JP7449526B2 - Composition for the production of equol derivatives - Google Patents

Description

The present invention relates to compositions for producing equol derivatives and methods for producing equol derivatives.

Equol is known to have the highest estrogenic activity among the metabolites of isoflavones contained in soybeans (Non-Patent Documents 1 and 2).
In addition, similar to equol, equol derivatives such as 5-hydroxyequol have been reported to have estrogenic activity and 3β-hydroxysteroid dehydrogenase inhibitory activity (Patent Document 1), and have been shown to have aldosterone and sugar By inhibiting the biosynthesis of corticoids, it is expected to be used to prevent or treat disorders of excess of these hormones.
Furthermore, in a report on a method for synthesizing isoflavan derivatives using an o-quinone methide-based method, it is disclosed that 3'-hydroxy equol can be obtained by a chemical synthesis method (Non-Patent Document 3).

On the other hand, an enzyme called flavin-dependent oxidase is known. It has been reported that the enzymes have different substrate specificities and actions depending on their type; for example, HpaB and HpaC possessed by Pseudomonas aeruginosa strain PAO1 exhibit activity against hydroxystilbene (resveratrol). A method for producing hydroxystilbene by allowing a protein having 50% or more homology at the amino acid level to HpaB and HpaC derived from Pseudomonas aeruginosa strain PAO1 to act on a substrate compound has been reported (Patent Documents 2 and 3). ).

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

Japanese Patent Application Publication No. 2003-81875 Japanese Patent Application Publication No. 2015-130819 Japanese Patent Application Publication No. 2017-074019

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

An object of the present invention is to provide a composition for producing an equol derivative and a method for producing an equol derivative.

As a result of extensive studies, the present inventors discovered that the above problems can be solved by using an equol-containing composition and an enzyme or microorganism that can convert equol in the equol-containing composition into an equol derivative, and have developed the present invention. Completed.
The present invention is as shown below.

[1] 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.
[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 a fermented alfalfa.
[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'-hydroxy equol or 6-hydroxy equol.
[5] A method for producing an equol derivative, which comprises a step of causing an equol-containing composition to act on an enzyme or microorganism that can convert equol in the equol-containing composition to an equol derivative.
[6] The step of allowing an enzyme or microorganism capable of converting equol in the equol-containing composition to act on the equol-containing composition comprises culturing the microorganism in a medium containing the equol-containing composition; The manufacturing method according to [5], which includes a step of producing an equol derivative from equol in the equol-containing composition by fermentation, and a step of recovering the produced equol derivative.
[7] The production method according to [5] or [6], wherein the equol derivative is 3'-hydroxy equol or 6-hydroxy equol.
[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 step of producing an equol derivative by acting on the 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 raw materials for food and drink products. A method for producing a food or drink containing an equol derivative, the method comprising:
[10] A step of producing an equol derivative by acting on the equol-containing composition with an enzyme or microorganism capable of converting equol in the equol-containing composition into an equol derivative, and a step of blending the equol derivative with a raw material for a pharmaceutical product. A method for producing a pharmaceutical product containing an equol derivative, including:
[11] The polynucleotide of (a) or (b) below.
(a) A polynucleotide encoding a protein consisting of one 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] The polynucleotide of (c) or (d) below.
(c) Equol consisting 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 one amino acid sequence selected from SEQ ID NOS: 1 to 8. A polynucleotide encoding a protein having an activity that can be converted into an equol derivative.
(d) A polynucleotide that encodes a protein that hybridizes with a DNA consisting of one base sequence selected from SEQ ID NOs: 9 to 16 under stringent conditions and has the activity of converting equol into an equol derivative.
[13] A vector into which the polynucleotide (a) or (b) described in [11] or the polynucleotide (c) or (d) described in [12] is inserted.

According to the present invention, a composition for producing an equol derivative and a method for producing an equol derivative can be provided. In addition, the known effects of equol derivatives can be obtained by the subject ingesting foods, drinks, and medicines containing the equol derivatives.

1 is an HPLC chromatogram showing the results of culturing equol and Escherichia coli into which a vector linked with a gene encoding HpaB _ro-3 has been introduced, according to one embodiment of the present invention. The upper graph is when the substrate is (S)-equol, and the lower graph is when the substrate is (R)-equol. 1 is a graph showing the amount of 3'-hydroxyequol produced by E. coli into which vectors linked with each gene are introduced, according to one embodiment of the present invention. White bars are when the substrate is (S)-equol, and black bars are when the substrate is (R)-equol. 1 is a graph showing the amount of 3'-hydroxy equol produced on a flask scale using E. coli into which a vector linked with a gene encoding HpaB _ro-3 according to one embodiment of the present invention is introduced. The left graph shows the case where the substrate is (S)-equol, and the right graph shows the case where the substrate is (R)-equol. 1 is an HPLC chromatogram showing the results of culturing equol and Escherichia coli into which a vector linked with a gene encoding HpaB _pl-1 has been introduced, according to one embodiment of the present invention. The upper graph is when the substrate is (S)-equol, and the lower graph is when the substrate is (R)-equol. 1 is a graph showing the amount of 6-hydroxyequol produced on a flask scale using E. coli into which a vector linked with a gene encoding HpaB _pl-1 according to one embodiment of the present invention is introduced. The left graph shows the case where the substrate is (S)-equol, and the right graph shows the case where the substrate is (R)-equol.

