BRPI0821816B1 - PROCESSES FOR PRODUCING EQUOL, TETRAHYDRODAIDZEIN AND DIHYDRODAIDZEIN - Google Patents

Abstract

ENZYME ASSOCIATED WITH EQUOL SYNTHESIS. An object of the present invention is to provide enzymes associated with equol synthesis, a gene encoding such enzymes, and a process for producing equol and its intermediates using the enzymes and genes. The present invention provides a dihydrodaidzein-synthesizing enzyme, tetrahydrodaidzein-synthesizing enzyme, equol-synthesizing enzyme, and genes encoding these enzymes. The present invention also provides a process for synthesizing dihydrodaidzein, tetrahydrodaidzein, and/or equol using these enzymes.

Description

Technical Field

[0001] The present invention relates to a polypeptide having an activity of synthesizing dihydrodaidzein using daidzein as a substrate, a polypeptide having an activity of synthesizing tetrahydrodaidzein using dihydrodaidzein as a substrate, and a polypeptide having an activity of synthesizing equol using tetrahydrodaidzein as a substrate, as well as a polynucleotide encoding these polypeptides. The invention also relates to processes for producing dihydrodaidzein, tetrahydrodaidzein, and equol using the above polypeptides, and a production apparatus used in the processes. The invention also provides techniques with respect thereto.

Background Technique

[0002] Isoflavone derivatives are believed to possess various physiological and pharmacological activities. For this reason, isoflavone derivatives are used as material for food and pharmaceutical products. As studies have revealed the functions of isoflavone derivatives, in this context there have been reports that the estrogen-like activity of isoflavone derivatives is not, as conveniently believed, due to the action of the isoflavone derivatives themselves, but rather due to strong estrogen-like activity. Estrogen found throughout the body displayed by equol, which is absorbed in the intestines after being released from various types of intestinal bacteria metabolizing (biosynthesizing) isoflavone derivatives to produce equol. In humans, not all individuals have the ability to produce equol in the intestines, and such ability to produce equol varies among different individuals. For example, some individuals may not have any equol-producing bacteria in their intestines, while others may have such bacteria but with only a limited ability to produce equol.

[0003] Consequently, it would be beneficial in countries with aging populations, such as Japan, to make effective use of equol in the body, particularly in consideration of chronic disorders such as osteoporosis affecting the elderly. Due to the fact that equol-producing bacteria are not found in all individuals, there is a need to find a synthesis of equol material that allows for efficient artificial equol production.

[0004] Under these circumstances, it is very important, in terms of providing a synthesis of equol material, to identify and use enzymes involved in the equol biosynthetic reaction series. However, no information is available regarding enzymes that produce or catalyze some of the intermediates involved in the biosynthetic reaction series. Consequently, identification of enzymes associated with the synthesis of such intermediates is desired.

Patent Document 1: JP-A-2006-296434 Description of the Invention Technical problem

[0005] It is an object of the present invention to provide an enzyme associated with the synthesis of dihydrodaidzein that can be used as an equol synthesis material. Specifically, the invention aims to provide a polypeptide having an activity of synthesizing dihydrodaidzein using daidzein as a substrate. The invention also aims to provide a polynucleotide encoding such a polypeptide, and techniques with respect to the synthesis of dihydrodaidzein using such a polypeptide.

[0006] It is another object of the present invention to provide an enzyme associated with the synthesis of tetrahydrodaidzein that can be used as an equol synthesis material. Specifically, the invention aims to provide a polypeptide having an activity of synthesizing tetrahydrodaidzein using dihydrodaidzein as a substrate. The invention also aims to provide a polynucleotide encoding such a polypeptide, and techniques with respect to the synthesis of tetrahydrodaidzein using such a polypeptide.

[0007] It is another object of the present invention to provide an enzyme associated with equol synthesis. Specifically, the invention aims to provide a polypeptide having an equol-synthesizing activity using tetrahydrodaidzein as a substrate. The invention also aims to provide a polynucleotide encoding such a polypeptide, and techniques with respect to the synthesis of equol using such a polypeptide.

[0008] It is another object of the present invention to provide a process for producing intermediates such as dihydrodaidzein and tetrahydrodaidzein generated in the production of equol from daidzein, and a process for producing equol using the obtained intermediates. The invention also aims to provide a production apparatus for use in production. Technical Solution

[0009] The inventors of the present invention conducted intensive studies to solve the above problems, and successfully isolated from equol-producing intestinal bacteria, an enzyme capable of synthesizing dihydrodaidzein, an enzyme capable of synthesizing tetrahydrodaidzein, and an enzyme capable of synthesizing equol, used as starting materials for equol synthesis, and revealed the structures of these enzymes. Furthermore, the inventors conducted other studies, and were successful in the artificial production of dihydrodaidzein, tetrahydrodaidzein, and equol, using the above enzymes. The present invention has been carried out in other studies based on these findings.

[00010] Specifically, in one aspect, the present invention provides:

[00011] Item A1. A polypeptide selected from: (Aa) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1; (Ab) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 with the substitution, deletion, insertion, and/or addition of one or more amino acids, and having an activity of synthesizing dihydrodaidzein using daidzein as a substrate; and (Ab) a polypeptide consisting of an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 1, and having an activity of synthesizing dihydrodaidzein using daidzein as a substrate.

[00012] Item A2. A polynucleotide selected from: (Ad) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4; (Ae) a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1; and (Af) a polynucleotide that hybridizes under stringent conditions with the complementary strand of the polynucleotide (Ad) or (Ae), and that encodes a polypeptide having an activity to synthesize dihydrodaidzein using daidzein as a substrate.

[00013] Item A3. An expression vector including the item A2 polynucleotide.

[00014] Item A4. A recombinant cell transformed with the item A3 expression vector.

[00015] Item A5. A recombinant cell according to item A4, wherein the recombinant cell is a bacterial prokaryotic cell.

[00016] Item A6. A recombinant cell according to item A5, wherein the bacterial prokaryotic cell belongs to the genus Lactococcus.

[00017] Item A7. A process for producing a polypeptide, comprising culturing a cell of any one of items A4 to A6 to obtain a polypeptide having an activity of synthesizing dihydrodaidzein using daidzein as a substrate.

[00018] Item A8. A polypeptide obtained by the process of item A7.

[00019] Item A9. A process for producing dihydrodaidzein, comprising having the polypeptide of Item A1 or A8, and NADPH and/or NADH acting on the daidzein.

[00020] Item A10. A process for producing dihydrodaidzein, comprising having the cell of any one of items A4 to A6 act on the daidzein.

[00021] Item A11. An antibody having the activity of the item A1 polypeptide, or the polypeptide encoded by the item A2 polynucleotide.

[00022] Item A12. An immunological method for detecting or measuring the item A1 polypeptide or the polypeptide encoded by the item A2 polynucleotide, the method comprising having the item A11 antibody contact the test sample.

[00023] Item A13. A method according to item A12, wherein the polypeptide to be detected or measured exists in a bacterial prokaryotic cell.

[00024] Item A14. A probe having a nucleotide sequence capable of hybridizing under stringent conditions to a polynucleotide encoding item A1 polypeptide, or item A2 polynucleotide.

[00025] Item A15. A primer having a nucleotide sequence capable of hybridizing under stringent conditions to a polynucleotide encoding the item A1 polypeptide, or to the item A2 polynucleotide.

[00026] Item A16. A method for detecting or measuring a polynucleotide encoding item A1 polypeptide, or item A2 polynucleotide, using item A14 probe.

[00027] Item A17. A method according to item A16, wherein the polypeptide to be detected or measured exists in a bacterial prokaryotic cell.

[00028] Item A18. A method according to item A16, comprising PCR amplification of all or part of a polynucleotide encoding the polypeptide of item A1, or the polynucleotide of item A2.

[00029] Item A19. A dihydrodaidzein synthesizing enzyme composition comprising the polypeptide of item A1, or a polypeptide encoded by the polynucleotide of item A2.

[00030] Item A20. A composition according to item A19, also comprising NADPH and/or NADH.

[00031] Item A21. A dihydrodaidzein synthetic composition, comprising: (Ai) the polypeptide of item A1, or a polypeptide encoded by the polynucleotide of item A2; (Aii) NADPH and/or NADH; and (Aiii) daidzein.

[00032] Item A22. A dihydrodaidzein synthesis composition, comprising: (Aiv) the cell of any one of items A4 to A6; and (Aiii) daidzein.

[00033] Item A23. A dihydrodaidzein synthesis kit, comprising: (Ai) the polypeptide of item A1, or a polypeptide encoded by the polynucleotide of item A2; (Aii) NADPH and/or NADH; and (Aiii) daidzein.

[00034] Item A24. A dihydrodaidzein synthesis kit, comprising: (Aiv) the cell of any one of items A4 to A6; and (Aiii) daidzein.

[00035] Item A25. An immunological measurement kit for measuring the item A1 polypeptide, or a polypeptide encoded by the item A2 polynucleotide,

[00036] The kit comprising at least the antibody of item A11.

[00037] Item A26. A PCR kit for detecting a polynucleotide encoding item A1 polynucleotide, or item A2 polynucleotide, the PCR kit comprising at least item A15 primer.

[00038] Item A27. A kit according to item A26, wherein the kit is to identify the cell containing the polynucleotide encoding the polypeptide of item A1, or the polynucleotide of item A2.

[00039] Item A28. A PCR kit in accordance with item A27, wherein the kit is for PCR.

[00040] Item A29. A dihydrodaidzein-synthesizing enzyme, consisting of the item A1 polypeptide.

[00041] Item B1. A polypeptide selected from: (Ba) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 7; (Bb) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 7 with the substitution, deletion, insertion, and/or addition of one or more amino acids, and having an activity of synthesizing tetrahydrodaidzein using dihydrodaidzein as a substrate; and (Bc) a polypeptide consisting of an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 7, and having an activity of synthesizing tetrahydrodaidzein using dihydrodaidzein as a substrate.

[00042] Item B2. A polynucleotide selected from: (Bd) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 10; (Be) a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 7; and (Bf) a polynucleotide that hybridizes under stringent conditions with the complementary strand of the polynucleotide (Bd) or (Be), and that encodes a polypeptide having an activity to synthesize tetrahydrodaidzein using dihydrodaidzein as a substrate.

[00043] Item B3. An expression vector including the item B2 polynucleotide.

[00044] Item B4. A recombinant cell transformed with the item B3 expression vector.

[00045] Item B5. A recombinant cell according to item B4, wherein the recombinant cell is a bacterial prokaryotic cell.

[00046] Item B6. A recombinant cell according to item B5, wherein the bacterial prokaryotic cell belongs to the genus Lactococcus.

[00047] Item B7. A process for producing a polypeptide, comprising culturing a cell of any one of items B4 to B6 to obtain a polypeptide having a tetrahydrodaidzein-forming activity using dihydrodaidzein as a substrate.

[00048] Item B8. a polypeptide obtained by the process of item B7.

[00049] Item B9. A process for producing tetrahydrodaidzein, comprising having the polypeptide of Item B1 or B8, and NADPH and/or NADH acting on the dihydrodaidzein.

[00050] Item B10. A process for producing tetrahydrodaidzein, comprising having the cell of any one of items B4 to B6 act on the dihydrodaidzein.

[00051] Item B11. An antibody having the activity of the item B1 polypeptide, or the polypeptide encoded by the item B2 polynucleotide.

[00052] Item B12. An immunological method for detecting or measuring the item B1 polypeptide or the polypeptide encoded by the item B2 polynucleotide, the method comprising having the item B11 antibody contact the test sample.

[00053] Item B13. A method according to item B12, wherein the polypeptide to be detected or measured exists in a bacterial prokaryotic cell.

[00054] Item B14. A probe having a nucleotide sequence capable of hybridizing under stringent conditions to a polynucleotide encoding item B1 polypeptide, or item B2 polynucleotide.

[00055] Item B15. A primer having a nucleotide sequence capable of hybridizing under stringent conditions to a polynucleotide encoding item B1 polypeptide, or item B2 polynucleotide.

[00056] Item B16. A method for detecting or measuring a polynucleotide encoding item B1 polypeptide, or item B2 polynucleotide, using item B14 probe.

[00057] Item B17. A method according to item B16, wherein the polypeptide to be detected or measured exists in a bacterial prokaryotic cell.

[00058] Item B18. A method according to item B16, comprising PCR amplification of all or part of a polynucleotide encoding the polypeptide of item B1, or the polynucleotide of item B2.

[00059] Item B19. The enzyme for synthesizing tetrahydrodaidzein composition, comprising the polypeptide of item B1, or a polypeptide encoded by the polynucleotide of item B2.

[00060] Item B20. A composition according to item B19, also comprising NADPH and/or NADH.

[00061] Item B21. A tetrahydrodaidzein synthetic composition, comprising: (Bi) the polypeptide of item B1, or a polypeptide encoded by the polynucleotide of item B2; (Bii) NADPH and/or NADH; and (Biii) dihydrodaidzein.

[00062] Item B22. A tetrahydrodaidzein synthetic composition, comprising: (Biv) the cell of any one of items B4 to B6; and (Biii) dihydrodaidzein.

[00063] Item B23. A tetrahydrodaidzein synthesis kit, comprising: (81) the item B1 polypeptide, or a polypeptide encoded by the item B2 polynucleotide; (811) NADPH and/or NADH; and (Biii) dihydrodaidzein.

[00064] Item B24. A tetrahydrodaidzein synthesis kit, comprising: (Biv) the cell of any one of items B4 to B6; and (Biii) dihydrodaidzein.

[00065] Item B25. An immunological measurement kit for measuring the item B1 polypeptide, or a polypeptide encoded by the item B2 polynucleotide, the kit comprising at least the item B11 antibody. Item B26. A PCR kit for detecting a polynucleotide encoding the item B1 polypeptide, or the item B2 polynucleotide, the PCR kit comprising at least the item B15 primer.

[00066] Item B27. A kit according to item B26, wherein the kit is to identify the cell containing the polynucleotide encoding the polypeptide of item B1, or the polynucleotide of item B2.

[00067] Item B28. A PCR kit in accordance with item B27, wherein the kit is for PCR.

[00068] Item B29. The enzyme for synthesizing tetrahydrodaidzein, which consists of the item B1 polypeptide.

[00069] Item C1. A polypeptide selected from: (Ca) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 13; (Cb) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 13 with the substitution, deletion, insertion, and/or addition of one or more amino acids, and having an activity to synthesize equol using tetrahydrodaidzein as a substrate; and (Cc) a polypeptide consisting of an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 13, and having an activity to synthesize equol using tetrahydrodaidzein as a substrate. Item C2. A polynucleotide selected from: (Cd) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 16; (Ce) a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 13; and (Cf) a polynucleotide that hybridizes under stringent conditions with the complementary strand of the polynucleotide (Cd) or (Ce), and that encodes a polypeptide having an activity to synthesize equol using tetrahydrodaidzein as a substrate.

[00070] Item C3. An expression vector including the item C2 polynucleotide.

[00071] Item C4. A recombinant cell transformed with the C3 item expression vector.

[00072] Item C5. A recombinant cell according to item C4, wherein the recombinant cell is a bacterial prokaryotic cell.

[00073] Item C6. A recombinant cell according to item C5, wherein the bacterial prokaryotic cell belongs to the genus Lacto-coccus.

[00074] Item C7. A process for producing a polypeptide, comprising culturing a cell of any one of C4 to C6 to obtain a polypeptide having an equol-forming activity using tetrahydrodaidzein as a substrate.

[00075] Item C8. A polypeptide obtained by the process of item C7.

[00076] Item C9. A process for producing equol, comprising having the polypeptide of Item C1 or C8 act on tetrahydrodaidzein.

[00077] Item C10. A process for producing equol, comprising having the cell of any one of C4 to C6 act on tetrahydrodaidzein.

[00078] Item C11. An antibody having the activity of the item C1 polypeptide, or the polypeptide encoded by the item C2 polynucleotide.

[00079] Item C12. An immunological method for detecting or measuring the item C1 polypeptide or the polypeptide encoded by the item C2 polynucleotide, the method comprising having the item C11 antibody contact the test sample.

[00080] Item C13. A method according to item C12, wherein the polypeptide to be detected or measured exists in a bacterial prokaryotic cell.

[00081] Item C14. A probe having a nucleotide sequence capable of hybridizing under stringent conditions to a polynucleotide encoding item C1 polypeptide, or item C2 polynucleotide.

[00082] Item C15. A primer having a nucleotide sequence capable of hybridizing under stringent conditions with a polynucleotide encoding item C1 polypeptide, or with item C2 polynucleotide.

[00083] Item C16. A method for detecting or measuring a polynucleotide encoding item C1 polypeptide, or item C2 polynucleotide, using item C14 probe.

[00084] Item C17. A method according to item C16, wherein the polypeptide to be detected or measured exists in a bacterial prokaryotic cell.

[00085] Item C18. A method according to item C16, comprising PCR amplification of all or part of a polynucleotide encoding the polypeptide of item C1, or the polynucleotide of item C2.

[00086] Item C19. An equol-synthesizing enzyme composition comprising the item C1 polypeptide, or a polypeptide encoded by the item C2 polynucleotide.

[00087] Item C20. An equol synthesis composition comprising: (Ci) the item C1 polypeptide, or a polypeptide encoded by the item C2 polynucleotide; and (Cii) tetrahydrodaidzein. Item C21. A synthesis of equol composition comprising: (Ciii) the cell of any one of items C4 to C6; and (Cii) tetrahydrodaidzein.

[00088] Item C22. An equol synthesis kit comprising: (Ci) the item C1 polypeptide, or a polypeptide encoded by the item C2 polynucleotide; and (Cii) tetrahydrodaidzein.

[00089] Item C23. An equol synthesis kit comprising: (Ciii) the cell of any one of items C4 to C6; and (Cii) tetrahydrodaidzein.

[00090] Item C24. An immunological measurement kit for measuring the polypeptide of item C1, or a polypeptide encoded by the polynucleotide of item C2, the kit comprising at least the antibody of item C11.

[00091] Item C25. A PCR kit for detecting a polynucleotide encoding the item C1 polypeptide, or the item C2 polynucleotide, the PCR kit comprising at least the item C15 primer.

[00092] Item C26. A kit according to item C25, wherein the kit is to identify the cell containing the polynucleotide encoding the polypeptide of item C1, or the polynucleotide of item C2.

[00093] Item C27. A kit in accordance with item C26, wherein the kit is for PCR.

[00094] Item C28. An equol-synthesizing enzyme, consisting of the item C1 polypeptide.

[00095] Item D1. A process for producing tetrahydrodaidzein comprising the following First Step and Second Step, the First Step comprising a step of having an enzyme consisting of one of the following polypeptides (Aa) to (Ac), and NADPH and/or NADH acting on the daidzein, thereby mode producing dihydrodaidzein, (Aa) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1; (Ab) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 with the substitution, deletion, insertion, and/or addition of one or more amino acids, and having an activity of synthesizing dihydrodaidzein using daidzein as a substrate; and (Ac) a polypeptide consisting of an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 1, and having an activity of synthesizing dihydrodaidzein using daidzein as a substrate, Second Step comprising a step of having an enzyme consisting of one of the following polypeptides (Ba) to (Bc) and NADPH and/or NADH act on dihydrodaidzein, thereby producing tetrahydrodaidzein, (Ba) a polypeptide comprising the amino acid sequence of SEQ ID NO: 7; (Bb) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 7 with the substitution, deletion, insertion, and/or addition of one or more amino acids, and having an activity of synthesizing tetrahydrodaidzein using dihydrodaidzein as a substrate; and (Bc) a polypeptide consisting of an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 7, and having an activity of synthesizing tetrahydrodaidzein using dihydrodaidzein as a substrate.

[00096] Item D2. A product containing tetrahydrodaidzein, produced by the process according to item D1.

[00097] Item D3. A process for producing equol comprising the following Second Step and Third Step, the Second Step comprising a step of having an enzyme consisting of one of the following polypeptides (Ba) to (Bc) and NADPH and/or NADH act on the dihydrodaidzein, thereby producing tetrahydrodaidzein, (Ba) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 7; (Bb) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 7 with the substitution, deletion, insertion, and/or addition of one or more amino acids, and having an activity of synthesizing tetrahydrodaidzein using dihydrodaidzein as a substrate; and (Bc) a polypeptide consisting of an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 7, and having an activity of synthesizing tetrahydrodaidzein using dihydrodaidzein as a substrate,

[00098] Third Step comprising a step of having an enzyme consisting of one of the following polypeptides (Ca) to (Cc) to act on tetrahydrodaidzein, thereby producing equol, (Ca) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 13; (Cb) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 13 with the substitution, deletion, insertion, and/or addition of one or more amino acids, and having an activity to synthesize equol using tetrahydrodaidzein as a substrate; and (Cc) a polypeptide consisting of an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 13, and having an activity to synthesize equol using tetrahydrodaidzein as a substrate.

[00099] Item D4. A product containing equol, produced by the process in accordance with item D3.

[000100] Item D5. A process for producing equol comprising First Step to Third Step.

[000101] Item D6. A product containing equol, produced by the process in accordance with item D5.

[000102] Item D7. An expression vector having at least one polynucleotide selected from the group consisting of the following (Ad) to (Af), (Bd) to (Bf), and (Cd) to (Cf), (Ad) a polynucleotide consisting of the sequence of nucleotide of SEQ ID NO: 4; (Ae) a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1; (Af) a polynucleotide that hybridizes under stringent conditions with the complementary strand of the polynucleotide (Ad) or (Ae), and that encodes a polypeptide having an activity to synthesize dihydrodaidzein using daidzein as a substrate; (Bd) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 10; (Be) a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 7; (Bf) a polynucleotide that hybridizes under stringent conditions with the complementary strand of the polynucleotide (Bd) or (Be), and that encodes a polypeptide having an activity to synthesize tetrahydrodaidzein using dihydrodaidzein as a substrate; (Cd) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 16; (Ce) a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 13; and (Cf) a polynucleotide that hybridizes under stringent conditions with the complementary strand of the polynucleotide (Cd) or (Ce), and that encodes a polypeptide having an activity to synthesize equol using tetrahydrodaidzein as a substrate.

[000103] Item D8. A recombinant cell transformed with the item 7 expression vector.

[000104] Item D9. A recombinant cell according to item 8, wherein the recombinant cell is a bacterial prokaryotic cell.

[000105] Item D10. A recombinant cell according to item 9, wherein the bacterial prokaryotic cell belongs to the genus Lactococcus.

[000106] Item D11. A process for producing dihydrodaidzein, tetrahydrodaidzein, and/or equol, comprising at least two of the following Fourth Step to Sixth Step,

[000107] Fourth Step comprising a step of having a recombinant cell comprising one of the following polynucleotides (Ad) to (Af) act on daidzein, thereby producing dihydrodaidzein, (Ad) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4; (Ae) a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1; and (Af) a polynucleotide that hybridizes under stringent conditions to the complementary strand of the polynucleotide (Ad) or (Ae), and that encodes a polypeptide having an activity to synthesize dihydrodaidzein using daidzein as a substrate,

[000108] Fifth Step comprising a step of having a recombinant cell comprising one of the following polynucleotides (Bd) to (Bf) act on dihydrodaidzein, thereby producing tetrahydrodaidzein, (Bd) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 10; (Be) a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 7; and (Bf) a polynucleotide that hybridizes under stringent conditions to the complementary strand of the polynucleotide (Bd) or (Be), and that encodes a polypeptide having an activity to synthesize tetrahydrodaidzein using dihydrodaidzein as a substrate,

[000109] Sixth Step comprising a step of having a recombinant cell comprising one of the following polynucleotides (Cd) to (Cf) act on tetrahydrodaidzein, thereby producing equol, (Cd) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 16; (Ce) a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 13; and (Cf) a polynucleotide that hybridizes under stringent conditions to the complementary strand of the polynucleotide (Cd) or (Ce), and that encodes a polypeptide having an activity to synthesize equol using tetrahydrodaidzein as a substrate, wherein at least two polynucleotides selected from the group consisting of (Ad) to (Af), (Bd) to (Bf), and (Cd) to (Cf) can exist in a single recombinant cell.

[000110] Item D12. A recombinant cell according to item 11, wherein the recombinant cell is a bacterial prokaryotic cell.

[000111] Item D13. A recombinant cell according to item 12, wherein the bacterial prokaryotic cell belongs to the genus Lactococcus.

[000112] Item D14. A product containing dihydrodaidzein, tetrahydrodaidzein, and/or equol, produced by any of the processes according to items D11 to D13.

[000113] Item D15. A device for producing dihydrodaidzein, tetrahydrodaidzein, and/or equol, comprising at least one of the following First to Third reaction vessels,

[000114] The first reaction vessel having a reaction feature in which an enzyme consisting of one of the polypeptides (Aa) to (Ac) is immobilized, a reaction vessel being used for the production of dihydrodaidzein from daidzein using the enzyme , the reaction feature being arranged in a position allowing contact with daidzein;

[000115] The second reaction vessel having a reaction feature in which an enzyme consisting of one of the polypeptides (Ba) to (Bc) is immobilized, a reaction vessel being used for the production of tetrahydrodaidzein from dihydrodaidzein using the enzyme , the reaction feature being disposed in a position allowing contact with dihydrodaidzein; It is

[000116] The third reaction vessel having a reaction feature in which an enzyme consisting of one of the polypeptides (Ca) to (Cc) is immobilized, a reaction vessel being used for the production of tetrahydrodaidzein equol using the enzyme, the reaction resource being arranged in a position allowing contact with tetrahydrodaidzein.

[000117] Item D16. A device for producing dihydrodaidzein, tetrahydrodaidzein, and/or equol, comprising at least one of the following Fourth to Sixth Reaction Vessels,

[000118] The Fourth Reaction Vessel having a reaction resource in which a recombinant cell comprising one of the polynucleotides (Ad) to (Af) is immobilized, a reaction vessel being used for the production of dihydrodaidzein from daidzein using a resource reaction feature, the reaction feature being arranged in a position allowing contact with daidzein;

[000119] Fifth Reaction Vessel having a reaction resource in which a recombinant cell comprising one of the polynucleotides (Bd) to (Bf) is immobilized, a reaction vessel being used for the production of tetrahydrodaidzein from dihydrodaidzein using a reaction, the reaction feature being disposed in a position allowing contact with dihydrodaidzein; It is

[000120] Sixth Reaction Vessel having a reaction vessel in which a recombinant cell comprising one of the polynucleotides (Cd) to (Cf) is immobilized, a reaction vessel being used for the production of tetrahydrodaidzein equol using a reaction vessel, the reaction feature being arranged in a position allowing contact with tetrahydrodaidzein.

Effects of the Invention

[000121] The present invention provides polypeptides capable of: (1) synthesizing dihydrodaidzein using daidzein as a substrate, (2) synthesizing tetrahydrodaidzein using dihydrodaidzein as a substrate, and (3) synthesizing equol using tetrahydrodaidzein as a substrate. Therefore, the present invention is useful for industrial synthesis of dihydrodaidzein and/or tetrahydrodaidzein, which are intermediates generated in the production of daidzein equol, as well as industrial synthesis of equol. The present invention will open the door to the industrial production of equol, without using the difficult-to-handle anaerobic bacterial strains traditionally used for equol production.

Best way to carry out the invention

[000122] In the following, all abbreviations used to represent the names of substances, including amino acids, polypeptides, base sequences, and nucleic acids, follow the nomenclature specified by IUPAC-IUB (IUPAC-IUB Communication on Biological Nomenclature, Eur. J Biochem., 138: 9 (1984)), the Guideline for Drafting Specifications containing Base Sequence or Amino Acid Sequence (Japanese Patent Office), and other conventional symbols commonly used in the field.

[000123] The following describes the present invention in detail.

A: dihydrodaidzein synthesizing enzyme

[000124] This section describes the dihydrodaidzein synthesizing enzyme (hereinafter also referred to as the E1 enzyme) in detail. Except as otherwise described, general explanation of the dihydrodaidzein-synthesizing enzyme is applied to a tetrahydrodaidzein-synthesizing enzyme and equol-synthesizing enzyme, described later.

A-1 Polypeptide

[000125] The present invention provides a polypeptide (hereinafter also referred to as "E1 polypeptide") for the synthesis of dihydrodaidzein using daidzein as a substrate. Specifically, the invention provides: (Aa) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1; for example, the range is 1 to 250, preferably 1 to 200, more preferably 1 to 150, more preferably 1 to 100, more preferably 1 to 50, more preferably 1 to 30, more preferably 1 to 15, more preferably 1 to 5, also preferably 1 to 4, even more preferably 1 to 3, and particularly preferably 1 or 2. (Ab) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 with the substitution, deletion, insertion, and/or or adding one or more amino acids, and having an activity of synthesizing dihydrodaidzein using daidzein as a substrate; and (Ab) a polypeptide consisting of an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 1, and having an activity of synthesizing dihydrodaidzein using daidzein as a substrate.

[000126] In polypeptide (Ab), the range of "one or more amino acids" is not particularly limited as long as the polypeptide has the activity of synthesizing dihydrodaidzein using daidzein as a substrate. Examples of polypeptide (Ab) include a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3. Compared to the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 2 contains three substituted amino acids. Compared to the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 3 contains ten substituted amino acids. The amino acid sequence of SEQ ID NO: 2 corresponds to the E1 polypeptide enzyme of Bacteroides ovatus strain E-23-15 (FERM BP-6435). The amino acid sequence of SEQ ID NO: 3 corresponds to the E1 polypeptide enzyme of Streptococcus constellatus A6G-225 (FERM BP-6437).

[000127] Furthermore, the type of amino acid substitution in polypeptide (Ab) is not particularly limited. However, from the point of view of preventing phenotypic change in the polypeptide, it is preferable that the substitution is made between analogous amino acids. Analogous amino acids can be classified as follows.Aromatic amino acid: Phe, Trp, Tyr Aliphatic amino acid: Ala, Leu, Ile, Val Polar amino acid: Gln, Asn Basic amino acid: Lys, Arg, His Acidic amino acid: Glu, Asp Amino acids with one group hydroxyl: Ser, Thr Amino acids with a short side chain: Gly, Ala, Ser, Thr, Met

[000128] In the polypeptide (Ab), it is preferable that the substitution, deletion, insertion, or addition of amino acid occurs in regions where the amino acid change does not have major effects on the higher order structures of the polypeptide, or where the change do not adversely affect the active center of the dihydrodaidzein synthesizing enzyme. Examples of such regions include regions of low conservation between the amino acid sequences of SEQ ID NOs: 1, 2, and 3, and vicinity thereof, and the N-terminal region or C-terminal region. Specific examples include leucine at position 45 , asparagine at position 80, valine at position 167, aspartic acid at position 231, isoleucine at position 233, arginine at position 435, aspartic acid at position 459, valine at position 462, arginine at position 528, serine at position 540, isoleucine at position 639, in the amino acid sequence of SEQ ID NO: 1, and adjacent regions of these amino acids. An "adjacent region" means regions where the activity-synthesizing enzyme of dihydrodaidzein is not affected. Examples include those within five amino acids of one of the exemplified amino acids, preferably those within four amino acids of one of the exemplified amino acids, more preferably those within three amino acids of one of the exemplified amino acids, even more preferably those within two amino acids of one of the exemplified amino acids. of the exemplified amino acids, particularly preferably those within an amino acid of one of the exemplified amino acids.

[000129] In an amino acid sequence of SEQ ID NO: 1, the sequence of amino acids at positions 112 to 116 is considered to correspond to the NADPH binding domain. As long as the functions of the NADPH domain are not inhibited, there may be substitution, deletion, insertion or addition of amino acids in the five amino acid sequence. In said sequence, the number of mutated amino acids is preferably not greater than three, more preferably not greater than two, and even more preferably one. Most preferred is no mutation in the amino acid sequence. Particularly, there is preferably no substitution, deletion, insertion or addition of the amino acids at positions 112, 115, and 116.

[000130] Histidine at position 260 in the amino acid sequence of SEQ ID NO: 1 is also considered to be associated with the proton relay site. Consequently, as long as the proton relay site functions are not inhibited, said histidine can be replaced by another amino acid, but is preferably unsubstituted.

[000131] In an amino acid sequence of SEQ ID NO: 1, the sequence of amino acids at positions 343 to 363 is considered to correspond to the Fe-S beam motif. As long as the functions of the motif are not inhibited, there may be substitution, deletion, insertion, or addition of an arbitrary amino acid in the sequence. It is however preferred that cysteines at positions 343, 346, 350, and 363 are not mutated. When the sequence has one or more amino acid substitutions at positions other than said three amino acids, it is preferred that the number of substituted amino acids is preferably not greater than four, more preferably not greater than three, even more preferably not greater than than two, and particularly preferably one. Most preferred is no substitution, deletion, insertion or addition of amino acids in the sequence.

[000132] In an amino acid sequence of SEQ ID NO: 1, the sequence of amino acids at positions 390 to 413 is considered to correspond to the FAD binding domain. Consequently, as long as the functions of the domain are not inhibited, there may be substitution, deletion, insertion or addition of an arbitrary amino acid in the sequence. It is however preferred that glycines at positions 390, 392, and 395 are not mutated. when the sequence has one or more amino acid substitutions, deletions, insertions or additions, the number of mutated amino acids is preferably no greater than four, more preferably no greater than three, even more preferably no greater than two, and particularly preferably one. Most preferred is no mutation in the amino acid sequence.

[000133] In an amino acid sequence of SEQ ID NO: 1, the sequence of amino acids at positions 512 to 540 is also considered to correspond to the FAD binding domain. Consequently, as long as the functions of the domain are not inhibited, there may be substitution, deletion, insertion or addition of an arbitrary amino acid in the sequence. It is however preferred that glycines at positions 512, 514, and 517 are not mutated. when the sequence has one or more amino acid substitutions, deletions, insertions or additions, the number of mutated amino acids is preferably no greater than four, more preferably no greater than three, even more preferably no greater than two, and particularly preferably one. Most preferred is no mutation in the amino acid sequence.

[000134] An alignment, which indicates the regions of amino acid sequences with possible functions as described above, is shown in Figure 27.

[000135] The substitution, deletion, insertion or addition of one or several amino acids in a specific amino acid sequence is possible by known techniques.

[000136] In polypeptide (Ab), the amino acid sequence has, for example, 60% or more identity with the amino acid sequence of SEQ ID NO: 1. It is preferred, however, that the amino acid sequence is generally 80 % or more, preferably 85% or more, more preferably 90% or more, further preferably 95% or more, even more preferably 98% or more, and particularly preferably 99% or more identity to the amino acid sequence of SEQ ID NO: 1.

[000137] Specifically, polypeptide (Ab) can be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 or a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3. The amino acid identity between the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2 is 99.5% (Blast2). The amino acid identity between the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 3 is 98.6% (Blast2). Accordingly, in a preferred embodiment of the invention, polypeptide (Ab) comprises an amino acid sequence having 98.6% or more, and more preferably 99.5%, identity to the amino acid sequence of SEQ ID NO: 1.

