Enzymatic reactions in reversed micelles

It has been recognised that enzymes in reversed micelles have potential for application in chemical synthesis. Before these expectations will be realised many problems must be overcome. This thesis deals with some of them.<br/>In Chapter 1 the present knowledge about reversed micelles and micellar enzymology is reviewed. Encapsulation of enzymes in reversed micelles enables the enzymatic conversion of apolar compounds. In the literature only a few cases have been reported of conversions of apolar compounds, and only initial enzyme activities were measured. In Chapters 3 and 4 of this thesis, the conversion of the apolar steroids progesterone and prednisone by 20β-hydroxysteroid dehydrogenase is described. In Chapter 3 it is shown that the reaction proceeds for at least nine hours, indicating that the steroid dehydrogenase used here is fairly stable in a reversed micellar environment under operational conditions. The stability of hydrogenase in reversed micelles was even higher than in an aqueous solution (Chapter 2).<br/>In order to function for longer periods of time, redox enzymes require a continuous and sufficient supply of reducing equivalents. Several systems have been described that provide reducing equivalents in aqueous solutions, but no such a system was known for reversed micellar media. In this thesis three methods for the generation of reducing equivalents in reversed micellar media have been applied. Chapter 2 describes a photochemical system for the vectorial transport of electrons from a donor in the continuous phase to an acceptor in the water pool of the reversed micelle. The spatial arrangement of the components is an important factor in determining the efficiency of such systems. The reducing equivalents thus generated can be converted to hydrogen by hydrogenase located in the water pool. In Chapter 3 a combined enzyme system consisting of hydrogenase and lipoamide dehydrogenase converts hydrogen gas into reducing equivalents in the form of NADH that are consumed by 20β-hydroxysteroid dehydrogenase during the conversion of apolar steroids. Based on the results of these two chapters, patent applications were filed in several countries, <u>e.g.</u> in Europe. A copy of this patent application is added as an Appendix. There, a third method is mentioned, <u>i.e.</u> the electrochemical regeneration of NADH and NAD(+).<br/>In Chapter 4 it is investigated how the composition of a reversed micellar medium affects the rate of conversion of apolar steroids by 20β-hydroxysteroid dehydrogenase. Evidence was obtained that the steroid concentration in the interphase dictates the rate of conversion. This concentration depends on the hydrophobicity of the substrate as compared to the hydrophobicity of the interphase and the hydrophobicity of the continuous phase. These observations are generalized to guidelines indicating that the difference between the hydrophobicity of the substrate and the interphase must be minimal to ensure a high substrate concentration in the interphase and that the difference between the hydrophobicity of the substrate and the hydrophobicity of the continuous phase must be maximal to keep the substrate concentration in the continuous phase as low as possible. These guidelines will prove useful to predict the optimal composition of a medium for enzymatic conversion of apolar compounds.<br/>Not only the composition of a reversed micellar medium with respect to organic solvent and cosurfactant is important, but also the charge of the surfactant used. In Chapter 5, evidence is presented that the charge of the surfactant head groups influences the kinetic parameters K <sub>m</sub> and k <sub>cat</sub> of enzymes in reversed micelles, either resulting in an increase or a decrease of activity. Furthermore, an expression is derived for the initial reaction rate of the enzymatic conversion of an apolar substrate in a pseudo two-phase system where the partition equilibrium of the substrate over the two phases can be shifted due to enzyme catalysis.<br/>In another section of the Discussion the advantages and disadvantages of reversed micellar media for the enzymatic conversion of apolar compounds are compared with those of other systems that have been proposed for that purpose.<br/>In conclusion, in a reversed micellar medium:<br/>- a highly organised photochemical system can be created (Chapter 2)<br/>- enzymes can be immobilized while retaining their activity (Chapters 1- 5)<br/>- enclosure of enzymes can lead to enhanced stability (Chapter 2)<br/>- enzymes experience an essentially aqueous micro-environment (Chapter 5)<br/>- enzymatic activity can be higher than in aqueous solution (Chapter 4 and 5)<br/>- apolar compounds can be converted enzymatically (Chapters 3, 4 and Appendix)<br/>- multi-enzymatic reactions can be performed for longer periods (Chapter 3)<br/>- cofactors can be regenerated with hydrogen and electricity (Chapter 3 and Appendix)<br/>- enzyme activity can be regulated by changing the composition of the interphase and continuous phase (Chapter 4)<br/>- the optimal composition for enzymatic conversion of any given apolar compound can be predicted (Chapter 4)<br/>- the thermodynamic equilibrium can be shifted in the desired direction (Chapter 5)<br/>- the product can be isolated while the other components can be recycled (Chapter 3).<p/&gt