CN116874537A - Preparation method of high-purity nicotinamide adenine dinucleotide - Google Patents
Preparation method of high-purity nicotinamide adenine dinucleotide Download PDFInfo
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- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 title claims abstract description 22
- 229950006238 nadide Drugs 0.000 title claims abstract description 22
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 title claims abstract description 20
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 239000012528 membrane Substances 0.000 claims abstract description 84
- 239000007788 liquid Substances 0.000 claims abstract description 58
- BAWFJGJZGIEFAR-NNYOXOHSSA-O NAD(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-O 0.000 claims abstract description 44
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 38
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 34
- 238000000034 method Methods 0.000 claims abstract description 26
- 238000001471 micro-filtration Methods 0.000 claims abstract description 16
- 238000000502 dialysis Methods 0.000 claims abstract description 15
- 238000006243 chemical reaction Methods 0.000 claims abstract description 14
- 238000001728 nano-filtration Methods 0.000 claims abstract description 12
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 11
- 238000011033 desalting Methods 0.000 claims abstract description 11
- 238000000909 electrodialysis Methods 0.000 claims abstract description 10
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 7
- 239000000919 ceramic Substances 0.000 claims abstract description 7
- 238000001914 filtration Methods 0.000 claims abstract description 5
- 238000004108 freeze drying Methods 0.000 claims abstract description 5
- 238000005406 washing Methods 0.000 claims abstract description 5
- 241001052560 Thallis Species 0.000 claims abstract description 3
- 229920002521 macromolecule Polymers 0.000 claims abstract description 3
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 10
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 10
- 235000011152 sodium sulphate Nutrition 0.000 claims description 10
- 239000002994 raw material Substances 0.000 claims description 2
- 239000012535 impurity Substances 0.000 abstract description 2
- 239000011148 porous material Substances 0.000 abstract description 2
- 238000004042 decolorization Methods 0.000 abstract 1
- 230000001276 controlling effect Effects 0.000 description 19
- 238000000746 purification Methods 0.000 description 13
- 238000000926 separation method Methods 0.000 description 12
- 238000007599 discharging Methods 0.000 description 8
- 239000000843 powder Substances 0.000 description 8
- 238000004128 high performance liquid chromatography Methods 0.000 description 7
- DAYLJWODMCOQEW-TURQNECASA-N NMN zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)([O-])=O)O2)O)=C1 DAYLJWODMCOQEW-TURQNECASA-N 0.000 description 6
- 239000008367 deionised water Substances 0.000 description 6
- 229910021641 deionized water Inorganic materials 0.000 description 6
- 238000005374 membrane filtration Methods 0.000 description 5
- 238000006555 catalytic reaction Methods 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000001953 recrystallisation Methods 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 239000011347 resin Substances 0.000 description 4
- 229920005989 resin Polymers 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 239000003513 alkali Substances 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 239000012295 chemical reaction liquid Substances 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 238000010612 desalination reaction Methods 0.000 description 3
- 238000009776 industrial production Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 102000009609 Pyrophosphatases Human genes 0.000 description 2
- 108010009413 Pyrophosphatases Proteins 0.000 description 2
- 102000004357 Transferases Human genes 0.000 description 2
- 108090000992 Transferases Proteins 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000003912 environmental pollution Methods 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 238000002834 transmittance Methods 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- 230000005730 ADP ribosylation Effects 0.000 description 1
- SRNWOUGRCWSEMX-KEOHHSTQSA-N ADP-beta-D-ribose Chemical group C([C@H]1O[C@H]([C@@H]([C@@H]1O)O)N1C=2N=CN=C(C=2N=C1)N)OP(O)(=O)OP(O)(=O)OC[C@H]1O[C@@H](O)[C@H](O)[C@@H]1O SRNWOUGRCWSEMX-KEOHHSTQSA-N 0.000 description 1
- 108020000946 Bacterial DNA Proteins 0.000 description 1
- BQOHYSXSASDCEA-KEOHHSTQSA-N Cyclic ADP-Ribose Chemical compound C([C@@H]1[C@H]([C@H]([C@@H](O1)N1C=2N=CN3C(C=2N=C1)=N)O)O)OP(O)(=O)OP(O)(=O)OC[C@@H]1[C@@H](O)[C@@H](O)[C@H]3O1 BQOHYSXSASDCEA-KEOHHSTQSA-N 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 230000033616 DNA repair Effects 0.000 description 1
