Enzyme and Microbial Technology 33 (2003) 278–285 Design of an immobilized preparation of catalase from Thermus thermophilus to be used in a wide range of conditions. Structural stabilization of a multimeric enzyme Aurelio Hidalgo a , Lorena Betancor a , Fernando Lopez-Gallego a , Renata Moreno b , José Berenguer b , Roberto Fernández-Lafuente a,∗ , José M. Guisán a,1 a Laboratorio de Tecnolog´ ıa Enzimática, Departamento de Biocatálisis, Instituto de Catálisis y Petroleoqu´ ımica-CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain b Centro de Biolog´ ıa Molecular “Severo Ochoa” CSIC-UAM, Campus UAM, Cantoblanco, 28049 Madrid, Spain Received 3 December 2002; accepted 3 May 2003 Abstract Crude extracts from Thermus thermophilus strains HB8 and HB27 with catalase activity were obtained. Activity measurements in the presence of azide suggested that Mn-catalase may be responsible for these activities, which was successfully stabilised by multipoint immobilization onto highly activated glyoxyl or glutaraldehyde agarose, while immobilization on poorly activated supports did not stabilize the enzyme. The most stable catalase preparation was obtained using extracts from T. thermophilus HB27 immobilized on glyoxyl agarose (compared to glutaraldehyde supports or glyoxyl derivatives of catalase from HB8 strain). However, the stability of the immobilized enzyme derivatives depended on enzyme concentration, suggesting that the dissociation of subunits could be playing a key role in the inactivation of the enzyme preparation. SDS–PAGE of this T. thermophilus HB27 catalase derivative confirmed that some protein subunits were not covalently attached to the support. In order to avoid this release of enzyme subunits to the supernatant, immobilized enzyme preparations were crosslinked with dextran aldehyde. Using partially oxidized dextrans, the full stabilization of the quaternary structure of these enzymes was achieved without severe detrimental effects on enzyme activity. Using the optimally stabilized derivative, dilution did not have any effect on the stability of the enzyme preparation. Thermal stability of the optimal derivatives was studied at different pH values and in the presence of organic cosolvents, reaching half-lives of 7 days in 50% acetonitrile or 50% dioxane at 40 ◦ C. Thus, these biocatalysts of immobilized catalase from T. thermophilus are very robust, stable and capable of functioning in a wide range of conditions. © 2003 Elsevier Inc. All rights reserved. Keywords: Thermostable enzymes; Covalent multipoint attachment; Dextran aldehyde; Stabilization of multimeric proteins; Glyoxyl agarose 1. Introduction Oxidases are useful enzymes in industrial biocatalysis (food, pharmaceutical, fine chemistry industries, etc.) since they are able to oxidize a wide range of substrates [1] with- out requiring any exogenous cofactor. Nevertheless, they produce hydrogen peroxide during the catalysis cycle which leads in many instances to the irreversible inactivation of the oxidase [2–4], and it may also promote non-desired oxidation of some substrates or products [5,6]. The imple- mentation of industrial processes involving oxidases would necessarily require this problem to be solved. Chemical catalysts carry out the decomposition of hydro- gen peroxide but their metallic nature may prove disadvan- ∗ Corresponding author. Fax: +34-91-585-4760. E-mail address: rfl@icp.csic.es (R. Fern´ andez-Lafuente). 1 Co-corresponding author. tageous if they release metal ions in the reaction medium or if they require harsher reaction conditions [7–12]. Al- ternatively, catalase may be used, since it is a widespread enzyme that catalyzes the decomposition of hydrogen per- oxide under mild conditions with no subproducts other than molecular oxygen and water. Therefore, a multienzymatic synthesis pathway may be used to obtain industrial prod- ucts using an oxidase coupled to a catalase [6], such as d-amino acid oxidase to produce -ketoacids [13,14] and glycerol-1-phosphate oxidase to produce dihidroxyacetone phosphate [15]. Additionally, catalase may be used on its own, in other industrial applications that require the removal of hydrogen peroxide, such as the treatment of textile bleach- ing effluents [16]. There are certain requirements an ideal catalase would have to meet in order to be used in the industry (e.g. high thermostability in a wide range of pH and stability in the presence of organic cosolvents) since the range of substrates 0141-0229/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0141-0229(03)00129-7