A fully enzymatic method for synthesizing NMN involving adenosine

Abstract

The present invention provides a total enzymatic synthesis method for NMN involving adenosine, which includes a production step of NMN by enzymatic reactions including a step of (A) reacting adenosine, phosphate, and sugars that can be metabolized by yeast cells with the catalytic action of yeast cells to produce ATP in the same reaction system, and (B) reacting nicotinamide, PRPP, and ATP with the action of NAMPT to produce NMN, ADP, and phosphate. In this way, efficient synthesis of NMN can be completed by unifying a series of reactions such as ATP production (regeneration), NMN synthesis, and ATP utilization within a single reaction system.

Description

[Technical Field]

[0001] The present invention relates to the technical field of β-nicotinamide mononucleotide (NMN) synthesis, and in particular to a fully enzymatic method for synthesizing NMN involving adenosine. [Background technology]

[0002] β-Nicotinamide mononucleotide (NMN) is the direct precursor for synthesizing nicotinamide adenine dinucleotide (NAD) in the body. Supplementing with NMN is the most effective way to increase the body's NAD content level and has broad, profound, and significant health implications for promoting normal metabolism. Because NAD levels decrease in elderly people and they are unable to obtain sufficient NMN from food, NMN is expected to become a widely applied supplement.

[0003] Currently, there are four conventional NMN synthesis techniques: fermentation, chemical synthesis, semi-synthesis, and full-enzyme synthesis. Fermentation requires the creation of a microbial strain capable of producing NMN, which is then mass-cultured and propagated to synthesize NMN in the bacterial cells. Because the basic activity of the key enzyme (NAMPT, nicotinamide phosphoribosyltransferase) responsible for catalytically synthesizing NMN in various species, including lower unicellular organisms, is generally low, it is extremely difficult to create a bacterial strain capable of efficiently expressing NMN. Furthermore, the NMN synthesis pathway is long and involves multiple enzymes and natural degrading enzymes, making efficient large-scale NMN production via fermentation extremely difficult, resulting in high process costs and a lack of market competitiveness. Chemical synthesis uses basic raw materials such as nicotinamide (or nicotinic acid), tetraacetyl ribose, and triphenylphosphine oxide to first synthesize nicotinamide riboside (NR) through chemical methods, and then phosphorylates NR to obtain NMN. The main problem with this method is that the second step, the chemical phosphorylation step, is flammable, explosive, and highly toxic. Large-scale industrialization would pose serious challenges to environmental protection and production safety supervision and management. Other issues include the presence of chemical enantiomer impurities, toxic raw materials, and residual solvents, creating persistent safety concerns for consumers regarding the long-term use of the product on the human body. The semi-synthetic method involves chemically synthesizing NR and then enzymatically phosphorylating it to obtain NMN. This method combines the advantages and disadvantages of the chemical and enzymatic methods, with the main issues being the risk of residual solvents and toxic components in the chemical method and the high cost of the enzymatic method's phosphorylation step, which requires expensive adenosine triphosphate (ATP). The fully enzymatic method uses nicotinamide, ribose, ATP, and other basic materials, and sequentially catalyzes a series of enzymes to form NMN. Although this method has the advantage of being environmentally friendly and safe, it has the drawback of high enzyme costs due to the need to express, purify, and immobilize various enzymes. Another major problem with the all-enzyme method is that it requires a large amount of ATP, which makes the method too expensive and prevents it from being widely used.

