CN1944634A - Hydrolyzed soy bean isoflavone glycosidase engineering strain, its construction method and its use - Google Patents
Hydrolyzed soy bean isoflavone glycosidase engineering strain, its construction method and its use Download PDFInfo
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- CN1944634A CN1944634A CN 200610045994 CN200610045994A CN1944634A CN 1944634 A CN1944634 A CN 1944634A CN 200610045994 CN200610045994 CN 200610045994 CN 200610045994 A CN200610045994 A CN 200610045994A CN 1944634 A CN1944634 A CN 1944634A
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Abstract
The present invention belongs to the field of enzyme genetic engineering technology, and is especially hydrolyzed soybean isoflavone glycosidase engineering strain and its construction method and use in producing hydrolyzed soybean isoflavone glycoside. The present invention obtains high activity hydrolyzed soybean isoflavone glycosidase engineering strain through culturing lactic acid bacteria containing beta-glucosidase, extracting glucosidase genome DNA, PCR proliferation, sequencing, subcloning the gene to T vector, final cloning to expression vector and expressing in colibacillus. The obtained engineering bacteria have high enzyme activity, low cost, obvious hydrolysis effect on soybean isoflavone glycoside and high safety, are used in functional food and possess wide application foreground.
Description
Technical field
The invention belongs to the gene engineering technology field of enzyme, particularly relate to hydrolyzed soy bean isoflavone glycosidase engineering strain and construction process thereof, and this bacterial strain is applied in the production of hydrolyzed soy bean isoflavone glucosides.
Background technology
Along with the raising of people's living standard, the enhancing of health care consciousness, the soybean isoflavones product more and more is subject to people's attention.The soybean isoflavones that contains in the soybean is a class important physical active substance, has stronger physiological function.Up to now, the isoflavones in the known soybean has 12 kinds of isomer, and wherein 3 kinds is the form of aglycon, and 9 kinds is the form of glucoside.Soybean isoflavones content in soybean only has about 5/1000ths, and 97%-98% exists with mating type soybean isoflavone glucoside form in the soybean isoflavones isomer, the 2%-3% of the aglycon form soybean isoflavones total amount of free type, but human body does not generally absorb the glucosides form of soybean isoflavones.Soybean isoflavone glucoside is hydrolyzed into the aglycon form under people's digestive ferment (special beta-glucosidase) effect, just can be absorbed.Studies have shown that the physiologically active of soybean isoflavones mainly is the activity of isoflavone genin.The hydrolysis of soybean isoflavone glucoside also becomes researcher primary study present situation.Soybean isoflavone glucoside glycosidic link under acidity or alkaline condition can rupture, and is decomposed into isoflavone genin and glucose, but alkaline condition hydrolysis gained isoflavone genin is very unstable, easily degraded.Therefore, people use acid hydrolysis means system isoflavone genin more at present.Acid hydrolysis mainly is a hydrochloric acid with acid.Adopt denseer hydrochloric acid (as 1-3molL more
-1), the acid-hydrolyzed efficient of higher temperature (98-100 ℃) is very high, has a lot of people to doubt for the stability of isoflavone genin under the acidic conditions.In addition, be difficult for carrying out industrialization under the strong acid condition, but also can bring industrial pollution.The enzymatic hydrolysis glucosides has just become the important means of glycoside hydrolysis, the enzyme hydrolysis condition gentleness, adopt weakly acidic buffered soln, isoflavone genin is volatility not more, is the very promising approach of the industrial preparation soybean isoflavone health food that is rich in isoflavone genin.Studying maximum soybean isoflavone glucoside hydrolases is exactly beta-glucosidase, and the endogenous beta-glucosidase hydrolytic activity that soybean self contains is not strong, and hydrolysis efficiency needs only 22%-29%, and the high active enzyme that adds capacity can make hydrolysis reach 100%.In fact discover,, just can make full use of soybean isoflavone glucoside as long as the hydrolysis degree of glucosides reaches 70%.Because the digestive ferment energy hydrolyzable moiety glucosides of minority is arranged in the human body.Along with development of science and technology, the enhancing of people's health care consciousness, the target that cost is low, hydrolysis effect good, safe lytic enzyme has become research.But most glycoside hydrolase beta-glucosidase comes from aspergillus class and the yeast now, though have very high hydrolysis efficiency, has potential safety hazard.
