CN101717432B - Polyglutamic acid and application thereof in processing medicine, cosmetics, food and water - Google Patents
Polyglutamic acid and application thereof in processing medicine, cosmetics, food and water Download PDFInfo
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- CN101717432B CN101717432B CN2009101543216A CN200910154321A CN101717432B CN 101717432 B CN101717432 B CN 101717432B CN 2009101543216 A CN2009101543216 A CN 2009101543216A CN 200910154321 A CN200910154321 A CN 200910154321A CN 101717432 B CN101717432 B CN 101717432B
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- polyglutamic acid
- glu glu
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Abstract
The invention discloses a polyglutamic acid and the application thereof in processing medicine, cosmetics, food and water, wherein the amino acid sequence thereof is presented as the sequence 2 in a sequence table or is acquired by adding, decreasing or replacing one or more amino acid residues in the sequence presented as the sequence 2 in a sequence table. Furthermore, the invention discloses a preparation method of the olyglutamic acid, the application thereof, and the like.
Description
Technical field
The invention belongs to biological technical field, particularly, the present invention relates to a kind of polyglutamic acid, its homogeneity is good, can be widely used in the application such as medicine, makeup, food and water treatment.In addition, the invention still further relates to preparation method and the application thereof etc. of said polyglutamic acid.
Background technology
Polyglutamic acids polypeptide can be applied to aspects such as medicine, makeup, food and water treatment as the polypeptide product of the water-soluble non-toxic (biodegradable) that has thickening, film forming, preserves moisture.Existing polyglutamic acids polypeptide comprises two types: one type is the polyglutamic acid that is formed by the polymerization of L-L-glutamic acid fully; For example one Chinese patent application CN1607962A, CN101300267A etc. are disclosed; Can be used as the carrier or the additive of medicine, makeup, food; But this type polyglutamic acid is formed by the polymerization of L-L-glutamic acid fully, and chemosynthesis is difficult to reach dalton's up to ten thousand easily (D) molecular weight, and passes through gene engineering expression; The identical amino-acid residue multiple of this type polypeptide expression amount can't be difficult to improve, and does not also improve the document public reported of its expression amount; Another kind of is by L-L-glutamic acid and D-L-glutamic acid alternating polymerization and the gamma-polyglutamic acid-that produces; Disclosed like one Chinese patent application N1464864A, CN1556859A, CN1644677A, CN1974432A, CN101061867A etc., it can be produced by fermentation of bacillus, but owing to be not to be directly to express the polypeptide that produces through encoding sox; But the result that a series of protein produce; Therefore gamma-polyglutamic acid-size and heterogeneity of producing of its fermentation, polymolecularity between 2-5 (referring to food engineering, 2009 (1): 23-26); That is to say; If in the demanding field of homogeneity (as, medicine) when using, the big and small gamma-polyglutamic acid-mixture of generation that needs to ferment is further purified and is kept the product of specified molecular weight; So not only with high costs, and make for the actual yield of the gamma-polyglutamic acid-of the specified molecular weight of final reservation much lower.
In order to overcome the defective of prior art; The inventor is through arduous research; Abandoned the structure of above two kinds of polyglutamic acids, designed a kind of new polyglutamic acid peptide sequence of the homogeneous of can the genetically engineered high yield preparing, laid the foundation for applying this polypeptide; More unexpectedly; Although this peptide species itself does not have anti-tumor activity, under the condition of puting together with taxol, can significantly strengthen the anti-tumor activity of taxol, and this peptide species still has the usefulness of removing heavy metal in the water body.
Summary of the invention
The technical problem that will solve of the present invention is to provide a kind of new polyglutamic acid peptide sequence of the homogeneous of can the genetically engineered high yield preparing, thereby the polyglutamic acid product application that can satisfy a large amount of lower costs is in the requirement in the demanding field of homogeneity.In addition, the present invention's technical problem that also will solve midbody (like nucleic acid, carrier, host cell etc.) of being to provide its preparation method, its preparation with and the product using and use.
