Mutant formic acid dehydrogenase and method for producing formic acid using same

Abstract

The present invention relates to mutant formic acid dehydrogenase and a method for producing formic acid using same, the mutant formic acid dehydrogenase according to the present invention comprising a substitution of an amino acid at position 434 in an amino acid sequence represented by SEQ ID NO: 1 by a negatively charged or non-polar amino acid.

Description

Mutant formic acid dehydrogenase and method for producing formic acid using the same

[0001] The present invention relates to a mutant formic acid dehydrogenase and a method for producing formic acid using the same.

[0002] The rapid development of fossil fuel-based industries is rapidly increasing the concentration of carbon dioxide in the atmosphere. Therefore, converting carbon dioxide into high-value chemicals is considered essential to slowing the rate of atmospheric carbon dioxide increase. Fortunately, carbon dioxide can produce environmentally friendly chemical platforms such as formic acid, dimethyl carbonate, and polymers. Among the many candidates for carbon dioxide production, formic acid is the most ideal chemical feedstock in terms of both economic and environmental benefits. Accordingly, research is being conducted in the field of application that aims to produce new energy through carbon dioxide reduction reactions, such as those involving formic acid dehydrogenase. Specifically, formic acid dehydrogenase can convert carbon dioxide, a greenhouse gas, into industrially valuable formic acid. While the production of formic acid from carbon dioxide has attracted significant attention due to its exceptional selectivity for formic acid, various challenges have hindered its practical application.

[0003] One of them is that although the existing formic acid dehydrogenase can produce formic acid using carbon dioxide, the efficiency of converting carbon dioxide into formic acid is very low, which limits the production of formic acid from by-product gases from the steelmaking industry, such as BFG, LDG, and COG. This is because the so-called product inhibition phenomenon occurs due to the formic acid produced when converting carbon dioxide into formic acid, and as the concentration of the produced formic acid increases, the activity of the enzyme is inhibited, resulting in a problem of decreased formic acid productivity. Therefore, it is necessary to develop an enzyme that is not inhibited by this formic acid product.

[0004] The background technology described above is something that the inventor possessed or acquired in the process of deriving the disclosure of the present application, and cannot necessarily be said to be a publicly known technology disclosed to the general public prior to the present application.

[0005] In order to solve the above-described problem, the present invention provides a mutant formic acid dehydrogenase that is not inhibited by formic acid products and a method for producing formic acid using the same.

[0006] However, the problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by a person having ordinary skill in the relevant technical field from the description below.

[0007] The mutant formic acid dehydrogenase according to the present invention includes a mutant formic acid dehydrogenase in which amino acid position 434 of the amino acid sequence described in SEQ ID NO: 1 is substituted with a negatively charged or nonpolar amino acid.

[0008] In one embodiment, the dehydrogenase may be derived from a strain of the genus Methylobacterium.

[0009] In one embodiment, the dehydrogenase may be formic acid dehydrogenase 1 derived from Methylobacterium extorquens AM1.

[0010] In one embodiment, the amino acid at position 434 may be an amino acid constituting a formic acid transport tunnel.

[0011] In one embodiment, the negatively charged amino acid may include aspartic acid, glutamic acid, or both, and the nonpolar amino acid may include at least one selected from the group consisting of glycine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, proline, and tryptophan.

[0012] In one embodiment, the dehydrogenase may be a mutant R434A comprising an amino acid sequence set forth in SEQ ID NO: 3.

[0013] In one embodiment, the dehydrogenase may be one whose activity is not reduced by formic acid.

[0014] A method for producing formic acid according to the present invention comprises the steps of: synthesizing a mutant formic acid dehydrogenase; and converting carbon dioxide into formic acid in the presence of the dehydrogenase.

