Method for expressing multi-subunit hydrogenase and producing formic acid by using formate dehydrogenase

Abstract

The present application belongs to the technical field of hydrogen conversion and energy storage, and particularly relates to a method for expressing multi-subunit hydrogenase and producing formic acid by using formate dehydrogenase. The present application uses a pBBR1MCS-1 vector, adds a Lac promoter in front of each subunit sequence of hydrogenase, so that each gene is controlled by a single promoter, and adds a His tag to each gene for subsequent purification of expressed protein. After that, the constructed vector is transformed into Ralstonia eutropha H16 competent cells to obtain an engineering bacterium and express hydrogenase in the engineering bacterium. The recombinantly expressed hydrogenase is combined with formate dehydrogenase to realize self-circulation regeneration of NAD + / NADH in the reaction system without additional NADH, thereby efficiently converting H2 and CO2 into formic acid in a green and economical manner, and realizing resource utilization of green hydrogen and high-value conversion of greenhouse gas CO2.

Description

Technical Field

[0001] This invention belongs to the field of hydrogen conversion and energy storage technology, specifically relating to a recombinant expression system capable of efficiently co-expressing multi-subunit hydrogenase, a recombinant engineered bacterium containing the system, and a method for directly synthesizing formic acid from hydrogen (H2) and carbon dioxide (CO2) by combining the hydrogenase with formic acid dehydrogenase. Background Technology

[0002] NADH, as a core reduced coenzyme in organisms, plays an irreplaceable role in redox enzymatic reactions, chiral compound synthesis, CO2 fixation, and biomanufacturing. However, the high cost of NADH and the shortcomings of traditional regeneration methods severely restrict the industrial application of NADH-dependent biocatalytic processes. Traditional cofactor regeneration methods (such as sacrificial substrate methods using glucose and formic acid) suffer from poor atom economy, numerous byproducts, and high separation costs; electrochemical and photochemical regeneration methods face challenges such as poor selectivity and insufficient stability.

[0003] Hydrogenases are a class of metalloenzymes capable of reversibly catalyzing the oxidation of H2 or the reduction of protons. Among them, NAD3... + Hydrogenases that are NAD+ dependent can use H2 as an electron donor to convert NAD+ into NAD+ under normal temperature and pressure. + This method efficiently reduces hydrogenase to NADH with 100% atom utilization and no byproducts, combining the advantages of high enzyme selectivity and the cleanliness of green hydrogen. However, most natural hydrogenases are multi-subunit complex enzymes, and achieving balanced and soluble expression of each subunit in common heterologous hosts such as E. coli has always been a technical challenge in this field. In particular, the oxygen-tolerant hydrogenase (SH hydrogenase) derived from Ralstonia eutropha has an active center composed of four subunits (HoxF, HoxU, HoxY, HoxH). Traditional co-expression strategies often require multiple plasmids or complex induction regulation, resulting in low expression efficiency and poor activity recovery, which is difficult to meet application requirements.

[0004] Therefore, developing a recombinant expression system capable of efficiently and evenly expressing multi-subunit hydrogenases and coupling it with CO2 reductases (such as formate dehydrogenase) to construct an H2+CO2→formate cascade reaction system without the need for external NADH is of great scientific significance and industrial value for the utilization of green hydrogen resources and the high-value conversion of CO2. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention provides a method for expressing multi-subunit hydrogenase and producing formic acid by combining it with formic acid dehydrogenase, aiming to solve some of the problems in the prior art or at least alleviate some of the problems in the prior art.

[0006] This invention is achieved by using an enzyme with the amino acid sequence shown in SEQ ID NO.1 in the catalytic preparation of formic acid or formate from CO2 or bicarbonate.

[0007] The present invention also provides a gene sequence encoding the amino acid sequence shown in SEQ ID NO.1, and the gene sequence is shown in SEQ ID NO.2.

[0008] The present invention also provides a formate dehydrogenase, wherein the amino acid sequence shown in SEQ ID NO.1 is mutated in any of the following ways: I123Y, I123F, G124F, G124M, G201Q, D222M, D222N, D222R, I123Y / D222M, I123F / D222M, I123Y / D222N, I123F / D222N, I123Y / D222R, or I123F / D222R.

[0009] Furthermore, formate dehydrogenase is NAD+. + Formate-dependent dehydrogenase.

[0010] The present invention also provides a recombinant genetically engineered bacterium that expresses formate dehydrogenase as described above.

[0011] This invention also provides the application of the above-mentioned formate dehydrogenase in catalyzing the preparation of formic acid or formate from CO2 or bicarbonate.

[0012] Furthermore, with CO2 or HCO3 - Using NADH as substrates, formic acid or formate is prepared by catalytic reduction using the formic acid dehydrogenase.

[0013] The present invention also provides a recombinant expression vector, wherein the recombinant expression vector uses the pBBR1MCS-1 vector as a backbone and simultaneously inserts four subunit coding genes of hydrogenase derived from Ralstonia eutropha H16: hoxF, hoxU, hoxY and hoxH into the same plasmid; each subunit coding gene is independently linked to a Lac promoter upstream, and each subunit coding gene is also linked to a histidine tag coding sequence.

[0014] Furthermore, the gene sequence of the hoxF subunit is shown in SEQ ID NO.21, and its protein sequence is shown in SEQ ID NO.22; the gene sequence of the hoxU subunit is shown in SEQ ID NO.31, and its protein sequence is shown in SEQ ID NO.32; the gene sequence of the hoxY subunit is shown in SEQ ID NO.33, and its protein sequence is shown in SEQ ID NO.34; the gene sequence of the hoxH subunit is shown in SEQ ID NO.35, and its protein sequence is shown in SEQ ID NO.36.

[0015] This invention also provides a method for constructing a recombinant expression vector:

[0016] S1: Using the gene DNA with GeneBank accession number M55230.1 as a template, the hydrogenase hoxF subunit was amplified using primers SEQ ID NO.23 and SEQ ID NO.24;

[0017] S2: The pBBR1MCS-1 plasmid was digested with BamHI restriction endonuclease to obtain the linearized pBBR1MCS-1 plasmid.

[0018] S3: The hoxF subunit gene fragment obtained in step S1 is ligated with the linearized pBBR1MCS-1 plasmid, and the positive recombinant pBBR1MCS-1-hoxF is screened and identified.

[0019] S4: Using the gene DNA with GeneBank accession number M55230.1 as a template, the hydrogenase hoxU subunit was amplified using primers SEQ ID NO.25 and SEQ ID NO.26;

[0020] S5: Digest the pBBR1MCS-1-hoxF plasmid obtained in step S3 using the XhoI restriction endonuclease to obtain the linearized pBBR1MCS-1-hoxF plasmid.

[0021] S6: The hoxU subunit gene fragment obtained in step S4 is ligated with the linearized pBBR1MCS-1-hoxF plasmid, and the positive recombinant pBBR1MCS-1-hoxFU is screened and identified.

[0022] S7: Using the gene DNA with GeneBank accession number M55230.1 as a template, the hydrogenase hoxY subunit was amplified using primers SEQ ID NO.27 and SEQ ID NO.28;

[0023] S8: Digest the pBBR1MCS-1-hoxFU plasmid obtained in step S6 using HindIII restriction endonuclease to obtain a linearized pBBR1MCS-1-hoxFU plasmid.

