CN101381382B - Method for producing deoxynucleotide acid or salt thereof - Google Patents
Method for producing deoxynucleotide acid or salt thereof Download PDFInfo
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- CN101381382B CN101381382B CN2007101213984A CN200710121398A CN101381382B CN 101381382 B CN101381382 B CN 101381382B CN 2007101213984 A CN2007101213984 A CN 2007101213984A CN 200710121398 A CN200710121398 A CN 200710121398A CN 101381382 B CN101381382 B CN 101381382B
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Abstract
The invention provides a method for separating and purifying deoxynucleotidyl or salt of thereof from a DNA enzymed solution or a dried deoxynucleotide water solution, which comprises the following steps of adjusting the PH value of the DNA enzymed solution or the dried deoxynucleotide water solution to 5.5 to 7.0; adding an anion exchange resin column; then eluting with a solution with big ionic strength to obtain deoxynucleotide or deoxynucleotide salt with the content greater than 90 percent after desalination.
Description
Technical field
The present invention relates to a kind of method of producing deoxynucleotide or its salt, relate in particular to a kind of method of separating separation and purification deoxynucleotide the aqueous solution of the dry thing of liquid or deoxynucleotide or its salt from deoxyribonuclease.
Background technology
Deoxynucleotide or its salt are to be raw material with thymus nucleic acid (DNA) traditionally, through 5 '-enzymolysis of phosphodiesterase obtains, has in fields such as medicine, reagent, fine chemistry industries very and use widely.Thymus nucleic acid generates deoxyadenylic acid (dAMP), dGMP (dGMP), deoxycytidylic acid(dCMP) (dCMP) and thymidylic acid four kinds of deoxynucleotides such as (TMP) through the biological enzyme hydrolysis reaction.The deoxynucleoside acid crude that enzymolysis obtained need can be used through separation and purification.The technology of existing production deoxynucleotide or its salt is: the DNA enzymolysis solution concentrates behind anion-exchange column, activated carbon column, needle-use activated carbon decolouring under the neutrallty condition, the dry product that gets.Needle-use activated carbon absorption property under acidic conditions is good, and decolorizing effect is obvious, and under neutral above condition, the needle-use activated carbon absorption property is general, and itself with some impurity understand strippings, decolorizing effect is not obvious, and causes the pollution of product.Thereby the finished product outward appearance is reddish yellow.Referring to Li Liangzhu, You Yongjin, Lu Shenghua, biochemical pharmacy is learned, and 1991, Chinese Medicine science and technology press, p.228-230; Li Liangzhu, Li Minghua, up-to-date biochemical drug technology of preparing, 2001, Chinese Medicine science and technology press, p.265-267; With internal organs biochemical pharmacy information center of Ministry of Commerce station, the animal biochemical pharmacy is learned, first version, and 1981, people's health is published Du, p.196-198.
Reinforcing yin essence ion exchange resin in the art methods is that the Cl type (is seen the Wu Chinese, Zhang Zhifen, the separating technology of 5 ' one deoxynucleotides in the mackerel milt, aquatic product journal, 2000,24 (3): p.275-279; Repair pretty roc, the research of enzyme process preparation 3 '-Nucleotide, Biochemistry and Molecular Biology.2005, East China University of Science's master thesis; And Qiu Weiran, Ding Qingbao, Gao Shuhong, the method for separating nucleotide from enzymolysis liquid of ribonuclease, 2000, East China University of Science), the OH type (sees Li Liangzhu, Li Minghua, up-to-date biochemical drug technology of preparing, 2001, Chinese Medicine science and technology press.P.265-267), formic acid type (seeing Cai Wucheng, Li Biyu, Li Yumin, Biochemistry Experiment technical tutorial, 1983, press of Fudan University).The ion exchange resin adsorptive capacity of these three kinds of types is big, but has adsorbed more non-deoxynucleoside acids material simultaneously.And disclosed art methods mostly is separating nucleotide greatly, but not deoxynucleotide.
