CN110256550A - A kind of recombinant spider silk protein fibrous material and preparation method thereof for the extraction of uranium from seawater - Google Patents
A kind of recombinant spider silk protein fibrous material and preparation method thereof for the extraction of uranium from seawater Download PDFInfo
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- CN110256550A CN110256550A CN201910580843.6A CN201910580843A CN110256550A CN 110256550 A CN110256550 A CN 110256550A CN 201910580843 A CN201910580843 A CN 201910580843A CN 110256550 A CN110256550 A CN 110256550A
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Abstract
The invention discloses a kind of preparation methods of recombinant spider silk protein fibrous material for the extraction of uranium from seawater, comprising the following steps: spider silk protein gene segment, extraction of uranium from seawater functional protein genetic fragment and flexible connection gene recombination are obtained extraction of uranium from seawater recombinant spider silk protein encoding gene；After be transferred to Escherichia coli, through protein expression, purifying obtain extraction of uranium from seawater recombinant spider silk protein liquid；Concentration makes its concentration 100-300mg/mL, the extraction of uranium from seawater recombinant spider silk protein liquid after concentration is promoted spinning solution, self assembly obtains extraction of uranium from seawater recombinant spider silk protein fiber；The pH of the spinning solution is 3-5.This method simple process, mild condition are easy to purify, and the extraction of uranium from seawater recombinant spider silk protein fibrous material prepared by this method, excellent in mechanical performance, and uranium absorption ability is strong and selectivity is high, can repeatedly use.
Description
Technical field
The present invention relates to field of material preparation more particularly to a kind of recombinant spider silk protein fibrous materials for the extraction of uranium from seawater
And preparation method thereof.
Background technique
The alpha-helix of beta sheet and Pro-rich that the spider's thread is usually made of alanine, and its second level knot closely piled up
Structure makes a kind of molecule spring structure.Due to having a wide range of applications in numerous areas latent with very high mechanical strength
Power, but due to natural spider silk limits throughput, seriously constrain the industrial application of spider silk.Artificial spider's thread protein fiber is solution
One of the important method of certainly natural spider's thread limits throughput, through the technology of genetic engineering, in microorganism, plant or even animal
Spider's thread protein is expressed, and is expected to realize the extensive use of the spider's thread by artificial means weaving spider's thread fiber, but due to lacking
To spider's thread protein Quality Research, early bird makes spider's thread technology and fails to be developed well, a kind of bionical until 2017
The exploitation of spider's thread protein spining technology, makes it possible large scale preparation spider's thread protein fiber.Nevertheless, at present to the spider's thread
The technology of preparing of azelon also rests on the level of pure spider's thread spinning, not yet uses the spider's thread and functional protein amalgamation and expression and makes
The research of standby functional protein fiber, can especially apply and be rarely reported in the spider's thread protein fiber in metal adsorption field.
And uranium is the basis of nuclear power industry development, uranium resource is mainly obtained from the uranium ore of land, however China is depleted uranium
Country, the new acquisition modes for developing uranium resource have great significance to the development of China's nuclear power industry, and the uranium in seawater
Resource reserve is 1000 times that there are resource reserve on land, resourceful.It is reported according to existing literature, mentioning uranium material at present is mostly base
In the organic polymer fibrous material of amidoxim functional group, but these materials are complicated there is synthesis technology or to propose uranium ability lower
The problems such as, and since URANIUM IN SEAWATER concentration is extremely low (3.3 μ g/L), interfering ion leads to poor selectivity more, and industrialization is also not implemented
Using.Therefore, exploitation is of great significance for extracting the recombinant spider silk protein fiber of URANIUM IN SEAWATER acyl metal ion.
Summary of the invention
In view of this, being solved existing the present invention provides a kind of recombinant spider silk protein SSUP fiber for the extraction of uranium from seawater
The limited problem with ion selectivity difference of uranium effect is inhaled with the presence of uranium material is mentioned.
