CN116574147A - Process for separating and purifying UMP conversion liquid by utilizing chromatographic technique - Google Patents
Process for separating and purifying UMP conversion liquid by utilizing chromatographic technique Download PDFInfo
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- CN116574147A CN116574147A CN202310542556.2A CN202310542556A CN116574147A CN 116574147 A CN116574147 A CN 116574147A CN 202310542556 A CN202310542556 A CN 202310542556A CN 116574147 A CN116574147 A CN 116574147A
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- 238000006243 chemical reaction Methods 0.000 title claims abstract description 59
- 239000007788 liquid Substances 0.000 title claims abstract description 46
- 238000000034 method Methods 0.000 title claims abstract description 42
- 230000008569 process Effects 0.000 title claims abstract description 30
- 238000004587 chromatography analysis Methods 0.000 title claims description 6
- 239000011347 resin Substances 0.000 claims abstract description 64
- 229920005989 resin Polymers 0.000 claims abstract description 64
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 52
- 239000003957 anion exchange resin Substances 0.000 claims abstract description 30
- 238000003795 desorption Methods 0.000 claims abstract description 29
- 239000011780 sodium chloride Substances 0.000 claims abstract description 26
- 239000012535 impurity Substances 0.000 claims abstract description 24
- 238000011068 loading method Methods 0.000 claims abstract description 21
- 238000005406 washing Methods 0.000 claims abstract description 21
- 238000001179 sorption measurement Methods 0.000 claims abstract description 17
- 230000008929 regeneration Effects 0.000 claims abstract description 5
- 238000011069 regeneration method Methods 0.000 claims abstract description 5
- 238000005342 ion exchange Methods 0.000 claims abstract description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 26
- 150000002500 ions Chemical class 0.000 claims description 13
- 239000002245 particle Substances 0.000 claims description 9
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims description 8
- 239000003795 chemical substances by application Substances 0.000 claims description 6
- 238000001728 nano-filtration Methods 0.000 claims description 6
- 125000003277 amino group Chemical group 0.000 claims description 5
- 229920000768 polyamine Polymers 0.000 claims description 4
- 125000001302 tertiary amino group Chemical group 0.000 claims description 4
- 125000000524 functional group Chemical group 0.000 claims description 3
- 239000011148 porous material Substances 0.000 claims description 3
- VILCJCGEZXAXTO-UHFFFAOYSA-N 2,2,2-tetramine Chemical compound NCCNCCNCCN VILCJCGEZXAXTO-UHFFFAOYSA-N 0.000 claims description 2
- 239000003054 catalyst Substances 0.000 claims description 2
- 238000004132 cross linking Methods 0.000 claims description 2
- 230000002255 enzymatic effect Effects 0.000 claims description 2
- FAGUFWYHJQFNRV-UHFFFAOYSA-N tetraethylenepentamine Chemical compound NCCNCCNCCNCCN FAGUFWYHJQFNRV-UHFFFAOYSA-N 0.000 claims description 2
- 125000001309 chloro group Chemical group Cl* 0.000 claims 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 14
- KURVIXMFFSNONZ-WFIJOQBCSA-L disodium;[(2r,3s,4r,5r)-5-(2,4-dioxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound [Na+].[Na+].O[C@@H]1[C@H](O)[C@@H](COP([O-])([O-])=O)O[C@H]1N1C(=O)NC(=O)C=C1 KURVIXMFFSNONZ-WFIJOQBCSA-L 0.000 abstract description 14
- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 14
- 238000004255 ion exchange chromatography Methods 0.000 abstract description 6
- 239000002253 acid Substances 0.000 abstract description 5
- 238000005516 engineering process Methods 0.000 abstract description 4
- 239000003513 alkali Substances 0.000 abstract description 3
- 150000001450 anions Chemical class 0.000 abstract description 3
- 238000010828 elution Methods 0.000 abstract description 2
- DJJCXFVJDGTHFX-XVFCMESISA-N uridine 5'-monophosphate Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(O)=O)O[C@H]1N1C(=O)NC(=O)C=C1 DJJCXFVJDGTHFX-XVFCMESISA-N 0.000 description 104
- 239000000243 solution Substances 0.000 description 48
- 239000011777 magnesium Substances 0.000 description 40
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 description 38
- 239000000047 product Substances 0.000 description 37
- 239000011734 sodium Substances 0.000 description 22
- 239000000523 sample Substances 0.000 description 19
- 238000000926 separation method Methods 0.000 description 10
- 238000002347 injection Methods 0.000 description 9
- 239000007924 injection Substances 0.000 description 9
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 7
- 239000003463 adsorbent Substances 0.000 description 7
- 239000000460 chlorine Substances 0.000 description 7
- 229910052801 chlorine Inorganic materials 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- DJJCXFVJDGTHFX-UHFFFAOYSA-N Uridinemonophosphate Natural products OC1C(O)C(COP(O)(O)=O)OC1N1C(=O)NC(=O)C=C1 DJJCXFVJDGTHFX-UHFFFAOYSA-N 0.000 description 6
- 230000002572 peristaltic effect Effects 0.000 description 6
- 239000012492 regenerant Substances 0.000 description 6
- FOGRQMPFHUHIGU-UHFFFAOYSA-N Uridylic acid Natural products OC1C(OP(O)(O)=O)C(CO)OC1N1C(=O)NC(=O)C=C1 FOGRQMPFHUHIGU-UHFFFAOYSA-N 0.000 description 5
- 238000013375 chromatographic separation Methods 0.000 description 5
