Mutant purine nucleoside phosphorylase

Abstract

To provide a mutant purine nucleoside phosphorylase capable of efficiently producing nicotinamide riboside.SOLUTION: Provided is a mutant purine nucleoside phosphorylase having one or more amino acid mutations in the amino acid sequence of a wild-type purine nucleoside phosphorylase having: (a) a specific amino acid sequence; (b) an amino acid sequence containing one or more amino acid substitutions, deletions, insertions or additions in the amino acid sequence of (a); or (c) an amino acid sequence having 90% or more sequence identity to the amino acid sequence of (a), and having a higher activity of producing nicotinamide riboside than the wild-type purine nucleoside phosphorylase.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to mutant purine nucleoside phosphorylases for the production of nicotinamide riboside. [Background technology]

[0002] Nicotinamide adenine dinucleotide (NAD) is a coenzyme that is ubiquitously present in living organisms, and either reduced or oxidized forms of NAD are used in redox reactions within cells. There are three major pathways for the synthesis of NAD within cells. The first is the de novo pathway using tryptophan as a raw material, the second is the salvage pathway from nicotinamide or nicotinic acid, and the third is the salvage pathway from nicotinamide riboside.

[0003] Mammals can biosynthesize nicotinamide adenine dinucleotide from a salvage pathway from nicotinamide or nicotinic acid, and from a salvage pathway from nicotinamide riboside.

[0004] In the salvage pathway from nicotinamide or nicotinic acid, nicotinic acid mononucleotide or nicotinamide mononucleotide is biosynthesized from nicotinamide or nicotinic acid and phosphoribosyl diphosphate (PRPP) by nicotinamide deaminase and nicotinic acid phosphoribosyltransferase. Nicotinamide adenine dinucleotide is then biosynthesized by ADPylation and amination by nicotinamide / nicotinic acid mononucleotide adenyltransferase.

[0005] In the salvage pathway from nicotinamide riboside, nicotinamide mononucleotide is biosynthesized by nicotinamide riboside kinase using adenosine triphosphate as a phosphate donor, and then nicotinamide adenine dinucleotide is biosynthesized by nicotinamide / nicotinic acid mononucleotide adenyltransferase.

[0006] Recent studies have reported that nicotinamide mononucleotide and nicotinamide riboside, which are precursors of nicotinamide adenine dinucleotide shown in the above biosynthetic pathway, increase the amount of nicotinamide adenine dinucleotide in cells, and as a result, are effective in improving metabolic disorders such as insulin resistance and dyslipidemia. It has also been reported that an increase in nicotinamide adenine dinucleotide reduces the production of β-amyloid, which causes Alzheimer's disease.

[0007] As a method for synthesizing nicotinamide riboside, organic synthesis and enzymatic synthesis have been reported so far. As an organic synthesis method, a method has been reported in which nicotinamide riboside in which the hydroxyl group of the ribose skeleton is protected is synthesized using tetraacetyl ribose as a starting material, and then a deprotection reaction is performed to obtain nicotinamide riboside (see, for example, Non-Patent Document 1). As an enzymatic synthesis method, a method has been reported in which nicotinamide riboside is obtained from ribose-1-phosphate and nicotinamide using an enzyme classified as pentosyltransferase (enzyme number EC2.4.2) (see, for example, Patent Document 1). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] US Patent Application Publication No. 2017 / 0121746 [Non-patent literature]

[0009] [Non-Patent Document 1] Makarov MV and Migaud ME, Syntheses and chemical properties of beta-nicotinamide riboside and its analogues and derivatives, Beilstein J. Org. Chem., 15, 401-430, 2019. Summary of the Invention [Problem to be solved by the invention]

[0010] In the organic synthesis method, both α- and β-forms of nicotinamide riboside are synthesized, which poses a problem in terms of stereoselectivity. On the other hand, the enzymatic synthesis method is superior to the organic synthesis method in that it does not require a protection and deprotection process and the stereoselectivity of the product is high. However, there is still room for improvement in the enzymatic synthesis of nicotinamide riboside in terms of production efficiency.

[0011] In view of the above circumstances, an object of the present invention is to provide a mutant purine nucleoside phosphorylase that can efficiently produce nicotinamide riboside. [Means for solving the problem]

[0012] The present invention includes the following aspects. [1] A mutant purine nucleoside phosphorylase having one or more amino acid mutations in the amino acid sequence of a wild-type purine nucleoside phosphorylase having: (a) the amino acid sequence set forth in SEQ ID NO: 1; (b) an amino acid sequence set forth in SEQ ID NO: 1 that includes one or more amino acid substitutions, deletions, insertions or additions; or (c) an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1; and having a higher activity of producing nicotinamide riboside than the wild-type purine nucleoside phosphorylase. [2] The mutant purine nucleoside phosphorylase according to [1], wherein the amino acid sequence of the wild-type purine nucleoside phosphorylase is the amino acid sequence set forth in SEQ ID NO:1. [3] A mutant purine nucleoside phosphorylase according to [1] or [2], having an amino acid mutation in at least one selected from the group consisting of amino acid residues corresponding to the 6th, 36th, 49th, 109th, 118th, 147th, 159th, 181st, 183rd, 186th, 201st, 207th, 237th, 238th, 239th, 242nd, 243rd, 249th, 251st, and 255th amino acid residues in the amino acid sequence set forth in SEQ ID NO:1. [4] A mutant purine nucleoside phosphorylase according to any one of [1] to [3], which comprises at least a mutation in an amino acid residue corresponding to the 186th and / or 207th amino acid residues in the amino acid sequence set forth in SEQ ID NO:1. [5] A mutant purine nucleoside phosphorylase according to any one of [1] to [4], which comprises at least a mutation in an amino acid residue corresponding to the 159th and / or 243rd amino acid residues in the amino acid sequence set forth in SEQ ID NO:1. [6] A mutant purine nucleoside phosphorylase according to any one of [1] to [5], which comprises at least a mutation in an amino acid residue corresponding to the 36th, 49th, 237th and / or 249th amino acid residues in the amino acid sequence set forth in SEQ ID NO:1. [7] A mutant purine nucleoside phosphorylase according to any one of [1] to [6], which comprises at least a mutation in an amino acid residue corresponding to the 6th and / or 201st amino acid residues in the amino acid sequence set forth in SEQ ID NO:1. [8] A mutant purine nucleoside phosphorylase according to any one of [1] to [7], which comprises at least a mutation in an amino acid residue corresponding to the 147th and / or 255th amino acid residues in the amino acid sequence set forth in SEQ ID NO:1. [9] The mutant purine nucleoside phosphorylase according to any one of [1] to [8], wherein Ala at the amino acid residue corresponding to the 159th amino acid in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with Glu, Ser, Asp, Gly or Arg.

[10] The mutant purine nucleoside phosphorylase according to any one of [1] to [9], wherein Asp at the amino acid residue corresponding to position 243 in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with Glu, Ser or Ala.

[11] The mutant purine nucleoside phosphorylase according to any one of [1] to

[10] , wherein Val, the amino acid residue corresponding to the sixth position in the amino acid sequence set forth in SEQ ID NO: 1, is substituted with Asp.

[12] The mutant purine nucleoside phosphorylase according to any one of [1] to

[11] , wherein Leu at the amino acid residue corresponding to the 201st position in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with Arg.

[13] A mutant purine nucleoside phosphorylase according to any one of [1] to

[12] , in which Asn at the amino acid residue corresponding to position 147 in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with Lys, Arg, Pro or Ala.

[14] A mutant purine nucleoside phosphorylase according to any one of [1] to

[13] , in which the Glu of the amino acid residue corresponding to the 255th amino acid in the amino acid sequence of SEQ ID NO: 1 is substituted with Gly, Ile, Leu, Arg, Lys, or Val.

[15] The mutant purine nucleoside phosphorylase according to any one of [1] to

[14] , which has an amino acid sequence as set forth in any one of SEQ ID NOs: 2 to 53.

[16] A nucleic acid encoding the mutant purine nucleoside phosphorylase according to any one of [1] to

[15] .

[17] A vector comprising the nucleic acid described in

[16] .

[18] A transformant comprising the vector described in

[17] .

[19] A method for producing a mutant purine nucleoside phosphorylase, comprising the steps of culturing the transformant described in

[18] in a medium to obtain a culture, and recovering the mutant purine nucleoside phosphorylase from the culture. Effect of the Invention

[0013] According to the present disclosure, it is possible to provide a mutant purine nucleoside phosphorylase that can efficiently produce nicotinamide riboside. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] [Mutant purine nucleoside phosphorylase] In one embodiment, the present invention provides a mutant purine nucleoside phosphorylase having one or more amino acid mutations in the amino acid sequence of a wild-type purine nucleoside phosphorylase having (a) the amino acid sequence set forth in SEQ ID NO: 1, (b) an amino acid sequence containing one or more amino acid substitutions, deletions, insertions or additions in the amino acid sequence set forth in SEQ ID NO: 1, or (c) an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, and having a higher activity of producing nicotinamide riboside than the wild-type purine nucleoside phosphorylase. Purine nucleoside phosphorylase is a type of pentosyltransferase, and its enzyme number is EC.2.4.2.1.

