Classifications

• • 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
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/70—Vectors or expression systems specially adapted for E. coli
• • 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.)
  • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
  • C12N9/0083—Miscellaneous (1.14.99)
• • 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
  • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group

Definitions

• the present disclosure relates to a method for producing a phenolic compound and a modified enzyme.
• Vanillin which is used as a flavoring agent in food and beverages and cosmetics, and as an intermediate in pharmaceuticals, is supplied to the market in chemically synthesized products as well as extracts from plants of the genus Vanilla of the Orchidaceae family.
• SDGs natural products and SDGs
• vanillin produced without the use of petroleum or chemical reagents is increasing. Therefore, attempts to produce vanillin using microorganisms and enzymes have been proposed. For example, a method for synthesizing vanillin from ferulic acid obtained from agricultural wastes such as rice bran and wheat bran using an enzymatic reaction has been reported.
• Non-Patent Documents 1 and 2 describe a first enzymatic reaction that converts ferulic acid as a starting material into 4-vinylguaiacol as an intermediate, and a second enzymatic reaction that converts this intermediate into vanillin.
• a method for synthesizing vanillin through an enzymatic reaction has been reported.
• Non-Patent Document 3 states that vanillin is produced through a first enzymatic reaction that converts ferulic acid as a starting material into feruloyl-coenzyme A as an intermediate, and a second enzymatic reaction that converts this intermediate into vanillin.
• a method to synthesize has been reported.
• Non-Patent Document 4 reports a method of converting ferulic acid to vanillin using a plant-derived hydrolase belonging to the cysteine proteinase family.
• Non-patent document 1 J Am Chem Soc, 140, 16001-16005 (2016)
• Non-patent document 2 ChemBioChem, 15, 2248-2254 (2014)
• Non-patent document 3 Biotechnol Lett, 27, 1829-1832 (2005)
• Non-patent document 4 Nat Commun, 5, 4037 (2014)
• Non-Patent Document 4 only qualitatively shows the activity of converting ferulic acid to vanillin because the activity of the enzyme used is weak, and does not show quantitative results.
• the present disclosure aims to provide a novel method for producing a phenol compound that utilizes an enzymatic reaction, and a novel enzyme that can be used for producing the phenol compound.
• a method for producing a compound represented by the following formula (2) from a compound represented by the following formula (1) comprising: A step of bringing an enzyme into contact with a compound represented by formula (1), A method for producing a phenolic compound, wherein the enzyme has an activity of cleaving a carbon-carbon double bond of a compound represented by formula (1) (provided that the compound represented by formula (1) is ferulic acid and the enzyme has SEQ ID NO. (Except when the protein has the amino acid sequence shown in 1).
• R 1 represents a hydrogen atom or an alkyl group
• R 2 each independently represents an alkoxy group, a hydroxy group, an alkyl group, or a halogen atom
• n is a number from 0 to 4.
• the 82nd amino acid residue is an amino acid residue other than a phenylalanine residue.
• the 124th amino acid residue is a tyrosine residue.
• the 157th amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is an amino acid residue other than lysine residue (a4) 332 in the amino acid sequence shown in SEQ ID NO: 1
• the 333rd amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is an amino acid residue other than valine residue (a5)
• the 333rd amino acid residue is an amino acid residue other than phenylalanine residue (a6)
• SEQ ID NO: In the amino acid sequence shown in 1, the 334th amino acid residue is an amino acid residue other than phenylalanine residue (b)
• Amino acid sequence satisfying the condition selected from (a1) to (a6) and an amino acid sequence of 70% or more have identity (excluding the amino acid sequence shown in SEQ ID NO: 1) ⁇ 4>
• the enzyme is an amino acid sequence that satisfies the condition (a1) and at least one condition selected from (a2) to (a6), or a protein that has an amino acid sequence identity of 70% or more with the amino acid
• the method for producing a phenol compound according to ⁇ 3> is a method for producing a phenol compound according to ⁇ 3>.
• ⁇ 5> The method for producing a phenol compound according to ⁇ 3> or ⁇ 4>, wherein in (a1), the 82nd amino acid residue is a tyrosine residue or a tryptophan residue.
• ⁇ 6> In (a4), (a5) or (a6), at least one amino acid residue selected from position 332, 333, or 334 is a polar amino acid residue, ⁇ 3> to ⁇ 5> A method for producing a phenol compound according to any one of the above.
