Photoenzymatic Hydroxylation of Ethylbenzene Catalyzed by Unspecific Peroxygenase: Origin of Enzyme Inactivation and the Impact of Light Intensity and Temperature

Article abstract of DOI:10.1002/cctc.201900610

Photoenzymatic cascades can be used for selective oxygenation of C?H-Bonds under mild conditions circumventing the hydrogen peroxide mediated peroxygenase inactivation via in situ H₂O₂ generation. Here, we report the “on demand” production of hydrogen peroxide via methanol assisted reduction of molecular oxygen using UV-illuminated titanium dioxide (Aeroxide P25) combined with the enantioselective hydroxylation of ethylbenzene to (R)-1-phenylethanole catalyzed by the Unspecific Peroxygenase from Agrocybe Aegerita. For the application of the system it is important to understand the influence of the reaction parameters to be able to optimize the system. Therefore, we systematically investigated product formation and enzyme inactivation as well as ROS formation (H₂O₂, ^.OH and ^.O₂^?) applying different light intensities and temperatures. As a result, Turnover Numbers up to 220 000, photonic efficiencies up to 13.6 % and production rates up to 0.9 mM h^?1 were achieved.

Full text of DOI:10.1002/cctc.201900610

Accepted Article
Title: Photoenzymatic Hydroxylation of Ethylbenzene catalyzed by
Unspecific Peroxygenase: Origin of Enzyme Inactivation and the
Impact of Light Intensity and Temperature
Authors: Bastien Oliver Burek, Sabrina Rose de Boer, Florian Tieves,
Wuyuan Zhang, Morten van Schie, Sebastian Bormann,
Miguel Alcalde, Dirk Holtmann, Frank Hollmann, Detlef W.
Bahnemann, and Jonathan Z. Bloh
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Photoenzymatic Hydroxylation of Ethylbenzene catalyzed by
Unspecific Peroxygenase: Origin of Enzyme Inactivation and the
Impact of Light Intensity and Temperature
Bastien O. Burek,+*[a,b] Sabrina R. de Boer,+[a] Florian Tieves,[c] Wuyuan Zhang,[c] Morten van Schie,[c]
Sebastian Bormann,[a] Miguel Alcalde,[d] Dirk Holtmann,[a] Frank Hollmann,[c] Detlef W. Bahnemann[b,e]
and Jonathan Z. Bloh*[a]
Abstract: Photoenzymatic cascades can be used for selective
oxygenation of C-H-Bonds under mild conditions circumventing the
hydrogen peroxide mediated peroxygenase inactivation via in situ
H2O2 generation. Here, we report the “on demand” production of
hydrogen peroxide via methanol assisted reduction of molecular
oxygen using UV-illuminated titanium dioxide (Aeroxide P25)
combined with the enantioselective hydroxylation of ethylbenzene to
(R)-1-phenylethanole catalyzed by the Unspecific Peroxygenase
from Agrocybe Aegerita. For the application of the system it is
important to understand the influence of the reaction parameters to
be able to optimize the system. Therefore, we systematically
investigated product formation and enzyme inactivation as well as
For the selective oxyfunctionalization of C-H- and C=C-bonds,
Unspecific Peroxygenases (UPOs, E.C. 1.11.2.1) are promising
enzymes catalyzing a variety of oxidation reactions[4–10] like
selective hydroxylations,[11–14] which can be used for example for
the synthesis of human drug metabolites.[15–17] Particularly, the
UPO from Agrocybe aegerita (AaeUPO)[9] available from
recombinant fermentation in Pichia pastoris[18,19] is an attractive
catalyst.
However, the production of AaeUPO is still economically and
environmentally challenging, therefore high enzymatic turnovers
are necessary to create truly green processes.[20] This is
particularly hampered by the instability of UPOs against their
own co-substrate H2O2.[7,21]
-
ROS formation (H2O2, •OH and •O2 ) applying different light
intensities and temperatures. As a result, Turnover Numbers up to
220 000, photonic efficiencies up to 13.6 % and production rates up
to 0.9 mM h-1 were achieved.
