Purine nucleoside phosphorylase mutant and preparation method for 2'-fluoro nucleoside

Abstract

Provided in the present invention are a purine nucleoside phosphorylase mutant and a preparation method for 2'-fluoro nucleoside. The purine nucleoside phosphorylase mutant comprises: (a) a mutant protein generated on the basis of a purine nucleoside phosphorylase wild-type enzyme GsPNP as shown in SEQ ID NO: 3, the mutation being selected from any one or more of the following mutations: L148, P19, Q41, etc.; or (b) a protein having 70% or more homology with the amino acid sequence defined in (a) and having purine nucleoside phosphorylase activity. The method can solve the problem in the prior art of low yield of 2'-fluoro nucleoside synthesized by enzyme catalysis, and is suitable for the field of enzyme catalysis.

Description

Purine nucleoside phosphorylase mutant and preparation method of 2'-fluoronucleoside

[0001] This application is based on the Chinese application with CN application number 202311341028.7 and application date October 17, 2023, and claims its priority. The disclosed content of the CN application is again introduced as a whole into this application. Technical Field

[0002] The present invention relates to the field of enzyme catalysis, and in particular to a preparation method of a purine nucleoside phosphorylase mutant and a 2'-fluoronucleoside. Background Art

[0003] Biotransformation methods for synthesizing nucleosides and their analogs can utilize nucleoside phosphorylases to convert inexpensive nucleosides into high-value nucleosides. Compared to chemical synthesis, these methods offer milder reaction conditions, a single product, and are environmentally friendly. Nucleoside phosphorylases, including pyrimidine nucleoside phosphorylase (PyNP) and purine nucleoside phosphorylase (PNP), are enzymes involved in the pyrimidine and purine metabolic pathways. They reversibly catalyze the phosphorolysis of nucleosides and can therefore be used synergistically in the biosynthesis of purine nucleosides and their analogs. For example, a one-pot process can be used to produce 2′-deoxyadenosine using PyNP and PNP to catalyze the conversion of 2′-deoxyuridine and adenine. Among them, PyNP can decompose 2′-deoxyuridine into 2′-deoxyribose-1-phosphate and uracil (Uracil), and another substrate adenine and 2′-deoxyribose-1-phosphate catalyzed by PyNP can be synthesized into 2′-deoxyadenosine by PNP.

[0004] However, for the biosynthesis of nucleoside analogs such as 2′-fluoro-2′-deoxyadenosine, the catalytic activity of wild-type PyNP and PNP is relatively low due to the non-natural substrate 2′-fluoro-2′-deoxyuridine, thus limiting the commercial production of 2′-fluoro-modified nucleoside monomers using biological methods. Zhou et al. used wild-type pyrimidine nucleoside phosphorylase from Thermus thermophilus (TtPyNP) and wild-type purine nucleoside phosphorylase from Deinococcus geothermalis (DgPNP) to react for 24 hours, but only less than 0.5 mM of 2′-fluoro-2′-deoxyadenosine was obtained (Recombinant purine nucleoside phosphorylases from thermophiles: preparation, properties and activity towards purine and pyrimidine nucleosides). Patent CN111534493 discloses a purine nucleoside phosphorylase (EcPNP) mutant derived from Escherichia coli, in which methionine at position 167 is mutated to arginine and phenylalanine at position 168 is mutated to alanine. Its activity is increased by 47.6% compared with the wild-type enzyme. However, the yield of 2′-fluoro-2′-deoxyadenosine prepared using this mutation is less than 1.5 mM, which cannot meet the needs of large-scale production.

[0005] Summary of the Invention

[0006] The main purpose of the present invention is to provide a purine nucleoside phosphorylase mutant and a preparation method of 2'-fluoronucleosides, so as to solve the problem of low yield of 2'-fluoronucleosides synthesized by enzyme in the prior art.

[0007] To achieve the above object, according to a first aspect of the present invention, a purine nucleoside phosphorylase mutant is provided, comprising: (a) a protein having a mutation based on the wild-type purine nucleoside phosphorylase GsPNP shown in SEQ ID NO: 3, wherein the mutation is selected from any one or more of the following mutations: L148, P19, Q41, T51, R56, T62, I69, S70, I73, T74, Q78, S79, Y80, N81, V82, Q83, A93, I94, K96, V98, K99, M107, T108, S109, or (b) a protein having greater than 70% homology to the amino acid sequence defined in (a) and having purine nucleoside phosphorylase activity.

[0008] Furthermore, in (a), the type of amino acid substituted at each position is independently selected from the following: L148P, P19M, P19K, Q41D, Q41G, Q41N, Q41T, T51F, T51W, T51Y, R56K, T62C, I69M, S70G, I73A, I73G, I73L, T74Q, Q78A, Q78K, S79T, Y80A, Y80P, Y80T, N81G, V82A, Q83G, A93S, I94A, I94L, I94N, K96E, K96P , K96S, V98E, V98H, V98L, V98M, V98Q, V98S, V98T, K99S, M107E, M107G, M107H, M107N, M107S, M107Y, T108I, T108L, T108 R, T108V, S109N, S113N, T119C, T119K, Y125F, Y125H, Y125K, Y125M, Y125Y, A126G, A126S, T128C, N130D, F131A, F131I, F 131L, F131P, F131S, F131T, F131W, K135E, Y138S, Y138L, K142R, K144A, K144L, L148A, L148G, L148Q, L148S, L148Y, V15 0E, V150P, S152D, S152G, S152H, S152K, S152N, S152P, V153S, F154L, F154K, A156E, F166I, F166P, F166S, F166T, F166V, K168E, A170E, A170R, Y172A, Y172C, Y172G, Y172L, Y172N, Y172Q, Y172R, V174D, V174G, V174L, V174M, V174N, V174Q, V174S, V174T, L175A, L175E, L175G, L175K, L175N, L175S, L175T, I205A, I205V or L206I; wherein, the letters before the numbers represent the original amino acids, and the letters after the numbers represent the mutated amino acids.

