Improved method for separating and purifying β-nicotinamide mononucleotide by macroporous adsorption resin
By adsorbing NMN near its isoelectric point using macroporous adsorption resin and eluting it with an alkaline solution, the problem of impurity removal in enzyme catalysis was solved, enabling the preparation and low-cost production of high-purity NMN.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SUZHOU LEAD BIOTECH CO LTD
- Filing Date
- 2023-12-21
- Publication Date
- 2026-07-24
AI Technical Summary
In the existing technology for preparing NMN by enzyme catalysis, there are complex impurities, especially nicotinic acid mononucleotide, which has similar properties to the product and is difficult to remove completely, resulting in low purification efficiency, high cost, and the possibility that organic solvent elution may lead to the decomposition of NMN.
NMN is adsorbed near its isoelectric point using macroporous adsorption resin, and then eluted by changing the ionization state of NMN with an alkaline solution, avoiding the use of organic solvents. Inorganic salts are recovered by nanofiltration concentration, thus reducing costs.
It has achieved the preparation of high-purity NMN with good impurity removal, high yield, avoidance of NMN decomposition, reduced production costs, and good environmental performance.
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Figure CN117720601B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biological product separation and purification, specifically relating to a method for the separation and purification of β-nicotinamide mononucleotide. Background Technology
[0002] β-Nicotinamide mononucleotide (NMN) is nicotinamide adenine dinucleotide (NAD). + As a precursor to NMN, recent studies have shown that by regulating the level of NMN in organisms, it can effectively prevent and treat various diseases caused by aging. It also has good repair and medical care effects on the treatment of age-related diseases such as Parkinson's disease, the regulation of insulin secretion, and the prevention and treatment of metabolic diseases such as diabetes and obesity.
[0003] Currently, large-scale production of NMN is mainly achieved through enzymatic reactions. Compared to chemical synthesis, enzymatic catalysis is more efficient, greener, and more environmentally friendly. However, the impurities in the enzyme-catalyzed reaction solution are often quite complex, containing a large amount of salts and divalent metal ions, as well as some by-reaction products, enzyme-catalyzed proteins, sugars, polyphosphate ions, and other impurities. Meanwhile, the market demands high purity NMN, and currently, the main methods for separating and purifying NMN are ion exchange and reverse chromatography.
[0004] Patent CN111424064A discloses a method for preparing high-purity NMN based on enzymatic reverse chromatography. The reaction solution is separated by gradient elution in reverse liquid chromatography, then concentrated by nanofiltration and crystallized to obtain a high-purity NMN product. However, reverse chromatography packing material is expensive, resulting in high costs.
[0005] Patent CN108026132A discloses a method for purifying NMN using an ion exchange method based on biocatalysis. This method involves treating the crude reaction solution prepared by biocatalysis with anion exchange resin, concentrating it by nanofiltration, then treating it with a chelating resin, concentrating it by nanofiltration, and finally freeze-drying it to obtain pure NMN. However, the ion exchange resin has limited effectiveness in removing impurities from the reaction solution. The large amount of salt in the system results in small sample loading, low purification efficiency, high regeneration water consumption, and increased waste.
[0006] Some side reactions occur during the enzyme reaction. Among them, the nicotinic acid mononucleotide produced by the decomposition of NMN has similar properties to the product. Furthermore, the process of adsorbing and eluting the product with ion exchange resin will lead to further decomposition of NMN, increasing the nicotinic acid mononucleotide impurities. These impurities are difficult to remove through crystallization, thus affecting the quality of the final product.
[0007] Therefore, it is necessary to develop a new separation and purification method to effectively separate impurities in NMN prepared by enzymatic methods. Summary of the Invention
[0008] This invention addresses the shortcomings of existing technologies by providing a method for separating and purifying β-nicotinamide mononucleotide using macroporous adsorption resin, thus solving the problems of high salt content, complex impurities, difficulty in removal, and low purification efficiency in enzyme reaction systems.
