
==== Front
Commun Chem
Commun Chem
Communications Chemistry
2399-3669
Nature Publishing Group UK London

1288
10.1038/s42004-024-01288-y
Article
H2-driven biocatalysis for flavin-dependent ene-reduction in a continuous closed-loop flow system utilizing H2 from water electrolysis
Lim Guiyeoul 1
Calabrese Donato 1
http://orcid.org/0009-0009-2395-0168
Wolder Allison 2
Cordero Paul R. F. 1
Rother Dörte 13
http://orcid.org/0000-0003-4140-002X
Mulks Florian F. 4
http://orcid.org/0000-0002-7889-9920
Paul Caroline E. 2
http://orcid.org/0000-0002-6601-6473
Lauterbach Lars lars.lauterbach@iamb.rwth-aachen.de

1
1 https://ror.org/04xfq0f34 grid.1957.a 0000 0001 0728 696X Institute of Applied Microbiology—iAMB RWTH Aachen University, Aachen, Germany
2 https://ror.org/02e2c7k09 grid.5292.c 0000 0001 2097 4740 Biocatalysis Section, Department Biotechnology, Delft University of Technology, Delft, The Netherlands
3 grid.8385.6 0000 0001 2297 375X Institute for Bio-and Geosciences 1: Biotechnology Forschungzentrum Jülich GmbH, Jülich, Germany
4 https://ror.org/04xfq0f34 grid.1957.a 0000 0001 0728 696X Institute of Organic Chemistry—iOC RWTH Aachen University, Aachen, Germany
7 9 2024
7 9 2024
2024
7 20029 4 2024
29 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by/4.0/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
Despite the increasing demand for efficient and sustainable chemical processes, the development of scalable systems using biocatalysis for fine chemical production remains a significant challenge. We have developed a scalable flow system using immobilized enzymes to facilitate flavin-dependent biocatalysis, targeting as a proof-of-concept asymmetric alkene reduction. The system integrates a flavin-dependent Old Yellow Enzyme (OYE) and a soluble hydrogenase to enable H2-driven regeneration of the OYE cofactor FMNH2. Molecular hydrogen was produced by water electrolysis using a proton exchange membrane (PEM) electrolyzer and introduced into the flow system via a designed gas membrane addition module at a high diffusion rate. The flow system shows remarkable stability and reusability, consistently achieving >99% conversion of ketoisophorone to levodione. It also demonstrates versatility and selectivity in reducing various cyclic enones and can be extended to further flavin-based biocatalytic approaches and gas-dependent reactions. This electro-driven continuous flow system, therefore, has significant potential for advancing sustainable processes in fine chemical synthesis.

Flavin-based biocatalysis using flavin mononucleotide (FMN) cofactor attracts significant attention for its application in asymmetric alkene reduction and various other reactions, however, the scale-up of flavin-based biocatalysis in flow remains unexplored. Here, the authors develop a closed-loop flow platform for H2-driven regeneration of cofactor FMNH2 and ene-reduction using immobilized Old Yellow Enzyme, achieving >99% conversion of ketoisophorone to levodione.

Subject terms

Biocatalysis
Immobilized enzymes
Oxidoreductases
Flow chemistry
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 955740. This project has received funding from the European Research Council (ERC) under the European Union&apos;s Horizon 2020 research and innovation programme (grant agreement n° 949910). This project is funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany´s Excellence Strategy – Cluster of Excellence 2186 „The Fuel Science Center” – ID: 390919832. Further funding was received through a Liebig Fellowship by the Fonds der Chemischen Industrie, grant number Li 210/01This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 955740. This project has received funding from the European Research Council (ERC) under the European Union&apos;s Horizon 2020 research and innovation programme (grant agreement n° 949910).issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Continuous flow biocatalysis is increasingly recognized as a sustainable manufacturing methodology within the pharmaceutical and fine chemicals sectors1–3. It offers improved control over reactions, minimizes waste generation, and enhances efficient use of energy4. The application of such systems is not limited to conventional chemical synthesis, it can be adapted to biocatalysis, using enzymes as catalysts to enable efficient syntheses under mild conditions5. Biocatalysis in continuous flow has become more feasible due to the advancements in flow chemistry and enzyme immobilization techniques6,7, harnessing the highly stereo- and regioselective catalytic power of enzymes. One of the most useful reactions for generating stereogenic centers involves the asymmetric reduction of activated C=C bonds8,9. This ene-reduction can be catalyzed by an ene reductase from the Old Yellow Enzyme family (OYE)10, which contains a prosthetic flavin mononucleotide (FMN) cofactor. Through trans-hydrogenation, OYE catalyzes the asymmetric reduction of α,β-unsaturated carbonyl compounds11. The commonly recognized process of regenerating enzyme-bound FMNH2 involves the reduced nicotinamide cofactor NAD(P)H, although OYE shows promiscuity with respect to the sources of reductant12–14, including receiving electrons from reduced free-flavins10,15,16. Flavin-based biocatalysis has recently attracted considerable attention not only for its application in C=C double bond reduction, but also for its applicability in epoxidation, hydroxylation and nitro group reduction, due to its low-cost cofactor compared to NAD(P)H17–19. Consequently, alternative regeneration methods such as photochemical and electrochemical approaches have been reported to regenerate the free flavins. These methods, however, are constrained by limitations, such as low conversion rates and stability20–24. In vivo ene-reduction in continuous flow using OYEs has demonstrated high conversion rates25,26. However, in vivo reactions pose challenges in downstream processing, as unwanted materials or contaminants can be co-purified. In- vitro biocatalysis in flow chemistry can significantly simplify the downstream process, and the potential for scaling up flavin-based biocatalysis through the application of flow chemistry remains unexplored4.

Molecular hydrogen (H2) serves as an ideal reductant since it does not produce any waste and can be produced by electrolysis of water using renewable energy. H2 can be reversibly oxidized by the soluble hydrogenase of Cupriavidus necator (SH) to reduce NAD+ to NADH27. This process involves a hydrogenase module, HoxHY, for H2 oxidation and a reductase module, HoxFUI2, for NAD+ reduction. Due to its 100% atomic efficiency28, SH/H2 presents as an excellent system for NADH cofactor regeneration29 compared to existing NAD(P)H-recycling systems, which often suffer from low activity or produce unwanted side products30. The native NAD+-reducing ability of SH can be further extended to NADPH through rational mutagenesis, allowing H2-driven NADPH regeneration31. Additionally, SH exhibits tolerance to ambient O2 condition32, making it a valuable tool for the recycling of NAD(P)H in various biocatalytic reactions for the production of amines and alcohols33–35. The application of hydrogenases has also been explored for regenerating synthetic analogous of NAD(P)H14 and reduced flavin cofactors17–19. Coupling of SH with the thermostable OYE from Thermus scotoductus (TsOYE)36 and styrene monooxygenase allowed the regeneration of FMNH2 for the reduction of C=C double bonds, and reduced flavin adenine dinucleotide (FADH2) for the epoxidation of styrenes, respectively, in small-volume batch reactions17. This work shows that SH is an atom-efficient regeneration system that uses H2 as electron source for regenerating not only NAD(P)H but also flavin cofactors. Compared to Hyd1 from E. coli18, which lacks a reductase module, the SH demonstrates nearly two orders of magnitude higher specific activity (5.8 U mg−1) for FMN reduction17. This indicates a significant contribution of the reductase module of SH to free FMN binding and reduction37,38.

