CN115353537A - NMNH purification process - Google Patents
NMNH purification process Download PDFInfo
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- CN115353537A CN115353537A CN202210907683.3A CN202210907683A CN115353537A CN 115353537 A CN115353537 A CN 115353537A CN 202210907683 A CN202210907683 A CN 202210907683A CN 115353537 A CN115353537 A CN 115353537A
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- 238000000746 purification Methods 0.000 title claims abstract description 25
- XQHMUSRSLNRVGA-TURQNECASA-N NMNH Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(O)=O)O1 XQHMUSRSLNRVGA-TURQNECASA-N 0.000 title 1
- 238000000034 method Methods 0.000 claims abstract description 30
- 239000003480 eluent Substances 0.000 claims abstract description 18
- 230000008569 process Effects 0.000 claims abstract description 17
- 239000011347 resin Substances 0.000 claims abstract description 17
- 229920005989 resin Polymers 0.000 claims abstract description 17
- 239000000047 product Substances 0.000 claims abstract description 15
- 239000012535 impurity Substances 0.000 claims abstract description 11
- 150000001450 anions Chemical class 0.000 claims abstract description 7
- 239000012043 crude product Substances 0.000 claims abstract description 7
- 238000001035 drying Methods 0.000 claims abstract description 7
- 238000011068 loading method Methods 0.000 claims abstract description 7
- 238000001914 filtration Methods 0.000 claims abstract description 6
- 150000002500 ions Chemical class 0.000 claims abstract description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 60
- 239000012528 membrane Substances 0.000 claims description 40
- 230000008929 regeneration Effects 0.000 claims description 36
- 238000011069 regeneration method Methods 0.000 claims description 36
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 32
- 238000001471 micro-filtration Methods 0.000 claims description 27
- 238000000108 ultra-filtration Methods 0.000 claims description 27
- 239000007788 liquid Substances 0.000 claims description 19
- 238000001728 nano-filtration Methods 0.000 claims description 19
- 235000002639 sodium chloride Nutrition 0.000 claims description 19
- 239000011780 sodium chloride Substances 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 14
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 12
- 239000011148 porous material Substances 0.000 claims description 11
- 238000010828 elution Methods 0.000 claims description 10
- 125000000129 anionic group Chemical group 0.000 claims description 7
- 239000001103 potassium chloride Substances 0.000 claims description 7
- 235000011164 potassium chloride Nutrition 0.000 claims description 7
- WMFOQBRAJBCJND-UHFFFAOYSA-M Lithium hydroxide Chemical compound [Li+].[OH-] WMFOQBRAJBCJND-UHFFFAOYSA-M 0.000 claims description 6
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 6
- 235000019270 ammonium chloride Nutrition 0.000 claims description 6
- 239000001632 sodium acetate Substances 0.000 claims description 6
- 235000017281 sodium acetate Nutrition 0.000 claims description 6
- 239000003957 anion exchange resin Substances 0.000 claims description 5
- 230000001172 regenerating effect Effects 0.000 claims description 3
- 239000000945 filler Substances 0.000 claims description 2
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 2
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims 3
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 8
- 238000004519 manufacturing process Methods 0.000 abstract description 7
- 230000003321 amplification Effects 0.000 abstract description 3
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 3
- 238000009776 industrial production Methods 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 91
- 238000006243 chemical reaction Methods 0.000 description 32
- 102000004190 Enzymes Human genes 0.000 description 13
- 108090000790 Enzymes Proteins 0.000 description 13
- 239000000758 substrate Substances 0.000 description 12
- 238000004108 freeze drying Methods 0.000 description 11
- 238000005406 washing Methods 0.000 description 10
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 8
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 8
- 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 description 7
- 238000001514 detection method Methods 0.000 description 7
- 238000004128 high performance liquid chromatography Methods 0.000 description 7
- 238000005119 centrifugation Methods 0.000 description 6
- 229950006238 nadide Drugs 0.000 description 6
- 230000003197 catalytic effect Effects 0.000 description 5
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 4
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- 238000006911 enzymatic reaction Methods 0.000 description 4
- 235000019253 formic acid Nutrition 0.000 description 4
- 238000010438 heat treatment Methods 0.000 description 4
