Biochemical Engineering Journal 43 (2009) 272–279 Contents lists available at ScienceDirect Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej Supermacroporous hydrophobic affinity cryogels for protein chromatography Fatma Yılmaz, Nilay Bereli, Handan Yavuz, Adil Denizli ∗ Department of Chemistry, Biochemistry Division, Hacettepe University, Ankara 06532, Turkey article info Article history: Received 28 July 2008 Received in revised form 20 September 2008 Accepted 16 October 2008 Keywords: Cryogels Protein purification Affinity adsorption Hydrophobic Interaction Chromatography Lysozyme abstract N-Methacryloyl-l-tryptophan (MATrp) containing poly(2-hydroxyethyl methacrylate) based supermacro- porous cryogel [PHEMATrp] was prepared for lysozyme purification form chicken egg white. MATrp was synthesized by reacting methacryloyl chloride with l-tryptophan methyl ester and provided hydrophobic functionality to the cryogel. PHEMATrp cryogel with 60–100 m pore size was obtained by free radi- cal polymerization of HEMA and MATrp having a specific surface area of 50m 2 /g. PHEMATrp cryogel was characterized by swelling studies, FTIR and SEM. The equilibrium swelling ratios of the cryogels were 7.18 g H 2 O/g for PHEMA and 6.99 g H 2 O/g for PHEMATrp. Lysozyme adsorption experiments were investigated under different conditions in continuous system (i.e., medium pH, flow-rate, protein concen- tration, temperature, salt type). Lysozyme adsorption capacity of PHEMA and PHEMATrp cryogels from aqueous solutions was estimated as 2.9 and 46.8mg/g (0.49 and 7.85mg/mL), respectively. Lysozyme molecules were desorbed with 0.5M ethylene glycol solution with 91% recovery. It was observed that PHEMATrp cryogel can be used without significant decrease in lysozyme adsorption capacity after five adsorption–desorption cycles. PHEMATrp cryogel was used for the purification of lysozyme from chicken egg white. Purity of lysozyme was estimated by SDS-PAGE. Possible denaturation of purified lysozyme was checked with fluorimetric measurements. Specific activity of the purified lysozyme was found as 43,140U/mg using Micrococcus lysodeikticus as substrate. © 2008 Elsevier B.V. All rights reserved. 1. Introduction The development of new techniques and methods for the sep- aration and purification of proteins has been essential for many of the recent advancements in biotechnology and biomedicine [1]. The purification of a protein is a prerequisite for its struc- tural and functional studies or its potential applications. A wide variety of purification techniques are available today, however, different types of chromatography have become dominant due to their high resolving power. In gel filtration chromatogra- phy, dye-affinity chromatography, ion-exchange chromatography, immobilized metal-ion affinity chromatography, affinity chro- matography, and hydrophobic interaction chromatography (HIC), the separation of a protein is dependent on it’s biological and physicochemical properties: molecular size, net charge, biospecific characteristics, and hydrophobicity [2–9]. HIC takes advantage of the hydrophobicity of proteins by pro- moting its separation on the basis of hydrophobic interactions between hydrophobic ligands and nonpolar regions on the surface of the proteins. The adsorption increases with high salt concentra- tion in the mobile phase and the elution is achieved by decreasing ∗ Corresponding author. E-mail address: denizli@hacettepe.edu.tr (A. Denizli). the salt concentration of the eluent. Therefore, the term of salt- promoted adsorption could be used for this type of chromatography [10]. In fact, HIC has been successfully used for separation pur- poses as it displays binding characteristics complementary to other chromatographic techniques. During the last few years, HIC has been studied by many researchers and today it is an established and powerful bioseparation technique in laboratory-scale, as well as industrial-scale, purification of proteins [11]. The development of a large variety of stationary phases for HIC has promoted a wide range of HIC applications in the purification of biomolecules, such as serum proteins, nuclear proteins, hormones, recombinant proteins, and enzymes [12]. A lot of different types of hydropho- bic molecules which have side chains of nonpolar amino acids such as alanine, methionine, tryptophan, and phenylalanine on their surfaces can be used as a ligand in HIC. These pseudospe- cific ligands have low binding constants (10 -4 to 10 -6 M -1 ) and, consequently, belong to weak-affinity ligands family. Nevertheless, they can exhibit selectivity resulting from the cumulative effects of multiple weak binding events such as electrostatic, hydrophobic, hydrogen binding, and van der Waals interactions with fast kinetics [13,14]. Lysozyme is a commercially valuable enzyme and has a widespread applications as a cell-disrupting agent for extraction of bacterial intracellular products, antibacterial agent in ophthal- mologic preparations, food additive in milk products, and as a drug 1369-703X/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2008.10.009