Colja Laane

Enzymatic conversion of apolar compounds in organic media using an NADH regenerating system and dihydrogen as reductant

A combined enzyme system, consisting of hydrogenase, lipoamide dehydrogenase and 20β‐hydroxysteroid dehydrogenase has been enclosed in reversed micelles. This system catalyzes the stereo‐ and site‐specific enzymatic reduction of apolar, poorly water‐soluble ketosteroids to their corresponding 20β‐hydroxyform using an in situ NADH‐regenerating enzyme system and H2 as ultimate reductant.

Purification of intracellular enzymes from whole bacterial cells using reversed micelles

A new, rapid pre-chromatography isolation procedure for intracellular enzymes from whole bacterial cells has been developed using reversed micelles. The method involves two relatively simple steps. In the first step, bacterial cells are disintegrated by the surfactants in the reversed micellar medium, and in the second step the liberated enzymes are extracted from the reversed micellar phase into an aqueous phase. The feasibility of using reversed micelles as a bioseparation tool has been demonstrated by following the activities and recoveries of three different dehydrogenases from Azotobacter vinelandii.

Design of reversed micellar media for the enzymatic synthesis of apolar compounds

Publisher Summary This chapter focuses on the reversed micellar enzymology of 20 β-hydroxysteroid dehydrogenase that catalyzes the reduction of various ketosteroids to the corresponding 20 β-hydroxysteroids at the expense of NADH. Reversed micelles are tiny oases of water stabilized in an organic solvent with the aid of surfactants. It shows that enzymes entrapped in these pools are capable of synthesizing apolar compounds—such as steroids, oxidized polyunsaturated fatty acids, and peptides. A reversed micellar medium consists of at least three components: an aqueous solution, a water-immiscible organic solvent, and a surfactant. The mechanism leading to the formation of reversed micelles is the tendency to extend the interfacial area until the concentrations of surfactants are sufficiently low to achieve a nonnegative interfacial tension. The results demonstrate that the reaction rate declines when the conversion is almost complete. Furthermore, at higher concentrations the reaction rate increases and the reaction proceeds steadily for >10 hr, indicating that the multienzyme system is stable for a considerable length of time.

Use of a bioelectrochemical cell for the synthesis of (bio)chemicals

Abstract A bioelectrochemical cell containing either d -glucose oxidase (β- d -glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) or xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2) plus dichlorophenol-indophenol as electron acceptor in one half-cell, and chloroperoxidase (chloride:hydrogen-peroxide oxidoreductase, EC 1.11.1.10) in the other half-cell is described. Due to a combination of chemical, biochemical and electrochemical reactions, electricity and specific (bio)chemicals can be produced in the cell simultaneously and in both compartments. Furthermore, the oxidases in a bioelectrochemical cell are not inactivated by H 2 O 2 and as a result the operational lifetimes of the oxidases were increased about five-fold.

Bioreduction of carboxylic acids by Pyrococcus furiosus in batch cultures

The reduction of aromatic and aliphatic (di)carboxylic acids to their corresponding aldehydes and alcohols by the hyperthermophilic organism Pyrococcus furiosus was investigated. The reduction was performed with P. furiosus cells growing in the presence of 1 mM acid with starch as a carbon and energy source at 90°C. The aromatic acids t-cinnamic and 3-phenylpropionic acid were reduced to their corresponding alcohols with the highest yields in the described batch cultures: 67 and 69%, respectively. The aliphatic acid reduced with the highest yield was hexanoic acid (yield: 38%). No aldehydes were detected during the reduction of acids, indicating that the reduction of aldehydes to alcohols is faster than the reduction of acids to aldehydes. Some aldehydes were both reduced to the corresponding alcohol and oxidized to the corresponding acid. Besides reduction of the unsaturated t-cinnamaldehyde to t-cinnamyl alcohol (63%), the double bond of t-cinnamaldehyde was also reduced by P. furiosus.

Application of hydrogenase in biotechnological conversions.

