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Characterization and surface properties of semi-synthetic redox enzymes for their application in biosensor devices

Objective

Chemically modifying redox enzymes of analytical interest by covalent attachment of coenzymes (FAD, NAD, NADP) and redox mediators to improve their properties for application in amperometric and/or combined electro-optic biosensors.
The initial objectives of the project include developing a method to purify flavin adenine dinucleotides (FAD) [N{6}-(2-aminoethyl)-FAD] by high performance liquid chromatography (HPLC), and the isolation, characterization and amino acid sequencing of reduced nicotinamide adenine dinucleotide (NADH) oxidase from Thermus Aquatiaus. Work to date has produced to the following results: from a reaction mixture partially purified N{6}-(2-aminoethyl)-FAD in mg amounts could be obtained;
a new purification procedure giving a higher yield for NADH oxidase was been devised and preliminary amino acid sequences of the first 8 amino acids have been obtained;
confirmation that the physicochemical properties are identical to previously purified NADH oxidase; effects of pH, temperature, substrate specificity and kinetic parameters were all identical.
The aim of the project is to contribute to bridging the existing gap with respect to the lack of knowledge of the particular enzymology concerned with the operational adaption and functioning of redox enzymes in biosensor devices. The results may lead to better functioning enzymes under the harsh conditions that arise from the direct proximity of interfacially acting enzymes to device surfaces, both for amperometric and electro-optic biosensors. The evaluation of various semi-synthetic redox enzymes derived by chemically modifying inherently stable (eg from thermophile microorganisms) flavoprotein oxidases and NAD(P) dependent dehydrogenases is premised. For covalent FMN- and FAD-oxidase conjugates, synthesized from apo-oxidases that originally bound flavin coenzymes noncovalently, we expect to achieve an improved compatibility with metalized (silver, gold or platinum), carbon and (semi) conducting surfaces (silicon dioxide, titanium dioxide, tin dioxide) by avoiding gradual loss of free FMN and FAD under s ress conditions. Covalent NAD(P)-dehydrogenase-NAD(P)H oxidator conjugates are anticipated to function as oxidases by intramolecular NAD(P) recycling. Attaching chemical electron relays to unmodified oxidases and all semi-synthetic redox enzymes may result in facilitated direct electron transport to amperometric devices and oxygen ion independency for oxidases.

Finding the most appropriate fixation mode for achieving optimal operational conditions for surface fixed semi-synthetic redox enzymes in amperometric and/or combined electro-optic biosensors will be supported by computer simulation of enzymatic redox reactions on surfaces.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

GBF - NATIONAL CENTRE FOR BIOTECHNOLOGY
Address
Mascheroder Weg 1
38124 Braunschweig
Germany

Participants (7)

AGRICULTURAL RESEARCH DEPARTMENT
Netherlands
Address
59,Bornsesteeg 59
6700 AA Wageningen
Cranfield Biotechnology Ltd
United Kingdom
Address

MK43 0AL Cranfield
EURO-DIAGNOSTICS BV - HEALTH BIOTECHNOLOGY
Netherlands
Address
Beyerinekweg
6802 EA Arnhem
SWISS FEDERAL INSTITUTE OF TECHNOLOGY ZUERICH
Switzerland
Address
Eth Hoenggerberg
8093 Zurich
UNIVERSITY COLLEGE DUBLIN
Ireland
Address
Belfield
4 Dublin
Universita degli Studi di Cagliari
Italy
Address
Via Università 40
09100 Cagliari
Wisstrans Umwelt GmbH
Germany
Address
Am Leinekanal 4
37073 Göttingen