[0015] As described later in the Examples, the inventors have clarified that the mutant purine nucleoside phosphorylase of this embodiment has a significantly higher activity of producing nicotinamide riboside than wild-type purine nucleoside phosphorylase. Therefore, the mutant purine nucleoside phosphorylase of this embodiment can efficiently produce nicotinamide riboside. Here, the activity of producing nicotinamide riboside means the activity of producing nicotinamide riboside from ribose-1-phosphate and nicotinamide.

[0016] The chemical formula of nicotinamide riboside (CAS number: 1341-23-7) is shown in the following formula (1). Nicotinamide riboside may be in a free form or in the form of a salt such as fluoride, chloride, bromide, iodide, formate, acetate, ascorbate, benzoate, carbonate, citrate, carbamate, formate, gluconate, lactate, methyl bromide, methyl sulfate, nitrate, phosphate, diphosphate, succinate, sulfate, trifluoroacetate, or maleate.

[0017] [ka]

[0018] The wild-type purine nucleoside phosphorylase is a purine nucleoside phosphorylase before mutation on which the mutant purine nucleoside phosphorylase of the present embodiment is based. The wild-type purine nucleoside phosphorylase of the present invention is a naturally occurring enzyme having an amino acid sequence derived from a microorganism such as a bacterium or a fungus, and is a purine nucleoside phosphorylase having any of the following amino acid sequences (a), (b), or (c): (a) the amino acid sequence set forth in SEQ ID NO: 1 (b) an amino acid sequence containing one or more amino acid substitutions, deletions, insertions or additions relative to the amino acid sequence set forth in SEQ ID NO:1; (c) an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1;

[0019] Here, the amino acid sequence of SEQ ID NO: 1 in (a) above is the amino acid sequence of wild-type purine nucleoside phosphorylase derived from Thermosporothrix hazakensis, a bacterium classified into the phylum Chloroflexi, class Ktedonobacteria, and genus Thermosporothrix, and has an NCBI accession number of GCE47107.1. An example of the base sequence of DNA encoding the wild-type purine nucleoside phosphorylase derived from Thermosporothrix hazakensis (the amino acid sequence is shown in SEQ ID NO: 1) is shown in SEQ ID NO: 122.

[0020] The amino acid sequence of (b) above comprises one or more amino acid substitutions, deletions, insertions or additions in the amino acid sequence of SEQ ID NO: 1. Here, "one or more" may mean, for example, 1 to 50, 1 to 30, 1 to 20, 1 to 10, or 1 to 5.

[0021] In the amino acid sequence of (c) above, the sequence identity to the amino acid sequence set forth in SEQ ID NO:1 is 90% or more, optionally 95% or more, or optionally 99% or more.

[0022] Here, examples of wild-type purine nucleoside phosphorylases represented by (b) or (c) above include purine nucleoside phosphorylase derived from Thermosporothrix hazakensis, a bacterium classified in the phylum Chloroflexi, class Ktedonobacteria, and genus Thermosporothrix (NCBI accession number PZW36453.1, the amino acid sequence of which is shown in SEQ ID NO: 123), and purine nucleoside phosphorylase derived from Thermosporothrix hazakensis sp.COM3 (NCBI accession number BBH88921.1, the amino acid sequence of which is shown in SEQ ID NO: 124).

[0023] The mutant purine nucleoside phosphorylase of this embodiment is preferably a mutant purine nucleoside phosphorylase having one or more amino acid mutations relative to a wild-type purine nucleoside phosphorylase having the amino acid sequence set forth in SEQ ID NO: 1 (a) among the amino acid sequences (a) to (c) above.

[0024] The mutant purine nucleoside phosphorylase of the present embodiment has one or more amino acid mutations with respect to the amino acid sequence of the wild-type purine nucleoside phosphorylase (preferably the amino acid sequence set forth in SEQ ID NO: 1), and the amino acid mutation may be a substitution, addition, insertion or deletion of an amino acid residue. Among them, the amino acid mutation is preferably a substitution of an amino acid residue. The number of amino acid mutations may be 1 to 100, 1 to 80, 1 to 50, 1 to 20, 1 to 15, 1 to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1.

[0025] Moreover, the mutant purine nucleoside phosphorylase of this embodiment consists of an amino acid sequence having 80% or more and less than 100% sequence identity to the amino acid sequence of the wild-type purine nucleoside phosphorylase (preferably the amino acid sequence set forth in SEQ ID NO: 1). The sequence identity of the amino acid sequence of the mutant purine nucleoside phosphorylase of this embodiment to the amino acid sequence of the wild-type purine nucleoside phosphorylase may be 85% or more, 90% or more, 95% or more, 97% or more, or 99% or more.

[0026] Here, the sequence identity of a subject amino acid sequence to a reference amino acid sequence can be determined, for example, as follows. First, the reference amino acid sequence and the subject amino acid sequence are aligned. Here, each amino acid sequence may contain gaps so as to maximize the sequence identity. Next, the number of matching amino acid residues in the reference amino acid sequence and the subject amino acid sequence is calculated, and the sequence identity can be determined according to the following formula. Sequence identity (%) = number of matching amino acid residues / total number of amino acid residues in the target amino acid sequence × 100

[0027] The sequence identity of amino acid sequences can be calculated by methods well known to those skilled in the art, for example, using Blastp (protein-protein BLAST), a search program of BLAST (Basic Local Alignment Search Tool).

[0028] The mutant purine nucleoside phosphorylase of the present embodiment preferably has an amino acid mutation at at least one selected from the group consisting of amino acid residues corresponding to the 6th, 36th, 49th, 109th, 118th, 147th, 159th, 181st, 183rd, 186th, 201st, 207th, 237th, 238th, 239th, 242nd, 243rd, 249th, 251st and 255th amino acid residues in the amino acid sequence of SEQ ID NO: 1 in the amino acid sequence of the wild-type purine nucleoside phosphorylase. As described later in the Examples, such mutant purine nucleoside phosphorylase has a significantly higher nicotinamide riboside production activity than wild-type purine nucleoside phosphorylase. Specific examples of such mutant purine nucleoside phosphorylase include mutant purine nucleoside phosphorylases having amino acid sequences as set forth in SEQ ID NO: 2 to 53.

[0029] Here, "an amino acid residue corresponding to the nth amino acid residue in the amino acid sequence set forth in SEQ ID NO:1" refers to an amino acid residue that is determined to correspond to the nth amino acid residue counted from the N-terminus of the amino acid sequence set forth in SEQ ID NO:1 by a method such as homology analysis.

[0030] Examples of the method of homology analysis include methods based on pairwise sequence alignment such as the Needleman-Wunsch method and the Smith-Waterman method, and methods based on multiple sequence alignment such as the ClustalW method. Based on these methods, a person skilled in the art can identify "the amino acid residue corresponding to the n-th amino acid residue in the amino acid sequence of SEQ ID NO: 1" in the amino acid sequence of a mutant purine nucleoside phosphorylase by using the amino acid sequence of SEQ ID NO: 1 as a reference sequence.

[0031] The mutant purine nucleoside phosphorylase of this embodiment preferably has an amino acid mutation in at least one selected from the group consisting of amino acid residues corresponding to the 6th, 159th, 201st and 243rd amino acid residues in the amino acid sequence shown in SEQ ID NO: 1, and preferably has an amino acid mutation in the amino acid residues corresponding to the 6th, 159th, 201st and 243rd amino acid residues in the amino acid sequence shown in SEQ ID NO: 1. The mutant purine nucleoside phosphorylase of this embodiment preferably has an amino acid mutation in at least one selected from the group consisting of amino acid residues corresponding to the 6th, 147th, 159th, 201st, 243rd and 255th amino acid residues in the amino acid sequence shown in SEQ ID NO: 1, and preferably has an amino acid mutation in the amino acid residues corresponding to the 6th, 147th, 159th, 201st, 243rd and 255th amino acid residues.

[0032] The mutant purine nucleoside phosphorylase of this embodiment may be a mutant purine nucleoside phosphorylase having, in addition to the mutations in amino acid residues corresponding to the 6th, 147th, 159th, 201st, 243rd and 255th amino acid residues in the amino acid sequence set forth in SEQ ID NO:1, at least one amino acid mutation selected from the amino acid residues corresponding to the 36th, 49th, 109th, 118th, 181st, 183rd, 238th, 239th, 242nd, 249th and 251st amino acid residues.

[0033] The mutant purine nucleoside phosphorylase of this embodiment may contain at least a mutation in an amino acid residue corresponding to the 186th and / or 207th amino acid residues in the amino acid sequence set forth in SEQ ID NO: 1. As described later in the Examples, such a mutant purine nucleoside phosphorylase has a nicotinamide riboside production activity that is at least about 1.6 times higher than that of a wild-type purine nucleoside phosphorylase.

[0034] The mutant purine nucleoside phosphorylase may have a mutation in the amino acid residue corresponding to the 186th amino acid residue in the amino acid sequence set forth in SEQ ID NO:1, may have a mutation in the amino acid residue corresponding to the 207th amino acid residue, may have mutations in the amino acid residues corresponding to the 186th and 207th amino acid residues, or may have mutations in an additional amino acid residue in addition to the amino acid residues corresponding to the 186th and 207th amino acid residues.

[0035] A specific example of a mutant purine nucleoside phosphorylase having mutations in the amino acid residues corresponding to the 186th and 207th amino acid residues in the amino acid sequence of SEQ ID NO:1 includes a mutant purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO:3.

