CN1177859C - Process for separating nucleotide from ribonucleic acid enzymolysis liquid using cationic exchanging resin - Google Patents
Process for separating nucleotide from ribonucleic acid enzymolysis liquid using cationic exchanging resin Download PDFInfo
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- CN1177859C CN1177859C CNB021368392A CN02136839A CN1177859C CN 1177859 C CN1177859 C CN 1177859C CN B021368392 A CNB021368392 A CN B021368392A CN 02136839 A CN02136839 A CN 02136839A CN 1177859 C CN1177859 C CN 1177859C
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Abstract
The present invention relates to a method for separating nucleotide from RNA enzymolysis solutions by using anion exchange resins. Mononucleotide with high purity is obtained by the primary separation and purification through the ordered arrangement and combination of the anion exchange resins and carbon columns. The method can obtain four nucleotides of 5'-AMP, 5'-GMP, 5'-CMP and 5'-UMP with high yield and high concentration, and the total yield of the separation can reach 90%. Most of impurities in the enzymolysis solutions are stopped into the anion exchange resins, or eluted when the separation is carried out. The method has the advantages of less resin dosage, low cost, high yield, good separation effect, high product purity, large-scale commercial production, etc.
Description
Technical field
The present invention relates to a kind of separation method, relate in particular to the method for a kind of employing ion exchange resin separating nucleotide from Yeast Nucleic Acid (RNA) enzymolysis solution.
Background technology
Nucleotide, as 5 '-adenylic acid (AMP) (be called for short 5 '-AMP), 5 '-guanylic acid (be called for short 5 '-GMP), 5 '-cytidylic acid (be called for short 5 '-CMP) and 5 '-uridylic acid (be called for short 5 '-UMP) and derivative be important biochemical industry raw material, can be used as foodstuff additive, protective foods and medicine intermediate.Generally, cultivate high nuclear yeast by continuously fermenting, therefrom extraction separation can obtain RNA then, also can separate to obtain RNA from cereuisiae fermentum or other yeast.With RNA nuclease P
1(or phosphodiesterase) enzymolysis separates enzymolysis solution then, can obtain said 5 '-adenylic acid (AMP), 5 '-guanylic acid, 5 '-cytidylic acid and 5 '-uridylic acid.At present, more existing bibliographical informations separation method, as:
Document: the production of ucleotides material and application, Institute of Microorganism, Academia Sinica compiles, Science Press: 1971, reported a kind of separation method, this method is by cation-anion resin bonded method, carry out the separation of Nucleotide, though this method can flash liberation go out 5 ' a large amount of-adenylic acid (AMP)s, but 5 '-guanylic acid, the separating effect of 5 '-cytidylic acid and 5 '-uridylic acid is unsatisfactory, the loss of 5 '-uridylic acid nearly 50%, the loss of 5 '-guanylic acid and 5 '-cytidylic acid nearly 40%, the production cycle is long, feed liquid is apt to deteriorate, and total separation yield rate is about 65%; The document has been reported another kind of separation method simultaneously, and this method directly adopts anionite-exchange resin to separate, and this method separation cycle is long, the easy mildew of upper prop liquid when temperature is high, and effluent volume is big, and water loss is many, does not have industrial application value.
Inventors etc. once reported with positive anionite-exchange resin and made up isolating method, Chinese patent application number: 00119589.1, solved isolating key issue, and product yield and purity are improved significantly.But its defective is that resin demand is big, and energy consumption and water consumption are bigger.
Along with the excavation of Nucleotide and derivative curative effect of medication thereof with as the brilliant application of health care, its demand increases day by day, and original production method can not satisfy people's needs, and relevant branch of industry is desirable to provide more efficiently production method.
Summary of the invention
The technical issues that need to address of the present invention are to disclose a kind of method with anionite-exchange resin separating nucleotide from enzymolysis liquid of ribonuclease, the method that comprises 5 '-adenylic acid (AMP), 5 '-guanylic acid, 5 '-cytidylic acid and 5 '-uridylic acid, to overcome the above-mentioned defective that prior art exists, satisfy people's needs.