Particularly, aspect first, the invention provides a kind of polyglutamic acid, its aminoacid sequence
A) shown in the sequence in the sequence table 2; Or
B) be to the sequence shown in the sequence in the sequence table 2 add, lack or replace one or several amino-acid residue and aminoacid sequence.
In this article, as do not particularly point out, term " polyglutamic acid ", " polyglutamic acid polypeptide " and " being rich in the polypeptide of polyglutamic acid " can exchange use.The aminoacid sequence of the polyglutamic acid of first aspect of the present invention can be to add on the basis of the sequence shown in the sequence 2 in sequence table, lack or replace one or several (preferred one to five; More preferably one to three) amino-acid residue and aminoacid sequence, and the polyglutamic acid of the homogeneous of still can the genetically engineered high yield preparing, still can under the condition of puting together with taxol, can significantly strengthen the anti-tumor activity of taxol and/or still have the usefulness of removing heavy metal in the water body.Those skilled in the art know; Import expression vector through the coding gene sequence of change known peptide and with it; Can prepare replacement, added or lack the polypeptide of amino-acid residue; These methods extensively are recorded in " molecular cloning experiment guide " documents well known in the art such as (Beijing: Science Press, 2002).In substituted amino-acid residue, preferably be substituted by other amino acid with original acid residue side chain similar performance, thereby more be able to keep original function active.The amino acid of side chain similar performance has hydrophobic amino acid (A respectively; I; L; M; F; P; W; Y; V); Hydrophilic amino acid (R; D; N; C; E; Q; G; H; K; S; T); Amino acid (the G of aliphatic lateral chain; A; V; L; I; P); Amino acid (the S of hydroxyl side chain; T; Y); Amino acid (the C of sulfur atom-containing side chain; M); Amino acid (the D that contains carboxylic acid and amide side chains; N; E; Q); Amino acid (the R that contains the basic group side chain; K; H); Amino acid (the H that contains the aromatic series side chain; F; Y; W).At present there has been the experimental technique that can check the polyglutamic acid function, in case of necessity also can be according to the described concrete experimental technique of the embodiment of the invention from through interpolation, disappearance or substituted aminoacid sequence, selecting.Of the present invention aspect first in, the aminoacid sequence of preferred said polyglutamic acid is shown in the sequence in the sequence table 2.
Aspect second, the invention provides a kind of nucleic acid molecule, its described polyglutamic acid in code book first aspect of invention.Nucleic acid molecule of the present invention can be a dna form, also can be rna form, preferred dna form.Dna form comprises the cDNA of natural cDNA and synthetic, and DNA can be coding strand or template strand.Through routine techniques, like the method for PCR method, recombination method or synthetic, those skilled in the art can be easy to obtain nucleic acid molecule of the present invention or its fragment.These sequences transform or are transfected into corresponding cell again in case acquisition can be cloned into carrier with it, breed through the host cell of routine then, therefrom separate obtaining a large amount of nucleic acid molecule.The nucleotide sequence of preferred nucleic acid molecule of the present invention is shown in the sequence in the sequence table 1.This preferred sequence has been optimized in colibacillary expression, makes that the expression ratio in e. coli bl21 (DE3) can account for 25% of total protein.
Aspect the 3rd, the invention provides a kind of carrier, it contains second described nucleic acid molecule in aspect of the present invention.Term among this paper " recombinant expression vector ", " expression vector "; Sometimes only claim in " carrier "; Can replace use alternately at this, be meant bacterial plasmid commonly used in this area, clay, phagemid, yeast plasmid, vegetable cell virus, animal virus and other various virus vector.The carrier that is suitable among the present invention includes but not limited to: the carrier (prokaryotic expression carrier) of in bacterium, expressing usefulness; The carrier of in yeast, expressing usefulness is (like pichia vector; Debaryomyces hansenii carrier etc.); Baculovirus vector in expressed in insect cells; In mammalian cell, express the carrier (vaccinia virus vector of usefulness; Retroviral vector; Adenovirus carrier; Adeno-associated virus carrier etc.); The various carriers of in plant, expressing the plant viral vector of usefulness and in mammal galactophore, expressing usefulness.In a word, duplicate as long as can in host cell, stablize, any plasmid and carrier all can use.The preferred expression carrier comprises selectable marker gene, like ampicillin resistance gene, tetracycline resistance gene, kalamycin resistance gene, streptomycin resistance gene, the chloramphenicol resistance gene of bacterium; Saccharomycetic neomycin resistance gene, Zeocin resistant gene, saccharomycetic defective selection marker, like His, Leu, Trp etc.; Eukaryotic neomycin resistance gene, Zeocin resistant gene, dihydrofolate reductase gene and fluorescent protein labeling gene etc.
