KR20180099380A - Recombinant e.coli producing equol and method for producing equol using thereof - Google Patents

Abstract

The present invention relates to a method of producing equol, dihydroequol, 5-hydroxy-equol, and 5-hydroxy-dehydroequol from daidzein or genistein in an efficient and selective manner by using a recombinant E. Coli in which Slackia isoflavoniconvertens-derived enzymes (daidzein reductase, dihydrodaidzein racemase, dihydrodaidzein reductase, and tetrahydrodaidzein reductase) are expressed. In addition, the microorganism strain and a biotransformed composition expressing 5-hydroxy-equol or 5-hydroxy-dehydroequol can be used as an antioxidant agent, an anticancer agent, and the like, and can be used in the prevention or treatment of climacteric disorders.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a recombinant E. coli producing an equol and a method for synthesizing an equol using the recombinant E. coli.

The present invention relates to a recombinant Escherichia coli producing equol and a method for synthesizing equol using the recombinant Escherichia coli.

It is known that some anaerobic microorganisms in intestinal microorganisms of humans metabolize isoflavones that people ingest through soybean plants and convert them into equol. The ecological synthesized microorganisms discovered so far are all bacteria, and most of them are bacteria such as Slackia sp., Eggerthella sp., Adlercreutizia sp. , And the exception is Lactococcus .

It is known that the equol produced by the enzymatic reaction of intestinal microorganism as described above only synthesizes the optical isomer of the S structure, which is absorbed by the human body and shows a phytoestrogen effect. Thus, steady ingestion of equol has been shown to alleviate the symptoms of women's menopausal symptoms, particularly osteoporosis, without symptoms of conventional menopausal symptoms such as breast cancer induction. This is because the molecular structure of equol has a similarity with human estrogen and is highly selective for α among estrogen receptors α and β. In addition, the efficacy of preventing the cardiovascular disease of (S) -equal and alleviating prostate cancer has been revealed (Non-Patent Document 1). It has been reported that 5-hydroxy-equol, an analogue of equol, is a plant-derived estrogen which is derived from isoflavone genistein and exhibits an antioxidative activity comparable to that of equol, though its biological efficacy is relatively small compared to equol (Non-Patent Document 2). In addition, it has been reported that dehydroequol, a 5-hydroxy-dehydroequol analogue synthesized in the present invention, inhibits the growth of prostate cancer and ovarian cancer cells, The results of the second phase test have shown that it is effective in inhibiting ovarian cancer that is resistant to chemotherapeutic agents (Non-Patent Document 3).

However, unlike a variety of efforts and studies for the synthesis of equol using intestinal microorganisms, synthetic efforts for 5-hydroxy-equol or 5-hydroxy-dihydroequ ator have been limited. It has been reported that some intestinal microbes biosynthesizing equol may synthesize 5-hydroxy-equol as follows. Genistein was found to be biosynthesized by 5-hydroxy-equol via dihydrogenentin using the Coryobacterium strain Mt1B8 identified in the rat's intestine and the slaky isois flavone converts identified in the human feces Non-Patent Document 4). However, the production of 5-hydroxy-equol using these strains is limited to the growth of microorganisms under anaerobic conditions, the production of 5-hydroxyequaurs is at least 15 hours after the microbial inoculation, the productivity is low, There is a limitation that it is difficult to accurately predict the final yield due to the absence of the material.

One of the isoflavones, genistein, was converted to 5-hydroxy-equol by the microorganisms Coriobacteriacae Strain Mt1B8 (16), Slackia isoflavoniconvertens (17), strain AUH-JLC159 There is a problem that the conversion and the conversion time are less than 80% when the amount of the substrate is 0.6 mmol / L or more (see Patent Document 1 ). On the other hand, there is no synthesis report for 5-hydroxy-dihydro etchol.

The synthesized 5-hydroxy-equol proved that DPPH radical scavenging effect is 20 times that of (S) -equal and increases life span and stress resistance of Caenorhabditis elegans (Non-Patent Document 5) . However, other physiological activities of 5-hydroxy-equol have not been elucidated.

Recently, four enzymes that are responsible for converting the isoflavone daidzein to the equol in bacteria of Lactococcus and Slaughter were first identified by the Annett Braune group in 2013 (Non-Patent Document 6). The enzyme is composed of three reducing enzymes and one isomerizing enzyme and is composed of a daidzein reductase, a dihydrodaidzein racemase, a dihydrodaidzein (dihydrodaidzein) reductase, and tetrahydrodaidzein reductase.

Chinese Patent Publication No. 103275884

 Nutrition Reviews, 69 (8), 432-448, 2011  J. Chin. Pharm. Sci., 23 (6), 378-384, 2014  Int. J. Gynecol. Cancer, 21 (4), 633-639, 2011  Applied and Environmental Microbiology, 74, 4847 (2008) / Applied and Environmental Microbiology, 75, 1740 (2009)  Conghui Zhang et al., J. Chin. Pharm. Sci., 23 (6): 378-384, 2014  Appl. Environ. Microbiol., 79 (11), 3494, 2013

It is an object of the present invention to provide a recombinant Escherichia coli expressing four enzymes derived from a slaky isoflavonikconvens.

Another object of the present invention is to provide a method for synthesizing equol, dihydroequor, 5-hydroxy-equol or 5-hydroxy-dihydroquercol from daidzein or genistein using the recombinant E. coli .

