CN117802004A - Preparation method of high-purity adenine nucleoside - Google Patents
Preparation method of high-purity adenine nucleoside Download PDFInfo
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- CN117802004A CN117802004A CN202410056513.8A CN202410056513A CN117802004A CN 117802004 A CN117802004 A CN 117802004A CN 202410056513 A CN202410056513 A CN 202410056513A CN 117802004 A CN117802004 A CN 117802004A
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- adenine nucleoside
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- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 title claims abstract description 288
- OIRDTQYFTABQOQ-UHFFFAOYSA-N ara-adenosine Natural products Nc1ncnc2n(cnc12)C1OC(CO)C(O)C1O OIRDTQYFTABQOQ-UHFFFAOYSA-N 0.000 title claims abstract description 68
- 238000002360 preparation method Methods 0.000 title claims abstract description 22
- 239000002126 C01EB10 - Adenosine Substances 0.000 claims abstract description 110
- 229960005305 adenosine Drugs 0.000 claims abstract description 110
- 238000000855 fermentation Methods 0.000 claims abstract description 73
- 230000004151 fermentation Effects 0.000 claims abstract description 73
- 239000000463 material Substances 0.000 claims abstract description 31
- 238000000034 method Methods 0.000 claims abstract description 30
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 29
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 28
- 239000012528 membrane Substances 0.000 claims abstract description 22
- 239000012535 impurity Substances 0.000 claims abstract description 18
- 244000063299 Bacillus subtilis Species 0.000 claims abstract description 17
- 235000014469 Bacillus subtilis Nutrition 0.000 claims abstract description 17
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- LRFVTYWOQMYALW-UHFFFAOYSA-N 9H-xanthine Chemical compound O=C1NC(=O)NC2=C1NC=N2 LRFVTYWOQMYALW-UHFFFAOYSA-N 0.000 claims description 36
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 27
- 239000008103 glucose Substances 0.000 claims description 27
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- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 18
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 18
- 229940075420 xanthine Drugs 0.000 claims description 18
- 238000012258 culturing Methods 0.000 claims description 17
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- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 15
- 239000002609 medium Substances 0.000 claims description 15
- LPUQAYUQRXPFSQ-DFWYDOINSA-M monosodium L-glutamate Chemical compound [Na+].[O-]C(=O)[C@@H](N)CCC(O)=O LPUQAYUQRXPFSQ-DFWYDOINSA-M 0.000 claims description 15
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- 229910002027 silica gel Inorganic materials 0.000 claims description 14
- 238000011218 seed culture Methods 0.000 claims description 13
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- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 claims description 12
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 9
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- 239000002054 inoculum Substances 0.000 claims description 9
- OZAIFHULBGXAKX-UHFFFAOYSA-N 2-(2-cyanopropan-2-yldiazenyl)-2-methylpropanenitrile Chemical compound N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 claims description 7
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- 239000008272 agar Substances 0.000 claims description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 6
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 6
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 6
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- 235000015278 beef Nutrition 0.000 claims description 6
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 6
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 239000012138 yeast extract Substances 0.000 claims description 6
- DBCAQXHNJOFNGC-UHFFFAOYSA-N 4-bromo-1,1,1-trifluorobutane Chemical compound FC(F)(F)CCCBr DBCAQXHNJOFNGC-UHFFFAOYSA-N 0.000 claims description 5
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical group CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 claims description 5
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- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 claims description 3
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 claims description 3
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims description 3
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 3
- 229930006000 Sucrose Natural products 0.000 claims description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 3
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- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 18
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- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 239000002777 nucleoside Substances 0.000 description 6
- 238000002834 transmittance Methods 0.000 description 6
- 229920002554 vinyl polymer Polymers 0.000 description 6
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- 150000003833 nucleoside derivatives Chemical class 0.000 description 4
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- ZKBQDFAWXLTYKS-UHFFFAOYSA-N 6-Chloro-1H-purine Chemical compound ClC1=NC=NC2=C1NC=N2 ZKBQDFAWXLTYKS-UHFFFAOYSA-N 0.000 description 2
- 229930024421 Adenine Natural products 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 229960000643 adenine Drugs 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
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- FWDBOZPQNFPOLF-UHFFFAOYSA-N ethenyl(triethoxy)silane Chemical compound CCO[Si](OCC)(OCC)C=C FWDBOZPQNFPOLF-UHFFFAOYSA-N 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- XHXFXVLFKHQFAL-UHFFFAOYSA-N phosphoryl trichloride Chemical compound ClP(Cl)(Cl)=O XHXFXVLFKHQFAL-UHFFFAOYSA-N 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- ACRCZEREEXQWEM-VQFZJOCSSA-N (2R,3R,4S,5R)-2-(6-aminopurin-9-yl)-5-(hydroxymethyl)oxolane-3,4-diol Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O.C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O ACRCZEREEXQWEM-VQFZJOCSSA-N 0.000 description 1
- JKGVLWCSUIXYMV-WTTYOQCCSA-N (2s)-2,6-diamino-n-[(2s,3r,4s,5r)-2-(6-aminopurin-9-yl)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]hexanamide Chemical compound C1=NC2=C(N)N=CN=C2N1[C@]1(NC(=O)[C@@H](N)CCCCN)O[C@H](CO)[C@@H](O)[C@H]1O JKGVLWCSUIXYMV-WTTYOQCCSA-N 0.000 description 1
- FPHJJCBLRAPJQJ-CRKDRTNXSA-N (2s,3r,4s,5r)-2-amino-2-(6-aminopurin-9-yl)-5-(hydroxymethyl)oxolane-3,4-diol Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@]1(N)O[C@H](CO)[C@@H](O)[C@H]1O FPHJJCBLRAPJQJ-CRKDRTNXSA-N 0.000 description 1
- 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 1
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- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
- 229930010555 Inosine Natural products 0.000 description 1
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 1