Evidence will be presented in this review article that the application of hydrogenase has large biotechnological possibilities. Our investigations show: Fast reaction of hydrogenase at an electrode surface to reduce H+; Photochemical production of H2 by hydrogenase by photosensitized Ru-complexes dissolved in reversed micellar membranes and vectorial H+ transport through the membrane to the water phase; The production of fine chemicals in reversed micelles by a system containing specific enzymes, hydrogenase and H2. The rules to obtain maximal conversion rates with this system will be presented.

Photosensitized electron transfer reactions in organized interfacial systems

Abstract The separation of photoproducts formed in photosensitized electron transfer reactions is essential for efficient energy conversion and storage. The organization of the components involved in the photoinduced process in interfacial systems leads to efficient compartmentalization of the products. Several interfacial systems, e.g. lipid bilayer membranes (vesicles), water-in-oil microemulsions and a solid SiO 2 colloidal interface, were designed to accomplish this goal. An electron transfer across a lipid bilayer membrane leading to the separation of the photoproducts at opposite sides of the membrane is facilitated by establishing a transmembrane potential and organizing the cotransport of cations with specific carriers. Colloidal SiO 2 particles provide a charged interface that interacts with charged photoproducts. By designing a system that results in oppositely charged photoproducts, a retardation of recombination by the charged interface can be produced. The photosensitized reduction of a neutral acceptor by positively charged sensitizers is described. The reactions are substantially enhanced in the SiO 2 colloid compared with in the homogeneous phase. The effect of the SiO 2 interface is attributed to a high surface potential that results in the separation of the intermediate photoproducts. The quantum yields of the photosensitized reactions are correlated with the interfacial surface potential and the electrical effects of other charged interfaces such as micelles are compared with those of SiO 2 . The possible utilization of the energy stored in the stabilized photoproducts in further chemical reactions is discussed. Special attention is given to the photodecomposition of water.

REGULATION AND PREDICTION OF ENZYME ACTIVITY IN REVERSED MICELLES

The rate of cholesterol oxidation has been studied in cholesterol oxidase containing reversed micellar media consisting of the surfactant cetyltrimethylammonium bromide (CTAB), the surfactant octanol, a buffered aqueous solution, and a variety of organic solvents. By varying the composition of the medium systematically it could be deduced that the rate of cholesterol oxidation obeys the same rules as described earlier for the conversion of apolar steroids by 20β-hydroxysteroid dehydrogenase in CTAB-hexanol-organic solvent reversed micelles (Hilhorst et al. 1984). The general applicability of these rules in optimizing biocatalysis in reversed micelles is discussed.

Bioelectrosynthesis of halogenated compounds using chloroperoxidase.

Abstract A bioelectrolytic system containing chloroperoxidase is described that chlorinates barbituric acid specifically and continuously into 5-chlorobarbituric acid. The system consists of an electrolytic cell, a hollow-fibre membrane reactor and an anion exchanger. The H2O2 produced in the electrolytic cell is consumed by chloroperoxidase in the membrane reactor and the product thus formed is subsequently scavenged by the anion exchanger. Besides producing H2O2 the electrolytic cell reverses the further enzymatic halogenation of 5-chlorobarbituric acid by dechlorination in an electro-reductive fashion.

Generation, Transport and Transfer of low-potential reducing equivalents in nitrogenase catalysis

Studies with nitrogenase systems, isolated from a variety of microorganisms, revealed that biological N2 fixation with the catalyst nitrogenase component I or. MoFe-protein needs another protein, nitrogenase component II or Fe-protein, anaerobic conditions, energy in the form of ATP and a strong reductant. Despite the specific demand for a strong reducing agent and an anaerobic environment for the site of the nitrogenase system, N2 fixation is observed in a variety of microorganisms, including obligate aerobes. There is little information about the absolute value of the redox potential necessary for nitrogenase activity. The midpoint potential of the Fe-protein of C.pasteurianum in the presence of MgATP or MgADP is approximately -400 mV. The MoFe-protein is completely reduced at -500 mV [1]. It has been demonstrated that the nitrogenase complexes isolated from A.vinelandii and C.vinosum show full activity at redox potentials below -500 mV and no significant activity at redox potentials higher than -430 mV [2–4]. It is clear that besides ATP the cell has to invest significant amounts of energy to generate the reducing power for N2 fixation.