Separation and purification of lysozyme by Reactive Green 19 immobilised membrane affinity chromatography

A poly(hydroxyethylmethacrylate)/chitosan (pHEMA-chitosan) composite membrane was prepared by UV-initiated photo-polymerisation. A dye ligand (i.e. Reactive Green 19; RG 19) was immobilised onto the membrane. The water content of the dye ligand-immobilised membrane was 50 ± 2% and the amount of immobilised Reactive Green 19 on the membrane was 0.865 μmol/ml. The rates of adsorption of lysozyme on plain composite and dye ligand-immobilised composite membranes were measured in a stirred cell. The adsorption capacity of the composite membrane was determined by changing pH and the concentration of lysozyme in the adsorption medium. Separation and purification of lysozyme from aqueous solution and egg white were investigated, respectively. The lysozyme adsorption capacity of the RG 19 immobilised membrane was 60.8 mg/ml. The adsorption capacity of the plain composite membrane was 7.2 mg/ml. Separation and purification were monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The lysozyme was purified 25.4-fold in a single step with a recovery 82%, as determined by HPLC. The membranes were stable when subjected to sanitization with sodium hydroxide after repeated adsorption-elution cycles.