Method for producing nicotinamide mononucleotide

Abstract

The present invention provides a method for producing nicotinamide mononucleotide, which uses one enzyme to produce nicotinamide mononucleotide from a nucleoside monophosphate, pyrophosphate, and nicotinamide as raw materials. The method for producing nicotinamide mononucleotide according to the present invention includes at least the following steps 1) and 2): 1) A first step of allowing substantially one enzyme to act on a nucleoside monophosphate and pyrophosphate to generate phosphoribosyl diphosphate; 2) A second step of allowing substantially only the above-mentioned one enzyme to act on the phosphoribosyl diphosphate as the product of the first step and nicotinamide to generate nicotinamide mononucleotide.

Description

Technical Field

[0001] The present invention relates to a method for producing nicotinamide mononucleotide. Background Art

[0002] In recent years, nicotinamide mononucleotide (hereinafter sometimes referred to as "NMN") has attracted attention as an anti-aging substance, and the demand for health foods and the like has expanded.

[0003] As a method for producing NMN, there are known (1) a production method based on organic synthesis, (2) a production method based on fermentation using yeast, (3) a production method based on an enzymatic method using an enzyme, and the like. Among these, the production method based on organic synthesis requires several stages of synthesis processes, and thus has problems of time and cost consumption. In addition, in the production method based on fermentation, the productivity of NMN is very poor, and large-scale culture equipment and the like are required.

[0004] Several NMN production methods based on an enzymatic method have been reported. Among them, Patent Documents 1 to 9 disclose a method for converting nicotinamide (hereinafter sometimes referred to as "NAM") and phosphoribosyl diphosphate (also referred to as phosphoribosyl pyrophosphate) (hereinafter sometimes referred to as "PRPP") into NMN using nicotinamide phosphoribosyltransferase (EC 2.4.2.12) (hereinafter sometimes referred to as "NAMPT").

[0005] Among these, Patent Documents 7 to 9 disclose the following method: using inosine monophosphate (IMP) and 5'-guanylic acid (GMP), which are low-cost nucleoside monophosphates, as raw materials, generating PRPP by hypoxanthine-guanine phosphoribosyltransferase (hypoxanthine phosphoribosyltransferase) (EC 2.4.2.8) (hereinafter sometimes referred to as "HGPRT" or "HPT"), and using this PRPP and NAM as raw materials, generating NMN by NAMPT, that is, a method for generating NMN from nucleoside monophosphates using two enzymes, HGPRT and NAMPT.

[0006] Prior Art Documents

[0007] Patent Documents

[0008] Patent Document 1: International Publication No. 2017 / 185549

[0009] Patent Document 2: International Publication No. 2018 / 023206

[0010] Patent Document 3: International Publication No. 2018 / 023207

[0011] Patent Document 4: International Publication No. 2018 / 023208

[0012] Patent Document 5: International Publication No. 2018 / 023209

[0013] Patent Document 6: International Publication No. 2019 / 065876

[0014] Patent Document 7: International Publication No. 2018 / 023210

[0015] Patent Document 8: Specification of US Patent Application Publication No. 2017 / 0121746

[0016] Patent Document 9: Specification of US Patent Application Publication No. 2021 / 0246476 Summary of the Invention

[0017] Problems to be Solved by the Invention

[0018] However, a method for producing NMN from nucleoside monophosphate using one enzyme is unknown.

[0019] An object of the present invention is to provide a method for producing nicotinamide mononucleotide, which uses one enzyme to produce nicotinamide mononucleotide from nucleoside monophosphate, pyrophosphate and nicotinamide as raw materials.

[0020] Means for Solving the Problems

[0021] The present inventors have found a reaction for producing nicotinamide mononucleotide using one enzyme from nucleoside monophosphate, pyrophosphate and nicotinamide as raw materials, thereby completing the present invention.

[0022] In order to achieve the above object, the present invention provides the following method for producing nicotinamide mononucleotide.

[0023] [1] A method for producing nicotinamide mononucleotide, which at least includes the following steps 1) and 2).

[0024] 1) A first step of allowing substantially one enzyme to act on nucleoside monophosphate and pyrophosphate to produce phosphoribosyl diphosphate

[0025] 2) A second step of allowing substantially only the above one enzyme to act on phosphoribosyl diphosphate, which is the product of the above first step, and nicotinamide to produce nicotinamide mononucleotide

[0026] [2] The production method according to the above [1], wherein the above first step and the second step are simultaneous steps.

[0027] [3] The production method as described in [1] or [2] above, wherein the enzyme is an enzyme belonging to pentosyltransferase (EC 2.4.2).

[0028] [4] The production method as described in [3] above, wherein the enzyme belonging to pentosyltransferase (EC 2.4.2) is an enzyme belonging to hypoxanthine phosphoribosyltransferase (EC 2.4.2.8).

[0029] [5] The production method as described in [4] above, wherein the enzyme belonging to hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) is any one of HPT-C, HPT-W, HPT-L, or a polypeptide comprising an amino acid sequence having 90% or more identity with their amino acid sequences.

[0030] [6] The production method as described in any one of [1] to [5] above, wherein the nucleoside monophosphate is inosinic acid, guanylic acid, or a mixture of inosinic acid and guanylic acid.

[0031] [7] The production method as described in any one of [1] to [6] above, wherein

[0032] part or all of the nucleoside monophosphate is inosinic acid,

[0033] the first step includes a step of allowing xanthine oxidase to act on hypoxanthine generated in this step.

[0034] [8] The production method as described in any one of [1] to [7] above, wherein the first step and the second step are steps of allowing the enzyme to act in the presence of Mg ions and / or Mn ions.

[0035] [9] A method for producing nicotinamide mononucleotide, wherein one or more enzymes belonging to hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) are allowed to act on phosphoribosyl diphosphate and nicotinamide to produce nicotinamide mononucleotide.

[0036]

[10] The production method as described in [9] above, wherein at least one of the enzymes belonging to hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) is any one of HPT-C, HPT-W, HPT-L, or a polypeptide comprising an amino acid sequence having 90% or more identity with their amino acid sequences.

[0037]

[11] The production method as described in [9] or

[10] above, wherein the enzyme is allowed to act in the presence of Mg ions and / or Mn ions.

[0038] Effects of the Invention

[0039] According to the present invention, a method for manufacturing nicotinamide mononucleotide can be provided, which uses one kind of enzyme to manufacture nicotinamide mononucleotide from nucleoside monophosphate, pyrophosphate, and nicotinamide as raw materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is a schematic diagram showing each process of the method for manufacturing nicotinamide mononucleotide according to an embodiment of the present invention.

[0041] Figure 2 It is a graph showing the HPLC elution behavior of the sample in Example 1.

[0042] Figure 3 It is a graph showing the MS / MS spectra of the sample in Example 14 and NMN. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0043] Hereinafter, the specific embodiments of the present invention (hereinafter referred to as "the present embodiment") will be described in detail. It should be noted that the present invention is not limited to the following embodiments, and various modifications can be made within the scope of its gist.

[0044] Figure 1 It is a schematic diagram showing each process of the method for manufacturing nicotinamide mononucleotide according to the present embodiment.

[0045] The method for manufacturing nicotinamide mononucleotide according to the present embodiment includes at least the following steps 1) and 2).

[0046] 1) The first step is to allow substantially one kind of enzyme to act on nucleoside monophosphate and pyrophosphate to generate phosphoribosyl diphosphate.

[0047] 2) The second step is to allow substantially only the above-mentioned one kind of enzyme to act on phosphoribosyl diphosphate, which is the product of the first step, and nicotinamide to generate nicotinamide mononucleotide.

[0048] The above first step can be carried out before the second step, but it is preferable to carry out the first step and the second step simultaneously. Here, "simultaneously" means "in the same reactor, the first step and the second step are carried out in parallel at the same time".

[0049] Nucleoside monophosphate is a kind of nucleotide, which is formed by the binding of the nitrogen atom of the base (such as purine base, pyrimidine base) to the 1-position carbon atom of the ribose analog (such as ribose, deoxyribose, etc.) and the binding of the 5-position carbon atom to the phosphate group. For example, it is AMP, GMP, IMP, XMP, CMP, UMP, OMP, dAMP, dGMP, dCMP, dTMP, etc.

[0050] As described above, the following methods have been reported so far: using an enzyme to generate PRPP from IMP or GMP as a nucleoside monophosphate and pyrophosphate (referred to as the first step in this specification), and allowing the enzyme to act on PRPP and NAM as its product (referred to as the second step in this specification), thereby generating NMN (Patent Documents 7 to 9).