[0009] The crude NMN solution prepared by enzymatic reaction has a complex composition, high salt content, and contains a large amount of phosphates and polyphosphate ions, as well as some by-reaction products, such as nicotinic acid mononucleotide, nicotinamide ribose (NR), 5-phosphate ribose, nicotinic acid, and unreacted raw materials such as nicotinamide and ribose. Nicotinic acid mononucleotide has a structure similar to the product and is difficult to remove completely using conventional methods such as ion exchange resins. Studies of NMN properties have revealed that NMN is an amphoteric molecule, existing as an inner salt at its isoelectric point and as an anion and cation at higher and lower isoelectric points, respectively. This invention utilizes this characteristic by selecting a suitable pH near the isoelectric point of nicotinamide mononucleotide (3.7) to allow NMN to exist in its inner salt form and adsorb onto the macroporous adsorption resin, while nicotinic acid mononucleotide remains in its anion form at this pH and is not adsorbed by the resin. Because NMN is an amphoteric molecule, the phosphate groups in its molecular structure have a certain pH buffer zone; the pH range of 3.5-4.8 remains within the isoelectric point buffer zone. A simple water wash can completely remove impurities such as phosphates, polyphosphate ions, nicotinic acid mononucleotides, and nicotinamide ribose from the system. After desorption, a high-purity NMN product can be obtained.
[0010] Conventional desorption methods use organic solvents such as methanol and ethanol. We found that the mixture of alcohol and water during desorption is significantly exothermic, and since NMN is a heat-sensitive substance, this may cause NMN decomposition during elution. Furthermore, the elution process generates a large amount of low-concentration ethanol solution, and ethanol recovery is energy-intensive and costly. This invention uses an alkaline solution instead of conventional organic solvents to elute the product. By forming a salt with NMN, the ionization state of NMN is altered, allowing the product to desorb from the resin. The entire process is essentially non-exothermic and avoids the use of organic solvents. The desorption solution is pH-adjusted and then concentrated by nanofiltration. The inorganic salts in the filtrate can be recovered as a byproduct, further reducing costs. This invention is simple in procedure, low in cost, and provides excellent impurity separation, significantly improving product purity.
[0011] Based on this, the present invention provides a method for the separation and purification of β-nicotinamide mononucleotide, characterized in that: Step 1: the pH value of the enzyme-catalyzed reaction solution containing β-nicotinamide mononucleotide is adjusted to 3.5-4.8 to obtain the purification solution;
[0012] Step 2: Pass the solution to be purified through a separation column packed with macroporous adsorption resin, wash off salts and impurities with pure water, then elute the product with an alkaline solution as the eluent, collect the eluent, and adjust the pH value to 3.5-4.0;
[0013] Step 3: Purify the eluent to obtain the purified β-nicotinamide mononucleotide product.
[0014] In one embodiment, the enzyme-catalyzed reaction solution containing β-nicotinamide mononucleotide in step one is a reaction solution obtained by synthesizing β-nicotinamide mononucleotide under the joint catalysis of D-ribose, nicotinamide, adenine nucleoside triphosphate ATP, and polyphosphate as raw materials and phosphoribosyl pyrophosphate synthase, ribokinase, and nicotinamide phosphoribosyltransferase.
[0015] In one embodiment, step one is as follows: add an adsorbent to the enzyme-catalyzed reaction solution containing β-nicotinamide mononucleotide, stir, filter, and then adjust the pH of the resulting filtrate to 3.5-4.8 to obtain the purified solution; or adjust the pH of the enzyme-catalyzed reaction solution containing β-nicotinamide mononucleotide to 3.5-4.8, add an adsorbent to the reaction solution, stir, filter, and obtain the purified solution.
[0016] In one implementation, step one is as follows: the pH of the enzyme-catalyzed reaction solution containing β-nicotinamide mononucleotide is adjusted to 2.5-3.0, an adsorbent is added and stirred, then filtered, and the pH of the resulting filtrate is adjusted to 3.5-4.8 to obtain the purified solution.
[0017] Adjusting the pH of the reaction solution to 2.5 to 3.0 beforehand allows the protein to denature and precipitate under acidic conditions, improving the impurity removal effect.
[0018] In one implementation, hydrochloric acid and / or NaOH are used to adjust the pH value in steps one and two.
[0019] In one embodiment, the adsorbent in step one is selected from activated carbon.
[0020] In one implementation, the amount of pure water used in step two is 2-4 times the column volume.
