Formate dehydrogenase mutant with improved electron transfer rate and application

Abstract

The invention provides a formate dehydrogenase mutant with improved electron transfer rate and application thereof, the formate dehydrogenase mutant takes an amino acid sequence as shown in SEQ ID NO.1 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identity with the SEQ ID NO.1 as a parent, and has site-saturated mutation corresponding to tyrosine at the 262nd site of the SEQ ID NO.1. The TsFDH mutant with improved electron transfer rate and stable catalytic activity is finally obtained by performing site-directed saturated mutation on formate dehydrogenase from thiobacillus.

Description

Technical Field

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

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

[0003] From Thiobacillus Thiobacillus sp. KNK65MA ) of formate dehydrogenase ( Ts FDH) is a NADH-dependent FDH that is oxygen-tolerant, easy to express, and has high yields, but its CO2-reducing enzyme activity is 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 the coenzyme NADH to form an enzyme-coenzyme complex, and then Ts The cysteine ​​at position 263 on the surface of FDH was mutated, and the thiol group on cysteine ​​was 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 electrocatalysts for the regeneration of NADH. The production of formic acid in this system was as high as 11.8 M / mU / h, which was the highest reported at the time (J CO2Util2019, 34, 568-575.). In 2021, Fonzo et al. used a nanostructured titanium nitride carrier to immobilize Ts FDH, the hierarchical TiN nanostructured support exhibits high surface area and wide pore size distribution, achieving high catalytic loading and using neutral red for NADH regeneration. In this CO2EER system, the production of formic acid is as high as 44.1 μmol / mg / h (ChemElectroChem 2021, 8(15), 2846-2857.). In both CO2EER systems, the TsDue to the low enzyme activity of FDH, the turnover rate of electroreduction of CO2 is lower than that of FDH that can directly transfer electrons (DET). Ts By constructing the electron transfer pathway from the FDH electron transfer exchange site to the electrode interface and building a DET system, it is expected to improve the CO2 turnover rate of the engineered CO2EER system. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a formate dehydrogenase mutant with improved electron transfer rate and its application. The present invention performs site-directed mutagenesis on formate dehydrogenase from Thiobacillus to finally obtain a TsFDH mutant with improved electron transfer rate and stable catalytic activity.

[0005] In one aspect of the present invention, a formate dehydrogenase mutant is provided, wherein the formate dehydrogenase mutant has an amino acid sequence as 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 a 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, and 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 SEQ ID NO:3.

[0008] In one embodiment of the present invention, the formate dehydrogenase mutant has direct electron transfer characteristics and can perform more efficient electron transfer on the electrode. In one embodiment of the present invention, the formate dehydrogenase mutant has an increased 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 to the Thiobacillus sp. KNK65MA) of a wild-type formate dehydrogenase, wherein the formate dehydrogenase mutant has improved enzyme kinetics.

[0012] In one embodiment of the present invention, compared to the Thiobacillus sp. KNK65MA ) of a wild-type formate dehydrogenase, wherein the formate dehydrogenase mutant has improved formate production.

[0013] In one embodiment of the present invention, compared to the Thiobacillus sp. KNK65MA ) of a wild-type formate dehydrogenase, wherein the formate production of the formate dehydrogenase mutant is increased by at least 20 times.

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

[0015] In a second aspect, the present invention provides a polynucleotide encoding the formate dehydrogenase mutant.

[0016] The third aspect of the present invention provides a vector comprising the above-mentioned polynucleotide.

[0017] In one embodiment of the present invention, the vector may be selected from plasmid, virus or yeast. In one embodiment of the present invention, the plasmid includes but is 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 comprises a promoter, and the promoter is operably linked to the polynucleotide.

[0019] In a fourth aspect, the present invention provides a host cell containing the above gene or expression vector, or capable of expressing the above 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 a fifth aspect, the present invention provides a method for preparing the mutant, comprising culturing the host cell to obtain the mutant.

