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WO2022186355A1 - Composition pour la production de 6,3'-dihydroxyequol - Google Patents

Composition pour la production de 6,3'-dihydroxyequol Download PDF

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Publication number
WO2022186355A1
WO2022186355A1 PCT/JP2022/009235 JP2022009235W WO2022186355A1 WO 2022186355 A1 WO2022186355 A1 WO 2022186355A1 JP 2022009235 W JP2022009235 W JP 2022009235W WO 2022186355 A1 WO2022186355 A1 WO 2022186355A1
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WIPO (PCT)
Prior art keywords
equol
enzyme
microorganism
containing composition
hydroxyequol
Prior art date
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PCT/JP2022/009235
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English (en)
Japanese (ja)
Inventor
彰収 松山
俊樹 古屋
大樹 野澤
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Daicel Corp
Tokyo University of Science
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Daicel Corp
Tokyo University of Science
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Priority to JP2023503957A priority Critical patent/JP7632853B2/ja
Publication of WO2022186355A1 publication Critical patent/WO2022186355A1/fr
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/24—Drugs for disorders of the endocrine system of the sex hormones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic

Definitions

  • the present disclosure relates to compositions for the production of 6,3'-dihydroxyequol.
  • Equol is known to have the highest estrogenic activity among metabolites of isoflavones contained in soybeans (Non-Patent Documents 1 and 2).
  • equol derivatives such as 5-hydroxyequol have also been reported to have estrogenic activity and 3 ⁇ -hydroxysteroid dehydrogenase inhibitory activity (Patent Document 1).
  • Patent Document 3 By inhibiting the biosynthesis of qualitative corticoids, it is expected to be used for the prevention or treatment of excess of these hormones.
  • 3'-hydroxyequol can be obtained by a chemical synthesis method (Non-Patent Document 3).
  • an enzyme called flavin-dependent oxidase is known.
  • the enzymes have different substrate specificities and actions depending on their types.
  • HpaB and HpaC possessed by the Pseudomonas aeruginosa PAO1 strain have been reported to exhibit activity against hydroxystilbene (resveratrol).
  • a method of producing hydroxystilbene by reacting a substrate compound with a protein having at least 50% homology at the amino acid level with HpaB and HpaC derived from the Pseudomonas aeruginosa PAO1 strain has been reported (Patent Documents 2 and 3). ).
  • Patent Document 4 a technology capable of converting equol in an equol-containing composition into 3'-hydroxyequol or 6-hydroxyequol using a predetermined enzyme or microorganism.
  • An object of the present disclosure is to provide at least a composition for producing 6,3'-dihydroxyequol.
  • equol-containing composition a first enzyme or first microorganism capable of converting equol in said equol-containing composition to 3'-hydroxyequol; and a second enzyme capable of converting equol in said equol-containing composition to 6-hydroxyequol. or a composition comprising a second microorganism.
  • equol-containing composition is equol, fermented soybean germ extract, fermented soybean hypocotyl, fermented soybean, or fermented alfalfa.
  • An equol-containing composition includes a first enzyme or a first microorganism capable of converting equol in the equol-containing composition into 3′-hydroxyequol, and converting equol in the equol-containing composition into 6-hydroxy equol.
  • the acting step is a step of culturing the first microorganism and the second microorganism in a medium containing the equol-containing composition to produce 6,3′-dihydroxyequol. 4>.
  • the present disclosure can have the effect of providing at least a composition for producing 6,3'-dihydroxyequol.
  • Example 2 HPLC chromatogram showing the results of Example 2, which is one embodiment of the present disclosure.
  • 1 represents (S)-equol
  • 2 represents (S)-3'-hydroxyequol
  • 3 represents (S)-6-hydroxyequol
  • 4 represents (S)-6,3'.
  • - indicates a dihydroxyequol.
  • 4 is a graph showing the results of Example 3, which is one embodiment of the present disclosure.
  • White circles in the figure indicate (S)-equol
  • black triangles indicate (S)-3-hydroxyequol
  • black squares indicate (S)-6-hydroxyequol
  • black circles indicate (S)-6,3. '-dihydroxyequol is shown.
  • FIG. 4 is a graph showing changes over time in the conversion of (S)-equol to (S)-3′-hydroxyequol by HpaB ro-3- expressing E. coli, shown in Patent Document 4.
  • FIG. White circles in the figure indicate (S)-equol, and black circles indicate (S)-3'-hydroxyequol.
  • 4 is a graph showing changes over time when (S)-equol is converted to (S)-6-hydroxyequol by E. coli expressing HpaB pl-1 , as shown in Patent Document 4.
  • FIG. White circles in the figure indicate (S)-equol, and black circles indicate (S)-6-hydroxyequol.
  • composition of the present disclosure is a concept that includes a mixture, regardless of whether its components are homogeneous or heterogeneous.
  • composition One aspect of the present disclosure provides an equol-containing composition, a first enzyme or first microorganism capable of converting equol in the equol-containing composition to 3′-hydroxyequol, and equol in the equol-containing composition.
  • a composition comprising a second enzyme or a second microorganism capable of converting to 6-hydroxyequol.
