Enzymatic agent for vanillin production and application thereof

Abstract

The present invention addresses the problem of providing a method for producing vanillin at low cost by bioconversion. The present invention relates to an enzymatic agent for vanillin production, the enzymatic agent containing a copper oxidase and capable of producing vanillin from isoeugenol. The present invention also relates to a method for producing vanillin, the method comprising treating isoeugenol with a copper oxidase. Furthermore, the present invention also relates to: a flavor improver for foods and beverages; a food or beverage; a vanillin-containing composition; and a flavoring agent.

Description

Enzyme preparation for vanillin production and its applications

[0001] The present invention relates to an enzyme preparation for vanillin production, a flavor enhancer for food and beverages, food and beverages, a method for producing vanillin, a vanillin-containing composition, and a flavoring agent.

[0002] Vanillin (4-hydroxy-3-methoxybenzaldehyde) is one of the chemical substances that give vanilla flavor and is widely used in food and beverages, fragrances, pharmaceuticals, cosmetics, etc. Vanillin is a substance found in vanilla seeds, but it can also be synthesized artificially.

[0003] Industrial methods for producing vanillin include chemical methods and bio-conversion methods (such as fermentation and enzymatic methods). Vanillin synthesized by bio-conversion methods is considered natural vanillin by various regulatory and legislative authorities. Phenols are used as starting materials for vanillin synthesis in bio-conversion methods.

[0004] For example, Patent Document 1 discloses a bioconversion method for producing vanillin, which involves converting the FfLSD protein to vanillin by reacting it with isoeugenol. Patent Document 2 discloses a method for producing vanillin from ferulic acid using a non-GMO mutant strain of Amycolaptosis sp. In the ferulic acid metabolic pathway, ferulic acid is nonoxidatively deacetylated to produce vanillin. This step is mediated by two enzymes: feruloyl coenzyme A (CoA) synthase encoded by the fcs gene and enoyl CoA hydratase / aldolase encoded by the ech gene. Transcription of these genes is induced only after the addition of ferulic acid, leading to vanillin production. Furthermore, Patent Document 3 discloses a reaction scheme in which aldehydes and ketones are oxidized in the presence of molecular oxygen, using at least one enzyme selected from peroxidase and laccase as a catalyst. In Patent Document 3, vanillin is produced in an example by reacting isoeugenol with peroxidase.

[0005] Japanese Patent No. 7387194, International Publication No. 2022 / 133269, JP 2010-532664

[0006] In recent years, the demand for natural vanillin has increased due to growing health consciousness. Therefore, the development of various manufacturing methods is being considered, and there is a need to establish a method for producing vanillin inexpensively through bioconversion.

[0007] Therefore, the present invention aims to provide a method for producing vanillin inexpensively by bioconversion.

[0008] Specifically, the present invention has the following configuration.

[0009] [1] An enzyme preparation for vanillin production, comprising copper oxidase, which produces vanillin from isoeugenol. [2] The enzyme preparation for vanillin production according to [1], wherein the copper oxidase is laccase derived from the genus Trametes. [3] A flavor enhancer for food and beverages, comprising copper oxidase, which produces vanillin from food and beverages containing isoeugenol. [4] The flavor enhancer for food and beverages according to [3], wherein the copper oxidase is laccase derived from the genus Trametes. [5] Food and beverages containing the flavor enhancer for food and beverages according to [3] or [4]. [6] A method for producing vanillin, comprising reacting isoeugenol with copper oxidase. [7] The method for producing vanillin according to [6], wherein the copper oxidase is laccase derived from the genus Trametes. [8] The method for producing vanillin according to [6] or [7], wherein the reaction of isoeugenol with copper oxidase is carried out under conditions of pH 7.5 or higher. [9] A vanillin-containing composition obtained by reacting the vanillin-producing enzyme preparation described in [1] or [2] with a raw material containing isoeugenol.

[10] A flavoring agent obtained by reacting the vanillin-producing enzyme preparation described in [1] or [2] with a raw material containing isoeugenol.

[0010] [A] Use of an enzyme preparation containing copper oxidase for producing vanillin from isoeugenol. [B] A method for using an enzyme preparation containing copper oxidase for producing vanillin from isoeugenol. [C] An enzyme preparation containing copper oxidase for use in the production of vanillin from isoeugenol.

[0011] According to the present invention, vanillin can be produced without going through a complex bioconversion process, thus enabling the inexpensive production of vanillin.

