阅读说明：本技术 一种烟酰胺腺嘌呤二核苷酸的制备方法 (Preparation method of nicotinamide adenine dinucleotide ) 是由 李斌 张超 刘发光 张晨晨 翁秋景 缪朱玲 于 2020-12-30 设计创作，主要内容包括：本发明提供一种烟酰胺腺嘌呤二核苷酸的制备方法,所述制备方法包括：向电渗析槽中加入烟酰胺单核苷酸、三磷酸腺苷、烟酰胺单核苷酸腺苷转移酶和水,开启电渗析设备,进行反应。所述制备方法通过电渗析法将反应的副产物焦磷酸从反应体系中移除,从而打破反应平衡,促使反应向正反应方向进行,能够提高产物NAD+的生成率,并降低反应后处理的难度。(The invention provides a preparation method of nicotinamide adenine dinucleotide, which comprises the following steps: adding nicotinamide mononucleotide, adenosine triphosphate, nicotinamide mononucleotide adenylyltransferase and water into an electrodialysis tank, and starting an electrodialysis device to perform reaction. According to the preparation method, the by-product pyrophosphoric acid generated in the reaction is removed from the reaction system through an electrodialysis method, so that the reaction balance is broken, the reaction is promoted to be carried out in the positive reaction direction, the generation rate of the product NAD + can be improved, and the difficulty in post-reaction treatment is reduced.)

1. A preparation method of nicotinamide adenine dinucleotide is characterized by comprising the following steps:

adding nicotinamide mononucleotide, adenosine triphosphate, nicotinamide mononucleotide adenyl transferase and water into an electrodialysis tank, starting an electrodialysis device, and reacting to obtain the nicotinamide adenine dinucleotide.

2. The method according to claim 1, wherein the mass ratio of nicotinamide mononucleotide, adenosine triphosphate, nicotinamide mononucleotide adenyltransferase and water is 1 (0.7-2.0): 0.1-10.0): 20-200;

preferably, the nicotinamide mononucleotide adenyl transferase comprises an immobilized nicotinamide mononucleotide adenyl transferase.

3. The method according to claim 1 or 2, wherein the stirring speed of the reaction is 100 to 300 rpm.

4. The method according to any one of claims 1 to 3, wherein the reaction temperature is 20 to 40 ℃.

5. The method according to any one of claims 1 to 4, wherein the reaction time is 4 to 25 hours.

6. A method as claimed in any one of claims 1 to 5, characterized in that the electrodialysis apparatus is operated continuously.

7. The production process according to any one of claims 1 to 5, wherein the electrodialysis device is opened at intervals;

preferably, the interval time of the interval opening is 1-2 h;

preferably, the duration time of the interval opening is 1-2 h.

8. The production method according to any one of claims 1 to 7, wherein the pH of the reaction system in the electrodialysis cell is 5 to 7;

preferably, the conductivity of the reaction system is less than 10000 mus/cm, preferably 3000-5000 mus/cm.

9. The production method according to any one of claims 1 to 8, characterized by comprising:

adding nicotinamide mononucleotide, adenosine triphosphate, nicotinamide mononucleotide adenyltransferase and water into an electrodialysis tank, wherein the mass ratio of nicotinamide mononucleotide, adenosine triphosphate, nicotinamide mononucleotide adenyltransferase and water is 1 (0.7-2.0) to (0.1-10.0) to (20-200), continuously starting electrodialysis equipment, controlling the conductivity of a reaction system in a reactor to be less than 10000 mus/cm and the pH to be 5-7, and reacting at 20-40 ℃ and 100-300 rpm for 4-25 hours to obtain the nicotinamide adenine dinucleotide.

10. The method of any one of claims 1 to 8, comprising the steps of:

adding nicotinamide mononucleotide, adenosine triphosphate, nicotinamide mononucleotide adenyltransferase and water into an electrodialysis tank, wherein the mass ratio of nicotinamide mononucleotide, adenosine triphosphate, nicotinamide mononucleotide adenyltransferase and water is 1 (0.7-2.0) to (0.1-10.0) to (20-200), starting electrodialysis equipment at intervals, the interval time is 1-2 h, the starting duration is 1-2 h, controlling the conductivity of a reaction system in a reactor to be less than 10000 mu s/cm, controlling the pH to be 5-7, and reacting at 20-40 ℃ and 100-300 rpm for 4-25 h to obtain the nicotinamide adenine dinucleotide.