• ⁇ 7> The method for producing a phenol compound according to any one of ⁇ 1> to ⁇ 6>, wherein the compound represented by formula (1) is ferulic acid or a ferulic acid alkyl ester.
• ⁇ 8> The method for producing a phenol compound according to any one of ⁇ 1> to ⁇ 7>, wherein the compound represented by formula (2) is vanillin or ethylvanillin.
• a method for producing a compound represented by the following formula (4) from a compound represented by the following formula (3) comprising: A step of bringing an enzyme into contact with a compound represented by formula (3),
• the enzyme is a protein having an amino acid sequence that satisfies at least one condition selected from the following (a1) to (a6) and (b), and is capable of cleaving the carbon-carbon double bond of the compound represented by formula (3).
• the 82nd amino acid residue is an amino acid residue other than a phenylalanine residue.
• the 124th amino acid residue is a tyrosine residue.
• the 157th amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is an amino acid residue other than lysine residue (a4) 332 in the amino acid sequence shown in SEQ ID NO: 1
• the 333rd amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is an amino acid residue other than valine residue (a5)
• the 333rd amino acid residue is an amino acid residue other than phenylalanine residue (a6)
• SEQ ID NO: In the amino acid sequence shown in 1, the 334th amino acid residue is an amino acid residue other than phenylalanine residue (b)
• Amino acid sequence satisfying the condition selected from (a1) to (a6) and an amino acid sequence of 70% or more have identity (excluding the amino acid sequence shown in SEQ ID NO: 1) ⁇ 11>
• the modified enzyme according to ⁇ 10> for use in a method for producing a phenol compound.
• ⁇ 12> A composition comprising the modified enzyme according to ⁇ 10> or ⁇ 11>.
• ⁇ 13> A polynucleotide encoding the modified enzyme according to ⁇ 10>.
• ⁇ 14> An expression vector comprising the polynucleotide according to ⁇ 13>.
• ⁇ 15> A transformant containing the expression vector according to ⁇ 14>.
• a novel method for producing a phenol compound that utilizes an enzymatic reaction, and a novel enzyme that can be used for producing a phenol compound are provided.
• FIG. 3 is a diagram showing the absorption spectrum of the product obtained in Example 1.
• 2 is a graph showing the quantitative results of the product obtained in Example 1.
• 2 is a graph showing the quantitative results of the product obtained in Example 2.
• 3 is a graph showing the quantitative results of the product obtained in Example 3.
• 3 is a graph showing the quantitative results of the product obtained in Example 4.
• 3 is a graph showing the quantitative results of the product obtained in Example 5.
• 3 is a graph showing the quantitative results of the product obtained in Example 6.
• 3 is a graph showing the quantitative results of the product obtained in Example 7.
• 3 is a graph showing the quantitative results of the product obtained in Example 8.
• 3 is a graph showing the quantitative results of the product obtained in Example 9.
• 3 is a graph showing the quantitative results of the product obtained in Example 10.
• a first embodiment of the present disclosure is a method for producing a compound represented by the following formula (2) from a compound represented by the following formula (1), comprising: A step of bringing an enzyme into contact with a compound represented by formula (1), A method for producing a phenolic compound, wherein the enzyme has an activity of cleaving a carbon-carbon double bond of a compound represented by formula (1) (provided that the compound represented by formula (1) is ferulic acid and the enzyme has SEQ ID NO. 1).
• R 1 represents a hydrogen atom or an alkyl group
• R 2 each independently represents an alkoxy group, a hydroxy group, an alkyl group, or a halogen atom
• n is a number from 0 to 4.
• a compound represented by formula (2) for example, vanillin
• a compound represented by formula (1) for example, ferulic acid
• the production efficiency is superior to the method of producing the compound represented by formula (2) from the compound represented by formula (1) via the generation of an intermediate.
• the above method can be carried out without using coenzymes, it is advantageous in terms of reducing production costs.
• the enzyme having the activity of cleaving the carbon-carbon double bond of the compound represented by formula (1) will also be referred to as a "specific enzyme".
• the specific enzyme has an activity of cleaving the carbon-carbon double bond that connects the aromatic ring of the compound represented by formula (1) and COOR 1 in the presence of oxygen molecules (O 2 ). Specifically, the specific enzyme catalyzes the reaction shown below.
• the specific enzyme is preferably an oxidative cleavage enzyme whose substrate is a compound having a carbon-carbon double bond.
• Specific examples of the oxidative cleavage enzyme include carotenoid oxidative cleavage enzyme, stilbene oxidative cleavage enzyme, isoeugenol oxidative cleavage enzyme, and 4-vinylguaiacol oxidative cleavage enzyme.
