Plasma-Driven in Situ Production of Hydrogen Peroxide for Biocatalysis

Article abstract of DOI:10.1002/cssc.201903438

Peroxidases and peroxygenases are promising classes of enzymes for biocatalysis because of their ability to carry out one-electron oxidation reactions and stereoselective oxyfunctionalizations. However, industrial application is limited, as the major drawback is the sensitivity toward the required peroxide substrates. Herein, we report a novel biocatalysis approach to circumvent this shortcoming: in situ production of H₂O₂ by dielectric barrier discharge plasma. The discharge plasma can be controlled to produce hydrogen peroxide at desired rates, yielding desired concentrations. Using horseradish peroxidase, it is demonstrated that hydrogen peroxide produced by plasma treatment can drive the enzymatic oxidation of model substrates. Fungal peroxygenase is then employed to convert ethylbenzene to (R)-1-phenylethanol with an ee of >96 % using plasma-generated hydrogen peroxide. As direct treatment of the reaction solution with plasma results in reduced enzyme activity, the use of plasma-treated liquid and protection strategies are investigated to increase total turnover. Technical plasmas present a noninvasive means to drive peroxide-based biotransformations.

Full text of DOI:10.1002/cssc.201903438

Accepted Article
Title: Plasma-driven in situ production of hydrogen peroxide for
biocatalysis
Authors: Abdulkadir Yayci, Álvaro Gómez Baraibar, Marco Krewing,
Elena Fernandez Fueyo, Frank Hollmann, Miguel Alcalde,
Robert Kourist, and Julia Elisabeth Bandow
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the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
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To be cited as: ChemSusChem 10.1002/cssc.201903438
Link to VoR: http://dx.doi.org/10.1002/cssc.201903438
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10.1002/cssc.201903438
ChemSusChem
Plasma-driven in situ production of hydrogen peroxide for biocatalysis
Abdulkadir Yayci[a] (ORCID: 0000-0003-4615-8981), Álvaro Gómez Baraibar[b] (ORCID: 0000-
0001-7191-7316), Marco Krewing[a] (ORCID: 0000-0002-5315-8755), Elena Fernandez
Fueyo[c] (ORCID: 0000-0002-5079-2240), Frank Hollmann[c] (ORCID: 0000-0003-4821-756X),
Miguel Alcalde[d] (ORCID: 0000-0001-6780-7616), Robert Kourist[b,e] (ORCID: 0000-0002-
2853-3525) and Julia E. Bandow[a]* (ORCID: 0000-0003-4100-8829)
[a]
Applied Microbiology, Faculty of Biology and Biotechnology
Ruhr University Bochum
Universitätsstraße 150, 44780 Bochum, Germany
[b]
Microbial Biotechnology, Faculty of Biology and Biotechnology
Ruhr-University Bochum
Universitätsstraße 150, 44780 Bochum, Germany
[c]
Department of Biotechnology
Delft University of Technology
Van der Maasweg 9, 2629 HZ Delft, The Netherlands
[d]
Department of Biocatalysis
Institute of Catalysis and Petrochemistry (CSIC)
Campus Cantoblanco, 28049 Madrid, Spain
[e]
current address:
Institute for Molecular Biotechnology
Graz University of Technology
Petersgasse 14, Graz, Austria
* Corresponding author: Julia E. Bandow, julia.bandow@rub.de
Supporting information for this article is given via a link at the end of the document.
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10.1002/cssc.201903438
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Abstract
Peroxidases and peroxygenases are promising classes of enzymes for biocatalysis because
of their ability to carry out one-electron oxidation reactions and stereoselective
oxyfunctionalizations. Industrial application is however limited, as the major drawback is the
sensitivity towards the required peroxide substrates. Herein, we report a novel biocatalysis
approach to circumvent this shortcoming: in situ production of H2O2 by dielectric barrier
discharge plasma. The discharge plasma can be controlled to produce hydrogen peroxide at
desired rates yielding desired concentrations. Using horseradish peroxidase, we demonstrated
that hydrogen peroxide produced by plasma treatment can drive the enzymatic oxidation of
model substrates. Fungal peroxygenase was then employed to convert ethylbenzene to (R)-
1-phenylethanol with an ee of >96 % using plasma-generated hydrogen peroxide. Since direct
treatment of the reaction solution with plasma resulted in reduced enzyme activity, the use of
plasma-treated liquid and protection strategies were investigated to increase total turnover.