[0024] S9: The hoxY subunit gene fragment obtained in step S7 is ligated with the linearized pBBR1MCS-1-hoxFU plasmid, and the positive recombinant pBBR1MCS-1-hoxFUY is screened and identified.

[0025] S10: Using the gene DNA with GeneBank accession number M55230.1 as a template, the hydrogenase hoxH subunit was amplified using primers SEQ ID NO.29 and SEQ ID NO.30;

[0026] S11: Digest the pBBR1MCS-1-hoxFUY plasmid obtained in step S9 using BamHI restriction endonuclease to obtain a linearized pBBR1MCS-1-hoxFUY plasmid.

[0027] S12: The hoxH subunit gene fragment obtained in step S10 is ligated with the linearized pBBR1MCS-1-hoxFUY plasmid, and the positive recombinant pBBR1MCS-1-hoxFUYH is screened and identified.

[0028] The present invention also provides a recombinant engineered bacterium containing the recombinant expression vector as described above.

[0029] Furthermore, the host cell of the recombinant engineered bacteria is Ralstonia eutropha H16.

[0030] This invention also provides a method for preparing a multi-subunit hydrogenase, comprising constructing a recombinant expression vector as described above; transforming the recombinant expression vector into host cells to obtain recombinant engineered bacteria; inducing expression in the recombinant engineered bacteria; and obtaining a crude enzyme solution of multi-subunit hydrogenase from the supernatant after cell lysis.

[0031] This invention also provides a method for catalyzing the reaction of H2 and CO2 to prepare formic acid, which involves the combined use of hydrogenase and formic acid dehydrogenase. H2 and CO2 are used as substrates in the reaction system, eliminating the need for additional NADH. The hydrogenase catalyzes the reaction of H2 and NAD... + NADH is generated, and formate dehydrogenase catalyzes the reaction of NADH and CO2 to produce formic acid and NAD. + To achieve NAD + / NADH self-regeneration.

[0032] Furthermore, the hydrogenase is an NADH-dependent hydrogenase.

[0033] Furthermore, the hydrogenase is a multi-subunit hydrogenase prepared using the above-described preparation method.

[0034] Furthermore, the amino acid sequence of the formate dehydrogenase is as shown in SEQ ID NO.1, or the amino acid sequence shown in SEQ ID NO.1 is mutated in any of the following ways: I123Y, I123F, G124F, G124M, G201Q, D222M, D222N, D222R, I123Y / D222M, I123F / D222M, I123Y / D222N, I123F / D222N, I123Y / D222R, or I123F / D222R.

[0035] This invention first obtained ArFDH from *Ancylobacter rudongensis* through gene mining. After codon optimization, gene synthesis, gene cloning, expression, and purification, the purified protein of this enzyme was successfully obtained. Studies of its enzymatic properties showed that the recombinant ArFDH exhibited an enzyme activity of 46.06 mU / mg, which is 1.55 times higher than the template enzyme TsFDH. The enzyme obtained in this invention can be stored at 4°C for over 72 hours while retaining 20% ​​of its activity. After rational design and modification, compared with wild-type ArFDH, the mutant I123F / D222M showed a 6.69-fold increase in kcat / Ka value, demonstrating the success of the mutation. The overall catalytic efficiency of the enzyme on the substrate was improved, and the substrate specificity was enhanced.

[0036] Secondly, this application uses the pBBR1MCS-1 vector as a base and, through genetic engineering experiments, successfully expressed four subunits of hydrogenase (hoxF, hoxU, hoxY, and hoxH) from Ralstonia eutropha H16 using a single plasmid. A Lac promoter was added to each subunit, allowing each subunit to be individually controlled by a Lac promoter, thus enabling efficient expression of each gene. Simultaneously, a histidine (His) tag was added to each gene to facilitate subsequent protein purification. Hydrogenases have great potential in green cofactor regeneration. This application not only provides a new pathway for multi-subunit heterologous expression of hydrogenases but also demonstrates that hydrogenases can catalyze the conversion of H2 to NADH.

[0037] Finally, H2, as a green energy source, and CO2, as a major greenhouse gas, are significant in their catalytic conversion into formic acid, a high-value compound. This application utilizes recombinantly expressed hydrogenases (using H2 and NAD) to... + (Based on NADH) and formate dehydrogenase (based on NADH and CO2 to produce formate and NAD) +( ) used in combination to achieve NAD in the reaction system + The self-circulating regeneration of NADH eliminates the need for additional NADH, thus efficiently converting H2 and CO2 into formic acid in a green and economical way. This simultaneously realizes the resource utilization of green hydrogen and the high-value conversion of greenhouse gas CO2.

[0038] In summary, the advantages and positive effects of this invention are as follows:

[0039] (1) This invention is the first to construct a recombinant expression vector in which the four subunits of hydrogenase (hoxF, hoxU, hoxY, hoxH) are independently configured with Lac promoters and His tags on the same plasmid.

[0040] (2) Heterologous co-expression of multi-subunit hydrogenases is a recognized technical challenge in this field. Existing technologies typically employ multi-plasmid co-transformation or fusion expression strategies, which suffer from problems such as expression imbalance, plasmid loss, and low activity. This invention achieves balanced soluble expression of all four subunits in Ralstonia eutropha H16 by independently adding a Lac promoter to each subunit, thus making transcription and translation independent of each subunit. Figure 11 (Four clear bands), and the specific activity of the purified hydrogenase reached 12.5 U / mg.

[0041] (3) The hydrogenase expression system provided by this invention is simple to operate and has high enzyme production efficiency, and can be directly used for the green regeneration of NADH. More importantly, this invention combines the hydrogenase with a highly active formic acid dehydrogenase (especially the mutant I123F / D222M) to construct a cascade reaction system of "H2 + CO2 → formic acid". This system does not require the addition of NADH, and can efficiently synthesize formic acid using only green hydrogen and greenhouse gases as raw materials under ambient temperature and pressure aqueous phase conditions (Example 7, product concentration 21.67 μg·mL). -1 This method provides a novel technological pathway for green hydrogen energy storage, CO2 resource utilization, and green synthesis of formic acid, and has clear prospects for industrial application. Attached Figure Description

[0042] Figure 1 The phylogenetic tree was constructed based on the amino acid sequences of two candidate enzymes (ArFDH and SnFDH) using the non-root neighbor-joining method. The relevant sequence information was obtained from NCBI.

