Patent Application
20230295814
The nicotinamide adenine dinucleotide redox couple, NAD+/NADH, and their phosphorylated counterparts, NADP+/NADPH, are ubiquitous cofactors in metabolic processes in all organisms. NAD+ and NADH are involved in redox reactions in glycolysis, fermentation, and the tricarboxylic acid (TCA) cycle in animal cells. The phosphorylated forms, NADP+ and NADPH, play a key part in photosystem I and the subsequent Calvin cycle for energy production in plant cells. NADP+/NADPH is also of great relevance to animal cells in fatty acid biosynthesis, pentose phosphate pathway, and as an antioxidant.
A critical step in most metabolic processes is the regeneration of these NAD(P)H cofactors. Metabolism is often a target of cancer therapies, and interconversion between NAD(P)+ and NAD(P)H is critical in these approaches. In addition to potential applications in artificial photosynthesis, these cofactors are critical in enzymatic biochemical reactions used in the pharmaceutical and chemical industries. For example, the commercial conversion of biomass to biofuels is primarily hampered by the cost of the cofactors (bulk cost in 2011 USD of $3000/mole ($4.52/g) for NADH and $215,000/mole ($288.82/g) for NADPH) and the challenge in recycling or regenerating them from their oxidized forms. Inexpensive production of butanol from lignocellulosic biomass via biofermentation, for instance, would be an economically appealing additive to gasoline were it not for the production cost. Direct regeneration of NADH/NADPH using photoelectrochemistry or photochemistry is also of interest for the development of next-generation biological solar converters, energy-storage devices, and artificial photosynthesis.
Methods for regeneration of NAD(P)H comprise enzymatic, chemical, electrochemical, and biological approaches. However, challenges in the artificial regeneration of NADH/NADPH include (1) the formation of inactive forms, which affect the purity of the product as determined by its utility, (2) need for high overpotentials, which can also lead to other non-useful products, and (3) requirement of expensive catalysts. Consequently, the development of effective cofactor regeneration systems is needed to reduce costs and increase the efficiency of the enzyme turnover in certain biocatalytic processes so that they can be conducted on an industrial scale. The compositions and methods discussed herein address these and other needs.
In accordance with the purposes of the disclosed compositions and methods as embodied and broadly described herein, the disclosed subject matter relates to nanostructured electrodes and methods of making and use thereof.
Additional advantages of the disclosed compositions and methods will be set forth in part in the description which follows, and in part will be obvious from the description. The advantages of the disclosed compositions and methods will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosed systems and methods, as claimed.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects of the disclosure, and together with the description, serve to explain the principles of the disclosure.
The devices, methods, and systems described herein may be understood more readily by reference to the following detailed description of specific aspects of the disclosed subject matter and the Examples included therein.
Before the present devices, methods, and systems are disclosed and described, it is to be understood that the aspects described below are not limited to specific synthetic methods or specific reagents, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.
Also, throughout this specification, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which the disclosed matter pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.
In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings.
Throughout the description and claims of this specification, the word “comprise” and other forms of the word, such as “comprising” and “comprises,” means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps.
As used in the description and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions, reference to “an agent” includes mixtures of two or more such agents, reference to “the component” includes mixtures of two or more such components, and the like.
“Optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. By “about” is meant within 5% of the value, e.g., within 4, 3, 2, or 1% of the value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.
“Exemplary” means “an example of” and is not intended to convey an indication of a preferred or ideal embodiment. “Such as” is not used in a restrictive sense, but for explanatory purposes.
It is understood that throughout this specification the identifiers “first” and “second” are used solely to aid in distinguishing the various components and steps of the disclosed subject matter. The identifiers “first” and “second” are not intended to imply any particular order, amount, preference, or importance to the components or steps modified by these terms.
Disclosed herein are nanostructured electrodes and methods of making and use thereof. For example, disclosed herein are nanostructured precursor electrodes comprising a copper substrate, a nanostructured copper oxide layer disposed on the copper substrate, and a nickel layer disposed on the nanostructured copper oxide layer. The term precursor electrode is used herein to refer to an electrode before it has undergone a photoelectrochemical modification process as disclosed herein. It is not meant to imply that the precursor electrode is not yet an electrode. Rather a precursor electrode is meant to refer to an electrode that has features or components that are available for photoelectrochemical reduction.
As used herein, “nanostructured” means any structure with one or more nanoscaled features. A nanoscale feature can be any feature with at least one dimension less than 1 micrometer (μm) in size (e.g., from 1 nm to less than 1 micrometer). For example, a nanoscaled feature can comprise a nanowire, nanotube, nanoparticle, nanopore, and the like, or combinations thereof. As such, the nanostructured copper oxide layer can comprise, for example, a nanowire, nanotube, nanoparticle, nanopore, or a combination thereof comprising copper oxide. In some examples, the nanostructured copper oxide layer can comprise copper oxide that is not nanoscaled but has been modified with a nanowire, nanotube, nanoparticle, nanopore, or a combination thereof. The nanostructured copper oxide layer can, for example, comprise Cu2O, CuO, CuO2, Cu2O3, or combination thereof.
The nanostructured copper oxide layer can, for example, have an average thickness of 50 nanometers (nm) or more (e.g., 60 nm or more, 70 nm or more, 80 nm or more, 90 nm or more, 100 nm or more, 125 nm or more, 150 nm or more, 175 nm or more, 200 nm or more, 225 nm or more, 250 nm or more, 275 nm or more, 300 nm or more, 350 nm or more, 400 nm or more, 450 nm or more, 500 nm or more, 600 nm or more, 700 nm or more, 800 nm or more, 900 nm or more, 1 micrometer (micron, μm) or more, 1.25 μm or more, 1.5 μm or more, 1.75 μm or more, 2 μm or more, 2.25 μm or more, 2.5 μm or more, 2.75 μm or more, 3 μm or more, 3.5 μm or more, 4 μm or more, 4.5 μm or more, 5 μm or more, 6 μm or more, 7 μm or more, 8 μm or more, 9 μm or more, 10 μm or more, 15 μm or more, 20 μm or more, 25 μm or more, 30 μm or more, 35 μm or more, 40 μm or more, 45 μm or more, 50 μm or more, 60 μm or more, 70 μm or more, 80 μm or more, 90 μm or more, 100 μm or more, 125 μm or more, 150 μm or more, 175 μm or more, 200 μm or more, 225 μm or more, 250 μm or more, 275 μm or more, 300 μm or more, 350 μm or more, 400 μm or more, 450 μm or more, 500 μm or more, 600 μm or more, 700 μm or more, 800 μm or more, or 900 μm or more).
In some examples, the nanostructured copper oxide layer can have an average thickness of 1000 μm or less (e.g., 900 μm or less, 800 μm or less, 700 μm or less, 600 μm or less, 500 μm or less, 450 μm or less, 400 μm or less, 350 μm or less, 300 μm or less, 275 μm or less, 250 μm or less, 225 μm or less, 200 μm or less, 175 μm or less, 150 μm or less, 125 μm or less, 100 μm or less, 90 μm or less, 80 μm or less, 70 μm or less, 60 μm or less, 50 μm or less, 45 μm or less, 40 μm or less, 35 μm or less, 30 μm or less, 25 μm or less, 20 μm or less, 15 μm or less, 10 μm or less, 9 μm or less, 8 μm or less, 7 μm or less, 6 μm or less, 5 μm or less, 4.5 μm or less, 4 μm or less, 3.5 μm or less, 3 μm or less, 2.75 μm or less, 2.5 μm or less, 2.25 μm or less, 2 μm or less, 1.75 μm or less, 1.5 μm or less, 1.25 μm or less, 1 μm or less, 900 nm or less, 800 nm or less, 700 nm or less, 600 nm or less, 500 nm or less, 450 nm or less, 400 nm or less, 350 nm or less, 300 nm or less, 275 nm or less, 250 nm or less, 225 nm or less, 200 nm or less, 175 nm or less, 150 nm or less, 125 nm or less, 100 nm or less, 90 nm or less, 80 nm or less, 70 nm or less, or 60 nm or less).
The average thickness of the nanostructured copper oxide layer can range from any of the minimum values described above to any of the maximum values described above. For example, the nanostructured copper oxide layer can have an average thickness of from 50 nm to 1000 μm (e.g., from 50 nm to 1 μm, from 1 μm to 100 μm, from 100 μm to 1000 μm, from 500 nm to 1000 μm, from 500 nm to 500 μm, from 50 nm to 100 μm, from 500 nm to 100 μm, from 1 μm to 50 μm, or from 1 μm to 10 μm). As used herein, the average thickness of the nanostructured copper oxide layer is determined using electron microscopy.
The copper substrate can, for example, comprise a high surface area substrate. For example, the copper substrate can have an average surface area of 1 μm2 or more (e.g., 5 μm2 or more; 10 μm2 or more; 50 μm2 or more; 100 μm2 or more; 500 μm2 or more; 1,000 μm2 or more; 5,000 μm2 or more; 10,000 μm2 or more; 50,000 μm2 or more; 100,000 μm2 or more; 500,000 μm2 or more; 1 mm2 or more; 5 mm2 or more; 10 mm2 or more; 50 mm2 or more; 100 mm2 or more; 500 mm2 or more; 1,000 mm2 or more; 5,000 mm2 or more; 10,000 mm2 or more; 50,000 mm2 or more; 100,000 mm2 or more; 500,000 mm2 or more; 1 m2 or more; 5 m2 or more; 10 m2 or more; or 50 m2 or more). In some examples, the copper substrate can have an average surface area of 100 m2 or less (e.g., 50 m2 or less; 10 m2 or less; 5 m2 or less; 1 m2 or less; 500,000 mm2 or less; 100,000 mm2 or less; 50,000 mm2 or less; 10,000 mm2 or less; 5,000 mm2 or less; 1,000 mm2 or less; 500 mm2 or less; 100 mm2 or less; 50 mm2 or less; 10 mm2 or less; 5 mm2 or less; 1 mm2 or less; 500,000 μm2 or less; 100,000 μm2 or less; 50,000 μm2 or less; 10,000 μm2 or less; 5,000 μm2 or less; 1,000 μm2 or less; 500 μm2 or less; 100 μm2 or less; 50 μm2 or less; 10 μm2 or less; or 5 μm2 or less). The average surface area of the copper substrate can range from any of the minimum values described above to any of the maximum values described above. For example, the copper substrate can have an average surface area of from 1 μm2 to 100 m2 (e.g., from 1 μm2 to 1,000 μm2; from 1,000 μm2 to 1 mm2; from 1 mm2 to 1,000 mm2; from 1,000 mm2 to 1 m2; from 1 m2 to 100 m2; from 1,000 μm2 to 100 m2; or from 1 mm2 to 100 m2).
In some examples, the copper substrate can comprise a porous copper substrate, such as a copper mesh. In some examples, the copper substrate can have an average thickness of from 1 micrometer (micron, μm) or more (e.g., 5 μm or more, 10 μm or more, 50 μm or more, 100 μm or more, 500 μm or more, 1 millimeter (mm) or more, 5 mm or more, 10 mm or more, 50 mm or more, 100 mm or more, or 500 mm or more). In some examples, the copper substrate can have an average thickness of 1 meter or less (e.g., 500 mm or less, 100 mm or less, 50 mm or less, 10 mm or less, 5 mm or less, 1 mm or less, 500 μm or less, 100 μm or less, 50 μm or less, 10 μm or less, or 5 μm or less). The average thickness of the copper substrate can range from any of the minimum values described above to any of the maximum values described above. For example, the copper substrate can have an average thickness of from 1 μm to 1 m (e.g., from 1 μm to 1 mm, from 1 mm to 1 m, from 1 μm to 10 μm, from 10 μm to 100 μm, from 100 μm to 1 mm, from 1 mm to 10 mm, from 10 mm to 100 mm, from 100 mm to 1 m, from 10 μm to 1 m, from 100 μm to 1 m, or from 1 μm to 500 mm).
The nickel layer can, for example, comprise metallic nickel. The nickel layer can, for example, have an average thickness of 1 nm or more (e.g., 2 nm or more, 3 nm or more, 4 nm or more, 5 nm or more, 6 nm or more, 7 nm or more, 8 nm or more, 9 nm or more, 10 nm or more, 15 nm or more, 20 nm or more, 25 nm or more, 30 nm or more, 35 nm or more, 40 nm or more, 45 nm or more, 50 nm or more, 60 nm or more, 70 nm or more, 80 nm or more, 90 nm or more, 100 nm or more, 125 nm or more, 150 nm or more, 175 nm or more, 200 nm or more, 225 nm or more, 250 nm or more, 275 nm or more, 300 nm or more, 325 nm or more, 350 nm or more, 375 nm or more, 400 nm or more, 425 nm or more, 450 nm or more, or 475 nm or more).