[0021] In one embodiment, the eluent used in step two is selected from ammonia, sodium hydroxide, and sodium acetate.
[0022] In one embodiment, the product is eluted using an alkaline solution of 0.01-1 mol / L in step two;
[0023] In one implementation, step three, the purification of the eluent, includes nanofiltration and crystallization.
[0024] In one implementation, in step three, the crystallization solvent is ethanol.
[0025] In one implementation, the method further includes step four: collecting and regenerating the eluent-containing solution separated during the purification process in step three.
[0026] In one implementation, the recycling and regeneration method in step four is reverse osmosis and / or electrodialysis.
[0027] Compared with existing technologies, this invention provides a purification process for β-nicotinamide mononucleotide synthesized by enzyme catalysis. By adjusting the pH of the solution, the ionization states of the product and impurities differ. The product is adsorbed by macroporous adsorption resin, and impurities and inorganic salts not adsorbed by the resin are washed off with water. The product is then eluted with an alkaline solution, yielding a purified NMN solution in high yield. This invention uses alkaline solution instead of conventional organic solvents for product elution. The ionization state of NMN is altered by the salt formation between the alkali and NMN, causing the product to desorb from the resin. The entire process is essentially non-exothermic and avoids the use of organic solvents. The desorbate is pH adjusted and then concentrated by nanofiltration. The inorganic salts in the filtrate can be recovered as a byproduct, further reducing costs. This invention has simple steps, low cost, and good impurity separation effect, significantly improving product purity. The adsorption and elution conditions of this invention are mild, avoiding the risk of product decomposition caused by strong acids during ion exchange resin elution, ensuring product purification effect. The simple steps and good impurity removal effect allow nicotinamide mononucleotide impurities to be controlled below 0.1%. The β-nicotinamide mononucleotide obtained by this invention has a purity of ≥99.8% and a yield of over 80%. Attached Figure Description
[0028] Figure 1 : Liquid phase spectrum of the crude β-nicotinamide mononucleotide reaction solution of this invention;
[0029] Figure 2 Liquid chromatography spectrum of the pure β-nicotinamide mononucleotide of Example 2 of this invention;
[0030] Figure 3 Liquid chromatography spectrum of the pure β-nicotinamide mononucleotide of Example 4 of this invention;
[0031] Figure 4 Liquid chromatography spectrum of the pure β-nicotinamide mononucleotide of Example 5 of this invention; Detailed Implementation
[0032] The present invention can be implemented through the following specific embodiments, but the present invention is not limited to the following embodiments.
[0033] Example 1:
[0034] Preparation of β-nicotinamide mononucleotide by enzymatic reaction
[0035] In a 30L reaction flask, 100g D-ribose, 97.4g nicotinamide, 3.66g ATP, 447.55g sodium hexametaphosphate, and 203g MgCl2·6H2O were added sequentially. 5L of 0.1M pH 9.0 TrisHCl buffer was added, and the mixture was stirred to dissolve. Then, 1% ribokinase, 1% pyrophosphate hydrolase, 3% phosphoribosyl pyrophosphate synthase, 1% adenosine kinase, 1% polyphosphate kinase, and 5% nicotinamide phosphoribosyltransferase were added. Finally, the volume was adjusted to 10L with pure water, and the reaction was carried out overnight at 35℃ with stirring. The pH was controlled at 8-8.5 using NaOH. The final molar yield was 92%. The HPLC chromatogram is shown below. Figure 1 As shown.
[0036] Example 2:
[0037] Macroporous adsorption resin separation and purification of β-nicotinamide mononucleotide, ammonia hydrolysis adsorption
[0038] An enzyme-catalyzed reaction produced a crude solution of β-nicotinamide mononucleotide (NMN) with a concentration of 18.8 g / L and nicotinic acid mononucleotide (NMN) of 0.486%. 5 L of the crude solution was taken, and the pH was adjusted to 2.5 with hydrochloric acid. Activated carbon was added for adsorption, and the solution was filtered. The filtrate was adjusted to pH 4.2 with sodium hydroxide and loaded onto a macroporous adsorption resin column. The concentration of the product in the effluent was measured. After loading, the resin column was eluted with 2 BV of pure water, and the conductivity of the effluent was monitored. The column was eluted with 0.2 mol / L ammonia solution, and the product eluent was collected. The temperature of the effluent was measured to be 20-23 °C. The concentration of the product in the effluent was monitored, and collection was stopped when the concentration dropped below 1 g / L.
