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Plasma-driven in situ H2O2 generation offers a potential sustainable route for oxidative biotransformations, but its application has been hindered by challenges such as insufficient H2O2 production and enzyme degradation from direct plasma exposure. This study establishes plasma-driven biocatalysis using a nanosecond pulsed microwave plasma torch (npMWPT) as a plasma source, highlighting its unique advantages over previously applied sources. npMWPT-driven biocatalysis addresses insufficient plasma-generated H2O2 production by enabling a continuous and ample H2O2 supply while decoupling plasma treatment from the enzymatic reaction. This setup significantly extends enzyme stability, thus supporting prolonged biocatalytic activity. We investigated the effectiveness of npMWPT-driven biocatalysis using both immobilized and free recombinant unspecific peroxygenase from Agrocybe aegerita (rAaeUPO) to convert ABTS and ethylbenzene. For ABTS, immobilized rAaeUPO demonstrated consistent reusability across five reaction cycles (TON 44,637 µmolABTS* µmol-1rAaeUPO). The decoupling of npMWPT-based H2O2 production resulted in a turnover number of 66,495 µmol product per µmol enzyme with ethylbenzene for free rAaeUPO, outperforming the use of immobilized rAaeUPO for the first time in plasma-driven biocatalysis (TTN of 22,116 µmol(R)1-PhOl µmol-1rAaeUPO). Mixing optimization further improved ethylbenzene conversion rates, although the continuous H2O2 flow eventually led to enzyme inactivation due to excess H2O2. These findings highlight the dual advantages of the npMWPT-driven biocatalysis in enhancing enzyme longevity and ensuring sustained H2O2 supply, creating a more favorable environment for biocatalysis. This proof-of-principle study demonstrates the successful integration of the npMWPT plasma source in plasma-driven biocatalysis, establishing a viable setup that provides a promising pathway for scaling up plasma-driven biocatalysis for complex biotransformations.
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| Release Date | 2025-10-23 |
| Identifier | 0f0329d3-7134-4a7b-9c56-ad1a36bb5705 |
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| Plasma Source Properties | The plasma-water reaction system consists of a coaxial plasma torch reactor (Heuermann HF-Technik GmbH, Germany) connected to a solid state microwave source (TRUMPF Hüttinger Microwave, Germany). The gas flow was fed into the torch using mass flow controllers (MFC, Vögtlin Instruments, Switzerland), and the liquid was fed via a 1.8 mm (o.d.) stainless steel tube connected to a water pump (1HM, Eldex, USA). A thermocouple was located approx. 2 cm away from the plasma zone to follow the temperature of the reaction environment. The quenching of the plasma zone was achieved using a metallic loop stainless steel tubing (1/8” i.d.) placed below the plasma-liquid interaction zone. The liquid product was collected downstream using a three-way valve that allows to take a specific amount of sample when required. An optical fiber connected to a UV-Vis detector (USB2000, Ocean Optics, USA) served as an optical emission spectrometer (OES), enabling real-time monitoring of species during the plasma-water interaction. A schematic representation of the experimental set up is shown in Supplementary Figure 1. The main parameters to stablish during pulsed operation of the microwave source are the time of active pulse (ton) and the time of inactive pulse (toff), where the sum of both represents the total pulse period. The fraction of ton over the total pulse period represents the duty cycle (DC) or frequency of pulsation. Furthermore, the specific energy input (SEI) used for the reaction can be estimated. |
