CN104031875A - Engineering bacteria for producing S-equol and application - Google Patents

Abstract

The invention discloses engineering bacteria for producing S-equol. The engineering bacteria for producing S-equol are obtained by cloning L-DDRC, L-DZNR, L-DHDR and L-THDR genes from Lactococcus sp. 20-92 to escherichia coli BL21 (D3) and transforming. The engineering bacteria for producing S-equol provided by the invention are simple in fermentation condition, convenient to use, stable in system, and wide in application; the engineering bacteria for producing S-equol have the ability of transforming daidzein in BHI and LB culture media into S-equol whether under an anaerobic condition or an aerobic condition, and have the ability of transforming bean pulp in BHI and LB into S-equol. Release of daidzein of the bean pulp in the BHI is facilitated.

Description

A kind of S-equol produces engineering bacteria and application

(1) technical field

The present invention relates to a kind of construction and application of engineering bacteria, particularly the S-equol of a kind of source and milk-acid bacteria produces engineering bacteria and Dai or dregs of beans is converted into the application of S-equol.

(2) background technology

Soybean isoflavones has various biological function, from 1980, a large amount of medical researches show, soybean isoflavones is except being combined with estrogen receptor, thereby effectively outside preventing osteoporosis, mammary cancer, carcinoma of endometrium, can also be combined with androgen receptor and reduce the generation of prostate cancer, and there are anti-oxidant, anti-hemolysis and anti-mycotic activity etc.On animal produces, large quantity research shows that soybean isoflavones can promote buck growth both at home and abroad, and enhancing body immunity, improves animal product quality, improves production performance etc.But the biological action of soybean isoflavones is to a certain extent owing to its meta-bolites Equol, and can human body be that Equol is that can decision soybean isoflavones more effectively bring into play the key factor that it reduces the chronic disease risk effects such as tumour, cardiovascular disorder, osteoporosis and climacteric syndrome by Daidezin metabolism.Therefore the effect of Equol is subject to common concern, becomes one of focus of current biological study.Equol is the important meta-bolites of soybean isoflavones, approximately has 30%～50% people soybean isoflavones can be changed into Equol and then excretion in crowd, but the factor that affects its conversion still imperfectly understands at present, and wherein intestinal microflora may be most important factor.

Isoflavonoid mainly exists to combine the glucosides soybean isoflavones mode of glycosyl in crude soya bean, i.e. glucosides daizeol (daidzin) and glucosides genistein (genistin).In mammalian body, the soybean isoflavones of glucosides form can not directly absorb through small bowel, therefore there is no physiologically active.And the mesostate daizeol aglycon (daidzein) forming after removal glycosyl part and genistein aglycon (genistein) etc. also only show weak estrogenic activity.Only by after intestinal bacteria specificity degraded, produce metabolic end product--Equol just has higher biologic activity.Compare with daizeol aglycon, the avidity that Equol is combined with estrogen receptor, anti-oxidant activity, anti-prostate cancer effect are stronger, and removing speed in blood plasma is slower.Therefore the biological activity of soybean isoflavones is more realized by its metabolic end product Equol.

The main method of producing at present Equol is chemosynthesis, and two problems of its existence are exactly: the Equol that (1) entero-bacte metabolism produces is all S-type, has biological activity; And take the Equol that Daidezin is raw material chemosynthesis, be the racemic compound that R-Equol and S-equol mix, R-Equol be do not have activated; (2) chemosynthesis needs metal catalyst, hydrogen, high pressure-temperature etc., high to production unit requirement, yields poorly, and cost is also higher.So can adopt the synthetic Equol of biological process to become the focus that everybody pays close attention to.The literature search of prior art is found, current and the closely-related patent of the present invention has " method that S-equol is prepared in acinetobacter calcoaceticus AUH-JLM455 and conversion thereof " (application number: 200810147314.9), " Equol produces bacterium and utilization " (application number: 201080007863.1), " composition that contains the milk-acid bacteria that produces Equol " (application number: 200480020952.4), " fermented product that contains the Equol generation microorganism that has maintained Equol generation ability and preparation method thereof " (application number: 200980136848.4), " a kind of Proteus mirabilis bacterial strain and soybean transformation aglycon thereof are produced the method for S-equol " (application number: 201210146746.4), " a kind of faecium and the method and the application that produce Equol " (application number: 201110086803.X), " clostridium bifermentans and microbial inoculum and application that a strain degraded Daidzein produces Equol " (application number: 201010513344.4).These patents are all to use separated single or a few bacterium hybrid anaerobic fermentation obtaining from enteron aisle to produce Equol above, but research finds that these bacterium major parts that are separated to are bacterium novel species, and different medium components and fermentation condition can affect it and whether can produce Equol, adding anaerobism cultivation etc. has relatively high expectations, so the method that adopts original isolate anaerobically fermenting to produce Equol not only wastes time and energy, also likely because medium component and culture condition former thereby affect Equol output and fermentation costs.

