CN113842671A - NTP/dNTP chromatographic separation method and system based on intelligent control - Google Patents
NTP/dNTP chromatographic separation method and system based on intelligent control Download PDFInfo
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Abstract
The invention discloses an NTP/dNTP chromatographic separation method and a system based on intelligent control, wherein NTP/dNTP chromatographic separation is carried out in a low-temperature chromatographic cabinet, real-time online detection and intelligent control are carried out according to a nucleic acid protein detector and an HPLC detector NTP/dNTP chromatographic separation process which are arranged outside the low-temperature chromatographic cabinet, and whether NTP/dNTP flows out in the processes of sample loading and washing is monitored through the real-time online detection of the nucleic acid protein detector, so that whether the processes run normally is known; the gradient elution process is controlled and NTP/dNTP is collected by timing on-line sampling and HPLC detector detection. The invention can avoid the degradation of nucleoside triphosphate caused by frequently opening a low-temperature chromatography cabinet for sampling or regulating instruments and the like, and simultaneously ensures the high-quality operation of the chromatography separation process.
Description
Technical Field
The invention belongs to the technical field of biochemical separation, and particularly relates to an NTP/dNTP chromatographic separation method and system based on intelligent control.
Background
Nucleoside Triphosphate (NTP) or deoxynucleoside triphosphate (dNTP), which can be used as raw material to produce corresponding triphosphate compound by biotransformation of Saccharomyces cerevisiae or Saccharomyces cerevisiae, or can be prepared by acetate kinase; for example: 5 '-Adenosine Triphosphate (ATP) can be generated by using 5' -Adenosine Monophosphate (AMP); 2 '-deoxy-5' -adenosine triphosphate (dATP) can be generated by using 2 '-deoxy-5' -adenylate (dAMP); 5 '-Cytidine Triphosphate (CTP) and the like can be produced by 5' -cytidine triphosphate (CMP).
The conventional preparation method of nucleoside triphosphate is as follows:
document 1: chinese patent CN 104762347B discloses a production method of Adenosine Triphosphate (ATP), which mainly comprises the steps of beer yeast freezing treatment, fermentation and invertase extraction, plate-and-frame filter pressing, microfiltration, fermentation and synthesis of adenosine triphosphate, nanofiltration and desalination, resin adsorption, resin washing and elution, ultrafiltration and protein and heat source removal, ethanol precipitation, drying and the like; the technology only uses 201X7 anion exchange resin in the experiment, and the removal of impurities uses microfiltration nanofiltration and ultrafiltration, which greatly increases the cost of the experiment.
Document 2: publication No. CN 104894192A proposes a preparation method of disodium adenosine triphosphate, magnesium sulfate is replaced by magnesium chloride to prevent the doping of new impurity ions, a single anion column is replaced by a serial column sample loading, the flow rate is improved, the sample loading time is shortened by more than 50%, and the yield is improved by more than 30%; this technique also uses only an anion exchange resin, and is an experiment performed under normal temperature conditions, and does not consider the problems such as instability of adenosine triphosphate under normal temperature conditions.
In both of the biological preparations of document 1 and document 2, it is necessary to remove nucleosides, bases, or unreacted nucleotides produced by side reactions, or diphosphoric acid compounds as intermediates, etc. by chromatographic separation with an anion exchange resin; since the triphosphate compounds are very susceptible to degradation, they are usually carried out in a low temperature chromatography cabinet; however, such frequent sampling, analysis, instrument control, separation and collection operations are very inconvenient, and too frequent sampling is very disadvantageous for keeping the chromatography cabinet at a low temperature.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide an NTP/dNTP chromatographic separation method and system based on intelligent control, through carrying out NTP/dNTP chromatographic separation in a low-temperature chromatographic cabinet and carrying out real-time online detection and intelligent control according to a nucleic acid protein detector and an HPLC detector which are arranged outside the low-temperature chromatographic cabinet, the degradation of nucleoside triphosphate caused by frequent opening of the low-temperature chromatographic cabinet sampling or regulating instrument and the like can be avoided, and meanwhile, the high-quality operation of the chromatographic separation process is ensured.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides an NTP/dNTP chromatographic separation method based on intelligent control, which comprises the following steps:
(1) loading, namely in a low-temperature chromatography cabinet, enabling NTP/dNTP reaction clear liquid to sequentially pass through a cation exchange resin column and an anion exchange resin column, and adsorbing NTP/dNTP into the anion exchange resin column;
(2) washing, namely washing the cation exchange resin column and the anion exchange resin column with deionized water or purified water which is 5-10 times of the volume of the resin column after sample loading is finished;
(3) and (3) performing gradient elution, namely disconnecting the cation exchange resin column, performing gradient elution on the anion exchange resin column, performing timing detection on the effluent liquid of the anion exchange resin column by adopting an HPLC (high performance liquid chromatography) detector, and collecting NTP/dNTP solution with the concentration of more than or equal to 5 g/L.