[0036] The mutant purine nucleoside phosphorylase of this embodiment may contain at least a mutation in an amino acid residue corresponding to the 159th and / or 243rd amino acid residues in the amino acid sequence set forth in SEQ ID NO: 1. As described later in the Examples, such a mutant purine nucleoside phosphorylase has a nicotinamide riboside production activity that is at least about 1.6 times higher than that of a wild-type purine nucleoside phosphorylase.

[0037] The mutant purine nucleoside phosphorylase may have a mutation in the amino acid residue corresponding to the 159th amino acid residue in the amino acid sequence set forth in SEQ ID NO:1, may have a mutation in the amino acid residue corresponding to the 243rd amino acid residue, may have mutations in the amino acid residues corresponding to the 159th and 243rd amino acid residues, or may have mutations in an additional amino acid residue in addition to the amino acid residues corresponding to the 159th and 243rd amino acid residues.

[0038] Specific examples of mutant purine nucleoside phosphorylases having mutations in at least the amino acid residues corresponding to the 159th and 243rd amino acid residues in the amino acid sequence of SEQ ID NO: 1 include mutant purine nucleoside phosphorylases having the amino acid sequences of SEQ ID NOs: 2, 4 to 53.

[0039] The mutant purine nucleoside phosphorylase of this embodiment may contain at least a mutation in an amino acid residue corresponding to the 36th, 49th, 237th and / or 249th amino acid residues in the amino acid sequence set forth in SEQ ID NO: 1. As described later in the Examples, such a mutant purine nucleoside phosphorylase has a nicotinamide riboside production activity that is at least about three times higher than that of a wild-type purine nucleoside phosphorylase.

[0040] The mutant purine nucleoside phosphorylase may have mutations in any combination of one, two, three or four amino acid residues selected from amino acid residues corresponding to the 36th, 49th, 237th and 249th amino acid residues in the amino acid sequence set forth in SEQ ID NO:1, or may have mutations in further amino acid residues in addition to the amino acid residues corresponding to the 36th, 49th, 237th or 249th amino acid residues.

[0041] Specific examples of mutant purine nucleoside phosphorylases having at least one mutation selected from amino acid residues corresponding to the 36th, 49th, 237th and 249th amino acid residues in the amino acid sequence set forth in SEQ ID NO: 1 include mutant purine nucleoside phosphorylases having the amino acid sequences set forth in SEQ ID NOs: 5, 43, 46, 47, 48 and 52.

[0042] The mutant purine nucleoside phosphorylase of this embodiment may contain at least a mutation in an amino acid residue corresponding to the 6th and / or 201st amino acid residues in the amino acid sequence set forth in SEQ ID NO: 1. As described later in the Examples, such a mutant purine nucleoside phosphorylase has a nicotinamide riboside production activity that is at least about 18 times higher than that of a wild-type purine nucleoside phosphorylase.

[0043] The mutant purine nucleoside phosphorylase may have a mutation in the amino acid residue corresponding to the 6th amino acid residue in the amino acid sequence set forth in SEQ ID NO:1, may have a mutation in the amino acid residue corresponding to the 201st amino acid residue, may have mutations in the amino acid residues corresponding to the 6th and 201st amino acid residues, or may have mutations in an additional amino acid residue in addition to the amino acid residues corresponding to the 6th and 201st amino acid residues.

[0044] Specific examples of mutant purine nucleoside phosphorylases having mutations in at least the amino acid residues corresponding to the 6th and 201st amino acid residues in the amino acid sequence of SEQ ID NO: 1 include mutant purine nucleoside phosphorylases having the amino acid sequences of SEQ ID NOs: 4, and 6 to 53.

[0045] The mutant purine nucleoside phosphorylase of this embodiment may contain at least a mutation in an amino acid residue corresponding to the 147th and / or 255th amino acid residues in the amino acid sequence set forth in SEQ ID NO: 1. As described later in the Examples, such a mutant purine nucleoside phosphorylase has a nicotinamide riboside production activity that is at least about 18 times higher than that of a wild-type purine nucleoside phosphorylase.

[0046] The mutant purine nucleoside phosphorylase may have a mutation in the amino acid residue corresponding to the 147th amino acid residue in the amino acid sequence set forth in SEQ ID NO:1, may have a mutation in the amino acid residue corresponding to the 255th amino acid residue, may have mutations in the amino acid residues corresponding to the 147th and 255th amino acid residues, or may have mutations in further amino acid residues in addition to the amino acid residues corresponding to the 147th and 255th amino acid residues.

[0047] Specific examples of mutant purine nucleoside phosphorylases having mutations in at least the amino acid residues corresponding to the 147th and 255th amino acid residues in the amino acid sequence of SEQ ID NO: 1 include mutant purine nucleoside phosphorylases having the amino acid sequences of SEQ ID NOs: 6 to 53.

[0048] In the mutant purine nucleoside phosphorylase of this embodiment, Ile at the amino acid residue corresponding to position 186 in the amino acid sequence set forth in SEQ ID NO:1 may be substituted with Thr.

[0049] In the mutant purine nucleoside phosphorylase of this embodiment, Ile at the amino acid residue corresponding to position 207 in the amino acid sequence set forth in SEQ ID NO: 1 may be substituted with Leu.

[0050] A specific example of a mutant purine nucleoside phosphorylase in which Ile at the amino acid residue corresponding to position 186 in the amino acid sequence of SEQ ID NO:1 is replaced with Thr, or Ile at the amino acid residue corresponding to position 207 is replaced with Leu, is a mutant purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO:3.

[0051] In the mutant purine nucleoside phosphorylase of this embodiment, Ala at the amino acid residue corresponding to position 36 in the amino acid sequence set forth in SEQ ID NO:1 may be substituted with Val.

[0052] In the mutant purine nucleoside phosphorylase of this embodiment, Ile at the amino acid residue corresponding to position 49 in the amino acid sequence set forth in SEQ ID NO:1 may be substituted with Val.

[0053] In the mutant purine nucleoside phosphorylase of this embodiment, Ser at the amino acid residue corresponding to position 237 in the amino acid sequence set forth in SEQ ID NO: 1 may be substituted with Asn.

[0054] In the mutant purine nucleoside phosphorylase of this embodiment, Asn of the amino acid residue corresponding to position 249 in the amino acid sequence set forth in SEQ ID NO: 1 may be substituted with Ser.

[0055] Specific examples of mutant purine nucleoside phosphorylases in which Ala at the amino acid residue corresponding to position 36 in the amino acid sequence of SEQ ID NO:1 is replaced with Val include mutant purine nucleoside phosphorylases having the amino acid sequences of SEQ ID NO:5 and SEQ ID NO:46.

[0056] Specific examples of mutant purine nucleoside phosphorylases in which Ile at the amino acid residue corresponding to position 49 in the amino acid sequence of SEQ ID NO:1 is replaced with Val include mutant purine nucleoside phosphorylases having the amino acid sequences of SEQ ID NOs:5, 43, 47, and 48.

[0057] A specific example of a mutant purine nucleoside phosphorylase in which Ser at the amino acid residue corresponding to position 237 in the amino acid sequence of SEQ ID NO:1 is replaced with Asn is a mutant purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO:5.

[0058] Specific examples of mutant purine nucleoside phosphorylases in which Asn at the amino acid residue corresponding to position 249 in the amino acid sequence of SEQ ID NO:1 is replaced with Ser include mutant purine nucleoside phosphorylases having the amino acid sequences of SEQ ID NOs:5 and 52.

[0059] In the mutant purine nucleoside phosphorylase of this embodiment, Ala of the amino acid residue corresponding to position 159 in the amino acid sequence set forth in SEQ ID NO: 1 may be substituted with Glu, Ser, Asp, Gly or Arg.

[0060] As described later in the Examples, such mutant purine nucleoside phosphorylase has significantly higher nicotinamide riboside producing activity than wild-type purine nucleoside phosphorylase.

[0061] Specific examples of mutant purine nucleoside phosphorylases in which Ala at the amino acid residue corresponding to position 159 in the amino acid sequence of SEQ ID NO: 1 is substituted with Glu include mutant purine nucleoside phosphorylases having the amino acid sequences of SEQ ID NOs: 2, 4 to 11, 16 to 31, 34, 39, and 41 to 53.

[0062] Specific examples of mutant purine nucleoside phosphorylases in which Ala at the amino acid residue corresponding to position 159 in the amino acid sequence of SEQ ID NO: 1 is replaced with Ser include mutant purine nucleoside phosphorylases having the amino acid sequences of SEQ ID NOs: 12, 32, and 33.

[0063] Specific examples of mutant purine nucleoside phosphorylases in which Ala at the amino acid residue corresponding to position 159 in the amino acid sequence of SEQ ID NO: 1 is replaced with Asp include mutant purine nucleoside phosphorylases having the amino acid sequences of SEQ ID NOs: 13, 35, and 36.

[0064] Specific examples of mutant purine nucleoside phosphorylases in which Ala at the amino acid residue corresponding to position 159 in the amino acid sequence of SEQ ID NO: 1 is replaced with Gly include mutant purine nucleoside phosphorylases having the amino acid sequences of SEQ ID NOs: 15 and 40.