Last sample pH in the art methods mostly is more than 8.0, and under this pH condition, adsorptive capacity is big, (sees the Wu Chinese but adsorbed more non-deoxynucleoside acids material simultaneously, Zhang Zhifen, the separating technology of 5 ' one deoxynucleotide in the mackerel milt, aquatic product journal, 2000,24 (3), p.275-279).In disclosed document, it is 5.0-8.0 that its pH of report is arranged, but it is used for separating nucleotide, but not deoxynucleotide (is seen Qiu Weiran, Yan Pengbo, Ding Qingbao is with the method for anionite-exchange resin separating nucleotide from enzymolysis liquid of ribonuclease, 2002, Shanghai Qiuzhiyou Bio-technology Co., Ltd).
Press for a kind of separation and purification deoxynucleotide of good decolorizing effect or the method for its salt in the prior art.
Summary of the invention
In order to solve the above-mentioned problems in the prior art, the inventor is through long-term deep research, realized purpose of the present invention by a kind of method of separating separation and purification deoxynucleotide the aqueous solution of liquid or the dry thing of deoxynucleotide or its salt from deoxyribonuclease is provided.
The invention provides a kind of method of separating separation and purification deoxynucleotide the aqueous solution of the dry thing of liquid or deoxynucleotide or its salt, comprise the steps: from deoxyribonuclease
A) regulate the pH value of aqueous solution of the dry thing of DNA enzymolysis solution or deoxynucleotide to 5.5-7.0; Last anion-exchange resin column;
B) with the big eluant solution of ionic strength; Then
C) after the desalination content greater than 90% deoxynucleotide or deoxynucleoside hydrochlorate.
In the methods of the invention, described anion-exchange resin column is preferably the strongly basic anion exchange resin post, more preferably acetic acid type strongly basic anion exchange resin post.
In a preferred embodiment of the invention, the inventive method also is included in steps A) step B afterwards) use the step of the acid pre-pickling anion-exchange resin column of 0.005-0.05M before.The acid that described prewashing is used is preferably selected from formic acid, acetic acid, hydrochloric acid, sulfuric acid and nitric acid, more preferably acetic acid.
In the methods of the invention, the solution that described ionic strength is big is preferably selected from the NaCl aqueous solution, the HCl aqueous solution, sodium acetate aqueous solution and composition thereof.
In another preferred embodiment of the inventive method, the elutriant or the additive method gained solution of anion-exchange column are regulated the pH value to 2.5-4.0, the activated carbon column of last certain volume, make it saturated, when supersaturation, an activated carbon column adsorpting pigment, effluent liquid are non-pigmented deoxynucleotide or its salt.This effluent liquid can through activated carbon column desalination, drying or additive method further refining product.
In another preferred embodiment of the inventive method, described reinforcing yin essence ion exchange resin is acetic acid type, and its treatment process is: use 0.5-2M HCl, and 0.2-2M NaOH, 0.5-2M acetic acid is handled successively.Be specially: 0.5-2M HCl handles more than 1 column volume, water wash to the pH value greater than 4; 0.5-2MNaOH handle more than 1 column volume, water wash to the pH value less than 11; 0.5-2M acetic acid is handled more than 1 column volume, water wash to the pH value greater than 4.With this root pillar on the solution of pH5.5-7.0, prewashing then, wash-out.
In the methods of the invention, the described sample pH5.5-7.0 that goes up can effectively remove most of non-deoxynucleoside acids material in the enzymolysis solution or the dry thing aqueous solution, and non-deoxynucleoside acids material can be further removed in the prewashing of described 0.005-0.05M acetic acid.
Be white in color or off-white color by deoxynucleotide or deoxynucleoside hydrochlorate outward appearance that the inventive method obtained, but injection.
Embodiment
Describe and explain the present invention in detail below in conjunction with specific embodiment.But the embodiment of the invention only limits to explain and explanation the present invention, and limits the scope of the invention never in any form.The institute that those skilled in the art will recognize that the specific embodiment of the invention changes, improvement, variant and equivalent all within the scope of the present invention.
