JP7002557B2 - Recombinant Escherichia coli that produces equol derivatives and methods for synthesizing equol derivatives using these - Google Patents

Description

The present invention relates to recombinant Escherichia coli that produces an equol derivative and a method for synthesizing an equol derivative using the recombinant Escherichia coli.

It is known that some anaerobic microorganisms among human intestinal microorganisms metabolize isoflavones ingested by humans through legumes and convert them into equol. All of the equol-synthesizing microorganisms discovered so far are bacteria, and most of them, such as Lactococcus sp., Eggerthella sp., And Adlercreutizia sp. Belongs to the genus Coriobacterium, with the exception of Lactococcus.

Equol produced by the enzymatic reaction of intestinal microorganisms as described above is known to synthesize only optical isomers of S structure, which are absorbed by the human body and exhibit a phytoestrogen effect. ing. Therefore, it has been demonstrated that constant intake of equol alleviates the onset and symptoms of female menopausal symptoms, especially osteoporosis, without the side effects of traditional menopausal treatments such as the induction of breast cancer. This effect is due to the fact that the molecular structure of equol is similar to that of human estrogen and that estrogen receptors α and β are highly selective for α. Not only that, (S) -equol has also been shown to be effective in preventing cardiovascular diseases and alleviating prostate cancer (Non-Patent Document 1). An analog of equol, 5-hydroxy-equol, is a substance derived from the isoflavone genistein, which has relatively less biological efficacy than equol but is superior to equol. It has been reported that it is a plant-derived estrogen exhibiting antioxidant properties (Non-Patent Document 2). In addition, it has been reported that dehydroequol inhibits the growth of prostate cancer and ovarian cancer cells, and as a result of the second clinical trial, it suppresses ovarian cancer that has become resistant to chemical anticancer drugs. It has been reported that it is effective in (Non-Patent Document 3).

However, unlike the various efforts and studies for equol synthesis utilizing gut microbiota, the effort for 5-hydroxy-equol synthesis was limited. However, it has been reported that some intestinal microorganisms that biosynthesize equol can synthesize 5-hydroxy-equol as follows. Using the coriobacterium Strine Mt1B8 identified in the rat intestine and the slackia isoflavoniconverter identified in human stool, it was revealed that genistein is biosynthesized via dihydrogenistein to 5-hydroxy-equol. (Non-Patent Document 4). However, the production of 5-hydroxyequol using such a strain has a limitation on the growth of microorganisms under anaerobic conditions, and 5-hydroxy-equol is produced at least 15 hours after inoculation of the microorganism. There is a limit that productivity is low and it is difficult to accurately predict the final yield due to the absence of reference materials.

One of the isoflavones, genistein, is converted to the microorganisms Coriobacteriaceae Strine Mt1B8 (16), Slackia isoflavoniconvertens (17), 5-lyen equol and 5-line AUH-JLC159. However, when the reaction and culture time is as long as 2 to 3 days, anaerobic reaction conditions are required, and the amount of the substrate genistein is 0.6 mmol / L or more, the conversion rate There is a problem that the amount is reduced to 80% or less (Patent Document 1).

The synthesized 5-hydroxy-equol has a DPPH radical scavenging effect 20 times that of (S) -equol, and has been shown to increase the lifespan and stress resistance of C. elegans nematodes (Caenorhabditis elegans). (Non-Patent Document 5). However, other physiological activities of 5-hydroxy-equol have not been revealed.

Recently, four enzymes responsible for converting isoflavone daidzein to equol from Lactococcus and Slackia bacteria were first identified in 2013 by the Annett Brane group (Non-Patent Document 6). The enzyme is composed of three reductases and one isomerizing enzyme. It is a reductase.

Chinese Patent Publication No. 103275884

NutritionReviews, 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 Microbialology, 74,4847 (2008) / Applied and Ambient Microbialology, 75,1740 (2009) Konghui Zhang et al. , J. Chin. Pharm. Sci. , 23 (6): 378-384, 2014 Apple. Environ. Microbiol. , 79 (11), 3494, 2013

An object of the present invention is to provide recombinant Escherichia coli expressing four enzymes derived from Slackia isoflavoniconverten.

Another object of the present invention is to provide a novel compound, 5-hydroxy-dihydroequol, for which there is no synthetic method.

Therefore, another object of the present invention is to provide a method for synthesizing equol, dihydroequol, 5-hydroxy-equol or 5-hydroxy-dihydroequol from daidzein or genistein using the recombinant Escherichia coli. be.

Another object of the present invention is to utilize recombinant Escherichia coli expressed by combining two or three of the four enzymes derived from Slackia isoflavoniconverten, 5-hydroxy-equol, dihydroequol and It is to provide a method for selectively and efficiently synthesizing 5-hydroxy-dihydroequol.

In order to achieve the above object, the present inventors have made a dihydrodizein reductase, a dihydrodizein reductase, a dihydrodizein reductase derived from the human gut microbiota Slackia isoflavoniconvertens. , Dihydrodaidzein reductase, recombinant Escherichia coli expressing tetrahydrodaidzein reductase, and recombinant Escherichia coli expressing two or three of the above four enzymes in combination were produced.

