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

Abstract

An aldehyde derivative of riboflavin was covalently attached by reductive alkylation to soluble polycationic supports. The flavopolymers so obtained were stable under operational conditions. The catalytic efficiency towards oxidation of NADH by these flavopolymers was demonstrated, and the kinetic parameters ( K , and k,,,) revealed an overall catalytic efficiency (k,,,/K,) 185-fold greater compared to riboflavin.Various factors affecting the chemical regeneration of NAD' from NADH such as pH, ionic strength, nature of the buffer etc. were studied. The most interesting result was the highly favourable influence of borate ions which increased the reaction rate by a factor 2 -4 compared to the other buffers.The flavopolymers are very effective for in situ recycling of NAD(P)+. With up to 300-fold NADH + NAD' conversions for the system using yeast alcohol dehydrogenase and up to 1500-fold NADPH + NADP' regenerations for the system using glucose-6-phosphate dehydrogenase. These flavopolymers are superior to previous chemical recycling systems.Enzyme technology has proved its economic and industrial potential by the successful development of a 'first generation' of enzymes such as hydrolases and isomerases, which do not require coenzymes.A new objective is now to develop what are already called the 'second generation' of enzyme reactors which catalyze the synthesis and the modification of the fine chemicals of high added value. These processes are mainly oxido-reduction reactions, and thus involve the use of enzymes requiring expensive coenzymes which are needed in stoichiometry amounts. Any economically feasible process will thus depend on the retention or recovery and the regeneration of the coenzymes. Regeneration of coenzymes required for oxidoreduction reactions has been accomplished mainly by enzymatic methods [1] rather than by non-enzymatic methods.The chemical methods of regenerating coenzymes have received less attention than the enzyme-dependent processes although their advantages are stability and low cost of re-

Cofactor Regeneration for Enzyme-Catalysed Synthesis

“…FMN covalently attached to a soluble support regenerated NAD (350 turnovers in 188 h) and NADP (1600 turnovers in 24 h) for the oxidation of ethanol and G6P, respectively (Montaine, Lenders and Crichton, 1987). The immobilized FMN can be used in high concentration but retained within a membrane reactor or recovered for reuse in subsequent reactions.…”

Flavin‐grafted poly(vinyl alcohol): Preparation and properties

“…The direct oxidation of NAD(P)H at the expense of molecular oxygen could be achieved with the aid of flavins, properly modified to enhance their catalytic ability. In fact, some flavin derivatives, containing the isoalloxazine ring, grafted on suitable macromolecules, both natural or synthetic, have been described,7 showing a NAD(P)H oxidase activity. These macromolecular flavoadducts show a certain degree of catalytic activity, more efficient than that of their physiological coenzyme counterparts, i.e., riboflavin, flavin mono nucleotide (FMN) and flavin adenine dinucleotide (FAD).…”