Enzyme engineering aspects of biocatalysis: Cofactor regeneration as example

Abstract

Reaction engineering is an important tool in the case of cofactor depending enzyme-catalyzed reactions. It allows the establishment of conditions resulting in lower product specific cofactor costs as compared with product-specific enzyme costs. This is shown for the stereospecific reduction of carbonyl compounds yielding chiral amino acids and alcohols. In continuous processes, cofactor costs can be reduced if the cofactor can be retained within the bioreactor or recycled into it after separation of the product. In case of readily water-soluble substrates it is even possible to recycle the cofactor during a single pass through a continuously operated reactor more than 4000 times because normally very low cofactor concentrations are sufficient to saturate the enzymes involved. L-tert-Leucine has been produced by reductive amination with a space-time yield of up to 366 g L(-1) d(-1) in a single continuously operated enzyme membrane reactor and a two-stage cascade. Total turnover number of the cofactor NAD(+) increased to 4230. (S)-1-Phenyl-2-propanol was obtained by reduction of the corresponding ketone in an membrane reactor with integrated extraction of the product. A new alcohol dehydrogenase from Rhodococcus erythropolis was used. A space-time yield of 63 g L(-1) d(-1) and a total turnover number of 1350 have been reached. L-Leucine has been produced using polymer-enlarged NADH. The total turnover number was 80,000 at a space-time yield of 214 g L(-1) d(-1).