[0012] The present invention will be described in detail below. The following descriptions of constituent elements may be based on representative embodiments or specific examples, but the present invention is not limited to such embodiments. In this specification, numerical ranges expressed using "~" mean a range that includes the numbers written before and after "~" as the lower and upper limits.

[0013] (Enzyme preparation for vanillin production) This embodiment relates to an enzyme preparation for vanillin production that contains copper oxidase and produces vanillin from isoeugenol. In this embodiment, vanillin can be produced in one step by reacting the vanillin-producing enzyme preparation containing copper oxidase with isoeugenol. Thus, in this embodiment, vanillin can be produced without going through a complex bioconversion process, and therefore vanillin can be manufactured at low cost.

[0014] The vanillin-producing enzyme preparation of this embodiment contains copper oxidase as an active ingredient. Copper oxidase oxidizes and decomposes the double bond of isoeugenol, adding an aldehyde group (-CHO) to the position of the double bond. The vanillin-producing enzyme preparation of this embodiment may be in powder, solid, gel, or liquid form.

[0015] The vanillin obtained in this embodiment is 4-hydroxy-3-methoxybenzaldehyde (CAS registration number: 121-33-5), which is known as a major component of vanilla flavor. Vanillin can be used in food and beverages, perfumes, cosmetics, personal care products, pharmaceutical intermediates, and the like.

[0016] In this embodiment, vanillin may be used in food and beverages, in which case it is preferable to produce vanillin by reacting a vanillin-producing enzyme preparation with a food or beverage containing isoeugenol. In this case, this embodiment may also relate to a flavor enhancer for food and beverages that contains copper oxidase and produces vanillin from a food or beverage containing isoeugenol. Furthermore, this embodiment may relate to a food or beverage containing a flavor enhancer for food and beverages, or to a food or beverage obtained by reacting a vanillin-producing enzyme preparation with a food or beverage containing isoeugenol.

[0017] Conventionally, vanillin has been produced by reacting isoeugenol with peroxidase. However, in such cases, the addition of hydrogen peroxide is necessary to allow the enzymatic reaction to proceed, and the vanillin obtained in such a reaction system is difficult to apply to food and beverages. On the other hand, the vanillin-producing enzyme preparation of this embodiment contains copper oxidase as an active ingredient, so the addition of hydrogen peroxide and other substances is unnecessary, and as a result, the vanillin obtained can be preferably used in food and beverages.

[0018] <Copper Oxidase> The vanillin-producing enzyme preparation of this embodiment contains copper oxidase. Copper oxidase is an oxidase containing copper, which is an enzyme that reduces oxygen by electrons to produce water molecules. Copper oxidase may be a copper-containing monooxidase or a copper-containing multi-copper oxidase (hereinafter also referred to as multi-copper oxidase). Examples of copper-containing monooxidases include dopamine-β-monooxygenase, peptidylglycine α-hydroxylated monooxygenase, tyrosinase, methane monooxygenase, etc.

[0019] The copper oxidase is preferably a multi-copper oxidase. A multi-copper oxidase is an enzyme that contains 2 to 8 copper atoms necessary for enzymatic activity in its molecule, and is an enzyme that electron-reduces oxygen to produce water molecules. The multi-copper oxidase is preferably at least one selected from phenol oxidase and bilirubin oxidase, more preferably phenol oxidase, and even more preferably polyphenol oxidase. Examples of multi-copper oxidases include laccase, bilirubin oxidase, ascorbic acid oxidase, ceruloplasmin, Fe3p, CueO, CotA, stellacyanin, tyrosinase, catechol oxidase, and nitrite reductase. Among these, the multi-copper oxidase is preferably at least one selected from the group consisting of laccase, bilirubin oxidase, ascorbic acid oxidase, and tyrosinase, and is particularly preferably laccase.

[0020] Laccase is an enzyme (EC 1.10.3.2) with phenol oxidase activity and can be used in any application that utilizes the oxidation reaction of a substrate and / or the various incidental chemical reactions resulting from the radical species of reaction intermediates produced by the oxidation reaction. Examples of such oxidation reactions or incidental chemical reactions include the oxidation of phenolic compounds such as o-,p-diphenols, urushiol, and laccol; the oxidation of aromatic amines such as p-phenylenediamine; the decomposition of lignin; and the crosslinking of proteins having easily oxidizable functional groups such as tyrosine side chains (phenolic hydroxyl groups), cysteine ​​side chains (sulfhydryl groups), lysine side chains (ε-amino groups), and histidine side chains (imidazole groups). This chemical modification of a substrate by the oxidation reaction and incidental chemical reactions of laccase can also be called "oxidative modification."

