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 mg/ 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 158 U/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. / Graduation date: 2004
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/27191 |
| Date | 19 March 2004 |
| Creators | Wu, Yawen |
| Contributors | Daeschel, Mark A. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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