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- MEFKEPWMEQBLKI-AIRLBKTGSA-N S-adenosyl-L-methioninate Chemical compound O[C@@H]1[C@H](O)[C@@H](C[S+](CC[C@H](N)C([O-])=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 MEFKEPWMEQBLKI-AIRLBKTGSA-N 0.000 description 1
- IHNHAHWGVLXCCI-FDYHWXHSSA-N [(2r,3r,4r,5s)-3,4,5-triacetyloxyoxolan-2-yl]methyl acetate Chemical compound CC(=O)OC[C@H]1O[C@@H](OC(C)=O)[C@H](OC(C)=O)[C@@H]1OC(C)=O IHNHAHWGVLXCCI-FDYHWXHSSA-N 0.000 description 1
- FJJCIZWZNKZHII-UHFFFAOYSA-N [4,6-bis(cyanoamino)-1,3,5-triazin-2-yl]cyanamide Chemical compound N#CNC1=NC(NC#N)=NC(NC#N)=N1 FJJCIZWZNKZHII-UHFFFAOYSA-N 0.000 description 1
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Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a preparation method of high-purity adenine nucleoside, utilize bacillus subtilis bcsw2023-1 as the fermentation strain, in the course of fermentation, adopt and feed supplement fermentation process of the compound carbon source of fed-batch to produce adenine nucleoside in the adenosine production stage, after fermentation, utilize the ceramic membrane to filter, ultrafiltration and filter, decolor, concentrate, crystallize, recrystalize method to get adenosine crude product; then adopting adenine nucleoside as a template to prepare a surface molecular imprinting chromatographic material, filling a Cheng Sepu column, preparing purified crude adenine nucleoside by a chromatographic column chromatography technology, and removing impurities in the purified crude adenine nucleoside; and finally crystallizing the purified adenine nucleoside to obtain a solid high-purity product. After crude extraction, the adenine nucleoside fermentation broth prepared by the method has the purity of more than 98.5 percent, and after purification by the special molecularly imprinted material, the adenine nucleoside fermentation broth has the purity of 99.9 percent.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and in particular relates to a preparation method for obtaining high-purity adenine nucleoside through crude extraction and molecular imprinting purification by utilizing bacillus subtilis to ferment and produce adenosine.
Background
Adenine nucleoside, abbreviated as Adenosine (Adenosine), chemical name 9-beta-D-ribofuranosyl adenine (9-beta-D-ribofuranosyl adenine), molecular formula C 10 H 13 N 5 O 4 The molecular weight is 267.24. Adenine nucleoside is ubiquitous in organisms, plays an important role in regulation and control in the physiological and biochemical processes, and has wide medicinal value. In China, the adenosine injection is taken as a new drug to be approved by registration of the SFDA, which provides a very wide development prospect for production and sales of adenosine products in China. In addition, adenosine is an important intermediate for synthesizing many nucleoside medicines, and can be used for producing many medicines such as nucleoside antibiotics such as high melon amino adenosine, lysyl amino adenosine, succinyl-5' -adenylate, S-adenosyl methionine and the like. Adenosine has long-term development prospect and wide market.
The production method of adenosine mainly includes enzyme method, chemical synthesis method and fermentation method, the enzyme method mainly adopts enzymolysis RNA, and utilizes phosphodiesterase to hydrolyze yeast RNA so as to obtain 4 kinds of nucleotide, then further adopts enzymolysis to remove phosphoric acid so as to obtain four kinds of nucleoside, and uses cation exchange resin to separate so as to obtain the adenosine. However, the hydrolysis method can obtain four nucleoside substances simultaneously, which brings inconvenience to the subsequent separation and extraction. The chemical synthesis process includes the reaction of hypoxanthine with phosphorus oxychloride to obtain 6-chloropurine, the condensation of 6-chloropurine with tetraacetylribose and ammonolysis to obtain adenosine. The two methods have the defects of high cost, serious pollution, serious limitation on popularization and application and the like. The report of producing adenosine by fermentation method is that the first report of producing adenosine by fermentation method is in Japan, after 20 years of research effort, the large-scale industrialized production stage is entered, the starting of producing adenosine by domestic fermentation method is late, the inosine producing strain is mainly used as the starting strain, the mutant strain with specific mark on genetic character is bred by using physical or chemical mutagenesis method to ferment and produce the adenosine, and compared with overseas, the method has the problems of lower yield, high cost and the like. Meanwhile, due to the limitation of purification cost, the existing adenine nucleosides in China have the problem of low purity (lower than 99.9 percent), and cannot meet the requirements of higher-level use scenes such as cell culture. The traditional method of adsorption by activated carbon and resin is difficult to remove a small amount of impurities (the content is lower than 0.1%) in the adenine nucleoside; while the traditional preparation chromatographic purification method can realize the removal of a small amount of impurities in the adenine nucleoside, the cost is high, and the method is not suitable for industrial production. It is therefore desirable to develop a low cost and efficient process for purifying adenine nucleosides.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for preparing high-purity adenosine by a fermentation method, and the method aims to solve the problems that after fermentation, the adenosine after crude extraction is further purified by a synthetic molecular imprinting material after the crude extraction is processed, so that the high-purity adenosine with the purity of more than 99.9 percent is obtained, and different application scenes of the adenosine are met.
The technical scheme for achieving the aim of the invention is as follows:
an adenine nucleoside-producing strain, the name of which is: bcsw2023-1, class naming: the preservation number of the bacillus subtilis (Bacillus subtilis) is CGMCC No.29120, and the preservation date is as follows: 2023, 11, 23, deposit unit: china general microbiological culture Collection center (CGMCC), preservation address: the institute of microorganisms of national academy of sciences of China, no. 1, no. 3, north Chen West Lu, the Korean region of Beijing.
A preparation method of high-purity adenine nucleoside comprises the following steps of
Comprising the step of producing an adenine-nucleoside-containing fermentation broth by fermentation using the strain of claim 1;
comprises the steps of purifying fermentation liquor containing adenine nucleoside to form crude adenosine;
comprises the step of removing impurities from the crude adenosine, and finally obtaining the high-purity adenine nucleoside product.
Moreover, the step of purifying the fermentation broth of the adenine nucleoside to form a crude adenosine product comprises the steps of: ceramic membrane filtration, ultrafiltration membrane filtration, decolorization, concentration, crystallization, and recrystallization steps.
