CN104805064B - High temperature resistant flavone aglycone invertase and its application - Google Patents
High temperature resistant flavone aglycone invertase and its application Download PDFInfo
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Abstract
High temperature resistant flavone aglycone invertase and its application, amino acid sequence is as shown in SEQ ID NO.1.High temperature resistant flavone aglycone conversion enzyme heat stability provided by the invention is good, resistant against high temperatures, all there is stronger catalyzed conversion ability to the flavone glycoside of different parent nucleus types and different glycosidic bond types, high temperature resistant flavone aglycone invertase provided by the invention is incubated after certain time through detection with typical flavone glycoside (isoquercitin, astragalin, daidzin, the wooden glycosides of dye), and flavone glycoside is almost completely converted into corresponding flavone aglycone (Quercetin, Kaempferol, daidzein and dye lignin).
Description
Technical field
The invention belongs to technique for gene engineering and biomedicine field, and in particular to a kind of high temperature resistant flavone aglycone invertase
And its a variety of flavone sugar glycosides compounds of application, especially enzymatic conversion method generate corresponding flavone aglycone.
Background technology
Flavone compound is a kind of pharmacological component being widely present in plant, the flavonoids of different types of structure
Compound is because the difference of the species and of bonding of its parent nucleus and connected glycosyl shows different physiologically actives and function, at present
Have more than the separated identification of 1500 kinds of different types of flavone compounds.Flavone compound in nature is mostly with its sugar
Base form is present.Research shows that the glycoside forms of Flavonoid substances are significantly better than its aglycon existence form in terms of solubility,
There is larger advantage in oral and absorption aspects.But energy is absorbed through cell membrane to it just because of its stronger hydrophily
Power brings strong influence, and its bioavilability is on the contrary not as good as corresponding flavone aglycone existence form, while flavone aglycone is for example
Quercetin is in anti-diabetic, anti-oxidant and suppression AChE（Acetylcholinesterase）Etc. pharmacological activity than its corresponding flavones
Glucosides (rutin and isoquercitin) has some superiority.Therefore, according to same flavone compound in the specific of different field
Purpose difference simultaneously considers its bioavilability and its pharmacological activity, selects different glucosides modified forms (retain glycosyl, go
Retain except part glycosyl or only flavone aglycone), have important practical significance.
At present, it is glycosylation engineered to flavone compound progress generally to have chemical method and bioanalysis.Use mostly both at home and abroad
Chemical method hydrolyzes flavone glycoside.Flavone glycoside glycosidic bond under acid and alkalescence condition can be broken, and be decomposed into aglycon and glycosyl,
Therefore people use sour means of hydrolysis and prepare flavone aglycone at present.However, not only reaction condition is violent for sour water solution, flavonoid glycoside is influenceed
The stability of member, and the fracture to glycosidic bond is without selectivity.Compared with chemical method, bioanalysis has selectivity strong, anti-
Answer mild condition, separating-purifying easily etc. outstanding feature.
For both at home and abroad conversion flavone glycoside relevant report we have found that：Using rutin as representative, when nineteen fifty-nine,
Westlake etc. [3], which screens several fungies, to be includedAspergillus flavus,A.niger,penicilliumSp.,pseudomonasSp. the beta-glucosidase of hydrolyzing rutin can be produced；Afterwards, Narikawa etc. fromPenicillium rugulosumIn IFO 7242 purifying obtain molecular weight be about 24.5kDa can only be using rutin or isoquercitin as substrate
Beta-glucosidase；Hendson etc. fromPseudomonas viridiflavaF62L clone to obtain can with the β of hydrolyzing rutin-
Glucuroide.In addition, in plantFagopyrum tataricumAndFagopyrum esculentumIn also purify
The beta-glucosidase for being capable of hydrolyzing rutin is arrived.But these beta-glucosidase catalytic efficiencies are preferable not enough, bar is catalyzed
Part is not met by the needs of some practical applications.Therefore it is considered that current bioanalysis conversion flavone glycoside generation flavonoid glycoside
Member still suffers from some limitations in the cost of transformation efficiency and its prepare with scale.