[0009] Furthermore, the above mutations include any of the following amino acid mutations: L148P+P19M, L148P+P19K, L148P+Q41D, L148P+Q41G, L148P+Q41N, L148P+Q41T, L148P+T51F, L148P+T51W, L148P+T51Y, L148P+R56K, L148P+T62C, L148P+I69M, L148P+S70G, L148P+I73A, L148P+I73G, L148P+I73L, L148P+T74Q, L148P+Q78A, L148P+Q78K, L148P+S79T, L148 P+Y80A, L148P+Y80P, L148P+Y80T, L148P+N81G, L148P+V82A, L148P+Q83G, L148P+A93S, L148P+I94A, L148P+I94L, L148P+I94N, L148P+K96E, L148P+K 96P, L148P+K96S, L148P+V98E, L148P+V98H, L148P+V98L, L148P+V98M, L14 8P+V98Q, L148P+V98S, L148P+V98T, L148P+K99S, L148P+M107E, L148P+M10 7G, L148P+M107H, L148P+M107N, L148P+M107S, L148P+M107Y, L148P+T108 I. L148P+T108L, L148P+T108R, L148P+T108V, L148P+S109N, L148P+S113N, L148P+T119C, L148P+T119K, L148P+Y125F, L148P+Y125H, L148P+Y125K, L1 48P+Y125M, L148P+Y125Y, L148P+A126G, L148P+A126S, L148P+T128C, L148 P+N130D, L148P+F131A, L148P+F131I, L148P+F131L, L148P+F131P, L148P+ F131S, L148P+F131T, L148P+F131W, L148P+K135E, L148P+Y138S, L148P+Y1 38L, L148P+K142R, L148P+K144A, L148P+K144L, L148P+V150E, L148P+V150 P, L148P+S152D, L148P+S152G, L148P+S152H, L148P+S152K, L148P+S152N,L148P+S152P、L148P+V153S、L148P+F154L、L148P+F154K、L148P+A156E、L148P+F166I、L148P+F166P、L148P+F166S、L148P+F166T、L148P+F166V、L148P+K168E、L148P+A170E、L148P+A170R、L148P+Y172A、L148P+Y172C、L148P+Y172G、L148P+Y172L、L148P+Y172N、L148P+Y172Q、L148P+Y172R、L148P+V174D、L148P+V174G、L148P+V174L、L148P+V174M、L148P+V174N、L148P+V174Q、L148P+V174S、L148P+V174T、L148P+L175A、L148P+L175E、L148P+L175G、L148P+L175K、L148P+L175N、L148P+L175S、L148P+L175T、L148P+I205A、L148P+I205V、L148P+L206I、L148P+P19K+M107G、L148P+P19M+F166P、L148P+Q41G+T119K、L148P+Q41D+A156E、L148P+Q41N+K96E、L148P+Q41T+T51W、L148P+Q41T+N81G、L148P+Q41T+T108R、 L148P+Q41T+L175K、L148P+T51F+F166I、L148P+T51W+V174S、L148P+T51Y+F166P、L148P+R56K+A126S、L148P+T62C+M107H、L148P+I69M+K135E、L148P+S70G+Y125H、L148P+I73A+S152N、L148P+I73A+M107Y、L148P+I73G+V98Q、L148P+T74Q+I94N、L148P+Q78A+V174S、L148P+Q78K+L175G、L148P+S79T+A93S、L148P+Y80A+F154L、L148P+Y80P+Y172A、L148P+Y80T+L206I、L148P+Q83G+S152D、L148P+V98E+V153S、L148P+V98L+V174L、L148P+V98M+F131I、L148P+V98H+K168E、L148P+V98S+Y172Q、L148P+M107S+S152K、L148P+T108L+V174D、L148P+T108V+L175K、L148P+S113N+S152H、L148P+T119C+S152G、L148P+Y125M+Y172L、L148P+A126G+Y172G、L148P+T128C+V174N、L148P+N130D+V150E、L148P+F131L+I205V、L148P+F131P+Y172C、L148P+F131W+L175S、L148P+Y138S+V174Q、L148P+F166S+V174M、L148P+F166V+L175E、L148P+P19K+T108V+Y125Y、L148P+P19M+M107N+F166P、L148P+Q41D+M107G+S152P、L148P+Q41N+Y80P+K144A、L148P+Q41T+M107H+V174L、L148P+Q41T+L175K+I73A、L148P+T51W+Y80P+V174S、L148P+T51Y+F131T+V174G、L148P+R56K+Q78A+F166S、L148P+T62C+Y125K+V150P、L148P+I73A+T108L+F131S、L148P+I73G+K96P+S152H、L148P+I73L+V98M+F166P、L148P+V82A+A93S+F131L、L148P+I69M+A156E+L175E、L148P+S70G+S113N+Y172R、L148P+S79T+A126G+S152D、L148P+I94A+F131P+V174M、L148P+I94L+Y138L+V174T、L148P+I94N+N130D+L206I、L148P+K96E+F131W+F166I、L148P+V98H+T119K+F166T、L148P+V98Q+Y138S+A170E、L148P+V98L+K135E+V174L