METHOD FOR SYNTHESIZING BETA-NICOTINAMIDE MONONUCLEOTIDE AND INTERMEDIATE THEREOF

Abstract

The present disclosure relates to a synthesis method of β-nicotinamide mononucleotide (β-NMN) and an intermediate thereof. In the present disclosure, phospholipid metabolism enzymes phospholipase D (PLD) and phospholipase C (PLC) widely present in the biosphere are used as catalysts to prepare β-NMN through two-step enzymolysis or one-pot synthesis; and an intermediate, namely phosphatidyl nicotinamide riboside (PNR), is obtained during the two-step enzymolysis. The present disclosure has simple reaction steps, low production cost, and environmental friendliness, and is suitable for large-scale industrial production.

Claims

1. A synthesis method of β-nicotinamide mononucleotide (β-NMN), comprising any one of (a) and (b): (a): two-step enzymolysis a1: with nicotinamide riboside (NR) and a phospholipid as substrates, conducting a catalytic reaction under the catalysis of a phospholipase D (PLD) in the presence of a calcium ion to produce phosphatidyl nicotinamide riboside (PNR); and a2: with the PNR produced in step a1 as a substrate, conducting a catalytic reaction under the catalysis of a phospholipase C (PLC) in the presence of a calcium ion to produce the β-NMN; (b): one-pot synthesis to prepare the β-NMN.

2. The synthesis method according to claim 1, wherein the one-pot synthesis to prepare the β-NMN is any one of (1) and (2): (1): with NR and a phospholipid as substrates, in the presence of a calcium ion, adding a PLD to allow a reaction, and after the reaction is completed, adding a PLC to allow a reaction to obtain the β-NMN; and (2): with NR and a phospholipid as substrates, in the presence of a calcium ion, adding a PLD and a PLC together to allow a reaction to obtain the β-NMN.

3. The synthesis method according to claim 1, wherein the PNR is I: ##STR00002## wherein R.sub.1 and R.sub.2 each are fatty acyl; natural phospholipids R.sub.1 and R.sub.2 are mostly long-chain fatty acids (LCFAs) that are preferably selected from the group consisting of C.sub.14-24 fatty acids and more preferably selected from the group consisting of C.sub.16 and C.sub.18 fatty acids; and further, R.sub.1 is any one selected from the group consisting of —C.sub.15H.sub.31, —C.sub.17H.sub.35, or —C.sub.17H.sub.33 and R.sub.2 is any one of a —C.sub.17H.sub.31, —C.sub.19H.sub.29, —C.sub.19H.sub.31, —C.sub.21H.sub.31, or —C.sub.17H.sub.33

4-10. (canceled)

11. The synthesis method according to claim 1, wherein the phospholipid is a natural phospholipid or a synthetic phospholipid; further preferably, a main component of the phospholipid is phosphatidylcholine and/or phosphatidylethanolamine; and further preferably, the phospholipid is lecithin.

12. The synthesis method according to claim 2, wherein the phospholipid is a natural phospholipid or a synthetic phospholipid; further preferably, a main component of the phospholipid is phosphatidylcholine and/or phosphatidylethanolamine; and further preferably, the phospholipid is lecithin.

13. The synthesis method according to claim 1, wherein the PLC is a PLC ubiquitous in organisms; and the PLC is preferably a broad-spectrum PLC and more preferably a phosphatidylinositol-specific phospholipase C (PIPLC).

14. The synthesis method according to claim 2, wherein the PLC is a PLC ubiquitous in organisms; and the PLC is preferably a broad-spectrum PLC and more preferably a phosphatidylinositol-specific phospholipase C (PIPLC).

15. The synthesis method according to claim 1, wherein the PLD is a PLD ubiquitous in organisms; and further preferably, the PLD is a PLD derived from a microorganism.

16. The synthesis method according to claim 2, wherein the PLD is a PLD ubiquitous in organisms; and further preferably, the PLD is a PLD derived from a microorganism.