The in- situ production of H2 by water electrolysis effectively minimizes the risks associated with handling large quantities of explosive H2. Photoelectrochemical oxidation of water has been applied to fuel enantioselective reduction of TsOYE as a cathodic reaction39. However, it has a limited efficiency due to low current densities (<1 mA cm−2) when coupling the photocatalysts with the biocatalysts, posing challenges for upscaling the reaction40. The use of electrical energy to drive NADH-dependent biocatalytic processes in a flow system, using H2 as a mediator, has been previously successfully demonstrated. In this process, electricity was used to split water via a simple two-electrode system to produce H2 and O241. The hydrogen gas was then introduced into the flow system by liquid-to-liquid gas transfer, facilitated by gas-permeable Teflon™ AF-2400 tubing41,42. Additionally, a commercial continuous-flow hydrogenation reactor has been applied in flow biocatalysis, designed for optimizing H2 availability which may become a relevant efficiency factor43. In this work, a simple and cost effective method of supplying different gases to the flow system was developed by integrating gas permeable tubing into a closed loop system. This approach allows continuous and efficient delivery of H2 and reduced cofactors without damaging the biocatalysts. For energy-efficient water electrolysis, proton exchange membrane (PEM) electrolyzer provide a suitable method to produce pure H2 with >60% voltage efficiency and >95% faradaic efficiency44. Compared to standard alkaline water electrolyzers, PEM electrolyzers can operate in high current densities, have instant current response, and can be coupled with renewable energy sources45. To introduce H2 produced from the PEM electrolyzer via gas-to-liquid transfer without bubble formation, tube-in-tube methods or the use of gas-permeable tubes in a gas addition module have been selected for application in flow chemistry46,47.

In this study, the main novelty is in development of a closed-loop flow platform for electro-driven flavin-dependent biocatalysis via H2 as a mediator produced by a commercial PEM electrolyzer in combination with a gas addition module. Thermostable TsOYE, as a reduced flavin-dependent model enzyme, was immobilised via coordination bonds and coupled to SH. To enable regeneration of the flavin cofactor, SH was immobilised by affinity binding and applied to the flow system. This system, which facilitates efficient gas-to-liquid H2 transfer, was thoroughly evaluated for biocatalyst immobilization, system stability, reusability, and adaptability with different substrates, establishing its potential for technical-scale flavin-dependent biocatalytic reactions.

Results and discussion

Gas addition module design and H2 supply for H2-driven flow biocatalysis

A closed-loop flow system was developed to serve as a versatile platform for electro-driven enzymatic reactions (Fig. 1). This system contained the H2-dependent SH for the recycling of reduced flavin FMNH2. H2 was supplied from water electrolysis by a commercially available lab-scale PEM electrolyzer with an IrRuOx anode and a mixed PtB/PtC (platinum black, platinum-supported carbon) cathode. The evolved H2 was fed into the flow system through gas-permeable membrane tubing within a constructed metal-encased gas addition module (see SI chapter 3). For H2 transfer from gas-to-liquid phase in the gas addition module, tubes made of polymethylvinylsiloxane (PVMS) with a high gas permeability and polytetrafluoroethylene (PTFE) as inert material were evaluated41,48. PVMS tubes with a membrane thickness of 250 µm and a length of 2 m rolled up in the gas addition module showed an H2 transfer rate of 0.647 µmol min−1 at 1 bar H2 pressure in a single pass through the tubing (see SI chapter 4). Therefore, it was favored for its high gas transfer efficiency. Conversely, PTFE tubing with a lower H2 transfer rate of 0.418 µmol min−1 was used as a non-reactive alternative in cases where chemical interactions with PVMS were observed.Fig. 1 Platform for electro-driven FMNH2-dependent asymmetric reduction of cyclic enones.

H2 is produced from a PEM electrolyzer using PtB/PtC as hydrogen evolution catalyst. A gas permeable tubing (PVMS or Teflon) transfers the H2 from the gas addition module to the flow system (17 mL, flow rate 2.6 mL min−1). H2 was supplied during biocatalysis to the gas addition module from the PEM electrolyzer (H2 10 mL min−1, 3.4 V, 0.89 A). Clark-type sensors for H2, optical sensors for O2, and temperature sensors and spectrophotometer (FMN) were integrated into the flow system to monitor online the interplay between enzymes and the electrolyzer. Due to overlapping of the substrates and FMNH2 absorbance peak, wavelength of 500 nm was used for FMN detection. The enzymes SH, and TsOYE were immobilized by Strep-Tactin XT 4Flow (down) and EziG beads (up), respectively, and packed into a column within the flow system. Reaction conditions: 17 mL, 50 mM Tris-HCl pH 8 at 20 °C containing SH (5 mg), TsOYE (6.5 mg), FMN (1 mM), catalase (5 mg) substrate 1 and 3 (25 mM), substrate 5 and 7 (5 mM). DMF as a cosolvent was added with the substrate at the ratio of 1:2. Conversions and ee values were determined by GC-FID. The flow system was at room temperature and the column with immobilized biocatalysts in temperature-controlled chamber was set at 30 °C. For upscale reaction, 3 was used at 18.5 mM concentrations.

Closed-loop flow biocatalysis reactions with immobilized enzymes

For facilitated product isolation and allowing reusability of enzymes in the flow setup, immobilization of SH and TsOYE simplifies product isolation and enables the recycling of enzymes within a flow system. However, adsorption of FMN was detected on the material Amberlite FP54TM, previously used in flow setup for SH immobilization49. Therefore, the Strep-Tactin XT 4flow resin was assessed as a potential alternative for application in flow chemistry, aimed at Strep-tagged SH immobilization (SH-Tactin)32,50.

Analogously, TsOYE was equipped with a 6xHis-tag on the N-terminal site and was immobilized on porosity glass metal ion affinity EziG beads from EnginZyme51. Different types of EziG beads varying in hydrophilicity were evaluated for TsOYE immobilization to assess performance (see SI chapter 5). Employing affinity immobilization techniques, His-tagged TsOYE with EziG beads and strep-tagged SH with Strep-Tactin were used in this proof-of-concept study to rationalize the purification and immobilization of both SH and TsOYE into a one-step process. This approach showed good residual activity and high immobilization yields (see SI chapter 2.4), while also simplifying the immobilization process compared to covalent bonding methods.

Subsequently, we investigated the electro-driven asymmetric reduction of cyclic enones using in -situ generated H2 within the enzymatic system. Given that the KM of SH is approximately 680 µM, we used 1 mM FMN to ensure an adequate concentration close to kcat of SH. To avoid reactive oxygen species (ROS) from the reaction of reduced FMNH2 with O2 diffusing into the flow system, which potentially damages metal-dependent enzymes52, catalase was introduced into the circulating system. The immobilized enzymes, SH and TsOYE, were packed into a column which was integrated into the flow reactor. To ensure higher immobilization yield of SH, it was essential for Strep-Tactin resin to be packed and settled for 24 h before immobilization. This led to a defined arrangement of the immobilized biocatalysts in the biotransformation unit where SH-Tactin is positioned beneath the TsOYE-EziG (Figs. 1, S2). Considering the relatively weaker binding affinity between the Strep-tag and Strep-Tactin compared to coordinate bonds, there is potential for SH leaching. This involves the possibility of SH circulating around the flow setup and being reintroduced into the packed bed reactor.

During the reaction, we conducted online monitoring of various parameters, such as H2 evolution and consumption, potential O2 leakage, and FMN reduction (Fig. 2). This was done to monitor each catalytic step and assess whether H2 concentrations and FMNH2 were sufficient to initiate and progress the reaction. In that way, the functionality of each enzyme was comprehensively assessed, ensuring that no parameter within the cascade was limiting. The time at which the reaction ended could be determined by observing the saturation of H2 and the low concentration of oxidised FMN at 19 h and 22.5 h respectively (Fig. 2). It is worth noting that such a monitoring system, as shown in Fig. 1, had previously only been used in-vivo during fermentations and one other in flow chemistry setup41. This novel application in our current study represents a significant advancement in the ability to monitor enzymatic processes and identification of potential bottlenecks.Fig. 2 On-line monitoring of H2, O2, and FMN during the electro-driven biotransformation.

The concentration of H2 (A, blue), O2 (A, green), and oxidized FMN (B, orange) were followed during 24 h of the transformation of 3 to 4 as exemplary biotransformation. H2 was supplied to the system with the rate of 11 mL min−1 by the PEM electrolyzer (3.4 V, 0.89 A) and gas addition module. The electrolysis was performed until H2 concentration reached a plateau and then FMN was added (black arrow). After H2 concentration started to increase again and FMN was fully reduced to FMNH2, substrate was added to ensure reducing conditions for TsOYE activity. Purple arrows indicate the activity of each enzyme: the activity of TsOYE is indicated by oxidizing FMNH2 to FMN, the activity of soluble hydrogenase (SH) is shown in the consumption of H2, and the reduction of FMN to FMNH2. The experiment was performed as described in SI 3.2.

