Method for producing phenol compound, and modified enzyme

Abstract

A method for producing a phenolic compound (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), the method for producing a compound represented by formula (2) from the compound represented by formula (1), the method for producing a phenolic compound includes a step of bringing an enzyme having an activity of cleaving a carbon-carbon double bond of a compound represented by formula (1) into contact with the compound represented by formula (1). In the formula (1) and the formula (2), R1 represents a hydrogen atom or an alkyl group, R2 independently represents an alkoxy group, a hydroxyl group, an alkyl group or a halogen atom, and n is a number from 0 to 4.

Description

Technical Field

[0001] The present invention relates to a method for producing a phenol compound and a modified enzyme. Background Art

[0002] Vanillin used as a food and / or beverage, a fragrance for cosmetics, an intermediate for pharmaceuticals, etc., is available in the market as an extract from a plant of the genus Vanilla of the orchid family, and chemically synthesized products. In recent years, due to the increased attention paid to natural products and SDGs, the demand for vanillin produced without using 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 using an enzyme reaction has been reported, and the ferulic acid is obtained from agricultural wastes such as rice bran and wheat bran.

[0003] For example, non-patent document 1 and non-patent document 2 report methods for synthesizing vanillin through the following enzyme reactions: a first enzyme reaction in which ferulic acid as a starting material is converted into 4-vinylguaiacol as an intermediate, and a second enzyme reaction in which the intermediate is converted into vanillin. Non-patent document 3 reports a method for synthesizing vanillin through the following enzyme reactions: a first enzyme reaction in which ferulic acid as a starting material is converted into feruloyl-CoA as an intermediate, and a second enzyme reaction in which the 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 ​​protease 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). Summary of the invention

[0005] The methods described in Non-Patent Documents 1 to 3 use two enzymes to perform two-step reactions, respectively, and therefore there is room for improvement in production efficiency. In Non-Patent Document 4, the activity of the enzyme used is weak, and therefore the activity of converting ferulic acid into vanillin can only be qualitatively shown, and no results are shown to a quantitative extent.

[0006] In view of the above circumstances, an object of the present invention is to provide a novel method for producing a phenol compound using an enzyme reaction, and a novel enzyme that can be used in the production of a phenol compound.

[0007] Specific means for solving the above-mentioned problems include the following embodiments. <1> A method for producing a phenolic compound (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), wherein a compound represented by formula (2) is produced from a compound represented by formula (1) below: The method for producing a phenol compound comprises the step of contacting an enzyme with a compound represented by formula (1). The enzyme has an activity of cleaving the carbon-carbon double bond of the compound represented by formula (1). [Chemistry 1] (Where R 1 represents a hydrogen atom or an alkyl group, R 2 Each independently represents an alkoxy group, a hydroxyl group, an alkyl group or a halogen atom, and n is a number from 0 to 4.) <2> The method for producing a phenol 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 phenol 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 an amino acid sequence identity of 70% or more with an amino acid sequence satisfying the conditions selected from (a1) to (a6), excluding the amino acid sequence represented by SEQ ID NO: 1. <4> The method for producing a phenolic compound according to <3>, wherein the enzyme is a protein having an amino acid sequence that satisfies condition (a1) and at least one condition selected from (a2) to (a6), or a protein having an amino acid sequence identity of 70% or more with the amino acid sequence. <5> The method for producing a phenol 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 phenol compound according to any one of <3> to <5>, wherein in (a4), (a5) or (a6), at least one amino acid residue selected from position 332, position 333 or position 334 is a polar amino acid residue. <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 ethyl vanillin. <9> A method for producing a phenol compound, comprising producing a compound represented by the following formula (4) from a compound represented by the following formula (3), The method for producing a phenol compound comprises the step of contacting an enzyme 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 has an activity of cleaving 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 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 the conditions selected from (a1) to (a6), excluding the amino acid sequence represented by SEQ ID NO: 1. [Chemistry 2] (Where R 1 Represents a hydrogen atom, an alkyl group, or COOR 3 , R 2 Each independently represents an alkoxy group, a hydroxyl group, an alkyl group or a halogen atom, and 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 satisfying at least one condition selected from the following (a1) to (a6) and (b), and has an activity of cleaving a carbon-carbon double bond of a phenol 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 the conditions selected from (a1) to (a6), excluding the amino acid sequence represented by SEQ ID NO: 1. <11> The modified enzyme according to <10>, which is used 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 described in <10>. <14> An expression vector comprising the polynucleotide described in <13>. <15> A transformant comprising the expression vector described in <14>. Effects of the Invention

