Method for producing nicotinamide mononucleotide
The present invention addresses the problem of providing a method for producing nicotinamide mononucleotide, that produces nicotinamide mononucleotide using a single enzyme and using nucleoside monophosphate, pyrophosphate, and nicotinamide as starting materials. This problem is solved by a nicotinamide mononucleotide production method that includes at least the following steps 1) and 2): 1) a first step of producing phosphoribosyl diphosphate by the action of substantially one enzyme on nucleoside monophosphate and pyrophosphate; and 2) a second step of producing nicotinamide mononucleotide by the action of only substantially the aforementioned one enzyme on nicotinamide and the phosphoribosyl diphosphate that is the product of the first step.
1. A method for making nicotinamide mononucleotide (NMN), which method comprises at least the following steps 1) and 2):
1) Reacting a hypoxanthine phosphoribosyltransferase (HPT, EC 2.4.2.8) in a reaction mixture with a nucleoside monophosphate at a concentration of 0.01-500 mM and pyrophosphate at a concentration of 0.001-100 mM to produce phosphoribosyl diphosphate (PRPP); and
2) Reacting said HPT in the reaction mixture with said PRPP and nicotinamide to produce nicotinamide mononucleotide (NMN) at a concentration of 0.01-500 mM;
wherein said nucleoside monophosphate is at least one member selected from the group consisting of adenosine monophosphate (AMP), 5′-guanylic acid (GMP), inosinic acid (IMP) and xanthosine monophosphate (XMP), said HPT is present in said reaction mixture in an amount of 0.01 U-1000 kU/L and said HPT is present in an amount of at least 90% of any enzymes present in said reaction mixture, wherein said at least 90% indicates enzyme protein weight or a unit of activity for the enzyme.
2. The method according to claim 1 , wherein said steps 1) and 2) are carried out simultaneously in the same reaction mixture.
3. The method according to claim 1 , wherein said reaction mixture comprises at least one member selected from the group consisting of a buffer, an enzyme stabilizer and a surfactant.
4. The method according to claim 2 , wherein said reaction mixture comprises at least one member selected from the group consisting of a buffer, an enzyme stabilizer and a surfactant.
5. The method according to claim 1 , wherein said HPT is at least one member selected from the group consisting of HPT-C(enzyme comprising the amino acid sequence given by SEQ ID NO: 7), HPT-W (enzyme comprising the amino acid sequence given by SEQ ID NO: 4), HPT-L (enzyme comprising the amino acid sequence given by SEQ ID NO: 11), and a polypeptide that contains an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of any one of HPT-C, HPT-W, and HPT-L.
6. The method according to claim 1 , wherein said nucleoside monophosphate is IMP, GMP, or a mixture of IMP and GMP.
7. The method according to claim 1 , wherein all or a portion of said nucleoside monophosphate is IMP, and
said step 1) includes a step of causing an action of xanthine oxidase on hypoxanthine produced in said step 1).
8. The method according to claim 1 , wherein said reaction mixture further comprises Mg ion and/or Mn ion.
9. A method for producing nicotinamide mononucleotide (NMN), which method comprises:
reacting at least one hypoxanthine phosphoribosyltransferase (HPT, EC 2.4.2.8) in a reaction mixture with phosphoribosyl diphosphate (PRPP) and nicotinamide to produce nicotinamide mononucleotide (NMN) at a concentration of 0.01-500 mM;
wherein said HPT is present in said reaction mixture in an amount of 0.01 U-1000 kU/L and said HPT is present in an amount of at least 90% of any enzymes present in said reaction mixture, wherein said at least 90% indicates enzyme protein weight or a unit of activity for the enzyme.
10. The method according to claim 9 , wherein said HPT is at least one member selected from the group consisting of HPT-C(enzyme comprising the amino acid sequence given by SEQ ID NO: 7), HPT-W (enzyme comprising the amino acid sequence given by SEQ ID NO: 4), HPT-L (enzyme comprising the amino acid sequence given by SEQ ID NO: 11), and a polypeptide that contains an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of any one of HPT-C, HPT-W, and HPT-L.
11. The method according to claim 9 , wherein said reaction mixture further comprises Mg ion and/or Mn ion.