CN1055202A - Technology by nucleic acid direct production nucleoside diphosphate - Google Patents
Technology by nucleic acid direct production nucleoside diphosphate Download PDFInfo
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- CN1055202A CN1055202A CN 90110394 CN90110394A CN1055202A CN 1055202 A CN1055202 A CN 1055202A CN 90110394 CN90110394 CN 90110394 CN 90110394 A CN90110394 A CN 90110394A CN 1055202 A CN1055202 A CN 1055202A
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Abstract
The invention discloses and a kind ofly nucleic acid is carried out phosphorolysis with Polyribonucleotide phosphorylase, the processing method of reaction direct production nucleoside diphosphate, have under higher concentration of substrate, the characteristics that directly prepare four kinds of nucleoside diphosphates, be suitable for large-scale industrial production, and advantages such as technology is simple, productive rate is high, cost is low, pollution-free are arranged.
Description
The present invention is to be raw material with nucleic acid, by the phosphorolysis of Polyribonucleotide phosphorylase (PNPase), and the technology of direct production nucleoside diphosphate.
Nucleoside diphosphate mainly contains four kinds, be adenosine diphosphate (ADP) (ADP), guanosine diphosphate(GDP) (GDP), cytidine diphosphate(CDP) (CDP) and uridine diphosphate (UDP) (UDP), its door all is important biochemical reagents, and wherein ADP and CDP produce broad spectrum antiviral-Polyinosinic-polycytidylic acid (PolyI: basic raw material C).
About the preparation method of nucleoside diphosphate, there are chemical synthesis, enzymic synthesis and ribonucleoside triphosphote edman degradation Edman several at present, now illustrate its technological process.Accompanying drawing 1 is the process flow sheet that enzymic synthesis and ribonucleoside triphosphote edman degradation Edman are produced nucleoside diphosphate, as can be seen from the figure, four kinds of nucleoside monophosphates is separately carried out with regard to needing from the synthetic beginning of enzymatic.Adopt the yield of this its nucleoside diphosphate of technology to have only about 12% of nucleic acid.Accompanying drawing 2 is the process flow sheet of nucleoside monophosphate chemosynthesis nucleoside diphosphate, and the yield that adopts this its nucleoside diphosphate of technology is about 35% of a nucleic acid.
These several processing methodes are the tired lock of more complicated all, and productive rate is low, the production cost height.Though chemical synthesis operation toxicity is big, the fraud of contaminate environment is arranged, be to adopt this technology more than production is gone up now.Utilize Polyribonucleotide phosphorylase (PNPase) that nucleic acid is carried out the phosphorolysis reaction, though some exploratory basic research works are also arranged, because the concentration very low (1-5mg/ml) of reaction solution amplifying nucleic acid, be unsuitable for mass-produced requirement, settled the present is not seen the report that adopts similar method to be used for the mass production nucleoside diphosphate.Purpose of the present invention is exactly at above existing problems, design a kind of industrial mass-produced production technique that directly prepares nucleoside diphosphate with nucleic acid that is fit to, have characteristics such as technology is simple, productive rate is high, cost is low, pollution-free, and can once prepare four kinds of nucleoside diphosphates simultaneously.
The present invention utilizes nucleotide phosphorylase (PNPase) that nucleic acid is carried out phosphorolysis reaction direct production nucleoside diphosphate, and accompanying drawing 3 is a process flow sheet of the present invention.
Technology of the present invention is achieved in that
Use 0.1-0.6Mol/L(PH8.2) phosphoric acid buffer, nucleic acid is made into the solution of 60-150g/L, and makes and contain KC10.1Mol/L, MgCl in the solution
20.01Mol/L, CuSO
40.004Mol/L, urea 0.3Mol/L, PNPase1000-3000 unit/L, 37 ± 2 ℃ were reacted 30-48 hour.After finishing reaction, the ratio in every gram resin absorption 4-5 gram nucleic acid directly goes up homemade D to reaction solution
296Macroporous anion exchange resin column, select the formic acid wash-out CDP of 0.02Mol/L for use, use the formic acid wash-out ADP of 0.15Mol/L again,, use 0.1Mol/L formic acid-0.1Mol/L sodium formate solution wash-out GDP at last with 0.01Mol/L formic acid-0.05Mol/L sodium formate solution wash-out UDP.Four kinds of nucleoside diphosphate solution that elute, transfer PH to 4.5-5.0 respectively, in homemade D on the ratio of every gram resin absorption 3 gram nucleoside diphosphates
001-ccMacroporous anion exchange resin column, with 0.05N hydrochloric acid wash-out, this is a concentration process.Elutriant adds 95% cold ethanol of 2.5-3.0 volume, makes the nucleoside diphosphate precipitation, centrifugal collecting precipitation, and vacuum-drying obtains finished product.
