Immobilization cross linking with glutaraldehyde

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. 

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],... 

A thermally stable NHase from Comamonas testosteroni 5-MGAM-4D (ATCC 55 744) [22] was recombinantly expressed in Escherichia coli, and the resulting transformant cells immobilized in alginate beads that were subsequently chemically cross-linked with glutaraldehyde and polyethylenimine. This immobilized cell catalyst (at 0.5 % dew per reaction volume) was added to an aqueous reaction mixture containing 32wt% 3-cyanopyridine at 25 °C, and a quantitative conversion to nicotinamide was obtained. The versatility of this catalyst system was further illustrated by a systematic study of substrates, which included... 

Wu et al. [183] studied the reversible hydrolysis of penicillin G into 6-aminopeni-cillanic acid (6-APA) and phenylacetic acid (PAA) in a chromatographic reactor. E. coli cells containing penicillin acylase (the catalyst) were immobilized by entrapment into gelatine and further cross-linking with glutaraldehyde. The ad-... 

Acetylcholineesterase and choline oxidase Immobilized by cross linking with glutaraldehyde vapor on the exposed end (diameter 0.2 mm) of a silica-sleeved Pt electrode. Detection limit for ACh and Ch were 20 and lOpmol, respectively. Sensor was used successfully in the FIA and for detection in HPLC. [84]... 

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]...

Immobilization of the respiratory chain from Escherichia coli by co-immobilization with gelatin, cross-linking with glutaraldehyde and attachment of the resulting film to an oxygen electrode NAD(P)H, lactate, succinate, malate, and 3-glycerophosphate [39]... 

MANAE-Agarose Cross-linked with glutaraldehyde One gram of the YLL immobilized onto MANAE agarose was incubated with 1% (v/v) glutaraldehyde solution in sodium phosphate buffer 5 mM pH 7 and 25°C for 1 h, under mild stirring. After that, the suspension was washed with 5 mM phosphate buffer and then filtered and stored at 4°C. The samples of the suspensions were withdrawn, and YLL activity was measured using /)NPB as substrate as described above. 

In the HER-100 (Omron Tateisi, Japan) an asymmetric cellulose acetate membrane is used, bearing LOD covalently bound by y-aminopropyltriethoxysilane and cross-linked with glutaraldehyde [371]. The membrane is highly selective for hydrogen peroxide. The analyzer is suitable for lactate assay in human serum. Pol)rurethane-immobilized LOD is used for whole blood lactate determination in both the Glukometer and in the ECA 20 (ESAT 6660) [372]. Dilution of the samples with a hypotonic buffer provides for both complete inhibition of glycolysis and immediate hemolysis. As shown by the correlation equations, the method appears to be fairly reliable ... 

Enzymes and ferrocene in 5-CyD can also be immobilized in polymer via cross-linking with glutaraldehyde. This configuration decreases the distance between redox centers of the enzyme and ferrocene - the electron-transfer mediator - and was shown to improve communication between the electrode and the redox centers ofGOx[76, 81, 84]. 

Intact cells adsorbed onto concanavalin-A-coated Sephadex or a form of Sephadex cross-linked with glutaraldehyde were used as specific immobilized, cell-surface carbohydrates in the purification of immunoglobulins by immuno-adsorption. °... 

Adsorption onto carbon and cross-linking with glutaraldehyde is unsuitable for the immobilization of p-D-galactosidase, since it results in loss of enzymic activity 34s p.D-Galactosidase immobilized on nylon tubing can be used for the continuous hydrolysis of lactose. An activity half-life of 100 days has been reported for p-D-galactosidase immobilized on feather-meal protein. ... 

The purified enzyme is soluble, and so would be difficult to recover from a reaction mixture. It must therefore be made insoluble again, that is to say immobilized, before it is used. A variety of methods are possible, ranging from its adsorption on to bentonite or on to a resin such as a polymethacrylate where it may be cross-linked with glutaraldehyde to form an insoluble matrix (cf. glucose isomerase, section 6.6.1), to its incorporation into fibres of cellulose triacetate. In any of these immobilized forms the amidohydrolase retains much, if not all, of its catalytic activity. The nature of the catalytic reactor depends largely on the form of the immobilized enzyme, but essentially the enzyme replaces the whole E. coli cells of the original process. When the hydrolysis is complete it can be easily recovered from the reaction mixture and re-used in tens, if not hundreds, of reactions. 

The properties of an amylase present in the thermophilic actinomycete Thermomonospora vulgaris have been reported. a-Amylase has been immobilized, with retention of enzymic activity, by adsorption onto a phenol-formaldehyde resin, by covalent attachment to activated, cross-linked poly(4-methacryloxybenzoic acid), and by cross-linking with glutaraldehyde. ... 

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