Method for producing taxifolin

Abstract

A method for enzymatically producing taxifolin, and the enzyme for that purpose are provided. [Solution] A method for producing taxifolin, comprising converting quercetin to taxifolin using at least one enzyme selected from the group consisting of an enzyme having the amino acid sequence of SEQ ID NO: 1, an enzyme having the amino acid sequence of SEQ ID NO: 2, an enzyme having the amino acid sequence of SEQ ID NO: 3, and an enzyme having an amino acid sequence that is at least 90% identical to any of SEQ ID NOs: 1 to 3.

Description

[Technical Field]

[0001] This invention relates to a method for enzymatically producing taxifolin, and to an enzyme for that purpose. [Background technology]

[0002] Taxifolin, also known as dihydroquercetin, is a flavonoid. It has been reported to possess various physiological activities, including antioxidant, anti-glycation, anti-inflammatory, blood flow improvement, and vascular protection properties. It is also being studied as a component of pharmaceuticals or foods for the prevention or improvement of conditions such as diabetes, hypertension, dementia, cerebrovascular disease, and cardiovascular disease. Taxifolin has a structure very similar to quercetin, another flavonoid, but possesses stronger antioxidant activity while exhibiting lower mutagenicity and toxicity. Taxifolin has a different three-dimensional structure from quercetin. In quercetin, the C-2 and C-3 atoms in the C heterocycle are linked by double bonds, whereas in taxifolin, these are single bonds. Therefore, taxifolin has four structural isomers.

[0003] Currently, taxifolin on the market is mainly produced by extraction from plants. For example, methods for extracting taxifolin from coniferous trees such as larch and from prickly pear cactus have been disclosed (Patent Documents 1-3). A method for obtaining taxifolin by extracting astilbins from plants and hydrolyzing them has also been disclosed (Patent Document 4). However, commercially available taxifolin is expensive, and much of it is derived from Siberian larch, raising concerns about resource sustainability.

[0004] Non-patent document 1 proposes a quercetin metabolic pathway in the intestinal bacterium Eubacterium ramulus, disclosing a pathway in which 3,4-dihydroxyphenylacetic acid and phloroglucinol are produced from quercetin via taxifolin and alfytonin. However, the enzymes involved in the conversion of quercetin to taxifolin in this metabolic pathway have not been reported.

[0005] Non-patent document 2 discloses an enzyme in the intestinal bacterium Flavonifractor plautii that converts apigenin to naringenin. This enzyme, named flavin reductase (FLR), is suggested to be involved in the conversion of the double bond between C-2 and C-3 of apigenin to a single bond through hydrogenation. Patent document 5 discloses the biosynthesis of taxifolin from quercetin using a mutant of FLR derived from Flavonifractor plautii. Patent document 6 discloses the conversion of quercetin to taxifolin using Lactobacillus bifermentans. On the other hand, none of these documents disclose that wild-type FLR is involved in the conversion of quercetin to taxifolin. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Special Publication No. 2014-503489 [Patent Document 2] Special Publication No. 2015-509979 [Patent Document 3] Patent No. 4418675 [Patent Document 4] Patent No. 4334834 [Patent Document 5] Chinese Published Patent Gazette No. 113755460 [Patent Document 6] Patent No. 7454958 [Non-patent literature]

[0007] [Non-Patent Document 1] Applied and Environmental Microbiology, 2001, 67(12):5558-5567 [Non-Patent Document 2] Nature Communications, 2021, 12:790 [Overview of the project] [Problems that the invention aims to solve]

[0008] This invention provides a method for enzymatically producing taxifolin, and an enzyme for that purpose. [Means for solving the problem]

