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Purification and stabilization of a glutamate dehygrogenase from Thermus thermophilus via oriented multisubunit plus multipoint covalent immobilization

Authors :
Bolívar Bolívar, Juan Manuel
Rocha-Martin, Javier
Mateo, Cesar
Cava, Felipe
Berenguer, Jose
Vega, Daniel
Fernandez-Lafuente, Roberto
Guisan, Jose M.
Bolívar Bolívar, Juan Manuel
Rocha-Martin, Javier
Mateo, Cesar
Cava, Felipe
Berenguer, Jose
Vega, Daniel
Fernandez-Lafuente, Roberto
Guisan, Jose M.
Publication Year :
2024

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).<br />Comunidad de Madrid<br />Ministerio de Ciencia<br />Fundación Ramón Areces<br />Depto. de Ingeniería Química y de Materiales<br />Fac. de Ciencias Químicas<br />TRUE<br />pub

Details

Database :
OAIster
Notes :
application/pdf, 1381-1177, English
Publication Type :
Electronic Resource
Accession number :
edsoai.on1429624874
Document Type :
Electronic Resource