Phenolic compound production method, and modified enzyme

Abstract

This phenolic compound production method is a method for producing a compound represented by formula (2) from a compound represented by formula (1), and comprises a step for bringing an enzyme into contact with the compound represented by formula (1), wherein the enzyme has an activity of cleaving the carbon-carbon double bond of the compound represented by formula (1) (excluding the case where the compound represented by formula (1) is ferulic acid and the enzyme is a protein having the amino acid sequence represented by SEQ ID NO: 1). In the formula, R1 represents a hydrogen atom or an alkyl group, R2 each independently represents an alkoxy group, a hydroxy group, an alkyl group or a halogen atom, and n is a number of 0-4.

Description

Method for producing phenolic compounds and modified enzymes

[0001] The present disclosure relates to methods for producing phenolic compounds and modified enzymes.

[0002] Vanillin, which is used as a flavoring agent for food and beverages and cosmetics, an intermediate for pharmaceuticals, and so on, is available on the market as an extract from the Vanilla genus of the Orchidaceae family, as well as chemically synthesized products. With the recent growing interest in natural products and the SDGs, demand for vanillin produced without the use of petroleum or chemical reagents has increased. Therefore, attempts to produce vanillin using microorganisms and enzymes have been proposed. For example, a method has been reported in which vanillin is synthesized from ferulic acid obtained from agricultural waste such as rice bran and wheat bran using an enzymatic reaction.

[0003] For example, Non-Patent Documents 1 and 2 report methods for synthesizing vanillin through a first enzymatic reaction in which ferulic acid as a starting material is converted into 4-vinylguaiacol as an intermediate, and a second enzymatic reaction in which this intermediate is converted into vanillin. Non-Patent Document 3 reports a method for synthesizing vanillin through a first enzymatic reaction in which ferulic acid as a starting material is converted into feruloyl-coenzyme A as an intermediate, and a second enzymatic reaction in which this intermediate is converted into vanillin. Non-Patent Document 4 reports a method for converting ferulic acid into vanillin using a plant-derived hydrolase belonging to the cysteine ​​proteinase family.

[0004] Non-patent document 1: J Am Chem Soc, 140, 16001-16005 (2018) 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)

[0005] The methods described in Non-Patent Documents 1 to 3 involve two-step reactions using two different enzymes, and therefore have room for improvement in terms of production efficiency. Non-Patent Document 4 only qualitatively demonstrates the activity of converting ferulic acid to vanillin, as the activity of the enzyme used is weak, and does not provide quantitative results.

[0006] In view of the above circumstances, an object of the present disclosure is to provide a novel method for producing phenolic compounds using an enzymatic reaction, and a novel enzyme that can be used for producing phenolic compounds.

[0007] Specific means for achieving the above object include the following embodiments: <1> 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 contacting the compound represented by formula (1) with an enzyme, wherein the enzyme has activity to cleave the carbon-carbon double bond of the compound represented by formula (1) (excluding the case where the compound represented by formula (1) is ferulic acid and the enzyme is a protein having the amino acid sequence represented by SEQ ID NO: 1). (In the formula, R １ represents a hydrogen atom or an alkyl group, R ２each independently represents an alkoxy group, a hydroxy group, an alkyl group, or a halogen atom, and n is a number from 0 to 4. <2> The method for producing a phenolic compound according to <1>, wherein the enzyme is an oxidative cleavage enzyme that uses a compound having a carbon-carbon double bond as a substrate. <3> The method for producing a phenolic compound according to <1> or <2>, wherein 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): (a1) In the amino acid sequence shown in SEQ ID NO: 1, the amino acid residue at position 82 is an amino acid residue other than a phenylalanine residue. (a2) In the amino acid sequence shown in SEQ ID NO: 1, the amino acid residue at position 124 is an amino acid residue other than a tyrosine residue. (a3) ​​In the amino acid sequence shown in SEQ ID NO: 1, the amino acid residue at position 157 is an amino acid residue other than a lysine residue. (a4) In the amino acid sequence shown in SEQ ID NO: 1, the amino acid residue at position 332 is an amino acid residue other than a valine residue. (a5) In the amino acid sequence shown in SEQ ID NO: 1, the amino acid residue at position 333 is an amino acid residue other than a phenylalanine residue. (a6) In the amino acid sequence shown in SEQ ID NO: 1, the amino acid residue at position 334 is an amino acid residue other than a phenylalanine residue. (b) Having 70% or more amino acid sequence identity with an amino acid sequence satisfying the condition selected from (a1) to (a6) (excluding the amino acid sequence shown in SEQ ID NO: 1). <4> The method for producing a phenolic compound according to <3>, wherein the enzyme has an amino acid sequence that satisfies the condition (a1) and at least one condition selected from (a2) to (a6), or a protein having 70% or more amino acid sequence identity with the amino acid sequence. <5> The method for producing a phenolic compound according to <3> or <4>, wherein in (a1), the amino acid residue at position 82 is a tyrosine residue or a tryptophan residue. <6> The method for producing a phenolic compound according to any one of <3> to <5>, wherein in (a4), (a5), or (a6), at least one amino acid residue selected from the amino acid residues at positions 332, 333, and 334 is a polar amino acid residue. <7> The method for producing a phenolic 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 phenolic compound according to any one of <1> to <7>, wherein the compound represented by formula (2) is vanillin or ethyl vanillin. <9> A method for producing a compound represented by formula (4) from a compound represented by formula (3), comprising the step of contacting the compound represented by formula (3) with an enzyme, wherein 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 has activity to cleave the carbon-carbon double bond of the compound represented by formula (3). (a1) In the amino acid sequence shown in SEQ ID NO: 1, the 82nd amino acid residue is an amino acid residue other than a phenylalanine residue. (a2) In the amino acid sequence shown in SEQ ID NO: 1, the 124th amino acid residue is an amino acid residue other than a tyrosine residue. (a3) ​​In the amino acid sequence shown in SEQ ID NO: 1, the 157th amino acid residue is an amino acid residue other than a lysine residue. (a4) In the amino acid sequence shown in SEQ ID NO: 1, the 332nd amino acid residue is an amino acid residue other than a valine residue. (a5) In the amino acid sequence shown in SEQ ID NO: 1, the 333rd amino acid residue is an amino acid residue other than a phenylalanine residue. (a6) In the amino acid sequence shown in SEQ ID NO: 1, the 334th amino acid residue is an amino acid residue other than a phenylalanine residue. (b) Having an amino acid sequence identity of 70% or more with an amino acid sequence satisfying a condition selected from (a1) to (a6) (excluding the amino acid sequence shown in SEQ ID NO: 1). (In the formula, R １ is a hydrogen atom, an alkyl group, or COOR ３ represents R ２ each independently represents an alkoxy group, a hydroxy group, an alkyl group, or a halogen atom; R ３represents a hydrogen atom or an alkyl group, and n is a number from 0 to 4.) <10> A modified enzyme that is a protein having an amino acid sequence that satisfies at least one condition selected from the following (a1) to (a6) and (b), and has the activity of cleaving the carbon-carbon double bond of a phenolic compound. (a1) In the amino acid sequence shown in SEQ ID NO: 1, the amino acid residue at position 82 is an amino acid residue other than a phenylalanine residue. (a2) In the amino acid sequence shown in SEQ ID NO: 1, the amino acid residue at position 124 is an amino acid residue other than a tyrosine residue. (a3) ​​In the amino acid sequence shown in SEQ ID NO: 1, the amino acid residue at position 157 is an amino acid residue other than a lysine residue. (a4) In the amino acid sequence shown in SEQ ID NO: 1, the amino acid residue at position 332 is an amino acid residue other than a valine residue. (a5) In the amino acid sequence shown in SEQ ID NO: 1, the amino acid residue at position 333 is an amino acid residue other than a phenylalanine residue. (a6) In the amino acid sequence shown in SEQ ID NO: 1, the amino acid residue at position 334 is an amino acid residue other than a phenylalanine residue. (b) Having an amino acid sequence identity of 70% or more with an amino acid sequence satisfying a condition selected from (a1) to (a6) (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 phenolic 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 comprising the expression vector according to <14>.