[0051] As the enzyme used in this first step, HGPRT is shown, and as the enzyme used in this second step, NAMPT is shown (Patent Documents 7 to 9). And it is also shown that the first step and the second step are carried out simultaneously (Examples 1 to 5 of Patent Document 7, Examples 32, 33, 54 to 56 of Patent Document 9).

[0052] However, even if the first step and the second step can be carried out simultaneously, so far, the case of catalyzing the reaction with one enzyme is still unknown, and at least two different enzymes need to be used. In the method for manufacturing a substance using an enzyme, reducing the types and amounts of enzymes is not only excellent in terms of cost, but also can simplify the control of the reaction system. In addition, it can simplify the purification of the product after the reaction, so it also significantly contributes to the improvement of the product quality.

[0053] The present inventors unexpectedly found that one enzyme can catalyze the reaction of the first step of generating PRPP from a nucleoside monophosphate and pyrophosphate, which was previously considered to be based on completely different enzymes, and the second step of generating NMN from PRPP and NAM as the product of the first step, and completed the Figure 1 shown method for manufacturing NMN including the first step and the second step by reacting with one enzyme.

[0054] Regarding the one enzyme mentioned here, as long as it is an enzyme that can catalyze the reaction of the first step of generating PRPP from a nucleoside monophosphate and pyrophosphate, and the second step of generating NMN from PRPP and NAM as the product of the first step, it can be a natural enzyme or a modified enzyme such as one that has been mutated, deleted, added, fused, etc. to change reactivity, stability, specificity, etc., but is not particularly limited. For example, an enzyme belonging to pentosyltransferase (EC 2.4.2) can be cited. Among them, for example, an enzyme belonging to hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) can be cited as a preferred enzyme. It should be noted that this specification also includes enzymes that are assigned different enzyme numbers in the current classification but were assigned the above enzyme numbers in the old classification.

[0055] The so-called activation of one enzyme means that there is one enzyme involved in the reaction that causes its activation, without excluding the presence of other enzymes that are not involved in the reaction that causes its activation. For example, the so-called action of one enzyme on nucleoside monophosphate and pyrophosphate to produce phosphoribosyl diphosphate means that there is one enzyme involved in the reaction of producing phosphoribosyl diphosphate from nucleoside monophosphate and pyrophosphate, and it also includes the case where other enzymes that are not involved in this reaction coexist.

[0056] Substantially one enzyme means that one enzyme accounts for 90% or more and the other coexisting enzymes account for 10% or less. Preferably, one enzyme accounts for 95% or more and the other coexisting enzymes account for 5% or less. More preferably, one enzyme accounts for 99% or more and the other coexisting enzymes account for 1% or less. In addition, the percentage display of the enzyme mentioned here can be any percentage display based on the protein weight of the enzyme or the enzyme unit (unit) as the activity unit of the enzyme. It should be noted that the other coexisting enzymes refer to one or more enzymes that can catalyze the reaction of the first step and / or the second step.

[0057] As an enzyme belonging to hypoxanthine phosphoribosyltransferase (EC 2.4.2.8), suitable enzymes can be exemplified by the enzyme "HPT-W (named by the inventors of this case)" formed by the amino acid sequence described in SEQ ID NO: 4, the enzyme "HPT-C (named by the inventors of this case)" formed by the amino acid sequence described in SEQ ID NO: 7, and the enzyme "HPT-L (named by the inventors of this case)" formed by the amino acid sequence described in SEQ ID NO: 11. Furthermore, polypeptides containing amino acid sequences having 70% or more, or 80% or more, or 90% or more, or 95% or more, or 98% or more identity with these amino acid sequences can also be exemplified.

[0058] Patent Documents 7 to 9 disclose a method for producing NMN using HGPRT and NAMPT as two enzymes with NAM, IMP or GMP, and pyrophosphate as raw materials, which does not imply the existence of an enzyme having both HGPRT activity and NAMPT activity, and does not recognize the discovery and development of such an enzyme as a subject. That is, even if the prior art up to now is combined, there is no hint of a method for producing NMN including the first step and the second step of reacting with one enzyme up to now.

[0059] In particular, the length of the peptide of hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) is about 200 amino acids, while the length of the peptide of nicotinamide phosphoribosyltransferase (EC 2.4.2.12) is about 450 amino acids, and there are significant structural differences. Therefore, it has not been envisaged so far that hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) has the same activity against nicotinamide as nicotinamide phosphoribosyltransferase (EC 2.4.2.12).

[0060] In the method for producing nicotinamide mononucleotide according to this embodiment, which at least includes the above-mentioned first step and the above-mentioned second step, in order to effectively produce nicotinamide mononucleotide, the conditions of the steps can be designed and optimized.

[0061] Effectively producing means producing in such a way that the amount, purity, etc. of the obtained product are increased compared to the raw materials input, the amount of enzyme, time, workload, work safety, and environmental burden. In particular, since both the first step and the second step are equilibrium reactions, it is important to design and optimize the conditions of the steps and effectively produce nicotinamide mononucleotide in such a way that the equilibrium shifts in the desired direction in order to increase the amount of the product.

[0062] The following will be described in detail. As the optimization of the conditions of the steps, it includes the type of enzyme, enzyme dosage form, enzyme concentration, type of substrate raw material, concentration and ratio of substrate raw material, reaction temperature, reaction time, dissolved oxygen concentration, pH, type and concentration of buffer solution, adjustment of ionic strength, addition of enzyme stabilizer, addition of product stabilizer, addition of surfactant, addition of organic solvent, addition of reaction cofactor and reaction promoting component, removal of by-products, etc.

