Biology subjects

Dirks, T.

Publications and source records attributed to Dirks, T..

3 recordsLinked to original sources

bioRxiv2025

Plasma-driven biocatalysis using the cytochrome P450 enzyme CYP152BSβ

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 (CYP152BS{beta}). CYP152BS{beta} 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 CYP152BS{beta} 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 [Formula], respectively. These reactions also served to assess compatibility of CYP152BS{beta} with plasma-driven biocatalysis regarding temperature and H2O2 operating windows. To establish CYP152BS{beta}-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 [Formula] 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.

biochemistry↗

bioRxiv2025

Iron-sulfur cluster proteins present the weak spot in plasma-treated Escherichia coli

Non-thermal atmospheric pressure plasmas have an antiseptic activity beneficial in different medical applications. In a genome-wide screening, hydrogen peroxide and superoxide were identified as key species contributing to the antibacterial effects of plasma while [FeS] cluster proteins emerged as potential cellular targets. We investigated the impact of plasma treatment on [FeS] cluster homeostasis in Escherichia coli treated for 1 min with the effluent of a microscale atmospheric pressure plasma jet ({micro}APPJ). Mutants defective in [FeS] cluster synthesis and maintenance lacking the SufBC2D scaffold protein complex or desulfurase IscS were hypersensitive to plasma treatment. Monitoring the activity of [FeS] cluster proteins of the tricarboxylic acid cycle (aconitase, fumarase, succinate dehydrogenase) and malate dehydrogenase (no [FeS] clusters), we identified cysteine, iron, superoxide dismutase, and catalase as determinants of plasma sensitivity. Survival rates, enzyme activity, and restoration of enzyme activity after plasma treatment were superior in mutants with elevated cysteine levels and in the wildtype under iron replete conditions. Mutants with elevated hydrogen peroxide and superoxide detoxification capacity over-expressing sodA and katE showed full protection from plasma-induced enzyme inactivation and survival rates increased from 34% (controls) to 87%. Our study indicates that metabolic and genetic adaptation of bacteria may result in plasma tolerance and resistance, respectively. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/631878v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@119626eorg.highwire.dtl.DTLVardef@18d2973org.highwire.dtl.DTLVardef@9c5568org.highwire.dtl.DTLVardef@1ab4e58_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG HighlightsO_LIEnzymes with [FeS] clusters are rapidly inactivated by plasma C_LIO_LIClusters damaged by plasma are repaired in vivo using iron and cysteine C_LIO_LIOver-expression of sodA and katE completely prevents disruption of [FeS] clusters by plasma C_LIO_LIPlasma resistance is increased threefold by SodA and KatE over-production C_LIO_LIPre-adaptation of E. coli to O2- increases plasma tolerance C_LI

microbiology↗

bioRxiv2025

The atmospheric pressure capillary plasma jet is well-suited to supply H2O2 for plasma-driven biocatalysis

Plasma-generated H2O2 can be used to fuel biocatalytic reactions that require H2O2 as co-substrate such as the conversion of ethylbenzene to (R)-1-phenylethanol ((R)-1-PhOl) catalyzed by unspecific peroxygenase from Agrocybe aegerita (rAaeUPO). Immobilization was recently shown to protect biocatalysts from inactivation by highly reactive plasma-produced species, however, H2O2 supply by the employed plasma sources ({micro}APPJ and DBD) was limiting for rAaeUPO performance. In this study we evaluated a recently introduced capillary plasma jet for suitability to supply H2O2 in situ. H2O2 production was modulated by varying the water concentration in the feed gas, providing a greater operating window for applications in plasma-driven biocatalysis. In a static system after 80 min of biocatalysis, a turnover number of 44,199 mol(R)-1-PhOl mol-1rAaeUPO was achieved without significant enzyme inactivation. By exchanging the reaction solution every 5 min, a total product yield of 122 {micro}mol (R)-1-PhOl was achieved in 700 min run time, resulting in a total turnover number of 174,209 mol(R)-1-PhOl mol-1rAaeUPO. We conclude that the capillary plasma jet, due to its flexibility regarding feed gas, admixtures, and power input, is well-suited for in situ H2O2 generation for plasma-driven biocatalysis tailoring to enzymes with high H2O2 turnover.

biochemistry↗