Immobilized formate dehydrogenase mutant and application thereof in electroenzymatic catalysis of CO2

Abstract

The invention relates to the technical field of biological catalysis, in particular to an immobilized formate dehydrogenase mutant and application of the immobilized formate dehydrogenase mutant in electroenzymatic catalysis of CO2. The formate dehydrogenase mutant comprises the following mutations on the basis of clostridium wild formate dehydrogenase: methionine at the 269th site is mutated into isoleucine, serine at the 285th site is mutated into alanine, and serine at the 637th site is mutated into alanine; the NCBI (National Center of Biotechnology Information) accession number of the wild formate dehydrogenase of the clostridium is WP082085143.1. The immobilized formate dehydrogenase mutant provided by the invention has better catalytic activity, temperature, pH stability and storage stability, and can be repeatedly utilized. The immobilized formate dehydrogenase mutant or an electrode material prepared from the immobilized formate dehydrogenase mutant can efficiently catalyze CO2 to be converted into formic acid or formate, which has important significance in the field of renewable energy utilization.

Description

Technical Field

[0001] The present invention relates to the field of biocatalysis technology, in particular to an immobilized formate dehydrogenase mutant and its electroenzymatic CO 2 Application in. Background Art

[0002] Greenhouse gas CO 2 Large-scale emissions have caused adverse effects such as global temperature rise, sea level rise, glacier melting, and land desertification, posing a serious threat to human survival. There are currently many ways to solve the greenhouse effect, such as promoting the use of renewable energy, improving energy utilization, or planting trees. Existing technologies currently focus on how to deal with CO 2 Reuse and convert CO 2 Recycling it into energy and other valuable chemicals will not only help reduce the carbon dioxide content in the atmosphere, but also provide a solution to the energy crisis. 2 It is a technical problem that needs to be solved urgently and has good development prospects.

[0003] CO 2 It can be converted into high value-added chemicals such as formic acid, formaldehyde and methanol. Formic acid is an important chemical raw material and is widely used in various industries such as chemical production, rubber manufacturing, textiles, printing and dyeing, and electroplating. 2 It has the characteristics of high selectivity, high efficiency and mild conditions for formic acid, which is superior to chemical catalysis, electrocatalysis and photoelectrocatalysis. Electroenzyme catalysis uses electricity as a reducing force to promote the reaction under mild conditions and achieve coenzyme regeneration, solving the problem of expensive coenzymes. However, the formate dehydrogenases reported so far still have problems such as low enzyme activity, poor stability and inability to be reused. And free enzymes face the challenges of poor environmental stability and limited recyclability. Therefore, immobilizing the enzyme on a solid phase carrier can improve its tolerance to operating conditions and maintain catalytic activity and stability. This method can achieve the reuse of enzymes, thereby promoting their industrial applications. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the present invention provides an immobilized formate dehydrogenase mutant and its electroenzymatic CO 2 Application in.

[0005] The present invention uses genetic engineering technology to obtain a high-activity formate dehydrogenase Cl FDH was used as a probe to discover a new formate dehydrogenase gene ( Cs The amino acid residues in the predicted conservative region of formate dehydrogenase (FDH) were mutated by rational design, and a formate dehydrogenase mutant with high catalytic activity was obtained through screening.

[0006] In a first aspect, the present invention provides a formate dehydrogenase mutant, wherein the formate dehydrogenase mutant is a formate dehydrogenase mutant in Clostridium ( Clostridium scatologenes ) The wild-type formate dehydrogenase 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 difficile Clostridium scatologenes ) The NCBI accession number of the 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 higher than the activity of the formate dehydrogenase before mutation, and can catalyze the conversion of carbon dioxide into formic acid or formate more efficiently.

[0010] In a second aspect, the present invention provides a nucleic acid for encoding the formate dehydrogenase mutant.

[0011] According to the amino acid sequence and codon rule 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. Based on the degeneracy of codons, 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 protection scope of the present invention.

[0012] The nucleic acid preferably comprises the nucleotide sequence shown in SEQ ID NO.2.

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

[0014] In a third aspect, the present invention provides a biological material, wherein the biological material comprises the nucleic acid; the biological material is an expression cassette, a vector or a transgenic cell.

[0015] The expression cassette of the present invention is a recombinant nucleic acid molecule obtained by operably linking the nucleic acid molecule with a transcription and / or translation regulatory element.

