CN103788105A - Separation and extraction method for epothilone B based on molecular imprinting adsorption technology - Google Patents
Separation and extraction method for epothilone B based on molecular imprinting adsorption technology Download PDFInfo
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- CN103788105A CN103788105A CN201410032697.0A CN201410032697A CN103788105A CN 103788105 A CN103788105 A CN 103788105A CN 201410032697 A CN201410032697 A CN 201410032697A CN 103788105 A CN103788105 A CN 103788105A
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- epothilone
- macroporous resin
- molecular imprinting
- methyl alcohol
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- QXRSDHAAWVKZLJ-OXZHEXMSSA-N Epothilone B Natural products O=C1[C@H](C)[C@H](O)[C@@H](C)CCC[C@@]2(C)O[C@H]2C[C@@H](/C(=C\c2nc(C)sc2)/C)OC(=O)C[C@H](O)C1(C)C QXRSDHAAWVKZLJ-OXZHEXMSSA-N 0.000 title claims abstract description 88
- HESCAJZNRMSMJG-HGYUPSKWSA-N epothilone A Natural products O=C1[C@H](C)[C@H](O)[C@H](C)CCC[C@H]2O[C@H]2C[C@@H](/C(=C\c2nc(C)sc2)/C)OC(=O)C[C@H](O)C1(C)C HESCAJZNRMSMJG-HGYUPSKWSA-N 0.000 title claims abstract description 88
- QXRSDHAAWVKZLJ-PVYNADRNSA-N epothilone B Chemical compound C/C([C@@H]1C[C@@H]2O[C@]2(C)CCC[C@@H]([C@@H]([C@@H](C)C(=O)C(C)(C)[C@@H](O)CC(=O)O1)O)C)=C\C1=CSC(C)=N1 QXRSDHAAWVKZLJ-PVYNADRNSA-N 0.000 title claims abstract description 88
- 238000001179 sorption measurement Methods 0.000 title claims abstract description 28
- 238000005516 engineering process Methods 0.000 title claims abstract description 19
- 238000000926 separation method Methods 0.000 title claims abstract description 17
- 238000000605 extraction Methods 0.000 title claims abstract description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 135
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 63
- 229920005989 resin Polymers 0.000 claims abstract description 52
- 239000011347 resin Substances 0.000 claims abstract description 52
- 239000007788 liquid Substances 0.000 claims abstract description 27
- 239000000287 crude extract Substances 0.000 claims abstract description 17
- 239000012528 membrane Substances 0.000 claims abstract description 11
- 239000013049 sediment Substances 0.000 claims abstract description 4
- 238000000034 method Methods 0.000 claims description 39
- 229920000344 molecularly imprinted polymer Polymers 0.000 claims description 35
- 238000000108 ultra-filtration Methods 0.000 claims description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 238000002425 crystallisation Methods 0.000 claims description 16
- 230000008025 crystallization Effects 0.000 claims description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 14
- 241000862997 Sorangium cellulosum Species 0.000 claims description 11
- 239000003463 adsorbent Substances 0.000 claims description 10
- 235000015097 nutrients Nutrition 0.000 claims description 10
- 239000000843 powder Substances 0.000 claims description 10
- 239000003795 chemical substances by application Substances 0.000 claims description 9
- 230000009514 concussion Effects 0.000 claims description 9
- 238000002156 mixing Methods 0.000 claims description 7
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 239000012535 impurity Substances 0.000 claims description 6
- DBCAQXHNJOFNGC-UHFFFAOYSA-N 4-bromo-1,1,1-trifluorobutane Chemical compound FC(F)(F)CCCBr DBCAQXHNJOFNGC-UHFFFAOYSA-N 0.000 claims description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 5
- 244000068988 Glycine max Species 0.000 claims description 5
- 235000010469 Glycine max Nutrition 0.000 claims description 5
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 claims description 5
- 229930006000 Sucrose Natural products 0.000 claims description 5
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 5
- STVZJERGLQHEKB-UHFFFAOYSA-N ethylene glycol dimethacrylate Substances CC(=C)C(=O)OCCOC(=O)C(C)=C STVZJERGLQHEKB-UHFFFAOYSA-N 0.000 claims description 5
- 238000000855 fermentation Methods 0.000 claims description 5
- 230000004151 fermentation Effects 0.000 claims description 5
- 239000008103 glucose Substances 0.000 claims description 5
- 238000009630 liquid culture Methods 0.000 claims description 5
- 239000000463 material Substances 0.000 claims description 5
