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2.3 Conclusions 37

dehydrogenase (Ml-LPD), was used to regenerate NAD + [74]. However, only
preliminary information on the synthetic application of this system is presently
available, which does not allow drawing definite conclusions on its effectivity.
As an alternative, the use of suitable redox mediators has been suggested that
are not only good substrates for laccase but also capable of rapidly oxidizing
the reduced nicotinamide cofactors. A coupled system using the redox mediator
Meldola’s blue (7-dimethylamino-1,2-benzophenoxazine) is shown in Scheme 2.6c
[75]. In fact, this mediator has a very high bimolecular rate constant with NAD(P)H
s ) and is very quickly oxidized by laccases as well. Efficient NADH
(20 000 M −1 −1
regeneration was first demonstrated by the gram-scale oxidation of the bile
acid named cholic acid to its 7-keto derivative catalyzed by a NADH-dependent
7α-hydroxysteroid dehydrogenase in a buffered aqueous reaction system. Space-
time yields of 5.8 mmol l −1 h −1 were achieved in the quantitative conversion of a
50 mM substrate solution. Interestingly, the same regeneration system could also
be applied in a biphasic system using the methyl ester of cholic acid as a substrate
dissolved in isopropylacetate as the organic solvent. Owing to the higher concen-
tration of starting substrate (0.2 M), the space-time yields were improved up to
−1
20 mmol l −1 h . Satisfactory TTNs of 100 and 180 were estimated for the reactions
in the homogeneous aqueous system and the biphasic system, respectively.

2.3
Conclusions

As far as the in situ regeneration of reduced nicotinamide cofactors is concerned,
different alternative enzymatic systems have been thoroughly investigated during
the last years, thus providing a quite clear picture of the advantages and drawbacks
for each of them. Moreover, it has been shown that the performance of the
overall production process can be optimized only by explicitly considering the
two coupled reactions, that is, the synthetic and the regenerating reactions, in
an integrated way. Therefore, the choice of the most suitable system for any
synthetic application cannot be based merely on the costs of the regenerating
enzyme and the co-substrate, but must also consider possible complications in the
downstream procedures because of the presence of the co-product, or the need of
performing very fast production cycles to avoid product degradation. Moreover, the
development of new biocatalysts by protein engineering, through either rational
or directed evolution approaches, in many cases has allowed to overcome the
limitations experienced with wild-type enzymes. In particular, the specificity for
the NADH or NADPH cofactors does not currently represent a big issue. In fact,
it has been shown that the switch of cofactor specificity can be accomplished
by simply mutating one or a few selected amino acid residues. Moreover, the
increasing number of available gene sequences in the databases allows the rapid
discovery of homologous enzymes of interest showing the desired specificity.
On the contrary, NAD(P)H oxidation methods have been less extensively studied
in the past, and there is still room for improvement for the enzymatic regeneration