An immobilized formate dehydrogenase mutant and its application in electroenzymatic CO2 catalysis

Abstract

This invention relates to the field of biocatalysis, and more particularly to an immobilized formate dehydrogenase mutant and its application in electroenzymatic catalysis of CO2. The formate dehydrogenase mutant is based on the wild-type formate dehydrogenase of *Clostridium perfringens* and contains the following mutations: methionine at position 269 is mutated to isoleucine, serine at position 285 is mutated to alanine, and serine at position 637 is mutated to alanine; the NCBI accession number for the wild-type formate dehydrogenase of *Clostridium perfringens* is WP_082085143.1. The immobilized formate dehydrogenase mutant provided by this invention exhibits superior catalytic activity, temperature, pH stability, and storage stability, and can be reused. The electrode material prepared using this immobilized formate dehydrogenase mutant can efficiently catalyze the conversion of CO2 into formic acid or formate salts, which is of great significance in the field of renewable energy utilization.

Description

Technical Field

[0001] This invention relates to the field of biocatalysis technology, and in particular to an immobilized formate dehydrogenase mutant and its application in electroenzymatic catalysis of CO2. Background Technology

[0002] The massive emission of the greenhouse gas CO2 has caused adverse effects such as global warming, sea-level rise, glacial melting, and desertification, posing a serious threat to human survival. Currently, there are various methods to address the greenhouse effect, such as promoting the use of renewable energy, improving energy efficiency, and afforestation. Existing technologies currently focus on how to reuse CO2, recycling it into energy and other valuable chemicals. This not only helps reduce atmospheric carbon dioxide levels but also provides a solution to the energy crisis. Therefore, the effective capture and utilization of CO2 is a pressing technological problem with promising development prospects.

[0003] CO2 can be converted into high-value-added chemicals such as formic acid, formaldehyde, and methanol. Formic acid is an important chemical raw material, widely used in various industries including chemical production, rubber manufacturing, textiles, printing and dyeing, and electroplating. Enzymatic reduction of CO2 to formic acid is superior to chemical catalysis, electrocatalysis, and photoelectrocatalysis due to its high selectivity, high efficiency, and mild conditions. Electroenzymatic catalysis utilizes electricity as a reducing force to promote the reaction under mild conditions and achieves coenzyme regeneration, solving the problem of expensive coenzymes. However, currently reported formic acid dehydrogenases still suffer from low enzyme activity, poor stability, and inability to be reused. Furthermore, free enzymes face challenges such as poor environmental stability and limited recyclability. Therefore, immobilizing the enzyme on a solid support can improve its tolerance to operating conditions and maintain its catalytic activity and stability. This method enables enzyme reuse, thereby promoting its industrial application. Summary of the Invention

[0004] To address the problems existing in the prior art, the present invention provides an immobilized formate dehydrogenase mutant and its application in electroenzymatic catalysis of CO2.

[0005] This invention utilizes genetic engineering techniques to develop a previously obtained high-activity formate dehydrogenase. Cl Using FDH as a probe, a novel formate dehydrogenase gene was discovered. Cs FDH (Follicular Unit Dehydrogenase) used rational design methods to mutate amino acid residues in a predicted conserved region of formate dehydrogenase, and after screening, obtained a formate dehydrogenase mutant with high catalytic activity.

[0006] In a first aspect, the present invention provides a formate dehydrogenase mutant, said formate dehydrogenase mutant being a mutant found in Clostridium difficile (Clostridium perfringens). Clostridium scatologenesBased on wild-type formate dehydrogenase, it contains the following mutations: methionine at position 269 is mutated to isoleucine, serine at position 285 is mutated to alanine, and serine at position 637 is mutated to alanine. The Clostridium ( Clostridium scatologenes The NCBI accession number for wild-type formate dehydrogenase is WP_082085143.1.

[0007] Furthermore, the formate dehydrogenase mutant comprises the amino acid sequence shown in SEQ ID NO.1.

[0008] The amino acid sequence shown in SEQ ID NO.1 is as follows: .

[0009] The formate dehydrogenase mutant described above has high catalytic activity, which is significantly improved compared to the activity of the original formate dehydrogenase, and can catalyze the conversion of carbon dioxide into formic acid or formate salt more efficiently.

[0010] Secondly, the present invention provides a nucleic acid for encoding the formate dehydrogenase mutant described above.

[0011] Based on the amino acid sequence and codon rules of the formate dehydrogenase mutant described above, those skilled in the art can obtain the nucleotide sequence of the nucleic acid molecule encoding the formate dehydrogenase mutant. Due to codon degeneracy, the nucleotide sequence of the nucleic acid molecule is not unique, and all nucleic acid molecules capable of encoding the formate dehydrogenase mutant are within the scope of protection of this invention.

[0012] The nucleic acid preferably includes a nucleotide sequence as shown in SEQ ID NO.2.

[0013] The nucleotide sequence shown in SEQ ID NO.2 is as follows:

[0014] Thirdly, the present invention provides biological materials, said biological materials including the aforementioned nucleic acids; said biological materials are expression cassettes, vectors, or transgenic cells.

