CN116121322A - Synthesis method of beta-nicotinamide mononucleotide - Google Patents
Synthesis method of beta-nicotinamide mononucleotide Download PDFInfo
- Publication number
- CN116121322A CN116121322A CN202310031039.9A CN202310031039A CN116121322A CN 116121322 A CN116121322 A CN 116121322A CN 202310031039 A CN202310031039 A CN 202310031039A CN 116121322 A CN116121322 A CN 116121322A
- Authority
- CN
- China
- Prior art keywords
- beta
- nicotinamide
- nicotinamide mononucleotide
- adenine dinucleotide
- synthesizing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
-
- 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
- C07H19/048—Pyridine radicals
Abstract
The invention discloses a method for synthesizing beta-nicotinamide mononucleotide, which belongs to the technical field of biosynthesis and specifically comprises the following steps: s1, adding an aqueous phase buffer solution with the pH value of 7-8 into a reaction container, adding a substrate beta-nicotinamide adenine dinucleotide and a preparation containing beta-nicotinamide adenine dinucleotide pyrophosphatase, and uniformly mixing; s2, reacting for 40-50min at the temperature of 30-40 ℃ to obtain a reaction solution; s3, purifying the reaction liquid to obtain the beta-nicotinamide mononucleotide. The invention utilizes the enzyme engineering technology to produce the beta-nicotinamide mononucleotide, simplifies the production steps, obviously improves the production efficiency and reduces the production cost. According to the invention, the reaction liquid is subjected to column purification by using different types of resin columns, so that inorganic salt in the product can be effectively reduced, and the quality and purity of the product are improved.
Description
Technical Field
The invention belongs to the technical field of biosynthesis, and particularly relates to a method for synthesizing beta-nicotinamide mononucleotide.
Background
Beta-nicotinamide mononucleotide (beta-NMN or NMN) is the most direct precursor of NAD+ (nicotinamide adenine dinucleotide, also called co-nucleotide), which is an important active substance involved in thousands of important physiological reactions such as cell metabolism, redox, protein transcription and the like. Since NAD+ has an excessively large molecular weight, it cannot be taken into cells by oral administration, and it is mainly dependent on cell synthesis in vivo, and the amount of synthesis is low. However, as research on the NAD+ precursor micromolecular substance beta-NMN shows, eating the beta-NMN can effectively promote the content of NAD in the body to be increased, and obviously inhibit metabolism caused by aging, so that the beta-NMN has great development potential and market prospect in the aspect of functional health-care food.
The conventional method for preparing beta-NMN in vitro is a chemical synthesis method (chemCommun.1999 (8): 29-730), which has the disadvantages of complex operation, more reaction steps, more intermediates, low yield, difficult purification of the product, etc., resulting in low purity of the product, and the use of dangerous chemicals and a large amount of organic solvents when preparing beta-nicotinamide mononucleotide by the chemical synthesis method is required, which severely damages the environment.
Therefore, the biosynthesis method is environment-friendly, for example, chinese patent application No. 201811606780.9 discloses a method for preparing NMN by using nicotinamide riboside transferase, and the method synthesizes beta-nicotinamide mononucleotide by using nicotinamide as a substrate under the catalysis of nicotinamide riboside transferase. In this way, the use of relatively expensive nicotinamide riboside or nicotinamide, riboside phosphate, and ATP results in a still high production cost, limiting the industrial mass production of β -NMN.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for synthesizing beta-nicotinamide mononucleotide.
The aim of the invention can be achieved by the following technical scheme:
the synthesis method of the beta-nicotinamide mononucleotide specifically comprises the following steps:
s1, adding an aqueous phase buffer solution with the pH value of 7-8 into a reaction container, adding a substrate beta-nicotinamide adenine dinucleotide and a preparation containing beta-nicotinamide adenine dinucleotide pyrophosphatase, and uniformly mixing;
s2, reacting for 40-50min at the temperature of 30-40 ℃ to obtain a reaction solution;
s3, purifying the reaction liquid to obtain the beta-nicotinamide mononucleotide.
Further, the aqueous buffer solution contains MgCl 2 Tris-Cl buffer of (C) MgCl in the aqueous buffer 2 The concentration of (C) is 10-200mM, and even more preferably 50-100mM.