• the specific enzyme may be an enzyme derived from a natural product, or may be an artificially modified or synthesized enzyme.
• the specific enzyme is a protein having an amino acid sequence that satisfies at least one condition selected from (a1) to (a6) and (b) below.
• the 82nd amino acid residue is an amino acid residue other than a phenylalanine residue.
• the 124th amino acid residue is a tyrosine residue.
• the 157th amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is an amino acid residue other than lysine residue (a4) 332 in the amino acid sequence shown in SEQ ID NO: 1
• the 333rd amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is an amino acid residue other than valine residue (a5)
• the 333rd amino acid residue is an amino acid residue other than phenylalanine residue (a6)
• SEQ ID NO: In the amino acid sequence shown in 1, the 334th amino acid residue is an amino acid residue other than phenylalanine residue (b) Amino acid sequence satisfying the condition selected from (a1) to (a6) and an amino acid sequence of 70% or more have identity (excluding the amino acid sequence shown in SEQ ID NO: 1)
• Non-Patent Document 1 describes that Ado exhibits the activity of converting 4-vinylguaiacol or isoeugenol, which is an intermediate obtained by converting the starting material ferulic acid, into vanillin.
• Ado exhibits the activity of converting 4-vinylguaiacol or isoeugenol, which is an intermediate obtained by converting the starting material ferulic acid, into vanillin.
• no case has been reported to date in which a starting material is directly converted to vanillin using Ado.
• Ado hardly exhibits any activity to convert ferulic acid into vanillin.
• the present inventors applied an enzyme obtained by converting an amino acid residue at a specific position of Ado to a different amino acid residue to a method for converting a compound represented by formula (1) into a compound represented by formula (2).
• a method for converting a compound represented by formula (1) into a compound represented by formula (2) As a result, it was found that the activity of converting the compound represented by formula (1) of Ado into vanillin was increased.
• the reason for this is, for example, that the amino acid residue converted at a specific position of Ado functions as an active center involved in the cleavage of the carbon-carbon double bond contained in the phenol compound.
• the enzyme obtained by converting the amino acid residue at a specific position of Ado to a different amino acid residue exhibits the activity of converting the compound represented by formula (1) into vanillin even if it is ferulic acid. did.
• the specific enzyme is a protein in which at least the 82nd, 124th, 157th, 332nd, 333rd, or 334th amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 has been changed.
• the number of amino acid residues to be converted is not particularly limited as long as it is one or more. From the viewpoint of conversion efficiency of the compound represented by formula (1), the number of amino acid residues to be converted in the amino acid sequence represented by SEQ ID NO: 1 is preferably 100 or less, and preferably 50 or less. More preferably, the number is 20 or less, even more preferably 10 or less.
• the specific enzyme has at least one amino acid residue among the 82nd and 124th, 157th, 332nd, 333rd, or 334th amino acid residues converted in the amino acid sequence shown in SEQ ID NO: 1. It may be a protein. In one embodiment, the specific enzyme may be a protein in which at least one amino acid residue among the 82nd, 332nd, 333rd, or 334th amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 has been changed. .
• the type of amino acid residue that replaces the 82nd amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is not particularly limited.
• the 82nd amino group residue is preferably an aromatic amino acid residue, more preferably a tyrosine residue or a tryptophan residue.
• the type of amino acid residue that replaces the 332nd, 333rd, or 334th amino acid residue in the amino acid sequence shown by SEQ ID NO: 1 is not particularly limited. From the viewpoint of conversion efficiency of the compound represented by formula (1), at least one amino acid residue selected from positions 332, 333, and 334 is a polar amino acid (basic amino acid, hydroxy amino acid, etc.) residue. is preferable, a basic amino acid (arginine, lysine or histidine) residue is more preferable, and an arginine residue is even more preferable.
• Specific examples of the specific enzyme include a protein having the amino acid sequence of SEQ ID NO: 2 in which the 82nd amino group residue in the amino acid sequence of SEQ ID NO: 1 is tyrosine residue Y (hereinafter also referred to as Ado-F82Y); A protein having the amino acid sequence of SEQ ID NO: 3 in which the 82nd amino group residue in the amino acid sequence of SEQ ID NO: 1 is tryptophan residue W (hereinafter also referred to as Ado-F82W); In the amino acid sequence of SEQ ID NO: 1, the 82nd amino acid residue is a tyrosine residue Y, and the 332nd amino acid residue is an amino acid residue X other than a valine residue, or the 333rd amino acid residue is A protein having the amino acid sequence of SEQ ID NO: 4 in which the amino acid residue X is other than the phenylalanine residue, or the 334th amino acid residue is an amino acid residue other than the phenylalanine residue (hereinafter referred to
