CN107417749B - Resin filler separation method of coenzyme I - Google Patents
Resin filler separation method of coenzyme I Download PDFInfo
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- CN107417749B CN107417749B CN201710709819.9A CN201710709819A CN107417749B CN 107417749 B CN107417749 B CN 107417749B CN 201710709819 A CN201710709819 A CN 201710709819A CN 107417749 B CN107417749 B CN 107417749B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/16—Purine radicals
- C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
- C07H19/207—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine adenine dinucleotide or nicotinamide-adenine dinucleotide
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
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Abstract
The invention belongs to the field of extraction of coenzyme I, and provides a resin filler separation method of coenzyme I, which comprises the steps of performing exchange on filtered cell filtrate containing the coenzyme I through a D945 macroporous resin column, then washing and eluting the cell filtrate, and collecting eluent. The method only adopts one resin filler to separate and purify the coenzyme I, can perform separation operation in the optimum PH range of the coenzyme I, reduces the generation rate of byproducts, and greatly improves the purity of the coenzyme I after resin separation.
Description
Technical Field
The invention relates to the field of extraction of coenzyme I, in particular to a resin filler separation method of coenzyme I.
Background
Coenzyme I (NAD), which is chemically named as nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide diphosphate or nicotinamide adenine dinucleotide, exists in mammals in two states of oxidation type (NAD +) and reduction type (NADH), is an important coenzyme in human body redox reaction, participates in various physiological activities such as metabolism of cell substances, energy synthesis, cell DNA repair and the like, and has an important effect on the immunity of organisms. Under the healthy state, the concentration of nicotinamide adenine dinucleotide in the human body is stable, and the normal functions of various cells are maintained. The concentration of nicotinamide adenine dinucleotide in the body determines the process and extent of cellular senescence, and a decrease in concentration accelerates the process of cellular senescence.
At present, the effective components of biological products are separated and purified by a resin separation method commonly used to obtain the target product with high purity. The reports of purifying coenzyme I by adopting a resin separation method are few, and the prior report proposes that a crude coenzyme I product is subjected to chromatographic separation by sequentially using 122# resin and 732# resin, and when the crude coenzyme I product is separated by using 122# resin, an eluent is ammonia water; when the mixture is separated by No. 732 resin, the eluent is distilled water; the pH is adjusted. This method has the following disadvantages: two kinds of resin are adopted for chromatographic separation, and the steps are complicated; the 122# resin is a weakly acidic phenolic aldehyde cation exchange resin, the application range of PH is required to be 4-14, the adsorption effect is good in a slightly alkaline environment, coenzyme I is most stable when the PH value is 2.5-3.5, and the coenzyme I is easy to deteriorate in the alkaline environment, so that the coenzyme I crude product is subjected to column elution by the 122# resin, an eluent is ammonia water, and under the condition, the coenzyme I is easy to deteriorate, a byproduct is generated, and the purity of the coenzyme I is influenced. There is therefore a need to find a suitable resin packing separation technique.
Disclosure of Invention
The invention aims to provide a novel resin filler separation technology. The invention adopts D945 macroporous resin to replace resin No. 122 and resin No. 732, and is used for separating and purifying coenzyme I. Compared with the 122# resin and the 732# resin, the D945 macroporous resin has the advantages of large specific surface area, strong adsorption force, high selectivity, high recycling rate, no need of PH regulation and mild condition requirements. The method only adopts one resin filler to separate and purify the coenzyme I, can perform separation operation in the optimum PH range of the coenzyme I, reduces the generation rate of byproducts, and greatly improves the purity of the coenzyme I after resin separation.
The specific scheme is as follows: a resin filler separation method of coenzyme I comprises the steps of subjecting filtered cell filtrate containing the coenzyme I to D945 macroporous resin column exchange, washing with water, eluting, and collecting eluent.
In the above-mentioned resin packing separation method of coenzyme I, the eluent is a 7-10vol% ethanol solution.
In the above-mentioned resin packing separation method of coenzyme I, the eluent is an ethanol solution of 8 to 9 vol%.
In the above-mentioned method for separating coenzyme I by resin packing, the water used in the washing operation is purified water.