Preferably, in the step (1):
the temperature in the low-temperature chromatography cabinet is 4-8 ℃; and/or
The cation exchange resin column is a regenerated Na-type cation exchange resin column; a Cl-type anion exchange resin column regenerated by the anion exchange resin column; and/or
In the sample loading process, a nucleic acid protein detector arranged outside the low-temperature chromatography cabinet is adopted to respectively carry out real-time online detection on the effluent liquid of the cation exchange resin column and the effluent liquid of the anion exchange resin column, and when the effluent liquid A260 of the anion exchange resin column is more than or equal to 1, the sample loading is finished.
Preferably, in the step (2):
and (2) carrying out real-time online detection on the effluent of the anion exchange resin column by adopting a nucleic acid protein detector arranged outside the low-temperature chromatography cabinet, and when A260 is less than 1, discharging waste, otherwise, collecting the effluent.
Preferably, in the step (3):
in the gradient elution process, a NaCl solution with the mass percentage concentration of 0-5% is adopted to carry out gradient elution on the anion exchange resin column; and/or
The collection process of the NTP/dNTP solution is as follows: in the process of timing detection of the effluent of the anion exchange resin column by using an HPLC (high performance liquid chromatography) detector, when NTP/dNTP is detected in the effluent of the anion exchange resin column and the concentration of the NTP/dNTP is more than or equal to 5g/L, collecting NTP/dNTP solution; when the concentration of NTP/dNTP in the effluent of the anion exchange resin column is less than 5g/L, stopping the collection.
Preferably, in the step (3), the NTP/dNTP solution collected after gradient elution is crystallized, filtered and vacuum-dried to obtain the NTP/dNTP product with the purity of more than or equal to 98%.
The second aspect of the present invention provides an intelligent chromatography separation system used in the intelligent control-based NTP/dNTP chromatography separation method according to the first aspect of the present invention, wherein the intelligent chromatography separation system comprises a low temperature chromatography cabinet, a detection mechanism and a control system;
a sample loading mechanism, a water washing mechanism, an elution mechanism, a column chromatography unit and a collector are arranged in the low-temperature chromatography cabinet; the column chromatography unit comprises a cation exchange resin column and an anion exchange resin column which are connected in series; the sample loading mechanism, the water washing mechanism and the elution mechanism are respectively communicated with the liquid inlets of the cation exchange resin column and the anion exchange resin column; the collector is communicated with the liquid outlet of the anion exchange resin column;
the detection mechanism detects effluent liquid of the cation exchange resin column and the anion exchange resin column and transmits detection information to the control system;
the control system receives the detection information of the detection mechanism, generates a water washing instruction, an elution instruction and a collection instruction according to the detection information, and outputs signals to the sample loading mechanism, the water washing mechanism, the elution mechanism, the column chromatography unit, the collector and the detection mechanism.
