Page 53 - Cascade_Biocatalysis_Integrating_Stereoselective_and_Environmentally_Friendly_Reactions

2.2 Advances in Cofactor Regeneration 29

acted as a conducting surface for the electron exchange, and generation of NADH
in the presence of H was demonstrated by the formation of lactate from pyruvate.
2
Although the results shown in this study provide only a proof-of-principle
demonstration of such a system, it may be foreseen that, given the availability
of several characterized hydrogenases [33], new hydrogenase–diaphorase cofactor
regeneration particles will be investigated in the future.
For example, a ferredoxin hydrogenase (EC 1.12.7.2) has been isolated recently
from the hyperthermophile Pyrococcus furiosus [38]. The performance of this
biocatalyst, which showed a remarkable stability under operative conditions, has
been investigated for the NADPH regeneration in the reduction of prochiral ketones
catalyzed by the thermophilic NADPH-dependent ADH from Thermoanaerobium
+
sp. Total turnover numbers (TTNs: mole product/mole consumed cofactor NADP )
of 100 and 160 could be estimated in the reduction of acetophenone and (2S)-
hydroxy-1-phenyl-propanone, respectively. As a side note, it should be mentioned
that, although the activity of the P. furiosus hydrogenase increased exponentially
◦
with temperature up to its maximum above 80 C, the reactions had to be performed
◦
at much lower temperature (40 C) because of the thermal instability of NADPH.
2.2.1.4 Glucose 6-Phosphate Dehydrogenase
The regeneration system based on the C1 oxidation of glucose 6-phosphate
(G6P) catalyzed by glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49),
forexample,the onefrom Leuconostoc mesenteroides, has been widely used in the
past on a laboratory scale. This was mainly due to the very high specific activity
−1
shown by this enzyme (≥550 U mg ) and due to its natural selectivity for the
+
reduction of the NADP cofactor. However, this enzymatic activity was never
applied in large-scale biocatalyzed reduction processes owing to both the high cost
of the substrate G6P and to the fact that the enzyme catalyzes the decomposition
of NAD(P)(H) at high concentrations [2].
In addition to the previously investigated methods for the in situ generation of
G6P in G6PDH recycling systems [39], an interesting cascade process compris-
ing the acid phosphatase from Shigella flexneri (Sf -Pho) was recently suggested
(Scheme 2.4) [40].
Although it is questionable whether this approach may be competitive to the sim-
pler glucose/GDH system, the three-enzyme one-pot cascade for the stereoselective
reduction of ketones catalyzed by an ADH, L. mesenteroides G6PDH and Sf -Pho,
showed a quite good efficiency, with TTNs for NADPH up to 3120 starting from
the cheap co-substrates: glucose and pyrophosphate (PP ). Interestingly, due to the
i
phosphate cycling shown in Scheme 2.4, only a limited amount of PP (apparently
i
only 1 equiv with respect to the Sf -Pho catalyst molecules) was needed initially to
phosphorylate the enzyme in order to start the reaction.

2.2.1.5 Alcohol Dehydrogenase
As previously mentioned, an ADH can be used for the in situ recycling of NAD(P)H
cofactors by exploiting the so-called substrate-coupled approach, that is, the coupling
of the reaction of interest with a secondary reaction running in the reverse direction