Nanosecond Pulsed Microwave Plasma Torch for Continuous In‐Line H$_2$O$_2$ Supply: A Path to Stable and Scalable Plasma‐Driven Biocatalysis

Plasma-driven H$_2$O$_2$ generation offers a potential sustainable route for biotransformations, but its application is hindered by challenges such as insufficient H$_2$O$_2$ production and enzyme degradation. This study establishes plasma-driven biocatalysis using a nanosecond pulsed microwave plasma torch (npMWPT), highlighting its unique advantages over previously applied sources. npMWPT-driven biocatalysis addresses insufficient plasma-generated H$_2$O$_2$ production by enabling continuous H$_2$O$_2$ supply while decoupling plasma treatment from enzymatic reactions. 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 reusability across five reaction cycles (turnover number TON 44,637 µmol$_{ABTS*}$ µmoL$^{−1}$$_{rAaeUPO}$). The decoupling of npMWPT-based H$_2$O$_2$ production resulted in a TON of 66,495 µmol$_{(R)-1-PhOl}$ µmoL$^{−1}$$_{rAaeUPO}$ with ethylbenzene for free rAaeUPO, outperforming the use of immobilized rAaeUPO for the first time in plasma-driven biocatalysis (TTN 22,116). ... mehrMixing optimization improved conversion rates, while observed enzyme inactivation under continuous operation revealed that H$_2$O$_2$ generation exceeded enzymatic consumption under the investigated conditions. The present study identifies supply-demand matching as the key optimization target for npMWPT-driven biocatalysis. This proof-of-principle work demonstrates the successful integration of spatially decoupled npMWPT H$_2$O$_2$ generation with peroxygenase-based biocatalysis and provides a basis for future process-controlled plasma-driven biocatalysis.