[0015] In one embodiment, the step of synthesizing the mutant formic acid dehydrogenase comprises the steps of: isolating a formic acid dehydrogenase comprising an amino acid sequence set forth in SEQ ID NO: 1 from a strain of the genus Methylobacterium; predicting a site to which formic acid attaches in the formic acid dehydrogenase; and forming a mutant by inducing a mutation in an amino acid that determines the site to which formic acid attaches; wherein the site to which formic acid attaches may be present in a formic acid transport tunnel.

[0016] In one embodiment, the amino acid may be located on the surface of the dehydrogenase or may have a size of 1.0 nm or more.

[0017] In one embodiment, the amino acid located on the surface of the dehydrogenase may be positively charged or polar.

[0018] According to one embodiment, the step of forming the mutant may be to replace amino acid position 434 of the amino acid sequence described in SEQ ID NO: 1 with at least one selected from the group consisting of aspartic acid, glutamic acid, glycine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, proline, and tryptophan.

[0019] In one embodiment, the variant may be variant R434A comprising the amino acid sequence set forth in SEQ ID NO: 3.

[0020] In one embodiment, the step of converting to formic acid may be such that the reduction in formic acid conversion efficiency is 30% or less.

[0021] The present invention can provide a mutant formic acid dehydrogenase and a method for producing formic acid using the same.

[0022] Specifically, the mutant formic acid dehydrogenase according to the present invention can provide a formic acid dehydrogenase treated with an amino acid mutation that has high enzyme activity and does not cause product inhibition by formic acid produced when converting carbon dioxide to formic acid, and a method for producing high-concentration formic acid with high formic acid productivity by converting carbon dioxide to formic acid using the same.

[0023] Figure 1 is a three-dimensional stereoscopic structure showing a transport tunnel of formic acid dehydrogenase for predicting the site where formic acid is attached in the method for producing formic acid according to the present invention.

[0024] Figure 2 is a graph showing the formic acid production efficiency according to the formic acid concentration of formic acid dehydrogenase according to an embodiment of the present invention.

[0025] Figure 3 is a graph showing the relative enzyme activity of formic acid dehydrogenase according to the concentration of formic acid according to an embodiment of the present invention.

[0026] Hereinafter, embodiments are described in detail with reference to the attached drawings. However, the embodiments may be modified in various ways, and the scope of the patent application is not limited or restricted by these embodiments. It should be understood that all modifications, equivalents, or alternatives to the embodiments are included within the scope of the patent application.

[0027] The terms used in the examples are for illustrative purposes only and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as "comprise" or "have" are intended to specify the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, but should be understood to not preclude the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0028] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiments pertain. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0029] In addition, when describing with reference to the attached drawings, the same components will be given the same reference numerals regardless of the drawing numbers, and redundant descriptions thereof will be omitted. When describing an embodiment, if it is determined that a detailed description of a related known technology may unnecessarily obscure the gist of the embodiment, the detailed description thereof will be omitted. In addition, when describing a component of an embodiment, terms such as first, second, A, B, (a), (b), etc. may be used. These terms are only to distinguish the component from other components, and the nature, order, or sequence of the component is not limited by the terms. When a component is described as being "connected," "coupled," or "connected" to another component, it should be understood that the component may be directly connected or connected to the other component, but another component may also be "connected," "coupled," or "connected" between each component.

[0030] Components included in one embodiment and components with common functions will be described using the same names in other embodiments. Unless otherwise stated, the descriptions given in one embodiment can be applied to other embodiments, and detailed descriptions will be omitted to the extent of overlap.

[0031]

[0032] The mutant formic acid dehydrogenase according to the present invention includes a mutant formic acid dehydrogenase in which amino acid position 434 of the amino acid sequence described in SEQ ID NO: 1 is substituted with a negatively charged or nonpolar amino acid.

[0033] Formic acid dehydrogenase is an enzyme that converts carbon dioxide into formic acid, and formic acid may include formic acid and formate. The mutant formic acid dehydrogenase according to the present invention is capable of producing high concentrations of formic acid from carbon dioxide, as it is free from the inhibitory effect of formic acid products compared to wild-type formic acid dehydrogenase.