[0065] Specific examples of mutant purine nucleoside phosphorylases in which Ala at the amino acid residue corresponding to position 159 in the amino acid sequence of SEQ ID NO: 1 is replaced with Arg include mutant purine nucleoside phosphorylases having the amino acid sequences of SEQ ID NOs: 14, 37, and 38.

[0066] In the mutant purine nucleoside phosphorylase of this embodiment, Asp of the amino acid residue corresponding to position 243 in the amino acid sequence set forth in SEQ ID NO: 1 may be substituted with Glu, Ser, or Ala.

[0067] As described later in the Examples, such mutant purine nucleoside phosphorylase has significantly higher nicotinamide riboside producing activity than wild-type purine nucleoside phosphorylase.

[0068] Specific examples of mutant purine nucleoside phosphorylases in which Asp at the amino acid residue corresponding to position 243 in the amino acid sequence of SEQ ID NO: 1 is replaced with Glu include mutant purine nucleoside phosphorylases having the amino acid sequences of SEQ ID NOs: 2, 4 to 18, 21 to 30, 37, 38, and 43 to 53.

[0069] Specific examples of mutant purine nucleoside phosphorylases in which Asp at the amino acid residue corresponding to position 243 in the amino acid sequence of SEQ ID NO: 1 is substituted with Ser include mutant purine nucleoside phosphorylases having the amino acid sequences of SEQ ID NOs: 19, 31 to 33, 39, and 40.

[0070] Specific examples of mutant purine nucleoside phosphorylases in which Asp at the amino acid residue corresponding to position 243 in the amino acid sequence of SEQ ID NO: 1 is replaced with Ala include mutant purine nucleoside phosphorylases having the amino acid sequences of SEQ ID NOs: 20, 34 to 36, 41, and 42.

[0071] In the mutant purine nucleoside phosphorylase of this embodiment, the Val of the amino acid residue corresponding to the 6th amino acid residue in the amino acid sequence set forth in SEQ ID NO: 1 may be substituted with Asp.

[0072] As described later in the Examples, such mutant purine nucleoside phosphorylase has significantly higher nicotinamide riboside producing activity than wild-type purine nucleoside phosphorylase.

[0073] Specific examples of mutant purine nucleoside phosphorylases in which Val at the sixth amino acid residue in the amino acid sequence of SEQ ID NO:1 is substituted with Asp include mutant purine nucleoside phosphorylases having the amino acid sequences of SEQ ID NOs:4, 6 to 53.

[0074] In the mutant purine nucleoside phosphorylase of this embodiment, Leu at the amino acid residue corresponding to position 201 in the amino acid sequence set forth in SEQ ID NO: 1 may be substituted with Arg.

[0075] As described later in the Examples, such mutant purine nucleoside phosphorylase has significantly higher nicotinamide riboside producing activity than wild-type purine nucleoside phosphorylase.

[0076] Specific examples of mutant purine nucleoside phosphorylases in which Leu at the amino acid residue corresponding to position 201 in the amino acid sequence of SEQ ID NO: 1 is substituted with Arg include mutant purine nucleoside phosphorylases having the amino acid sequences of SEQ ID NOs: 4, and 6 to 53.

[0077] In the mutant purine nucleoside phosphorylase of this embodiment, Asn of the amino acid residue corresponding to position 147 in the amino acid sequence set forth in SEQ ID NO: 1 may be substituted with Lys, Arg, Pro, or Ala.

[0078] As described later in the Examples, such mutant purine nucleoside phosphorylase has significantly higher nicotinamide riboside producing activity than wild-type purine nucleoside phosphorylase.

[0079] Specific examples of mutant purine nucleoside phosphorylases in which Asn at the amino acid residue corresponding to position 147 in the amino acid sequence of SEQ ID NO: 1 is replaced with Lys include mutant purine nucleoside phosphorylases having the amino acid sequences of SEQ ID NOs: 6 to 8, 12 to 32, 34, 35, 37, 39 to 41, and 43 to 53.

[0080] Specific examples of mutant purine nucleoside phosphorylases in which Asn at the amino acid residue corresponding to position 147 in the amino acid sequence of SEQ ID NO: 1 is replaced with Arg include mutant purine nucleoside phosphorylases having the amino acid sequences of SEQ ID NOs: 9, 33, and 42.

[0081] Specific examples of mutant purine nucleoside phosphorylases in which Asn at the amino acid residue corresponding to position 147 in the amino acid sequence of SEQ ID NO: 1 is replaced with Pro include mutant purine nucleoside phosphorylases having the amino acid sequences of SEQ ID NOs: 10 and 36.

[0082] Specific examples of mutant purine nucleoside phosphorylases in which Asn at the amino acid residue corresponding to position 147 in the amino acid sequence of SEQ ID NO: 1 is replaced with Ala include mutant purine nucleoside phosphorylases having the amino acid sequences of SEQ ID NOs: 11 and 38.

[0083] In the mutant purine nucleoside phosphorylase of this embodiment, Glu of the amino acid residue corresponding to position 255 in the amino acid sequence set forth in SEQ ID NO: 1 may be substituted with Gly, Ile, Leu, Arg, Lys, or Val.

[0084] As described later in the Examples, such mutant purine nucleoside phosphorylase has significantly higher nicotinamide riboside producing activity than wild-type purine nucleoside phosphorylase.

[0085] Specific examples of mutant purine nucleoside phosphorylases in which Glu at the amino acid residue corresponding to position 255 in the amino acid sequence of SEQ ID NO: 1 is replaced with Gly include mutant purine nucleoside phosphorylases having the amino acid sequences of SEQ ID NOs: 6 to 20, 26, and 43 to 53.

[0086] Specific examples of mutant purine nucleoside phosphorylases in which Glu at the amino acid residue corresponding to position 255 in the amino acid sequence of SEQ ID NO: 1 is substituted with Ile include mutant purine nucleoside phosphorylases having the amino acid sequences of SEQ ID NOs: 22, 28, and 34 to 36.

[0087] Specific examples of mutant purine nucleoside phosphorylases in which Glu at the amino acid residue corresponding to position 255 in the amino acid sequence of SEQ ID NO: 1 is replaced with Leu include mutant purine nucleoside phosphorylases having the amino acid sequences of SEQ ID NOs: 21, 31 to 33.

[0088] Specific examples of mutant purine nucleoside phosphorylases in which the Glu at the amino acid residue corresponding to position 255 in the amino acid sequence of SEQ ID NO: 1 is replaced with Arg include mutant purine nucleoside phosphorylases having the amino acid sequences of SEQ ID NOs: 24, 29, 39 and 40.

[0089] Specific examples of mutant purine nucleoside phosphorylases in which Glu at the amino acid residue corresponding to position 255 in the amino acid sequence of SEQ ID NO: 1 is replaced with Lys include mutant purine nucleoside phosphorylases having the amino acid sequences of SEQ ID NOs: 23, 27, 37, and 38.

[0090] Specific examples of mutant purine nucleoside phosphorylases in which the Glu at the amino acid residue corresponding to position 255 in the amino acid sequence of SEQ ID NO: 1 is replaced with Val include mutant purine nucleoside phosphorylases having the amino acid sequences of SEQ ID NOs: 25, 30, 41, and 42.

[0091] In the mutant purine nucleoside phosphorylase of this embodiment, the Val of the amino acid residue corresponding to the 109th amino acid residue in the amino acid sequence set forth in SEQ ID NO: 1 may be substituted with Ala.

[0092] A specific example of a mutant purine nucleoside phosphorylase in which Val at the amino acid residue corresponding to the 109th amino acid residue in the amino acid sequence set forth in SEQ ID NO:1 is replaced with Ala is a mutant purine nucleoside phosphorylase having the amino acid sequence set forth in SEQ ID NO:44.

[0093] In the mutant purine nucleoside phosphorylase of this embodiment, Pro of the amino acid residue corresponding to position 118 in the amino acid sequence set forth in SEQ ID NO:1 may be substituted with Leu.

[0094] A specific example of a mutant purine nucleoside phosphorylase in which Pro at the amino acid residue corresponding to position 118 in the amino acid sequence of SEQ ID NO:1 is replaced with Leu is a mutant purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO:45.

[0095] In the mutant purine nucleoside phosphorylase of this embodiment, the Thr of the amino acid residue corresponding to position 181 in the amino acid sequence set forth in SEQ ID NO: 1 may be substituted with Ala, Ser, Val, Tyr, or Gly.

[0096] A specific example of a mutant purine nucleoside phosphorylase in which the Thr at the amino acid residue corresponding to position 181 in the amino acid sequence of SEQ ID NO:1 is replaced with Ala is a mutant purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO:8.

[0097] A specific example of a mutant purine nucleoside phosphorylase in which the Thr at the amino acid residue corresponding to the 181st position in the amino acid sequence of SEQ ID NO:1 is replaced with Ser is a mutant purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO:7.

[0098] A specific example of a mutant purine nucleoside phosphorylase in which the Thr at the amino acid residue corresponding to the 181st position in the amino acid sequence of SEQ ID NO:1 is replaced with Val is a mutant purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO:18.

[0099] A specific example of a mutant purine nucleoside phosphorylase in which the Thr at the amino acid residue corresponding to the 181st position in the amino acid sequence of SEQ ID NO:1 is replaced with Tyr is a mutant purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO:16.