It is another object of the present invention to provide a recombinant Escherichia coli expressing a combination of two or three enzymes out of four enzymes derived from a slaky isoflavonikonvirtense. The recombinant Escherichia coli expressing 5-hydroxy-equol, And a process for selectively and efficiently synthesizing 5-hydroxy-dihydrocodeol.

In order to achieve the above object, the present inventors have found that a daidzein reductase derived from Slackia isoflavoniconvertens , which is intestinal microorganism of human, a dihydrodaidzein racemase (dihydrodaidzein racemase) ), A dihydrodaidzein reductase, a tetrahydrodaidzein reductase, and a recombinant Escherichia coli expressing a combination of two or three enzymes among the four enzymes. .

In addition, the recombinant E. coli of the present invention can be used as a whole cell biocatalyst for biotransformation of equol or dehydroequol from daidzein, Hydroxy-equol or 5-hydroxy-dihydrocourea. ≪ / RTI >

The present invention relates to a process for the biosynthesis of equol, dihydroequol, 5-hydroxy-equol or 5-hydroxy-dihydroequor with a substrate concentration of from about 0.2 to about 1 mmol / can do.

In addition, the present invention can selectively synthesize 5-hydroxy-equol, dihydroequor or 5-hydroxy-dihydroequoir.

The present invention also provides for the selective synthesis of 5-hydroxy-equol, dihydroequol and 5-hydroxy-dihydroequccol under aerobic conditions in hours to hours, It can be used for a variety of applications such as foods and pharmaceuticals through efficient mass production.

In addition, when the recombinant E. coli of the present invention is applied to a reaction system using a whole cell catalyst as an additional oxidoreductase, various hydroxy-isoflavonoids can be synthesized, which is highly valuable in research.

In addition, the compound synthesized by the present invention can be used for prevention and treatment of menopausal diseases, and can be used as an anticancer agent for the treatment of prostate cancer and ovarian cancer. It can also be used as an antioxidant in the form of a cosmetic or food additive.

1 is a chart showing the yield of (S) -equal with the concentration of initial daidzein.
FIG. 2 is a graph showing the effect of the equol, the dehydroequol, the 5-hydroxy-equol and the 5-hydroxy-dihydroxy compound, which are recombinant Escherichia coli expressing all four enzymes, (5-hydroxy-dehydroequol) synthesis reaction.
FIG. 3 is a graph showing the results of reaction of genistein (GSN), dihydrogenistein (DHG), 5-hydroxy-equol, 5OH-EQ, (ΜM) of 5-hydroxy-dehydroequol, 5-OH-DEQ in the reaction system.
FIG. 4 is a schematic diagram of a synthetic reaction compartmentalized using recombinant E. coli 1 and 2 expressing two enzymes.
FIG. 5 is a graph showing the results of reaction of genistein (GSN), dihydrogenistein (DHG), 5-hydroxy-equol, 5OH-EQ, (ΜM) of 5-hydroxy-dehydroequol, 5-OH-DEQ in the reaction system.
Figure 6 shows the amount of protein in the cell extract of recombinant E. coli referred to in Figures 2 and 4 by SDS-PAGE.
FIG. 7 is a chart for measuring the ellipticity using a circular dichroism dispersion system in a wavelength range of 210 to 320 nm. FIG.
Figure 8 shows the concentration (μM) of dehydroequol in the reaction system with the reaction time using recombinant E. coli expressing two or three enzymes.
FIG. 9 is a chart showing the substrate conversion and the yield of 5-hydroxy-dihydroequoir according to the concentration of the initial genistein.
Figure 10 (a) shows the mass spectrum and molecular structure of the 5-hydroxy-equol analyzed by EI-MS and (b) is the mass spectrum and molecular structure of 5-hydroxy-dihydroequour.
11 is a graph showing the concentration of (S) - equol in the reaction system with time in the reaction solution containing polyethylene glycol (PEG) or polyvinyl pyrrolidone (PVP) It is a chart.

The term used in the present invention is commonly used in the art and anyone skilled in the art can understand the meaning thereof, but here is briefly described as follows:

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

(2) Vector - means a polynucleotide consisting of single strand, double strand, circular or super stranded DNA or RNA. The vector consists of a cloned origin, a promoter, a ribosome binding site, a nucleic acid cassette, and a terminator operatively linked at appropriate distances to produce a recombinant protein. The gene encoding the recombinant protein to be expressed is inserted into the nucleic acid cassette.

(3) Cell Extract - refers to the supernatant obtained by disrupting the microorganism expressing the recombinant protein and removing the solid cell debris with a centrifuge. The term "microorganism extract" means a microorganism extract in which some or all of a reducing enzyme such as a daidzein reductase, a dihydroididase, a racemizing enzyme, a dihydroididase reductase, or a tetrahydroididase is expressed in the present invention.

(4) Whole Cell Reaction - This refers to a reaction using whole cell without extracting or purifying the enzyme by using cell extract to disrupt cells containing specific enzyme.