Preferably, the detection mechanism comprises a nucleic acid protein detector, a sampling dilution mechanism and an HPLC detector which are arranged outside the low-temperature chromatography cabinet;
the nucleic acid protein detector is communicated with the liquid outlets of the cation exchange resin column and the anion exchange resin column, and a one-way valve is arranged on a pipeline between the nucleic acid protein detector and the cation exchange resin column and the anion exchange resin column;
the sampling dilution mechanism is communicated with a liquid outlet of the anion exchange resin column, and a timing switch is arranged on a pipeline between the sampling dilution mechanism and the anion exchange resin column;
the HPLC detector is connected with the sampling dilution mechanism.
Preferably, the intelligent chromatography separation system further comprises a monitoring system arranged outside the low-temperature chromatography cabinet.
The invention has the following beneficial effects:
1. according to the NTP/dNTP chromatographic separation method and system based on intelligent control, NTP/dNTP chromatographic separation is carried out in the low-temperature chromatographic cabinet, real-time online detection and intelligent control are carried out according to the nucleic acid protein detector and the HPLC detector which are arranged outside the low-temperature chromatographic cabinet, the degradation of nucleoside triphosphate caused by frequent opening of the low-temperature chromatographic cabinet for sampling or regulating instruments and the like is avoided, meanwhile, the traceable chromatographic separation process is ensured, and the high-quality operation is realized;
2. according to the NTP/dNTP chromatographic separation method and system based on intelligent control, whether NTP/dNTP flows out in the processes of sample loading and water washing is monitored through real-time online detection of a nucleic acid protein detector, and therefore whether the processes run normally is known; the gradient elution process is controlled and NTP/dNTP is collected by timing on-line sampling and HPLC detector detection.
Drawings
Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:
FIG. 1 is a schematic diagram of an intelligent chromatographic separation system for an intelligent control-based NTP/dNTP chromatographic separation method according to the present invention;
FIG. 2 is a logic diagram of an intelligent chromatographic separation system for the intelligent control-based NTP/dNTP chromatographic separation method;
FIG. 3 is a flow chart of the intelligent control-based NTP/dNTP chromatographic separation method of the invention.
Detailed Description
In order to better understand the technical scheme of the invention, the technical scheme of the invention is further explained by combining the embodiment.
With reference to fig. 1, 2 and 3, the general concept of the present invention is as follows: because NTP/dNTP (such as 4 nucleoside triphosphates of NTP, 5 '-ATP, 5' -CTP, 5 '-GTP, 5' -UTP, 4 deoxynucleoside triphosphates of dNTP, 5 '-dATP, 5' -dCTP, 5 '-dGTP and 5' -dUTP) are unstable at normal temperature and are easy to degrade, the chromatographic separation of NTP/dNTP is usually required to be carried out in the low-temperature chromatographic cabinet 100, and the frequent sampling or regulating of instruments, separation and collection and the like in the chromatographic separation process are considered; therefore, in the invention, a regenerated cation exchange resin column 105 and an anion exchange resin column 106 are placed in a low-temperature chromatography cabinet 100, liquid inlets of the cation exchange resin column 105 and the anion exchange resin column 106 are respectively connected with a sample loading mechanism, a water washing mechanism and an elution mechanism, the cation exchange resin column 105 and the anion exchange resin column 106 are connected in series and then connected with a waste discharge tank and a collector 107, wherein the waste discharge tank can also be placed outside the low-temperature chromatography cabinet 100 and connected with the cation exchange resin column 105 and the anion exchange resin column 106 by pipelines; then the cation exchange resin column 105 and the anion exchange resin column 106 are respectively connected with a nucleic acid protein detector 108, a sampling dilution mechanism 110 and an HPLC detector 109 (high performance liquid chromatography detector) outside the low-temperature chromatography cabinet 100; the whole chromatographic separation process is carried out in the low-temperature chromatographic cabinet 100, and meanwhile, the A260 in the effluent liquid of the cation exchange resin column 105 and the anion exchange resin column 106 is detected on line in real time by the nucleic acid protein detector 108, so that whether NTP/dNTP flows out in the sample loading and water washing processes is monitored, and whether the sample loading and water washing processes run normally is known; detecting the effluent of the anion exchange resin column 106 by an HPLC detector 109, and controlling the elution process and the collection process, specifically: and (3) sampling and diluting at regular time by using a sampling and diluting mechanism 110, putting the diluted effluent into an automatic sampler of an HPLC detector 109 for detection and analysis, and judging whether NTP/dNTP is generated and the concentration of NTP/dNTP according to a detected chromatogram so as to control the elution process and the collection process.
