A formate dehydrogenase mutant with improved electron transfer rate and its application

Abstract

The present invention provides a formate dehydrogenase mutant with improved electron transfer rate and its application. The formate dehydrogenase mutant uses the amino acid sequence shown in SEQ ID NO.1 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identity with SEQ ID No.1 as the parent, and has a site-directed saturation mutation corresponding to tyrosine at position 262 of SEQ ID NO.1. By performing site-directed saturation mutation on formate dehydrogenase from Thiobacillus, the present invention finally obtains an Ts FDH mutant with improved electron transfer rate and stable catalytic activity.

Description

Technical Field

[0001] The present invention relates to the technical field of oxidoreductases, and particularly relates to a formate dehydrogenase mutant with improved electron transfer rate and its application. Background Art

[0002] Carbon dioxide enzymatic electroreduction reaction (CO2EER) is an electro-driven CO2 reduction process catalyzed by immobilized or non-immobilized enzymes in the cathode chamber of a bioelectrochemical system. It has mild reaction conditions, a single product with high purity, mainly formate ([ BiotechnoI Adv.2023 , 63 , 108098; ACS Catal.2018 , 8 (5), 4429-4437.). Formate is a stable carbon dioxide reduction intermediate and can be used as a liquid energy carrier in fuel cells, a hydrogen storage material, or a raw material for synthesizing fine chemicals (ACS Catal. 2019, 9 (6), 5584-5589.). Thus, the formate production reaction of CO2EER has broad application potential in the field of carbon fixation.

[0003] Formate dehydrogenase ( Thiobacillus sp. KNK65MA ) derived from Thiobacillus Ts FDH) is an NADH-dependent FDH, which is oxygen-tolerant, easy to express, and has a high yield. However, its CO2-reducing enzyme activity is relatively low. Zhu Zhiguang et al. constructed a hybrid CO2 electroreduction system. In this system, Ts FDH uses polyethylene glycol as a swing arm to couple with coenzyme NADH to form an enzyme-coenzyme complex. Then, cysteine is mutated at the surface fixed-point 263 site of Ts FDH, and the thiol group on cysteine is cross-linked with copper nanoparticles electrodeposited on the electrode surface to achieve the immobilization of the enzyme-coenzyme complex. At the same time, copper nanoparticles can be used as an electrocatalyst for the regeneration of NADH. The formate production in this system is as high as 11.8 M / mU / h, achieving the highest reported at that time (J CO2Util 2019, 34, 568-575.). In 2021, Fonzo et al. first used a nanostructured titanium nitride support to immobilize Ts FDH. The hierarchical TiN nanostructured support exhibits a high surface area and a wide pore size distribution, achieving a high catalytic load, and using neutral red for the regeneration of NADH. In this CO2EER system, the formate production is as high as 44.1 μmol / mg / h (ChemElectroChem 2021, 8(15), 2846-2857.). In the two CO2EER systems, limited by TsThe low enzyme activity of FDH results in a lower turnover rate of electrochemically reduced CO2 compared to FDH with direct electron transfer (DET). Therefore, if an electron transfer pathway from the FDH electron transfer exchange site to the electrode interface can be discovered and a DET system can be constructed, it is expected to increase the CO2 turnover rate of the engineered CO2 EER system. Ts If an electron transfer pathway from the FDH electron transfer exchange site to the electrode interface can be discovered and a DET system can be constructed, it is expected to increase the CO2 turnover rate of the engineered CO2 EER system. Summary of the Invention

[0004] Aiming at the deficiencies in the prior art, the present invention provides a formate dehydrogenase mutant with improved electron transfer rate and its application. By performing site-directed mutagenesis on formate dehydrogenase from Thiobacillus, a TsFDH mutant with improved electron transfer rate and stable catalytic activity is finally obtained.

[0005] On the one hand, the present invention provides a formate dehydrogenase mutant, which uses the amino acid sequence shown in SEQ ID NO.1 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identity with SEQ ID No.1 as the parent, and has a site-directed saturation mutation corresponding to tyrosine at position 262 of SEQ ID NO.1.

[0006] In one embodiment of the present invention, the site-directed saturation mutation includes any one of T262H, T262G, T262R, T262K, T262E, T262D, T262Q, T262N, T262M, T262C, T262S, T262W, T262Y, T262F, T262P, T262I, T262L, T262V, T262A. Preferably, the site-directed mutation is T262I.

