Formate dehydrogenase and application of mutant thereof in carbon dioxide reduction

Abstract

The invention belongs to the technical field of enzyme catalysis, and particularly relates to formate dehydrogenase and application of a mutant thereof in carbon dioxide reduction. According to the present invention, the ArFDH derived from the Ancylobacter rudongensis is obtained through the gene mining, and the ArFDH derived from the Ancylobacter rudongensis is obtained; the purified protein of the enzyme is successfully obtained through the steps of codon optimization, gene synthesis, gene cloning, expression purification and the like. The enzyme activity of the recombinant ArFDH reaches 46.06 U / g, which is 1.55 times higher than that of the template enzyme TsFDH with the highest activity reported. Compared with a wild type ArFDH, the mutant I123F / D222M has the advantages that the catalytic efficiency on HCO3 <-> / CO2 is improved by 6.69 times, the substrate catalytic efficiency of enzyme is integrally improved, and the substrate specificity is enhanced. The invention not only provides a new way for catalyzing and utilizing CO2 through a biological enzyme method, but also lays a foundation stone for constructing sustainable artificial carbon cycle.

Description

Technical Field

[0001] This invention belongs to the field of carbon dioxide (CO2) resource utilization technology, and particularly relates to a catalytic enzyme (formate dehydrogenase, FDH) for the catalytic reduction of CO2 to formic acid and its application. Specifically, it relates to a novel formic acid dehydrogenase obtained through gene mining and its rationally designed mutant, as well as the application of the enzyme in the efficient catalytic reduction of CO2 to formic acid under mild conditions. Background Technology

[0002] As a significant greenhouse gas, CO2's substantial impact on global climate change is increasingly attracting attention. With the escalating environmental problems such as global warming, governments worldwide are placing greater emphasis on CO2 emission control and resource utilization, with the latter becoming a particularly hot research area globally. Capturing CO2 from the atmosphere and converting it into high-value organic compounds has become a cutting-edge topic in biotechnology and synthetic biology. In this process, biocatalysis technology has garnered significant attention due to its advantages, including mild reaction conditions, high conversion efficiency, and contribution to carbon recycling.

[0003] Formate dehydrogenases (FDHs) are heterogeneous proteins widely found in eukaryotes and prokaryotes that catalyze the breakdown of formate into CO2 and water. These enzymes were initially used primarily as coenzyme regeneration systems in the synthesis of chiral compounds. Recent studies have discovered that some formate dehydrogenases can reversibly catalyze the conversion reaction between formate and CO2 (HCOO). - CO2+ H + + 2e - Formic acid, as an important chemical raw material and high-value-added product, has broad application prospects in the green economy industry. Formic acid dehydrogenase can efficiently and specifically convert CO2 into formic acid under mild conditions of normal temperature and pressure without producing toxic byproducts, thus showing great potential in the resource utilization of CO2. However, known natural FDH generally suffers from problems such as low catalytic activity, poor affinity for CO2, and insufficient thermal stability, making it difficult to meet the needs of industrial applications.

[0004] Microbial resources are a diverse and abundant natural treasure trove on Earth, containing a wealth of untapped biocatalytic potential. Despite significant progress in microbiology research, the physiological functions, metabolic pathways, and application potential of many microorganisms in industry, the environment, and biotechnology still require in-depth exploration and full utilization. Gene mining technology, with its high efficiency, precision, and strong targeting capabilities, has become an important tool for discovering novel enzyme resources; while molecular docking technology is widely used to predict small molecule-protein and protein-protein interaction sites and can efficiently assess binding affinity. Combining these two technologies can not only accelerate the discovery of novel enzymes but also significantly improve the accuracy of target enzyme prediction. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention provides a formate dehydrogenase for CO2 reduction and its application, 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 encoding an enzyme with an amino acid sequence as shown in SEQ ID NO.1 and a nucleotide sequence as 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 the formate dehydrogenase as described above, or integrates the gene sequence as described above.

[0011] Furthermore, the host of the genetically engineered bacteria is Escherichia coli, and the vector is the pET series vector.

[0012] 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.

[0013] Furthermore, with CO2 or HCO3 - Using NADH as substrates, formic acid or formate is prepared by catalytic reduction using the formic acid dehydrogenase.