[0009] The following is provided as a representative embodiment of the present invention. [1] A method for producing taxifolin, The method involves converting quercetin to taxifolin using at least one enzyme selected from the group consisting of an enzyme having the amino acid sequence of SEQ ID NO: 1, an enzyme having the amino acid sequence of SEQ ID NO: 2, an enzyme having the amino acid sequence of SEQ ID NO: 3, and an enzyme having an amino acid sequence that is at least 90% identical to any of SEQ ID NOs: 1 to 3, and having the activity to convert the double bond between C-2 and C-3 in the C ring of a flavonoid into a single bond. method. [2] The method according to [1], comprising converting quercetin to taxifolin using at least one enzyme selected from the group consisting of an enzyme having the amino acid sequence of SEQ ID NO: 1 and an enzyme having an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 and having the activity to convert quercetin to taxifolin. [3] The method according to [1], comprising converting quercetin to taxifolin using at least one enzyme selected from the group consisting of an enzyme having the amino acid sequence of SEQ ID NO: 3, and an enzyme having an amino acid sequence that is at least 90% identical to SEQ ID NO: 3 and having the activity to convert quercetin to taxifolin. [4] The method according to any one of [1] to [3], wherein the conversion of quercetin to taxifolin is carried out in the presence of at least one selected from the group consisting of FMN oxidoreductase and FAD oxidoreductase. [5] The method according to any one of [1] to [4], wherein the conversion of quercetin to taxifolin is carried out under anaerobic conditions. 〔6〕The method according to any one of 〔1〕 to 〔5〕, wherein the taxifolin contains 50% by mass or more of (2R,3R)-taxifolin. 〔7〕An enzyme comprising the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence that is at least 90% identical thereto, and having an activity to convert the double bond between C-2 and C-3 in the C ring of a flavonoid into a single bond. 〔8〕A polynucleotide encoding the enzyme according to 〔7〕. 〔9〕A vector encoding the polynucleotide according to 〔8〕.

Advantages of the Invention

[0010] The present invention provides an enzyme capable of converting quercetin into taxifolin. The present invention also provides a method for producing taxifolin from quercetin using the enzyme. The present invention enables the production of taxifolin without relying on extraction from plants, and provides taxifolin stably and at low cost.

Brief Description of the Drawings

[0011] [Figure 1] Conversion of quercetin to taxifolin by FpFLR in Example 1. The bars in the figure indicate the taxifolin concentration (left axis) in the supernatant of the reaction solution after 16, 18, and 24 hours of reaction, and the black circles represent the conversion rate of quercetin to taxifolin (right axis). [Figure 2] Conversion of quercetin to taxifolin by ErFLR in Example 2. The taxifolin concentration in the supernatant of the reaction solution after 1.5 to 11 hours of reaction is shown. [Figure 3] HPLC chromatogram of the reaction solution after 6 hours of enzyme reaction. Quercetin+ErFLR6h: Quercetin-containing reaction solution containing ErFLR, Quercetin+pET21a6h: Quercetin-containing reaction solution not containing ErFLR, ErFLR6h: ErFLR-containing quercetin-free reaction solution. [Figure 4]HPLC chromatogram of the reaction solution 6 hours after the enzyme reaction. 0h: Before reaction, Quercetin+ErFLR: Quercetin-containing reaction solution including ErFLR, Quercetin+ErFLR+Fre: Quercetin-containing reaction solution including ErFLR and LpFre, Quercetin+Fre: Quercetin-containing reaction solution including LpFre, ErFLR+Fre: Quercetin-free reaction solution including ErFLR and LpFre. [Figure 5] Relative activity of ErFLR in the conversion reaction of quercetin to taxifolin under different pH conditions. [Figure 6] Relative activity of ErFLR at different reaction temperatures in the conversion of quercetin to taxifolin by ErFLR. [Figure 7] Contents of quercetin and taxifolin in the reaction solution at each reaction time in the conversion reaction of quercetin to taxifolin by ErFLR. [Figure 8] Conversion of quercetin to taxifolin by LbFLR in Example 4. The concentration of taxifolin in the reaction supernatant after 18 hours of reaction is shown. [Figure 9] Conversion reaction of apigenin to naringenin by FLR in Example 5. A: FpFLR, B: ErFLR. [Modes for carrying out the invention]

[0012] In this specification, "at least 90%" with respect to the identity of amino acid sequences and nucleotide sequences means identity of 90% or more, preferably 92% or more, more preferably 93% or more, even more preferably 94% or more, even more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more.

[0013] In this specification, "one or several" as used with respect to the deletion, substitution, addition, or insertion of amino acids in an amino acid sequence may be, for example, 1 to 30, preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, even more preferably 1 to 4, even more preferably 1 to 3, and even more preferably 1 to 2. Also in this specification, "one or several" as used with respect to the deletion, substitution, addition, or insertion of nucleotides in a nucleotide sequence may be, for example, 1 to 90, preferably 1 to 60, more preferably 1 to 30, even more preferably 1 to 15, even more preferably 1 to 12, even more preferably 1 to 9, even more preferably 1 to 6, and even more preferably 1 to 3. In this specification, "addition" of an amino acid or nucleotide includes the addition of one or several amino acids or nucleotides to one end and both ends of a sequence.

