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Purification and stabilization of a glutamate dehygrogenase fromThermus thermophilusvia oriented multisubunit plus multipoint covalent immobilization

Journal of Molecular Catalysis B Enzymatic
Publication Date
DOI: 10.1016/j.molcatb.2008.12.010
  • Thermophilic Enzymes
  • Multimeric Enzymes
  • Enzyme Stabilization
  • Enzyme Immobilization
  • Protein Purification
  • Oriented Immobilization
  • Multipoint Covalent Attachment
  • Biology


Abstract The immobilization of a glutamate dehydrogenase from Thermus thermophilus (GDH) on glyoxyl agarose beads at pH 7 has permitted to perform the immobilization, purification and stabilization of this interesting enzyme. It was cloned in Escherichia coli and a first thermal shock of the crude preparation destroyed most mesophilic multimeric proteins. Glyoxyl agarose can only immobilize enzymes via a multipoint and simultaneous attachment. Therefore, only proteins having several terminal amino groups in a position that permits their interaction with a flat surface can be immobilized. GDH became rapidly immobilized at pH 7 and its multimeric structure became stabilized as evidenced by SDS-PAGE. This derivative was stable at acidic pH value while the non-stabilized enzyme was very unstable under these conditions due to subunit dissociation. After immobilization, a further incubation at pH 10 improved enzyme stability under any inactivating conditions by increasing the enzyme–support bonds. In fact, GDH immobilized at pH 7 and incubated at pH 10 preserved more activity than GDH directly immobilized at pH 10 (50% versus 15% after 24 h of incubation) and was also more stable (1.5- to 3-fold, depending on the conditions). This method could be extended to any other multimeric enzyme expressed in mesophilic hosts.

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