Summary of the invention
The present invention seeks to utilize genetic engineering means to make up a kind of new engineering strain,, can solve the safety problem of active hydrolysis enzyme source again in the hope of can the effectively hydrolyzing soybean isoflavone glucoside.
Another object of the present invention provides the construction process of this bacterial strain.
Another purpose of the present invention provides the purposes of this bacterial strain.
The representative strain colon bacillus of above-mentioned engineering bacteria (Escherichia coli) has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), deposit number: CGMCCNo.1619, preservation day: on February 16th, 2006.
Problem to be solved by this invention is exactly to select a strain to carry out zymologic property research from the milk-acid bacteria that produces beta-glucosidase, extract the genomic dna of this bacterial strain beta-glucosidase then, and with this gene subclone to pET 28a (+) carrier, in intestinal bacteria, realize at last efficiently expressing.
Engineering bacteria feature of the present invention is that it is as follows that the karyomit(e) of this bacterial strain comprises effective kinds of protein beta-glucosidase gene group dna sequence dna table:
1 atgactgaca ttaccacggc cggctctttg gaaagtccgc gttatctgac ttacactttg
61 gatggcaagg atggcaaagt agctgggatg ttcgccagct ccttgccaaa gggcgctaag
121 gggaagattt ttgacaatga atactacccg aaccatgtag cgatcgattt ttaccatcac
181 tacaaggaag acatcaagat gttcgcggac atgggcttta aggtattcag aacttccatt
241 gcctggacgc ggattttccc aaccggtgaa gaagacaagc caaaccagga agggctggac
301 ttctaccgca gagtctttga agaattgaag aagaacggga ttgaaccatt agttacgatt
361 tcccactatg aagacccatt ggctttaggc gaaaagtaca acgactggca agaccgcaag
421 atgattgacc tctacgttaa gtacgcgacg accttgttca aggaatacaa ggacctggtt
481 aagtactggc tgaccttcaa cgaaatcaac tcatctttga tgttcttgaa gctggtgggt
541 gatggcaagg tatctgatgc agattaccaa aaggcttacc aaaagctgca ccaccaattc
601 gttgcttccg ctaaggcagt tgttgcaggc cacaagatca acccagactt catgatcggg
661 aacatgattg ccggttctgt ttactaccca ggcactcctg atccaaagga tgctttggct
721 gctcgctatg aagaagaatt gagccagctt tactgtgctg acgcgcaagc taagggtgag
781 tatccaagtt ttgccaagcg cttatgggat gaacacaatg ttcatttgaa gattgaagat
841 ggcgaccttg aagtcatgaa ggaaggtaaa gttgacatgt acaccttctc atactacatg
901 tcaaacatgg ttaccaccca tgatgttggc gaaaaggcta agggcaactt cgctgccggc
961 gctaagaacc catatcttga atactctgaa tggggctggt caactgaccc agacggcttg
1021 caactgtact tggaaaagat gtatgaccgt tatggcatcc caatgatggt ggtggaaaat
1081 ggtcttggtg ccgttgataa gctagaagat ggtactgttc atgacgatta ccggattgac
1141 tacttgagaa agcacatcaa ggcgatggac aaggcagttg aacatggggt tgacctgcgt
1201 gcctacacga cttggggctg cattgactgc gtttctgctg gaactggtca aatgtccaag
1261 cggtatggct tcatctacgt tgaccgtgat gacaagggcg aaggtacgct taagcgcctg
1321 cctaaggatt catactactg gtaccaaaag gttatcgctt caaacggcga tgaattataa
The aminoacid sequence table of the enzyme that beta-glucosidase gene group dna sequence dna is derived
1MTDITTAGSLESPRYLTYTLDGKDGKVAGMFASSLPKGAKGKIFDNEYYPNHVAIDFYHH