Technical plasmas present a non-invasive means to drive peroxide-based biotransformations.
Introduction
Cold plasmas have a variety of different applications, ranging from surface preparation in
material sciences to treating skin infections in plasma medicine.[1–3] In general, plasmas are
generated by accelerating free electrons by applying an electric field to ambient air or a defined
gas mixture. Collisions of high-temperature electrons with atoms or molecules in the gas phase
lead to the formation of excited species, radicals, and metastables, which in turn react to form
other species. In fact, for ambient air over 600 different reaction mechanisms are postulated.[4]
Because the electric field provides the light electrons with high kinetic energy, but not the
heavier particles, the overall temperature of the plasma is ambient (hence the term non-thermal
plasma). The plasma-generated species as well as the UV-photons stemming from the
relaxation of excited species are able to interact with gases or liquids exposed to the plasma.
One of the species generated in high amounts in plasma-treated liquids is hydrogen peroxide
(H2O2)[5], which can be a valuable oxidant for enzymatic conversions.
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Peroxidases and peroxygenases are heme-containing enzymes that upon activation by H2O2
can perform a multitude of natural functions, e.g. condensation of biopolymers[6], immune
defense[7], or detoxification of highly reactive H2O2[8]. However, peroxidases, which perform
one-electron oxidations, and especially peroxygenases (performing both one-electron
oxidations and most remarkably, two-electron oxidation reactions) are also remarkable
enzymes for different biotechnological purposes. In particular, the 2-electron C-H
oxyfunctionalization reactions carried out by peroxygenases are raising great interest for
synthetic chemistry.[9] Nevertheless, using these biocatalysts on a larger scale is challenging,
mainly because the substrate H2O2 also leads to inactivation of the enzymes when present at
high concentrations.[10] In a commercial setting, this would require enzyme replacement after
few reaction cycles or it would lead to strong dilution of the reaction solution if H2O2 is added
at low concentrations. Both strategies are typically little profitable. An alternative to using stock
solutions is the generation of H2O2 in situ. To this effect, several different strategies have been
developed, such as the use of enzyme cascades[11,12], light activated flavins[13–15], or
photocatalysts[16–18]. All of these strategies require the addition of extra components to the
reaction, incurring additional costs, especially in the case of enzymes and flavins. Another
approach uses immersed electrodes and electrochemistry to produce H2O2.[19–22] While this
system does not rely on the addition of components to the solution, electrodes are immersed
which may trigger precipitation of buffer salts and enzymes. A non-invasive in situ approach
that allows to control H2O2 levels would present a significant advantage.
Here, we report on the use of a novel, non-invasive approach to fuel H2O2-based
biotransformations: the in situ generation of H2O2 using a dielectric barrier discharge (DBD)
plasma (Fig. 1). The DBD device is operated in a surface discharge mode so that only the
plasma comes into contact with the reaction solution. Plasma parameters are readily tunable
and can be tailored to the needs of the enzyme employed. For instance, frequency, voltage,
and power density influence H2O2 production and can be adjusted.[23] With regard to
temperature reaction conditions are mild since the non-thermal plasma used here causes
negligible heating.[24] A proof-of-principle study was performed with horseradish peroxidase
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(HRP) and the model substrate guaiacol. Plasma stability of the enzyme, optimal plasma
parameters, and plasma-triggered side reactions were investigated. Enzyme protection
strategies were tested, including protein immobilization, which, by placing enzymes at a
distance from the plasma-liquid interface, protects proteins from the most reactive species.
The biotechnological potential with regard to selectivity was evaluated using the evolved
recombinant unspecific peroxygenase from Agrocybe aegerita (rAaeUPO), one of the most
promising enzymes for peroxide-dependent oxyfunctionalization chemistry.[25]
Results and Discussion
Proof of principle
The concept of plasma-driven biocatalysis is illustrated in Fig. 1. DBD plasmas induce
formation of reactive oxygen and nitrogen species (RONS), including H2O2. Peroxidases or
peroxygenases then utilize the supplied H2O2 to produce valuable products from precursors,
i.e. oxidized organic substances.
Figure 1. General scheme of plasma-driven biocatalysis. A dielectric barrier discharge is
generating a non-thermal plasma that interacts with the liquid, thus forming reactive oxygen
-
and nitrogen species (RONS), e.g. peroxynitrite (ONOO-), superoxide (O2 ), or H2O2. Some of
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the species can further react to other reactive particles, most of which represent toxicants.