[0063] Optimization of the type of enzyme means that, in order to effectively produce nicotinamide mononucleotide, an appropriate type of enzyme is selected from a group of enzymes capable of catalyzing the reactions of the first and second steps according to the type of substrate raw material. For example, optimization can be carried out by selecting an enzyme belonging to pentosyltransferase (EC 2.4.2). In addition, optimization can be carried out by selecting an enzyme belonging to any of the following enzymes from the enzymes belonging to pentosyltransferase (EC 2.4.2): purine nucleotide phosphorylase (EC 2.4.2.1), pyrimidine nucleotide phosphorylase (EC 2.4.2.2), uridine phosphorylase (EC 2.4.2.3), thymidine phosphorylase (EC 2.4.2.4), nucleoside ribosyltransferase (EC 2.4.2.5), nucleoside deoxyribosyltransferase (EC 2.4.2.6), adenine phosphoribosyltransferase (EC 2.4.2.7), hypoxanthine phosphoribosyltransferase (EC 2.4.2.8), uracil phosphoribosyltransferase (EC 2.4.2.9), orotate phosphoribosyltransferase (EC 2.4.2.10), nicotinic acid phosphoribosyltransferase (EC 6.3.4.21, former enzyme number: EC 2.4.2.11), nicotinamide phosphoribosyltransferase (EC 2.4.2.12), methionine adenosyltransferase (EC 2.5.1.6, former enzyme number: EC 2.4.2.13), aminophosphoribosyltransferase (EC 2.4.2.14), guanosine phosphorylase (EC 2.4.2.15), uric acid ribonucleoside phosphorylase (EC 2.4.2.16), ATP phosphoribosyltransferase (EC 2.4.2.17), anthranilate phosphoribosyltransferase (EC 2.4.2.18), nicotinic acid nucleotide diphosphorylase (carboxylating) (EC 2.4.2.19), dioxotetrahydropyrimidine phosphoribosyltransferase (EC 2.4.2.20), nicotinic acid nucleotide dimethylbenzimidazole phosphoribosyltransferase (EC 2.4.2.21), xanthine phosphoribosyltransferase (EC 2.4.2.22), deoxyuridine phosphorylase (EC 2.4.2.2, EC 2.4.2.3, EC 2.4.2.4., former enzyme number: EC 2.4.2.23), 1,4-β-D-xylan synthase (EC 2.4.2.24), flavone apiosyltransferase (EC 2.4.2.25), protein xylosyltransferase (EC 2.4.2.26), dTDP-dihydrostreptose-streptidine-6-phosphate dihydrostreptosyltransferase (EC 2.4.2.27), S-methyl-5'-thioadenosine phosphorylase (EC 2.4.2.28), tRNA-guanosine 34 transglycosylase (EC 2.4.2.29), NAD + ADP-ribosyltransferase (EC 2.4.2.30), NAD +- Protein - arginine ADP - ribosyltransferase (EC 2.4.2.31), polyprenylphosphate D - xylosyltransferase (EC 2.4.2.32), polyprenylxylosylphosphate - protein xylosyltransferase (EC 2.4.2.33), indolylacetylinositol arabinosyltransferase (EC 2.4.2.34), flavonol - 3 - O - glycoside xylosyltransferase (EC 2.4.2.35), NAD + - Diphthamide ADP - ribosyltransferase (EC 2.4.2.36), NAD +- Diazoreductase ADP-D-ribosyltransferase (EC 2.4.2.37), glycoprotein 2-β-D-xylosyltransferase (EC 2.4.2.38), xyloglucan 6-xylosyltransferase (EC 2.4.2.39), zeatin O-β-D-xylosyltransferase (EC 2.4.2.40), xylose-galacturonic acid β-1,3-xylosyltransferase (EC 2.4.2.41), UDP-D-xylose:β-D-glucoside α-1,3-D-xylosyltransferase (EC 2.4.2.42), lipid A 4-amino-4-deoxy-L-arabinose transferase (EC 2.4.2.43), S-methyl-5'-thioinosine phosphorylase (EC 2.4.2.44), geranyl diphosphate phosphoribosyltransferase (EC 2.4.2.45), galactan 5-O-arabinofuranosyltransferase (EC2.4.2.46), arabinofuran 3-O-arabinotransferase (EC 2.4.2.47), tRNA-guanine 15 transglycosylase (EC2.4.2.48), norspermidine phosphoribosyltransferase (EC 2.4.2.49), cyanidin 3-O-galactoside 2”-O-xylosyltransferase (EC 2.4.2.50), anthocyanin 3-O-glucoside 2”'-O-xylosyltransferase (EC 2.4.2.51), ribose triphosphate - dephospho-CoA synthase (EC 2.4.2.52), undecaprenyl phosphate 4-deoxy-4-formamido-L-arabinose transferase (EC 2.4.2.53), β-ribofuranosyl 5' phosphate synthase (EC 2.4.2.54), nicotinic acid D-ribonucleotide:phenol phosphate-D-ribosyltransferase (EC 2.4.2.55), kaempferol 3-O-xylosyltransferase (EC 2.4.2.56), AMP phosphorylase (EC 2.4.2.57), hydroxyproline O-arabinofuranosyltransferase (EC 2.4.2.58), sulfide-dependent adenosine diphosphate thiazole synthase (EC 2.4.2.59), cysteine-dependent adenosine diphosphate thiazole synthase (EC2.4.2.60), α-dystroglycan β1,4-xylosyltransferase (EC 2.4.2.61), xylosyl α-1,3-xylosyltransferase (EC 2.4.2.62), EGF domain serine xylosyltransferase (EC 2.4.2.63), NAD + - Protein arginine ADP-ribosyltransferase (EC 2.4.2.B12), NAD +- Protein arginine ADP-ribosyltransferase (EC 2.4.2.B13), (KDO)2-lipid IV(A) 4-amino-4-deoxy-L-arabinopyranosyltransferase (EC 2.4.2.B4). Additionally, optimization can be carried out by selecting hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) from any of the following bacteria: Archaeoglobus veneficus, Artemia sp., Bos taurus, Caldanaerobacter subterraneus subsp.Tengcongensis), Hydrogenothermus marinus, Chinese hamster (Cricetulus griseus), Cryptosporidium parvum, Escherichia coli, Red junglefowl (Gallus gallus), Giardia intestinalis, Giardia intestinalis Portland, Halobacterium salinarum, Haloferax volcanii, Homo sapiens, Clostridium thermocellum, Clostridium thermocellum DSM 1237, Legionella pneumophila, Leishmania donovani, Leishmania tarentolae, Southern river otter (Lontra longicaudis), Methanococcus voltae, Northern elephant seal (Mirounga angustirostris), House mouse (Mus musculus), Mycobacterium tuberculosis, Mycobacterium tuberculosis H37Rv, Plasmodium chabaudi, Plasmodium falciparum, Plasmodium lophurae, Pyrococcus horikoshii, Brown rat (Rattus norvegicus), Sulfolobus solfataricus, Sulfolobus solfataricus P2, Saccharomyces cerevisiae, Salmonella enterica subsp.Salmonella enterica serovar Typhimurium, Salmonella enterica subsp. enterica serovar Typhimurium LT2, Schistosoma mansoni, Schizosaccharomyces pombe, Streptomyces cyanogenus, Sus scrofa, Thermus thermophilus, Thermus thermophilus HB8 / ATCC 27634 / DSM 579, Toxoplasma gondii, Tritrichomonas suis, Trypanosoma cruzi. Additionally, optimization can be carried out by directly selecting any of the above enzymes, or by selecting an enzyme produced by genetic recombination organisms such as an enzyme obtained by producing an enzyme modified by deletion, insertion, addition, fusion, etc. in the amino acid sequence. When selecting any one of the above HPT-W, HPT-C, and HPT-L, it is preferable to select an enzyme produced as described in the following examples, but an enzyme produced by other genetically recombined bacteria can also be selected.

[0064] When the nucleoside monophosphate of the substrate raw material is IMP, GMP, or a mixture thereof, it is preferable to select an enzyme belonging to any one of hypoxanthine phosphoribosyltransferase (EC 2.4.2.8), nicotinate phosphoribosyltransferase (EC 6.3.4.21), and nicotinamide phosphoribosyltransferase (EC 2.4.2.12).

[0065] When the nucleoside monophosphate of the substrate raw material is AMP or a substance containing AMP, it is preferable to select an enzyme belonging to any one of adenine phosphoribosyltransferase (EC 2.4.2.7), nicotinate phosphoribosyltransferase (EC 6.3.4.21), and nicotinamide phosphoribosyltransferase (EC 2.4.2.12).

[0066] When the nucleoside monophosphate of the substrate raw material is UMP or a substance containing UMP, it is preferable to select an enzyme belonging to any one of uracil phosphoribosyltransferase (EC 2.4.2.9), nicotinate phosphoribosyltransferase (EC 6.3.4.21), and nicotinamide phosphoribosyltransferase (EC 2.4.2.12).

[0067] When the nucleoside monophosphate of the substrate raw material is OMP or a substance containing OMP, it is preferable to select an enzyme belonging to any one of orotate phosphoribosyltransferase (EC 2.4.2.10), nicotinate phosphoribosyltransferase (EC 6.3.4.21), and nicotinamide phosphoribosyltransferase (EC 2.4.2.12).

[0068] When the nucleoside monophosphate of the substrate raw material is XMP or a substance containing XMP, it is preferable to select an enzyme belonging to any one of xanthine phosphoribosyltransferase (EC 2.4.2.22), nicotinate phosphoribosyltransferase (EC 6.3.4.21), and nicotinamide phosphoribosyltransferase (EC 2.4.2.12).

[0069] Regarding the optimization of the enzyme dosage form, select the optimal enzyme dosage form under each condition from live microorganisms containing the enzyme, inactivated microorganisms containing the enzyme, crude extracts or crude extracts of microorganisms containing the enzyme, purified powder enzymes or liquid enzymes, enzymes polymerized using aggregating agents such as linkers, enzymes immobilized on carriers such as polystyrene, acrylamide, and agarose, etc. In addition, the carrier immobilized with the enzyme can be filled in a column, and the reaction solution of the process can be passed through and circulated.

[0070] Regarding the optimization of the enzyme concentration, for the concentration of the enzyme that acts on nucleoside monophosphate, pyrophosphate and acts on PRPP and NAM as its products, select the optimal concentration while considering various other conditions. Regarding the protein concentration of the enzyme, for example, it can be optimized by selecting an appropriate concentration from the range of 0.001 mg to 100 g / L, preferably 1 mg to 100 g / L, more preferably 1 to 100 g / L; regarding the activity concentration of the enzyme, for example, it can be optimized by selecting an appropriate concentration from the range of 0.01 U to 1000 kU / L, preferably 1 U to 100 kU / L, more preferably 10 U to 10 kU / L, etc.

[0071] Regarding the optimization of the type of substrate raw material, as long as nucleoside monophosphate can be used as the substrate raw material to generate PRPP through an enzymatic reaction, for example, it can be IMP, GMP, AMP, UMP, OMP, XMP, or a mixture of two or more of them, and select the optimal nucleoside monophosphate while considering various other conditions. In addition, as long as nucleoside monophosphate, pyrophosphate, and NAM are in this form during the enzymatic reaction in the reaction solution of each process, when they are put in as raw materials, they can be their salts, such as sodium salts, potassium salts, ammonium salts, hydrates, etc., and can be optimized by selecting the optimal form of the raw material while considering various other conditions.