[0008] According to the present invention, a novel method for producing a phenol compound using an enzyme reaction and a novel enzyme that can be used for producing a phenol compound can be provided. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 This is a diagram showing the absorption spectrum of the product obtained in Example 1. Figure 2 This is a graph showing the quantitative results of the product obtained in Example 1. Figure 3 This is a graph showing the quantitative results of the product obtained in Example 2. Figure 4 This is a graph showing the quantitative results of the product obtained in Example 3. Figure 5 This is a graph showing the quantitative results of the product obtained in Example 4. Figure 6 This is a graph showing the quantitative results of the product obtained in Example 5. Figure 7 This is a graph showing the quantitative results of the product obtained in Example 6. Figure 8 This is a graph showing the quantitative results of the product obtained in Example 7. Fig. 9 This is a graph showing the quantitative results of the product obtained in Example 8. Fig.10 This is a graph showing the quantitative results of the product obtained in Example 9. Fig.11 This is a graph showing the quantitative results of the product obtained in Example 10. DETAILED DESCRIPTION

[0010] Hereinafter, embodiments of the present invention will be described. These descriptions and examples illustrate the present invention and do not limit the scope of the present invention. In the present specification, a numerical range expressed using "to" indicates a range including the numerical values ​​described before and after "to" as the minimum value and the maximum value, respectively.

[0011] <First embodiment> The first embodiment of the present invention is a method for producing a phenolic compound (excluding the case where the compound represented by formula (1) is ferulic acid and the enzyme is a protein having an amino acid sequence represented by SEQ ID NO: 1), wherein a compound represented by the following formula (2) is produced from a compound represented by the following formula (1): The method for producing a phenol compound comprises the step of contacting an enzyme with a compound represented by formula (1). The enzyme has an activity of cleaving the carbon-carbon double bond of the compound represented by formula (1).

[0012] [Chemistry 3]

[0013] In the formula, R 1 represents a hydrogen atom or an alkyl group, R 2 Each independently represents an alkoxy group, a hydroxyl group, an alkyl group or a halogen atom, and n is a number of 0-4.

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

[0015] The specific enzyme has the ability to cleave the aromatic ring and COOR of the compound represented by formula (1) in the presence of oxygen molecules (O2). 1 Specifically, the specific enzyme catalyzes the reaction shown below.

[0016] [Chemistry 4]

[0017] The specific enzyme is preferably an oxidative lyase that uses a compound having a carbon-carbon double bond as a substrate. Specific examples of the oxidative lyase include carotenoid oxidative lyase, stilbene oxidative lyase, isoeugenol oxidative lyase, and 4-vinylguaiacol oxidative lyase. The specific enzyme may be an enzyme derived from a natural substance or an enzyme artificially modified or synthesized.

[0018] In one aspect of the present 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) having an amino acid sequence identity of 70% or more with an amino acid sequence satisfying the conditions selected from (a1) to (a6), excluding the amino acid sequence represented by 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 Thermothelomyces thermophila (hereinafter also referred to as Ado, GenBANK accession No. XP_003665585). Non-patent document 1 states that Ado exhibits activity in converting 4-vinylguaiacol or isoeugenol as intermediates obtained by converting ferulic acid as a starting material into vanillin. However, there has been no report to date on the direct conversion of a starting material into vanillin using Ado. Furthermore, as shown in Examples described later, it was found that Ado showed almost no activity of converting ferulic acid into vanillin.

[0020] The present inventors applied an enzyme to a method for converting a compound represented by formula (1) into a compound represented by formula (2), wherein the enzyme is obtained by converting an amino acid residue at a specific position of Ado into a different amino acid residue. As a result, it was found that the activity of Ado in converting the compound represented by formula (1) 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 that participates in cleaving a carbon-carbon double bond contained in the phenol compound. Furthermore, it was found that an enzyme obtained by replacing the amino acid residue at a specific position of Ado with a different amino acid residue exhibits an activity of converting the compound represented by formula (1) into vanillin even if it is ferulic acid.

[0021] The specific enzyme is a protein obtained by converting at least the 82nd, 124th, 157th, 332nd, 333rd or 334th amino acid residue in the amino acid sequence shown in SEQ ID NO:1. In the amino acid sequence shown in SEQ ID NO: 1, the number of amino acid residues to be converted is 1 or more and is not particularly limited. From the viewpoint of the conversion efficiency of the compound shown in formula (1), the number of amino acid residues to be converted in the amino acid sequence shown in SEQ ID NO: 1 is preferably 100 or less, more preferably 50 or less, further preferably 20 or less, and particularly preferably 10 or less. In one embodiment, the specific enzyme may be a protein in which at least one of the amino acid residues at position 82 and position 124, position 157, position 332, position 333 or position 334 in the amino acid sequence shown in SEQ ID NO: 1 is converted. In one embodiment, the specific enzyme may be a protein in which at least one of the amino acid residues at position 82 and position 332, position 333, or position 334 in the amino acid sequence shown in SEQ ID NO: 1 is converted.

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

[0023] There is no particular limitation on the type of amino acid residue that replaces the amino acid residue at position 332, position 333 or position 334 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), at least one amino acid residue selected from positions 332, 333 and 334 is preferably a polar amino acid residue (basic amino acid, hydroxy amino acid, etc.), more preferably a basic amino acid residue (arginine, lysine or histidine), and still 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 82nd amino acid residue 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, wherein 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, wherein the amino acid residue at position 82 in the amino acid sequence of SEQ ID NO: 1 is a tyrosine residue Y, the amino acid residue at position 332 is an amino acid residue X other than a valine residue, the amino acid residue at position 333 is an amino acid residue X other than a phenylalanine residue, and the amino acid residue at position 334 is an amino acid residue X other than a phenylalanine residue (hereinafter also referred to as Ado-F82Y-V332X-F333X-F334X); and A protein having an amino acid sequence identity of 70% or more to the amino acid sequence of SEQ ID NOs: 1 to 3.