[0016] The vectors of the present invention include but are not limited to plasmid vectors, viral vectors, transposons, etc. Among them, plasmid vectors include expression vectors and cloning vectors.

[0017] The transgenic cells of the present invention do not include transgenic cells that have the ability to independently develop into a complete individual, that is, do not include animal species and plant species. For example, it can be Escherichia bacteria or yeast, and more preferably Escherichia coli.

[0018] In a fourth aspect, the present invention provides a recombinant genetically engineered bacterium, which 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 is provided, which expresses the formate dehydrogenase mutant described above and can be used to produce the formate dehydrogenase mutant described above.

[0020] In a fifth aspect, the present invention provides any one of the following uses 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) In the preparation of electroenzymatic CO 2 Application in the preparation of electrode materials for formic acid or formates; (3) Application in catalyzing the conversion of carbon dioxide into formic acid or formate; (4) Application in the production of formic acid or formates 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 carrier; Preferably, the immobilization carrier is a HOF material; And / or, the mass ratio of the immobilized carrier to the formate dehydrogenase mutant is 1:(0.28-1.39).

[0022] In some embodiments of the present invention, the immobilized carrier is a HOF material. Preferably, it is HOF-101. HOF-101 is a new type of HOF material. The metal-free property of HOF makes it more biocompatible than MOF materials. The present invention prepares an immobilized enzyme by immobilizing the formate dehydrogenase mutant with HOF-101, which significantly improves the stability (including temperature stability, pH stability and storage stability) of the formate dehydrogenase mutant, and solves the problem that the free enzyme cannot be recycled. The obtained immobilized enzyme has high catalytic activity, high stability and reusability, and has good performance on substrate. 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 can still maintain 90% of its initial activity; after being reused 10 times, the activity can still maintain 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 the formate dehydrogenase mutant with an immobilized carrier, so that the formate dehydrogenase mutant is adsorbed and encapsulated in the pore size of the immobilized carrier, for example, by a pore diffusion method.

[0024] In an eighth aspect, the present invention provides a method for electroenzymatically catalyzing CO 2 An electrode for preparing formic acid or formate, wherein the electrode comprises 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, there is provided a method for electroenzymatic catalysis of CO 2 A carbon cloth electrode for preparing formic acid comprises a carbon cloth electrode and a catalyst coated on the surface of the carbon cloth electrode, wherein the catalyst is the above-mentioned immobilized formate dehydrogenase. Preferably, the loading amount of the catalyst in the electrode is controlled to be 0.6-1.5 mg / cm 2 .

[0027] In some other embodiments of the present invention, there is provided a method for electroenzymatically catalyzing CO 2A glassy carbon electrode for preparing formic acid comprises a glassy carbon electrode and a catalyst coated on the surface of the glassy carbon electrode, wherein the catalyst is the above-mentioned immobilized formate dehydrogenase. Preferably, the loading amount of the catalyst in the electrode is controlled to be 0.6-1.5 mg / cm 2 .

[0028] In a ninth aspect, the present invention provides the immobilized formate dehydrogenase or the electrode in catalyzing CO 2 Application in the preparation of formic acid or formates.

[0029] In a tenth aspect, the present invention provides a method for preparing formic acid, the method comprising: 2 As a substrate, the immobilized formate dehydrogenase or the electrode is used to perform electroenzymatic catalysis of CO 2 Converted to formic acid.

[0030] The present invention has the following beneficial effects: 1. The present invention is in Clostridium ( Clostridium scatologenes ) A formate dehydrogenase mutant was obtained based on the wild-type formate dehydrogenase, which has a higher catalytic activity and a specific enzyme activity of 896 mU / mg. Cl FDH has 89 times the activity of the enzyme, which is higher than that of Clostridium difficile before mutation ( Clostridium scatologenes ) Formate dehydrogenase is 5 times more active than formate and can efficiently catalyze the conversion of carbon dioxide into formic acid or formate.