Design of the present invention is such:
The present invention adopts two anion-exchange resin columns, with the enzymolysis solution upper prop of connecting, stepwise elution.Because 5 '-AMP, 5 '-GMP, 5 '-CMP and 5 '-UMP has phosphate, under the neutral condition, under the condition as pH=6.5～7.5, phosphate is dissociated, and has negative charge, so and anionite-exchange resin carry out ion-exchange, be adsorbed.
The ultimate principle of two anion-exchange resin column wash-outs is such: because 5 '-dissociation constant of AMP, 5 '-GMP and 5 '-CMP amino is respectively 3.70,2.30 and 4.24,5 '-the phosphate dissociation constant of AMP, 5 '-GMP, 5 '-CMP, 5 '-CMP is respectively 0.89,0.70,0.80,1.02.When washing with acidic solution, 5 '-CMP electronegative a little less than, therefore earlier eluted; Using instead can be with 5 than strongly acidic solution '-AMP elutes.When taking off earlier than strongly-acid and salts solution, owing to purine and styrene resin have the affinity reason, 5 '-UMP prior to 5 '-GMP eluted.
Therefore adopt the method for stepwise elution, can be successively with 5 '-CMP, 5 '-AMP, 5 '-UMP, 5 '-GMP elutes, four kinds of elutriants are adsorbed in respectively on four charcoal posts, and then respectively 4 kinds of Nucleotide are eluted with basic solution, so can isolate the higher Nucleotide of purity by high once yield.
In the past, used the anionite-exchange resin separation method, because in order to obtain separating effect preferably, Nucleotide upper prop total amount is often very low, is generally 5% of total exchange capacity.And the present invention is in order to save cost, improving post imitates, adopt two placed in-line methods of cloudy post, during last sample, first post exchange capacity state that reaches capacity, second post mainly act on be increase by 5 '-separating effect of CMP, 5 '-AMP and 5 '-UMP, and adopt stepwise elution with the spissated method of charcoal, thereby remedied the isolating defective of former anionite-exchange resin.
Principal character of the present invention is that by the ordered arrangement combination of anionite-exchange resin and charcoal post, the flash liberation purifying obtains the high purity mononucleotide, and it is few to have resin demand, and cost is low, isolating mononucleotide concentration height, disengaging time is short, and product is difficult for advantages such as pollution.
Method of the present invention in turn includes the following steps:
1. will contain four kinds of 5-Nucleotide, the enzymolysis solution of other Nucleotide (as 5 '-t-inosinic acid) and nucleosides impurity such as (as adenosine, guanosine, cytidine, uridine, inosines) flows through two strong anion-exchange resin posts according to the order of sequence, 4 kinds of 5 '-Nucleotide is attracted in two strong anion-exchange resin posts on a small quantity, 5 '-AMP, 5 '-GMP, 5 '-UMP, 5 '-CMP is attracted in first strong anion-exchange resin post.
2. with two strong anion-exchange resin posts of acidic solution washing, elutriant flows into the first charcoal post, make wherein 5 '-CMP is adsorbed in the first charcoal post, uses the basic solution wash-out first charcoal post then, collect elutriant, can obtain purity higher 5 '-CMP solution;
3. use than two strong anion-exchange resin posts of strongly acidic solution washing, elutriant flows into the second charcoal post, make wherein 5 '-AMP is adsorbed in the second charcoal post, uses the alkaline solution wash-out then, can obtain purity higher 5 '-AMP solution;
4. use than two strong anion-exchange resin posts of strongly-acid and salts solution washing, elutriant flows into the 3rd charcoal post, make wherein 5 '-UMP is adsorbed in the 3rd charcoal post, uses the alkaline solution wash-out then, can obtain purity higher 5 '-UMP solution.
5. with stronger first anionite-exchange resin of solution washing of ionic strength, elutriant flows into the 4th charcoal post, make wherein 5 '-GMP is adsorbed in the 4th charcoal, uses the basic solution wash-out then, can obtain purity higher 5 '-GMP solution.
The method of the isolated solution that contains four kinds of Nucleotide by routine concentrated and make with extra care, can obtain purity and be the high purity product more than 98%.