- 102000011990 Sirtuin Human genes 0.000 description 1
- 108050002485 Sirtuin Proteins 0.000 description 1
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 108010031234 carbon monoxide dehydrogenase Proteins 0.000 description 1
- 108091092356 cellular DNA Proteins 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 238000011034 membrane dialysis Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000006479 redox reaction Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
Classifications
-
- 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
-
- 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
Abstract
The invention discloses a preparation method of high-purity nicotinamide adenine dinucleotide, which comprises the following steps: filtering the NAD+ reaction solution prepared by a biocatalysis method by a ceramic membrane to remove thalli, thereby obtaining a micro-filtration solution; removing macromolecular substances from the micro-filtration liquid through an ultrafiltration organic membrane to obtain ultrafiltration clear liquid; intercepting NAD+ from the ultrafiltration clear liquid through an ultrafiltration organic membrane to remove ATP and ADP, and carrying out dialysis water washing by using pure water to obtain ultrafiltration turbid liquid; decolorizing the ultrafiltered turbid liquid by using active carbon to obtain decolorized liquid; passing the medium decolorization solution through an electrodialysis device to obtain desalted solution; concentrating the medium desalting solution by a nanofiltration organic membrane to obtain a concentrated solution with the concentration of 15-30 g/L; the purified NAD+ finished product is obtained after the medium concentrated solution is subjected to freeze drying treatment, and impurities in a reaction system are precisely separated by adopting ultrafiltration, nanofiltration membranes and electrodialysis devices with proper pore diameters, so that the purity and the content of the NAD+ product are finally improved.
Description
Technical Field
The invention relates to the technical field of biochemistry, in particular to a preparation method of high-purity nicotinamide adenine dinucleotide.
Background
Nicotinamide adenine dinucleotide (abbreviated as NAD+), also known as co-dehydrogenase I or coenzyme I. Used as a coenzyme in the redox reaction, as a donor for the ADP-ribose moiety in the ADP-ribosylation reaction, and as a precursor for the second messenger molecule, cyclic ADP-ribose. NAD+ also serves as a substrate for bacterial DNA ligase and a group of enzymes called sirtuins that use NAD+ to remove acetyl groups from proteins. Nicotinamide adenine dinucleotide participates in various physiological activities such as cellular material metabolism, energy synthesis, cellular DNA repair and the like, and plays an important role in the immunity of organisms.
The current mainstream purification process mostly adopts means such as ion exchange resin purification and recrystallization, but the production process is not easy to control, the production efficiency, the product purity and the yield are low, and the market demands can not be met. The preparation method of oxidized coenzyme I and nicotinamide adenine dinucleotide of Chinese patent CN105131065B and CN105481923B both disclose a separation and purification method of NAD+, wherein resin columns are mainly used for separation and purification. The method for separating and purifying by using the resin column is complex in operation, long in time consumption, and high in cost, and a large amount of reagents such as acid, alkali and salt are needed for treating the resin, so that the method is not beneficial to large-scale industrial production, and has the advantages of high environmental pollution. Besides, the application of Chinese patent No. CN111065644B for preparing high-purity NAD+ discloses a separation and purification method for NAD+ by mainly adopting a method of recrystallizing poor solvents such as ethanol and the like. The separation and purification method has the advantages of complex flow, long extraction period, difficult control, large amount of reagents such as acid, alkali, alcohol and the like, and is unfavorable for large-scale industrial production, and has larger environmental pollution and higher cost.
Disclosure of Invention
The invention aims to provide a preparation method of high-purity nicotinamide adenine dinucleotide, impurities in a reaction system can be precisely separated by adopting ultrafiltration, nanofiltration membrane and electrodialysis device with proper pore diameters, and the purity and content of NAD+ products are finally improved, so that the problems in the background technology can be solved.