Many strategies are known for a H2O2 supply in adequate
concentrations, especially in situ generation by reduction of
molecular oxygen is often applied e.g. via chemical,[22,23]
enzymatic,[24–29] electrochemical[30–36] or photochemical[37–45]
approaches to alleviate the instability issue, see Table S4 for a
detailed comparison of the methods on the example of AaeUPO
catalyzed hydroxylation of ethylbenzene.
Introduction
The heterogeneous photocatalytic approach has the advantage
that it is utilizing light as the only energy source and the catalyst
can be easily separated after the reaction. Furthermore, the
maximum possible H2O2 concentration is limited intrinsically by
the equilibrium between photocatalytic generation and
degradation, cf. Scheme 1, which creates an additional safety
margin against peroxide runaway.[46] However, the enzyme
performed poorly with only low turnovers when water was
employed as the electron donor.[38] Introduction of methanol as
sacrificial electron donor and radical scavenger dramatically
enhanced the enzyme stability.[37]
Enzymes are typically the catalyst of choice for atom economic
and highly selective reactions and offer a nature-inspired
approach for developing sustainable synthetic routes.[1] Process
development and enzyme evolution already led to many novel
industrial applications.[2,3]
[a]
Dr. J. Z. Bloh, B. O. Burek, S. R. de Boer
Chemical Technology Group and
Dr. D. Holtmann, S. Bormann
Industrial Biotechnology Group,
DECHEMA Forschungsinstitut,
Theodor-Heuss-Allee 25, 60486 Frankfurt am Main (Germany)
E-mail: bloh@dechema.de, burek@dechema.de
Prof. Dr. D. W. Bahnemann, B. O. Burek
Institut für Technische Chemie
[b]
[c]
[d]
[e]
[+]
Leibniz Universität Hannover
Callinstraße 3, 30167 Hannover (Germany)
Dr. F. Hollmann, M. van Schie, F. Tieves, W. Zhang
Department of Biotechnology
Delft University of Technology
Van der Maasweg 9, 2629HZ Delft (The Netherlands)
Dr. M. Alcalde
Department of Biocatalysis
Institute of Catalysis, CSIC
28049 Madrid (Spain)
Prof. Dr. D. W. Bahnemann
Scheme 1. Reactions occurring during the methanol assisted photocatalytic
reduction of molecular oxygen to hydrogen peroxide over UV-illuminated TiO2
in aqueous phase.
Laboratory “Photoactive Nanocomposite Materials”
Saint-Petersburg State University
Ulyanovskaya str. 1, Peterhof, Saint-Petersburg, 198504 (Russia)
These authors contributed equally to this work.
Supporting information for this article is given via a link at the end of
the document.
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As shown in Scheme 1 there are several radical intermediates
appearing during the photocatalytic reaction as a result of the
one-electron transfers. Since those reactive oxygen species
(ROS) are known to affect the stability of enzymes in
complete enzyme inactivation within two hours. Adding the
photocatalyst provokes an even faster decay of active enzyme.
However, the addition of an alcohol, e.g. 2-propanol or methanol,
reduces the inactivation rate drastically. In both cases (with and
without an alcohol) the inactivation occurs slower in the
presence of ethylbenzene as a substrate. Nonetheless, even
with an added alcohol and the substrate the inactivation is still
about four times faster compared to the deactivation observed
with the enzyme stirred in the dark.
general,[47,48]
a
detailed study of the interaction in
photoenzymatic reactions might lead to an understanding of how
to counteract the inactivation.
In this study we focus on the optimization of the photoenzymatic
system on the basis of our previous work on the photocatalytic
H2O2 production using pure titanium dioxide without noble
metals. As a model reaction the methanol assisted selective
hydroxylation of ethylbenzene catalyzed by AaeUPO is studied,
cf. Scheme 2. Methanol is an attractive electron donor since
formaldehyde and formate are oxidized very fast,[49] and
therefore gaseous CO2 is the only significant side product which
leaves the reaction mixture and does not further complicate the
downstream processing. The enzymatic reaction is studied very
well and therefore allows for a good comparison.[24,26,30,37–40,50]
1.0
0.8
0.6
no UV
only UV
TiO2
0.4
0.2
0.0
TiO2+S
TiO2+iPrOH
TiO2+iPrOH+S
TiO2+MeOH+S
OH
TiO2, h
AaeUPO
CO2
O2
H O
CH3OH
+
+
2
0
1
2
3
4
5
6
+
+
time / h
Scheme 2. Reaction equation of the photoenzymatic hydroxylation of
ethylbenzene catalyzed by a cascade of TiO2 and AaeUPO.
