Novel biobattery and its use for powering applications

Abstract

Described are enzyme fuel cells and batteries comprising an anode with a multicopper oxidase enzyme immobilized with a phenolic substrate.

Description

NOVEL BIOBATTERY AND ITS USE FOR POWERING APPLICATIONSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application 63 / 505,259, filed May 31, 2023, and is hereby incorporated by reference in its entirety.REFERENCE TO A SEQUENCE LISTING

[0002] The Sequence Listing written in file name 614149SEQLIST.xml is 7.6 kilobytes, was created on May 24, 2024, and is hereby incorporated by reference.BACKGROUND

[0003] Biomedical devices, including implantable devices provide many important therapeutic functions and require small yet powerful and long-lasting power sources. Currently, biomedical devices use traditional batteries, such as lithium-ion batteries, which have many drawbacks. Traditional batteries contain metals and toxic reagents which are not biocompatibility, lack environmentally sustainability and have size limitations based on the necessary lifespan.

[0004] Enzyme fuel cells, which utilize enzymes as catalysts to produce electricity under physiological conditions, have great promise for powering biomedical devices with non-toxic reagents. However, existing enzyme fuel cells have many limitations which have prevented their practical implementation in clinical settings, including stability, the availability of fuels for the electrode, and low power output. There remains a great need for practical and functional enzyme fuel cells capable of powering biomedical devices. The enzyme fuel cells and BioBatteries disclosed herein address these limitations.BRIEF SUMMARY

[0005] Disclosed herein are anodes comprising oxidoreductase enzymes immobilized with a substrate, enzyme fuel cells and batteries comprising said anodes, and methods for making enzyme fuel cells.

[0006] One embodiment is an anode comprising an electroconductive material, wherein an oxidoreductase enzyme is immobilized on the anode and a substrate immobilized on the anode, wherein the substrate can be oxidized by the enzyme. In embodiments, said oxidoreductaseenzyme is a multi copper oxidase. Tn some embodiments, the multicopper oxidase is selected from the group consisting of laccase, bilirubin oxidase, ascorbic acid oxidase, tyrosinase, and multicopper oxidase derived from Pyrobaculum aerophilum. In one embodiment, the laccase is derived from Trametes versicolor or Aspergillus sp. In another embodiment, the bilirubin oxidase is derived from Myrothecium verrucaria.

[0007] In embodiments, disclosed herein, the substrate is formed by depositing a crosslinking reagent. In one embodiment, the cross-linking reagent is glutaraldehyde. In some embodiments, the substrate is a phenolic compound. In one embodiment, the phenolic compound is derived from lignin.

[0008] In some embodiments, the electroconductive material is selected from the group consisting of carbon paper, glassy carbon, carbon nanotubes, gold, and palladium.

[0009] Provided herein is a fuel cell comprising said anode and a cathode, wherein the cathode comprises an electroconductive material and an electron acceptor to reduce oxygen, wherein the anode and the cathode are in a solution, wherein the cathode is contact with dissolved oxygen.

[0010] In some embodiments, the electron acceptor of the cathode comprises the reduced status of the electron acceptor molecules which can transfer electron to oxygen spontaneously. In some embodiments, the electron acceptor of the cathode comprises a phenazine compound. In one embodiment, the phenazine compound comprises phenazine ethosulfate (PES) or phenazine methosulfate (PMS). In some embodiments, the phenazine compound is amine-reactive. In one embodiment, the amine-reactive phenazine compound is N-hydroxysuccinimidylester 1-propoxy- 5-ethylphenazinium ethyl sulfate (arPES).

[0011] In other embodiments, the electron acceptor of the cathode comprises a metal complex. In one embodiment, the metal complex is an osmium complex or a ruthenium complex.

[0012] In some embodiments, the electron acceptor of the cathode is immobilized on a protein. In one embodiment, the protein is selected from the group consisting of bovine serum albumin (BSA), glucose dehydrogenase (GDH), lactate dehydrogenase (LDH), glucose oxidase (GOx), and lactate oxidase (LOx).

[0013] In further embodiments, the electron acceptor of the cathode is an oxidase enzyme. In some embodiments, the oxidase enzyme of the cathode is a multicopper oxidase. In some embodiments, the multi copper oxidase of the cathode is selected from the group laccase, bilirubinoxidase, ascorbic acid oxidase, and multicopper oxidase derived from Pyrobaculum aerophilum. In one embodiment, the multicopper oxidase of the cathode is laccase derived from Trametes versicolor or Aspergillus sp. In another embodiment, the multicopper oxidase of the cathode is bilirubin oxidase derived from Myrothecium verrucaria.

[0014] An embodiment is said fuel cell, wherein the anode and / or the cathode comprise an enzyme with an amino acid sequence set forth in any one SEQ ID NOs: 1-4, provided the enzyme of the cathode is different than the enzyme of the anode.

[0015] Another embodiment is said fuel cell, wherein the electroconductive material of the cathode is selected from the group consisting of carbon paper, glassy carbon, carbon nanotubes, gold, and palladium.

[0016] In some embodiments, the solution of the anode and the solution of the cathode are in the same environment. In other embodiments, the solution of the anode and the solution of the cathode are in separate environments and connected by a salt bridge.

[0017] An additional embodiment is a battery comprising said fuel cell and a container. In some embodiments, the battery comprises more than one fuel cell. In one embodiment, the fuel cells are in a series configuration. In other embodiments, the fuel cells are in a parallel configuration. In some embodiments, the fuel cells are stacked. In some embodiments, said battery comprises a biodegradable material.

[0018] Another embodiment is a method of making said fuel cell comprising: (a) preparing the anode by depositing the enzyme and the substrate on an electrode comprising an electroconductive material; and (b) preparing the cathode by depositing the electron acceptor on an electrode comprising an electroconductive material. In some embodiments, the method further comprises the deposition of a cross-linking reagent on the anode and / or cathode. In one embodiment, the cross-linking reagent deposited on the anode and / or cathode is glutaraldehyde.BRIEF DESCRIPTION OF FIGURES

[0019] Figure 1 shows a representative scheme of an enzyme biofuel cell (created with BioRender.com).

[0020] Figures 2A-2B show two different enzyme fuel cells with a multicopper oxidase enzyme immobilized on the anode and an oxygen reducing cathode (created with BioRender.com). Figure 2A shows an enzyme fuel cell with the anode and cathode in the same environment. Figure 2Bshows an enzyme fuel cell where the anode and cathode are kept in separate environments and connected via a salt bridge.

[0021] Figures 3A-3D shows enzyme fuel cells in a stacked configuration using a multicopper oxidase enzyme anode and oxygen-reducing cathode to create a BioBattery (created with BioRender.com). Figure 3A shows the anode end of the BioBattery with a multicopper oxidase enzyme immobilized by a hydrogel on an electroconductive material. Figure 3B shows the cathode end of the BioBattery with an oxygen-reducing molecule on an electroconductive material. Figure 3C shows exploded and closed views of an individual BioBattery setup with a cathode, anode, and salt bridge. Figure 3D shows a closed view of a stacked BioBattery setup.

[0022] Figure 4A shows the power curve and Figure 4B shows the current density curve of a Pyrobaculum aerophilum multicopper oxidase (PaMCO) anode and an amine reactive phenazine ethosulfate (arPES) cathode, showing the successful generation of power and current transfer.

