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.
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Fig. 2 Kinetics of H2O2 accumulation and substrate conversion by HRP during direct plasma treatment.
Figure 8. Production of (R)-1-phenylethanol with immobilized rAaeUPO using plasma-treated KPi buffer (250 mm) treated for several cycles
Supplementary Figure 5. HRP inactivation kinetics at different protein concentrations during treatment.
Supplementary Figure 12. Relative activity of rAaeUPO after plasma treatment in the presence of SodA from E. coli.
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|Plasma Source Name|
|Plasma Source Application|
|Plasma Source Specification|
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V(RMS) = 13.5 kV, trigger frequency = 300 Hz, diameter=20 mm
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Aqueous sample of different volumes (40-200) μl on glass support placed on grounded support. Distance between sample and DBD was 2 mm.
|Plasma Medium Name|
|Plasma Medium Properties|| |
|Plasma Target Name|
|Plasma Target Properties|| |
Horseradish peroxidase (P8375); unspecific Peroxygenase from Agrocybe aegerita, purified from Pichia pastoris expression strain; both enzymes in 100 mM KPi buffer, pH 7
English (United States)
|Public Access Level|| |
potential copyright conflict with publisher
|Contact Name|| |
Bandow, Julia E.
Data and Resources
- Fig. 2 Kinetics of H2O2 accumulation and substrate conversion by HRP during direct plasma treatment.xlsx
Samples were placed onto glass slides and treated for the indicated amount...
- Figure 3. HRP inactivation by plasma treatmentxlsx
Plasma treatment was performed
with 110 mL HRP (1 kUmL@1) in KPi...
- Figure 4. CD spectra of HRP after exposure to plasmaxlsx
HRP (110 mL) was
treated as described above for activity measurements...
- Figure 5. Decrease in protein concentration and heme content.xlsx
was determined using the Bradford method....
- Figure 6. Influence of voltage and frequency on HRP after 1 min plasma treatmentxlsx
HRP was plasma-treated at 10 UmL@1 for 1 min and activity was
- Figure 7. Immobilization of HRP protects against plasma-mediated damagexlsx
Glutaraldehyde-activated polymethacrylate beads (Relizyme HA403) were...
- Figure 8. Production of (R)-1-phenylethanol with immobilized rAaeUPO using plasma-treated KPi buffer (250 mm) treated for several cyclesxlsx
of the reaction vial, that is, buffer without enzyme, was...
- Supplementary Figure 1. Background activity of direct biocatalysis using HRP.xlsx
Reactions of 100 μl were run without enzyme, without plasma treatment, or...
- Supplementary Figure 2. Comparison of active and inactive HRP in direct catalysis.xlsx
order to extract the heme from HRP, acidified enzyme solution was...
- Supplementary Figure 3. Spectrum of reaction products of HRP and guaiacol.xlsx
of 0.1 U ml-1 HRP and 5 mM of guaiacol in 50 mM KPi buffer...
- Supplementary Figure 4. Conversion of substrates by plasma in the absence of enzyme.xlsx
100 μl of 100 mM pyrogallol, 10 mM guaiacol, and 20 mM L-DOPA in 100 mM KPi...
- Supplementary Figure 5. HRP inactivation kinetics at different protein concentrations during treatment.xlsx
HRP was diluted to different concentrations and treated with plasma for the...
- Supplementary Figure 6. Influence of SodA on HRP activity.xlsx
SodA was added to the
standard reaction mixture (10 U ml-1, 5 mM...
- Supplementary Figure 7. Relative activity of HRP after plasma treatment with and without mannitol.xlsx
Plasma treatment was performed with 100 μl of 10 U ml-1 HRP with or
- Supplementary Figure 8. Relative activity of free and immobilized HRP.xlsx
was carried out as described in the Methods section....
- Supplementary Figure 9. Analysis of reaction products of rAaeUPO and plasma-treated buffer.xlsx
A reaction mixture of 300 μl plasma-treated buffer, 5 μl ethylbenzene, and...
- Supplementary Figure 10. Relative activity of rAaeUPO after incubation in plasmatreated buffer.xlsx
110 μl of buffer were treated for 10 min as described before and H2O2
- Supplementary Figure 11. Relative activity of rAaeUPO after plasma treatment.xlsx
exposure was performed as described for HRP. Enzyme solution...
- Supplementary Figure 12. Relative activity of rAaeUPO after plasma treatment in the presence of SodA from E. coli.xlsx
Plasma treatment of rAaeUPO was performed either without
or in the...
- Supplementary Figure 13. Inactivation of immobilized rAaeUPO.xlsx
was diluted to 1 μM and 100 μl were treated with...
- Supplementary Figure 14. Relative activity of rAaeUPO before and after immobilization.xlsx
Activities of free and immobilized rAaeUPO were measured using 2.5 mM ABTS,...
- Supplementary Figure 15. Conversion of ethylbenzene to (R)-1-phenylethanol with immobilized rAaeUPO.xlsx
Either 110 μl of plasma-exposed (5 min treatment time) KPi buffer