METHODS AND APPARATUS FOR PERFORMING ELECTROCHEMICAL AND ENZYMATIC REACTIONS

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Application No. 63/430,735 filed 7 Dec. 2022 and entitled METHOD FOR PERFORMING COUPLED ENZYMATIC ELECTROCATALYTIC REACTIONS which is hereby incorporated herein by reference for all purposes.

FIELD

This invention relates generally to apparatuses and methods for performing electrochemical and enzymatic reactions. Specific embodiments provide electrochemical cells and methods which apply such cells for regenerating cofactors for use in enzymatic reactions.

BACKGROUND

Enzyme catalysis enables challenging chemical reactions to be performed with high activity and selectivity at ambient conditions. The industrial use of enzymes for chemical production currently represents a $100 billion USD industry. A major application of enzyme catalysis is for enantioselective synthesis in the pharmaceutical industry, with ⅔ of commercial chiral products involving enzyme catalysis. Up to 25% of known classes of enzymes require costly stoichiometric cofactors such as nicotinamide adenine dinucleotide (NAD⁺) and its reduced form (NADH). These stoichiometric cofactors provide hydride as a source of both protons and electrons. These cofactors must also be continuously regenerated in situ for viable catalysis.

Cofactor regeneration in industry is currently only performed with secondary enzymes (e.g. formate and glucose dehydrogenase) that catalyze NADH formation from NAD⁺. These systems require separation of water-soluble co-substrates and their byproducts, which results in high regeneration costs and limits their application to the synthesis of high-value products. The development of alternative cofactor regeneration methods that do not require separation of co-substrates and byproducts is a current challenge for the development of low cost enzymatic catalytic systems with expanded industrial applications.

The inventors have recognized a general need for improved apparatuses and methods for performing coupled enzymatic electrocatalytic reactions. There is a particular need for improved methods and cells for regenerating enzyme cofactors for use in enzymatic reactions.

SUMMARY

This application has a number of aspects. These include, without limitation:

• • methods and apparatuses for performing coupled electrochemical and enzymatic reactions.
  • methods and apparatuses for regenerating cofactors in parallel with enzyme catalysis.

One aspect of the invention relates to methods for performing electrochemical and enzymatic reactions. The method comprises applying an electrical potential between an anode and a metallic membrane, electrochemically dissociating, at the anode, a hydrogen-containing compound to form hydrogen ions, transporting the hydrogen ions to a first surface of the metallic membrane and at the first surface, and reducing the hydrogen ions to form hydrogen atoms. The hydrogen atoms may then diffuse through the metallic membrane to an opposing second surface of the metallic membrane. At the second surface, electrons may be transferred to the hydrogen atoms to form hydride ions. The hydride ions may then react with oxidized cofactors in the enzyme compartment to form reduced cofactors.

In some embodiments, the method further comprises supplying a flow of enzymes and substrates into the enzyme compartment. The enzymes and the reduced cofactors may react with the substrates to yield one or more products and oxidized cofactors. The oxidized cofactors may be released into the enzyme compartment. The released oxidized cofactors may transport within the enzyme compartment toward the second surface of the metallic membrane. The released oxidized cofactors may react with the hydride ions to form the reduced cofactors for further enzymatic reactions.

The transferring of the electron from the metallic membrane at the second surface to the hydrogen atoms to form hydride ions is performed in a solvent. In some embodiments, the solvent comprises a proton source. In some example embodiments, the solvent comprises water. In some example embodiments, the solvent comprises a buffer solution. In some embodiments, the solvent is maintained at a pH of about 6 to about 10.

One aspect of the invention relates to apparatuses for performing electrochemical and enzymatic reactions. The apparatus comprises an electrochemical compartment and an enzyme compartment separated by a metallic membrane. The metallic membrane serves as two or more of, or all of: 1) a cathode of the electrochemical cell; 2) a hydrogen-selective layer; 3) a catalyst which helps to promote a desired chemical reaction in the enzyme compartment; and 4) a separator which separates a solvent and reactants in the electrochemical compartment from a different solvent or solvents and reactants used in the enzyme compartment. The apparatus comprises an anode exposed in the electrochemical compartment, adapted to oxidize a hydrogen-containing compound to form hydrogen ions.

In some embodiments, the metallic membrane comprises a layer of palladium or a palladium alloy. In some embodiments, the palladium or palladium alloy of the membrane is coated with one or more layers of co-catalyst. The one or more layers of co-catalyst may comprise another metal, such as palladium or platinum black, or mixtures thereof. In some embodiments, the one or more layers of co-catalyst comprises a porous support layer applied on one or both surfaces of the metallic membrane.

One non-limiting example application of the methods and apparatuses is in the regeneration of nicotinamide adenine dinucleotide (NAD(H)). The electrochemical dissociation of a hydrogen-containing compound at the anode forms hydrogen ions. The hydrogen ions may be reduced to form hydrogen atoms on a first surface of the metallic membrane. The hydrogen atoms may then diffuse through the metallic membrane to reach a second surface of the metallic membrane in the enzyme compartment. At the second surface, hydride ions are formed by transferring electrons to the hydrogen atoms. The oxidized form of the nicotinamide adenine dinucleotide (NAD⁺) that is present in the enzyme compartment may be reduced to form NADH by reacting NAD⁺ with the hydride ions. The reduced NADH may then be used by the enzyme in the enzyme compartment in the enzymatic reaction with the substrate to form one or more products. The cofactor regeneration may thus be performed in tandem with the enzymatic reaction.

Further aspects and example embodiments are illustrated in the accompanying drawings and/or described in the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings illustrate non-limiting example embodiments of the invention.

FIG. 1 is a schematic illustration showing an apparatus for performing paired electrochemical and enzymatic reactions according to an example embodiment of this invention.

FIG. 2A is a flow chart showing the steps of a method for regenerating a cofactor using the FIG. 1 electrochemical cell according to an example embodiment of the invention. FIG. 2B is a flow chart showing the steps of a method for converting a substrate into a product using an enzyme and the regenerated cofactor according to an example embodiment of the invention.

FIG. 3A is a schematic diagram illustrating the reactions that are proposed to occur in enzymatic NADH regeneration.

FIG. 3B is a schematic diagram illustrating the reactions that are proposed to occur in prior art electrocatalytic NADH regeneration.

FIG. 3C is a schematic diagram illustrating the reactions that are proposed to occur in NADH regeneration using the FIG. 1 apparatus (referred to herein as “BioPMR”) and FIGS. 2A and 2B methods.

FIG. 4 is a schematic diagram illustrating a 3D-printed reactor design of the BioPMR.

FIG. 5A is a schematic diagram illustrating the cell setup and the reactions that are proposed to occur in the BioPMR in the generation of NADH.

FIG. 5B is a plot showing the reaction profile for 1,4-NADH generation with Pd black catalyst and Pt-coated catalyst (Pt/Pd black) in tris buffer at pH 9.0.

FIG. 5C is a plot showing 1,4-NADH conversion in 3 h with Pt-coated catalyst as a function of buffer pH (phosphate buffer for pH 6.5, tris for pH 7-9).

FIG. 6A illustrates the reactions that are proposed to occur in the BioPMR in an enzymatic reaction which involves reduction of propionic aldehyde with alcohol dehydrogenase (ADH) in phosphate buffer at pH 6.5. The enzymatic reaction mixture contains 1 mg/mL BSA.

FIG. 6B is a plot of conversion (%) as a function of time (h) in the performance of the FIG. 6A enzymatic reaction.

FIG. 6C illustrates the reactions that are proposed to occur in the BioPMR in an enzymatic reaction which involves reduction of pyruvic acid with lactate dehydrogenase (LDH) in tris buffer at pH 7. The enzymatic reaction mixture contains 1 mg/mL BSA.

FIG. 6D is a plot of conversion (%) as a function of time (h) in the performance of the FIG. 6C enzymatic reaction.

FIG. 6E illustrates the reactions that are proposed to occur in the BioPMR in an enzymatic reaction which involves reductive amination of pyruvic acid with alanine dehydrogenase (AlaDH) in tris buffer at pH 9. The enzymatic reaction mixture contains 1 mg/mL BSA.

FIG. 6F is a plot of conversion (%) as a function of time (h) in the performance of the FIG. 6E enzymatic reaction.

