WO2022220325A1 - Genetically recombined formate dehydrogenase or immobilized, genetically recombined formate dehydrogenase, and formate production method using same - Google Patents
Genetically recombined formate dehydrogenase or immobilized, genetically recombined formate dehydrogenase, and formate production method using same Download PDFInfo
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- WO2022220325A1 WO2022220325A1 PCT/KR2021/005554 KR2021005554W WO2022220325A1 WO 2022220325 A1 WO2022220325 A1 WO 2022220325A1 KR 2021005554 W KR2021005554 W KR 2021005554W WO 2022220325 A1 WO2022220325 A1 WO 2022220325A1
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- WIPO (PCT)
- Prior art keywords
- formic acid
- dehydrogenase
- histidine
- formate
- nta
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
- C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
- C12Y102/01002—Formate dehydrogenase (1.2.1.2)
-
- C—CHEMISTRY; METALLURGY
- C25—ELECTROLYTIC OR ELECTROPHORETIC PROCESSES; APPARATUS THEREFOR
- C25B—ELECTROLYTIC OR ELECTROPHORETIC PROCESSES FOR THE PRODUCTION OF COMPOUNDS OR NON-METALS; APPARATUS THEREFOR
- C25B3/00—Electrolytic production of organic compounds
- C25B3/01—Products
- C25B3/07—Oxygen containing compounds
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
Definitions
- 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.
- formic acid is the most ideal chemical raw material in terms of economic and environmental benefits.
- 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.
- DFFC direct formate fuel cell
- 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.
- 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.
- 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.
- 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.
- 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).
- His-tag histidine-tagged formic acid dehydrogenase
- 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:
- 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.
- 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).
- protein refers to a polymer composed of two or more amino acids linked by amide bonds (or peptide bonds).
- 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).
- 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.
- 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.
- 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 FDH1 ⁇ (Formate Dehydrogenase 1 alpha subunit) and an endogenous gene encoding FDH1 ⁇ (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.
- fdh1A which is an endogenous gene encoding FDH1 ⁇
- fdh1B an endogenous gene encoding FDH1 ⁇
- Methylobacterium extorquens AM1 an endogenous gene encoding FDH1 ⁇
- Methylobacterium extorquens AM1 an endogenous gene encoding FDH1 ⁇
- Methylobacterium extorquens AM1 wild-type Methylobacterium extorquens AM1 compared to formic acid production amount and production efficiency
- 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.
- the exogenous gene encoding FDH1 may be introduced into a microorganism by a genetic material carrier.
- the genetic material carrier may be a vector.
- 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.
- 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.
- 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. .
- 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.
- 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. horrorquens , M. fujisawaense , M. gnaphalii, M.
- M. iners 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
- M. zatmanii M. zatmanii It may be at least one selected from the group consisting of.
- 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 .
- 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 ⁇ subunit (GenBank accession No. ACS42636.1) and MeFDH I ⁇ subunit (GenBank accession No. ACS42635.1).
- 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.
- 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 (%).
- 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.
- 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).
- PEG polyethylene glycol
- 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.
- His-tag histidine-tagged 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.
- 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 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.
- 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.
- composition for preparing formic acid may further include 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.
- 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.
- 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.
- FDH1 is known to have a W-pterin guanidine dinucleotide instead of molybdenum (Mo) as a prosthetic group.
- FDH1 ⁇ contains a bis-tungstopterin guanine dinucleotide cofactor, three 4Fe4S clusters, and one 2Fe2S cluster.
- FDH1 ⁇ contains a flavin mononucleotide (FMN), NAD, and one 4Fe4S cluster and one 2Fe2S.
- Air-purified FDH1 ⁇ can be successfully applied to the regeneration of NADH using methyl viologen (MV), an artificial electron transporter, in an electrochemical reduction system.
- MV methyl viologen
- the formic acid dehydrogenase may be immobilized on Ni-NTA (Nickel-Nitrilotriacetic Acid).
- Ni-NTA Nickel-Nitrilotriacetic acid
- Ni-NTA resin Ni-NTA affinity resin
- Ni-NTA column column (column)
- Ni-NTA affinity column Ni-NTA agarose
- Ni-NTA bead Ni-NTA agarose bead
- Ni-NTA magnetic bead Ni-NTA magnetic bead
- the Ni-NTA may be a Ni-NTA agarose bead.
- 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 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. .
- 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.
- 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.
- the electrochemical carbon dioxide reduction system it is possible to produce formic acid from carbon dioxide without microorganisms or enzymes.
- the histidine-labeled formic acid dehydrogenase 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.
- a cathode such as copper, graphite, carbon felt, and carbon fiber
- an anode such as platinum
- Ag/ A reference electrode such as AgCl
- 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.
- 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.
- the formic acid dehydrogenase may be immobilized on Ni-NTA (Nickel-Nitrilotriacetic Acid), and the Ni-NTA may be within the aforementioned range.
- Ni-NTA Nickel-Nitrilotriacetic Acid
- 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.
- the formic acid dehydrogenase may be immobilized on Ni-NTA (Nickel-Nitrilotriacetic Acid), and the Ni-NTA may be within the aforementioned range.
- Ni-NTA Nickel-Nitrilotriacetic Acid
- 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.
- 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.
- the formic acid dehydrogenase may be immobilized on Ni-NTA (Nickel-Nitrilotriacetic Acid), and the Ni-NTA may be within the aforementioned range.
- Ni-NTA Nickel-Nitrilotriacetic Acid
- 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.
- the formic acid dehydrogenase may be immobilized on Ni-NTA (Nickel-Nitrilotriacetic Acid), and the Ni-NTA may be within the above-described range.
- Ni-NTA Nickel-Nitrilotriacetic Acid
- 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.
- 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.
- 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.
- 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.
- the electrochemical carbon dioxide reduction system can be modified in a manner well known to those skilled in the art, if necessary.
- the gas including carbon dioxide may include oxygen.
- 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.
- 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.
- FIG. 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).
- FIG. 3 is a view showing the FDH1-his tag immobilized on Ni-NTA agarose bead.
- FIG. 4 is a diagram showing a standard curve for measuring an extinction coefficient for measuring the concentration of a reduced EV electron transporter.
- 5a to 5c are diagrams showing Lineweaver-Burk plots for the carbon dioxide reduction reaction.
- FIG. 6 is a diagram comparing the rate of carbon dioxide reduction and formic acid oxidation of enzymes.
- K eq rate constant
- FIG. 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 (dots and lines: formic acid productivity, bar: Faraday efficiency).
- 11 is a view showing the results of formic acid production according to long-term operation.
- FIG. 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 despite the abandonment of oxygen.
- 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 ( ⁇ -subunit), ACS42635.1 ( ⁇ -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.
- SLIC sequence and ligation-independent cloning
- 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 ⁇ l of the mixture was added to 100 ⁇ l of water-soluble E. coli DH5 ⁇ (RBC), and the DH5 ⁇ E. coli was incubated on ice for 20 minutes. Then, 950 ⁇ l of LB medium was added, and incubated at 37° C. for 16 hours.
- RBC water-soluble E. coli DH5 ⁇
- FDH1 ⁇ , and/or FDH1 ⁇ gene of Methylobacterium extorquens AM1 was amplified with DNA located in the front and rear portions.
- the primers used for cloning are shown in Table 1 below.
- 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.
- 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.
- the knock-out microorganism was expressed with a recombinant plasmid.
- 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 FDH1 ⁇ .
- Table 1 below shows bacterial strains and plasmids for knock-out or recombinant expression.
- the basic culture medium of the microorganisms is 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).
- 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
- /L NaH 2 PO 4 .2H 2 O As selective antibiotics for screening recombinant microorganisms, 50 ⁇ g/mL rifamicin (Rif), 50 ⁇ g/mL kanamicin (Kan), or 10 ⁇ g/mL tetracycline (Tet) was used.
- All of the above microorganisms were cultured at 26°C and 200 rpm in a volume of 200 mL in a 1L Erlenmeyer shake flask.
- 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. Thereafter, the supernatant was recovered by centrifugation at 4,611 g for 30 minutes after pulverization using a cell disrupter. Then, the supernatant was recovered using Ni-NTA agarose beads (Bio-Rad, Cat No.
- 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
- FDH1-his tag enzyme SEQ ID NO: 10
- FDH1 beta subunit SEQ ID NO: 11
- 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.
- EV ethyl viologen
- 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).
- 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.
- 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 4 Formic acid production through carbon dioxide reduction using an electrochemical reactor
- FIG. 9 The schematic diagram of the electrochemical reactor used in this example was shown in FIG. 9 below.
- 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.
- 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.
- samples were taken from the cathode at regular intervals and the concentration of formic acid generated was measured using HPLC.
- Example 4 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.
- the formic acid productivity was repeatedly measured using the enzyme immobilized on the Ni-NTA agarose bead recovered after 220 hours of reaction.
- 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.
- Example 6 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.
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Abstract
An aspect provides a genetically recombined, histidine-tagged formate dehydrogenase or immobilized, histidine-tagged formate dehydrogenase. The genetically recombined, histidine-tagged formate dehydrogenase according to an aspect can reduce carbon dioxide to effectively produce formate at high yield. Furthermore, when immobilized, the formate dehydrogenase allows production of a high concentration of formate in an electrochemical reactor without decreasing in yield for a long period of time. The formate dehydrogenase is not significantly affected by dissolved oxygen levels in addition to being reusable, thus greatly contributing to the industries of carbon dioxide removal or formate production.
Description
유전자 재조합 포름산 탈수소효소 또는 고정화된 유전자 재조합 포름산 탈수소효소 및 그들을 이용한 개미산의 제조 방법에 관한 것이다.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.