(3) summary of the invention

The present invention is directed to current biological method and produce the limitation of S-equol, built a kind of effective, the simple S-equol generation of fermentation condition engineering bacteria simple to operate, stable.And utilize this engineering bacteria that the throughput of S-equol is verified and applied.

The technical solution used in the present invention is:

The invention provides a kind of S-equol and produce engineering bacteria, described engineering bacteria be by L-DDRC, the L-DZNR, L-DHDR and the L-THDR gene clone that derive from milk-acid bacteria (Lactococcus sp.) 20-92 to e. coli bl21 (D3), transform and to obtain S-equol and produce engineering bacteria; The nucleotides sequence of described L-DDRC is classified as shown in SEQ ID NO:1, the nucleotides sequence of L-DZNR is classified as shown in SEQ ID NO:2, the nucleotides sequence of L-DHDR is classified as shown in SEQ ID NO:3, and the nucleotides sequence of L-THDR is classified as shown in SEQ ID NO:4.

Further, S-equol generation engineering bacteria of the present invention builds as follows:

At the N of L-DDRC gene end and C end, add respectively BamH I and Not I restriction enzyme site, at the N of L-DZNR gene end and C end, add respectively Bgl II and Kpn I restriction enzyme site, at the N of L-DHDR gene end and C end, add respectively BamH I and Not I restriction enzyme site, at the N of L-THDR gene end and C end, add respectively EcoR V and Not I restriction enzyme site; Utilize the synthetic method of artificial full gene synthetic above gene respectively, during synthetic, at the two ends of above gene, add respectively EcoR V restriction enzyme site, after full gene synthetic, with EcoR V, carry out enzyme and cut and respectively L-DDRC, L-DZNR, L-DHDR and L-THDR are cloned into carrier pUC57 afterwards, obtain plasmid pUC57-L-DDRC, pUC57-L-DZNR, pUC57-L-DHDR and pUC57-L-THDR; With BamH I and Not I, plasmid pUC57-L-DDRC and pETDuet-1 are carried out respectively after double digestion, glue reclaims enzyme and cuts product, then utilizes the product connecting after test kit cuts back to close enzyme to connect into a cyclic plasmid pETDuet-1-L-DDRC; With BamH I and Not I, plasmid pUC57-L-DHDR and pCDFDuet-1 are carried out respectively after double digestion, glue reclaims enzyme and cuts product, then utilizes the product connecting after test kit cuts back to close enzyme to connect into a cyclic plasmid pCDFDuet-1-L-DHDR; With Bgl II and Kpn I, to plasmid pUC57-L-DZNR and pETDuet-1-L-DDRC, carry out respectively after double digestion, glue reclaims enzyme and cuts product, then utilizes the product connecting after test kit cuts back to close enzyme to connect into a cyclic plasmid pETDuet-1-L-DDRC-DZNR; With EcoR V and Kpn I, to plasmid pUC57-L-THDR and pCDFDuet-1-L-DHDR, carry out respectively after double digestion, glue reclaims enzyme and cuts product, then utilizes the product connecting after test kit cuts back to close enzyme to connect into a cyclic plasmid pCDFDuet-1-L-DHDR-THDR; Then plasmid pETDuet-1-L-DDRC-DZNR and pCDFDuet-1-L-DHDR-THDR are transformed into escherichia coli expression Host Strains BL21 (D3) simultaneously, obtain recombinant bacterial strain; Recombinant bacterial strain is seeded on the LB culture medium flat plate containing final concentration 50 μ g/ml Pyocianils and final concentration 50 μ g/ml Streptomycin sulphates, 37 ℃ of aerobic cultivation 24h, screening has the clone of Pyocianil and streptomycin resistance simultaneously, obtains the engineering bacteria of producing S-equol.