[0031] 2. The stability of the immobilized enzyme prepared by combining the formate dehydrogenase mutant provided by the present invention with the HOF material is significantly improved, including significantly improved temperature stability (after being stored at 30°C-55°C for 1 hour, the residual enzyme activity is significantly higher than that of the free enzyme); in the pH range of 5.0-11.5, it exhibits higher stability than the free enzyme (after being stored 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 absorption and conversion of CO 2 production practice application; and the immobilized enzyme molecule has higher structural stability, which greatly improves the storage stability of the immobilized enzyme (after being stored at 4°C for 30 days, it still maintains 90% of the initial activity, while under the same conditions, the activity of the free enzyme only maintains 40% of the initial activity); in addition, the immobilized enzyme solves the problem that the free enzyme cannot be recycled (the activity of the immobilized formate dehydrogenase of the present invention can still be maintained at 85% of the initial activity after 10 repeated uses), which is conducive to the practical application of formate dehydrogenase.

[0032] 3. Enzyme-electrochemical coupling catalysis of CO by using the immobilized formate dehydrogenase of the present invention and the carbon cloth electrode or glassy carbon electrode prepared therefrom 2The production of formic acid or formate can realize the recycling of coenzyme NADH without the need for additional hydrogen, greatly reducing production costs and improving production safety. The immobilized formate dehydrogenase provided by the present invention has good industrialization prospects and is of great value in the field of renewable energy utilization. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0034] Figure 1 HOF-101 (a) and immobilized formate dehydrogenase mutant provided in Example 4 of the present invention Cs FDH M Scanning electron micrograph of @HOF-101 (b).

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

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

[0038] Figure 5 The immobilized enzyme provided in Example 8 of the present invention Cs FDH M @HOF-101's reuse count graph.

[0039] Figure 6 CO provided in Example 11 of the present invention 2 HPLC detection spectrum of conversion to formic acid.

[0040] Figure 7 The immobilized enzyme provided in Example 11 of the present invention Cs FDH M @HOF-101 Preparation of carbon cloth electrode electroenzymatic catalysis of CO 2Diagram of the process of producing formic acid. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] The experimental methods involved in the following examples, unless otherwise mentioned, are all conventional methods in the art, for example, reference may be made to experimental manuals in the art, or the conditions recommended in the manufacturer's instructions.

[0043] Unless otherwise specified, the experimental materials and reagents involved in the following examples can be obtained from commercial sources, for example: The general development process of the present invention is as follows: The present invention uses genome mining technology to identify the previously reported high-activity formate dehydrogenase Cl The protein sequence of FDH was used as a probe, and BLAST analysis was performed in various genomic data. A series of hypothetical formate dehydrogenases with genomic information sources and no expression identification were found from the query results. Evolutionary trees were constructed and analyzed for these sequences, and then representative gene sequences were selected. The selected genes were codon optimized and then fully synthesized, and expression screening was performed. Among them, the gene from Clostridium ( Clostridium scatologenes ) genome, a new type of highly active formate dehydrogenase ( Cs FDH, to the formate dehydrogenase ( Cs FDH) was used as the parent for computer-assisted rational design. The parent formate dehydrogenase ( Cs The three-dimensional structure of FDH was analyzed by using Consensus Finder to analyze the amino acid conservation of the protein, and CAVER was used to analyze its substrate channel to preliminarily determine the mutation site. Then, the protein ( Cs FDH) and substrates and coenzymes were subjected to semi-flexible molecular docking simulations, and the three-dimensional structure of the obtained complex was analyzed using Pymol and Discovery Studio software to exclude the key amino acid residues in the active center pocket. Finally, the 269th methionine (Met) was mutated to isoleucine (Ile), the 285th serine (Ser) was mutated to alanine (Ala), and the 637th serine (Ser) was mutated to alanine (Ala). The mutation was achieved through improved whole plasmid amplification technology, and finally the CO was successfully obtained. 2Mutants with high catalytic activity Cs FDH M The mutant was further fixed to the HOF material HOF-101 to obtain the immobilized enzyme. Cs FDH M @HOF-101, enhancing its stability and reusability, and by immobilizing the enzyme Cs FDH M @HOF-101, carbon cloth electrode or glassy carbon electrode prepared by immobilized enzyme for enzyme-electrochemical coupling catalysis of CO 2 Production of formic acid. The carbon cloth electrode prepared by immobilized enzyme catalyzes CO 2 The production of formic acid reached 69 mM after 12 h.

