Stabilization of the quaternary structure of a hexameric alpha-galactosidase from Thermus sp. T2 by immobilization and post-immobilization techniques Benevides C. Pessela b , Cesar Mateo a , Miguel Filho b , Alfonso V. Carrascosa b , Roberto Fernandez-Lafuente a, * , Jose M. Guisa ´n a, * a Departamento de Biocata ´lisis, Instituto de Cata ´lisis-CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain b Departamento de Microbiologia, Instituto de Fermentaciones Industriales-CSIC, C/ Juan de la Cierva 3, 28006 Madrid, Spain Received 6 August 2007; received in revised form 16 November 2007; accepted 19 November 2007 Abstract An alpha-galactosidase from Thermus sp. T2, a hexameric protein, has been immobilized on cyanogen bromide agarose, retaining its activity almost intact, but without any significant improvement in enzyme stability. In fact, enzyme subunits could be desorbed from the immobilized preparation by boiling the solution in the presence of SDS (detected by SDS-PAGE) and a dependence of the enzyme stability on the enzyme concentration could be detected under certain conditions. The further cross-linking of this immobilized preparation with aldehyde–dextran permitted to improve the enzyme stability, avoiding the release of enzyme subunits to the reaction medium that could produce enzyme inactivation or food contamination. # 2007 Elsevier Ltd. All rights reserved. Keywords: Multimeric enzymes; Enzyme stabilization; Cross-linking; Aldehyde–dextran 1. Introduction Alpha-galactosidase enzymes are widely present in micro- organisms, plants, and animals. Some of them have been purified and characterized [1]. These enzymes catalyze the hydrolysis of 1,6 linked a-galactose residues from oligosac- charides such as melibiose (galactose-a-1,6-glucose), raffinose (galactose-1,6-a-sucrose), and stachyose (galactose-a-raffi- nose) and from polymeric galactomannans [2–4]. Alpha- galactose oligosaccharides are known to cause flatulence and digestive disorders. Considering the increasing demand and production of soybean milk, that presents some of these alpha- galactose oligosaccharides, the industrial interest of these enzymes in food technology is evident [5]. This has greatly increased the number of patents on this topic in the recent years [6,7]. Moreover, the enzyme may have some other uses, for example in the sugar beet industry, alpha-galactosidases have been used to increase the sucrose yield by removing raffinose, which makes the crystallization of beet sugar more complex [8– 10]. Recently we have reported the cloning and production of an alpha-galactosidase from Thermus sp. T2 [11]. The enzyme has high activity under a wide rage of conditions (from pH 5 to 10, at a wide range of temperatures, being the optimal T at around 70 8C at pH 7). The industrial use of an enzyme usually requires the use of an immobilized formulation. This way the design of the reaction is easier and the immobilized enzyme may be reused. Additionally, in order to use enzymes in food technology processes, it is convenient to use them in an immobilized form, to prevent food contamination by the enzyme (that also may contain proteins) causing allergic reactions [12]. The use of multimeric enzymes in food technology may be a bit more complex. Only immobilization protocols that can stabilize the quaternary structure of the enzyme can prevent the contamination of food by enzyme subunits. Moreover, in many instances the first step of the inactivation of multimeric enzymes is the dissociation of the enzyme subunits [13] that cause the inactivation of the immobilized enzyme under diluted www.elsevier.com/locate/procbio Process Biochemistry 43 (2008) 193–198 * Corresponding authors. Tel.: +34 91 585 4809; fax: +34 91 585 4760. E-mail addresses: rfl@icp.csic.es (R. Fernandez-Lafuente), jmguisan@icp.csic.es (J.M. Guisa ´n). 1359-5113/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2007.11.013