JP2024059796A - Composition for the production of equol derivatives - Google Patents

Abstract

The present invention provides a composition for producing an equol derivative, and a method for producing an equol derivative. The present invention provides a composition comprising an equol-containing composition and an enzyme or microorganism capable of converting equol in the equol-containing composition into an equol derivative. [Selected Figure] Figure 1

Description

The present invention relates to a composition for producing equol derivatives and a method for producing equol derivatives.

Equol is known to have the highest estrogenic activity among the isoflavone metabolites contained in soybeans (Non-Patent Documents 1 and 2).
Similarly to equol, equol derivatives such as 5-hydroxyequol have been reported to have estrogen-like activity and 3β-hydroxysteroid dehydrogenase inhibitory activity (Patent Document 1). By inhibiting the biosynthesis of aldosterone and glucocorticoids, they are expected to be useful in the prevention or treatment of excesses of these hormones.
Furthermore, in a report on a method for synthesizing isoflavan derivatives using an o-quinone methide-based technique, it has been disclosed that 3'-hydroxyequol can be obtained by chemical synthesis (Non-Patent Document 3).

On the other hand, enzymes called flavin-dependent oxidases are known. The substrate specificity and action of the enzymes vary depending on the type. For example, it has been reported that HpaB and HpaC possessed by Pseudomonas aeruginosa PAO1 strain are active against hydroxystilbene (resveratrol). A method for producing hydroxystilbene by allowing a substrate compound to react with a protein having 50% or more homology at the amino acid level with HpaB and HpaC derived from Pseudomonas aeruginosa PAO1 strain has been reported (Patent Documents 2 and 3).

However, there is no known method for obtaining equol derivatives from equol through biosynthesis (fermentation) using enzymes or microorganisms.

JP 2003-81875 A JP 2015-130819 A JP 2017-074019 A

Adlercreutz, H., The Lancet Oncol., 3, 364-373 (2002) Duncan, A. M. et al., Best Pract. Res. Clin. Endocrinol. Metab., 17, 253-271 (2003) Tetrahedron Letters (2008), 49(18), 2974-2978

The present invention aims to provide a composition for producing equol derivatives and a method for producing equol derivatives.

As a result of extensive research, the inventors have discovered that the above problems can be solved by using an equol-containing composition and an enzyme or microorganism capable of converting the equol in the equol-containing composition into an equol derivative, and have completed the present invention.
The present invention is as follows.

[1] A composition comprising an equol-containing composition and an enzyme or microorganism capable of converting equol in the equol-containing composition into an equol derivative.
[2] The composition according to [1], wherein the equol-containing composition is equol, a fermented soybean germ extract, a fermented soybean hypocotyl, a fermented soybean, or an alfalfa fermented product.
[3] The composition according to [1] or [2], wherein the enzyme or microorganism is a flavin-dependent oxidase or a microorganism having equol oxidation activity.
[4] The composition according to any one of [1] to [3], wherein the equol derivative is 3'-hydroxyequol or 6-hydroxyequol.
[5] A method for producing an equol derivative, comprising the step of treating an equol-containing composition with an enzyme or microorganism capable of converting equol in the equol-containing composition to an equol derivative.
[6] The manufacturing method according to [5], wherein the step of subjecting the equol-containing composition to an enzyme or microorganism capable of converting equol in the equol-containing composition to an equol derivative comprises a step of culturing the microorganism in a medium containing the equol-containing composition to produce the equol derivative by fermentation from the equol in the equol-containing composition, and a step of recovering the produced equol derivative.
[7] The method according to [5] or [6], wherein the equol derivative is 3'-hydroxyequol or 6-hydroxyequol.
[8] The production method according to any one of [5] to [7], wherein the enzyme or microorganism is a flavin-dependent oxidase or a microorganism having equol oxidation activity.
[9] A method for producing a food or beverage containing an equol derivative, comprising: a step of producing an equol derivative by treating an equol-containing composition with an enzyme or microorganism capable of converting equol in the equol-containing composition to an equol derivative; and a step of blending the equol derivative with ingredients of the food or beverage.
[10] A method for producing a pharmaceutical product containing an equol derivative, comprising the steps of: producing an equol derivative by treating an equol-containing composition with an enzyme or microorganism capable of converting equol in the equol-containing composition to an equol derivative; and blending the equol derivative with a pharmaceutical raw material.
[11] A polynucleotide represented by the following (a) or (b):
(a) a polynucleotide encoding a protein consisting of an amino acid sequence selected from SEQ ID NOs: 1 to 8;
(b) a polynucleotide comprising one base sequence selected from SEQ ID NOs: 9 to 16.
[12] A polynucleotide represented by the following (c) or (d):
(c) a polynucleotide encoding a protein having the activity of converting equol into an equol derivative, the protein consisting of an amino acid sequence selected from SEQ ID NOs: 1 to 8, in which one or more amino acids are substituted or deleted, or one or more amino acids are inserted or added.
(d) a polynucleotide that hybridizes under stringent conditions with DNA consisting of one base sequence selected from SEQ ID NOs: 9 to 16 and encodes a protein having the activity of converting equol into an equol derivative.
[13] A vector into which the polynucleotide (a) or (b) according to [11], or the polynucleotide (c) or (d) according to [12] is inserted.

The present invention provides a composition for producing an equol derivative and a method for producing an equol derivative. In addition, a subject can obtain the known effects of the equol derivative by ingesting a food, drink, or medicine containing the equol derivative.

HPLC chromatograms showing the results of culturing E. coli containing a vector carrying a gene encoding HpaB _ro-3 and equol according to one embodiment of the present invention. The upper graph is for the case where the substrate is (S)-equol, and the lower graph is for the case where the substrate is (R)-equol. Graph showing the amount of 3'-hydroxyequol produced by E. coli containing vectors linking each gene according to one embodiment of the present invention, where the white bars are for when the substrate is (S)-equol and the black bars are for when the substrate is (R)-equol. Graphs showing the amount of 3'-hydroxyequol produced on a flask scale using E. coli carrying a vector containing a gene encoding HpaB _ro-3 according to one embodiment of the present invention. The graph on the left is for when the substrate is (S)-equol, and the graph on the right is for when the substrate is (R)-equol. HPLC chromatograms showing the results of culturing E. coli containing a vector carrying a gene encoding HpaB _pl-1 and equol according to one embodiment of the present invention. The upper graph is for the case where the substrate is (S)-equol, and the lower graph is for the case where the substrate is (R)-equol. Graphs showing the amount of 6-hydroxyequol produced on a flask scale using E. coli carrying a vector containing a gene encoding HpaB _pl-1 according to one embodiment of the present invention. The graph on the left is for when the substrate is (S)-equol, and the graph on the right is for when the substrate is (R)-equol.

The present invention will be described in detail below.
The composition described herein is a concept that includes a mixture, regardless of whether the components are homogeneous or heterogeneous.

(Composition)
One aspect of the present invention is a composition comprising an equol-containing composition and an enzyme or microorganism capable of converting equol in the equol-containing composition into an equol derivative.