And these problems depend primarily on the catalysis characteristics and the aspect factor of enzyme protein expression amount two of enzyme.Accordingly, we recognize
To screen the flavone glycoside hydrolase and its gene suitable for a variety of flavones parent nucleus and different glycosidic bond types from molecular level, gram
Grand and high efficient expression glycosidase genes, suitable living things catalysis and the technique of conversion are established, will be expected to can effectively solve aglycon Huang
The technical bottleneck of ketone living things catalysis.The present invention obtains a kind of high temperature resistant flavone aglycone turn by screening corresponding Thermophilic Bacteria genome
Change enzyme SiBGL and to its codon optimize improve its encode mRNA structures stability reach high efficient expression, while this kind
High temperature resistant flavone aglycone invertase SiBGL has to different flavones parent nucleus and the glucosides of bonding of different connection form widely to be urged
Change conversion capability, this allows this kind of high temperature resistant flavone aglycone invertase to be widely used in the conversions of a variety of flavone glycosides.
In addition, the enzyme has higher glucose tolerance, and it is larger that its high temperature resistant and excellent heat endurance also make it that it has
Industrial applications potentiality.
The content of the invention
The technical problem of solution：The invention provides a kind of high temperature resistant flavone aglycone invertase and its application, and pass through base
Because engineering technology obtains efficient and single-minded catalytic performance high temperature resistant flavone aglycone invertase SiBGL, recombinase SiBGL is to more
Kind flavone sugar glycosides compound all has stronger catalytic capability, while is resistant to the glucose of higher concentration, can reduce to enzyme
The feedback inhibition of vigor.
Technical scheme：A kind of high temperature resistant flavone aglycone invertase, amino acid sequence is as shown in SEQ ID NO.1.
The nucleotides of the high temperature resistant flavone aglycone invertase is encoded, nucleotide sequence is as shown in SEQ ID NO.2.
The preparation method of the high temperature resistant flavone aglycone invertase, the nucleotides inserted shown in SEQ ID NO.2 is expressed
Carrier obtains recombinant plasmid, by recombinant plasmid transformed Host Strains, purifies and obtains through its induced expression and destination protein.
The preparation of high temperature resistant flavone aglycone invertase comprises the following steps that：
1）, basisSulfolobus islandicusGenome interior coding high temperature resistant flavone aglycone invertase SiBGL genes
For template, the encoding gene is optimized according to e. coli codon Preference, improves the steady of encoding gene mRNA structures
It is qualitative, the nucleotides with the sense primer with the nucleotide sequence shown in SEQ ID NO.3 and shown in SEQ ID NO.4
Sequence downstream primer expands, and PCR expands to obtain high temperature resistant flavone aglycone invertase SiBGL DNA fragmentation SEQ ID NO.2；
2）, by obtained high temperature resistant flavone aglycone invertase DNA fragmentation and vector plasmid pET-20b respectively with Nde I and
Xho I carry out double digestion, and connection obtains the recombinant expression plasmid containing high temperature resistant flavone aglycone invertase gene；
3）, by step 2）Obtained recombinant expression plasmid translation table reaches Host Strains JM109 (DE3), adds derivant in 30
High temperature resistant flavone aglycone conversion expression of enzymes is induced at DEG C, thalline is collected by centrifugation, through Ni after broken thalline2+Affinity column purifies
Obtain high temperature resistant flavone aglycone invertase.
Include the recombinant plasmid for the DNA fragmentation for encoding the high temperature resistant flavone aglycone invertase.
Application of the high temperature resistant flavone aglycone invertase in flavone aglycone is prepared.
The specific method of application is that high temperature resistant flavone aglycone invertase is different in pH 4-8,40 DEG C -95 DEG C of temperature, enzymolysis
Corresponding flavone aglycone is prepared in species flavone glycoside.
The pH is preferably 5.5, and temperature is preferably 90 DEG C.
The flavone glycoside is flavonols glucosides or osajin glucosides.
The flavonols glucosides is isoquercitin or astragalin；The osajin glucosides is daidzin or dye wood
Glycosides.
Beneficial effect：（1）High temperature resistant flavone aglycone conversion enzyme heat stability of the present invention is good, resistant against high temperatures；
（2）Flavones of the high temperature resistant flavone aglycone invertase of the present invention to different parent nucleus types and different glycosidic bond types
Glucosides all has stronger catalyzed conversion ability, high temperature resistant flavone aglycone invertase of the present invention and typical flavone glycoside
(isoquercitin, astragalin, daidzin, the wooden glycosides of dye) is almost completely converted into after being incubated certain time through detection, flavone glycoside
Corresponding flavone aglycone (Quercetin, Kaempferol, daidzein and dye lignin).
