Cross enzyme immobilization

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. 

In summary, enzyme immobilization is extremely important in the scale-up of many biocatalytic processes. The preferred method for pharmaceutical production involves covalent binding through cross-linking or attachment to a support. Noncovalent attachment is less attractive, but it is heavily utihzed owing to the commercial availabihty of industrial quantities of some enzymes immobilized using this technique. 

Like many other useful discoveries, enzyme immobilization by cross-linking was actually an unintended by-product of another research project. In 1964, Florante Quiocho and Frederic Richards at Yale university cross-linked crystals of carboxy-peptidase-A with glutaraldehyde (pentane-1,5-dial), hoping to get stable crystals for X-ray diffraction studies. They noted that these cross-linked enzyme crystals (now... 

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... 

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

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]...

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. 

Figure 3.1 is a scanning electron microscopy (SEM) photograph of Novozym 435 before and after immobilization of CALB on the matrix (Lewatit). It is obvious that after immobilization, the enzyme has been adsorbed on the surface of the matrix and the surface has been saturated. This observation confirms the results of synchrotron infrared microspectroscopy performed at amide band wavelength on Novozym 435 (Figure 3.2) [5, 6], The researchers measured the intensity of the amide band across the cross-section of a Novozym 435 bead and attributed the presence of amide groups to the location of the enzyme immobilized on the bead. They showed that distribution of CALB on the bead is not homogenous and it mostly saturates the surface of the beads and hardly enters the center. The CALB enzyme is a globular protein with dimensions of 30 A x 40 A x 50 A [10], whereas...

Soluble copolymers of albumin and L-glutamate dehydrogenase have been prepared by glutaraldehyde cross-linking. The kinetic and electron microscopic properties of the soluble derivatives were compared with data available concerning the enzyme immobilized within proteic films. 

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