Continuous regeneration of NAD(P)⁺ by flavins covalently bound to sepharose

Abstract

Various flavins, FMN, FAD, and acriflavin, were immobilized to Sepharose using several different coupling methods. The only product stable enough to permit extended studies was acriflavin coupled to epoxy-substituted Sepharose. The nonenzymic oxidizing capacity towards NAD(P) H was investigated and a 25% specific activity, compared to that of free acriflavin, was observed. The reduced acriflavin was immediately auto-reoxidized in air and could thus be reused. It was shown that acriflavin-Sepharose preparations function as NAD(P)H oxidizing agents in a number of different dehydrogenase systems including lactate dehydrogenase (LDH), alcohol dehydrogenase (ADH), malate dehydrogenase (MDH), alanine dehydrogenase (alaDH), and glutamate dehydrogenase (GDH). The amount of expensive coenzyme necessary for high product formation of such systems was thereby markedly reduced.

Use of a polymer‐bound flavin derivative for the rapid regeneration of NAD(P)+ from NAD(P)H in dehydrogenase systems

“…These results are superior to others previously described with free or immobilized recycling systems [2,18,19], however, the long-term stability of the enzyme should be improved which could probably be obtained by immobilizing the later on an adequate support.…”

“…In 1976, Mansson et al [2] showed that this latter difficulty can be easily overcome by the immobilization on a particulate support of a chemical oxidant, a flavin derivative, commonly used as a regenerating reagent of 1,4-dihydronicotinamide.…”

Cofactor Regeneration for Enzyme-Catalysed Synthesis

“…TTN for NAD have reached 1125. Polymerized FMN (Chambers et al, 1974) and immobilized acriflavin (Mansson et al , 1976) have also been used to regenerate NAD photochemically.…”

Immobilized Coenzymes in General Ligand Affinity Chromatography and Their Use as Active Coenzymes

“…Alternative procedures include electrochemical regeneration, a method still in its infancy, like the chemical approach utilizing artificial electron acceptors and donors. I n a recent example of the latter method, an electron acceptor, acriflavin, was immobilized on a gel to allow subsequent facile removal from the assay mixture (145). In this form it was used for the continuous nonenzymic oxidation of NADH.…”