[0043] Figure 2 This is a demonstration of the ArFDH model structure; a: HCO3 - b: Molecular docking results of NADH and ArFDH; - A schematic diagram of the interaction between NADH and ArFDH;

[0044] Figure 3 It is the amino acid sequence of wild-type formate dehydrogenase ArFDH;

[0045] Figure 4 This is a schematic diagram of the construction of the pET28a-fdh recombinant plasmid;

[0046] Figure 5 This is the result of agarose gel electrophoresis of the pET28a-fdh recombinant plasmid;

[0047] Figure 6 This is the result of SDS-PAGE detection of formate dehydrogenase protein expression; Lane M: Protein Marker; Lane 1: Supernatant of lysed E. coli BL21(DE3) / pET28a cells; Lane 2: Supernatant of lysed E. coli BL21(DE3) / pET28a-fdh cells; Lanes 3-8: Formate dehydrogenase purified by nickel column;

[0048] Figure 7 This is the HPLC chromatogram of formic acid in the ArFDH catalytic reaction products;

[0049] Figure 8 The results are as follows: (a) Effect of temperature on the enzyme activity of mutant I123F / D222M. The enzyme solution was placed in 0.1 M PBS (pH 7.0) buffer and incubated at 20℃, 30℃, 37℃, 50℃, 60℃ and 70℃ for 20 minutes, respectively, and the residual enzyme activity was measured; (b) Thermal kinetics of mutant I123F / D222M. The enzyme solution was placed in 0.1 M PBS (pH 7.0) buffer and incubated at 20℃, 30℃, 37℃, 50℃, 60℃ and 70℃, respectively, and samples were taken at different time points to measure the residual enzyme activity; (c) Effect of pH and buffer system on the enzyme activity of mutant I123F / D222M. The residual enzyme activity was measured in 0.1 M PBS buffer (pH 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0) at 30°C; (d) pH stability of mutant I123F / D222M. The enzyme solution was placed in 0.1 M PBS buffer (pH 5.5–8.0) and stored at 4°C. Samples were taken at different time points to determine the residual enzyme activity.

[0050] Figure 9 This is a schematic diagram of the construction of the pBBR1MCS-1-hoxFUYH vector;

[0051] Figure 10These are the results of agarose gel assay for single enzyme digestion of pBBR1MCS-1-hoxFUYH plasmid; Lane M: DL15000 DNA Marker, Lane 1: pBBR1MCS-1-hoxFUYH HindIII digestion product, Lane 2: pBBR1MCS-1 HindIII digestion product.

[0052] Figure 11 This is the SDS-PAGE electrophoresis result of Ralstonia eutropha H16 hydrogenase expression; lane M is the protein marker, lane 1 is the supernatant of the lysate induced by Ralstonia eutropha H16 / pBBR1MCS-1, lane 2 is the supernatant of the lysate induced by Ralstonia eutropha H16 / pBBR1MCS-1-hoxFUYH, and obvious protein expression bands can be seen. Lane 5 is the purified hydrogenase product, and four obvious bands can be seen from top to bottom, corresponding to HoxF, HoxU, HoxY, and HoxH (HoxF: 66.79 kDa, HoxH: 54.87 kDa, HoxU: 26.18 kDa, HoxY: 22.88 kDa). Detailed Implementation

[0053] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0054] Based on the information contained in this application, various changes to the precise description of the invention can be readily made by those skilled in the art without departing from the spirit and scope of the appended claims. It should be understood that the scope of the invention is not limited to the defined processes, properties, or components, as these embodiments and other descriptions are merely illustrative of specific aspects of the invention. In fact, various modifications to embodiments of the invention that will be apparent to those skilled in the art or related fields are covered within the scope of the appended claims.

[0055] To better understand the invention and not to limit its scope, all figures indicating amounts, percentages, and other numerical values ​​used in this application should, in all cases, be understood to be modified by the word "about". Therefore, unless specifically stated otherwise, the numerical parameters listed in the specification and appended claims are approximate values ​​and may vary depending on the desired properties being sought. Each numerical parameter should at least be considered as obtained based on reported significant figures and through conventional rounding methods. In this invention, "about" means within 10%, preferably within 5%, of a given value or range.

[0056] Unless otherwise specified, all embodiments of the present invention are based on ambient temperature conditions. Ambient temperature refers to the natural room temperature during the four seasons, without additional cooling or heating treatment. Generally, ambient temperature is controlled between 10 and 30°C, preferably between 15 and 25°C. The abbreviations are as follows: "min" represents minutes, "s" represents seconds, "U" represents enzyme activity units, "mM" represents millimoles per liter, "M" represents moles per liter, "rpm" represents revolutions per minute, "mol" represents moles, "μg" represents micrograms, "mg" represents milligrams, "g" represents grams, "μL" represents microliters, "mL" represents milliliters, "bp" represents base pairs, and Kan50 indicates that the culture medium contains 50 μg / mL kanamycin.

[0057] Taq DNA polymerase, bacterial genome extraction kit, plasmid extraction kit, FastPure gel DNA extraction mini kit, one-step cloning kit, Mut Express II rapid mutation kit, and DL 5000 DNA molecular weight standards were all purchased from Nanjing Vazyme Biotechnology Co., Ltd. Gene synthesis, primer synthesis, and sequencing were all performed by Shanghai Sangon Biotech Co., Ltd. In the examples, experimental methods without specific conditions were generally performed under standard conditions, such as those described in *Molecular Cloning: A Laboratory Manual* (Chinese version) (J. Sambrook and MR. Green, eds., translated by He Fuchu, 4th edition, Beijing: Science Press, 2017) and the methods described in the New England Biolabs (NEB) kits.

[0058] This invention first combines gene mining technology with molecular docking technology to efficiently and accurately obtain formate dehydrogenase (ArFDH) from *Ancylobacter rudongensis*. This enzyme can catalyze the reduction of CO2 to formic acid and was successfully expressed at a high level in *E. coli*. Subsequently, ArFDH was directionally modified using a semi-rational design method, ultimately obtaining the I123Y / D222M mutant with excellent catalytic performance.

[0059] Secondly, to address the issue that hydrogenase multi-subunits cannot be simultaneously expressed using a single plasmid in E. coli, a method is provided...

[0060] The pBBR1MCS-1-hoxFUYH gene tandem recombination expression vector. This invention uses a hydrogenase (hoxFUYH) from Ralstoniaeutropha. This enzyme is not only unaffected by oxygen stress but also maintains excellent catalytic performance under aerobic conditions. Moreover, this enzyme uses H2 as a substrate to generate NADH, bypassing glycolysis and the tricarboxylic acid cycle in cells, and does not use glucose as a substrate, showing great promise in "green hydrogen" applications.

[0061] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.

[0062] Example 1: Gene Mining

[0063] TsFDH is a highly active FDH (GeneBank accession number: AB106890.1), encoded by Thiobacillus sp. KNK65MA, and can be used as a probe to search for novel FDHs. In this invention, TsFDH was used as a probe enzyme to perform a BLAST search for novel FDHs in NCBI, successfully screening two candidate enzymes: formate dehydrogenase (ArFDH) from Ancylobacter rudongensis and formate dehydrogenase (SnFDH) from Starkeya novella.

[0064] The obtained target genes were analyzed using MEGA 6.0 software, and a model was constructed as follows: Figure 1 The phylogenetic tree shown reveals that formate dehydrogenase can be roughly divided into four branches, with the target enzyme and probe enzyme belonging to the same branch and exhibiting high homology.

[0065] The candidate protein sequences obtained through screening were used to predict their protein structures using AlphaFold, and the molecular docking software Discovery Studio-2019 was used to perform HCO3--- - Molecular docking was performed using NADH as a substrate. The docking results showed that ArFDH had a stronger affinity for the substrate, with His333 forming stable hydrogen bonds with the substrate, which is more conducive to protein-substrate binding. Ultimately, ArFDH was selected as the research target, and its molecular docking results are shown below. Figure 2 The GenBank number for ArFDH is SCW94487, and its amino acid sequence is shown in SEQ ID NO.1.