FIG. 7A is a schematic diagram illustrating example conditions used for cofactor regeneration and the reactions proposed to occur in the BioPMR. The enzyme solution contains 1 mg/mL BSA.

FIG. 7B is a schematic diagram illustrating example conditions used for cofactor regeneration and the reactions proposed to occur in conventional electrocatalytic regeneration methods. The enzyme solution contains 1 mg/mL BSA.

FIG. 7C is a plot of LDH activity (%) as a function of time (h) for both the FIGS. 7A and 7B cell setups.

FIG. 8A is a schematic diagram illustrating example conditions used in studies involving hydrogen permeation in different pH conditions, and the reactions proposed to occur in the BioPMR. The metallic membrane used comprises a Pt-coated catalyst.

FIG. 8B is a plot of H₂ ion current that represents H₂ gas evolution as a function of pH for both tris-buffer and phosphate-buffer used as the solvent in the enzyme compartment.

FIG. 9 is a schematic diagram illustrating the reactions that are proposed to occur in the formation of NADH in the BioPMR.

FIG. 10A is a schematic diagram illustrating example conditions used in NADH generation from NAD⁺ in tris buffer at pH 9, and the reactions proposed to occur in the BioPMR.

FIG. 10B is a plot of UV/Vis absorption spectra of NADH generation as a function of time.

FIG. 11A is a schematic diagram illustrating example conditions used in NADH generation from NAD⁺ in tris buffer at pH 9, and the reactions proposed to occur in the BioPMR.

FIG. 11B is a plot of absorbance (Abs) at 340 nm as a function of time from an ex-situ LDH assay study.

FIG. 12 is a 1H NMR spectra of NADH generation from 1.5 mM NAD⁺ in tris buffer at pH 9.

FIG. 13 is an ESI-MS of NADH generation from 1.5 mM NAD⁺ in tris buffer at pH 9.

FIG. 14A is a schematic diagram illustrating example conditions for studying NADH stability as a function of pH.

FIG. 14B is a plot of 1,4-NADH concentration (%) at each of pH 6.5 (phosphate buffer), pH 7 (tris buffer), and pH 9 (tris buffer) as a function of time.

FIG. 15A is a schematic diagram illustrating example conditions for comparing NADH generation from NAD⁺ in both tris buffer (pH 9) and DMSO.

FIG. 15B is a plot of 1,4 NADH conversion (%) as a function of time.

FIG. 16A is a plot of voltage (V vs. SHE) as a function of time (s) for NADH regeneration in the BioPMR at pH 6.5.

FIG. 16B is a plot of voltage (V vs. SHE) as a function of time (s) for NADH regeneration in the BioPMR at pH 7. The voltage range for typical electrochemical systems are shown in the shaded section.

FIG. 16C is a plot of voltage (V vs. SHE) as a function of time (s) for NADH regeneration in the BioPMR at pH 9.

FIG. 17 is a 1H NMR spectra of 1-propanol formation from propionic aldehyde.

FIG. 18 is a 1H NMR spectra of lactate formation from pyruvate.

FIG. 19 is a 1H NMR spectra of alanine formation from pyruvate.

FIG. 20 is a plot showing CV measurements in tris buffer at pH 9 without NAD⁺ and with 4.5 mM NAD⁺. The measurements were taken with a Pt-coated Pd foil working electrode, a Ag/AgCl reference electrode, and a Pt mesh counter electrode. A scan rate of 250 mV/s was used and the cell was sparged with Argon prior to the measurement.

DETAILED DESCRIPTION

Throughout the following description, specific details are set forth in order to provide a more thorough understanding of the invention. However, the invention may be practiced without these particulars. In other instances, well known elements have not been shown or described in detail to avoid unnecessarily obscuring the invention. Accordingly, the specification and drawings are to be regarded in an illustrative, rather than a restrictive sense.

Definitions

“BioPMR” refers to a “biocatalytic palladium membrane reactor” that includes an electrochemical compartment and enzyme compartment separated by a palladium membrane further comprising a transition metal catalyst.

“Cofactor” is a substance that acts in conjunction with an enzyme in a catalytic reaction. Cofactors may comprise inorganic ions, such as metal ions, and/or organic compounds. One non-limiting example of a cofactor is nicotinamide adenine dinucleotide (NAD(H)).

“Cofactor regeneration” refers to a method of converting a cofactor from its oxidized form to its corresponding reduced form. The regenerated cofactors that are in the reduced form may be used in enzymatic reactions. For example, the regeneration of the cofactor nicotinamide adenine dinucleotide (NAD(H)) refers to the conversion from NAD⁺ to NADH.

“Hydrogen” is any isotope of the element with atomic number 1.

“Hydrogen ion” is ionized hydrogen (H⁺). A proton is an example of a hydrogen ion.

“Hydride ions” or “hydrogen anion” is a negative ion of hydrogen (H).

“Palladium” is used herein broadly and comprises various composition of matter, including alloys and other combinations of palladium metal with other materials. For example, a “palladium membrane” may be formed by electrodepositing one or more layers of palladium onto a substrate (which may be a palladium foil, or a porous polymer). In one example, the substrate may be a rolled Pd wafer bar. Without being bound to any particular theory, the electrodeposited palladium may provide increased surface area that may increase the rate of reaction. Any suitable method for depositing palladium on a substrate, membrane foil, or other dense hydrogen selective material may be used.

“Porosity” is defined as the ratio of the volume of pores in a material to the total volume of the material.

“Transition metal” refers to a chemical element that has valence electrons, i.e., electrons that can participate in the formation of chemical bonds, in two shells. In other words, transition metals are elements with partially filled d orbitals. Transition metals are located in the d-block of the periodic table, occupying groups 3 to 12 on the periodic table.

Example Embodiments

Aspects of the invention relate to methods and apparatuses for performing electrochemical and enzymatic reactions. In some embodiments, the methods and apparatuses involve regenerating a cofactor in parallel with enzyme catalysis. Such methods and apparatus may involve pairing one or more electrochemical reactions that produce hydride ions, and a chemical reaction which uses the hydride ions as a reactant in the regeneration of a cofactor. The regenerated cofactor may be used to assist an enzyme in the catalysis of a substrate to yield one or more products. The methods and apparatuses do not require the use of any secondary enzymes, co-substrates, and/or mediators in the cofactor regeneration process. In some embodiments, oxygen gas (O₂) is formed as a byproduct. In some embodiments, the cofactors regenerated from the present methods are free or substantially free from undesired forms of the cofactor. Undesired forms of the cofactor may include dimeric forms of the cofactor. For example, (NAD)₂ dimer is an undesired and inactive form of a reduced product that is typically formed in conventional electrochemical NADH regeneration processes.

The methods may be performed in systems that include an electrochemical cell that is operative to produce hydrogen ions. The hydrogen ions may be reduced to hydrogen atoms. The hydrogen atoms may undergo an electron transfer reaction to produce hydride ions. The hydride ions may participate in a reaction with a cofactor to regenerate the cofactor. The cofactor may participate in a catalysis reaction to assist an enzyme in the conversion of a substrate to one or more products.

Overview of Apparatus and Methods for Cofactor Regeneration

FIG. 1 is a schematic diagram that illustrates an example system 10 that includes an electrochemical cell 11 for regenerating cofactors.

Cell 11 comprises a hydrogen selective membrane 12 that separates an electrochemical compartment 14 from an enzyme compartment 18. Electrochemical compartment 14 may be operated as described below to generate hydrogen ions and to supply the hydrogen ions to hydrogen selective membrane 12. In some embodiments, hydrogen selective membrane 12 serves as two or more of, or all of: 1) a cathode of the cell; 2) a hydrogen-selective layer; 3) a catalyst which helps to promote a desired chemical reaction in enzyme compartment 18; and 4) a separator which separates a solvent and reactants in electrochemical compartment 14 from a different solvent or solvents and reactants used in enzyme compartment 18.

Hydrogen selective membrane 12 is operative to pass atomic hydrogen into enzyme compartment 18. Membrane 12 selectively allows absorbed hydrogen atoms 32 to pass through membrane 12 while membrane 12 essentially blocks passage of all other ions, electrolytes and solvents. Membrane 12 may be referred to as a “hydrogen selective layer”.

