Enzyme and Microbial Technology 40 (2007) 310–315 Modulation of the catalytic properties of multimeric -galactosidase from E. coli by using different immobilization protocols Benevides C.C. Pessela, Gisella Dellamora-Ortiz 1 , Lorena Betancor, Manuel Fuentes, Jose M. Guis´ an ∗ , Roberto Fernandez-Lafuente ∗ Departamento de Biocat ´ alisis, Instituto de Cat ´ alisis-CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain Received 1 June 2005; received in revised form 18 April 2006; accepted 24 April 2006 Abstract Different immobilized preparations of the -galactosidase from E. coli were obtained by using different supports and immobilization strategies (bearing glyoxyl, epoxy, or BrCN groups or by glutaraldehyde crosslinking on matrices containing primary amino groups). In all cases, the immobilization yield was 100% with activity recoveries between more than 50% and almost 100% (using o-NPG as substrate). The immobilization varied the specificity of the enzyme. While the enzyme immobilized on Eupergit 250L decreased its activity to a 70% in the hydrolysis of o-NPG, this immobilized preparation exhibited an increment in the enzyme activity against lactose by a factor of 2. The different derivatives exhibited very different synthetic activity/hydrolytic activity ratios (Vs/Vh) in transglycosylation reactions and was strongly modulated by the experimental conditions. Thus, Vs/Vh values lower than 0.1 were obtained with the enzyme immobilized on BrCN at 4 ◦ C and pH 7, while the soluble enzyme gave a ratio value of 0.46 and the enzyme immobilized on Eupergit 250L gave a ratio of 0.8. Under similar conditions, again experimental conditions presented different effects on the different enzyme preparations: Eupergit C immobilized enzyme and soluble enzyme improve the Vs/Vh ratio when decreasing the temperature while BrCN immobilized enzyme suffered a great decrease in this ratio. These results suggest that the properties of this enzyme may be strongly modulated during immobilization, and that using a battery of immobi- lization strategies is possible to prepare immobilized enzymes with very different properties. © 2006 Elsevier Inc. All rights reserved. Keywords: Multimeric enzymes; Modulation via immobilization; Oligosaccharides; Transglycosylation 1. Introduction Immobilization of enzymes is usually necessary to employ the enzymes in an industrial reactor, because this permits the reuse of the biocatalyst, simplifies the reaction design, etc. [1–3]. Bearing this in mind, it seems interesting to couple the immo- bilization to the solution of other enzyme flaws. Thus, if the immobilization is properly designed, the enzyme stability may also be increased by multipoint [4,5], or multi-subunit [6–8] attachment. Even more interestingly, it has been recently shown that immobilization may be used to improve other enzyme cat- alytic properties, for example reducing enzyme inhibition [9,10]. ∗ Corresponding authors. Tel.: +34 91 585 48 09; fax: +34 91 585 47 60. E-mail addresses: jmguisan@icp.csic.es (J.M. Guis´ an), rfl@icp.csic.es (R. Fernandez-Lafuente). 1 Present address: Universidade Federal do Rio de Janeiro, Faculdade de Far- macia, Departamento de Farmacos, CEP: 21941-590, Rio de Janeiro, Brazil. In general, it is accepted that immobilization of proteins pro- mote distortions of the enzyme structure, that drives to a decrease in the enzyme activity. However, these distortions may be used to alter the enzyme regio and enantioselectivity [11–16]. Multimeric enzymes are very complex enzymes, whose active center may be determined by the exact assembly of the different enzyme subunits (Scheme 1) [17–19]. This means that if we can immobilize these enzymes using different proto- cols, involving different regions of the protein, the immobiliza- tion may produce small distortions on the subunit assembling, altering the active center shape and producing “immobilized enzymes” with different properties. In order to achieve this goal, the only requirement is to have a battery of very differ- ent immobilization protocols (that may imply different enzyme areas) and to control the immobilization processes (to control the support–enzyme interaction), in order to produce different changes in the active center of the multimeric enzyme. In this manuscript, we will present a first study on the alter- ation of the properties of the tetrameric -galactosidase from 0141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2006.04.015