- 239000002773 nucleotide Substances 0.000 description 5
- 125000003729 nucleotide group Chemical group 0.000 description 5
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 description 4
- IERHLVCPSMICTF-XVFCMESISA-N cytidine 5'-monophosphate Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(O)=O)O1 IERHLVCPSMICTF-XVFCMESISA-N 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 229910019142 PO4 Inorganic materials 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000005265 energy consumption Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- RQFCJASXJCIDSX-UUOKFMHZSA-N guanosine 5'-monophosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O RQFCJASXJCIDSX-UUOKFMHZSA-N 0.000 description 3
- 235000013928 guanylic acid Nutrition 0.000 description 3
- 238000004811 liquid chromatography Methods 0.000 description 3
- 239000010452 phosphate Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 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 description 2
- HNSDLXPSAYFUHK-UHFFFAOYSA-N 1,4-bis(2-ethylhexyl) sulfosuccinate Chemical compound CCCCC(CC)COC(=O)CC(S(O)(=O)=O)C(=O)OCC(CC)CCCC HNSDLXPSAYFUHK-UHFFFAOYSA-N 0.000 description 2
- 229930183912 Cytidylic acid Natural products 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 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
- 239000007864 aqueous solution Substances 0.000 description 2
- 238000005341 cation exchange Methods 0.000 description 2
- IERHLVCPSMICTF-UHFFFAOYSA-N cytidine monophosphate Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(COP(O)(O)=O)O1 IERHLVCPSMICTF-UHFFFAOYSA-N 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- UNXNGGMLCSMSLH-UHFFFAOYSA-N dihydrogen phosphate;triethylazanium Chemical compound OP(O)(O)=O.CCN(CC)CC UNXNGGMLCSMSLH-UHFFFAOYSA-N 0.000 description 2
- 239000000796 flavoring agent Substances 0.000 description 2
- 235000019634 flavors Nutrition 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- MBABOKRGFJTBAE-UHFFFAOYSA-N methyl methanesulfonate Chemical compound COS(C)(=O)=O MBABOKRGFJTBAE-UHFFFAOYSA-N 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000035515 penetration Effects 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- GETQZCLCWQTVFV-UHFFFAOYSA-N trimethylamine Chemical compound CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 description 2
- 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 description 1
- 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 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical class [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 240000005979 Hordeum vulgare Species 0.000 description 1
- 235000007340 Hordeum vulgare Nutrition 0.000 description 1
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial 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
- 230000002210 biocatalytic effect Effects 0.000 description 1
- -1 ca is obtained 2+ Substances 0.000 description 1
- 239000003729 cation exchange resin Substances 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 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 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 239000012527 feed solution Substances 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 239000004226 guanylic acid Substances 0.000 description 1
- 239000000413 hydrolysate Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-M hydroxide Chemical group [OH-] XLYOFNOQVPJJNP-UHFFFAOYSA-M 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 229910001425 magnesium ion Inorganic materials 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000012898 sample dilution Substances 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 229940035893 uracil Drugs 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
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
- C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P10/00—Technologies related to metal processing
- Y02P10/20—Recycling
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- 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)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention discloses a process for separating and purifying UMP conversion liquid by utilizing chromatographic technology, which comprises the steps of loading UMP conversion liquid into a chromatographic column containing anion exchange resin for ion exchange, and collecting effluent after three steps of adsorption, impurity washing and desorption. The invention utilizes the adsorption and ion exchange mechanism and adopts the fixed bed technology to realize Ca removal in one step 2+ 、Mg 2+ 、ATP + 、AMP + Plasma and UR ‑ 、PO 4 3‑ And (3) separating and purifying to obtain UMP sodium salt products for the purpose of plasma anions. The whole process does not use acid and alkali, ca in the product liquid 2+ 、Mg 2+ 、PO 4 3‑ The content is lower than the detection limit of ion chromatography, the yield of UMP products reaches more than 99%, the HPLC purity reaches 100%, and Ca 2+ 、Mg 2+ 、PO 4 3‑ 、ATP + 、AMP + 、UR ‑ The removal rate of the plasma reaches more than 99 percent. NaCl is selected as desorbent, so that the elution of the product and the regeneration of the resin are completed simultaneously, and the process is simplified.
Description
Technical Field
The invention belongs to the technical field of biological separation, and particularly relates to a process for separating and purifying UMP conversion liquid by utilizing a chromatographic technique.