Substrate scope of flow system

A range of substrates was chosen according to the substrate spectrum of TsOYE to assess the adaptability of the flow system in 17 mL volume with various substrates (Fig. 1)16. Cyclohexenone (1) was first selected for its high water solubility and relatively high conversion53. In a 17 mL flow reaction, 25 mM of (1) yielded a 35% conversion to cyclohexanone (2) within 15 h, while no further conversion was detected. This conversion rate is consistent with previous batch experiments17, but this yield is lower compared to other reactions using NADPH53 (Table S4). The lower conversion rate could be due to the propensity of (1) for Michael's addition with water54. This combined with the potential for FMNH2 to generate H2O2 in the presence of O2 traces during substrate injection or sampling32,38,55 could have contributed to side reactions and resulted to low conversions. Our detection of hydroxylated side products via GC-MS in the flow system suggested a potential issue with side reactions (see SI chapter 14.3). Side products were also observed when SH and TsOYE were entrapped in an enzymatic membrane reactor (EMR) with a cutoff membrane of 30 kDa as an alternative immobilization technique (see SI chapter 6)56. (1) yielded a 28% conversion to (2) after 6 days in an EMR. While the utilization of EMR extended the enzymatic activity, it also increased the likelihood of side product formation due to its longer reaction time, rendering it unsuitable for this application.

Subsequently, the reduction of ketoisophorone (3) to levodione (4) was evaluated in the flow system due to the pharmaceutical significance of levodione as a building block. Complete conversion of 25 mM (3) to (4) was achieved 22 h after addition of substrate (>99%, Fig. 1, Figure S6). Despite constantly attaining high conversion rate, the flow reaction produced (4) with a relatively low optical purity, i.e., from ee = 77% R at 2 h after substrate addition to ee = 39% R at 22 h (see SI chapter 7). This decrease of optical purity has been linked to non-enzymatic racemization in water due to keto-enol tautomerization during sample preparations or to prolonged reaction conditions17,57,58, as well as the non-enzymatic reduction of (3) to racemic product (4) by free FMNH221. Additionally, it has been demonstrated that a lower concentration of the enzyme contributes to higher optical purity, which introduces the possibility of reversibility of the reaction by the enzyme57.

(S)- and (R)-carvone (5, 7) were also included to assess the performance of the flow system with chiral centers. The same flow volume, 17 mL, was used with these chiral substrates (5, 7), but at a reduced concentration of 5 mM due to their low solubility in aqueous solution. It is important to note that during the reaction, loss of substrate due to adsorption on PVMS tubing was detected by GC-FID. Hence, PTFE was used as the tubing for H2 transfer for reactions with carvone as substrate. After 16 h substrate addition, (5) resulted in 15% formation of the product (1R, 4S)-dihydrocarvone (6) (ee = 68%), while (7) exhibited an 11% conversion to (1R, 4R)-dihydrocarvone (8) (ee = 74%) (Fig. 1). In flow reaction, substrate conversion was similar to the batch reaction17. However, lower optical purity was observed, which can be also attributed to product racemization due to keto-enol tautomerization59,60.

Reusability of immobilized biocatalysts and upscaling

Reusability of the immobilized enzymes and the scalability of the reaction in flow setup was then investigated. Product (4) was synthesized from (3) with a maintained product formation of >99% throughout seven cycles without replacing the enzymes due to high enzyme loading (Fig. S7), demonstrating reusability of the system (see SI chapter 8). The biocatalysts utilized in these flow reactions achieved a total turnover number (TTN) of 1.01 × 105 for SH-Tactin and 1.69 × 104 for TsOYE-EziG (see SI chapter 9, Table S3). Overall, the consistently high conversion rates (>99%) in multiple cycles, along with the high TTN, especially for sophisticated oxidoreductases, illustrate the remarkable stability of the biocatalysts, indicating that Strep-Tactin XT 4Flow resin and EziG beads are robust immobilization carriers for SH and TsOYE, respectively. These observations corroborate previous findings, where EziG beads served as a stable enzyme carrier for imine reductase (IRED) and IRED-EziG was reused for up to 14 cycles61. The high TTN of SH-Tactin and TsOYE-EziG highly suggests that stable enzymes have the potential to be used in upscaled flow reactions.

We also determined the Faradaic efficiency for the electro-driven reduction of (3) to (4) in the flow system. With an applied voltage of 3.3 V and a current of 500 mA, the Faradaic efficiency of the flow system was calculated to be 0.15% (see SI chapter 12). The limited electron contribution to product formation compared to other work39 stems from the requirement of H2 gas outflow from the gas addition module, which is a drawback for commercially available PEM electrolyzers that have difficulty in managing back pressure if the module remains closed. Additionally, the commercial PEM electrolyzer only facilitated stable electrolysis of water at high voltage (>3 V) to overcome the internal resistance in the cell. To increase Faradaic efficiency or the energy efficiency of the system, zero gap cells can be used where lower amount of electrical energy is used for the production of exact amount of H2 that is needed for the biocatalytic reaction62,63.

Finally, we increased the volume of the flow reaction to 185 mL to demonstrate the scalability of the system. This was done by adding 168 mL segment that increases the volume without any headspace to inhibit O2 introduction to the system. Compound (3) was used as substrate due its high conversion in the flow setup (Fig. 1). Product formation of above 99% was achieved after 77 h (see SI chapter 10), resulting to a theoretical yield of 527 mg (4). The employed procedure ensured a practical product isolation. Simple extraction of the collected dispersion with diethyl ether yielded 89% (471 mg) of (4) in 96% purity. The TTN of mol product per mol SH-Tactin and TsOYE-EziG in the 185 mL upscaled reaction reached up to 3.2 × 105 and 2.6 × 104, respectively, with indications that further upscaling can possibly yield even higher TTN. This approach slightly exceeds the performance of previous biphasic batch reactions for (4) production in TsOYE-TTN as well as by an order of magnitude the TTN achieved by the Hyd1 hydrogenase from E. coli18,64. The high TTN values for SH-Tactin and TsOYE-EziG demonstrate that immobilizing Strep-tagged SH with Strep-Tactin resin and 6xHis-tagged TsOYE with EziG beads are optimal methods for significantly enhancing the robustness of biocatalysts in flavin-dependent biocatalytic reactions, surpassing the performance of previous biphasic batch reactions for (4) production in TsOYE-TTN. Furthermore, the SH TTN during FMN recycling was an order of magnitude lower than during NADH recycling, indicating the physiological electron acceptor improves enzyme stability41.

The specific activity of SH for reducing FMN is 5.8 U × mg−1, with a KM,FMN of 680 μM17. Thus, we decided to use 1 mM FMN in the reaction, which resulted in a slightly lower TTN of mol product per mol FMN in comparison to other studies (see Table S4). It is worth noting that catalase (~5000 U) was added every day to mitigate potential inhibition by ROS, ensuring full product conversion. This highlights the adverse impact of ROS, particularly H2O2, on enzymatic activity. E factor was also quantified to compare the waste generated by product65,66. 17 mL and 185 mL reactions were calculated to E factor of 6.0 and 5.7, respectively (see SI chapter 13), which represents low waste production compared to other FMNH2 regeneration systems19. This approach to electro-driven flavin-based biocatalysis in a flow system demonstrates a promising pathway for scalable and efficient biotransformations.

