CN103819522B - A kind of method of purifying citicoline in her Sa yeast bio conversion fluid eastwardly - Google Patents
A kind of method of purifying citicoline in her Sa yeast bio conversion fluid eastwardly Download PDFInfo
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- CN103819522B CN103819522B CN201410068841.6A CN201410068841A CN103819522B CN 103819522 B CN103819522 B CN 103819522B CN 201410068841 A CN201410068841 A CN 201410068841A CN 103819522 B CN103819522 B CN 103819522B
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Abstract
The present invention discloses the method for purifying citicoline in a kind of her Sa yeast bio conversion fluid eastwardly.Issatchenkia orientalis CDP-c bioconversion broth is passed through a kind of anion-exchange resin column by the method, first fall non-adsorbent with deionized water drip washing, adopt 0.01 ~ 0.50M salts solution wash-out CDP-c, 0.10 ~ 1.00M salts solution wash-out cytidylic acid again, the disposable CDP-c that can obtain high purity (> 99%), high yield (> 88%), and the cytidylic acid of recyclable non-complete reaction.The present invention enormously simplify sepn process, easy and simple to handle, and cost is low, environmental protection and saving, is adapted to industrialization scale operation.
Description
Technical field
The present invention relates to the separation purification method of cytidine diphosphate, particularly relate to the method for purifying citicoline in her Sa yeast bio conversion fluid eastwardly, belong to technical field of bioseparation.
Background technology
Cytidine diphosphate have another name called CDP-C (Cytidine-5 '-diphosphate choline, CDP-c, CDP-C), the important as precursors of phospholipid metabolism and the intermediate of Phosphatidylcholine biosynthesis, for the coenzyme that Yelkin TTS biosynthesizing is required, significant curative effect (Sugino, Biochimica et Biophysica Acta, 1960.40:425-434) is had to central nervous system damage model and encephalopathic.Research proves that CDP-C can promote biological metabolism, especially biosynthesizing reaction (Roe J.H., Journal of Biological Chemistry, 1954.210 (2): 703-707.) of phosphatide.The 1950's kennedy(Kennedy E.P., Journal of the American Chemical Society, 1955.77 (1): 250-251.) people such as doctor finds and determines its molecular structure, Rossiter(Rossiter after a while, R.., I.McLeod, and K.Strickland, Canadian Journal of Biochemistry and Physiology, 1957.35 (11): 945-951.) etc. its function is illustrated.The sixties, Wu Tian company of Japan researches and develops and is used for the treatment of the adjoint disturbance of consciousness such as cerebral trauma, brain operation, succeed clinically, its commodity are called Nicholin, middle translated name Ni Keling (Li Liangzhu, Li Mingye, up-to-date biochemical drug technology of preparing .2001: China Medical Science Press .).Thus, the research about cytidine diphosphate worldwide launches.China, from the seventies in last century, develops the Technology (Wang Yongzhi, Wei Yahui, Nie Lifang, northwest pharmaceutical journal, 2009.24 (3): 235-236.) utilizing yeast cell to produce cytidine diphosphate; East China University of Science ox red magnitude people, by the cerevisiae of Kappa-Carraginan-konjaku-polysaccharide gum blend embedding, for the synthesis of Cytidine Diphosphate Choline (Niu Hongxing after freeze thawing treatment, Qiu Weiran, Ding Qingbao, East China University of Science's journal, 2002.).
Biological synthesis process is prepared in the reaction solution of cytidine diphosphate, phosphorylcholine calcium containing protein, pigment, non-complete reaction, cytidylic acid, phosphoric acid salt, magnesium sulfate and the material such as glucose metabolism by product and cytidine, these impurity bring a lot of difficulty to the separation and purification of cytidine diphosphate.The removal that cytidine diphosphate needs through thalline and albumen is extracted from above-mentioned reaction solution, 769 charcoal absorptions, ion exchange resin adsorbs, stepwise elution, step (the Huang Ying such as carbon dust decolouring, Chinese Journal of Pharmaceuticals, 1985.1:001.), process is complicated, yield only has 45%, loss is serious, reacts remaining cytidylic acid and is not recycled, be not suitable for industrial mass production.
Summary of the invention
For the deficiency that prior art exists, the applicant, through Improvement, provides the method for purifying citicoline in a kind of her Sa yeast bio conversion fluid eastwardly.The present invention only utilizes a kind of anion-exchange resin column achieve the CDP-c of separating high-purity, high yield in disposable her Sa yeast bio conversion fluid eastwardly and reclaim non-complete reaction cytidylic acid, easy and simple to handle, process is simple, and cost is low, is adapted to industrialization scale operation.
