Heat-resistant glucose dehydrogenase and application thereof in enzyme fuel cell

Abstract

The invention discloses heat-resistant glucose dehydrogenase and application thereof in an enzyme fuel cell, and belongs to the crossing field of bioelectrochemistry and new energy technologies. According to the invention, firstly, a heat-resistant glucose dehydrogenase TeGDH is screened, and the heat-resistant glucose dehydrogenase TeGDH is subjected to heterologous expression in escherichia coli BL21 (DE3) and shows good heat stability and catalytic activity; teGDH is dissolved in an anolyte containing an electron mediator anthraquinone-2, 6-disulfonic acid disodium salt, and the heat-resistant high-energy enzyme fuel cell is prepared by adopting a non-immobilized system; the maximum power density of the battery at 50 DEG C reaches 1.74 mW / cm, and the battery has good thermal stability and long-term operation performance; the invention combines big data mining, enzyme engineering and electrochemical technologies, provides a novel enzyme fuel cell solution with high activity, high stability and low cost, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to a heat-resistant glucose dehydrogenase and its application in enzyme fuel cells, particularly its application in enzyme fuel cells, which belongs to the interdisciplinary field of bioelectrochemistry and new energy technology. Background Technology

[0002] Biofuel cells are a special type of fuel cell that uses organic matter as fuel and directly or indirectly utilizes enzymes as catalysts to generate electricity. They possess characteristics not found in conventional fuel cells and can be divided into enzyme fuel cells (EBFCs) that directly use enzymes and microbial fuel cells that indirectly utilize enzymes within living organisms. Enzyme fuel cells offer more advantages and greater potential for various applications. Unlike microbial fuel cells that use whole-cell microorganisms as biocatalysts, enzyme fuel cells do not have cell membranes to restrict mass transfer and can achieve high enzyme loads without the dilution effect of other biomolecules, potentially resulting in higher power output. Enzyme fuel cells use biological enzymes as catalysts, which are low-cost, renewable, and biodegradable. Through the catalysis of the substrate, they produce water and degradable byproducts. Low cost and wide availability are also major advantages of enzyme fuel cells. They can use common small molecules such as formic acid, methanol, ethanol, and glucose, as well as other biomass-related sugars, fats, and starches as fuels, making enzyme fuel cells an economically viable energy solution. Glucose is the most widely used fuel in enzyme fuel cells. In addition, enzyme fuel cells operate under mild conditions, can be carried out at room temperature and pressure, have good biocompatibility, and the reaction solutions are mostly close to neutral. The conditions are simple and will not cause damage to the battery system. This provides the prerequisite for implanting enzyme fuel cells into living organisms, enabling the use of oxygen and glucose in the blood to generate electricity as a power source for devices such as pacemakers.

[0003] Currently, glucose oxidase is one of the most commonly used enzymes in enzyme fuel cells, catalyzing the oxidation of glucose to gluconic acid, and is widely used in glucose-based enzyme fuel cells. However, it also has significant drawbacks. For example, glucose oxidase requires oxygen to catalyze the oxidation of glucose, and in low-oxygen or anaerobic environments (such as implantable devices, closed systems, and high altitudes), the reaction rate drops sharply, leading to unstable battery output. Glucose oxidase requires a two-step reaction (first oxidizing glucose, then reducing O2 to H2O2), resulting in a longer electron transfer path and relatively lower efficiency. The generated H2O2 is corrosive, damaging the enzyme itself (leading to inactivation), destroying electrode materials (such as carbon nanotubes and conductive polymers), and is toxic to biological tissues, making it unsuitable for long-term implantation. In contrast, glucose dehydrogenase has significant advantages over glucose oxidase. Glucose dehydrogenase does not require oxygen as an electron acceptor, making it highly adaptable to low-oxygen or anaerobic environments (such as implantable devices, high altitudes, and closed systems), and suitable for implantable devices. It can directly dehydrogenate glucose to gluconolactone and rapidly transfer electrons through coenzymes, resulting in a higher reaction rate, and it does not generate H2O2. Glucose dehydrogenase is gradually becoming a replacement for glucose oxidase, showing broad application prospects in the field of enzyme fuel cells.

[0004] However, in recent years, enzyme fuel cells have faced challenges in improving power output and stability. High activity and stability of the enzymes used are crucial for achieving high power and stability in enzyme fuel cells. The reaction system of an enzyme fuel cell also determines its overall performance. Enzyme immobilization by embedding enzymes in polymer matrices or inorganic frameworks on electrode surfaces is an effective method of enzyme modification. However, this often results in the masking or denaturation of enzyme active sites, significantly reducing actual activity. Furthermore, substrates and products must diffuse within the solid phase, causing severe mass transfer resistance and limiting power density. Moreover, the cell fabrication process is complex, involving electrode modification, cross-linking, and film formation, each step of which can potentially deactivate the enzyme. Therefore, developing a heat-resistant glucose dehydrogenase suitable for high-energy batteries is of significant guiding importance for the industrial application of high-energy, high-power, and high-stability enzyme fuel cells. Summary of the Invention

[0005] In order to solve some technical problems existing in the prior art, the present invention provides a glucose dehydrogenase and its application in biofuel cells, especially its application in heat-resistant high-energy enzyme fuel cells.

