CN113621010A - Method for separating and extracting cytidine from fermentation liquor - Google Patents
Method for separating and extracting cytidine from fermentation liquor Download PDFInfo
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- CN113621010A CN113621010A CN202110942265.3A CN202110942265A CN113621010A CN 113621010 A CN113621010 A CN 113621010A CN 202110942265 A CN202110942265 A CN 202110942265A CN 113621010 A CN113621010 A CN 113621010A
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- cytidine
- resin
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- cation exchange
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- UHDGCWIWMRVCDJ-UHFFFAOYSA-N 1-beta-D-Xylofuranosyl-NH-Cytosine Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 UHDGCWIWMRVCDJ-UHFFFAOYSA-N 0.000 title claims abstract description 108
- UHDGCWIWMRVCDJ-PSQAKQOGSA-N Cytidine Natural products O=C1N=C(N)C=CN1[C@@H]1[C@@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-PSQAKQOGSA-N 0.000 title claims abstract description 108
- UHDGCWIWMRVCDJ-ZAKLUEHWSA-N cytidine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-ZAKLUEHWSA-N 0.000 title claims abstract description 108
- 238000000034 method Methods 0.000 title claims abstract description 43
- 238000000855 fermentation Methods 0.000 title claims abstract description 31
- 230000004151 fermentation Effects 0.000 title claims abstract description 31
- 239000013078 crystal Substances 0.000 claims abstract description 77
- 239000011347 resin Substances 0.000 claims abstract description 34
- 229920005989 resin Polymers 0.000 claims abstract description 34
- 239000003480 eluent Substances 0.000 claims abstract description 22
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 claims abstract description 20
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000003729 cation exchange resin Substances 0.000 claims abstract description 15
- 239000007788 liquid Substances 0.000 claims abstract description 15
- 238000001179 sorption measurement Methods 0.000 claims abstract description 12
- YPNVIBVEFVRZPJ-UHFFFAOYSA-L silver sulfate Chemical compound [Ag+].[Ag+].[O-]S([O-])(=O)=O YPNVIBVEFVRZPJ-UHFFFAOYSA-L 0.000 claims abstract description 10
- 229910000367 silver sulfate Inorganic materials 0.000 claims abstract description 10
- 239000012528 membrane Substances 0.000 claims description 56
- 238000005406 washing Methods 0.000 claims description 54
- 239000012535 impurity Substances 0.000 claims description 38
- 239000012466 permeate Substances 0.000 claims description 36
- 238000001728 nano-filtration Methods 0.000 claims description 32
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 23
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 23
- 239000000919 ceramic Substances 0.000 claims description 23
- 238000010828 elution Methods 0.000 claims description 22
- 238000001914 filtration Methods 0.000 claims description 19
- 230000008569 process Effects 0.000 claims description 17
- 238000003756 stirring Methods 0.000 claims description 9
- 238000001816 cooling Methods 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 239000002904 solvent Substances 0.000 claims description 6
- 230000004048 modification Effects 0.000 claims description 5
- 238000012986 modification Methods 0.000 claims description 5
- SCYULBFZEHDVBN-UHFFFAOYSA-N 1,1-Dichloroethane Chemical compound CC(Cl)Cl SCYULBFZEHDVBN-UHFFFAOYSA-N 0.000 claims description 3
- UOCLXMDMGBRAIB-UHFFFAOYSA-N 1,1,1-trichloroethane Chemical compound CC(Cl)(Cl)Cl UOCLXMDMGBRAIB-UHFFFAOYSA-N 0.000 claims description 2
- JEGUKCSWCFPDGT-UHFFFAOYSA-N h2o hydrate Chemical compound O.O JEGUKCSWCFPDGT-UHFFFAOYSA-N 0.000 claims description 2
- 238000005341 cation exchange Methods 0.000 abstract description 5
- 238000001035 drying Methods 0.000 description 19
- 238000002425 crystallisation Methods 0.000 description 14
- 239000012452 mother liquor Substances 0.000 description 13
- 238000000967 suction filtration Methods 0.000 description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 12
- 230000008025 crystallization Effects 0.000 description 12
- 239000012530 fluid Substances 0.000 description 12
- 238000005070 sampling Methods 0.000 description 12
- 239000012465 retentate Substances 0.000 description 10
- 238000000926 separation method Methods 0.000 description 10
- 239000000049 pigment Substances 0.000 description 8
- 238000001291 vacuum drying Methods 0.000 description 8
- 238000011068 loading method Methods 0.000 description 7
- 238000002156 mixing Methods 0.000 description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- 238000001704 evaporation Methods 0.000 description 6
- 230000008020 evaporation Effects 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 239000008399 tap water Substances 0.000 description 6
- 235000020679 tap water Nutrition 0.000 description 6
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- 150000001768 cations Chemical class 0.000 description 4
- 239000007791 liquid phase Substances 0.000 description 4
- 230000000813 microbial effect Effects 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000001308 synthesis method Methods 0.000 description 3