In order to achieve the above purpose, the present invention provides the following technical solutions:
a preparation method of high-purity nicotinamide adenine dinucleotide is shown in figure 1, and comprises the following steps:
step 1: filtering the NAD+ reaction solution prepared by a biocatalysis method by a ceramic membrane to remove thalli, thereby obtaining a micro-filtration solution;
step 2: removing macromolecular substances from the micro-filtration liquid in the step 1 through an ultrafiltration organic membrane to obtain ultrafiltration clear liquid;
step 3: intercepting NAD+ from the ultrafiltered clear liquid in the step 2 through an ultrafiltered organic membrane to remove ATP and ADP, and performing dialysis water washing by using pure water to obtain ultrafiltered turbid liquid;
step 4: decolorizing the ultrafiltration turbid liquid in the step 3 by activated carbon to obtain decolorized liquid;
step 5: passing the decolorized solution obtained in the step 4 through an electrodialysis device to obtain desalted solution;
step 6: concentrating the desalted liquid obtained in the step 5 by using a nanofiltration organic membrane to obtain concentrated liquid with the concentration of 15-30 g/L;
step 7: and (3) freeze-drying the concentrated solution in the step (6) to obtain a purified NAD+ finished product.
Further, the NAD+ content in the NAD+ reaction solution is 30-100g/L.
Further, the specification of the microfiltration ceramic membrane is 10000-30000 daltons, the specification of the ultrafiltration organic membrane is 5000-10000 daltons, the specification of the ultrafiltration organic membrane is 3000-5000 daltons, and the specification of the nanofiltration organic membrane is 100-400 daltons.
Furthermore, the membrane passing frequency in the step 1 is controlled at 50HZ, the membrane feeding pressure is 0.32MPa, the membrane discharging pressure is 0.28MPa, and 0.2-0.6 times of pure water is adopted for dialysis.
Furthermore, the membrane passing frequency in the step 2 is controlled at 30HZ, the membrane feeding pressure is 2.0MPa, the membrane discharging pressure is 3MPa, and 0.2-0.6 times of pure water is adopted for dialysis.
Furthermore, in the step 3, the membrane passing frequency is controlled at 30HZ, the membrane inlet pressure is 2.0MPa, the membrane outlet pressure is 1.5MPa, and 0.2-0.6 times of pure water is adopted for dialysis.
Further, the addition amount of the active carbon in the step 4 is 2% of the total raw material mass, and the active carbon is stirred for 1h at the rotating speed of 300 rpm/min under the condition of 30 ℃ and is filtered by a microporous filter membrane of 0.45 mu m.
Further, 5L of 5g/L sodium sulfate solution is prepared in the step 5 and added into a polar water tank; preparing 1g/L sodium sulfate solution 5L and adding into a concentrated water tank; 5kg of material is added into a desalting solution tank, the voltage is 23V, the initial current is recorded, and after the current is 0 along with the time change, the desalting solution is discharged.
Further, in the step 6, the membrane passing frequency is controlled at 30HZ, the membrane feeding pressure is 2.0MPa, and the membrane discharging pressure is 1.5MPa.
Further, the concentrated solution in the step 7 is pre-frozen for 4 hours at the temperature of minus 60 ℃, and is dried for 20 hours in vacuum, the vacuum degree is less than or equal to 50Pa, and the residual moisture is less than or equal to 2 percent.
Compared with the prior art, the invention has the beneficial effects that:
according to the invention, the method of ultrafiltration membrane and nanofiltration membrane separation and purification and electrodialysis desalination and purification are adopted to purify and separate NAD+ reaction liquid, so that the method of resin column elution and recrystallization commonly used at present is replaced, the separation efficiency is improved, the acid washing and alkali washing steps are avoided, and the generation of a large amount of wastewater is reduced. The method can improve the purity and the yield of the NAD+ product, has low technical cost, can be repeatedly used, is suitable for large-scale industrial production, is environment-friendly, has simple and easy-to-operate process and lower production cost, and has remarkable industrial application value and environmental benefit.
Drawings
FIG. 1 is a flow chart of a preparation method of the invention;
FIG. 2 is a first peak diagram of the detection of NAD+ content and purity in the solution by high performance liquid chromatography according to example 1 of the present invention;
FIG. 3 is a second peak diagram of the detection of NAD+ content and purity in the solution by HPLC according to example 1 of the present invention;
FIG. 4 is a first peak diagram of the detection of NAD+ content and purity in the solution by high performance liquid chromatography in example 2 of the present invention;
FIG. 5 is a second peak diagram of the detection of NAD+ content and purity in the solution by high performance liquid chromatography according to example 2 of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The NAD+ reaction solutions treated in the following examples and comparative examples were: the NAD+ reaction solution is prepared by adopting a biological enzyme catalysis method and adopting NMN and ATP-2Na as substrates and adopting nicotinamide mononucleotide adenylate transferase catalysis and pyrophosphatase.