61YKEDIKMFADMGFKVFRTSIAWTRIFPTGEEDKPNQEGLDFYRRVFEELKKNGIEPLVTI
121SHYEDPLALGEKYNDWQDRKMIDLYVKYATTLFKEYKDLVKYWLTFNEINSSLMFLKLGD
181GKVSDADYQKAYQKLHHQFVASAKAVVAGHKINPDFMIGNMIAGSVYYPGTPDPKDALAA
241RYEEELSQLYCADAQAKGEYPSFAKRLWDEHNVHLKIEDGDLEVMKEGKVDMYTFSYYMS
301NMVTTHDVGEKAKGNFAAGAKNPYLEYSEWGWSTDPDGLQLYLEKMYDRYGIPMMVVENG
361LGAVDKLEDGTVHDDYRIDYLRKHIKAMDKAVEHGVDLRAYTTWGCIDCVSAGTGQMSKR
421YGFIYVDRDDKGEGTLKRLPKDSYYWYQKVIASNGDEL
The construction process of a kind of high reactivity hydrolysis soy bean isoflavone glycosidase engineering strain provided by the invention, realize having following steps by the following technical solutions:
At first be to select the milk-acid bacteria that produces beta-glucosidase for use;
Second step: be amplification bgl A gene (beta-glucosidase gene) from the milk-acid bacteria of selecting;
The 3rd step: be according to requirement of experiment design primer;
The 4th step: from the milk-acid bacteria that provides, extract genomic dna;
The 5th step: with the genomic dna is template, and BglA F/BglA R is a primer, uses TaKaRa LA Taq
TM(CodeNo.DRR002A) carry out pcr amplification purpose fragment.Re-use TaKaRa Agarose Gel DNA Purification KitVer.2.0 (Code No.DV805A) and cut glue recovery pcr amplified fragment, called after CTA480-I;
The 6th step: use the Ligation Mix among the TaKaRa DNA Ligation Kit (Code No.D6023), with CTA480-I with after pMD18-T Simple carrier (Code No.D103A) is connected, thermal transition to E.coli CompetentCells JM109 (Code No.D9052), spread plate incubated overnight thalline.Choosing colony, behind the extraction plasmid, name CTA480-T-1, CTA480-T-2.
The 7th step: expression vector pET28a (+) is carried out double digestion with EcoR I/HindIII, use TaKaRa Agarose GelDNA Purification Kit Ver.2.0 (Code No.DV805A) to cut glue and reclaim carrier part DNA, called after VectorDNA;
The 8th step: the CTA480-T-1 plasmid that will contain the BglA goal gene carries out double digestion with EcoR I/HindIII, use TaKaRa Agarose Gel DNA Purification Kit Ver.2.0 (Code No.DV805A) to cut the dna fragmentation that glue reclaims about 1.4kb, called after Insert DNA;
The 9th step: use the Ligation Mix among the TaKaRa DNA Ligation Kit (Code No.D6023), with Vector DNA with after Insert DNA is connected, thermal transition to E.coli Competent Cell JM109 (Code No.D9052), spread plate incubated overnight thalline.Choosing colony, behind the extraction plasmid, plasmid name CTA480-P-1 and CTA480-P-2;
The tenth step: plasmid CTA480-P-1 and CTA480-P-2 are carried out enzyme respectively with EcoR I/HindIII cut evaluation, agarose gel electrophoresis, plasmid CTA480-P-2 meets the requirements.
The 11 step: get CTA480-P-2 respectively and pET28a (+) plasmid 100ng goes in 100ul BL21 (DE3) competent cell, 100ul is coated with flat board, and picking list colony inoculation is being led cultivation, the fragmentation of collection bacterium, and SDS-PAGE detects;
The 12 step: purifying beta-glucosidase liquid, measure enzyme and live.