[0004] Of the four conventional methods for synthesizing NMN, the fully enzymatic method is a natural synthesis method found in the body, and has clear advantages in terms of safety and environmental protection. If its production costs can be appropriately reduced, it should be the most competitive method in the market. This method uses nicotinamide, ribose, ATP, etc. as its main raw materials and produces NMN through three enzymatic reactions: (1) ribose is converted to ribose-5-phosphate by catalysis with ribokinase (RK), (2) ribose-5-phosphate is converted to phosphoribosyl diphosphate (PRPP) by catalysis with phosphoribosyl pyrophosphate transferase (RPPK), and (3) PRPP and nicotinamide (NAM) are combined by the action of nicotinamide phosphoribosyltransferase (NAMPT) to produce NMN. All three steps of the fully enzymatic method require the consumption of ATP. In the first two steps, ATP acts as a substrate, providing a phosphate group, producing ADP and AMP, respectively. In the third step, ATP is hydrolyzed to provide energy, producing ADP and phosphate. Due to the high cost of ATP, production practices require the coupling of an additional enzymatic reaction to repeatedly utilize ATP (AMP → ADP → ATP). These two reactions must be catalyzed by two specific enzymes, and polyphosphate (e.g., pyrophosphate, tripolyphosphate, or hexametaphosphate) must provide the phosphate group as a substrate. In addition to ATP, accumulated phosphate also accumulates. Because the large amount of accumulated phosphate can affect subsequent reactions, a phosphate removal step is required during the process, which also affects the ATP recovery rate. Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide an all-enzymatic synthesis method for NMN involving adenosine that maintains the advantages of the conventional all-enzymatic method while simplifying the purification process for NMN products and reducing raw material costs, thereby resulting in low production costs. Compared to conventional all-enzyme methods, the present invention uses inexpensive adenosine instead of ATP, and introduces yeast cells during the reaction to convert adenosine to ATP in accordance with energy metabolism. This combines with the conventional all-enzyme method to achieve repeated use of ATP, eliminating the ATP recovery process, and using the phosphate formed in the conventional all-enzyme method as a reactant, thereby eliminating the phosphate removal process. One object of the present invention is to provide a fully enzymatic method for synthesizing NMN involving adenosine, which thus simplifies the NMN product purification process. One object of the present invention is to provide a method for fully enzymatically synthesizing NMN involving adenosine, which, compared to the conventional fully enzymatic method, uses inexpensive adenosine instead of ATP, and introduces yeast cells into the reaction to convert adenosine to ATP according to energy metabolism. This method combines the conventional fully enzymatic method to achieve repeated use of ATP and reduce consumption of the ATP raw material. Furthermore, because the price of adenosine is much lower than that of ATP, the raw material cost of the corresponding NMN product is significantly reduced, resulting in lower production costs. Compared to the conventional method of consuming ATP as a raw material, the present invention uses inexpensive adenosine instead of ATP, and introduces yeast cells into the reaction to convert adenosine into ATP according to energy metabolism, thereby combining this with the conventional all-enzyme method to achieve repeated use of ATP, and the phosphate formed in the conventional all-enzyme method can be used as a reactant. In other words, after separating and purifying the NMN product, other reactants and products can be reused, reducing emissions. One object of the present invention is to provide a method for fully enzymatically synthesizing NMN involving adenosine, which makes the production of the corresponding NMN product environmentally friendly and reduces environmental costs. One object of the present invention is to provide a fully enzymatic method for synthesizing NMN involving adenosine, which uses nicotinamide, ribose, adenosine, phosphate, and sugars that can be metabolized by yeast cells as raw materials, and uses RK, RPPK, NAMPT, and yeast cells as catalysts, to unify ATP production, NMN synthesis, and ATP utilization within a single reaction system, thereby completing the efficient synthesis of NMN and ensuring the efficiency of NMN product synthesis while essentially completely consuming the various reactants, thereby reducing their excretion. This method is therefore simpler, easier to implement, and less costly than conventional fully enzymatic methods. [Means for solving the problem]

