Method for preparing NMN
By using multiple enzymatic reactions under normal temperature, normal pressure and neutral conditions, the problems of high cost and low efficiency of existing NMN preparation methods have been successfully solved, and efficient and low-cost NMN preparation is achieved, which is suitable for large-scale industrial production.
Patent Information
- Application Number
- CN202510090596.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-20
- Publication Date
- 2025-04-29
AI Technical Summary
The existing NMN preparation methods have problems such as high cost, low production efficiency, and threats to product purity and safety, which limits their application in the high-end pharmaceutical field.
Ribose phosphate is synthesized by enzymatic reactions such as ribose, polyphosphate, RK enzyme and PPK1 enzyme under normal temperature, normal pressure and neutral conditions, and nicotinamide, PRPPS enzyme, NMPRT enzyme, etc. are added on the basis of it, and the efficient synthesis of NMN is achieved through multiple enzymatic reactions.
This method significantly reduces production costs, improves production efficiency through the recycling of ATP, and is carried out under neutral conditions, reducing equipment investment and energy consumption, and achieving high conversion and efficient NMN preparation.
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Figure CN119899883A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biopharmaceutical technology, and more specifically, to a method for preparing NMN. Background Art
[0002] In the booming development of modern biopharmaceutical research, nicotinamide mononucleotide (NMN) has gradually become a focus of attention. NMN occupies an extremely critical position in the metabolic process of biological cells. After being adenylated by nicotinamide nucleotide adenylyltransferase through a specific biochemical reaction, it can be smoothly converted into nicotinamide adenine dinucleotide (NAD, i.e. coenzyme I) necessary for cell survival. In this process, NMN also directly participates in the adenosine transfer activity in the body. Its content level in biological cells has a direct and important impact on the concentration of NAD, and thus plays an indispensable role in the core link of biological cell energy generation.
[0003] As scientific research continues to deepen, the huge potential of NMN in the field of healthcare is constantly being tapped. Numerous studies have shown that NMN has a positive performance in delaying the aging process, coping with Parkinson's and other elderly diseases, fine-tuning insulin secretion, and affecting mRNA expression. This makes its application prospects in the pharmaceutical and health care products market extremely broad, and the market demand for NMN is showing a rapid growth trend.
[0004] However, the existing NMN preparation methods all have defects to varying degrees. Although the yeast fermentation method can achieve the production of NMN to a certain extent, organic solvents will inevitably remain during the production process. These residual substances may pose a potential threat to the purity and safety of the product, limiting its application in the field of high-end medicine. Although the chemical synthesis method has the advantage of a relatively clear synthesis route, it has the problem of high cost in actual operation, and it is easy to produce chiral compounds during the synthesis process. The subsequent complex separation and purification procedures not only increase the production cost, but also reduce the production efficiency. Although the traditional biocatalytic method has certain advantages in environmental protection, it does not contain organic solvent residues and does not have chirality problems. The prepared NMN is similar to the same type of substance in the body, but because it usually uses nicotinamide and 5'phosphoribosyl-1'-pyrophosphate (PRPP) or AMP or ribose as substrates for catalytic reactions, the market price of PRPP is expensive and the supply source is extremely limited. This leads to the high cost of preparing NMN using this biocatalytic method, and faces huge obstacles in large-scale industrial production and market promotion and application.
[0005] In summary, in view of the important position of NMN in the biomedical and health industries and the limitations of existing preparation methods, a new NMN preparation method is proposed that can effectively overcome the defects of existing technologies to meet the growing market demand and promote the application and development of NMN in a wider range of fields. Summary of the invention
[0006] In order to overcome the above-mentioned defects of the prior art, the present invention provides a method for preparing NMN to solve the problems raised in the above-mentioned background technology.