17. The synthesis method according to claim 1, wherein: a molar ratio of the NR to the phospholipid in a1 is 1:10 to 10:1; further preferably, the molar ratio of the NR to the phospholipid in a1 is 1:5 to 5:1; and further more preferably, the molar ratio of the NR to the phospholipid in a1 is 1:2 to 3:1; a concentration of the calcium ion in a reaction system is 0.01 g/L to 20 g/L, and further preferably, the concentration of the calcium ion in the reaction system is 0.5 g/L to 5 g/L; and the catalytic reaction of the PLD is conducted at a temperature of 20° C. to 70° C. and a pH of 4.5 to 7.5; and further preferably, the catalytic reaction is conducted at a temperature of 40° C. to 60° C. and a pH of 5.0 to 6.5.

18. The synthesis method according to claim 2, wherein: a molar ratio of the NR to the phospholipid in a1 is 1:10 to 10:1; further preferably, the molar ratio of the NR to the phospholipid in a1 is 1:5 to 5:1; and further more preferably, the molar ratio of the NR to the phospholipid in a1 is 1:2 to 3:1; a concentration of the calcium ion in a reaction system is 0.01 g/L to 20 g/L, and further preferably, the concentration of the calcium ion in the reaction system is 0.5 g/L to 5 g/L; and the catalytic reaction of the PLD is conducted at a temperature of 20° C. to 70° C. and a pH of 4.5 to 7.5; and further preferably, the catalytic reaction is conducted at a temperature of 40° C. to 60° C. and a pH of 5.0 to 6.5.

19. The synthesis method according to claim 1, wherein during the catalytic reaction of the PLD, an additive is further added, and the additive comprises, but is not limited to, one or more selected from the group consisting of n-hexane, n-heptane, and isopropanol; and based on a weight proportion, a content of the additive is 0% to 50% when the additive is n-hexane or n-heptane and is 0% to 30% when the additive is isopropanol.

20. The synthesis method according to claim 2, wherein during the catalytic reaction of the PLD, an additive is further added, and the additive comprises, but is not limited to, one or more selected from the group consisting of n-hexane, n-heptane, and isopropanol; and based on a weight proportion, a content of the additive is 0% to 50% when the additive is n-hexane or n-heptane and is 0% to 30% when the additive is isopropanol.

21. The synthesis method according to claim 1, wherein in a2, a concentration of the calcium ion in a reaction system is 0.01 g/L to 20 g/L, and further preferably, the concentration of the calcium ion in the reaction system is 0.1 g/L to 2 g/L; and the catalytic reaction of the PLC is conducted at a temperature of 20° C. to 70° C. and a pH of 4.0 to 7.0, and further preferably, the catalytic reaction is conducted at a temperature of 40° C. to 55° C. and a pH of 5.0 to 7.0.

22. The synthesis method according to claim 2, wherein in a2, a concentration of the calcium ion in a reaction system is 0.01 g/L to 20 g/L, and further preferably, the concentration of the calcium ion in the reaction system is 0.1 g/L to 2 g/L; and the catalytic reaction of the PLC is conducted at a temperature of 20° C. to 70° C. and a pH of 4.0 to 7.0, and further preferably, the catalytic reaction is conducted at a temperature of 40° C. to 55° C. and a pH of 5.0 to 7.0.

23. The synthesis method according to claim 21, wherein during the catalytic reaction of the PLC, an alkane and/or a short-chain alcohol are/is further added as an additive; the alkane is one or two selected from the group consisting of n-hexane and n-heptane and the short-chain alcohol is one or two selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, and pentanol; and based on a weight proportion, a content of the additive is 0% to 80% when the additive is an alkane and is 0% to 30% when the additive is a short-chain alcohol.

24. The synthesis method according to claim 22, wherein during the catalytic reaction of the PLC, an alkane and/or a short-chain alcohol are/is further added as an additive; the alkane is one or two selected from the group consisting of n-hexane and n-heptane and the short-chain alcohol is one or two selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, and pentanol; and based on a weight proportion, a content of the additive is 0% to 80% when the additive is an alkane and is 0% to 30% when the additive is a short-chain alcohol.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0050] FIG. 1 shows action sites of the PLD and PLC of the present disclosure.

[0051] FIG. 2 is a schematic diagram illustrating the sequenced catalytic reactions of the PLD and PLC of the present disclosure.

[0052] FIG. 3 shows liquid chromatography (LC) spectra for a reaction between NR and a phospholipid under the catalysis of the PLD of the present disclosure to produce PNR (before and after the reaction).

[0053] FIG. 4 shows LC spectra for the production of β-NMN under the catalysis of the PLC of the present disclosure from the PNR produced by the hydrolysis catalyzed by the PLD (standards of NR and NMN, and samples from an NMN reaction solution).

