Modification and Immobilization of Proteins Enzymes

Immobilization of Proteins and Enzymes onto Functionalized Polypropylene Surfaces by a Gaseous Plasma Modification Technique... 

As schematically depicted in Figure 5, two different routes are available for immobilizing biotin-labeled enzymes on the support through avidin-biotin complexation. The first procedure employs the biotin-modified surface on which biotin-labeled enzymes are immobilized through avidin as binder protein. For this procedure, the covalent linkage of biotin onto the surface of a carbon electrode and the preparation of biotin-labeled lipid bilayer on electrode have been studied. An alternative way involves the direct modification of an electrode surface with avidin. If avidin could be immobilized directly without loss of the binding activity to biotin, biotin-labeled enzymes could be loaded more easily on the electrode surface. 

In order to construct functional microspheres by modification of the surface with adsorbed proteins, e.g., enzymes and antibodies, the conformation and orientation of adsorbed proteins must be controlled to keep them as active as free proteins. If hydrophilic particles are used as a carrier, they hardly suffer nonspecific adsorption, but even antibody cannot be adsorbed. In this case, antibody is immobilized on the particles by chemical reactions such as those mentioned in the previous section (9). 

Thus, rather than trial-and-error development of functionality, it should be possible to design functionality based on the principles of protein structure and function and the specificities of the enzymes used for modification. Use of immobilized exo- and endopeptidases in such technology could be especially attractive for the reasons listed in Table I, particularly since problems associated with autolysis would be eliminated and the extent of proteolytic reactions could be controlled with some precision. 

Endopeptidases. Our expanding understanding of the relationship between structure and functionality of food proteins presents the opportunity for designing functionality into proteins by selective, specific proteolytic modification. Control of reaction and prevention of autolysis offered by immobilization are essential to establish the conditions for a highly selective modification. Hydrolysis at specific positions in the primary structure of proteins could be coupled with resynthesis of peptide bonds by selection of conditions, for example, as in the plastein reaction. By careful choice of enzymes and conditions according to the characteristics of the substrate proteins, it may be possible to design new structures from known food proteins. 

The chemical modification of redox enzymes with electron relay groups permits the mediated electron transfer and the electrical wiring of the proteins [83-85] (Figure 5A). The covalent attachment of electron-relay units at the protein periphery, as well as inner sites, yields short inter-relay electron-transfer distances. Electron hopping or tunneling between the periphery and the active site allows electrical communication between the redox enzyme and its environment. The simplest systems of this kind involve electron relay-functionalized enzymes diffusionally communicating with electrodes [83], but more complex assemblies including immobilized enzymes have also been reported. 

Figure 39. Electrical communication between an enzyme redox center and a photoexcited species attaining light-induced biocatalyzed transformations (A) direct electrical wiring of the protein by its chemical modification with tethered electron-relay units (B) electrical communication by the immobilization of the protein into a redox-functionalized polymer matrix.

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