Technical scheme of the present invention is as follows:
The method of purifying citicoline in her Sa yeast bio conversion fluid, comprises the steps: eastwardly
(1) be that 0.1 ~ 10g/L(is preferably 2 ~ 5g/L by CDP-c content) Issatchenkia orientalis bioconversion broth by highly basic or weak base anion-exchange resin post;
(2) upper prop is complete, with anion-exchange resin column described in deionized water drip washing, rinses out non-adsorbent;
(3) wash post with the salts solution of 0.01 ~ 0.50M, until column outlet place effluent liquid is no longer containing CDP-c, collects elutriant, obtain high-purity C DP-c solution;
(4) wash post with the salts solution of 0.10 ~ 1.00M, until column outlet place effluent liquid is no longer containing cytidylic acid, collect elutriant, evaporation concentration, dialysis tubing desalts, freeze-drying, reclaims and obtains cytidylic acid.
Particularly, step (1) described highly basic or weak base anion-exchange resin post are selected from macropore polystyrene strongly basic anion exchange resin post or macropore polystyrene weak base anion-exchange resin post, comprise 717, D301, D318, granularity 0.3 ~ 1.2mm >=95%, upper colume content is 5 ~ 40%(mass ratio that post exchanges total amount), be preferably 10 ~ 15%(mass ratio), post aspect ratio is 10:1 ~ 50:1, is preferably 10:1 ~ 20:1.
Particularly, on step (1) described highly basic or weak base anion-exchange resin post, column flow rate is 0.2 ~ 2mL/min, is preferably 0.5 ~ 1.5mL/min.
Preferably, step (3) described concentration of salt solution is 0.02 ~ 0.20M.
Preferably, step (4) described concentration of salt solution is 0.20 ~ 0.50M.
Particularly, step (3) and (4) described wash-out salts solution are selected from NaCl, NH
4hCO
3, NaHCO
3, NH
4cl, Na
2cO
3, KCl, KHCO
3, K
2cO
3, be preferably NaCl, NaHCO
3or Na
2cO
3.
The present invention also comprise step (3) gained CDP-c solution is concentrated after, desalted by the dialysis tubing of molecular weight cut-off 100, freeze-drying, obtains the step of purity more than 98% cytidine diphosphate solid.
The present invention is also included in after CDP-c separation and purification terminates, and described anion-exchange resin column salt, bronsted lowry acids and bases bronsted lowry is rinsed respectively the step recycled.
Advantageous Effects of the present invention is:
The present invention with her Sa yeast bio cytidine diphosphate conversion fluid of east for purification raw material, only adopt a kind of highly basic or weak base anion-exchange resin chromatography by most of species adsorbs wherein on pillar, utilize each material with the difference of resin-bonded ability, meticulously grope to optimize wash-out concentration and condition, finally realize low concentration (0.01 ~ 0.50M, be preferably 0.02 ~ 0.20M) salts solution wash-out cytidine diphosphate, higher concentration (0.10 ~ 1.00M, be preferably 0.20 ~ 0.50M) salts solution wash-out thuja acid, reach disposable purifying citicoline and reclaim the object that reaction remains cytidylic acid.Bioconversion broth of the present invention only needed a post can obtain high purity (> 99%), the cytidine diphosphate of high yield (> 88%), and the cytidylic acid of the non-complete reaction of recyclable major part, other impurity are all removed, compared with prior art, easy and simple to handle, process is simple, environmental protection and saving, cost is low, product yield is high, good separating effect, overcome existing CDP-c separating technology to realize through the combination of two or more separator columns, CDP-c and the cytidylic acid rate of recovery low, the defect that sepn process is loaded down with trivial details, be adapted to industrialization scale operation.
Accompanying drawing explanation
Fig. 1 is CDP-c separation purifying technique schematic flow sheet of the present invention.
Embodiment
Below in conjunction with crossing embodiment, the specific embodiment of the present invention is specifically described.
In following examples 1 ~ embodiment 3, Issatchenkia orientalis cytidine diphosphate bio-transformation liquid and preparation method thereof is as follows:
Take glucose as energy donor, the energy requirement of thalline is provided on the one hand, on the other hand for cytidine diphosphate synthetase series provides ATP; By add one in potassium ion, magnesium ion and mn ion or combination change metabolic fluxes to, improve the regeneration efficiency of ATP, thus match with cytidine diphosphate enzyme system speed, realize efficiently preparing cytidine diphosphate.(concrete preparation method is see following patent document: Zhang Liang, stone Kweiyang, outstanding Cuiping, Deng. a strain Issatchenkia orientalis and resting cell thereof produce the method for cytidine diphosphate: China, CN102286386A [P] .2011-12-21.), wherein Issatchenkia orientalis (I.orientalis) Z1 is purchased from Chinese classical collection culture collection center, and bacterium numbering is: CCTCC NO:2011272.