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0054] The substances mentioned above are tested. [0055] 1. Sampling and analysis of NR and β-NMN during the reaction.

[0056] In the reaction, the substrate NR and the product β-NMN are water-soluble compounds with strong polarity and need to be analyzed with a reversed-phase column.

[0057] A high-performance liquid chromatography (HPLC) analysis method for NR and β-NMN is as follows:

[0058] Chromatographic column: Agilent SB-C18 (5 μm, 4.6×250 mm); and detection wavelength: 254 nm.

[0059] Mobile phases: mobile phase A and mobile phase B, which are combined according to Table 1 to conduct gradient elution with an initial flow rate of 0.8 mL/min and a column temperature of 30° C. Mobile phase A:water (1.36 g of monopotassium phosphate (MKP) is dissolved in 1 L of water, and a pH is adjusted with phosphoric acid to 2.5); and mobile phase B:methanol.

TABLE-US-00001 Time/min A % B % 0.01 95 5 5 95 5 11 5 95 15 5 95 16 95 5 25 95 5 [0060] 2. Sampling and analysis of the phospholipid and PNR during the reaction.

[0061] FIG. 4 shows the analysis of the substrate phospholipid and PNR in a sample taken during the reaction. During the reaction, the substrate phospholipid and the product PNR are water-insoluble compounds with weak polarity and need to be analyzed with a normal-phase column. An HPLC analysis method for the two is as follows: chromatographic column: silica gel column Si60 (5 μm, 4.5×250 mm); detection wavelength: 205 nm; analysis at room temperature; mobile phase: acetonitrile-methanol-85% phosphoric acid aqueous solution (100:10:1.8, V/V/V); isocratic constant-velocity analysis; and flow rate: 1.0 mL/min

SPECIFIC EXAMPLES

Example 1

Preparation of PNR

[0062] 1,000 L of a mixture of water, n-heptane, and isopropanol in a ratio of 5:4:1 was added to a reactor, then 150 g of a phospholipid (with 50% phosphatidylcholine-containing lecithin as an example) was added, and emulsification and dissolution were conducted; 50 kg of NR was added, and then 0.8 kg of calcium chloride was added; a resulting mixture was stirred and heated to 50° C. to 55° C. until the mixture was uniformly emulsified, and a pH was adjusted to 5.0 to 6.0 with a hydrochloric acid or sodium hydroxide solution; an appropriate amount of PLD was added to allow a reaction for about 2 h; and after the reaction was completed, a resulting reaction system was allowed to stand, and a resulting water-insoluble phase was separated to obtain 83 kg of PNR.

Example 2

Preparation of PNR

[0063] 2,000 L of a mixture of water and isopropanol in a ratio of 8.5:1.5 was added to a reactor, then 500 g of a phospholipid (with 50% phosphatidylcholine-containing lecithin as an example) was added, and emulsification and dissolution were conducted; 120 kg of NR was added, and then 10 kg of calcium chloride was added; a resulting mixture was stirred and heated to 45° C. to 50° C. until the mixture was uniformly emulsified, and a pH was adjusted to 5.6 to 6.4 with a hydrochloric acid or sodium hydroxide solution; an appropriate amount of PLD was added to allow a reaction for about 1 h; and after the reaction was completed, a resulting reaction system was allowed to stand, and a resulting water-insoluble phase was separated to obtain 264 kg of PNR.

Example 3

Preparation of PNR

[0064] 2,000 L of drinking water was added to a reactor, then 400 kg of a phospholipid (with 50% phosphatidylcholine-containing lecithin as an example) was added, and emulsification and dissolution were conducted; 140 kg of NR was added, and then 3 kg of calcium chloride was added; a resulting mixture was stirred and heated to 45° C. to 50° C. until the mixture was uniformly emulsified, and a pH was adjusted to 5.5 to 6.0 with a hydrochloric acid or sodium hydroxide solution; an appropriate amount of PLD was added to allow a reaction for about 4 h; and after the reaction was completed, a resulting reaction system was allowed to stand, and a resulting water-insoluble phase or water-insoluble substance was separated to obtain 218 kg of PNR.

