Epoxy-Amino Groups: A New Tool for Improved Immobilization of Proteins by the Epoxy Method Cesar Mateo, Rodrigo Torres, Gloria Ferna ´ ndez-Lorente, Claudia Ortiz, Manuel Fuentes, Aurelio Hidalgo, Fernando Lo ´ pez-Gallego, Olga Abian, Jose M. Palomo, Lorena Betancor, Benevides C. C. Pessela, Jose ´ M. Guisan,* and Roberto Ferna ´ ndez-Lafuente* Departamento de Biocata ´ lisis, Instituto de Catalisis, CSIC, Campus Universidad Autonoma, Cantoblanco, 28049 Madrid, Spain Received December 17, 2002; Revised Manuscript Received March 3, 2003 The properties of a new commercially available amino-epoxy support (amino-epoxy-Sepabeads) for immobilizing enzymes have been compared to those of conventional epoxy supports. The new support has a layer of epoxy groups over a layer of ethylenediamine that is covalently bound to the support. Thus, this support has a great anionic exchanger power and a high number of epoxy groups. We have found a number of advantages to this new heterofunctional support. Immobilization proceeds at low ionic strength using amino epoxy Sepabeads while requiring high ionic strength using conventional monofunctional epoxy supports. Immobilization is much more rapid using amino-epoxy supports than employing conventional epoxy supports. The possibility of achieving immobilized preparations in which the enzyme orientation may be different to that obtained using the traditional hydrophobic supports (with likely effects in terms of activity or stability). Stability of the immobilized enzyme has been found to be much higher using the new support than in preparations using the conventional ones in many cases. Here we show some examples of these advantages using different enzymes (beta-galactosidases, lipase, glutaryl acylase, invertase, and glucoamylase). Introduction Epoxy-activated supports seem to be almost-ideal systems to develop very easy protocols for enzyme immobilization. 1-5 Epoxy groups are very stable at neutral pH values even in wet conditions, and hence, commercial supports can be stored for long periods of time, and they can be prepared quite far from the place where the enzyme has to be immobilized. Furthermore, epoxy supports are able to react with different nucleophilic groups on the protein surface (e.g., amino, hydroxy, or thiol moieties) to form extremely strong linkages (secondary amino bonds, ether bonds, and thioether bonds) with minimal chemical modification of the protein (e.g., pK values of the new secondary amino groups are very similar to those of the preexisting primary amino groups). However, epoxy groups actually are hardly reactive for enzyme immobilization under mild experimental conditions (neutral pH, low ionic strength). 6-8 Thus, the immobilization on this kind of supports take place through a two step mechanism: in the first step, the adsorption of the protein on the support surface is promoted 4,9 (Scheme 1). For this reason, most commercial supports designed for protein immobilization have a fairly hydrophobic nature, and the recommended immobilization conditions on these supports include the use of high ionic strength (to force the hydro- phobic adsorption of the proteins). In a second step, the protein which has been previously adsorbed is covalently attached to the epoxy groups present in the support surface. Occasionally, this may promote some stability problems on the immobilized enzyme, that may be, at least partially solved by a suitable chemical blocking protocol. 10 Even with * To whom correspondence should be addressed. Dr. Roberto Ferna ´ndez- Lafuente/Jose ´ M. Guisan, Instituto de Catalisis, CSIC, Campus Universidad Auto ´noma, 28049 Madrid, Spain. Fax: 34 91 585 47 60. Phone: 34 91 585 48 09. E-mail: rfl@icp.csic.es/jmguisan@icp.csic.es. Scheme 1. Mechanism of Immobilization of Proteins on Epoxy Supports a a The covalent reaction between soluble enzyme and epoxy support is extremely slow. However, the covalent reaction between previously adsorbed proteins and the support proceeds at very high apparent concentrations of reactive groups on the support and on the protein surface (an “intramolecular reaction”) giving a high covalent immobilization rate. 772 Biomacromolecules 2003, 4, 772-777 10.1021/bm0257661 CCC: $25.00 © 2003 American Chemical Society Published on Web 04/10/2003