KR102222517B1 - Biocatalytic compositions comprising monooxygenases and method for the preparation of hydroxy-equol derivatives using the same - Google Patents

Abstract

An object of the present invention is to provide a method for efficiently synthesizing an equol hydroxide derivative from equol using a monooxygenase or a biocatalyst containing a monooxygenase. In particular, it provides a method of using the wild type and mutations of tyrosinase derived from microorganisms, cytochrome P450 and 4-hydroxyphenylacetate 3-hydroxylase for regioselective hydroxylation of equol.

Description

Biocatalyst composition containing monooxygenase and method for producing equol hydroxide derivatives using the same {BIOCATALYTIC COMPOSITIONS COMPRISING MONOOXYGENASES AND METHOD FOR THE PREPARATION OF HYDROXY-EQUOL DERIVATIVES USING THE SAME}

The present invention relates to a method for producing an equol hydroxide derivative from equol using a biocatalyst composition containing a microorganism-derived monooxygenase. In addition, the present invention relates to a biocatalyst composition comprising a mutant monooxygenase for selectively synthesizing 3'-hydroxy-equol or 6-hydroxy-equol from equol.

It is known that some anaerobic bacteria among human intestinal microbes metabolize isoflavones consumed by humans through legumes and bioconvert them into equols. And the equol synthesizing microorganisms all bacteria found so far, typically slash Escherichia genus (Slackia sp.), Egg Stavanger telra in (Eggerthella sp.), Most of the nose Rio bacteria such as Adler Crew jjiah in (Adlercreutizia sp.) belongs in, there are also exceptionally Lactococcus genus (Lactococcus) origin. Equol synthesized by the enzymatic reaction of such intestinal microbes is only synthesized with optical isomers of the S structure.

S-equol is known to be effective in relieving women's menopausal diseases, preventing osteoporosis, preventing ovarian or prostate cancer, preventing cardiovascular disease, and preventing hair loss as a kind of phytoestrogens (Non-Patent Document 1). In addition, 5-hydroxy-equol, a derivative in which a hydroxyl group is added to carbon 5 of equol, is known to have a liver cancer inhibitory effect and an antioxidant effect. However, if S-Equol is absorbed into the human body Ortho-hydroxylated in the liver to produce various ortho-hydroxylated equols, but their synthetic pathways or biological functions in the human body are unknown. In order to more actively study the biological function of ortho-hydroxylated equol, there is a need for an efficient synthesis method for ortho-hydroxylated equol.

Meanwhile, various monooxygenases have been reported in nature. Among them, tyrosinase, cytochrome P450, and 4-hydroxyl as enzymes that induce ortho-hydroxylation in secondary metabolites derived from plants, for example, aromatic plant secondary metabolites such as flavonoids and stilbenoids. Flavin-containing monooxygenases such as phenylacetate 3-hydroxylase are known. In particular, in the case of tyrosinase, a catechol-type structural substance from monophenol-type structural substances such as daidzein, genistein, apigenin, and phloretin by inhibiting the secondary oxidation reaction. An example of selectively producing is reported by the present inventor (Patent Document 1), and an example in which daidzein, genistein, and resveratrol are ortho-hydroxylated has been reported by the present inventor (Non-Patent Document 2).

In the case of cytochrome P450, the substrate recognition site of cytochrome P450 derived from the genus Streptomyces , Bacillus , or Nocardia is altered to be specific to the 3'- or 6-position of daidzein. An example of increasing the efficiency of the ortho-hydroxylation reaction has been reported.In addition, bioconversion studies on the 6- or 8-position specific ortho-hydroxylation reaction of isoflavones using the cytochrome P450 activity of live bacteria of Aspergillus have been reported. It has been reported (Non-Patent Document 3). However, the production of catechol-type substances using cytochrome P450 has several disadvantages such as lower catalytic activity and coenzyme requirement than tyrosinase.

In the case of 4-hydroxyphenylacetate 3-hydroxylase (HpaBC), using recombinant E. coli expressing HpaBC as a whole cell catalyst, such as umbelliferone, resveratrol, and naringenin. An example of ortho-hydroxylating a plant-derived secondary metabolite has been reported by the present inventor (Non-Patent Document 4).

On the other hand, the preparation method for ortho-hydroxyl equol has not been reported yet. However, there is an example in which equol hydroxide was produced from isoflavin hydroxide such as 3'-hydroxydidezein, 6-hydroxydidezein, and 8-hydroxydidezein by the present inventors using a recombinant microorganism. . However, in this case, there is a problem that the price of isoflavone hydroxide used as a substrate for the reaction is expensive (80 to 40 million won/g), and it is difficult to produce it on an industrial scale. From this point of view, a method of synthesizing ortho-hydroxylated equol from equol using a biocatalyst composition containing monooxygenase may be a viable alternative. However, there are no reports or inventions related to a method for synthesizing ortho-hydroxylated equol from equol using a biocatalyst.

Unexamined Patent Publication No. 10-2018-0079233 (2018.07.10) Registered Patent Publication No. 10-1566672 (2015.11.02) Unexamined Patent Publication No. 10-2018-0099380 (2018.09.05)

R.L. Jackson et al., Emerging evidence of the health benefits of S-equol, an estrogen receptor β agonist. Nutrition Reviews, 69(8), 432-448 (2011) S.-H. Lee et al., Using tyrosinase as a monophenol monooxygenase: A combined strategy for effective inhibition of melanin formation. Biotehcnol. Bioeng. 113(4), 735-743 (2016) P.-G. Lee et al., Recent advances in the microbial hydroxylation and reduction of soy isoflavones, FEMS Microbiology Letters, 365 (19), fny195 (2018). Y. Lin et al., Biotechnological production of plant-specific hydroxylated phenylpropanoids. Biotehcnol. Bioeng. 111(9), 1895-1899 (2014)

Under such circumstances, development of a biocatalyst capable of synthesizing ortho-hydroxylated equol by introducing a hydroxyl group directly into equol is required. There are no reported examples. An object of the present invention is to provide a biocatalyst composition comprising a monooxygenase and a method for producing an equol hydroxide derivative, particularly ortho-hydroxylated equol, from equol using the same.

In addition, another object of the present invention is to provide a biocatalyst composition comprising a mutant monooxygenase for the selective synthesis of 3'-hydroxy-equol or 6-hydroxy-equol in ortho-hydroxyl-equol. will be.

In order to achieve the above object, the present inventors produced recombinant E. coli each expressing a gene encoding a tyrosinase, cytochrome P450 or 4-hydroxyphenylacetate 3-hydroxylase among known monooxygenases.

In addition, in order to increase the selectivity to 3'-hydroxy-equol or 6-hydroxy-equol in ortho-hydroxyl equol, the present inventors have developed a mutant tyrosinase and a mutant 4-hydroxyphenylacetate 3-hydroxyla. Each enzyme was prepared, and recombinant E. coli each expressing a gene encoding these mutant enzymes was prepared.