[0006] According to one aspect of the present invention, the method for total enzymatic synthesis of NMN involving adenosine according to the present invention includes the following steps in the same reaction system: (A) reacting adenosine, phosphate, and sugars that can be metabolized by yeast cells catalyzed by the yeast cells to produce ATP; (B) A step of producing NMN by an enzymatic reaction, which includes a step of reacting nicotinamide, PRPP, and ATP with the action of NAMPT to produce NMN, ADP, and phosphate. In one embodiment, step (B) further comprises reacting ribose-5-phosphate and ATP with RPPK catalysis to produce PRPP and AMP. In one embodiment, the adenosine-mediated total enzymatic synthesis method for NMN further comprises the step of converting AMP and phosphate to ADP by the action of yeast cells. In one embodiment, the step (B) further comprises reacting ribose and ATP with the catalytic action of RK to produce ribose-5-phosphate and ADP. In one embodiment, in the reaction system of the fully enzymatic method for synthesizing NMN involving adenosine, RK, RPPK, and NAMPT are present in at least one initial form selected from the group consisting of a liquid enzyme form and an immobilized enzyme form. In one embodiment, in the reaction system of the total enzymatic synthesis method of NMN involving adenosine, the molar ratio of adenosine to ribose is in the range of 0.01 to 1. In one embodiment, in the reaction system of the total enzymatic method for synthesizing NMN involving adenosine, the molar ratio of ribose to phosphate ranges from 1 to 20. In one embodiment, the adenosine-mediated total enzymatic synthesis method for NMN further comprises the step of regenerating ATP by the action of yeast cells using ADP and phosphate. In one embodiment, in the reaction system of the method for totally enzymatically synthesizing NMN involving adenosine, the sugar that can be metabolized by yeast cells is at least one selected from glucose, sucrose, starch, and glycerin. In one embodiment, in the reaction system of the total enzymatic method for synthesizing NMN involving adenosine, the yeast cells are yeast cells capable of oxidative phosphorylation metabolism. In one embodiment, in the reaction system of the method for total enzymatic synthesis of NMN involving adenosine, the yeast cells are at least one species selected from Pichia yeast and Saccharomyces cerevisiae. In one embodiment, a metal ion is further added to the reaction system of the fully enzymatic method for synthesizing NMN involving adenosine. In one embodiment, in the reaction system of the method for totally enzymatically synthesizing NMN involving adenosine, the added metal ion is at least one selected from magnesium ions and manganese ions. In one embodiment, in the reaction system of the total enzymatic synthesis method of NMN involving adenosine, the molar ratio of adenosine to nicotinamide is in the range of 0.01 to 1. In one embodiment, in the reaction system of the total enzymatic method for synthesizing NMN involving adenosine, the molar ratio of nicotinamide to phosphate ranges from 1 to 20. In one embodiment, in the reaction system for the adenosine-related total enzymatic synthesis of NMN, the yeast cells are frozen and preserved wet yeast. In one embodiment, at least one organic reagent selected from toluene and n-butanol is further added to the reaction system of the fully enzymatic method for synthesizing NMN involving adenosine. In one embodiment, step (A) is performed before step (B), thereby providing ATP to the reaction of step (B), thereby forming a reaction state in which step (A) and step (B) mutually promote each other in the same reaction system. DETAILED DESCRIPTION OF THE INVENTION

[0007] The following description is intended to disclose the present invention and enable those skilled in the art to realize the present invention. The preferred embodiments in the following description are merely illustrative, and other obvious modifications may occur to those skilled in the art. The basic principles of the present invention defined in the following description may be applied to other embodiments, modifications, improvements, equivalents, and other technical means without departing from the spirit and scope of the present invention. It should be understood by those skilled in the art that in the disclosure of the present invention, the use of terms such as "longitudinal," "lateral," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," etc., to indicate direction or positional relationships is merely for the purpose of facilitating and simplifying the description of the present invention, and does not indicate or suggest that the devices or elements shown must have a particular orientation, be configured, or operate in a particular orientation, and therefore such terms should not be construed as limiting the present invention. It should be noted that the term "one" should be understood to mean "at least one" or "one or more," i.e., in one embodiment, the number of an element may be one, and in another embodiment, the number of the element may be multiple, and the term "one" should not be understood to limit the number.

[0008] Compared to conventional all-enzyme methods, the present invention uses inexpensive adenosine instead of ATP, and introduces yeast cells into the reaction to convert adenosine to ATP in accordance with energy metabolism. This combines this with the conventional all-enzyme method to achieve repeated use of ATP, and provides a total enzymatic method for synthesizing NMN involving adenosine, in which phosphate formed by the conventional all-enzyme method is used as a reactant.

[0009] Specifically, the above-mentioned fully enzymatic method for synthesizing NMN involving adenosine uses nicotinamide, ribose, adenosine, phosphate, and sugars that can be metabolized by yeast cells as raw materials, and RK, RPPK, NAMPT, and yeast cells as catalysts. ATP production, NMN synthesis, and ATP utilization are all carried out within a single reaction system, enabling efficient NMN synthesis. The reaction equation is nicotinamide + ribose + sugars that can be metabolized by yeast cells + adenosine + phosphate + O → NMN + ATP + CO + H O. In this reaction system, yeast cells provide energy through the dehydrogenation and oxidation of sugars that can be metabolized by yeast cells during oxidative phosphorylation metabolism, promoting the binding of phosphate and adenosine to produce adenylic acid (AMP), which then produces ADP and ATP. The ATP then promotes the production of PRPP from ribose, which then reacts with NAM to produce NMN. The ADP, AMP, adenosine, and phosphates that are both present and produced in the reaction system are automatically converted into ATP by the yeast cells and can continue to participate in the reaction. Compared to the conventional all-enzyme method, the phosphates that are formed can be used as reactants, eliminating the phosphate removal process, and enabling repeated use of ATP, eliminating the ATP recovery process and reducing consumption of ATP raw materials. In this way, the involvement of adenosine simplifies the purification process for NMN products.