In addition, the recombinant Escherichia coli of the present invention can be utilized as a whole cell catalyst (wole cell biocatalist) for bioconverting equol or dihydroequol from daidzein, and genistein is 5-hydroxy-equol or It can be utilized as a whole cell catalyst for selective bioconversion to 5-hydroxy-dihydroequol.

The present invention biosynthesizes equol, dihydroequol, 5-hydroxy-equol or 5-hydroxy-dihydroequol at a substrate concentration of about 0.2 to about 1 mmol / L and a substrate conversion rate of 95% under aerobic conditions. be able to.

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

In addition, since the present invention can carry out the selective synthesis of 5-hydroxy-equol, dihydroequol and 5-hydroxy-dihydroequol in aerobic conditions every few hours, the production cost is reduced and the product is high. It is possible to acquire added value, and through efficient mass production, it can be used for various applications such as foods and pharmaceuticals.

In addition, when the recombinant Escherichia coli of the present invention is applied to an additional oxidoreductase for a reaction system using a whole cell catalyst, various hydroxy-isoflavonoids can be synthesized, which is of high research value. ..

In addition, the compound 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 cosmetics or food additives.

FIG. 1 is a chart showing the yield of (S) -equol according to the concentration of initial daidzein. FIG. 2 shows equol, dihydroequol, 5-hydroxy-equol (5-hydroxy-equol) and 5-hydroxy-dihydroequol (5) using recombinant Equol expressing all four enzymes. -Hydroxy-dehydroequol) It is a schematic diagram of a synthetic reaction. FIG. 3 shows genistein (GSN), dihydrogenistein (DHG), 5-hydroxy-equol (5-hydroxy-equol, 5OH-EQ), and 5-hydroxy-based on the reaction time in the synthetic reaction of FIG. The concentration (μM) in the reaction system of dihydroequol (5-hydroxy-dehydropool, 5-OH-DEQ) is shown. FIG. 4 is a schematic diagram of a synthetic reaction partitioned using recombinant Escherichia coli 1 and 2 expressing two enzymes. FIG. 5 shows genistein (GSN), dihydrogenistein (DHG), 5-hydroxy-equol (5-hydroxy-equol, 5OH-EQ), and 5-hydroxy depending on the reaction time in the synthetic reaction of FIG. -The concentration (μM) in the reaction system of dihydroequol (5-hydroxy-dehydroequol, 5-OH-DEQ) is shown. FIG. 6 shows the amount of protein in the cell extracts of recombinant E. coli mentioned in FIGS. 2 and 4 by SDS-PAGE. FIG. 7 is a chart in which the ellipticity was measured using a circular dichroism dispersion system in the wavelength range of 210 to 320 nm. FIG. 8 shows the concentration (μM) of dihydroequol in the reaction system according to the reaction time using recombinant Escherichia coli expressing two or three enzymes. FIG. 9 is a chart showing the substrate conversion rate and the yield of 5-hydroxy-dihydroequol depending on the concentration of initial genistein. FIG. 10 (a) shows the mass spectrum and molecular structure of 5-hydroxy-equol analyzed by EI-MS, and FIG. 10 (b) shows the mass spectrum and molecular structure of 5-hydroxy-dihydroequol. FIG. 11 shows the concentration of (S) -equol in the reaction system in a reaction solution to which polyethylene glycol (polyethylene glycol, hereinafter, PEG) or polyvinylpyrrolidone (polyvinylpyrrolidone, hereinafter, PVP), which is a water-soluble polymer, is added, by time. It is a chart shown.

The terms used in the present invention are commonly used in the art and can be understood by anyone skilled in the art, but are briefly described herein. Then:

(1) Expression: It means that a recombinant protein is produced in a microorganism containing a vector containing a recombinant gene.

(2) Vector: means a polynucleotide consisting of single-stranded, double-stranded, round or supercoiled DNA or RNA. Vectors are composed of replication origins, promoters, ribosome binding sites, nucleic acid cassettes, terminations, etc. that are operably linked at appropriate distances so that recombinant proteins can be produced. The gene to be coded for the expressed recombinant protein is inserted at the position of the nucleic acid cassette.

(3) Cell extract: means a supernatant after disrupting a microorganism expressing a recombinant protein and then removing solid cell residues with a centrifuge. In the present invention, it means a microbial extract in which a part or all of daidzein reductase, dihydrodaidzein racemicase, dihydrodaidzein reductase, and tetrahydrodaidzein reductase is expressed.

(4) Whole cell reaction: A reaction in which a cell containing a specific enzyme is disrupted and a cell extract is used, or a complete cell is used without separating and purifying the enzyme.

The present invention relates to didzein reductase, dihydrodaidzein reductase, dihydrodizein reductase, and tetrahydrodizein reductase, a tetrahydrodizein reductase, and tetrahydrodizein reductase.