There are no limitations on the form of the equol-containing composition, so long as the equol in the equol-containing composition is converted to an equol derivative by an enzyme or microorganism capable of converting the equol into an equol derivative.
Examples of the equol-containing composition include fermented products of legumes such as soybeans, kidney beans, broad beans, peanuts, chickpeas, kudzu, red clover, alfalfa, licorice, etc. In other words, if the legume is soybeans, the equol-containing composition is a fermented product of soybeans themselves (soybean fermented product).
In addition, the equol-containing composition may be a fermented product of a part of soybean, such as a fermented product of soybean germ parts (fermented soybean germ product) or a fermented product of soybean hypocotyl parts (fermented soybean hypocotyl product) in the case of soybean among the legumes. Furthermore, in the case of soybean germ fermentation, it may be a fermented product obtained by fermenting an extract obtained from soybean germ parts (fermented soybean germ extract product).
The soybean fermented product is a fermented product obtained by fermenting whole soybeans, and is different from fermented products obtained by fermenting a part of soybeans, such as a soybean germ fermented product obtained by using soybean germs and a soybean hypocotyl fermented product obtained by using soybean hypocotyls.

As an example of the mode of fermentation, in the case of obtaining a fermented product of soybeans themselves (soybean fermented product), the legume plant is prepared, and then Aspergillus oryzae is added thereto and fermented to obtain a fermented product. As for the mode of preparation, soybeans themselves may be prepared as they are, or soybeans may be prepared which have been subjected to a heating treatment, drying treatment, or steaming treatment and then mashed ....

The equol-containing composition may be a fermented product of isoflavones derived from the legumes, such as isoflavone aglycones obtained by fermenting isoflavones derived from soybeans with an enzyme such as β-glucosidase or a microorganism.

The equol-containing composition may be equol, and may be either (R)-equol or (S)-equol.

In this embodiment, the enzyme capable of converting equol in the equol-containing composition to an equol derivative is not limited as long as it can convert equol in the equol-containing composition to an equol derivative.

The enzyme may be an enzyme produced by a microorganism, or an enzyme obtained without relying on microbial production.
In the case of an enzyme produced by a microorganism, the microorganism may be a microorganism that originally expresses the enzyme, or a microorganism that has been made to express the enzyme by known techniques such as genetic recombination.
When an enzyme produced by a microorganism is to be recovered, a known method can be used as the recovery method. For example, after culturing a microorganism, the microorganism is collected by a method such as filtration or centrifugation, and the microorganism is washed with a buffer solution or physiological saline, and the enzyme can be recovered by, for example, physical treatment (e.g., freeze-thaw treatment, ultrasonic treatment, pressure treatment, osmotic pressure difference treatment, grinding treatment, etc.), biochemical treatment (e.g., treatment with a cell wall-lytic enzyme such as lysozyme, etc.), or chemical treatment (e.g., contact treatment with a surfactant, etc.), either alone or in combination. The recovered enzyme may then be subjected to separation and purification treatment.

Examples of enzymes that can convert equol in the equol-containing composition to an equol derivative include oxidoreductases (dehydrogenases, cytochromes, catalases, oxidases, oxygenases (e.g., flavin-dependent oxidases, etc.), fatty acid desaturases, etc.), transferases (acyltransferases, phosphotransferases, aminotransferases, etc.), protein degrading enzymes (proteases), lipid degrading enzymes (lipases), carbohydrate degrading enzymes (amylase, lysozyme, β-galactosidase, etc.), phosphate degrading enzymes (nucleases, phosphatases, restriction enzymes), hydrolases (urease, lactonase, ATP hydrolase, etc.), lyases (carbonic hydratase, pyruvate decarboxylase, etc.), isomerases (racemases, phosphoglycerate phosphomutase, glucose-6-phosphate isomerase, etc.), and synthetic enzymes (DNA ligase, aminoacyl-tRNA synthetases, acyl-CoA synthetases, carboxylases, etc.).
Preferably it is an oxygenase, more preferably a flavin-dependent oxidase.

Flavin-dependent oxidases include flavin-dependent monooxygenases, and enzymes that supply reduced flavins to flavin-dependent oxidases include enzymes consisting of NAD(P)H and flavin oxidoreductases. The former includes HpaB, and the latter includes HpaC.
As HpaB, HpaB _derived from Pseudomonas aeruginosa PAO1 (SEQ ID NO: 1) is preferable.
), HpaB _ec (sequence number 2) derived from Escherichia coli BL21 (DE3), HpaB _pl-1 (sequence number 3), HpaB _pl-2 (sequence number 4), and HpaB _pl-3 (sequence number 5) derived from Photorhabdus luminescens sub sp. laumondii TTO1, and HpaB _ro-1 (sequence number 6), HpaB _ro-2 (sequence number 7), and HpaB _ro-3 (sequence number 8) derived from Rhodococcus opacus B4.

HpaB _pa (SEQ ID NO: 1) derived from Pseudomonas aeruginosa PAO1 may be an amino acid sequence that has 80% or more, preferably 90% or more, and more preferably 95% or more identity to the amino acid sequence represented by SEQ ID NO: 1, as long as it is capable of converting equol in an equol-containing composition into an equol derivative.
The same applies to each HpaB (SEQ ID NOs: 2 to 8) other than the above-mentioned HpaB _pa .

The microorganism capable of converting equol in the equol-containing composition to an equol derivative is not limited in type, so long as it can convert equol in the equol-containing composition to an equol derivative, and is preferably a microorganism having equol oxidizing activity (equol oxygenase), more preferably a microorganism expressing the flavin-dependent oxidase.
The microorganism may be a microorganism that is originally capable of converting equol to an equol derivative, or may be a microorganism that has been modified by known techniques such as genetic recombination so that it can convert equol to an equol derivative. There are no limitations on the genus or species to which the microorganism belongs, and examples of the microorganism include Escherichia coli, yeast, and filamentous fungi.

The equol derivative is not particularly limited, but is preferably hydroxylated equol or methoxylated equol, such as 3'-hydroxyequol, 5-hydroxyequol, 3'-methoxyequol, 6-methoxyequol, and 6-hydroxyequol.
More preferred are hydroxides, such as 3'-hydroxyequol, 5-hydroxyequol, and 6-hydroxyequol, and even more preferred are 3'-hydroxyequol and 6-hydroxyequol.
When _HpaBpa , _HpaBec , HpaBpl _-2 , HpaBpl _-3 , HpaBro _-1 , HpaBro _-2 , or HpaBro _-3 is used as the flavin-dependent oxidase, equol is converted to 3'-hydroxyequol, and when HpaBpl _-1 is used as the flavin-dependent oxidase, equol is converted to 6-hydroxyequol.

(Production method)
Another aspect of the present invention is a method for producing an equol derivative, comprising the step of treating an equol-containing composition with an enzyme or microorganism capable of converting equol in the equol-containing composition to an equol derivative.

The above explanations are incorporated herein with respect to the equol-containing composition in this embodiment, and the enzymes and microorganisms capable of converting equol in the equol-containing composition into an equol derivative.

