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

Plasma-driven biocatalysis utilizes in situ HO production by atmospheric pressure plasmas to drive HO-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 HO operating windows. With heptanoic acid as the decoy molecule and HO from stock solution, guaiacol and ABTS conversion yielded 18.28 and 21.13 nmol product min nmol, respectively. We then supplied HO using a capillary plasma jet operated with 1280 ppm HO 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 mol was reached, demonstrating that plasma-driven biocatalysis can be extended to HO-dependent enzymes beyond rAaeUPO to expand the product range.