Physical entrapment, enzyme stabilization

The success of the enzyme electrode depends, in part, on the immobilization of the enzyme layer. The objective is to provide intimate contact between the enzyme and the sensing surface while maintaining (and even improving) the enzyme stability. Several physical and chemical schemes can thus be used to immobilize the enzyme onto the electrode. The simplest approach is to entrap a solution of the... 

Both chemical and physical methods may be used to immobilize biocatalysts while retaining or modifying their activity, selectivity, or stability. Among the techniques used for immobilization of enzymes are physical adsorption, covalent bonding, ionic binding, chelation, cross-linking, physical entrapment, microencapsulation, and retention in permselective membrane reactors. The mode of immobilization employed for a particular application depends not only on the specific choice of enzyme and support, but also on the constraints imposed by the microenvironment associated with the application. 

Another problem with the development of implantable sensors is the need to calibrate the sensor ex vivo. This requires a high enzyme stability since the sensor has to be calibrated before implantation. The longest lifetime reported for enzymes immobilized in an implanted sensor was between 6 and 10 days (Shichiri et al., 1987). Neither physical entrapment nor chemical binding and crosslinking of GOD have provided a higher stability for continuously operated glucose sensors. 

All these immobilization techniques run the risk of altering activity compared to the native enzyme. Improved activity is occasionally reported, but this is the exception. The immobilization techniques fisted above are in approximate order of loss in activity. Physical entrapment normally causes no change. Adsorption will distort the shape of the molecule compared to the native state. The effect of covalent bonding depends on the location of the bond relative to an active site. If remote from the site, it may have no effect. The chemical nature of the support can affect activity. Crosslinking requires two covalent attachments per enzyme molecule and is thus likely to distort the shape of the enzyme to the point that catalytic activity is lost. Such distortions are even more likely, but not inevitable, for coagulated or flocculated enzymes. On the positive side, immobilization tends to stabilize enzymes against deactivation. 

In addition to nucleic acids, enzymes have also been incorporated into conducting a polymer matrix for biosensor applications. As a transducer, a CP can convert the chemical response into an electric current. To enhance the sensitivity and the response time, fabrication of CPs/enzyme nanocomposites with large surface area is a meaningful objective. Syu and Chang demonstrated the immobilization of urease onto PPy nanotubes over carbon paper substrate by a physical entrapment approach [124]. The composite electrodes exhibited a detection sensitivity for the determination of urea of 53.74 mVdecade and a detection limit on the urea concentration of 1.0 pM. Furthermore, the composite electrode shows rapid response, storage stability and reusability. Lipase can also be covalently immobilized... 

Pig. 1. Physical entrapment of enzymes (left) results in high degrees of activity retention but leaves the enz5mies susceptible to leeching and denaturation. Covalent immobilization (right) often results in an initial decrease in activity but imparts drastically better stability to the enzyme by reducing the susceptibihty to denaturation. 

Immobilization Theory. Chemical immobilization of enzymes results in the enhanced stability of the enzyme in the presence of harsh conditions, such as pH extremes, high temperature, solvents, or a variety of other potential environmental conditions that might otherwise denature or inhibit the catalytic activity of the enzyme. Additionally, immobilization enables development of materials that can be inserted and removed from reactors, reducing the need for separation to remove enzymes from solutions during purification. Several types of chemistries exist for immobilization of enzymes. Typically, the two main types of immobilization strategies are physical entrapment or entanglement, and covalent immobilization. These immobilization schemes are illustrated in Figure 1. 

Physical entrapment is now rarely used because the presence of the enzyme in solution does not give good long-term enzymatic activity. Chemical immobilization, using covalent bonds, ensures a greater long-term enzymatic stability [24], and a number of examples of this will now be described. 

The behavior of immobilized enzymes differs from that of dissolved enzymes because of the effects of the support material, or matrix, as well as conformational changes in the enzyme that result from interactions with the support and covalent modification of amino acid residues. Properties observed to change significantly upon immobilization include specific activity, pH optimum, Km, selectivity, and stability.23 Physical immobilization methods, especially entrapment and encapsulation, yield less dramatic changes in an enzyme s catalytic behavior than chemical immobilization methods or adsorption. The reason is that entrapment and encapsulation result in the enzyme remaining essentially in its native conformation, in a hydrophilic environment, with no covalent modification. 

A major disadvantage of the gel entrapment route to immobilization is the potential for physical loss of the enzyme as time elapses. To circumvent this problem, cross-linking agents, such as A,A -methylene-bis-acrylamide or glutaraldehyde, may be used to more firmly immobilize the enzyme or to provide mechanical stability. However, the more rigid the matrix the greater is the possibility that diffusional resistance to transport of reactants (substrates) to the site of the enzyme and of products out of the gel will limit the reaction rate. 

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