[0010] Furthermore, in one embodiment of the present invention, the above-mentioned fully enzymatic method for synthesizing NMN involving adenosine uses nicotinamide, ribose, phosphate, adenosine, sucrose, and magnesium ions as raw materials, RK, RPPK, NAMPT, and budding yeast as catalysts, and reacts them in an aqueous solution at a neutral initial pH while stirring and exposing to air. In this way, ATP production, NMN synthesis, and ATP utilization occur within a single reaction system, with the various reactants essentially completely consumed. The corresponding reaction system is simple, easy to implement, low-cost, and environmentally friendly, resulting in lower environmental costs.

[0011] In another embodiment of the present invention, NMN is produced by a one-pot method using nicotinamide, D-ribose, adenosine, etc. as substrates, budding yeast, RK magnetically immobilized enzyme, RPPK magnetically immobilized enzyme, and NAMPT magnetically immobilized enzyme. Adenosine (final concentration: 50 mM), dipotassium hydrogen phosphate (final concentration: 330 mM), potassium dihydrogen phosphate (final concentration: 70 mM), sucrose (final concentration: 120 mM), magnesium chloride (final concentration: 50 mM), manganese chloride (final concentration: 5 mM), and 300 g of budding yeast were added in that order to a 1 L reaction system, and after thorough stirring to dissolve, the reaction temperature was controlled at 37°C and fermentation was carried out for 1 hour. To the yeast fermentation broth, nicotinamide (final concentration: 100 mM), D-ribose (final concentration: 50 mM), 100 g of magnetically immobilized ribokinase enzyme, 100 g of magnetically immobilized phosphoribosyl diphosphate kinase enzyme, and 300 g of magnetically immobilized nicotinamide phosphoribosyltransferase enzyme (all three of the above magnetically immobilized enzymes were provided by Hong Kong Life Science Research Institute Limited) were added, and the mixture was stirred at 300 rpm to react. The reaction temperature was controlled at 37°C, and the reaction pH was controlled at 6.0 with 3 M sodium hydroxide using an automatic titrator. The concentration of NMN was detected by high-performance liquid chromatography during the reaction, and the reaction was completed within 4 hours, yielding 13.77 g of NMN, with a reaction conversion rate of 82.4%.

[0012] It should be noted that, in order to reduce the amount of enzyme used and the complexity of the reaction, the reaction intermediates ribose-5-phosphate and PRPP of the present invention can be directly added to the reaction system as reaction substrates (raw materials). If ribose-5-phosphate is used as the raw material instead of ribose, there is no need to use ribokinase (RK), and if PRPP is used as the raw material instead of ribose, there is no need to use two enzymes, RK and RPPK.

[0013] In another embodiment of the present invention, the above-mentioned total enzymatic synthesis method of NMN involving adenosine uses phosphoribosyl diphosphate, nicotinamide, adenosine, etc. as substrates, and produces NMN in a one-pot method using yeast and nicotinamide phosphoribosyltransferase. To a 1 L reaction system, nicotinamide to a final concentration of 100 mM, phosphoribosyl diphosphate to a final concentration of 50 mM, adenosine to a final concentration of 50 mM, dipotassium hydrogen phosphate to a final concentration of 330 mM, potassium dihydrogen phosphate to a final concentration of 70 mM, sucrose to a final concentration of 120 mM, magnesium chloride to a final concentration of 50 mM, manganese chloride to a final concentration of 5 mM, 300 g of Saccharomyces cerevisiae, and 300 U of liquid nicotinamide phosphoribosyltransferase were added in that order and stirred thoroughly to dissolve. The reaction temperature was then controlled at 37°C, the reaction pH was controlled at 6.0, and the reaction was carried out with stirring at 300 rpm. The concentration of NMN was detected by high-performance liquid chromatography during the reaction. The reaction was completed within 4 hours, yielding 15.44 g of NMN, with a reaction conversion rate of 92.4%.