Through enzyme activity determination, the enzyme work of new bacterial strain is 10 times that former bacterial strain enzyme is lived.This bacterial strain is applied to the hydrolyzed soy bean isoflavone glucosides, and the glycoside hydrolysis rate can reach more than 80%, and hydrolysis effect obtains very big improvement.
According to the resulting engineering bacteria of aforesaid method, bacterial strain is rod-short at microscopically.
The present invention selects for use the milk-acid bacteria that contains beta-glucosidase to cultivate, extract the genomic dna of glucuroide and it is carried out pcr amplification, order-checking, this gene subclone to the T carrier, be cloned into expression vector at last, in intestinal bacteria, express, obtain high reactivity hydrolysis soy bean isoflavone glycosidase engineering strain.The engineering bacteria enzyme that the present invention obtains is lived high, and price is low, is applied to the hydrolyzed soy bean isoflavone glucosides, the improvement that hydrolysis effect obtains, bacterium source has good security simultaneously,, is with a wide range of applications and economic implications in functional foodstuff is used as engineering strain hydrolysis glucosides.
Description of drawings
Fig. 1 extracts genomic dna agarose gel electrophoresis figure from milk-acid bacteria; Numeral and letter among the figure are represented respectively:
M：DNA Marker DL2,000
1: genomic dna
Fig. 2 is CTA480-I agarose gel electrophoresis figure; Numeral and letter among the figure are represented respectively:
M：DNA Marker DL2,000
1：CTA480-I
Fig. 3 is plasmid agarose gel electrophoresis figure; Numeral and letter among the figure are represented respectively:
The 1:CTA480-T-1 plasmid
The 2:CTA480-T-2 plasmid
M：λ-Hind III DNA Marker
Fig. 4 is Vector DNA agarose gel electrophoresis figure; Numeral and letter among the figure are represented respectively:
M：λ-Hind III DNA Marker
1：Vector DNA
Fig. 5 is the building process of plasmid CTA480-T-1, CTA480-T-2;
Fig. 6 is Insert DNA agarose gel electrophoresis figure; Numeral and letter among the figure are represented respectively:
M：DNA Marker DL2,000
1：Insert DNA
Fig. 7 is plasmid agarose gel electrophoresis figure; Numeral and letter among the figure are represented respectively:
M：λ-Hind III DNA Marker
1：CTA480-P-1
2：CTA480-P-2
Fig. 8 is plasmid CTA480-P-1 and CTA480-P-2 building process; Numeral and letter among the figure are represented respectively:
Fig. 9 is that plasmid CTA480-P-1 and CTA480-P-2 enzyme are cut evaluation agarose gel electrophoresis figure; Numeral and letter among the figure are represented respectively:
M1：λ-Hind III DNA Marker
1:CTA480-P-1 EcoR I/HindIII cuts
2:CTA480-P-2 EcoR I/HindIII cuts
M2：DNA Marker DL2,000
Figure 10 is the goal gene solubility expression; Numeral and letter among the figure are represented respectively:
M
1：protein MW marker(High)
The full cell of 1:pET28a (+)
2:pET28a (+) supernatant
3:pET28a (+) precipitation
The full cell of 4:CTA480-P-2
The 5:CTA480-P-2 supernatant
The 6:CTA480-P-2 precipitation
M
2: protein MW marker (Low) 0.1OD quite goes up sample.
Figure 11 is the survey alive of purifying and enzyme, M1, and M2 Mark standard protein, the numeral among the figure is represented respectively:
1: the beta-glucosidase standard substance
2: the beta-glucosidase after the expression through the DEAE purifying
3: through the secondarily purified beta-glucosidase of dextran G-200
Figure 12 is 40% soybean isoflavone glucoside high performance liquid phase figure;
Figure 13 is 40% soybean isoflavone glucoside hydrolysis high performance liquid phase figure;
Figure 14 is 40% soybean isoflavone glucoside and hydrolysis high performance liquid phase coincidence design sketch.