[0023] Figure 5A shows the power curve and Figure 5B shows the current density curve of a Trametes versicolor laccase anode and arPES cathode, showing the successful generation of power and current transfer.

[0024] Figure 6A shows the power curve and Figure 6B shows the current density curve of a Myrothecium verrucaria bilirubin oxidase (BOD) anode and arPES cathode, showing the successful generation of power and current transfer.

[0025] Figure 7A shows the power curve and Figure 7B shows the current density curve of a Myrothecium verrucaria bilirubin oxidase (BOD) + BSA anode and arPES cathode, showing the successful generation of power and current transfer.

[0026] Figure 8A shows the power curve and Figure 8B shows the current density curve of a Pyrobaculum aerophilum multicopper oxidase (PaMCO) anode and Trametes versicolor laccase cathode, showing the successful generation of power and current transfer. This system shows the successful development of an enzyme fuel cell with a laccase cathode and validates a dual enzyme approach of a laccase type enzyme combined with another enzyme on the anode electrode surface.

[0027] Figure 9A shows the power curve and Figure 9B shows the current density curve of a Pyrobaculum aerophilum multicopper oxidase (PaMCO) anode and Myrothecium verrucaria bilirubin oxidase (BOD) cathode, revealing the successful generation of power and current transfer. This system shows the successful development of an enzyme fuel cell with a multicopper oxidase anode and BOD cathode.

[0028] Figures 10A-10B show characterization of BioBatteries with a Pyrobaculum aerophilum multicopper oxidase (PaMCO) anode and arPES cathode for single cell, double cell, and triple cell setups. Figure 10A shows open circuit potential (OCP) measurements at 500 kQ of BioBatteries in series configurations to optimize power output. Figure 10B shows current output measurements at 8 kQ of BioBatteries in parallel configurations to optimize current output.

[0029] Figures 11A-11H show power density and current density curves for various anode and cathode combinations in the biobattery. Figures 11A and 11B show different multicopper oxidase anodes (laccase, BOD, and PaMCO) while using arPES-BSA as a cathode. Figures 11C and 11D display different cathodes (Laccase, BOD and arPES-BSA) against a constant anode (PaMCO). Figures HE and 11F compare anodes with one, two and three layers of the PaMCO enzyme deposited. Figures 11G and 11H show a single cell compared to two or three cells in a series configuration.

[0030] Figure 12 shows an example of the biobattery using Carbon Toray paper as the base substrate.DETAILED DESCRIPTION

[0031] As described herein, enzyme fuel cells have been developed employing multicopper oxidases immobilized with a fuel source on an anode and various electron acceptors, including a second multicopper oxidase enzyme immobilized on a cathode to reduce oxygen. BioBatteries comprising the fuel cells described herein have advantages over existing enzyme fuel cells, including fuel availability, enhanced performance throughout the operational lifespan, and the ability to modulate power and current output by stacking individual cells in various configurations. The enzyme fuel cells and BioBatteries disclosed herein have useful potential applications as non-toxic, environmentally sustainable batteries for implantable devices and medical devices.

[0032] The presently disclosed subject matter will now be described more fully hereinafter. However, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited tothe specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. In other words, the subject matter described herein covers all alternatives, modifications, and equivalents. In the event that one or more of the incorporated literature, patents, and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in this field. All publications, patent applications, patents and other references mentioned herein are incorporated by reference into their entirety.I. Overview

[0033] Enzyme fuel cells are an attractive subset of fuel cells that utilize enzymes as catalysts to produce electricity by utilizing physiological substrates and other readily available materials to continuously provide power for a variety of applications. Enzyme fuel cells are of particular interest to the field of implantable and medical devices due to their potential to activate and power medical devices without the need for potentially toxic materials. Small, long-lasting batteries capable of consistently generating sufficient electricity to power biomedical devices from enzyme fuel cells composed of biologically compatible materials would provide many advantages over currently available technology.

[0034] However, various issues have prevented the widespread adoption of enzyme fuel cells for practical applications, including lack of stability, biocompatibility, mismatches between output voltage, availability of physiological substrates, and the necessity to provide fuels to the electrode. Like other fuel cells, enzyme fuel cells contain an anode and a cathode to produce electricity. The difference in electric potentials of the oxidation reaction at the anode and the reduction reaction at the cathode generates an electric potential and the transfer of electrons from the anode to the cathode generates an electric current. Both the electric potential and electric current determine the theoretical power output of a fuel cell.

[0035] In reported enzyme fuel cells, the anode contains a sugar oxidoreductase (e.g., glucose oxidase, glucose dehydrogenase, cellobiose dehydrogenase, or fructose dehydrogenase) or metabolite-oxidizing enzymes (e g., lactate oxidase, lactate dehydrogenase, and cholesterol oxidase) which oxidizes its physiological substrate as a fuel and the cathode reduces ambientoxygen to water using multicopper oxidases (e.g., laccases, bilirubin oxidase, and ascorbic acid oxidase) (Figure 1). One issue with this approach is that the physiological substrates (e.g., glucose, lactate, etc.) used as fuel for the anode are necessary in a liquid solution and must by supplied from a biological fluid. These physiological substrates can have variable concentrations, are not always readily available and can be diffusion limited to the surface of the skin. Furthermore, previous enzyme fuel cells have often relied on the reduction of Fe3+to Fe2+, which cause biocompatibility issues due to leaching metal ions. Additionally, mismatches between the output voltage from insufficient voltage differences can limit the function of enzyme fuel cells. The novel enzyme biofuel cell and BioBattery disclosed herein addresses these limitations.

[0036] Disclosed herein, enzyme fuel cells have been developed with multicopper oxidase enzymes as the anode catalyst which oxidize a substrate (e.g., phenol compounds formed by the cross-linking of gluteraldehyde and / or lignin-derived compounds) immobilized on the anode with the enzyme.II. Definitions

[0037] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the present application and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. The terminology used in the description of the invention herein is for the purpose of describing particular aspects only and is not intended to be limiting of the invention. In case of a conflict in terminology, the present specification is controlling.

[0038] As used herein, the term “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0039] The term “or” refers to any one member of a particular list.

[0040] Unless otherwise apparent from the context, the term “about” encompasses values within a standard margin of error of measurement (e.g., SEM) of a stated value or variations ± 0.5%, 1%, 5%, or 10% from a specified value.

[0041] The singular forms of the articles “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a protein” or “at least one protein” can include a plurality of proteins, including mixtures thereof.

[0042] As used herein, “about” means within a statistically meaningful range of a value or values such as a stated concentration, length, molecular weight, pH, sequence identity, time frame, temperature or volume. Such a value or range can be within an order of magnitude, typically within 20%, more typically within 10%, and even more typically within 5% of a given value or range. The allowable variation encompassed by “about” will depend upon the particular system under study, and can be readily appreciated by one of skill in the art.

[0043] Compositions or methods “comprising” or “including” one or more recited elements may include other elements not specifically recited. For example, a composition that “comprises” or “includes” a protein may contain the protein alone or in combination with other ingredients.

[0044] As used herein, the term “physiological conditions” refers to the range of conditions of temperature, pH, and tonicity (or osmolality) normally encountered within tissues in the body of a living human.