• SEQ ID NO: 4 Amino acid sequence of Ado-F82Y-V332X-F333X-F334X
• the amino acid sequence identity with the amino acid sequence satisfying the conditions selected from (a1) to (a6) may be 70% or more, 75% or more, 80% or more , 85% or more, 85% or more, or 90% or more.
• the amino acid sequence identity with the amino acid sequence satisfying the conditions selected from (a1) to (a6) may be 70% or more, 75% or more, 80% or more , 85% or more, 85% or more, or 90% or more.
• the amino acid sequence identity of the protein (b) is determined such that the number of amino acid residues that match the amino acid sequence that satisfies the conditions selected from (a1) to (a6) is the largest. It is expressed as a percentage of the value obtained by dividing the total number of amino acid residues whose sequences match when aligned by the total number of amino acid residues in the amino acid sequence of (b).
• Amino acid sequence identity searches can be performed using search programs well known to those skilled in the art, such as BLAST (Basic Local Alignment Search Tool).
• the total number of amino acid residues of the specific enzyme may be the same as or different from the total number (603) of the amino acid sequence of SEQ ID NO: 1.
• the total number Y of amino acid residues of the specific enzyme may satisfy the following formula.
• Z1 and Z2 are each independently a number from 0 to 10, preferably a number from 0 to 5, and more preferably a number from 0 to 2. 603-Z1 ⁇ Y ⁇ 603+Z2
• R 1 represents a hydrogen atom or an alkyl group
• R 2 each independently represents an alkoxy group, a hydroxy group, an alkyl group, or a halogen atom
• n is a number from 0 to 4.
• R 1 is preferably a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, more preferably a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, and preferably a hydrogen atom, a methyl group, or an ethyl group. More preferred.
• R 2 is each independently preferably an alkoxy group having 1 to 5 carbon atoms, a hydroxy group, an alkyl group having 1 to 5 carbon atoms, or a halogen atom, and more preferably an alkoxy group having 1 to 3 carbon atoms. , methoxy group or ethoxy group is more preferable.
• n is preferably a number from 0 to 2, and more preferably 1. When n is a number of 1 or more, it is preferable that at least one of R 2 is at the ortho position with respect to the phenolic hydroxyl group.
• R 2 each independently represents an alkoxy group, a hydroxy group, an alkyl group, or a halogen atom
• n is a number from 0 to 4.
• R 2 is each independently preferably an alkoxy group having 1 to 5 carbon atoms, a hydroxy group, an alkyl group having 1 to 5 carbon atoms, or a halogen atom, and more preferably an alkoxy group having 1 to 3 carbon atoms.
• methoxy group or ethoxy group is more preferable.
• n is preferably a number from 0 to 2, and more preferably 1. When n is a number of 1 or more, it is preferable that at least one of R 2 is at the ortho position with respect to the phenolic hydroxyl group.
• Specific examples of the compound represented by formula (1) include ferulic acid (R 1 is a hydrogen atom, R 2 is a methoxy group, n is 1, and R 2 is in the ortho position to the phenolic hydroxyl group); ), ferulic acid alkyl esters (compounds in which R 1 is an alkyl group, R 2 is a methoxy group, n is 1, and R 2 is in the ortho position to the phenolic hydroxyl group), etc. It will be done.
• the compound represented by formula (2) examples include vanillin (a compound in which R 2 is a methoxy group, n is 1, and R 2 is in the ortho position with respect to the phenolic hydroxyl group), ethyl vanillin (R Examples include compounds in which 2 is an ethoxy group, n is 1, and R 2 is at the ortho position with respect to the phenolic hydroxyl group.
• vanillin is obtained as the compound represented by formula (2).
• the number of enzymes used in the method of this embodiment may be one or two or more.
• the number of compounds represented by formula (1) used in the method of this embodiment may be one or two or more.
• the method of carrying out the step of bringing an enzyme into contact with the compound represented by formula (1) is not particularly limited.
• the reaction may be carried out in a reaction solution containing the compound represented by formula (1), an enzyme, and an aqueous medium.
• Conditions for carrying out the step of bringing an enzyme into contact with the compound represented by formula (1) are not particularly limited.
• the temperature of the reaction solution may be selected from the range of 10°C to 90°C
• the pH of the reaction solution may be selected from the range of 2 to 13