Figure 1. Time courses of the residual enzyme activity towards ABTS during
different conditions: 100 mM phosphate, pH 7, 100 nM AaeUPO, stirred in the
dark (▲); all other experiments were irradiated (365 nm, 656 µE L-1 min-1), no
TiO2 (★); 0.1 g L-1 TiO2 (■); 10 vol.-% 2-propanol, 0.1 g L-1 TiO2 (▼); 10 vol.-
% 2-propanol, 0.1 g L-1 TiO2, 10 mM ethylbenzene (♦); 10 vol.-% methanol,
0.1 g L-1 TiO2 (●) and 200 nM AaeUPO, 2 g L-1 TiO2, 10 mM ethylbenzene(◄).
With the systematic investigation of the formation rates of
hydrogen peroxide, hydroxyl- and superoxide radicals we
provide insights about the complex relationship between
photocatalytic enzyme inactivation and photoenzymatic
production. Furthermore, detailed analysis of the reaction
conditions, in particular light intensity, temperature and enzyme
concentration, is used to show how the process parameters
turnover number, production rate and efficiency can be
optimized.
(UV: UV-light, S: substrate
= ethylbenzene, iPrOH: 2-propanol, MeOH:
methanol) The corresponding inactivation rates are shown in Table S1.
Presumably, the direct inactivation by ultraviolet light is strongly
suppressed in the presence of TiO2 as due to its high extinction
coefficient the light is absorbed almost immediately[51] and the
enzyme is effectively shielded from the radiation. Nonetheless,
the radiative deactivation mechanism cannot be ruled out
completely. However, the three-fold increase in inactivation rate
Results and Discussion
observed upon addition of TiO2 strongly implies that
photocatalytic process is responsible.
a
The photocatalytic H2O2 production with TiO2-based catalysts
results in low equilibrium concentrations which are ideal for a
coupled process with peroxygenases. However, previously
reported photoenzymatic coupling of Au-TiO2 with AaeUPO
showed unexpectedly high enzyme inactivation which cannot be
solely explained by H2O2-based inactivation as the delivered
concentration is too low.[37,38]
To identify reasons for this we investigated the inactivation of
AaeUPO in more detail using pure TiO2 (Aeroxide P25®, Evonik)
as a photocatalyst. For this purpose, the residual enzyme
activity towards 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic
acid) (ABTS) has been measured under different conditions over
time, cf. Figure 1.
As already stated during the photocatalytic hydrogen peroxide
generation, hydroxyl and superoxide radicals may be formed
which are very strong oxidants. The enzyme inactivation rate
linearly increases with the employed photon flux, further
confirming that light associated processes are responsible (cf.
Figure 2). Unfortunately, all three candidates for the inactivation,
UV light, hydroxyl radicals (•OH) and superoxide radicals (•O2 )
-
increase linearly with the light intensity, making an unambiguous
correlation impossible. However, while the superoxide formation
rate surpasses the deactivation rate by more than three orders
•
of magnitude, OH formation and enzyme deactivation rates are
While the enzyme remains quite stable stirred in the dark, UVA-
irradiation (365 nm) of the solution without photocatalyst leads to
more closely correlated. This can be exemplified by their ratio
which is relatively low and approximately constant
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(kF(•OH)/rdeact = 5) over the whole investigated range of light
intensity.
compared to methanol oxidation, while the enzyme is inactivated
five times faster in the former case. Thus, •O2- seems to have no
significant impact on the enzyme deactivation in this reaction.
This also explains the observation that a reduction of the
superoxide concentration in the reaction through the use of
superoxide dismutase (SOD) did not improve the overall enzyme
stability.[38] Additionally, as the light intensity is the same in both
cases, yet the inactivation differs by a factor of almost six, direct
inactivation by UV light could only account for a minor
1400000
-
kF ( •O2
)
1200000
1000000
800000
600000
400000
200000
kdeact (AaeUPO)
kF (•OH)
•
contribution. So, OH seem to be damaging and inactivating the
enzyme, as there is a good correlation between the inactivation
rates and the corresponding •OH formation rates. Those radicals
are also attributed to oxidize the heme in the H2O2 mediated
inactivation mechanism of AaeUPO.[21] It should also be noted
that direct transfer of photogenerated holes from TiO2 to the
enzyme followed by water addition yields the same result as an
hydroxyl radical attack and is therefore virtually indistinguishable
in aqueous media. However, due to steric effects a direct
photocatalytic oxidation should predominantly take place at the
enzyme´s side chains, while •OH might also directly attack within
the enzyme´s active site.
