Heterogeneous systems/immobilized enzymes

An immobilized enzyme-carrier complex is a special case that can employ the methodology developed for evaluation of a heterogeneous cat ytic system. The enzyme complex also has external diffusional effects, pore diffusional effects, and an effectiveness factor. When carried out in aqueous solutions, heat transfer is usually good, and it is safe to assume that isothermal conditions prevail for an immobihzed enzyme complex. 

Flow cells may also act as reactors. In BL, enzymes may be immobilized inside the cell either by chemical bonding on the inner surface or by entrapping the enzyme as a heterogeneous system by mechanical ways. This approach has the advantage of low consumption of expensive reagents and enhancement of their stability, which is usually low. Many bioluminescent reactions have utilized the benefit of this process. The flow cell is also used as a reactor in the case of electrogenerated chemiluminescence (ECL) when used with FI manifolds. Some of these applications are included in Table 4. 

BRADLEY A. SAVILLE is an Associate Professor of Chemical Engineering at i the University of Toronto. He received his B.Sc. and Ph.D. in chemical engi-neering at the University of Alberta. He is the author or co-author of over 25 research articles on enzyme kinetics, pharmacokinetics, heterogeneous reactions in biological systems, and reactors for immobilized enzymes. He is a member, of the Chemical Institute of Canada, the Canadian Society of Chemical Engineering, and Professional Engineers Ontario. 

Considerable progress has been made within the last decade in elucidating the effects of the microenvironment (such as electric charge, dielectric constant and lipophilic or hydrophilic nature) and of external and internal diffusion on the kinetics of immobilized enzymes (7). Taking these factors into consideration, quantitative expressions have been derived for the kinetic behavior of relatively simple enzyme systems. In all of these derivations the immobilized enzymes were treated as simple heterogeneous catalysts. 

This study employed conventional diffusion-reaction theory, showing that with diffusion-limited reactions the internal effectiveness factor of a heterogeneous catalyst is inversely related to the Thiele modulus. Using a standard definition of the Thiele modulus [100], the observed reaction rate of an immobilized-enzyme reaction will vary with the square root of the immobilized-enzyme concentration in a diffusion-limited system. In this case, a plot of the reaction rate versus the enzyme loading in the catalyst formulation will be nonlinear. 

In fermentation reactors, cell growth is promoted or maintained to produce metabolite, biomass, transformed substrate, or purified solvent. Systems based on macro-organism cultures are usually referred as tissue cultures. Those based on dispersed non-tissue forming cultures of micro-organisms are loosely referred as microbial reactors. In enzyme reactors, substrate transformation is promoted without the life-support system of whole cells. Frequently, these reactors employ immobilized enzymes, where an enzyme is supported on inert solids so that it can be reused in the process. Virtually all bioreactors of technological importance deal with a heterogeneous system involving more than two phases. 

Alternatively, if introduction of water in the system is not considered a problem (e.g. if water is used as a solvent or cosolvent in a homogenous or heterogeneous, two-phase, system), the enzyme can be added as an aqueous formulation. Advantages are also reported on the use of reversed micelles (detergent micelles containing aqueous enzyme in organic media). In any case, for all reaction conditions, the use of an immobilized enzyme preparation should be considered as outlined below. 

Microreactors can be used for either gas-phase or liquid-phase reactions, whether catalyzed or uncatalyzed. Heterogeneous catalysts (or immobilized enzymes) can be coated onto the channel wall, although on occasion the metal wall itself can act as the catalyst. Gas-liquid contacting can be effected in the microchannels by either bubbly or slug flow of gas, an annular flow of liquid, or falling liquid films along the vertical channel walls. Contact between two immiscible liquids is also possible. The use of microreactor systems in the area of biotechnology shows much promise, not only for analytical purposes but also for small-scale production systems. 

Another case of heterogeneous systems refers to immobilized enzymes. The kinetic behaviour of a bound enzyme can differ significantly from that of the same enzyme in free solution. The properties of an enzyme can be modified by suitable choice of the immobilisation protocol, whereas the same method may have appreciably different effects on different enzymes. These changes may be due to conformational alterations within the enzyme, immobilisation procedure, the presence and nature of the immobilisation support. The advantages of immobilised enzymes are for instance in reusability and possibility to use continuous mode. 

Effective (or apparent) kinetics of the immobilized enzyme is that directly determined from the observed behavior. Effective and apparent seem opposite concepts, but this is not so, since it is effective from the standpoint of the enzyme user (it is what one gets), but apparent from the standpoint of the enzyme since it does not reflect its actual catalytic potential which is obscured by the heterogeneous nature of the system. 

Engasser J, Horvath C (1973) Effect of internal diflusion in heterogeneous enzyme systems evril-uation of true kinetic partimeters and substrate diflusivity. J Theor Biol 42 137-155 Engasser JM, Horvath C (1976) Diffusion and kinetics with immobilized enzymes. In > ngard L, Katchalski E, Goldstein L (eds). Immobilized enzyme principles. Academic Press, New York, pp 127-220... 

New immunoassay concepts were described in which immobilized affinity ligands are used in unique flow-through systems (flow-injection immunoassay, FIIA). These systems are exclusively heterogeneous. Both immobilization of a specific antibody or a hapten on the solid support is possible. In most cases the scheme of the assay used is based on a sequential competitive enzyme immu-noas.say procedure [66], [67],... 

This technique of immobilized enzymes combines the unique features of both forms of catalysis the specificity of the enzymes with the stability and ease of handling and storing of supported heterogeneous catalysts. Furthermore, immobilized enzymes can be reused and are applicable to flow systems. Consequently, they find increasing applications in several analytical areas and in medicine. 

Fig. 6.70 Substrate and product profiles in an immobilized enzyme system as a consequence of partition and mass transfer limitations. (From A. Illanes, R. Femandez-Lafuente, JM Guisan, L. Wilson, Heterogeneous enzyme kinetics, in A. Illanes (Ed.), Enzyme Biocatalysis, Springer, 2008, pp. 155-203. Copyright 2008 Springer).

In Section 8.3.3 several examples of cascade systems involving homogeneous catalytic and enzymatic reactions were presented. There are also examples when heterogeneous catalytic reactions are combined with enzymatic catalysis. Fig. 8.35 shows a combination of a heterogeneous catalytic reaction (hydrogenation of a ketone over a supported metal catalyst) and an enzymatic one (acylation of obtained R-alcohol into the corresponding to R-acetate over an immobilized enzyme, Upase). 

Fig. 3 Generic FIIA system. A heterogeneous format is shown. The antibodies are immobilized in the immunoreactor. The analyte and the labeled Ag (in this case with an enzyme) are passed through the system and the competition step takes place. The flow of the substrate solution through the system allows the determination of the amount of bound labeled Ag, which is then detected and measured...

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