[000138] Amino acid sequence identity can be calculated using analysis tools such as, for example, FASTA, BLAST, PSI-BLAST, SSEARCH, or other types of commercially available software or via telecommunications lines (the Internet). Specifically, a BLAST query is generally performed under the following initial conditions to calculate the identity (%) of the amino acid sequence. BLAST 2.1 advanced: Program: blastp Expected value: 10 Filters: all OFF Matrix: BLOSUM62 Gap existence cost: 11 (default) Per residue gap cost: 1 (default) Lambda ratio: 0.85 (default) Other parameters (default)

[000139] Considering polypeptides (Ab) and (Ac), the activity to synthesize dihydrodaidzein using daidzein as a substrate can be confirmed as follows. First, a polypeptide of interest is added to a substrate solution of the composition below at a concentration of 0.001 mg/mL, the solution is then incubated at 37°C for 2 hours to check for the presence or absence of dihydrodaidzein in the solution. . The presence of dihydrodaidzein in the solution after incubation is used as an indicator of the activity of the polypeptide to synthesize dihydrodaidzein using daidzein as a substrate. Composition of Substrate Solution 0.1 M potassium phosphate buffer 1 mM PMSF (phenylmethylsulfonyl fluoride) 2 mM dithiothreitol 5 mM sodium hydrosulfite 2 mM NADPH or 2 mM NADH 40 μM daidzein PH 7.0

Enzyme Property

[000140] Since the E1 polypeptide has an enzyme activity to synthesize dihydrodaidzein using daidzein as a substrate, the E1 polypeptide is also referred to as the E1 enzyme. The E1 enzyme is activated by the presence of a reducing agent such as Na2S2O4 and metal ions such as Fe2+ and Mn2+. Furthermore, the E1 enzyme requires NADPH or NADH as a coenzyme. The ideal temperature for the E1 enzyme is around 30°C, and the ideal pH is 7.0. The E1 enzyme can not only synthesize dihydrodaidzein using daidzein as a substrate, but also synthesize daidzein from dihydrodaidzein as a reverse reaction.

[000141] The E1 polypeptide can be produced by genetic engineering techniques to be described later or by isolation and purification of microorganisms capable of producing it. Furthermore, common chemical synthesis methods can be used to produce the E1 polypeptide, based on the amino acid sequence information of SEQ ID NO: 1, 2, or 3. Such chemical synthesis methods include phase peptide synthesis methods common liquid or solid phase.

[000142] The following describes a method for the isolation and purification of E1 polypeptide from a microorganism capable of producing E1 polypeptide. First, the cells of a microorganism capable of producing the E1 polypeptide are disrupted to obtain a raw extract of the microorganism. Here, cells can be disrupted by methods used in common cell disruption processes, such as disruption using a French press, a cell mill, and other disruptors, and sonication in a hypotonic solution. The raw extract can be supplemented with an appropriate buffer. When the E1 polypeptide is anaerobically conditioned, it is desirable to add a suitable reducing agent to the raw extract to keep the E1 polypeptide active. To improve purity, the raw extract can also be purified through processes such as ammonium sulfate precipitation, organic solvent precipitation using ethanol or others, and isoelectric precipitation. An E1 polypeptide-containing fraction can then be obtained by subjecting the crude extract to processes such as ion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, various types of affinity chromatography, reversed-phase chromatography, and column chromatography. of hydroxyapatite. These chromatography processes may use an open column, or HPLC may be used, as required. The purity of the fraction including the E1 polypeptide can be easily estimated by visualization through electrophoresis, and particularly, through SDS-PAGE. Confirmation of the E1 polypeptide is also possible through amino acid sequence analysis, mass spectrometry using a mass spectrometer such as MALDI-TOF MS, ESI Q-TOF MS, and MALDI Q-TOF MS, and fingerprinting of the peptide mass, for example.

[000143] Here, the microorganism capable of producing the E1 polypeptide is preferably cultivated in a medium containing a desired amount of daidzein (e.g., but not limited to, a medium containing at least 0.01 μg/mL of daidzein) in terms efficient production of the E1 polypeptide.

[000144] The E1 polypeptide can be monomeric, or dimeric or polymeric, since it can synthesize dihydrodaidzein. Furthermore, to improve stability and other characteristics, the E1 polypeptide can be modified as required by adding polyethylene glycol or a sugar chain.

[000145] The E1 polypeptide can serve as a catalyst that converts daidzein (a substrate) into dihydrodaidzein. Dihydrodaidzein is also converted to equol by tetrahydrodaidzein-synthesizing enzyme and equol-synthesizing enzyme as described below. Equol is believed to exhibit several physiological activities in the body. In this regard, the E1 polypeptide, capable of supplying the material for equol synthesis, is considered important.

A-2. Polynucleotide

[000146] The present invention also provides a polynucleotide (hereinafter also referred to as "E1 polynucleotide") that encodes a polypeptide having an activity of synthesizing dihydrodaidzein using daidzein as a substrate. Specifically, the invention provides: (Ad) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4; (Ae) a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1; and (Af) a polynucleotide that hybridizes under stringent conditions with the complementary strand of the polynucleotide (Ad) or (Ae), and that encodes a polypeptide having an activity to synthesize dihydrodaidzein using daidzein as a substrate.

[000147] The amino acid sequence of SEQ ID NO: 1 corresponds to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 4. The amino acid sequence of SEQ ID NO: 2 corresponds to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 5. The amino acid sequence of SEQ ID NO: 3 corresponds to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 6.

[000148] Considering the polynucleotide (Af), the phrase "hybridizes under stringent conditions" describes hybridization between two polynucleotide fragments under ordinary hybridization conditions such as described in Sambrook et al. in Molecular Cloning: A laboratory Manual (1989) , Cold Spring Harbor Laboratory Press, New York, USA. More specifically, "stringent conditions" means hybridization in 6.0 x SSC at about 45°C, and washing with 2.0 x SSC at 50°C. Examples of (Af) polynucleotide include the nucleotide sequence of SEQ ID NO: 5 and the nucleotide sequence of SEQ ID NO: 6.

[000149] The "polynucleotide that hybridizes under stringent conditions" generally has above a certain level of identity with the nucleotide sequence of the polynucleotide used as a probe. The identity is, for example, 60% or more, preferably 70% or more, more preferably 80% or more, also preferably 90% or more, even more preferably 95% or more, and particularly preferably 98% or more. The identity of the nucleotide sequence can be calculated using analytical instruments such as, for example, FASTA, BLAST, PSI-BLAST, SSEARCH, or other types of software available either commercially or via telecommunications lines (the Internet). Specifically, a BLAST query is generally performed under the following initial conditions to calculate the identity (%) of the nucleotide sequence. BLAST 2.1 advanced: Program: blastn Parameters: basic

[000150] The base sequence homology between the nucleotide sequence of SEQ ID NO: 4 and the nucleotide sequence of SEQ ID NO: 5 is 99.6% (Blast2). The base sequence homology between the nucleotide sequence of SEQ ID NO: 4 and the nucleotide sequence of SEQ ID NO: 6 is 97.6% (Blast2). Accordingly, in a preferred embodiment of the invention, a polynucleotide (Af) comprises a base sequence having 97.6% homology, and more preferably 99.6% homology, to the base sequence of SEQ ID NO: 4.

[000151] Considering the polynucleotide (Af), the "activity of synthesizing dihydrodaidzein using daidzein as a substrate" can be confirmed by the method used for the polypeptides (Ab) and (Ac).

[000152] The E1 polynucleotide can be produced or obtained through chemical DNA synthesis methods, based on the sequence information of SEQ ID NOs: 4, 5, and 6. Generally, the E1 polynucleotide can be easily produced or obtained through of common genetic engineering techniques (see, e.g., Molecular Cloning 2d Ed, Cold Spring Harbor Lab. Press (1989); and Zoku Seikagaku Jikken Kouza, Gene Kennkyu-hou I, II, III, the Japanese Biochemical Society (1986) ).

[000153] An example of such chemical DNA synthesis methods is a solid phase synthesis method using the phosphoramidite method, which may employ an autosynthesizer.

[000154] In a specific example of common genetic engineering techniques, a cDNA library is prepared by a customary method from a suitable source expressing the E1 polynucleotide, and the library is analyzed for the desired clones using a suitable probe or specific antibody to the E1 polynucleotide (see, for example, Proc. Natl. Acad. Sci., USA., 78, 6613 (1981); Science, 222, 778 (1983)).

[000155] The source of cDNA is not particularly limited since it is an organism expressing the E1 polynucleotide. Specific examples include microorganisms capable of equol production, preferably lactic acid bacteria, bacteria belonging to the genera Bacteroides and Streptococci capable of equol production, more preferably Lactococcus garvieae, Bacteroides ovatus and Streptococcus constellatus capable of equol production, also preferably fecal Lactococcus garvieae capable of equol production, and particularly preferably Lactococcus strain 20-92, Bacteroides ovatus strain E-23-15, Streptococcus constellation A6G-225 (FERM BP-10036, FERM BP-6435, and FERM BP-6437, respectively; deposited with the International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan), fecal Lactococcus garvieae strains capable of equol production.

[000156] Usual methods can be used for procedures including the separation of total RNA, the separation and purification of mRNA, and the preparation and cloning of cDNA. The cDNA library analysis method for a polynucleotide of the present invention is not particularly limited, and a customary method can be used. For example, polypeptides produced from cDNA can be analyzed by selecting corresponding cDNA clones by immunological analysis using a polypeptide-specific antibody. Additionally, analysis can be done using hybridization techniques such as plaque hybridization and colony hybridization, which use probes that specifically bind to target nucleotide sequences. Furthermore, a combination of these different techniques can be used.

[000157] Generally, the probe may be, for example, chemically synthesized DNA obtained based on information regarding the nucleotide sequence of the E1 polynucleotide (for example, the nucleotide sequence of SEQ ID NO: 4, 5 , or 6). Furthermore, a probe used for analysis can be a sense primer and/or an antisense primer designed based on the base sequence information of a polynucleotide of the present invention.

[000158] The E1 polynucleotide can be suitably obtained by the PCR method (Science, 130, 1350 (1985)), or by variants of the PCR method, such as a DNA or RNA amplification method. Any difficulty in analyzing the library for full-length cDNA can be avoided by using appropriate techniques such as the RACE method (Rapid amplification of cDNA ends, Experimental Medicine, 12(6), 35 (1994)); and particularly the 5'-RACE method (M.A. Frohman, et al., Proc. Natl. Acad. Sci., USA., 8, 8998 (1988)). The Race method and the 5'-RACE method are useful for obtaining the eukaryotic E1 polypeptide.

[000159] The primers used for the PCR method can be appropriately designated based on the sequence information of the E1 polynucleotide. Such primers can be synthesized using a customary method. As noted above, isolation and purification of amplified DNA or RNA fragments can be performed by a customary method such as gel electrophoresis and hybridization.

[000160] The E1 polynucleotide easily allows stable mass production of the polynucleotide Product (the polypeptide), using common genetic engineering techniques. The successful isolation of the E1 polynucleotide by the present invention will open the door to the industrial production of equol, without use of the difficult-to-handle anaerobic bacterial strains traditionally used for equol production.

A-3. Expression Vector

[000161] An expression vector of the present invention is not particularly limited since it includes the E1 polynucleotide and is capable of expressing the E1 polynucleotide. Generally, it is appropriately selected according to the host cell type.

[000162] When the host cell is the prokaryotic cell, the expression vector can be, for example, an expression plasmid vector, replicable in the host cell, prepared by adding promoters and an SD base sequence (Shine-Dalgarno) upstream of the polynucleotide to cause expression of the polynucleotide. A specific example is an expression plasmid using a PL promoter, a T7 promoter, and a lac promoter. Other examples of preferable bacterial expression vectors include plasmid pKK233-2 and plasmid pKK233-3 using a tac promoter or trc promoter. These are non-limiting examples, and other known bacterial strains and vectors can be used, as well.

[000163] When the host cell is a eukaryotic cell, the expression vector may generally include promoters upstream of the polynucleotide to be expressed, and other specific sequences including an RNA splicing site, a polyadenylation site, and a termination sequence of transcription. The expression vector may also include an origin of replication. There are several well-known eukaryotic vectors useful for polynucleotide insertion. Examples of such suitable eukaryotic vectors include pCD and pCMV. Other examples include pMSG and pSVL, which make use of the MMTV or SV40 late promoter as required. These are not limiting examples, and several known eukaryotes and vectors can be used, as well.

A-4. Recombinant cell

[000164] The present invention provides a recombinant cell (transformant) transformed with an expression vector including the E1 polynucleotide.

[000165] The host cell used for the recombinant cell can be a prokaryotic cell or a eukaryotic cell.

[000166] Suitable examples of a prokaryotic host cell include: bacterial prokaryotic cells of the genus Lactococcus, such as lactic acid bacteria; and bacterial prokaryotic cells such as, for example, Escherichia coli, Streptomyces, Bacillus subtilis, Streptococcus, and Staphylococcus, which can grow under aerobic conditions.

[000167] Examples of a eukaryotic host cell include: eukaryotic microorganisms such as yeast and Aspergillus; insect cells such as Drosophila S2 and Spodoptera Sf9; and animal and plant cells such as L cells, CHA cells, COS cells, HeLa cells, C127 cells, BALB/c3T3 cells (including mutant strains lacking dihydrofolate reductase or thymidine kinase), BHK21 cells, HEK293 cells, melanoma cells of Bowes, and oocytes.

[000168] The method used to introduce the expression vector into the host cell is not particularly limited, and a variety of common methods can be used. For example, the expression vector can be introduced into the host cell according to the methods of many standard laboratory manuals, including, for example, Davis et al., Basic Methods in Molecular Biology, 1986, and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. Specific examples include calcium phosphate transfection, DEAE-dextran-mediated transfection, transvection, microinjection, lipid-mediated transfection cationic, electroporation, transduction, scrape charge, ballistic introduction, and infection.

[000169] Because the recombinant cell is capable of producing the E1 polypeptide (a dihydrodaidzein-converting enzyme), it can be used to produce a dihydrodaidzein-converting enzyme. The recombinant cell can also be used to produce dihydrodaidzein in a cellular form.

A-5. Polypeptide Production using Recombinant Cell

[000170] The E1 polypeptide can be produced by culturing a recombinant cell into which the E1 polynucleotide is introduced and collecting the E1 polypeptide from the cell and culture.

[000171] The culture can be subculture or batch culture using a medium suitable for the host. Cells can be cultured until an adequate amount of the E1 polypeptide is obtained, using the level of the E1 polypeptide inside and outside the recombinant cells as an index.

[000172] The culture medium can be appropriately selected from common media according to the type of host cell used. The culture can be incubated under development conditions suitable for the host cell.

[000173] The resulting E1 polypeptide can optionally be separated and purified through various types of separation techniques utilizing the physical and chemical properties of the E1 polypeptide (see, for example, Biochemistry Data Book II, pp. 1175-1259, first edition, first publication, June 23, 1980, Tokyo Kagaku Dozin Co., Ltd.; Biochemistry, 25(25), 8274 (1986); and Eur. J. Biochem., 163, 313 (1987)). Specifically, the E1 polypeptide can be separated and purified, for example, as in the "method for isolation and purification of the E1 polypeptide from microorganisms capable of producing the E1 polypeptide" described in Section A-1 under the heading "Polypeptide".

A-6. Production of Dihydrodaidzein using E1 polypeptide

[000174] The present invention provides a process for producing dihydrodaidzein using E1 polypeptide. In the production process, daidzein can be converted into dihydrodaidzein with the E1 polypeptide acting on daidzein in the presence of NADPH and/or NADH. Preferably, daidzein can be converted to dihydrodaidzein by having the E1 polypeptide act on daidzein in the presence of NADPH.

[000175] Since the enzyme synthesizing the dihydrodaidzein activity of the E1 polypeptide is activated by the presence of Mn2+ and Fe2+, the E1 peptide should preferably have the E1 polypeptide acting on the daidzein in the presence of Mn2+ and/or Fe2+. The concentration of Mn2+ and/or Fe2+ in a reaction solution is not particularly limited since the enzyme activity of the E1 polypeptide can be activated. A concentration of Fe2+ is preferably 2mM and more, more preferably 2mM to 100mM, more preferably 10mM to 40mM. The concentration of Mn2+ is preferably 0.2 μM and more, more preferably 0.2 μM to 100 mM and even more preferably 1.0 μM to 40 mM.

[000176] The reaction used in the production process can be performed in a suitable buffer. Examples of such suitable buffers include phosphate buffer, carbonate buffer, acetate buffer, Tris buffer, and borate buffer. The pH condition of a reaction can be appropriately selected so as not to inactivate the desired enzyme activity of the E1 polypeptide. For example, the reaction can be carried out in a pH range of preferably 5.0 to 10.0, and more preferably 6.0 to 8.0.

[000177] In the reaction, an appropriate amount of protease inhibitor such as PMSF or EDTA can be added as required. Furthermore, considering that the polypeptide derives from anaerobic bacteria, an appropriate amount of reducing agent, such as DTT, 2ME, DET, and Na2S2O4 can be added as well.

[000178] The reaction used in the production process is carried out under such conditions in which, for example, each component is added to a medium in the concentration ranges shown below at the beginning of the reaction in order to prepare a mixture. The mixture is incubated for 0.5 to 10 hours, preferably 1 to 6 hours, and more preferably 2 to 4 hours at temperatures of 20 to 45°C, preferably 25 to 40°C, and most preferably 30 to 38°C. The initial concentrations of each component are as follows:

[000179] The polypeptide content is 0.0001 to 1.0% by weight, preferably 0.001 to 0.1% by weight, and more preferably 0.001 to 0.01% by weight;

[000180] The daidzein content is 0.0001 to 10.0% by weight, preferably 0.001 to 1.0% by weight, and more preferably 0.001 to 0.1% by weight;

[000181] The content of NADPH and/or NADH is 0.01 to 5% by weight, preferably 0.05 to 1% by weight, and more preferably 0.1 to 0.5% by weight.

[000182] The present invention also provides a mixture of material for the synthesis of dihydrodaidzein, specifically, a composition for synthesizing dihydrodaidzein including (Ai) E1 polypeptide, (Aii) NADPH and/or NADH, and (Aiii) daidzein. Additionally, the present invention provides a mixture of material for the synthesis of dihydrodaidzein, specifically a composition of dihydrodaidzein synthesis material including (Ai) E1 polypeptide, (Aii) NADPH and/or NADH, (Aiii) daidzein, and ( Aiv) Mn2+ and/or Fe2+. By incubating the composition under the above-mentioned conditions, the daidzein contained in the composition can be converted to dihydrodaidzein. The synthetic material composition corresponds to the aforementioned material mixture used to initiate a reaction to produce dihydrodaidzein. Furthermore, the concentrations of the E1 polypeptide, NADPH and/or NADH, and daidzein in the composition, and other components that can be added to the synthetic starting material composition are essentially as in a reaction system (a mixture of starting materials at the beginning of the reaction) used in the above-mentioned production process.

[000183] The present invention also provides a kit for synthesizing dihydrodaidzein, specifically, a dihydrodaidzein synthesis kit including (Ai) E1 polypeptide, (Aii) NADPH and/or NADH, and (Aiii) daidzein. Additionally, the present invention provides a kit for synthesizing dihydrodaidzein, specifically a dihydrodaidzein synthesis kit including (Ai) E1 polypeptide, (Aii) NADPH and/or NADH, (Aiii) daidzein, and (Aiv) Mn2+ and/or Fe2+ . The components may optionally be stored separately in the synthesis kit in order to conveniently allow the synthesis of dihydrodaidzein from daidzein under the above conditions. The synthesis kit may also include a buffer as required. A synthesis kit may also include any necessary instruments or operating manuals that help perform the synthesis of dihydrodaidzein.

A-7. Composition of dihydrodaidzein synthesizing enzyme

[000184] The present invention also provides an enzyme for synthesizing the dihydrodaidzein composition including the E1 polypeptide. An enzyme composition can be suitably used as a dihydrodaidzein synthesizing enzyme in the process of producing dihydrodaidzein using E1 polypeptide.

[000185] The enzyme composition can be a raw purified E1 polypeptide, or an enzyme composition can be a raw or purified purified E1 polypeptide formulated with a suitable support.

[000186] The proportion of the E1 polypeptide in an enzyme composition is not particularly limited since the composition can be used as a dihydrodaidzein synthesizing enzyme in the dihydrodaidzein production process. Specifically, the E1 polypeptide content is, for example, 0.001 to 20.0% by weight, preferably 0.005 to 5.0% by weight, and more preferably 0.01 to 1.0% by weight, with respect to the total an enzyme composition.

[000187] The enzyme composition may include NADPH and/or NADH, which act as a coenzyme of the E1 polypeptide. When contained in an enzyme composition, the proportion of NADPH and/or NADH, although not particularly limited, is 0.0005 to 25.0% by weight, preferably 0.005 to 5.0% by weight, and more preferably 0.01 at 2.5% by weight, with respect to the total of an enzyme composition.

[000188] Furthermore, in a preferred embodiment, an enzyme composition may include Mn2+ and/or Fe2+. When contained in an enzyme composition, the proportion of Mn2+ and/or Fe2+ is not particularly limited since the E1 polypeptide dihydrodaidzein activity-synthesizing enzyme can be activated. The proportion of Fe2+ is preferably 2mM or more, more preferably 2mM to 100mM, and even more preferably 10mM to 40mM with respect to the total composition. The proportion of Mn2+ is preferably 0.2 μM or more, more preferably 0.2 μM to 100 mM, and even more preferably 1.0 μM to 40 mM with respect to the total composition.

[000189] To improve the stability of the polypeptide, an enzyme composition may also include antioxidants such as sulfite, ascorbic acid, a-tocopherol, and cysteine, in addition to the E1 polypeptide. Furthermore, to ensure the preservability of an enzyme composition, an enzyme composition may include preservatives such as p-hydroxybenzoates, chlorobutanol, benzyl alcohol, 2-phenylethyl alcohol, dehydroacetic acid, and sorbic acid, as required.

A-8. Dihydrodaidzein Production Process Using Recombinant Cell

[000190] The present invention provides a process for producing dihydrodaidzein using recombinant cells including the E1 polynucleotide. Specifically, in the production process, daidzein is converted into dihydrodaidzein with the recombinant cell acting on the daidzein.

[000191] The reaction used in the production process is carried out under conditions that allow the recombinant cell to survive, and the daidzein to be converted into dihydrodaidzein.

[000192] Specifically, appropriate amounts of recombinant cells and daidzein are added and cultured in a medium that allows development of the recombinant cells.

[000193] The medium used in the production process is suitably selected from various types of conventional media according to the type of cell used as the host recombinant cell.

[000194] The medium can be supplemented with appropriate amounts of protease inhibitors such as PMSF and EDTA, as required. Furthermore, considering that the polypeptide is derived from anaerobic bacteria, appropriate amounts of reducing agents such as DTT, 2ME, DET, and Na2S2O4 can also be added. When using a recombinant cell, the medium can be supplemented with NADPH and/or NADH as required, although it is not essential. Furthermore, the medium can be supplemented with Mn2+ and/or Fe2+.

[000195] Specifically, the production process is performed as follows. First, the recombinant cells are inoculated into medium containing 0.001 to 1 wt%, preferably 0.01 to 1 wt%, and more preferably 0.01 to 0.5 wt% daidzein, and the culture is incubated under conditions permissive temperature ranges for 6 to 30 hours, preferably 7 to 24 hours, and more preferably 7 to 18 hours.

[000196] The present invention also provides a mixture of material for synthesizing dihydrodaidzein, specifically, a composition of dihydrodaidzein synthesis material containing (Aiv) the recombinant cell and (Aiii) daidzein. By cultivating the synthesis material composition under the above-mentioned conditions, the dihydrodaidzein in the composition can be converted to dihydrodaidzein. The composition corresponds to the above-mentioned starting material mixture used to initiate a reaction to produce dihydrodaidzein. Furthermore, the concentrations of the recombinant cell and daidzein in the composition, and other components that can be added to a synthetic starting material composition, are essentially as in the conditions used in the foregoing production process.

[000197] The present invention also provides a kit for synthesizing dihydrodaidzein, specifically, a dihydrodaidzein synthesis kit including (Aiv) the recombinant cell and (Aiii) daidzein. Additionally, the present invention provides a kit for synthesizing dihydrodaidzein, specifically a dihydrodaidzein synthesis kit including (Aiv) the recombinant cell, (Aiii) daidzein, and (Aiv) Mn2+ and/or Fe2+. The synthesis kit may include the recombinant cell and daidzein separately as required, in order to conveniently allow synthesis of dihydrodaidzein from daidzein under the above-mentioned conditions. The synthesis kit may also include a buffer or medium, as required. The synthesis kit may also include any necessary instruments or operating manuals that help perform dihydrodaidzein synthesis.

[000198] The recombinant cells included in the synthesis kit can be preserved in it using known methods. There are several known recombinant cell preservation techniques. For example, there is a method in which recombinant cells are preserved at 4 to 25°C after being treated with a lyophilizer to evacuate an ampoule by storing the recombinant cells in a solvent such as dimethylformamide. Another example is a liquid nitrogen method, in which cells are suspended in a preservation medium supplemented with 10% glycerol, which is then stored in a specific ampoule and maintained in a liquid nitrogen tank (at -150 to - 196°C).

A-9. Antibody Having Affinity to the Polypeptide

[000199] The present invention also provides an antibody (an IgG antibody) having affinity to the E1 polypeptide.

[000200] The monoclonal antibody can be prepared using a usual method. Specifically, the method described in Harlow, H. and Lane, D. in Antibody: Laboratory Manual, Cold Spring Harbor Lab., New York, pp. 139-240 (1988) can be used.

[000201] The polyclonal antibody can also be prepared using a usual method. Specifically, the method described in, for example, Cell Engineering Experiment Protocol, Department of Oncology, The Institute of Medical Science, The University of Tokyo, 1992, pp. 155-173, can be used.

[000202] The polyclonal IgG antibody and the monoclonal IgG antibody can be purified by common methods, such as a sulfuric acid ammonium precipitation method, and protein A chromatography.

A-10. Immunological Method for the Detection or Measurement of the Polypeptide

[000203] The present invention also provides an immunological method for detecting or measuring the E1 polypeptide using the antibody. Specifically, the immunological method is performed by having the antibody be in contact with the test sample. More specifically, the polypeptide in the test sample can be detected or measured as follows. First, the antibody is established to be in contact with the test sample to check for the presence of the E1 polypeptide in the test sample. If present, the antibody specifically binds to the E1 polypeptide. Next, the antibody bound to the E1 polypeptide is detected and optionally quantified.

[000204] Here, the test sample is a sample used for the detection or measurement of the E1 polypeptide. Because the E1 polypeptide is expected to exist in bacterial prokaryotic cells, the immunological method is suitable for the detection and measurement of the E1 polypeptide residing in bacterial prokaryotic cells. In detecting and measuring the E1 polypeptide in the cell, the test sample can be prepared from ruptured cells, or cells subjected to protein purification after rupture.

[000205] Techniques for detecting or measuring a target polypeptide by the immunological method using the antibody are known, and it would be obvious to the skilled artisan appropriately to establish various conditions suitable for the immunological method. For example, methods such as radioimmunoassay and ELISA can be used under appropriately established conditions.

[000206] The present invention also provides an immunological detection kit including the antibody, used for the detection or measurement of the E1 polypeptide. The detection kit may also include a standard E1 polypeptide, as required. A detection kit may also include, as required, additional reagents that facilitate easy detection of the E1 polypeptide under the foregoing conditions. The detection kit may also include any necessary instruments or operating manuals that facilitate easy detection of the E1 polypeptide.

A-11. Method for Detection or Measurement of Polynucleotide Encoding the Polypeptide

[000207] The present invention also provides a method for detecting or measuring the E1 polynucleotide. Specifically, the method is performed by bringing an E1 polynucleotide binding probe into contact with the test sample. To be more specific, the E1 polynucleotide in the test sample can be detected or measured as follows. First, the probe is brought into contact with the test sample to hybridize with the test sample, which occurs when the E1 polynucleotide is present in the test sample. Next, the presence or absence of the double strand is detected, and the double strand, if present, is optionally quantified.

[000208] Here, the test sample is a sample used for the detection or measurement of the E1 polynucleotide. Because the E1 polynucleotide is expected to exist in bacterial prokaryotic cells, the method is suitable for the detection and measurement of the E1 polynucleotide residing in bacterial prokaryotic cells. In detecting and measuring the E1 polynucleotide in the cell, the test sample can be prepared from ruptured cells, or cells subjected to nucleic acid purification after rupture.

[000209] The probe used in the method has a nucleotide sequence that can hybridize with the polynucleotide under stringent conditions. As used herein, "stringent conditions" describe, for example, conditions common to probes and primers, and specifically, conditions described in Section A-2 above.

[000210] The probe may be chemically synthesized based on information regarding the nucleotide sequence of the E1 polynucleotide (e.g., the nucleotide sequence of SEQ ID NO: 4, 5, or 6), or it may be an E1 polynucleotide previously produced or a fragment of the E1 polynucleotide. The probe may be labeled or unlabeled, although labeled probes are generally used. Furthermore, when the probe is used as a PCR primer (a sense primer or an antisense primer), a probe size is, for example, about 10 to 40 nucleotides, and preferably about 20 to 30 nucleotides.

[000211] Examples of methods for specifically detecting the polynucleotide include: plaque hybridization, colony hybridization, Southern blotting, Northern blotting, and the PCR method. From a sensitivity standpoint, a PCR method that uses the probes as primers to amplify part or all of the E1 polynucleotide is preferably used.

[000212] The PCR method can be, for example, RT-PCR method, or various types of variant methods used in the technique. The PCR method can be used to test the presence and quantity of the E1 polynucleotide. Examples of such PCR methods include a competitive assay such as MSSA (Kinoshita, M., et al., CCA, 228, 83-90 (1994)), and the PCR-SSCP method, which is a known method of detecting mutations based on changes in the mobility of single-stranded DNA due to different high-order structures (Orita, M., et al., Genomics, 5, 874-879 (1989)).

[000213] The present invention also provides a kit for detecting or measuring E1 polynucleotide, specifically an E1 polynucleotide detection kit including the probe. For ease of detection of the E1 polynucleotide under the above-mentioned conditions, the detection kit may include additional or other reagents as required, in addition to the probe. Furthermore, the detection kit can be used to identify cells containing the E1 polynucleotide. From the point of view of enabling accurate detection, the detection kit may preferably be provided as a kit for detection performance using PCR.

B. Tetrahydrodaidzein Synthesizing Enzyme B-1. Polypeptide

[000214] The present invention provides a polypeptide (hereinafter also referred to as "E2 polypeptide") for the synthesis of tetrahydrodaidzein using dihydrodaidzein as a substrate. Specifically, the invention provides: (Ba) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 7; (Bb) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 7 with the substitution, deletion, insertion, and/or addition of one or more amino acids, and having an activity of synthesizing tetrahydrodaidzein using dihydrodaidzein as a substrate; and (Bc) a polypeptide consisting of an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 7, and having an activity of synthesizing tetrahydrodaidzein using dihydrodaidzein as a substrate.

[000215] In the polypeptide (Bb), the range of "one or more amino acids" is not particularly limited since the polypeptide has the activity of synthesizing tetrahydrodaidzein using dihydrodaidzein as a substrate. For example, the range is 1 to 50, preferably 1 to 30, more preferably 1 to 15, more preferably 1 to 5, also preferably 1 to 4, even more preferably 1 to 3, and particularly preferably 1 or 2.

[000216] Examples of (Bb) polypeptide include a polypeptide consisting of the amino acid sequence of SEQ ID NO: 8 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 9. Compared to the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8 contains two substituted amino acids and an amino acid sequence having 24 amino acids at the N-terminus. Compared to the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 9 contains 20 substituted amino acids and loses 1 amino acid. The amino acid sequence of SEQ ID NO: 8 corresponds to the E2 enzyme polypeptide of Bacteroides ovatus strain E-23-15 (FERM BP-6435). The amino acid sequence of SEQ ID NO: 9 corresponds to the E2 enzyme polypeptide of A6G-225 from Streptococcus constellatus (FERM BP-6437).

[000217] In the (Bb) polypeptide, the substitution, deletion, insertion, or addition of amino acid can be made as in the substitution, deletion, insertion, or addition of amino acid in the E1 polypeptide described in Section A-1. It is preferred that the amino acid substitution, deletion, insertion, or addition in the polypeptide (Bb) be in regions where the amino acid change does not have major effects on the higher order structures of the polypeptide, or where the change does not adversely affect the active center of a tetrahydrodaidzein synthesizing enzyme. Examples of such regions include weakly conserved regions between the amino acid sequences of SEQ ID NOs: 7, 8, and 9, and vicinity thereof, and the N-terminal region or C-terminal region. Specific examples include valine at position 7, proline at position 8, valine at position 26, leucine at position 36, arginine at position 46, aspartic acid at position 94, glutamic acid at position 101, glycine at position 126, isoleucine at position 137, glutamine at position 156, lysine at position 157, aspartic acid at position 159, alanine at positions 160 and 171, cysteine at position 185, serine at position 221, alanine at position 233, valine at position 241, serine at position 258, isoleucine at position 266, and valine at position position 286, in the amino acid sequence of SEQ ID NO: 7; and adjacent regions of these amino acids. The "adjacent region" means regions where the activity-synthesizing enzyme of tetrahydrodaidzein is not affected. Examples of said regions include amino acids within five amino acids of one of the exemplified amino acids, preferably those within four amino acids of one of the exemplified amino acids, more preferably those within three amino acids of one of the exemplified amino acids, even more preferably those within two amino acids from one of the exemplified amino acids, and particularly preferably an amino acid from one of the exemplified amino acids.

[000218] In the amino acid sequence of SEQ ID NO: 7, the sequence of amino acids at positions 38 to 45 is considered to correspond to the NADPH binding domain. Consequently, since the functions of the domain are not inhibited, there may be substitution, deletion, insertion, or addition of an arbitrary amino acid in the sequence. It is preferred, however, that threonine at position 38, glycine at position 39, glycine at position 43, and glycine at position 45 are not mutated. When the sequence contains one or more amino acid substitutions, deletions, insertions, or additions, the number of mutated amino acids is preferably no more than four, more preferably no more than three, even more preferably no more than two. and more preferably one.

[000219] In the amino acid sequence of SEQ ID NO: 7, the sequence of amino acids at positions 115 to 118 is considered to correspond to the highly conserved motif among the SDR family. When the sequence contains one or more amino acid substitutions, deletions, insertions, or additions in the sequence, the number of mutated amino acids is preferably no more than three, more preferably no more than two, and even more preferably one. Most preferred is no mutation in the sequence.

[000220] In the amino acid sequence of SEQ ID NO: 7, serine at position 168, histidine at position 182, and lysine at position 186 are considered to be associated with the center of activity of the E2 enzyme. Consequently, since enzyme activity is not inhibited, the three amino acids can be replaced by other arbitrary amino acids. It is preferred, however, that these amino acids are not mutated.

[000221] In the amino acid sequence of SEQ ID NO: 7, the sequence of amino acids at positions 212 to 217 is considered to be associated with binding to cofactors. Consequently, since the function is not inhibited, there can be substitution, deletion, insertion, or addition of an arbitrary amino acid in the sequence. It is however preferred that the proline at position 212, glycine at position 213, and threonine at position 217 are not mutated. When the sequence contains one or more amino acid substitutions, deletions, insertions, or additions, the number of mutated amino acids is preferably no more than three, more preferably no more than two, and even more preferably one. Particularly preferred is no mutation in the sequence. An alignment, which indicates the regions of amino acid sequences with possible functions as described above, is shown in Fig. 28.