  • the equol-containing composition comprises a first enzyme or a first microorganism capable of converting equol in the equol-containing composition to 3′-hydroxyequol, and converting equol in the equol-containing composition to 6-hydroxyequol.
  • a first enzyme or a first microorganism capable of converting equol in the equol-containing composition to 3′-hydroxyequol, and converting equol in the equol-containing composition to 6-hydroxyequol.
  • the aspect is not limited.
  • Examples of the equol-containing composition include fermented leguminous plants such as soybeans, kidney beans, fava beans, peanuts, chickpeas, kudzu, red clover, alfalfa, and licorice.
  • the legume is soybean, it is a fermented product of soybean itself (fermented soybean product).
  • a fermented soybean germ portion (fermented soybean germ) or a fermented soybean hypocotyl portion (fermented soybean hypocotyl) may be used. It may be a fermented product of a part of soybeans, such as Furthermore, taking fermented soybean germ as an example, it may be a fermented product (fermented soybean germ extract) obtained by fermenting an extract obtained from a portion of soybean germ.
  • the fermented soybean product is a fermented product obtained by fermenting the whole soybean. Like the fermented soybean hypocotyl, it is different from a fermented product obtained by fermenting a part of soybeans.
  • the soybeans themselves are prepared according to a conventional method, and koji mold is added to ferment them to obtain a fermented product.
  • the soybeans themselves may be prepared raw, or may be ground after being subjected to heat treatment, drying treatment, steaming treatment, etc., or may be heated after being ground.
  • a processed, dried, or steamed product may be prepared.
  • the mode of obtaining the fermented soybean germ, the fermented soybean hypocotyl, and the fermented soybean germ extract can also follow a conventional method.
  • the equol-containing composition may be one obtained by allowing an enzyme to act on the legume-derived isoflavones, or a fermented product obtained by fermentation using microorganisms.
  • an enzyme to act on the legume-derived isoflavones
  • a fermented product obtained by fermentation using microorganisms.
  • isoflavone aglycones obtained by allowing enzymes such as ⁇ -glucosidase to act on soybean-derived isoflavones, and microorganisms having ⁇ -glucosidase activity on soybean-derived isoflavones are used. Examples include isoflavone aglycones obtained by fermentation.
  • the equol-containing composition may be equol.
  • equol may be (R)-equol or (S)-equol.
  • the aspect is Not restricted.
  • the enzyme can similarly hydroxylate the 3'-position when the substrate is 6-hydroxyequol instead of equol. That is, the enzyme can convert equol in the equol-containing composition to 3'-hydroxyequol and 6-hydroxyequol to 6,3'-dihydroxyequol.
  • the enzyme may be an enzyme produced by a microorganism or an enzyme obtained without production by a microorganism (for example, an enzyme obtained by a chemical synthesis method).
  • the microorganism may be a microorganism that originally expresses the enzyme, or a microorganism that has been made to express the enzyme by a known technique such as genetic recombination. good too.
  • a known method can be used as the recovery method.
  • the microorganisms are collected by a method such as filtration or centrifugation, washed with a buffer solution, physiological saline, or the like, and subjected to physical treatment (e.g., freeze-thaw treatment, ultrasonic treatment, etc.). , pressure treatment, osmotic pressure difference treatment, grinding treatment, etc.), biochemical treatment (e.g., treatment with cell wall-dissolving enzyme such as lysozyme), chemical treatment (e.g., contact treatment with surfactant, etc.), Enzymes can be recovered singly or in combination. The recovered enzyme may then be subjected to separation or purification treatment.
  • physical treatment e.g., freeze-thaw treatment, ultrasonic treatment, etc.
  • biochemical treatment e.g., treatment with cell wall-dissolving enzyme such as lysozyme
  • chemical treatment e.g., contact treatment with surfactant, etc.
  • Enzymes can be recovered singly or in combination. The recovered enzyme may then
  • Examples of the first enzyme capable of converting equol in the equol-containing composition to 3'-hydroxyequol include oxidoreductases (dehydrogenase, cytochrome, catalase, oxidase, oxygenase (e.g., flavin-dependent oxidase, etc.), fatty acid desaturase, etc.), transferase (acyltransferase, phosphotransferase, aminotransferase, etc.), protease (protease), lipid decomposition enzyme (lipase), carbohydrate decomposition enzyme (amylase, lysozyme, ⁇ - galactosidase, etc.), phosphatase (nuclease, phosphatase, restriction enzyme), hydrolase (urease, lactonase, ATP hydrolase, etc.), releasing enzyme (carbonic hydratase, pyruvate decarboxylase, etc.), isomerase ( race
  • HpaB pa SEQ ID NO: 1 derived from Pseudomonas aeruginosa PAO1
  • HpaB ec SEQ ID NO: 2 derived from Escherichia coli BL21 (DE3)
  • HpaB pl-2 SEQ ID NO: 4
  • HpaB ro-1 SEQ ID NO: 6
  • HpaB ro-2 SEQ ID NO: 7
  • HpaB ro-3 SEQ ID NO: 8 from Rhodococcus opacus B4.
  • examples of the flavin reductase that supplies reduced flavin to the flavin-dependent oxidase include those described in Patent Document 4. That is, Pseudomonas aeruginosa PAO1-derived HpaC pa (SEQ ID NO: 17) can be mentioned.