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is the required accompanying drawing used in technology description to be briefly described.
Fig. 1 is that high temperature resistant flavone aglycone of the present invention converts enzymatic conversion typical flavone glycoside (isoquercitin, Chinese milk vetch
Glycosides, daidzin, the wooden glycosides of dye) the corresponding flavone aglycone of generation (Quercetin, Kaempferol, daidzein and dye lignin) hydrolysis circuit
Figure.
Fig. 2 is the Purity result figure that embodiment 2 purifies high temperature resistant flavone aglycone invertase；Wherein swimming lane M is albumen
Marker (is purchased from Thermo scientific companies, article No. 2661), and swimming lane 1 is that PET-20b converts Host Strains blank control
Full cell pyrolysis liquid；Swimming lane 2 is full cell pyrolysis liquid swimming lane after induced expression；3 be 85 DEG C heat treatment 15 minutes after crude enzyme liquid swim
Road；4 be high temperature resistant flavone aglycone invertase pure enzyme protein.
Fig. 3 is the qualitative determination result figure of the high temperature resistant flavone aglycone invertase of the present invention of embodiment 3, and wherein a is most
Suitable reaction pH measurement result figure, abscissa pH, ordinate are enzyme activity, unit %；B is the survey of optimal reactive temperature
Determine result figure, abscissa is temperature, degrees Celsius（℃）, ordinate is enzyme activity, unit %.
Fig. 4 show the monose rejection coefficient Ki measure knots of the high temperature resistant flavone aglycone invertase of the present invention of embodiment 4
Fruit is schemed.Abscissa is the addition of glucose in reaction system, and unit mM, ordinate is enzyme activity, unit %.
Fig. 5 A are that high temperature resistant flavone aglycone invertase SiBGL hydrolyzes generation Quercetin to isoquercitin under the differential responses time
Change of component situation carry out HPLC analysis result figures；
Fig. 5 B are that high temperature resistant flavone aglycone invertase SiBGL hydrolyzes generation Kaempferol to astragalin under the differential responses time
Change of component situation carry out HPLC analysis result figures；
Fig. 5 C are that high temperature resistant flavone aglycone invertase SiBGL hydrolyzes generation daidzein to daidzin under the differential responses time
Change of component situation carry out HPLC analysis result figures；
Fig. 5 D are that high temperature resistant flavone aglycone invertase SiBGL contaminates lignin to contaminating wooden glycosides enzyme hydrolysis generation under the differential responses time
Change of component situation carry out HPLC analysis result figures.
Embodiment
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described,
Obviously, described embodiment is only part of the embodiment of the present invention, rather than whole embodiments.Based on the reality in the present invention
Example is applied, the every other embodiment that those of ordinary skill in the art are obtained under the premise of creative work is not made, is all belonged to
In the scope of protection of the invention.
The invention provides a kind of high temperature resistant flavone aglycone invertase, its amino acid sequence is as shown in SEQ ID NO.1, life
Entitled SiBGL.
Present invention provides the genetic fragment for encoding high temperature resistant flavone aglycone invertase SiBGL of the present invention.Due to
The degeneracy of codon, there may be a variety of nucleotides that can encode high temperature resistant flavone aglycone invertase of the present invention
Sequence (as shown in SEQ ID NO.2).
In some embodiments, the invention provides described coding high temperature resistant flavone aglycone invertase SiBGL base
Because of fragment, its nucleotide sequence is as shown in SEQ ID NO.2.
In order to prepare zymoprotein of the present invention, the system of high temperature resistant flavone aglycone invertase of the present invention is additionally provided
Preparation Method.
In some embodiments, high temperature resistant flavone aglycone invertase SiBGL of the present invention preparation method：Obtain
The genetic fragment of coding high temperature resistant flavone aglycone invertase of the present invention, DNA fragmentation insertion expression vector is weighed
Group plasmid, by recombinant plasmid transformed expressive host bacterium, under suitable inducer concentrations at 30 DEG C induced expression, isolate and purify i.e.
.