In this embodiment, the step of subjecting the equol-containing composition to an enzyme capable of converting equol in the equol-containing composition to an equol derivative includes a step of reacting the equol in the equol-containing composition with the enzyme in a solution containing the equol-containing composition.

Examples of solutions containing the equol-containing composition include water, organic solvents, and two-phase mixtures of an organic solvent and an aqueous medium.
Examples of the organic solvent include ethyl acetate, butyl acetate, toluene, chloroform, and n-hexane.
Examples of the aqueous medium include ethanol and acetone.

Additionally, an inclusion compound may be added to increase the solubility of the substrate equol.
Examples of the inclusion compound include α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, cluster dextrin (highly branched cyclic dextrin), and their analogues, such as methyl-β-cyclodextrin, trimethyl-β-cyclodextrin, and hydroxypropyl-β-cyclodextrin.
The amount of the inclusion compound added is, in terms of molar ratio, typically 0.1 equivalents or more, preferably 0.5 equivalents or more, and more preferably 1.0 equivalents or more relative to the equol in the equol-containing composition, and is typically 5.0 equivalents or less, preferably 2.5 equivalents or less, and more preferably 2.0 equivalents or less.

The reaction temperature is preferably 20°C to 45°C, more preferably 25°C to 40°C, and further preferably 30°C to 37°C.
The reaction time is preferably 8 to 340 hours, more preferably 12 to 170 hours, and even more preferably 16 to 120 hours.
The pH of the reaction solution is preferably 4 to 10, more preferably 6 to 8.

In this embodiment, when an enzyme produced by a microorganism is used as the enzyme capable of converting equol in the equol-containing composition to an equol derivative, the method may include a step of recovering the enzyme from the microorganism that produces the enzyme prior to the step of allowing the enzyme to act on the equol-containing composition, and may include a step of culturing the microorganism that produces the enzyme prior to that.

The step of culturing the microorganism that produces the enzyme is not particularly limited as long as it is carried out under conditions that allow the microorganism to produce the enzyme. For example, the conditions for culturing the microorganism in a medium containing an equol-containing composition, which will be described later, can be mentioned.

The process of recovering the enzyme from the microorganism that produces the enzyme may include a process of collecting the microorganism by a method such as filtration or centrifugation, and then a process of washing the microorganism with a buffer solution, physiological saline, or the like. Examples of the washing process include a process of physical treatment (e.g., freeze-thaw treatment, ultrasonic treatment, pressure treatment, osmotic pressure difference treatment, grinding treatment, etc.), a biochemical treatment (e.g., treatment with a cell wall-lytic enzyme such as lysozyme, etc.), and a chemical treatment (e.g., contact treatment with a surfactant, etc.), which may be used alone or in combination. In addition, a process of purifying or concentrating the enzyme may be included thereafter.

In this embodiment, the step of subjecting the equol-containing composition to a microorganism capable of converting equol in the equol-containing composition to an equol derivative includes a step of causing the microorganism to convert equol in the equol-containing composition to an equol derivative in a solution containing the equol-containing composition.
For example, this is a step of culturing the microorganism in a medium containing an equol-containing composition and producing an equol derivative from equol in the equol-containing composition by fermentation.

A solution containing an equol-containing composition may be a solution in which the microorganisms can grow or not grow.

Examples of the solution in which the microorganisms can grow include culture media, which may be minimal or synthetic media. Commercially available media include, for example, ANAEROBE BASAL BROTH (ABB medium) manufactured by Oxoid, Wilkins-Chalgren Anaerobe Broth (CM0643) manufactured by Oxoid, GAM medium, modified GAM medium, brain heart infusion medium, LB medium, and the like manufactured by Nissui Pharmaceutical Co., Ltd.

Water-soluble organic matter can be added to the medium as a carbon source. Examples of water-soluble organic matter include sugars such as glucose, arabinose, sorbitol, fructose, mannose, sucrose, trehalose, xylose, galactose, starch, starch hydrolysates, molasses, and blackstrap molasses; natural carbohydrates such as wheat and corn; alcohols such as glycerol, methanol, and ethanol; hydrocarbons such as normal paraffin; organic acids such as valeric acid, butyric acid, propionic acid, acetic acid, formic acid, fumaric acid, gluconic acid, pyruvic acid, and citric acid; and amino acids such as glycine, glutamine, and asparagine.
The concentration of the organic matter can be appropriately adjusted for efficient growth, and is generally in the range of 0.1 to 10 wt/vol %.

In addition to the carbon source, the medium may contain a nitrogen source, which may be any of various nitrogen compounds that can be used in normal fermentation.
Preferred inorganic nitrogen sources include ammonium salts, nitrates, urea, and the like, and more preferred are ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium hydrogen phosphate, ammonium nitrate, sodium nitrate, potassium nitrate, and sodium nitrate.
Examples of organic nitrogen sources include amino acids, milk casein, casamino acids, corn steep liquor, yeast extract, peptones (e.g., polypeptone N, soybean peptone, etc.), meat extracts (e.g., Ehrlich bonito extract, Lab-Remco powder, bouillon, etc.), seafood extracts, liver extracts, digestive serum powder, fish oil, etc.

In addition to carbon and nitrogen sources, growth and activity can sometimes be enhanced by adding cofactors such as vitamins and inorganic compounds such as various salts to the medium. For example, the following are examples of microbial growth cofactors derived from plants and animals, such as inorganic compounds, vitamins, and fatty acids.

Inorganic compounds Vitamins Potassium dihydrogen phosphate Biotin Magnesium sulfate Folic acid Manganese sulfate Pyridoxine Sodium chloride Thiamine Cobalt chloride Riboflavin Calcium chloride Nicotinic acid Zinc sulfate Pantothenic acid Copper sulfate Vitamin B12
Alum, Thiooctic acid, Sodium molybdate, p-aminobenzoic acid, Potassium chloride, Vitamin K
Boric acid, etc. Nickel chloride Sodium tungstate Sodium selenate Ammonium ferrous sulfate Sodium acetate trihydrate Magnesium sulfate heptahydrate Manganese sulfate tetrahydrate

Furthermore, growth may be improved by adding known reducing agents such as L-cysteine (hydrochloride), thioglycolic acid, ascorbic acid, mercaptoacetic acid, thiolacetic acid, glutathione, sodium sulfide, sodium sulfide, sulfite, thioglycolic acid, and rutin, reducing activators such as L-cystine, and enzymes that break down reactive oxygen species such as catalase and superoxide mutase to the medium.

As a culture method, for example, conventional culture methods such as shaking culture, aeration and agitation culture, continuous culture, and fed-batch culture can be used.

The gas phase and aqueous phase during culture preferably do not contain air or oxygen, and may, for example, contain nitrogen and/or hydrogen in any ratio, or nitrogen and/or carbon dioxide in any ratio, and preferably are gas phases and aqueous phases containing hydrogen. The proportion of hydrogen in the gas phase is usually 0.5% or more, preferably 1.0% or more, more preferably 2.0% or more, since this promotes the production of equol derivatives, while it is usually 100% or less, preferably 20% or less, more preferably 10% or less.