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 239000012295 chemical reaction liquid Substances 0.000 description 3
- 239000012510 hollow fiber Substances 0.000 description 3
- 239000008213 purified water Substances 0.000 description 3
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 108090000698 Formate Dehydrogenases Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 1
- 229930003537 Vitamin B3 Natural products 0.000 description 1
- FZAQROFXYZPAKI-UHFFFAOYSA-N anthracene-2-sulfonyl chloride Chemical compound C1=CC=CC2=CC3=CC(S(=O)(=O)Cl)=CC=C3C=C21 FZAQROFXYZPAKI-UHFFFAOYSA-N 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- -1 disodium salt Chemical class 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000012055 fruits and vegetables Nutrition 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000006241 metabolic reaction Methods 0.000 description 1
- 235000005152 nicotinamide Nutrition 0.000 description 1
- 239000011570 nicotinamide Substances 0.000 description 1
- 229960003966 nicotinamide Drugs 0.000 description 1
- 229940101270 nicotinamide adenine dinucleotide (nad) Drugs 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- BYTCDABWEGFPLT-UHFFFAOYSA-L potassium;sodium;dihydroxide Chemical compound [OH-].[OH-].[Na+].[K+] BYTCDABWEGFPLT-UHFFFAOYSA-L 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 150000003141 primary amines Chemical group 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 150000003512 tertiary amines Chemical group 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 150000003698 vitamin B derivatives Chemical class 0.000 description 1
- 239000011708 vitamin B3 Substances 0.000 description 1
- 235000019160 vitamin B3 Nutrition 0.000 description 1
Images
Classifications
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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
- C07H19/048—Pyridine radicals
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Separation Using Semi-Permeable Membranes (AREA)
Abstract
The invention discloses a NMNH purification process, which comprises the following steps: (1) filtering the NMNH crude product; (2) Loading the product filtered in the step (1) on an anion resin column, and eluting the ion column by using an impurity eluent and an NMNH (N-methyl-N-NH) eluting eluent in sequence after the loading is finished to obtain an eluent; (3) And (3) concentrating and drying the eluent obtained in the step (2) to obtain purified NMNH. The yield of the purification method can reach more than 75%, the purity of the purified NMNH can reach more than 98%, the rapid amplification in industrial production is ensured, and the market competitiveness is strong; the use of organic reagents is reduced, after the process is used, the obtained product has no organic reagent residue, large-scale equipment and organic reagents are not needed in the whole production process, the investment is low, heavy pollution is avoided, and the process is green and environment-friendly.
Description
Technical Field
The invention belongs to the field of chemical industry, and particularly relates to a purification process of high-purity NMNH.
Background
NMNH is an abbreviation for the reduction of nicotinamide adenine mononucleotide (β -nicotinamide mononucleotide, reduced form, disodium salt), a naturally occurring biologically active reducing nucleotide. Because niacinamide belongs to vitamin B3, NMN belongs to the category of vitamin B derivatives, and is widely involved in various biochemical reactions of human bodies and closely related to immunity and metabolism.
The substances inherent in the human body are also rich in some fruits and vegetables. In humans NMNH is the most direct precursor of NAD +, whose function is represented by NAD +. NAD + is also called coenzyme I, and is known as nicotinamide adenine dinucleotide, and is involved in thousands of reactions in every cell. In various cellular metabolic reactions, nicotinamide Adenine Dinucleotide (NAD) molecules play an important role and are an important support for maintaining the viability of cells.
At present, no enterprises produce NMNH in large batch in the market, and mainly some NMNH synthesized in laboratories is used for research and development; the synthesis quantity is small, the cost is high, and organic reagents and other production processes are used; the NMNH produced by the ionic resin has the following advantages:
1) The production is green and environment-friendly, and the edible and non-toxic inorganic salt is used; 2) The production cost is low, the used solvent is aqueous solution, the cost is low, and the solvent does not need to be recycled; 3) The production process is easy to amplify, and the process uses the ionic resin, is not influenced by equipment and process and is easy to quickly amplify.