- 239000002245 particle Substances 0.000 claims description 5
- 229920001592 potato starch Polymers 0.000 claims description 5
- VVWRJUBEIPHGQF-UHFFFAOYSA-N propan-2-yl n-propan-2-yloxycarbonyliminocarbamate Chemical compound CC(C)OC(=O)N=NC(=O)OC(C)C VVWRJUBEIPHGQF-UHFFFAOYSA-N 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- 239000005720 sucrose Substances 0.000 claims description 5
- 235000013619 trace mineral Nutrition 0.000 claims description 5
- 239000011573 trace mineral Substances 0.000 claims description 5
- 150000001408 amides Chemical class 0.000 claims description 3
- 229920002492 poly(sulfone) Polymers 0.000 claims description 3
- 229920002239 polyacrylonitrile Polymers 0.000 claims description 3
- 238000001291 vacuum drying Methods 0.000 claims description 3
- 230000001580 bacterial effect Effects 0.000 claims description 2
- 239000007787 solid Substances 0.000 claims description 2
- 238000007654 immersion Methods 0.000 claims 1
- 230000008901 benefit Effects 0.000 abstract description 7
- 238000001914 filtration Methods 0.000 abstract description 3
- 229920000642 polymer Polymers 0.000 abstract description 2
- 238000002360 preparation method Methods 0.000 abstract description 2
- 238000011031 large-scale manufacturing process Methods 0.000 abstract 1
- 239000012153 distilled water Substances 0.000 description 7
- 239000000706 filtrate Substances 0.000 description 4
- XKMLYUALXHKNFT-UUOKFMHZSA-N Guanosine-5'-triphosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XKMLYUALXHKNFT-UUOKFMHZSA-N 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000009776 industrial production Methods 0.000 description 3
- 238000005374 membrane filtration Methods 0.000 description 3
- 239000000178 monomer Substances 0.000 description 3
- 230000008719 thickening Effects 0.000 description 3
- 102000029749 Microtubule Human genes 0.000 description 2
- 108091022875 Microtubule Proteins 0.000 description 2
- 229930012538 Paclitaxel Natural products 0.000 description 2
- 239000002246 antineoplastic agent Substances 0.000 description 2
- 229940041181 antineoplastic drug Drugs 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 210000004688 microtubule Anatomy 0.000 description 2
- 229960001592 paclitaxel Drugs 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 2
- MQLACMBJVPINKE-UHFFFAOYSA-N 10-[(3-hydroxy-4-methoxyphenyl)methylidene]anthracen-9-one Chemical compound C1=C(O)C(OC)=CC=C1C=C1C2=CC=CC=C2C(=O)C2=CC=CC=C21 MQLACMBJVPINKE-UHFFFAOYSA-N 0.000 description 1
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000003120 macrolide antibiotic agent Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
- C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
- C07D493/04—Ortho-condensed systems
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin
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- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines Containing Plant Substances (AREA)
- Saccharide Compounds (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention provides a separation and extraction method for epothilone B based on a molecular imprinting adsorption technology. The separation and extraction method comprises the following steps: adopting macroporous resin to adsorb fermented liquid; then extracting the adsorbed macroporous resin by using ethyl acetate; filtering extracting liquid by using an ultra-filtering membrane to obtain a crude extract; adding an epothilone B molecular imprinting polymer into the crude extract to specifically adsorb the epothilone B; vibrating on a shaking table, and centrifuging and collecting lower-layer sediment; eluting the sediment by using methanol for a plurality of times and collecting eluting liquid; re-crystallizing to obtain the epothilone B. The separation and extraction method has the characteristics of simplicity and convenience in operation, good selectivity, low pollution and the like. The separation and extraction method is simple and feasible in operation steps, few in separation steps, low in cost and high in yield; the loss of the epothilone B in other complicated preparation flows is avoided; more importantly, the separation and extraction method is convenient for industrial large-scale production and has very wide application prospect and economic benefits.
