Fundamentals of Biochemical Engineering Part 1 - Bailey J., Ollis D. 1989

Application of enzyme-catalyzed reactions
Technological processes involving immobilized enzymes
Cofactor utilization and regeneration

Along with their application in Affinity Chromatography, Cofactors are also of considerable interest due to their ability to maintain enzyme activity. In the absence of cofactors, only two out of the six enzyme classes are capable of exhibiting catalytic activity. Obviously, large-scale industrial use of the Enzymes belonging to the other four classes requires efficient Methods FOR PRODUCING, separating, and isolating sufficient quantities of organic cofactors. Furthermore, the efficient utilization and regeneration of Coenzymes call for specialized reactors and catalytic units. To date, many of these challenges remain unresolved, although intensive research is underway in this field to unlock the immense practical and scientific potential of thoroughly developed cofactor technology. Here, we will briefly Touch upon only a few of the problems that arise in the design of enzyme-coenzyme reactors; more detailed information on cofactor Applications can be found in the suggested reading at the end of the chapter.

If an enzyme and a coenzyme are enclosed within hollow fibers featuring semipermeable walls through which both substrate and reaction products can pass, the enzymatic reaction can be carried out without the loss of either the enzyme or the coenzyme. Fig. 4.15 illustrates a laboratory-scale Reactor based on this principle. Clearly, given sufficient activity and Stability of the catalytic system, such a design can also serve as the basis for engineering a larger-scale process. Specifically, this type of reactor has been used to study the two-step ethanol oxidation reaction proceeding in the presence of NAD:

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Obviously, this process can proceed continuously only if NAD⁺ is constantly regenerated via The oxidation of NADH; for this purpose, the enzyme diaphorase (derived from porcine Heart) was added to the reaction mixture contained within the hollow fibers. Diaphorase catalyzes the oxidation of NADH by oxygen, with the regeneration of the cofactor NAD⁺ also producing H₂O₂. Because hydrogen peroxide inactivates many enzymes, the enzyme system is supplemented with yet another enzyme, catalase. Naturally, this approach can be extended to other enzymatic reactions as well. At the same time, if the Molecular Weight of the substrate is close to that of the coenzyme(s) and enzymes, some Modification of the scheme will be required. (Explain why.) If the coenzyme molecules are relatively small, their loss via ultrafiltration can be prevented, for instance, by covalently binding them to a soluble polymer such as polyethylene glycol, dextran, or polylysine.

FIG. 4.15. Schematic diagram of a laboratory hollow-fiber semipermeable reactor for continuous Processing catalyzed by an enzyme-coenzyme system.

We will now examine processes catalyzed by enzyme-coenzyme systems in which the enzymes and/or their corresponding coenzymes are bound to insoluble Supports. Any catalyst of this type must ensure, first, physical contact between the enzyme and the coenzyme and, second, the potential for cofactor regeneration. At least three different approaches can be outlined to solve problems of this type.

1. The coenzyme can be immobilized, while the enzyme and all substances required for regeneration are introduced in dissolved form. In this case, the reaction mixture exiting the reactor will contain all reactants except for the coenzyme.

2. The enzyme can be immobilized, leaving the coenzyme in solution; consequently, the coenzyme will also pass into the reaction products. Other Enzymes and substrates required for coenzyme regeneration can be fed into the reactor as a solution. Alternatively, the regeneration process can be carried out in a separate reactor, which may also utilize immobilized enzymes.

3. Finally, the enzyme and coenzyme molecules can be linked by a long, flexible chain and this enzyme-coenzyme complex can then be immobilized (a "tethered coenzyme").

The First and Second approaches have been implemented in laboratory experiments, whereas the potential of the third approach is still merely being explored. When scaling up to industrial production, the choice between the first and second approaches will be dictated by a range of technological and economic factors, the most important being the operational ease, efficiency, and cost of recovering and reusing the enzyme, coenzyme, and/or the regeneration System of the latter. If, for instance, none of these reaction mixture components can be reused, the first approach may prove more practical, given that most coenzymes are significantly more expensive than any other component in the mixture.

The high efficiency of cofactor utilization and regeneration in living Cells suggests an alternative approach to developing processes catalyzed by enzyme-coenzyme systems. This approach involves the immobilization of whole living cells followed by the exploitation of their natural systems for cofactor synthesis and regeneration. However, the practical Structure/175.html">Implementation of this outwardly appealing approach has not been particularly successful thus far. In particular, problems related to maintaining the stability of immobilized cells and introducing the required enzyme-coenzyme systems into easily immobilizable, active cells remain unsolved. The latter problem could, in principle, be resolved using Introduction/32.html">Genetic Engineering techniques that allow a Gene encoding the required enzyme to be inserted into The Genome of a suitable host Cell; The Cell would then essentially act as a repository for the cofactor and its corresponding regeneration system. Another fundamental challenge encountered when working with immobilized cells is associated with regulating Transport Across the cell envelope. Much like the enzyme-coenzyme catalytic system immobilized by a semipermeable membrane, a living cell must allow substrates to enter its interior, release reaction products into the extracellular environment, and at the same time retain enzymes, cofactors, and other substances essential for the normal functioning of intracellular catalytic systems.