In conclusion, the NMNH production process has the characteristics of environmental protection, simplicity, easy amplification and the like, and the produced NMNH has low cost and is beneficial to market competition.
Disclosure of Invention
The invention aims to provide a NMNH purification process.
The technical scheme adopted by the invention is as follows:
the application provides a NMNH separation and purification process, which comprises the following steps:
(1) Filtering the NMNH crude product;
(2) Loading the product filtered in the step (1) on an anion resin column, and eluting the ion column by using an impurity eluent and an eluent for eluting NMNH (N-methyl-N-NH) in sequence after the loading is finished to obtain an eluent;
(3) And (3) concentrating and drying the eluent obtained in the step (2) to obtain purified NMNH.
In some embodiments of the invention, the pH of the crude NMNH product is adjusted to 7.0-11.0 prior to step (1).
In some embodiments of the present invention, the filtration in step (1) is specifically: centrifugation, microfiltration or ultrafiltration.
In some embodiments of the invention, the centrifugation speed is 2000 to 4000r/min.
In some embodiments of the invention, the microfiltration membrane has a pore size of 0.2 to 0.45 μm.
In some embodiments of the invention, the ultrafiltration membrane has a pore size of 5 to 10kd.
In some embodiments of the invention, the ultrafiltration membrane is a pore size wound membrane or a hollow fiber membrane.
In some embodiments of the invention, the process further comprises a pretreatment step of the anion exchange resin: equilibrating 2.0-3.0BV of anionic resin with sodium hydroxide solution pH =8.5 ± 0.5.
In some embodiments of the present invention, the anionic resin effective functional group can be a primary amine functional group, a quaternary amine functional group, a tertiary amine functional group, and the like, and more preferably the effective functional group is a quaternary amine functional group.
In some embodiments of the invention, the mass ratio of the resin filler to NMNH is 20 to 35.
In some embodiments of the invention, the chromatography column has a diameter to height ratio of 1:6-10.
In some embodiments of the invention, the sample application rate is 0.8-1.5BV/h; after loading, the sample was rinsed with pure water for about 3BV.
In some embodiments of the invention, the impurity eluent is 0.01-0.1mol/L monovalent salt such as sodium chloride, potassium chloride, ammonium chloride, etc.; sodium chloride is preferred.
In some embodiments of the invention, the impurity eluent leaches 1.0-4.0BV; used for washing NMN and degrading impurities.
In some embodiments of the invention, the eluent for elution of NMNH is 0.1-0.5mol/L monovalent salt such as sodium acetate, ammonium chloride, sodium chloride, potassium chloride, etc., preferably sodium acetate.
In some embodiments of the invention, NMNH is eluted with an eluent that elutes NMNH, the eluate is collected, and fractions greater than or equal to 98% are qualified by detection.
In some embodiments of the invention, the process further comprises a step of regenerating the anion exchange resin, in particular, sequentially regenerating the anion exchange resin by using the regeneration liquid I and the regeneration liquid II.
In some embodiments of the invention, the regeneration liquid I is 0.5-3.0mol/L sodium hydroxide potassium hydroxide, lithium hydroxide solution, and the regeneration volume is 2.0-4.0BV.
In some embodiments of the invention, the regeneration liquid II is a 0.5-3.0mol/L solution of sodium chloride, ammonium chloride or potassium chloride, and the regeneration volume is 2.0-4.0BV.
In some embodiments of the invention, the regeneration process is performed by first rinsing with 0.5-3.0mol/L sodium hydroxide for 2.0-4.0BV, rinsing with pure water until the pH is less than or equal to 10.0, rinsing with 0.5-3.0mol/L sodium chloride solution for 2.0-4.0BV, and rinsing with pure water until the pH is less than or equal to 9.0.
In some embodiments of the invention, the concentration treatment comprises, in order, microfluidization, ultrafiltration and nanofiltration.
In some embodiments of the invention, the microfiltration membrane has a pore size of 0.2 to 0.45 μm; the aperture of the ultrafiltration membrane during ultrafiltration is 5-10kd, and the aperture of the nanofiltration membrane during nanofiltration is 100-300dal.
