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10.1002/adsc.201801312
Advanced Synthesis & Catalysis
to drive the equilibrium reaction. Alcohol oxidases,
on the other hand, utilise molecular oxygen as
oxidant, yielding H2O2 as stoichiometric by-product
in an irreversible fashion. Hazardous hydrogen
peroxide can easily be dismutated using catalase.
Hence, AOx-catalysed oxidations appear more
suitable from an environmental point-of-view (i.e.
yielding less waste products) as compared to ADH-
catalysed ones. Therefore, we drew our attention to
the aryl alcohol oxidase from Pleurotus eryngii
(PeAAOx)[8].
ceased rather quickly due to acidification of the
aqueous layer caused by autohydrolysis.
We chose dodecane as organic phase. Figure 1
shows a representative time course of a reaction in
the biphasic reaction system. Pleasingly, full
conversion of the starting material into the desired
product (49 g l-1 organic phase) was observed within
24h. The nominal catalytic performance of PeAAOx
in the biphasic system (TF of 14.3 s-1 within the first
5 h) was somewhat lower compared to the
monophasic reaction (TF of 22.8 s-1, Figure S2),
which most likely is attributed to phase transfer
limitations of substrates (trans-hex-2-en-1-ol and
O2).[10] Comparative experiments revealed that O2
transfer in to the aqueous reaction buffer was overall
rate limiting (Figure S5).
The enzyme was heterologously expressed in
Escherichia coli, in vitro reactivated and purified (see
SI for a detailed description).
Both, the substrate and product of the reaction are
sparingly soluble in aqueous reaction mixtures (130
and 60 mM in the reaction buffer used here,
respectively). Therefore we evaluated the so-called
Here, a hydrophobic organic phase serves both, as
substrate reservoir and product sink enabling overall
high reagent loadings as demonstrated previously for
various reactions.[9]
Nevertheless, PeAAOx performed more than
650.000 catalytic turnovers corresponding to a
catalyst loading of less than 0,0002 mol-% or almost
900 gproduct g-1PeAAOx, respectively. The values for
catalase are 0,00002 mol-% and 8166 gproduct g-1
respectively.
,
Catalase
The 2LPS also contributes to minimise enzyme
inhibition by the product and undesired side reactions
of the aldehyde in the aqueous phase.
In a first set of experiments we further elucidated
the operational window for PeAAOx in terms of
optimal pH and temperature and mechanical and
solvent stability. Regarding the optimal pH, PeAAOx
is active in a broad pH range, displaying the highest
activity between pH 5 and 8 (Figure S1). Based on
these results, pH 7 was selected for further
experiments due to its compatibility with the activity
of catalase (required for the dismutation of H2O2).
PeAAOx exhibits the maximum activity at 30oC with
a turnover frequency of 25 s-1. Above this
temperature the activity dropped dramatically, with a
o
25 fold decrease at 40 C (TF < 1s-1, Figure S2). The
decrease of the activity at elevated temperatures is
most likely attributed to thermal denaturation of the
biocatalyst. We therefore conducted all further
experiments at 20oC as a compromise between high
activity and stability. 2LPSs are frequently plagued
by diffusion limitations over the phase border, which
can be addressed by e.g. vigorous mixing to increase
the surface area. We therefore investigated the
robustness of PeAAOx against mechanical stress
(Figure S3). Pleasingly, the enzyme was seemingly
not affected by high shaking velocities.
Figure 1. PeAAOx-catalysed oxidation of trans-2-hexen-
1-ol () to trans-2-hexen-1-al () using a biphasic
(2LPS) reaction system. Conditions: T= 20oC, shaking rate
= 1000 rpm; aqueous phase: 0.5 ml of 50 mM KPi (pH 7),
[PeAAOx] = 0.75 µM, [Catalase] = 720 U ml-1 (0.1 µM);
organic phase: 0.5 ml of dodecane, [trans-2-hexen-1-ol] =
500 mM; phase ratio: 1:1 (v/v).
Finally, also the stability of PeAAOx in the
presence of various organic solvents was determined
(Figure S4). Hydrophobic solvents such as isooctane
or dodecane were tolerated well by the enzyme and
initial rates up to 13 turnovers per second were
achieved. Using toluene gave no catalytic conversion
at all. Possibly, -stacking interactions of the
aromatic ring with the flavin prosthetic group resulted
in a strong competitive inhibition of PeAAOx.[8b]
Quite surprisingly, even ethyl acetate was tolerated
by PeAAOx as organic phase even though reactions
Another advantage of the 2LPS approach lies with
the facile downstream processing as simple phase
separation is sufficient to separate the dodecane-
product mixture (in case of full conversion as e.g.
shown in Figure 1) from the aqueous reaction buffer.
Chromatographic and/or destillative separation of the
o
solvent (dodecane, bp = 214 C) from the product
(trans-2-hexenal, bp = 145 oC) is straightforward.
2
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