- KCURWTAZOZXKSJ-IAIGYFSYSA-N 4-amino-1-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidin-2-one;hydrochloride Chemical compound Cl.O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 KCURWTAZOZXKSJ-IAIGYFSYSA-N 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- -1 cytosine nucleoside Chemical class 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 230000003068 static effect Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000007738 vacuum evaporation Methods 0.000 description 2
- RZZPDXZPRHQOCG-OJAKKHQRSA-M CDP-choline(1-) Chemical compound O[C@@H]1[C@H](O)[C@@H](COP([O-])(=O)OP([O-])(=O)OCC[N+](C)(C)C)O[C@H]1N1C(=O)N=C(N)C=C1 RZZPDXZPRHQOCG-OJAKKHQRSA-M 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 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 description 1
- 241001085205 Prenanthella exigua Species 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000002259 anti human immunodeficiency virus agent Substances 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 229960001284 citicoline Drugs 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Natural products NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 238000004042 decolorization Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012269 metabolic engineering Methods 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 238000010587 phase diagram Methods 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002718 pyrimidine nucleoside Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002910 solid waste Substances 0.000 description 1
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 1
- 229940045145 uridine Drugs 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/06—Pyrimidine radicals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Saccharide Compounds (AREA)
Abstract
The invention discloses a method for separating and extracting cytidine from fermentation liquor, which comprises the steps of adsorbing and eluting liquid containing cytidine by cation exchange resin, and concentrating and crystallizing the obtained eluent to obtain cytidine crystals; wherein the cation exchange resin is obtained by modifying NH-1 resin with silver sulfate and/or sulfuric acid. The cation exchange column used in the invention is modified NH-1 resin, and the cytidine adsorption capacity of the cation exchange column reaches 0.3g/g, so that the resin dosage is reduced, and the acid-base dosage and the operation time are indirectly saved.
Description
Technical Field
The invention belongs to the technical field of biochemical separation, and particularly relates to a method for separating and extracting cytidine from fermentation liquor.
Background
Cytidine is pyrimidine nucleoside, an intermediate of citicoline, and an intermediate of a plurality of antiviral, antitumor, anti-AIDS drugs and the like. Cytidine is widely used in various industries such as food, health products, cosmetics, and medicines. Cytidine is especially an important intermediate for drug synthesis in modern medicine, and as the application range of cytidine is continuously expanded, the demand for cytidine is increased, and the preparation research of cytidine is naturally deepened. At present, the development of a low-cost cytosine nucleoside production separation and purification process which can be applied in a large scale is urgently needed.
At present, the production of cytidine at home and abroad mainly comprises a chemical synthesis method, an enzymatic synthesis method and a microbial fermentation synthesis method. The microbial fermentation synthesis method is mainly used, and many reports are reported on how to breed efficient and high-yield microbial strains, but the research reports on the large-scale separation and purification process of cytidine are less. Therefore, the present invention provides a method for separating and extracting cytidine from a fermentation broth.
Disclosure of Invention
The purpose of the invention is as follows: the technical problem to be solved by the invention is to provide a method for separating and extracting cytidine aiming at the defects of the prior art.
In order to solve the technical problems, the invention discloses a method for separating and extracting cytidine, which comprises the steps of adsorbing and eluting liquid containing cytidine by cation exchange resin, and concentrating and crystallizing the obtained eluent to obtain cytidine crystals.
Wherein the cation exchange resin is obtained by modifying NH-1 resin with silver sulfate and/or sulfuric acid.
Wherein the adsorption capacity of the cation exchange resin is 0.2-0.4 g/g; preferably, the adsorption amount of the cation exchange resin is 0.3 g/g.
Wherein the dosage of the silver sulfate is 2-8% of the mass of the NH-1 resin; preferably, the dosage of the silver sulfate is 4 to 6 percent of the mass of the NH-1 resin; preferably, the amount of silver sulfate used is 5% by mass of the NH-1 resin.
Wherein the dosage of the sulfuric acid is 10-20 times of the volume of the resin.
Wherein, the solvent in the modification process is dichloroethane and/or trichloroethane.
Wherein the volume ratio of the NH-1 resin to the solvent is 1: (6-20).