[0014] In another embodiment of the present invention, the adenosine-related total enzymatic synthesis method for NMN uses nicotinamide, ribose-5-phosphate, adenosine, and other substrates to produce NMN in a one-pot process using budding yeast, phosphoribosyl diphosphate kinase, and nicotinamide phosphoribosyltransferase. To a 1-L reaction system, adenosine (final concentration: 50 mM), dipotassium hydrogen phosphate (final concentration: 330 mM), potassium dihydrogen phosphate (final concentration: 70 mM), sucrose (final concentration: 120 mM), magnesium chloride (final concentration: 50 mM), manganese chloride (final concentration: 5 mM), and 300 g of budding yeast were added in that order, and the mixture was thoroughly stirred to dissolve. The reaction temperature was then controlled at 37°C, and fermentation was carried out for 1 hour. To the yeast fermentation broth, nicotinamide (final concentration: 100 mM), ribose-5-phosphate (final concentration: 50 mM), 300 U of liquid nicotinamide phosphoribosyltransferase, and 300 U of phosphoribosyl diphosphate kinase were added, and the mixture was stirred at 300 rpm to react. The reaction temperature was controlled at 37°C, and the reaction pH was controlled at 6.0 with 3M sodium hydroxide using an automatic titrator. The NMN concentration was detected during the reaction by high-performance liquid chromatography, and the reaction was completed within 6 hours, yielding 14.42 g of NMN, with a reaction conversion rate of 86.3%.

[0015] To further illustrate the present invention, the method for totally enzymatically synthesizing NMN involving adenosine described in the present invention comprises the following steps in the same reaction system: (A) reacting adenosine, phosphate, and sugars that can be metabolized by yeast cells catalyzed by the yeast cells to produce ATP; (B) A step of producing NMN by an enzymatic reaction, which includes a step of reacting nicotinamide, PRPP, and ATP with the action of NAMPT to produce NMN, ADP, and phosphate. Furthermore, the above step (B) further includes a step of reacting ribose-5-phosphate and ATP with the catalytic action of RPPK to produce PRPP and AMP. It should be noted that the above-mentioned fully enzymatic method for synthesizing NMN involving adenosine further includes the step of converting AMP and phosphate into ADP by the action of yeast cells. Furthermore, the above step (B) further comprises a step of reacting ribose and ATP with the catalytic action of RK to produce ribose-5-phosphate and ADP. In the reaction system of the above-mentioned total enzymatic synthesis method of NMN involving adenosine, RK, RPPK, and NAMPT exist in at least one initial form selected from the liquid enzyme form and the immobilized enzyme form.

[0016] In particular, in the reaction system of the above-mentioned total enzymatic synthesis method of NMN involving adenosine, the molar ratio of adenosine to ribose ranges from 0.01 to 1. Furthermore, in the reaction system of the above-mentioned total enzymatic synthesis method of NMN involving adenosine, the molar ratio of ribose to phosphate ranges from 1 to 20. It should be noted that the above-mentioned fully enzymatic method for synthesizing NMN involving adenosine further includes a step of regenerating ATP by the action of yeast cells using ADP and phosphate.

[0017] In the reaction system of the above-mentioned fully enzymatic method for synthesizing NMN involving adenosine, sugars that can be metabolized by yeast cells include, but are not limited to, a single sugar or a mixture of sugars selected from glucose, sucrose, starch, and glycerin. Furthermore, in the reaction system of the above-mentioned total enzymatic synthesis method of NMN involving adenosine, the yeast cells are various yeast cells capable of oxidative phosphorylation metabolism, such as Pichia yeast or Saccharomyces cerevisiae. Preferably, metal ions such as magnesium ions and manganese ions may be further added to the reaction system of the above-mentioned total enzymatic synthesis method of NMN involving adenosine. Preferably, in the reaction system of the above-mentioned total enzymatic synthesis method of NMN involving adenosine, the molar ratio of adenosine to nicotinamide is in the range of 0.01 to 1. Preferably, in the reaction system of the above-mentioned total enzymatic synthesis method of NMN involving adenosine, the molar ratio of nicotinamide to phosphate is in the range of 1-20. It should be noted that in the reaction system of the above-mentioned method for totally enzymatically synthesizing NMN involving adenosine, the yeast cells may be frozen and stored wet yeast. In particular, in the reaction system of the above-mentioned total enzymatic synthesis method of NMN involving adenosine, an organic reagent such as toluene or n-butanol may be further added. It should be noted that in some embodiments of the present invention, step (A) is carried out before step (B) in the reaction process, thereby providing ATP to the reaction of step (B), so that step (A) and step (B) form a reaction state in which they mutually promote each other in the same reaction system.

[0018] As will be understood by those skilled in the art, the above examples are merely illustrative, and by combining features of different examples with each other, embodiments can be obtained that are easily conceivable based on the contents disclosed in the present invention but are not explicitly pointed out in the above description. It should be understood by those skilled in the art that the embodiments of the present invention shown in the above description are merely illustrative and do not limit the present invention. The objects of the present invention have already been fully and effectively achieved. The function and structural principles of the present invention have been shown and explained in the examples, and any variations or modifications can be made to the embodiments of the present invention without departing from the above principles.