[0008] According to the present disclosure, there are provided a novel method for producing a phenolic compound using an enzymatic reaction, and a novel enzyme that can be used for producing a phenolic compound.

[0009] FIG. 1 is a diagram showing the absorption spectrum of the product obtained in Example 1. FIG. 2 is a graph showing the quantification results of the product obtained in Example 1. FIG. 3 is a graph showing the quantification results of the product obtained in Example 2. FIG. 4 is a graph showing the quantification results of the product obtained in Example 3. FIG. 5 is a graph showing the quantification results of the product obtained in Example 4. FIG. 6 is a graph showing the quantification results of the product obtained in Example 5. FIG. 7 is a graph showing the quantification results of the product obtained in Example 6. FIG. 8 is a graph showing the quantification results of the product obtained in Example 8. FIG. 9 is a graph showing the quantification results of the product obtained in Example 9. FIG. 10 is a graph showing the quantification results of the product obtained in Example 10.

[0010] Hereinafter, embodiments of the present disclosure will be described. These descriptions and examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention. In this specification, numerical ranges indicated using "to" indicate ranges that include the numerical values ​​before and after "to" as the minimum and maximum values, respectively.

[0011] <First Embodiment> 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), the method comprising a step of contacting the compound represented by formula (1) with an enzyme, wherein the enzyme has activity to cleave the carbon-carbon double bond of the compound represented by formula (1) (excluding the case where the compound represented by formula (1) is ferulic acid and the enzyme is a protein having the amino acid sequence represented by SEQ ID NO: 1).

[0012]

[0013] In the formula, 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; and n is a number from 0 to 4.

[0014] According to the above method, a compound represented by formula (2) (e.g., vanillin) can be produced from a compound represented by formula (1) (e.g., ferulic acid) without generating an intermediate. Therefore, the production efficiency is superior to a method of producing a compound represented by formula (2) from a compound represented by formula (1) via generating an intermediate. Furthermore, the above method can be carried out without using a coenzyme, which is advantageous in terms of reducing production costs. Hereinafter, an enzyme having the activity of cleaving the carbon-carbon double bond of a compound represented by formula (1) is also referred to as a "specific enzyme."

[0015] The specific enzyme reacts with oxygen molecules (O ２ ) in the presence of the aromatic ring of the compound represented by formula (1) and COOR １ Specifically, the specific enzyme catalyzes the reaction shown below.

[0016]

[0017] The specific enzyme is preferably an oxidative cleavage enzyme that uses a compound having a carbon-carbon double bond as a substrate. 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 an artificially modified or synthesized enzyme.

[0018] In one aspect of this embodiment, the specific enzyme is a protein having an amino acid sequence that satisfies at least one condition selected from the following (a1) to (a6) and (b): (a1) In the amino acid sequence shown in SEQ ID NO: 1, the amino acid residue at position 82 is an amino acid residue other than a phenylalanine residue; (a2) In the amino acid sequence shown in SEQ ID NO: 1, the amino acid residue at position 124 is an amino acid residue other than a tyrosine residue; (a3) ​​In the amino acid sequence shown in SEQ ID NO: 1, the amino acid residue at position 157 is an amino acid residue other than a lysine residue; (a4) In the amino acid sequence shown in SEQ ID NO: 1, the amino acid residue at position 332 is an amino acid residue other than a valine residue; (a5) In the amino acid sequence shown in SEQ ID NO: 1, the amino acid residue at position 333 is an amino acid residue other than a phenylalanine residue; (a6) In the amino acid sequence shown in SEQ ID NO: 1, the amino acid residue at position 334 is an amino acid residue other than a phenylalanine residue; (b) The specific enzyme has an amino acid sequence identity of 70% or more with an amino acid sequence that satisfies the condition selected from (a1) to (a6) (excluding the amino acid sequence shown in SEQ ID NO: 1).

[0019] The protein having the amino acid sequence shown in SEQ ID NO: 1 is an oxidase derived from the fungus Thermotheromyces thermophila (hereinafter also referred to as Ado, GenBANK accession No. XP_003665585). Non-Patent Document 1 describes that Ado exhibits the activity of converting 4-vinylguaiacol or isoeugenol, which are intermediates obtained by converting the starting material ferulic acid, into vanillin. However, there have been no reports to date of the direct conversion of a starting material into vanillin using Ado. Furthermore, as shown in the Examples below, it has been found that Ado exhibits almost no activity in converting ferulic acid into vanillin.

[0020] The present inventors applied an enzyme obtained by converting an amino acid residue at a specific position in Ado to a different amino acid residue to a method for converting a compound represented by formula (1) to a compound represented by formula (2), and found that the activity of Ado to convert the compound represented by formula (1) to vanillin was increased. One possible reason for this is that the amino acid residue converted at the specific position in Ado functions as an active center involved in cleavage of the carbon-carbon double bond contained in the phenolic compound. Furthermore, it was found that the enzyme obtained by converting an amino acid residue at a specific position in Ado to a different amino acid residue exhibits the activity to convert the compound represented by formula (1) to vanillin even when the compound is ferulic acid.