[0007] To achieve the above object, the present invention provides the following technical solution: a method for preparing NMN, comprising the following steps:
[0008] S1: Select a suitable reaction container, mix ribose, polyphosphate, and RK enzyme, and add high-energy substances ATP and PPK1 enzyme to construct a reaction system, promote ribose and polyphosphate to be catalyzed by RK enzyme, and synthesize ribose phosphate under the action of ATP and PPK1 enzyme to form a reaction solution;
[0009] S2: Take the reaction solution obtained in S1, add nicotinamide, polyphosphate, PRPPS enzyme, and NMPRT enzyme to form a new reaction system to achieve the synthesis of NMN.
[0010] Preferably, the reaction temperature in steps S1 and S2 is 35-38° C. and is under normal pressure.
[0011] Preferably, in S1, 20% sodium hydroxide solution is used to control the pH, and the pH is adjusted to 5.0-8.0.
[0012] Preferably, the reaction time in S1 is 1-1.5 h.
[0013] Preferably, in S2, a 20% sodium carbonate solution is first used to adjust the pH, and then a 20% sodium hydroxide solution is used to adjust the pH when the reaction solution in SI is added, and the pH is adjusted to 5.0-8.0.
[0014] Preferably, the reaction time in S2 is 2-4h.
[0015] Preferably, the specific method steps for converting to ribose phosphorylation in S1 include:
[0016] First, a phosphate solution with a concentration of 50 mM is prepared in a reaction container;
[0017] Then, appropriate amounts of magnesium chloride, ATP and substrate ribose were added, and the mixture was kept warm and stirred;
[0018] Then, the pH value of the reaction system was detected, and the pH was adjusted with an alkaline solution, and then RK enzyme and PPK1 enzyme were added, followed by adding water and stirring evenly;
[0019] Finally, sodium polyphosphate was added at a uniform rate and completed within 40 minutes. The entire reaction process was carried out under normal pressure with insulation and pH was controlled with 20% sodium hydroxide solution. After a certain reaction time, the substrate was completely converted.
[0020] Preferably, the specific method steps for converting into NMN in S2 include:
[0021] First, add 50 mM phosphate buffer to the reaction vessel;
[0022] Subsequently, appropriate amounts of magnesium chloride and nicotinamide are added, and the mixture is stirred and kept warm;
[0023] Then, the pH value of the reaction system was detected, and the pH was adjusted with an alkaline solution, and then PRPPS enzyme, PPK1 enzyme, PPK2 enzyme and PRPP enzyme were added and stirred evenly.
[0024] Finally, the reaction was stirred and kept warm under normal pressure, and the reaction solution in S1 was evenly added. During the reaction, the pH was controlled with an alkaline solution. After a certain reaction time, the conversion was completed and NMN was prepared.
[0025] Technical effects and advantages of the present invention:
[0026] 1. During the reaction, ATP is recycled in the first and second steps of the reaction by PPK1 and PPK2 enzymes, which greatly reduces the consumption of ATP, effectively reduces production costs, and improves production efficiency.
[0027] 2. The entire preparation process is carried out under normal temperature, normal pressure and neutral conditions, which has relatively low requirements on equipment, reduces equipment investment costs, and also reduces energy consumption. In addition, the operation is relatively simple, easy to control and implement, and is conducive to large-scale industrial production.
[0028] 3. The conversion rate of synthesizing ribose phosphate in the first step is greater than 99%, and the conversion rate of synthesizing NMN in the second step is greater than 65%. The high conversion rate ensures the effective utilization of raw materials, reduces raw material waste, improves production efficiency, reduces production costs, and makes the product more competitive in the market. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] Figure 1 It is the reaction diagram of the present invention. DETAILED DESCRIPTION
[0030] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without making any creative work shall fall within the scope of protection of the present invention.
[0031] As attached Figure 1 As shown, the present invention provides a method for preparing NMN, comprising the following steps:
[0032] S1: Select a suitable reaction container, mix ribose, polyphosphate, and RK enzyme, and add high-energy substances ATP and PPK1 enzyme to construct a reaction system, promote ribose and polyphosphate to be catalyzed by RK enzyme, and synthesize ribose phosphate under the action of ATP and PPK1 enzyme to form a reaction solution;
[0033] S2: Take the reaction solution obtained in S1, add nicotinamide, polyphosphate, PRPPS enzyme, and NMPRT enzyme to form a new reaction system to achieve the synthesis of NMN.