Example 4

Preparation of PNR

[0065] 2,000 L of a mixture of water, n-hexane, and isopropanol in a ratio of 6:3:1 was added to a reactor, then 240 g of a phospholipid (with 50% phosphatidylcholine-containing lecithin as an example) was added, and emulsification and dissolution were conducted; 20 kg of NR was added, and then 4 kg of calcium chloride was added; a resulting mixture was stirred and heated to 55° C. to 60° C. until the mixture was uniformly emulsified, and a pH was adjusted to 5.0 to 6.0 with a hydrochloric acid or sodium hydroxide solution; an appropriate amount of PLD was added to allow a reaction for about 1 h; and after the reaction was completed, a resulting reaction system was allowed to stand, and a resulting water-insoluble phase was separated to obtain 63 kg of PNR.

Example 5

Preparation of PNR

[0066] 1,000 L of drinking water was added to a reactor, then 200 kg of a phospholipid (with 60% phosphatidylcholine-containing lecithin as an example) was added, and emulsification and dissolution were conducted; 150 kg of NR was added, and then 0.5 kg of calcium chloride was added; a resulting mixture was stirred and heated to 45° C. to 50° C. until the mixture was uniformly emulsified, and a pH was adjusted to 5.5 to 6.0 with a hydrochloric acid or sodium hydroxide solution; an appropriate amount of PLD was added to allow a reaction for about 4 h; and after the reaction was completed, a resulting reaction system was allowed to stand, and a resulting water-insoluble phase or water-insoluble substance was separated to obtain 115 kg of PNR.

Example 6

Preparation of PNR

[0067] 3,000 L of a mixture of water, n-heptane, and isopropanol in a ratio of 7.5:2:0.5 was added to a reactor, then 500 kg of a phospholipid (with 30% phosphatidylcholine-containing lecithin as an example) was added, and emulsification and dissolution were conducted; 100 kg of NR was added, and then 20 kg of calcium chloride was added; a resulting mixture was stirred and heated to 47° C. to 52° C. until the mixture was uniformly emulsified, and a pH was adjusted to 5.5 to 6.0; an appropriate amount of PLD was added to allow a reaction for about 12 h; and after the reaction was completed, a resulting reaction system was allowed to stand, and a resulting water-insoluble phase was separated to obtain 163 kg of PNR.

Example 7

Synthesis Method of β-NMN

[0068] 150 kg of the PNR from Examples 1 and 6 was taken, 100 kg of water, 10 kg of n-heptane, 5 kg of isopropanol, and 200 g of calcium chloride were added, a resulting mixture was thoroughly stirred and heated to 43° C. to 48° C., and a pH was adjusted to 5.5 to 6.0; and an appropriate amount of PLC was added to allow a reaction for about 2 h to obtain 52 kg of β-NMN.

Example 8

Synthesis Method of β-NMN

[0069] 100 kg of PNR from Examples 3 and 5 was taken, 200 kg of drinking water and 0.5 kg of calcium chloride were added, a resulting mixture was thoroughly stirred and heated to 45° C. to 50° C., and a pH was adjusted to 6.0 to 6.5; and an appropriate amount of PLC was added to allow a reaction for about 4 h to obtain 34 kg of β-NMN.

Example 9

Synthesis Method of β-NMN

[0070] 200 kg of PNR from Example 2 was taken, 880 kg of drinking water, 120 kg of isopropanol, and 2 kg of calcium chloride were added, a resulting mixture was thoroughly stirred and heated to 43° C. to 48° C., and a pH was adjusted to 6.0 to 6.5; and an appropriate amount of PLC was added to allow a reaction for about 8 h to obtain 70 kg of β-NMN.

Example 10

Synthesis Method of β-NMN

[0071] 50 kg of the PNR from Example 4 was taken, 100 kg of water, 7 kg of n-hexane, 3 kg of isopropanol, and 80 g of calcium chloride were added, a resulting mixture was thoroughly stirred and heated to 45° C. to 50° C., and a pH was adjusted to 6.0 to 6.5; and an appropriate amount of PLC was added to allow a reaction for about 1 h to obtain 16 kg of β-NMN.

Example 11

Synthesis Method of β-NMN

[0072] 100 kg of PNR from Examples 3 and 5 was taken, 300 kg of drinking water, 30 kg of butanol, and 2 kg of calcium chloride were added, a resulting mixture was thoroughly stirred and heated to 45° C. to 50° C., and a pH was adjusted to 6.5 to 7.0; and an appropriate amount of PLC was added to allow a reaction for about 2 h to obtain 34 kg of β-NMN.