Other species such as H2O2, however, can serve as a reactant to fuel biocatalysis.
The DBD device used in this study can be ignited in air, eliminating the need for expensive
feed gases like helium or argon, which are commonly used to operate other plasma devices.
Also, the source generates H2O2 in the treated liquid at rates adequate for the enzymes
employed here. HRP was used as the model enzyme in the proof-of-principle experiment since
it is well-studied, highly stable, and commercially available.[26–28] Without further purification
(Rz>2.5), HRP was dissolved in phosphate buffer and, after addition of the chromogenic
substrate guaiacol, treated directly with the DBD device operated in ambient air (Fig. 2). In the
presence of H2O2, HRP oxidizes guaiacol to tetraguaiacol which exhibits an Amax at λ = 470
nm. With increasing treatment time, A470 rises, indicating successful production of
tetraguaiacol. Plasma treatment of guaiacol alone or incubation of HRP with guaiacol without
plasma treatment did not result in tetraguaiacol production (Supplementary Fig. 1). When
inactivated HRP (HRP from which heme had been extracted with ethyl acetate) was used, no
product was observed either, indicating that enzyme activity is strictly required for guaiacol
conversion (Supplementary Fig. 2). H2O2 concentration was measured concomitantly and was
found to increase linearly by approximately 0.1 mM min-1 for the reaction volume tested here.
This is in agreement with previous reports on plasma-based H2O2 production.[29]
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Figure 2. Kinetics of H2O2 accumulation and substrate conversion by HRP during direct
plasma treatment with the CINOGY PlasmaDerm DBD device. Samples were placed onto
glass slides and treated for the indicated amount of time. Black: 100 µl of 50 mM KPi buffer
were treated with plasma. Immediately after treatment, 20 µl of the samples were mixed with
180 µl A. dest. and H2O2 concentrations determined using the Spectroquant Hydrogen
Peroxide kit (Merck) and photometrical measurements at 455 nm. Red: direct conversion of
guaiacol (5 mM) with plasma was carried out by treating 100 µl of KPi buffer containing 0.1 U
ml-1 HRP. Production of tetraguaiacol was followed at λ = 470 nm. The data shown represents
means of three independent experiments.
HRP inactivation mechanisms
While the H2O2 supply is steady for the treatment times tested, the tetraguaiacol production
rate declines at prolonged exposure (Fig. 2). The observed decrease in tetraguaiacol
production rates at 5 min could be caused by degradation of the enzyme or further modification
of the product during plasma exposure. Indeed, the absorption spectrum of the plasma-
generated biocatalysis product of guaiacol conversion differed from the product formed with
exogenously added H2O2 (Supplementary Fig. 3). For alternative HRP substrates pyrogallol
and L-DOPA we observed oxidation to the final product by plasma treatment even in the
absence of enzyme (Supplementary Fig. 4).
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Inactivation of enzymes with the plasma source used here was shown before, e.g. with RNase
A which was fully inactivated after 5 min of treatment.[29] Activity loss of heme-containing
proteins, and specifically HRP, has also been studied[30–32], albeit not with the plasma device
used here. When treated with an argon discharge, HRP was fully inactivated within 30 min.
However, the plasma device used in this work was specifically designed for use in
dermatology[33] and could be characterized by slower inactivation kinetics. It is worth
mentioning here that it depends on the enzyme how deleterious the effects of plasmas are.
Some enzymes even exhibited increased activity after plasma treatment.[34,35]
In order to assess HRP inactivation by the plasma treatment performed in this work, HRP was
treated with the DBD plasma and activity determined subsequently ex situ with a defined
amount of H2O2 (Fig. 3). HRP activity decreased with increasing plasma treatment time,
resulting in only residual activity after 10 min of treatment. HRP inactivation was found to be
largely independent of protein concentration during treatment (Supplementary Fig. 5).
Figure 3. HRP inactivation by plasma treatment. Plasma treatment was performed with 110 µl
1 kU ml-1 HRP in KPi (100 mM, pH 6.5) buffer without guaiacol. Activity was measured by
diluting the treated samples to 0.1 U ml -1 in KPi buffer including 5 mM of guaiacol and
subsequent addition of H2O2 at a final concentration of 0.25 mM. Guaiacol conversion was
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immediately monitored at λ = 470 nm and activity calculated from the initial slope during the
first 30 s. From the same treated samples, absorption spectra were measured with a 1:5
dilution in KPi. The inset shows the Soret band of HRP with Amax at ~ 403 nm. Spectra are
displayed with the untreated sample as blank, thereby showing a decrease of absorbance at
the Soret peak. Data was recorded in triplicates for both activity measurements and spectra.
