Covalent binding, immobilization mechanism

Like cross-linking procedures, covalent binding to a resin typically utilizes accessible amino groups or carboxylic acids exposed on the enzyme. Fortunately, these common residues (Glu, Asp, Lys) are in general found on the surface (whereas more hydrophobic residues are in the interior). If not directly involved in the catalytic mechanism, the residues can be used for immobilization without significantly affecting the catalytic activity. However, as with other immobilization methods, some loss of specific activity can be expected due to distortion of the structure (loss of mobility), shielding of the active site and so on. 

References about immobilized enzyme reactors for environmental purposes are scarce (Katchalski-Katzir and Kraemer 2000). The concept is based on the immobilization of the enzyme onto a support by covalent binding or ionic interaction. The feasibility of the immobilized enzyme reactors is determined by the following requirements i) the specific activity of the derivative (units of enzyme per g of support) should be as high as possible ii) the support or membrane should be applied with a secondary function, such as the separation of substrates or products and iii) the support should have good mechanical resistance and minimum interaction with the substrates or products. Additionally, the immobilization process should be simple and inexpensive. 

The properties of supported enzyme preparations are governed by the properties of both the enzyme and the carrier material. The interaction between the two provides an immobilized enzyme with specific chemical, biochemical, mechanical and kinetic properties. The support (carrier) can be a synthetic organic polymer, a biopolymer or an inorganic solid. Enzyme-immobilized polymer membranes are prepared by methods similar to those for the immobilized enzyme, which are summarized in Fig. 22.7 (a) molecular recognition and physical adsorption of biocatalyst on a support membrane, (b) cross-linking between enzymes on (a), (c) covalent binding between the biocatalyst and the membrane, (d) ion complex formation between the biocatalyst and the membrane, (e) entrapment of the biocatalyst in a polymer gel membrane, (f) entrapment and adsorption of biocatalyst in the membrane, (g) entrapment and covalent binding between the biocatalyst and the membrane, (h) entrapment and ion complex formation between the biocatalyst and the membrane, (i) entrapment of the biocatalyst in a pore of an UF membrane, (j) entrapment of the biocatalyst in a hollow-fiber membrane, (k) entrapment of biocatalyst in microcapsule, and (1) entrapment of the biocatalyst in a liposome. 

Fig. 8 Carboxylic groups activation process and then antibody immobilization mechanisms. (a) Immobilization via the more reactive amine groups of the antibody (direct covalent binding) (b) Immobilization via the hydrophobic zone of the antibody (hydro-phobic pre-adsorptioh) (c) Immobilization via the positive charged richest zone of the...

Hemoproteins are a broad class of redox-proteins that act as cofactors, e.g. cytochrome c, or as biocatalysts, e.g. peroxidases. Direct ET between peroxidases such as horseradish peroxidase, lactoperoxidase," or chloropcroxidasc"" and electrode surfaces, mainly carbonaceous materials, were extensively studied. The mechanistic aspects related with the immobilized peroxidases on electrode surfaces and their utilization in developing biosensor devices were reviewed in detail. The direct electrical contact of peroxidases with electrodes was attributed to the location of the heme site at the exterior of the protein that yields close contact with the electrode surface even though the biocatalyst is randomly deposited on the electrode. For example, it was reported " that non-oriented randomly deposited horseradish peroxidase on a graphite electrode resulted in 40-50% of the adsorbed biocatalyst in an electrically contacted configuration. For other hemoproteins such as cytochrome c it was found that the surface modification of the electrodes with promoter units such as pyridine units induced the binding of the hemoproteins in an orientation that facilitated direct electron transfer. By this method, the promoter sites induce a binding-ET process-desorption mechanism at the modified electrode. Alternatively, the site-specific covalent attachment of hemoproteins such as cytochrome c resulted in the orientation of the protein on the electrode surfaces and direct ET communication. ... 

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