[0022] In one embodiment of the present invention, the following steps are included: (1) Inoculate the above host cell monoclone into 5 mL of liquid LB medium and culture at 37°C overnight to obtain seed solution; (2) Inoculate the seed liquid into LB medium at a 1% inoculum amount and culture at 37°C until the biomass OD600 reaches 0.6-0.8. Then add the inducer and continue to culture at 16°C for 12-16 h. Collect the cells by centrifugation. (3) The cells collected by centrifugation were subjected to high-pressure homogenization and the broken liquid was centrifuged at 8000 rpm at 4°C for 25 min to obtain a crude enzyme solution; (4) The crude enzyme solution was subjected to nickel column affinity chromatography to obtain pure enzyme of TsFDH mutant.

[0023] In one embodiment of the present invention, the target gene (sequence shown in SEQ ID NO: 2) is subjected to site-directed mutagenesis by PCR site-directed mutagenesis.

[0024] In one embodiment of the present invention, the following steps are included: (1) designing a mutation primer, the sequence of which is shown in SEQ ID NO: 5-42. (2) using a template DNA containing the target gene for PCR amplification, and using the designed mutation primer to amplify a fragment of the target gene. (3) transforming a host cell for expression, and screening to obtain the above mutant.

[0025] In a sixth aspect, the present invention provides an enzyme electrode for enzyme electrocatalytic reduction, comprising a metal electrode and a formate dehydrogenase mutant coated on the surface of the metal electrode.

[0026] In one embodiment of the present invention, the metal electrode is selected from an ITO electrode, a FTO electrode, a glassy carbon electrode, a gold electrode, a carbon electrode or a carbon cloth electrode.

[0027] 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 .

[0028] In one embodiment of the present invention, the formate dehydrogenase mutant is coated on the surface of a metal electrode and incubated to allow the mutant to bind to the electrode surface; then, the surface active sites on the metal electrode that are not bound to the formate dehydrogenase mutant are blocked to obtain the enzyme electrode.

[0029] According to a seventh aspect of the present invention, an enzyme-electrochemical reactor is provided, comprising the above-mentioned enzyme electrode, wherein the enzyme electrode is a working electrode.

[0030] In an eighth aspect, the present invention provides a method for catalytic reduction reaction using the above enzyme electrode or enzyme electroreactor, comprising contacting the enzyme electrode or enzyme electroreactor with a reaction solution and generating an electric current for reaction, wherein the reaction solution comprises coenzyme NADH, a buffer, and a substrate. The substrate is a substrate for formate dehydrogenase. The buffer comprises at least one of a phosphate buffer, a carbonate buffer, a Tris-HCl buffer, a PBS buffer, and the like.

[0031] In an embodiment of the present invention, an enzyme electrode or an enzyme-electric reactor is contacted with a reaction solution to generate an electric current for reaction to prepare formic acid, wherein the reaction solution includes coenzyme NADH, PBS buffer, and NaHCO3.

[0032] In a ninth aspect, the present invention provides the use of the formate dehydrogenase mutant, polynucleotide, vector, host cell, enzyme electrode, enzyme-electric reactor in the preparation of fuel cells, hydrogen storage materials, fine chemicals (including methanol, ethanol or other organic acid / alcohol compounds), and biofuels.

[0033] Compared with the prior art, the present invention has the following beneficial effects: The invention performs site-directed saturation mutation on formate dehydrogenase derived from Thiobacillus, and finally obtains a TsFDH mutant with improved electron transfer rate and stable catalytic activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 To test the performance of TsFDH mutant / M / Au on CO2 reduction; Figure 2 is the enzyme kinetic properties of TsFDH mutant; Figure 3 The optimal mutant enzyme electrocatalytic formic acid production performance of TsFDH. DETAILED DESCRIPTION

[0035] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0036] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0037] "Wild" refers to a naturally occurring or wild-type form of a protein, or refers to a naturally occurring or wild-type form of an amino acid in a naturally occurring protein. Ts The sequence of FDH is shown in SEQ ID NO: 1. The term "mutation" as used herein includes replacing one 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 one embodiment of the present invention, the sequence of the TsFDH mutant is shown in SEQ ID NO: 3 and SEQ ID NO: 4.