In following examples, said anionite-exchange resin is the 711 type strongly basic anion exchange resins that Shanghai Huazhen Science and Technology Co., Ltd. produces, and said gac is the 769 type gacs that the precious gac of Shanghai evil spirit company limited produces.
Embodiment 1
The 711 resin anion(R.A) posts of 20L are washed 1 column volume with 0.5M HCl, water wash to the pH value be 5.0; 0.5M NaOH handles 1 column volume, water wash to the pH value be 11.0; 0.5M acetic acid is handled 1 column volume, water wash to the pH value be 5.0.
With the 250L weight percent is that 1% DNA enzymolysis solution filters, and removes solid particulate and albumen, regulates pH value of filtrate to 5.5 with 6M HCl; Last 711 resin anion(R.A) posts after last sample is intact, with 0.5M NaCl aqueous solution wash-out, are collected elutriant 200L.With the elutriant desalination, concentrate, after the drying the Sodium Deoxyribomuleotide of content 90.5%.
Embodiment 2
The 711 resin anion(R.A) posts of 20L are washed 2 column volumes with 1M HCl, water wash to the pH value be 7.0; 1M NaOH handles 2 column volumes, water wash to the pH value be 9.0; 1M acetic acid is handled 2 column volumes, water wash to the pH value be 7.0.
With the 250L weight percent is that 1% DNA enzymolysis solution filters, and removes solid particulate and albumen, regulates pH value of filtrate to 6.0 with 6M HCl; Last 711 resin anion(R.A) posts after last sample is intact, with 0.5M sodium acetate aqueous solution wash-out, are collected elutriant 250L.With the elutriant desalination, concentrate, after the drying the Sodium Deoxyribomuleotide of content 93%.
Embodiment 3
The 711 resin anion(R.A) posts of 20L are washed 3 column volumes with 2M HCl, water wash to the pH value be 6.0; 2M NaOH handles 3 column volumes, water wash to the pH value be 10.0; 2M acetic acid is handled 3 column volumes, water wash to the pH value be 6.0.
With the 250L weight percent is that 1% DNA enzymolysis solution filters, and removes solid particulate and albumen, regulates pH value of filtrate to 7.0 with 6M HCl; Last 711 resin anion(R.A) posts after last sample is intact, with 0.5M HCl aqueous solution wash-out, are collected elutriant 90L.With the elutriant desalination, concentrate, after the drying the Sodium Deoxyribomuleotide of content 95%.
Embodiment 4
The 711 resin anion(R.A) posts of 20L are washed 1 column volume with 0.5M HCl, water wash to the pH value be 5.0; 0.5M NaOH handles 1 column volume, water wash to the pH value be 11.0; 0.5M acetic acid is handled 1 column volume, water wash to the pH value be 5.0.
With the 250L weight percent is that 1% DNA enzymolysis solution filters, and removes solid particulate and albumen, regulates pH value of filtrate to 5.5 with 6M HCl; Last 711 resin anion(R.A) posts are after last sample is intact, with acetic acid prewashing 4 column volumes of 0.005M; Use 0.5M HCl aqueous solution wash-out then, collect elutriant 90L.With the elutriant desalination, concentrate, after the drying the Sodium Deoxyribomuleotide of content 92%.
Embodiment 5
The 711 resin anion(R.A) posts of 20L are washed 2 column volumes with 1M HCl, water wash to the pH value be 7.0; 1M NaOH handles 2 column volumes, water wash to the pH value be 9.0; 1M acetic acid is handled 2 column volumes, water wash to the pH value be 7.0.
With the 250L weight percent is that 1% DNA enzymolysis solution filters, and removes solid particulate and albumen, regulates pH value of filtrate to 6.0 with 6M HCl; Last 711 resin anion(R.A) posts are after last sample is intact, with acetic acid prewashing 4 column volumes of 0.01M; Use 0.5M sodium acetate aqueous solution wash-out then, collect elutriant 250L.With the elutriant desalination, concentrate, after the drying the Sodium Deoxyribomuleotide of content 95%.