Conclusion

In this study, we established a closed-loop flow system using immobilized biocatalysts to convert electrical energy into chemical energy for production of fine chemicals. This flow system demonstrates adaptability to a range of substrates, scalability, and serves as an electro-driven system for regenerating flavin cofactors. It can be implemented to diverse biocatalytic processes that are dependent on reduced flavins such as styrene monooxygenase, unspecific peroxygenase-catalyzed hydroxylation via H2O2 from reduced flavin re-oxidation with O2, and nitroreductases10,17–19. Apart from improving the H2-availability for H2-driven biocatalysis with a PEM electrolyzer and highly gas-permeable tubing PVMS, this study introduces Strep-Tactin resin and highlights EziG beads as stable enzyme carriers suitable for flavin-dependent biocatalytic applications in a flow setup. It was highly advantageous that Strep-Tactin resin did not show adsorption of FMN, unlike Amberlite resin, an alternative for immobilizing SH49. Additionally, using coordination bonds between the EziG beads and the 6xHis-tag on the enzyme, high residual activity was maintained while demonstrating remarkable stability, compared to other methods such as covalent irreversible immobilization. However, the use of aqueous solutions led to side reactions, notably in cyclohexenone conversion, with observed low optical purity of the chiral products. The subsequent challenge will be to incorporate immiscible organic solvents to the flow setup to increase the solubility of substrates in water and the address the poor optical purity of chiral products57. TsOYE-EziG can be further investigated regarding its stability in organic solvents within a micro-aqueous environment, following previous studies on TsOYE immobilized via adsorption on Celite67.

It is also crucial to understand the significant differences in enzyme behavior between flow and batch reactions, including effects on activity, stability, and stereoselectivity, in order to further fine-tune conditions and preserve desired enzyme functionalities in flow reactions, especially in large scales. Overall, this scalable platform for electro-driven flow biocatalysis demonstrates high potential for chemical synthesis and can be flexibly adapted to other gas-dependent enzymes by attachment of corresponding gas bottles to the gas addition module. These enzymes include O2-dependent P450 monooxygenase, CH4-/O2-dependent soluble methane monooxygenases, or CO2-dependent formate dehydrogenases for hydroxylation and CO2 fixation reactions, respectively. The integration of advanced monitoring systems, effective biocatalyst immobilization, and careful selection of reaction conditions have provided a robust basis for further investigation and optimization of enzymatic processes in flow systems.

Methods

Plasmid construction, growth conditions, and protein purification

TsOYE (Thermos scotoductus SA-01 ATCC 700910) was recombinantly produced in E. coli BL21 Gold (DE3) cells with the corresponding plasmid in a pET-28a(+) vector, with an N-terminal His-tag (NdeI/EcoRI). For TsOYE purification, 50 mL pre-culture of LB medium containing 50 μg/mL of kanamycin was inoculated with toothpick of a colony from transformed cells with plasmid pET-28a(+)-TsOYE and incubated overnight at 37 °C and 180 rpm. Overexpression was carried out in 2 L of TB medium supplemented with 50 μg/mL of kanamycin. The main cultures were inoculated with 1% of pre-culture and grown at 37 °C and 180 rpm. When an OD600 of ~0.6 was reached (~2 h 30 min), 0.1 mM of IPTG was added. After induction, cultures were incubated overnight at 30 °C and 180 rpm. Cells were harvested by centrifugation (18,692 × g for 30 min at 4 °C). The obtained cell pellets (~27 g wet pellet mass) were washed and suspended in approximately 30 mL MOPS-NaOH buffer (20 mM, pH 7.0), supplemented with a spatula spoon tip of DNase and MgCl2, and one pill of EDTA-free cOmpleteTM protease inhibitor cocktail. Cells were disrupted using Multi Shot Cell Disruption System (1 cycle at 1.36 kbar pressure) and the cell debris was separated from the crude extract by centrifugation (18,692 × g for 30 min at 4 °C). The obtained supernatant was incubated in 50 mL falcon tubes in a water bath at 70 °C for 1 h 30 min. Precipitated proteins were separated by centrifugation at 38,759 × g for 30 min at 4 °C. (See SI chapter 2.2 for TsOYE amino acid and nucleotide sequence respectively).

The production and purification of NAD+-reducing hydrogenase (SH) was performed as described in Lauterbach et al. (2013)32.

Immobilization of biocatalysts

Strep-Tactin® XT 4Flow resin and EziG beads were used to immobilized the enzymes. For SH immobilization, the Strep-Tactin® XT 4Flow resin was first packed inside the C 10/10 column (Cytiva) and let settle for 1 day at 4 °C. Purified SH fused with a Strep-tag was loaded into the column bed at 3.3 mg SH per g resin. For TsOYE immobilization, the carrier used was EziG Amber beads. In a 50 mL centrifuge tube, TsOYE fused with 6×His-tag was loaded to the carrier (14.4 mg g−1 EziG), followed by incubation by overnight at room temperature in a roller shaker (30 rpm). The immobilized TsOYE in EziG was then resuspended and added to the C 10/10 column. Catalase was added on to the top (2,000 U) before closing. The C 10/10 column, with SH-Tactin underneath and TsOYE-EziG on the top, was used as the biotransformation unit and was attached to the flow setup. For further detailed steps see SI chapter 3.1.

Reactions in flow setup

Each experiment in flow setup was done in step-reactions to check the functionality of the gas addition module, SH-Tactin, and TsOYE-EziG. The flow volume (Tris-HCl 50 mM, pH 8, 30 °C) was first saturated with H2 by starting the PEM electrolyzer to 0.89 A to produce rate of 11 mL min−1 H2 gas. The generated H2 was added to the flow volume through the gas-addition module via the gas-permeable tubing (SI chapter 3). Once H2 saturation was observed, 1 mL of H2-saturated stock solution of FMN (concentration depending on the flow volume) was added to the flow volume to prevent O2 from entering the system. After observing reduction of FMN to FMNH2 and checking the functionality of SH, substrate was added (Fig. 2). The consumption of H2 after free FMNH2 was depleted was indicative of TsOYE-EziG and SH-Tactin activities. Upon saturation of H2 was observed, the reaction was stopped and the flow volume was stored for analysis.

For 17 mL flow reactions, flow volume with 1 mM FMN, 2000 U catalase, and substrate (either 25 mM ketoisophorone, 25 mM cyclohexenone, or 5 mM (S)- or (R)-carvone). For the 185 mL upscaled flow volume, the reaction involved a mobile phase with 500 µM FMN, ~5000 U catalase (added every day), and 18.5 mM of substrate ketoisophorone. A segment of 168 mL was added to the existing 17 mL to increases the volume without any headspace. For further detailed information of reaction by substrate see SI chapters 3.2 to 3.5.

Analytics

Compound analysis was carried out on Thermo Scientific Trace GC Ultra instrument with a AS 3000 auto-sampler coupled with flame ionization detector (FID) using helium as carrier gas. Products were confirmed by reference standards. Details of the column and temperature programs used are given in Table S6. All measurements were calibrated with calibration curves. Only conversion rate was quantified with GC-FID. Enantiomeric excess (ee %) of compounds was assessed with Shimadzu GC-2010 equipped with an FID using helium as carrier gas (see SI chapter 14.2). Details of the column and temperature programs used are given in Table S7. Time between experiments and GC measurements was several days which also explained the poor enantiomeric excess obtained (racemization of certain products occurred). Gas chromatography-mass spectrometry was carried out on a Thermo Scientific Trace GC Ultra instrument coupled with a Thermo Scientific ISQ mass selective detector (MSD) and an additional flame ionization detector. Injector temperature: 250 °C. Split mode with a split ratio of 10. Detector temperature: 320 °C. Electron ionization of the analyte with 70 eV acceleration voltage. For further information see SI chapter 14.3. Nuclear magnetic resonance spectra were recorded on a BRUKER Avance Neo 600 spectrometer at 26 °C and were analyzed with the software Mnova 14.2.3 (Mestrelab Research). Chemical shifts δ were reported in parts per million (ppm). The residual solvent signals were used for referencing 1H NMR spectra (7.26 ppm for CDCl3)68,69. A relaxation delay of D1 = 25 s was used for quantitative 1H NMR experiments. For further information see SI chapter 15.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Supplementary information

Peer Review File

Supplementary Information

Reporting Summary

Supplementary information

The online version contains supplementary material available at 10.1038/s42004-024-01288-y.

Acknowledgements

We thank EnginZyme (Stockholm, Sweden) for EziGTM beads. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 955740, from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No. 949910) and from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany´s Excellence Strategy—Cluster of Excellence 2186 “The Fuel Science Center”—ID: 390919832. Further funding was received through a Liebig Fellowship by the Fonds der Chemischen Industrie, grant number Li 210/01 (F.F.M.).

