Cofactor Regeneration in Artificial Enzyme Membranes: Potentialities for Analytical and Reactor Applications
Immobilization and stabilization of enzyme systems by various methods (l–2) have increased the possibilities in enzyme technology (3). These methods permit the use of enzymes in biochemical reactors (4). One of the limiting steps in the further development of enzyme technology is the regeneration of cofactors, especially the pyridine nucleotide cofactors which are involved in a large number of metabolic pathways. For obvious reasons the stoichiometric consumption of cofactors has to be avoided in enzyme reactors. Wykes et al (5) have described the use of a soluble high molecular weight derivative of NAD which can be retained in a continuous flow reactor by an ultrafiltration membrane. They also described an insoluble NAD derivative. Also, Davies and Mosbach (6) have described the application of soluble immobilized NAD in an enzyme electrode and in model enzyme reactors. According to Wykes et al (5) the immobilization of NAD cannot be justified and they concluded that it is better to use free NAD in a batch reactor. Recently, Coughlin et al (7) have described the electrochemical regeneration of NAD from its reduced form, but this requires a two step process. The present paper deals with the immobilization of cofactor on the active site of an enzyme and its regeneration with oxygen using as an electron carrier phenazine methosulfate (PMS). The cofactor requiring enzyme is transformed into an enzyme bearing a prosthetic group. In this technique alcohol dehydrogenase (ADH) and NAD were immobilized together in a protein membrane by a previously described method (8). The active NAD-ADH membranes were used both in an electrode and in a reactor.
KeywordsEnzyme Electrode Enzyme Technology Cofactor Regeneration Phenazine Methosulfate Continuous Flow Reactor
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