[0066] 1 MAKILCVLYD DPIDGYPTTY ARDDLPKIDH YPGGQTLPTP KAVDFTPGHL

[0067] 51 LGSVSGALGL RKYLESNGHT LVVTSDKDGP NSVFEKELVD ADIVISQPFW

[0068] 101 PAYLTPERIA KAKNLKLALT AGIGSDHVDL QSAIDRNITV AEVTYCNSIS

[0069] 151 VAEHVVMMIL GLVRNYLPSH DWARQGGWNI ADCVAHSYDL EAMSVGTVAA

[0070] 201 GRIGLAVLRR LAPFDVKLHY TDHRLPDAV EKELNLTWHA SREEMYPHCD

[0071] 251 VVTLNCPLHP ETEHMINDET LKLFKRGAYI VNTARGKLCD RDAVARALEN

[0072] 301 GQLAGYAGDV WFPQPAPADH PWRTMKWNGM TPHISGTSLS AQARYAAGTR

[0073] 351 EILECFFEGR AIRDEYLIVQ GGALAGTGAH SYSKGNATGG SEEAAKFKKA

[0074] 401 V

[0075] The Arfdh gene was codon optimized, and the optimized gene sequence (see SEQ ID NO.2) was sent to Shanghai Sangon Biotech Co., Ltd. for synthesis.

[0076] Example 2 Construction of recombinant plasmid and expression of target protein

[0077] See the schematic diagram of recombinant plasmid construction. Figure 4 The primers used in this application for amplifying the target gene and constructing the FDH mutant are listed in Table 1, where the restriction enzyme sites are underlined.

[0078] Table 1 Primer Sequences

[0079]

[0080] The formate dehydrogenase gene fdh was amplified using the target gene synthesized by Shanghai Sangon Biotech Co., Ltd. as a template, under the following specific conditions:

[0081]

[0082] The PCR temperature program was designed as follows:

[0083]

[0084] The target gene was recovered using the FastPure gel DNA extraction mini kit.

[0085] The pET28a plasmid was linearized by digesting it with BamHI and HindIII restriction endonucleases from NEB.

[0086] Double enzyme digestion system:

[0087]

[0088] Double digestion was performed by water bath at 37°C for 2 h. The double digestion products were then recovered by gel electrophoresis. The concentration was estimated based on the gel electrophoresis results, and the concentration of the linearized pET28a plasmid was approximately 60 ng / μL, while the concentration of the target gene fdh was approximately 135 ng / μL.

[0089] Subsequently, the amplified gene fragment was ligated into the linearized pET28a plasmid using a one-step cloning kit. 10 µL of the ligation product was transformed into chemically competent *E. coli* DH5α cells, incubated on ice for 30 min, subjected to heat shock at 42°C for 60 s, incubated on ice for 2 min, and then 950 μL of LB broth was added. The cells were gently shaken at 37°C for 1 h. 100 μL of the bacterial suspension was spread onto Kan50 LB plates and incubated overnight at 37°C. Positive recombinants were identified by screening.

[0090] Target fragment and linearized vector linkage system:

[0091]

[0092] Using pET28a-fdh as a template, mutants were obtained using the Mut Express II rapid mutation kit. For mutants with more than one mutation site, the PCR product of the previously obtained mutation site was used as a template for site-directed mutagenesis at the corresponding sites.

[0093] PCR reaction system:

[0094]

[0095] The PCR temperature program was designed as follows:

[0096]

[0097] The amplification product contains the original template plasmid. To prevent the formation of false positive transformants after transformation, the point-mutated amplification product is digested with DpnI to remove the methylated template plasmid.

[0098] DpnI digestion system:

[0099]

[0100] The digestion product of the obtained mutant was thermally transferred into competent Escherichia coli DH5α cells. The recombinant plasmid was extracted from the positive strain and the obtained mutant plasmid was transformed into E. coli BL21(DE3).

[0101] The recombinant E. coli BL21(DE3) strain was cultured in 50 mL LB medium containing 50 μg / mL kanamycin at 37 °C and 220 rpm for 3 hours. When the OD of the recombinant strain... 600nm When the expression level reached 0.6–0.8, 0.5 mM IPTG was added, and expression was induced for 20 hours at 16 °C and 180 rpm. Subsequently, the induced cells were collected by centrifugation at 6000 × g for 10 minutes, washed twice with dipotassium hydrogen phosphate buffer (PBS, 100 mM, pH 7.0), and lysed using sonication. The cell lysate was then centrifuged at 12000 × g at 4 °C for 20 minutes, and the supernatant was purified to a homogeneous state by nickel chelate affinity chromatography. Finally, protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and protein concentration was determined using the Bradford method. To facilitate subsequent enzyme activity assays, the purified enzyme solutions of ArFDH and various mutant FDH were diluted to a concentration of 200 μg / mL using 0.1 mol / L pH 7.0 PBS buffer.

[0102] The identification results of the recombinant plasmid pET28a-fdh are shown in the figure. Figure 5 Lane 4 contains the product of double enzyme digestion of the pET28a-fdh plasmid, which is consistent with the target gene fdh (1206 bp); Lane 5 contains the PCR amplification product of the pET28a-fdh plasmid, confirming that the fdh gene has been successfully inserted into pET28a.

[0103] like Figure 6In the expression of ArFDH, the target protein was introduced into E. coli BL21(DE3). After 12% SDS-PAGE analysis, it was found that compared with lane 1, lane 2 showed a clear band at a molecular weight of 43 kDa, which indicates that ArFDH has been successfully expressed in E. coli.

[0104] In this embodiment, it was found that ArFDH with optimized codons, after being transferred into a series of Escherichia coli host bacteria, can achieve higher expression of soluble proteins. Figure 6 Lanes 3 to 8 in figure a show the collection results of ArFDH, I123Y, I123F, G124M, G124F, and G201Q after purification with 250 mM imidazole elution using a nickel column. Figure 6 Lanes 1 to 8 in Figure b show the collection results of D222M, D222N, D222R, I123Y / D222M, I123F / D222M, I123Y / D222N, I123F / D222N, and I123Y / D222R after purification with 250 mM imidazole elution using a nickel column. The results demonstrate that high-purity formate dehydrogenase and formate dehydrogenase mutants can be obtained through nickel column purification for subsequent enzymological studies.

[0105] Example 3 Study on Enzymatic Properties

[0106] NAD + The principle of the formate-dependent dehydrogenase activity assay is that NADH has an absorption peak at 340 nm (εNADH, 340 nm = 6220 M). -1 cm -1 Furthermore, each molecule of NADH consumed can catalyze… It is converted into formic acid.