• the reaction time may be selected from the range of 1 minute to 1 month. You may choose.
• the enzyme used in the method of this embodiment may be in the form of a transformant that expresses the enzyme.
• a second embodiment of the present disclosure is a method for producing a compound represented by the following formula (4) from a compound represented by the following formula (3), comprising: A step of bringing an enzyme into contact with a compound represented by formula (3),
• the enzyme is a protein having an amino acid sequence that satisfies at least one condition selected from the above-mentioned (a1) to (a6) and (b), and has a carbon-carbon double bond of the compound represented by formula (3).
• This is a method for producing a phenol compound having cleaving activity.
• R 1 represents a hydrogen atom, an alkyl group, or COOR 3
• R 2 each independently represents an alkoxy group, a hydroxy group, an alkyl group, or a halogen atom
• R 3 represents a hydrogen atom or an alkyl group
• n is It is a number from 0 to 4.
• a compound represented by formula (4) can be produced from a compound represented by formula (3). Furthermore, since the above method can be carried out without using coenzymes, it is advantageous in terms of reducing production costs.
• R 1 is a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, or COOR 3
• R 3 is preferably a hydrogen atom or an alkyl group having 1 to 5 carbon atoms
• 1 is a hydrogen atom, an alkyl group having 1 to 3 carbon atoms, or COOR 3
• R 3 is preferably a hydrogen atom or an alkyl group having 1 to 3 carbon atoms
• R 1 is a hydrogen atom, a methyl group, or an ethyl group.
• COOH, COOMe or COOEt are more preferred.
• R 2 is each independently preferably an alkoxy group having 1 to 5 carbon atoms, a hydroxy group, an alkyl group having 1 to 5 carbon atoms, or a halogen atom, and more preferably an alkoxy group having 1 to 3 carbon atoms. , methoxy group or ethoxy group is more preferable.
• n is preferably a number from 0 to 2, and more preferably 1. When n is a number of 1 or more, it is preferable that at least one of R 2 is at the ortho position with respect to the phenolic hydroxyl group.
• R 2 is preferably each independently an alkoxy group having 1 to 5 carbon atoms, a hydroxy group, an alkyl group having 1 to 5 carbon atoms, or a halogen atom; It is more preferable that it is an alkoxy group of 3, and even more preferable that it is a methoxy group or an ethoxy group.
• n is preferably a number from 0 to 2, and more preferably 1. When n is a number of 1 or more, it is preferable that at least one of R 2 is at the ortho position with respect to the phenolic hydroxyl group.
• Specific examples of the compound represented by formula (3) include ferulic acid (R 1 is COOR 3 , R 2 is a methoxy group, R 3 is a hydrogen atom, n is 1, and R 2 is ferulic acid alkyl ester (R 1 is COOR 3 , R 2 is a methoxy group, R 3 is an alkyl group, n is 1, R 2 is in the ortho position to the phenolic hydroxyl group), 4-vinylguaiacol (R 1 is a hydrogen atom, R 2 is a methoxy group, n is 1, and R 2 is in the ortho position to the phenolic hydroxyl group) (a compound in which R 1 is a methyl group, R 2 is a methoxy group, n is 1, and R 2 is in an ortho position with respect to a phenolic hydroxyl group), etc. It will be done.
• the compound represented by formula (4) include vanillin (a compound in which R 2 is a methoxy group, n is 1, and R 2 is in the ortho position with respect to the phenolic hydroxyl group), ethyl vanillin (R Examples include compounds in which 2 is an ethoxy group, n is 1, and R 2 is at the ortho position with respect to the phenolic hydroxyl group.
• the enzyme used in the second embodiment is an enzyme included in the specific enzymes used in the first embodiment, and has an amino acid sequence that satisfies the conditions selected from (a1) to (a6) and (b). It is a protein.
• the above enzyme has an activity of cleaving the carbon-carbon double bond connecting the aromatic ring of the compound represented by formula (3) and R 1 in the presence of oxygen molecules (O 2 ).
• the enzyme used in the second embodiment catalyzes the reaction shown below.
• the enzyme used in the method of the present embodiment has the activity of converting a compound in which R 1 is COOR 3 in formula (3) to a compound represented by formula (4), and in addition, in formula (3), R 1 is a hydrogen atom. Or, it has an activity of converting a compound that is an alkyl group into a compound represented by formula (4). Furthermore, the activity of the enzyme to convert the compound represented by formula (3) into the compound represented by formula (4) converts the compound represented by formula (3) of Ado into the compound represented by formula (4). superior to activity.
• the above enzymes are also useful in multi-step synthetic methods such as converting starting materials such as ferulic acid into intermediates such as 4-vinylguaiacol and isoeugenol, and then converting the intermediates into target substances such as vanillin. It is.