The recombinant E. coli of the present invention can be used in a method of synthesizing an equol hydroxylated derivative from equol.

Specifically, the present invention provides the following.

(1) Monooxygenase, a recombinant microorganism containing a gene encoding a monooxygenase, or a biocatalyst for producing an equol hydroxide derivative from equol containing a cell culture medium or cell extract containing the recombinant microorganism Composition.

(2) The biocatalyst composition according to (1), wherein the monooxygenase is a flavin-containing monooxygenase containing 4-hydroxyphenylacetate 3-hydroxylase, tyrosinase, or cytochrome P450.

(3) In (2), the tyrosinase is

Tyrosinase represented by the amino acid sequence of SEQ ID NO: 1, or

In the tyrosinase represented by the amino acid sequence of SEQ ID NO: 1, the 209 th arginine is substituted with glycine, alanine, cysteine, serine, threonine, tyrosine or phenylalanine.

(4) The biocatalyst composition according to (3), wherein the tyrosinase is a tyrosinase derived from Bacillus megaterium.

(5) The biocatalyst composition according to (2), wherein the cytochrome P450 is cytochrome P450 represented by the amino acid sequence of SEQ ID NO: 2.

(6) In (2), the 4-hydroxyphenylacetate 3-hydroxylase is an oxygenase component (HpaB) and 4-hydroxyphenylacetate of 4-hydroxyphenylacetate 3-hydroxylase. It contains the reductase component (HpaC) of 3-hydroxylase, and the HpaB is

HpaB represented by the amino acid sequence of SEQ ID NO: 3, or

In HpaB represented by the amino acid sequence of SEQ ID NO: 3, the biocatalyst composition is a mutant HpaB in which the 292 th threonine, 464 th serine or 469 th arginine is substituted with glycine, alanine, valine, leucine or isoleucine.

(7) The biocatalyst composition according to (1), wherein the microorganism is E. coli.

(8) The equol hydroxide derivative according to (1) is 3'-hydroxy-equol, 6-hydroxy-equol, 8-hydroxy-equol, and 6, 3'-dihydroxy-equol. Biocatalyst composition which is one or more compounds selected from the group consisting of ol.

(9) The biocatalyst according to (3), wherein the equol hydroxide derivative contains 3'-hydroxy-equol, 6-hydroxy-equol, and 6, 3'-dihydroxy-equol. Composition.

(10) The tyrosinase according to (9), in the tyrosinase represented by SEQ ID NO: 1, the 209 th arginine is a mutant tyrosinase substituted with glycine, alanine, cysteine, serine, threonine, tyrosine or phenylalanine,

The mutant tyrosinase is a biocatalyst composition, characterized in that the selectivity for 3'-hydroxy-equol compared to 6-hydroxy-equol is 1.2 to 6 times improved compared to the tyrosinase represented by SEQ ID NO: 1.

(11) The biocatalyst composition according to (5), wherein the equol hydroxide derivative contains 3'-hydroxy-equol, 6-hydroxy-equol, and 8-hydroxy-equol.

(12) In (6), the HpaB is HpaB represented by the amino acid sequence of SEQ ID NO: 3,

The biocatalyst composition wherein the equol hydroxide derivative comprises 3'-hydroxy-equol, 6-hydroxy-equol, and 6, 3'-dihydroxy-equol.

(13) In (6), the HpaB is a mutant HpaB in which the 292 th threonine, the 464 th serine or the 469 th arginine is substituted with glycine, alanine, valine, leucine or isoleucine in HpaB represented by the amino acid sequence of SEQ ID NO: 3 ,

The 4-hydroxyphenylacetate 3-hydroxylase containing the mutant HpaB is selected for 6-hydroxy-equol compared to 3'-hydroxy-equol and 6, 3'-dihydroxy-equol. A biocatalyst composition, characterized in that it is improved by 2 to 16 times compared to 4-hydroxyphenylacetate 3-hydroxylase containing HpaB represented by SEQ ID NO: 3.

(14) A method for producing an equol hydroxide derivative, comprising reacting a reactant containing equol in the presence of the biocatalyst composition of any one of (1) to (13) to obtain an equol hydroxide derivative.

(15) The method according to (14), wherein the reactant contains a reducing agent in order to suppress oxidation of the produced equol hydroxide derivative.

(16) One or more compounds according to (15), wherein the reducing agent is selected from the group consisting of NAD(P)H, L-ascorbic acid, glutathione, dithiothreitol, and cysteine. Way.

(17) The step of (14), further comprising the step of reacting a reactant containing daidzein to obtain equol, obtaining equol from the reactant containing daidzein, and a reactant comprising equol A method, characterized in that the step of obtaining the equol hydroxide derivative from is carried out at the same time.

The present invention uses a recombinant E. coli containing microorganism-derived tyrosinase, cytochrome P450 or 4-hydroxyphenylacetate 3-hydroxylase as a biocatalyst, and 3'-hydroxy-equol from S-equol, 6-hydroxy-equol, 8-hydroxy-equol and 6,3'-dihydroxy-equol can be biosynthesized.

In addition, the present invention improved the selectivity for 3'-hydroxy-equol by up to 6 times compared to wild-type tyrosinase by using recombinant E. coli containing mutant tyrosinase as a biocatalyst.

In addition, the present invention uses a recombinant E. coli containing a mutant 4-hydroxyphenylacetate 3-hydroxylase as a biocatalyst to achieve selectivity for 6-hydroxy-equol. It was improved up to 16 times compared to roxylase.

In addition, by combining the recombinant E. coli containing the mutant 4-hydroxyphenylacetate 3-hydroxylase and the recombinant E. coli synthesizing S-equol from Daidzein prepared in the prior invention of the present inventor (Patent Document 3) When one-pot synthesis was carried out, it was possible to produce 6-hydroxy-equol from daidzein at a maximum of 244 mg/L.

The present invention proposes an efficient and economical method for selectively synthesizing 3'-hydroxy-equol or 6-hydroxy-equol from S-equol. It enables physiological research and use as food or medicine.