[0015] The expression cassette of the present invention is a recombinant nucleic acid molecule obtained by operatively linking the nucleic acid molecule with transcriptional and / or translational regulatory elements.

[0016] The vectors described in this invention include, but are not limited to, plasmid vectors, viral vectors, and transposons. Among them, plasmid vectors include expression vectors and cloning vectors.

[0017] The transgenic cells described in this invention do not include transgenic cells capable of independently developing into a complete individual, that is, they do not include animal or plant varieties. For example, they may be Escherichia coli bacteria or yeast, more preferably Escherichia coli.

[0018] Fourthly, the present invention provides a recombinant genetically engineered bacterium that expresses the formate dehydrogenase mutant, or the nucleic acid, or carries a vector containing the nucleic acid; preferably, the recombinant genetically engineered bacterium is Escherichia coli or yeast.

[0019] In some embodiments of the present invention, a recombinant Escherichia coli expressing the formate dehydrogenase mutant described above is provided, which can be used to produce the formate dehydrogenase mutant described above.

[0020] Fifthly, the present invention provides any one of the following applications of the formate dehydrogenase mutant, the nucleic acid, the biological material, or the recombinant genetically engineered bacteria: (1) Application in the preparation of immobilized formate dehydrogenase; (2) Application in the preparation of electrode materials for the electroenzymatic catalysis of CO2 to produce formic acid or formate salt; (3) Application in the catalytic conversion of carbon dioxide into formic acid or formate salts; (4) Application in the production of formic acid or formate using carbon dioxide; (5) Application in the construction of engineered bacteria for converting carbon dioxide to produce formic acid or formate.

[0021] In a sixth aspect, the present invention provides an immobilized formate dehydrogenase, wherein the immobilized formate dehydrogenase comprises the formate dehydrogenase mutant and an immobilization vector; Preferably, the immobilization carrier is a HOF material; And / or, the mass ratio of the immobilized vector to the formate dehydrogenase mutant is 1:(0.28-1.39).

[0022] In some embodiments of the present invention, the immobilization carrier is an HOF material. Preferably, it is HOF-101. HOF-101 is a novel HOF material; the metal-free nature of HOF gives it better biocompatibility than MOF materials. The present invention prepares an immobilized enzyme by immobilizing the formate dehydrogenase mutant with HOF-101, significantly improving the stability of the formate dehydrogenase mutant (including temperature stability, pH stability, and storage stability), while solving the problem of the inability to recycle free enzymes. The resulting immobilized enzyme possesses high catalytic activity, high stability, and reusability, and exhibits good substrate properties. Km The value is 45 mM. Kcat / Km The value is 0.15 s -1 ·mM -1 ; for coenzymes Km The value is 0.4 mM. Kcat / Km The value is 43 s -1 ·mM -1 After being placed in PBS buffer (100 mM, pH 7.0) and stored at 4°C for 30 days, it still retains 90% of its initial activity; after 10 reuses, the activity still retains 85% of the initial activity.

[0023] In a seventh aspect, the present invention provides a method for preparing the immobilized formate dehydrogenase, the method comprising: mixing a formate dehydrogenase mutant with an immobilization carrier, such that the formate dehydrogenase mutant is adsorbed and encapsulated within the pores of the immobilization carrier. For example, a diffusion method through pores.

[0024] Eighthly, the present invention provides an electrode for the electro-enzymatic catalysis of CO2 to prepare formic acid or formate, the electrode comprising an electrode body and a catalyst coated on the surface of the electrode body; The catalyst comprises the immobilized formate dehydrogenase.

[0025] Furthermore, the electrode body is a carbon cloth electrode or a glassy carbon electrode.

[0026] In some embodiments of the present invention, a carbon cloth electrode for the electroenzymatic preparation of formic acid from CO2 is provided, comprising a carbon cloth electrode and a catalyst coated on the surface of the carbon cloth electrode, wherein the catalyst is the aforementioned immobilized formic acid dehydrogenase. Preferably, the catalyst loading in the electrode is controlled at 0.6-1.5 mg / cm³. 2 .

[0027] In other embodiments of the present invention, a glassy carbon electrode for the electroenzymatic preparation of formic acid from CO2 is provided, comprising a glassy carbon electrode and a catalyst coated on the surface of the glassy carbon electrode, said catalyst being the aforementioned immobilized formic acid dehydrogenase. Preferably, the catalyst loading in the electrode is controlled at 0.6-1.5 mg / cm³.2 .

[0028] In a ninth aspect, the present invention provides the application of the immobilized formate dehydrogenase or the electrode described herein in the catalytic preparation of formic acid or formate salt from CO2.

[0029] In a tenth aspect, the present invention provides a method for preparing formic acid, the method comprising: using CO2 as a substrate, electro-enzymatically catalyzing the conversion of CO2 into formic acid using the immobilized formic acid dehydrogenase described above or the electrode described above.

[0030] The present invention has the following beneficial effects: 1. This invention relates to Clostridium difficile (Clostridium perfringens) Clostridium scatologenes A formate dehydrogenase mutant was obtained by mutation based on wild-type formate dehydrogenase. This mutant exhibits high catalytic activity, with a specific enzyme activity reaching 896 mU / mg, and serves as a probe. Cl FDH has 89 times the enzyme activity and is a precursor to the mutated Clostridium difficile. Clostridium scatologenes Formate dehydrogenase is 5 times more active than enzymes and can efficiently catalyze the conversion of carbon dioxide into formic acid or formate salts.

