Method for synthesizing beta-nicotinamide mononucleotide through enzymatic conversion
By using raw materials such as nicotinamide ribose, acetyl phosphate diamine salt and disodium adenosine triphosphate salt, combined with enzyme liquid catalysis of nicotinamide nucleoside kinase and phosphate transferase, the cost of β-nicotinamide single nucleotide preparation is solved. The problems of high, low conversion rate and many by-products have achieved an efficient and low-cost synthesis process and are suitable for large-scale production.
Patent Information
- Application Number
- CN202411497953.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-25
- Publication Date
- 2025-01-10
AI Technical Summary
The existing preparation methods for β-nicotinamide single nucleotides have problems such as high cost, low conversion rate and many by-products, making it difficult to achieve industrial production.
Using nicotinamide ribose, acetyl phosphate diamine salt and adenosine triphosphate disodium salt as raw materials, the reaction conditions are adjusted to achieve low cost, high conversion rate and fewer sub-parameters by catalyzing the enzyme liquid of nicotinamide nucleoside kinase and phosphate transferase. Product synthesis process.
The high conversion rate (90%) and high purity (99.8%) of β-nicotinamide single nucleotides were achieved, which reduced the cost of raw materials, simplified the subsequent treatment process, and was suitable for large-scale production.
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Figure CN119286959A_ABST
Abstract
Description
Technical Field
[0001] The embodiments of the present application relate to the technical field of biotechnology synthesis, and in particular to a method for synthesizing β-nicotinamide mononucleotide by enzymatic conversion. Background Art
[0002] β-Nicotinamide mononucleotide (β-NMN or NMN) is the precursor of NAD+ (nicotinamide adenine dinucleotide, also known as coenzyme 1). As the research on NMN is carried out in depth, it is found that it has a variety of biological functions, such as therapeutic effects on cardiovascular and brain diseases, elderly neurodegenerative diseases, and anti-aging. At present, NMN has great development potential and market prospects in functional health foods. Some developed countries have approved it to enter the market as a health food raw material, such as Geneport. This prompted researchers to conduct deeper research and development on the preparation process of β-nicotinamide mononucleotide.
[0003] There are many reports on the preparation methods of β-nicotinamide mononucleotide, mainly including chemical synthesis, enzymatic method (semi-enzymatic method and full-enzymatic method) and fermentation method. Different methods have different disadvantages, as follows:
[0004] Chemical synthesis produces racemates, making it difficult to separate isomers. In addition, the raw materials are expensive and the separation cost is high, making it difficult to achieve industrial production.
[0005] The fermentation method uses niacinamide or nicotinamide as raw materials and relies on microbial fermentation. Although the cost is reduced, the fermentation level is low, the subsequent processing is difficult, and it is difficult to obtain qualified products.
[0006] The whole enzyme method uses D-ribose and nicotinamide as starting materials, and catalyzes the reaction to obtain NMN under the action of ribokinase, phosphoribosyl pyrophosphate synthetase and nicotinamide phosphoribosyltransferase. This method has a large number of intermediates, which is difficult to separate and purify, has a low overall yield and a high production cost.
[0007] The semi-enzymatic method is an effective method that is widely used in industrial production. It uses nicotinamide riboside (NR) as the starting material and obtains NMN in one step under the action of nicotinamide riboside kinase and ATP. However, the semi-enzymatic method has the problem of large ATP dosage, and as the equimolar reaction of ATP and nicotinamide riboside proceeds, a large amount of byproduct AMP will be produced, which increases the workload of subsequent processing, has a long reaction time, and a low conversion rate of only about 90%, with a total yield of 75%.
[0008] Therefore, how to provide a new process flow with low cost, high conversion rate and few by-products is of great significance for the production of β-nicotinamide mononucleotide. Summary of the invention
[0009] In order to solve the above problems, the first aspect of the invention of the present application aims to provide a method for synthesizing β-nicotinamide mononucleotide by enzymatic conversion, by selecting the enzyme and the phosphate donor, so that the synthesis process of β-nicotinamide mononucleotide has the characteristics of low cost, high conversion rate and few by-products.