[0025] (SEQ ID NO: 1: amino acid sequence of Ado) *.

[0026] (SEQ ID NO: 2: Amino acid sequence of Ado-F82Y) MAHIHDLAPEVSNYSSGRLTPPTPVRFPRTPVFASMNKPCRFEGDVFDLEVSGAIPPDIDGTFFRVQPDHRFPPLFEDDIHYNGDGSVTAIRISGGHADLRQRYVRTERYLLETRARRSLFGRYRNPWTDNESVRGVIRTASNTNVVFWRGALLAMKEDGPPFAMDPVTLETLGRYDFEGQILSPTFTAHPKIDPDTGEMVCFAYEAGGDGSDCSVDVAVWTVDADGKKVEECWYKAPFAGMIHDCGITKNWVVLPLTPIKMDLERMKRGGNKFAWDPSEDQWYGVVPRRGAKSDDIIWFRADNGFHGHVAGCYELPSGEIVFDLTVADGNVFFFFPPDDNITPPADGVAKRNRLSSPTVRWIFDPKAKKSAIRTEAAGDADIWVADERVKPALTWPTNGEFSRIDDRYVTKPYRHFWQAVVDPTRPYDFEKCGPPAGGLFNCLGHYTWSDQNYHHGHNTGDPSGDGRSNGSAEEATAGKFGLQDVYFAGPTMTFQEPTFIPRQGAAEGEGYLIALLNHLDELRNDVVIFEARNLGKGPLAVIHLPLKLKLGLHGNWVDSREIEAWRRRRAENGDVGPLRVAKEPLPWQKKFAAAAQNGSNGV*。

[0027] (Sequence No. 3: Amino acid sequence of Ado - F82W) MAHIHDLAPEVSNYSSGRLTPPTPVRFPRTPVFASMNKPCRFEGDVFDLEVSGAIPPDIDGTFFRVQPDHRFPPLFEDDIHWNGDGSVTAIRISGGHADLRQRYVRTERYLLETRARRSLFGRYRNPWTDNESVRGVIRTASNTNVVFWRGALLAMKEDGPPFAMDPVTLETLGRYDFEGQILSPTFTAHPKIDPDTGEMVCFAYEAGGDGSDCSVDVAVWTVDADGKKVEECWYKAPFAGMIHDCGITKNWVVLPLTPIKMDLERMKRGGNKFAWDPSEDQWYGVVPRRGAKSDDIIWFRADNGFHGHVAGCYELPSGEIVFDLTVADGNVFFFFPPDDNITPPADGVAKRNRLSSPTVRWIFDPKAKKSAIRTEAAGDADIWVADERVKPALTWPTNGEFSRIDDRYVTKPYRHFWQAVVDPTRPYDFEKCGPPAGGLFNCLGHYTWSDQNYHHGHNTGDPSGDGRSNGSAEEATAGKFGLQDVYFAGPTMTFQEPTFIPRQGAAEGEGYLIALLNHLDELRNDVVIFEARNLGKGPLAVIHLPLKLKLGLHGNWVDSREIEAWRRRRAENGDVGPLRVAKEPLPWQKKFAAAAQNGSNGV*。

[0028] (Amino acid sequence of Serial No. 4: Ado-F82Y-V332X-F333X-F334X) *.

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

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

[0031] The amino acid sequence identity of the protein of (b) in the present invention is expressed as a percentage of the value obtained by arranging the amino acid sequences satisfying the conditions selected from (a1) to (a6) in such a way that the number of amino acid residues with identical sequences is the largest, and dividing the total number of amino acid residues with identical sequences by the total number of amino acid residues in the amino acid sequence of (b). The amino acid sequence identity search can be performed using a search program well known to those skilled in the art, such as BLAST (Basic Local Alignment Search Tool).

[0032] 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 mathematical formula. 603-Z1≤Y≤603+Z2 In the formula, Z1 and Z2 are each independently a number of 0-10, preferably a number of 0-5, and more preferably a number of 0-2.

[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 hydroxyl group, an alkyl group or a halogen atom, and n is a number of 0-4.

[0034] R 1 It 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 still more preferably a hydrogen atom, a methyl group or an ethyl group. R 2 Each independently is preferably an alkoxy group having 1 to 5 carbon atoms, a hydroxyl 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 still more preferably a methoxy group or an ethoxy group. n is preferably a number of 0 to 2, and more preferably 1. When n is a number greater than or equal to 1, R 2 At least one of the groups is located in the ortho position relative to the phenolic hydroxyl group.