[0006] To achieve the above-mentioned technical objectives, the present invention provides the following technical solution: The present invention first provides a thermostable glucose dehydrogenase, wherein the amino acid sequence of the glucose dehydrogenase is the sequence shown in SEQ ID No: 1, or an amino acid sequence that has at least 90% identity with the sequence shown in SEQ ID No: 1 and has glucose dehydrogenase activity.

[0007] The sequence of SEQ ID NO:1 is shown below: MAPLCQTASSSYDYIVVGGGTSGLVVANRLSENPNVSVLVIEAGDSVYNNANVTNVNGYGLAFGTPIDWQYQSTNQTYAGNTRQTLRAGKALGGTSTINGMAYTRAQDVQIDAWAAIGNDGWDWSSLWPYYLKSEAFTAPNQTQ RAAGASYNPAYHGVTGPLHVGFIEMQPNNLSSILNQTYQALGVPWTEDVNGGKMRGYNFFPSTVDDAADVREDAARAYYYPFESRPNLRVMLNTLANRIVWKNETSGGNVTADGVEVTPLNGTVCRIQANNEVILSAGSLRSPGI LELSGVGNPSILNKYNIPVKVNLPTVGENMQDQMYNDASAEGYSAIAGTKSVAYPSVTDLFGNRTSAVAASVQNQLAQYAEAAANQSQGTMKASDLQRLFQIQYDLIFKQEVPIAEIITYPTGNTLAAGYWGLLPFARGSVHIAS ADPTVQPVINPNYFMFDWDVQQQIGTAKFIRNLYKTAPLSSLVKNETEPGSAVPEGASDSVWEAWLKETYRSNFHPVGTAAIMPRSIGGVVDERLRVYGTANVRVVDASVLPFQICGHLTSTLYAVAERASDFLKEDAARLGNLE Furthermore, the glucose dehydrogenase has a purification tag fused to its N-terminus or C-terminus. Preferably, the purification tag includes a 6×his tag, and the amino acid sequence of the glucose dehydrogenase fused with the purification tag is shown in SEQ ID No: 4.

[0008] The present invention also provides a polynucleotide sequence encoding the thermostable glucose dehydrogenase, the polynucleotide sequence comprising the sequence shown in SEQ ID No: 2 or 5.

[0009] The present invention also provides an enzyme fuel cell anolyte, wherein the anolyte contains the heat-resistant glucose dehydrogenase.

[0010] Furthermore, the anolyte also contains the electron mediator anthraquinone-2,6-disulfonic acid disodium salt, HEPES buffer, and Mg. 2+ Mn 2+ Na + One or more metal ion sources.

[0011] Preferably, the concentration of anthraquinone-2,6-disulfonic acid disodium salt in the anolyte is 5 mM, the concentration of hydroxyethylpiperazine ethanesulfonic acid in the HEPES buffer is 100 mM and the pH value is 7.4, and the metal ion source includes magnesium chloride, manganese chloride and sodium chloride, wherein the concentration of magnesium chloride is 10 mM, the concentration of manganese chloride is 0.5 mM and the concentration of sodium chloride is 100 mM.

[0012] More preferably, the thermostable glucose dehydrogenase activity in the anolyte is 20 U (the amount of 2,6-dichlorophenolindophenol sodium salt reduced every 10 minutes at pH 7.4 and 30°C is defined as one unit of glucose dehydrogenase activity, U).

[0013] The present invention also provides the application of the glucose dehydrogenase or the anolyte in the preparation of enzyme fuel cells.

[0014] The present invention also provides an enzyme fuel cell, the enzyme fuel cell comprising an anode chamber, a cathode chamber and a membrane disposed therebetween, the anode chamber containing the anolyte, wherein glucose dehydrogenase and electron mediators in the anolyte are in a non-immobilized state.

[0015] Furthermore, the anode chamber of the enzyme fuel cell is provided with an anode electrode, which is a carbon-based electrode loaded with 2,3-dichloronaphthoquinone and multi-walled carbon nanotubes; the cathode chamber is provided with a cathode electrode and contains a cathode electrolyte, which is a carbon felt and the cathode electrolyte is a PBS buffer containing potassium ferricyanide.

[0016] The anode electrode is a carbon paper substrate with a multi-walled carbon nanotube layer loaded on it, and 2,3-dichloronaphthoquinone is loaded on the multi-walled carbon nanotube layer. The anode electrode is prepared by: dropping a multi-walled carbon nanotube solution onto carbon paper and drying it to obtain multi-walled carbon nanotube-modified carbon paper; then dropping a 2,3-dichloronaphthoquinone solution onto the multi-walled carbon nanotube-modified carbon paper and drying it to obtain the anode electrode of the battery. Preferably, the concentration of the multi-walled carbon nanotube solution is 5 mg / mL, and the solvent in the solution is dimethylformamide; the concentration of the 2,3-dichloronaphthoquinone solution is 10 mM, and the solvent is ethanol.