The present invention will be explained in detail below.
The composition described herein is a concept that includes a mixture, regardless of whether the components thereof 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 the equol in the equol-containing composition to an equol derivative.

The form of the equol-containing composition is not limited as long as the equol in the equol-containing composition is converted into an equol derivative by an enzyme or microorganism capable of converting equol into an equol derivative.
Examples of the equol-containing composition include fermented leguminous plants such as soybean, kidney bean, fava bean, groundnut, chickpea, arrowroot, red clover, alfalfa, and licorice. That is, if the legume is a soybean, it is a fermented product of soybean itself (fermented soybean product).
In addition, the equol-containing composition may be a fermented product of soybean germ (fermented soybean germ) or a fermented product of soybean hypocotyl (fermented soybean hypocotyl), for example of soybean among legumes. It may also be a fermented product of a portion of soybeans, such as Furthermore, taking a fermented soybean germ as an example, it may be a fermented product obtained by fermenting an extract obtained from a soybean germ portion (fermented soybean germ extract).
The fermented soybean product is a fermented product obtained by fermenting whole soybeans, and includes fermented soybean germs obtained using soybean germs, and soybean embryos obtained using soybean hypocotyls. It is different from fermented products obtained by fermenting part of the soybean, such as fermented cobs.

For example, in the case of obtaining a fermented product of soybean itself (fermented soybean product), examples of the mode of fermentation include preparing the legume, adding koji mold to it, and fermenting it to obtain a fermented product. It will be done. As for the mode of preparation, the soybeans themselves may be prepared raw, or may be ground after being subjected to heat treatment, drying treatment, or steaming treatment, or after being ground. It may also be prepared after being subjected to heat treatment, drying treatment, steaming treatment, or the like.

Further, the equol-containing composition may be a fermented product obtained by fermenting isoflavones derived from the legume family plant. For example, in the case of soybeans, examples include isoflavone aglycones obtained by allowing enzymes such as β-glucosidase or microorganisms to act on soybean-derived isoflavones.

Further, the equol-containing composition may be equol, and may be (R)-equol or (S)-equol.

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

The enzyme may be an enzyme produced by a microorganism or an enzyme obtained without relying on production by a microorganism.
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. Good too.
When an enzyme produced by a microorganism is recovered, a known method can be used as a recovery method. For example, after culturing microorganisms, the microorganisms are collected by methods such as filtration or centrifugation, and the microorganisms are washed with a buffer solution, physiological saline, etc., and then subjected to physical treatment (e.g., freeze-thaw treatment, ultraviolet treatment, etc.). (sonic treatment, pressure treatment, osmotic pressure difference treatment, grinding treatment, etc.), biochemical treatment (for example, treatment with cell wall lytic enzymes such as lysozyme, etc.), chemical treatment (for example, contact treatment with surfactants, etc.) can be used alone or in combination to recover the enzyme. The recovered enzyme may then be subjected to separation and purification treatments.

Enzymes that can convert equol in the equol-containing composition into equol derivatives include, for example, oxidoreductases (dehydrogenases, cytochromes, catalases, oxidases, oxygenases (e.g., flavin-dependent oxidases, etc.), fatty acid desaturases, etc.), Transferases (acyltransferases, phosphotransferases, aminotransferases, etc.), proteolytic enzymes (proteases), lipid-degrading enzymes (lipases), carbohydrate-degrading enzymes (amylase, lysozyme, β-galactosidase, etc.), phosphorylases (nuclease, phosphatase, restriction enzyme), hydrolase (urease, lactonase, ATP hydrolase, etc.), elimination enzyme (carbonic hydratase, pyruvate decarboxylase, etc.), isomerase (racemase, phosphoglycerate phosphomutase, glucose) -6-phosphate isomerase, etc.), synthetic enzymes (DNA ligase, aminoacyl-tRNA synthetase, acyl-CoA synthetase, carboxylase, etc.).
Oxygenase is preferred, and flavin-dependent oxidase is more preferred.

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 oxidoreductase. Ru . The former includes HpaB, and the latter includes HpaC.
HpaB is preferably HpaB _pa derived from Pseudomonas aeruginosa PAO1 (SEQ ID NO: 1
), HpaB _ec (SEQ ID NO: 2) derived from Escherichia coli BL21 (DE3), HpaB _pl-1 (SEQ ID NO: 3), HpaB _pl-2 (SEQ ID NO: 4), HpaB _pl- derived from Photorhabdus luminescens sub sp. laumondii TTO1. ₃ (SEQ ID NO: 5), HpaB _ro-1 (SEQ ID NO: 6), HpaB _ro-2 (SEQ ID NO: 7), and HpaB _ro-3 (SEQ ID NO: 8) derived from Rhodococcus opacus B4.

HpaB _pa (SEQ ID NO: 1) derived from Pseudomonas aeruginosa PAO1 is 80% or more, preferably 90% or more, of the amino acid sequence represented by SEQ ID NO: 1, as long as equol in the equol-containing composition can be converted into an equol derivative. Preferably, the amino acid sequences may have an identity of 95% or more.
This also applies to each HpaB (SEQ ID NOs: 2 to 8) other than the above-mentioned HpaB _pa .

