KR20220141146A - A genetically modified formate dehydrogenase or an immobilized genetically modified formate dehydrogenase and an preparing method of formic acid using them - Google Patents

Abstract

One aspect provides a genetically recombinant histidine-labeled formic acid dehydrogenase or an immobilized histidine-labeled formic acid dehydrogenase. According to one aspect, the genetically recombinant histidine-labeled formic acid dehydrogenase can efficiently produce formic acid in high yield by reducing carbon dioxide. Furthermore, when the formic acid dehydrogenase is immobilized, the genetically recombinant histidine-labeled formic acid dehydrogenase can prepare high-concentration formic acid without a decrease in efficiency for a long time in an electrochemical reactor. In addition, the genetically recombinant histidine-labeled formic acid dehydrogenase is not only reusable, but is not significantly affected by dissolved oxygen concentration, and thus can greatly contribute to industries in the field of carbon dioxide removal or formic acid preparation.

Description

A GENETICALLY MODIFIED FORMATE DEHYDROGENASE OR AN IMMOBILIZED GENETICALLY MODIFIED FORMATE DEHYDROGENASE AND AN PREPARING METHOD OF FORMIC ACID USING THEM}

It relates to a recombinant formic acid dehydrogenase or an immobilized recombinant formic acid dehydrogenase and a method for producing formic acid using the same.

The rapid development of the industry using fossil fuels is rapidly increasing the concentration of carbon dioxide in the atmosphere. In addition, a number of papers have confirmed that the increase in atmospheric carbon dioxide is a decisive factor in causing global warming through the greenhouse effect. Therefore, the conversion of carbon dioxide into high value-added chemicals is assumed to be an essential technology to slow the rate of increase in atmospheric carbon dioxide. Fortunately, carbon dioxide is a promising renewable resource for producing environmentally friendly chemical platforms such as formic acid, dimethyl carbonate, and polymers.

Among the many candidates that can be produced from carbon dioxide, formic acid is the most ideal chemical raw material in terms of economic and environmental benefits. In particular, the direct formate fuel cell (DFFC), which converts formic acid into electrical energy, can easily store energy produced from various renewable energy sources such as wind power, solar power, and hydro power as formic acid, so it is a solution for renewable energy storage. is being presented In addition, since formic acid is non-flammable, non-toxic, and inert in the environment, there is an advantage that it can be safely transported than hydrogen gas.

Despite promising potential, existing technologies for producing high value-added compounds from carbon dioxide have fatal limitations, requiring harsh reaction conditions, rare noble metal catalysts, and expensive reducing agents such as hydrogen gas and hydrides. Although electrocatalysis of carbon dioxide is a promising alternative given the relatively low cost of electrical energy, some chemical electrocatalysts produce hydrogen gas and carbon monoxide, which are by-products at high rates during the conversion of carbon dioxide to formic acid, resulting in insufficient reaction selectivity and consequently It shows the risks of the process. Therefore, formic acid production from carbon dioxide by an enzyme-based electrochemical reaction is receiving more attention due to its excellent formic acid selectivity. However, in the case of an enzyme-based electrochemical reaction, despite these advantages, it has not been put to practical use due to various problems. Among them, the biggest factor is low oxygen stability, and in the case of a metal-containing enzyme with a high carbon dioxide reduction reaction rate, it is extremely unstable to oxygen and thus easily loses its activity. In addition, both the reaction of reducing carbon dioxide and the reaction of re-oxidizing the generated formic acid are catalyzed by the enzyme, resulting in a reaction equilibrium, which makes it difficult to produce a high concentration of formic acid.

Accordingly, the histidine tag was recombined with formate dehydrogenase derived from Methylobacterium extorquens AM1 containing tungsten, and the recombinant enzyme was immobilized on Ni-NTA agarose bead. A method that can be easily and quickly prepared with an enzyme catalyst at the same time as separation and a long-term operation in an electrochemical reactor to produce a high concentration of formic acid and at the same time lead to the development of the immobilized enzyme capable of continuous reuse.

Korean Patent No. 10-1564315

One aspect is to provide a histidine-tagged formic acid dehydrogenase (Formate Dehydrogenase).

Another aspect is to provide a histidine-tagged (His-tag) formic acid dehydrogenase (Formate Dehydrogenase) immobilized on Ni-NTA (Nickel-Nitrilotriacetic Acid).

Another aspect is to provide a composition for preparing formic acid comprising histidine-labeled (His-tag) formic acid dehydrogenase (Formate Dehydrogenase).

Another aspect is to provide an electrochemical reaction system for producing formic acid including histidine-tagged formic acid dehydrogenase (Formate Dehydrogenase).

Another aspect is to provide an apparatus for producing formic acid comprising histidine-labeled (His-tag) formic acid dehydrogenase (Formate Dehydrogenase).

Another aspect is to provide a filter for removing carbon dioxide including histidine-tagged formic acid dehydrogenase (Formate Dehydrogenase).

Another aspect is to provide a method for producing formic acid comprising the step of contacting a gas containing carbon dioxide and an electron transporter to histidine-labeled (His-tag) formic acid dehydrogenase (Formate Dehydrogenase).

One aspect provides a histidine-tagged formic acid dehydrogenase (Formate Dehydrogenase).

The formic acid dehydrogenase can be converted into formic acid by contact with carbon dioxide. Specifically, formic acid dehydrogenase can catalyze the reaction of Scheme 1 below to prepare formic acid:

[Scheme 1]

CO ₂ + 2H ⁺ + EM (reduced state) -> HCOOH + EM (oxidized state).

In one aspect, the formic acid dehydrogenase may be derived from nature, and may be obtained by various protein synthesis methods well known in the art. As an example, it may be prepared using a gene recombination and protein expression system, or it may be prepared by a method of synthesizing in vitro through chemical synthesis such as protein synthesis, a cell-free protein synthesis method, and the like. Also, as an example, the formate dehydrogenase may be a peptide, an extract of a plant-derived tissue or cell, or a product obtained by culturing a microorganism (eg, bacteria or fungi, and particularly yeast).

The term “protein” refers to a polymer composed of two or more amino acids linked by amide bonds (or peptide bonds).

The term “expression” refers to the process by which a polypeptide is produced from a structural gene. The process involves the transcription of a gene (polynucleotide) into mRNA and the translation of such mRNA into polypeptide (protein)(s).

The term "recombinant" refers to a cell in which the cell replicates, expresses a heterologous nucleic acid, or expresses a peptide, heterologous peptide or protein encoded by the heterologous nucleic acid. The recombinant cell may express a gene or a gene fragment not found in the natural form of the cell in either the sense or antisense form. Also, 　recombinant 　 cells can express 　 genes found in cells in a natural state, but the 　 genes are modified and re-introduced into the cell by artificial means.

In one aspect, the formic acid dehydrogenase may be produced by genetic recombination, and specifically, the formic acid dehydrogenase is Methylobacterium extorquens AM1-derived FDH1 (Formate Dehydrogenase 1) as,

The methylobacterium extorquens AM1 is an endogenous gene encoding FDH1α (Formate Dehydrogenase 1 alpha subunit) and an endogenous gene encoding FDH1β (Formate Dehydrogenase 1 beta subunit) is deleted,

It may be a recombinant microorganism into which an exogenous gene encoding FDH1 derived from Methylobacterium extorquens AM1 and a histidine label (His-tag) has been introduced.

When re-introduced by knocking out fdh1A, which is an endogenous gene encoding FDH1α, and fdh1B, an endogenous gene encoding FDH1β, in Methylobacterium extorquens AM1, and transforming the gene fdh1 encoding FDH1, wild-type Methylobacterium extorquens AM1 compared to formic acid production amount and production efficiency can be increased. When the gene encoding fdh1 is transformed and artificially expressed without knock-out pretreatment in wild-type Methylobacterium extorquens AM1, the production amount and production efficiency of formic acid as the recombinant microorganisms do not appear.

In one aspect, the exogenous gene encoding FDH1 may be introduced into a microorganism by a genetic material carrier. The genetic material carrier may be a vector.

The term "vector" refers to a DNA preparation containing a DNA sequence operably linked to suitable regulatory sequences capable of expressing the DNA in a suitable host. A vector can be a plasmid, a phage particle, or simply a potential genomic insert. Upon transformation into an appropriate host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself. As plasmid is currently the most commonly used form of vector, "plasmid" and "vector" are sometimes used interchangeably herein. The vector used in the present invention may include other forms of vectors known or having an equivalent function as known in the art.

In one embodiment, the vector may include a PmxaF promoter. The vector including the P _mxaF promoter is well known to those skilled in the art, and for example, may be pCM110, and pCM110 into which the histidine-labeled formic acid dehydrogenase is introduced may be a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 9. .

In addition, as the vector, any known to a person skilled in the art may be used as long as it is suitable for introducing an exogenous gene into a cell (specifically, a microorganism).

The formic acid dehydrogenase is about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 92% or more, about 95% or more, about 97, respectively, with the amino acid sequence of formic acid dehydrogenase. % or greater, about 98% or greater, or about 99% or greater sequence homology. The formic acid dehydrogenase may be an isoenzyme of formic acid dehydrogenase, for example, the formic acid dehydrogenase is methylobacterium sp., Candida sp. Thiobacillus sp. ), Ralstonia sp., and Rhodobacter sp. may be derived from microorganisms of at least one genus selected from the group consisting of.