[0017] The cathode electrolyte contains 1 M potassium ferricyanide, 0.05 M potassium chloride, 0.05 M disodium hydrogen phosphate, and 0.015 M potassium dihydrogen phosphate.

[0018] The non-immobilized system described in this invention refers to a system in which both the enzyme and the electron mediator are dissolved or dispersed in the anolyte and are not fixed to the electrode surface by means of covalent bonds, physical adsorption, or embedding.

[0019] The fuel cell reaction system provided by this invention preferably occupies 5 mL of space. The anode and cathode chambers are cylindrical plexiglass chambers with a net volume of 7 mL, tightly assembled by four bolts. A Nafion 117 membrane is used to separate the anode and cathode chambers. The anode and cathode are connected to the outside of the chambers using titanium wires for testing.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: In the field of biomanufacturing, the discovery and use of thermostable enzymes from thermophilic microorganisms is of great significance for improving enzyme stability and reducing enzyme production costs. This is because it simplifies the enzyme purification process, extends the enzyme's shelf life, enhances the ability of enzyme fuel cells to operate under a wider range of conditions, and also improves enzyme stability. This invention, through big data mining, obtained a thermostable glucose dehydrogenase, named TeGDH, and has found it to be well-suited for use in enzyme fuel cells.

[0021] (1) Based on the provided heat-resistant glucose dehydrogenase, the present invention constructs a heat-resistant high-energy enzyme fuel cell using a non-immobilized system. The enzyme fuel cell constructed in the present invention directly adds the enzyme to the electrolyte with anthraquinone-2,6-disulfonic acid disodium salt as an electron mediator to release enzyme activity. By leaving the enzyme and electron mediator in the solution to diffuse freely, only a thin layer of mediator or conductive nanostructure is modified on the electrode surface, allowing electrons to efficiently transfer between the enzyme-mediator-electrode, instead of binding the enzyme itself to the electrode. This maintains the enzyme activity and ensures mass transfer efficiency, releasing the natural catalytic potential of the enzyme. It combines the stability of the heat-resistant enzyme with the high efficiency of the non-immobilized system, significantly breaking through the performance bottleneck of traditional immobilization technology.

[0022] (2) The glucose dehydrogenase obtained through big data mining provided by this invention is used in the preparation of a heat-resistant high-energy sugar fuel cell. The resulting fuel cell exhibits excellent heat resistance. Experimental verification shows that the heat-resistant glucose dehydrogenase (TeGDH) provided by this invention retains enzyme activity within the range of 20-70℃, and its activity exceeds 70 U / mL at 50℃. The enzyme-based fuel cell preparation method of this invention also has the following advantages: It has abundant raw materials: common glucose can be used as battery fuel, which is low in cost, widely available and easy to obtain.

[0023] Mild operating conditions: The prepared enzyme biofuel cells can be carried out at room temperature and pressure. The reaction solutions are mostly close to neutral, and the conditions are simple and will not cause damage to the battery system. This also provides the prerequisite for implanting EBFCs into living organisms.

[0024] It has good biocompatibility: enzyme biofuel cells can be implanted into the human body to generate electricity using the oxygen and glucose inherent in human blood, which can then be used as a power source for devices such as pacemakers.

[0025] Green and environmentally friendly: Traditional fuel cells mostly use precious metals such as Pt / C as catalysts, while enzyme biofuel cells use biological enzymes as catalysts. Through the catalysis of the substrate, water and degradable products are produced, which is non-toxic, green and sustainable.

[0026] Furthermore, the enzyme used in this invention to prepare the enzyme fuel cell has high output power, with a maximum power density of up to 1.74 mW / cm³. 2 The battery exhibits good stability and can operate stably at a high temperature of 70℃. The prepared enzyme fuel cell system is simple to construct and has a relatively low cost, providing a feasible path for the next generation of high-energy, high-power, and high-stability enzyme fuel cells. Attached Figure Description

[0027] Figure 1 This is the phylogenetic tree constructed in Example 1, where the glucose dehydrogenase (TeGDH) of the present invention is shown in the red box.

[0028] Figure 2 This is a comparison of enzyme activity before and after purification in Example 2.

[0029] Figure 3 This invention presents the results of a comparison of the activities of glucose dehydrogenase (TeGDH) with other different types of glucose dehydrogenases based on big data mining.

[0030] Figure 4 The cyclic voltammetry curves of the enzyme fuel cell constructed based on the glucose dehydrogenase (TeGDH) of the present invention at different temperatures are shown in Example 3.

