Enzyme and Microbial Technology 40 (2007) 1160–1166 Effect of the support and experimental conditions in the intensity of the multipoint covalent attachment of proteins on glyoxyl-agarose supports: Correlation between enzyme–support linkages and thermal stability Justo Pedroche a,∗ , Maria del Mar Yust a , Cesar Mateo b,∗∗ , Roberto Fern´ andez-Lafuente b , Julio Gir ´ on-Calle a , Manuel Alaiz a , Javier Vioque a , Jos´ e M. Guis´ an b , Francisco Mill´ an a a Instituto de la Grasa, CSIC, Avda. Padre Garc´ ıa Tejero 4, 41012 Sevilla, Spain b Departamento de Biocat ´ alisis, Instituto de Cat ´ alisis, CSIC, Campus UAM Cantoblanco, 28049 Madrid, Spain Received 23 May 2006; received in revised form 22 August 2006; accepted 29 August 2006 Abstract The immobilization of trypsin, chymotrypsin and carboxypeptidase A using 4 and 10% glyoxyl-agarose beads at different times of incubation was investigated. Enzyme loadings of 30 mg/mL gel for trypsin and chymotrypsin, and 2 mg/mL gel for carboxypetidase A were used. Immobilization rates were very rapid in both supports and reactions were completed after 1 h of reaction. Final residual activities at these concentrations were around 60% for trypsin and chymotrypsin, and 50% for carboxypeptidase A. Comparison of the thermal stability of the soluble and immobilized enzymes revealed that immobilization by binding to 10% glyoxyl-agarose yielded the most stable enzymatic activities. Reaction with this support yielded immobilized trypsin, chymotrypsin, and carboxypeptidase A that were 4700, 10,000, and 1000 times more stable than the soluble enzymes, respectively. It was observed that the number of lysine residues that took part in the immobilization process was a consequence of the type of support and reaction time of the experimental conditions, and that the increasing of the thermal stability of the derivatives was correlated with a increasing number of lysines residues involved in a multipoint covalent attachment. © 2006 Elsevier Inc. All rights reserved. Keywords: Immobilized enzymes; Glyoxyl-agarose; Thermal stability; Trypsin; Chymotrypsin; Carboxypeptidase A 1. Introduction Proteases represent a class of enzymes with important roles in physiological process. Besides this, commercially they are extremely important, accounting for about 60% of the total worldwide sale of enzymes. In terms of evolution, these enzymes are responsible of many essential biological processes, but nowa- days they are also involved in a wide variety of applications, mainly in detergent and food industries [1–3]. However, most proteolytic enzymes can degrade themselves and therefore, they are difficult to recycle. Any immobilization protocol of proteins inside a porous support can solve these problems because this concerns the fixation of individual molecules of enzyme, which ∗ Corresponding author. Tel.: +34 954611550; fax: +34 954616790. ∗∗ Corresponding author. Tel.: +34 915854760; fax: +34 915854809. E-mail addresses: jjavier@cica.es (J. Pedroche), ce.mateo@icp.csic.es (C. Mateo). reduces losses of activity by autolysis and increases the half-life of the enzymes [4–7]. Moreover, if the immobilization system is properly designed, can also enhance the stability of enzymes by “rigidification” of their three-dimensional structures, which results in a higher resistance to conformational changes induced by heat, organic solvents or pH [8–11]. Several procedures have been employed to achieve stabi- lization of enzymes, including genetic and protein engineering techniques [12–17]. However, immobilization of enzymes on porous solid carriers (silica, alumina, glass, agarose and cellu- lose) [18–20] by different methods (covalent and ionic attach- ment or physical adsorption) is probably the most used strategy to insolubilize and improve the stability of enzymes [21–23]. In this sense, glyoxyl-agarose beads have been successfully employed for the immobilization–stabilization of many different enzymes, resulting in high stabilization factors and high preser- vation of enzymatic activities [24]. As commented by Mateo et al. [24] and Graz ´ u et al. [25], the first immobilization in these supports is already a multipoint 0141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2006.08.023