In some embodiments of the invention, the microfiltration membrane is a hollow fiber membrane; the ultrafiltration membrane is a pore-size roll-type membrane or a hollow fiber membrane.
In some embodiments of the invention, the concentration of NMNH after concentration is in the range of 100 to 250g/L, preferably 100 to 200g/L, which is advantageous for lyophilization.
In some embodiments of the invention, the drying process is vacuum freeze drying. The drying mode makes NMNH less prone to degradation during drying.
In some embodiments of the invention, nanofiltration concentration is followed by 3-6 washes for the removal of low molecular weight reagents during purification.
The invention has the beneficial effects that:
the method has the advantages that the anion resin is used for purifying the NMNH, the process is simple, the yield of the purification method can reach more than 75%, the purity of the purified NMNH can reach more than 98%, the rapid amplification of the NMNH in industrial production is guaranteed, and the market competitiveness is strong; and the application uses the ion exchange method of the anion tree, reduces the use of organic reagents, and after the process is used, the obtained product has no organic reagent residue, and the whole production process does not need large-scale equipment and organic reagents, so that the investment is low, the heavy pollution is avoided, and the environment is protected.
Drawings
Fig. 1 is a high performance liquid spectrum of NMN before the enzymatic reaction of NMNH in example 1.
FIG. 2 is a high performance liquid chromatogram after the NMNH enzymatic reaction in example 1.
FIG. 3 is a high performance liquid chromatography spectrum of the NMNH anion resin after purification in example 2.
Fig. 4 is a high performance liquid chromatogram of NMNH of example 2 after lyophilization.
Detailed Description
The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and other embodiments obtained by those skilled in the art without inventive efforts are within the protection scope of the present invention based on the embodiments of the present invention.
Example 1
The preparation method of the NMNH reaction solution (crude NMNH product) is as follows:
a substrate solution of 1.4kg of NMN, 0.4kg of formic acid and 1.0kg of Tris-HCl buffer was added to a 100L reactor, and the pH was adjusted to 7.0 to 8.0 with 1.0mol/L hydrochloric acid. Then adding catalytic enzyme, wherein the addition amounts are respectively as follows: 5.0ml/L (crude enzyme solution/substrate solution) of the supernatant crude enzyme solution of the mutant V54N/A121K/E228K is metered to 60L, the stirring speed is set to be 150rpm, the reaction temperature is controlled to be 30 ℃, and the pH value is kept to be 7.0-8.0. The pH value is controlled to be 7-8 by 6.0mol/L sodium hydroxide solution in the reaction process, a solution containing a crude product is obtained after 4 hours of reaction, and a substrate solution before the reaction and a reaction solution after the reaction are detected by HPLC (high performance liquid chromatography), wherein a high performance liquid spectrum of NMN before the enzymatic reaction is shown in figure 1, and a high performance liquid spectrum of NMNH after the enzymatic reaction is shown in figure 2.
The catalytic enzyme is formate dehydrogenase, which is disclosed in patent document CN114752572A, and other examples have the same theory.
The obtained NMNH reaction solution was used for purification experiments:
60L of NMNH reaction liquid, wherein the NMNH reaction liquid contains 1.2kg of NMNH, the purity is 93.5%, the pH value is =7.49, the pH value is adjusted to 9.15 by 6.0mol/L sodium hydroxide, 60L of clear liquid containing 1.19kgNMNH is obtained through centrifugation, and the rotation speed of a centrifuge is 3000r/min; the clarified NMNH solution was microfiltered through 1 microfiltration membrane having a pore size of 0.2um of 100mm 1100mm to obtain 80L of 1.19kg of post-microfiltration NMNH solution, and then the microfiltered solution was ultrafiltered through 1 membrane having a cut-off molecular weight of 5kd and a size of 100mm 1100mm to obtain 100L of solution containing 1.18kg of NMNH after ultrafiltration.