In the present invention, a DNA sequence corresponding to a daidzein reducing enzyme, a dihydrodaidzein rasemizing enzyme, a dihydrodaidzein reducing enzyme, or a tetrahydrodaidzein reducing enzyme derived from Slackia isoflavoniconvertens is subjected to a polymerase chain reaction (polymerase chain reaction). After amplification through PCR), it was placed in a pRSFDuet or pCDFDuet vector, respectively, and this was put into E.I. It was expressed as his-tag (6 histidine) in colli.

The recombinant Escherichia coli can be utilized in synthesizing equol or dihydroequol from the substrate daidzein.

In addition, the recombinant Escherichia coli can be utilized in synthesizing 5-hydroxy-equol or 5-hydroxy-dihydroequol from the substrate genistein.

The method for synthesizing equol or 5-hydroxy-equol can be utilized in synthesizing (S) -equol or 5-hydroxy- (S) -equol.

More specifically, the recombinant Escherichia coli can be utilized as a whole cell catalyst for bioconverting equol and dehydroequol from daidzein, and genistein is 5-hydroxyl. It can be utilized as a whole cell catalyst for bioconversion to -equol (5-hydroxy-equol) and 5-hydroxy-dihydroequol (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 suppress the oxidation of the product, and the reducing agents include NAD (P) H, L-ascorbic acid and glutathione. (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 a 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 ° C, more preferably about 25 to about 30 ° C. 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, still more preferably about 100 to about 200 rpm.

Glucose and glycerol can be used as the carbon source in the equol synthesis reactor, and the concentration of glucose or glycerol is preferably about 1 to about 5 (w / v)%, preferably about 1.5 to about. 4 (w / v)% is more preferable, and about 2 to about 3 (w / v)% is further preferable. The concentration of the recombinant microorganism of the present invention is preferably about 1 to 100, more preferably about 5 to about 50, still more preferably about 10 to about 30 in optical density (OD).

The substrate of the equol synthesis reaction system includes genistein, glycitein, daidzein, ortho-hydroxy-daidzein, depending on the type of isoflavane or isoflavane to be synthesized. Ortho-hydroxy-daidzein), ortho-hydroxy-genistein (ortho-hydroxy-genistein) and the like can be used.

In order to improve the solubility of the substrate in the reaction solution, polyethylene glycol (polyethylene glycol), polyvinylpyrrolidone, polyvinyl alcohol (polyvinyl alcohol), and β-cyclodextrin (β-cyclodextrin), methyl Cyclodextrin (methyl-β-cyclodextrin), 2-hydroxypropyl-β-cyclodextrin (2- (Hydroxypolyethylene) -β-cyclodextrin) and the like can be utilized.

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

FIG. 2 shows from daidzein and genistine to daidzein redictase (DZNR), dihydrodaidzein racemase (DDRC), dihydrodaidzein reductase (dihydradedeidzeinhidedzein red) It is a schematic diagram of the synthesis reaction of equol, dihydroequol, 5-hydroxy-equol and 5-hydroxy-dihydroequol using recombinant Equol expressing).

As shown in FIG. 2, daidzein is converted to (R) -dihydrodaidzein by daidzein reductase (DZNR), which is (S)-by dihydrodaidzein racemization enzyme (DDRC). Converted to dihydrodaidzein. (S) -Dihydrodaidzein is converted to (3S, 4R) -trans-tetrahydrodaidzein by dihydrodaidzein reductase (DHDR), which is converted to equol by tetrahydrodaidzein reductase (THDR) or dihydro by dehydration. Converted to equol.

Also, as shown in FIG. 2, genistein is converted to (R) -dihydrogenistein by daidzein reductase (DZNR), which is converted to (R) -dihydrogenistein by dihydrodaidzein racemization enzyme (DDRC) (S). )-Converted to dihydrogenistein. (S) -Dihydrodaidzein reductase (DHDR) converts to (3S, 4R) -trans-tetrahydrogenistein, which can be converted to 5-hydroxy-equol by tetrahydrodaidzein reductase (THDR). , Converted to 5-hydroxy-dihydroequol by dehydration.

In the present invention, daidzein redicase, dihydrodaidzein racemase, dihydrodaidzein redase, tetrahydrodaidzein redase, and tetrahydrodaidzein reductase, tetrahydrodizein reductase, tetrahydrodaidzein reductase, and tetrahydrodaidzein reductase. Concerning a method for selectively synthesizing 5-hydroxy-equol from the substrate genistein using recombinant Escherichia coli in which the daidzein reductase is overexpressed 5 times or more as compared with the remaining 3 enzymes. It is a thing.

Specifically, 5-hydroxy-equol can be selectively synthesized by utilizing recombinant Escherichia coli in which the tetrahydrodaidzein reductase is overexpressed 5 times or more, preferably 10 times or more as compared with the remaining three enzymes. Can be done.

This is because, as shown in FIG. 2, increasing the expression level of tetrahydrodaidzein reductase increases the rate of synthesizing 5-hydroxy-equol from tetrahydrogenistein to produce 5-hydroxy-dihydroequol. Is competitively hindered.