[0072] Regarding the optimization of the concentration and ratio of the substrate raw materials, for the concentration and ratio of nucleoside monophosphate and pyrophosphate in the reaction solution at the start of the first process, the optimal concentration and ratio can be selected while considering various other conditions; for the concentration and ratio of nucleoside monophosphate, pyrophosphate, and NAM in the reaction solution at the start of the second process, optimization can be carried out while considering various other conditions. As the concentration and ratio in the reaction solution at the start of the first process, for example, the concentration of nucleoside monophosphate can be appropriately selected from the range of 0.01 - 500 mM, preferably 0.1 - 20 mM, the concentration of pyrophosphate can be appropriately selected from the range of 0.001 - 100 mM, preferably 0.02 - 10 mM, and the ratio of the concentration of nucleoside monophosphate to pyrophosphate can be appropriately selected from the range of 1:0.001 - 100, for example. For the concentration and ratio in the reaction solution at the start of the second process, for example, the concentration of nucleoside monophosphate can be appropriately selected from the range of 0 - 500 mM, preferably 0.1 - 20 mM, the concentration of pyrophosphate can be selected from the range of 0 - 100 mM, the concentration of NAM can be appropriately selected from the range of 0.01 - 500 mM, preferably 0.1 - 20 mM, and the ratio of the concentration of nucleoside monophosphate to pyrophosphate to NAM can be appropriately selected from the range of 0 - 10:0 - 100:1, for example. For example, when the first process and the second process are started simultaneously and IMP is used as the nucleoside monophosphate, the appropriate value can be selected from the range of 0.1 - 10:0.001 - 100:1 as the ratio of the concentration of IMP to pyrophosphate to NAM. Additionally, sometimes concentration or dilution is carried out during the process, or additional raw materials are added, and the concentration and ratio of the substrate raw materials are changed for optimization.

[0073] Regarding the optimization of the reaction temperature, when the first process is carried out before the second process, the temperature of each process can be optimized separately while considering various other conditions; when the first process and the second process are carried out simultaneously, the temperature of this process can be optimized while considering various other conditions. For example, research can be carried out on a constant temperature or a case where the temperature changes midway within the range of 0 - 70°C, preferably 25 - 65°C, and the optimal temperature and the optimal temperature change program are selected for optimization.

[0074] Regarding the optimization of the reaction time, when the first process is carried out before the second process, the time of each process can be optimized separately while considering various other conditions; when the first process and the second process are carried out simultaneously, the time of this process can be optimized while considering various other conditions. For example, selection can be made within the range of 1 - 240 hr, preferably 1 - 72 hr for optimization.

[0075] Regarding the optimization of the dissolved oxygen concentration, when the first process is carried out before the second process, the oxygen concentration in the reaction solution in each process can be optimized while considering various other conditions; when the first process and the second process are carried out simultaneously, the oxygen concentration in the reaction solution of this process can be optimized while considering various other conditions. For example, within the range of 0 to 14.15 mg / L, the constant concentration or the case of changing the concentration midway in the process can be studied, and the optimal concentration and the optimal concentration change program can be selected to carry out the optimization.

[0076] Regarding the optimization of the pH, when the first process is carried out before the second process, the pH in the reaction solution in each process can be optimized while considering various other conditions; when the first process and the second process are carried out simultaneously, the pH in the reaction solution of this process can be optimized while considering various other conditions. For example, within the range of pH 4 to 11, the constant pH or the case of changing the pH midway in the process can be studied, and the optimal pH and the optimal pH change program can be selected to carry out the optimization.

[0077] Regarding the optimization of the type and concentration of the buffer solution, when the first process is carried out before the second process, the type and concentration of the buffer solution in each process can be optimized while considering various other conditions; when the first process and the second process are carried out simultaneously, the type and concentration of the buffer solution in this process can be optimized while considering various other conditions. As the type of the buffer solution, for example, it can be selected from citric acid buffer solution, tartaric acid buffer solution, acetic acid buffer solution, carbonic acid buffer solution, phosphoric acid buffer solution, boric acid buffer solution, Good buffer solution, Tris buffer solution, Bis-Tris buffer solution, ammonium buffer solution, triethylamine buffer solution, glycine buffer solution, Mcilvaine buffer solution, buffer solutions composed of combinations thereof, etc., and the optimal type and concentration can be selected within the range of 1 to 500 mM concentration of the buffer solution to carry out the optimization.

[0078] Regarding the optimization of the adjustment of the ionic strength, when the first process is carried out before the second process, the adjustment of the ionic strength in each process can be carried out while considering various other conditions; when the first process and the second process are carried out simultaneously, the adjustment of the ionic strength in this process can be carried out while considering various other conditions. As the method for adjusting the ionic strength, for example, it can be adjusted by the concentration of the buffer solution in the reaction solution, adding salts such as NaCl, KCl, (NH 4 ) 2 SO 4 etc. For the buffer solution, the concentration is selected within the range of 1 to 500 mM, for NaCl, the concentration is selected within the range of 0 to 2 M, for KCl, the concentration is selected within the range of 0 to 2 M, for (NH 4 ) 2 SO4 Select the concentration in the range of 0 to 1 M to set the optimal ionic strength, and optimization can be carried out accordingly.

[0079] Regarding the optimization of the addition of enzyme stabilizers, in order to maintain the activity of the enzyme in the process, the type and concentration of the enzyme stabilizer can be selected while considering various other conditions, and added to the reaction solution in the first process or the second process. As the enzyme stabilizer, for example, NaCl, KCl, (NH 4 ) 2 SO 4 Select the concentration in the range of 0.1 to 0.5 M, select the concentration of glycerol, ethylene glycol, sucrose, trehalose, sorbitol, mannitol, ethanol in the range of 0.1 to 50%, and select the concentration of surfactants, skim milk, soy protein, whey, casein, albumin in the range of 0.001 to 1%, and add them to the reaction solution, and optimization can be carried out accordingly.

[0080] Regarding the optimization of the addition of product stabilizers, in order to prevent the products in the first process and the second process from being affected by heat, oxygen, co-existing substances, co-existing enzymes, etc. and reducing the yield due to accidental decomposition, the type and concentration of the product stabilizer can be selected while considering various other conditions, and added to the reaction solution in the first process or the second process. As the product stabilizer, for example, appropriately select from chelating agents such as EDTA, reducing agents such as catalase, mercaptoethanol, DTT or thioglycerol, sodium sulfite, etc., and add them to the reaction solution, and optimization can be carried out accordingly.

[0081] Regarding the optimization of the addition of surfactants, in order to shift the equilibrium of the reaction to promote the enzyme reaction in the first process or the second process and increase the product amount, the type and concentration of the surfactant can be selected while considering various other conditions, and added to the reaction solution in the first process or the second process. As the surfactant, for example, it can be appropriately selected from anionic surfactants, cationic surfactants, amphoteric surfactants, non-ionic surfactants, saponins, phospholipids, peptides, biosurfactants (glycolipid series, acyl peptide series, phospholipid series, fatty acid series, polymer series), etc. and added to the reaction solution, and optimization is carried out accordingly.

[0082] Regarding the optimization of the addition of organic solvents, in order to shift the equilibrium of the reaction to promote the enzyme reaction in the first process or the second process and increase the product amount, the type and concentration of the organic solvent can be selected while considering various other conditions, and added to the reaction solution in the first process or the second process. As the organic solvent, for example, it can be appropriately selected from methanol, ethanol, isopropanol, hexane, etc. and added to the reaction solution, and optimization is carried out accordingly.

[0083] Regarding the optimization of the addition of reaction cofactors and reaction-promoting components, for the cofactors required for the enzyme-based reaction in the first or second step, or for the reaction-promoting components that activate the reaction to shorten the reaction time and / or reduce the amount of added enzyme, the type and concentration of the reaction cofactor or reaction-promoting component can be selected while considering various other conditions and added to the reaction solution in the first or second step. As the reaction cofactor or reaction-promoting component, for example, Mg compounds such as MgCl 2 and MgSO 4 , Mn compounds such as MnCl 2 , Fe compounds such as FeCl 3 and FeCl 2 , Zn compounds such as ZnCl 2 , Co compounds, Mo compounds, Cu compounds, Ag compounds, Al compounds, Ca compounds, Ni compounds, etc. can be appropriately selected or combined in the range of 0.001 to 100 mM, preferably 0.1 to 100 mM, more preferably 2 to 50 mM, etc., and added to the reaction solution to optimize it accordingly.

