Immobilization-stabilization of a new recombinant glutamate dehydrogenase from Thermus thermophilus

Bolivar, Juan MDepartamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica, CSIC, Campus UAM, Madrid, Spain (författare)

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.

Cava, FelipeCBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain(Swepub:umu)feca0003 (författare)
Mateo, CesarDepartamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica, CSIC, Campus UAM, Madrid, Spain (författare)
Rocha-Martín, JavierDepartamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica, CSIC, Campus UAM, Madrid, Spain (författare)
Guisán, Jose MDepartamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica, CSIC, Campus UAM, Madrid, Spain (författare)
Berenguer, JoséCBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain (författare)
Fernandez-Lafuente, RobertoDepartamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica, CSIC, Campus UAM, Madrid, Spain (författare)
Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica, CSIC, Campus UAM, Madrid, SpainCBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain (creator_code:org_t)

Ingår i:Applied Microbiology and Biotechnology: Springer Science and Business Media LLC80:1, s. 49-580175-75981432-0614

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