In currently preferred embodiments, hydrogen selective membrane 12 comprises or consists of a metallic membrane. In the following description, membrane 12 is described as “metallic membrane 12”.

In some embodiments, including the embodiment illustrated in FIG. 1, a first surface 22 of metallic membrane 12 is exposed to electrochemical compartment 14 and an opposing second surface 24 of metallic membrane 12 is exposed to enzyme compartment 18.

An anode 28 is exposed to electrochemical compartment 14. Anode 28 may comprise platinum metal, for example. Other suitable materials may be used as anode 28. For example, metals such as palladium metal and metal oxides such as a nickel oxide (NiO_x) or ruthenium (IV) oxide (RuO₂) may be used for anode 28. Carbonaceous materials such as graphite may also be used as anode 28.

A reference electrode may be exposed to electrochemical compartment 14. Ag/AgCl, Cu/CuSO₄, SCE may for example be used as reference electrode. The reference electrode may be positioned between anode 28 and metallic membrane 12.

A power source 26 is connected to apply a potential difference between anode 28 and metallic membrane 12. Metallic membrane 12 serves as a cathode.

Power source 26 may be configured to maintain a desired electric current between metallic membrane 12 and anode 28 and/or maintain a potential difference between metallic membrane 12 and anode 28 at a desired level or in a desired range.

An ion exchange membrane may optionally be arranged to divide electrochemical compartment 14 into two parts. Ion exchange membrane is a membrane that is selectively permeable to certain dissolved ions while blocking other ions or neutral molecules. In example embodiments, ion exchange membrane is a cation exchange membrane. For example, membrane may comprise a commercially available cation exchange membrane such as those marketed under the product name Nafion™. In example embodiments, ion exchange membrane is selectively permeable to hydrogen ions.

A suitable electrolyte 60 is supplied to electrochemical compartment 14. A suitable electrolyte 60 facilitates a first electrochemical reaction 50 at anode 28 by providing electrons to reactant 56 to yield hydrogen ions (H⁺). The suitable electrolyte 60 may also facilitate a second electrochemical reaction 64 at metallic membrane 12 by providing a medium within which hydrogen ions 48 travel to metallic membrane 12 to yield atomic hydrogens 32. The pH at electrochemical compartment 14 can range from 0 to 14. Suitable electrolytes may be an acid or a base. Non-limiting examples of suitable electrolytes include H₂SO₄, HCl, H₃PO₄, KHCO₃, KOH.

In embodiments in which an ion exchange membrane is arranged within the electrochemical compartment 14 for separating anode 28 from metallic membrane 12, electrochemical compartment 14 may define an anode chamber and a cathode chamber. A suitable anolyte may be supplied to the anode chamber to facilitate first electrochemical reaction 50. A suitable catholyte may be supplied to the cathode chamber to facilitate second electrochemical reaction 64. The same or different electrolyte solutions may be used as the anolyte and the catholyte.

One or more reactants 56 are supplied to electrochemical compartment 14 to participate in first electrochemical reaction 50 at anode 28 to yield hydrogen ions 48. In some embodiments, first electrochemical reaction 50 is an oxidation reaction.

In some embodiments, reactant 56 comprises water (H₂O). In such embodiments, the electrochemical dissociation of water at anode 28 yields oxygen gas 47 and hydrogen ions 48. First electrochemical reaction 50 may however comprise any other oxidation reaction which produces hydrogen ions (H⁺).

Hydrogen ions 48 are released into electrochemical compartment 14 and may migrate to metallic membrane 12. If an ion exchange membrane is present, hydrogen ions 48 may migrate through the ion exchange membrane to reach metallic membrane 12. Hydrogen ions 48 participate in second electrochemical reaction 64 at metallic membrane 12 to yield hydrogen atoms 32. Second electrochemical reaction 64 may occur at first surface 22 of metallic membrane 12. Second electrochemical reaction 64 is a reduction reaction. The hydrogen atoms are absorbed into metallic membrane 12 and permeate through metallic membrane 12 to second surface 24 where they are available to participate in one or more reactions within enzyme compartment 18. In doing so, hydrogen atoms 32 transition from first surface 22 into the bulk of a lattice 34, and transition to the opposing second surface 24 in enzyme compartment 14. Once hydrogen atoms 32 reach enzyme compartment 14, hydrogen atoms 32 may undergo an electron transfer reaction 65 at metallic membrane 12 to yield hydride ions 49. Electron transfer reaction 65 may take place at second surface 24. Hydride ions 49 may be released into enzyme compartment 18 where they are available for reaction with cofactors 36.

Without being bound to theory, water is hypothesized to assist with the charge separation of hydrogen atoms at second surface 24 of metallic membrane 12 to yield hydrogen ions and hydride ions, in accordance with the following chemical equation (Eq. 1):

H·+H·→H⁺+H⁻  (Eq. 1)

In particular, it is hypothesized that hydrogen atoms 32 that are diffused through metallic membrane 12 to enzyme compartment 18 may bind to second surface 24 of metallic membrane 12, forming a Pt—H bond. A water molecule may bind to such surface hydrogen. Heterolytic cleavage of the Pt—H bond may then occur on second surface 24 of metallic membrane 12, forming H₃O⁺ and releasing an electron. The released electron may be migrated into the bulk of lattice 34 of metallic membrane 12, forming a reduced metal surface. The electron may be transferred from the reduced metal to surface-bound hydrogen atoms 32 to form hydride ions 49 in electron transfer reaction 65.

Enzyme compartment 18 contains a cofactor 36 and a suitable solvent 66. Cofactor 36 that is supplied to enzyme compartment 18 may be in an oxidized form (referred to herein as “oxidized cofactors 37”) and/or in a corresponding reduced form (referred to herein as “reduced cofactors 38”). As used herein, a cofactor is in its “oxidized” form if the molecule is in a state in which it loses one or more electrons. A cofactor is in its “reduced” form if the molecule is in a state in which it gains one or more electrons. A reduced cofactor may serve as a mediator which shuttles electrons and protons between the enzyme and substrate in an enzymatic reaction. One non-limiting example of a cofactor is nicotinamide adenine dinucleotide. The oxidized form of nicotinamide adenine dinucleotide is NAD⁺ and its reduced form is NADH. NAD⁺ reduces to NADH with the addition of a proton and two electrons. The chemical reaction that is known to occur is shown in Eq. 2 below.

NAD⁺+2e⁻+H⁺→NADH  (Eq. 2)

Cofactors 36 undergo a chemical reaction 80 by reacting with hydride ions 49 produced in electron transfer reaction 65. Chemical reaction 80 may involve reacting oxidized cofactors 37 with hydride ions 49 to yield reduced cofactors 38. In some embodiments, chemical reaction 80 occurs at second surface 24. Chemical reaction 80 may be catalyzed by the material of metallic membrane 12 and/or by a catalyst provided on or adjacent to second surface 24 of metallic membrane 12. Reduced cofactors 38 may be released in enzyme compartment 18. In some embodiments, reduced cofactors 38 are discharged from electrochemical cell 11. The discharged reduced cofactors 38 may optionally be processed (e.g., by purification, etc.) and be supplied to a downstream process and apparatus for performing enzyme catalysis.

The enzyme catalysis may be performed in enzyme compartment 18. In some embodiments, one or more enzymes 68 and substrates 70 are supplied to the enzyme compartment 18. Reduced cofactors 38 may assist enzyme 68 in a chemical reaction 74 with substrate 70 to yield one or more products 78. Reduced cofactors 38 may serve to shuttle electrons and protons between enzyme 68 and substrate 70. Reduced cofactors 38 are oxidized to oxidized cofactors 37 in chemical reaction 74. Oxidized cofactors 37 may be released in enzyme compartment 18 after participating in chemical reaction 74. Oxidized cofactors 37 may participate in chemical reaction 80 to regenerate the cofactor to yield reduced cofactors 38, which can be available for use in further chemical reactions 74.