화석연료를 이용하는 산업 전반의 빠른 발전은 대기중 이산화탄소의 농도를 급속하게 증가시키고 있다. 게다가, 다수의 논문들이 대기중 이산화탄소의 증가가 온실효과를 통한 지구온난화를 발생시키는 결정적인 요소임을 확인한 바 있다. 따라서 이러한 이산화탄소를 고부가가치의 화학물질로 전환하는 것은 대기중 이산화탄소 증가의 속도를 늦추는데 필수적인 기술로 상정되고 있다. 다행히도, 이산화탄소는 개미산, 디메틸 카보네이트, 고분자 등 환경 친화적인 화학 플랫폼을 생산하는 유망한 재생 가능한 자원이다.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.
이산화탄소로부터 생산할 수 있는 많은 후보 물질들 중에서 개미산은 경제적 및 환경적 이익 측면에서 가장 이상적인 화학 원료이다. 특히, 개미산을 전기에너지로 전환시키는 직접적 개미산 연료셀(direct formate fuel cell; DFFC)은 풍력, 태양력, 수력 등 다양한 재생 에너지원에서 생산된 에너지를 개미산으로 쉽게 저장할 수 있기 때문에 재생 에너지 저장의 해결책으로 제시되고 있다. 또한 개미산은 환경에서 불연성, 무독성, 및 불활성이므로 수소 가스보다 안전하게 수송이 가능한 장점이 있다. 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.
유망한 잠재력에도 불구하고, 이산화탄소로부터 고부가가치의 화합물을 생산하는 기존의 기술은 가혹한 반응 조건과 희귀한 귀금속 촉매 및 수소 가스, 수소화물 등과 같은 값 비싼 환원제를 요구하는 치명적인 한계를 갖고 있다. 상대적으로 낮은 전기 에너지 비용을 고려할 때, 이산화탄소의 전기 촉매 반응이 유망한 대안일지라도, 일부 화학 전기 촉매는 이산화탄소의 개미산 전환 중에 높은 비율로 부산물인 수소 가스 및 일산화탄소를 생산하기 때문에 불충분한 반응선택성과 이에 따른 공정의 위험성을 보여주고 있는 실정이다. 따라서 효소 기반의 전기화학반응에 의한 이산화탄소로부터의 개미산 생산은 탁월한 개미산 선택성으로 인하여 더 많은 관심을 받고 있다. 그러나 효소 기반의 전기화학반응의 경우 이러한 장점에도 불구하고 여러가지 문제점으로 인하여 실용화되지 못하고 있다. 그중 가장 큰 요인은 낮은 산소안정성으로 이산화탄소 환원반응속도가 큰 금속 함유 효소의 경우 산소에 극히 불안정하여 쉽게 활성을 잃는 단점이 있다. 또한 이산화탄소를 환원하는 반응과 생성된 개미산을 다시 산화하는 반응 양 반응을 모두 효소가 촉매하여 결과적으로 반응 평형이 존재하고 이로 인하여 고농도의 개미산 생산이 어려운 단점이 존재한다. 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.
이에, 텅스텐을 포함하는 메틸로박테리움 엑스토르쿠엔스(Methylobacterium extorquens) AM1 유래 포름산 탈수소효소(formate dehydrogenase)에 히스티딘 태그(Histidine tag)를 재조합시킨 후, 재조합 효소를 Ni-NTA agarose bead에 고정화시켜 분리와 동시에 효소 촉매로 쉽고 빠르게 제조할 수 있는 방법 및 전기화학반응기에서 장기간 운전을 통하여 고농도의 개미산을 생산하고 동시에 연속적인 재사용이 가능한 상기 고정화된 효소를 개발하기에 이르렀다.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.
일 양상은 히스티딘 표지(His-tag)된 포름산 탈수소효소(Formate Dehydrogenase)를 제공하는 것이다.One aspect is to provide a histidine-tagged formic acid dehydrogenase (Formate Dehydrogenase).
다른 양상은 Ni-NTA(Nickel-Nitrilotriacetic Acid)에 고정화된 히스티딘 표지(His-tag)된 포름산 탈수소효소(Formate Dehydrogenase)를 제공하는 것이다.Another aspect is to provide a histidine-tagged (His-tag) formic acid dehydrogenase (Formate Dehydrogenase) immobilized on Ni-NTA (Nickel-Nitrilotriacetic Acid).
또 다른 양상은 히스티딘 표지(His-tag)된 포름산 탈수소효소(Formate Dehydrogenase)를 포함하는 개미산 제조용 조성물을 제공하는 것이다.Another aspect is to provide a composition for preparing formic acid comprising histidine-labeled (His-tag) formic acid dehydrogenase (Formate Dehydrogenase).
또 다른 양상은 히스티딘 표지(His-tag)된 포름산 탈수소효소(Formate Dehydrogenase)를 포함하는 개미산 제조용 전기화학반응 시스템을 제공하는 것이다.Another aspect is to provide an electrochemical reaction system for producing formic acid including histidine-tagged formic acid dehydrogenase (Formate Dehydrogenase).
또 다른 양상은 히스티딘 표지(His-tag)된 포름산 탈수소효소(Formate Dehydrogenase)를 포함하는 개미산 제조용 장치를 제공하는 것이다.Another aspect is to provide an apparatus for producing formic acid comprising histidine-labeled (His-tag) formic acid dehydrogenase (Formate Dehydrogenase).
또 다른 양상은 히스티딘 표지(His-tag)된 포름산 탈수소효소(Formate Dehydrogenase)를 포함하는 이산화탄소 제거용 필터를 제공하는 것이다.Another aspect is to provide a filter for removing carbon dioxide including histidine-tagged formic acid dehydrogenase (Formate Dehydrogenase).
또 다른 양상은 히스티딘 표지(His-tag)된 포름산 탈수소효소(Formate Dehydrogenase)에 이산화탄소를 포함하는 기체 및 전자전달체를 접촉시키는 단계를 포함하는 개미산의 제조 방법을 제공하는 것이다.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).
일 양상은 히스티딘 표지(His-tag)된 포름산 탈수소효소(Formate Dehydrogenase)를 제공한다.One aspect provides a histidine-tagged formic acid dehydrogenase (Formate Dehydrogenase).
상기 포름산 탈수소효소는 이산화탄소와 접촉하여 개미산으로 전환시킬 수 있다. 구체적으로 포름산 탈수소효소는 하기 반응식 1의 반응을 촉매하여 개미산을 제조할 수 있다: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:
[반응식 1][Scheme 1]
CO2 + 2H+ + EM(환원된 상태) -> HCOOH + EM(산화된 상태).CO 2 + 2H + + EM (reduced state) -> HCOOH + EM (oxidized state).
일 양상에 있어서, 상기 포름산 탈수소효소는 천연으로부터 유래될 수도 있고, 당해 분야에서 널리 공지된 다양한 단백질 합성 방법으로 획득할 수 있다. 일례로서, 유전자 재조합과 단백질 발현 시스템을 이용하여 제조하거나 단백질 합성과 같은 화학적 합성을 통하여 시험관 내에서 합성하는 방법 및 무세포 단백질 합성법 등으로 제조될 수 있다. 또한, 일례로서, 상기 포름산 탈수소효소는 펩티드, 식물 유래 조직이나 세포의 추출물, 미생물(예를 들어 세균류 또는 진균류, 그리고 특히 효모)의 배양으로 얻어진 생산물일 수 있다.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).
용어 "단백질(Protein)"는 아마이드 결합 (또는 펩티드 결합)으로 연결된 2개 이상의 아미노산으로 이루어진 폴리머를 의미한다.The term “protein” refers to a polymer composed of two or more amino acids linked by amide bonds (or peptide bonds).
용어 "발현(expression)"은 폴리펩티드(polypeptide)가 구조 유전자로부터 생산되는 과정을 지칭한다. 상기 과정은 유전자(폴리뉴클레오티드)의 mRNA로의 전사, 및 이러한 mRNA의 폴리펩티드(단백질)(들)로의 해독을 포함한다.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).
용어 "재조합"은 세포가 이종의 핵산을 복제하거나, 상기 핵산을 발현하거나 또는 펩티드, 이종의 펩티드 또는 이종의 핵산에 의해 코딩된 단백질을 발현하는 세포를 지칭하는 것이다. 재조합 세포는 상기 세포의 천연 형태에서는 발견되지 않는 유전자 또는 유전자 절편을, 센스 또는 안티센스 형태 중 하나로 발현할 수 있다. 또한 재조합 세포는 천연 상태의 세포에서 발견되는 유전자를 발현할 수 있으며, 그러나 상기 유전자는 변형된 것으로써 인위적인 수단에 의해 세포 내 재도입된 것이다.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.
일 양상에 있어서, 상기 포름산 탈수소효소는 유전자 재조합으로 제조된 것일 수 있고, 구체적으로, 상기 포름산 탈수소효소는 메틸로박테리움 엑스토르쿠엔스(Methylobacterium extorquens) AM1 유래 FDH1(Formate Dehydrogenase 1)으로서,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,
상기 메틸로박테리움 엑스토르쿠엔스 AM1은 FDH1α(Formate Dehydrogenase 1 alpha subunit)를 코딩하는 내인성 유전자 및 FDH1β(Formate Dehydrogenase 1 beta subunit)를 코딩하는 내인성 유전자가 결실되고,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,
메틸로박테리움 엑스토르쿠엔스 AM1 유래 FDH1 및 히스티딘 표지(His-tag)를 코딩하는 외인성 유전자가 도입된 재조합 미생물일 수 있다.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.
