Objectif Understanding of structure function relationships between redox enzymes, mediators and electrodes. Synthesis of stable and less toxic mediators. Procedures to allow long term operational stability for chiral product formation in a preparative scale. Nicotinamide adenine dinucleotide phosphate (NADP) dependent oxidoreductases which represent approximately two thirds of the known oxidoreductases can be used in preparative organic synthesis only if the regeneration of the pyridine nucleotides can be achieved at a reasonable cost. Suitable reductive regeneration processes exist. The practical feasability of oxidative processes are demonstrated in this project.In a wide variety of aerobic and anaerobic microorganisms enzyme activities exist which reversibly transfer electrons between artificial 1-electron mediators and pyridine nucleotides. Theses enzymes are known as viologen accepting pyridine nucleotide oxido reductases (VAPOR). They are interesting because they are catalysts for pyridine nucleotide regeneration and because of the mechanistic aspects, especially with the change between 1-electron and 2-electron transfer steps. The suitability of these VAPOR activities was tested with respect to pyridine nucleotide regeneration and preparative formation of chiral compounds and 3 noncommercially available viologens have been synthesized.Gram positive methanol utilisers appear to have a unique set of enzymes when compared to their gram-negative counterparts. Amylolatopis methanolica appears to contain 2 novel types of alcohol dehydrogenase with bound nicotinamide adenine dinucleotide (NAD) and NADP functioning as a cofactor a formaldehyde dehydrogenase, and a formate ester hydrolase. The mechanistic aspects of these enzymes and their enantioselectivity are unknown. Work on the complete characterisation of these enzymes is in progress.Enoate reductase and an alcohol dehydrogenase have been studied.A new approach is under development for a preparative electrochemical cell. Glucose oxidase is able to communicate directly with an electrode via a conducting polymer. The enzyme is adsorbed to electrodes consisting of a platinum coated membrane on which a layer of polpyrole is applied. Thus the enzyme is in direct communication with the electrode via the conducting polymer. An advantage of this system is that there is no need for a mediator in the reaction medium.Combinations of redox enzymes, mediators and coenzymes will be tested. The results will be evaluated in order to select a limited number of enzymes, mediators and coenzymes which are to be looked on with respect to homogenous and heterogeneous bioelectrocatalysis. The group at the university in Delft will screen for new enzymes containing pyrroloquinoline quinone (PQQ) as a cofactor (=quinoproteins). The group at the Technical University in Munich detected enzymes accepting viologens, which will be studied for their use in electrobiocatalysis. Both universities will try to purify and study the respective enzymes and assess their capability for the production of high added value compounds. Further enzymes will be a 3-ketoacid reductase, discovered at Munich, which forms a chiral synthon for L-carnitine synthesis, and new types of alcohol dehydrogenases. The Dutch university will proof the discovered quinoprotein alcohol dehydrogenase (enantioselective for glycidol or solketal) with novel mediators. New analytical methods will be developed for the determination of the alcohols and amines. Future plans include: synthesis of PQQ analogues and derivatives and recombinations with apoenzymes; interaction studies of the holoenzymes with the mediators developed; coupling of the modified PQQ's to an electrode and reconstitution with apoenzymes. Interactions between mediators and isolated enzymes will be studied by spectroscopic techniques (mainly NMR, EPR and Moessbauer spectroscopy) by the group at CTQB in Portugal. This group with its experience in spectroscopic techniques (mainly NMR and EPR) will collect data for the elucidation of structural properties of the active center and for structure function relationships of these new enzymes. This knowledge is a prerequisite for rational cofactor modelling and optimization procedures. The group will start with enoate reductase and hydroxycarboxylate viologen oxidoreductase, two new enzymes which were purified by the group in Munich, and alcohol- and aldehyde dehydrogenase purified by the group in Delft. The partners from TNO and ATO Will use the enzymes to test their electrochemical behaviour and long term stability, with emphasis on modelling for ATO. At first catalytically active enzyme/mediator and/or coenzyme combinations delivered by the two universities will be tested on electroactivity by cyclic voltammetry (homogeneous bioelectrochemistry). Champ scientifique natural scienceschemical sciencesorganic chemistryaldehydesnatural scienceschemical scienceselectrochemistrybioelectrochemistrynatural scienceschemical sciencesorganic chemistryalcoholsnatural sciencesbiological sciencesgeneticsnucleotidesnatural sciencesbiological sciencesbiochemistrybiomoleculesproteinsenzymes Programme(s) FP2-BRIDGE - Specific research and technological development programme (EEC) in the field of biotechnology (BRIDGE), 1990-1994 Thème(s) Data not available Appel à propositions Data not available Régime de financement CSC - Cost-sharing contracts Coordinateur TECHNICAL UNIVERSITY OF MUNICH Contribution de l’UE Aucune donnée Adresse Lichtenbergstrasse 4 85747 Garching Allemagne Voir sur la carte Coût total Aucune donnée Participants (4) Trier par ordre alphabétique Trier par contribution de l’UE Tout développer Tout réduire Business Unit of TNO Built Environment and Geosciences Pays-Bas Contribution de l’UE Aucune donnée Adresse Van Mourik Broekmanweg 6 2628 XE DELFT Voir sur la carte Liens Site web Opens in new window Coût total Aucune donnée Dienst Landbouwkundig Onderzoek Pays-Bas Contribution de l’UE Aucune donnée Adresse 6,Haagsteeg 6708 PM Wageningen Voir sur la carte Coût total Aucune donnée Technische Universiteit Delft Pays-Bas Contribution de l’UE Aucune donnée Adresse 136,Julianalaan 2628 BL Delft Voir sur la carte Coût total Aucune donnée Universidade Nova de Lisboa Portugal Contribution de l’UE Aucune donnée Adresse 6,Quinta do Marqués 2780 Oeiras Voir sur la carte Coût total Aucune donnée