[0014] In this specification, the identity of amino acid sequences and nucleotide sequences can be determined using the BLAST algorithm (Pro.Natl.Acad.Sci.USA.,1993,90:5873-5877). Based on this BLAST algorithm, programs called BLASTN, BLASTX, BLASTP, TBLASTN, and TBLASTX have been developed (J.Mol.Biol.,1990,215:403-410). When using these programs, the default parameters of each program can be used. The specific methods of these analysis methods are publicly known (see [www.ncbi.nlm.nih.gov]).

[0015] The present invention provides a method for enzymatically producing taxifolin. In the method for producing taxifolin of the present invention (hereinafter also simply referred to as "the method of the present invention"), taxifolin is produced from quercetin in one step by an enzymatic reaction using quercetin as a substrate. The presumed reaction equation of the enzymatic reaction is as shown in the following formula [1], and quercetin is converted to taxifolin by converting the double bond between C-2 and C-3 in the C ring of quercetin to a single bond. The enzyme involved in the above enzymatic reaction is referred to herein as flavin reductase (FLR).

[0016] [ka]

[0017] Taxifolin has four structural isomers, specifically (2R,3R)-taxifolin, (2S,3R)-taxifolin, (2R,3S)-taxifolin, and (2S,3S)-taxifolin. Of these, (2R,3R)-taxifolin is the main component of commercially available larch-derived taxifolin and is also referred to as natural-type taxifolin in this specification. The taxifolin produced by the method of the present invention is mainly natural-type taxifolin. Preferably, the taxifolin produced by the method of the present invention contains 50% by mass or more of natural-type taxifolin.

[0018] Examples of FLRs used in the method of the present invention include enzymes consisting of the amino acid sequence of SEQ ID NO: 1, enzymes consisting of the amino acid sequence of SEQ ID NO: 2, enzymes consisting of the amino acid sequence of SEQ ID NO: 3, and enzymes consisting of amino acid sequences that are at least 90% identical to any of SEQ ID NOs: 1 to 3. Any one of these or any two or more can be used in combination. The FLRs used in the method of the present invention are enzymes that have the activity to convert quercetin to taxifolin, and more specifically, enzymes that can hydrogenate the double bond between C-2 and C-3 in the C ring of flavonoids such as quercetin to convert it to a single bond.

[0019] The enzyme consisting of the amino acid sequence of Sequence ID No. 1 is Eubacterium ramulus (e.g., DSM16296 and DSM15684). T It can be isolated from ). The enzyme consisting of the amino acid sequence of Sequence ID No. 2 is Flavonifractor plautii (e.g., ATCC49531 T and ATCC29863 T It can be isolated from ). The enzyme consisting of the amino acid sequence of SEQ ID NO: 3 can be isolated from Lactobacillus bifermentans (e.g., JCM1094). T It can be isolated from ). In this specification, FLRs consisting of the amino acid sequence of SEQ ID NO: 1 and an amino acid sequence that is at least 90% identical thereto may be collectively referred to as ErFLR, FLRs consisting of the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence that is at least 90% identical thereto may be collectively referred to as FpFLR, and FLRs consisting of the amino acid sequence of SEQ ID NO: 3 and an amino acid sequence that is at least 90% identical thereto may be collectively referred to as LbFLR. As shown in the examples below, ErFLR and LbFLR are enzymes whose function as flavin reductases, that is, their activity to convert the double bond between C-2 and C-3 in the flavonoid C ring into a single bond, and their ability to convert quercetin to taxifolin, was previously unknown.

[0020] The FLR used in the method of the present invention can be produced according to known methods. For example, the FLR can be chemically synthesized based on its amino acid sequence. Alternatively, the FLR can be isolated from microorganisms expressing them, as described above. Or, the FLR used in the method of the present invention can be expressed from the gene encoding the FLR. The polynucleotide of the gene encoding the FLR may be chemically synthesized based on its amino acid sequence, or it can be isolated from a microorganism and amplified. For example, the polynucleotide of the gene encoding the FLR consisting of the amino acid sequences of SEQ ID NOs: 1-3 can be isolated from Eubacterium ramulus, Flavonifractor plautii, or Lactobacillus bifermentans, as described above.

[0021] Alternatively, the polynucleotide of the gene encoding the FLR can be produced by introducing mutations into the gene isolated or synthesized by the above procedure using known methods such as ultraviolet irradiation or site-directed mutagenesis. For example, a mutant polynucleotide can be obtained by introducing a mutation into a polynucleotide encoding any of the amino acid sequences of SEQ ID NOs: 1 to 3 using a known method. By examining the activity of the polypeptide expressed from the obtained mutant polynucleotide and selecting a mutant polynucleotide encoding a polypeptide that functions as an FLR, the polynucleotide of the gene encoding the FLR used in the method of the present invention can be obtained.