In addition to the inactivation rate, also the productivity is of high
importance for an efficient process. Both parameters in
conjunction govern the enzymatic TON that can be achieved in a
given system. As methanol showed the best performance
regarding both parameters (cf. Figures S11-13), further
optimization strategies were carried out using this alcohol.
200
150
100
50
0
0
1000
2000
3000
4000
5000
6000
photon flux density / µE L-1 min-1
Figure 2. Comparison of AaeUPO deactivation rates (♦, a) with hydroxyl (■,
b) and superoxide (●, c) generation rates in dependence of the light intensity.
Reaction conditions: 5 vol.-% methanol, 2 g L-1TiO2, 0.1 M phosphate, pH 7,
25 °C and (a): 10 mM ethylbenzene, 200 nM AaeUPO, (b): 0.1 M coumarin or
(c): 0.5 mM NBT.
Furthermore, if an alcohol is added, it acts as a sacrificial
electron donor (further called sacrificial reagent, SR)
competitively suppressing the water oxidation, cf. Scheme 1.
•
In a next step, we wanted to intensify the reaction to increase
the overall productivity. We could show earlier that H2O2
formation can be directly manipulated by the incident photon
flux.[51] Consequently we investigated the effect of light intensity
on the photoenzymatic reaction. As already mentioned, the
enzyme inactivation rate linearly increases with the employed
photon flux (cf. Figure 3).
This selectively inhibits the formation of OH radicals, which are
an intermediate of the water oxidation reaction. Alcohols are
also known to act as radical scavengers.[52] This justified a more
precise investigation of the radicals forming during the
photocatalytic process using different electron donors to identify
the main culprit for the enzyme inactivation (cf. Table 1).
Fortunately, the formation rate of the desired product (R)-1-
phenylethanol also rises with the light intensity. In accordance
with the higher H2O2 generation rate the product formation
increases linearly up to a photon flux of 656 µE L-1 min-1 and
reached to its maximum at approximately 2223 μE L-1 min-1 as
shown in Figure 3. Above this light intensity, the product
formation rate stagnates even though the H2O2 generation still
increases further beyond this point. Consequently, both the
photonic efficiency and the H2O2 efficiency begin to decline at
higher light intensity. The exponential losses regarding photonic
efficiencies can be attributed to the inhomogeneous light
distribution inside the reactor, which we explained earlier for
photocatalytic H2O2 generation.[51] In the photoenzymatic
reactions these losses are even more pronounced due to the
stagnation of product formation at high light intensities. So, there
has to be a compromise between productivity and (photonic and
enzymatic) efficiency to find the optimal light intensity. However,
in the presented system employing a photon flux above
2223 µE L-1 min-1 is a waste of energy. Since the enzyme
degradation still continues to increase linearly, the stagnation of
Table 1. Effect of different electron donors on the photoenzymatic system and
ROS generation. Reaction conditions: 5 vol.-% alcohol (in case of water as e--
donor no alcohol), 2 g L-1 TiO2, 0.1 M phosphate, pH 7, 25 °C. (PE: (R)-1-
phenylethanol, n.d.: not determined)
[a]
-
kF(PE)[a]
/ mM h-1
kdeact
kF(•OH)[b]
/ nM h-1
TON[a]
kF(•O2 )[b]
e--donor
/ nm h-1
/ nM h-1
2.8 ∙105
11.4 ∙105
n.d.
water
0.20
0.46
0.33
0.46
52
9
732 000
240
4 000
40 000
30 000
31 000
methanol
2-propanol
t-butanol
12
17
220
530
n.d.
[a] 200 nM AaeUPO, 656 µE L-1 min-1. [b] for a more precise result the radical
formation was investigated at a higher light intensity of 5934 µE L-1 min-1.