Author contributions

Conceptualization: L.L.; Experiments: G.L., D.C., A.W., F.F.M.; Investigation and analysis: G.L., D.C., A.W., F.M., P.R.F.C., A.D., D.R., C.E.P., L.L.; Writing—original draft: G.L., D.C., P.R.F.C.; Writing—editing and review: G.L., D.C., A.W., F.F.M., P.R.F.C., D.R., D.C.P., L.L.; Supervision and funding: L.L. All authors contributed to the discussion. All authors have given approval to the final version of the manuscript.

Peer review

Peer review information

Communications Chemistry thanks László Poppe and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Funding

Open Access funding enabled and organized by Projekt DEAL.

Data availability

Raw and metadata for this contribution are available at 10.5281/zenodo.10798153.

Competing interests

The authors declare no competing interests.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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References

1. Meyer L-E Hobisch M Kara S Process intensification in continuous flow biocatalysis by up and downstream processing strategies Curr. Opin. Biotechnol. 2022 78 102835 10.1016/j.copbio.2022.102835 36332339
Meyer, L.-E., Hobisch, M. & Kara, S. Process intensification in continuous flow biocatalysis by up and downstream processing strategies. Curr. Opin. Biotechnol. 78, 102835 (2022).36332339 10.1016/j.copbio.2022.102835
2. Ley SV On being green: can flow chemistry help? Chem. Rec. 2012 12 378 390 10.1002/tcr.201100041 22711555
Ley, S. V. On being green: can flow chemistry help? Chem. Rec. 12, 378–390 (2012).22711555 10.1002/tcr.201100041
3. Tang, Z., Oku, Y. & Matsuda, T. Application of immobilized enzymes in flow biocatalysis for efficient synthesis. Org. Process Res. Dev. 28, 1308–1326 (2024).
4. Britton J Majumdar S Weiss GA Continuous flow biocatalysis Chem. Soc. Rev. 2018 47 5891 5918 10.1039/C7CS00906B 29922795
Britton, J., Majumdar, S. & Weiss, G. A. Continuous flow biocatalysis. Chem. Soc. Rev. 47, 5891–5918 (2018).29922795 10.1039/C7CS00906B
5. Reetz MT Biocatalysis in organic chemistry and biotechnology: past, present, and future J. Am. Chem. Soc. 2013 135 12480 12496 10.1021/ja405051f 23930719
Reetz, M. T. Biocatalysis in organic chemistry and biotechnology: past, present, and future. J. Am. Chem. Soc. 135, 12480–12496 (2013).23930719 10.1021/ja405051f
6. Romero-Fernández M Paradisi F Protein immobilization technology for flow biocatalysis Curr. Opin. Chem. Biol. 2020 55 1 8 10.1016/j.cbpa.2019.11.008 31865258
Romero-Fernández, M. & Paradisi, F. Protein immobilization technology for flow biocatalysis. Curr. Opin. Chem. Biol. 55, 1–8 (2020).31865258 10.1016/j.cbpa.2019.11.008
7. Tamborini L Fernandes P Paradisi F Molinari F Flow bioreactors as complementary tools for biocatalytic process intensification Trends Biotechnol. 2018 36 73 88 10.1016/j.tibtech.2017.09.005 29054312
Tamborini, L., Fernandes, P., Paradisi, F. & Molinari, F. Flow bioreactors as complementary tools for biocatalytic process intensification. Trends Biotechnol. 36, 73–88 (2018).29054312 10.1016/j.tibtech.2017.09.005
8. Stuermer R Hauer B Hall M Faber K Asymmetric bioreduction of activated C=C bonds using enoate reductases from the old yellow enzyme family Curr. Opin. Chem. Biol. 2007 11 203 213 10.1016/j.cbpa.2007.02.025 17353140
Stuermer, R., Hauer, B., Hall, M. & Faber, K. Asymmetric bioreduction of activated C=C bonds using enoate reductases from the old yellow enzyme family. Curr. Opin. Chem. Biol. 11, 203–213 (2007).17353140 10.1016/j.cbpa.2007.02.025
9. Toogood HS Structure-based insight into the asymmetric bioreduction of the C=C double bond of alpha, beta-unsaturated nitroalkenes by pentaerythritol tetranitrate reductase Adv. Synth. Catal. 2008 350 2789 2803 10.1002/adsc.200800561 20396603
Toogood, H. S. et al. Structure-based insight into the asymmetric bioreduction of the C=C double bond of alpha, beta-unsaturated nitroalkenes by pentaerythritol tetranitrate reductase. Adv. Synth. Catal. 350, 2789–2803 (2008).20396603 10.1002/adsc.200800561
10. Toogood HS Gardiner JM Scrutton NS Biocatalytic reductions and chemical versatility of the old yellow enzyme family of flavoprotein oxidoreductases ChemCatChem 2010 2 892 914 10.1002/cctc.201000094
Toogood, H. S., Gardiner, J. M. & Scrutton, N. S. Biocatalytic reductions and chemical versatility of the old yellow enzyme family of flavoprotein oxidoreductases. ChemCatChem 2, 892–914 (2010).10.1002/cctc.201000094
11. Stott K Saito K Thiele DJ Massey V Old yellow enzyme. The discovery of multiple isozymes and a family of related proteins J. Biol. Chem. 1993 268 6097 6106 10.1016/S0021-9258(18)53224-5 8454584
Stott, K., Saito, K., Thiele, D. J. & Massey, V. Old yellow enzyme. The discovery of multiple isozymes and a family of related proteins. J. Biol. Chem. 268, 6097–6106 (1993).8454584 10.1016/S0021-9258(18)53224-5
12. Toogood HS Knaus T Scrutton NS Alternative hydride sources for ene‐reductases: current trends ChemCatChem 2014 6 951 954 10.1002/cctc.201300911
Toogood, H. S., Knaus, T. & Scrutton, N. S. Alternative hydride sources for ene‐reductases: current trends. ChemCatChem 6, 951–954 (2014).10.1002/cctc.201300911
13. Knaus T Better than nature: nicotinamide biomimetics that outperform natural coenzymes J. Am. Chem. Soc. 2016 138 1033 1039 10.1021/jacs.5b12252 26727612
Knaus, T. et al. Better than nature: nicotinamide biomimetics that outperform natural coenzymes. J. Am. Chem. Soc. 138, 1033–1039 (2016).26727612 10.1021/jacs.5b12252
14. Reeve HA A hydrogen-driven biocatalytic approach to recycling synthetic analogues of NAD(P)H Chem. Commun. 2022 58 10540 10543 10.1039/D2CC02411J
Reeve, H. A. et al. A hydrogen-driven biocatalytic approach to recycling synthetic analogues of NAD(P)H. Chem. Commun. 58, 10540–10543 (2022).10.1039/D2CC02411J
15. Zhang W Hollmann F Nonconventional regeneration of redox enzymes—a practical approach for organic synthesis? Chem. Commun. 2018 54 7281 7289 10.1039/C8CC02219D
Zhang, W. & Hollmann, F. Nonconventional regeneration of redox enzymes—a practical approach for organic synthesis? Chem. Commun. 54, 7281–7289 (2018).10.1039/C8CC02219D
16. Bernard J van Heerden E Arends IWCE Opperman DJ Hollmann F Chemoenzymatic reduction of conjugated C=C double bonds ChemCatChem 2012 4 196 199 10.1002/cctc.201100312
Bernard, J., van Heerden, E., Arends, I. W. C. E., Opperman, D. J. & Hollmann, F. Chemoenzymatic reduction of conjugated C=C double bonds. ChemCatChem 4, 196–199 (2012).10.1002/cctc.201100312
17. Al-Shameri A Willot SJ-P Paul CE Hollmann F Lauterbach L H2 as a fuel for flavin- and H2O2-dependent biocatalytic reactions Chem. Commun. 2020 56 9667 9670 10.1039/D0CC03229H
