CN110382701A - Produce the recombination bacillus coli of female horse amphyl and the equol derivative synthesizing process using it - Google Patents
Produce the recombination bacillus coli of female horse amphyl and the equol derivative synthesizing process using it Download PDFInfo
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- CN110382701A CN110382701A CN201880014592.9A CN201880014592A CN110382701A CN 110382701 A CN110382701 A CN 110382701A CN 201880014592 A CN201880014592 A CN 201880014592A CN 110382701 A CN110382701 A CN 110382701A
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- C12N15/09—Recombinant DNA-technology
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Abstract
Expression be the present invention relates to the use of from enzyme (the daidzein reductase (daidzein reductase) of isoflavones conversion Si Naike Salmonella (Slackia isoflavoniconvertens), dihydro daidzein racemase (dihydrodaidzein racemase), dihydro daidzein reductase (dihydrodaidzein reductase), tetrahydro daidzein reductase (tetrahydrodaidzein reductase)) recombination bacillus coli and from daidzein or genistein (genistein) selectivity and effectively synthesize equol, dehydroequol, 5- hydroxyl-equol (5-h ) and the method for 5- hydroxy-dehydro equol (5-hydroxy-dehydroequol) ydroxy-equol.Also, it is used to express mentioned microorganism bacterial strain and 5- hydroxyl-equol or the bioconversion composition of 5- hydroxy-dehydro equol can be used as antioxidant, anticancer agent etc., and can be used for preventing or treating climacteric syndrome.
Description
Technical field
The present invention relates to the recombination bacillus coli of production female horse amphyl and utilize its female horse amphyl synthesis side
Method.
Background technique
Isoflavones that some anaerobes metabolism mankind in human intestine absorbs by leguminous plant is simultaneously biological by it
It is converted into equol.Up to the present the equol synthesized micro-organism found is all bacterium, typically, most of to belong to red stinkbug
Pseudomonas, such as have Si Naike Bordetella (Slackia sp.), Iger hereby Bordetella (Eggerthella sp.), Adler gram
Roy thatch Pseudomonas (Adlercreutizia sp.), exception also have from galactococcus (Lactococcus).
The equol generated by the enzymatic reaction of enteric microorganism as described above is only closed with the optical isomer of S configuration
At, and it generates phytoestrogen (phytoestrogen) effect and being absorbed by the body.Therefore, in continuous ingestion female horse
In the case where phenol, the morbidity and symptom of Menopause symptom, especially osteoporosis can be alleviated, without such as breast cancer
The side effect of the previous menopause symptom therapeutic agents such as induction.This effect is swashed since the molecular structure and human body of equol are female
Element have similitude, and in estrogen receptor alpha and β α selectivity it is very high.Moreover, (S)-equol, which has, can prevent the heart
Vascular diseases and the effect of alleviate prostate cancer (non-patent literature 1).5- hydroxyl-equol as equol analog comes
Derived from the substance of the genistein (genistein) as isoflavones, it was reported that, it is a kind of phytoestrogen, biological function
Effect is lower than equol, but has inoxidizability more better than equol (non-patent literature 2).Also, it was reported that dehydroequol
(dehydroequol) it can inhibit the growth of prostate cancer and ovarian cancer cell, phase ii clinical trial is the results show that anti-to chemistry
Cancer agent, which has indefatigable oophoroma, has inhibitory effect (non-patent literature 3).
However, with the various effort and research made to use enteric microorganism to synthesize equol on the contrary, in order to close
The effort made at 5- hydroxyl-equol is restricted.It reports as follows, only some enteric microorganism of biosynthesis equol
5- hydroxyl-equol can be synthesized.It is identified using the red stinkbug bacterium Strain Mt1B8 identified in mouse enteron aisle and in human faecal mass
Isoflavones convert Si Naike Salmonella, genistein through dihydro genistein biology synthesize 5- hydroxyl-equol (non-patent literature
4).But there is limitation, microorganism to growth microorganism under anaerobic using these bacterial strains production 5- hydroxyl equol
Just start to produce 5- hydroxyl equol after at least 15 hours after inoculation, therefore productivity is low, due to lacking standard substance, and deposit
In the limitation for being difficult to Accurate Prediction ultimate yield.
It was reported that the genistein of one of isoflavones passes through microorganism red stinkbug Bacteriaceae Strain Mt1B8
(Coriobacteriaceae Strain Mt1B8) (16), isoflavones convert Si Naike Salmonella (Slackia
Isoflavoniconvertens) (17), strain AUH-JLC159 bioconversion be at 5- hydroxyl-equol, but react and
Incubation time is long, i.e., 2~3 days, and need anaerobic reaction condition, the amount of the genistein as substrate be 0.6mmol/L with
In the case where upper, there is a problem of (patent document 1) that conversion ratio drops to 80% or less.
It is (S)-that 5- hydroxyl-equol 1,1- diphenyl -2- trinitrophenyl-hydrazine (DPPH) group of synthesis, which captures effect,
20 times of equol have been demonstrated service life and the resistance to compression that can increase Caenorhabditis elegans (Caenorhabditis elegans)
Property (non-patent literature 5).But other physiological activity of 5- hydroxyl-equol are not illustrated.
Recently, Annett Braune group is in first identified in 2013 in the thin of lactococcus and Si Naike Bordetella
Have the function of for isoflavones daidzein (daidzein) being converted to four kinds of enzymes (non-patent literature 6) of equol in bacterium.On
It states enzyme to be made of three kinds of reductases and a kind of isomerase, they are respectively daidzein reductase (daidzein
Reductase), dihydro daidzein racemase (dihydrodaidzein racemase), dihydro daidzein reductase
(dihydrodaidzein reductase), tetrahydro daidzein reductase (tetrahydrodaidzein reductase).
Existing technical literature
Patent document
Patent document 1: Chinese patent publication the 103275884th
Non-patent literature
Non-patent literature 1:Nutrition Reviews, 69 (8), 432-448,2011
Non-patent literature 2:J.Chin.Pharm.Sci., 23 (6), 378-384,2014
Non-patent literature 3:Int.J.Gynecol.Cancer, 21 (4), 633-639,2011
Non-patent literature 4:Applied and Environmental Microbiology, 74,4847 (2008)/
Applied and Environmental Microbiology, 75,1740 (2009)
Non-patent literature 5:Conghui Zhang et al., J.Chin.Pharm.Sci., 23 (6): 378-384,2014
Non-patent literature 6:Appl.Environ.Microbiol., 79 (11), 3494,2013.