In addition, the methylobacterium genus microorganisms are, for example, M. adhesivum ( M. adhaesivum ), M. aerolatum ( M. aerolatum ), M. aminoborans ( M. aminovorans ), M Aquaticum ( M. aquaticum ), M. brachiatum ( M. brachiatum ), M. brachythecii ( M. brachythecii ), M. bullatum ( M. bullatum ), M. cerastii ( M. cerastii ) , M. dankookense , M. extorquens , M. fujisawaense , M. gnaphalii, M. goe Shin Kense ( M. goesingense ), M. gossipiicola ( M. gossipiicola ), M. gregans ( M. gregans ), M. haplocladii ( M. haplocladii ), M. hispanicum ( M. hispanicum ) ), M. iners , M. isbiliense , M. jeotgali , M. komagatae, M. longum M. longum ), M. marchantiae ( M. marchantiae ), M. mesophilicum ( M. mesophilicum ), M. nodulans ( M. nodulans ), M. organophilum ( M. organophilum ), M M. oryzae , M. oxalidis , M. persicinum, M. phyllosphaerae , M. phyllosphaerae , M. phyllostachyos ), M. platani , M. podarium , M. populi , M. pseudosasae, M. pseudosasae . M. pseudosasicola ), M. radic M. radiotolerans, M. rhodesianum , M. rhodesianum , M. rhodinum, M. salsuginis , M. soli , M. Suomiense ( M. suomiense ), M. tardum ( M. tardum ), M. tarhaniae ( M. tarhaniae ), M. thiocyanatum ( M. thiocyanatum ), M. thuringienceae ( M. thuringiense ), M. trifolii ( M. trifolii ), M. variabile ( M. variabile ) and M. zatmanii ( M. zatmanii ) It may be at least one selected from the group consisting of.

Specifically, the formic acid dehydrogenase is methylobacterium extorquens ( Methylobacterium extorquens ), Ralstonia eutropha , Candida boidinii ) Thiobacillus sp. KNK65MA and It may be derived from at least one microorganism selected from the group consisting of Rhodobacter capsulatus .

More specifically, the formic acid dehydrogenase may be formic acid dehydrogenase I derived from Methylobacterium extorquens AM1 (ATCC 14781, GenBank accession No. CP001150.1).

The Methylobacterium extorquens AM1-derived formic acid dehydrogenase I may consist of MeFDH I α subunit (GenBank accession No. ACS42636.1) and MeFDH I β subunit (GenBank accession No. ACS42635.1). And, the methylobacterium extorquens ( Methylobacterium extorquens )-derived formic acid dehydrogenase I is fdh1A (GenBank accession, CP0011510.1: 5169596-5172565, Methylobacterium extorquens AM1, complete genome) and fdh1B (GenBank accession, CP0011510.1) :5167825-5169543, Methylobacterium extorquens AM1, complete genome) may be a polypeptide encoded.

The term "homology" is intended to indicate a degree of similarity with a wild-type amino acid sequence, and the comparison of such homology can be performed using a comparison program well known in the art, and the homology between two or more sequences is It can be calculated as a percentage (%).

The term "stability" may refer to storage stability (eg, room temperature storage stability) as well as in vivo stability to protect the formate dehydrogenase from attack by in vivo protein cleaving enzymes.

In addition, in order to obtain better chemical stability, enhanced pharmacological properties (half-life, absorption, potency, potency, etc.), altered specificity (e.g., broad spectrum of biological activity), reduced antigenicity, the N of the formate dehydrogenase - or a protecting group may be bonded to the C-terminus. The protecting group may be an acetyl group, a fluorenyl methoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, or polyethylene glycol (PEG). As long as it is a component capable of enhancing , it may be included without limitation.

In addition, the formic acid dehydrogenase may additionally include a targeting sequence, a tag, and an amino acid sequence prepared for a specific purpose for the labeled residue in addition to the histidine label. The phosphorus (histidine-labeled) formate dehydrogenase may be a polypeptide consisting of the amino acid sequences of SEQ ID NOs: 10 and 11.

Another aspect provides a histidine-tagged (His-tag) formic acid dehydrogenase (Formate Dehydrogenase) immobilized on Ni-NTA (Nickel-Nitrilotriacetic Acid).

The "histidine label", "formic acid dehydrogenase", etc. may be within the above-mentioned range.

When the histidine-labeled formic acid dehydrogenase is immobilized on Ni-NTA, the efficiency of the histidine-labeled formic acid dehydrogenase is not lowered for a long period of time compared to the unimmobilized histidine-labeled formic acid dehydrogenase, and formic acid production is possible, and not only can be reused, but also depending on the dissolved oxygen concentration The decrease in activity may be small.

In one aspect, the Ni-NTA is Ni-NTA (Nickel-Nitrilotriacetic acid), but is not limited as long as it includes Ni-NTA, for example, Ni-NTA resin, Ni-NTA affinity resin, Ni -NTA column (column), Ni-NTA affinity column, Ni-NTA agarose, Ni-NTA bead, Ni-NTA agarose bead, may be Ni-NTA magnetic bead, etc., specifically, the Ni-NTA is Ni-NTA agarose It can be a bead.

Another aspect provides a composition for preparing formic acid comprising histidine-labeled (His-tag) formic acid dehydrogenase (Formate Dehydrogenase).

The "histidine label", "formic acid dehydrogenase", "formic acid", etc. may be within the above-described range.

In one aspect, the composition for preparing formic acid may further include an electron transporter.

When the composition further comprises an electron transporter, the formic acid production efficiency can be increased.

The electron transporter may be a natural electron transporter or an artificial electron transporter. In addition, the electron transporter may be at least one electron transporter selected from the group consisting of an electron transporter having a viologen group and an electron transporter having an adenine dinucleotide group, for example, an alkyl viologen ( alkyl viologen), benzyl viologen, NAD (nicotinamide adenine dinucleotide), and FAD (flavin adenine dinucleotide) may be at least one electron transporter selected from the group consisting of, wherein the alkyl viologen is methyl Viologen (methyl viologen), ethyl viologen (ethyl viologen), propyl viologen (propyl viologen) and the like may be included.

Specifically, the electron transporter is selected from the group consisting of methyl viologen, ethyl viologen, benzyl viologen, nicotinamide adenine dinucleotide (NAD) and flavin adenine dinucleotide (FAD). It may be at least one electron transporter, and more specifically, it may be at least one electron transporter selected from the group consisting of methyl viologen and ethyl viologen.

On the other hand, FDH1 is known to have a W-pterin guanidine dinucleotide instead of molybdenum (Mo) as a prosthetic group. Specifically, FDH1α contains a bis-tungstopterin guanine dinucleotide cofactor, three 4Fe4S clusters, and one 2Fe2S cluster. FDH1β contains a flavin mononucleotide (FMN), NAD, and one 4Fe4S cluster and one 2Fe2S. Air-purified FDH1β can be successfully applied to the regeneration of NADH using methyl viologen (MV), an artificial electron transporter, in an electrochemical reduction system.

Also, in one aspect, the formic acid dehydrogenase may be immobilized on Ni-NTA (Nickel-Nitrilotriacetic Acid).

The Ni-NTA is Ni-NTA (Nickel-Nitrilotriacetic acid), and is not limited as long as it includes Ni-NTA, but for example, Ni-NTA resin, Ni-NTA affinity resin, Ni-NTA column (column) ), Ni-NTA affinity column, Ni-NTA agarose, Ni-NTA bead, Ni-NTA agarose bead, Ni-NTA magnetic bead, etc., specifically, the Ni-NTA may be a Ni-NTA agarose bead.

When the histidine-labeled formic acid dehydrogenase is immobilized on Ni-NTA, the efficiency of the histidine-labeled formic acid dehydrogenase is not lowered for a long period of time compared to the unimmobilized histidine-labeled formic acid dehydrogenase, and formic acid production is possible, and not only can be reused, but also depending on the dissolved oxygen concentration The decrease in activity may be small.

Also, in one aspect, the pH of the composition may be 5.0 to 7.5, 5.0 to 7.0, 5.0 to 6.5, 5.5 to 7.5, 5.5 to 7.0, 5.5 to 6.5, 6.0 to 7.5, 6.0 to 7.0 or 6.0 to 6.5. .

If the pH of the composition is less than 5.0, the reduction rate of carbon dioxide (formic acid production rate) may be lowered, and if the pH is greater than 7.5, the carbon dioxide reduction rate (formic acid production rate) is lowered, and the oxidation rate of formic acid is to be increased Can be, formic acid production yield may be reduced.

Another aspect provides an electrochemical reaction system for producing formic acid comprising histidine-tagged formic acid dehydrogenase (Formate Dehydrogenase).

The "histidine label", "formic acid dehydrogenase", "formic acid", etc. may be within the above-described range.

The histidine-labeled formic acid dehydrogenase can produce formic acid from carbon dioxide using the electrochemically produced electrons without an expensive electron donor such as NADH, unlike a general enzyme. Therefore, any system capable of supplying electrons to the formic acid dehydrogenase can be used to produce formic acid, and as such a system, for example, an electrical system in which an electric current flows directly from the battery by injecting an electrode or an electric current by a redox reaction There are electrochemical (chemical cells) systems that can generate and supply electrons. Therefore, in one aspect, the formic acid dehydrogenase may be applied in an electrical or electrochemical system to produce formic acid.

In one aspect, the electrochemical system may be an electrochemical carbon dioxide reduction system. The electrochemical carbon dioxide reduction system refers to a system capable of reducing carbon dioxide to formic acid using a chemical cell principle. The electrochemical conversion technology can reduce carbon dioxide even at room temperature and pressure conditions, and there is no emission of chemicals because water and carbon dioxide are mainly used, and the system is simple and easy to modularize. According to the electrochemical carbon dioxide reduction system, it is possible to produce formic acid from carbon dioxide without microorganisms or enzymes. However, as can be seen in this example, when using the histidine-labeled formic acid dehydrogenase according to the aspect, it is possible to produce formic acid from carbon dioxide with higher yield and efficiency. The electrochemical carbon dioxide reduction system includes, for example, a cathode such as copper, graphite, carbon felt, and carbon fiber and an anode such as platinum, as illustrated in FIG. 9 , and Ag/ A reference electrode such as AgCl may be further included. However, the electrochemical carbon dioxide reduction system can be modified in a manner well known to those skilled in the art, if necessary.

The histidine-labeled formic acid dehydrogenase can not only produce formic acid very effectively using electrons supplied from the negative electrode, but also has a very stable property to oxygen in the air. Among the known biocatalysts, there were some that could directly use the electrons supplied from the cathode, but they were very unstable to oxygen and thus practically impossible to apply. Therefore, the formic acid dehydrogenase according to an aspect can be used very usefully because it can be applied to an actual industrial process.

