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

  • Juan M. Bolivar
  • Felipe Cava
  • Cesar Mateo
  • Javier Rocha-Martín
  • Jose M. GuisánEmail author
  • José Berenguer
  • Roberto Fernandez-Lafuente
Biotechnologically Relevant Enzymes and Proteins


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 α-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°C, but at acidic pH values, the dissociation of enzyme subunits produced the rapid enzyme inactivation even at 25°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°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.


Redox enzymes Thermophilic enzymes hyperstabilization Multipoint immobilization Multimeric enzyme stabilization Cofactor regeneration Glutamate biosensor 



This work has been supported by grants of code S0505/PPQ/0344 of the Comunidad Autónoma de Madrid (CAM) to J. Berenguer and J.M. Guisán and grants BIO2007-60245 (J. Berenguer) and CTQ2005-02420/PPQ (R. Fernández-Lafuente) from the Ministry of Education and Science (MEC). A Ramón y Cajal Contract, MEC (César Mateo). An institutional grant from Fundación Ramón Areces to CBMSO is also acknowledged. J.M. Bolívar and J.Rocha-Martín are the holders of a Ph.D. fellowship from CAM, and F. Cava holds a contract from the MEC. We thank A. Berenguer for his help during the writing of this paper.


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Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Juan M. Bolivar
    • 1
  • Felipe Cava
    • 2
  • Cesar Mateo
    • 1
  • Javier Rocha-Martín
    • 1
  • Jose M. Guisán
    • 1
    Email author
  • José Berenguer
    • 2
  • Roberto Fernandez-Lafuente
    • 1
  1. 1.Departamento de Biocatálisis. Instituto de Catálisis y Petroleoquímica, CSICCampus UAM—CantoblancoMadridSpain
  2. 2.Centro de Biología Molecular Severo Ochoa (CSIC-UAM)Campus UAM—CantoblancoMadridSpain

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