[0107] The enzyme activity assay method and conditions are as follows: Prepare 0.2 M pH 7.0 phosphate buffer, degas the solution, and then purge the phosphate buffer with CO2 gas for 1 h to dissolve sufficient CO2 substrate in the solution. This operation needs to be repeated before each enzyme activity assay. The reaction system volume is 2 mL, containing enzyme (20 μg), NADH (1 mM), 1 mL pH 7.0 phosphate buffer, and pure water to bring the volume to 2 mL. The specific operation steps are as follows: Add 1 mL phosphate buffer and 850 µL pure water to a quartz cuvette, place the enzyme solution in a 0.5 mL EP tube, and preheat in a 30℃ water bath for 2 min. After the preheating, place the quartz cuvette in a UV spectrophotometer, add 100 µL of enzyme solution and 50 µL of NADH, and start recording the change in NADH at 340 nm. After reacting for 5 minutes, determine the remaining NADH content. One unit of enzyme activity (U) is defined as: under the above reaction conditions, the consumption of 1 μM NADH / NAD per minute.+ The required amount of enzyme.

[0108] The enzyme activity detection results of wild-type ArFDH and each mutant are shown in Table 2.

[0109] Table 2. Results of formate dehydrogenase activity assay

[0110]

[0111] The enzymatic properties of formate dehydrogenase include optimal reaction temperature, thermal stability, optimal reaction pH, pH stability, and enzyme reaction kinetics. For optimal reaction temperature, thermal stability, and optimal reaction pH, pH stability was determined by varying the reaction temperature and pH. The optimal reaction temperature was determined by incubating cuvettes containing the reaction solution and EP tubes containing the enzyme solution in constant temperature water baths at 4℃, 20℃, 30℃, 37℃, 50℃, and 60℃ for 5 min, followed by enzyme activity assay. Thermal stability was determined by incubating EP tubes containing the enzyme solution in constant temperature water baths at 4℃, 20℃, 30℃, 37℃, 50℃, and 60℃, taking samples at regular intervals, and then detecting enzyme activity. The optimal reaction pH was determined by replacing the pH 7.0 phosphate buffer used in the enzyme activity assay system with PBS at pH 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0, respectively, and then detecting enzyme activity according to the enzyme activity assay. pH stability was determined by dissolving the purified enzyme in PBS at pH values ​​of 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0, respectively, and then detecting the enzyme activity according to the enzyme activity assay method.

[0112] For the determination of kinetic parameters at different substrate concentrations, the specific experimental method involves modifying the substrate concentration only, based on the existing enzyme activity system and detection method. When measuring NADH substrate, enzyme activity was detected at concentrations of 1 mM, 2 mM, 3 mM, 4 mM, and 5 mM. At that time, NaHCO3 was used as the reaction substrate, and the enzyme activity was detected at substrate concentrations of 50 mM, 100 mM, 150 mM, 200 mM, 250 mM and 300 mM.

[0113] Formic acid detection in the enzyme catalysis system: The catalytic reaction solution of recombinant ArFDH was filtered through a 0.22 µm filter membrane and detected by Agilent 1100 high performance liquid chromatography.

[0114] Mobile phase A: 0.05 M phosphate buffer (pH 2.8): Weigh 7.8 g Adjust the pH to 2.8 with phosphoric acid, bring the volume to 1 L, filter through a 0.22 μm filter membrane, and degas for 20 min. Mobile phase B: degas with chromatographic grade methanol for 20 min.

[0115] The HPLC detection conditions were as follows: Eclipse XDB-C18 column (5 μm, 150 mm × 4.6 mm), injection volume 20 μL, flow rate 0.8 mL / min, column temperature 25℃, detector: UV detector, detection wavelength 210 nm, total flow rate of 35% mobile phase A and 65% mobile phase B 0.8 mL / min, detection for 5 min.

[0116] Liquid chromatography detection results as follows Figure 7 As shown, a distinct formic acid characteristic absorption peak appears at a retention time of approximately 2.1 s.

[0117] The kinetic parameters of formate dehydrogenase, including Km and kcat, were obtained using the Michaelis-Menten equation; all kinetic parameters were obtained by fitting data using Origin 2018 software.

[0118] By studying the catalytic rate of ArFDH for different substrate concentrations, the catalytic effects of recombinant ArFDH and mutant formate dehydrogenase on... And the kinetic parameters of NADH. ArFDH for It has good affinity (Table 3) and can catalyze efficiently. Efficient conversion to formic acid.

[0119] Table 3 Kinetic parameters of ArFDH and various mutants

[0120]

[0121] k cat / K m The improvement reflects the enhancement of the enzyme's catalytic ability. Table 3 shows that the catalytic abilities of the single-point mutants I123Y, I123F, G201Q, and D222M, and the double-point mutants I123Y / D222M, I123F / D222M, I123Y / D222N, I123F / D222N, and I123F / D222R are all significantly improved compared to ArFDH. In particular, the kcat / Km of the mutant I123F / D222M is significantly improved compared to the wild-type recombinant ArFDH, increasing from 0.072 to 0.482, a 6.69-fold increase. This proves the correctness of the experimental procedure combining theory and experiment, successfully modifying formate dehydrogenase and improving its catalytic performance. The single-point mutation D222R resulted in a decrease in catalytic efficiency to 58% of the wild type, while the catalytic efficiency of the double-point mutant I123Y / D222R, formed by introducing I123Y, was restored to 89% of the wild type. This indicates that the I123Y mutation not only improves catalytic efficiency itself but also compensates for the adverse effects of D222R, producing a synergistic effect.

[0122] Enzymatic characteristics of mutant I123F / D222M were studied, such as Figure 8 As shown in a and b, the optimal reaction temperature for the purified mutant I123F / D222M is 30℃, with enzyme activities of 85% and 92% at 20℃ and 37℃, respectively. Even after incubation at 60℃ for 5 minutes, its activity remained above 50%. However, when the temperature rose to 70℃, the enzyme activity was almost completely lost, indicating that 70℃ may be the limiting temperature for ArFDH. Figure 8 As shown in c and d, the optimal pH for mutant I123F / D222M is 7.0, but pH has little effect on enzyme activity. In the pH range of 5.5 to 8.0, its residual activity exceeds 60%.

[0123] Example 4: Construction of the pBBR1MCS-1-hoxFUYH vector

[0124] 1. Cloning of the hydrogenase hoxF subunit and construction of the pBBR1MCS-1-hoxF vector

[0125] The hydrogenase hoxFUYH from *Ralstonia eutropha* is a highly active, oxygen-stable hydrogenase (GeneBank accession number: M55230.1), composed of four subunits: HoxF, HoxU, HoxY, and HoxH. The HoxFU subunits collectively form the diaphorase unit, which contains NAD+. + The HoxFUYH subunit is the reducing catalytic center; it acts as a hydrogenase unit responsible for H2 conversion. This invention amplifies the hoxFUYH gene using designed primers and inserts each subunit into the multiple cloning site of the pBBR1MCS-1 vector using homologous recombination. A schematic diagram of the vector construction is shown below. Figure 1 As shown.

[0126] The gene sequence of the HoxF subunit of the hydrogenase of Ralstonia eutropha H16 is shown in SEQ ID NO.21, and the protein sequence is shown in SEQ ID NO.22.

[0127] The primers used to amplify the target gene in this application are listed in Table 4. Lowercase letters indicate homologous sequences complementary to the vector, restriction enzyme sites are underlined, His tags are shown in italics, and Lac promoters are indicated by wavy lines.

