Enhanced activity of an immobilized lipase promoted by site-directed chemical modification with polymers Ce ´ sar A. Godoy a , Blanca de las Rivas a , Marco Filice a , Gloria Ferna ´ ndez-Lorente a,b , Jose M. Guisan a, *, Jose M. Palomo a, * a Departamento de Biocata ´lisis, Instituto de Cata ´lisis (CSIC), Marie Curie 2, Campus UAM Cantoblanco, 28049 Madrid, Spain b Departamento de Microbiologı´a, Instituto de Fermentaciones Industriales, C/Juan de la Cierva 3, 28006 CSIC, Madrid, Spain 1. Introduction One important parameter for the application of biological catalysts in chemical industries is the specific activity displayed towards natural or unnatural substrates [1–5]. Different strategies such as site-directed mutagenesis or molecular directed evolution have been used to improve this enzyme property [6,7]. Chemical modification of amino acid side chains has been also broadly used to introduce a high variety of groups [8]. Most strategies for chemical protein modification rely on the nucleophilic side chains of amino acids such as aspartic and glutamic acids and especially on lysines [9], which are frequently targeted with amine-reactive end-groups such as activated esters, isocyanates, or through reductive amination [10]. However, a protein may contain several internal lysine residues in addition to the N-terminal amine. Therefore, this approach is often nonspecific, and the resultant protein–molecule conjugate is heterogeneous in the number and location of the molecules. Hence, creating well defined adducts is important, and site-specific modification of the protein is a better approach to prepare such biomolecules. The strategy must be both site-selective and efficient under protein compatible conditions: aqueous media, low to ambient temperature, and at or near pH 7. Moreover, the reaction must tolerate salts and surfactants commonly used to stabilize proteins [11]. Various creative methods have been explored to obtain site- specific bioconjugates, [12,13] and cysteine represents a conve- nient target for selective modification owing to the strong nucleophilic side chain sulfhydryl that enables thiol-disulfide exchange [14]. Conjugation of synthetic polymers to proteins by covalent attachment has been shown to significantly improve different properties such as stability, biocompatibility, etc. [15]. Addition- ally, the attachment of a polymer chain can be used to modulate protein activity and an interesting method to prepare polymer bioconjugates would be the application of cysteine chemistry using tailor-made polymers as protein-reactive initiators [16]. Furthermore, the modification performed on solid phase presents advantages on the preparation of protein–polymer conjugates, such as the use of excess of polymer, easy control of the reaction, easy separation of proteins and polymers, etc. [17]. This site-directed introduction of polymers is of particular importance in lipases, which present a complex catalytic Process Biochemistry 45 (2010) 534–541 ARTICLE INFO Article history: Received 25 August 2009 Received in revised form 3 November 2009 Accepted 23 November 2009 Keywords: Chemical modification Thiol-disulfide exchange Tailor-made polymers Lipase Activity Lipase hyperactivation ABSTRACT The activity of a lipase from Geobacillus thermocatenulatus (BTL2) can be greatly improved by site- directed chemical modification of a single external Cys64. This residue is placed in the proximity of the region where the lid is allocated when the lipase exhibits its open and active form. Thiol group of Cys64 was modified by thiol-disulfide exchange with pyridyldisulfide poly-aminated-dextrans or mono- carboxylated-polyethyleneglycol. The modification was performed on the covalently immobilized lipase on CNBr-agarose or glyoxyl-agarose. The activity of modified derivatives was strongly dependent on the immobilized preparation, the polymer used and the substrate assayed. For example, the modification with PEG-COOH of BTL2 immobilized on glyoxyl-agarose increased 5-fold the enzyme activity towards the hydrolysis of 2-O-butyryl-2-phenylacetic acid. However, the modification with 3-(2-pyridyldithio)- propionyl-dextran-NH 2 reduced the activity to 40%. The fact that the modified enzymes can be inhibited by an irreversible inhibitor much more rapidly than the unmodified ones suggested that the main effect of the modification is to somehow stabilize the open form of the lipase. ß 2009 Elsevier Ltd. All rights reserved. * Corresponding authors. Fax: +34 91 5854760. E-mail addresses: jmguisan@icp.csic.es (Guisan), josempalomo@icp.csic.es (J.M. Palomo). Contents lists available at ScienceDirect Process Biochemistry journal homepage: www.elsevier.com/locate/procbio 1359-5113/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2009.11.014