Further, the formulation comprises purified beta-nicotinamide adenine dinucleotide pyrophosphatase and/or recombinant cells comprising beta-nicotinamide adenine dinucleotide pyrophosphatase, which are microbial cells.
Further, the microbial cells are escherichia coli, saccharomyces cerevisiae or pichia pastoris.
Further, the purification treatment process comprises the following steps: the pH value of the reaction solution is regulated to 2.5-3.0 by hydrochloric acid, then the reaction solution flows through a separation column filled with strong-alkaline anion exchange resin, water is used for eluting, the pH value of the obtained effluent is regulated to 7.5-8.0 by sodium hydroxide aqueous solution, then the obtained effluent flows through the separation column filled with strong-acid cation exchange resin, water is used for eluting, the obtained effluent is subjected to nanofiltration, desalted beta-nicotinamide mononucleotide aqueous solution is obtained, and the beta-nicotinamide mononucleotide is obtained through crystallization and drying.
Further, the concentration of the hydrochloric acid is 2mol/L.
Further, the strongly basic anion exchange resin is D202 resin and the strongly acidic cation exchange resin is D001 resin.
Further, the concentration of the sodium hydroxide aqueous solution is 1mol/L.
The invention has the beneficial effects that:
the invention utilizes the enzyme engineering technology to produce the beta-nicotinamide mononucleotide, simplifies the production steps, obviously improves the production efficiency and reduces the production cost.
According to the invention, the reaction liquid is subjected to column purification by using different types of resin columns, so that inorganic salt in the product can be effectively reduced, and the quality and purity of the product are improved.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, 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.
Example 1
The synthesis method of the beta-nicotinamide mononucleotide specifically comprises the following steps:
s1, adding a water phase buffer solution with the pH value of 78 into a reaction container, adding a substrate beta-nicotinamide adenine dinucleotide and a preparation containing beta-nicotinamide adenine dinucleotide pyrophosphatase, and uniformly mixing;
s2, reacting for 40min at the temperature of 30 ℃ to obtain a reaction solution;
s3, purifying the reaction liquid to obtain the beta-nicotinamide mononucleotide.
The aqueous phase buffer solution contains MgCl 2 Tris-Cl buffer of (C) MgCl in the aqueous buffer 2 Is 100mM.
The preparation comprises purified beta-nicotinamide adenine dinucleotide pyrophosphatase and/or recombinant cells containing the beta-nicotinamide adenine dinucleotide pyrophosphatase, wherein the recombinant cells are microbial cells.
The microbial cells are escherichia coli, saccharomyces cerevisiae or pichia pastoris.
The purification treatment process comprises the following steps: the pH value of the reaction solution is regulated to 2.5 by hydrochloric acid, then the reaction solution flows through a separation column filled with strong-alkaline anion exchange resin, water is used for eluting, the obtained effluent liquid is regulated to 7.5 by sodium hydroxide aqueous solution, then the reaction solution flows through a separation column filled with strong-acid cation exchange resin, water is used for eluting, the obtained effluent liquid is subjected to nanofiltration, and desalted beta-nicotinamide mononucleotide aqueous solution is obtained, and then crystallization and drying are carried out, so that the beta-nicotinamide mononucleotide is obtained.
The concentration of the hydrochloric acid is 2mol/L.
The strong base anion exchange resin is D202 resin, and the strong acid cation exchange resin is D001 resin.
The concentration of the sodium hydroxide aqueous solution is 1mol/L.
Example 2
The synthesis method of the beta-nicotinamide mononucleotide specifically comprises the following steps:
s1, adding an aqueous phase buffer solution with the pH value of 7.5 into a reaction container, adding a substrate beta-nicotinamide adenine dinucleotide and a preparation containing beta-nicotinamide adenine dinucleotide pyrophosphatase, and uniformly mixing;
s2, reacting for 45min at 35 ℃ to obtain a reaction solution;
s3, purifying the reaction liquid to obtain the beta-nicotinamide mononucleotide.
The aqueous phase buffer solution contains MgCl 2 Tris-Cl buffer of (C) MgCl in the aqueous buffer 2 Is 100mM.
The preparation comprises purified beta-nicotinamide adenine dinucleotide pyrophosphatase and/or recombinant cells containing the beta-nicotinamide adenine dinucleotide pyrophosphatase, wherein the recombinant cells are microbial cells.