상기 메틸로박테리움 엑스토르쿠엔스 AM1에서 FDH1α를 코딩하는 내인성 유전자인 fdh1A 및 FDH1β를 코딩하는 내인성 유전자인 fdh1B를 녹-아웃하고, FDH1을 코딩하는 유전자 fdh1을 형질전환하여 재도입하는 경우, 야생형 메틸로박테리움 엑스토르쿠엔스 AM1에 비해 개미산 제조량 및 제조 효율이 증가할 수 있다. 야생형 메틸로박테리움 엑스토르쿠엔스 AM1에 녹-아웃 전처리 없이 fdh1 코딩하는 유전자를 형질전환하여 인위적으로 발현시키는 경우, 상기 재조합 미생물과 같은 개미산 제조량 및 제조 효율이 나타나지 않는다.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.
일 양상에 있어서, 상기 FDH1을 코딩하는 외인성 유전자는 유전 물질 전달체에 의해서 미생물 내에 도입된 것일 수 있다. 상기 유전 물질 전달체는 벡터일 수 있다.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.
용어 "벡터 (vector)"는 적합한 숙주 내에서 DNA를 발현시킬 수 있는 적합한 조절 서열에 작동가능하게 연결된 DNA 서열을 함유하는 DNA 제조물을 의미한다. 벡터는 플라스미드, 파지 입자, 또는 간단하게 잠재적 게놈 삽입물일 수 있다. 적당한 숙주로 형질전환 되면, 벡터는 숙주 게놈과 무관하게 복제하고 기능할 수 있거나, 또는 일부 경우에 게놈 그 자체에 통합될 수 있다. 플라스미드가 현재 벡터의 가장 통상적으로 사용되는 형태이므로, 본 원에서 "플라스미드(plasmid)" 및 "벡터 (vector)"는 때로 상호 교환적으로 사용된다. 본 발명에서 사용되는 벡터는 당업계에 알려진 또는 알려지게 되는 바와 동등한 기능을 갖는 벡터의 다른 형태를 포함할 수 있다.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.
일 구체예에서, 상기 벡터는 PmxaF 프로모터를 포함하는 것일 수 있다. 상기 PmxaF 프로모터를 포함하는 벡터는 통상의 기술자에게 잘 알려져 있고, 예를 들어, pCM110일 수 있고, 히스티딘 표지된 포름산 탈수소효소가 도입된 pCM110은 서열번호 9의 염기 서열로 이루어진 폴리뉴클레오티드일 수 있다.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. .
또한, 상기 벡터는 외인성 유전자를 세포(구체적으로, 미생물)로 도입시키기 위해 적합한 것이라면 통상의 기술자에게 알려진 어떤 것이든 이용될 수 있다.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).
상기 포름산 탈수소효소는 포름산 탈수소효소의 아미노산 서열과 각각 약 70% 이상, 약 75% 이상, 약 80% 이상, 약 85%이상, 약 90% 이상, 약 92% 이상, 약 95% 이상, 약 97% 이상, 약 98% 이상, 또는 약 99% 이상의 서열 상동성을 갖는 단백질을 포함할 수 있다. 상기 포름산 탈수소효소는 포름산 탈수소효소의 동질효소일 수 있고, 예를 들어, 상기 포름산 탈수소효소는 메틸로박테리움속(Methylobacterium sp.), 칸디다속(Candida sp.) 싸이오바실러스속(Thiobacillus sp.), 랄스토니아속(Ralstonia sp.) 및 로도박터속(Rhodobacter sp.)으로 이루어진 군에서 선택되는 적어도 하나의 속의 미생물 유래일 수 있다.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.
또한, 상기 메틸로박테리움 속 미생물은 예를 들어, M. 아드하에시붐(M. adhaesivum), M. 아에로라툼(M. aerolatum), M. 아미노보란스(M. aminovorans), M. 아쿠아티쿰(M. aquaticum), M. 브라치아툼(M. brachiatum), M. 브라치테시이(M. brachythecii), M. 불라툼(M. bullatum), M. 세라스티(M. cerastii), M. 단코오켄세(M. dankookense), M. 엑스토르쿠엔스(M. extorquens), M. 푸지사와덴세(M. fujisawaense), M. 그나팔리이(M. gnaphalii), M. 고에신켄세(M. goesingense), M. 고씨피이콜라(M. gossipiicola), M. 그레간스(M. gregans), M. 하플로클라디이(M. haplocladii), M. 히스파니쿰(M. hispanicum), M. 이네르스 (M. iners), M. 이스빌리엔세(M. isbiliense), M. 제오트갈리(M. jeotgali), M. 코마가타에(M. komagatae), M. 롱굼(M. longum), M. 마르찬티아에(M. marchantiae), M. 메소필리쿰(M. mesophilicum), M. 노둘란스(M. nodulans), M. 오르가노필럼(M. organophilum), M. 오리자에(M. oryzae), M. 옥살리디스(M. oxalidis), M. 페르시치눔(M. persicinum), M. 필로스파에라에(M. phyllosphaerae), M. 필로스타키오스(M. phyllostachyos), M. 플라타니 (M. platani), M. 포다리움(M. podarium), M. 포푸리(M. populi), M. 슈도사사에(M. pseudosasae), M. 슈도나시콜라(M. pseudosasicola), M. 라디오톨레란스(M. radiotolerans), M. 로데시아눔(M. rhodesianum), M. 로디눔(M. rhodinum), M. 살수지니스(M. salsuginis), M. 솔리(M. soli), M. 수오미엔세(M. suomiense), M. 타르둠(M. tardum), M. 타르하니아에(M. tarhaniae), M. 티오시아나툼(M. thiocyanatum), M. 투린지엔세(M. thuringiense), M. 트리폴리이(M. trifolii), M. 바리아빌레(M. variabile) 및 M. 자트마니이(M. zatmanii)로 이루어진 군에서 선택되는 적어도 하나일 수 있다.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.
구체적으로, 상기 포름산 탈수소효소는 메틸로박테리움 엑스토르쿠엔스(Methylobacterium extorquens), 랄스토니아 유트로파(Ralstonia eutropha), 칸디다 보이디니(Candida boidinii) 싸이오바실러스속(Thiobacillus sp.) KNK65MA 및 로도박터 캡슐레이터스(Rhodobacter capsulatus)로 이루어진 군에서 선택되는 적어도 하나의 미생물 유래일 수 있다.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 .
보다 구체적으로 상기 포름산 탈수소효소는 메틸로박테리움 엑스토르쿠엔스(Methylobacterium extorquens) AM1(ATCC 14781, GenBank accession No. CP001510.1) 유래 포름산 탈수소효소 I일 수 있다.More specifically, the formic acid dehydrogenase may be formic acid dehydrogenase I derived from Methylobacterium extorquens AM1 (ATCC 14781, GenBank accession No. CP001150.1).
상기 메틸로박테리움 엑스토르쿠엔스(Methylobacterium extorquens) AM1 유래 포름산 탈수소효소 I은 MeFDH I α subunit(GenBank accession No. ACS42636.1) 및 MeFDH I β subunit(GenBank accession No. ACS42635.1)으로 이루어질 수 있고, 상기 메틸로박테리움 엑스토르쿠엔스(Methylobacterium extorquens) 유래 포름산 탈수소효소 I은 fdh1A(GenBank accession, CP001510.1:5169596-5172565, Methylobacterium extorquens AM1, complete genome) 및 fdh1B(GenBank accession, CP001510.1:5167825-5169543, Methylobacterium extorquens AM1, complete genome)가 코딩하는 폴리펩티드일 수 있다.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.
용어 "상동성(Homology)"은 야생형 아미노산 서열과의 유사한 정도를 나타내기 위한 것으로서, 이러한 상동성의 비교는 당업계에서 널리 알려진 비교 프로그램을 이용하여 수행할 수 있으며, 2개 이상의 서열간 상동성을 백분율(%)로 계산할 수 있다.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 (%).
상기 용어 "안정성"은 생체 내 단백질 절단 효소의 공격으로부터 상기 포름산 탈수소효소를 보호하는 인 비보(in vivo) 안정성뿐만 아니라, 저장 안정성(예컨대, 상온 저장 안정성)도 의미하는 것일 수 있다. 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.
또한, 보다 나은 화학적 안정성, 강화된 약리 특성(반감기, 흡수성, 역가, 효능 등), 변경된 특이성(예를 들어, 광범위한 생물학적 활성 스펙트럼), 감소된 항원성을 획득하기 위하여, 상기 포름산 탈수소효소의 N- 또는 C-말단에 보호기가 결합되어 있을 수 있다. 상기 보호기는 아세틸기, 플루오레닐 메톡시 카르보닐기, 포르밀기, 팔미토일기, 미리스틸기, 스테아릴기 또는 폴리에틸렌글리콜(PEG)일 수 있으나, 상기 포름산 탈수소효소의 개질, 특히 포름산 탈수소효소의 안정성을 증진시킬 수 있는 성분이라면, 제한없이 포함될 수 있다.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.
아울러, 상기 포름산 탈수소효소는 히스티딘 표지 외에도 표적화 서열, 태그 (tag), 표지된 잔기를 위한 특정 목적으로 제조된 아미노산 서열도 추가적으로 포함할 수 있고, 구체적으로, His-tag 말단의 단백질과 결합된 형태인(히스티딘 표지된) 포름산 탈수소효소는 서열번호 10 및 11의 아미노산 서열로 이루어진 폴리펩티드일 수 있다.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.
다른 양상은 Ni-NTA(Nickel-Nitrilotriacetic Acid)에 고정화된 히스티딘 표지(His-tag)된 포름산 탈수소효소(Formate Dehydrogenase)를 제공한다.Another aspect provides a histidine-tagged (His-tag) formic acid dehydrogenase (Formate Dehydrogenase) immobilized on Ni-NTA (Nickel-Nitrilotriacetic Acid).
상기 "히스티딘 표지", "포름산 탈수소효소" 등은 전술한 범위 내일 수 있다.The "histidine label", "formic acid dehydrogenase", etc. may be within the above-mentioned range.
