Peter J. Halling

Reaction rate with suspended lipase catalyst shows similar dependence on water activity in different organic solvents.

We have studied the effect of thermodynamic water activity (a W) on the initial rate of esterification catalysed by an immobilised lipase (Lipozyme) suspended in an organic reaction mixture. The catalyst and the organic phase were separately pre-equilibrated to the same aw value. The rate shows similar dependence on aw in reaction mixtures based on five different organic solvents ranging in polarity from pentan-3-one to hexane, and in a liquid reactant mixture. There is a maximum at aw about 0.5, with a decline to 30-70% at aw of either 0.9 or less than 0.01. When the rates are presented in terms of water concentration in the organic phase (or total water content of the system), the maxima for the various solvents come at very different positions, reflecting the widely varying solubilities of water in the organic phase.

Biocatalyst behaviour in low-water systems

An improving understanding of the parameters that affect biocatalyst activity, specificity and stability in low-water non-aqueous media make reliable predictions about the behaviour of such systems increasingly feasible. Here, we discuss some of the key factors, such as control of water activity, and the effects of solvent on K m and protein-ionization state, that must be addressed in order to obtain predictable results.

Salt hydrates for water activity control with biocatalysts in organic media

Addition of solid salt hydrate(s) to the reaction mixture is a convenient method of water activity control. This article discusses the theoretical background to their use, and gives a compilation of data on the water activity values produced by 48 hydrate pairs of possible use in this application.

Water activity fails to predict critical hydration level for enzyme activity in polar organic solvents: Interconversion of water concentrations and activities

Abstract When studying biocatalysts in organic media, it is useful to be able to convert between water concentration and thermodynamic water activity ( a w ). We have obtained relationships for this purpose based on published vapor-liquid equilibrium data for a range of commonly used polar solvents. Their use is illustrated by the reanalysis of literature data for the activity of an immobilized laccase. Kinetic constants measured as a function of water concentration can thus be related to a w . We show that the critical water content for significant V m values is characterized by a similar a w (about 0.4) in five different alcohols; however, the critical a w becomes progressively higher in other water-miscible solvents in the order: dioxane, acetonitrile, tetrahydrofuran, acetone (critical a w about 0.8). This indicates that such polar solvents probably have important direct effects on the enzyme.

Effects of water on equilibria catalysed by hydrolytic enzymes in biphasic reaction systems

Abstract In the literature on hydrolase-catalysed synthetic reactions in aqueous-organic biphasic systems, it has been stated that low water concentrations contribute to favourable shifts in equilibrium. It is argued here that this is not a sufficient condition for mass action effects of water. A simple method of treating such equilibria is suggested, using the thermodynamic activity of water. Only when its activity is significantly reduced below 1 can water contribute to a shift of equilibrium in favour of synthetic products. Such a reduction is not necessarily obtained by creating a biphasic system, however low the water content, but requires that the aqueous phase becomes a very concentrated solution/dispersion of hydrophilic species.

Kinetics of lipase-catalyzed esterification in organic media : correct model and solvent effects on parameters

The Ping-Pong model (incl. alcohol inhibition) is not the correct model to describe the kinetics of a lipase-catalyzed esterification reaction. The first product, water, is always present at the start of the reaction. This leads to an equation with one extra parameter. This new equation fits our experimental data on the esterification of sulcatol and fatty acids in toluene, catalyzed by Candida rugosa lipase. The new model does not significantly improve the mean square of the fit; however, using a model which can be expected to be more correct, results in the conclusion that a larger part of the differences can be explained by substrate solvation. For comparison of the kinetic constants in different solvents, it is essential to make corrections for solvation. The deviation from the average corrected kinetic constant shows to what extent differences can be explained by substrate solvation and an effect on the enzyme. We have made corrections for solvation with the new model for the esterification in toluene, hexane, trichloroethane, and diisopropyl ether. This has resulted in kinetic constants that deviate less from the average value.

Use of salt hydrates to buffer optimal water level during lipase catalysed in synthesis in organic media: a practical procedure for organic chemists

Enzyme catalyzed reactions in mainly organic media depend very much on the amount of water in the system. We have shown that addition of appropriate solid salt hydrates to the reaction mixture is a simple and convenient method to obtain optimal water level conditions throughout the reaction. As a model reaction the esterification of butanoic acid with butanol catalysed by lipase from Candida rugosa was chosen. Variations in the amount of enzyme, in the solvent and in the concentration of reactants were made.

High-affinity binding of water by proteins is similar in air and in organic solvents

Published data for water adsorption by proteins suspended in organic solvents (of interest as enzyme reaction mixtures) have been converted to a basis of thermodynamic water activity (aw). The resulting adsorption isotherms have been compared with those known for proteins equilibrated with water from a gas phase. This comparison can show any effects of the solvent on the interaction between the protein and water at the molecular level. At lower water contents (aw less than about 0.4), similar adsorption isotherms are found in each solvent and in the gas phase; differences are probably less than the likely errors. Hence, it may be concluded that the presence of an organic solvent has little effect on the interaction between proteins and tightly bound water; on a molecular scale there is probably little penetration of the primary hydration layer by solvent molecules, even fairly polar ones such as EtOH. At higher aw values, there are differences between the isotherms which probably are significant. Nonpolar solvents increase the amount of water bound by the enzyme (at fixed aw), while polar solvents (mainly EtOH) may reduce the amount of water bound by the enzyme, presumably by occupying part of the secondary hydration layers in place of water.

Substrate specificity and kinetics of Candida rugosa lipase in organic media

The fatty acid specificity of lipase from Candida rugosa during the esterification of saturated fatty acids and sulcatol in toluene has been studied. The true kinetic parameters are obtained by fitting the experimental data to a Ping-Pong kinetic model that includes alcohol inhibition. The fitted parameter values are compared with apparent values that would be obtained from restricted data sets in which one of the substrate concentrations was kept constant. It has been found that in reactions inhibited by alcohol the true Ping-Pong parameters can be significantly different from the apparent ones. Corrections for solvation are made by using activities instead of concentrations to fit the kinetic parameters. Though activity coefficients, estimated using the UNIFAC group contribution method, vary by over 25% for changing concentrations in the same solvent, their use did not improve the fit to the data. This contrasts with what has been found in comparisons of different solvents, where the differences in activity coefficients are much larger.

Guidelines for reporting of biocatalytic reactions.

Enzymes and whole cells are being increasingly applied in research and industry, but the adoption of biocatalysis relies strongly on useful scientific literature. Unfortunately, too many published papers lack essential information needed to reproduce and understand the results. Here, members of the scientific committee of the European Federation of Biotechnology Section on Applied Biocatalysis (ESAB) provide practical guidelines for reporting experiments. The document embraces the recommendations of the STRENDA initiative (Standards for Reporting Enzymology Data) in the context of pure enzymology and provides further guidelines and explanations on topics of crucial relevance for biocatalysis. In particular, guidelines are given on issues such as the selectivity, specificity, productivity and stability of biocatalysts, as well as on methodological problems related to reactions in multiphase systems. We believe that adoption and use of these guidelines could greatly increase the value and impact of published work in biocatalysis, and hence promote the further growth of applications.