[0031] Figure 5 The results represent the optimized reaction conditions for the enzyme fuel cell constructed based on the glucose dehydrogenase (TeGDH) of this invention in Example 3; wherein, Figure 5 a represents the maximum power density variation of the enzyme fuel cell constructed in this invention at different temperatures. Figure 5 b represents the change in maximum power density of the enzyme fuel cell constructed in this invention under conditions of adding different concentrations of glucose.

[0032] Figure 6 The maximum power density (solid line) and polarization (dashed line) curves of the enzyme fuel cell (non-immobilized enzyme fuel cell) constructed based on the glucose dehydrogenase (TeGDH) of the present invention in Example 3 are shown.

[0033] Figure 7 The results show the thermal stability test of the enzyme fuel cell constructed based on the glucose dehydrogenase (TeGDH) of the present invention in Example 3.

[0034] Figure 8The maximum power density (solid line) and polarization (dashed line) curves of the immobilized enzyme fuel cell constructed based on the glucose dehydrogenase (TeGDH) of this invention are shown. Detailed Implementation

[0035] The technical solutions of the present invention will be further described below through specific embodiments. It should be understood that these embodiments are preferred solutions of the present invention and are intended to aid in understanding the technical content of the present invention, rather than limiting the scope of protection of the present invention. Without departing from the basic concept of the present invention, various substitutions, equivalent improvements, or combinations can be made by those skilled in the art, and all such substitutions, equivalent improvements, or combinations should be considered to fall within the scope of protection of the present invention.

[0036] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0037] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0038] Example 1: Gene mining and phylogenetic analysis For those derived from thermophilic anaerobic bacteria Thermoanaerobacter ethanolicus The draft genome of strain JW200 (GenBank assembly accession number: GCA_000023565.1) was annotated de novo. Gene prediction and preliminary functional annotation were performed using NCBI's Prokaryotic Genome Annotation Process (PGAP). To further obtain functional clues at the level of conserved protein domains, all protein sequences predicted by PGAP were submitted to InterProScan for comprehensive analysis. This analysis integrated models from multiple professional databases such as Pfam, SMART, CDD, and PROSITE, aiming to comprehensively identify conserved domains, functional sites, and protein family characteristics contained in the sequences. The analysis results clearly showed that the N-terminus of the proposed glucose dehydrogenase (denoted as A0A1E1GL61, TeGDH) contains a typical Rossmann fold structure (corresponding to the NAD(P)+ binding domain), while the C-terminus contains a highly conserved catalytic domain of the short-chain dehydrogenase / reductase (SDR) family, with its core catalytic tetrad being "Ser-Tyr-Lys-Asn". This crucial discovery not only structurally confirms that the protein belongs to the short-chain dehydrogenase superfamily, but also provides direct and important structural biology evidence for subsequent speculation on its catalytic function (such as glucose dehydrogenase activity).

[0039] Based on the above analysis, an open reading frame encoding a short-chain dehydrogenase was successfully identified, as shown in SEQ ID No: 3: MAPLCQTASSSYDYIVVGGGTSGLVVANRLSENPNVSVLVIEAGDSVYNNANVTNVNGYGLAFGTPIDWQYQSTNQTYAGNTRQTLRAGKALGGTSTINGMAYTRAQDVQIDAWAAIGNDGWDWSSLWPYYLKSEAFTAPNQTQ RAAGASYNPAYHGVTGPLHVGFIEMQPNNLSSILNQTYQALGVPWTEDVNGGKMRGYNFFPSTVDDAADVREDAARAYYYPFESRPNLRVMLNTLANRIVWKNETSGGNVTADGVEVTPLNGTVCRIQANNEVILSAGSLRSPG ILELSGVGNPSILNKYNIPVKVNLPTVGENMQDQMYNDASAEGYSAIAGTKSVAYPSVTDLFGNRTSAVAASVQNQLAQYAEAAANQSQGTMKASDLQRLFQIQYDLIFKQEVPIAEIITYPTGNTLAAGYWGLLPFARGSVHI ASADPTVQPVINPNYFMFDWDVQQQIGTAKFIRNLYKTAPLSSLVKNETEPGSAVPEGASDSVWEAWLKETYRSNFHPVGTAAIMPRSIGGVVDERLRVYGTANVRVVDASVLPFQICGHLTSTLYAVAERASDFLKEDAARLGN The estimated amino acid sequence was subjected to a BLASTP homology search in the UniProt database (https: / / www.uniprot.org / , select UniProtKB database), with an E-value threshold of ≤1×10⁻⁶. -10 The results showed that the sequence was similar to that known to originate from Bacillus megaterium. Bacillus megaterium The sequence similarity of the glucose dehydrogenase (UniProt accession number: P67848) was 78%, and the key catalytic tetrad (Ser-152, Tyr-167, Lys-171, Asn-191) and the hypothesized substrate-binding residues (such as Arg-244, Asp-256) were completely conserved in the sequence alignment. This finding strongly supports its functional hypothesis as a glucose dehydrogenase from both the perspectives of sequence homology and conservation of key functional sites, hence it was named TeGDH.