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

[0045] The present invention uses the previously obtained high-activity formate dehydrogenase Cl The protein sequence of FDH was used as a probe, and BLAST analysis was performed with various genomic sequences as search objects. From the query results, a series of hypothetical formate dehydrogenases with genomic information sources and not yet expressed and identified were found. Evolutionary trees were constructed and analyzed for these sequences, and then three representative gene sequences were selected for full gene synthesis after codon optimization. After comparative analysis, Clostridium senegalense , Clostridium drakei and Clostridium scatologenes The three potential formate dehydrogenases from the source were further studied and named Cse FDH, Cd FDH and Cs FDH.

[0046] by E.coli The codon usage frequency of K12 strain was used as a reference, and the OPTIMIZER server was used to optimize the codons of the potential formate dehydrogenase gene sequence. Bam HI and Nde I restriction site, and then commissioned Qingke Biotechnology Co., Ltd. to synthesize the whole gene. The synthesized formate dehydrogenase encoding gene was connected to the pET28a plasmid to obtain the recombinant vector pET28a- Cse FDH, pET28a- Cd FDH and pET28a- Cs FDH.

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

[0048] The present invention uses the formate dehydrogenase with the highest catalytic activity among the three formate dehydrogenases screened in Example 1 Cs FDH was used as the parent for computer-assisted rational design. First, the parent formate dehydrogenase ( Cs The three-dimensional structure of FDH was analyzed by using Consensus Finder and CAVER to analyze the substrate channel, and the mutation sites were preliminarily determined. Cs The three-dimensional structure of FDH was subjected to semi-flexible molecular docking analysis with the substrate sodium bicarbonate and the coenzyme NADH, and then observed and analyzed using PyMol and Discovery studio software to determine the substrate channel of formate dehydrogenase and the key amino acid residues and metal ions that interact with the substrate and coenzyme, and exclude the key amino acid residues in the active center pocket. Finally, the 269th methionine (Met) was mutated to isoleucine (Ile), the 285th serine (Ser) was mutated to alanine (Ala), and the 637th serine (Ser) was mutated to alanine (Ala). The mutated formate dehydrogenase (named Cs FDH M ) is shown in SEQ ID NO.2, and its amino acid sequence is shown in SEQ ID NO.1.

[0049] The mutation was carried out by using improved whole plasmid amplification technology to recombinant vector pET28a- Cs FDH was used as a template, and a reverse universal primer (R) was designed at about 3.2 kb downstream of the vector cloning site. Forward mutation primers F1, F2, and F3 were designed at about 15 bp upstream and downstream of the three mutation sites. A pair of forward and reverse primers were used for the first round of PCR amplification, and the product of the first round was used as a large primer to amplify the whole plasmid. After two rounds of PCR amplification, Dpn I digested the template to obtain a recombinant vector with one mutation site; then, the recombinant vector with one mutation site was used as a template, and the remaining two pairs of forward and reverse primers were used for the second and third amplifications according to the above method to obtain a recombinant vector pET28a- Cs FDH M The above primer sequences are shown in Table 1.

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

[0051] Example 3 Construction of recombinant genetically engineered bacteria expressing formate dehydrogenase mutants and induced expression and analysis of formate dehydrogenase mutants

[0052] 1. Recombinant vector pET28a- Cs FDH M Transform into host cells: recombinant vector pET28a- Cs FDH M The cells were transformed into competent E. coli BL21 (DE3) 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 monoclonal colonies.

[0053] 2. Screening and identification of recombinant genetically engineered bacteria: Pick a single clone colony and place it in 5 mL of LB liquid culture medium containing 50 μg / mL ampicillin, culture it overnight at 37°C and 220 rpm, identify the bacterial solution by PCR the next day, and extract the plasmid for use Bam HI and Nde I was double-enzyme digested and identified. According to the electrophoresis results, it was judged that the clone contained a gene fragment with the same size as the target gene, and the clone was preliminarily identified as a positive clone. The positive clone was then sent to Shanghai Biotechnology for sequence determination, and the sequencing results further showed that the clone colony was the target genetic engineering bacteria.

[0054] 3. Induced expression: Select the identified single colony of the recombinant genetically engineered bacteria obtained above and inoculate it into 5 mL of LB liquid culture medium containing 50 μg / mL ampicillin, and culture it overnight at 37°C and 220 rpm; use a pipette to inoculate the product of overnight culture into 100 mL of LB liquid culture medium at a 2% inoculation rate; then, culture it at 37°C and 200 rpm for 2.5 h, and finally add IPTG with a final concentration of 0.1 mmol / L for induction, and culture it at 16°C and 220 rpm for 20 h to obtain the induced expression bacterial solution (fermentation broth).