Description
Technical field
The present invention relates to a kind of method of the separation and Extraction epothilone B that membrane filtration technique is combined with molecular imprinting, be specifically related to a kind of epothilone B separating and extracting method based on molecular imprinting adsorption technology.
Background technology
Epothilone B (Epothilone B) is the class Macrolide secondary metabolite that microorganism sorangium cellulosum produces, and molecular formula is C
27h
41nO
6s, relative molecular weight is 506.25, fusing point is 93 ℃-94 ℃, has multiple biological activity, can be used as antibiotic medicine, is a study hotspot of current antitumor drug.
Research finds that epothilone B not only has the mechanism of action identical with taxol, can promote GTP (GTP (guanosine triphosphate)) dependency tubulin polymerization to form microtubule, and microtubule is had to stabilization.And thering is the characteristic more superior than taxol, water-soluble and great pharmaceutical potential simple in structure, good, makes people drop into huge enthusiasm and studies, to faster its exploitation being become to antitumor drug.Investigator puts into a lot of energy in and separation and Extraction synthetic to it, but, for the separating-purifying of ebormycine, currently used method all has following defect and deficiency: step is too loaded down with trivial details, make the efficiency of operation compared with low and process expense is very high, efficiency of pcr product is lower, and cost is higher, is especially difficult to realize industrialized scale operation.Thereby research is the novel method of separation and purification ebormycine effectively, to reduce the cost of ebormycine, tool is of great significance.
Along with the progress of biochemical isolation technique, various advanced persons' equipment and process is played a greater and greater role aborning, and the particularly development of membrane technique and application greatly taken on a new look various biochemical separating technologies.Membrane filtration technique is according to its pore size, can stop the solute of the various different molecular weights in solution compared with usual method, have without the conversion of phase, do not need to add that chemical reagent, filter membrane are difficult for stopping up and the advantage such as durable, to reach object concentrated or that separate.
Molecular imprinting is the emerging technology growing up in separation field in recent ten years.Molecularly imprinted polymer has predetermined selectivity, identification specifically to target molecule, target molecule can be separated from mixing solutions.And there is the advantages such as anti-adverse environment ability is strong, good stability, long service life, make the example in the existing successful Application of medicine separation method.
Summary of the invention
The object of this invention is to provide a kind of epothilone B separating and extracting method based on molecular imprinting adsorption technology, it is easy and simple to handle, and separating step is few, selectivity is high, separation efficiency significantly promotes compared with conventional art, has greatly reduced cost for purification, and is suitable for large-scale industrial production.
For achieving the above object, the present invention adopts following technical scheme:
(1) resin adsorbs fermented liquid: will in sorangium cellulosum liquid medium within, ferment, and add treated macroporous resin in liquid nutrient medium, after fermentation 120～150h, filter and obtain polymeric adsorbent; Wherein, the add-on of macroporous resin is the 2-5% of liquid nutrient medium volume;
(2) ethyl acetate lixiviate: with ethyl acetate lixiviate polymeric adsorbent, collect vat liquor;
(3) use ultrafiltration membrance filter vat liquor: it is that 1000～5000 ultrafiltration membrance filter is to remove macromole impurity that vat liquor is adopted to molecular weight cut-off; And use ethyl acetate filter wash, merging filtrate, obtains crude extract;
(4) add molecularly imprinted polymer: in crude extract, add epothilone B molecularly imprinted polymer to carry out specific adsorption epothilone B, centrifugal and collect lower sediment thing after concussion on shaking table, with methyl alcohol, throw out is carried out to repeatedly wash-out again, collect elutriant; Wherein, the add-on of epothilone B molecularly imprinted polymer is 5～10% of crude extract volume;
(5) crystallization: elutriant is carried out to vacuum concentration, obtain light yellow concentrated solution; Concentrated solution is carried out to low temperature crystallization at-20～-10 ℃, obtain white powder crystallization, be epothilone B.
In described step (1), sorangium cellulosum bacterial strain number is ATCC25532 or ATCC25569; Liquid culture based component is potato starch 2.5～3.0g/L, sucrose 0.7～1.0g/L, glucose 0.2～0.5g/L, soybean cake powder 1.7～2.0g/L, MgSO
47H
2o2.3～2.5g/L, CaCl
23.0～3.5g/L, EDTA-Fe
3+2mL/L, trace element 0.5～1.0mL/L, pH value is 7.2; The model of macroporous resin is XAD-16 macroporous resin.
