Journal of Biotechnology 125 (2006) 85–94 Improvement of the stability of alcohol dehydrogenase by covalent immobilization on glyoxyl-agarose Juan M. Bolivar, Lorena Wilson 1 , Susana Alicia Ferrarotti 2 , Jos´ e M. Guis´ an, Roberto Fern´ andez-Lafuente ∗ , Cesar Mateo ∗ Departamento de Biocatalisis, Instituto de Catalisis-CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain Received 21 October 2005; received in revised form 10 January 2006; accepted 24 January 2006 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 20 h 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. © 2006 Elsevier B.V. All rights reserved. Keywords: Enzyme redox reactions; Cofactor regeneration; Inactivation by gas bubbles; Enzyme rigidification; Stabilization of multimeric enzymes ∗ Corresponding authors. Tel.: +34 91 585 48 09; fax: +34 91 585 47 60. E-mail addresses: rfl@icp.csic.es (R. Fern´ andez-Lafuente), ce.mateo@icp.csic.es (C. Mateo). 1 Present address: School of Biochemical Engineering, Universidad Cat´ olica de Valpara´ ıso, Chile. 2 Present address: Universidad Nacional de Luj´ an, Departamento de Ciencias B´ asicas, Luj´ an, Buenos Aires, Argentina. 0168-1656/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2006.01.028