Adenosine-Involved Fully Enzymatic Synthesis Method for NMN

Abstract

An adenosine-involved fully enzymatic synthesis method for NMN includes the steps under the same reaction system: (A) reacting, under the catalytic action of a yeast cell, adenosine, phosphate and sugar that is metabolizable by a yeast cell, so as to generate ATP; and (B) generating NMN by an enzymatic reaction including the step of under the action of NAMPT, reacting nicotinamide, PRPP and ATP to generate NMN, ADP and phosphate. In this way, a series of reactions such as the generation (regeneration) of ATP, the synthesis of NMN and the utilization of ATP are performed in one reaction system in a unified manner, and thus efficient synthesis of NMN can be implemented.

Claims

1. An adenosine-involved fully enzymatic synthesis method for NMN which comprises the following steps in a same reaction system: (A) generating ATP by the reaction of adenosine, phosphate and carbohydrate which is capable of being metabolized by yeast cells under the catalysis of the yeast cells; and (B) producing NMN by enzymatic reaction which comprises a step of reacting nicotinamide, PRPP and ATP to produce NMN, ADP and phosphate under the action of NAMPT.

2. The adenosine-involved fully enzymatic synthesis method for NMN according to claim 1, wherein the step (B) further comprises a step of reacting 5-ribose-phosphate and ATP to produce PRPP and AMP under the catalysis of RPPK.

3. The adenosine-involved fully enzymatic synthesis method for NMN according to claim 2, wherein the adenosine-involved fully enzymatic synthesis method for NMN further comprises a step of converting AMP and phosphate into ADP under the action of the yeast cells.

4. The adenosine-involved fully enzymatic synthesis method for NMN according to claim 2, wherein the step (B) further comprises a step of reacting ribose and ATP to produce 5-phosphate ribose and ADP under the catalysis of RK.

5. The adenosine-involved fully enzymatic synthesis method for NMN according to claim 4, wherein in the reaction system of the adenosine-involved fully enzymatic synthesis method for NMN, RK, RPPK and NAMPT exist in at least one original form of liquid enzyme form and immobilized enzyme form.

6. The adenosine-involved fully enzymatic synthesis method for NMN according to claim 5, wherein in the reaction system of the adenosine-involved fully enzymatic synthesis method for NMN, the molar ratio of adenosine to ribose ranges from 0.01 to 1.

7. The adenosine-involved fully enzymatic synthesis method for NMN according to claim 6, wherein in the reaction system of the adenosine-involved fully enzymatic synthesis method for NMN, the molar ratio of ribose to phosphate ranges from 1 to 20.

8. The adenosine-involved fully enzymatic synthesis method for NMN according to claim 1, wherein the adenosine-involved fully enzymatic synthesis method for NMN further comprises a step of regenerating ATP by ADP and phosphate under the action of the yeast cells.

9. The adenosine-involved fully enzymatic synthesis method for NMN according to claim 8, wherein in the reaction system of the adenosine-involved fully enzymatic synthesis method for NMN, the carbohydrate metabolized by the yeast cells is selected from at least one of glucose, sucrose, starch and glycerol.

10. The adenosine-involved fully enzymatic synthesis method for NMN according to claim 8, wherein in the reaction system of the adenosine-involved fully enzymatic synthesis method for NMN, the yeast cells are yeast cells capable of oxidative phosphorylation metabolism.

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

12. The adenosine-involved fully enzymatic synthesis method for NMN according to claim 8, wherein metal ion is further added to the reaction system of the adenosine-involved fully enzymatic synthesis method for NMN.

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

14. The adenosine-involved fully enzymatic synthesis method for NMN according to claim 8, wherein in the reaction system of the adenosine-involved fully enzymatic synthesis method for NMN, the molar ratio of adenosine to nicotinamide ranges from 0.01 to 1.

15. The adenosine-involved fully enzymatic synthesis method for NMN according to claim 8, wherein in the reaction system of the adenosine-involved fully enzymatic synthesis method for NMN, the molar ratio of nicotinamide to phosphate ranges from 1 to 20.

16. The adenosine-involved fully enzymatic synthesis method for NMN according to claim 8, wherein in the reaction system of the adenosine-involved fully enzymatic synthesis method for NMN, the yeast cells are wet yeasts once being stored cryogenically.