Technical Field

The invention belongs to the technical field of pharmacy, and relates to a preparation method of nicotinamide adenine dinucleotide.

Background

Nicotinamide adenine dinucleotide (NAD +) is a coenzyme for transferring protons, participates in various physiological activities such as substance metabolism, energy synthesis and DNA repair, and has an important effect on the immunity of an organism. There are several diseases that have been shown to be associated with imbalances in the concentration of NAD + in the body.

At present, NAD + has been widely used for treating diseases, and its synthesis methods mainly include chemical methods, chemical enzyme methods and biological fermentation methods. Chemical methods have long reaction routes and require expensive reagents and large amounts of organic solvents, and therefore, enzymatic chemical methods and biological fermentation methods are often used for the industrial production of NAD +.

Lvxiangqu et al (Luxiangqu, Zuiquan, Xuxu, et al. expression, purification and activity determination of Nicotinamide mononucleotide adenylyltransferase [ J ]. second university of Leguminosae, 2010(11): 1251-.

The NMNAT is reported to catalyze the reaction of NMN and ATP to synthesize NAD + to be a reversible reaction, which generates pyrophosphoric acid as a by-product, when the concentration of pyrophosphate in the reaction system reaches a certain level, the reaction can reach an equilibrium, thus being unfavorable for the complete conversion of NMN into NAD +.

Lu (Dongmei Lu, Guiqiu Xie, Renjun Gao Cloning, Purification, and catalysis of inorganic pyrophosphatase from the hyperthermophilic mechanism of supercoccus horikoshii [ J ]. Protein Expression and Purification,2014,99:94-98.) and the like use pyrophosphatase to decompose pyrophosphoric acid, thereby removing pyrophosphoric acid in the reaction catalyzing the synthesis of NAD + from NMN and ATP, and promoting the reaction to proceed toward the synthesis of NAD +, but the additional addition of pyrophosphatase increases the cost, and also generates other byproducts, reduces the yield of NAD + and increases the difficulty of post-treatment.

In conclusion, the preparation method which is simple in process, low in cost and capable of achieving high conversion rate is provided, and the preparation method has important significance for the field of NAD + preparation.

Disclosure of Invention

Aiming at the defects and actual requirements of the prior art, the invention provides the preparation method of the nicotinamide adenine dinucleotide, which is simple to operate, and can improve the conversion rate of the reaction and reduce the difficulty of the post-treatment of the reaction.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a preparation method of nicotinamide adenine dinucleotide, which comprises the following steps:

adding nicotinamide mononucleotide, adenosine triphosphate, nicotinamide mononucleotide adenyl transferase and water into an electrodialysis tank, starting an electrodialysis device, and reacting to obtain the nicotinamide adenine dinucleotide.

According to the invention, Nicotinamide adenine dinucleotide (NAD +) can catalyze synthesis of Nicotinamide Mononucleotide (NMN) and Adenosine Triphosphate (ATP) by Nicotinamide mononucleotide adenylyltransferase (NMNAT).

Preferably, the mass ratio of nicotinamide mononucleotide, adenosine triphosphate, nicotinamide mononucleotide adenyltransferase and water is 1 (0.7-2.0): (0.1-10.0): (20-200), including but not limited to 1:0.9:0.2:25, 1:1.2:0.3:40, 1:1.5:1.5:60, 1:1.6:5.5:100, 1:1.7:7.5:150 or 1:1.9:9.5: 180.

According to the invention, through controlling the mass ratio of the nicotinamide mononucleotide to the nicotinamide mononucleotide adenylyltransferase to be 1 (0.1-10.0), the nicotinamide mononucleotide adenylyltransferase can be efficiently catalyzed to synthesize the nicotinamide mononucleotide adenylyltransferase by the nicotinamide mononucleotide and the adenosine triphosphate, and waste caused by meaningless excessive use of the nicotinamide mononucleotide adenylyltransferase can be avoided.

Preferably, the nicotinamide mononucleotide adenyl transferase comprises an immobilized nicotinamide mononucleotide adenyl transferase.