[0035] In the compound represented by formula (2), R 2 Each independently represents an alkoxy group, a hydroxyl group, an alkyl group or a halogen atom, and n is a number of 0-4. R 2 Each independently is preferably an alkoxy group having 1 to 5 carbon atoms, a hydroxyl 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 still more preferably a methoxy group or an ethoxy group. n is preferably a number of 0 to 2, and more preferably 1. When n is a number greater than or equal to 1, R 2 At least one of the groups is located 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, R 2 is methoxy, n is 1, R2 Compounds located at an ortho position relative to the phenolic hydroxyl group), alkyl ferulate (R 1 is an alkyl group, R 2 is methoxy, n is 1, R 2 A compound located at an ortho position relative to the phenolic hydroxyl group) etc.

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

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

[0039] The enzyme used in the method of the present embodiment may be only one kind or two or more kinds. The compound represented by formula (1) used in the method of the present embodiment may be only one kind or two or more kinds. The method for carrying out the step of contacting the enzyme with the compound represented by formula (1) is not particularly limited. For example, it can 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 enzyme with the compound represented by formula (1) are not particularly limited. For example, the temperature of the reaction solution can be selected from the range of 10°C to 90°C, the pH of the reaction solution can be selected from the range of 2 to 13, and the reaction time can be selected from the range of 1 minute to 1 month. The enzyme used in the method of the present embodiment may be in a state of a converter or the like expressing the enzyme.

[0040] <Second embodiment> A second embodiment of the present invention is a method for producing a phenol compound, wherein a compound represented by the following formula (4) is produced from a compound represented by the following formula (3): The method for producing a phenol compound comprises the step of contacting an enzyme with a compound represented by formula (3). The enzyme is a protein having an amino acid sequence satisfying at least one condition selected from the group consisting of (a1) to (a6) and (b) above, and has an activity of cleaving the carbon-carbon double bond of the compound represented by formula (3).

[0041] [Chemistry 5]

[0042] In the formula, R 1Represents a hydrogen atom, an alkyl group, or COOR 3 , R 2 Each independently represents an alkoxy group, a hydroxyl group, an alkyl group or a halogen atom, and R 3 represents a hydrogen atom or an alkyl group, and n is a number of 0 to 4.

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

[0044] In the compound represented by formula (3), R 1 A hydrogen atom, an alkyl group having 1 to 5 carbon atoms, or COOR 3 , R 3 is a hydrogen atom or an alkyl group having 1 to 5 carbon atoms; more preferably R 1 A hydrogen atom, an alkyl group with 1 to 3 carbon atoms, or COOR 3 , R 3 is a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; R 1 More preferably, it is a hydrogen atom, a methyl group, an ethyl group, COOH, COOMe or COOEt. R 2 Each independently is preferably an alkoxy group having 1 to 5 carbon atoms, a hydroxyl 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 still more preferably a methoxy group or an ethoxy group. n is preferably a number of 0 to 2, and more preferably 1. When n is a number greater than or equal to 1, R 2 At least one of the groups is located in the ortho position relative to the phenolic hydroxyl group.

[0045] In the compound represented by formula (4), R 2 Each independently is preferably an alkoxy group having 1 to 5 carbon atoms, a hydroxyl 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 still more preferably a methoxy group or an ethoxy group. n is preferably a number of 0 to 2, and more preferably 1. When n is a number greater than or equal to 1, R 2 At least one of the groups is located 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 For COOR 3 , R 2 is methoxy, R 3 is a hydrogen atom, n is 1, R 2Compounds located at an ortho position relative to the phenolic hydroxyl group), alkyl ferulate (R 1 For COOR 3 , R 2 is methoxy, R 3 is an alkyl group, n is 1, R 2 The compound located at the ortho position relative to the phenolic hydroxyl group), 4-vinylguaiacol (R 1 is a hydrogen atom, R 2 is methoxy, n is 1, R 2 relative to the phenolic hydroxyl group is located in the ortho position), isoeugenol (R 1 is methyl, R 2 is methoxy, n is 1, R 2 A compound located at an ortho position relative to the phenolic hydroxyl group) etc.

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

[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 has the ability to cleave the aromatic ring and R of the compound represented by formula (3) in the presence of oxygen molecules (O2). 1 Activity of bonding carbon-carbon double bonds. Specifically, the enzyme used in the second embodiment catalyzes the reaction shown below.

[0050] [Chemistry 6]

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

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

[0053] The enzyme used in the method of the present embodiment may be only one kind or two or more kinds. The compound represented by formula (3) used in the method of this embodiment may be only one kind or two or more kinds. The method for carrying out the step of contacting the enzyme with the compound represented by formula (3) is not particularly limited. For example, it can 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 enzyme with the compound represented by formula (3) are not particularly limited. For example, the temperature of the reaction solution can be selected from the range of 10°C to 90°C, the pH of the reaction solution can be selected from the range of 2 to 13, and the reaction time can be selected from the range of 1 minute to 1 month. The enzyme used in the method of the present embodiment may be in a state of a converter or the like expressing the enzyme.