In some examples, the nickel layer can have an average thickness of 500 nm or less (e.g., 475 nm or less, 450 nm or less, 425 nm or less, 400 nm or less, 375 nm or less, 350 nm or less, 325 nm or less, 300 nm or less, 275 nm or less, 250 nm or less, 225 nm or less, 200 nm or less, 175 nm or less, 150 nm or less, 125 nm or less, 100 nm or less, 90 nm or less, 80 nm or less, 70 nm or less, 60 nm or less, 50 nm or less, 45 nm or less, 40 nm or less, 35 nm or less, 30 nm or less, 25 nm or less, 20 nm or less, 15 nm or less, 10 nm or less, 5 nm or less, 4 nm or less, 3 nm or less, or 2 nm or less).
The average thickness of the nickel layer can range from any of the minimum values described above to any of the maximum values described above. For example, the nickel layer can have an average thickness of from 1 nm to 500 nm (e.g., from 1 nm to 400 nm, from 1 nm to 300 nm, from 1 nm to 200 nm, from 1 nm to 100 nm, or from 1 nm to 50 nm). As used herein, the average thickness of the nickel layer is determined using electron microscopy.
Also disclosed herein are nanostructured electrodes comprising a copper substrate, a nanostructured copper oxide layer disposed on the copper substrate, and an active layer disposed on the nanostructured copper oxide layer, wherein the active layer comprises copper and nickel.
The copper substrate can, for example, have an average surface area of from 1 μm2 to 100 m2. In some examples, the copper substrate can comprise a copper mesh. The nanostructured copper oxide layer can, for example, have an average thickness of from 50 nm to 1000 μm (e.g., from 500 nm to 500 μm, or from 1 μm to 10 μm). In some examples, the nanostructured copper oxide layer can comprise Cu2O, CuO, CuO2, Cu2O3, or combination thereof.
The active layer can, for example, comprise metallic copper and metallic nickel. In some examples, the active layer consists essentially of copper and nickel. In some examples, the active layer consists of copper and nickel. In some examples, the active layer is substantially free of copper oxide.
The active layer can, for example, have an average thickness of 1 nm or more (e.g., 2 nm or more, 3 nm or more, 4 nm or more, 5 nm or more, 6 nm or more, 7 nm or more, 8 nm or more, 9 nm or more, 10 nm or more, 15 nm or more, 20 nm or more, 25 nm or more, 30 nm or more, 35 nm or more, 40 nm or more, 45 nm or more, 50 nm or more, 60 nm or more, 70 nm or more, 80 nm or more, 90 nm or more, 100 nm or more, 125 nm or more, 150 nm or more, 175 nm or more, 200 nm or more, 225 nm or more, 250 nm or more, 275 nm or more, 300 nm or more, 325 nm or more, 350 nm or more, 375 nm or more, 400 nm or more, 425 nm or more, 450 nm or more, or 475 nm or more).
In some examples, the active layer can have an average thickness of 500 nm or less (e.g., 475 nm or less, 450 nm or less, 425 nm or less, 400 nm or less, 375 nm or less, 350 nm or less, 325 nm or less, 300 nm or less, 275 nm or less, 250 nm or less, 225 nm or less, 200 nm or less, 175 nm or less, 150 nm or less, 125 nm or less, 100 nm or less, 90 nm or less, 80 nm or less, 70 nm or less, 60 nm or less, 50 nm or less, 45 nm or less, 40 nm or less, 35 nm or less, 30 nm or less, 25 nm or less, 20 nm or less, 15 nm or less, 10 nm or less, 5 nm or less, 4 nm or less, 3 nm or less, or 2 nm or less).
The average thickness of the active layer can range from any of the minimum values described above to any of the maximum values described above. For example, the active layer can have an average thickness of from 1 nm to 500 nm (e.g., from 1 nm to 400 nm, from 1 nm to 300 nm, from 1 nm to 200 nm, from 1 nm to 100 nm, or from 1 nm to 50 nm). As used herein, the average thickness of the active layer is determined using electron microscopy.
The active layer can, for example, be formed by photoelectrochemically modifying a precursor nanostructured electrode, the nanostructured precursor electrode comprising the copper substrate, the nanostructured copper oxide layer disposed on the copper substrate, and a nickel layer disposed on the nanostructured copper oxide layer. The nickel layer can, for example, comprise metallic nickel. In some example, the nickel layer can have an average thickness of from 1 nm to 500 nm (e.g., from 1 nm to 100 nm, or from 1 nm to 50 nm). The nanostructured electrodes described herein can, for example, comprise a photoelectrochemically modified product of a nanostructured precursor electrode, the nanostructured precursor electrode comprising a copper substrate, a nanostructured copper oxide layer disposed on the copper substrate, and a nickel layer disposed on the nanostructured copper oxide layer. The nanostructured precursor electrode can, for example, comprise any of the nanostructured precursor electrodes described herein.
Also described herein are nanostructured electrodes comprising a photoelectrochemically modified product of a nanostructured precursor electrode, the nanostructured precursor electrode comprising a copper substrate, a nanostructured copper oxide layer disposed on the copper substrate, and a nickel layer disposed on the nanostructured copper oxide layer. The nanostructured precursor electrode can, for example, comprise any of the nanostructured precursor electrodes described herein.
For example, the copper substrate can have an average surface area of from 1 μm2 to 100 m2. In some examples, the copper substrate can comprise a copper mesh. The nanostructured copper oxide layer can, for example comprise Cu2O, CuO, CuO2, Cu2O3, or combination thereof. In some examples, the nanostructured copper oxide layer can have an average thickness of from 50 nanometers (nm) to 1000 micrometers (microns, μm) (e.g., from 500 nm to 500 μm, or from 1 μm to 10 μm). The nickel layer can, for example comprise metallic nickel. In some examples, the nickel layer can have an average thickness of from 1 nm to 500 nm (e.g., from 1 nm to 100 nm, or from 1 nm to 50 nm).
Also described herein are nanostructured electrodes for the direct regeneration of NADH, NADPH, or combination thereof using photoelectrochemistry or photochemistry with a low overpotential, wherein the nanostructured electrode comprises a high surface area substrate, a nanostructured layer comprising a p-type semiconductor disposed on the high surface area substrate, and an active layer comprising a hydrogen capture material disposed on the nanostructured layer. The high surface area substrate, the nanostructured layer, the active layer, or a combination thereof can, for example, comprise an inexpensive and/or abundant material. The nanostructured electrode for the direct regeneration of NADH, NADPH, or combination thereof can, for example, comprise any of the nanostructured precursor electrodes described herein or any of the nanostructured electrodes described herein.
Also described herein are methods of making the nanostructured precursor electrodes and nanostructured electrodes described herein. For example, also disclosed herein are methods of making the nanostructured precursor electrodes described herein, the methods comprising depositing the nanostructured copper oxide layer on the copper mesh and subsequently depositing the nickel layer on the nanostructured copper oxide layer. The nanostructured copper oxide layer can, for example, be deposited by electrodeposition, chemical bath deposition, physical vapor deposition, atomic layer deposition, spray pyrolysis, chemical vapor deposition, or a combination thereof.
In some examples, the methods comprise electrodepositing the nanostructured copper oxide layer on the copper mesh. In some examples, the electrodeposition is performed at room temperature.
Electrodepositing the nanostructured copper oxide layer can, for example, comprise submersing the copper mesh in a solution comprising copper and applying a potential to the copper mesh. In some examples, the potential is −0.1 V vs. Ag/AgCl or less (e.g., −0.2 V or less, −0.3 V or less, −0.4 V or less, or −0.5 V or less). In some examples, the potential is −0.6 V vs. Ag/AgCl or more (e.g., −0.5 V or more, −0.4 V or more, −0.3 V or more, or −0.2 V or more). The potential can range from any of the minimum values described above to any of the maximum values described above. For example, the potential can be from −0.1 Volts (V) vs. Ag/AgCl to −0.6 V vs. Ag/AgCl (e.g., from −0.1 V to −0.4 V, from −0.4 V to −0.6 V, from −0.1 V to −0.5 V, from −0.2 V to −0.6 V, or from −0.2 V to −0.5 V). The potential can, for example, be −0.5 V vs. Ag/AgCl.
In some examples, the potential is applied for an amount of time of 1 minute or more (e.g., 2 minutes or more, 3 minutes or more, 4 minutes or more, 5 minutes or more, 10 minutes or more, 15 minutes or more, 20 minutes or more, 30 minutes or more, 45 minutes or more, 1 hour or more, 1.5 hour or more, 2 hours or more, 2.5 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 8 hours or more, or 10 hours or more). In some examples, the potential is applied for an amount of time of 12 hours or less (e.g., 10 hours or less, 8 hours or less, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, 2.5 hours or less, 2 hours or less, 1.5 hours or less, 1 hour or less, 45 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less). The amount of time the potential is applied can range from any of the minimum values described above to any of the maximum values described above. For example, the potential can be applied for an amount of time of from 1 minute to 12 hours (e.g., from 1 minute to 10 minutes, from 10 minutes to 1 hour, from 1 hour to 12 hours, from 10 minutes to 12 hours, or from 1 minute to 4 hours). The potential can, for example, be applied for 2 hours.
The solution comprising copper can comprise a solution of a copper compound, such as CuSO4. In some examples, he solution comprising copper can comprise a solution of a copper compound such as that described by Zhu et al. “Seed layer-assisted chemical bath deposition of CuO films on ITO-coated glass substrates with tunable crystallinity and morphology”, Chemistry of Materials, 2014, 26, 2960-2966. The solution comprising copper can, for example, have a concentration of copper of 0.1 molar (M) or more (e.g., 0.15 M or more, 0.2 M or more, 0.25 M or more, 0.3 M or more, 0.35 M or more, 0.4 M or more, 0.45 M or more, 0.5 M or more, 0.6 M or more, 0.7 M or more, 0.8 M or more, or 0.9 M or more). In some examples, the solution comprising copper can have a concentration of copper of 1 M or less (e.g., 0.9 M or less, 0.8 M or less, 0.7 M or less, 0.6 M or less, 0.5 M or less, 0.45 M or less, 0.4 M or less, 0.35 M or less, 0.3 M or less, 0.25 M or less, or 0.2 M or less). The concentration of copper in the solution comprising copper can range from any of the minimum values described above to any of the maximum values described above. For example, the solution comprising copper can have a concentration of copper of from 0.1 M to 1 M (e.g., from 0.1 M to 0.5 M, from 0.5 M to 1 M, from 0.1 M to 0.3 M, from 0.3 M to 0.6 M, from 0.6 M to 1 M, or from 0.4 M to 0.5 M).
In some examples, the solution comprising copper can have a pH of 9 or more (e.g., 9.5 or more, 10 or more, 10.5 or more, or 11 or more). In some examples, the solution comprising copper can have a pH or 12 or less (e.g., 11.5 or less, 11 or less, 10.5 or less, or 10 or less). The pH of the solution comprising copper can range from any of the minimum values described above to any of the maximum values described above. For example, the solution comprising copper can have a pH of from 9 to 12 (e.g., from 9 to 10.5, from 10.5 to 12, from 9 to 10, from 10 to 11, from 11 to 12, from 10 to 12, from 9 to 11, or from 9.5 to 11.5).
The nickel layer can be deposited, for example, by thin film processing techniques, such as sputtering, pulsed layer deposition, molecular beam epitaxy, evaporation, atomic layer deposition, or combinations thereof. In some examples, depositing the nickel layer comprises sputtering, such as DC sputtering. In some examples, depositing the nickel layer is performed under vacuum and/or an inert atmosphere (e.g., Ar, N2, etc.).
The nickel layer can, for example, be deposited for an amount of time of 1 hour or more (e.g., 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 8 hours or more, 10 hours or more, 12 hours or more, 14 hours or more, 16 hours or more, 18 hours or more, 20 hours or more, 22 hours or more, 24 hours or more, 30 hours or more, 36 hours or more, 42 hours or more, 48 hours or more, 60 hours or more, 72 hours or more, 84 hours or more, or 96 hours or more). In some examples, the nickel layer can be deposited for 100 hours or less (e.g., 96 hours or less, 84 hours or less, 72 hours or less, 60 hours or less, 48 hours or less, 42 hours or less, 36 hours or less, 30 hours or less, 24 hours or less, 22 hours or less, 20 hours or less, 18 hours or less, 16 hours or less, 14 hours or less, 12 hours or less, 10 hours or less, 8 hours or less, 6 hours or less, 5 hours or less, 4 hours or less, or 3 hours or less). The amount of time the nickel layer is deposited for can range from any of the minimum values described above to any of the maximum values described above. For example, the nickel layer can be deposited for an amount of time of from 1 hour to 100 hours (e.g., from 1 hour to 48 hours, from 48 hours to 100 hours, from 1 hour to 24 hours, from 24 hours to 60 hours, from 60 hours to 100 hours, from 6 hours to 100 hours, from 1 hour to 96 hours, from 6 hours to 96 hours, or from 10 hours to 100 hours). In some examples, the nickel layer can be deposited for 96 hours.