[0021] Laccase is found in plants, fungi, bacteria, and animals. In particular, the laccase used in the present invention is preferably laccase derived from fungi or bacteria. Specifically, examples include laccases derived from genera such as Aspergillus, Neurospora, Podospora, Botrytis, Collybia, Fomes, Lentinus, Pleurotus, Pycnoporus, Pyricalaria, Trametes, Rhizoctonia, Rigidoporus, Coprinus, Psatyrella, Myceliophtera, Schtalidium, Polyporus, Phlebia, Coriolus, and Bacillus. Among these, laccase derived from the genus Trametes is particularly preferred. Using laccase derived from the genus Trametes can effectively increase the amount of vanillin produced from isoeugenol.

[0022] Laccase can be prepared from the culture medium of the microorganisms from which laccase is derived, as described above. Specific preparation methods include recovering laccase from the culture medium or cells of the above-mentioned microorganisms. For example, when using laccase-secreting microorganisms, the cells are recovered from the culture medium by filtration, centrifugation, etc., as needed, and then the enzyme is separated and / or purified. When using non-laccase-secreting microorganisms, the cells are recovered from the culture medium by filtration, centrifugation, etc., as needed, and then the cells are crushed by pressurization, sonication, etc., to expose the enzyme, and then the enzyme is separated and / or purified. The method for separating and / or purifying the enzyme is not particularly limited, and known protein separation and / or purification methods can be employed, such as centrifugation, UF concentration, salting out, and various chromatography methods using ion exchange resins. The separated and / or purified enzyme can be powdered by drying methods such as freeze-drying or vacuum drying, and powdering can also be performed using appropriate excipients and / or drying aids in the drying method. Furthermore, the separated and / or purified enzymes can be liquefied by adding appropriate additives and sterilizing by filtration.

[0023] Commercially available laccases can also be used. Preferred examples of commercial products include Trametes sp. derived laccase from Amano Enzyme Co., Ltd., Laccase from Trametes versicolor from Merck & Co., Ltd., Laccase from Aspergillus sp. from Merck & Co., Ltd., and Laccase YK1 from Heat-Resistant Enzyme Research Institute Co., Ltd.

[0024] <Isoeugenol> Isoeugenol is 2-methoxy-4-(prope-1-en-1-yl)phenol (CAS registration number: 97-54-1). Isoeugenol has two isomers: trans-isoeugenol ((E)-isoeugenol) and cis-isoeugenol ((Z)-isoeugenol). The isoeugenol used in this embodiment may be either trans-isoeugenol or cis-isoeugenol, or a mixture of isomers of trans-isoeugenol and cis-isoeugenol. Isoeugenol may be present in a solution containing water, or it may be contained in a dried powder.

[0025] Isoeugenol is a compound found in plants. Therefore, the enzymatic reaction system of this embodiment may contain other components found in the plant from which isoeugenol was extracted. For example, when isoeugenol is extracted from rice bran, the enzymatic reaction system may contain phytochemicals such as γ-oryzanol, phenolic acids such as ferulic acid and p-coumaric acid, antioxidants such as tocopherols, and nutrients such as proteins, dietary fiber, and lipids. Similarly, when isoeugenol is extracted from clove products, the enzymatic reaction system may contain eugenol, terpenes such as β-caryophyllene, and aromatic components such as vanillin.

[0026] (Method for producing vanillin) This embodiment relates to a method for producing vanillin, which includes reacting isoeugenol with copper oxidase. In this embodiment, vanillin can be produced from isoeugenol in a single step without going through a complex bioconversion process.

[0027] Examples of copper oxidases used in the manufacturing method of this embodiment include the copper oxidases described above, and among them, the copper oxidase is preferably a laccase derived from the genus Trametes.

[0028] In the step of reacting isoeugenol with copper oxidase (enzyme treatment step), the isoeugenol content is not particularly limited, but is preferably 0.1% by mass or more, more preferably 0.5% by mass or more, even more preferably 1.0% by mass or more, even more preferably 1.5% by mass or more, and particularly preferably 2.0% by mass or more. By setting the isoeugenol content to be above the above lower limit, the enzyme reaction rate can be effectively increased. Furthermore, in the enzyme treatment step, the upper limit of the isoeugenol content is not particularly limited, but is preferably 30% by mass or less, preferably 10% by mass or less, more preferably 7.0% by mass or less, and even more preferably 5.0% by mass or less.