Macroporous resin is adopted with the following method and is processed in described step (1): first, soak macroporous resin with the methyl alcohol of 3～5 times of macroporous resin volumes, after shaking table concussion 12-16h, methyl alcohol is outwelled in taking-up, then is washed till without methyl alcohol taste with pure water; And then soak after 12-16h with methyl alcohol, remove methyl alcohol and be washed till without methyl alcohol taste with pure water.
The consumption of the middle ethyl acetate of described step (2) is 3～5 times of macroporous resin volume, and the time of lixiviate is 24-48h.
In described step (3), the material of ultra-filtration membrane is polysulfones, polyacrylonitrile or polymeric amide.
In described step (3), the pressure of ultrafiltration membrance filter is 0.1-0.6MPa, temperature 0-40 ℃, and flow velocity is 5-10mL/min.
In described step (4), the particle diameter of epothilone B molecularly imprinted polymer is 40-60um; In step (4), the rotating speed of shaking table is 100～160r/min, and the concussion time is 24-48h; Centrifugal in step (4) is the centrifugal 20～30min of rotating speed with 2500～3000r/min at room temperature.
In described step (4), epothilone B molecularly imprinted polymer is made by following methods: after epothilone B, methacrylic acid, ethylene glycol dimethacrylate are mixed according to mol ratio 1:4:20, being dissolved in volume ratio is in the ethanol of 4:1 and the mixing solutions of methyl alcohol again, after stirring, adds azo-bis-isobutyl cyanide; Fill N
2after reaction 12-14h, grind to obtain particulate solid, finally use methanol-eluted fractions epothilone B; Wherein, Diisopropyl azodicarboxylate is 5mg:1mol with the ratio of epothilone B.
The molecularly imprinted polymer of sloughing epothilone B is through methanol wash, then after vacuum-drying, can also add when the separation and Extraction as the specific adsorption agent of epothilone B, and reusing number of times is 2～5 times.
40～45 ℃ of concentrated temperature in described step (5), vacuum tightness-0.085～-0.09MPa.
Compared with prior art, the present invention has following beneficial effect: the method for membrane filtration technique separation and Extraction epothilone B provided by the present invention, a micromolecular feature from epothilone B upon adsorption, selecting ultra-filtration membrane is filtration medium, under certain pressure, macromolecular substance is trapped within the liquid feeding side of ultra-filtration membrane, only allow the small-molecule substance of epothilone B and small part to pass through, thereby effectively reach the object separating, and the method has easy and simple to handle, selectivity is good, the features such as low pollution.
In the present invention, adopt epothilone B molecularly imprinted polymer, on the one hand because this imprinted polymer has efficient selective and specific recognition ability, thereby can carry out specific adsorption to epothilone B, it is separated from mixing solutions, the epothilone B purity making is higher, and operation steps is simple; Molecularly imprinted polymer only need can be reused through drying treatment on the other hand, has so just greatly reduced the production cost of separation and Extraction ebormycine medicine.
In the present invention, operation steps is simple, and separating step is few, with low cost, and productive rate is higher, has avoided the loss of epothilone B in other complicated preparation flow, the more important thing is and be convenient to industrialized production to have very wide application prospect and economic benefit.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in further detail, and the explanation of the invention is not limited.
Embodiment 1
(1) resin adsorbs fermented liquid: will in sorangium cellulosum ATCC25532 liquid medium within, ferment, and add treated macroporous resin XAD-16 in liquid nutrient medium, after fermentation 150h, filter and obtain polymeric adsorbent, then clean with distilled water the sorangium cellulosum of removing polymeric adsorbent surface adhesion;
Wherein, 2% of the volume that the add-on of described macroporous resin is liquid nutrient medium;
Described liquid culture based component is potato starch 2.5～3.0g/L, sucrose 0.7～1.0g/L, glucose 0.2～0.5g/L, soybean cake powder 1.7～2.0g/L, MgSO
47H
2o2.3～2.5g/L, CaCl
23.0～3.5g/L, EDTA-Fe
3+2mL/L, trace element 0.5～1.0mL/L, pH value is 7.2.