Example 12

Synthesis Method of β-NMN

[0073] 100 kg of PNR from Examples 3 and 5 was taken, 300 kg of drinking water, 30 kg of ethanol, and 2 kg of calcium chloride were added, a resulting mixture was thoroughly stirred and heated to 45° C. to 50° C., and a pH was adjusted to 6.5 to 7.0; and an appropriate amount of PLC was added to allow a reaction for about 2 h to obtain 33 kg of β-NMN.

Example 13: Preparation of β-NMN through one-pot synthesis in which enzymes were added successively

[0074] 2,000 L of a mixture of water, n-heptane, and isopropanol in a ratio of 6:4:1 was added to a reactor, then 360 g of a phospholipid (with 50% phosphatidylcholine-containing lecithin as an example) was added, and emulsification and dissolution were conducted; 120 kg of NR was added, and then 2 kg of calcium chloride was added; a resulting mixture was stirred and heated to 47° C. to 52° C. until the mixture was homogeneous, and a pH was adjusted to 5.5 to 6.0; and an appropriate amount of PLD was added to allow a reaction for about 2 h; and then PLC was added, a temperature was adjusted to 43° C. to 48° C., and a reaction was conducted for about 4 h to obtain 71 kg of β-NMN.

Example 14

Preparation of β-NMN Through One-Pot Synthesis in Which Enzymes Were Added Successively

[0075] 3,000 L of a mixture of water and isopropanol in a ratio of 8:2 was added to a reactor, then 450 g of a phospholipid (with 50% phosphatidylcholine-containing lecithin as an example) was added, and emulsification and dissolution were conducted; 90 kg of NR was added, and then 6 kg of calcium chloride was added; a resulting mixture was stirred and heated to 48° C. to 53° C. until the mixture was uniformly emulsified, and a pH was adjusted to 5.5 to 6.0; an appropriate amount of PLD was added to allow a reaction for about 2 h; and then PLC was added, a pH was adjusted to 6.0 to 6.5, and a reaction was conducted for about 4 h to obtain 92 kg of β-NMN.

Example 15

Preparation of β-NMN Through One-Pot Synthesis in Which Enzymes Were Added Successively

[0076] 2,000 L of drinking water was added to a reactor, then 400 kg of a phospholipid (with 50% phosphatidylcholine-containing lecithin as an example) was added, and emulsification and dissolution were conducted; 90 kg of NR was added, and then 1.5 kg of calcium chloride was added; a resulting mixture was stirred and heated to 45° C. to 50° C. until the mixture was uniformly emulsified, and a pH was adjusted to 5.5 to 6.0 with a hydrochloric acid or sodium hydroxide solution; an appropriate amount of PLD was added to allow a reaction for about 3 h; and then PLC was added, a temperature was adjusted to 43° C. to 48° C. and a pH was adjusted to 6.0 to 6.5, and a reaction was conducted for about 6 h to obtain 83 kg of β-NMN.

Example 16

Preparation of β-NMN Through One-Pot Synthesis in Which Enzymes Were Added Successively

[0077] 3,000 L of a mixture of water, n-hexane, and isopropanol in a ratio of 6:3:1 was added to a reactor, then 500 kg of a phospholipid (with 50% phosphatidylcholine-containing lecithin as an example) was added, and emulsification and dissolution were conducted; 120 kg of NR was added, and then 5 kg of calcium chloride was added; a resulting mixture was stirred and heated to 55° C. to 60° C. until the mixture was uniformly emulsified, and a pH was adjusted to 5.5 to 6.0 with a hydrochloric acid or sodium hydroxide solution; an appropriate amount of PLD was added to allow a reaction for about 2 h; and then PLC was added, a temperature was adjusted to 43° C. to 48° C. and a pH was adjusted to 6.0 to 6.5, and a reaction was conducted for about 3 h to obtain 99 kg of (3-NMN.