The method for creating such an environment for the gas phase and aqueous phase during culture is not particularly limited, but may be, for example, a method of replacing the gas phase with the gas before culture, a method of supplying the gas from the bottom of the culture vessel and/or supplying the gas to the gas phase part of the culture vessel during culture, or a method of bubbling the aqueous phase with the gas before culture. As the hydrogen, hydrogen gas may be used as it is. Alternatively, a source of hydrogen such as formic acid and/or a salt thereof may be added to the culture medium, and hydrogen may be generated during culture by the action of the microorganism.

The amount of ventilation is, for example, 0.005 to 2 vvm, and preferably 0.05 to 0.5 vvm. The mixed gas can also be supplied in the form of nanobubbles.
The culture temperature is preferably 20°C to 45°C, more preferably 25°C to 40°C, and even more preferably 30°C to 37°C.
The pressurization conditions for the culture vessel are not particularly limited as long as they allow growth, and are, for example, 0.001 to 1 MPa, preferably 0.01 to 0.5 MPa.
The culture time is usually 8 to 340 hours, preferably 12 to 170 hours, more preferably 16 to 120 hours.
The pH of the medium at the start of the culture is usually 4 to 10, preferably 6 to 8.

Furthermore, the production of equol derivatives may be promoted by adding a surfactant, adsorbent, inclusion compound, etc. to the medium.
Examples of surfactants include SDS, TritonX-100, Tween80, and Adekanol, and can be added at about 0.001 g/L to 10 g/L.
Examples of the adsorbent include cellulose and its derivatives; dextrin; hydrophobic adsorbents such as the Diaion HP series and Sepabeads series manufactured by Mitsubishi Chemical Corporation; and the Amberlite XAD series manufactured by Organo Corporation.
Examples of the inclusion compound include α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, cluster dextrin (highly branched cyclic dextrin), and their analogues, such as methyl-β-cyclodextrin, trimethyl-β-cyclodextrin, and hydroxypropyl-β-cyclodextrin.
The amount of the inclusion compound added is, in terms of molar ratio, usually 0.1 equivalents or more, preferably 0.5 equivalents or more, and more preferably 1.0 equivalents or more, relative to the equol in the equol-containing composition, and is usually 5.0 equivalents or less, preferably 2.5 equivalents or less, and more preferably 2.0 equivalents or less.

This embodiment may include, for example, a step of quantifying the obtained equol derivative. The quantification method may follow a conventional method. For example, a portion of the culture medium may be taken, appropriately diluted, thoroughly stirred, and then filtered using a membrane such as a polytetrafluoroethylene (PTFE) membrane to remove insoluble matter, followed by quantifying the amount by high performance liquid chromatography.

This embodiment may also include a step of recovering the obtained equol derivative. The recovery step includes a purification step, a concentration step, etc. Purification treatments in the purification step include sterilization of microorganisms by heat or the like; sterilization by microfiltration (MF), ultrafiltration (UF), etc.; removal of solids and polymeric substances; extraction with organic solvents, ionic liquids, etc.; extraction with hydrophobic adsorbents, ion exchange resins,
Treatments such as adsorption and decolorization can be carried out using an activated carbon column, etc. Concentration treatments in the concentration step include concentration using an evaporator, a reverse osmosis membrane, etc.
Furthermore, a solution containing the equol derivative can be powdered by freeze-drying, spray-drying, etc. In the powdering process, an excipient such as lactose, dextrin, corn starch, etc. can be added.

Examples of solutions in which the microorganisms cannot grow include salt solutions and buffer solutions. An example of a salt solution is physiological saline. Examples of buffer solutions include phosphate buffer, Tris-HCl buffer, citrate-phosphate buffer, citrate buffer, MOPS buffer, acetate buffer, and glycine buffer. The pH and concentration of the buffer can be appropriately adjusted according to conventional methods.

Such a solution in which the microorganism cannot grow can be used, for example, when the microorganism is a resting cell. The term "resting cell" refers to a cell that is not growing and is obtained by removing medium components from a cultured microorganism by centrifugation or other procedures, washing with a salt solution or buffer, and suspending the cell in the same liquid as the washing liquid. In this embodiment, the term refers to a cell that has at least a metabolic system capable of producing an equol derivative from equol.

The case where the solution may be either one in which the microorganism can grow or one in which the microorganism cannot grow is, for example, a case where an immobilized microorganism is used as the microorganism, which is a microorganism immobilized by a known method such as a polyacrylamide gel method, a sulfur-containing polysaccharide gel method (carrageenan gel method), an alginic acid gel method, or an agar gel method.
In this specification, such gels that fix microorganisms are also defined as solutions.

Another aspect of the present invention is a method for producing a pharmaceutical product containing an equol derivative, comprising the steps of: producing an equol derivative by treating an equol-containing composition with an enzyme or microorganism capable of converting equol in the equol-containing composition into an equol derivative; and blending the equol derivative with a pharmaceutical raw material.

The process for producing the equol derivative is the same as that described above. In this embodiment, it is preferable to include a process for recovering the produced equol derivative after the process for producing the equol derivative.

The raw materials for pharmaceuticals can be any commonly used raw materials for pharmaceuticals, and there are no particular restrictions on the timing of their addition.

The pharmaceuticals produced can be used as pharmaceuticals for preventing or treating diseases that can be prevented or treated by ingestion or administration of equol derivatives. The dosage form can be selected according to the disease to be prevented or treated, the form of use of the pharmaceutical, the route of administration, etc. Examples include internal medicines such as tablets, granules, powders, capsules, soft capsules, and syrups; external medicines such as emulsions, suspensions, ointments, creams, lotions, gels, sprays, patches, poultices, liniments, aerosols, ointments, packs, inhalants, and suppositories; and injections. These various preparations can be formulated according to the usual method using known auxiliary agents that can be normally used in the pharmaceutical formulation technology field, such as fillers, bulking agents, excipients, binders, moisturizing agents, disintegrants, surfactants, lubricants, colorants, flavoring agents, dissolving agents, suspending agents, and coating agents, in addition to the main drug, as necessary. In addition, colorants, preservatives, fragrances, flavoring agents, sweeteners, and other pharmaceuticals may be contained in the pharmaceuticals.

The content of the equol derivative in the total amount of the pharmaceutical product is not particularly limited, but is preferably a content that allows the desired effect of the equol derivative to be obtained when the pharmaceutical product is ingested or administered.
The content is preferably 0.001 to 50% by mass, and more preferably 0.01 to 50% by mass.

Another aspect of the present invention is a method for producing a food or beverage containing an equol derivative, comprising the steps of: producing an equol derivative by acting on an equol-containing composition with an enzyme or microorganism capable of converting equol in the equol-containing composition to an equol derivative; and blending the equol derivative with ingredients for the food or beverage.

The process for producing the equol derivative is the same as that described above. In this embodiment, it is preferable to include a process for recovering the produced equol derivative after the process for producing the equol derivative.

The ingredients of the food and beverage products can be any ingredients that are commonly used for food and beverage products, and there are no particular restrictions on the time of mixing.

