Crude extracts from Thermus thermophilus strains HB8 and 141327 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. (C) 2003 Elsevier Inc. All rights reserved.