[0031] 2. The immobilized enzyme prepared by combining the formate dehydrogenase mutant provided by this invention with HOF material exhibits significantly improved stability, including significantly improved temperature stability (after storage at 30℃-55℃ for 1 hour, the residual enzyme activity is significantly higher than that of the free enzyme); it shows higher stability than the free enzyme within the pH range of 5.0-11.5 (after storage at pH 6.0 for 1 hour, the residual enzyme activity can reach 100%, and it has better adaptability in neutral and alkaline environments), which is more conducive to the practical application of CO2 absorption and conversion in production; moreover, the immobilized enzyme molecule has higher structural stability, which greatly improves the storage stability of the immobilized enzyme (after storage at 4℃ for 30 days, it still retains 90% of the initial activity, while under the same conditions, the activity of the free enzyme only retains 40% of the initial activity); in addition, the immobilized enzyme solves the problem that the free enzyme cannot be recycled (after 10 reuses, the immobilized formate dehydrogenase of this invention can still maintain 85% of the initial activity), which is beneficial to the practical application of formate dehydrogenase.

[0032] 3. By utilizing the immobilized formate dehydrogenase of this invention and the electrode materials prepared therefrom, such as carbon cloth electrodes or glassy carbon electrodes, for enzyme-electrocoupling catalysis of CO2 to produce formate or formate salts, the coenzyme NADH can be recycled without the need for additional hydrogen gas, greatly reducing production costs and improving production safety. The immobilized formate dehydrogenase provided by this invention has good industrialization prospects and is of significant value in the field of renewable energy utilization. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0034] Figure 1 The HOF-101(a) and immobilized formate dehydrogenase mutant provided in Example 4 of this invention are examples of this invention. Cs FDH M Scanning electron microscope image of @HOF-101(b).

[0035] Figure 2 The free enzyme and immobilized enzyme provided in Example 6 of this invention. Cs FDH M Temperature stability comparison chart of @HOF-101.

[0036] Figure 3 The free enzyme and immobilized enzyme provided in Example 6 of this invention. Cs FDH M pH stability comparison chart of @HOF-101.

[0037] Figure 4 The free enzyme and immobilized enzyme provided in Example 7 of this invention. Cs FDH M Storage stability comparison chart for @HOF-101.

[0038] Figure 5 The immobilized enzyme provided in Example 8 of this invention Cs FDH M A graph showing the number of times @HOF-101 can be reused.

[0039] Figure 6 This is the HPLC detection chromatogram of CO2 converted to formic acid provided in Example 11 of the present invention.

[0040] Figure 7 The immobilized enzyme provided in Example 11 of this invention Cs FDH M A process diagram of the electroenzymatic catalysis of CO2 to formic acid using a carbon cloth electrode prepared with @HOF-101. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0042] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0043] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available, for example: The general research and development process of this invention is as follows: This invention utilizes genome mining technology, with previously reported high-activity formate dehydrogenase... Cl Using FDH protein sequences as probes, BLAST analysis was performed on various genomic data to identify a series of presumed formate dehydrogenases with genomic information sources but not yet expressed or identified. Phylogenetic trees were constructed and analyzed from these sequences, and representative gene sequences were selected. After codon optimization, the selected genes were synthesized and their expression was screened. Sequences from Clostridium difficile (Clostridium perfringens) were used as probes. Clostridium scatologenes A novel, highly active formate dehydrogenase was discovered in the genome of [a specific organism / organism]. Cs FDH), with formate dehydrogenase ( Cs Computer-aided rational design of parents using FDH. Parental formate dehydrogenase (FDH) was predicted using AlphaFold 2. Cs The three-dimensional structure of FDH was determined, and the amino acid conservation of the protein was analyzed using Consensus Finder. Its substrate channels were analyzed using CAVER to preliminarily identify mutation sites. Then, the protein (FDH) was analyzed using Autodock 4.2 software. Cs Semi-flexible molecular docking simulations were performed using FDH, substrate, and coenzyme. The three-dimensional structure of the obtained complex was analyzed using Pymol and Discovery Studio software to exclude key amino acid residues in the active site pocket. Finally, methionine (Met) at position 269 was mutated to isoleucine (Ile), serine (Ser) at position 285 was mutated to alanine (Ala), and serine (Ser) at position 637 was mutated to alanine (Ala). These mutations were achieved using an improved whole-plasmid amplification technique, ultimately successfully yielding a mutant with high catalytic activity against CO2. Cs FDH M The mutant was further immobilized with HOF material HOF-101 to obtain an immobilized enzyme.Cs FDH M @HOF-101 enhances its stability and reusability, and through enzyme immobilization Cs FDH M @HOF-101, carbon cloth electrodes or glassy carbon electrodes prepared with immobilized enzymes were used for enzyme-electrocoupling catalysis of CO2 to formic acid production. Among them, the yield of CO2 to formic acid catalyzed by carbon cloth electrodes prepared with immobilized enzymes reached 69 mM after 12 h.