By above-mentioned sepn process, can high yield, high density ground obtains 5 '-AMP, 5 '-GMP, 5 '-CMP and 5 '-four kinds of Nucleotide of UMP, total separation yield rate can reach 90%, and the most of impurity in the enzymolysis solution is trapped within the anionite-exchange resin, or washes away when separated.
The said method of the present invention has that resin demand is few, cost is low, yield is high, good separating effect, products obtained therefrom purity is high and advantage such as suitable large-scale industrial production.
Description of drawings.
Fig. 1 is a schema.
Embodiment
As seen from Figure 1, the said method of the present invention in turn includes the following steps:
1. contain four kinds of 5 '-Nucleotide nuclear, other Nucleotide (as 5 '-t-inosinic acid) and nucleosides impurity pH values such as (as adenosine, guanosine, cytidine, uridine, inosines) are that 5～8 enzymolysis solution flows through the first strong anion-exchange resin post 1 and the second strong anion-exchange resin post 2 according to the order of sequence on a small quantity.
The upper prop total amount is the 16-30% of the total exchange capacity of resin, and (mol ratio), the best are 20～25%, i.e. 10 liters of commutative approximately 1.2～2 kilograms of Nucleotide of anionite-exchange resin, and the best is the 1.6-1.8 kilogram, the volume ratio of post 1 and post 2 is 1～(2: 1);
The upper prop linear velocity is 0.11～0.45 meter/hour, and the best is 0.3～0.36 meter/hour;
Nucleotide content is about 0.5-2% in the upper prop liquid, and the best is 0.8-1.2wt%.
After upper prop finishes,, make to remain in two impurity such as nucleosides in the strong anion-exchange resin post and wash off preferably with the abundant drip washing of deionized water.
2. with acidic solution such as 0.005mol/L～0.02mol/L formic acid washing strong anion-exchange resin post, as 5 '-when CMP solution flows out, connect the first charcoal post 3 immediately, make 5 '-CMP all is adsorbed in the first charcoal post, when 5 '-after CMP all is washed till charcoal post 3 from post 1, post 2, disconnect post 2, post 3, and with basic solution washing column 3, just can get 5 '-CMP solution.
3. use than strongly acidic solution, 0.05mol/L two strong anion-exchange resin posts 1 of～0.2mol/L formic acid series connection washing and 2, when effluent liquid be 5 '-during AMP, connect the second charcoal post 4, when post 2 outlet do not have 5 '-during AMP, stop wash-out, and with basic solution washing column 4, just can get 5 '-AMP solution.
4. use more highly acid salts solution such as 0.05mol/L～0.2mol/L formic acid and 0.05mol/L～-0.2mol/L sodium formiate wash-out post 1, post 2, when post 2 outlet occur 5 '-during UMP, be connected with the 3rd charcoal post 5 immediately, and with basic solution washing column 5, can get 5 '-UMP solution.
5. use 1%～5% sodium chloride solution washing column 1 of stronger ionic strength solution such as pH=1.0～3.0, wash-out 5 '-GMP is sent washings into the 4th charcoal post 6, makes 5 '-GMP be adsorbed in the 4th charcoal post 6, and with basic solution washing column 6, just can get 5 '-GMP solution.
Said basic solution comprises inorganic alkali solutions such as sodium hydroxide, potassium hydroxide, yellow soda ash, salt of wormwood, and its concentration is 0.1-1mol/L.
Four kinds of mononucleotides of gained are slightly through concentrating, and crystallization gets final product to such an extent that purity is 5 '-Nucleotide more than 98%.
Said anionite-exchange resin for the Shanghai synthetic resin plant produce 717 or 201 * 8, a kind of in the strong-basicity styrene Zeo-karb such as U.S. Amberlite IRA-900; the order number is suitable with the 80-200 order; 100-120 order preferably; the used charcoal of charcoal post 3,4,5,6 is the K15 charcoal produced of 769 charcoals produced of Shanghai gac factory or brilliance timber mill, Beijing preferably, and the order number is the 15-50 order.
By above-mentioned sepn process, can high yield, high density ground obtains 5 '-AMP, 5 '-GMP, 5 '-CMP and 5 '-four kinds of Nucleotide of UMP, total separation yield rate can reach 90%, and the most of impurity in the enzymolysis solution is trapped within post 1 and the post 2, or washes away when separated.The method of the isolated solution that contains four kinds of Nucleotide by routine concentrated and make with extra care, can obtain purity and be the high purity product more than 98%.