[0054] <Third embodiment> A third embodiment of the present invention is a modified enzyme which is a protein having an amino acid sequence satisfying at least one condition selected from the group consisting of (a1) to (a6) and (b) above and has an activity of cleaving a carbon-carbon double bond of a phenol compound.

[0055] The details and preferred embodiments of the modified enzyme are the same as those of the specific enzyme described in the first embodiment. Examples of uses of the modified enzyme include a method for producing a phenol compound (for example, the method according to the first embodiment or the second embodiment described above).

[0056] The third embodiment also includes a composition comprising the above-mentioned modified enzyme, a polynucleotide encoding the above-mentioned modified enzyme, an expression vector comprising the polynucleotide, and a transformant comprising the expression vector.

[0057] The composition containing the modified enzyme includes, for example, the modified enzyme and an aqueous medium. The composition may include a buffer, a pH adjuster, a surfactant, an organic solvent, and the like as needed. The modified enzyme contained in the composition may be in the form of a converter or the like expressing the modified enzyme.

[0058] Examples of the polynucleotide encoding the modified enzyme include DNA and RNA. The expression vector containing the polynucleotide encoding the modified enzyme may contain, in addition to the polynucleotide, a promoter, a terminator, a region encoding a drug resistance gene, and the like. 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. Examples of hosts for introducing expression vectors include prokaryotic cells such as Escherichia bacteria such as Escherichia coli, Corynebacterium bacteria, and Bacillus bacteria, and eukaryotic cells such as Saccharomyces bacteria, Pichia bacteria, and Aspergillus bacteria, etc. Among them, Escherichia bacteria are preferred, and Escherichia coli is more preferred. The medium used for culturing the host can be selected from commonly used medium such as LB medium. By subjecting the transformant to treatments such as disruption or dissolution, a disrupted product or a dissolved product containing the modified enzyme can be obtained. Example

[0060] Hereinafter, the above-mentioned embodiment will be described by way of examples, but the above-mentioned embodiment is not limited to these examples.

[0061] [Preparation Example 1 of Converter] The ado gene (sequence number 5) was prepared by chemical synthesis, and the ado gene encodes the oxidase Ado having the amino acid sequence shown in sequence number 1. The ado gene was used as a template, and PCR was performed using primers of sequence numbers 6 and 7 and KOD One PCRMaster Mix (manufactured by Toyobo). The amplified product was cut using restriction endonucleases BamHI and NotI, and connected to the pETDuet-1 vector (manufactured by Novagen) to prepare pETDado. The prepared pETDado was introduced into Escherichia coli BL21 Star (DE3) (manufactured by ThermoFisher) by the heat shock method to prepare recombinant Escherichia coli. The prepared recombinant Escherichia coli was inoculated in LB medium containing 50 μg / ml of ampicillin and cultured at 30°C for 6 hours. After 6 hours, isopropyl-β-D- was added to a final concentration of 0.1 mM, and FeCl2 was added to a final concentration of 1 mM, and cultured at 16°C for 15 hours to induce gene expression. After the cells were collected, the cells were washed with a phosphate buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) and frozen for storage. Through the above steps, expression cells of Escherichia coli into which the gene encoding Ado was introduced were obtained.

[0062] [Preparation Example 2 of Converter] Prepare ado-F82Y gene (SEQ ID NO. 8), which encodes oxidase Ado-F82Y having the amino acid sequence shown in SEQ ID NO. 2. Specifically, pETDado was used as a template, and primers of SEQ ID NO. 9 and SEQ ID NO. 10 and KOD-Plus-Mutagenesis Kit (manufactured by Toyobo) were used to perform PCR to prepare pETDado-F82Y in which the ado-F82Y gene was connected to the pETDuet-1 vector. Using pETDado-F82Y, the same steps as in Preparation Example 1 were used to obtain expression cells of Escherichia coli into which the gene encoding Ado-F82Y was introduced.

[0063] [Preparation Example 3 of Converter] Prepare ado-F82W gene (sequence number 11), which encodes oxidase Ado-F82W having the amino acid sequence shown in sequence number 3. Specifically, pETDado was used as a template, and primers of sequence number 12 and sequence number 13 and KOD-Plus-Mutagenesis Kit (manufactured by Toyobo) were used to perform PCR to prepare pETDado-F82W in which the ado-F82W gene was connected to the pETDuet-1 vector. Using pETDado-F82W, the same steps as in Preparation Example 1 were used to obtain expression cells of Escherichia coli into which the gene encoding Ado-F82W was introduced.

[0064] [Preparation Example 4 of Converter] A gene group (sequence number 14) is prepared, which encodes an oxidase group Ado-F82Y-V332X-F333X-F334X having the amino acid sequence shown in sequence number 4 in a state in which mutations are randomly introduced at at least one of positions 332, 333 and 334 except position 82 of Ado. Specifically, pETDado-F82Y plasmid was used as a template, and PCR was performed using a mixed primer of sequence number 15 and a primer of sequence number 16 and KOD-Plus-Mutagenesis Kit (manufactured by Toyobo) to prepare a plasmid group pETDado-F82Y-V332X-F333X-F334X in which the ado-F82Y-V332X-F333X-F334X gene group was connected to the pETDuet-1 vector. These plasmid groups were introduced into Escherichia coli BL21 Star (DE3) (manufactured by ThermoFisher) by a heat shock method to obtain expression bacteria of Escherichia coli introduced with the gene group encoding Ado-F82Y-V332X-F333X-F334X.

