The CYP152-family P450 enzyme CypC of Bacillus subtilis converts non-natural substrates in plasma-driven biocatalysis

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, we here employed CypC from Bacillus subtilis 168 (synonyms: YbdT, P450BSβ), an integral enzyme of surfactin and fengycin biosynthesis. CypC naturally hydroxylates medium and long-chain carboxylic acids. With short-chain carboxylic acids as decoy molecules, it also converts non-natural substrates such as ethylbenzene. We optimized production and heme loading of CypC and established guaiacol and ABTS-based reactions to assess compatibility of CypC with plasma-driven biocatalysis regarding temperature and H₂O₂ operating windows. With heptanoic acid as the decoy molecule and H₂O₂ from stock solution, guaiacol and ABTS conversion yielded 18.28 and 21.13 nmol product min^-1 nmol^-1_CypC, respectively. We then supplied H₂O₂ using a capillary plasma jet operated with 1280 ppm H₂O in helium to convert ethylbenzene with immobilized CypC in a rotating bed reactor (5 ml reaction volume). After 120 min run time, a turnover number (TON) of 18.82 mol_(R)-1-PhOl mol^-1_CypC was reached, demonstrating that plasma-driven biocatalysis can be extended to H₂O₂-dependent enzymes beyond rAaeUPO to expand the product range.