、L148P+M107E+S152N+L175K、L148P+T108R+A126S+K168E、L148P+S109N+F131I+F154L、L148P+T119C+Y125H+K135E、L148P+F131A+K142R+V174S、L148P+S152G+F166V+I205V、L148P+P19K+K96P+V150P+A126S、L148P+P19M+M107N+F166P+V174T、L148P+Q41D+T108L+K144A+V153S、L148P+Q41G+T51Y+S113N+F166I、L148P+Q41N+T51F+A93S+F131A、L148P+Q41N+Y80A+S152G+L175E、L148P+Q41T+Y80A+V150E+Y172A、L148P+Q41T+I73A+S113N+L175K、L148P+T51W+Y80P+Y125M+V174S、L148P+T51Y+I73A+Y125K+L142R、L148P+R56K+M107H+A126G+L206I、L148P+T62C+V98Q+M107G+S152P、L148P+I69M+F131W+L175K+L206I、L148P+I73G+F131L+S152K+Y172C、L148P+I73L+I94A+T119C+S152D、L148P+T74Q+V98S+T108I+I205V、L148P+Q78A+S152P+F166V+L175G、L148P+Q78K+I94L+L175E+L206I、L148P+S79T+K96E+Y138L+V174M、L148P+Y80T+M107S+S152H+Y172R、L148P+N81G+Y125Y+F131T+A156E、L148P+Q83G+M107Y+A126S+K168E、L148P+V98L+T119K+K135E+V174L、L148P+V98T+M107E+Y138S+I205A、L148P+Y125H+F131I+S152H+L175T、L148P+P19M+T51Y+M107N+F166P+V174T、L148P+Q41T+I73A+A126S+L175K+S113N、L148P+T51W+Y80P+Y125M+S152P+V174S、L148P+I73L+Y80T+M107S+S152H+Y172R、L148P+Q78A+S152P+F166V+L175G+I205V、L148P+V98L+T119K+K135E+F166P+V174L、L148P+P19M+T51Y+M107N+S152H+F166P+V174T、L148P+Q41D+V98L+T119K+K135E+F166P+V174L、L148P+Q41T+I73A+A126S+K144A+L175K+S113N、L148P+T51W+Y80P+M107N+S113N+F166V+I205A、 L148P+T51W+Y80P+Y125M+S152P+V174S+L206I、L148P+R56K+I73L+Y80T+M107S+S152H+Y172R、L148P+Q78A+M107S+S152P+F166V+L175G+I205V、L148P+P19M+T51Y+I73A+M107N+S152H+F166P+V174T、L148P+Q41D+V98L+T119K+K135E+S152H+F166P+V174L、L148P+Q41T+I73A+S113N+A126S+K144A+L175K+I205V、L148P+T51W+Y80P+M107N+S113N+F166V+L175G+I205A、L148P+T51W+Y80P+V98L+Y125M+S152P+V174S+L206I、L148P+R56K+I73L+Y80T+M107S+T119K+S152H+Y172R、L148P+Q78A+M107S+Y125H+S152P+F166V+L175G+I205V、L148P+P19M+Q41T+I73A+S113N+A126S+K144A+L175K+I205V、L148P+P19M+T51Y+I73A+V98T+M107N+S152H+F166P+V174T、L148P+Q41D+V98L+T119K+K135E+S152H+F166P+V174L+I205V、L148P+T51W+Y80P+V98T+M107N+S113N+F166V+L175G+I205A、L148P+T51W+Y80P+V98L+Y125M+S152P+F166V+V174S+L206I、L148P+R56K+I73L+Y80T+M107S+T119K+S152H+Y172R+L206I、L148P+Q78A+M107S+S113N+Y125H+S152P+F166V+L175G+I205V、L148P+P19M+Q41T+I69M+I73A+S113N+A126S+K144L+L175K+I205V、L148P+P19M+T51Y+I73A+V98T+M107N+S152H+F166P+V174T+L206I、L148P+Q41D+S70G+V98L+T119K+K135E+S152H+F166P+V174L+I205V, L148P+T51W+I69M+Y80P+V98T+M107N+S113N+F166V +L175G+I205A, L148P+T51W+Y80P+V98L+Y125M+K144L+S152P+F166V+V174S+L206I, L148P+R56K+I73L+Y80T+M107S+T119 K+K144A+S152H+Y172R+L206I, L148P+Q78A+V98L+M107S+S113N+Y125H+S152P+F166V+L175G+I205V, L148P+P19M+Q41T+ I69M+I73A+S113N+Y125F+A126S+T128C+N130D+K144L+L175K+I205V, L148P+P19M+Q41D+T51Y+I73A+V98T+M107N+A126G+ K135E+S152H+F166P+V174T+L206I, L148P+Q41D+S70G+V98L+T108L+T119K+Y125F+K135E+K144A+S152H+F166P+V174L+I 205V, L148P+T51W+I69M+Y80P+V98T+M107N+S113N+N130D+K142R+F154L+F166V+L175G+I205A, L148P+T51W+T74Q+Y80P+V 98L+T108I+S113N+Y125M+K144L+S152P+F166V+V174S+L206I、L148P+R56K+I73L+Y80T+V98M+M107S+T119K+Y125F+F131S+K144A+S152H+Y172R+L206I、or L148P+I69M+Q78A+V98L+M107S+S113N+Y125H+N130D+K144A+S152P+F166V+L175G+I205V。