Summary of the invention
Technical problem
The object of the present invention is to provide a kind of recombinations of four kinds of expression enzymes from isoflavones conversion Si Naike Salmonella
Escherichia coli.
Also, it is a further object of the present invention to provide a kind of 5- hydroxy-dehydro equol as new compound,
And its synthetic method was absolutely not suggested.
For this purpose, another object of the present invention is to provide a kind of using above-mentioned recombination bacillus coli and from daidzein or
Genistein synthesizes equol, dehydroequol, the method for 5- hydroxyl-equol or 5- hydroxy-dehydro equol.
Also, further object of the invention is, provides a kind of how convert this from isoflavones using combinational expression
The recombination bacillus coli of two or three of enzyme in four kinds of enzymes of kirschner bacterium carrys out selectivity and effectively synthesizes 5- hydroxyl-female horse
The method of phenol, dehydroequol and 5- hydroxy-dehydro equol.
Solution to problem
To achieve the goals above, the present inventor is prepared for expression from the isoflavones turn as human intestinal microorganisms
Change daidzein reductase, the dihydro daidzein racemization of Si Naike Salmonella (Slackia isoflavoniconvertens)
Enzyme, dihydro daidzein reductase, the recombination bacillus coli of tetrahydro daidzein reductase and the above-mentioned four kinds of enzymes of combinational expression
In two or three of enzyme recombination bacillus coli.
Also, recombination bacillus coli of the invention can be used as being biologically converted into equol (equol) or de- from daidzein
The whole-cell catalyst (whole cell biocatalyst) of hydrogen equol, and can be used as selectively giving birth to genistein
Object is converted into the whole-cell catalyst of 5- hydroxyl-equol or 5- hydroxy-dehydro equol.
The effect of invention
The present invention is under conditions of the concentration of aerobic condition, substrate is about 0.2mmol/L to about 1mmol/L, with 95%
The substrate transformation rate can biosynthesis equol, dehydroequol, 5- hydroxyl-equol or 5- hydroxy-dehydro equol.
Also, optionally biosynthesis 5- hydroxyl-equol, dehydroequol or 5- hydroxy-dehydro are female by the present invention
Horse phenol.
Also, the present invention can selectively be synthesized 5- hydroxyl-equol as unit of a few hours, taken off under aerobic condition
Hydrogen equol and 5- hydroxy-dehydro equol, therefore producing cost can be reduced and obtain high added value, and can be by having
Effect is mass produced to be suitable for the various uses of food and drug.
Also, additional oxygen is applied recombination bacillus coli of the invention to be used as in the reaction system of whole-cell catalyst
In the case where changing reductase, various hydroxy-isoflavones can be synthesized, therefore researching value is high.
Also, the compound synthesized through the invention can be used for preventing or treating climacteric syndrome, and before can be used as treatment
The anticancer agent of column gland cancer, oophoroma.Also, it can be used as the antioxidant of cosmetics or food additives form.
Detailed description of the invention
Fig. 1 is the chart for showing the yield of (S)-equol of the concentration according to original soy aglycon.
Fig. 2 is to utilize equol, dehydroequol, the 5- hydroxyl-equol (5- for expressing all four recombination bacillus colis
) and the schematic diagram of 5- hydroxy-dehydro equol (5-hydroxy-dehydroequol) synthetic reaction hydroxy-equol.
Fig. 3 is shown to be contaminated according to the reaction time in genistein (genistein, GSN), dihydro in the synthetic reaction of Fig. 2
Expect that lignin (dihydrogenistein, DHG), 5- hydroxyl-equol (5-hydroxy-equol, 5OH-EQ), 5- hydroxyl-are de-
Concentration (μM) in the reaction system of hydrogen equol (5-hydroxy-dehydroequol, 5-OH-DEQ).
Fig. 4 is the signal of the synthetic reaction divided using the recombination bacillus coli 1 and recombination bacillus coli 2 of two kinds of enzymes of expression
Figure.
Fig. 5 is shown in the synthetic reaction of Fig. 4 according to the reaction time in genistein, dihydro genistein, 5- hydroxyl-female
Horse phenol, 5- hydroxy-dehydro equol reaction system in concentration (μM).
Fig. 6 is to be shown in Fig. 2 and Fig. 4 using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
Albumen quality in the cell extract of the recombination bacillus coli referred to.
Fig. 7 is that the chart of circular dichroism dispersion meter measurement ellipticity is utilized under the wave-length coverage of 210~320nm.
Fig. 8 is shown using the recombination bacillus coli for expressing two or three of enzyme according to the reaction time in dehydroequol
Concentration (μM) in reaction system.
Fig. 9 is the yield for showing the substrate transformation rate and 5- hydroxy-dehydro equol according to the concentration of initial dye lignin
Chart.
(first) of Figure 10 partially using the mass spectrum of electron impact mass spectra (EI-MS) analysis and is divided for 5- hydroxyl-equol
Minor structure, (second) are partially the mass spectrum and molecular structure of 5- hydroxy-dehydro equol.
Figure 11 is to show adding the polyethylene glycol (polyethylene as water soluble polymer according to the time
Glycol, hereinafter referred to as " PEG ") or polyvinylpyrrolidone (polyvinyl pyrrolidone, hereinafter referred to as " PVP ")
The chart of concentration in the reaction system of (S)-equol in reaction solution.
Specific embodiment
Term used in the present invention is usually used in the art, and those skilled in the art can manage
Its meaning is solved, is briefly described below in the present specification:
(1) it expresses: referring in the microorganism of the carrier comprising recombination and produce recombinant protein.