FIG. 1 shows 3'-hydroxy-equol, 6-hydroxyl, wherein S-equol is ortho-hydroxylated equol by being hydrated by tyrosinase, cytochrome P450 or 4-hydroxyphenylacetate 3-hydroxylase. -Equol, 8-hydroxy-equol or 6,3'-dihydroxy-equol is a schematic diagram of the bioconversion reaction.
FIG. 2 shows an ortho-hydroxylation reaction of S-equol using a biocatalyst containing a wild-type tyrosinase derived from Bacillus megaterium, and then the reaction product was subjected to High Performance Liquid Chromatography (HPLC). This is the result of analysis. As a result of the analysis, 3'-hydroxy-equol (3'-OHE), 6-hydroxy-equol (6-OHE) or 6,3'-dihydroxy-equol (6,3' -diOHE) was confirmed to be synthesized.
Figure 3 is a diagram showing the ortho-hydroxyl position selectivity for S-equol of the R209C, R209F or R209S mutant enzyme of tyrosinase derived from Bacillus megaterium compared to wild-type tyrosinase. Compared to the wild type, the 3'-OHE/6-OHE hydroxyl selectivity was improved by 6 times for R209C, 3 times for R209F, and 1.2 times for R209S.
4 is a recombinant Escherichia coli expressing cytochrome P450 CYP102G4 derived from Streptomyces ketilia and derived from Escherichia coli. The result of bioconversion of S-equol to ortho-hydroxyl equol using recombinant E. coli expressing 4-hydroxyphenylacetate 3-hydroxylase as a biocatalyst was analyzed by gas chromatography.
5 is a mass analyzed by gas chromatography/mass spectrometry after trimethyl silylation of the ortho-hydroxyl equol synthesized in FIGS. 2 and 4 with BSTFA ( N , O-bis(trimethylsilyl)trifluoroacetamide) It's a spectrum.
Figure 6 shows the ortho-hydroxyl position selectivity for S-equol of the T292A, S464A or S469A mutant enzyme of 4-hydroxyphenylacetate 3-hydroxylase derived from Escherichia coli wild-type 4-hydroxyphenylacetate 3-hydroxyl. It is a diagram showing the comparison of roxylase. Compared to the wild type, the hydroxyl selectivity for 6-OHE was improved about 2 times for S464A and S469A, and about 16 times for T292A.
7 is a mutant 4-hydroxyphenylacetate 3-hydroxylase T292A synthesizing 6-hydroxy-equol from S-equol of the present invention, S- in daidzein prepared in the previous paper of the present inventor. It is a chart that records the concentration of each isoflavone derivative over time after mixing with recombinant E. coli (Patent Document 3) that synthesizes equol, and proceeding with one-pot synthesis.

Hereinafter, the present invention will be described in more detail.

The nomenclature used in this specification and the experimental methods described below are well known and commonly used in the art. In addition, unless otherwise defined, all technical and scientific terms used in the present specification have the same meaning as commonly understood by a skilled expert in the technical field to which the present invention belongs, and anyone skilled in the art can understand the meaning. There will be, but briefly described in this specification as follows:

(1) Expression: It means the production of a recombinant protein in a microorganism containing a vector containing a recombinant gene.

(2) Vector: refers to a polynucleotide consisting of single-stranded, double-stranded, circular or super-stranded DNA or RNA. The vector is composed of an origin of replication, a promoter, a ribosome binding site, a nucleic acid cassette, a terminator, etc. that are operably linked at an appropriate distance to produce a recombinant protein. The gene encoding the recombinant protein to be expressed is inserted at the position of the nucleic acid cassette.

(3) Cell extract: refers to the supernatant after crushing the microorganism expressing the recombinant protein and removing solid cell debris with a centrifuge.

(4) Whole cell reaction: refers to a reaction using a cell extract by disrupting cells containing a specific enzyme, or using whole cells without separating and purifying the enzyme.

(5) Transformation: refers to the modification of the genotype of a host cell by the introduction of a foreign gene, and refers to the introduction of a foreign gene into the host cell regardless of the method used for the transformation. This is any method known to those skilled in the art for gene introduction, for example, bombardment with DNA-filled particles, transformation using protoplasts, microinjection of DNA, electroporation, conjugation of competent cells. Or transformation, chemical or Agrobacteria-mediated transformation. In addition, the foreign gene may be introduced into a cell under the assistance of a vector or free nucleic acid (eg, DNA, RNA), and may be inserted into the host genome by recombination or exist in a free form in the cell.

The biocatalyst composition of the present invention comprises a monooxygenase, a recombinant microorganism containing a gene encoding a monooxygenase, or a cell culture medium or cell extract containing the recombinant microorganism. In addition, the biocatalyst composition of the present invention includes a mutant monooxygenase, a recombinant microorganism comprising a gene encoding a mutant monooxygenase, or a cell culture medium or cell extract containing the recombinant microorganism. The recombinant microorganism is preferably a recombinant E. coli.

The monooxygenase of the present invention refers to an enzyme that introduces one hydroxyl group into a substrate, and a flavin-containing monooxygenase including 4-hydroxyphenylacetate 3-hydroxylase, Tyrosinase or cytochrome P450.

And the tyrosinase is derived from that microorganism, the microorganism comprises a Bacillus (Bacillus), in Aspergillus (Aspergilus), in Agaricus (Agaricus) and Streptomyces genus cis (Streptomyces). Preferably, the tyrosinase is derived from the genus Bacillus, more preferably, is derived from Bacillus megaterium.

The cytochrome P450 is derived from a microorganism, and the microorganisms include the genus Bacillus , the genus Streptomyces , the genus Pseudomonas , and the genus Candida . Preferably, the cytochrome P450 is Streptomyces cattleya , Streptomyces avermitilis, Streptomyces coelicolor , Bacillus megaterium , Pseudomonas putida , Candida tropicalis (Candida tropicalis), more preferably, is derived from Streptomyces ketilia.

The 4-hydroxyphenyl acetate, 3-hydroxy xylanase may be derived from a microorganism, said microorganism including the genus Escherichia (Escherichia), Pseudomonas species (Pseudomonas) and keulrep when in Ella (Klebsiella). Preferably, the 4-hydroxyphenylacetate 3-hydroxylase is Escherichia coli , Thermus thermophiles , Pseudomonas acidovorans , Pseudomonas ovalis , , Pseudomonas putida , Klebsiella pneumonia (Klebsiella pneumonia) , more preferably from E. coli.

The 4-hydroxyphenylacetate 3-hydroxylase of the present invention comprises the oxygenase component (HpaB) of 4-hydroxyphenylacetate 3-hydroxylase and 4-hydroxyphenylacetate 3-hydroxylase. It means that the reductase component (HpaC) is expressed and functions together, and is also referred to as HpaBC. Here, HpaB may refer to wild-type HpaB or mutant HpaB.

The mutant 4-hydroxyphenylacetate 3-hydroxylase of the present invention means that the mutant HpaB and wild-type HpaC are expressed and functioned together, and the position of the altered amino acid in the mutant HpaB is based on the amino acid sequence of wild-type HpaB. .

In the present invention, the wild-type tyrosinase derived from Bacillus megaterium is represented by the amino acid sequence of SEQ ID NO: 1, and the wild-type cytochrome P450 derived from Streptomyces ketilia is represented by the amino acid sequence of SEQ ID NO: 2, derived from E. coli The resulting wild-type HpaB is represented by the amino acid of SEQ ID NO: 3, and the wild-type HpaC derived from E. coli is represented by the amino acid of SEQ ID NO: 4.