Aspect the 4th, the invention provides a kind of host cell, it is characterized in that said cell contains the described carrier of third aspect of the present invention, perhaps said cell transforms or transfection with the described nucleic acid molecule in second aspect of the present invention.Host cell can be a prokaryotic cell prokaryocyte, also can be eukaryotic cell, as, bacterial cell, yeast cell, vegetable cell, insect cell, mammalian cell etc.Host cell promptly constitutes through engineering approaches cell or cell strain after transforming or transfection contains the gene order of encoding fusion protein according to the invention, can be used for producing required fusion rotein.Those skilled in the art can select appropriate carriers, host cell rightly; And how know carrier high-efficiency ground is transformed or is transfected in the host cell; Method therefor includes but not limited to: Calcium Chloride Method, electroporation are used for bacterial cell; Electroporation and protoplastis fusion method are used for yeast cell, and liposome, coprecipitation of calcium phosphate, electro fusion method and microinjection are used for eukaryotic cells such as mammalian cell.Preferred host cell of the present invention is e. coli bl21 (DE3).This preferred host cell cooperates with the nucleotide sequence shown in the sequence 1, can efficiently express polyglutamic acid of the present invention.
Aspect the 5th; The invention provides the method for preparing first said polyglutamic acid in aspect of the present invention; It comprises, with the described polyglutamic acid in the present invention first aspect of described host cell expression the present invention, the 4th aspect, the said polyglutamic acid of separation and purification then.
Aspect the 6th, the invention provides a kind of compsn, it comprises the said polyglutamic acid in first aspect of the present invention.Usually, can also comprise at least a other carrier in the compsn, for example saline water, damping fluid and/or deionized water.Wherein, compsn can be pharmaceutical composition, makeup, food or water treatment additive.For example, the present invention preferably provides a kind of pharmaceutical composition, and it comprises the said polyglutamic acid in first aspect of the present invention as active drug carrier, and more preferably wherein said active medicine is a taxol.And for example, the present invention preferably provides a kind of makeup, and it comprises the carrier of the said polyglutamic acid in first aspect of the present invention as the cosmetic activeconstituents.And for example, the present invention preferably provides a kind of food, and it comprises the carrier of the said polyglutamic acid in first aspect of the present invention as functional food ingredients.And for example, the present invention preferably provides a kind of water treatment additive, and it comprises the sorbent material of the said polyglutamic acid in first aspect of the present invention as heavy metal ion, and more preferably wherein said is the Pb ion.
Aspect the 7th, the invention provides the application of the said polyglutamic acid in first aspect of the present invention in preparation medicine, makeup, food or water treatment additive.
The beneficial effect that the present invention obtains has: expression amount is high; Homogeneity is good; Can significantly strengthen the anti-tumor activity of taxol; And/or, kept usefulness with heavy metal in the removing water body.
Description of drawings
Fig. 1 is for be the SDS-PAGE electrophorogram of polyglutamic acid expression strain nutrient solution, wherein on the swimming lane of left side kind be molecular weight marker (be followed successively by 66kD from top to bottom, 45kD, 35kD, 25kD, 18.4kD, 14.4kD); Appearance is the nutrient solution before inducing with IPTG on the swimming lane 1; Appearance is the nutrient solution after inducing with IPTG on the swimming lane 2.
Fig. 2 is the SDS-PAGE electrophorogram of purified polyglutamic acid, wherein on the swimming lane of left side appearance be molecular weight marker (be followed successively by from top to bottom and be followed successively by 66kD from top to bottom, 45kD, 35kD, 25kD, 18.4kD, 14.4kD); What centre and right side swimming lane were gone up appearance respectively is the purified polyglutamic acid that two different batches obtain.
