Enzymatic reactions in reversed micelles

Enzymatic Reactions in Reversed Micelles at Low Solubilized Water Concentrations... 

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,... 

Hakoda, M., Enomoto, A., Hoshino, T., Shiragami, N. 1996. Electroultrafiltration bioreactor for enzymatic reaction in reversed micelles. J. Ferment. Bioeng. 82, 361-365. 

Self-replicating micelles aqueous micelles and enzymatically driven reactions in reverse micelles. J. Am. Chem. Soc., 113, 8204-9. 

Bachmann, P. A., Walde, P, Luisi, P. L. and Long, J. (1991). Self-replicating micelles aqueous micelles and enzymatically driven reactions in reverse micelles. Journal of the American Chemical Society, 113, 8204-9. 

Discussion. Temelli asked how the addition of ethanol and the creation of a mixed phase would affect a reaction. King replied that patents exist on multiphase systems containing ethanol or glycerol with SC-COj. Debenedetti inquired about opportunities for enzymatic reactions involving reverse micelles. King replied he had seen some theses related to this topic. Debenedetti and Johnston discussed why one would want to carry out reactions in carbon dioxide that could just as well be carried out in water the answer is that follow-up separation may be easier in the first case, and water-insoluble substrates could be made to react at the interface. Schneider stated that the word solvation should not be used... 

Such a system was used successfully to solubilize enzymes within the inner core of reversed micelles without significant loss of activity (27). Besides its use to study enzymatic reactions in organic solvents with poorly water-soluble substrates (27), reversed-micellar systems have also been developed for the isolation and recovery of solubilized proteins... 

In this section we will discuss briefly some theoretical aspects related to the design and realization of compartmentalized reactions, mainly derived from our experience with biochemical systans. As a general premise, we remade that although an ever inoeasing number of published work on this subject (mono- or multi-enzymatic reactions inside liposomes, fatty add vesicles, polymersomes, as well as w/o or w/o/w compartments ) a full understanding of the physico-chemical details of compartmentalized reactions has not been reached. A related field, which has a longer tradition, i.e. that one of enzymes in reverse micelles has revealed interesting compartmentation-dependent mechanisms. ... 

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. 

Reaction acceleration in reverse micelles may reach factors of 10 (or more), as against about 10 in normal micelles, and is caused, as in aqueous micellar catalysis, by local concentration effects and orientations and interactions in the microenvironment of the micellar cavity. Because reverse micelles are normally used for enzymatic reactions, we defer a treatment of this combinatorial strategy until later. 

Free or immobilized enzymes have been exploited already in a number of systems. Here, biocatalysis may take place in reversed micelles or in an aqueous phase in contact with an organic solvent. In a powdered state some enzymes are able to function in pure organic solvents. Furthermore, modified enzymes such as polymer bound enzymes or surfactant-coated enzymes have been developed so that they can solubilize in organic solvents to overcome diffusion limitation. The advantages of enzymatic reactions using organic solvents can be briefly summarized as follows ... 

Fig. 6. Schematic representation of an enzymatic reaction in a reverse micellar system under steady state conditions and excess of empty micelles. E,S, and P represent the enzyme the substrate, and the product, respectively. The circles represent the water pools of the reverse micelles (cross-section) (from Ref. [88])... 

One may be brought to ask the question, which of the two K , values represents more closely the physics of the system. In fact, this is the question which we have asked ourselves in some of our earlier papers, generally opting for since the enzymatic reaction takes place in the water microphase. The question is important, because K, is a measure of the affinity of the enzyme for the substrate, and accepting the K value, would imply that the enzyme in reverse micelles has a rather low affinity. 

The enzymatic reaction in PFPE/SCCO2 reverse micelles was found to follow the classical Michaelis-Menten kinetics, whereas the activity was a strong function of the molar ratio of H2O-PEPE. The optimum ChOx activity occurred when the molar ratio of H2O-PFPE exceeded 12. The values of the apparent first-order rate constant (A cat,app) were comparable with that obtained in AOT/ isooctane system. In contrast, the authors observed that the best case for the apparent Michaelis constant (A mapp) was -twofold better than that reported for reverse micelles in liquid isooctane. There was no significant change in A cat,app or /fm,app when the CO2 pressure was changed between 100 and 260 bar. 

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],... 

Microemulsions or reverse micelles are composed of enzyme-containing, surfactant-stabiHzed aqueous microdroplets in a continuous organic phase. Such systems may be considered as a kind of immobilization in enzymatic synthesis reactions. 

The kinetics of the piperidino-defluorination reaction of l-fluoro-2,4-dinitrobenzene have been studied in non-aqueous reverse micelles consisting of ethylene glycol-AOT-/ -heptane or DMF-AOT-n-heptane. The reaction, which is not base catalysed, is accelerated when DMF, a non-hydrogen bond donor solvent, is used in the micelle core.22 Catalysis by human glutathione 5-transferase M la-la of the reaction of glutathione with 1-chloro- and l-fluoro-2,4-dinitrobenzenes has been investigated. Much stronger enzymatic catalysis was observed in the case of the dechlorination reaction than for the defluorination and a transition-state model was proposed 23... 

The rapid development of biotechnology during the 1980s provided new opportunities for the application of reaction engineering principles. In biochemical systems, reactions are catalyzed by enzymes. These biocatalysts may be dispersed in an aqueous phase or in a reverse micelle, supported on a polymeric carrier, or contained within whole cells. The reactors used are most often stirred tanks, bubble columns, or hollow fibers. If the kinetics for the enzymatic process is known, then the effects of reaction conditions and mass transfer phenomena can be analyzed quite successfully using classical reactor models. Where living cells are present, the growth of the cell mass as well as the kinetics of the desired reaction must be modeled [16, 17]. 

Recently, the investigations of nitrobenzisoxazoles mainly 6-nitrobenzisoxazole-3-carboxilate ions have received considerable interest due to their participation in reverse micellar systems [679-682], Reverse micelles are of considerable interest as reaction media because they are powerful models for biological compartmental-ization, enzymatic catalysis, and separation of biomolecules. Solutions of ionic surfactants in apolar media may contain reverse micelles, but they may also contain ion pairs or small clusters with water of hydration [679], Molecular design of nonlinear optical organic materials based on 6-nitrobenzoxazole chromophores has been developed [451],... 

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