The present invention additionally provides a kind of comprising coding high temperature resistant flavone aglycone invertase gene piece of the present invention simultaneously
The recombinant plasmid of section, described recombinant plasmid is pET-SiBGL.
Translation table of the present invention up to Host Strains be E. coli expression strains, including Rosetta series and BL21,
JM109 series bacterial strains.In a preferred embodiment, host cell is JM109 (DE3) bacterial strain.
Induced expression described in the preparation method of high temperature resistant flavone aglycone invertase of the present invention isolate and purify specially in
Lower expressive host bacterium of 30 DEG C of Fiber differentiations containing recombinant plasmid of suitable derivant (IPTG) concentration, collects thalline ultrasonic disruption,
Supernatant affinity chromatography is taken to obtain fusion protein.
Present invention also offers a kind of method for preparing flavone aglycone, high temperature resistant flavone aglycone specially of the present invention turns
Change enzyme digested respectively under the conditions of lower 5.5,95 DEG C of pH 4 kinds of typical flavone glycosides (isoquercitin, astragalin, daidzin,
Contaminate wooden glycosides) respectively hydrolysis prepares corresponding flavone aglycone (Quercetin, Kaempferol, daidzein and contaminate lignin).
High temperature resistant flavone aglycone invertase SiBGL of the present invention efficiently can connect with parent nucleus and different type glycosidic bond
The flavone sugar glycosides compound connect, is efficiently prepared corresponding flavone aglycone.
For a further understanding of the present invention, with reference to embodiment, the present invention will be described in detail, wherein, such as without special
Illustrate, the various reaction reagents being related in embodiment can be commercially available by commercial channel；Unless otherwise specified, embodiment
In the concrete operations that are related to referring to《The Molecular Cloning:A Laboratory guide third edition》.
Embodiment 1：The acquisition of high temperature resistant flavone aglycone invertase gene of the present invention and recombinant plasmid pET-SiBGL's
Structure
High temperature resistant flavone aglycone invertase SiBGL gene codons optimize and recombinant plasmid pET-SiBGL structure
According to knownSulfolobus islandicusWhole genome sequence（Accession number：NC_012588.1）Screen
To coding high temperature resistant flavone aglycone invertase SiBGL of the present invention genetic fragment, due to this kind of Thermophilic Bacterium codon
Preference and expressive host e. coli bl21 (DE3) there is different, it is possible to this kind extremely heat-resisting flavone aglycone turn
The high efficient expression of hdac protein impacts, therefore our coding bases according to the codon preferences of Host Strains to destination protein
Because carrying out codon optimization, while optimize coding high temperature resistant flavone aglycone invertase SiBGL mRNA structures, it is stable to improve it
Property, the coding high temperature resistant flavone aglycone invertase SiBGL genetic fragments after optimization are by Nanjing Genscript Biotechnology Co., Ltd.
Synthesis：Nucleotides with the sense primer with the nucleotide sequence shown in SEQ ID NO.3 and shown in SEQ ID NO.4
Sequence downstream primer expands, and PCR expands to obtain high temperature resistant flavone aglycone invertase SiBGL DNA fragmentation SEQ ID NO.2；Will
Obtained high temperature resistant flavone aglycone invertase DNA fragmentation and vector plasmid pET-20b carries out double enzymes with Nde I and Xho I respectively
Cut, connection obtains the recombinant expression plasmid pET-SiBGL containing high temperature resistant flavone aglycone invertase gene.
Embodiment 2：High temperature resistant flavone aglycone invertase SiBGL of the present invention preparation
Recombinant plasmid pET-SiBGL is converted into e. coli jm109 (DE3) Host Strains（Purchased from Novagen companies）,
Contain kanamycins（50 μg/mL）LB flat boards（LB culture mediums：The g/L of tryptone 10, yeast extract 5 g/L, NaCl
5 g/L, the g/L of agar 15）It is upper to pass through 37 DEG C of overnight incubations, transformant is chosen into 200 mL LB culture mediums（50 μ g/mL cards
That mycin）37 DEG C, when 200 rpm shaken cultivations to OD600 are 0.6, add the thio pyrroles of final concentration of 0.5 mM isopropyl ss-D-
Mutter galactoside（IPTG）Derivant, 30 DEG C culture 6 h, with high speed freezing centrifuge by nutrient solution at 4 DEG C, with 13,000
Rpm centrifuges 15 min, collects thalline.