The invention provides a kind of described S-equol and produce the application of engineering bacteria in preparing S-equol, described is applied as: take Dai or dregs of beans as substrate, S-equol is produced to seed liquor that engineering bacteria obtains through enlarged culturing and with the inoculum size of volumetric concentration 5%, be seeded to BHI substratum or LB substratum, under aerobic or anaerobic condition, cultivate 24h for 37 ℃, to the IPTG that adds final concentration 25mg/L in nutrient solution, 37 ℃ are continued to cultivate after 6～72h, get nutrient solution separation and purification, obtain S-equol; Described BHI substratum final concentration consists of: peptone 10g/L, dehydration calf brain soak powder 12.5g/L, dehydration beef heart infusion 5.0g/L, sodium-chlor 5.0g/L, glucose 2.0g/L, Sodium phosphate dibasic 2.5g/L, and solvent is water, and pH value is 7.0; Described LB substratum final concentration forms: Tryptones 10g/L, yeast extract 5.0g/L, sodium-chlor 10g/L, and solvent is water, pH value is 7.0.

The starting point concentration of described Dai is 50 μ g/ml.

The starting point concentration of described dregs of beans is 15g/L.

Described S-equol produces the application of engineering bacteria in preparing S-equol to carry out as follows: (1) seed culture: S-equol is produced to engineering bacteria and be seeded in the BHI substratum that contains final concentration 50 μ g/ml Pyocianils and final concentration 50 μ g/ml Streptomycin sulphates, 37 ℃ standing, anaerobism is cultivated after 24h, obtains seed liquor; (2) fermentation culture and conversion reaction: Dai or dregs of beans are added to BHI substratum or LB substratum, then with the inoculum size of volumetric concentration 5%, inoculate seed liquor, under aerobic or anaerobic condition, cultivate 24h for 37 ℃, to the IPTG (inserting the expression of gene to induce external source) that adds final concentration 25mg/L in nutrient solution, 37 ℃ are continued to cultivate after 6～72h, get nutrient solution separation and purification, obtain S-equol; The starting point concentration of described Dai is 50 μ g/ml, and the starting point concentration of described dregs of beans is 15g/L.

The method of described nutrient solution separation and purification is: get the nutrient solution of 1ml, centrifugal 3 minutes of 8000r/min, obtains supernatant a; Get 900 μ l supernatant a to another clean 2ml EP pipe, then in pipe, add 900 μ l ethyl acetate, fully mix rear standing 5min, centrifugal 5 minutes of 5000r/min, obtains supernatant b and precipitation b; Get 900 μ l supernatant b to another clean 2ml EP pipe; Get precipitation b and add the ethyl acetate repeated centrifugation 1 time of equivalent, obtain supernatant c; Supernatant b and supernatant c are mixed, move on in 2ml centrifuge tube, 45 ℃ of vacuum freezings are condensed into powder; Add 200 μ l anhydrous methanols to dissolve concentrated powder to this EP pipe, and through 0.22 μ m polyvinyladine floride filtering with microporous membrane, filtrate is S-equol.

Supernatant a of the present invention, supernatant b, supernatant c are supernatant liquor, for the ease of distinguishing the supernatant liquor difference of different step acquisition, name, and letter itself does not have implication.

The checking of the engineering bacteria of the production S-equol that the present invention is constructed comprises: (1) is used the method validation Equol of PCR and double digestion to produce gene L-DDRC, L-DZNR, L-DHDR and L-THDR and has really been cloned into same Host Strains BL21 (D3) simultaneously; (2) engineering bacteria that uses HPLC method proved to invent constructed production S-equol has the ability that Dai or dregs of beans is converted into S-equol really; (3) to invent the doubtful S-equol spectrum peak that constructed engineering bacteria transforms out Dai be S-equol to the further proved of liquid-matter coupling really.

The invention has the beneficial effects as follows: provide that a kind of fermentation condition is simple, easy to use, stable system, widely used S-equol produce engineering bacteria, described S-equol produces no matter engineering bacteria is under anaerobism or aerobic conditions, all has the ability that the Dai in BHI and LB substratum is transformed to S-equol; This project bacterium has the ability that the dregs of beans in BHI and LB is converted into S-equol; The Daidzein that this project bacterium is conducive to dregs of beans in BHI discharges.

(4) accompanying drawing explanation

Fig. 1 is the structure schematic diagram that in embodiment 1, milk-acid bacteria source S-equol produces engineering bacteria.