[0045] Other definitions are provided below.III. CompositionsEnzymes

[0046] Provided herein, oxidoreductase enzymes catalyze the oxidization of a substrate at the anode of an enzyme fuel cell. In embodiments, anode oxidoreductases are multicopper oxidases. Multicopper oxidases (MCOs) belong to a protein superfamily of enzymes which catalyze the four-electron reduction of oxygen to water by the oxidation of various substrates. They catalyze the one-electron oxidation of a broad range of compounds including substituted phenols, arylamines and aromatic thiols to the corresponding radicals. MCOs contain at least four copper atoms, which are classified as type 1 (Tl), type 2 (T2) or type 3 (T3). The oxidation of a substrate starts at a Tl mononuclear copper center, then electrons are transferred internally to the trinuclear copper center T2 / T3 where oxygen (02) is reduced by four electrons, yielding two water molecules. MCOs are capable of direct electron transfer to an electrode when they are immobilized on the electrode. MCOs including laccase, bilirubin oxidase, tyrosinase, and ascorbic acid oxidasehave been used as the catalyst for the reduction of oxygen at the cathode of enzyme fuel cells but have not been used as anode catalysts.

[0047] Disclosed herein, isolated oxidoreductase enzymes are used to catalyze the oxidation of an immobilized substrate at the anode of an enzyme fuel cell. Previously, multicopper oxidase enzymes have not been used to catalyze the anode reaction of enzyme fuel cells due to intrinsic oxidase activity (e.g., transferring electrons to oxygen) which competes with the desired transfer of electrons to the anode to generate current. Without wishing to be bound by theory, it is believed that the process of immobilizing the substrate with the oxidoreductase enzyme on the electrode promotes dehydrogenase activity over oxidase activity (e.g., electrons are transferred to the electrode instead of oxygen), even for enzymes which would otherwise have oxidase activity. In embodiments, the anode catalyst belongs to a family of multicopper oxidase enzymes. In some embodiments, the multicopper oxidase catalyzing the oxidation of a substrate at the anode is a multicopper oxidase derived from Pyrobaculum aerophilum, a laccase, a bilirubin oxidase, tyrosinase, or an ascorbic acid oxidase. In embodiments, the enzyme catalyzing the oxidation of an immobilized substrate at the anode is a wild-type multicopper oxidase or a mutant multicopper oxidase. In some embodiments, the mutant multicopper oxidase harbors one or more mutations to improve catalytic activity towards a substrate (e.g., Phe290Ile (SEQ ID NO: 1), Phe290Ala, Phe290Leu, or Phe290Val mutations of multicopper oxidase derived from Pyrobaculum aerophilum).

[0048] As used herein, “isolated,” with respect to a polypeptide (and also a polynucleotide), means a molecule (e.g., polypeptide, protein or polynucleotide) isolated from its natural environment or prepared using synthetic methods such as those known to one of skill in the art. Complete purification is not required in either case. The molecules described herein can be isolated and purified from normally associated material in conventional ways, such that in the purified preparation the molecule is the predominant species in the preparation. At the very least, the degree of purification is such that extraneous material in the preparation does not interfere with use of the molecule in the manner disclosed herein. The molecule is at least about 85% pure; alternatively, at least about 90% pure, alternatively, at least about 95% pure; and alternatively, at least about 99% pure.

[0049] As used herein, the term “oxidoreductase” refers to any member of a class of enzymes that catalyst the removal of hydrogen atoms and electrons from a substrate.Oxidoreductases can act as oxidases, which use oxygen as an electron acceptor, or act as dehydrogenases, which use an electron acceptor other than oxygen (e.g., NAD+or FAD).

[0050] As used herein, “oxidase activity” refers to the ability of an enzyme to catalyze the removal of hydrogen and electrons from a substrate and transfer electrons to oxygen (e.g., reducing oxygen to water). The oxidase activity may be assayed using 2,2'-azino-bis(3- ethylbenzothiazoline-6-sulphonic acid) (ABTS) as a substrate by monitoring the increase in absorbance at 420nm based on the formation of reduced ABTS, and / or by any method known in the art.

[0051] As used herein, “dehydrogenase activity” or “Dh activity” refers to the ability of an enzyme to catalyze the removal of hydrogen and electrons from a substrate and transfer electrons to an electron acceptor other than oxygen (e.g., an anode). It should be known that both oxidases and dehydrogenases oxidize their substrates, but different in the identity of the electron acceptor. For an enzyme fuel cell, it is desirable for an anode catalyst to have enhanced dehydrogenase activity over oxidase activity, as disclosed herein by the immobilization of anode enzymes with a substrate (e.g., crosslinked glutaraldehyde) to improve the transfer of electrons to the anode over oxygen. However, oxidase activity is beneficial for the cathode reaction, wherein electrons are transferred to oxygen as the electron acceptor.

[0052] The term “wild-type” refers to entities having a structure and / or activity as found in a normal (as contrasted with mutant, diseased, altered, or so forth) state or context. Wild-type genes and polypeptides often exist in multiple different forms (e.g., alleles). A “wild-type amino acid residue” at a given position refers to the amino acid present at a given position in a wild-type polypeptide.

[0053] As used herein, “mutant,” when used in connection with a polypeptide or protein such as an enzyme, means a variant containing a substitution in one or more of the amino acid residues on the polypeptide or protein at the indicated position(s). Mutant also is used for a polynucleotide encoding such a mutant polypeptide or protein.

[0054] As used herein, “a position corresponding to” means the position of an amino acid residue in a query amino acid sequence that is aligned with the amino acid residue in a reference amino acid sequence using software such as AlignX of Vector NTI with default parameters (available from Invitrogen; see, Lu & Moriyama (2004) Brief Bioinform. 5:378-88). Thus, “amino acid (AA) residue at a position corresponding to the position Y of the amino acid sequence setforth in SEQ ID NO: X” means the AA residue in a query amino acid sequence that is aligned with AA Y of SEQ ID NO: X when the query amino acid sequence is aligned with SEQ ID NO: X using AlignX of Vector NTI with default parameters. It should be noted that the AA Y of SEQ ID NO: X itself is also encompassed by this term.

[0055] As used herein, “catalytic activity” refers to the ability of an enzyme to catalyze the oxidation of a substrate immobilized on an electrode. The “oxidation of a substrate” may be used interchangeably with “dehydrogenation.”

[0056] In some embodiments, the enzyme catalyzing the oxidation of an immobilized substrate at the anode is a multi copper oxidase derived from Pyrobaculum aer ophilum. In one embodiment, the enzyme catalyzing the oxidation of an immobilized substrate at the anode is a wild-type multicopper oxidase derived from Pyrobaculum aerophilum comprising the amino acid sequence set forth in SEQ ID NO: 5.

