Enzyme Immobilization Via Crosslinking Protocols

Fig. 4. Multipoint covalent attachment of proteins.

the support, after which other subunits of each enzyme molecule can be cross-linked to the immobilized subunits via bifunctional, or polyfunctional, cross-linking agents (e.g., glutaraldehyde, aldehyde-dextran). In this way, complex multimeric structures can be fully stabilized (4,5) (see Fig. 5).

4.3.2. CLEAs or CLECs of Multimeric Enzymes

The aggregation or crystallization of multimeric enzymes with further cross-linking of the aggregated or crystallized beads with bifunctional or polyfunctional reagents should prevent the dissociation of subunits. These protocols for immobilization of enzymes with no support may be very powerful tools for greatly improving the stability of multimeric enzymes (23).

4.4. Hyperactivation of Lipases by Selective Adsorption on Hydrophobic Surfaces

The adsorption of lipases at very low ionic strength onto hydrophobic supports can promote the immobilization and stabilization of the open structure of lipases (see Fig. 6) (24-26). In this way, these immobilized lipases may exhibit a very important hyperactivation—at least towards small and hydrophobic substrates— associated with the immobilization process .

4.5. Physiochemical Modification of Immobilized Enzymes

Immobilized enzymes can be easily modified with chemical reagents (27). The process of modification can be easily controlled by ending the reaction via fast separation of immobilized enzymes and reagents. Intermolecular chemical modifications are then impossible, and the potential aggregations promoted by the modi-

Polyfunctional Reagent Glutaraldehyde
Fig. 5. Stabilization of multimeric enzymes: multisubunit covalent immobilization plus additional cross-linking.
Multisubunit Complex Enzyme
Fig. 6. Hyperactivation of lipases by interfacial activation on hydrophobic supports.

_ Organic solvent molecules

_ Organic solvent molecules

Immobilised Enzymes
Fig. 7. Generation of hyperhydrophilic environments surrounding immobilized enzymes

fication—via hydrophobic or electrostatic interactions—are also impossible. Modifications of immobilized enzymes producing very different protein surfaces, however, can be performed, (e.g., hydrophobization of enzyme surfaces, physical adsorption of hyperhydrophilic polymers, or chemical cross-linking of surface residues).

As a consequence of chemical modification, the conformational changes involved in inactivation or catalysis of immobilized enzymes can be strongly modified. In this way, some improvements in stability or selectivity may be observed.

4.6. Generation of Hyperhydrophilic Microenvironments Surrounding Every Molecule of Immobilized Derivatives

Coimmobilization of enzymes and very high concentrations of very high-molecular-weight (MW) hydrophilic polymers can promote the generation of new hyperhydrophilic microenvironments that surround every molecule of immobilized enzyme (see Fig. 7). These microenvironments can promote partition phenomena of hydrophobic substances (e.g., cosolvents, substrates, products) between the bulk solution and the environment where every molecule of the immobilized enzyme is placed. Again, stability and catalytic behavior of these immobilized enzymes can become strongly modified and improved (28-30).

5. Immobilization of the Enzymes, Reaction Media, Reactor, and Application of the Immobilized Derivative

Obviously, the preparation of suitable immobilized enzyme derivatives strongly depends on the further utilization of such derivatives.

5.1. The Reaction Medium

Enzyme biotransformations can be carried out in very different reaction media such as aqueous media, anhydrous organic solvents, supercritical fluid, and ionic liquids. The supports and the immobilization protocols are strongly dependent on the reaction medium in which the immobilized enzyme is going to be used or tested.

5.2. The Reactors

Enzyme biotransformations can be carried out in very different types of reactors, such as stirred tanks, packed beads, fluidized beds, and basket reactors. The mechanics and hydrodynamics of immobilized derivatives and supports are strongly related to the reactor to be used.

5.3. Applications

Immobilized enzymes are highly versatile may be used in many applications, such as chemical reactors for fine chemistry, repeated reaction cycles in food chemistry, different types of devices (for one or many reaction cycles) in analytical chemistry, and therapeutic applications (e.g., inside humans for long periods of time). The supports, immobilization protocols, and properties of immobilized derivatives will be very dependent on the particular application.

6. Basic Applications of Immobilized Enzymes and Proteins

Immobilization of enzymes and other proteins may also be useful at the laboratory scale for the evaluation of critical properties of enzymes and proteins (e.g., assembly of multimeric enzymes, studies of unfolding and refolding of proteins, studies of enzymes attached to cofactors, studies on protein-protein interactions). The full retention of all structural and functional properties of immobilized enzymes, even the most negative, has to be critical. In this way, immobilization protocols have to cover quite a range of structural and functional necessities ranging from unaltered immobilized enzymes (see Fig. 8) to those that have been greatly modified and improved (10,31).

7. Immobilization of Other Biomacromolecules

Different protocols for enzyme immobilization can be useful for the immobilization of other biomolecules such as antibodies (32,33) and DNA probes (34,35). On one hand, antibodies can be immobilized on different supports (pre-existing porous supports, magnetic nanoparticles) with a correct orientation through their glycosylated chains of their Fc regions (32,33). This immobilization is similar to the immobilization of other glycosylated enzymes. On the other hand, DNA probes (i.e., those containing amino or thiol groups inserted in one terminus) could also be covalently immobilized (see Subheading 6.) for pure immobilization of enzymes. In both cases, the final surface of the support should be fully inert in order to prevent undesirable adsorption of antigens or DNA (35,37). These "tailor-made" immobilizations may be critical in very recent studies in genomics (e.g., molecular hybridization of DNA under experimental conditions favoring the "perfect match") and proteomics (e.g., interaction of antibodies with traces of antigens and protein-protein interactions).

Protein Immobilization

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