The present invention relates to a process for the production of a pharmaceutical composition comprising a daidzein reductase, a dihydrodaidzein racemase, a dihydrodaidzein reductase and a tetrahydrodaidzein reductase, Lt; RTI ID = 0.0 > E. coli. ≪ / RTI >

In the present invention, there is provided a method for producing a compound of formula (I), which is a reducing enzyme which is a sideroid derived from Slackia isoflavoniconvertens , a racemizing enzyme which is a dihydroidide, a reducing enzyme which is a dihydroidide, The DNA sequence was amplified by polymerase chain reaction (PCR) and inserted into pRSFDuet or pCDFDuet vector, which was expressed in E. coli as his-tag (6 histidine).

The recombinant E. coli can be used to synthesize equol or dihydrocequal from the substrate daidzein.

In addition, the recombinant E. coli can be used to synthesize 5-hydroxy-equol or 5-hydroxy-dihydrocequal from the substrate, genistein.

The equol or 5-hydroxy-equol synthesis method can be used to synthesize (S) -equol or 5-hydroxy- (S) -equol.

More specifically, the recombinant E. coli can be used as a whole cell biocatalyst for biotransformation of equol and dehydroequol from daidzein, and also can be used as a whole cell biocatalyst, It can be used as a whole-cell catalyst for biotransformation into 5-hydroxy-equol and 5-hydroxy-dehyroequol.

In the equol synthesis reaction using the recombinant Escherichia coli of the present invention as a whole cell catalyst, a reducing agent is added to inhibit the oxidation of the product, and NAD (P) H, L-ascorbic acid, glutathione, Dithiothreitol, cysteine and the like can be used. The concentration of the added reducing agent is preferably about 10 to about 100 times the initial substrate concentration. The reaction solution contains potassium phosphate buffer solution for pH buffering action, and the concentration of potassium phosphate is preferably about 50 to about 400 mM. The reaction temperature is preferably about 18 to about 37 캜, more preferably about 25 to about 30 캜. 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 stirring speed of the reactor is preferably about 50 to about 400 rpm, more preferably about 80 to about 250 rpm, and even more preferably about 100 to about 200 rpm.

The concentration of glucose or glycerol is preferably about 1 to about 5 (w / v)%, more preferably about 1.5 to about 4 (w / v)%, , More preferably from about 2 to about 3 (w / v)%. The concentration of the recombinant microorganism of the present invention is preferably about 1 to 100, more preferably about 5 to about 50, and even more preferably about 10 to about 30, in optical density (O.D.).

As the substrate of the equol synthetic reaction system, depending on the kind of isoflavan or isoflavene to be synthesized, genistein, glycitein, daidzein, ortho- hydroxy-daidzein (ortho -hydroxy-daidzein), ortho-can be used genistein (ortho -hydroxy-genistein) such as -hydroxy.

In order to improve the solubility of the substrate in the reaction solution, it is preferable to use polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol and beta-cyclodextrin, methyl beta-cyclodextrin methyl-beta-cyclodextrin, 2- hydroxypropyl-beta-cyclodextrin, and the like.

FIG. 1 is a graph showing the yield of (S) -equal according to the concentration of the initial daidzein. As shown in FIG. 1, as the concentration of the initial daidzein increases, the concentration of (S) The yield (%) decreases, but the (S) - equol (mM) produced tends to increase.

FIG. 2 is a graph showing the results obtained from daidzein and genistein by using a daidzein reductase (DZNR), a dihydrodaidzein racemase (DDRC), a dihydrodaidzein reductase (DHDR) Scheme of Synthesis of Equol, Dihydroequal, 5-Hydroxy-Equol and 5-Hydroxy-Dihydrocodeol Using Recombinant Escherichia coli Expressing Tetrahydrodaidzein Reductase (THDR) to be.

As shown in FIG. 2, daidzein is converted to (R) -dihydrodaidzein by a daidzein reductase (DZNR), which is a racemizing enzyme (DDRC), a dihydroidide, (S) -dihydroidide < / RTI > (S) -dihydrodaidzein is converted to (3S, 4R) -trans-tetrahydroidadein by a reductase which is a dihydroidide (DHDR), which is a tetrahydroidade reductase (THDR) , Or converted to dihydrocourea by dehydration.

Also, as shown in FIG. 2, genistein is converted to (R) -dihydrogenentanthin by a dideoxynucleotide reductase (DZNR), which is converted by racemic dehydratase (DDRC) (S) -dihydrogenentyne. (S) -dihydrogenentanthine was converted to (3S, 4R) -trans-tetrahydrogenenustine by a reductase which is a dihydroidide (DHDR), which is converted to 5- Hydroxy-equol, or converted to 5-hydroxy-dihydrocourea by dehydration.

The present invention relates to a process for the production of a pharmaceutical composition comprising a daidzein reductase, a dihydrodaidzein racemase, a dihydrodaidzein reductase and a tetrahydrodaidzein reductase, , And 5-hydroxy-equol was selectively produced from the substrate, genistein, by using recombinant E. coli having a tetrahydrodaidzein reductase, which is over-expressed 5 times more than the other three enzymes, Lt; / RTI >

Specifically, 5-hydroxy-equol can be selectively synthesized by using a recombinant E. coli overexpressing 5-fold or more, preferably 10-fold or more over the other three enzymes of the reductase, which is a tetrahydrolide.

This is because, as shown in FIG. 2, when the amount of expression of the reducing enzyme, a tetrahydroidide, is increased, the rate of synthesizing 5-hydroxy-equol from tetrahydrogenidyne is increased and 5-hydroxy-dihydro This is because the production of Kuol is competitive.

