Plasma-driven biocatalysis using the cytochrome P450 enzyme CYP152BSβ

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 (CYP152BSβ). CYP152BSβ 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 CYP152BSβ 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 , respectively. These reactions also served to assess compatibility of CYP152BSβ with plasma-driven biocatalysis regarding temperature and H2O2 operating windows. To establish CYP152BSβ-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 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. Competing Interest Statement J.E.B. and A.Y. are coauthors of the following patent: J. Bandow, A. Yayci, M. Krewing, R. Kourist, A. Gomez Baraibar. Plasma-driven Biocatalysis. International patent WO/2020/007576, published 09.01.2020.