[0057] Wild-type multicopper oxidase from Pyrobaculum aerophilumMTGEVKRPETSAPVPPLIKEATYIEATASGYMAEGVLNPTIILRRGQRVDMTLKN KLTEPTIVHWHGFDVNWHNDAHPSFAITPGESYNYSFDVVNRAGTYLYHPHPHG LTAKQFYMGQLGLVIVEDSGSDLGFKYGVNDLPLVISDRRFIGGAPVYNPTPME MIAGFLGNAVLVNGVKDAVFKLSGGSYRLRLVNGSNARLYMLSIVKKNGDVVP MRLIAVDQGFLARPIEVRALFLAPAERAEVVVELGEGVYLLKNTPFDPMHLEMG HGMQEALPEGSEYTIATFLVEGKGEAVPVEALSDPPPEPPKPTRTRRFALSLSGM QWTINGMFWNASNPLFEHVSVEGVELWEIVNDKASMPHPMHLHGFPMWIIERK DSPRQVAELAVDNRGRLPTDLGLKDTVLIWPGETVKIVVNFDAKKRGQLFPFHC HNLEHEDGGMMINIAVK (SEQ ID NO: 5)

[0058] In some embodiments, the enzyme catalyzing the oxidation of an immobilized substrate at the anode is a multicopper oxidase derived from Pyrobaculum aerophilum harboring one or more mutations to improve catalytic activity. In one embodiment, the mutation to improve catalytic activity is Phe290Ile, which corresponds to position 262 of the wild-type amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the multicopper oxidase derived from Pyrobaculum aerophilum harboring a Phe290Ile (or F290I) mutation to improve catalytic activity comprises the amino acid sequence set forth in SEQ ID NO: 1.

[0059] Multicopper oxidase from Pyrobaculum aerophilum with Phe290IleMTGEVKRPETSAPVPPLIKEATYIEATASGYMAEGVLNPTIILRRGQRVDMTLKN KLTEPTIVHWHGFDVNWHNDAHPSFAITPGESYNYSFDVVNRAGTYLYHPHPHG LTAKQFYMGQLGLVIVEDSGSDLGFKYGVNDLPLVISDRRFIGGAPVYNPTPME MIAGFLGNAVLVNGVKDAVFKLSGGSYRLRLVNGSNARLYMLSIVKKNGDVVP MRLIAVDQGFLARPIEVRALFLAPAERAEVVVELGEGVYLLKNTPIDPMHLEMG HGMQEALPEGSEYTIATFLVEGKGEAVPVEALSDPPPEPPKPTRTRRFALSLSGM QWTINGMFWNASNPLFEHVSVEGVELWEIVNDKASMPHPMHLHGFPMWIIERK DSPRQVAELAVDNRGRLPTDLGLKDTVLIWPGETVKIVVNFDAKKRGQLFPFHC HNLEHEDGGMMINIAVK (SEQ ID NO: 1)

[0060] In some embodiments, the enzyme catalyzing the oxidation of an immobilized substrate at the anode is a laccase (EC 1.10.3.2). In some embodiments, the laccase is derived from Trametes versicolor. In one embodiment, the Trametes versicolor laccase comprises the amino acid sequence set forth in SEQ ID NO: 2.

[0061] Laccase from Trametes versicolorHWHGFFQAGTNWADGPAFVNQCPIASGHSFLYDFHVPDQAGTFWYHSHLSTQY CDGLRGPFVVYDPKDPHASRYDVDNESTVITLTDWYHTAARLGPRFPLGADATL INGLGRSASTPTAALAVINVQHGKRYRFRLVSISCDPNYTFSIDGHNLTVIEVDGI NSQPLLVDSIQIFAAQRYSFVLNANQTVGNYWVRANPNFGTVGFAGGINSAILRY RGAPVAEPTTTQTTSVIPLIETNLHPLARMPVPGSPTPGGVDKALNLAFNFNGTNF FINNATFTPPTVPVLLQILSGAQTAQDLLPAGSVYPLPAHSTIEITLPATALAPGAP HPFHLHGHAFAVVRSAGSTTYNYNDPIFRDVVSTGTPAAGDNVTIRFQTDNPGP WFLHCHIDFHL (SEQ ID NO: 2)

[0062] In some embodiments, the enzyme catalyzing the oxidation of an immobilized substrate at the anode is a bilirubin oxidase (BOD) (EC 1.3.3.5). In some embodiments, the bilirubin oxidase is derived from Myrothecium verrucaria. In one embodiment, the Myrothecium verrucaria bilirubin oxidase comprises the amino acid sequence set forth in SEQ ID NO: 3.

[0063] Bilirubin oxidase from Myrothecium verrucariaMFKHTLGAAALSLLFNSNAVQASPVPETSPATGHLFKRVAQISPQYPMFTVPLPIP PVKQPRLTVTNPVNGQEIWYYEVEIKPFTHQVYPDLGSADLVGYDGMSPGPTFQ VPRGVETVVRFINNAEAPNSVHLHGSFSRAAFDGWAEDITEPGSFKDYYYPNRQ S ARTLW YHDHAMHIT AENA YRGQ AGL YMLTDP AED ALNLP SGYGEFDIPMILT SKQYTANGNLVTTNGELNSFWGDVIHVNGQPWPFKNVEPRKYRFRFLDAAVSRS FGLYFADTDAIDTRLPFKVIASDSGLLEHPADTSLLYISMAERYEWFDFSDYAG KTIELRNLGGSIGGIGTDTD YDNTDKVMRF VVADDTTQPDT S VVP ANLRD VPFP S PTTNTPRQFRFGRTGPTWTINGVAFADVQNRLLANVPVGTVERWELINAGNGWT HPIHIHLVDFKVISRTSGNNARTVMPYESGLKDVVWLGRRETVVVEAHYAPFPG VYMFHCHNLIHEDHDMMAAFNATVLPDYGYNATVFVDPMEELWQARPYELG EFQAQSGQFSVQAVTERIQTMAEYRPYAAADE (SEQ ID NO: 3)

[0064] In some embodiments, the enzyme catalyzing the oxidation of an immobilized substrate at the anode is an ascorbic acid oxidase (EC 1.10.3.3).

[0065] In some embodiments, the cathode of an enzyme fuel cell provided herein comprises an oxidase enzyme to catalyze the reduction of oxygen. In some embodiments the enzyme of the cathode comprises a multicopper oxidase, such as a multicopper oxidase derived from Pyrobaculum aerophilum, a laccase, a bilirubin oxidase, or an ascorbic acid oxidase. It should be known that when the anode comprises a multicopper oxidase derived from Pyrobaculum aerophilum, the cathode may comprise another enzyme in the family of multicopper oxidases, but the enzyme of the cathode must be different from the multicopper oxidase derived from Pyrobaculum aerophilum.

[0066] In some embodiments, the enzyme catalyzing the reduction of oxygen at the cathode is bilirubin oxidase derived from Myrothecium verrucaria. In one embodiment, the Myrothecium verrucaria bilirubin oxidase comprises the amino acid sequence set forth in SEQ ID NO: 3.

[0067] In some embodiments, the enzyme catalyzing the reduction of oxygen at the cathode is laccase derived from Aspergillus sp. In one embodiment, the Aspergillus sp. is Aspergillus niger and the laccase comprises the amino acid sequence set forth in SEQ ID NO: 4.

