In a glovebox (Glove Box Technology) under a protective N₂ atmosphere (O₂ < 10 ppm), carbon particles (BP2000) were suspended in deoxygenated Tris-HCl buffer (50 mM, pH 8.0) to make a 20 mg ml⁻¹ slurry. Larger carbon agglomerates were dispersed, then sonicated twice for 30 min to give an ink-like slurry. The slurry was allowed to cool on ice. The designated amounts of Hyd1, SH and BP2000 slurry were combined in a centrifuge tube, mixed using a pipette and allowed to incubate at 4°C for 60 min. The catalyst slurry was centrifuged (12,000 × g), and the supernatant was then withdrawn using a pipette to remove any unadsorbed enzyme. The pellet was resuspended in deoxygenated Tris-HCl buffer (50 mM, pH 8.0) and separated into aliquot samples as needed. Samples were flash-frozen and stored at −80°C.

Activity assays were also performed in an anaerobic glovebox in deoxygenated Tris-HCl buffer (100 mM, pH 8.0) in a UV–Vis cuvette that was sealed with a rubber septum pierced with two needles to allow gas flow in and out. The concentration of NADH was monitored over time by the absorbance at 340 nm (ε = 6.22 mM⁻¹cm⁻¹) associated with the reduced cofactor, NADH.

A solution of 800 μl of 0.1 mM NAD⁺ in Tris-HCl (100 mM, pH 8.0) was saturated with H₂ by bubbling for 10 min. The inlet needle was next adjusted such that it was streaming H₂ through the cuvette headspace rather than through the solution. The H₂-saturated solution (0.2 ml) was used to transfer the carbon-supported catalyst (see General Enzyme Immobilization Procedure for Batch Studies) into the cuvette by a fresh syringe and needle, and the resulting slurry was mixed once by gently syringing up and down. The reaction was monitored by UV–Vis, and the concentration of NADH (determined from A_340 nm) was plotted against time to determine the specific activity from the linear phase of reaction.

Acetophenone reduction reactions were prepared on the benchtop in a N₂-flushed Asynt OCTO mini parallel reactor, in which each reaction tube contained a magnetic stirrer bar. Solutions of NAD⁺ (0.5 mM) were prepared in Tris-HCl (40 mM, pH 8.0) and flushed with N₂ followed by H₂ before use. Stock solutions of acetophenone (2 M) in DMSO were added to the reaction mixtures to give the desired concentrations. The Asynt OCTO reactor was kept at room temperature under a steady flow (1 bar) of H₂ throughout the reaction setup. H₂-saturated NAD⁺ solution was added to each reaction tube, followed by SH/C or SH + Hyd1/C, ADH-105 and acetophenone in DMSO in the designated order of addition and amounts described below. The parallel reactor was left to stir at 350 r.p.m. at room temperature under a steady flow of H₂ for the specified reaction time, during which aliquots (<50 μl) were collected and prepared for GC analysis by extraction into ethyl acetate (800 μl), after which 600 μl of the organic layer was dried over Na₂SO₄ and analysed by chiral-phase GC-FID. An additional 20 μL acetophenone (2 M in DMSO) was added at 19 h using a needle and syringe.

Biocatalytic cartridges (200 µl reactor volume) were prepared by two different methods. 1) A CatCart™ cartridge (30 × 4 mm) was packed and sealed with activated charcoal using a cartridge packer (ThalesNano). The required mixture of enzymes was manually pumped into the pre-sealed cartridge via syringe. The cartridge was then stored at 4°C for 1 h, then installed in the H-Cube and flushed with Tris-HCl buffer (50 mM, pH 8.0) to remove any unadsorbed enzyme. 2) A CatCart™ cartridge was filled with activated charcoal, and the required mixture of enzymes was then added into the cartridge before it was sealed. The cartridge was sealed using the cartridge packer, and was agitated on a vortex mixer and then stored at 4°C for 1 h. The column was then installed in the H-Cube and was flushed with Tris-HCl buffer (50 mM, pH 8.0) to remove any unadsorbed enzyme. Further experimental details are provided in the Supporting Information, Supplementary Table S1.