  • the amino acid sequence of HpaB pa derived from Pseudomonas aeruginosa PAO1 is 80% or more, preferably 90%, the amino acid sequence represented by SEQ ID NO: 1, as long as the equol in the equol-containing composition can be converted to 3'-hydroxyequol. It may be an amino acid sequence having an identity of 95% or more, more preferably 95% or more. This is the same for each HpaB and HpaC pa other than HpaB pa described above.
  • the first microorganism capable of converting equol in the equol-containing composition to 3′-hydroxyequol is not limited in its aspect as long as it can convert equol in the equol-containing composition to 3′-hydroxyequol.
  • the microorganism is preferably a microorganism having equol oxidation activity (equol oxygenase), more preferably a microorganism expressing the flavin-dependent oxidase.
  • the microorganism can similarly hydroxylate the 3'-position when the substrate is 6-hydroxyequol instead of equol. That is, the microorganism can convert equol in the equol-containing composition to 3'-hydroxyequol and 6-hydroxyequol to 6,3'-dihydroxyequol.
  • the microorganism may be a microorganism that can originally convert equol to 3′-hydroxyequol, or a microorganism that has been modified by known techniques such as genetic recombination so that equol can be converted to 3′-hydroxyequol. microorganisms. Although the classification, genus, species, etc. of such microorganisms are not limited, bacteria (such as Escherichia coli) and fungi (such as yeast and filamentous fungi) are preferred.
  • the second enzyme capable of converting equol in the equol-containing composition to 6-hydroxyequol is not limited as long as it can convert equol in the equol-containing composition to 6-hydroxyequol.
  • the enzyme can also hydroxylate the 6-position when the substrate is 3'-hydroxyequol instead of equol. That is, the enzyme can convert equol in the equol-containing composition to 6-hydroxyequol and 3'-hydroxyequol to 6,3'-dihydroxyequol.
  • the enzyme may be an enzyme produced by a microorganism or an enzyme obtained without production by a microorganism (for example, an enzyme obtained by a chemical synthesis method).
  • the microorganism may be a microorganism that originally expresses the enzyme, or a microorganism that has been made to express the enzyme by a known technique such as genetic recombination. good too.
  • a known method can be used as the recovery method.
  • the microorganisms are collected by a method such as filtration or centrifugation, washed with a buffer solution, physiological saline, or the like, and subjected to physical treatment (e.g., freeze-thaw treatment, ultrasonic treatment, etc.). , pressure treatment, osmotic pressure difference treatment, grinding treatment, etc.), biochemical treatment (e.g., treatment with cell wall-dissolving enzyme such as lysozyme), chemical treatment (e.g., contact treatment with surfactant, etc.), Enzymes can be recovered singly or in combination. The recovered enzyme may then be subjected to separation or purification treatment.
  • physical treatment e.g., freeze-thaw treatment, ultrasonic treatment, etc.
  • biochemical treatment e.g., treatment with cell wall-dissolving enzyme such as lysozyme
  • chemical treatment e.g., contact treatment with surfactant, etc.
  • Enzymes can be recovered singly or in combination. The recovered enzyme may then
  • Enzymes capable of converting equol in the equol-containing composition to 6-hydroxyequol include, for example, oxidoreductases (dehydrogenase, cytochrome, catalase, oxidase, oxygenase (e.g., flavin-dependent oxidase, etc.), fatty acid desaturase, etc.).
  • oxidoreductases dehydrogenase, cytochrome, catalase, oxidase, oxygenase (e.g., flavin-dependent oxidase, etc.), fatty acid desaturase, etc.
  • transferase (acyltransferase, phosphotransferase, aminotransferase, etc.), protease (protease), lipid-degrading enzyme (lipase), carbohydrate-degrading enzyme (amylase, lysozyme, ⁇ -galactosidase, etc.), phosphor Acidase (nuclease, phosphatase, restriction enzyme), hydrolase (urease, lactonase, ATP hydrolase, etc.), releasing enzyme (carbonic hydratase, pyruvate decarboxylase, etc.), isomerase (racemase, phosphoglycerate) phosphomutase, glucose-6-phosphate isomerase, etc.), synthetase (DNA ligase, aminoacyl-tRNA synthetase, acyl-CoA synthetase, carboxylase, etc.). Oxygenase is preferred, and flavin-dependent
  • flavin-dependent oxidases examples include those described in Patent Document 4. That is, Photorhabdus luminescens sub sp. laumondii TTO1-derived HpaB pl-1 (SEQ ID NO: 3) can be mentioned.
  • examples of the flavin reductase that supplies reduced flavin to the flavin-dependent oxidase include those described in Patent Document 4. That is, Pseudomonas aeruginosa PAO1-derived HpaC pa (SEQ ID NO: 17) can be mentioned.
  • the amino acid sequence of HpaB pl-1 derived from Photorhabdus luminescens sub sp. laumondii TTO1 is 80% or more the amino acid sequence represented by SEQ ID NO: 3 as long as the equol in the equol-containing composition can be converted to 6-hydroxyequol. , preferably 90% or more, more preferably 95% or more, amino acid sequences. This is the same for HpaCpa .