In the invention, physical or chemical methods are adopted to transform nicotinamide mononucleotide adenylyltransferase into immobilized enzyme, the transformed immobilized nicotinamide mononucleotide adenylyltransferase is insoluble in water but still has enzyme activity, and the NAD + is efficiently catalytically synthesized in a space range containing high-concentration NMN and ATP, so that the NAD synthesis efficiency is improved to a certain extent. The preparation method of the required immobilized nicotinamide mononucleotide adenyl transferase comprises the following steps:

(1) the sequencing-verified recombinant plasmid (Nmnat-pET28a +) was transformed into competent cells and plated on LB (100. mu.g/mL) kanamycin agarose plates and incubated overnight at 37 ℃;

(2) the positive clones were inoculated into 2 XYT or TB culture medium containing kanamycin (75. mu.g/mL) and chloramphenicol (37. mu.g/mL), cultured overnight at 37 ℃ with shaking at 200r/min, diluted to a medium containing the same antibiotic at a ratio of 1:20, and cultured to OD₆₀₀When the temperature is 0.6 ℃, adding IPTG, and inducing at 28 ℃ or 37 ℃;

(3) collecting 1mL of bacterial solution, centrifuging at 13400 Xg for 1min, collecting the bacteria, and suspending the bacteria in lysis buffer (20mmol/L Tris-HCl, pH 7.5, 2mM Mg)²⁺And 0.7mM Mn²⁺) In 200W, cells are ultrasonically cracked, and ultrasonic treatment is carried out for 30min at intervals of 9s for 1 s. Centrifuging lysate at 4 deg.C and 15000 Xg for 50min, collecting supernatant, mixing with sodium alginate solution at volume ratio of 1:1, and slowly adding into 0.2mol/L NaCl and 0.2mol/L CaCl under stirring₂In the solution, controlling the diameter of particles to be 2-3 mm, stirring for more than 45min, filtering, collecting particles, and placing the particles in 0.2mol/L NaCl and 0.2mol/L CaCl₂Storing in the solution at 4 deg.C for use, and filtering to obtain immobilized nicotinamide mononucleotide adenyl transferase.

Preferably, the stirring speed of the reaction is 100-300 rpm, including but not limited to 120rpm, 150rpm, 180rpm, 200rpm, 240rpm, 260rpm or 280 rpm.

Preferably, the reaction temperature is 20-40 ℃, including but not limited to 21 ℃, 22 ℃, 23 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃ or 40 ℃.

Preferably, the reaction time is 4-25 h, including but not limited to 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h or 24 h.

Preferably, the electrodialysis apparatus is switched on continuously.

Preferably, the electrodialysis device is opened at intervals.

Preferably, the interval time of the interval opening is 1-2 h, including but not limited to 1.2h, 1.4h, 1.6h or 1.8 h.

Preferably, the duration of the interval opening is 1-2 h, including but not limited to 1.2h, 1.4h, 1.6h or 1.8 h.

Preferably, the pH value of a reaction system in the electrodialysis tank is 5-7.

Preferably, the conductivity of the reaction system is less than 10000 μ s/cm, including but not limited to 8000 μ s/cm, 7000 μ s/cm, 6000 μ s/cm, 5000 μ s/cm, 3000 μ s/cm or 2500 μ s/cm, preferably 3000-5000 μ s/cm.

As a preferred technical scheme, the preparation method of the nicotinamide adenine dinucleotide comprises the following steps:

adding nicotinamide mononucleotide, adenosine triphosphate, nicotinamide mononucleotide adenyltransferase and water into an electrodialysis tank, wherein the mass ratio of nicotinamide mononucleotide, adenosine triphosphate, nicotinamide mononucleotide adenyltransferase and water is 1 (0.7-2.0): (0.1-10.0): (20-200), continuously starting electrodialysis equipment, reacting at 20-40 ℃ and 100-300 rpm for 4-25 h, and obtaining the nicotinamide adenine dinucleotide, wherein the conductivity of a reaction system in a reactor is less than 10000 mus/cm, the pH is 5-7.