Example 1:
the purification process of the NAD+ reaction solution is as follows:
1. microfiltration:
performing microfiltration on NAD+ reaction liquid by using a ceramic membrane (10000 daltons), controlling the membrane filtration frequency at 50HZ, controlling the flow rate at 60L/h, controlling the membrane inlet pressure at about 0.32MPa, the membrane outlet pressure at about 0.28MPa, controlling the clear liquid flow meter to be less than 1L/min, controlling the clear liquid outflow speed at 210ml/min, and adding 0.4 times deionized water for dialysis to obtain microfiltration clear liquid and turbid liquid;
2. first ultrafiltration:
passing the micro-filtration liquid through an ultrafiltration membrane (10000 daltons), controlling the membrane passing frequency at 30HZ, the membrane feeding pressure at 3.0MPa, the membrane discharging pressure at about 2.0MPa, the clear liquid flow rate at 220ml/min, and then adding 0.4 times deionized water for dialysis to obtain ultrafiltration clear liquid and turbid liquid;
3. first ultrafiltration:
then passing the ultrafiltration clear liquid through an ultrafiltration membrane 2 (3000 daltons), controlling the membrane passing frequency at 30HZ, the membrane feeding pressure at 2.0MPa, the membrane outlet pressure at about 1.5MPa, the clear liquid flow rate at 230ml/min, and then adding 0.4 times deionized water for dialysis to obtain ultrafiltration clear liquid and turbid liquid;
4. decoloring:
decolorizing with 2% of active carbon, stirring at 400rpm in water bath at 30deg.C for 1 hr, and vacuum filtering to obtain decolorized solution with 95% light transmittance;
5. desalting:
(1) Preparing 5g/L sodium sulfate solution and adding 5L sodium sulfate solution into a polar water tank; preparing 1g/L sodium sulfate solution 5L and adding into a concentrated water tank; adding about 5kg of materials into a desalting solution tank;
(2) Starting a raw water pump, a concentrated water pump and a polar water pump; the pressure and the flow are regulated through a valve, the flow of the concentrated water tank is controlled to be slightly higher than 600L/h, the flow of the desalted liquid tank is controlled to be slightly lower than 600L/h, the valve of the polar water tank is slightly opened, and the pressure is extremely low;
(3) And (3) electrifying, wherein the regulated voltage is about 23V, the initial current is recorded, the current is 0 along with the time change, the electrifying is continued for 10 minutes, the electrifying is ended, the three pumps are closed, and the liquid in the desalting liquid tank is discharged.
6. Concentrating:
concentrating the desalted liquid subjected to electrodialysis treatment by using a 100 dalton nanofiltration organic membrane, controlling the membrane passing frequency at 30HZ, controlling the flow rate at 7L/min, and controlling the membrane feeding pressure at about 2.0MPa and the membrane discharging pressure at about 1.5 MPa; concentrating until the concentration of the turbid liquid NMN is about 15%, and discarding clear liquid;
7. and (3) freeze drying:
pre-freezing the concentrated solution at-60 ℃ for 4 hours, extracting and drying for 20 hours, wherein the vacuum degree is less than or equal to 50Pa, and the residual moisture is less than or equal to 2 percent, so that NAD+ freeze-dried powder is obtained, namely an NAD+ finished product.
The content and purity of the purified NAD+ product were measured by high performance liquid chromatography, and the yield was calculated, and the results are shown in FIG. 2-FIG. 3, table 1 and Table 2.