Background
The 5' -uracil nucleotide (hereinafter referred to as uridylic acid, UMP) is a single nucleotide composed of uracil, ribose and phosphate. The medicinal uridylic acid is a mixture of disodium salts, is white or yellow powder, has delicate flavor, is easily dissolved in water, and is difficultly dissolved in organic solvents such as ethanol, acetone and the like. Currently, the production technology of uridylic acid mainly comprises nuclease hydrolysis, chemical synthesis and biocatalysis. Among them, the biocatalytic method has become a hot spot in recent years. Its advantage is: compared with other methods, the biocatalysis method not only shortens the production period, but also greatly increases the yield of the uridylic acid. And the reaction system is simple, and generally only a substrate, a surfactant, an enzyme prosthetic group (such as magnesium ion) and a certain amount of pH regulator are needed. Therefore, uridine-cytidine enzyme (UCK) can catalyze the phosphorylation of uridine and cytidine into uridylic acid and cytidylic acid as important catalysts in the metabolic compensation pathway of nucleotides in organisms in the production of UMP conversion solution. The UMP conversion solution contains Na + ，Mg 2+ UMP, small amount of PO 4 3- And the like, and the pH value of the conversion solution is about 2. Depending on the theoretical concentration profile of UMP in solution, different forms exist, as shown in FIG. 1. As can be seen, UMP is used in most cases at pH2 - In the form of a gel. Because the components in the enzymatic hydrolysate are complex, the traditional method for separating UMP conversion solution generally adopts 1 cation exchange column to separate 4 mononucleotides [1] Respectively obtaining dilute solutions of UMP, cytidylic acid (CMP) and Adenylate (AMP) and mixed solutions of guanylic acid (GMP) and CMP; and adjusting the pH value of UMP, separating, purifying and concentrating by using an oxyhydrogen type anion column. The method has low resin utilization rate, 4 nucleotides are not easy to completely separate, and the hydroxyl anion exchange resin exchanges groups OH in the purification process - Easy to be matched with Mg 2+ A precipitate forms, causing the column to clog, resulting in excessive resource consumption. For example, shongling [2] A new cation exchange resin was studied, 4 nucleotides could be separated on the cation column, but there was little distance between the chromatographic peaks of each product, resulting in some difficulty in effluent product collection. Li Deying [3] Using cation-exchange columns in seriesUnder the acidic condition, the UMP is firstly adsorbed AMP, CMP, GMP, and the UMP which is not adsorbed flows through a weak alkaline resin column and a strong alkaline resin column and is eluted, concentrated and dried to obtain UMP products, the process is more complicated, and excessive acid and alkali are consumed in the regeneration of the resin.
Reference is made to:
[1]DEODA A J,SINGHAL R S.5'-Phosphodiesterase(5'-PDE)from germinated barley for hydrolysis of RNA to produce flavour nucleotides[J].Bioresour Technol,2003,88(3):245-50.
[2] research [ D ] of separation and purification process of Shortlinping 5' -uridylic acid; university of south Beijing industry, 2003.
[3]Tanaka,Release of intracellularly stored 5-phosphodiesterase with preserved plant.Biotech Bioengineering 1985.
Disclosure of Invention
Aiming at the defects of complicated experimental process and high energy consumption in the prior art, the invention provides a process for separating and purifying UMP conversion liquid by utilizing a chromatographic technique so as to solve the problem of Ca in UMP conversion liquid 2+ 、Mg 2+ 、ATP + 、AMP + 、UR - 、PO 4 3- Difficult to remove.
In order to solve the technical problems, the invention adopts the following technical scheme:
a process for separating and purifying UMP conversion liquid by chromatography comprises loading UMP conversion liquid into chromatographic column containing anion exchange resin for ion exchange, adsorbing, washing impurities, desorbing, collecting effluent, and obtaining UMP sodium salt.
Wherein, the UMP conversion liquid is UMP liquid which is generated by removing two phosphate radicals from UTP through enzymolysis and is obtained through sodium filtration, and Ca is added in the preparation process of the UMP conversion liquid 2+ 、Mg 2+ And the auxiliary factors are added, and after acid adjustment, the pH value of the whole feed liquid is 1-3 and is basically about pH2, so the whole UMP conversion liquid contains UMP - ，UR - 、PO 4 3- 、Mg 2+ ，Ca 2+ 、Na + 、ATP + 、AMP + . The purpose of nanofiltration is mainly to remove a large amount of PO 4 3 、Ca 2+ ，Mg 2+ ，Na + ，UR - For example, nanofiltration membranes with a molecular weight cut-off of 300Da can be selected, and UMP conversion solution is subjected to nanofiltration treatment and contains substantially UMP - And part of the remaining UR - 、PO 4 3- 、Mg 2+ 、Ca 2+ 、Na + ，ATP + 、AMP + 。
Preferably, the UMP conversion solution, mg 2+ The content of Na is 0.1 mg/L-3 g/L + The content of UMP is 1g/L to 30g/L - The content of Ca is 10g/L to 70g/L 2+ The content of (2) is 0.1 mg/L-3 g/L, AMP + The content of ATP is 2g/L to 10g/L + The content of (3) is 2 g/L-10 g/L, UR - The content of (2) is 3 g/L-10 g/L, PO 4 3- The content of (C) is 0.1 mg/L-1 g/L.
More preferably, the UMP conversion solution, mg 2+ The content of Na is 0.3g/L to 0.7g/L + The content of UMP is 10g/L to 20g/L - The content of Ca is 40g/L to 60g/L 2+ 0.06g/L to 0.08g/L, AMP + The content of ATP is 2.5 g/L-3.5 g/L + The content of (3) is 2 g/L-3 g/L, UR - The content of (2-4 g/L) PO 4 3- The content of (C) is 0.1 mg/L-0.4 mg/L.