[0038] A "polynucleotide" is a single- 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 a combination of natural and synthetic molecules.

[0039] "Amino acid mutation" refers to replacing an amino acid residue at a given position (e.g., a naturally occurring amino acid residue present in wild-type TsFDH (e.g., SEQ ID NO: 1)) with another amino acid residue (e.g., other than a 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); therefore, an amino acid substitution at T262 refers to replacing the naturally occurring tyrosine with any amino acid residue other than asparagine.

[0040] "Corresponding to an amino acid residue (X) in (a specified sequence)" refers to an amino acid in a polypeptide of interest that aligns with the equivalent amino acid in the specified sequence when the polypeptide and the sequence are optimally aligned. For example, the amino acid corresponding to a position in SEQ ID NO: 1 can be determined using an alignment algorithm such as BLAST. In some embodiments, by comparing SEQ ID NO: 1 to another Ts FDH sequences were aligned to determine the "correspondence" of amino acid positions.

[0041] 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 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 located in vitro or in vivo. For example, a host cell can be located in a transgenic animal or transgenic plant. A host cell can, for example, be transformed with a heterologous polynucleotide.

[0042] "Vector" refers to a nucleic acid that contains a coding sequence and sequences necessary for the expression of the coding sequence. A vector can be viral or non-viral. A "plasmid" is a non-viral vector, such as a nucleic acid molecule that encodes 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, retroviral, adenoviral, lentiviral, and adeno-associated viral vectors.

[0043] "Direct electron transfer" refers to the fact that when the formate dehydrogenase mutant catalyzes the substrate (CO2), electrons are directly transferred to the electron acceptor (such as a metal electrode) through the active site of the enzyme without relying on traditional cofactors, thereby achieving direct electron transfer.

[0044] Example 1 Ts Preparation of FDH mutant plasmid Based on the mutant plasmid from previous work (glutamic acid at position 263 has been mutated to cysteine, marked as the wild type in the present invention), site-directed saturation mutagenesis at position 262 was performed: The required cells and reagents are as follows: the expression plasmid pET-20b(+) was obtained from our laboratory, and E. coli Top 10 and BL21(DE3) were all homemade and can also be purchased; the nickel affinity chromatography filler was purchased from Sigma and was loaded into the column as needed for protein purification; β-nicotinamide adenine dinucleotide (NAD + ) and β-nicotinamide adenine dinucleotide coenzyme reduced (NADH) were purchased from Aladdin Company, and the other reagents were domestically produced or imported of analytical grade.

[0045] Site-directed mutagenesis primers were used to generate mutant plasmid pET-20- Ts The target mutant was obtained by PCR amplification using FDH as a template. The PCR amplification procedure was as follows: pre-denaturation at 98°C for 2min; denaturation at 98°C for 10s, annealing at 56°C for 30s, extension at 72°C for 90s, 25 cycles; extension at 72°C for 5min. The above products were digested with DpnI at 37°C for 1 h, purified using a DNA reagent purification kit, and then transformed into E. coli competent Top 10 to obtain positive clones. After the single clones were correctly sequenced, different mutants were obtained, and the recombinant plasmids of 20 mutants were extracted for use. The specific primer sequences are shown in Table 1: Table 1 Primers used for site-directed mutagenesis

[0046] Example 2 Ts Expression and screening of FDH mutants The obtained in Example 1 Ts The FDH mutant plasmid was transformed into Escherichia coli BL21 (DE3) for Ts FDH mutant expression. The specific steps are as follows: (1) Expression Ts Preparation of Escherichia coli strains of FDH mutants. The screened positive strains were streaked on LB solid medium (100 μg / mL ampicillin) until a single clone grew out, and a single clone was picked and inoculated into liquid LB medium, and cultured in a shaker at 37°C and 200 rpm for 12-16 h to obtain seed liquid.

