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CSIC QUI012- METHOD FOR THE IMMOBILIZATION OF A GLUTAMATE DEHYDROGENASE
Two Spanish Research Institutes have developed a new method for the immobilization of a glutamate dehydrogenase from Thermus thermophilus. The enzyme preparation obtained is extremely stable under a wide range of conditions (e.g., pH 4-10), using NADP(H) and NAD(H) with good activities. It is suitable as a cofactor-regenerator enzyme in oxidation and reduction processes and also as a biosensor. The institutes are interested in establishing collaborations with industrial partners.
Specification sheet:
DESCRIPTION

The present invention describes the preparation of a glutamate dehydrogenase immobilized-stabilized derivative (GDH) from Thermus thermophilus, via multipoint covalent attachment.

The gene codifying for the GDH has been first cloned and expressed in E. coli. We then obtained a GDH which recognizes NAD, NADH, NADP and NADPH, and which catalyzes both the oxidation of the glutamic acid or the reduction of the alfa-keto-glutaric acid during the regeneration of the reduced and the oxidized cofactors respectively.

The soluble enzyme is very stable from pH 6 to pH10, even at 60ºC, but in acidic pHs, which is very interesting in the recycling of reduced cofactors, it inactivates due to the dissociation of its subunits and it becomes a very unstable enzyme even at room temperature. Its immobilization in glyoxyl supports allows the stabilization of both, its multimeric structure and the structure of each monomer, and increases the range of conditions where the enzyme can be used.

This immobilized preparation is stable in the presence of solvents, at high temperatures or with drastic pHs values, including pHs below 5. Therefore, it is an excellent candidate to be used as a cofactor regeneration enzyme in oxido-redox processes or as a biosensor for detection of glutamic acid, heavy metals, ammonia, urea….

INNOVATIVE ASPECTS

The GDH from Thermus thermophilus has not been described previously (although the gene has been identified), and the enzyme has been never immobilized before neither its multimeric structure stabilized. Its high termostability under a broad range of conditions and its ability to use NAD(H) and NADP(H) and to regenerate the cofactors in both directions, makes this immobilized enzyme a good alternative to the existing ones.

COMPETITIVE ADVANTAGES

The stability will permit to explore more drastic conditions, moreover the immobilized enzyme stability will save cost in the biocatalysts.

KEYWORDS

Cofactor regeneration, detection of glutamic, stability, immobilized enzymes, NAD, NADP

PATENT

P200702294 applied for 2007-08-16

CONTACT

Beatriz Lara
email: blara@orgc.csic.es
phone: +34 91 585 49 62

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