Embodiment
Below in conjunction with accompanying drawing, to foundation embodiment provided by the invention, feature and effect, describe in detail as the back: amplification bgl A gene from the high-yield beta-glucosidase milk-acid bacteria that filters out, and with this gene subclone to pET 28a (+) carrier (carrier is a pET series), and in intestinal bacteria, realize to express.
1. design and synthesize following primer according to requirement of experiment:
BglA F： 5′GAATTCATGACTGACATTACCACGGC 3′ (26mer)
BglA R： 5′AAGCTTTTACGCTTTAATTTTATAA 3′ (26mer)
CTA480 T-R：5′TTAGCCTTTTCGCCAACATC 3′ (20mer)
BglA f1： 5′TTTGGCTGCTCGCTATGAAG 3′ (20mer)
BglA r1： 5′CTTCATAGCGAGCAGCCAAA 3′ (20mer)
BglA r2： 5′AATCATCTTGCGGTCTTGCC 3′ (20mer)
2. use DNAiso (DNA extraction reagent) from milk-acid bacteria, to extract genomic dna.Get 1 μ l and carry out agarose gel electrophoresis, the result is shown in Figure 1;
3. be template with the genomic dna, BglA F/BglA R is a primer, uses TaKaRa LA Taq
TM(CodeNo.DRR002A) carry out pcr amplification purpose fragment.Re-use TaKaRa Agarose Gel DNA Purification KitVer.2.0 (Code No.DV805A) and cut glue recovery pcr amplified fragment, called after CTA480-I.Get 1 μ l and carry out agarose gel electrophoresis, the result as shown in Figure 2;
4. CTA480-I is checked order with BglA R/BglA f1/BglA r1/BglA r2 primer.
5. use the Ligation Mix among the TaKaRa DNA Ligation Kit (Code No.D6023), with CTA480-I with after pMD18-T Simple carrier (Code No.D103A) is connected, thermal transition to E.coli Competent Cells JM109 (Code No.D9052), spread plate incubated overnight thalline.
6. choosing colony behind the extraction plasmid, is got 1 μ l and is carried out agarose gel electrophoresis, and the result is illustrated in fig. 3 shown below:
7. choose plasmid CTA480-T-1, CTA480-T-2, building process such as Fig. 5 are that primer carries out dna sequencing with BcaBEST Primer M13-47/BcaBEST Primer RV-M/CTA480T-R, and plasmid CTA480-T-1 meets the requirements.
8. expression vector pET28a (+) is carried out double digestion with EcoR I/HindIII, use TaKaRa Agarose Gel DNAPurification Kit Ver.2.0 (Code No.DV805A) to cut glue and reclaim carrier part DNA, called after Vector DNA.Get 1 μ l and carry out agarose gel electrophoresis, the result as shown in Figure 4:
9. the CTA480-T-1 plasmid that will contain the BglA goal gene carries out double digestion with EcoR I/HindIII, use TaKaRaAgarose Gel DNA Purification Kit Ver.2.0 (Code No.DV805A) to cut the dna fragmentation that glue reclaims about 1.4kb, called after Insert DNA.Get 1 μ l and carry out agarose gel electrophoresis, the result as shown in Figure 6:
10. use the Ligation Mix among the TaKaRa DNA Ligation Kit (Code No.D6023), with VectorDNA with after Insert DNA is connected, thermal transition to E.coli Competent Cell JM109 (Code No.D9052), spread plate incubated overnight thalline.
11. choosing colony behind the extraction plasmid, is got 1 μ l and carried out agarose gel electrophoresis, the result is shown in Figure 7:
Cut evaluation 12. plasmid CTA480-P-1 and CTA480-P-2 (building process such as Fig. 8) are carried out enzyme respectively with EcoR I/HindIII, the agarose gel electrophoresis result as shown in Figure 9;
13. get CTA480-P-2 respectively and pET28a (+) plasmid 100ng goes in 100ul BL21 (DE3) competent cell, 100ul is coated with flat board.
