S. Kochius et al. / Journal of Molecular Catalysis B: Enzymatic 103 (2014) 61–66
65
of substrate although the overall reaction will be thermodynami-
cally favourable [23]. The only effective method for minimising the
effects of product inhibition is to remove the product from the reac-
tion system. To overcome the product inhibition continuous reactor
concepts, e.g. based on in situ product removal [24] or, after further
optimisation, the above mentioned immobilisation technique onto
the carrier Amberlite FPA 54 would be promising reaction systems.
Table 2 summarises results achieved by using different settings
and set-ups for the conversion of meso-2,3-butanediol into (R)-
acetoin with coupled electrochemical cofactor regeneration. Using
soluble ADH-9 was shown as even higher productivity compared
to using immobilised form (entries 1 and 2). In a simple glass reac-
2
−1
tor with an electrode surface-to-volume ratio of 0.1 cm mL an
increase of enzyme concentration had nearly no effect onto produc-
tivities (entries 2 and 3). Using the 3-dimensional electrochemical
with an adequate surface-to-volume ratio up to 6000 cm mL
improved productivities can be achieved.
−
1
Fig. 3. Conversion of meso-2,3-butandiol with 5 U mL soluble ADH-9 in electro-
2
−1
−
1
chemical reactor. Addition of enzyme (5 U mL ) after 12.5, 25 and 37.5 h. Value
of the pH was controlled and adjusted over the whole experiment. ᭹: Meso-2,3-
butandiol, ꢁ: (S)-Acetoin, ꢀ: (R)-Acetoin.
4. Conclusion
fold increased enzyme concentration was investigated (Table 2,
entry 3). The initial as well as the overall productivity was more or
less constant in the reactions. Therefore, it can be assumed that the
regeneration system was the limiting step of the reaction system.
One of the main features of electroenzymatic production systems
are limitations caused by the heterogeneous character of the elec-
trochemical step. An enhanced surface of electrode should improve
the cofactor regeneration. Therefore, a 3-dimensional packed bed
electrode was used as an electrochemical reactor. Initially, the con-
Oxidations are central transformations in organic chemistry.
In the past decades, tremendous advances in transition metal-
and organocatalytic oxidation and oxyfunctionalisations have been
achieved. Next to these, biocatalysis is emerging as additional
pillar for environmentally benign oxidation catalysis [25]. ADHs
are enjoying increasing interest as versatile and selective biocat-
alysts in the context of both, academic research and industrial
implementation [15]. On the way to the practical application of
ADH in preparative synthesis different topics must be addressed.
The identification of a suitable enantioselective enzyme is always
the starting point for the development of a novel bioprocess. In
the present study, an alcohol dehydrogenase was identified to
be (S)-selective in the oxidation of meso-2,3-butanediol to (R)-
acetoin. This provides an unique feature compared to published
conversions of 2,3-butanediol with dehydrogenases (Table 3). The
substrate of the bioconversion 2,3-butanediol is produced as a nat-
ural fermentation product by several bacteria and several species
of yeast. Product stereo specificity is found to vary from organ-
ism to organism [26]. K. pneumonia is known to produce a mixture
of meso- and (S,S)-stereoisomers whereas P. polymyxa produces
(R,R)-2,3-butanediol at optical purities as high as 98%. Nielsen et al.
−
1
version of meso-2,3-butanediol was driven by using 1 U mL sol-
−
1
uble ADH-9. The initial and average productivity were 4.8 mM h
−1
and 1.52 mM h (Table 2, entry 4). Compared to the reaction using
0 U mL ADH-9 in the standard electrochemical reaction cell the
−
1
1
productivity was even higher, although the enzyme concentra-
tion was 10 times lower by using the electrochemical flow cell
(Table 2, entries 3 and 4). This demonstrates that the overall reac-
tion in the standard cell was limited by the electrode surface. The
application of an electrochemical flow cell with enhanced elec-
trode area and an improved ratio between the reaction volume and
the electrode surface overcomes this limitation. In the next step a
slight improvement of the productivity was achieved with 5-fold
increased enzyme concentration (Table 2, entry 5). The electroen-
−
1
engineered an E. coli strain which was able to produce up to 1.1 g L
−
1
zymatic conversion in the electrochemical reactor with 5 U mL
meso-2,3-butanediol [26]. Production of meso-2,3-butanediol from
glucose was also reported in recombinant E. coli harbouring a
fragment of the K. pneuominae IAM 1063 genome encoding the
meso-2,3-butanediol biosynthetic pathway [27]. The application
soluble ADH-9 is shown in Fig. 3. Within 50 h 65% of the substrate
was converted to (R)-acetoin, resulting in a product concentration
of about 50 mM. The conversion demonstrates again the need for an
efficient regeneration system resulting in a driving force for the pro-
of an engineered E. coli resulted in product concentration up
−
1
−1
duction of (R)-acetoin. The initial productivity was 5.7 mM h and
to 15.7 g L
meso-2,3-butanediol [28]. The discovery of the (S)-
−1
the average productivity was 2 mM h . The decreasing substrate
concentration fits well with the time course of the produced (R)-
acetoin. After 12.5 h the product formation stopped. It was assumed
that this might be due to enzyme degradation, so additional enzyme
was dosed into the reaction system. Immediately product forma-
tion proceeded. In contrast to the results regarding the stability of
the enzyme found in the standard cell the enzyme was shown to
be merely stable up to a particular time. As the enzyme degraded
after 12.5 h fresh enzyme was added each 12.5 h. A slight chemical
racemisation of the product into the (S)-enantiomer was observed.
The optically active acetoin undergoes racemisation at a basic pH
selective enzyme enables a novel production process of (R)-acetoin
from meso-2,3-butanediol. Admittedly, the produced acetoin con-
centration of 50 mM is much lower as the concentrations which can
be reached in fermentative processes [3–5]. However, the enzy-
matic route facilitates the production of a valuable enantiopure
product. Certainly, a production process of (R)-acetoin with whole-
cell system over expressing a meso-2,3-butanediol dehydrogenase
would be possible. Due to the cofactor challenge, possible formation
of side-products as well as back reactions a process with isolated
enzymes seems to be more convenient.
The oxidation of reduced cofactors NADH is a key point in
preparative application of dehydrogenase for alcohol oxidation
[30]. Besides the economical and ecological impact of a cofactor
regeneration system, most enzymatic oxidations are thermody-
namically unfavourable. Only efficient cofactor regeneration can
shift the ratio of the oxidised and reduced cofactor in the direction
of NAD+ and therefore enable a preparative oxidation process. The
applied electrochemical reaction system was shown to be a suitable
[
22]. As the product formation decreased after 35 h and an addition
of fresh enzyme had nearly no effect as well as protein denat-
uration was not observed a product inhibition was assumed. To
+
investigate an inhibition an NAD -assay with 50 mM acetoin was
performed. In this assay no cofactor conversion takes place. This
product inhibition cannot be overcome through a further increase
of the cofactor regeneration system or by higher concentrations