The method of expression of the recombinant E. coli and the method of synthesizing 5-hydroxy-equol using the same are a method for producing recombinant Escherichia coli expressing all four of the enzymes derived from the slaky iso flavonicona vector, This is the same as the synthesis method.

FIG. 6 is a graph showing the amount of protein in the cell extract of recombinant E. coli mentioned in FIGS. 2 and 4 by SDS-PAGE and showing the expression levels of four enzymes when the four recombinant enzymes were expressed in a single E. coli , A recombinant Escherichia coli expressing a daidzein reductase and a dihydroididase as a single vector, and a recombinant Escherichia coli expressing a reductase which is a dihydroidide, a reductase, a tetrahydrolide, as a single vector The expression level of the enzyme was increased. In FIG. 6, 1: DDDT represents the case where the four recombinant enzymes mentioned in FIG. 2 are expressed in one E. coli, and 2: DD represents the case of the redox enzyme, which is a diiodine reducing enzyme and a dihydroidide, 3: DT corresponds to a cell extract of a recombinant Escherichia coli expressing a recombinant Escherichia coli, which is a dihydroidide, and a reducing enzyme, which is a tetrahydrolide, as one vector. .

FIG. 5 shows the concentration (μM) of the reaction system in the reaction system of the reaction time with respect to the reaction time of the genistein, dihydrogenistein and 5-hydroxy-equol, and the expression amount of the reducing enzyme, tetrahydroidide, Using the system (FIG. 6, 2 and 3) expressing recombinant E. coli increased more than twice, the yield and productivity of 5-hydroxy-equol and 5- Indicating that the selectivity to 5-hydroxy-equol versus hydroxy-dihydroequ ateol can be increased.

The present invention also relates to a recombinant Escherichia coli 1 expressing a daidzein reductase and a dihydrodaidase (dihydrodaidzein racemase), a tetrahydrodaidzein reductase and a tetrahydroidide The present invention relates to a method for selectively synthesizing 5-hydroxy-equol from a substrate, genistein, using recombinant Escherichia coli 2 expressing tetrahydrodaidzein reductase.

5-hydroxy-equol can be selectively synthesized from genistein by a biotransformation system in which the recombinant Escherichia coli 1 and 2 are mixed with a whole-cell catalyst.

The expression method of recombinant Escherichia coli 1 and 2 of the present invention and the method of synthesizing 5-hydroxy-equol using the same according to the present invention are characterized by a method of expressing recombinant enzyme expressing all four of the enzymes derived from slaky iso flavonicona And equol synthesis method using the same.

The mixed reaction system of recombinant Escherichia coli 1 and 2 is not limited to the selective production of 5-hydroxy-equol but also to the production of 6-methoxy-equol and various hydroxy- ) And the like.

FIG. 4 is a graph showing the relationship between a recombinant E. coli expressing a daidzein reductase (DZNR) and a dihydrodaidzein racemase (DDRC) as a single vector and a dihydrodaidzein reductase (DHDR), and tetrahydrodaidzein reductase (THDR) as one vector, into a recombinant E. coli.

According to FIG. 4, the recombinant Escherichia coli 1 converts genistein to (S) -dihydrogenentanthine and the (S) -dihydrogenentanthin is converted to 5-hydroxy- (S) , Indicating that selective recombination of 5-hydroxy-equol from genistein by recombinant E. coli 1 and 2 is possible.

The whole cell reaction system in which the reaction is compartmentalized with different recombinant E. coli is advantageous in that the productivity and final yield of 5-hydroxy- (S) -equal is improved and the production of 5-hydroxy-dihydroequ ~ (S) -equol is increased by reducing the selectivity to 5-hydroxy- (S) -equol.

The selective synthesis of the 5-hydroxy-equol can selectively synthesize 5-hydroxy- (S) -equoir.

Specifically, 5-hydroxy- (S) -equol can be selectively synthesized using recombinant Escherichia coli overexpressing 5-fold or more of a tetrahydrodaidzein reductase, or recombinant Escherichia coli 1 and 2 have.

In this regard, FIG. 7 is a chart for measuring the ellipticity using a circular dichroism dispersion system in a wavelength range of 210 to 320 nm in order to determine the optical structure of 5-hydroxy-equol synthesized using the recombinant E. coli. The 5-hydroxy-equol biosynthesized by recombinant E. coli of the invention has an S-form at the 3-carbon.

Furthermore, the present invention provides a method for producing a recombinant vector comprising the steps of expressing a daidzein reductase, a dihydrodaidase, a dihydrodaidzein racemase, and a dihydrodaidze reductase, the present invention relates to a method for selectively synthesizing dihydroquercol from a substrate zidene using recombinant E. coli that does not express a tetrahydrodaidzein reductase.

Specifically, the dihydroquercol can be selectively synthesized from daidzein using the recombinant E. coli as a whole cell catalyst.

Addition of boric acid to the reaction solution for synthesizing the dihydrocoureth promotes the dehydration reaction of (3S, 4R) -trans-tetrahydroidadein, so that dihydroquercol can be synthesized with high yield. The amount of boric acid added is preferably about 50 mM to about 200 mM.