The competent cell that transforms is carried out blue hickie screening, the individual white single colony inoculation of picking 4-6 contains LB nutrient solution (concrete composition 1g Tryptones, 1g sodium-chlor, the 0.5g yeast extract powder of the plain 50ug/ml of that enzyme of card at random, add 100ml distilled water, PH=7.2), 37 ℃ of concussions of spending the night are cultivated; The ratio of activatory kind daughter bacteria with 5%-8% is inoculated in the LB substratum 37 ℃ of enlarged culturing.Be cultured to logarithmic phase (OD
600Be 0.6-0.8) to add IPTG be 1mmol/L to final concentration, inducing culture 2-4 hour, the centrifugal 10-15min of 6000-8000r/min collected thalline.Sampling adds 1 * SDS sample-loading buffer, carries out SDS-PAGE according to ordinary method and detects.Goal gene solubility expression such as Figure 10, detected result is as follows:
| Induce preceding absorbancy | Absorbancy before the collection bacterium | The PBS addition | |
| pET28a(+) | 0.736 | 1.053 | 210ul |
| CTA480-P-2 | 0.610 | 1.725 | 350ul |
Experimental result finds to have in the thalline a large amount of albumen to exist, and the strain construction success is described.
14. carry out purifying and enzyme activity determination
Living through the enzyme of the new bacterial strain that makes up of enzyme activity determination is 34U, is 10 times that former bacterial strain enzyme is lived, and enzyme biopsy survey method is as follows:
A. adopting Genistoside is the enzyme activity determination of substrate:
With Genistoside is substrate, genistein is the relative enzyme activity determination method of product: the substrate solution of getting 0.1ml (is used 0.02mol/L, it is 15mg/ml that the pH5.0HAVc-NaAc damping fluid is made into concentration with Genistoside), the crude enzyme liquid that adds 0.1ml, mixing, behind the reaction appropriate time, add the ethyl acetate extraction 1h of 2 times of volumes under 40 ℃, get thing 10ul and make thin-layer chromatography (TLC).Thin layer chromatography board needs before use at 110 ℃ of activation 30-60min, and developping agent is a chloroform: butanone: methyl alcohol: water (10: 7: 1: 1), launch about 6cm, volatilize solvent, with the scanning of dual-wavelength lamellar scanning instrument, calculate enzyme activity according to the integrated value of speck area.The definition of enzyme activity unit is: under the said determination condition, the enzyme amount that per hour discharges the 1nmol genistein is an enzyme activity unit (U).
B. with pNPG the enzyme activity determination of substrate
Get the pNPG (using 0.02mol/L, the acetate buffer solution preparation of Ph5.0) of 0.4ml 1mM, in 40 ℃ of following preheating 5min, add the enzyme liquid of 0.1ml, reaction 30min to the NaCO3 solution of reaction solution adding 2.5ml 1mol/L, surveys the OD value down in the 405nm wavelength.Enzyme activity is defined as under these conditions, and per hour to produce the enzyme amount of 1umol p-NP be an enzyme activity unit to hydrolysis substrate.
C. with Ginsenoside Rd the enzyme activity determination of substrate
Get the substrate solution of 0.1ml and (use 0.02mol/L, pH5.0 HAc-NaAc damping fluid is made into concentration 10mg/ml with Rd), the enzyme liquid that adds 0.1ml, mixing, after reacting appropriate time under 40 ℃, in reaction system, add the water-saturated n-butanol extraction 1h of 2 times of volumes, get extraction phase 10ul and make thin-layer chromatography (TLC).Thin layer chromatography board needs before use 110 ℃ of activation 30-60 minute, and developping agent is a chloroform: methyl alcohol: water (70: 30: 5), launch about 6cm, and volatilize solvent, spray 10% sulfuric acid heating colour developing.With the scanning of dual-wavelength lamellar scanning instrument, calculate enzyme activity according to the integrated value of speck area.The enzyme activity unit definition is under the said determination condition, per hour discharges the enzyme amount of 1nmol ginsenoside product, is defined as an enzyme activity unit.