[000222] In the polypeptide (Bc), the amino acid sequence has, for example, 60% or more identity with the amino acid sequence of SEQ ID NO: 7. It is preferred, however, that the amino acid sequence generally has 80% or more, preferably 85% or more, more preferably 90% or more, also preferably 95% or more, even more preferably 98% or more, and particularly preferably 99% or more identity to the amino acid sequence of SEQ ID NO: 7.

[000223] Specifically, the (Bc) polypeptide can be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 8 or a polypeptide consisting of the amino acid sequence of SEQ ID NO: 9. The identity between the amino acid sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 8 is 99.3% (Blast2). The identity between the amino acid sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 9 is 93.0% (Blast 2). Accordingly, in a preferred embodiment of the invention, the (Bc) polypeptide comprises an amino acid sequence having 93.0% or more, and more preferably 99.3%, identity to the amino acid sequence of SEQ ID NO: 7.

[000224] Considering the polypeptides (Bb) and (Bc), the activity of synthesizing tetrahydrodaidzein using dihydrodaidzein as a substrate can be confirmed as follows. First, a polypeptide of interest is added to a substrate solution of the composition below at a concentration of 0.001 mg/mL. The solution is then incubated at 37°C for 2 hours to check for the presence or absence of tetrahydrodaidzein in a solution. The presence of tetrahydrodaidzein in a solution after incubation is used as an indicator of polypeptide activity to synthesize tetrahydrodaidzein using dihydrodaidzein as a substrate. Substrate Solution Composition 0.1 M potassium phosphate buffer (pH 7.0) 1 mM PMSF (phenylmethylsulfonyl fluoride) 2 mM dithiothreitol 5 mM sodium hydrosulfite 2 mM NADPH 2 mM NADH dihydrodaidzein a 40μM

Enzyme Property

[000225] Since the E2 polypeptide has an enzyme activity to synthesize tetrahydrodaidzein using dihydrodaidzein as a substrate, the E2 polypeptide is also referred to as the E2 enzyme. The E2 enzyme requires NADPH or NADH as a coenzyme. The ideal temperature of the E2 enzyme is in the vicinity of 37°C, and the ideal pH is 4.5. The E2 enzyme can not only synthesize tetrahydrodaidzein using dihydrodaidzein as a substrate, but also synthesize dihydrodaidzein from tetrahydrodaidzein as a reverse reaction.

[000226] Similar to the E1 polypeptide, the E2 polypeptide can be produced by genetic engineering techniques based on the nucleotide sequence information of SEQ ID NO: 10, 11, or 12. In addition, common chemical synthesis methods can be used to produce the E2 polypeptide, based on the amino acid sequence information of SEQ ID NO: 7, 8, or 9. The E2 polypeptide can also be produced by isolating and purifying microorganisms capable of producing it. These methods can be performed in accordance with the description in the previous section A-1.

[000227] The microorganism capable of producing E2 polypeptide can be cultivated in a medium containing a desired amount of dihydrodaidzein, and additionally, daidzein. The E2 polypeptide can also be produced in this way in microorganisms capable of producing the E2 polypeptide.

[000228] The E2 polypeptide can be monomeric, or dimeric or polymeric, since it can synthesize tetrahydrodaidzein. Furthermore, to improve stability and other characteristics, the E2 polypeptide can be modified as required by the addition of polyethylene glycol or a sugar chain.

[000229] The E2 polypeptide can serve as a catalyst that converts dihydrodaidzein (a substrate) into tetrahydrodaidzein. Tetrahydrodaidzein is also converted to equol by equol-synthesizing enzyme as described below. Equol is believed to exhibit several physiological activities in the body. In this regard, the E2 polypeptide, capable of providing a starting material for equol synthesis, is considered important. The present invention therefore provides an enzyme for synthesizing tetrahydrodaidzein including any of the polypeptides (Ba) to (Bc).

B-2. Polynucleotide

[000230] The present invention also provides a polynucleotide (hereinafter also referred to as "E2 polynucleotide") that encodes a polypeptide having an activity of synthesizing tetrahydrodaidzein using dihydrodaidzein as a substrate. Specifically, the invention provides: (Bd) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 10; (Be) a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 7; and (Bf) a polynucleotide that hybridizes under stringent conditions with the complementary strand of the polynucleotide (Bd) or (Be), and that encodes a polypeptide having an activity to synthesize tetrahydrodaidzein using dihydrodaidzein as a substrate.

[000231] The amino acid sequence of SEQ ID NO: 7 corresponds to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 10. The amino acid sequence of SEQ ID NO: 8 corresponds to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 11. The amino acid sequence of SEQ ID NO: 9 corresponds to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 12.

[000232] Considering the polynucleotide (Bf), the phrase "hybridizes under stringent conditions" is synonymous with the phrase "hybridizes under stringent conditions" in Section A-2. Examples of polynucleotide (Bf) include the nucleotide sequence of SEQ ID NO: 11 and the nucleotide sequence of SEQ ID NO: 12. Base sequence homology between the nucleotide sequence of SEQ ID NO: 10 and the sequence of nucleotide of SEQ ID NO: 11 is 99.7% (Blast2). The base sequence homology between the nucleotide sequence of SEQ ID NO: 10 and the nucleotide sequence of SEQ ID NO: 12 is 91.0% (Blast2). Accordingly, in a preferred embodiment of the invention, a polynucleotide (Bf) comprises the base sequence having 91.0% homology, and more preferably 99.7% homology to the base sequence of SEQ ID NO: 10.

[000233] Considering the polynucleotide (Bf), the "activity of synthesizing tetrahydrodaidzein using dihydrodaidzein as a substrate" can be confirmed by the same method used for polypeptides (Bb) and (Bc).

[000234] The E2 polynucleotide can be produced or obtained by chemical synthesis methods or genetic engineering techniques, based on the sequence information of SEQ ID NO: 10, 11, or 12. Specifically, the methods described for the E1 polynucleotide in Section A-2 may be employed. The method can be modified or changed as required.

[000235] The source of E2 polynucleotide cDNA is not particularly limited since it is a microorganism expressing the E2 polynucleotide. Specific examples include microorganisms capable of equol production, preferably lactic acid bacteria, bacteria belonging to the genera Bacteroides and Streptococci capable of equol production, more preferably Lactococcus garvieae, Bacteroides ovatus and Streptococcus constellatus capable of equol production, also preferably Lactococcus fecal garvieae capable of equol production, and particularly preferably Lactococcus strain 20-92, Bacteroides ovatus strain E-23-15, Streptococcus constellatus A6G-225 (FERM BP-10036, FERM BP-6435, and FERM BP-6437 , respectively; filed with the International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan), fecal Lactococcus garvieae strains capable of equol production.

[000236] Expression of E2 polynucleotide using common genetic engineering techniques easily allows stable mass production of the polynucleotide Product (the polypeptide). The successful isolation of E2 polynucleotide by the present invention will open the door to industrial production of equol, without use of the difficult-to-handle anaerobic bacterial strains traditionally used for equol production.

B-3. Expression Vector

[000237] An expression vector of the present invention is not particularly limited since it includes the E2 polynucleotide and is capable of expressing the E2 polynucleotide. Generally, it is appropriately selected according to the host cell type, similar to the expression vector including the E1 polynucleotide. Specifically, the host cell described in Section A-3 can be used.

B-4. Recombinant Cell

[000238] The present invention provides a recombinant cell (transformant) transformed with an expression vector including the E2 polynucleotide. The host cell used for the recombinant cell may be those described in Section A-4, without limitation. Additionally, the expression vector can be introduced into the host cell by the method described in Section A-4.

[000239] Because the recombinant cell is capable of producing E2 polypeptide (a tetrahydrodaidzein-converting enzyme), it can be used to produce a tetrahydrodaidzein-converting enzyme. The recombinant cell can also be used to produce tetrahydrodaidzein in a cellular form.

B-5. Polypeptide Production using Recombinant Cell

[000240] The E2 polypeptide can be produced by culturing a recombinant cell into which the E2 polynucleotide is introduced and collecting the E2 polypeptide from the cell and culture. Recombinant cells can be cultured as described in Section A-5.

B-6. Production of Tetrahydrodaidzein using the E2 polypeptide

[000241] The present invention provides a process for producing tetrahydrodaidzein using the E2 polypeptide. In the production process, dihydrodaidzein can be converted into tetrahydrodaidzein with the E2 polypeptide acting on dihydrodaidzein in the presence of NADPH and/or NADH.

[000242] The reaction used in the production process can be performed in a suitable buffer such as that described in Section A-6.

[000243] The reaction used in the production process is carried out under such conditions in which, for example, each component is added to a medium in the concentration ranges shown below at the beginning of the reaction in order to prepare a mixture. The mixture is incubated for 0.5 to 10 hours, preferably 1 to 6 hours, and more preferably 2 to 4 hours at temperatures that do not alter or inactivate the starting materials, including the E2 polypeptide and dihydrodaidzein, and the product, including tetrahydrodaidzein. The reaction temperature is not particularly limited. For example, when the reaction temperature is 0°C or lower, the reaction uses, for example, a buffer that does not freeze at such reaction temperatures. The reaction temperature is, for example, preferably 0 to 45°C, and more preferably 0 to 37°C. Considering tetrahydrodaidzein, the compound exists in either a cis form or a trans form. However, the formation of cis and trans configurations can be controlled by variable reaction conditions such as reaction temperature and reaction time. For example, a mixture of cis- and trans-tetrahydrodaidzeins can be produced by setting the reaction temperature at 0°C, or the reaction can be controlled to facilitate the trans form by setting the reaction temperature at 37°C.

[000244] The concentration of each component in the above production process are as follows.

[000245] The E2 polypeptide content is 0.0001 to 1.0% by weight, preferably 0.001 to 0.1% by weight, and more preferably 0.001 to 0.01% by weight; the dihydrodaidzein content is 0.0001 to 10.0% by weight, preferably 0.001 to 1.0% by weight, and more preferably 0.001 to 0.1% by weight; and the NADPH and/or NADH content is 0.01 to 5% by weight, preferably 0.05 to 1% by weight, and more preferably 0.1 to 0.5% by weight.

[000246] The present invention also provides a mixture of material for the synthesis of tetrahydrodaidzein. Specifically, the composition of tetrahydrodaidzein synthesis material including (Bi) E2 polypeptide, (Bii) NADPH and/or NADH, and (Biii) dihydrodaidzein. By incubating the composition under the above-mentioned conditions, the dihydrodaidzein contained in the synthetic starting material composition can be converted to tetrahydrodaidzein. The composition corresponds to the above-mentioned starting material mixture used to initiate the reaction to produce tetrahydrodaidzein. Furthermore, the concentrations of E2 polypeptide, NADPH and/or NADH, and dihydrodaidzein in the composition, and other components that can be added to the synthetic starting material composition are essentially as in a reaction system (a mixture of starting material at the beginning of the reaction) used in the above-mentioned production process.

[000247] The present invention also provides a kit for synthesizing tetrahydrodaidzein. Specifically, a tetrahydrodaidzein synthesis kit including (Bi) E2 polypeptide, (Bii) NADPH and/or NADH, and (Biii) dihydrodaidzein. The synthesis kit may include the components in compartments when required, to conveniently allow the synthesis of tetrahydrodaidzein from dihydrodaidzein under the foregoing conditions. The synthesis kit may also include a buffer when required. The synthesis kit may also include any necessary tools or operating manuals that assist in carrying out the synthesis of tetrahydrodaidzein.

B-7. Tetrahydrodaidzein Composition Synthesizing Enzyme

[000248] The present invention also provides the enzyme for synthesizing the tetrahydrodaidzein composition including E2 polypeptide. The enzyme composition can be suitably used as a tetrahydrodaidzein synthesizing enzyme in the process of producing tetrahydrodaidzein using E2 polypeptide.

[000249] The enzyme composition may be a crude purified E2 polypeptide, or an enzyme composition may be a crude or purified purified E2 polypeptide formulated with a suitable support. The support does not have any adverse effect on E2 polypeptide activity, and is used in an adequate amount.

[000250] The proportion of E2 polypeptide in an enzyme composition is not particularly limited as long as the composition can be used as a tetrahydrodaidzein synthesizing enzyme in the tetrahydrodaidzein production process. Specifically, the E2 polypeptide content is, for example, 0.001 to 20.0% by weight, preferably 0.005 to 5.0% by weight, and more preferably 0.01 to 1.0% by weight, with respect to the total an enzyme composition.

[000251] The enzyme composition may include NADPH and/or NADH, which acts as an E2 polypeptide coenzyme. When contained in an enzyme composition, the proportion of NADPH and/or NADH, although not particularly limited, is 0.005 to 50.0% by weight, preferably 0.05 to 10.0% by weight, and more preferably 0.1 at 5.0% by weight, with respect to the total of an enzyme composition.

[000252] To improve the stability of the polypeptide and/or to ensure the preservation capacity of an enzyme composition, the enzyme composition may also include various antioxidants and preservatives described in Section A-7, in addition to the E2 polypeptide.

B-8. Tetrahydrodaidzein Production Process Using Recombinant Cell

[000253] The present invention provides a process for producing tetrahydrodaidzein using recombinant cells including E2 polynucleotide. Specifically, in the production process, the dihydrodaidzein that is converted into tetrahydrodaidzein having the recombinant cell acts on a dihydrodaidzein.

[000254] The reaction used in the production process is carried out under conditions that allow the recombinant cell to survive, and the dihydrodaidzein to be converted into tetrahydrodaidzein.

[000255] Specifically, appropriate amounts of recombinant cells and dihydrodaidzein are added and cultivated in a medium that allows the growth of the recombinant cells.

[000256] The medium used in the production process is suitably selected from various types of conventional media according to the type of cell used as the host recombinant cell.

[000257] The medium can be supplemented with appropriate amounts of protease inhibitors such as PMSF and EDTA, when required. Furthermore, considering that the polypeptide is derived from anaerobic bacteria, appropriate amounts of reducing agents such as DTT, 2ME, DET, and Na2S2O4 can also be added. When using the recombinant cell, the medium can be supplemented with NADPH and/or NADH when required, although it is not essential.

[000258] Specifically, the production process is carried out as follows. First, the recombinant cells are inoculated into medium containing 0.001 to 1 wt%, preferably 0.01 to 0.5 wt%, and more preferably 0.01 to 0.1 wt% dihydrodaidzein, and the culture is incubated under permissive temperature conditions for 7 to 30 hours, preferably 15 to 24 hours, and more preferably 17 to 20 hours. Here, the permissive temperature conditions are not particularly limited, and are essentially as in the previous section B-6 under the heading "Production of Tetrahydrodaidzein Using E2 Polypeptide".

[000259] The present invention also provides the mixture of material for synthesizing tetrahydrodaidzein, specifically, a composition of tetrahydrodaidzein synthesis material containing (Biv) the recombinant cell and (Biii) dihydrodaidzein. By cultivating the composition under the above-mentioned conditions, the dihydrodaidzein in the composition can be converted to tetrahydrodaidzein. The composition corresponds to the previous starting material mixture used to initiate a reaction to produce tetrahydrodaidzein. Furthermore, the concentrations of the recombinant cell and dihydrodaidzein in a synthetic starting material composition, and other components that can be added to a synthetic starting material composition, are essentially as in the conditions used in the previous production process.

[000260] The present invention also provides a kit for synthesizing tetrahydrodaidzein. Specifically, a tetrahydrodaidzein synthesis kit including (Biv) the recombinant cell and (Biii) dihydrodaidzein. The synthesis kit may include the recombinant cell and dihydrodaidzein separately when required, to conveniently allow the synthesis of tetrahydrodaidzein from dihydrodaidzein under the above-mentioned conditions. The synthesis kit may also include a buffer or medium when required. The synthesis kit may also include any necessary tools or operating manuals that assist in carrying out the synthesis of tetrahydrodaidzein.

[000261] The recombinant cells included in the synthesis kit can be preserved here by known methods. There are several recombinant cell preservation techniques. For example, there is a method in which recombinant cells are preserved at 4 to 25°C after being treated with a lyophilizer to evacuate the ampoule that stores the recombinant cells in a solvent such as dimethylformamide. Another example is a liquid nitrogen method, in which cells are suspended in a preservation medium supplemented with 10% glycerol, which is then stored in a specific ampoule and maintained in a tank of liquid nitrogen (at -150 to - 196°C).

B-9. Antibody Having Affinity to the Polypeptide

[000262] The present invention also provides an antibody (an IgG antibody) having affinity for the E2 polypeptide.

[000263] The monoclonal antibody and polyclonal antibody can be prepared by a usual method. Specifically, these antibodies can be prepared by the method described in Section A-9.

B-10. Immunological Method for the Detection or Measurement of Polypeptide

[000264] The present invention also provides an immunological method for detecting or measuring E2 polypeptide using the antibody. Specifically, the immunological method can be carried out according to the method described in Section A-10.

[000265] The present invention also provides an immunological detection kit including antibody, used for the detection or measurement of E2 polypeptide. The detection kit may also include a standard E2 polypeptide when required. The detection kit may also include, when required, additional reagents that facilitate easy detection of E2 polypeptide under the foregoing conditions. A detection kit may also include any necessary tools or operating manuals that facilitate easy E2 polypeptide detection.

B-11. Method for Detection or Measurement of Polynucleotide Encoding Polypeptide

[000266] The present invention also provides a method for detecting or measuring E2 polynucleotide. Specifically, the method is performed by having an E2 Polynucleotide binding probe contact the test sample in accordance with the method described in Section A-11.

[000267] The present invention also provides a kit for detecting or measuring E2 polynucleotide, specifically an E2 polynucleotide detection kit including the probe. For ease of detection of E2 polynucleotide under the above conditions, the detection kit may include additional reagents or the like when required, in addition to the probe. Furthermore, the detection kit can be used to identify cells containing E2 polynucleotide. From the point of view of enabling accurate detection, the detection kit may preferably be provided as a kit for carrying out detection using PCR.

C. Equol Synthesizing Enzyme C-1. Polypeptide

[000268] The present invention provides a polypeptide (hereinafter also referred to as "E3 polypeptide") for the synthesis of equol using tetrahydrodaidzein as a substrate. Specifically, the invention provides: (Ca) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 13; (Cb) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 13 with the substitution, deletion, insertion, and/or addition of one or more amino acids, and having an activity to synthesize equol using tetrahydrodaidzein as a substrate; and (Cc) a polypeptide consisting of an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 13, and having an activity to synthesize equol using tetrahydrodaidzein as a substrate.

[000269] In the polypeptide (Cb), the range of "one or more amino acids" is not particularly limited as long as the polypeptide has the activity to synthesize equol using tetrahydrodaidzein as a substrate. For example, the range is 1 to 200, preferably 1 to 150, more preferably 1 to 100, more preferably 1 to 50, more preferably 1 to 45, more preferably 1 to 40, more preferably 1 to 30, more preferably 1 to 15, more preferably 1 to 5, also preferably 1 to 4, even more preferably 1 to 3, and particularly preferably 1 or 2.

[000270] Examples of (Cb) polypeptide include a polypeptide consisting of the amino acid sequence of SEQ ID NO: 14 or a polypeptide consisting of the amino acid sequence of SEQ ID NO: 15. Compared to the amino acid sequence of SEQ ID NO: 13, the amino acid sequence of SEQ ID NO: 14 contains two substituted amino acids. Compared to the amino acid sequence of SEQ ID NO: 13, the amino acid sequence of SEQ ID NO: 15 contains 42 substituted amino acids and an additional amino acid (glutamic acid) at the C-terminus. The amino acid sequence of SEQ ID NO: 14 corresponds to the Bacteroides ovatus E-23-15 filament E3 enzyme polypeptide (FERM BP-6435). The amino acid sequence of SEQ ID NO: 15 corresponds to the Streptococcus constellatus E3 enzyme polypeptide A6G-225 (FERM BP-6437).

[000271] In the (Cb) polypeptide, "the substitution, deletion, insertion, or addition of amino acid" can be done as in the substitution, deletion, insertion, or addition of amino acid in E1 polypeptide described in Section A-1. It is preferable that the amino acid substitution, deletion, insertion, or addition in the polypeptide (Cb) be in regions where the amino acid change does not have major effects on the higher order structures of the polypeptide, or where the change does not affect the active site of the enzyme. of equol synthesis. Examples of such regions include poorly conserved regions between the amino acid sequences of SEQ ID NOs: 13, 14, and 15, and neighborhoods thereof, and the N-terminal region or C-terminal region. Specific examples include glutamic acid at position 3, arginine at position 28, glutamic acid at position 29, arginine at position 32, asparagine at position 61, isoleucine at position 80, asparagine at position 92, aspartic acid at position 112, alanine at position 119, asparagine at position 129, aspartic acid at position 172, alanine at position 174, serine at position 204, glutamic acid at position 206, threonine at position 223, valine at position 230, proline at position 244, tyrosine at position 246, threonine at position 280, arginine at position 282, alanine at position 285, valine at position 307, alanine at position 322, glutamic acid at position 347, glycine at position 359, serine at position 360, alanine at position 366, leucine at position 367, isoleucine at position 368, valine at position 372, aspartic acid at position 373, threonine at position 374, alanine at position 377, alanine at position 380, aspartic acid at position 381, glutamine at position 399, proline at position 403, methionine at position 404, valine at position 405, glutamic acid at position 406, glycine at position 407, arginine at position 426, valine at position 434, alanine at position 436, tyrosine at position 438, and alanine at position 440, in the amino acid sequence of SEQ ID NO: 13; and adjacent regions of these amino acids. The "adjacent region" means regions where equol-synthesizing enzyme activity is not affected. Examples of said regions include amino acids in five amino acids of one of the exemplified amino acids, preferably those in four amino acids of one of the exemplified amino acids, more preferably those in three amino acids of one of the exemplified amino acids, still more preferably those in two amino acids of one of the exemplified amino acids. exemplified, and particularly preferably an amino acid of one of the exemplified amino acids. Preferable examples of regions with low preservation capacity and their neighbors include the region corresponding to positions 25 to 35, the region corresponding to positions 170 to 177, the region corresponding to positions 201 to 208, the region corresponding to positions 201 to 208, the region corresponding to positions 201 to 208, the region corresponding to positions to positions 242 to 248, the region corresponding to positions 276 to 289, the region corresponding to positions 355 to 385, the region corresponding to positions 396 to 409, and the region corresponding to positions 431 to 443, in the sequence of amino acid of SEQ ID NO: 13.

[000272] In the amino acid sequence of SEQ ID NO: 13, the sequence ranging from the amino acid at position 14 to the amino acid at position 19 is considered to correspond to the FAD binding domain. Consequently, as long as the functions of the domain are not inhibited, there may be substitution, deletion, insertion, or addition of an arbitrary amino acid in the sequence. It is, however, preferred that glycine at position 14, glycine at position 16, and glycine at position 19 are not mutated. In substitution, deletion, insertion, or addition of amino acid in the sequence, the plurality of mutated amino acids are preferably no more than 3, more preferably no more than 2, even more preferably 1. More preferred is no mutation in the sequence.

[000273] Fig. 29 shows an alignment of the amino acid sequences of SEQ ID Nos. 13, 14 and 15.

[000274] In polypeptide (Cc), the amino acid sequence has, for example, 60% or more identity with the amino acid sequence of SEQ ID NO: 13. It is preferred that the amino acid sequence is generally 80% or more, preferably 85% or more, more preferably 90% or more, also preferably 95% or more, even more preferably 98% or more, and particularly preferably 99% or more identity with the amino acid sequence of SEQ ID NO: 13.

[000275] Specifically, (Cc) polypeptide can be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 14 or a polypeptide consisting of the amino acid sequence of SEQ ID NO: 15. The amino acid identity between the amino acid sequence of SEQ ID NO: 13 and the amino acid sequence of SEQ ID NO: 14 is 99.6% (Blast2). The amino acid identity between the amino acid sequence of SEQ ID NO: 13 and the amino acid sequence of SEQ ID NO: 15 is 90.9% (Blast 2). Accordingly, in a preferred embodiment of the invention, (Bc) polypeptide comprises an amino acid sequence having 90.9% or more, and more preferably 99.6%, identity with the amino acid sequence of SEQ ID NO: 13.

[000276] Considering the polypeptides (Cb) and (Cc), the activity to synthesize equol using tetrahydrodaidzein as a substrate can be confirmed as follows. First, a polypeptide of interest is added to a substrate solution of the composition below at a concentration of 0.001 mg/mL. The solution is then incubated at 37°C for 2 hours to check for the presence or absence of equol in a solution. The presence of equol in a solution after incubation is used as an indicator of polypeptide activity to synthesize equol using tetrahydrodaidzein as a substrate. Substrate Solution Composition 0.1 M potassium phosphate buffer (pH 7.0) 1 mM PMSF (phenylmethylsulfonyl fluoride) 2 mM dithiothreitol 5 mM sodium hydrosulfite 40 uM dihydrodaidzein

Enzyme Property

[000277] Since E3 polypeptide has an enzyme activity to synthesize equol using tetrahydrodaidzein as a substrate, E3 polypeptide is also referred to as E3 enzyme. The ideal E3 enzyme temperature is around 23 to 37°C, and the ideal pH is 4.5. The E3 enzyme can also synthesize tetrahydrodaidzein from equol.

[000278] Similar to E1 polypeptide and E2 polypeptide, E3 polypeptide can be produced by genetic engineering techniques based on the formation of a nucleotide sequence of SEQ ID NO: 16, 17, or 18. In addition, synthetic methods Common chemistry can be used to produce E3 polypeptide, based on the amino acid sequence information of SEQ ID NO: 13, 14, or 15. The E3 polypeptide can also be produced by isolating and purifying microorganisms capable of producing it. These methods can be carried out in accordance with the description in the previous section A-1.

[000279] The microorganism capable of producing E3 polypeptide can be cultivated in a medium containing a desired amount of tetrahydrodaidzein. The E3 polypeptide can also be produced in this way in microorganisms capable of producing E3 polypeptide.

[000280] The E2 polypeptide can be monomeric, or dimeric or polymeric, as long as it can synthesize tetrahydrodaidzein. Furthermore, to improve stability and other characteristics, the E3 polypeptide can be modified when required by adding polyethylene glycol or a sugar chain.

[000281] The E3 polypeptide can serve as a catalyst that converts tetrahydrodaidzein (a substrate) into equol. Equol is believed to exhibit various physiological activities in the body. In this regard, the E3 polypeptide, capable of providing equol, is considered important. The present invention therefore provides the equol-synthesizing enzyme including any of the polypeptides (Ca) to (Cc).

C-2. Polynucleotide

[000282] The present invention also provides a polynucleotide (hereinafter also referred to as "E3 polynucleotide") that encodes a polypeptide having an activity of synthesizing equol using tetrahydrodaidzein as a substrate. Specifically, the invention provides: (Cd) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 16; (Ce) a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 13; and (Cf) a polynucleotide that hybridizes under stringent conditions with the complementary strand of the polynucleotide (Cd) or (Ce), and that encodes a polypeptide having an activity to synthesize equol using tetrahydrodaidzein as a substrate.

[000283] The amino acid sequence of SEQ ID NO: 13 corresponds to the amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 16. The amino acid sequence of SEQ ID NO: 14 corresponds to the amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 17. The amino acid sequence of SEQ ID NO: 15 corresponds to the amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 18.

[000284] Considering polynucleotide (Cf), the phrase "hybridizes under stringent conditions" is synonymous with the phrase "hybridizes under stringent conditions" in Section A-2. Examples of polynucleotide (Cf) include the nucleotide sequence of SEQ ID NO: 17 and the nucleotide sequence of SEQ ID NO: 18. Base sequence homology between the nucleotide sequence of SEQ ID NO: 16 and the nucleotide sequence of SEQ ID NO: 17 is 99.8% (Blast2). The homology between the nucleotide sequence of SEQ ID NO: 16 and the nucleotide sequence of SEQ ID NO: 18 is 85.2% (Blast2). Accordingly, in a preferred embodiment of the invention, a polynucleotide (Cf) comprises a base sequence having 85.2% homology, and more preferably 99.8% homology, to the base sequence of SEQ ID NO: 16.

[000285] Considering polynucleotide (Cf), the "activity of synthesizing equol using tetrahydrodaidzein as a substrate" can be confirmed by the same method used for polypeptides (Cb) and (Cc).

[000286] E3 polynucleotide can be produced or obtained by chemical DNA synthesis methods or genetic engineering techniques, based on the sequence information of SEQ ID NO: 16, 17 or 18. Specifically, the method described for E1 polynucleotide in Sec. A-2 may be employed.

[000287] The source of E3 polynucleotide cDNA is not particularly limited as long as it is a microorganism expressing the E3 polynucleotide. Specific examples include microorganisms capable of producing equol, preferably lactic acid bacteria, bacteria belonging to the genera Bacteroides and Streptococcus capable of producing, more preferably Lactococcus garvieae, Bacteroides ovatus, and Streptococcus constellatus capable of producing equol, also preferably Lactococcus garvieae fecal and particularly preferably Lactococcus 20-92 filament, Bacteroides ovatus E-23-15 filament, Streptococcus constellatus A6G-225 (FERM BP-10036, FERM BP-6435, and FERM BP-6437, respectively; deposited with the International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan), fecal Lactococcus garvieae filaments capable of producing equol.

[000288] Expression of E3 polynucleotide using common genetic engineering techniques easily allows mass, stable production of the polynucleotide product (the polypeptide). The successful isolation of E3 polynucleotide by the present invention will open the door to the industrial production of equol, without using the difficult to manipulate anaerobic bacterial filaments traditionally used for equol production.

C-3. Expression Vector

[000289] An expression vector of the present invention is not particularly limited since it includes the E3 polynucleotide and is capable of expressing the E3 polynucleotide. Generally, it is appropriately selected according to the host cell type, similar to the expression vector including the E1 polynucleotide. Specifically, the host cell described in Section A-3 can be used.

C-4. Recombinant Cell

[000290] The present invention provides a recombinant cell (transformant) transformed with an expression vector including the E3 polynucleotide. The host cell used for the recombinant cell can be any of those described in Section A-4. Additionally, the expression vector can be introduced into the host cell by the method described in Section A-4.

[000291] Because the recombinant cell is capable of producing E3 polypeptide (an equol-converting enzyme), it can be used to produce an equol-converting enzyme. The recombinant cell can also be used to produce equol in a cellular form.

C-5. Polypeptide Production using Recombinant Cell

[000292] The E3 polypeptide can be produced by culturing a recombinant cell into which the E3 polynucleotide is introduced, and collecting the E3 polypeptide from the cell and culturing. Recombinant cells can be cultured as described in Section A-5.

C-6. Equol production using E3 polypeptide

[000293] The present invention provides a process for producing equol using E3 polypeptide. In the production process, tetrahydrodaidzein can be converted into equol with the E3 polypeptide acting on tetrahydrodaidzein.

[000294] The reaction used in the production process can be performed in a suitable buffer. Specifically, the buffers described in Section A-6 may be used.

[000295] The reaction used in the production process is carried out under such conditions in which, for example, each component is added to a medium in the concentration ranges described below at the beginning of the reaction in order to prepare a mixture. The mixture is incubated for 0.5 to 10 hours, preferably 1 to 6 hours, and most preferably 2 to 4 hours at temperatures that do not alter or inactivate the starting materials, including the E3 polypeptide and tetrahydrodaidzein, and the product, including equol. . The reaction temperature is not particularly limited. For example, when the reaction temperature is 0°C or lower, the reaction uses, for example, a buffer that does not freeze at such reaction temperatures. The reaction temperature is, for example, preferably 0 to 45°C, and more preferably 0 to 37°C.

[000296] The E3 polypeptide content is 0.0001 to 1.0% by weight, preferably 0.001 to 0.1% by weight, and more preferably 0.001 to 0.01% by weight; and the tetrahydrodaidzein content is 0.0001 to 10.0% by weight, preferably 0.001 to 1.0% by weight, and more preferably 0.001 to 0.1% by weight.

[000297] The present invention also provides a mixture of material for the synthesis of equol, specifically, a synthesis of equol composition material including (Ci) E3 polypeptide, (Cii) tetrahydrodaidzein. By incubating the composition under the above conditions, the tetrahydrodaidzein contained in the composition can be converted to equol. The composition corresponds to the preceding starting material mixture used to initiate the reaction to produce equol. Furthermore, the concentrations of E3 polypeptide, and tetrahydrodaidzein in the composition, and other components that can be added to the composition are essentially as in a reaction system (a mixture of starting material at the beginning of the reaction) used in the preceding production process.

[000298] The present invention also provides a kit for synthesizing equol, specifically, an equol synthesis kit including (Ci) E3 polypeptide and (Cii) tetrahydrodaidzein. The synthesis kit may include the components in compartments as required in order to conveniently allow synthesis of tetrahydrodaidzein equol under the foregoing conditions. The synthesis kit may also include a buffer as required. The synthesis kit may also include any necessary instruments or operating manuals that help perform equol synthesis.

C-7. Composition of equol synthesizing enzyme

[000299] The present invention also provides an equol-synthesizing enzyme composition including the E3 polypeptide. An enzyme composition can be suitably used as an equol synthesizing enzyme in the process of producing equol using E3 polypeptide.

[000300] The enzyme composition can be a raw purified E3 polypeptide, or an enzyme composition can be a raw or purified purified E3 polypeptide formulated with a suitable support. The support does not have any adverse effect on E3 polypeptide activity, and is used in an adequate amount.

[000301] The proportion of E3 polypeptide in an enzyme composition is not particularly limited since the composition can be used as an equol-synthesizing enzyme in the equol production process. Specifically, the E3 polypeptide content is, for example, 0.001 to 20.0 wt%, preferably 0.005 to 5.0 wt%, and more preferably 0.01 to 1.0 wt%, with respect to the total an enzyme composition.

[000302] To improve the stability of the polypeptide and/or to ensure the preservability of an enzyme composition, an enzyme composition may also include various antioxidants and preservatives described in Section A-7, in addition to the E3 polypeptide.

C-8. Equol Production Process Using Recombinant Cell

[000303] The present invention provides a process for producing equol using recombinant cells including E3 polynucleotide. Specifically, in the production process, tetrahydrodaidzein is converted into equol and the recombinant cell acts on the tetrahydrodaidzein to convert.

[000304] The reaction used in the production process is carried out in an environment that allows the recombinant cell to survive, and the tetrahydrodaidzein to be converted into equol.

[000305] Specifically, appropriate amounts of recombinant cells and tetrahydrodaidzein are added and cultured in a medium that allows development of the recombinant cells.

[000306] The medium used in the production process is suitably selected from various types of conventional media according to the type of cell used as the host recombinant cell.

[000307] The medium can be supplemented with appropriate amounts of protease inhibitors such as PMSF and EDTA, as required. Furthermore, considering that the polypeptide is derived from anaerobic bacteria, appropriate amounts of reducing agents such as DTT, 2ME, DET, and Na2S2O4 can also be added. When using a recombinant cell, the medium can be supplemented with NADPH and/or NADH as required, although it is not essential.

[000308] Specifically, the production process is performed as follows. First, the recombinant cells are inoculated into medium containing 0.001 to 1 wt%, preferably 0.01 to 0.5 wt%, and more preferably 0.01 to 0.1 wt% tetrahydrodaidzein, and the culture is incubated under permissive temperature conditions for 7 to 30 hours, preferably 15 to 24 hours, and more preferably 17 to 20 hours. Here, the permissive temperature conditions are not particularly limited, and are essentially as in the previous section C-6.