  • the second microorganism capable of converting equol in the equol-containing composition to 6-hydroxyequol is not limited in its aspect as long as it can convert equol in the equol-containing composition to 6-hydroxyequol.
  • the microorganism is preferably a microorganism having equol oxidation activity (equol oxygenase), more preferably a microorganism expressing the flavin-dependent oxidase.
  • the microorganism can similarly hydroxylate the 6-position when the substrate is 3'-hydroxyequol instead of equol. That is, the microorganism can convert equol in the equol-containing composition to 6-hydroxyequol and 3'-hydroxyequol to 6,3'-dihydroxyequol.
  • the microorganism may be a microorganism that can originally convert equol to 6-hydroxyequol, or a microorganism that has been modified by known techniques such as genetic recombination so that equol can be converted to 6-hydroxyequol.
  • the classification, genus, species, etc. of such microorganisms are not limited, bacteria (such as Escherichia coli) and fungi (such as yeast and filamentous fungi) are preferred.
  • Another aspect of the present disclosure provides, in an equol-containing composition, a first enzyme or first microorganism capable of converting equol in the equol-containing composition to 3′-hydroxyequol, and A method for producing 6,3'-dihydroxyequol, comprising a step of acting with a second enzyme or a second microorganism capable of converting equol to 6-hydroxyequol.
  • the equol-containing composition in this embodiment a first enzyme or a first microorganism capable of converting equol in the equol-containing composition to 3′-hydroxyequol, and converting equol in the equol-containing composition to 6-hydroxy
  • the second enzyme or the second microorganism capable of converting to equol the explanation given above is used.
  • the step of allowing the equol-containing composition to react with the first enzyme capable of converting equol in the equol-containing composition to 3′-hydroxyequol is carried out in a solution containing the equol-containing composition. and a step of reacting equol in the substance with the enzyme.
  • the step of allowing the equol-containing composition to react with the first enzyme capable of converting the equol in the equol-containing composition into 3′-hydroxyequol will be described. The same applies to the step of allowing a second enzyme capable of converting equol in the equol-containing composition to 6-hydroxyequol.
  • 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 organic solvents include ethyl acetate, butyl acetate, toluene, chloroform, n-hexane and the like.
  • Examples of aqueous media include ethanol and acetone.
  • an inclusion compound may be added to the solution in order to increase the solubility of the substrate equol.
  • inclusion compounds include ⁇ -cyclodextrin, ⁇ -cyclodextrin, ⁇ -cyclodextrin, cluster dextrin (highly branched cyclic dextrin), and analogues thereof.
  • examples include methyl- ⁇ -cyclodextrin, trimethyl- ⁇ -cyclodextrin, hydroxypropyl- ⁇ -cyclodextrin and the like.
  • the amount of the clathrate compound to be added to the solution is usually 0.1 equivalent or more, preferably 0.5 equivalent or more, more preferably 1.5 equivalent or more, in terms of total molar ratio to equol in the equol-containing composition. It is 0 equivalents or more, and is usually 5.0 equivalents or less, preferably 2.5 equivalents or less, more preferably 2.0 equivalents or less.
  • the reaction temperature is preferably 20°C to 45°C, more preferably 25°C to 40°C, still 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 4-10, more preferably 6-8.
  • a step of recovering the enzyme from the enzyme-producing microorganism may be included prior to the step of allowing the enzyme to act, and prior to that, a step of culturing the enzyme-producing microorganism may be included.
  • the step of culturing the microorganism that produces the enzyme is not particularly limited as long as it is performed under conditions that allow the microorganism to produce the enzyme. Examples thereof include conditions for culturing the microorganism in a medium containing an equol-containing composition, which will be described later.
  • the step of recovering the enzyme from the enzyme-producing microorganism may include a step of collecting the microorganism by a method such as filtration or centrifugation, and then washing the microorganism with a buffer solution, physiological saline, or the like. may include the step of Furthermore, thereafter, 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-dissolving enzyme such as lysozyme, etc.) ), chemical treatment (eg, contact treatment with a surfactant, etc.), which may be used alone or in combination to recover the enzyme. Moreover, a step of purifying the enzyme, a step of concentrating the enzyme, or the like may be included thereafter.
  • physical treatment e.g., freeze-thaw treatment, ultrasonic treatment, pressure treatment, osmotic pressure difference treatment, grinding treatment, etc.
  • biochemical treatment
  • the step of allowing the equol-containing composition to act on the first microorganism capable of converting equol in the equol-containing composition to 3′-hydroxyequol includes: , converting equol in the equol-containing composition to 3′-hydroxyequol.
  • it is a step of culturing the microorganism in a medium containing an equol-containing composition and producing 3′-hydroxyequol from the equol in the equol-containing composition by fermentation.
  • the step of allowing the equol-containing composition to react with the first microorganism capable of converting the equol in the equol-containing composition into 3′-hydroxyequol will be described. The same applies to the step of allowing a second microorganism capable of converting equol in the equol-containing composition to 6-hydroxyequol.
  • the solution containing the equol-containing composition may be a solution in which the microorganisms can grow or a solution in which the microorganisms cannot grow.