[0040] To clarify the evolutionary position of TeGDH, a phylogenetic analysis was further performed. The 79 highly homologous sequences obtained from the BLASTP search (covering different genera and with E-values ​​<1×10⁻⁶) were analyzed. -50 The TeGDH sequence, along with the data, was imported into the MEGA X software platform (version 11.0.13; https: / / www.megasoftware.net / ). Multiple sequence alignment was performed using the software's built-in ClustalW program. Default parameters were used to ensure the accuracy of alignment for key catalytic and coenzyme binding sites. Based on the alignment results, a phylogenetic tree was constructed using Neighbor-Joining (NJ), with a Poisson model used to correct for distances. Missing data were handled using pairwise deletion. To assess the reliability of the phylogenetic tree nodes, the Bootstrap resampling value was set to 1000 times.

[0041] The constructed phylogenetic tree clearly divides all sequences into five main evolutionary branches (Group 1-Group 5) based on sequence similarity. Phylogenetic localization shows that TeGDH is robustly clustered in Group 2, where other members are glucose oxidoreductases with flavin adenine dinucleotide (FAD) as a coenzyme. Furthermore, the bootstrap support for this branch is 100, indicating a high degree of confidence in the clustering results and suggesting that TeGDH and other members in Group 2 may have originated from a recent common ancestor. The phylogenetic tree was visualized and annotated using the built-in graphical tools in MEGA X software, as shown below. Figure 1 As shown, in this embodiment, the phylogenetic tree is visualized using the built-in graphical tools of MEGA X, with each branch name and Bootstrap value labeled, and a high-resolution vector graphic is output for result display. Multiple sequence alignment is performed using the built-in ClustalW program. Based on the alignment results, a phylogenetic tree is constructed using Neighbor-Joining (NJ). To assess the reliability of the topology, the Bootstrap repeat sampling value is set to 1000.

[0042] The amino acid sequence of the thermostable glucose dehydrogenase screened in this invention is shown in SEQ ID No: 1.

[0043] The nucleotide sequence encoding the thermostable glucose dehydrogenase is shown in SEQ ID No: 2:

[0044] The amino acid sequence of the thermostable glucose dehydrogenase with a 6×His tag is shown in SEQ ID No: 4: .

[0045] The nucleotide sequence encoding the thermostable glucose dehydrogenase shown in SEQ ID No: 4 is shown in SEQ ID No: 5:

[0046] Example 2: TeGDH Heterologous Expression and Purification

[0047] (1) Heterologous expression of TeGDH: The nucleotide sequence of TeGDH was synthesized by a biotechnology company (Sangon Biotech (Shanghai) Co., Ltd.). pET29b was used as the expression vector, and Escherichia coli BL21(DE3) was used as the host. Both the expression vector and Escherichia coli were purchased from Sangon Biotech (Shanghai) Co., Ltd. LB medium (containing 10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride) was used as the culture medium.

[0048] First, TeGDH-containing colonies were inoculated into 50 mL of LB medium, and 50 µg / mL kanamycin was added. The mixture was then incubated overnight at 37°C and 180 rpm. The incubated medium was then transferred to a 1 L Erlenmeyer flask containing 500 mL of LB medium, 50 µg / mL kanamycin, 1 mM MgCl2, 1 mM CaCl2, and 10 mM glucose (filtered and sterilized). The colonies were further incubated at 37°C and 180 rpm for approximately 7 hours, followed by induction with 0.3 mM IPTG. Incubation was continued for 20 hours with agitation at room temperature to maintain aerobic conditions. Next, the bacterial culture was aliquoted into 100 mL centrifuge tubes, centrifuged, and the supernatant was removed. 50 mL of Tris-HCl buffer (50 mM, pH 7.4) was added to each tube, and the mixture was centrifuged again to remove the supernatant. This process was repeated three times to remove the LB medium. The resulting precipitate was the bacterial sludge, which could be frozen at 80°C for further use. TeGDH was extracted using an ultrasonic disruption method. Specifically, TeGDH-containing cells in a cryotube were resuspended in 20 mL of Tris-HCl buffer and disrupted by sonication (30% amplitude, 15 seconds on, 30 seconds off, for a total of 11 minutes). Cell debris was removed by centrifugation (10,000 rpm, 30 minutes, 4°C), and the supernatant was then collected to obtain the crude TeGDH enzyme solution.