Fig. 2 is the PCR qualification result figure that S-equol produces engineering bacteria.Use respectively PCR primer pET Up1 and pET Down1 (swimming lane 1,5), PCR primer DuetUP2 and T7terminator (swimming lane 2,4,6 and 8) and PCR primer PCDFUp1 and PCDF Down1 (swimming lane 3 and 7) to carry out the detection of pcr amplification rear electrophoresis engineering bacteria plasmid and empty carrier.

Fig. 3 is the Western blot qualification result figure that S-equol produces engineering bacteria.Swimming lane 1 and 2 represents that respectively these protein samples derive from the intestinal bacteria that contain empty carrier pETDuet-1 and pCDFDuet-1 and the intestinal bacteria that contain engineering bacteria plasmid pETDuet-1-L-DDRC-DZNR and pCDFDuet-1-L-DHDR-THDR.A is the result that the monoclonal antibody of the anti-His label of use detects.B is the result that the monoclonal antibody of the anti-S label of use detects.

Fig. 4 is that high performance liquid chromatography (HPLC) detects the collection of illustrative plates that engineering bacteria transforms production S-equol; A is S-equol standard substance, and B is that S-equol produces engineering bacterium fermentation liquid.

Fig. 5 is the Mass Spectrometric Identification collection of illustrative plates of engineering bacterium fermentation liquid spectrum absorption peak in the time of HPLC9.5 minute, A is the positively charged ion scintigram of S-equol standard substance, B is the negative ion scintigram of S-equol standard substance, C is engineering bacterium fermentation liquid positively charged ion scintigram in the time of HPLC9.5 minute, and D is engineering bacterium fermentation liquid negative ion scintigram in the time of HPLC9.5 minute.

Fig. 6 is the output figure of the production S-equol of engineering bacteria under different culture media and different culture condition.

Fig. 7 is that engineering bacteria utilizes dregs of beans to transform the output figure that generates S-equol.

(5) embodiment

Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:

Embodiment 1: milk-acid bacteria source Equol produces the structure of engineering bacteria

The present invention has built a kind of S-equol by the method for using gene synthetic to be connected with double digestion and has produced engineering bacteria, and Equol produces the structure of engineering bacteria, mainly comprises the following steps:

1. milk-acid bacteria source Equol produces the synthetic of gene

Adopt the method for full gene synthetic will to derive from the L-DDRC (nucleotides sequence is classified as shown in SEQ ID NO:1) of lactic bacterium strains (Lactococcus sp.) 20-92 respectively, L-DZNR (nucleotides sequence is classified as shown in SEQ ID NO:2), L-DHDR (nucleotides sequence is classified as shown in SEQ ID NO:3) and L-THDR (nucleotides sequence is classified as shown in SEQ ID NO:4) gene clone are to the EcoR V restriction enzyme site of PUC57 carrier (Jin Sirui bio tech ltd), and add respectively BamH I and Not I restriction enzyme site at the N of L-DDRC gene end and C end, at the N of L-DZNR gene end and C end, add respectively Bgl II and Kpn I restriction enzyme site, at the N of L-DHDR gene end and C end, add respectively BamH I and Not I restriction enzyme site, at the N of L-THDR gene end and C end, add respectively EcoR V and Not I restriction enzyme site.DNA sequencing result shows, the present invention is in full accord with the generation of the Equol of lactic bacterium strains 20-92 L-DDRC, L-DZNR, L-DHDR and L-THDR gene by these genes of synthetic.

2.S-Equol produces the structure of plasmid and engineering bacteria

Use the latter linked method of double digestion respectively that L-DDRC gene and L-DZNR gene clone is upper to the facultative expression vector PETDuet-1 of intestinal bacteria (purchased from Chinese plasmid vector strain cell pnca gene preservation center), obtain plasmid PETDuet-1-L-DDRC-DZNR; L-DHDR gene and L-THDR gene clone are gone up to (purchased from Chinese plasmid vector strain cell pnca gene preservation center) (Fig. 1) to the facultative expression vector PCDFDuet-1 of intestinal bacteria, obtain plasmid PCDFDuet-1-L-DHDR-THDR.Then utilize the method for chemical conversion to be transformed into escherichia coli expression Host Strains BL21 (D3) (purchased from Quan Shijin Bioisystech Co., Ltd) plasmid PETDuet-1-L-DDRC-DZNR and PCDFDuet-1-L-DHDR-THDR simultaneously.Then on the LB flat board that contains Pyocianil (50 μ g/ml) and Streptomycin sulphate (50 μ g/ml), screening has the clone of these two kinds of antibiotic resistances simultaneously, obtains S-equol and produces engineering bacteria.