In one embodiment of the present invention, a mutant tyrosinase in which the 209 th arginine of the amino acid sequence of the tyrosinase derived from Bacillus megaterium is substituted with glycine, alanine, cysteine, serine, threonine, tyrosine or phenylalanine is prepared. Preferably, the mutant tyrosinase comprises a substitution of cysteine (R209C), serine (R209S) or phenylalanine (R209F) of the 209 th arginine, more preferably, a substitution of cysteine or phenylalanine, More preferably, it includes substitution with cysteine.

In another embodiment of the present invention, the 292 th threonine, 464 th serine or 469 th arginine of the amino acid sequence of 4-hydroxyphenylacetate 3-hydroxylase derived from E. coli is glycine, alanine, valine, leucine, or isoleucine. A substituted mutant 4-hydroxyphenylacetate 3-hydroxylase is prepared. Preferably, the mutant 4-hydroxyphenylacetate 3-hydroxylase is a substitution of threonine at 292 to alanine (T292A), substitution of serine at 464 to alanine (S464A), or alanine at arginine at 469 And the substitution of (R469A).

The biocatalyst composition is used in a reaction for synthesizing an equol hydroxide derivative from S-equol. The equol hydroxide derivative is preferably ortho-hydroxyl equol, but is not limited thereto, and the ortho-hydroxyl equol is 3'-hydroxy-equol, 6-hydroxy-equol, 8-hydroxy -Equol and 6, 3'-dihydroxy-equol.

The recombinant microorganism can be used as a whole cell catalyst. Whole-cell catalysts are used in many biocatalytic reactions, and have the advantage that the reaction proceeds under mild conditions at room temperature and pressure, and has excellent reaction specificity. It is more advantageous to perform the biocatalytic process using whole cells in a case where a coenzyme is absolutely necessary, a process involving a reaction of several steps, or when the activity of the enzyme is significantly reduced during purification. In addition, even when the product is purified after completion of the reaction, it is advantageous because the recombinant microorganism used as whole cells can be selectively separated using a centrifuge.

In one embodiment of the present invention, ortho-hydroxyl equol is synthesized from S-equol using recombinant E. coli as a whole cell catalyst. In order to suppress further oxidation of the produced ortho-hydroxylated equol, a reducing agent may be added to the reactant, and a substance capable of coordinating with the produced ortho-hydroxylated equol may be added.

As the reducing agent, NAD(P)H, L-ascorbic acid, glutathione, dithiothreitol, cysteine, and the like may be used, but are not limited thereto.

The concentration of the reducing agent to be added is preferably about 1 to about 100 times the initial substrate concentration, more preferably 1 to 30 times, and more preferably 1 to 10 times. The concentration of the added coordination precursor is preferably 1 to 10000 times, more preferably 50 to 600 times, and more preferably 100 to 500 times the concentration of S-equol.

The reaction solution may contain a buffer solution for pH buffering. As the buffer, Tris buffer, HEPES (N-[2-hydroethyl] piperazine-N'-[2-ethanesulfonic acid]) 　 buffer, borate buffer, phosphate buffer, etc. may be used, It is not limited thereto. As the buffer solution, a phosphate buffer solution is preferable, and a potassium phosphate buffer solution is more preferable. The concentration of the buffer solution is preferably about 50 to about 200 mM.

The pH of the reaction solution is preferably about 5 to about 10, more preferably about 6 to about 9, and even more preferably about 7 to about 8. The reaction temperature is preferably about 18 to about 37°C, more preferably about 25 to about 37°C. The stirring speed of the reactor is preferably about 50 to about 400 rpm, more preferably about 80 to about 250 rpm, more preferably about 100 to about 200 rpm.

Glucose or glycerol may be used as a carbon source in the equol synthesis reactor. The concentration of glucose or glycerol is preferably about 1 to about 5 (w/v)%, more preferably about 1 to about 3 (w/v)%. The concentration of the recombinant microorganism of the present invention has an optical density (Optical Density, hereinafter abbreviated as OD) of 600 nm, preferably about 1 to about 100, more preferably about 5 to about 50, and more preferably about 10 to about 20. .

In the reaction, polyethylene glycol or polyvinyl pyrrolidone may be used to improve the solubility of the substrate.

In one embodiment of the present invention, a combination of the recombinant strain produced in the present invention and the recombinant E. coli (tDDDT) producing S-equol from Daidzein disclosed in the prior invention of the present inventor (Patent Document 3) is produced. As a catalyst, 6-hydroxy-equol is selectively synthesized from daidzein via a single vessel reaction. A reducing agent is added to the reaction to inhibit further oxidation of the product. As the reducing agent, NAD(P)H, L-ascorbic acid, glutathione, dithiothreitol, cysteine, and the like may be used, but are not limited thereto. Other reaction conditions are as described above.

Hereinafter, a specific method of the present invention will be described in detail as examples, but the technical scope of the present invention is not limited to these examples.

[Example]

[Production Example 1]: Preparation of recombinant E. coli expressing tyrosinase

After amplifying the tyrosinase DNA sequence derived from Bacillus megaterium through PCR, put it in pet28a or petDuet vector, respectively, and this in E. coli as His-tag (6 histidine-tag), respectively. Expressed. The expression method is as follows. The plasmid into which the tyrosinase base sequence was inserted was transformed into BL21 competent cells, and then incubated in an incubator at 37° C. in an LB solid medium containing an antibiotic suitable for each plasmid. Thereafter, one colony obtained in the LB solid medium was inoculated into 5 mL LB liquid medium containing antibiotics and incubated at a speed of 200 rpm in a 37°C incubator for 8 hours. The obtained culture solution was inoculated to 1 v% (500 uL) in 50 mL of a fresh liquid medium containing antibiotics, and further cultured in a 37°C incubator. When the cell OD 600 nm reached 0.6, 2 mM IPTG and 1 mM CuSO ₄ were added, and protein expression was induced by incubating for 17 hours at a rate of 200 rpm in an 18 °C incubator. After culturing for 17 hours, the cell culture solution was centrifuged at 4000 rpm to obtain cells, which were washed with 5 mL PBS. After washing, the whole cell reaction was carried out in tris-HCl buffer or borate buffer, and other cell extracts were prepared through ultrasonic treatment. For the in vitro whole cell reaction, the receptor portion of the cell extract was used as it is, or a protein purified by a conventional histack purification method was used, which is specifically specified in each of the Examples.

[Preparation Example 2]: Preparation of recombinant E. coli containing mutant tyrosinase and mutant 4-hydroxyphenylacetate 3-hydroxylase

In the present invention, in order to prepare a mutant enzyme for tyrosinase derived from Bacillus megaterium, the 209 th arginine residue at the substrate binding site in the tyrosinase (wild type tyrosinase) represented by the amino acid sequence of SEQ ID NO: 1 was identified. I did. Then, each mutant tyrosinase was prepared in which the corresponding residue was substituted with cysteine, phenylalanine, or serine.

