Enzyme immobilization on supports

FIGURE 4 Activity of glucose-6-phosphate dehydrogenase as a function of time for ( ) the enzyme immobilized on the polyphosphazene/ alumina support and (o) in the presence of free enzyme and non-activated support. (From Ref. 23.)... 

Enzymes are usually used in aqueous or organic media and the temperature is limited to 40 °C to preserve enzyme activity as a consequence the reactions need very long times. With enzymes immobilized on solid supports [80] it is possible to operate at higher temperatures. 

Fig. 37.5. Imaging of enzymes immobilized on electrode surfaces, (a) Ox-idoreductase immobilized on insulators (imaging and quantification possible) (b) oxidoreductase immobilized on conductor the feedback may be caused by the electrode or the enzymes and (c) GC mode experiment does not depend on the nature of the support surface.

Gianfreda and Bollag (1994) investigated the behavior of laccase and peroxidase in the presence of a montmorillonite, a kaolinite, and a silt loam soil. They observed considerable variation in the retained activities of the two enzymes immobilized on the different supports as well as variation in the amount of each enzyme sorbed (Table 2.10). Interestingly enough, laccase immobilized on montmorillonite showed a higher specific activity (118%) than that of the free enzyme. This may be attributed to the steric modification of the immobilized enzyme or possibly due to the catalytic ability of montmorillonite itself. Their studies showed that the performance of these enzymes is significantly affected by soil mineral colloids. 

Svec, F. (2006) Less common applications of monoliths I. Microscale protein mapping with proteolytic enzymes immobilized on monolithic supports. Electrophoresis, 27, 947. 

While no attempts were made to compare the binding capacity of matrices, enzyme immobilized on either support were shown to exhibit full catalytic activity, improvement in stability, and no alteration in Km, Vmax and Kj values. The preparation obtained by direct covalent coupling of carboxypeptidase to support however exhibited relatively lower activity. It was also observed that orientation of the antibody on protein A supports may not after all enhance its enzyme binding activity. The author reasoned that even the most accessible antibody molecule may not bind to more than one enzyme molecule due to the large dimensions of the latter. 

Table 3. Native and affinity-tail bearing enzymes immobilized on metal chelate supports...

These profound effects on enzyme activity necessitate the adjustment of the experimental conditions for optimal results in EIA. It is not correct to establish optimum conditions in the liquid phase and to expect that the same conditions yield optimal results with the enzyme immobilized on a polystyrene microplate. Chemical modification of the support to immobilize one of the immunoreactants may also have pronounced effects. Unfortunately, such important and essential features have been ignored for various EIA. 

Because free-enzyme porcine pancreatic lipase had much lower activity in SCCO2 than in organic solvents, the authors decided instead to test immobilized lipases. Macroporous resin-supported lipase from M. miehei had fair activity but poor enantioselectivity. Porcine pancreatic lipase immobilized on supports had the greatest activity if the support was highly hydrophilic Sephadex G-25 and Bio-gel P6 were selected. Enantioselectivity was 83% for the (5)-gycidyl butyrate at 25-30% conversion, comparable to results in organic solvents. Several chiral alcohols were studied by Cernia et al. (80) as substrates for kinetic resolution by esterification catalyzed by silica-supported lipase from Pseudomonas sp. [Eq. (5)] ... 

It is possible to use enzymes immobilized on solid supports (either mineral or organic) of adequate pH in dry media [154] and, consequently, to operate at higher temperatures than in aqueous or organic media. Two main enzymatic systems including lipases can be used ... 

Information about the secondary structures (a-helices, /5-sheets, random coil) can be useful for understanding conformation changes of proteins upon the immobilization process. More specifically, circular dichroism (CD) [70] and FT-IR spectroscopy [56, 58, 61, 71-73] have been applied to study the structural characteristics of various proteins adsorbed on mineral surfaces. Kondo and coworkers [70] have studied the modification in a-helix content of proteins adsorbed on ultrafine silica particles with CD and found a decrease upon immobilization. Circular dichroism is not usually used because this technique is applicable only for the study of enzymes immobilized on nano-sized mineral particles due to problems arising from light scattering effects. On the other hand, infrared spectroscopy does not suffer from light scattering perturbations and has thus been used for the study of the conformation of proteins when they are immobilized on solid supports [57, 58]. 

Enzymes immobilized on an insoluble support also belong to two-phase systems. They have been mentioned earlier and are not discussed further at this point. 

O. Hemandez-Justiz, R. Fernandez-La-fuente, J. M. Terrini, Guisan, Use of aqueous two-phase systems for in situ extraction of water soluble antibiotics during their synthesis by enzymes immobilized on porous supports, Biotech. Bioeng. 1998, 59, 1, 73-79. 

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