The dull and stereotyped final concentration of LB consists of: Tryptones 10g/L, yeast extract 5.0g/L, sodium-chlor 10g/L, and solvent is water, pH value is 7.0.

Concrete operation step is as follows: with BamH I (precious biotechnology (Dalian) company limited) and Not I (precious biotechnology (Dalian) company limited), plasmid pUC57-L-DDRC and pETDuet-1 are carried out respectively to double digestion, utilize glue to reclaim test kit (purchased from Hangzhou BIOER Technology Co., Ltd) enzyme is cut after product recovery, the product that utilizes DNA fragmentation to connect after test kit (precious biotechnology (Dalian) company limited) cuts back to close enzyme connects into a cyclic plasmid pETDuet-1-L-DDRC; With BamH I and Not I, plasmid pUC57-L-DHDR and pCDFDuet-1 are carried out respectively after double digestion, glue reclaims enzyme and cuts product, then utilizes the product connecting after test kit cuts back to close enzyme to connect into a cyclic plasmid pCDFDuet-1-L-DHDR; With Bgl II (precious biotechnology (Dalian) company limited) and Kpn I (precious biotechnology (Dalian) company limited), to plasmid pUC57-L-DZNR and pETDuet-1-L-DDRC, carry out respectively after double digestion, glue reclaims enzyme and cuts product, then utilizes the product connecting after test kit cuts back to close enzyme to connect into a cyclic plasmid pETDuet-1-L-DDRC-DZNR; With EcoR V (precious biotechnology (Dalian) company limited) and Kpn I, to plasmid pUC57-L-THDR and pCDFDuet-1-L-DHDR, carry out respectively after double digestion, glue reclaims enzyme and cuts product, then utilizes the product connecting after test kit cuts back to close enzyme to connect into a cyclic plasmid pCDFDuet-1-L-DHDR-THDR; Then plasmid pETDuet-1-L-DDRC-DZNR and pCDFDuet-1-L-DHDR-THDR are transformed into escherichia coli expression Host Strains BL21 (D3) (Beijing Quanshijin Biotechnology Co., Ltd) simultaneously, obtain recombinant bacterial strain; Recombinant bacterial strain is seeded on the LB culture medium flat plate containing final concentration 50 μ g/ml Pyocianils and final concentration 50 μ g/ml Streptomycin sulphates, 37 ℃ of aerobic cultivation 24h, screening has the clone of Pyocianil and streptomycin resistance simultaneously, obtains the engineering bacteria of producing S-equol.

3.S-Equol produces the checking of engineering bacteria

In order further to verify whether successfully plasmid PETDuet-1-L-DDRC-DZNR and PCDFDuet-1-L-DHDR-THDR are transformed into escherichia coli expression Host Strains BL21 (D3), this project bacterium is seeded to LB substratum, after 37 ℃ of overnight incubation, collect thalline after centrifugal 3 minutes for 8000 revs/min.Part thalline extracts for bacterial plasmid, and another part extracts for bacterioprotein.Use plasmid extraction kit (purchased from Hangzhou BIOER Technology Co., Ltd) to extract the plasmid in this project bacterium, and adopt the method for PCR electrophoresis to detect, result as shown in Figure 2.Adopt bacterioprotein extracting solution (Shanghai Sheng Gong biotechnology company limited) to extract the whole protein of this project bacterium, and use respectively the monoclonal antibody of anti-His label and anti-S albumen label to carry out Western blot detection, result as shown in Figure 3.

PCR system and condition: TaKaRa Ex Taq (5U/ μ l) (precious biotechnology (Dalian) company limited) 0.3 μ l; 10 * Ex Taq Buffer (Mg2+Plus), 5 μ l; DNTP Mixture (each 2.5mM) 4 μ l; Plasmid template 2 μ l; Primer 1:10pM; Primer 2: 10pM; Sterile purified water is supplied 50 μ l.