TABLE 1
TABLE 2
| Detecting items | Index (I) | Results |
| Appearance of | White-like powder | White powder |
| Purity of | ≥98.0％ | 98.8％ |
| Moisture content | ≤5％ | 1.76％ |
| Na+ | ＜1％ | ＜0.02％ |
Embodiment case 2:
1. microfiltration:
performing microfiltration on NAD+ reaction liquid by using a ceramic membrane (30000 daltons), controlling the membrane filtration frequency at 50HZ, controlling the flow rate at 60L/h, feeding the membrane at about 0.32MPa, discharging the membrane at about 0.28MPa, controlling the flow rate of clear liquid to be less than 1L/min, controlling the outflow speed of clear liquid to be 300ml/min, and adding deionized water with the concentration of 0.3 times for dialysis to obtain microfiltration clear liquid and turbid liquid;
2. first ultrafiltration:
passing the micro-filtration liquid through an ultrafiltration membrane (10000 daltons), controlling the membrane passing frequency at 30HZ, the membrane feeding pressure at 3.0MPa, the membrane discharging pressure at about 2.0MPa, the clear liquid flow rate at 180ml/min, and then adding 0.5 times deionized water for dialysis to obtain ultrafiltration clear liquid and turbid liquid;
3. second ultrafiltration:
then passing the ultrafiltration clear liquid through an ultrafiltration membrane (5000 daltons), controlling the membrane passing frequency at 30HZ, the membrane feeding pressure at 2.0MPa, the membrane outlet pressure at about 1.5MPa, the clear liquid flow rate at 320ml/min, and then adding 0.3 times deionized water for dialysis to obtain ultrafiltration clear liquid and turbid liquid;
4. decoloring:
decolorizing with 2% of active carbon, stirring at 400rpm in water bath at 30deg.C for 1 hr, and vacuum filtering to obtain decolorized solution with 95% light transmittance;
5. desalting:
(1) Preparing 5g/L sodium sulfate solution and adding 5L sodium sulfate solution into a polar water tank; preparing 1g/L sodium sulfate solution 5L and adding into a concentrated water tank; adding about 5kg of materials into a desalting solution tank;
(2) Starting a raw water pump, a concentrated water pump and a polar water pump; the pressure and the flow are regulated through a valve, the flow of the concentrated water tank is controlled to be slightly higher than 600L/h, the flow of the desalted liquid tank is controlled to be slightly lower than 600L/h, the valve of the polar water tank is slightly opened, and the pressure is extremely low;
(3) And (3) electrifying, wherein the regulated voltage is about 23V, the initial current is recorded, the current is 0 along with the time change, the electrifying is continued for 10 minutes, the electrifying is ended, the three pumps are closed, and the liquid in the desalting liquid tank is discharged.
6. Concentrating:
concentrating the desalted liquid subjected to electrodialysis treatment by using a 100 dalton nanofiltration organic membrane, controlling the membrane passing frequency at 30HZ, controlling the flow rate at 7L/min, and controlling the membrane feeding pressure at about 2.0MPa and the membrane discharging pressure at about 1.5 MPa; concentrating until the concentration of the turbid liquid NMN is about 15%, and discarding clear liquid;
7. and (3) freeze drying:
pre-freezing the concentrated solution at-60 ℃ for 4 hours, extracting and drying for 20 hours, wherein the vacuum degree is less than or equal to 50Pa, and the residual moisture is less than or equal to 2 percent, so that NAD+ freeze-dried powder is obtained, namely an NAD+ finished product.
The content and purity of the purified NAD+ product were measured by high performance liquid chromatography, and the yield was calculated, and the results are shown in FIGS. 4 to 5, tables 3 and 4.
TABLE 3 Table 3
TABLE 4 Table 4
| Detecting items | Index (I) | Results |
| Appearance of | White-like powder | White powder |
| Purity of | ≥98.0％ | 98.5％ |
| Moisture content | ≤5％ | 1.34％ |
| Na+ | ＜1％ | ＜0.02％ |
Comparative example 1
The NAD+ reaction solution prepared by adopting a biological enzyme catalysis method and adopting NMN and ATP-2Na as substrates and nicotinamide mononucleotide adenylate transferase catalysis and pyrophosphatase is separated by adopting a commonly used recrystallization method, and the content and purity of NAD+ in the solution are detected by high performance liquid chromatography and are shown in tables 5 and 6.
TABLE 5
TABLE 6
| Detecting items | Index (I) | Results |
| Appearance of | White-like powder | White powder |
| Purity of | ≥98.0％ | 95.1％ |
| Moisture content | ≤5％ | 1.53％ |
| Na+ | ＜1％ | ＜0.05％ |
As is clear from tables 1 to 6, in examples 1 and 2, the separation and purification of NAD+ reaction solution by ultrafiltration membrane and nanofiltration membrane separation and electrodialysis desalination purification of the present invention were performed to obtain NAD+ product with purity higher than 98% and yield higher than 80%, and in comparative example 1, the separation was performed by conventional recrystallization method with purity of 95.1% and yield higher than 71%, and thus it was found that the separation and purification of NAD+ reaction solution by ultrafiltration membrane and nanofiltration membrane separation and electrodialysis desalination purification of the present invention was performed to improve separation efficiency and purity and yield of NAD+ product.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should be covered by the protection scope of the present invention by making equivalents and modifications to the technical solution and the inventive concept thereof.
