Continuous flow biocatalysis has been established as a key concept in fine chemical production by employing immobilization techniques to reuse the biocatalysts in flow. The success lies in using the biocatalysts enantioselective and regioselective abilities to produce optically pure chemicals in a more sustainable way. With the upcoming development of molecular biological tools that can further increase activity and solve the weak stability issue of the biocatalysts, many more can be applied in flow reactions which has high potential for industrial applications. However due to the necessity of reduced cofactors has hindered the application of biocatalysts in industry or upscaled reaction.

To address the challenges in upscaling the reaction, using O₂-tolerant soluble hydrogenase (SH) natively from Cupriavidus Necator has emerged as a powerful cofactor regeneration method that can use H₂ as an electron source^1,2,3. The SH not only can regenerate NAD(P)H cofactor but also has been demonstrated to work as a regeneration method for flavin cofactors such as FMN and FAD⁴. Therefore, reduced flavin cofactor regeneration method can be coupled with many industrially interesting biocatalysts that are dependent on reduced flavins such as styrene monooxygenase, unspecific peroxygenase- catalysed hydroxylation via H₂O₂ from reduced flavin re-oxidation with O₂, and nitroreductases. SH can be a great alternative as it can use cheaper free flavin cofactors which in reduced state can transfer the necessary electrons to the flavin in the active site. In this case because SH uses H₂ as an electron source, and if H₂ is constantly added it can also strip off the remaining O₂ that can responsible for the spontaneous production of H₂O₂ by reacting with FMNH₂.

While using SH as a cofactor regeneration method for flavin dependent biocatalytic reactions offers clear advantages, it is not without limitations. One major issue is that using Amberlite an anionic resin as a conventional immobilization method for SH via adsorption cannot be used in flow chemistry reactions as it also shows adsorption with the flavin cofactors⁵. Another significant limitation is the necessity of H₂ in the flow system, where direct bubbling of the reaction can restrict the flow and deactivate the biocatalysts that are immobilized.

In response to these challenges, we have developed a flow chemistry setup that utilizes two innovative concepts: using the affinity between Strep-tagged SH and StrepTactin® resin as an immobilization method which does not interact with the flavin cofactor in the system, and the usage of gas addition module that adds H₂ that is produced from electrolysis of water to the system using gas permeable membrane tubing without disrupting the immobilized biocatalysts, realizing electro-driven biocatalysis.  

For demonstration of the flavin dependent reaction in the flow setup, we have used an Old Yellow Enzyme from Thermus Scotoductus (TsOYE) which can reduce the double carbon bond of enones⁶. TsOYE was His-tagged and immobilized with EziG beads via coordination bonds. Immobilized SH and TsOYE were packed in a column and attached to the flow setup (Figure 1). Range of cyclic enones were chosen as a substrate for the demonstration of the setup (Figure 2). One of substrate ketoisophorone in 25 mM concentration showed full conversion in 17 mL volume. We also observed production of side products from substrate cyclohexenone and racemization of product from substrate ketoisophorone both due to keto-enol tautomerization.

Additionally, the scale of the reaction was increased via adding addition flow volume up to 185 mL which also showed full conversion (Figure 2). Easy isolation of product Levodione was possible due to its high conversion rate, and we were able to obtain 0.471 g of product from theoretical yield of 0.527 g (Figure 3).

One of our bottlenecks that we faced during the project was working with hydrogen gas but also investigating the what is really happening in the reaction. This was resolved by incorporating the H₂, and O₂ sensor to the system to check the functionality of the enzymes. Also, identifying the chemical reactions such as formation of side products was done by using the right analytical methods. Overall, we have advanced the field of continuous flow biocatalysis by incorporating H₂ from electrolysis of water into the flow system to fuel flavin mediated reaction through SH. Aspects such as how the biocatalytic reactions would differ in flow from batch reactions were also investigated. 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.  

1.      Lauterbach, L., Lenz, O., & Vincent, K. A. (2013). H2-driven cofactor regeneration with NAD(P)+-reducing hydrogenases. FEBS Journal, 280(13), 3058–3068. https://doi.org/10.1111/febs.12245

2.      Lauterbach, L., & Lenz, O. (2013). Catalytic production of hydrogen peroxide and water by oxygen-tolerant [NiFe]-hydrogenase during H2 cycling in the presence of O2. Journal of the American Chemical Society, 135(47), 17897–17905. https://doi.org/10.1021/ja408420d

3.      Al-Shameri, A., Petrich, M. C., junge Puring, K., Apfel, U. P., Nestl, B. M., & Lauterbach, L. (2020). Powering Artificial Enzymatic Cascades with Electrical Energy. Angewandte Chemie - International Edition, 59(27), 10929–10933. https://doi.org/10.1002/anie.202001302

4.      Al-Shameri, A., Willot, S. J. P., Paul, C. E., Hollmann, F., & Lauterbach, L. (2020). H2as a fuel for flavin- And H2O2-dependent biocatalytic reactions. Chemical Communications, 56(67), 9667–9670. https://doi.org/10.1039/d0cc03229h

5.      Herr, N., Ratzka, J., Lauterbach, L., Lenz, O., & Ansorge-Schumacher, M. B. (2013). Stability enhancement of an O2-tolerant NAD+-reducing [NiFe]-hydrogenase by a combination of immobilisation and chemical modification. Journal of Molecular Catalysis B: Enzymatic, 97, 169–174. https://doi.org/10.1016/j.molcatb.2013.06.009

6.      Opperman, D. J., Sewell, B. T., Litthauer, D., Isupov, M. N., Littlechild, J. A., & van Heerden, E. (2010). Crystal structure of a thermostable Old Yellow Enzyme from Thermus scotoductus SA-01. Biochemical and Biophysical Research Communications, 393(3), 426–431. https://doi.org/10.1016/j.bbrc.2010.02.011