[0039] The eluent was adjusted to pH 3.5-4.0 with hydrochloric acid, concentrated to 150 g / L by nanofiltration, and 2 L of ethanol was added. A large amount of solid precipitated out. The solid was filtered and dried under vacuum at 30 °C to obtain 87.5 g of white solid, with a yield of 93.1% and a purity of 99.93%, containing 0.024% nicotinic acid mononucleotide.
[0040] The nanofiltration filtrate was concentrated using a reverse osmosis membrane, followed by electrodialysis to obtain ammonia and hydrochloric acid. The HPLC chromatogram is shown below. Figure 2 As shown.
[0041] Example 3:
[0042] Macroporous adsorption resin separation and purification of β-nicotinamide mononucleotide, ammonia hydrolysis adsorption
[0043] An enzyme-catalyzed reaction produced a crude solution of β-nicotinamide mononucleotide (NMN) with a concentration of 18.8 g / L and nicotinic acid mononucleotide (NMN) of 0.486%. 5 L of the crude solution was taken, and the pH was adjusted to 2.5 with hydrochloric acid. Activated carbon was added for adsorption, and the solution was filtered. The filtrate was adjusted to pH 4.2 with sodium hydroxide and loaded onto a macroporous adsorption resin column. The concentration of the product in the effluent was measured. After loading, the resin column was eluted with 2 BV of pure water, and the conductivity of the effluent was monitored. The column was eluted with 0.4 mol / L ammonia solution, and the product eluent was collected. The temperature of the effluent was measured to be 20-25 °C. The concentration of the product in the effluent was monitored, and collection was stopped when the concentration dropped below 1 g / L.
[0044] The eluent was adjusted to pH 3.5-4.0 with hydrochloric acid, concentrated to 150 g / L by nanofiltration, and 2 L of ethanol was added. A large amount of solid precipitated out. The solid was filtered and dried under vacuum at 30 °C to obtain 86.2 g of white solid, with a yield of 91.7% and a purity of 99.93%, containing 0.023% nicotinic acid mononucleotide.
[0045] The nanofiltration filtrate is concentrated using a reverse osmosis membrane and then treated by electrodialysis to obtain ammonia and hydrochloric acid.
[0046] Example 4:
[0047] β-Nicotinamide mononucleotide was separated and purified using macroporous adsorption resin, followed by desorption using sodium hydroxide.
[0048] The crude product solution obtained from the enzyme-catalyzed reaction had a concentration of 18.8 g / L and contained 0.486% nicotinic acid mononucleotide. 5 L of the crude product solution was taken, and the pH was adjusted to 3.0 with hydrochloric acid. Activated carbon was added for adsorption, and the solution was filtered. The filtrate was adjusted to pH 4.2 with sodium hydroxide and loaded onto a macroporous adsorption resin column. The concentration of the product in the effluent was measured. After loading, the resin column was eluted with 2 BV of pure water, and the conductivity of the effluent was monitored. Elution was performed with 0.02 mol / L sodium hydroxide solution, and the product eluent was collected. The temperature of the effluent was measured to be 20-25℃. The concentration of the product in the effluent was monitored, and collection was stopped when the concentration dropped below 1 g / L.
[0049] The eluent was adjusted to pH 3.5-4.0 with hydrochloric acid, concentrated to 150 g / L by nanofiltration, and 2 L of ethanol was added. A large amount of solid precipitated out. The solid was filtered and dried under vacuum at 30 °C to obtain 84.5 g of white solid, with a yield of 89.8%, a purity of 99.92%, and 0.025% nicotinic acid mononucleotide.
[0050] The nanofiltration filtrate was concentrated using a reverse osmosis membrane, followed by electrodialysis to obtain sodium hydroxide and hydrochloric acid. The HPLC chromatogram is shown below. Figure 3 As shown.
[0051] Example 5:
[0052] Macroporous adsorption resin separation and purification of β-nicotinamide mononucleotide, followed by sodium acetate desorption.