| Plasma Source Procedure | To enable effective integration of the npMWPT with biocatalysis, it was first necessary to establish plasma operating conditions that ensured consistent H2O2 production to drive the subsequent enzymatic biotransformation reactions. Thus, preliminary investigations aimed to select appropriate plasma operating parameters, including average input power, pulsation time, and gas and liquid flow rates. A constant set of microwave parameters was established, consisting of a pulsation duration (ton) of 500 ns and interpulse time of (toff) of 1167 ns, corresponding to a duty cycle of 30%. The total argon gas flow rate was kept constant at 8.7 l min-1, and the water flow rate was held constant at 2.5 ml min-1. Under these conditions, the specific energy input (SEI) was estimated at approximately 1.03 kJ l-1gas. Overall, the selected parameters yielded plasma-treated liquid containing H2O2 concentrations of 1.97 +/- 0.09 mmol l-1. The plasma-liquid treatment was operated continuously, and liquid samples were collected over a 4 h period of operation to verify the stability of the liquid product (more details are included in Supplementary Table 1). The gas temperature was measured at around 2 cm away from the plasma-liquid reaction environment with values of 45 °C +/- 1, aligned with low-temperature non-equilibrium plasma conditions. Additionally, all the experiments were carried out using a cooling coil downstream of the plasma zone, which further reduced the liquid product's temperature down to room temperature. The optical emission spectroscopy analysis of the plasma-water interaction zone revealed the presence of OH (A-X) (306 to 315 nm), hydrogen Balmer lines H (656 nm), H (485 nm), and H (420 to 430 nm); and atomic oxygen lines (777 and 844 nm). |
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| Plasma Medium Properties | The total argon gas flow rate was kept constant at 8.7 l min-1 |
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| Contact Name | Tim Dirks |
| Plasma Target Properties | 100 mM potassium phosphate buffer containing substrate and (immobilized) enzyme rAaeUPO. |
| Plasma Target Procedure | rAaeUPO was purified as described previously. For immobilization, amino (HA403 M) functionalized beads (Resindion, Italy) were used. In a suitable vessel, 500 mg beads were weighed and washed thrice with 5 ml potassium phosphate buffer (100 mmol l-1, pH 7). Carrier materials were incubated for 3 h in the presence of 0.5 % (w/v) glutaraldehyde in a total volume of 5 ml potassium phosphate buffer (100 mmol l-1, pH 7). After incubation, beads were washed thrice with potassium phosphate buffer to remove the glutaraldehyde. Finally, enzyme (2 nmol) was added, and immobilization was carried out overnight at 8°C with overhead shaking.
Binding efficiency
Enzyme binding efficiency was evaluated by measuring residual enzyme activity in the supernatant after immobilization. Using 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as substrate, either rAaeUPO (2 nmol) or supernatant after immobilization was added to 100 mmol l-1sodium citrate buffer (pH 5) containing ABTS (5 mmol l-1). An equivalent volume of H2O2 (2 mmol l-1) was added to initiate the reaction, while product formation was monitored at 405 nm using a microplate reader (Biotek Epoch, Germany). Final concentrations were 2.5 mmol l-1 ABTS, 1 mmol l-1 H2O2, and 50 mmol l-1 citrate. Unimmobilized rAaeUPO served as a control.
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| Plasma Diagnostic Properties | The experimental conditions were kept constant at 8.7 L/min of argon, 2.5 mL/min of H2O, 500 W of input power with 500 ns of t_on and 1167 of t_off. The argon region is extended from 700 to 850 nm. The visibility of the H and some Ar lines were limited by the spectral resolution of the UV-Vis detector, overcoming the maximum intensity. |
| Public Access Level | Public |
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Data and Resources
- Figure 1: Substrate (ABTS) conversion performing plasma-driven biocatalysis using immobilized rAaeUPO with npMWPT.xlsx
Substrate (ABTS) conversion performing plasma-driven biocatalysis using...
Download - Figure 2 Ethylbenzene conversion using npMWPT-treated water in combination with free rAaeUPOxlsx
Plasma-treated water (1.5 ml) was mixed with 50 mmol l-1 ethylbenzene and 50...
Download - Figure 3: Ethylbenzene conversion using continuous flow of npMWPT-treated water in combination with free rAaeUPO. xlsx
Ethylbenzene (50 mmol l-1) was mixed with free rAaeUPO (0.1 nmol) in a total...
Download - Figure 4: Substrate (ethylbenzene) conversion performing plasma-driven biocatalysis using immobilized rAaeUPO with npMWPTxlsx
Conversion of ethylbenzene with immobilized rAaeUPO was analyzed by loading...
Download - Supplementary Figure 4: Ethylbenzene conversion using continuous flow of npMWPT-treated water in combination with free rAaeUPOxlsx
Ethylbenzene (50 mmol l-1) was mixed with free rAaeUPO (0.1 nmol) in a total...
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