[0010] Furthermore, the purine nucleoside phosphorylase mutants include proteins having 80% or more, 85% or more, more preferably 95% or more, and even more preferably 99% or more homology to the amino acid sequence defined in (a) and having purine nucleoside phosphorylase activity;

[0011] Preferably, the purine nucleoside phosphorylase mutant includes: the protein shown in SEQ ID NO: 5, or a protein having more than 80% homology with the protein shown in SEQ ID NO: 5 and having purine nucleoside phosphorylase activity.

[0012] In order to achieve the above object, according to a second aspect of the present invention, a DNA molecule is provided, which encodes the above purine nucleoside phosphorylase mutant.

[0013] In order to achieve the above object, according to the third aspect of the present invention, a recombinant plasmid is provided, wherein the recombinant plasmid is connected to the above DNA molecule.

[0014] In order to achieve the above object, according to a fourth aspect of the present invention, a host cell is provided, wherein the host cell contains the above DNA molecule or recombinant plasmid.

[0015] Further, the host cell includes a prokaryotic cell; preferably, the prokaryotic cell includes Escherichia coli.

[0016] In order to achieve the above object, according to the fifth aspect of the present invention, a method for preparing 2′-fluoronucleosides is provided, which comprises: using the above-mentioned purine nucleoside phosphorylase to catalyze the binding of phosphorylated 2′-fluoropentose and substrate base to prepare 2′-fluoronucleosides.

[0017] Furthermore, the preparation method of phosphorylated 2′-fluoropentose includes: using nucleoside phosphorylase to catalyze the cleavage of substrate nucleosides having a 2′-fluoropentose structure into phosphorylated 2′-fluoropentose and free bases; preferably, the nucleoside phosphorylase includes pyrimidine nucleoside phosphorylase, thymidine phosphorylase, uridine phosphorylase or 5′-methylthioadenosine phosphorylase.

[0018] Furthermore, the substrate nucleoside includes 2′-fluoro-2′-deoxyuridine, 2′-fluoro-2′-deoxycytidine or 2′-deoxy-2′-fluorothymidine.

[0019] Furthermore, the substrate base includes modified or unmodified adenine, guanine, thymine, cytosine, or modified uracil; preferably, the substrate base includes adenine, guanine or 2-aminoadenine.

[0020] Furthermore, the 2′-fluoro nucleoside includes a nucleoside containing a 2′-fluoro-2′-deoxypentose structure; preferably, the 2′-fluoro nucleoside includes 2′-fluoro-2′-deoxyadenosine, 2′-fluoro-2′-deoxy-2-aminoadenosine, 2′-fluoro-2′-deoxyguanosine or 2′-fluoro-2′-deoxycytidine; or, the 2′-fluoro nucleoside is selected from a 2′-fluoro-2′-deoxynucleoside having a substituent on the base structure.

[0021] The technical solution of the present invention utilizes the aforementioned purine nucleoside phosphorylase mutant to efficiently catalyze the phosphorylation of 2′-fluoropentoses and the binding of different substrate bases to produce 2′-fluoronucleosides. Compared to the wild-type enzyme, the purine nucleoside phosphorylase mutant exhibits superior enzymatic activity and a higher conversion rate and efficiency in producing 2′-fluoronucleoside products. This significantly reduces enzyme usage, thereby lowering production costs and making it suitable for industrial scale-up production. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0023] FIG1 shows a schematic diagram of a chemical reaction for preparing 2′-fluoronucleosides by using purine nucleoside phosphorylase to catalyze the phosphorylation of 2′-fluoropentose and the combination with the substrate base according to an embodiment of the present invention.

[0024] FIG2 shows a schematic diagram of a chemical reaction using nucleoside phosphorylase to catalyze the cleavage of substrate nucleosides having 2′-fluoropentose into phosphorylated 2′-fluoropentose and free bases according to an embodiment of the present invention.

[0025] FIG3 shows an HPLC spectrum of preparing 2′-fluoro-2′-deoxyadenosine using 2′-fluoro-2′-deoxyuridine and adenine as substrates according to Example 4 of the present invention. DETAILED DESCRIPTION

[0026] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0027] Explanation of terms:

[0028] 2′-Fluoronucleoside: A nucleoside in which the hydroxyl group on the 2-carbon atom of the pentose sugar is replaced by a fluorine atom.

[0029] As mentioned in the background art, the yield and output of 2′-fluoronucleosides prepared by enzyme catalysis are both low, making it difficult to meet the requirements of industrial production. Therefore, in this application, the inventors attempted to develop a purine nucleoside phosphorylase mutant with higher enzyme activity. Through directed evolution, a purine nucleoside phosphorylase mutant was obtained, whose catalytic activity was increased by about 20 times compared to the wild type, reducing the amount of enzyme used, thereby reducing production costs and being applied to large-scale production of related products. It can efficiently prepare the target product 2′-fluoronucleoside, and thus proposed a series of protection schemes for this application.

[0030] In a first typical embodiment of the present application, a purine nucleoside phosphorylase mutant is provided, comprising: (a) a protein having a mutation based on the wild-type purine nucleoside phosphorylase GsPNP shown in SEQ ID NO: 3, wherein the mutation is selected from any one or more of the following mutations: L148, P19, Q41, T51, R56, T62, I69, S70, I73, T74, Q78, S79, Y80, N81, V82, Q83, A93, I94, K96, V98, K99, M107, T108, S109, S113, T 119, Y125, A126, T128, N130, F131, K135, Y138, L142, K144, V150, S152, V153, F154, A156, F166, K168, A170, Y172, V174, L175, I205 or L206; (b) a protein that has more than 70% homology to the amino acid sequence defined in (a) and has purine nucleoside phosphorylase activity.

[0031] In a preferred embodiment, in (a), the type of amino acid substituted at each site is independently selected from the following:

[0032] L148P, P19M, P19K, Q41D, Q41G, Q41N, Q41T, T51F, T51W, T51Y, R56K, T62C, I69M, S70G, I73A, I73G, I73L, T74Q, Q78 A. Q78K, S79T, Y80A, Y80P, Y80T, N81G, V82A, Q83G, A93S, I94A, I94L, I94N, K96E, K96P, K96S, V98E, V98H, V98L, V98 M, V98Q, V98S, V98T, K99S, M107E, M107G, M107H, M107N, M107S, M107Y, T108I, T108L, T108R, T108V, S109N, S113N, T119C, T119K, Y125F, Y125H, Y125K, Y125M, Y125Y, A126G, A126S, T128C, N130D, F131A, F131I, F131L, F131P, F131S , F131T, F131W, K135E, Y138S, Y138L, K142R, K144A, K144L, L148A, L148G, L148Q, L148S, L148Y, V150E, V150P, S15 2D, S152G, S152H, S152K, S152N, S152P, V153S, F154L, F154K, A156E, F166I, F166P, F166S, F166T, F166V, K168E, A1 70E, A170R, Y172A, Y172C, Y172G, Y172L, Y172N, Y172Q, Y172R, V174D, V174G, V174L, V174M, V174N, V174Q, V174S, V174T, L175A, L175E, L175G, L175K, L175N, L175S, L175T, I205A, I205V or L206I; wherein, the letters before the numbers represent the original amino acids, and the letters after the numbers represent the mutated amino acids.