The microbial cells are escherichia coli, saccharomyces cerevisiae or pichia pastoris.
The purification treatment process comprises the following steps: the pH value of the reaction solution is regulated to 2.8 by hydrochloric acid, then the reaction solution flows through a separation column filled with strong-alkaline anion exchange resin, water is used for eluting, the obtained effluent liquid is regulated to 7.8 by sodium hydroxide aqueous solution, then the reaction solution flows through a separation column filled with strong-acid cation exchange resin, water is used for eluting, the obtained effluent liquid is subjected to nanofiltration, and desalted beta-nicotinamide mononucleotide aqueous solution is obtained, and then crystallization and drying are carried out, so that the beta-nicotinamide mononucleotide is obtained.
The concentration of the hydrochloric acid is 2mol/L.
The strong base anion exchange resin is D202 resin, and the strong acid cation exchange resin is D001 resin.
The concentration of the sodium hydroxide aqueous solution is 1mol/L.
Example 3
The synthesis method of the beta-nicotinamide mononucleotide specifically comprises the following steps:
s1, adding a water phase buffer solution with the pH value of 8 into a reaction container, adding a substrate beta-nicotinamide adenine dinucleotide and a preparation containing beta-nicotinamide adenine dinucleotide pyrophosphatase, and uniformly mixing;
s2, reacting for 50min at the temperature of 40 ℃ to obtain a reaction solution;
s3, purifying the reaction liquid to obtain the beta-nicotinamide mononucleotide.
The aqueous phase buffer solution contains MgCl 2 Tris-Cl buffer of (C) MgCl in the aqueous buffer 2 Is 200mM.
The preparation comprises purified beta-nicotinamide adenine dinucleotide pyrophosphatase and/or recombinant cells containing the beta-nicotinamide adenine dinucleotide pyrophosphatase, wherein the recombinant cells are microbial cells.
The microbial cells are escherichia coli, saccharomyces cerevisiae or pichia pastoris.
The purification treatment process comprises the following steps: the pH value of the reaction solution is adjusted to 3.0 by hydrochloric acid, then the reaction solution flows through a separation column filled with strong-alkaline anion exchange resin, water is used for eluting, the obtained effluent is adjusted to 8.0 by sodium hydroxide aqueous solution, then the reaction solution flows through a separation column filled with strong-acid cation exchange resin, water is used for eluting, the obtained effluent is subjected to nanofiltration, and desalted beta-nicotinamide mononucleotide aqueous solution is obtained, and then crystallization and drying are carried out, so that the beta-nicotinamide mononucleotide is obtained.
The concentration of the hydrochloric acid is 2mol/L.
The strong base anion exchange resin is D202 resin, and the strong acid cation exchange resin is D001 resin.
The concentration of the sodium hydroxide aqueous solution is 1mol/L.
The above detailed description describes the analysis method according to the present invention. It should be noted that the above description is only intended to help those skilled in the art to better understand the method and idea of the present invention, and is not intended to limit the related content. Those skilled in the art may make appropriate adjustments or modifications to the present invention without departing from the principle of the present invention, and such adjustments and modifications should also fall within the scope of the present invention.
Claims (8)
1. The synthesis method of the beta-nicotinamide mononucleotide is characterized by comprising the following steps of:
s1, adding an aqueous phase buffer solution with the pH value of 7-8 into a reaction container, adding a substrate beta-nicotinamide adenine dinucleotide and a preparation containing beta-nicotinamide adenine dinucleotide pyrophosphatase, and uniformly mixing;
s2, reacting for 40-50min at the temperature of 30-40 ℃ to obtain a reaction solution;
s3, purifying the reaction liquid to obtain the beta-nicotinamide mononucleotide.
2. The method for synthesizing beta-nicotinamide mononucleotide according to claim 1, wherein the aqueous buffer solution contains MgCl 2 Tris-Cl buffer of (C) MgCl in the aqueous buffer 2 The concentration of (2) is 10-200mM.
3. The method of claim 1, wherein the preparation comprises purified β -nicotinamide adenine dinucleotide pyrophosphatase and/or recombinant cells containing β -nicotinamide adenine dinucleotide pyrophosphatase, and wherein the recombinant cells are microbial cells.
4. A method of synthesizing a β -nicotinamide mononucleotide according to claim 3, wherein the microbial cell is escherichia coli, saccharomyces cerevisiae, or pichia pastoris.