(2) carrier: refer to by single-stranded, double-strand, circle or supercoil DNA (DNA) or ribonucleic acid (RNA)
The polynucleotides of composition.Carrier by replication orgin, promoter, ribosome bind site, nucleic acid cassette and terminate object form, they
Appropriately distance is operably connected to generate recombinant protein.The gene for encoding recombinant protein to be expressed is inserted into nucleic acid cassette position
It sets
(3) cell extract: referring to after being crushed the microorganism of recombinant protein to be expressed, thin using centrifuge removal solid
Supernatant after born of the same parents' fragment.Refer to some or all daidzein reductases of expression, dihydro daidzein racemization in the present invention
Enzyme, dihydro daidzein reductase, tetrahydro daidzein reductase microorganism extracts.
(4) full cell effect: refer to by the broken cell comprising certain enzyme and using cell extract or using not
Isolate and purify the reaction of the complete cell entirety of enzyme.
The present invention relates to for expressing daidzein reductase, dihydro daidzein racemase, the reduction of dihydro daidzein
The recombination bacillus coli of enzyme and tetrahydro daidzein reductase.
In the present invention, it is expanded and is corresponded to by polymerase chain reaction (polymerase chain reaction, PCR)
The daidzein reductase of Si Naike Salmonella (Slackia isoflavoniconvertens), two are converted in deriving from isoflavones
Hydrogen daidzein racemase, dihydro daidzein reductase, tetrahydro daidzein reductase DNA sequence after, point
Be not put into pRSFDuet or pCDFDuet carrier, and in Escherichia coli (E.coli) with His label (his-tag) (6 groups of ammonia
Sour (6histidine)) it is expressed.
Above-mentioned recombination bacillus coli can be utilized synthesizing in equol or dehydroequol from the daidzein as substrate.
Also, above-mentioned recombination bacillus coli can synthesize 5- hydroxyl-equol or 5- hydroxyl from the genistein as substrate
It is utilized in base-dehydroequol.
Above-mentioned equol or 5- hydroxyl-equol synthetic method can be in synthesis (S)-equol or 5- hydroxyl-(S)-female horses
It is utilized in phenol.
More specifically, above-mentioned recombination bacillus coli can be used as from daidzein bioconversion equol and dehydroequol
Whole-cell catalyst, also, can be used as converting genistein biology to 5- hydroxyl-equol and 5- hydroxy-dehydro equol
The whole-cell catalyst of (5-hydroxy-dehyroequol).
Recombination bacillus coli of the invention is being used as in the equol synthetic reaction of whole-cell catalyst, also by addition
Former agent inhibits the oxidation of product, and NAD (P) H, L-AA, glutathione can be used as reducing agent
(glutathione), dithiothreitol (DTT) (dithiothreitol), cysteine (cysteine) etc..Preferably, addition also
The concentration of former agent is initial substrate concentration about 10 again to about 100 times.Reaction solution contains slow for the potassium phosphate of pH buffer function
Fliud flushing, it is preferable that the concentration of potassium phosphate is about 50mM to about 400mM.Preferably, reaction temperature is about 18 DEG C to about 37 DEG C, more
It preferably, is about 25 DEG C to about 30 DEG C.Preferably, the pH of reaction solution is about 5 to about 10, it is highly preferred that being about 6 to about 9, more
It preferably, is about 7 to about 8.Preferably, the mixing speed of reactor is about 50rpm to about 400rpm, it is highly preferred that being about
80rpm to about 250rpm, it is further preferred that being about 100rpm to about 200rpm.
Glucose and glycerol can be used as the carbon source in above-mentioned equol synthesis reactor, it is preferable that glucose or glycerol
Concentration is about 1 (w/v) % to about 5 (w/v) %, it is highly preferred that be about 1.5 (w/v) % to about 4 (w/v) %, it is further preferred that
It is about 2 (w/v) % to about 3 (w/v) %.The concentration of recombinant microorganism of the invention is about optical density (OD) (Optical
Density, following O.D.) 1 to optical density (OD) 100, it is highly preferred that being about O.D.5 to about O.D.50, it is further preferred that being about
O.D.10 to about O.D.30.
According to the type of the isoflavan to be synthesized (isoflavan) or isoflavones (isoflavene), as above-mentioned female
Genistein, Glycitein (glycitein), daidzein, adjacent hydroxyl soybean can be used in the substrate of horse phenol synthetic reaction system
Aglycon (ortho-hydroxy-daidzein), adjacent hydroxyl-genistein (ortho-hydroxy-genistein) etc..
In order to improve the solubility of the reaction solution to above-mentioned substrate, polyethylene glycol (polyethylene can be used
Glycol), polyvinylpyrrolidone (polyvinyl pyrrolidone), polyvinyl alcohol (polyvinyl alcohol) and β-
Cyclodextrin (β-cyclodextrin), methyl beta-cyclodextrin (methyl- β-cyclodextrin), 2- hydroxypropylβ-cyclodextrin
(2- (Hydroxypropyl)-β-cyclodextrin) etc..
Fig. 1 is the chart for showing the yield of (S)-equol of the concentration according to original soy aglycon, as shown in Figure 1,
(S)-equol yield (%) is reduced as the concentration of original soy aglycon increases, but (S)-equol (mM) of production
Tend to increase.
Fig. 2 is to show to express daidzein reductase (daidzein using from daidzein and genistein
Reductase:DZNR), dihydro daidzein racemase (dihydrodaidzein racemase:DDRC), dihydro daidzin
First reductase (dihydrodaidzein reductase:DHDR), tetrahydro daidzein reductase
Equol, dehydroequol, the 5- hydroxyl-of the recombination bacillus coli of (tetrahydrodaidzein reductase:THDR)
The schematic diagram of equol and 5- hydroxy-dehydro equol synthetic reaction.
As shown in Figure 2 above, daidzein is converted into (R)-dihydro daidzein by daidzein reductase (DZNR),
It is converted into (S)-dihydro daidzein by dihydro daidzein racemase (DDRC).(S)-dihydro daidzein passes through dihydro
Daidzein reductase (DHDR) be converted into (3S, 4R)-it is trans--tetrahydro daidzein, pass through tetrahydro daidzein reductase
(THDR) it is converted into equol, or dehydroequol is converted by dehydration.
Also, as shown in Figure 2 above, genistein is converted into (R)-dihydro dyestuff by daidzein reductase (DZNR)
Lignin is converted into (S)-dihydro genistein by dihydro daidzein racemase (DDRC).(S)-dihydro genistein is logical
Cross dihydro daidzein reductase (DHDR) be converted into (3S, 4R)-it is trans--tetrahydro genistein, pass through tetrahydro daidzein
Reductase (THDR) is converted into 5- hydroxyl-equol, or is converted into 5- hydroxy-dehydro equol by dehydration.