[0128] Table 4 Primer Sequences

[0129]

[0130] Using the extracted gene DNA from Ralstonia eutropha H16 as a template, the hoxF subunit of hydrogenase was amplified using the hoxF-F and hoxF-R primers in Table 4 (the PCR system and procedure were the same as the amplification method of the fdh gene in Example 2). A His tag for subsequent protein purification was added to the reverse primer.

[0131] The pBBR1MCS-1 plasmid was digested using the BamHI restriction endonuclease from NEB to obtain a linearized pBBR1MCS-1 plasmid.

[0132] Double enzyme digestion system:

[0133]

[0134] Double digestion was performed by water bath at 37°C for 2 h. The double digestion products were then recovered by gel electrophoresis. The concentration was estimated based on the gel electrophoresis pattern, and the concentration of the linearized pBBR1MCS-1 plasmid was approximately 106 ng / μL, and the concentration of the target gene hoxF was approximately 105 ng / μL.

[0135] Subsequently, the amplified gene fragment was ligated into the linearized pBBR1MCS-1 plasmid using a one-step cloning kit (ligation system as in Example 2). 10 µL of the ligation product was transformed into chemically competent *E. coli* DH5α cells, incubated on ice for 30 min, subjected to heat shock at 42°C for 60 s, incubated on ice for 2 min, and then 950 μL of LB broth was added. The cells were gently shaken at 37°C for 1 h. 100 μL of the bacterial suspension was spread onto LB agar plates containing chloramphenicol Cm20 and incubated overnight at 37°C. Positive recombinant pBBR1MCS-1-hoxF was identified through screening.

[0136] 2. Cloning of the hydrogenase hoxU subunit and construction of the pBBR1MCS-1-hoxFU vector

[0137] The gene sequence of the HoxU subunit of the hydrogenase from Ralstonia eutropha H16 is shown in SEQ ID NO. 31, and the protein sequence is shown in SEQ ID NO. 32. Using extracted gene DNA from Ralstonia eutropha as a template, the hoxU subunit of the hydrogenase was amplified using the hoxU-F and hoxU-R primers listed in Table 4 (the PCR system and procedure were consistent with the amplification method for the fdh gene in Example 2). A Lac promoter responsible for HoxU subunit expression was designed in the forward primer, and a His tag for subsequent protein purification was added to the reverse primer.

[0138] The pBBR1MCS-1-hoxF plasmid constructed in step 1 was digested using the NEB XhoI restriction endonuclease to obtain a linearized pBBR1MCS-1-hoxF plasmid.

[0139] Double enzyme digestion system:

[0140]

[0141] Double digestion was performed by water bath at 37°C for 2 h. The double digestion products were then recovered by gel electrophoresis. The concentration was estimated based on the gel electrophoresis pattern, and the concentration of the linearized pBBR1MCS-1-hoxF plasmid was approximately 86 ng / μL, and the concentration of the target gene hoxU was approximately 123 ng / μL.

[0142] Subsequently, the amplified gene fragment was ligated into the linearized pBBR1MCS-1-hoxF plasmid using a one-step cloning kit (ligation system as in Example 2). 10 µL of the ligation product was transformed into chemically competent *E. coli* DH5α cells, incubated on ice for 30 min, subjected to heat shock at 42°C for 60 s, incubated on ice for 2 min, and then 950 μL of LB broth was added. The cells were gently shaken at 37°C for 1 h. 100 μL of the bacterial suspension was spread onto chloramphenicol Cm20 LB agar plates and incubated overnight at 37°C. Positive recombinant pBBR1MCS-1-hoxFU was identified through screening.

[0143] 3. Cloning of the hydrogenase hoxY subunit and construction of the pBBR1MCS-1-hoxFUY vector

[0144] The gene sequence of the HoxY subunit of the hydrogenase of Ralstonia eutropha H16 is shown in SEQ ID NO.33, and the protein sequence is shown in SEQ ID NO.34.

[0145] Using the extracted gene DNA from Ralstonia eutropha H16 as a template, the HoxY subunit of hydrogenase was amplified using the hoxY-F and hoxY-R primers in Table 4 (the PCR system and procedure were the same as the amplification method of the fdh gene in Example 2). A Lac promoter responsible for HoxH subunit expression was designed in the forward primer, and a His tag for subsequent protein purification was added to the reverse primer.

[0146] The pBBR1MCS-1-hoxFU plasmid constructed in step 2 was digested using HindIII restriction endonuclease from NEB to obtain a linearized pBBR1MCS-1-hoxFU plasmid.

[0147] Double enzyme digestion system:

[0148]

[0149] Double digestion was performed by water bath at 37°C for 2 h. The double digestion products were then recovered by gel electrophoresis. The concentration was estimated based on the gel electrophoresis pattern, and the concentration of the linearized pBBR1MCS-1-hoxFU plasmid was approximately 145 ng / μL, and the concentration of the target gene hoxY was approximately 93 ng / μL.

[0150] Subsequently, the amplified gene fragment was ligated into the linearized pBBR1MCS-1-hoxFU plasmid using a one-step cloning kit. 10 µL of the ligation product was transformed into chemically competent *E. coli* DH5α cells, incubated on ice for 30 min, subjected to heat shock at 42°C for 60 s, incubated on ice for 2 min, and then 950 μL of LB broth was added. The cells were gently shaken at 37°C for 1 h. 100 μL of the bacterial suspension was spread onto LB agar plates containing chloramphenicol Cm20 and incubated overnight at 37°C. Positive recombinants, pBBR1MCS-1-hoxFUY, were identified through screening.

[0151] 4. Cloning of the hydrogenase hoxH subunit and construction of the pBBR1MCS-1-hoxFUYH vector

[0152] The gene sequence of the HoxH subunit of the hydrogenase of Ralstonia eutropha H16 is shown in SEQ ID NO.35, and the protein sequence is shown in SEQ ID NO.36.

[0153] Using the extracted gene DNA from Ralstonia eutropha H16 as a template, the hoxH subunit of hydrogenase was amplified using the hoxH-F and hoxH-R primers in Table 4 (the PCR system and procedure were the same as the amplification method of the fdh gene in Example 2). A Lac promoter responsible for the expression of the hoxH subunit was designed in the forward primer, and a His tag for subsequent protein purification was added to the reverse primer.

[0154] pBBR1MCS-1-hoxFUY constructed in step 3 was digested using the BamHI restriction enzyme from NEB to obtain linearized pBBR1MCS-1-hoxFUY.

[0155] Double enzyme digestion system:

[0156]

[0157] Double digestion was performed by water bath at 37°C for 2 h. The double digestion products were then recovered by gel electrophoresis. The concentration was estimated based on the gel electrophoresis pattern. The concentration of the linearized pBBR1MCS-1-hoxFUY plasmid was approximately 103 ng / μL, and the concentration of the target gene hoxU was approximately 143 ng / μL.

[0158] Subsequently, the amplified gene fragment was ligated into the linearized pBBR1MCS-1-hoxFUY plasmid using a one-step cloning kit. 10 µL of the ligation product was transformed into chemically competent *E. coli* DH5α cells, incubated on ice for 30 min, subjected to heat shock at 42°C for 60 s, incubated on ice for 2 min, and then 950 μL of LB broth was added. The cells were gently shaken at 37°C for 1 h. 100 μL of the bacterial suspension was spread onto LB agar plates containing chloramphenicol Cm20 and incubated overnight at 37°C. Positive recombinants, pBBR1MCS-1-hoxFUYH, were identified through screening.