And the step of removing impurities from the crude adenosine comprises the following steps: filling chromatographic columns with an adenine nucleoside molecular imprinting chromatographic material, and obtaining adenine nucleoside after column chromatography.
Moreover, the preparation method of the adenine nucleoside molecularly imprinted chromatographic material comprises the following steps: introducing vinyl groups on the surface of porous silica gel, and then placing the porous silica gel modified with vinyl groups, methacrylic acid based acrylic acid, ethylene glycol dimethacrylate and adenine nucleoside in acetonitrile solvent by adopting a one-pot method, initiating by azodiisobutyronitrile, stirring and polymerizing to obtain the adenine nucleoside molecular imprinting chromatographic material.
And the pore diameter of the ceramic membrane is 50-100nm.
Moreover, the molecular weight cut-off of the ultrafiltration membrane is 3000-5000Da.
Moreover, the step of fermenting the bcsw2023-1 comprises:
(1) slant culture: inoculating bacillus subtilis bcsw2023-1 to an activation inclined plane, and culturing for 20-28h at 30-33 ℃;
(2) seed culture: scraping the thalli on the inclined plane by using sterile purified water, smashing the thalli by using glass beads, and inoculating the thalli to a seed culture medium according to 1-5% of inoculum size, and carrying out constant-temperature shaking culture for 6-10 hours at the temperature of 30-33 ℃ and at the speed of 210-230rpm to obtain fermentation seed liquid;
(3) batch fermentation culture: transferring into fermentation tank containing fermentation medium at 31-35deg.C with dissolved oxygen maintained at 10-40% and NH concentration of 20% 3 ·H 2 Regulating pH with O to 6.0-7.0, culturing for 40-50 hr, and culturing in thallus OD 600 * 20. After reaching 1.0-1.5, and when the pH value begins to rebound to 7.0-7.5, starting feeding the composite carbon source, and feeding the composite carbon source for 3-10 hours before the fermentation end point;
moreover, the slant solid culture medium comprises the following components in g/L: 1-3 parts of glucose, 5-10 parts of peptone, 5-10 parts of beef extract, 2-5 parts of yeast powder, 1-2.5 parts of NaCl, 0.01-0.05 part of xanthine, 0.02-0.06 part of histidine, 20-30 parts of agar and 7.0-7.2 parts of pH;
the seed culture medium comprises the following components in g/L: 10-15 parts of glucose, 3-8 parts of yeast powder, 4-10 parts of monosodium glutamate, 5-8 parts of peptone and KH 2 PO 4 0.5-0.8,MgSO 4 ·7H 2 0.2-0.8 of O, 2-6 of urea, 0.015-0.03 of xanthine, 0.01-0.03 of histidine and pH value of 6.5-6.8;
the liquid fermentation medium comprises the following components in g/L: edible glucose 8-12, yeast extract 12-15, monosodium glutamate 8-10, K 2 HPO 4 1-2, xanthine 0.01-0.03, histidine 0.01-0.03, mgSO 4 ·7H 2 O 1-4,MnSO 4 0.003-0.005, FeSO 4 ·7H 2 O 0.003-0.005,CaCl 2 1 to 1.5, 0.5 to 0.8 of ammonium sulfate; 10-20 parts of corn steep liquor mL/L, 10-30 parts of soybean meal hydrolysate mL/L and pH of 6.4-7.0.
In addition, the amount of the composite carbon source added in each time of fermentation of the strain is 10-30 mL/h, the reducing sugar content in the fermentation liquid is maintained to be 1.0-3.0%, and the composite carbon source comprises any two of glucose, glycerol, lactose and sucrose.
The invention has the advantages and beneficial effects that:
1. the microbial strain used in the invention can still ensure that the production capacity of the microbial strain is not reduced after continuous passage and expansion culture, thereby laying a good foundation for industrial production.
2. The invention is a fermentation production process, can produce adenosine more efficiently and greenly, and prevents irreversible pollution of chemical reagents to the environment.
3. The invention maintains the residual sugar content in the fermentation process to be basically stable by continuously adding the composite carbon source in the fermentation process, so that the yield of the adenine nucleoside is greatly improved to 45.0g/L, and compared with the non-supplement process, the yield is improved by 30 percent.
4. After crude extraction, the adenosine fermentation broth prepared by the method has the purity of over 98.5 percent, and after purification by the special molecularly imprinted material, the adenosine fermentation broth has the purity of 99.9 percent.
Drawings
FIG. 1 is a chromatogram of 200ppm adenosine on a common C18 chromatographic column.
FIG. 2 is an enlarged view of a chromatogram of 200ppm adenosine on a common C18 chromatographic column.
FIG. 3 is a chromatogram of 200ppm adenosine on an adenosine surface molecularly imprinted chromatographic column.
FIG. 4 is an enlarged view of a chromatogram of 200ppm adenosine on an adenosine surface molecularly imprinted chromatographic column.
FIG. 5 is a chromatogram of a test prior to purification of an adenosine sample.
FIG. 6 is a graph of a test chromatogram after adenosine sample purification.
The strain names are as follows: bcsw2023-1, class naming: the preservation number of the bacillus subtilis (Bacillus subtilis) is CGMCC No.29120, and the preservation date is as follows: 2023, 11, 23, deposit unit: china general microbiological culture Collection center, preservation address: the institute of microorganisms of national academy of sciences of China, no. 1, no. 3, north Chen West Lu, the Korean region of Beijing.
Detailed Description
The invention is described below with reference to specific examples. Those skilled in the art will appreciate that the specific ratios of materials, process conditions, and results thereof described in these specific examples are intended to be illustrative of the present invention only and are not intended to limit the scope of the present invention in any way.
A process for preparing high-purity adenine nucleoside includes such steps as adding the composite carbon source, continuous flowing to obtain the fermented liquid of adenosine, ceramic membrane separation, ultrafiltering, decoloring, crystallizing, recrystallizing, and molecular imprinting.