[0049] (2) Purification of crude TeGDH enzyme solution: TeGDH contains a 6×His tag during synthesis, where the histidine residues can bind with immobilized Ni. 2+ Coordinate bonds are formed, allowing the enzyme to be specifically adsorbed onto a nickel column using a Ni-NTA column. The enzyme is then competitively eluted using imidazole, and finally, a desalting column is used to remove the imidazole, yielding high-purity TeGDH. First, the Ni-NTA column (1 mL column volume) was washed with ultrapure water to remove ethanol. Then, the column was equilibrated with binding buffer (containing 20 mM imidazole, 1.2 M NaCl, 80 mM Tris-HCl, pH 7.4). Next, 10 mL of crude TeGDH enzyme solution was loaded and held for 10 min. Then, the column was washed sequentially with 10 mL of binding buffer, followed by 5 mL of washing buffer (containing 240 mM imidazole, 2 M NaCl, 80 mM Tris-HCl, pH 7.4). Finally, the protein was eluted with 3 mL of elute buffer (containing 1 M imidazole, 1 M NaCl, 40 mM Tris-HCl, pH 7.4) to obtain imidazole-containing TeGDH. Next, the desalting column was filled with binding buffer and allowed to completely enter the column bed. This process was repeated four times to equilibrate the column. Then, the imidazole-containing TeGDH was added to the desalting column, eluted with 3 mL of elution buffer, and collected to obtain high-purity TeGDH.

[0050] (3) TeGDH enzyme activity assay: TeGDH enzyme activity was determined using the sodium 2,6-dichlorophenolindophenol (DCIP) method. 3 mL of the reaction mixture contained 50 mM Tris-HCl buffer (pH 7.4), 0.14 mM methyl phenazine sulfate, 0.07 mM sodium 2,6-dichlorophenolindophenol, and 300 mM glucose. The purified TeGDH enzyme (0.1 mL) was added to initiate the enzymatic reaction, which was carried out at a constant temperature in a water bath. The absorbance of the reactants at 600 nm was monitored using a UV-Vis spectrophotometer. One unit of dehydrogenase activity (U) was defined as the amount of DCIP reduced by TeGDH every 10 minutes at pH 7.4 and 30°C. Comparison of enzyme activity before and after purification is shown below. Figure 2 As shown in the figure, the activity of purified TeGDH was increased in the range of 20-70℃, with the most significant purification effect at 50℃. Before purification, the enzyme activity was only about 16 U / mL, and after purification, it increased by 4 times to about 70 U / mL.

[0051] (4) Comparison of enzyme activities of different types of glucose dehydrogenase To further examine the activity advantages of the enzymes screened in this invention, this embodiment compares the enzymes screened in this invention with the activities of other glucose dehydrogenases. Based on the phylogenetic tree in Example 1, different types of glucose dehydrogenases from the ends of branches in Group 1, Group 2, Group 3, Group 4, and Group 5 were selected, including glucose dehydrogenases numbered P13006, A0A1E1GL61 (i.e., TeGDH), M5C0X7, A0A135UZR9, M5C6E8, R7SZD5, A0A7Y1Z2A0, and A0A485IEJ1 (the aforementioned numbers are the accession numbers of each enzyme in the UniProt database, https: / / www.uniprot.org / , selecting the UniProtKB database), for enzyme expression, purification, and activity determination (referring to the methods described in (1) to (3) of Example 2). After enzyme activity determination and comparison (results are as follows...), enzyme expression, purification, and activity determination were performed. Figure 3 As shown in the figure, enzymes M5C0X7 and R7SZD5 exhibited the highest initial enzyme activity, reaching 19 U / mL and 18 U / mL, respectively, while A0A1E1GL61 (TeGDH) ranked third at 16 U / mL. After heat treatment at 50°C for 5 hours, A0A1E1GL61 (TeGDH) showed the highest enzyme activity of 14 U / mL, which is more than three times the activity of the other enzymes. Therefore, the selection of TeGDH as a heat-resistant glucose dehydrogenase for sugar battery applications in this invention is reasonable.

[0052] Example 3: Construction and application of TeGDH-based enzyme fuel cell (1) Preparation of the anode electrode Carbon paper was soaked in acetone for 8 hours, then transferred to 3M hydrochloric acid solution for 12 hours, and this process was repeated 2-3 times. Finally, it was rinsed with ultrapure water until the pH value reached approximately 7, and then dried in an oven to obtain pretreated carbon paper. Multi-walled carbon nanotubes (MWCNTs) were dissolved in dimethylformamide to prepare a MWCNT (5 mg / mL) solution, and sonicated for 30 minutes to ensure complete dissolution. 2,3-Dichloronaphthoquinone (2,3-dichloronaphthoquinone) was dissolved in ethanol to prepare a 10 mM 2,3-dichloronaphthoquinone solution, and sonicated for 30 minutes to ensure complete dissolution. Then, 1 mL of the MWCNT solution was deposited onto the pretreated carbon paper, and after drying, MWCNT-modified carbon paper was obtained. 1 mL of the 2,3-dichloronaphthoquinone solution was then deposited onto the MWCNT-modified carbon paper, and after drying, a usable anode electrode was obtained.