The above-mentioned method for expressing recombinant Escherichia coli and the method for synthesizing 5-hydroxy-equol using the above-mentioned method for producing recombinant Escherichia coli expressing all four enzymes derived from Slackia isoflavoniconvertense and the above-mentioned method for producing recombinant Escherichia coli are used. It is the same as the method of synthesizing equol.

FIG. 6 shows the amount of protein in the cell extract of recombinant Escherichia coli mentioned in FIGS. 2 and 4 by SDS-PAGE, and the expression levels of the four enzymes include four recombinant enzymes. In recombinant Escherichia coli expressing dizein reductase and dihydrodizein lasemilyase in one vector and recombinant Escherichia coli expressing dihydrodizein reductase and tetrahydrodizein reductase in one vector than when expressed in one E. coli. The expression level of the enzyme increased. In FIG. 6, 1: DDDT is the case where the four recombinant enzymes mentioned in FIG. 2 are expressed in one Escherichia coli, and 2: DD is the case where the dizein reductase and the dihydrodizein resembling enzyme are expressed in FIG. Corresponds to the cell extract of recombinant Escherichia coli expressed in one vector, 3: DT corresponds to the cell extract of recombinant Escherichia coli expressing dihydrodizein reductase and tetrahydrodizein reductase in one vector in FIG. do.

FIG. 5 shows the in-reaction system concentration (μM) of genistein, dihydrogenistein, and 5-hydroxy-equol according to the reaction time, and the expression level of the tetrahydrodaidzein reductase is 5 as compared with the remaining three enzymes. Utilizing a system expressing more than doubled recombinant Equol (2 and 3 in FIGS. 6), 5-hydroxy-equol yield and production compared to FIG. 2 for recombinant Equol expressing all four enzymes. It shows that sex and 5-hydroxy-equol selectivity for 5-hydroxy-dihydroequol can be increased.

In addition, the present invention also comprises recombinant Equol 1 expressing daidzein redictase and dihydrodaidzein racemase, tetrahydrodaidzein reductase and tetrahydrodaidzein redizein reddazein reddazein reddazein reddazein reddazein reddazein. It relates to a method for selectively synthesizing 5-hydroxy-equol from the substrate genistein by utilizing the expressed recombinant Escherichia coli 2.

5-Hydroxy-equol can be selectively synthesized from genistein by a bioconversion reaction system in which recombinant Escherichia coli 1 and 2 are mixed as a whole cell catalyst.

In the expression method of recombinant Escherichia coli 1 and 2 of the present invention and the method for synthesizing 5-hydroxy-equol using the same, the expression of the recombinant enzyme expressing all four enzymes derived from Slackia isoflavoni-convertence described above is expressed. It is the same as the method and the method for synthesizing equol using it.

The mixed reaction system of recombinant Equol 1 and 2 includes not only selective production of 5-hydroxy-equol, but also equol, 6-methoxy-equol (6-Methoxyxy-equol), and various hydroxy-equol (Hydroxy-). It can be applied to the production of equol) and the like.

FIG. 4 shows recombinant Escherichia coli expressing daidzein reductase (DZNR) and dihydrodizein reductase (DDRC) in a single vector, and dihydrodizein reductase (dihydrodizein reductase) tetra, dihydrodizein reductase. Shown shows a whole cell reaction system in which the reaction is partitioned with other recombinant Escherichia coli expressing reductase (ttrahydrodizein redutase: THDR) in one vector.

According to FIG. 4, genistein is converted to (S) -dihydrogenistein by the recombinant E. coli 1, and (S) -dihydrogenistein is converted to 5-hydroxy- (S) -equol by the recombinant E. coli 2. Therefore, it is shown that 5-hydroxy-equol can be selectively synthesized from genistein by recombinant E. coli 1 and 2.

A whole-cell reaction system in which the reaction is partitioned by different recombinant E. coli is advantageous in that it improves the productivity and final yield of 5-hydroxy- (S) -equol and is a by-product 5-hydroxy-. Reducing the production of dihydroequol is advantageous in that it increases the selectivity of 5-hydroxy- (S) -equol.

The method for selectively synthesizing 5-hydroxy-equol can selectively synthesize 5-hydroxy- (S) -equol.

Specifically, recombinant Escherichia coli in which tetrahydrodaidzein reductase is overexpressed 5-fold or more, or recombinant Escherichia coli 1 and 2 are used to selectively select 5-hydroxy- (S) -equol. Can be synthesized.

In this regard, FIG. 7 utilizes a circular dichroism dispersion system in the wavelength range of 210-320 nm to know the optical structure of 5-hydroxy-equol synthesized using the recombinant E. coli. It is a chart in which the ellipticity was measured, and it can be confirmed that the 5-hydroxy-equol biosynthesized by the recombinant Escherichia coli of the present invention has an S-form at carbon No. 3.

Furthermore, the present invention expresses daidzein redictase, dihydrodaidzein racemase and dihydrodaidzein redicase, and does not express tetrahydrodaidzein redidase. It relates to a method for selectively synthesizing dihydroequol from the substrate daidzein.