The fastest inactivation is observed in the case of water
oxidation which also coincides with the highest generation rate
of hydroxyl radicals. On the contrary, the formation rate of
superoxide seems to behave inversely proportional to the
•
inactivation rate. In case of water oxidation, less O2- is detected
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productivity lead to a pronounced decrease in the TON with
higher photon flux after the abovementioned inflection point.
the peroxide concentration should be higher as a result of its
overproduction which should lead to higher enzyme reaction
rates. Nonetheless, to exclude a limitation of the enzyme
concentration here, we carried out reactions varying the enzyme
concentrations at three different light intensities (cf. Figure 4).
3.0
kF ((R)-1-Phenylethanol)
At low light intensity (22 µE L-1 min-1) the product formation
corresponds directly to the respective H2O2 formation rate in the
investigated range of enzyme amount, indicating that here, the
reaction is entirely limited by the amount of supplied peroxide.
However, at higher photon fluxes the product formation rate
never reaches the H2O2 formation rate. Initially, the product
formation rate increases linearly with the enzyme concentration
up to about 50 nM, thereafter no further significant change is
observed. At this enzyme concentration, the product formation
rate plateaus at approx. 0.9 mM h-1 for both higher light
intensities studies. This appears to be invariant of the H2O2
generation rate which differs by about 50% for those two cases.
In this regime the reaction appears to be neither controlled by
the amount of available peroxide nor by the enzyme
concentration. The reasons for this limit of productivity remain
unknown for now.
In line with the productivity increases, likewise the photonic
efficiency can be increased by using higher enzyme
concentrations up to the aforementioned limit of about 50 nM.
However, the overall photonic efficiency is predominantly
governed by the efficiency of H2O2 generation which is most
efficient at lower light intensity due to kinetic constraints in the
"bright spots" that appear at high light intensity.[51]
40
kF (H2O2)
kdeact (AaeUPO)
2.5
2.0
1.5
1.0
0.5
0.0
30
20
10
0
0
1000
2000
3000
4000
5000
6000
photon flux density / µE L-1 min-1
100
80
60
40
20
0
maximal possible TON
TON
14
12
10
8
50000
40000
30000
20000
10000
0
H2O2-efficiency
photonic efficiency
6
4
2
0
At all three studied light intensities the enzyme deactivation rises
linearly with the enzyme concentration. Since the inactivation is
likely caused by hydroxyl radials whose concentration is
constant at a given light intensity this indicates a second order
inactivation reaction, linearly dependent on both the
concentrations of the enzyme and hydroxyl radicals. A higher
enzyme concentration (above 50 nM) therefore also leads to
significantly lower TONs, as this only increases the enzyme
inactivation rate but not the overall product formation rate. To
reach high TON a good balance between H2O2 generation and
enzyme amount is necessary, and neither may exceed the
respective limits of effect. This is illustrated in Figure 4 which
shows that the highest TON was reached at a moderate light
intensity (moderate H2O2 generation) and low enzyme
concentration (5 nM AaeUPO).
0
1000
2000
3000
4000
5000
6000
photon flux density / µE L-1 min-1
Figure 3. Reaction data determined through variation of the light intensity.
AaeUPO deactivation rates (♦, a), hydrogen peroxide (■) and product (●, a)
generation rates in dependence of the light intensity (left) and derived turnover
numbers (★), hydrogen peroxide efficiency (◄) and photonic efficiency of
product formation (▲) (right). Reaction conditions: 5 vol.-% methanol, 2 g L-1
TiO2, 0.1 M phosphate, pH 7, 25 °C and (a) 10 mM ethylbenzene, 200 nM
AaeUPO. The maximal possible TON during these experiments derives from
the substrate concentration of 10 mM.
