title: Use of physicochemical tools to determine the choice of optimal enzyme: stabilization of D-amino acid oxidase.
abstract: An evaluation of the stability of several forms (including soluble and two immobilized preparations) of d-amino acid oxidases from Trigonopsis variabilis (TvDAAO) and Rhodotorula gracilis (RgDAAO) is presented here. Initially, both soluble enzymes become inactivated via subunit dissociation, and the most thermostable enzyme seemed to be TvDAAO, which was 3-4 times more stable than RgDAAO at a protein concentration of 30 microg/mL. Immobilization on poorly activated supports was unable to stabilize the enzyme, while highly activated supports improved the enzyme stability. Better results were obtained when using highly activated glyoxyl agarose supports than when glutaraldehyde was used. Thus, multisubunit immobilization on highly activated glyoxyl agarose dramatically improved the stability of RgDAAO (by ca. 15,000-fold) while only marginally improving the stability of TvDAAO (by 15-20-fold), at a protein concentration of 6.7 microg/mL. Therefore, the optimal immobilized RgDAAO was much more stable than the optimal immobilized TvDAAO at this enzyme concentration. The lower stabilization effect on TvDAAO was associated with the inactivation of this enzyme by FAD dissociation that was not prevented by immobilization. Finally, nonstabilized RgDAAO was marginally more stable in the presence of H(2)O(2) than TvDAAO, but after stabilization by multisubunit immobilization, its stability became 10 times higher than that of TvDAAO. Therefore, the most stable DAAO preparation and the optimal choice for an industrial application seems to be RgDAAO immobilized on glyoxyl agarose.
authors.affiliation: Laboratorio de Tecnología Enzimática, Departamento de Biocatálisis, ICP-CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain.
authors.forename: Lorena
authors.initials: L
authors.lastname: Betancor
authors.forename: Aurelio
authors.initials: A
authors.lastname: Hidalgo
authors.forename: Gloria
authors.initials: G
authors.lastname: Fernández-Lorente
authors.forename: Cesar
authors.initials: C
authors.lastname: Mateo
authors.forename: Verónica
authors.initials: V
authors.lastname: Rodríguez
authors.forename: Manuel
authors.initials: M
authors.lastname: Fuentes
authors.forename: Fernando
authors.initials: F
authors.lastname: López-Gallego
authors.forename: Roberto
authors.initials: R
authors.lastname: Fernández-Lafuente
authors.forename: José M
authors.initials: JM
authors.lastname: Guisan
mesh_terms: D003605:D-Amino-Acid Oxidase / Q000737:chemistry* / Q000145:classification*; D004789:Enzyme Activation; D004795:Enzyme Stability; D006861:Hydrogen Peroxide / Q000737:chemistry; D007527:Isoenzymes / Q000737:chemistry; D008970:Molecular Weight; D011489:Protein Denaturation; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry; D011786:Quality Control; D012248:Rhodotorula / Q000737:chemistry / Q000201:enzymology*; D004718:Saccharomycetales / Q000737:chemistry / Q000201:enzymology*; D013696:Temperature
chemical_list: D007527:Isoenzymes; D006861:Hydrogen Peroxide; D003605:D-Amino-Acid Oxidase; D017027:Protein Tyrosine Phosphatases