D. with the salicin enzyme activity determination of substrate
Get 1.0% salicin of 1.0ml 0.05mol/L pH5.0 acetate buffer solution preparation, add the 1.0ml enzyme liquid of purifying, 50 ℃ of reaction 30min, 100 ℃ of water-bath 5min behind the adding 2.5mlDNS reagent, use the deionized water constant volume to 5.0ml behind the cool to room temperature, measure OD530, with every milliliter of enzyme solution, enzymolysis salicin generation 1mg glucose is an enzyme activity unit in the 30min under these conditions.
Hydrolysis glucosides compliance test result as shown in figure 11
Extract the soybean isoflavones product, content is approximately 40%.Carry out enzymatic hydrolysis experiment: according to 1: 1 volume mixture, is that concentration of substrate is 10mg/ml with enzyme liquid and substrate solution, mixing, and 50 ℃ of shaking baths reaction 1.5h, the hexyl acetate with 2 times of volumes extracts then, and extract concentrates evaporate to dryness, detects with HPLC.Hydrolysis result is seen Figure 12,13,14.
The hydrolysis glucosides found that soybean isoflavone glucoside is not having before the hydrolysis, and glucosides content accounts for more than 90% of total glucosides, the content of sweet unit seldom, but the content of aglycon accounts for more than 80% of total glucosides, the hydrolysis requirement to glucosides that reaches after hydrolysis.
Claims (4)
1, a kind of hydrolyzed soy bean isoflavone glucosides engineering bacteria is characterized in that: the deposit number of bacterial strain colon bacillus (Escherichia coli): CGMCCNo.1619.
2, a kind of hydrolyzed soy bean isoflavone glucosides engineering bacteria as claimed in claim 1 is characterized in that: it is as follows that the karyomit(e) of this bacterial strain contains effective kinds of protein beta-glucosidase gene group dna sequence dna table:
1 atgactgaca ttaccacggc cggctctttg gaaagtccgc gttatctgac ttacactttg
61 gatggcaagg atggcaaagt agctgggatg ttcgccagct ccttgccaaa gggcgctaag
121 gggaagattt ttgacaatga atactacccg aaccatgtag cgatcgattt ttaccatcac
181 tacaaggaag acatcaagat gttcgcggac atgggcttta aggtattcag aacttccatt
241 gcctggacgc ggattttccc aaccggtgaa gaagacaagc caaaccagga agggctggac
301 ttctaccgca gagtctttga agaattgaag aagaacggga ttgaaccatt agttacgatt
361 tcccactatg aagacccatt ggctttaggc gaaaagtaca acgactggca agaccgcaag
421 atgattgacc tctacgttaa gtacgcgacg accttgttca aggaatacaa ggacctggtt
481 aagtactggc tgaccttcaa cgaaatcaac tcatctttga tgttcttgaa gctggtgggt
541 gatggcaagg tatctgatgc agattaccaa aaggcttacc aaaagctgca ccaccaattc
601 gttgcttccg ctaaggcagt tgttgcaggc cacaagatca acccagactt catgatcggg
661 aacatgattg ccggttctgt ttactaccca ggcactcctg atccaaagga tgctttggct
721 gctcgctatg aagaagaatt gagccagctt tactgtgctg acgcgcaagc taagggtgag
781 tatccaagtt ttgccaagcg cttatgggat gaacacaatg ttcatttgaa gattgaagat
841 ggcgaccttg aagtcatgaa ggaaggtaaa gttgacatgt acaccttctc atactacatg
901 tcaaacatgg ttaccaccca tgatgttggc gaaaaggcta agggcaactt cgctgccggc
961 gctaagaacc catatcttga atactctgaa tggggctggt caactgaccc agacggcttg
1021 caactgtact tggaaaagat gtatgaccgt tatggcatcc caatgatggt ggtggaaaat
1081 ggtcttggtg ccgttgataa gctagaagat ggtactgttc atgacgatta ccggattgac
1141 tacttgagaa agcacatcaa ggcgatggac aaggcagttg aacatggggt tgacctgcgt
1201 gcctacacga cttggggctg cattgactgc gtttctgctg gaactggtca aatgtccaag
1261 cggtatggct tcatctacgt tgaccgtgat gacaagggcg aaggtacgct taagcgcctg
1321 cctaaggatt catactactg gtaccaaaag gttatcgctt caaacggcga tgaattataa。
3, the construction process of the described engineering strain of claim 1, it is characterized in that: this method comprises the steps: to extract the genomic dna of this bacterial strain beta-glucosidase, and with this gene as goal gene, by subclone behind the pcr amplification to the T carrier, be cloned among the coli expression carrier at last, obtaining the purpose bacterial strain.