[0055] The induced expression bacterial solution was divided into 50 mL centrifuge tubes, centrifuged at 8000 rpm and 4°C for 5 min, the bacterial cells were collected, and each was washed twice with 50 mL of deionized water, and the bacterial cells were collected under the same conditions. Each of the above bacterial cells was suspended in 100 mL of lysis buffer (PBS buffer, pH = 7.0), and the cells were broken by a high-pressure homogenizer at 4°C and 900 bar for 3 minutes. Centrifuged at 12000 rpm and 4°C for 15 min, and the supernatant and precipitate were collected respectively. The supernatant was purified by Ni-Agarose medium affinity chromatography, and the expression morphology of the target protein was analyzed by SDS-PAGE.

[0056] 4. Enzyme activity determination: Use a UV spectrophotometer to measure the change in the absorbance of NADH at 340 nm (εNADH, 340 nm = 6.22 mM-1 cm -1 ). The reaction was initiated by adding formate dehydrogenase and monitored for 1 minute. The reaction system for enzyme activity determination was 1 mL: containing 0.1 mM NADH, 100 mM sodium bicarbonate, 100 mM PBS buffer, pH=7.0. After the enzyme solution was diluted appropriately, 100 μL was added to the above reaction system incubated at 30°C for 2 min. The change in absorbance within 1 min was measured at 340 nm. Under this condition, the amount of enzyme required to consume 1 μmol NADH per minute is defined as 1U. 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: the volume of the reaction solution (mL), 6220: molar extinction coefficient (L*mol -1 *cm -1 ), L: optical path distance (cm).

[0057] It was determined that formate dehydrogenase mutant Cs FDH M The specific activity of the pure enzyme reached 896 mU / mg, which is a good probe Cl FDH (10mU / mg) was 89 times higher than that of formate dehydrogenase before mutation. Cs FDH (the difference between the coding gene sequence and the sequence shown in SEQ ID NO.2 is that the 269th codon is a methionine codon, the 285th codon is a serine codon, and the 637th codon is a serine codon. The recombinant vector pET28a- Cs The construction of FDH was described in Example 2) as a control. The results showed that formate dehydrogenase before mutation Cs The specific activity of FDH was 179 mU / mg, and the formate dehydrogenase mutant Cs FDH M The specific activity (896 mU / mg) of formate dehydrogenase before mutation Cs 5 times of FDH.

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

[0059] 1. Synthesis using an improved mild de novo protein-directed assembly method Cs FDH M @HOF-101. The first step was to prepare the formate dehydrogenase mutant at a concentration of 2 mg / ml using PBS buffer (100 mM, pH = 7.0). Cs FDH M Then 20 mg H 4TBAPy was dissolved in 2 mL DMF by ultrasonication. Subsequently, 18 mL Cs FDH M The solution (1 mg / ml) was stirred continuously at 25 °C for 20 min to promote the formation of the composite material. After the stirring process was completed, the obtained Cs FDH M @HOF-101 composite material. The centrifugation conditions were: 25°C, 12000 rpm, 15 min. After collecting the supernatant, the collected precipitate was washed three times with PBS buffer (100mM, pH=7.0), centrifuged and dried under the same conditions to remove any residual solvent and unreacted precursors. The HOF material immobilized formate dehydrogenase mutant (abbreviated as immobilized formate dehydrogenase) was obtained. Cs FDH M @HOF-101.

[0060] All supernatants collected after centrifugation were combined and the Cs FDH M The content of Cs FDH M The content comparison can be used to obtain the immobilized on the carrier HOF-101 Cs FDH M The solid loading amount can be calculated from this. The calculation method of the solid loading amount is as follows:

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

[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. From the image, we can see that the initial synthesized HOF-101 and the immobilized Cs FDH M The HOF-101 after the treatment all had a clear rod-like structure, indicating that the morphology of HOF-101 was not affected by Cs FDH M of fixation is destroyed.