[0044] Example 1: Gene mining of formate dehydrogenase encoding gene and construction of recombinant vector

[0045] This invention uses a previously obtained formate dehydrogenase with high activity. Cl Using FDH protein sequences as probes, BLAST analysis was performed on various genomic sequences to identify a series of presumed formate dehydrogenases with genomic information sources but not yet expressed or identified. Phylogenetic trees were constructed and analyzed from these sequences, and then three representative gene sequences were selected, codon optimized, and the entire genome synthesized. Comparative analysis was then conducted to select... Clostridium senegalense , Clostridium drakei and Clostridium scatologenes The three potential formate dehydrogenases from this source will be further investigated and named accordingly. Cse FDH, Cd FDH and Cs FDH.

[0046] by E.coli Using the codon usage frequency of strain K12 as a reference, codon optimization of potential formate dehydrogenase gene sequences was performed using the OPTIMIZER server. Codons were added upstream and downstream of the gene. Bam HI and Nde After the I restriction site was cut, the entire gene was synthesized by Qingke Biotechnology Co., Ltd. The synthesized formate dehydrogenase encoding gene was ligated into the pET28a plasmid, yielding the recombinant vector pET28a- Cse FDH, pET28a- Cd FDH and pET28a- Cs FDH.

[0047] Example 2: Rational modification of formate dehydrogenase and construction of mutant recombinant vector

[0048] The present invention selects the formate dehydrogenase with the highest catalytic activity from the three formate dehydrogenases screened in Example 1. Cs FDH was used for computer-aided rational design of the parents. First, AlphaFold 2 was used to predict the parental formate dehydrogenase (FDH). CsThe three-dimensional structure of FDH was obtained, and the amino acid conservation of the protein was analyzed using Consensus Finder. Its substrate channels were analyzed using CAVER to preliminarily determine mutation sites. AutoDock 4.2 software was used to sequentially... Cs The three-dimensional structure of FDH was analyzed using semi-flexible molecular docking with the substrate sodium bicarbonate and the coenzyme NADH. Further analysis using PyMol and Discovery Studio software identified the substrate channel of formate dehydrogenase and its key amino acid residues and metal ions involved in its interaction with the substrate and coenzyme, excluding key amino acid residues in the active site pocket. Finally, methionine (Met) at position 269 was mutated to isoleucine (Ile), serine (Ser) at position 285 was mutated to alanine (Ala), and serine (Ser) at position 637 was mutated to alanine (Ala). The resulting formate dehydrogenase (named...) Cs FDH M The gene encoding ) is shown in SEQ ID NO.2, and its amino acid sequence is shown in SEQ ID NO.1.

[0049] The mutation was performed using an improved whole-plasmid amplification technique with the recombinant vector pET28a- Cs Using FDH as a template, a reverse universal primer (R) was designed approximately 3.2 kb downstream of the vector cloning site. Forward mutant primers F1, F2, and F3 were designed approximately 15 bp upstream and downstream of the three mutation sites. A first round of PCR amplification was performed using a pair of forward and reverse primers. The product from the first round was then used as a large primer for amplification of the entire plasmid. After two rounds of PCR amplification, the plasmid was then... Dpn First, the template is digested to obtain a recombinant vector with one mutated site. Then, using the recombinant vector with one mutated site as a template, the remaining two pairs of forward and reverse primers are used for a second and third amplification according to the above method, thus obtaining the recombinant vector pET28a- with three mutated sites. Cs FDH M The primer sequences are shown in Table 1.

[0050] Table 1 Primers for obtaining formate dehydrogenase mutants by amplification of the whole plasmid

[0051] Example 3: Construction of recombinant genetically engineered bacteria expressing formate dehydrogenase mutant, and induction and analysis of formate dehydrogenase mutant.

[0052] 1. Recombinant vector pET28a- Cs FDH M Transformation into host cells: The recombinant vector pET28a- Cs FDH MThe cells were transformed into Escherichia coli BL21(DE3) competent cells by heat shock, and 0.5 mL of LB liquid medium was added. After incubation at 37°C and 200 rpm for 1 h, the cells were spread on LB solid plates containing 50 μg / mL ampicillin and cultured at 37°C for 12-16 h to obtain single colonies.

[0053] 2. Screening and identification of recombinant genetically engineered bacteria: Single colonies were picked and cultured overnight at 37°C and 220 rpm in 5 mL LB broth containing 50 μg / mL ampicillin. The bacterial culture was then identified by PCR the following day, and plasmids were extracted for use. Bam HI and Nde I performed double enzyme digestion identification, and the electrophoresis results showed that it contained a gene fragment of the same size as the target gene, and the clone was initially identified as a positive clone. Then, the positive clone was sent to Shanghai Sangon Biotech for sequencing, and the sequencing results further confirmed that the clone colony was the target genetically engineered bacteria.