[0100] A specific example of a mutant purine nucleoside phosphorylase in which the Thr at the amino acid residue corresponding to the 181st position in the amino acid sequence of SEQ ID NO: 1 is replaced with Gly is a mutant purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO: 17.

[0101] In the mutant purine nucleoside phosphorylase of this embodiment, Gln at the amino acid residue corresponding to position 183 in the amino acid sequence set forth in SEQ ID NO: 1 may be substituted with His.

[0102] Specific examples of mutant purine nucleoside phosphorylases in which Gln at the amino acid residue corresponding to position 183 in the amino acid sequence of SEQ ID NO: 1 is replaced with His include mutant purine nucleoside phosphorylases having the amino acid sequences of SEQ ID NOs: 47, 48, and 49.

[0103] In the mutant purine nucleoside phosphorylase of this embodiment, Asn of the amino acid residue corresponding to position 238 in the amino acid sequence set forth in SEQ ID NO: 1 may be substituted with Ser.

[0104] Specific examples of mutant purine nucleoside phosphorylases in which Asn at the amino acid residue corresponding to position 238 in the amino acid sequence of SEQ ID NO: 1 is replaced with Ser include mutant purine nucleoside phosphorylases having the amino acid sequences of SEQ ID NOs: 26, 27, 28, 29, and 30.

[0105] In the mutant purine nucleoside phosphorylase of this embodiment, Glu at the amino acid residue corresponding to position 239 in the amino acid sequence set forth in SEQ ID NO: 1 may be substituted with Gly.

[0106] Specific examples of mutant purine nucleoside phosphorylases in which the Glu at the amino acid residue corresponding to position 239 in the amino acid sequence of SEQ ID NO: 1 is replaced with Gly include mutant purine nucleoside phosphorylases having the amino acid sequences of SEQ ID NOs: 48 and 50.

[0107] In the mutant purine nucleoside phosphorylase of this embodiment, Glu at the amino acid residue corresponding to position 251 in the amino acid sequence set forth in SEQ ID NO: 1 may be substituted with Gly.

[0108] A specific example of a mutant purine nucleoside phosphorylase in which the Glu at the amino acid residue corresponding to the 251st position in the amino acid sequence of SEQ ID NO:1 is replaced with Gly is a mutant purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO:53.

[0109] [Method of producing nicotinamide riboside] In one embodiment, the present invention provides a method for producing nicotinamide riboside, comprising the step of contacting the mutant purine nucleoside phosphorylase described above with ribose-1-phosphate and nicotinamide in an aqueous medium, thereby producing nicotinamide riboside. As described later in the Examples, the method of this embodiment allows efficient production of nicotinamide riboside. Here, "contact" may mean that the mutant purine nucleoside phosphorylase, ribose-1-phosphate and nicotinamide are present in the same reaction system or culture system.

[0110] In the production method of this embodiment, the transformant expressing mutant purine nucleoside phosphorylase described below may be used as it is for the production of nicotinamide riboside. Alternatively, a crude extract of the transformant may be used for the production of nicotinamide riboside. Alternatively, mutant purine nucleoside phosphorylase may be collected from the transformant and used for the production of nicotinamide riboside. The crude extract of the transformant can be obtained by a method commonly used in this field, such as freezing and thawing, ultrasonic disruption, or dissolution of the transformant.

[0111] Ribose-1-phosphate may be a compound represented by the following formula (2) (D-ribose-1-phosphate) or a salt thereof. Examples of the salt of ribose-1-phosphate include salts formed with ammonium, magnesium, etc., and more specifically, examples thereof include ribose-1-phosphate diammonium salt (a salt containing two ammonium atoms per ribose-1-phosphate molecule).

[0112] [ka]

[0113] Nicotinamide is a compound represented by the following formula (3) (nicotinamide) or a derivative thereof. Examples of the nicotinamide derivative include a compound in which one or more substituents are bonded to the pyridine ring of nicotinamide. Examples of the substituent include a hydroxy group, an alkyl group, an aryl group, an alkoxy group, a carboxy group, an aldehyde group, a cyano group, and a halogen atom. In the method of the present embodiment, nicotinamide and a derivative of nicotinamide may be used in combination.

[0114] [ka]

[0115] In the production method of this embodiment, the mutant purine nucleoside phosphorylase used may be of one type alone or of two or more types in combination.

[0116] When ribose-1-phosphate, nicotinamide, and mutant purine nucleoside phosphorylase are contacted in an aqueous medium, the phosphate group of ribose-1-phosphate is replaced with nicotinamide by an enzymatic reaction of the mutant purine nucleoside phosphorylase to produce nicotinamide riboside.

[0117] The method of contacting ribose-1-phosphate, nicotinamide, and mutant purine nucleoside phosphorylase in an aqueous medium is not particularly limited. For example, a method of dissolving ribose-1-phosphate, nicotinamide, and mutant purine nucleoside phosphorylase in an aqueous medium in sequence can be mentioned. The order of dissolving ribose-1-phosphate, nicotinamide, and mutant purine nucleoside phosphorylase in an aqueous medium is not particularly limited. For example, mutant purine nucleoside phosphorylase may be dissolved in an aqueous medium in which ribose-1-phosphate and nicotinamide are dissolved.

[0118] The aqueous medium is not particularly limited as long as it contains water and can dissolve ribose-1-phosphate, nicotinamide, and mutant purine nucleoside phosphorylase. If necessary, the aqueous medium may contain a buffer, a pH adjuster, a surfactant, an organic solvent, an inorganic salt, a nucleoside compound, and the like. Examples of the buffer include sodium acetate buffer, ammonium acetate buffer, Tris (trishydroxymethylaminomethane) buffer, and MES (2-morpholinoethanesulfonic acid) buffer. Among these, at least one buffer selected from the group consisting of sodium acetate buffer, ammonium acetate buffer, and MES buffer is preferred.

[0119] The pH of the aqueous medium is not particularly limited, but from the viewpoint of suppressing the decomposition of the produced nicotinamide riboside and promoting the enzymatic reaction of the mutant purine nucleoside phosphorylase, it is preferably 7.0 or less, more preferably 6.7 or less, and even more preferably 6.5 or less. Moreover, from the viewpoint of promoting the enzymatic reaction of the mutant purine nucleoside phosphorylase, the pH of the aqueous medium is preferably 4.0 or more, more preferably 4.5 or more, and even more preferably 5.0 or more.

[0120] The temperature of the aqueous medium is not particularly limited, but from the viewpoint of promoting the enzyme reaction, it is preferably 5°C to 50°C, more preferably 10°C to 35°C, and even more preferably 10°C to 25°C.

[0121] The time for contacting ribose-1-phosphate, nicotinamide, and mutant purine nucleoside phosphorylase in an aqueous medium is not particularly limited, and is preferably 0.5 to 72 hours, more preferably 1 to 48 hours, and even more preferably 3 to 24 hours.

[0122] [Nucleic acid, vector, transformant] In one embodiment, the present invention provides a nucleic acid encoding the mutant purine nucleoside phosphorylase described above. The nucleic acid is not particularly limited as long as it encodes the mutant purine nucleoside phosphorylase described above, and may be DNA or RNA. The nucleic acid of this embodiment may have a base sequence or the like added thereto that encodes an affinity tag for purifying the mutant purine nucleoside phosphorylase. The affinity tag is not particularly limited, and examples thereof include His tag, PA tag, and FLAG tag.

[0123] The nucleic acid may be incorporated into a vector. The vector may be an expression vector. In addition to the nucleic acid encoding the mutant purine nucleoside phosphorylase, the expression vector may further include regions such as a promoter, a terminator, and a region encoding a drug resistance gene. The expression vector may be a plasmid or an integrative vector. The expression vector may be a viral vector or a vector for a cell-free system.

[0124] The transformant can be obtained by introducing the above-mentioned nucleic acid or vector into a host. Examples of the host include prokaryotic cells such as Escherichia bacteria such as E. coli, Corynebacterium bacteria, and Bacillus bacteria, and eukaryotic cells such as Saccharomyces bacteria, Pichia bacteria, and Aspergillus bacteria. Among these, Escherichia bacteria are preferred, and E. coli is more preferred.

[0125] For example, a transformant expressing the mutant purine nucleoside phosphorylase can be obtained by transforming a host with the above-mentioned expression vector. The transformant can be cultured in an appropriate medium depending on the host. For example, when the host is Escherichia coli, the medium can be selected from commonly used media such as LB medium. The above-mentioned mutant purine nucleoside phosphorylase can be obtained by culturing the transformant in an appropriate medium and inducing expression as necessary.

[0126] [Method for producing mutant purine nucleoside phosphorylase] In one embodiment, the present invention provides a method for producing a mutant purine nucleoside phosphorylase, comprising the steps of culturing the above-mentioned transformant in a medium to obtain a culture, and recovering mutant purine nucleoside phosphorylase from the culture. The production method of this embodiment makes it possible to produce mutant purine nucleoside phosphorylase. The culture includes cultured cells, culture supernatant, cultured bacterial cells, disrupted cultured cells, or disrupted cultured bacterial cells.

[0127] The culture conditions for the transformant are not particularly limited, but examples include conditions of 20° C. to 37° C., 5 hours to 72 hours, and pH 6 to 9.