[0029] When a vanillin-producing enzyme preparation is reacted with isoeugenol, the amount of copper oxidase contained in the enzyme preparation is not particularly limited, but it is preferable to add the vanillin-producing enzyme preparation so that the copper oxidase activity is 10 U or more per 1 g of isoeugenol. From the viewpoint of further increasing the amount of vanillin produced, it is preferable to add the vanillin-producing enzyme preparation so that the copper oxidase activity per 1 g of isoeugenol is preferably 100 U or more, more preferably 1000 U or more, even more preferably 2000 U or more, even more preferably 3500 U or more, even more preferably 8000 U or more, even more preferably 12000 U or more, and particularly preferably 15000 U or more. There is no particular upper limit to the amount of vanillin-producing enzyme preparation added, but it is preferable to add the vanillin-producing enzyme preparation such that the copper oxidase activity per 1 g of isoeugenol is preferably 1,000,000 U or less, more preferably 800,000 U or less, even more preferably 500,000 U or less, even more preferably 300,000 U or less, even more preferably 200,000 U or less, and even more preferably 100,000 U or less.

[0030] The enzymatic activity of copper oxidase is measured by the following method. First, a substrate solution is prepared by mixing 0.25 mol / L phenol reagent, 0.009 mol / L 4-aminoantipyrine reagent, and 1 mol / L acetic acid / sodium acetate buffer (pH 4.5) in a volume ratio of 1:1:0.5. 2.5 ml of this substrate solution is placed in a cuvette, preheated to 30°C, then 0.5 ml of enzyme solution is added and stirred. The mixture is incubated at 30°C, and the absorbance at 505 nm is measured after 10 seconds and 40 seconds. When the absorbance increases by 0.1 per minute, the amount of enzyme contained in 1 mL of the reaction solution is defined as 1 unit (U).

[0031] The step of reacting isoeugenol with copper oxidase (enzyme treatment step) is preferably carried out under conditions where the pH is 4 or higher, more preferably 5 or higher, even more preferably 6 or higher, even more preferably 6.5 or higher, even more preferably 7 or higher, even more preferably 7.1 or higher, even more preferably 7.3 or higher, particularly preferably 7.5 or higher, and most preferably 7.7 or higher. In other words, the pH of the reaction solution in which isoeugenol is reacted with copper oxidase is preferably 4 or higher, more preferably 5 or higher, even more preferably 6 or higher, even more preferably 6.5 or higher, even more preferably 7 or higher, even more preferably 7.1 or higher, even more preferably 7.3 or higher, particularly preferably 7.5 or higher, and most preferably 7.7 or higher. The upper limit of the pH of the reaction solution is not particularly limited, but for example, the pH is preferably 10 or less, more preferably 9.7 or less, even more preferably 9.5 or less, even more preferably 9.3 or less, and even more preferably 9.1 or less. In particular, the pH in the enzyme treatment step is preferably 7.5 or higher. By carrying out the enzyme treatment reaction under such basic conditions, the oxidative polymerization reaction of isoeugenol can be suppressed, and the efficiency of vanillin production can be increased.

[0032] To maintain the pH of the reaction solution within the above range, it is preferable to add a buffer that can maintain a predetermined pH in the reaction solution. Examples of buffers that can be used include Bis-Tris buffer, acetate-NaOH buffer, phosphate buffer, glycine-NaOH buffer, formic acid-NaOH buffer, etc.

[0033] The step of reacting isoeugenol with copper oxidase (enzyme treatment step) is preferably carried out in an aqueous solvent. Specifically, the reaction solution preferably contains substantially no organic solvents, and the content of organic solvents relative to the total mass of the reaction solution in the step of reacting isoeugenol with copper oxidase (enzyme treatment step) is preferably 10% by mass or less, more preferably 5% by mass or less, even more preferably 1% by mass or less, and particularly preferably 0.1% by mass or less.

[0034] The reaction time, temperature, etc., for reacting isoeugenol with copper oxidase are not particularly limited. The reaction temperature is, for example, 10 to 80°C, preferably 20 to 70°C, and more preferably 40 to 60°C. The reaction time is, for example, 30 minutes to 72 hours, preferably 2 hours to 48 hours, and more preferably 10 hours to 30 hours. By setting the reaction temperature and reaction time outside of the above ranges, the efficiency of vanillin production can be increased. These reaction conditions are appropriately selected depending on the type of enzyme preparation used. The optimal reaction conditions can be determined through preliminary experiments.

