Complete reactivation of immobilized derivatives of a trimeric glutamate dehydrogenase from Thermus thermophillus Juan M. Bolivar, Javier Rocha-Martin, Cesar Godoy, Rafael C. Rodrigues, Jose M. Guisan * Departamento de Biocata ´lisis, Instituto de Cata ´lisis (CSIC), Campus UAM Cantoblanco, 28049 Madrid, Spain 1. Introduction The relatively low enzyme stability under industrially relevant conditions is one of the problems that are hindering the implementation of these interesting biocatalysts [1–4]. In an industrial reactor, enzymes are usually employed in an immobilized form [5–7]. In this sense, the reactivation of these enzymes (after their partial inactivation) in these industrial reactors would increase the operational half-life of the biocatalyst [8]. At first glance, the reactivation of immobilized enzymes may have some advantages compared to the reactivation of free enzymes. For example, immobilized enzymes cannot aggregate during any step of inactivation or reactivation [9]. In fact, in many instances enzymes having a poly-His tag and that are produced as inclusion bodies, are unfolded and refolded after their immobiliza- tion on IMAC columns to get an active molecule [10–12]. Moreover, if an intense multipoint covalent attachment of the enzyme and the support has been achieved, not only is the enzyme more stable [1,13], but also these points, fixed to a rigid support, act as reference points that help to reach higher activity recoveries of the enzyme [8,14–17]. Multimeric enzymes are an especially complex case. These enzymes are produced ‘‘in vivo’’ as individual monomers that later assemble by a set of multipoint non-covalent and weak interac- tions (that globally are strong enough to keep the multimer assembled), the dissociation of subunits being in many instances the main reason for enzyme inactivation [18,19]. Reactivation of these enzymes needs not only to get the correct folding of each individual monomer, but also the correct assembly of the multimer. There are many examples of unfolding–refolding of multimeric soluble enzymes, in many cases with good results [20– 25]. However, there are no reports on the reactivation of immobilized multimeric enzymes. Penicillin G acylase and chymotrypsin are two enzymes that have been reactivated in soluble [26,27] and immobilized [15–17] form, but these enzymes are produced as a monomer and are processed later, being not ‘‘real’’ multimeric enzymes [28–32]. Process Biochemistry 45 (2010) 107–113 ARTICLE INFO Article history: Received 21 April 2009 Received in revised form 19 July 2009 Accepted 21 August 2009 Keywords: Multimeric enzyme stabilization Aldehyde-dextan Multisubunit immobilization Enzyme crosslinking Oriented immobilization Enzyme reactivation ABSTRACT First, the enzyme immobilized on cyanide bromide agarose beads (CNBr) (that did not involve all enzyme subunits in the immobilization) has been crosslinked with aldehyde-dextran. This preparation did not any longer release enzyme subunits and become fully stable at pH 4 and 25 8C. Then, the stabilities of many different enzyme preparations (enzyme immobilized on CNBr, that derivative further crosslinked with aldehyde-dextran, enzyme immobilized on highly activated amino- epoxy supports, GDH immobilized on supports having a few animo groups and many epoxy groups, GDH immobilized on glyoxyl-agarose beads at pH 7, and that preparation further incubated at pH 10, and finally the enzyme immobilized on this support directly at pH 10) were compared at pH 4 and high temperatures, conditions where both dissociation and distortion play a relevant role in the enzyme inactivation. The most stable preparation was that prepared at pH 7 and incubated at pH 10, followed by GDH immobilized on amino and epoxy supports and the third one was the enzyme immobilized on glyoxyl-agarose at pH 10. The incubation of all enzyme preparations in saturated guanidine solutions produced the full inactivation of all enzyme preparations. When not all enzyme subunits were immobilized, activity was not recovered at all. Among the other derivatives, only glyoxyl preparations (the most inert supports and those where a more intense multipoint covalent attachment were expected) gave significant reactivation when re-incubated in aqueous medium. After optimization of the reactivation conditions, the enzyme immobilized at pH 7 and later incubated at pH 10 recovered 100% of the enzyme activity. ß 2009 Published by Elsevier Ltd. * Corresponding author. Tel.: +34 91 585 4809/5478; fax: +34 91 585 4760. E-mail address: jmguisan@icp.csic.es (J.M. Guisan). Contents lists available at ScienceDirect Process Biochemistry journal homepage: www.elsevier.com/locate/procbio 1359-5113/$ – see front matter ß 2009 Published by Elsevier Ltd. doi:10.1016/j.procbio.2009.08.014