Wherein, NH-1 resin is mixed with solvent and kept for 0.5 to 2 hours at the temperature of between 50 and 70 ℃; adding silver sulfate and/or sulfuric acid, maintaining at 70-90 deg.C for 2-4 hr, maintaining at 90-100 deg.C for 2-3 hr, filtering, and washing.
Wherein the washing is to neutral.
Wherein the washing is washing with any one or more organic solvents selected from methanol, ethanol and acetone
The static adsorption experiment after the modification by the method shows that the adsorption capacity of the resin is improved to 0.3g/g from the original 0.09 g/g.
Preferably, the method for separating and extracting cytidine comprises the following steps:
(1) filtering the cytidine fermentation liquor by a ceramic membrane to remove solid impurities to obtain a first permeate;
(2) adsorbing the first permeate obtained in the step (1) by using cation exchange resin, washing impurities by using water and/or ammonia water, eluting by using ammonia water, continuously detecting by using HPLC (high performance liquid chromatography) in the process, and directly switching to cytidine elution when detecting that cytidine flows out to obtain eluent;
(3) synchronously performing nanofiltration and decoloration on the eluent obtained in the step (2) through a nanofiltration membrane to obtain a second permeate;
(4) and (4) concentrating the second permeate obtained in the step (3) through a nanofiltration membrane, carrying out vacuum evaporation concentration, adjusting the pH of the obtained concentrated solution, adding a small amount of seed crystals as an inducer, stirring, cooling, crystallizing, filtering, washing and drying to obtain cytidine crystals.
In the step (1), cytidine is derived from microbial fermentation liquor, uridine high-yield strains obtained through system metabolic engineering are cultured in a 50L fermentation tank, and the initial yield of cytidine is 30-60 g/L. The fermentation liquor contains a large amount of thalli, culture medium, pigment and other impurities, and the separation difficulty is high.
In the step (1), the cytidine fermentation liquor is filtered by a ceramic membrane after the pH is adjusted; preferably, the pH is 2-3.5.
In the step (1), the aperture of the ceramic membrane is 50-200nm, and the pressure of the ceramic membrane is 0.1-0.3 MPa.
In the step (1), the filtering temperature is normal temperature.
In the step (2), the sample loading speed of the first permeation solution to the cation exchange resin is 1-2 BV/h.
In the step (2), in the impurity washing process, the concentration of ammonia water is 0-0.05 mol/L; preferably, the concentration of ammonia is 0.03 mol/L.
In the step (2), the flow rate of the impurity washing is 1-2 BV/h.
In the step (2), the volume of the impurities is 4-14 BV.
In the step (2), the concentration of ammonia water is 0.05-0.1mol/L in the elution process; preferably, the concentration of ammonia is 0.06 mol/L.
In the step (2), the flow rate of elution is 1-2 BV/h.
In the step (2), the elution volume is 10-30 BV; preferably, elution is ended until the elution is spotted with no cytidine present.
In the step (3), the molecular weight cut-off of the nanofiltration membrane is 300-800 Da.
In the step (3), the pressure of the nanofiltration membrane is 1-3 MPa.
In the step (4), the molecular weight cut-off of the nanofiltration membrane is 60-200 Da; preferably, the molecular weight cut-off of the nanofiltration membrane is 60-120 Da; preferably, the nanofiltration membrane has a molecular weight cut-off of 90 Da.
In the step (4), the vacuum degree of the vacuum evaporation concentration is less than 100mbar, and the water bath temperature is 45-70 ℃; preferably, the bath temperature is 50 ℃.
In the step (4), the concentration is up to 800 g/L; preferably, the concentration is up to 400-; further preferably, the concentration is to 600 g/L.
In the step (4), the pH is 5-10; preferably, the pH is 6-8; further preferably, the pH is 7.
In the step (4), the rotation speed of the stirring is 100-400 rpm.
In the step (4), crystal precipitation occurs after the seed crystal is added and stirred for 1-2 h.
In the step (4), the temperature is reduced to 0-10 ℃; preferably, the temperature is reduced to 2-6 ℃; further preferably, the temperature is reduced to 4 ℃.
In the step (4), the temperature is reduced by adopting a gradient temperature reduction method; preferably, crystallization is completed within 6-12 h.
In the step (4), the crystal system is a water system, so that the workshop reconstruction and production cost is reduced.
In the step (4), the washing is to wash the crystals by using an ethanol solution with the concentration of 95-99%, and the crystals are collected by centrifugation or suction filtration, so that the purity of the crystals can be improved, and the influence of impurities in the crystallization mother liquor can be reduced.