상기 히스티딘 표지된 포름산 탈수소효소는 Ni-NTA에 고정화된 경우, 고정화되지 않은 히스티딘 표지된 포름산 탈수소효소에 비해 장기간 동안 효율이 떨어지지 않고 개미산 제조가 가능하며, 재사용이 가능할 뿐만 아니라, 용존 산소 농도에 따라 활성 감소가 적을 수 있다.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.
일 양상에 있어서, 상기 Ni-NTA는 Ni-NTA(Nickel-Nitrilotriacetic acid)로서, Ni-NTA를 포함한다면 제한되지 않으나, 예를 들어, Ni-NTA resin(수지), Ni-NTA affinity resin, Ni-NTA column(컬럼), Ni-NTA affinity column, Ni-NTA agarose, Ni-NTA bead, Ni-NTA agarose bead, Ni-NTA magnetic bead 등일 수 있고, 구체적으로, 상기 Ni-NTA는 Ni-NTA agarose bead일 수 있다.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.
또 다른 양상은 히스티딘 표지(His-tag)된 포름산 탈수소효소(Formate Dehydrogenase)를 포함하는 개미산 제조용 조성물을 제공한다.Another aspect provides a composition for preparing formic acid comprising histidine-labeled (His-tag) formic acid dehydrogenase (Formate Dehydrogenase).
상기 "히스티딘 표지", "포름산 탈수소효소", "개미산" 등은 전술한 범위 내일 수 있다.The "histidine label", "formic acid dehydrogenase", "formic acid", etc. may be within the above-described range.
일 양상에 있어서, 상기 개미산 제조용 조성물은 전자전달체를 더 포함할 수 있다.In one aspect, the composition for preparing formic acid may further include an electron transporter.
상기 조성물은 전자전달체를 추가로 포함할 경우, 보다 개미산 제조 효율이 증가될 수 있다.When the composition further comprises an electron transporter, the formic acid production efficiency can be increased.
상기 전자전달체는 천연 전자전달체 또는 인공 전자전달체일 수 있다. 또한, 상기 전자전달체는 바이올로젠(viologen)기를 가지는 전자전달체 및 아데닌 디뉴클레오티드(adenine dinucleotide)기를 가지는 전자전달체로 이루어진 군에서 선택되는 적어도 하나의 전자전달체일 수 있고, 예를 들어, 알킬 바이올로젠(alkyl viologen), 벤질 바이올로젠(benzyl viologen), NAD(nicotinamide adenine dinucleotide) 및 FAD(Flavin adenine dinucleotide)로 이루어진 군에서 선택되는 적어도 하나의 전자전달체일 수 있고, 상기 알킬 바이올로젠(alkyl viologen)은 메틸 바이올로젠(methyl viologen), 에틸 바이올로젠(ethyl viologen), 프로필 바이올로젠(propyl viologen) 등을 포함할 수 있다.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.
구체적으로, 상기 전자전달체는 메틸 바이올로젠(Methyl viologen), 에틸 바이올로젠(Ethyl viologen), 벤질 바이올로젠(Benzyl viologen), NAD(nicotinamide adenine dinucleotide) 및 FAD(Flavin adenine dinucleotide)로 이루어진 군에서 선택되는 적어도 하나의 전자전달체일 수 있고, 보다 구체적으로, 메틸 바이올로젠(methyl viologen) 및 에틸 바이올로젠(ethyl viologen)로 이루어진 군에서 선택되는 적어도 하나의 전자전달체일 수 있다.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.
한편, FDH1은 보결분자단으로 몰리브덴(Mo) 대신 W-프테린 구아니딘 디뉴클레오티드를 보유하는 것으로 알려져 있다. 구체적으로, FDH1α는 비스-텅스토프테린 구아닌 디뉴클레오티드 공동인자(bis-tungstopterin guanine dinucleotide cofactor), 3 개의 4Fe4S 클러스터, 및 한 개의 2Fe2S 클러스터를 포함한다. FDH1β는 플래빈 모노뉴클레오티드 (FMN), NAD, 및 한 개의 4Fe4S 클러스터 및 한 개의 2Fe2S를 보유한다. 대기 중에서 정제된 FDH1β는 전기 화학적 환원 시스템에서 인공 전자전달체인 메틸 바이올로젠(MV)을 이용하여 NADH의 재생에 성공적으로 적용될 수 있다.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.
또한, 일 양상에 있어서, 상기 포름산 탈수소효소는 Ni-NTA(Nickel-Nitrilotriacetic Acid)에 고정화된 것일 수 있다.Also, in one aspect, the formic acid dehydrogenase may be immobilized on Ni-NTA (Nickel-Nitrilotriacetic Acid).
상기 Ni-NTA는 Ni-NTA(Nickel-Nitrilotriacetic acid)로서, Ni-NTA를 포함한다면 제한되지 않으나, 예를 들어, Ni-NTA resin(수지), Ni-NTA affinity resin, Ni-NTA column(컬럼), Ni-NTA affinity column, Ni-NTA agarose, Ni-NTA bead, Ni-NTA agarose bead, Ni-NTA magnetic bead 등일 수 있고, 구체적으로, 상기 Ni-NTA는 Ni-NTA agarose bead일 수 있다.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.
상기 히스티딘 표지된 포름산 탈수소효소는 Ni-NTA에 고정화된 경우, 고정화되지 않은 히스티딘 표지된 포름산 탈수소효소에 비해 장기간 동안 효율이 떨어지지 않고 개미산 제조가 가능하며, 재사용이 가능할 뿐만 아니라, 용존 산소 농도에 따라 활성 감소가 적을 수 있다.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.
또한, 일 양상에 있어서, 상기 조성물의 pH는 5.0 내지 7.5, 5.0 내지 7.0, 5.0 내지 6.5, 5.5 내지 7.5, 5.5 내지 7.0, 5.5 내지 6.5, 6.0 내지 7.5, 6.0 내지 7.0 또는 6.0 내지 6.5일 수 있다.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. .
상기 조성물의 pH가 5.0 미만인 경우, 이산화탄소의 환원 속도(개미산 생성 속도)가 저하될 수 있고, pH가 7.5 초과인 경우, 이산화탄소 환원 속도(개미산 생성 속도)는 저하되고, 개미산의 산화 속도는 증가될 수 있어, 개미산 제조 수율이 저하될 수 있다.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.
또 다른 양상은 히스티딘 표지(His-tag)된 포름산 탈수소효소(Formate Dehydrogenase)를 포함하는 개미산 제조용 전기화학반응 시스템을 제공한다.Another aspect provides an electrochemical reaction system for producing formic acid comprising histidine-tagged formic acid dehydrogenase (Formate Dehydrogenase).
상기 "히스티딘 표지", "포름산 탈수소효소", "개미산" 등은 전술한 범위 내일 수 있다.The "histidine label", "formic acid dehydrogenase", "formic acid", etc. may be within the above-described range.
상기 히스티딘 표지된 포름산 탈수소효소는 일반적인 효소와 달리 NADH와 같은 값비싼 전자 공여체 없이도 전기 화학적으로 생산된 전자를 이용하여 이산화탄소로부터 개미산을 생산할 수 있다. 따라서 상기 포름산 탈수소효소에 전자를 공급할 수 있는 시스템이라면 개미산을 생산하는데 이용될 수 있으며, 그러한 시스템으로는 예를 들어, 전극을 주입하여 전지로부터 직접 전류를 흘려주는 전기적 시스템 또는 산화 환원 반응에 의해 전류를 생성하고 전자를 공급할 수 있는 전기 화학적 (화학 전지) 시스템이 있다. 따라서, 일 양상에서 상기 포름산 탈수소효소는 개미산을 생산하기 위하여 전기적 또는 전기화학적 시스템에서 적용되는 것일 수 있다.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.
일 양상에서, 상기 전기화학적 시스템은 전기 화학적 이산화탄소 환원 시스템일 수 있다. 전기 화학적 이산화탄소 환원 시스템이란, 화학전지 원리를 이용하여 이산화탄소를 개미산으로 환원시킬 수 있는 시스템을 의미한다. 전기 화학적 전환 기술은 상온, 상압 조건에서도 이산화탄소 환원 반응이 가능하고, 물과 이산화탄소를 주로 이용하므로 화학물질의 배출이 없으며, 시스템이 간단하고 모듈화가 용이한 장점을 갖는다. 전기 화학적 이산화탄소 환원 시스템에 의하면 미생물이나 효소가 없이도 이산화탄소로부터 개미산 생성이 가능하다. 그러나 본 실시예에서 살필 수 있듯이, 상기 일 양상에 따른 히스티딘 표지된 포름산 탈수소효소를 사용하는 경우, 보다 높은 수율 및 효율로 이산화탄소로부터 개미산 생성이 가능하다. 상기 전기 화학적 이산화탄소 환원 시스템은 도 9에 도식된 바와 같이, 예를 들어, 구리, 흑연(graphite), 탄소 펠트(carbon felt), 및 탄소 섬유와 같은 음극 및 백금과 같은 양극을 포함하고, Ag/AgCl와 같은 기준 전극을 더 포함할 수 있다. 그러나 상기 전기 화학적 이산화탄소 환원 시스템은 필요에 따라 통상의 기술자에게 잘 알려진 방식으로 얼마든지 변형 가능하다.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.
상기 히스티딘 표지된 포름산 탈수소효소는 음극에서 공급되는 전자를 이용하여 매우 효과적으로 개미산을 생산할 수 있을 뿐만 아니라, 공기 중 산소에 매우 안정한 특성을 갖는다. 기존에 알려진 생촉매들 중 음극에서 공급된 전자를 직접 이용할 수 있는 것이 있었으나, 이들은 매우 산소에 불안정하여 실제 적용이 불가능하였다. 따라서, 일 양상에 따른 포름산 탈수소효소는 실제 산업화 공정에 적용이 가능하므로 매우 유용하게 이용될 수 있을 것이다.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.