Described macroporous resin is adopted with the following method and is processed: first, soak macroporous resin with the methyl alcohol of 3 times of macroporous resin volumes, after shaking table concussion 12h, take out and outwell methyl alcohol, then be washed till without methyl alcohol taste with pure water; And then soak after 12h with methyl alcohol, remove methyl alcohol and be washed till without methyl alcohol taste with pure water; Macroporous resin is XAD-16 type macroporous resin.
(2) ethyl acetate lixiviate: with after the macroporous resin 24h cleaning through distilled water in ethyl acetate lixiviate step (1), collect vat liquor; Wherein the consumption of ethyl acetate is 3 times of macroporous resin volume;
(3) use ultrafiltration membrance filter vat liquor: it is 1000 ultrafiltration membrance filter that vat liquor is adopted to molecular weight cut-off, removes macromole impurity, and ultrafiltration pressure is 0.3MPa, 20 ℃ of temperature, coutroi velocity 5mL/min, and with 50mL ethyl acetate filter wash 1 time, merging filtrate, obtains crude extract; Wherein the material of ultra-filtration membrane is polyacrylonitrile.
(4) add molecularly imprinted polymer: in crude extract, add epothilone B molecularly imprinted polymer to carry out specific adsorption epothilone B, on the shaking table that is 100r/min at rotating speed, shake after 24h; At room temperature, the centrifugal 20min of 2500r/min, the centrifugal and molecularly imprinted polymer of epothilone B of having collected absorption in liquid, carries out wash-out with methyl alcohol, collects elutriant;
Wherein, the add-on of epothilone B molecularly imprinted polymer is about 5% of crude extract volume; The particle diameter of epothilone B molecularly imprinted polymer is 40-60um.
Described epothilone B molecularly imprinted polymer is adopted with the following method and is obtained: by template molecule (epothilone B), function monomer (methacrylic acid), after linking agent (ethylene glycol dimethacrylate) mixes according to mol ratio 1:4:20, being dissolved in volume ratio is in the ethanol of 4:1 and the mixing solutions of methyl alcohol again, stirs and adds pore-creating agent azo-bis-isobutyl cyanide; Fill N
2reaction 12h, finally uses methanol-eluted fractions template molecule (epothilone B); Wherein, Diisopropyl azodicarboxylate is 5mg:1mol with the ratio of epothilone B;
(5) crystallization: elutriant is carried out to vacuum concentration, and thickening temperature is 40 ℃, and vacuum tightness is-0.085MPa, obtain light yellow concentrated solution, concentrated solution is put into clean crystallizer-20 ℃ low temperature crystallization, can obtain a large amount of white powder crystallizations in crystallizing dish bottom, be epothilone B.
Embodiment 2
(1) resin adsorbs fermented liquid: will in sorangium cellulosum ATCC25569 liquid medium within, ferment, and add treated macroporous resin XAD-16 in liquid nutrient medium, after fermentation 120h, filter and obtain polymeric adsorbent, then clean with distilled water the sorangium cellulosum of removing polymeric adsorbent surface adhesion;
Wherein, 5% of the volume that the add-on of described macroporous resin is liquid nutrient medium;
Described liquid culture based component is potato starch 2.5～3.0g/L, sucrose 0.7～1.0g/L, glucose 0.2～0.5g/L, soybean cake powder 1.7～2.0g/L, MgSO
47H
2o2.3～2.5g/L, CaCl
23.0～3.5g/L, EDTA-Fe
3+2mL/L, trace element 0.5～1.0mL/L, pH value is 7.2.
Described macroporous resin is adopted with the following method and is processed: first, soak macroporous resin with the methyl alcohol of 5 times of macroporous resin volumes, after shaking table concussion 16h, take out and outwell methyl alcohol, then be washed till without methyl alcohol taste with pure water; And then soak after 16h with methyl alcohol, remove methyl alcohol and be washed till without methyl alcohol taste with pure water; Macroporous resin is XAD-16 type macroporous resin.
(2) ethyl acetate lixiviate: with after the macroporous resin 48h cleaning through distilled water in ethyl acetate lixiviate step (1), collect vat liquor; Wherein the consumption of ethyl acetate is 5 times of resin volume;
(3) use ultrafiltration membrance filter vat liquor: it is 2000 ultrafiltration membrance filter that vat liquor is adopted to molecular weight cut-off, removes macromole impurity, and ultrafiltration pressure is 0.6MPa, 40 ℃ of temperature, coutroi velocity 10mL/min, and with ethyl acetate filter wash 3 times, each 50mL, merging filtrate, obtains crude extract; Wherein the material of ultra-filtration membrane is polymeric amide.