[0128] In the production method of this embodiment, in the step of recovering mutant purine nucleoside phosphorylase from the culture, the mutant purine nucleoside phosphorylase may be recovered from the transformant, or the mutant purine nucleoside phosphorylase may be recovered from the medium.

[0129] In the step of recovering the mutant purine nucleoside phosphorylase, a crude extract of the mutant purine nucleoside phosphorylase may be obtained, or the mutant purine nucleoside phosphorylase may be purified.

[0130] The mutant purine nucleoside phosphorylase may be purified by affinity chromatography using the above-mentioned affinity tag, or by various types of chromatography usually used for purifying proteins. EXAMPLES

[0131] The present disclosure will now be described in more detail with reference to examples, but the present disclosure is not limited to the following examples.

[0132] [Experimental Example 1] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO: 1) A gene encoding purine nucleoside phosphorylase (WT: wild-type purine nucleoside phosphorylase) having the amino acid sequence of SEQ ID NO:1 was obtained by artificial gene synthesis and digested with restriction enzymes EcoRI / HindIII. The DNA fragment and pUC18 (Takara Bio) were then ligated using Ligation High (Toyobo) and introduced into Escherichia coli DH5α strain to obtain a transformant. This transformant was cultured in LB medium containing ampicillin (100 μg / mL), the plasmid was extracted, and plasmid No. 1 into which a gene encoding purine nucleoside phosphorylase (PNP) having the amino acid sequence of SEQ ID NO:1 was inserted was obtained.

[0133] [Experimental Example 2] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO:2) Using plasmid No. 1 as a template, PCR amplification was performed using a forward primer (name "A159E_Fw", SEQ ID NO: 54), a reverse primer (name "A159E_Rv", SEQ ID NO: 55), and Gflex Polymerase (Takara Bio). The reaction conditions for PCR amplification were (1) 94°C for 2 minutes, (2) 98°C for 10 seconds, (3) 60°C for 30 seconds, (4) 68°C for 3 minutes, and 25 cycles of (2) to (4).

[0134] DpnI (Takara Bio) was added to the PCR reaction solution containing the obtained DNA fragment, and the mixture was incubated at 37°C for 1 hour. The DNA fragment was purified using NucleoSpin Gel and PCR Clean-up (Takara Bio) and then introduced into Escherichia coli DH5α strain to obtain a transformant. The transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted.

[0135] PCR amplification was carried out in the same manner as above, except that this plasmid was used as a template and a forward primer (name "D243E_Fw", SEQ ID NO: 56) and a reverse primer (name "D243_Rv", SEQ ID NO: 57) were used. Subsequently, DpnI treatment and spin column purification were carried out in the same manner as above, and the obtained DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant. This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 2 into which a gene encoding PNP having the amino acid sequence of SEQ ID NO: 2 was inserted.

[0136] [Experimental Example 3] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO:3) A DNA fragment was obtained in the same manner as above, except that plasmid No. 1 was used as a template and a forward primer (name "I186T_Fw", SEQ ID NO: 58) and a reverse primer (name "I186T_Rv", SEQ ID NO: 59) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant, and the plasmid was extracted.

[0137] Next, a DNA fragment was obtained in the same manner as above, except that this plasmid was used as a template and a forward primer (name "I207L_Fw", SEQ ID NO: 60) and a reverse primer (name "I207L_Rv", SEQ ID NO: 61) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0138] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 3 into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:3 was inserted.

[0139] [Experimental Example 4] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO: 4) A DNA fragment was obtained in the same manner as above, except that plasmid No. 2 was used as a template and a forward primer (name "V6D_Fw", SEQ ID NO: 62) and a reverse primer (name "V6D_Rv", SEQ ID NO: 63) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant, and the plasmid was extracted.

[0140] Next, a DNA fragment was obtained in the same manner as above, except that this plasmid was used as a template and a forward primer (name "L201R_Fw", SEQ ID NO: 64) and a reverse primer (name "L201R_Rv", SEQ ID NO: 65) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0141] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 4, into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:4 was inserted.

[0142] [Experimental Example 5] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO:5) A DNA fragment was obtained in the same manner as above, except that plasmid No. 2 was used as a template and a forward primer (name "A36V_Fw", SEQ ID NO: 66) and a reverse primer (name "A36V_Rv", SEQ ID NO: 67) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant, and the plasmid was extracted.

[0143] Next, a DNA fragment was obtained in the same manner as above, except that this plasmid was used as a template and a forward primer (named "I49V_Fw", SEQ ID NO: 68) and a reverse primer (named "I49V_Rv", SEQ ID NO: 69) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant, and the plasmid was extracted.

[0144] Next, a DNA fragment was obtained in the same manner as above, except that this plasmid was used as a template and a forward primer (name "N249S_Fw", SEQ ID NO: 70) and a reverse primer (name "N249S_Rv", SEQ ID NO: 71) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant, and the plasmid was extracted.

[0145] Next, a DNA fragment was obtained in the same manner as above, except that this plasmid was used as a template and a forward primer (named "S237N_Fw", SEQ ID NO: 72) and a reverse primer (named "S237N_Rv", SEQ ID NO: 73) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0146] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 5 into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:5 was inserted.

[0147] [Experimental Example 6] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO:6) A DNA fragment was obtained in the same manner as above, except that plasmid No. 4 was used as a template and a forward primer (name "N147K_Fw", SEQ ID NO: 74) and a reverse primer (name "N147K_Rv", SEQ ID NO: 75) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant, and the plasmid was extracted.

[0148] Next, a DNA fragment was obtained in the same manner as above, except that this plasmid was used as a template and a forward primer (name "E255G_Fw", SEQ ID NO: 76) and a reverse primer (name "E255G_Rv", SEQ ID NO: 77) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0149] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 6, into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:6 was inserted.

[0150] [Experimental Example 7] (Construction of expression plasmid for purine nucleoside phosphorylase having the amino acid sequences of SEQ ID NOs: 7 and 8) A DNA fragment was obtained in the same manner as above, except that plasmid No. 6 was used as a template and a forward primer (name "T181N_Fw", SEQ ID NO: 78) and a reverse primer (name "T181N_Rv", SEQ ID NO: 79) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant, and the plasmid was extracted.

[0151] Subsequently, DNA sequence analysis was performed to select plasmids into which the desired mutation had been introduced, and plasmids No. 7 and 8 into which genes encoding PNPs having the amino acid sequences of SEQ ID NOs: 7 and 8 were inserted were obtained.

[0152] [Experimental Example 8] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO:9) A DNA fragment was obtained in the same manner as above, except that plasmid No. 6 was used as a template and a forward primer (name "N147R_Fw", SEQ ID NO: 80) and a reverse primer (name "N147R_Rv", SEQ ID NO: 81) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0153] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 9, into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:9 was inserted.

[0154] [Experimental Example 9] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO: 10) A DNA fragment was obtained in the same manner as above, except that plasmid No. 9 was used as a template and a forward primer (name "N147P_Fw", SEQ ID NO: 82) and a reverse primer (name "N147P_Rv", SEQ ID NO: 83) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0155] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 10, into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:10 was inserted.

[0156] [Experimental Example 10] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO: 11) A DNA fragment was obtained in the same manner as above, except that plasmid No. 9 was used as a template and a forward primer (name "N147A_Fw", SEQ ID NO: 84) and a reverse primer (name "N147A_Rv", SEQ ID NO: 85) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0157] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 11, into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:11 was inserted.

[0158] [Experimental Example 11] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO: 12) A DNA fragment was obtained in the same manner as above, except that plasmid No. 6 was used as a template and a forward primer (name "A159S_Fw", SEQ ID NO: 86) and a reverse primer (name "A159S_Rv", SEQ ID NO: 87) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0159] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 12, into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:12 was inserted.

[0160] [Experimental Example 12] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO: 13) A DNA fragment was obtained in the same manner as above, except that plasmid No. 12 was used as a template and a forward primer (name "A159D_Fw", SEQ ID NO: 88) and a reverse primer (name "A159D_Rv", SEQ ID NO: 89) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0161] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 13, into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:13 was inserted.

[0162] [Experimental Example 13] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO: 14) A DNA fragment was obtained in the same manner as above, except that plasmid No. 12 was used as a template and a forward primer (name "A159R_Fw", SEQ ID NO: 90) and a reverse primer (name "A159R_Rv", SEQ ID NO: 91) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0163] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 14, into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:14 was inserted.

[0164] [Experimental Example 14] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO: 15) A DNA fragment was obtained in the same manner as above, except that plasmid No. 12 was used as a template and a forward primer (name "A159G_Fw", SEQ ID NO: 92) and a reverse primer (name "A159G_Rv", SEQ ID NO: 93) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0165] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 15, into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:15 was inserted.

[0166] [Experimental Example 15] (Construction of expression plasmids for purine nucleoside phosphorylases having the amino acid sequences of SEQ ID NOs: 16, 17, and 18) A DNA fragment was obtained in the same manner as above, except that plasmid No. 6 was used as a template and a forward primer (name "Th-P181N-F", SEQ ID NO: 94) and a reverse primer (name "Th-P181N-R", SEQ ID NO: 95) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant, and the plasmid was extracted.

