Immobilizing, during adsorption

Commercial yeast invertase (Bioinvert ) was immobilized by adsorption on anion-exchange resins, collectively named Dowex (1x8 50-400,1x4 50-400, and 1x2 100-400). Optimal binding was obtained at pH 5.5 and 32°C. Among different polystyrene beads, the complex Dowex-1x4-200/invertase showed a yield coupling and an immobilization coefficient equal to 100%. The thermodynamic and kinetic parameters for sucrose hydrolysis for both soluble and insoluble enzyme were evaluated. The complex Dowex/inver-tase was stable without any desorption of enzyme from the support during the reaction, and it had thermodynamic parameters equal to the soluble form. The stability against pH presented by the soluble invertase was between 4.0 and 5.0, whereas for insoluble enzyme it was between 5.0 and 6.0. In both cases, the optimal pH values were found in the range of the stability interval. The Km and Vmax for the immobilized invertase were 38.2 mM and 0.0489 U/mL, and for the soluble enzyme were 40.3 mM and 0.0320 U/mL. 

All reactive functions, apart from epoxide, require a preliminary amine activation step prior to chemical coupling or a reduction after grafting step (aldehyde). The quantities of chemically immobilized ODN are in the same order of magnitude than those grafted during adsorption (less than 1 mg nr2) which let one suppose that the amine functions carried by the bases also react... 

In one limiting case the inhibiting molecule is immobilized upon adsorption and subsequently buried as an inclusion during metal deposition. In this instance the rate constant, fcino is simply related to the neighboring metal deposition rate to obtain [140] ... 

There are two basic procedures for enzyme immobilization by adsorption. Both share the same first steps preparation of the immobilization matrices (discussed above) and the aqueous enzyme solution. Typically, the aqueous solution is fairly concentrated in enzyme (approximately 5 to 40 mg/ml) and buffered (approximately 10 mM) to the optimal pH value of the enzyme. In addition, necessary co-factors, such as NAD(P)+ or NAD(P)H, should be included. Also, the presence of albumin or high-molecular-weight PEG at concentrations of 2 mg per g of matrix have been shown effective in protecting enzymes during the water removal stage of immobi-lization. °° Furthermore, the presence of sorbitol can increase activity and reduce side reactions, such as hydrolysis. -It is recommended that the aqueous solutions be centrifuged prior to use to remove any nonsolubilized matter. 

By comparing the remaining activity of the intermediate state , expressed by tt, for the two biocatalysts obtained at pH 7.0 and different initial enzyme activities in the supernatant, it is possible to evaluate the formation of lipase multilayers during adsorption. The intermediate of CALB-7A, CALB immobilized at pH 7 using Ea= 40 U/ml, has higher activity (ai=59.2%) than the intermediate of CALB-7B (oi = 14.3%), CALB immobilized at pH 7 using 0=280 U/ml, probably because of the formation of multilayers on CALB-7B. Due to this enzyme aggregation, lipase is weakly linked, and it is quickly deactivated [10], As it can be seen, CALB-7A is 4.6-fold more stable than CALB-7B. 

In the present work, green coconut fiber was successfiilly used to immobilize lipase B from C. antarctica by adsorption. During adsorption studies, it was observed that adsorption equilibrium was achieved afler a contact time of 2 h (in case of o=30 U/ml and Eo=(0 U/ml) or 6 h ( o=90 U/ml). Moreover, an improvement of hydrolytic activity of immobilized CALB is also observed with increasing concentrations of lipase offered to immobilization. This increase in activity is due to formation of multilayers, confirmed by thermal stability essays. Two plateaus of enzyme activity were observed when the pH of lipase solution during adsorption was varied in the range studied. This behavior is typical of an ionic support At 50 and 60 °C, the adsorbed enzyme was, respectively, 2- and 92-fold more stable than the soluble enzyme. At 60 °C, however, Novozyme 435 s stability was higher than that of CALB-7A. TUIer 10 h of incubation at 60 °C, Novozyme 435 retained more than 70% of its initial activity, whereas CALB-7A retained only 50%. Last but not least operational stabilities studies of butyl butyrate synthesis, compared to a commercial derivative, showed that C7U..B-7A is a suitable biocatalyst to be used in the synthesis of flavors. 

Fig. 8.7. Sensor response as a function of antigen concentration (filled squares, filled triangles, and filled diamonds are for 3, 5, and 1 x 10 B g ml-1 of the antibody, respectively, immobilized during polymerization. Asterisks and times are for 3 and 1 x 10 " g ml of the antibody, respectively, immobilized by physical adsorption after the polymerization. Filled circle is for the control experiment where no antibody was immobilized) [7] - Reproduced by permission of the Royal Society of Chemistry...

Immobilization by adsorption of P. laminosum and photosynthetic electron transport activity The immobilization was carried out successfully. This seems to indicate that the possible toxic compounds released during the polymerisation reaction can be removed by autoclaving the foams. 

Immobilization by adsorption and entrapment of A. azollae and ammonia production The immobilization was carried out successfully using both techniques. Ammonia production by immobilized and freely suspended cultures of A. azollae was measured during a period of 8 hours (samples were kept under continuous illumination or darkness) with or without MSX (fig.2). The results clearly showed that ammonia production by immobilized cells was much higher than free-living cells in agreement with previous works (7). Unlike the case of immobilized cells (table 2), no ammonia production was found in the absence of MSX with free-living cells (fig.2). 

Since many metallic catalysts have high adsorption affinities, we often find that certain poison molecules are adsorbed in an immobile form after only a very few collisions with the catalyst surface. In this situation, the outer periphery of the catalyst particle will be completely poisoned while the inner shell will be completely free of poison. The thickness of the poisoned shell grows with prolonged exposure to poison molecules until the pellet is completely deactivated. During the poisoning process, the boundary between active and deactivated regions is relatively sharp. 

In this context the lipase was immobilized on a support which also adsorbed water and propionic acid. During the reaction, the water caused a decrease of the reaction rate. While the water adsorption on the catalyst results in a reversible decrease of the enzyme activity, an excessive accumulation of water in the bulk mobile phase resulted in rapid irreversible deactivation of the enzyme. 

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