또한, 일 양상에 있어서, 상기 포름산 탈수소효소는 Ni-NTA(Nickel-Nitrilotriacetic Acid)에 고정화된 것일 수 있고, 상기 Ni-NTA는 전술한 범위 내일 수 있다.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.
상기 히스티딘 표지된 포름산 탈수소효소는 Ni-NTA에 고정화된 경우, 고정화되지 않은 히스티딘 표지된 포름산 탈수소효소에 비해 장기간 동안 효율이 떨어지지 않고 개미산 제조가 가능하며, 재사용이 가능할 뿐만 아니라, 용존 산소 농도에 따라 활성 감소가 적을 수 있다.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.
또 다른 양상은 히스티딘 표지(His-tag)된 포름산 탈수소효소(Formate Dehydrogenase)를 포함하는 개미산 제조용 장치를 제공한다.Another aspect provides a device for producing formic acid comprising a histidine-tagged (His-tag) formic acid dehydrogenase (Formate Dehydrogenase).
상기 "히스티딘 표지", "포름산 탈수소효소", "개미산" 등은 전술한 범위 내일 수 있다.The "histidine label", "formic acid dehydrogenase", "formic acid", etc. may be within the above-described range.
또한, 일 양상에 있어서, 상기 포름산 탈수소효소는 Ni-NTA(Nickel-Nitrilotriacetic Acid)에 고정화된 것일 수 있고, 상기 Ni-NTA는 전술한 범위 내일 수 있다.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.
상기 히스티딘 표지된 포름산 탈수소효소는 Ni-NTA에 고정화된 경우, 고정화되지 않은 히스티딘 표지된 포름산 탈수소효소에 비해 장기간 동안 효율이 떨어지지 않고 개미산 제조가 가능하며, 재사용이 가능할 뿐만 아니라, 용존 산소 농도에 따라 활성 감소가 적을 수 있다.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.
또 다른 양상은 히스티딘 표지(His-tag)된 포름산 탈수소효소(Formate Dehydrogenase)를 포함하는 이산화탄소 제거용 필터를 제공한다.Another aspect provides a filter for removing carbon dioxide including histidine-tagged formic acid dehydrogenase (Formate Dehydrogenase).
상기 "히스티딘 표지", "포름산 탈수소효소", "이산화탄소" 등은 전술한 범위 내일 수 있다.The "histidine label", "formic acid dehydrogenase", "carbon dioxide", etc. may be within the above-described range.
상기 히스티딘 표지된 포름산 탈수소효소는 전술한 바와 같이, 이산화탄소를 환원시켜 개미산을 제조함으로써 이산화탄소는 제거하되, 유용물질인 개미산을 생성할 수 있다.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.
또한, 일 양상에 있어서, 상기 포름산 탈수소효소는 Ni-NTA(Nickel-Nitrilotriacetic Acid)에 고정화된 것일 수 있고, 상기 Ni-NTA는 전술한 범위 내일 수 있다.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.
상기 히스티딘 표지된 포름산 탈수소효소는 Ni-NTA에 고정화된 경우, 고정화되지 않은 히스티딘 표지된 포름산 탈수소효소에 비해 장기간 동안 효율이 떨어지지 않고 이산화탄소 제거가 가능하며, 재사용이 가능할 뿐만 아니라, 용존 산소 농도에 따라 활성 감소가 적을 수 있다.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.
상기 필터는 담배 필터, 공기청정기 필터 등 이산화탄소가 발생되는 장소에서 다양한 필터에 적용될 수 있으며, 나아가 유해 가스 처리 기술이 필요한 산업 현장, 공기 내 유해 물질 살균/제거 정화기술 및 시스템, 차량, 열차 등 이동수단 내 실내 공기질 관리를 위한 처리시설 및 관련기술, 환기효율 및 경제적 환기를 위한 기술 및 장치, 공기청정기, 에어컨, 환풍기 등과 같은 실내공기 정화장치 등에 포함될 수 있다. 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.
또 다른 양상은 히스티딘 표지(His-tag)된 포름산 탈수소효소(Formate Dehydrogenase)에 이산화탄소를 포함하는 기체 및 전자전달체를 접촉시키는 단계를 포함하는 개미산의 제조 방법을 제공한다.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).
상기 "히스티딘 표지", "포름산 탈수소효소", "이산화탄소", "전자전달체", "개미산" 등은 전술한 범위 내일 수 있다.The "histidine label", "formic acid dehydrogenase", "carbon dioxide", "electron transporter", "formic acid", etc. may be within the above-described range.
상기 이산화탄소를 포함하는 기체를 상기 포름산 탈수소효소에 접촉시킴으로써 이산화탄소를 환원시켜 개미산을 제조할 수 있으며, 구체적인 이산화탄소의 환원에 따른 개미산 제조 메커니즘은 전술한 범위 내일 수 있다.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.
일 양상에 있어서, 상기 포름산 탈수소효소는 Ni-NTA(Nickel-Nitrilotriacetic Acid)에 고정화된 것일 수 있고, 상기 Ni-NTA는 전술한 범위 내일 수 있다.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.
상기 히스티딘 표지된 포름산 탈수소효소는 Ni-NTA에 고정화된 경우, 고정화되지 않은 히스티딘 표지된 포름산 탈수소효소에 비해 장기간 동안 효율이 떨어지지 않고 개미산 제조가 가능하며, 재사용이 가능할 뿐만 아니라, 용존 산소 농도에 따라 활성 감소가 적을 수 있다.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.
상기 전자전달체는 천연 전자전달체 또는 인공 전자전달체일 수 있다. 또한, 상기 전자전달체는 바이올로젠(viologen)기를 가지는 전자전달체 및 아데닌 디뉴클레오티드(adenine dinucleotide)기를 가지는 전자전달체로 이루어진 군에서 선택되는 적어도 하나의 전자전달체일 수 있고, 예를 들어, 알킬 바이올로젠(alkyl viologen), 벤질 바이올로젠(benzyl viologen), NAD(nicotinamide adenine dinucleotide) 및 FAD(Flavin adenine dinucleotide)로 이루어진 군에서 선택되는 적어도 하나의 전자전달체일 수 있고, 상기 알킬 바이올로젠(alkyl viologen)은 메틸 바이올로젠(methyl viologen), 에틸 바이올로젠(ethyl viologen), 프로필 바이올로젠(propyl viologen) 등을 포함할 수 있다.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.
구체적으로, 상기 전자전달체는 메틸 바이올로젠(Methyl viologen), 에틸 바이올로젠(Ethyl viologen), 벤질 바이올로젠(Benzyl viologen), NAD(nicotinamide adenine dinucleotide) 및 FAD(Flavin adenine dinucleotide)로 이루어진 군에서 선택되는 적어도 하나의 전자전달체일 수 있고, 보다 구체적으로, 메틸 바이올로젠(methyl viologen) 및 에틸 바이올로젠(ethyl viologen)로 이루어진 군에서 선택되는 적어도 하나의 전자전달체일 수 있다.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.
일 양상에 있어서, 상기 접촉하는 단계는 전기화학반응 시스템 내에서 이루어지는 것일 수 있다.In one aspect, the contacting may be performed in an electrochemical reaction system.
상기 전기화학반응 시스템은 전술한 범위 내일 수 있고, 그 중 상기 전기 화학적 이산화탄소 환원 시스템은 도 9에 도식된 바와 같이, 예를 들어, 구리, 흑연(graphite), 탄소 펠트(carbon felt), 및 탄소 섬유와 같은 음극 및 백금과 같은 양극을 포함하고, Ag/AgCl와 같은 기준 전극을 더 포함할 수 있다. 그러나 상기 전기 화학적 이산화탄소 환원 시스템은 필요에 따라 통상의 기술자에게 잘 알려진 방식으로 얼마든지 변형 가능하다.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.
또한, 일 양상에 있어서, 상기 이산화탄소를 포함하는 기체는 산소를 포함하는 것일 수 있다.Also, in one aspect, the gas including carbon dioxide may include oxygen.
전술한 바와 같이, 일반적인 미생물 또는 효소와 달리 일 양상에 따른 히스티딘 표지(His-tag)된 포름산 탈수소효소(Formate Dehydrogenase)는 산소 또는 용존 산소 농도에 따라 효소 활성이 저하되지 않을 수 있어, 이산화탄소로부터 개미산을 고효율 및 고수율로 환원시켜 제조할 수 있다.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.
일 양상에 따라 유전적으로 재조합된 히스티딘 표지된 포름산 탈수소효소는 이산화탄소를 환원시켜 개미산을 효율적으로 높은 수율로 제조할 수 있으며, 나아가, 상기 포름산 탈수소효소를 고정화시키는 경우, 전기화학반응기에서 장기간 동안 효율이 떨어지지 않고 고농도의 개미산 제조가 가능하고, 재사용이 가능할 뿐만 아니라, 용존 산소 농도에도 크게 영향을 받지 않아, 이산화탄소 제거 또는 개미산 제조 분야의 산업에 크게 이바지할 수 있다.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.
도 1은 FDH1-his tag 생산용 knock out 균주의 준비 과정을 나타낸 도이다.1 is a diagram showing the preparation process of a knock-out strain for FDH1-his tag production.
도 2는 Ni-NTA agarose를 이용하여 정제한 FDH1-his tag 효소의 SDS PAGE 결과를 나타낸 도이다(S: lysate; FT: flow-through of Ni-NTA resin; UW: unbounded washing, E1: primary elute of FDH1 E2: secondary elute of FDH1).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).
도 3은 Ni-NTA agarose bead에 고정화된 FDH1-his tag를 나타낸 도이다.3 is a view showing the FDH1-his tag immobilized on Ni-NTA agarose bead.