Specifically, the recombinant Escherichia coli can be used as a whole cell catalyst to selectively synthesize dihydroequol from daidzein.

When boric acid is added to the reaction solution for synthesizing dihydroequol, the dehydration reaction of (3S, 4R) -trans-tetrahydrodaidzein is promoted, so that dihydroequol can be synthesized in a high yield. The amount of boric acid added is preferably about 50 mM to about 200 mM.

In addition, the present invention expresses daidzein reductase, dihydrodaidzein racemase, dihydrodaidzein reductase, and tetrahydrodaidzein reductase, which does not express tetrahydrodaidzein reductase. It relates to a method for selectively synthesizing 5-hydroxy-dihydroequol from the substrate genistein.

Specifically, the recombinant Escherichia coli can be used as a whole cell catalyst to selectively synthesize 5-hydroxy-dihydroequol from genistein.

The method for expressing recombinant Escherichia coli of the present invention and the method for synthesizing dihydroequol or 5-hydroxy-dihydroequol using the same are the above-mentioned recombinant enzymes expressing all four enzymes derived from Slackia isoflavoniconvertense. It is the same as the expression method and the method for synthesizing equol using it.

FIG. 8 shows recombinant Escherichia coli (DZNR + DHDR) expressing daidzein redactase (DZNR), dihydrodaidzein redactase (DHDR), and daidzein reductase (daidzein reductase, daidzein reduct). (Dihydraidzein racemase, DDRC), recombinant Escherichia coli (DZNR + DDRC + DHDR) expressing dihydrodaidzein reductase (DHDR), and a hydro reaction with dizein over time. It is a chart which showed the concentration in the reaction system of equol. As shown in FIG. 8, the recombinant E. coli can be used to selectively synthesize dihydroequol from daidzein without the production of equol, and the recombinant E. coli (DZNR + DDRC) expressing the three enzymes can be synthesized. When + DHDR) is used, the concentration of dihydroequol produced is higher than that of recombinant Escherichia coli (DZNR + DHDR) expressing the two enzymes.

FIG. 9 shows the substrate conversion rate according to the concentration of initial genistein and the substrate conversion rate when a whole cell reaction was carried out with genistein using recombinant Equol expressing daidzein reducing enzyme, dihydrodaidzein lasemigenase, and dihydrodaidzein reducing enzyme. It is a chart showing the yield of 5-hydroxy-dihydroequol, and as shown in FIG. 9, the recombinant Escherichia coli is used to selectively synthesize 5-hydroxy-dihydroequol from genistein. Can be done.

The recombinant Escherichia coli of the present invention can be used as a whole cell catalyst. Whole-cell catalysts are used in many biocatalytic reactions and have the advantage of allowing the reaction to proceed under mild conditions at room temperature and pressure and having excellent reaction specificity. In cases where a coenzyme is absolutely necessary, a process involving a multi-step reaction, or a case where the activity of the enzyme is significantly reduced during purification, it is more advantageous to carry out the biocatalyst process using whole cells. be. In addition, it is advantageous because the recombinant Escherichia coli used as whole cells can be selectively separated by using a centrifuge even when the product is purified after the reaction is completed.

The method for synthesizing the synthesized equol, dihydroequol, 5-hydroxy-equol or 5-hydroxy-dihydroequol can proceed under aerobic conditions, and is therefore milder than the conventional technique for synthesizing equol under anaerobic conditions. Equol can be synthesized under certain conditions. Therefore, the synthetic method of the present invention does not require the apparatus or technique used to provide anaerobic conditions in the prior art.

The method for synthesizing the synthesized equol, dihydroequol, 5-hydroxy-equol or 5-hydroxy-dihydroequol can use a substrate of about 0.2 to about 5 mmol / L.

If the concentration of the substrate daidzein or genistein is less than 0.2 mmol / L, the amount of equol, dihydroequol, 5-hydroxy-equol and 5-hydroxy-dihydroequol synthesized is insufficient or or Purification costs can also be high, and above 5 mmol / L, the conversion of the substrate can be reduced and additional purification costs can be incurred.

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

However, when the reaction system further contains a water-soluble polymer, equol, dihydroequol, 5-hydroxy-equol or 5-hydroxy-dihydroequol can be obtained in high yield even if the concentration of the substrate exceeds 1 mmol / L. The substrate concentration is preferably about 3-5 mmol / L because it can be synthesized.

The method for synthesizing equol or dihydroequol has a conversion rate of daidzein of 95% or more, and the method for synthesizing 5-hydroxy-equol or 5-hydroxy-dihydroequol has a conversion rate of genistein of 95% or more.

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

Further, in the synthesis method of the present invention, the conversion rate of the substrate can 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), 5-hydroxy-equol (5OH-EQ) in the reaction system according to the reaction time, and the reaction time is 10. Over time alone, substrate conversion has been shown to be greater than or equal to 95%.

The method for synthesizing equol, dihydroequol, 5-hydroxy-equol or 5-hydroxy-dihydroequol can further include a water-soluble polymer in the reaction system. Further, as the water-soluble polymer, a water-soluble polymer for food additives can be used.