[0167] Subsequently, DNA sequence analysis was performed to select plasmids into which the desired mutations had been introduced, and plasmids Nos. 16, 17, and 18 into which genes encoding PNPs having the amino acid sequences of SEQ ID NOs: 16, 17, and 18 were inserted were obtained.

[0168] [Experimental Example 16] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO: 19) A DNA fragment was obtained in the same manner as above, except that plasmid No. 6 was used as a template and a forward primer (name "D243S_Fw", SEQ ID NO: 96) and a reverse primer (name "D243S_Rv", SEQ ID NO: 97) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0169] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 19, into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:19 was inserted.

[0170] [Experimental Example 17] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO: 20) A DNA fragment was obtained in the same manner as above, except that plasmid No. 19 was used as a template and a forward primer (name "D243A_Fw", SEQ ID NO: 98) and a reverse primer (name "D243A_Rv", SEQ ID NO: 99) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0171] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 20, into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:20 was inserted.

[0172] [Experimental Example 18] (Construction of expression plasmids for purine nucleoside phosphorylases having the amino acid sequences of SEQ ID NOs: 21, 22, 23, 24, and 25) A DNA fragment was obtained in the same manner as above, except that plasmid No. 6 was used as a template and a forward primer (name "Th-P255N-F", SEQ ID NO: 100) and a reverse primer (name "Th-P255N-R", SEQ ID NO: 101) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant, and the plasmid was extracted.

[0173] Subsequently, DNA sequence analysis was performed to select plasmids into which the desired mutations had been introduced, and plasmids Nos. 21, 22, 23, 24, and 25 into which genes encoding PNPs having the amino acid sequences of SEQ ID NOs: 21, 22, 23, 24, and 25 had been inserted were obtained.

[0174] [Experimental Example 19] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO:26) A DNA fragment was obtained in the same manner as above, except that plasmid No. 6 was used as a template and a forward primer (name "N238S_Fw", SEQ ID NO: 102) and a reverse primer (name "N238S_Rv", SEQ ID NO: 103) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0175] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 26, into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:26 was inserted.

[0176] [Experimental Example 20] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO:27) A DNA fragment was obtained in the same manner as above, except that plasmid No. 23 was used as a template and a forward primer (name "N238S_Fw", SEQ ID NO: 102) and a reverse primer (name "N238S_Rv", SEQ ID NO: 103) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0177] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 27, into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:27 was inserted.

[0178] [Experimental Example 21] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO:28) A DNA fragment was obtained in the same manner as above, except that plasmid No. 22 was used as a template and a forward primer (name "N238S_Fw", SEQ ID NO: 102) and a reverse primer (name "N238S_Rv", SEQ ID NO: 103) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0179] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 28, into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:28 was inserted.

[0180] [Experimental Example 22] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO:29) A DNA fragment was obtained in the same manner as above, except that plasmid No. 24 was used as a template and a forward primer (name "N238S_Fw", SEQ ID NO: 102) and a reverse primer (name "N238S_Rv", SEQ ID NO: 103) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0181] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 29, into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:29 was inserted.

[0182] [Experimental Example 23] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO: 30) A DNA fragment was obtained in the same manner as above, except that plasmid No. 25 was used as a template and a forward primer (name "N238S_Fw", SEQ ID NO: 102) and a reverse primer (name "N238S_Rv", SEQ ID NO: 103) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0183] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 30 into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:30 was inserted.

[0184] [Experimental Example 24] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO:31) A DNA fragment was obtained in the same manner as above, except that plasmid No. 21 was used as a template and a forward primer (name "D243S_Fw", SEQ ID NO: 96) and a reverse primer (name "D243S_Rv", SEQ ID NO: 97) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0185] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 31, into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:31 was inserted.

[0186] [Experimental Example 25] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO: 32) A DNA fragment was obtained in the same manner as above, except that plasmid No. 31 was used as a template and a forward primer (name "A159S_Fw", SEQ ID NO: 86) and a reverse primer (name "A159S_Rv", SEQ ID NO: 87) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0187] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 32, into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:32 was inserted.

[0188] [Experimental Example 26] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO: 33) A DNA fragment was obtained in the same manner as above, except that plasmid No. 32 was used as a template and a forward primer (name "N147R_Fw", SEQ ID NO: 80) and a reverse primer (name "N147R_Rv", SEQ ID NO: 81) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0189] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 33, into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:33 was inserted.

[0190] [Experimental Example 27] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO: 34) A DNA fragment was obtained in the same manner as above, except that plasmid No. 22 was used as a template and a forward primer (name "D243A_Fw", SEQ ID NO: 98) and a reverse primer (name "D243A_Rv", SEQ ID NO: 99) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0191] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 34, into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:34 was inserted.

[0192] [Experimental Example 28] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO: 35) A DNA fragment was obtained in the same manner as above, except that plasmid No. 34 was used as a template and a forward primer (name "A159D_Fw", SEQ ID NO: 88) and a reverse primer (name "A159D_Rv", SEQ ID NO: 89) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0193] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 35, into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:35 was inserted.

[0194] [Experimental Example 29] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO: 36) A DNA fragment was obtained in the same manner as above, except that plasmid No. 35 was used as a template and a forward primer (name "N147P_Fw", SEQ ID NO: 82) and a reverse primer (name "N147P_Rv", SEQ ID NO: 83) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0195] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 36, into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:36 was inserted.

[0196] [Experimental Example 30] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO:37) A DNA fragment was obtained in the same manner as above, except that plasmid No. 23 was used as a template and a forward primer (name "A159R_Fw", SEQ ID NO: 90) and a reverse primer (name "A159R_Rv", SEQ ID NO: 91) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0197] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 37, into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:37 was inserted.

[0198] [Experimental Example 31] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO: 38) A DNA fragment was obtained in the same manner as above, except that plasmid No. 37 was used as a template and a forward primer (name "N147A_Fw", SEQ ID NO: 84) and a reverse primer (name "N147A_Rv", SEQ ID NO: 85) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0199] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 38, into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:38 was inserted.

[0200] [Experimental Example 32] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO:39) A DNA fragment was obtained in the same manner as above, except that plasmid No. 24 was used as a template and a forward primer (name "D243S_Fw", SEQ ID NO: 96) and a reverse primer (name "D243S_Rv", SEQ ID NO: 97) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0201] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 39, into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:39 was inserted.

[0202] [Experimental Example 33] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO: 40) A DNA fragment was obtained in the same manner as above, except that plasmid No. 39 was used as a template and a forward primer (name "A159G_Fw", SEQ ID NO: 92) and a reverse primer (name "A159G_Rv", SEQ ID NO: 93) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0203] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 40, into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:40 was inserted.

[0204] [Experimental Example 34] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO: 41) A DNA fragment was obtained in the same manner as above, except that plasmid No. 25 was used as a template and a forward primer (name "D243A_Fw", SEQ ID NO: 98) and a reverse primer (name "D243A_Rv", SEQ ID NO: 99) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0205] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 41, into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:41 was inserted.

[0206] [Experimental Example 35] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO: 42) A DNA fragment was obtained in the same manner as above, except that plasmid No. 41 was used as a template and a forward primer (name "N147R_Fw", SEQ ID NO: 80) and a reverse primer (name "N147R_Rv", SEQ ID NO: 81) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0207] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 42, into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:42 was inserted.

[0208] [Experimental Example 36] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO: 43) A DNA fragment was obtained in the same manner as above, except that plasmid No. 6 was used as a template and a forward primer (name "I49V_Fw", SEQ ID NO: 104) and a reverse primer (name "I49V_Rv", SEQ ID NO: 105) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0209] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 43, into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:43 was inserted.

[0210] [Experimental Example 37] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO: 44) A DNA fragment was obtained in the same manner as above, except that plasmid No. 6 was used as a template and a forward primer (name "V109A_Fw", SEQ ID NO: 106) and a reverse primer (name "V109A_Rv", SEQ ID NO: 107) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0211] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 44, into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:44 was inserted.

[0212] [Experimental Example 38] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO: 45) A DNA fragment was obtained in the same manner as above, except that plasmid No. 6 was used as a template and a forward primer (name "P118L_Fw", SEQ ID NO: 108) and a reverse primer (name "P118L_Rv", SEQ ID NO: 109) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0213] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 45, into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:45 was inserted.

[0214] [Experimental Example 39] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO: 46) A DNA fragment was obtained in the same manner as above, except that plasmid No. 6 was used as a template and a forward primer (name "A36V_Fw", SEQ ID NO: 110) and a reverse primer (name "A36V_Rv", SEQ ID NO: 111) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0215] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 46, into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:46 was inserted.

[0216] [Experimental Example 40] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO: 47) A DNA fragment was obtained in the same manner as above, except that plasmid No. 43 was used as a template and a forward primer (name "Q183H_Fw", SEQ ID NO: 112) and a reverse primer (name "Q183H_Rv", SEQ ID NO: 113) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0217] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 47, into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:47 was inserted.

[0218] [Experimental Example 41] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO: 48) A DNA fragment was obtained in the same manner as above, except that plasmid No. 47 was used as a template and a forward primer (name "E239G_Fw", SEQ ID NO: 114) and a reverse primer (name "E239G_Rv", SEQ ID NO: 115) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0219] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 48 into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:48 was inserted.