Claims (10)
1. The preparation method of the high-purity nicotinamide adenine dinucleotide is characterized by comprising the following steps of:
step 1: filtering the NAD+ reaction solution prepared by a biocatalysis method by a ceramic membrane to remove thalli, thereby obtaining a micro-filtration solution;
step 2: removing macromolecular substances from the micro-filtration liquid in the step 1 through an ultrafiltration organic membrane to obtain ultrafiltration clear liquid;
step 3: intercepting NAD+ from the ultrafiltered clear liquid in the step 2 through an ultrafiltered organic membrane to remove ATP and ADP, and performing dialysis water washing by using pure water to obtain ultrafiltered turbid liquid;
step 4: decolorizing the ultrafiltration turbid liquid in the step 3 by activated carbon to obtain decolorized liquid;
step 5: passing the decolorized solution obtained in the step 4 through an electrodialysis device to obtain desalted solution;
step 6: concentrating the desalted liquid obtained in the step 5 by using a nanofiltration organic membrane to obtain concentrated liquid with the concentration of 15-30 g/L;
step 7: and (3) freeze-drying the concentrated solution in the step (6) to obtain a purified NAD+ finished product.
2. The method for preparing high-purity nicotinamide adenine dinucleotide according to claim 1, wherein the NAD+ content in the NAD+ reaction solution is 30-100g/L.
3. The method for preparing high-purity nicotinamide adenine dinucleotide according to claim 1, wherein the specification of the microfiltration ceramic membrane is 10000-30000 daltons, the specification of the ultrafiltration organic membrane is 5000-10000 daltons, the specification of the ultrafiltration organic membrane is 3000-5000 daltons, and the specification of the nanofiltration organic membrane is 100-400 daltons.
4. The method for preparing high-purity nicotinamide adenine dinucleotide as claimed in claim 1, wherein the membrane passing frequency in the step 1 is controlled at 50HZ, the membrane inlet pressure is 0.32MPa, the membrane outlet pressure is 0.28MPa, and 0.2-0.6 times of pure water is adopted for dialysis.
5. The method for preparing high-purity nicotinamide adenine dinucleotide as claimed in claim 1, wherein the membrane passing frequency in the step 2 is controlled at 30HZ, the membrane inlet pressure is 2.0MPa, the membrane outlet pressure is 3MPa, and 0.2-0.6 times of pure water is adopted for dialysis.
6. The method for preparing high-purity nicotinamide adenine dinucleotide as claimed in claim 1, wherein the membrane passing frequency in the step 3 is controlled at 30HZ, the membrane inlet pressure is 2.0MPa, the membrane outlet pressure is 1.5MPa, and 0.2-0.6 times of pure water is adopted for dialysis.
7. The method for preparing high-purity nicotinamide adenine dinucleotide as claimed in claim 1, wherein the addition amount of the active carbon in the step 4 is 2% of the total raw material mass, and the active carbon is stirred for 1h at a rotating speed of 300 rpm/min under the condition of 30 ℃ and filtered by a microporous filter membrane of 0.45 μm.
8. The method for preparing high-purity nicotinamide adenine dinucleotide as claimed in claim 1, wherein 5g/L sodium sulfate solution 5L is prepared in the step 5 and added into the polar water tank; preparing 1g/L sodium sulfate solution 5L and adding into a concentrated water tank; 5kg of nicotinamide adenine dinucleotide solution was added to the desalting solution tank, the voltage was 23V, the initial current was recorded, and after the current was 0 over time, the desalting solution was discharged.
9. The method for preparing high-purity nicotinamide adenine dinucleotide according to claim 1, wherein the membrane passing frequency in the step 6 is controlled at 30HZ, the membrane inlet pressure is 2.0MPa, and the membrane outlet pressure is 1.5MPa.
10. The method for preparing high-purity nicotinamide adenine dinucleotide as claimed in claim 1, wherein the concentrated solution in the step 7 is pre-frozen at-60 ℃ for 4 hours, vacuum-dried for 20 hours, the vacuum degree is less than or equal to 50Pa, and the residual moisture is less than or equal to 2%.
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