Examples of the water-soluble polymer include polyethylene glycol (polyethylene glycol), polyvinylpyrrolidone (polyvinyl pyrrolidone), polyvinyl alcohol (polyvinyl alcohol), β-cyclodextrin (β-cyclodextrin), and methyl-β-cyclodextrin. -Β-cyclodextrin), 2-hydroxypropyl-β-cyclodextrin (2- (Hydroxypolypyl) -β-cyclodextrin) and the like can be utilized. Preferably, polyethylene glycol, polyvinyl alcohol or polyvinylpyrrolidone can be utilized.

The inclusion of additional water-soluble polymers in the reaction system increases the solubility of the substrate in the reaction system, resulting in a final yield of equol, dihydroequol, 5-hydroxy-equol or 5-hydroxy-dihydroequol produced. Can increase.

FIG. 11 is a chart showing the concentration of (S) -equol in the reaction system in the reaction solution obtained by further adding polyethylene glycol or polyvinylpyrrolidone, which is a water-soluble polymer, to the reaction solution of Example 1 over time. As shown in FIG. 11, if the reaction solution further contains polyethylene glycol (PEG) or polyvinylpyrrolidone (PVP), equol should be synthesized in high yield even with a 5 mmol / L substrate. Can be done.

The present invention also relates to equol, dihydroequol, 5-hydroxy-equol or 5-hydroxy-dihydroequol, which are produced by the synthetic method of the present invention mentioned above.

In particular, 5-hydroxy-dihydroequol produced by the synthetic method of the present invention is a novel compound previously unknown and can be represented by the following chemical formula.

The present invention is capable of synthesizing (S) -equol with a high substrate conversion rate under aerobic conditions, and because the synthesized equol has a molecular structure similar to estrogen, it is a conventional treatment for menopausal symptoms such as induction of breast cancer. Without the side effects of the agent, menopausal symptoms in women, especially the onset and symptoms of osteoporosis, can be alleviated, cardiovascular disease can be prevented, and breast cancer can be alleviated.

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

In addition, since the present invention can carry out the selective synthesis of 5-hydroxy-equol, dihydroequol and 5-hydroxy-dihydroequol in aerobic conditions every few hours, the production cost is reduced and the product is high. It is possible to acquire added value, and through efficient mass production, it can be used for various applications such as foods and pharmaceuticals.

In addition, when the recombinant Escherichia coli of the present invention is applied to an additional oxidoreductase for a reaction system using a whole cell catalyst, various hydroxy-isoflavonoids can be synthesized, which is of high research value. ..

Specifically, as the oxidoreductase, tyrosinase, cytochrome P450, flavin monooxygenase and the like can be used, and 3'-hydroxy-equol, 6-hydroxy-equol, 8-hydroxy-equol, 6,3'-dihydroxy can be used. -Hydroxy-isoflavonoids such as 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 as an anticancer agent for the treatment of prostate cancer and ovarian cancer. Can be used. It can also be used as an antioxidant in the form of cosmetics or food additives.

Hereinafter, the 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.

Method for producing recombinant E. coli

Dizein reductase (DZNR), dihydrodizein reductase (DDRC), dihydrodizein reductase (dihydradedizein reductase: DhDR), dihydrodizein reductase (DhDR), tetra After amplification through PCR, it was placed in a pRSFDuet or pCDFDuet vector, respectively, and this was put into E.I. It was expressed as his-tag (6 histidine) in colli.

Specifically, the plasmid into which the base sequence of the enzyme was inserted was transformed into BL21 competent cell, and then cultured in an LB solid medium containing an antibiotic suitable for the plasmid for 12 hours in a 37 ° C. incubator. One colony was inoculated into 3 mL LB liquid medium containing antibiotics and cultured in a 37 ° C. incubator at a rate of 200 rpm for 12 hours. 2v% (1 mL) of this culture was inoculated into a new LB liquid medium containing 50 mL of antibiotics. O. of the culture solution. D. When (600 nm) reached 0.6 to 0.8, 0.1 mM IPTG and 0.1 mM MnSO ₄ were added to induce protein expression in an incubator at 18 ° C. at 200 rpm for 18 hours. After 18 hours, cells were centrifuged at 4000 rpm and washed with 25 mL PBS to prepare.

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

A 10-1000 ml glassware is used as the reactor, and the reactor is supplied with the recombinant Escherichia coli and the substrate having the following concentrations.

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

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

0.2-5 mM daidzein was added to O.D. D. Using recombinant Escherichia coli expressing the above four enzymes of 10, the reaction was allowed to proceed according to the reaction method 1. The substrate, intermediate, and product are extracted with ethyl acetate (EA) of 4 times or more, the solvent is removed using a centrifugal vacuum purifier, and then the solvent is redissolved in a certain amount of methanol. High performance-liquid chromatography confirmed the reactivity and concentration of the substance. As shown in FIG. 1, the yield of (S) -equol is 95% or more at 0.2 mM, 80% or more at 0.4 and 0.6 mM, 50% or more at 2 mM, and 20% or more at 5 mM. The yield could be confirmed.