The food and drink products produced may contain water, proteins, carbohydrates, lipids, vitamins, minerals, organic acids, organic bases, fruit juice, flavors, etc. as main components.
Examples of proteins include animal and vegetable proteins such as whole milk powder, skim milk powder, partially skim milk powder, casein, soy protein, egg protein, and meat protein, as well as hydrolysates thereof, and butter.
Examples of carbohydrates include sugars, modified starches (dextrin, soluble starch, British starch, oxidized starch, starch esters, starch ethers, etc.), and dietary fibers.
Examples of lipids include vegetable oils and fats such as lard, safflower oil, corn oil, rapeseed oil, coconut oil, fish oil, fractionated oils thereof, hydrogenated oils, and interesterified oils.
Examples of vitamins include vitamin A, carotenes, B vitamins, vitamin C, D vitamins, vitamin E, K vitamins, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline, and folic acid.
Examples of minerals include calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, selenium, and whey minerals.
Examples of organic acids include malic acid, citric acid, lactic acid, and tartaric acid.
These components may be used in combination of two or more kinds, or may be synthetic products.

Specific examples of the foods and beverages to be produced include general foods and beverages, as well as foods for specified health uses, nutritional supplements, functional foods, and foods for the sick. There are no particular limitations on the form of these foods and beverages, but specific examples include main dishes such as bread and noodles; side dishes such as cheese, ham, sausages, and processed seafood products; beverages such as fruit juice drinks, carbonated drinks, and milk drinks; luxury items such as cookies, cakes, jellies, puddings, candies, and yogurt; and supplements such as capsules (soft capsules and hard capsules), tablets, granules, powders, jellies, liposome preparations, and nutritional drinks. Of these foods and beverages, supplements are preferred, and capsules are more preferred.

The content of the equol derivative in the total amount of the food or beverage produced is not particularly limited, but it is preferable that the content be such that the effect of the equol derivative can be obtained when the food or beverage is ingested. The content of the equol derivative in the total amount of the food or beverage is usually 0.0001 to 50% by mass, preferably 0.001 to 50% by mass, and more preferably 0.01 to 50% by mass.

When the food or beverage is a supplement, it may be in the form of a solid, gel, or liquid, for example, various processed foods and beverages, powders, tablets, pills, capsules, jellies, granules, etc.

The supplement may also contain additives such as excipients such as dextrin, preservatives such as vitamin C, flavorings such as vanillin, colorants such as safflower pigment, monosaccharides, oligosaccharides and polysaccharides (e.g., glucose, fructose, sucrose, saccharose, and carbohydrates containing these), acidulants, flavorings, oils and fats, emulsifiers, whole milk powder, or agar. Two or more of these ingredients may be used in combination, or may be synthetic products.

Another aspect of the present invention is a method for producing a cosmetic product containing an equol derivative, comprising the steps of: producing an equol derivative by acting on an equol-containing composition with an enzyme or microorganism capable of converting equol in the equol-containing composition to an equol derivative; and blending the equol derivative with a cosmetic ingredient.

The process for producing the equol derivative is the same as that described above. In this embodiment, it is preferable to include a process for recovering the produced equol derivative after the process for producing the equol derivative.

As the raw materials for cosmetics, raw materials for cosmetics that are commonly used can be used, and the timing of their blending is not particularly limited.
Examples of cosmetics produced include creams, milky lotions, skin lotions, packs, cleansers, makeup cosmetics, scalp and hair products, oils, lip products, lipsticks, foundations, eyeliners, blushers, mascara, eye shadows, manicures and pedicures (and removers), shampoos, rinses, hair treatments, permanents, hair dyes, shaving products, soaps (hand soaps, body soaps, facial cleansers), and the like.

The content of the equol derivative in the total amount of the cosmetic product is not particularly limited, but it is preferable that the content be such that the desired effect of the equol derivative can be obtained when the cosmetic product is used. The content of the equol derivative in the total amount of the cosmetic product is usually 0.000001 to 10% by mass, preferably 0.00001 to 10% by mass, and more preferably 0.0001 to 10% by mass.

Another aspect of the present invention is a method for producing a feed containing an equol derivative, comprising the steps of: producing an equol derivative by treating an equol-containing composition with an enzyme or microorganism capable of converting equol in the equol-containing composition into an equol derivative; and blending the equol derivative with feed ingredients.
The feed of this embodiment includes feed (pet food) for companion animals such as dogs and cats.

The process for producing the equol derivative is the same as that described above. In this embodiment, it is preferable to include a process for recovering the produced equol derivative after the process for producing the equol derivative.

As the raw materials for the feed, any raw materials for feed that are commonly used can be used, and the timing of their addition is not particularly limited.
Specific examples of feed ingredients include grains such as corn, wheat, barley, rye, etc.; bran, rice bran, etc.; meal, such as corn gluten meal, corn jam meal, etc.; animal feeds, such as skim milk powder, whey, fish meal, bone meal, etc.; yeasts, such as brewer's yeast; calcium compounds, such as calcium phosphate, calcium carbonate, etc.; vitamins; fats and oils; amino acids; sugars, etc.

The content of the equol derivative relative to the total amount of the produced feed is not particularly limited, but it is preferable that the content is such that the effect of the equol derivative can be obtained when the feed is ingested.
The content is preferably 0.0001 to 10% by mass, and more preferably 0.001 to 10% by mass.

Another aspect of the present invention is a polynucleotide of (a) or (b) below:
(a) a polynucleotide encoding a protein consisting of an amino acid sequence selected from SEQ ID NOs: 1 to 8;
(b) a polynucleotide comprising one base sequence selected from SEQ ID NOs: 9 to 16.

SEQ ID NO: 1 is the amino acid sequence of the enzyme _HpaBpa encoded by the gene _hpaBpa (ORF No. PA4091) of flavin-dependent oxidase derived from Pseudomonas aeruginosa PAO1.
SEQ ID NO: 2 is the amino acid sequence of the enzyme _HpaBec encoded by the gene _hpaBpa (ORF No. B21_04188) of flavin-dependent oxidase derived from Escherichia coli BL21 (DE3).
SEQ ID NO: 3 is the amino acid sequence of the enzyme HpaB _pl-1 encoded by the flavin-dependent oxidase gene hpaB _pl-1 (ORF No. plu0246) derived from Photorhabdus luminescens subsp. laumondii TTO1.
SEQ ID NO: 4 is the amino acid sequence of the enzyme HpaB _pl-2 encoded by the flavin-dependent oxidase gene (ORF No. plu0975) derived from Photorhabdus luminescens subsp. laumondii TTO1.
SEQ ID NO:5 is the amino acid sequence of the enzyme HpaB _pl-3 encoded by the flavin-dependent oxidase gene (ORF No. plu4027) derived from Photorhabdus luminescens subsp. laumondii TTO1.
SEQ ID NO: 6 is the amino acid sequence of the enzyme HpaB _ro-1 encoded by the flavin-dependent oxidase gene (ORF No. ROP_20940) derived from Rhodococcus opacus B4.
SEQ ID NO: 7 is the amino acid sequence of the enzyme HpaB _ro-2 encoded by the flavin-dependent oxidase gene (ORF No. ROP_22410) derived from Rhodococcus opacus B4.
SEQ ID NO: 8 is the amino acid sequence of the enzyme HpaB _ro-3 encoded by the flavin-dependent oxidase gene (ORF No. ROP_37410) derived from Rhodococcus opacus B4.