[0034] The above mutant formic acid dehydrogenase is one in which a portion of the amino acid sequence of the formic acid dehydrogenase described in sequence number 1 is replaced with a different amino acid.

[0035] The above substitution refers to synthesizing a mutation by replacing an amino acid at a specific position in the amino acid sequence with another amino acid. Any method used in the technical field of the present invention may be used as long as it does not deviate from the purpose of the present invention. For example, it may be a saturated mutagenesis method, a random mutagenesis method, a site-directed mutagenesis method, etc. Preferably, it may be a site-directed mutagenesis method.

[0036] The mutant formic acid dehydrogenase according to the present invention can produce formic acid, which is one of the useful products, from carbon dioxide, and therefore can be used in a technology for reducing carbon dioxide emissions.

[0037]

[0038] In one embodiment, the dehydrogenase may be derived from a strain of the genus Methylobacterium.

[0039] In one embodiment, the dehydrogenase may be formic acid dehydrogenase 1 derived from Methylobacterium extorquens AM1.

[0040] The formic acid dehydrogenase 1 (MeFDH1) derived from the above Methylobacterium extorquens AM1 may include a MeFDH1 alpha subunit consisting of an amino acid sequence of sequence number 1 and a MeFDH1 beta subunit consisting of an amino acid sequence of sequence number 2.

[0041]

[0042] In one embodiment, the amino acid at position 434 may be an amino acid constituting a formic acid transport tunnel.

[0043] Formic acid, generated from carbon dioxide, can move out of the enzyme through the formic acid transport tunnel. However, if formic acid binds to the formic acid transport tunnel, its entry and exit are inhibited, acting as a non-competitive inhibitor of the dehydrogenase, reducing the efficiency of converting carbon dioxide to formic acid. Therefore, by substituting some of the amino acids that make up the formic acid transport tunnel to prevent formic acid from binding to the formic acid transport tunnel, formic acid does not act as a non-competitive inhibitor, allowing for the production of high concentrations of formic acid without inhibition by formic acid.

[0044]

[0045] In one embodiment, the negatively charged amino acid may include aspartic acid, glutamic acid, or both, and the nonpolar amino acid may include at least one selected from the group consisting of glycine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, proline, and tryptophan.

[0046] Amino acid position 434 of the amino acid sequence described in sequence number 1 is a positively charged or polar amino acid, and can inhibit binding of formic acid when substituted with a negatively charged amino acid or non-polar amino acid.

[0047]

[0048] In one embodiment, the dehydrogenase may be a mutant R434A comprising an amino acid sequence set forth in SEQ ID NO: 3.

[0049] The above mutant refers to a protein or protein having a mutation in the sequence of at least one amino acid among the sequences of a polypeptide constituting the target protein. Specifically, it refers to a protein produced by mutation of any one or more amino acid sequences of formic acid dehydrogenase. The mutant R434A may include a MeFDH1 alpha subunit having an amino acid sequence of SEQ ID NO: 3 and a MeFDH1 beta subunit having an amino acid sequence of SEQ ID NO: 4, in which amino acid arginine (Arginine 434, R434) at position 434 of the amino acid sequence described in SEQ ID NO: 1 is substituted with alanine (Alanine 434, A434).

[0050] Arginine, a polar amino acid, possesses a positive charge and thus can form an ionic bond with formic acid, which possesses a negative charge, leading to allosteric product inhibition. Therefore, substituting arginine with the nonpolar and smaller alanine eliminates the charge, thereby suppressing allosteric product inhibition. This effect mitigates product inhibition by the produced formic acid, enabling the production of high-concentration formic acid with high productivity.

[0051]

[0052] In one embodiment, the dehydrogenase may be one whose activity is not reduced by formic acid.