As a preferred technical scheme, the preparation method of the nicotinamide adenine dinucleotide comprises the following steps:

adding nicotinamide mononucleotide, adenosine triphosphate, nicotinamide mononucleotide adenyltransferase and water into an electrodialysis tank, wherein the mass ratio of nicotinamide mononucleotide, adenosine triphosphate, nicotinamide mononucleotide adenyltransferase and water is 1 (0.7-2.0) to (0.1-10.0) to (20-200), starting electrodialysis equipment at intervals, the interval time is 1-2 h, the starting duration is 1-2 h, controlling the conductivity of a reaction system in a reactor to be less than 10000 mu s/cm, controlling the pH to be 5-7, and reacting at 20-40 ℃ and 100-300 rpm for 4-25 h to obtain the nicotinamide adenine dinucleotide.

Compared with the prior art, the invention has at least the following beneficial effects:

(1) according to the preparation method, the by-product pyrophosphoric acid generated in the reaction is removed from the reaction system through an electrodialysis method, so that the reaction balance is broken, the reaction is promoted to be carried out in the positive reaction direction, the generation rate of the reaction product NAD + is improved, and the generation rate of the product NAD + is higher than 73.5 percent and is 96 percent at most;

(2) according to the method, the pH value of a reaction system in the reactor is controlled to be 5-7, the conductivity is 3000-5000 mu s/cm, the electrodialysis equipment is continuously started or is started at intervals of 1-2 h, the generation of the product NAD + is improved, the generation rate of the product NAD + is higher than 92%, and in addition, the difficulty of post-treatment is reduced due to the removal of pyrophosphoric acid;

(3) the preparation method provided by the invention is simple to operate, high in conversion rate, easy to separate the product, and wide in application prospect in preparation of nicotinamide adenine dinucleotide.

Drawings

FIG. 1 shows the detection result of pyrophosphate in the reaction solution before starting the electrodialysis apparatus;

FIG. 2 is a detection result of pyrophosphoric acid in the reaction solution after the electrodialysis device is turned on;

FIG. 3 shows the results of detection of pyrophosphate in the solution in the separation vessel.

Detailed Description

For the purpose of facilitating an understanding of the present invention, the present invention will now be described by way of examples. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

The electrodialysis device in the embodiment of the invention is an EX-10 electrodialysis experimental device of Hangzhou blue environmental technology GmbH.

The preparation method of the immobilized nicotinamide mononucleotide adenyl transferase comprises the following steps:

(1) transformation of sequencing-verified recombinant plasmid (Nmnat-pET28a +) whose sequence is referred to in the study of Luxian et al (Ludwig, Zuiqu, Xuxuweng, et al., Nicotinamide mononucleotide adenyltransferase expression, purification and activity assay [ J ] second Leguminosae, 2010(11): 1251-1254.) into E.coli DH 5. alpha. competent cells and plated on LB (100. mu.g/mL) kanamycin agarose plates and incubated overnight at 37 ℃;

(2) inoculating the positive clone into 2 XYT culture solution containing 75. mu.g/mL kanamycin and 37. mu.g/mL chloramphenicol, shaking-culturing at 37 deg.C and 200r/min overnight, diluting at a ratio of 1:20 into 2 XYT culture solution containing the same antibiotic, and culturing to OD₆₀₀When the temperature is 0.6 ℃, adding IPTG (isopropyl thiogalactoside), and inducing at 37 ℃;

(3) collecting 1mL of the induced bacterial solution, centrifuging at 13400 Xg for 1min to collect the bacteria, and resuspending the bacteria in 20mmol/L Tris-HCl (pH 7.5, containing 2mM Mg)²⁺And 0.7mM Mn²⁺) Performing ultrasonic lysis on cells at 200W for 30min at a gap of 9s for 1s, centrifuging lysate at 4 deg.C for 50min at 15000 Xg, collecting supernatant, mixing with sodium alginate solution at a volume ratio of 1:1, and slowly adding 0.2mol/L NaCl and 0.2mol/L CaCl into the mixed solution with stirring by using an injector₂In the solution, controlling the particle diameter to be 2-3 mm, stirring for 45min, filtering and collecting particles, and placing the particles in 0.2mol/L NaCl and 0.2mol/L CaCl₂Storing in solution at 4 deg.C for use, and filtering to obtain immobilized nicotinamide mononucleotide adenylyltransferase(immobilized NMNAT).