The invention uses the strain name: bcsw2023-1, class naming: the preservation number of the bacillus subtilis (Bacillus subtilis) is CGMCC No.29120, and the preservation date is as follows: 2023, 11, 23, deposit unit: china general microbiological culture Collection center (CGMCC), preservation address: the strains used in the following examples were Bacillus subtilis bcsw2023-1 strain, which was used in the following examples, and was written in the national institute of microbiology, national institute of sciences, no. 3, north Chen, west Lu, 1, beijing, and the area of Chapter.
The method comprises the following operation steps:
(1) Slant culture: inoculating bacillus subtilis bcsw2023-1 to an activation inclined plane, and culturing for 20-28h at 30-33 ℃;
(2) Seed culture: scraping the thalli on the inclined plane by using sterile purified water, smashing the thalli by using glass beads, and inoculating the thalli to a seed culture medium according to 1-5% of inoculum size, and carrying out constant-temperature shaking culture for 6-10 hours at the temperature of 30-33 ℃ and at the speed of 210-230rpm to obtain fermentation seed liquid;
(3) Batch fermentation culture: transferring into fermentation tank containing fermentation medium at 31-35deg.C with dissolved oxygen maintained at 10-40% and NH concentration of 20% 3 ·H 2 Regulating pH with O to 6.0-7.0, culturing for 40-50 hr, and culturing in thallus OD 600 * 20. After reaching 1.0-1.5, and when the pH value begins to rebound to 7.0-7.5, the composite carbon source is fed in, and 3-10 hours before the fermentation end point is fed in.
OD 600 * 20. The absorbance of the bacterial culture at 600nm was measured, and the cell 20 was diluted.
(4) And (3) ceramic membrane filtration: filtering the fermentation broth with ceramic membrane (membrane pore diameter 50-100 nm), wherein the pressure of the inlet and outlet membranes is 0.3 and 0.4Mpa, the pH is 6.0-7.0, and the temperature is 50-65deg.C; the bacterial protein is recovered by filtering bacterial slurry with a plate frame, and the filtrate is added into an ultrafiltration membrane for further filtration.
(5) Ultrafiltering the ceramic membrane filtrate with organic membrane (membrane cut-off molecular weight of 3000-5000) to remove small molecular protein in the fermentation broth and obtain ultrafiltrate.
(6) Adding 0.1-1% active carbon into the ultrafiltrate, pH4.0-5.0, decolorizing at 50-60deg.C for 0.5-1 hr, and plate-frame filtering to obtain decolorized solution.
(7) The decolorized solution enters a vacuum concentrator for concentration, the temperature is 60-65 ℃, the vacuum degree is-0.085-0.090 Mpa, and the decolorized solution is concentrated to a concentrated solution with the adenosine concentration of 100-120 g/L.
(8) Pumping the concentrated solution into a freezing crystallization tank, cooling to 5-10deg.C, and crystallizing for 4-6 hr; and (5) centrifugally separating by using a centrifugal machine to obtain the crude adenosine.
(9) Dissolving the crude adenosine with concentration of 20g/L, adding 0.1-0.5% active carbon for decolorization, pH4.0, decolorizing at 60deg.C for half an hour, and plate-frame filtering to obtain decolorized solution.
(10) Concentrating the decolorized solution in a vacuum concentrator at 65deg.C under vacuum degree of-0.085 Mpa to obtain concentrated solution with adenosine concentration of 100-120 g/L; pumping the concentrated solution into a freezing crystallization tank, cooling to 5-10deg.C, and crystallizing for 4-6 hr; separating the crystallization liquid by a centrifugal machine to obtain an adenosine solution of a product with the adenosine purity of 98.5 percent.
(11) Purifying: preparing chromatographic column from the surface molecular imprinting material of adenosine, separating saturated solution prepared from crude adenosine by column chromatography, and collecting liquid with retention time corresponding to adenosine chromatographic peak.
(12) And (3) crystallization: crystallizing the purified adenine nucleoside to obtain a solid high-purity product.
Wherein, the inclined plane solid culture medium comprises the following components in g/L: 1-3 parts of glucose, 5-10 parts of peptone, 5-10 parts of beef extract, 2-5 parts of yeast powder, 1-2.5 parts of NaCl, 0.01-0.05 part of xanthine, 0.02-0.06 part of histidine, 20-30 parts of agar and 7.0-7.2 parts of pH;
the seed culture medium comprises the following components in g/L: 10-15 parts of glucose, 3-8 parts of yeast powder, 4-10 parts of monosodium glutamate, 5-8 parts of peptone and KH 2 PO 4 0.5-0.8,MgSO 4 ·7H 2 0.2-0.8 of O, 2-6 of urea, 0.015-0.03 of xanthine, 0.01-0.03 of histidine and pH value of 6.5-6.8;
the liquid fermentation medium comprises the following components in g/L: edible glucose 8-12, yeast extract 12-15, monosodium glutamate 8-10, K 2 HPO 4 1-2, xanthine 0.01-0.03, histidine 0.01-0.03, mgSO 4 ·7H 2 O 1-4,MnSO 4 0.003-0.005, FeSO 4 ·7H 2 O 0.003-0.005,CaCl 2 1 to 1.5, 0.5 to 0.8 of ammonium sulfate; 10-20 parts of corn steep liquor mL/L, 10-30 parts of soybean meal hydrolysate mL/L and pH of 6.4-7.0.
The amount of the composite carbon source fed in each time in the step (3) is 10-30 mL/h, and the reducing sugar content in the fermentation liquor is maintained to be 1.0-3.0%. The composition of the composite carbon source is any two of glucose, glycerol, lactose and sucrose.
The invention also provides a preparation method of the adenine nucleoside molecularly imprinted material, which comprises the following steps:
firstly, introducing vinyl groups on the surface of porous silica gel, then adopting a one-pot method, placing the porous silica gel modified with vinyl groups, methacrylic acid based acrylic acid, ethylene glycol dimethacrylate and adenine nucleoside in an acetonitrile solvent, initiating by azodiisobutyronitrile, stirring, and polymerizing to obtain the molecularly imprinted material of adenine nucleoside. The invention relates to a preparation method of an adenine nucleoside molecularly imprinted material, which comprises the following steps:
the preparation method of the first adenine nucleoside molecularly imprinted material comprises the following steps:
step 1, preparation of vinyl modified porous silica gel: 10g of 5 μm 100A spherical porous silica gel is placed in a 250ml reaction kettle, 6g of triethoxyvinylsilane and 150ml of toluene are added, and the mixture is heated and refluxed for 17 hours. Filtering to obtain the vinyl modified porous silica gel.