In the step (4), the drying is forced air drying, double cone drying or vacuum drying; preferably, the drying is vacuum drying; preferably, the vacuum is 0 to 200 mbar; preferably, the vacuum is 10-100 mbar.
In the step (4), the drying temperature is 40-100 ℃; preferably, the temperature of the drying is 50-80 ℃.
In the step (4), preferably, the yield is improved by secondary crystallization or nested crystallization.
The purity of the cytidine obtained by the method is more than 99 percent, and the total yield is 90 to 95 percent.
Has the advantages that: compared with the prior art, the invention has the following advantages:
(1) compared with the existing crystallization method of cytidine, the cytidine hydrochloride is firstly formed and then crystallized for the second time to obtain cytidine, and the cytidine crystal is obtained by adopting a one-step separation direct crystallization method, so that the process of obtaining the secondary crystallization of a crude cytidine hydrochloride product is omitted.
(2) The cation exchange column used in the invention is modified NH-1 resin, and the cytidine adsorption capacity of the cation exchange column reaches 0.3g/g, so that the resin dosage is reduced, and the acid-base dosage and the operation time are indirectly saved.
(3) In the invention, cytidine is adsorbed on a cation exchange column NH-1, the impurity washing concentration of an ammonia water solution is determined to be 0.03mol/L and the elution concentration is determined to be 0.06mol/L, and the optimal process parameters are determined and stably operated.
(4) The invention adopts the 300-800Da nanofiltration membrane for decolorization, can obtain clear light yellow solution, and adopts the 60-200Da membrane for concentration, and the process has no solid waste, high speed, high yield, no pollution and low cost.
(5) The crystallization system in the invention is a water system, and no reagent is added, thereby greatly reducing the crystallization cost. The crystallization process of the invention is completed within 12h to obtain bright white crystals, the yield of one-time crystallization is more than 50%, the yield of crystals is 90-95% after multiple crystallization or nested crystallization, meanwhile, the purity of the crystals is more than 99%, and the product quality, yield and cost are higher than those of the prior art.
(6) The method not only successfully realizes the problem of difficult separation of more impurities, but also has the advantages of high efficiency, low energy consumption, low cost, good quality, high yield and the like.
Drawings
The foregoing and/or other advantages of the invention will become further apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.
FIG. 1 shows a crystalline product of cytidine.
FIG. 2 shows the cytidine crystal structure.
FIG. 3 is a liquid phase purity detection profile.
FIG. 4 is a liquid phase diagram of the original fermentation liquid.
Detailed Description
The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.
The NH-1 resin described in the following examples was synthesized by Nanjing university of industry and was disclosed in Chinese patent CN106632519B, a process for separating nucleotides by continuous ion exchange chromatography, and other patents.
The cytidine-containing fermentation broth in the following examples is prepared by converting glucose, corn steep liquor, vitamins, inorganic salts, yeast extract and the like with escherichia coli to form cytidine-containing fermentation broth (with cytidine content of 32 g/L); the fermentation broth contains cytidine, and its components mainly comprise sugar, nucleic acid, salt, pigment, amino acids, protein, organic acid, etc., and its liquid phase is shown in FIG. 4.
The modified cation exchange resin NH-1 described in the following examples was prepared by mixing NH-1 resin with dichloroethane at a resin to solution volume ratio of 1: 6, maintaining the temperature at 70 ℃ for 1h, adding a silver sulfate catalyst accounting for 5 percent of the mass of the resin and 10 times of the volume of sulfuric acid, maintaining the temperature at 70 ℃ for 2h, heating to 90 ℃ for 3h, filtering to obtain modified cation resin, continuously washing the cation resin to be neutral by deionized water, then washing the cation resin by acetone, and washing the cation resin by water for later use. Static adsorption experiments show that the adsorption capacity of the modified resin is improved to 0.3g/g from the original 0.09g/g resin.
Example 1:
(1) adjusting pH of fermentation liquor containing cytidine to 2, filtering with 50nm ceramic membrane, collecting permeate, washing the retentate with tap water until the retentate contains no cytidine, and mixing to obtain ceramic membrane permeate.
(2) Loading the ceramic membrane permeate obtained in the step (1) to modified cation exchange resin NH-1215 g, adsorbing, washing impurities, eluting, and collecting eluent; the sampling rate is 1BV/h, and the sampling amount is 64g of cytidine fermentation broth clear liquid; in the impurity washing process, the concentration of ammonia water is 0.01mol/L, the flow rate is 1BV/h, and the impurity washing volume is 8 BV; in the elution process, the concentration of ammonia water is 0.05mol/L, the flow rate is 1BV/h, and the volume of elution is 14 BV.