[0035] In the step of reacting isoeugenol with copper oxidase (enzyme treatment step), it is preferable to supply oxygen. When supplying oxygen, it is preferable to actively supply air to the reaction system using a pump or the like. By sharing oxygen with the reaction system, the production efficiency when generating vanillin from isoeugenol can be more effectively increased.

[0036] By using the production method of this embodiment, vanillin can be produced. One aspect of the method for producing vanillin of the present invention includes the following steps (1) and (2). Note that an enzyme inactivation step may be added after step (2). (1) Step of preparing a raw material containing isoeugenol. (2) Step of treating the prepared raw material with an enzyme agent for vanillin production.

[0037] (Vanillin-containing composition) This embodiment may relate to a vanillin-containing composition obtained by allowing the above-described enzyme agent for vanillin production to act on a raw material containing isoeugenol. Further, this embodiment may relate to a flavor agent obtained by allowing the above-described enzyme agent for vanillin production to act on a raw material containing isoeugenol. The vanillin-containing composition and the flavor agent can be used in food and beverages, perfumes, cosmetics, personal care products, pharmaceutical intermediates, etc. Among them, the vanillin-containing composition and the flavor agent of this embodiment are preferably used in food and beverages. This embodiment may relate to food and beverages containing the vanillin-containing composition and the flavor agent.

[0038] The features of the present invention will be further specifically described with reference to the following examples. The materials, amounts used, ratios, treatment details, treatment procedures, etc. shown in the following examples can be appropriately changed without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be construed as being limited by the specific examples shown below.

[0039]

[0040]

[0041] (Method for Measuring Enzyme Activity) The activity value of multi-copper oxidase (laccase) was measured by the following method. The reagents used for measuring the activity value of multi-copper oxidase were prepared based on the test method for measuring the activity of polyphenol oxidase in the 9th edition of the Japanese Pharmacopoeia of Food Additives. Specifically, a 0.25 mol / L phenol test solution, a 0.009 mol / L 4-aminoantipyrine test solution, and a 1 mol / L acetic acid / sodium acetate buffer solution (pH 4.5) were mixed at a volume ratio of 1:1:0.5 to obtain a substrate solution. 2.5 ml of this substrate solution was taken into a cuvette, preheated at 30°C, and then 0.5 ml of an enzyme solution diluted to a predetermined concentration was added, stirred, incubated at 30°C, and the absorbance at 505 nm was measured after 10 seconds and 40 seconds. Under these conditions, when the absorbance increased by 0.1 per minute, the amount of enzyme contained in 1 mL of the reaction solution was defined as 1 unit (U).

[0042] <Test Example 1> 100 ml of a substrate solution was prepared and mixed in a medium bottle so as to have the composition shown in Table 3. The phosphate buffer solution described in Table 3 was prepared by mixing a 100 mM KH ２ HPO ４ solution and a 100 mM KH ２ PO ４ solution to a pH of 8.0.

[0043]

[0044] Trametes sp. multi-copper oxidase (laccase) manufactured by Amano Enzyme Inc. was added to the substrate solution. At this time, it was added so that the multi-copper oxidase activity was 20,000 U per 1 g of the substrate, and stirring was carried out under the conditions of 50°C and 200 rpm. The reaction was carried out while supplying air to a part of the substrate solution with an air pump (Air Compact Plus manufactured by Mizusaku Co., Ltd.). After 24 hours, 4 times the amount of methanol was added to the reaction solution to stop the reaction. The reaction stop solution was measured by HPLC, and the amount of vanillin produced was quantified. The vanillin concentration (mM) is shown in Table 4.

[0045] HPLC Measurement Conditions Column: Eclipse XDB-C18 Eluent: A solution prepared by mixing methanol:water:ethyl acetate at a volume ratio of 60:37:3 Flow rate: 0.3 mL / min Temperature: 40°C Analyzer: UV280 nm

[0046]

[0047] <Test Example 2> 5 ml of substrate solution was prepared in a test tube to match the composition shown in Table 5 and mixed. The phosphate buffer listed in Table 5 is 100 mM K ２ HPO ４ Solution and 100 mM KH ２ PO ４ The solution was prepared by mixing it to a pH of 8.0.