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

[0066] [Chemistry 7]

[0067] After the reaction, 5N 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 Prominence LC-20 system (manufactured by Shimadzu Corporation) as an apparatus and XTerra MS C18 IS (manufactured by Waters Corporation) as a column. The developing solvent was 0.1% formic acid aqueous solution (solution A) and methanol (solution B). After the flow rate was 0.5 ml / min, the ratio of solution A was 95% and solution B was 5% from 0 to 3 minutes, and then the ratio of solution B was increased to 40% from 3 to 4 minutes, the ratio of solution B was increased to 80% from 4 to 14 minutes, and the ratio of solution B was increased to 100% from 14 to 15 minutes by a linear gradient. In addition, after the flow of solution B was 100% from 15 to 18 minutes, the ratio of solution B was reduced to 5% from 18 to 19 minutes by a linear gradient, and the solution B was flowed at a ratio of 5% from 19 to 22 minutes to elute the compound.

[0068] The results of HPLC analysis showed that a peak corresponding to the product was detected at a retention time of 10.5 minutes in the reaction solution containing the expression cells of Escherichia coli obtained in Preparation Examples 2 and 3. The retention time of the peak of the product was consistent with the retention time of the peak of the vanillin standard. A peak corresponding to the product was detected at a retention time of 10.5 minutes in the reaction solution containing the expression cells of Escherichia coli obtained in Preparation Example 1. The results of the absorption spectrum of the product are shown in Figure 1 .like Figure 1 As shown, the absorption spectrum (A) of the product detected after the reaction is consistent with the absorption spectrum (B) of the vanillin standard. Therefore, it is determined that the product is vanillin. The results of quantification of vanillin as a product by HPLC analysis (n=3) are shown in Figure 2 .like Figure 2 As shown, when Ado was used in the enzyme reaction, no vanillin was produced. On the other hand, when Ado-F82Y was used in the enzyme reaction, 0.023 mM of vanillin was produced, and when Ado-F82W was used in the enzyme reaction, 0.007 mM of vanillin was produced.

[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 an equal amount of ethyl ferulate (the following compound), and the reaction was carried out at 30° C. for 18 hours with shaking, and HPLC analysis was performed.

[0070] [Chemistry 8]

[0071] The results of HPLC analysis showed that a peak corresponding to the product was detected at a retention time of 10.5 minutes in the reaction solution containing the expression cells of Escherichia coli obtained in each of Preparation Examples 1, 2 and 3. The retention time of the peak of the product was consistent with the retention time of the peak of the vanillin standard. The results of quantification of vanillin as a product by HPLC analysis (n=3) are shown in Figure 3 .like Figure 3 As shown, when Ado was used in the enzyme reaction, 0.075 mM of vanillin was produced, when Ado-F82Y was used in the enzyme reaction, 0.376 mM of vanillin was produced, and when Ado-F82W was used in the enzyme reaction, 0.234 mM of 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 an equal amount of 4-vinylguaiacol (the following compound), and the reaction was carried out at 30° C. for 2 hours with shaking, and HPLC analysis was performed.

[0073] [Chemistry 9]

[0074] The results of HPLC analysis showed that a peak corresponding to the product was detected at a retention time of 10.5 minutes in the reaction solution containing the expression cells of Escherichia coli obtained in each of Preparation Examples 1, 2 and 3. The retention time of the peak of the product was consistent with the retention time of the peak of the vanillin standard. The results of quantification of vanillin as a product by HPLC analysis (n=3) are shown in Figure 4 .like Figure 4 As shown, when Ado was used, 1.032 mM of vanillin was produced, when Ado-F82Y was used, 5.408 mM of vanillin was produced, and when Ado-F82W was used, 1.565 mM of vanillin was produced.

[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 an equal amount of isoeugenol (the following compound), and the mixture was reacted at 30° C. for 2 hours with shaking, and then analyzed by HPLC.

[0076] [Chemistry 10]

[0077] The results of HPLC analysis showed that a peak corresponding to the product was detected at a retention time of 10.5 minutes in the reaction solution containing the expression cells of Escherichia coli obtained in each of Preparation Examples 1, 2 and 3. The retention time of the peak of the product was consistent with the retention time of the peak of the vanillin standard. The results of quantification of vanillin as a product by HPLC analysis (n=3) are shown in Figure 5 .like Figure 5 As shown, when Ado was used, 1.934 mM of vanillin was produced, when Ado-F82Y was used, 5.105 mM of vanillin was produced, and when Ado-F82W was used, 2.602 mM of vanillin was produced.