Claims

1. In the same reaction system, adenosine, phosphate, sugars that can be metabolized by yeast cells, yeast cells, NAMPT, nicotinamide, and PRPP are added and reacted in one pot, and the following steps are carried out: (A) reacting adenosine, phosphate, and sugars that can be metabolized by the yeast cells catalyzed by the yeast cells to produce ATP; (B) producing NMN by an enzymatic reaction, which includes reacting nicotinamide, PRPP, and ATP with the action of NAMPT to produce NMN, ADP, and phosphate; A method for the total enzymatic synthesis of NMN involving adenosine, comprising:

2. 2. The method for fully enzymatically synthesizing NMN involving adenosine according to claim 1, wherein step (B) further comprises a step of reacting ribose-5-phosphate and ATP under the catalytic action of RPPK to produce PRPP and AMP.

3. 3. The method for fully enzymatically synthesizing NMN involving adenosine according to claim 2, further comprising the step of converting AMP and phosphate to ADP by the action of yeast cells.

4. 3. The method for totally enzymatically synthesizing NMN involving adenosine according to claim 2, wherein step (B) further comprises a step of reacting ribose and ATP under the catalytic action of RK to produce ribose-5-phosphate and ADP.

5. 5. The method for totally enzymatically synthesizing NMN involving adenosine according to claim 4, wherein in the reaction system of the method for totally enzymatically synthesizing NMN involving adenosine, RK, RPPK and NAMPT are present in at least one initial form selected from the group consisting of a liquid enzyme form and an immobilized enzyme form.

6. 6. The method for totally enzymatically synthesizing NMN involving adenosine according to claim 5, wherein the molar ratio of adenosine to ribose in the reaction system of the method for totally enzymatically synthesizing NMN involving adenosine is in the range of 1:0.01 to 1:

1.

7. 7. The method for totally enzymatically synthesizing NMN involving adenosine according to claim 6, wherein the molar ratio of ribose to phosphate in the reaction system of the method for totally enzymatically synthesizing NMN involving adenosine is in the range of 1:1 to 20.

8. 8. The method for total enzymatic synthesis of NMN involving adenosine according to claim 1, further comprising the step of regenerating ATP by the action of yeast cells using ADP and phosphate.

9. The method for total enzymatic synthesis of NMN involving adenosine according to claim 8, wherein in the reaction system of the method for total enzymatic synthesis of NMN involving adenosine, the sugars that can be metabolized by yeast cells are at least one selected from glucose, sucrose, and starch.

10. 9. The method for totally enzymatically synthesizing NMN involving adenosine according to claim 8, wherein the yeast cells in the reaction system of the method for totally enzymatically synthesizing NMN involving adenosine are yeast cells capable of oxidative phosphorylation metabolism.

11. 9. The method for total enzymatic synthesis of NMN involving adenosine according to claim 8, wherein the yeast cells in the reaction system of the method for total enzymatic synthesis of NMN involving adenosine are at least one species selected from Pichia yeast and Saccharomyces cerevisiae.

12. 9. The method for fully enzymatically synthesizing NMN involving adenosine according to claim 8, wherein a metal ion is further added to the reaction system of the method for fully enzymatically synthesizing NMN involving adenosine.

13. The method for total enzymatic synthesis of NMN involving adenosine according to claim 12, wherein the metal ion added in the reaction system of the method for total enzymatic synthesis of NMN involving adenosine is at least one selected from magnesium ions and manganese ions.

14. 9. The method for totally enzymatically synthesizing NMN involving adenosine according to claim 8, wherein the molar ratio of nicotinamide to phosphate in the reaction system of the method for totally enzymatically synthesizing NMN involving adenosine is in the range of 1:1 to 1:

20.

15. 9. The method for totally enzymatically synthesizing NMN involving adenosine according to claim 8, wherein the yeast cells in the reaction system of the method for totally enzymatically synthesizing NMN involving adenosine are frozen and stored wet yeast cells.

16. 9. The method for totally enzymatically synthesizing NMN involving adenosine according to claim 8, wherein at least one organic reagent selected from the group consisting of toluene and n-butanol is further added to the reaction system of the method for totally enzymatically synthesizing NMN involving adenosine.

17. 9. The method for total enzymatic synthesis of NMN involving adenosine according to claim 8, wherein step (A) is carried out before step (B), thereby providing ATP for the reaction of step (B), thereby forming a reaction state in which steps (A) and (B) promote each other in the same reaction system.