[0034] Specifically, the specific method steps for converting to ribose phosphorylation in S1 include:
[0035] First, a phosphate solution with a concentration of 50 mM is prepared in a reaction container;
[0036] Then, appropriate amounts of magnesium chloride, ATP and substrate ribose were added, and the mixture was kept warm and stirred;
[0037] Then, the pH value of the reaction system is detected, and the pH is adjusted to 5.0-8.0 with an alkaline solution, and then RK enzyme and PPK1 enzyme are added, followed by adding water and stirring evenly;
[0038] Finally, sodium polyphosphate is added at a uniform rate and completed within 40 minutes. The entire reaction process is carried out under normal pressure with insulation, and the pH is controlled at 5.0-8.0 with 20% sodium hydroxide solution. After a certain reaction time, the substrate is completely converted.
[0039] The specific method steps for converting into NMN in S2 include:
[0040] First, add 50 mM phosphate buffer to the reaction vessel;
[0041] Subsequently, appropriate amounts of magnesium chloride and nicotinamide are added, and the mixture is stirred and kept warm;
[0042] Then, the pH value of the reaction system is detected, and the pH is adjusted to 5.0-8.0 with an alkaline solution, and then PRPPS enzyme, PPK1 enzyme, PPK2 enzyme and PRPP enzyme are added and stirred evenly.
[0043] Finally, the reaction was stirred and kept warm under normal pressure, and the reaction solution in S1 was evenly added. During the reaction, the pH was controlled at 5.0-8.0 with an alkaline solution. After a certain reaction time, the conversion was completed and NMN was prepared.
[0044] In specific implementation, under normal temperature, pressure and neutral conditions, the RK enzyme catalyzes the reaction between ribose and polyphosphate, ATP provides energy to drive the reaction, and the PPK1 enzyme plays a key role in the reaction system, which can promote the recycling of ATP. Specifically, when ATP is hydrolyzed to produce ADP, the PPK1 enzyme can use polyphosphate to re-phosphorylate ADP into ATP, maintain a relatively stable concentration of ATP in the reaction system, and ensure that the reaction continues to obtain energy supply, so that ribose is efficiently converted into ribose phosphate, and the conversion rate is greater than 99%;
[0045] Also at room temperature, pressure, and neutral environment, PRPPS enzymes, NMPRT enzymes, and the like work together to react with ribose phosphorylation generated by S1 and added nicotinamide as substrates. ATP again provides energy for the reaction, and the PPK2 enzyme is responsible for the recycling of ATP. During the reaction, when ATP decomposes to produce ADP, the PPK2 enzyme prompts polyphosphate to react with ADP to regenerate ATP. By regulating the pH value of the reaction system, the pH is first adjusted to 8 with a 20% sodium carbonate solution, and then maintained with a sodium hydroxide solution to ensure that each enzyme is in a suitable active environment, so that the reaction is completed within 2-4 hours, and the conversion rate is greater than 65%.
[0046] Example
[0047] The first step is to transform into:
[0048] (1) Prepare 20 L of 50 mM phosphate buffer in a 50 L reactor;
[0049] (2) Add 300 g magnesium chloride, 5 g ATP, and 1 kg substrate ribose, and stir at 35 °C;
[0050] (3) Detect the pH value of the reaction system, adjust the pH to 8.0 with 20% sodium hydroxide solution, add 10g RK enzyme and 10g PPK1 enzyme, add water and adjust the volume to 30L, and stir evenly;
[0051] (4) 4 L of sodium polyphosphate was added at a constant rate and the addition was completed within 40 min. The entire reaction process was carried out at 38° C. and the pH was controlled at 8.0 with 20% sodium hydroxide solution. After 1 h, 200 μl of the sample was taken and centrifuged and then placed on a plate for reaction analysis. After 1-1.5 hours, the substrate was completely converted to form a reaction solution.