(3) And (3) nano-filtering the eluent obtained in the step (2) by using a nano-filtration membrane of 300Da to remove macromolecular impurities such as pigments and the like, continuously washing the trapped fluid until the trapped fluid does not contain cytidine, and collecting the nano-filtration membrane permeate.
(4) Concentrating the permeate obtained in the step (3) to 50g/L through a nanofiltration membrane of 60Da, concentrating through evaporation to obtain cytidine with the concentration of 400g/L, adjusting the pH to 5.0 with hydrochloric acid, stirring at room temperature of 20 ℃ for 1h with seed crystal continuously at 200rpm, separating out crystals, and cooling to 4 ℃ in a gradient manner for 10 h.
(5) And (4) carrying out suction filtration on the crystals obtained in the step (4), removing the mother liquor, adding absolute ethyl alcohol, washing twice, and carrying out suction filtration to obtain the crystals.
(6) And (3) putting the crystals obtained in the step (5) into a vacuum drying oven at 40 ℃, and drying for 4h under 10mbar to obtain 35.6g of cytidine crystals, wherein the yield of cytidine once is 55.6%, the obtained cytidine product is shown in figure 1, the crystal structure is shown in figure 2, and the liquid phase is shown in figure 3.
(7) After the mother liquor obtained in the step (5) is crystallized for the second time and the third time, 15.62g of cytidine crystals and 6.45g of cytidine crystals are obtained after drying respectively, the purity of the cytidine crystals is 99.9%, and the total yield is 90.1%.
Comparative example 1
(1) Adjusting pH of fermentation liquor containing cytidine to 2, filtering with 50nm ceramic membrane, collecting permeate, washing the retentate with tap water until the retentate contains no cytidine, and mixing to obtain ceramic membrane permeate.
(2) Loading the ceramic membrane permeate obtained in the step (1) to 7650 g of cation exchange resin 001 x, adsorbing, washing impurities, eluting, and collecting eluent; the sampling rate is 1.5BV/h, and the sampling amount is 64g of cytidine fermentation broth clear liquid; in the impurity washing process, the concentration of ammonia water is 0.01mol/L, the flow rate is 1.5BV/h, and the impurity washing volume is 10 BV; in the elution process, the concentration of ammonia water is 0.05mol/L, the flow rate is 1BV/h, and the volume of elution is 15 BV.
(3) And (3) nano-filtering the eluent obtained in the step (2) by using a nano-filtration membrane of 300Da to remove macromolecular impurities such as pigments and the like, continuously washing the trapped fluid until the trapped fluid does not contain cytidine, and collecting the nano-filtration membrane permeate.
(4) Concentrating the permeate obtained in the step (3) to 50g/L through a nanofiltration membrane of 60Da, concentrating through evaporation to obtain cytidine with the concentration of 400g/L, adjusting the pH to 5.0 with hydrochloric acid, stirring at room temperature of 20 ℃ for 1h with seed crystal continuously at 200rpm, separating out crystals, and cooling to 4 ℃ in a gradient manner for 10 h.
(5) And (4) carrying out suction filtration on the crystals obtained in the step (4), removing the mother liquor, adding absolute ethyl alcohol, washing twice, and carrying out suction filtration to obtain the crystals.
(6) And (3) putting the crystals obtained in the step (5) into a vacuum drying oven at 40 ℃, and drying for 4 hours at 10mbar to obtain 30.7g of cytidine crystals, wherein the primary yield of cytidine is 47.9%.
(7) And (3) crystallizing the mother liquor obtained in the step (5) for two times and three times, and drying the crystals to obtain 15.65g and 7.4g of cytidine crystals respectively, wherein the purity of the cytidine crystals is 98.8 percent, and the total yield of the cytidine crystals is 84 percent.
The change of the resin causes the reduction of the adsorption amount and the separation degree, the increase of the resin usage amount, the increase of the volume of the whole eluent, the longer the flow time, and the more the acid-base amount of the regenerated resin.
Comparative example 2
(1) Adjusting pH of fermentation liquor containing cytidine to 2, filtering with 50nm ceramic membrane, collecting permeate, washing the retentate with tap water until the retentate contains no cytidine, and mixing to obtain ceramic membrane permeate.