In some embodiments, a suitable solvent 66 comprises a source of proton (H⁺). Solvent 66 may comprise a protic solvent. Solvent 66 may comprise an inorganic solvent and/or an organic solvent. In some embodiments, solvent 66 comprises water. In some embodiments, solvent 66 comprises a buffer solution that is suitable for facilitating reactions 65, 74, 80 occurring within enzyme compartment 18. The buffer solution may comprise a weak acid and its conjugate base, or a weak base and its conjugate acid. In some embodiments, the pH of solvent 66 is in the range of from about 5 to 14, and in some embodiments, in the range of from 6 to 12, and in some embodiments, in the range of from about 6 to about 10, and in some embodiments, greater than about 6. Suitable buffers include for example, tris-buffer saline (TBS), phosphate buffered saline (PBS), and the like.

Basic conditions (i.e., higher pH) within the enzyme compartment may provide stability to the cofactor, thereby advantageously promotes higher rate and/or yield in the formation of the reduced cofactors in the chemical reaction between the oxidized cofactors and the hydride ions. A higher rate of formation of the reduced cofactors may facilitate uptake of hydride ions at the metallic membrane. This may reduce undesired hydrogen gas (H₂) formation in the enzyme compartment. For example, hydrogen gas may be formed by the desorption of H species (i.e., H⁺+H⁻) as H₂ (i.e., H⁺+H⁻→H_2(g).

With the sole exception of hydrogen which can be transported through metallic membrane 12, cofactors 36, solvent 66, (and if present, enzymes 68 and substrates 70) in enzyme compartment 18 can be kept isolated from reactant 56 and electrolyte 60 in electrochemical compartment 14. The near complete isolation provided by metallic membrane 12 allows different materials and/or conditions (e.g., temperature, pH, ionic strength, and/or catalyst, etc.) to be provided at opposite sides of metallic membrane 12. The separation of enzyme compartment 18 from electrochemical compartment 14 also isolates the enzyme from the electric field and/or resistive heating at the electrodes exposed in electrochemical compartment 14 (e.g., anode 28 and/or the reference electrode), advantageously reducing the likelihood of enzyme inactivation or denaturing occurring at the electrodes.

In some embodiments, cofactors 36 are molecules which have reversible redox chemistry. Cofactor 36 may be a molecule that can be reversibly oxidized and reduced. In such embodiments, cofactor 36 assists with oxidations and reductions of substrates by acting as mediator that shuttles electrons and protons between the enzyme and the substrate in enzymatic reactions. Cofactor 36 may be a redox cofactor. The present methods and apparatuses may be used to regenerate any suitable cofactors. Such suitable cofactors in their corresponding reduced form may be hydride sources. Cofactors 36 may for example be a dihydropyridine (e.g., nicotinamide adenine dinucleotide (NAD(H)), nicotinamide adenine dinucleotide phosphate (NADP((H)), etc.) and/or a flavin (e.g., flavin adenine dinucleotide (FAD(H)), flavin mononucleotide (FMN(H)), etc.). In one example embodiment, cofactor 36 comprises nicotinamide adenine dinucleotide (NAD⁺). The reduced form of NAD⁺ is NADH. The present methods and apparatuses are not limited to the regeneration of NADH.

Example Constructions for Metallic Membrane 12

Metallic membrane 12 is made of a material which is selectively permeable to absorbed hydrogen atoms 32.

Metallic membrane 12 may serve as all of: 1) a cathode; 2) a hydrogen selective layer which allows passage of hydrogen atoms (i.e. any isotope of hydrogen) and blocks other reactants including hydrogen ions and hydride ions; 3) a physical barrier which separates cofactors, solvent, enzymes and substrates in the enzyme compartment from the different solution or solutions used in the electrochemical compartment; and 4) a catalyst which helps to promote chemical reaction 80 in the enzyme compartment. The physical barrier advantageously allows for the use of materials and/or conditions in the enzyme compartment to be different from the materials and/or conditions present in the electrochemical compartment. The physical barrier also isolates the enzyme present in the enzyme compartment from the electrochemical compartment, thereby reducing the likelihood of enzyme inactivation or denaturing occurring at the electrodes.

Metallic membrane 12 is made up of at least one metal. The metal, may for example, have a crystalline lattice that provides interstitial sites that can accept hydrogen atoms. In example embodiments, metallic membrane 12 is made from palladium (Pd) metal. Palladium is highly selective for passing hydrogen and is impermeable to most practical solvents and electrolytes. Palladium metal has a face centered cubic crystal lattice that is capable of hosting hydrogen atoms up to a hydrogen/palladium ratio (H:Pd) of approximately 0.7 (PdH_0.7). Another example metal that may be used as metallic membrane 12 is a hydrogen permeable palladium alloy. Examples of palladium alloys that may be used to make metallic membrane 12 include but are not limited to: Pd—Ag, Pd—Sn, Pd—Au, Pd—Pb, Pd—B, Pd—Pt, Pd—Rh, Pd—Ni and Pd—Cu. Other metals that have high permeability to hydrogen include niobium, vanadium and tantalum.

In some embodiments, metallic membrane 12 is formed of one or more layers. The one or more layers may be formed by electrodeposition and/or sputtering. The one or more layers may comprise a metal such as palladium or a palladium alloy and/or one or more layers of co-catalyst 42.

In some embodiments, one or more layers of catalyst 42 is applied on first surface 22 and/or second surface 24 of metallic membrane 12 to promote chemical reactions 65 and 80 in enzyme compartment 18. Catalyst 42 may be called a “co-catalyst”. Co-catalyst 42 may be porous. Co-catalyst 42 may for example be a porous support layer. Co-catalyst 42 may be heterogeneous.

In some embodiments, co-catalyst 42 comprises one or more transition metals. “Transition metals” include elements that have (or readily form) partially filled d-orbitals, for example those located in groups 3-12 of the periodic table. Examples of suitable elements that may be used as co-catalyst 42 include but are not limited to palladium, platinum, gold, iridium, and ruthenium. In one example embodiment, co-catalyst 42 comprises palladium, platinum black, or mixtures thereof.

In some embodiments, the thickness of the layer of co-catalyst 42 is in the range of from 3 nm to 20 nm, including any value therebetween, such as 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, etc.

In some embodiments, metallic membrane 12 comprises a self-supporting membrane made of a hydrogen selective material as described herein. The membrane may, for example, have the form of a sheet, plate, corrugated sheet or plate, casting or the like. In some embodiments, metallic membrane 12 is formed on or attached to a permeable substrate that helps to support metallic membrane 12.

In example embodiments, metallic membrane 12 is formed by electrodepositing one or more layers of palladium on one or both sides of a palladium foil or a hydrogen selective membrane. Such membrane 12 may be called a “Pd/Pd membrane”. Without being bound to theory, the electrodepositing of one or more layers of palladium may result in a morphology that provides greater surface area of the membrane. The one or more layers may for example be made of palladium electrodeposited from a solution comprising a palladium salt. In example embodiments, the palladium salt comprises palladium chloride (PdCl₂).

Any suitable method for electro-depositing and/or sputter-depositing palladium salt and/or the layer of co-catalyst on a hydrogen selective membrane may be used. In an example electro-deposition process, an Ag/AgCl electrode is used as a reference electrode and a Pt mesh electrode is used as the counter electrode. The electrodeposition may be performed in an acidic PdCl₂ solution. For example, the solution may comprise 15.9 mM PdCl₂ dissolved in 1M HCl. Roughly −0.1 V vs. Ag/AgCl potential is applied to the electrodes. The electrodeposition is complete when a desired thickness of palladium has been deposited. Completion may be determined by measuring a charge passed in the electrodeposition circuit. For example, some satisfactory electrodeposited palladium layers were made by terminating the electrodeposition when 18.5 C of charge (7.38 C/cm²) of the membrane had passed in the circuit.

In some embodiments, an electrodeposition current in the range of about 20 mA to about 100 mA is applied to electrodeposit a co-catalyst on metallic membrane 12. The magnitude of the electrical current may be set based on the type of co-catalyst to be deposited. For example, the electrical current may be maintained at about 30 mA in embodiments in which gold and or platinum are selected as the co-catalyst and 70 mA in embodiments in which iridium is selected as the co-catalyst.

In some embodiments, the one or more layers of co-catalyst 42 is sputter-deposited at a rate in a range of from about 0.1 nm/s to about 1 mm/s. In some example embodiments, the co-catalyst is sputter-deposited at a rate of about 0.2 nm/s.

In some example embodiments, membrane 12 comprises a palladium foil. The palladium foil is coated with electrodeposited palladium black (Pd black) at second surface 24. The Pd black layer is further coated with sputtered platinum (Pt). The platinum (Pt) is 10 nm thick.