Nucleic acid raw material of the present invention selects for use the dephosphorization degreasing to take off the nucleic acid of secondary structure.The unit concentration of the concentration of phosphoric acid buffer and PNPase is that increase with nucleic acid raw material concentration increases in the technology, thereby concentration that can not determine them is on a value.The reaction times of reaction solution, generally in the time of 25-28 hour, the growing amount maximum of nucleoside diphosphate in unit time, then descend gradually, also had growing amount to 50 hours, thereby can't determine to react time of end, temperature of reaction, enzyme concentration, enzyme are lived or the like and all to be affected the length in reaction times, are 34-48 hour so finish the Best Times scope of reaction.
In a word, the present invention is under higher concentration of substrate, carry out directly four kinds of nucleoside diphosphates of preparation of phosphorolysis with PNPase, be suitable for fairly large industrial production, and (yield is about 60% of a nucleic acid to have that technology is simple, productive rate is high, reach as high as 80%), cost low (reducing by 1000 yuan/kilogram at least), advantage such as pollution-free than present production cost, the application on industrial production then has very important meaning.
Claims (4)
1, a kind of Polyribonucleotide phosphorylase (PNPase) that utilizes carries out phosphorolysis reaction, the processing method of producing nucleoside diphosphate to nucleic acid.The invention is characterized in: the phosphoric acid buffer with 0.1-0.6Mol/L (PH8.2) is made into the solution of 60-150g/L to nucleic acid, and makes and contain KClMol/L in the solution; MgCl
20.01Mol/L, CuSO
40.004Mol/L, urea 0.3Mol/L, PNPase1000-3000 unit/L, 37 ± 2 ℃ of reactions 34-48 hour, finish reaction after, separate concentrate, precipitation, drying, get product.
2, processing method according to claim 1 is characterized in that: nucleic acid raw material is selected the nucleic acid of dephosphorization degreasing secondary structure for use.
According to the described processing method of claim 1.2, it is characterized in that 3, separation method is in the ratio of every gram resin absorption 4-5 gram nucleic acid, and reaction solution is directly gone up homemade D
296Macroporous anion exchange resin column, select for use 0.02Mol/L formic acid wash-out CDP, use the formic acid wash-out ADP of 0.15Mol/L again,, use 0.1Mol/L formic acid-0.Mol/L sodium formate solution wash-out GDP at last with 0.01Mol/L formic acid-0.05Mol/L sodium formate solution wash-out UDP.
4, processing method according to claim 3 is characterized in that, spissated method is four kinds of nucleoside diphosphate solution that elute, and transfers PH to 4.5-5.0 respectively, in homemade D on the ratio of every gram resin absorption 3 gram nucleoside diphosphates
001-ccMacroporous anion exchange resin column is with 0.05N hydrochloric acid wash-out.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 90110394 CN1055202A (en) | 1990-12-30 | 1990-12-30 | Technology by nucleic acid direct production nucleoside diphosphate |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 90110394 CN1055202A (en) | 1990-12-30 | 1990-12-30 | Technology by nucleic acid direct production nucleoside diphosphate |
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| CN1055202A true CN1055202A (en) | 1991-10-09 |
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| CN 90110394 Pending CN1055202A (en) | 1990-12-30 | 1990-12-30 | Technology by nucleic acid direct production nucleoside diphosphate |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101792786A (en) * | 2010-03-04 | 2010-08-04 | 南京工业大学 | Method for synthesizing cytidine phosphoryl compound by directional catalysis |
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1990
- 1990-12-30 CN CN 90110394 patent/CN1055202A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101792786A (en) * | 2010-03-04 | 2010-08-04 | 南京工业大学 | Method for synthesizing cytidine phosphoryl compound by directional catalysis |
| CN101792786B (en) * | 2010-03-04 | 2012-07-11 | 南京工业大学 | Method for synthesizing cytidine phosphoryl compound by directional catalysis |
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