Referring to fig. 1 and 2, the intelligent chromatography separation system for the intelligent control-based NTP/dNTP chromatography separation method of the present invention includes a low-temperature chromatography cabinet 100, a detection mechanism, and a control system 111; wherein a sample loading mechanism, a water washing mechanism, an elution mechanism, a column chromatography unit and a collector 107 are arranged in the low-temperature chromatography cabinet 100; the column chromatography unit comprises a cation exchange resin column 105 and an anion exchange resin column 106 which are connected in series, and a one-way valve 6 is arranged between the cation exchange resin column 105 and the anion exchange resin column 106; the sample loading mechanism, the water washing mechanism and the elution mechanism are respectively communicated with the liquid inlets of the cation exchange resin column 105 and the anion exchange resin column 106; the collector 107 is communicated with the liquid outlet of the anion exchange resin column 106; the detection mechanism detects effluent liquid of the cation exchange resin column 105 and effluent liquid of the anion exchange resin column 106 and transmits detection information to the control system 111; the control system 111 receives the detection information of the detection mechanism, generates a water washing instruction, an elution instruction and a collection instruction according to the detection information, and outputs signals to the sample loading mechanism, the water washing mechanism, the elution mechanism, the column chromatography unit, the collector 107 and the detection mechanism.
As shown in fig. 1, the sample loading mechanism includes a sample loading tank 101, a sample loading pump and a one-way valve 1; the water washing mechanism comprises a water washing tank 102, a water washing pump and a one-way valve 2; the elution mechanism includes a first elution tank 103, a second elution tank 104, and an elution pump and one-way valve 3. Wherein the sample loading mechanism, the water washing mechanism and the elution mechanism are respectively communicated with the liquid inlets of the cation exchange resin column 105 and the anion exchange resin column 106 of the column chromatography unit, a one-way valve 4 is arranged on a pipeline between the sample loading mechanism, the water washing mechanism, the elution mechanism and the cation exchange resin column 105, and a one-way valve 5 is arranged on a pipeline between the sample loading mechanism, the water washing mechanism, the elution mechanism and the anion exchange resin column 106. The collector 107 is respectively communicated with the liquid outlets of the cation exchange resin column 105 and the anion exchange resin column 106 of the column chromatography unit, and a one-way valve 8 is arranged on a pipeline between the collector 107 and the cation exchange resin column 105 and the anion exchange resin column 106 of the column chromatography unit.
Referring to fig. 1, in the column chromatography unit, both the cation exchange resin column 105 and the anion exchange resin column 106 are regenerated by NaOH or HCl, wherein the cation exchange resin column 105 is a regenerated Na-type cation exchange resin column 105; anion exchange resin column 106 regenerated Cl-type anion exchange resin column 106.
Referring to fig. 1, the detection mechanism includes a nucleic acid protein detector 108, a sampling dilution mechanism 110 and an HPLC detector 109 disposed outside the low-temperature chromatography cabinet 100; wherein the nucleic acid protein detector 108 is communicated with the liquid outlets of the cation exchange resin column 105 and the anion exchange resin column 106, and a one-way valve 7 is arranged on a pipeline between the nucleic acid protein detector 108 and the cation exchange resin column 105 and the anion exchange resin column 106; the sampling dilution mechanism 110 is communicated with the liquid outlet of the anion exchange resin column 106, and a timing switch 9 is arranged on a pipeline between the sampling dilution mechanism 110 and the anion exchange resin column 106; the HPLC detector 109 is connected to the sample dilution mechanism 110, and is configured to detect an effluent diluted by the sample dilution mechanism 110.