In addition, in order to produce a mutant 4-hydroxyphenylacetate 3-hydroxylase derived from E. coli, the 292 th threonine residue at the substrate binding site in HpaB (wild type HpaB) represented by the amino acid sequence of SEQ ID NO: 3, 464 th The serine residue and the 469th arginine residue were identified. Then, in wild-type HpaB, each mutant 4-hydroxyphenylacetate 3-hydroxylase in which the 292 th threonine residue, the 464 th serine residue, or the 469 th arginine residue was substituted with alanine was prepared. Construction of a vector including the gene of mutant tyrosinase and mutant 4-hydroxyphenylacetate 3-hydroxylase was constructed according to the site-directed mutagenesis method generally widely used in the art. A vector containing the prepared mutant tyrosinase and mutant 4-hydroxyphenylacetate 3-hydroxylase was transformed into E. coli to obtain a recombinant strain, and in the case of tyrosinase, the enzyme was expressed using the recombinant strain. After that, the cell extract obtained by disrupting the cells or purified tyrosinase was used as a biocatalyst, and in the case of 4-hydroxyphenylacetate 3-hydroxylase, a whole cell reaction was used.

[Example 1]: Synthesis of ortho-hydroxyl equol using tyrosinase as a biocatalyst

After the tyrosinase derived from Bacillus megaterium was expressed in the recombinant E. coli strain according to Preparation Example 1, the cells were pulverized to obtain a cell extract. Thereafter, a reaction solution consisting of 20 μL of a cell extract containing wild-type tyrosinase, 5 mM ascorbic acid, 200 mM borate buffer (pH 9), and 0.2 mM S-equol was added at 37° C., 200 rpm for 1 hour. Reacted for a while. After the reaction was completed, 40 μL of 1 N hydrochloric acid and 1 mL of ethyl acetate were added to 0.5 mL of the reaction solution to extract the reaction product, and the product was confirmed by High Performance Liquid Chromatography (HPLC). As a result, the conversion rate of S-equol was 40%, and as main products, 3'-hydroxy-equol (3'-OHE), 6-hydroxy-equol (6-OHE) and 6,3'- It was confirmed by HPLC that dihydroxy-equol (6,3'-diOHE) was produced (FIG. 2). The conversion rate represents "(concentration of reduced substrate/concentration of initial substrate) x 100 (%)".

[Example 2]: Synthesis of ortho-hydroxyl equol using mutant tyrosinase as a biocatalyst

Synthesis of ortho-hydroxyl equol from S-equol was performed under the same reaction conditions as in Example 1 using the mutant tyrosinase R209C, R209F and R209S prepared according to Preparation Example 2. As a result, compared to the wild-type tyrosinase, it was confirmed that the selectivity of the mutant tyrosinase to 3'-hydroxy-equol increased 1.2 to 6 times (FIG. 3).

[Example 3]: Synthesis of ortho-hydroxyl equol using cytochrome P450 or 4-hydroxyphenylacetate 3-hydroxylase as a biocatalyst

Recombinant E. coli expressing wild-type cytochrome P450 CYP102G4 derived from Streptomyces ketilia and recombinant E. coli expressing wild-type 4-hydroxyphenylacetate 3-hydroxylase derived from E. coli were used as biocatalysts. After setting the OD of the microorganism to 10 in mM potassium phosphate buffer, a reaction solution containing 0.2 mM S-equol, 2% glucose (w/v), 1% glycerol (v/v), and 10 mM ascorbic acid was added. The reaction was carried out for 18 hours while stirring at 200 rpm. As a result, when cytochrome P450 was used as a biocatalyst, 3'-hydroxy-equol, 6-hydroxy-equol and 8-hydroxy-equol were obtained as products, whereas 4-hydroxyphenylacetate 3-hydroxyl When roxylase was used as a biocatalyst, 6-hydroxy-equol and 6,3'-dihydroxy-equol were obtained as products (Fig. 4).

Since the ortho-hydroxyl equol synthesized in the present invention is the result of bioconversion for the first time using an enzyme, trimethylsilylation with BSTFA (N , O- Bis(trimethylsilyl)trifluoroacetamide), followed by gas chromatography/mass spectrometry. Analysis was performed to identify the exact hydroxyl position (FIG. 5).

[Example 4]: Synthesis of ortho-hydroxyl equol using mutant 4-hydroxyphenylacetate 3-hydroxylase as a biocatalyst

A bioconversion reaction of S-equol was attempted using E. coli expressing the mutant 4-hydroxyphenylacetate 3-hydroxylase prepared according to Preparation Example 2 as a biocatalyst. Specifically, conversion rate and production concentration of the produced ortho-hydroxyl-S-equol were measured after 4 hours reaction using 0.2 mM S-equol as a substrate, and the remaining reaction conditions were carried out in the same manner as in Example 3. As a result, compared to wild-type 4-hydroxyphenylacetate 3-hydroxylase, the selectivity of the mutant 4-hydroxyphenylacetate 3-hydroxylase to 6-hydroxy-equol is about 2 to 16 times. It could be confirmed that it increased. The selectivity is that of 6-hydroxy equol (6-OHE) compared to 3'-hydroxy equol (3'-OHE) and 6,3'-dihydroxy-equol (6,3'-diOHE). It shows the ratio of the amount produced [that is, selectivity = 6-OHE/(3'-OHE + 6,3'-diOHE)] (Fig. 6).

[Example 5]: Selective synthesis of daidzein to 6-hydroxy-equol through a single vessel reaction

Proceeding at once by combining the conventional method of producing S-equol from Daidzein using the recombinant strain disclosed in Patent Document 3 and the method of the present invention for producing an equol hydroxide derivative from S-equol In order to check whether it is possible to do, the following experiment was performed.

Specifically, according to Preparation Example 2, a mutant 4-hydroxyphenylacetate 3-hydroxylase T292A enzyme was prepared, which was transformed into E. coli to produce a recombinant strain (tHpaBC-T292A). In addition, using the recombinant Escherichia coli (tDDDT) (OD ₆₀₀ = 20) producing S-equol from Daidzein disclosed in Patent Document 3 and the tHpaBC-T292A (OD ₆₀₀ = 20) as biocatalysts, 0.2 M phosphoric acid 1 mM daidzein was added to a reaction solution containing potassium buffer pH 8.0, glucose 2% (w/v), glycerol 1% (v/v), and ascorbic acid 10 mM, followed by reaction for 24 hours. After completion of the reaction, the reaction solution was extracted with ethyl acetate and analyzed by gas chromatography-mass spectrometry (GC-MS). As a result of the analysis, the conversion rate of daidzein was 99% or more, and 6-hydroxy-equol was produced up to 244 mg/L in 12 hours (FIG. 7).