[0010] In order to achieve the above object, the present invention provides the following technical solutions:
[0011] A method for synthesizing β-nicotinamide mononucleotide by enzyme conversion, comprising taking a composition comprising nicotinamide ribose, acetylphosphodiamine salt and adenosine triphosphate disodium salt as a raw material, converting the raw material under the catalytic action of an enzyme solution to obtain β-nicotinamide mononucleotide, wherein the enzyme solution comprises nicotinamide riboside kinase and phosphotransferase.
[0012] In some embodiments, the raw material includes the following components in parts by weight:
[0013] Nicotinamide Riboside 50-90 parts
[0014] Acetylphosphodiamine salt 70-150 parts
[0015] 1-4 parts of adenosine triphosphate disodium salt
[0016] In some embodiments, the enzyme activity ratio of nicotinamide riboside kinase to the phosphotransferase in the enzyme solution is 1:(0.1-100) based on the enzyme activity ratio.
[0017] In some embodiments, the raw material is mixed with water to prepare an aqueous solution, the pH of the aqueous solution is adjusted to 6.5-7.5, and an enzyme solution is added to carry out a conversion reaction.
[0018] In some embodiments, the temperature of the conversion reaction is 25-35° C., and the reaction time is less than or equal to two hours.
[0019] In some embodiments, the preparation of the enzyme solution comprises:
[0020] The nicotinamide riboside kinase and the phosphotransferase are obtained from fermented bacteria by centrifugation, and then washed and prepared into a 25% bacterial solution, and the bacterial solution is treated by a homogenizer low-temperature crushing method;
[0021] Among them, the homogenization pressure is 80MPa, and homogenization is performed 2-3 times.
[0022] In some embodiments, the purification step of β-nicotinamide mononucleotide is also included:
[0023] The reaction solution obtained by completely converting the raw material under the action of the enzyme solution is adjusted to pH 2.5-3.0, and then subjected to membrane filtration after standing to obtain a membrane filtration reaction solution;
[0024] The membrane filtered reaction solution is adjusted to weak acidity and passed through an anion and cation exchange column and nanofiltered to remove salt to obtain a desalted solution, and the desalted solution is concentrated, filtered, crystallized, and dried to obtain β-nicotinamide mononucleotide.
[0025] In some embodiments, in the step of adjusting the membrane filtration reaction solution to weak acidity and passing it through an anion and cation exchange column and nanofiltration to remove salt from the anion and cation exchange resin, the membrane filtration reaction solution is exchanged through the cation column to obtain a cation column eluate,
[0026] The pH of the cation column eluate is adjusted to 5.5-6.5, and then passed through an anion exchange column to obtain an anion column effluent.
[0027] In some embodiments, in the step of adjusting the membrane filtered reaction solution to weak acidity and passing it through an anion and cation exchange column and nanofiltration to remove salt from anion and cation exchange resins, the cation exchange resin is preferably a weakly acidic cation exchange resin XRK540, and the anion exchange resin is preferably a weakly basic anion exchange resin XRK209.
[0028] In some embodiments, the desalted solution is concentrated to a concentration of 15-25% under reduced pressure at low temperature, ethanol is added for crystallization, the crystals are grown at 0-5° C. for 5 hours, filtered, and dried to obtain purified β-nicotinamide mononucleotide (NMN).
[0029] Based on the above technical solutions, the present invention has the following technical effects:
[0030] 1. The present invention provides a method for synthesizing β-nicotinamide mononucleotide by enzyme conversion, wherein nicotinamide ribose is phosphorylated in a system in which a phosphate donor is present to obtain β-nicotinamide mononucleotide. The enzyme used is a composite enzyme solution composed of nicotinamide riboside kinase and phosphotransferase, and acetylphosphodiamidate is selected as the phosphate donor. Acetylphosphodiamidate itself has the characteristics of low cost, and compared with other phosphate donors, it has a low residual effect, which effectively simplifies the subsequent impurity removal process.
