Micelles enzyme-catalyzed reactions

Free energy of micellization, 24 130 Free enzyme-catalyzed reactions ionic liquids in, 26 897-898 Free fatty acids, 70 802-804, 825-826 removal of, 70 807 as soap bar additives, 22 742-743 Free-flow agents, in sodium chloride (salt), 22 808... 

It appears from a survey of the literature that the essential properties of micelles in nonpolar solvents are understood, namely their stability and variations of size, the dissociation behavior, and their solubilizing capacities. Reverse micelles can dissolve relatively large amounts of water (1-10% w/v depending on emulsion formula) as well as polar solutes and, of course, water-soluble compounds. Consequently, they can be used as media for a number of reactions, including enzyme-catalyzed reactions. Very few attempts to investigate such reverse micelles at subzero temperatures are known, in spite of the fact that hydrocarbon solutions present very low freezing points. 

From bisubstrate, kinetic analysis with a transferase from hen oviduct that, under the conditions of the assay, formed only GlcNAc-PP-Dol, it followed that both dolichol phosphate and UDP-GlcNAe have to he bound to the enzyme before release of the product occurs.52 However, the fact that only partially purified preparations have thus far been obtained (the preparations may also still be contaminated with substrates and product), together with experimental difficulties in handling both the substrate dolichol phosphate (which, furthermore, is not one compound, see the earlier discussion) and the unstable enzyme (enveloped in micelles of detergent), make difficult a sensible interpretation and comparison of the kinetic parameters detenuined for the different enzvme-preparations. The solubilized enzymes catalyzing reactions 1,2, and 3 have in common their alkaline pH optima and dependence on Mg2+ or Mn2+ ions. The latter fact makes (ethylenedinitrilo)tetraacetic acid (EDTA) a reversible inhibitor of enzyme activity and an important experimental tool. 

Proteins and enzymes have been successfully entrapped in surfactant-solubilized water pools in organic solvents [268-278]. Furthermore, many reversed-micelle-entrapped enzymes retained their activity and could be used for peptide synthesis [273,274]. That the water pools corresponding to very small w-values exhibited freezing points Mow — 50°C enabled both the enzyme structures and the rates of enzyme-catalyzed reactions to be investigated at low temperatures. These studies much aided the development of cryoenzymology [279, 180],... 

The effects of macromolecules other than surfactants on the rates of organic reactions have been investigated extensively (Morawetz, 1965). In many cases, substrate specificity, bifunctional catalysis, competitive inhibition, and saturation (Michaelis-Menten) kinetics have been observed, and therefore these systems also serve as models for enzyme-catalyzed reactions and, in these and other respects, resemble micellar systems. Indeed, in some macromolecular systems micelle formation is very probable or is known to occur, and in others mixed micellar systems are likely. Recent books and reviews should be consulted for a more detailed description of macromolecular systems and for their applicability as models for enzymatic catalysis and other complex interactions (Morawetz, 1965 Bruice and Benkovic, 1966 Davydova et al., 1968 Winsor, 1968 Jencks, 1969 Overberger and Salamone, 1969). 

Very large rate enhancements by functional micelles have been observed, and sometimes, as in some deacylations, they are comparable in magnitude to those observed in enzyme catalyzed reactions, and it is important to understand the source of these rate enhancements. 

These reverse micelles, or water pools, also provide excellent media for enzyme-catalyzed reactions [141,142], and recently the details of some enzymic reactions have been examined at low temperatures using supercooled reverse micelles... 

Reverse micelles are normally used in enzyme-catalyzed reactions. The water in the core of the micelle is called the water pool. At a constant surfactant concentration, the amount of water introduced determines the micellar size. The nature of the entrapped water in reverse micelles has been a subject of considerable debate. At low amounts of water, it is thought that most of it is bound, leading to low enzyme activity. At higher amounts, the water becomes more free with a resultant increase in enzymatic activity. 

The conditions required to favor esterification can be obtained in different manners. It is possible to add a water-miscible solvent that will lower the water concentration and increase the solubility of organic substrates and products. It is also possible to work in a two-phase system with a non-water-miscible solvent, which will serve as a reservoir for the substrates and products. This can be achieved either with macroscopic phases or with highly dispersed systems such as reversed micelles. In the above-mentioned cases, the enzyme-catalyzed reaction takes place in the aqueous phase or at the phase interface. The enzyme can be dissolved in this phase or immobilized by covalent attachment to a solid carrier... 

TABLE 4 Third-Order Nonlinear Optical Properties of Polymers Synthesized by Enzyme-Catalyzed Reactions in Reverse Micelle Media... 

Enzymes when hosted in reverse micelles can catalyze reactions that are not favored in aqueous media. Products of high-added value can be thus produced in these media. The potential technical and commercial applications of enzyme-containing microemulsions as microreactors are mainly linked to their unique physicochemical properties. The potential biotechnological applications of microemulsions with immobilized biocatalysts such as enzymes are described in Chapter 12 by Kunz and coworkers and in Chapter 13 by Xenakis and coworkers. 

Micelle-catalyzed reactions are somewhat similar to enzyme-catalyzed reactions the proper choice of surfactant brings about a rate increase of up to 1000-fold, and the diameter of micelles is 30-50 A, similar in size to globular enzymes. Micelle-catalyzed reactions can be treated in a manner analogous to the reaction scheme for enzymatic catalysis, as will be shown below. 