[0033] In a preferred embodiment, the above mutations include any one of the following amino acid mutations: L148P+P19M, L148P+P19K, L148P+Q41D, L148P+Q41G, L148P+Q41N, L148P+Q41T, L148P+T51F, L148P+T51W, L148P+T51Y, L148P+R56K, L148P+T62C, L148P+I69M, L148P+S70G, L148P+I73A, L148P+I73G, L148P+I73L, L148P+T74Q, L148P+Q78A, L148P+Q78K, L148P+S79T, L148P+Y80A, L148P+Y80P, L148 P+Y80T, L148P+N81G, L148P+V82A, L148P+Q83G, L148P+A93S, L148P+I94A, L148P+I94L, L148P+I94N, L148P+K96E, L148P+K9 6P, L148P+K96S, L148P+V98E, L148P+V98H, L148P+V98L, L148P+V98M, L148P+V98Q, L148P+V98S, L148P+V98T, L148P+K99S, L 148P+M107E, L148P+M107G, L148P+M107H, L148P+M107N, L148P+M107S, L148P+M107Y, L148P+T108I, L148P+T108L, L148P+T1 08R, L148P+T108V, L148P+S109N, L148P+S113N, L148P+T119C, L148P+T119K, L148P+Y125F, L148P+Y125H, L148P+Y125K, L14 8P+Y125M, L148P+Y125Y, L148P+A126G, L148P+A126S, L148P+T128C, L148P+N130D, L148P+F131A, L148P+F131I, L148P+F131 L, L148P+F131P, L148P+F131S, L148P+F131T, L148P+F131W, L148P+K135E, L148P+Y138S, L148P+Y138L, L148P+K142R, L148P +K144A, L148P+K144L, L148P+V150E, L148P+V150P, L148P+S152D, L148P+S152G, L148P+S152H, L148P+S152K, L148P+S152N,L148P+S152P、L148P+V153S、L148P+F154L、L148P+F154K、L148P+A156E、L148P+F166I、L148P+F166P、L148P+F166S、L148P+F166T、L148P+F166V、L148P+K168E、L148P+A170E、L148P+A170R、L148P+Y172A、L148P+Y172C、L148P+Y172G、L148P+Y172L、L148P+Y172N、L148P+Y172Q、L148P+Y172R、L148P+V174D、L148P+V174G、L148P+V174L、L148P+V174M、L148P+V174N、L148P+V174Q、L148P+V174S、L148P+V174T、L148P+L175A、L148P+L175E、L148P+L175G、L148P+L175K、L148P+L175N、L148P+L175S、L148P+L175T、L148P+I205A、L148P+I205V、L148P+L206I、L148P+P19K+M107G、L148P+P19M+F166P、L148P+Q41G+T119K、L148P+Q41D+A156E、L148P+Q41N+K96E、L148P+Q41T+T51W、L148P+Q41T+N81G、L148P+Q41T+T108R、L148P+Q41T+L175K、L148P+T51F+F166I、L148P+T51W+V174S、L148P+T51Y+F166P、L148P+R56K+A126S、L148P+T62C+M107H、L148P+I69M+K135E、L148P+S70G+Y125H、L148P+I73A+S152N、L148P+I73A+M107Y、L148P+I73G+V98Q、L148P+T74Q+I94N、L148P+Q78A+V174S、L148P+Q78K+L175G、L148P+S79T+A93S、L148P+Y80A+F154L、L148P+Y80P+Y172A、L148P+Y80T+L206I、L148P+Q83G+S152D、L148P+V98E+V153S、L148P+V98L+V174L、L148P+V98M+F131I、L148P+V98H+K168E、L148P+V98S+Y172Q、L148P+M107S+S152K、L148P+T108L+V174D、L148P+T108V+L175K、L148P+S113N+S152H、L148P+T119C+S152G、L148P+Y125M+Y172L、L148P+A126G+Y172G、L148P+T128C+V174N、L148P+N130D+V150E、L148P+F131L+I205V、L148P+F131P+Y172C、L148P+F131W+L175S、L148P+Y138S+V174Q、L148P+F166S+V174M、L148P+F166V+L175E、L148P+P19K+T108V+Y125Y、L148P+P19M+M107N+F166P、L148P+Q41D+M107G+S152P、L148P+Q41N+Y80P+K144A、L148P+Q41T+M107H+V174L、L148P+Q41T+L175K+I73A、L148P+T51W+Y80P+V174S、L148P+T51Y+F131T+V174G、L148P+R56K+Q78A+F166S、L148P+T62C+Y125K+V150P、L148P+I73A+T108L+F131S、L148P+I73G+K96P+S152H、L148P+I73L+V98M+F166P、L148P+V82A+A93S+F131L、L148P+I69M+A156E+L175E、L148P+S70G+S113N+Y172R、L148P+S79T+A126G+S152D、L148P+I94A+F131P+V174M、L148P+I94L+Y138L+V174T、L148P+I94N+N130D+L206I、L148P+K96E+F131W+F166I、L148P+V98H+T119K+F166T、L148P+V98Q+Y138S+A170E、L148P+V98L+K135E+V174L、L148P+M107E+S152N+L175K、L148P+T108R+A126S+K168E、L148P+S109N+F131I+F154L、L148P+T119C+Y125H+K135E、L148P+F131A+K142R+V174S、L148P+S152G+F166V+I205V、 