HRP depends on a heme cofactor for activity that absorbs at λ ~ 403 nm (Soret band). To
assess integrity of the heme cofactor, absorption spectra were recorded for the protein
samples that were tested for activity (inset of Fig. 3). In congruence with previous reports[30]
,
absorption of the Soret band declined after plasma exposure, indicating that the heme is
-
modified. Heme has been shown to be attacked by ROS, e.g. O2 radicals and H2O2[36,37], both
of which represent major components of the discharge and the treated liquid. H2O2 is needed
-
as a substrate for the peroxidation reaction, but O2 may only act as toxicant. We attempted to
-
eliminate O2 radicals using superoxide dismutase A (SodA) from Escherichia coli, an enzyme
-
that converts O2 to H2O2. Superoxide dismutases are among the fastest enzymes, operating
near the diffusion limit of substrate supply.[38,39] SodA was added to the reaction solution prior
to plasma treatment. No difference in catalytic efficiency of HRP was observed (Supplementary
-
Fig. 6). Therefore, we conclude that either the concentration of O2 in plasma treated samples
is insignificant for heme degradation or that SodA is immediately inactivated by plasma-
generated species and thus cannot provide protection to HRP.
Heme-binding can also induce conformational changes in apoproteins.[40] Since it was shown
previously that plasma discharges can impact the structural integrity of proteins[29,31,41,42], we
investigated the structure of HRP upon plasma exposure using circular dichroism (CD)
spectroscopy (Fig. 4). No conformational changes of HRP were detected even at treatment
times of 5 min which render HRP largely inactive. This indicated that damage to the heme
moiety did not negatively affect HRP structure. The fact that no structural changes were
observed also allowed conclusions on another structural feature, namely disulfide bonds. HRP
contains eight cysteines, all of which are engaged in disulfide bonds, that contribute to the high
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enzyme stability.[26] For RNase A, a highly stable enzyme with four disulfide bonds, oxidation
of cysteines to their sulfenic and sulfonic acids was observed upon DBD treatment which
resulted in significant unfolding.[29] If such amino acid modifications also occurred in HRP, they
did not seem to affect the overall protein fold.
Figure 4. CD spectra of HRP after exposure to plasma. 110 µl HRP were treated as described
above for activity measurements and diluted 1:5, which corresponds to 0.2 mg ml-1 for the
untreated sample. Immediately after treatment, the sample was transferred to a suitable
cuvette and subjected to CD measurements. CD spectra were normalized with respect to
protein concentration as determined by the Bradford method.
The specific heme content (heme per protein) of peroxidases is defined by the Rz value
(A403/A275). Because plasma-generated species also absorb in the UV region[43], specific heme
content in this case was calculated as the quotient of A403 and protein concentration as
measured by Bradford assay (Fig. 5). Dependent on the plasma exposure time, protein
concentration decreased. This phenomenon was observed previously for HRP as well as other
enzymes treated in aqueous solution, e.g. hemoglobin, myoglobin, and BSA.[31,44] In fact, it has
been shown that treatment with the plasma source used in this study leads to the cleavage of
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peptide bonds and thus protein degradation[44]. This protein degradation was partially inhibited
by the addition of mannitol, which had been described as •OH scavenger.[45] Protein
fragmentation will lead to activity loss. However, since the CD spectra indicate that the
structure of the HRP remaining after 5 min treatment was still intact and the rate of heme
degradation for HRP exceeded the decrease in HRP concentration, we conclude that heme
damage, rather than structural changes or protein degradation, is the primary cause for HRP
inactivation.
Figure 5. Decrease in protein concentration and heme content. Protein concentration was
determined using the Bradford method. Specific heme content was set as absorbance at λ =
403 (see Fig. 3) divided by protein concentration and displayed in arbitrary units. Protein
concentration was determined in triplicates.