도 4는 환원된 EV 전자전달체의 농도를 측정하기 위한 흡광 계수 측정 표준 곡선을 나타낸 도이다.4 is a diagram showing a standard curve for measuring an extinction coefficient for measuring the concentration of a reduced EV electron transporter.
도 5a 내지 5c는 이산화탄소 환원 반응에 대한 Lineweaver-Burk plot을 나타낸 도이다.5a to 5c are diagrams showing Lineweaver-Burk plots for the carbon dioxide reduction reaction.
도 6은 효소들의 이산화탄소 환원 및 개미산 산화 반응속도 비교한 도이다.6 is a diagram comparing the rate of carbon dioxide reduction and formic acid oxidation of enzymes.
도 7은 효소별 이산화탄소 환원 및 개미산 산화 소모 속도 비율 상수(Keq)를 나타낸 도이다.7 is a diagram showing the rate constant (K eq ) of carbon dioxide reduction and formic acid oxidation consumption rate by enzyme.
도 8은 pH에 따른 FDH1-his tag의 CO2 환원 및 formate 산화 반응의 속도 변화를 나타낸 도이다(CO2 환원 (closed symbols), formate 산화 (open symbols)).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)).
도 9는 FDH1-his tag 효소를 이용하여 이산화탄소를 환원시켜 개미산을 생산하기 위한 전기화학반응기 모식도를 나타낸 도이다.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.
도 10은 전압 변화에 따른 개미산 생산속도 및 패러데이 효율을 나타낸 도이다(점과 선: 개미산 생산성, 막대: 패러데이 효율).10 is a diagram showing formic acid production rate and Faraday efficiency according to voltage change (dots and lines: formic acid productivity, bar: Faraday efficiency).
도 11은 장기간 운전에 따른 개미산 생산 결과를 나타낸 도이다.11 is a view showing the results of formic acid production according to long-term operation.
도 12는 고정화된 효소의 반복적인 재사용 가능성을 입증한 도이다.12 is a diagram demonstrating the repeatability of the immobilized enzyme.
도 13은 용존 산소에 따른 효소의 잔류 활성 및 이를 통한 효소의 용존 산소에 대한 저항성을 추정한 도이다.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.
도 14는 산소 포기에도 불구하고 개미산 생산 가능함을 나타낸 도이다.Figure 14 is a diagram showing that it is possible to produce formic acid despite the abandonment of oxygen.
이하 본 발명을 실시예를 통하여 보다 상세하게 설명한다. 그러나, 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다. 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.
실시예Example
실시예 1. FDH1-His tag 재조합 효소의 제조Example 1. Preparation of FDH1-His tag recombinant enzyme
재조합 미생물을 제조하기 위해, 메틸로박테리움 엑스토르쿠엔스 AM1(ATCC 14781, GenBank accession No. CP001510.1)를 이용하여 하기 도 1에 도시된 바에 따라 클로닝하여 변형시켰다. 메틸로박테리움 엑스토르쿠엔스 AM1은 3개의 암호화된 개미산 탈수소 효소 유전자 (fdh1, fdh2, fdh3)를 보유하고 있으며, 3 개의 개미산 탈수소 효소 유전자 중에서, FDH1에 대한 fdh1 (GenBank accession No. ACS42636.1(α-subunit), ACS42635.1(β-subunit)) 유전자는 개미산의 전세포 산화과정 중에 주요한 역할을 하는 것으로 알려져 메틸로박테리움 엑스토르쿠엔스 AM1의 재조합 미생물을 제작하기 위해 선택되었다.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.
하기의 클로닝에는 모두 one-step SLIC(sequence and ligation-independent cloning)을 적용하였다. SLIC은 T4 DNA 중합 효소를 엑소뉴클레아제로 사용한다. 벡터를 제한효소 및 DNA 증폭기에 의해서 선형화 및 증폭시키고, NEB 2.1 버퍼 (B7202S, BioLabs) 및 T4 중합효소를 첨가하여 실온에서 2.5분 동안 인큐베이션한 다음, 즉시 10 분간 얼음에 인큐베이션하였다. 이후 상기 혼합물 1 ㎕를 100㎕의 수용성 대장균 DH5α (RBC)에 첨가하고, 상기 DH5α 대장균을 20 분간 얼음에서 인큐베이션하였다. 그런 다음 950㎕의 LB 배지를 추가하여, 37℃에서 16 시간 동안 배양하였다.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.
구체적으로 재조합 미생물을 제조하기 위해, 먼저 결실시키고자 하는 유전자에 따라 메틸로박테리움 엑스토르쿠엔스 AM1의 FDH1α, 및/또는 FDH1β 유전자(GenBank accession No. ACS42636.1(α-subunit), ACS42635.1(β-subunit))의 앞 부분과 뒷 부분에 위치한 DNA를 증폭시켰다. 클로닝에 사용된 프라이머는 하기 표 1과 같았다.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.
| 프라이머primer | 서열order | 서열번호SEQ ID NO: |
| fdh1α 녹아웃 upstream Ffdh1α knockout upstream F |
5'-gccgccatatgcatccatggtacc CCGGCGGGTCGATGCGGTTGGAAA-3'5'-gccgccatatgcatccatggtacc CCGGCGGGTCGATGCGGTTGGAAA-3' |
1One |
| fdh1α 녹아웃 upstream Rfdh1α knockout upstream R |
5'-cacctgacgtctagatctgaattc TGGCCCGCGACCTCACCGCGAACTACTT-3'5'-cacctgacgtctagatctgaattc TGGCCCGCGACCTCACCGCGAACTACTT-3' |
22 |
| fdh1α 녹아웃 downstream Ffdh1α knockout downstream F |
5'-tggtcggctggatcctctagtgagctc TCTACGCCGAGGGCGTGAACGGACC-3'5'-tggtcggctggatcctctagtgagctc TCTACGCCGAGGGCGTGAACGGACC-3' |
33 |
| fdh1α 녹아웃 downstream Rfdh1α knockout downstream R |
5'-gatccagcttatcgataccgcgggccc GAGGTGCCGATAGGCGTGGCGCGA-3'5'-gatccagcttatcgataccgcgggccc GAGGTGCCGATAGGCGTGGCGCGA-3' |
44 |
| fdh1β 녹아웃 upstream Ffdh1β knockout upstream F |
5'-gccgccatatgcatccatggtacc AATCTCTGTGTCCGCGCCT-3'5'-gccgccatatgcatccatggtacc AATCTCTGTGTCCGCGCCT-3' |
55 |
| fdh1β 녹아웃 upstream Rfdh1β knockout upstream R |
5'-cacctgacgtctagatctgaattc GCTTCACCGCGTTCTTGAGGAA-3'5'-cacctgacgtctagatctgaattc GCTTCACCGCGTTCTTGAGGAA-3' |
66 |
| fdh1β 녹아웃 downstream Ffdh1β knockout downstream F |
5'-tggtcggctggatcctctagtgagctc GGCAGAGGTCTCGCCGTTGT-3'5'-tggtcggctggatcctctagtgagctc GGCAGAGGTCTCGCCGTTGT-3' |
77 |
| fdh1β 녹아웃 downstream Rfdh1β knockout downstream R |
5'-gatccagcttatcgataccgcgggccc GACGCGACCTGTGTTCCAACTAA-3'5'-gatccagcttatcgataccgcgggccc GACGCGACCTGTGTTCCAACTAA-3' |
88 |
상기 증폭된 DNA를 pC184(Addgene plasmid 46012)의 loxP 및 카나마이신 유전자의 양측에 삽입하여 클로닝하였다. 상기 클로닝된 pC184를 메틸로박테리움 엑스토르쿠엔스 AM1에 형질전환시켰다. 메틸로박테리움 엑스토르쿠엔스 AM1은 pCM184로 형질전환되었을 때, 대립 유전자 교환을 일으켜 loxP와 카나마이신 유전자를 획득하지만 FDH1의 유전자 서열을 부분적으로 상실하게 된다. 상기 메틸로박테리움 엑스토르쿠엔스 AM1에 pCM157(Addgene plasmid 45863)을 형질전환시키고, pCM157에서 발현된 cre 재조합 효소에 의해서 loxP부위 사이의 카나마이신 유전자를 부위 특이적 재조합 (site-specific recombination)으로 추출하여 녹-아웃된 미생물을 생산하였다. 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.
이후 필요에 따라, 상기 녹-아웃된 미생물에 재조합 플라스미드를 발현시켰다. 구체적으로, 상기 특정 유전자가 녹-아웃된 메틸로박테리움 엑스토르쿠엔스 AM1에 FDH1 유전자를 포함하는 pCM110(서열번호 9)으로 형질전환시켜 FDH1 또는 FDH1α의 발현을 다시 회복시켰다.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.