The methods can, in some examples, further comprise cleaning the copper mesh prior to depositing the nanostructured copper oxide layer thereon. Cleaning the copper mesh can, for example, comprise submersing the copper mesh in a first solvent and sonicating for a first amount of time, and subsequently removing the copper mesh from the first solvent and rinsing the copper mesh with deionized water.
The first solvent can, for example, comprise tetrahydrofuran (THF), N-methyl-2-pyrrolidone (NMP), dimethylformamide (DMF), N-methylformamide, formamide, dichloromethane (CH2Cl2), ethylene glycol, polyethylene glycol, glycerol, alkane diol, ethanol, methanol, propanol, isopropanol, water, acetonitrile, chloroform, toluene, methyl acetate, ethyl acetate, acetone, hexane, heptane, tetraglyme, propylene carbonate, diglyme, dimethyl sulfoxide (DMSO), dimethoxyethane, xylene, dimethylacetamide, or combinations thereof. In some examples, the first solvent comprises ethanol.
The first amount of time can, for example, be 1 minute or more (e.g., 2 minutes or more, 3 minutes or more, 4 minutes or more, 5 minutes or more, 10 minutes or more, 15 minutes or more, 20 minutes or more, 30 minutes or more, 40 minutes or more, 45 minutes or more, 50 minutes or more, or 55 minutes or more). In some examples, the first amount of time can be 1 hour or less (e.g., 55 minutes or less, 50 minutes or less, 45 minutes or less, 40 minutes or less, 35 minutes or less, 30 minutes or less, 25 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less). The first amount of time can range from any of the minimum values described above to any of the maximum values described above. For example, the first amount of time can be from 1 minute to 1 hour (e.g., from 1 minute to 30 minutes, from 30 minutes to 1 hour, from 1 minute to 20 minutes, from 20 minutes to 40 minutes, from 40 minutes to 1 hour, from 5 minutes to 1 hour, from 1 minute to 50 minutes, or from 5 minutes to 50 minutes). In some examples, the first amount of time is 10 minutes.
In some examples, cleaning the copper mesh can further comprise, after rinsing the copper mesh with deionized water, submersing the copper mesh in a second solvent and sonicating for a second amount of time, and subsequently removing the copper mesh from the first solvent and rinsing the copper mesh with deionized water. The second solvent can be the same as or different than the first solvent. In some examples, second solvent is the same as the first solvent. In some examples, the second solvent comprises ethanol. The second amount of time can, for example, be from 1 minute to 1 hour. In some examples, the second amount of time can be the same as the first amount of time. In some examples, the second amount of time is 10 minutes.
In some examples, cleaning the copper mesh can further comprise, after rinsing the copper mesh with deionized water, submersing the copper mesh in a third solvent and sonicating for a third amount of time, and subsequently removing the copper mesh from the first solvent and rinsing the copper mesh with deionized water. The third solvent can be the same as or different than the first solvent and/or the second solvent. In some examples, the third solvent is different than the first solvent and the second solvent. In some examples, the third solvent comprises deionized water. The third amount of time can, for example, be from 1 minute to 1 hour. In some examples, the third amount of time is the same as the first amount of time and/or the second amount of time. In some examples, the third amount of time is 10 minutes.
In some examples, cleaning the copper mesh can further comprise, after rinsing the copper mesh with deionized water, drying the copper mesh.
Also disclosed herein are methods of making any of the nanostructured electrodes described herein, the methods comprising photoelectrochemically modifying any of the nanostructured precursor electrodes described herein. In some examples, the methods can further comprise making the nanostructured precursor electrode, for example using any of the methods described herein.
Photoelectrochemically modifying the nanostructured precursor electrode can, for example, comprise irradiating the nanostructured precursor electrode in conjunction with (e.g., concurrently with) performing potentiostatic electrolysis on the nanostructured precursor electrode. In some examples, the photoelectrochemical modification is performed while the nanostructured precursor substrate is submersed in a sodium phosphate buffer.
Irradiating the nanostructured precursor electrode can, for example, comprise exposing the nanostructured precursor electrode to electromagnetic radiation, wherein the nanostructured precursor electrode has a bandgap energy and at least a portion of the electromagnetic radiation has an energy greater than or equal to the bandgap energy.
The electromagnetic radiation can, for example, be provided by a light source. The light source can be any type of light source. Examples of suitable light sources include natural light sources (e.g., sunlight) and artificial light sources (e.g., incandescent light bulbs, light emitting diodes, gas discharge lamps, arc lamps, lasers, etc.). In some examples, the electromagnetic radiation comprises sunlight. In some examples, the light source can comprise a laser. In some examples, irradiating the nanostructured precursor electrode can comprise exposing the nanostructured precursor electrode to a coherent light source, such as a 10 mW, 532 nm laser.
The nanostructured precursor electrode can, for example, be irradiated for an amount of time of 1 minute or more (e.g., 2 minutes or more, 3 minutes or more, 4 minutes or more, 5 minutes or more, 10 minutes or more, 15 minutes or more, 20 minutes or more, 30 minutes or more, 45 minutes or more, 1 hour or more, 1.5 hour or more, 2 hours or more, 2.5 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 8 hours or more, or 10 hours or more). In some examples, the nanostructured precursor electrode can be irradiated for an amount of time of 12 hours or less (e.g., 10 hours or less, 8 hours or less, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, 2.5 hours or less, 2 hours or less, 1.5 hours or less, 1 hour or less, 45 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less). The amount of time the nanostructured precursor electrode is irradiated can range from any of the minimum values described above to any of the maximum values described above. For example, the nanostructured precursor electrode can be irradiated for an amount of time of from 1 minute to 12 hours (e.g., from 1 minute to 10 minutes, from 10 minutes to 1 hour, from 1 hour to 12 hours, from 10 minutes to 12 hours, or from 1 minute to 4 hours). In some example, the nanostructured precursor electrode is irradiated for an hour.
Performing potentiostatic electrolysis on the precursor electrode can, for example, comprise applying a potential of −0.75 V vs. Ag/AgCl or less (e.g., −0.8 V or less, −0.9 V or less, −1 V or less, −1.1 V or less, −1.2 V or less, −1.3 V or less, or −1.4 V or less). In some examples, the potential can be −1.5 V vs. Ag/AgCl or more (e.g., −1.4 V or less, −1.3 V or less, −1.2 V or less, −1.1 V or less, −1 V or less, −0.9 V or less, or −0.8 V or less). The potential can range from any of the minimum values described above to any of the maximum values described above. For example, the potential can be from −0.75 V to −1.5 V vs. Ag/AgCl (e.g., from −0.75 V to −1.1 V, from −1.1 V to −1.5 V, from −0.75 V to −1 V, from −1 V to −1.25 V, from −1.25 V to −1.5 V, from −0.75 V to −1.25 V, or from −0.9 V to −1.2 V). In some examples, the potential is −0.75 V vs. Ag/AgCl.
The potential can, for example, be applied for an amount of time of 1 minute or more (e.g., 2 minutes or more, 3 minutes or more, 4 minutes or more, 5 minutes or more, 10 minutes or more, 15 minutes or more, 20 minutes or more, 30 minutes or more, 45 minutes or more, 1 hour or more, 1.5 hour or more, 2 hours or more, 2.5 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 8 hours or more, or 10 hours or more). In some examples, the potential is applied for an amount of time of 12 hours or less (e.g., 10 hours or less, 8 hours or less, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, 2.5 hours or less, 2 hours or less, 1.5 hours or less, 1 hour or less, 45 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less). The amount of time the potential is applied can range from any of the minimum values described above to any of the maximum values described above. For example, the potential can be applied for an amount of time of from 1 minute to 12 hours (e.g., from 1 minute to 10 minutes, from 10 minutes to 1 hour, from 1 hour to 12 hours, from 10 minutes to 12 hours, or from 1 minute to 4 hours). In some examples, the potential is applied for 1 hour.
Also disclosed herein are methods of making any of the nanostructured electrodes described herein, the methods comprising making a nanostructured electrode precursor electrode (e.g., using any of the methods described herein) and photoelectrochemically modifying the nanostructured precursor electrode to form the nanostructured electrode (e.g., using any of the methods described herein), wherein the nanostructured precursor electrode comprises the copper substrate, the nanostructured copper oxide layer disposed on the copper substrate, and a nickel layer disposed on the nanostructured copper oxide layer.
Also described herein are methods of use of any of the nanostructured precursor electrodes described herein and/or any of the nanostructured electrodes described herein. For example, also described herein are methods of use of any of the nanostructured precursor electrodes described herein or any of the nanostructured electrodes described herein, the method comprising using the nanostructured precursor electrode or the nanostructured electrode to directly regenerate NADH, NADPH, or a combination thereof photochemically or photoelectrochemically.
In some examples, the method can comprise directly regenerating NADH, NADPH, or a combination thereof photoelectrochemically using a low overpotential. The methods can, for example, comprise applying an overpotential to the nanostructured precursor electrode or the nanostructured electrode in the presence of a solution comprising NAD(P)+ to electrochemically regenerate NAD(P)H. As used herein, the term NAD(P)H includes NADH, NADPH, and combinations thereof. Similarly, as used herein, the term NAD(P)+ includes NAD+, NADP+, and combinations thereof.
The overpotential applied can, for example, be −0.75 V vs. Ag/AgCl or less (e.g., −0.8 V or less, −0.9 V or less, −1 V or less, −1.1 V or less, −1.2 V or less, −1.3 V or less, or −1.4 V or less). In some examples, the overpotential can be −1.5 V vs. Ag/AgCl or more (e.g., −1.4 V or less, −1.3 V or less, −1.2 V or less, −1.1 V or less, −1 V or less, −0.9 V or less, or −0.8 V or less). The overpotential can range from any of the minimum values described above to any of the maximum values described above. For example, the overpotential can be from −0.75 V to −1.5 V vs. Ag/AgCl (e.g., from −0.75 V to −1.1 V, from −1.1 V to −1.5 V, from −0.75 V to −1 V, from −1 V to −1.25 V, from −1.25 V to −1.5 V, from −0.75 V to −1.25 V, or from −0.9 V to −1.2 V). In some examples, the overpotential is −0.75 V vs. Ag/AgCl.
The overpotential can, for example, be applied for an amount of time of 1 minute or more (e.g., 2 minutes or more, 3 minutes or more, 4 minutes or more, 5 minutes or more, 10 minutes or more, 15 minutes or more, 20 minutes or more, 30 minutes or more, 45 minutes or more, 1 hour or more, 1.5 hour or more, 2 hours or more, 2.5 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 8 hours or more, or 10 hours or more). In some examples, the overpotential is applied for an amount of time of 12 hours or less (e.g., 10 hours or less, 8 hours or less, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, 2.5 hours or less, 2 hours or less, 1.5 hours or less, 1 hour or less, 45 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less). The amount of time the overpotential is applied can range from any of the minimum values described above to any of the maximum values described above. For example, the overpotential can be applied for an amount of time of from 1 minute to 12 hours (e.g., from 1 minute to 10 minutes, from 10 minutes to 1 hour, from 1 hour to 12 hours, from 10 minutes to 12 hours, or from 1 minute to 4 hours).
In some examples, the methods can further comprise irradiating the nanostructured precursor electrode or the nanostructured electrode concurrently with applying the overpotential. In some examples, the methods can comprise irradiating any of the nanostructured precursor electrodes described herein concurrently with applying the overpotential which converts the nanostructured precursor electrode to any of the nanostructured electrodes described herein in situ.
Irradiating the nanostructured precursor electrode or the nanostructured electrode can, for example, comprise exposing the nanostructured precursor electrode or the nanostructured electrode to electromagnetic radiation, wherein the nanostructured precursor electrode or the nanostructured electrode has a bandgap energy and at least a portion of the electromagnetic radiation has an energy greater than or equal to the bandgap energy.
The electromagnetic radiation can, for example, be provided by a light source. The light source can be any type of light source. Examples of suitable light sources include natural light sources (e.g., sunlight) and artificial light sources (e.g., incandescent light bulbs, light emitting diodes, gas discharge lamps, arc lamps, lasers, etc.). In some examples, the electromagnetic radiation comprises sunlight. In some examples, the light source can comprise a laser. In some examples, irradiating the nanostructured precursor electrode can comprise exposing the nanostructured precursor electrode to a 10 mW, 532 nm laser.