Ortho-hydroxyl-equol synthesized in the present invention (3'-hydroxy-equol, 6-hydroxy-equol, 8-hydroxy-equol, 6,3'-dihydroxy-equol) is female As a hormone analogue, it can be used as a dietary supplement to relieve menopausal symptoms. In addition, it can be applied as a functional raw material for cosmetics having antioxidant and anti-aging functions.

The mutant tyrosinase and the mutant 4-hydroxyphenylacetate 3-hydroxylase produced in the present invention are plant secondary metabolites other than S-equol, such as flavonoids, isoflavonoids, stilbenoids, etc. It can be used as a biocatalyst for an efficient regioselective hydroxylation reaction.

<110> SEOUL NATIONAL UNIVERSITY R&DB FOUNDATION <120> Biocatalyst compositions comprising monooxygenases and method for the preparation of hydroxy equol derivatives using the same <130> P-001 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 297 <212> PRT <213> Bacillus megaterium <400> 1 Met Ser Asn Lys Tyr Arg Val Arg Lys Asn Val Leu His Leu Thr Asp 1 5 10 15 Thr Glu Lys Arg Asp Phe Val Arg Thr Val Leu Ile Leu Lys Glu Lys 20 25 30 Gly Ile Tyr Asp Arg Tyr Ile Ala Trp His Gly Ala Ala Gly Lys Phe 35 40 45 His Thr Pro Pro Gly Ser Asp Arg Asn Ala Ala His Met Ser Ser Ala 50 55 60 Phe Leu Pro Trp His Arg Glu Tyr Leu Leu Arg Phe Glu Arg Asp Leu 65 70 75 80 Gln Ser Ile Asn Pro Glu Val Thr Leu Pro Tyr Trp Glu Trp Glu Thr 85 90 95 Asp Ala Gln Met Gln Asp Pro Ser Gln Ser Gln Ile Trp Ser Ala Asp 100 105 110 Phe Met Gly Gly Asn Gly Asn Pro Ile Lys Asp Phe Ile Val Asp Thr 115 120 125 Gly Pro Phe Ala Ala Gly Arg Trp Thr Thr Ile Asp Glu Gln Gly Asn 130 135 140 Pro Ser Gly Gly Leu Lys Arg Asn Phe Gly Ala Thr Lys Glu Ala Pro 145 150 155 160 Thr Leu Pro Thr Arg Asp Asp Val Leu Asn Ala Leu Lys Ile Thr Gln 165 170 175 Tyr Asp Thr Pro Pro Trp Asp Met Thr Ser Gln Asn Ser Phe Arg Asn 180 185 190 Gln Leu Glu Gly Phe Ile Asn Gly Pro Gln Leu His Asn Arg Val His 195 200 205 Arg Trp Val Gly Gly Gln Met Gly Val Val Pro Thr Ala Pro Asn Asp 210 215 220 Pro Val Phe Phe Leu His His Ala Asn Val Asp Arg Ile Trp Ala Val 225 230 235 240 Trp Gln Ile Val His Arg Asn Gln Asn Tyr Gln Pro Met Lys Asn Gly 245 250 255 Pro Phe Gly Gln Asn Phe Arg Asp Pro Met Tyr Pro Trp Asn Thr Thr 260 265 270 Pro Glu Asp Val Met Asn His Arg Lys Leu Gly Tyr Val Tyr Asp Ile 275 280 285 Glu Leu Arg Lys Ser Lys Arg Ser Ser 290 295 <210> 2 <211> 1068 <212> PRT <213> Streptomyces cattleya <400> 2 Met Ser Pro Thr Pro His Ser Ala Ser Gly Thr Thr Gly Ala Ala Ala 1 5 10 15 Ala Thr Pro Gly Ala Ala Ser Pro Ala Pro Pro Val Pro Val Ala Asp 20 25 30 Ile Ser Asp Thr Gly Phe Gly Thr Thr Pro Ile Gln Gln Ala Met Ala 35 40 45 Leu Ala Arg Glu His Gly Pro Val Phe Arg Arg Arg Phe Gly Thr Phe 50 55 60 Glu Ser Leu Leu Val Gly Ser Val Asp Ala Val Thr Glu Leu Cys Asp 65 70 75 80 Asp Glu Arg Phe Val Lys Ala Val Gly Pro Val Leu Thr Asn Val Arg 85 90 95 Gln Ile Ala Gly Asp Gly Leu Phe Thr Ala Tyr Asn Asp Glu Pro Asn 100 105 110 Trp Ala Lys Ala His Asp Ile Leu Leu Pro Ala Phe Ala Leu Ser Ser 115 120 125 Met His Thr Tyr His Pro Thr Met Leu Arg Val Ala Lys Arg Leu Ile 130 135 140 Ala Ala Trp Asp Thr Ala Leu Ala Asp Gly Ala Pro Val Asp Val Ala 145 150 155 160 Asp Asp Met Thr Arg Met Thr Leu Asp Thr Ile Gly Leu Ala Gly Phe 165 170 175 Gly Tyr Asp Phe Gly Ser Phe Arg Arg Gly Glu Pro His Pro Phe Val 180 185 190 Ala Ala Met Val Arg Gly Leu Leu His Ser Gln Ala Leu Leu Ser Arg 195 200 205 Lys Ala Asp Asp Gly Val Asp His Ser Ala Ala Asp Glu Ala Phe Arg 210 215 220 Ala Asp Asn Ala Tyr Leu Ala Gln Val Val Asp Glu Val Ile Glu Ala 225 230 235 240 Arg Arg Ala Ser Gly Glu Thr Gly Thr Asp Asp Leu Leu Gly Leu Met 245 250 255 Leu Gly Ala Pro His Pro Ser Asp Gly Thr Pro Leu Asp Ala Ala Asn 260 265 270 Ile Arg Asn Gln Val Ile Thr Phe Leu Ile Ala Gly His Glu Thr Thr 275 280 285 Ser Gly Ala Leu Ser Phe Ala Leu Tyr Tyr Leu Ala Lys Asn Pro Ala 290 295 300 Val Leu Arg Arg Ala Gln Ala Glu Val Asp Ala Leu Trp Gly Asp Asp 305 310 315 320 Pro Asp Pro Glu Pro Asp Tyr Thr Asp Val Gly Arg Leu Thr Tyr Val 325 330 335 Arg Gln Val Leu Asn Glu Ala Leu Arg Leu Trp Pro Thr Ala Ala Ala 340 345 350 Phe Gly Arg Gln Ala Val Thr Asp Thr Val Leu Asp Gly Arg Val Pro 355 360 365 Met Arg Ala Gly Asp Thr Ala Leu Val Leu Thr Pro Val Leu His Arg 370 375 380 Asp Pro Val Trp Gly Asp Asn Val Glu Ala Phe Asp Pro Glu Arg Phe 385 390 395 400 Ser Pro Glu Arg Glu Ala Ala Arg Pro Val His Ala Phe Lys Pro Phe 405 410 415 Gly Thr Gly Glu Arg Ala Cys Ile Gly Arg Gln Phe Ala Leu His Glu 420 425 430 Ala Val Met Leu Leu Gly Met Leu Ile His Arg Tyr Arg Phe Leu Asp 435 440 445 His Ala Asp Tyr Arg Leu Arg Val Arg Glu Thr Leu Thr Leu Lys Pro 450 455 460 Asp Gly Phe Thr Leu Lys Leu Ala Arg Arg Thr Ser Ala Asp Arg Val 465 470 475 480 Arg Thr Val Ala Ser Arg Ala Ala Glu Gly Thr Ala Gly Gln Asp Ala 485 490 495 Gly Leu Pro Thr Thr Ala Arg Pro Gly Thr Thr Leu Thr Val Leu His 500 505 510 Gly Ser Asn Leu Gly Ala Cys Arg Glu Phe Ala Ala Gly Leu Ala Asp 515 520 525 Leu Gly Glu Arg Cys Gly Phe Glu Thr Thr Val Ala Pro Leu Asp Ala 530 535 540 Tyr Arg Ala Gly Asp Leu Pro Arg Thr Ser Pro Val Val Val Val Ala 545 550 555 560 Ala Ser Tyr Asn Gly Arg Pro Thr Asp Asp Ala Ala Gly Phe Val Ser 565 570 575 Trp Leu Glu Gln Ala Gly Pro Gly Ala Ala Asp Gly Val Arg Tyr Ala 580 585 590 Val Leu Gly Val Gly Asp Arg Asn Trp Ala Ala Thr Tyr Gln Lys Val 595 600 605 Pro Thr Leu Ile Asp Glu Arg Leu Ala Glu Cys Gly Ala Thr Arg Leu 610 615 620 Leu Glu Arg Ala Ala Ala Asp Ala Ala Gly Asp Leu Ala Gly Thr Val 625 630 