SEQ ID NO:9 is the nucleotide sequence of the gene _hpaBpa , which is a flavin-dependent oxidase gene derived from Pseudomonas aeruginosa PAO1 and corresponds to ORF No. PA4091.
SEQ ID NO: 10 is the nucleotide sequence of the gene _hpaBec , which is a flavin-dependent oxidase gene derived from Escherichia coli BL21 (DE3) and corresponds to ORF No. B21_04188.
SEQ ID NO: 11 is the nucleotide sequence of the gene hpaB _pl-1 , which is a flavin-dependent oxidase gene derived from Photorhabdus luminescens subsp. laumondii TTO1 and corresponds to ORF No. plu0246.
SEQ ID NO: 12 is the nucleotide sequence of the gene hpaB _pl-2 , which is a flavin-dependent oxidase gene derived from Photorhabdus luminescens subsp. laumondii TTO1 and corresponds to ORF No. plu0975.
SEQ ID NO: 13 is the nucleotide sequence of the gene hpaB _pl-3 , which is a flavin-dependent oxidase gene derived from Photorhabdus luminescens subsp. laumondii TTO1 and corresponds to ORF No. plu4027.
SEQ ID NO: 14 is the nucleotide sequence of the gene hpaB _ro-1 , which is a flavin-dependent oxidase gene derived from Rhodococcus opacus B4 and corresponds to ORF No. ROP_20940.
SEQ ID NO: 15 is the nucleotide sequence of the gene hpaB _ro-2 , which is a flavin-dependent oxidase gene derived from Rhodococcus opacus B4 and corresponds to ORF No. ROP_22410.
SEQ ID NO: 16 is the nucleotide sequence of the gene hpaB _ro-3 , which is a flavin-dependent oxidase gene derived from Rhodococcus opacus B4 and corresponds to ORF No. ROP_37410.

Another aspect of the present invention is a polynucleotide represented by (c) or (d) below:
(c) a polynucleotide encoding a protein having the activity of converting equol into an equol derivative, the protein consisting of an amino acid sequence selected from SEQ ID NOs: 1 to 8, in which one or more amino acids are substituted or deleted, or one or more amino acids are inserted or added.
(d) a polynucleotide that hybridizes under stringent conditions with DNA consisting of one base sequence selected from SEQ ID NOs: 9 to 16 and encodes a protein having the activity of converting equol into an equol derivative.

"1 to multiple" preferably means 1 to 50, more preferably 1 to 30, even more preferably 1 to 10, and even more preferably 1 to 5. The same applies when amino acids are added to the N-terminus and/or C-terminus.

Conservative substitution is preferable. Conservative substitution is, for example, between Phe, Trp, and Tyr when the substitution site is an aromatic amino acid, between Leu, Ile, and Val when the substitution site is a hydrophobic amino acid, between Gln and Asn when the substitution site is a polar amino acid, and between Lys when the substitution site is a basic amino acid.
Conservative substitutions refer to substitutions between Asp and Glu when they are acidic amino acids, and between Ser and Thr when they are amino acids having a hydroxyl group. Specific examples of conservative substitutions include substitutions of Ala with Ser or Thr, Arg with Gln, His or Lys, Asn with Glu, Gln, Lys, His or Asp, Asp with Asn, Glu or Gln, Cys with Ser or Ala, Gln with Asn, Glu, Lys, His, Asp or Arg, Glu with Gly, Asn, Gln, Lys or Asp, Gly with Pro, His with Asn, Lys, Gln, Arg or Tyr, Ile with L substitution of eu, Met, Val or Phe; substitution of Leu with Ile, Met, Val or Phe; substitution of Lys with Asn, Glu, Gln, His or Arg; substitution of Met with Ile, Leu, Val or Phe; substitution of Phe with Trp, Tyr, Met, Ile or Leu; substitution of Ser with Thr or Ala; substitution of Thr with Ser or Ala; substitution of Trp with Phe or Tyr; substitution of Tyr with His, Phe or Trp; and substitution of Val with Met, Ile or Leu.

The amino acid sequence to be inserted is not particularly limited as long as it maintains the activity of converting equol to an equol derivative. Furthermore, the added amino acid sequence may be, for example, an amino acid sequence of a different origin that serves as a fluorescent protein or a tag protein for use in quantifying expression or separation, so long as it maintains the activity of converting equol to an equol derivative. If it is a fluorescent protein, the protein can be tracked, and if it is a tag protein, it can be separated and purified. Either can be done according to the usual method.

A polynucleotide that hybridizes with DNA under stringent conditions refers to a DNA that hybridizes with one or more selected sequences of at least 20, preferably at least 30, for example 40, 60 or 100 consecutive bases of one base sequence selected from SEQ ID NOs: 9 to 16, using, for example, an ECL direct nucleic acid labeling and detection system (Amersham Pharmaica Biotech) under the conditions described in the manual (wash: 42°C, primary wash buffer containing 0.5xSSC).

Furthermore, a protein having the activity of converting equol into an equol derivative, which consists of an amino acid sequence in which one or more amino acids are substituted or deleted, or one or more amino acids are inserted or added in an amino acid sequence selected from SEQ ID NOs: 1 to 8, may be a protein that has a homology therewith, preferably of 30% or more, and more preferably of 50% or more.
Furthermore, the protein encoded by DNA that hybridizes under stringent conditions with DNA consisting of one of the base sequences selected from SEQ ID NOs: 9 to 16 and having the activity of converting equol to an equol derivative may be a protein that has a homology therewith, preferably of 30% or more, more preferably of 50% or more.
For example, a protein homology search can be performed using the DNA Databank of JAPAN (DDBJ) with FASTA.
This can be done using the BLAST program, etc.

The polynucleotide encoding the protein can be produced using known genetic engineering techniques and molecular biology techniques.

Another aspect of the present invention is a vector into which the polynucleotide of (a), (b), (c), or (d) is inserted.
The vector and the microorganism to be transformed with the vector are not particularly limited, and those used in known genetic engineering techniques and molecular biological techniques can be used.

The present invention will be specifically explained using the following examples, but the present invention is not limited to these examples.

Example 1 Selection of Microorganisms Microorganisms were selected based on the genome sequence information of microorganisms that possess flavin-dependent oxidase.
Focusing on microorganisms that possess a large number of flavin-dependent oxidases, we selected Photorhabdus luminescens sub sp. laumondii TTO1 as a representative of Gram-negative bacteria and Rhodococcus opacus B4 as a representative of Gram-positive bacteria.
As a control, Pseudomonas aeruginosa, which possesses a flavin-dependent oxidase that has been reported to be active against hydroxystilbene (resveratrol), was used.
PAO1 and Escherichia coli BL21 (DE3) were used.