Also, in one aspect, the formic acid dehydrogenase may be immobilized on Ni-NTA (Nickel-Nitrilotriacetic Acid), and the Ni-NTA may be within the aforementioned range.

When the histidine-labeled formic acid dehydrogenase is immobilized on Ni-NTA, the efficiency of the histidine-labeled formic acid dehydrogenase is not lowered for a long period of time compared to the unimmobilized histidine-labeled formic acid dehydrogenase, and formic acid production is possible, and not only can be reused, but also depending on the dissolved oxygen concentration The decrease in activity may be small.

Another aspect provides a device for producing formic acid comprising a histidine-tagged (His-tag) formic acid dehydrogenase (Formate Dehydrogenase).

The "histidine label", "formic acid dehydrogenase", "formic acid", etc. may be within the above-described range.

Also, in one aspect, the formic acid dehydrogenase may be immobilized on Ni-NTA (Nickel-Nitrilotriacetic Acid), and the Ni-NTA may be within the aforementioned range.

When the histidine-labeled formic acid dehydrogenase is immobilized on Ni-NTA, the efficiency of the histidine-labeled formic acid dehydrogenase is not lowered for a long period of time compared to the unimmobilized histidine-labeled formic acid dehydrogenase, and formic acid production is possible, and not only can be reused, but also depending on the dissolved oxygen concentration The decrease in activity may be small.

Another aspect provides a filter for removing carbon dioxide including histidine-tagged formic acid dehydrogenase (Formate Dehydrogenase).

The "histidine label", "formic acid dehydrogenase", "carbon dioxide", etc. may be within the above-described range.

As described above, the histidine-labeled formic acid dehydrogenase removes carbon dioxide by reducing carbon dioxide to produce formic acid, but can produce formic acid, a useful material.

Also, in one aspect, the formic acid dehydrogenase may be immobilized on Ni-NTA (Nickel-Nitrilotriacetic Acid), and the Ni-NTA may be within the aforementioned range.

When the histidine-labeled formic acid dehydrogenase is immobilized on Ni-NTA, the efficiency of the histidine-labeled formate dehydrogenase is not lowered for a long period of time compared to the unimmobilized histidine-labeled formate dehydrogenase, and carbon dioxide removal is possible, reuse is possible, and depending on the dissolved oxygen concentration The decrease in activity may be small.

The filter can be applied to various filters in places where carbon dioxide is generated, such as cigarette filters and air purifier filters, and furthermore, in industrial sites requiring harmful gas treatment technology, sterilization/removal purification technology and systems for harmful substances in the air, vehicles, trains, etc. It may be included in treatment facilities and related technologies for indoor air quality management in Sudan, technologies and devices for ventilation efficiency and economical ventilation, and indoor air purification devices such as air purifiers, air conditioners, and fans.

Another aspect provides a method for producing formic acid comprising the step of contacting a gas containing carbon dioxide and an electron transporter to histidine-labeled (His-tag) formic acid dehydrogenase (Formate Dehydrogenase).

The "histidine label", "formic acid dehydrogenase", "carbon dioxide", "electron transporter", "formic acid", etc. may be within the above-described range.

Formic acid can be produced by reducing carbon dioxide by contacting the gas containing carbon dioxide with the formic acid dehydrogenase, and a specific formic acid production mechanism according to the reduction of carbon dioxide may be within the above-described range.

In one aspect, the formic acid dehydrogenase may be immobilized on Ni-NTA (Nickel-Nitrilotriacetic Acid), and the Ni-NTA may be within the above-described range.

When the histidine-labeled formic acid dehydrogenase is immobilized on Ni-NTA, the efficiency of the histidine-labeled formic acid dehydrogenase is not lowered for a long period of time compared to the unimmobilized histidine-labeled formic acid dehydrogenase, and formic acid production is possible, and not only can be reused, but also depending on the dissolved oxygen concentration The decrease in activity may be small.

The electron transporter may be a natural electron transporter or an artificial electron transporter. In addition, the electron transporter may be at least one electron transporter selected from the group consisting of an electron transporter having a viologen group and an electron transporter having an adenine dinucleotide group, for example, an alkyl viologen ( alkyl viologen), benzyl viologen, NAD (nicotinamide adenine dinucleotide), and FAD (flavin adenine dinucleotide) may be at least one electron transporter selected from the group consisting of, wherein the alkyl viologen is methyl Viologen (methyl viologen), ethyl viologen (ethyl viologen), propyl viologen (propyl viologen) and the like may be included.

Specifically, the electron transporter is selected from the group consisting of methyl viologen, ethyl viologen, benzyl viologen, nicotinamide adenine dinucleotide (NAD) and flavin adenine dinucleotide (FAD). It may be at least one electron transporter, and more specifically, it may be at least one electron transporter selected from the group consisting of methyl viologen and ethyl viologen.

In one aspect, the contacting may be performed in an electrochemical reaction system.

The electrochemical reaction system may be within the above-described range, among which the electrochemical carbon dioxide reduction system is as illustrated in FIG. 9 , for example, copper, graphite, carbon felt, and carbon It includes a cathode such as a fiber and an anode such as platinum, and may further include a reference electrode such as Ag/AgCl. However, the electrochemical carbon dioxide reduction system can be modified in a manner well known to those skilled in the art, if necessary.

Also, in one aspect, the gas including carbon dioxide may include oxygen.

As described above, unlike general microorganisms or enzymes, histidine-tagged formic acid dehydrogenase (Formate Dehydrogenase) according to an aspect may not decrease enzyme activity depending on oxygen or dissolved oxygen concentration, so from carbon dioxide to formic acid can be prepared by reducing it with high efficiency and high yield.

According to an aspect, the genetically recombined histidine-labeled formic acid dehydrogenase can reduce carbon dioxide to efficiently produce formic acid in high yield, and furthermore, when the formic acid dehydrogenase is immobilized, the efficiency is increased for a long time in the electrochemical reactor. It is possible to manufacture a high concentration of formic acid without falling, and it is not only reusable, but also not greatly affected by the dissolved oxygen concentration, which can greatly contribute to the industry in the field of carbon dioxide removal or formic acid manufacturing.

1 is a diagram showing the preparation process of a knock-out strain for FDH1-his tag production.
Figure 2 is a diagram showing the SDS PAGE result of the FDH1-his tag enzyme purified using Ni-NTA agarose (S: lysate; FT: flow-through of Ni-NTA resin; UW: unbounded washing, E1: primary elute of FDH1 E2: secondary elute of FDH1).
3 is a view showing the FDH1-his tag immobilized on Ni-NTA agarose bead.
4 is a diagram showing a standard curve for measuring an extinction coefficient for measuring the concentration of a reduced EV electron transporter.
5 is a diagram showing a Lineweaver-Burk plot for a carbon dioxide reduction reaction.
6 is a diagram comparing the rate of carbon dioxide reduction and formic acid oxidation of enzymes.
7 is a diagram showing the rate constant (K _eq ) of carbon dioxide reduction and formic acid oxidation consumption rate by enzyme.
8 is a diagram showing the rate change of the CO ₂ reduction and formate oxidation reaction of the FDH1-his tag according to pH (CO ₂ reduction (closed symbols), formate oxidation (open symbols)).
9 is a diagram showing a schematic diagram of an electrochemical reactor for producing formic acid by reducing carbon dioxide using the FDH1-his tag enzyme.
10 is a diagram showing formic acid production rate and Faraday efficiency according to voltage change (black line: formic acid productivity, blue bar: Faraday efficiency).
11 is a view showing the results of formic acid production according to long-term operation.
12 is a diagram demonstrating the repeatability of the immobilized enzyme.
13 is a diagram illustrating residual activity of an enzyme according to dissolved oxygen and estimating the resistance of the enzyme to dissolved oxygen through this.
Figure 14 is a diagram showing that it is possible to produce formic acid in spite of the abandonment of oxygen.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes of the present invention, and the scope of the present invention is not limited to these examples.

Example

Example 1. Preparation of FDH1-His tag recombinant enzyme

To prepare a recombinant microorganism, Methylobacterium extorquens AM1 (ATCC 14781, GenBank accession No. CP001150.1) was cloned and modified as shown in FIG. 1 below. Methylobacterium extorquens AM1 has three encoded formic acid dehydrogenase genes (fdh1, fdh2, fdh3), and among the three formic acid dehydrogenase genes, fdh1 for FDH1 (GenBank accession No. ACS42636.1) (α-subunit), ACS42635.1 (β-subunit)) gene was selected to construct a recombinant microorganism of Methylobacterium extorquens AM1, known to play a major role during the whole-cell oxidation of formic acid.

For the following cloning, one-step SLIC (sequence and ligation-independent cloning) was applied. SLIC uses T4 DNA polymerase as an exonuclease. The vector was linearized and amplified by restriction enzymes and DNA amplifier, NEB 2.1 buffer (B7202S, BioLabs) and T4 polymerase were added and incubated for 2.5 minutes at room temperature, and then immediately incubated on ice for 10 minutes. Then, 1 μl of the mixture was added to 100 μl of water-soluble E. coli DH5α (RBC), and the DH5α E. coli was incubated on ice for 20 minutes. Then, 950 μl of LB medium was added, and incubated at 37° C. for 16 hours.

