Enzyme water content

There are many pharmaceutical applications for the modification of one enantiomer over another, and to this end, many have studied these selective reactions in carbon dioxide. Glowacz et al. (1996) studied the enzymatic hydrolysis of triolein and its partial glycerides and found that stereoselectivity depends on reaction time and enzyme water content. They suggest that the water content varies the local environment of the enzyme in carbon dioxide and changes the local pH value. Rantakyla et al. (1996) also found that the hydrolysis of one stereoisomer over another was water-dependent. They studied the hydrolysis of 3-(4-methoxyphenyl)glycidic acid methylester and found that the 2S,3R enantiomer hydrolyzed more than fivefold faster than the 2R3S form. 

Structure depend on the enzyme water content, the enzyme/substrate ratio, the monomer/substrate ratio, and the reaction temperature. 

Glowacz et al. [36] examined the stereoselectivity of PPL during enzymatic hydrolysis of triolein and its partial glycerides in the presence of SCCO2. The water content of the immobilized lipase was varied. The stereoselectivity depends on the reaction time, the substrates, and the enzyme water content. The authors suggested that the effect of the enzyme water content on the activity and selectivity of PPL is based on a modification of the microenvironment of the enzyme by tiie solution of CO2 in water, causing a decrease of the pH value. The PPL with 1.5% water content showed a preference for the sn-3 position. With 5% water content only the racemate of these glycerides could be detected. The lipase with 15% water content showed an sn-3 enantioselectivity only in the analysis of the dioleins and 1,2-dioleins were performed preferentially. 

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 low content of water in these formulations promotes improved stabilization of enzyme and bleach additives. The combination of LAS and AE in a low-water-content formulation is effective at solubilizing enzymes and preserving enzyme stability when the sum of the LAS and water levels ranges between 25% and 45% [53],... 

Several reports have indicated that enzymes are more thermostable in organic solvents than in water. The high thermal stability of enzymes in organic solvents, especially in hydrophobic ones and at low water content, was attributed to increased conformational rigidity and to the absence of nearly all the covalent reactions causing irreversible thermoinactivation in water [23]. 

Enantiomeric or specific synthesis of cyanohydrin is influenced by the reaction medium, cyanide source, water content, buffer pH, enzyme, and temperature during the HNL-catalyzed reaction. To maximize the enantiomeric excess of the cyanohydrin product, care must be taken to minimize the parallel chemical (nonenzymatic) condensation and racemi-zation of products. 

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],... 

Y.L. Khmelnitsky, A.V. Levashov, N.L. Klyachko, and K. Martinek, Engineering biocatalytic systems in organic media with low water content. Enzyme Microb. Technol. 10, 710-724 (1988). 

Initially, the sol gel compositions were optimized using Congo red dye as the dopant because of its optical properties. This facilitates monitoring of the release process by optical spectroscopy. Next, the gels were evaluated for their stabilization and release of subtilisin. These sol gel matrices bring about controlled release of the encapsulated enzyme molecules as a response to a change in the water content of the medium (Figure 2.20).15... 

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] >... 

The use of water-miscible organic solvent-water mixtures is a particularly attractive method for use with cofactor-dependent enzymes due to its simphcity. The high water content can allow dissolution of both enzyme and cofactor, whilst the water-miscible solvent can provide a dual role in both substrate dissolution and as a cosubstrate for cofactor recycling (substrate-coupled cofactor recycling).The asymmetric reduction of a ketone intermediate of montelukast using an engineered ADH in the presence of 50 % v/v isopropanol offers a powerful demonstration of this methodology (Scheme 1.55). 

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). 

There have been a number of reports of the use of enzymes in the extraction of oils from sources such as fish, rape seed, yeast, palms, and soya beans. Celluloses and pectinases are used in pdm oil extraction. In soya bean and fish, much oil has been found to be associated with protein, so that addition of proteases increases the yield of oil and protein. Use of thermostable proteases is preferred, but m general the use of enzymes is limited by the minimal water contents of the various process streams. Trichoderma uride and A niger celluloses, hemicellulases and proteases have been used to extract hydrocarbons from Euphorbia plants 39 40) and similar enzymes used to extract sapogenins from Helleborus 41). 

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.

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