title: Improvement of the stability of alcohol dehydrogenase by covalent immobilization on glyoxyl-agarose.
abstract: Immobilization of alcohol dehydrogenase (ADH) from Horse Liver inside porous supports promotes a dramatic stabilization of the enzyme against inactivation by air bubbles in stirred tank reactors. Moreover, immobilization of ADH on glyoxyl-agarose promotes additional stabilization against any distorting agent (pH, temperature, organic solvents, etc.). Stabilization is higher when using highly activated supports, they are able to immobilize both subunits of the enzyme. The best glyoxyl derivatives are much more stable than conventional ADH derivatives (e.g., immobilized on BrCN activated agarose). For example, glyoxyl immobilized ADH preserved full activity after incubation at pH 5.0 for 20h at room temperature and conventional derivatives (as well as the soluble enzyme) preserved less than 50% of activity after incubation under the same conditions. Moreover, glyoxyl derivatives are more than 10 times more stable than BrCN derivatives when incubated in 50% acetone at pH 7.0. Multipoint covalent immobilization, in addition to multisubunit immobilization, seems to play an important stabilizing role against distorting agents. In spite of these interesting stabilization factors, immobilization hardly promotes losses of catalytic activity (keeping values near to 90%). This immobilized preparation is able to keep good activity using dextran-NAD(+). In this way, ADH glyoxyl immobilized preparation seems to be suitable to be used as cofactor-recycling enzyme-system in interesting NAD(+)-mediated oxidation processes, catalyzed by other immobilized dehydrogenases in stirred tank reactors.
authors.affiliation: Departamento de Biocatalisis, Instituto de Catalisis-CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain.
authors.forename: Juan M
authors.initials: JM
authors.lastname: Bolivar
authors.forename: Lorena
authors.initials: L
authors.lastname: Wilson
authors.forename: Susana Alicia
authors.initials: SA
authors.lastname: Ferrarotti
authors.forename: José M
authors.initials: JM
authors.lastname: Guisán
authors.forename: Roberto
authors.initials: R
authors.lastname: Fernández-Lafuente
authors.forename: Cesar
authors.initials: C
authors.lastname: Mateo
mesh_terms: D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D004795:Enzyme Stability / Q000187:drug effects; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism*; D006038:Glyoxylates / Q000737:chemistry* / Q000494:pharmacology; D006736:Horses; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology; D017434:Protein Structure, Tertiary / Q000187:drug effects; D012685:Sepharose / Q000737:chemistry* / Q000494:pharmacology; D012997:Solvents / Q000737:chemistry / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors
chemical_list: D004800:Enzymes, Immobilized; D006038:Glyoxylates; D012997:Solvents; C070399:glyoxyl agarose; D012685:Sepharose; D000426:Alcohol Dehydrogenase