Enzyme glutaraldehyde

Many types of mono-, bi-, and multifunctional coupling reagents are available for labeling antibodies or antigens with an enzyme. Glutaraldehyde, carbodiimide, N-succinimidyl-3-(2-pyridyldithio)propionate, and periodate oxidation of carbohydrate moieties to form active dialdehydes are several commonly used approaches in the preparation of enzyme conjugates (104-106). 

Copolymerization with acrylamide after maleylation of the enzyme Glutaraldehyde- or carbodi-imide-mediated reaction with glycidyl methacrylate-ethylene dimethacrylate copolymers or amino-derivatives thereof Entrapment between a glass electrode and a membrane... 

Immobilization. The fixing property of PEIs has previously been discussed. Another appHcation of this property is enzyme immobilization (419). Enzymes can be bound by reactive compounds, eg, isothiocyanate (420) to the PEI skeleton, or immobilized on soHd supports, eg, cotton by adhesion with the aid of PEIs. In every case, fixing considerably simplifies the performance of enzyme-catalyzed reactions, thus faciHtating preparative work. This technique has been appHed to glutaraldehyde-sensitive enzymes (421), a-glucose transferase (422), and pectin lyase, pectin esterase, and endopolygalacturonase (423). 

Two types of immobilization are used for immobilizing glucose isomerase. The intracellular enzyme is either immobilized within the bacterial cells to produce a whole-ceU product, or the enzyme is released from the cells, recovered, and immobilized onto an inert carrier. An example of the whole-ceU process is one in which cells are dismpted by homogenization, cross-linked with glutaraldehyde, flocculated using a cationic flocculent, and extmded (42). 

In a second example, a cell—gelatin mixture is cross-linked with glutaraldehyde (43). When soluble enzyme is used for binding, the enzyme is first released from the cell, then recovered and concentrated. Examples of this type of immobilization include binding enzyme to a DEAE-ceUulose—titanium dioxide—polystyrene carrier (44) or absorbing enzyme onto alumina followed by cross-linking with glutaraldehyde (45,46). 

Because enzymes can be intraceUularly associated with cell membranes, whole microbial cells, viable or nonviable, can be used to exploit the activity of one or more types of enzyme and cofactor regeneration, eg, alcohol production from sugar with yeast cells. Viable cells may be further stabilized by entrapment in aqueous gel beads or attached to the surface of spherical particles. Otherwise cells are usually homogenized and cross-linked with glutaraldehyde [111-30-8] to form an insoluble yet penetrable matrix. This is the method upon which the principal industrial appHcations of immobilized enzymes is based. 

The cross-linking method relies on bifimctional reagents to form intermolecular linkages between the enzyme molecules to render them insoluble. Often albumin is added as an extender and glutaraldehyde is most commonly employed. This material can then be either formed as a free standing membrane or applied to the inner surface of the dialysis membrane... 

Purified MeHNL was crystallized by the sitting-drop vapor-diffusion method. The 10-20 mm bipyramidal crystals formed were cross-linked with glutaraldehyde and used as biocatalyst for the synthesis of optically active cyanohydrins. The cross-linked crystals were more stable than Celite-immobilized enzymes when incubated in organic solvents, especially in polar solvents. After six consecutive batch reactions in dibutyl ether, the remaining activity of the cross-linked crystals was more than 70 times higher than for the immobilized enzymes. Nevertheless, the specific activity of the cross-linked crystals per milligram protein was reduced compared with the activity of Celite-immobilized enzymes [53],... 

In their method, after co-adsorption of enzymes and magnetite nanoparticles, enzyme molecules were cross-linked via glutaraldehyde treatment. The HMMS... 

Fig. 4.25 I mmobilization of enzyme molecules and magnetite nanoparticles in hierarchically ordered mesocellular mesoporous silica with the aid of cross-linking via glutaraldehyde treatment.

Scheme 1 Covalent enzyme immobilization Aminopropyl-modified silica reacts with glutaraldehyde and the N-terminus of the enzyme.

Figure 6. Cross-linking of enzymes with glutaraldehyde.

Glutaraldehyde was one of the first and still is one of the most commonly used crosslinking agents available for creating antibody-enzyme conjugates. The crosslinking process using... 

In a fume hood, add 10 pi of 25 percent glutaraldehyde (Sigma) per ml of antibody/enzyme solution. Mix well. 

Figure 20.7 Glutaraldehyde antibody-enzyme crosslinking procedures usually produce a wide range of high-molecular-weight complexes, some of which may precipitate from solution.

Purify the activated enzyme from excess glutaraldehyde by gel filtration using a desalting resin or by dialysis against PBS, pH 6.8. 

---