[0063] 2. Determination of the reduction activity of the immobilized enzyme: Use a UV spectrophotometer to measure the change in the absorbance of NADH at 340 nm (εNADH, 340 nm = 6.22 mM -1 cm -1 ). By adding a certain amount of immobilized formate dehydrogenase Cs FDH M @HOF-101 initiated the reaction and monitored for 1 minute. The reaction system for enzyme activity determination 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 amount of immobilized formate dehydrogenase Cs FDH M @HOF-101 was added to the above reaction system. The change in absorbance within 1 min was measured at 340 nm. Under this condition, the amount of enzyme required to consume 1 μmol NADH per minute was defined as 1 U.

[0064]

[0065] EW: OD in 1 minute 340 The change value, V: the volume of the 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 of optimal fixation conditions

[0067] 1. Optimal carrier dosage

[0068] In order to determine the optimal dosage of the carrier HOF-101 used for immobilizing formate dehydrogenase, 5, 10, 15, 20 and 25 mg of HOF-101 were added to the immobilized formate dehydrogenase. 4 TBAPy was dissolved in 2 mL DMF by ultrasonication, and 18 mL Cs FDH M The solution (1 mg / ml) was used to immobilize formate dehydrogenase, and then the activity of formate dehydrogenase was determined under different HOF-101 dosages. Cs FDH M The solid loading and Cs FDH M @HOF-101 activity. Finally, it was immobilized Cs FDH M The optimal amount of HOF-101 required was 20 mg / 18 mL of 1 mg / mL formate dehydrogenase mutant solution. At this time, the immobilization amount of the carrier HOF-01 was 78 mg / g.

[0069] 2. Optimal fixation time

[0070] The present invention adds a certain amount of H 4 After TBAPy was dissolved in 2 mL DMF by ultrasonication, it was mixed with 1 mg / mL formate dehydrogenase mutant solution, and the immobilization capacity was investigated at immobilization times of 10, 20, 40, and 60 min to determine the optimal immobilization time. The optimal immobilization time for formate dehydrogenase mutant was finally determined to be 20 min.

[0071] Example 6 Immobilized enzyme Cs FDH M Characterization of enzymatic properties of @HOF-101

[0072] 1. Temperature stability

[0073] The present invention is to evaluate the free formate dehydrogenase mutant ( Cs FDH M ) and immobilized enzymes Cs FDH M @HOF-101 stability at different temperatures, respectively Cs FDH M and Cs FDH M @HOF-101 was kept at 20-55℃ (5℃ interval) for 1h, and then the residual enzyme activity was measured at the optimal reaction temperature. The residual enzyme activity after 1h of ice bath was 100%, so as to investigate the free Cs FDH M and Cs FDH M @HOF-101 temperature stability. The test results are as follows Figure 2 As shown, after being stored at 30℃-55℃ for 1h, Cs FDH M @HOF-101 residual enzyme activity is relatively free Cs FDH M Got a big improvement.

[0074] 2. pH stability

[0075] The present invention will free Cs FDH M and Cs FDH M @HOF-101 was placed in a buffer solution with a pH value of 2.5-11.5 (intervals of 0.5 and 1) on ice for 1 h, and then Cs FDH M and Cs FDH M@HOF-101 was used to determine the residual enzyme activity under the optimal conditions, with the untreated value as 100% to determine the free Cs FDH M and Cs FDH M @HOF-101 stability at different pH values. The test results are as follows Figure 3 As shown, the fixed Cs FDH M @HOF-101 is relatively free in the pH range of 5.0-11.5 Cs FDH M It showed better stability. After being stored at pH 6.0 for 1 hour, the residual enzyme activity still reached 100%, and it also had better adaptability in neutral and alkaline environments. Cs FDH M @HOF-101 is more conducive to the absorption and conversion of CO 2 practical applications.