Wherein the anion exchange resin is I-type or II-type strong alkaline anion exchange resin taking styrene as a framework, or polyamine type alkaline anion exchange resin, or gel type anion exchange resin.
Wherein the polyamine comprises triethylenetetramine and/or tetraethylenepentamine.
Wherein the functional groups of the anion exchange resin are quaternary amino groups and/or tertiary amino groups, the content of the quaternary amino groups is 1.0-2.5 mmol/g of anion exchange resin, and the content of the tertiary amino groups is 1.0-2.5 mmol/g of anion exchange resin; the exchangeable ions of the anion exchange resin are chloride ions, the content of the chloride ions is 1.0-2.5 mmol/g, and the volume total exchange capacity is more than or equal to 1.35mmol/mL; the degree of crosslinking of the anion exchange resin is 3-10%.
Wherein the particle diameter of the anion exchange resin is 0.1-0.8 mm, the water content is 42-48%, and the wet true density is 1.03-1.18 g/cm 3 Specific surface area of 100-2000 m 2 Per gram, the pore volume is 0.51-1.33 cm 3 And/g, the aperture is 1-200 nm.
The anion exchange resin includes, but is not limited to, the anion exchange resins described above.
Wherein the sample loading rate of the adsorption is 0.3-1.5 BV/h. The loading amount is sufficient to saturate the resin, and the resin is adsorbed to saturation by continuously increasing the loading amount, so as to enable UMP - Excessive loss is caused in the residual liquid and the impurity washing liquid; wherein the saturation point (C/C) of the penetration curve in the fixed bed penetration experiment can be calculated 0 =1) to determine whether the resin is saturated in adsorption.
Wherein the impurity-washing agent is water, the water consumption is 0.8-1 BV, and the flow rate is 0.3-1.5 BV/h.
Wherein, the desorption and desorbing agent is 0.1M-3M NaCl water solution (preferably 1.5M NaCl water solution), the dosage of the desorbing agent is 0.5-3 BV, and the flow rate is 0.3-1.5 BV/h.
Wherein, after the desorption is completed, the resin is completely converted into chlorine type, no regeneration operation is performed, and NaCl in the resin gap is washed away by pure water before the resin is reused, and the use amount and the flow rate of the pure water are not particularly limited, and the preferred use amount and the preferred flow rate of the pure water are 0.5-3 BV and 0.3-1.5 BV/h.
In the above process flow, the adsorption (Ca-containing 2+ 、Mg 2+ 、Na + 、ATP + 、AMP + 、UR - 、PO 4 3- 、UMP - The UMP conversion liquid loading process), impurity washing (UMP conversion liquid with small gaps between resin particles) and desorption (UMP sodium salt product liquid) are all carried out at normal temperature.
In the above process flow, the fixed bed column separation device used in the invention consists of 1 resin column filled with adsorbent, peristaltic pump and automatic part collector, and the whole device is prepared by switching mobile phaseThe system is divided into three steps of adsorption, impurity washing and desorption, and the three steps are switched in sequence. Advanced UMP conversion solution is treated by resin column to adsorb UMP - After saturation, a raffinate (Ca 2 + 、Mg 2+ 、Na + 、ATP + 、AMP + 、UR - 、PO 4 3- ). Then pure water is switched to wash impurities, and the material in gaps of resin particles is removed, wherein the material in gaps of resin mainly contains Ca 2+ 、Mg 2+ 、Na + 、ATP + 、AMP + 、UR - 、PO 4 3- Contains a trace amount of UMP - . And (3) carrying out desorption by using NaCl aqueous solution, collecting adsorbed UMP sodium salt product liquid, completely converting the resin into chlorine after the desorption is finished, and washing NaCl in a resin column gap by using pure water, wherein the adsorbent can be reused.
The beneficial effects are that: compared with the prior art, the invention has the following advantages:
(1) The chromatographic separation operation is carried out at room temperature, so that the energy consumption is greatly reduced.
(2) The desorbent NaCl is used, and NaCl is selected as the desorbent, so that the elution of the product and the regeneration of the resin are finished simultaneously, the process is simplified, the energy consumption in the UMP separation process is greatly reduced, and the scale clean production of UMP is realized. The resin can be reused, has a certain pigment removing effect, has low running cost and can be directly amplified.
(3) Through a single resin column, the whole process does not use acid and alkali, ca is obtained 2+ 、Mg 2+ 、ATP + 、AMP + 、UR - 、PO 4 3- Separating the impurities from UMP in one step, and Ca in the product liquid 2+ 、Mg 2+ 、PO 4 3- The content is lower than the detection limit of ion chromatography, the purity and quality of UMP products are ensured, the yield can reach more than 99 percent, the HPLC purity can reach 100 percent, and Ca 2+ 、Mg 2+ 、PO 4 3- 、ATP + 、AMP + 、UR - The removal rate of the plasma reaches more than 99 percent.
Drawings
The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings and detailed description.
FIG. 1 shows the theoretical concentration profile of UMP in a solution in which different forms exist.
Fig. 2 is a process flow diagram of the present invention.
FIG. 3 is a graph showing the decoloring effect of the present invention.