Example 17

Preparation of β-NMN Through One-Pot Synthesis in Which Enzymes Were Added Successively

[0078] 2,000 L of drinking water was added to a reactor, then 350 kg of a phospholipid (with 60% phosphatidylcholine-containing lecithin as an example) was added, and emulsification and dissolution were conducted; 120 kg of NR was added, and then 1 kg of calcium chloride was added; a resulting mixture was stirred and heated to 48° C. to 53° C. until the mixture was uniformly emulsified, and a pH was adjusted to 5.5 to 6.0 with a hydrochloric acid or sodium hydroxide solution; an appropriate amount of PLD was added to allow a reaction for about 4 h; and then PLC was added, a temperature was adjusted to 43° C. to 48° C. and a pH was adjusted to 6.0 to 6.5, and a reaction was conducted for about 1 h to obtain 86 kg of β-NMN.

Example 18

Preparation of β-NMN Through One-Pot Synthesis in Which Enzymes Were Added Successively

[0079] 5,000 L of a mixture of water, n-heptane, and isopropanol in a ratio of 7.5:2:0.5 was added to a reactor, then 1,000 kg of a phospholipid (with 40% phosphatidylcholine-containing lecithin as an example) was added, and emulsification and dissolution were conducted; 150 kg of NR was added, and then 20 kg of calcium chloride was added; a resulting mixture was stirred and heated to 47° C. to 52° C. until the mixture was uniformly emulsified, and a pH was adjusted to 5.5 to 6.0; an appropriate amount of PLD was added to allow a reaction for about 6 h; and then PLC was added, a temperature was adjusted to 43° C. to 48° C. and a pH was adjusted to 6.0 to 6.5, and a reaction was conducted for about 6 h to obtain 157 kg of β-NMN.

Example 19

Preparation of β-NMN Through One-Pot Synthesis in Which Enzymes Were Added Simultaneously

[0080] 3,000 L of a mixture of water and isopropanol in a ratio of 9:1 was added to a reactor, then 500 g of a phospholipid (with 50% phosphatidylcholine-containing lecithin as an example) was added, and emulsification and dissolution were conducted; 100 kg of NR was added, and then 3 kg of calcium chloride was added; a resulting mixture was stirred and heated to 45° C. to 50° C. until the mixture was uniformly emulsified, and a pH was adjusted to 5.8 to 6.3; and PLD and PLC were added simultaneously to allow a reaction for about 4 h to obtain 100 kg of β-NMN.

Example 20

Preparation of β-NMN Through One-Pot Synthesis in Which Enzymes Were Added Simultaneously

[0081] 2,000 L of drinking water was added to a reactor, then 400 kg of a phospholipid (with 50% phosphatidylcholine-containing lecithin as an example) was added, and emulsification and dissolution were conducted; 80 kg of NR was added, and then 2 kg of calcium chloride was added; a resulting mixture was stirred and heated to 45° C. to 50° C. until the mixture was uniformly emulsified, and a pH was adjusted to 5.8 to 6.3 with a hydrochloric acid or sodium hydroxide solution; and PLD and PLC were added simultaneously to allow a reaction for about 5 h to obtain 82 kg of β-NMN.

Example 21

Preparation of β-NMN Through One-Pot Synthesis in Which Enzymes Were Added Simultaneously

[0082] 2,000 L of drinking water was added to a reactor, then 300kg of a phospholipid (with 60% phosphatidylcholine-containing lecithin as an example) was added, and emulsification and dissolution were conducted; 72 kg of NR was added, and then 1 kg of calcium chloride was added; a resulting mixture was stirred and heated to 45° C. to 50° C. until the mixture was uniformly emulsified, and a pH was adjusted to 5.8 to 6.3 with a hydrochloric acid or sodium hydroxide solution; and PLD and PLC were added simultaneously to allow a reaction for about 3 h to obtain 74 kg of β-NMN.

Example 22

Preparation of β-NMN Through One-Pot Synthesis in Which Enzymes Were Added Simultaneously

[0083] 5,000 L of drinking water was added to a reactor, then 1,000 kg of a phospholipid (with 40% phosphatidylcholine-containing lecithin as an example) was added, and emulsification and dissolution were conducted; 175 kg of NR was added, and then 10 kg of calcium chloride was added; a resulting mixture was stirred and heated to 45° C. to 50° C. until the mixture was uniformly emulsified, and a pH was adjusted to 5.8 to 6.3 with a hydrochloric acid or sodium hydroxide solution; and PLD and PLC were added simultaneously to allow a reaction for about 8 h to obtain 167 kg of β-NMN.