• the number of enzymes used in the method of this embodiment may be one or two or more.
• the number of compounds represented by formula (3) used in the method of this embodiment may be one or two or more.
• the method of carrying out the step of bringing an enzyme into contact with the compound represented by formula (3) is not particularly limited.
• the reaction may be carried out in a reaction solution containing the compound represented by formula (3), an enzyme, and an aqueous medium.
• Conditions for carrying out the step of bringing an enzyme into contact with the compound represented by formula (3) are not particularly limited.
• the temperature of the reaction solution may be selected from the range of 10°C to 90°C
• the pH of the reaction solution may be selected from the range of 2 to 13
• the reaction time may be selected from the range of 1 minute to 1 month. You may choose.
• the enzyme used in the method of this embodiment may be in the form of a transformant that expresses the enzyme.
• a third embodiment of the present disclosure is a protein having an amino acid sequence that satisfies at least one condition selected from (a1) to (a6) and (b) described above, and which has a carbon-carbon double bond of a phenolic compound. It is a modified enzyme that has cleaving activity.
• modified enzyme Details and preferred aspects of the modified enzyme are the same as those of the specific enzyme described in the first embodiment.
• Applications of the modified enzyme include methods for producing phenolic compounds (for example, the method according to the first embodiment or the second embodiment described above).
• the third embodiment also includes a composition containing the above modification enzyme, a polynucleotide encoding the above modification enzyme, an expression vector containing this polynucleotide, and a transformant containing this expression vector.
• a composition containing a modified enzyme includes, for example, a modified enzyme and an aqueous medium. If necessary, the composition may contain a buffer, a pH adjuster, a surfactant, an organic solvent, and the like.
• the modified enzyme contained in the composition may be in the form of a transformant that expresses the modified enzyme.
• Polynucleotides encoding modifying enzymes include DNA and RNA.
• An expression vector containing a polynucleotide encoding a modification enzyme may further contain regions such as a promoter, a terminator, and a region encoding a drug resistance gene in addition to the polynucleotide.
• the expression vector may be a plasmid or an integral vector.
• the expression vector may be a viral vector or a cell-free vector.
• a transformant containing an expression vector can be obtained by transforming a host using the expression vector and culturing the transformed host.
• hosts for introducing the expression vector include prokaryotic cells such as bacteria of the genus Escherichia such as Escherichia coli, bacteria of the genus Corynebacterium, and bacteria of the genus Bacillus, and bacteria of the genus Saccharomyces, bacteria of the genus Pichia, and bacteria of the genus Aspergillus.
• Examples include eukaryotic cells. Among these, Escherichia bacteria are preferred, and Escherichia coli is more preferred.
• the medium for culturing the host can be selected from commonly used media such as LB medium.
• a crushed product or a lysed product containing the modified enzyme may be obtained by subjecting the transformant to treatments such as crushing and lysis.
• ado gene (SEQ ID NO: 5) encoding the oxidase Ado having the amino acid sequence shown by SEQ ID NO: 1 was produced by chemical synthesis.
• PCR was performed using the primers of SEQ ID NO: 6 and SEQ ID NO: 7 and KOD One PCR Master Mix (manufactured by Toyobo), the amplified product was cut with restriction enzymes BamHI and NotI, and the pETDuet-1 vector ( Novagen) to create pETDado.
• the prepared pETDado was introduced into E.
• E. coli BL21 Star (DE3) (manufactured by ThermoFisher) by the heat shock method to produce recombinant E. coli.
• the produced recombinant E. coli was inoculated into LB medium containing 50 ⁇ g/ml ampicillin and cultured at 30° C. for 6 hours. After 6 hours, isopropyl- ⁇ -D-thiogalactopyranoside was added to a final concentration of 0.1 mM and FeCl 2 was added to a final concentration of 1 mM, and the cells were cultured at 16°C for 15 hours to induce gene expression. .
• the bacterial cells were washed with a phosphate buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , pH 7.4) and stored frozen.
• a phosphate buffer 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , pH 7.4
• ado-F82Y gene (SEQ ID NO: 8) encoding the oxidase Ado-F82Y having the amino acid sequence shown by SEQ ID NO: 2 was produced. Specifically, PCR was performed using pETDado as a template, primers of SEQ ID NO: 9 and SEQ ID NO: 10, and KOD-Plus-Mutagenesis Kit (manufactured by Toyobo), and the ado-F82Y gene was ligated to the pETDuet-1 vector. pETDado-F82Y was created. Using pETDado-F82Y and following the same procedure as in Preparation Example 1, an expression cell of E. coli into which the gene encoding Ado-F82Y had been introduced was obtained.