[0053] Electrochemical characterization of enzyme fuel cell anode material: A three-electrode system was used for cyclic voltammetry (CV) testing, with the anode electrode prepared in step (1) as the experimental electrode, the saturated calomel electrode as the reference electrode, and the platinum wire electrode as the counter electrode. The reaction system was 5 mL. Figure 4Cyclic voltammograms of the battery at different temperatures (30°C to 60°C) are presented. It can be observed that the current response significantly increases with increasing temperature, especially at 50°C, where the current density reaches its highest level and the catalytic performance is optimal. Furthermore, the curves at all temperatures show good reversibility, indicating good compatibility between the electrode material and the enzymatic reaction.

[0054] (2) Construction of enzyme fuel cells The overall output capacity of the enzyme fuel cell was tested using a two-electrode system with a reaction volume of 5 mL. The enzyme-dynamite battery constructed in this invention includes an anode chamber, a cathode chamber, and a membrane disposed between them. The anode and cathode chambers are cylindrical plexiglass chambers with a net volume of 7 mL, tightly assembled with four bolts. A Nafion 117 membrane was used to separate the anode and cathode chambers. Titanium wires were used to connect the anode and cathode outside the chambers for testing.

[0055] The anode chamber contains an anolyte in which glucose dehydrogenase and electron mediators are in a non-immobilized state. The anolyte contains 20 U TeGDH, as well as 5 mM anthraquinone-2,6-disulfonic acid disodium salt, HEPES buffer, and Mg. 2+ Mn 2+ Na + One or more metal ion sources are used. In this embodiment, the concentration of hydroxyethylpiperazine ethanesulfonic acid in the HEPES buffer is 100 mM and the pH value is 7.4. The metal ion source includes magnesium chloride, manganese chloride and sodium chloride, wherein the concentration of magnesium chloride is 10 mM, the concentration of manganese chloride is 0.5 mM and the concentration of sodium chloride is 100 mM.

[0056] The cathode chamber is equipped with a cathode electrode and contains a cathode electrolyte. The cathode electrode is a carbon felt, and the cathode electrolyte is a PBS buffer containing potassium ferricyanide. The cathode electrolyte contains 1 M potassium ferricyanide, 0.05 M potassium chloride, 0.05 M disodium hydrogen phosphate, and 0.015 M potassium dihydrogen phosphate.

[0057] (3) Performance testing and optimization of enzyme fuel cells The constructed enzyme fuel cell, using green, environmentally friendly, readily available, and low-cost glucose as a substrate, has broad application prospects in portable green power sources, implantable medical devices, and other fields. Temperature and substrate concentration are two key parameters affecting the performance of the enzyme fuel cell, directly influencing its power density, stability, and overall efficiency. It is necessary to test the performance of the enzyme fuel cell at different temperatures and substrate concentrations to determine the optimal temperature and concentration for the reaction. In this embodiment, the reaction conditions are optimized to achieve a more efficient and stable enzyme fuel cell.

[0058] like Figure 5 As shown in Figure a, the output power of the enzyme fuel cell was tested using the variable resistance method (a conventional technique). By changing the resistance of an external precision variable resistance box, the steady-state operating voltage across the resistance box was measured when an external resistor of a corresponding value was connected. A series of voltage-current data points were calculated to determine the battery's output power. The battery was placed in a water bath to simulate different operating temperatures. The test results show that the battery's output power is highest at 50℃, and it maintains a high power density even at higher temperatures (70℃), demonstrating excellent thermal stability. This result is consistent with... Figure 4 The results shown in the CV experiment, where the best catalytic performance was observed at 50°C, are consistent with the results, further validating the stability advantage of TeGDH at high temperatures.

[0059] like Figure 5 As shown in b, the effect of adding different concentrations of glucose on the battery output power was tested (using the same method as above). The results show that as the glucose concentration increases, the battery power density gradually increases, but tends to saturate after the glucose concentration reaches 500 mM. This is because at high concentrations, the mass transfer resistance of the reactants increases, limiting further power increases. This result has guiding significance for optimizing glucose concentration in practical applications.

[0060] After optimizing the reaction conditions, it was found that the TeGDH-based enzyme fuel cell exhibits the best performance at a reaction temperature of 50℃ and a substrate concentration of 500 mM. Figure 6 The polarization and power density curves of the enzyme fuel cell under these conditions are shown, with a maximum power density of approximately 1.74 mW / cm². 2 .

[0061] Furthermore, in this embodiment, the thermal stability of the enzyme fuel cell was tested: Figure 7 The long-term operational stability of the constructed enzyme fuel cell was demonstrated at 30℃, 50℃, and 70℃. The results showed that the current density of the enzyme fuel cell at each temperature decreased only slightly with time over 60 hours, indicating good stability. Particularly at 50℃, the current density retention rate remained high after 60 hours, further validating the stability advantage of TeGDH at high temperatures.