[0007] In one embodiment of the present invention, the amino acid sequence of the formate dehydrogenase mutant is as shown in SEQ ID NO:3, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity with that shown in SEQ ID NO:3.

[0008] In one embodiment of the present invention, the formate dehydrogenase mutant has the property of direct electron transfer and can perform more efficient electron transfer on the electrode. In one embodiment of the present invention, the formate dehydrogenase mutant has an improved electron transfer rate.

[0009] In one embodiment of the present invention, the formate dehydrogenase mutant can increase the turnover rate of CO2.

[0010] In one embodiment of the present invention, the formate dehydrogenase mutant has stable enzyme catalytic activity.

[0011] In one embodiment of the present invention, compared with that from Thiobacillus Thiobacillus sp. KNK65MAThe wild-type formate dehydrogenase of (), and the formate dehydrogenase mutant has improved enzyme kinetic performance.

[0012] In one embodiment of the present invention, compared with the wild-type formate dehydrogenase derived from Thiobacillus ( Thiobacillus sp. KNK65MA ), the formate dehydrogenase mutant has increased formic acid production.

[0013] In one embodiment of the present invention, compared with the wild-type formate dehydrogenase derived from Thiobacillus ( Thiobacillus sp. KNK65MA ), the formic acid production of the formate dehydrogenase mutant is increased by at least 20-fold.

[0014] In one embodiment of the present invention, the formic acid production of the formate dehydrogenase mutant can reach 0.025 mM / g / h.

[0015] In the second aspect of the present invention, there is provided a polynucleotide encoding the above-mentioned formate dehydrogenase mutant.

[0016] In the third aspect of the present invention, there is provided a vector containing the above-mentioned polynucleotide.

[0017] In one embodiment of the present invention, the vector can be selected from plasmids, viruses or yeasts. In one embodiment of the present invention, the plasmids include but are not limited to pET series, pACYC series or pGEX series. Preferably, the plasmid is selected from pET-20b(+).

[0018] In one embodiment of the present invention, the vector contains a promoter, and the promoter is operably linked to the above-mentioned polynucleotide.

[0019] In the fourth aspect of the present invention, there is provided a host cell containing the above-mentioned gene or expression vector, or capable of expressing the above-mentioned mutant.

[0020] In one embodiment of the present invention, the host cell is selected from bacteria or fungi. In one embodiment of the present invention, the host cell is Escherichia coli.

[0021] In the fifth aspect of the present invention, there is provided a method for preparing the above-mentioned mutant, including culturing the above-mentioned host cell to obtain the mutant.

[0022] In one embodiment of the present invention, it includes the following steps:

[0023] (1) Inoculate the above-mentioned host cell monoclonal into 5 mL of liquid LB medium and culture overnight at 37 °C to obtain a seed solution;

[0024] (2) Inoculate the seed solution into LB medium at an inoculation amount of 1%, culture at 37 °C until the biomass OD600 reaches 0.6 - 0.8, then add an inducer and continue to culture at 16 °C for 12 - 16 h, and collect the thalli by centrifugation;

[0025] (3) The centrifugally collected cells were disrupted by high-pressure homogenization, and the disrupted solution was centrifuged at 8000 rpm at 4 °C for 25 min to obtain a crude enzyme solution.

[0026] (4) The crude enzyme solution was subjected to nickel column affinity chromatography to obtain the pure enzyme of the TsFDH mutant.

[0027] In one embodiment of the present invention, site-directed mutagenesis was performed on the target gene (the sequence shown in SEQ ID NO: 2) by PCR site-directed mutagenesis.

[0028] In one embodiment of the present invention, the following steps are included: (1) Design mutant primers, the sequences of which are shown in SEQ ID NO: 5-42. (2) Use the template DNA containing the target gene for PCR amplification, and use the designed mutant primers to amplify the fragment of the target gene. (3) Transform the host cell for expression and screen to obtain the above-mentioned mutant.

[0029] In the sixth aspect of the present invention, an enzyme electrode for enzyme electrocatalytic reduction is provided, which includes a metal electrode and a formate dehydrogenase mutant covering the surface of the metal electrode.

[0030] In one embodiment of the present invention, the metal electrode is selected from an ITO electrode, an FTO electrode, a glassy carbon electrode, a gold electrode, a carbon electrode or a carbon cloth electrode.