Al-Shameri, A., Willot, S. J.-P., Paul, C. E., Hollmann, F. & Lauterbach, L. H2 as a fuel for flavin- and H2O2-dependent biocatalytic reactions. Chem. Commun. 56, 9667–9670 (2020).10.1039/D0CC03229H
18. Joseph Srinivasan S E. coli nickel-iron hydrogenase 1 catalyses non-native reduction of flavins: demonstration for alkene hydrogenation by old yellow enzyme ene-reductases Angew. Chem. Int. Ed. 2021 60 13824 13828 10.1002/anie.202101186
Joseph Srinivasan, S. et al. E. coli nickel-iron hydrogenase 1 catalyses non-native reduction of flavins: demonstration for alkene hydrogenation by old yellow enzyme ene-reductases. Angew. Chem. Int. Ed. 60, 13824–13828 (2021).10.1002/anie.202101186
19. Ramirez MA H2-driven reduction of flavin by hydrogenase enables cleaner operation of nitroreductases for nitro-group to amine reductions Front. Catal. 2022 2 906694 10.3389/fctls.2022.906694
Ramirez, M. A. et al. H2-driven reduction of flavin by hydrogenase enables cleaner operation of nitroreductases for nitro-group to amine reductions. Front. Catal. 2, 906694 (2022).10.3389/fctls.2022.906694
20. Grau MM Photoenzymatic reduction of C=C double bonds Adv. Synth. Catal. 2009 351 3279 3286 10.1002/adsc.200900560
Grau, M. M. et al. Photoenzymatic reduction of C=C double bonds. Adv. Synth. Catal. 351, 3279–3286 (2009).10.1002/adsc.200900560
21. Taglieber A Schulz F Hollmann F Rusek M Reetz MT Light-driven biocatalytic oxidation and reduction reactions: scope and limitations Chembiochem 2008 9 565 572 10.1002/cbic.200700435 18288667
Taglieber, A., Schulz, F., Hollmann, F., Rusek, M. & Reetz, M. T. Light-driven biocatalytic oxidation and reduction reactions: scope and limitations. Chembiochem 9, 565–572 (2008).18288667 10.1002/cbic.200700435
22. Hollmann F Hofstetter K Habicher T Hauer B Schmid A Direct electrochemical regeneration of monooxygenase subunits for biocatalytic asymmetric epoxidation J. Am. Chem. Soc. 2005 127 6540 6541 10.1021/ja050997b 15869268
Hollmann, F., Hofstetter, K., Habicher, T., Hauer, B. & Schmid, A. Direct electrochemical regeneration of monooxygenase subunits for biocatalytic asymmetric epoxidation. J. Am. Chem. Soc. 127, 6540–6541 (2005).15869268 10.1021/ja050997b
23. Mifsud M Photobiocatalytic chemistry of oxidoreductases using water as the electron donor Nat. Commun. 2014 5 3145 10.1038/ncomms4145 24473192
Mifsud, M. et al. Photobiocatalytic chemistry of oxidoreductases using water as the electron donor. Nat. Commun. 5, 3145 (2014).24473192 10.1038/ncomms4145
24. Ruinatscha R Dusny C Buehler K Schmid A Productive asymmetric styrene epoxidation based on a next generation electroenzymatic methodology Adv. Synth. Catal. 2009 351 2505 2515 10.1002/adsc.200900291
Ruinatscha, R., Dusny, C., Buehler, K. & Schmid, A. Productive asymmetric styrene epoxidation based on a next generation electroenzymatic methodology. Adv. Synth. Catal. 351, 2505–2515 (2009).10.1002/adsc.200900291
25. Valotta A Malihan-Yap L Hinteregger K Kourist R Gruber-Woelfler H Design and investigation of a photocatalytic setup for efficient biotransformations within recombinant cyanobacteria in continuous flow ChemSusChem 2022 15 e202201468 10.1002/cssc.202201468 36069133
Valotta, A., Malihan-Yap, L., Hinteregger, K., Kourist, R. & Gruber-Woelfler, H. Design and investigation of a photocatalytic setup for efficient biotransformations within recombinant cyanobacteria in continuous flow. ChemSusChem 15, e202201468 (2022).36069133 10.1002/cssc.202201468
26. Szczepańska E Ene-reductase transformation of massoia lactone to δ-decalactone in a continuous-flow reactor Sci. Rep. 2021 11 18794 10.1038/s41598-021-97585-w 34552113
Szczepańska, E. et al. Ene-reductase transformation of massoia lactone to δ-decalactone in a continuous-flow reactor. Sci. Rep. 11, 18794 (2021).34552113 10.1038/s41598-021-97585-w
27. Lauterbach L Lenz O How to make the reducing power of H2 available for in vivo biosyntheses and biotransformations Curr. Opin. Chem. Biol. 2019 49 91 96 10.1016/j.cbpa.2018.11.020 30544016
Lauterbach, L. & Lenz, O. How to make the reducing power of H2 available for in vivo biosyntheses and biotransformations. Curr. Opin. Chem. Biol. 49, 91–96 (2019).30544016 10.1016/j.cbpa.2018.11.020
28. Lauterbach L Lenz O Vincent KAH 2-driven cofactor regeneration with NAD(P)+-reducing hydrogenases FEBS J. 2013 280 3058 3068 10.1111/febs.12245 23497170
Lauterbach, L., Lenz, O. & Vincent, K. A. H. 2-driven cofactor regeneration with NAD(P)+-reducing hydrogenases. FEBS J. 280, 3058–3068 (2013).23497170 10.1111/febs.12245
29. Ratzka J Lauterbach L Lenz O Ansorge-Schumacher MB Systematic evaluation of the dihydrogen-oxidising and NAD+-reducing soluble [NiFe]-hydrogenase from Ralstonia eutropha H16 as a cofactor regeneration catalyst Biocatal. Biotranformation 2011 29 246 252 10.3109/10242422.2011.615393
Ratzka, J., Lauterbach, L., Lenz, O. & Ansorge-Schumacher, M. B. Systematic evaluation of the dihydrogen-oxidising and NAD+-reducing soluble [NiFe]-hydrogenase from Ralstonia eutropha H16 as a cofactor regeneration catalyst. Biocatal. Biotranformation 29, 246–252 (2011).10.3109/10242422.2011.615393
30. Mordhorst S Andexer JN Round, round we go—strategies for enzymatic cofactor regeneration Nat. Prod. Rep. 2020 37 1316 1333 10.1039/D0NP00004C 32582886
Mordhorst, S. & Andexer, J. N. Round, round we go—strategies for enzymatic cofactor regeneration. Nat. Prod. Rep. 37, 1316–1333 (2020).32582886 10.1039/D0NP00004C
31. Preissler J Dihydrogen‐driven NADPH recycling in imine reduction and P450‐catalyzed oxidations mediated by an engineered O2 ‐tolerant hydrogenase ChemCatChem 2020 12 4853 4861 10.1002/cctc.202000763
Preissler, J. et al. Dihydrogen‐driven NADPH recycling in imine reduction and P450‐catalyzed oxidations mediated by an engineered O2 ‐tolerant hydrogenase. ChemCatChem 12, 4853–4861 (2020).10.1002/cctc.202000763
32. Lauterbach L Lenz O Catalytic production of hydrogen peroxide and water by oxygen-tolerant NiFe-hydrogenase during H2 cycling in the presence of O2 J. Am. Chem. Soc. 2013 135 17897 17905 10.1021/ja408420d 24180286
Lauterbach, L. & Lenz, O. Catalytic production of hydrogen peroxide and water by oxygen-tolerant NiFe-hydrogenase during H2 cycling in the presence of O2. J. Am. Chem. Soc. 135, 17897–17905 (2013).24180286 10.1021/ja408420d
33. Holzer AK Asymmetric biocatalytic amination of ketones at the expense of NH3 and molecular hydrogen Org. Lett. 2015 17 2431 2433 10.1021/acs.orglett.5b01154 25946312
Holzer, A. K. et al. Asymmetric biocatalytic amination of ketones at the expense of NH3 and molecular hydrogen. Org. Lett. 17, 2431–2433 (2015).25946312 10.1021/acs.orglett.5b01154