It the present invention relates to the use of expression daidzein reductase, dihydro daidzein racemase, the reduction of dihydro daidzein
Enzyme and tetrahydro daidzein reductase and by the tetrahydro daidzein reductase in four kinds of above-mentioned enzymes compared to its excess-three kind enzyme mistake
The recombination bacillus coli of 5 times of expression or more selectively synthesizes 5- hydroxyl-equol method from the genistein as substrate.
Specifically, if being overexpressed 5 times or more compared to its excess-three kind enzyme using tetrahydro daidzein reductase, it is preferable that
It is overexpressed 10 times or more of recombination bacillus coli, then optionally synthesizes 5- hydroxyl-equol.
As shown in Fig. 2, this is because increasing if increasing the expression quantity of tetrahydro daidzein reductase from tetrahydro dyestuff
Lignin synthesizes 5- hydroxyl-equol speed, to competitively inhibit the generation of 5- hydroxy-dehydro equol.
The expression of above-mentioned recombination bacillus coli and 5- hydroxyl-equol and synthetic method and as described above using it
Expression from isoflavones conversion Si Naike Salmonella all four enzymes recombination bacillus coli preparation method and utilize its
Equol synthetic method it is identical.
Fig. 6 is to be shown in Fig. 2 and Fig. 4 using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
Albumen quality in the cell extract of the recombination bacillus coli referred to is big compared at one in the expression quantity of four kinds of enzymes
When expressing four kinds of recombinases in enterobacteria, with carrier expression daidzein reductase and dihydro daidzein racemase
Recombination bacillus coli and big with the recombination of carrier expression dihydro daidzein reductase and tetrahydro daidzein reductase
The expression quantity of enzyme increases in enterobacteria.In Fig. 6,1:DDDT is referred in Fig. 2 at one four kinds of expression in escherichia coli
The case where when recombinase, 2:DD correspond to being disappeared in Fig. 4 with a carrier expression daidzein reductase and dihydro daidzein
The cell extract of the recombination bacillus coli of enzyme is revolved, 3:DT, which corresponds in Fig. 4, expresses dihydro daidzein also with a carrier
The cell extract of the recombination bacillus coli of protoenzyme and tetrahydro daidzein reductase.
Fig. 5 is shown according to the reaction time in genistein, dihydro genistein, 5- hydroxyl-equol, 5- hydroxy-dehydro
Concentration (μM) in the reaction system of equol, if the expression quantity of tetrahydro daidzein reductase is increased compared to its excess-three kind enzyme
Add 5 times or more of recombination bacillus coli for (the 2 of Fig. 6 and 3) in system, then with the recombination bacillus coli of expressing all four enzymes
Related Fig. 2 is compared, and can be increased 5- hydroxyl-equol yield and productivity and is directed to the 5- of 5- hydroxy-dehydro equol
Hydroxyl-equol selectivity.
Also, it the present invention relates to the use of the recombination large intestine bar of expression daidzein reductase and dihydro daidzein racemase
The recombination bacillus coli 2 of bacterium 1 and expression tetrahydro daidzein reductase and tetrahydro daidzein reductase is from the dye as substrate
Material lignin selectively synthesizes 5- hydroxyl-equol method.
It can be by the way that above-mentioned recombination bacillus coli 1 and recombination bacillus coli 2 to be mixed into the bioconversion of whole-cell catalyst
Reaction system selectively synthesizes 5- hydroxyl-equol from genistein.
The expression of recombination bacillus coli 1 and recombination bacillus coli 2 of the invention and the 5- hydroxyl-equol for utilizing it
Synthetic method and expression as described above convert the method and benefit of all four recombinases of Si Naike Salmonella from isoflavones
It is identical with its equol synthetic method.
The hybrid reaction system of above-mentioned recombination bacillus coli 1 and recombination bacillus coli 2 can be applied not only to 5- hydroxyl-female horse
The selectivity of phenol produces, and can be applied to equol, 6- methoxyl group-equol (6-Methoxy-equol) and a variety of hydroxyls-
In the production of equol (Hydroxy-equol) etc..
Fig. 4 shows the recombination large intestine by expressing daidzein reductase, dihydro daidzein racemase with a carrier
Bacillus and another recombination bacillus coli that dihydro daidzein reductase, tetrahydro daidzein reductase are expressed with a carrier
The full cell effect system that reaction is divided.
According to above-mentioned Fig. 4, genistein is converted into (S)-dihydro genistein, (S)-by above-mentioned recombination bacillus coli 1
Dihydro genistein is converted into 5- hydroxyl-(S)-equol by above-mentioned recombination bacillus coli 2, therefore, can be by recombinating large intestine
Bacillus 1 and recombination bacillus coli 2 selectively synthesize 5- hydroxyl-equol from genistein.
The full cell effect system for being divided reaction by different recombination bacillus colis is in the life of 5- hydroxyl-(S)-equol
Production property and ultimate yield are improved aspect favorably, and by reducing the 5- hydroxy-dehydro equol as by-product
It generates outstanding to 5- hydroxyl-(S)-equol selectivity aspect to increase.
The method for selectively synthesizing above-mentioned 5- hydroxyl-equol optionally synthesizes 5- hydroxyl-(S)-equol.
Specifically, the recombination bacillus coli using 5 times of tetrahydro daidzein reductase overexpression or more or above-mentioned recombination
Escherichia coli 1 and recombination bacillus coli 2 selectively synthesize 5- hydroxyl-(S)-equol.
Related to this, Fig. 7 is to understand 5- hydroxyl-equol optics knot using the synthesis of above-mentioned recombination bacillus coli
Structure, and using the chart of circular dichroism dispersion meter measurement ellipticity under the wave-length coverage of 210~320nm, this can be identified through
5- hydroxyl-equol of the recombination bacillus coli biosynthesis of invention has S- configuration in No. 3 carbon.