[0022] Furthermore, it is preferable that the FLR-coding gene prepared by the above procedure be optimized for codon usage frequency in the cells into which the gene is introduced and expressed. Information on codons used by various organisms can be obtained from the Codon Usage Database (www.kazusa.or.jp / codon / ).

[0023] The obtained FLR-encoding gene can be introduced into host cells to obtain transformed cells, and FLR can be expressed by culturing these transformed cells. While bacteria such as Escherichia coli, fungi, and algae are preferred as host cells, they are not particularly limited. A vector containing the FLR-encoding gene can be used for gene introduction into host cells. The type of vector used for introduction can be appropriately selected depending on the type of host cell, the cloning method, the gene expression method, etc. For example, when expressing FLR from a gene on a vector located outside the cell's genome, an expression vector is preferably used. The polynucleotide of the FLR-encoding gene is incorporated into an appropriate vector, and the vector containing the polynucleotide is introduced into host cells. Known methods such as electroporation, particle gun (gene gun) method, competent cell method, protoplast method, calcium phosphate coprecipitation method, Agrobacterium tumefaciens-mediated transformation (ATMT) method and its modifications (Appl. Environ. Microbiol., 2009, 75:5529-5535) can be used for vector introduction into cells. In this case, if an appropriate marker gene is incorporated into the vector, transformed cells into which a vector containing the gene encoding FLR has been introduced can be selected using the expression of the marker as an indicator.

[0024] The conditions for culturing the transformed cells for FLR expression can be appropriately selected depending on the type of host cell. Alternatively, FLR expression from the gene encoding FLR can be performed in a cell-free system. The expressed FLR can be isolated and, if necessary, purified according to known methods for the isolation or purification of proteins.

[0025] In the method of the present invention, the quercetin used as the substrate for FLR can be extracted from vegetables or fruits such as onions, apples, and broccoli, or a commercially available product can be used.

[0026] In the method of the present invention, the quercetin (substrate) concentration in the reaction solution for the enzymatic reaction in which quercetin is converted to taxifolin by FLR is preferably 0.01 to 1000 mM as an initial concentration. The enzyme (FLR) concentration in the reaction solution is preferably 0.0001 to 10 mM as an initial concentration.

[0027] In the method of the present invention, the reaction solution for the enzymatic reaction that converts quercetin to taxifolin contains, in addition to quercetin (substrate) and FLR, flavin mononucleotide or flavin adenine dinucleotide or both (e.g., FMN, FMNH2, FAD, FADH2), NADH, NADPH, NAD + , NADP + It can contain coenzymes such as glucose dehydrogenase (GDH), glucose, etc. FLR is thought to reduce quercetin to taxifolin by utilizing the reaction that oxidizes FMNH2 or FADH2 to FMN or FAD. NADH and NADPH are involved in the reaction that converts FMN or FAD to its reduced form (FMNH2 or FADH2). GDH and glucose are involved in the NAD + and NADP + It is involved in the reaction that reduces to produce NADH and NADPH.

[0028] In the method of the present invention, the enzymatic reaction for converting quercetin to taxifolin by the FLR is preferably carried out in the presence of FMN oxidoreductase (NAD(P)H-dependent FMN reductase) and / or FAD oxidoreductase (NAD(P)H-dependant FAD reductase). In the following description of the present specification, FMN oxidoreductase and FAD oxidoreductase are also collectively referred to as "Fre". Fre promotes the reaction of converting oxidized FMN or FAD to a reductant (FMNH2 or FADH2). By including Fre in the reaction solution, the concentration of FMNH2 or FADH2 in the reaction solution increases, and the conversion reaction of quercetin to taxifolin by FLR is promoted. The conversion reaction of quercetin to taxifolin in the method of the present invention in the presence of Fre is presumed to be carried out in the reaction system of the following formula [2].

[0029]

Chemical formula

[0030] Examples of Fre include oxidoreductases belonging to EC1.5.1.41. More specific examples of Fre include an enzyme consisting of the amino acid sequence of SEQ ID NO: 4 and an enzyme consisting of the amino acid sequence of SEQ ID NO: 5. Any one or any combination of two or more of the above-exemplified Fres can be used in the method of the present invention. For example, the Fre used in the method of the present invention may be at least one selected from the group consisting of FMN oxidoreductase and FAD oxidoreductase. Preferably, but not limited to, the Fre used in the method of the present invention is at least one selected from the group consisting of an enzyme consisting of the amino acid sequence of SEQ ID NO: 4 and an enzyme consisting of the amino acid sequence of SEQ ID NO: 5.

