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. © 1996 John Wiley & Sons, Inc.

Reactor Operation and Scale‐Up of Whole Cell Baeyer‐Villiger Catalyzed Lactone Synthesis

“…On the other hand, processes employing isolated CHMO have extra cost burdens associated with enzyme isolation. Also, since NADPH is used by the enzyme on a mole for mole basis and its cost prohibits addition of excess quantities, the implementation of an in situ cofactor recycling system is essential (Willetts et al, 1991; Kragl et al, 1996).…”

Stabilization of NAD‐dependent formate dehydrogenase from Candida boidinii by site‐directed mutagenesis of cysteine residues

“…In order to perform enzymatic reductions on a large scale, a production and downstream process has been established [9]. However, the enzyme shows a fairly rapid inactivation under biotransformation conditions [10] resulting in higher costs for the biocatalyst. Therefore, the fdh gene from C. boidinii was cloned and overexpressed in Escherichia coli .…”

Study on Mesophilic and Thermophilic Alcohol Dehydrogenases in Gas-Phase Reaction

“…In addition, the STY of the chiral phenylethanol achieved in this study with both enzymes was even higher than that obtained with the most successful industrial liquid‐phase biotransformation involving cofactor regeneration such as the synthesis of l ‐leucine (STY 214 g L ‐ 1 d ‐ 1 ) and l ‐phenylalanine (STY 366 g L ‐ 1 d ‐ 1 ) (25) Even if a direct comparison of STY using a different set of reactor, medium, and product seems meaningless, it demonstrates that the critical benchmarking values of 100 g L ‐ 1 d ‐ 1 are achieved and enzymatic gas‐phase catalysis may be applied on a large scale.…”

“…The reduction of prochiral ketones to chiral alcohols in gas phase using S‐ADH from Thermoanaerobacter sp. (ADH T) definitely showed higher STY (620 g L ‐ 1 d ‐ 1 ) than the same reaction in aqueous phase using S‐ADH from Rhodococcus erythropolis (READH) with the STY of about 63 g L ‐ 1 d ‐ 1 (25).…”