Due to containing His-tag labels in recombinant plasmid pET-SiBGL, pass through His Bind Purification Kit
（Purchased from Novagen companies）Purified, the recombinase purified.Specific operation process：
A. the processing of sample
（1）By washed thalline, it is resuspended with 1 × Binding Buffer 8mL, supersonic wave wall breaking.
（2）After broken wall, 13,000 g centrifuge 30 min, and it is sample to take supernatant.
（3）Because this kind of albumen has preferable heat endurance, therefore can (after being incubated 15min at 70 DEG C) by heat treatment
Most of foreign protein is denatured to form infusible precipitate, 13,000 g centrifuge 30 min, and it is the sample after being heat-treated to take supernatant.
B. pillar is handled
（1）1 mL fillers are taken to fill post.
（2）With 3mL sterile washing pillar.
（3）Pillar is washed with 5mL 1 × Charge Buffer.
（4）Pillar is washed with 3mL 1 × Binding Buffer.
C. loading
（1）Sample is added into pillar, coutroi velocity 6 drop per minute.
（2）Pillar is washed with 31 × Binding of mL Buffer, removes uncombined protein.
（3）Pillar is washed with the 4 mL eluents for containing 20 mM imidazoles, except foreigh protein removing.
（4）Pillar is washed with the eluent of 80 mmol/L imidazoles, destination protein is eluted.
（5）Pillar is washed with 41 × Strip of mL Buffer.
The high temperature resistant flavone aglycone invertase purified by this process, identified by SDS-PAGE electrophoresis poststaining resistance to
High temperature flavone aglycone invertase SiBGL purity, as a result as shown in Figure 2.
From Fig. 2 results, SiBGL genes expression quantity in Host Strains JM109 (DE3) is higher, is eliminated after heat treatment
Part foreign protein, afterwards destination protein by HisTag labels after purification, high temperature resistant flavone aglycone invertase in its eluent
SiBGL purity is higher, there is single band at 55kDa, reaches the pure rank of electrophoresis.
Embodiment 3：High temperature resistant flavone aglycone invertase SiBGL of the present invention qualitative determination
1st, the assay method of enzyme activity
The 20 mmol/L p-nitrophenyl α-D- arabinofuranosidase glucosides of μ L, 5 μ L of reaction system 100（pNPG）Middle addition
The mmol/L citrate-phosphate disodium hydrogen buffer solutions of 85 μ L 100（pH 6.0）, first 90oC is incubated 3 min, adds 10 μ
L enzyme liquids（It is diluted to suitable multiple）10 min are reacted, the μ L of sodium carbonate liquor 600 that 1 mol/L is added after colour developing are terminated instead
Should.Light absorption value is determined under 405 nm.Enzyme activity unit (U) is defined as：Under condition determination, 1 μm of ol p- of generation per minute
Enzyme amount required for nitrophenol is 1 enzyme activity unit.
2nd, optimal reaction pH measure
In different pH（3.0-7.5,100 mmol/L citrate-phosphate disodium hydrogen buffer solutions）Under the conditions of, 90 DEG C of difference
Enzyme activity is determined, as a result as shown in Figure 3 a.
From Fig. 3 results, the optimal reaction pH of high temperature resistant flavone aglycone invertase of the present invention is 5.5, and
Remain to show higher enzyme activity between pH5-7, so this kind of enzyme reaction advantageous pH range is wider.
3rd, the measure of optimal reactive temperature
In the range of 60-100 DEG C, every 5 DEG C, enzyme activity is determined respectively.Buffer as 100 mmol/L citrate-phosphates hydrogen two
Sodium buffer solution, pH 6.0, as a result as shown in Figure 3 b.
From Fig. 3 results, the optimal reactive temperature of high temperature resistant flavone aglycone invertase of the present invention is 95 DEG C, and should
Kind enzyme still has higher enzyme activity between 80-100 DEG C.
Embodiment 4：High temperature resistant flavone aglycone invertase of the present invention determines to arabinose and glucose tolerance.
Assay method：In identical reaction system (100 μ L, 10mM pNPG, 50mM pH5.5 citrate-phosphate hydrogen
Disodium buffer solution;) in add glucose to different final concentrations, it is yellow that high temperature resistant of the present invention is determined under optimum reaction conditionses
Ketoside member invertase activity, as a result as shown in Figure 4.