• ado-F82W gene (SEQ ID NO: 11) encoding the oxidase Ado-F82W having the amino acid sequence shown by SEQ ID NO: 3 was produced. Specifically, PCR was performed using pETDado as a template, primers of SEQ ID NO: 12 and SEQ ID NO: 13, and KOD-Plus-Mutagenesis Kit (manufactured by Toyobo), and the ado-F82W gene was ligated to the pETDuet-1 vector. pETDado-F82W was created. Using pETDado-F82W and following the same procedure as in Preparation Example 1, an expression cell of E. coli into which the gene encoding Ado-F82W was introduced was obtained.
• Example 4 of preparation of transformant An oxidizing enzyme group Ado-F82Y-V332X-F333X- having the amino acid sequence shown in SEQ ID NO: 4 in which a mutation is randomly introduced into at least one of the 332nd, 333rd, and 334th positions in addition to the 82nd position of Ado.
• a gene group (SEQ ID NO: 14) encoding F334X was created.
• PCR was performed using the pETDado-F82Y plasmid as a template, the mixed primer of SEQ ID NO: 15, the primer of SEQ ID NO: 16, and the KOD-Plus-Mutagenesis Kit (manufactured by Toyobo), and ado-F82Y-V332X- A plasmid group pETDado-F82Y-V332X-F333X-F334X was created in which the F333X-F334X gene group was ligated to the pETDuet-1 vector.
• plasmid groups were introduced into Escherichia coli BL21 Star (DE3) (manufactured by ThermoFisher) by the heat shock method to obtain expression cells of Escherichia coli into which a gene group encoding Ado-F82Y-V332X-F333X-F334X was introduced. .
• Example 1 40 mg/ml of expressed E. coli cells obtained in each of Preparation Examples 1 to 3 (final concentration, calculated as wet bacterial weight), 10 mM of ferulic acid (compound below) as a substrate compound, and 10% (v) of dimethyl sulfoxide. 250 ⁇ l of a reaction solution containing 100 mM Tris-HCl buffer (pH 9.5) was prepared. Thereafter, the mixture was reacted at 30° C. for 18 hours with shaking.
• HPLC analysis was performed using a Prominence LC-20 system (manufactured by Shimadzu Corporation) as an apparatus and XTerra MS C18 IS (manufactured by Waters Corporation) as a column. A 0.1% formic acid aqueous solution (liquid A) and methanol (liquid B) were used as developing solvents.
• the ratio of liquid B was increased to 40% from 3 to 4 minutes, and from 4 to 14 minutes, the ratio of liquid B was increased.
• the ratio was increased to 80%, and the ratio of liquid B was increased to 100% over a period of 14 to 15 minutes by a linear gradient.
• the ratio of liquid B was decreased to 5% from 18 to 19 minutes by a linear gradient, and the ratio of liquid B was 5% from 19 to 22 minutes, and the compound was eluted.
• Example 2 A reaction solution was prepared in the same manner as in Example 1, except that the substrate compound was changed from ferulic acid to the same amount of ethyl ferulate (compound below), and the reaction solution was reacted for 18 hours with shaking at 30°C, and HPLC analysis was performed. carried out.
• Ado was used in the enzyme reaction
• the concentration was 0.075mM
• Ado-F82Y was used in the enzyme reaction
• Ado-F82W was used in the enzyme reaction
• the concentration was 0.234mM. Vanillin was produced.
• Example 3 A reaction solution was prepared in the same manner as in Example 1, except that the substrate compound was changed from ferulic acid to the same amount of 4-vinylguaiacol (compound below), and the reaction solution was reacted for 2 hours with shaking at 30°C, followed by HPLC analysis. was carried out.
• Example 4 A reaction solution was prepared in the same manner as in Example 1, except that the substrate compound was changed from ferulic acid to the same amount of isoeugenol (compound below), and the reaction solution was reacted for 2 hours with shaking at 30 ° C., and HPLC analysis was performed. did.
• the compound represented by formula (2) can be converted from the compound represented by formula (1) by using an enzyme having the activity of cleaving the carbon-carbon double bond of the compound represented by formula (1). It was found that the compound can be produced in a one-step enzymatic reaction. Furthermore, when an enzyme having the amino acid sequence of SEQ ID NO: 2 or 3 is used, the amount of the compound represented by formula (2) produced increases compared to when using an enzyme having the amino acid sequence of SEQ ID NO: 1. I found out that it does.
• Example 5 The expressed E. coli cells prepared in Production Example 4 were streaked onto an LB solid medium containing ampicillin (50 ⁇ g/ml), isopropyl- ⁇ -D-thiogalactopyranoside (0.1 mM), and FeCl 2 (1 mM). and cultured at 20°C for 24 hours. Approximately 80 mg/ml of grown E. coli expressed cells (final concentration, wet bacterial weight), ferulic acid (10 mM), dimethyl sulfoxide (10% (v/v)), Tris-HCl buffer 100 mM (pH 9.5) ) was prepared and shaken at 30°C for 24 hours.