[000309] The present invention also provides a mixture of material for synthesizing equol, specifically, a synthesis of equol composition material containing (Ciii) the recombinant cell and (Cii) tetrahydrodaidzein. By cultivating the composition under the above conditions, the tetrahydrodaidzein in the composition can be converted to equol. The composition corresponds to the preceding starting material mixture used to initiate the reaction to produce equol. Furthermore, the concentrations of the recombinant cell and tetrahydrodaidzein in the composition, and other components that may be added to the composition, are essentially as under the conditions used in the foregoing production process.

[000310] The present invention also provides a kit for synthesizing equol, specifically, an equol synthesis kit including (Ciii) the recombinant cell and (Cii) tetrahydrodaidzein. The synthesis kit may include the recombinant cell and tetrahydrodaidzein in compartments as required, in order to conveniently allow synthesis of dihydrodaidzein equol under the foregoing conditions. The synthesis kit may also include a buffer or medium, as required. The synthesis kit may also include any necessary instruments or operating manuals that help perform equol synthesis.

[000311] The recombinant cells included in the synthesis kit can be preserved in it using known methods. There are several known recombinant cell preservation techniques. For example, there is a method in which recombinant cells are preserved at 4 to 25 °C after being treated with a lyophilizer to evacuate an ampoule storing the recombinant cells in a solvent such as dimethylformamide. Another example is a liquid nitrogen method, in which cells are suspended in a preservation medium supplemented with 10% glycerol, which is then stored in a specific ampoule and maintained in a liquid nitrogen tank (at -150 to - 196°C).

C-9. Antibody Having Affinity to the Polypeptide

[000312] The present invention also provides an antibody (an IgG antibody) having affinity to the E3 polypeptide.

[000313] Monoclonal antibody and polyclonal antibody can be prepared using a usual method. Specifically, these antibodies can be produced by the methods described in Section A-9.

C-10. Immunological Method for the Detection or Measurement of the Polypeptide

[000314] The present invention also provides an immunological method for detecting or measuring the E3 polypeptide using the antibody. Specifically, the immunological method can be performed according to the method described in Section A-10.

[000315] The present invention also provides an immunological detection kit including the antibody, used for the detection or measurement of the E3 polypeptide. The detection kit may also include a standard E3 polypeptide, as required. The detection kit may also include, as required, additional reagents that facilitate easy detection of E3 polypeptide under the foregoing conditions. The detection kit may also include any necessary instruments or operating manuals that facilitate easy detection of E3 polypeptide.

C-11. Method for Detection or Measurement of Polynucleotide Encoding the Polypeptide

[000316] The present invention also provides a method for detecting or measuring E3 polynucleotide. Specifically, the method is performed by contacting an E3 polynucleotide binding probe with the test sample in accordance with the method described in Section A-11.

[000317] The present invention also provides a kit for detecting or measuring E3 polynucleotide, specifically an E3 polynucleotide detection kit including the probe. For ease of detection of the E3 polynucleotide under the foregoing conditions, the detection kit may include additional or other reagents as required, in addition to the probe. Furthermore, the detection kit can be used to identify cells containing the E3 polynucleotide. From the point of view of enabling accurate detection, the detection kit may preferably be provided as a kit for detection performance using PCR.

D. Process for producing equol using enzymes E1 - E3, or intermediate of those D-I-1. Process for producing tetrahydrodaidzein comprising First Stage and Second Stage

[000318] The present invention provides a process for producing tetrahydrodaidzein comprising the following First Step and Second Step (hereinafter, this process is occasionally referred to as the "First Production Method"). First Stage produces dihydrodaidzein from daidzein. Second Step produces tetrahydrodaidzein from dihydrodaidzein.

[000319] The First Step comprises a step of inducing an enzyme consisting of one of the following polypeptides (Aa) to (Ac), and NADPH and/or NADH acting on daidzein, thereby producing dihydrodaidzein. (Aa) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1. (Ab) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 with the substitution, deletion, insertion, and/or addition of one or more amino acids, and having an activity of synthesizing dihydrodaidzein using daidzein as a substrate. (Ac) a polypeptide consisting of an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 1, and having an activity of synthesizing dihydrodaidzein using daidzein as a substrate.

[000320] The Second Step comprises a step of having an enzyme consisting of one of the following polypeptides (Ba) to (Bc), and NADPH and/or NADH acting on dihydrodaidzein, thereby producing tetrahydrodaidzein. (Ba) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 7. (Bb) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 7 with the substitution, deletion, insertion, and/or addition of one or more amino acids, and having an activity of synthesizing tetrahydrodaidzein using dihydrodaidzein as a substrate. (Bc) a polypeptide consisting of an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 7, and having an activity of synthesizing tetrahydrodaidzein using dihydrodaidzein as a substrate.

[000321] The First Production Method of the present invention allows production of tetrahydrodaidzein from dihydrodaidzein, and equol from tetrahydrodaidzein.

First step

[000322] In the First Step, daidzein is converted into dihydrodaidzein by having an enzyme consisting of the E1 polypeptide acting on daidzein in the presence of NADPH and/or NADH. The First Step reaction is carried out by incubation in a solution containing an enzyme consisting of the E1 polypeptide, daidzein, and NADPH and/or NADH at temperatures that do not alter or inactivate the starting materials, including the polypeptide and daidzein, and the product, including dihydrodaidzein; namely as conditions detailed in the previous section A-6.

[000323] When the First Step and the Second Step described below are carried out under the same conditions, the reaction is carried out under such conditions that the components satisfy the concentration ranges as shown below in a reaction system at the beginning of the reaction (in a mixture of starting materials at the start of the reaction), and that the mixture is incubated for 0.5 to 10 hours, preferably 1 to 6 hours, and more preferably 2 to 4 hours, at 15 to 45 °C, preferably 25 to 40°C, and more preferably 30 to 38°C. With this arrangement, both the First and Second Steps are efficiently carried out. The content of the enzyme consisting of the E1 polypeptide is 0.0001 to 1.0% by weight, preferably 0.001 to 0.1% by weight, and more preferably 0.001 to 0.01% by weight. The daidzein content is 0.0001 to 10.0% by weight, preferably 0.001 to 1.0% by weight, and more preferably 0.001 to 0.1% by weight. The NADPH and/or NADH content is 0.01 to 5% by weight, preferably 0.05 to 1% by weight, and more preferably 0.1 to 0.5% by weight.

[000324] The source of daidzein used as a substrate in the First Step is not limited. For example, commercially available daidzeins or daidzeins produced or synthesized by appropriate methods can be used.

[000325] Furthermore, for example, a composition of dihydrodaidzein synthesis material containing (Ai) an enzyme consisting of the E1 polypeptide; (Aii) NADPH and/or NADH; and (Aiii) daidzein can be used as a mixture of starting materials in the production of dihydrodaidzein in the First Step. By incubating the composition under the above conditions, the daidzein in the composition is converted to dihydrodaidzein. A composition is specifically described in previous sections A-6 and A-7.

Second stage

[000326] In the Second Step, dihydrodaidzein is converted into tetrahydrodaidzein by having an enzyme consisting of the E2 polypeptide acting on dihydrodaidzein in the presence of NADPH and/or NADH.

[000327] The reaction in the Second Step is carried out by incubation in a solution containing an enzyme consisting of the E2 polypeptide, dihydrodaidzein, and NADPH and/or NADH at temperatures that do not alter or inactivate the starting materials, including the enzyme consisting of the E2 polypeptide and dihydrodaidzein, and the product including tetrahydrodaidzein; namely as conditions detailed in previous section B-6.

[000328] Considering tetrahydrodaidzein, the compound exists in a cis form or a trans form. The formation of cis and trans configurations can be controlled by variable reaction conditions such as reaction temperature and reaction time. For example, a mixture of cis- and trans-tetrahydrodaidzeins can be produced by setting the reaction temperature at 0°C, or the reaction can be controlled to facilitate the trans form by setting the reaction temperature at 37°C.

[000329] When the First Step and Second Step are carried out under the same conditions, the reaction is carried out by incubation under the previously mentioned conditions to carry out the First Step and Second Step under the same conditions, which are detailed in the previous explanation of the First Stage. With this arrangement, both the First and Second Steps are efficiently carried out. In this case, the contents of the enzyme consisting of the E2 polypeptide, dihydrodaidzein, and NADPH and/or NADH are adjusted as follows: the content of the enzyme consisting of the E2 polypeptide is 0.0001 to 1.0% by weight, preferably 0.001 to 0.1% by weight, and more preferably 0.001 to 0.01% by weight; the dihydrodaidzein content is 0.0001 to 10.0% by weight, preferably 0.001 to 1.0% by weight, and more preferably 0.001 to 0.1% by weight; and the NADPH and/or NADH content is 0.01 to 5% by weight, preferably 0.05 to 1% by weight, and more preferably 0.1 to 0.5% by weight.

[000330] However, when carried out in the presence of the enzyme consisting of the E1 polypeptide or the enzyme consisting of the E2 polypeptide, the First Step and Second Step require the use of NADPH to increase the efficiency of the dihydrodaidzein production reaction. The concentration of NADPH is determined based on the previously mentioned conditions to perform the First Step and Second Step under the same conditions, which are detailed in the previous explanation of the First Step. The concentration of NADH, which is used with NADPH, is 0.01 to 5% by weight, preferably 0.05 to 1% by weight, more preferably 0.1 to 0.5% by weight.

[000331] The source of dihydrodaidzein used as a substrate in the Second Step is not limited.

[000332] For example, dihydrodaidzein produced in the First Daidzein Step can be used as a substrate for the Second Step. Dihydrodaidzein is used either in the form of the dihydrodaidzein-containing solution as produced in the First Step, or in a crudely refined state or appropriately refined state.

[000333] Commercially available dihydrodaidzeins or dihydrodaidzeins synthesized by appropriate methods can also be used.

[000334] Furthermore, the synthesis of a tetrahydrodaidzein material composition containing (Bi) an enzyme consisting of the E2 polypeptide; (Bii) NADPH and/or NADH; and (Biii) dihydrodaidzein can be used as a mixture of starting materials in the production of tetrahydrodaidzein in the Second Step. By incubating the composition under the above conditions, the dihydrodaidzein in the composition is converted to tetrahydrodaidzein. A synthetic composition is specifically described in previous sections A-6 and A-7.

[000335] In the Second Step, it is preferable to use dihydrodaidzein, produced in the First Step using daidzein as a substrate; however, commercially available dihydrodaidzein, a synthetic starting material composition, an enzyme composition, etc. can be mixed with the dihydrodaidzein produced in the First Stage.

E1 Polypeptide and E2 Polypeptide

[000336] First Production Method of the present invention uses the enzyme consisting of the E1 polypeptide as detailed in the previous section A-1, and the enzyme consisting of the E2 polypeptide as detailed in the previous section B-1.

D-I-2. Product containing tetrahydrodaidzein, produced by the First Production Method

[000337] The present invention provides a product containing tetrahydrodaidzein, produced by the previous process comprising First Step and Second Step (First Production Method).

[000338] As described above, the First Production Method of the present invention allows production of dihydrodaidzein from daidzein, and tetrahydrodaidzein from dihydrodaidzein.

[000339] Consequently, the product produced by the First Production Method of the present invention contains not only tetrahydrodaidzein, but also dihydrodaidzein and/or daidzein.

[000340] The product of the present invention can be used in the form of the solution as produced in the First Production Method, or it can be a tetrahydrodaidzein product obtained crudely or appropriately by refining the solution.

[000341] The product of the present invention can be incorporated into food, drinks, or makeup product materials, medicinal products, etc., or can be used as a substrate.

D-I-3. Equol production process comprising Second Stage and Third Stage

[000342] The present invention provides a process for producing equol comprising the following Second Step and Third Step (hereinafter, this process is occasionally referred to as the "Second Production Method"). The Second Step produces tetrahydrodaidzein from dihydrodaidzein. The Third Step produces tetrahydrodaidzein equol.

[000343] Second Step comprises a step of having an enzyme consisting of one of the following polypeptides (Ba) to (Bc), and NADPH and/or NADH acting on dihydrodaidzein, thereby producing tetrahydrodaidzein. (Ba) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 7. (Bb) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 7 with the substitution, deletion, insertion, and/or addition of one or more amino acids, and having an activity of synthesizing tetrahydrodaidzein using dihydrodaidzein as a substrate. (Bc) a polypeptide consisting of an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 7, and having an activity of synthesizing tetrahydrodaidzein using dihydrodaidzein as a substrate.

[000344] Third Step comprises a step of having an enzyme consisting of one of the following (Ca) to (Cc) polypeptides acting on tetrahydrodaidzein, thereby producing equol. (Ca) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 13. (Cb) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 13 with the substitution, deletion, insertion, and/or addition of one or more amino acids, and having an activity to synthesize equol using tetrahydrodaidzein as a substrate. (Cc) a polypeptide consisting of an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 13, and having an activity to synthesize equol using tetrahydrodaidzein as a substrate.

[000345] Second Production Method of the present invention allows production of tetrahydrodaidzein equol, and tetrahydrodaidzein equol. Second stage

[000346] Second Step of Second Production Method of the present invention is the same as the Second Step of First Production Method.

[000347] When the Second Step and the Third Step described below are carried out under the same conditions, the reaction is carried out under such conditions that the components satisfy the concentration ranges below in a reaction system at the beginning of the reaction (in a mixture of starting materials at the start of the reaction), and that the mixture is incubated for 0.5 to 10 hours, preferably 1 to 6 hours, and more preferably 2 to 4 hours, at 15 to 45°C, preferably 25 to 40°C , and more preferably 30 to 38°C. With this arrangement, both the First and Second Steps are efficiently carried out.

[000348] The content of the enzyme consisting of the E2 polypeptide is 0.0001 to 1.0% by weight, preferably 0.001 to 0.1% by weight, and more preferably 0.001 to 0.01% by weight. The dihydrodaidzein content is 0.0001 to 10.0% by weight, preferably 0.001 to 1.0% by weight, and more preferably 0.001 to 0.1% by weight. The NADPH and/or NADH content is 0.01 to 5% by weight, preferably 0.05 to 1% by weight, and more preferably 0.1 to 0.5% by weight.

Third Stage

[000349] The Third Step induces an enzyme consisting of the E3 polypeptide to act on tetrahydrodaidzein, thereby converting tetrahydrodaidzein into equol.

[000350] The reaction in the Third Step is carried out by incubation in a solution containing an enzyme consisting of the E3 polypeptide, and tetrahydrodaidzein at temperatures that do not alter or inactivate the starting materials, including the enzyme consisting of the polypeptide. E3 peptide and tetrahydrodaidzein, and the product, including equol; namely as conditions detailed in the previous section A-6.

[000351] When the Second Step and Third Step are carried out under the same conditions, the reaction is carried out by means of incubation under the previously mentioned conditions to carry out the Second Step and Third Step under the same conditions, which are detailed in the previous explanation of the Second stage. With this arrangement, both the Second and Third Steps are efficiently carried out. In this case, the contents of the enzyme consisting of the E3 polypeptide, tetrahydrodaidzein, and NADPH and/or NADH are adjusted as follows: the content of the enzyme consisting of the E3 polypeptide is 0.0001 to 1.0% by weight, preferably 0.001 to 0. 1% by weight, and more preferably 0.001 to 0.01% by weight; the tetrahydrodaidzein content is 0.0001 to 10.0% by weight, preferably 0.001 to 1.0% by weight, and most preferably 0.001 to 0. 1% by weight; It is

[000352] The content of NADPH and/or NADH is 0.01 to 5% by weight, preferably 0.05 to 1% by weight, and more preferably 0.1 to 0.5% by weight.

[000353] The source of tetrahydrodaidzein used as a substrate in the Third Step is also not limited.

[000354] For example, tetrahydrodaidzein produced in the Second Step of dihydrodaidzein can be used as a substrate for the Third Step. Tetrahydrodaidzein is used either in the form of a solution containing tetrahydrodaidzein as produced in the Second Step, or in a crudely refined state or appropriately refined state.

[000355] Commercially available tetrahydrodaidzeins or tetrahydrodaidzeins synthesized by appropriate methods can also be used.

[000356] Furthermore, a synthesis of tetrahydrodaidzein material composition containing (Ci) an enzyme consisting of the E3 polypeptide; and (Cii) tetrahydrodaidzein can be used as a mixture of starting materials in the production of tetrahydrodaidzein in the Third Step. By incubating the composition under the above conditions, the tetrahydrodaidzein in the composition is converted to equol. A composition is specifically described in previous sections C-6 and C-7.

[000357] In the Third Step, it is preferable to use tetrahydrodaidzein, produced in the Second Step using dihydrodaidzein as a substrate; however, commercially available tetrahydrodaidzein, a synthetic starting material composition, an enzyme composition, etc. can be mixed with the tetrahydrodaidzein produced in the Second Stage.

E2 Polypeptide and E3 Polypeptide

[000358] The Second Production Method of the present invention uses an enzyme consisting of the E2 polypeptide described in the previous section B-1, and an enzyme consisting of the E3 polypeptide described in the previous section C-1.

D-I-4. Product containing equol, produced by the Second Production Method

[000359] The present invention provides a product containing equol, produced by the previous process comprising Second Stage and Third Stage (Second Production Method).

[000360] As described above, the Second Production Method of the present invention allows production of tetrahydrodaidzein from dihydrodaidzein, and equol from tetrahydrodaidzein.

[000361] Consequently, the product produced by the Second Production Method of the present invention contains not only equol, but also tetrahydrodaidzein and/or dihydrodaidzein.

[000362] The product of the present invention can be used in the form of the solution as produced in the Second Production Method, or it can be an equol Product obtained crudely or appropriately by refining the solution.

[000363] The product of the present invention can be incorporated into foods, drinks, makeup product materials, medicinal products, etc., or can be used as a substrate.

D-I-5. Equol production method comprising First Step to Third Step

[000364] The present invention provides a method of producing equol comprising First Step, Second Step and Third Step (hereinafter, this process is occasionally referred to as "Third Production Method").

[000365] The Third Production Method of the present invention allows production of dihydrodaidzein from daidzein, tetrahydrodaidzein from dihydrodaidzein, and equol from tetrahydrodaidzein.

First step

[000366] In the First Step of the Third Production Method of the present invention, daidzein is converted into dihydrodaidzein by having an enzyme consisting of the E1 polypeptide acting on daidzein in the presence of NADPH and/or NADH. The First Step of Third Production Method of the present invention is the same as the aforementioned First Step.

[000367] When the First Step and Second Step are carried out under the same conditions, when the First Step and Third Step are carried out under the same conditions, or when the First Step to the Third Step are carried out under the same conditions, the reaction is carried out through incubation under the above-mentioned conditions/concentrations to perform the First Step and Second Step under the same conditions, which are detailed in the previous explanation of the First Step. With this arrangement, both the First and Second Steps are efficiently carried out.

Second stage

[000368] In the Second Step of the Third Production Method of the present invention, dihydrodaidzein is converted into tetrahydrodaidzein by having an enzyme consisting of the E2 polypeptide acting on dihydrodaidzein in the presence of NADPH and/or NADH. The Second Step of the Third Production Method of the present invention is the same as the previously mentioned Second Step.

[000369] When the First Step and Second Step are carried out under the same conditions, or when the First Step to the Third Step are carried out under the same conditions, the reaction is carried out by means of incubation under the above-mentioned conditions/concentrations to carry out the First Stage and Second Stage under the same conditions, which are detailed in the previous explanation of the Second Stage. With this arrangement, both the First and Second Steps are efficiently carried out.

[000370] Furthermore, when the First Step and Second Step are performed in the presence of the enzyme consisting of the E1 polypeptide and the enzyme consisting of the E2 polypeptide, or when the First Step to the Third Step are performed in the presence of the enzyme consisting of the E1 polypeptide, the enzyme consisting of the E2 polypeptide, and the enzyme consisting of the E3 polypeptide, the reaction is performed by incubating in the presence of the enzyme consisting of the E1 polypeptide and the enzyme consisting of the E2 polypeptide, under the above-mentioned conditions/concentrations, which is detailed in explanation of the Second Stage, for example.

[000371] When the Second Step and Third Step are carried out under the same conditions, the reaction is carried out by means of incubation under the above-mentioned conditions/concentrations to carry out the Second Step and Third Step under the same conditions, which are detailed in the explanation of the Second Stage. With this arrangement, both the Second and Third Steps are efficiently carried out.

Third Stage

[000372] In the Third Step of the Third Production Method of the present invention, tetrahydrodaidzein is converted into equol by having an enzyme consisting of the E3 polypeptide acting on tetrahydrodaidzein in the presence of NADPH and/or NADH, thereby converting tetrahydrodaidzein into equol . The Third Step of the Third Production Method of the present invention is the same as the aforementioned Third Step.

[000373] When the First Step and Third Step are carried out under the same conditions, or when the First Step to the Third Step are carried out under the same conditions, the reaction is carried out by means of incubation under the previously mentioned conditions for carrying out the First Step and Second Stage under the same conditions, which are detailed in the previous explanation of the First Stage. With this arrangement, both the First and Second Steps are efficiently carried out. In this case, the contents of the enzyme consisting of the E3 polypeptide, tetrahydrodaidzein, and NADPH and/or NADH are adjusted as follows: the content of the enzyme consisting of the E3 polypeptide is 0.0001 to 1.0% by weight, preferably 0.001 to 0. 1% by weight, and more preferably 0.001 to 0.01% by weight; the tetrahydrodaidzein content is 0.0001 to 10.0% by weight, preferably 0.001 to 1.0% by weight, and more preferably 0.001 to 0.1% by weight; and the NADPH and/or NADH content is 0.01 to 5% by weight, preferably 0.05 to 1% by weight, and more preferably 0.1 to 0.5% by weight.

[000374] However, when carried out in the presence of the enzyme consisting of the E1 polypeptide and the enzyme consisting of the E3 polypeptide, the First Step and Third Step require the use of NADPH to increase the efficiency in the dihydrodaidzein production reaction using the enzyme consisting of the E1 polypeptide. Similarly, when carried out in the presence of the enzyme consisting of the E1 polypeptide, the enzyme consisting of the E2 polypeptide, and the enzyme consisting of the E3 polypeptide, the First Step through the Third Step require the use of NADPH to increase the efficiency of the dihydrodaidzein production reaction. using the enzyme consisting of the E1 polypeptide. In this case, the concentration of NADPH is determined based on the previously mentioned conditions to carry out the First Step and Second Step under the same conditions, which are detailed in the previous explanation of the First Step. The concentration of NADH, which is used with NADPH, is determined based on the previously mentioned conditions for carrying out reaction in the presence of the enzyme consisting of the E1 polypeptide, the enzyme consisting of the E2 polypeptide, which are detailed in the previous explanation of the Second Step, and as previously mentioned conditions to carry out the Second Step and Third Step under the same conditions, which are detailed in the previous explanation of the Second Step.

E1 to E3 polypeptides

[000375] The Third Production Method of the present invention uses the enzyme consisting of the E1 polypeptide, the enzyme consisting of the E2 polypeptide, and the enzyme consisting of the E3 polypeptide. Peptides E1 to E3 are the same as those described above.

D-I-6. Product containing equol, produced by the Third Production Method

[000376] The present invention provides an equol-containing product, produced by the previous process comprising First Step to Third Step (Third Production Method).

[000377] As described above, the Third Production Method of the present invention allows production of dihydrodaidzein from daidzein, tetrahydrodaidzein from dihydrodaidzein, and equol from tetrahydrodaidzein.

[000378] Consequently, the product produced by the Third Production Method of the present invention contains not only equol, but also tetrahydrodaidzein, dihydrodaidzein and/or daidzein.

[000379] The product of the present invention can be used in the form of the solution as produced in the Third Production Method, or it can be a tetrahydrodaidzein product obtained crudely or appropriately by refining the solution.

[000380] The product of the present invention can be incorporated into food, drinks, or makeup product materials, medicinal products, etc., or can be used as a substrate.

D-I-7. Process for producing dihydrodaidzein, tetrahydrodaidzein and/or equol, comprising at least two from the Fourth Step to the Sixth Step

[000381] The present invention provides a process for producing dihydrodaidzein, tetrahydrodaidzein, and/or equol, comprising at least two of the following Fourth Step to Sixth Step (hereinafter, this process is occasionally referred to as "Fourth Production Method"). The Fourth Step produces dihydrodaidzein from daidzein. The Fifth Step produces tetrahydrodaidzein from dihydrodaidzein, and the Sixth Step produces equol from tetrahydrodaidzein.

[000382] The Fourth Step comprises a step of having a recombinant cell consisting of one of the following (Ad) to (Af) polynucleotides acting on daidzein, thereby producing dihydrodaidzein. (Ad) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4; (Ae) a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1; and (Af) a polynucleotide that hybridizes under stringent conditions with the complementary strand of the polynucleotide (Ad) or (Ae), and that encodes a polypeptide having an activity to synthesize dihydrodaidzein using daidzein as a substrate.

[000383] The Fifth Step comprises a step of having a recombinant cell consisting of one of the following (Bd) to (Bf) polynucleotides acting on dihydrodaidzein, thereby producing tetrahydrodaidzein. (Bd) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 10; (Be) a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 7; and (Bf) a polynucleotide that hybridizes under stringent conditions with the complementary strand of the polynucleotide (Bd) or (Be), and that encodes a polypeptide having an activity to synthesize tetrahydrodaidzein using dihydrodaidzein as a substrate.

[000384] The Sixth Step comprises a step of having a recombinant cell consisting of one of the following (Cd) to (Cf) polynucleotides acting on tetrahydrodaidzein, thereby producing equol. (Cd) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 16; (Ce) a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 13; and (Cf) a polynucleotide that hybridizes under stringent conditions with the complementary strand of the polynucleotide (Cd) or (Ce), and that encodes a polypeptide having an activity to synthesize equol using tetrahydrodaidzein as a substrate.

[000385] The Fourth Production Method of the present invention produces dihydrodaidzein, tetrahydrodaidzein and/or equol by appropriately combining the Fourth to Sixth Steps.

[000386] The Fourth Production Method of the present invention comprises at least two Fourth Steps, Fifth Steps and Sixth Steps.

[000387] For example, the Fourth Production Method of the present invention that comprises the Fourth Step and Fifth Step and does not comprise the Sixth Step produces dihydrodaidzein from daidzein, and tetrahydrodaidzein from dihydrodaidzein.

[000388] For example, the Fourth Production Method of the present invention that comprises Fifth Step and Sixth Step and does not comprise the Fourth Step produces tetrahydrodaidzein from dihydrodaidzein, and equol from tetrahydrodaidzein.

[000389] And, for example, the Fourth Production Method of the present invention which comprises the entirety of the Fourth Step, Fifth Step and Sixth Step produces dihydrodaidzein from daidzein, tetrahydrodaidzein from dihydrodaidzein, and tetrahydrodaidzein equol.

[000390] Each recombinant cell used for the Fourth Production Method of the present invention may contain one or more polynucleotides selected from the group consisting of (Ad) to (Af), (Bd) to (Bf) and (Cd) to ( Cf).

[000391] For example, when the cell contains two polynucleotides: a polynucleotide selected from the group consisting of (Ad) to (Af), and a polynucleotide selected from the group consisting of (Bd) to (Bf), the Fourth Step and Fifth Step can be performed with this cell, that is, the Fourth Step and Fifth Step can be performed with a recombinant cell type.

[000392] Similarly, when the cell contains three polynucleotides: a polynucleotide selected from the group consisting of (Ad) to (Af), a polynucleotide selected from the group consisting of (Bd) to (Bf), and a polynucleotide selected from the group consisting of (Cd) to (Cf), the Fourth Step to the Sixth Step can be performed with this cell, that is, the Fourth Step to the Sixth Step can be performed with a recombinant cell type.

[000393] In the Fourth Production Method of the present invention, the Fourth Step to the Sixth Step can also be carried out using a recombinant cell containing two polynucleotides: a polynucleotide selected from the group consisting of (Ad) to (Af), a polynucleotide cleotide selected from the group consisting of (Bd) to (Bf); and another recombinant cell containing a polynucleotide selected from the group consisting of (Cd) to (Cf).

[000394] Similarly, in the Fourth Production Method of the present invention, the Fourth Step to Sixth Step can also be carried out using a recombinant cell containing two polynucleotides: A polynucleotide selected from the group consisting of (Ad) to (Af), a polynucleotide selected from the group consisting of (Cd) to (Cf); and another recombinant cell containing a polynucleotide selected from the group consisting of (Bd) to (Bf).

Fourth Stage

[000395] The Fourth Step induces a recombinant cell having the E1 polynucleotide as detailed in section A-2 to act on daidzein, thereby converting daidzein into dihydrodaidzein.

[000396] The reaction in the Fourth Step can be carried out under the same conditions as those in the First Step. More specifically, the recombinant cell is inoculated into the culture medium containing 0.001 to 1% by weight, preferably 0.01 to 1% by weight, more preferably 0.01 to 0.5% by weight of daidzein, and incubated for 6 to 30 hours, preferably 7 to 24 hours, more preferably 7 to 18 hours at a temperature allowing cell development. The temperature is not limited, and can be the temperature adopted in the First Stage.

Recombinant cell used in the Fourth Step

[000397] Any recombinant cell can be used in the Fourth Step since the cell contains the E1 polynucleotide and is capable of expressing the E1 polynucleotide. The recombinant cells detailed in section A-4 can be used in the step.

Production of Enzyme Consisting of E1 Polypeptide Using Recombinant Cell Possessing E1 Polynucleotide

[000398] The enzyme consisting of the E1 polypeptide can be produced by culturing the recombinant cell after introducing the E2 polynucleotide and collecting the E1 polypeptide from the cell culture. More specifically, the E1 polypeptide can be produced by culturing the recombinant cell under the conditions detailed in section A-5.

Fifth Stage

[000399] The Fifth Step induces a recombinant cell having the E2 polynucleotide as detailed in section B-2 to act on dihydrodaidzein, thereby converting dihydrodaidzein into tetrahydrodaidzein.

[000400] The reaction in the Fifth Step can be carried out under the same conditions as those in the Second Step. More specifically, the recombinant cell is inoculated into the culture medium containing 0.001 to 1% by weight, preferably 0.01 to 0.5% by weight, more preferably 0.01 to 0.1% by weight daidzein, and incubated for 7 to 30 hours, preferably 15 to 24 hours, more preferably 17 to 20 hours at a temperature allowing cell development. The temperature is not limited, and can be the temperature adopted in the Second Stage.

Recombinant cell used in Fifth Stage

[000401] Any recombinant cell can be used in the Fifth Step since the cell contains the E2 polynucleotide described in section B-2 and is capable of expressing the E2 polynucleotide. An example is a recombinant cell detailed in section B-4.

Production of Enzyme Consisting of E2 Polypeptide Using Recombinant Cell Possessing E2 Polynucleotide

[000402] The enzyme consisting of the E2 polypeptide can be produced by culturing the recombinant cell after introducing the E2 polynucleotide and by collecting the E2 polypeptide from the cells and/or culture.

[000403] The same ways and conditions as those in the section "Production of enzyme consisting of E1 polypeptide using recombinant cell having E1 polynucleotide" can be used for incubation of recombinant cell provided with E2 polynucleotide, culture medium, and isolation/purification of E2 polypeptide.

Sixth Stage

[000404] The Sixth Step induces a recombinant cell having the E3 polynucleotide as detailed in section C-2 to act on tetrahydrodaidzein, thereby converting tetrahydrodaidzein into equol.

[000405] The reaction in the Sixth Step can be carried out under the same conditions as those in the Third Step. More specifically, the recombinant cell is inoculated into the culture medium containing 0.001 to 1% by weight, preferably 0.01 to 0.5% by weight, more preferably 0.01 to 0.1% by weight of tetrahydrodaidzein, and incubated for 7 to 30 hours, preferably 15 to 24 hours, more preferably 17 to 20 hours at a temperature allowing cell development. The temperature is not limited, and may be the temperature adopted in the Third Stage.

[000406] The source of tetrahydrodaidzein used as a substrate in the Sixth Step is also not limited.

[000407] For example, tetrahydrodaidzein produced in the Fifth Step of dihydrodaidzein can be used as a substrate for Sixth Step. Tetrahydrodaidzein is used either in the form of a solution containing tetrahydrodaidzein as produced in the Fifth Step, or in a crudely refined state or appropriately refined state.

[000408] Commercially available tetrahydrodaidzeins or tetrahydrodaidzeins synthesized by appropriate methods can also be used.

[000409] Furthermore, the synthesis of tetrahydrodaidzein material composition containing recombinant cells containing E3 polynucleotide; and tetrahydrodaidzein can be used as a mixture of starting materials in the production of tetrahydrodaidzein in the Sixth Step. By incubating the composition under the above conditions, the tetrahydrodaidzein in the composition is converted to equol. The same conditions and restrictions as above are applied to determine the concentrations of the recombinant cells and tetrahydrodaidzein in the composition, other ingredients added to the composition, reaction condition, etc.

[000410] In the Sixth Step, it is preferable to use tetrahydrodaidzein, produced in the Fifth Step using dihydrodaidzein as a substrate; however, commercially available tetrahydrodaidzein, a synthetic starting material composition, an enzyme composition, etc. can be mixed with the tetrahydrodaidzein produced in the Fifth Stage.

Recombinant cell used in the Sixth Step

[000411] Any recombinant cell can be used in the Sixth Step since the cell contains the E3 polynucleotide described in section C-2 and is capable of expressing the E3 polynucleotide. An example is a recombinant cell detailed in section C-4.

Production of Enzyme Consisting of E3 Polypeptide Using Recombinant Cell Possessing E3 Polynucleotide

[000412] The enzyme consisting of the E3 polypeptide can be produced by culturing the recombinant cell after introducing the E3 polynucleotide and by collecting the E3 polypeptide from the cells and/or culture.

[000413] The same ways and conditions as those in the section "Production of enzyme consisting of E1 polypeptide using recombinant cell having E1 polynucleotide" can be used for incubation of recombinant cell provided with E3 polynucleotide, culture medium, and isolation/purification of E3 polypeptide.

E1 to E3 polynucleotides

[000414] The Fourth Production Method of the present invention uses at least one of E1 to E3 polynucleotides, which are detailed in the previous sections A-2, B-2 and C-2, respectively.

D-I-8. Product containing dihydrodaidzein, tetrahydrodaidzein and/or equol, produced by the Fourth Production Method

[000415] The present invention provides a product containing dihydrodaidzein, tetrahydrodaidzein and/or equol, produced by the preceding Fourth Production Method.

[000416] As described above, the Fourth Production Method of the present invention allows production of dihydrodaidzein, tetrahydrodaidzein, and/or equol by appropriately combining the Fourth to Sixth Steps.

[000417] Consequently, the product produced by the Fourth Production Method of the present invention contains dihydrodaidzein, tetrahydrodaidzein, and/or equol.

[000418] The product of the present invention can be used in the form of the solution as produced in the Fourth Production Method, or it can be a dihydrodaidzein product, a tetrahydrodaidzein product or an equol product obtained by coarsely or suitably purifying the solution.