[0031] In addition, the ADP generated during the reaction is converted into ATP for recycling by the action of acetyl diammonium phosphate and phosphokinase, which greatly reduces the use of ATP and reduces costs. In addition, there are fewer by-products, which facilitates the simplification of post-processing processes. The total yield reaches 90% and the purity reaches 99.8%.
[0032] The method for preparing β-nicotinamide mononucleotide of the present invention improves the conversion rate, shortens the reaction time, reduces the cost of raw materials, and is suitable for large-scale production of β-nicotinamide mononucleotide. BRIEF DESCRIPTION OF THE DRAWINGS
[0033] Figure 1 This is the finished product spectrum of β-nicotinamide mononucleotide (NMN) in Example 1 of the present invention;
[0034] Figure 2 This is the spectrum of the β-nicotinamide mononucleotide (NMN) reference substance in Example 1 of the present invention;
[0035] Figure 3 is the substrate NR reference spectrum;
[0036] Figure 4 is the ATP reference spectrum;
[0037] Figure 5 is the ADP reference spectrum;
[0038] Figure 6 This is the AMP reference spectrum. DETAILED DESCRIPTION
[0039] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below in conjunction with specific embodiments. The present invention provides preferred embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thoroughly understood.
[0040] Before further describing various embodiments of the compounds / compositions and methods of the present disclosure in more detail through exemplary descriptions, examples and results, it should be understood that the embodiments of the present disclosure are not limited to the details of the methods and compositions described in the following description in application. The description provided herein is for illustrative purposes only and is not to be interpreted in a limiting sense. The inventive concepts of the present disclosure can have other embodiments or can be practiced or implemented in various ways. Therefore, the language used herein is intended to give the broadest scope and meaning; and the embodiments are intended to be exemplary, not exhaustive, and are not intended to limit the present disclosure to these specific embodiments. Moreover, it should be understood that the wording and terminology used herein are for the purpose of description and should not be considered restrictive unless otherwise stated. In addition, in the following detailed description, many specific details are listed in order to provide a more thorough understanding of the present disclosure.
[0041] However, it is obvious to those skilled in the art that the embodiments of the present disclosure can be practiced without these specific details. In other cases, features known to those skilled in the art are not described in detail to avoid unnecessary complex descriptions. It is intended that all substitutions, replacements, modifications and equivalents obvious to those skilled in the art are included within the scope of the present disclosure. According to the present disclosure, all compounds / compositions disclosed herein and their preparation methods, applications and uses can be prepared and implemented without undue experimentation.
[0042] Thus, although the compounds / compositions and methods of the present disclosure have been described according to specific embodiments, it will be apparent to those skilled in the art that changes may be made to the formulations, compounds or compositions and / or methods, as well as the steps or order of steps of the methods described herein, without departing from the spirit and scope of the inventive concepts of the present disclosure.
[0043] As used herein, any reference to "one embodiment" or "an embodiment" means that a particular element, feature, structure, or characteristic described in conjunction with the implementation is included in at least one embodiment. The phrase "in one embodiment" appearing in multiple places in the specification does not necessarily refer to the same embodiment.
[0044] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present disclosure shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
[0045] Example 1
[0046] Preparation of enzyme solution
[0047] The wet cells were separated from the bacterial fermentation liquid containing nicotinamide riboside kinase and phosphotransferase by a high-speed centrifuge, washed twice with pure water to obtain the enzyme wet cells, and stored in a freezer at -10°C.
[0048] Thaw the wet bacteria, prepare a 25% concentration of wet bacteria liquid with purified water, use a homogenizer to break the cell wall at low temperature, homogenize at a pressure of 80 MPa, homogenize 2-3 times, and refrigerate at 0-5℃ for later use.
[0049] Preparation of β-nicotinamide mononucleotide (NMN) reaction solution
[0050] 100g of pure water was added to the reactor, stirring was started, 50g of NR, 4g of disodium adenosine triphosphate, 4.5g of MgCl2·6H2O, and 70g of diammonium acetylphosphate were added in sequence, pH was adjusted to 6.5 after dissolving, enzyme solution was added, the reaction started, and samples were taken every 30 minutes to detect the conversion rate. The complete conversion of NR was the end point of the reaction, and the conversion reaction solution was obtained.