• reaction solution is analyzed by thin -layered chromatography (Aluminum TLC Plate, SILICA GEL COATED WITH FLUORESCENT INDICATOR F254, MERCK), Excellent recombinant Escherichia coli that generates a lot of phosphorus ( M13, M26, M40, M41, M56, M57, M69, M70, M79) were selected.
• the selected superior recombinant Escherichia coli was inoculated into LB medium containing ampicillin (50 ⁇ g/ml) and cultured at 30° C. for 6 hours.
• isopropyl- ⁇ -D-thiogalactopyranoside and FeCl2 were added to the medium to a final concentration of 0.1 mM and 1 mM, respectively, and cultured at 16°C for 15 hours to determine gene expression. guided. After bacterial collection, the bacterial cells were washed with a phosphate buffer (containing 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , pH 7.4) and stored frozen. Expression cells of superior recombinant E.
• a phosphate buffer containing 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , pH 7.4
• modified enzyme M40 which has a particularly high conversion efficiency of ferulic acid to vanillin, revealed that the 332nd amino acid residue is an arginine residue, the 333rd amino acid residue is an isoleucine residue, and the 334th amino acid residue is an isoleucine residue.
• the amino acid residue was a leucine residue.
• the reason for the high conversion efficiency of the modified enzyme M40 is that, for example, an electrostatic interaction is formed between the carboxy group of ferulic acid and the 332nd arginine residue located in the vicinity, resulting in the conversion of the enzyme to ferulic acid. This may be due to an increase in affinity.
• Example 6 Regarding the expression cells of E. coli prepared in Preparation Example 4, a reaction solution was prepared in the same manner as in Example 5, and shaken at 30° C. for 24 hours. Furthermore, the reaction solution was analyzed by thin layer chromatography, and excellent recombinant E. coli that produced more vanillin than Ado-F82Y recombinant E. coli was selected. Next, a reaction solution was prepared in the same manner as in Example 5 using the selected superior recombinant E. coli. The reaction solution was reacted for 24 hours with shaking at 30° C., and then subjected to HPLC analysis. The results are shown in FIG.
• Example 7 Regarding the bacterial cells expressing the modified enzyme M258 that showed high activity in Example 6, the reaction solution was prepared in the same manner as in Example 5, except that the substrate compound was changed from ferulic acid to the same amount of p-coumaric acid (compound below). was prepared, reacted for 24 hours with shaking at 30°C, and subjected to HPLC analysis.
• Example 8 A reaction solution was prepared in the same manner as in Example 5, except that the substrate compound was changed from ferulic acid to the same amount of sinapinic acid (compound below) for the bacterial cells expressing the modified enzyme M258 that showed high activity in Example 6. The mixture was reacted at 30° C. for 24 hours with shaking, and then subjected to HPLC analysis.
• Example 9 Regarding the bacterial cells expressing the modified enzyme M258 that showed high activity in Example 6, the reaction solution was prepared in the same manner as in Example 5, except that the substrate compound was changed from ferulic acid to the same amount of conifer alcohol (compound below). The mixture was prepared and reacted for 24 hours with shaking at 30°C, and then subjected to HPLC analysis.
• Example 10 Regarding the bacterial cells expressing the modified enzyme M258 that showed high activity in Example 6, the production of vanillin from ferulic acid was examined by changing the reaction conditions. Specifically, 80 mg/ml of cells expressing modified enzyme M258 (final concentration, calculated as wet cell weight), ferulic acid (10 mM, 20 mM, or 30 mM), butyl acetate (10% (v/v)), A reaction solution (250 ⁇ l) containing Tris-HCl buffer (100 mM, pH 10.5) was prepared, reacted at 30° C. for 48 hours, and then subjected to HPLC analysis.

Landscapes

• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Organic Chemistry (AREA)
• Engineering & Computer Science (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Biotechnology (AREA)
• General Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Microbiology (AREA)
• Biomedical Technology (AREA)
• Biochemistry (AREA)
• Molecular Biology (AREA)
• Medicinal Chemistry (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Physics & Mathematics (AREA)
• Biophysics (AREA)
• Plant Pathology (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Enzymes And Modification Thereof (AREA)

Priority Applications (3)

Applications Claiming Priority (4)

Publications (1)

Family

ID=89706417

Family Applications (1)

Country Status (4)

Cited By (1)

Citations (5)

• 2023
  • 2023-07-25 CN CN202380055107.3A patent/CN119768523A/zh active Pending
  • 2023-07-25 WO PCT/JP2023/027278 patent/WO2024024806A1/ja not_active Ceased
  • 2023-07-25 JP JP2024537764A patent/JPWO2024024806A1/ja active Pending
  • 2023-07-25 EP EP23846536.3A patent/EP4545642A4/en active Pending

Patent Citations (5)

Non-Patent Citations (8)

Also Published As

Similar Documents

Legal Events