PCR primer

pET?Up1：5-‘ATGCGTCCGGCGTAGA-3’

pET?Down1：5-‘GATTATGCGGCCGTGTACAA-3’

DuetUp2：5-‘TTGTACACGGCCGCATAATC-3’

T7Terminator：5-‘GCTAGTTATTGCTCAGCGG-3’

pCDF?Up1：5-‘GGATCTCGACGCTCTCCCT-3’

pCDF?Down1：5-‘GATTATGCGGCCGTGTACAA-3’

PCR condition: 94 ℃, 4 minutes, 1 circulation; 94 ℃, 1 minute, 50 ℃, 1 minute, 72 ℃, 1.5 minutes, 35 circulations; 72 ℃, 15 minutes, 1 circulation;

Western blot detects

Bacterioprotein extracts: use bacterial cellular protein lysate (life work biotechnology (Shanghai) limited-liability company) to extract bacterium whole protein.Protein electrophorese: use 15%Tris-HCl pre-prepared colloid (life work biotechnology (Shanghai) limited-liability company) 150V electrophoresis 70 minutes.

Western blotting: use 10 * High molecules western transfer buffer (life work biotechnology (Shanghai) limited-liability company) 100V transferring film 60 minutes.Then use novel one step process Fast W B test kit and highly sensitive ECL luminescence reagent to detect.

In this project bacterium, contain as can be seen from Figure 24 electrophoretic bands consistent with L-DDRC, L-DZNR, L-DHDR and L-THDR gene size.Illustrate successfully these 4 gene clones to escherichia coli expression Host Strains BL21 (D3).We utilize the method for Western blot the basically identical protein bands of 4 albumen sizes and the albumen size of L-DDRC, L-DZNR, L-DHDR and L-THDR genes encoding to be detected as seen from Figure 3, illustrate that these 4 genes can express simultaneously in engineering bacteria.

Embodiment 2: milk-acid bacteria source S-equol produces the functional verification of engineering bacteria

The present invention carries out functional verification by the ability of using HPLC and the coupling of liquid-matter to produce S-equol to engineering bacteria, and the functional verification that S-equol produces engineering bacteria mainly comprises the following steps:

1.S-Equol produces the preparation of engineering bacteria

50 μ l engineering bacterias being preserved to liquid from-80 ℃ of refrigerators is inoculated in the brain heart infusion liquid nutrient medium (BHI) that 5ml contains Pyocianil (50 μ g/ml) and Streptomycin sulphate (50 μ g/ml), be placed in Electrotek anaerobism workstation (purchased from Britain Electrotek company), after 37 ℃ of standing cultivation 24h, obtain seed liquor, for inoculation.Preserve liquid and preparation method thereof: by after centrifugal 3 minutes of 8000 revs/min of the bacterium liquid of 2ml overnight incubation, abandon supernatant, then toward the glycerine that adds 1ml20% in pipe, put into-80 ℃ of Refrigerator stores after fully that bacterium is resuspended.

BHI substratum final concentration consists of: peptone 10g/L, dehydration calf brain soak powder 12.5g/L, dehydration beef heart infusion 5.0g/L, sodium-chlor 5.0g/L, glucose 2.0g/L, Sodium phosphate dibasic 2.5g/L, and solvent is water, and pH value is 7.0.

2. ferment with substratum and fermentation condition

250 μ g Dais are added in the BHI substratum (liquid amount is 30ml) of 5ml, then seed liquor are inoculated with the inoculum size of volumetric concentration 5%, at 37 ℃ in anaerobism workstation standing cultivation 24h, obtain fermented liquid.

3. in engineering bacterium fermentation liquid, the HPLC of S-equol content detects

To step 2, cultivate the IPTG aqueous solution that adds 5 μ L25mg/ml in the fermented liquid after 24h, 37 ℃ are continued to cultivate after 48h, getting 1ml nutrient solution manages to 1.5ml EP, 8000 rpms after centrifugal 3 minutes, 900 μ L supernatant a are transferred to a new 2.0ml EP pipe, and then every pipe adds the ethyl acetate of equivalent, fully mixes latter standing 5 minutes, 5000 rpms after centrifugal 5 minutes, get supernatant b to another clean 2mL EP pipe; Get the ethyl acetate of equivalent centrifugal 1 time again for lower floor's liquid remaining after supernatant b, obtain supernatant c, then supernatant b and supernatant c are mixed, 45 ℃ of frozen centrifugations are condensed into powder, add 200 μ L anhydrous methanol dissolved powders, and filter through 0.22 μ m polyvinyladine floride millipore filtration (upper Haixing County sub-scavenging material factory), filtrate is detected for HPLC.Take S-equol standard substance (purchased from Daicel medicine chiral technology (Shanghai) Co., Ltd.) as contrast.