The purification column was 200mm 1500mm in diameter and packed with 35kg of anionic resin. 100L were first equilibrated with sodium hydroxide solution pH = 8.4. And (3) putting 160L of NMNH solution after ultrafiltration on a purification column, and controlling the speed to be 1.0BV/H in the whole process. After the NMNH solution is applied, 120L of impurities are washed by 0.05mol/L sodium chloride, and then washed by 0.2mol/L sodium acetate to obtain 130L of NMNH with qualified volume and 0.95kg, the purity is 99.3% after detection, and a high performance liquid chromatogram after anion resin purification is shown in figure 3; performing microfiltration with pore diameter of 0.2 μm, ultrafiltration with molecular weight cutoff of 5kd, and nanofiltration washing for 5 times, and concentrating to obtain 8L of concentrated solution, wherein the nanofiltration membrane is 2540 nanofiltration membrane with molecular weight cutoff of 100Dal, NMNH of the concentrated solution is 0.94kg, and purity is 99.2%; cooling the obtained concentrated solution to below-45 deg.C, and maintaining at below-45 deg.C for 2-4 hr; vacuumizing, and when the vacuum pressure is less than or equal to 50Pa, heating at the speed of 2 ℃/h until the temperature of the product is raised to 25 ℃, and keeping the constant temperature for 6-8 hours; completing freeze-drying until the change in vacuum pressure is less than or equal to 3.0pa within 5 min; after freeze-drying, 0.93kg of NMNH with a purity of 99.0% and a yield of 77.5% was obtained, and the HPLC chromatogram after freeze-drying is shown in FIG. 4.
And (3) eluting the regeneration of the purification column by using 0.8mol/L sodium hydroxide solution for 4.0BV, eluting the regeneration column by using pure water until the pH is =9.8 after elution, centrifuging the regeneration column by using 1.0mol/L sodium chloride solution for 3.0BV, eluting the regeneration column by using pure water until the pH of the flow-through liquid is =9.0 after elution, and reserving the regeneration column for later use.
Example 2
The preparation method of the NMNH reaction solution (crude NMNH product) is as follows:
500L of purified water was added to a 1000L reaction vessel, and 16kg of NMN, 4.6kg of formic acid, and 11.4kg of a substrate solution of Tris-HCl buffer were sequentially added thereto, and the pH was adjusted to 7.0 to 8.0 with 1.0mol/L of hydrochloric acid. Then adding catalytic enzyme, wherein the addition amounts are respectively as follows: 5.0ml/L (crude enzyme solution/substrate solution) of the crude enzyme solution of the supernatant of the mutant V54N/A121K/E228K is metered to 600L, the stirring speed is set to be 150rpm, the reaction temperature is controlled to be 30 ℃, and the pH value is kept to be 7.0-8.0. In the reaction process, 6.0mol/L sodium hydroxide solution is used for controlling the pH value to be 7-8, after 4 hours of reaction, solution containing crude products is obtained, and the substrate solution before the reaction and the reaction solution after the reaction are 14.3kg through HPLC detection. The purity is 93.5%, the pH =7.65, the pH is adjusted to 9.35 by 6.0mol/L sodium hydroxide, 600L clear solution containing 14.1kgNMNH is obtained by centrifugation, and the rotation speed of a centrifuge is 3000r/min; and (3) microfiltration of the clarified NMNH solution by using 4-branch microfiltration membranes, wherein the pore diameter of each microfiltration membrane is 0.2um, and the size of each microfiltration membrane is 100mm 1100mm, thus obtaining 700L of post-microfiltration NMNH solution 14.1kg, and ultrafiltration of the solution after microfiltration by using 4 membranes, wherein the cut-off molecular weight of each ultrafiltration membrane is 5kd, and the size of each ultrafiltration membrane is 100mm 1100mm, and 900L of solution containing 14.0kg of NMNH is obtained after ultrafiltration.