[0031] In the method of the present invention, the concentration of Fre in the reaction solution is preferably 0.0001 to 10 mM at the initial concentration. Also, in the reaction solution, the concentrations of FMN / FMNH2 and FAD / FADH2 are preferably 0.01 to 10 mM, and NAD + / NADH, NADP + The concentration of NADPH is preferably 1 to 100 mM, the concentration of GDH is preferably 1 to 1000 U mL, and the concentration of glucose is preferably 10 to 1000 mM (all initial concentrations of the reaction solution).

[0032] In the method of the present invention, the enzymatic reaction in which quercetin is converted to taxifolin by FLR is carried out under anaerobic conditions. Preferred reaction conditions are a pH of 4.5 to 9.0, preferably 7.0 to 8.5, a temperature of 10 to 70°C, preferably 30 to 60°C, a reaction time of 4 hours or more, preferably 6 hours or more, and 24 hours or less from an economic standpoint.

[0033] Taxifolin can be recovered from the reaction solution by known methods such as liquid-liquid separation using an organic solvent or chromatography using silica gel or a C18 column. The produced taxifolin is rich in natural taxifolin, preferably 50% by mass or more. Furthermore, natural taxifolin can be recovered from the taxifolin produced by the method of the present invention by known methods such as the silica gel chromatography described above.

[0034] The taxifolin obtained in this invention can be used in the manufacture of pharmaceuticals, cosmetics, foods, supplements, feeds, etc., for humans or non-human animals. The type and form of such pharmaceuticals, cosmetics, foods, supplements, and feeds are not particularly limited. [Examples]

[0035] The present invention will be described in more detail below using examples, but the technical scope of the present invention is not limited to the following examples.

[0036] Example 1: Taxifolin production using FpFLR (1) Preparation of enzymes Flavonifractor plautii strain JCM32125, preserved at Kyoto University T(i.e., ATCC29863) T From ), the polynucleotides of the gene encoding FLR (FpFLR) consisting of the amino acid sequence of SEQ ID NO: 2 were isolated and amplified. The obtained polynucleotides were introduced into E. coli to create recombinant E. coli (E. coli Rosetta 2(DE3) / pET21a-FpFLR) expressing the FpFLR of SEQ ID NO: 2.

[0037] E. coli Rosetta 2(DE3) / pET21a-FpFLR was pre-cultured in 4 mL of LB medium, then cultured in 30 mL of LB medium until the OD reached 0.4, and FpFLR expression was induced by IPTG, followed by centrifugation (12000 g, 20 mins). The precipitated culture was diluted in twice the volume of 200 mM Tris-HCl buffer, and cell-free extract (FpFLR CFE) was prepared by sonication. In addition, E. coli Rosetta 2(DE3) / pET21b-Lp0146 transformed with Fre (SEQ ID NO: 4, referred to as LpFre) derived from lactic acid bacteria (Lactiplantibacillus plantarum) was cultured in the same manner as above, centrifuged, and cell-free extract (LpFre CFE) was prepared by sonication of the resulting culture.

[0038] (2) Conversion of quercetin to taxifolin The reaction mixture was prepared by adding 1 mL of Reaction Mixture (see Table 1) to the total volume of obtained FpFLR CFE and LpFre CFE. The enzymatic reaction was carried out under anaerobic conditions at 37°C for 16, 18, or 24 hours while shaking the reaction mixture. After the reaction, methanol was added to stop the reaction, the supernatant was collected by centrifugation, and the supernatant was analyzed by HPLC. The procedure for the enzymatic reaction and the conditions for HPLC analysis are shown in Tables 1 and 2 below.

[0039] [Table 1]

[0040] [Table 2]

[0041] HPLC analysis of the reaction supernatant confirmed that up to approximately 37% of quercetin was converted to taxifolin by the enzymatic reaction (Figure 1).

[0042] (3) Analysis of taxifolin stereoisomers The three-dimensional structure of taxifolin obtained in (2) above was analyzed by HPLC. After investigating the HPLC conditions, a new condition was found that allowed for the separation of the four stereoisomers of taxifolin in a single analysis, and this was used for the three-dimensional structure analysis in this example. The procedure for the three-dimensional structure analysis is shown in Table 3 below, and the results of the analysis are shown in Table 4.