635 640 Arg Gly Phe Gly Glu Ala Leu Arg Arg Ala Leu Leu Ala Glu Tyr Gly 645 650 655 Asp Pro Asp Ser Val Gly Ala Val Ala Gly Ala Glu Asp Gly Tyr Glu 660 665 670 Val Thr Glu Val Thr Gly Gly Pro Leu Asp Ala Leu Ala Ala Arg His 675 680 685 Glu Val Val Ala Met Thr Val Thr Glu Thr Gly Asp Leu Ala Asp Leu 690 695 700 Thr His Pro Leu Gly Arg Ser Lys Arg Phe Val Arg Leu Ala Leu Pro 705 710 715 720 Asp Gly Ala Thr Tyr Arg Thr Gly Asp His Leu Ala Val Leu Pro Ala 725 730 735 Asn Asp Pro Ala Leu Val Glu Arg Ala Ala Arg Leu Leu Gly Ala Asp 740 745 750 Pro Asp Thr Val Leu Gly Val Arg Ala Arg Arg Pro Gly Arg Gly Thr 755 760 765 Leu Pro Val Asp Arg Pro Val Thr Val Arg Glu Leu Leu Thr Tyr His 770 775 780 Leu Glu Leu Ser Asp Pro Ala Thr Ala Ala Gln Ile Ala Val Leu Ala 785 790 795 800 Asp Arg Asn Pro Cys Pro Pro Glu Gln Ala Glu Leu Lys Lys Leu Ala 805 810 815 Pro Gly Arg Ala Ser Val Leu Asp Leu Val Glu Arg Tyr Pro Ala Leu 820 825 830 Thr Gly Arg Leu Asp Trp Pro Thr Val Leu Gly Thr Leu Leu Pro Gln 835 840 845 Ile Arg Ile Arg His Tyr Ser Val Ser Ser Ser Pro Ala Val Ser Pro 850 855 860 Gly His Val Asp Leu Met Val Ser Leu Leu Glu Ala Asp Gly Arg Arg 865 870 875 880 Gly Thr Gly Ser Gly His Leu His Arg Val Arg Pro Gly Asp Val Val 885 890 895 Tyr Ala Arg Val Ala Pro Cys Arg Glu Ala Phe Arg Ile Ala Ala Gly 900 905 910 Asp Glu Val Pro Val Val Met Val Ala Ala Gly Thr Gly Leu Ala Pro 915 920 925 Phe Arg Gly Ala Val Ala Asp Arg Val Ala Leu Arg Ser Ala Gly Arg 930 935 940 Glu Leu Ala Pro Ala Leu Leu Tyr Phe Gly Cys Asp His Pro Glu Val 945 950 955 960 Asp Phe Leu His Ala Ala Glu Leu Arg Gly Ala Glu Ala Ala Gly Ala 965 970 975 Val Ser Leu Arg Pro Ala Phe Ser Ala Ala Pro Asp Gly Asp Val Arg 980 985 990 Phe Val Gln His Arg Ile Ala Ala Glu Ala Asp Glu Val Trp Ser Leu 995 1000 1005 Leu Lys Gly Gly Ala Arg Val Tyr Val Cys Gly Asp Gly Ser Arg Met 1010 1015 1020 Ala Pro Gly Val Arg Glu Ala Phe Thr Ala Leu Tyr Ala Ser Arg Thr 1025 1030 1035 1040 Gly Ala Thr Ala Glu Gln Ala Ala Gly Trp Leu Ala Asp Leu Val Ala 1045 1050 1055 Arg Gly Arg Tyr Val Glu Asp Val Tyr Ala Ala Gly 1060 1065 <210> 3 <211> 520 <212> PRT <213> Escherichia coli <400> 3 Met Lys Pro Glu Asp Phe Arg Ala Ser Thr Gln Arg Pro Phe Thr Gly 1 5 10 15 Glu Glu Tyr Leu Lys Ser Leu Gln Asp Gly Arg Glu Ile Tyr Ile Tyr 20 25 30 Gly Glu Arg Val Lys Asp Val Thr Thr His Pro Ala Phe Arg Asn Ala 35 40 45 Ala Ala Ser Val Ala Gln Leu Tyr Asp Ala Leu His Lys Pro Glu Met 50 55 60 Gln Asp Ser Leu Cys Trp Asn Thr Asp Thr Gly Ser Gly Gly Tyr Thr 65 70 75 80 His Lys Phe Phe Arg Val Ala Lys Ser Ala Asp Asp Leu Arg Gln Gln 85 90 95 Arg Asp Ala Ile Ala Glu Trp Ser Arg Leu Ser Tyr Gly Trp Met Gly 100 105 110 Arg Thr Pro Asp Tyr Lys Ala Ala Phe Gly Cys Ala Leu Gly Ala Asn 115 120 125 Pro Gly Phe Tyr Gly Gln Phe Glu Gln Asn Ala Arg Asn Trp Tyr Thr 130 135 140 Arg Ile Gln Glu Thr Gly Leu Tyr Phe Asn His Ala Ile Val Asn Pro 145 150 155 160 Pro Ile Asp Arg His Leu Pro Thr Asp Lys Val Lys Asp Val Tyr Ile 165 170 175 Lys Leu Glu Lys Glu Thr Asp Ala Gly Ile Ile Val Ser Gly Ala Lys 180 185 190 Val Val Ala Thr Asn Ser Ala Leu Thr His Tyr Asn Met Ile Gly Phe 195 200 205 Gly Ser Ala Gln Val Met Gly Glu Asn Pro Asp Phe Ala Leu Met Phe 210 215 220 Val Ala Pro Met Asp Ala Asp Gly Val Lys Leu Ile Ser Arg Ala Ser 225 230 235 240 Tyr Glu Met Val Ala Gly Ala Thr Gly Ser Pro Tyr Asp Tyr Pro Leu 245 250 255 Ser Ser Arg Phe Asp Glu Asn Asp Ala Ile Leu Val Met Asp Asn Val 260 265 270 Leu Ile Pro Trp Glu Asn Val Leu Ile Tyr Arg Asp Phe Asp Arg Cys 275 280 285 Arg Arg Trp Thr Met Glu Gly Gly Phe Ala Arg Met Tyr Pro Leu Gln 290 295 300 Ala Cys Val Arg Leu Ala Val Lys Leu Asp Phe Ile Thr Ala Leu Leu 305 310 315 320 Lys Lys Ser Leu Glu Cys Thr Gly Thr Leu Glu Phe Arg Gly Val Gln 325 330 335 Ala Asp Leu Gly Glu Val Val Ala Trp Arg Asn Thr Phe Trp Ala Leu 340 345 350 Ser Asp Ser Met Cys Ser Glu Ala Thr Pro Trp Val Asn Gly Ala Tyr 355 360 365 Leu Pro Asp His Ala Ala Leu Gln Thr Tyr Arg Val Leu Ala Pro Met 370 375 380 Ala Tyr Ala Lys Ile Lys Asn Ile Ile Glu Arg Asn Val Thr Ser Gly 385 390 395 400 Leu Ile Tyr Leu Pro Ser Ser Ala Arg Asp Leu Asn Asn Pro Gln Ile 405 410 415 Asp Gln Tyr Leu Ala Lys Tyr Val Arg Gly Ser Asn Gly Met Asp His 420 425 430 Val Gln Arg Ile Lys Ile Leu Lys Leu Met Trp Asp Ala Ile Gly Ser 435 440 445 Glu Phe Gly Gly Arg His Glu Leu Tyr Glu Ile Asn Tyr Ser Gly Ser 450 455 460 Gln Asp Glu Ile Arg Leu Gln Cys Leu Arg Gln Ala Gln Ser Ser Gly 465 470 475 480 Asn Met Asp Lys Met Met Ala Met Val Asp Arg Cys Leu Ser Glu Tyr 485 490 495 Asp Gln Asn Gly Trp Thr Val Pro His Leu His Asn Asn Asp Asp Ile 500 505 510 Asn Met Leu Asp Lys Leu Leu Lys 515 520 <210> 4 <211> 170 <212> PRT <213> Escherichia coli <400> 4 Met Gln Leu Asp Glu Gln Arg Leu Arg Phe Arg Asp Ala Met Ala Ser 1 5 10 15 Leu Ser Ala Ala Val Asn Ile Ile Thr Thr Glu Gly Asp Ala Gly Gln 20 25 30 Cys Gly Ile Thr Ala Thr Ala Val Cys Ser Val Thr Asp Thr Pro Pro 35 40 45 Ser Leu Met Val Cys Ile Asn Ala Asn Ser Ala Met Asn Pro Val Phe 50 55 60 Gln Gly Asn Gly Lys Leu Cys Val Asn Val Leu Asn His Glu Gln Glu 65 70 75 80 Leu Met Ala Arg His Phe Ala Gly Met Thr Gly Met Ala Met Glu Glu 85 90 95 Arg Phe Ser Leu Ser Cys Trp Gln Lys Gly Pro Leu Ala Gln Pro Val 100 105 110 Leu Lys Gly Ser Leu Ala Ser Leu Glu Gly Glu Ile Arg Asp Val Gln 115 120 125 Ala Ile Gly Thr His Leu Val Tyr Leu Val Glu Ile Lys Asn Ile Ile 130 135 140 Leu Ser Ala Glu Gly His Gly Leu Ile Tyr Phe Lys Arg Arg Phe His 145 150 155 160 Pro Val Met Leu Glu Met Glu Ala Ala Ile 165 170