[0084] Regarding the optimization of by-product removal, the conditions for removing alkali from the reaction solution can be selected in the first step and the conditions for removing pyrophosphate from the reaction solution can be selected in the second step while considering various other conditions. In the first step, hypoxanthine is removed when the nucleoside monophosphate is IMP, guanine is removed when it is GMP, and adenine is removed when it is AMP.

[0085] Regarding the removal of hypoxanthine, it can be removed by converting it to xanthine and uric acid using xanthine oxidase or xanthine dehydrogenase. When using xanthine oxidase, the generated hydrogen peroxide is removed using catalase or peroxidase, whereby the removal of hypoxanthine can be carried out more effectively. When using xanthine dehydrogenase, the generated NADH is converted to NAD using NADH oxidase, etc., whereby the removal of hypoxanthine can be carried out more effectively. Regarding xanthine oxidase, it can be added at an enzyme concentration of 0.01 to 100 U / mL, preferably 0.1 to 10 U / mL, such as XTO-212 (product number) manufactured by Toyobo Co., Ltd., and the above conversion reaction can be carried out at a reaction temperature of 0 to 70 °C, preferably 25 to 65 °C.

[0086] Regarding the removal of guanine, it can be achieved by converting guanine to xanthine using guanine deaminase and then removing xanthine.

[0087] Regarding the removal of adenine, it can be achieved by converting adenine to hypoxanthine using adenine deaminase and then removing hypoxanthine.

[0088] In addition, since the solubilities of hypoxanthine, guanine, and adenine in water are all very low, the reaction can be carried out at a high temperature, followed by cooling and precipitation. After removal by centrifugation or filtration, the temperature is restored to a high temperature, and the above operations are repeated for removal. For example, the high temperature can be in the range of 37 to 65 °C, and the cooling can be in the range of 0 to 10 °C. In addition, since the solubilities of hypoxanthine, guanine, and adenine in water are all very low, water can be evaporated, etc., for concentration to cause precipitation. After removal by centrifugation or filtration, water is added again for restoration, and the above operations are repeated for removal. It can also be removed by extraction and liquid separation in organic solvents such as hexane and chloroform. Additionally, hypoxanthine, guanine, and adenine can be adsorbed onto a resin, etc., for removal.

[0089] In the case where the nucleoside monophosphate is a mixture of IMP and GMP in the first step, the above methods can be appropriately combined for use.

[0090] Regarding the pyrophosphate generated in the second step, when the first step is carried out before the second step, it can be simply removed. When the first step and the second step are carried out simultaneously, it needs to be removed after the first step has been sufficiently carried out. Sufficiently carrying out the first step means that, for example, 10% or more, preferably 30% or more, more preferably 50% or more, further preferably 70% or more, and most preferably 90% or more of the nucleoside monophosphate as the input raw material has been converted into bases.

[0091] Regarding the removal of pyrophosphate, there are the following methods: a method of adding inorganic diphosphatase (EC 3.6.1.1) etc. to the reaction solution for hydrolysis into phosphoric acid; a method of adding a cation for precipitation (for example, a method of adding an excessive amount of Mg ions, Mn ions, or Ca ions for precipitation). Adding an excessive amount of Mg ions, Mn ions, or Ca ions means adding an excessive concentration of Mg compounds, Mn compounds, or Ca compounds of 50 mM or more, preferably 100 mM or more, more preferably 200 mM or more, further preferably 500 mM or more, and most preferably 1000 mM or more to the reaction solution. Additionally, pyrophosphate can be adsorbed onto a resin, etc., for removal.

[0092] According to the present embodiment, a method for manufacturing nicotinamide mononucleotide can be provided, which uses one enzyme with nucleoside monophosphate, pyrophosphate, and nicotinamide as raw materials to manufacture nicotinamide mononucleotide. Therefore, according to the present embodiment, a method for manufacturing nicotinamide mononucleotide can be provided, which manufactures nicotinamide mononucleotide from a small number of relatively inexpensive raw materials through a simple process.

[0093] In addition, according to another embodiment, a method for producing nicotinamide mononucleotide can be provided, in which an enzyme belonging to hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) is allowed to act on phosphoribosyl diphosphate and nicotinamide to produce nicotinamide mononucleotide. When this production method is used in the production method of the above-described embodiment including the above first and second steps, the enzyme belonging to hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) that functions is one kind (including the case of substantially one kind as described above). In other cases, for example, when implementing a production method consisting only of the second step, the enzyme can be plural kinds. For example, two or more enzymes selected from HPT-C, HPT-W, HPT-L, and polypeptides having an amino acid sequence with 90% or more identity to these amino acid sequences can be allowed to act. It should be noted that as described above for substantially one enzyme, the one or two or more enzymes here are enzymes belonging to hypoxanthine phosphoribosyltransferase (EC 2.4.2.8), and the other co-existing enzymes are one or two or more enzymes capable of catalyzing the above reaction (corresponding to the second step) for producing nicotinamide mononucleotide. When allowing one or two or more enzymes belonging to hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) to act, other co-existing enzymes that are 10% or less, preferably 5% or less, more preferably 1% or less of the entire enzyme catalyzing the above reaction can also be co-existing. In addition, in addition to one or two or more enzymes belonging to hypoxanthine phosphoribosyltransferase (EC 2.4.2.8), other enzymes that do not catalyze the above reaction (corresponding to the second step) for producing nicotinamide mononucleotide can also co-exist.

[0094] The following will specifically describe the examples of the present invention, but the present invention is not limited by any of them.

[0095] Examples

[0096] [Example 1: HPLC-based detection of bases, nucleoside monophosphates, nicotinamide, and nicotinamide mononucleotide]

[0097] [Preparation of sample solution]

[0098] The following samples were dissolved in pure water at the recorded concentrations to prepare sample solutions.

[0099] 0.05 mg / mL IMP

[0100] 0.05 mg / mL GMP

[0101] 0.05 mg / mL NMN

[0102] 0.02 mg / mL hypoxanthine

[0103] 0.02 mg / mL guanine

[0104] 0.02 mg / mL NAM

[0105] [Detection conditions]

[0106] HPLC system: Shimadzu LC-20A

[0107] Detection: UV254 nm

[0108] Column: YMC-Triart C18

[0109] TA12S05-1546WT 150 mm x 4.6 mm (YMC Co., Japan)

[0110] Flow rate, mode, column temperature: 1 mL / min, isocratic, 37 °C

[0111] Mobile phase: 10 mM potassium dihydrogen phosphate

[0112] Sample injection: 5 μL

[0113] [Results]

[0114] The results are shown in Figure 2 and Table 1. Figure 2 The elution behavior is shown (horizontal axis: elution time (minutes), vertical axis: detection intensity (mV)). The retention times of each sample are as described in Table 1, and it can be seen that the peaks are separated except for hypoxanthine and guanine.

[0115] [Table 1]

[0116] Sample Delay Time (minutes) NMN 2.615 GMP 4.352 IMP 4.670 Hypoxanthine 6.027 Guanine 6.027 NAM 12.616

[0117] [Example 2: Production of HGPRT]

[0118] (1) Preparation of a transformant inserted with a plasmid of Escherichia coli-derived HGPRT

[0119] (1.1) Confirmation of the sequence of Escherichia coli-derived HGPRT

[0120] Using the chromosomal DNA of Escherichia coli strain W3110 as a template, PCR was performed using a sense primer (sequence number 1), an antisense primer (sequence number 2), and KOD PLUS NEO (model: KOD-401, Toyobo Co., Ltd.) to amplify the HGPRT gene, and a PCR product was obtained. The obtained PCR product was inserted into pCR-Blunt II-Topo (Thermo Fisher) using the Zero Blunt TOPO PCR cloning kit. Subsequently, the nucleotide sequence of the obtained PCR product was confirmed by sequencing. The nucleotide sequence of HGPRT from Escherichia coli strain W3110 and the amino acid sequence deduced from this nucleotide sequence are shown in sequence numbers 3 and 4, respectively.

[0121] (1.2) Preparation of a transformant of a plasmid inserted with Escherichia coli-derived HGPRT

[0122] Using pCR-Blunt II-Topo inserted with the Escherichia coli-derived HGPRT gene as a template, PCR was performed using a sense primer (sequence number 5), an antisense primer (sequence number 6), and KOD PLUS NEO (model: KOD-401, Toyobo Co., Ltd.) to amplify the HGPRT gene having the AAGGAGATATACAT sequence before the start codon at the 5'-end, the CATCACCATCACCATCAC sequence encoding a His-tag immediately after the start codon of HGPRT, and the GGATCCGAATTCGAGC sequence immediately after the stop codon on the 3'-end side, and a PCR product was obtained. The obtained PCR product was inserted into the NdeI-BamHI site of the pET-21a(+) vector (Novagen), an expression vector, by the In-Fusion method using the In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain a plasmid for HGPRT / pET21a(+) expression. In this expression plasmid, a nucleotide sequence encoding a His-tag was added to the 5'-end of the HGPRT gene. This expression plasmid was introduced into One shot BL21(DE3) Chemically Competent E.coli (Invitrogen) to obtain a transformant HGPRT / pET-21a(+) / BL21(DE3) having a recombinant vector (this recombinant vector contains a polynucleotide encoding Escherichia coli-derived HGPRT).