[0021] The specified enzyme is a protein obtained by converting at least the amino acid residues at positions 82, 124, 157, 332, 333, or 334 in the amino acid sequence shown in SEQ ID NO: 1. The number of converted amino acid residues in the amino acid sequence shown in SEQ ID NO: 1 is not particularly limited, as long as it is one or more. From the viewpoint of the conversion efficiency of the compound shown in Formula (1), the number of converted amino acid residues in the amino acid sequence shown in SEQ ID NO: 1 is preferably 100 or less, more preferably 50 or less, even more preferably 20 or less, and particularly preferably 10 or less. In one embodiment, the specified enzyme may be a protein obtained by converting the amino acid residues at positions 82 and at least one of the amino acid residues at positions 124, 157, 332, 333, or 334 in the amino acid sequence shown in SEQ ID NO: 1. In one embodiment, the specified enzyme may be a protein obtained by converting the amino acid residues at positions 82 and at least one of the amino acid residues at positions 332, 333, or 334 in the amino acid sequence shown in SEQ ID NO: 1.

[0022] There are no particular limitations on the type of amino acid residue substituting the amino group residue at position 82 in the amino acid sequence shown in SEQ ID NO: 1. From the viewpoint of the conversion efficiency of the compound shown in formula (1), the amino group residue at position 82 is preferably an aromatic amino acid residue, more preferably a tyrosine residue or a tryptophan residue.

[0023] There are no particular limitations on the type of amino acid residue substituting the amino group residue at position 332, 333, or 334 in the amino acid sequence shown in SEQ ID NO: 1. From the viewpoint of the conversion efficiency of the compound shown by formula (1), at least one amino acid residue selected from positions 332, 333, and 334 is preferably a polar amino acid residue (basic amino acid, hydroxyamino acid, etc.), more preferably a basic amino acid residue (arginine, lysine, or histidine), and even more preferably an arginine residue.

[0024] Specific examples of the specific enzyme include a protein having the amino acid sequence of SEQ ID NO: 2, in which the amino acid residue at position 82 in the amino acid sequence of SEQ ID NO: 1 is a 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 amino acid residue at position 82 in the amino acid sequence of SEQ ID NO: 1 is a tryptophan residue W (hereinafter, also referred to as Ado-F82W); a protein having the amino acid sequence of SEQ ID NO: 4, in which the amino acid residue at position 82 in the amino acid sequence of SEQ ID NO: 1 is a tyrosine residue Y and the amino acid residue at position 332 is an amino acid residue other than a valine residue X, the amino acid residue at position 333 is an amino acid residue other than a phenylalanine residue X, or the amino acid residue at position 334 is an amino acid residue other than a phenylalanine residue X (hereinafter, also referred to as Ado-F82Y-V332X-F333X-F334X); and proteins having 70% or more amino acid sequence identity with the amino acid sequences of SEQ ID NOs: 1 to 3.

[0025] (SEQ ID NO: 1: Amino acid sequence of Ado) MAHIHDLAPEVSNYSSGRLTPPPTPVRFPRTPVFASMNKPCRFEGDVFDLEVSGAIPPDIDGTFFRVQPDHRFPPLFEDDIHFNGDGSVTAIRISGGGHADLRQRYVRTERYLLETRARRSLFGRYRNPWTDNESVRGVIRTASNTNVVFWRG ALLAMKEDGPPFAMDPVTLETLGRYDFEGQILSPTFTAHPKIDPDTGEMVCFAYEAGGDGSDCSDVDVAVWTVDAD GKKVEECWYKAPFAGMIHDCGITKNWVVLPLTPIKMDLERMKRGGNKFAWDPSEDQWYGVVPRRGAKSDDIIWFRA DNGFHGHVAGCYELPSGEIVFDLTVADGNVFFFFPPDDNITPPADGVAKRNRLSSPTVRWIFDPKAKKSAIRTEA AGDADIWVADERVKPALTWPTNGEFSRIDDRYVTKPYRHFWQAVVDPTRPYDFEKCGPPAGGLFNCLGHYTWSDQN YHHGHNTGDPSGDGRSNGSAEEATAGKFGLQDVYFAGPTMTFQEPTFIPRQGAAEGEGYLIALLNHLDELRNDVV IFEARNLGKGPLAVIHLPLKLKLGLHGNWVDSREIEAWRRRRAENGDVGPLRVAKEPLPWQKKFAAAAQNGSNGV*

[0026] (SEQ ID NO: 2: Amino acid sequence of Ado-F82Y) MAHIHDLAPEVSNYSSGRLTPPPTPVRFPRTPVFASMNKPCRFEGDVFDLEVSGAIPPDIDGTFFRVQPDHRFPPLFEDDIHYNGDGSVTAIRISGGGHADLRQRYVRTERYLLETRARRSLFGRYRNPWTDNESVRGVIRTASNTNVVFWRG ALLAMKEDGPPFAMDPVTLETLGRYDFEGQILSPTFTAHPKIDPDTGEMVCFAYEAGGDGSDCSDVDVAVWTVDAD GKKVEECWYKAPFAGMIHDCGITKNWVVLPLTPIKMDLERMKRGGNKFAWDPSEDQWYGVVPRRGAKSDDIIWFRA DNGFHGHVAGCYELPSGEIVFDLTVADGNVFFFFPPDDNITPPADGVAKRNRLSSPTVRWIFDPKAKKSAIRTEA AGDADIWVADERVKPALTWPTNGEFSRIDDRYVTKPYRHFWQAVVDPTRPYDFEKCGPPAGGLFNCLGHYTWSDQN YHHGHNTGDPSGDGRSNGSAEEATAGKFGLQDVYFAGPTMTFQEPTFIPRQGAAEGEGYLIALLNHLDELRNDVV IFEARNLGKGPLAVIHLPLKLKLGLHGNWVDSREIEAWRRRRAENGDVGPLRVAKEPLPWQKKFAAAAQNGSNGV*

[0027] (SEQ ID NO: 3: Amino acid sequence of Ado-F82W) MAHIHDLAPEVSNYSSGRLTPPPTPVRFPRTPVFASMNKPCRFEGDVFDLEVSGAIPPDIDGTFFRVQPDHRFPPLFEDDIHWNGDGSVTAIRISGGGHADLRQRYVRTERYLLETRARRSLFGRYRNPWTDNESVRGVIRTASNTNVVFWRG ALLAMKEDGPPFAMDPVTLETLGRYDFEGQILSPTFTAHPKIDPDTGEMVCFAYEAGGDGSDCSDVDVAVWTVDAD GKKVEECWYKAPFAGMIHDCGITKNWVVLPLTPIKMDLERMKRGGNKFAWDPSEDQWYGVVPRRGAKSDDIIWFRA DNGFHGHVAGCYELPSGEIVFDLTVADGNVFFFFPPDDNITPPADGVAKRNRLSSPTVRWIFDPKAKKSAIRTEA AGDADIWVADERVKPALTWPTNGEFSRIDDRYVTKPYRHFWQAVVDPTRPYDFEKCGPPAGGLFNCLGHYTWSDQN YHHGHNTGDPSGDGRSNGSAEEATAGKFGLQDVYFAGPTMTFQEPTFIPRQGAAEGEGYLIALLNHLDELRNDVV IFEARNLGKGPLAVIHLPLKLKLGLHGNWVDSREIEAWRRRRAENGDVGPLRVAKEPLPWQKKFAAAAQNGSNGV*