[0031] In one embodiment of the present invention, the loading amount of the formate dehydrogenase mutant on the metal electrode is 0.1-0.4 mg / cm 2 .

[0032] In one embodiment of the present invention, the formate dehydrogenase mutant is coated on the surface of the metal electrode and incubated to make the mutant bind to the electrode surface; subsequently, the surface active sites on the metal electrode that are not bound to the formate dehydrogenase mutant are blocked to obtain the enzyme electrode.

[0033] In the seventh aspect of the present invention, an enzyme electrolytic reactor is further provided, which includes the above-mentioned enzyme electrode, and the enzyme electrode is a working electrode.

[0034] In the eighth aspect of the present invention, a method for performing a catalytic reduction reaction using the above-mentioned enzyme electrode or enzyme electrolytic reactor is provided, which includes bringing the enzyme electrode or enzyme electrolytic reactor into contact with the reaction solution and generating an electric current for the reaction. The reaction solution includes coenzyme NADH, a buffer solution, and a substrate. The substrate is a substrate of formate dehydrogenase. The buffer solution includes at least one of a phosphate buffer solution, a carbonate buffer solution, a Tris-HCl buffer solution, a PBS buffer solution, etc.

[0035] In an embodiment of the present invention, an enzyme electrode or an enzyme electroreactor is contacted with a reaction solution to generate an electric current for the reaction to prepare formic acid. The reaction solution includes coenzyme NADH, PBS buffer, and NaHCO3.

[0036] In a ninth aspect of the present invention, there is provided the use of the above-mentioned formate dehydrogenase mutant, polynucleotide, vector, host cell, enzyme electrode, and enzyme electroreactor in the preparation of fuel cells, hydrogen storage materials, fine chemicals (including methanol, ethanol, or other organic acid / alcohol compounds), and biofuels.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] In the present invention, site-directed saturation mutagenesis is performed on formate dehydrogenase derived from Thiobacillus, and finally a TsFDH mutant with improved electron transfer rate and stable catalytic activity is obtained. Description of the Drawings

[0039] Figure 1 Performance test of the TsFDH mutant / M / Au for CO2 reduction;

[0040] Figure 2 Enzyme kinetic performance of the TsFDH mutant;

[0041] Figure 3 Enzyme electrocatalytic formic acid production performance of the optimal TsFDH mutant. Detailed Embodiments

[0042] The technical solutions of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are only for illustrative explanation of the present invention and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0043] Unless otherwise specified, the raw materials and reagents used in the following embodiments are all commercially available products or can be prepared by known methods.

[0044] "Wild" refers to a naturally occurring or wild-type form of a protein, or a naturally occurring or wild-type form of an amino acid in a naturally occurring protein. In an embodiment of the present invention, the sequence of wild-type Ts FDH is shown in SEQ ID NO:1. The term "mutation" as used herein includes replacing a natural amino acid with a single new amino acid, or replacing two or more natural amino acids with two or more new amino acids. In an embodiment of the present invention, the sequences of the TsFDH mutants are shown in SEQ ID NO:3 and SEQ ID NO:4.

[0045] "Polynucleotide" is a single-stranded or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases, typically read from the 5' to the 3' end. Polynucleotides include RNA and DNA and can be isolated from natural sources, synthesized in vitro, or prepared from combinations of natural and synthetic molecules.

[0046] "Amino acid mutation" refers to the replacement of an amino acid residue at a given position (e.g., a naturally occurring residue in wild-type TsFDH (e.g., SEQ ID NO:1)) with another amino acid residue (e.g., other than the naturally occurring residue). For example, the naturally occurring amino acid residue at position 262 of the wild-type TsFDH sequence (SEQ ID NO:1) is tyrosine (T) (T262); thus, an amino acid substitution at T262 refers to the replacement of the naturally occurring tyrosine with any amino acid residue other than asparagine.

[0047] "Corresponding to the amino acid residue (X) in (designated sequence)" refers to the amino acid in the polypeptide of interest that aligns with the equivalent amino acid of the designated sequence when the polypeptide and the sequence are optimally aligned. For example, the amino acid corresponding to the position of SEQ ID NO:1 can be determined using an alignment algorithm such as BLAST. In some embodiments, the "correspondence" of amino acid positions is determined by aligning SEQ ID NO:1 with another Ts FDH sequence.