[0068] Laccase from Aspergillus nigerNTPWQGYDINTNYYETIPQTNVVREYWFDIVNTTAALDGVERPVLLVNGQFPGP TIEANWGDTVKVHVTNRMENNGTAIHFHGIRQLYNNQMDGVAALTQCPVPPNS SYTYVWRAEEYGSSWYHSHFSLQAWEGVFGGILIHGPSTAEYDHDLGMVFLND WSHQTVDEMYQSVLESQNPPHFQTGLINGSNIWVTADNQTVGRRFQTEFVPGQR YRLRLVNAAMHTHFRFSIDNHDLTVIASDFVPIVPFTTNNVPIGMGQRYDIIVTAN Q APDNYWIRAIPQ SFC SDNANSDNIKGVLHYEGA ADNSDPT STKWD YGDDIQCLDFSLDELVPWLALDADIGGAQMAESDVDFTPFGDVPLYLWTMGGNALNISWKD PTLQQTFEDPDKMDWKASQGVIEAAIPNKWTVLVVQTDLPVPHPIHLHGHDFYL LAQGFGQFNPQNVTLKTHNPPRRDTALMTAATPENGGGGYMVIGFPADNPGVW LIHCHIGFHATEGF AQQI VERQ SEFNTFF SEDLLENTCD AWDEYAKVNP YGHQ YR ALAGPYESGI (SEQ ID NO: 4)

[0069] In embodiments, the isolated enzyme of the anode and / or cathode may be purified by any of the known purification techniques including, but not limited to, ion exchange column chromatography, affinity chromatography, liquid chromatography, filtration, ultrafiltration, salt precipitation, solvent precipitation, immunoprecipitation, gel electrophoresis, isoelectric electrophoresis and dialysis.

[0070] It should be understood that the numbering of the amino acid sequence for the enzymes herein begins at an initial Met and that the enzymes may or may not have the signal peptide. Examples of amino acid sequences for the fuel cell enzymes include, those having at least 90% sequence identity to any one of SEQ ID NOs: 1-5. In some embodiments, the enzyme of the anode and / or cathode has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to any one of SEQ ID NOs: 1-5. In some embodiments, the enzyme of the anode and / or the cathode comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-5.

[0071] “Sequence identity” or “identity” in the context of two polynucleotides or polypeptide sequences refers to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity.” Means for making this adjustment are well known to those of skill in the art. Typically, this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and anon-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC / GENE (Intelligenetics, Mountain View, California).

[0072] “Percentage of sequence identity” refers to the value determined by comparing two optimally aligned sequences (greatest number of perfectly matched residues) over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. Unless otherwise specified (e.g., the shorter sequence includes a linked heterologous sequence), the comparison window is the full length of the shorter of the two sequences being compared.

[0073] Unless otherwise stated, sequence identity / similarity values refer to the value obtained using GAP Version 10 using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof. “Equivalent program” includes any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.

[0074] The term “conservative amino acid substitution” refers to the substitution of an amino acid that is normally present in the sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine, or leucine for another non-polar residue. Likewise, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, or between glycine and serine. Additionally, the substitution of a basic residue such as lysine, arginine, or histidine for another, or the substitution of one acidic residue such as asparticacid or glutamic acid for another acidic residue are additional examples of conservative substitutions. Examples of nonconservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, or methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and / or a polar residue for a non-polar residue. Typical amino acid categorizations are summarized below.Alanine Ala A Nonpolar Neutral 1.8Arginine Arg R Polar Positive -4.5Asparagine Asn N Polar Neutral -3.5Aspartic acid Asp D Polar Negative -3.5Cysteine Cys C Nonpolar Neutral 2.5Glutamic acid Glu E Polar Negative -3.5Glutamine Gin Q Polar Neutral -3.5Glycine Gly G Nonpolar Neutral -0.4Histidine His H Polar Positive -3.2Isoleucine He I Nonpolar Neutral 4.5Leucine Leu L Nonpolar Neutral 3.8Lysine Lys K Polar Positive -3.9Methionine Met M Nonpolar Neutral 1.9Phenylalanine Phe F Nonpolar Neutral 2.8Proline Pro P Nonpolar Neutral -1.6Serine Ser S Polar Neutral -0.8Threonine Thr T Polar Neutral -0.7Tryptophan Trp W Nonpolar Neutral -0.9Tyrosine Tyr Y Polar Neutral -1.3Valine Vai V Nonpolar Neutral 4.2Fuel Sources

[0075] One challenge with existing enzyme fuel cells which utilize physiological substrates in solution is the availability of the enzyme’s substrate to power the fuel cell. Provided herein are enzyme fuel cells utilizing an oxidoreductase enzyme to catalyze the oxidation of an immobilized substrate at the anode. The immobilized substrate at the anode is the fuel source for the enzyme fuel cell and the oxidation of the substrate allows for electrons to be transferred to the anode. The enzyme fuel cells disclosed herein are provided with a fuel source which is consistently available at the point of the reaction, allowing for the enzyme fuel cell to remain at maximum power throughout the duration of its operational lifespan. The design of an enzyme fuel cell withan enzyme immobilized with its substrate on the anode as disclosed herein provides practical advantages over previous enzyme fuel cells which rely on the availability of a substrate in solution.

[0076] The term “substrate” refers to a molecule which is oxidized during the enzymatic reaction of the anode. The term “physiological substrate” refers to a biological molecule such as a sugar or metabolite (e.g., glucose, fructose, lactate, cholesterol, etc.) which is oxidized during an enzymatic reaction.

[0077] The term “immobilized” refers to a compound which is attached to a surface (e.g., electrode). Immobilization can be performed through adsorption, cross-linking, covalent bonding and / or affinity-tag binding.

[0078] The term “cross-linking” refers to a polymerization reaction of a cross-linking reagent. A “cross-linking reagent” refers to a molecule that contains two or more reactive ends capable of forming covalent bonds.

[0079] In some embodiments, the fuel source immobilized on the electrode is formed by depositing a cross-linking reagent. In some embodiments, the cross-linking reagent comprises aldehyde functional groups. In some embodiments, the cross-linking reagent is glutaraldehyde. Glutaraldehyde may react with basic amino acid residues (e.g., lysine) of a protein or enzyme (e.g., multicopper oxidase and / or BSA deposited on an electrode). There is no general scientific agreement on the reactive species of glutaraldehyde, which can form many variable intermediates and products during the cross-linking process, which are not well characterized. Without wishing to be bound by theory, it is believed that the cross-linking of glutaraldehyde forms phenolic compounds, which can be oxidized as a fuel source by multicopper oxidase enzymes to power enzyme fuel cells.

[0080] Multicopper oxidases disclosed herein as anode catalysts for enzyme fuel cells can oxidize various substrates, such as phenolic compounds. In embodiments, the fuel source immobilized on the anode is a phenolic compound.

[0081] The term “phenolic compound” refers to a molecule comprising phenol (hydroxybenzene). A phenolic compound can be a polymer formed from the cross-linking of glutaraldehyde. Phenolic compounds can be large molecules with additional functional groups and can include multiple phenol moieties, such as lignin.

[0082] In some embodiments, the phenolic fuel source is derived from lignin. It may be desired with respect to enzyme fuel cells to use a sustainable and non-toxic fuel source. Lignin, aclass of phenolic polymers naturally produced by a variety of plants and biomass, is a plentiful, renewable, and non-hazardous material, making it a desirable fuel source for an enzyme fuel cell.Electron Acceptor of the Cathode

[0083] Disclosed herein, enzyme fuel cells comprise an electron acceptor on the cathode to reduce oxygen. In some embodiments, the electron acceptor comprises an oxidase enzyme disclosed herein. In embodiments, electron acceptors can be prepared by conjugating an electron acceptor molecule to a protein or enzyme. In some embodiments, the conjugation can be performed with an amine reactive moiety (e.g., NHS ester).

[0084] In other embodiments the electron acceptor is a molecule other than an enzyme. In some embodiments, the electron acceptor is a phenazine compound.

[0085] The term “phenazine compound” refers to a molecule comprising a 9,10- di azaanthracene moiety. A phenazine compound may be a salt.