One aspect of the present invention provides a kind of preparation method of recombinant spider silk protein fibrous material for the extraction of uranium from seawater, including
Following steps:
(1) genetic recombination: by spider silk protein gene segment, extraction of uranium from seawater functional protein genetic fragment and flexible linking group
Because obtaining extraction of uranium from seawater recombinant spider silk protein encoding gene by chemical synthesis；
(2) gene expression: mentioning uranium spider's thread protein encoding gene for extraction of uranium from seawater recombination and be transferred to Escherichia coli, trains in LB liquid
Base overnight incubation is supported, is inoculated with and expands culture, then inducing expression culture afterwards, last thalline were collected by centrifugation；
(3) purifying protein: use buffer washing thalline, after cracked with lysate, supernatant is collected by centrifugation and goes forward side by side
Row purifying, successively obtains extraction of uranium from seawater recombinant spider silk protein with gradient elution；
(4) self assembly spinning: making its concentration 100-300mg/mL for extraction of uranium from seawater recombinant spider silk protein centrifugal concentrating, with
Extraction of uranium from seawater recombinant spider silk protein liquid after concentration is promoted spinning solution by certain fltting speed, and self assembly obtains extraction of uranium from seawater recombination
Spider's thread protein fiber；The pH of the spinning solution is 3-5.
Preferably, in step (1), the nucleotide series such as SEQ of the extraction of uranium from seawater recombinant spider silk protein encoding gene
Shown in No.4.
Preferably, in step (2), the lysate is 8M Urea, 100mM NaH2PO4With 100mM Tris.
Preferably, in step (3), shown in the amino acid series table SEQ No.5 of the extraction of uranium from seawater recombinant spider silk protein.
Preferably, in step (3), the concentration of the gradient eluent is respectively the urine of 6M, 4M, 2M, 1M, 0.5M and 0M
Element, glycerol of the eluent containing 10% mass can get high purity protein.
Preferably, in step (4), the spinning solution includes 100mM sodium phosphate, 100mM sodium acetate and 200mM citric acid.
Preferably, in step (4), fltting speed 0.6mL/h, the pH of spinning solution is 4.
Preferably, selecting syringe needle is that the 1mL syringe of 24G is promoted.
Using a kind of preparation method of extraction of uranium from seawater recombinant spider silk protein SSUP fibrous material provided by the present invention, pass through
Chimeric recombination obtains extraction of uranium from seawater recombinant spider silk protein gene, after induced in Bacillus coli expression and generate the extraction of uranium from seawater and recombinate the spider's thread
Albumen, by self assembly spinning after purification, simple process, mild condition is easy to purify, reaction it is controllable and can magnanimity prepare, at
This is low.A kind of new approaches not only are provided for artificial spinning, but also mentions uranium for natural sea-water and provides a kind of high-efficiency adsorbent.
Another aspect of the present invention also provides a kind of recombinant spider silk protein fibrous material for the extraction of uranium from seawater, and tensile strength can
Up to 128.26MPa, up to 2.4 times are stretched, is resistant to complicated marine environment damage；Further, this extraction of uranium from seawater recombinates spider's thread egg
It is hydrogel spline structure under white fiber material moisture state, is tight structure under drying regime, is mentioned for the entrance and combination of uranyl
Intermolecular space abundant and binding force are supplied, extractability is up to 12.33mg/g in 3.5 days in natural sea-water, to seawater
Uranium ion has strong adsorption capacity；Selectivity height (significantly larger than maximum rival-vanadium ion), responds fast (5min) and can
It repeatedly uses, long service life.