[0053] The conventional wild-type formic acid dehydrogenase containing sequence number 1 had a problem in that the formic acid produced while converting carbon dioxide into formic acid acted as a non-competitive inhibitor for the enzyme, resulting in a very low efficiency of formic acid conversion, thereby reducing productivity.

[0054] The mutant formic acid dehydrogenase according to the present invention can prevent formic acid from acting as a non-competitive inhibitor by replacing a key amino acid of the formic acid transport tunnel, so that the efficiency of formic acid conversion does not decrease over time.

[0055]

[0056] A method for producing formic acid according to the present invention comprises the steps of: synthesizing a mutant formic acid dehydrogenase; and converting carbon dioxide into formic acid in the presence of the dehydrogenase.

[0057] Formic acid may be produced by conversion from carbon dioxide via dehydrogenase.

[0058]

[0059] In one embodiment, the step of synthesizing the mutant formic acid dehydrogenase comprises the steps of: isolating a formic acid dehydrogenase comprising an amino acid sequence represented by SEQ ID NO: 1 from a strain of the genus Methylobacterium; predicting a site to which formic acid attaches in the formic acid dehydrogenase; and forming a mutant by inducing a mutation in an amino acid that determines the site to which formic acid attaches; wherein the site to which formic acid attaches may be present in a formic acid transport tunnel.

[0060] The formic acid dehydrogenase comprising the amino acid sequence described in the above sequence number 1 may be MeFDH1.

[0061] The step of predicting the site where the formic acid binds may be by predicting the movement path of the formic acid and analyzing the formic acid transport tunnel. In other words, the site where the formic acid binds may be present in the formic acid transport tunnel, and the site where the formic acid binds may mean a site where the activity of the formic acid transport tunnel is reduced by binding of the formic acid, thereby causing the formic acid to act as a non-competitive inhibitor to the enzyme. Therefore, the inhibition phenomenon of formic acid may occur depending on the binding at a site where the formic acid can act as a non-competitive inhibitor, and the inhibition phenomenon by formic acid can be suppressed by mutating a specific amino acid at the site where the formic acid binds so that the formic acid cannot bind.

[0062] To predict the site of attachment of formic acid, a computer can be used to extract various candidates, and through simulation, amino acids that maintain overall activity and increase stability can be ultimately derived from among the extracted candidates. Specifically, when mutations are induced in the above amino acid candidate group, it is desirable to consider a method that interferes with the binding of formic acid without affecting the protein folding of the resulting mutant or the enzymatic activity. Various prediction programs or techniques that can implement a three-dimensional structure and produce results through simulation can be utilized, and amino acids present in the formic acid transport tunnel can be selected accordingly. However, the formic acid attachment site is not necessarily limited to the above-mentioned content, and various known methods or techniques can be used to select the site of attachment of formic acid.

[0063] Figure 1 is a three-dimensional stereoscopic structure showing the transport tunnel of formic acid dehydrogenase for predicting the site of attachment of formic acid in the method for producing formic acid according to the present invention. Referring to Figure 1, a 3D image of formic acid dehydrogenase (MeFDH1) and the formic acid transport tunnel can be confirmed. In this way, the path through which carbon dioxide and formic acid move within formic acid dehydrogenase can be predicted through the 3D image, and after predicting the formic acid transport tunnel, the site of attachment of formic acid can be predicted.

[0064] The site of attachment of formic acid can be determined by an amino acid, and the step of forming the mutant involves inducing a mutation in at least a portion of the amino acid sequence of the formic acid dehydrogenase described in SEQ ID NO: 1, wherein the mutation is preferably an amino acid substitution. By substituting with a different amino acid, a decrease in the activity of the formic acid transport tunnel can be suppressed.

[0065]

[0066] In one embodiment, the amino acid may be located on the surface of the dehydrogenase or may have a size of 1.0 nm or more.