[0062] To demonstrate the advantages of using a non-immobilized system in enzyme fuel cells, a comparative experiment was conducted in this embodiment to compare the performance of immobilized enzyme fuel cells with that of TeGDH cells. First, TeGDH bioanodes were prepared by depositing 1 mL of multi-walled carbon nanotube solution (5 mg / mL) onto pretreated carbon paper (using the same method as in step (1)). After drying, multi-walled carbon nanotube-modified carbon paper was obtained. 1 mL of 2,3-dichloronaphthoquinone solution (10 mM) was deposited onto the multi-walled carbon nanotube-modified carbon paper. After drying, 1.5 mL of a mixture containing 50 mM tris(hydroxymethyl)aminomethane-hydrochloric acid buffer (pH 7.4), 20 U TeGDH, and 0.6 mg / mL dopamine was deposited onto the electrode and allowed to dry at room temperature. Next, an immobilized enzyme fuel cell was constructed using a two-electrode system. The reaction volume was 5 mL. The anolyte consisted of 5 mM anthraquinone-2,6-disulfonic acid disodium salt, 100 mM hydroxyethylpiperazine ethanesulfonic acid (pH 7.4), 10 mM magnesium chloride, 0.5 mM manganese chloride, and 100 mM sodium chloride. The catholyte consisted of 1 M potassium ferricyanide, 0.05 M potassium chloride, 0.05 M disodium hydrogen phosphate, and 0.015 M potassium dihydrogen phosphate. The anode and cathode chambers were 7 mL net volume acrylic cylindrical chambers, tightly assembled with four bolts. A Nafion 117 membrane was used to separate the anode and cathode chambers. Titanium wires were used to connect the anode and cathode outside the chambers for testing.

[0063] The performance of the immobilized enzyme fuel cell was tested under optimal conditions (50℃, 500mM glucose), and the results are as follows. Figure 8 As shown, the battery's maximum power density is only 0.046 mW / cm². 2 The output performance is only 1 / 30 of that of immobilized enzyme fuel cells under the same conditions. This shows that the immobilized enzyme fuel cell prepared by the present invention can significantly release the activity of enzymes, thereby improving the output performance of enzyme fuel cells.

[0064] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A thermostable glucose dehydrogenase, characterized in that, The amino acid sequence of the glucose dehydrogenase is the sequence shown in SEQ ID No: 1, or an amino acid sequence that has at least 90% identity with the sequence shown in SEQ ID No: 1 and has glucose dehydrogenase activity.

2. The thermostable glucose dehydrogenase according to claim 1, characterized in that, The glucose dehydrogenase has a purification tag fused to its N-terminus or C-terminus, and the purification tag includes a 6×his tag.

3. A polynucleotide encoding the glucose dehydrogenase of any one of claims 1-2, wherein the polynucleotide sequence comprises the sequence shown in SEQ ID No: 2 or 5.

4. An anode electrolyte for an enzyme fuel cell, wherein the anode electrolyte contains the heat-resistant glucose dehydrogenase as described in claim 1 or 2.

5. The electrolyte according to claim 4, characterized in that, The anolyte also contains the electron mediator anthraquinone-2,6-disulfonic acid disodium salt, HEPES buffer, and Mg. 2+ Mn 2+ Na + One or more metal ion sources.

6. The electrolyte according to any one of claims 4-5, characterized in that, The activity of the heat-resistant glucose dehydrogenase in the anolyte is 20 U. The concentration of anthraquinone-2,6-disulfonic acid disodium salt in the anolyte is 5 mM. The HEPES buffer solution contains 100 mM hydroxyethylpiperazine ethanesulfonic acid at a pH of 7.

4. The metal ion source includes magnesium chloride, manganese chloride, and sodium chloride, wherein the concentration of magnesium chloride is 10 mM, the concentration of manganese chloride is 0.5 mM, and the concentration of sodium chloride is 100 mM.

7. The use of the glucose dehydrogenase according to any one of claims 1-2 or the electrolyte according to any one of claims 4-6 in the preparation of an enzyme fuel cell.

8. An enzyme fuel cell, characterized in that, The enzyme fuel cell includes an anode chamber, a cathode chamber, and a membrane disposed therebetween. The anode chamber contains the anolyte according to any one of claims 5-7, wherein glucose dehydrogenase and electron mediators in the anolyte are in a non-immobilized state.

9. The enzyme fuel cell according to claim 8, characterized in that, The enzyme fuel cell has an anode chamber equipped with an anode electrode, which is a carbon-based electrode loaded with 2,3-dichloronaphthoquinone and multi-walled carbon nanotubes; the cathode chamber is equipped with a cathode electrode and contains a cathode electrolyte, which is a carbon felt and the cathode electrolyte is a PBS buffer containing potassium ferricyanide.

10. The enzyme fuel cell according to claim 9, characterized in that, The anode electrode is based on a carbon paper substrate and loaded with a multi-walled carbon nanotube layer, on which 2,3-dichloronaphthoquinone is loaded. The cathode electrolyte contains 1 M potassium ferricyanide, 0.05 M potassium chloride, 0.05 M disodium hydrogen phosphate, and 0.015 M potassium dihydrogen phosphate.

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