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34 2 New Trends in the In Situ Enzymatic Recycling of NAD(P)(H) Cofactors

as regenerating enzyme in coupled oxidation reactions are currently available, thus
making a complete evaluation of its applicative potential difficult. Also, the NOX
from Lactobacillus plantarum presents promising features, such as being active and
stable over a broad pH range [63]. Moreover, this enzyme was engineered in order
to accept and oxidize NADPH as well as NADH. However, also in this case, the
exploitation of this novel NOX in recycling the oxidized nicotinamide cofactors has
not been deeply investigated up to now.
More information is available instead for the water-forming NOX from Strep-
tococcus mutans (Sm-NOX2) [51, 66]. In fact, engineered enzyme variants capable
of oxidizing both NADH and NADPH have been recently patented by DSM
[67]. Specifically, NAD(P)H oxidation catalyzed by the best Sm-NOX2 mutant
(Asp194His/Gly200Lys) was coupled to the enantioselective oxidation of 50 mM
(RS)-1-phenylethanol catalyzed either by the R-specific NADPH-dependent ADH
from L. brevis or by the S-specific NADH-dependent ADH from Candida parapsilo-
sis. In both cases, using a cofactor concentration of 1 mM, the reaction was almost
complete in 25 h and TTNs for the cofactor were around 24.
Interestingly, about 10-fold higher TTN values were obtained when using the
NOX from T. kodakarensis in the same reaction [48]. Specifically, the enantiose-
lective oxidation for resolution of racemic 1-phenylethanol was catalyzed by either
the R-specific NADPH-dependent ADH from Lactobacillus kefir (Lk-ADH) or the
S-specific NADH-dependent ADH from Rhodococcus erythropolis (Re-ADH). In this
study, different substrate, cofactor and enzyme concentrations, and temperatures
were tested in order to optimize the reaction conditions. Best results were obtained
by using Re-ADH that achieved complete oxidation of the (S)-enantiomer to the
corresponding ketone starting from a 120-mM racemic 1-phenylethanol solution.
◦
The reaction carried out in the presence of 0.2 mM cofactor at 45 C resulted in
a TTN for the cofactor of almost 300. The performance of reactions catalyzed by
Lk-ADH at a substrate concentration of 20 mM and using a cofactor concentration
of 2 mM was far less satisfactorily (TTN = 5).

2.2.2.3 Alcohol Dehydrogenase
Concerning the exploitation of the substrate-coupled strategy in ADH-catalyzed
oxidation reactions, the complete oxidation of the majority of the substrates of
interest is again hampered by equilibrium issues. However, a quasi-irreversibility
of the coupled reduction reaction was recently achieved by using small ketones
bearing electron-withdrawing groups (EWGs), such as chloroacetone or methyl
acetoacetate, as co-substrates (Scheme 2.5) [68, 69]. It has been subsequently sug-
gested that this may be because of the stabilization of the formed alcohols by
strong intramolecular interactions with the formation of a H-bonding network
[70]. Moreover, as the co-substrates are required in at least stoichiometric amounts
to achieve complete conversions of the target substrate, this strategy concomi-
tantly leads to a significant reduction of consumed organic reagents and formed
by-products.