L148P+P19K+K96P+V150P+A126S、L148P+P19M+M107N+F166P+V174T、L148P+Q41D+T108L+K144A+V153S、L148P+Q41G+T51Y+S113N+F166I、L148P+Q41N+T51F+A93S+F131A、L148P+Q41N+Y80A+S152G+L175E、L148P+Q41T+Y80A+V150E+Y172A、L148P+Q41T+I73A+S113N+L175K、L148P+T51W+Y80P+Y125M+V174S、L148P+T51Y+I73A+Y125K+L142R、L148P+R56K+M107H+A126G+L206I、L148P+T62C+V98Q+M107G+S152P、L148P+I69M+F131W+L175K+L206I、L148P+I73G+F131L+S152K+Y172C、L148P+I73L+I94A+T119C+S152D、L148P+T74Q+V98S+T108I+I205V、L148P+Q78A+S152P+F166V+L175G、L148P+Q78K+I94L+L175E+L206I、L148P+S79T+K96E+Y138L+V174M、L148P+Y80T+M107S+S152H+Y172R、L148P+N81G+Y125Y+F131T+A156E、L148P+Q83G+M107Y+A126S+K168E、L148P+V98L+T119K+K135E+V174L、L148P+V98T+M107E+Y138S+I205A、L148P+Y125H+F131I+S152H+L175T、L148P+P19M+T51Y+M107N+F166P+V174T、L148P+Q41T+I73A+A126S+L175K+S113N、L148P+T51W+Y80P+Y125M+S152P+V174S、L148P+I73L+Y80T+M107S+S152H+Y172R、L148P+Q78A+S152P+F166V+L175G+I205V、L148P+V98L+T119K+K135E+F166P+V174L、L148P+P19M+T51Y+M107N+S152H+F166P+V174T、L148P+Q41D+V98L+T119K+K135E+F166P+V174L、L148P+Q41T+I73A+A126S+K144A+L175K+S113N、L148P+T51W+Y80P+M107N+S113N+F166V+I205A、L148P+T51W+Y80P+Y125M+S152P+V174S+L206I、L148P+R56K+I73L+Y80T+M107S+S152H+Y172R、L148P+Q78A+M107S+S152P+F166V+L175G+I205V、L148P+P19M+T51Y+I73A+M107N+S152H+F166P+V174T、L148P+Q41D+V98L+T119K+K135E+S152H+F166P+V174L、L148P+Q41T+I73A+S113N+A126S+K144A+L175K+I205V、L148P+T51W+Y80P+M107N+S113N+F166V+L175G+I205A、L148P+T51W+Y80P+V98L+Y125M+S152P+V174S+L206I、L148P+R56K+I73L+Y80T+M107S+T119K+S152H+Y172R、L148P+Q78A+M107S+Y125H+S152P+F166V+L175G+I205V、L148P+P19M+Q41T+I73A+S113N+A126S+K144A+L175K+I205V、L148P+P19M+T51Y+I73A+V98T+M107N+S152H+F166P+V174T、L148P+Q41D+V98L+T119K+K135E+S152H+F166P+V174L+I205V、L148P+T51W+Y80P+V98T+M107N+S113N+F166V+L175G+I205A、L148P+T51W+Y80P+V98L+Y125M+S152P+F166V+V174S+L206I、L148P+R56K+I73L+Y80T+M107S+T119K+S152H+Y172R+L206I、L148P+Q78A+M107S+S113N+Y125H+S152P+F166V+L175G+I205V、L148P+P19M+Q41T+I69M+I73A+S113N+A126S+K144L+L175K+I205V、L148P+P19M+T51Y+I73A+V98T+M107N+S152H+F166P+V174T+L206I、L148P+Q41D+S70G+V98L+T119K+K135E+S152H+F166P+V174L+I205V, L148P+T51W+I69M+Y80P+V98T+M107N+S113N+F166V+L175G+I205A, L148P+T51W+Y80P+V98L+Y125M+K144L+S152 P+F166V+V174S+L206I, L148P+R56K+I73L+Y80T+M107S+T119K+K144A+S152H+Y172R+L206I, L148P+Q78A+V98L+M 107S+S113N+Y125H+S152P+F166V+L175G+I205V, L148P+P19M+Q41T+I69M+I73A+S113N+Y125F+A126S+T128C+N1 30D+K144L+L175K+I205V, L148P+P19M+Q41D+T51Y+I73A+V98T+M107N+A126G+K135E+S152H+F166P+V174T+L206I , L148P+Q41D+S70G+V98L+T108L+T119K+Y125F+K135E+K144A+S152H+F166P+V174L+I205V, L148P+T51W+I69M+Y 80P+V98T+M107N+S113N+N130D+K142R+F154L+F166V+L175G+I205A, L148P+T51W+T74Q+Y80P+V98L+T108I+S113N +Y125M+K144L+S152P+F166V+V174S+L206I、L148P+R56K+I73L+Y80T+V98M+M107S+T119K+Y125F+F131S+K144A+S152H+Y172R+L206I、or L148P+I69M+Q78A+V98L+M107S+S113N+Y125H+N130D+K144A+S152P+F166V+L175G+I205V。