[000419] The product of the present invention can be incorporated into food, drinks, or makeup product materials, medicinal products, etc., or can be used as a substrate.

D-II-1. A device for producing dihydrodaidzein, tetrahydrodaidzein and/or equol, provided with at least one of the First Reaction Vessel to the Third Reaction Vessel

[000420] The present invention provides a device for producing dihydrodaidzein, tetrahydrodaidzein and/or equol, provided with at least one of the following First Reaction Vessel and Third Reaction Vessel (hereinafter, this device is occasionally referred to as "First Reaction Device" Production ").

First Reaction Vessel

[000421] The First Reaction Vessel is a reaction vessel having a reaction medium (hereinafter, this medium is occasionally referred to as "reaction medium 1") in which an enzyme consisting of one of (Aa) to (Ac) polypeptides (E1 polypeptides) is immobilized. This reaction vessel is used for production of dihydrodaidzein from daidzein using the polypeptide. In this reaction vessel, a reaction medium is arranged in a position allowing contact with the daidzein.

Second Reaction Vessel

[000422] The Second Reaction Vessel is a reaction vessel having a reaction medium (hereinafter, this medium is occasionally referred to as "reaction medium 2") in which an enzyme consisting of one of (Ba) to (Bc) polypeptides (E2 polypeptides) is immobilized. This reaction vessel is used for production of tetrahydrodaidzein from dihydrodaidzein using the polypeptide. In a reaction vessel, a reaction medium is arranged in a position allowing contact with the dihydro-daidzein.

Third Reaction Vessel

[000423] The Third Reaction Vessel is a reaction vessel having a reaction medium (hereinafter, this medium is occasionally referred to as "reaction medium 3") in which an enzyme consisting of one of (Ca) to (Cc) polypeptides (E3 polypeptides) is immobilized. This reaction vessel is used for production of tetrahydrodaidzein equol using the polypeptide. In a reaction vessel, a reaction medium is arranged in a position allowing contact with the tetrahydrodaidzein.

[000424] The First Production Device of the present invention allows production of dihydrodaidzein, tetrahydrodaidzein and/or equol by appropriately combining the First to Third Reaction Vessels.

[000425] The First Production Device of the present invention has at least one of the First Reaction Vessel, Second Reaction Vessel and Third Reaction Vessel.

[000426] For example, when the First Production Device of the present invention has the First Reaction Vessel, and does not have the Second Reaction Vessel and Third Reaction Vessel, the First Production Device of the present invention allows production of dihydrodaidzein of daidzein.

[000427] For example, when the First Production Device of the present invention has the Second Reaction Vessel, and does not have the First Reaction Vessel and Third Reaction Vessel, the First Production Device of the present invention allows production of tetrahydrodaidzein of dihydrodaidzein.

[000428] For example, when the First Production Device of the present invention has the Third Reaction Vessel, and does not have the First Reaction Vessel and Second Reaction Vessel, the First Production Device of the present invention allows production of tetrahydrodaidzein equol .

[000429] For example, when the First Production Device of the present invention has the First Reaction Vessel and Second Reaction Vessel, and does not have the Third Reaction Vessel, the First Production Device of the present invention allows production of dihydrodaidzein from daidzein , and dihydrodaidzein tetrahydrodaidzein.

[000430] For example, when the First Production Device of the present invention has the Second Reaction Vessel and Third Reaction Vessel, and does not have the First Reaction Vessel, the First Production Device of the present invention allows production of tetrahydrodaidzein from dihydrodaidzein , and tetrahydrodaidzein equol.

[000431] For example, when the First Production Device of the present invention has the First Reaction Vessel, Second Reaction Vessel, and Third Reaction Vessel, the First Production Device of the present invention allows production of dihydrodaidzein from daidzein, tetrahydrodaidzein from dihydrodaidzein, and tetrahydrodaidzein equol.

[000432] In the First Production Device of the present invention, a single reaction vessel can have two of a reaction medium 1 to 3.

[000433] For example, when reaction medium 1 and 2 are provided in a single reaction vessel, the respective reactions carried out in the First Reaction Vessel and Second Reaction Vessel can be carried out in one reaction vessel. Furthermore, for example, when a reaction medium 1 to 3 are provided in a single reaction vessel, the respective reactions carried out in the First to Third Reaction Vessels can be carried out in one reaction vessel.

[000434] As long as the desired effect is ensured, the way of mounting this reaction medium to a reaction vessel is not limited.

[000435] Furthermore, when the First Production Device of the present invention has at least two of a reaction medium 1 to 3 and multiple different reaction vessels, the reaction vessels are connected through a supply means.

[000436] For example, when the First Production Device of the present invention has the First Reaction Vessel and Second Reaction Vessel, and does not have the Third Reaction Vessel, in which the First Reaction Vessel and Second Reaction Vessel are units independent and the First Reaction Vessel and Second Reaction Vessel contain a reaction resource 1 and 2, respectively; the First Reaction Vessel and Second Reaction Vessel are connected via a supply facility to supply a dihydrodaidzein product, produced in the First Reaction Vessel, to the Second Reaction Vessel.

[000437] Furthermore, for example, when the First Production Device of the present invention comprises a reaction medium 1 and 2 in a reaction vessel, and a reaction medium 3 in another reaction vessel; a reaction vessel containing a reaction medium 1 and 2 and a reaction vessel containing a reaction medium 3 are connected via a supply facility for supplying a product, produced in a reaction vessel containing a reaction medium 1 and 2, to a reaction vessel containing a reaction medium 3.

[000438] The product may be a solution form as produced, or it may be a dihydrodaidzein product etc. obtained by coarsely or adequately purifying the solution.

Reaction Vessel

[000439] The shapes, sizes, materials, etc. from the First Reaction Vessel to the Third Reaction Vessel provided in the First Production Device of the present invention are not limited, as each vessel is capable of containing a reaction medium, and suitably producing dihydrodaidzein, tetrahydrodaidzein and/or equol.

Reaction Medium

[000440] The reaction medium 1 to 3 to be used in the First Production Device of the present invention are not limited since each of them contains a respective immobilized enzyme, and is capable of appropriately producing dihydrodaidzein, tetrahydrodaidzein and/or equol.

[000441] The respective enzyme immobilized in each reaction medium can be crudely or adequately (purely) purified. Immobilization of an enzyme consisting of one of the polypeptides in a reaction medium is carried out using a known method.

[000442] For example, when the enzyme is immobilized on a carrier, the carrier is not limited since the desired activity of an enzyme can be exhibited. Examples of the carrier include a carrier having a functional group connectable with an enzyme through covalent bonding, such as amino groups, carboxyl groups, or hydroxyl groups; and a carrier connectable with an enzyme consisting of the polypeptide through a linker. The shape of the bearer is not limited. The carrier, working group, and connector etc. are appropriately selected according to the known technique adopted for immobilization of an enzyme on the carrier. Immobilization of an enzyme consisting of the polypeptide on the carrier is also carried out using a known method.

Means of Supply

[000443] The supply means provided in the First Production Device of the present invention is not limited since it is capable of connecting multiple different reaction vessels used in the First Production Device of the present invention, and capable of producing dihydrodaidzein, tetrahydrodaidzein and/or equol in the First Production Device of the present invention. The means of delivery is carried out by a suitable means in accordance with a suitable known technology.

E1 to E3 polypeptide

[000444] E1 to E3 polypeptides used in the First Production Device of the present invention are the same as those above.

Production of dihydrodaidzein in the First Reaction Vessel

[000445] The First Reaction Vessel induces an enzyme consisting of the E1 polypeptide immobilized in a reaction medium 1 to act on daidzein in the presence of NADPH and/or NADH, thereby converting daidzein into dihydrodaidzein. The enzyme consisting of the E1 polypeptide can be immobilized in a reaction medium together with NADPH and/or NADH that serves as a coenzyme of an enzyme consisting of the E1 polypeptide. The reaction in the First Reaction Vessel is carried out according to the method detailed in the previous explanation of "First Step".

Production of tetrahydrodaidzein in the Second Reaction Vessel

[000446] The Second Reaction Vessel induces an enzyme consisting of the E2 polypeptide immobilized in a reaction medium 2 to act on dihydrodaidzein in the presence of NADPH and/or NADH, thereby converting dihydrodaidzein into tetrahydrodaidzein. The enzyme consisting of the E2 polypeptide can be immobilized in a reaction medium together with NADPH and/or NADH that serves as a coenzyme of the enzyme consisting of the E2 polypeptide. The reaction in the Second Reaction Vessel is carried out according to the method detailed in the previous explanation of "Second Step".

Equol production in the Third Reaction Vessel

[000447] The Third Reaction Vessel induces an enzyme consisting of the E3 polypeptide immobilized in a reaction medium 3 to act on tetrahydrodaidzein, thereby converting tetrahydrodaidzein into equol. The reaction in the Third Reaction Vessel is carried out according to the method detailed in the previous explanation of "Third Step".

D-II-2. A device for producing dihydrodaidzein, tetrahydrodaidzein and/or equol, comprising at least one of the Fourth to Sixth Reaction Vessels

[000448] The present invention provides a device for producing dihydrodaidzein, tetrahydrodaidzein and/or equol, comprising at least one of the following Fourth to Sixth Reaction Vessels (hereinafter, this device is occasionally referred to as "Second Production Device").

Fourth Reaction Vessel

[000449] The Fourth Reaction Vessel is a reaction vessel having a reaction medium (hereinafter, this medium is occasionally referred to as "reaction medium 4") in which a recombinant cell consisting of one of (Ad) to (Af ) polynucleotides (E1 polynucleotides) is immobilized. This reaction vessel is used for production of dihydrodaidzein from daidzein using the polynucleotide. In this reaction vessel, a reaction medium is arranged in a position allowing contact with daidzein.

Fifth Reaction Vessel

[000450] The Fifth Reaction Vessel is a reaction vessel having a reaction medium (hereinafter, this medium is occasionally referred to as "reaction medium 5") in which a recombinant cell consisting of one of (Bd) to (Bf ) polynucleotides (E2 polynucleotides) is immobilized. This reaction vessel is used for production of tetrahydrodaidzein from dihydrodaidzein using the polynucleotide. In a reaction vessel, a reaction medium is arranged in a position allowing contact with dihydrodaidzein.

Sixth Reaction Vessel

[000451] The Sixth Reaction Vessel is a reaction vessel having a reaction medium (hereinafter, this medium is occasionally referred to as "reaction medium 6") in which a recombinant cell consisting of one of (Cd) to (Cf ) polynucleotides (E3 polynucleotides) is immobilized. This reaction vessel is used for production of tetrahydrodaidzein equol using the polynucleotide. In a reaction vessel, a reaction medium is arranged in a position allowing contact with tetrahydrodaidzein.

[000452] The Second Production Device of the present invention allows production of dihydrodaidzein, tetrahydrodaidzein and/or equol by appropriately combining the Fourth to Sixth Reaction Vessels.

[000453] The Second Production Device of the present invention has at least one of the Fourth Reaction Vessel, Fifth Reaction Vessel and Sixth Reaction Vessel.

[000454] For example, when the Second Production Device of the present invention has a Fourth Reaction Vessel, and does not have the Fifth Reaction Vessel and Sixth Reaction Vessel, the Second Production Device of the present invention allows production of dihydrodaidzein from daidzein.

[000455] For example, when the Second Production Device of the present invention has the Fifth Reaction Vessel, and does not have the Fourth Reaction Vessel and Sixth Reaction Vessel, the Second Production Device of the present invention allows production of tetrahydrodaidzein of dihydrodaidzein.

[000456] For example, when the Second Production Device of the present invention has the Sixth Reaction Vessel, and does not have the Fourth Reaction Vessel and Fifth Reaction Vessel, the Second Production Device of the present invention allows production of tetrahydrodaidzein equol .

[000457] For example, when the Second Production Device of the present invention has the Fourth Reaction Vessel and Fifth Reaction Vessel, and does not have the Sixth Reaction Vessel, the Second Production Device of the present invention allows production of dihydrodaidzein. from daidzein, and tetrahydrodaidzein from dihydrodaidzein.

[000458] For example, when the Second Production Device of the present invention has the Fifth Reaction Vessel and Sixth Reaction Vessel, and does not have the Fourth Reaction Vessel, the Second Production Device of the present invention allows production of tetrahydrodaidzein of dihydrodaidzein, and equol of tetrahydrodaidzein.

[000459] For example, when the Second Production Device of the present invention has the Fourth Reaction Vessel, Fifth Reaction Vessel, and Sixth Reaction Vessel, the Second Production Device of the present invention allows production of dihydrodaidzein from daidzein, tetrahydrodaidzein from dihydrodaidzein, and tetrahydrodaidzein equol.

[000460] In the Second Production Device of the present invention, a single reaction vessel can have two of a reaction medium 4 to 6.

[000461] For example, when reaction medium 4 and 5 are provided in a single reaction vessel, the respective reactions carried out in the Fourth Reaction Vessel and Fifth Reaction Vessel can be carried out in one reaction vessel. Furthermore, for example, when a reaction medium 4 to 6 are provided in a single reaction vessel, the respective reactions carried out in the Fourth to Sixth Reaction Vessels can be carried out in one reaction vessel.

[000462] Insofar as the desired effect is ensured, the way of mounting this reaction medium to a reaction vessel is not limited.

[000463] Furthermore, when the Second Production Device of the present invention has at least two of one of the reaction means 4 to 6 and multiple different reaction vessels, the reaction vessels are connected through a supply means.

[000464] For example, when the Second Production Device of the present invention has the Fourth Reaction Vessel and Fifth Reaction Vessel, and does not have the Sixth Reaction Vessel, wherein the Fourth Reaction Vessel and the Fifth Reaction Vessel are independent units and the Fourth Reaction Vessel and Fifth Reaction Vessel contain a 4 and 5 reaction resource, respectively; the Fourth Reaction Vessel and Fifth Reaction Vessel are connected via a supply facility to supply a dihydrodaidzein product, produced in the Fourth Reaction Vessel, to the Fifth Reaction Vessel.

[000465] Furthermore, for example, when the second production device of the present invention comprises reaction methods 4 and 5 in one reaction vessel, and reaction methods 6 in another reaction vessel; the reaction vessel containing reaction methods 4 and 5 and the reaction vessel containing reaction methods 6 are connected by a supply means to supply a product, produced in the reaction vessel containing reaction methods 4 and 5, to the reaction vessel containing reaction methods 6.

[000466] The product may be a solution form when produced, or it may be a dihydrodaidzein product, etc. obtained roughly or appropriately by purifying the solution.

Reaction vessel

[000467] The shapes, sizes, materials, etc. from the Fourth Reaction Vessel to the Sixth Reaction Vessel provided in the Second Production Device of the present invention are not limited, provided that each vessel is capable of containing a reaction medium, and appropriately producing dihydrodaidzein, tetrahydrodaidzein and/or equol.

Means of Reaction

[000468] Reaction media 4 to 6 to be used in the Second Production Device of the present invention are not limited as long as each contains a respective immobilized recombinant cell, and is capable of appropriately producing dihydrodaidzein, tetrahydrodaidzein and/or equol.

[000469] Immobilization of the recombinant cell in reaction methods is not limited as long as the desired activity of the recombinant cell can be exhibited, and carried out using a known method. The recombinant cell can be incubated or incubated in the reaction media. The conditions for incubating the recombinant cells are determined according to the previous explanations of the "Fourth Step", "First Step", and "Sixth Step".

Means of Supply

[000470] The supply means provided in the Second Production Device of the present invention are not limited as long as they are capable of connecting different plural reaction vessels used in the Second Production Device of the present invention, and capable of producing dihydrodaidzein, tetrahydrodaidzein and/or equol in the Second Production Device of the present invention. The supply means are realized by a suitable means according to a suitable known technology.

Recombinant Cells

[000471] The recombinant cells used in the Second Production Device of the present invention are the same as those detailed in the previous sections "Recombinant Cells used in the Fourth Step", "Recombinant Cell used in the First Step", and "Recombinant Cell used in the Sixth Step ".

E1 to E3 polypeptide

[000472] The E1 to E3 polynucleotides used in the Second Production Device of the present invention are the same as those above.

Dihydrodaidzein production in the fourth reaction vessel

[000473] The fourth reaction vessel converts daidzein to dihydrodaidzein using a recombinant cell consisting of immobilized E1 polynucleotide in reaction methods 4. The reaction in the fourth reaction vessel is carried out according to the method detailed in the previous explanation of "Fourth Step".

Production of tetrahydrodaidzein in the Fifth reaction vessel

[000474] The Fifth reaction vessel converts dihydrodaidzein to tetrahydrodaidzein using a recombinant cell consisting of E2 polynucleotide immobilized in reaction methods 5. The reaction in the Fifth reaction vessel is carried out according to the method detailed in the previous explanation of " First step".

Equol production in the sixth reaction vessel

[000475] The Sixth reaction vessel converts tetrahydrodaidzein to equol using a recombinant cell consisting of E3 polynucleotide immobilized in reaction methods 6. The reaction in the Sixth reaction vessel is carried out according to the method detailed in the previous explanation of " Sixth Stage".

D-II-3. Device for producing dihydrodaidzein, tetrahydrodaidzein and/or equol, comprising at least one of the First to Third reaction vessels, and at least one of the Fourth to Sixth reaction vessels

[000476] The present invention provides a device for producing dihydrodaidzein, tetrahydrodaidzein and/or equol, comprising at least one of the First to Third reaction vessels, and at least one of the Fourth to Sixth reaction vessels (hereinafter, this device is occasionally referred to as the "Third Production Device").

[000477] The combinations of reaction vessels, reaction media in the Third Production Device are made in accordance with those of the previous First and Second Production Devices. The reaction vessels, reaction media, polynucleotides, recombinant cells, and reaction processes in the reaction vessels in the Third Production Device are the same as those for the previous First and Second Production Devices.

Examples Example A Reference example A1

[000478] A Lactococcus 20-92 strain (FERM BP-10036) was inoculated into a liquid amplification medium containing daidzein (a modified GAM broth medium (Nissui Pharmaceutical Co. Ltd.) into which daidzein was added in an amount of 10 μg/mL), and the culture was incubated at 37°C for appropriate hours of 7 to 18 hours under anaerobic conditions (BBL Gas Pack systems). After incubation, cells were collected by centrifugation and cryogenically preserved for use in the Examples below.

Example A1: Confirmation of Dihydrodaidzein Biosynthesis Activity in the Centrifuged Supernatant of Disrupted Cellular Material, and Confirmation of NADPH Dependence

[000479] The frozen cells (67mL x 2 bottle) were thawed, and centrifuged at 4°C at 8,000rpm for 10 minutes. The pellet was used in the test below. First, the pellet was suspended in 2 ml of a 0.1 M potassium phosphate solution containing 1 mM PMSF (Wako Pure Chemical Industries, Ltd.), 2 mM DTT (Wako Pure Chemical Industries, Ltd.) and sodium hydrosulfide. 5mM sodium (Wako Pure Chemical Industries, Ltd.). A suspension was supplied to two 2ml screw cap tubes (Assist) and pre-supplemented with 0.1mM zirconia/silica beads (BioSpec Products, Inc.). Cells were disrupted using FastPrep® FP100A (Thermo ELECTRON CORPORATION) (6500rpm, 10 seconds, ice cooling x8). The resulting liquid was centrifuged to obtain the supernatant. The disrupted cell suspension was centrifuged at about 10,000 rpm at 4°C for 10 minutes to obtain a supernatant, which was then diluted to 4.5 mL with 0.1 M potassium phosphate solution containing PMSF at 1 mM, 2 mM DTT and 5 mM sodium hydrosulfide. This was used as an enzyme source.

[000480] An enzyme reaction mixture of the composition below was prepared, and the mixture was incubated at 37°C for 2 hours. After incubation, 3 mL of ethyl acetate was added to the enzyme reaction mixture for extraction. After drying, the extract was dissolved by the mobile phase (eluent). The dissolved product was analyzed by HPLC. As the standard solution for HPLC Analysis, a mixed solution of daidzein (2 μg/mL; Funakoshi Corporation), equol (2 μg/mL; Funakoshi Corporation), dihydrodaidzein (2 μg/mL; Toronto Research Chemicals Inc.) was used . Composition of Enzyme Reaction Mixture Disrupted cell supernatant (enzyme source): 250 μl Sterile water, NADH (100mM) or NADPH (100mM): 5 μl Dihydrodaidzein (1 mg/ml): 10 μl Potassium phosphate buffer a 0.1 M pH 7/ 1mM DTT / 5mM sodium hydrosulfide: 735 μl Total: 1,000 μl

[000481] The results are shown in FIG. 1. The results confirmed the presence of dihydrodaidzein biosynthesis activity in the centrifuged supernatant of the disrupted cellular material. It was also confirmed that the conversion of daidzein to dihydrodaidzein is dependent on the coenzyme NADH.

Example A2: Purification of Dihydrodaidzein Synthesizing Enzyme

[000482] Lactococcus strain 20-92 cells were incubated for 18 hours in a modified GAM broth medium (Nissui Pharmaceutical Co. Ltd.), 67 ml per incubation bottle. Cultured cells from nine such bottles were centrifuged, and suspended in a 0.1 M potassium phosphate buffer (pH 7) containing 2 mM DTT (1,4-Dithiothreitol, Merck), 5 mM sodium hydrosulfide (Wako Pure Chemical Industries, Ltd.) and proteinase inhibitor (EDTA-free complete proteinase inhibitor cocktail, Roche Diagnostics). The suspension was supplied to three 2 ml tubes with screw caps (Assist) which previously contained 0.1 mM zirconia/silica beads (BioS- pec Products, Inc.). Cells were disrupted using FastPrep® FP100A (Thermo ELECTRON CORPORATION) (6500rpm, 20 seconds x 4). The resulting liquid was centrifuged to obtain the supernatant. Lactococcus 20-92 strain cells were incubated for 18 hours in the same liquid medium, 200 ml per incubation bottle. Cells cultured from eight such bottles were centrifuged to obtain the supernatant equivalent to eight bottles.

[000483] Purification was carried out using a 0.1 M potassium phosphate buffer (pH 7.0) ("Buffer A", below) containing 1mM PMSF (phenylmethylsulfonyl fluoride, Sigma Aldrich), sodium hydrosulfide at 2 mM DTT and 5 mM. The disrupted supernatant of the cells was mixed with Buffer A containing the same amount of 2 M ammonium sulfate, and placed on columns in micro bio spin columns (x11, Bio-Red Laboratories, Inc.) filled with fast-flow butyl sepha- rose 4 (gel was about 0.3 ml per bottle, GE Healthcare) and equilibrated with Buffer A containing 1 M ammonium sulfate. After being washed with Buffer A containing 1 M ammonium sulfate and rinsed twice subsequently with 0.75 ml of Buffer A containing 0.5 M ammonium sulfate, 0.75 ml and then 0.5 ml of Buffer A was added to elute a fraction having a dihydrodaidzein synthesis activity. In the following, the eluate obtained by adding 0.75 ml of Buffer A is referred to as an eluate I, and the eluate obtained by adding 0.5 ml of Buffer A is referred to as an eluate II.

[000484] 0.3 ml and 0.2 ml of Buffer A containing 3.4M ammonium sulfate were added to eluate I (with 0.75 ml of Buffer A) and eluate II (with 0.5 ml of Buffer A), respectively. Then, the two eluates were mixed to be subjected to HPLC using TSKgel Ether-5PW (TOSOH) column equilibrated with an eluent containing 1 M ammonium sulfate. 0.5 ml of the mixed solution was poured into the column 2 to 9 times , followed by washing at a flow rate of 0.1 ml/min using an eluent containing 1 M ammonium sulfate (Eluent B). Next, the mixing ratio of Eluent B to Eluent A was changed using a program so that the ammonium sulfate concentration linearly decreased to 0M (Eluent A) in 15 minutes. Enzymatic activity was observed at the elution position of ammonium sulfate at about 0.6 M to 0.35 M, and the total eluate was about 3.5 ml. The following shows the conditions in HPLC. Protein absorption at 280 nm was observed. Column: TSKgel Ether-5PW Flow Rate: 0.05 to 0.1 ml/min in sample supply, 0.1 ml/min in elution Eluent A: 0.1 M potassium phosphate buffer (pH 7, 0)/ 2mM DTT / 2.5mM sodium hydrosulfide / 1% 2-propanol Eluent B: Eluent A containing 1M ammonium sulfate

[000485] The eluate from the elution position where enzymatic activity was observed was diluted with Buffer A, and the diluted liquid was subjected to the microspinning column loaded with 2'5' ADP Sepharose 4B (GE Healthcare; the gel was about 0. 3 ml per bottle), equilibrated with Buffer A. After washing the column with Buffer A, the fraction having dihydrodaidzein synthesis activity was eluted with 0.7 ml, and then 0.6 ml of liquid from the Buffer composition A of pH 7.5 containing 20 mM NADPH.

[000486] The eluate obtained was subjected to HPLC using a Mono Q column (GE Healthcare) equilibrated with Eluent C (pH 7.5). Eluent C was supplied to the column at a flow rate = 0.1 ml/min. After washing, the mixing ratio of Eluent C to Eluent D was changed to run a program to linearly convert to 0.65 M NaCl over 32.5 minutes. The following shows the conditions in HPLC. Protein absorption at 280 nm was observed.column: Mono Q PC 1.6/5 Eluent C: 0.1M potassium phosphate buffer pH 7.5/3mM DTT/2.5mM sodium hydrosulfide/1% of 2-propanol Eluent D: Eluent C containing 1 M NaCl

[000487] Enzymatic activity was observed in a 0.4 to 0.46 M NaCl fraction (fraction no. 28 to 30).

[000488] Fig. 2 shows the result of Mono Q HPLC and enzymatic activity. Fig. 3 shows the SDS-PAGE result for fractions Nos. 27 to 31. According to these results, a band of 70 kDa was observed in the fraction having a dihydrodaidzein synthesis activity under reducing condition.

Example A3: Determination of the N-terminal amino acid sequence

[000489] 30 μl of 0.1% trifluoroacetic acid (TFA) was added to 70μl of fraction, no. 29 having a dihydrodaidzein synthesis activity obtained by Mono Q HPLC (100μl in total).

[000490] A ProSorb cartridge PVDF membrane (Applied Biosystems Japan) was moistened with 10 μl of methanol, and the preceding mixed liquid was added. After water absorption using a ProSorb filter (Applied Biosystems Japan), the membrane was dried, and then cut using a membrane punch (Applied Biosystems Japan). The membrane was washed five times with 20% methanol and dried. The membrane was subjected to N-terminal amino acid sequence analysis using a protein sequencer (Applied Biosystems, Procise 494cLC), thereby finding a continuous amino acid sequence having the following 22 residues.Met Lys Asn Lys Phe Tyr Pro Lys Thr Phe Glu Arg Gly Tyr Ile Gly Asn Leu Glu Val Glu Asn (SEQ ID NO: 19)

Example A4: Determination of amino acid sequence

[000491] The enzyme protein exhibiting a dihydrodaidzein synthesis activity was fragmented using a digestive enzyme into a peptide. The internal amino acid sequence was found by analyzing the amino acid sequence at the N-Terminal of the peptide.

(1) Sample preparation

[000492] Lactococcus 20-92 strain cells were incubated for 18 hours in a modified GAM broth medium (Nissui Pharmaceutical Co. Ltd.), 200 ml per culture bottle. Three strains of the cultured cells were centrifuged and suspended in a 0.1 M potassium phosphate buffer (pH 7.0) containing 2 mM DTT (1,4-Dithiothreitol, Merck), 5 mM sodium hydrosulfide (Wako Pure Chemical Industries, Ltd.) and proteinase inhibitor (Complete Proteinase Inhibitor Cocktail EDTA-Free, Roche Diagnostics). The suspension was then transferred to a 2 ml tube (Assist) that has a screw cap and contains 0.1 mM zirconia/silica beads (BioSpec Pro-ducts, Inc.). Cells were disrupted using FastPrep® FP100A (Thermo ELECTRON CORPORATION) (6500 rpm, 20 seconds x4). The resulting liquid was centrifuged to obtain the supernatant.

[000493] A 0.1 M potassium phosphate buffer (pH 7) ("Buffer A", below) containing 1 mM PMSF (phenylmethylsulfonyl fluoride, Sigma Aldrich), 2 mM DTT and sodium hydrosulfide 5 mM was used in the following process. The disrupted cell supernatant was mixed with Buffer A containing the equivalent amount of 2 M ammonium sulfate, and placed on a micro bio spin column (x3, Bio-Red Laboratories, Inc.) loaded with Fast Flow Butyl Sepharose 4 (the gel was about 0.3 ml per bottle, GE Healthcare), equilibrated with Buffer A containing 1 M ammonium sulfate. After being washed with Buffer A containing 1 M ammonium sulfate and subsequently rinsed twice with 0.75 ml of Buffer A containing 0.5 M ammonium sulfate, 0.75 ml of Buffer A was added twice to elute a fraction having a dihydrodaidzein synthesis activity.

[000494] The micro bio spinning column loaded with 2'5'ADP Sepharose 4B was equilibrated with Buffer A, and then 1.5 ml of the previously eluted solution was added. After five hours of washing with 0.75 ml of Buffer A, elution was carried out using 0.75 ml, and then 0.45 ml of Buffer A containing 20 mM NADPH. Eluates were mixed and desalted and concentrated to 5 μl in a microcentrifuge concentration tube (NANOSEP 10K OMEGA, Pall Life Sciences).

[000495] The concentrated solution was mixed with a double concentrated SDS-PAGE sample buffer containing 2-mercaptoethanol; and the mixture was heated for 7 minutes at 90°C before being subjected to SDS-PAGE according to the Laemmuli method. SuperSep HG 10 to 20% (Wako Pure Chemical Industries, Ltd.) was used as an electrophoresis gel plate. After staining with Colloidal Blue (Invitrogen) and subsequent destaining with Milli-Q water, an electrophoresis band at 70 kDa was excised. As a control, an equivalent and equimolar sized gel was cut from the same portion in a lane in the non-protein electrophoresis area, and processed in the same manner. Another group of cells was processed in the same way, and the cells were transferred to a PVDF membrane after SDS-PAGE. Next, a band at the same position was subjected to N-terminal amino acid sequence analysis to confirm consistency with the sequence of fraction #29 on Mono Q HPLC.

(2) Generation of reduced enzyme and carboxamidemethyl digestion

[000496] The excised gels were cut into pieces, decolorized using a 50% aqueous solution of acetonitrile, dehydrated by acetonitrile, and dried with a centrifugal thickener (SpeedVac A160, Savant). After 100 mM aqueous ammonium hydrogencarbonate solution containing 55 mM DTT was added, the reduction was carried out for one hour at 56 ° C. After removing a DTT solution, a 100 mM aqueous ammonium hydrogencarbonate solution containing 100 mM iodoacetamide was added, and the light-blocked mixture was gently stirred for 30 minutes to generate carboxamidemethyl. After removing the reaction reagent, the gel was washed sequentially with a 50% aqueous solution of acetonitrile, acetonitrile, a 100 mM aqueous solution of ammonium hydrogen carbonate, and acetonitrile, before being dried in a centrifugal thickener. 20 mM Tris-HCL buffer (pH 9) containing 2 μg of Achromobacter protease I (Wako Pure Chemical Industries, Ltd.) and 0.02% Tween 20 was added, and digestion was carried out at 37°C for seven hours. The centrifuged supernatant was transferred to another tube, and the gel was mixed with 60% acetonitrile - 0.1% TFA aqueous solution and heated at 30°C for 20 minutes. The heated mixture was centrifuged for 10 minutes x three times to obtain peptide fragments. The supernatant was collected to be filtered using Ultrafree-MC (0.22 μm, Amicon), followed by centrifugal concentration.

(3) Peptide Mapping

[000497] Using reversed-phase HPLC, the peptide resulting from enzyme digestion was isolated. Column: μRPC C2/C18 SC2.1/10 (GE Healthcare Bio Science) Flow Rate: 0.1 ml/min Eluent E: 0.05% TFA Eluent F: 90% acetonitrile/0.04% TFA Elution Program: 0 minute: 5% F 3 minutes: 5% F 43 minutes: 65% F 48 minutes: 100% F 68 minutes: 100% F Fraction: 30 μl Detection: 215 nm

[000498] For example, "0 minute 5% B" means use of an Eluent containing Eluent E and Eluent F in an amount of 95% and 5%, respectively, at time 0 in elution.

(4) Internal amino acid sequence analysis

[000499] In comparison with the control chromatogram, an enzyme-protein derived peptide peak exhibiting dihydrodaidzein synthesis activity was selected, and N-terminal amino acid sequence analysis was performed using a protein sequencer ( Applied Biosystems, Process 492HT). After separation using reversed-phase HPLC was initiated, elution was initiated in 20.6 minutes. The following shows the amino acid sequence (Peptide 1, below) of the elution peak. Phe Asp Glu Pro Val Tyr Pro Gln Ala Glu (SEQ ID NO: 20)

[000500] The following shows the amino acid sequence (Peptide 2, below) of the elution peak that started at 22.1 minutes. Ala Ser Arg Met Val Met Asp Ala Val His Glu Gly Tyr Ile Ala Gly (SEQ ID NO :21)

[000501] The elution peak that started at 26.6 minutes was a peptide that was not specifically removed; however, as shown below, its N-terminus had glycine which is the 13th residue from the N-Terminal of said enzyme protein (the following amino acid sequence is called Peptide 3, below).Gly Tyr Ile Gly Asn Leu Glu Val Glu Asn Arg Ala Ile Arg Met Pro Met (SEQ ID NO: 22)

[000502] Figure 4 shows the result of peptide mapping in example A4, and amino acid sequences corresponding to the respective peaks. Peak 1 (20.6 minutes) Peak 2 (22.1 minutes) Peak 3 (26.6 minutes)

Example A5: Amplification of N-Terminal dihydrodaidzein synthesis enzyme gene and partial amino acid sequences of purified polypeptide

[000503] Based on the N-Terminal and partial amino acid sequences obtained in Examples A3 and A4, a degenerative primer was designed and created. Using the genome DNA of the Lactococcus 20-92 strain as a template, amplification of the gene encoding the dihydrodaidzein synthesis enzyme was attempted via degenerative PCR.

(1) Purification of Lactococcus 20-92 strain genome DNA

[000504] The Lactococcus 20-92 strain incubated in a 40 mL modified GAM broth culture medium (Nissui Pharmaceutical Co. Ltd.) under an anaerobic condition was centrifuged for ten minutes at 5000 rpm, 4°C. The culture medium was removed by decantation, and the bacterial cells were collected. Cells were immediately suspended in 11 mL of B1 solution (containing 200 μg/mL RNase) in the QIAGEN Genomic DNA Buffer Set (Qiagen), and incubated for 16 hours at 37°C after being mixed with 300 μL of RNase solution. Lysozyme (100 mg/mL), and 500 μL of QIAGEN Proteinase K solution (Qiagen). Then, after being mixed with 4mL of solution B2 and mixing thoroughly several times, a three-hour incubation was carried out at 50°C.