[0014] In this application, the Arfdh gene sequence from *Ancylobacter rudongensis* was obtained 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 U / g, which is 1.55 times higher than the activity of the most widely studied and reported template enzyme TsFDH from *Thiobacillus sp.*. 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 to wild-type ArFDH, the mutant I123F / D222M showed a higher kJ / g content. cat / K m The value increased by 6.69 times, demonstrating the success and effectiveness of the rational design. The overall catalytic efficiency of the enzyme on the substrate was improved, and the substrate specificity was enhanced. Formate dehydrogenase has great potential in the resource utilization of CO2. This application not only provides a new approach for the catalytic utilization of CO2 through biological enzymes, but also lays the foundation for building a sustainable "artificial carbon cycle".

[0015] In summary, compared with the prior art, the present invention has the following significant beneficial effects and innovations:

[0016] 1. This invention provides a novel source of biocatalysts. Using gene mining technology, this invention, for the first time, isolated and functionally validated ArFDH (SEQ ID NO.1) from *Ancylobacter rudongensis*. The amino acid sequence of this enzyme is unique compared to any known FDH.

[0017] 2. This invention utilizes rational design based on structural information to perform site-directed mutagenesis on ArFDH. Compared to wild-type ArFDH, the optimal double-site mutant I123F / D222M exhibits better CO2 / HCO3 ratio. - The catalytic efficiency was increased by 6.69 times, and the activity of the currently reported highest-activity template enzyme TsFDH (29.71 U / g) was significantly increased to 305.54 U / g (an increase of 10.28 times). More importantly, as shown in Table 3, the catalytic activity of the two-point mutants (such as I123Y / D222R) is not a simple summation of the effects of their corresponding single-point mutants (increased activity of I123Y and decreased activity of D222R), but rather exhibits a complex synergistic or compensatory effect.

[0018] 3. This invention provides an efficient, mild, and sustainable pathway for the resource utilization of CO2. The FDH and its mutants provided by this invention can efficiently catalyze the conversion of CO2 to formic acid in an aqueous system at room temperature and pressure, with a green process and no toxic byproducts. Enzymatic properties studies show that ( Figure 8The mutant I123F / D222M has a wide pH range (5.5-8.0, residual activity >60%) and good thermal stability (retaining >50% activity after 5 minutes at 60℃), laying the foundation for its industrial application. Attached Figure Description

[0019] 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.

[0020] 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;

[0021] Figure 3 It is the amino acid sequence of wild-type formate dehydrogenase ArFDH;

[0022] Figure 4 This is a schematic diagram of the construction of the pET28a-fdh recombinant plasmid;

[0023] Figure 5 This is the result of agarose gel electrophoresis of the pET28a-fdh recombinant plasmid;

[0024] 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;

[0025] Figure 7 This is the HPLC chromatogram of formic acid in the ArFDH catalytic reaction products;

[0026] Figure 8The 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 in 0.1 M PBS buffer (pH 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0) was measured 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. Detailed Implementation

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] Plasmid extraction kits, FastPure gel DNA extraction mini kits, one-step cloning kits, MutExpress II rapid mutation kits, 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 New England Biolabs (NEB) kits.

[0032] This invention discloses a formate dehydrogenase for CO2 reduction and its applications. Firstly, gene mining and molecular docking techniques were combined to efficiently and accurately obtain formate dehydrogenase (ArFDH) from *Ancylobacter rudongensis*. This enzyme catalyzes the reduction of CO2 to formic acid and was successfully expressed at high levels in *E. coli*. Subsequently, ArFDH was directionally modified using a semi-rational design method, ultimately obtaining the I123Y / D222M mutant with excellent catalytic performance. This research provides a new method for the discovery and modification of novel enzymes and offers new ideas for the resource utilization of CO2.

[0033] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.

