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BIOCATALYSIS BY NOVEL METAL CLUSTERS AND HYDROGENASE (STRUCTURE REACTIVITY AND IMMOBILIZATION)

Objective

PROTEINS CONTAINING METAL IONS DO CATALYSE MANY IMPORTANT BIOLOGICAL REACTIONS, NAMELY THOSE INVOLVING TRANSFER OF ELECTRONS. IT IS ANTICIPATED THAT MODIFICATIONS OF THE METAL CLUSTERS OF THESE PROTEINS WILL OPEN NEW CATALYTIC POSSIBILITIES THAT CAN BE USED IN MULTIPHASE BIOCATALYSIS AND THE DEVELOPMENT OF NOVEL BIOREACTORS. THE DESIGN OF SUCH CATALYSTS OBTAINED BY A BIOSYNTHETIC ROUTE MAY TURN OUT TO BE ADVANTAGEOUS IN COMPARISON WITH CHEMICAL SYNTHETIC ONES AND CAN THUS SUBSTANTIALLY EXPAND THE FIELD OF BIOINORGANIC CHEMISTRY.
Iron sulphur proteins are involved as main catalysts in bioconversion processes (hydrogen evolution, nitrogen and carbon monoxide fixation, photosynthesis, respiration, etc). The metal active centres of different iron sulphur proteins (simple and complex, eg rubredoxin, ferredoxins and hydrogenase) were characterised and chemically modified, in order to produce new active structures with novel, and/or enhanced catalytic properties. A new concept of assisted inorganic synthesis by a protein template was developed for the synthesis of mixed metal clusters. The reactivity and stability of the newly formed catalysts and hydrogenase (free and immobilised) were tested by measuring hydrogen evolution/production, deuterium(2)/hydrogen(+) exchange (mass spectrometry) and hydrogenation activity. Another important goal was to develop an adequate system to test hydrogenation activity in a multiphase biocatalyst system with immobilised hydrogenase or bacterial cells.

Iron sulfur proteins are involved as main catalysts in bioconversion processes (hydrogen evolution, nitrogen and carbon monoxide fixation, photosynthesis, respiration, etc). The metal active centres of different iron sulfur proteins, simple and complex, (eg, rubredoxin, ferredoxins and hydrogenase) were characterized and chemically modified, in order to produce new active structures with novel, and/or enhanced catalytic properties. A new concept of assisted inorganic synthesis by a protein template was developed for the synthesis of mixed metal clusters. The reactivity and stability of the newly formed catalysts and hydrogenase (free and immobilized) were tested by measuring hydrogen evolution and production, deuterium protium exchange (mass spectroscopy) and hydrogenation activity.

The following results were obtained (from research into the structure, reactivity and immobilization of metalloproteins:
isomorphous substitutions (cobalt and nickel) of the iron centre in rubredoxins:
chemical and spectroscopic characterization;
testing biocatalytic performance and mimicking of bacterial hydrogenase activity (model design);
assisted inorganic synthesis of novel mixed metal clusters of the type M, 3 iron-4 sulphur (M = cobalt, nickel, zinc, cadmium, calcium);
spectroscopic studies involving electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR) and Moessbauer methods;
characterization of the metal sites involved in bacterial hydrogen production and consumption;
differentiation of hydrogenase types by their spectroscopic, catalytic and inhibition parameters;
biocatalysis in organic media using hydrogenase encapsulated in micellar systems;
kinetics of both free and immobilized hydrogenases;
stability of the hydrogen evolving system in reversed micellar systems (multiphase system) using purified hydrogenase and whole cells.
THE RESEARCH WILL DEAL WITH TWO ASPECTS : IMMOBILIZATION PROCEDURES AND MULTIPHASE BIOCATALYSIS. METALLOPROTEINS WITH A MODIFIED METAL CLUSTER, AS OBTAINED BY THE GROUP OF DR MOURA IN THIS PROJECT, WILL BE IMMOBILIZED BY THE FOLLOWING METHODS : COVALENT LINKAGE TO A POROUS SILICA SUPPORT OR MICROENCAPSULATION IN REVERSED MICELLES. THE EFFECT OF THIS IMMOBILIZATION ON THE RETENTION OF THE ENZYMATIC ACTIVITY AND THE OPERATION STABILITY OF THESE ENZYMES WILL BE STUDIED. IN A MODEL SYSTEM, WITH ACETYLENE GAS AS A SUBSTRATE, HYDROGENATION REACTIONS WILL BE STUDIED IN A MULTIPHASE SYSTEM ULTIMATELY LEADING TO THE DESIGN OF A MULTIPHASE BIOREACTOR.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

INSTITUTO SUPERIOR TECNICO
Address
Av Rovisco Pais
1049-001 Lisboa
Portugal

Participants (2)

Association pour la Recherche en Bioénergie Solaire
France
Address
Centre D'études De Cadarache Sere-ders
13108 Saint-paul-lez-durance
UNIVERSIDADE NOVA DE LISBOA
Portugal
Address

Liboa