하기의 표 1은 박테리아 균주 및 녹-아웃 또는 재조합 발현을 위한 플라스미드를 나타낸 것이다. Table 1 below shows bacterial strains and plasmids for knock-out or recombinant expression.
| Strain/PlasmidStrain/Plasmid | Relevant propertiesRelevant properties |
| Strainsstrains | |
| M. extorquens AM1 M. extorquens AM1 | Rifamycin resistance, wild typeRifamycin resistance, wild type |
| M. extorquens AM1 (△fdh1α) M. extorquens AM1 ( Δfdh1α ) | △fdh1α M. extorquens AM1 △fdh1α M. extorquens AM1 |
| M. extorquens AM1 (△fdh1α/β) M. extorquens AM1 ( Δfdh1α/ β) | △fdh1α/β M. extorquens AM1 △fdh1α/ β M. extorquens AM1 |
| PlasmidsPlasmids | |
| pCM184pCM184 | Allelic exchange vector (AmpR, TetR, KanR)Allelic exchange vector (Amp R , Tet R , Kan R ) |
| pCM184-△fdh1α pCM184 -Δfdh1α | pCM184 for fdh1α knockoutpCM184 for fdh1α knockout |
| pCM184-△fdh1βpCM184- △fdh1 β | pCM184 for fdh1β knockoutpCM184 for fdh1 β knockout |
| pCM157pCM157 | Cre recombinase expression vector (TetR) Cre recombinase expression vector (Tet R ) |
| pCM110pCM110 | Recombinant expression vector (TetR), P mxaF promoterRecombinant expression vector (Tet R ), P mxaF promoter |
| pCM110-MeFDH1-HispCM110-MeFDH1-His | MeFDH1 homologous expression vector, containing C-terminus His tag of alpha subunitMeFDH1 homologous expression vector, containing C-terminus His tag of alpha subunit |
상기 미생물들의 기본적인 배양 배지는 탄소공급원으로 16 g/L 숙신산 및 최소염 배지(minimal salt medium) (1.62 g/L NH4Cl, 0.2 g/L MgSO4, 2.21 g/L K2HPO4, 및 1.25 g/L NaH2PO4·2H2O)를 포함한다. 재조합 미생물을 선별하기 위한 선택적 항생제로는 50 ㎍/mL 리파미신 (Rif), 50 ㎍/mL 카나미신 (Kan), 또는 10 ㎍/mL 테트라시클린 (Tet)을 사용하였다. 상기 미생물들은 모두 1L 에를렌마이어 쉐이크 플라스크 중에 200 mL의 부피로 하여 26℃, 200 rpm에서 배양하였다. 세포 농도가 OD600 기준으로 0.5에서 0.8이 될 때 0.5% 메탄올을 첨가하여 목표로 하는 FDH1-his tag 효소를 발현하였다. 세포는 총 48시간을 배양하였으며, 7,000 rpm 15분간 원심분리하여 배양된 세포를 획득하였다. 이후 이를 세포파쇄기를 이용하여 분쇄한 후 4,611 g 30분간 원심분리하여 상등액을 회수한 후 이를 Ni-NTA agarose bead (Bio-Rad, Cat No. 7321010)을 이용하여 FDH1-his tag(FDH1 alpha subunit-His-tag의 아미노산 서열: 서열번호 10; FDH1 beta subunit: 서열번호 11)만을 선택적으로 고정화시킨다. 고정화시킨 후 imidazole 용액을 이용하여 탈리 회수한 FDH1-hist tag 효소의 SDS PAGE를 이용하여 확인한 결과 상당히 고농도의 효소가 잘 분리정제되어 생산된 것으로 나타났다(도 2). 구체적으로, Ni-NTA agarose bead에 고정화된 효소의 구조는 하기 도 3과 같았다.The basic culture medium of the microorganisms is 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 above 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. Thereafter, the supernatant was recovered by centrifugation at 4,611 g for 30 minutes after pulverization using a cell disrupter. Then, the supernatant was recovered using Ni-NTA agarose beads (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.
실시예 2. 재조합 효소의 이산화탄소의 환원 및 개미산 산화 반응 속도 측정Example 2. Reduction of carbon dioxide and formic acid oxidation reaction rate measurement of recombinant enzymes
재조합 FDH1-his tag 효소의 이산화탄소 환원 및 개미산 산화 반응 속도는 ethyl viologen(EV)을 전자전달체로 이용하여 측정되었다. 환원된 형태의 ethyl viologen의 농도는 가시광선흡광계를 이용하여 600 nm에서 측정되었으며, 흡광계수는 10.220 mM-1cm-1로 결정되었다(도 4). 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).
이산화탄소 환원반응은 50 mM potassium phosphate 버퍼 용액 (pH 6.3)에 3 ~ 100 mM potassium bicarbonate, 0.03 ~ 0.12 mM 환원된 EV를 투입한다. 이 때 potassium bicarbonate가 일정부분 이산화탄소와 평형을 이루기 때문에 이산화탄소 공급원이 된다. 개미산 산화반응은 50 mM potassium phosphate 버퍼 용액(pH 6.3)에 0.5 ~ 30 mM potassium formate, 0.5 ~ 4 mM 산화된 상태의 EV를 투입한다. 모든 반응은 10 ㎕의 순수하게 정제된 FDH1-his tag 효소를 투입하여 30℃에서 1분 간 반응을 진행하고 이 때, 변화하는 환원된 형태의 EV 농도를 가시광선흡광계를 이용하여 측정하였으며, 이 결과를 이용하여 Lineweaver-Burk plot을 그리고 이로부터 효소 반응속도 상수들을 추정하였으며, 다른 알려진 비교 효소들에 대해서도 동일하게 실험을 진행하여 진행하였다(도 5a 내지 5c 및 도 6). 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 input. For all reactions, 10 μl of purely purified FDH1-his tag enzyme was added and the reaction was performed 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. 5A to 5C and 6 ).
그 결과, MeFDH1(≡FDH1-his tag)은 이산화탄소를 환원하는 반응속도가 개미산을 산화소모하는 반응속도에 비하여 58배 정도 빠르나 RcFDH는 5.3배로 약 1/10 수준이며, ReFDH, TsFDH, CbFDH 등은 도리어 개미산을 산화소모하는 반응속도가 훨씬 빨라 개미산 생성 농도가 극히 낮았다(도 6 및 7). 이로 미루어 보면 현재까지 보고된 어떠한 효소에 비해서도 이산화탄소 환원을 통하여 개미산을 고농도로 고속으로 효율적으로 생산할 수 있을 것으로 예측되었다. 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.
실시예 3. FDH1-his tag 재조합 효소의 pH 의존도 및 개미산 생성을 위한 최적의 pH 결정Example 3. Determination of optimum pH for pH dependence and formic acid production of FDH1-his tag recombinant enzyme
pH 5.5 ~ pH 6.1 부분의 pH 영역은 200 mM의 citrate/citric acid buffer을 이용하였으며, 그 외 pH는 200 mM의 potassium phosphate buffer를 이용하였으며, 30℃에서 1분 간 효소 활성을 측정하였다. 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.
그 결과, CO2 환원을 통하여 개미산을 생산하기에 적절한 pH 6.3 부근이며 pH가 그 이상 및 그 이하에서는 상당히 감소하는 것을 볼 수 있으며, pH가 증가하여 pH 7 에서 개미산 산화 소모 속도가 최대가 되는 것을 알 수 있었다(도 8). 따라서 개미산을 생산하기 가장 좋은 pH는 6.3임을 알 수 있었다. 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.
실시예 4. 전기화학반응기를 이용한 이산화탄소 환원을 통한 개미산 생산Example 4. Formic acid production through carbon dioxide reduction using an electrochemical reactor
본 실시예에서 사용된 전기화학반응기 모식도는 하기 도 9와 같았다. 전기화학반응기는 음극 부분(30 mL)에는 carbon felt 전극 (2 X 1.5 cm), 기준 전극(Ag/AgCl, MF-2079, BASi)이 200 mM-potassium phosphate (pH 7), 10 mM의 ethyl viologen이 포함된 용액에 설치되어 있고, 양극 부분에는 물 분해 반응을 위한 Pt wire가 장착되었으며, 100 mM의 H2SO4 용액으로 채워져 있다. 양극에서 생성된 양성자의 이송을 위하여 양성자 선택성 막 (Nafion® 115 membrane, 0.005 inch)이 설치되었다. 반응을 위하여 60 U FDH1-his tag 효소가 음극 부분에 투입되었으며, 이산화탄소를 연속적으로 공급하였다. 전압은 기준 전극(수소 전극, Reversible hydrogen electrode) 대비 - 0.01 ~ - 0.36 V까지 변화시키면서 일정하게 유지하면서 반응을 시키면서 소모되는 전류량을 연속적으로 측정하였다. 또한 일정기간마다 cathode 부분에서 샘플을 취하여 HPLC을 이용하여 생성된 개미산 농도를 측정하였다.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.
전압을 올려가면서 생성되는 개미산의 생산속도 및 패러데이 효율을 측정한 결과, 도 10과 같았다. 과전압이 거의 없는 상태에서도 개미산 생산속도가 3 mM/hr 수준으로 나타난 것을 볼 때 효소가 활성화에너지 장벽없이 매우 효율적으로 개미산을 합성한다는 것을 알 수 있었다. 또한, 전압을 증가시킬 경우 비례하여 개미산 생산성이 증가하지만 - 0.2 V 이상에서는 더 증가하지는 않아서 다른 요인이 제한 인자인 것을 알 수 있다. 모든 경우에 개미산 생성 패러데이 효율은 거의 100 % 인 바, 소모된 전자는 모두 완벽하게 오직 개미산 합성에만 사용되었다는 것을 알 수 있었다.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.
실시예 5. 전기화학반응기를 이용한 이산화탄소 환원에 의한 개미산 생산 장기 운전 시험Example 5. Long-term operation test for formic acid production by carbon dioxide reduction using an electrochemical reactor
고정화된 효소와 free 용해 상태의 FDH1-his tag 효소를 이용하여 실시예 4에서 정해진 조건하에서 개미산 생산 실험을 장기 운전을 실시하였으며 그 결과를 도 11에 나타내었다. 투입된 전압은 - 0.164 V vs. RHE 였으며 생산된 개미산의 농도가 높아질 경우 버퍼의 중화 능력을 초과하기 때문에 pH 조절 장치를 이용하여 6 M KOH를 투입하여 반응 도중에 pH 6.4 를 유지하도록 하였다. 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.