[0053] The crude product solution obtained from the enzyme-catalyzed reaction had a concentration of 18.8 g / L and contained 0.486% nicotinic acid mononucleotide. 5 L of the crude product solution was taken, and the pH was adjusted to 3.0 with hydrochloric acid. Activated carbon was added for adsorption, and the solution was filtered. The filtrate was adjusted to pH 4.2 with sodium hydroxide and loaded onto a macroporous adsorption resin column. The concentration of the product in the effluent was measured. After loading, the resin column was eluted with 2 BV of pure water, and the conductivity of the effluent was monitored. Elution was performed with 0.1 mol / L sodium acetate solution, and the product eluent was collected. The temperature of the effluent was monitored as 18-22℃. The concentration of the product in the effluent was monitored, and collection was stopped when the concentration dropped below 1 g / L.
[0054] The eluent was concentrated to 150 g / L by nanofiltration. The pH of the concentrate was then adjusted to 3.5-4.0 with hydrochloric acid. 2 L of ethanol was added, and a large amount of solid precipitated out. The solid was filtered and dried under vacuum at 30 °C to obtain 79.8 g of white solid, with a yield of 84.9% and a purity of 99.89%, containing 0.033% nicotinic acid mononucleotide.
[0055] The nanofiltration filtrate was concentrated using a reverse osmosis membrane, followed by further concentration and crystallization to obtain solid sodium acetate. The HPLC chromatogram is shown below. Figure 4 As shown.
[0056] Comparative Example 1:
[0057] β-Nicotinamide mononucleotide was separated and purified using macroporous adsorption resin, followed by ethanol elution.
[0058] A crude β-nicotinamide mononucleotide (NMN) solution (5 L, concentration 18.8 g / L) and nicotinic acid mononucleotide (0.486%) were prepared. The reaction solution was adjusted to pH 3.0 with hydrochloric acid, activated carbon was added for adsorption, and the solution was filtered. The filtrate was adjusted to pH 4.3 with sodium hydroxide and loaded onto a macroporous adsorption resin column. The concentration of the product in the effluent was monitored. After loading, the resin column was eluted with 2.0 BV of pure water, and the conductivity of the effluent was monitored. The product was eluted with 50% ethanol solution, and the eluent was collected. The temperature of the effluent was monitored at 32-36℃. The concentration of the product in the effluent was monitored, and collection was stopped when the concentration dropped below 1 g / L. The eluent was concentrated to 150 g / L by nanofiltration, and 2 L of ethanol was added. A large amount of solid precipitated out. The solid was filtered, dried under vacuum at 30℃, and 81 g of white solid was obtained, with a yield of 86.2% and a purity of 99.93%, containing 0.025% nicotinic acid mononucleotide.
[0059] Comparative Example 2:
[0060] β-Nicotinamide mononucleotide was separated and purified using macroporous adsorption resin, followed by ethanol elution.
[0061] A crude β-nicotinamide mononucleotide (NMN) solution (5 L, concentration 18.8 g / L) and nicotinic acid mononucleotide (0.486%) were prepared. The reaction solution was adjusted to pH 3.0 with hydrochloric acid, activated carbon was added for adsorption, and the solution was filtered. The filtrate was adjusted to pH 4.2 with sodium hydroxide and loaded onto a macroporous adsorption resin column. The concentration of the product in the effluent was monitored. After loading, the resin column was eluted with 2 BV of pure water, and the conductivity of the effluent was monitored. The product was eluted with 70% ethanol solution, and the eluent was collected. The temperature of the effluent was monitored at 40-45℃. The concentration of the product in the effluent was monitored, and collection was stopped when the concentration dropped below 1 g / L. The eluent was concentrated to 150 g / L by nanofiltration, and 1.6 L of ethanol was added. A large amount of solid precipitated out. The solid was filtered and dried under vacuum at 30℃ to obtain 75.7 g of white solid, with a yield of 80.5% and a purity of 99.91%, containing 0.021% nicotinic acid mononucleotide.
[0062] The HPLC purity and impurities of the crude reaction solution, the products of Examples 2-4, and Comparative Examples 1-3 were analyzed, and the purification yields were calculated. Specific results are shown in Table 1.