[Example 2] Construction of recombinant E. coli expressing flavin-dependent oxidase
The genes encoding the eight flavin-dependent oxidases were each ligated into the pETDuet-1 vector (Novagen).
Specifically, the gene hpaB _pa derived from Pseudomonas aeruginosa PAO1, the gene hpaB _ec derived from Escherichia coli BL21 (DE3), three genes hpaB _pl-1 , hpaB _pl-2 , and hpaB pl _-3 derived from Photorhabdus luminescens sub sp. laumondii TTO1, and three genes hpaB _ro-1 , hpaB _ro-2 , and hpaB _ro-3 derived from Rhodococcus opacus B4 were synthesized.

For _hpaBpa , the gene was amplified by PCR using primers of SEQ ID NOs: 17 and 18, and then digested with restriction enzymes BspHI and BamHI, followed by ligation into the pETDuet-1 vector.
For _hpaBec , the gene was amplified by PCR using primers of SEQ ID NOs: 19 and 20, digested with restriction enzymes BspHI and BamHI, and ligated into the pETDuet-1 vector.
For hpaB _pl-1 , the gene was amplified by PCR using primers of SEQ ID NOs: 21 and 22, digested with restriction enzymes PciI and BamHI, and ligated into the pETDuet-1 vector.
For hpaB _pl-2 , the gene was amplified by PCR using primers of SEQ ID NOs: 23 and 24, digested with restriction enzymes BspHI and BamHI, and ligated into the pETDuet-1 vector.
For hpaB _pl-3 , the gene was amplified by PCR using primers of SEQ ID NOs: 25 and 26, digested with restriction enzymes BspHI and BamHI, and ligated into the pETDuet-1 vector.
For hpaB _ro-1 , the gene was amplified by PCR using primers of SEQ ID NOs: 27 and 28, digested with restriction enzymes NdeI and MunI, and ligated into the pETDuet-1 vector.
For hpaB _ro-2 , the gene was amplified by PCR using primers of SEQ ID NOs: 29 and 30, digested with restriction enzymes NdeI and MunI, and ligated into the pETDuet-1 vector.
For hpaB _ro-3 , the gene was amplified by PCR using primers of SEQ ID NOs: 31 and 32, digested with restriction enzymes NdeI and MunI, and ligated into the pETDuet-1 vector.

In addition, a gene encoding a flavin reductase that supplies reduced flavin to the flavin-dependent oxidase was ligated to the pCDFDuet-1 vector (Novagen). Specifically, the enzyme gene derived from P. aeruginosa PAO1 (the gene corresponding to ORF no. PA4092, designated hpaC) was synthesized,
After amplification by PCR using primers of SEQ ID NO: 33 and 34, the gene was digested with restriction enzymes NdeI and KpnI and ligated into the pCDFDuet-1 vector. The plasmid with the flavin-dependent oxidase gene ligated thereto was transformed into E.
The plasmid was introduced into the E. coli BL21 Star (DE3) strain.

[Example 3] Evaluation of activity of flavin-dependent oxidase towards equol The recombinant E. coli thus prepared was inoculated into LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl (pH 7.0)) containing 50 μg/ml ampicillin and 50 μg/ml streptomycin, and cultured for 6 hours at 30° C. After 6 hours, 1 mM isopropyl-β-D-thiogalactopyranoside was added and the culture was further continued for 15 hours at 25° C. to induce gene expression.
After harvesting, the cells were washed with 50 mM potassium phosphate buffer (pH 7.5) containing 10% glycerol (v/v) and 1.5% Tween 80 (v/v), and the collected cells were used for activity evaluation.
A reaction solution (250 μl) containing 10% glycerol (v/v), 1.5% Tween 80 (v/v), and 50 mM potassium phosphate buffer (pH 7.5) was prepared and then shaken at 30° C. for 24 hours.

After the reaction, 5 μl of 5N HCl, 250 μl of H ₂ O, and 500 μl of methanol were added to prepare a sample for HPLC analysis. HPLC analysis was performed using a Prominence, LC-20 system (Shimadzu Corporation) and a column (XTerra MS C18 IS, column length 4.6 × 20 mm, particle size 3.5 μm, Waters Corporation). The developing solvent was 0.1% formic acid solution (liquid A) and methanol (liquid B). After flowing at 5% B solution from 0 to 3 minutes at a flow rate of 0.5 ml/min, the concentration of B solution 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%. After flowing at 100% B solution from 15 to 18 minutes, the concentration of B solution was decreased linearly from 18 to 19 minutes to 5% and from 19 to 22 minutes to elute the sample.

As a result, when (S)-equol was used as a substrate, a conversion product was detected at a retention time of 13.2 minutes by HPLC analysis in the reaction mixtures of HpaB _pa , HpaB _ec , HpaB _pl-2 , HpaB _{pl-3, HpaB ro-1} , and HpaB _{ro-3 . As a representative example, the HPLC chromatogram of the reaction mixture of HpaB ro-3} is shown in the top of Figure 1. On the other hand, when (R)-equol was used as a substrate, a conversion product was detected at a retention time of 13.2 minutes by HPLC analysis in the reaction mixtures of HpaB _pa , HpaB _ec , HpaB _pl-2 , HpaB _pl-3 , HpaB _ro-1 , HpaB ro- ₂ , and HpaB _ro-3 . As a representative example, the HPLC chromatogram of the reaction mixture of HpaB _ro-3 is shown in the bottom of Figure 1.

Analysis of the conversion product by LC-MS suggested that it was a hydroxylated form of equol. Furthermore, when the conversion product was purified and analyzed by NMR, it was identified as 3'-hydroxyequol, hydroxylated at the 3' position. The results of the NMR and LC-MS analyses are shown below.

^1H NMR (400 MHz, [ _D4 ]methanol): δ=2.75 (m, 2H, H-4), 2.90 (m, 1H, H-3), 3.79 (m. 1H, H-2), 4.10 (m, 1H, H-2), 6.13 (d, J=2.4 Hz, 1H, H-8), 6.22 (dd, J=8.2, 2.4 Hz, 1H, H-6), 6.50 (dd, J=8.1, 2.0 Hz, 1H, H-6'), 6.60 (d, J=2.0 Hz, 1H, H-2'), 6.63 (d, J=8.1 Hz, 1H, H-5'), 6.78 (d, J=8.2 Hz, 1H, H-5); ^13C NMR (400 MHz, [D ₄ ]methanol): δ=157.6 (C-7), 156.3 (C-8a), 146.5 (C-3'), 145.2 (C-4'), 134.7 (C-1'), 131.2 (C-5), 119.6 (C-6'), 116.5 (C-5'), 115.4 (C-2'), 114.6 (C-4a), 109.1 (C-6), 103.8 (C-8), 72.3 (C-2), 39.6 (C-3), 33.1 (C-4) ; MS (ESI) (m/z): calculated for C ₁₅ H ₁₃ O ₄ [MH] ^- , 257.08138; found, 257.08146.