[0047] (2) Induced expression and screening of mutant enzymes. The induced expression conditions of 20 mutant enzymes and the wild type TsThe induction expression conditions of FDH are the same. The overnight cultured seed solution was inoculated into LB medium at 1% inoculum, cultured at 37°C until the biomass OD600 reached 0.6-0.8, and then the inducer isopropyl-β-D-thiogalactoside (IPTG) was added at a final concentration of 0.1 mM for induction at 16°C for 12-16h, and the bacteria were collected by centrifugation at 4°C. The collected bacteria were added to the disruption solution (50 mM PBS, pH 7.0) for high-pressure homogenization (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 added to the nickel affinity chromatography column, and the impurities were eluted with the equilibration buffer (50 mM PBS, pH 7.0, containing 20mM imidazole), and then the target protein was eluted with the 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 below 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), T262 62C-E263C (C), T262S-E263C (S), T262W-E263C (W), T262Y-E263C (Y), T262F-E263C (F), T 262P-E263C (P), T262I-E263C (I), T262L-E263C (L), T262V-E263C (V), T262A-E263C (A), Example 3 Ts Characterization of electrocatalytic performance of FDH mutants A gold electrode was selected as the base electrode, and the gold electrode was polished with Al2O3 and electrochemically activated in 0.5 M H2SO4. The scanning range was -0.3 V to 1.5 V, the scanning rate was 50 mV / s, and the number of scanning circles was 20. After that, it was ultrasonically cleaned with ultrapure water and ethanol for 5 min each, and dried with nitrogen. 10 ul 2 g / L of the above-mentioned active mutant was absorbed and dropped onto the surface of the Au electrode, incubated at 37 °C overnight, and marked as FDH / Au. The next day, the unbound mutant was washed with ultrapure water, dried with N2, and FDH / Au was immersed in 1 mM 6-mercapto-1-hexanol (MCH) solution for 1 h, waiting for MCH to block the unbound active sites on the gold electrode surface, recorded as FDH / M / Au, and set aside. First, all mutant electrodes were subjected to cyclic voltammetry scanning, with a scanning range of -0.9 V to 0 V, a scanning rate of 1 mV / s, and an electrolyte of 0.1 M PBS (pH 7.5). 50 mM sodium bicarbonate was added and CO2 was continuously introduced for two hours. After the scanning was stable, the last cycle was taken as the final analysis basis. The reduction current of CO2 of different mutant enzyme electrodes was compared, and mutant electrodes that can directly transfer electrons were selected based on the size of the reduction current ( Figure 1 ).

[0048] Depend on Figure 1 It can be seen that T262I-E263C (I) showed a significantly increased current value, indicating that the mutant has enhanced electron transfer ability and can perform more efficient direct electron transfer at the electrode.

[0049] Example 4 Ts Enzyme kinetics test of FDH mutants Ts The principle of FDH mutant activity determination is as follows: the reaction substrate NaHCO3 (CO2) is Ts Under the catalysis of FDH, the absorbance of its cofactor NADH at 340 nm will decrease. The concentration change of NADH can be calculated based on the molar extinction coefficient, thus calculating Ts FDH enzyme activity.

[0050] Under this detection condition, the extinction coefficient of NADH is 6.22 mM -1 cm -1 .

[0051] Enzyme activity is defined as mg / mL at 37°C. Ts The amount of enzyme required for FDH to catalyze the consumption of 1 μmol of NADH is 1 U.

[0052] The specific steps are as follows: (1) Final concentration of 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.

[0053] (2) Add the above reaction solution to a 96-well plate, 250 μL per well, and react at 25°C for 40 min.

[0054] (3) GraphPad Prism software was used to calculate the enzyme kinetic parameters by nonlinear regression. Figure 2 shown.

[0055] The results show that all Ts All FDH mutants had higher Vmax and lower Km, indicating that these sites had little effect on the enzymatic performance.