[0123] (2) Preparation of Escherichia coli-derived HGPRT (HPT-W)

[0124] (2.1) Induced Expression of HGPRT in the Transformant and Preparation of Crude Enzyme Solution

[0125] Take one colony of the transformant obtained in the above (1.2), inoculate it into an LB liquid medium (5 mL) containing 50 μg / mL ampicillin, and culture it in a test tube at about 30 °C for about 22 hours. Add the culture (1.6 mL) to a liquid medium containing 50 μg / mL ampicillin (6% Overnight Express Instant TB Medium (Merck), 0.1% ADEKANOL LG-109 (manufactured by ADEKA), containing 1% glycerol) (1.6 L), and culture it in a fermenter at 30 °C, with aeration at 1.6 L / min and a rotation speed of 650 rpm for about 24 hours. Centrifuge the culture to collect the bacteria, resuspend the obtained bacterial cells in Solution A (20 mM potassium phosphate buffer (pH 7.0), 0.3 M NaCl), solubilize the bacterial cells by ultrasonic disruption, and then centrifuge to obtain a crude enzyme solution. TM

[0126] (2.2) Purification of HPT-W

[0127] Pack Chelating Sepharose Fast Flow (GE Healthcare) into a column and immobilize Ni. 2+ After that, equilibrate it with Solution A. Add the above crude enzyme solution to the obtained column to adsorb HPT-W. Wash the column with Solution A, and then elute HPT-W with a linear gradient of Solution A and Solution A containing 0.4 M imidazole for 10 column volumes (CV). Concentrate the obtained active fraction of HPT-W using a pen-type ultrafiltration (UF) module (Asahi Kasei Chemicals Corporation) until it reaches 1 / 10 of the original volume, and then desalt it in a PD-10 column (GE Healthcare) equilibrated with 10 mM potassium phosphate buffer (pH 7.0) to obtain an enzyme solution of HPT-W.

[0128] (3) Preparation of HPT-C and HPT-L

[0129] (3.1) Preparation of HGPRT (HPT-C) from Hungateiclostridium thermocellum

[0130] ​Using the synthetic gene as a polynucleotide (SEQ ID NO: 8) encoding HGPRT (SEQ ID NO: 7) derived from Hungateiclostridium thermocellum as a template, PCR was performed using a sense primer (SEQ ID NO: 9) and an antisense primer (SEQ ID NO: 10), and inserted into the pET-21a(+) vector using the In-fusion method. Except for this, the production of transformants, the induced expression of HGPRT, and purification were carried out in the same manner as HPT-W, and a solution of HGPRT (HPT-C) derived from Hungateiclostridium thermocellum with a His tag at the N-terminus was obtained.

[0131] (3.2) Preparation of HGPRT (HPT-L) derived from Archaeoglobus veneficus

[0132] Using the synthetic gene as a polynucleotide (SEQ ID NO: 12) encoding HGPRT (SEQ ID NO: 11) derived from Archaeoglobus veneficus as a template, PCR was performed using a sense primer (SEQ ID NO: 13) and an antisense primer (SEQ ID NO: 14), and inserted into the pET-21a(+) vector using the In-fusion method. Except for this, the production of transformants, the induced expression of HGPRT, and purification were carried out in the same manner as HPT-W, and a solution of HGPRT (HPT-L) derived from Archaeoglobus veneficus with a His tag at the N-terminus was obtained.

[0133] (4) Activity assay of HPT-C, HPT-W and HPT-L

[0134] Regarding the enzyme activity of HPT-C, HPT-W and HPT-L against inosine monophosphate, the hypoxanthine generated by the action of HGPRT was converted into uric acid using xanthine dehydrogenase, and the measurement was based on the change in absorbance at 340 nm of the resulting NADH. The amount of enzyme that produces 1 μmol of hypoxanthine in 1 minute at 37 °C was defined as 1 U. The measurement was carried out using a Hitachi 7080 automatic analyzer (Hitachi High-Technologies Corporation). The composition of the activity assay reagent, the composition of the enzyme dilution solution, and the measurement parameters of the automatic analyzer are as follows.

[0135] [Composition of activity assay reagent]

[0136] 20 mM Tris-HCl pH 7.5 (Merck)

[0137] 5 mM Inosine monophosphate (Merck)

[0138] 5 mM disodium phosphate (FUJIFILM Wako Pure Chemical Corporation)

[0139] 5 mM NAD (Oriental Yeast Co., Ltd.)

[0140] 5 mM MgCl 2 (FUJIFILM Wako Pure Chemical Corporation)

[0141] 5 U / mL XDH II (Xanthine dehydrogenase: T-134, Asahi Kasei Pharma Corporation)

[0142] [Composition of enzyme dilution solution]

[0143] 20 mM Tris-HCl pH 7.5 (Merck)

[0144] [Measurement parameters of the automatic analyzer]

[0145] Analysis method Rate-A

[0146] Measurement wavelength (secondary / principal) 405 nm / 340 nm

[0147] Reaction time 5 minutes

[0148] Measurement points 10 - 13

[0149] Sample volume 5 μL

[0150] Reagent volume for activity measurement 150 μL

[0151] [Example 3: Generation of base from nucleoside monophosphate and pyrophosphate based on HGPRT (first step)]

[0152] [Preparation of reaction solution]

[0153] Dissolve the following reagent components in pure water at the indicated concentrations and mix them to prepare the reaction solution.

[0154] 50 mM Tris-HCl (pH 8.0)

[0155] 5 mM Pyrophosphate

[0156] 5 mM Nucleoside monophosphate (IMP or GMP)

[0157] 20 mM MgSO 4

[0158] 2.5 U / mL HGPRT (HPT-C, HPT-W or HPT-L)

[0159] [Measurement]

[0160] After reacting at 37°C for 1 hour, it was diluted 20-fold with pure water, filtered using a membrane with a molecular weight cut-off of 10,000, and the filtrate was analyzed by HPLC.

[0161] [Detection conditions]

[0162] Performed in the same manner as in Example 1.

[0163] [Results]

[0164] The peak areas of IMP, hypoxanthine, GMP, and guanine are shown in Table 2. It can be seen that all three HGPRTs catalyzed the reactions from IMP to hypoxanthine and from GMP to guanine.

[0165] [Table 2]

[0166]

[0167] [Example 4: Generation of NMN from NAM and PRPP based on HGPRT or NAMPT (second step)]

[0168] [Preparation of reaction solution]

[0169] The following reagent components were dissolved in pure water at the recorded concentrations and mixed to prepare a reaction solution.

[0170] 100 mM of each buffer shown in Table 3

[0171] 2 mM NAM

[0172] 2 mM PRPP

[0173] 20 mM MgSO 4

[0174] 2.5 U / mL HGPRT (HPT-C, HPT-W, or HPT-L)

[0175] Or 0.2 μg / mL NAMPT (SPR0514 manufactured by MERCK)

[0176] [Measurement]

[0177] After reacting at 37°C for 2 hours, it was diluted 8-fold with pure water, filtered using a membrane with a molecular weight cut-off of 10,000, and the filtrate was analyzed by HPLC.

[0178] [Detection conditions]

[0179] Performed in the same manner as in Example 1.

[0180] [Results]

[0181] The peak areas of NMN and NAM are shown in Table 3. It can be seen that HPT-C catalyzed the reaction from NAM to NMN in buffers with pH 5 and 6, HPT-W catalyzed the reaction from NAM to NMN in buffers with pH 5, 6, 7, 8, and 9, HPT-L catalyzed the reaction from NAM to NMN in the buffer with pH 5, and NAMPT catalyzed the reaction from NAM to NMN in buffers with pH 6, 7, 8, and 9.

[0182] [Table 3]

[0183]

[0184] [Example 5: Generation of nucleoside monophosphate from base and PRPP based on HGPRT (reverse reaction of the first step)]

[0185] [Preparation of reaction solution]

[0186] Dissolve the following reagent components in pure water at the recorded concentrations, mix them, and prepare the reaction solution.