[0028] (SEQ ID NO: 4: Amino acid sequence of Ado-F82Y-V332X-F333X-F334X) MAHIHDLAPEVSNYSSGRLTPPPTPVRFPRTPVFASMNKPCRFEGDVFDLEVSGAIPPDIDGTFFRVQPDHRFPPLFEDDIHYNGDGSVTAIRISGGGHADLRQRYVRTERYLLETRARRSLFGRYRNPWTDNESVRGVIRTASNTNVVFWRG ALLAMKEDGPPFAMDPVTLETLGRYDFEGQILSPTFTAHPKIDPDTGEMVCFAYEAGGDGSDCSDVDVAVWTVDAD GKKVEECWYKAPFAGMIHDCGITKNWVVLPLTPIKMDLERMKRGGNKFAWDPSEDQWYGVVPRRGAKSDDIIWFRA DNGFHGHVAGCYELPSGEIVFDLTVADGNXXXFFPPDDNITPPADGVAKRNRLSSPTVRWIFDPKAKKSAIRTEA AGDADIWVADERVKPALTWPTNGEFSRIDDRYVTKPYRHFWQAVVDPTRPYDFEKCGPPAGGLFNCLGHYTWSDQN YHHGHNTGDPSGDGRSNGSAEEATAGKFGLQDVYFAGPTMTFQEPTFIPRQGAAEGEGYLIALLNHLDELRNDVV IFEARNLGKGPLAVIHLPLKLKLGLHGNWVDSREIEAWRRRRAENGDVGPLRVAKEPLPWQKKFAAAAQNGSNGV*

[0029] When the specified enzyme is the protein (b), the amino acid sequence identity with an amino acid sequence satisfying a condition selected from (a1) to (a6) may be 70% or more, or may be 75% or more, 80% or more, 85% or more, 85% or more, or 90% or more.

[0030] When the specified enzyme is the protein (b), the amino acid sequence identity with an amino acid sequence satisfying a condition selected from (a1) to (a6) may be 70% or more, or may be 75% or more, 80% or more, 85% or more, 85% or more, or 90% or more.

[0031] In the present disclosure, the amino acid sequence identity of the protein (b) is expressed as a percentage by aligning the amino acid sequences that satisfy the conditions selected from (a1) to (a6) so as to maximize the number of identical amino acid residues, and dividing the total number of identical amino acid residues by the total number of amino acid residues in the amino acid sequence (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).

[0032] The total number of amino acid residues in the specific enzyme may be the same as or different from the total number (603) of amino acid residues in the amino acid sequence of SEQ ID NO: 1. The total number Y of amino acid residues in the specific enzyme may satisfy the following formula: In the formula, Z1 and Z2 each independently represent 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

[0033] In the compound represented by formula (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; and n is a number from 0 to 4.

[0034] 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 even more preferably a hydrogen atom, a methyl group, or an ethyl group. ２ are 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, more preferably an alkoxy group having 1 to 3 carbon atoms, and even more preferably a methoxy group or an ethoxy group. n is preferably a number from 0 to 2, more preferably 1. When n is a number of 1 or more, R ２ It is preferred that at least one of the following is in the ortho position relative to the phenolic hydroxyl group.

[0035] In the compound represented by formula (2), R ２R each independently represents an alkoxy group, a hydroxy group, an alkyl group or a halogen atom, and n is a number from 0 to 4. ２ are 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, more preferably an alkoxy group having 1 to 3 carbon atoms, and even more preferably a methoxy group or an ethoxy group. n is preferably a number from 0 to 2, more preferably 1. When n is a number of 1 or more, R ２ It is preferred that at least one of the following is in the ortho position relative to the phenolic hydroxyl group.

[0036] Specific examples of the compound represented by formula (1) include ferulic acid (R １ is a hydrogen atom, and R ２ is a methoxy group, n is 1, and R ２ is in the ortho position relative to the phenolic hydroxyl group), ferulic acid alkyl ester (R １ is an alkyl group, and R ２ is a methoxy group, n is 1, and R ２ is in the ortho position relative to the phenolic hydroxyl group).

[0037] Specific examples of the compound represented by formula (2) include vanillin (R ２ is a methoxy group, n is 1, and R ２ is in the ortho position relative to the phenolic hydroxyl group), ethyl vanillin (R ２ is an ethoxy group, n is 1, and R ２ is in the ortho position relative to the phenolic hydroxyl group).

[0038] When ferulic acid or a ferulic acid alkyl ester is used as the compound represented by formula (1), vanillin is obtained as the compound represented by formula (2).

[0039] The method of this embodiment may use one or more types of enzymes. The method of this embodiment may use one or more types of compounds represented by formula (1). The method of this embodiment may use one or more types of compounds represented by formula (1). The method of carrying out the step of contacting the compound represented by formula (1) with the enzyme is not particularly limited. For example, the step may be carried out in a reaction solution containing the compound represented by formula (1), the enzyme, and an aqueous medium. The conditions for carrying out the step of contacting the compound represented by formula (1) with the enzyme are not particularly limited. For example, 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, and the reaction time may be selected from the range of 1 minute to 1 month. The enzyme used in the method of this embodiment may be in the form of a transformant that expresses the enzyme.

[0040] Second Embodiment 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), the method comprising a step of contacting the compound represented by formula (3) with an enzyme, wherein 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 activity to cleave the carbon-carbon double bond of the compound represented by formula (3).

[0041]

[0042] In the formula, R １ is a hydrogen atom, an alkyl group, or COOR ３ represents 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, and n is a number from 0 to 4.

[0043] According to the above method, a compound represented by formula (4) can be produced from a compound represented by formula (3). Furthermore, the above method can be carried out without using a coenzyme, which is advantageous in terms of reducing production costs.