The purification column diameter x high is 500mm 2500mm, packed with anionic resin 400kg. First equilibrate 2.0BV with sodium hydroxide solution pH = 8.6. Putting 1500L NMNH solution after ultrafiltration on a purification column, and controlling the speed to be 1.5BV/H in the whole process. After the NMNH solution is applied, washing impurities by 0.05mol/L sodium chloride for 2.5BV, and then washing by 0.2mol/L sodium acetate to obtain 1800L of NMNH with 12.3kg and purity of 99.0 percent by detection; carrying out microfiltration with the aperture of 0.2 mu m, ultrafiltration with the molecular weight cutoff of 5kd, nanofiltration washing for 5 times, and concentrating to obtain 72L of concentrated solution, wherein the nanofiltration membrane is an 8040 nanofiltration membrane with the molecular weight cutoff of 100Dal, the NMNH content of the concentrated solution is 12.1kg, and the purity is 99.0%; cooling the obtained concentrated solution to below-45 deg.C, and maintaining at below-45 deg.C for 2-4 hr; vacuumizing, when the vacuum pressure is less than or equal to 50Pa, heating at the speed of 2 ℃/h until the temperature of the product is raised to 25 ℃, and keeping the constant temperature for 6-8 hours; completing freeze-drying until the change in vacuum pressure is less than or equal to 3.0pa within 5 min; 11.9kg of NMNH with a purity of 99.0% and a yield of 83.2% was obtained by freeze-drying.
And (3) eluting the regeneration of the purification column by using 1.5mol/L sodium hydroxide solution for 3.0BV, eluting the regeneration column by using pure water until the pH is =9.5 after elution, centrifuging the regeneration column by using 1.0mol/L sodium chloride solution for 2.0BV, eluting the regeneration column by using pure water until the pH of the flow-through liquid is =9.0 after elution, and reserving the regeneration column for later use.
Example 3
The preparation method of the NMNH reaction solution (crude NMNH product) is as follows:
500L of purified water was added to a 1000L reaction vessel, and 16kg of NMN, 4.6kg of formic acid, and 11.4kg of a substrate solution of Tris-HCl buffer were sequentially added thereto, and the pH was adjusted to 7.0 to 8.0 with 1.0mol/L of hydrochloric acid. Then adding catalytic enzyme, wherein the addition amounts are respectively as follows: 5.0ml/L (crude enzyme solution/substrate solution) of the supernatant crude enzyme solution of the mutant V54N/A121K/E228K is metered to 600L, the stirring speed is set to be 150rpm, the reaction temperature is controlled to be 30 ℃, and the pH value is kept to be 7.0-8.0. In the reaction process, 6.0mol/L sodium hydroxide solution is used for controlling the pH value to be 7-8, after 4 hours of reaction, solution containing crude products is obtained, 14.5kg of substrate solution before reaction and reaction liquid after reaction are detected through HPLC, the purity is 94.5 percent, the pH value is =7.45, the pH value is adjusted to 9.35 by 6.0mol/L sodium hydroxide, 600L clear solution containing 14.4kgNMNH is obtained through centrifugation, and the rotating speed of a centrifuge is 3000r/min; and (3) microfiltration of the clarified NMNH solution by using 4-branch microfiltration membranes, wherein the pore diameter of each microfiltration membrane is 0.2um, and the size of each microfiltration membrane is 100mm 1100mm, thus obtaining 700L of post-microfiltration NMNH solution 14.3kg, and ultrafiltration of the solution after microfiltration by using 4 membranes, wherein the cut-off molecular weight of each ultrafiltration membrane is 5kd, and the size of each ultrafiltration membrane is 100mm 1100mm, and 900L of solution containing 14.3kg of NMNH is obtained after ultrafiltration.
