Conclusion

The main feature of textiles modified by enzymes immobilized according to the biotin-avidin molecular recognition process as compared to the covalent linkage, is the much higher efficiency of the former. This efficiency is the result of the lower deactivation of the enzymes immobilized in this way, and can be attributed to the creation of microenvironments that are presumably favorable to the preservation of the protein structure of the enzyme and of the accessibility of the catalytic site.

Conversely, the covalent linkage ensure a very high content of immobilized enzymes onto the textile fibers; however, most of the covalently enzymes bound were shown to be inactive.

Urease Electrodialysis Coupling

We present in the last part of this chapter an application of the concept of enzyme-modified textiles based on urease immobilization in a coupling process (reaction-separation), the separation being achieved by coupling to an electromembrane process. Various technologies have been proposed for the recycling of wastewater (Dean 1991). In many cases removal of urea is difficult (for example, rejection by reverse osmosis membranes is weak; Lee and Lueptow 2001). However, one approach to handling urea is to hydrolyze it with urease immobilized on a support material (Schussel and Atwater 1995). Urea was shown to be efficiently removed from aqueous solutions by combining a catalytic reaction and ionic migration through an ion-exchange membrane in an electrodialysis cell. (Huang and Chen 1992, 1993). As urease allows the hydrolysis of urea with the production of ammonium and carbonate ions, these produced cations and anions can be separated by electrodialysis across cation- and anion-exchange membranes.

As an indication of the efficiency of this coupling process, urea and its by-products were totally removed from the treated aqueous solutions. Moreover, the migration through the membrane of ionic reaction products has an additional advantage, since it avoided the kinetic inhibition of the enzymatic reaction due to the back reaction.

Carboxylic textiles were modified by immersion in NaOH and urea solution (1 M) for 12 h to graft amino groups onto carboxylic acid molecules. Biotin was attached to this amino group using N-hydroxy-succinimide biotin as a coupling reagent (0.4mg/ml for 12 h at room temperature). Anion exchange textiles were modified directly by immersion in a sulfo-N-hydroxy-succinimide biotin (0.4 mg/ml) aqueous solution for 12 h.

A biotinylated urease was prepared by adding a fixed amount (2 mg/ml) of urease solution (EC 3.5.1.5, type III) from Jack Bean, in phosphate buffer 100 mM, pH 7.5, to 100 times mole excess of biotin-amidocaproate N-hydroxysuccinimide ester.

The biotin-modified textile support immersed in a 2 ml avidin aqueous solution (1 mg/ml)for 1 hwasthenimmersedin 2 ml ofbiotinylatedenzyme solution (2 mg/ml) for 1 hand then washed again with distilled water before the attachment of biotinylated urease.

A laboratory cell for assays of catalytic substrate transformation composed of three compartments separated by two ion-exchange membranes was used for the coupling with electrodialysis (Dejean et al. 1997). The modified textile was squeezed between the anion- and the cation-exchange membranes in the central compartment. The solution flowed continuously between both membranes across the textile enzyme membrane as in a frontal filtration.

A urea (10-2M) solution circulated in the central compartment while molar sodium hydroxide solution and molar sulfuric acid solution circulated in anodic and cathodic compartments, respectively. Urease catalyzes the hydrolysis of urea producing ammonium and carbonate ions according to reaction (1). The determination of the activities of the free and immobilized urease was carried out by measuring of the ammonium production with blood urea nitrogen (BUN) acid reagent and BUN color reagent (Sigma product).

Figure 20 shows the specific activity of immobilized urease as compared to free enzyme. It is clear that immobilization via avidin-biotin technology

Fig. 20. Specific activity of free urease and immobilized urease on the textile support (1 enzymatic unit corresponds to the degradation of 1 |M of urea at 20 °C in phosphate buffer pH = 8.5)

on the textile support allows the maintenance of the catalytic activity of the enzyme. The electrical field was applied to the electrodialysis cell with the textile inserted in the central compartment and clipped between the two ion-exchange membranes (Fig. 21).

Figures 22 and 23 depict the ammonium concentration evolution in the cathodic and central compartments, respectively. Three current densities were used (1, 5, and 10 mA/cm2). In the central compartment, the ammonium concentration produced by the enzymatic reaction decreases as the current density increases. The cations are transported through the cation-exchange membrane to the cathodic compartment owing to the driving force of electric field. In the central compartment, ammonium and carbonate ions are produced by the enzymatic reaction. When the current is increased in the electrodialysis cell the ammonium ion concentration decreases as the concentration in the cathodic compartment increases, because of the efficiency of the transport process across the cation-exchange membrane due to the driving force of the electrical field. Moreover, the efficiency of the transport of the ions produced by the enzymatic reaction is also demonstrated since no ammonium ions are detected in the anodic compartment. A weak leakage of ammonium through the anion-exchange membrane is detected at high current density after 90 min of working (only 1% of total ammonium concentration). The values of pH are kept constant during the process in anodic and cathodic compartments due to the high

Fig. 21. Scheme of electrodialysis cell incorporating an enzyme modified textile. AEM: anion exchange membrane , CEM: cation exchange membrane
Fig. 22. Ammonium concentration in cathodic compartment versus time of electrodialysis and applied current density

Fig. 23. Ammonium concentration in central compartment versus time of elctrodialysis and applied current density

Fig. 23. Ammonium concentration in central compartment versus time of elctrodialysis and applied current density volumeusedandtheweakcurrentdensity. Conversely, thepHinthecentral compartment is changed (initially pH 8.5, dropping to pH 7 after 90 min of the process).

In conclusion, in the prospect of achieving a complete removal of specific compounds such as organic pollutants by an enzymatic process represents an interesting example whereby the coupling between a chemical reaction process and membrane separation technology allows the complete treatment of a liquid phase.

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