High performance liquid chromatography conditions: NUCLEOTIEDUER C18 Pyramid, 4.6mm × 250mm, 5 μm; flow rate 1.0mL/min, ultraviolet detection at 260nm, column temperature: 30 ℃, injection volume: 10 μ L, mobile phase: 0.1% TFA.

Example 1

This example provides a method for preparing NAD +, the method comprising:

adding 4L of water, 80g of NMN and 120g of ATP into an electrodialysis tank, adding 20g of immobilized NMNAT at 100rpm and 25 ℃, starting an electrodialysis device, controlling the pH value to be 5.0 +/-0.2, removing pyrophosphoric acid generated by the reaction, continuously starting the electrodialysis device, and keeping the conductivity of a reaction system in a reaction cell to be 3000 mu s/cm; reaction was carried out for 15h, samples were taken and the product NAD + formation was determined using High Performance Liquid Chromatography (HPLC).

The production rate of NAD + as a product is shown in Table 1.

Example 2

This example provides a method for preparing NAD +, the method comprising:

adding 4L of water, 80g of NMN and 120g of ATP into an electrodialysis tank, adding 20g of immobilized NMNAT at 300rpm and 20 ℃, starting an electrodialysis device, controlling the pH value to be 6.0 +/-0.2, removing pyrophosphoric acid generated by reaction, starting the electrodialysis device at intervals of 1h, keeping the running time of each time for 1h, and keeping the conductivity of a reaction system in a reaction cell at 4000 mus/cm; the reaction was carried out for 25h, samples were taken and the rate of production of NAD + was determined using High Performance Liquid Chromatography (HPLC). The immobilized enzyme (which can be used) is recovered from the reaction liquid by filtration, and the filtrate is subjected to nanofiltration concentration and crystallization to obtain the product NAD + with the purity of 99.1 percent.

The production rate of NAD + as a product is shown in Table 1.

Example 3

This example provides a method of preparing NAD, the method comprising:

adding 4L of water, 20g of NMN and 40g of ATP into an electrodialysis tank, adding 200g of NMNAT at 300rpm and 35 ℃, starting an electrodialysis device, controlling the pH value to be 7.0 +/-0.2, removing pyrophosphoric acid generated by reaction, starting the electrodialysis device at intervals of 2 hours, keeping the running time of each time for 2 hours, and keeping the conductivity of a reaction system in a reaction cell to be 5000 mus/cm; reaction was carried out for 8h, samples were taken and the product NAD + formation was determined using High Performance Liquid Chromatography (HPLC).

The production rate of NAD + as a product is shown in Table 1.

Example 4

This example provides a method for preparing NAD +, the method comprising:

adding 4L of water, 200g of NMN and 140g of ATP into an electrodialysis tank, adding 20g of immobilized NMNAT at 300rpm and 40 ℃, starting an electrodialysis device, controlling the pH value to be 4.0 +/-0.2, removing pyrophosphoric acid generated by reaction, starting the electrodialysis device at intervals of 1.5h, keeping the running time of each time for 1.5h, and keeping the conductivity of a reaction system in a reaction tank to be 3000 mu s/cm; reaction was carried out for 4h, samples were taken and the product NAD + formation was determined using High Performance Liquid Chromatography (HPLC).

The production rate of NAD + as a product is shown in Table 1.

Example 5

The difference from example 1 is only that the pH value is controlled to 8.0. + -. 0.2 in step (2), and the rest is the same as example 1.

Example 6

The difference from example 1 was only that the conductivity was controlled to 8000. mu.s/cm in step (2), and the rest was the same as example 1.

Example 7

The difference from example 1 was only that the conductivity was controlled to 7000. mu.s/cm in step (2), and the rest was the same as example 1.

Example 8

The difference from example 1 was only that the conductivity was controlled to 6000. mu.s/cm in step (2), and the other examples were the same as example 1.

Example 9

The only difference compared with example 2 is that the electrodialysis device of step (2) is opened at an interval of 3h, and the other steps are the same as example 2.