17. The adenosine-involved fully enzymatic synthesis method for NMN according to claim 8, wherein at least one organic reagent of toluene and n-butanol is further added to the reaction system of the adenosine-involved fully enzymatic synthesis method for NMN.

18. The adenosine-involved fully enzymatic synthesis method for NMN according to claim 8, wherein the step (A) is initiated before the step (B) to provide ATP for the reaction of the step (B), so as to form a state in which the step (A) and the step (B) are in the same reaction system to be beneficial to promote each other.

Description

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0031] The following description is disclosed to enable any person skilled in the art to make and use the present invention. Preferred embodiments are provided in the following description only as examples and modifications will be apparent to those skilled in the art. The general principles defined in the following description would be applied to other embodiments, alternatives, modifications, equivalents, and applications without departing from the spirit and scope of the present invention.

[0032] Those skilled in the art should understand that, in the disclosure of the present invention, terminologies of “longitudinal,” “lateral,” “upper,” “front,” “back,” “left,” “right,” “perpendicular,” “horizontal,” “top,” “bottom,” “inner,” “outer,” and etc. that indicate relations of directions or positions are based on the relations of directions or positions shown in the appended drawings, which are only to facilitate descriptions of the present invention and to simplify the descriptions, rather than to indicate or imply that the referred device or element is limited to the specific direction or to be operated or configured in the specific direction. Therefore, the above-mentioned terminologies shall not be interpreted as confine to the present invention.

[0033] It is understandable that the term “a” or “an” should be understood as “at least one” or “one or more”. In other words, in some embodiments, the number of an element can be one and in other embodiment the number of the element can be more than one. The term “a” or “an” is not construed as a limitation of quantity.

[0034] The present invention provides an adenosine-involved fully enzymatic synthesis method for NMN, compared with the conventional fully enzymatic method, the adenosine-involved fully enzymatic synthesis method for NMN of the present invention adopts cheap adenosine instead of ATP, and yeast cells are introduced in the reaction to convert adenosine into ATP according to energy metabolism, so that it can combine the conventional enzymatic method to realize the reuse of ATP and utilize the phosphate formed by the conventional conventional fully enzymatic method as a reactant.

[0035] More specifically, the adenosine-involved fully enzymatic synthesis method for NMN adopts nicotinamide, ribose, adenosine, phosphate, and carbohydrate that can be metabolized by yeast cells as raw materials, and uses RK, RPPK, NAMPT, and yeast cells as catalysts, the generation of ATP, the synthesis of NMN and the utilization of ATP are unified in one reaction system, and the efficient synthesis of NMN can be completed. The reaction formula is: nicotinamide+ribose+carbohydrate that can be metabolized by yeast cells+adenosine+Phosphate+O.sub.2.fwdarw.NMN+ATP+CO.sub.2+H.sub.2O. In this reaction system, yeast cells use the dehydrogenation and oxidation of carbohydrate that can be metabolized by yeast cells to provide energy to drive the combination of phosphate and adenosine to generate adenosine monophosphate (AMP) through the metabolic process of oxidative phosphorylation, and subsequently generate ADP and ATP, then ATP activates ribose to generate PRPP, and PRPP reacts with NAM to produce NMN. The original and generated ADP, AMP, adenosine and phosphate in the reaction system can also be automatically converted into ATP by yeast cells to continue to participate in the reaction. Compared with the conventional fully enzymatic method, the formed phosphate can be used as a reactant, so that the removal process of phosphate is eliminated, and the reuse of ATP can be realized without the recovery process of ATP and the consumption of ATP raw materials is reduced, so the purification process of the NMN product is simplified based on the participation of the adenosine.

[0036] Furthermore, in one embodiment of the present invention, the adenosine-involved fully enzymatic synthesis method for NMN adopts nicotinamide, ribose, phosphate, adenosine, sucrose and magnesium ions as raw materials, and adopts RK, RPPK, NAMPT and Saccharomyces cerevisiae are used as catalysts. In aqueous solution, the initial pH is in the neutral range, and the reaction is carried out in contact with air and under stirring. Then the generation of ATP, the synthesis of NMN and the utilization of ATP are carried out in one reaction system, and various reactants can be substantially completely consumed, and the corresponding reaction system is simple and easy, with low cost, and is environmentally friendly and has low environmental cost.

