Progress in Scaling up and Streamlining a Nanoconfined, Enzyme‐Catalyzed Electrochemical Nicotinamide Recycling System for Biocatalytic Synthesis

Abstract An electrochemically driven nicotinamide recycling system, referred to as the ‘electrochemical leaf’ has unique attributes that may suit it to the small‐scale industrial synthesis of high‐value chemicals. A complete enzyme cascade can be immobilized within the channels of a nanoporous electrode, allowing complex reactions to be energized, controlled and monitored continuously in real time. The electrode is easily prepared by depositing commercially available indium tin oxide (ITO) nanoparticles on a Ti support, resulting in a network of nanopores into which enzymes enter and bind. One of the enzymes is the photosynthetic flavoenzyme, ferredoxin NADP+ reductase (FNR), which catalyzes the quasi‐reversible electrochemical recycling of NADP(H) and serves as the transducer. The second enzyme is any NADP(H)‐dependent dehydrogenase of choice, and further enzymes can be added to build elaborate cascades that are driven in either oxidation or reduction directions through the rapid recycling of NADP(H) within the pores. In this Article, we describe the measurement of key enzyme/cofactor parameters and an essentially linear scale‐up from an analytical scale 4 mL reactor with a 14 cm2 electrode to a 500 mL reactor with a 500 cm2 electrode. We discuss the advantages (energization, continuous monitoring that can be linked to a computer, natural enzyme immobilization, low costs of electrodes and low cofactor requirements) and challenges to be addressed (optimizing minimal use of enzyme applied to the electrode).


FNR Expression
A vector (aLICator pLATE 51) containing the gene encoding Nterminal Histagged FNR from Chlamydomonas reinhardtii was used to transform Escherichia coli cells (BL21 (DE3))which were subsequently plated on Lysongeny broth (LB) agar containing ampicillin at 100 µgmL 1 . Positive transformants were selected by resistance to ampicillin. A single colony was used to inoculate 100 mL LB media containing ampicillin at 100 µg mL 1 and grown shaking (200 rpm), overnight at 37 ℃.
This was used to inoculate 500 mL of LB containing ampicillin at 100 µg mL 1 and grown at 37 ℃, 200 rpm for approximately 3 hours at which point they were induced by addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM and grown for a further 3 -4 hours. The cells were then harvested by centrifugation and the pellets resuspended in cold cell resuspension buffer (50 mM HEPES; 150 mM NaCl; 10% V/V Glycerol pH 7.4) and stored at -80 ℃ until purification.

FNR Purification
The cells were disrupted using a French press at 20 psi and centrifuged at 45000 rpm for 1 hour.
The supernatant was retained and purification of FNR was carried out using a Ni 2+ HisTrap HP affinity column (GE Healthcare); fractions containing FNR were selected based on the absorbance at 280 nm and 460 nm. The fractions were pooled and concentrated using a 4 mL centrifugal filters (Amicon® Ultra4 Merck) to a final volume of approximately 2 mL. The concentrated protein was passed through a desalting column (PD10 Ge Healthcare) to remove imidazole, portioned into single use aliquots and flash frozen in liquid nitrogen before storing at 80 ℃.

GLDH Expression
Under sterile conditions, competent cells (BL21 (DE3)) were transformed with the HIStag E. coli GDH plasmid (previously constructed by first, colony PCR to amplify the GDH gene using E. coli BL21 De3 cells as template, followed by ligation-independent cloning into an N-terminal Histag vector (Thermo Scientific aLICator system #K1251). Colonies were grown on LB agar containing ampicillin at 100 µg mL 1 . A single colony was used to inoculate 500 mL LB media containing ampicillin at 100 µg mL 1 and also IPTG to a final concentration of 1 mM, and grown 16 h at 37 ℃, 200 rpm.

GLDH Purification
The cells were centrifuged at 6000 rpm (4 ℃) for 30 minutes, resuspended in cell resuspension buffer (pH 7.4) containing 50 mM HEPES, 150 mM NaCl, 10% glycerol and stored at 80 ℃. Upon thawing, the cells were disrupted using a French press at 20 psi and centrifuged at 45000 rpm, (4 ℃) for 1 hour. The supernatant was loaded on a Ni 2+ HisTrap HP affinity column (GE Healthcare) using Buffer A (50 mM HEPES, 500 mM NaCl, 1 mM DTT, pH 7.4) and Buffer B (50 mM HEPES, 500 mM NaCl, 250 mM Imidazole, 1 mM DTT pH 7.4). A linear (0100%) imidazole gradient was used; fractions were selected based on the absorbance at 280 nm and also the enzyme activity tested by solution assay using a UV/Vis spectrophotometer (Perkin Elmer, Lambda 19). The fractions were pooled and concentrated using a 10K centrifugal filter (Amicon® Ultra4 Merck) and dialysed overnight against 1 L of dialysis buffer: 50 mM HEPES, 150 mM NaCl, 1 mM DTT and 10% glycerol. Protein aliquots (20 µL) were flash frozen in liquid nitrogen and stored at 80 ℃.

Electrophoretic deposition (EPD) method
In the EPD method, ITO nanopowder (Sigma-Aldrich < 50 nm) is suspended in a solution of I2 in acetone and sonicated for at least 45 min. Two electrodes, one of which is the piece of Ti foil to be modified, are held in parallel in the ITO suspension at a close distance apart (ca. 1 cm). [1][2] A 10 V potential is applied between the two electrodes for 5-10 min. The ITO nanoparticles migrate to the cathode and form a thin porous layer. The electrode is dried thoroughly in air before the next stage in which enzymes are bound. The EPD method works particularly well for Ti foil which has a flat face and is easily performed in the lab for surfaces up to 150 cm 2 . The average coverage of ITO on Ti foil is approximately 1 mg / cm 2 .

Figure S9 L-glutamic acid crystal after product separation
First, the post-reaction solution is filtered using filter paper. This step removes fine insoluble impurities that consist mainly of dislodged ITO. Decolorization is then performed by adding 10% mass/vol activated charcoal to the solution and stirring for half an hour. The charcoal is then removed by filtration, leaving a clean and colourless solution, the volume of solution reduced from 500 mL to 25 mL by rotary evaporation, and the pH adjusted to 3.2 with HCl. At this pH, L-glutamic acid is in its neutral (acid) form that has very low solubility at low temperature (3.53 g L 1 in H2O at 5.5 ℃) and high solubility at high temperature (122.8 g L 1 in H2O at 100 ℃). The white suspension is heated to 85 ℃ to produce a clear, transparent solution. The solution is then cooled down to room temperature and placed in a cold room (4 ℃). Finally, the crystalline product is filtered off and dried at 80 ℃. The NMR spectrum indicates a purity of over 99% (shown in Figure S9). The mass of pure product (1.86 g) represents a recovery rate of approximately 66% from the product in the cell solution, which could be improved by further work up.  The depreciation of research equipment is not taken into calculation and only key consumables are considered. The enzyme preparation protocol can still be further optimized.

Cost of making the (FNR+GLDH)@ITO/Ti electrode in the laboratory
Therefore, by the general calculation the enzyme cost is GBP 3.05 / mol (discount) and GBP 7.37 / mol (commercial). For a 10 cm 2 ITO/Ti electrode loaded with 20 nmol enzymes (FNR and GLDH in this case), the cost is approximately GBP 1.594 (discount) and 4.76 (commercial).