The purification column diameter x high is 500mm 2500mm, packed with anionic resin 400kg. 1000L were first equilibrated with sodium hydroxide solution pH = 8.3. And (3) putting 1500L of NMNH solution after ultrafiltration on a purification column, and controlling the speed to be 500L/h in the whole process. After the NMNH solution is added, 1500L of impurities are washed by 0.05mol/L sodium chloride, and then 0.3mol/L sodium acetate is used for washing to obtain 2000L of NMNH with qualified volume and 12.6kg, and the purity is 99.6% through detection; carrying out microfiltration with the aperture of 0.2 mu m, ultrafiltration with the molecular weight cutoff of 5kd, nanofiltration washing for 5 times, and concentrating to obtain 69L of concentrated solution, wherein the nanofiltration membrane is an 8040 nanofiltration membrane with the molecular weight cutoff of 100Dal, the NMNH content of the concentrated solution is 12.5kg, and the purity is 99.0%; cooling the obtained concentrated solution to below-45 deg.C, and maintaining at below-45 deg.C for 2-4 hr; vacuumizing, when the vacuum pressure is less than or equal to 50Pa, heating at the speed of 2 ℃/h until the temperature of the product is raised to 25 ℃, and keeping the constant temperature for 6-8 hours; completing freeze-drying until the change in vacuum pressure is less than or equal to 3.0pa within 5 min; 12.3kg of NMNH with a purity of 99.0% and a yield of 84.8% was obtained after freeze-drying.
And (3) eluting the regeneration of the purification column by using 1.5mol/L sodium hydroxide solution for 3.0BV, eluting the regeneration column by using pure water until the pH is =9.5 after elution, centrifuging the regeneration column by using 1.0mol/L sodium chloride solution for 2.0BV, eluting the regeneration column by using pure water until the pH of the flow-through liquid is =9.0 after elution, and reserving the regeneration column for later use.
Example 4
The preparation method of the NMNH reaction solution (crude NMNH product) is as follows:
500L of purified water was added to a 1000L reaction vessel, and 16kg of NMN, 4.6kg of formic acid, and 11.4kg of a substrate solution of Tris-HCl buffer were sequentially added thereto, and the pH was adjusted to 7.0 to 8.0 with 1.0mol/L of hydrochloric acid. Then adding catalytic enzyme, wherein the addition amounts are respectively as follows: 5.0ml/L (crude enzyme solution/substrate solution) of the crude enzyme solution of the supernatant of the mutant V54N/A121K/E228K is metered to 600L, the stirring speed is set to be 150rpm, the reaction temperature is controlled to be 30 ℃, and the pH value is kept to be 7.0-8.0. In the reaction process, 6.0mol/L sodium hydroxide solution is used for controlling the pH value to be 7-8, after 4 hours of reaction, solution containing crude products is obtained, and the substrate solution before the reaction and the reaction solution after the reaction are 14.1kg through HPLC detection. The purity is 90.3 percent, the pH is =7.75, the pH is adjusted to 9.45 by 6.0mol/L sodium hydroxide, 1150L clear liquid containing 14.0kgNMNH is obtained by centrifugation, and the rotation speed of a centrifuge is 3000r/min; the clarified NMNH solution was microfiltered through 4 microfiltration membranes with pore size of 0.2um of 100mm 1100mm to obtain 700L of post-microfiltration NMNH solution 14.0kg, and then the microfiltered solution was ultrafiltered through 4 membranes with cut-off molecular weight of 5kd and size of 100mm 1100mm to obtain 900L of solution containing 14.0kg of NMNH.
The purification column diameter x high is 500mm 2500mm, packed with anionic resin 400kg. 1000L were first equilibrated with sodium hydroxide solution pH = 8.6. And (3) putting 1500L of NMNH solution after ultrafiltration on a purification column, and controlling the speed to be 2.5BV/H in the whole process. After the NMNH solution is coated, washing 1100L of impurities by using 0.05mol/L potassium chloride, and then washing by using 0.4mol/L sodium chloride to obtain 1600L of NMNH with the qualified volume of 11.5kg, wherein the purity is 98.6% by detection; performing microfiltration with pore diameter of 0.2 μm, ultrafiltration with molecular weight cutoff of 5kd, and nanofiltration washing for 5 times, and concentrating to obtain 65L concentrated solution, wherein the nanofiltration membrane is 8040 nanofiltration membrane with molecular weight cutoff of 100Da, NMNH of the concentrated solution is 11.5kg, and the purity is 98.6%; cooling the obtained concentrated solution to below-45 deg.C, and maintaining at below-45 deg.C for 2-4 hr; vacuumizing, when the vacuum pressure is less than or equal to 50Pa, heating at the speed of 2 ℃/h until the temperature of the product is raised to 25 ℃, and keeping the constant temperature for 6-8 hours; completing freeze-drying until the change in vacuum pressure is less than or equal to 3.0pa within 5 min; 11.3kg of NMNH with a purity of 98.5% and a yield of 80.1% was obtained by freeze-drying.