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

[0077] The final concentration of sodium bicarbonate was fixed at 250 mmol / L, and 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). Cs FDH M @HOF-101, tested under optimal conditions Cs FDH M @HOF-101 The corresponding enzymatic reaction rate at different coenzyme concentrations. Cs FDH M @HOF-101 Michaelis constant for coenzyme NADH K m and reaction rate K cat Similarly, 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). Cs FDH M @HOF-101, tested under the optimal conditions determined Cs FDH M @HOF-101 The initial velocity of the enzymatic reaction at different substrate concentrations. This is used to complete the Michaelis constant of the substrate sodium bicarbonate. K m and reaction rate K catThe measured data were fitted nonlinearly using Origin 9.0 software to obtain Cs FDH M @The Michaelis constants of HOF-101 for different substrates K m and reaction rate K cat The immobilization value was measured Cs FDH M @HOF-101 substrate 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] The present 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. The enzyme activity was measured every 5 days to detect Cs FDH M @HOF-101 storage stability. Figure 4 After 30 days of storage, the free Cs FDH M and Cs FDH M @HOF-101 activity changes. It can be seen that after being stored at 4°C for 30 days, Cs FDH M @HOF-101 still maintains 90% of its initial activity, while under the same conditions, free Cs FDH M The activity of the immobilized enzyme only retained 40% of its initial activity. Cs FDH M @HOF-101 improves the structural stability of the enzyme molecule, making it free Cs FDH MThe storage stability was greatly improved after immobilization.

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

[0081] The present invention is to determine Cs FDH M @HOF-101 reusability, continuous measurement Cs FDH M @HOF-101 activity was measured 10 times, and after each measurement Cs FDH M @HOF-101 was centrifuged and washed with PBS buffer (100 mM, pH 7.0) before the next enzyme activity test. In practical applications, the water solubility of enzyme molecules makes them difficult to recycle. Immobilizing FDH on a carrier can solve this problem. Immobilized enzyme Cs FDH M @HOF-101 is reusable as Figure 5 After 10 times of reuse, Cs FDH M @HOF-101 can still maintain 85% of its initial activity, indicating that Cs FDH M Immobilization on HOF-101 can resolve free Cs FDH M The problem of not being able to recycle Cs FDH M practical applications become possible.

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

[0083] The present invention weighs 0.1 g of the prepared immobilized enzyme Cs FDH M @HOF-101 was placed in a mortar, and then 5 mL of 50% ethanol solution was added to fully grind the mixed slurry, and then 0.5 mL of Nafion solution was added to continue fully grinding. The fully ground slurry was evenly coated on one side of a 10 mm*10 mm carbon cloth with a brush and dried at 25 °C to obtain the immobilized enzyme. Cs FDH M @HOF-101 prepared carbon cloth electrode, the catalyst loading in the electrode is 0.85 mg / cm 2 .

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

[0085] In the present invention, the carbon cloth in Example 9 is replaced with a glassy carbon electrode (GCE, d=3 mm), and the immobilized enzyme is dispersed and fixed on the surface of the glassy carbon electrode by the same method as in Example 9. Cs FDH M @HOF-101 film, thereby obtaining immobilized enzyme Cs FDH M Glassy carbon electrode prepared by @HOF-101, the catalyst loading in the electrode is 1.0 mg / cm 2 .

[0086] Example 11 Electroenzyme catalysis of CO 2 Converted to formic acid

[0087] 1. Use Cs FDH M @HOF-101 Carbon cloth electrode catalyzes CO 2 Converted to formic acid

[0088] The catalytic method is carried out in an H-type electrolytic cell. The working electrode is Cs FDH M @HOF-101 carbon cloth electrode, counter electrode is platinum sheet (5 mm×5 mm×0.1 mm), reference electrode is Ag / AgCl 3 (3.5 M KCl 6×65 mm, +198 mVvs. NHE). A proton exchange membrane (Nafion115, Φ30 mm, 0.125 mm thick) was placed between the anode chamber and the cathode chamber. The electrochemical workstation was connected to three electrodes for data analysis and the voltage was set to -0.75 V. The anode chamber was filled with 1 mM sulfuric acid, and the generated protons entered the cathode chamber through the proton exchange membrane. CO was bubbled into the cathode chamber (working electrode and reference electrode) at a flow rate of 40 mL / min. 2 , 35 mL of phosphate buffer (100 mM pH 7.0) and 0.1 mM NADH were placed in the cathode chamber and gently stirred with a magnetic stirrer. After sampling from the cathode chamber at different reaction times (2h, 4h, 6h, 8h, 10h and 12h) and filtering, detection was performed using a high performance liquid chromatography system (HPLC; Shimadzu LC-2020), a Shim-pack GIST C18 column (5µm, 4.6 mm×250 mm, Shimadzu) and a UV detector. The temperature of the column and detector was set to 35°C. The mobile phase was 5 mM sulfuric acid at a flow rate of 1 mL / min. The formic acid generated by the reaction was detected at 210 nm and eluted at 2.3 min. The HPLC detection spectrum is shown below Figure 6 shown.