As shown in fig. 1 and fig. 2, the control system 111 includes a sample loading controller, a water washing controller, and an elution controller; the sample loading controller controls the opening and closing of the one-way valve 1 according to a sample loading instruction of the control system 111, so as to control the start and the end of sample loading, wherein the sample loading instruction comprises sample loading time, sample loading quantity, flow rate, opening and closing of the one-way valve 1 and the like; the water washing controller controls the opening and closing of the one-way valve 2 according to a water washing instruction of the control system 111, so as to control the starting and the ending of the water washing, wherein the water washing instruction comprises the opening and closing of the one-way valve 2, the usage amount of deionized water, the flow rate and the like; the elution controller controls the opening and closing of the one-way valve 3 according to an elution instruction of the control system 111, so as to control the beginning and the end of the elution process, wherein the elution instruction comprises the type, the concentration, the usage amount and the flow rate of the eluent, the opening and closing of the one-way valve 3, the one-way valve 5 and the like; the collectors 107 collect the eluted NTPs/dntps into the corresponding collectors 107 according to the collection instruction of the control system 111.
Referring to fig. 1, in order to facilitate remote control, the intelligent chromatography separation system further includes a monitoring system disposed outside the low temperature chromatography cabinet 100.
Referring to fig. 1, 2 and 3, the intelligent control-based NTP/dNTP chromatographic separation method provided by the present invention comprises the following steps:
(1) loading, in a low-temperature chromatography cabinet 100, the NTP/dNTP reaction clear solution passes through a cation exchange resin column 105 and an anion exchange resin column 106 in sequence, and the NTP/dNTP is adsorbed into the anion exchange resin column 106;
the specific process is as follows: firstly, filtering NTP/dNTP reaction liquid generated by biological catalysis to remove yeast, and removing macromolecular cell autolysate by ultrafiltration to obtain NTP/dNTP reaction clear liquid; the whole chromatographic separation process is carried out in a low-temperature chromatographic cabinet 100 at 4-8 ℃, NTP/dNTP reaction clear liquid is added into a sample loading mechanism in the low-temperature chromatographic cabinet 100, a control system 111 controls the sample loading mechanism to carry out sample loading, the NTP/dNTP reaction clear liquid in the sample loading mechanism sequentially passes through a cation exchange resin column 105 and an anion exchange resin column 106 which are connected in series in the sample loading process, proteins, pigments, cations and the like in the NTP/dNTP reaction clear liquid are adsorbed in the cation exchange resin column 105, and NTP/dNTP in the NTP/dNTP reaction clear liquid is adsorbed in the anion exchange resin column 106; meanwhile, in the process, the effluent of the anion exchange resin column 106 is detected on line in real time by using the nucleic acid protein detector 108, and when A260 in the effluent is more than or equal to 1, the anion exchange resin column 106 is indicated to be saturated in adsorption, and the sample loading is finished; otherwise, it indicates that the anion exchange resin column 106 is not saturated by adsorption, and the effluent liquid is discharged; wherein the cation exchange resin column 105 is a regenerated Na-type cation exchange resin column 105; a Cl-type anion exchange resin column 106 regenerated from the anion exchange resin column 106;
(2) washing, namely washing the cation exchange resin column 105 and the anion exchange resin column 106 by using deionized water or purified water with the volume 5-10 times that of the resin column after the sample loading is finished;
the specific process is as follows: after the sample loading is finished, the control system 111 controls the water washing mechanism to carry out water washing, deionized water or purified water with the volume 5-10 times of that of the resin column is adopted to carry out water washing on the cation exchange resin column 105 and the anion exchange resin column 106, the water washing is stopped automatically, meanwhile, the nucleic acid protein detector 108 is adopted to carry out real-time online detection on the effluent liquid of the anion exchange resin column 106, when A260 is less than 1, the effluent liquid is considered to have no NTP/dNTP flowing out, the waste can be discharged normally, otherwise, the effluent liquid is considered to have NTP/dNTP, the effluent liquid needs to be collected at the moment, and signals are transmitted to the control system 111 to adjust the sample loading amount or increase the resin and the like. In the process of loading and washing with water, since nucleoside, base, etc. are not generally adsorbed on the anion exchange resin column 106, they are discharged to the outside of the column with washing with water, etc.
