Process Biochemistry 47 (2012) 26–33 Contents lists available at SciVerse ScienceDirect Process Biochemistry jo u rn al hom epa ge: www .elsevier.com/locate/procbio Immobilization of catalase via adsorption onto metal-chelated affinity cryogels Nalan Tüzmen a,∗ , Tülden Kalburcu b , Adil Denizli c a Department of Chemistry, Faculty of Science, Dokuz Eylul University, 35600 Buca, Izmir, Turkey b Department of Chemistry, Faculty of Science and Arts, Aksaray University, Aksaray, Turkey c Department of Chemistry, Faculty of Science, Hacettepe University, Ankara, Turkey a r t i c l e i n f o Article history: Received 11 January 2011 Received in revised form 26 September 2011 Accepted 27 September 2011 Available online 12 October 2011 Keywords: Catalase Cryogel IMAC Enzyme immobilization Adsorption Dye ligand a b s t r a c t A poly (acrylamide-allylglycidyl ether) [p(AAm-AGE)] cryogel was prepared by radical polymerization of acrylamide (AAm) and allylglycidyl ether (AGE). Cibacron Blue F3GA (CB) was covalently attached to the p(AAm-AGE) cryogel via the reaction between the chloride groups of the reactive dyes and the epoxide groups of the AGE. The CB-attached p(AAm-AGE) cryogel was chelated with Fe 3+ ions. This immobi- lized metal ion affinity chromatography (IMAC) cryogel carrying 25.8 ± 2.0 mol Fe 3+ ions was used in adsorption studies to interrogate the effects of pH, protein initial concentration, flow rate, temperature and ionic strength on enzyme activity. Maximum adsorption capacities were found to be 75.7 ± 1.2 mg/g for p(AAm-AGE)–CB–Fe 3+ cryogels and 60.6 ± 1.0 mg/g for p(AAm-AGE)–CB cryogels, respectively. The adsorbed amounts of catalase per unit mass of cryogel reached a plateau value at about 1.5 mg/mL at pH 6.0. The K m values were found to be 0.73 ± 0.02 g/L for the free catalase and 0.18 ± 0.02 g/L for the immobilized catalase. The V max value of free catalase (2.0 × 10 3 U/mg enzyme) was found to be lower than that of the immobilized catalase (2.5 × 10 3 U/mg enzyme). It was also observed that the enzyme could be repeatedly adsorbed and desorbed onto the p(AAm-AGE)–CB–Fe 3+ cryogel. © 2011 Elsevier Ltd. All rights reserved. 1. Introduction Most industrial applications of enzymes are carried out using immobilized systems, facilitating the recovery and reuse of the biocatalyst. Immobilization confers additional stability to a vari- ety of enzymes against several forms of denaturation. Enzymes have been immobilized on various supports (e.g., membranes, poly- meric materials, hollow fibers, or microbeads) either by adsorption, entrapment, or covalent binding [1]. Among the immobilization techniques, noncovalent immobilization methods, such as metal- chelated adsorption, are potentially more commercially useful than other methods because adsorption is simpler, less expensive and retains a high level of catalytic activity. Another important advan- tage of this method is the possibility of reusing the enzyme and support material for different purposes because of the reversibility of the method [2]. Support materials used in enzyme immobilization are classified as inorganic supports, synthetic polymers or natural macro- molecules [3]. Polymeric materials make suitable candidates due to their reactive functional groups, good mechanical properties, ease of preparation method and ability to accommodate bio- friendly components for improved biocompatibility [4]. Polymeric ∗ Corresponding author. Tel.: +90 232 4128702; fax: +90 232 4534188. E-mail address: nalan.tuzmen@deu.edu.tr (N. Tüzmen). gels have applications in many different areas of biotechnology, including use as chromatographic materials, carriers for the immo- bilization of molecules and cells, matrices for electrophoresis and immunodiffusion, and as a gel basis for solid culture media [5]. One of the new types of polymer gels with considerable poten- tial in biotechnology is ‘cryogels’ [6]. Cryogels are an excellent alternative for protein purification with many advantages such as large pores, short diffusion paths, low pressure drops, and very short times for both adsorption and elution. Cryogels are also low-cost materials and can be disposed after a single use, avoid- ing cross-contamination between batches [7]. There are many examples of the use of cryogels as matrices for immobilized biopolymers (enzymes, polysaccharides, nucleic acids) in cases where the unique properties of cryogels give better results than traditional gel carriers [8]. In recent years, immobilized metal ion affinity chromatography (IMAC) has become a widespread analyt- ical and preparative separation method for therapeutic proteins, peptides, nucleic acids, hormones, and enzymes [9–12]. IMAC introduces a new approach for generating selectively interacting materials on the basis of their affinities for chelated metal ions. First described by Porath et al. in 1975 [13], IMAC exploits the noncovalent, specific interactions between proteins, nucleic acids, and other biomolecules with immobilized metal ions and the sur- rounding solute molecules. The benefits of IMAC-ligand stability, which include high protein loading capacity, rapid purification, mild elution conditions, simple regeneration and low cost [14,15], 1359-5113/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2011.09.021