Detailed description of the invention
Fig. 1 is the recombination schematic diagram of the extraction of uranium from seawater recombinant spider silk protein gene of the embodiment of the present invention one；
Fig. 2 is the electrophoretogram of the extraction of uranium from seawater recombinant spider silk protein SSUP of the embodiment of the present invention one；
Fig. 3 is the circular dichroism spectrogram of the extraction of uranium from seawater recombinant spider silk protein SSUP of the embodiment of the present invention one；
Fig. 4 is pattern of the extraction of uranium from seawater recombinant spider silk protein SSUP fiber of the embodiment of the present invention one under moisture state
Figure；
Fig. 5 is the SEM figure of the extraction of uranium from seawater recombinant spider silk protein SSUP fiber of the embodiment of the present invention one in the dry state；
Fig. 6 is spinning effect of the extraction of uranium from seawater recombinant spider silk protein of the embodiment of the present invention one in different pH spinning solutions；
Fig. 7 be the embodiment of the present invention one extraction of uranium from seawater recombinant spider silk protein SSUP fiber different uranium concentrations (2,4,8,
The adsorption effect figure in simulated seawater 16ppm)；
Fig. 8 is that the extraction of uranium from seawater recombinant spider silk protein SSUP fiber of the embodiment of the present invention one is adsorbed in simulated seawater-taken off
The repeating effect figure of uranium；
Fig. 9 is that the extraction of uranium from seawater recombinant spider silk protein SSUP fiber of the embodiment of the present invention one is containing different metal ions
Selective absorption effect picture in natural sea-water；
Figure 10 is absorption of the extraction of uranium from seawater recombinant spider silk protein SSUP fiber of the embodiment of the present invention one in natural sea-water
Effect picture.
Specific embodiment
The principles and features of the present invention are described below, and illustrated embodiment is served only for explaining the present invention, is not intended to
It limits the scope of the invention.
Spider silk protein gene segment TuSp2-RP (SEQ No.1) and functional protein genetic fragment (SEQ No.3) are by giving birth to
Work bioengineering (Shanghai) limited liability company is synthesized by conventional method, and this will not be repeated here.
A kind of embodiment one: recombinant spider silk protein fibrous material for the extraction of uranium from seawater, comprising the following steps:
(1) genetic recombination: such as schematic diagram 1, spider silk protein gene segment TuSp2-RP (SEQ No.1) and the extraction of uranium from seawater
Functional protein genetic fragment (SEQ No.3) is closed by flexible connection gene Linker (SEQ No.2) using existing conventional method
At acquisition extraction of uranium from seawater recombinant spider silk protein SSUP encoding gene (SEQ No.4)；
Wherein, the nucleotides sequence list of TuSp2-RP is as shown in SEQ No.1 (270bp):
MEFAKCMYELVLKSANSLGVLNPHLIANNIYQSIVSNLDILHSSVMINLYANAMARNLFHEGFLNLDN
AATLAKKCANDMEAFAKKMVET；
The nucleotides sequence list of Linker is as shown in SEQ No.2: GGGGGSGGGGSGGGGS；
Shown in the nucleotides sequence list of extraction of uranium from seawater functional protein gene such as SEQ No.3 (237bp):
LDCRERIEKDLEDLEKELMEMKSIKLSDDEEAVVERALNYRDDSVYYLEKGDHITSFGCITYAEGLLD
SLRmLHRIIEG；
Shown in extraction of uranium from seawater recombinant spider silk protein gene such as SEQ No.4 (558bp):
MEFAKCMYELVLKSANSLGVLNPHLIANNIYQSIVSNLDILHSSVMINLYANAMARNLFHEGFLNLDN
AATLAKKCANDMEAFAKKMVETGGGGGSGGGGSGGGGSLDCRERIEKDLEDLEKELMEMKSIKLSDDEEAVVERAL
NYRDDSVYYLEKGDHITSFGCITYAEGLLDSLRmLHRIIEG。
(2) gene expression: extraction of uranium from seawater recombinant spider silk protein gene plasmid is transferred to Escherichia coli pET-22b, and is being contained
There is the LB liquid medium (37 DEG C, 180rpm) of ampicillin (50 μ g/mL of concentration) to be incubated overnight, the bacterium solution of overnight incubation with
1% ratio is inoculated in LB culture medium of the 500mL containing ampicillin (50g/mL), and expands in 26 DEG C of constant-temperature table 180rpm
Big culture, as light absorption value OD600Reach 0.8, the isopropylthiogalactoside IPTG of final concentration of 0.4mM is added, at 20 DEG C
180rpm revolving speed overnight incubation and induced fusion protein expression in constant-temperature table, finally at 4 DEG C, 10000rpm revolving speed is centrifuged
15min collects thallus.
