Synthesis of Phenylalanine-Containing Hydrophobic Beads for Lysozyme Adsorption

Abstract

Hydrophobic interaction chromatography (HIC) takes advantage of the hydrophobicity of proteins by promoting its separation on the basis of hydrophobic interactions between immobilized hydrophobic ligands and nonpolar regions on the surface of the proteins. In this study, poly(2-hydroxyethyl methacrylate) poly(HEMA) beads having methacryloylamido-phenylalanine (MAPA) ligand, as a comonomer, providing hydrophobic functionality to the adsorbent were prepared. MAPA was synthesized by reacting methacryloyl chloride with L-phenylalanine. Spherical beads with an average size of 150-200 μm were obtained by suspension polymerization of HEMA and MAPA, conducted in an aqueous dispersion medium. The beads had a specific surface area of 19.1 m 2 /g, and were characterized by means of swelling studies, pore size analysis, elemental analysis, FTIR, NMR, and SEM. Beads with a swelling ratio of 68% and containing 3.2 mmol MAPA/g were used for the separation of lysozyme. Adsorption studies were performed under different conditions in a batch system (i.e., medium pH, protein concentration, temperature, and ionic strength). Lysozyme adsorption capacity of poly(HEMA) and poly(HEMA-MAPA) beads were 2.1 and 114.3 mg/g, respectively. It was observed that after 5 adsorption-desorption cycles poly(HEMA-MAPA) beads can be used without significant loss in lysozyme adsorption capacity. Purification of lysozyme from egg white was also investigated. Purification of lysozyme was monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The purity of the desorbed lysozyme was about 82% with recovery about 74%. The specific activity of the desorbed lysozyme was high as 42.800 U/mg.