The observed product formation rates of up to about 0.9 mM h-1
are far below the catalytic turnover of the enzyme at that
concentration (TOF=410 s-1 resulting in 295 mM h-1).[13] This will
of course be somewhat diminished by the low equilibrium
concentrations of H2O2. However, at the higher light intensities
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5802 µE L-1 min-1
2223 µE L-1 min-1
22 µE L-1 min-1
2.4
kF(H2O2) @ 5802 µE L-1 min-1
40
30
20
10
0
5802 µE L-1 min-1
2223 µE L-1 min-1
22 µE L-1 min-1
2.0
1.6
1.2
0.8
0.4
0.0
kF(H O ) @ 2223 µE L-1 min-1
2
2
kF(H O2) @ 22 µE L-1 min-1
2
0
100
200
300
400
0
100
200
300
400
c(AaeUPO) / nM
c(AaeUPO) / nM
250000
200000
150000
100000
50000
0
5802 µE L-1 min-1
2223 µE L-1 min-1
22 µE L-1 min-1
5802 µE L-1 min-1
2223 µE L-1 min-1
22 µE L-1 min-1
10
1
0,1
0
100
200
300
400
0
100
200
300
400
c(AaeUPO) / nM
c(AaeUPO) / nM
Figure 4. Reaction data determined through variation of the enzyme
concentration at three different light intensities: 5802 µE L-1 min-1 (■),
2223 µE L-1 min-1 (●) and 22 µE L-1 min-1 (▲). Reaction Conditions: 5 vol.-%
methanol, 2 g L-1 TiO2, 0.1 M phosphate, pH 7, 25 °C and 10 mM
ethylbenzene.
productivity are contradictory. For an overall efficient process a
pareto optimization is necessary in order to find an acceptable
compromise.
Another very important parameter in photoenzymatic reactions is
the temperature, as the photocatalytic H2O2 productivity in the
presence of an alcohol can be increased by heating the
reactor.[51] This can be directly attributed to the better mass
transport from and to the photocatalysts surface as well as
improvements in the charge transfer kinetics. The product
formation rate in the photoenzymatic system is also increasing
with the temperature, even over-proportionally in comparison
with the H2O2 formation (cf. Figure 5). Therefore, the photonic
efficiency increases as well. However, while at low temperatures
full conversion has been reached and the enzyme was still
active, verified by ABTS activity and further product formation
after addition of the substrate, at high temperatures the yield
dropped to 30% at 50°C (see Table S2 and Figure S4 for further
details). This can be attributed to increased thermal
inactivation[18] of the enzyme which increases slightly with the
temperature up to 40 °C and abruptly rises above this
temperature. Consequently, the TON slightly decreases with the
temperature and sharply drops above 40 °C.
These findings highlight that unfortunately, the conditions
required for high photonic efficiency, high enzyme TON and high
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250000
200000
150000
100000
50000
0
0.9
80
70
60
50
40
30
20
10
0
kF ((R)-1-Phenylethanol)
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
kF (H2O2)
kdeact (AaeUPO)
0
50000
100000
150000
200000
250000
0
10
20
30
40
50
experimentally determined TON
temperature / °C
Figure 6. Comparison of the experimentally determined turnover number with
the estimated turnover number calculated by the fraction of product generation
and enzyme deactivation rates. The dotted line marks the angle bisector
where estimated and determined TON are equal.
6
5
4
3
2
1
0
60000
50000
40000
30000
20000
10000
0
Even though the theoretical TON is slightly overrated, it is
possible to estimate the TON of the system with acceptable
precision within the first hours of the reaction with this simple
approximation. This will facilitate a faster screening for optimized
reaction conditions.
TON
photonic efficiency
0
10
20
30
40
50
temperature / °C
Conclusions
Figure 5. Reaction data determined through variation of the temperature.
AaeUPO deactivation rates (♦, a), hydrogen peroxide (■) and product (●, a)
generation rates in dependence of the temperature (left) and derived turnover
numbers (★) and photonic efficiency of product formation (▲) (right). Reaction
conditions: 5 vol.-% methanol, 2 g L-1TiO2, 0.1 M phosphate, pH 7, 696 µE L-
1 min-1 and (a) 10 mM ethylbenzene, 200 nM AaeUPO.
In the present study the inactivation pathways of AaeUPO in a
photoenzymatic reaction with TiO2 as photocatalyst were studied
in detail. While the observed inactivation rate is the sum of many
effects such as temperature and UV radiation, the main
degradation pathway was identified to be associated with photo-
generated hydroxyl radicals. Since these are mainly formed as
an intermediate in the oxidation of water, using an alternative
electron donor such as an alcohol strongly suppresses the
hydroxyl radical formation and increases enzyme stability.