Embodiment 1
With 3000 milliliters of weight percents is that 1% RNA solution adds 300 milliliters of nuclease P 1s (enzyme activity is 110u/ml), in 65 ℃ of following enzyme digestion reactions 2 hours, to filter, solid particulate and albumen are removed in ultrafiltration, regulate the pH to 8.0 of filtrate with 6N NaOH, wherein, contain 4.6 the gram 5 '-AMP, 6.4 the gram 5 '-GMP, 4.6 restrain 5 '-CMP, 4.8 restrain 5 '-UMP, flow through placed in-line two anion-exchange columns then according to the order of sequence, linear velocity is 0.36m/h;
The size of post 1,2 is respectively 1. 16 * 1000mm, 1. 16 * 700mm, and filling 201 * Final 8 alkali resin anion(R.A) (C1 type) of regenerating is 150 milliliters and 60 milliliters respectively, and post 3,4,5,6 is of a size of 1. 16 * 700mm, adorns 80 milliliters of 769 charcoals respectively.
Upper prop finishes, wash post with 500 ml deionized water, use 2000 milliliters of 0.02mol/L formic acid solution washing columns 1,2 then, when effluent liquid contains 5 '-CMP, be connected with the first charcoal post 3 immediately, make 5 '-CMP is adsorbed in charcoal post 3, when post 2 outlets 5 '-CMP no longer flows out, with 0.1mol/LNaOH wash-out charcoal post, collect elutriant 150ml, wherein contain 4.2 grams, 5 '-CMP.
Use 0.1mol/L formic acid-0.1mol/L sodium formiate washing column 1,2 again, make post 2 effusive 5 '-UMP is adsorbed in the 3rd post 5, the same method is collected elutriant, wherein contain 4.3 grams 5 '-UMP.
Use the 3%NaCl wash-out post 1 of pH=3.0 instead, when post 1 outlet has 5 '-GMP to flow out, be connected with the 4th charcoal post 6 immediately, later the same, collect elutriant, wherein contain 5.8 grams, 5 '-GMP.
5 '-Nucleotide total separation yield rate is 91%, wherein, 5 '-the AMP yield is 93.4%, and 5 '-GMP is 90.6%, and 5 '-CMP is 91.3%, 5 '-UMP is 90%.
With the each several part solution of above-mentioned collection respectively by concentrate, after the crystallization, drying, can get 3.87 the gram 5 '-AMP, 6.1 the gram 5 '-GMP (sodium salt contains 15% crystal water), 3.78 the gram 5 '-CMP, 5.1 restrain 5 '-UMP (sodium salt contains 25% crystal water), its content is more than 98%.
With 3000 milliliters enzymolysis solutions (wherein, contain 4.6 grams 5 '-AMP, 6.4 grams, 5 '-GMP, 4.6 grams, 5 '-CMP, 4.8 grams 5 '-UMP) flow through series connection according to the order of sequence by two radical ion exchange columns, linear velocity is 0.6m/h;
The size of post 1,2 is respectively 1. 16 * 1000mm, and 1. 16 * 700mm loads Amberlite IRA-900 (120 order) strong base anion resins of regenerating respectively, load 150 milliliters, 60 milliliters of post 2 fillings, post 3,4,5,6 is of a size of 1. 16 * 700mm, adorns K15 gac 90ml respectively.
Upper prop finishes, and washes post with 500 ml deionized water.All the other operations are the same.In post 3 can get 4.3 the gram 5 '-CMP, yield is 93.4%; Post 4 can get 4.18 the gram 5 '-AMP, yield is 91%; Post 5 can 4.41 restrain 5 '-UMP, yield is 92%; Post 6 can get 5.765 '-the GMP gram, yield is 90%.
With the each several part solution of above-mentioned collection respectively by concentrate, after the crystallization, drying, can obtain 3.76 the gram 5 '-AMP, 6.1 the gram 5 '-GMP (sodium salt contains 15% crystal water), 3.95 the gram 5 '-CMP, 5.3 restrain 5 '-UMP (sodium salt contains 25% crystal water), its content is more than 98%.