FIG. 4 is an HPLC chart (sample diluted 100-fold) of UMP sodium salt conversion.
Fig. 5 is an HPLC diagram (100-fold dilution of sample) of UMP sodium salt product solution.
FIG. 6 is an ion chromatogram of UMP sodium salt conversion solution (200-fold sample dilution).
Fig. 7 is an ion chromatogram (sample undiluted) of UMP sodium salt product solution.
Detailed Description
The UMP concentration and Mg concentration in the conversion solution are measured using the external standard method in the following examples 2+ And (5) detecting.
Ca 2+ 、Mg 2+ 、Na + 、PO 4 3- The plasma chromatographic conditions were:
1) A detector: thermo Scientific-CD detector;
2) Chromatographic column: dionex Ionpac TM CS12A/AS22；
3) A suppressor: dionex TM CDRS 600 4mm/ADRS 600 4mm；
4) Mobile phase: 20mmol/L dimethyl sulphonic acid/4.5 mmol/L sodium carbonate and 1.4mmol/L sodium bicarbonate;
5) Flow rate: 1mL/min;
6) Column temperature: 25 ℃;
7) Sample injection volume: 25. Mu.L.
Ca 2+ 、Mg 2+ 、Na + In (2) and step (PO) 4 3- Similar):
1) Equilibrium of the chromatographic column: the chromatographic column is flushed by the prepared 20mmol/L dimethyl sulfonic acid at the flow rate of 1mL/min, the column incubator is opened, the baseline is collected, and when the baseline of the CD signal value tends to be below 0.5, the balance is ended.
2) And (3) detecting a sample: according to ion chromatographic conditions, writing a sample injection sequence and a method, placing the standard substance and the sample subjected to membrane treatment on corresponding positions of an automatic sample injector according to the sample injection sequence, starting sample injection and collecting map information.
UMP、ATP + 、AMP + 、UR - The chromatographic conditions are as follows:
1) A detector: agilent 1260 type high performance liquid chromatograph-uv detector (254 nm);
2) Chromatographic column: ZORBAX SB-AQ liquid chromatographic column
3) Mobile phase: triethylamine phosphate buffer at pH 6.5: methanol=91: 9, a step of performing the process;
4) Flow rate: 0.7mL/min;
5) Column temperature: 25 ℃;
6) Sample injection volume: 20. Mu.L.
The detection method comprises the following steps:
1) Equilibrium of the chromatographic column: a triethylamine phosphate buffer with a mobile phase pH of 6.5 was prepared: methanol=91: 9, filtering with a mixed microporous filter membrane with the pore diameter of 0.22 μm, and performing ultrasonic treatment for 30min. Flushing the chromatographic column with the treated mobile phase at a flow rate of 0.7mL/min, simultaneously opening a column incubator, starting to collect a baseline, and ending the balance when the baseline is on a straight line.
2) And (3) detecting a sample: writing a sample injection sequence and a method according to chromatographic conditions, placing the standard substance and the sample subjected to membrane treatment on corresponding positions of an automatic sample injector according to the sample injection sequence, starting sample injection and collecting map information.
The yield of UMP in the product liquid was calculated using the following method:
C 1 : the concentration of UMP in the product liquid;
v 2 : the volume of the product liquid;
c: concentration of UMP in the loading solution;
v: the volume of the loading solution.
The Ca in the product liquid was calculated using the following method 2+ 、Mg 2+ 、ATP + 、AMP + 、UR - 、PO 4 3- Plasma removal rate:
C 0 : ca in the sample solution 2+ 、Mg 2+ 、ATP + 、AMP + 、UR - 、PO 4 3- Is a concentration of (2);
C 2 : ca in the product liquid 2+ 、Mg 2+ 、ATP + 、AMP + 、UR - 、PO 4 3- Is a concentration of (3).
In the following examples, the chlorine anion exchange resin (HY 06) used was a type I strongly basic anion resin having styrene as a skeleton. The functional group of the resin is trimethylamine, and the content of the amino group is 1.0-2.5 mmol/g anion exchange resin; the exchangeable ions are chloride ions, and the content of the chloride ions is 1.0-2.5 mmol/g anion exchange resin; the ion exchange resin has a particle size of 0.4-0.7 mm, a water content of 42.00-48.00% and a wet true density of 1.07-1.10g/cm 3 The volume total exchange capacity is more than or equal to 1.35mmol/mL. The resin manufacturer can synthesize the resin by himself according to the above conditions.
The separation device used in the following examples consists of 1 resin column filled with adsorbent, peristaltic pump and automatic part collector, and the whole system is divided into three steps of adsorption, impurity washing and desorption by switching mobile phases, and the steps are sequentially switched to make UMP conversion solution be absorbed by resin column - After saturation, pure water is switched to wash impurities, ca is collected at the outlet of the adsorption section 2+ 、Mg 2+ 、Na + 、ATP + 、AMP + 、UR - 、PO 4 3- And then switching the NaCl aqueous solution for desorption, and collecting UMP sodium salt product liquid. The regenerant is desorbent NaCl, and after the desorption is completed, the NaCl in the gaps of the resin column is washed out by pure water.
Example 1: obtaining UMP conversion liquid P1.