(3) And (3) performing gradient elution, namely disconnecting the cation exchange resin column 105, eluting the anion exchange resin column 106, performing timing detection on the effluent of the anion exchange resin column 106 by using an HPLC (high performance liquid chromatography) detector 109, and collecting NTP/dNTP solution with the concentration being more than or equal to 5 g/L.
The specific process is as follows: after the water washing is finished, the control system 111 controls the elution mechanism to carry out elution, closes the one-way valve 2, opens the one-way valve 3, disconnects the cation exchange resin column 105, adopts NaCl solution with the mass percentage concentration of 0-5% to carry out gradient elution on the anion exchange resin column 106, simultaneously adopts an HPLC detector 109 to carry out timing detection on effluent liquid of the anion exchange resin column 106, judges whether NTP or dNTP is eluted according to the peak appearance time of NTP or dNTP in a chromatogram detected by the HPLC detector 109, and calculates the concentration of NTP or dNTP, wherein the concentration C of NTP or dNTP is the peak area of a real mapping map of a certain nucleoside triphosphate multiplied by the standard concentration of the nucleoside triphosphate/the standard map peak area of the nucleoside triphosphate;
when NTP or dNTP is detected in the effluent liquid of the anion exchange resin column 106 and the concentration is more than or equal to 5g/L, NTP or dNTP solution starts to be collected, and the concentration of NTP or dNTP gradually rises until the concentration is reduced to be less than 5 g/L; when the concentration of NTP or dNTP in the effluent of the anion exchange resin column 106 is less than 5g/L, the collection is stopped.
And transmitting the analysis result to a control system 111, controlling the collector 107 to distribute and collect by the control system 111, finally collecting the part of NTP or dNTP with the purity of 95% or more than 98%, merging the collecting pipes, adding 2-3 times of 95% ethanol, crystallizing, filtering, and drying in vacuum to obtain the high-purity NTP or dNTP product with the purity of more than or equal to 98%.
The intelligent control-based NTP/dNTP chromatographic separation method and system of the invention are further described in the following by combining specific examples.
Example 1
In this embodiment, the chromatographic separation of 5' -ATP is performed by using the intelligent chromatographic separation system shown in fig. 1, and the specific process is as follows:
70g of quick-frozen beer yeast is added into 1L of reaction liquid containing 40mmol/L adenosine, 200mmol/L phosphate buffer solution (pH is 6.5), 200mmol/L glucose and 5mmol/L magnesium phosphate, and the reaction is carried out for 4 hours at 32 ℃, wherein the molar conversion rate can reach 90 percent, and the reaction liquid still contains unreacted adenosine, intermediate AMP, ADP and the like. The 5 '-ATP reaction solution prepared above was filtered to remove yeast, and a 5' -ATP reaction clear solution was obtained.
The method comprises the steps of loading, washing and gradient elution through an intelligent chromatographic separation system, wherein the dosage of cation exchange resin is 100ml (Na type), the dosage of deionized water in the washing process is 1-2L, the flow rate is 100ml/h, the dosage of anion exchange resin is 200ml (Cl type), and the eluent is subjected to gradient elution by adopting NaCl with the mass percentage concentration of 0-5%, and the flow rate is 20-30 ml/h. Collecting partial effluent liquid with initial concentration of 5g/L and final concentration of 5g/L according to the report of a control system after chromatographic separation, mixing, adding 3 times of 95% ethanol with volume of 260ml and average concentration of 5' -ATP of 60g/L, stirring at 28 ℃ for 1-2 h under heat preservation, separating out ATPNa salt crystal, filtering, and drying in vacuum to obtain 14g of ATPNa with content of 98%.
Example 2
In this embodiment, the chromatographic separation of 5' -dATP is performed by using the intelligent chromatographic separation system shown in fig. 1, and the specific process is as follows:
70g of quick-frozen lager brewing yeast was added to 1L of a reaction mixture containing 40mmol/L of dAMP, 200mmol/L of phosphate buffer (pH 6.5), 200mmol/L of glucose and 5mmol/L of magnesium phosphate, and the mixture was reacted at 32 ℃ for 4 hours to achieve a molar conversion of 85%. The solution was filtered to remove yeast to obtain a clear solution.