Reactions of the high temperature resistant flavone aglycone invertase SiBGL of the present invention in glucose final concentration 500mM as shown in Figure 4
In system, there is nearly 50% residual enzyme activity, its Ki coefficient is 500mM, and the high temperature resistant flavone aglycone invertase is in reaction system
When between the middle final concentration of 0-150mM of glucose, there is obvious activation.
Embodiment 5：High temperature resistant flavone aglycone invertase SiBGL of the present invention converts 4 kinds of typical flavone glycoside (different Mongolian oaks
Pi Su, astragalin, daidzin, the wooden glycosides of dye) respectively hydrolysis prepare corresponding flavone aglycone (Quercetin, Kaempferol, daidzein
With dye lignin)
Above-mentioned flavonoid standard items are purchased from Chengdu Man Site bio tech ltd.
HPLC testing conditions are:Agilent 1260 Infinity；DAD detectors Detection wavelength is 203nm, and column temperature is
30 DEG C, flow rate of mobile phase is 1.0mL/min (A:1% formic acid water, B:Methanol；0min, A:B is 50:50；10min, A:B is 30:
70；12min, A:B is 50:50；15min, A:B is 50:50.)
1. enzymatic conversion isoquercitin, astragalin generation Quercetin, Kaempferol.
Enzymatic conversion reaction system is 100 μ L, and wherein isoquercitin, astragalin concentration is respectively 2g/L, and enzyme addition is
0.7U/mL, react and carried out in the case where 5.5,95 DEG C of pH, composition detection is carried out using HPLC to the sample of differential responses time respectively.
As the testing result shown in Fig. 5 A, 5B：It is that detection has isoquercitin and astragalin hydrolysis generation Quercetin after 10min is reacted
And Kaempferol, and as the extension in reaction time, conversion ratio improve.After 70min and 60min is reacted, isoquercitin and purple cloud
English glycosides is almost completely converted to Quercetin and Kaempferol, yield 92%.
2. enzymatic conversion daidzin and the wooden glycosides generation daidzein of dye and dye lignin.
Enzymatic conversion reaction system is 100 μ L, and wherein daidzin, the wooden glycosides concentration of dye are respectively 2g/L, and enzyme addition is
0.35U/mL, react and carried out in the case where 5.5,95 DEG C of pH, the sample of differential responses time is carried out into go-on-go using HPLC respectively
Survey.It is that detection has daidzein and dye lignin generation, and prolonging with the reaction time after 10min is reacted as shown in Fig. 5 C, 5D
Long, conversion ratio gradually steps up.After 60min is reacted, daidzin and the wooden glycosides of dye are almost completely converted into daidzein and dye lignin,
Yield is 95%.
SEQUENCE LISTING
<110>Nanjing Forestry University
Guangyuan Bel's natural bioactivity substance engineering and technological research institute
Sichuan Jin Beier Eco Science Technologies development corporation, Ltd.
<120>High temperature resistant flavone aglycone invertase and its application
<130>
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 489
<212> PRT
<213>Artificial sequence
<400> 1
Met Tyr Ser Phe Pro Lys Asn Phe Arg Phe Gly Trp Ser Gln Ala Gly
1 5 10 15