[0037] In another embodiment of the present invention, nicotinamide, D-ribose and adenosine are used as substrates, and Saccharomyces cerevisiae, RK magnetic immobilized enzyme, RPPK magnetic immobilized enzyme and NAMPT magnetic immobilized enzyme are used for one-pot production of NMN. Add adenosine with a final concentration of 50 mM, 330 mM dipotassium hydrogen phosphate, 70 mM potassium dihydrogen phosphate, 120 mM sucrose, 50 mM magnesium chloride, 5 mM manganese chloride, and 300 g Saccharomyces cerevisiae in 1 L reaction system. After fully stirring and dissolving, control the reaction temperature to be 37° C., and the fermentation is carried out for one hour. Add nicotinamide with a final concentration of 100 mM, 50 mM D-ribose, 100 g ribokinase magnetically immobilized enzyme, 100 g ribose phosphate pyrophosphate kinase magnetically immobilized enzyme, and 300 g nicotinamide phosphoribosyltransferase magnetically immobilized enzyme in the above yeast fermentation broth Lyase (the above three magnetically immobilized enzymes are all provided by Hong Kong Life Science and Technology Research Institute Co., Ltd.), stir at 300 rpm, control the reaction temperature at 37° C., and use an automatic titrator to control the reaction pH to 6.0 with 3M sodium hydroxide. During the reaction process, the NMN concentration is detected by the high performance liquid chromatography, and the reaction is completed within four hours, and 13.77 g of NMN is obtained from the reaction, and the reaction conversion rate is 82.4%.

[0038] It is worth mentioning that, in order to reduce the amount of enzyme used and simplify the complexity of the reaction, the reaction intermediates 5-phosphate ribose and PRPP involved in the present invention can also be directly put into the reaction system as the substrate (raw material) of the reaction. If ribose 5-phosphate is used instead of ribose as the raw material, ribokinase (RK) is not needed; if PRPP is used instead of ribose as the raw material, the two enzymes RK and RPPK are not needed.

[0039] In another embodiment of the present invention, the adenosine-involved fully enzymatic synthesis method for NMN adopts 5-phosphoribosyl 1-pyrophosphate, nicotinamide and adenosine as substrates, and uses yeast and nicotinamide phosphoribosyl transfererase enzyme to carry out one-pot production of NMN. Add nicotinamide with a final concentration of 100 mM, 50 mM 5-phosphoribosyl 1-pyrophosphate, 50 mM adenosine, 330 mM dipotassium hydrogen phosphate, 70 mM potassium dihydrogen phosphate, 120 mM Sucrose, 50 mM magnesium chloride, 5 mM manganese chloride, 300 g Saccharomyces cerevisiae, with 300 U nicotinamide phosphoribosyl transferase liquid enzyme, after fully stirring and dissolving, control the reaction temperature to 37° C., the reaction pH to 6.0, 300 rpm to stir the reaction, and the concentration of NMN is detected by the high performance liquid chromatography, and the reaction is completed within four hours, and 15.44 g of NMN is obtained from the reaction, and the reaction conversion rate is 92.4%.

[0040] In another embodiment of the present invention, the adenosine-involved fully enzymatic synthesis method for NMN adopts nicotinamide, 5-phosphate ribose and adenosine as substrates, adopts Saccharomyces cerevisiae and ribose phosphate pyrophosphate kinase, nicotinamide phosphoribosyl transferase enzyme to carry out one-pot production of NMN. Add adenosine with a final concentration of 50 mM, 330 mM dipotassium hydrogen phosphate, 70 mM potassium dihydrogen phosphate, 120 mM sucrose, 50 mM magnesium chloride, 5 mM manganese chloride, and 300 g brewing yeast into a 1 L reaction system, after fully stirring and dissolving, control the reaction temperature at 37° C. and ferment for one hour. Add nicotinamide with a final concentration of 100 mM, 50 mM ribose-5-phosphate, 300 U nicotinamide phosphoribosyltransferase liquid enzyme, 300 U ribose phosphate pyrophosphokinase to the above yeast fermentation broth, stir the reaction at 300 rpm, and control the reaction temperature at 37° C., use an automatic titrator to control the reaction pH to 6.0 with 3M sodium hydroxide. During the reaction process, the NMN concentration is detected by the high performance liquid chromatography, and the reaction is completed within six hours, and 14.42 g of NMN is obtained from the reaction, and the reaction conversion rate is 86.3%.