With the sealed CatCart™ biocatalyst cartridge installed in the H-Cube reactor, a liquid feed (Knauer H-Cube piston pump) containing NAD⁺ (1–5 mM) in Tris-HCl buffer (50 mM, pH 8.0) was pumped through the H-Cube with the H₂ that was 1) generated by built-in water reservoir and electrolyzer (gas feed flow rate was set to 7–12 ml min⁻¹); or 2) from a H₂ cylinder pumped in by Vapourtec SF-10 peristaltic pumps (gas feed flow rate 0.6–1.2 ml min⁻¹) using PTFE tubing (1/16″ OD, 1/32″ ID) and Tefzel™ fittings and mixed with H-Cube pump liquid solution at a Y-piece (PEEK, 1/4–28, 0.02″ thru-hole, IDEX). The liquid feed flow rate was set to 0.05–3 ml min⁻¹ to give a 0.8–30 s residence time (t _Res) through the biocatalyst cartridge. All reactions were performed at room temperature. Samples were collected using a fraction collector (BioRad 2110), and reactor volumes were combined to obtain a sufficient amount of material for analysis by UV–Vis spectroscopy. The ratio of absorbance at 260 and 340 nm was used to calculate the extent of conversion (using previously described calibration curves) (Reeve et al., 2015). Further experimental details are provided in the Supporting Information, Supplementary Table S2.

An appropriate sealed CatCart™ was prepared according to the procedures described in Preparation of Biocatalyst Cartridges for Hydrogenation Flow Reactor Studies. This was then installed in the H-Cube reactor and a liquid feed (Knauer H-Cube piston pump) containing sodium pyruvate (10–20 mM) and NAD⁺ (1 mM) in Tris-HCl buffer (50 mM, pH 8.0) was pumped through the H-Cube with the H₂ that was 1) generated by built-in water reservoir and electrolyzer (gas feed flow rate was set to 7–12 ml min⁻¹); or 2) obtained from a H₂ cylinder pumped in by Vapourtec SF-10 peristaltic pumps (gas feed flow rate 0.6–1.2 ml min⁻¹) using PTFE tubing (1/16″ OD, 1/32″ ID) and Tefzel™ fittings and mixed with H-Cube pump liquid solution at a Y-piece (PEEK, ¼-28, 0.02 thru-hole, IDEX).

The liquid feed flow rate was set to 0.05–1 ml min⁻¹ to give a 0.8–18 s t _Res through the biocatalytic cartridge. Samples were collected using a fraction collector (BioRad, 2110), and reactor volumes were combined to obtain a sufficient amount of material for analysis by ¹H NMR spectroscopy. Reaction progress was determined by comparison to analytical standards and literature data. Further experimental details are provided in the Supporting Information, Supplementary Table S2.

An appropriate sealed CatCart™ was prepared according to the procedures described in Preparation of Biocatalyst Cartridges for Hydrogenation Flow Reactor Studies. This was then installed in the H-Cube reactor and a liquid feed (Knauer H-Cube piston pump) containing sodium pyruvate 92 mM) and NAD⁺ (1.7 mM) in Tris-HCl buffer (50 mM, pH 8.0) was pumped through the H-Cube with the H₂ that was generated by the built-in water reservoir and electrolyzer (gas feed flow rate was set to 12 ml min⁻¹); the liquid feed flow rate was set to 0.05 ml min⁻¹ to give a 1 s t _Res through the biocatalyst cartridge. The reaction solution was recirculated through the reactor and aliquots were removed periodically for analysis by ¹H NMR spectroscopy. Reaction progress was determined by comparison to analytical standards and literature data. Further experimental details are provided in the Supporting Information, Supplementary Table S2.

To study the immobilized biocatalysts in continuous flow, enzyme-modified carbon particles were tested in the hydrogenation flow reactor within flow cartridges, prepared as described in Preparation of Biocatalyst Cartridges for Hydrogenation Flow Reactor Studies. Activated charcoal (>0.15 mm) was used as the support because a larger carbon particle diameter was required over BP2000 (∼10 nm) to avoid carbon leaching through the frits. For each of the flow results, parameters such as mass of enzyme and mass of carbon are specified in the relevant figure caption and experimental details are summarized in Supplementary Table S1 and Table S2 of the Supporting Information. An example of SH immobilization on carbon showing 100% immobilization yield and negligible leaching over 18 h is given in Supplementary Table S3 of the Supporting Information. First, we conducted a proof-of-concept experiment to determine the suitability of the reactor for H₂-driven biocatalysis (Figure 4) by testing SH/C in a flow cartridge for the conversion of NAD⁺ to NADH.