(3) protein purification: bacterium is washed with the PBS buffer solution and phosphate-buffered salt PBS of the X-100 containing 1%Triton respectively
Body 3 times, with lysate (8M Urea, 100mM NaH2PO4, 100mM Tris, pH=8.0) and cracking thallus 2h, in 4 DEG C of condition
Under with 10000rpm revolving speed be centrifuged 40min, collect supernatant, supernatant is loaded on protein purification Ni-NTA column, with 4 columns
Eluent (8M Urea, the 100mM NaH of volume2PO4, 100mM Tris, pH=6.3) and washing albumen, successively use various concentration
The eluent of urea (6M, 4M, 2M, 1M, 0.5M, 0M, pH=8.0 contain 10% glycerol) carries out on-column refolding to albumen, finally
With eluent (the 100mM NaH containing 400mM imidazoles2PO4, 100mM Tris, pH=7.5) and the elution extraction of uranium from seawater recombination spider's thread
Protein S SUP (SEQ No.5) can be purified according to the thallus for calculating every 1L step (2) and be obtained the 40mg extraction of uranium from seawater recombination spider's thread
Albumen；
(4) self assembly spinning: it is centrifuged 30min concentrated seawater under 4200rpm revolving speed with the molecule of 10kDa retention column and mentions uranium
Recombinant spider silk protein SSUP obtains the extraction of uranium from seawater recombinant spider silk protein SSUP liquid that concentration is 150mg/mL, injects 1mL syringe
In, the syringe needle of 24G is selected, being 0.6mL/h with fltting speed with pressure pump promotes extraction of uranium from seawater recombinant spider silk protein SSUP liquid
PH=4 spinning solution (100mM sodium phosphate (PH>5.5), 100mM sodium acetate (pH4.0-5.5) and 200mM citric acid (pH<
4.0) extraction of uranium from seawater recombinant spider silk protein SSUP fiber is obtained in).
Embodiment two: the extraction of uranium from seawater recombinant spider silk protein SSUP liquid of embodiment one and its characterization and test of fiber
As Fig. 2 confirms that its theoretical molecular weight is by the recombinant spider silk protein SSUP of SDS-PAGE electrophoresis analysis purifying
21.1kDa；Such as Fig. 3, by using circular dichroism spectrometer (Chirascan, Applied of the wavelength between 190-260nm
Photophysics Ltd), in conjunction with software cd NN analysis result to determine that Secondary structure forms；By using liquid phase
Chromatography-tandem mass spectrum (LC-MS) recombinant spider silk protein SSUP is to identify that protein molecular weight for 20.8kDa, also confirmed its ammonia
Base acid series of tables such as SEQ No.5.
After extraction of uranium from seawater recombinant spider silk protein SSUP liquid self assembly is spun to recombinant spider silk protein SSUP fiber, use
The micro-structure of Hitachi S-4800 scanning electron microscope observation extraction of uranium from seawater recombinant spider silk protein SSUP fiber；Such as Fig. 4, sea
It is 220 μm that water, which mentions diameter of the uranium recombinant spider silk protein SSUP fiber under moisture state, and hydrophilic hydrogel spline structure is presented, adopts
Its tensile strength is tested up to 128.26MPa with the micro-processor controlled electronic universal tester of Gaopin, can be elongated 2.4 times；
Such as Fig. 5, extraction of uranium from seawater recombinant spider silk protein SSUP fiber is 21 μm in the dry state, shows as fine and close structure.
Embodiment three: with embodiment one the difference is that it is 50mg/mL, 100mg/ that concentration, which is respectively adopted, in step (4)
The extraction of uranium from seawater recombinant spider silk protein SSUP liquid of mL, 150mg/mL, 300mg/mL carry out self assembly spinning under the same conditions.It is logical
Crossing experimental result would know that, extraction of uranium from seawater recombinant spider silk protein SSUP liquid concentration be 100-300mg/ml, can self assembly spinning,
When concentration is 50mg/ml, spinning failure, wherein optimal concentration is 150mg/ml, is easy to be self-assembled into silk and fiber production is high.