[0159] Example 5: Enzyme digestion identification and target expression of the pBBR1MCS-1-hoxFUYH recombinant plasmid

[0160] The pBBR1MCS-1-hoxFUYH plasmid extracted from Example 4 was digested with HindIII and digested at 37°C for 2 h. The digestion products were detected by agarose gel electrophoresis.

[0161]

[0162] The identification results of the recombinant plasmid pBBR1MCS-1-hoxFUYH are shown in the figure. Figure 10 The product of single enzyme digestion of pBBR1MCS-1-hoxFUYH plasmid in lane 1 was about 10,000 bp, consistent with the theoretical size (9,489 bp); the product of single enzyme digestion of pBBR1MCS-1 plasmid in lane 3 was about 5,000 bp, confirming that the hydrogenase hoxFUYH gene has been successfully inserted into pBBR1MCS-1.

[0163] The pBBR1MCS-1-hoxFUYH plasmid was transformed into Ralstonia eutropha H16. 10 µL of the plasmid was transferred into chemocompetent Ralstonia eutropha H16 cells, incubated on ice for 30 min, subjected to heat shock at 42°C for 60 s, incubated on ice for 2 min, and then 950 μL of LB broth was added. The cells were gently shaken at 37°C for 1 h. 100 μL of the bacterial suspension was spread onto LB agar plates containing chloramphenicol Cm20 and incubated overnight at 37°C. Positive recombinants were identified by selection.

[0164] The recombinant *Ralstonia eutropha* H16 / pBBR1MCS-1-hoxFUYH strain was cultured overnight in 25 mL LB medium containing 20 μg / mL chloramphenicol at 30°C and 220 rpm to obtain a seed culture. One mL of the seed culture was inoculated into 50 mL LB medium containing 20 μg / mL chloramphenicol and cultured for 3 hours at 30°C and 220 rpm. When the OD of the recombinant strain... 600nm When the expression level reached 0.6–0.8, 0.5 mM IPTG was added, and expression was induced for 20 hours at 16°C and 180 rpm. Subsequently, the induced cells were collected by centrifugation at 6000×g for 10 minutes, washed twice with 0.1 M pH 7.0 Tris-HCl, and lysed using sonication at 300 W for 3 seconds followed by a 5-second pause for 10 minutes. The cell lysate was then centrifuged at 12000×g at 4°C for 20 minutes, and the supernatant was collected as the crude hydrogenase solution (hoxFUYH).

[0165] The supernatant was loaded into a nickel gravity column equilibrated with 0.1 M pH 7.0 Tris-HCl buffer. First, unbound proteins were washed away with 0.1 M pH 7.0 Tris-HCl buffer. Second, proteins were eluted sequentially with 25 mM, 50 mM, and 100 mM imidazole solutions. Third, the target protein was eluted with 250 mM imidazole solution, and the eluent was collected. Finally, protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and protein concentration was determined using the Bradford method. To facilitate subsequent enzyme activity assays, the purified enzyme solution was diluted to a concentration of 200 μg / mL with 0.1 mol / L pH 7.0 Tris-HCl buffer.

[0166] like Figure 11 The target protein was introduced into Ralstonia eutropha H16 for expression. 12% SDS-PAGE analysis revealed distinct bands in lane 2 at molecular weights of 67, 55, 26, and 23 kDa, compared to lane 1, indicating successful expression of the hydrogenase hoxFUYH in Ralstonia eutropha H16. Lane 5 showed the purified hydrogenase, with the four subunits HoxF, HoxU, HoxY, and HoxH clearly visible. These results demonstrate that high-purity hydrogenase can be obtained through nickel column purification for subsequent enzymological studies.

[0167] Example 6 Study on Enzymatic Properties

[0168] The principle of the NADH-dependent hydrogenase activity assay is that NADH has an absorption peak at 340 nm (εNADH, 340 nm = 6220 M). -1 cm -1 Furthermore, for every 1 molecule of H2 consumed, 2 molecules of NADH are generated. Therefore, the enzyme activity of hydrogenase can be defined based on the amount of NADH increased.

[0169] The enzyme activity assay method and conditions were as follows: A 0.2 M pH 7.0 Tris-HCl buffer solution was prepared, degassed, and then H2 gas was bubbled into the 0.2 M pH 7.0 Tris-HCl buffer solution for 1 h to dissolve sufficient H2 substrate in the solution. This procedure was repeated before each enzyme activity assay. The reaction system volume was 2 mL, containing enzyme (20 μg), riboflavin (FMN, 10 mM), and NAD+. + (5 mM), 1 mL pH 7.0 Tris-HCl buffer, and pure water to bring the volume to 2 mL. The specific steps are as follows: Add 1 mL of 7.0 Tris-HCl buffer and 790 µL of pure water to a quartz cuvette. Place the enzyme solution in a 0.5 mL EP tube and preheat in a 30°C water bath for 2 min. After the preheating period, place the quartz cuvette in a UV spectrophotometer and add 100 µL of enzyme solution, 10 µL of FMN, and 100 µL of NAD. + Changes in NADH were recorded at 340 nm initially, and the reaction was allowed to proceed for 5 minutes. The amount of NADH produced was then measured. One unit of enzyme activity (U) was defined as the amount of enzyme required to produce 1 μM NADH per minute under the above reaction conditions. The specific activity of the hydrogenase was calculated to be 12.5 U / mg.

[0170] Example 7 Application

[0171] Hydrogenase uses H2 as a substrate to convert NAD+ + The formate dehydrogenase mutant I123F / D222M consumes NADH during the catalytic production of formic acid from CO2. Purified hydrogenase and formate dehydrogenase are added to the reaction system along with H2, CO2, and NAD. + The substrate was tested to determine whether formic acid was produced in the final product, in order to demonstrate that the hydrogenase has the ability to produce the cofactor NADH.

[0172] The specific experimental conditions are as follows: Prepare a 0.2 M pH 7.0 Tris-HCl buffer solution, degas the solution, and then bubble CO2 and H2 gases into the 0.2 M pH 7.0 Tris-HCl buffer solution for 1 h to dissolve sufficient amounts of the reaction substrate CO2 and H2. This operation needs to be repeated before each enzyme activity assay. The reaction system volume is 2 mL, containing formate dehydrogenase (mutant I123F / D222M), hydrogenase (20 μg each), riboflavin (FMN, 10 mM), and NAD+. + (5 mM), 1 mL pH 7.0 Tris-HCl buffer, and pure water to bring the volume to 2 mL. The specific steps are as follows: Add 1 mL 7.0 Tris-HCl buffer, 690 µL pure water, 10 µL FMN, and 100 µL NAD. + Add the enzymes to a 5 mL centrifuge tube. Place the formate dehydrogenase and hydrogenase in a 0.5 mL EP tube, respectively. Preheat the 5 mL centrifuge tube and the 0.5 mL EP tube in a 30°C water bath for 2 min. After the warming process, add 100 µL each of the formate dehydrogenase and hydrogenase enzyme solutions to the reaction system in the 5 mL centrifuge tube. Place the reaction system in a 30°C water bath and time the reaction for 5 min. After the reaction, boil the 5 mL centrifuge tube in a boiling water bath for 1 min to inactivate the enzymes and terminate the reaction.