34. Lonsdale TH H2-driven biotransformation of n-octane to 1-octanol by a recombinant Pseudomonas putida strain co-synthesizing an O2-tolerant hydrogenase and a P450 monooxygenase Chem. Commun. 2015 51 16173 16175 10.1039/C5CC06078H
Lonsdale, T. H. et al. H2-driven biotransformation of n-octane to 1-octanol by a recombinant Pseudomonas putida strain co-synthesizing an O2-tolerant hydrogenase and a P450 monooxygenase. Chem. Commun. 51, 16173–16175 (2015).10.1039/C5CC06078H
35. Al-Shameri A Synthesis of N-heterocycles from diamines via H2-driven NADPH recycling in the presence of O2 Green. Chem. 2019 21 1396 1400 10.1039/C8GC03798A
Al-Shameri, A. et al. Synthesis of N-heterocycles from diamines via H2-driven NADPH recycling in the presence of O2. Green. Chem. 21, 1396–1400 (2019).10.1039/C8GC03798A
36. Opperman DJ Crystal structure of a thermostable old yellow enzyme from Thermus scotoductus SA-01 Biochem. Biophys. Res. Commun. 2010 393 426 431 10.1016/j.bbrc.2010.02.011 20138824
Opperman, D. J. et al. Crystal structure of a thermostable old yellow enzyme from Thermus scotoductus SA-01. Biochem. Biophys. Res. Commun. 393, 426–431 (2010).20138824 10.1016/j.bbrc.2010.02.011
37. Lauterbach L The hydrogenase subcomplex of the NAD + ‐reducing [NiFe] hydrogenase from Ralstonia eutropha—insights into catalysis and redox interconversions Eur. J. Inorg. Chem. 2011 2011 1067 1079 10.1002/ejic.201001053
Lauterbach, L. et al. The hydrogenase subcomplex of the NAD + ‐reducing [NiFe] hydrogenase from Ralstonia eutropha—insights into catalysis and redox interconversions. Eur. J. Inorg. Chem. 2011, 1067–1079 (2011).10.1002/ejic.201001053
38. Lauterbach L Idris Z Vincent KA Lenz O Catalytic properties of the isolated diaphorase fragment of the NAD+-reducing NiFe-hydrogenase from Ralstonia eutropha PloS one 2011 6 e25939 10.1371/journal.pone.0025939 22016788
Lauterbach, L., Idris, Z., Vincent, K. A. & Lenz, O. Catalytic properties of the isolated diaphorase fragment of the NAD+-reducing NiFe-hydrogenase from Ralstonia eutropha. PloS one 6, e25939 (2011).22016788 10.1371/journal.pone.0025939
39. Son EJ Carbon nanotube–graphitic carbon nitride hybrid films for flavoenzyme‐catalyzed photoelectrochemical cells Adv. Funct. Mater. 2018 28 1705232 10.1002/adfm.201705232
Son, E. J. et al. Carbon nanotube–graphitic carbon nitride hybrid films for flavoenzyme‐catalyzed photoelectrochemical cells. Adv. Funct. Mater. 28, 1705232 (2018).10.1002/adfm.201705232
40. Choi DS Kim J Hollmann F Park CB Solar‐assisted eBiorefinery: photoelectrochemical pairing of oxyfunctionalization and hydrogenation reactions Angew. Chem. Int. Ed. Engl. 2020 132 16020 16024 10.1002/ange.202006893
Choi, D. S., Kim, J., Hollmann, F. & Park, C. B. Solar‐assisted eBiorefinery: photoelectrochemical pairing of oxyfunctionalization and hydrogenation reactions. Angew. Chem. Int. Ed. Engl. 132, 16020–16024 (2020).10.1002/ange.202006893
41. Al-Shameri A Powering artificial enzymatic cascades with electrical energy Angew. Chem. Int. Ed. 2020 59 10929 10933 10.1002/anie.202001302
Al-Shameri, A. et al. Powering artificial enzymatic cascades with electrical energy. Angew. Chem. Int. Ed. 59, 10929–10933 (2020).10.1002/anie.202001302
42. O’Brien M Baxendale IR Ley SV Flow ozonolysis using a semipermeable Teflon AF-2400 membrane to effect gas-liquid contact Org. Lett. 2010 12 1596 1598 10.1021/ol100322t 20218640
O’Brien, M., Baxendale, I. R. & Ley, S. V. Flow ozonolysis using a semipermeable Teflon AF-2400 membrane to effect gas-liquid contact. Org. Lett. 12, 1596–1598 (2010).20218640 10.1021/ol100322t
43. Poznansky, B., Cleary, S. E., Thompson, L. A., Reeve, H. A. & Vincent, K. A. Boosting the productivity of H2-driven biocatalysis in a commercial hydrogenation flow reactor using H2 from water electrolysis. Front. Chem. Eng. 3, 718257 (2021).
44. Lagadec MF Grimaud A Water electrolysers with closed and open electrochemical systems Nat. Mater. 2020 19 1140 1150 10.1038/s41563-020-0788-3 33020614
Lagadec, M. F. & Grimaud, A. Water electrolysers with closed and open electrochemical systems. Nat. Mater. 19, 1140–1150 (2020).33020614 10.1038/s41563-020-0788-3
45. Carmo M Fritz DL Mergel J Stolten D A comprehensive review on PEM water electrolysis Int. J. Hydrog. Energy 2013 38 4901 4934 10.1016/j.ijhydene.2013.01.151
Carmo, M., Fritz, D. L., Mergel, J. & Stolten, D. A comprehensive review on PEM water electrolysis. Int. J. Hydrog. Energy 38, 4901–4934 (2013).10.1016/j.ijhydene.2013.01.151
46. Mallia CJ Baxendale IR The use of gases in flow synthesis Org. Process Res. Dev. 2016 20 327 360 10.1021/acs.oprd.5b00222
Mallia, C. J. & Baxendale, I. R. The use of gases in flow synthesis. Org. Process Res. Dev. 20, 327–360 (2016).10.1021/acs.oprd.5b00222
47. Greiner L Membrane aerated hydrogenation: enzymatic and chemical homogeneous catalysis Adv. Synth. Catal. 2003 345 679 683 10.1002/adsc.200303031
Greiner, L. et al. Membrane aerated hydrogenation: enzymatic and chemical homogeneous catalysis. Adv. Synth. Catal. 345, 679–683 (2003).10.1002/adsc.200303031
48. Grushevenko EA Borisov IL Volkov AV High-selectivity polysiloxane membranes for gases and liquids separation (a review) Pet. Chem. 2021 61 959 976 10.1134/S0965544121090103
Grushevenko, E. A., Borisov, I. L. & Volkov, A. V. High-selectivity polysiloxane membranes for gases and liquids separation (a review). Pet. Chem. 61, 959–976 (2021).10.1134/S0965544121090103
49. Herr N Ratzka J Lauterbach L Lenz O Ansorge-Schumacher MB Stability enhancement of an O2-tolerant NAD+-reducing [NiFe]-hydrogenase by a combination of immobilisation and chemical modification J. Mol. Catal. B Enzym. 2013 97 169 174 10.1016/j.molcatb.2013.06.009
Herr, N., Ratzka, J., Lauterbach, L., Lenz, O. & Ansorge-Schumacher, M. B. Stability enhancement of an O2-tolerant NAD+-reducing [NiFe]-hydrogenase by a combination of immobilisation and chemical modification. J. Mol. Catal. B Enzym. 97, 169–174 (2013).10.1016/j.molcatb.2013.06.009
50. Cha J Lee J Jeon BW Kim YH Kwon I Real flue gas CO2 hydrogenation to formate by an enzymatic reactor using O2- and CO-tolerant hydrogenase and formate dehydrogenase Front. Bioeng. Biotechnol. 2023 11 1265272 10.3389/fbioe.2023.1265272 37854886
Cha, J., Lee, J., Jeon, B. W., Kim, Y. H. & Kwon, I. Real flue gas CO2 hydrogenation to formate by an enzymatic reactor using O2- and CO-tolerant hydrogenase and formate dehydrogenase. Front. Bioeng. Biotechnol. 11, 1265272 (2023).37854886 10.3389/fbioe.2023.1265272
51. Thompson MP A generic platform for the immobilisation of engineered biocatalysts Tetrahedron 2019 75 327 334 10.1016/j.tet.2018.12.004
