The use of NAD(H)-dependent oxidoreductases as catalysts for large-scale asymmetric syntheses of a host of specialty chemicals requirs efficient and economical procedure for in situ regeneration of the expensive cofactor. Multienzyme reaction systems with a simultaneous coenzyme regeneration have been developed for the production of several L-amino acids in a continuously operated membrane reactor. For L-leucine production from $\alpha$-ketoisocaproate, leucine dehydronase (L-leucine : NADH oxidoreductase, EC1.4.1.9, from bacillus species), formate dehydrogenase (formate : $NAD^+$ oxidoreductase, EC1.2.1.2, from pseudomonas oxalaticus), and $NAD^+$ covalently bound to water-soluble dextran (dextran-$NAD^+$) as the catalytic enzyme, coenzyme regeneration enzyme, and coenzyme, respectively, were used in a hollow fiber enzyme reactor. The sustrates, formate and $\alpha$-ketoisocaproic acid, were pumped through a membrane reactor containing the FDH, LeuDH, and dextran-$NAD^+$ : the product, L-leucine, was continuously ultrafiltered and collected while the high molecular weight enzymes and cofactor analog were retained in the reactor. The experiment was carried out in phospate buffer containing 30 mmol/L sodium $\alpha$-ketoisocaproate and 0.3 mol/L ammonium formate with a residence time of about 1 h. The activity of LeuDH and FDH used was about 5 unit/mL.
The continuous production of L-leucine in a hollow fiber enzyme reactor for 34 hrs resulted in a space-time yield of 78.2 g/L/d (or 596 mmol/L/d) with a mean substrate conversion of 76%. The reaction was finished by loss of enzyme activity. Either the enzymes were degraded or cleaved into subunits. The loss of enzymes would be due to irreverseble adsorption onto the membrane. The turnover number of cofactor-ratio of moles of product to moles of NAD(H) - exceeded 8,500. Without the regeneration system, the $NAD^+$ cost per kg leucine production is \$1215/kg. By mean of the regeneration method, the cost reduced to 14 cents/kg, an enormous 10,000-fold reduction. As a result, the high cofactor costs in oxidoreductases reactor had been overcome with efficient in situ regeneration. The cofactor regeneration scheme for NAD(H) had been succesful to the extent that cofactor was no longer the dominant cost in leucine production.