The nanostructured precursor electrode or the nanostructured electrode can, for example, be irradiated for an amount of time of 1 minute or more (e.g., 2 minutes or more, 3 minutes or more, 4 minutes or more, 5 minutes or more, 10 minutes or more, 15 minutes or more, 20 minutes or more, 30 minutes or more, 45 minutes or more, 1 hour or more, 1.5 hour or more, 2 hours or more, 2.5 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 8 hours or more, or 10 hours or more). In some examples, the nanostructured precursor electrode or the nanostructured electrode can be irradiated for an amount of time of 12 hours or less (e.g., 10 hours or less, 8 hours or less, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, 2.5 hours or less, 2 hours or less, 1.5 hours or less, 1 hour or less, 45 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less). The amount of time the nanostructured precursor electrode or the nanostructured electrode is irradiated can range from any of the minimum values described above to any of the maximum values described above. For example, the nanostructured precursor electrode or the nanostructured electrode can be irradiated for an amount of time of from 1 minute to 12 hours (e.g., from 1 minute to 10 minutes, from 10 minutes to 1 hour, from 1 hour to 12 hours, from 10 minutes to 12 hours, or from 1 minute to 4 hours).
The methods can, for example, provide 1,4-NAD(P)H with a purity of 66% or more (e.g., 67% or more, 68% or more, 69% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more). As used herein, the purity is determined as the percentage of the product of the method that comprises 1,4-NAD(P)H produced relative to the sum of the amounts of inactive forms of NAD(P)H, such as dimers (e.g., (NAD(P)H)2), inactive isomers (e.g. 1,6-NAD(P)H), and the like, or combinations thereof.
The composition of the nanostructured precursor electrode (e.g., composition of layers, thickness of layers), the composition of the nanostructured electrode (e.g., composition of layers, thickness of layers), the irradiation conditions (e.g., electromagnetic radiation source, irradiation time and/or power), the overpotential conditions (e.g., overpotential applied, time overpotential is applied), or a combination thereof can be selected to minimize the production of inactive forms of NAD(P)H using the methods, e.g., to increase the selective production of 1,4-NAD(P)H over the inactive forms of NAD(P)H and thereby increase the purity.
In some examples, the method produces a product that is substantially free of (NAD(P)H)2, 1,6-NAD(P)H, or a combination thereof. In some examples, the method produces a product that consists essentially of 1,4-NADP(H). In some examples, the method produces a product that consists of 1,4-NAD(P)H.
Also disclosed herein are methods of making biofuels, the methods comprising synthesizing the biofuel via biofermentation of a biomass concurrently with regeneration of NAD(P)H, wherein the NAD(P)H is regenerated using any of the methods described herein using any of the nanostructured precursor electrodes described herein and/or any of the nanostructured electrodes described herein.
The term “biomass,” as used herein, refers to living or dead biological material that can be used in one or more of the disclosed methods. In the disclosed methods the “biomass” can comprise any cellulosic and/or lignocellulosic biomass and can include materials comprising cellulose, and optionally hemicellulose, lignin, starch, oligosaccharides and/or monosaccharides, their mixtures, and breakdown products (e.g., metabolites). Biomass can also comprise additional components, such as protein and/or lipid. Biomass can be derived from a single source, or biomass can comprise a mixture derived from more than one source. Some specific examples of suitable biomasses that can be used in the disclosed methods include, but are not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper manufacture, yard waste, wood, and forestry waste. Additional examples of suitable types of biomass include, but are not limited to, corn grain, corn cobs, crop residues such as corn husks, corn stover, grasses, wheat, wheat straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, soy, components obtained from milling of grains, trees (e.g., pine), branches, roots, leaves, wood chips, wood pulp, sawdust, shrubs and bushes, vegetables, fruits, flowers, animal manure, and multi-component feed. In some examples, the biomass can comprise a lignocellulosic biomass.
Lignocellulosic biomass typically comprises three major components: cellulose, hemicellulose, and lignin, along with some extractive materials (Sjostorm, E. Wood Chemistry: Fundamentals and Applications, 2nd ed., 1993, New York.). Depending on the source, their relative compositions usually vary to certain extent. Cellulose is the most abundant polymer on Earth and enormous effort has been put into understanding its structure, biosynthesis, function, and degradation (Stick, R. V. Carbohydrates—The Sweet Molecules of Life, 2001, Academic Press, New York.). Cellulose is a polysaccharide comprising a linear chain of several hundred to over ten thousand β(1→4) linked D-glucose units. The chains are hydrogen bonded either in parallel or anti-parallel manner which imparts more rigidity to the structure, and a subsequent packaging of bound-chains into microfibrils forms the ultimate building material of the nature.
Hemicellulose is the principal non-cellulosic polysaccharide in lignocellulosic biomass. Hemicellulose is a branched heteropolymer, consisting of different sugar monomers with 500-3000 units. Hemicellulose is usually amorphous.
Lignin is the most complex naturally occurring high-molecular weight polymer (Hon, D. N. S.; Shiraishi, N., Eds., Wood and Cellulosic Chemistry, 2nd ed., 2001, Marcel Dekker, Inc., New York.). Lignin is relatively hydrophobic and aromatic in nature, but lacks a defined primary structure. Softwood lignin primarily comprises guaiacyl units, and hardwood lignin comprises both guaiacyl and syringyl units. Cellulose content in both hardwood and softwood is about 43±2%. Typical hemicellulose content in wood is about 28-35 wt %, depending on type of wood. Lignin content in hardwood is about 18-25% while softwood may contain about 25-35% of lignin.
Examples of biofuels include, but are not limited to, methane, ethanol, propanol, methanol, butanol, and biodiesel. In some examples, the biofuel comprises butanol.
Also disclosed herein are methods of use of any of the nanostructured precursor electrodes described herein and/or any of the nanostructured electrodes described herein, the methods comprising using the nanostructured precursor electrode and/or the nanostructured electrode for artificial photosynthesis, conversion of a biomass to a biofuel, solar energy conversion, energy storage, or a combination thereof. Also disclosed herein are methods of use of any of the nanostructured precursor electrodes described herein and/or any of the nanostructured electrodes described herein, the methods comprising using the nanostructured precursor electrode and/or the nanostructured electrode in a pharmaceutical application, a chemical application, an energy storage device, of a combination thereof.
A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
The examples below are intended to further illustrate certain aspects of the systems and methods described herein, and are not intended to limit the scope of the claims.
The following examples are set forth below to illustrate the methods and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.
Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C. or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of measurement conditions, e.g., component concentrations, temperatures, pressures and other measurement ranges and conditions that can be used to optimize the described process.
The direct electrochemical regeneration of the cofactor 1,4-NADPH at a Ni coated Cu20 derived cathode is demonstrated. Enzymatic assay reveals the purity of electrolysis products at a low overpotential (−0.75 V vs. Ag/AgCl) to be 74.1±1.0% and 89.1±2.8% 1,4-NADPH for two separate electrodes. Temporal kinetic studies suggest that an initial light-assisted surface restructuring process, in which the semiconducting Cu2O is converted back to a catalytic form of Cu, is necessary to observe NADP+ reduction. Material analysis indicates that the Ni-sputtering step used to prepare the electrode results in significant surface morphology changes that undergird the catalytic capabilities of the electrode.
BACKGROUND The nicotinamide adenine dinucleotide redox couple, NAD+/NADH, and their phosphorylated counterparts, NADP+/NADPH, are ubiquitous cofactors in metabolic processes in all organisms. In animal cells, NAD+ and NADH are cofactors involved in the redox reactions in glycolysis as well as in the TriCarboxylic Acid (TCA) cycle (also commonly known as the citric acid cycle) (Van Linden M R et al. The Biochemist, 2015, 37(1), 9-13). The corresponding phosphorylated forms, NADP+ and NADPH, play a key part in photosynthesis, particularly in photosystem I and the subsequent Calvin cycle for energy production in plant cells (Blankenship R. Molecular Mechanisms of Photosynthesis. Hoboken: Wiley. 2014). From an industrial standpoint, nicotinamides are of interest due to their relevancy to biocatalysis. Biocatalysis, the usage of enzymes isolated from biological material, is commonly applied in the chemical and pharmaceutical industry to synthesize drugs, biofuels, and other marketable chemical compounds (Bornscheuer U T et al. Nature, 2012, 485(7397), 185-194). Oxidoreductases, a large class of enzymes, typically require NAD(P)+/NAD(P)H as cofactors to facilitate the transfer of both electrons and hydrogen to and from substrate molecules in biocatalytic processes (Uppada V et al. Current Science, 2014, 106(7), 946-957; Wilson J et al. Molecular biology of the cell. 5th ed. New York: Garland. 2015, 159). For example, NADH is commonly used as a cofactor with various alcohol dehydrogenases to convert aldehydes to alcohol, a key step in the production of biofuels. In the process, NADH is converted to NAD+ and the reaction stops until more NADH is supplied. The current high cost of the reduced forms of the cofactors, NADH and NADPH, however, makes it economically unfeasible to use stoichiometric amounts of cofactor in biocatalysis. The bulk (not retail) cost of a single mole of NADH in 2011 dollars was approximately $3,000 ($4.52 per gram) and $215,000 ($288.82 per gram) for NADPH (Faber K. Biotransformations in Organic Chemistry. Springer Berlin Heidelberg. 2011. pp. 31-313). Consequently, the development of effective cofactor regeneration systems is needed to reduce costs and increase the efficiency of the enzyme turnover in certain biocatalytic processes so that they can be conducted on an industrial scale.
Electrochemical Cofactor Regeneration Techniques. Of the studied techniques for NAD(P)H regeneration (enzymatic, chemical, electrochemical, biological) (Chenault H K et al. Applied Biochemistry and Biotechnology, 2007, 14(2), 147-197), electrochemical methods have shown promise due to the economic availability of electricity and the direct supply of electrons without the addition of co-substrates (Wang X et al. Chem, 2017, 2(5), 621-654; Weckbecker A et al. Biosystems Engineering I. Springer Berlin Heidelberg. 2010. pp. 195-242). In the direct electrochemical reduction of NAD(P)+ to NAD(P)H, the reaction generally proceeds in two steps. First, the NAD(P)+ accepts a single electron and is reduced to the intermediate radical form, NAD(P)* with an unpaired electron in the valence shell of the carbon-4 atom. The radical is then further reduced by the addition of another electron and protonated by H+ to form NAD(P)H, as seen in Scheme 1 (Ali I et al. International Journal of Electrochemical Science, 2013, 8(3), 4283-4304). Unfortunately, the intermediate radical formed following the first reduction step can quickly dimerize with another C4 radical and thereby prevent generation of NAD(P)H that could subsequently be used by an enzyme (Ali I et al. International Journal of Electrochemical Science, 2013, 8(3), 4283-4304). Additionally, protonation of the intermediate could lead to 1,6-NAD(P)H (Moiroux J et al. Journal of Electroanalytical Chemistry, 1985, 194(1), 99-108), which is also not a substrate for subsequent biocatalytic transformations. Very high cathodic overpotentials (negative biases ca.<−1.00 V vs. Ag/AgCl) are typically required for the reduction and protonation of the intermediate radical NADP* and the overall purity (the relative amount of enzymatically active 1,4-NADPH formed vs. inactive products) can be dependent on the electrode material in addition to the electrode potential (Damian A et al. Chemical and Biochemical Engineering Quarterly, 2007, 21(1), 21-32; Ali I et al. Electrochemistry Communications, 2011, 13(6), 562-565; Ali I et al. Chemical Engineering Journal, 2012, 188, 173-180; Ali I et al. Journal of Molecular Catalysis A: Chemical, 2014, 387, 86-91; Ullah N et al. Materials Chemistry and Physics, 2015, 149, 413-417; Ali I et al. The Canadian Journal of Chemical Engineering, 2017, 9999, 1-6; Burnett J N et al. Biochemistry, 1965, 4(10), 2060-2064). For example, Omanovich et al. have shown that the purity of NADH formed at bare Au and Cu electrodes is 32% and 52% respectively at a high overpotential of −1.1 V vs. saturated calomel electrode (−1.06 V vs. Ag/AgCl) (Damian A et al. Chemical and Biochemical Engineering Quarterly, 2007, 21(1), 21-32). The purity, however, tends to maximize at some optimal overpotential; purity of products tends to be low at low overpotential and extreme overpotential for a given electrode material (Damian A et al. Chemical and Biochemical Engineering Quarterly, 2007, 21(1), 21-32; Ali I et al. Journal of Molecular Catalysis A: Chemical, 2014, 387, 86-91). Therefore, reaction product selectivity is a key issue in electrochemical regeneration of NAD(P)H.
