Adsorption, enzyme immobilization

Table 1 shows the enzyme immobilization results. By adsorption procedure, it is possible to observe that no enzyme was retained on the N-ITQ-6 material after... 

In view of the conductive and electrocatalytic features of carbon nanotubes (CNTs), AChE and choline oxidases (COx) have been covalently coimmobilized on multiwall carbon nanotubes (MWNTs) for the preparation of an organophosphorus pesticide (OP) biosensor [40, 41], Another OP biosensor has also been constructed by adsorption of AChE on MWNTs modified thick film [8], More recently AChE has been covalently linked with MWNTs doped glutaraldehyde cross-linked chitosan composite film [11], in which biopolymer chitosan provides biocompatible nature to the enzyme and MWNTs improve the conductive nature of chitosan. Even though these enzyme immobilization techniques have been reported in the last three decades, no method can be commonly used for all the enzymes by retaining their complete activity. 

These results demonstrate several useful properties of the C. fimi CBDs for enzyme immobilization and purification. Presumably, the CBDs of other bacterial cellulases (Figure 3) can be used in similar ways. It remains to be seen whether these differ significantly in their affinity or capacity for adsorption if so, certain CBDs may be preferable for specific purposes. 

Adsorption on solid matrices represents a quite simple and inexpensive method for enzyme immobilization. Enzyme dispersion is improved, reducing the diffusion limitations and favoring the accessibility of substrate to the enzyme [12]. On the other hand, because of the weak binding, the system can suffer from catalyst leaching, and there is little stabilization of the enzyme. The most common carriers... 

Enzymes, immobilized, are attached to a solid support by adsorption or chemical binding or mechanical entrapment in the pores of a gel structure, yet retain most of their catalytic powers... 

Four methods have been developed for enzyme immobilization (1) physical adsorption onto an inert, insoluble, solid support such as a polymer (2) chemical covalent attachment to an insoluble polymeric support (3) encapsulation within a membranous microsphere such as a liposome and (4) entrapment within a gel matrix. The choice of immobilization method is dependent on several factors, including the enzyme used, the process to be carried out, and the reaction conditions. In this experiment, an enzyme, horseradish peroxidase (donor H202 oxidoreductase EC 1.11.1.7), will be imprisoned within a polyacrylamide gel matrix. This method of entrapment has been chosen because it is rapid, inexpensive, and allows kinetic characterization of the immobilized enzyme. Immobilized peroxidase catalyzes a reaction that has commercial potential and interest, the reductive cleavage of hydrogen peroxide, H202, by an electron donor, AH2 ... 

In recent years the electrochemistry of the enzyme membrane has been a subject of great interest due to its significance in both theories and practical applications to biosensors (i-5). Since the enzyme electrode was first proposed and prepared by Clark et al. (6) and Updike et al. (7), enzyme-based biosensors have become a widely interested research field. Research efforts have been directed toward improved designs of the electrode and the necessary membrane materials required for the proper operation of sensors. Different methods have been developed for immobilizing the enzyme on the electrode surface, such as covalent and adsorptive couplings (8-12) of the enzymes to the electrode surface, entrapment of the enzymes in the carbon paste mixture (13 etc. The entrapment of the enzyme into a conducting polymer has become an attractive method (14-22) because of the conducting nature of the polymer matrix and of the easy preparation procedure of the enzyme electrode. The entrapment of enzymes in the polypyrrole film provides a simple way of enzyme immobilization for the construction of a biosensor. It is known that the PPy-... 

The non-aqueous lipase system for flavor esters developed by our group used components and preparative techniques for enzyme immobilization, that would not only be cost effective and simple but also meet regulatory requirements. The enzyme could have been immobilized by a number of methods however for the Intended application only (i) adsorption (11) ionic bonding or (lii) glutaraldehyde cross-linking would be... 

As discussed in sec 3, CNTs have been extensively used to develop pesticide sensors with higher sensitivity and longer stability. In this section we discuss about the design and the development of CNT based pesticide sensors. Joshi et al. reported the detection of OP compounds at a disposable biosensor with AChE-functionalized acid purified multi-wall carbon nanotubes (MCNTs) modified SPE [10]. The degree of inhibition of AChE by OP compounds was determined by measuring the electro oxidation current of the thiocholine generated by the AChE catalyzed hydrolysis of ATCh. The large surface area and electro-catalytic activity of MWCNTs lowered the over potential for thiocholine oxidation to + 0.2 Y. Further, mediators were not used in this case and enzyme immobilization was done by physical adsorption. 

Figure 4.1. Enzyme immobilization methods, (a) Nonpolymerizing, (b) cross-linking, (c) adsorption, (d) entrapment, and (e) encapsulation.

Lee outlines three different physical methods that are commonly utilized for enzyme immobilization. Enzymes can be adsorbed physically onto a surface-active adsorbent, and adsorption is the simplest and easiest method. They can also be entrapped within a cross-linked polymer matrix. Even though the enzyme is not chemically modified during such entrapment, the enzyme can become deactivated during gel formation and enzyme leakage can be problematic. The microencapsulation technique immobilizes the enzyme within semipermeable membrane microcapsules by interfacial polymerization. All of these methods for immobilization facilitate the reuse of high-value enzymes, but they can also introduce external and internal mass-transfer resistances that must be accounted for in design and economic considerations. 

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