Further, it is big that expression daidzein reductase, dihydro daidzein racemase and dihydro be the present invention relates to the use of
Beans aglycon reductase is selected without expressing the recombination bacillus coli of tetrahydro daidzein reductase from the daidzein as substrate
The method for synthesizing dehydroequol to property.
Specifically, above-mentioned recombination bacillus coli can selectively to synthesize from daidzein as whole-cell catalyst de-
Hydrogen equol.
If adding boric acid into the reaction solution for synthesizing above-mentioned dehydroequol, promote (3S, 4R)-it is trans--tetrahydro soybean
The dehydration of aglycon, therefore can be to synthesize dehydroequol in high yield.Preferably, the amount of the boric acid of addition be about 50mM extremely
About 200mM.
Also, it the present invention relates to the use of expression daidzein reductase, dihydro daidzein racemase and dihydro daidzin
First reductase without express tetrahydro daidzein reductase recombination bacillus coli from the genistein as substrate selectively
The method for synthesizing 5- hydroxy-dehydro equol.
Specifically, above-mentioned recombination bacillus coli can be used as whole-cell catalyst selectively to synthesize 5- from genistein
Hydroxy-dehydro equol.
The expression of the recombination bacillus coli of aforementioned present invention and female using its dehydroequol or 5- hydroxy-dehydro
Horse phenol synthetic method and expression as described above convert the recombinase of all four enzymes of Si Naike Salmonella from isoflavones
Expression and using its equol synthetic method it is identical.
Fig. 8 is the recombination bacillus coli shown when using expression daidzein reductase, dihydro daidzein reductase
(DZNR+DHDR) and expression daidzein reductase, dihydro daidzein racemase, the recombination of dihydro daidzein reductase are big
When enterobacteria (DZNR+DDRC+DHDR) carries out full cell effect to daidzein, according to the reaction time in the anti-of dehydroequol
Answer the chart of the concentration in system.It is taken off as shown in figure 8, selectively being synthesized using above-mentioned recombination bacillus coli from daidzein
Hydrogen equol is utilized without generating equol, and compared to the recombination bacillus coli (DZNR+DHDR) of two kinds of enzymes of expression
The concentration for expressing the dehydroequol of recombination bacillus coli (DZNR+DDRC+DHDR) Shi Shengcheng of three kinds of enzymes is high.
Fig. 9 is to show to restore when using expression daidzein reductase, dihydro daidzein racemase, dihydro daidzein
When the recombination bacillus coli of enzyme carries out full cell effect to genistein, according to the substrate transformation rate of the concentration of initial dye lignin
And the chart of the yield of 5- hydroxy-dehydro equol, as shown in figure 9, being selected using above-mentioned recombination bacillus coli from genistein
Synthesize to selecting property 5- hydroxy-dehydro equol.
Recombination bacillus coli of the invention can be used as whole-cell catalyst.Whole-cell catalyst is anti-for a variety of biocatalysis
Ying Zhong is reacted under normal temperature and pressure conditions, and has the advantages that response characteristic is outstanding.It is being necessarily required to coenzyme or with more
The process of the reaction of a step or purifying when enzyme activity substantially reduce the case where etc. in, using full cell be more advantageous into
Row biocatalysis.Also, when purified product after the completion of reaction, the weight of full cell is used as using the separation of centrifuge alternative
Group Escherichia coli and it is advantageous.
Equol, dehydroequol, 5- hydroxyl-equol or the 5- hydroxy-dehydro equol synthetic method of above-mentioned synthesis
It can be carried out under aerobic condition, therefore compared with the prior art for synthesizing equol under anaerobic, it can be in mild condition
Lower synthesis equol.Therefore, synthetic method of the invention do not need device for providing anaerobic condition in the prior art or
Technology.
The equol of above-mentioned synthesis, dehydroequol, 5- hydroxyl-equol or 5- hydroxy-dehydro equol synthetic method
The substrate of about 0.2mmol/L to about 5mmol/L can be used.
In the case where the concentration of daidzein or genistein as substrate is less than 0.2mmol/L, the female horse of synthesis
Phenol, dehydroequol, the amount of 5- hydroxyl-equol and 5- hydroxy-dehydro equol are insufficient or purifying expense may also be got higher,
In the case where greater than 5mmol/L, since the substrate transformation rate is reduced, and it is likely to occur additional purifying expense.
Preferably, the concentration of substrate is about 0.2mmol/L to 1mmol/L, it is highly preferred that the concentration of substrate is about
0.8mmol/L to about 1mmol/L.
But in the case where reaction system includes also water soluble polymer, even if the concentration of substrate is greater than 1mmol/L,
It also can be therefore, excellent to synthesize equol, dehydroequol, 5- hydroxyl-equol or 5- hydroxy-dehydro equol in high yield
Selection of land, the concentration of substrate are about 3mmol/L to 5mmol/L.
The conversion ratio of above-mentioned equol or the daidzein of dehydroequol synthetic method is 95% or more, above-mentioned 5- hydroxyl
The conversion ratio of base-equol or the genistein of 5- hydroxy-dehydro equol synthetic method is 95% or more.
Above-mentioned conversion ratio is indicated by (concentration/initial substrate concentration of the substrate of reduction) × 100 (%).
Also, synthetic method of the invention is 0.6mmol/L or more in the concentration of substrate, and the substrate transformation rate may be
95% or more.
Fig. 3 and Fig. 5 is shown according to the reaction time in genistein, dihydro genistein, 5- hydroxyl-equol reactant
Concentration (μM) in system, in the reaction time, the substrate transformation rate is 95% or more after 10 hours.
In above-mentioned equol, dehydroequol, 5- hydroxyl-equol or 5- hydroxy-dehydro equol synthetic method, instead
Answering also may include water soluble polymer in system.Also, above-mentioned water soluble polymer can be used food addition with water-soluble high score
Son.
For example, can be pasted using polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol and β-ring as water soluble polymer
Essence, methyl beta-cyclodextrin, 2- hydroxypropylβ-cyclodextrin etc..Preferably, using polyethylene glycol, polyvinyl alcohol or polyvinyl pyrrole
Alkanone.
By further including water soluble polymer in the reaction system, the solubility of the substrate in reaction system increases,
Therefore the ultimate yield of the equol of preparation, dehydroequol, 5- hydroxyl-equol or 5- hydroxy-dehydro equol may increase
Add.