The microorganism that can convert the equol in the equol-containing composition into an equol derivative is not limited in its mode as long as it can convert the equol in the equol-containing composition into an equol derivative. The microorganism is preferably a microorganism having equol oxidizing activity (equol oxygenase), and more preferably a microorganism expressing the flavin-dependent oxidase.
The microorganism may be a microorganism that is originally capable of converting equol into an equol derivative, or may be a microorganism that has been modified by known techniques such as genetic recombination so as to be able to convert equol into an equol derivative. good. The genus or species to which the microorganism belongs is not limited, and examples include Escherichia coli, yeast, and filamentous fungi.

The equol derivatives are not particularly limited, but are preferably hydroxylated equol or methoxylated equol, such as 3'-hydroxy equol, 5-hydroxy equol, 3'-methoxy equol, 6-methoxy equol, 6-hydroxy equol. etc.
More preferred are hydroxides, such as 3'-hydroxy equol, 5-hydroxy equol, and 6-hydroxy equol, and even more preferred are 3'-hydroxy equol and 6-hydroxy equol.
Furthermore, when HpaB _pa , HpaB _ec , HpaB _pl-2 , HpaB _pl-3 , HpaB _ro-1 , HpaB _ro-2 , or HpaB _ro-3 is used as the flavin-dependent oxidase, equol is - Converted to hydroxy equol. Furthermore, when HpaB _pl-1 is used as a 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, which includes the step of causing an equol-containing composition to act on an enzyme or microorganism that can convert equol in the equol-containing composition to an equol derivative.

Regarding the equol-containing composition in this embodiment, and the enzyme and microorganism that can convert equol in the equol-containing composition into an equol derivative, the above explanations are used.

In this embodiment, the step of acting on the equol-containing composition with an enzyme capable of converting equol in the equol-containing composition into an equol derivative is performed in a solution containing the equol-containing composition. It includes a step of reacting with an enzyme.

Examples of the solution containing the equol-containing composition include water, an organic solvent, and a two-phase mixed system 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.

In addition, clathrate compounds may be added to increase the solubility of equol, which is a substrate.
Examples of the inclusion compound include α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, cluster dextrin (highly branched cyclic dextrin), and analogs thereof. Examples include methyl-β-cyclodextrin, trimethyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, and the like.
The amount of the clathrate compound added is usually 0.1 equivalent or more, preferably 0.5 equivalent or more, more preferably 1.0 equivalent or more, based on the total molar ratio of equol in the equol-containing composition. On the other hand, it is usually 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, even more preferably 30°C to 37°C.
The reaction time is preferably 8 to 340 hours, more preferably 12 to 170 hours, 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 and obtained from a microorganism is used as the enzyme capable of converting equol in the equol-containing composition into an equol derivative, the step of causing the enzyme to act on the equol-containing composition is performed. The method may include a step of recovering the enzyme from a microorganism that produces the enzyme, and may also include a step of culturing the microorganism that produces the enzyme.

The process 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, conditions for culturing the microorganism in a medium containing an equol-containing composition, which will be described later, may be mentioned.

The step of recovering the enzyme from the microorganism that produces the enzyme includes a step of collecting the microorganism by a method such as filtration or centrifugation, and then a step of washing the microorganism with a buffer solution, physiological saline, etc. That's fine. The washing step includes, for example, physical treatment (for example, freeze-thaw treatment, ultrasonic treatment, pressure treatment, osmotic pressure difference treatment, grinding treatment, etc.), biochemical treatment (for example, treatment with cell wall lytic enzymes such as lysozyme) treatment, etc.), chemical treatment (for example, contact treatment with a surfactant, etc.), and these may be used alone or in combination. Further, after that, a step of purifying the enzyme, a step of concentrating it, etc. may be included.

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

The solution containing the equol-containing composition may be a solution in which the microorganisms can proliferate or a solution in which the microorganisms cannot proliferate.

Examples of the solution in which the microorganism can grow include a medium, and the medium may be a minimal medium or a synthetic medium. 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, and Brain medium manufactured by Nissui Pharmaceutical Co., Ltd. Examples include heart infusion medium and LB medium.

Additionally, water-soluble organic matter can be added to the medium as a carbon source. Examples of water-soluble organic substances include sugars such as glucose, arabinose, sorbitol, fructose, mannose, sucrose, trehalose, xylose, galactose, starch, starch hydrolyzate, 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; Glycine, glutamine, Examples include amino acids such as asparagine.
The concentration of the organic matter can be adjusted as appropriate for efficient growth. Generally, it is in the range of 0.1 to 10 wt/vol%.

In addition to the carbon sources mentioned above, a nitrogen source can be added to the medium. As the nitrogen source, various nitrogen compounds that can be used in normal fermentation can be used.
Preferred inorganic nitrogen sources include ammonium salts, nitrates, urea and the like, more preferably ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium hydrogen phosphate, ammonium nitrate, sodium nitrate, potassium nitrate and sodium nitrate.
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, Rub-Lemco powder, bouillon). etc.), seafood extract, liver extract, digested serum powder, fish oil, etc.

Furthermore, 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. Examples of microbial growth cofactors derived from animals and plants include inorganic compounds, vitamins, fatty acids, and the like.