Specifically, in order to prepare a recombinant microorganism, first, depending on the gene to be deleted, FDH1α, and/or FDH1β gene of Methylobacterium extorquens AM1 (GenBank accession No. ACS42636.1 (α-subunit), ACS42635. 1(β-subunit)) was amplified with DNA located in the front and rear portions. The primers used for cloning are shown in Table 1 below.

primer order SEQ ID NO: fdh1α knockout upstream F 5'-gccgccatatgcatccatggtaccCCGGCGGGTCGATGCGGTTGGAAA-3' One fdh1α knockout upstream R 5'-cacctgacgtctagatctgaattcTGGCCCGCGACCTCACCGCGAACTACTT-3' 2 fdh1α knockout downstream F 5'-tggtcggctggatcctctagtgagctcTCTACGCCGAGGGCGTGAACGGACC-3' 3 fdh1α knockout downstream R 5'-gatccagcttatcgataccgcgggcccGAGGTGCCGATAGGCGTGGCGCGA-3' 4 fdh1β knockout upstream F 5'-gccgccatatgcatccatggtaccAATCTCTGTGTCCGCGCCT-3' 5 fdh1β knockout upstream R 5'-cacctgacgtctagatctgaattcGCTTCACCGCGTTCTTGAGGAA-3' 6 fdh1β knockout downstream F 5'-tggtcggctggatcctctagtgagctcGGCAGAGGTCTCGCCGTTGT-3' 7 fdh1β knockout downstream R 5'-gatccagcttatcgataccgcgggcccGACGCGACCTGTGTTCCAACTAA-3' 8

The amplified DNA was cloned by inserting it into both sides of the loxP and kanamycin genes of pC184 (Addgene plasmid 46012). The cloned pC184 was transformed into Methylobacterium extorquens AM1. When Methylobacterium extorquens AM1 is transformed with pCM184, it undergoes an allele exchange to acquire loxP and kanamycin genes, but partially loses the gene sequence of FDH1. The Methylobacterium extorquens AM1 was transformed with pCM157 (Addgene plasmid 45863), and the kanamycin gene between the loxP sites was extracted by site-specific recombination by the cre recombinase expressed in pCM157. to produce knock-out microorganisms.

Then, if necessary, the knock-out microorganism was expressed with a recombinant plasmid. Specifically, Methylobacterium extorquens AM1, in which the specific gene was knocked out, was transformed with pCM110 (SEQ ID NO: 9) containing the FDH1 gene to restore expression of FDH1 or FDH1α.

Table 1 below shows bacterial strains and plasmids for knock-out or recombinant expression.

Strain/Plasmid Relevant properties strains M. extorquens AM1 Rifamycin resistance, wild type M. extorquens AM1 ( Δfdh1α ) △fdh1α M. extorquens AM1 M. extorquens AM1 ( Δfdh1α/β ) △fdh1α/β M. extorquens AM1 Plasmids pCM184 Allelic exchange vector (Amp ^R , Tet ^R , Kan ^R ) pCM184 -Δfdh1α pCM184 for fdh1α knockout pCM184 -Δfdh1β pCM184 for fdh1β knockout pCM157 Cre recombinase expression vector (Tet ^R ) pCM110 Recombinant expression vector (Tet ^R ), P _mxaF promoter pCM110-MeFDH1-His MeFDH1 homologous expression vector, containing C-terminus His tag of alpha subunit

The basic culture medium of the microorganisms was 16 g/L succinic acid and a minimal salt medium (1.62 g/L NH ₄ Cl, 0.2 g/L MgSO 4 , 2.21 g/LK ₂ HPO ₄ , and 1.25 g) as a carbon source. /L NaH ₂ PO ₄ .2H ₂ O). As selective antibiotics for screening recombinant microorganisms, 50 μg/mL rifamicin (Rif), 50 μg/mL kanamicin (Kan), or 10 μg/mL tetracycline (Tet) was used. All of the microorganisms were cultured at 26°C and 200 rpm in a volume of 200 mL in a 1L Erlenmeyer shake flask. When the cell concentration changed from 0.5 to 0.8 based on OD600, 0.5% methanol was added to express the target FDH1-his tag enzyme. Cells were cultured for a total of 48 hours, and cultured cells were obtained by centrifugation at 7,000 rpm for 15 minutes. Then, the supernatant was recovered by centrifugation at 4,611 g for 30 minutes after pulverization using a cell disrupter, and then the supernatant was recovered using Ni-NTA agarose bead (Bio-Rad, Cat No. 7321010) with FDH1-his tag (FDH1 alpha subunit- Only the amino acid sequence of His-tag: SEQ ID NO: 10; FDH1 beta subunit: SEQ ID NO: 11) was selectively immobilized. After immobilization, it was confirmed by using SDS PAGE of the FDH1-hist tag enzyme that was desorbed and recovered using imidazole solution. As a result, it was found that a fairly high concentration of the enzyme was well separated and purified (FIG. 2). Specifically, the structure of the enzyme immobilized on Ni-NTA agarose beads was shown in FIG. 3 below.

Example 2. Reduction of carbon dioxide and formic acid oxidation reaction rate measurement of recombinant enzymes

The rate of carbon dioxide reduction and formic acid oxidation of the recombinant FDH1-his tag enzyme was measured using ethyl viologen (EV) as an electron transporter. The concentration of the reduced form of ethyl viologen was measured at 600 nm using a visible light absorptometer, and the extinction coefficient was determined to be 10.220 mM ^-1 cm ^-1 (FIG. 4).

For carbon dioxide reduction reaction, 3 ~ 100 mM potassium bicarbonate, 0.03 ~ 0.12 mM reduced EV is added to 50 mM potassium phosphate buffer solution (pH 6.3). At this time, since potassium bicarbonate is in equilibrium with carbon dioxide to a certain extent, it becomes a carbon dioxide source. For the oxidation of formic acid, 0.5 ~ 30 mM potassium formate, 0.5 ~ 4 mM oxidized EV in 50 mM potassium phosphate buffer solution (pH 6.3) is injected. For all reactions, 10 μl of purely purified FDH1-his tag enzyme was added and the reaction was carried out at 30° C. for 1 minute. At this time, the changing concentration of reduced EV was measured using a visible light absorptometer, Using this result, a Lineweaver-Burk plot was drawn and enzyme reaction rate constants were estimated therefrom, and the same experiment was performed for other known comparative enzymes ( FIGS. 5 and 6 ).

As a result, MeFDH1 (≡FDH1-his tag) has a reaction rate of 58 times faster than that of oxidizing and consuming formic acid in reducing carbon dioxide, but RcFDH is 5.3 times, which is about 1/10. On the contrary, the reaction rate of oxidizing and consuming formic acid was much faster, and the concentration of formic acid production was extremely low ( FIGS. 6 and 7 ). Judging from this, it was predicted that it would be possible to efficiently produce formic acid at high concentration and high speed through carbon dioxide reduction compared to any enzyme reported so far.

Example 3. Determination of optimum pH for pH dependence and formic acid production of FDH1-his tag recombinant enzyme

For the pH range of pH 5.5 to pH 6.1, 200 mM citrate/citric acid buffer was used, and for other pHs, 200 mM potassium phosphate buffer was used, and enzyme activity was measured at 30° C. for 1 minute.

As a result, it is close to pH 6.3, which is suitable for producing formic acid through CO ₂ reduction, and it can be seen that the pH is significantly reduced above and below it, and the formic acid oxidation consumption rate is the maximum at pH 7 as the pH increases. was found (FIG. 8). Therefore, it was found that the best pH to produce formic acid was 6.3.

Example 4. Formic acid production through carbon dioxide reduction using an electrochemical reactor

The schematic diagram of the electrochemical reactor used in this example was shown in FIG. 9 below. In the electrochemical reactor, a carbon felt electrode (2 X 1.5 cm) and a reference electrode (Ag/AgCl, MF-2079, BASi) in the cathode part (30 mL) are 200 mM-potassium phosphate (pH 7), 10 mM ethyl viologen It is installed _{in a} solution containing A proton-selective membrane (Nafion® 115 membrane, 0.005 inch) was installed to transport the protons generated at the anode. For the reaction, 60 U FDH1-his tag enzyme was added to the negative electrode, and carbon dioxide was continuously supplied. The voltage was changed from -0.01 to -0.36 V compared to the reference electrode (reversible hydrogen electrode) and the amount of current consumed during the reaction was continuously measured while maintaining it constant. In addition, samples were taken from the cathode at regular intervals and the concentration of formic acid generated was measured using HPLC.

As a result of measuring the production rate and Faraday efficiency of formic acid generated while increasing the voltage, it was shown in FIG. 10 . Seeing that the formic acid production rate was 3 mM/hr even in the absence of overvoltage, it was found that the enzyme synthesizes formic acid very efficiently without an activation energy barrier. In addition, when the voltage is increased, the productivity of formic acid increases proportionally, but it does not increase further above 0.2 V, so it can be seen that other factors are the limiting factors. In all cases, the Faraday efficiency of formic acid generation was almost 100%, and it could be seen that all the consumed electrons were completely used only for formic acid synthesis.

Example 5. Long-term operation test for formic acid production by carbon dioxide reduction using an electrochemical reactor

Using the immobilized enzyme and the FDH1-his tag enzyme in a free dissolved state, a long-term operation was performed for the formic acid production experiment under the conditions set in Example 4, and the results are shown in FIG. 11 . The applied voltage is - 0.164 V vs. It was RHE, and when the concentration of formic acid produced increases, it exceeds the neutralization ability of the buffer, so 6 M KOH was added using a pH control device to maintain pH 6.4 during the reaction.

As a result, in the case of an unimmobilized FDH1-his tag recombinase, after 50 hours of reaction, 600 mM formic acid was generated after the production of formic acid no longer, whereas in the case of an enzyme immobilized on Ni-NTA agarose bead (Fig. 3) 200 hours It can be seen that formic acid is continuously produced until then, and about 1.7 M formic acid is produced, and it can be seen that the Faraday efficiency is maintained at 100% level despite long-term operation (FIG. 11). In the case of an unimmobilized enzyme, it is presumed that the activity of the enzyme was deactivated under the influence of shear stress caused by carbon dioxide aeration, whereas the immobilized enzyme is found to be resistant to shear stress due to such gas aeration.

Example 6. Demonstration of reuse performance using immobilized enzyme

In order to prove the reuse performance using the immobilized enzyme, the formic acid productivity was repeatedly measured using the enzyme immobilized on the Ni-NTA agarose bead recovered after 220 hours of reaction.

As a result, it can be seen that the formic acid productivity of the enzyme was not observed to decrease even in the experiment repeated three times (FIG. 12). From this, it was confirmed that the stability of the immobilized enzyme was greatly increased, and it was found that formic acid could be economically produced from carbon dioxide in the future.

Example 7. Residual activity change according to dissolved oxygen concentration change and stability test of enzyme immobilized enzyme according to air injection

The residual activity of the enzyme according to the dissolved oxygen concentration was measured.