Embodiment 6
The 711 resin anion(R.A) posts of 20L are washed 3 column volumes with 2M HCl, water wash to the pH value be 6.0; 2M NaOH handles 3 column volumes, water wash to the pH value be 10.0; 2M acetic acid is handled 3 column volumes, water wash to the pH value be 6.0.
With the 250L weight percent is that 1% DNA enzymolysis solution filters, and removes solid particulate and albumen, regulates pH value of filtrate to 7.0 with 6M HCl; Last 711 resin anion(R.A) posts are after last sample is intact, with acetic acid prewashing 4 column volumes of 0.05M; Use 0.5M HCl aqueous solution wash-out then, collect elutriant 90L.With the elutriant desalination, concentrate, after the drying the Sodium Deoxyribomuleotide of content 98%.
Embodiment 7
With gac 1M sodium-hydroxide treatment. boiled 15 minutes, filter is done, and the acid treatment of 1M salt is used in water flushing 5 times again, boils 15 minutes, and filter is done, water flushing 5 times, dress post.Adorn the activated carbon column of 2L and 20L respectively, be numbered activated carbon column 1 and activated carbon column 2 accordingly.
The resin anion(R.A) elutriant that obtains among the embodiment 1 is regulated pH value to 2.5, and through activated carbon column 1, effluent liquid is non-pigmented Sodium Deoxyribomuleotide.
With activated carbon column on the effluent liquid 2, after last sample was intact, water washed two column volumes, desalination again.Use ethanol-ammoniacal liquor wash-out V (ammoniacal liquor): V (95% ethanol): V (distilled water)=3: 95: 47 again, collect elutriant 150L.Be concentrated into syrupy shape, the 20L water dissolution, 10%NaOH transfers pH to 7.0, vacuum, after the drying the Sodium Deoxyribomuleotide of content 90.5%, outward appearance is off-white color.
Embodiment 8
With gac 1M sodium-hydroxide treatment. boiled 15 minutes, filter is done, and the acid treatment of 1M salt is used in water flushing 5 times again, boils 15 minutes, and filter is done, water flushing 5 times, dress post.Adorn the activated carbon column of 2L and 20L respectively, be numbered activated carbon column 1 and activated carbon column 2 accordingly.
The resin anion(R.A) elutriant that obtains among the embodiment 4 is regulated pH value to 3.0, and through activated carbon column 1, effluent liquid is non-pigmented Sodium Deoxyribomuleotide.
With activated carbon column on the effluent liquid 2, after last sample was intact, water washed two column volumes, desalination again.Use ethanol one ammoniacal liquor wash-out V (ammoniacal liquor): V (95% ethanol): V (distilled water)=3: 95: 47 again, collect elutriant 150L.Be concentrated into syrupy shape, the 20L water dissolution, 10%NaOH transfers pH to 7.0, gets the Sodium Deoxyribomuleotide of content 92% after the vacuum-drying, and outward appearance is off-white color.
Embodiment 9
With gac 1M sodium-hydroxide treatment. boiled 15 minutes, filter is done, and the acid treatment of 1M salt is used in water flushing 5 times again, boils 15 minutes, and filter is done, water flushing 5 times, dress post.Adorn the activated carbon column of 2L and 20L respectively, be numbered activated carbon column 1 and activated carbon column 2 accordingly.
The resin anion(R.A) elutriant that obtains among the embodiment 4 is regulated pH value to 4.0, and through activated carbon column 1, effluent liquid is non-pigmented Sodium Deoxyribomuleotide.
With activated carbon column on the effluent liquid 2, after last sample was intact, water washed two column volumes, desalination again.Use ethanol-ammoniacal liquor wash-out V (ammoniacal liquor): V (95% ethanol): V (distilled water)=3: 95: 47 again, collect elutriant 150L.Be concentrated into syrupy shape, the 20L water dissolution, 10%NaOH transfers pH to 7.0, gets the Sodium Deoxyribomuleotide of content 98% after the vacuum-drying, and outward appearance is white in color.