[0086] In some embodiments, the phenazine compound comprises phenazine ethosulfate (PES) or phenazine methosulfate (PMS). In some embodiments the phenazine compound is aminereactive.

[0087] The term “amine-reactive” refers to a chemical group which will react with a primary amine moiety (e.g., lysine residue). Amine-reactive chemical groups include N- hydroxysuccinimide esters (NHS ester) and imidoesters.

[0088] In some embodiments the amine-reactive phenazine compound is N- hydroxysuccinimidylester l-propoxy-5-ethylphenazinium ethyl sulfate (arPES).

[0089] In other embodiments, the electron acceptor comprises a metal complex. In some embodiments the metal complex is an osmium complex or a ruthenium complex.

[0090] In some embodiments, the electron acceptor is immobilized on a protein. In some embodiments the protein is bovine serum albumin (BSA), glucose dehydrogenase (GDH), lactate dehydrogenase (LDH), glucose oxidase (GOx), and / or lactate oxidase (LOx).IV. DevicesEnzyme Fuel Cells

[0091] Disclosed herein are enzyme fuel cells utilizing an oxidoreductase enzyme and a substrate (e.g., fuel source) immobilized on an anode, providing a constant source of fuel andpower. The enzyme catalyzes the oxidation of a substrate (e.g., dehydrogenation of phenol), transferring electrons to the anode, while the cathode reduces oxygen. The cathode is in a solution (e.g. aqueous) with dissolved oxygen. Ambient oxygen from a physiological environment is sufficient to catalyze the cathode half reaction. See Figure 2A-2B.

[0092] In embodiments, an anode oxidoreductase catalyst belongs to a family of multicopper oxidase enzymes including but not limited to a multicopper oxidase derived from Pyrobaculum aerophilum, a laccase, a bilirubin oxidase, or an ascorbic acid oxidase provided herein.

[0093] The cathode and anode of the enzyme fuel cell provided herein comprise an electroconductive material. In some embodiments, the electroconductive material comprises carbon paper, glassy carbon, carbon nanotubes, gold, and / or palladium. In some embodiments, the cathode and / or anode comprises conductive ink.

[0094] The term “electroconductive material” refers to a substance capable of transmitting electricity.

[0095] The oxidoreductase catalyzing the anode reaction, the electron acceptor of the cathode (e.g., phenazine compound, metal complex or enzyme), and the substrate (e.g., fuel source) are immobilized on the electrodes. The immobilization may be performed by any method known in the art (e.g., cross-linking with glutaraldehyde). In some embodiments, the substrate (e g., fuel source) may be formed through the cross-linking process (e.g., phenolic compounds formed by the cross-linking of glutaraldehyde). In some embodiments, another substrate may be provided in addition to the cross-linking reagent (e.g., lignin-derived materials).

[0096] In some embodiments, another protein may be immobilized on the anode in addition to the oxidoreductase enzyme (e.g., BSA). In some embodiments, a protein may be immobilized on the cathode in addition to the electron acceptor (e.g., phenazine compound, metal complex, or oxidase enzyme). In some embodiments, the enzyme, substrate, electron acceptor, and / or protein of the anode and / or cathode may be immobilized in a hydrogel.

[0097] In embodiments, the anode and the cathode are in a solution, which may contain electrolytes. In some embodiments, the anode and cathode are in the same environment (Figure 2A). The solution containing the cathode comprises dissolved oxygen, which may be ambient oxygen from physiological conditions. In other embodiments the anode and cathode are in separateenvironments and are connected by a salt bridge (Figure 2B). The salt bridge can be composed of any material known in the art.BioBattery

[0098] A BioBattery is provided comprising any of the enzyme fuel cells disclosed herein. The BioBattery comprises an enzyme fuel cell provided herein for the consistent generation of power from an oxidoreductase enzyme catalyzing the oxidation of a substrate immobilized on an anode while ambient oxygen is reduced at a cathode. See Figures 3A-3D. As shown in Figure 3A, the anode may comprise any of the oxidoreductase enzymes, fuel sources, and electroconductive materials disclosed herein immobilized on an electrode. The cathode may comprise any of the electron acceptors immobilized on an electrode as disclosed herein (e.g., phenazine compounds, metal complexes, or oxidase enzymes) to reduce oxygen (Figure 3B).

[0099] As shown in Figure 3C, the BioBattery disclosed herein comprises an anode and a cathode of said enzyme fuel cell enclosed in a container. In some embodiments, the BioBattery may comprise a salt bridge between the anode and the cathode. The BioBattery may be contained by any material known in the art. In some embodiments the BioBattery comprises a biodegradable material. A BioBattery comprising a biodegradable material disclosed herein has the advantage of environmental sustainability and reduced toxicity compared to existing batteries.

[0100] The term “biodegradable material” refers to a substance which can be decomposed by microorganisms through composting or by normal environmental processes.

[0101] It may be desired with respect to a battery to modulate power and current output for powering biomedical devices. The design of the BioBattery disclosed herein allows for modification of the number of enzyme fuel cells and the configuration connecting multiple fuel cells to adjust power and current output based on the needs of the biomedical device.

[0102] In some embodiments the BioBattery comprises one enzyme fuel cell disclosed herein. In other embodiments, the BioBattery comprises more than one enzyme fuel cell disclosed herein. As seen in Figure 3D, multiple enzyme fuel cells can be stacked to increase the current output. In some embodiments, multiple enzyme fuel cells are connected in series configurations. In other embodiments, multiple enzyme fuel cells are connected in parallel configurations. In embodiments, batteries comprising multiple stacked enzyme fuel cells can be used to power biomedical devices.

[0103] The disclosed subject matter is further described in the following non-limiting Examples. It should be understood that these Examples, while indicating preferred embodiments of the subject matter, are given by way of illustration only.EXAMPLESExample 1: Enzyme fuel cells with multicopper oxidase anodes and a phenazine ethosulfate cathode

[0104] Various multicopper oxidase enzymes were tested as anode catalysts, immobilized with glutaraldehyde as the fuel source on the anode. For the cathode, phenazine ethosulfate was used as the electron acceptor to reduce oxygen. The phenazine ethosulfate was prepared by reacting N-hydroxysuccinimidylester l-propoxy-5-ethylphenazinium ethyl sulfate, an aminereactive phenazine ethosulfate (arPES) with bovine serum albumin (BSA), forming the conjugate arPES-BSA. The N-hydroxysuccinimidyl ester (NHS) moiety reacts directly with a primary amine (e.g., a lysine residue of BSA) and allows for a rapid one-step conjugation to a protein.

[0105] Anodes and cathodes were prepared by first cleaning Toray Carbon Paper electrodes by spraying with deionized water. Next, Ketjenblack Ink was prepared by mixing Ketjenblack powder with triton and deionized water, and then dispersed via ultrasonication.

[0106] For the cathode, arPES was conjugated to BSA via incubated centrifugation. Afterwards, arPES-BSA, potassium phosphate buffer (PPB), and Ketjenblack ink were mixed and spotted onto the Toray Carbon Paper electrode. The electrode was then dried and cross-linked by vapor deposition of glutaraldehyde. Afterwards, the electrode was dipped in Tris-HCl and stored in PPB until use.

[0107] Electrodes were tested in an electrochemical cell containing 100 mM PPB. Both electrodes were placed in the electrochemical cell, connected to a potentiostat, and the open circuit potential was measured as different resistance was applied.