(4) add molecularly imprinted polymer: in crude extract, add epothilone B molecularly imprinted polymer to carry out specific adsorption epothilone B, on the shaking table that is 150r/min at rotating speed, shake after 48h; At room temperature, the centrifugal 30min of 3000r/min, the centrifugal and molecularly imprinted polymer of epothilone B of having collected absorption in liquid, carries out wash-out with methyl alcohol, collects elutriant;
Wherein, the add-on of epothilone B molecularly imprinted polymer is about 10% of crude extract volume; The particle diameter of epothilone B molecularly imprinted polymer is 40-60um.
Described epothilone B molecularly imprinted polymer is adopted with the following method and is obtained: by template molecule (epothilone B), function monomer (methacrylic acid), after linking agent (ethylene glycol dimethacrylate) mixes according to mol ratio 1:4:20, being dissolved in volume ratio is in the ethanol of 4:1 and the mixing solutions of methyl alcohol again, adds pore-creating agent azo-bis-isobutyl cyanide after stirring; Fill N
2reaction 13h, finally uses methanol-eluted fractions template molecule (epothilone B); Wherein, Diisopropyl azodicarboxylate is 5mg:1mol with the ratio of epothilone B;
(5) crystallization: above-mentioned merging epothilone B component is carried out to vacuum concentration, and thickening temperature is 45 ℃, and vacuum tightness is-0.09MPa to obtain light yellow concentrated solution; Concentrated solution is put into clean crystallizer-10 ℃ low temperature crystallization, can obtain a large amount of white powder crystallizations in crystallizing dish bottom, be epothilone B.
Embodiment 3
(1) resin adsorbs fermented liquid: will in sorangium cellulosum ATCC25532 liquid medium within, ferment, and add treated macroporous resin XAD-16 in liquid nutrient medium, after fermentation 140h, filter and obtain polymeric adsorbent, then clean with distilled water the sorangium cellulosum of removing polymeric adsorbent surface adhesion;
Wherein, 4% of the volume that the add-on of described macroporous resin is liquid nutrient medium;
Described liquid culture based component is potato starch 2.5～3.0g/L, sucrose 0.7～1.0g/L, glucose 0.2～0.5g/L, soybean cake powder 1.7～2.0g/L, MgSO
47H
2o2.3～2.5g/L, CaCl
23.0～3.5g/L, EDTA-Fe
3+2mL/L, trace element 0.5～1.0mL/L, pH value is 7.2.
Described macroporous resin is adopted with the following method and is processed: first, soak macroporous resin with the methyl alcohol of 4 times of macroporous resin volumes, after shaking table concussion 14h, take out and outwell methyl alcohol, then be washed till without methyl alcohol taste with pure water; And then soak after 13h with methyl alcohol, remove methyl alcohol and be washed till without methyl alcohol taste with pure water; Macroporous resin is XAD-16 type macroporous resin.
(2) ethyl acetate lixiviate: with after the macroporous resin 33h cleaning through distilled water in ethyl acetate lixiviate step (1), collect vat liquor; Wherein the consumption of ethyl acetate is 4 times of macroporous resin volume;
(3) use ultrafiltration membrance filter vat liquor: it is 5000 ultrafiltration membrance filter that vat liquor is adopted to molecular weight cut-off, removes macromole impurity, and ultrafiltration pressure is 0.1MPa, 0 ℃ of temperature, coutroi velocity 7mL/min, and with ethyl acetate filter wash 2 times, each 50mL, merging filtrate, obtains crude extract; Wherein the material of ultra-filtration membrane is polysulfones.
(4) add molecularly imprinted polymer: in crude extract, add epothilone B molecularly imprinted polymer to carry out specific adsorption epothilone B, on the shaking table that is 160r/min at rotating speed, shake after 30h; At room temperature, the centrifugal 25min of 2700r/min, the centrifugal and molecularly imprinted polymer of epothilone B of having collected absorption in liquid, carries out wash-out with methyl alcohol, collects elutriant;
Wherein, the add-on of epothilone B molecularly imprinted polymer is about 7% of crude extract volume; The particle diameter of epothilone B molecularly imprinted polymer is 40-60um.