Optimizing plasma treatment conditions
For the proof of concept study with HRP, the parameters for plasma operation (pulse amplitude
13.5 kV, trigger frequency 300 Hz) were chosen simply by applying the same parameters as
in previous studies.[29,46] In an effort to optimize operating conditions for longevity of HRP, a
range of amplitudes and frequencies between 11.5 and 17.5 kV and 150 and 700 Hz,
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respectively, was tested (Fig. 6). To this end, HRP activity was measured immediately after 1
min of plasma treatment. The activity measurement was decoupled from plasma treatment of
the enzyme-containing solution in order to circumvent the issue of plasma-induced product
modification discussed above (Fig. 2) that may obstruct interpretation of the results. Changes
in trigger frequency showed little to no effect on HRP activity (Fig. 6), while HRP inactivation
increased with increasing applied voltage. A very similar dependency of protein activity on
discharge voltage was shown previously for tomato peroxidase, even with similar kinetics,
although a different DBD plasma was used.[47]
Figure 6. Influence of voltage and frequency on HRP after 1 min of plasma treatment. HRP
was plasma-treated at 10 U ml-1 for 1 min and activity was subsequently measured ex situ as
described above. Untreated samples were set to 100%. The graph shows mean values of
three independent replicates.
An increase in applied voltage leads to higher electron temperatures in the gas phase and in
turn increased ROS production, so that a higher density of toxicants is expected in the liquid.
It was for instance shown previously that •OH production in plasma-treated liquid depends on
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the specific energy transferred, which can be manipulated either by treatment time or by the
voltage applied.[23] Addition of the •OH scavenger mannitol during plasma treatment did not
have a significant effect on HRP lifetime (Supplementary Fig. 7). We speculate that other
RONS such as atomic oxygen (•O), UV radiation, and/or the applied electric field[48,49] might
impact enzyme activity.
Another approach to extending HRP lifetime was immobilization of the enzyme to non-reactive
support beads. Immobilization was carried out by reacting HRP with glutaraldehyde-activated
polymer beads. Immobilized HRP exhibited only ~10% of the activity of the free enzyme
(Supplementary Fig. 8). However, when plasma treated, HRPimmobilized retained its activity
significantly longer. After 5 min of treatment, immobilized HRP showed 41.8% activity
compared to the untreated HRPimmobilized, while free enzyme activity was reduced to 0.5% to
10% (results presented in Fig. 7 and Fig. 3, respectively).
Figure 7. Immobilization of HRP protects against plasma-mediated damage. Glutaraldehyde-
activated polymethacrylate beads (Relizyme HA403) were incubated with HRP over night to
yield an immobilized HRP solution corresponding to 20 U ml-1. For both enzyme formulations,
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100 µl were treated at 13.5 kV and 300 Hz. Free enzyme activity was measured as described
above. Activity assays for immobilized HRP were conducted with constant shaking to provide
sufficient substrate delivery to the macroscopic beads, using 5 mM guaiacol and 0.25 mM
H2O2. The displayed relative activities were calculated by relating the activities of treated
samples to their respective untreated controls. Data represent means of three replicates.
The comparably high robustness of immobilized enzyme might be attributed to spatial
separation. Most recombination reactions in plasma-treated liquids are thought to occur at or
-
in close proximity to the liquid-gas interface.[50] RONS like singlet oxygen (1ΔgO2), O2 , and •OH
have average diffusion distances in the nm-µm range.[51] The liquid droplets in our experimental
setup are approximately 2 mm in height and the protein-loaded beads sink to the bottom of the
sample. This creates a protein-free buffer zone that allows most short-lived toxicants to react
and form less harmful species before they can interact with the protein. However, the
recombination reactions at the plasma-liquid interface are experimentally very challenging to
address. Most studies, therefore, address this question based on numerical modelling
(reviewed in [52]). According to the models, many of the reactive species have short lifetimes in
the liquid phase, e.g. in the ns-µs range for •OH. Thus, a spatial gradient forms in the liquid
phase such that the more reactive species penetrate less deep before they react further.[53,54]
The models are congruent with a mm of liquid column being sufficient to protect proteins from
exposure to the most reactive species.
Selectivity in plasma-driven biocatalysis
In order to assess the potential of plasma-driven biocatalysis, we felt it necessary to investigate
if plasma-based H2O2 production interferes with the stereoselectivity of enzymatic reactions.