Intramolecularity or induced intramolecularity is an important feature of many enzyme-catalyzed reactions. Micellar systems provide models for understanding the effects of changes in the microreaction environment on reaction rates of bioorganic reactions. There are only a limited number of reports on systematic studies on the effects of micelles on the rates of intramolecular reactions. 

In a system containing surfactant, S, substrate, X, and enzyme, E, the amount of substrate available for an enzyme-catalyzed reaction will be reduced if the substtate was hydrophobic and sequestered within the aggregates of surfactant molecules. This reduction will also depend on the nature of the surfactant. Moreover, the ability of the enzyme to perform at its best may be reduced if it is adsorbed onto the micellar surface. These effects will become more pronounced if the concentration of the surfactant is greater than the critical micelle concentration (cmc). 

Pseudophase Model and Enzyme-Catalyzed Reaction Kinetics in Reverse Micelles... 

When binding of a substrate molecule at an enzyme active site promotes substrate binding at other sites, this is called positive homotropic behavior (one of the allosteric interactions). When this co-operative phenomenon is caused by a compound other than the substrate, the behavior is designated as a positive heterotropic response. Equation (6) explains some of the profile of rate constant vs. detergent concentration. Thus, Piszkiewicz claims that micelle-catalyzed reactions can be conceived as models of allosteric enzymes. A major factor which causes the different kinetic behavior [i.e. (4) vs. (5)] will be the hydrophobic nature of substrate. If a substrate molecule does not perturb the micellar structure extensively, the classical formulation of (4) is derived. On the other hand, the allosteric kinetics of (5) will be found if a hydrophobic substrate molecule can induce micellization. 

Lewis-acid catalysis of Diels-Alder reactions involving bidentate dienophiles in water is possible also if the beneficial effect of water on the catalyzed reaction is reduced relative to pure water. There are no additional effects on endo-exo selectivity. As expected, catalysis by Cu ions is much more efficient than specific-acid catalysis.Using a-amino acids as chiral ligands, Lewis-acid enan-tioselectivity is enhanced in water compared to organic solvents. Micelles, in the absence of Lewis acids, are poor catalysts, but combining Lewis-acid catalysis and micellar catalysis leads to a rate accelaration that is enzyme-like. 

The core of a micelle and the bilayer of a vesicle are comparable with a liquid-crystalline phase and can influence the stereoregularity of asymmetrically catalyzed reactions. Self-organization and the neighborhood of hydrophilic and hydrophobic regions are close to those of natural systems and we designate this as membrane mimetic or enzyme mimetic chemistry [45]. The large field of artificial enzymes was recently reviewed by Murakami et al. [46]. 

The use of microemulsions or reverse micelles as media for chemical and enzymatic reactions has been reviewed in recent years [20,37,38]. Microemulsions, including those based on organogels, are also useful media for enzyme-catalyzed synthetic reactions [37,39-43] and for preparation of nanoparticles [44]. In a very different direction, Vanag and Hanazaki [45] showed that the ferroin-catalyzed Belousov -Zhabitinskii oscillatory reaction exhibits frequency-multiplying bifurcations in reverse AOT microemulsions in octane, A clear understanding of reactivity in microemulsions and insight into how to optimize the experimental conditions requires kinetic models with predictive power. We focus attention primarily on this problem. 

This section describes the horseradish peroxidase-catalyzed synthesis of both homo- and copolymers of aromatic polymers based on phenols, naphthols, aniline, and their derivatives. Syntheses of novel optically active polymers are studied by changing the environment in which the enzyme functions, along with the organization of the monomers in the reaction mixture. To this objective, enzyme-catalyzed polymer syntheses are carried out in bulk monophasic conditions in which the solvent is miscible with water, biphasic solvent systems in which the solvents used for the syntheses are not miscible with water, and oil-in-water system in the presence of a detergent called reverse micelles. These experimental approaches are shown schematically in Fig. 4. 

Micellization of a copolymer, that may be considered as a comb-graft copolymer was studied by Watterson and co-workers [174]. This copolymer, with C10-C12 alkyl side chains, synthesized through enzyme-catalyzed condensation reaction, is of the following structure ... 

The determination of the enzyme activity as a function of the composition of the reaction medium is very important in order to find the optimal reaction conditions of an enzyme-catalyzed synthesis. However, the correlation between the reaction media properties and their effects on enzymatic reactions in reverse micelles is still unclear,... 

As mentioned above, cytochromes were among the first enzymes that were successfully encapsulated in reverse micelles. In a study that appeared in 1979, cytochrome c, a small enzyme catalyzing several reactions and showing peroxidase activity by oxidation of various electron donors, was solubilized at room... 

So far, some oxidoreductase-catalyzed reactions have been examined in reversed micelles. The enzyme activity and stability that depend largely on the microemulsion composition, mostly the water-to-stirfactant ratio (ivo), are often comparable to values in aqueous media. Orlich et al. reported the application of reverse micelles for ADH-catalyzed reduction of less water-soluble ketones in an FDHcontained water, cyclohexane, and Marlipal 013-16 as the surfactant. The reaction rate of ADH for the reduction of 2-heptanone in reverse micellar medium was increased up to 12 times compared to aqueous medium [84], The improved enzymes stability was observed at optimal Wq. Finally, it was possible to perform successful semibatch experiments reducing 2-butanone with full conversion and enantioselectivity [85]. 

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