[0044] In the compound represented by formula (3), R １ is a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, or COOR ３And R ３ is preferably a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, and R １ is a hydrogen atom, an alkyl group having 1 to 3 carbon atoms, or COOR ３ And R ３ is more preferably a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, and R １ More preferably, R is a hydrogen atom, a methyl group, an ethyl group, COOH, COOMe, or COOEt. ２ are 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, more preferably an alkoxy group having 1 to 3 carbon atoms, and even more preferably a methoxy group or an ethoxy group. n is preferably a number from 0 to 2, more preferably 1. When n is a number of 1 or more, R ２ It is preferred that at least one of the following is in the ortho position relative to the phenolic hydroxyl group.

[0045] In the compound represented by formula (4), R ２ are 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, more preferably an alkoxy group having 1 to 3 carbon atoms, and even more preferably a methoxy group or an ethoxy group. n is preferably a number from 0 to 2, more preferably 1. When n is a number of 1 or more, R ２ It is preferred that at least one of the following is in the ortho position relative to the phenolic hydroxyl group.

[0046] Specific examples of the compound represented by formula (3) include ferulic acid (R １ COOR ３ and R ２ is a methoxy group, and R ３ is a hydrogen atom, n is 1, and R ２ is in the ortho position relative to the phenolic hydroxyl group), ferulic acid alkyl ester (R １ COOR ３ and R ２ is a methoxy group, and R ３is an alkyl group, n is 1, and R ２ is in the ortho position relative to the phenolic hydroxyl group), 4-vinylguaiacol (R １ is a hydrogen atom, and R ２ is a methoxy group, n is 1, and R ２ is in the ortho position relative to the phenolic hydroxyl group), isoeugenol (R １ is a methyl group, and R ２ is a methoxy group, n is 1, and R ２ is in the ortho position relative to the phenolic hydroxyl group).

[0047] Specific examples of the compound represented by formula (4) include vanillin (R ２ is a methoxy group, n is 1, and R ２ is in the ortho position relative to the phenolic hydroxyl group), ethyl vanillin (R ２ is an ethoxy group, n is 1, and R ２ is in the ortho position relative to the phenolic hydroxyl group).

[0048] The enzyme used in the second embodiment is an enzyme included in the specific enzyme used in the first embodiment, and is a protein having an amino acid sequence that satisfies the conditions selected from (a1) to (a6) and (b).

[0049] The enzyme reacts with molecular oxygen (O ２ ), the aromatic ring of the compound represented by formula (3) and R １ Specifically, the enzyme used in the second embodiment catalyzes the reaction shown below.

[0050]

[0051] The details and preferred aspects of the enzyme used in the method of this embodiment are the same as the details and preferred aspects of the specific enzyme described in the first embodiment.

[0052] The enzyme used in the method of this embodiment is represented by the formula (3) １ COOR ３In addition to the activity of converting a compound represented by formula (4) into a compound represented by formula (3), １ is a hydrogen atom or an alkyl group to a compound represented by formula (4). Furthermore, the activity of the enzyme to convert a compound represented by formula (3) to a compound represented by formula (4) is superior to the activity of Ado to convert a compound represented by formula (3) to a compound represented by formula (4). For this reason, the enzyme is also useful in multi-step synthesis methods in which a starting material such as ferulic acid is converted to an intermediate such as 4-vinylguaiacol or isoeugenol, and the intermediate is then converted to a target substance such as vanillin.

[0053] The method of this embodiment may use one or more types of enzymes. The method of this embodiment may use one or more types of compounds represented by formula (3). The method of this embodiment may use one or more types of compounds represented by formula (3). The method of carrying out the step of contacting the compound represented by formula (3) with the enzyme is not particularly limited. For example, the step may be carried out in a reaction solution containing the compound represented by formula (3), the enzyme, and an aqueous medium. The conditions for carrying out the step of contacting the compound represented by formula (3) with the enzyme are not particularly limited. For example, 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, and the reaction time may be selected from the range of 1 minute to 1 month. The enzyme used in the method of this embodiment may be in the form of a transformant that expresses the enzyme.

[0054] <Third Embodiment> A third embodiment of the present disclosure is a modified enzyme that 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 the activity of cleaving the carbon-carbon double bond of a phenolic compound.

[0055] The details and preferred aspects of the modified enzyme are the same as those of the specific enzyme described in embodiment 1. The modified enzyme can be used in a method for producing a phenolic compound (for example, the method according to the first or second embodiment described above).

[0056] The third embodiment also encompasses a composition containing the modified enzyme, a polynucleotide encoding the modified enzyme, an expression vector containing the polynucleotide, and a transformant containing the expression vector.

[0057] A composition containing a modified enzyme includes, for example, the modified enzyme and an aqueous medium. If necessary, the composition may contain a buffer, a pH adjuster, a surfactant, an organic solvent, etc. The modified enzyme contained in the composition may be in the form of a transformant that expresses the modified enzyme.

[0058] Polynucleotides encoding the modified enzymes include DNA and RNA. An expression vector containing a polynucleotide encoding the modified 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 integrative vector. The expression vector may be a viral vector or a vector for a cell-free system.

[0059] A transformant containing an expression vector can be obtained by transforming a host with the expression vector and culturing the transformed host. Hosts into which the expression vector can be introduced include prokaryotic cells such as Escherichia bacteria (e.g., Escherichia coli), Corynebacterium bacteria, and Bacillus bacteria, and eukaryotic cells such as Saccharomyces bacteria, Pichia bacteria, and Aspergillus bacteria. Among these, Escherichia bacteria are preferred, and E. coli is more preferred. The medium for culturing the host can be selected from commonly used media such as LB medium. The transformant may be subjected to disruption, lysis, or other treatment to obtain a disrupted or lysed product containing the modified enzyme.

[0060] The above-described embodiment will be described below with reference to examples, but the above-described embodiment is not limited to these examples.

[0061] [Transformant Preparation Example 1] The ado gene (SEQ ID NO: 5) encoding the oxidase Ado having the amino acid sequence shown in SEQ ID NO: 1 was prepared by chemical synthesis. Using the ado gene as a template, PCR was performed using primers of SEQ ID NO: 6 and SEQ ID NO: 7 and KOD One PCR Master Mix (Toyobo). The amplified product was cleaved with restriction enzymes BamHI and NotI and ligated into the pETDuet-1 vector (Novagen) to prepare pETDado. The prepared pETDado was introduced into Escherichia coli BL21 Star (DE3) (ThermoFisher) by the heat shock method to prepare recombinant E. coli. The prepared recombinant E. coli was inoculated into LB medium containing 50 μg / ml ampicillin and cultured at 30 ° C for 6 hours. After 6 hours, isopropyl-β-D-thiogalactopyranoside was added to a final concentration of 0.1 mM, 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. After harvesting the cells, they were resuspended in phosphate buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na ２ HPO ４ , 2 mM KH ２ P.O. ４ The cells were washed with PBS (pH 7.4) and stored frozen. Through the above steps, expression cells of E. coli into which the Ado-encoding gene had been introduced were obtained.