Figure 11 is to show the poly- second further added in the reaction solution of embodiment 1 according to the time as water soluble polymer
The chart of concentration in the reaction system of (S)-equol in the reaction solution of glycol or polyvinylpyrrolidone.Such as Figure 11 institute
Show, in the case where reaction solution further includes polyethylene glycol (PEG) or polyvinylpyrrolidone (PVP), even if using
The substrate of 5mmol/L, also can be to synthesize equol in high yield.
Also, the present invention relates to the equol prepared by synthetic method present invention as described above, dehydroequol,
5- hydroxyl-equol or 5- hydroxy-dehydro equol.
In particular, the 5- hydroxy-dehydro equol of synthetic method preparation through the invention is previous unknown novel compounds
Object can be indicated by following chemical formula.
The present invention can synthesize (S)-equol under aerobic condition with the high conversion of substrate, and the equol of synthesis has
With molecular structure as estrogens, therefore Menopause symptom can be relieved, especially the morbidity acute disease shape of osteoporosis,
Without the side effect of the previous menopause symptom therapeutic agents such as such as breast cancer induction, and prevent cardiovascular disease, before can be relieved
Column gland cancer.
Also, the dehydroequol synthesized in the present invention can be used as the anticancer agent for treating prostate cancer, oophoroma.
Also, the present invention it is female can to carry out 5- hydroxyl-equol, dehydrogenation under aerobic condition as unit of a few hours
The selectively synthesis of horse phenol and 5- hydroxy-dehydro equol, therefore producing cost can be reduced and obtain high added value, and
It can be suitable for the various uses of food and drug by being effectively mass produced.
Also, additional oxygen is applied recombination bacillus coli of the invention to be used as in the reaction system of whole-cell catalyst
In the case where changing reductase, various hydroxy-isoflavones can be synthesized, therefore researching value is high.
Specifically, tyrosinase can be used as oxidoreducing enzyme.Cytochrome P450 or flavine monooxygenase etc. can close
At hydroxy-isoflavone, such as 3'- hydroxyl-equol, 6- hydroxyl-equol, 8- hydroxyl-equol, 6,3'- dihydroxy-equol.
Also, the 5- hydroxyl-equol or 5- hydroxy-dehydro equol synthesized through the invention can be used for preventing or treating
Climacteric syndrome, and can be used as treating the anticancer agent of prostate cancer, oophoroma.Also, it can be used as cosmetics or food additives
The antioxidant of form.
Hereinafter, specific method of the invention is described in detail by embodiment, but technical scope of the invention is not
It is defined in this.
The preparation method of recombination bacillus coli
It is big to correspond to daidzein reductase, dihydro daidzein racemase, dihydro by polymerase chain reaction amplification
Beans aglycon reductase, tetrahydro daidzein reductase DNA sequence after, be respectively put into pRSFDuet or
In pCDFDuet carrier, and expressed in Escherichia coli with His label (6 histidine).
Specifically, the plasmid (plasmid) for being inserted into the base sequence of above-mentioned enzyme is transformed into BL21 competent cell
After (competent cell), respectively in the LB solid medium of the antibiotic comprising corresponding plasmid in 37 DEG C of incubator
Culture 12 hours.By a colony inoculation in the LB liquid medium for wrapping antibiotic 3mL, and in 37 DEG C of incubator
With speed culture 12 hours of 200rpm.This culture solution of 2v% (1mL) is inoculated in the new LB liquid of the antibiotic comprising 50mL
In body culture medium.If the O.D. (600nm) of culture solution reaches 0.6~0.8, the isopropylthiogalactoside of 0.1mM is added
(IPTG) and the MnSO of 0.1mM4, and with 200rpm inducible protein matter expression 18 hours in 18 DEG C of incubator.After 18 hours,
Cell is centrifuged with 4000rpm and is cleaned with the phosphate buffer of 25mL (PBS) to prepare.
Utilize the production method (reaction method 1) of the equol in the full cell effect system of recombination bacillus coli of the invention
The transparent glass reactor that 10~1000mL is used as reactor, following concentration is provided into reactor
Recombination bacillus coli and substrate.
Into reaction, 10 times of L-AA of addition initial substrate concentration includes as reducing agent, and in reaction solution
The kaliumphosphate buffer for pH buffer function of 200mmol/L concentration, reaction temperature are 30 DEG C.The pH of reaction solution is 8, reaction
The mixing speed of device is 150rpm.Use glucose or glycerol as the carbon source in reactor of the invention, glucose or glycerol
Concentration be 2 (w/v) %, the concentration of recombinant microorganism is O.D.10 or O.D.20.
Embodiment
Embodiment 1: the recombination bacillus coli of all four enzymes of equol conversion is participated in using expression and from daidzein
Synthesize (S)-equol
For the daidzein of 0.2~5mM, using the recombination bacillus coli of four kinds of above-mentioned enzymes of expression O.D.10 with it is anti-
The identical mode of method 1 is answered to be reacted.Substrate, intermediate, product with 4 times or more of ethyl acetate (ethyl acetate, with
It is known as " EA " down) it extracts, after centrifugation pressure reducer removal solvent, re-dissolved in the methanol of specified amount and by efficient liquid
The reactivity and concentration of phase chromatography confirmation substance.(the S)-equol yield indicated as shown in figure 1, can confirm the yield at 0.2mM
It is 95% or more, is 80% or more at 0.4mM, 0.6mM, be 50% or more at 2mM, is 20% or more at 5mM.
Embodiment 2: the recombination bacillus coli of all four enzymes of equol conversion is participated in using expression and from genistein
Synthesize 5- hydroxyl-equol or 5- hydroxy-dehydro equol
For 500 μM of genisteins (Fig. 2), using the recombination bacillus coli of four kinds of above-mentioned enzymes of expression O.D.10 with
The identical mode of reaction method 1 is reacted.Substrate, intermediate, product with 4 times or more of ethyl acetate (ethyl acetate,
Hereinafter referred to as " EA ") it extracts, after centrifugation pressure reducer removal solvent, re-dissolved in the methanol of specified amount and by efficient
The reactivity and concentration of liquid chromatogram confirmation substance.99% or more the genistein conversion of 500 μM of the confirmation when reacting 6 hours,
And generate the 5- hydroxy-dehydro equol of 5- hydroxyl-equol of 97mg/L, 22mg/L.