As a result, it can be seen that the residual activity of the enzyme decreases when exposed to atmospheric oxygen for a long time, but it is generally resistant to oxygen at a level containing a trace amount in the level in which carbon dioxide is captured and the residual activity is not reduced at all. was found (FIG. 13). Based on this, it was found that formic acid could be smoothly produced by using carbon dioxide containing a certain amount of oxygen as a substrate.

In addition, under the conditions of Example 6, the effect on formic acid production was confirmed assuming that air was arbitrarily abandoned and injected in the middle of the reaction.

As a result, it was confirmed that the instantaneous air injection stops the production of formic acid, but when the air injection is stopped and carbon dioxide is injected again, formic acid is continuously produced (FIG. 14). Judging from this, it was found that since air injected in a short period of time does not completely inhibit the activity of the immobilized enzyme, the enzyme can continuously resist this and produce formic acid.

<110> UNIST(ULSAN NATIONAL INSTITUTE OF SCIENCE AND TECHNOLOGY) <120> A GENETICALLY MODIFIED FORMATE DEHYDROGENASE OR AN IMMOBILIZED GENETICALLY MODIFIED FORMATE DEHYDROGENASE AND AN PREPARING METHOD OF FORMIC ACID USING THEM <130> UTP21106 <130> UTP21106 KoPatentIn 3.0 <210> 1 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> fdh1 alpha knock-out upstream F <400> 1 gccgccatat gcatccatgg taccccggcg ggtcgatgcg gttggaaa 48 <210> 2 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> fdh1 alpha knock-out upstream R <400> 2 cacctgacgt ctagatctga attctggccc gcgacctcac cgcgaactac tt 52 <210> 3 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> fdh1 alpha knock-out down stream F <400> 3 tggtcggctg gatcctctag tgagctctct acgccgaggg cgtgaacgga cc 52 <210> 4 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> fdh1 alpha knock-out downstream R <400> 4 gatccagctt atcgataccg cgggcccgag gtgccgatag gcgtggcgcg a 51 <210> 5 <211> 43 <212> DNA <213> Artificial Sequence <220> <2 23> fdh1 beta knock-out upstream F <400> 5 gccgccatat gcatccatgg taccaatctc tgtgtccgcg cct 43 <210> 6 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> fdh1 beta knock-out upstream R < 400> 6 cacctgacgt ctagatctga attcgcttca ccgcgttctt gaggaa 46 <210> 7 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> fdh1 beta knock-out downstream F <400> 7 tggtcggctg gatcctctag tgagctcggc agaggt 47 210> 8 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> fdh1 beta knock-out downstream R <400> 8 gatccagctt atcgataccg cgggcccgac gcgacctgtg ttccaactaa 50 <210> 9 <211> 10907 <212> DNA <213> Artificial Sequence <220> <223> pCM110-MeFDH1-His tag <400> 9 gaccctttcc gacgctcacc gggctggttg ccctcgccgc tgggctggcg gccgtctatg 60 gccctgcaaa cgcgccagaa acgccgtcga agccgtgtgc gagacaccgc ggccgccggc 120 gttgtggata cctcgcggaa aacttggccc tcactgacag atgaggggcg gacgttgaca 180 cttgaggggc cgactcaccc ggcgcggcgt tgacagatga ggggcaggct cgatttcggc 240 cggcgacgtg gagctggcca gcctcgcaaa tcggcgaaaa cg cctgattt tacgcgagtt 300 tcccacagat gatgtggaca agcctgggga taagtgccct gcggtattga cacttgaggg 360 gcgcgactac tgacagatga ggggcgcgat ccttgacact tgaggggcag agtgctgaca 420 gatgaggggc gcacctattg acatttgagg ggctgtccac aggcagaaaa tccagcattt 480 gcaagggttt ccgcccgttt ttcggccacc gctaacctgt cttttaacct gcttttaaac 540 caatatttat aaaccttgtt tttaaccagg gctgcgccct gtgcgcgtga ccgcgcacgc 600 cgaagggggg tgccccccct tctcgaaccc tcccggcccg ctaacgcggg cctcccatcc 660 ccccaggggc tgcgcccctc ggccgcgaac ggcctcaccc caaaaatggc agccaagctg 720 atccatatgt aagatttaaa tgcaaccgtt ttttcggaag gaaatgatga cctcgtttcc 780 accgaattag cttgcatgcc tgcaggtcga ctctagatca cctccttaag cttcccgctt 840 ggtcgggccg cttcgcgagg gcccgttgac gacaacggtg cgatgggtcc cggccccggt 900 caagacgatg ccaatacgtt gcgacactac gccttggcac ttttagaatt gccttatcgt 960 cctgataaga aatgtccgac cagctaaaga catcgcgtcc aatcaaagcc tagaaaatat 1020 aggcgaaggg acgctaataa gtctttcata agaccgcgca aatctaaaaa tatccttaga 1080 ttcacgatgc ggcacttcgg atgacttccg agcgagcctg gaacctcaga aaaacgtct g 1140 agagataccg cggatcctaa gggcgaattc cagcacactg gcggccgtta ctagaaataa 1200 ttttgtttaa ctttaagaag gagatataca tatgagtgag gcaagtggga ctgtccggag 1260 cttcgcgcat ccgggccgtg gccgtaacgt cgcccgcgcc gtgccgaagg ggcgtcaggt 1320 cgatccccac gccaaggtcg agatcgagga gctgctcggc acccgcccgc gccagcgcga 1380 cctgctgatc gagcacctgc acctgatcca agacacctac ggccagatca gcgccgatca 1440 tctcgcggcg ctggccgacg agatgagcct cgccttcgcc gaggtgttcg agaccgcgac 1500 cttctacgcg catttcgatg tggtgaagga gggcgaggcc gacatcccgc gcctgacgat 1560 ccgggtttgc gacagcatca cctgcgccat gttcggcgcc gacgagctgc tggagacgct 1620 gcagcgcgag ctggcctcgg atgcggtccg cgtcgtgcgc gcgccctgtg tcggcctgtg 1680 cgaccacgcc ccggcggtcg aggtcgggca caacttcctg caccgggccg acctcgcctc 1740 cgtgcgcgcc gcggtcgagg ccgaggacac ccacgcccac atccccactt acgtcgatta 1800 cgacgcctac cgggccggcg gcggctacgc gaccctggag cggctgcgca gcggcgaact 1860 gccggtcgat gacgtgctga aggtgctcga cgacggcggc ctgcgcggcc tcggcggcgc 1920 cggctttccc accggccgca agtggcgctc cgtgcgcggc gagcccggac cccggctgat 1980 ggcggtcaac ggcgacgagg gcgagcccgg caccttcaag gatcagctct acctcaacac 2040 cgatccgcac cgctttctgg aaggcatgct gatcggcgcc cacgtcgtcg aggccgccga 2100 cgtctacatc tacctgcgcg acgagtaccc gatctcccgc gagatcctgg cccgcgagat 2160 cgcgaagctc cccgagggcg gcacccgcat ccacctgcgc cgtggggccg gcgcctatat 2220 ctgcggcgag gaatcctcgc tgatcgagtc gctggagggc aagcgcggcc tgccgcggca 2280 caagccgccc ttccccttcc aggtcggcct gttcaaccgg ccgacgctga tcaacaacat 2340 cgagacgctg ttctgggtgc gcgacctgat cgagcgcggc gccgaatggt ggaagagcca 2400 cgggcgcaac ggccgcgtcg gcctgcgctc gtactcggtt tcgggccggg tcaaggagcc 2460 gggcgtcaag ctcgcgcccg ccggcctgac catccaggaa ctcatcgacg agtattgcgg 2520 cggcatctct gacggccaca gcttcgcggc ctacctgccg ggcggagcct cgggcggcat 2580 cctgccggcc tcgatgaacg acatcccgct cgatttcggc acgctggaaa aatacggctg 2640 cttcatcggc tcggccgcgg tcgtgatcct gtccgatcag gacgatgtgc gcggtgccgc 2700 gttgaacctg atgaagttct tcgaggacga gtcctgcggg cagtgcacgc cctgccgctc 2760 gggcacgcag aaggcccgca tgctgatgga gaacggcgtg tgggacaccg atctcctcgg 2820 cgagc tggcg caatgcatgc gcgacgcctc gatctgcggt ctcggtcagg cggcctcgaa 2880 ccccgtcagc accgtgatca agtacttccc tgatctcttc ccggagccgc gggccgtggc 2940 ggccgagtga cgcccagccc gacgatgtct cctcacccga tgccagagcc tggagcggac 3000 gcatgagcaa cggccccgaa ccgcacggca acaagatcga acagcccgag atccgcgccg 3060 acgaacgtca ggatgccggc gggccggcaa atggcgcacc atcgacctcc ggcggcgcct 3120 actcgcaggg cgccaagtcg ggtggccagg ccgcgccgga tccgtccggc tcctacggca 3180 tcaaggacgc cccggtcgcg cccgcgacca tcgccttcga gttcgacggc caacaggtcg 3240 aggcccagcc cggcgagacg atctgggcgg tcgccaagcg ccttggcacg catatcccgc 3300 atctctgcca caagcccgat cccggctacc gcccggacgg caattgccgc gcctgcatgg 3360 tcgagatcga gggcgagcgc gtgctcgccg cctcgtgcaa gcgcacgccc gccatcggca 3420 tgaaggtgaa gtcggccacc gagcgcgcca ccaaggcccg cgccatggtg ctcgaactgc 3480 tcgtggccga tcagcccgag cgcgcgacct cgcatgaccc gtcgtcgcat ttctgggtgc 3540 aggccgacgt cctcgatgtg accgagagcc gcttcccggc ggccgagcgc tggaccagcg 3600 acgtcagcca cccggcgatg agcgtcaatc tcgacgcctg catccagtgc aatctctgtg 3660 tccgcgcctg ccgcgaggtt caggtcaacg acgtgatcgg catggcctac cgcgccgcgg 3720 gctccaaggt cgtgtttgac ttcgacgatc cgatgggtgg ctccacctgc gtggcctgcg 3780 gcgagtgcgt ccaagcctgc ccgaccgggg cgctgatgcc ggccgcctat ctcgacgcaa 3840 accagacccg gacggtctat cccgaccgcg aggtgaagtc gctctgcccc tattgcggcg 3900 tcggctgcca agtctcctac aaggtcaagg acgagcgcat cgtctacgcc gagggcgtga 3960 acggaccggc caaccagaac cggctctgcg tgaagggccg cttcggcttc gactacgtcc 4020 accaccccca ccgcctgacg gtgccgctga tccgcttgga gaacgtgccc aaggacgcca 4080 acgatcaggt cgatccggcg aacccctgga cgcatttccg cgaggcgacc tgggaagagg 4140 cgctcgaccg cgcggcgggc ggcctgaagg cgatccgtga caccaacggg cgcaaggcgc 4200 tggcgggctt cggctcggcc aagggttcga acgaggaggc ctacctcttc cagaagctcg 4260 