  • the solution in which the microorganism can grow includes, for example, a medium, and the medium may be a minimal medium or a synthetic medium. If it is a commercially available medium, for example, ANAEROBE BASAL BROTH (ABB medium) manufactured by Oxoid, Wilkins-Chalgren Anaerobe Broth (CM0643) manufactured by Oxoid, GAM medium manufactured by Nissui Pharmaceutical Co., Ltd., modified GAM medium, brain heart infusion medium, LB medium and the like.
  • ABB medium ANAEROBE BASAL BROTH
  • CM0643 Wilkins-Chalgren Anaerobe Broth
  • GAM medium manufactured by Nissui Pharmaceutical Co., Ltd.
  • modified GAM medium brain heart infusion medium
  • LB medium and the like.
  • a water-soluble organic substance can be added to the medium as a carbon source.
  • water-soluble organic substances 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; Amino acids such as asparagine and the like can be mentioned.
  • the concentration of the organic matter can be appropriately adjusted for efficient growth. Generally, it ranges from 0.1 to 10 wt/vol%.
  • a nitrogen source can be added to the medium.
  • Various nitrogen compounds that can be used in normal fermentation can be used as the nitrogen source.
  • Preferred inorganic nitrogen sources include ammonium salts, nitrates, urea and the like, and more preferred examples include 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 (eg, polypeptone N, soybean peptone, etc.), meat extracts (eg, Ehrlich bonito extract, Rab-Remco powder, bouillon etc.), fish and shellfish extract, liver extract, digested serum powder, fish oil and the like.
  • cofactors such as vitamins and inorganic compounds such as various salts may be added to the medium to enhance growth and activity.
  • examples of microbial growth cofactors derived from animals and plants, such as inorganic compounds, vitamins, and fatty acids, include the following.
  • 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.
  • Magnesium sulfate heptahydrate Manganese sulfate tetrahydrate
  • reducing agents such as L-cysteine (hydrochloride), thioglycolic acid, ascorbic acid, mercaptoacetic acid, thiolacetic acid, glutathione, sodium sulfide, sodium sulfide, sulfites, thioglycolic acid, rutin, etc.
  • a reduction activator such as L-cystine and an enzyme that decomposes reactive oxygen species such as catalase and superoxide mutase may improve growth.
  • Usual culture methods such as shaking culture, aeration stirring culture, continuous culture, and fed-batch culture can be used as the culture method.
  • the gas phase and aqueous phase during cultivation preferably do not contain air or oxygen, for example, they may contain nitrogen and/or hydrogen in any ratio, or nitrogen and/or carbon dioxide in any ratio. and preferably a gas phase or aqueous phase 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 the oxidation of the 3'-position is promoted. % or less, preferably 20% or less, more preferably 10% or less.
  • the method of creating such an environment for the gas phase and aqueous phase during culture but for example, a method of replacing the gas phase with the gas before culture, and in addition, a method of removing gas from the bottom of the incubator during culture.
  • a method of supplying the gas and/or a method of supplying the gas to the gas phase portion of the incubator, and a method of bubbling the water phase with the gas before culture can be adopted.
  • hydrogen gas may be used as it is.
  • a source of hydrogen such as formic acid and/or a salt thereof may be added to the medium, and hydrogen may be produced 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.
  • the mixed gas can be supplied as nanobubbles.
  • the culture temperature is preferably 20°C to 45°C, more preferably 25°C to 40°C, still more preferably 30°C to 37°C.
  • the pressurizing condition of the incubator is not particularly limited as long as the conditions allow growth, and is, for example, 0.001 to 1 MPa, preferably 0.01 to 0.5 MPa.
  • Cultivation 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.
  • a surfactant include, for example, SDS, TritonX-100, Tween80, and adecanol, and can be added in an amount of about 0.001 g/L to 10 g/L.
  • adsorbents include cellulose and its derivatives; dextrin; Diaion HP series and Sepabeads series, which are hydrophobic adsorbents manufactured by Mitsubishi Chemical Corporation; and Amberlite XAD series manufactured by Organo Corporation.
  • inclusion compounds include ⁇ -cyclodextrin, ⁇ -cyclodextrin, ⁇ -cyclodextrin, cluster dextrin (highly branched cyclic dextrin), and analogues thereof.
  • examples include methyl- ⁇ -cyclodextrin, trimethyl- ⁇ -cyclodextrin, hydroxypropyl- ⁇ -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, more preferably 1.0 equivalent or more in terms of total molar ratio to equol in the equol-containing composition. On the other hand, it is usually 5.0 equivalents or less, preferably 2.5 equivalents or less, more preferably 2.0 equivalents or less.
  • solutions in which the microorganisms cannot grow include salt solutions and buffer solutions.
  • salt solutions include physiological saline and the like.
  • buffers include phosphate buffer, Tris-HCl buffer, citrate-phosphate buffer, citrate buffer, MOPS buffer, acetate buffer, glycine buffer and the like. The pH and concentration of the buffer solution can be appropriately adjusted according to a conventional method.
  • a stationary body is a microorganism that has been suspended in the same liquid as the washing liquid after removing the medium components from the cultured microorganism by centrifugation or the like, washing it with a salt solution or a buffer solution, and is in a non-proliferating state.
