Dirks Tim, Klopsch Sabrina, Stoesser Davina, Trenkle Sophie Desdemona, Yayci Abdulkadir, Schüttler Steffen, Golda Judith, Bandow Julia Elisabeth
Applied Microbiology, Faculty of Biology and Biotechnology, Ruhr University Bochum, Bochum, Germany.
Plasma Interface Physics, Faculty of Physics and Astronomy, Ruhr University Bochum, Bochum, Germany.
Appl Microbiol Biotechnol. 2025 Sep 2;109(1):193. doi: 10.1007/s00253-025-13568-1.
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.
等离子体驱动的生物催化利用大气压等离子体原位产生羟基自由基（HO）来驱动依赖HO的酶促反应。我们之前已经利用来自高大环柄菇的重组非特异性过氧酶（rAaeUPO）建立了等离子体驱动的生物催化体系，用于从乙苯生产（R）-1-苯乙醇。在此，我们使用了来自枯草芽孢杆菌168的CypC（同义词：YbdT、P450BSβ），它是表面活性素和丰原素生物合成的一个整合酶。CypC天然地催化中长链羧酸的羟基化反应。以短链羧酸作为诱饵分子时，它还能转化非天然底物，如乙苯。我们优化了CypC的生产和血红素负载，并建立了基于愈创木酚和ABTS的反应，以评估CypC在温度和HO操作窗口方面与等离子体驱动的生物催化的兼容性。以庚酸作为诱饵分子，使用储备溶液中的HO时，愈创木酚和ABTS的转化率分别为18.28和21.13 nmol产物·min⁻¹·nmol⁻¹。然后，我们使用在氦气中含有1280 ppm HO运行的毛细管等离子体射流来提供HO，以在旋转床反应器（5 ml反应体积）中用固定化的CypC转化乙苯。运行120分钟后，周转数（TON）达到18.82 mol·mol⁻¹，这表明等离子体驱动的生物催化可以扩展到rAaeUPO之外的依赖HO的酶，以扩大产物范围。
