Integrated electro-biocatalysis in flow system

RWTH Publications (RWTH Aachen)

Abstract

Despite the growing demand for efficient and sustainable chemical processes, the development of scalable biocatalytic systems for the sustainable fine chemical production remains a major challenge. This limitation arises primarily from the dependence of many oxidoreductases on stoichiometric supply of reducing power in the form of reduced cofactors. H2-driven cofactor regeneration by using hydrogenases offers a cheap and green solution and has been applied in various biotechnological approaches both in-vivo and in-vitro. While electro-driven biocatalytic reactions have been demonstrated by green H2 coupled with hydrogenase, the engineering requirements per oxidoreductase and scale-up, as well as systematic understanding of the relevant biocatalytic parameters for process optimization, remain largely unexplored. This work demonstrates the use of green hydrogen from water electrolysis to drive biocatalytic reactions involving NAD(P)H- and flavin-dependent cofactors in flow systems using electricity. The flow platform was employed not only to enable reaction scale-up, but also to systematically investigate individual biocatalytic steps through the integration of H2/O2 sensorsand online spectrophotometric analysis. This approach enabled improved process efficiency while providing mechanistic insight into the biocatalytic reactions through an improved understanding of the interplay between electrocatalysts and biocatalysts. Therefore, this research actively studies by investigating the benefits of flow chemistry, biocatalysis, and leverage electrochemical methods to demonstrate target-oriented synthesis using biocatalysis that can use electricity to fuel the reaction, which aligns with green chemistry principles. This thesis is divided into three chapters: Firstly, a closed-loop flow setup was designed for electro-driven, FMNH2-dependent biocatalytic ene-reduction. Soluble hydrogenase (SH), immobilized on Strep-Tactin XT 4Flow resin, and the enoatereductase TsOYE, immobilized on EziG beads, were integrated into a column as a packed-bed flow reactor. Necessary H2 generated by a PEM electrolyzer was transferred into the flow volume through agas-addition module, allowing highly efficient non-segmented gas addition without direct bubbling. The system enabled full reduction of ketoisophorone to levodione, reaching >99% conversion at 25 mM substrate concentration within 22 h. The immobilized biocatalysts retained efficient activity over seven consecutive reaction cycles, again maintaining >99% conversion. In the upscaled 185 mL reaction, levodione was obtained in 89 % isolated yield (471 mg, 96% purity; theoretical yield 527 mg). The corresponding TTNs reached 1.01×105 for SH and 1.69×104 for TsOYE during seven consecutive reaction cycles, reusing the immobilized biocatalysts. The TTN was further increased to 3.2×105 forSH and 2.6×104 for TsOYE in the upscaled reaction, highlighting the robustness of both immobilized enzymes under flow operation. Secondly, a closed-loop flow setup was developed for electro-driven lactone synthesis by coupling the enoate reductase XenB with cyclohexanone monooxygenase (CHMO) and H2-driven cofactor regeneration by SH. The flow system was equipped with two separate gas-addition modules, establishing an H2 phase for the immobilized SH and XenB, followed by an O2 phase for the immobilized CHMO, with dissolved O2 maintained at 20–40%. Two cofactor-matched cascade designswere investigated: an NADH-based system with wild-type SH and an NADH-switched CHMO variant, and an NADPH-based system with the stabilized CHMO M16DS variant and engineered NADPH-SH. In batch, the initial NADH-based cascade reached only 35% conversion, and troubleshooting identified caprolactone instability and catalase-related side reactions as major causes of poor mass balance. After switching to CHMO M16DS and NADPH, full conversion of 10 mM cyclohexanone to ε-caprolactone was achieved in batch. In flow, however, both cascade configurations remained limited, reaching 17%conversion in the NADPH-based system and 13% in the NADH-based system. Onlinespectro photometric monitoring showed that leaching of the immobilisation resin, Amberlite FPA54, interfered with measurements at 370 nm. Meanwhile, flavin leaching from the immobilised XenB-CHMO module complicated spectrophotometric interpretation, indicating unproductive flavinrecycling. This led to potential ROS formation upon contact with O₂, further impairing cascade performance and product stability. Thirdly, a flow setup was designed for electro-driven O-demethylation in upscaled reaction while addressing the inhibitory side product formaldehyde. SH was immobilized on Amberlite FPA54 by adsorption and coupled to the Rieske oxygenase VanA/VanB, which was entrapped in a dialysis membrane to preserve the proximity and flexibility required for efficient electron transfer. The membrane-entrapped oxygenase module was operated at 30 °C and kept open to allow continuouse vaporation and removal of formaldehyde. H2 and O2 produced by PEM water electrolysis were introduced through two separate gas-addition modules equipped with different gas-permeable tubings. Among the investigated O-demethylase systems, VanAB showed the highest robustness and was therefore selected for flow application. In the closed-loop flow setup, >99% conversion of 50 mMvanillate to protocatechuate was achieved within 5 h. The immobilized/entrapped biocatalysts reached TTNs of up to 3.5 × 105 for SH and 5.0 × 103 for VanAB. In addition, the system achieved a Faradaic efficiency of 18% after 5 h, while the electron contribution to product formation reached 62% at an earlier time point. Overall, this work demonstrates that PEM water electrolysis can be effectively integrated with H2-driven cofactor regeneration and immobilized oxidoreductases to realize electro-driven biocatalysis incontinuous closed-loop flow systems. Across the three case studies, the platform proved suitable for FMN-dependent ene-reduction, nicotinamide-dependent redox cascades, and O-demethylation, while also enabling reaction scale-up, online monitoring, and improved process control. The most mature systems were the TsOYE-based ene-reduction and the VanAB-based O-demethylation, both of which delivered high conversions together with high TTNs, underscoring the operational stability of the immobilized biocatalysts. For future development, further improvements should focus on increasing Faradaic efficiency, reducing gas losses, and strengthening the integration between electrolysis and biotransformation. In the lactone cascade in particular, improving CHMO stability, reducing uncoupling, and controlling product degradation will be essential to achieve full cascade performance in flow. More broadly, the modular reactor concept established here provides a basis for extending H2-driven electro-biocatalysis to other multi-enzyme cascades, especially those involving O2-dependent oxidoreductases and complex redox networks. This opens a route toward scalable and selective synthesis in which electricity and water serve as the ultimate source of reducing power for chemical synthesis using biocatalysts.

Authors 1

1. Guiyeoul Lim corresponding Aachen

   RWTH Aachen University

   Affiliation as printed

   RWTH Aachen