Improvements in selectivity at bare electrodes can be made by modifying electrode surfaces with materials capable of catalyzing the protonation step in the reduction of the nicotinamide radical. For instance, Omanovich et al. have demonstrated that precious metals including Pt and Ru as well as the transition metal Ni can increase the 1,4-NAD(P)H selectivity toward 100%; they hypothesized that this was the result of the catalyst material adsorbing hydrogen atoms at the electrode surface to assist in the protonation and physically blocking dimerization (Damian A et al. Chemical and Biochemical Engineering Quarterly, 2007, 21(1), 21-32; Ali I et al. The Canadian Journal of Chemical Engineering, 2017, 9999, 1-6; Azem A et al. Journal of Molecular Catalysis A: Chemical, 2004, 219(2), 283-299).
In addition, organic materials such as L-histidine and cholesterol have also been shown to increase purity when attached to bare metallic electrodes (Baik S H et al. Biotechnology Techniques, 1999, 13(1), 1-5; Long Y T et al. Journal of Electroanalytical Chemistry, 1997, 440(1-2), 239-242). These synthetic organic materials, however, lack the robustness that is offered by metallic catalysts in solid, inorganic cofactor regeneration systems (Ali I et al. International Journal of Electrochemical Science, 2013, 8(3), 4283-4304). Indirect electrochemical methods, which involve the use of a redox mediator to reduce NAD(P)+ in a single step, can also block the formation of inactive dimers (Kohlmann C et al. Journal of Molecular Catalysis B: Enzymatic, 2008, 51(3-4), 57-72). Although these indirect systems operate at lower overpotentials, they typically require complicated organometallic Rh3+ complexes capable of direct hydride transfer as redox mediators (Wienkamp V R. Angewandte Chemie, 1982, 94(10), 8249; Ruppert R et al. Tetrahedron Letters, 1987, 28(52), 6583-6586; Hildebrand F et al. Advanced Synthesis and Catalysis, 2008, 350(6), 909-918; Tan B et al. Journal of the Electrochemical Society, 2014, 162(3), H102-H107; Vuorilehto K et al. Bioelectrochemistry, 2004, 65(1), 1-7). Less complicated redox mediators, such as methyl viologen or flavin adenine dinucleotide (FAD) can be used, but only in a coupled enzymatic reaction with NAD(P)+ (Kim M H et al. Biotechnology Letters, 2004, 26(1), 21-26; Jayabalan R et al. Bioresource Technology, 2012, 123, 686-689). Thus, there is merit in developing simple and effective, inorganic electrochemical systems capable of direct NAD(P)H regeneration.
Despite the improvements to ‘all-solid’, direct electrochemical systems, high overpotentials, and thus more electrical energy, are still required to drive the cofactor regeneration process. The unique energy band structure of semiconducting electrodes can provide an avenue for lowering the overpotential and replacing electrical energy with light energy in a direct photoelectrochemical cofactor regeneration system. Before embarking on this topic, the basic physics of semiconductor-electrolyte interface is described first.
Semiconductor-Electrolyte Interfaces. When a semiconductor is placed in contact with an electrolyte solution, the system establishes electrochemical equilibrium following a temporary current flow as the Fermi levels of the semiconductor and the electrolyte align (
SCOPE. The majority of the research into photoelectrochemistry has been restricted to cells used for the electrolysis of water into H2 and O2 or for power generation (Gratzel M. Nature, 2001, 4/4(6861), 338). The usage of semiconducting photocathodes for the direct reduction of NAD(P)+ has largely been unexplored. There is only one example of direct photoelectrochemical NAD(P)H regeneration at a semiconducting surface in literature. Stufano et. al. utilized p doped GaAs (p-GaAs) and p-GaP photocathodes for direct NADH regeneration (Stufano P et al. ChemElectroChem, 2017, (614), 1-9). They reported that the illuminated p-GaAs surface produced only dimeric species, while the p-GaP generated no reaction products whatsoever during electrolysis (Stufano P et al. ChemElectroChem, 2017, (614), 1-9). The authors speculated that the UV light used to excite the band-gap of p-GaP caused any reaction products to dissociate back to NAD+ (Stufano P et al. ChemElectroChem, 2017, (614), 1-9). Only after modification of the surface with the precious metals Ru and Pt, was enzymatically active NADH formed at the p-GaAs electrode (Stufano P et al. ChemElectroChem, 2017, (614), 1-9). The authors reported moderately high purity of 75% utilizing a Pt coated-p-GaAs electrode at a low overpotential of −0.75 V vs. Ag/AgCl (Stufano P et al. ChemElectroChem, 2017, (614), 1-9). The authors did not investigate the corrosion behavior of the electrodes.
Another potential candidate for a photocathode is the inexpensive and earth-abundant material, cuprous oxide. Cuprous oxide possesses a direct band gap in the visible light region (1.9-2.4 eV) and a conduction band that lies negative of the NAD(P)+/NAD(P)H formal potential (
Materials and Methods
Electrode Preparation. The cuprous oxide photocathodes were prepared by a single step electrochemical process. A solution containing 0.48 M CuSO4 3 M Lactic Acid was prepared using copper sulfate pentahydrate (Sigma-209198), concentrated lactic acid (Ricca RABL0010-500A) and deionized water (18.2 MΩ·cm, Milli-Q). The pH of the solution was then adjusted to 11 with the addition of concentrated NaOH. The electrodeposition process was carried out potentiostatically at −0.5 V vs. Ag/AgCl (3 M NaCl, Basi MF-2052) and room temperature (20-25° C.) in a two-compartment cell separated by an agarose bridge (2% agar, Invitrogen 15110-019) and a coiled Pt wire counter electrode. The cathode chamber was filled with cupric lactate solution and the anode solution was filled with potassium phosphate buffer solution to prevent any Pt degradation or Cu adsorption. The Cu2O electrodeposition reaction is given in equation 1.1, where L represents the lactate anion.
2CuL2+2OH−+2e−→Cu2O+H2O+4L− 1.1
Cu 100 mesh substrates (Alfa Aesar 45186) were chosen as the working electrodes as they are less expensive and provide a higher surface area to volume ratio than planar foils. Prior to deposition, Cu substrates were cleaned by sonication in 200 proof ethanol for 10 minutes followed by sonication in deionized water for 10 minutes. The substrates were dump rinsed in deionized water in between each sonication step. Finally, substrates were dried with compressed clean air. Ni modified Cu2O electrodes were prepared by DC sputtering (Ebtec) a Ni target with Ar gas. The sputtering chamber height and inner diameter are 15 cm and 11.3 cm, respectively. The cathode (Ni target) and anode have diameters of 5.1 cm and 6.6 cm, respectively. Sputtering was conducted at 340 V at a working distance of approximately 3 cm between the sample and Ni target. Electrodes were sputtered for 96 hours on one side and 24 hours on the other. The Ni target was polished with 100 grit sandpaper before each sputtering session. Wires were attached to the Ni sputtered Cu2O electrodes with conductive silver epoxy (MG Chemicals 8331) before being used in electrolysis experiments to reduce any uncompensated contact resistances (IR drops) in electrode potential measurements.
NADPH Regeneration. Bulk electrolysis of NADP+ solutions was carried out in a custom quartz H-cell with glass frit separator to mitigate light attenuation through the walls of the cell. Starting solutions of 1.5 mM NADP+ (Sigma-10128031001) were prepared in either a 0.5 M potassium-phosphate buffer (pH 7) or a 0.5 M sodium-phosphate buffer (pH 8). Phosphate buffers at pH 7 and 8 were selected due to their electrochemical conductivity and their supporting NADPH stability 9 Vuorilehto K et al. Bioelectrochemistry, 2004, 65(1), 1-7). The potentiostatic electrolysis was performed in 3-electrode setup with an Ag/AgCl (3 M NaCl) reference and Pt mesh (Alfa Aesar 10283) counter electrode. The Cu2O photocathode was illuminated with a 10 mW (532 nm, frequency doubled YAG) laser light source. All photocathodes had approximately a 1 cm2 illuminated geometric surface area and were illuminated on the side on which Ni was sputtered for 96 hours. To discern the purity and activity of the reaction products, enzymatic assay (see Description of Enzymatic Assay) was performed using Lactobacillus brevis alcohol dehydrogenase (Lb-ADH). All experiments were carried out by holding the Cu2O cathode at a fixed electrode potential of −0.75 V (Ag/AgCl). Any geometric electrode areas are calculated assuming a grid of perfectly cylindrical wire.
The geometric surface area of meshes were calculated by assuming that the Cu wires in the mesh form a perfect orthogonal grid of cylinders (
Assuming that the open space on the edges of the mesh has width O, the number of wires running horizontally and vertically can be estimated as follows in equations A.C.1 and A.C.2. Here, vertical running wires are denoted NW and horizontal running wires are denoted NL.
Now that the number of vertical and horizontal wires is estimated, the surface area of these wires is now calculated assuming perfectly cylindrical geometry. The surface area of vertical wires and horizontal wires is denoted as SL and SW, respectively. Only half of the circular end caps on the vertical wires are solution-immersed, hence the factor of ¼ present in equation A.C.3.
Finally, the wire crossings are assumed to cover up approximately D2 on both cylindrical wires forming the intersection. Thus, the total, solution immersed geometric surface area (Stot) can be estimated in equation A.C.5.
S
tot
=S
W
+S
L−2NWNLD2 A.C.5
Description of Enzymatic Assay. The purity of electrolysis products was determined based on enzymatic assay. Here Lb-ADH was employed as the enzyme to catalyze the reduction of butyraldehyde to butanol with the concomitant oxidation of NADPH to NADP+ (
After measuring the decrease in absorbance, the purity can be estimated by the method developed by Omanovich et al. applied to NADPH (Damian A et al. Chemical and Biochemical Engineering Quarterly, 2007, 21(1), 21-32; Ali I et al. The Canadian Journal of Chemical Engineering, 2017, 9999, 1-6; Azem A et al. Journal of Molecular Catalysis A: Chemical, 2004, 219(2), 283-299). The purity, Q, is calculated according to equation 2.1. The ratio between the initial and final 340 nm absorbance determines the fraction of total products that are enzymatically active. The initial absorbance of electrolysis products prior to initiating the reaction is denoted as A0 and the absorbance after the reaction is completed (i.e. at a steady state) is denoted as Af. The difference between the initial and final absorbance in the assay is just A1,4-NADPH and the initial absorbance is A1,4-NADPH+A1,6-NADPH+A(NADP)
The assay entails withdrawing an aliquot of electrolysis products and adding it to butyraldehyde. The initial absorbance at 340 nm is recorded. ADH is then added to the cuvette and mixed by inversion. The absorbance is continuously measured over time until a steady state is reached. The absorbance at steady state is then recorded and Q is calculated. The initial absorbance is adjusted according to the dilution upon adding the ADH. All absorbances are normalized (blanked) to a time zero aliquot that is made prior to beginning electrolysis. All spectrophotometric measurements were performed using a Thermo Scientific Evolution 300 UV-Vis spectrophotometer in quartz cuvettes. The enzymatic assay was first evaluated on an NADPH standard (MP Biomedicals 02101167) before being used to quantitate the purity of any electrolysis products.