Example IV: with embodiment one the difference is that in step (4), using the spinning buffer and not of different pH
Self assembly spinning is carried out to extraction of uranium from seawater recombinant spider silk protein with fltting speed, is learnt from the experimental result of table 1 and Fig. 6, in pH
=4 spinning buffer, the azelon obtained with fltting speed 0.6mL/h self assembly spinning is continuous and uniform diameter, explanation
The pH value of spinning buffer is most important to the self assembly of azelon.
The spinning buffer and fltting speed of 1 difference pH of table is to extraction of uranium from seawater recombinant spider silk protein fibre spinning result
Embodiment five: the extraction of uranium from seawater recombinant spider silk protein SSUP fiber applications of embodiment one are in extraction of uranium from seawater measure of merit
Weighing azelon material made from 10mg above-mentioned steps and being immersed in uranium concentration is respectively 2,4,8 and 16ppm
In 30mL seawater, adsorption time 1h measures absorption front and back using ICP-OES inductively coupled plasma bulk optics emission spectrometer
The concentration of uranium, adsorption capacity calculation formula such as formula (1)
Wherein qtIndicate the amount that uranium is extracted in the t time, C0 indicates the concentration of initial uranium；CtIt indicates in the dense of t moment uranium
Degree；V indicates the volume of test seawater；The quality of m expression azelon material.
One, uranium dynamic absorption is tested:
By extraction of uranium from seawater recombinant spider silk protein SSUP fibrous material made from embodiment one in 30mL simulated seawater (uranium concentration
Respectively 2,4,8,16ppm) in carry out absorption test, such as Fig. 7, quick uranium absorption is shown in initial 5min,
30min reaches balance, and uranium saturated extent of adsorption respectively reaches 6.77mg/g, 11.47mg/g, 15.10mg/g and 15.91mg/g, says
Extraction of uranium from seawater recombinant spider silk protein SSUP fibrous material prepared by embodiment one is illustrated to respond fastly the uranium ion in seawater, has
Good suction-operated.
Two, the reperformance test of uranium absorption:
Extraction of uranium from seawater recombinant spider silk protein SSUP fibrous material made from 10mg embodiment one is added into uranium in 30mL first
In simulated seawater, until calculating saturated extent of adsorption by using formula (1), afterwards at room temperature using 100mM EDTA (pH=8.0)
The uranium ion in conjunction with fiber is eluted, and elution efficiency is analyzed by the concentration of uranium in measurement elution solution.It strips every time
Cheng Hou, extraction of uranium from seawater recombinant spider silk protein SSUP fiber are washed with deionized three times, rear to repeat to inhale uranium step, from table 2 and Fig. 8
As a result it is found that extraction of uranium from seawater recombinant spider silk protein SSUP fibrous material of the invention has good repeat performance, make
91.3% is kept respectively with the initial uranium absorption capacity after 5 circulations, shows that SSUP fiber can repeatedly use extraction seawater
In uranium ion, in natural sea-water have longer service life.