[0108] First, multicopper oxidase derived from Pyrobaculum aerophilum (PaMCO) (SEQ ID NO: 1) was tested as an anode catalyst. To prepare the anode, a mixture of Ketjenblack ink, the PaMCO enzyme (SEQ ID NO: 1), and PPB was prepared and spotted onto the Toray Carbon Paper electrode. In one optimized protocol, 4 pL of Ketjenblack was used with 20 pl of enzyme (e.g., a 1 :5 ratio of Ketenjblack ink to enzyme) and 36 pl of PPB. Ratios of Ketenjblack ink to enzyme of 1 : 1 to 1 :20 were tested. The electrode was dried and cross-linked by vapor deposition ofglutaraldehyde. Afterwards, the electrode was dipped in Tris-HCl and stored in PPB until use. The resulting power output is shown in Figure 4A and the current density is shown in Figure 4B.

[0109] Next, laccase from Trametes versicolor was tested as an anode catalyst (SEQ ID NO: 2). A mixture of Ketjenblack ink, laccase from Trametes versicolor (SEQ ID NO: 2), and PPB was prepared and spotted onto the Toray Carbon Paper electrode. The electrode was then dried and cross-linked by vapor deposition of glutaraldehyde. Afterwards, the electrode was dipped in Tris-HCl and then stored in PPB until use. The resulting power output is shown in Figure 5A and the current density is shown in Figure 5B.

[0110] Then, bilirubin oxidase (BOD) from Myrothecium verrucaria (SEQ ID NO: 3) was tested as an anode catalyst. A mixture of Ketjenblack ink, bilirubin oxidase from Myrothecium verrucaria (SEQ ID NO: 3), and PPB was prepared and spotted onto the Toray Carbon Paper electrode. The electrode is then dried and cross-linked by vapor deposition of glutaraldehyde. Afterwards, the electrode was dipped in Tris-HCl and then stored in PPB until use. The resulting power output is shown in Figure 6A and the current density is shown in Figure 6B.

[0111] Additionally, an anode was prepared with bilirubin oxidase from Myrothecium verrucaria immobilized with BSA. A mixture of Ketjenblack ink, bilirubin oxidase from Myrothecium verrucaria (SEQ ID NO: 3) combined with BSA, and PPB was prepared and spotted onto the Toray Carbon Paper electrode. The electrode is then dried and cross-linked by vapor deposition of glutaraldehyde. Afterwards, the electrode was dipped in Tris-HCl and then stored in PPB until use. The resulting power output is shown in Figure 7A and the current density is shown in Figure 7B.

[0112] The results indicated that fuel cells utilizing multicopper oxidase derived from Pyrobaculum aerophilum (PaMCO), laccase, and bilirubin oxidase as anode catalysts all successfully generated power and transferred current. The various multicopper oxidase enzymes tested catalyzed the oxidation of glutaraldehyde as a fuel source immobilized on the anode with a phenazine sulfate electron donor to reduce oxygen on the cathode, giving working enzyme fuel cells. The fuel cells with bilirubin oxidase as the anode enzyme provided more power than the fuel cells with PaMCO and laccase as the anode enzymes. Furthermore, the immobilization of bilirubin oxidase with BSA on the anode improved the power output of the fuel cell.Example 2: Dual enzyme fuel cells with multicopper oxidase anodes and cathodes

[0113] Instead of using a phenazine sulfate compound as the electron acceptor on the cathode, the development of a dual enzyme fuel cell with a second multicopper oxidase reducing oxygen at the cathode was explored. Enzyme fuel cells utilizing a multicopper oxidase enzyme immobilized with glutaraldehyde as the fuel source on the anode were tested with a second different enzyme to reduce oxygen at the cathode. Multicopper oxidase derived from Pyrobaculum aerophilum was used as the anode catalyst with laccase and bilirubin oxidase, which were both tested as cathode catalysts.

[0114] Anodes and cathodes were prepared by first cleaning Toray Carbon Paper electrodes by spraying with deionized water. Next, Ketjenblack Ink was prepared by mixing Ketjenblack powder with triton and deionized water, and then dispersed via ultrasonication.

[0115] To prepare the anode, a mixture of Ketjenblack ink, the PaMCO enzyme (SEQ ID NO: 1), and PPB was prepared and spotted onto the Toray Carbon Paper electrode, as shown in Figure 12. The electrode was dried and cross-linked by vapor deposition of glutaraldehyde. Afterwards, the electrode was dipped in Tris-HCl and stored in PPB until use.

[0116] Electrodes were tested in an electrochemical cell containing 100 mM PPB. Both electrodes were placed in the electrochemical cell, connected to a potentiostat, and the open circuit potential was measured as different resistance was applied.

[0117] For one dual enzyme fuel cell, laccase from Aspergillus sp (SEQ ID NO: 4) was tested as the cathode catalyst. To fabricate the cathode, 40.1 pL of laccase from Trametes versicolor (SEQ ID NO: 2), 13.3 pL of PPB, and 6.7 pL of Ketjenblack ink were mixed and spotted onto the Toray Carbon Paper electrode. The electrode was then dried and cross-linked by vapor deposition of glutaraldehyde. Afterwards, the electrode was dipped in Tris-HCl and then stored in PPB until use. The resulting power output is shown in Figure 8A and the current density is shown in Figure 8B, indicating the successful generation of power and current transfer. This system shows the successful development of an enzyme fuel cell with a laccase cathode and validates the use of a dual enzyme approach of a laccase type enzyme combined with another enzyme on the anode electrode surface.

[0118] For another dual enzyme fuel cell, bilirubin oxidase from Myrothecium verrucaria (SEQ ID NO: 3) was tested as the cathode enzyme. For the cathode, bilirubin oxidase from Myrothecium verrucaria, PPB, and Ketjenblack ink were mixed and spotted onto the Toray Carbon Paper electrode. The electrode was then dried and cross-linked by vapor deposition ofglutaraldehyde. Afterwards, the electrode was dipped in Tris-HCl and then stored in PPB until use. The resulting power output is shown in Figure 9A and the current density is shown in Figure 9B, indicating the successful generation of power and current transfer. This system shows the successful development of a dual enzyme fuel cell with a bilirubin oxidase cathode.

[0119] A summary of the maximum power of the various enzyme fuel cell configurations tested is provided in Table 1.Table 1: Summary of EBFC configurations as well as their associated max powerExample 3: Enzyme fuel cells designed for stacking and connection in series or parallel configurations

[0120] In order to meet system requirements, multiple enzyme fuel cells can be stacked and connected to adjust current and power output. Batteries were fabricated with multiple fuel cells connected in series or in parallel.

[0121] The anode and cathode were prepared by cleaning gold disk electrodes (GDEs). GDEs were polished using 0.3 and 0.05 pm aluminum powder followed by soaking in piranha (3 : 1 sulfuric acid to hydrogen peroxide) for 1 hour, then each electrode was electrochemically cleaned using cyclic voltammetry in a solution of 50 mM KOH, sweeping between 0 V-1.2 V. Next, Ketjenblack Ink was prepared by mixing Ketjenblack powder with triton and DI water, and then dispersed via ultrasonication.

[0122] To prepare cathodes, arPES was conjugated to BSA via incubated centrifugation. Afterwards, arPES-BSA, PPB, and Ketjenblack ink were mixed and spotted onto the GDE surface.The electrode was then dried and cross-linked by vapor deposition of glutaraldehyde. Afterwards, the electrode was dipped in Tris-HCl and stored in PPB until use.