By forming as described above, the genistein of 1mM is reacted by reaction method 1, as can be known from Fig. 3,
The substrate transformation rate is 95% or more in 10 hours reaction time, and can produce 5- hydroxyl-equol, 63mg/L of 199mg/L
5- hydroxy-dehydro equol.
Embodiment 3: selectively synthesizing the schematic diagram such as Fig. 4 using 5- hydroxyl-equol of the reaction system divided,
The recombination bacillus coli 1 and expression dihydro daidzin of daidzein reductase and dihydro daidzein racemase will be expressed by establishing
The recombination bacillus coli 2 of first reductase and tetrahydro daidzein reductase is mixed into the bioconversion reactant of whole-cell catalyst
System.When reacting the genistein of 1mM according to reaction method 1, as shown in figure 5, when being confirmed by liquid chromatogram according to reaction
Between substrate product concentration.The substrate transformation rate is 99% or more within 10 hours reaction time in this reaction system, and raw
Produce 5- hydroxyl-equol of 231mg/L, the 5- hydroxy-dehydro equol of 12mg/L.Compare the knot of production result and embodiment 1
Fruit, 5- hydroxyl-equol productivity increase by 1.6 times, are defined as 5- hydroxyl equol concentration/5- hydroxy-dehydro equol
Concentration increase by 500 for 5- hydroxyl-equol selectivity.
Also, in order to compare the expression quantity of four kinds of enzymes, the recombination large intestine bar that is crushed by ultrasonication in embodiment 2
The cell extract of recombination bacillus coli 1 and recombination bacillus coli 2 in bacterium and embodiment 3, by cell extract with
After 13000rpm is centrifuged 30 minutes under conditions of 4 DEG C, as shown in fig. 6, passing through sodium dodecyl sulfate-polypropylene acrylamide gel
Electrophoretic analysis supernatant.
The expression quantity as a result, for four kinds of enzymes is analyzed, compared to Escherichia coli used in embodiment 2, in embodiment 3
The two kinds of recombination bacillus coli kinds used increase.Wherein, the expression quantity of tetrahydro daidzein reductase dramatically increases, compared to
The Escherichia coli of embodiment 1 are crushed liquid, increase by 20 times or more, and the expression quantity compared to its excess-three kind enzyme is overexpressed 5 times or more.
The analysis of embodiment 4:5- hydroxyl-equol optical isomer
In order to determine the 5- hydroxyl-equol optical texture synthesized with recombination bacillus coli of the invention, justified
Dichroic astigmatism spectrum analysis.Wave-length coverage is estimated in 210~320nm, and has used circular dichroism dispersion meter ChirascanTM
(Britain applies optical physics company (Applied to plus CD spectrometer (ChirascanTM plus CD spectrometer)
Photophysics, UK)).After sample is dissolved in pure methanol with the concentration of 1.5mg/ml, it is measured.
Measurement result as illustrated in the graph of figure 7, the feminine gender cotton effect with higher at 238nm, at 270nm
Positive cotton effect with higher.Therefore, the 5- hydroxyl of recombination bacillus coli biosynthesis of the invention can be identified through
Equol has S- configuration in No. 3 carbon.
Embodiment 5: the selectively synthesis of dehydroequol
For biosynthesis dehydroequol, in the recombination bacillus coli of embodiment 1, it is prepared for missing tetrahydro daidzin
First reductase, and the recombination for containing or not contain dihydro daidzein racemase (DZNR+DDRC+DHDR, following DDD bacterial strain) is big
Enterobacteria (DZNR+DHDR, following DD bacterial strain).Using above-mentioned Escherichia coli and according to reaction method 1, and carry out with daidzin
Member is the reaction of substrate, and the boric acid of 200mM is further added into reaction solution.As a result, confirmation only generates dehydroequol,
Without generate equol (Fig. 8), specifically, when with the concentration of original soy aglycon be 0.5mM start biological respinse and carry out 2
When hour, 28 μM of dehydroequol is synthesized in DD bacterial strain, and 60 μM of dehydroequol is synthesized in DDD bacterial strain.
The selectively synthesis of embodiment 6:5- hydroxy-dehydro equol
For biosynthesis 5- hydroxy-dehydro equol, in the recombination bacillus coli of embodiment 2, prepares in plasmid and lack
Lose the recombination bacillus coli of tetrahydro daidzein reductase.Using above-mentioned Escherichia coli and according to reaction method 1, and carried out with
Genistein is the reaction of substrate, and confirmation only generates 5- hydroxy-dehydro equol, without generating 5- hydroxyl-equol (figure
9).When being started with the concentration of initial dye lignin for 0.2mM, 0.4mM, 0.6mM, 1.0mM, bioconversion reacts and to carry out 36 small
It constantly, is 99% or more in the conversion ratio of 0.6mM or less genistein, the conversion ratio of genistein is 97% in 1.0mM.5-
The conversion yields of hydroxy-dehydro equol are respectively 44%, 35%, 27%, 39%.
The quality analysis of embodiment 7:5- hydroxyl-equol and 5- hydroxy-dehydro equol
5- hydroxyl-the equol synthesized in embodiment 3 and embodiment 6 is identified by gas chromatograph-mass spectrometer (GC-MS)
And 5- hydroxy-dehydro equol, the results are shown in Figure 10.