Step 2, preparing a surface molecular imprinting material: 10g of 5 μm 100A vinyl spherical porous silica gel is placed in a 250ml reaction kettle, 150ml of acetonitrile, 6g of methacrylic acid, 1.5g of ethylene glycol dimethacrylate and 0.6g of adenine nucleoside are added, 100mg of azobisisobutyronitrile is added, stirring is carried out at 60 ℃ and the reaction is carried out for 24 hours. Filtering to obtain the 5 μm 100A adenine nucleoside surface molecular imprinting material.
The preparation method of the second adenine nucleoside molecularly imprinted material comprises the following steps:
step 1, preparation of vinyl modified porous silica gel: 10g of 10 μm, 200A spherical porous silica gel is placed in a 250ml reaction kettle, and then 6g of triethoxyvinylsilane, 150ml of toluene are added and heated to reflux for reaction for 17 hours. Filtering to obtain the vinyl modified porous silica gel.
Step 2, preparing a surface molecular imprinting material: 10g of a 10 μm 200A vinyl spherical porous silica gel is placed in a 250ml reaction kettle, 180ml of acetonitrile, 5g of methacrylic acid, 1.2g of ethylene glycol dimethacrylate and 0.5g of adenine nucleoside are added, 80mg of azobisisobutyronitrile is added, stirring is carried out at 60 ℃ and the reaction is carried out for 24 hours. Filtering out to obtain the surface molecular imprinting material of the 10 mu m 200A adenine nucleoside.
Adenine nucleoside (hereinafter referred to as adenosine) has a relatively high polarity and is not suitable for normal phase chromatography. On the reverse phase chromatographic column, impurities (as shown in fig. 2, the recovery time is 5.889min and 7.360min respectively) which are close to the adenosine peak are difficult to remove, which means that the impurities have similar physicochemical properties, and the impurities are difficult to separate by adopting common purification means (such as recrystallization, activated carbon adsorption and the like) without great loss of the adenosine. The traditional reversed phase preparation chromatographic technology can realize separation of adenosine and impurities, but has lower separation degree, so that the sample quantity of high purity obtained by single preparation is smaller, the cost is high, and the industrial production of high purity adenosine with purity of more than 99.9% is limited.
The surface molecular imprinting material of the adenosine is adopted, so that the selective adsorption capacity of the adenosine to the specificity of the adenosine can be utilized, the separation degree of the adenosine and impurities is increased, the single preparation sample quantity is improved, and the production cost of the high-purity adenosine is greatly reduced. Compared with fig. 4 and fig. 2, the difference between the retention time of the main peak and the front and back impurity peaks in fig. 4 is larger, for example, in fig. 4, the time distance between the adenosine peak and the back impurity peak is larger than that of a common C18 chromatographic column, and the separation degree of adenosine and impurities can be larger than that of a chromatographic column prepared by using a surface molecular imprinting adsorption material of adenosine.
As shown in fig. 5 and 6, the purity of adenosine can be easily improved from 99.8% to 99.9% by using a chromatographic column made of a surface molecularly imprinted adsorption material of adenosine.
The analytical methods used for the adenosine content in the examples below were as follows:
liquid chromatograph: a qingbo Hua Yexiang chromatograph;
chromatographic column: A. a common C18 liquid chromatography column (5 μm, 4.6X1250 mm),
B. surface molecular imprinting chromatographic column (5 μm, 4.6X1250 mm) of adenosine;
column temperature: 25 ℃; a detector: an ultraviolet detector (254 nm);
mobile phase: the mobile phase comprises phases a and B, wherein: a is 0.01M KH 2 PO 4 B is acetonitrile, A: volume ratio b=92:8, 8; flow rate: 1.0 mL/min; sample injection amount: 20. mu L;
sample: 200ppm aqueous adenosine solution.
The analytical methods used for the adenosine content in the examples below were as follows: the instrument is adopted: waters UHPLC; chromatographic column: adenosine surface molecular imprinting chromatographic column (5 μm, 4.6X1250 mm); column temperature: 30. the temperature is lower than the temperature; a detector: an ultraviolet detector (254 nm); the mobile phase includes items a and B, wherein: a is 0.01M KH 2 PO 4 B is acetonitrile, A: b=92: 8, 8; flow rate: 1.0 mL/min; sample injection amount: 20. mu L; sample: 200ppm aqueous adenosine solution.
Example 1
A preparation method of high-purity adenine nucleoside comprises the following steps:
the slant culture medium formula is calculated in g/L: glucose 2, peptone 7, beef extract 8, yeast powder 3, naCl 2, agar 30 and pH 7.0.
Seed medium formulation in g/L: 10-15 parts of glucose, 3-8 parts of yeast powder, 4-10 parts of monosodium glutamate, 5-8 parts of peptone and KH 2 PO 4 0.5-0.8,MgSO 4 ·7H 2 0.2-0.8 of O, 2-6 of urea, 0.015-0.03 of xanthine, 0.01-0.03 of histidine and 6.5-6.8 of pH value; wherein, the formula of the seed culture medium is preferably as follows: glucose 11, yeast powder 6, monosodium glutamate 7, peptone 7 and KH 2 PO 4 0.6, MgSO 4 ·7H 2 O0.5, urea 4, xanthine 0.02, histidine 0.02, pH 6.6.
Fermentation medium is calculated in g/L: glucose 10, yeast extract 13, monosodium glutamate 9,K for oral administration 2 HPO 4 1.5 xanthine 0.02, L-histidine 0.02, mgSO 4 ·7H 2 O 3,MnSO 4 0.004,FeSO 4 ·7H 2 O 0.004,CaCl 2 1.3, ammonium sulfate 0.6 corn steep liquor 15 mL/L, soybean meal hydrolysate 15 mL/L.