And (3) eluting the regeneration of the purification column for 2.0BV by using 1.5mol/L sodium hydroxide solution, eluting the regeneration column for 2.0BV by using pure water until the pH value is =9.5 after elution is finished, centrifuging the regeneration column for 2.0BV by using 1.0mol/L sodium chloride solution, eluting the regeneration column for 2.0BV by using pure water until the pH value of the flow-through solution is =9.0 after elution is finished, and reserving the regeneration column for later use.
The present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.
Claims (10)
1. A process for purification of NMNH, comprising the steps of:
(1) Filtering the NMNH crude product;
(2) Loading the product filtered in the step (1) on an anion resin column, and eluting the ion column by using an impurity eluent and an NMNH (N-methyl-N-NH) eluting eluent in sequence after the loading is finished to obtain an eluent;
(3) And (3) concentrating and drying the eluent obtained in the step (2) to obtain purified NMNH.
2. The method as claimed in claim 1, wherein the crude NMNH product is adjusted to a pH of 7.0-11.0 prior to filtration in step (1).
3. The method of claim 1, wherein the process further comprises a step of pretreating the anion exchange resin by: the anionic resin was equilibrated with sodium hydroxide solution at pH 8.5. + -. 0.5.
4. The method according to claim 1, characterized in that the mass ratio of the resin filler and NMNH is 10-15.
5. The method according to claim 1, wherein the sample application rate is 0.8-1.5BV/h.
6. The method of claim 1, wherein the impurity eluent is: 0.01-0.1mol/L of sodium chloride, potassium chloride and ammonium chloride.
7. The method according to claim 1, wherein the eluent for elution of NMNH is 0.1-0.5mol/L sodium acetate, ammonium chloride, sodium chloride, potassium chloride.
8. The method according to claim 1, wherein the process further comprises the step of regenerating the anion exchange resin, wherein a regeneration liquid I and a regeneration liquid II are sequentially used in the regeneration process, and after the regeneration, the regeneration is performed by rinsing with pure water until the pH value is less than or equal to 9.0; preferably, the regeneration liquid I is 0.5-3.0mol/L sodium hydroxide, potassium hydroxide or lithium hydroxide solution, and the regeneration volume is 2.0-4.0BV; more preferably, the regeneration liquid II is 0.5-3.0mol/L sodium chloride, ammonium chloride or potassium chloride solution, and the regeneration volume is 2.0-4.0BV.
9. The method according to claim 1, wherein the concentration is a filtration concentration, in particular comprising microfiltration, ultrafiltration and nanofiltration; preferably, the pore diameter of the microfiltration membrane used for microfiltration is 0.2-0.45 μm; the aperture of the ultrafiltration membrane used for ultrafiltration is 5-10kd, and the aperture of the nanofiltration membrane used for nanofiltration is 100-300dal.
10. The method of claim 1, wherein the drying process is vacuum freeze drying.
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| CN113490676A (en) * | 2019-11-19 | 2021-10-08 | 清华大学 | Synthesis method of NMN derivative and medical application of NMN and derivative thereof |
| WO2021214299A1 (en) * | 2020-04-24 | 2021-10-28 | Nuvamid Sa | Nicotinamide mononucleotide and nicotinamide riboside derivatives and use thereof in the treatment of viral infections and respiratory complications, in particular caused by influenzavirus or coronavirus |
| CN114660187A (en) * | 2020-12-23 | 2022-06-24 | 安徽古特生物科技有限公司 | Preparation method of beta-nicotinamide mononucleotide |
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| CN108431015A (en) * | 2016-12-14 | 2018-08-21 | 邦泰生物工程(深圳)有限公司 | A kind of purifying process of NADPH |
| CN113490676A (en) * | 2019-11-19 | 2021-10-08 | 清华大学 | Synthesis method of NMN derivative and medical application of NMN and derivative thereof |
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