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 Thiooctoic acid Sodium molybdate p-aminobenzoic acid Potassium chloride Vitamin K
Boric acid, etc. Nickel chloride Sodium tungstate Sodium selenate Ferrous ammonium sulfate Sodium acetate trihydrate Magnesium sulfate heptahydrate Manganese sulfate tetrahydrate

In addition, known reducing agents such as L-cysteine (hydrochloride), thioglycolic acid, ascorbic acid, mercaptoacetic acid, thiol acetic acid, glutathione, sodium sulfide, sodium sulfide, sulfite, thioglycolic acid, and rutin may be added to the medium. Growth may be improved by adding a reducing agent such as L-cystine or an enzyme that decomposes active oxygen species such as catalase or superoxide mutase.

As a culture method, for example, normal culture methods such as shaking culture, aerated 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 may contain nitrogen and/or carbon dioxide in any ratio. A gas phase or an aqueous phase containing hydrogen is preferred. 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 it promotes the production of equol derivatives, while usually 100%. It is preferably 20% or less, more preferably 10% or less.

There are no particular restrictions on the method of creating such an environment for the gas phase and aqueous phase during culture, but examples include a method of replacing the gas phase with the gas before culturing, and a method of replacing the gas phase with the gas before culturing. Methods such as supplying the gas and/or supplying the gas to the gas phase portion of the incubator, and bubbling the aqueous phase with the gas before culturing can be used. As the hydrogen, hydrogen gas may be used as it is. Alternatively, a hydrogen raw material such as formic acid and/or its salt may be added to the medium, and hydrogen may be generated during culture by the action of microorganisms.

The ventilation amount is, for example, 0.005 to 2 vvm, preferably 0.05 to 0.5 vvm. Moreover, the mixed gas can also be supplied as nanobubbles.
The culture temperature is preferably 20°C to 45°C, more preferably 25°C to 40°C, even more preferably 30°C to 37°C.
The pressurizing conditions of 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 culture is usually 4-10, preferably 6-8.

Furthermore, the production of equol derivatives may be promoted by adding surfactants, adsorbents, clathrate compounds, etc. to the culture medium.
Examples of the surfactant include SDS, Triton
Examples of the adsorbent include cellulose and its derivatives; dextrin; hydrophobic adsorbents Diaion HP series and Sepabead series manufactured by Mitsubishi Chemical Corporation; Amberlite XAD series manufactured by Organo Co., Ltd., and the like.
Examples of the inclusion compound include α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, cluster dextrin (highly branched cyclic dextrin), and analogs thereof. Examples include methyl-β-cyclodextrin, trimethyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, and the like.
The amount of the clathrate compound to be added is usually 0.1 equivalent or more, preferably 0.5 equivalent or more, and more preferably 1.0 equivalent or more, based on the total molar amount of equol in the equol-containing composition. On the other hand, it 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 quantitative method can follow a conventional method. For example, a portion of the culture solution is collected, diluted appropriately, stirred well, and then filtered using a membrane such as a polytetrafluoroethylene (PTFE) membrane to remove insoluble matter, which is then subjected to high performance liquid chromatography. Examples include quantifying.

Further, this embodiment may include a step of recovering the obtained equol derivative. The recovery process includes a purification process, a concentration process, and the like. Purification treatments in the purification process include sterilization of microorganisms by heat, etc.; sterilization by microfiltration (MF), ultrafiltration (UF), etc.; removal of solids and polymer substances; extraction with organic solvents, ionic liquids, etc. ; Treatments such as adsorption and decolorization using hydrophobic adsorbents, ion exchange resins, activated carbon columns, etc. can be performed. In addition, examples of the concentration treatment in the concentration step include concentration using an evaporator, a reverse osmosis membrane, and the like.
Furthermore, a solution containing an equol derivative can be pulverized by freeze drying, spray drying, or the like. In powdering, excipients such as lactose, dextrin, cornstarch, etc. can also be added.

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

Such a solution when the microorganism cannot grow can be used, for example, when the microorganism is a quiescent cell. A quiescent bacterial cell is a bacterial cell that has been removed from a cultured microorganism by centrifugation or other operations, washed with a salt solution or buffer solution, and suspended in the same solution as the washing solution, and is in a non-proliferating state. It refers to a bacterial cell, and in this embodiment, it refers to a bacterial cell that has at least a metabolic system capable of producing equol derivatives from equol.

An example of a case where the solution may be a solution in which the microorganism can grow or a solution in which the microorganism cannot grow is a case where an immobilized microorganism is used as the microorganism. Immobilized microorganisms are microorganisms that have been immobilized using known methods such as polyacrylamide gel method, sulfur-containing polysaccharide gel method (carrageenan gel method), alginate gel method, agar gel method, etc. .
In addition, in this specification, such a gel etc. which immobilize microorganisms are also defined as a solution.

Another aspect of the present invention is a step of producing an equol derivative by causing an equol-containing composition to act on an enzyme or a microorganism capable of converting equol in the equol-containing composition into an equol derivative, and a raw material for the equol derivative and a pharmaceutical product. This is a method for producing a pharmaceutical containing an equol derivative, including the step of blending.

Regarding the process of producing the equol derivative, the description of the method for producing an equol derivative, which has already been explained, is referred to. In addition, in this aspect, it is preferable to include a step of recovering the produced equol derivative after the step of producing the equol derivative.