Example Methods for Cofactor Regeneration and Enzyme Catalysis

FIG. 2A is a flow chart illustrating the steps of an example method 100 of regenerating cofactors. FIG. 2B is a flow chart illustrating the steps of an example method 100 of performing an enzymatic reaction using the cofactors regenerated in FIG. 2A. The cofactor regeneration may be performed in tandem with the performing of the enzymatic reaction. Referring to FIG. 2A, in block 112, an electrical current and/or potential is applied between an anode and a metallic membrane which serves as a cathode. In block 114, a hydrogen-containing compound such as water is supplied at an anode exposed in an electrochemical compartment. In block 116, the hydrogen-containing compound undergoes an oxidation reaction to produce hydrogen ions (H⁺). The hydrogen ions (H⁺) migrate toward the metallic membrane (block 118). In block 120, the hydrogen ions (H⁺) undergo a reduction reaction on a first surface of the metallic membrane to form hydrogen atoms. The hydrogen atoms pass through the metallic membrane as absorbed hydrogen atoms and appear on a second surface of the metallic membrane (block 122). The hydrogen atoms may undergo an electron transfer reaction on a second surface of the metallic membrane to form hydride ions (block 124). In block 126, a solvent comprising an oxidized cofactor is supplied to an enzyme compartment. The oxidized cofactor undergoes a chemical reaction by reacting with the hydride ions to produce a reduced cofactor (block 128). The chemical reaction may occur on the second surface of the metallic membrane.

In some embodiments, the reduced cofactor is discharged from the enzyme compartment. The reduced cofactor may be provided to a process or apparatus downstream of the electrochemical cell for use in enzyme catalysis.

Referring to FIG. 2B, in some embodiments, the enzyme catalysis is performed within the enzyme compartment. In such embodiments, one or more enzymes and substrates are supplied to the enzyme compartment (block 130). The enzyme participates in a chemical reaction with the substrate to yield one or more products (block 132). The reduced cofactor may facilitate the chemical reaction by mediating the transfer of electrons and protons from the enzyme to the substrate. In the chemical reaction in block 132, the reduced cofactor converts to the oxidized cofactor. The oxidized cofactor may be released into the enzyme compartment where it becomes available for regeneration by reacting with a hydride ion in block 126. The one or more products may be removed from the enzyme compartment for downstream processing and/or storage.

In some embodiments, the hydrogen ions (H⁺) produced at the anode in block 116 migrate to an ion exchange membrane and pass through the ion exchange membrane before migrating to the metallic membrane to participate in the reduction reaction at block 120.

Method 100 may be tuned to optimize one or more of product selectivity, current efficiency and reaction rate of each of the electrochemical reactions and chemical reactions by adjusting one or more of:

• • characteristics of the metallic membrane such as the particular metal or metals used to make the membrane hydrogen selective and its surface area, density and thickness, and/or
  • additional catalysts present; and/or
  • conditions of the flow cell such as temperature, pH, pressure, etc.; and/or
  • the type of solvent and electrolyte in the area where each reaction takes place; and/or
  • flow rate of the cofactors and/or enzyme and/or substrate and/or solvent; and/or
  • flow rate and/or composition of the reactants and/or solvent and/or electrolyte; and/or
  • electrical operating conditions such as the applied electrical potential; and/or
  • characteristics of the cathode and/or anode electrodes such as the material and method of fabrication; and/or
  • nature of the cathode and/or anode catalyst;
  • presence and/or characteristics of the ion exchange membrane such as the thickness, porosity, etc.; and/or
  • etc.

At least some of these factors may be separately optimized for each of the electrochemical and enzymatic reactions to achieve high rates of formation of the products and/or high selectivity of the desired products at each of the electrochemical and the enzyme compartments. The physical barrier provided by metallic membrane 12 advantageously allows the electrochemical and chemical reaction conditions in electrochemical compartment 14 and enzyme compartment 18 to be controlled independently. Examples of conditions that can be independently controlled are: catalysts, choice of solvent, choice of electrolytes or other additives, etc. Within limits imposed by the physical design of metallic membrane 12, it may be possible to independently control temperature and/or pressure on either side of metallic membrane 12.

In some embodiments, electrochemical cell 11 is maintained at a temperature in a range of from about 25° C. to about 80° C., including any value therebetween such as 30° ° C., 35° C., 40° ° C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., etc. In some embodiments, electrochemical cell 11 is heated and/or maintained at a physiological temperature. The physiological temperature may for example be in the range of from about 30° ° C. to about 45° C., and in some embodiments, from about 30° C. to about 40° C., and in some embodiments, about 37° C.

In some embodiments, the pH maintained at enzyme compartment 18 is in a range of from about 5 to about 14, and in some embodiments, in the range of from about 7 to about 12, and in some embodiments, in the range of from about 8 to about 11, and in some embodiments, in the range of from about 6 to about 10.

In some embodiments, the concentration of oxidized cofactors 37 maintained in enzyme compartment 18 is less than the concentration of the enzymes 68 and/or substrates 70 maintained in enzyme compartment 18. A lower concentration of oxidized cofactors 37 may improve the rate of the enzymatic reaction.

In some embodiments, enzyme compartment 18 is maintained in an atmosphere that is free or substantially free of oxygen. In some embodiments, an inert gas is supplied to enzyme compartment 18. In such embodiments, enzyme compartment 18 is maintained in an inert gas atmosphere. The inert gas may for example be Argon (Ar) and/or Nitrogen gas (N), or the like.

The apparatus as described herein may be operated at low pressure (e.g., atmospheric pressure), or pressures above or below atmospheric pressure.

The invention is further described with reference to the following specific examples, which are not meant to limit the invention, but rather to further illustrate it.

EXAMPLES

An electrochemical cell of the type illustrated in FIG. 1 and the method illustrated in FIGS. 2A and 2B were used to regenerate enzymatic cofactors, and to use the regenerated enzymatic cofactors in enzymatic reactions. The enzymatic cofactor used in the Examples is nicotinamide adenine dinucleotide or NAD⁺. The reduced form of NAD⁺ is NADH. NAD⁺ is the oxidized cofactor 37. NADH is the reduced cofactor 38. The electrochemical cell 11 used in these Examples are hereinafter referred to as a “BioPMR cell” or “BioPMR”.

Example 1: Apparatus Design

FIG. 4 is a schematic diagram illustrating the BioPMR cell used in the Examples. The BioPMR cell comprises an electrochemical compartment 14 and an enzyme compartment 18. The electrochemical compartment 14 contains a 3-electrode cell comprising a Ag/AgCl reference electrode, a Pt mesh counter electrode as anode 28 (where water oxidation occurs), a 25-μm thick Pd foil working electrode as metallic membrane 12 (where proton reduction and hydrogen absorption occur). Electrolyte 60 comprises 8 mL of 1 M H₂SO₄. The Pd foil also functions as a hydrogen-selective membrane separating electrochemical compartment 14 from enzyme compartment 18. The enzyme compartment 18 (where the hydrogen atom that diffuses through the membrane reacts with NAD⁺) contains buffer solution (10 mL of 0.1 M phosphate or tris buffer) as solvent 66. The enzyme compartment 18 also comprises 1.5 mM NAD⁺, enzyme, and 1.5 mM substrate. The second surface 24 of the metallic membrane 12 (i.e., the side of the palladium membrane facing the enzyme compartment 18) is coated with electrodeposited Pd black to increase the active area. The Pd black layer is further coated with sputtered Pt. The sputtered Pt is about 10-nm thick.