[0054] 3. Induction of expression: Select the identified recombinant genetically engineered bacteria obtained above and inoculate them into 5 mL of LB liquid medium containing 50 μg / mL ampicillin. Incubate overnight at 37℃ and 220 rpm. Inoculate the overnight culture product into 100 mL of LB liquid medium at a 2% inoculation rate using a pipette. Then, incubate at 37℃ and 200 rpm for 2.5 h. Finally, add IPTG to a final concentration of 0.1 mmol / L for induction and incubate at 16℃ and 220 rpm for 20 h to obtain the induced bacterial culture (fermentation broth).

[0055] The induced bacterial cultures were aliquoted into 50 mL centrifuge tubes and centrifuged at 8000 rpm and 4°C for 5 min. The bacterial cells were collected and washed twice with 50 mL of deionized water under the same conditions. Each bacterial cell was resuspended in 100 mL of lysis buffer (PBS buffer, pH 7.0) and homogenized using a high-pressure homogenizer at 4°C and 900 bar for 3 minutes. After centrifugation at 12000 rpm and 4°C for 15 min, the supernatant and precipitate were collected separately. The supernatant was purified by Ni-Agarose affinity chromatography, and the expression morphology of the target protein was analyzed by SDS-PAGE.

[0056] 4. Enzyme activity assay: The change in absorbance of NADH was measured at 340 nm using a UV spectrophotometer (εNADH, 340 nm = 6.22 mM). -1 cm -1The reaction was initiated by adding formate dehydrogenase and monitored for 1 minute. The reaction system for enzyme activity assay was 1 mL containing 0.1 mM NADH, 100 mM sodium bicarbonate, 100 mM PBS buffer, pH=7.0. 100 μL of the enzyme solution was added to the above reaction system after appropriate dilution and incubation at 30℃ for 2 min. The change in absorbance was measured at 340 nm over 1 minute. Under these conditions, the amount of enzyme required to consume 1 μmol of NADH per minute was defined as 1 U. The enzyme activity calculation formula is: U=EW·V·1000 / 6220·L=EW / 6.22, where EW: OD at 1 min. 340 The change value, V: volume of reaction solution (mL), 6220: molar extinction coefficient (L*mol). -1 *cm -1 L: Optical path distance (cm).

[0057] The formate dehydrogenase mutant was determined to be... Cs FDH M The specific activity of the purified enzyme reached 896 mU / mg, which is suitable for probes. Cl FDH (10 mU / mg) was 89 times higher than that of formate dehydrogenase before the mutation. Cs The FDH coding gene sequence differs from the sequence shown in SEQ ID NO.2 in that codon 269 is a methionine codon, codon 285 is a serine codon, and codon 637 is a serine codon. The recombinant vector pET28a- Cs The FDH was constructed as described in Example 2 (as a control). Results showed that the pre-mutant formate dehydrogenase... Cs The specific activity of FDH is 179 mU / mg, formate dehydrogenase mutant. Cs FDH M The specific activity (896 mU / mg) was that of premutated formate dehydrogenase. Cs Five times that of FDH.

[0058] Example 4 Immobilized formate dehydrogenase mutant Cs FDH M Preparation of @HOF-101

[0059] 1. Synthesized using a modified, mild de novo protein-directed assembly method. Cs FDH M @HOF-101. Step 1: Prepare a 2 mg / ml formate dehydrogenase mutant using PBS buffer (100 mM, pH=7.0). Cs FDH M Solution. Next, 20 mg of H4TBAPy was dissolved in 2 mL of DMF by sonication. Then, 18 mL of DMF was added.Cs FDH M The solution (1 mg / ml) was stirred continuously at 25°C for 20 minutes to promote the formation of the composite material. After the stirring process was completed, the resulting product was collected by centrifugation. Cs FDH M @HOF-101 composite material. Centrifugation conditions: 25℃, 12000 rpm, 15 min. After collecting the supernatant, the collected precipitate was washed three times consecutively with PBS buffer (100 mM, pH=7.0), centrifuged under the same conditions, and dried to remove any residual solvent and unreacted precursors. The HOF material immobilized formate dehydrogenase mutant (referred to as immobilized formate dehydrogenase) was obtained. Cs FDH M @HOF-101.

[0060] Combine all the supernatants collected after centrifugation and determine their concentrations. Cs FDH M The content of [the substance], compared with the initial addition [of the substance]. Cs FDH M The content comparison can be used to obtain the content immobilized on the carrier HOF-101. Cs FDH M The amount of load can be calculated from this. The method for calculating the load is as follows:

[0061] Wherein, Co and Cs refer to the initial enzyme solution added during the immobilized enzyme reaction and the mixed supernatant after the immobilized enzyme reaction, respectively. Cs FDH M The concentration (mg / ml); Vo and Vs represent the volumes (mL) of the initial enzyme solution added to the immobilized enzyme reaction and the mixed supernatant after the immobilized enzyme reaction, respectively; m represents the mass (mg) of the carrier used.

[0062] Figure 1 a and b are HOF-101 and immobilized formate dehydrogenase mutants, respectively. Cs FDH M Scanning electron microscope image of @HOF-101. The image shows the initially synthesized HOF-101 and the immobilized... Cs FDH M All subsequent HOF-101 samples exhibit a clear rod-like structure, indicating that the morphology of HOF-101 has not changed due to [the following text is incomplete and requires further context]. Cs FDH M Its fixation was disrupted.