To this end, the rAaeUPO was used, an enzyme capable of performing stereoselective
oxidations of hydrocarbons.[12,55] For rAaeUPO, total turnover numbers (TTNs) of >10,000 have
been reported, indicating its remarkable stability.[56–58] As model reaction we chose the well-
characterized oxidation of ethylbenzene to (R)-1-phenylethanol, a reaction that would provide
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proof that a value-adding reaction can be driven by plasma. First, H2O2 was generated by
treating a set volume of 100 µl KPi buffer with plasma for 5 min and then adding it to a solution
containing rAaeUPO and ethylbenzene (indirect treatment). After 10 min reaction time, another
100 µl of treated buffer were added, and after further 10 min reaction time, another 100 µl of
treated buffer were added prior to allowing a final 10 min reaction time. Formation of (R)-1-
phenylethanol was determined, yielding a final concentration of 0.46 mM (Table 1).
Table 1. Production of (R)-1-phenylethanol from ethylbenzene starting with a solution of 1 µM
rAaeUPO and 5 µl of ethylbenzene. 100 µl increments of buffer solutions were treated for the
indicated amounts of time and added to the reaction solution. After 10 min of incubation under
agitation, the next increment of treated buffer was added. In total, three increments were added
per sample. TON: turnover number. Data represent means and standard deviations of three
replicates.
treatment time per
final (R)-1-phenylethanol
concentration [mM]
0
buffer composition
TON
increment [min]
0
5
KPi 250 mM
KPi 250 mM
KPi 250 mM
KPi 250 mM
KPi 250 mM
KPi 1 M
0
0.46 ± 0.01
4576
6935
9709
13787
8279
6993
9170
13493
7831
10
15
20
5
0.69 ± 0.25
0.97 ± 0.17
1.38 ± 0.13
0.83 ± 0.06
5
KPi 50 mM
Tris 50 mM
HEPES 50 mM
MES 50 mM
0.7 ± 0.03
5
0.92 ± 0.01
5
1.35 ± 0.03
5
0.78 ± 0.01
Treatment times for each increment were then varied to change H2O2 concentrations. Product
formation was linearly correlated with the treatment time. The calculated turnover number of
13,787 indeed is on the order of previously reported TTNs of rAaeUPO.[55] Negative controls
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without enzyme, plasma treatment, or ethylbenzene did not result in any detectable product
formation (Supplementary Fig. 9). The product was optically pure with an ee ranging between
96 % to >99 %, which clearly demonstrates that the hydroxylation is indeed an enzymatic
reaction (Supplementary Fig. 9). We also tested whether other long-living RONS present in
plasma-treated buffer have an impact on rAaeUPO activity. The enzyme was added to plasma-
treated buffer or to H2O2 diluted to the same concentration as detected in the plasma-treated
sample. After 2 min incubation, substrate was added and the enzyme activity assay performed
using only the H2O2 already present in the sample (Supplementary Fig. 10). There was no
difference in enzyme inactivation rates, showing that other long-living plasma-induced species,
such as ONOO- or NO2, do not have a significant effect on enzyme lifetime.
Different buffer salts were tested for their influence on the reaction yield. The best results were
obtained with HEPES buffer yielding about twice as much product than with KPi, followed by
MES buffer and Tris buffer (Tab. 1). It has been reported that UV-irradiated HEPES produces
H2O2, which may be one possible explanation for the observed increase since the employed
plasma source emits UV photons.[59] Another possible explanation is that plasma-generated
-
[60]
peroxynitrite (from the reaction of O2 and •NO) can react with HEPES to form H2O2.
Having established that plasma-treated buffer is a suitable source of H2O2 for biocatalysis with
rAaeUPO, we investigated suitability of rAaeUPO for direct plasma-driven biocatalysis. 100 µl
of 1 µM enzyme in 250 mM KPi buffer was mixed with 5 µl of ethylbenzene and plasma-treated
for 5 min, yielding 0.05 mM of (R)-1-phenylethanol. Compared to supplying H2O2 from plasma-
treated KPi buffer incrementally, plasma-driven biocatalysis gave a 11 % yield, indicating either
enzyme inactivation, substrate limitation (the substrate is poorly soluble in aqueous solution
and forms an organic phase on top of the reaction solution), or degradation of the product (R)-
1-phenylethanol by short-lived plasma species. Like HRP, rAaeUPO relies on a heme cofactor.
Inactivation kinetics of rAaeUPO were similar to those of HRP (Supplementary Fig. 11). The
addition of E. coli SodA also did not have a significant effect on the lifetime of rAaeUPO when
exposed to plasma (Supplementary Fig. 12). And like for HRP, when rAaeUPO was
immobilized using the same carrier, the relative activity after plasma treatment compared to
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untreated samples was significantly higher than for the free enzyme (Supplementary Fig. 11
and 13). Immobilized rAaeUPO retained approx. 39% activity compared to the free enzyme
(Supplementary Fig. 14), indicating that immobilization has less of an impact on enzyme
activity than for HRP (approximately 10% activity after immobilization (Supplementary Fig. 8)).