  • it refers to a microbial organism having at least a metabolic system capable of producing 3′-hydroxyequol from equol.
  • the case where the microorganisms may or may not grow in a solution is, for example, the case where immobilized microorganisms are used as the microorganisms.
  • immobilized microorganisms refers to microorganisms immobilized using known methods such as the polyacrylamide gel method, the sulfur-containing polysaccharide gel method (carrageenan gel method), the alginic acid gel method, the agar gel method, and the like. . In the present disclosure, such a gel or the like that immobilizes microorganisms is also defined as a solution.
  • This embodiment may include, for example, a step of quantifying the obtained 6,3'-dihydroxyequol.
  • a quantification method can follow a conventional method. For example, a portion of the culture solution is collected, diluted as appropriate, stirred well, filtered using a membrane such as polyterolafluoroethylene (PTFE) membrane, and insoluble matter is removed, followed by high-performance liquid chromatography. For example, it can be quantified.
  • PTFE polyterolafluoroethylene
  • This embodiment may also include a step of recovering the resulting 6,3'-dihydroxyequol.
  • the recovery process includes a purification process, a concentration process, and the like.
  • Purification processes in the purification process include sterilization of microorganisms by heat, etc.; sterilization by microfiltration (MF), ultrafiltration (UF), etc.; removal of solids and macromolecular substances; extraction by organic solvents, ionic liquids, etc. ; treatment such as adsorption and decolorization using a hydrophobic adsorbent, ion exchange resin, activated carbon column, or the like can be performed.
  • Concentration processing in the concentration step includes concentration using an evaporator, a reverse osmosis membrane, and the like.
  • a solution containing 6,3'-dihydroxyequol can be powdered by freeze-drying, spray-drying, or the like.
  • excipients such as lactose, dextrin, cornstarch and the like can also be added.
  • Another aspect of the present disclosure provides, in an equol-containing composition, a first enzyme or first microorganism capable of converting equol in the equol-containing composition to 3′-hydroxyequol, and A step of producing 6,3'-dihydroxyequol by reacting with a second enzyme or a second microorganism capable of converting equol to 6-hydroxyequol, and producing 6,3'-dihydroxyequol and food or drink.
  • the explanation of the method for producing 6,3'-dihydroxyequol already explained is used.
  • the step of recovering the produced 6,3′-dihydroxyequol is included, and the recovered 6,3′-dihydroxyequol and food and drink are It is preferable to set it as the process of mix
  • the raw materials of the food and drink As for the raw materials of the food and drink, the raw materials of the food and drink that are normally used can be used, and there are no particular restrictions on the timing of mixing.
  • the food and drink to be produced may contain water, protein, sugar, lipid, vitamins, minerals, organic acids, organic bases, fruit juices, flavors and the like as main ingredients.
  • proteins include animal and plant proteins such as whole milk powder, skim milk powder, partially skim milk powder, casein, soybean protein, chicken egg protein and meat protein, hydrolysates thereof, and butter.
  • Carbohydrates include saccharides, processed starch (dextrin, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), dietary fiber, and the like.
  • Lipids include, for example, vegetable oils such as lard, safflower oil, corn oil, rapeseed oil, coconut oil, fish oil, their fractionated oils, hydrogenated oils and transesterified oils.
  • vitamins include vitamin A, carotene, vitamin B group, vitamin C, vitamin D group, vitamin E, vitamin K group, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline. , and folic acid.
  • Minerals include, for example, calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, selenium, and whey minerals.
  • Organic acids include, for example, malic acid, citric acid, lactic acid, and tartaric acid. These components may be used in combination of two or more, or may be synthetic products.
  • manufactured food and drink include general food and drink, as well as foods for specified health uses, dietary supplements, functional foods, and foods for the sick.
  • the form of these food and drink is not particularly limited, but specifically, main dishes such as bread and noodles; side dishes such as cheese, ham, wieners and processed seafood products; Beverages;
  • luxury goods such as cookies, cakes, jellies, puddings, candies, and yogurt;
  • Capsules soft capsules, hard capsules), tablets, granules, powders, jellies, liposome formulations, supplements such as nutritional drinks, etc. be.
  • the content of 6,3′-dihydroxyequol relative to the total amount of the food or drink to be produced is not particularly limited, but the content is such that the effect of 6,3′-dihydroxyequol can be obtained when the food or drink is ingested.
  • the content of 6,3'-dihydroxyequol is usually 0.0001 to 50% by mass, preferably 0.001 to 50% by mass, more preferably 0.01 to 50% by mass, based on the total amount of food and drink.
  • the food or drink When the food or drink is a supplement, its form may be solid, gel or liquid. Examples include various processed food and drink, powders, tablets, pills, capsules, jellies and granules. etc. can be used.
  • supplements include 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 saccharose and carbohydrates containing these), acidulants, flavorings, fats and oils, emulsifiers, whole milk powder, and agar may be added. These components may be used in combination of two or more, or may be synthetic products.