[0052] The second step of conversion:
[0053] (1) Add about 30 L of 50 mM potassium phosphate buffer to a 100 L reactor;
[0054] (2) Add 300 g of magnesium chloride and 900 g of nicotinamide, and stir at 38°C;
[0055] (3) Detect the pH value of the reaction system and adjust the pH to 8 with 20% Na2CO3 solution, then add 20g PRPPS enzyme, 30g PPK1 enzyme, 30g PPK2 enzyme and 30g PRPP enzyme and stir evenly.
[0056] (4) The reaction was stirred at 38°C and the first step reaction solution was evenly added. During the reaction, the pH was controlled at 8 with 20% sodium hydroxide solution. The total reaction volume was about 91 L. Samples were taken at intervals after 1 h and detected by HPLC. The conversion was completed in 3.5 hours and the NMN concentration of the reaction conversion solution was about 18 g / L.
[0057] Among them, the second step conversion liquid phase is as follows:
[0058]
[0059] From the above table, we can see that sampling was carried out every half an hour after 1 hour, and the NMN peak area hardly changed after 3-3.5 hours. Therefore, the conversion was basically completed in this time period. And through the proportion of NMN peak area, we can see that the conversion rate is greater than 65%, and the concentration of NMN reaches 18.00g / L.
[0060] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims
1. A method for preparing NMN, characterized in that: The following steps are involved: S1: Select a suitable reaction container, mix ribose, polyphosphate, and RK enzyme, and add high-energy substances ATP and PPK1 enzyme to construct a reaction system, promote ribose and polyphosphate to be catalyzed by RK enzyme, and synthesize ribose phosphate under the action of ATP and PPK1 enzyme to form a reaction solution; S2: Take the reaction solution obtained in S1, add nicotinamide, polyphosphate, PRPPS enzyme, and NMPRT enzyme to form a new reaction system to achieve the synthesis of NMN.
2. A method for preparing NMN according to claim 1, characterized in that: The reaction temperature in the steps S1 and S2 is 35-38° C. and is under normal pressure.
3. A method for preparing NMN according to claim 1, characterized in that: In the S1, 20% sodium hydroxide solution is used to control the pH, and the pH is adjusted to 5.0-8.
0.
4. A method for preparing NMN according to claim 1, characterized in that: The reaction time in S1 is 1-1.5h.
5. A method for preparing NMN according to claim 1, characterized in that: In the S2, the pH is first regulated by a 20% sodium carbonate solution, and then a 20% sodium hydroxide solution is used to regulate the pH when the reaction solution in the SI is added, and the pH is adjusted to 5.0-8.
0.
6. A method for preparing NMN according to claim 1, characterized in that: The reaction time in S2 is 2-4h.
7. A method for preparing NMN according to claim 1, characterized in that: The specific method steps for converting to ribose phosphorylation in S1 include: First, a phosphate solution with a concentration of 50 mM is prepared in a reaction container; Then, appropriate amounts of magnesium chloride, ATP and substrate ribose were added, and the mixture was kept warm and stirred; Then, the pH value of the reaction system was detected, and the pH was adjusted with an alkaline solution, and then RK enzyme and PPK1 enzyme were added, followed by adding water and stirring evenly; Finally, sodium polyphosphate was added at a uniform rate and completed within 40 minutes. The entire reaction process was carried out under normal pressure with insulation and pH was controlled with 20% sodium hydroxide solution. After a certain reaction time, the substrate was completely converted.
8. A method for preparing NMN according to claim 1, characterized in that: The specific method steps for converting into NMN in S2 include: First, add 50 mM phosphate buffer to the reaction vessel; Subsequently, appropriate amounts of magnesium chloride and nicotinamide are added, and the mixture is stirred and kept warm; Then, the pH value of the reaction system was detected, and the pH was adjusted with an alkaline solution, and then PRPPS enzyme, PPK1 enzyme, PPK2 enzyme and PRPP enzyme were added and stirred evenly; Finally, the reaction was stirred and kept warm under normal pressure, and the reaction solution in S1 was evenly added. During the reaction, the pH was controlled with an alkaline solution. After a certain reaction time, the conversion was completed and NMN was prepared.