The BioPMR may perform a wide range of NADH-dependent enzymatic transformations on different reagents under ideal operating conditions with water and electricity as the primary inputs and benign gases as the primary outputs. For example, it can be used to: i) electrochemically-regenerate NADH from NAD⁺ (see Example 2) and; ii) perform NADH regeneration in parallel with enzyme catalysis (see Example 3). The BioPMR has several advantages over conventional enzymatic and electrochemical cofactor regeneration methods. First, the BioPMR does not require any secondary enzymes, co-substrates, or mediators. Second, the only major waste product in the BioPMR is oxygen gas generated at the anode. Both advantages are in contrast to formate and glucose dehydrogenase systems that require separation of water-soluble co-substrates and byproducts, or electrochemical cofactor regeneration systems that often require mediators to suppress inactive NAD₂ formation. Both advantages minimize the purification steps required for isolation of the desired product. There are additional advantages of the BioPMR that stem from the separation of the electrochemical and enzymatic processes. Typical electrochemical cofactor regeneration systems with both processes occurring in the same reaction mixture necessitate compromise with regard to the ideal operating conditions (e.g., pH, temperature, ionic strength, and catalyst choice) for each process. Enzyme inactivation or denaturation can also occur from the electric field or resistive heating at the electrodes in these systems. In the BioPMR, the Pd foil separates the two compartments and enables optimal conditions for each process to be used while also isolating the enzyme from the electric field of the cell. Moreover, the separation of the BioPMR enables a lower overpotential for NADH formation at biologically-relevant pH values (pH 6.5-9.0) relative to a typical electrochemical cofactor regeneration system (−0.3 V vs SHE (see e.g., FIGS. 16A-C) and −0.6 to −1.7 V vs. SHE, respectively).

Example 2: Proof of Concept

The inventors have demonstrated that the BioPMR enables generation of NADH from NAD⁺ using water as a proton and electron source. A solution of 1.5 mM NAD⁺ and 0.1 M tris buffer (pH_measured=9.0) was added to the enzyme compartment 18 and 1 M H₂SO₄ was added to the electrochemical compartment 14 (FIG. 5A). The cell 11 was configured as described in Example 1 (FIG. 4) and heated to physiological temperature (37° C.) in an oven. Galvan ostatic electrolysis was performed (lapplied=−50 mA·cm⁻²) under Ar gas. The formation of the biologically-active NADH isomer (i.e. 1,4-NADH) in the enzyme compartment was initially confirmed by UV-Vis absorption spectroscopy (λmax=340 nm, FIGS. 10A-B) and a lactatedehydrogenase (LDH) assay (FIGS. 11A-B). The percentage of 1,4-NADH formed was then quantified by 1H NMR spectroscopy as a function of time (FIG. 5B, FIG. 12). This experiment was performed for both a Pd black catalyst and a Pt-coated catalyst (FIG. 5A). The yield of 1,4-NADH after 3 h was ˜26% with the Pd black catalyst, and ˜67% with the Pt-coated catalyst (FIG. 5B). No NAD₂ dimer was observed by 1H NMR (FIG. 12) and ESI-MS (FIG. 13).

Example 3: Three Enzymatic Processes

To study enzyme catalysis in the BioPMR with concurrent 1,4-NADH regeneration, three different relevant enzymatic processes were examined: i) aldehyde reduction (FIGS. 6A,B); ii) ketone reduction (FIGS. 6C,D), and; iii) reductive amination (FIGS. 6E,F). These enzymatic reactions were chosen because each has a different optimum operating pH. Each reaction mixture consisted of 5 units of enzyme and 1 mg/mL bovine serum albumin (BSA) with 0.1 M of the appropriate buffer in the enzyme compartment. The electrochemical compartment was filled with 1 M H₂SO₄ as the electrolyte. All enzymatic studies were performed at 37° C. for 3 h with an applied current density of −50 mA·cm⁻²(V_avg=−0.3±0.05 V vs. SHE, FIGS. 16A-C). Product conversion was tracked as a function of time by 1H NMR (FIGS. 6A-6F, FIGS. 17-19). All enzymes showed high yields (65-100%) and conversion rates for their respective substrates. Control experiments with no added NAD⁺ and no enzyme confirmed that the NADH-dependent enzymes were responsible for product formation (FIGS. 6A-6F). Additionally, the enzymatic aldehyde reduction was performed with an excess amount of substrate relative to NAD⁺ (1:5 stoichiometric ratio) leading to full conversion in 4 h (FIG. 20).

Example 4: Enzyme Stability

Two additional experiments were performed with LDH under similar conditions, one in the BioPMR (FIG. 7A) and one under conventional electrocatalytic regeneration conditions (FIG. 7B), to test if isolation of an enzyme from the electrochemical process in the BioPMR increases the stability of such enzyme. Both experiments were performed under electrolysis with LDH in 0.1 M tris (pH 7). The appropriate potential (−0.8 V vs. SHE) for NAD⁺ reduction in the electrocatalytic experiment was identified by cyclic voltammetry (CV, FIG. 20). The activity of LDH was lost after 30 min for electrocatalytic regeneration conditions while BioPMR conditions retained activity comparable to the control in which no electrolysis was performed (FIG. 7C).

Example 5: Mechanism of NADH Regeneration

FIGS. 8A and 8B are results of the pH-dependent hydrogen permeation studies to illustrate the mechanism of NADH regeneration in the BioPMR. In general, the mechanism of formation of the reduced product (NADH) involves disproportionation of H atoms (i.e., H·+H·→H⁺+H⁻) on the catalyst surface. These charged H species (i.e., H⁺+H⁻) desorb as H₂ (i.e., H⁺+H⁻→+H_2(g)), if H⁻ does not react with NAD⁺. An earlier study demonstrated that the amount of H_2(g) formed on the Pt-coated Pd catalyst in the enzyme compartment is governed by the desorption process. The study demonstrated that H₂ formation is favored at lower pH values.

The BioPMR cell was connected to an atmospheric mass spectrometer (atm-MS), with gas sampling of the enzyme compartment (FIGS. 8A-B). The cell was filled with electrolyte (1 M H₂SO₄) in the electrochemical compartment and buffer only (1 M tris or phosphate) in the enzyme compartment. Electrolysis was performed (l_applied=−50 mA·cm⁻²) and the amount of H₂ gas generated at the catalyst surface was recorded at steady-state (˜1 h). A series of these measurements were performed at sequential buffer pH values (pH_tris=3.4, 7, 8, 9; pH_phosphate=3.4, 6, 7, 8). The inventors observed that lower pH values resulted in higher H₂ gas generation for both tris and phosphate buffer (FIG. 8B). The pH 3.4 data points extend outside of the buffering pH range.

The inventors believe that NADH formation from NAD⁺ proceeds via one of the following mechanisms: a) electron transfer and proton transfer (2e⁻+H⁺); b) electron transfer and hydrogen transfer (e⁻ and H), or; c) hydride transfer (H⁻). Electrocatalytic NADH generation follows a) or b). NADH formation in the BioPMR is unlikely to undergo mechanism a) and b) because the surface of the catalyst is isolated from the electrochemical potential of the cell (FIG. 3C), thus any pathway involving direct e-transfer is believed to be unlikely. This assertion is also supported by the absence of NAD₂ formation in the BioPMR, which occurs via direct electron transfer, while the presence of surface bound hydride is supported by the pH-dependence of H₂ gas generation experiments. These experiments demonstrated that higher H⁺ concentrations resulted in faster H₂ gas generation. Therefore, the presence of charged hydrogen species (i.e., H⁺ and H⁻) on the catalyst surface was believed to be responsible for the accelerated H₂ gas generation at low pH. This acceleration would not be expected if the desorption-limited hydrogen evolution was solely occurring through charge neutral processes (e.g., H·+H·→H₂). Charged hydrogen species are formed on the Pt surface. This process is believed to occur through water molecules binding with surface hydrogen followed by heterolytic cleavage of the Pt—H bond. This forms H₃O⁺ and an electron (e⁻) is transferred to the Pt bulk. Such a mechanism can be conceptualized as a reverse Volmer process (H⁺+e⁻→H·). Comparable experiments in DMSO support that water may facilitate NADH regeneration to occur (FIGS. 15A,B). Based on the experimental evidence, The inventors proposed that the hydride transfer involves water-assisted charge separation of H-atoms at the Pt-coated catalyst surface (e.g., H·+H·→H⁺+H⁻). The proposed reactions are schematically illustrated in FIG. 9.

Methods and Materials

The following materials and methods were used in performing the Examples.