[0043] [Table 3]

[0044] [Table 4]

[0045] As shown in Table 4, approximately 94% of the taxifolin obtained by the enzymatic reaction using FpFLR was the natural (2R,3R)-taxifolin. This demonstrates that natural taxifolin can be selectively produced using FpFLR.

[0046] Example 2: Taxifolin production using ErFLR (1) Preparation of enzymes Eubacterium ramulus (DSM15684) TWe searched for flavodoxin family enzyme genes in the genome of (purchased from the German Microbial Cell Culture Collection) and found a gene encoding FLR (ErFLR) consisting of the amino acid sequence of SEQ ID NO: 1. The sequence identity between SEQ ID NO: 1 and SEQ ID NO: 2 was 60.8%. ErFLR, consisting of the amino acid sequence of SEQ ID NO: 1, is an enzyme whose function as a flavin reductase, i.e., the activity of converting quercetin to taxifolin, was not previously known.

[0047] Eubacterium ramulus (DSM15684) T Polynucleotides of the gene encoding ErFLR were isolated and amplified from ). The obtained polynucleotides were introduced into E. coli to create recombinant E. coli (E. coli Rosetta 2(DE3) / pET21a(+)-ErFLR) expressing ErFLR of SEQ ID NO: 1. The recombinant E. coli was cultured using the same procedure as in Example 1 to express ErFLR, then centrifuged, and the resulting culture was sonicated to prepare a Cell Free Extract (ErFLR CFE). A Cell Free Extract (pET21a CFE) of E. coli into which an empty vector had been introduced was prepared using the same procedure. LpFre CFE was also prepared using the same procedure as in Example 1.

[0048] (2) Conversion of quercetin to taxifolin by ErFLR A reaction solution was prepared by adding 1 mL of Reaction Mixture to the total volume of ErFLR CFE and LpFre CFE obtained. Reaction Mixture composition: 0.8 mM FMN, 0.8 mM FAD, 8 mM NADH, 8 mM NADPH, 1 mM quercetin, 50 mM potassium phosphate buffer (pH 6.9). The enzymatic reaction was carried out at 37°C for 1.5, 4, 6, 9, or 11 hours under anaerobic conditions while shaking the reaction solution. After the reaction, methanol was added to stop the reaction, and the supernatant was collected by centrifugation. Analysis of the supernatant by HPLC using the same procedure as in Example 1 confirmed that up to approximately 6% of quercetin was converted to taxifolin by the enzymatic reaction (Figure 2).

[0049] (3) Comparison with an empty vector Instead of ErFLR CFE, the enzymatic reaction was carried out using pET21a CFE, which does not contain ErFLR and was obtained from E. coli introduced with an empty vector, following the same procedure as in (2). Separately, the enzymatic reaction was carried out with a reaction mixture without quercetin. The reaction time was 6 hours. The supernatant of the reaction mixture was analyzed by HPLC. The HPLC chromatogram is shown in Figure 3. No conversion of quercetin to taxifolin was observed in the reaction mixture containing pET21a CFE.

[0050] (4) The effect of Fre The enzymatic reaction was carried out using reaction solutions with or without ErFLR CFE or LpFre CFE, following the same procedure as in (2). Separately, the enzymatic reaction was carried out with a reaction solution without quercetin. The reaction time was 6 hours. The supernatant of the reaction solution was analyzed by HPLC. The HPLC chromatogram is shown in Figure 4. Conversion of quercetin to taxifolin was observed in reaction solutions containing ErFLR CFE alone and in combination with LpFre CFE, and the amount of taxifolin produced was similar in both reaction solutions.

[0051] (5) Analysis of taxifolin stereoisomers The three-dimensional structure of the taxifolin obtained in (2) above was analyzed by HPLC using the same procedure as in Example 1. The results of the analysis are shown in Table 5. The taxifolin obtained by the enzymatic reaction using ErFLR contained approximately 68% of the natural form (2R,3R)-taxifolin, but also contained the isomers (2S,3S)-taxifolin, (2R,3S)-taxifolin, and (2S,3R)-taxifolin. The taxifolin obtained by ErFLR mainly contained the natural form of taxifolin, but had a higher isomer ratio compared to that obtained by FpFLR.

[0052] [Table 5]

[0053] Example 3: Enzymatic properties of ErFLR As described above, ErFLR is a novel flavin reductase that has the activity to convert quercetin to taxifolin. In this example, the enzymatic properties of ErFLR were investigated. Recombinant Escherichia coli Rosetta 2(DE3) / pET28a(+)-ErFLR expressing His-tagged ErFLR was subjected to an automated protein expression induction system (Overnight Express). TM Using an Autoinduction System (Merck), the cells were incubated at 30°C for 20 hours to express ErFLR. After centrifugation, the resulting culture was sonicated to prepare Cell Free Extract (ErFLR CFE). ErFLR was purified by affinity purification of the CFE.

