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

A rotating ring-disk electrode (RRDE) has been used to study heterogeneous catalytic reactions involving an immobilized enzyme. Glucose oxidase (E.C. 1.1.3.4.) has been immobilized by covalent attachment to a variety of disk electrode supports resulting in the rotating ring-disk enzyme electrode (RRDEE). Covalent attachment to graphitic oxide, platinum and carbon paste has been achieved using the bifunctional reagents glutaraldehyde or 1-ethyl-3(3-dimethyl amino-propyl)-carbodiimide. By varying the electrode rotation speed, the effects of external substrate mass transport on the rate of enzymatic catalysis have been investigated. Extremely small diffusion boundary layers (ca. 10-25 μm) at the disk catalytic support, under conditions of well defined and reproducible hydrodynamics, facilitate this investigation. The rate enzymatic catalysis is evaluated by spectrophotometrically monitoring the formation of the product H₂O₂ in the bulk solution. Peroxide may also be conveniently monitored amperometrically at the concentric platinum ring giving a steady-state response proportional to the substrate concentration. It is assumed that the enzyme obeys Michaelis-Menten kinetics. The intrinsic value of the heterogeneous apparent Michaelis-Menten constant (K'(m)) has been determined from Lineweaver-Burk plots for both methods of product detection. Catalysis limited rates are observed only when the electrode rotation speed is high (ω ≥ 1600 rpm) as established from linear Lineweaver-Burk plots and by calculating characteristic dimensionless parameters relating the ratio of the rate of catalysis to substrate mass transfer (e.g. the Damkoehler number and reaction velocity parameter). A careful characterization of the "immobilized enzyme layer" was necessary to evaluate the RRDEE as a viable model for these investigations. The specific activity of the immobilized enzyme was measured and related to the disk supports, immobilization procedures, and subsequent enzyme loading. Specific activities range from Whereas the effects of external mass transfer resistances may be eliminated at high rotation speeds, any internal or intra-enzyme layer resistances may not. Using chronoamperometric techniques for several disk electroactive species, it was shown that the the effect diffusion coefficients of small molecules within the enzyme layer are approximately 25 - 50% of the bulk solution value. K'(m) determined from the ring amperometric measurements was found to be slightly larger than values determined from the bulk solution detection of product and was attributed to the interenzyme diffusion of product. This, however, was shown not to interfere with product detection under conditions of catalysis limited rates.

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Thesis (M.S. in chemical engineering)--Washington State University, December 2006. / Includes bibliographical references.

Enzyme immobilization as a technique attaches free forms of enzyme molecules to stationary support materials to permit enzymes to be reused several times. Bovine trypsin as a model enzyme was immobilized onto controlled pore glass (CPG) beads to investigate the optimum conditions for immobilization, as well as the physico-chemical properties of the immobilized enzyme versus the free form of the enzyme. At pH 9, about 60% of the enzyme protein incubated with CPG was immobilized onto the CPG, and immobilized bovine trypsin activity was determined as 0.265 BAPNA U/g CPG beads. The immobilized bovine trypsin showed lower affinity for its substrate, lower susceptibility to inhibition by soybean trypsin inhibitor and higher thermal stability, while the optimum pH and optimum temperature values were shifted to higher values compared to those of the free enzyme. The immobilized enzyme was evaluated for its capacity to extract carotenoproteins from shrimp shell. After 11 re-uses, the immobilized enzyme retained about 77% of its initial activity, and the total yield of the product from the same immobilized trypsin was 4.3 times higher than a single use of the same amount of the free enzyme. Cunner fish is a cold water adapted, stomachless teleost fish. Cunner fish trypsin possesses some unique properties compared with homologous trypsins from (i) species acclimated to warm temperature regimes, and (ii) species with functionally distinct-stomachs. Cunner fish trypsin was extracted from pancreatic tissue, and immobilized onto CPG beads using glutaraldehyde as cross-linking reagent. The influence of enzyme loading, the properties of the immobilized enzyme in terms of specific activities, and responses to pH and temperature were investigated. The kinetic properties and operational stability of the immobilized cunner trypsin were studied as well. The pH optimum of the immobilized fish trypsin shifted from pH 8.5 to pH 9, and the temperature optimum also increased from 45ºC to 50ºC versus the free form of the cunner enzyme. The catalytic efficiencies (Vmax/Km) of the immobilized fish trypsin were determined for both amidase and esterase reactions, using BAPNA and TAME as substrates and were found to be greater than those of immobilized bovine trypsin. Thus, the immobilized cunner fish trypsin had a higher catalytic capacity for the hydrolysis of both the amide and ester substrates. The operational stability of immobilized fish trypsin was studied by extracting carotenoprotein from shrimp shell. The immobilized fish trypsin retained 75% of its initial hydrolytic capacity after 11 re-uses, and the yield obtained was over 20% higher than that of immobilized bovine trypsin. When the immobilized cunner fish trypsin was applied to digest native pectin methylesterase (PME), it exhibited greater capacity to inactivate the PME than immobilized bovine trypsin. The inactivation efficiency of the immobilized fish trypsin was 20% higher than that of the immobilized bovine trypsin. The inactivation of PME was influenced by PME concentration, incubation time and temperature. In general, higher temperature, longer incubation period, and lower initial PME concentration produced more PME inactivation. PME inactivated by immobilized fish trypsin and bovine trypsin regained part of its activity during storage at 4ºC. The initial PME concentration affected the reactivation period. The kinetic studies indicated that the inactivation rate constants increased and D-values (time to inactivate 90% of the enzyme) decreased with increasing temperature for both immobilized fish trypsin and bovine trypsin. The activation energy (Ea) of PME inactivation by the immobilized fish trypsin was lower than that by the immobilized bovine trypsin, which explains why the immobilized fish trypsin had higher catalytic capacity at various temperatures than immobilized bovine trypsin.

Thesis (Ph.D.) - University of St Andrews, March 2008. / Restricted until 14th March 2009.

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