Page 48 - Cascade biocatalysis

24 2 New Trends in the In Situ Enzymatic Recycling of NAD(P)(H) Cofactors

All other types of enzymatic recycling of the NAD(P)(H) cofactors are based
on an enzyme-coupled cascade approach that requires the concurrent use of an
ancillary enzyme and stoichiometric quantities of a sacrificial co-substrate. This
approach poses several requirements to the regeneration system in order to be
both practical and cheap. Just to mention some of them, regenerating enzymes
and co-substrates should be easily available, inexpensive, and stable both during
storage and under process conditions. Possibly, the recycling reaction should help
overcoming undesired thermodynamic equilibria occurring in product formation,
for example, by being irreversible. Moreover, the co-product formed in the coupled
reaction should be easily separated from the target product.
In this chapter, an overview of the more recent attempts of solving the limitations
of the already established enzymatic recycling methods will be provided, together
with some selected examples of conceptually novel systems.

2.2
Recent Advancements in the Enzymatic Methods for the Recycling of NAD(P)(H)
Coenzymes and Novel Regeneration Systems

2.2.1
In Situ Regeneration of Reduced NAD(P)H Cofactors

2.2.1.1 Formate Dehydrogenase and Glucose Dehydrogenase
The most frequently used enzymatic systems for the regeneration of the reduced
form of nicotinamide cofactors are those based on the use of formate dehydrogenase
(FDH, EC 1.2.1.2) and glucose dehydrogenase (GDH, EC 1.1.1.47) in the presence
of their respective substrates, that is, formate and glucose. In fact, in the last
decades, they have been employed most in different reductive reactions, from a
laboratory scale up to an industrial scale [2].
Both systems share the advantage of using practically irreversible reactions
on cheap co-substrates, being thus suitable for strongly driving the reaction
equilibrium of reversible reactions, for example, those catalyzed by ADHs, toward
product formation under economically acceptable conditions.
Although the formate/FDH system is, in principle, the most attractive for
large-scale applications, the co-product carbon dioxide being very easily removed
from the reaction mixture, the catalytic and operational features of native FDH
enzymes are far from being optimal. In fact, they are usually characterized by a very
low specific activity, limited chemical and thermal stability, and strict preference
for the NADH cofactor. As these facts hamper the wide application of FDHs in
the development of novel industrial synthetic processes, a huge effort has been
carried out in the last years to improve the performance of this biocatalyst, mainly
by protein engineering [4].
The chemical stability of FDHs toward oxidative stress and reactive reagents
such as α-haloketones has been enhanced by site-directed mutagenesis of solvent-
accessible cysteine residues [5–7]. In some cases, for example, in the case of
Candida boidinii FDH, the introduced mutations resulted in a significant decrease