Glutaraldehyde enzymes with

Figure 6. Cross-linking of enzymes with glutaraldehyde.

Differential scanning calorimetiy (DSC) of native and crosslinked -D-g ucosidase. The sample of glutaraldehyde-stabilized -D-glucosidase was produced essentially as detailed earlier (77), i.e., by treatment of the enzyme with glutaraldehyde at pH 9.0 in borate for... 

PEI (polyethylene imine) is often used as an additive when crosslinking enzymes with glutaraldehyde. Explain why. 

The stability of covalent bonds formed in method A have been tested by washing off the immobilized enzyme with detergents.40 This study indicated that glutaraldehyde reacts quickly with the APTS surface with the formation of stable bonds. The enzymes adsorb to the activated surface, with the majority being adsorbed within the first minute. Stable covalent bonds are formed between the glutaraldehyde and the enzyme. 

The fluoride formed was indicated at a fluoride sensitive electrode. The substrate, 4-fluorophenol, has a high reaction rate and a favorable diffusion behavior. The authors observed no interferences by ascorbic acid or uric acid glutathione interfered above 0.87 mg/ml. When immobilized by glutaraldehyde crosslinking with BSA, the enzymes were stable for 30 days. 

For direct fixation of GOD to platinum electrodes, Yao (1983) and Castner and Wingard (1984) treated the sensor surface with amino-propylsilane and crosslinked the enzyme with glutaraldehyde and BSA to the alkylamine groups. The main advantage of this sensor type is its low response time up to 100 measurements could be performed per hour. 

Aromatic amines are activators of horseradish peroxidase. Kulys and Vidziunaite (1983) adsorbed HRP together with GOD on a carbon electrode and crosslinked the enzymes with glutaraldehyde to assemble a sensor for aromatic amines. H2O2 is produced in the presence of glucose, acting as cosubstrate in the HRP-catalyzed oxidation of ferrocyanide. The ferricyanide formed was reduced back to ferrocyanide at an electrode potential of +10 mV vs SCE, the current being limited by the... 

On adding a reagent enzyme with two or more reactive groups to the solution, the protein globules are cross-linked with the formation of a space lattice. The compounds most often used for this purpose are glutaraldehyde (1), hexamethylene diisocyanate (2), adipimidate (3), diazobenzidine-3,3-dianisidine (4), 4,4-diisothiocyanatediphenyl-2,2-disulfonic acid (5), and sym-trichlortriazine (6) ... 

Reaction with agarose cyclic imido-carbonate or with an aminohexanoyl derivative of cross-linked agarose after modification of the enzyme with periodate and 1,2-diaminoethane or glycyl-L-tyrosine Co-immobilized with catalase on glutaraldehyde-activated controlled pore glass... 

Many enzyme immobilization techniques developed in connection with the preparation of heterogeneous biocatalysts have been applied for the construction of enzyme electrodes as well as for other types of biosensors. The immobilization of enzymes or other biocomponents on different membranes is most frequently realized by crosslinking agents, first of all by glutaraldehyde, and with the addition of bovine serum albumin [169] or other proteins, with l,8-diamino-4-aminomethyloc-tane, [170], etc. 

Glutaraldehyde cross-linked on silica-alumina with (or without) nickel, silicon carbide, or Kieselguhr Glutaraldehyde cross-linked with albumin Reaction of the periodate-oxidized enzyme with poly(4-aminostyrene)... 

Active immobilized forms of fS-D-glucosidase have been prepared by reaction of the enzyme with cellulose cyclic imidocarbonate, glutaraldehyde-treated beads of polyacrylamide, copolymers of acrylamide and 2-hydroxyethyl-methacrylate treated with cyanogen bromide, and copolymers of acrylamide and acrylic acid, and by entrapment of the enzyme in beads of polyacrylamide. 

Figure 2.27 Scheme of mechanism of covalent binding of enzyme with glutaraldehyde. 

Ion-sensitive field-effect transistors (ISFETs) were also used to fabricate poten-tiometric biosensors for OP compounds. Nyamsi-Hendji et al. have constructed a differential ISFET-based system. Acetyl or butyryl cholinesterase with BSA was fixed from solution to the surface of one pH-FET by glutaraldehyde treatment. Inhibition by solutions of diisopropyl fluorophosphate (10 -10 M) was almost complete in about 30 min. However, reactivation of the inhibited enzyme with 1 mM 2-PAM was insufficient (30-40 %), making the system of limited usefulness for repeated analyses. Stripping off the used enzyme membrane and recoating the FET is a time-consuming process. 

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. 

---