Claims (10)
1. a method of separating separation and purification deoxynucleotide the aqueous solution of the dry thing of liquid or deoxynucleotide or its salt from deoxyribonuclease comprises the steps:
A) pH value of aqueous solution of regulating the dry thing of DNA enzymolysis solution or deoxynucleotide is to 5.5-7.0, last anion-exchange resin column;
B) with the big eluant solution of ionic strength, the solution that described ionic strength is big is selected from the NaCl aqueous solution, the HCl aqueous solution, sodium acetate aqueous solution and composition thereof; Then
C) after the desalination content greater than 90% deoxynucleotide or deoxynucleoside hydrochlorate.
2. method according to claim 1 is characterized in that this method also is included in steps A) step B afterwards) use the step of the acid pre-pickling anion-exchange resin column of 0.005-0.05M before.
3. method according to claim 2 is characterized in that the acid that described prewashing is used is selected from formic acid, acetic acid, hydrochloric acid, sulfuric acid and nitric acid.
4. according to claim 2 or 3 described methods, it is characterized in that the acid that described prewashing is used is acetic acid.
5. method according to claim 1 and 2 is characterized in that, described anion-exchange resin column is the strongly basic anion exchange resin post.
6. method according to claim 5 is characterized in that, described anion-exchange resin column is 711 type strongly basic anion exchange resins.
7. method according to claim 1 and 2 is characterized in that, described anion-exchange resin column is handled with 0.5-2M HCl, 0.2-2M NaOH, 0.5-2M acetic acid successively.
8. method according to claim 1 and 2 is characterized in that, described anion-exchange resin column is handled more than 1 column volume with 0.5-2M HCl successively, water wash to the pH value greater than 4; Handle more than 1 column volume with 0.5-2M NaOH, water wash to the pH value less than 11; Handle more than 1 column volume with 0.5-2M acetic acid, water wash to the pH value greater than 4.
9. method according to claim 1 and 2 is characterized in that, this method also comprises the steps: the elutriant of anion-exchange resin column is regulated the pH value to 2.5-4.0, and last activated carbon column makes its supersaturation, obtains non-pigmented deoxynucleotide or its salt.
10. method according to claim 1 and 2 is characterized in that this method also comprises the purification step through the activated carbon column desalination.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2007101213984A CN101381382B (en) | 2007-09-05 | 2007-09-05 | Method for producing deoxynucleotide acid or salt thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007101213984A CN101381382B (en) | 2007-09-05 | 2007-09-05 | Method for producing deoxynucleotide acid or salt thereof |
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| CN101381382A CN101381382A (en) | 2009-03-11 |
| CN101381382B true CN101381382B (en) | 2011-06-22 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102382150B (en) * | 2010-09-06 | 2015-05-20 | 南通秋之友生物科技有限公司 | Preparation method of high-purity mixed sodium deoxyribonucleotide |
| CN114874275B (en) * | 2021-09-24 | 2023-08-01 | 上海蔚之星生物科技有限公司 | Deoxynucleotide chromatographic separation method and preparation of deoxynucleotide sodium bulk drug |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1408719A (en) * | 2002-09-05 | 2003-04-09 | 上海秋之友生物科技有限公司 | Process for separating nucleotide from ribonucleic acid enzymolysis liquid using cationic exchanging resin |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1408719A (en) * | 2002-09-05 | 2003-04-09 | 上海秋之友生物科技有限公司 | Process for separating nucleotide from ribonucleic acid enzymolysis liquid using cationic exchanging resin |
Non-Patent Citations (2)
| Title |
|---|
| 吴汉民等.鲐鱼鱼精中5′- 脱氧核苷酸的分离工艺.《水产学报》.2000,第24卷(第3期),275-279. * |
| 王光柱.5"-混合脱氧单核苷酸分离纯化的工艺研究.《中国优秀博硕士学位论文全文数据库(硕士)工程科技I辑》.中国学术期刊(光盘版)电子杂志社,2006,(第9期),B016-160. * |
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