Materials

Pd (99.95%) was obtained from Silver Gold Bull as a 1 oz wafer bar. β-Nicotinamide adenine dinucleotide hydrate (>99%), β-Nicotinamide adenine dinucleotide, reduced disodium salt hydrate (≥97%, HPLC), propionic aldehyde (reagent grade, 97%), ammonium chloride, bovine serum albumin, L-lactic dehydrogenase from rabbit muscle, alcohol dehydrogenase from Saccharomyces cerevisiae, L-alanine dehydrogenase from Bacillus subtilis, sulfuric acid (95.0-98.0%), H2O2 solution (30 wt. % in H₂O) and dimethylsulfone (quantitative NMR standard, TraceCERT) were purchased from Sigma Aldrich and used as received. Sodium pyruvate (≥9%) was purchased from Fisher Scientific. Nitric acid (68-70%) was purchased from VWR. Pt gauze (52 mesh, 99.9%) and Pt wire (0.5 mm, 99.95%) were obtained from Alfa Aesar. Ag/AgCl reference electrodes (RE5B) were purchased from BASi.

Cell Design

The three cell cores (FIG. 4) were printed by a stereolithography 3D printer. The cores were composed of high temperature resistant resin (Formlabs proprietary resin). The membrane was sealed between the cores by Viton ring gaskets, and the assembly was clamped together by two waterjet cut ¼ in thick aluminum end plates connected by four M4 stainless steel bolts. Quick-turn polycarbonate couplings (¼-28 in) were used to feed gas inlet/outlet ports on the enzyme cell (threads were printed in the core).

Electrochemistry

A Metrohm Autolab PGSTAT302N/PGSTAT204M potentiostat was used for electrochemical experiments. To set up the electrocatalytic palladium membrane reactor, a palladium foil was fitted between the enzyme and electrochemical compartment of the cell. Viton ring gaskets were used to seal both the palladium foil in place and to prevent leaking. The enzyme compartment was filled with 10 mL of tris or phosphate buffer. The electrochemical compartment was filled with 8 mL of 1 M H₂SO₄. A Ag/AgCl reference electrode (3.0 M NaCl) and a Pt mesh counter electrode were inserted into the electrochemical compartment. All potentials are reported versus SCE unless stated otherwise. Electrolysis was conducted galvanostatically at −50 mA/cm² (V_avg=−0.3 V vs. SCE) for BioPMR experiment. The uncompensated resistance was 1-2Ω for all experiments, and no IR correction was used. The thickness of the foil was 25 μm and the geometric surface area of the foil was 2.25 cm². Cyclic voltammetry (CV) measurements were performed under an Argon atmosphere with a Pt-coated Pd working electrode, a Ag/AgCl reference electrode, Pt mesh counter electrode, and a scan rate of 250 mV/s.

Pd Foil and Pd-Black Catalyst Preparation

In these Examples, Pd foils were rolled from a 1 oz palladium wafer bar to a thickness of 25 μm. The thickness of the foil was measured by a Mitutoyo digital micrometer to an accuracy of +/−1 μm. The 25 μm thick palladium foil was then annealed in Ar at 850° C. for 1.5 hours and stored in air. Prior to use, the annealed foils were cleaned by soaking the foil in a 0.5:0.5:1 vol. % concentration HNO₃:H₂O:30% H₂O₂ for about 20 minutes.

The foil was removed from the cleaning solution and rinsed with ultrapure water. The Pd-black catalyst was immediately electrodeposited onto the surface. The cell was assembled as described above, however, the cathode compartment was filled with an electrodeposition solution (15.9 mM PdCl₂ in 1 M HCl) in lieu of the 1 M H₂SO₄ electrolyte. A voltage of −0.1 V vs. SCE was applied to the Pd foil working electrode to reduce the Pd ions in solution. The electrodeposition was stopped when 18.5 C of charge (7.38 C/cm²) had been passed. This additional catalyst layer increases the surface area of the enzyme side of the palladium foil up to 250-fold that increases the hydrogenation rate. Immediately following electrodeposition, the foils were thoroughly rinsed with MilliQ water, covered in a 4″ diameter petri dish to maintain cleanliness, and stored in ambient conditions.

Pt-Coated Membrane Preparation

The catalyst-coated membranes were prepared by sputter deposition. The electrodeposited palladium foils were secured against the deposition plate of a Leica EM MED020 coating system using Kapton tape. The chamber was sealed, and a vacuum was applied to achieve a base pressure of 2×10⁻⁵ mbar (which required ˜20 minutes). After the base pressure was reached, argon was continuously flowed into the chamber to maintain a pressure of 1×10⁻² mbar. The plasma was ignited, and voltage was adjusted to maintain a constant sputter current of 30 mA. Following a 30 s pre-sputter step, the target shutter was opened and 10-nm platinum was sputtered onto the electrodeposited palladium catalyst. The sputtering rate for every metal was 0.2 nm/s, as determined by in situ quartz crystal microbalance. Following deposition, the shutter was closed, chamber vented, and the foil was removed from the deposition plate. The foils were used for NADH regeneration and enzymatic reduction/reductive amination experiments without any further processing. The catalyst-coated membranes were used for up to 20 reactions. The entire catalyst layer of Pt-coated Pd black was removed with concentrated nitric acid then the foil was cleaned (described above), replated, and reused. Each palladium foil was used for ˜5 cleaning cycles.

Buffer Preparation

Phosphate buffers were prepared by dissolving the appropriate amounts of disodium and monosodium phosphate salts in deionized water. Tris (tris(hydroxymethyl)aminomethane) buffer was prepared by dissolving tris salts in deionized water. The pH for these buffers was then adjusted with addition of the appropriate amounts of 1 molar aqueous solutions of NaOH and/or HCl.

NADH Generation

NADH regeneration was performed in a 0.1 M phosphate or tris buffer at pH 6.5, 7 and 9. All reactions were carried out in an oven at 37° C. under Argon. The enzyme compartment of the 3D-printed cell with a magnetic stir bar was filled with 1.5 mM NAD⁺ and 10 mg bovine serum albumin (BSA) in buffer (10 mL) or dry DMSO. 1 M H₂SO₄ electrolyte solution (8 mL) was added to the electrochemical compartment and a constant reductive current of −50 mA was applied for 3 h. NADH reaction mixture was stirred at a constant rate (400 rpm) throughout the experiment. Reaction aliquots were sampled every 0.5 h to monitor the reaction progression of NADH generation by UV/Vis and 1H NMR spectroscopy. UV/Vis absorption spectra of reaction mixture were collected by using a Jasco J-815 spectrometer. All measurements were performed at room temperature with the sample perpendicular to the light path. A tris buffer blank was used as the baseline measurement. Solution temperature and pH were checked before and after the reaction. Electrospray mass spectrometry was recorded with a Waters ZQ mass spectrometer equipped with ESI ion source.

Enzyme Catalysis

All enzyme catalyzed reactions were carried out in the oven at 37° C. under Argon gas. The enzyme compartment, containing a magnetic stir bar, was filled with 1.5 mM of NAD⁺, 10 mg BSA, 1.5 mM of substrates (propionic aldehyde for aldehyde reduction, sodium pyruvate for ketone reduction, pyruvate and 25 mM NH₄Cl for reductive amination), and 5 units of corresponding enzyme (alcohol dehydrogenase for aldehyde reduction, lactate dehydrogenase for ketone reduction, and L-alanine dehydrogenase for reductive amination) in the buffer (10 mL). Alcohol reduction was performed in 0.1 M phosphate buffer at pH 6.5. Ketone reduction was performed in 0.1 M tris buffer at pH 7. Reductive amination was performed in 0.1 M tris buffer at pH 9.1 M H₂SO₄ electrolyte solution (8 mL) was added to an electrochemical compartment and a constant reductive current of −50 mA was applied for 3 h. Reaction mixture was stirred at a constant rate (400 rpm) throughout the experiment. Reaction aliquots were sampled every 0.5 h to monitor the reaction progression of product formation by 1H NMR spectroscopy.

Product Quantification

Proton nuclear magnetic resonance (1H NMR) was used for product quantification for NADH generation and enzyme catalysis. 460 μL of the reaction mixture (1.5 mM) and 40 μL 0.2 M dimethylsulfone internal standard in D₂O were added to an NMR tube. 1H NMR spectra were acquired on a Bruker Avance 400 dir, 400 inv, or 400 sp spectrometer at 298 K. Relative concentrations were determined by comparing the dimethyl group of dimethylsulfone.

