No description available.

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.

No description available.

No description available.

Thesis (Ph. D.)--University of Washington, 1990. / Vita. Includes bibliographical references (leaves [287]-307).

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

Thesis (Ph. D.)--Washington State University, August 2007. / Includes bibliographical references (p. 113-118).

A new immobilization technique has been developed in our lab using a new inert support material. Immobilized enzymes by the new method have a number of salient features of high activity, low cost and large catalytic area in comparison with the traditional immobilized enzymes. This new immobilization technique has potential to be widely applied for other enzymes. / Another new application of the immobilization method is to produce low lactose milk using immobilized lactase. A large population of people, especially the Asian, has intolerance to milk because of the lack of lactase in intestines. Pre-treatment of milk with immobilized lactase effectively reduces the milk lactose to an acceptable level for those people. / Enzyme was introduced for industrial application as early as nineteenth century. Immobilized enzymes have been developed since early 1960s. In recent years, immobilized enzymes have expanded their applications in many fields, food industry in particular, thanks to their superiority over the soluble forms: multiple or repetitive use; easier separation from reaction system; continuous production process; longer half-life and predictable decay rates. / Glucoamylase was also immobilized by the method. The differences between soluble and immobilized enzyme were compared. The potential using the immobilized glucoamylase in production of glucose syrup in replacement of liquid glucoamylase in current industrial process was assessed. / In this thesis, I have established the optimal immobilization conditions for three food enzymes, glucose isomerase, glucoamylase and lactase. The parameters including temperature, pH, protein capacity, and the ratios of cross-linker and adsorption agent have been studied in details. The applications of the consequently produced immobilized enzymes were studied and compared with the current industrial processes. / The novel immobilized glucose isomerase was applied to convert corn glucose into High Fructose Corn Syrup (HFCS) containing higher concentration of fructose than the current HFCS and achieved much better productivity than the existing immobilized glucose isomerases. / Shen, Dong. / "August 2007." / Advisers: Jun Wang; Kwok Kueng Ho. / Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 1000. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 175-194). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

Microfluidic biosensors and bioreactors based on immobilized biomaterials are described in this dissertation. Photocrosslinkable hydrogel or polymeric microbeads were used as a supporting matrix for immobilizing E.coli or enzymes in a microfluidic device. This dissertation covers a microfluidic bioreactor based on hydrogel-entrapped E.coli, a microfluidic biosensor based on an array of hydrogel-entrapped enzymes, and a microfluidic bioreactor based on microbead-immobilized enzymes. Hydrogel micropatches containing E.coli were fabricated within a microfluidic channel by in-situ photopolymerization. The cells were viable in the hydrogel micropatch and their membranes could be porated by lysating agents. Entrapment of viable cells within hydrogels, followed by lysis, could provide a convenient means for preparing biocatalysts without the need for enzyme extraction and purification. Our results suggested that hydrogel-entrapped cells, immobilized within microfluidic channels, can act as sensors for small molecules and as bioreactors for carrying out reactions. A microfluidic biosensor based on an array of hydrogel-entrapped enzymes could be used to simultaneously detect different concentrations of the same analyte or multiple analyte in real time. The concentration of an enzyme inhibitor could be quantified using the same basic approach. Isolations of the microchannels within different microfluidic channels could eliminate the possibility of cross talk between enzymes. Finally, we characterized microfluidic bioreactors packed with microbead-immobilized enzymes that can carry out sequential, two-step enzyme-catalyzed reactions under flow conditions. The overall efficiency of the reactors depended on the spatial relationship of the two enzymes immobilized on the beads. Digital simulations confirmed the experimental results.