5. The method for synthesizing beta-nicotinamide mononucleotide according to claim 1, wherein the purification treatment in step S3 comprises the following steps: the pH value of the reaction solution is regulated to 2.5-3.0 by hydrochloric acid, then the reaction solution flows through a separation column filled with strong-alkaline anion exchange resin, water is used for eluting, the pH value of the obtained effluent is regulated to 7.5-8.0 by sodium hydroxide aqueous solution, then the obtained effluent flows through the separation column filled with strong-acid cation exchange resin, water is used for eluting, the obtained effluent is subjected to nanofiltration, desalted beta-nicotinamide mononucleotide aqueous solution is obtained, and the beta-nicotinamide mononucleotide is obtained through crystallization and drying.
6. The method for synthesizing β -nicotinamide mononucleotide according to claim 5, wherein the concentration of hydrochloric acid is 2mol/L.
7. The method according to claim 5, wherein the strongly basic anion exchange resin is D202 resin and the strongly acidic cation exchange resin is D001 resin.
8. The method for synthesizing a β -nicotinamide mononucleotide according to claim 5, wherein the concentration of the aqueous sodium hydroxide solution is 1mol/L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310031039.9A CN116121322A (en) | 2023-01-10 | 2023-01-10 | Synthesis method of beta-nicotinamide mononucleotide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310031039.9A CN116121322A (en) | 2023-01-10 | 2023-01-10 | Synthesis method of beta-nicotinamide mononucleotide |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116121322A true CN116121322A (en) | 2023-05-16 |
Family
ID=86307617
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310031039.9A Pending CN116121322A (en) | 2023-01-10 | 2023-01-10 | Synthesis method of beta-nicotinamide mononucleotide |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116121322A (en) |
-
2023
- 2023-01-10 CN CN202310031039.9A patent/CN116121322A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104450806A (en) | Method for preparing sodium gluconate with enzymatic method | |
| WO2017133242A1 (en) | Method for extracting 1,5-pentanediamine from solution system containing 1,5-pentanediamine salt | |
| CN109369734A (en) | The method that chemical catalysis isomery glucose prepares industrial fructose | |
| CN111574358A (en) | Preparation process of potassium diformate | |
| CN116121322A (en) | Synthesis method of beta-nicotinamide mononucleotide | |
| CN111705096A (en) | Method for producing beta-nicotinamide mononucleotide by enzyme conversion method | |
| US20220363704A1 (en) | Method for preparing nicotinamide mononucleotide by using nicotinamide as raw material | |
| CN106591275A (en) | Method for preparing calcium gluconate through co-immobilization of glucose oxidase and catalase | |
| CN110698536A (en) | Novel method for producing glutathione by adopting fermentation method | |
| FI97393B (en) | Preparation of glyoxylic acid by enzymatic oxidation of glycolic acid | |
| WO2023103543A1 (en) | Method for preparing nuclease p1 | |
| CN111849959B (en) | Method for preparing cycloastragaloside by using co-immobilized double-enzyme catalytic astragaloside IV | |
| CN101130798A (en) | Method for preparing sodium L-aspartate by bio-enzyme | |
| CN110372606B (en) | Method for separating and purifying cytosine from microbial fermentation liquor | |
| CN111118079A (en) | Composite powder containing calcium gluconate and fructo-oligosaccharide and preparation method thereof | |
| CN114369129A (en) | Synthetic method of chlorinated nicotinamide ribose | |
| WO2014146242A1 (en) | Enzymatic preparation method for oxidized coenzyme ii | |
| CN106119307B (en) | Preparation method of alpha-ketoglutaric acid | |
| CN112608960A (en) | Preparation method of uridine diphosphate glucuronic acid | |
| CN105949250A (en) | Preparation method of alpha-2,3-sialyllactulose | |
| JPS62253388A (en) | Improved production of l-calnitine | |
| CN1188114A (en) | Preparation of gluconic acid by hydrogen peroxide oxidizing process | |
| CN110776535A (en) | Preparation method of disodium xanthate | |
| CN101104862B (en) | Method for synthesizing D-arylglycine by using heterogeneous enzyme to catalytically hydrolyzing 5-arylhydantoin | |
| CN116590360B (en) | Method suitable for enzymatic synthesis of sitagliptin |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |