Plasma-driven biocatalysis using the cytochrome P450 enzyme CYP152_BSβ

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 (CYP152_BSβ). CYP152_BSβ 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 CYP152_BSβ 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 CYP152_BSβ with plasma-driven biocatalysis regarding temperature and H₂O₂ operating windows. To establish CYP152_BSβ-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.