Described epothilone B molecularly imprinted polymer is adopted with the following method and is obtained: by template molecule (epothilone B), function monomer (methacrylic acid), after linking agent (ethylene glycol dimethacrylate) mixes according to mol ratio 1:4:20, being dissolved in volume ratio is in the ethanol of 4:1 and the mixing solutions of methyl alcohol again, adds pore-creating agent azo-bis-isobutyl cyanide after stirring; Fill N
2reaction 14h, finally uses methanol-eluted fractions template molecule (epothilone B); Wherein, Diisopropyl azodicarboxylate is 5mg:1mol with the ratio of epothilone B;
(5) crystallization: elutriant is carried out to vacuum concentration, and thickening temperature is 40 ℃, and vacuum tightness-0.085MPa obtains light yellow concentrated solution; / 10th left and right of initial volume will be concentrated into; Concentrated solution is put into clean crystallizer-15 ℃ low temperature crystallization, can obtain a large amount of white powder crystallizations in crystallizing dish bottom, be epothilone B.
In the present invention, slough the molecularly imprinted polymer of epothilone B after methyl alcohol fully washs, then after vacuum-drying, can also add when the separating-purifying as the specific adsorption agent of epothilone B, reusing number of times is 2～5 times.
The invention provides a kind of epothilone B separating and extracting method based on molecular imprinting adsorption technology, its step is through macroporous resin adsorption, and distilled water cleans removes the thalline that resin surface adheres to, and ethyl acetate obtains vat liquor after resolving; First vat liquor removes the impurity such as macromole through ultra-filtration membrane; Carry out specific adsorption epothilone B through molecularly imprinted polymer again, resolve with methyl alcohol again after centrifugal; Obtain the crystal of epothilone B finally by mistake crystallization.The present invention has the following advantages: efficient selective, and simple to operate, the feature such as separating step is few, and cost is low, low pollution, the prior favorable method that is to provide large-scale industrial production, has more wide industrial applications prospect and economic benefit.
Method operation steps provided by the present invention is simple, has high-level efficiency, low cost, and highly purified advantage, can adapt to large-scale industrial production.
Claims (10)
1. the epothilone B separating and extracting method based on molecular imprinting adsorption technology, is characterized in that, comprises the following steps:
(1) resin adsorbs fermented liquid: will in sorangium cellulosum liquid medium within, ferment, and add treated macroporous resin in liquid nutrient medium, after fermentation 120～150h, filter and obtain polymeric adsorbent; Wherein, the add-on of macroporous resin is the 2-5% of liquid nutrient medium volume;
(2) ethyl acetate lixiviate: with ethyl acetate lixiviate polymeric adsorbent, collect vat liquor;
(3) use ultrafiltration membrance filter vat liquor: by vat liquor adopt molecular weight cut-off be 1000～5000 ultrafiltration membrance filter to remove macromole impurity, obtain crude extract;
(4) add molecularly imprinted polymer: in crude extract, add epothilone B molecularly imprinted polymer to carry out specific adsorption epothilone B, centrifugal and collect lower sediment thing after concussion on shaking table, with methyl alcohol, throw out is carried out to repeatedly wash-out again, collect elutriant; Wherein, the add-on of epothilone B molecularly imprinted polymer is 5～10% of crude extract volume;
(5) crystallization: elutriant is carried out to vacuum concentration, obtain light yellow concentrated solution; Concentrated solution is carried out to low temperature crystallization at-20～-10 ℃, obtain white powder crystallization, be epothilone B.
2. a kind of epothilone B separating and extracting method based on molecular imprinting adsorption technology according to claim 1, is characterized in that, in described step (1), sorangium cellulosum bacterial strain number is ATCC25532 or ATCC25569; Liquid culture based component is potato starch 2.5～3.0g/L, sucrose 0.7～1.0g/L, glucose 0.2～0.5g/L, soybean cake powder 1.7～2.0g/L, MgSO
47H
2o2.3～2.5g/L, CaCl
23.0～3.5g/L, EDTA-Fe
3+2mL/L, trace element 0.5～1.0mL/L, pH value is 7.2; The model of macroporous resin is XAD-16 macroporous resin.