A calibration curve was created using the purified conversion products, and the results of quantifying the conversion products in the reaction solutions of each substrate and each enzyme are shown in Figure 2. The white bars are for the case where the substrate is (S)-equol, and the black bars are for the case where the substrate is (R)-equol. HpaB _pa and HpaB _ec have been reported to be active against resveratrol, but the activity of these enzymes against equol was weak. On the other hand, HpaB _pl-2 and HpaB _ro-3 , which have not been reported previously, showed high activity against equol, and HpaB _ro-3 in particular retained extremely high activity.

Furthermore, when (S)-equol or (R)-equol was used as a substrate, a conversion product was detected at a retention time of 12.8 minutes in HPLC analysis of the reaction mixture of HpaB _pl-1 . The HPLC chromatogram of the reaction mixture of HpaB _pl-1 and (S)-equol is shown in the upper graph of Figure 4, and the HPLC chromatogram of the reaction mixture of HpaB _pl-1 and (R)-equol is shown in the lower graph of Figure 4.

Analysis of the conversion product by LC-MS suggested that it was a hydroxylated form of equol. Furthermore, when the conversion product was purified and analyzed by NMR, it was identified as 6-hydroxyequol, hydroxylated at the 6th position. The results of the NMR and LC-MS analyses are shown below.

1H NMR (400 MHz, [D ₄ ]methanol): δ=2.79 (m, 2H, H-4), 3.02 (m, 1H, H-3), 3.84 (m. 1H, H-2), 4.12 (m, 1H, H-2), 6.24 (s, 1H, H-8), 6.48 (s, 1H, H-5), 6.73 (dd,
J=6.6, 2.0 Hz, 2H, H-3'), 7.06 (dd, J=6.6, 1.8 Hz, 2H, H-2'); 13C NMR (400 MHz, [D ₄ ]methanol): δ=157.3 (C-4'), 148.7 (C-8a), 145.5 (C-7), 140.1 (C-6), 134.1 (C-1'), 129.4 (C-2'), 116.6 (C-5), 116.4 (C-3'), 113.8 (C-4a), 104.4 (C-8),
72.1 (C-2), 39.6 (C-3), 33.2 (C-4) ; MS (ESI) (m/z ₎ : calculated for _C15H13O4 [MH] ^- , 257.08138; found, _257.08161 .

[Example 4] Flask-scale production of 3'-hydroxyequol Using HpaBro _-3 , which showed the highest activity in Example 3, 3'-hydroxyequol synthesis was attempted on a flask scale.
A 20 ml reaction solution containing (R)-equol or (R)-equol, 4% dimethyl sulfoxide (v/v), 10% glycerol (v/v), 1.5% Tween 80 (v/v), and 200 mM potassium phosphate buffer (pH 7.5) was prepared and shaken at 30°C.

The results are shown in Figure 3. The graph on the left is for the case where the substrate was (S)-equol, and the graph on the right is for the case where the substrate was (R)-equol. When (S)-equol was used as the substrate, 3.5 mM (0.90 g/L) of the hydroxyl form was produced in 90 minutes, and when (R)-equol was used as the substrate, 1.7 mM (0.44 g/L) was produced in 90 minutes. From the above, it was demonstrated that 3'-hydroxyequol can be produced efficiently on a flask scale.

[Example 5] Flask-scale production of 6-hydroxyequol
Using HpaB _pl-1 , we attempted to synthesize 6-hydroxyequol on a flask scale. Here, in order to efficiently express and solubilize HpaB _pl-1 , we used a bacterial cell in which HpaB _pl-1 was co-expressed with chaperonins and cochaperonins. Specifically, the bacterial cell was 125 g/l (wet cell weight equivalent).
A 20 ml reaction solution containing 10 mM (S)-equol or (R)-equol, 4% (v/v), glycerol 10% (v/v), Tween 80 1.5% (v/v), and 200 mM potassium phosphate buffer (pH 7.5) was prepared and shaken at 30°C.

The results are shown in Figure 5. The graph on the left is for when the substrate was (S)-equol, and the graph on the right is for when the substrate was (R)-equol. When (S)-equol was used as the substrate, 2.2 mM (0.57 g/L) of the hydroxyl form was produced in 12 hours, and when (R)-equol was used as the substrate, 1.0 mM (0.26 g/L) was produced in 12 hours. From the above, it was demonstrated that 6-hydroxyequol can be produced efficiently on a flask scale.

Claims (13)

A composition comprising an equol-containing composition and an enzyme or microorganism capable of converting the equol in the equol-containing composition into an equol derivative. The composition according to claim 1, wherein the equol-containing composition is equol, a fermented soybean germ extract, a fermented soybean hypocotyl, a fermented soybean, or an alfalfa fermented product. The composition according to claim 1 or 2, wherein the enzyme or microorganism is a flavin-dependent oxidase or a microorganism having equol oxidation activity. The composition according to any one of claims 1 to 3, wherein the equol derivative is 3'-hydroxyequol or 6-hydroxyequol. A method for producing an equol derivative, comprising the step of subjecting an equol-containing composition to the action of an enzyme or microorganism capable of converting equol in the equol-containing composition to an equol derivative. the step of subjecting the equol-containing composition to an enzyme or microorganism capable of converting equol in the equol-containing composition to an equol derivative comprises a step of culturing the microorganism in a medium containing the equol-containing composition and producing the equol derivative from equol in the equol-containing composition by fermentation;
and recovering the produced equol derivative.
The method according to claim 5 . The method according to claim 5 or 6, wherein the equol derivative is 3'-hydroxyequol or 6-hydroxyequol. The method according to any one of claims 5 to 7, wherein the enzyme or microorganism is a flavin-dependent oxidase or a microorganism having equol oxidation activity. A method for producing a food or beverage containing an equol derivative, comprising the steps of: producing an equol derivative by treating an equol-containing composition with an enzyme or microorganism capable of converting equol in the equol-containing composition into an equol derivative; and blending the equol derivative with ingredients for the food or beverage. A method for producing a pharmaceutical product containing an equol derivative, comprising the steps of: producing an equol derivative by treating an equol-containing composition with an enzyme or microorganism capable of converting equol in the equol-containing composition into an equol derivative; and blending the equol derivative with a pharmaceutical raw material. A polynucleotide represented by (a) or (b) below:
(a) a polynucleotide encoding a protein consisting of an amino acid sequence selected from SEQ ID NOs: 1 to 8;
(b) a polynucleotide comprising one base sequence selected from SEQ ID NOs: 9 to 16. A polynucleotide represented by (c) or (d) below:
(c) a polynucleotide encoding a protein having the activity of converting equol into an equol derivative, the protein consisting of an amino acid sequence selected from SEQ ID NOs: 1 to 8, in which one or more amino acids are substituted or deleted, or one or more amino acids are inserted or added.
(d) a polynucleotide that hybridizes under stringent conditions with DNA consisting of one base sequence selected from SEQ ID NOs: 9 to 16 and encodes a protein having the activity of converting equol into an equol derivative. A vector into which the polynucleotide (a) or (b) according to claim 11, or the polynucleotide (c) or (d) according to claim 12 is inserted.

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