[0048] The term "host cell" refers to any cell capable of replicating and / or transcribing and / or translating a heterologous polynucleotide. Thus, a "host cell" refers to any prokaryotic cell (including but not limited to Escherichia coli (E. coli)) or eukaryotic cell (including but not limited to yeast cells, mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells), whether in vitro or in vivo. For example, a host cell can be present in a transgenic animal or transgenic plant. A host cell can be transformed, for example, with a heterologous polynucleotide.

[0049] "Vector" refers to a nucleic acid that contains a coding sequence and the sequences necessary to express the coding sequence. A vector can be viral or non-viral. A "plasmid" is a non-viral vector, e.g., a nucleic acid molecule encoding a gene and / or regulatory elements required for gene expression. A "viral vector" is a nucleic acid of viral origin that is capable of transporting another nucleic acid into a cell. When present in an appropriate environment, a viral vector is capable of directing the expression of one or more proteins encoded by one or more genes carried by the vector. Examples of viral vectors include but are not limited to retroviruses, adenoviruses, lentiviruses, and adeno-associated virus vectors.

[0050] "Direct electron transfer" means that when the formate dehydrogenase mutant catalyzes the substrate (CO2), electrons are directly transferred from the active site of the enzyme to the electron acceptor (such as a metal electrode) without relying on traditional cofactors, thus achieving direct electron transfer.

[0051] Example 1 Ts Preparation of FDH mutant plasmid

[0052] Based on the mutant plasmid from the previous work (glutamate at position 263 has been mutated to cysteine, labeled as the wild type in this invention), site-directed saturation mutagenesis was performed at position 262:

[0053] The required cells and reagents are as follows: The expression plasmid pET-20b(+) was obtained from our laboratory, and Escherichia coli Top 10 and BL21(DE3) were both homemade and can also be purchased; The nickel affinity chromatography packing material was purchased from Sigma Corporation and was packed into a column according to needs during protein purification; β-Nicotinamide adenine dinucleotide (NAD + ) and β-Nicotinamide adenine dinucleotide coenzyme reduced form (NADH) were purchased from Aladdin Corporation, and the rest of the reagents were all domestic or imported analytical pure.

[0054] Using site-directed mutagenesis primers, the target mutant was obtained by PCR amplification with the mutant plasmid pET-20- Ts FDH containing the previous work in the laboratory as a template. The PCR amplification program is as follows: Pre-denaturation at 98°C for 2 min; Denaturation at 98°C for 10 s, annealing at 56°C for 30 s, extension at 72°C for 90 s, 25 cycles; Extension at 72°C for 5 min. The above product was digested with DpnI enzyme at 37°C for 1 h, purified using a DNA reagent purification kit, and then the product was transformed into Escherichia coli competent Top 10 to obtain positive clones. After picking monoclonal colonies and correct sequencing, different mutants were obtained, and at the same time, the recombinant plasmids of 20 mutants were extracted for standby. The specific primer sequences are shown in Table 1:

[0055] Table 1 Primers used for site-directed mutagenesis

[0056]

[0057] Example 2 Ts Expression and screening of FDH mutants

[0058] The Ts FDH mutant plasmid obtained in Example 1 was transformed into Escherichia coli BL21(DE3) for Ts FDH mutant expression. The specific operation steps are as follows:

[0059] (1) Expression TsPreparation of Escherichia coli strains of FDH mutants. The screened positive strains were streaked on LB solid medium (100 μg / mL ampicillin) until monoclonal colonies grew out. Monoclonal colonies were picked and inoculated into liquid LB medium, and cultured in a shaker at 37 °C and 200 rpm for 12 - 16 h to obtain a seed solution.

