Bolívar Bolívar, Juan ManuelRocha-Martin, JavierMateo, CesarCava, FelipeBerenguer, JoseVega, DanielFernandez-Lafuente, RobertoGuisan, Jose M.2024-01-152024-01-152009Bolivar, J. M., Rocha-Martin, J., Mateo, C., Cava, F., Berenguer, J., Vega, D., Fernandez-Lafuente, R., & Guisan, J. M. (2009). Purification and stabilization of a glutamate dehygrogenase from Thermus thermophilus via oriented multisubunit plus multipoint covalent immobilization. Journal of Molecular Catalysis B: Enzymatic, 58(1-4), 158-163. https://doi.org/10.1016/J.MOLCATB.2008.12.0101381-117710.1016/j.molcatb.2008.12.010https://hdl.handle.net/20.500.14352/93132The 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).engAttribution-NonCommercial-NoDerivatives 4.0 Internationaljournal articlehttps://www.doi.org/10.1016/j.molcatb.2008.12.010https://www.sciencedirect.com/science/article/pii/S138111770800266X?via%3Dihubrestricted access577.1Thermophilic enzymesMultimeric enzymesEnzyme stabilizationEnzyme immobilizationProtein purificationOriented immobilizationMultipoint covalent attachmentIngeniería químicaBioquímica (Química)BiotecnologíaQuímica2302 Bioquímica3302 Tecnología Bioquímica3303 Ingeniería y Tecnología Químicas