Photoinduced charge separation and enzyme reactions in reversed micelles

R.M.D. Verhaert

Research output: Thesisinternal PhD, WU


In this thesis the performance and coupling of two types of reaction, photoinduced charge separation and enzymatic conversion were studied in reversed micelles. Reversed micelles are 1 to 10 nm sized water droplets dispersed in an organic solution. The dispersant is a detergent (cationogenic, nonionogenic or anionogenic). In some solutions an additional compound (cosurfactant) is necessary to stabilize the droplets. In the studies presented here aliphatic alcohols were used for this purpose. The composition of reversed micelles can be optimized to yield both an efficient photoinduced charge separation and a high enzyme turnover number.<p>In Chapter 2 a photosystem was coupled to the reduction of α-βunsaturated carboxylates by enoate reductase (EC The photosystem was composed of a porphyrin as photosensitizer, tributylamine as electrondonor and methylviologen as electronacceptor. The photoinduced enzymatic system was active for over 100 hours. A 100% conversion could be accomplished. Systematic studies to the relation between the efficiency of the photosystem and the composition of the reversed micellar solution showed that an increase in cosurfactant concentration and cosurfactant polarity induced a higher yield of charge separation. The location of a porphyrin as a function of the composition of the reversed micellar solution was studied. The polarity of the environment of a porphyrin could very well be probed by <sup><font size="-2">19</font></SUP>F-NMR. The results showed that the porphyrin was located in the interfacial region of the reversed micelle. Modification of the porphyrin with a hexadecyl tail anchored the porphyrin. Although distinct differences between the environment and mobility of the tailed and untailed porphyrin could be detected, the efficiencies of photosystems using these porphyrins were similar. Thus neither the location, nor the mobility of the porphyrin but the concentration of alcohol in the reversed micellar solution is the main factor determining the yield of the photoinduced charge separation. An increased alcohol concentration in the medium is known to increase the flexibility of the micelles and to affect the partitioning of the electron donor. Therefore, the availability of the donor and the flexibility of the reversed micellar system have to be optimized to obtain a highly efficient photosystem. The spectroscopic properties of those porphyrins are affected by the interfacial location of the molecule. An increasing resolution of the fluorescence emission spectrum and a transient fluorescence rise in detergent solutions indicate that after excitation an intramolecular rearrangement occurs.<p>The enzyme performance in photoinduced enzymatic reactions is determined by its stability and its activity. Increasing the chain length of the cosurfactant increased the stability of the enzyme in its oxidized state, but the stability of the reduced enzyme was lowered. The activity of enoate reductase increased with increasing cosurfactant polarity, but the relation with the concentration of cosurfactant in the reversed micellar solution was unexpected (Chapter 2). Therefore the kinetics of enoate reductase were studied in great detail using a water soluble substrate. A model was derived taking into account that both substrate supply and substrate concentration in the microheterogeneous reversed micellar solution deviate from those in aqueous solutions. Not only the, unaltered, intrinsic kinetic parameters of the enzyme, but also the reversed micellar concentration, the intramicellar substrate concentration and the rate and efficiency of the exchange of reversed micellar contents were shown to determine the enzyme activity. This model could be used to predict the experimental enoate reductase results in reversed micelles prepared with an anionogenic, a cationogenic and a nonionogenic detergent (Chapter 4.2).<p>The enzyme kinetic study was extended to the case of 20β-hydroxysteroid dehydrogenase (EC This enzyme catalyzes the reduction of a variety of 20β-ketosteroids. It could be demonstrated that partitioning of the substrate between the aqueous phase and the organic phase was the main factor affecting the Michaelis constant observed in reversed micelles. These results could also be interpreted with the model presented in Chapter 4.1.<p>The implications of both the kinetic model and the study to photoinduced charge separation are discussed in Chapter 5. It was shown that the electron transfer in forward direction emerges from the triplet state of the porphyrin and strongly depends on the flexibility of the system. For further optimization the redox potentials of the components, especially that of the porphyrin, need to be considered, too. With respect to the enzymatic conversion the model presented in Chapter 4 was used to explain the dependency of enzyme activity on the cosurfactant concentration. It is stressed that because of the general validity of the principles used to derive it, the model describing the enzymatic reactions in reversed micelles can be equally applied to other microheterogeneous systems.<p>Finally some results of this thesis are listed as guidelines to optimize photoinduced enzymatic reactions in reversed micelles.<p><TT></TT>
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Schaafsma, T.J., Promotor
Award date15 Sep 1989
Place of PublicationS.l.
Publication statusPublished - 1989


  • fermentation
  • food biotechnology
  • enzymes
  • immobilization
  • biotechnology
  • chemical industry
  • biochemistry

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