However, when immobilized rAaeUPO was directly treated in-solution in the presence of
ethylbenzene for 5 min, no product was formed. Since the enzyme still showed 75% activity
after 5 min of plasma exposure (Supplementary Fig. 13), we attribute the lack of conversion to
substrate limitation, possibly caused by poor substrate solubility and insufficient mixing or by
evaporation of the ethylbenzene during the treatment.
To overcome the limits of the direct exposure (presumably the substrate limitation) while
retaining the benefit of the in situ approach of keeping the final volume constant, ex situ
plasma-treatment was combined with the use of immobilized rAaeUPO (Fig. 8). After the
addition of the substrate, a volume of 100 µl of the solution from the liquid column above the
beads was taken from the vial, treated with the DBD for 5 min, and returned to the reaction.
Using this strategy, the same amount of product was obtained as for the addition of plasma-
treated buffer, but without diluting the reaction solution. For reactions with poorly soluble
substrates, the ex situ treatment of reaction solutions thus presents a viable option with the
decisive advantage of avoiding sample dilution in synthetic applications.
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Figure 8. Production of (R)-1-phenylethanol with immobilized rAaeUPO using plasma-treated
KPi buffer (250 mM) treated for several cycles. Supernatant of the reaction vial, i.e. buffer
without enzyme, was treated as mentioned before and added back to the container. This was
repeated for several cycles as indicated. Turnover of ethylbenzene to (R)-1-phenylethanol was
allowed to take place for 30 min after a new cycle was initiated. (R)-1-PhOl: (R)-1-
phenylethanol.
The product obtained using immobilized rAaeUPO and several cycles of plasma-treated buffer
presented an ee of > 99 %, indicating that the plasma treatment did neither modify nor
racemize (R)-1-phenylethanol, nor did it change selectivity of the enzyme. Up to seven
treatment cycles were tested, resulting in an accumulation of (R)-1-phenylethanol to
concentrations of up to 1.26 mM, which is in the range of product obtained with 60 min
cumulated treatment time in the ex situ approach (Tab. 1). To investigate if immobilized
rAaeUPO that was exposed to plasma-treated liquids can be reused, the supernatant was
extracted before new plasma-treated buffer and substrate were added. In all of the 8 cycles
the same amount of (R)-1-phenylethanol was generated (Supplementary Fig. 15), indicating
that enzyme activity is not impaired by repeated exposure to plasma-treated buffer.
Conclusion
In this work, we present the first non-invasive in situ method to generate H2O2 for biocatalysis
using a cold plasma device. Advantageous to this system is the ability to fine-tune the H2O2
production rate without changing the setup. We show that limitations in enzyme stability under
direct plasma treatment of the reaction solution can be overcome by enzyme immobilization.
Plasma-driven biocatalysis may present a path forward for peroxidase and peroxygenase
catalyzed stereoselective oxyfunctionalization reactions. Furthermore, our results suggest that
enzyme immobilization is a useful tool for studying the interaction mechanisms of plasma,
liquids, and proteins.
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10.1002/cssc.201903438
ChemSusChem
Experimental Section
Enzymes
HRP was purchased from Sigma (P8375, Rz > 2.5) and stored in 100 mM KPi, pH 6.5. SodA
(Uniprot P00448) was obtained from an E. coli strain harbouring a His6-sodA fusion plasmid
(E. coli BL21 DE3 pASK-IBA+::his6-sodA) by following standard protocols for cultivation, cell
lysis, and purification as described in the supporting information. rAaeUPO (Uniprot B9W4V6)
was purified as described before.[61] Briefly, culture supernatant of a Pichia pastoris strain
expressing rAaeUPO and secreting it into the culture medium was subjected to micro-filtration
prior to use.
Plasma source and treatment
The plasma source used for all experiments was the CINOGY PlasmaDerm system (CINOGY,
Duderstadt, Germany). For a detailed review of the plasma source, including power
calculations, see[33]. Unless indicated otherwise, all plasma treatments were performed with
standard conditions at room temperature and in ambient air: electrode diameter, 20 mm; pulse
amplitude, 13,5 kV; trigger frequency, 300 Hz. Plasma exposure of liquid samples of 110 µl
volume was performed using PTFE-coated glass slides. Since plasma treatment increases
hydrophilicity of the glass surface, the liquid was contained in PTFE wells to prevent spreading.
