Microemulsions enzymatic reactions

Enzyme activity is highly dependent on the composition, and as a consequence on different microstructures of the microemulsion. Up to the present, no suitable theory exists to explain the correlation between the reaction media properties and their effects on enzymatic reactions in microemulsions. All experimental results on enzymatic reactions show that the activity is greatly affected by the structure of the microemulsion. 

A common technique for separating the water and the oil in a microemulsion is a temperature-induced phase separation, yielding an excess water phase (increasing the temperature) or an excess oil phase (decreasing the temperature). It is the simplest way to separate the oil and the water. Nevertheless this method is quite time consuming and often not complete. In case of enzymatic reaction, a change in temperature can lead to a loss of enzyme stabiUty. 

Holmes et al. (1998) performed two enzymatic reactions, the lipase-catalyzed hydrolysis of y>-nitrophenol butyrate and lipoxygenase-catalyzed peroxidation of linoleic acid, in w/c microemulsions stabilized by a fluorinated two-chained sulfosuccinate surfactant (di-HCF4). The activity of both enzymes in the w/c microemulsion environment was found to be essentially equivalent to that in a water/heptane microemulsion stabilized by Aerosol OT, a surfactant with the same headgroup as di-HCF4. The buffer 2-(A-morpholino)ethanesulfonic acid (MES) was used to fix the pH in the range 5-6. 

The structure and dynamics of inverse (water in oil) micellar solutions and microemulsions are of interest because of the unique properties of the water core, the view that such micelles may serve as models of enzyme active sites, and the potential use of inverse micelles as hosts for enzymatic reactions (80-82). 

In some cases, substrates and enzymes are not soluble in the same solvent. To achieve efficient substrate conversion, a large interface between the immiscible fluids has to be established, by the formation of microemulsions or multiple-phase flow that can be conveniently obtained in microfluidic devices. Until now only a couple of examples are published in which a two-phase flow is used for biocatalysis. Goto and coworkers [431] were first to study an enzymatic reaction in a two-phase flow in a microfluidic device, in which the oxidation ofp-chlorophenol by the enzyme laccase (lignin peroxidase) was analyzed (Scheme 4.106). The surface-active enzyme was solubilized in a succinic acid aqueous buffer and the substrate (p-chlorophenol) was dissolved in isooctane. The transformation ofp-chlorophenol occurred mainly at... 

Microemulsions are used as reaction media for a variety of chemical reactions. The aqueous droplets of water-in-oil micro emulsions can be regarded as minireactors for the preparation of nanoparticles of metals and metal salts and particles of the same size as the starting microemulsion droplets can be obtained [1-3]. Polymerisation in micro emulsions is an efficient way to prepare nanolatexes and also to make polymers of very high molecular weight. Both discontinuous and bicontinuous micro emulsions have been used for the purpose [4]. Microemulsions are also of interest as media for enzymatic reactions. Much work has been done with lipase-catalysed reactions and water-in-oil microemulsions have been found suitable for ester synthesis and hydrolysis, as well as for transesterification [5,6]. 

Holmes et al. reported the first enzyme catalyzed reactions in water-in-CO2 microemulsions (67). Two reactions, a lipase-catalyzed hydrolysis and a lipoxygenase-catalyzed peroxidation, were demonstrated in water-in-C02 microemulsions using the surfactant di(l/7,l/7,5/7-octafluoro- -pentyl) sodium sulfosuccinate (di-HCF4). A major concern of enzymatic reactions in CO2 is the pH of the aqueous phase, which is approximately 3 when there is contact with CO2 at elevated pressures. Holmes et al. examined the ability of various buffers to maintain the pH of the aqueous solution in contact with CO2. The biological buffer 2-(A-morpholino)ethanesulfonic acid sodium salt (MES) was the most effective, able to maintain a pH of 5, depending on the pressure, temperature, and buffer concentration. The activity of the enzymes in the water-in-C02 microemulsions was comparable to that in a water-in-heptane microemulsion stabilized by the surfactant AOT, which contains the same head group as di-HCF4. 

Moniruzzaman M, Kamiya N, Nakashima K et al (2008) Water in ionic liquid microemulsions as a new medium for enzymatic reactions. Green Chem 10 497-500... 

Khmelnitsky, Y. L., HUhorst, R., and Veeger, C., Detergentless microemulsions as a media for enzymatic reactions cholesterol oxidation catalyzed by cholesterol oxidase, Eur. J. Biochem., 176, 265-271, 1988. 

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. 

Nucleophilic substitution reactions are another class of reactions that are easily conducted in these microemulsions. Finally, enzymatic reactions using supercritical CO2 microemulsions may be of high interest because of the general biocompatibility of the enzymes with the two primary solvents, water and CO2. 

The choice of surfactant is of importance for the rate of many enzymatic reactions in microemulsions. For instance, it has been found that whereas lipase-catalyzed hydrolysis of triglycerides is rapid in microemulsions based on AOT, it is extremely sluggish when... 