tccgcctcgg cttcggcacc aacaacgtcg atcactgcac gcgcctgtgc cacgcctcgt 4320 cggtggcggc gctgatggag ggcctgaatt ccggcgccgt caccgctccc ttctcggcag 4380 cgctcgacgc cgaggtcatc gtcgtcatcg gcgccaaccc gaccgtgaac catccggtcg 4440 cggcgacctt cctcaagaac gcggtgaagc agcgcggcgc caagctgatc atcatggacc 4500 cgcggcgcca gacgct ctcg cgccacgcct atcggcacct cgccttccgc cccggctcgg 4560 acgtggcgat gctcaacgcg atgctcaacg tgatcgtcac ggagggcctc tacgacgagc 4620 agtacatcgc cggctacacc gagaacttcg aggctctgcg cgagaagatc gtcgacttca 4680 cgccggagaa gatggcctcg gtctgcggca tcgacgccga gaccctgcgc gaggtcgccc 4740 ggctctacgc ccgggccaag tcgtcgctca tcttctgggg catgggcgtc agccagcacg 4800 tgcacggcac cgacaactcg cgctgcctga tcgcgctcgc cctcatcacc ggccagatcg 4860 gccggcccgg caccggcctg cacccgttgc gcggccagaa caacgtccag ggcgcgtccg 4920 atgccggcct gatcccgatg gtctacccgg actatcagtc ggtcgagaag gacgcggtgc 4980 gtgagctgtt cgaggagttc tgggggcagt ccctcgatcc tcagaagggc ctcaccgtgg 5040 tcgagatcat gcgcgcgatc cacgcgggcg agatccgggg catgttcgtc gagggcgaga 5100 acccggcgat gtccgacccc gacctcaacc atgcccgcca cgcgctggcg atgctcgacc 5160 atctcgtggt gcaggacctg ttcctgacgg agacggcctt ccacgccgac gtggtgctgc 5220 cggcctcggc ctttgccgag aaagccggga ccttcaccaa caccgaccgg cgcgtgcaga 5280 tcgcccagcc cgtcgtcgcc cctccgggcg atgcgcgcca ggattggtgg atcatccagg 5340 aactggcccg acgcctcgac c tcgactgga actacggcgg cccggccgac atcttcgccg 5400 agatggcgca ggtgatgccg tccttgaaca acatcacctg ggagcggctg gagcgcgagg 5460 gggcggtgac ctatccggtc gatgccccgg accagcccgg caacgagatc atcttctatg 5520 ccggcttccc gaccgagagc ggtcgcgcca agatcgtgcc cgcggcgatc gtgccgccgg 5580 acgaggtgcc ggacgacgag ttcccgatgg tgctctcgac cggccgcgtg ctcgaacact 5640 ggcacacggg ctcgatgacc cggcgcgcgg gcgtgctcga cgcgctggag ccggaggcgg 5700 tggccttcat ggcacccaag gagctctacc ggctcggtct ccggcccggc gggtcgatgc 5760 ggttggaaac acggcgcggc gccgtcgtgt tgaaggtgcg ctccgaccgg gacgtgccga 5820 tcggcatgat cttcatgccc ttctgctacg cggaagccgc cgccaacctt ctgaccaacc 5880 ccgccctcga ccccctcgga aagattcccg agttcaaatt ctgcgcagcc cgcgtcgtcc 5940 ccgcggaggc tgcgccgatg gccgccgagc accaccacca ccaccactaa ggatccggct 6000 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca 6060 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata 6120 tccggatatc ccgcaagagg cccggcagta ccggcataac caagcctatg cctacagcat 6180 ccagggtgac ggtgccgagg atgacga tga gcgcattgtt agatttcata cacggtgcct 6240 gactgcgtta gcaatttaac tgtgataaac taccgcatta aagcttatcg atagatccgt 6300 tcagggcatc aaatgcagac tcgtttctga cacttgcaga ctggatctat ctcatctgcg 6360 caaggcagaa cgtgaagacg gccgccctgg acctcgcccg cgagcgccag gcgcacgagg 6420 ccggcgcgcg gacccgcgcc acggcccacg agcggacgcc gcagcaggag cgccagaagg 6480 ccgccagaga ggccgagcgc ggccgtgagg cttggacgct agggcagggc atgaaaaagc 6540 ccgtagcggg ctgctacggg cgtctgacgc ggtggaaagg gggaggggat gttgtctaca 6600 tggctctgct gtagtgagtg ggttgcgctc cggcagcggt cctgatcaat cgtcaccctt 6660 tctcggtcct tcaacgttcc tgacaacgag cctccttttc gccaatccat cgacaatcac 6720 cgcgagtccc tgctcgaacg ctgcgtccgg accggcttcg tcgaaggcgt ctatcgcggc 6780 ccgcaacagc ggcgagagcg gagcctgttc aacggtgccg ccgcgctcgc cggcatcgct 6840 gtcgccggcc tgctcctcaa gcacggcccc aacagtgaag tagctgattg tcatcagcgc 6900 attgacggcg tccccggccg aaaaacccgc ctcgcagagg aagcgaagct gcgcgtcggc 6960 cgtttccatc tgcggtgcgc ccggtcgcgt gccggcatgg atgcgcgcgc catcgcggta 7020 ggcgagcagc gcctgcctga agctgcgggc at tcccgatc agaaatgagc gccagtcgtc 7080 gtcggctctc ggcaccgaat gcgtatgatt ctccgccagc atggcttcgg ccagtgcgtc 7140 gagcagcgcc cgcttgttcc tgaagtgcca gtaaagcgcc ggctgctgaa cccccaaccg 7200 ttccgccagt ttgcgtgtcg tcagaccgtc tacgccgacc tcgttcaaca ggtccagggc 7260 ggcacggatc actgtattcg gctgcaactt tgtcatgatt gacactttat cactgataaa 7320 cataatatgt ccaccaactt atcagtgata aagaatccgc gcgttcaatc ggaccagcgg 7380 aggctggtcc ggaggccaga cgtgaaaccc aacatacccc tgatcgtaat tctgagcact 7440 gtcgcgctcg acgctgtcgg catcggcctg attatgccgg tgctgccggg cctcctgcgc 7500 gatctggttc actcgaacga cgtcaccgcc cactatggca ttctgctggc gctgtatgcg 7560 ttggtgcaat ttgcctgcgc acctgtgctg ggcgcgctgt cggatcgttt cgggcggcgg 7620 ccaatcttgc tcgtctcgct ggccggcgcc actgtcgact acgccatcat ggcgacagcg 7680 cctttccttt gggttctcta tatcgggcgg atcgtggccg gcatcaccgg ggcgactggg 7740 gcggtagccg gcgcttatat tgccgatatc actgatggcg atgagcgcgc gcggcacttc 7800 ggcttcatga gcgcctgttt cgggttcggg atggtcgcgg gacctgtgct cggtgggctg 7860 atgggcggtt tctcccccca cgctccgttc ttcgccgc gg cagccttgaa cggcctcaat 7920 ttcctgacgg gctgtttcct tttgccggag tcgcacaaag gcgaacgccg gccgttacgc 7980 cgggaggctc tcaacccgct cgcttcgttc cggtgggccc ggggcatgac cgtcgtcgcc 8040 gccctgatgg cggtcttctt catcatgcaa cttgtcggac aggtgccggc cgcgctttgg 8100 gtcattttcg gcgaggatcg ctttcactgg gacgcgacca cgatcggcat ttcgcttgcc 8160 gcatttggca ttctgcattc actcgcccag gcaatgatca ccggccctgt agccgcccgg 8220 ctcggcgaaa ggcgggcact catgctcgga atgattgccg acggcacagg ctacatcctg 8280 cttgccttcg cgacacgggg atggatggcg ttcccgatca tggtcctgct tgcttcgggt 8340 ggcatcggaa tgccggcgct gcaagcaatg ttgtccaggc aggtggatga ggaacgtcag 8400 gggcagctgc aaggctcact ggcggcgctc accagcctga cctcgatcgt cggacccctc 8460 ctcttcacgg cgatctatgc ggcttctata acaacgtgga acgggtgggc atggattgca 8520 ggcgctgccc tctacttgct ctgcctgccg gcgctgcgtc gcgggctttg gagcggcgca 8580 gggcaacgag ccgatcgctg atcgtggaaa cgataggcct atgccatgcg ggtcaaggcg 8640 acttccggca agctatacgc gccctagaat tgtcaatttt aatcctctgt ttatcggcag 8700 ttcgtagagc gcgccgtgcg tcccgagcga tactgagcga agc aagtgcg tcgagcagtg 8760 cccgcttgtt cctgaaatgc cagtaaagcg ctggctgctg aacccccagc cggaactgac 8820 cccacaaggc cctagcgttt gcaatgcacc aggtcatcat tgacccaggc gtgttccacc 8880 aggccgctgc ctcgcaactc ttcgcaggct tcgccgacct gctcgcgcca cttcttcacg 8940 cgggtggaat ccgatccgca catgaggcgg aaggtttcca gcttgagcgg gtacggctcc 9000 cggtgcgagc tgaaatagtc gaacatccgt cgggccgtcg gcgacagctt gcggtacttc 9060 tcccatatga atttcgtgta gtggtcgcca gcaaacagca cgacgatttc ctcgtcgatc 9120 aggacctggc aacgggacgt tttcttgcca cggtccagga cgcggaagcg gtgcagcagc 9180 gacaccgatt ccaggtgccc aacgcggtcg gacgtgaagc ccatcgccgt cgcctgtagg 9240 cgcgacaggc attcctcggc cttcgtgtaa taccggccat tgatcgacca gcccaggtcc 9300 tggcaaagct cgtagaacgt gaaggtgatc ggctcgccga taggggtgcg cttcgcgtac 9360 tccaacacct gctgccacac cagttcgtca tcgtcggccc gcagctcgac gccggtgtag 9420 gtgatcttca cgtccttgtt gacgtggaaa atgaccttgt tttgcagcgc ctcgcgcggg 9480 attttcttgt tgcgcgtggt gaacagggca gagcgggccg tgtcgtttgg catcgctcgc 9540 atcgtgtccg gccacggcgc aatatcgaac aaggaaagct gcatttcct t gatctgctgc 9600 ttcgtgtgtt tcagcaacgc ggcctgcttg gcctcgctga cctgttttgc caggtcctcg 9660 ccggcggttt ttcgcttctt ggtcgtcata gttcctcgcg tgtcgatggt catcgacttc 9720 gccaaacctg ccgcctcctg ttcgagacga cgcgaacgct ccacggcggc cgatggcgcg 9780 ggcagggcag ggggagccag ttgcacgctg tcgcgctcga tcttggccgt agcttgctgg 9840 accatcgagc cgacggactg gaaggtttcg cggggcgcac gcatgacggt gcggcttgcg 9900 atggtttcgg catcctcggc ggaaaacccc gcgtcgatca gttcttgcct gtatgccttc 9960 cggtcaaacg tccgattcat tcaccctcct tgcgggattg ccccgactca cgccggggca 10020 atgtgccctt attcctgatt tgacccgcct ggtgccttgg tgtccagata atccacctta 10080 tcggcaatga agtcggtccc gtagaccgtc tggccgtcct tctcgtactt ggtattccga 10140 atcttgccct gcacgaatac cagctccgcg aagtcgctct tcttgatgga gcgcatgggg 10200 acgtgcttgg caatcacgcg caccccccgg ccgttttagc ggctaaaaaa gtcatggctc 10260 tgccctcggg cggaccacgc ccatcatgac cttgccaagc tcgtcctgct tctcttcgat 10320 cttcgccagc agggcgagga tcgtggcatc accgaaccgc gccgtgcgcg ggtcgtcggt 10380 gagccagagt ttcagcaggc cgcccaggcg gcccaggtcg ccattgat gc gggccagctc 10440 gcggacgtgc tcatagtcca cgacgcccgt gattttgtag ccctggccga cggccagcag 10500 gtaggcctac aggctcatgc cggccgccgc cgccttttcc tcaatcgctc ttcgttcgtc 10560 tggaaggcag tacaccttga taggtgggct gcccttcctg gttggcttgg tttcatcagc 10620 catccgcttg ccctcatctg ttacgccggc ggtagccggc cagcctcgca gagcaggatt 10680 cccgttgagc accgccaggt gcgaataagg gacagtgaag aaggaacacc cgctcgcggg 10740 tgggcctact tcacctatcc tgcccggctg acgccgttgg atacaccaag gaaagtctac 10800 acgaaccctt tggcaaaatc ctgtatatcg