  • Another aspect of the present disclosure provides, in an equol-containing composition, a first enzyme or first microorganism capable of converting equol in the equol-containing composition to 3′-hydroxyequol, and A step of producing 6,3'-dihydroxyequol by reacting with a second enzyme or a second microorganism capable of converting equol to 6-hydroxyequol, and the 6,3'-dihydroxyequol and a pharmaceutical material
  • a method for producing a pharmaceutical containing 6,3'-dihydroxyequol, comprising the step of blending
  • the explanation of the method for producing 6,3'-dihydroxyequol already explained is used.
  • the step of recovering the produced 6,3′-dihydroxyequol is included, and the recovered 6,3′-dihydroxyequol and raw materials for pharmaceuticals are included. It is preferable to set it as a step of blending.
  • the raw materials of pharmaceuticals As for the raw materials of pharmaceuticals, the raw materials of pharmaceuticals that are commonly used can be used, and there is no particular limitation on the timing of their blending.
  • the manufactured drug can be used as a drug for the prevention or treatment of diseases that can be prevented or treated by ingestion or administration of 6,3'-dihydroxyequol.
  • the dosage form can be selected according to the disease to be prevented or treated, the mode of use of the drug, the route of administration, and the like.
  • medicines for internal use such as tablets, granules, powders, capsules, soft capsules, syrups; emulsions, suspensions, ointments, creams, lotions, gels, sprays, patches, poultices , liniments, aerosols, ointments, packs, inhalants, external medicines such as suppositories; and injections.
  • compositions may be added to the main drug in accordance with conventional methods, if necessary, with fillers, extenders, excipients, binders, moisturizing agents, disintegrants, surfactants, lubricants, coloring agents, and flavoring agents. , solubilizing agents, suspending agents, coating agents, and other known auxiliary agents that can be commonly used in the technical field of pharmaceutical preparation.
  • solubilizing agents suspending agents, coating agents, and other known auxiliary agents that can be commonly used in the technical field of pharmaceutical preparation.
  • coloring agents, preservatives, flavoring agents, flavoring agents, sweetening agents, etc., and other pharmaceutical agents may be contained in this pharmaceutical product.
  • the content of 6,3'-dihydroxyequol relative to the total amount of the drug is not particularly limited, but the content should be such that the desired effect of 6,3'-dihydroxyequol can be obtained when the drug is taken or administered. is preferred.
  • the content of 6,3'-dihydroxyequol relative to the total amount of the drug is usually 0.0001-50% by mass, preferably 0.001-50% by mass, more preferably 0.01-50% by mass.
  • SEQ ID NO: 1 is the amino acid sequence of the enzyme HpaB pa encoded by the Pseudomonas aeruginosa PAO1-derived flavin-dependent oxidase gene hpaB pa (ORF No. PA4091).
  • SEQ ID NO: 2 is the amino acid sequence of the enzyme HpaB ec encoded by the Escherichia coli BL21 (DE3)-derived flavin-dependent oxidase gene hpaB pa (ORF No. B21_04188).
  • 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.
  • 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 Rhodococcus opacus B4-derived flavin-dependent oxidase gene (ORF No. ROP_20940).
  • SEQ ID NO: 7 is the amino acid sequence of the enzyme HpaB ro-2 encoded by the Rhodococcus opacus B4-derived flavin-dependent oxidase gene (ORF No. ROP_22410).
  • SEQ ID NO: 8 is the amino acid sequence of the enzyme HpaB ro-3 encoded by the Rhodococcus opacus B4-derived flavin-dependent oxidase gene (ORF No. ROP_37410).
  • SEQ ID NO: 17 is the amino acid sequence of the enzyme HpaC pa , which is derived from Pseudomonas aeruginosa PAO1 and encoded by the flavin reductase gene (ORF no. PA4092) that supplies reduced flavin to flavin-dependent oxidase.
  • SEQ ID NO: 9 is the base sequence of the gene hpaB pa , which is a Pseudomonas aeruginosa PAO1-derived flavin-dependent oxidase gene and corresponds to ORF No. PA4091.
  • SEQ ID NO: 10 is the nucleotide sequence of hpaBec , a gene of Escherichia coli BL21 (DE3)-derived flavin-dependent oxidase, corresponding to ORF No. B21_04188.
  • SEQ ID NO: 11 is the gene of Photorhabdus luminescens sub sp.
  • SEQ ID NO: 12 is a gene of Photorhabdus luminescens sub sp. laumondii TTO1-derived flavin-dependent oxidase, which is the base sequence of gene hpaB pl-2 corresponding to ORF No. plu0975.
  • SEQ ID NO: 13 is the gene of Photorhabdus luminescens sub sp. laumondii TTO1-derived flavin-dependent oxidase, and is the base sequence of gene hpaB pl-3 corresponding to ORF No. plu4027.
  • SEQ ID NO: 14 is a Rhodococcus opacus B4-derived flavin-dependent oxidase gene, which is the base sequence of the gene hpaB ro-1 corresponding to ORF No. ROP_20940.
  • SEQ ID NO: 15 is the base sequence of the gene hpaB ro-2 , which is a Rhodococcus opacus B4-derived flavin-dependent oxidase gene and corresponds to ORF No. ROP_22410.
  • SEQ ID NO: 16 is the base sequence of hpaB ro-3 gene, which is a Rhodococcus opacus B4-derived flavin-dependent oxidase gene and corresponds to ORF No. ROP_37410.