UMP conversion solution P1 is provided by Nanjing Hokkimen Biotechnology Limited liability company, and is prepared by removing two phosphate radicals from UTP by enzymolysis to obtain UMP feed liquid, wherein Ca is added in the process 2+ 、Mg 2+ And the auxiliary factors are equal, and after the acid is regulated, the pH value of the whole liquid is about 2, so the whole UMP liquid contains Ca 2+ 、Mg 2+ 、Na + 、ATP + 、AMP + 、UR - 、PO 4 3- 、UMP - . The UMP feed liquid is subjected to sodium filtration treatment to obtain UMP conversion liquid which basically contains Ca 2+ 、Mg 2+ 、Na + 、ATP + 、AMP + 、UR - 、PO 4 3- 、UMP - 。
Four experiments were repeated according to the above procedure to obtain four batches of UMP conversion solution, respectively.
In the first conversion solution, mg 2+ The content is 0.3g/L, na + The content is 15g/L, ca 2+ Is 0.07g/L, AMP + Is 2.5g/L, ATP + The content of (3 g/L), UR - The content of (2.5 g/L) PO 4 3- Is 0.2mg/L UMP - The content is 50g/L;
in the second conversion solution, mg 2+ The content is 0.5g/L, na + The content is 10g/L, ca 2+ Is 0.06g/L, AMP + Is 3g/L, ATP + The content of (3 g/L), UR - The content of (2) is 3g/L, PO 4 3- Is 0.1mg/L, UMP - The content is 60g/L;
in the third conversion solution, mg 2+ The content is 0.4g/L, na + The content is 14g/L, ca 2+ Is 0.08g/L, AMP + Is 3.3g/L, ATP + The content of (3) is 2.8g/L, UR - Is 4g/L, PO 4 3- Is 0.3mg/L, UMP - The content is 45g/L;
in the fourth conversion solution, mg 2+ The content of Na is 0.65g/L + The content is 16g/L, ca 2+ The content of (3) is0.07g/L，AMP + Is 3.4g/L, ATP + The content of (3) is 2.9g/L, UR - Is 4.3g/L PO 4 3- Is 0.4mg/L UMP - The content is 53g/L.
In the following examples 2 to 5, the whole separation device system is divided into three steps of adsorption, impurity washing and desorption, wherein the feeding rate of the adsorption section is 1.35 to 1.5BV, the pure water consumption of the impurity washing section is 0.8BV, the impurity washing flow rate is 1.5BV/h, the desorption section is desorbed by using 1.5M NaCl solution, the volume of desorbent is 1.5BV, the desorption flow rate is 1.5BV/h, and UMP product liquid is collected after the desorption is completed. The regenerant, namely desorbent NaCl, after the desorption was completed, the NaCl in the resin column gap was washed out with 1BV of pure water at a flow rate of 0.3BV/h, and finally the resin was completely converted into chlorine type.
Example 2: chromatographic separation of UMP conversion solution P1
A separation system consisting of 1 resin column packed with adsorbent, peristaltic pump and automatic partial collector was used. The resin column was packed with 0.6L of resin (HYO 6) having a diameter of 4cm and a height of 56cm. Loading the first UMP conversion solution P1 pretreated in the embodiment 1 into a column, wherein the loading volume is 1.6BV, the loading rate is 1.5BV/h, dividing the whole system into three steps of adsorption, impurity washing and desorption by switching mobile phases, switching sequentially, and allowing the advanced UMP conversion solution to be adsorbed by a resin column - After saturation, a raffinate (Ca 2+ 、Mg 2+ 、Na + 、ATP + 、AMP + 、UR - 、PO 4 3- ) And then 0.8BV of pure water is switched for impurity washing to obtain a small amount of UMP conversion liquid in gaps of resin particles, then 1.5BV of NaCl solution is used for desorption, and the adsorbed UMP product is collected. The regenerant, namely desorbent NaCl, after the desorption was completed, the NaCl in the resin column gap was washed out with 1BV of pure water at a flow rate of 0.3BV/h, and finally the resin was completely converted into chlorine type. UMP detection by HPLC of collected product effluent - 、ATP + 、AMP + 、UR - Concentration, detection of Ca by ion chromatography 2+ 、Mg 2+ 、PO 4 3- Concentration of plasma, ca 2+ 、Mg 2+ 、PO 4 3- Concentration below ion chromatographic limit, ATP + 、AMP + 、UR - The concentration of the UMP sodium salt is lower than the detection limit of liquid chromatography, the yield of UMP sodium salt products reaches 99%, the purity of HPLC reaches 100%, and Ca 2+ 、Mg 2+ 、PO 4 3- The removal rate of the plasma reaches 99.4 percent. From the HPLC and ion chromatograms, FIG. 4 shows an HPLC chromatogram of a nanofiltration of a UMP conversion diluted 100 times, and from FIG. 4, it can be seen that there are some impurity peaks (such as ATP + 、AMP + 、UR - ) These impurity peaks overlap slightly with UMP product peaks, so that the automatic integration of the chromatographic peaks deviates slightly, and as can be seen from the UMP sodium salt product chromatogram of FIG. 5, UMP has a purity of 100%, impurities (ATP + 、AMP + 、UR - ) Completely removed. Meanwhile, FIG. 6 is an ion chromatogram of a UMP conversion solution diluted 200 times after nanofiltration, and FIG. 7 is an ion chromatogram of an undiluted UMP sodium salt product, and FIG. 6 and FIG. 7 are compared to each other to see Mg in the UMP sodium salt product 2+ Plasma (Na removal) + Outside) was almost 0, and it was also shown that the purification effect of the present experiment was good.