Pass through the 5- hydroxyl-equol and 5- hydroxyl-two of the above-mentioned recombinant microorganism synthesis of electron impact mass spectra analysis and utilization
Hydrogen-equol.Electron impact mass spectra has used the super gas chromatograph of the TRACE GC of Thermo company/ITQ1100 (TRACE
GC Ultra gas chromatograph/ITQ1100).In positive mode, using non-polar capillary column (TR-5ms),
Helium flow velocity is 1mL/min, entrance (inlet), mass transfer line (mass transfer line) and ion source (ion source)
Temperature is respectively 250 DEG C, 275 DEG C, 230 DEG C.The oven temperature of gas chromatograph is maintained at a temperature of 65 DEG C after five minutes,
250 DEG C are risen to the rate of 3 DEG C/min.Ionization voltage is 70eV, and the mass range measured is 50~600amu.
Electron impact mass spectra analyzes result (Chang, Y.C., and identical as the spectrum of the DHD of previous report
M.G.Nair.J.Natr.Proc.58:1892-1896,1995；Wahala, K., A.Salakka, and H.Adlercreutz
.Proc.Soc.Exp.Biol.Med.217:293-29,1998).The electron impact mass spectra of the compound of synthesis shows [M+H]+
Molecular ion peak and m/z 256 as expected at C15H12O4 (DHD) it is consistent.Other quasi-molecular ions of the compound of synthesis are
137(100)、120(52)、91(36)、65(16)。
Embodiment 8: the synthesis of equol in the reaction system additionally comprising water soluble polymer
In order to improve daidzein to the solubility of reaction solution, in addition to including dimethyl sulfoxide (dimethyl
Sulfoxide, following DMSO) and additionally comprising except polyethylene glycol (PEG) or polyvinylpyrrolidone (PVP), with reality
The identical mode of example 1 is applied to be reacted.
Specifically, in order to increase the equol yield in the daidzein of 5mM, in the water for further including 5% (w/v)
It is reacted in the reactor of soluble macromolecular polyethylene glycol or polyvinylpyrrolidone, in the anti-of addition polyvinylpyrrolidone
In the case where answering, the equol (yield is 99% or more, 1.16g/L) of 5mM is synthesized in reaction in 12 hours, is adding poly- second
In the case where the reaction of glycol, the equol of 3.7mM is synthesized in reaction in 24 hours (yield is 74% or more, 0.9g/L).
According to above-described embodiment 1 and embodiment 2, four kinds of enzymes of Si Naike Salmonella can be converted from isoflavones by expressing
Recombination bacillus coli, under aerobic condition within time a few hours with high yield from daidzein synthesize (S)-equol, or
Person synthesizes 5- hydroxyl-equol and 5- hydroxy-dehydro equol from genistein.
In above-described embodiment 3, embodiment 5 and embodiment 6, the recombination of two or three of enzyme in four kinds of enzymes of combination is utilized
Escherichia coli can respectively selectively synthesize 5- hydroxyl-equol, dehydroequol or 5- hydroxy-dehydro equol.
Also, it can be identified through above-described embodiment 4 and embodiment 7, according to the weight of embodiment 2, embodiment 3 and embodiment 6
Group Escherichia coli can synthesize 5- hydroxyl-(S)-equol or 5- hydroxy-dehydro equol.
Also, in above-described embodiment 8, compared to the reaction system (embodiment 1) for being free of water soluble polymer, reactant
The yield for the equol that system further includes water soluble polymer is more excellent.
Claims (16)
1. a kind of recombination bacillus coli, which is characterized in that for express daidzein reductase, dihydro daidzein racemase,
Dihydro daidzein reductase and tetrahydro daidzein reductase.
2. a kind of synthetic method, which is characterized in that using the recombination bacillus coli of claim 1 from as the daidzein of substrate
Synthesize equol.
3. a kind of synthetic method, which is characterized in that using the recombination bacillus coli of claim 1 from as the genistein of substrate
Synthesize 5- hydroxyl-equol or 5- hydroxy-dehydro equol.
4. a kind of synthetic method, which is characterized in that utilize expression daidzein reductase, dihydro daidzein racemase, dihydro
Daidzein reductase and tetrahydro daidzein reductase and by the tetrahydro daidzein reductase in four kinds of above-mentioned enzymes compared to
The recombination bacillus coli that its excess-three kind enzyme is overexpressed 5 times or more selectively synthesizes 5- hydroxyl-from the genistein as substrate
Equol.
5. a kind of synthetic method, which is characterized in that recombinated using expression daidzein reductase and dihydro daidzein racemase
The recombination bacillus coli of Escherichia coli and expression dihydro daidzein reductase and tetrahydro daidzein reductase is from as substrate
Genistein selectively synthesize 5- hydroxyl-equol.
6. a kind of synthetic method, which is characterized in that utilize expression daidzein reductase, dihydro daidzein racemase and dihydro
Daidzein reductase is selected without expressing the recombination bacillus coli of tetrahydro daidzein reductase from the daidzein as substrate
Synthesize dehydroequol to selecting property.
7. a kind of synthetic method, which is characterized in that utilize expression daidzein reductase, dihydro daidzein racemase and dihydro
Daidzein reductase is selected without expressing the recombination bacillus coli of tetrahydro daidzein reductase from the genistein as substrate
Synthesize to selecting property 5- hydroxy-dehydro equol.
8. synthetic method according to claim 4 or 5, which is characterized in that above-mentioned 5- hydroxyl-equol is 5- hydroxyl-
(S)-equol.
9. the synthetic method according to any one of claim 2 to 7, which is characterized in that use above-mentioned recombination bacillus coli
Make whole-cell catalyst.
10. the synthetic method according to any one of claim 2 to 7, which is characterized in that carried out under aerobic condition.
11. the synthetic method according to any one of claim 2 to 7, which is characterized in that use the bottom of 0.2~1mmol/L
Object.
12. the synthetic method according to any one of claim 2 to 7, which is characterized in that the substrate transformation rate be 95% with
On.
13. the synthetic method according to any one of claim 2 to 7, which is characterized in that also comprising water-soluble in reaction system
Property macromolecule.
14. synthetic method according to claim 13, which is characterized in that above-mentioned water soluble polymer is added as food and used
Water soluble polymer is polyethylene glycol or polyvinylpyrrolidone.
15. a kind of 5- hydroxy-dehydro equol, which is characterized in that synthesized by the synthetic method of claim 3 or 7.
16. a kind of 5- hydroxy-dehydro equol, which is characterized in that indicated by following chemical formula:
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| JP7449526B2 (en) * | 2018-12-07 | 2024-03-14 | 学校法人東京理科大学 | Composition for the production of equol derivatives |
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| PYUNG-GANG LEE等: "P212A Mutant of Dihydrodaidzein Reductase Enhances (S)-Equol Production and Enantioselectivity in a Recombinant Escherichia coli Whole-Cell Reaction System.", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
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