The method comprises inoculating Bacillus subtilis as initial strain to activated slant, culturing at 31deg.C for 22 hr, scraping thallus on slant with sterile purified water, breaking glass beads, inoculating into shake flask seed solution according to 1-5% inoculum size, culturing at 31deg.C at 200 rpm for 9 hr, and culturing at 15% (v)Transferring the inoculum size of/v) to a 5L fermenter containing a fermentation medium, maintaining dissolved oxygen at a temperature of 32deg.C and a temperature of 10-40%, and transferring the inoculum size to a fermentation tank containing a fermentation medium with NH 3 ·H 2 And (3) regulating the pH by O, maintaining the pH at 7.1, and feeding a composite carbon source formed by combining glucose and glycerol until the pH starts to rebound so as to control the content of residual sugar in the fermentation liquor to be 2.0%, stopping feeding 5 hours before the fermentation is finished, fermenting 45. 45h, and enabling the adenosine yield to reach 45g/L.
The filtrate light transmittance is 98% after the filtration of the fermentation liquor is filtered by a ceramic membrane with the aperture of 50 nm.
Filtering the ceramic membrane filtrate by an ultrafiltration membrane with a molecular weight of 5000Da, removing the impurity proteins and most pigments to obtain filtrate, and measuring the purity of the adenosine to be 95%.
Adding 0.4% medicinal grade active carbon into the ultrafiltrate, pH4.0, temperature 50 deg.C, decolorizing for 1 hr, plate-frame filtering to obtain decolorized solution, and measuring light transmittance of the decolorized solution 99%, and purity of decolorized solution adenosine 95.8%.
And (5) concentrating the decolorized solution in a vacuum concentrator at 63 ℃ and a vacuum degree of-0.087 Mpa until the concentration of the adenosine is 110g/L.
Pumping the concentrated solution into a freezing crystallization tank, cooling to 6 ℃, and crystallizing for 5 hours; and (5) centrifugally separating by using a centrifugal machine to obtain the crude adenosine.
Dissolving crude adenosine with concentration of 20g/L, adding 0.2% active carbon for decolorizing, pH4.0, decolorizing at 60deg.C for half an hour, and plate-frame filtering to obtain decolorized solution.
And (3) feeding the decolorized solution into a vacuum concentrator for concentration, wherein the temperature is 65 ℃, the vacuum degree is-0.085 Mpa, and the decolorized solution is concentrated into a concentrated solution with the concentration of 100g/L of adenosine.
Pumping the concentrated solution into a freezing crystallization tank, cooling to 6 ℃, and crystallizing for 5 hours; separating the crystallization liquid by a centrifuge to obtain a crystallized adenosine solid with the adenosine purity of 98.5%, and preparing the crystallized solid into a saturated solution for later use.
Purifying: preparing chromatographic column from adenosine surface molecularly imprinted material, separating saturated solution prepared by crystallizing solid adenosine by column chromatography (chromatographic column is filled with first adenine nucleoside molecularly imprinted material), and collecting liquid with retention time corresponding to adenosine chromatographic peak.
Crystallizing: concentrating and crystallizing the purified adenosine solution to obtain a solid high-purity product. The purity was found to be 99.91%.
Referring to fig. 5 and 6, the purity of the adenosine can be improved to more than 99.90% through the preparation and purification of the adenine nucleoside molecularly imprinted material by column chromatography.
Example 2
A preparation method of high-purity adenine nucleoside comprises the following steps:
the slant culture medium formula is calculated in g/L: glucose 2, peptone 7, beef extract 8, yeast powder 3, naCl 2, agar 30 and pH 7.0.
Seed medium formulation in g/L: 10-15 parts of glucose, 3-8 parts of yeast powder, 4-10 parts of monosodium glutamate, 5-8 parts of peptone and KH 2 PO 4 0.5-0.8,MgSO 4 ·7H 2 0.2-0.8 of O, 2-6 of urea, 0.015-0.03 of xanthine, 0.01-0.03 of histidine and 6.5-6.8 of pH;
the seed culture medium preferably comprises the following formula: glucose 11, yeast powder 6, monosodium glutamate 7, peptone 7 and KH 2 PO 4 0.6 MgSO 4 ·7H 2 O0.5, urea 4, xanthine 0.02, histidine 0.02, pH 6.6.
Fermentation medium is calculated in g/L: glucose 10, yeast extract 13, monosodium glutamate 9 and KH for oral administration 2 PO 4 1.5 xanthine 0.02, L-histidine 0.02, mgSO 4 ·7H 2 O 3,MnSO 4 0.004,FeSO 4 ·7H 2 O 0.004,CaCl 2 1.3, ammonium sulfate 0.6, corn steep liquor 15 mL/L, soybean meal hydrolysate 15 mL/L.
Inoculating bacillus subtilis bcsw2023-1 serving as an initial strain to an activated inclined plane, culturing at 31 ℃ for 22h, scraping thalli on the inclined plane by using sterile purified water, smashing glass beads, inoculating into shake flask seed liquid according to 1-5% of inoculation amount, and culturing at 200 rpm for 9h at 31 ℃; transferring to 5L fermenter containing fermentation medium at a 15% (v/v) inoculum size, maintaining dissolved oxygen at 32 deg.C and 10-40%, and adding NH 3 ·H 2 O adjusts the pH, the pH is maintained at 7.1, and the mixture is waited to pAnd when H starts to rebound, feeding a composite carbon source formed by combining glucose and glycerol so as to control the content of residual sugar in the fermentation liquid to be 2.0%, stopping feeding until 5 hours before fermentation is finished, fermenting 45H, and enabling the adenosine yield to reach 45.2g/L.
The fermentation broth was filtered through a ceramic membrane with a pore size of 50nm, and the filtrate transmittance was 98% after filtration.
The ceramic membrane filtrate is filtered by an ultrafiltration membrane with a molecular weight of 5000Da, and the impurity proteins and most pigments are removed to obtain filtrate, and the purity of the adenosine is measured to be 95%.
