Protein-surface interactions immobilized proteins/enzymes

Both the nature of protein-surface interactions and inherent properties of a specific enzyme will contribute to the catalytic activity of an immobilized biocatalyst. Adsorption of an enzyme onto a surface can induce conformational changes which affect the rate and specificity of the catalyst. The total amount of enzyme loading, enzyme distribution within the immobilization support, and microenvironment surroimding the supported enzyme can all influence enzyme-catalyst activity, specificity and stability. ... 

Another approach has been to immobilize proteins within arrays of microfabricated polyacrylamide gel pads (Arenkov et al., 2000). Nanoliters of protein solutions are transferred to 100 x 100 x 20-pM gel pads and assayed with antibodies that are labeled with a fluorescent tag. Antigen imbedded in the gel pads can be detected with high sensitivity and specificity (Arenkov et al., 2000). Furthermore, enzymes such as alkaline phosphatase can be immobilized in the gel pads and enzymatic activity is readily detected upon the addition of an indicator substrate. The main advantage of the use of the threedimensional gel pad for fixation of proteins is the large capacity for immobilized molecules. In addition, the pads in the array are separated from one another by a hydrophobic surface. Thus, each pad behaves as a small test tube for assay of protein-protein interactions and enzymatic reactions (Arenkov et al., 2000). The disadvantage of the method is the need to microfabricate the array of gel pads in that microfabrication is... 

FIGURE 5.6 Schematic representation of the immunosensor based on a Protein A-GEB biocomposite as a transducer, (a) Immobilization of RlgG on the surface via interaction with Protein A, (b) competitive immunoassay using anti-RIgG and biotinylated anti-RIgG, (c) enzyme labeling using HRP-streptavidin and (d) electrochemical enzyme activity determination. (Reprinted from [31] with permission from Elsevier.)... 

Sensitivity The limit of detection for a certain compound is determined by its affinity constant or binding constant to the interaction partner immobilized to the surface. Therefore, the choice of the capture compound affects remarkably the assay performance. Because of the opportunity to create antibodies against different kind of compounds, e.g., peptides, proteins, enzymes, vitamins, environmental pollutants, herbicides, fungicides, etc., they are frequently used as a versatile tool for sensor assay developments. 

The distinction between substrate binding and supersubstrate binding as it has developed from studies on lipolytic enzymes has far-reaching implications. Many of the substrates of interacellular metabolism are partially immobilized, i.e., they are parts of biological membranes. The enzymes acting on such substrates are much larger than the substrate molecules, and the reactive center of the enzyme occupies only a small part of the surface of the enzymic protein. The rest of the protein must interact with the environment and in particular with the matrix in which... 

IMA is based upon affinity of surface functional groups of protein for the immobilized metal ions. The strength of association between the chelated and bound metal ion and the residues on protein surface may be quite high and even approach the strength of interaction between enzyme and cofactor/inhibitor or even that between antigen and antibody [140]. Considering that affinity of pro-... 

The non-specific adsorption of proteins on carbon nanotubes is an interesting phenomenon but represents a relatively less controllable mode of protein-CNT interaction. Moreover, in non-covalent immobilization process, the immobilized protein is in equilibrium between the surface of the carbon nanotubes and the solution and can therefore be gradually detached from the nano-material surface, a phenomenon called protein leakage [127]. To prevent the leaching of enzymes, covalent bonds have been used to attach the enzyme molecules to the nanostructured materials, which lead to more robust and predictable conjugation. Experimental evidences prove that proteins can be immobilized either in their hollow cavity or on the surface of carbon nanotubes [130]. 

Physical adsorption relies on non-specific physical interaction between the enzyme protein and the surface of the matrix. In this method only weak bonds are involved, e.g. hydrogen bonds, multiple salt linkages, van der Waals forces, which on one hand is less desruptive towards enzyme, while on the other hand leads to desorption from the surfaces once there are changes in temperature, pH, ionic strength etc. Physical forces are non-specific and further adsorption of other proteins or other substances can occur as the immobilized enzyme is used which may alter the properties of the immobilized enzyme. 

The adsorption method is the simplest one and is often used in bioelectrocatalysis research. Essentially it involves the incubation of protein in the carrier suspension with the subsequent washing of the nonadsorbed protein. Adsorption of proteins on different types of surfaces is effected due to electrostatic, hydrophobic, and dispersion interactions. The most popular carriers are carbon, soot, clays, aluminum oxide, silica gel, and glass. The optimal inert carrier is glass. It has recently been shown that porous glass with calibrated pore size can be used for immobilization of enzymes by adsorption. An interesting method of immobilization by adsorption has been proposed in which lipid is first adsorbed on carbon or silica gel and then the enzyme is adsorbed on the so-called soft surface of the lipid. 

Chitosan is widely used as supports for enzyme and cell immobilization due to its appropriate characteristics. Immobilization is the process in which the enzyme, cells or organelles is confined in a definite position thus rendering an insoluble form that retains the catalytic activity and can be reused several times. The enzymes or cells are bound to the carrier material via reversible surface interactions. The forces involved are van der Waals forces and ionic and H-bonding interaction as well as hydrophobic forces. Chitosan support being a positively charged polymer binds negatively charged proteins bind easily [43, 70]. 

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