Distance between dielectric and sample apex was kept constant at approximately 2 mm for all
samples. All subsequent analyses were performed immediately after treatment unless noted
otherwise.
H2O2 measurement
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10.1002/cssc.201903438
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Samples were analysed and calibrated with a commercially available test kit following the
manufacturer’s instructions (Spectroquant Hydrogen Peroxide, Merck). After a 10 min reaction
time, absorption at 455 nm was measured. Calibration was performed with serial dilutions from
0-200 µM made from H2O2 stock.
Ex-situ activity assays
HRP was added to a guaiacol solution in 100 mM KPi (pH 6.5). To start the reaction, the same
volume of 1 mM H2O2 was added and the absorption at 470 nm immediately monitored with a
plate reader (Biotek µQuant, Bad Friedrichshall, Germany). Final concentrations were usually
0.1 U ml-1 HRP, 5 mM guaiacol, 50 mM KPi, and 0.5 mM H2O2. For rAaeUPO activity
measurements, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) was used as
chromogenic substrate. Final concentrations in this case were 40 nM rAaeUPO, 2.5 mM ABTS,
50 mM sodium acetate buffer (pH 5.5), and 1 mM H2O2. Activity was determined based on the
slope of the linear region of the absorption measurement plot.
CD spectroscopy
HRP was treated as described above and immediately mixed with four parts of 100 mM KPi.
After transfer to a cuvette, CD spectra were recorded with a Jasco J-815 CD spectrometer
(Jasco, Pfungstadt, Germany) with the following parameters: range, 190-300 nm; data interval,
0.1 nm; bandwidth, 2 nm; accumulations, 5. KPi buffer was used as blank. Samples were then
extracted from the cuvette and subjected to a Bradford assay performed according to
manufacturer’s instructions (RotiNanoquant Kit, Roth, Karlsruhe, Germany). The ellipticity was
corrected for the protein concentration as described before.[62]
Immobilization
HRP and rAaeUPO were immobilized with Relizyme HA403 M beads (Resindion, Binasco,
Italy). To this end, 10 mg of beads were activated by incubating in KPi buffer (pH 7) with 0.4%
glutaraldehyde for 1 h. After washing twice with deionized water, up to 5 mg of enzyme were
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10.1002/cssc.201903438
ChemSusChem
added in 1 ml buffer. Immobilization was carried out overnight at room temperature under
constant shaking. Binding efficiency was determined by measuring the protein concentration
in the supernatant after incubation and was found be >80% in all cases.
Analysis of rAaeUPO catalysis products
Buffer volumes of 110 µl were treated with the DBD plasma as described for HRP for different
amounts of time. Treated buffer was then allowed to rest for five minutes for short-lived reactive
species to react. 100 µl of this treated buffer was combined with 5 µl of ethylbenzene and 50
µl of a 1 µM rAaeUPO solution and incubated for 10 min at 30 °C and 600 rpm. Then, another
100 µl of treated buffer was added and the reaction was incubated for another 10 minutes.
This was followed by a third addition of treated buffer and incubation. The final reaction volume
(355 µl) was extracted with 300 µl ethyl acetate containing 2 mM of 1-octanol as injection
standard. The organic phase was dried with MgSO4 and measured with a Shimadzu 2010 1
system containing a Hydrodex β-6TBDM column (Macherey-Nagel, Germany). The column
was heated at 120 °C for 20 minutes. Final concentrations of (R)-1-phenylethanol were
determined by applying a standard curve that was measured with racemic 1-phenylethanol.
Acknowledgements
We thank Cinogy for kindly providing the plasma source and Britta Schubert for technical
assistance. We gratefully acknowledge funding by the German Research foundation to JEB
(CRC1316-1 and BA 4193/7-1).
Keywords: biocatalysis • peroxidase • peroxides • peroxygenase • plasma chemistry
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10.1002/cssc.201903438
ChemSusChem
Table of contents
Ride the lightning: Non-thermal plasma was applied for non-invasive production of hydrogen
peroxide to be used in biocatalysis. Side effects of plasma exposure can be alleviated by
enzyme immobilization. Enantiomerically pure (R)-1-phenylethanol was produced with
plasma-generated H2O2 and AaeUPO.
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