Validation of Absorbance Measurement. Before testing on electrolysis products, the enzymatic assay was first evaluated on a prepared NADPH standard (MP Biomedicals 02101167). The first NADPH standard was prepared at a concentration of 150 μM in a 0.5 M potassium phosphate buffer (pH 7). An initial 340 nm absorbance measurement is made on a reference (blank) sample containing only phosphate buffer and 10 mM butyraldehyde (substrate) (
Reference to
Effects of Cuvette Mixing. The effects of the cuvette mixing technique used in the assay were also explored on a standard. It was hypothesized that, since the enzyme must bind both to NADPH and butyraldehyde for reaction progression, the cuvette mixing technique could be important to optimize the duration of the reaction. To explore this notion, two additional 100 μM NADPH standards were prepared and assayed as described earlier. One standard was left unmixed after addition of ADH and the other was inverted 3 times prior to absorbance measurement. In
Results Using the Enzymatic Assay
ADH Concentration Dependence of Assay. The enzymatic assay which was previously evaluated with NADPH standards was then tested on bulk NADP+ electrolysis products. Since electrolysis products will contain a mixture of NADP+ and NADPH in solution, an ADH concentration study was performed to determine if the presence of NADP+ would inhibit the enzyme performance. Electrolysis was performed at illuminated Ni/Cu2O electrodes in 0.5 M potassium phosphate buffer until a measurable absorbance signal (0.1-0.3) in the approximately linear regime (
A total of four enzymatic assays were carried out at ADH concentrations of 0.415 μM, 0.83 μM, 1.99 μM and 3.98 μM. For each ADH concentration, the purity Q was calculated for the same electrolysis products (i.e. the same sample) and plotted as a function of ADH concentration. From
Effects of Dilution in Assay. The effects of sample dilution on the assay were also explored. Electrolysis was performed at a Ni/Cu2O electrode in potassium phosphate buffer solution until the 340 nm absorbance reached the non-linear region (Abs>0.3). The sample was then diluted by a factor 1/6 in the final step of the enzymatic assay; the dilution factor is 1/6 accounting for all species in the cuvette in the final step of the assay. An enzyme concentration of 1.99 μM was utilized. The reactant volumes and assay results for this experiment are given in Table 3 and
The fact that a purity value over 100% was obtained in
Correcting the Purity Values. By performing a least-squares linear fit to the NADP+ calibration (
The actual value of the correction factor will depend on the magnitude of the dilution used as well as the linear fit. For this assay, the sample was diluted by 1/6 and its initial maximum combined NADP+ and NADPH concentration was 1500 μM prior to dilution. The concentration of the reference sample is diluted from 1500 μM to 1440 μM after addition of butyraldehyde. If the predicted absorbance value from the least squares fit is denoted as A′([NADP+]) and the measured data is denoted as A([NADP+]), then the correction factor for this experiment will be Cf=A′(1440 μM)−A′(248.6 μM). The final concentration is 248.6 μM to account for the fact that a dilution exactly by 1/6 cannot be achieved when pipetting to whole microliter amounts (see Table 3).
A new Q value can now be calculated and compared to that of
Referring back to Table 1 and Table 2, it can be seen that the end point sample is also slightly diluted compared to the to sample in obtaining the Q vs. [ADH] data in
Uncertainty Propagation for Dilution Correction Factor in Enzymatic Assay
To calculate an error for the corrected purity values described above, the uncertainty in the correction factor must first be propagated. As a concrete example, the 1/6 dilution is considered where Cf=A′(1440 μM)−A′(248.6 μM). To start, the uncertainties in A′(1440 μM) and A′(248.6 μM) are added in quadrature. This gives the uncertainty in Cf as δCf=√{square root over ((δA′(1440 uM))2+(δA′(248.6 uM))2)}. Before this quantity can be calculated, the uncertainties in the predicted absorbances must be known. These can be estimated by forming the 95% (α=0.05) prediction interval at the two absorbances (concentrations) of interest. This can be calculated as follows in equation A.B.1.
In the above equation, tα/
NADP+ Reduction Kinetics
Temporal Studies of NADP Reduction. Time-course studies were employed to qualitatively elucidate the effects of light (532 nm laser irradiation) in the reduction of the NADP+. The progress of the reaction was monitored spectrophotometrically by measuring absorbance at 340 nm as a function of time. The experiments were conducted by performing bulk electrolysis and sampling small-volume aliquots from the electrochemical cell and measuring their absorbance at regular 15-minute intervals. To avoid reactant depletion, the aliquots were returned to the cell prior to the next measurement. Additionally, care was taken to aliquot from the same location and the solution was stirred to avoid concentration gradient effects in the measurement. Electrolysis experiments were performed potentiostatically at −0.75 V (Ag/AgCl) in 0.5 M sodium phosphate buffer solution. For experiments conducted in the dark, i.e. in the absence of laser irradiation, a heavy black sheet was placed over the experimental apparatus.
The first time-course study utilized a Ni/Cu2O/Cu electrode, which was under illumination for the entire duration of the experiment (
The second possibility was explored by performing electrolysis at a Ni/Cu2O/Cu electrode entirely in the dark (curve (d),
The kinetics of the cofactor regeneration using the electro-reduced Ni/Cu2O/Cu electrode were then compared to the bare substrate Cu mesh and Ni coated Cu mesh. Surprisingly, both the bare substrate mesh as well as the Ni coated Cu mesh show no activity for electrochemical NADP+ reduction at −0.75 V (Ag/AgCl) (
Investigation of the Role of Irradiation. The role of the light in the reduction reaction was further corroborated by examining the chronoamperometric current traces, one of which is shown in
Photocurrent measurements were also performed to further investigate the role of laser irradiation in the NADP+ reduction process (
From the data in
Chemically, since the experiment is carried out in inert buffer, the photo-generated electrons could potentially split the H2O solvent or reduce Cu2O (assuming negligible amounts of other redox species, such as dissolved O2 gas) if there is no recombination. Once again, no bubbling is observed, so the measured photocurrent is attributed to reduction of the oxide layer. Furthermore, it can be seen that the dark trace (the top portion of the current oscillations) does not show purely capacitive charging behavior (neglecting the transient portions). Ideal semiconductors should act as capacitors and show a constant charging current depending on the scan rate since IC=C dV/dt in the absence of faradaic reactions (Bott A W et al. Current Separations, 1998, 17(3), 87-91). With the electrode described herein, this current steadily increases as electrode potential decreases and then sharply increases beyond −0.7 V (Ag/AgCl). The presence of this current corroborates the dark reduction observed in
Enzymatic Assay
Improved Quantification with the Enzymatic Assay. The enzymatic assay employed here, originally developed by Omanovich et al for NADH, is what is commonly applied in the literature for the determination of nicotinamide purity (Damian A et al. Chemical and Biochemical Engineering Quarterly, 2007, 21(1), 21-32; Ali I et al. The Canadian Journal of Chemical Engineering, 2017, 9999, 1-6; Azem A et al. Journal of Molecular Catalysis A: Chemical, 2004, 219(2), 283-299). The work here has demonstrated a host of issues with this purity quantification scheme. The primary concern is the fact that NADP+, the oxidized nicotinamide, also absorbs at 340 nm along with NADPH. Consequently, the absorbance measurement in the assay primarily includes contributions from NADPH, NADP+ and any inactive forms such as the (NADP)2 dimer. This ultimately will lead to errors in the calculation of Q in the event of impure electrolysis products. This expectation is illustrated mathematically assuming the inactive products consist of (NADP)2 dimer, no dilutions are performed in the assay and the enzyme turns over all NADPH molecules supplied to it. Each measurement includes background subtraction of the reference sample.
In this notation, A represents the absorbance with the superscript denoting the step in the enzymatic assay and the subscript denoting the species contributing the absorbance. In measurement step 2, an aliquot is being measured directly from the electrochemical cell which contains a mixture of NADP+, NADPH including potentially some dimer. The signal attributed to any residual NADP+ is then lumped into an error term denoted by ∈. The enzyme is then added in measurement 3 and the reaction is run to completion. It is presumed that all NADPH is converted back to NADP+, leaving only NADP+ and dimer contributions in the measurement. Once again, the NADP+ signal is lumped into an error term. This leads to the more physically realizable definition of Q in equation 4.8. The initial and final absorbances in equation 2.1, which were assumed to be only inactive species and NADPH, now include additional error terms. It is clear in equation 4.8 that if the electrolysis products are pure then Q=1 since ANADP+0=ANADP+2 and ANADP
The more experimentally accurate definition of Q in equation 4.8 can be compared to the idealized definition in equation 2.1 by analyzing it as an inequality. Since ∈2≤∈1 (equality in the case of totally impure electrolysis products), the experimentally accurate definition is expected to yield smaller values than the idealized definition. An inequality can now be set up with equation 4.8 and equation 2.1.
The inequality in equation 4.9 can then be multiplied by (ANADPH1+A(NADP)
Enzyme Stability and Thermodynamics
Another assumption in the enzymatic assay is that the enzyme can utilize all NADPH molecules in the reaction. In evaluating the assay on even just a standard NADPH solution, it was found that if the NADPH concentration were too high for a given ADH concentration, the reaction would cease with residual absorbance at 340 nm, implying that some NADPH remains even when the standard is pure. This problem was exacerbated by introducing NADP+, causing Q to depend on ADH concentration. Two potential causes are enzyme deactivation and thermodynamic equilibrium.
Lb-ADH has been demonstrated to be active in potassium phosphate buffer, however, the concentration of the buffer and pH can have adverse effects on its stability. Higher K-PBS buffer concentrations and higher pH tends to reduce the half-life of Lb-ADH. The optimum conditions have been shown to be [K-PBS]<0.5 M and 6.5<pH<7, showing that the tests here were operating at the limits of these ranges (Kohlmann C et al. Green Chemistry, 2011, 13(6), 1430-1436; Leuchs S et al. Green Chemistry, 2013, 15(1), 167-176). The reversibility of the enzymatic reaction raises a roadblock. Lb-ADH can be used to both oxidize NADPH and reduce NADP+, thus the final nicotinamide concentrations in the assay should be governed by the equilibrium constant, K, of the reaction, given in equation 4.10. This implies that the reaction should not convert all NADPH molecules into NADP+ molecules, only to the degree in which K can be satisfied at a given temperature.
If the reaction is in equilibrium at a given temperature, a few strategies can be used to help drive the reaction to the right-hand side, i.e. favoring production of NADP+. Examining the reaction in
At present, separate experiments (two different electrodes) have yielded purity values of 89.1% and 74.1% in the enzymatic assay, confirming that the electrodes are capable of producing enzymatically active NADPH. These results are impressive compared to what is reported in the literature.
NADP+ Reduction Kinetics
Photoelectrochemistry of Cu2O. The time studies collected in
Carrier Transport Mechanisms for Cu2O Reduction. Examination of the time studies in
In addition to the possibility of inversion layer formation at the semiconductor electrolyte interface, the dark behavior could also be explained by examining the metal (Cu back contact) semiconductor interface. In this view, the semiconductor forms a depletion layer at the metal contact initially at thermal equilibrium. The potentiostat then applies a large negative bias to the metal contact. If the bias is sufficient enough, the Fermi level of the Cu back contact can rise above that of the conduction band of the semiconductor in the bulk. Electrons residing in Cu can then tunnel through the band gap and into a lower energy state in the conduction band of the Cu2O. Once there, the electron can diffuse into the space-charge region near the electrolyte interface and potentially reduce the oxide layer. The tunneling effect is similar to the operation of a Zener diode. This is illustrated in the band diagram in
One other finding in the time study data presented in this work is that, while it appears the electrode is being reduced back to Cu, the substrate Cu is unable to reduce NADP+. This is the case with or without a sputtered Ni coating. Other preliminary experiments have shown negligible activity at a Cu2O electrode with no Ni coating (see
Characterization of Electrode Material. Analysis by SEM imaging and EDS on the electrodes reveals some interesting information. Four separate conditions were imaged by SEM: bare substrate Cu, bare Cu2O/Cu (electrodeposited Cu2O only, no Ni sputtered), untreated Ni/Cu2O/Cu (sputtered Ni on electrodeposited Cu2O), and treated Ni/Cu2O/Cu (photoelectrochemically reduced Ni/Cu2O/Cu). The bare Cu2O/Cu, untreated Ni/Cu2O/Cu, and treated Ni/Cu2O/Cu samples were each collected from the same electrode during the electrode preparation. The corresponding SEM images are displayed in
The overall elemental compositions of the treated and untreated electrodes were confirmed through the use of energy dispersive X-ray electron spectroscopy (EDS). Here, for the untreated and treated Ni/Cu2O/Cu electrodes, the emitted X-rays confirmed the presence of Ni along with Cu and O comprising the oxide layer. The 10 kV high energy electron beam (
The SEM images and EDS results presented here show that sputtering process is an important step in nano-structuring the electrode surface. After sputtering, a significant amount of surface roughness is introduced. Therefore, the catalytic NADP+ reduction properties can be the combined results of creating additional electrochemical surface area through the sputtering process and providing a strong Ni-Hydrogen bond for the eventual protonation of the NADP+ molecule. Next, potential mechanisms for the observed changes in surface morphologies are explored.
The contrast in morphology between the Cu2O/Cu sample and the Ni/Cu2O/Cu sample suggests that Ni bombardment of the surface could be responsible for destroying the crystalline structure of as-is Cu2O. One potential explanation is that the sputtered Ni is ionized to Niz− on impact (where z is the number of excess electrons). The electric field in the chamber then accelerates the negatively charged Ni ion toward the anode, where it eventually strikes the sample and locally modifies the surface. Considering the degree to which the surface is modified and the EDS spectra, a substantial amount of Ni likely exists on the surface of the Ni/Cu2O/Cu a electrodes. If a complete and contiguous Ni layer is forming, the Cu2O layer may not be directly interacting with an aqueous species in its reduction. The Cu2O could potentially donate its oxygen atom to the Ni, forming an intermediate NiO layer which is then further reduced back to Ni. This chemical mechanism is depicted in Scheme 3.