tgcgaaaaag gatggatata ccgaaaaaat 10860 cgctataatg accccgaagc agggttatgc agcggaaaag atccgtc 10907 <210> 10 <211> 995 <212> PRT <213> Artificial Sequence <220> <223> FDH1 alpha subunit-His-tag <400> 10 Met Ser Asn Gly Pro Glu Pro His Gly Asn Lys Ile Glu Gln Pro Glu 1 5 10 15 Ile Arg Ala Asp Glu Arg Gln Asp Ala Gly Gly Pro Ala Asn Gly Ala 20 25 30 Pro Ser Thr Ser Gly Gly Ala Tyr Ser Gln Gly Ala Lys Ser Gly Gly 35 40 45 Gln Ala Ala Pro Asp Pro Ser Gly Ser Tyr Gly Ile Lys Asp Ala Pro 50 55 60 Val Ala Pro Ala Thr Ile Ala Phe Glu Phe Asp Gly Gln Gln Val Glu 65 70 75 80 Ala Gln Pro Gly Glu Thr Ile Trp Ala Val Ala Lys Arg Leu Gly Thr 85 90 95 His Ile Pro His Leu Cys His Lys Pro Asp Pro Gly Tyr Arg Pro Asp 100 105 110 Gly Asn Cys Arg Ala Cys Met Val Glu Ile Glu Gly Glu Arg Val Leu 115 120 125 Ala Ala Ser Cys Lys Arg Thr Pro Ala Ile Gly Met Lys Val Lys Ser 130 135 140 Ala Thr Glu Arg Ala Thr Lys Ala Arg Ala Met Val Leu Glu Leu Leu Leu 145 150 155 160 Val Ala Asp Gln Pro Glu Arg Ala Thr Ser His Asp Pro Ser Ser His 165 170 175 Phe Trp Val Gln Ala Asp Val Leu Asp Val Thr Glu Ser Arg Phe Pro 180 185 190 Ala Ala Glu Arg Trp Thr Ser Asp Val Ser His Pro Ala Met Ser Val 195 200 205 Asn Leu Asp Ala Cys Ile Gln Cys Asn Leu Cys Val Arg Ala Cys Arg 210 215 220 Glu Val Gln Val Asn Asp Val Ile Gly Met Ala Tyr Arg Ala Ala Gly 225 230 235 240 Ser Lys Val Val Phe Asp Phe Asp Asp Pro Met Gly Gly Ser Thr Cys 245 250 255 Val Ala Cys Gly Glu Cys Val Gln Ala Cys Pro Thr Gly Ala Leu Met 260 265 270 Pro Ala Ala Tyr Leu Asp Ala Asn Gln Thr Arg Thr Val Tyr Pro Asp 275 280 285 Arg Glu Val Lys Ser Leu Cys Pro Tyr Cys Gly Val Gly Cys Gln Val 290 295 300 Ser Tyr Lys Val Lys Asp Glu Arg Ile Val Tyr Ala Glu Gly Val Asn 305 310 315 320 Gly Pro Ala Asn Gln Asn Arg Leu Cys Val Lys Gly Arg Phe Gly Phe 325 330 335 Asp Tyr Val His His Pro His Arg Leu Thr Val Pro Leu Ile Arg Leu 340 345 350 Glu Asn Val Pro Lys Asp Ala Asn Asp Gln Val Asp Pro Ala Asn Pro 355 360 365 Trp Thr His Phe Arg Glu Ala Thr Trp Glu Glu Ala Leu Asp Arg Ala 370 375 380 Ala Gly Gly Leu Lys Ala Ile Arg Asp Thr Asn Gly Arg Lys Ala Leu 385 390 395 400 Ala Gly Phe Gly Ser Ala Lys Gly Ser Asn Glu Glu Ala Tyr Leu Phe 405 410 415 Gln Lys Leu Val Arg Leu Gly Phe Gly Thr Asn Asn Val Asp His Cys 420 425 430 Thr Arg Leu Cys His Ala Ser Ser Val Ala Ala Leu Met Glu Gly Leu 435 440 445 Asn Ser Gly Ala Val Thr Ala Pro Phe Ser Ala Ala Leu Asp Ala Glu 450 455 460 Val Ile Val Val Ile Gly Ala Asn Pro Thr Val Asn His Pro Val Ala 465 470 475 480 Ala Thr Phe Leu Lys Asn Ala Val Lys Gln Arg Gly Ala Lys Leu Ile 485 490 495 Ile Met Asp Pro Arg Arg Gln Thr Leu Ser Arg His Ala Tyr Arg His 500 505 510 Leu Ala Phe Arg Pro Gly Ser Asp Val Ala Met Leu Asn Ala Met Leu 515 520 525 Asn Val Ile Val Thr Glu Gly Leu Tyr Asp Glu Gln Tyr Ile Ala Gly 530 535 540 Tyr Thr Glu Asn Phe Glu Ala Leu Arg Glu Lys Ile Val Asp Phe Thr 545 550 555 560 Pro Glu Lys Met Ala Ser Val Cys Gly Ile Asp Ala Glu Thr Leu Arg 565 570 575 Glu Val Ala Arg Leu Tyr Ala Arg Ala Lys Ser Ser Leu Ile Phe Trp 580 585 590 Gly Met Gly Val Ser Gln His Val His Gly Thr Asp Asn Ser Arg Cys 595 600 605 Leu Ile Ala Leu Ala Leu Ile Thr Gly Gln Ile Gly Arg Pro Gly Thr 610 615 620 Gly Leu His Pro Leu Arg Gly Gln Asn Asn Val Gln Gly Ala Ser Asp 625 630 635 640 Ala Gly Leu Ile Pro Met Val Tyr Pro Asp Tyr Gln Ser Val Glu Lys 645 650 655 Asp Ala Val Arg Glu Leu Phe Glu Glu Phe Trp Gly Gln Ser Leu Asp 660 665 670 Pro Gln Lys Gly Leu Thr Val Val Glu Ile Met Arg Ala Ile His Ala 675 680 685 Gly Glu Ile Arg Gly Met Phe Val Glu Gly Glu Asn Pro Ala Met Ser 690 695 700 Asp Pro Asp Leu Asn His Ala Arg His Ala Leu Ala Met Leu Asp His 705 710 715 720 Leu Val Val Gln Asp Leu Phe Leu Thr Glu Thr Ala Phe His Ala Asp 725 730 735 Val Val Leu Pro Ala Ser Ala Phe Ala Glu Lys Ala Gly Thr Phe Thr 740 745 750 Asn Thr Asp Arg Arg Val Gln Ile Ala Gln Pro Val Val Ala Pro Pro 755 760 765 Gly Asp Ala Arg Gln Asp Trp Trp Ile Ile Gln Glu Leu Ala Arg Arg 770 775 780 Leu Asp Leu Asp Trp Asn Tyr Gly Gly Pro Ala Asp Ile Phe Ala Glu 785 790 795 800 Met Ala Gln Val Met Pro Ser Leu Asn Asn Ile Thr Trp Glu Arg Leu 805 810 815 Glu Arg Glu Gly Ala Val Thr Tyr Pro Val Asp Ala Pro Asp Gln Pro 820 825 830 Gly Asn Glu Ile Ile Phe Tyr Ala Gly Phe Pro Thr Glu Ser Gly Arg 835 840 845 Ala Lys Ile Val Pro Ala Ala Ile Val Pro Pro Asp Glu Val Pro Asp 850 855 860 Asp Glu Phe Pro Met Val Leu Ser Thr Gly Arg Val Leu Glu His Trp 865 870 875 880 His Thr Gly Ser Met Thr Arg Arg Ala Gly Val Leu Asp Ala Leu Glu 885 890 895 Pro Glu Ala Val Ala Phe Met Ala Pro Lys Glu Leu Tyr Arg Leu Gly 900 905 910 Leu Arg Pro Gly Gly Ser Met Arg Leu Glu Thr Arg Arg Gly Ala Val 915 920 925 Val Leu Lys Val Arg Ser Asp Arg Asp Val Pro Ile Gly Met Ile Phe 930 935 940 Met Pro Phe Cys Tyr Ala Glu Ala Ala Ala Asn Leu Leu Thr Asn Pro 945 950 955 960 Ala Leu Asp Pro Leu Gly Lys Ile Pro Glu Phe Lys Phe Cys Ala Ala 965 970 975 Arg Val Val Pro Ala Glu Ala Ala Pro Met Ala Ala Glu His His His 980 985 990 His His His 995 <210> 11 <211> 572 <212> PRT <213> Artificial Sequence <220> <223> FDH1 beta subunit <400> 11 Met Ser Glu Ala Ser Gly Thr Val Arg Ser Phe Ala His Pro Gly Arg 1 5 10 15 Gly Arg Asn Val Ala Arg Ala Val Pro Lys Gly Arg Gln Val Asp Pro 20 25 30 His Ala Lys Val Glu Ile Glu Glu Leu Leu Gly Thr Arg Pro Arg Gln 35 40 45 Arg Asp Leu Leu Ile Glu His Leu His Leu Ile Gln Asp Thr Tyr Gly 50 55 60 Gln Ile Ser Ala Asp His Leu Ala Ala Leu Ala Asp Glu Met Ser Leu 65 70 75 80 Ala Phe Ala Glu Val Phe Glu Thr Ala Thr Phe Tyr Ala His Phe Asp 85 90 95 Val Val Lys Glu Gly Glu Ala Asp Ile Pro Arg Leu Thr Ile Arg Val 100 105 110 Cys Asp Ser Ile Thr Cys Ala Met Phe Gly Ala Asp Glu Leu Leu Glu 115 120 125 Thr Leu Gln Arg Glu Leu Ala Ser Asp Ala Val Arg Val Val Arg Ala 130 135 140 Pro Cys Val Gly Leu Cys Asp His Ala Pro Ala Val Glu Val Gly His 145 150 155 160 Asn Phe Leu His Arg Ala Asp Leu Ala Ser Val Arg Ala Ala Val Glu 165 170 175 Ala Glu Asp Thr His Ala His Ile Pro Thr Tyr Val Asp Tyr Asp Ala 180 185 190 Tyr Arg Ala Gly Gly Gly Tyr Ala Thr Leu Glu Arg Leu Arg Ser Gly 195 200 205 Glu Leu Pro Val Asp Asp Val Leu Lys Val Leu Asp Asp Gly Gly Leu 210 215 220 Arg Gly Leu Gly Gly Ala Gly Phe Pro Thr Gly Arg Lys Trp Arg Ser 225 230 235 240 Val Arg Gly Glu Pro Gly Pro Arg Leu Met Ala Val Asn Gly Asp Glu 245 250 255 Gly Glu Pro Gly Thr Phe Lys Asp Gln Leu Tyr Leu Asn Thr Asp Pro 260 265 270 His Arg Phe Leu Glu Gly Met Leu Ile Gly Ala His Val Val Gl u Ala 275 280 285 Ala Asp Val Tyr Ile Tyr Leu Arg Asp Glu Tyr Pro Ile Ser Arg Glu 290 295 300 Ile Leu Ala Arg Glu Ile Ala Lys Leu Pro Glu Gly Gly Thr Arg Ile 305 310 315 320 His Leu Arg Arg Gly Ala Gly Ala Tyr Ile Cys Gly Glu Glu Ser Ser 325 330 335 Leu Ile Glu Ser Leu Glu Gly Lys Arg Gly Leu Pro Arg His Lys Pro 340 345 350 Pro Phe Pro Phe Gln Val Gly Leu Phe Asn Arg Pro Thr Leu Ile Asn 355 360 365 Asn Ile Glu Thr Leu Phe Trp Val Arg Asp Leu Ile Glu Arg Gly Ala 370 375 380 Glu Trp Trp Lys Ser His Gly Arg Asn Gly Arg Val Gly Leu Arg Ser 385 390 395 400 Tyr Ser Val Ser Gly Arg Val Lys Glu Pro Gly Val Lys Leu Ala Pro 405 410 415 Ala Gly Leu Thr Ile Gln Glu Leu Ile Asp Glu Ty r Cys Gly Gly Ile 420 425 430 Ser Asp Gly His Ser Phe Ala Ala Tyr Leu Pro Gly Gly Ala Ser Gly 435 440 445 Gly Ile Leu Pro Ala Ser Met Asn Asp Ile Pro Leu Asp Phe Gly Thr 450 455 460 Leu Glu Lys Tyr Gly Cys Phe Ile Gly Ser Ala Ala Val Val Ile Leu 465 470 475 480 Ser Asp Gln Asp Asp Val Arg Gly Ala Ala Leu Asn Leu Met Lys Phe 485 490 495 Phe Glu Asp Glu Ser Cys Gly Gln Cys Thr Pro Cys Arg Ser Gly Thr 500 505 510 Gln Lys Ala Arg Met Leu Met Glu Asn Gly Val Trp Asp Thr Asp Leu 515 520 525 Leu Gly Glu Leu Ala Gln Cys Met Arg Asp Ala Ser Ile Cys Gly Leu 530 535 540 Gly Gln Ala Ala Ser Asn Pro Val Ser Thr Val Ile Lys Tyr Phe Pro 545 550 555 560Asp Leu Phe Pro Glu Pro Arg Ala Val Ala Ala Glu 565 570

