Microenvironment effect immobilized enzymes

Immobilization onto a solid support, either by surface attachment or lattice entrapment, is the more widely used approach to overcome enzyme inactivation, particularly interfacial inactivation. The support provides a protective microenvironment which often increases biocatalyst stability, although a decrease in biocata-lytic activity may occur, particularly when immobilization is by covalent bonding. Nevertheless, this approach presents drawbacks, since the complexity (and cost) of the system is increased, and mass transfer resistances and partition effects are enhanced [24]. For those applications where enzyme immobilization is not an option, wrapping up the enzyme with a protective cover has proved promising [21]. 

As with most heterogeneous catalysts, it is often difficult to characterize immobilized enzymes at a molecular level. Most immobilized preparations are often complex mixtures with a distribution of chemically modified protein species. The gross catalytic properties observed are a composite of those of a range of differentially modified individual proteins, often irregularly distributed within the sample. Mass transfer limitations and microenvironment effects further complicate characterization. 

As far as enzyme immobilization is concerned, the biocompatibility of support is another important requirement [120-123], as the biocompatible surface can reduce some non-biospecific enzyme-support interactions, create a specific microenvironment for the enzymes and thus provide substantial benefits to the enzyme activity [124], To increase the biocompatibility of the support, various surface modification protocols have often been used to introduce a biofriendly interface on the support surface, such as coating, adsorption, self-assembly and graft polymerization. Among these methods, it is relatively easy and effective to directly tether natural macromolecules on the support surface to form a biomimetic layer for enzyme immobilization. In fact, this protocol has been used in tissue engineering recently [125-127]. 

In order to make a useful biosensor, enzyme has to be properly attached to the transducer with maintained enzyme activity. This process is known as enzyme immobilization. The choice of immobilization method depends on many factors such as the nature of the enzyme, the type of transducer used, the physiochemical properties of analyte, and the operating conditions [73]. The major requirement out of all these is its maximum activity in immobilized microenvironment. Enzyme-based electrodes provide a tool to combine selectivity of enzyme toward particular analyte and the analytical power of electrochemical devices. The amperometric transducers are highly compatible when enzymes such as urease, generating electro-oxidizable ions, are used [74]. The effective fabrication of enzyme biosensor based on how well the enzyme bounds to the transducer surface and remains there during use. The enzyme molecules dispersed in solutions will have a freedom of their movement randomly. Enzyme immobilization is a technique that prohibits this freedom of movement of enzyme molecules. There are four basic methods of immobilizing enzymes on support materials [75] and they are physical adsorption, entrapment, covalent bonding, and cross-linking, as shown in the Fig. 36. 

Considerable progress has been made within the last decade in elucidating the effects of the microenvironment (such as electric charge, dielectric constant and lipophilic or hydrophilic nature) and of external and internal diffusion on the kinetics of immobilized enzymes (7). Taking these factors into consideration, quantitative expressions have been derived for the kinetic behavior of relatively simple enzyme systems. In all of these derivations the immobilized enzymes were treated as simple heterogeneous catalysts. 

Several thermodynamic and kinetic behaviors of enzyme-catalyzed reactions performed in ILs, with respect to enzymatic reactions carried out in conventional solvents, could lead to an improvement in the process performance [34—37]. ILs showed an over-stabilization effect on biocatalysts [38] on the basis of the double role played by these neoteric solvents ILs could provide an adequate microenvironment for the catalytic action of the enzyme (mass transfer phenomena and active catalytic conformation) and if they act as a solvent, ILs may be regarded as liquid immobilization supports, since multipoint enzyme-1L interactions (hydrogen. Van der Waals, ionic, etc.) may occur, resulting in a flexible supramolecular not able to maintain the active protein conformation [39]. Their polar and non-coordinating properties hold considerable potential for enantioselective reactions since profound effects on reactivities and selectivities are expected [40]. In recent years attention has been focused on the appUcation of ILs as reaction media for enantioselective processes [41—43]. 

Analytical usefulness of immobilized bioluminescent assays depends on properties of their immobilized enzymes. The most popular application of immobilized bioluminescent systems is for analysis and monitoring of chemical and biochemical analytes and environmental pollutants. The wide range of analytes measured and monitored by immobilized bioluminescent systems has been reviewed. Stability, sensitivity, precision, and effects of interfering substances and the microenvironment are also discussed. 

The other important phenomenon that, in addition to the mass transfer, occurs when enzymes become heterogeneous catalysts, is the partitioning of substrates, products, inhibitors, metal and hydrogen ions between a bulk solution and a carrier. An elegant and simple theory describing the effect of microenvironment inside the particles of immobilized enzymes on their kinetics, has been developed by the group... 

If gel formation occurs, enzymes are effectively immobilized without meaningful changes in their microenvironment.31 Moreover, their settlement is partic-... 

The many circumstances leading to the Henri equation for enzyme conversion of soluble substrates are first noted, followed by some kinetic forms for particulate and polymer hydrolysis. Effects common to immobilized enzyme systems are summarized. Illustrative applications discussed Include metabolic kinetics, lipid hydrolysis, enzymatic cell lysis, starch liquefaction, microenvironment influences, colloidal forces, and enzyme deactivation, all topics of interest to the larger themes of kinetics and thermodynamics of microbial systems. 

Partitioning effects the equilibrium substrate, or effector concentrations within the support may be different from those in the bulk solution. Such effects, related to the chemical nature of the support material, may arise from electrostatic or hydrophobic interactions between the matrix and low-molecular weight species present in the medium, leading to a modified microenvironment, l.e., to different concentrations of substrate, product or effector, hydrogen and hydroxyl ions, etc., in the domain of the immobilized enzyme particle. 

Microenvironmental effects on the intrinsic catalytic parameters of the enzyme such effects due to the perturbation of the catalytic pathway of the enzymic reaction would reflect events arising from the fact that enzyme-substrate interactions occur in a different microenvironment when an enzyme is immobilized on a solid support. 

When diffusion of a substrate to an immobilized enzyme is relatively slow, the concentration of substrate is lower in the microenvironment than in the macroenvironment the resulting reduction in enzymic activity is referred to as diffusional inhibition, a factor attenuated by an inhibitor of the enzyme. Consequently, inhibition by products is more pronounced in the presence, than in the absence, of diffusional resistances at a given concentration of product in the macroenvironment. The anti-energistic interaction between chemical and diffusional inhibitions, however, makes the activity of a bound enzyme less sensitive to changes in product concentration in the macroenvironment. The interplay between the various types of product and diffusional inhibitions was illustrated graphically. Another consequence of the anti-energistic interaction is that the activity of the boimd enzyme at steady state is affected less by an inhibitor in the presence, than in the absence, of diffusion-limited transfer of the substrate. These effects also apply to membrane-bound enzymes in cellular milieu. 

The e ffect of the gel on the reactions within it is a clear example of a "microenvironment". Other effects which pol3mers have been suggested to exert on immobilized enzyme activity include excluded volimie (25,26), conformation... 

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