[0062] [Transformant Preparation Example 2] An ado-F82Y gene (SEQ ID NO: 8) encoding the oxidase Ado-F82Y having the amino acid sequence shown in SEQ ID NO: 2 was prepared. Specifically, using pETDado as a template, PCR was performed using primers shown in SEQ ID NO: 9 and SEQ ID NO: 10 and a KOD-Plus-Mutagenesis Kit (Toyobo) to prepare pETDado-F82Y in which the ado-F82Y gene was ligated to the pETDuet-1 vector. Using pETDado-F82Y, expression cells of E. coli into which a gene encoding Ado-F82Y had been introduced were obtained according to the same procedure as in Preparation Example 1.

[0063] [Transformant Preparation Example 3] An ado-F82W gene (SEQ ID NO: 11) encoding the oxidase Ado-F82W having the amino acid sequence shown in SEQ ID NO: 3 was prepared. Specifically, using pETDado as a template, PCR was performed using primers shown in SEQ ID NO: 12 and SEQ ID NO: 13 and a KOD-Plus-Mutagenesis Kit (Toyobo) to prepare pETDado-F82W in which the ado-F82W gene was ligated to the pETDuet-1 vector. Using pETDado-F82W, expression cells of E. coli into which a gene encoding Ado-F82W had been introduced were obtained according to the same procedure as in Preparation Example 1.

[0064] [Transformant Preparation Example 4] A gene group (SEQ ID NO: 14) encoding the oxidase group Ado-F82Y-V332X-F333X-F334X having the amino acid sequence shown in SEQ ID NO: 4 in which a mutation was randomly introduced into at least one of positions 332, 333, and 334 in addition to position 82 of Ado was prepared. Specifically, using the pETDado-F82Y plasmid as a template, PCR was performed using the mixed primer of SEQ ID NO: 15 and the primer of SEQ ID NO: 16 and the KOD-Plus-Mutagenesis Kit (manufactured by Toyobo Co., Ltd.), and the ado-F82Y-V332X-F333X-F334X gene group linked to the pETDuet-1 vector to prepare the plasmid group pETDado-F82Y-V332X-F333X-F334X. These plasmids were introduced into Escherichia coli BL21 Star (DE3) (ThermoFisher) by the heat shock method to obtain expression cells of Escherichia coli into which the genes encoding Ado-F82Y-V332X-F333X-F334X had been introduced.

[0065] Example 1 A reaction solution (250 μl) was prepared containing 40 mg / ml (final concentration, wet cell weight) of the expressed Escherichia coli cells obtained in each of Preparation Examples 1 to 3, 10 mM ferulic acid (compound below) as a substrate compound, 10% (v / v) dimethyl sulfoxide, and 100 mM Tris-HCl buffer (pH 9.5). The mixture was then reacted at 30° C. for 18 hours with shaking.

[0066]

[0067] After the reaction, 5 N HCl (5 μl) and ethanol (250 μl) were added to 250 μl of the reaction solution to prepare a sample for HPLC analysis. HPLC analysis was performed using a Prominence LC-20 system (Shimadzu Corporation) and an XTerra MS C18 IS column (Waters Corporation). The developing solvents were 0.1% formic acid aqueous solution (Solution A) and methanol (Solution B). After flowing at a flow rate of 0.5 ml / min with a ratio of 95% Solution A and 5% Solution B from 0 to 3 minutes, the ratio of Solution B was increased linearly from 3 to 4 minutes to 40%, from 4 to 14 minutes to 80%, and from 14 to 15 minutes to 100%. Furthermore, after flowing 100% solution B from 15 to 18 minutes, the ratio of solution B was decreased linearly to 5% from 18 to 19 minutes, and then the ratio of solution B was increased to 5% from 19 to 22 minutes to elute the compound.

[0068] As a result of HPLC analysis, a peak corresponding to the product was detected at a retention time of 10.5 minutes in the reaction solutions containing the expressed E. coli cells obtained in Preparation Examples 2 and 3. The retention time of this product peak matched the retention time of the peak for authentic vanillin. In the reaction solution containing the expressed E. coli cells obtained in Preparation Example 1, no peak corresponding to the product was detected at a retention time of 10.5 minutes. The results of measuring the absorption spectrum of the product are shown in Figure 1. As shown in Figure 1, the absorption spectrum (A) of the product detected after the reaction matched the absorption spectrum (B) of authentic vanillin. This identified the product as vanillin. The results of quantification of the vanillin product by HPLC analysis (n=3) are shown in Figure 2. As shown in Figure 2, no vanillin was produced when Ado was used in the enzymatic reaction, whereas 0.023 mM vanillin was produced when Ado-F82Y was used in the enzymatic reaction, and 0.007 mM vanillin was produced when Ado-F82W was used in the reaction.

[0069] [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 (the compound below). The reaction was carried out at 30°C for 18 hours with shaking, and then HPLC analysis was carried out.

[0070]

[0071] As a result of HPLC analysis, a peak corresponding to the product was detected at a retention time of 10.5 minutes in the reaction solutions containing the expressed E. coli cells obtained in Preparation Examples 1, 2, and 3. The retention time of this product peak matched the retention time of the peak for authentic vanillin. The results of quantification of vanillin as the product by HPLC analysis (n=3) are shown in FIG. 3. As shown in FIG. 3, when Ado was used in the enzymatic reaction, 0.075 mM vanillin was produced, when Ado-F82Y was used in the enzymatic reaction, 0.376 mM vanillin was produced, and when Ado-F82W was used in the enzymatic reaction, 0.234 mM vanillin was produced.

[0072] 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 (the compound below). The reaction was carried out at 30°C for 2 hours with shaking, and then HPLC analysis was carried out.

[0073]

[0074] As a result of HPLC analysis, a peak corresponding to the product was detected at a retention time of 10.5 minutes in the reaction solutions containing the expressed E. coli cells obtained in Preparation Examples 1, 2, and 3. The retention time of this product peak matched the retention time of the peak of authentic vanillin. The results of quantification of the vanillin product by HPLC analysis (n=3) are shown in Figure 4. As shown in Figure 4, 1.032 mM vanillin was produced with Ado, 5.408 mM with Ado-F82Y, and 1.565 mM with Ado-F82W.

[0075] [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 (the compound below). The reaction was carried out at 30°C for 2 hours with shaking, and then HPLC analysis was carried out.

