Water content, enzyme activity

Although provides a useful reference scale for predicting the effect of water on enzyme activity, the adsorption (or retention) of water in the solid and fluid phases strongly varies with a. The adsorption/retention data is usually plotted in terms of water adsorbed by the solid phase vs. water retained in the solvent, or water adsorbed (retained) vs. a. Typically, the solvent water content increases linearly with for lipophilic solvents, sharply at high (approximately 0.6) for slightly polar solvents, - and sharply at low for very polar solvents. In addition, water adsorption is strongly influenced by the types and concentration of substrates. Profiles similar to those just described for slightly polar solvents exist for solid-phase (lyophilized or immobilized) enzymes. Typically, water... 

When either the organic solvent or the ionic liquid is used as pure solvent, proper control over the water content, or rather the water activity, is of crucial importance, as a minimum amount is necessary to maintain the enzyme s activity. For ionic liquids, a reaction can be operated at constant water activity by use of the same methods as established for organic solvents [17]. [BMIM][PFg] or [BMIM][(CF3S02)2N], for example, may be used as pure solvents and in biphasic systems. Water-miscible ionic liquids, such as [BMIM][BF4] or [MMIM][MeS04], can be used in the second case. 

Iborra and co-workers (Entry 8) examined the transesterification of N-acetyl-i-tyrosine ethyl ester in different ionic liquids and compared their stabilizing effect relative to that found with 1-propanol as solvent [36]. Despite the fact that the enzyme activity in the ionic liquids tested reached only 10 to 50 % of the value in 1-propanol, the increased stability resulted in higher final product concentrations. Fixed water contents were used in both studies. 

Further studies of Pseudomonas sp. lipase revealed a strong influence of the water content of the reaction medium (Entry 20) [48]. To be able to compare the enzyme activity and selectivity as a function of the water present in solvents of different polarities, it is necessary to use the water activity (a ) in these solvents. We used the... 

Lipases have also been used as initiators for the polymerization of lactones such as /3-bu tyro lac tone, <5-valerolactone, e-caprolactone, and macrolides.341,352-357 In this case, the key step is the reaction of lactone with die serine residue at the catalytically active site to form an acyl-enzyme hydroxy-terminated activated intermediate. This intermediate then reacts with the terminal hydroxyl group of a n-mer chain to produce an (n + i)-mer.325,355,358,359 Enzymatic lactone polymerization follows a conventional Michaelis-Menten enzymatic kinetics353 and presents a controlled character, without termination and chain transfer,355 although more or less controlled factors, such as water content of the enzyme, may affect polymerization rate and the nature of endgroups.360... 

The investigation of enzymes in water-miscible organic solvents trivially called non-aqueous enzymology about 20 years ago became an independent part of modem biochemistry and enzymology [174-176], In concentrated organic solvents, with the water content less than 10-15%, the enzymes are rather stable and can even retain their activity [176, 177], Recent studies even demonstrated improvement of the enzyme activity in concentrated organic solvents [178],... 

Hi) Enzyme activity is arrested by adding 10.0 ml of 0.02 N NaOH to each tube. Remove them from the water-bath and mix the contents thoroughly,... 

The aqueous cores of reverse micelles are of particular interest because of their analogy with the water pockets in bioaggregates and the active sites of enzymes. Moreover, enzymes solubilized in reverse micelles can exhibit an enhanced catalytic efficiency. Figure B4.3.1 shows a reverse micelle of bis(2-ethylhexyl)sulfosuccinate (AOT) in heptane with three naphthalenic fluorescent probes whose excited-state pK values are much lower than the ground-state pK (see Table 4.4) 2-naphthol (NOH), sodium 2-naphthol sulfonate (NSOH), potassium 2-naphthol-6,8-disulfonate (NSOH). The spectra and the rate constants for deprotonation and back-recombination (determined by time-resolved experiments) provide information on the location of the probes and the corresponding ability of their microenvironment to accept a proton , (i) NDSOH is located around the center of the water pool, and at water contents w = [H20]/[A0T] >... 

Usually, activities of enzymes (hydrogenases included) are investigated in solutions with water as the solvent. However, enhancement of enzyme activity is sometimes described for non-aqueous or water-limiting surroundings, particular for hydrophobic (or oily) substrates. Ternary phase systems such as water-in-oil microemulsions are useful tools for investigations in this field. Microemulsions are prepared by dispersion of small amounts of water and surfactant in organic solvents. In these systems, small droplets of water (l-50nm in diameter) are surrounded by a monolayer of surfactant molecules (Fig. 9.15). The water pool inside the so-called reverse micelle represents a combination of properties of aqueous and non-aqueous environments. Enzymes entrapped inside reverse micelles depend in their catalytic activity on the size of the micelle, i.e. the water content of the system (at constant surfactant concentrations). 

The methods of gel synthesis, immobilization of monomer conjugated enzyme, assay of enzyme activity, and determination of gel water content have been published elsewhere (4,5). A schematic of the synthesis is shown in Fig. 1. The gel compositions are identified as NA-100" (100% NIPAAm), "NA-95" (95% NIPAAm, 5% AAm), NA-90 (90% NIPAAm, 10% AAm) and "NA-85" (85% NIPAAm, 15% AAm) all are based on mole percents of monomers. Total monomer concentration was always 1.75 M. The experiment to determine the temperature dependence of enzyme activity was carried out after the enzyme reversibility experiment. 

Figure 8. The change of enzyme activity at 40 C in the NA series of gels (first cycle) as a function of the gel water contents at 40 C.

In RMs, enzyme properties will depend on essentially three factors the structure of RMs, the dynamics of RMs, and the distribution of enzyme in RMs. Based on an understanding of these aspects, Bru et al. [183] have developed a model, as summarized below, which simulates the dependence of enzyme activity on water content. 

The importance of the particular compartmentation in this field is made apparent by a series of interesting and partly still unexplained effects. For example, when the amount of water is varied in the reverse micellar solution, the maximum enzyme activity - even in the case of hydrolases - is not observed with higher water-content values, but with relatively low amounts of water. In addition, the local pH - due to the constraints of the water pool - is anomalous with respect to the pH value in water (El Seoud, 1984 Luisi and Straub, 1984). 

Martinek et al. (1981) also studied stabilization in systems of low water content. Several enzymes have been microencapsulated into reversed micelles formed by surfactants in apolar organic solvents (see Chapter 9). The enzymes retained their catalytic activity and substrate specificity. 

The scope and limitations of biocatalysis in non-conventional media are described. First, different kinds of non-conventional reaction media, such as organic solvents, supercritical fluids, gaseous media and solvent-free systems, are treated. Second, enzyme preparations suitable for use in these media are described. In several cases the enzyme is present as a solid phase but there are methods to solubilise enzymes in non-conventional media, as well. Third, important reaction parameters for biocatalysis in non-conventional media are discussed. The water content is of large importance in all non-conventional systems. The effects of the reaction medinm on enzyme activity, stabihty and on reaction yield are described. Finally, a few applications are briefly presented. 

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