As raw materials for pharmaceuticals, commonly used pharmaceutical raw materials can be used, and the time of blending is not particularly limited.

The manufactured drug can be used as a drug for preventing or treating diseases that can be prevented or treated by ingesting or administering equol derivatives. Further, the dosage form can be selected depending on the disease to be prevented or treated, the usage form of the drug, the administration route, etc. For example, internal medicines such as tablets, granules, powders, capsules, soft capsules, and syrups; emulsions, suspensions, ointments, creams, lotions, gels, sprays, patches, and poultices. , liniments, aerosols, ointments, packs, inhalants, suppositories, and other external pharmaceuticals; injections, and the like. These various preparations are prepared by adding fillers, extenders, excipients, binders, humectants, disintegrants, surfactants, lubricants, coloring agents, and flavorings to the main drug according to conventional methods. It can be formulated using known adjuvants commonly used in the field of pharmaceutical formulation technology, such as solubilizing agents, suspending agents, and coating agents. In addition, coloring agents, preservatives, fragrances, flavoring agents, sweeteners, etc., and other drugs may be contained in this drug.

The content of the equol derivative relative to the total amount of the drug is not particularly limited, but it is preferably such that the desired effect of the equol derivative can be obtained when the drug is ingested or administered. The content of the equol derivative based on the total amount of the drug is usually 0.0001 to 50% by weight, preferably 0.001 to 50% by weight, and more preferably 0.01 to 50% by weight.

Another aspect of the present invention is a step of producing an equol derivative by causing an equol-containing composition to act on an enzyme or microorganism capable of converting equol in the equol-containing composition into an equol derivative, and a step of producing an equol derivative with a food or drink. This is a method for producing a food or drink containing an equol derivative, including a step of blending the equol derivative with a raw material.

Regarding the process of producing the equol derivative, the description of the method for producing an equol derivative, which has already been explained, is referred to. In addition, in this aspect, it is preferable to include a step of recovering the produced equol derivative after the step of producing the equol derivative.

As the raw material for the food and drink, commonly used raw materials for food and drink can be used, and the time of blending is not particularly limited.

The food and drink products produced may have water, protein, carbohydrates, lipids, vitamins, minerals, organic acids, organic bases, fruit juice, flavors, etc. as main components.
Examples of proteins include whole milk powder, skim milk powder, partially skim milk powder, casein, soybean protein, egg protein, meat protein, and other animal and vegetable proteins, their hydrolysates, butter, and the like.
Examples of carbohydrates include saccharides, modified starches (in addition to dextrin, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), dietary fiber, and the like.
Examples of the 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 transesterified oils.
Examples of vitamins include vitamin A, carotenes, vitamin B group, vitamin C, vitamin D group, vitamin E, vitamin K group, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, and choline. , folic acid, etc.
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 types, or may be synthetic products.

Specific examples of the foods and drinks produced include general foods and drinks, as well as foods for specified health uses, nutritional supplements, functional foods, and foods for the sick. The form of these foods and drinks is not particularly limited, but specifically, main dishes such as breads and noodles; side dishes such as cheese, ham, sausages, and processed seafood products; fruit juice drinks, carbonated drinks, milk drinks, etc. Beverages; Luxury items such as cookies, cakes, jellies, puddings, candies, and yogurt; Examples include capsules (soft capsules, hard capsules), tablets, granules, powders, jellies, liposome preparations, supplements such as nutritional drinks, etc. Ru. Among these food and drink products, supplements are preferred, and capsules are more preferred.

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

If the food or drink is a supplement, it may be in the form of a solid, gel, or liquid, such as various processed food and drink, powder, tablet, pill, capsule, jelly, and granule. It can be in the form of

Supplements also contain excipients such as dextrin, preservatives such as vitamin C, flavoring agents such as vanillin, pigments such as safflower pigment, monosaccharides, oligosaccharides, and polysaccharides (e.g., glucose, fructose, sucrose, Additives such as sucrose (and carbohydrates containing these), acidulants, fragrances, oils and fats, emulsifiers, whole milk powder, or agar may be blended. These components may be used in combination of two or more types, or may be synthetic products.

Another aspect of the present invention is a step of producing an equol derivative by causing an equol-containing composition to act on an enzyme or microorganism capable of converting equol in the equol-containing composition into an equol derivative, and a raw material for making the equol derivative and cosmetics. A method for producing cosmetics containing an equol derivative, the method comprising the step of blending a cosmetic product containing an equol derivative.

Regarding the process of producing the equol derivative, the description of the method for producing an equol derivative, which has already been explained, is referred to. In addition, in this aspect, it is preferable to include a step of recovering the produced equol derivative after the step of producing the equol derivative.

As raw materials for cosmetics, commonly used raw materials for cosmetics can be used, and the time of blending is not particularly limited.
Cosmetics manufactured include, for example, creams, emulsions, lotions, packs, cleaning agents, makeup cosmetics, scalp and hair products, oils, lipsticks, foundations, eyeliners, blushers, mascaras, and eyelashes. Examples include shadow, manicure/pedicure (and remover), shampoo, conditioner, hair treatment, permanent agent, hair dye, shaving agent, soap (hand soap, body soap, facial cleanser), etc.