The present invention also provides a method for producing a recombinant vector comprising the steps of expressing a daidzein reductase, a dihydrodaidase, a dihydrodaidzein racemase and a dihydrodaidzein reductase, dihydroquinol is selectively synthesized from a substrate, genistein, using recombinant E. coli that does not express a tetrahydrodaidzein reductase.

Specifically, 5-hydroxy-dihydroquercol can be selectively synthesized from genistein using the recombinant E. coli as a whole cell catalyst.

The method of expression of the recombinant E. coli of the present invention and the method of synthesizing dihydrococcal or 5-hydroxy-dihydroequaucol using the same are characterized in that the recombinant Escherichia coli expressing all four of the enzymes derived from the slaky iso flavonicona The expression of the enzyme and the method of synthesizing equol using the same.

FIG. 8 is a graph showing the relationship between recombinant E. coli (DZNR + DHDR) and daidzein reductase (DZNR) expressing daidzein reductase (DZNR), dihydrodaidzein reductase (DHDR) (DZNR + DRDR + DHDR) expressing a dihydrodaidzein racemase (DDRC) and a dihydrodaidzein reductase (DHDR) were used to express the dideoxynucleotides And the concentration of dihydroequaol in the reaction system according to the reaction time when the cell reaction is performed. As shown in FIG. 8, the recombinant Escherichia coli can be used to selectively synthesize dihydrocourea from daidzein without generating an equol, and to utilize recombinant Escherichia coli (DZNR + DRDR + DHDR) expressing three enzymes , The concentration of dihydrocourea produced from recombinant E. coli (DZNR + DHDR) expressing two enzymes is high.

FIG. 9 is a graph showing the results obtained when a whole cell reaction was performed on genistein using a daidzein reductase, a racemizing enzyme as a dihydroidide, and a recombinant Escherichia coli expressing a reductase as a dihydroidide, As shown in FIG. 9, the recombinant E. coli can be used to selectively synthesize 5-hydroxy-dihydroquercol from genistein, which is a chart showing the conversion rate and the yield of 5-hydroxy-dihydroequor.

The recombinant E. coli of the present invention can be used as a whole-cell catalyst. The whole cell catalyst is used for many biocatalytic reactions and has an advantage of promoting the reaction under mild conditions of normal temperature and normal pressure and excellent in reaction specificity. It is more advantageous to carry out the biocatalytic process using the whole cell in the case that coenzyme is necessary or the activity accompanied by the reaction of several stages or the activity of the enzyme is remarkably decreased upon purification. In addition, it is advantageous that the recombinant E. coli used as the host cell can be selectively separated using a centrifuge even when the product is purified after completion of the reaction.

The method of synthesizing the above-synthesized equol, dihydroequoir, 5-hydroxy-equol or 5-hydroxy-dihydroequaucol can be carried out under aerobic conditions, so that, compared to the prior art of synthesizing equol in anaerobic conditions Equol can be synthesized under mild conditions. Therefore, the synthesis method of the present invention does not require the devices or techniques used to provide anaerobic conditions in the prior art.

The synthesized equol, dihydroequoir, 5-hydroxy-equol or 5-hydroxy-dihydroequaucol synthesis method may employ a substrate of about 0.2 to about 5 mmol / L.

When the concentration of the substrate zidene or genistein is less than 0.2 mmol / L, the amount of equol, dihydroequoir, 5-hydroxy-equol and 5-hydroxy-dihydroequaol synthesized is insufficient The purification cost may be increased, and if it exceeds 5 mmol / L, the conversion rate of the substrate may decrease, resulting in further purification costs.

Preferably, the concentration of the substrate is about 0.2 to 1 mmol / L, more preferably the concentration of the substrate is about 0.8 to about 1 mmol / L.

However, when a water-soluble polymer is additionally contained in the reaction system, even if the concentration of the substrate exceeds 1 mmol / L, equol, dihydroequor, 5-hydroxy- equol or 5-hydroxy- Can be synthesized with a high yield, the concentration of the substrate is preferably about 3 to 5 mmol / L.

The method for synthesizing equol or dihydroequaucol has a conversion rate of dideoxyne of 95% or more, and the method for synthesizing 5-hydroxy-equol or 5-hydroxy-dihydroequorin has a conversion rate of genistein of 95% to be.

The conversion rate (reduced substrate concentration / initial substrate concentration) X 100 (%).

In the synthesis method of the present invention, the conversion of the substrate may be 95% or more even when the concentration of the substrate is 0.6 mmol / L or more.

3 and 5 show the concentration (μM) of genistein (GSN), dihydrogenistein (DHG) and 5-hydroxy-equol (5OH-EQ) The conversion rate of the substrate is more than 95% in 10 hours.

The method for synthesizing the equol, dihydroequol, 5-hydroxy-equol or 5-hydroxy-dihydroequaucol may further comprise a water-soluble polymer in the reaction system.

Examples of the water-soluble polymer include polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol and beta-cyclodextrin, methyl-beta-cyclodextrin, -cyclodextrin, 2- hydroxypropyl-beta-cyclodextrin and the like can be used. Preferably, polyethylene glycol, polyvinyl alcohol or polyvinylpyrrolidone can be used.

Since the solubility of the substrate in the reaction system is increased by further including a water-soluble polymer in the reaction system, the final yield of the produced equol, dihydroequor, 5-hydroxy-equol or 5-hydroxy- Can be increased.