Example 23

Preparation of β-NMN Through One-Pot Synthesis in Which Enzymes Were Added Simultaneously

[0084] 3,000 L of a mixture of water, n-heptane, and isopropanol in a ratio of 6:3:1 was added to a reactor, then 750 kg of a phospholipid (with 50% phosphatidylcholine-containing lecithin as an example) was added, and emulsification and dissolution were conducted; 200 kg of NR was added, and then 7 kg of calcium chloride was added; a resulting mixture was heated to 45° C. to 50° C., and a pH was adjusted to 5.8 to 6.3 with a hydrochloric acid or sodium hydroxide solution; and PLD and PLC were added simultaneously to allow a reaction for about 12 h to obtain 128 kg of β-NMN.

Example 24

Preparation of β-NMN Through One-Pot Synthesis in Which Enzymes Were Added Simultaneously

[0085] 3,000 L of drinking water was added to a reactor, then 550 kg of a phospholipid (with 55% phosphatidylcholine-containing lecithin as an example) was added, and emulsification and dissolution were conducted; 300 kg of NR was added, and then 2 kg of calcium chloride was added; a resulting mixture was stirred and heated to 45° C. to 50° C. until the mixture was uniformly emulsified, and a pH was adjusted to 5.8 to 6.3 with a hydrochloric acid or sodium hydroxide solution; and PLD and PLC were added simultaneously to allow a reaction for about 4 h to obtain 121 kg of β-NMN.

Example 25

Preparation of β-NMN Through One-Pot Synthesis in Which Enzymes Were Added Successively

[0086] 1,000 L of drinking water was added to a reactor, then 20 kg of a phospholipid (with 60% phosphatidylcholine-containing lecithin as an example) was added, and emulsification and dissolution were conducted; 36 kg of NR was added, and then 0.1 kg of calcium chloride was added; a resulting mixture was stirred and heated to 45° C. to 50° C. until the mixture was uniformly emulsified, and a pH was adjusted to 5.8 to 6.3 with a hydrochloric acid or sodium hydroxide solution; and PLD and PLC were added simultaneously to allow a reaction for about 2 h to obtain 4.1 kg of β-NMN.

Example 26

Preparation of β-NMN Through One-Pot Synthesis in Which Enzymes Were Added Successively

[0087] 5,000 L of a mixture of water, n-heptane, and isopropanol in a ratio of 7.5:2:0.5 was added to a reactor, then 800 kg of a phospholipid (with 70% phosphatidylcholine-containing lecithin as an example) was added, and emulsification and dissolution were conducted; 250 kg of NR was added, and then 100 kg of calcium chloride was added; a resulting mixture was stirred and heated to 45° C. to 50° C. until the mixture was uniformly emulsified, and a pH was adjusted to 5.8 to 6.3 with a hydrochloric acid or sodium hydroxide solution; and PLD and PLC were added simultaneously to allow a reaction for about 6 h to obtain 222 kg of β-NMN.

Example 27

Preparation of β-NMN Through One-Pot Synthesis in Which Enzymes Were Added Simultaneously

[0088] 3,000 L of a mixture of water and isopropanol in a ratio of 7:3 was added to a reactor, then 600 g of a phospholipid (with 50% phosphatidylcholine-containing lecithin as an example) was added, and emulsification and dissolution were conducted; 100 kg of NR was added, and then 2 kg of calcium chloride was added; a resulting mixture was stirred and heated to 45° C. to 50° C. until the mixture was uniformly emulsified, and a pH was adjusted to 5.8 to 6.3 with a hydrochloric acid or sodium hydroxide solution; and PLD and PLC were added simultaneously to allow a reaction for about 5 h to obtain 105 kg of β-NMN.

Example 28

Preparation of β-NMN Through One-Pot Synthesis in Which Enzymes Were Added Simultaneously

[0089] 3,000 L of water was added to a reactor, then 600 g of a phospholipid (with 50% phosphatidylcholine-containing lecithin as an example) was added, and emulsification and dissolution were conducted; 45 kg of NR was added, and then 1 kg of calcium chloride was added; a pH was adjusted to 5.8 to 6.3 with a hydrochloric acid or sodium hydroxide solution; and PLD and PLC were added simultaneously to allow a reaction for about 2 h to obtain 26 kg of β-NMN.