A reaction solution containing NAD⁺ (1 mM) in buffer was pumped into the hydrogenation reactor at a range of flow rates (0.3, 1 or 3 ml min⁻¹), where it was combined with H₂ from the built-in water electrolysis H₂ generator (approximately 12 ml min⁻¹). As the gas flow rate made the major contribution to the total flow rate, this equated to t _Res values through the reactor of 1, 0.9 or 0.8 s. The system pressure was set to 2, 10 or 50 bar. All of these parameters were tested across a single experiment on one biocatalytic cartridge, and the results are shown in Figure 5.

Quantitative conversion to NADH was achieved for liquid flow rates of 0.3 and 1 ml min⁻¹ at 2 bar. At 3 ml min⁻¹, the highest TOF of 223 min⁻¹ was achieved (70% conversion), suggesting that this approach has overcome previous limitations of H₂ availability in flow which were observed in earlier work (Thompson et al., 2020a). Increasing the pressure to 10 bar at 1 ml min⁻¹ showed lower conversion (65%) compared with that at 2 bar; however, further increasing the system pressure to 50 bar had little effect on the conversion (60%). This demonstrated the stability of the biocatalytic cartridge at elevated pressure and, although the enzyme system does not require high pressure for operation, it shows the potential for use in processes where pressure is needed to keep up with H₂ demand, or for multistep continuous processes in which higher pressures may be required for other reaction steps.

Having established the validity of the hydrogenation reactor for biocatalysis, we next examined intensifying the substrate throughput by performing experiments using higher concentrations of NAD⁺ over a longer period of time with the SH/C cartridge (see Supporting Information, Supplementary Figure S3). High conversion (>98%) of NAD⁺ (5 mM) to NADH is shown for 6.3 h and the system operated at an overall productivity of 108 g_NADH g_SH ⁻¹ an E factor of 0.9.

To determine whether addition of Hyd1 to the activated charcoal would display a boosting effect, as seen in batch conditions, we performed an activity lifetime comparison of SH/C against SH + Hyd1/C in continuous flow, Figure 6A. A cartridge containing SH/C and a cartridge containing SH + Hyd1/C were prepared with a decreased SH loading compared with the previous flow experiments, with the aim of putting the catalyst under strained conditions (<70% conversion). This allowed for differences between the two systems to be easily recognized. The cartridges were both tested under conditions of NAD⁺ (5 mM) in buffer, at 30°C, and at a liquid flow rate 0.05 ml min⁻¹. Results in Figure 6A show that the SH/C is almost fully deactivated after 17 h (3,300 reactor volumes), displaying a TTN of 47,200 and an overall system productivity of 197 g_NADH g_SH ⁻¹. This deactivation is similar to that which was observed when SH was immobilized onto carbon nanotube-lined columns and used for NADH generation in continuous flow (Thompson et al., 2020a).

Promisingly, after 61 h (11,895 reactor volumes) the SH + Hyd1/C was still displaying a TOF of 60 min⁻¹ and had reached a TTN of 294,000 with an overall system productivity of 671 g_NADH g_SH ⁻¹. This clearly demonstrated that the addition of Hyd1 enhances the catalytic stability of the system. It is probable that the hydrogenase module of the SH denatures at a faster rate than the NAD⁺ reductase module, which leads to the decrease in activity displayed by the SH/C. The Hyd1 is able to provide supplementary electrons from H₂ oxidation to the NAD⁺ reductase module by conduction through the activated charcoal, thus boosting the overall cofactor regeneration activity of the dual-enzyme system.

It was also found that the SH/C deactivates rapidly at >40°C in the hydrogenation flow reactor (see Supplementary Figure S4, Supporting Information). In contrast Hyd1 has excellent temperature stability (Joseph Srinivasan et al., 2021). To determine the activity lifetime of SH + Hyd1/C at elevated temperatures, three identical biocatalytic cartridges containing SH + Hyd1/C were prepared and were tested under conditions of NAD⁺ (5 mM) in buffer, at a liquid flow rate 0.1 ml min⁻¹ in the flow reactor at 30°C, 45°C or 55°C, respectively. At 1 h, all cartridges showed comparable activity. However, at 4.6 h, the cartridge at 55°C had fully deactivated reaching an overall TTN of 34,600 for the NAD⁺ reductase module. At 21.7 h, the cartridge at 45°C had displayed an average TOF of 85 min⁻¹, 83% of the average TOF displayed by the cartridge at 30°C at the same time point. The respectable performance of the system at 45°C suggests that, in the future, it could be combined with thermophilic NADH-dependent enzymes or work in cascade with other bio- and chemocatalysts that require elevated temperatures.