[000505] The culture was centrifuged for ten minutes at 5000 rpm, 4°C. The supernatant was poured onto a QIAGEN Genomic-tip 500/G column (Qiagen) equilibrated with QBT solution so that the genomic DNA adhered to the column. After washing the column twice with 30 mL of QC solution, genome DNA was eluated from the column using 15 mL of QF; then 10.5 mL of isopropanol was added to the salt outside the DNA. The precipitated strand-type genome DNA was placed in a 1.5 mL microtube, washed with 75% ethanol, and air-dried, before being dissolved in 250 μL of a TE solution (0 .4 μg/μL). The concentration of the genome DNA solution thus obtained was measured, and the solution was then adjusted to 40 ng/μL by adding a TE solution. This DNA solution was used as a PCR template.

(2) Design and Production of Degenerative Primer

[000506] In the degenerative primer design, to reduce the number of degeneration, the most frequent codon in each amino acid was used for the sequence at the 5' end according to the codon usage information (Codon Usage Database http ://www.kazusa.or.jp/codon/) from Lactococcus garvieae. Furthermore, a scrambled base was used for the 3' end sequence to avoid imbalance with the dihydrodaidzein synthesis enzyme gene.

[000507] The following degenerative primer was designed and created based on the N-terminal sequence: MKNKFYPKTFERGYIGN-LEVEN determined in example A3, to be used for degenerative PCR. E1-terminal of N-31: TGAAGAATAANTTNTAYCC- NAARACNTTYGA (SEQ ID NO: 23) (In SEQ ID NO: 23, "N" in the 11th and 14th positions represents inosine, and "N" in the 20th and 26th positions represents adenine, guanine , cytosine or thymine.) E1-terminal of N-37: TGAAGAATAANTTNTAYCC- NAARACNTTYGARRGNGG (SEQ ID NO: 24) (In SEQ ID NO: 24, "N" in the 11th, 14th, 20th and 26th positions represents inosine, and "N" " in the 35th position represents adenine, guanine, cytosine or thymine.) E1-terminal of N-F32: ATGAAGAATAAGTTTTAYCC-NAARACNTTYGA (SEQ ID NO: 25) (In SEQ ID NO: 25, "N" in the 21st and 27th positions represent adenine, guanine, cytosine or thymine.)

[000508] Furthermore, the following degenerative primer was designed and created based on the internal sequence Peptide 2: ASRMVMDA-VHEGYIAG determined in example A4, to be used for degenerative PCR. E1-internal-RP1: CCTGCAATATAACCTTCATGTACNG-CRTCCATNACCAT (SEQ ID NO: 26) (In SEQ ID NO: 26, "N" in the 24th and 33rd positions represents adenine, guanine, cytosine or thymine.)

[000509] All oligo DNAs used as the primers of the present Example were produced by Sigma-Aldrich Japan K.K.

(3) Amplification of the dihydrodaidzein synthesis enzyme gene through degenerative PCR.

[000510] Using the produced degenerative primer, dihydrodaidzein synthesis enzyme gene amplification by degenerative PCR mode was attempted.

[000511] The following are the combinations of degenerative primers used in degenerative PCR. (i) E1-Terminal of N-31 and E1-internal-RP1 (ii) E1-Terminal of N-37 and E1-internal-RP1 (iii) E1-Terminal of N-F32 and E1-internal-RP1

[000512] In amplification of the dihydrodaidzein synthesis enzyme gene through degenerative PCR, Ex-Taq DNA polymerase (Takara Bio Inc.) was used. The amplification program was: 95°C, 2min (95°C, 45 sec; 38°C to 54°C, 30sec; 72°C, 2 min) x 50 cycles, and 72°C, 3 min. In consideration of the imbalance with the degenerative primer genome DNA, annealing was performed in 5 stages, starting at 38°C, with a temperature increase of 4°C before each additional stage, up to 54°C. After the PCR reaction, 1/10 of 10xDye was added to the PCR product. 6 μL of the product was electrophoresed using 0.8% agarose gel. In the agarose electrophoresis in the present Example, staining was performed using ethidium bromide; and À/StyI (Nippongene Co. Ltd.) and 100 bp ladder (Toyobo Co. Ltd.) were used as molecular weight markers.

[000513] Figure 5 shows the result of electrophoresis of degenerative PCR product. For the combination (E1-Terminal of N-F32 and E1-internal-RP1) of the degenerative primer (iii) in (3) of the present Example, about 1.9 kb of amplification was observed in the DNA fragment at any of the temperatures of annealing. The most significant increase was observed when the annealing temperature was 54°C.

(4) Determination of the base sequence of amplified DNA fragment

[000514] The amplified DNA fragment of about 1.9kb (annealing temperature = 54°C) was excised from the agarose gel, and purified using a Gel extraction kit (Qiagen). The purified DNA fragment was inserted into a pT7-Blue Cloning Vector (Novagen) to determine the base sequence.

[000515] The obtained DNA base sequence was analyzed using SEQUENCHER DNA sequence assembly software (Gene Codes Inc, USA), with the result that the DNA fragment contained the base sequences corresponding to the amino acid sequences of Peptides 1 and 3 found in example A4.

Example A6: Determination of total base sequence of dihydrodaidzein synthesis enzyme gene

[000516] To determine the 5' end and 3' end sequences of the 1.9kb dihydrodaidzein synthesis enzyme gene obtained in example A5 to determine the entire base sequences, rapid amplification of cDNA terminal sequence (5' -, 3'-RACE) was performed using the Lactococcus 20-92 strain genome DNA library as a template.

(1) Genome DNA library production

[000517] The genome DNA of the Lactococcus 20-92 strain purified in example A5 was fragmented through 16 hours of digestion at 37°C, using restriction endonuclease (BamHI, EcoRI, HindIII, KpnI, PstI, SacI, SalI, Sau3AI , XhoI: products of Takara Bio Inc.). After processing with phenol/chloroform, the fragment was purified by ethanol sedimentation. The purified genome DNA fragments were ligated with pUC19 cloning vectors, which were previously excised by corresponding restriction endonuclease and dephosphorylated by Shrimp Alkaline Phosphatase (Takara Bio Inc.), using the TaKaRa Ligation kit var.2.1 (Takara Bio Inc.), thereby producing a genome DNA library.

(2) Rapid amplification of cDNA terminal sequence (5'-RACE, 3'-RACE)

[000518] The genome DNA library (ligation reaction liquid) was diluted 20 times with sterilized water. Amplification of the 5'-end and 3'-end Sequences of the dihydrodaidzein synthesis enzyme gene through rapid amplification of cDNA Terminal Sequences (5'-RACE, 3'-RACE) was attempted using a 1 μL template.

(2-1) Launcher

[000519] The following are the combinations of primers used for 5'-RACE, 3'-RACE, respectively. (2-1-1) 5'-RACE Prime-PCR: E1-RACE-N-P1 and pUC19-FP-1, E1-RACE-N- P1 and pUC19-RP-1 Built-in PCR: E1-RACE-N -P2 and pUC19-FP-2, E1-RACE- N-P2 and pUC19-RP-2 (2-1-2) 3'-RACE Primer-PCR: E1-RACE-RP2-1 and pUC19-FP-1 , E1-RACE- RP2-1 and pUC19-RP-1 Built-in PCR: E1-RACE-RP2-2 and pUC19-FP-2, E1-RACE- RP2-2 and pUC19-RP-2

(2-1-3) primer sequence used

[000520] The following are the primer sequences used for 5'-RACE, 3'-RACE, respectively. Primers for Vector: pUC19-FP-1: ACACAGGAAACAGCTATGACCATGATTACG (SEQ ID NO: 27) pUC19-RP-1: AGCTGGCGAAAGGGGGATGTGCTG- CAAGGC (SEQ ID NO: 28) pUC19-FP-2: ATGATTACGCCAAGCTTGCATGCCTG- CAGG (SEQ ID NO: 29) pUC19-RP-2: CCAGTCACGACGTTTGTAAAACGACGG- CCAG (SEQ ID NO: 30)

[000521] Primers for dihydrodaidzein synthesis enzyme gene E1-RACE-N-P1: ATGCGGATCGCTCGGTTCTCGAC- CTCTAGGTTAC (SEQ ID NO: 31) E1-RACE-RP2-1: ATCGAGGAGAAGTGCGAGGAC- GTCAGGGTCATC (SEQ ID NO: 32) E1-RACE-N-P2: TTCTCGACCTCTAGGTTAC- CGATGTAGCCGC (SEQ ID NO: 33) E1-RACE-RP2-2: ACGTCAGGGTCATCGG- CATCGGCGACTGCAAG (SEQ ID NO: 34)

[000522] All oligo DNAs used as primers in the present Example were produced by Sigma-Aldrich Japan K.K.

(2-2) Amplification of the 5' end and 3' end sequences of the dihydrodaidzein synthesis enzyme gene using 5'-RACE and 3'-RACE

[000523] Ex-Taq DNA polymerase (Takara Bio Inc.) was used for 5'-RACE, 3'-RACE. Prime-PCR and Embedded PCR were performed using the same amplification program: 95°C 2min, (95°C 45 seconds, 60°C 30 seconds, 72°C 1 minute) x 30 cycles, 72°C 3 minutes. In First-PCR, 1 μL (40 ng) of the genome DNA dilution liquid prepared in the aforementioned manner was used as a template. In Embedded PCR, 0.5 μL of Prime-PCR product was used.

[000524] After the Embedded PCR reaction, 1/10 of 10xday was added to the PCR product, and 5μL of the mixture was electrophoresed with 0.8% agarose gel. The observed amplification of the DNA Fragment was about 1.2kb (SacI) and about 1.0kb (Sau3AI) in 5'-RACE, and about 0.6kb (SacI) and 0.3kb (KpnI) in 3 '-RACE.

[000525] In agarose electrophoresis in the present Example, staining was performed using ethidium bromide (Nippongene Co. Ltd.); and À/StyI (Nippongene Co. Ltd.) and 100 bp ladder (Toyobo Co. Ltd.) were used as molecular weight markers.

[000526] The amplified DNA fragments were excised from the agarose gel, and purified using a Gel Extraction Kit (Qiagen). The base sequences of the purified DNA fragments were determined by direct sequencing using the primers used for amplification, with the result that the dihydrodaidzein synthesis enzyme gene sequence was observed in those DNA fragments of about 1.2 kb(SacI ) and about 1.0 kb(Sau3AI) in 5'-RACE, and those of about 0.6kb(SacI) in 3'-RACE.

(3) Determination of the total base sequence of dihydrodaidzein synthesis enzyme gene

[000527] The DNA base sequence obtained by the degenerative PCR in example A5 and the rapid amplification of the cDNA terminal sequence in (2) of the present Example were subjected to the assembly assay using SEQUENCHER DNA sequence assembly software (Gene Codes Inc, USA). As a result, the 3548 bp genome structure in the vicinity of the dihydrodaidzein synthesis enzyme gene was obtained. This revealed that the dihydrodaidzein synthesis enzyme gene was a polypeptide consisting of 1935 nucleotides and 644 amino acids.

[000528] The entire amino acid sequences obtained by the Base Sequences, and the partial amino acid sequences obtained by the amino acid sequence were collected, with the result that all partial amino acid sequences obtained by the amino acid sequence were assigned to the amino acid sequences integers obtained by the Base Sequences.

(4) Confirmation of base sequence in the coating region

[000529] In the sequence obtained in (3) of the present Example, many portions were observed with the clones having inconsistent bases caused by error in incorporating the DNA polymerase bases in PCR amplification. Therefore, using the first strand of cDNA as a template, the region (2368bp) containing the coding region of the dihydrodaidzein synthesis enzyme gene was amplified by PCR using Easy-A®High-Fidelity PCR Cloning Enzyme (Stratagene ), which is High-Fidelity DNA Polymerase. The following are the amplification primers used above. E1-conf- NP:TGCCGGTGCAATGGCTGACATCATGTTCAACCTG (SEQ ID NO: 35) E1-conf- CP:TCCTCCATCGTTCCTCCAATCAGTAAGACACGCG (SEQ ID NO: 36)

[000530] The DNA fragment obtained was purified using a Gel Extraction kit (Qiagen), and confirmation and final sequence determination was made using direct sequence.

Example A7: Induction of dihydrodaidzein synthesis enzyme gene expression by adding daidzein to amplification culture solution

[000531] As shown in example A1, when daidzein, which is a substrate, is added to the amplification culture solution of the Lactococcus 20-92 strain, the cultured cells exhibit a dihydrodaidzein synthesis activity. This leads to an assumption that the addition of daidzein to the amplification culture solution induces transcription of the dihydrodaidzein synthesis enzyme gene, and also induces translation into protein. The following test was performed based on this assumption. Two cDNA samples derived from the Lactococcus 2092 strain were incubated in a culture medium containing daidzein and in a culture medium without daidzein; they were subjected to RT-PCR to find out whether dihydrodaidzein synthesis enzyme gene expression was induced by the addition of daidzein into the amplification culture solution.

(1) Extraction of total DNA from Lactococcus 20-92 strain and purification of DNA Extraction

[000532] Extraction of total DNA from the Lactococcus 20-92 strain and purification were carried out as follows.

[000533] The sealed Lactococcus 20-92 strain maintained at 4°C was incubated for eight hours in a modified GAM broth culture medium, which contains 10mg/L daidzein (Funakoshi Co., Ltd.) or no daidzein, in 37°C under an anaerobic condition. 25mL of the culture solution was transferred to a 50mL tube, and centrifuged for ten minutes at 3500rpm, 4°C. The culture medium was removed by decantation and the cells were collected. The collected cells were immediately frozen by liquid nitrogen. Then, total RNA was extracted and purified using 1 mL of TRIzol solution (Invitrogen) according to the instruction. Purified full-length RNA was processed by DNase I (Invitrogen) to remove genome DNA, and used for first-strand cDNA synthesis.

(2) Synthesis of first-strand cDNA from full-length RNA

[000534] From 2 μg of the entire RNA thus processed by DNaseI, first strand cDNA (reverse transcription) was synthesized using SuperScript® First Strand Synthesis System for RT-PCR (Invitrogen). First-strand cDNA synthesis was performed using Random Hexamer mixture as an extension primer according to the instruction. Furthermore, to confirm that the DNase I-processed full-length RNA used for first-strand cDNA synthesis did not contain genome DNA, another reaction was performed at the same time without reverse transcription. The final reaction liquid was used as a template for RT-PCR.

[000535] The following give the four types of final reaction liquids thus prepared. 1. Whole RNA reverse transcription product derived from bacterial cells incubated in a medium containing daidzein (DZN(+)RT(+)) 2. Whole RNA reverse transcription product derived from bacterial cells incubated in a medium containing none daidzein (DZN(-)RT(+)) 3. Non-reverse transcription product of full-length RNA derived from bacterial cells incubated in a medium containing daidzein (DZN(+)RT(-)) 4. Non-reverse transcription product reverse whole RNA derived from bacterial cells incubated in a medium with no daidzein (DZN(-)RT(-))

(3) Confirmation of dihydrodaidzein synthesis enzyme gene expression using RT-PCR

[000536] The dihydrodaidzein synthesis enzyme gene was amplified through RT-PCR using the four final reaction products as detailed in (2) of the present Example as models. The expression amounts were compared with each other. As a control, another RT-PCR was performed at the same time using the 16S-ribosome RNA sequence as a control gene.

[000537] The following are RT-PCR amplification conditions and a primer sequence for each gene amplification.

[000538] Ex-Taq DNA polymerase (Takara Bio Inc.) was used for RT-PCR. The amplification program was: 95°C 2 minutes, (95°C 30 seconds, 56°C 20 seconds, 72°C 30 seconds) x 30 cycles, 72°C 2min. As a model, 1 μL of each final reaction liquid as in (2) of the present example was used.

[000539] The following shows a primer sequence used by the RT-PCR of the present Example and the size of the DNA Fragment to be amplified.

[000540] Dihydrodaidzein synthesis enzyme: 239bp E1- FP:CTACATCGGTAACCTAGAGGTCG (SEQ ID NO: 37) E1-RP:CCGTGCTGCTTGATGGTCTTTGC (SEQ ID NO: 38)

[000541] 16S-ribosome RNA sequence: 326bp Gar-16S-Ribo-FP:TGCGTAGATATATGGAGGAAC (SEQ ID NO: 39) Gar-16S-Ribo-RP:CTTATCTCTAAGGATAGCACG (SEQ ID NO: 40)

[000542] The primer used for amplification of the 16S-ribosomal RNA sequence was created based on the FLG12 16S ribosomal RNA gene sequence of the Lactococcus garvieae strain, partial sequence (Accession No. AF352163-66) on the basis of DNA data (GenBank) provided by the National Center for Biotechnology Information: http://www.ncbi.nlm.nih.gov/

[000543] 1/10 of 10xday was added to the PCR Product, and 5μL of the mixture was electrophoresed with 1.5% agarose gel. Amplification of each DNA fragment was observed.

[000544] In agarose electrophoresis in the present Example, staining was performed using ethidium bromide (Nippongene Co. Ltd.); and À/StyI (Nippongene Co. Ltd.) and 100 bp ladder (Toyobo Co. Ltd.) were used as molecular weight markers.

[000545] Figure 6 shows the results.

[000546] As evident from the result of amplification of dihydrodaidzein synthesis enzyme gene through RT-PCR, efficient gene expression was observed only in the reverse transcription product of full-length RNA derived from bacterial cells incubated in a medium with daidzein. The same level of expression was observed in the amplification of 16S-ribosome RNA sequence used as a control, regardless of daidzein incorporation. Furthermore, since both the dihydrodaidzein synthesis enzyme gene and the 16S-ribosome RNA sequence were not amplified in the non-reverse transcription product, it was confirmed that the full-length DNase I-processed RNA used for the synthesis of first-strand cDNA does not contain genome DNA. These results showed that the addition of daidzein to the medium induced the expression of dihydrodaidzein synthesis enzyme gene mRNA.

Example A8: Expression of Recombinant E1 Polypeptide using Escherichia coli, and Confirmation of Dihydrodaidzein Synthesis Activity

[000547] The pET system, a recombinant protein expression system using Escherichia coli, was used to express E1 polypeptide, and its dihydrodaidzein synthesis activity was confirmed.

(1) Preparation of E1 Polypeptide Expression Vector

[000548] To prepare an E1 Polypeptide expression vector (pET21-E1-His), DNA in the E1 nucleotide open reading frame was amplified by PCR.

[000549] The following amplification primers were prepared based on the E1 nucleotide sequence determined in example A6.exp.E1 pet F Nde: AGCTCATATGAAGAACAAGTTCTA- TCCGAA (SEQ ID NO: 41) exp.E1 pet His: AATCGAATTCCTACAGGTT- GCAGCCAGCGATGT (SEQ ID NO: 42)

[000550] For insertion into pET21a (Novagen), amplification primers exp.E1 pet F Nde and exp.E1 pet His were designed to include NdeI and EcoRI restriction sequences, respectively.

[000551] A PCR reaction was performed using 25 μL of a reaction mixture containing the primers, 5 pmol each; dNTP, 5 nmol each; Genomic DNA from the Lactococcus 20-92 strain purified in example A5, 40 ng; 10 x buffer for KOD-plus DNA polymerase (Toyobo Co., Ltd.), 2.5 μL; KOD-Plus DNA polymerase, 0.3 U (Toyobo Co., Ltd.). Amplification program: 95°C for 3 min, (94°C for 30 sec, 60°C for 30 sec, 68°C for 2 min) x 30 cycles, 68°C for 7 min. PCR device: GeneAmpPCR System 9700 (Applied Biosystems). Analysis of a portion of the PCR reaction mixture by agarose gel electrophoresis detected a band of an expected size. The entire PCR Product was collected by a QIAGEN PCR Purification kit (Qiagen).

[000552] The DNA fragments thus collected were cut with NdeI and EcoRI restriction enzymes, and subjected to agarose gel electrophoresis. Then, a portion containing the target band was excised, and purified and collected with a Qiagen Gel Extraction kit (Qiagen). After collection, DNA fragments were ligated at 16°C overnight with NdeI and EcoRI digested pET21a using a DNA Ligation kit ver.2.1 (Takara Bio Inc.). The bound reaction mixture was then used to transform Escherichia coli strain JM109 (Takara Bio Inc.).

[000553] The transformant was grown at 37°C overnight on an LB medium agar plate (GIBCO) containing ampicillin (50 μg/mL). The resulting single colonies were grown overnight in 3 mL LB medium (GIBCO) containing ampicillin (50 μg/mL). Then, plasmid DNA was extracted using a PI-100 plasmid autoextractor (KURABO).

[000554] The base sequence of the DNA inserted into the plasmid was sequenced by the pigment terminator method. This confirmed the successful insertion of E1 polynucleotide when inserted. In this way, pET21-E1-His was obtained. In this example, the DNA sequence was determined using ABI3700 DNA sequencer (Applied Biosystems).

(2) Preparation of Recombinant, and Expression and Confirmation of Recombinant E1 Polypeptide in Escherichia coli'

[000555] Plasmid pET21a (negative control) and plasmid pET21-E1-His expressing recombinant E1 Polypeptide were used to transform the Escherichia coli strain BL21 (DE3) (Novagen). To obtain single colonies, transformants were grown overnight at 37°C on an LB medium agar plate containing ampicillin (50 μg/mL).

[000556] Each E. coli BL21 (DE3) transformant was grown overnight at 37°C in a 3 mL liquid LB medium containing ampicillin (50 μg/mL). Then, 0.5 mL of the culture was pre-cultured for 3 hours (until the OD at 630 nm was approximately 0.4) by adding 50 mL of liquid LB medium containing the same concentration of ampicillin. After adjusting the final concentration to 1 mM by adding IPTG (isopropyl-p-thiogalactopyranoside; Wako Pure Chemical Industries, Ltd.), the culture was further incubated at 37°C for 4 hours.

[000557] After incubation, cells were harvested by centrifugation (6,000 rpm, 4°C, 15 minutes) using an Avanti HP25 (Beckman Coulter). Subsequent procedures were performed on ice. After removing the supernatant (the medium), cells were suspended in 1 mL of 0.1 M potassium phosphate buffer (pH 7.0; KPB-PDH) containing 1 mM PMSF, 2 mM DTT, and hydrosulfide. 5 mM sodium. The cell suspension was then placed in a 2 mL auxiliary tube loaded with 0.7 mL of zirconia-silica beads (BioSpec Products, Inc.) and 400 μL of KPB-PDH. Cells were then disrupted by two repeated cycles of a 20 second, 6,500 rpm treatment and 3 minute ice cooling using a FastPrep® (Thermo Electron Corporation). As a result, a suspension of ruptured cells was obtained.

[000558] The expression of recombinant E1 Polypeptide in E. coli was confirmed by SDS-polyacrylamide-gel electrophoresis (SDS-PAGE).

[000559] 2.5 μL of 5x sample buffer (125 mM Tris-HCl (pH 2.5)/25% glycerol/5% SDS/5% 2-mercaptoethanol/0.5% BPB) was added to 10 μL of the disrupted cell suspension. After thermal denaturation at 98°C for 5 minutes, the suspension was cooled with ice, and 5 μL was electrophoresed by SDS-PAGE. SDS-PAGE was performed with a commercially available gel plate (SuperSep® 5-20%; Wako Pure Chemical Industries, Ltd.), and Quick CBB (Wako Pure Chemical Industries, Ltd.) for staining. The Prestained XL-Ladder Broad strip (Apro Science) was used as a molecular weight marker.

[000560] The SDS-PAGE results are shown in FIG. 7. A recombinant E1 Polypeptide with a molecular weight of about 70 kDa was confirmed in the disrupted cell suspension derived from the pET21-E1-His transformant.

(3) Confirmation of dihydrodaidzein synthesis activity of E1 Polypeptide obtained from Recombinant

[000561] The ruptured cell suspension obtained in (2) of this Example was used as an enzyme source to measure the conversion activity of daidzein to dihydrodaidzein. As a result, activity was confirmed in the expressed recombinant E1 Polypeptide.

[000562] In this example, the measurement of the conversion activity of daidzein to dihydrodaidzein was carried out as follows.

[000563] An enzyme reaction mixture of the composition below was prepared, and the mixture was incubated at 37°C for 2 hours.

[000564] After incubation, 3 mL of ethyl acetate was added to the enzyme reaction mixture for extraction. After drying, the extract was dissolved by the mobile phase (eluent). The dissolved product was analyzed by HPLC to measure the daidzein and dihydrodaidzein contents in the enzyme reaction mixture.

[000565] The results of the HPLC analysis are shown in FIG. 8. In the disrupted cell suspension derived from pET21-E1-His plasmid transformant expressing the recombinant E1 polypeptide, daidzein (substrate) added to the enzyme reaction mixture was converted to dihydrodaidzein whereas dihydrodaidzein was not detected in the enzyme reaction mixture. enzyme reaction in pET21a transformant (negative control).

[000566] These results show that the recombinant E1 polypeptide has the activity to synthesize daidzein dihydrodaidzein.

Example B Reference Example B1 Synthesis of cis-tetrahydrodaidzein and trans-tetrahydrodaidzein

[000567] Cis-tetrahydrodaidzein and trans-tetrahydrodaidzein were produced according to the reaction flow below. The compounds are designated as. Compound 1 (daidzein): 4',7-dihydroxy isoflavone Compound 2: 4',7-diacetoxy isoflavone Compound 3: 4',7-diacetoxy isoflavan-4-one Compound 4: cis- isoflavan-4-ol 4',7-diacetoxy Compound 5: trans-4',7-diacetoxy isoflavan-4-ol Compound 6: cis-tetrahydrodaidzein Compound 7: trans-tetrahydrodaidzein Formula 1

Synthesis of compound 2

[000568] 0.76 mL (8.0 mmol) of acetic anhydride was added to a pyridine solution (5 mL) containing 500 mg (1.97 mmol) of daidzein (Compound 1), and the mixture was stirred at 60 °C for 2 hours. After adding a minute amount of methanol, the reaction mixture was transferred into 3 N hydrochloric acid. After dilution with water, the resulting solid precipitate was filtered and rinsed with water. The solid was then air-dried at room temperature to obtain 609 mg of a white, powdered Compound 2 (1.80 mmol, 91% product).

[000569] Compound 2: 1H NMR (250 MHz, CDCh) δ (ppm): 2.33 (3H, s), 2.37 (3H, s), 7.13-7.22 (3H, m), 7.32 (1H, d, J = 2.3 Hz), 7.54-7.63 (2H, m), 8.01 (1H, s), 8.33 (1H, d, J = 8, 8 Hz).

Synthesis of compound 3

[000570] A methanol (6 mL)-ethyl acetate (6 mL) suspension containing 400 mg (1.18 mmol) of Compound 2 and 150 mg of 10% palladium-carbon (hydrated, about 50% by weight ) was stirred at room temperature for 2 hours under a hydrogen atmosphere. The reaction mixture was filtered through Celite, and the residues were washed with ethyl acetate. The residues obtained by concentration of the filtrate were purified by silica gel column chromatography (silica gel: dichloromethane/ethyl acetate = 100/0 - 19/1) to obtain 312 mg of a white, powdered Compound 3 (0.917 mmol, 78% of product). Compound 3: 1H NMR (250 MHz, CDCl3) δ (ppm): 2.29 (3H, s), 2.32 (3H, s), 3.93-4.04 (1H, m), 4.60 -4.75 (2H, m), 6.76-6.84 (2H, m), 7.04-7.13 (2H, m), 7.27-7.35 (2H, m), 7 .93-8.01 (1H, m).

Synthesis of Compound 4 and Compound 5

[000571] 11 mg (0.29 mmol) of sodium borohydride were added to a methanol (1 mL)-dichloromethane (1 mL) solution containing 100 mg (0.294 mmol) of Compound 3 at 0 ° C. After stirring the reaction mixture at 0°C for 30 minutes, 2 mL of 1 N hydrochloric acid and 50 mL of water were successively added, followed by extraction with ethyl acetate. The organic layer was successively washed with saturated sodium bicarbonate, water and saturated brine, followed by drying with anhydrous sodium sulfate. The solvent was distilled, and the resulting residues were purified by silica gel column chromatography (silica gel: dichloromethane/ethyl acetate = 19/1 - 3/1). The resulting diastereomer mixture was purified by medium pressure column chromatography (Yamazen, Ultra Pack SI-40B: n-hexane/ethyl acetate = 3/2) to obtain 44 mg of a colorless, acicular Compound 4 (0 .13 mmol, 44% production), and 26 mg of a colorless, acicular Compound 5 (75 mmol), 26% production).

[000572] Compound 4: 1H NMR (250 MHz, CDCI3) δ (ppm): 1.80 (1H, d, J = 4.0 Hz), 2.30 (3H, s), 2.31 (3H, s), 3.32 (1H, td, J = 3.5, 11.5 Hz), 4.32 (1H, ddd, J = 1.3, 3.5, 10.5 Hz), 4.59 (1H, dd, J = 10.5, 11.5 Hz), 4,764.83 (1H, m), 6.64-6.73 (2H, m), 7.06-7.14 (2H, m ), 7.27-7.35 (3H, m).

[000573] Compound 5: 1H NMR (250 MHz, CDCl3) δ (ppm): 2.02 (1H, d, J = 5.5 Hz), 2.29 (3H, s), 2.30 (3H, s), 3.11-3.24 (1H, m), 4.26 (1H, dd, J = 9.0, 11.3 Hz), 4.37 (1H, dd, J = 3.8, 11.3 Hz), 4.92 (1H, dd, J = 5.5, 7.8 Hz), 6.62 (1H, d, J = 2.3 Hz), 6.72 (1H, dd, J = 2.3, 8.5 Hz), 7.04-7.12 (2H, m), 7.21-7.30 (2H, m), 7.47 (1H, d, J = 8, 5 Hz).

Synthesis of compound 6

[000574] 55 mg (1.0 mmol) of sodium methoxide was added to a methanol solution (4 mL) containing 116 mg (0.339 mmol) of Compound 4, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was neutralized by adding an ion exchange resin (DOWEX 50 x 8W, ammonium form). After filtering the resin, the solid obtained by concentrating the filtrate was washed with methanol to obtain 62 mg of a white, powdered compound 6 (0.24 mmol, 71% production).

[000575] Compound 6: 1H NMR (250 MHz, DMSO-d6) δ (ppm): 3.03 (1H, td, J = 3.3, 12.0 Hz), 4.00-4.15 (1H , m), 4.31-4.51 (2H, m, including 4.39, dd, J = 10.3, 12.0 Hz), 4.96 (1H, d, J = 5.8 Hz) , 6.18 (1H, d, J = 2.3 Hz), 6.32 (1H, dd, J = 2.3, 8.3 Hz), 6.69 (2H, d, J = 8.5 Hz), 7.00 (1H, d, J = 8.3 Hz), 7.09 (2H, d, J= 8.5 Hz), 9.19 (1H, br s), 9.31 (1H , br s).

Synthesis of Compound 7

[000576] 0.73 mL (0.73 mmol) of 1N sodium hydroxide was added to a methanol suspension (2 mL) containing 84 mg (0.25 mmol) of Compound 5, and the mixture was stirred in room temperature for 2.5 hours. Then, saturated ammonium chloride water was added to the reaction mixture, followed by extraction with ethyl acetate. The organic layer was successively washed with saturated sodium bicarbonate water and saturated saline, followed by drying with anhydrous sodium sulfate. By distilling the solvent, 64 mg of a white, powdered Compound 7 (0.25 mmol, quantitative yield) were obtained.

[000577] Compound 7: 1H NMR (250 MHz, DMSO-d6) δ (ppm): 2,822.96 (1H, m), 4.04-4.22 (2H, m), 4.60 (1H, t , J = 7.0 Hz), 5.18 (1H, d, J = 7.0 Hz), 6.14 (1H, d, J = 2.3 Hz), 6.34 (1H, dd, J = 2.3, 8.5 Hz), 6.67 (2H, d, J = 8.5 Hz), 7.04 (2H, d, J = 8.5 Hz), 7.16 (1H, d , J = 8.5 Hz), 9.21 (1H, s), 9.28 (1H, s).

Reference Example B2

[000578] A Lactococcus 20-92 strain (FERM BP-10036) was inoculated into a liquid amplification medium containing daidzein, and the culture was incubated at 37°C for 7 to 24 hours under anaerobic conditions. After incubation, cells were collected and cryogenically preserved for use in the Examples below.

Example B1: Confirmation of Tetrahydrodaidzein Production

[000579] The following experiments were conducted to confirm that tetrahydrodaidzein is the equol biosynthesis intermediate.

(1) Preparation of Disrupted Cellular Material

[000580] Lactococcus 20-92 strain cells preserved at -80°C were quickly thawed. Cells were suspended by buffering, and centrifuged at 4°C (8,000 rpm x 5 min) using a VC960 centrifuge (Taitec). After removing the supernatant, 0.1 M potassium phosphate buffer/1 mM PMSF (phenylmethanesulfonyl fluoride)/2 mM DTT (Dithiothreitol)/5 mM sodium hydrosulfide (pH 7.0) were added to obtain a cell suspension. The cell suspension was placed in a 2 ml tube prepared to include zirconia-silica beads (about 0.8 ml, 0.1 mm, 1 lb; Wakenyaku Co., Ltd.), and the tube was charged with 0.1 M potassium phosphate/ 1 mM PMSF/ 2 mM DTT/ 5 mM sodium hydrosulfide solution (pH 7.0) to charge the tube almost completely. For disruption, the tube was closed and kept on ice, where a cycle of 6,500 rpm x 20 sec of centrifugation and cooling on ice was repeated four times using FastPrep®-FP100A (Thermo Electron Corporation). The resulting disrupted cellular material was used as an enzyme source in an enzyme reaction.

(2) Enzyme reaction

[000581] A 1 ml enzyme reaction mixture of the composition below, containing 0.1 ml of the disrupted cellular material obtained in (1) was prepared, and the mixture was incubated at 37°C for 2 hours. After incubation, 3 mL of ethyl acetate was added to the resulting enzyme reaction product for extraction. The product was dried to prepare a sample for HPLC analysis. Enzyme Reaction Mix Composition 0.1 M Potassium Phosphate Buffer / 1 mM PMSF / 2 mM DTT / 5 mM Sodium Hydrosulfide (pH 7.0) 2 mM NADPH 2 mM NADH 0 μg/mL of dihydrodaidzein or tetrahydrodaidzein

(3) Production of tetrahydrodaidzein from dihydrodaidzein by enzyme reaction

[000582] FIG. 9 shows the results of HPLC analysis of the enzyme reaction product obtained using dihydrodaidzein as a substrate and the disrupted cellular material as an enzyme source. FIG. 9 also shows the results of HPLC analysis of the tetrahydrodaidzein synthesized in Reference Example B1. The results show that the enzyme reaction product obtained using dihydrodaidzein as a substrate and disrupted cellular material as an enzyme source includes an intermediate having a retention time corresponding to the retention time of trans-tetrahydrodaidzein, confirming the production of trans-tetrahydrodaidzein.

(4) Production of Equol from tetrahydrodaidzein by enzyme reaction

[000583] FIG. 10 shows the results of HPLC analysis of the enzyme reaction product obtained using cis-tetrahydrodaidzein or trans-tetrahydrodaidzein as a substrate and the disrupted cellular material as an enzyme source. As is clear from FIG. 10, equol was produced from both compounds, showing that tetrahydrodaidzein is used as a substrate in equol biosynthesis, both in the cis and trans forms.