(2) Loading the ceramic membrane permeate obtained in the step (1) to original cation exchange resin NH-1600 g, adsorbing, washing impurities, eluting, and collecting eluent; the sampling rate is 1.5BV/h, and the sampling amount is 64g of cytidine fermentation broth clear liquid; in the impurity washing process, the concentration of ammonia water is 0.01mol/L, the flow rate is 1.5BV/h, and the impurity washing volume is 10 BV; in the elution process, the concentration of ammonia water is 0.05mol/L, the flow rate is 1BV/h, and the volume of elution is 15 BV.
(3) And (3) nano-filtering the eluent obtained in the step (2) by using a nano-filtration membrane of 300Da to remove macromolecular impurities such as pigments and the like, continuously washing the trapped fluid until the trapped fluid does not contain cytidine, and collecting the nano-filtration membrane permeate.
(4) Concentrating the permeate obtained in the step (3) to 50g/L through a nanofiltration membrane of 60Da, concentrating through evaporation to obtain cytidine with the concentration of 400g/L, adjusting the pH to 5.0 with hydrochloric acid, stirring at room temperature of 20 ℃ for 1h with seed crystal continuously at 200rpm, separating out crystals, and cooling to 4 ℃ in a gradient manner for 10 h.
(5) And (4) carrying out suction filtration on the crystals obtained in the step (4), removing the mother liquor, adding absolute ethyl alcohol, washing twice, and carrying out suction filtration to obtain the crystals.
(6) And (3) putting the crystals obtained in the step (5) into a vacuum drying oven at 40 ℃, and drying for 4 hours at 10mbar to obtain 32.64g of cytidine crystals, wherein the primary yield of cytidine is 51%.
(7) And (3) crystallizing the mother liquor obtained in the step (5) for two times and three times, and drying the crystals to obtain 15.68g and 8g of cytidine crystals respectively, wherein the purity of the cytidine crystals is 99 percent, and the total yield of the cytidine crystals is 88 percent.
The adsorption amount is reduced due to the unmodified resin, the use amount of the resin is increased, the volume of the whole eluent is increased, the flow time is prolonged, and the acid-base amount of the regenerated resin is increased.
Example 2:
(1) adjusting pH of cytidine-containing fermentation liquor to 3.5, filtering with 200nm ceramic membrane, collecting permeate, washing the retentate with tap water until the retentate contains no cytidine, and mixing to obtain ceramic membrane permeate.
(2) Loading the ceramic membrane permeate obtained in the step (1) to modified cation exchange resin NH-1215 g, adsorbing, washing impurities, eluting, and collecting eluent; the sampling rate is 1.5BV/h, and the sampling amount is 64g of cytidine fermentation broth clear liquid; in the impurity washing process, the concentration of ammonia water is 0.05mol/L, the flow rate is 1.5BV/h, and the impurity washing volume is 10 BV; in the elution process, the concentration of ammonia water is 0.1mol/L, the flow rate is 2BV/h, and the volume of elution is 12 BV.
(3) And (3) nano-filtering the eluent obtained in the step (2) by using a nano-filtration membrane of 800Da to remove macromolecular impurities such as pigments and the like, continuously washing the trapped fluid until the trapped fluid does not contain cytidine, and collecting the nano-filtration membrane permeate.
(4) Concentrating the permeate obtained in the step (3) to 70g/L through a nanofiltration membrane of 200Da, concentrating through evaporation to obtain cytidine with concentration of 600g/L, adjusting pH to 10.0, adjusting the room temperature to 20 ℃, adding seed crystals, continuously stirring at 400rpm for 2h, separating out crystals, and cooling to 1 ℃ in a gradient manner for 10 h.
(5) And (4) carrying out suction filtration on the crystals obtained in the step (4), removing the mother liquor, adding absolute ethyl alcohol, washing twice, and carrying out suction filtration to obtain the crystals.
(6) And (3) putting the crystals obtained in the step (5) into a vacuum drying oven at 40 ℃, and drying for 4 hours at 10mbar to obtain 41.2g of cytidine crystals, wherein the primary yield of cytidine is 64.3%.
(7) And (3) crystallizing the mother liquor obtained in the step (5) for two times and three times, and drying the crystals to obtain 14.64g and 5g of cytidine crystals respectively, wherein the purity of the cytidine crystals is 99.1 percent, and the total yield of the cytidine crystals is 95 percent.
Example 3:
(1) adjusting pH of fermentation liquid containing cytidine to 2.5, filtering with 100nm ceramic membrane, collecting permeate, washing the retentate with tap water until the retentate contains no cytidine, and mixing to obtain ceramic membrane permeate.