An even more pronounced picture of the interplay between
productivity and inactivation can be demonstrated by their ratio
(kF(PE) / kdeact(AaeUPO)), which represents a kind of theoretical
or estimated TON. As shown in Figure 6 there is a good
correlation between the experimentally determined TON and the
theoretical TON. In most of the experiments the theoretical TON
slightly overestimates the reached TON. This is plausible as the
productivity gets lower if the level of the active enzyme is below
a certain concentration (e.g. below 50 nM for the above
presented moderate and high light intensities, cf. Figure 4).
Hence, the ratio of product formation rate and the enzyme
deactivation rate is only equal to the TON in the linear regime of
product formation assuming a linear degradation which was
observed in all reactions.
Furthermore, the effects of light intensity and temperature on the
system were evaluated. While an increase in both of these
parameters positively affects the photocatalytic H2O2 generation
rate, this cannot be fully exploited in the photoenzymatic system
at the moment as also the hydroxyl radical mediated inactivation
rises with the light intensity while at the same time the
effectiveness of the enzyme to utilize the higher peroxide
generation diminishes. Thermal inactivation at elevated
temperatures limits that strategy as well. Both of these
limitations will be faced in the future with optimized
photocatalysts and via immobilization of the enzyme.
Generally, a good harmonization between the photo and the
enzymatic system is necessary to improve the overall
performance, however there will always have to be
a
compromise between productivity, enzymatic stability and
efficiency.
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Agilent) for the separation. To determine the concentrations, 150 μL
samples of the reaction mixture were taken at defined reaction times.
Subsequently, the samples were extracted three times each with 50 μL
ethyl acetate containing 5 mM dodecane as internal standard. The
concentrations were calculated according to calibration with authentic
standards using the same extraction method.
Nonetheless, we could reach turnover numbers of up to 220 000,
space time yields of up to 2.6 g L-1 d-1 and photonic efficiencies
of up to 13.6 % which are a promising improvement compared to
the state of the art and present a new benchmark for this
reaction with photoenzymatic systems. Although the
performance remains still slightly below enzymatic or
electrochemical H2O2 production, the presented results built the
base for further optimization on the way to an industrial
applicability of photoenzymatic reactions.
Enzyme Activity Measurement
The relative activity of AaeUPO was determined using the oxidation of
ABTS to its green radical which can be analyzed via UV-Vis (λ = 405 nm).
Current drawbacks like the illumination and scalability issues
can be addressed by the use of flow-reactors[53] or internal
illumination with wirelessly powered light devices.[54] Future
studies will also focus on photocatalysts with less OH radical
production to enhance the overall enzyme stability.[55]
A
mixture of 20 μL H2O2 (0.3 mM) and 200 μL ABTS (2 mM) in
phosphate buffer (0.1 M, pH 5) were added to 20 μL of the supernatant of
a centrifugated sample from the reaction mixture. Immediately after the
addition the change of absorbance over the time was measured in a
microplate reader (PowerWave HT, BioTek) and the initial linear regime
was analyzed.
•
Quantification of ROS
Experimental Section
Hydrogen peroxide was quantified using the HRP-catalyzed
Chemicals
stoichiometric dimerization of POHPAA which yields
a fluorescent
product.[56] In defined temporal intervals samples were taken from the
illuminated reaction suspension and filtered through a syringe filter
(0.2 µm, PVDF, Roth) to remove the TiO2 particles. Lyophilized powder
of HRP (1 mg, 163 u mg-1) and freshly recrystallized POHPAA (4 mg)
were both dissolved in TRIS buffer (12.5 mL, 1.0 M, pH 8.8) respectively.
A 100 µL H2O2-containing sample was mixed with 12.5 µL of each
solution for 30 min and the fluorescence signal (λex = 315 nm,
λem = 406 nm, 25°C) was measured in a microplate reader (SynergyMx,
BioTek). The concentrations were calculated according to calibration with
an authentic H2O2 standard.
TiO2 (Aeroxide P25, Evonik), dodecane, 3,3'-(3,3'-dimethoxy-4,4'-
biphenylylene)bis-[2-(4-nitrophenyl)5-phenyl-2H-tetrazolium
chloride
(NBT), Coumarin, 7-Hydroxycoumarin, Horseradish peroxidase (HRP),
2,2'-azino-di (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), (R)- and (S)-
1-phenylethanol were purchased from Sigma-Aldrich. Ethyl acetate,
methanol and t-butanol have been supplied by Carl Roth and
ethylbenzene, 2-propanol, acetophenone by Merck EMD. p-
hydroxyphenylacetic acid (POHPAA) and TRIS-buffer were purchased
from Alfa Aesar, H2O2 from VWR. If not mentioned otherwise, all
chemicals were of analytical grade and used without a further purification.