In the resin filler separation method of coenzyme I, the cell filtrate containing the coenzyme I is subjected to column-loading exchange at the speed of 20-30 ml/min.
In the above-mentioned resin filler separation method of coenzyme I, the volume ratio of the D945 macroporous resin to water in the water washing operation is 1: 2-4.
In the above method for separating coenzyme I by resin filler, the washing speed in the washing operation is 20-30 ml/min.
In the above method for separating coenzyme I by resin packing, the elution speed in the elution operation is 15-20 ml/min.
In the above method for separating coenzyme I by resin filler, the method specifically comprises:
s1, extracting coenzyme I from fresh yeast: breaking the cell wall of fresh yeast by adopting a temperature difference method to obtain cell sap, microfiltering the cell sap by using a ceramic membrane to obtain a microfiltrate, continuously ultrafiltering to obtain an ultrafiltrate, and nanofiltering to obtain a nanofiltration liquor, wherein the nanofiltration liquor is a cell filtrate containing coenzyme I;
s2, column loading: exchanging the nano filtrate obtained in the S1 through a D945 macroporous resin column;
s3, washing: after completion of S2, washing with purified water;
s4, elution: and after S3 is finished, eluting with ethanol solution to obtain eluent.
In the resin filler separation method of coenzyme I, the aperture of the ceramic membrane is 100nm, the cut-off molecular weight of the ultrafiltration membrane is 2000, the nanofiltration membrane is a polyether sulfone membrane, and the cut-off molecular weight is 300;
in S4, the concentration of coenzyme I in the eluent is detected by a chromatograph, the signal peak value change on the chromatogram is observed at any time, the collection is started when the response value is in a straight-line rising trend and rises to 500-.
The invention has the beneficial effects that:
the D945 macroporous resin is selected to elute and separate the coenzyme I after nanofiltration, so that inorganic salt and a part of organic impurities are effectively removed, and the purity of the coenzyme I is greatly improved; and the D945 macroporous resin regeneration method is simple, convenient to operate, energy-saving and suitable for industrial application.
Detailed Description
The technical solution of the present invention will be described in further detail with reference to the following embodiments, but the present invention is not limited thereto.
In order to more clearly illustrate the present invention, the following examples are given to illustrate the advantages of the present invention.
Example 1
A novel elution method of coenzyme I comprises the following steps:
s1, extracting coenzyme I from fresh yeast: breaking the wall of fresh yeast by a temperature difference method to obtain cell sap A. Microfiltering the cell sap A with 100nm ceramic membrane to obtain microfiltered solution B, continuously ultrafiltering with ultrafiltration membrane with MWCO of 2000D to obtain ultrafiltrate C, and passing through nanofiltration membrane with MWCO of 300D to obtain nanofiltration solution D.
S2, column loading: and (4) feeding the nanofiltration liquid D obtained in the S1 to a D945 macroporous resin column at the speed of 20 ml/min.
S3, washing: after completion of S2, the column was washed with purified water at a rate of 23ml/min, which was 2 times the volume of the resin column.
S4, elution: and after S3 is finished, eluting with 7% ethanol solution at the speed of 15ml/min to obtain eluent E. And in the elution process, an ultraviolet detector is used for monitoring in real time, the signal peak value change on the chromatogram is observed at any time, the collection is started when the response value is in a straight-line rising trend and rises to 500, and the collection is stopped when the signal peak value 500 has a certain peak value and is in a stable state.
Example 2
A novel elution method of coenzyme I comprises the following steps:
s1, extracting coenzyme I from fresh yeast: breaking the wall of fresh yeast by a temperature difference method to obtain cell sap A. Microfiltering the cell sap A with 100nm ceramic membrane to obtain microfiltered solution B, continuously ultrafiltering with ultrafiltration membrane with MWCO of 2000D to obtain ultrafiltrate C, and passing through nanofiltration membrane with MWCO of 300D to obtain nanofiltration solution D.
S2, column loading: and (4) feeding the nanofiltration liquid D obtained in the S1 to a D945 macroporous resin column at the speed of 25 ml/min.
S3, washing: after completion of S2, the column was washed with purified water at a rate of 20ml/min, which was 2.5 times the volume of the resin column.