[0060] (2) Induction expression and screening of mutant enzymes. The induction expression conditions of 20 mutant enzymes were the same as those of the wild-type Ts FDH. The overnight cultured seed solution was inoculated into LB medium at an inoculation amount of 1%, and cultured at 37 °C until the biomass OD600 reached 0.6 - 0.8. Then, an inducer isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.1 mM was added and induced at 16 °C for 12 - 16 h. The cells were collected by centrifugation at 4 °C. The collected cells were added to a lysis solution (50 mM PBS, pH 7.0) and homogenized by high pressure (pressure 1000 bar) for 1 min, and then centrifuged at 8000 rpm at 4 °C for 25 min to collect the supernatant. The supernatant was directly loaded onto a nickel affinity chromatography column, and the impurity proteins were eluted using a balance buffer (50 mM PBS, pH 7.0, containing 20 mM imidazole), and then the target protein was eluted using an elution buffer (50 mM PBS, pH 7.0 containing 200 mM imidazole). The target protein was concentrated by centrifugation in a centrifuge tube and the imidazole concentration was diluted to less than 0.1 mM. The 20 mutant enzymes were named T262H-E263C (H), T262G-E263C (G), T262R-E263C (R), T262K-E263C (K), T262E-E263C (E), T262D-E263C (D), T262Q-E263C (Q), T262N-E263C (N), T262M-E263C (M), T262C-E263C (C), T262S-E263C (S), T262W-E263C (W), T262Y-E263C (Y), T262F-E263C (F), T262P-E263C (P), T262I-E263C (I), T262L-E263C (L), T262V-E263C (V), T262A-E263C (A),

[0061] Example 3 Ts Electrocatalytic performance characterization of FDH mutants

[0062] A gold electrode was selected as the base electrode. The gold electrode was polished with Al2O3 and electrochemically activated in 0.5 M H2SO4. The scanning range was from -0.3 V to 1.5 V, the scanning rate was 50 mV / s, and the number of scanning cycles was 20. Then, it was ultrasonically cleaned with ultrapure water and ethanol for 5 min each in turn and dried with nitrogen. 10 ul of the above-mentioned viable mutant at a concentration of 2 g / L was aspirated and drop-coated onto the surface of the Au electrode, incubated overnight at 37 °C, and labeled as FDH / Au. The next day, the unbound mutants were washed with ultrapure water and dried with N2. The FDH / Au was immersed in a 1 mM 6-mercapto-1-hexanol (MCH) solution for 1 h to wait for MCH to block the active sites on the surface of the unbound gold electrode, denoted as FDH / M / Au, for use. First, cyclic voltammetry scans were performed on all mutant electrodes. The scanning range was from -0.9 V to 0 V, the scanning rate was 1 mV / s, the electrolyte was 0.1 M PBS (pH 7.5), 50 mM sodium bicarbonate was added, and CO2 was continuously bubbled through for two hours. After the scan was stable, the last cycle was taken as the basis for final analysis. The reduction currents of different mutant enzyme electrodes for CO2 were compared, and the mutant electrodes capable of direct electron transfer were screened according to the magnitude of the reduction current ( Figure 1 ).

[0063] As Figure 1 can be seen, T262I-E263C (I) showed a significantly increased current value, indicating that this mutant has enhanced electron transfer ability and can perform more efficient direct electron transfer on the electrode.

[0064] Example 4 Ts Enzymatic kinetic performance test of FDH mutants

[0065] Ts The principle of FDH mutant activity determination is as follows: The reaction substrate NaHCO3 (CO2) is Ts catalyzed by FDH, and the absorbance value of its cofactor NADH at 340 nm will show a decreasing change. According to the molar extinction coefficient, the concentration change of NADH is calculated, and thus Ts the FDH enzyme activity is calculated.

[0066] Under this detection condition, the extinction coefficient of NADH is 6.22 mM -1 cm -1 .

[0067] The enzyme activity is defined as the amount of enzyme required to catalyze the consumption of 1 μmol NADH per milligram of Ts FDH at 37 °C is 1 U.

[0068] The specific operation steps are as follows:

[0069] (1) Final concentration of the reaction solution: 50 mM NaHCO3, 0.1, 0.2, 0.4, 0.8, 1.2, 1.6, 2.0, 2.5, 3.0, 4.0 mM NADH solution, 100 mg / L Ts FDH mutants.

[0070] (2) Add the above reaction solution to a 96-well plate, 250 μL per well, and react at 25 °C for 40 min.

[0071] (3) Use GraphPad Prism software to calculate the kinetic parameters of the enzyme by non-linear regression. The results are as Figure 2 shown.

[0072] The results show that all Ts FDH mutants have relatively high Vmax and low Km, indicating that these sites have little effect on the enzymatic properties.