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

Another aspect of the present invention is a step of producing an equol derivative by causing an equol-containing composition to act on an enzyme or microorganism capable of converting equol in the equol-containing composition into an equol derivative, and a step of producing an equol derivative using the equol derivative and raw materials for feed. This is a method for producing feed containing an equol derivative, including the step of blending.
The feed of this embodiment includes feed for pets such as dogs and cats (pet food).

Regarding the process of producing the equol derivative, the description of the method for producing an equol derivative, which has already been explained, is referred to. In addition, in this aspect, it is preferable to include a step of recovering the produced equol derivative after the step of producing the equol derivative.

As the raw material for the feed, commonly used raw materials for feed can be used, and there are no particular restrictions on the time of blending.
Specific examples of raw materials for feed include grains such as corn, wheat, barley, and rye; brans such as bran and rice bran; lees such as corn gluten meal and corn jam meal; skim milk powder, whey, fish meal, and bone meal. Animal feeds; yeasts such as brewer's yeast; calciums such as calcium phosphate and calcium carbonate; vitamins; oils and fats; amino acids; saccharides and the like.

The content of the equol derivative in the total amount of the feed to be produced is not particularly limited, but it is preferably such a content that the effect of the equol derivative can be obtained when the feed is ingested. The content of equol derivatives based on the total amount of feed is usually 0.00001 to 10% by mass, preferably 0.0001 to 10% by mass, and more preferably 0.001 to 10% by mass.
It is.

Another aspect of the present invention is the following polynucleotide (a) or (b).
(a) A polynucleotide encoding a protein consisting of one 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 HpaB _pa encoded by the flavin-dependent oxidase gene hpaB _pa (ORF No. PA4091) derived from Pseudomonas aeruginosa PAO1.
SEQ ID NO: 2 is the amino acid sequence of the enzyme HpaB _ec encoded by the flavin-dependent oxidase gene hpaB _pa (ORF No. B21_04188) 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 sub sp. 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 sub sp. 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 sub sp. 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 gene for flavin-dependent oxidase derived from Pseudomonas aeruginosa PAO1, and is the base sequence of the gene hpaB _pa corresponding to ORF No. PA4091.
SEQ ID NO: 10 is a gene for a flavin-dependent oxidase derived from Escherichia coli BL21 (DE3), and is the base sequence of the gene hpaB _ec corresponding to ORF No. B21_04188.
SEQ ID NO: 11 is a gene for a flavin-dependent oxidase derived from Photorhabdus luminescens sub sp. laumondii TTO1, and is the base sequence of the gene hpaB _pl-1 corresponding to ORF No. plu0246.
SEQ ID NO: 12 is the gene for flavin-dependent oxidase derived from Photorhabdus luminescens sub sp. laumondii TTO1, and is the base sequence of the gene hpaB _pl-2 corresponding to ORF No. plu0975.
SEQ ID NO: 13 is a gene for a flavin-dependent oxidase derived from Photorhabdus luminescens sub sp. laumondii TTO1, and is the base sequence of the gene hpaB _pl-3 corresponding to ORF No. plu4027.
SEQ ID NO: 14 is the flavin-dependent oxidase gene derived from Rhodococcus opacus B4, and is the base sequence of the gene hpaB _ro-1 corresponding to ORF No. ROP_20940.
SEQ ID NO: 15 is a gene for a flavin-dependent oxidase derived from Rhodococcus opacus B4, and is the base sequence of the gene hpaB _ro-2 corresponding to ORF No. ROP_22410.
SEQ ID NO: 16 is the nucleotide sequence of the gene hpaB _ro-3 , which is a gene for flavin-dependent oxidase derived from Rhodococcus opacus B4 and corresponds to ORF No. ROP_37410.

Another aspect of the present invention is the polynucleotide of (c) or (d) below.
(c) Equol consisting 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 one amino acid sequence selected from SEQ ID NOS: 1 to 8. A polynucleotide encoding a protein having an activity that can be converted into an equol derivative.
(d) A polynucleotide that encodes a protein that hybridizes with a DNA consisting of one base sequence selected from SEQ ID NOs: 9 to 16 under stringent conditions and has the activity of converting equol into an equol derivative.

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

Conservative substitutions are preferred, and conservative substitutions include between Phe, Trp, and Tyr when the substitution site is an aromatic amino acid, and between Leu, Ile, and Tyr when the substitution site is a hydrophobic amino acid. Between Val, Gln and Asn for polar amino acids, and Lys for basic amino acids.
, Arg, and His; in the case of an acidic amino acid, between Asp and Glu; and in the case of an amino acid with a hydroxyl group, between Ser and Thr. Specifically, conservative substitutions include Ala to Ser or Thr, Arg to Gln, His, or Lys, Asn to Glu, Gln, Lys, His, or Asp, Asp to Asn, Substitution of Glu or Gln, substitution of Cys with Ser or Ala, substitution of Gln with Asn, Glu, Lys, His, Asp or Arg, substitution of Glu with Gly, Asn, Gln, Lys or Asp, substitution of Gly with Substitution of Pro, substitution of His with Asn, Lys, Gln, Arg or Tyr, substitution of Ile with Leu, Met, Val or Phe, substitution of Leu with Ile, Met, Val or Phe, substitution of Lys with Asn, Substitution of 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 Substitutions include Ser or Ala, Trp to Phe or Tyr, Tyr to His, Phe or Trp, and Val to Met, Ile or Leu.