The method comprises the steps of loading, washing and eluting through an intelligent chromatographic separation system, wherein the dosage of cation exchange resin is 100ml (Na type), the dosage of deionized water in the washing process is 1-2L, the flow rate is 100ml/h, the dosage of anion exchange resin is 200ml (Cl type), and the eluent is subjected to gradient elution by adopting NaCl with the mass percentage concentration of 0-5%, and the flow rate is 20-30 ml/h. Collecting partial effluent liquid with initial concentration of 5g/L and final concentration of 5g/L according to a report of a control system after chromatographic separation, mixing, adding 3 times of 95% ethanol with volume of 260ml and average concentration of 5' -dATP of 60g/L, stirring at 28 ℃ for 1-2 h at the constant temperature, separating out dATPNa salt crystals, filtering, and drying in vacuum to obtain 10g dATPNa with content of 98.2%.
Example 3
In this embodiment, the chromatographic separation of 5' -CTP is performed by using the intelligent chromatographic separation system shown in fig. 1, and the specific process is as follows:
a reaction mixture containing 40mmol/L of CMP, 200mmol/L of phosphate buffer (pH 6.5), 200mmol/L of glucose and 5mmol/L of magnesium phosphate was prepared in 1L, and 70g of quick-frozen lager brewing yeast was added thereto, and the other conditions were the same as in example 1. The CTP can be obtained finally, and the content is 98.1 percent.
Example 4
In this example, the chromatographic separation of 5' -UTP was performed, and the intelligent chromatographic separation system shown in FIG. 1 was used in the separation process to change CMP in example 3 to UMP, and under the same conditions, 8.5g of UTP with a content of 98.3% was finally obtained.
Example 5
In this example, the chromatographic separation of 5' -GTP was carried out by using the intelligent chromatographic separation system shown in FIG. 1 in the separation process, changing CMP in example 3 to GMP, and under the same conditions, 7.8g of GTP, having a content of 98%, was finally obtained.
dCTP and dGTP can also be produced by the same method as described above.
According to the NTP/dNTP chromatographic separation method and system based on intelligent control, NTP/dNTP chromatographic separation is carried out in the low-temperature chromatographic cabinet, real-time online detection and intelligent control are carried out according to a nucleic acid protein detector and an HPLC detector which are arranged outside the low-temperature chromatographic cabinet, the degradation of nucleoside triphosphate caused by frequent opening of the low-temperature chromatographic cabinet for sampling or regulating instruments and the like is avoided, and meanwhile, high-quality operation in the chromatographic separation process is guaranteed; the invention monitors whether NTP/dNTP flows out in the processes of sample loading and water washing through the real-time online detection of the nucleic acid protein detector, thereby knowing whether the processes run normally; the elution process is controlled and NTP/dNTP is collected by timing on-line sampling and detection by an HPLC detector.
It should be understood by those skilled in the art that the above embodiments are only for illustrating the present invention and are not to be used as a limitation of the present invention, and that changes and modifications to the above described embodiments are within the scope of the claims of the present invention as long as they are within the spirit and scope of the present invention.
Claims (8)
1. An NTP/dNTP chromatographic separation method based on intelligent control is characterized by comprising the following steps:
(1) loading, namely in a low-temperature chromatography cabinet, enabling NTP/dNTP reaction clear liquid to sequentially pass through a cation exchange resin column and an anion exchange resin column, and adsorbing NTP/dNTP into the anion exchange resin column;
(2) washing, namely washing the cation exchange resin column and the anion exchange resin column with deionized water or purified water which is 5-10 times of the volume of the resin column after sample loading is finished;
(3) and (3) performing gradient elution, namely disconnecting the cation exchange resin column, performing gradient elution on the anion exchange resin column, performing timing detection on the effluent liquid of the anion exchange resin column by adopting an HPLC (high performance liquid chromatography) detector, and collecting NTP/dNTP solution with the concentration of more than or equal to 5 g/L.