High-efficient liquid phase chromatogram condition:

Liquid chromatographic system: Waters2695; Chromatographic column: SunFireTM C185 μ m (4.6mm * 205mm column).Moving phase: 0.01% formic acid (50%), methyl alcohol (20%) and acetonitrile (30%); Elution program: isocratic elution 15min; Flow velocity 0.8mL/min; 30 ± 2 ℃ of column temperatures, 7 ℃ of sample temperatures; Detect wavelength: S-equol 205nm, Dai 254nm.

From the HPLC detected result of Fig. 4, can find out, compare with S-equol standard substance, the engineering bacteria that the present invention builds has detected the generation of S-equol after by fermentation in its fermented liquid.Compare with S-equol standard substance, in engineering bacterium fermentation, in the time of approximately 9.5 minutes, corresponding spectral absorption peak also can be detected, illustrate that it is the ability of S-equol that this project bacterium has Dai conversion production.

4. in engineering bacterium fermentation liquid, the liquid chromatography-mass spectrography of S-equol is identified.

HPLC is detected and shows that the fermented sample that contains S-equol carries out further liquid-matter and identifies.

Liquid-matter coupling testing conditions

Instrument model: Agilent6460 triplex tandem level Four bar high performance liquid chromatography GC-MS

HPLC chromatographic condition: the same

Mass spectrum condition: ESI ion source, plus or minus ion scan, sweep limit is 100～1000amu, dry gas temperature: 325 ℃; Dry gas flow: 5L/min; Spraying gun pressure: 45Psi; Sheath gas temperature: 350 ℃; Sheath gas flow: 11L/min; Capillary voltage: 3000V (+), 3500 (-); Atomization gas pressure voltage: 0 (+), 500 (-); Cracking voltage: 135V.

The spectrum peak sample occurring while collecting in engineering bacterium fermentation liquid HPLC9.5 minute is analyzed for positively charged ion and negatively charged ion mass spectrometric detection.As can be seen from Figure 5, in fermented liquid, the molecular weight of the corresponding chemical substance in this peak and S-equol standard substance is in full accord, can conclude that thus the spectrum peak occurring in the time of 9.5 minutes in its fermented liquid of the constructed engineering bacteria of the present invention is exactly S-equol.Further illustrate the constructed engineering bacteria of the present invention and there is the ability that Dai is converted into Equol.

The application that embodiment 3 milk-acid bacteria source S-equols produce engineering bacteria

1.S-Equol produces the preparation of engineering bacteria

Seed culture:

50 μ l engineering bacterias being preserved to liquid from-80 ℃ of refrigerators is inoculated in the brain heart infusion liquid nutrient medium that 5ml contains Pyocianil (50 μ g/ml) and Streptomycin sulphate (50 μ g/ml) (BHI), be placed in anaerobism workstation, 37 ℃ of standing overnight incubation, obtain seed liquor, for inoculation.

2. ferment with substratum and fermentation condition

(1) using Dai as substrate, add in BHI substratum, Dai is 0.05g/L at BHI substratum final concentration, then the seed liquor that the inoculum size of volumetric concentration 5% of take obtains step 1 is inoculated (liquid amount is 30ml), at 37 ℃, in anaerobism workstation, cultivate 24h, the IPTG aqueous solution that adds 5 μ L (account for nutrient solution volume 0.1%) 25mg/ml, 37 ℃ are continued after cultivation 24,48h, 72h and 96h, adopt the method for embodiment 2 steps 3 to carry out HPLC detection.

(2) under similarity condition, take Dai as substrate, take LB substratum as nutrient solution, the final concentration of Dai in LB substratum is 0.05g/L, the same step of culture condition and detection method (1).LB substratum final concentration forms: Tryptones 10g/L, yeast extract 5.0g/L, sodium-chlor 10g/L, and solvent is water, pH value is 7.0.

(3) under similarity condition, take dregs of beans as substrate, take BHI substratum as nutrient solution, the final concentration of dregs of beans in BHI substratum is 15g/L, the same step of culture condition and detection method (1).

(4) culture condition of step (1)-(3) is changed into respectively under aerobic condition, 37 ℃ of common constant incubators are cultivated the corresponding time.