11 is a graph showing the concentration of (S) - equol in the reaction system in the reaction solution obtained by further adding polyethylene glycol or polyvinylpyrrolidone as a water-soluble polymer to the reaction solution of Example 1 with time. As shown in FIG. 11, when polyethylene glycol (PEG) or polyvinylpyrrolidone (PVP) is further added to the reaction solution, equol can be synthesized at a high yield even when a substrate of 5 mmol / L is used .

The present invention can synthesize (S) -equal with a high substrate conversion rate under aerobic conditions, and since the synthesized equol has a molecular structure similar to that of estrogen, it can be administered to women without the side effects of conventional treatments for menopausal symptoms such as breast cancer It can alleviate menopausal symptoms, especially the onset and symptoms of osteoporosis, prevent cardiovascular disease and relieve prostate cancer.

In addition, the dihydroequaldehyde synthesized in the present invention can be used as an anticancer agent for the treatment of prostate cancer and ovarian cancer.

The present invention also provides for the selective synthesis of 5-hydroxy-equol, dihydroequol and 5-hydroxy-dihydroequccol under aerobic conditions in hours to hours, It can be used for a variety of applications such as foods and pharmaceuticals through efficient mass production.

Further, when an additional oxidoreductase is applied to a reaction system using the recombinant E. coli of the present invention as a whole-cell catalyst, various hydroxy-isoflavonoids can be synthesized, which is worth research.

Specifically, tyrosinase, cytochrome P450 or flavin monooxygenase can be used as an oxidoreductase, and 3'-hydroxy-equol, 6-hydroxy-equol, 8-hydroxy- Hydroxy-isoquinone, such as 6,3'-dihydroxy-equol, can be synthesized.

In addition, 5-hydroxy-equol or 5-hydroxy-dihydroequol synthesized by the present invention can be used for the prevention and treatment of menopausal diseases and can be used as an anticancer agent for the treatment of prostate cancer and ovarian cancer. It can also be used as an antioxidant in the form of a cosmetic or food additive.

Hereinafter, specific methods of the present invention will be described in detail by way of examples, but the technical scope of the present invention is not limited to these examples.

Method for producing recombinant E. coli

(DDR), a dihydrodaidzein reductase (DHDR), a tetrahydrodaidzein (tetrahydrodaidzein), and a tetrahydrodaidzein reductase reductase: THDR) was amplified by PCR and inserted into pRSFDuet or pCDFDuet vector, which was expressed in E. coli as his-tag (6 histidine).

Specifically, the plasmid containing the inserted nucleotide sequence of the enzyme was transformed into BL21 competent cells and cultured in LB solid medium containing antibiotics for 12 hours at 37 ° C. One colony was inoculated into 3 mL LB broth containing antibiotics and cultured at 37 ° C for 12 hours at 200 rpm. 2 v% (1 mL) of this culture was inoculated into fresh LB broth containing 50 mL of antibiotics. When the OD (600 nm) of the culture reached 0.6 to 0.8, 0.1 mM IPTG and 0.1 mM MnSO4 were added and protein expression was induced at 18 rpm for 18 hours at 200 rpm in an incubator. After 18 h, the cells were centrifuged at 4000 rpm and washed with 25 mL PBS.

The method for producing equol in the whole cell reaction system using the recombinant E. coli of the present invention (reaction method 1)

10 ~ 1000 ml glass beaker is used as a reactor, and the following concentrations of the recombinant E. coli and the substrate are supplied to the reactor.

In the reaction, L-ascorbic acid as a reducing agent is added at an initial substrate concentration of 10 times, and the reaction solution contains potassium phosphate buffer having a concentration of 200 mmol / L for pH buffering reaction, and the reaction temperature is 30 占 폚. The pH of the reaction solution was 8, and the stirring speed of the reactor was 150 rpm. Glucose or glycerol was used as a carbon source in the reactor of the present invention, the concentration of glucose or glycerol was 2 (w / v)%, and the concentration of recombinant microorganism was OD 10 or OD 20.

[Example]

[Example 1] Synthesis of (S) -equanol from dideoxyne using recombinant Escherichia coli expressing all four enzymes involved in equol conversion.

0.2 to 5 mM of daidzein was prepared according to OD. The reaction was carried out according to Reaction Method 1 using recombinant Escherichia coli expressing the above four enzymes. Substrate, intermediate, and product were extracted with ethyl acetate (EA) over four times, and the solvent was removed using centrifugal decompressor. Then, the methanol was re-dissolved and the reactivity and concentration of the substance were determined by high performance liquid chromatography Respectively. As in the (S) -equal yield shown in FIG. 1, yields were found to be 95% or more at 0.2 mM, 80% at 0.4, 0.6 mM, 50% at 2 mM, and 20% at 5 mM.

[Example 2] Synthesis of 5-hydroxy-equol or 5-hydroxy-dihydrocodeol from genistein using recombinant Escherichia coli expressing all four enzymes involved in equol conversion.