Claims (8)
1. with the method for anionite-exchange resin separating nucleotide from enzymolysis liquid of ribonuclease, it is characterized in that in turn including the following steps:
1. be that 5～8 enzymolysis solutions that contain four kinds of 5 '-Nucleotide flow through two strong anion-exchange resin posts according to the order of sequence, 4 kinds of 5 '-Nucleotide are attracted in two strong anion-exchange resin posts with the pH value;
2. with acidic solution washing strong anion-exchange resin post, elutriant flows into the first charcoal post, make wherein 5 '-CMP is adsorbed in the first charcoal post, uses the alkaline alcohol solution wash-out first charcoal post then, collects elutriant, collect 5 '-CMP solution;
3. use than strongly acidic solution washing strong anion-exchange resin post, elutriant flows into the second charcoal post, make wherein 5 '-AMP is adsorbed in the second charcoal post, uses the alkaline solution wash-out then, collect 5 '-AMP solution;
4. use than strongly-acid and salts solution washing strong anion-exchange resin post, elutriant flows into the 3rd charcoal post, make wherein 5 '-UMP is adsorbed in the 3rd charcoal post, uses the alkaline solution wash-out then, collect 5 '-UMP solution;
5. with stronger first anionite-exchange resin of solution washing of ionic strength, elutriant flows into the 4th charcoal post, make wherein 5 '-GMP is adsorbed in the 4th charcoal, uses the basic solution wash-out then, collect 5 '-GMP solution;
The strong anion-exchange resin post is the strong-basicity styrene cation exchange resin column;
Acidic solution is the formic acid of 0.005mol/L～0.02mol/L;
Than strongly acidic solution is the formic acid of 0.05mol/L～0.2mol/L;
Than strongly-acid and salts solution is the formic acid of 0.05mol/L～0.2mol/L and the sodium formiate of 0.05mol/L～0.2mol/L;
The solution that ionic strength is stronger is 1%～5% sodium chloride solution of pH=1.0～3.0;
Said basic solution comprises a kind of alkaline solution in sodium hydroxide, potassium hydroxide, yellow soda ash or the salt of wormwood, and its concentration is 0.1-1mol/L.
2. method according to claim 1 is characterized in that the upper prop total amount is the 16-30% of the total exchange capacity of resin, mol ratio.
3. method according to claim 2 is characterized in that the upper prop total amount is 20～25% of the total exchange capacity of resin, mol ratio.
4. method according to claim 1 is characterized in that the upper prop linear velocity is 0.11～0.45 meter/hour.
5. method according to claim 1 is characterized in that the upper prop linear velocity is 0.3～0.36 meter/hour.
6. method according to claim 1 is characterized in that nucleotide content is 0.5-2wt% in the upper prop liquid.
7. method according to claim 1 is characterized in that nucleotide content is 0.8-1.2wt% in the upper prop liquid.
8. according to each described method of claim 1～7, it is characterized in that upper prop finishes after, with the abundant drip washing of deionized water.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101418327B (en) * | 2008-11-21 | 2012-09-05 | 大连珍奥生物技术股份有限公司 | Novel production process of high-purity 5' nucleotide |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101381382B (en) * | 2007-09-05 | 2011-06-22 | 上海丽珠制药有限公司 | Method for producing deoxynucleotide acid or salt thereof |
| 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 |
| CN102924551B (en) * | 2012-11-30 | 2016-02-24 | 通辽梅花生物科技有限公司 | A kind of method extracting guanosine from fermented liquid |
| CN108752405B (en) * | 2018-05-16 | 2021-03-23 | 南通秋之友生物科技有限公司 | Method for separating nucleotide by ion exchange resin combined chromatography |
| 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 |
| CN113831379B (en) * | 2021-09-24 | 2023-06-30 | 上海蔚之星生物科技有限公司 | RNA enzymolysis liquid chromatographic separation method and system based on intelligent control |
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2002
- 2002-09-05 CN CNB021368392A patent/CN1177859C/en not_active Expired - Lifetime
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101418327B (en) * | 2008-11-21 | 2012-09-05 | 大连珍奥生物技术股份有限公司 | Novel production process of high-purity 5' nucleotide |
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