4, the purposes of the described engineering strain of claim 1 is characterized in that: this bacterial strain is used for the hydrolyzed soy bean isoflavone glucosides.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102382788A (en) * | 2010-09-01 | 2012-03-21 | 中国农业大学 | Lactic acid bacterium containing isoflavone synthetase (IFS)-cytochromes P450 reductase (CPR) fusion gene and application thereof |
| CN103140588A (en) * | 2010-07-12 | 2013-06-05 | 吉利亚尼股份公司 | Fermented soya based mixture comprising isoflavones- aglicones, equol and lunasil, process for the preparation and uses thereof in food, medical and cosmetic fields |
| CN103789331B (en) * | 2013-04-26 | 2015-08-26 | 中山大学 | A kind of beta-glucosidase gene of effectively hydrolyzing soybean isoflavone glucoside and application |
| CN113005157A (en) * | 2021-03-29 | 2021-06-22 | 吉林大学 | Preparation method of soybean isoflavone aglycone |
| CN113558193A (en) * | 2021-06-24 | 2021-10-29 | 苏州大学 | Preparation method of fermented beverage rich in soybean isoflavone aglycone |
-
2006
- 2006-03-08 CN CN 200610045994 patent/CN1944634A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103140588A (en) * | 2010-07-12 | 2013-06-05 | 吉利亚尼股份公司 | Fermented soya based mixture comprising isoflavones- aglicones, equol and lunasil, process for the preparation and uses thereof in food, medical and cosmetic fields |
| CN103140588B (en) * | 2010-07-12 | 2015-09-30 | 吉利亚尼股份公司 | Comprise the mixture based on fermented soybean, its preparation method and the related application at food, medicine and cosmetic field thereof of isoflavones-aglycon, Equol and Lu Na element (lunasin) |
| CN102382788A (en) * | 2010-09-01 | 2012-03-21 | 中国农业大学 | Lactic acid bacterium containing isoflavone synthetase (IFS)-cytochromes P450 reductase (CPR) fusion gene and application thereof |
| CN102382788B (en) * | 2010-09-01 | 2014-07-09 | 中国农业大学 | Lactic acid bacterium containing isoflavone synthetase (IFS)-cytochromes P450 reductase (CPR) fusion gene and application thereof |
| CN103789331B (en) * | 2013-04-26 | 2015-08-26 | 中山大学 | A kind of beta-glucosidase gene of effectively hydrolyzing soybean isoflavone glucoside and application |
| CN113005157A (en) * | 2021-03-29 | 2021-06-22 | 吉林大学 | Preparation method of soybean isoflavone aglycone |
| CN113558193A (en) * | 2021-06-24 | 2021-10-29 | 苏州大学 | Preparation method of fermented beverage rich in soybean isoflavone aglycone |
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