[0054] The optimal conditions for the enzymatic reaction to convert quercetin to taxifolin were investigated using purified ErFLR. The reaction mixture consisted of 40 μg / mL ErFLR, 0.8 mM FMN, 0.8 mM FAD, 8 mM NADH, 8 mM NADPH, 1 mM quercetin, 1.4 mM cysteine, and a pH adjuster. The pH of the reaction mixture was adjusted to 2.5–11.0 using citrate / sodium citrate, acetate / sodium acetate, potassium dihydrogen phosphate / dipotassium hydrogen phosphate, tris hydrochloride, and sodium carbonate / sodium bicarbonate as pH adjusters. The enzymatic reaction was carried out under anaerobic conditions while shaking the reaction mixture. The reaction temperature was varied from 10–50°C, and the reaction time from 0–30 hours.

[0055] (1) pH Figure 5 shows the enzyme activity under different pH conditions (37°C, 22 hours). The enzyme activity in Figure 5 is relative activity, with the activity at pH 8.0 set as 100%. Maximum enzyme activity was observed at pH 8.0, at which point 7.2% of quercetin was converted to taxifolin.

[0056] (2) Reaction temperature Figure 6 shows the enzyme activity under different temperature conditions (pH 8.0, 22 hours). The enzyme activity in Figure 6 is relative activity, with the activity at 50°C set as 100%. Maximum enzyme activity was observed at 50°C.

[0057] (3) Reaction time Figure 7 shows the content of the substrate (quercetin) and product (taxifolin) in the reaction solution at each reaction time (50°C, pH 8.0). With increasing reaction time, the quercetin content decreased while the taxifolin content increased, with 7.8% of the quercetin being converted to taxifolin after 22 hours of reaction. No further increase in taxifolin content was observed after 22 hours.

[0058] Based on the above, the optimal conditions for the conversion of quercetin to taxifolin by ErFLR were 50°C, pH 8.0, and 22 hours. Under these conditions, we found that quercetin could be converted to a maximum of 8.5% taxifolin.

[0059] Example 4: Taxifolin production using LbFLR (1) Preparation of enzymes Lactobacillus bifermentans (JCM1094 T We searched for flavodoxin family enzyme genes in the genome of (purchased from the RIKEN BioResource Research Center) and found a gene encoding FLR (LbFLR) consisting of the amino acid sequence of SEQ ID NO: 3. The sequence identity between SEQ ID NO: 1 and SEQ ID NO: 3 was 53.7%, and the sequence identity between SEQ ID NO: 2 and SEQ ID NO: 3 was 61.5%. LbFLR, consisting of the amino acid sequence of SEQ ID NO: 3, is an enzyme whose function as a flavin reductase, i.e., the activity of converting quercetin to taxifolin, was not previously known.

[0060] Lactobacillus bifermentans (JCM1094 T The polynucleotides of the gene encoding LbFLR were isolated and amplified from ). The obtained polynucleotides were introduced into E. coli to create recombinant E. coli (E. coli Rosetta 2(DE3) / pET21a(+)-LbFLR) expressing LbFLR of SEQ ID NO: 3. These recombinant E. coli were then subjected to an automated protein expression induction system (Overnight Express). TMLbFLR was expressed by culturing the cells at 30°C for 20 hours using an Autoinduction System (Merck). The culture was then centrifuged, and the resulting cell-free extract (LbFLR CFE) was prepared by sonication.

[0061] (2) Preparation of ErFre Eubacterium ramulus (DSM15684) T We created recombinant E. coli Rosetta 2(DE3) / pET21a(+)-ErFre by transforming it with Fre (SEQ ID NO: 5, referred to as ErFre) derived from ). This recombinant E. coli expresses His-tagged ErFre. This recombinant E. coli was then subjected to an automated protein expression induction system (Overnight Express). TM Using an Autoinduction System (Merck), the cells were incubated at 30°C for 20 hours to express ErFre. After centrifugation, the resulting culture was sonicated to prepare Cell Free Extract (ErFre CFE). ErFre was purified by affinity purification of the CFE.