[0051] Extraction of β-nicotinamide mononucleotide (NMN)
[0052] The conversion reaction solution was acidified to adjust the pH to 2.5-3.0, allowed to stand for 2 hours, and membrane filtration was started to obtain a membrane filtered reaction solution.
[0053] The reaction solution was filtered through a membrane and adjusted to pH 5.5 and loaded onto a positive column at a flow rate of 1 BV / hour. After washing with water, it was eluted with 0.3 M acid and the eluate was collected to obtain the positive column eluate.
[0054] After the cation column eluate is adjusted to pH 5.5-6.5, it is applied to the anion column at a flow rate of 1 BV / hour, and the effluent is collected to obtain the anion column effluent.
[0055] The pH of the anion column effluent was adjusted to 3.0, and nanofiltration was performed to desalinate to obtain a desalted solution. The desalted solution was concentrated under reduced pressure to 1 / 3 of the volume, and activated carbon was added for decolorization for 30 minutes at a temperature of 25-35°C. After filtration, it was further concentrated to 15-25%, transferred to a crystallization tank, and 95% ethanol was added for crystallization. The crystals were grown at 0-5°C for 5 hours, filtered, washed, and dried to obtain 45.75 g of a finished product of β-nicotinamide mononucleotide (NMN), with a total yield of 85%, a purity of 99.7%, and a content of 99.2%.
[0056] Example 2
[0057] The difference from Example 1 is that the mass of nicotinamide riboside is 70 g, the mass of acetylphosphodiamine salt is 98 g, and the mass of adenosine triphosphate disodium salt is 4 g.
[0058] Example 3
[0059] The difference from Example 1 is that the mass of nicotinamide riboside is 90 g, the mass of acetylphosphodiamine salt is 127 g, and the mass of adenosine triphosphate disodium salt is 1 g.
[0060] Comparative Example 1
[0061] The difference from Example 3 is that acetyl phosphate diamine salt is replaced by acetyl phosphate dipotassium salt.
[0062] Add 100g pure water into the reactor, start stirring, add 90g NR, 1g ATP disodium salt, 4.5g MgCl2·6H2O, 141.6g acetyl phosphate dipotassium salt in sequence, adjust the pH to 6.5 after dissolving, add enzyme solution, start the reaction, take samples every 30 minutes to detect the conversion rate. The complete conversion of NR is the end point of the reaction, and the conversion reaction solution is obtained.
[0063] Comparative Example 2
[0064] The difference from Example 1 is that the phosphotransferase is replaced by polyphosphate kinase.
[0065] Add 100g pure water into the reactor, start stirring, add 50g NR, 50g ATP disodium salt, 8.3g MgCl2·6H2O, 25g sodium hexametaphosphate in sequence, adjust to pH 7.5 after dissolving, add enzyme solution, start the reaction, take samples every 30 minutes to detect the conversion rate. The complete conversion of NR is the end point of the reaction, and the conversion reaction solution is obtained.
[0066] Taking Example 1 as an example, the β-nicotinamide mononucleotide product obtained in Example 1 was analyzed by high performance liquid chromatography to calculate the purity, content and corresponding conversion rate of the product. Figure 1 to Figure 2 shown.
[0067] It should be noted that the extraction process of β-nicotinamide mononucleotide in Examples 2-3 and Comparative Examples 1-2 is the same as that in Example 1. The weight, total yield, purity and content of the β-nicotinamide mononucleotide product are calculated according to the method of Example 1. The specific data are as follows:
[0068] Table.1 Quality table of finished products of various embodiments and comparative examples
[0069] Finished product weight Total yield purity content Example 1 45.75g 85％ 99.7％ 99.2％ Example 2 69.21g 86％ 99.72％ 99.5％ Example 3 91.05g 88％ 99.8％ 100.12％ Comparative Example 1 77.6g 75％ 98.48％ 99.01％ Comparative Example 2 41.96g 73％ 98.51％ 99.21％
[0070] The above contents are merely examples and illustrations of the structure of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, and these obvious replacement forms all belong to the protection scope of the present invention.