[0048]

[0049] Trametes sp. multi-copper oxidase (laccase) manufactured by Amano Enzyme Co., Ltd. was added to the substrate solution. The multi-copper oxidase activity was adjusted to 4000 U, 20000 U, or 40000 U per 1 g of substrate, and the reaction was carried out with stirring at 50°C and 200 rpm. After 24 hours, methanol was added in an amount four times the volume of the reaction solution to stop the reaction. The amount of vanillin produced was measured by HPLC in the stop solution to quantify the amount of vanillin produced. Table 6 shows the ratio of vanillin production under each reaction condition, with the amount of vanillin produced at an enzyme addition rate of 20000 U / g-substrate and a reaction time of 24 hours set to 1.

[0050] HPLC measurement conditions: Column: Eclipse XDB-C18; Eluent: Solution of methanol, water, and ethyl acetate mixed in a volume ratio of 60:37:3; Flow rate: 0.3 mL / min; Temperature: 40°C; Analyzer: UV 280 nm

[0051]

[0052] <Test Example 3> A substrate solution was prepared in a medium bottle to a composition shown in Table 7, and mixed. At that time, when the enzyme reaction was carried out at pH 4 or pH 5, an acetic acid-NaOH buffer solution was added; when it was carried out at pH 6, pH 7 or pH 8, a phosphate buffer solution was added; when it was carried out at pH 9 or pH 10, a glycine-NaOH buffer solution was added to adjust the pH. The acetic acid-NaOH buffer solution described in Table 7 was prepared by dissolving 1.2 g of acetic acid in an appropriate amount of purified water, mixing sodium hydroxide so that the pH became 4.0 or 5.0, and then making up to 200 ml with purified water. The phosphate buffer solution described in Table 7 was prepared by mixing 100 mM K ２ HPO ４ solution and 100 mM KH ２ PO ４ solution so that the pH became 6.0, 7.0 or 8.0. The glycine-NaOH buffer solution described in Table 7 was prepared by dissolving 1.5 g of glycine in an appropriate amount of purified water, mixing sodium hydroxide so that the pH became 9.0 or 10.0, and then making up to 200 ml with purified water.

[0053] <得

[0054] Trametes sp. multi-copper oxidase (laccase) manufactured by Amano Enzyme Inc. was added to the substrate solution. At this time, it was added so that the multi-copper oxidase activity became 20,000 U per 1 g of the substrate, and stirring was carried out under the conditions of 50 °C and 200 rpm, and the reaction was carried out while supplying air with an air pump (Air-Max 12000 C-4 manufactured by Mizusaku Co., Ltd.). After 24 hours, 4 times the amount of methanol was added to the reaction solution to stop the reaction. The reaction stop solution was measured by HPLC to quantify the vanillin production amount. Table 8 shows the ratio of the vanillin production amount under each reaction condition when the vanillin production amount at pH 8.0 was taken as 1.

[0055] HPLC measurement conditions Column: Eclipse XDB-C18 Eluent: A solution prepared by mixing methanol: water: ethyl acetate in a volume ratio of 60:37:3 Flow rate: 0.3 mL / min Temperature: 40 °C Analyzer: UV280 nm

[0056]

Claims

1. A vanillin-producing enzyme preparation containing copper oxidase, which produces vanillin from isoeugenol.

2. The vanillin-producing enzyme preparation according to claim 1, wherein the copper oxidase is a laccase derived from the genus Trametes.

3. A flavor enhancer for food and beverages that contains copper oxidase and generates vanillin from food and beverages containing isoeugenol.

4. The flavor enhancer for food and beverages according to claim 3, wherein the copper oxidase is a laccase derived from the genus Trametes.

5. Food or beverage containing the flavor enhancer for food or beverage according to claim 3 or 4.

6. A method for producing vanillin, comprising reacting isoeugenol with copper oxidase.

7. The method for producing vanillin according to claim 6, wherein the copper oxidase is a laccase derived from the genus Trametes.

8. The method for producing vanillin according to claim 6 or 7, wherein the reaction of the isoeugenol with the copper oxidase is carried out under conditions of pH 7.5 or higher.

9. A vanillin-containing composition obtained by reacting the vanillin-producing enzyme preparation according to claim 1 or 2 with a raw material containing isoeugenol.

10. A flavoring agent obtained by reacting the vanillin-producing enzyme agent according to claim 1 or 2 with a raw material containing isoeugenol.

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