The amino acid sequence to be inserted is not particularly limited as long as it retains the activity of converting equol into an equol derivative. In addition, as long as the added amino acid sequence retains the activity of converting equol into equol derivatives, amino acids of different origins can be added, such as fluorescent proteins and tag proteins used for expression quantification and separation. An array may be added. If it is a fluorescent protein, it can be tracked, and if it is a tag protein, it can be separated and purified. All can be carried out according to conventional methods.

A polynucleotide that hybridizes with DNA under stringent conditions refers to at least 20, preferably at least 30, for example 40, 60 or 100 consecutive base sequences selected from SEQ ID NOS: 9 to 16. One or more selected sequences were used as probe DNA, and the DNA was incubated under the conditions described in the manual (wash: 42°C, 0.5×SSC) using, for example, the ECL direct nucleic acid labeling and detection system (manufactured by Amersham Pharmaica Biotech). This refers to DNA that hybridizes in the primary wash buffer containing the primary wash buffer.

Furthermore, equol is 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 one amino acid sequence selected from SEQ ID NOs: 1 to 8. The protein having the activity of converting into an equol derivative may be a protein having preferably 30% or more, more preferably 50% or more homology thereto.
Preferably, a protein encoded by a DNA that hybridizes under stringent conditions with a DNA consisting of one base sequence selected from SEQ ID NOs: 9 to 16 and having an activity capable of converting equol into an equol derivative is preferred. may be proteins having a homology of 30% or more, more preferably 50% or more.
Protein homology searches can be performed using FASTA, for example, using DNA Databank of JAPAN (DDBJ).
This can be done using programs such as BLAST program and BLAST program.

As a method for producing a polynucleotide encoding the protein, known genetic engineering techniques and molecular biological techniques can be used.

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 biology techniques can be used.

The present invention will be specifically described below using Examples, but the present invention is not limited to these Examples.

[Example 1] Selection of microorganisms Microorganisms were selected based on genome sequence information of microorganisms that possess flavin-dependent oxidases.
Focusing on microorganisms that possess many 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 comparison, Pseudomonas aeruginosa, which possesses a flavin-dependent oxidase that has been reported to exhibit activity against hydroxystilbene (resveratrol), was also used.
PAO1 and Escherichia coli BL21 (DE3) were used.

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

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

Furthermore, a gene encoding a flavin reductase that supplies reduced flavin to a flavin-dependent oxidase was ligated to the pCDFDuet-1 vector (manufactured by Novagen). Specifically, we synthesized the enzyme gene derived from P. aeruginosa PAO1 (the gene corresponding to ORF no. PA4092, expressed as hpaC),
After amplification by PCR using primers of SEQ ID NOs: 33 and 34, the product was digested with restriction enzymes NdeI and KpnI, and ligated to pCDFDuet-1 vector. A plasmid containing a flavin-dependent oxidase gene and a plasmid containing a flavin reductase gene were used to generate E. coli by heat shock method.
coli BL21 Star (DE3) strain.

[Example 3] Evaluation of the activity of flavin-dependent oxidase against equol The prepared recombinant Escherichia coli was cultured in LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl (pH 7.0)) and cultured at 30°C for 6 hours. After 6 hours, 1 mM isopropyl-β-D-thiogalactopyranoside was added, and the cells were further cultured at 25°C for 15 hours to induce gene expression.
After bacterial collection, 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. . Specifically, bacterial cells 50 g/l (wet weight equivalent), 10 mM (S)-equol or (R)-equol, 4% dimethyl sulfoxide (v/v)
After preparing 250 μl of a reaction solution containing 10% glycerol (v/v), 1.5% Tween 80 (v/v), and 50 mM potassium phosphate buffer (pH 7.5), shake at 30°C for 24 hours. did.

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 Prominence, LC-20 system (Shimadzu) and column (XTerra MS C18 IS, column length 4.6 x 20 mm, particle size 3.5 μm, Waters). The developing solvents used were 0.1% formic acid aqueous solution (liquid A) and methanol (liquid B). After flowing with 5% liquid B from 0 to 3 minutes at a flow rate of 0.5 ml/min, and then increasing liquid B to 40% from 3 to 4 minutes. The B solution was increased to 80% from 4 to 14 minutes, and to 100% from 14 to 15 minutes by a linear gradient. Furthermore, after flowing with 100% B solution from 15 to 18 minutes, the B solution was lowered to 5% by a linear gradient from 18 to 19 minutes, and then 5% B solution was passed from 19 to 22 minutes to elute the sample. .

As a result, when (S)-equol was used as a substrate, in the reaction solution of HpaB _pa , HpaB _ec , HpaB _pl-2 , HpaB _ro-1 , and HpaB _ro-3 , HPLC analysis showed that the conversion product had a retention time of 13.2 minutes. was detected. As a representative example, the HPLC chromatogram of the HpaB _ro-3 reaction solution is shown at the top of Figure 1. On the other hand, when (R)-equol was used as a substrate, HPLC was performed in the reaction solution of HpaB _pa , HpaB _ec , HpaB _pl-2 , HpaB _pl -3 , HpaB _ro-1 , HpaB _ro- 2 , and HpaB _ro-3 . Analysis detected a conversion product at a retention time of 13.2 minutes. As a representative example, the HPLC chromatogram of the HpaB _ro-3 reaction solution is shown at 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'-hydroxy equol, which is hydroxylated at the 3' position. The results of NMR analysis and LC-MS analysis are shown below.