S4, elution: after S3, eluting with 7.5% ethanol solution at a rate of 17ml/min to obtain eluent E. And in the elution process, an ultraviolet detector is used for monitoring in real time, the signal peak value change on the chromatogram is observed at any time, the collection is started when the response value is in a straight-line rising trend and rises to 560, and the collection is stopped when the signal peak value 700 has a certain peak value and is in a stable state.
Example 3
A novel elution method of coenzyme I comprises the following steps:
s1, extracting coenzyme I from fresh yeast: breaking the wall of fresh yeast by a temperature difference method to obtain cell sap A. Microfiltering the cell sap A with 100nm ceramic membrane to obtain microfiltered solution B, continuously ultrafiltering with ultrafiltration membrane with MWCO of 2000D to obtain ultrafiltrate C, and passing through nanofiltration membrane with MWCO of 300D to obtain nanofiltration solution D.
S2, column loading: the nanofiltration solution D obtained in S1 was loaded on a D945 macroporous resin column at a rate of 22.5 ml/min.
S3, washing: after completion of S2, the column was washed with purified water at a rate of 25.5ml/min, which was 3 times the volume of the resin column.
S4, elution: after S3 is finished, 8% ethanol solution is used for elution at the speed of 18.5ml/min, and eluent E is obtained. And in the elution process, an ultraviolet detector is used for monitoring in real time, the signal peak value change on the chromatogram is observed at any time, the collection is started when the response value is in a straight-line rising trend and rises to 750, and the collection is stopped when the signal peak value 600 has a certain peak value and is in a stable state.
Example 4
A novel elution method of coenzyme I comprises the following steps:
s1, extracting coenzyme I from fresh yeast: breaking the wall of fresh yeast by a temperature difference method to obtain cell sap A. Microfiltering the cell sap A with 100nm ceramic membrane to obtain microfiltered solution B, continuously ultrafiltering with ultrafiltration membrane with MWCO of 2000D to obtain ultrafiltrate C, and passing through nanofiltration membrane with MWCO of 300D to obtain nanofiltration solution D.
S2, column loading: the nanofiltration solution D obtained in S1 was loaded on a D945 macroporous resin column at a rate of 27.5 ml/min.
S3, washing: after completion of S2, the column was washed with purified water at a rate of 30ml/min, which was 3.5 times the volume of the resin column.
S4, elution: and after S3 is finished, eluting with 9% ethanol solution at the speed of 20ml/min to obtain eluent E. And in the elution process, an ultraviolet detector is used for monitoring in real time, the signal peak value change on the chromatogram is observed at any time, the collection is started when the response value is in a straight-line rising trend and rises to 850, and the collection is stopped when the signal peak value 550 has a certain peak value and is in a stable state.
Example 5
A novel elution method of coenzyme I comprises the following steps:
s1, extracting coenzyme I from fresh yeast: breaking the wall of fresh yeast by a temperature difference method to obtain cell sap A. Microfiltering the cell sap A with 100nm ceramic membrane to obtain microfiltered solution B, continuously ultrafiltering with ultrafiltration membrane with MWCO of 2000D to obtain ultrafiltrate C, and passing through nanofiltration membrane with MWCO of 300D to obtain nanofiltration solution D.
S2, column loading: and (4) feeding the nanofiltration liquid D obtained in the S1 to a D945 macroporous resin column at the speed of 30 ml/min.
S3, washing: after completion of S2, the column was washed with purified water at a rate of 27ml/min, which was 4 times the volume of the resin column.
S4, elution: and after S3 is finished, eluting with 10% ethanol solution at the speed of 16ml/min to obtain eluent E. And in the elution process, an ultraviolet detector is used for monitoring in real time, the signal peak value change on the chromatogram is observed at any time, the collection is started when the response value is in a straight-line rising trend and rises to 1000, and the collection is stopped when the signal peak value is 650 with a certain peak value and is in a stable state.
Test example 1
The content of coenzyme I in the eluate obtained in examples 1 to 5 was measured and compared with the results of separation in resin No. 122 and resin No. 732.
