Immobilization techniques enzyme entrapment

With these assumptions in hand, interpretation of real assay data involves plotting a model-derived value for concentration of NAD at the enzyme surface (NAD ). The value for the Mnad+ can be fitted to allow the Lineweaver-Burk plot to intercept the x-axis at a value that yields the value of Km as determined in solution. The value for Vmax is then read as the intercept at the y-axis (Figure 12.2). This approach permits derivation of a Vmax for the electrode that is independent of the effects of mass transfer. If one further assumes that the immobilization process does not affect the turnover rate of the immobilized enzyme (relative to its activity in solution), then this value of Vmax (which represents the total activity of all bound enzyme) can also be used to estimate the amount of immobilized enzyme. This model can be particularly useful when fabricating electrodes using immobilization techniques that entrap a fraction of enzyme from bulk solutions, such as direct physical absorption or co-immobilization within gels. 

All these immobilization techniques run the risk of altering activity compared with the native enzyme. Improved activity is occasionally reported, but this is the exception. The immobilization techniques listed above are in approximate order of loss in activity. Physical entrapment normally causes no change. Adsorption will distort the shape of the molecule compared with the native... 

Methods for Immobilization of enzymes on Insoluble supports can be generally classified as using adsorption, entrapment or covalent attachment techniques (Table II). In a few cases, enzymes have also been rendered Insoluble by covalent crosslinking however, more often, enzymes are crossllnked within entrapped cells. These methods have been reviewed extensively in the past [for example, see refs. 

The mode of immobilization, as well as the source and extent of purification of the enzyme, are important factors in determining the lifetime of the bio-catalyst. Generally, the lifetime of a soluble enzyme electrode is about one week or 25-50 assays, and the physically entrapped polyacrylamide electrodes are satisfactory for about 50-100 assays, depending primarily on the degree of care exercised in the preparation of the polymer. The chemically attached enzyme can be kept for years, if used infrequendy. In frequent use, the GOD electrode has a lifetime of over one year and can be used for over 1000 assays. For 1-amino acid oxidase or uricase (100) biosensors, about 200-1000 assays per electrode can be obtained, depending on the immobilization technique. 

Fig. 5. Correlation between heat response and reaction rate of cephalosporin C transformation by immobilized D-amino acid oxidase of Trigonopsis variabilis. Enzyme immobilization techniques entrapment in polyacrylamide gel ( ), cells cross-linked with glutaraldehyde ( ), cells entrapped in polyacrylamide gel (a) [28]...

Lee outlines three different physical methods that are commonly utilized for enzyme immobilization. Enzymes can be adsorbed physically onto a surface-active adsorbent, and adsorption is the simplest and easiest method. They can also be entrapped within a cross-linked polymer matrix. Even though the enzyme is not chemically modified during such entrapment, the enzyme can become deactivated during gel formation and enzyme leakage can be problematic. The microencapsulation technique immobilizes the enzyme within semipermeable membrane microcapsules by interfacial polymerization. All of these methods for immobilization facilitate the reuse of high-value enzymes, but they can also introduce external and internal mass-transfer resistances that must be accounted for in design and economic considerations. 

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 immobilization technique proposed is nanoentrapment into NPs. In this method, a water-in-oil microemulsion system is used for the fabrication of NPs and for the dispersion of enzyme. This procedure leads to the creation of discrete NPs through polymerization in the water phase or on the interface, in which the enzyme is dispersed [195, 196], One of the challenges of this approach is the difficulty in controlling the size of reverse micelles, as well as the number of enzyme molecules within each reverse micelle, which will directly affect the final properties of enzyme-entrapped nanoparticles [6],... 

Enzymes, when immobilized in spherical particles or in films made from various polymers and porous materials, are referred to as immobilized enzymes. Enzymes can be immobilized by covalent bonding, electrostatic interaction, cross-linking of the enzymes, entrapment in a polymer network, among other techniques. In the case of batch reactors, the particles or films of immobilized enzymes can be reused after having been separated from the solution after reaction by physical means, such as sedimentation, centrifugation, and filtration. Immobilized enzymes can also be used in continuous fixed-bed reactors, fluidized reactors, and membrane reactors. 

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. 

Most of the membrane segregated enzyme systems previously examined suffer some constitutive drawbacks which limit their yield and area of application. When enzymes are entrapped within the sponge of asymmetric membranes, product and substrate mass transfer occur mainly by a diffusive mechanism reactor performance is then controlled only by means of the amount and kind of charged enzyme, and the fluid dynamics of the solution in the core of the fibers. UF or RO fluxes, moreover, result in enzyme losses. Enzyme crosslinking in the membrane pores can reduce these losses, but it can lead to an initial activity loss, as compared to that of the native enzyme. Of course, once the enzyme is deactivated, it makes the reactor useless for further operation. Such immobilization techniques are seldom useful for microbial cells due to their large size. 

During the past fifteen years there has been a great deal of research in the area of immobilized enzymes. This research effort has been focused in three major areas. First is the form of the enzyme to be immobilized, whether the enzyme is purified or is contained within whole cells. The second has been in the area of immobilization techniques, whether by physical eibsorp-tion, entrapment, or chemical bonding. The third area of research has been in the development of various types of carriers that can support the enzyme and be used in various types of reactors. 

As one can easily imagine from the above-mentioned examples, it is high time to develop biological nanodevices based on LbL techniques. We here summarize recent developments on enzyme-encapsulated LbL devices and related functions where encapsulated does not always entail entrapment within spherical structures but generally includes immobilization of enzymes within LbL structures. 

Most of the above problems can be overcome by using immobilized enzymes in other words, enzymes which are confined in a well-defined region of space by means of a selective membrane, or immobilized by, for example, absorption or entrapment within the polymeric matrix of a membrane. Enzymes are prohibited, due to their molecular size, from diffusing out or permeating through the membrane, while substrates and products can readily permeate the membrane. The enzymes retain their catalytic properties and can be repeatedly and continuously used. Traditional immobilization techniques are summarized in Rg. 1.1. They include adsorption on a surface, covalent binding to an insoluble support, co-polymerization with a proteic carrier, encapsulation in a membrane shell and confinement in a gel. A comparison between some different techniques is also reported in Table 1.2. Hollow fibre membranes are commonly used for membrane reactors... 

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