(2) Loading the ceramic membrane permeate obtained in the step (1) to modified cation exchange resin NH-1215 g, adsorbing, washing impurities, eluting, and collecting eluent; the sampling rate is 2BV/h, and the sampling amount is 64g of cytidine to obtain fermentation liquor; in the impurity washing process, the concentration of ammonia water is 0.03mol/L, the flow rate is 2BV/h, and the impurity washing volume is 14 BV; in the elution process, the concentration of ammonia water is 0.06mol/L, the flow rate is 1.5BV/h, and the volume of elution is 13 BV.
(3) And (3) nano-filtering the eluent obtained in the step (2) by using a nano-filtration membrane of 500Da to remove macromolecular impurities such as pigments and the like, continuously washing the trapped fluid until the trapped fluid does not contain cytidine, and collecting the nano-filtration membrane permeate.
(4) Concentrating the permeate obtained in the step (3) to 60g/L through a nanofiltration membrane of 90Da, concentrating through evaporation to obtain a cytidine concentration of 500g/L, adjusting the pH to 7.0, adjusting the room temperature to 20 ℃, adding seed crystals, continuously stirring at 400rpm for 1.5h, separating out crystals, and carrying out gradient cooling to 4 ℃ for 10 h.
(5) And (4) carrying out suction filtration on the crystals obtained in the step (4), removing the mother liquor, adding absolute ethyl alcohol, washing twice, and carrying out suction filtration to obtain the crystals.
(6) And (3) putting the crystals obtained in the step (5) into a vacuum drying oven at 40 ℃, and drying for 4 hours at 10mbar to obtain 38.1g of cytidine crystals, wherein the primary yield of cytidine is 59.5%.
(7) After the mother liquor obtained in the step (5) is crystallized for the second time and the third time, 15.28g of cytidine crystals and 6.27g of cytidine crystals are obtained after drying, the purity of the cytidine crystals is 99.6%, and the total yield is 93.2%.
Example 4:
(1) adjusting pH of fermentation liquid containing cytidine to 3.0, filtering with 150nm ceramic membrane, collecting permeated liquid, washing trapped liquid with tap water until the trapped liquid contains no cytidine, and mixing to obtain ceramic membrane permeated liquid.
(2) Loading the ceramic membrane permeate obtained in the step (1) to modified cation exchange resin NH-1215 g, adsorbing, washing impurities, eluting, and collecting eluent; the sampling rate is 1.5BV/h, and the sampling amount is 64g of cytidine fermentation broth clear liquid; in the impurity washing process, the concentration of ammonia water is 0.02mol/L, the flow rate is 1.5BV/h, and the impurity washing volume is 11 BV; in the elution process, the concentration of ammonia water is 0.07mol/L, the flow rate is 1BV/h, and the volume of elution is 12.5 BV.
(3) And (3) nano-filtering the eluent obtained in the step (2) by using a nano-filtration membrane of 400Da to remove macromolecular impurities such as pigments and the like, continuously washing the trapped fluid until the trapped fluid does not contain cytidine, and collecting the nano-filtration membrane permeate.
(4) Concentrating the permeate obtained in the step (3) to 65g/L through a nanofiltration membrane of 150Da, concentrating through evaporation to obtain cytidine with the concentration of 450g/L, adjusting the pH to 7.0, adjusting the room temperature to 20 ℃, adding seed crystals, continuously stirring at 400rpm for 0.5h, separating out crystals, and carrying out gradient cooling to 10 ℃ for 10 h.
(5) And (4) carrying out suction filtration on the crystals obtained in the step (4), removing the mother liquor, adding absolute ethyl alcohol, washing twice, and carrying out suction filtration to obtain the crystals.
(6) And (3) putting the crystals obtained in the step (5) into a vacuum drying oven at 40 ℃, and drying for 4 hours at 10mbar to obtain 36.1g of cytidine crystals, wherein the primary yield of the glycoside is 56.4%.
(7) After the mother liquor obtained in the step (5) is crystallized for the second time and the third time, 15.63g of cytidine crystals and 6.45g of cytidine crystals are obtained after drying respectively, the purity of the cytidine crystals is 99.4%, and the total yield is 90.9%.
The method innovatively adopts fermentation liquor to directly pass through a membrane, adopts three sets of membrane systems to carry out coarse separation to remove thalli and macromolecular impurities, and carries out one-step refined chromatographic separation by cation exchange resin synthesized in a laboratory, wherein the maximum cytidine adsorption amount is 0.3 g/g; and the optimal impurity concentration and elution concentration are determined: in the impurity washing process, the concentration of ammonia water is 0.03 mol/L; in the elution process, the concentration of ammonia water is 0.06 mol/L. Concentrating the eluent, adding a crystal seed water system for crystallization, and washing and drying crystals to obtain the product. The whole separation yield is more than 90 percent, the purity is higher than 99 percent, the yield is better than that of the prior art, and the separation cost is low.