[0067] The above amino acid may be an amino acid located on the surface of the dehydrogenase while existing in the formic acid transport tunnel in order to find an amino acid residue to which formic acid is attached, or an amino acid having a size of 1.0 nm or larger.

[0068] Since amino acids located on the surface can bind to formic acid, they can be replaced with other amino acids to prevent formic acid from binding.

[0069] Since large amino acids can interfere with the entry and exit of formic acid, they can be replaced with other amino acids to facilitate the entry and exit of formic acid.

[0070]

[0071] In one embodiment, the amino acid located on the surface of the dehydrogenase may be positively charged or polar.

[0072] The amino acids that determine the binding sites of formic acid, located in the transport tunnel and on the enzyme surface, may be positively charged or polar and bind to formic acid, and the bound formic acid may act as a non-competitive inhibitor of the enzyme.

[0073]

[0074] According to one embodiment, the step of forming the mutant may be to replace amino acid position 434 of the amino acid sequence described in SEQ ID NO: 1 with at least one selected from the group consisting of aspartic acid, glutamic acid, glycine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, proline, and tryptophan.

[0075] Amino acid position 434 of the amino acid sequence described in the above sequence number 1 may be arginine. By replacing arginine with the above amino acid, binding of formic acid can be inhibited so that formic acid cannot act as an inhibitor in the formic acid transport tunnel, and thus inhibition by formic acid can be prevented.

[0076]

[0077] In one embodiment, the variant may be variant R434A comprising the amino acid sequence set forth in SEQ ID NO: 3.

[0078] The variant R434A including the amino acid sequence described in the above sequence number 3 may be one in which the 434th amino acid arginine (Arginine 434, R434) of the amino acid sequence described in the above sequence number 1 is substituted with alanine (Alanine 434, A434).

[0079] Arginine, a polar amino acid, possesses a positive charge and thus can form an ionic bond with formic acid, which possesses a negative charge, leading to allosteric product inhibition. Therefore, substituting arginine with the nonpolar and smaller alanine eliminates the charge, thereby suppressing allosteric product inhibition. This effect mitigates product inhibition by the produced formic acid, enabling the production of high-concentration formic acid with high productivity.

[0080]

[0081] In one embodiment, the step of converting to formic acid may be such that the reduction in formic acid conversion efficiency is 30% or less.

[0082] Conventional formic acid dehydrogenases have a problem in that the formic acid produced during the conversion of carbon dioxide to formic acid acts as a non-competitive inhibitor of the dehydrogenase, reducing the efficiency of formic acid conversion and thus reducing productivity. In other words, as formic acid conversion progresses over time, the resulting formic acid prevents the production of new formic acid.

[0083] The above formic acid conversion efficiency represents the formic acid produced relative to the carbon dioxide injected, and may be reduced by 30% or less when comparing the initial formic acid conversion efficiency and the late formic acid conversion efficiency. The late formic acid conversion efficiency refers to the conversion efficiency when the amount of formic acid produced increases over time, for example, when 2 hours or more have passed since the reaction or when the concentration of the produced formic acid is 20 mM or more, and the reduced degree may be expressed as (late formic acid conversion efficiency / early formic acid conversion efficiency) X 100 (%). Preferably, it may be 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less, and since there is little inhibition by the formic acid product, a high concentration of formic acid can be produced.

[0084] The mutant formic acid dehydrogenase according to the present invention may have an activity that is at least 10% higher than that of the formic acid dehydrogenase before mutation treatment. The activity may be formic acid conversion efficiency, and the excellent activity may produce a high concentration of formic acid.

[0085]

[0086] The mutant formic acid dehydrogenase according to the present invention has resistance to high concentrations of formic acid due to mutation of a specific amino acid, so that the product inhibition phenomenon by formic acid produced when converting carbon dioxide to formic acid does not occur, and accordingly, regardless of the production of formic acid, the activity of the enzyme that converts formic acid from carbon dioxide is increased, so that formic acid productivity increases, and high concentrations of formic acid can be produced.