[0089] Cyclic voltammetry (CV) was used to detect the information of charge transfer in the redox reaction on the electrode surface. The voltage range was -0.8~0.2V, the scan rate was 20 mV / s, and 6 cycles were performed with or without the addition of coenzyme NADH until the current stabilized. In addition, cyclic voltammetry was performed at different scan rates of 10, 20, 50, 100 and 200 mV / s to study the diffusion process of the redox reaction. According to the results of cyclic voltammetry, the reduction reaction was carried out at -0.75V to generate formic acid.

[0090] After 12 h of reaction, the formic acid production can reach 69 mM. Figure 7 shown.

[0091] 2. Use Cs FDH M @HOF-101 Glassy Carbon Electrode Catalyzes CO 2 Converted to formic acid

[0092] The catalytic method described in step 1 above Cs FDH M @HOF-101 carbon cloth electrode replaced by Cs FDH M @HOF-101 glassy carbon electrode, catalyzed CO using the same method as in 1 above 2 The qualitative and quantitative detection methods are the same as above.

[0093] 3. Immobilize Cs FDH M @HOF-101 is added to the cathode chamber to catalyze CO 2

[0094] The catalytic method described in step 1 above Cs FDH M @HOF-101 The carbon cloth electrode was replaced with a graphite rod (Φ6×90 mm), and 0.5 g of immobilized enzyme was added to the cathode chamber. Cs FDH M @HOF-101, and gently stir with a magnetic stirrer. The rest is the same as the catalytic method 1 above to catalyze CO 2 The qualitative and quantitative detection methods are the same as above.

[0095] In summary, the formate dehydrogenase mutant of the present invention Cs FDH M The immobilized enzyme has high catalytic activity and high stability. The immobilized enzyme of the present invention and the electrode material prepared therefrom can be used for electroenzymatic conversion of carbon dioxide into formic acid, have good application prospects, and provide a win-win strategy for alleviating 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, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions 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 a mutant of Clostridium ( Clostridium scatologenes ) The wild-type formate dehydrogenase 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 difficile Clostridium scatologenes ) The NCBI accession number of the wild-type formate dehydrogenase is WP_082085143.

1.

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

1.

3. A nucleic acid, characterized in that The nucleic acid is used to encode the formate dehydrogenase mutant according to claim 2; the nucleic acid preferably comprises the nucleotide sequence shown in SEQ ID NO.

2.

4. A biomaterial, characterized in that: The biological material comprises the nucleic acid according to claim 3; the biological material is an expression cassette, a vector or a transgenic cell.

5. A recombinant genetically engineered bacterium, characterized in that: The recombinant genetically engineered bacteria expresses the formate dehydrogenase mutant according to claim 1 or 2, or comprises the nucleic acid according to claim 3, or carries a vector comprising the nucleic acid according to claim 3; preferably, the recombinant genetically engineered bacteria is Escherichia coli or yeast.

6. Any one of the following uses of the formate dehydrogenase mutant according to claim 1 or 2, the nucleic acid according to claim 3, the biological material according to claim 4, or the recombinant genetically engineered bacteria according to claim 5: (1) Application in the preparation of immobilized formate dehydrogenase; (2) Application in the preparation of electrode materials for electroenzymatic CO2 catalysis to produce formic acid or formate; (3) Application in catalyzing the conversion of carbon dioxide into formic acid or formate; (4) Application in the production of formic acid or formates using carbon dioxide; (5) 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 according to claim 1 or 2 and an immobilization carrier; Preferably, the immobilization carrier is a HOF material; And / or, the mass ratio of the immobilized carrier to the formate dehydrogenase mutant is 1:(0.28-1.39).

8. The method for preparing immobilized formate dehydrogenase according to claim 7, characterized in that: The method comprises: mixing a formate dehydrogenase mutant with an immobilized carrier, so that the formate dehydrogenase mutant is adsorbed and encapsulated in the pore size of the immobilized carrier.

9. An electrode for preparing formic acid or formate by electroenzymatic catalysis of CO2, 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 according to claim 7.

10. Use of the immobilized formate dehydrogenase according to claim 7 or the electrode according to claim 9 in catalyzing CO2 to produce formic acid or formate.

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