The present invention provides a method and a concept for separating and extracting cytidine from fermentation broth, and a method and a way for implementing the technical scheme are numerous, and the above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, a plurality of improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention. All the components not specified in the present embodiment can be realized by the prior art.
Claims (10)
1. A method for separating and extracting cytidine is characterized in that liquid containing cytidine is adsorbed and eluted by cation exchange resin, and the obtained eluent is concentrated and crystallized to obtain cytidine crystals;
wherein the cation exchange resin is obtained by modifying NH-1 resin with silver sulfate and/or sulfuric acid; the adsorption capacity of the cation exchange resin is 0.2-0.4 g/g.
2. The method according to claim 1, wherein the amount of silver sulfate is 2-8% of the mass of the NH-1 resin; the dosage of the sulfuric acid is 10-20 times of the volume of the resin.
3. The method according to claim 1, characterized in that the solvent in the modification process is dichloroethane and/or trichloroethane; the volume ratio of the NH-1 resin to the solvent is 1: (6-20).
4. The method of claim 1, wherein the NH-1 resin is mixed with a solvent and maintained at 50-70 ℃ for 0.5-2 h; adding silver sulfate and/or sulfuric acid, maintaining at 70-90 deg.C for 2-4 hr, maintaining at 90-100 deg.C for 2-3 hr, filtering, and washing.
5. The method of claim 1, comprising the steps of:
(1) filtering the cytidine fermentation liquor by a ceramic membrane to obtain a first permeate;
(2) adsorbing the first permeate obtained in the step (1) by using cation exchange resin, washing impurities by using water and/or ammonia water, and eluting by using ammonia water to obtain an eluent;
(3) nanofiltration of the eluent obtained in the step (2) by a nanofiltration membrane to obtain a second permeate;
(4) and (4) concentrating the second permeate obtained in the step (3) through a nanofiltration membrane, adjusting the pH value, stirring, cooling and crystallizing to obtain a cytidine crystal.
6. The method according to claim 5, wherein in the step (2), the concentration of the ammonia water in the impurity washing process is 0-0.05 mol/L; in the elution process, the concentration of ammonia water is 0.05-0.1 mol/L.
7. The method as claimed in claim 5, wherein in step (3), the nanofiltration membrane has a molecular weight cut-off of 300-800 Da.
8. The method as claimed in claim 5, wherein in the step (4), the molecular weight cut-off of the nanofiltration membrane is 60-200 Da.
9. The method as claimed in claim 5, wherein in step (4), the concentration is up to 800g/L at 400-.
10. The method according to claim 5, wherein in the step (4), the pH is 5 to 10; the temperature is reduced to 0-10 ℃.
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| CN105348346A (en) * | 2015-12-14 | 2016-02-24 | 山东凯盛新材料有限公司 | Refining method of 5'-cytidine acid |
| CN106632519A (en) * | 2016-12-12 | 2017-05-10 | 南京工业大学 | Technology for separating nucleotide by adopting continuous ion exchange chromatography technique |
| CN108358989A (en) * | 2018-04-24 | 2018-08-03 | 苏州华赛生物工程技术有限公司 | A method of isolating and purifying cytidine from microbial fermentation solution |
| CN108440624A (en) * | 2018-04-24 | 2018-08-24 | 苏州华赛生物工程技术有限公司 | A kind of method that environment-friendly type isolates and purifies cytidine from microbial fermentation solution |
| CN111087432A (en) * | 2020-01-10 | 2020-05-01 | 南京高新工大生物技术研究院有限公司 | Separation and extraction method of N-acetylneuraminic acid |
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| CN105348346A (en) * | 2015-12-14 | 2016-02-24 | 山东凯盛新材料有限公司 | Refining method of 5'-cytidine acid |
| CN106632519A (en) * | 2016-12-12 | 2017-05-10 | 南京工业大学 | Technology for separating nucleotide by adopting continuous ion exchange chromatography technique |
| CN108358989A (en) * | 2018-04-24 | 2018-08-03 | 苏州华赛生物工程技术有限公司 | A method of isolating and purifying cytidine from microbial fermentation solution |
| CN108440624A (en) * | 2018-04-24 | 2018-08-24 | 苏州华赛生物工程技术有限公司 | A kind of method that environment-friendly type isolates and purifies cytidine from microbial fermentation solution |
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