[0062] (3) Conversion of quercetin to taxifolin by LbFLR The entire amount of LbFLR CFE obtained and 40 μg of ErFre were combined with 1 mL of Reaction Mixture to prepare the reaction solution. The composition of the Reaction Mixture was the same as in Example 1, except that it contained 1 mM quercetin as a substrate. The enzymatic reaction was carried out at 37°C for 18 hours under anaerobic conditions while shaking the reaction solution. After the reaction, methanol was added to stop the reaction, and the supernatant was collected by centrifugation. Analysis of the supernatant by HPLC using the same procedure as in Example 1 confirmed that approximately 20% of the quercetin was converted to taxifolin by the enzymatic reaction (Figure 8).

[0063] (4) Analysis of taxifolin stereoisomers The three-dimensional structure of the taxifolin obtained in (3) above was analyzed by HPLC using the same procedure as in Example 1. The results of the analysis are shown in Table 6. The taxifolin obtained by the enzymatic reaction using LbFLR contained approximately 83% of the natural (2R,3R)-taxifolin, but also contained the isomers (2S,3S)-taxifolin, (2R,3S)-taxifolin, and (2S,3R)-taxifolin. The taxifolin obtained by LbFLR mainly contained the natural taxifolin, but had a higher isomer ratio compared to that obtained by FpFLR.

[0064] [Table 6]

[0065] Example 5: FLR-mediated conversion activity of apigenin to naringenin Cell Free Extract containing FpFLR (FpFLR CFE) and Cell Free Extract containing LpFre (LpFre CFE) were prepared using the same procedure as in Example 1. Cell Free Extract containing ErFLR (ErFLR CFE) was prepared using the same procedure as in Example 2.

[0066] The reaction solution was prepared by adding 1 mL of Reaction Mixture to the total volume of obtained FpFLR CFE and LpFre CFE. The composition of the Reaction Mixture was the same as in Example 1, except that it contained 1 mM apigenin instead of 1 mM quercetin as a substrate. The enzymatic reaction was carried out at 37°C for 2 to 24 hours under anaerobic conditions while shaking the reaction solution. After the reaction, methanol was added to stop the reaction, and the supernatant was collected by centrifugation.

[0067] The reaction solution was prepared by adding 1 mL of Reaction Mixture to the total volume of the obtained ErFLR CFE. The composition of the Reaction Mixture was the same as in Example 2, except that it contained 1 mM apigenin instead of 1 mM quercetin as a substrate. The enzymatic reaction was carried out at 37°C for 2 to 24 hours under anaerobic conditions while shaking the reaction solution. After the reaction, methanol was added to stop the reaction, and the supernatant was collected by centrifugation.

[0068] Each supernatant obtained was analyzed by HPLC using the same procedure as in Example 1. Figures 9A and 9B show the naringenin content in the reaction solution containing FpFLR CFE (A) and the reaction solution containing ErFLR CFE (B).

Claims

1. A method for producing taxifolin, The method involves converting quercetin to taxifolin using at least one enzyme selected from the group consisting of an enzyme having the amino acid sequence of SEQ ID NO: 1, an enzyme having the amino acid sequence of SEQ ID NO: 2, an enzyme having the amino acid sequence of SEQ ID NO: 3, and an enzyme having an amino acid sequence that is at least 90% identical to any of SEQ ID NOs: 1 to 3, and having the activity to convert the double bond between C-2 and C-3 in the C ring of a flavonoid into a single bond. method.

2. The method according to claim 1, comprising converting quercetin to taxifolin using at least one enzyme selected from the group consisting of an enzyme having the amino acid sequence of SEQ ID NO: 1, and an enzyme having an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 and having the activity to convert quercetin to taxifolin.

3. The method according to claim 1, comprising converting quercetin to taxifolin using at least one enzyme selected from the group consisting of an enzyme having the amino acid sequence of SEQ ID NO: 3, and an enzyme having an amino acid sequence that is at least 90% identical to SEQ ID NO: 3 and having the activity to convert quercetin to taxifolin.

4. The method according to claim 1, wherein the conversion of quercetin to taxifolin is carried out in the presence of at least one enzyme selected from the group consisting of FMN oxidoreductase and FAD oxidoreductase.

5. The method according to claim 1, wherein the conversion of quercetin to taxifolin is carried out under anaerobic conditions.

6. The method according to claim 1, wherein the taxifolin contains 50% by mass or more of (2R,3R)-taxifolin.

7. An enzyme comprising the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence that is at least 90% identical to these, and having the activity to convert the double bond between C-2 and C-3 in the C ring of a flavonoid into a single bond.

8. A polynucleotide encoding the enzyme according to claim 7.

9. A vector encoding a polynucleotide according to claim 8.

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