Phe Gln Ser Glu Met Gly Thr Pro Gly Ser Glu Asp Pro Asn Thr Asp
20 25 30
Trp Tyr Lys Trp Val His Asp Pro Glu Asn Ile Ala Ala Gly Leu Val
35 40 45
Ser Gly Asp Leu Pro Glu Asn Gly Pro Gly Tyr Trp Gly Asn Tyr Lys
50 55 60
Thr Phe His Asp Asn Ala Gln Lys Met Gly Leu Lys Met Ala Arg Leu
65 70 75 80
Asn Val Glu Trp Ser Arg Ile Phe Pro Asn Pro Leu Pro Lys Pro Gln
85 90 95
Asn Phe Asp Glu Ser Lys Gln Asp Val Thr Glu Val Glu Ile Asn Gln
100 105 110
Asn Glu Leu Arg Arg Leu Asp Glu His Ala Asn Lys Asp Ala Leu Asn
115 120 125
His Tyr Arg Glu Ile Phe Lys Asp Leu Lys Ser Arg Gly Ile Tyr Phe
130 135 140
Ile Leu Asn Met Tyr His Trp Pro Leu Pro Ser Trp Leu His Asp Pro
145 150 155 160
Ile Arg Val Arg Arg Gly Asp Leu Ser Gly Pro Thr Gly Trp Leu Ser
165 170 175
Thr Arg Thr Val Tyr Glu Phe Ala Arg Phe Ser Ala Tyr Ile Ala Trp
180 185 190
Lys Phe Asp Asp Leu Val Asp Glu Tyr Ser Thr Met Asn Glu Pro Asn
195 200 205
Val Val Gly Gly Leu Gly Tyr Val Gly Val Lys Ser Gly Phe Pro Pro
210 215 220
Gly Tyr Leu Ser Phe Glu Leu Ser Arg Lys Ala Met Tyr Asn Ile Ile
225 230 235 240
Gln Ala His Val Arg Ala Tyr Asp Gly Ile Lys Ser Val Ser Lys Lys
245 250 255
Pro Ile Gly Ile Ile Tyr Ala Asn Ser Ser Phe Gln Pro Leu Thr Glu
260 265 270
Lys Asp Met Glu Ala Val Glu Met Ala Glu Tyr Asp Asn Arg Trp Ala
275 280 285
Phe Phe Asp Ala Ile Ile Arg Gly Glu Ile Met Lys Gly Ser Glu Lys
290 295 300
Val Val Arg Asp Asp Leu Arg Gly Arg Leu Asp Trp Ile Gly Val Asn
305 310 315 320
Tyr Tyr Thr Arg Thr Val Val Lys Lys Thr Glu Lys Gly Tyr Val Ser
325 330 335
Leu Gly Gly Tyr Gly His Gly Cys Glu Arg Asn Ser Val Ser Leu Ala
340 345 350
Gly Leu Pro Thr Ser Asp Phe Gly Trp Glu Phe Phe Pro Glu Gly Leu
355 360 365
Tyr Asp Val Leu Thr Lys Tyr Trp Asn Arg Tyr His Leu His Met Tyr
370 375 380
Val Thr Glu Asn Gly Ile Ala Asp Asp Ala Asp Tyr Gln Arg Pro Tyr
385 390 395 400
Tyr Leu Val Ser His Val Tyr Gln Val His Arg Ala Ile Asn Ser Ser
405 410 415
Ala Asp Val Arg Gly Tyr Leu His Trp Ser Leu Ala Asp Asn Tyr Glu
420 425 430
Trp Ala Ser Gly Phe Ser Met Arg Phe Gly Leu Leu Lys Val Asp Tyr
435 440 445
Gly Thr Lys Arg Leu Tyr Trp Arg Pro Ser Ala Leu Val Tyr Arg Glu
450 455 460
Ile Ala Thr Asn Gly Gly Ile Thr Asp Glu Ile Glu His Leu Asn Ser
465 470 475 480
Val Pro Pro Ile Arg Pro Leu Arg His
485
<210> 2
<211> 1470
<212> DNA
<213>Artificial sequence
<400> 2
atgtattcgt tcccgaaaaa cttccgcttt ggttggtcgc aggcaggctt ccagtctgaa 60
atgggcacgc cgggctctga agatccgaat accgattggt ataaatgggt gcatgacccg 120
gaaaacatcg cggccggcct ggttagcggt gatctgccgg aaaacggccc gggttattgg 180
ggtaattaca aaacgtttca cgacaacgca cagaaaatgg gcctgaaaat ggcccgtctg 240
aatgttgaat ggtcacgcat ttttccgaat ccgctgccga aaccgcagaa cttcgatgaa 300
tcgaaacaag acgtcaccga agtggaaatc aaccagaatg aactgcgtcg cctggatgaa 360
catgcaaaca aagacgctct gaatcactac cgtgaaatct tcaaagatct gaaaagccgc 420
ggtatttatt tcatcctgaa catgtaccat tggccgctgc cgtcttggct gcacgatccg 480
atccgtgtcc gtcgcggtga cctgagcggt ccgaccggtt ggctgtctac ccgtacggtg 540
tatgaatttg cgcgcttcag tgcatacatt gcttggaaat ttgatgacct ggtggatgaa 600
tattccacga tgaacgaacc gaatgtggtt ggcggtctgg gctatgtggg tgttaaatca 660
ggctttccgc cgggttacct gtcattcgaa ctgtcgcgta aagcgatgta taatattatc 720