Adding 0.5%medicinal grade active carbon into the ultrafiltrate, pH4.0, temperature 50 ℃, decolorizing for 1h, plate-frame filtering to obtain decolorized solution, and measuring the light transmittance of the decolorized solution to 99.7%, wherein the purity of the decolorized solution adenosine is 96.1%.
The decolorized solution enters a vacuum concentrator for concentration, the temperature is 63 ℃, the vacuum degree is-0.087 Mpa, and the decolorized solution is concentrated to a concentrated solution with the concentration of 115g/L of adenosine.
Pumping the concentrated solution into a freezing crystallization tank, cooling to 5 ℃, and crystallizing for 5 hours; and (5) centrifugally separating by using a centrifugal machine to obtain the crude adenosine.
Dissolving the crude adenosine with concentration of 22g/L, adding 0.3% active carbon for decolorization, pH4.5, decolorizing at 60deg.C for half an hour, and plate-frame filtering to obtain decolorized solution.
The decolorized solution enters a vacuum concentrator for concentration at 65 ℃ and the vacuum degree of-0.085 Mpa until the concentration of the adenosine is 100g/L.
Pumping the concentrated solution into a freezing crystallization tank, cooling to 6 ℃, and crystallizing for 5 hours; separating the crystallization liquid by a centrifuge to obtain a crystallized adenosine solid with the adenosine purity of 98.7%, and preparing the crystallized solid into a saturated solution for later use.
Purifying: preparing chromatographic column from adenosine surface molecularly imprinted material, separating saturated solution prepared by coarse extracted adenosine by column chromatography (chromatographic column is filled with second adenine nucleoside molecularly imprinted material), and collecting liquid with retention time corresponding to adenosine chromatographic peak.
And (3) crystallization: concentrating and crystallizing the purified adenosine solution to obtain a solid high-purity product. The purity was found to be 99.95%.
Example 3
A preparation method of high-purity adenine nucleoside comprises the following steps:
the slant culture medium formula is calculated in g/L: glucose 2, peptone 7, beef extract 8, yeast powder 3, naCl 2, agar 30 and pH 7.0.
Seed medium formulation in g/L: 10-15 parts of glucose, 3-8 parts of yeast powder, 4-10 parts of monosodium glutamate, 5-8 parts of peptone and KH 2 PO 4 0.5-0.8,MgSO 4 ·7H 2 0.2-0.8 of O, 2-6 of urea, 0.015-0.03 of xanthine, 0.01-0.03 of histidine and 6.5-6.8 of pH;
the seed culture medium is preferably prepared from the following formula: glucose 11, yeast powder 6, monosodium glutamate 7, peptone 7 and KH 2 PO 4 0.6,MgSO 4 ·7H 2 O0.5, urea 4, xanthine 0.02, histidine 0.02, pH 6.6.
Fermentation medium is calculated in g/L: glucose 10, yeast extract 13, monosodium glutamate 9,K for oral administration 2 HPO 4 1.5 xanthine 0.02, L-histidine 0.02, mgSO 4 ·7H 2 O 3,MnSO 4 0.004,FeSO 4 ·7H 2 O 0.004,CaCl 2 1.3, ammonium sulfate 0.6, corn steep liquor 15 mL/L, soybean meal hydrolysate 15 mL/L.
Inoculating Bacillus subtilis bcsw2023-1 as starting strain to activated slant, culturing at 31deg.C for 22 hr, scraping thallus on slant with sterile purified water, breaking glass beads, inoculating into shake flask seed liquid according to 1-5% of inoculum size, culturing at 200rpm for 9h, transferring into 5L fermentation tank containing fermentation medium according to 15% (v/v) of inoculum size, maintaining dissolved oxygen at 32deg.C at 10-40%, and culturing with NH 3 ·H 2 And regulating the pH value by O, maintaining the pH value at 7.1, and feeding a composite carbon source formed by combining glucose and glycerol until the pH value starts to rebound so as to control the content of residual sugar in the fermentation liquor to be 2.0%, stopping feeding 5 hours before the fermentation is finished, fermenting for 45 hours, and enabling the adenosine yield to reach 45.2g/L.
The fermentation broth was filtered through a ceramic membrane with a pore size of 50nm, and the filtrate transmittance was 98% after filtration.
The ceramic membrane filtrate is filtered by an ultrafiltration membrane with a molecular weight of 5000Da, and the impurity proteins and most pigments are removed to obtain filtrate, and the purity of the adenosine is measured to be 95%.
Adding 0.6 per mill medicinal grade active carbon into the ultrafiltrate, pH4.0, decolorizing at 50deg.C for 1 hr, and plate-frame filtering to obtain decolorized solution, wherein the light transmittance of the decolorized solution is 99.9%, and the adenosine purity of the decolorized solution is 96.8%.
Concentrating the decolorized solution in a vacuum concentrator at 63 deg.C under vacuum degree of-0.087 Mpa until the concentration of adenosine is 120g/L, cooling to 5 deg.C, and crystallizing for 5 hr; and (5) centrifugally separating by using a centrifugal machine to obtain the crude adenosine.
Dissolving the crude adenosine with the concentration of 25g/L, adding 0.4% active carbon for decolorization, wherein the pH is 4.5, the temperature is 60 ℃, and the decolorization is carried out for half an hour, and the plate and frame filtration is carried out to obtain decolorized solution.
The decolorized solution enters a vacuum concentrator for concentration, the temperature is 65 ℃, the vacuum degree is-0.085 Mpa, and the decolorized solution is concentrated to a concentrated solution with the concentration of adenosine of 100g/L.
Pumping the concentrated solution into a freezing crystallization tank, cooling to 6 ℃, and crystallizing for 5 hours; separating the crystallization liquid by a centrifuge to obtain a crystallized adenosine solid with the adenosine purity of 98.6%, and preparing the crystallized solid into a saturated solution for later use.