[0056] Example 5 Ts Performance test of electrocatalytic formic acid production by FDH optimal mutant enzyme Immerse a 1 cm×1 cm Cu sheet or other metal catalyst capable of regenerating NADH in 1 mL 2 g / L optimal mutant T262I or wild type (WT), incubate overnight at 4°C, and label as T262I / Cu and WT / Cu. Wash unbound mutants with ultrapure water the next day, blow dry with N2, immerse T262I / Cu and WT / Cu in 1 mM 6-mercapto-1-hexanol (MCH) solution for 1 h, wait for MCH to block unbound active sites on the Cu electrode surface, and record as T262I / M / Au and WT / M / Cu for later use. Immerse T262I / M / Au and WT / M / Cu in 5 mL PBS (pH 6.0) solution containing 50 mM NaHCO3, 0.1 mM NADH, sample every 12 h, and measure formic acid content by HPLC. The results are as follows: Figure 3 The results showed that the formate production of the optimal mutant T262I-E263C (I) was 0.025 mM / g / h, which was about 20 times that of the wild type.

[0057] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

[0058] SEQ ID NO.1 sequence: MAKILCVLYDDPVDGYPKTYARDDLPKIDHYPGGQTLPTPKAIDFTPGQLLGSVSGELGLRKYLEANGHTFVVTSDKDGPDSVFEKELVDADVVISQPFWPAYLTPERIAKAKNLKLALTAGIGSDHVDLQSAIDRGITVAEVTYCNSISVAEHVVMMILGLVRNYIPSHDWARKGGWNIADCVEHSYDLEGMTVGSVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPEAVEKELGLVWHDTREDMYPHCDVVTLNVPLHPETCHMINDETLKLFKRGAYIVNTARGKLADRDAIVRAIESGQLAGYAGDVWFPQPAPKDHPWRTMKWEGMTPHISGTSLSAQARYAAGTREILECFFEGRPIRDEYLIVQGGALAGTGAHSYSKGNATGGSEEAAAKFKKAG SEQ ID NO.2 SEQ ID NO: ATGGCCAAGATCCTGTGCGTGCTGTACGACGACCCCGTGGACGGCTACCCCAAGAGCCTAC GCCAGAGACGACCTGCCCAAGATCGACCACTACCCCGGCGGCCAGACCCTGCCCACCCC AAGGCCATCGACTTCACCCCCGGCCAGCTGCTGGGCAGCGTGAGCGGCGAGCTGGGCCTG AGAAAGTACCTGGAGGCCAACGGCCACACCTTCGTGGTGACCAGCGACAAGGACGGCCCC GACAGCGTGTTCGAGAAGGAGCTGGTGGACGCCGACGTGGTGATCAGCCAGCCCTTCTGG CCCGCCTACCTGACCCCCGAGAGAATCGCCAAGGCCAAGAACCTGAAGCTGGCCCTGACC GCCGGCATCGGCAGCGACCACGTGGACCTGCAGAGCGCCATCGACAGAGGCATCACCGTG GCCGAGGTGACCTACTGCAACAGCATCAGCGTGGCCGAGCACGTGGTGATGATGATCCTG GGCCTGGTGAGAAACTACATCCCCAGCCACGACTGGGCCAGAAAGGGCGGCTGGAACATC GCCGACTGCGTGGAGCACAGCTACGACCTGGAGGGCATGACCGTGGGCAGCGTGGCCGCC GGCAGAATCGGCCTGGCCGTGCTGAGAAGACTGGCCCCCTTCGACGTGAAGCTGCACTAC ACCGACAGACACAGACTGCCCGAGGCCGTGGAGAAGGAGCTGGGCCTGGTGTGGCACGAC ACCAGAGAGGACATGTACCCCCACTGCGACGTGGTGACCCTGAACGTGCCCCTGCACCCC GAGACCTGCCACATGATCAACGACGAGACCCTGAAGCTGTTCAAGAGAGGCGCCTACATC GTGAACACCGCCAGAGGCAAGCTGGCCGACAGAGACGCCATCGTGAGAGCCATCGAGAGC GGCCAGCTGGCCGGCTACGCCGGCGACGTGTGGTTCCCCCAGCCCGCCCCCAAGGACCAC CCCTGGAGAACCATGAAGTGGGAGGGCATGACCCCCCACATCAGCGGCACCAGCCTGAGC GCCCAGGCCAGATACGCCGCCGGCACCAGAGAGATCCTGGAGTGCTTCTTCGAGGGCAGA CCCATCAGAGACGAGTACCTGATCGTGCAGGGCGGCGCCCTGGCCGGCACCGGCGCCCAC AGCTACAGCAAGGGCAACGCCACCGGCGGCAGCGAGGAGGCCGCCAAGTTCAAGAAGGCC GGC Sequence of SEQ ID NO.3: MAKILCVLYDDPVDGYPKTYARDDLPKIDHYPGGQTLPTPKAIDFTPGQLLGSVSGELGLRKYLEANGHTFVVTSDKDGPDSVFEKELVDADVVISQPFWPAYLTPERIAKAKNLKLALTAGIGSDHVDLQSAIDRGITVAEVTYCNSISVAEHVVMMILGLVRNYIPSHDWARKGGWNIADCVEHSYDLEGMTVGSVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPEAVEKELGLVWHDTREDMYPHCDVVTLNVPLHPEICHMINDETLKLFKRGAYIVNTARGKLADRDAIVRAIESGQLAGYAGDVWFPQPAPKDHPWRTMKWEGMTPHISGTSLSAQARYAAGTREILECFFEGRPIRDEYLIVQGGALAGTGAHSYSKGNATGGSEEAAAKFKKAG SEQ ID NO.4 SEQ ID NO: MAKILCVLYDDPVDGYPKTYARDDLPKIDHYPGGQTLPTPKAIDFTPGQLLGSVSGELGLRKYLEANGHTFVVTSDKDGPDSVFEKELVDADVVISQPFWPAYLTPERIAKAKNLKLALTAGIGSDHVDLQSAIDRGITVAEVTYCNSISVAEHVVMMILGLVRNYIPSHDWARKGGWNIADCVEHSYDLEGMTVGSVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPEAVEKELGLVWHDTREDMYPHCDVVTLNVPLHPEKCHMINDETLKLFKRGAYIVNTARGKLADRDAIVRAIESGQLAGYAGDVWFPQPAPKDHPWRTMKWEGMTPHISGTSLSAQARYAAGTREILECFFEGRPIRDEYLIVQGGALAGTGAHSYSKGNATGGSEEAAAKFKKAG