caggcgcatg ttcgcgccta cgatggtatt aaatcagtct cgaaaaaacc gatcggcatt 780
atctatgcca atagctcttt ccaaccgctg accgaaaaag atatggaagc agttgaaatg 840
gctgaatacg ataaccgttg ggcgtttttc gacgccatta tccgcggcga aattatgaaa 900
ggttctgaaa aagtcgtgcg tgatgacctg cgtggccgcc tggattggat cggtgtgaat 960
tattacaccc gcacggttgt gaagaaaacc gaaaaaggtt atgtcagtct gggcggttac 1020
ggccatggtt gcgaacgcaa cagcgtgtct ctggcaggtc tgccgacgtc cgattttggt 1080
tgggaatttt tcccggaagg cctgtatgac gttctgacca aatattggaa tcgttaccat 1140
ctgcacatgt acgtcacgga aaacggtatc gcggatgacg ccgattatca gcgcccgtat 1200
tacctggttt cacatgtcta ccaagtgcac cgtgcaatta atagttccgc tgatgttcgc 1260
ggctatctgc actggtcgct ggcggacaac tacgaatggg ccagtggctt ttccatgcgt 1320
ttcggtctgc tgaaagtgga ttatggtacc aaacgtctgt actggcgccc gagcgcactg 1380
gtttatcgcg aaattgctac gaacggcggt atcaccgatg aaattgaaca cctgaactcc 1440
gtgccgccga ttcgcccgct gcgccattaa 1470
<210> 3
<211> 25
<212> DNA
<213>Artificial sequence
<400> 3
catatgtatt cgttcccgaa aaact 25
<210> 4
<211> 24
<212> DNA
<213>Artificial sequence
<400> 4
ctcgagatgg cgcagcgggc gaat 24
Claims (1)
1. the high temperature resistant flavone aglycone invertase of nucleotide coding the answering in flavone aglycone is prepared as shown in SEQ ID NO.2
With, it is characterised in that high temperature resistant flavone aglycone invertase digests variety classes flavone sugar under conditions of pH 5.5,90 DEG C of temperature
Corresponding flavone aglycone is prepared in glycosides, and the flavone glycoside is daidzin or the wooden glycosides of dye in osajin glucosides.
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| TWI580784B (en) * | 2016-01-13 | 2017-05-01 | 國立臺南大學 | Method of manufacturing methoxy-isoflavones by biotransformation and use thereof |
| CN107964556B (en) * | 2018-01-09 | 2021-05-07 | 皖西学院 | Method for producing astragalin by converting kaempferol through microorganisms |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1174839A (en) * | 1996-06-11 | 1998-03-04 | 蛋白质技术国际公司 | Recovery of isoflavones from soy molasses |
| CN1395573A (en) * | 2000-01-11 | 2003-02-05 | 比奥雷克斯健康有限公司 | Extraction of flavonoids |
| CN102093326A (en) * | 2010-12-22 | 2011-06-15 | 晨光生物科技集团股份有限公司 | Method for extracting and refining ginkgo flavone from ginkgo leaves |
| CN104328098A (en) * | 2014-09-28 | 2015-02-04 | 江苏康缘药业股份有限公司 | [Beta]-glucosidase, preparation method and application thereof |
-
2015
- 2015-02-10 CN CN201510070692.1A patent/CN104805064B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1174839A (en) * | 1996-06-11 | 1998-03-04 | 蛋白质技术国际公司 | Recovery of isoflavones from soy molasses |
| CN1395573A (en) * | 2000-01-11 | 2003-02-05 | 比奥雷克斯健康有限公司 | Extraction of flavonoids |
| CN102093326A (en) * | 2010-12-22 | 2011-06-15 | 晨光生物科技集团股份有限公司 | Method for extracting and refining ginkgo flavone from ginkgo leaves |
| CN104328098A (en) * | 2014-09-28 | 2015-02-04 | 江苏康缘药业股份有限公司 | [Beta]-glucosidase, preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| beta-galactosidase [Sulfolobus islandicus];NCBI;《GenBank》;20130526;Accesion WP_012712281 * |
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| CN104805064A (en) | 2015-07-29 |
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