2 recombinant spider silk protein SSUP fibrous material of table repeats to inhale uranium performance
Three, the selectivity test of uranium absorption:
It chooses 14 kinds and competes related metal ion (VO with uranyl in natural sea-water3-, Fe3+, Co2+, Ni2+, Cu2+, Zn2+,
Ba2+, Mn2+, Sr2+, Cr3+, Na+, Mg2+, Ca2+And K) natural sea-water (being derived from island near the boundary of the South Sea) is added, wherein day
Na in right seawater+, Mg2+, Ca2+And K+Original concentration is kept, other competition metal ions are 100 times of original concentration, by 20mg
Extraction of uranium from seawater recombinant spider silk protein SSUP fibrous material is added in the solution of the above-mentioned preparation of 500mL after absorption 1h, is surveyed with ICP-MS
Determine the concentration of each metal ion in solution.Such as Fig. 9 the result shows that, even if competing ions excessive 2.0 × 105Times, other ions and
The binding force of extraction of uranium from seawater recombinant spider silk protein SSUP fibrous material is still weak to cross uranyl ion, even uranyl ion competition is most
Strong vanadium ion has also embodied excellent selectivity, this, which puts, proposes the outstanding aobvious protrusion of uranium material comparison with existing, also furtherly
Extraction of uranium from seawater recombinant spider silk protein SSUP fibrous material of the present invention is illustrated and proposes uranium selectivity with outstanding.Further as schemed
10, extraction of uranium from seawater recombinant spider silk protein SSUP fibrous material is placed in natural sea-water and is impregnated, saturation time is 3.5 days, and uranium is inhaled
Receipts amount reaches 12.33mg/g.
In conclusion using a kind of recombinant spider silk protein SSUP fibrous material for the extraction of uranium from seawater provided by the present invention
Preparation method, simple process, mild condition is easy to purify, can prepare and industrialize in batches.The one kind prepared by this method
For the recombinant spider silk protein SSUP fibrous material of the extraction of uranium from seawater, tensile strength is up to 2.4 up to 128.26MPa, stretching ratio
Times, there is specific adsorption capacity to uranium, selectivity is high, and can repeatedly use, and the service life is long.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent substitution, improvement and etc. done be should be included within the scope of the present invention.
Sequence table
<110>University Of Hainan
<120>a kind of recombinant spider silk protein fibrous material and preparation method thereof for the extraction of uranium from seawater
<160> 5
<170> SIPOSequenceListing 1.0
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atggagtttg cgaagtgtat gtatgaactt gtgcttaaat cagcaaactc cttgggcgta 60
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ctgcactctt cagtgatgat aaacctctac gctaacgcga tggccagaaa tctttttcat 180
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gaagcctttg ccaagaagat ggttgaaaca gga 273
<210> 2
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<213>artificial sequence (unknown)
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gattgccgtg aacgcattga aaaagacctg gaagacctgg aaaaagaact gatggaaatg 60
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gatgacagtg tctattacct ggaaaaaggc gatcatatta cctcctttgg ttgtatcacg 180
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atggagtttg cgaagtgtat gtatgaactt gtgcttaaat cagcaaactc cttgggcgta 60
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gaacgtgccc tgaattatcg cgatgacagt gtctattacc tggaaaaagg cgatcatatt 480
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20 25 30
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35 40 45
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50 55 60
Asn Leu Asp Asn Ala Ala Thr Leu Ala Lys Lys Cys Ala Asn Asp Met
65 70 75 80
Glu Ala Phe Ala Lys Lys Met Val Glu Thr Gly Gly Gly Gly Gly Ser
85 90 95
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Asp Cys Arg Glu Arg
100 105 110
Ile Glu Lys Asp Leu Glu Asp Leu Glu Lys Glu Leu Met Glu Met Lys
115 120 125
Ser Ile Lys Leu Ser Asp Asp Glu Glu Ala Val Val Glu Arg Ala Leu
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145 150 155 160
Thr Ser Phe Gly Cys Ile Thr Tyr Ala Glu Gly Leu Leu Asp Ser Leu
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180 185
Claims (9)
1. a kind of preparation method of the recombinant spider silk protein fibrous material for the extraction of uranium from seawater, which is characterized in that including following step
It is rapid:
(1) genetic recombination: spider silk protein gene segment, extraction of uranium from seawater functional protein genetic fragment and flexible connection gene are closed
At acquisition extraction of uranium from seawater recombinant spider silk protein encoding gene；
(2) gene expression: being transferred to Escherichia coli for recombinant spider silk protein encoding gene, in LB liquid medium overnight incubation, after
It is inoculated with and expands culture, then inducing expression culture, last thalline were collected by centrifugation；
(3) purifying protein: use buffer washing thalline, after cracked with lysate, supernatant is collected by centrifugation and carries out pure
Change, successively obtains extraction of uranium from seawater recombinant spider silk protein with gradient elution；
(4) self assembly spinning: extraction of uranium from seawater recombinant spider silk protein centrifugal concentrating is made into its concentration 100-300mg/mL, with certain
Extraction of uranium from seawater recombinant spider silk protein liquid after concentration is promoted spinning solution by fltting speed, and self assembly obtains the extraction of uranium from seawater and recombinates the spider's thread
Azelon；The pH of the spinning solution is 3-5.