[0063] 2. Immobilized enzyme reducing activity assay: The change in absorbance of NADH was measured at 340 nm using a UV spectrophotometer (εNADH, 340 nm = 6.22 mM). -1 cm -1 By adding a certain mass of immobilized formate dehydrogenase... Cs FDH M The reaction was initiated using @HOF-101 and monitored for 1 minute. The reaction system for enzyme activity assay was 1 mL containing 0.1 mM NADH, 100 mM sodium bicarbonate, 100 mM PBS buffer, pH 7.0, and incubated at 30°C for 2 min. A certain mass of immobilized formate dehydrogenase was then added. Cs FDH M @HOF-101 was added to the above reaction system. The change in absorbance was measured at 340 nm over 1 minute. Under these conditions, the amount of enzyme required to consume 1 μmol of NADH per minute was defined as 1 U.

[0064]

[0065] EW: OD in 1 minute 340 The change value, V: volume of reaction solution (mL), 6220: molar extinction coefficient (L*mol). -1 *cm -1 L: optical path distance (cm), m: mass of immobilized formate dehydrogenase.

[0066] Example 5: Screening for optimal fixed conditions

[0067] 1. Optimal carrier dosage

[0068] To determine the optimal amount of HOF-101 carrier used for immobilizing formate dehydrogenase, 5, 10, 15, 20, and 25 mg of H4TBAPy were ultrasonically dissolved in 2 mL of DMF, and then 18 mL of DMF was added to each solution. Cs FDH M Formate dehydrogenase was immobilized in a solution (1 mg / ml), and then the effects of different HOF-101 dosages were measured. Cs FDH M Solid load and Cs FDH M The activity of @HOF-101 was ultimately immobilized. Cs FDH M The optimal amount of HOF-101 required is 20 mg / 18 mL of a 1 mg / mL formate dehydrogenase mutant solution. At this point, the immobilization capacity of the HOF-01 vector is 78 mg / g.

[0069] 2. Optimal immobilization time

[0070] In this invention, a certain amount of H4TBAPy was ultrasonically dissolved in 2 mL of DMF, and then mixed with a 1 mg / mL formate dehydrogenase mutant solution. The immobilization load was then investigated at immobilization times of 10, 20, 40, and 60 min to determine the optimal immobilization time. The optimal immobilization time for the formate dehydrogenase mutant was ultimately determined to be 20 min.

[0071] Example 6 Immobilized enzyme Cs FDH M Enzymatic characterization of @HOF-101

[0072] 1. Temperature stability

[0073] This invention aims to evaluate free formate dehydrogenase mutants ( Cs FDH M ) and immobilized enzymes Cs FDH M The stability of @HOF-101 at different temperatures, respectively, free Cs FDH M and Cs FDH M @HOF-101 was incubated at 20–55°C (in 5°C intervals) for 1 hour, and then the residual enzyme activity was measured at the optimal reaction temperature. The residual enzyme activity after 1 hour of ice bath storage was taken as 100% to examine the free enzyme activity. Cs FDH M and Cs FDH M Temperature stability of @HOF-101. Measurement results are as follows: Figure 2 As shown, after being stored at 30℃-55℃ for 1 hour, Cs FDH M The residual enzyme activity of @HOF-101 is relatively higher than that of free enzymes. Cs FDH M Significant improvements have been made.

[0074] 2. pH stability

[0075] This invention will free Cs FDH M and Cs FDH M @HOF-101 was incubated on ice for 1 h in buffer solutions with pH values ​​ranging from 2.5 to 11.5 (intervals of 0.5 and 1), and then in free... Cs FDH M and Cs FDH MThe residual enzyme activity of each enzyme was measured under the optimal conditions of @HOF-101, with the untreated value set at 100%, to determine the free enzyme activity. Cs FDH M and Cs FDH M The stability of @HOF-101 at different pH values. The test results are as follows: Figure 3 As shown, immobilized Cs FDH M @HOF-101 is relatively free in the pH range of 5.0-11.5. Cs FDH M It exhibited better stability. After storage at pH 6.0 for 1 hour, the residual enzyme activity still reached 100%, and it also showed better adaptability in neutral and alkaline environments. Therefore, the immobilized enzyme... Cs FDH M @HOF-101 is more conducive to the practical application of CO2 absorption and conversion.

[0076] 3. Determination of Michaelis constant and related kinetic parameters

[0077] The final concentration of sodium bicarbonate was fixed at 250 mmol / L. The final concentration of coenzyme NADH in the enzyme activity assay system was gradually increased (0.02, 0.06, 0.08, 0.1, 0.14, 0.18, 0.2 mM), and a certain mass of [unspecified ingredient] was added. Cs FDH M @HOF-101, detected under optimal conditions. Cs FDH M The corresponding enzymatic reaction rates of @HOF-101 at different coenzyme concentrations were thus determined. Cs FDH M @HOF-101 Michaelis constant for coenzyme NADH K m and reaction rate K cat The concentration of coenzyme NADH was fixed at 5 mmol / L, and the final concentration of sodium bicarbonate solution in the enzyme activity assay system was gradually increased (10, 25, 50, 75, 100, 150 mM), with a certain mass of [missing information - likely a specific ingredient or component] added. Cs FDH M @HOF-101, tested under the determined optimal conditions. Cs FDH M @HOF-101 represents the initial rate of the enzymatic reaction at different substrate concentrations. This is used to determine the Michaelis constant of the substrate sodium bicarbonate. K m and reaction rate K catValue determination. The measured data were nonlinearly fitted using Origin 9.0 software to obtain... Cs FDH M @HOF-101 Michael constants for different substrates K m and reaction rate K cat Value. The immobilization was measured. Cs FDH M @HOF-101 for substrates K m The value is 45 mM. K cat / K m The value is 0.15 s -1 ·mM -1 ; for coenzymes K m The value is 0.4 mM. K cat / K m The value is 43 s -1 ·mM -1 .