[0076]

[0077] As a result of HPLC analysis, a peak corresponding to the product was detected at a retention time of 10.5 minutes in the reaction solutions containing the expressed E. coli cells obtained in Preparation Examples 1, 2, and 3. The retention time of this product peak matched the retention time of the peak of authentic vanillin. The results of quantification of the vanillin product by HPLC analysis (n=3) are shown in Figure 5. As shown in Figure 5, 1.934 mM vanillin was produced with Ado, 5.105 mM with Ado-F82Y, and 2.602 mM with Ado-F82W.

[0078] The results of the above examples demonstrate that the compound represented by formula (2) can be produced in a single enzymatic reaction 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). Furthermore, it was found that the amount of the compound represented by formula (2) produced was greater when the enzyme having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3 was used than when the enzyme having the amino acid sequence of SEQ ID NO: 1 was used.

[0079] Example 5 The expression cells of Escherichia coli prepared in Preparation Example 4 were incubated in a 500-mL culture medium containing ampicillin (50 μg / ml), isopropyl-β-D-thiogalactopyranoside (0.1 mM), FeCl ２The resulting mixture was streaked onto LB solid medium containing 1 mM E. coli and cultured for 24 hours at 20° C. A reaction solution (50 μl) containing approximately 80 mg / ml (final concentration, calculated as wet cell weight) of the grown E. coli cells, ferulic acid (10 mM), dimethyl sulfoxide (10% (v / v)), and 100 mM Tris-HCl buffer (pH 9.5) was prepared and shaken at 30° C. for 24 hours. Furthermore, the reaction solution was analyzed by thin layer chromatography (Aluminum TLC plate, silica gel coated with fluorescent indicator F254, Merck), and superior recombinant E. coli (M13, M26, M40, M41, M56, M57, M69, M70, M79) that produced more vanillin than the Ado-F82Y recombinant E. coli were selected. Next, the selected superior recombinant E. coli was inoculated into LB medium containing ampicillin (50 μg / ml) and cultured at 30°C for 6 hours. After 6 hours, isopropyl-β-D-thiogalactopyranoside was added to a final concentration of 0.1 mM, FeCl ２ was added to the medium to a final concentration of 1 mM, and the medium was cultured at 16°C for 15 hours to induce gene expression. After harvesting the cells, they were diluted with phosphate buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na ２ HPO ４ , 2 mM KH ２ P.O. ４ The cells were washed with a solution containing 1000 mg / ml of purified E. coli (containing 1000 mg / ml of purified E. coli), pH 7.4, and then frozen and stored. A reaction solution (250 μl) containing 40 mg / ml (final concentration, calculated as wet cell weight) of expressed cells of the excellent recombinant E. coli, ferulic acid (10 mM), dimethyl sulfoxide (10% (v / v)), and Tris-HCl buffer (100 mM, pH 9.5) was prepared and shaken at 30°C for 24 hours. 5 N HCl (5 μl) and ethanol (250 μl) were then added to the reaction solution (250 μl) to prepare a sample for HPLC analysis, and HPLC analysis was performed in the same manner as in Example 1. For comparison, HPLC analysis was also performed on a sample of Ado-F82Y. The results are shown in FIG. 6.

[0080] As shown in FIG. 6, it was found that the sample prepared from the expression cells of E. coli into which the gene group encoding Ado-F82Y-V332X-F333X-F334X had been introduced was capable of synthesizing 0.2 mM or more of vanillin.

[0081] Analysis of the amino acid sequence of the modified enzyme M40, which has a particularly high conversion efficiency of ferulic acid to vanillin, revealed that the amino acid residue at position 332 was an arginine residue, the amino acid residue at position 333 was an isoleucine residue, and the amino acid residue at position 334 was a leucine residue. One possible reason for the high conversion efficiency of the modified enzyme M40 is that, for example, electrostatic interaction is formed between the carboxyl group of ferulic acid and the nearby arginine residue at position 332, increasing the affinity of the enzyme for ferulic acid.

[0082] Example 6 A reaction solution was prepared in the same manner as in Example 5 using the expression cells of E. coli prepared in Preparation Example 4, and the reaction solution was shaken at 30°C for 24 hours. The reaction solution was then analyzed by thin-layer chromatography, and superior recombinant E. coli strains that produced more vanillin than the Ado-F82Y recombinant E. coli were selected. Next, a reaction solution was prepared in the same manner as in Example 5 using the selected superior recombinant E. coli strains. The reaction solution was reacted for 24 hours with shaking at 30°C, and then subjected to HPLC analysis. The results are shown in Figure 7.

[0083] As shown in FIG. 7, among the samples prepared from expression cells of E. coli into which a gene group encoding Ado-F82Y-V332X-F333X-F334X had been introduced, there was a sample (M258) that was capable of synthesizing more than 5 mM vanillin.

[0084] Analysis of the amino acid sequence of the modified enzyme with a high conversion efficiency of ferulic acid to vanillin revealed that the amino acid residues at positions 332, 333, and 334 were substituted with various amino acid residues. The results are shown in Table 1. This demonstrates that introducing mutations into these amino acid residues is effective in increasing the activity of Ado against ferulic acid.

[0085]

[0086] Example 7 For the bacterial cells expressing the modified enzyme M258 that showed high activity in Example 6, 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 p-coumaric acid (the compound described below). The reaction was carried out at 30°C for 24 hours with shaking, and then HPLC analysis was performed.

[0087]

[0088] As a result of HPLC analysis, a peak corresponding to the product was detected at a retention time of 6.16 minutes in the reaction solution containing the expressed bacterial cells of M258. The retention time of this product peak matched the retention time of the peak of a standard sample of p-hydroxybenzaldehyde (the compound described below). The results of quantification of the product p-hydroxybenzaldehyde by HPLC analysis (n=3) are shown in Figure 8. As shown in Figure 8, when M258 was used in the enzymatic reaction, 0.43 mM p-hydroxybenzaldehyde was produced.

[0089]

[0090] [Example 8] For the expressing cells of the modified enzyme M258 that showed high activity in Example 6, 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 sinapic acid (the compound described below). The reaction was carried out at 30°C for 24 hours with shaking, and HPLC analysis was performed.

[0091]

[0092] As a result of HPLC analysis, a peak corresponding to the product was detected at a retention time of 11.5 minutes in the reaction solution containing E. coli cells expressing M258. The retention time of this product peak matched the retention time of the peak of a standard syringaldehyde (compound described below). The results of quantification of the product syringaldehyde by HPLC analysis (n=3) are shown in Figure 9. As shown in Figure 9, when M258 was used in the enzymatic reaction, 8.6 mM syringaldehyde was produced.