2. a kind of preparation method of the recombinant spider silk protein fibrous material for the extraction of uranium from seawater according to claim 1, special
Sign is, in the step (1), the nucleotide sequence of the extraction of uranium from seawater recombinant spider silk protein encoding gene such as SEQ No.4 institute
Show.
3. a kind of preparation method of the recombinant spider silk protein fibrous material for the extraction of uranium from seawater according to claim 1, special
Sign is, in the step (2), the lysate is 8M Urea, 100mM NaH2PO4With 100mM Tris.
4. a kind of preparation method of the recombinant spider silk protein fibrous material for the extraction of uranium from seawater according to claim 1, special
Sign is, in the step (3), shown in the amino acid sequence table SEQ No.5 of the extraction of uranium from seawater recombinant spider silk protein.
5. a kind of preparation method of the recombinant spider silk protein fibrous material for the extraction of uranium from seawater according to claim 1, special
Sign is that in the step (3), the concentration of the gradient eluent is respectively the urea of 6M, 4M, 2M, 1M, 0.5M and 0M, institute
State glycerol of the eluent containing 10% mass.
6. a kind of preparation method of the recombinant spider silk protein fibrous material for the extraction of uranium from seawater according to claim 1, special
Sign is, in the step (4), the spinning solution includes 100mM sodium phosphate, 100mM sodium acetate and 200mM citric acid.
7. a kind of preparation method of the recombinant spider silk protein fibrous material for the extraction of uranium from seawater according to claim 1, special
Sign is, in the step (4), fltting speed 0.6mL/h, the pH of spinning solution is 4.
8. a kind of preparation method of the recombinant spider silk protein fibrous material for the extraction of uranium from seawater according to claim 1, special
Sign is, in the step (4), selecting syringe needle is that the 1mL syringe of 24G is promoted.
9. a kind of method according to claim 1-8 is prepared fine for the recombinant spider silk protein of the extraction of uranium from seawater
Tie up material.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112844325A (en) * | 2020-09-15 | 2021-05-28 | 海南大学 | Specific DNA hydrogel material for extracting uranium from seawater and preparation method thereof |
| CN112920279A (en) * | 2021-03-09 | 2021-06-08 | 海南大学 | Anti-biological fouling type polymer peptide hydrogel material for extracting uranium from seawater and preparation method and application thereof |
| WO2022258499A1 (en) * | 2021-06-10 | 2022-12-15 | Amsilk Gmbh | Silk polypeptide formulation comprising urea |
-
2019
- 2019-06-29 CN CN201910580843.6A patent/CN110256550A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| YIHUI YUAN ET AL.: "Ultrafast and Highly Selective Uranium Extraction from Seawater by Hydrogel-like Spidroin-based Protein Fiber", 《ANGEW. CHEM.》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112844325A (en) * | 2020-09-15 | 2021-05-28 | 海南大学 | Specific DNA hydrogel material for extracting uranium from seawater and preparation method thereof |
| CN112844325B (en) * | 2020-09-15 | 2022-01-28 | 海南大学 | Specific DNA hydrogel material for extracting uranium from seawater and preparation method thereof |
| CN112920279A (en) * | 2021-03-09 | 2021-06-08 | 海南大学 | Anti-biological fouling type polymer peptide hydrogel material for extracting uranium from seawater and preparation method and application thereof |
| WO2022258499A1 (en) * | 2021-06-10 | 2022-12-15 | Amsilk Gmbh | Silk polypeptide formulation comprising urea |
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