[0078] The results of the above examples show that the compound represented by formula (2) can be produced from the compound represented by formula (1) by a single-step enzyme reaction by using an enzyme having an activity of cleaving the carbon-carbon double bond of the compound represented by formula (1). Furthermore, it was found that when the enzyme having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3 was used, the amount of the compound represented by formula (2) produced increased compared to 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 streaked in LB solid medium containing ampicillin (50 μg / ml), isopropyl-β-D-thiogalactopyranoside (0.1 mM), and FeCl2 (1 mM), and cultured at 20° C. for 24 hours. A reaction solution (50 μl) composed of about 80 mg / ml (final concentration, wet cell weight conversion) of the expression cells of the proliferating Escherichia coli, ferulic acid (10 mM), dimethyl sulfoxide (10% (v / v)), and 100 mM (pH 9.5) of Tris-HCl buffer was prepared and shaken at 30° C. for 24 hours. Furthermore, the reaction solution was analyzed using thin layer chromatography (Aluminum TLC plate, silica gel coated with fluorescent indicator F254, manufactured by Merck), and excellent recombinant Escherichia coli (M13, M26, M40, M41, M56, M57, M69, M70, M79) that produced more vanillin than Ado-F82Y recombinant Escherichia coli were selected. Next, the selected excellent recombinant Escherichia coli was inoculated in 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 the medium to a final concentration of 0.1 mM, and FeCl2 was added to a final concentration of 1 mM, and cultured at 16°C for 15 hours to induce gene expression. After collecting the bacteria, the cells were washed with phosphate buffer (containing 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) and frozen for storage. A reaction solution (250 μl) composed of 40 mg / ml (final concentration, wet cell weight conversion) of excellent recombinant Escherichia coli expression cells, 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. Then, 5N HCl (5 μl) and ethanol (250 μl) were 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 the sample of Ado-F82Y. The results are shown in Figure 6 .

[0080] like Figure 6 As shown, it was found that the sample prepared from the expression cells of Escherichia coli introduced with the gene group encoding Ado-F82Y-V332X-F333X-F334X was able to synthesize 0.2 mM or more of vanillin.

[0081] The amino acid sequence of the modified enzyme M40, which has a particularly high conversion efficiency of ferulic acid to vanillin, was analyzed, and it was found 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. The reason why the modified enzyme M40 has a high conversion efficiency is, for example, that the carboxyl group of ferulic acid forms an electrostatic interaction with the arginine residue at position 332 located nearby, thereby increasing the affinity of the enzyme for ferulic acid.

[0082] [Example 6] Regarding the expression bacteria of Escherichia coli prepared in Preparation Example 4, a reaction solution was prepared in the same manner as in Example 5, and the mixture was shaken at 30°C for 24 hours. Furthermore, the reaction solution was analyzed by thin layer chromatography, and excellent recombinant Escherichia coli that produced more vanillin than the Ado-F82Y recombinant Escherichia coli was selected. Next, a reaction solution was prepared in the same manner as in Example 5 using the selected excellent recombinant Escherichia coli. The reaction solution was shaken at 30°C for 24 hours, and HPLC analysis was performed. The results are shown in Figure 7 .

[0083] like Figure 7 As shown, among the samples prepared from expression cells of Escherichia coli introduced with the gene group encoding Ado-F82Y-V332X-F333X-F334X, there was a sample (M258) capable of synthesizing more than 5 mM of vanillin.

[0084] The amino acid sequence of the modified enzyme with high conversion efficiency of ferulic acid to vanillin was analyzed, and as a result, the amino acid residues at positions 332, 333, and 334 were replaced by various amino acid residues. The results are shown in Table 1. It can be seen that the introduction of mutations to these amino acid residues is effective for improving the activity of Ado against ferulic acid.

[0085] [Table 1]

[0086] [Example 7] Regarding the expression bacteria of the modified enzyme M258 showing 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 an equal amount of p-coumaric acid (the following compound), and the reaction was carried out at 30°C with shaking for 24 hours, and HPLC analysis was performed.

[0087] [Chemistry 11]

[0088] The results of HPLC analysis showed that a peak corresponding to the product was detected at a retention time of 6.16 minutes in the reaction solution containing the expression bacteria of M258. The retention time of the peak of the product was consistent with the retention time of the peak of the p-hydroxybenzaldehyde (hereinafter referred to as the compound) standard. The results of quantification of p-hydroxybenzaldehyde as a product by HPLC analysis (n=3) are shown in Figure 8 .like Figure 8 As shown, when M258 was used in the enzyme reaction, 0.43 mM of p-hydroxybenzaldehyde was generated.

[0089] [Chemistry 12]

[0090] [Example 8] Regarding the expression bacteria of the modified enzyme M258 showing 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 an equal amount of sinapinic acid (the following compound), and the reaction was carried out at 30°C with shaking for 24 hours, and HPLC analysis was performed.

[0091] [Chemistry 13]

[0092] The results of HPLC analysis showed that in the reaction solution containing the expression cells of Escherichia coli M258, a peak corresponding to the product was detected at a retention time of 11.5 minutes. The retention time of the peak of the product was consistent with the retention time of the peak of the syringaldehyde (hereinafter referred to as the compound) standard. The results of quantitative analysis of the product syringaldehyde by HPLC (n=3) are shown in Fig. 9 .like Fig. 9 As shown, when M258 was used in the enzyme reaction, 8.6 mM of syringaldehyde was generated.