  • SEQ ID NO: 18 is a flavin reductase gene derived from Pseudomonas aeruginosa PAO1 and supplying reduced flavin to a flavin-dependent oxidase, and is the base sequence of the gene hpaC pa corresponding to ORF no. PA4092.
  • Example 1 Preparation of recombinant Escherichia coli expressing flavin-dependent oxidases
  • the gene hpaB pl-1 derived from Photorhabdus luminescens sub sp. laumondii TTO1 (a gene corresponding to ORF No. plu0246.
  • the amino acid sequence is the amino acid sequence shown in SEQ ID NO: 3 and Rhodococcus opacus B4.
  • the derived gene hpaB ro-3 (a gene corresponding to ORF No. ROP_37410, the amino acid sequence is the amino acid sequence shown in SEQ ID NO: 8) was synthesized.
  • hpaB pl-1 was amplified by PCR using primers of SEQ ID NOs: 19 and 20, cleaved with restriction enzymes PciI and BamHI, and ligated to pETDuet-1 vector (manufactured by Novagen) to prepare pETDhpaB pl-1 . did.
  • hpaB ro-3 was amplified by PCR using the primers of SEQ ID NOS: 21 and 22, cleaved with restriction enzymes NdeI and MunI, and ligated to pETDuet-1 vector to prepare pETDhpaB ro-3 .
  • the gene hpaC pa derived from Pseudomonas aeruginosa PAO1 (a gene corresponding to ORF no. PA4092; the amino acid sequence is the amino acid sequence shown in SEQ ID NO: 17) was transferred to pCDFDuet-1 vector (manufactured by Novagen).
  • the ligated plasmid pCDFDhpaCpa was used.
  • each of pETDhpaB pl-1 and pETDhpaB ro-3 was introduced into Escherichia coli BL21 Star (DE3) together with pCDFDhpaC pa by the heat shock method.
  • pETDhpaB pl-1 a plasmid pGro7 (manufactured by Takarabio) ligated with a chaperonin-encoding gene was also introduced to promote soluble expression of the protein.
  • Example 2 Analysis of reaction between flavin-dependent oxidase and (S)-equol Escherichia coli harboring pETDhpaB pl-1 , pCDFDhpaC pa and pGro7 were cultured and induced for expression as follows.
  • the recombinant E. coli was placed 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 of isopropyl- ⁇ -D-thiogalactopyranoside was added and cultured at 15°C for 15 hours to induce gene expression. After collection, the cells were washed with 50 mM potassium phosphate buffer (pH 7.5) containing 10% (v/v) glycerol, and the collected cells were used for the reaction.
  • Escherichia coli harboring pETDhpaB ro-3 and pCDFDhpaC pa were cultured and induced for expression as follows.
  • the produced recombinant E. coli was inoculated into LB medium containing 50 ⁇ g/ml ampicillin and 50 ⁇ g/ml streptomycin and cultured at 30° C. for 6 hours. After 6 hours, 1 mM of isopropyl- ⁇ -D-thiogalactopyranoside was added and cultured at 25°C for 15 hours to induce gene expression. After collection, the cells were washed with 50 mM potassium phosphate buffer (pH 7.5) containing 10% (v/v) glycerol, and the collected cells were used for the reaction.
  • 50 mM potassium phosphate buffer pH 7.5
  • Example 3 Flask-scale (S)-6,3'-dihydroxyequol production
  • flask-scale (S)-6,3'-dihydroxyequol production was attempted.
  • (S)-equol was converted to (S)-6,3'-dihydroxyequol via (S)-3'-hydroxyequol or (S)-6-hydroxyequol (Fig. 2 ).
  • the production of (S)-6,3'-dihydroxyequol reached 8.7 mM (2.4 g/l) in 24 hours. Also, no by-products were detected (Fig. 1). From the above, it was clarified that (S)-6,3'-dihydroxyequol can be efficiently produced on a flask scale.
  • Patent Document 4 when E. coli expressing HpaB ro-3 is reacted with (S)-equol, (S)-3'-hydroxyequol is produced (Fig. 3), and E. coli expressing HpaB pl-1 ( When reacted with S)-equol, (S)-6-hydroxyequol was produced (Fig. 4), but neither enzyme produced (S)-6,3'-dihydroxyequol alone. In this study, (S)-6,3'-dihydroxyequol was produced by simultaneously reacting E. coli expressing HpaB ro-3 and E. coli expressing HpaB pl-1 with (S)-equol.

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Abstract

Un problème abordé par la présente invention, au moins, est de fournir une composition pour la production de 6,3'-dihydroxyequol. Le problème est résolu par une composition qui comprend une composition contenant de l'équol, une première enzyme ou un premier micro-organisme capable de convertir l'équol dans la composition contenant de l'équol en 3'-hydroxyequol, et une seconde enzyme ou un second micro-organisme capable de convertir l'équol dans la composition contenant de l'équol en 6-hydroxyequol.
PCT/JP2022/009235 2021-03-03 2022-03-03 Composition pour la production de 6,3'-dihydroxyequol Ceased WO2022186355A1 (fr)

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