Claims (17)

As a biocatalyst composition for producing an equol hydroxide derivative from equol, comprising a monooxygenase, a recombinant microorganism containing a gene encoding a monooxygenase, or a cell culture solution or cell extract containing the recombinant microorganism ,
The monooxygenase is 4 containing an oxygenase component of 4-hydroxyphenylacetate 3-hydroxylase (HpaB) and a reductase component of 4-hydroxyphenylacetate 3-hydroxylase (HpaC). -Hydroxyphenylacetate 3-hydroxylase,
In HpaB represented by the amino acid sequence of SEQ ID NO: 3, HpaB is a mutant HpaB in which the 292 th threonine, the 464 th serine or the 469 th arginine is substituted with alanine,
The equol hydroxide derivative is at least one compound selected from the group consisting of 3'-hydroxy-equol, 6-hydroxy-equol, and 6, 3'-dihydroxy-equol. The biocatalyst composition according to claim 1, wherein the microorganism is E. coli. The method of claim 1,
The 4-hydroxyphenylacetate 3-hydroxylase containing the mutant HpaB is selected for 6-hydroxy-equol compared to 3'-hydroxy-equol and 6, 3'-dihydroxy-equol. A biocatalyst composition, characterized in that it is improved by 2 to 16 times compared to 4-hydroxyphenylacetate 3-hydroxylase containing HpaB represented by SEQ ID NO: 3. A method for producing an equol hydroxide derivative, comprising the step of reacting a reactant containing equol to obtain an equol hydroxide derivative in the presence of the biocatalyst composition of any one of claims 1 to 3. 5. The method of claim 4, wherein the reactant comprises a reducing agent to inhibit oxidation of the resulting equol hydroxide derivative. The method of claim 5, wherein the reducing agent is at least one compound selected from the group consisting of NAD(P)H, L-ascorbic acid, glutathione, dithiothreitol, and cysteine. The method of claim 4, further comprising the step of obtaining equol by reacting a reactant comprising daidzein, obtaining equol from the reactant including daidzein, and equol from the reactant comprising daidzein A method, characterized in that the steps of obtaining the hydroxide derivative are carried out simultaneously. delete delete delete delete delete delete delete delete delete delete

Images