Hydroxyl radicals were quantified using the reaction of coumarin to its
fluorescent product 7-hydroxycoumarin.[57] In defined temporal intervals
samples were taken from the reaction mixture containing 0.1 M coumarin.
Removement of TiO2 was done by centrifugation. The fluorescence
(λex = 323 nm and λem = 450 nm) of 100 µL of the supernatant was
measured in a microplate reader at 25 °C. The concentrations were
calculated according to calibration with an authentic 7-hydroxycoumarin
standard and the known yield (6.1 %) of 7-hydroxycoumarin from the
reaction of coumarin with hydroxyl radicals.
Enzyme Preparation
The enzyme was produced via heterologous expression in Pichia
pastoris following a previously reported procedure.[20] The concentration
of the enzyme was determined using CO difference spectra analysis as
also described previously.[20]
Reaction Set Up
Superoxide radicals were quantified using the decay of absorption of
All photoreactions were carried out in a 14 mL borosilicate glass reactor
with a cylindrical shape and a circular illumination window with a
diameter of 2 cm, cf. Figure S1. The temperature was kept constant by
circulating water through an outer jacket. Illumination was provided by
365 nm LED lamps (either M365LP1, Thorlabs or LEDMOD365.1050.V2,
Omicron) through collimating optics for an almost homogeneous
illumination of the window. All reactions were carried out in potassium
phosphate buffer (0.1 M, pH 7) under magnetic stirring. The solutions
were pre-saturated with O2 before adding the reagents/enzyme, and
connected to an O2 containing balloon afterwards. This closed reaction
system minimized evaporative losses of ethylbenzene. The photon flux
densities have been determined via ferrioxalate actinometry as reported
earlier.[51]
NBT during the reaction with O2 to the corresponding di-formazan.[58]
Samples were taken from the reaction mixture containing NBT (0.5 mM)
and centrifuged to remove TiO2 and the solid di-formazan. The
absorbance of 100 µL of the supernatant (λmax = 380 nm) was measured
in a microplate reader (PowerWave HT, BioTek).
•
-
Acknowledgements
SRdB, BOB and JZB gratefully acknowledge financial support by
German Research Foundation (DFG, Grant No: BL 1425/1-1).
FH thanks for financial support by the European Research
Council (ERC Consolidator Grant No. 648026).
Reactant Quantification by GC
Ethylbenzene, acetophenone as well as (R)- and (S)-1-phenylethanol
were quantified via gas chromatography with a flame ionization detector
(GC-FID, Focus, Thermo Scientific) using nitrogen as carrier gas and a
chiral column (Chirasil-DEX-CB column, 25 m, 0.25 mm, 0.25 µm,
Keywords: peroxygenases • photocatalysis • hydrogen peroxide
• light intensity • enzyme inactivation
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Entry for the Table of Contents (Please choose one layout)
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We present a systematic
Bastien O. Burek*, Sabrina R. de Boer,
Florian Tieves, Wuyuan Zhang, Morten
van Schie, Sebastian Bormann, Miguel
Alcalde, Dirk Holtmann, Frank
Hollmann, Detlef W. Bahnemann and
Jonathan Z. Bloh*
investigation of a photoenzymatic
reaction cascading photocatalytic in
situ H2O2 generation over TiO2 and
peroxygenase catalyzed
hydroxylation of ethyl benzene
utilizing AaeUPO. Therefore, product
formation and enzyme inactivation
as well as ROS formation (H2O2, •OH
Page No. – Page No.
Photoenzymatic Hydroxylation of
Ethylbenzene catalyzed by
Unspecific Peroxygenase: Origin of
Enzyme Inactivation and the Impact
of Light Intensity and Temperature
-
and •O2 ) under variation of light
intensity and temperature were
investigated, resulting in Turnover
Numbers up to 220 000, photonic
efficiencies up to 13.6 % and
production rates up to 0.9 mM h-1.
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