[0123] To prepare anodes, a mixture of Ketjenblack ink, multicopper oxidase derived from Pyrobaculum aerophilum (SEQ ID NO: 1), and PPB was prepared and spotted onto the GDE surface. The electrode was dried and cross-linked by vapor deposition of glutaraldehyde. Afterwards, the electrode was dipped in Tris-HCl and stored in PPB until use.

[0124] Each anode and cathode pair represent an individual enzyme fuel cell. A battery was prepared with a single enzyme fuel cell and batteries were prepared with two or three enzyme fuel cells connected to provide stacked double or triple enzyme fuel cells. First, batteries were tested with the enzyme fuel cells in a series configuration. As shown in Figure 10A, the open circuit potential measurements increased with the added enzyme fuel cells when tested at 500 kQ. Batteries were also tested with enzyme fuel cells connected in a parallel configuration. Figure 10B shows the current output increased with the added enzyme fuel cells when tested at 8 kQ. These results show that enzyme biofuel cells can be stacked and connected with multiple cells in various configurations and tailored for specific power and current requirements. Thus, these enzymes have great potential for powering implantable and medical devices with non-toxic materials in a sustainable manner.

[0125] Additionally, various enzyme fuel cells were designed using both multiple anode layers via drop casting, as well as stacking the cells in series. The results shown in Figures 11A-11H demonstrate increased power and current density. Figures 11A-11B display the power and current density, respectively, of the standalone systems, alternating various anodes and cathodes. These combinations include multicopper oxidase derived from Pyrobaculum aerophilum (SEQ ID NO: 1), Myrothecium verrucaria bilirubin oxidase (SEQ ID NO: 3), and Trametes versicolor laccase (SEQ ID NO: 2) as anodes evaluated against arPES modified BSA as the cathode all immobilized onto Toray paper. Figures 11C-11D show the power and current density, respectively, of the arPES modified BSA, Myrothecium verrucaria bilirubin oxidase (SEQ ID NO: 3), and Trametes versicolor laccase (SEQ ID NO: 2) as cathodes against multicopper oxidase derived from Pyrobaculum aerophilum (SEQ ID NO: 1) as the anode. In addition, Figures 11E-11H show the increased power and current density of the biobattery from both increasing the number of layers on the anode (Figures 11E-11F, respectively), or by stacking the biobatteries in series (Figures 11G-11H, respectively).

[0126] Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which the inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Claims

CLAIMSWhat is claimed is:

1. An anode comprising an electroconductive material, wherein an oxidoreductase enzyme is immobilized on the anode and a substrate immobilized on the anode, wherein the substrate can be oxidized by the enzyme.

2. The anode of claim 1, wherein the oxidoreductase enzyme is a multicopper oxidase.

3. The anode of claim 2, wherein the multicopper oxidase is selected from the group consisting of laccase, bilirubin oxidase, tyrosinase, ascorbic acid oxidase, and multicopper oxidase derived from Pyrobaculum aerophilum.

4. The anode of claim 3, wherein the laccase is derived from Trametes versicolor or Aspergillus sp.

5. The anode of claim 3, wherein the bilirubin oxidase is derived from Myrothecium verrucaria.

6. The anode of any one of claims 1-5, wherein the substrate is formed by depositing a cross-linking reagent.

7. The anode of claim 6, wherein the cross-linking reagent is glutaraldehyde.

8. The anode of any one of claims 1-7, wherein the substrate is a phenolic compound.

9. The anode of claim 8, wherein the phenolic compound is derived from lignin.

10. The anode of any one of claims 1-9, wherein the electroconductive material is selected from the group consisting of carbon paper, glassy carbon, carbon nanotubes, gold, and palladium.

11. A fuel cell comprising the anode of any one of claims 1-10 and a cathode, wherein the cathode comprises an electroconductive material and an electron acceptor to reduce oxygen,wherein the anode and the cathode are in a solution, wherein the cathode is contact with dissolved oxygen.

12. The fuel cell of claim 11, wherein the electron acceptor of the cathode comprises a phenazine compound.

13. The fuel cell of claim 12, wherein the phenazine compound comprises phenazine ethosulfate (PES) or phenazine methosulfate (PMS).

14. The fuel cell of claim 12 or 13, wherein the phenazine compound is amine-reactive.

15. The fuel cell of claim 14, wherein the amine-reactive phenazine compound is N- hydroxysuccinimidylester l-propoxy-5-ethylphenazinium ethyl sulfate (arPES).

16. The fuel cell of claims 11, wherein the electron acceptor of the cathode comprises a metal complex.

17. The fuel cell of claim 16, wherein the metal complex is an osmium complex or a ruthenium complex.

18. The fuel cell of any one of claims 11-17, wherein the electron acceptor of the cathode is immobilized on a protein.

19. The fuel cell of claim 18, wherein the protein is selected from the group consisting of bovine serum albumin (BSA), glucose dehydrogenase (GDH), lactate dehydrogenase (LDH), glucose oxidase (GOx), and lactate oxidase (LOx).

20. The fuel cell of claim 11, wherein the electron acceptor of the cathode is an oxidase enzyme.

21. The fuel cell of claim 20, wherein the oxidase enzyme of the cathode is a multicopper oxidase.

22. The fuel cell of claim 21, wherein the multicopper oxidase of the cathode is selected from the group laccase, bilirubin oxidase, tyrosinase, ascorbic acid oxidase, and multicopper oxidase derived from Pyrobaculum aerophilum.

23. The fuel cell of claim 22, wherein the multicopper oxidase of the cathode is laccase derived from Trametes versicolor or Aspergillus sp.

24. The fuel cell of claim 22, wherein the multicopper oxidase of the cathode is bilirubin oxidase derived from Myrothecium verrucaria.

25. The fuel cell of any one of claims 1-24, wherein the anode and / or the cathode comprise an enzyme with an amino acid sequence set forth in any one SEQ ID NOs: 1-4.

26. The fuel cell of any one of claims 11-25, wherein the electroconductive material of the cathode is selected from the group consisting of carbon paper, glassy carbon, carbon nanotubes, gold, and palladium.

27. The fuel cell of any one of claims 11-26, wherein the solution of the anode and the solution of the cathode are in the same environment.

28. The fuel cell of any one of claims 11-27, wherein the solution of the anode and the solution of the cathode are in separate environments and connected by a salt bridge.

29. A battery comprising the fuel cell of any one of claims 11-28 and a container.

30. The battery of claim 29, comprising more than one fuel cell.

31. The battery of claim 30, wherein the fuel cells are in a series configuration.

32. The battery of claim 30, wherein the fuel cells are in a parallel configuration.

33. The battery of any one of claims 30-32, wherein the fuel cells are stacked.

34. The battery of any one of claims 29-32, comprising a biodegradable material.

35. A method of making the fuel cell of any one of claims 11-28, said method comprising:(a) preparing the anode by depositing the enzyme and the substrate on an electrode comprising an electroconductive material; and(b) preparing the cathode by depositing the electron acceptor on an electrode comprising an electroconductive material.

36. The method of claim 35, comprising the deposition of a cross-linking reagent on the anode and / or cathode.

37. The method of claim 36, wherein the cross-linking reagent deposited on the anode and / or cathode is glutaraldehyde.

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