[0034] Example 1: Gene Mining

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 1 MAKILCVLYD DPIDGYPTTY ARDDLPKIDH YPGGQTLPTP KAVDFTPGHL

[0039] 51 LGSVSGALGL RKYLESNGHT LVVTSDKDGP NSVFEKELVD ADIVISQPFW

[0040] 101 PAYLTPERIA KAKNLKLALT AGIGSDHVDL QSAIDRNITV AEVTYCNSIS

[0041] 151 VAEHVVMMIL GLVRNYLPSH DWARQGGWNI ADCVAHSYDL EAMSVGTVAA

[0042] 201 GRIGLAVLRR LAPFDVKLHY TDHRLPDAV EKELNLTWHA SREEMYPHCD

[0043] 251 VVTLNCPLHP ETEHMINDET LKLFKRGAYI VNTARGKLCD RDAVARALEN

[0044] 301 GQLAGYAGDV WFPQPAPADH PWRTMKWNGM TPHISGTSLS AQARYAAGTR

[0045] 351 EILECFFEGR AIRDEYLIVQ GGALAGTGAH SYSKGNATGG SEEAAKFKKA

[0046] 401 V

[0047] 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.

[0048] Example 2 Construction of recombinant plasmid and expression of target protein

[0049] 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.

[0050] Table 1 Primer Sequences

[0051]

[0052] 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:

[0053] PCR reaction system:

[0054]

[0055] The PCR temperature program was designed as follows:

[0056]

[0057] The target gene was recovered using the FastPure gel DNA extraction mini kit.

[0058] The pET28a plasmid was linearized by digesting it with BamHI and HindIII restriction endonucleases from NEB.

[0059] Double enzyme digestion system:

[0060]

[0061] 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.

[0062] 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.

[0063] Target fragment and linearized vector linkage system:

[0064]

[0065] 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.

[0066] PCR reaction system:

[0067]

[0068] The PCR temperature program was designed as follows:

[0069]

[0070] 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.

[0071] DpnI digestion system:

[0072]

[0073] 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).

[0074] 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 with 0.1 mol / L pH 7.0 PBS buffer.

[0075] 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.

[0076] like Figure 6 In 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.

[0077] 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 6Lanes 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.

[0078] Example 3 Study on Enzymatic Properties

[0079] 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 HCO3- - (CO2) is converted into formic acid.

[0080] 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.

[0081] The enzyme activity detection results of wild-type ArFDH and each mutant are shown in Table 2.

[0082] Table 2. Results of formate dehydrogenase activity assay

[0083]

[0084] 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.

[0085] For the determination of kinetic parameters at different substrate concentrations, the specific experimental method is to modify only the substrate concentration based on the original enzyme activity system and detection method. When measuring NADH substrate, enzyme activity was detected at substrate concentrations of 1 mM, 2 mM, 3 mM, 4 mM, and 5 mM. The substrate HCO3 was also measured. - When CO2 is present, NaHCO3 is used as the reaction substrate, and enzyme activity is detected at substrate concentrations of 50 mM, 100 mM, 150 mM, 200 mM, 250 mM and 300 mM.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] By studying the catalytic rate of ArFDH for different substrate concentrations, the catalytic rate of ArFDH and its mutant formate dehydrogenase on CO2 (HCO3-) was determined. - The kinetic parameters of ArFDH and NADH. The effect of ArFDH on CO2 (HCO3) - It has good affinity (Table 3) and can efficiently catalyze CO2 (HCO3-) - Efficient conversion of ) to formic acid.

[0092] Table 3 Kinetic parameters of ArFDH and various mutants

[0093]

[0094] 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.

[0095] Enzymatic characteristics of mutant I123F / D222M were studied, such as Figure 8As 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%.

[0096] 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. Application of enzymes with amino acid sequences as shown in SEQ ID NO.1 in the catalytic preparation of formic acid or formate from CO2 or bicarbonate.

2. A gene encoding an enzyme with the amino acid sequence shown in SEQ ID NO.1, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

2.

3. A formate dehydrogenase, characterized in that: 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.

4. The formate dehydrogenase according to claim 3, characterized in that: The formate dehydrogenase is NAD. + Formate-dependent dehydrogenase.

5. A recombinant genetically engineered bacterium, characterized in that: The recombinant genetically engineered bacteria expresses the formate dehydrogenase as described in claim 3, or integrates the gene sequence as described in claim 2.

6. The recombinant genetically engineered bacterium according to claim 5, characterized in that: The host of the genetically engineered bacteria is Escherichia coli, and the vector is a pET series vector.

7. The application of the formate dehydrogenase as described in claim 3 in catalyzing the preparation of formic acid or formate salt from CO2 or bicarbonate.

8. The application according to claim 7, characterized in that: CO2 or HCO3 - Using NADH as substrates, formic acid or formate is prepared by catalytic reduction using the formic acid dehydrogenase.

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