Enzymes for H₂-driven cofactor recycling were next coupled to a model C=O bond reduction, the conversion of pyruvate to lactate by lactate dehydrogenase (LDH), Scheme 1. The biocatalyst cartridge was prepared by co-immobilizing LDH with SH on the activated carbon. The hydrogenation flow reactor was set up with a single liquid feed containing pyruvate (10 mM) and NAD⁺ (1 mM) in Tris-HCl buffer (50 mM, pH 8.0). The liquid flow rate was set to 0.1, 0.5 or 1 ml min⁻¹, equating to a t _Res of 1.7, 1.6 or 1.5 s, when combined with a H₂ flow rate of 7 ml min⁻¹. For each set of conditions, the reactor was allowed to reach steady-state and then multiple fractions were collected for analysis of pyruvate/lactate content by ¹H NMR spectroscopy. Results of this experiment are presented in Figure 7. The conversion of pyruvate to lactate reached a steady-state conversion of >96% at 0.1 ml min⁻¹ liquid flow rate, which was maintained for 100 min. The steady-state conversions achieved at 0.5 ml min⁻¹ and 1 ml min⁻¹ were 69 and 22%, respectively, resulting in a SH TTN of 47,200 across the whole experiment.

Having established activity of the system for pyruvate to lactate conversion, the system was once again set up to determine whether Hyd1 would provide a similar boosting effect. To compare the activity lifetime of the two enzyme combinations, a cartridge containing SH + LDH/C and a cartridge containing SH + Hyd1+LDH/C were prepared. They were both tested under conditions of pyruvate (20 mM), NAD⁺ (1 mM) in buffer at 30°C, at a range of liquid flow rates (0.05 and 0.1 ml min⁻¹) in the hydrogenation flow reactor, Figure 8.

The results in Figure 8 show a similar trend to that observed in the NADH generation experiments shown in Figure 6A. The SH + LDH/C was almost entirely deactivated after 21.5 h, displaying a SH TTN of 156,400 (average TOF = 121 min⁻¹) and an overall system productivity of 102 g_lactate g_SH ⁻¹. The SH + Hyd1+LDH/C was still displaying catalytic TOF of 180 min⁻¹ after 46.8 h of operation and reached a TTN of 679,800 (average TOF = 243 min⁻¹), demonstrating an overall system productivity of 444 g_lactate g_SH ⁻¹. The initial catalytic TOFs of the SH + LDH/C and the SH + Hyd1+LDH/C systems were 860 min⁻¹and 1,013 min⁻¹, respectively. The rapid consumption of the regenerated NADH by the LDH appears to drive an increase in SH catalytic performance for both of the H₂-driven cofactor recycling systems; this is likely enhanced further by immobilizing the enzymes alongside each other on the carbon (Reeve et al., 2015).

Overall, the continuous flow system demonstrates an improvement in catalytic efficiency over the batch studies due to the intensification of conditions achievable in the flow reactor, namely increased H₂ availability, high enzyme-to-substrate ratio, and consequently short t _Res. To achieve higher conversions, one option would be to increase the catalyst bed volume, but this would have required more enzyme, and at present the SH and Hyd1 are both produced at laboratory scale although scale-up efforts are underway in separate research. Therefore a multi-pass set-up was explored in a closed loop reactor in which the reaction solution is recirculated through the hydrogenation flow reactor from a single, ambient pressure reactor vessel and combined constantly with the H₂ stream from the electrolyzer. This was tested at gram scale for the conversion of pyruvate to lactate. A cartridge containing SH + Hyd1+LDH/C was prepared. A 100 ml solution of pyruvate (92 mM), NAD⁺ (1.7 mM) in buffer at 30°C was circulated through the hydrogenation flow reactor at a liquid flow rate 0.5 ml min⁻¹ (complete pass = 200 min, t _Res = 1 s). Time points were taken for analysis to measure the course of the reaction. The results in Figure 9 show that the closed loop, multi-pass flow system ran over a total of 67 h (20 complete reaction solution passes, giving overall t _Res = 20 s), producing 0.525 g of lactate and demonstrating a SH TTN of 766,100 at an overall productivity of 500 g_lactate g_SH ⁻¹. The cofactor recycling system displayed a maximum TOF of 1,060 min⁻¹ (63,600 h⁻¹), which is at the top end of typical rates achieved for the metal-catalyzed transfer hydrogenation of ketones (10²–10⁵ h⁻¹) (Štefane and Požgan, 2016).