[0093] [Chemistry 14]

[0094] [Example 9] Regarding the expression cells of the modified enzyme M258 showing 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 an equal amount of coniferyl alcohol (the following compound), reacted at 30°C with shaking for 24 hours, and analyzed by HPLC.

[0095] [Chemistry 15]

[0096] The results of HPLC analysis showed that a peak corresponding to the product was detected at a retention time of 10.6 minutes in the reaction solution containing the expression cells of Escherichia coli M258. The retention time of the peak of the product was consistent with the retention time of the peak of the vanillin standard. The results of quantification of vanillin as a product by HPLC analysis (n=3) are shown in Fig.10 .like Fig.10 As shown, when M258 was used in the enzyme reaction, 3.4 mM of vanillin was produced.

[0097] [Example 10] Regarding the expression cells of the modified enzyme M258 showing high activity in Example 6, the reaction conditions were changed to study the production of vanillin from ferulic acid. Specifically, a reaction solution (250 μl) composed of 80 mg / ml (final concentration, wet cell weight conversion) of the expression cells of 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, reacted at 30°C for 48 hours, and HPLC analysis was performed.

[0098] The results of quantification of vanillin as a product by HPLC analysis (n=3) are shown in Fig.11 .like Fig.11As shown, the concentration of vanillin as a product increased with the increase in the concentration of ferulic acid as a substrate, and 16.0 mM (2.44 g / L) of vanillin was generated from 30 mM ferulic acid. The above results suggest that vanillin can be produced on a gram per liter scale using the modified enzyme of the present invention.

[0099] The disclosures of Japanese Patent Application No. 2022-120869 and Japanese Patent Application No. 2022-158419 are incorporated herein by reference in their entirety. For all documents, patent applications, and technical standards described in this specification, each document, patent application, and technical standard is incorporated by reference to the same extent as if it were specifically and separately described, and is incorporated herein by reference.

Claims

1. A method for producing a phenol compound, characterized in that: The compound represented by the following formula (2) is prepared from the compound represented by the following formula (1), The method for producing a phenol compound comprises the step of contacting an enzyme with a compound represented by formula (1). The enzyme has an activity of cutting the carbon-carbon double bond of the compound represented by formula (1). The method for producing a phenolic compound does not include 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 sequence number 1, In formula (1) and (2), R 1 represents a hydrogen atom or an alkyl group, R 2 Each independently represents an alkoxy group, a hydroxyl group, an alkyl group or a halogen atom, and n is a number of 0-4.

2. The method for producing a phenol compound according to claim 1, wherein The enzyme is an oxidative lyase that uses a compound having a carbon-carbon double bond as a substrate.

3. The method for producing a phenol compound according to claim 1, 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 an amino acid sequence identity of 70% or more with an amino acid sequence satisfying the conditions selected from (a1) to (a6), excluding the amino acid sequence represented by SEQ ID NO:

1.

4. The method for producing a phenol compound according to claim 3, wherein The enzyme is a protein having an amino acid sequence satisfying condition (a1) and at least one condition selected from (a2) to (a6), or a protein having an amino acid sequence identity of 70% or more with the amino acid sequence.

5. The method for producing a phenol compound according to claim 3, wherein In the above (a1), the amino acid residue at position 82 is a tyrosine residue or a tryptophan residue.

6. The method for producing a phenol compound according to claim 3, wherein In (a4), (a5) or (a6), at least one amino acid residue selected from position 332, position 333 or position 334 is a polar amino acid residue.

7. The method for producing a phenol compound according to claim 1, wherein The compound represented by formula (1) is ferulic acid or alkyl ferulic acid 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 phenol compound, characterized in that: The compound represented by the following formula (4) is prepared from the compound represented by the following formula (3), The method for producing a phenol compound comprises the step of contacting an enzyme with a compound represented by formula (3). The enzyme is a protein having an amino acid sequence that satisfies at least one of the following conditions (a1) to (a6) and (b), and has an activity of cleaving 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 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 the conditions selected from (a1) to (a6), excluding the amino acid sequence represented by SEQ ID NO: 1; In formula (3) and (4), R 1 Represents a hydrogen atom, an alkyl group, or COOR 3 , R 2 Each independently represents an alkoxy group, a hydroxyl group, an alkyl group or a halogen atom, and R 3 represents a hydrogen atom or an alkyl group, and n is a number of 0 to 4.

10. A modified enzyme, characterized in that A protein having an amino acid sequence that satisfies at least one of the following conditions (a1) to (a6) and (b), and having an activity of cleaving a 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 the conditions selected from (a1) to (a6), excluding the amino acid sequence represented by SEQ ID NO:

1. The modified enzyme according to claim 10, which is used in a method for producing a phenol compound.

12. A composition comprising the modified enzyme according to claim 10.

13. A polynucleotide encoding the modified enzyme according to claim 10.

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

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

Images