For the ease of understanding, the present invention has quoted open source literature, and these documents are in order more clearly to describe the present invention, its in full content all include this paper in and carry out reference.Below will the present invention be described in detail through concrete embodiment.What need particularly point out is that these descriptions only are exemplary descriptions, do not constitute limitation of the scope of the invention.According to the argumentation of this specification sheets, many variations of the present invention, change have been obviously all concerning one of ordinary skill in the art.
Embodiment
The said experimental technique of following examples; Do not specify; According to " molecular cloning experiment guide " (2002, the third edition, Science Press), " cell experiment guide " (calendar year 2001; Science Press) etc. the described method of laboratory manual is carried out, and perhaps carries out according to the reagent of specifically using in the experiment, the specification sheets that manufacturer provided or the handbook of instrument.
The construction and expression of embodiment 1. polyglutamic acid expression vectors
In order to help the expression of polyglutamic acid in intestinal bacteria; We according to oneself empirical design in intestinal bacteria the polyglutamic acid gene order of optimization expression; It and entrusts Shanghai to give birth to worker company this gene order of synthetic through the commercial channel shown in sequence 2.After obtaining the synthetic gene order of this purchase, we are reflected at above synthetic sequence two ends through following PCR and introduce EcoRI and XhoI restriction enzyme site and enteropeptidase point of contact respectively:
Upstream primer: sequence 3;
Downstream primer: sequence 4;
PCR system: H2O 34 μ L, 10 * PCR damping fluid, 5 μ L, 25mM MgSO4 2 μ L; 2mM dNTP5 μ L; Upstream primer (being diluted to 50uM concentration) 1 μ L, downstream primer (being diluted to 50uM concentration) 1 μ L, synthetic polyglutamic acid gene (being diluted to 10ng/uL concentration) 1 μ L; KOD PLUS archaeal dna polymerase (Toyoba) 1 μ L, TV 50 μ L;
The PCR condition: at first 94 ℃ of sex change are 5 minutes, 25 circulations then (each circulation be 94 ℃ 30 seconds, 50 ℃ 30 seconds, 68 ℃ 30 seconds), last 68 ℃ 2 minutes.
Then, electrophoresis pcr amplification product on sepharose, the nucleic acid fragment of about 400bp size is reclaimed in rubber tapping.With EcoRI and XhoI the double digestion PCR product that should reclaim and pET28a+ plasmid (can available from Invitrogen company) respectively; PCR product after enzyme cut is connected with the T4 ligase enzyme with the pET28a plasmid, is built into transformed into escherichia coli DH5 α cell behind the polyglutamic acid expression vector pET28-pGlu (can available from ATCC).Get the e.colidh5 extracting plasmid of conversion, after order-checking detects sequence clone the correct sequence of polyglutamic acid is arranged, this plasmid is transformed in the e. coli bl21 (DE3) (can available from ATCC), obtain the polyglutamic acid expression strain thus.
Get the polyglutamic acid expression strain and be inoculated among the LB liquid nutrient medium 100mL, after 37 ℃ of joltings were cultivated 6 hours, the 1mol/L IPTG that adds 10uL induced, and continued to cultivate 4 hours in 37 ℃.Respectively get bacterium liquid 50 μ L before and after inducing, use the 12%SDS-PAGE electrophoresis.Electrophoresis result is as shown in Figure 1; Induce the back that tangible polyglutamic acid band of expression is arranged on the position about 15kD; Its expression amount accounts for more than 25% of tropina total amount, shows that nucleotide sequence (sequence 2), expression vector (pET28-pGlu) and host bacterium (e. coli bl21 (DE3)) that we select for use have constituted the protokaryon efficient expression system.
