water thermodynamic activity (a(w)) by combining the use of salt hydrates and two-phase systems with concentrated solutes in the aqueous phase. When free water was added, activity reached a maximum at a defined water content, but this maximum increased with buffer content, suggesting that there was competition for water with the buffer salts from which the enzyme was lyophilized. Alternatively, a range of salt hydrates was used, each able to fix the water activity (a(w)) at a different value. By providing water to the organic solvent phase in this way, the dependency of enzyme activity upon a(w) was investigated and shown to be independent of buffer concentration. However, the optimum a(w) was uncertain because the available a(w) range for salt hydrates is < or = 0.90. Investigation of the remaining water activity range was made possible by using an a(w) depressor (sorbitol) to lower the a(w) of a two-phase system. The optimum a(w) for the bilirubin oxidase activity in this two-phase system was a(w) = 0.936, independent of buffer concentration. The study therefore confirmed the need to control the water 'available' to low-water systems and the dependence of enzyme activity on water thermodynamic activity (a(w)) not water content.     

Polyvinyl alcohol--trypsin as a catalyst for amino acid ester synthesis in organic media

The bovine trypsin-catalyzed synthesis of N-alpha-benzoyl-DL-arginine esters from N-benzoyl-DL-arginine were studied in various organic solvents. Trypsin was immobilized to polyvinyl alcohol (PVA) by adsorption from its aqueous solutions. Immobilized enzyme showed higher catalytic activities than free enzyme for amino acid esterification in ethanol. The yield of ester is strongly dependent upon the PVA/trypsin ratio and water content in the reaction medium. The rate and equilibrium constant of the ester formation reaction are also dependent on water content.     

Pressure affects enzyme function in organic media

Pressure affects enzyme function in nonaqueous media. Activation volumes have been determined and provide evidence that the primary effect of pressure is to enhance the stripping of water off an enzyme in polar organic solvents and leads to decreased enzymatic activity. Activation volumes of subtilisin Carlsberg in organic solvents, particularly with the enzyme hydrated, have a larger magnitude than activation volumes determined in aqueous solutions. This study provides further evidence that enzymatic activity in polar organic solvents is dominated by the interaction of enzyme-bound water with the solvent. From a practical standpoint, however, the results of this study suggest that enzymatic catalysis in organic solvents may be controlled by the combined effects of pressure and enzyme hydration. (c) 1993 John Wiley & Sons, Inc.     

Enzymes in Low Water Systems

Water is fundamental for enzyme action and for formation of the three-dimensional structure of proteins. Hence, it may be assumed that studies on the interplay between water and enzymes can yield insight into enzyme function and formation. This has proven correct, because the numerous studies that have been made on the behavior of water-soluble and membrane enzymes in systems with a low water content (reverse micelles or enzymes suspended in nonpolar organic solvents) have revealed properties of enzymes that are not easily appreciated in aqueous solutions. In the low water systems, it has been possible to probe the relation between solvent and enzyme kinetics, as well as some of the factors that affect enzyme thermostability and catalysis. Furthermore, the studies show that low water environments can be used to stabilize conformers that exhibit unsuspected catalytic properties, as well as intermediates of enzyme function and formation that in aqueous media have relatively short life-times. The structure of enzymes in these unnatural conditions is actively being explored.     

Thermodynamics of some nucleic acid bases and nucleosides in water, and their transfer to aqueous glucose and sucrose solutions at 298.15 K

The partial molar heat capacities and volumes of some of the constituents of nucleic acids have been determined in water and 1 molal aqueous glucose and sucrose solutions in order to elucidate the nature of interactions occurring between various nucleic acid bases, nucleosides and the sugar (glucose and sucrose) molecules. The results have been explained in terms of the contributions from hydrophobic interactions, hydrophilic interactions and the hydrogen bonding between the solute and solvent molecules. The results have also been compared with those of amino acids and peptides in aqueous glucose and sucrose solutions.     

A three-dimensional solubility parameter approach to nonaqueous enzymology

Widespread commercial application of enzymes as catalysts for specialty or commodity chemical synthesis will require their use in nonaqueous systems. While a number of non-aqueous enzyme applications have been demonstrated, the lack of useful rules for predicting enzyme-solvent interactions has hindered the development of this technology. Both Hildebrand and solvent hydrophobicity (octanol-water partition coefficient) parameters have been used previously to correlate and predict enzyme activity in nonaqueous systems, with some success, but any single-parameter approach is inherently limited in its ability to reflect the spectrum of possible enzyme-solvent interactions. Therefore, this study evaluates the three-dimensional solubility parameter space, as proposed by Hansen, to correlate and predict enzyme activity in microaqueous, miscible, and biphasic nonaqueous systems. Preliminary results suggest that Hansen parameters may be useful for correlating nonaqueous enzyme activity, and that the dispersive and polar parameters may be disproportionately important in single-phase microaqueous systems. The Hansen hydrogen-bonding parameter appears to be the only parameter yet evaluated capable of correlating the water requirement for enzyme activity in microaqueous systems, suggesting that water affects protein structure through enthalpic rather than entropic processes in nonaqueous systems. Insufficient data are available for miscible and biphasic systems, but it is proposed that enzyme activity may correlate with the average solubility parameters of miscible systems and of the aqueous phase in biphasic systems.     

Effect of water activity on enzyme hydration and enzyme reaction rate in organic solvents

The effects of water on enzyme (protein) hydration and catalytic efficiency of enzyme molecules in organic solvents have been analyzed in terms of the thermodynamic activity of water, which has been estimated by the NRTL or UNIFAC equations. When the amount of water bound to the enzyme was plotted as a function of water activity, the water adsorption isotherms obtained from the water-solvent liquid mixtures were similar to the reported water-vapor adsorption isotherms of proteins. The water adsorption of proteins from the organic media was not significantly dependent on the properties of the solvents or the nature of the proteins. It is also shown that there is a linear relationship between the logarithm of the enzyme reaction rate and water activity. However, the dependence of the enzyme reaction rate on water activity was found to be different depending on the properties of the solvent. The relationship between water activity and other solvent parameters such as solvent hydrophobicity and the solubility of water in the solvent is also discussed.