Copper oxide-based cathode for direct NADPH regeneration

Nearly a fourth of all enzymatic activities is attributable to oxidoreductases, and the redox reactions supported by this vast catalytic repertoire sustain cellular metabolism. In many biological processes, reduction depends on hydride transfer from either reduced nicotinamide adenine dinucleotide (NADH) or its phosphorylated derivative (NADPH). Despite longstanding efforts to regenerate NADPH by various methods and harness it to support chemoenzymatic synthesis strategies, the lack of product purity has been a major deterrent. Here, we demonstrate that a nanostructured heterolayer Ni–Cu2O–Cu cathode formed by a photoelectrochemical process has unexpected efficiency in direct electrochemical regeneration of NADPH from NADP+. Remarkably, two-thirds of NADP+ was converted to NADPH with no measurable production of the inactive (NADP)2 dimer and at the lowest reported overpotential [− 0.75 V versus Ag/AgCl (3 M NaCl) reference]. Sputtering of nickel on the copper-oxide electrode nucleated an unexpected surface morphology that was critical for high product selectivity. Our results should motivate design of integrated electrolyzer platforms that deploy this heterogeneous catalyst for direct electrochemical regeneration of NADH/NADPH, which is central to design of next-generation biofuel fermentation strategies, biological solar converters, energy-storage devices, and artificial photosynthesis.

= 0 + 2.303 log 10 ∏ ∏ (S2) In equation S2, 0 is the standard reduction potential of the redox couple, is the universal gas constant, is the temperature, is the number of electrons involved in the half-reaction, is Faraday's constant, and are the concentrations of oxidized and reduced species, respectively, and and are the stoichiometric coefficients of oxidized and reduced species, respectively. All non-pH determining species, e.g. Cu2O or NADP+, are considered to be at unit concentrations. The standard reduction potential of the Cu2O/Cu couple was taken to be -0.360 V (Standard Hydrogen Electrode) and the formal potential (pH 7) of the NADP+/NADPH couple was taken to be -0.320 V (Standard Hydrogen Electrode).

Calculation of selectivity of product, Q
The selectivity, , of cofactor regeneration products given by Eq. 1 may be re-written as follows.
The numerator in Eq. S3 is the decrease in absorbance following the butyraldehyde reduction reaction with lbADH, which is the absorbance due to 1,4-NADPH only. The denominator represents the absorbance of all cofactor regeneration products with non-negligible absorbance at 340 nm excitation, i.e. 1,4-NADPH, (NADP)2 and isomers such as 1,6-NADPH. Invoking the Beer-Lambert Law and denoting the extinction coefficient as and concentrations as [•], Eq. S3 takes the following form.
Finally, if the extinction coefficients are taken to be approximately equal, the selectivity is the ratio of the concentration of enzymatically active 1,4-NADPH to the sum total of the concentrations of cofactor regeneration products.
Overexpression and purification of LbADH enzyme Escherichia coli BL-21 (DE3) cells were transformed with pACYC-LbADH. A single bacterial colony was used to inoculate 2.5 mL of LB medium supplemented with 35 µg/ml chloramphenicol and grown overnight at 37°C with shaking. This overnight seed culture was used to inoculate 250 mL of fresh LB medium containing the appropriate antibiotics as mentioned above. These cells were grown at 37°C with shaking until the OD600 reached 0.6 and were induced with 1 mM IPTG for an additional 3 h. Following IPTG induction, the cells were harvested by centrifugation and the cell pellets were stored at -80°C until further use.
Purification of LbADH was achieved using immobilized metal-affinity chromatography (IMAC). A 250-mL cell pellet obtained after overexpression, was thawed on ice, re-suspended in 16 mL lysis buffer [95% buffer A, (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM DTT); 5% buffer B, (buffer A + 500 mM imidazole)] containing 80 µl bacterial protease arrest (G Biosciences, USA) and sonicated (2s on 5s off, 50% amplitude). After centrifugation of the crude lysate (24,000 g, 30 min, 4°C), the supernatant was applied to 1-mL of 50% slurry of nickel-Sepharose resin (50% slurry) (Nickel Sepharose 6 fast flow, GE Healthcare, Sweden) that had been pre-equilibrated with 5 mL equilibration buffer (95% buffer A + 5% buffer B, without DTT) and mixed gently by nutating at 24°C for 10 min. The resin was allowed to settle down and the supernatant was collected and labeled as the flow-through. The unbound proteins were removed by mixing the resin with 10 ml wash buffer [90% buffer A + 10% buffer B] for 5 min and allowing the resin to settle. The supernatant was collected and labeled as the wash fraction. After washing, the ADH protein was eluted in five successive elution steps each with 0.5 ml elution buffer with increasing imidazole concentration (100 to 500 mM). At each elution step, the resin was mixed with 0.5 mL elution buffer and the clear supernatant after centrifugation was collected separately to serve as the elution fraction. The purity of each fraction was checked by SDS-PAGE analysis and fractions primarily containing ADH were pooled together. The pooled fractions were passed through a SpinX column to remove any adventitiously co-eluted Ni-Sepharose beads and the flow-through was subjected to dialysis for 16 h at 4°C 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 1 mM DTT. The concentration of the final protein was determined using its molar extinction coefficient (20,065 M -1 cm -1 ) at 280 nm and the final preparation in 10% glycerol was stored at -80°C in small aliquots for subsequent biochemical enzyme assays. From a 250-mL culture, we obtained ~13 mg of recombinant ADH. Supplementary Fig. S1: Possible products from regeneration of NADPH. This is Fig. 2 from ref.[10] and is reproduced here with no changes, for ease of reading; A depicts a general biocatalyzed reaction showing NADPH being oxidized as a product is formed; B shows the desirable pathway for cofactor regeneration; C is the pathway for formation of the inactive dimer; D is the pathway for forming the inactive isomer.

SUPPLEMENTARY FIGURES
Supplementary Fig. S2: Schematic of two-compartment cell with an agar bridge used to electrodeposit copper oxide on a Cu mesh electrode. Ten-mL beakers were used for both working and counter-electrode compartments. Cupric lactate solution was used in the working electrode compartment for electrodeposition (right) and potassium phosphate buffer (pH 7) was used in the counter-electrode compartment (left).