[0220] [Experimental Example 42] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO: 49) A DNA fragment was obtained in the same manner as above, except that plasmid No. 6 was used as a template and a forward primer (name "Q183H_Fw", SEQ ID NO: 112) and a reverse primer (name "Q183H_Rv", SEQ ID NO: 113) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0221] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 49, into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:49 was inserted.

[0222] [Experimental Example 43] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO:50) A DNA fragment was obtained in the same manner as above, except that plasmid No. 6 was used as a template and a forward primer (name "E239G_Fw", SEQ ID NO: 114) and a reverse primer (name "E239G_Rv", SEQ ID NO: 115) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0223] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 50, into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:50 was inserted.

[0224] [Experimental Example 44] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO:51) A DNA fragment was obtained in the same manner as above, except that plasmid No. 6 was used as a template and a forward primer (name "G242D_Fw", SEQ ID NO: 116) and a reverse primer (name "G242D_Rv", SEQ ID NO: 117) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0225] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 51, into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:51 was inserted.

[0226] [Experimental Example 45] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO:52) A DNA fragment was obtained in the same manner as above, except that plasmid No. 6 was used as a template and a forward primer (name "N249S_Fw", SEQ ID NO: 118) and a reverse primer (name "N249S_Rv", SEQ ID NO: 119) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0227] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 52, into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:52 was inserted.

[0228] [Experimental Example 46] (Construction of an expression plasmid for purine nucleoside phosphorylase having the amino acid sequence of SEQ ID NO:53) A DNA fragment was obtained in the same manner as above, except that plasmid No. 6 was used as a template and a forward primer (name "E251G_Fw", SEQ ID NO: 120) and a reverse primer (name "E251G_Rv", SEQ ID NO: 121) were used. This DNA fragment was introduced into Escherichia coli DH5α strain to obtain a transformant.

[0229] This transformant was cultured in LB medium containing ampicillin (100 μg / mL), and the plasmid was extracted to obtain plasmid No. 53, into which a gene encoding PNP having the amino acid sequence of SEQ ID NO:53 was inserted.

[0230] [Experimental Example 47] (Preparation of Purine Nucleoside Phosphorylase) Plasmids No. 1 to 53 expressing purine nucleoside phosphorylase having the amino acid sequences of SEQ ID NO: 1 to 53 were introduced into E. coli W3110 strain to obtain the desired transformants. These transformants were inoculated into 20 mL of medium (tryptone 20 g / L, yeast extract 10 g / L, sodium chloride 5 g / L, iron sulfate heptahydrate 0.2 g / L, pH 7.5) and cultured at 30°C, 200 rpm, and 18 hours. The entire culture solution obtained was collected and centrifuged at 8000 rpm for 15 minutes to collect the cells. The collected cells were suspended in ion-exchanged water and then frozen and thawed at -20°C to obtain a purine nucleoside phosphorylase solution.

[0231] [Experimental Example 48] (Evaluation of nicotinamide riboside production activity) Purine nucleoside phosphorylases having the amino acid sequences of SEQ ID NOs: 1 to 53 were evaluated for their nicotinamide riboside producing activity.

[0232] 0.49 g of ribose-1-phosphate diammonium salt, 0.53 g of nicotinamide, 0.89 g of magnesium chloride hexahydrate, and 0.24 g of ammonium chloride were dissolved in an aqueous medium (0.5 M MES buffer, pH 6.0) to prepare 10 g of a reaction raw material solution.

[0233] To 0.9 g of this reaction raw material liquid, 0.1 g of each of the purine nucleoside phosphorylase solutions having the amino acid sequences of SEQ ID NOs: 1 to 53 was added, and the enzyme reaction was carried out at a reaction temperature of 15°C. The amount of nicotinamide riboside produced was measured to evaluate the nicotinamide riboside production activity.

[0234] The amount of nicotinamide riboside produced was measured using a high-performance liquid chromatograph (manufactured by JASCO) under the following conditions: flow rate: 1.0 mL / min, eluent: 10 mM phosphoric acid-methanol:water = 1:9, analytical column: Develosil ODS-MG-5, 4.6 μm × 250 mm (Nomura Chemical), detection method: UV 254 nm.

[0235] The evaluation results of the nicotinamide riboside production activity are shown in Tables 1 and 2 below. In Tables 1 and 2, "X number Y" in the mutation pattern represents an amino acid mutation, where X represents the amino acid before the mutation, the number represents the position of the amino acid counted from the N-terminus, and Y represents the amino acid after the mutation. "NR" represents nicotinamide riboside. The amount of NR produced (relative value) was calculated by the following formula. NR production amount (relative value) = NR production amount after 24 hours by purine nucleoside phosphorylase of SEQ ID NO: 2 to 53 (mutant type) / NR production amount after 24 hours by purine nucleoside phosphorylase of SEQ ID NO: 1 (wild type)

[0236] These results demonstrated that the nicotinamide riboside production activity was dramatically improved by introducing substitution mutations into amino acid residues corresponding to the 6th, 36th, 49th, 109th, 118th, 147th, 159th, 181st, 183rd, 186th, 201st, 207th, 237th, 238th, 239th, 242nd, 243rd, 249th, 251st, and 255th amino acid residues in the amino acid sequence of wild-type purine nucleoside phosphorylase set forth in SEQ ID NO:1.

[0237] [Table 1]

[0238] [Table 2] [Industrial Applicability]

[0239] According to the present disclosure, it is possible to provide a mutant purine nucleoside phosphorylase that can efficiently produce nicotinamide riboside.

Claims

1. (a) the amino acid sequence set forth in SEQ ID NO:1; (b) an amino acid sequence comprising one or more amino acid substitutions, deletions, insertions or additions in the amino acid sequence set forth in SEQ ID NO:1; or (c) an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO:1; A mutant purine nucleoside phosphorylase having one or more amino acid mutations in the amino acid sequence of a wild-type purine nucleoside phosphorylase having the formula: and having a higher activity of producing nicotinamide riboside than the wild-type purine nucleoside phosphorylase.

2. The mutant purine nucleoside phosphorylase according to claim 1, wherein the amino acid sequence of the wild-type purine nucleoside phosphorylase is the amino acid sequence set forth in SEQ ID NO:

1.

3. The mutant purine nucleoside phosphorylase according to claim 1, having an amino acid mutation in at least one selected from the group consisting of amino acid residues corresponding to the 6th, 36th, 49th, 109th, 118th, 147th, 159th, 181st, 183rd, 186th, 201st, 207th, 237th, 238th, 239th, 242nd, 243rd, 249th, 251st and 255th amino acid residues in the amino acid sequence set forth in SEQ ID NO:

1.

4. 4. The mutant purine nucleoside phosphorylase according to claim 3, which comprises at least a mutation in the amino acid residues corresponding to the 186th and / or 207th amino acid residues in the amino acid sequence set forth in SEQ ID NO:

1.

5. 4. The mutant purine nucleoside phosphorylase according to claim 3, which comprises at least a mutation in an amino acid residue corresponding to the 159th and / or 243rd amino acid residues in the amino acid sequence set forth in SEQ ID NO:

1.

6. 6. The mutant purine nucleoside phosphorylase according to claim 3 or 5, which comprises at least a mutation in an amino acid residue corresponding to the 36th, 49th, 237th and / or 249th amino acid residues in the amino acid sequence set forth in SEQ ID NO:

1.

7. 6. The mutant purine nucleoside phosphorylase according to claim 3 or 5, which comprises at least a mutation in the amino acid residues corresponding to the 6th and / or 201st amino acid residues in the amino acid sequence set forth in SEQ ID NO:

1.

8. 8. The mutant purine nucleoside phosphorylase according to claim 7, which comprises at least a mutation in the amino acid residues corresponding to the 147th and / or 255th amino acid residues in the amino acid sequence set forth in SEQ ID NO:

1.

9. 6. The mutant purine nucleoside phosphorylase according to claim 5, wherein Ala of the amino acid residue corresponding to the 159th amino acid residue in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with Glu, Ser, Asp, Gly or Arg.

10. 6. The mutant purine nucleoside phosphorylase according to claim 5, wherein Asp of the amino acid residue corresponding to the 243rd position in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with Glu, Ser or Ala.

11. 8. The mutant purine nucleoside phosphorylase according to claim 7, wherein Val at the sixth amino acid residue in the amino acid sequence set forth in SEQ ID NO:1 is substituted with Asp.

12. 8. The mutant purine nucleoside phosphorylase according to claim 7, wherein Leu at the 201st amino acid residue in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with Arg.

13. 9. The mutant purine nucleoside phosphorylase according to claim 8, wherein Asn of the amino acid residue corresponding to the 147th position in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with Lys, Arg, Pro or Ala.

14. 9. The mutant purine nucleoside phosphorylase according to claim 8, wherein Glu of the amino acid residue corresponding to the 255th position in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with Gly, Ile, Leu, Arg, Lys or Val.

15. The mutant purine nucleoside phosphorylase according to claim 3, having an amino acid sequence set forth in any one of SEQ ID NOs: 2 to 53.

16. A nucleic acid encoding the mutant purine nucleoside phosphorylase according to claim 1 or 3.

17. A vector comprising the nucleic acid of claim 16.

18. A transformant comprising the vector according to claim 17.

19. Cultivating the transformant according to claim 18 in a medium to obtain a culture; and recovering the mutant purine nucleoside phosphorylase from the culture.

Images