[0078] Example 7 Immobilized enzyme Cs FDH M Storage stability of @HOF-101

[0079] This invention will Cs FDH M @HOF-101 was placed in PBS buffer (100 mM, pH 7.0) and stored at 4°C for 30 days. Changes in enzyme activity were measured every 5 days to detect changes. Cs FDH M Storage stability of @HOF-101. Figure 4 After 30 days of preservation, free Cs FDH M and Cs FDH M Changes in the activity of @HOF-101. It can be seen that after storage at 4°C for 30 days, Cs FDH M @HOF-101 retained 90% of its initial activity, while under the same conditions, free... Cs FDH M The activity of the immobilized enzyme was only 40% of its initial activity. The results indicate that the immobilized enzyme... Cs FDH M @HOF-101 improves the structural stability of enzyme molecules, making free... Cs FDH MThe storage stability was greatly improved after immobilization.

[0080] Example 8 Immobilized enzyme Cs FDH M @HOF-101's reusability

[0081] This invention is for the determination Cs FDH M @HOF-101 reusability, continuous measurement Cs FDH M The activity of @HOF-101 was measured 10 times, and after each measurement... Cs FDH M After centrifugation using @HOF-101, the enzyme was washed with PBS buffer (100 mM, pH 7.0) before the next enzyme activity assay. In practical applications, the water solubility of enzyme molecules makes them difficult to recover and reuse; immobilizing FDH on a carrier can solve this problem. Immobilized enzyme Cs FDH M The reusability of @HOF-101 is as follows: Figure 5 As shown. After 10 reuses, Cs FDH M @HOF-101 retains 85% of its initial activity, indicating that... Cs FDH M Immobilization on HOF-101 can solve the problem of free radicals. Cs FDH M The inability to recycle makes Cs FDH M This makes practical applications possible.

[0082] Example 9 Cs FDH M @Preparation of HOF-101 carbon cloth electrode

[0083] In this invention, 0.1 g of the prepared immobilized enzyme was weighed. Cs FDH M @HOF-101 was placed in a mortar, and then 5 mL of 50% ethanol solution was added. The mixture was thoroughly ground, and then 0.5 mL of Nafion solution was added and the mixture was ground again. The thoroughly ground mixture was then evenly coated onto one side of a 10 mm * 10 mm carbon cloth using a brush and dried at 25 ℃ to obtain the immobilized enzyme. Cs FDH M A carbon cloth electrode prepared with @HOF-101 was used, with a catalyst loading of 0.85 mg / cm³. 2 .

[0084] Example 10 Cs FDHM Preparation of @HOF-101 glassy carbon electrode

[0085] In this invention, the carbon cloth in Example 9 is replaced with a glassy carbon electrode (GCE, d=3 mm), and the same method as in Example 9 is used to form a dispersed and immobilized enzyme on the surface of the glassy carbon electrode. Cs FDH M @HOF-101 membrane, thus obtaining immobilized enzyme Cs FDH M A glassy carbon electrode prepared using HOF-101 was used, with a catalyst loading of 1.0 mg / cm³. 2 .

[0086] Example 11 Electro-enzymatic conversion of CO2 to formic acid

[0087] 1. Use ​ FDH M @HOF-101 carbon cloth electrode catalyzes the conversion of CO2 to formic acid.

[0088] The catalytic method is carried out in an H-type electrolyzer. The working electrode is... ​ FDH M The electrode used was a HOF-101 carbon cloth electrode, with a platinum sheet as the counter electrode (5 mm × 5 mm × 0.1 mm) and an Ag / AgCl3 reference electrode (3.5 M KCl 6 × 65 mm, +198 mV vs. NHE). A proton exchange membrane (Nafion 115, Φ30 mm, 0.125 mm thick) was placed between the anode and cathode chambers. An electrochemical workstation was connected to the three electrodes used for data analysis and set to -0.75 V. The anode chamber was filled with 1 mM sulfuric acid, and the generated protons passed through the proton exchange membrane into the cathode chamber. CO2 was bubbled into the cathode chamber (working and reference electrodes) at a flow rate of 40 mL / min. The cathode chamber was filled with 35 mL of phosphate buffer (100 mM pH 7.0) and 0.1 mM NADH, and gently stirred with a magnetic stirrer. Samples were taken from the cathode chamber at different reaction times (2 h, 4 h, 6 h, 8 h, 10 h, and 12 h), filtered, and detected using a high-performance liquid chromatography (HPLC; Shimadzu LC-2020) system, a Shim-pack GIST C18 column (5 µm, 4.6 mm × 250 mm, Shimadzu), and a UV detector. The column and detector temperatures were set to 35 °C. The mobile phase was 5 mM sulfuric acid at a flow rate of 1 mL / min. The formic acid generated in the reaction was detected at 210 nm and eluted at 2.3 min. The HPLC chromatogram is shown below. ​ As shown.