SCFs are an environmentally friendly alternative to organic solvents as media for biocatalysis. A key feature of biocatalysis in SCFs is the tunability of the medium [75]. Enzymatic activity in SCFs has been proven and well documented [76]. Limiting factors, which may affect enzymatic activity in supercritical solvent systems, have been identified and are well characterized. A major limitation to the broader use of SCFs is their inability to dissolve a wide range of hydrophilic and ionic compounds, which greatly impedes their ability to carry out biolransformation with polar substrates. The interest in water-in-SCF microemulsion as reaction media stems from the fact that in such systems high concentrations of both polar and apolar molecules can be dissolved within the dispersed aqueous and continuous SCF phases, respectively. 

By employing DLS and electrochemical methods (using potassium hexacyanofer-rate K Fe (CN) and ferrocene used as electroactive probes), regions in [bmim] [PFg]-in-water microemulsion were located. On an average, the microemulsion droplet size was approximately 3 nm. The synthesized microemulsion proficiently solubilized a lipophilic dye, 4-(p-nitrophenylazo)-pyrocatechol (NAP) and also vitamin Kj with prospects in biological extractions, and also as solvent for enzymatic reactions. 

Water/TX-100/[C mim][PF ] microemulsions were used as reaction media in enzymatic reactions. The catalytic activities of alcohol dehydrogenase in this ternary system were determined, and it was found to be greatly improved as compared with those in pure [Bmim][PF ] [62].The same system was used in order to analyze the effect on the catalytic activity of lignin peroxidase and laccase [63]. The catalytic behavior and stability of lipases from Candida rugosa, Chromobacterium viscosum, and Thermomyces lanuginosa in these microemulsions were investigated and compared to other microheterogeneous media used so far for enzyme-catalyzed reactions [64]. 

The addition of alcohol, as cosurfactant, to the [Cgmim][TfjN]/AOT/water system leads to stable w/IL microemulsions. DLS and protein solubilization experiments confirm the existence of an aqueous nanoenvironment in the IL phase of [C mirnTf N]/ AOT/l-hexanol/water microemulsions [67]. The kinetics of the enzymatic reactions were performed in this quaternary system. Specifically, lipase-catalyzed hydrolysis of p-nitrophenyl butyrate (p-NPB) was used as a model reaction [68]. In a similar way, the hpase-catalyzed hydrolysis of p-NPB was investigated to evaluate the catalytic efficiency in water/AOT/Triton X-100/[C mim][PFJ [69]. A large single-phase microemulsion region can be obtained from the combination of two surfactants in IL. 

Lipase-Catalyzed Hydrolysis Goto and coworkers [63] proposed an approach for carrying out enzymatic reactions in water-in-IL microemulsion. 

In another stndy, Monirnzzaman et al. [215] explored the use of w/IL microemulsions comprised anionic surfactant, AOT/hydrophobic IL [C mim] [TfjN] (l-octyl-3-methyl imidazolium bis(trifluoromethylsnlfonyl)amide)/water/l-hexanol as the reaction medinm for the enzymatic oxidation of pyrogallol catalyzed by HRP. The results demonstrated that the rate of HRP-catalyzed reactions in IL microemnlsions increases significantly compared with that obtained in conventional oil microemnlsions. It was concluded that a w/IL microemulsion may be a very promising system for performing enzymatic reactions with HRP in ILs media. According to them, the findings will be of valne for the development of ILs... 

Since the pioneering work of Martinek et al. [1] more than 30 years ago, numerous studies have been published, where enzymatic reactions were investigated in surfactant containing media. Most of them deal with reverse microemulsions, in which the enzyme is entrapped in water nanodroplets that are dispersed in an external oil pseudophase. Surfactants and cosurfactants stabilize the whole system by forming an interfacial layer between the aqueous and the hydrophobic part. 

The kinetics of enzymatic reactions in microemulsions obey, as a rule, the classic Michaelis-Menten equation [6,26,35], but difhculties arise in interpreting the results because of the distribution of reactants, products, and enzyme molecules among the microphases of the microemulsion [8,36-38], In addition, there are some enzymes in reverse micelles that exhibit enhanced activity as compared to that expressed in water this has given rise to the concept of superactivity [6,26,39], The superactivity has been explained in terms of the state of water in reverse micelles, the increased rigidity of the enzymes caused by the surfactant layer, and the enhanced substrate concentration at the enzyme microenvironment [36,40],... 

Reverse micelles have been associated with the idea of microreactors for enzymatic reactions, when snbstrates and/or products are lipophilic and low water content is desired. Microemnlsions provide an enormous interfacial area through which the conversion of hydrophobic snbstrates can be catalyzed. Increasing the interfacial area is of great technological interest because it results in the increase in the number of substrate molecules available to react. Enzymes in w/o microemulsions offer considerable advantages as a reaction medium is used for biocatalytic transformations ... 

Miyake, Y, Owari, T., Matsuura, K., Teramoto, 1993. M. Enzymatic reaction in water-in-oil microemulsions. Part 1. Rate of hydrolysis of a hydrophilic substrate Acetylsalicylic acid. J. Chem. Soc. Faraday Trans. 89, 1993-1999. 

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