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

500 μM genistein (Fig. 2. Genistein) was added to O.D. D. Using recombinant Escherichia coli expressing the above four enzymes of 10, the reaction was allowed to proceed according to the reaction method 1. Substrates, intermediates and products are extracted with at least 4x ethyl acetate (EA), the solvent is removed using a centrifugal decompressor and then redissolved in a certain amount of methanol for high performance liquid chromatography. Chromatography confirmed the reactivity and concentration of the substance. It was confirmed that at 6 hours of the reaction, 500 μM genistein was converted by 99% or more to produce 5-hydroxy-equol 97 mg / L and 5-hydroxy-dihydroequol 22 mg / L.

Through the composition as described above, 1 mM genistein was added to the reaction through Reaction Method 1, and as can be confirmed from FIG. 3, the substrate conversion rate was 95% or more in the reaction time of only 10 hours, and 5-hydroxy-equol 199 mg. It was possible to produce / L, 5-hydroxy-dihydroequol 63 mg / L.

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

As shown in the schematic diagram of FIG. 4, recombinant Escherichia coli 1 expressing daidzein reductase and dihydrodaidzein reductase and recombinant Escherichia coli 2 expressing dihydrodaidzein reductase and tetrahydrodaidzein reductase are mixed as a whole cell catalyst. A bioconversion reaction system was constructed. When the reaction was carried out with 1 mM genistein according to the reaction method 1, the concentrations of the substrate and the product according to the reaction time were confirmed by liquid chromatography as shown in FIG. This reaction system produced 5-hydroxy-equol 231 mg / L and 5-hydroxy-dihydroequol 12 mg / L with a substrate conversion rate of 99% or more with a reaction time of only 6 hours. When the production results are compared with the results of Example 1, the productivity of 5-hydroxy-equol is increased by 1.6 times and is defined by the concentration of 5-hydroxy-equol / the concentration of 5-hydroxy-dihydroequol. The productivity of 5-hydroxy-equol increased 5-fold.

Further, in order to compare the expression levels of the four enzymes, the cell extracts of recombinant Escherichia coli in Example 2 and recombinant Escherichia coli 1 and 2 in Example 3 were crushed and prepared by ultrasonic treatment, and cell extraction was performed. The material was centrifuged for 30 minutes at 13000 rpm and 4 ° C., and then a supernatant was obtained and analyzed by SDS-PAGE as shown in FIG.

As a result of the analysis, the expression levels of the four enzymes were all increased in the two recombinant E. coli used in Example 3 as compared with the E. coli used in Example 2. Among them, the expression level of tetrahydrodaidzein reductase was significantly increased, increased by 20 times or more as compared with the Escherichia coli crushed solution of Example 1, and overexpressed by 5 times or more as compared with the expression levels of the remaining three enzymes. ..

[Example 4]: Analysis of optical isomers of 5-hydroxy-equol

Circular dichroism dispersion spectrum analysis was advanced to determine the optical structure of 5-hydroxy-equol synthesized in the recombinant E. coli of the present invention. The measurement wavelength range was 210 to 320 nm, and a circular dichroism dispersometer Chirascan ^TM plus CD spectrometer (Applied Photophysics, UK) was used. The sample was measured after dissolving it in pure methanol at a concentration of 1.5 mg / ml.

The measurement results are shown graphically in FIG. 7, showing a high magnitude negative Cotton effect at 238 nm and a high magnitude positive Cotton effect at 270 nm. Therefore, it was confirmed that the 5-hydroxyequol biosynthesized by the recombinant Escherichia coli of the present invention has an S-form at carbon No. 3.

[Example 5]: Selective synthesis of dihydroequol

In order to biosynthesize dihydroequol, the recombinant E. coli of Example 1 is deficient in tetrahydrodaidzein reductase and contains or does not contain dihydrodaidzein racemized enzyme (DZNR + DDRC + DHDR, hereinafter, DDD strain) or not (DZNR + DHDR). , Hereinafter, DD strain) was prepared. Using the Escherichia coli, a reaction using daidzein as a substrate was allowed to proceed according to Reaction Method 1, and 200 mM boric acid was further added to the reaction solution. As a result, it was confirmed that only dihydroequol was produced without producing equol (Fig. 8). Specifically, the bioconversion reaction was started at an initial daidzein concentration of 0.5 mM and reacted for 2 hours. In this case, 28 μM dihydroequol was synthesized in the DD strain and 60 μM dihydroequol was synthesized in the DDD strain.

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

In order to biosynthesize 5-hydroxy-dihydroequol, recombinant Escherichia coli lacking the tetrahydrodaidzein reductase from the plasmid was prepared in the recombinant Escherichia coli of Example 2. Using the Escherichia coli, the reaction using genistein as a substrate was carried out according to the reaction method 1, and it was confirmed that only 5-hydroxy-dihydroequol was produced without producing 5-hydroxy-equol (FIG. 9). .. When the initial genistein concentration was 0.2, 0.4, 0.6, 1.0 mM and the bioconversion reaction was started and reacted for 36 hours, the conversion rate of genistein was 99% or more at 0.6 mM or less. There was 97% at 1.0 mM. The conversion yields of 5-hydroxy-dihydroequol were 44, 35, 27 and 39%, respectively.