Step 1: Cu2O+2e−⇄2Cu+O2−
Step 2: Ni+O2−⇄NiO+2e−
Step 3: NiO+H2O+2e−⇄Ni+2OH−
Net Reaction: Cu2O+H2O+2e−⇄2Cu+2OH−
Scheme 3. Chemical mechanism for the reduction of Cu2O forming an NiO intermediate.
Transmission electron microscopy (TEM) and cross-section EDS can provide insight on the nature of each surface layer in the electrode. Additionally, electron energy-loss spectroscopy (EELS) can provide information about the oxidation states of the electrode's constituent elements before and after electro-reduction. A complete materials characterization can clarify the origin of the electrode's catalytic properties upon sputtering and photoelectrochemical reduction.
NADP+ Reduction Mechanism. Two potential mechanisms are proposed to describe the overall catalytic NADP+ reduction mechanism at the electro-reduced Ni/Cu2O/Cu electrodes (denoted as OD-Cu—Ni in the mechanism). In the first (Scheme 4), the commonly observed Volmer type mechanism is proposed for the adsorption of hydrogen onto Ni coated locations on the electrode (Gong M et al. Nano Research, 2016, 9(1), 28-46). An NADP+ molecule then adsorbs onto a neighboring site on the electrode. The adsorbed NADP+ is radicalized upon accepting an electron from the electrode. The nearby adsorbed hydrogen is then transferred chemically to the nearby NADP+ to generate NADPH, which then desorbs back into the electrolyte. This can be described as hydrogenation of the radical. Structurally, the reaction would proceed in step 1 followed by 2c) in Scheme 1 where the electron and proton transfer happen simultaneously via the hydrogenation. The first electron transfer neutralizes the positively charged nitrogen atom and the second fills the valence shell of the carbon-4 atom.
Step 1: OD-Cu-Ni+H2O+e−⇄OD-Cu—Ni-Hads+OH−
Step 2: OD-Cu—Ni NADP+⇄OD-Cu—Ni-NADPads+
Step 3: OD-Cu—Ni-NADPads++e−⇄OD-Cu—Ni-NADPads+
Step 4: OD-Cu—Ni-NADPads++OD-Cu—Ni-Hads⇄OD-Cu—Ni-NADPHads+OD-Cu—Ni
Step 5: OD-Cu—Ni-NADPHads⇄OD-Cu-Ni+NADPH
Scheme 4. Potential mechanism for the reduction of NADP+ observed at the Ni/Cu2O/Cu electrode after reduction of the oxide layer. OD-Cu—Ni denotes an available site for surface adsorption on the electrode.
The second mechanism (steps labeled 3b and 4b in Scheme 5) proceeds in a similar fashion as the first, with the hydrogen and electron transfer order reversed. The adsorbed NADP+ molecule accepts the adsorbed hydrogen to form NADPH*+, and then receives an additional electron from the electrode before desorption (
Step 3b: OD-Cu—Ni-NADPads++OD-Cu—Ni—Hads ⇄OD-Cu—Ni-NADPads*++OD-Cu—Ni
Step 4b: OD-Cu—Ni-NADPads*++e−⇄OD-Cu—Ni-NADPHads
Scheme 5. Alternate steps 3 and 4 for NADP+ reduction.
CONCLUSIONS. Several results regarding electrochemical cofactor regeneration at electro-reduced Ni/Cu2O/Cu electrodes have been presented in this work. It was found that laser irradiation of the Ni/Cu2O/Cu photocathode assists in developing an electrocatalyst for NADP+ reduction by photoelectrochemically reducing the oxide layer in the Ni/Cu2O/Cu electrode. The activity of the electrochemically-produced reaction products was confirmed via the spectrophotometric enzymatic assay. The exact purity of the reaction products, however, is not clear. Using the method of Omanovich et al., the enzymatic assay gave values of 74.1±1.0% and 89.1±2.8% (where the error bar arises due to dilution correction) for two different electrodes. When examining the procedure in a more rigorous mathematical manner, it was seen that there is additional uncompensated uncertainty that arises due to the NADP+ absorbance at 340 nm. Basic thermodynamic consideration of the assay also suggests that the ADH should not turn over all the 1,4-NADPH, just enough to satisfy its equilibrium constant. Analytical methods such as mass spectrometry or nuclear magnetic resonance spectroscopy can accurately characterize the purity of reaction products and confirm or deny the presence of dimer/inactive isomer.
Temporal studies monitoring progress of NADP+ reduction at Ni/Cu2O/Cu electrodes showed that the reaction proceeded with or without light, with an initial dormant period with no product formation. Meanwhile, a substantial cathodic current is passed during the dormant period, indicating that the oxide layer must be reduced to a certain degree before the NADP+ reduction can proceed. The dead period was mitigated by turning on the light source (532 nm laser irradiation) or pre-electro-reducing the electrode in buffer prior to adding NADP+. This implies that any photoelectrochemistry is reserved for reducing the oxide layer rather than conversion of the NADP+. Interestingly, the electro-reduced Ni/Cu2O/Cu electrode shows significantly stronger activity for reducing NADP+ at low overpotential compared to the Cu substrate (with and without Ni coat) and uncoated Cu2O. Preliminary materials characterization suggests this catalytic behavior is due to the combined effects of sputtered Ni generating an increased electrode surface area and creating a favorable metal-hydrogen bond on the solution side. A more detailed materials characterization can more fully elucidate the catalytic properties of the nanoheterostructure, especially after being electro-reduced. Fundamental physical differences exist between the native substrate Ni/Cu and the oxide-derived Ni/Cu electrodes and understanding these can assist in fine tuning of the catalytic properties of the electrode for cofactor regeneration or other potential electrochemical processes.
A Ni/Cu2O/Cu nanoheterostructure electrode has been developed for the direct electrochemical reduction of NADP+ to enzymatically active NADPH. This electrode offers strong advantages in its low material cost and two-step manufacturing process compared to other precious metal decorated electrodes. The capability of low overpotential and direct NADPH regeneration reduces the electrical energy input cost compared to other high-overpotential systems and provides a simple experimental setup for regenerating NADPH in solution. Understanding of the sources of the catalytic nature of the electrode demonstrated here can allow for the electrochemical regeneration of cofactors to be optimized and potentially become viable as an electrode for industrial scale cofactor regeneration.
Nearly a fourth of the proteome consists of oxidoreductases, and the redox reactions supported by this vast catalytic repertoire sustain cellular metabolism. In the majority of 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, lack of product purity has been a major deterrent. Herein, a nanostructured heterolayer Ni—Cu2O—Cu cathode formed by a photoelectrochemical process is shown to have unexpected efficacy 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]. The remainder of the inactive product is likely an isomer (e.g., 1,6-NADPH) arising from the electrode-adjacent layers. Sputtering of nickel on the copper-oxide electrode nucleated an unexpected surface morphology (as uncovered by SEM and XPS studies) that contributed to high product selectivity. These results can motivate design of integrated electrolyzer platforms that deploy this heterogeneous catalyst for direct electrochemical regeneration of NADH/NADPH, which is central to next-generation biofuel fermentation strategies, biological solar converters, energy-storage devices, and artificial photosynthesis.
INTRODUCTION. Electron transfer is key to all cellular metabolism as redox reactions undergird the work performed by all living organisms (VanLinden M R et al. Biochem. 2015, 37(1), 9-13; Johnson M P. Essays in Biochemistry, 2016, 60, 255-273). During the exergonic oxidation of nutrients and foods, catabolic processes first capture electrons in the form of coenzymes (e.g., reduced form of nicotinamide adenine dinucleotide, NADH) and then generate cellular energy currencies by harnessing the electromotive force during electron transport from these reduced coenzymes to oxygen. In contrast, specialized coenzymes (e.g., reduced form of nicotinamide adenine dinucleotide phosphate, NADPH) are used to support reductive syntheses during anabolism. Metabolism is often a target of cancer therapies (Liberti M V et al. Trends Biochem Sci. 2016, 41(3), 211-218).
Reduced coenzymes also have potential application in bioinorganic artificial photosynthesis (Zhang T. Science, 2016, 350(6262), 738-739; Wu Y A et al. Nature Energy, 2019, 4, 957-968), and are important for biocatalyst-centered synthetic efforts in the pharmaceutical and (bio)chemical industries (Faber K. Biotransformations in Organic Chemistry. Springer Berlin Heidelberg, 2011. pp. 31-313; Uppada V et al. Current Science, 2014, 106(7), 946-957; Bornscheuer U T et al. Nature, 2012, 485, 185-193; Robinson P K. Essays in Biochemistry, 2015, 59, 1-41; Wang X et al. Chem, 2017, 2, 621-654; Hofler G T et al. ChemBioChem Comm. 2018, 19, 2344-2347). However, economic viability of these approaches depends on lowering the cost of reduced coenzymes. Inexpensive production of butanol from lignocellulosic biomass via biofermentation, for instance, would be an economically appealing additive to gasoline were it not for the cost (Zhang Y et al. J. Ind. Microbiol. Biotechnol. 2014, 41, 1505-1516). This roadblock is address herein by exploiting a nanostructured Ni—Cu2O—Cu heterolayer material for photoelectrochemical regeneration of NADPH.
NAD(P)H has been regenerated by enzymatic, chemical, electrochemical, and biological approaches (Faber K. Biotransformations in Organic Chemistry. Springer Berlin Heidelberg, 2011. pp. 31-313; Wang X et al. Chem, 2017, 2, 621-654; Hofler G T et al. ChemBioChem Comm. 2018, 19, 2344-2347). Among these methods, electrochemical regeneration is the most direct, requiring few to no intermediate steps (Faber K. Biotransformations in Organic Chemistry. Springer Berlin Heidelberg, 2011. pp. 31-313; Wang X et al. Chem, 2017, 2, 621-654). However, challenges in the artificial regeneration of NADH/NADPH include: (i) the formation of inactive forms such as the (NADP)2 dimer and the inactive isomer 1,6-NAD(P)H, which affect the purity of the product as determined by its utility (
Material and characterization. CuO and Cu2O are both p-type semiconductors (1.3 eV 1.7 eV and 2.0-2.5 eV) (Paracchino A et al. Nature Materials, 2011, 10, 456-461; Yang Y et al. Scientific Reports, 2016, 6, 35158). The photo-galvanic properties of interfaces between semiconductors and electrolytes have been extensively studied since Edmond Becquerel's first report in 1839 (Becquerel E. Compte Rendu des Seances de L'Academie des Sciences, 1839, IX(3), 145-149; Garrison A. Journal of Physical Chemistry, 1924, 28(3), 279-284; Gratzel M. Nature, 2001, 414, 338-344). Copper-oxide based electrodes have since been used for hydrogen production (Paracchino A et al. Nature Materials, 2011, 10, 456-461; Dubale A A et al. Journal of Materials A, 2015, 3, 12482-12499; Pan L et al. Nature Catalysis, 2018, 1, 412-420), photoelectrocatalytic conversion of CO to liquid fuels (ethanol, acetate, and n-propanol) (Li C W et al. Nature, 2014, 508, 504-507), and solar energy conversion (Zhu C et al. Chemistry of Materials, 2014, 26, 2960-2966; Zhang T. Science, 2015, 350(6262), 738; Garcia-Esparza A T et al. Journal of Materials Chemistry A, 2014, 2, 7389-7401). The Becquerel effect has been used in photoelectrocatalytic regeneration of the cofactor NADH, but with exotic and expensive materials (Pt-modified p-GaAs semiconductor electrodes) (Stufano P et al. ChemElectroChem. 2017, 4, 1066-1073). Herein, a photoelectrochemically surface-modified Ni—Cu2O—Cu electrode was used to regenerate NADPH from NADP+ using inexpensive materials and a facile process. The choice of this cofactor was in part due to the availability of an enzymatic assay based on Lactobacillus brevis alcohol dehydrogenase (LbADH) to evaluate the efficacy of the process based on the activity of the product, NADPH (Halloum I et al. Fermentation, 2015, 1, 24-37).