The separation and purification of embodiment 2. polyglutamic acids
Thalline to inducing culture among the embodiment 1 carries out the separation and purification operation, and the polyglutamic acid of results purifying uses for further experiment.Concrete steps are: with resuspended 1 gram thalline, the ultrasonication on ice of Tris-HCl (pH 8.0) lysate of 10mL 50mM concentration.4 degrees centigrade 10,000 rev/mins centrifugal 20 minutes, collecting precipitation.Then, deposition is thoroughly washed with the lysate that contains 2M urea of 3 times of volumes (30mL), 4 ℃ 10000 rev/mins centrifugal 10 minutes, abandon supernatant, thoroughly dissolve 4 ℃ of 10000 rev/mins of centrifugal depositions that discard with the lysate that contains 8M urea of 3 times of volumes.With the inclusion body lysate that obtains with Q-Sepharose Fast Flow post (available from GE company) purifying; Successively carry out gradient elution with A liquid (containing 8mol/L urea, 25mmol/L Tris-HCl (pH 9.0), 1mmol/L EDTA and 1mmol/L DTT) and B liquid (0.5mol/L NaCl, 8mol/L urea, 25mmol/L, Tris-HCl (pH 9.0), 1mmol/L EDTA and 1mmol/L DTT); The 280nm ultraviolet monitoring; Be eluted to and no longer go out the peak; Substep is collected elutriant, carries out SDS-PAGE, collects the elutriant of 15kD.Then the elutriant of collecting is crossed Sephedex G-25 post (available from GE company) and slough the salt ion in the elutriant.Then, according to the guidance of manufacturers instruction, at 20 ℃, 20mM Tris-HCl (pH7.6), under the condition of 100mMNaCl, with the rEK of 1 μ l (recombinant enterokinase can be given birth to worker company available from Shanghai) with polyglutamic acid (having the partial sequence on the carrier) reaction 8 hours.Then, flow through NTA post (available from Merck company) that huge legendary turtle closes Ni of reaction solution is cut albumen completely to prevent to sneak into not enzyme.At last, the effluent 1ml in a last step was crossed Q-Sepharose Fast Flow column purification, condition is with 50mM Tris-HCl damping fluid balance, and with 0 to 1mol/L NaCl gradient elution, collection contains the elution peak of polyglutamic acid.
Purifying through above step; The SDS-PAGE electrophoresis result of the polyglutamic acid of the purifying that obtains is as shown in Figure 2, and in the result that two different batches purification experiment obtain, the polyglutamic acid purity of purifying is all more than 95%; And the molecular weight homogeneity is good, and repeatability is good as a result.
The preparation and the medicinal application thereof of embodiment 3. polyglutamic acids-taxol conjugate
The polyglutamic acid 23g of embodiment 2 preparations is joined in the round-bottomed flask, add N, dinethylformamide 180ml stirred 10 minutes, added taxol 14.33g and N then, and N-dimethyl aminopyridine 0.4g stirred 10 minutes.Slowly drip N down at 20 ℃, N-DIC (in 1 hour, adding 2.4g) also stirs, and stirs then 3 hours.Reaction mixture is cooled to 5 ℃～10 ℃ and keep this temperature, drips the sodium-chlor cold soln 0.5L of 10% (w/w) then.Accomplish after the adding sodium chloride solution, the hydrochloric acid soln that drips 1mol/L reaches 2.5 (needing hydrochloric acid soln 20ml approximately) until pH value of reactants, stirs 30 minutes at 5 ℃～10 ℃, filters polyglutamic acid-taxol conjugate that collecting precipitation comes out then.To precipitate with water washing 3 times, lyophilize is 24 hours in Freeze Drying Equipment.Then, pulverize dried solid piece, be suspended in the 500ml acetonitrile) and stirred 2 hours, filter then, and with acetonitrile washing precipitation 2 times.After being deposited under the vacuum dry 24 hours, obtain polyglutamic acid-taxol conjugate 28.7g, according to 1H NMR (d6 DMSO), the content of taxol is 36 weight % in polyglutamic acid-taxol conjugate.Carry out following experimentation on animals then:
The nude mice left side rib side inoculated with subcutaneous injections 10 in 6 ages in week
7Individual's lung cancer MAD109 cell (can be available from ATCC, the U.S.).Inoculation back the 6th day; The nude mice random packet; On the same group mouse every day 1 independent abdominal injection 1mL blank solvent PBS, 1mL taxol (dosage is 10mg/kg), 1mL polyglutamic acid (dosage is 16mg/kg) and 1mL polyglutamic acid-taxol conjugate (dosage is 10mg/kg, in taxol) respectively not.Record growth of tumor situation utilizes length * wide * high volume calculated to represent the size of tumour, and the result is shown in table 3.1.The result shows; Although itself does not have therapeutic activity polyglutamic acid; But polyglutamic acid-taxol conjugate still has therapeutic activity; And use polyglutamic acid-taxol conjugate and do not have significant difference with respect to using taxol for the clinical therapeutic efficacy in the short duration of tumor suppression, still unexpected is significantly to strengthen in 12 days later restraining effect to tumor growth of administration.