[0063]
[0064] Table 1
[0065] Analysis of the above data shows that the crude solutions in both the examples and the comparative examples exhibited good impurity removal after purification with macroporous adsorption resin. The examples used alkaline solutions of certain concentrations, such as ammonia, sodium hydroxide, and sodium acetate, for desorption, with the temperature of the desorption solution controlled at around 20°C. In contrast, the comparative examples used ethanol of varying concentrations for desorption; higher ethanol concentrations resulted in higher desorption solution temperatures, lower product yields, and higher nicotinamide proportions. Since NMN is a thermosensitive substance, its degradation accelerates at temperatures above 40°C. Therefore, when using ethanol for desorption, the exothermic reaction between ethanol and water causes localized degradation of NMN. The examples used an alkaline solution to elute the product by adjusting the pH to change the ionization state of NMN. This elution process was non-exothermic and under mild conditions. After elution, the pH was adjusted to 3.5-4.0 with hydrochloric acid, followed by nanofiltration, concentrating the product while simultaneously removing salt. The inorganic salts in the filtrate, including ammonia, hydrochloric acid, and sodium hydroxide, were recovered via electrodialysis and can be reused as raw materials, significantly reducing raw material costs, minimizing wastewater generation, and promoting environmental friendliness.
[0066] The above embodiments are merely the preferred embodiments of the present invention and do not limit the scope of protection of the present invention. All non-substantial changes and adjustments made by those skilled in the art based on the present invention are within the scope of protection claimed by the present invention.
Claims
1. A method for the isolation and purification of β-nicotinamide mononucleotide, characterized in that: Step 1: Adjust the pH of the enzyme-catalyzed reaction solution containing β-nicotinamide mononucleotide to 3.5-4.8 to obtain the purification solution; Step 2: Pass the solution to be purified through a separation column packed with macroporous adsorption resin, wash off salts and impurities with pure water, then elute the product with an alkaline solution as the eluent, collect the eluent, and adjust the pH value to 3.5-4.0; Step 3: Purify the eluent to obtain the purified β-nicotinamide mononucleotide product.
2. The method as described in claim 1, characterized in that, The enzyme-catalyzed reaction solution containing β-nicotinamide mononucleotide mentioned in step one is a reaction solution obtained by synthesizing β-nicotinamide mononucleotide under the combined catalysis of D-ribose, nicotinamide, adenine nucleoside triphosphate ATP, and polyphosphate as raw materials and phosphoribosyl pyrophosphate synthase, ribokinase, and nicotinamide phosphoribosyltransferase.
3. The method as described in claim 1, characterized in that, Step one is as follows: Add an adsorbent to the enzyme-catalyzed reaction solution containing β-nicotinamide mononucleotide, stir, and then filter. Adjust the pH of the resulting filtrate to 3.5-4.8 to obtain the purified solution. Alternatively, adjust the pH of the enzyme-catalyzed reaction solution containing β-nicotinamide mononucleotide to 3.5-4.8, add an adsorbent to the reaction solution, stir, and then filter. The resulting filtrate is the purified solution.
4. The method as described in claim 1, characterized in that, Step one is as follows: Adjust the pH of the enzyme-catalyzed reaction solution containing β-nicotinamide mononucleotide to 2.5-3.0, add adsorbent, stir and filter, then adjust the pH of the resulting filtrate to 3.5-4.8 to obtain the purified solution.
5. The method as described in claim 1, characterized in that, In steps one and two, hydrochloric acid and / or NaOH are used to adjust the pH value.
6. The method as described in claim 3, characterized in that, The adsorbent used in step one is selected from activated carbon.
7. The method as described in claim 1, characterized in that, The eluent used in step two is selected from ammonia, sodium hydroxide, and sodium acetate.
8. The method as described in claim 1, characterized in that, In step two, the product is eluted using an alkaline solution of 0.01-1 mol / L.
9. The method as described in claim 1, characterized in that, In step three, the purification of the eluent includes nanofiltration and crystallization.
10. The method according to any one of claims 1-9, characterized in that, It also includes step four: collecting and regenerating the solution containing the eluent separated during the purification process in step three.