[0089] Cyclic voltammetry (CV) was used to detect charge transfer information at the electrode surface during redox reactions. The voltage range was −0.8–0.2 V, and the scan rate was 20 mV / s. Six cycles were performed with or without NADH coenzyme until the current stabilized. Furthermore, cyclic voltammetry was performed at different scan rates of 10, 20, 50, 100, and 200 mV / s to investigate the diffusion process of the redox reaction. Based on the cyclic voltammetry results, the reduction reaction at -0.75 V yielded formic acid.

[0090] After 12 hours of reaction, the formic acid yield can reach 69 mM, such as ​ As shown.

[0091] 2. Use ​ FDH M @HOF-101 glassy carbon electrode catalyzes the conversion of CO2 to formic acid.

[0092] The catalytic method described in step 1 above ​ FDH M @HOF-101 carbon cloth electrode replaced with ​ FDH M The @HOF-101 glassy carbon electrode catalyzes CO2 using the same method as described in section 1 above. Qualitative and quantitative detection methods are also the same.

[0093] 3. Immobilization ​ FDH M @HOF-101 is added to the cathode chamber to catalyze CO2.

[0094] The catalytic method described in step 1 above ​ FDH M The HOF-101 carbon cloth electrode was replaced with a graphite rod (Φ6×90 mm), and an additional 0.5g of immobilized enzyme was added to the cathode chamber. ​ FDH M @HOF-101 and gently stir with a magnetic stirrer. The rest of the catalytic method for CO2 is the same as described in section 1 above. Qualitative and quantitative detection methods are the same as above.

[0095] In summary, the formate dehydrogenase mutant of the present invention ​ FDH M The immobilized enzyme exhibits high catalytic activity and high stability. The immobilized enzyme and the electrode material prepared therefrom can be used for electrocatalytic conversion of carbon dioxide to formic acid, showing promising application prospects and providing a win-win strategy for mitigating global warming and promoting the use of renewable energy.

[0096] Finally, it should be noted that the above embodiments 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 foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A formate dehydrogenase mutant, characterized in that, The formate dehydrogenase mutant is based on the wild-type formate dehydrogenase of Clostridium difficile with only the following mutations: methionine at position 269 is mutated to isoleucine, serine at position 285 is mutated to alanine, and serine at position 637 is mutated to alanine. The NCBI accession number for the wild-type formate dehydrogenase of Clostridium perfringens is WP_082085143.

1.

2. A nucleic acid, characterized in that, The nucleic acid is used to encode the formate dehydrogenase mutant of claim 1.

3. A biomaterial, characterized in that, The biomaterial includes the nucleic acid described in claim 2; the biomaterial is an expression cassette, vector, or transgenic cell.

4. A recombinant genetically engineered bacterium, characterized in that, The recombinant genetically engineered bacteria express the formate dehydrogenase mutant of claim 1, or contain the nucleic acid of claim 2, or carry a vector containing the nucleic acid of claim 2.

5. The recombinant genetically engineered bacteria according to claim 4, characterized in that, The recombinant genetically engineered bacteria are Escherichia coli or yeast.

6. Any one of the following applications of the formate dehydrogenase mutant of claim 1, the nucleic acid of claim 2, the biological material of claim 3, or the recombinant genetically engineered bacteria of claim 4 or 5: (1) Application in the preparation of immobilized formate dehydrogenase; (2) Application in the preparation of electrode materials for the electroenzymatic catalysis of CO2 to produce formic acid or formate salt; (3) Application in the production of formic acid or formate using carbon dioxide; (4) Application in the construction of engineered bacteria for converting carbon dioxide to produce formic acid or formate.

7. An immobilized formate dehydrogenase, characterized in that, The immobilized formate dehydrogenase comprises the formate dehydrogenase mutant of claim 1 and the immobilized vector.

8. The immobilized formate dehydrogenase according to claim 7, characterized in that, The immobilization carrier is HOF material; And / or, the mass ratio of the immobilized vector to the formate dehydrogenase mutant is 1:(0.28-1.39).

9. The method for preparing immobilized formate dehydrogenase according to claim 7 or 8, characterized in that, The method includes: mixing a formate dehydrogenase mutant with an immobilized carrier, such that the formate dehydrogenase mutant is adsorbed and encapsulated in the pores of the immobilized carrier.

10. An electrode for the electroenzymatic catalysis of CO2 to prepare formic acid or formate, characterized in that, The electrode comprises an electrode body and a catalyst coated on the surface of the electrode body; The catalyst comprises the immobilized formate dehydrogenase as described in claim 7 or 8.

11. The use of the immobilized formate dehydrogenase of claim 7 or 8 or the electrode of claim 10 in the catalytic preparation of formic acid or formate salt from CO2.

Images