[0187] 50 mM Tris-HCl (pH 8.0)

[0188] 1 mM base (hypoxanthine or guanine)

[0189] 1 mM PRPP

[0190] 5 mM MgSO 4

[0191] 2.5 U / mL HGPRT (HPT-C, HPT-W or HPT-L)

[0192] [Measurement]

[0193] After reacting at 37 °C for 1 hr, dilute 4-fold with pure water, filter through a membrane with a molecular weight cut-off of 10,000, and perform HPLC analysis on the filtrate.

[0194] [Detection conditions]

[0195] Perform in the same manner as in Example 1.

[0196] [Results]

[0197] The peak areas of IMP, hypoxanthine, GMP, and guanine are shown in Table 4. It can be seen that all three types of HGPRT catalyzed the reactions from hypoxanthine to IMP and from guanine to GMP.

[0198] [Table 4]

[0199]

[0200] [Example 6: Generation of NAM from NMN and pyrophosphate based on HGPRT or NAMPT (reverse reaction of the second step)]

[0201] [Preparation of reaction solution]

[0202] Dissolve the following reagent components in pure water at the recorded concentrations and mix them to prepare a reaction solution.

[0203] 50 mM Tris-HCl (pH 8.0)

[0204] 5 mM Pyrophosphate

[0205] 5 mM NMN

[0206] 20 mM MgSO 4

[0207] 2.5 U / mL HGPRT (HPT-C, HPT-W or HPT-L)

[0208] Or 0.2 μg / mL NAMPT (SPR0514 manufactured by MERCK)

[0209] [Measurement]

[0210] After reacting at 37 °C for 1 hr, dilute 20-fold with pure water, filter through a membrane with a molecular weight cut-off of 10,000, and perform HPLC analysis on the filtrate.

[0211] [Detection conditions]

[0212] Perform in the same manner as in Example 1.

[0213] [Results]

[0214] The peak areas of NMN and NAM are shown in Table 5. It can be seen that all three types of HGPRT and NAMPT catalyzed the reaction from NMN to NAM.

[0215] [Table 5]

[0216]

[0217] [Example 7: Generation of NMN from IMP, pyrophosphate, and NAM based on HGPRT (simultaneous performance of the first and second steps)]

[0218] [Preparation of reaction solution]

[0219] Dissolve the following reagent components in pure water at the recorded concentrations and mix them to prepare a reaction solution.

[0220] 100 mM Each buffer shown in Table 6

[0221] 20 mM IMP

[0222] 2 mM Pyrophosphate

[0223] 20 mM NAM

[0224] 20 mM MgSO 4

[0225] 2.5 U / mL HGPRT (HPT-C or HPT-W)

[0226] [Determination]

[0227] After reacting at 37 °C for 2 h, it was diluted 40-fold with pure water, filtered using a membrane with a molecular weight cut-off of 10,000, and the filtrate was analyzed by HPLC.

[0228] [Detection Conditions]

[0229] Performed in the same manner as in Example 1.

[0230] [Results]

[0231] The peak areas of NMN, NAM, IMP, and hypoxanthine are shown in Table 6. It was found that both HPT-C and HPT-W (excluding HPT-W at pH 6) catalyzed the reaction from IMP, pyrophosphate, NAM to NMN.

[0232] [Table 6]

[0233]

[0234] [Example 8: Addition of XOD in the production reaction of NMN from IMP, pyrophosphate, and NAM based on HGPRT (the first and second steps are carried out simultaneously)]

[0235] [Preparation of Reaction Solution]

[0236] The following reagent components were dissolved in pure water at the indicated concentrations and mixed to prepare a reaction solution.

[0237] 100 mM Each buffer shown in Table 7

[0238] 5 mM IMP

[0239] 5 mM Pyrophosphate

[0240] 5 mM NAM

[0241] 20 mM MgSO 4

[0242] 2.5 U / mL HPT-C (without addition of XOD)

[0243] or 2.5 U / mL HPT-C (added with 50 U / mL XOD (XTO-212 manufactured by Toyobo Co., Ltd.))

[0244] [Measurement]

[0245] After reacting at 37 °C for 5 hours, it was diluted 20-fold with pure water, filtered using a membrane with a molecular weight cut-off of 10,000, and the filtrate was analyzed by HPLC.

[0246] [Detection conditions]

[0247] The same procedure as in Example 1 was carried out.

[0248] [Results]

[0249] The peak areas of NMN, NAM, IMP, and hypoxanthine are shown in Table 7. It was found that the addition of XOD promoted the reaction from IMP, pyrophosphate, NAM to NMN.

[0250] [Table 7]

[0251]

[0252] [Example 9: Addition of XOD in the production reaction of NMN from IMP, pyrophosphate, and NAM based on HGPRT (the first and second steps are carried out simultaneously)]

[0253] [Preparation of reaction solution]

[0254] The following reagent components were dissolved in pure water at the recorded concentrations and mixed to prepare a reaction solution.

[0255] 100 mM acetate (pH 5)

[0256] 1 mM IMP

[0257] 0.2 mM pyrophosphate

[0258] 1 mM NAM

[0259] 50 mM MgSO 4

[0260] 2.5 U / mL HGPRT (HPT-C, HPT-W, or HPT-L)

[0261] 10 U / mL XOD (XTO-212 manufactured by Toyobo Co., Ltd.)

[0262] [Measurement]

[0263] After reacting at 37 °C or 65 °C for 1 hour, it was diluted 4-fold with pure water, filtered using a membrane with a molecular weight cut-off of 10,000, and the filtrate was analyzed by HPLC.

[0264] [Detection conditions]

[0265] Carry out in the same manner as in Example 1.

[0266] [Results]

[0267] The peak areas of NMN, NAM, IMP, and hypoxanthine are shown in Table 8. It can be seen that HGPRT catalyzed the reaction from IMP, pyrophosphate, NAM to NMN at 37 °C and 65 °C.

[0268] [Table 8]

[0269]

[0270] [Example 10: Addition of XOD in the production reaction of NMN from a mixture of IMP and GMP, pyrophosphate, and NAM based on HGPRT (the first and second steps are carried out simultaneously)]

[0271] [Preparation of reaction solution]

[0272] Dissolve the following reagent components in pure water at the recorded concentrations and mix them to prepare a reaction solution.

[0273] 100 mM acetate (pH 5)

[0274] 0.5 mM IMP

[0275] 0.5 mM GMP

[0276] 0.2 mM pyrophosphate

[0277] 1 mM NAM

[0278] 50 mM MgSO 4

[0279] 2.5 U / mL HGPRT (HPT-C, HPT-W, or HPT-L)

[0280] 10 U / mL XOD (XTO-212 manufactured by Toyobo Co., Ltd.)

[0281] [Measurement]

[0282] After reacting at 37 °C or 65 °C for 1 hr, dilute 4-fold with pure water, filter using a membrane with a molecular weight cut-off of 10,000, and perform HPLC analysis on the filtrate.

[0283] [Detection conditions]

[0284] Carry out in the same manner as in Example 1.

[0285] [Results]

[0286] The peak areas of NMN, NAM, IMP, and GMP are shown in Table 9. It can be seen that HGPRT catalyzed the reaction of a mixture of IMP and GMP, pyrophosphate, NAM to NMN at 37 °C and 65 °C.

[0287] [Table 9]

[0288]

[0289] [Example 11: Adding XOD to the production reaction of NMN from IMP, pyrophosphate, and NAM based on HGPRT and changing the ratios of IMP, pyrophosphate, and NAM (the first and second steps are carried out simultaneously)]

[0290] [Preparation of reaction solution]

[0291] Dissolve the following reagent components in pure water at the recorded concentrations, mix them, and prepare the reaction solution.

[0292] 100 mM acetate (pH 5)

[0293] IMP at each concentration shown in Table 10

[0294] Pyrophosphate at each concentration shown in Table 10

[0295] NAM at each concentration shown in Table 10

[0296] 50 mM MgSO 4

[0297] 2.5 U / mL HPT-C

[0298] 10 U / mL XOD (XTO-212 manufactured by Toyobo Co., Ltd.)

[0299] [Measurement]

[0300] After reacting at 37 °C for 3 hours, dilute the reaction solution of 1 mM NAM 4-fold with pure water, dilute the reaction solution of 0.5 mM NAM 2-fold, and do not dilute the reaction solution of 0.2 mM NAM. Filter through a membrane with a molecular weight cut-off of 10,000 and perform HPLC analysis on the filtrate.

[0301] [Detection conditions]

[0302] Carry out in the same manner as in Example 1.