[0073] Example 5 Ts Performance test of electrocatalytic formic acid production by the optimal FDH mutant enzyme

[0074] Immerse a 1 cm × 1 cm Cu sheet or other metal catalysts capable of regenerating NADH in 1 mL of 2 g / L optimal mutant T262I or wild type (WT), incubate overnight at 4 °C, label as T262I / Cu, WT / Cu. The next day, wash the unbound mutants with ultrapure water and dry with N2. Immerse T262I / Cu and WT / Cu in 1 mM 6-mercapto-1-hexanol (MCH) solution for 1 h, waiting for MCH to block the active sites on the surface of the unbound Cu electrode, denoted as T262I / M / Au and WT / M / Cu, for use. Immerse T262I / M / Au and WT / M / Cu in 5 mL of PBS (pH 6.0) solution containing 50 mM NaHCO3, 0.1 mM NADH, sample every 12 h, and measure the formic acid content by HPLC. The results are as Figure 3 shown. The results show that the formic acid production of the optimal mutant T262I-E263C (I) is 0.025 mM / g / h, which is about 20 times that of the wild type.

[0075] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the purpose and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

[0076] SEQ ID NO.1 sequence:

[0077] MAKILCVLYDDPVDGYPKTYARDDLPKIDHYPGGQTLPTPKAIDFTPGQLLGSVSGELGLRKYLEANGHTFVVTSDKDGPDSVFEKELVDADVVISQPFWPAYLTPERIAKAKNLKLALTAGIGSDHVDLQSAIDRGITVAEVTYCNSISVAEHVVMMILGLVRNYIPSHDWARKGGWNIADCVEHSYDLEGMTVGSVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPEAVEKELGLVWHDTREDMYPHCDVVTLNVPLHPETCHMINDETLKLFKRGAYIVNTARGKLADRDAIVRAIESGQLAGYAGDVWFPQPAPKDHPWRTMKWEGMTPHISGTSLSAQARYAAGTREILECFFEGRPIRDEYLIVQGGALAGTGAHSYSKGNATGGSEEAAKFKKAG

[0078] SEQ ID NO.2 sequence:

[0079] ATGGCCAAGATCCTGTGCGTGCTGTACGACGACCCCGTGGACGGCTACCCCAAGACCTAC

[0080] GCCAGAGACGACCTGCCCAAGATCGACCACTACCCCGGCGGCCAGACCCTGCCCACCCCC

[0081] AAGGCCATCGACTTCACCCCCGGCCAGCTGCTGGGCAGCGTGAGCGGCGAGCTGGGCCTG

[0082] AGAAAGTACCTGGAGGCCAACGGCCACACCTTCGTGGTGACCAGCGACAAGGACGGCCCC

[0083] GACAGCGTGTTCGAGAAGGAGCTGGTGGACGCCGACGTGGTGATCAGCCAGCCCTTCTGG

[0084] CCCGCCTACCTGACCCCCGAGAGAATCGCCAAGGCCAAGAACCTGAAGCTGGCCCTGACC

[0085] GCCGGCATCGGCAGCGACCACGTGGACCTGCAGAGCGCCATCGACAGAGGCATCACCGTG

[0086] GCCGAGGTGACCTACTGCAACAGCATCAGCGTGGCCGAGCACGTGGTGATGATGATCCTG

[0087] GGCCTGGTGAGAAACTACATCCCCAGCCACGACTGGGCCAGAAAGGGCGGCTGGAACATC

[0088] GCCGACTGCGTGGAGCACAGCTACGACCTGGAGGGCATGACCGTGGGCAGCGTGGCCGCC

[0089] GGCAGAATCGGCCTGGCCGTGCTGAGAAGACTGGCCCCCTTCGACGTGAAGCTGCACTAC

[0090] ACCGACAGACACAGACTGCCCGAGGCCGTGGAGAAGGAGCTGGGCCTGGTGTGGCACGAC

[0091] ACCAGAGAGGACATGTACCCCCACTGCGACGTGGTGACCCTGAACGTGCCCCTGCACCCC

[0092] GAGACCTGCCACATGATCAACGACGAGACCCTGAAGCTGTTCAAGAGAGGCGCCTACATC

[0093] GTGAACACCGCCAGAGGCAAGCTGGCCGACAGAGACGCCATCGTGAGAGCCATCGAGAGC

[0094] GGCCAGCTGGCCGGCTACGCCGGCGACGTGTGGTTCCCCCAGCCCGCCCCCAAGGACCAC

[0095] CCCTGGAGAACCATGAAGTGGGAGGGCATGACCCCCCACATCAGCGGCACCAGCCTGAGC

[0096] GCCCAGGCCAGATACGCCGCCGGCACCAGAGAGATCCTGGAGTGCTTCTTCGAGGGCAGA

[0097] CCCATCAGAGACGAGTACCTGATCGTGCAGGGCGGCGCCCTGGCCGGCACCGGCGCCCAC

[0098] AGCTACAGCAAGGGCAACGCCACCGGCGGCAGCGAGGAGGCCGCCAAGTTCAAGAAGGCC

[0099] GGC

[0100] Sequence of SEQ ID NO.3:

[0101] MAKILCVLYDDPVDGYPKTYARDDLPKIDHYPGGQTLPTPKAIDFTPGQLLGSVSGELGLRKYLEANGHTFVVTSDKDGPDSVFEKELVDADVVISQPFWPAYLTPERIAKAKNLKLALTAGIGSDHVDLQSAIDRGITVAEVTYCNSISVAEHVVMMILGLVRNYIPSHDWARKGGWNIADCVEHSYDLEGMTVGSVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPEAVEKELGLVWHDTREDMYPHCDVVTLNVPLHPEICHMINDETLKLFKRGAYIVNTARGKLADRDAIVRAIESGQLAGYAGDVWFPQPAPKDHPWRTMKWEGMTPHISGTSLSAQARYAAGTREILECFFEGRPIRDEYLIVQGGALAGTGAHSYSKGNATGGSEEAAKFKKAG

[0102] Sequence of SEQ ID NO.4:

[0103] MAKILCVLYDDPVDGYPKTYARDDLPKIDHYPGGQTLPTPKAIDFTPGQLLGSVSGELGLRKYLEANGHTFVVTSDKDGPDSVFEKELVDADVVISQPFWPAYLTPERIAKAKNLKLALTAGIGSDHVDLQSAIDRGITVAEVTYCNSISVAEHVVMMILGLVRNYIPSHDWARKGGWNIADCVEHSYDLEGMTVGSVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPEAVEKELGLVWHDTREDMYPHCDVVTLNVPLHPEKCHMINDETLKLFKRGAYIVNTARGKLADRDAIVRAIESGQLAGYAGDVWFPQPAPKDHPWRTMKWEGMTPHISGTSLSAQARYAAGTREILECFFEGRPIRDEYLIVQGGALAGTGAHSYSKGNATGGSEEAAKFKKAG

Claims

1. A formate dehydrogenase mutant, characterized in that: The formate dehydrogenase mutant uses the amino acid sequence shown in SEQ ID NO.1 as the parent, and site-directed saturation mutagenesis is performed on tyrosine at position 262 of SEQ ID NO:1; The formate dehydrogenase mutant is derived from Thiobacillus ( Thiobacillus sp. ) KNK65MA and has formate dehydrogenase activity; The site-directed saturation mutagenesis is selected from any one of T262R, T262W, T262F, T262I, T262V, T262A.

2. The formate dehydrogenase mutant according to claim 1, characterized in that: The amino acid sequence of the formate dehydrogenase mutant is as shown in SEQ ID NO:

3.

3. A polynucleotide, characterized in that: The polynucleotide encodes the formate dehydrogenase mutant according to claim 1 or 2.

4. A carrier, characterized in that: Comprising the polynucleotide according to claim 3.

5. A host cell, characterized in that: The host cell contains the vector according to claim 4, and the host cell is a non-plant cell.

6. An enzyme electrode for enzyme electrocatalytic reduction, characterized in that: Comprising a metal electrode and the formate dehydrogenase mutant according to claim 1 or 2 covering the surface of the metal electrode.

7. An enzyme electroreactor, characterized in that: Comprising the enzyme electrode according to claim 6, and the enzyme electrode is a working electrode.

8. A method for catalyzing a reduction reaction, comprising contacting the enzyme electrode according to claim 6 or the enzyme electroreactor according to claim 7 with a reaction solution and generating an electric current for the reaction, wherein the reaction solution comprises coenzyme NADH, a buffer solution, and a substrate; the substrate is a substrate of formate dehydrogenase; the buffer solution comprises at least one of a phosphate buffer solution, a carbonate buffer solution, a Tris-HCl buffer solution, and a PBS buffer solution.

9. Use of the formate dehydrogenase mutant according to claim 1 or 2, the polynucleotide according to claim 3, the vector according to claim 4, the host cell according to claim 5, the enzyme electrode according to claim 6, and the enzyme electroreactor according to claim 7 in the preparation of fuel cells, hydrogen storage materials, and biofuels.

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