[0041] To further illustrate the present invention, the adenosine-involved fully enzymatic synthesis method for NMN of the present invention comprises the following steps in the same reaction system: [0042] (A) generating ATP by the reaction of adenosine, phosphate and carbohydrate which is capable of being metabolized by yeast cells under the catalysis of the yeast cells; and [0043] (B) producing NMN by enzymatic reaction, comprising the step of reacting nicotinamide, PRPP and ATP to produce NMN, ADP and phosphate under the action of NAMPT.

[0044] Furthermore, the step (B) further comprises a step of reacting 5-ribose-phosphate and ATP to produce PRPP and AMP under the catalysis of RPPK.

[0045] It is worth mentioning that the adenosine-involved fully enzymatic synthesis method for NMN further comprises a step of converting AMP and phosphate into ADP under the action of the yeast cells.

[0046] In addition, the step (B) further comprises a step of reacting ribose and ATP to produce 5-phosphate ribose and ADP under the catalysis of RK.

[0047] It is worth mentioning that in the reaction system of the adenosine-involved fully enzymatic synthesis method for NMN, RK, RPPK and NAMPT exist in at least one original form of liquid enzyme form and immobilized enzyme form.

[0048] Particularly, in the reaction system of the adenosine-involved fully enzymatic synthesis method for NMN, the molar ratio of adenosine to ribose ranges from 0.01 to 1.

[0049] Furthermore, in the reaction system of the adenosine-involved fully enzymatic synthesis method for NMN, the molar ratio of ribose to phosphate ranges from 1 to 20.

[0050] It is worth mentioning that the adenosine-involved fully enzymatic synthesis method for NMN further comprises a step of regenerating ATP by ADP and phosphate under the action of yeast cells.

[0051] It can be understood that in the reaction system of the adenosine-involved fully enzymatic synthesis method for NMN, the carbohydrate metabolized by yeast cells is selected from at least one of glucose, sucrose, starch, glycerol, and the combination thereof.

[0052] Furthermore, in the reaction system of the adenosine-involved fully enzymatic synthesis method for NMN, the yeast cells are yeast cells capable of oxidative phosphorylation metabolism, such as Pichia pastoris and Saccharomyces cerevisiae.

[0053] Alternatively, metal ion is further added to the reaction system of the adenosine-involved fully enzymatic synthesis method for NMN, and the added metal ion can be magnesium ion or manganese ion.

[0054] Preferably, in the reaction system of the adenosine-involved fully enzymatic synthesis method for NMN, the molar ratio of adenosine to nicotinamide ranges from 0.01 to 1.

[0055] Preferably, in the reaction system of the adenosine-involved fully enzymatic synthesis method for NMN, the molar ratio of nicotinamide to phosphate ranges from 1 to 20.

[0056] Preferably in the reaction system of the adenosine-involved fully enzymatic synthesis method for NMN, the yeast cells are wet yeasts once being stored cryogenically.

[0057] Preferably, at least one organic reagent of toluene and n-butanol is further added to the reaction system of the adenosine-involved fully enzymatic synthesis method for NMN.

[0058] It is worth mentioning that, in some embodiments of the present invention, the step (A) is initiated before the step (B) to provide ATP for the reaction of the step (B), so as to form a state in which the step (A) and the step (B) are in the same reaction system to be beneficial to promote each other.

[0059] One skilled in the art will understand that the embodiment of the present invention as shown in the drawings and described above is exemplary only and not intended to be limiting.

[0060] It will thus be seen that the objects of the present invention have been fully and effectively accomplished. The embodiments have been shown and described for the purposes of illustrating the functional and structural principles of the present invention and are subject to change without departure from such principles. Therefore, this invention includes all modifications encompassed within the spirit and scope of the following claims.