[0034] In a preferred embodiment, the purine nucleoside phosphorylase mutant comprises a protein having 80% or more, 85% or more, more preferably 95% or more, and even more preferably 99% or more homology to the amino acid sequence defined in (a), and having purine nucleoside phosphorylase activity; preferably, the purine nucleoside phosphorylase mutant comprises the protein shown in SEQ ID NO: 5, or a protein having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, 99.8% or 99.9% or more homology to the protein shown in SEQ ID NO: 5 and having purine nucleoside phosphorylase activity.

[0035] The above amino acid mutations were all experimentally explored in the examples of this application. Compared with the parent having the amino acid sequence shown in SEQ ID NO: 3, they all have the activity of catalyzing the binding of phosphorylated 2′-fluoropentose and substrate base to prepare 2′-fluoronucleosides. The above mutation sites are all mutations made around the amino acid active site. Such mutations can improve the mutant's ability to bind to the substrate and / or catalytic ability. For mutations away from the active site, the catalytic ability of the enzyme is less affected, so it is possible to obtain a protein with 80% or more homology to the above amino acid sequence and the same catalytic activity.

[0036] The term "identity" used herein refers to the "homology" between amino acid sequences, that is, the total ratio of identical amino acid residues in an amino acid sequence. The homology of amino acid sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

[0037] Proteins with 70%, 75%, 80%, 85%, 90%, 95%, or more than 99% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more, or even 99.9% or more) homology and the same function, whose active sites, active pockets, active mechanisms, protein structures, etc. are most likely the same as those of the protein provided by sequence (a), are homologous proteins obtained by amino acid mutations.

[0038] As used herein, amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0039] Substitution and replacement rules generally refer to the fact that amino acids with similar properties will have similar effects when substituted with each other. For example, conservative amino acid substitutions may occur in the homologous proteins mentioned above. "Conservative amino acid substitutions" include but are not limited to:

[0040] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0041] Substitution of bulky hydrophobic amino acids (Phe, Tyr, Trp) with other bulky hydrophobic amino acids;

[0042] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0043] Amino acids with polar and uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar and uncharged side chains.

[0044] Those skilled in the art may also perform conservative substitutions on amino acids according to amino acid substitution rules well known to those skilled in the art, such as the "blosum62 scoring matrix" in the prior art.

[0045] The "AlphaFold2-Multimer" used in this application is a publicly available artificial intelligence model that can predict the conformation of protein complexes. Its predictions of protein 3D structures are very close to those observed in real experiments using instruments such as cryo-electron microscopy. This allows for the acquisition of relatively realistic protein structures, thus guiding the study of protein structure and activity.

[0046] In a second typical embodiment of the present application, a DNA molecule is provided, which encodes the above-mentioned purine nucleoside phosphorylase mutant.

[0047] Preferably, the DNA molecule comprises the nucleotide sequence shown in SEQ ID NO:6.

[0048] In a third typical embodiment of the present application, a recombinant plasmid is provided, wherein the recombinant plasmid is connected to the above-mentioned DNA molecule.

[0049] The DNA can encode the purine nucleoside phosphorylase mutant and can be linked to a recombinant plasmid to form a circular DNA. Both the DNA and the recombinant plasmid can be transcribed and translated under the action of RNA polymerase, ribosomes, tRNA, etc. to obtain the purine nucleoside phosphorylase mutant.

[0050] In a fourth typical embodiment of the present application, a host cell is provided, wherein the host cell contains the above-mentioned DNA molecule or the above-mentioned recombinant plasmid.

[0051] The host cells can be used to replicate recombinant plasmids and transcribe and translate DNA molecules carried on the recombinant plasmids, thereby obtaining a large number of purine nucleoside phosphorylase mutants. Using existing technologies, the host cells can be fragmented for protein purification, followed by crude enzyme catalysis after fragmentation, or other methods to obtain purine nucleoside phosphorylase mutants, and subsequently catalyze substrate nucleosides. The host cells are non-plant-derived.

[0052] In a preferred embodiment, the host cell comprises a prokaryotic cell; preferably, the prokaryotic cell comprises Escherichia coli.

[0053] In a fifth typical embodiment of the present application, a method for preparing 2′-fluoronucleosides is provided, which comprises: using the above-mentioned purine nucleoside phosphorylase to catalyze the phosphorylation of 2′-fluoropentose and the binding of the substrate base to prepare 2′-fluoronucleosides.

[0054] Using the above preparation method and the above purine nucleoside phosphorylase (PNP), the phosphate group on the phosphorylated 2′-fluoropentose is replaced with a substrate base, thereby obtaining a 2′-fluoronucleoside bound to the substrate base. The above reaction (Step 2) is shown in Figure 1. The structures of the substrate base and the phosphorylated 2′-fluoropentose include, but are not limited to, the structure in Figure 1.

[0055] In a preferred embodiment, the preparation method of phosphorylated 2′-fluoropentose comprises: using nucleoside phosphorylase to catalyze the cleavage of a substrate nucleoside having a 2′-fluoropentose structure into phosphorylated 2′-fluoropentose and a free base; preferably, the nucleoside phosphorylase includes but is not limited to pyrimidine nucleoside phosphorylase, thymidine phosphorylase, uridine phosphorylase or 5′-methylthioadenosine phosphorylase.

[0056] In the above preparation method, the nucleoside phosphorylase includes, but is not limited to, the pyrimidine nucleoside phosphorylase from Thermus thermophilus or a mutant thereof (having 80% or greater homology) as shown in SEQ ID NO: 1, and also includes nucleoside phosphorylases (NP) from other sources. The above reaction (Step 1) is shown in FIG2 , and the structure of the substrate nucleoside having a 2′-fluoropentose includes, but is not limited to, the structure shown in FIG2 .

[0057] The above-mentioned nucleoside phosphorylase includes, but is not limited to, pyrimidine nucleoside phosphorylase (PyNP), thymidine phosphorylase (TP), uridine phosphorylase (UP), or 5′-methylthioadenosine phosphorylase (MTAP, 5′-methylthioadenosine phosphorylase, EC2.4.2.28), and also includes, but is not limited to, enzymes with the same function in other prior art. In the process of preparing phosphorylated 2′-fluoropentose, the phosphorylase is selected according to the type of substrate having the substrate nucleoside of 2′-fluoropentose.

[0058] Different phosphorylases act on different substrate structures. In this preparation method, the type of nucleoside phosphorylase used can be flexibly selected based on the affinity of the nucleoside phosphorylase and the substrate known in the prior art (Kamel S, Thiele I, Neubauer P, et al. Thermophilic nucleoside phosphorylases: Their properties, characteristics and applications [J]. Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics, 2019, 1868 (2): 140304.). The above-mentioned reaction of catalyzing the substrate nucleoside having 2'-fluoropentose into phosphorylated 2'-fluoropentose can be carried out simultaneously with the reaction of catalyzing the phosphorylated 2'-fluoropentose into 2'-fluoronucleoside at the same time, in the same container, and under the same reaction conditions. Therefore, the above-mentioned preparation method can achieve a one-pot preparation of 2'-fluoronucleosides. The above preparation method has high conversion rate and efficiency, can significantly reduce the amount of enzyme used, thereby reducing production costs, and is suitable for industrial scale-up production.