Example B2: Confirmation of Tetrahydrodaidzein Biosynthesis Activity in Centrifuged Supernatant of Disrupted Cellular Material, and Confirmation of NADH or NADPH Dependence

[000584] Frozen cells were thawed and centrifuged at 4°C for 15 minutes (5,000 x G). The pellet was used in the test below. First, the pellet (wet weight 2.7 g) was suspended in 10 ml of a 0.1 M potassium phosphate solution containing 1 mM PMSF and 5 mM sodium hydrosulfide. After preheating to 37°C for 5 minutes, lysozyme was added in an amount of 100 mg per gram of pellet (wet weight) to cause a reaction at 37°C for 1.5 hours. Then, an equivalent amount of 0.1 M potassium phosphate dissolution was added to the reaction mixture, and the mixture was vigorously stirred with a vortex mixer after adding zirconia-silica beads (3 ml). The cells were then disrupted with a sonicator (Branson Sonifier Cell Disruptor 200) (3 cycles of a 5-minute sonication and a 2-minute rest). The disrupted cell suspension was centrifuged at approximately 10,000 x G for 15 minutes to obtain a supernatant, which was then used as an enzyme source.

[000585] An enzyme reaction mixture of the composition below was prepared, and was incubated at 37°C for 2 hours. After incubation, 5 mL of ethyl acetate was added to the enzyme reaction product for extraction. Then, the product was dried to prepare a sample for HPLC analysis.

[000586] The results are shown in FIG. 11. The results confirmed the presence of tetrahydrodaidzein biosynthesis activity in the centrifuged supernatant of the ruptured cellular material. It was also confirmed that the conversion of dihydrodaidzein to tetrahydrodaidzein is dependent on the coenzyme NADH, and much more strongly on NADPH.

Example B3: Purification of Tetrahydrodaidzein Synthesizing Enzyme

[000587] Cells of the Lactococcus 20-92 strain were incubated for 20 hours in 67 ml of a liquid amplification medium containing daidzein contained in an incubation bottle. Cultured cells from ten such bottles were centrifuged and suspended in a 0.02 M potassium phosphate buffer (pH 7; hereafter "Buffer A") containing 1 mM PMSF (phenylmethylsulfonyl fluoride) and 4 mM DTT ( Dithiothreitol). Cells were disrupted with a French press (SLM Instruments Inc.) six times at 10 kPa, and the disrupted cell suspension was centrifuged to obtain a supernatant. Separately, Lactococcus 20-92 strain cells were incubated for 18 hours in 200 ml of liquid medium contained in an incubation bottle. Cultured cells from five such bottles were similarly disrupted with a French press, and a supernatant was obtained after centrifugation. These supernatants, 38 ml and 47 ml, were mixed, and 82 ml of the mixture was fed to red-Sepharose (about 7 ml) equilibrated with buffer A. After washing the red-Sepharose with 150 ml of Buffer A, the mixture was eluted with Buffer A containing 10 mM NADPH (20 ml/fraction). Each fraction was used as an enzyme source, and the tetrahydrodaidzein biosynthesis activity was measured under the same conditions as the enzyme reaction conditions of Example B2 (using NADPH as a coenzyme). As a result, active fractions No.: 1 to 5 were obtained.

[000588] Fractions No.: 1 to 5 having tetrahydrodaidzein biosynthesis activity were concentrated by ultrafiltration using Amicon Ultra Centrifugal UFC801024 (MW Cut: 10,000) to obtain a concentrate (about 2.1 ml). The concentrate was separated into three portions and fed to HPLC using TSKgel Phenyl-5PW (Tosoh) after mixing each portion with an equivalent amount of Buffer A containing 3 M ammonium sulfate. HPLC conditions are as follows. The protein was assayed by measuring absorption at 280 nm. Column: TSKgel Phenyl-5PW Flow Rate: 1 ml/min Fraction: 2 ml/2 min/fraction Eluent A: 0.02 M potassium phosphate buffer pH 7/ 1 mM DTT / 2.5 mM sodium hydrosulfide /0.5% isoPrOH Eluent B: Eluent A containing 1 M ammonium sulfate

[000589] HPLC results revealed that tetrahydrodaidzein synthesis activity is present in a wide range of fractions (fraction no.: 15 and subsequent fractions, a retention time of 30 minutes and longer). Once this result was determined, HPLC fractions Nos: 19 to 22 were mixed and concentrated by ultrafiltration (Amicon Ultra Centrifugal UFC801024; MW Cut: 10,000). From about 130 μl of the concentrate, 100 μl was fed to gel filtration HPLC using TSKgel G2000SWXL (Tosoh), under the following conditions.

[000590] FIG. 12 shows the gel filtration HPLC results. FIG. 13 shows the SDS-PAGE results of each fraction performed under reduced conditions. The results showed bands of 28 kDa and 32 kDa under reduced conditions in fraction No:7, a major fraction of tetrahydrodaidzein synthesis activity.

Example B4: Tetrahydrodaidzein Amino Acid Sequence Synthesizing Enzyme Analysis

[000591] The fraction (fraction no. 7) having tetrahydrodaidzein synthesis activity obtained in example B3 was used as a sample for MS analysis. More specifically, the sample was separated by SDS-PAGE, and the bands were excised. The excised bands were reduced by in-gel alkylation, and digested with in-gel trypsin. Trypsin-deferred peptides were collected and purified for LC-MS analysis. Data acquired by LC-MS analysis were analyzed by de novo sequencing using the MS analysis support software PICOS® (Infocom) to calculate and estimate the amino acid sequences of the peptides. Specifically, this was performed according to the procedures below.

(1) Experiment Materials

[000592] The experiment used the following materials:

[000593] SuperSep HG 10/20% (Wako Pure Chemical Industries, Ltd.); Flamingo Gel stain kit (Bio-Rad); TCEP (Tris[2-carboxyethyl]phosphine) (Pierce); molecular weight marker (Apro Science); DTT (Calbiochem); iodoacetamide (Wako Pure Chemical Industries, Ltd.); acetonitrile (Kanto Kagaku); trypsin (Promega); TFA (Pierce); ammonium bicarbonate (Sigma); ammonium water (Merck); formic acid (Kanto Kagaku); Empore Cation-SR Disk (Sumitomo 3M); MonoCap concentration column (GL Science); MonoCap for Nano Flow 0.1 x 150 mm (GL Science); FortisTip (AMR); SpeedVac Concentrator (SAVANT); HTS-PAL autosampler (CTC-Analytics); Chorus 220 (CTC-Analytics); QSTAR Pulsar i (Applied Biosystems); and PICOS® software (Infocom).

(2) SDS-Polyacrylamide Gel Electrophoresis

[000594] 1 μl of 100 mM TCEP was added to 20 μl of the fraction (fraction no. 7) having tetrahydrodaidzein synthesis activity obtained in example B3. After reduction at 70°C for 10 minutes, the entire sample was applied to SuperSep HG, and SDS-PAGE was performed by the usual method. Following electrophoresis, the gel was stained with Flamingo Gel staining kit (Bio-Rad) (see FIG. 14). Then, the bands LG1 and LG2 that appeared after staining were each cut to a size of about 1 mm2. The excised gels were washed with 100 mM ammonium bicarbonate solution, dehydrated with acetonitrile, and dried and solidified with a SpeedVac Concentrator.

(3) Trypsin Digestion Gel

[000595] A solution of DTT (1.54 mg/ml in 100 mM ammonium bicarbonate) was added to the dried gels, and the mixture was incubated at 55°C for 45 minutes to cause reduction. After discarding the DTT solution, an iodoacetamide solution (10.1 mg/ml in 100 mM ammonium bicarbonate) was added, and the mixture was incubated at room temperature for 30 minutes in the dark. After discarding the solution, the gels were successively washed with a 50% acetonitrile solution, a 100% acetonitrile solution, a 100 mM ammonium bicarbonate solution, and a 100% acetonitrile solution, followed by drying and solidification with a SpeedVac Concentrator. Then, a small amount of trypsin solution (12.5 μg/ml in 50 mM ammonium bicarbonate) was added to the dried gels to soak the gels for 45 minutes on ice. After impregnation, the excess trypsin solution was removed, and a 50 mM ammonium bicarbonate solution was added until the gels were immersed. Then, a reaction was carried out at 37°C for 16 hours.

(4) Amino Acid Sequence Analysis by Mass Spectrometry

[000596] The trypsin-digested peptides were collected, and pretreated by purifying the peptides with a simple column loaded with Empore Cation-SR Disk at the pipette tip. Trypsin peptide digests were collected by washing with a 0.1% TFA/90% acetonitrile solution. In the single column, the sample was first treated with a balanced solution of 0.1% TFA/2% acetonitrile. After sample absorption, the column was washed with a 0.1% TFA/90% acetonitrile solution, and the sample was eluted with a 5% ammonia/30% acetonitrile solution. After elution, the digested peptides were dried and concentrated with a SpeedVac Concentrator. The pH of the digested peptide solution was adjusted to about 3 by adding TFA, and the sample was adjusted on the HTS-PAL autosampler. The sample pool in HTS-PAL was then column washed by loading it into a sample concentration column located in the LC-MS injector valve. The sample in the concentration column was separated with an analytical column using nanoHPLC-Chorus 220, and analyzed with QSTAR Pulsar after ionization with the FortisTip set on the analytical column. The LC-MS analysis conditions are as follows.

MS

[000597] NanoESI Positive Mode, information-dependent acquisition mode (m/z = 400 to 1,400, in 25 counts, state of charge = 2 to 4); 4 experiments/1 cycle: Experiment 1 (TOF-MS, m/z = 400 to 1,400, accumulation time = 1 sec); Experiments 2 to 4 (Positive Product Ion, m/z = 100 to 1,400, accumulation time = 2 sec).

[000598] Data acquired by LC-MS for each band LG1 and LG2 (see FIG. 14) were analyzed by de novo sequencing using PICOS® software to estimate the amino acid sequences of the digested peptides.

Example B5: Peripheral Genomic DNA Sequence Analysis of Dihydrodaidzein Synthesizing Enzyme (E1) Gene (1) Genomic DNA Library Preparation for Inverse PCR

[000599] The genomic DNA of the Lactococcus strain 20-92 (FERM BP-10036) purified according to Example A5 was digested with restriction enzymes (BamHI, EcoRI, HindIII, KpnI, PstI, SacI, SalI, Sau3AI, XhoI; all available from Takara Bio) at 37°C for 16 hours to obtain DNA Fragments. After treatment with phenol-chloroform, the fragments were purified by ethanol precipitation. Purified genomic DNA fragments were self-ligated using TaKaRa Ligation kit ver. 2.1 (Takara Bio). Each binding solution was diluted tenfold with sterilized water to prepare a genomic DNA library for inverse PCR.

(2) Inverse PCR

[000600] 1 μL (40 ng equivalent) of the genomic DNA library for inverse PCR obtained in (1) was used as a template to amplify downstream and upstream regions of the genomic DNA close to the E1 polynucleotide, using inverse PCR. The fragments treated with PstI and XhoI were used as template DNA for inverse PCR to amplify the upstream region. For inverse PCR to amplify the downstream region, fragments treated with HindIII, PstI, SacI, and XhoI were used as template DNA. TaKaRa LA Taq (Takara Bio) was used for inverse PCR. The first PCR was performed using 20 μL of a reaction mixture containing: 1 x PCR buffer (free Mg2+); primers, 0.5 nM each; dNTP, 0.5 mM each; MgCl2, 2.5 mM; and TaKaRa LA Taq, 0.2 U. 1 μL (40 ng) of a diluted genomic DNA library solution was used as a template. Amplification program: 98°C for 1 min, (95°C for 10 sec, 62°C for 10 sec, 68°C for 10 min) x 35 cycles, 68°C for 15 min. The subsequent embedded PCR was performed using 0.5 μL of the first PCR Product as a template, and 30 μL of a reaction mixture containing: 1 x PCR buffer (free Mg2+); primers, 0.5 nM each; dNTP, 0.5 mM each; MgCl2, 2.5 mM; and TaKaRa LA Taq, 0.3 U. Amplification program: 98°C for 1 min, (95°C for 10 sec, 62°C for 10 sec, 68°C for 10 min) x 30 cycles, 68°C for 15 minutes.

(2-1) Starters

[000601] The primer groups used for inverse PCR are as follows. (2-1-1) Upstream side Prime-PCR: RACE-N-P3-1 and E1-Bub-N-P1 Built-in PCR: RACE-N-P3-2 and E1-Bub-N-P2 (2- 1-2) Downstream side Prime-PCR: RACE-C-P3-1 and E1-Bub-C-P1 Embedded PCR: RACE-C-P3-2 and E1-Bub-C-P2 (2-2) Sequences from Launcher

[000602] The sequences of the primers used for amplification of the upstream and downstream sides by inverse PCR are as follows.

[000603] Primer sequences for amplification of the upstream side RACE-N-P3-1: ATGGAGATAGTGCCGCTGGCAAGG- CAACGGCAC (SEQ ID NO: 43) RACE-N-P3-2: TCAACGAAGACTCGATTTGAG- CGAGAGGCGAGG (SEQ ID NO: 44) E1- Bub-N-P1: ACGTGGAACCGGCATCGTGTTCATG- GACAAC (SEQ ID NO: 45) E1-Bub-N-P2: GCGTGACCCAGTTCCACATGTCGGAC- TGTC (SEQ ID NO: 46) Primer sequences for amplification of the RACE-C-P3-1 downstream side : GACATCCCGTTCGAGCGCAG- GATCACCCATGAG (SEQ ID NO: 47) RACE-C-P3-2: AGGATCACCCATGAGCGCATCGC- TATCATGGAC (SEQ ID NO: 48) E1-Bub-C-P1: CATCGCTCTTGCAGTCGTTGTCCAG- GAAGTCC (SEQ ID NO: 49) E1-Bub -C-P2: TTGTCCAGGAAGTCCATCGCGTACAC- GACGGAG (SEQ ID NO: 50)

[000604] Note that all oligo DNAs used as amplification primers in this Example were synthesized by Sigma-Aldrich Japan.

(3) Purification and Base Sequence Determination of Amplified Inverse PCR, Peripheral Genomic DNA Fragments of Dihydrodaidzein Synthesizing Enzyme Gene

[000605] 10 x day was added to the Embedded PCR product obtained in (2), in an amount of 1/10 of the Embedded PCR product. Then, 5 μL was electrophoresed in 0.8% agarose gel. This confirmed the amplification of 0.5 kb (PstI) and 3.5 kb (XhoI) DNA fragments in the upstream region, and 1 kb (HindIII), 1 kb (SacI), and 2.5 kb (XhoI) of DNA Fragments in the downstream region. In this example, agarose electrophoresis used ethidium bromide (Nippon Gene) for staining, and À/StyI (Nippon Gene) and 100 bp ladder (Toyobo) as molecular weight markers. The amplified DNA fragments were excised from the agarose gel, and purified using a QIAGEN Gel Extraction Kit (Qiagen). The base sequences of the purified DNA fragments were then determined by the direct sequence method and the walking method, using the primers used for amplification.

(4) Genome Sequence Analysis and ORF (Open Reading Frame) Estimation

[000606] The DNA sequences obtained in (3) were subjected to assembly analysis using the SEQUENCHER sequence assembly software (Gene Codes Inc, USA). Furthermore, ORF estimation was done. The analysis determined the 6,685 bp sequence in the peripheral genome region including the E1 enzyme gene. FIG. 15 schematically illustrates the analyzed peripheral genome structure including the dihydrodaidzein synthesizing enzyme gene. ORF estimation found three ORFs upstream of the E1 enzyme gene (the N-terminal unidentified in one of the ORFs), and one ORF on the downstream side. The upstream ORFs were designated US (upstream) 1, US2, and US3, in that order, from the dihydrodaidzein synthesizing enzyme. The downstream ORF was designated DS (downstream) 1.

Example B6: Comparison of Digested Peptide Sequences with Genome Sequence by LC-MS Analysis

[000607] The estimated amino acid sequences obtained in example B4 were compared with the peripheral genomic DNA dihydrodaidzein (E1) synthesizing enzyme gene sequence data determined in example B5. As a result, some of the sequences mainly obtained from LG2 matched the polypeptide sequence inferred from the nucleotide sequence of ORF-US2. This suggests the possibility that the ORF-US2 polypeptide may be the tetrahydrodaidzein-synthesizing enzyme. The digested peptide sequences that matched the ORF-US2 polypeptide are shown below. FIGS. 16-1, 16-2, and 16-3 show the data obtained by LC-MS.Table 1

[000608] In the table above, m/z represents the mass to charge ratio, z the number of charges, Mass the mass of the peptide, and Peptide the estimated amino acid sequence. Score represents the exact percentage of the sequence calculation performed by the PICOS software (100% being maximally accurate). Note that even though isoleucine (I) and leucine (L) have the same molecular weight and are indistinguishable, they are both denoted by X in the digested peptide sequences combining the ORF-US2 polypeptide.

Example B7: Synthesis of ORF-US2 Polypeptide using Acellular Protein Synthesis System and Confirmation of Tetrahydrodaidzein Synthesis Activity

[000609] The ORF-US2 polypeptide was synthesized using the acellular protein synthesis system (PURESYSTEM Classic II mini; Post Genome Institute Co., Ltd.), and its tetrahydrodaidzein synthesis activity was confirmed.

(1) DNA template preparation

[000610] By a two-step PCR, the ORF-US2 polynucleotide DNA template was prepared for acellular protein synthesis. (1-1) Starters

[000611] The primers used for PCR to prepare ORF-US2 DNA template are as follows. E2-invitroTS-FP1: ACTTTAAGAAGGAGATATACCAATGG- CACAGGAAGTCAAAGTCC (SEQ ID NO: 57) AAGAAGGAGATATACCA (SEQ ID NO: 59)

[000612] E2-invitroTS-FP1 and E2-invitroTS-RP were synthesized by Sigma-Aldrich Japan based on the nucleotide sequence of ORF-US2 determined in example B5. The universal primer was obtained from the ligation of PURESYSTEM Classic II mini (Post Genome Institute Co., Ltd.).

(1-2) Preparation of template DNA by Two-Step PCR

[000613] The template DNA used for ORF-US2 polypeptide synthesis was prepared by a two-step PCR according to the manual. In this example, the PCR used Easy-A® High-Fidelity PCR Cloning Enzyme (DNA polymerase, Stratagene), and GeneAmp PCR System 9700 (PCR device, Applied Biosystems).

[000614] In the first PCR, the genomic DNA of the Lactococcus 2092 strain was used as a template to amplify the ORF-US2 nucleotide, using the primers E2-invitroTS-FP1 and E2-invitroTS-RP determined in (1-1). The resulting PCR product (ORF-US2 nucleotide) was used as a template to perform the second PCR, using the universal primer and E2-invitroTS-RP. 300 μL (50 μL x 6) of the PCR product was purified using PCR Purification kit (Qiagen), and used as template DNA (template DNA for ORF-US2 polypeptide synthesis) to synthesize ORF-US2 polypeptide. The First and Second PCR were performed under the following conditions.

[000615] First PCR: A 50 μL reaction mixture containing amplification primers, 10 pmol each; dNTP, 2.5 pmol each; Genomic DNA derived from Lactococcus strain 20-92, 40 ng; Easy-A buffer (Stratagene); and Easy-A® High-Fidelity PCR Cloning Enzyme, 2U (Stratagene). Amplification program: 95°C for 2 min (95°C for 45 sec, 58°C for 20 sec, 72°C for 1 min) x 30 cycles, 72°C for 3 min.

[000616] Second PCR: A 50 μL reaction mixture containing amplification primers, 10 pmol each; dNTP, 2.5 pmol each; first PCR reaction mix, 0.5 μL; Easy-A buffer; and Easy-A® High-Fidelity PCR Cloning Enzyme, 2U. Amplification program: 95°C for 2 minutes, (95°C for 45 seconds, 45°C for 20 seconds, 72°C for 1 minute) x 5 cycles, (95°C for 45 seconds, 60°C for 20 seconds, 72°C for 1 minute) x 25 cycles, 72°C for 3 minutes.

(2) Acellular Protein Synthesis of ORF-US2 Polypeptide and Confirmation of Tetrahydrodaidzein Synthesis Activity

[000617] 25 μL of solution A and 10 μL of solution B of PURESYSTEM Classic II mini, preserved at -80°C, were thawed on ice and mixed together. Next, 0.6 μg (60 ng/μL, 10 μL) of ORF-US2 polypeptide synthesis template DNA (including a T7 promoter sequence and a ribosome binding sequence 5' upstream of the initiator codon) prepared by second PCR was added. The total volume was adjusted to 50 μL by adding sterilized water, and the mixture was incubated at 37°C for 90 minutes to synthesize a target polypeptide. As a positive control of protein synthesis using this system, 0.5 μg (0.2 μg/μL, 2 μL) of dihydrofolate reductase (DHFR) synthesis template DNA, provided as a linkage of PURESYSTEM Classic II mini, was used. No template DNA was added in a negative control, and only sterile water was used. For activity measurement, 40 μL of the reaction mixture was added to an enzyme reaction buffer of the composition below, and the mixture was incubated at 37 ° C for 6 hours. After the reaction, the enzyme reaction mixture was extracted with 3 mL of ethyl acetate. The extract was dried and dissolved in phoresis buffer, and tetrahydrodaidzein in the enzyme reaction mixture was assayed by HPLC analysis. Enzyme Reaction Buffer Composition 0.1 M potassium phosphate buffer/ 1 mM PMSF/ 2 mM DTT/ 5 mM sodium hydrosulfide (pH 7.0) 2 mM NADPH 2 mM NADH 10 μg /mL of dihydrodaidzein

[000618] The samples with the dihydrofolate reductase (DHFR) synthesis template DNA successfully expressed the DNA protein, whereas no protein expression was observed in the samples with no template DNA. These results suggest that the experiment was undoubtedly appropriate.

[000619] The results are shown in FIG. 17. The reaction mixture expressing ORF-US2 polypeptide had a peak for tetrahydrodaidzein at about a 6.7 minute position 6 hours post-reaction, whereas no tetrahydrodaidzein peak was observed in the reaction mixture with the synthesis protein (NC) and the reaction mixture expressing dihydrofolate reductase (DHFR). Furthermore, while the reaction mixture expressing ORF-US2 polypeptide had a reduced level of dihydrodaidzein (substrate) due to the biosynthesis of tetrahydrodaidzein, a reduction of dihydrodaidzein was not observed in the reaction mixture as in protein synthesis (NC) and in the reaction mixture expressing dihydrofolate reductase (DHFR). These results show that the ORF-US2 polypeptide had the activity of synthesizing tetrahydrodaidzein from dihydrodaidzein. It can therefore be said that the ORF-US2 polypeptide corresponds to the E2 polypeptide.

Example B8: Expression of Recombinant ORF-US2 Polypeptide Using Escherichia coli, and Confirmation of Tetrahydrodaidzein Synthesis Activity

[000620] The pET system, a recombinant protein expression system using Escherichia coli, was used to express ORF-US2 polypeptide, and its tetrahydrodaidzein synthesis activity was confirmed.

(1) Preparation of ORF-US2 Polypeptide Expression Vector

[000621] To prepare an ORF-US2 polypeptide expression vector (pET21-US2), DNA in the open reading frame region of ORF-US2 Polypeptide was amplified by PCR.

[000622] The following amplification primers were prepared based on the ORF-US2 polypeptide sequence determined in example B5. exp.US2 pet F Nde: TATACATATGGCACAGGAAGT- CAAAGTC (SEQ ID NO: 60) exp.US2 pet: AATCGAATTCCTAGACCTCGATCTCG- CCCTGC (SEQ ID NO: 61)

[000623] For insertion into pET21a (Novagen), amplification primers exp.US2 pet F Nde and exp.US2 pet were designed to include NdeI and EcoRI restriction sequences, respectively.

[000624] A PCR reaction was performed using 25 μL of a reaction mixture containing the primers, 5 pmol each; dNTP, 5 nmol each; Lactococcus 20-92 strain genomic DNA, 40 ng; 10 x buffer for KOD-plus DNA polymerase (Toyobo), 2.5 μL; KOD-Plus DNA polymerase, 0.3 U (Toyobo). Amplification program: 95°C for 3 min, (94°C for 30 sec, 60°C for 30 sec, 68°C for 1 min) x 30 cycles, 68°C for 7 min. PCR device: GeneAmpPCR System 9700 (Applied Biosystems). Analysis of a portion of the PCR reaction mixture by agarose gel electrophoresis detected a band of an expected size. The entire PCR product was collected by a QIAGEN PCR purification kit (Qiagen).

[000625] The DNA fragments thus collected were cut with restriction enzymes NdeI and EcoRI, and subjected to agarose gel electrophoresis. Then, a portion containing the target band was excised, and purified and collected with a Qiagen Gel Extraction kit (Qiagen). After collection, DNA fragments were ligated at 16°C overnight with pET21a digested with NdeI and EcoRI, using a DNA Ligation Kit ver.2.1 (Takara Bio). The bound reaction mixture was then used to transform Escherichia coli strain JM109 (Takara Bio).

[000626] The transformant was grown at 37°C overnight on an LB medium agar plate (GIBCO) containing ampicillin (50 μg/mL). The resulting single colonies were grown overnight in 3 mL LB medium (GIBCO) containing ampicillin (50 μg/mL). Then, plasmid DNA was extracted using a PI-100 plasmid autoextractor (KURABO).

[000627] The base sequence of the DNA inserted into the plasmid was sequenced using the terminator method. This confirmed the successful polynucleotide insertion of ORF-US2, as intended. In this example, the DNA sequence was determined using ABI3700 DNA sequencer (Applied Biosystems).

(2) Expression and Confirmation of Recombinant ORF-US2 Polypeptide in Escherichia coli

[000628] The recombinant ORF-US2 polypeptide expression plasmid pET21-US2 and plasmid pET21a (negative control) were used to transform the Escherichia coli strain BL21 (DE3) (Novagen). To obtain single colonies, transformants were grown overnight at 37°C on an LB medium agar plate containing ampicillin (50 μg/mL).

[000629] Each E. coli BL21 (DE3) transformant was grown overnight at 37°C in a 3 mL liquid LB medium containing ampicillin (50 μg/mL). Then, 0.5 ml of the culture was pre-cultured for 3 hours (until the OD at 630 nm was about 0.4) by adding 50 ml of liquid LB medium containing the same concentration of ampicillin. After adjusting the final concentration to 1 mM by adding IPTG (isopropyl-p-thiogalactopyranoside; Wako Pure Chemical Industries, Ltd.), the culture was also incubated at 37°C for 4 hours.

[000630] After incubation, cells were collected by centrifugation (6,000 rpm, 4°C, 15 min) using an Avanti HP25 (Beckman Coulter). Subsequent procedures were performed on ice. After removing the supernatant (the medium), cells were suspended in 1 mL of 0.1 M potassium phosphate buffer (pH 7.0; KPB-PDH) containing 1 mM PMSF, 2 mM DTT, and hydrosulfide. 5 mM sodium. The cell suspension was then placed in a 2 mL auxiliary tube loaded with 0.7 mL of zirconia-silica beads (BioSpec Products, Inc.) and 400 μL of KPB-PDH. The cells were then disrupted by two repeated cycles of a 20 second, 6,500 rpm treatment and 3 minute ice cooling using a FastPrep® (Thermo Electron Corporation). As a result, a suspension of ruptured cells was obtained.

[000631] Expression of recombinant ORF-US2 polypeptide in E. coli was confirmed by SDS-polyacrylamide-gel electrophoresis (SDS-PAGE).

[000632] 5 μL of 5x sample buffer (125 mM Tris-HCl (pH 6.5)/25% glycerol/5% SDS/5% 2-mercaptoethanol/0.5% BPB) was added to 20 μL of the disrupted cell suspension. After thermal denaturation at 98°C for 5 minutes, the suspension was cooled with ice, and 10 μL was electrophoresed by SDS-PAGE. SDS-PAGE was performed with a commercially available gel plate (SuperSep® 5-20%; Wako Pure Chemical Industries, Ltd.), and Quick CBB (Wako Pure Chemical Industries, Ltd.) for staining. Prestained XL-Ladder Broad (Apro Science) was used as a molecular weight marker.

[000633] The results of SDS-PAGE are shown in FIG. 18. A recombinant polypeptide with a molecular weight of about 29 kDa was confirmed in the disrupted cell suspension derived from transformant pET21-US2.

(3) Confirmation of Recombinant ORF-US2 Polypeptide Dihydrodaidzein Synthesis Activity

[000634] The ruptured cell suspension obtained in (2) of this Example was used as an enzyme source to measure the conversion activity of dihydrodaidzein to tetrahydrodaidzein. As a result, activity was confirmed in the expressed recombinant ORF-US2 polypeptide.

[000635] In this example, the measurement of the conversion activity of dihydrodaidzein to tetrahydrodaidzein was carried out as follows.

[000636] An enzyme reaction mixture of the composition below was prepared, and the mixture was incubated at 0°C for 2 hours.

[000637] After incubation, 3 mL of ethyl acetate was added to the enzyme reaction mixture for extraction. After drying, the extract was dissolved by the mobile phase (eluent). The dissolved product was analyzed by HPLC to measure the contents of dihydrodaidzein and cis-trans-tetrahydrodaidzeins in the enzyme reaction mixture.

[000638] HPLC analysis results are shown in FIG. 19. In the disrupted cell suspension derived from plasmid pET21-US2 transformant expressing the recombinant ORF-US2 polypeptide, dihydrodaidzein (substrate) added to the enzyme reaction mixture was converted to cis-tetrahydrodaidzein (c-THD) and trans -tetrahydrodaidzein (t-THD), whereas tetrahydrodaidzein was not detected in the enzyme reaction mixture in the pET21a transformant (negative control).

[000639] These results show that recombinant ORF-US2 polypeptide has the activity of synthesizing tetrahydrodaidzein from dihydrodaidzein. It can therefore be said that the recombinant ORF-US2 polypeptide corresponds to the E2 polypeptide.

Example C Example C1: Confirmation of Biosynthetic Activity of Tetrahydrodaidzein Bacterial Cell Equol

[000640] A Lactococcus 20-92 strain (FERM BP-10036) was inoculated into a liquid amplification medium containing tetrahydrodaidzein (a modified GAM broth medium (NISSUI PHARMACEUTICAL CO., LTD.) to which cis- or trans -tetrahydrodaidzein (organically synthesized by Otsuka Pharmaceutical Co., Ltd.: see Reference Example B1) was added in an amount of 10 μg/mL), and the culture was incubated at 37°C for 18 hours under anaerobic conditions (using BBL Gas Pack systems). After incubation, 1 mL of the culture was immediately placed in a glass centrifuge tube with a lid, and 3 mL of ethyl acetate was added to this for extraction. The product was dried to prepare a sample for HPLC analysis. As the standard solution for HPLC analysis, a mixed solution of daidzein (2 μg/mL; Funakoshi Corporation), equol (2 μg/mL; Funakoshi Corporation), dihydrodaidzein (2 μg/mL; Trend Research Chemicals Inc.) , cis -tetrahydrodaidzein and trans -tetrahydrodaidzein (2 μg/mL; both chemically synthesized by Otsuka Pharmaceutical Co., Ltd.) was used.

[000641] The results are shown in Fig. 20. Fig. 20 shows that the Lactococcus 20-92 strain has an activity to biosynthesize equol of both cis- and trans-tetrahydrodaidzeins (Fig. 20, middle and lower graphs). Note that in the figures, daidzein is abbreviated as DZN, dihydrodaidzein as DD, cis-tetrahydrodaidzein as c-THD, trans-tetrahydrodaidzein as t-THD, and equol as EQL.

Example C2: Search and Identification of Equol Synthesizing Enzyme by Recombinant Protein Expression System using Escherichia coli

[000642] The pET system (Novagen), a recombinant protein expression system using Escherichia coli, was used to express polypeptides each corresponding to three ORF polynucleotides (ORF-US3, US1, and DS1) identified in the synthesizing enzyme gene of dihydrodaidzein of peripheral genomic DNA sequence (E1) determined in example B5, in Escherichia coli. The activity to catalyze the conversion of equol to tetrahydrodaidzein was examined to screen for the equol-synthesizing enzyme (E3).

(4) Preparation of ORF Polypeptide Expression Vector

[000643] To prepare a polypeptide expression vector for each ORF (ORF-US3, US1, and DS1), the polynucleotide in the open reading frame region of each polypeptide was amplified by PCR and inserted into the pET21a vector (Novagen).

(1-1) Amplification Starter

[000644] The following amplification primers were prepared based on the peripheral genomic DNA sequence of dihydrodaidzein synthesizing enzyme (E1) determined in example B5. Polypeptide of ORF-US3 exp.US3 F: TATACATATGGCAGAATTCGATGTTGAG (SEQ ID NO: 62) exp.US3 R: CCGCAAGCTTCTACATAGTGGAGATCGCG- TGG ORF-US (SEQ ID NO: 63) Polypeptide of ORF-US1 exp.US1 F: TATACATATGTTCAAGGGTCCACAGGGC (SEQ ID NO: 64) US1 exp. 67)

[000645] For insertion into pET21a (Novagen), amplification primers exp.US3 F, exp.US1 F, and exp.DS1 F were designed to include NdeI restriction sequence, exp.US3 R to include HindIII restriction sequence , and exp.US1R and exp.DS1 R to include the EcoRI restriction sequence.

(1-2) Amplification of Each ORF Polynucleotide

[000646] A PCR reaction was performed using 25 μL of a reaction mixture containing the primers, 5 pmol each; dNTP, 5 nmol each; Genomic DNA from Lactococcus strain 20-92 purified in example A5, 40 ng; 10 x buffer for KOD-plus DNA polymerase (Toyobo Co., Ltd.), 2.5 μL; KOD-Plus DNA polymerase, 0.3 U (Toyobo Co., Ltd.). Amplification program: 95°C for 3 min, (94°C for 30 sec, 60°C for 30 sec, 68°C for 2 min) x 30 cycles, 68°C for 7 min. PCR device: GeneAmp PCR System 9700 (Applied Biosystems). Analysis of a portion of the PCR reaction mixture by agarose gel electrophoresis detected a band of an expected size for each primer. The entire PCR product was collected by a QIAGEN PCR Purification kit (Qiagen).

(1-3) Preparation of ORF Polypeptide Expression Vector

[000647] The ORF-US3 polynucleotide fragments collected in process (1-2) were cut with NdeI and HindIII restriction enzymes, and the ORF-US1 and ORF-DS1 polynucleotide fragments were cut with NdeI and HindIII restriction enzymes. EcoRI. The fragments were subjected to agarose gel electrophoresis. Then, a portion containing the target band was excised, and purified and collected with a Qiagen Gel Extraction kit (Qiagen). After collection, polynucleotide fragments were ligated at 16°C overnight with pET21a digested with NdeI and HindIII or EcoRI, using a DNA Ligation kit ver. 2.1 (Takara Bio). The bound reaction mixture was then used to transform Escherichia coli strain JM109 (Takara Bio) by a usual method. The obtained transformant was grown at 37°C overnight on an LB medium agar plate (GIBCO) containing ampicillin (50 μg/mL). The resulting single colonies were grown overnight in 3 mL LB medium (GIBCO) containing ampicillin (50 μg/mL). Then, plasmid DNA was extracted using a PI-100 plasmid autoextractor (KURABO).