Purifying: the chromatographic column is prepared by adopting the surface molecular imprinting material of the adenosine, then the saturated solution prepared by the adenosine after crude extraction is separated by column chromatography (the chromatographic column is filled with the first adenine nucleoside molecular imprinting material), and the liquid with retention time corresponding to the adenosine chromatographic peak (highest peak) is collected).
And (3) crystallization: concentrating and crystallizing the purified adenosine solution to obtain a solid high-purity product. The purity was found to be 99.92%.
As can be seen from the examples: the fermentation method of the fed-batch carbon source can improve the yield of the adenosine, and the purity of the adenine nucleoside can reach more than 98.5% by using the traditional extraction method, so that the method has good application value and scene. The purity of the adenine nucleoside can reach more than 99.9 percent by combining molecular imprinting separation.
Claims (10)
1. An adenine nucleoside-producing strain, characterized in that: the strain names are as follows: bcsw2023-1, class naming: the preservation number of the bacillus subtilis (Bacillus subtilis) is CGMCC No.29120, and the preservation date is as follows: 2023, 11, 23, deposit unit: china general microbiological culture Collection center, preservation address: the institute of microorganisms of national academy of sciences of China, no. 1, no. 3, north Chen West Lu, the Korean region of Beijing.
2. A preparation method of high-purity adenine nucleoside is characterized in that: comprises the following steps of
Comprising the step of producing an adenine-nucleoside-containing fermentation broth by fermentation using the strain of claim 1;
comprises the steps of purifying fermentation liquor containing adenine nucleoside to form crude adenosine;
comprises the step of removing impurities from the crude adenosine, and finally obtaining the high-purity adenine nucleoside product.
3. The method for producing high-purity adenine nucleoside as claimed in claim 2, wherein: the step of purifying the fermentation broth of the adenine nucleoside to form a crude adenosine product comprises the following steps: ceramic membrane filtration, ultrafiltration membrane filtration, decolorization, concentration, crystallization, and recrystallization steps.
4. The method for producing high-purity adenine nucleoside as claimed in claim 2, wherein: the step of removing impurities from the crude adenosine comprises the following steps: filling chromatographic columns with an adenine nucleoside molecular imprinting chromatographic material, and obtaining adenine nucleoside after column chromatography.
5. The method for producing high-purity adenine nucleoside as claimed in claim 4, wherein: the preparation method of the adenine nucleoside molecular imprinting chromatographic material comprises the following steps: introducing vinyl groups on the surface of porous silica gel, and then placing the porous silica gel modified with vinyl groups, methacrylic acid based acrylic acid, ethylene glycol dimethacrylate and adenine nucleoside in acetonitrile solvent by adopting a one-pot method, initiating by azodiisobutyronitrile, stirring and polymerizing to obtain the adenine nucleoside molecular imprinting chromatographic material.
6. The method for producing high-purity adenine nucleoside as claimed in claim 3, wherein: the aperture of the ceramic membrane is 50-100nm.
7. The method for producing high-purity adenine nucleoside as claimed in claim 3, wherein: the molecular weight cut-off of the ultrafiltration membrane is 3000-5000Da.
8. The method for producing high-purity adenine nucleoside as claimed in claim 2, wherein: the bcsw2023-1 fermentation step comprises the following steps:
(1) slant culture: inoculating bacillus subtilis bcsw2023-1 to an activation inclined plane, and culturing for 20-28h at 30-33 ℃;
(2) seed culture: scraping the thalli on the inclined plane by using sterile purified water, smashing the thalli by using glass beads, and inoculating the thalli to a seed culture medium according to 1-5% of inoculum size, and carrying out constant-temperature shaking culture for 6-10 hours at the temperature of 30-33 ℃ and at the speed of 210-230rpm to obtain fermentation seed liquid;
(3) batch fermentation culture: transferring into fermentation tank containing fermentation medium at 31-35deg.C with dissolved oxygen maintained at 10-40% and NH concentration of 20% 3 ·H 2 Regulating pH with O to 6.0-7.0, culturing for 40-50 hr, and culturing in thallus OD 600 * 20. After reaching 1.0-1.5, and when the pH value begins to rebound to 7.0-7.5, the composite carbon source is fed in, and 3-10 hours before the fermentation end point is fed in.
9. The method for producing high-purity adenine nucleoside as claimed in claim 8, wherein:
the slant culture adopts a slant solid culture medium, and the slant solid culture medium comprises the following components in g/L: 1-3 parts of glucose, 5-10 parts of peptone, 5-10 parts of beef extract, 2-5 parts of yeast powder, 1-2.5 parts of NaCl, 0.01-0.05 part of xanthine, 0.02-0.06 part of histidine, 20-30 parts of agar and 7.0-7.2 parts of pH;
the seed culture adopts a slant solid culture medium, and the seed culture medium comprises the following components in g/L: 10-15 parts of glucose, 3-8 parts of yeast powder, 4-10 parts of monosodium glutamate, 5-8 parts of peptone and KH 2 PO 4 0.5-0.8,MgSO 4 ·7H 2 0.2-0.8 of O, 2-6 of urea, 0.015-0.03 of xanthine, 0.01-0.03 of histidine and pH value of 6.5-6.8;
the batch fermentation culture adopts a liquid fermentation culture medium, wherein the liquid fermentation culture medium comprises the following components in g/L: edible glucose 8-12, yeast extract 12-15, monosodium glutamate 8-10, K 2 HPO 4 1-2, xanthine 0.01-0.03, histidine 0.01-0.03, mgSO 4 ·7H 2 O 1-4,MnSO 4 0.003-0.005, FeSO 4 ·7H 2 O 0.003-0.005,CaCl 2 1 to 1.5, 0.5 to 0.8 of ammonium sulfate; 10-20 parts of corn steep liquor mL/L, 10-30 parts of soybean meal hydrolysate mL/L and pH of 6.4-7.0.
10. The method for producing high-purity adenine nucleoside as claimed in claim 2, wherein: the amount of the composite carbon source fed in each time in the strain fermentation is 10-30 mL/h, the reducing sugar content in the fermentation liquid is maintained to be 1.0-3.0%, and the composite carbon source comprises any two of glucose, glycerol, lactose and sucrose.
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