Enzyme Activity Assay

All enzyme stability experiments were carried out in an oven at 37° C. under Argon. 10 μL (˜245 units) LDH and 30 mg BSA in 30 mL Tris buffer at pH 7.0 were prepared and divided into 3×10 mL solutions. For the BioPMR platform, the enzyme compartment was filled with LDH solution and a stir bar. 1 M H₂SO₄ electrolyte solution (8 mL) was added to the electrochemical compartment and a constant current of −50 mA (V_ave=−0.3 V vs. SCE) was applied for 3 h. For the electrocatalytic platform, the enzyme compartment was empty, and the Pt-coated side of the membrane was facing the electrochemical compartment. LDH solution was added to the electrochemical compartment and a constant potential of −0.8 V vs. SCE was applied for 3 h. The LDH solution was stirred at a constant rate (400 rpm) throughout the experiment. Reaction aliquots were sampled every 0.5 h to monitor the LDH stability in each platform. These samples were then diluted 100 times in the assay buffer (0.1 M Tris buffer pH 7.0, 2 mM NADH) and incubated for 2 minutes. The reactions were then initiated by addition of sodium pyruvate (final concentration of 3.5 mM) and the rate of each reaction was determined based on the rate of the consumption of NADH, by monitoring the absorbance at 340 nm. The concentration of the remaining active enzyme after each time point was thus quantified, since the rate of these reactions are directly proportional to the enzyme concentration under these conditions. All the assays were performed at least in triplicates and in 96-well half-area plated [Corning] and monitored with a Synergy H1 plate reader [BioTek].

pH Dependent H₂ Gas Permeation Experiment

An ESS CatalySys atmospheric mass spectrometer was used for pH dependent hydrogen permeation experiments. Hydrogen permeation experiments were conducted with 1 M sulfuric acid (H₂SO₄) in each electrochemical compartment and phosphate or tris buffer at their buffering pH in the enzyme compartment. H₂ generation outside of their buffering pH (pH 3.4) was also measured to examine if the pH dependency extends outside of their buffering pH. The palladium foil was placed between the electrochemical and enzyme compartments with the Pt-coated surface facing into the enzyme compartment and a constant current of −50 mA was applied. The production of gaseous H₂ (2 m/z) in the enzyme compartment with constant stirring was monitored by atmospheric mass spectrometry (atm MS) with a flow rate of 10 mL/min into the instrument. Permeation experiments were repeated at least 3 times. The ion current value was taken once the signal had equilibrated (˜1 h). The average value for at least 3 experiments is reported, with error bars representing one standard deviation of the mean.

REFERENCES

The following documents describe related technologies. Embodiments of the present technology may incorporate features as described in these references. All of the following references are hereby incorporated herein by reference as if fully set forth herein for all purposes.

PATENT DOCUMENTS

WO 2019/144239 Methods and Apparatus for Performing Chemical and Electrochemical Reactions.

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Interpretation of Terms

Unless the context clearly requires otherwise, throughout the description and the claims:

• • “comprise”, “comprising”, and the like are to be construed in an inclusive sense, as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”;
  • “connected”, “coupled”, or any variant thereof, means any connection or coupling, either direct or indirect, between two or more elements; the coupling or connection between the elements can be physical, logical, or a combination thereof;
  • “herein”, “above”, “below”, and words of similar import, when used to describe this specification, shall refer to this specification as a whole, and not to any particular portions of this specification;
  • “or”, in reference to a list of two or more items, covers all of the following interpretations of the word: any of the items in the list, all of the items in the list, and any combination of the items in the list;
  • the singular forms “a”, “an”, and “the” also include the meaning of any appropriate plural forms. These terms (“a”, “an”, and “the”) mean one or more unless stated otherwise;
  • “and/or” is used to indicate one or both stated cases may occur, for example A and/or B includes both (A and B) and (A or B);
  • “approximately” when applied to a numerical value means the numerical value ±10%;
  • where a feature is described as being “optional” or “optionally” present or described as being present “in some embodiments” it is intended that the present disclosure encompasses embodiments where that feature is present and other embodiments where that feature is not necessarily present and other embodiments where that feature is excluded. Further, where any combination of features is described in this application this statement is intended to serve as antecedent basis for the use of exclusive terminology such as “solely,” “only” and the like in relation to the combination of features as well as the use of “negative” limitation(s)” to exclude the presence of other features; and
  • “first” and “second” are used for descriptive purposes and cannot be understood as indicating or implying relative importance or indicating the number of indicated technical features.

Words that indicate directions such as “vertical”, “transverse”, “horizontal”, “upward”, “downward”, “forward”, “backward”, “inward”, “outward”, “left”, “right”, “front”, “back”, “top”, “bottom”, “below”, “above”, “under”, and the like, used in this description and any accompanying claims (where present), depend on the specific orientation of the apparatus described and illustrated. The subject matter described herein may assume various alternative orientations. Accordingly, these directional terms are not strictly defined and should not be interpreted narrowly.

Where a range for a value is stated, the stated range includes all sub-ranges of the range. It is intended that the statement of a range supports the value being at an endpoint of the range as well as at any intervening value to the tenth of the unit of the lower limit of the range, as well as any subrange or sets of sub ranges of the range unless the context clearly dictates otherwise or any portion(s) of the stated range is specifically excluded. Where the stated range includes one or both endpoints of the range, ranges excluding either or both of those included endpoints are also included in the invention.

Certain numerical values described herein are preceded by “about”. In this context, “about” provides literal support for the exact numerical value that it precedes, the exact numerical value ±5%, as well as all other numerical values that are near to or approximately equal to that numerical value. Unless otherwise indicated a particular numerical value is included in “about” a specifically recited numerical value where the particular numerical value provides the substantial equivalent of the specifically recited numerical value in the context in which the specifically recited numerical value is presented. For example, a statement that something has the numerical value of “about 10” is to be interpreted as: the set of statements:

• • in some embodiments the numerical value is 10;
  • in some embodiments the numerical value is in the range of 9.5 to 10.5; and if from the context the person of ordinary skill in the art would understand that values within a certain range are substantially equivalent to 10 because the values with the range would be understood to provide substantially the same result as the value 10 then “about 10” also includes:
  • in some embodiments the numerical value is in the range of C to D where C and D are respectively lower and upper endpoints of the range that encompasses all of those values that provide a substantial equivalent to the value 10

Specific examples of systems, methods and apparatus have been described herein for purposes of illustration. These are only examples. The technology provided herein can be applied to systems other than the example systems described above. Many alterations, modifications, additions, omissions, and permutations are possible within the practice of this invention. This invention includes variations on described embodiments that would be apparent to the skilled addressee, including variations obtained by: replacing features, elements and/or acts with equivalent features, elements and/or acts; mixing and matching of features, elements and/or acts from different embodiments; combining features, elements and/or acts from embodiments as described herein with features, elements and/or acts of other technology; and/or omitting combining features, elements and/or acts from described embodiments.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any other described embodiment(s) without departing from the scope of the present invention.

Any aspects described above in reference to apparatus may also apply to methods and vice versa.

Any recited method can be carried out in the order of events recited or in any other order which is logically possible. For example, while processes or blocks are presented in a given order, alternative examples may perform routines having steps, or employ systems having blocks, in a different order, and some processes or blocks may be deleted, moved, added, subdivided, combined, and/or modified to provide alternative or subcombinations. Each of these processes or blocks may be implemented in a variety of different ways. Also, while processes or blocks are at times shown as being performed in series, these processes or blocks may instead be performed in parallel, simultaneously or at different times.

Various features are described herein as being present in “some embodiments”. Such features are not mandatory and may not be present in all embodiments. Embodiments of the invention may include zero, any one or any combination of two or more of such features. All possible combinations of such features are contemplated by this disclosure even where such features are shown in different drawings and/or described in different sections or paragraphs. This is limited only to the extent that certain ones of such features are incompatible with other ones of such features in the sense that it would be impossible for a person of ordinary skill in the art to construct a practical embodiment that combines such incompatible features. Consequently, the description that “some embodiments” possess feature A and “some embodiments” possess feature B should be interpreted as an express indication that the inventors also contemplate embodiments which combine features A and B (unless the description states otherwise or features A and B are fundamentally incompatible). This is the case even if features A and B are illustrated in different drawings and/or mentioned in different paragraphs, sections or sentences.

It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions, omissions, and sub-combinations as may reasonably be inferred. The scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.