[0087]

[0088] Hereinafter, the present invention will be described in more detail by way of examples.

[0089] However, the following examples are only intended to illustrate the present invention, and the content of the present invention is not limited to the following examples.

[0090]

[0091] Example

[0092] A mutant formic acid dehydrogenase that is not inhibited by formic acid was prepared.

[0093] First, in order to find out the factor that inhibits the carbon dioxide reduction reaction of formic acid dehydrogenase (MeFDH1), an experiment was conducted in which the activity of MeFDH1 was measured by adding increasing concentrations of formic acid in the carbon dioxide reduction reaction. Fig. 2 is a graph showing the efficiency of formic acid production according to the concentration of formic acid of formic acid dehydrogenase according to an example of the present invention. Referring to Fig. 2, it can be confirmed that as the concentration of artificially added formic acid increases, the activity of MeFDH1 in the carbon dioxide reduction reaction decreases. In addition, according to the Lineweaver-Burk plot of the carbon dioxide reduction reaction of MeFDH1 according to the concentration of formic acid, it was found that formic acid acts as a non-competitive inhibitor of this reaction. Since a non-competitive inhibitor reduces the activity of the enzyme by binding to a site other than the active site, it can be confirmed that in order to solve this problem, it is necessary to find the site where formic acid binds to MeFDH1 and prevent the inhibitor from binding.

[0094] Experiments have shown that formic acid inhibits the carbon dioxide reduction reaction of MeFDH1 at concentrations above 114.7 mM, and usually K of MeFDH1 M The value is 1 mM and the K of formic acid I Since the value is 114.7 mM, it can be predicted that formic acid binds to a specific part of the formic acid transport tunnel and continues to accumulate, inhibiting its exit from the enzyme. Therefore, it was found that if we can find the site where formic acid binds in the formic acid transport tunnel and prevent it from binding, we can resolve the phenomenon of formic acid inhibiting the carbon dioxide reduction reaction of MeFDH1.

[0095] After predicting the path that carbon dioxide and formic acid move within MeFDH1 and predicting the formic acid transport tunnel, we looked into where formic acid would bind, and used two methods to find the key amino acid site in the formic acid transport tunnel that reduces the activity of formic acid produced by the carbon dioxide reduction reaction of MeFDH1. First, among the amino acids that make up the formic acid transport tunnel, the positively charged or polar amino acids located on the enzyme surface were changed to negatively charged or nonpolar amino acids to prevent formic acid from binding. Second, among the amino acids that make up the formic acid transport tunnel, the large amino acids were changed to alanine to reduce the size so that formic acid can easily enter and exit. The mutations used in the experiment are shown in Table 1 below. The mutant dehydrogenase in Table 1 below is a mutant generated as a result of an amino acid substitution of MeFDH1 containing sequence number 1. For example, if threonine 433 (T433) is substituted with aspartic acid 433 (D433), it can be expressed as T433D.

[0096] Location Dehydrogenase sequence (MeFDH1 sequence (7VW6)) Mutant dehydrogenase (MeFDH1 mutant) Characteristic Surface T433T433D Negative charge T433V Nonpolar R434R434E Negative charge R434W Nonpolar R434ANonpolar S639S639D Negative charge charge)S639VNonpolarAllH431H431ASizeT433T433AR434R434AH437H437AS450S450AR630R630AG631G631AQ632Q632AQ636Q636AS639S639AD640D640A

[0097] Formic acid was artificially added to the carbon dioxide reduction reaction of the mutants in Table 1 to measure changes in enzyme activity. Figure 3 is a graph showing the relative enzyme activity of formic acid dehydrogenase according to the concentration of formic acid according to an example of the present invention. Referring to Figure 3, the carbon dioxide reduction reaction activity of the mutants was lower than that of the wild type (WT) or decreased as the concentration of formic acid increased, but the activity of the mutants with a mutated R434 amino acid did not change significantly even when formic acid was added. This indicates that the R434 amino acid is an amino acid that is significantly involved in the inhibitory effect of formic acid. To prevent formic acid from attaching to the R434 amino acid, the experiment was conducted after changing it to various negatively charged or nonpolar amino acids. As a result, the R434A mutant had higher activity than the other mutants and its activity hardly changed with the concentration of formic acid. It can be seen that the R434A mutant has little inhibitory effect of formic acid, so it will increase formic acid production by MeFDH1.

