Enzyme immobilization support characteristics

More recently, a new series of water dispersed anionic polymers, the AQ 29D, 38D and 55D polymers were released by Eastman Kodak. Since that time, these polymers were used as electrode modifier (12, 13), as covering membrane (14) and as support for enzyme immobilization (15, 16). AQ polymers are high molecular weights (14,000 to 16,000 Da) sulfonated polyester type polymers (17, 18). Their possible structures have been recently presented (18). The AQ polymer serie shows many interesting characteristics useful for the fabrication of biosensors. They are water dispersed polymers and thus compatible with enzymatic activity. They have sulfonated pendant groups similar to Nafion and they can act as a membrane barrier for anionic interferring substances and they offer the possibility to immobilize redox mediators by ion exchange. 

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]. 

The choice of a suitable immobilization method for a given enzyme and appHcation is based on a number of considerations including previous experience, new experiments, enzyme cost and productivity, process demands, chemical and physical stabiHty of the support, approval and safety issues regarding support, and chemicals used. Enzyme characteristics that greatly influence the approach include intra- or extraceUular location size surface properties, eg, charge/pl, lysine content, polarity, and carbohydrate and active site, eg, amino acids or cofactors. The size, charge, and polarity of the substrate should also be considered. 

In the last decade there were many papers published on the study of enzyme catalyzed reactions performed in so-called chromatographic reactors. The attractive feature of such systems is that during the course of the reaction the compounds are already separated, which can drive the reaction beyond the thermodynamic equilibrium as well as remove putative inhibitors. In this chapter, an overview of such chromatographic bioreactor systems is given. Besides, some immobilization techniques to improve enzyme activity are discussed together with modern chromatographic supports with improved hydrodynamic characteristics to be used in this context. 

The characteristics of a support material are of great importance to the measured enzyme activity [79, 101]. Hydrophobic carriers have a low ability to attract water, thus leaving more available for the enzyme, hence Wehtje et al. [102, 103] have shown that celite is a suitable carrier for the PaHnl to yield an immobilized form of the enzyme. In contrast, controlled pore glass (CPG) and Sephadex G25 were found to be less well suited to enzyme support as, using these systems, cyanohydrin synthesis was significantly reduced (over 30%). Sephadex also promoted the spontaneous addition of HCN to benzaldehyde [102]. A series of batch experiments showed that if the solvent (diisopropyl ether) surrounding the immobilised PaHnl contained insufficient water (i. e. less than 2 %), it would be extracted from the enzyme preparation and consequently enzyme activity was lost [102]. These results were the basis for the production... 

Nature also prefers this method by keeping enzymes within cells, on cell walls, or adsorbed ( bound ) on large molecules, which are said to be immobilized. We therefore need to consider carefully the binding of catalysts on supports. The creation of structures and configurations of catalyst, support, and reactants and products to facilitate mass transfer and to provide access of reactants to the catalyst are essential features in designing a reaction system that has appropriate mass transfer characteristics. 

In this polymerization, the biofunctional component (enzyme) can be concentrated in an interfacial area between the frozen ice crystal and the supercooled monomer phase, and immobilized by molecular entanglement between the enzyme and polymer molecules. This is a different procedure for fixation from the usual entrapping method with a crosslinked structure in a gel. Therefore, we may call this procedure the adhesion-method to distinguish it from the usual entrapping. This term was extended to cover the use of the usual synthetic polymers including hydrophobic polymers as the supports. One of the characteristic properties of products obtained in this way was that there is a maximum activity at a certain monomer concentration. The maximum activity is observed when the increased inner surface area is balanced by the increased leakage of enzyme and these occur with a decrease of monomer concentration. Immobilization by physical entrapping was also studied by Rosiak [26], Carenza [27] and Ha [28]. 

From a practical point of view, a prolonged operation of a reactor with enzyme in suspension is not feasible. Procedures to retain the enzyme in the tubular reactor have been developed in order to maintain high enzymatic activity and to avoid enzyme washout. A plug-flow reactor operated with immobilized enzyme is known either as a fixed bed reactor or a fluidized bed reactor, depending on the characteristics of the flow pattern and the immobilized enzyme. Since mechanical stirring is not required in plug flow reactors, the support material is not damaged by the impeller, which may be a drawback in CSTR with immobilized enzyme. 

A variety of physical and chemical methods have evolved for immobilizing enzymes on or within solid supports. Kennedy and Cabral employed a variation of the scheme in Fig. 1 to classify techniques for immobilization of enzymes.f Judicious choice of the support is essential not only for the stability of immobilized enzymes, but also for the operational characteristics of the device containing the immobilized enzyme and the economic viability of the intended application. The discussion below and the information in Table 1 indicate some of the criteria employed in selecting a mode of immobilization. 

Industrial scale processes involving immobilized enzymes are normally carried out in fixed-bed reactors. Hence, the desired characteristics of the catalyst support are closely akin to those for the heterogenous catalysts commonly employed in the chemical industry, namely ... 

Candida antarctica Lipase B (CALB) is atfracting increasing attention as a biocatalyst for the synthesis of low molar mass and polymeric molecules. Almost all publications on immobilized CALB use the commercially available catalyst Novozym 435, which consists of CALB physically adsorbed onto a macroporous acrylic polymer resin (Lewatit VP OC 1600, Bayer). Primarily, commercial uses of CALB are limited to production of high-priced specialty chemicals because of the high cost of commercially available CALB preparations Novozym 435 (Novozymes A/S) and Chirazyme (Roche Molecular Biochemicals). Studies to better correlate enzyme activity to support parameters will lead to improved catalysts that have acceptable price-performance characteristics for an expanded range of industrial processes. 

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