Claims (22)

Histidine-tagged formic acid dehydrogenase (Formate Dehydrogenase).
The method according to claim 1, wherein the formic acid dehydrogenase is derived from a microorganism of the genus Methylobacterium, formic acid dehydrogenase.
The method according to claim 1, wherein the formic acid dehydrogenase is Methylobacterium extorquens ( Methylobacterium extorquens ) It is derived from AM1, formic acid dehydrogenase.
The method according to claim 1, wherein the formic acid dehydrogenase is produced by genetic recombination, formic acid dehydrogenase.
The method according to claim 1, wherein the formic acid dehydrogenase is Methylobacterium extorquens (Methylobacterium extorquens ) AM1-derived FDH1 (Formate Dehydrogenase 1) as,
The methylobacterium extorquens AM1 is an endogenous gene encoding FDH1α (Formate Dehydrogenase 1 alpha subunit) and an endogenous gene encoding FDH1β (Formate Dehydrogenase 1 beta subunit) is deleted,
Formic acid dehydrogenase, a recombinant microorganism into which an exogenous gene encoding FDH1 derived from Methylobacterium extorquens AM1 and a histidine label (His-tag) has been introduced.
The method according to claim 1, wherein the formic acid dehydrogenase is a polypeptide consisting of the amino acid sequences of SEQ ID NOs: 10 and 11, formic acid dehydrogenase.
Histidine-tagged Formate Dehydrogenase immobilized in Nickel-Nitrilotriacetic Acid (Ni-NTA).
The method according to claim 7, wherein the Ni-NTA is Ni-NTA agarose beads, formic acid dehydrogenase.
Histidine-labeled (His-tag) formic acid production composition comprising formic acid dehydrogenase (Formate Dehydrogenase).
The method according to claim 9, wherein the formic acid dehydrogenase is Ni-NTA (Nickel-Nitrilotriacetic Acid) will be immobilized in, formic acid production composition.
The method according to claim 9, wherein the pH of the composition is 5.0 to 7.5, formic acid preparation composition.
Histidine-labeled (His-tag) formic acid production electrochemical reaction system comprising formic acid dehydrogenase (Formate Dehydrogenase).
The electrochemical reaction system for producing formic acid according to claim 12, wherein the formic acid dehydrogenase is immobilized in Ni-NTA (Nickel-Nitrilotriacetic Acid).
Histidine-labeled (His-tag) formic acid production device comprising formic acid dehydrogenase (Formate Dehydrogenase).
The apparatus of claim 14, wherein the formic acid dehydrogenase is immobilized on Ni-NTA (Nickel-Nitrilotriacetic Acid).
A filter for removing carbon dioxide containing histidine-labeled formic acid dehydrogenase (Formate Dehydrogenase).
The method according to claim 16, wherein the formic acid dehydrogenase is Ni-NTA (Nickel-Nitrilotriacetic Acid) will be immobilized on, carbon dioxide removal filter.
A method for producing formic acid comprising the step of contacting a gas containing carbon dioxide and an electron transporter to histidine-labeled (His-tag) formic acid dehydrogenase (Formate Dehydrogenase).
The method for producing formic acid according to claim 18, wherein the formic acid dehydrogenase is immobilized on Ni-NTA (Nickel-Nitrilotriacetic Acid).
The method for producing formic acid according to claim 18, wherein the electron transporter is a compound having a viologen group.
The method for producing formic acid according to claim 18, wherein the contacting step is made in an electrochemical reaction system.
The method for producing formic acid according to claim 18, wherein the gas containing carbon dioxide includes oxygen.

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