Example 3: chromatographic separation of UMP conversion solution P1
A separation system consisting of 1 resin column packed with adsorbent, peristaltic pump and automatic partial collector was used. The resin column was packed with 0.6L of resin (HYO 6) having a diameter of 4cm and a height of 56cm. Loading the second UMP conversion solution P1 pretreated in the embodiment 1 into a column, wherein the loading volume is 1.35BV, the loading rate is 1.5BV/h, dividing the whole system into three steps of adsorption, impurity washing and desorption by switching mobile phases, switching sequentially, and allowing the advanced UMP conversion solution to be adsorbed by a resin column - After saturation, a raffinate (Ca 2+ 、Mg 2+ 、Na + 、ATP + 、AMP + 、UR - 、PO 4 3- ) And then 0.8BV of pure water is switched for impurity washing to obtain a small amount of UMP conversion liquid in gaps of resin particles, then 1.5BV of NaCl solution is used for desorption, and the adsorbed UMP product is collected. The regenerant is desorbent NaCl, after the desorption is completed, the NaCl in the gap between the resin columns is washed out by pure water of 1BV, and the flow rate is0.3BV/h, and finally the resin is completely converted into chlorine type. UMP detection by HPLC of collected product effluent - 、ATP + 、AMP + 、UR - Concentration, detection of Ca by ion chromatography 2+ 、Mg 2+ 、PO 4 3- Concentration of plasma, ca 2+ 、Mg 2+ 、PO 4 3- Concentration below ion chromatographic limit, ATP + 、AMP + 、UR - The concentration of (C) is lower than the detection limit of liquid chromatography, the yield of UMP products reaches 99%, the purity of HPLC reaches 100%, and Ca 2+ 、Mg 2+ 、PO 4 3- The removal rate of the plasma reaches 99.8 percent.
Example 4: chromatographic separation of UMP conversion solution P1
A separation system consisting of 1 resin column packed with adsorbent, peristaltic pump and automatic partial collector was used. The resin column was packed with 0.6L of resin (HYO 6) having a diameter of 4cm and a height of 56cm. Loading the pretreated UMP conversion solution P1 of the third batch of the embodiment 1 into a column, wherein the loading volume is 1.8BV, the loading rate is 1.5BV/h, dividing the whole system into three steps of adsorption, impurity washing and desorption by switching mobile phases, switching sequentially, and enabling the advanced UMP conversion solution to be adsorbed by a resin column - After saturation, a raffinate (Ca 2+ 、Mg 2+ 、Na + 、ATP + 、AMP + 、UR - 、PO 4 3- ) And then 0.8BV of pure water is switched for impurity washing to obtain a small amount of UMP conversion liquid in gaps of resin particles, then 1.5BV of NaCl solution is used for desorption, and the adsorbed UMP product is collected. The regenerant, namely desorbent NaCl, after the desorption was completed, the NaCl in the resin column gap was washed out with 1BV of pure water at a flow rate of 0.3BV/h, and finally the resin was completely converted into chlorine type. UMP detection by HPLC of collected product effluent - 、ATP + 、AMP + 、UR - Concentration, detection of Ca by ion chromatography 2+ 、Mg 2+ 、PO 4 3- Concentration of plasma, ca 2+ 、Mg 2+ 、PO 4 3- Concentration below ion chromatographic limit, ATP + 、AMP + 、UR - Is lower than that of liquid chromatographyLimit, UMP product yield up to 99%, HPLC purity up to 100%, ca 2+ 、Mg 2+ 、PO 4 3- The removal rate of the plasma reaches 99.4 percent.
Example 5: chromatographic separation of UMP conversion solution P1
A separation system consisting of 1 resin column packed with adsorbent, peristaltic pump and automatic partial collector was used. The resin column was packed with 0.6L of resin (HYO 6) having a diameter of 4cm and a height of 56cm. Loading the pretreated UMP conversion solution P1 of the fourth batch of the embodiment 1 into a column, wherein the loading volume is 1.5BV, the loading rate is 1.5BV/h, dividing the whole system into three steps of adsorption, impurity washing and desorption by switching mobile phases, switching sequentially, and enabling the advanced UMP conversion solution to be adsorbed by a resin column - After saturation, a raffinate (Ca 2+ 、Mg 2+ 、Na + 、ATP + 、AMP + 、UR - 、PO 4 3- ) And then 0.8BV of pure water is switched for impurity washing to obtain a small amount of UMP conversion liquid in gaps of resin particles, then 1.5BV of NaCl solution is used for desorption, and the adsorbed UMP product is collected. The regenerant, namely desorbent NaCl, after the desorption was completed, the NaCl in the resin column gap was washed out with 1BV of pure water at a flow rate of 0.3BV/h, and finally the resin was completely converted into chlorine type. UMP detection by HPLC of collected product effluent - 、ATP + 、AMP + 、UR - Concentration, detection of Ca by ion chromatography 2+ 、Mg 2+ 、PO 4 3- Concentration of plasma, ca 2+ 、Mg 2+ 、PO 4 3- Concentration below ion chromatographic limit, ATP + 、AMP + 、UR - The concentration of (C) is lower than the detection limit of liquid chromatography, the yield of UMP products reaches 99%, the purity of HPLC reaches 100%, and Ca 2+ 、Mg 2+ 、PO 4 3- The removal rate of the plasma reaches 99.7 percent.