Copper oxide-based cathodes were prepared by a single-step electrochemical process on copper 100 mesh substrates (Alfa Aesar 45186, woven from 0.11 mm dia. Wire, 1 cm2). Copper mesh was selected as a working electrode because it is inexpensive and provides a higher surface area to volume ratio compared to planar foils. Prior to electrodeposition, the meshes were cleaned by two 10-min sequential sets of sonication in absolute ethanol and de-ionized water for another 10 min. The meshes were dump rinsed in de-ionized water between each sonication step. Finally, the meshes were dried with clean, compressed air. The copper oxide layer was electrodeposited using a solution containing 0.48 M CuSO4 and 3 M lactic acid. This solution was prepared using copper sulfate pentahydrate (Sigma-209198), concentrated lactic acid (Ricca RABL0010-500A) and deionized water (18.2 Me-cm, Milli-Q) (Yang Y et al. Scientific Reports 2016, 6(1), 35158). The pH of the solution was then adjusted to 11 by adding pure NaOH. The electrodeposition process was carried out potentiostatically (Gamry Interface 1000) at −0.5 V vs Ag/AgCl (3 M NaCl, Basi MF-2052) for 2 h at room temperature (20-25° C.) in a two-compartment cell (
The electrodeposited material was reddish in appearance, a characteristic typical of Cu2O. This nanostructured heterolayer cathode was then further modified by DC sputter deposition (EBTEC Co.). A thin nickel coating was deposited onto the Cu2O—Cu heterostructure by sputtering a Ni target (5.1 cm diameter, polished with 100 grit sandpaper before deposition) in Ar gas (˜600 mTorr) at 340 V, 12.5 mA at a working distance of 3 cm between the sample and Ni target. Since the reduction of NADP+ to NADPH was desired, Ni was selected because of its well documented ability to adsorb hydrogen (Gong M et al. Nano Research! 2016, 9(1), 28-46). The sputtering process was carried out for 96 h after which the sample was overturned, and the sputtering process repeated for 24 h. Wires (22 AWG tinned Cu) were then attached to the Ni—Cu2O—Cu electrode with conductive silver epoxy (MG Chemicals 8331) in order to decrease any uncompensated contact resistance (i.e. IR drops) in subsequent measurements of the electrode potential.
Surface modification of the cathode. In the final step of electrode fabrication, the Ni—Cu2O—Cu electrode is exposed to 10 mW, 532 nm unfocused laser radiation while immersed in sodium phosphate (0.5 M, pH 8). This process was conducted in a customized quartz H-cell with a glass-frit separator to minimize light attenuation through the walls of the cell (
The features characteristic of CuO are evident in
A cross section of the surface-modified electrode was prepared by focused ion beam (FIB) and imaged using the SEM. EDS analysis of the surface layers (
Direct electrochemical regeneration of NADPH at low overpotential.
Bulk electrolyses of 1.5 mM NADP+ solutions (Sigma 10128031001) were performed in the same apparatus used for photoelectrochemical surface modification of the cathode, and with the same buffer (
Regenerated NADPH does not contain the inactive dimer. The purity of the product of electrochemical regeneration (NADPH) was investigated using the LbADH enzyme (see Methods below) (Halloum I et al. Fermentation, 2015, 1, 24-37). When this enzyme was added to commercially obtained NADPH (60 μM), near-complete conversion to NADP+ was observed (Q>99%, Equation S3-Equation S5,
Fourier-transform ion cyclotron resonance mass spectrometry (Bruker 15T FT-ICR MS) with matrix-assisted desorption/ionization (MALDI) in both negative- and positive-ion modes was used to determine the composition of the products of cofactor regeneration. α-cyano-4-hydroxy-cinnamic acid) was used as the matrix. This instrument is capable of ultrahigh resolution (>106) and mass accuracy (<1 ppm).
The above results highlight the favorable cofactor regeneration outcome obtained with the unexpected morphology and properties of the oxide-derived copper-nickel (odCu-Ni) surface, produced by photoelectrochemical surface modification of the nanostructured heterolayer Ni—Cu2O—Cu cathode. A proposed mechanism based on the results presented here is:
(odCu—Ni)+H2O+e(odCu—Ni)+Hads+OH− (1)
(odCu—Ni)+NADP+(odCu—Ni)+NADPads+ (2)
(odCu—Ni)+NADPads++e−(odCu—Ni)+NADPads+ (2)
(odCu—Ni)NADPads++Hads(odCu—Ni)+NADPHads (4)
(odCu—Ni)+NADPHads(odCu—Ni)+NADPH (5)
where the subscript ads denotes a species adsorbed on the odCu-Ni surface, and the superscript * denotes an excited state. The first step (Equation 1) is simply the commonly observed Volmer-type mechanism for adsorption of H on Ni (Gong M et al. Nano Research! 2016, 9(1), 28-46)]. The steps indicated in Equation 2-Equation 4 represent hydrogenation with an adsorbed H atom, of the adsorbed NADP+ which is subsequently radicalized after accepting an electron from the electrode. The resulting NADPH is then desorbed back into the electrolyte (Equation 5). Alternatively, Equation 3-Equation 4 may be replaced by:
(odCu—Ni)+NADPads++Hads(odCu—Ni)+NADPHads*+ (6)
(odCu—Ni)+NADPHads*++e−⇄(dCu—Ni)+NADPHads (7)
after which NADPH desorbs according to Equation 5. These reaction pathways could all equally lead to formation of the 1,6-NADPH inactive isomer. The inactive dimer was not detected in the FT-ICR MS MALDI spectra. These two results together indicate that the inactive product is likely the isomer which could form adjacent to the cathode sheath where higher electric fields are expected.
CONCLUSIONS. A nanostructured Ni—Cu2O—Cu heterolayer material was developed and photoelectrochemical regeneration of the important cofactor, NADPH, was demonstrated. Sputtering of nickel on a copper oxide electrode produced an unexpectedly desirable surface morphology leading to high product selectivity. The demonstrated properties of this cathode for direct electrochemical regeneration of NADPH are at the lowest reported overpotential (−0.75 V versus Ag/AgCl (3M NaCl) reference). Materials characterization by SEM, EDS, XPS, and XRD all confirm that sputtering of Ni on the heterolayer Cu2O—Cu mesh cathode leads additionally to formation of CuO in the surface nanolayers. Furthermore, after photoelectrochemical surface modification, oxygen is depleted from the surface layers of the electrode to generate a Ni—Cu nanolayer. The NADPH product electrochemically regenerated from NADP+ using this nanostructured heterolayer cathode is found to be free of any dimers, unlike previously reported with direct electrochemical cofactor regeneration using more expensive and exotic electrode materials or even a Cu mesh electrode with the same sputtered Ni overcoat. The concomitant ability of the cathode to adsorb a hydrogen and donate an electron to NADP+ appears to greatly diminish the propensity of the radicalized NADP+ to form an inactive (NADP)2 dimer. Despite the unexpected payoffs from the new electrode, an LbADH enzyme assay revealed the presence of another inactive product, possibly an isomer such as 1,6-NADPH that may be generated in the electrode-adjacent layers in the electrochemical H-cell. Previous work (Damian A et al. Chem. Biochem. Eng. Q. 2007, 21, 21-32), as with the present work utilized static (non-flowing media in their experiments, which can lead to accumulation of inactive products. In practice, production of the unwanted inactive isomer could be suppressed by using a flow cell rather than static or batch processes. Recent work on electrochemical regeneration of the non-phosphorylated cofactor NADH, using multiwalled carbon nanotubes grown on a stainless steel mesh and decorated with nickel nanoparticles, showed a recovery of 98% but at elevated cathode potentials of −1.168 V (vs. Ag/AgCl (3M NaCl)) (Ali I et al. The Canadian Journal of Chemical Engineering. 2018, 96(1), 68-73). A potential higher than −0.75 V used in this work with the nanostructured heterolayer Ni—Cu2O—Cu electrode, that can produce higher yields of active NADPH may therefore exist, and should be further explored. Beyond significantly advancing cofactor regeneration, the electrode material and associated photoelectrochemical surface modification process reported here could advance processes for water-splitting for hydrogen generation, artificial photosynthesis (Zhang T. Science, 2015, 350(6262), 738), and synthesis of biofuels such as butanol via biofermentation (Agu C V et al. Journal of Industrial Microbiology & Biotechnology. 2016, 43(9), 1215-1226; Ujor V et al. Applied Microbiology and Biotechnology, 2014, 98, 6511-6521; Zhang Y et al. J. of Industrial Microbiology & Biotechnology. 2014, 41, 1505-1516).
Spectrophotometric measurements. All spectrophotometric measurements were carried out with a Thermo Scientific Evolution 300 UV-Vis spectrophotometer using the VisionPro software. All measurements were conducted in fixed wavelength mode with an integration time of 3 s and a wavelength bandwidth of 1 nm. Samples were pipetted into 10 mm pathlength micro-quartz cuvettes (Fisher) and mixed by inversion prior to measurement. Errors were determined from the standard deviation of 30 sequential absorbance measurements on the same aliquot, for each time point.
LbADH assay to determine the turnover number. The LbADH activity assay was performed as described elsewhere (Halloum I et al. Fermentation, 2015, 1, 24-37), albeit with some modifications. All assays were carried out at 37° C. in a 30-μl total reaction. Typically, the reaction mix contained 5 mM butyraldehyde, 0.25 mM NADPH in 50 mM sodium phosphate (pH 8). The reaction was initiated by the addition of 1.5 μl of 0.29 μM recombinant ADH to 28.5 μl reaction mix. From the 30-μl reaction that was assembled, 28-μl was immediately transferred to a 384-well microplate and the absorbance at 340 nm was monitored real-time using a SpectraMax M5 (Molecular Devices) Microplate Reader (integration time of 1000 ms; settle time of 300 ms). This continuous spectrophotometric readout enabled calculation of initial velocities. Linear regression analysis of NADPH generated as a function of time was used to calculate the initial velocity (0.97≤r2≤0.99). For each assay, a control assay was performed that included all the assay components except the ADH. Using activities determined from initial velocity measurements with 5 mM butyraldehyde substrate, the kcat was determined to be 214±5 min−1, consistent with the value reported earlier (Halloum I et al. Fermentation, 2015, 1, 24-37).
LbADH assay for detecting electrochemically regenerated NADPH. The LbADH enzyme assay for determination of NADPH selectivity in electrochemical regeneration was performed by first collecting a to aliquot of NADP+ solution (1.5 mM, 325 μL) prior to running the experiment. This sample was then mixed with 13 μL 0.25 M butyraldehyde and diluted with sodium phosphate buffer solution (pH 8) to yield a final concentration of 9.3 mM butyraldehyde in a total volume of 350 μL. This butyraldehyde concentration was chosen to ensure adequate amount of substrate for complete turnover of all regenerated 1,4-NADPH. The A340 measurement of this sample served as a reference for baseline subtraction (Aref). Three-hundred and twenty-five μL of regenerated cofactor was then collected and mixed with 13 μL 0.25 M butyraldehyde substrate and its initial absorbance at 340 nm (A0) was recorded. Twelve μL 58 μM LbADH was then added to the regenerated cofactor/butyraldehyde mixture to yield a final concentration of ˜2 μM ADH, and its absorbance at 340 nm was monitored until a steady state was reached (Af). The selectivity, Q, of the sample was calculated according to Equation A1, where the constant, α, represents the factor by which the regenerated cofactor/butyraldehyde mixture is diluted upon addition of LbADH:
Inclusion of the constant accounts for any decrease in absorbance upon addition of LbADH solely by virtue of dilution. To ensure applicability of Beer's Law, only regenerated cofactor samples with an initial absorbance less than 0.25 (˜70 μM NADPH) were considered (
Calculation of redox potentials. The electrodeposition reaction used for depositing copper oxide on the copper mesh is given by:
2CuL2+2OH−+2e−→Cu2O+H2O+4L− (S1)
where L represents the lactate anion. All redox potentials at pH 8 are computed via the Nernst equation:
where E0 is the standard reduction potential of the redox couple, R is the universal gas constant, T is the temperature, n is the number of electrons involved in the half-reaction, F is Faraday's constant, cox and cred are the concentrations of oxidized and reduced species, respectively, and γox and γred are the stoichiometric coefficients of oxidized and reduced species, respectively. All non-pH determining species, e.g. Cu2O or NAP+, 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 Equation A1 may be re-written as follows.
The numerator in Equation 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 εε and concentrations as [·], Equation 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 (2 s on 5 s 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, ˜13 mg of recombinant ADH was obtained.
Other advantages which are obvious and which are inherent to the invention will be evident to one skilled in the art. It will be understood that certain features and sub-combinations are of utility and may be employed without reference to other features and sub-combinations. This is contemplated by and is within the scope of the claims. Since many possible embodiments may be made of the invention without departing from the scope thereof, it is to be understood that all matter herein set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense.
The methods of the appended claims are not limited in scope by the specific methods described herein, which are intended as illustrations of a few aspects of the claims and any methods that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of the methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative method steps disclosed herein are specifically described, other combinations of the method steps also are intended to fall within the scope of the appended claims, even if not specifically recited. Thus, a combination of steps, elements, components, or constituents may be explicitly mentioned herein or less, however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated.
This application claims the benefit of priority to U.S. Provisional Application No. 63/059,051 filed Jul. 30, 2020, which is hereby incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US21/43962 | 7/30/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63059051 | Jul 2020 | US |