Table 3.1 different dosing regimes is implemented the volume of back mouse tumor
The water treatment applications of embodiment 4. polyglutamic acids
The polyglutamic acid 2g of embodiment 2 preparations is dissolved in the 1mol/L NaOH solution, and adjustment pH to 6.0 also is settled to 100ml, is mixed with the polyglutamic acid sodium solution of 2% (w/v) thus, adds the polyglutamic acid sodium solution that deionized water is diluted to 0.016% (w/v) then.Get Pb (NO
3)
2Prepare concentration with deionized water and be respectively 0.10,0.15,0.20,0.30,0.40 and Pb (the NO of 0.50mmol/L
3)
2Solution is respectively got 15ml and is mixed with the polyglutamic acid sodium solution of 25ml 0.016% (w/v) respectively, so adjusts pH to pH 6.0 with 10% NaOH solution, adds deionized water and is settled to 50ml.Leave standstill and accomplished chelating in 30 minutes, use ultra-filtration membrane (available from Nanjing Kaimi Science and Technology Co., Ltd.) ultrafiltration of molecular weight cut-off then as 10kD.Pb content in the mass spectrometric detection filtered solution, the result is shown in table 4.1.The result shows, although polyglutamic acid of the present invention does not form the crosslinked throw out of traditional gamma-polyglutamic acid-, has still kept harmful heavy metal ionic abilities such as absorption Pb ion.
Pb (the NO of table 4.1 different concns
3)
2The Pb ionic concn of solution before and after polyglutamic acid is handled
| Pb ionic concn (mmol/L) before handling | Handle back Pb ionic concn (mmol/L) |
| 0.10 | 0.01 |
| 0.15 | 0.01 |
| 0.20 | 0.01 |
| 0.30 | 0.01 |
| 0.40 | 0.01 |
| 0.50 | 0.03 |
Sequence table
< 110>sharp loyal, the king
< 120>polyglutamic acid and the application in medicine, makeup, food and water treatment thereof
<160>4
<170>PatentIn version 3.3
<210>1
<211>366
<212>DNA
< 213>artificial sequence
<220>
< 223>polyglutamic acid encoding sox
<400>1
atggaggaag aggaggagga agacgaggaa gaggaggagg aacctcctga ggaagaggag 60
gaggaacgtg aggaagagga ggaggaacat accgaggaag aggaggagga agacgaggaa 120
gaggaggagg aacagcagga ggaagaggag gaggaacatg aggaagagga ggaggaacct 180
tgtgaggaag aggaggagga actcgaggaa gaggaggagg aaaaccatga ggaagaggag 240
gaggaagccg aggaagagga ggaggaatgt catgaggaag aggaggagga acgtgaggaa 300
gaggaggagg aattcgacga ggaagaggag gaggaacgtg aggaagagga ggaggaagac 360
ggttaa 366
<210>2
<211>121
<212>PRT
< 213>artificial sequence
<220>
< 223>polyglutamic acid polypeptide
<400>2
Met Glu Glu Glu Glu Glu Glu Asp Glu Glu Glu Glu Glu Glu Pro Pro
1 5 10 15
Glu Glu Glu Glu Glu Glu Arg Glu Glu Glu Glu Glu Glu His Thr Glu
20 25 30
Glu Glu Glu Glu Glu Asp Glu Glu Glu Glu Glu Glu Gln Gln Glu Glu
35 40 45
Glu Glu Glu Glu His Glu Glu Glu Glu Glu Glu Pro Cys Glu Glu Glu
50 55 60
Glu Glu Glu Leu Glu Glu Glu Glu Glu Glu Asn His Glu Glu Glu Glu
65 70 75 80
Glu Glu Ala Glu Glu Glu Glu Glu Glu Cys His Glu Glu Glu Glu Glu
85 90 95
Glu Arg Glu Glu Glu Glu Glu Glu Phe Asp Glu Glu Glu Glu Glu Glu
100 105 110
Arg Glu Glu Glu Glu Glu Glu Asp Gly
115 120
<210>3
<211>42
<212>DNA
< 213>artificial sequence
<220>
< 223>upstream primer
<400>3