As follows with analysing and detecting method involved by embodiment: protein adopts Xylene Brilliant Cyanine G determination of color, calcium ions and magnesium ions adopts chromium black T indicator to measure, and CDP-c and cytidylic acid adopt HPLC method to measure.
Embodiment 1
300mL Issatchenkia orientalis cytidine diphosphate bioconversion broth, containing CDP-c1350mg, cytidylic acid 650mg, phosphoric acid salt 3750mg, phosphorylcholine calcium 500mg, vitriol 900mg.
(1) above-mentioned cytidine diphosphate bioconversion broth is passed through 717HCO
3 -type anion-exchange resin column, column dimension 20 × 300mm, column volume 60mL, upper column flow rate 0.5mL/min, upper colume content is the 13.5%(mass ratio that post exchanges total amount).
(2) upper prop is complete, with 300mL deionized water drip washing exchange column to rinse out non-adsorbent, until effluent liquid is water entirely.
(3) wash post with 0.03M sodium chloride solution, until column outlet place effluent liquid is no longer containing CDP-c, collects elutriant 200mL, obtain high-purity C DP-c solution.
(4) wash post with 0.3M sodium chloride solution, until column outlet place effluent liquid is no longer containing cytidylic acid, collect elutriant 150mL.
(5) the 200mL CDP-c elutriant that step (3) is collected is concentrated into 30mL, desalted by the dialysis tubing of molecular weight cut-off 100, freeze-drying, cytidine diphosphate solid 1153mg can be obtained, yield is 85.4%, cytidine diphosphate purity 99% after testing, does not detect phosphoric acid salt and magnesium ion, and protein detection is negative; 150mL cytidylic acid elutriant step (3) collected does same process, obtains cytidylic acid 581mg, and yield is 89.4%.
Embodiment 2
1000mL Issatchenkia orientalis cytidine diphosphate bioconversion broth, containing cytidine diphosphate 4500mg, cytidylic acid 2039mg, phosphoric acid salt 11380mg, phosphorylcholine calcium 1520mg, vitriol 2750mg.
(1) above-mentioned cytidine diphosphate bioconversion broth is passed through 717HCO
3 -type anion-exchange resin column, column dimension 30 × 500mm, column volume 200mL, upper column flow rate 1.0mL/min.Upper colume content is the 12.8%(mass ratio that post exchanges total amount).
(2) upper prop is complete, with 1000mL deionized water drip washing exchange column to rinse out non-adsorbent, until effluent liquid is water entirely.
(3) wash post with 0.04M sodium chloride solution, until column outlet place effluent liquid is no longer containing CDP-c, collects elutriant 800mL, obtain high-purity C DP-c solution.
(4) wash post with 0.2M sodium chloride solution, until column outlet place effluent liquid is no longer containing cytidylic acid, collect elutriant 650mL.
(5) the 800mL CDP-c elutriant that step (3) is collected is concentrated into 100mL, desalted by the dialysis tubing of molecular weight cut-off 100, freeze-drying, cytidine diphosphate solid 4025mg can be obtained, yield is 89.4%, cytidine diphosphate purity 99% after testing, does not detect phosphoric acid salt and magnesium ion, and protein detection is negative; 650mL cytidylic acid elutriant step (3) collected does same process, obtains cytidylic acid 1837mg, and yield is 90.1%.
Embodiment 3
300mL cytidine diphosphate bioconversion broth, containing cytidine diphosphate 1350mg, cytidylic acid 650mg, phosphoric acid salt 3750mg, phosphorylcholine calcium 500mg, vitriol 900mg.
(1) above-mentioned cytidine diphosphate bioconversion broth is passed through D301HCO
3 -type anion-exchange resin column, column dimension 20 × 300mm, column volume 60mL, upper column flow rate 0.5mL/min.Upper colume content is the 11.3%(mass ratio that post exchanges total amount).
(2) upper prop is complete, with 300mL deionized water drip washing exchange column to rinse out non-adsorbent, until effluent liquid is water entirely.
(3) wash post with 0.03M sodium chloride solution, until column outlet place effluent liquid is no longer containing CDP-c, collects elutriant 200mL, obtain high-purity C DP-c solution.
(4) wash post with 0.3M sodium chloride solution, until column outlet place effluent liquid is no longer containing cytidylic acid, collect elutriant 150mL.
(5) the 200mL CDP-c elutriant that step (3) is collected is concentrated into 30mL, desalted by the dialysis tubing of molecular weight cut-off 100, freeze-drying, cytidine diphosphate solid 1051mg can be obtained, yield is 77.9%, cytidine diphosphate purity 98% after testing, does not detect phosphoric acid salt and magnesium ion, and protein detection is negative; 150mL cytidylic acid elutriant step (3) collected does same process, obtains cytidylic acid 573mg, and yield is 88.2%.