¹ H NMR (400 MHz, [D ₄ ]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); ¹³ C 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 ( 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 solution of each substrate and each enzyme are shown in Figure 2. White bars are when the substrate is (S)-equol, and black bars are when the substrate is (R)-equol. HpaB _pa and HpaB _ec have been reported to exhibit activity 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 so far, showed high activity against equol, and HpaB _ro-3 in particular maintained very high activity.

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

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-hydroxy equol with hydroxylation at the 6-position. The results of NMR analysis and LC-MS analysis 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,
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 C ₁₅ H ₁₃ O ₄ [MH] ^- , 257.08138; found, 257.08161.

[Example 4] Production of 3'-hydroxy equol on a flask scale Using HpaB _ro-3 , which showed the highest activity in Example 3, 3'-hydroxy equol synthesis was attempted on a flask scale. Specifically, bacterial cells 125 g/l (wet bacterial weight equivalent), 10 mM (S)
-equol or (R)-equol, 4% dimethyl sulfoxide (v/v), 10% glycerol (v/v), 1.5% Tween 80 (v/v), 200 mM potassium phosphate buffer (pH 7.5). After preparing 20 ml of the reaction solution according to the composition, it was shaken at 30°C.

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

[Example 5] 6-hydroxy equol production on flask scale
We attempted to synthesize 6-hydroxyequol on a flask scale using HpaB _pl-1 . Here, in order to efficiently solubilize and express HpaB _pl-1 , we used bacterial cells in which HpaB _pl-1 was coexpressed with chaperonin and cochaperonin. Specifically, bacterial cells: 125 g/l (converted to wet bacterial weight)
, (S)-equol or (R)-equol 10 mM, dimethyl sulfoxide 4% (v/v), glycerol 10% (v/v), Tween 80 1.5% (v/v), potassium phosphate buffer 200 After preparing 20 ml of a reaction solution having a composition of mM (pH 7.5), it was shaken at 30°C.

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

Claims (6)

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, the composition comprising:
The equol derivative is 3'-hydroxy equol or 6-hydroxy equol,
The enzyme or microorganism is a flavin-dependent oxidase, or a microorganism that expresses the flavin-dependent oxidase,
A composition, wherein the flavin-dependent oxidase comprises an amino acid sequence having 90 % or more identity with any of the amino acid sequences of SEQ ID NOs: 1 to 8, and is an enzyme capable of converting the equol into the 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 a fermented alfalfa. A method for producing an equol derivative, the method comprising the step of acting on an equol-containing composition with an enzyme or microorganism capable of converting equol in the equol-containing composition into an equol derivative,
The equol derivative is 3'-hydroxy equol or 6-hydroxy equol,
The enzyme or microorganism is a flavin-dependent oxidase, or a microorganism that expresses the flavin-dependent oxidase,
The production method, wherein the flavin-dependent oxidase comprises an amino acid sequence having 90 % or more identity with any of the amino acid sequences of SEQ ID NOs: 1 to 8, and is an enzyme capable of converting the equol into the equol derivative. The manufacturing method according to claim 3,
The step of causing an enzyme or microorganism capable of converting equol in the equol-containing composition into an equol derivative to act on the equol-containing composition comprises culturing the microorganism in a medium containing the equol-containing composition, and converting the equol-containing composition into Including the process of producing equol derivatives from equol in substances by fermentation,
The microorganism is a microorganism that expresses the flavin-dependent oxidase,
The flavin-dependent oxidase comprises an amino acid sequence having 90 % or more identity with any of the amino acid sequences of SEQ ID NOs: 1 to 8, and is an enzyme capable of converting the equol into the equol derivative;
Including the step of recovering the produced equol derivative,
Production method. A step 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 into an equol derivative, and a step of blending the equol derivative with raw materials for food and drink products. , a method for producing a food or drink containing an equol derivative, comprising:
The equol derivative is 3'-hydroxy equol or 6-hydroxy equol,
The enzyme or microorganism is a flavin-dependent oxidase, or a microorganism that expresses the flavin-dependent oxidase,
The production method, wherein the flavin-dependent oxidase comprises an amino acid sequence having 90 % or more identity with any of the amino acid sequences of SEQ ID NOs: 1 to 8, and is an enzyme capable of converting the equol into the equol derivative. A step 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 into an equol derivative; and a step of blending the equol derivative with a raw material for a pharmaceutical product. A method for producing a pharmaceutical containing an equol derivative, the method comprising:
The equol derivative is 3'-hydroxy equol or 6-hydroxy equol,
The enzyme or microorganism is a flavin-dependent oxidase, or a microorganism that expresses the flavin-dependent oxidase,
The production method, wherein the flavin-dependent oxidase comprises an amino acid sequence having 90 % or more identity with any of the amino acid sequences of SEQ ID NOs: 1 to 8, and is an enzyme capable of converting the equol into the equol derivative.

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