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bioRxiv · 10.1101/2025.01.09.631812

Plasma-driven biocatalysis using the cytochrome P450 enzyme CYP152BSβ

Dirks, T.·Klopsch, S.·Stoesser, D.·Trenkle, S. D.·Yayci, A.·Schüttler, S.·Golda, J.·Bandow, J. E.
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Abstract

Plasma-driven biocatalysis utilizes in situ H2O2 production by atmospheric pressure plasmas to drive H2O2-dependent enzymatic reactions. Having previously established plasma-driven biocatalysis using recombinant unspecific peroxygenase from Agrocybe aegerita (rAaeUPO) to produce (R)-1-phenylethanol from ethylbenzene (ETBE), we here employed CYP152 from Bacillus subtilis (CYP152BS{beta}). CYP152BS{beta} naturally hydroxylates medium and long-chain carboxylic acids, and, with short-chain carboxylic acids as decoy molecules, also converts non-natural substrates such as ETBE. To produce active CYP152BS{beta} overexpression and heme loading were optimized. The conversion of the non-natural substrates guaiacol and ABTS with heptanoic acid as decoy molecule and H2O2 from stock solution yielded 18.28 and 21.13 nmol product min-1 [Formula], respectively. These reactions also served to assess compatibility of CYP152BS{beta} with plasma-driven biocatalysis regarding temperature and H2O2 operating windows. To establish CYP152BS{beta}-based plasma-driven biocatalysis, immobilized enzyme in a rotating bed reactor (5 ml reaction volume) was then supplied with H2O2 from a capillary plasma jet operated with 1280 ppm H2O in helium. After a 120 min run time a turnover number (TON) of 18.82 mol(R)-1-PhOl [Formula] was reached. We conclude that plasma-driven biocatalysis can be extended to other H2O2-dependent enzymes. Future efforts will be directed at increasing the TON and product range.

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Dirks, T., Klopsch, S., Stoesser, D., Trenkle, S. D., Yayci, A., Schüttler, S., Golda, J., Bandow, J. E.. 2025-01-11. Plasma-driven biocatalysis using the cytochrome P450 enzyme CYP152BSβ. https://doi.org/10.1101/2025.01.09.631812

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