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

The 5-hydroxy-equol and 5-hydroxy-dihydroequol synthesized in Examples 3 and 6 were identified through a gas chromatography-mass spectrometer (GC-MS) and the results are shown in FIG.

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

The results of EI-MS analysis were shown to be identical to the previously reported spectra of DHD (Chang, YC, and MG Nair. J. Natr. Proc. 58: 1892- 1896, 1995; Wahala, K., A. Salakka, and H. Adlercreutz. Proc. Soc. Exp. Biol. Med. 217: 293-29, 1998). As expected, the EI-MS of the synthesized compound showed a [M + H] ⁺ molecular ion peak consistent with C ₁₅ H ₁₂ O ₄ (DHD) at m / z 256. Other ion peaks of the synthesized compound were 137 (100), 120 (52), 91 (36), 65 (16).

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

Same as Example 1 except that it contains dimethyl sulfoxide (DMSO) and further contains polyethylene glycol (PEG) or polyvinylpyrrolidone (PVP) in order to improve the solubility of daidzein in the reaction solution. The reaction was allowed to proceed.

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

According to Examples 1 and 2, recombinant Escherichia coli expressing four enzymes derived from Slackia isoflavoniconverten was used in aerobic conditions within a few hours in high yield from daidzein (S)-. Equol could be synthesized and 5-hydroxy-equol and 5-hydroxy-dihydroequol could be synthesized from genistein.

In Examples 3, 5 and 6, recombinant Escherichia coli in which two or three of the four enzymes are combined is used to generate 5-hydroxy-equol, dihydroequol or 5-hydroxy-dihydroequol. Each could be selectively synthesized.

It was also confirmed through Examples 4 and 7 that the recombinant E. coli according to Examples 2, 3 and 6 synthesize 5-hydroxy- (S) -equol or 5-hydroxy-dihydroequol.

Further, in Example 8, it was confirmed that the yield of equol was superior to that of the reaction system (Example 1) containing no water-soluble polymer by further containing the water-soluble polymer in the reaction system. We were able to.

Claims (11)

Surakkia iso Flavobacterium Nikon barman scan (Slackia isoflavoniconvertens) derived from daidzein reductase (daidzein reductase), dihydrodaidzein racemase (dihydrodaidzein racemase), dihydrodaidzein reductase (dihydrodaidzein reductase) and tetrahydrodaidzein reductase (tetrahydrodaidzein reductase) expressing , 5-Hydroxy-equol is selected from the substrate genistein using recombinant Escherichia coli that overexpresses tetrahydrodaidzein redactase 5 times or more compared to the remaining 3 enzymes. How to synthesize Recombinant Escherichia coli expressing daidzein redactase and dihydrodaidzein racemase derived from slackia isoflavoniconvertens, and recombinant E. coli derived from slackia isoflavoniconverse. It expresses daidzein reductase and tetrahydrodaidzein reductase, and among the above four enzymes, it expresses tetrahydrodaidzein reductase (tetrahydzein reductase) and tetrahydrodaidzein reductase (three times more than the remaining three enzymes). A method for selectively synthesizing 5-hydroxy-equol from the substrate, genistein, using a recombinant E. coli. Daidzein redactase derived from Slackia isoflavoniconvertens, dihydrodaidzein racemase, dihydrodaidzein racemase, and dihydrodaidzein reductase dihydrodaidzein reductase. A method for selectively synthesizing dehydroequol from the substrate daidzein using recombinant Escherichia coli that does not express tetrahydrodaidzein reductase derived from isoflavoniconvertens. Daidzein redactase derived from Slackia isoflavoniconvertens, dihydrodaidzein racemase, dihydrodaidzein racemase, and dihydrodaidzein reductase dihydrodaidzein reductase. A method for selectively synthesizing 5-hydroxy- dehydroequol from the substrate genistein using recombinant Escherichia coli that does not express tetrahydrodaidzein reductase derived from isoflavoniconvertens. The method according to claim 1 or 2, wherein the 5-hydroxy-equol is 5-hydroxy- (S) -equol. The method in which the recombinant Escherichia coli is used as a whole cell catalyst according to any one of claims 1 to 4. The method according to any one of claims 1 to 4, wherein the process proceeds under aerobic conditions. The method for synthesizing using a substrate of 0.2 to 1 mmol / L in any one of claims 1 to 4. The method according to any one of claims 1 to 4, wherein the substrate conversion rate is 95% or more. The method according to any one of claims 1 to 4, wherein the reaction system further contains a water-soluble polymer. The method according to claim 10, wherein the water-soluble polymer is a water-soluble polymer for food additives, and is polyethylene glycol or polyvinylpyrrolidone.

Images