500 [mu] M of genistein (Fig. The reaction was carried out according to Reaction Method 1 using recombinant Escherichia coli expressing the above four enzymes. Substrate, intermediate, and product were extracted with ethyl acetate (EA) more than four times, and the solvent was removed using centrifugal decompressor, and the reactivity and concentration of the substance were confirmed by high performance liquid chromatography using a certain amount of methanol. At 6 hours of reaction, 500 μM of genistein was converted by 99% or more, and it was confirmed that 5-hydroxy-equol 97 mg / L and 5-hydroxy-dihydroequccol 22 mg / L were produced.

As shown in FIG. 3, the conversion of the substrate was 95% or more and the conversion rate of 5-hydroxy-equol was 199 mg / L And 63 mg / L of 5-hydroxy-dihydrocodeol.

[Example 3]: Selective synthesis of 5-hydroxy-equol using a partitioned reaction system

As shown in the schematic diagram of FIG. 4, recombinant Escherichia coli 1 expressing a daidzein reductase and a racemized enzyme, a dihydroidide, and a recombinant Escherichia coli 2 expressing a reducing enzyme, a reducing hydrolase and a tetrahydrolide, A biocatalytic reaction system was constructed by mixing with a cell catalyst. When 1 mM of genistein was reacted according to Reaction Scheme 1, the concentration of the substrate and product according to the reaction time was confirmed by liquid chromatography as shown in FIG. In this reaction system, the conversion of the substrate was 99% or more, the 231 mg / L of 5-hydroxy-equol and 12 mg / L of 5-hydroxy-dihydroquercol in a reaction time of 6 hours. The productivity of the 5-hydroxy-equol increased 1.6-fold when the production results were compared with the results of Example 1, and the productivity of the 5-hydroxy-equaol / 5-hydroxy-dihydroequal The selectivity to 5-hydroxy-equol increased 5-fold.

In order to compare the expression amounts of the four enzymes, the recombinant E. coli in Example 2 and the cell extracts of recombinant E. coli 1 and 2 in Example 3 were prepared by disruption by ultrasonic treatment, and the cell extract was centrifuged at 13000 rpm And centrifuged at 4 ° C. The supernatant was obtained and analyzed by SDS-PAGE as shown in FIG. 6.

As a result of the analysis, the expression amounts of the four enzymes were increased in both recombinant E. coli used in Example 3 than in E. coli used in Example 2. Among them, the expression amount of a reducing enzyme such as tetrahydroidide was remarkably increased, and it was increased more than 20 times as compared with that of the Escherichia coli disruptant of Example 1, and was over 5 times more than the expression amount of the remaining three enzymes.

[Example 4]: Optical isomer analysis of 5-hydroxy-equol

To determine the optical structure of the 5-hydroxy-equol synthesized in the recombinant E. coli of the present invention, the original dichroism spectrum analysis was carried out. The measurement wavelength range was from 210 to 320 nm, and a circular dichroic dispersion ChirascanTM plus CD spectrometer (Applied Photophysics, UK) was used. The sample was dissolved in pure methanol at a concentration of 1.5 mg / ml and then measured.

The measurement results are shown graphically in FIG. 7, showing a high negative cotton effect at 238 nm and a positive cotton effect at 270 nm. Therefore, it was confirmed that 5-hydroxyequaur biosynthesized by the recombinant E. coli of the present invention had an S-form at the carbon number 3.

[Example 5]: Selective synthesis of dihydrocourea

In order to biosynthesize dihydroquercol, the reductase, which is a tetrahydroidide, in the recombinant E. coli of Example 1 is deficient and contains a racemizing enzyme (DZNR + DDRC + DHDR, hereinafter referred to as DDD strain) Recombinant Escherichia coli (DZNR + DHDR, hereinafter referred to as DD strain). The E. coli was reacted with dideazine as a substrate according to Reaction Scheme 1, and 200 mM boric acid was further added to the reaction solution. As a result, it was confirmed that only dihydroquat was produced without equol production (FIG. 8). Specifically, when the biosynthesis reaction was initiated at a concentration of 0.5 mM, 28 μM, and 60 μM of DDD in the DDD strain.

[Example 6]: Selective synthesis of 5-hydroxy-dihydrocodeol

In order to biosynthesize 5-hydroxy-dihydrocodeol, a recombinant Escherichia coli in which a reducing enzyme, a tetrahydroidide, was deleted in the plasmid in the recombinant Escherichia coli of Example 2 was prepared. Using the above-mentioned Escherichia coli, the reaction was carried out according to the reaction method 1 using the genistein as a substrate, and it was confirmed that only 5-hydroxy-dihydroquercol was produced without generating 5-hydroxy-equol (FIG. Conversion rate of genistein was more than 99% at the concentration below 0.6 mM and 97% at 1.0 mM when the biotransformation was initiated at 0.2, 0.4, 0.6 and 1.0 mM for the initial 36 hours. The conversion yields of 5-hydroxy-dihydrocodeol were 44, 35, 27 and 39%, respectively.

[Example 7] Mass spectrometry of 5-hydroxy-equol and 5-hydroxy-dihydroequor

The 5-hydroxy-equol and 5-hydroxy-dihydroequoir synthesized in Examples 3 and 6 were identified by gas chromatography-mass spectrometry (GC-MS) .