The content detection is carried out according to the national quality standard of coenzyme I (WS-10001(HD-0877) -2002), and the results are as follows:
as can be seen from the above table, the present invention has a high content and less side reactions.
The above description is only exemplary of the invention, and any modification, equivalent replacement, and improvement made within the spirit and scope of the present invention should be considered within the scope of the present invention.
Claims (7)
1. A resin filler separation method of coenzyme I is characterized in that cell filtrate containing the coenzyme I after filtration is subjected to D945 macroporous resin column exchange, washed and eluted, and eluent is collected to obtain the coenzyme I-containing resin filler separation method;
the method specifically comprises the following steps:
s1, extracting coenzyme I from fresh yeast: breaking the cell wall of fresh yeast by adopting a temperature difference method to obtain cell sap, microfiltering the cell sap by using a ceramic membrane to obtain a microfiltrate, continuously ultrafiltering to obtain an ultrafiltrate, and nanofiltering to obtain a nanofiltration liquor, wherein the nanofiltration liquor is a cell filtrate containing coenzyme I;
s2, column loading: exchanging the nano filtrate obtained in the S1 through a D945 macroporous resin column;
s3, washing: after completion of S2, washing with purified water;
s4, elution: after S3, eluting with ethanol solution to obtain eluent;
in S4, detecting the concentration of coenzyme I in the eluent by a chromatograph, observing the peak value change of the signal on the chromatogram at any time, starting to collect when the response value is in a straight-line rising trend and rises to 500-1000, and stopping collecting when the peak value of the signal is 500-700 and is in a stable state;
the eluent is 7-10vol% ethanol solution.
2. The method of separating coenzyme I by using a resin filler according to claim 1, characterized in that the eluent is an ethanol solution of 8 to 9 vol%.
3. The method for separating coenzyme I by using resin packing as claimed in claim 1, wherein the cell filtrate containing coenzyme I is subjected to column-loading exchange at a rate of 20 to 30 ml/min.
4. The method for separating coenzyme I by using resin packing as claimed in claim 1, wherein the volume ratio of the D945 macroporous resin to water in the water washing operation is 1: 2-4.
5. The method of separating a coenzyme I resin filler according to claim 4, wherein the washing speed in the washing operation is 20 to 30 ml/min.
6. The method of separating coenzyme I according to claim 1, wherein the elution speed in the elution step is 15 to 20 ml/min.
7. The method for separating the resin filler of coenzyme I according to claim 6, wherein the pore diameter of the ceramic membrane is 100nm, the cut-off molecular weight of the ultrafiltration membrane is 2000, the nanofiltration membrane is a polyether sulfone membrane, and the cut-off molecular weight is 300.
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| CN108484707A (en) * | 2018-04-10 | 2018-09-04 | 开封康诺药业有限公司 | The technique of trifluoroacetic acid in a kind of removal Coenzyme I |
| CN109836469B (en) * | 2019-02-27 | 2021-01-12 | 开封康诺药业有限公司 | Coenzyme I decoloring process |
| CN114262355A (en) * | 2021-12-27 | 2022-04-01 | 江苏诚信药业有限公司 | Purification method of nicotinamide adenine dinucleotide |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876759A (en) * | 2012-10-29 | 2013-01-16 | 尚科生物医药（上海）有限公司 | Preparation method of nicotinamide adenine dinucleotide |
| CN105131065A (en) * | 2015-07-16 | 2015-12-09 | 合肥平光制药有限公司 | Preparation method of oxidizing-type coenzyme I |
| CN105481923A (en) * | 2015-12-30 | 2016-04-13 | 平光制药股份有限公司 | Preparation method of nicotinamide adenine dinucleotide |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876759A (en) * | 2012-10-29 | 2013-01-16 | 尚科生物医药（上海）有限公司 | Preparation method of nicotinamide adenine dinucleotide |
| CN105131065A (en) * | 2015-07-16 | 2015-12-09 | 合肥平光制药有限公司 | Preparation method of oxidizing-type coenzyme I |
| CN105481923A (en) * | 2015-12-30 | 2016-04-13 | 平光制药股份有限公司 | Preparation method of nicotinamide adenine dinucleotide |
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