3. in engineering bacterium fermentation liquid, the HPLC of S-equol content detects

Fermentation results shows, no matter the constructed engineering bacteria of the present invention is under aerobic or anaerobic condition, all the Dai in BHI substratum and LB substratum can be converted into S-equol (Fig. 6).As can be seen from Figure 7, the constructed engineering bacteria of the present invention also has the ability (Fig. 7) of directly utilizing dregs of beans to transform production S-equol.

Claims (6)

1. a S-equol produces engineering bacteria, it is characterized in that described engineering bacteria be by L-DDRC, the L-DZNR, L-DHDR and the L-THDR gene clone that derive from milk-acid bacteria (Lactococcus sp.) 20-92 to e. coli bl21 (D3), transform and to obtain S-equol and produce engineering bacteria; The nucleotides sequence of described L-DDRC is classified as shown in SEQ ID NO:1, the nucleotides sequence of L-DZNR is classified as shown in SEQ ID NO:2, the nucleotides sequence of L-DHDR is classified as shown in SEQ ID NO:3, and the nucleotides sequence of L-THDR is classified as shown in SEQ ID NO:4.

2. described in a claim 1, S-equol produces the application of engineering bacteria in preparing S-equol, it is characterized in that described being applied as: take Dai or dregs of beans as substrate, S-equol is produced to seed liquor that engineering bacteria obtains through enlarged culturing and with the inoculum size of volumetric concentration 5%, be seeded to BHI substratum or LB substratum, under aerobic or anaerobic condition, cultivate 24h for 37 ℃, to the IPTG that adds final concentration 25mg/L in nutrient solution, 37 ℃ are continued to cultivate after 6～72h, get nutrient solution separation and purification, obtain S-equol; Described BHI substratum final concentration consists of: peptone 10g/L, dehydration calf brain soak powder 12.5g/L, dehydration beef heart infusion 5.0g/L, sodium-chlor 5.0g/L, glucose 2.0g/L, Sodium phosphate dibasic 2.5g/L, and solvent is water, and pH value is 7.0; Described LB substratum final concentration forms: Tryptones 10g/L, yeast extract 5.0g/L, sodium-chlor 10g/L, and solvent is water, pH value is 7.0.

3. S-equol produces the application of engineering bacteria in preparing S-equol as claimed in claim 2, and the starting point concentration that it is characterized in that described Dai is 50 μ g/ml.

4. S-equol produces the application of engineering bacteria in preparing S-equol as claimed in claim 2, and the starting point concentration that it is characterized in that described dregs of beans is 15g/L.

5. S-equol produces the application of engineering bacteria in preparing S-equol as claimed in claim 2, application described in it is characterized in that is carried out as follows: (1) seed culture: S-equol is produced to engineering bacteria and be seeded in the BHI substratum that contains final concentration 50 μ g/ml Pyocianils and final concentration 50 μ g/ml Streptomycin sulphates, 37 ℃ standing, anaerobism is cultivated after 24h, obtains seed liquor; (2) fermentation culture and conversion reaction: Dai or dregs of beans are added to BHI substratum or LB substratum, then with the inoculum size of volumetric concentration 5%, inoculate seed liquor, under aerobic or anaerobic condition, cultivate 24h for 37 ℃, to the IPTG that adds final concentration 25mg/L in nutrient solution, 37 ℃ are continued to cultivate after 6～72h, get nutrient solution separation and purification, obtain S-equol; The starting point concentration of described Dai is 50 μ g/ml, and the starting point concentration of described dregs of beans is 15g/L.

6. application as claimed in claim 2, is characterized in that the method for described nutrient solution separation and purification is: get the nutrient solution of 1ml, centrifugal 3 minutes of 8000r/min, obtains supernatant a; Get 900 μ l supernatant a to another clean 2ml EP pipe, then in pipe, add 900 μ l ethyl acetate, fully mix rear standing 5min, centrifugal 5 minutes of 5000r/min, obtains supernatant b and precipitation b; Get 900 μ l supernatant b to another clean 2ml EP pipe; Get precipitation b and add the ethyl acetate repeated centrifugation 1 time of equivalent, obtain supernatant c; Supernatant b and supernatant c are mixed, move on in 2ml centrifuge tube, 45 ℃ of vacuum freezings are condensed into powder; Add 200 μ l anhydrous methanols to dissolve concentrated powder to this EP pipe, and through 0.22 μ m polyvinyladine floride filtering with microporous membrane, filtrate is S-equol.