Thompson, M. P. et al. A generic platform for the immobilisation of engineered biocatalysts. Tetrahedron 75, 327–334 (2019).10.1016/j.tet.2018.12.004
52. Torella JP Efficient solar-to-fuels production from a hybrid microbial-water-splitting catalyst system Proc. Natl Acad. Sci. Usa. 2015 112 2337 2342 10.1073/pnas.1424872112 25675518
Torella, J. P. et al. Efficient solar-to-fuels production from a hybrid microbial-water-splitting catalyst system. Proc. Natl Acad. Sci. Usa. 112, 2337–2342 (2015).25675518 10.1073/pnas.1424872112
53. Jongkind, E. P. J. et al. Synthesis of chiral amines via a bi‐enzymatic cascade using an ene‐reductase and amine dehydrogenase. ChemCatChem. 14, e202101576 (2022).
54. Resch V Seidler C Chen B-S Degeling I Hanefeld U On the Michael addition of water to α,β‐unsaturated ketones using amino acids Eur. J. Org. Chem. 2013 2013 7697 7704 10.1002/ejoc.201301230
Resch, V., Seidler, C., Chen, B.-S., Degeling, I. & Hanefeld, U. On the Michael addition of water to α,β‐unsaturated ketones using amino acids. Eur. J. Org. Chem. 2013, 7697–7704 (2013).10.1002/ejoc.201301230
55. Halliwell, B. & Gutteridge, J. M. C. Free Radicals in Biology and Medicine (Oxford University Press, 2015).
56. Kragl U Gygax D Ghisalba O Wandrey C Enzymatic two‐step synthesis of N‐acetyl‐neuraminic acid in the enzyme membrane reactor Angew. Chem. Int. Ed. 1991 30 827 828 10.1002/anie.199108271
Kragl, U., Gygax, D., Ghisalba, O. & Wandrey, C. Enzymatic two‐step synthesis of N‐acetyl‐neuraminic acid in the enzyme membrane reactor. Angew. Chem. Int. Ed. 30, 827–828 (1991).10.1002/anie.199108271
57. Fryszkowska A Asymmetric reduction of activated alkenes by pentaerythritol tetranitrate reductase: specificity and control of stereochemical outcome by reaction optimisation Adv. Synth. Catal. 2009 351 2976 2990 10.1002/adsc.200900574 20396613
Fryszkowska, A. et al. Asymmetric reduction of activated alkenes by pentaerythritol tetranitrate reductase: specificity and control of stereochemical outcome by reaction optimisation. Adv. Synth. Catal. 351, 2976–2990 (2009).20396613 10.1002/adsc.200900574
58. Fu Y Castiglione K Weuster-Botz D Comparative characterization of novel ene-reductases from cyanobacteria Biotechnol. Bioeng. 2013 110 1293 1301 10.1002/bit.24817 23280373
Fu, Y., Castiglione, K. & Weuster-Botz, D. Comparative characterization of novel ene-reductases from cyanobacteria. Biotechnol. Bioeng. 110, 1293–1301 (2013).23280373 10.1002/bit.24817
59. Zhang W The chemo-catalytic racemization of lactic acid enantiomer derived from biomass via keto-enol tautomerization Ind. Crop. Prod. 2023 193 116260 10.1016/j.indcrop.2023.116260
Zhang, W. et al. The chemo-catalytic racemization of lactic acid enantiomer derived from biomass via keto-enol tautomerization. Ind. Crop. Prod. 193, 116260 (2023).10.1016/j.indcrop.2023.116260
60. Russell GF Hills JI Odor differences between enantiomeric isomers Science 1971 172 1043 1044 10.1126/science.172.3987.1043 5573953
Russell, G. F. & Hills, J. I. Odor differences between enantiomeric isomers. Science 172, 1043–1044 (1971).5573953 10.1126/science.172.3987.1043
61. Gand M A NADH-accepting imine reductase variant: Immobilization and cofactor regeneration by oxidative deamination J. Biotech. 2016 230 11 18 10.1016/j.jbiotec.2016.05.006
Gand, M. et al. A NADH-accepting imine reductase variant: Immobilization and cofactor regeneration by oxidative deamination. J. Biotech. 230, 11–18 (2016).10.1016/j.jbiotec.2016.05.006
62. Rad R A hybrid bioelectrochemical system coupling a zero-gap cell and a methanogenic reactor for carbon dioxide reduction using a wastewater-derived catholyte Cell Rep. Phys. Sci. 2023 4 101526 10.1016/j.xcrp.2023.101526
Rad, R. et al. A hybrid bioelectrochemical system coupling a zero-gap cell and a methanogenic reactor for carbon dioxide reduction using a wastewater-derived catholyte. Cell Rep. Phys. Sci. 4, 101526 (2023).10.1016/j.xcrp.2023.101526
63. Baek G Rossi R Saikaly PE Logan BE High-rate microbial electrosynthesis using a zero-gap flow cell and vapor-fed anode design Water Res. 2022 219 118597 10.1016/j.watres.2022.118597 35609490
Baek, G., Rossi, R., Saikaly, P. E. & Logan, B. E. High-rate microbial electrosynthesis using a zero-gap flow cell and vapor-fed anode design. Water Res. 219, 118597 (2022).35609490 10.1016/j.watres.2022.118597
64. Nett N A robust and stereocomplementary panel of ene-reductase variants for gram-scale asymmetric hydrogenation Mol. Catal. 2021 502 111404 10.1016/j.mcat.2021.111404
Nett, N. et al. A robust and stereocomplementary panel of ene-reductase variants for gram-scale asymmetric hydrogenation. Mol. Catal. 502, 111404 (2021).10.1016/j.mcat.2021.111404
65. Sheldon RA The E factor 25 years on: the rise of green chemistry and sustainability Green. Chem. 2017 19 18 43 10.1039/C6GC02157C
Sheldon, R. A. The E factor 25 years on: the rise of green chemistry and sustainability. Green. Chem. 19, 18–43 (2017).10.1039/C6GC02157C
66. Wu Y Paul CE Hollmann F Mirror, mirror on the wall, which is the greenest of them all? A critical comparison of chemo- and biocatalytic oxyfunctionalisation reactions Green. Carbon 2023 1 227 241 10.1016/j.greenca.2023.10.004
Wu, Y., Paul, C. E. & Hollmann, F. Mirror, mirror on the wall, which is the greenest of them all? A critical comparison of chemo- and biocatalytic oxyfunctionalisation reactions. Green. Carbon 1, 227–241 (2023).10.1016/j.greenca.2023.10.004
67. Villa R Ferrer-Carbonell C Paul CE Biocatalytic reduction of alkenes in micro-aqueous organic solvent catalysed by an immobilised ene reductase Catal. Sci. Technol. 2023 13 5530 5535 10.1039/D3CY00541K 38013840
Villa, R., Ferrer-Carbonell, C. & Paul, C. E. Biocatalytic reduction of alkenes in micro-aqueous organic solvent catalysed by an immobilised ene reductase. Catal. Sci. Technol. 13, 5530–5535 (2023).38013840 10.1039/D3CY00541K
68. Fulmer GR NMR Chemical shifts of trace impurities: common laboratory solvents, organics, and gases in deuterated solvents relevant to the organometallic chemist Organometallics 2010 29 2176 2179 10.1021/om100106e
Fulmer, G. R. et al. NMR Chemical shifts of trace impurities: common laboratory solvents, organics, and gases in deuterated solvents relevant to the organometallic chemist. Organometallics 29, 2176–2179 (2010).10.1021/om100106e
69. Gottlieb HE Kotlyar V Nudelman A NMR Chemical shifts of common laboratory solvents as trace impurities J. Org. Chem. 1997 62 7512 7515 10.1021/jo971176v 11671879
Gottlieb, H. E., Kotlyar, V. & Nudelman, A. NMR Chemical shifts of common laboratory solvents as trace impurities. J. Org. Chem. 62, 7512–7515 (1997).11671879 10.1021/jo971176v