Claims
1. A method for synthesizing β-nicotinamide mononucleotide by enzymatic conversion, characterized in that: A composition including nicotinamide ribose, acetylphosphodiamine salt and adenosine triphosphate disodium salt is used as a raw material, and the raw material is converted into β-nicotinamide mononucleotide under the catalytic action of an enzyme solution, wherein the enzyme solution includes nicotinamide riboside kinase and phosphotransferase.
2. The method for synthesizing β-nicotinamide mononucleotide by enzymatic conversion according to claim 1, characterized in that: The raw materials include the following components in parts by mass: Nicotinamide Riboside 50-90 parts Acetylphosphodiamine salt 70-150 parts 1-4 parts of adenosine triphosphate disodium salt.
3. The method for synthesizing β-nicotinamide mononucleotide by enzymatic conversion according to claim 1, characterized in that: In terms of enzyme activity ratio, the enzyme activity ratio of nicotinamide riboside kinase to the phosphotransferase in the enzyme solution is 1:(0.1-100).
4. The method for synthesizing β-nicotinamide mononucleotide by enzymatic conversion according to claim 1, characterized in that: The raw materials are mixed with water to prepare an aqueous solution, the pH of the aqueous solution is adjusted to 6.5-7.5, and an enzyme solution is added to carry out a conversion reaction.
5. The method for synthesizing β-nicotinamide mononucleotide by enzymatic conversion according to claim 1, characterized in that: The temperature of the conversion reaction is 25-35° C., and the reaction time is less than or equal to two hours.
6. The method for synthesizing β-nicotinamide mononucleotide by enzymatic conversion according to claim 1, characterized in that: The preparation of the enzyme solution comprises: The nicotinamide riboside kinase and the phosphotransferase are obtained from fermented bacteria by centrifugation, and then washed and prepared into a 25% bacterial solution, and the bacterial solution is treated by a homogenizer low-temperature crushing method; Among them, the homogenization pressure is 80MPa, and homogenization is performed 2-3 times.
7. The method for synthesizing β-nicotinamide mononucleotide by enzymatic conversion according to claim 1, characterized in that: Also included is the purification step of β-nicotinamide mononucleotide: The reaction solution obtained by completely converting the raw material under the action of the enzyme solution is adjusted to pH 2.5-3.0, and then subjected to membrane filtration after standing to obtain a membrane filtration reaction solution; The membrane filtered reaction solution is adjusted to weak acidity and passed through an anion and cation exchange column and nanofiltered to remove salt to obtain a desalted solution, and the desalted solution is concentrated, filtered, crystallized, and dried to obtain β-nicotinamide mononucleotide.
8. The method for synthesizing β-nicotinamide mononucleotide by enzymatic conversion according to claim 1, characterized in that: In the step of adjusting the membrane filtration reaction liquid to weak acidity and passing it through an anion and cation exchange column and nanofiltration to remove salt from the anion and cation exchange resin, the membrane filtration reaction liquid is exchanged through the cation column to obtain a cation column eluate, The pH of the cation column eluate is adjusted to 5.5-6.5, and then passed through an anion exchange column to obtain an anion column effluent.
9. The method for synthesizing β-nicotinamide mononucleotide by enzymatic conversion according to claim 8, characterized in that: In the step of adjusting the membrane filtration reaction liquid to weak acidity and passing it through anion and cation exchange columns and nanofiltration to remove salt from anion and cation exchange resins, the cation exchange resin is preferably a weakly acidic cation exchange resin XRK540, and the anion exchange resin is preferably a weakly basic anion exchange resin XRK209.
10. The method for synthesizing β-nicotinamide mononucleotide by enzymatic conversion according to claim 8, characterized in that: The desalted solution is concentrated at low temperature and reduced pressure to a concentration of 15-25%, ethanol is added for crystallization, the crystals are grown at 0-5° C. for 5 hours, filtered and dried to obtain purified β-nicotinamide mononucleotide.