Above-mentioned experiment results proved: utilize that the inventive method is disposable can obtain the CDP-c that can obtain high purity (> 99%), high yield (> 88%), and the cytidylic acid of recyclable non-complete reaction.
Above-described is only the preferred embodiment of the present invention, the invention is not restricted to above embodiment.Be appreciated that the oher improvements and changes that those skilled in the art directly derive without departing from the spirit and concept in the present invention or associate, all should think and be included within protection scope of the present invention.
Claims (10)
1. the method for purifying citicoline in her Sa yeast bio conversion fluid eastwardly, is characterized in that comprising the steps:
(1) be that the Issatchenkia orientalis bioconversion broth of 0.1 ~ 10g/L is by highly basic or weak base anion-exchange resin post by CDP-c content;
(2) upper prop is complete, with anion-exchange resin column described in deionized water drip washing, rinses out non-adsorbent;
(3) wash post with the salts solution of 0.01 ~ 0.04M, until column outlet place effluent liquid is no longer containing CDP-c, collects elutriant, obtain high-purity C DP-c solution;
(4) wash post with the salts solution of 0.2 ~ 1.00M, until column outlet place effluent liquid is no longer containing cytidylic acid, collect elutriant, evaporation concentration, dialysis tubing desalts, freeze-drying, reclaims and obtains cytidylic acid.
2. the method for purifying citicoline as claimed in claim 1, it is characterized in that: step (1) described highly basic or weak base anion-exchange resin post are selected from macropore polystyrene strongly basic anion exchange resin post or macropore polystyrene weak base anion-exchange resin post, upper colume content is 5 ~ 40% (mass ratioes) of the total exchange capacity of post, and post aspect ratio is 10:1 ~ 50:1.
3. the method for purifying citicoline as claimed in claim 2, it is characterized in that: described macropore polystyrene strongly basic anion exchange resin post or macropore polystyrene weak base anion-exchange resin post comprise 717, D301, D318, granularity 0.3 ~ 1.2mm >=95%.
4. the method for purifying citicoline as claimed in claim 2, it is characterized in that: on described macropore polystyrene strongly basic anion exchange resin post or macropore polystyrene weak base anion-exchange resin post, colume content is 10 ~ 15% (mass ratioes) of the total exchange capacity of post, and post aspect ratio is 10:1 ~ 20:1.
5. the method for purifying citicoline as claimed in claim 1, is characterized in that: on step (1) described highly basic or weak base anion-exchange resin post, column flow rate is 0.2 ~ 2mL/min.
6. the method for purifying citicoline as claimed in claim 1, is characterized in that: step (3) described concentration of salt solution is 0.02 ~ 0.04M.
7. the method for purifying citicoline as claimed in claim 1, is characterized in that: step (4) described concentration of salt solution is 0.20 ~ 0.50M.
8. the method for purifying citicoline as claimed in claim 1, is characterized in that: step (3) and (4) described wash-out salts solution are selected from NaCl, NH
4hCO
3, NaHCO
3, NH
4cl, Na
2cO
3, KCl, KHCO
3, K
2cO
3.
9. the method for purifying citicoline as claimed in claim 1, characterized by further comprising after step (3) gained CDP-c solution is concentrated, desalted by the dialysis tubing of molecular weight cut-off 100, freeze-drying, can obtain the cytidine diphosphate solid that purity is 98%.
10. the method for purifying citicoline as claimed in claim 1, characterized by further comprising after CDP-c separation and purification terminates, and described anion-exchange resin column salt, bronsted lowry acids and bases bronsted lowry is rinsed respectively the step recycled.
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| CN109836468A (en) * | 2017-11-24 | 2019-06-04 | 苏州华赛生物工程技术有限公司 | A method of the purifying citicoline sodium from microbial fermentation solution |
| CN111349131A (en) * | 2018-12-21 | 2020-06-30 | 上海渔霁生物技术有限公司 | Method for refining cytidylic acid derivative on large scale by HPLC |
| CN113831378B (en) * | 2021-09-24 | 2023-06-30 | 上海蔚之星生物科技有限公司 | Intelligent control-based citicoline chromatographic separation method |
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Effective date of registration: 20160620 Address after: 214000, No. 88, Huicheng Road, Changan street, Huishan District, Jiangsu, Wuxi Patentee after: Wuxi Tai Da Da Da machine equipment Co., Ltd. Address before: No. 1800 road 214122 Jiangsu Lihu Binhu District City of Wuxi Province Patentee before: Jiangnan University |