[000648] The base sequence of the DNA inserted into the plasmid was sequenced by the pigment terminator method. This confirmed the successful insertion of each polynucleotide, as intended. In this way, pET-US3, pET-US1, and pET-DS1 were obtained. In this example, the DNA sequence was determined using ABI3700 DNA sequencer (Applied Biosystems).

(5) Expression of Each Recombinant Polypeptide in Escherichia coli' (2-1) Preparation of Escherichia coli BL21 Transformant

[000649] The recombinant ORF polypeptide expression plasmids pET-US3, pET-US1, pET-DS1, and pET21a (negative control) were used to transform the Escherichia coli strain BL21 (DE3) (Novagen) by a usual method . To obtain single colonies, transformants were grown overnight at 37°C on an LB medium agar plate containing ampicillin (50 μg/mL).

(2-2) Induction of Recombinant Polypeptide Expression

[000650] Each E. coli BL21 (DE3) transformant was grown overnight at 37°C in a 3 mL liquid LB medium containing ampicillin (50 μg/mL). Then, 0.5 mL of the culture was pre-cultured for 3 hours (until the OD at 630 nm was approximately 0.4 to 0.7) by adding 50 mL of liquid LB medium containing the same concentration of ampicillin. After adjusting the final concentration to 1 mM by adding IPTG (isopropyl-p-thiogalactopyranoside; Wako Pure Chemical Industries, Ltd.), the culture was further incubated at 37°C for 4 hours. In this way, recombinant polypeptide expression in Escherichia coli was induced.

(2-3) Preparation of Disrupted Cell Suspension

[000651] After induction of expression in process (2-2), cells were collected by centrifugation (6,000 rpm, 4°C, 15 minutes) using an Avanti HP25 (Beckman Coulter). Subsequent procedures were performed on ice. After removing the supernatant (the medium), cells were suspended in 1 mL of 0.1 M potassium phosphate buffer (pH 7.0; hereafter abbreviated as KPB-PDH) containing 1 mM PMSF, 2 µM DTT mM, and 5 mM sodium hydrosulfide. The cell suspension was then placed in a 2 mL auxiliary tube loaded with 0.7 mL of zirconia-silica beads (BioSpec Products, Inc.) and 400 μL of KPB-PDH. The cells were then disrupted by two repeated cycles of a 20 second, 6,500 rpm treatment and 3 minute ice cooling, using a FastPreTM (Thermo Electron Corporation). As a result, a suspension of ruptured cells was obtained.

(2-4) Confirmation of Recombinant Polypeptide Expression by SDS-Polyacrylamide-Gel Electrophoresis (SDS-PAGE)

[000652] The expression of each recombinant ORF polypeptide in E. coli was confirmed by SDS-PAGE. 5 μL of 5x sample buffer (125 mM Tris-HCl (pH 6.5)/25% glycerol/5% SDS/5% 2-mercaptoethanol/0.5% BPB) was added at 20°C. μL of the ruptured cell suspension obtained in process (2-3). After thermal denaturation at 98°C for 5 minutes, the suspension was cooled with ice, and 4 μL was electrophoresed by SDS-PAGE. SDS-PAGE was performed with a commercially available gel plate (SuperSep® 5-20%; Wako Pure Chemical Industries, Ltd.), and Quick CBB (Wako Pure Chemical Industries, Ltd.) for staining. The Prestained XL-Ladder Broad strip (Apro Science) was used as a molecular weight marker. The SDS-PAGE results are shown in FIG. 21. Expression of recombinant ORF-US3 and ORF-DS1 polypeptides with molecular weights of about 52 kDa and 50 kDa was confirmed in disrupted cell suspensions derived from pET-US3 and pET-DS1 transformants, respectively. No expression of recombinant ORF-US1 polypeptide was observed in the disrupted cell suspension derived from pET-US1 transformant.

Example C3: Measurement of Recombinant ORF Polypeptide Equol Synthesis Activity.

[000653] A ruptured cell suspension obtained in example C2 was used as an enzyme source to measure the activity of converting tetrahydrodaidzein to equol. In this example, measurement of the conversion activity of tetrahydrodaidzein to equol was performed as follows.

[000654] An enzyme reaction mixture of the composition below was prepared, and the mixture was incubated at 37°C for 1 hour.

[000655] After incubation, 3 mL of ethyl acetate was added to the enzyme reaction mixture for extraction. After drying, the extract was dissolved by the mobile phase (eluent). The dissolved product was analyzed by HPLC to measure the contents of dihydrodaidzein, cis-tetrahydrodaidzein, trans-tetrahydrodaidzein, and equol in the enzyme reaction mixture. As the standard solution for HPLC analysis, a mixed solution of daidzein (2 μg/mL; Funakoshi Corporation), equol (2 μg/mL; Funakoshi Corporation), dihydrodaidzein (2 μg/mL; Trend Research Chemicals Inc.), cis -tetrahydrodaidzein and trans-tetrahydrodaidzein (2 μg/mL; both chemically synthesized by Otsuka Pharmaceutical Co., Ltd.) was used.

[000656] As a result of HPLC analysis, in the disrupted cell suspension derived from plasmid pET-US3 transformant expressing the recombinant ORF-US3 polypeptide, the tetrahydrodaidzein added to the enzyme reaction mixture was converted to equol ( Fig. 22). Additionally, the conversion activity for dihydrodaidzein, which is a precursor of tetrahydrodaidzein, was confirmed during the equol biosynthetic reaction series (Fig. 22). As for transformants pET-US1 and pET-DS1, and transformant pET21a (negative control), only tetrahydrodaidzein was detected in the enzyme reaction mixture. Note that in the figures, aidzein is abbreviated as DZN, dihydrodaidzein as DD, cis-tetrahydrodaidzein as c-THD, trans-tetrahydrodaidzein as t-THD, and equol as EQL.

[000657] These results show that ORF-US3 polypeptide has the activity of biosynthesizing tetrahydrodaidzein equol. Therefore, it is said that the recombinant ORF-US3 polypeptide corresponds to the E3 polypeptide.

Example D Example D1: Expression and Purification of Enzyme Related to the Production of Recombinant His-Tagged Equol Using Escherichia coli

[000658] When using a pET system (Novagen), which is a recombinant protein expression system using Escherichia coli, three enzymes related to the production of His-tagged equol: dihydrodaidzein (E1) synthesis enzyme, etetrahydrodaidzein (E2), equol synthesis enzyme (E3), were expressed, followed by affinity purification using the His-labeled protein purification column (Ni).

(6) Production of His-Tagged Enzyme Expression Vector

[000659] To produce the expression vectors for the enzymes (E1, E2, E3), a polynucleotide in each open reading frame region was amplified by PCR and inserted into a pET21a vector (Novagen).

(1-1) Amplification Starter

[000660] Based on the genome sequence in the vicinity of the dihydrodaidzein synthesis enzyme (E1) found in example B5, the following amplification primers were produced. E1 enzyme labeled by His exp.E1 pet F Nde:AGCTCATATGAAGAACAAGTTCTATCCGAA (Sequence Number:41) exp.E1 pet His:AATCGAATTCGTACAGGTTGCAGCCAGCGATGT (Sequence Number:42) E2 enzyme labeled by His exp.US2 pet F Nde:TATACATATGGCACAGGAAGTCAAAGTC (Sequence Number :60) exp.E2 pet His:AATCGAATTCGAGACCTCGATCTCGCCCTGC (Sequence Number:68) E3 enzyme labeled by His exp.US3 F:TATACATATGGCAGAATTCGATGTTGAG (Sequence Number:62) exp.E3 R His:CCGCAAGCTTGTACATAGTGGAGATCGCGTGG (Sequence Number:69)

[000661] For insertion into pET21a (Novagen), amplification primers: exp.E1 pet F Nde, exp.US2 pet F, and exp.US3 F were designed to each include a restriction enzyme NdeI cleavage site sequence ; exp.E1 pet His and exp.E2 pet His were designed to include an EcoRI cleavage site sequence; and exp.E3 R His was designed to include a HindIII cleavage site sequence.

(1-2) Amplification of His-Tagged Enzyme Polynucleotides

[000662] A PCR reaction was performed using 25 μL of a reaction mixture containing the primers, 5 pmol each; dNTP, 5 nmol each; Genomic DNA from Lactococcus strain 20-92 (FERM BP-10036) purified in example A5, 40 ng; 10* buffer for KOD-plus DNA polymerase (Toyobo), 2.5 μL; KOD-Plus DNA polymerase (Toyobo) 0.3 U, using an amplification program: 95°C for 3 minutes, (94°C for 30 sec, 60°C for 30 sec, 68°C for 2 minutes) * 30 cycles, 68°C for 7 minutes (PCR device: GeneAmpPCR System 9700 (Applied Biosystems)). Analysis of a portion of the PCR reaction mixture by agarose gel electrophoresis detected a band of an expected size. Total PCR product was removed by QIAGEN PCR Purification kit (Qiagen).

(1-3) Production of His-Tagged Enzyme Polypeptide Expression Vector

[000663] The His-labeled enzyme polynucleotide fragments collected in (1-2) were cut with restriction enzymes NdeI and EcoRI, and subjected to agarose gel electrophoresis. Then, a portion containing the target band was excised, purified, and collected with Qiagen Gel Extraction kit (Qiagen). After collection, polynucleotide fragments were ligated at 16°C overnight with pET21a digested with NdeI and EcoRI, using DNA Ligation Kit ver.2.1 (Takara Bio). With the reaction mixture turned on, transformation of the Escherichia coli strain JM109 (Takara Bio) was carried out in a general method. The transformant thus obtained was grown at 37°C overnight on an LB medium agar plate (GIBCO) containing ampicillin (50 μg/mL). The resulting single colonies were grown overnight in 3 mL LB medium (GIBCO) containing ampicillin (50 μg/mL). Then, plasmid DNA was extracted using PI-100 plasmid autoextractor (KURABO).

[000664] The base sequence of the DNA inserted into the plasmid was sequenced by the pigment terminator method. This confirmed the successful polynucleotide insertion of ORF-US2 as intended. pET-E1-His, pET-E2-His, and pET-E3-His were obtained. In this example, the DNA sequence was determined using ABI3700 DNA sequencer (Applied Biosystems).

(7) Expression and Affinity Purification of Each His-Tagged Recombinant Enzyme Polypeptide in Escherichia coli' (2-1) Production of Escherichia coliBL21 transformant

[000665] By using the plasmid pET-E1-His, pET-E2-His, pET-E3-His to express the His-labeled enzyme polypeptide, Escherichia coli strains BL21(DE3) (Novagen) were transformed using a method general. Transformants were grown at 37°C overnight on LB medium agar plates (GIBCO) containing ampicillin (50μg/mL), thereby obtaining single colonies.

(2-2) Purification of His-Tagged E1 and E2 Recombinant Enzyme Polypeptide (2-2-1) Cultivation of Escherichia coli and Induction of Expression of His-Tagged E1 and E2 Recombinant Enzyme Polypeptides

[000666] Each of the E. coli BL21 (DE3) transformants, respectively transformed by pET-E1-His and pET-E2-His, was grown overnight at 37°C in a 10 mL liquid LB medium containing amps. cylin (50 μg/mL). Then, 7.5 mL of the culture was pre-cultured for 2 hours (until the OD at 600 nm was approximately 0.5) by adding 150 mL of liquid LB medium containing the same concentration of ampicillin. After adjusting the final concentration to 0.5 mM by adding IPTG (isopropyl-β-thiogalactopyranoside; Wako Pure Chemical Industries, Ltd.), the culture was also incubated at 30°C for 4 hours while being gently shaken. , thereby inducing the expression of His-labeled E1 and E2 recombinant enzyme polypeptides in Escherichia coli.

(2-2-2) Lysate Preparation

[000667] After induction of expression in (2-2), the culture was centrifuged (6000 rpm, 4°C, 10 minutes) using Avanti HP25 (beckman coulter) to collect the cells, thereby obtaining recombinant E1 enzyme polypeptides and His-tagged E2 expressed in Escherichia coli, respectively in amounts of 0.66 g and 0.73 g. A Bugbuster Protein Extraction solution (Novagen) was added to the obtained cells in an amount of 15 mL per gram (wet weight) of the cell. The mixture was suspended gently with a pipette, and Lysozyme (SIGMA) and Benzonase (Novagen) were added to the suspension in amounts of 2000 units/mL, and 25 units (1 μL)/mL, respectively. Then, the mixture was slowly stirred with a rotor (RT-50:Taitec) at room temperature for 30 minutes, thereby obtaining a lysate (Lysate A). Lysate A was centrifuged (8000 rpm, 4°C, 15 minutes) with Avanti HP25 (beckman coulter) to separate a supernatant (Lysate B).

(2-2-3) Affinity Purification of E1 and E2 His-Tagged Enzyme Polypeptides

[000668] When using His GraviTrap (GE Healthcare Bioscience) as a His-labeled protein purification column, affinity purification of the His-labeled enzyme polypeptides E1 and E2 was performed according to the instruction manual except for some modifications . More specifically, His GraviTrap was equilibrated with ice-cold 10 mL binding buffer, and total Lysate B prepared in (2-2-2) was poured into the mixture so that the target His-labeled E1 or E2 enzymes were adhered to. His GraviTrap for freefall. Therefore, His GraviTrap was cleaned twice with ice-cold 10 mL cleaning buffer, followed by elution of the target His E1 and E2 labeled enzymes from His GraviTrap using 3 mL elution buffer. DTT (Dithiothreitol: Wako Pure Chemical Industries, Ltd.) was added to the eluate so that the final concentration was 3 mM. The mixture was divided into 300 μL microtubes. A part of the microtubes was examined for their E1 and E2 enzymatic activities in the respective eluates. The eluate was preserved at 4°C until it was used for enzyme testing.

[000669] The compositions of the binding buffer, the binding buffer, and the elution buffer used for purification were shown below.

[000670] Binding buffer: 20 mM Tris-HCl, 20 mM Imidazole (Wako Pure Chemical Industries, Ltd.), 0.5M NaCl (Wako Pure Chemical Industries, Ltd.), 1 mM DTT (Dithiothreitol: Wako Pure Chemical Industries, Ltd.), 1 mM PMSF (phenylmethylsulfonyl fluoride: Wako Pure Chemical Industries, Ltd.)

[000671] Cleaning Buffer: 20 mM Tris-HCl, 60 mM Imidazole, 0.5M NaCl, 1mM DTT (Dithiothreitol), 1mM PMSF (phenylmethylsulfonyl fluoride)

[000672] Elution Buffer: 20 mM Tris-HCl, 500 mM Imidazole, 0.5M NaCl, 1 mM DTT (Dithiothreitol), 1 mM PMSF (phenylmethylsulfonyl fluoride).

(2-3) Purification of His-Tagged E3 Recombinant Enzyme Polypeptide (2-3-1) Cultivation and Induction of Escherichia coli His-Tagged E3 Recombinant Enzyme Polypeptide Expression

[000673] The E. coli BL21 (DE3) transformant, transformed by pET-E3-His, was grown overnight at 37°C in a 50 mL liquid LB medium containing ampicillin (50 μg/mL). Then, 20 mL of the culture was pre-cultured for 3 hours (until the OD at 600 nm was approximately 0.4) by adding 1 L of liquid LB medium containing the same concentration of ampicillin. After adjusting the final concentration to 0.5 mM by adding IPTG (isopropyl-β-thiogalactopyranoside; Wako Pure Chemical Industries, Ltd.), the culture was also incubated at 37°C for 4 hours while being shaken, thereby inducing the expression of His-tagged recombinant E3 enzyme polypeptide in Escherichia coli. After expression induction, the culture was centrifuged (6000 rpm, 4°C, 10 minutes) using Avanti HP25 (beckman coulter) to collect the cells. 0.1 M potassium phosphate buffer containing 1 mM PMSF (phenylmethylsulfonyl fluoride), 2 mM DTT (Wako Pure Chemical Industries, Ltd.), and 5 mM sodiohydrosulfide (Buffer A, below: pH 7.0 ) was added for suspension. After centrifugation (6000 rpm, 4°C, 10 minutes), the culture was maintained at -80°C.

(2-3-2) Lysate Preparation

[000674] Cells preserved at -80°C were quickly thawed and centrifuged (10,000xg 15 min). Cells were suspended by adding 25 mL of new Buffer A. For disruption, cells were then disrupted by three repeated cycles of a 20 second, 6,500 rpm treatment and 3 minute ice cooling, using FastPrep® (Thermo Electron Corporation). The resulting disrupted cellular material was centrifuged (10,000 Xg, 15 minutes). The supernatant was separated to obtain a lysate.

(2-3-3) Affinity Purification of His-Tagged E3 Recombinant Enzyme Polypeptide

[000675] 4 mL of the lysate obtained in (2-3-3) was supplied to His-Trap HP (GE Healthcare Bioscience), which is a His-tag fusion protein purification column, to purify the recombinant enzyme polypeptide His-tagged E3 under the purification conditions below. Protein was measured based on absorption at 280 nm. Fraction using a fraction collector was started after Absorption at 280 nm in passing through the fraction decreased to the reference value. Flow Rate: 1 ml/min Fraction: 2 ml/2 min/Fraction Eluent A: 0.1 M potassium phosphate buffer (pH 7)/0.5 M NaCl /1% isoPrOH Eluent B: Eluent A containing 0.5M Imidazole Elution Program:

[000676] E3 synthesis activity was observed in Fractions No.7 to 9. The active fractions obtained by a plurality of purifications were pooled, mixed with glycerin at a ratio of 8%, kept at -28°C, and dissolved when required to be used for the experiment.

Example D2: Synthesis of Tetrahydrodaidzein from Daidzein Using Recombinant His-Tagged E1 and E2 Enzymes

[000677] By using the His-labeled recombinant E1 and E2 enzymes obtained in the (2-2-3) process of Example D1 as an enzyme source, an enzyme reaction mixture of the composition below was prepared, and was reacted at 37 °C for 2 hours to synthesize tetrahydrodaidzein from daidzein. Additionally, reactions using His-labeled recombinant E1 or E2 enzyme solely as an enzyme source were conducted at the same time.

[000678] After incubation, 3 mL of ethyl acetate (Wako Pure Chemical Industries, Ltd.) was added to the enzyme reaction mixture obtained for extraction. After drying, the extract was dissolved by the mobile phase (eluent). The dissolved product was analyzed by HPLC to measure the contents of cis- and trans-tetrahydrodaidzeins in the enzyme reaction mixture.

[000679] As the standard solution for HPLC analysis, a mixed solution of daidzein (2 μg/mL; Funakoshi Corporation), equol (2 μg/mL; Funakoshi Corporation), dihydrodaidzein (2 μg/mL; Trend Research Chemicals Inc. ), cis-tetrahydrodaidzein (2 μg/mL; Reference Example B1) and trans-tetrahydrodaidzein (2 μg/mL; Reference Example B1) were used.

[000680] FIG. 23 shows the results of HPLC analysis. When using the mixture of His-labeled recombinant E1 and E2 enzymes as an enzyme source, cis- and trans-tetrahydrodaidzeins were confirmed in the product. When using the recombinant His-labeled E1 or E2 enzyme solely as an enzyme source, however, cis- or trans-tetrahydrodaidzeins were not confirmed in the product.

Example D3: Synthesis of Dihydrodaidzein Equol Using Recombinant His-Tagged E2 and E3 Enzymes

[000681] When using the His-labeled recombinant E2 and E3 enzymes obtained in the processes (2-2-3) and (2-3-3) of Example D1 as an enzyme source, an enzyme reaction mixture of the composition below was prepared, and was reacted at 37°C for 2 hours to synthesize dihydrodaidzein equol. Additionally, reactions using His-labeled recombinant E1 or E2 enzyme solely as an enzyme source were conducted at the same time.

[000682] After incubation, 3 mL of ethyl acetate (Wako Pure Chemical Industries, Ltd.) was added to the enzyme reaction mixture obtained for extraction. After drying, the extract was dissolved by the mobile phase (eluent). The dissolved product was analyzed by HPLC to measure the equol content in the enzyme reaction mixture.

[000683] FIG. 24 shows the results of HPLC analysis. When using the mixture of His-labeled recombinant E2 and E3 enzymes as an enzyme source, equol was confirmed in the product. When using the recombinant His-labeled E2 or E3 enzyme solely as an enzyme source, however, equol was not confirmed in the product.

Example D4: Synthesis of Daidzein Equol Using Recombinant His-Tagged E1, E2, and E3 Enzymes

[000684] When using the His-labeled recombinant enzymes E1, E2, and E3 obtained in processes (2-2-3) and (2-3-3) of Example D1 as an enzyme source, an enzyme reaction mixture of the composition below was prepared, and was reacted at 37°C for 2 hours to synthesize daidzein equol. Additionally, reactions using the recombinant His-labeled E1, E2, or E3 enzyme solely as an enzyme source were conducted at the same time.

[000685] After incubation, 3 mL of ethyl acetate (Wako Pure Chemical Industries, Ltd.) was added to the enzyme reaction mixture obtained for extraction. After drying, the extract was dissolved by the mobile phase (eluent). The dissolved product was analyzed by HPLC to measure the equol content in the enzyme reaction mixture.

[000686] FIG. 25 shows the HPLC Analysis results. When using the recombinant His-tagged E1, E2, or E3 enzyme solely as an enzyme source, equol was not confirmed in the product. When using the mixture of His-labeled E1, E2, and E3 recombinant enzymes as an enzyme source, however, equol was confirmed in the product.

Example E: Influence of metal ions on dihydrodaidzein synthesis activity of His-labeled E1 Recombinant Enzyme

[000687] By using the recombinant His-labeled E1 enzyme (E1-His) expressed in Escherichia coli, purified by Ni-Sepharose, as an enzyme source, the influence of metal ions on the conversion activity of daidzein to dihydrodaidzein was examined . Various metals (MnCh2H2O, FeSO4-7H2O, CaCh2H2O, Zn(CH3COO)2-2H2O, CoSO4-7H2O, MgSO4-7H2O, NiSO4-6H2O) were dissolved in distilled water to obtain a concentration of 100 mM. By using the metal ion solution, an enzyme reaction mixture of the composition below was prepared, and it was reacted at 37°C for 2 hours to measure the activity.

[000688] After incubation, 3 mL of ethyl acetate was added to the enzyme reaction mixture obtained for extraction. After drying, the extract was dissolved by the mobile phase (eluent). The dissolved product was analyzed by HPLC to measure the daidzein and dihydrodaidzein contents in the enzyme reaction mixture. As a result, it was confirmed that Mn2+ and Fe2+ stimulated the activity (Fig. 26).

Brief Description of the Drawings

[000689] FIG. 1 shows the results of HPLC analysis in example A1. Three upper chromatographic: Enzyme reaction products using no coenzymes (control), and NADH and NADPH as coenzymes. Bar diagram: Peak areas corresponding to dihydrodaidzein in control (no coenzyme) and in samples using NADH and NADPH as coenzymes.

[000690] FIG. 2 shows the Mono Q HPLC results in example A2 (top), and the dihydrodaidzein synthesizing enzyme activity of each fraction (bottom). Enzyme activity is shown as a peak area corresponding to dihydrodaidzein using daidzein as a substrate.

[000691] FIG. 3 shows the SDS-PAGE results in example A2 (left part), and the dihydrodaidzein synthesizing enzyme activity of each fraction (right part). Enzyme activity is shown as a peak area corresponding to dihydrodaidzein using daidzein as a substrate. SuperSep HG 10-20% (Wako Pure Chemical Industries, Ltd.) was used as an electrophoresis gel plate.

[000692] FIG. 4 shows the peptide mapping results in example A4, and peptide amino acid sequences corresponding to the peaks. The numbers next to the upper HPLC analysis diagram arrows correspond to 1:FDEPVYPQAE, 2:ASRMVMDAVHEGYIAG, and 3:GYIGNLEVENRAIRMPM respectively.

[000693] FIG. 5 shows the results of agarose electrophoresis of degenerative PCR product in example A5. (1) E1-N-terminal-31 and E1-internal-RP1, (2) E1-N-terminal-37 and E1-internal-RP1, (3) E1-N-terminal-F32 and E1-internal-RP1 ; marker M1: À/Styl, M2: 100 bp ladder.

[000694] FIG. 6 shows the results of agarose electrophoresis of RT-PCR product in example A7. Top: dihydrodaidzein synthase; bottom: Ribosomal-RNA.

[000695] FIG. 7 shows the results of SDS-PAGE in example A8.

[000696] FIG. 8 shows the results of HPLC analysis in example A8. Top: Control; central part: pET-E1-His; bottom: pET21a.

[000697] FIG. 9 shows the results of HPLC analysis in example B1. Top diagram: Cis-tetrahydrodaidzein (REF-000312); central diagram: Enzyme reaction product using dihydrodaidzein as a substrate and disrupted cellular material as an enzyme source; lower diagram: Trans-tetrahydrodaidzein (REF-000313).

[000698] FIG. 10 shows the results of HPLC analysis in example B1. Top two diagrams: Enzyme reaction product using cis-tetrahydrodaidzein (REF-000312) as a substrate and disrupted cellular material as an enzyme source; bottom two diagrams: Enzyme reaction product using trans-tetrahydrodaidzein (REF-000313) as a substrate and disrupted cellular material as an enzyme source.

[000699] FIG. 11 shows the results of HPLC analysis in example B2. Top three diagrams: Enzyme reaction products using no coenzymes (control), and NADH and NADPH as coenzymes. Bar diagram: Peak areas corresponding to tetrahydrodaidzein in control (no coenzyme) and in samples using NADH and NADPH as coenzymes.

[000700] FIG. 12 shows the results of HPLC gel filtration analysis in example B3. Top graph: The results for standard protein; central graph: Enzyme activity of each fraction shown as a peak area corresponding to the product tetrahydrodaidzein; lower graph: Absorption intensity (280 nm) corresponding to the protein of each fraction.

[000701] FIG. 13 shows the results of SDS-PAGE in example B3.

[000702] FIG. 14 shows the results of SDS-PAGE in example B4.

[000703] FIG. 15 shows a schematic illustration of a peripheral genome structure of dihydrodaidzein synthesizing enzyme gene (E1) determined in example B5.

[000704] FIG. 16-1 shows LC-MS analysis data, and an example of a digested peptide amino acid sequence inferred from the LC-MS data in example B6; L, an amino acid residue denoted by X in the description. The top figure is for the peptide TPGVAASVADEXK, and the bottom figure is for the peptide MPGAPVFGK.

[000705] FIG. 16-2 shows LC-MS analysis data, and an example of a digested peptide amino acid sequence inferred from the LC-MS data in example B6; L, an amino acid residue denoted by X in the description. The top figure is for the peptide KXXXTG-TTK and the bottom figure is for the peptide VTQEXXCAHGAFVCGSGR.

[000706] FIG. 16-3 shows LC-MS analysis data, and an example of a digested peptide amino acid sequence (WXSPEE-SVGQR) inferred from the LC-MS data in example B6; L, an amino acid residue denoted by X in the description.

[000707] FIG. 17 shows the results of HPLC analysis in example B7. Top left diagrams: Enzyme reaction product using dihydrodaidzein as a substrate and a reaction mixture expressing ORF-US2 polypeptide as an enzyme source (ORF-US2); upper right diagrams: Enzyme reaction product using a protein-free reaction mixture as an enzyme source (NC). Bar diagram: Peak areas corresponding to dihydrodaidzein (DD) and tetrahydrodaidzein (THD) produced by enzyme reactions in NC (in protein synthesis), and in samples using DHFR (reaction mixture expressing dihydrofolate reductase) and ORF- US2 (Reaction mix expressing the ORF-US2 polypeptide) as an enzyme source. FIG. 18 shows the results of SDS-PAGE in example B8.

[000708] FIG. 19 shows the results of HPLC analysis in example B8. Top diagram: Enzyme reaction product using dihydrodaidzein as a substrate, and a disrupted cell suspension derived from plasmid transformant pET21-US2 expressing the recombinant ORF-US2 polypeptide as an enzyme source (pET21-US2); bottom diagram: Enzyme reaction product using dihydrodaidzein as a substrate, and a disrupted cell suspension derived from pET21a transformant (negative control) as an enzyme source (pET21a). Bar diagram: Peak areas corresponding to dihydrodaidzein (DD) and cis-(c-THD) and trans-tetrahydrodaidzeins (t-THD) produced by enzyme reactions in samples using disrupted cell suspensions derived from reaction mixture expressing ORF- polypeptide US2 and pET21a transformant as an enzyme source.

[000709] FIG. 20 shows the results of HPLC analysis in example C1.

[000710] FIG. 21 shows the results of SDS-PAGE in example C2.

[000711] FIG. 22 shows the results of HPLC analysis in example C3. Top: Standard; central part: pET-US3; lower part: pET21a.

[000712] FIG. 23 shows the results of HPLC analysis in example D2. First part from the top: Standard; second: E1 and E2; third: E1; Wednesday: E2.

[000713] FIG. 24 shows the results of HPLC analysis in example D3. First part from the top: Standard; second: E2 and E3; third: E2; Wednesday: E3.

[000714] FIG. 25 shows the results of HPLC analysis in example D4. First part from the top: Standard; second: E1, E2 and E3; third: E1; fourth: E2; Thursday: E3.

[000715] FIG. 26 shows the effects of metal ions on E1 enzyme activity.

[000716] FIG. 27 shows the alignment of amino acid sequences of SEQ ID NOs: 1, 2 and 3.

[000717] FIG. 28 shows the alignment of amino acid sequences of SEQ ID NOs: 7, 8 and 9.

[000718] FIG. 29 shows the alignment of amino acid sequences of SEQ ID NOs: 13, 14 and 15. Sequence Listing Free Text SEQ ID NO: 23 is the E1-N-terminal-31 primer base sequence. SEQ ID NO: 24 is the base sequence of primer E1-N-terminal-37. SEQ ID NO: 25 is the E1-N-terminal-F32 primer base sequence. SEQ ID NO: 26 is the base sequence of primer E1-internal-RP1. SEQ ID NO: 27 is the pUC19-FP-1 primer base sequence. SEQ ID NO: 28 is the pUC19-RP-1 primer base sequence. SEQ ID NO: 29 is the pUC19-FP-2 primer base sequence. SEQ ID NO: 30 is the pUC19-RP-2 primer base sequence. SEQ ID NO: 31 is the base sequence of primer E1-RACE-N-P1. SEQ ID NO: 32 is the base sequence of primer E1-RACE-RP2-1. SEQ ID NO: 33 is the base sequence of primer E1-RACE-N-P2. SEQ ID NO: 34 is the base sequence of primer E1-RACE-RP2-2. SEQ ID NO: 35 is the base sequence of primer E1-conf-NP. SEQ ID NO: 36 is the base sequence of E1-conf-CP primer. SEQ ID NO: 37 is the E1-FP primer base sequence. SEQ ID NO: 38 is the E1-RP primer base sequence. SEQ ID NO: 39 is the Gar-16S-Ribo-FP primer base sequence. SEQ ID NO: 40 is the Gar-16S-Ribo-RP primer base sequence. SEQ ID NO: 41 is the base sequence of primer exp.E1 pet F Nde. SEQ ID NO: 42 is the exp primer base sequence. E1 pet His. SEQ ID NO: 43 is the base sequence of primer RACE-N-P3-1. SEQ ID NO: 44 is the RACE-N-P3-2 primer base sequence. SEQ ID NO: 45 is the E1-Bub-N-P1 primer base sequence. SEQ ID NO: 46 is the base sequence of primer E1-Bub-N-P2. SEQ ID NO: 47 is the base sequence of primer RACE-C-P3-1. SEQ ID NO: 48 is the base sequence of primer RACE-C-P3-2. SEQ ID NO: 49 is the base sequence of primer E1-Bub-C-P1. SEQ ID NO: 50 is the base sequence of primer E1-Bub-C-P2. SEQ ID NO: 57 is the base sequence of E2-invitroTS-FP primer. SEQ ID NO: 58 is the base sequence of E2-invitroTS-RP primer. SEQ ID NO: 59 is the Universal-Primer base sequence. SEQ ID NO: 60 is the base sequence of primer exp.US2 pet F Nde. SEQ ID NO: 61 is the exp primer base sequence. US2 pet. SEQ ID NO: 62 is the base sequence of primer exp.US3 F. SEQ ID NO: 63 is the base sequence of primer exp.US3 R. SEQ ID NO: 64 is the base sequence of primer exp.US1 F SEQ ID NO: 65 is the base sequence of primer exp.US1 R. SEQ ID NO: 66 is the base sequence of primer exp.DS1 F. SEQ ID NO: 67 is the base sequence of primer exp.DS1 A.

Claims (12)

1. Process for producing equol, characterized by the fact that it comprises the step of having an enzymatic composition for synthesizing equol that acts on tetrahydrodaidzein, wherein the composition for synthesizing equol comprises a polypeptide consisting of the amino acid sequence of SEQ ID NO : 13, hydrosulfite and NADPH and/or NADH. 2. Process for producing equol, according to claim 1, characterized by the fact that the equol synthesis enzymatic composition further comprises dithiothreitol. 3. Process for producing tetrahydrodaidzein, which is used as an intermediate in the synthesis of equol by the process as defined in claim 1 or 2, characterized in that it comprises an enzymatic composition for the synthesis of tetrahydrodaidzein that acts on dihydrodaidzein, in which the Tetrahydrodaidzein synthesis enzymatic composition comprises a polypeptide consisting of the amino acid sequence of SEQ ID NO: 7, hydrosulfite and NADPH and/or NADH. 4. Process for producing tetrahydrodaidzein, according to claim 3, characterized by the fact that the enzymatic composition for synthesizing tetrahydrodaidzein further comprises dithiothreitol. 5. Process for producing dihydrodaidzein, which is used as an intermediate in the synthesis of equol by the process as defined in claim 1 or 2, characterized in that it comprises an enzymatic composition for synthesizing dihydrodaidzein that acts on daidzein, wherein the composition of dihydrodaidzein synthesis enzyme comprises a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, hydrosulfite and NADPH and/or NADH. 6. Process for producing dihydrodaidzein, according to claim 5, characterized by the fact that the enzymatic composition for synthesizing dihydrodaidzein further comprises dithiothreitol. 7. Process for producing tetrahydrodaidzein, according to claim 3, characterized by the fact that it optionally comprises the following First Step and Second Step, wherein the First Step comprises a step of having an enzyme consisting of a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, hydrosulfite and NADPH and/or NADH that acts on daidzein, thus producing dihydrodaidzein; wherein the Second Step comprises a step of having an enzyme consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO: 7, hydrosulfite and NADPH and/or NADH that acts on dihydrodaidzein, thereby producing tetrahydrodaidzein. 8. Process according to claim 7, characterized by the fact that both the First Step and the Second Step further comprise dithiothreitol. 9. Process according to claim 1 or 2, characterized in that it further comprises a step of having an enzyme consisting of a polypeptide consisting of the amino acid sequence of SEQ ID NO: 7, hydrosulfite and NADPH and/or NADH that acts into dihydrodaidzein, thus producing tetrahydrodaidzein. 10. Process according to claim 9, characterized by the fact that it further comprises dithiothreitol. 11. Process according to claim 1 or 2, characterized in that it further comprises a step of having an enzyme consisting of a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, hydrosulfite and NADPH and/or NADH that acts into daidzein, thus producing dihydrodaidzein. 12. Process according to claim 11, characterized by the fact that it further comprises dithiothreitol.