ttggaattcg acgacgacga caagatggag gaagaggagg ag 42
<210>4
<211>25
<212>DNA
< 213>artificial sequence
<220>
< 223>downstream primer
<400>4
ttgctcgagt taaccgtctt cctcc 25
Claims (9)
1. polyglutamic acid, its aminoacid sequence is shown in the sequence in the sequence table 2.
2. nucleic acid molecule, the described polyglutamic acid of its coding claim 1.
3. the described nucleic acid molecule of claim 2, its nucleotide sequence is shown in the sequence in the sequence table 1.
4. carrier, it contains claim 2 or 3 described nucleic acid molecule.
5. a host cell is characterized in that, said cell contains the described carrier of claim 4, and perhaps said cell transforms or transfection with claim 2 or 3 described nucleic acid molecule.
6. the method for preparing the described polyglutamic acid of claim 1, it comprises, with the described polyglutamic acid of the described host cell expression of claim 5 claim 1, the said polyglutamic acid of separation and purification then.
7. compsn, it comprises the described polyglutamic acid of claim 1.
8. the described compsn of claim 7, it is pharmaceutical composition, makeup, food or water treatment additive.
9. the application of the described polyglutamic acid of claim 1 in preparation medicine, makeup, food or water treatment additive.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101543216A CN101717432B (en) | 2009-11-26 | 2009-11-26 | Polyglutamic acid and application thereof in processing medicine, cosmetics, food and water |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101543216A CN101717432B (en) | 2009-11-26 | 2009-11-26 | Polyglutamic acid and application thereof in processing medicine, cosmetics, food and water |
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| Publication Number | Publication Date |
|---|---|
| CN101717432A CN101717432A (en) | 2010-06-02 |
| CN101717432B true CN101717432B (en) | 2012-01-25 |
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| CN105028864B (en) * | 2015-06-30 | 2018-10-30 | 朱丹 | A kind of black tea extract buccal tablet and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1164533A (en) * | 1997-01-09 | 1997-11-12 | 云南汉德技术发展有限公司 | Water-soluble anticancer compound-polyamino acid toxol ester |
| CN1974432A (en) * | 2005-12-02 | 2007-06-06 | 东海生物科技股份有限公司 | Use of gamma- polyglutamic acid, gamma- polyglutamate and/ or its aqueous gel for removing heavy metal and dissolving calcium and/ or magnesium scale |
-
2009
- 2009-11-26 CN CN2009101543216A patent/CN101717432B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1164533A (en) * | 1997-01-09 | 1997-11-12 | 云南汉德技术发展有限公司 | Water-soluble anticancer compound-polyamino acid toxol ester |
| CN1974432A (en) * | 2005-12-02 | 2007-06-06 | 东海生物科技股份有限公司 | Use of gamma- polyglutamic acid, gamma- polyglutamate and/ or its aqueous gel for removing heavy metal and dissolving calcium and/ or magnesium scale |
Non-Patent Citations (1)
| Title |
|---|
| 王军等.γ-聚谷氨酸的合成、化学修饰及其应用进展.《化学与生物工程》.2008,第25卷(第4期),17-20. * |
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| CN101717432A (en) | 2010-06-02 |
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