Use of physicochemical tools to determine the choice of optimal enzyme: stabilization of D-amino acid oxidase.
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.
Betancor
Lorena
L
Laboratorio de Tecnología Enzimática, Departamento de Biocatálisis, ICP-CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain.
Hidalgo
Aurelio
A
Fernández-Lorente
Gloria
G
Mateo
Cesar
C
Rodríguez
Verónica
V
Fuentes
Manuel
M
López-Gallego
Fernando
F
Fernández-Lafuente
Roberto
R
Guisan
José M
JM
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
D007527:Isoenzymes; D006861:Hydrogen Peroxide; D003605:D-Amino-Acid Oxidase; D017027:Protein Tyrosine Phosphatases