3. a kind of epothilone B separating and extracting method based on molecular imprinting adsorption technology according to claim 1, it is characterized in that, in described step (1), macroporous resin is adopted with the following method and is processed: first, with the methyl alcohol immersion macroporous resin of 3～5 times of macroporous resin volumes, after shaking table concussion 12-16h, methyl alcohol is outwelled in taking-up, then is washed till without methyl alcohol taste with pure water; And then soak after 12-16h with methyl alcohol, remove methyl alcohol and be washed till without methyl alcohol taste with pure water.
4. a kind of epothilone B separating and extracting method based on molecular imprinting adsorption technology according to claim 1, is characterized in that, the consumption of the middle ethyl acetate of described step (2) is 3～5 times of macroporous resin volume, and the time of lixiviate is 24-48h.
5. a kind of epothilone B separating and extracting method based on molecular imprinting adsorption technology according to claim 1, is characterized in that, in described step (3), the material of ultra-filtration membrane is polysulfones, polyacrylonitrile or polymeric amide.
6. a kind of epothilone B separating and extracting method based on molecular imprinting adsorption technology according to claim 1, is characterized in that, in described step (3), the pressure of ultrafiltration membrance filter is 0.1-0.6MPa, temperature 0-40 ℃, and flow velocity is 5-10mL/min.
7. a kind of epothilone B separating and extracting method based on molecular imprinting adsorption technology according to claim 1, is characterized in that, in described step (4), the particle diameter of epothilone B molecularly imprinted polymer is 40-60um; In step (4), the rotating speed of shaking table is 100～160r/min, and the concussion time is 24-48h; Centrifugal in step (4) is the centrifugal 20～30min of rotating speed with 2500～3000r/min at room temperature.
8. a kind of epothilone B separating and extracting method based on molecular imprinting adsorption technology according to claim 1, it is characterized in that, in described step (4), epothilone B molecularly imprinted polymer is made by following methods: after epothilone B, methacrylic acid, ethylene glycol dimethacrylate are mixed according to mol ratio 1:4:20, being dissolved in volume ratio is in the ethanol of 4:1 and the mixing solutions of methyl alcohol again, after stirring, adds azo-bis-isobutyl cyanide; Fill N
2after reaction 12-14h, grind to obtain particulate solid, finally use methanol-eluted fractions epothilone B; Wherein, Diisopropyl azodicarboxylate is 5mg:1mol with the ratio of epothilone B.
9. a kind of epothilone B separating and extracting method based on molecular imprinting adsorption technology according to claim 1, it is characterized in that, slough the molecularly imprinted polymer of epothilone B through methanol wash, then after vacuum-drying, can also add when the separation and Extraction as the specific adsorption agent of epothilone B, reusing number of times is 2～5 times.
10. a kind of epothilone B separating and extracting method based on molecular imprinting adsorption technology according to claim 1, is characterized in that, 40～45 ℃ of concentrated temperature in described step (5), vacuum tightness-0.085～-0.09MPa.
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Citations (4)
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| WO2006098585A1 (en) * | 2005-03-15 | 2006-09-21 | Sungkyunkwan University Foundation Corporate Collaboration | Method for preparing a useful secondary metabolite by effective elimination of biological by-products |
| CN102373252A (en) * | 2011-11-04 | 2012-03-14 | 陕西科技大学 | Fermentation production process of Epothilone B |
| CN102432753A (en) * | 2011-09-02 | 2012-05-02 | 陕西科技大学 | Preparation method of epothilone B molecularly imprinted polymer |
| CN103243134A (en) * | 2013-04-15 | 2013-08-14 | 陕西科技大学 | Fermentation production method based on epothilone B metabolic pathways |
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| WO2006098585A1 (en) * | 2005-03-15 | 2006-09-21 | Sungkyunkwan University Foundation Corporate Collaboration | Method for preparing a useful secondary metabolite by effective elimination of biological by-products |
| CN102432753A (en) * | 2011-09-02 | 2012-05-02 | 陕西科技大学 | Preparation method of epothilone B molecularly imprinted polymer |
| CN102373252A (en) * | 2011-11-04 | 2012-03-14 | 陕西科技大学 | Fermentation production process of Epothilone B |
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