2. The intelligent control-based NTP/dNTP chromatographic separation method according to claim 1, wherein in step (1):
the temperature in the low-temperature chromatography cabinet is 4-8 ℃; and/or
The cation exchange resin column is a regenerated Na-type cation exchange resin column; a Cl-type anion exchange resin column regenerated by the anion exchange resin column; and/or
In the sample loading process, a nucleic acid protein detector arranged outside the low-temperature chromatography cabinet is adopted to respectively carry out real-time online detection on the effluent liquid of the cation exchange resin column and the effluent liquid of the anion exchange resin column, and when the effluent liquid A260 of the anion exchange resin column is more than or equal to 1, the sample loading is finished.
3. The intelligent control-based NTP/dNTP chromatographic separation method according to claim 1, wherein in step (2):
and (2) carrying out real-time online detection on the effluent of the anion exchange resin column by adopting a nucleic acid protein detector arranged outside the low-temperature chromatography cabinet, and when A260 is less than 1, discharging waste, otherwise, collecting the effluent.
4. The intelligent control-based NTP/dNTP chromatographic separation method according to claim 1, wherein in step (3):
in the gradient elution process, a NaCl solution with the mass percentage concentration of 0-5% is adopted to elute the anion exchange resin column; and/or
The collection process of the NTP/dNTP solution is as follows: in the process of timing detection of the effluent of the anion exchange resin column by using an HPLC (high performance liquid chromatography) detector, when NTP/dNTP is detected in the effluent of the anion exchange resin column and the concentration of the NTP/dNTP is more than or equal to 5g/L, collecting NTP/dNTP solution; when the concentration of NTP/dNTP in the effluent of the anion exchange resin column is less than 5g/L, stopping the collection.
5. The NTP/dNTP chromatographic separation method based on intelligent control of claim 1, wherein in the step (3), the NTP/dNTP solution collected after elution is crystallized, filtered and dried in vacuum to obtain the NTP/dNTP product with the purity not less than 98%.
6. An intelligent chromatographic separation system used in the intelligent control-based NTP/dNTP chromatographic separation method according to any one of claims 1-5, wherein the intelligent chromatographic separation system comprises a low temperature chromatographic cabinet, a detection mechanism and a control system;
a sample loading mechanism, a water washing mechanism, an elution mechanism, a column chromatography unit and a collector are arranged in the low-temperature chromatography cabinet; the column chromatography unit comprises a cation exchange resin column and an anion exchange resin column which are connected in series, and a one-way valve is arranged between the cation exchange resin column and the anion exchange resin column; the sample loading mechanism, the water washing mechanism and the elution mechanism are respectively communicated with the liquid inlets of the cation exchange resin column and the anion exchange resin column; the collector is communicated with the liquid outlet of the anion exchange resin column;
the detection mechanism detects effluent liquid of the cation exchange resin column and the anion exchange resin column and transmits detection information to the control system;
the control system receives the detection information of the detection mechanism, generates a water washing instruction, an elution instruction and a collection instruction according to the detection information, and outputs signals to the sample loading mechanism, the water washing mechanism, the elution mechanism, the column chromatography unit, the collector and the detection mechanism.
7. The intelligent chromatographic separation system according to claim 6, wherein the detection mechanism comprises a nucleic acid protein detector, a sampling dilution mechanism and an HPLC detector which are arranged outside the low-temperature chromatographic cabinet;
the nucleic acid protein detector is communicated with the liquid outlets of the cation exchange resin column and the anion exchange resin column, and a one-way valve is arranged on a pipeline between the nucleic acid protein detector and the cation exchange resin column and the anion exchange resin column;
the sampling dilution mechanism is communicated with a liquid outlet of the anion exchange resin column, and a timing switch is arranged on a pipeline between the sampling dilution mechanism and the anion exchange resin column;
the HPLC detector is connected with the sampling dilution mechanism.
8. The intelligent chromatographic separation system of claim 6 further comprising a monitoring system external to the cryo-chromatographic cabinet.
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