The 5-hydroxy-equol and 5-hydroxy-dihydro-equol synthesized from the recombinant microorganism were analyzed by EI-MS. The EI-MS was a TRACE GC Ultra gas chromatograph / ITQ1100 from Thermo. In the positive mode, a non-polar capillary column (TR-5 ms) was used. The helium gas flow rate was 1 ml / min and the inlet, mass transfer line and ion source temperatures were 250, 275 and 230 ° C, respectively. The oven temperature of the gas chromatograph is maintained at 65 ° C for 5 minutes and then increases to 250 ° C at a rate of 3 ° C / min. The ionization voltage was 70 eV and the measured mass range was 50 to 600 amu.

The results of the EI-MS analysis were the same as those of the previously reported DHD (Chang, YC, and MG Nair. J. Natr. Proc. 58: 1892-1896, 1995; Wahala, K., A. Salakka, and H. Adlercreutz, Proc. Soc. Exp Biol., 217: 293-29, 1998). EI-MS of the synthesized compound showed a molecular ion peak of [M + H] < + > consistent with C15H12O4 (DHD) at m / z 256 as expected. The other ion peaks of the synthesized compounds were 137 (100), 120 (52), 91 (36) and 65 (16).

[Example 8] Synthesis of equol in a reaction system further containing a water-soluble polymer

Except that it contains dimethyl sulfoxide (DMSO) and further contains polyethylene glycol (PEG) or polyvinylpyrrolidone (PVP) in order to improve the solubility in the reaction solution of the daidzein, The reaction was carried out in the same manner as in Example 1

Specifically, in order to increase the equol yield at 5 mM of daidzein, the reaction proceeded in a reactor further comprising water-soluble polymer PEG or PVP 5% (w / v). In the case of PVP-added reaction, 5 mM equol was synthesized (yield: 99% or more, 1.16 g / L) in 12 hours. In the case of PEG-added reaction, 3.7 mM of equol was synthesized in 24 hours of reaction time (yield: 74 % Or more, 0.9 g / L).

According to the above Examples 1 and 2, the recombinant Escherichia coli expressing four enzymes derived from Slaqia isoflavonik konvartens was able to produce (S) -equanol from daidzein in a high yield within a few hours under aerobic conditions Or synthesized 5-hydroxy-equol and 5-hydroxy-dihydroquercol from genistein.

In Examples 3, 5 and 6, recombinant E. coli combined with two or three enzymes among the four enzymes was used to synthesize 5-hydroxy-equol, dihydroequor or 5-hydroxy-dihydro Respectively.

Also, it can be seen from Examples 4 and 7 that the recombinant Escherichia coli according to Examples 2, 3 and 6 synthesized 5-hydroxy- (S) -equol or 5-hydroxy-dihydrocodeol there was.

Further, in Example 8, it was confirmed that the yield of equol was superior to the reaction system not containing a water-soluble polymer (Example 1) by further containing a water-soluble polymer in the reaction system.

Claims (13)

A recombinant yeast that expresses a daidzein reductase, a dihydrodaidzein racemase, a dihydrodaidzein reductase, and a tetrahydrodaidzein reductase, Escherichia coli. A method for synthesizing equol or dihydroequoir from a substrate zidene using the recombinant E. coli of claim 1. A process for synthesizing 5-hydroxy-equol or 5-hydroxy-dihydrocodeol from a substrate, genistein, using the recombinant E. coli of claim 1. A dihydrodaidzein reductase, a dihydrodaidzein reductase, and a tetrahydrodaidzein reductase, which are expressed as a dihydrogenase, a dihydrazine reductase, a dihydrodaidzein racemase, a dihydrodaidzein reductase and a tetrahydrodaidzein reductase, A method of selectively synthesizing 5-hydroxy-equol from a substrate, genistein, using recombinant Escherichia coli overexpressing five times or more of tetrahydrodaidzein reductase, which is a tetrahydrodaidzein reductase, among the four enzymes . Recombinant Escherichia coli expressing daidzein reductase and dihydrodaidzein racemase, tetrahydrodaidzein reductase and tetrahydrodaidzein reductase (hereinafter referred to as " reductase " ) Is expressed by using the recombinant Escherichia coli to selectively synthesize 5-hydroxy-equol from the substrate, genistein. A daidzein reductase, a dihydrodaidzein racemase, and a dihydrodaidzein reductase, and a tetrahydrodaidzein reductase, which is a tetrahydroidide, A method for selectively synthesizing dihydroquercol from a substrate zidene using recombinant E. coli that does not express. A daidzein reductase, a dihydrodaidzein racemase, and a dihydrodaidzein reductase, and a tetrahydrodaidzein reductase, which is a tetrahydroidide, A method of selectively synthesizing 5-hydroxy-dihydrocodeol from a substrate, genistein, using recombinant E. coli that does not express. The method according to claim 4 or 5,
Wherein the 5-hydroxy-equol is 5-hydroxy- (S) -equoir. 8. The method according to any one of claims 2 to 7,
Wherein said recombinant E. coli is used as a whole-cell catalyst. 8. The method according to any one of claims 2 to 7,
Lt; RTI ID = 0.0 > aerobic < / RTI > condition. 8. The method according to any one of claims 2 to 7,
A method of synthesizing using a substrate of 0.2 to 1 mmol / L. 8. The method according to any one of claims 2 to 7,
Wherein the conversion of the substrate is 95% or more. 8. The method according to any one of claims 2 to 7,
Characterized in that the reaction system further comprises a water-soluble polymer.

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