[0098]

[0099] Although the embodiments have been described above, those skilled in the art will appreciate that various technical modifications and variations can be applied based on the above. For example, appropriate results can still be achieved even if the described techniques are performed in a different order than described, and / or components of the described systems, structures, devices, circuits, etc. are combined or combined in a different manner than described, or are replaced or substituted with other components or equivalents.

[0100] Therefore, other implementations, other embodiments, and equivalents to the claims also fall within the scope of the claims described below.

[0101] Attach an electronic file of the sequence list

[0102] (C: / KipoNet / NKEditor / appfile / APC-2023-0722_Specification.xml)

Claims

1. Amino acid position 434 of the amino acid sequence described in sequence number 1 is replaced with a negatively charged or nonpolar amino acid. Mutant formic acid dehydrogenase.

2. In paragraph 1, The above dehydrogenase is derived from a strain of the genus Methylobacterium. Mutant formic acid dehydrogenase.

3. In paragraph 1, The above dehydrogenase is formic acid dehydrogenase 1 derived from Methylobacterium extorquens AM1. Mutant formic acid dehydrogenase.

4. In paragraph 1, The above amino acid number 434 is an amino acid that constitutes a formic acid transport tunnel. Mutant formic acid dehydrogenase.

5. In paragraph 1, The above negatively charged amino acid comprises aspartic acid, glutamic acid or both, The above nonpolar amino acid comprises at least one selected from the group consisting of glycine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, proline and tryptophan. Mutant formic acid dehydrogenase.

6. In paragraph 1, The above dehydrogenase is a mutant R434A comprising an amino acid sequence described as sequence number 3. Mutant formic acid dehydrogenase.

7. In paragraph 1, The above dehydrogenase is not deactivated by formic acid. Mutant formic acid dehydrogenase.

8. Step of synthesizing mutant formic acid dehydrogenase; and A step of converting carbon dioxide into formic acid in the presence of the above dehydrogenase; Method for producing formic acid.

9. In paragraph 8, The steps for synthesizing the above mutant formic acid dehydrogenase are: It comprises the steps of isolating a formic acid dehydrogenase comprising an amino acid sequence described as sequence number 1 from a strain of the genus Methylobacterium; the step of predicting a site to which formic acid is attached in the formic acid dehydrogenase; and the step of forming a mutant by inducing a mutation in an amino acid that determines the site to which formic acid is attached. The above-mentioned place where the formic acid is attached is located in the formic acid transport tunnel. Method for producing formic acid.

10. In paragraph 9, The above amino acid is located on the surface of the dehydrogenase, or Having a size of 1.0 nm or more, Method for producing formic acid.

11. In paragraph 10, The amino acid located on the surface of the above dehydrogenase is positively charged or polar. Method for producing formic acid.

12. In paragraph 9, The step of forming the above mutant is to replace amino acid 434 of the amino acid sequence described in sequence number 1 with at least one selected from the group consisting of aspartic acid, glutamic acid, glycine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, proline, and tryptophan. Method for producing formic acid.

13. In paragraph 9, The above mutant is mutant R434A, which comprises an amino acid sequence described as sequence number 3. Method for producing formic acid.

14. In paragraph 8, The step of converting to formic acid is such that the reduction in formic acid conversion efficiency is 30% or less. Method for producing formic acid.

Images