[0173] First, formic acid was identified as the main product in the reaction by liquid chromatography. Then, the formic acid content in the reaction product was quantitatively detected using a colorimetric reaction method. Reagent preparation: Solution 1: Weigh 0.5 g of citric acid and 10 g of acetamide and dissolve them in 100 mL of isopropanol solution. Solution 2: 30% sodium acetate, weigh 30 g of sodium acetate and dissolve it in 100 mL of pure water. Preparation of formic acid standard solution: Weigh 0.01 g of formic acid and dilute to 100 mL to obtain 1000 µg / mL. Experimental method: Pipette 50 µL of formic acid standard solution, add 100 µL of Solution 1 and 4 µL of Solution 2 sequentially, add 360 µL of acetic anhydride, mix thoroughly by pipetting, and let stand at room temperature for 2 h. Then, use an ELISA reader to detect the absorbance of the sample at 515 nm. Table 5 shows the detection results of the formic acid standard; this data was used to construct a standard curve.

[0174] Table 5. Results of formic acid concentration detection

[0175]

[0176] This experimental method was used to detect formic acid in the reaction system after the reaction was completed, and the OD was measured. 515 The nm value was 0.013 ± 0.002. Based on the standard curve of formic acid, the concentration of formic acid in the reaction system was calculated to be 21.67 μg·mL. -1Experiments have shown that hydrogenase can catalyze NAD+ dehydratase. + The generation of NADH provides a cofactor for the catalytic reaction of formate dehydrogenase, and also proves the feasibility of using hydrogenase and formate dehydrogenase in the same reaction system to catalyze the conversion of H2 and CO2 into formate.

[0177] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A recombinant expression vector, characterized in that: The recombinant expression vector uses the pBBR1MCS-1 vector as a backbone, and simultaneously inserts four subunit coding genes of hydrogenase from Ralstonia eutropha: hoxF, hoxU, hoxY and hoxH into the same plasmid; each subunit coding gene is independently linked to a Lac promoter upstream, and each subunit coding gene is also linked to a histidine tag coding sequence.

2. The recombinant expression vector according to claim 1, characterized in that: The gene sequence of the hoxF subunit is shown in SEQ ID NO.21, and its protein sequence is shown in SEQ ID NO.22; the gene sequence of the hoxU subunit is shown in SEQ ID NO.31, and its protein sequence is shown in SEQ ID NO.32; the gene sequence of the hoxY subunit is shown in SEQ ID NO.33, and its protein sequence is shown in SEQ ID NO.34; the gene sequence of the hoxH subunit is shown in SEQ ID NO.35, and its protein sequence is shown in SEQ ID NO.

36.

3. The method for constructing a recombinant expression vector as described in claim 1, characterized in that: S1: Using the gene DNA with GeneBank accession number M55230.1 as a template, the hydrogenase hoxF subunit was amplified using primers SEQ ID NO.23 and SEQ ID NO.24; S2: The pBBR1MCS-1 plasmid was digested with BamHI restriction endonuclease to obtain the linearized pBBR1MCS-1 plasmid. S3: The hoxF subunit gene fragment obtained in step S1 is ligated with the linearized pBBR1MCS-1 plasmid, and the positive recombinant pBBR1MCS-1-hoxF is screened and identified. S4: Using the gene DNA with GeneBank accession number M55230.1 as a template, the hydrogenase hoxU subunit was amplified using primers SEQ ID NO.25 and SEQ ID NO.26; S5: Digest the pBBR1MCS-1-hoxF plasmid obtained in step S3 using the XhoI restriction endonuclease to obtain the linearized pBBR1MCS-1-hoxF plasmid. S6: The hoxU subunit gene fragment obtained in step S4 is ligated with the linearized pBBR1MCS-1-hoxF plasmid, and the positive recombinant pBBR1MCS-1-hoxFU is screened and identified. S7: Using the gene DNA with GeneBank accession number M55230.1 as a template, the hydrogenase hoxY subunit was amplified using primers SEQ ID NO.27 and SEQ ID NO.28; S8: Digest the pBBR1MCS-1-hoxFU plasmid obtained in step S6 using HindIII restriction endonuclease to obtain a linearized pBBR1MCS-1-hoxFU plasmid. S9: The hoxY subunit gene fragment obtained in step S7 is ligated with the linearized pBBR1MCS-1-hoxFU plasmid, and the positive recombinant pBBR1MCS-1-hoxFUY is screened and identified. S10: Using the gene DNA with GeneBank accession number M55230.1 as a template, the hydrogenase hoxH subunit was amplified using primers SEQ ID NO.29 and SEQ ID NO.30; S11: Digest the pBBR1MCS-1-hoxFUY plasmid obtained in step S9 using BamHI restriction endonuclease to obtain a linearized pBBR1MCS-1-hoxFUY plasmid. S12: The hoxH subunit gene fragment obtained in step S10 is ligated with the linearized pBBR1MCS-1-hoxFUY plasmid, and the positive recombinant pBBR1MCS-1-hoxFUYH is screened and identified.

4. A recombinant engineered bacterium, characterized in that: The recombinant engineered bacteria contain the recombinant expression vector as described in any one of claims 1-3.

5. The recombinant engineered bacteria according to claim 4, characterized in that: The host cell for the recombinant engineered bacteria is Ralstonia eutropha H16.

6. A method for preparing a multi-subunit hydrogenase, characterized in that: Construct a recombinant expression vector as described in any one of claims 1-3; transform the recombinant expression vector into host cells to obtain recombinant engineered bacteria; induce expression in the recombinant engineered bacteria; and obtain a crude enzyme solution of multi-subunit hydrogenase from the supernatant after cell lysis.

7. A method for preparing formic acid by catalytic reaction of hydrogen and CO2, characterized in that: By combining hydrogenase with formate dehydrogenase, H2 and CO2 are used as substrates in the reaction system, eliminating the need for additional NADH. The hydrogenase catalyzes the reaction of H2 and NAD. + NADH is generated, and formate dehydrogenase catalyzes the reaction of NADH and CO2 to produce formic acid and NAD. + To achieve NAD + / NADH self-regeneration.

8. The method for preparing formic acid by catalytic reaction of H2 and CO2 according to claim 7, characterized in that: The hydrogenase is an NADH-dependent hydrogenase.

9. The method for preparing formic acid by catalytic reaction of H2 and CO2 according to claim 7, characterized in that: The hydrogenase is a multi-subunit hydrogenase prepared using the preparation method of claim 6.

10. The method for preparing formic acid by catalytic reaction of H2 and CO2 according to claim 7, characterized in that: The amino acid sequence of the formate dehydrogenase is shown in SEQ ID NO.1, or the amino acid sequence shown in SEQ ID NO.1 can be mutated in any of the following ways: I123Y, I123F, G124F, G124M, G201Q, D222M, D222N, D222R, I123Y / D222M, I123F / D222M, I123Y / D222N, I123F / D222N, I123Y / D222R, or I123F / D222R.

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