[0093]

[0094] [Example 9] For the expressing cells of the modified enzyme M258 that showed high activity in Example 6, 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 conifer alcohol (the compound described below). The reaction was carried out at 30°C for 24 hours with shaking, and HPLC analysis was performed.

[0095]

[0096] As a result of HPLC analysis, a peak corresponding to the product was detected at a retention time of 10.6 minutes in the reaction solution containing E. coli cells expressing M258. The retention time of this product peak matched the retention time of the peak of authentic vanillin. The results of quantification of the vanillin product by HPLC analysis (n=3) are shown in Figure 10. As shown in Figure 10, when M258 was used in the enzymatic reaction, 3.4 mM vanillin was produced.

[0097] Example 10 The production of vanillin from ferulic acid was investigated by changing the reaction conditions for the bacterial cells expressing the modified enzyme M258, which showed high activity in Example 6. Specifically, a reaction solution (250 μl) containing 80 mg / ml (final concentration, calculated as wet bacterial cell weight) of the bacterial cells expressing the modified enzyme M258, ferulic acid (10 mM, 20 mM, or 30 mM), butyl acetate (10% (v / v)), and Tris-HCl buffer (100 mM, pH 10.5) was prepared and reacted at 30° C. for 48 hours, followed by HPLC analysis.

[0098] The results of quantification of the product vanillin by HPLC analysis (n=3) are shown in Figure 11. As shown in Figure 11, the concentration of the product vanillin increased as the concentration of the substrate ferulic acid increased, and 16.0 mM (2.44 g / L) vanillin was produced from 30 mM ferulic acid. These results suggest that the modified enzyme of the present disclosure enables vanillin production on a gram per liter scale.

[0099] The disclosures of Japanese Patent Application Nos. 2022-120869 and 2022-158419 are incorporated herein by reference in their entirety. All documents, patent applications, and technical standards mentioned herein are incorporated by reference to the same extent as if each individual document, patent application, and technical standard was specifically and individually indicated to be incorporated by reference.

Claims

1. A method for producing a compound represented by the following formula (2) from a compound represented by the following formula (1), The method comprises the step of contacting an enzyme with a compound represented by formula (1), The enzyme is a protein having an amino acid sequence that satisfies at least one of the following conditions (a1), (a4), (a5), and (a6) or condition (b), and has activity to cleave the carbon-carbon double bond of the compound represented by formula (1). (a1) the 82nd amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is a tyrosine or tryptophan residue; (a4) the 332nd amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is an arginine, threonine, or lysine residue; (a5) the 333rd amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is a residue of isoleucine, glutamine, serine, histidine, glycine, valine, or leucine; (a6) The 334th amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is a residue of leucine, serine, arginine, cysteine, isoleucine, glycine, asparagine, or methionine. (b) satisfying at least one of the conditions selected from (a1), (a4), (a5), and (a6), and having an amino acid sequence identity of 70% or more with an amino acid sequence satisfying at least one of the conditions selected from (a1), (a4), (a5), and (a6) (excluding the amino acid sequence shown in SEQ ID NO: 1). 【Chemistry 1】 (In the formula, 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, and n is a number from 0 to 4.

2. The method for producing a phenolic compound according to claim 1, wherein the enzyme is an oxidative cleavage enzyme which utilizes a compound having a carbon-carbon double bond as a substrate.

3. The method for producing a phenolic compound according to claim 1, wherein the enzyme is a protein having an amino acid sequence that satisfies the condition (a1) or an amino acid sequence that satisfies the condition (a1) and also satisfies the condition (b).

4. The method for producing a phenolic compound according to claim 1, wherein the enzyme is a protein having an amino acid sequence that satisfies at least one condition selected from (a4), (a5) and (a6), or an amino acid sequence that satisfies at least one condition selected from (a4), (a5) and (a6) and also satisfies condition (b).

5. The method for producing a phenolic compound according to any one of claims 1 to 4, wherein the amino acid sequence identity in (b) is 80% or more.

6. The method for producing a phenolic compound according to any one of claims 1 to 4, wherein the amino acid sequence identity in (b) is 90% or more.

7. The method for producing a phenolic compound according to claim 1, 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 claim 1, wherein the compound represented by formula (2) is vanillin or ethyl vanillin.

9. A method for producing a compound represented by the following formula (4) from a compound represented by the following formula (3), The method includes contacting an enzyme with a compound represented by formula (3), The method for producing a phenolic compound, wherein the enzyme is a protein having an amino acid sequence that satisfies at least one of the following conditions (a1), (a4), (a5), and (a6) or condition (b), and has activity to cleave the carbon-carbon double bond of a compound represented by formula (3). (a1) the 82nd amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is a tyrosine or tryptophan residue; (a4) the 332nd amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is an arginine, threonine, or lysine residue; (a5) the 333rd amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is a residue of isoleucine, glutamine, serine, histidine, glycine, valine, or leucine; (a6) The 334th amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is a residue of leucine, serine, arginine, cysteine, isoleucine, glycine, asparagine, or methionine. (b) satisfying at least one of the conditions selected from (a1), (a4), (a5), and (a6), and having an amino acid sequence identity of 70% or more with an amino acid sequence satisfying at least one of the conditions selected from (a1), (a4), (a5), and (a6) (excluding the amino acid sequence shown in SEQ ID NO: 1). 【Chemistry 2】 (In the formula, R １ is a hydrogen atom, an alkyl group or COOR ３ represents 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, and n is a number from 0 to 4.

10. A modified enzyme which is a protein having an amino acid sequence that satisfies at least one of the conditions selected from (a1), (a4), (a5), and (a6) below, or condition (b), and has the activity of cleaving the carbon-carbon double bond of a phenolic compound. (a1) the 82nd amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is a tyrosine or tryptophan residue; (a4) the 332nd amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is an arginine, threonine, or lysine residue; (a5) the 333rd amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is a residue of isoleucine, glutamine, serine, histidine, glycine, valine, or leucine; (a6) The 334th amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is a residue of leucine, serine, arginine, cysteine, isoleucine, glycine, asparagine, or methionine. (b) satisfying at least one of the conditions selected from (a1), (a4), (a5), and (a6), and having an amino acid sequence identity of 70% or more with an amino acid sequence satisfying at least one of the conditions selected from (a1), (a4), (a5), and (a6) (excluding the amino acid sequence shown in SEQ ID NO: 1).

11. The modified enzyme of claim 10 for use in a method for producing a phenolic compound.

12. A composition comprising the modified enzyme of claim 10.

13. A polynucleotide encoding the modified enzyme described in claim 10.

14. An expression vector comprising the polynucleotide of claim 13.

15. A transformant comprising the expression vector according to claim 14.

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