Immobilization of hen egg white lysozyme by the sole histidine residue to polystyrene beads through peptide spacers

Lysozyme is a natural antimicrobial agent that is effective against many food spoilage and pathogenic microorganisms by disintegrating their cell walls. Immobilization of lysozyme has attractive applications for use in the food industry: (1) The enzyme could be readily separated from treated foods and beverages and re-used while the foods could still be claimed additive-free; (2) It could impart stable antimicrobial capability to the surface of food packaging polymers.; In this study, a novel method is described for the preparation of a highly active immobilized lysozyme system. The method addressed three key issues in the covalent attachment of a biological active protein to an insoluble support: (1) The protein should be attached to the matrix by the fewest possible bonds to minimize conformational change; (2) The binding site(s) on the enzyme to the supports should be located as far as practical from its active center and be nonessential for its tertiary structure; (3) The binding method should minimize the steric interference between the support and the immobilized enzyme.; Using polystyrene resin beads as support matrix, peptide spacers of various lengths composed of 6-aminocaproic acid were synthesized with the solid phase peptide synthesis method. Then the amino terminals of the spacers were derivatized with bromoacetyl bromide and coupled to the protein's only histidine residue (His-15) that is nonessential for its lytic activity.; Immobilized lysozyme with a spacer composed of three 6-aminocaproic acid units displayed the best lytic result against lyophilized M. lysodeikticus cells: 2736 U/g resin with a protein load of 2.21 mgl resin. Retained activity was 14.2% of that of the free enzyme. Preparations with longer spacers yielded higher protein load yet the retained activity remained at about 14% level. A control consisted of random coupling of lysozyme to polystyrene beads without spacer gave an activity of 158U/G with a protein load of 1.24 mg/g resin and 1.4% of retained activity.; Properties of the immobilized lysozyme system were studied, including stability, effect of pH, surface characteristics of the support. A kinetics study of the system using Eadie-Hofstee plot demonstrated strong external diffusion effects, which resulted in deviation from classic Michaelis-Menton kinetic behavior.