Supermacroporous hydrophobic affinity cryogels for protein chromatography

N-Methacryloyl-L-tryptophan (MATrp) containing poly(2-hydroxyethyl methacrylate) based supermacroporous 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 mu m pore size was obtained by free radical 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 7.18g H2O/g for PHEMA and 6.99 g H2O/g for PHEMATrp. Lysozyme adsorption experiments were investigated under different conditions in continuous system (i.e., medium pH, flow-rate, protein concentration, temperature, salt type). Lysozyme adsorption capacity of PHEMA and PHEMATrp cryogels from aqueous solutions was estimated as 2.9 and 46.8 mg/g (0.49 and 7.85 mg/mL), respectively. Lysozyme molecules were desorbed with 0.5 M 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 43,140 U/mg using Micrococcus lysodeikticus as substrate. (C) 2008 Elsevier B.V. All rights reserved.