Claims

1. A formate dehydrogenase mutant, characterized in that: The formate dehydrogenase mutant has an amino acid sequence as shown in SEQ ID NO.1 or an amino acid sequence having at least 99% identity with SEQ ID NO.1 as a parent, and has a site-directed saturation mutation corresponding to 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 mutation includes any one of T262R, T262W, T262F, T262I, T262V, and T262A.

2. A formate dehydrogenase mutant according to claim 1, characterized in that: The amino acid sequence of the formate dehydrogenase mutant is shown in SEQ ID NO: 3, or an amino acid sequence having at least 99% identity with SEQ ID NO: 3; The formate dehydrogenase mutant is derived from Thiobacillus ( Thiobacillus sp. KNK65MA ), and has formate dehydrogenase activity.

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 of claim 3.

5. A host cell, characterized in that: The host cell contains the vector according to claim 4.

6. An enzyme electrode for enzyme electrocatalytic reduction, characterized in that: The invention comprises a metal electrode and the formate dehydrogenase mutant according to claim 1 or 2 which is covered on the surface of the metal electrode.

7. An enzyme electroreactor, characterized in that: It comprises the enzyme electrode as claimed in claim 6, wherein the enzyme electrode is a working electrode.

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

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-electric reactor according to claim 7 in the preparation of fuel cells, hydrogen storage materials, fine chemicals, and biofuels.

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