Comparative example 1
As in example 5, the same batch of feed solution, the same sample injection amount, flow rate and other conditions were used, the chlorine anion exchange resin HY06 was replaced with the strongly basic anion exchange resins 201x7 and D301, respectively, the presence of UMP was detected in the product solution collected at the outlet of the adsorption section, the UMP product yield in the collected solution of the 201x7 resin was 75%, and the UMP product yield in the collected solution of the D301 resin was 71%, indicating that neither resin could purify UMP product well in the UMP conversion solution.
The invention provides a method and a thought of a process for separating and purifying UMP conversion liquid by utilizing chromatographic technology, and the method and the way for realizing the technical scheme are numerous, the above description is only a preferred embodiment of the invention, and it should be pointed out that a plurality of improvements and modifications can be made to those skilled in the art without departing from the principle of the invention, and the improvements and modifications are also considered as the protection scope of the invention. The components not explicitly described in this embodiment can be implemented by using the prior art.
Claims (11)
1. A process for separating and purifying UMP conversion liquid by utilizing chromatographic technique is characterized in that UMP conversion liquid is loaded into a chromatographic column containing anion exchange resin for ion exchange, and effluent liquid is collected after three steps of adsorption, impurity washing and desorption.
2. The process of claim 1, wherein the UMP conversion solution is UMP solution obtained by enzymatic removal of two phosphate groups from UTP and nanofiltration, and comprises Mg 2+ 、Na + 、UMP - 、Ca 2+ 、AMP + 、ATP + UR-and PO 4 3- ，pH1～3。
3. The process of claim 2, wherein said UMP conversion solution, mg 2+ The content of Na is 0.1 mg/L-3 g/L + The content of UMP is 1g/L to 30g/L - The content of Ca is 10g/L to 70g/L 2+ The content of (2) is 0.1 mg/L-3 g/L, AMP + The content of ATP is 2g/L to 10g/L + The content of the catalyst is 2 g/L-10 g/L, the content of UR-is 2 g/L-10 g/L, PO 4 3- The content of (C) is 0.1 mg/L-1 g/L.
4. The process of claim 1 wherein the anion exchange resin is a strong base anion exchange resin of type I or type II, or a polyamine type, or a gel type anion exchange resin.
5. The process of claim 4 wherein the polyamine comprises triethylenetetramine and/or tetraethylenepentamine.
6. The process according to claim 4 or 5, wherein the functional groups of the anion exchange resin are quaternary amine groups and/or tertiary amine groups, the content of the quaternary amine groups is 1.0-2.5 mmol/g of anion exchange resin, and the content of the tertiary amine groups is 1.0-2.5 mmol/g of anion exchange resin; the exchangeable ions of the anion exchange resin are chloride ions, the content of the chloride ions is 1.0-2.5 mmol/g, and the volume total exchange capacity is more than or equal to 1.35mmol/mL; the degree of crosslinking of the anion exchange resin is 3-10%.
7. The process according to claim 1, wherein the anion exchange resin has a particle size of 0.1 to 0.8mm, a water content of 42 to 48%, and a wet true density of 1.03 to 1.18g/cm 3 Specific surface area of 100-2000 m 2 Per gram, the pore volume is 0.51-1.33 cm 3 And/g, the aperture is 1-200 nm.
8. The process of claim 1 wherein the adsorption is carried out at a loading rate of 0.3 to 1.5BV/h.
9. The process according to claim 1, wherein the impurity-washing agent is water, the water is used in an amount of 0.8-1 BV, and the flow rate is 0.3-1.5 BV/h.
10. The process of claim 1, wherein the desorbing and desorbing agent is 0.1M to 3M aqueous NaCl, the desorbing agent is used in an amount of 0.5 to 3BV, and the flow rate is 0.3 to 1.5BV/h.
11. The process of claim 10 wherein after desorption is completed, the resin is completely converted to chlorine form without regeneration and the resin is washed free of resin interstitial NaCl with pure water prior to reuse.
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| CN1872867A (en) * | 2006-06-12 | 2006-12-06 | 南京工业大学 | Method for separating 5' nucleoside triphosphate continuously |
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| US20170130254A1 (en) * | 2014-03-26 | 2017-05-11 | Nanjing University Of Technology | Nucleotide production process |
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| CN1872867A (en) * | 2006-06-12 | 2006-12-06 | 南京工业大学 | Method for separating 5' nucleoside triphosphate continuously |
| CN101781346A (en) * | 2010-03-10 | 2010-07-21 | 南京工业大学 | Method for separating uridylic acid from biocatalytic conversion solution |
| US20170130254A1 (en) * | 2014-03-26 | 2017-05-11 | Nanjing University Of Technology | Nucleotide production process |
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