[0303] [Results]

[0304] The peak areas of NMN, NAM, and IMP are shown in Table 10. It can be seen that NMN was generated at various ratios of IMP, pyrophosphate, and NAM.

[0305] [Table 10]

[0306]

[0307] [Example 12: Add XOD to the production reaction of generating NMN from GMP, pyrophosphate, and NAM based on HGPRT, and change the ratios of GMP, pyrophosphate, and NAM (the first process and the second process are carried out simultaneously)]

[0308] [Preparation of reaction solution]

[0309] Dissolve the following reagent components in pure water at the recorded concentrations, and mix them to prepare a reaction solution.

[0310] 100 mM acetate (pH 5)

[0311] 1 mM GMP

[0312] Pyrophosphate at each concentration shown in Table 11

[0313] 1 mM NAM

[0314] 50 mM MgSO 4

[0315] 2.5 U / mL HPT-C

[0316] 10 U / mL XOD (XTO-212 manufactured by Toyobo Co., Ltd.)

[0317] [Measurement]

[0318] After reacting at 37 °C for 6 hours, dilute the reaction solution 4-fold with pure water, filter it through a membrane with a molecular weight cut-off of 10,000, and perform HPLC analysis on the filtrate.

[0319] [Detection conditions]

[0320] Carry out in the same manner as in Example 1.

[0321] [Results]

[0322] The peak areas of NMN, NAM, and GMP are shown in Table 11. It can be seen that NMN was generated at various ratios of GMP, pyrophosphate, and NAM.

[0323] [Table 11]

[0324]

[0325] [Example 13: Add XOD to the production reaction of generating NMN from IMP, pyrophosphate, and NAM based on HGPRT, and change Mg ions to Mn ions (the first process and the second process are carried out simultaneously)]

[0326] [Preparation of reaction solution]

[0327] Dissolve the following reagent components in pure water at the recorded concentrations, mix them, and prepare a reaction solution.

[0328] 100 mM acetate (pH 5)

[0329] 1 mM IMP

[0330] 0.2 mM pyrophosphate

[0331] 1 mM NAM

[0332] 50 mM MgSO 4 or 50 mM MnCl 2

[0333] 2.5 U / mL HPT-C

[0334] 10 U / mL XOD (XTO-212 manufactured by Toyobo Co., Ltd.)

[0335] [Measurement]

[0336] After reacting at 37 °C for 3 hours, dilute the reaction solution 4-fold with pure water, filter it using a membrane with a molecular weight cut-off of 10,000, and perform HPLC analysis on the filtrate.

[0337] [Detection conditions]

[0338] Perform in the same manner as in Example 1.

[0339] [Results]

[0340] The peak areas of NMN, NAM, and IMP are shown in Table 12. It can be seen that even when the Mg ions are replaced with Mn ions, NMN is produced.

[0341] [Table 12]

[0342]

[0343] [Example 14: Confirmation of the molecular weight and structure of nicotinamide mononucleotide generated from nucleoside monophosphate, pyrophosphate, and nicotinamide based on HGPRT]

[0344] [Preparation of reaction solution]

[0345] Dissolve the following reagent components in pure water at the recorded concentrations, mix them, and prepare a reaction solution.

[0346] 100 mM acetate (pH 5)

[0347] 1 mM IMP

[0348] 0.2 mM pyrophosphate

[0349] 1 mM NAM

[0350] 50 mM MgSO 4

[0351] 2.5 U / mL HPT-C

[0352] 10 U / mL XOD (XTO-212 manufactured by Toyobo Co., Ltd.)

[0353] [Measurement]

[0354] After reacting at 37 °C for 3 hours, the reaction solution was diluted 4-fold with pure water, filtered using a membrane with a molecular weight cut-off of 10,000, and subjected to LC-MS / MS analysis.

[0355] [Analysis Conditions]

[0356] LC system: ACQUITY UPLC I-Class manufactured by Waters K.K., Japan

[0357] MS: micrOTOF-Q III manufactured by Bruker Daltonics GmbH

[0358] Measurement conditions

[0359] Source type: ESI, Scan range: 50 - 1,000 m / z, Ion polarity: positive

[0360] Capillary: 4,500 V, End plate: 500 V, Nebulizer: 1.2 bar

[0361] Dry heater: 200 °C, Dry gas: 6.0 L / min

[0362] Software: DataAnalysis Ver.4.3 manufactured by Bruker Daltonics GmbH

[0363] NIST MS Search Ver.2.2 manufactured by the National Institute of Standards and Technology, USA

[0364] Column: ACQUITY UPLC BEH C18 2.1 mm I.D.×50 mm, 1.7 μm

[0365] Column temperature: Constant temperature near 40 °C

[0366] Mobile phase: 10 mmol / L ammonium formate solution (pH 4.6)

[0367] Flow rate: 0.2 mL / min

[0368] Sample injection: 5 μL or 20 μL

[0369] [Results]

[0370] Molecular weight measurement value [M+H] of the substance (sample) eluted with the same delay time as the NMN generated in this reaction + was 335.0637, and it was confirmed to be consistent with NMN using the Figure 3 MS / MS spectrum shown

[0371] Sequence Listing Free Text

[0372] Sequence No. 1: Sense primer used in (1.1) of Example 2

[0373] Sequence No. 2: Antisense primer used in (1.1) of Example 2

[0374] Sequence No. 3: Base sequence of the gene encoding HPT-W

[0375] Sequence No. 4: Amino acid sequence of HPT-W

[0376] Sequence No. 5: Sense primer used in (1.2) of Example 2

[0377] Sequence No. 6: Antisense primer used in (1.2) of Example 2

[0378] Sequence No. 7: Amino acid sequence of HPT-C

[0379] Sequence No. 8: Base sequence of the gene encoding HPT-C

[0380] Sequence No. 9: Sense primer used in (3.1) of Example 2

[0381] Sequence No. 10: Antisense primer used in (3.1) of Example 2

[0382] Sequence No. 11: Amino acid sequence of HPT-L

[0383] Sequence No. 12: Base sequence of the gene encoding HPT-L

[0384] Sequence No. 13: Sense primer used in (3.2) of Example 2

[0385] Sequence No. 14: Antisense primer used in (3.2) of Example 2

Claims

1. A method for manufacturing nicotinamide mononucleotide, which at least includes the following steps 1) and 2): 1) The first step, causing substantially one kind of enzyme to act on nucleoside monophosphate and pyrophosphate to generate phosphoribosyl diphosphate; 2) The second step, causing substantially only the above-mentioned one kind of enzyme to act on phosphoribosyl diphosphate, which is the product of the first step, and nicotinamide to generate nicotinamide mononucleotide, The nucleoside monophosphate is AMP, GMP, IMP, XMP or a mixture of two or more of them, the one kind of enzyme is an enzyme belonging to hypoxanthine phosphoribosyltransferase (EC 2.4.2.8), and the enzyme belonging to hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) is any one of enzyme HPT-C formed by the amino acid sequence recited in SEQ ID NO: 7, enzyme HPT-W formed by the amino acid sequence recited in SEQ ID NO: 4, and enzyme HPT-L formed by the amino acid sequence recited in SEQ ID NO: 11; The term "substantially" means that in the percentage display based on the protein weight of the enzyme or the enzyme unit as the activity unit of the enzyme, the one kind of enzyme is 95% or more, and the other co-existing enzymes are 5% or less.

2. The manufacturing method according to claim 1, wherein, The first step and the second step are steps carried out simultaneously.

3. The manufacturing method according to claim 1 or 2, wherein, The nucleoside monophosphate is IMP, GMP, or a mixture of IMP and GMP.

4. The manufacturing method according to claim 1 or 2, wherein, Part or all of the nucleoside monophosphate is IMP, The first step includes a step of causing xanthine oxidase to act on hypoxanthine generated in this step.

5. The manufacturing method according to claim 1 or 2, wherein, The first step and the second step are steps of causing the enzyme to act in the presence of Mg ions and / or Mn ions.

6. A method for manufacturing nicotinamide mononucleotide, wherein, Causing one or more kinds of enzymes belonging to hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) to act on phosphoribosyl diphosphate and nicotinamide to manufacture nicotinamide mononucleotide, At least one of the enzymes belonging to hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) is any one of enzyme HPT-C formed by the amino acid sequence recited in SEQ ID NO: 7, enzyme HPT-W formed by the amino acid sequence recited in SEQ ID NO: 4, and enzyme HPT-L formed by the amino acid sequence recited in SEQ ID NO:

11.

7. The manufacturing method according to claim 6, wherein, Causing the enzyme to act in the presence of Mg ions and / or Mn ions.

Images