Comparative example 1

This comparative example provides a method of preparing NAD +, the method comprising the steps of:

(1) adding 4L of water, 80g of NMN and 120g of ATP into a reaction tank, adding 20g of immobilized NMNAT at 300rpm and 20 ℃, and controlling the pH value to be 6.0 +/-0.2;

(2) the reaction was carried out for 25h, samples were taken and the rate of production of NAD + was determined using High Performance Liquid Chromatography (HPLC).

The production rate of NAD + as a product is shown in Table 1.

TABLE 1

Numbering	Rate of production of NAD +)
		Example 1	95％
Example 2	96％
		Example 3	92％
Example 4	78％
		Example 5	74.7％
Example 6	73.5％
		Example 7	82％
Example 8	86.7％
		Example 9	84.8％
Comparative example 1	62％

As shown in Table 1, in examples 1-9, the production rate of NAD + was higher than 73.5% and at most 96% by the electrodialysis method, while in the comparative example, the production rate of NAD + was only 62% without the electrodialysis method, which indicates that the present invention removes pyrophosphoric acid, which is a by-product of the reaction, from the reaction system by the electrodialysis method, thereby breaking the reaction equilibrium, and promoting the reaction to proceed in the direction of the forward reaction, thereby increasing the production rate of NAD + which is a reaction product.

In the embodiment 1-3, the pH of the reaction system in the reactor is controlled to be 5-7, the conductivity is 3000-5000 mu s/cm, the electrodialysis device is continuously opened or opened at intervals of 1-2 h, and the generation rate of the reaction product NAD + is higher than 92%, compared with the embodiment 4 and the embodiment 5 in which the pH of the reaction system in the reactor is not controlled to be 5-7, the generation rate of the reaction product NAD + is lower and is only 78% at most; in the embodiment 6-8, the conductivity of the reaction system in the uncontrolled reactor is 3000-5000 mus/cm, and the generation rate of the product NAD + is only 86.7% at most; in the embodiment 9, the electrodialysis equipment is not controlled to be continuously started or started at intervals of 1-2 h, and the generation rate of the product NAD + is 84.8%; the synthesis of the above results show that the invention can further improve the generation rate of the product NAD + by controlling the pH and the conductivity of the reaction system in the reactor and the starting mode of the electrodialysis equipment.

Test example 1

The test example detects the effect of removing the pyrophosphoric acid in the reaction system by the electrodialysis device.

Taking example 1 as an example, the electrodialysis apparatus had a plurality of cells, and each reaction material was charged into one cell to carry out the reaction, the cell being a reaction cell, and after the electrodialysis apparatus was turned on, the pyrophosphate in the reaction cell was transferred to an adjacent cell, the cell being a separation cell. Respectively taking 10mL of the reaction solution before the electrodialysis equipment is started, 10mL of the reaction solution after the electrodialysis equipment is started and 10mL of the solution in the separation tank of the electrodialysis equipment, respectively dripping 1 drop of 0.1% silver nitrate solution, and observing the turbid condition of the solution.

The reaction solution before the electrodialysis device was turned on was turbid (as shown in FIG. 1), indicating that pyrophosphate was present in a high concentration in the reaction solution, the reaction solution after the electrodialysis device was turned on was clear (as shown in FIG. 2), indicating that pyrophosphate was not contained or contained in a lower concentration, the solution in the separation tank was turbid (as shown in FIG. 3), indicating that pyrophosphate was contained in a high concentration, and it was shown that the present invention was able to effectively remove pyrophosphate from the reaction system using the electrodialysis method.

In conclusion, the preparation method of nicotinamide adenine dinucleotide disclosed by the invention couples the enzyme reaction and the removal of the byproduct pyrophosphoric acid together through an electrodialysis method, so that the reaction balance is broken, the reaction is promoted to be carried out in the positive reaction direction, the generation rate of the NAD + product is improved, the generation rate of the NAD + product is higher than 73.5 percent and is 96 percent at most, the difficulty of post-treatment is reduced, and the preparation method has a wide application prospect in preparation of nicotinamide adenine dinucleotide.

The applicant states that the present invention is illustrated by the above examples to show the detailed process equipment and process flow of the present invention, but the present invention is not limited to the above detailed process equipment and process flow, i.e. it does not mean that the present invention must rely on the above detailed process equipment and process flow to be implemented. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.