그 결과, 고정화하지 않은 FDH1-his tag 재조합효소의 경우 50시간 반응 후 600 mM 개미산 생성 이후 더 이상 개미산을 생성하지 못하는 데 반하여, Ni-NTA agarose bead에 고정화된 효소의 경우(도 3) 200시간 이후까지 지속적으로 개미산이 생산되어 약 1.7 M 개미산이 생산됨을 볼 수 있으며, 장기간의 운전에도 불구하고 패러데이 효율은 100 % 수준을 유지하는 것을 볼 수 있었다(도 11). 이는 고정화되지 않은 효소의 경우 이산화탄소 포기에 따른 전단응력 등의 영향으로 효소의 활성이 실활되었기 때문으로 추정되며, 반면 고정화된 효소의 경우 이러한 기체 포기에 의한 전단 응력에 내성이 있는 것으로 파악된다. 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.
실시예 6. 고정화된 효소를 이용한 재사용 성능 입증Example 6. Demonstration of reuse performance using immobilized enzyme
고정화된 효소를 이용하여 재사용 성능을 입증하기 위해, 220 시간 반응 후 회수된 Ni-NTA agarose bead에 고정화된 효소를 이용하여 반복적으로 개미산 생산성을 측정하였다.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.
그 결과, 3회 반복된 실험에서도 효소의 개미산 생산성이 감소되는 것이 관찰되지 않았음을 알 수 있다(도 12). 이로 미루어보아 고정화된 효소의 안정성이 크게 증가된 것을 확인할 수 있었으며 이는 향후 경제적으로 개미산을 이산화탄소로부터 생산할 수 있음을 알 수 있었다.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.
실시예 7. 용존산소농도 변화에 따른 잔류 활성 변화 및 공기 주입에 따른 효소 고정화된 효소의 안정성 시험Example 7. Residual activity change according to dissolved oxygen concentration change and stability test of enzyme immobilized enzyme according to air injection
용존 산소 농도에 따른 효소의 잔류 활성을 측정하였다.The residual activity of the enzyme according to the dissolved oxygen concentration was measured.
그 결과, 대기중의 산소와 장시간 노출할 경우 효소의 잔류 활성이 감소하는 것을 알 수 있으나, 일반적으로 이산화탄소가 포집된 수준에 미량 포함되는 수준의 산소에는 저항성을 가지고 전혀 잔류 활성이 감소되지 않는 것을 알 수 있었다(도 13). 이로 미루어 일정부분 산소가 포함된 이산화탄소를 기질로 이용하여 개미산을 원활하게 생산 가능하다는 것을 알 수 있었다.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.
또한 실시예 6의 조건하에서 반응 중간에 임의로 공기가 포기되어 주입된 경우를 가정할 때 개미산 생산에 미치는 영향을 확인하였다.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.
그 결과, 순간적인 공기 주입은 개미산 생산을 멈추게 하지만 공기 주입을 멈추고 다시 이산화탄소를 주입할 경우 개미산이 지속적으로 생산되는 것을 확인할 수 있었다(도 14). 이로 미루어 볼 때 단기간에 주입되는 공기는 고정화된 효소의 활성을 완전하게 저해하지 못하기 때문에 효소는 지속적으로 이에 저항하면서 개미산을 생산할 수 있음을 알 수 있었다.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.
Claims (22)
- 히스티딘 표지(His-tag)된 포름산 탈수소효소(Formate Dehydrogenase).Histidine-tagged formic acid dehydrogenase (Formate Dehydrogenase).
- 청구항 1에 있어서, 상기 포름산 탈수소효소는 메틸로박테리움 속 미생물로부터 유래된 것인, 포름산 탈수소효소.The method according to claim 1, wherein the formic acid dehydrogenase is derived from a microorganism of the genus Methylobacterium, formic acid dehydrogenase.
- 청구항 1에 있어서, 상기 포름산 탈수소효소는 메틸로박테리움 엑스토르쿠엔스(Methylobacterium extorquens) AM1으로부터 유래된 것인, 포름산 탈수소효소.The method according to claim 1, wherein the formic acid dehydrogenase is Methylobacterium extorquens ( Methylobacterium extorquens ) It is derived from AM1, formic acid dehydrogenase.
- 청구항 1에 있어서, 상기 포름산 탈수소효소는 유전자 재조합으로 제조된 것인, 포름산 탈수소효소.The method according to claim 1, wherein the formic acid dehydrogenase is produced by genetic recombination, formic acid dehydrogenase.
- 청구항 1에 있어서, 상기 포름산 탈수소효소는 메틸로박테리움 엑스토르쿠엔스(Methylobacterium extorquens) AM1 유래 FDH1(Formate Dehydrogenase 1)으로서,The method according to claim 1, wherein the formic acid dehydrogenase is Methylobacterium extorquens (Methylobacterium extorquens ) AM1-derived FDH1 (Formate Dehydrogenase 1) as,상기 메틸로박테리움 엑스토르쿠엔스 AM1은 FDH1α(Formate Dehydrogenase 1 alpha subunit)를 코딩하는 내인성 유전자 및 FDH1β(Formate Dehydrogenase 1 beta subunit)를 코딩하는 내인성 유전자가 결실되고,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,메틸로박테리움 엑스토르쿠엔스 AM1 유래 FDH1 및 히스티딘 표지(His-tag)를 코딩하는 외인성 유전자가 도입된 재조합 미생물인, 포름산 탈수소효소.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.
- 청구항 1에 있어서, 상기 포름산 탈수소효소는 서열번호 10 및 11의 아미노산 서열로 이루어진 폴리펩티드인, 포름산 탈수소효소.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.
- Ni-NTA(Nickel-Nitrilotriacetic Acid)에 고정화된 히스티딘 표지(His-tag)된 포름산 탈수소효소(Formate Dehydrogenase).Histidine-tagged Formate Dehydrogenase immobilized in Nickel-Nitrilotriacetic Acid (Ni-NTA).
- 청구항 7에 있어서, 상기 Ni-NTA는 Ni-NTA agarose bead인, 포름산 탈수소효소.The method according to claim 7, wherein the Ni-NTA is Ni-NTA agarose beads, formic acid dehydrogenase.
- 히스티딘 표지(His-tag)된 포름산 탈수소효소(Formate Dehydrogenase)를 포함하는 개미산 제조용 조성물.Histidine-labeled (His-tag) formic acid production composition comprising formic acid dehydrogenase (Formate Dehydrogenase).
- 청구항 9에 있어서, 상기 포름산 탈수소효소는 Ni-NTA(Nickel-Nitrilotriacetic Acid)에 고정화된 것인, 개미산 제조용 조성물.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.
- 청구항 9에 있어서, 상기 조성물의 pH는 5.0 내지 7.5인, 개미산 제조용 조성물.The method according to claim 9, wherein the pH of the composition is 5.0 to 7.5, formic acid preparation composition.
- 히스티딘 표지(His-tag)된 포름산 탈수소효소(Formate Dehydrogenase)를 포함하는 개미산 제조용 전기화학반응 시스템.Histidine-labeled (His-tag) formic acid production electrochemical reaction system comprising formic acid dehydrogenase (Formate Dehydrogenase).
- 청구항 12에 있어서, 상기 포름산 탈수소효소는 Ni-NTA(Nickel-Nitrilotriacetic Acid)에 고정화된 것인, 개미산 제조용 전기화학반응 시스템.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).
- 히스티딘 표지(His-tag)된 포름산 탈수소효소(Formate Dehydrogenase)를 포함하는 개미산 제조용 장치.Histidine-labeled (His-tag) formic acid production device comprising formic acid dehydrogenase (Formate Dehydrogenase).
- 청구항 14에 있어서, 상기 포름산 탈수소효소는 Ni-NTA(Nickel-Nitrilotriacetic Acid)에 고정화된 것인, 개미산 제조용 장치.The apparatus of claim 14, wherein the formic acid dehydrogenase is immobilized on Ni-NTA (Nickel-Nitrilotriacetic Acid).
- 히스티딘 표지(His-tag)된 포름산 탈수소효소(Formate Dehydrogenase)를 포함하는 이산화탄소 제거용 필터.A filter for removing carbon dioxide containing histidine-labeled formic acid dehydrogenase (Formate Dehydrogenase).
- 청구항 16에 있어서, 상기 포름산 탈수소효소는 Ni-NTA(Nickel-Nitrilotriacetic Acid)에 고정화된 것인, 이산화탄소 제거용 필터.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.
- 히스티딘 표지(His-tag)된 포름산 탈수소효소(Formate Dehydrogenase)에 이산화탄소를 포함하는 기체 및 전자전달체를 접촉시키는 단계를 포함하는 개미산의 제조 방법.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).
- 청구항 18에 있어서, 상기 포름산 탈수소효소는 Ni-NTA(Nickel-Nitrilotriacetic Acid)에 고정화된 것인, 개미산의 제조 방법.The method for producing formic acid according to claim 18, wherein the formic acid dehydrogenase is immobilized on Ni-NTA (Nickel-Nitrilotriacetic Acid).
- 청구항 18에 있어서, 상기 전자전달체는 바이올로젠(viologen)기를 갖는 화합물인, 개미산의 제조 방법.The method for producing formic acid according to claim 18, wherein the electron transporter is a compound having a viologen group.
- 청구항 18에 있어서, 상기 접촉하는 단계는 전기화학반응 시스템 내에서 이루어지는 것인, 개미산의 제조 방법.The method for producing formic acid according to claim 18, wherein the contacting step is made in an electrochemical reaction system.
- 청구항 18에 있어서, 상기 이산화탄소를 포함하는 기체는 산소를 포함하는 것인, 개미산의 제조 방법.The method for producing formic acid according to claim 18, wherein the gas containing carbon dioxide includes oxygen.
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| KR20180076288A (en) * | 2016-12-27 | 2018-07-05 | 한국과학기술원 | A recombinant microorganism introduced exotic genes and a method for producing useful product from carbon dioxide and formic acid using the recombinant microorganism |
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| KR20220141146A (en) | 2022-10-19 |
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