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Immobilization-stabilization of a new recombinant glutamate dehydrogenase from Thermus thermophilus
Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica, CSIC, Campus UAM, Madrid, Spain.
CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica, CSIC, Campus UAM, Madrid, Spain.
Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica, CSIC, Campus UAM, Madrid, Spain.
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2008 (English)In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 80, no 1, 49-58 p.Article in journal (Refereed) Published
Abstract [en]

The genome of Thermus thermophilus contains two genes encoding putative glutamate dehydrogenases. One of these genes (TTC1211) was cloned and overexpressed in Escherichia coli. The purified enzyme was a trimer that catalyzed the oxidation of glutamate to alpha-ketoglutarate and ammonia with either NAD+ or NADP+ as cofactors. The enzyme was also able to catalyze the inverse reductive reaction. The thermostability of the enzyme at neutral pH was very high even at 70 degrees C, but at acidic pH values, the dissociation of enzyme subunits produced the rapid enzyme inactivation even at 25 degrees C. The immobilization of the enzyme on glyoxyl agarose permitted to greatly increase the enzyme stability under all conditions studied. It was found that the multimeric structure of the enzyme was stabilized by the immobilization (enzyme subunits could be not desorbed from the support by boiling it in the presence of sodium dodecyl sulfate). This makes the enzyme very stable at pH 4 (e.g., the enzyme activity did not decrease after 12 h at 45 degrees C) and even improved the enzyme stability at neutral pH values. This immobilized enzyme can be of great interest as a biosensor or as a biocatalyst to regenerate both reduced and oxidized cofactors.

Place, publisher, year, edition, pages
2008. Vol. 80, no 1, 49-58 p.
Keyword [en]
edox enzymes, thermophilic enzymes hyperstabilization, multipoint immobilization, multimeric enzyme stabilization, cofactor regeneration, glutamate biosensor
National Category
Microbiology
Identifiers
URN: urn:nbn:se:umu:diva-81866DOI: 10.1007/s00253-008-1521-3ISI: 000257485900006PubMedID: 18545996OAI: oai:DiVA.org:umu-81866DiVA: diva2:658614
Available from: 2013-10-22 Created: 2013-10-22 Last updated: 2017-12-06Bibliographically approved

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Cava, Felipe

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