[0059] In a preferred embodiment, the substrate nucleoside includes but is not limited to 2′-fluoro-2′-deoxyuridine, 2′-fluoro-2′-deoxycytidine or 2′-deoxy-2′-fluorothymidine or 2′-fluoro nucleosides with other modifications; preferably, the substrate nucleoside includes 2′-fluoro-2′-deoxyuridine.

[0060] In a preferred embodiment, the substrate base includes modified or unmodified adenine, guanine, thymine, cytosine, or modified uracil; preferably, the substrate base includes adenine, guanine or 2-aminoadenine.

[0061] In a preferred embodiment, the 2′-fluoro nucleoside includes a nucleoside containing a 2′-fluoro-2′-deoxypentose structure. Preferably, the 2′-fluoro nucleoside includes but is not limited to 2′-fluoro-2′-deoxyadenosine, 2′-fluoro-2′-deoxy-2-aminoadenosine, 2′-fluoro-2′-deoxyguanosine or 2′-fluoro-2′-deoxycytidine; alternatively, the 2′-fluoro nucleoside is selected from a 2′-fluoro-2′-deoxynucleoside having a substituent on the base structure.

[0062] The substituents on the above-mentioned base structure or the modification of the above-mentioned substrate base include, but are not limited to, the introduction of a methyl group (-CH3), a carbamoyl group (-CH2NH2), a sugar group, a phosphonic acid group (-PO4), a sulfhydryl group (-SH), a nitro group (-NO2), a hydroxyl group (-OH) or deamination (-NH2) on the base structure, thereby achieving modifications such as methylation, carbamoylation, glycosylation, phosphorylation, thiolation, nitration, oxidation or deamination of the nucleoside.

[0063] Methylation: The modification of a nucleotide base by the addition of a methyl group. For example, in DNA, cytosine can be methylated to form 5-methylcytosine.

[0064] Aminomethylation: A modification of a nucleotide base by the addition of a carbamyl group. This modification can occur on cytosine and adenine bases.

[0065] Glycosylation: The modification of a nucleotide base by the addition of a sugar moiety. For example, adenosine can be glycosylated.

[0066] Phosphorylation: The modification of a nucleotide base by the addition of a phosphate group. Phosphorylation can occur on cytosine, adenine, and guanine bases.

[0067] Sulfuration: A modification of a nucleotide base by the addition of a sulfur group. Sulfuration can occur on cytosine, adenine, and guanine bases.

[0068] Nitration: The modification of a nucleotide base by the addition of a nitrate group. This modification can occur on both cytosine and adenine bases.

[0069] Oxidation: The modification of a nucleotide base by the addition of an oxygen group. For example, in DNA, guanine is oxidized to form 8-oxoguanine (8-oxoG).

[0070] Deamination: A modification of a nucleotide base by the loss of an amino group. This modification can occur on cytosine, adenine, and guanine bases.

[0071] The beneficial effects of the present application will be further explained in detail below with reference to specific embodiments.

[0072] Example 1 Construction of TtPyNP expression strain

[0073] The protein sequence of a pyrimidine nucleoside phosphorylase from Thermus thermophilus was obtained from NCBI (Accession No. BAD71594), as shown in SEQ ID NO: 1. After codon optimization, the DNA sequence encoding the enzyme was obtained, as shown in SEQ ID NO: 2, and cloned into the expression vector pET28a(+) to generate the plasmid pET28a-TtPyNP. The pET28a-TtPyNP plasmid was transformed into competent Escherichia coli BL21(DE3) to generate a monoclonal strain.

[0074] Example 2 Construction of GsPNP expression strain

[0075] The protein sequence of purine nucleoside phosphorylase from Geobacillus stearothermophilus was obtained from NCBI (Accession No. BAA13510), as shown in SEQ ID NO: 3. After codon optimization, the DNA sequence encoding the enzyme was obtained, as shown in SEQ ID NO: 4, and cloned into the expression vector pET28a(+) to generate the plasmid pET28a-GsPNP. The pET28a-GsPNP plasmid was transformed into competent Escherichia coli BL21(DE3) to obtain a monoclonal strain.

[0076] Example 3 Enzyme Solution Preparation

[0077] Protein expression: The wild-type and mutant seed liquids were inoculated into a test tube containing 5 mL of Luria-Bertani liquid medium (containing 50 μg / mL kanamycin), cultured at 37°C for 16 h, and then inoculated into a 2 L shake flask containing 500 mL of Luria-Bertani liquid medium at a 1% inoculum size and cultured at 37°C until the OD 600 When the pH value was 0.6, IPTG was added to a final concentration of 0.1 M to induce expression at 20°C for 18 h. The bacterial culture was centrifuged at 7000 rpm for 10 min to collect the cells and stored at -20°C for later use.

[0078] Preparation of enzyme solution: Weigh 0.1 g of the bacterial sludge prepared above, resuspend it in 1 mL of 2 mM potassium phosphate buffer with a pH of 7.5, shake and mix, and then disrupt the bacterial suspension using an ultrasonic disruptor (power: 30%, time: 5 min).

[0079] HPLC detection method: After the reaction is completed, an equal volume of DMSO is added to terminate the reaction. The sample is diluted 2-fold and then analyzed by HPLC. The column used is an Atlantis T3 column (4.6 mm x 150 mm). The mobile phase is 0.1% TFA / methanol at a flow rate of 1 mL / min at 40°C. The detection wavelength is 254 nm, and the detection time is 15 min.

[0080] Example 4: Use of GsPNP mutants in the synthesis of 2′-fluoro-2′-deoxyadenosine

[0081] Site-directed and multi-point mutagenesis were performed using the GsPNP template shown in SEQ ID NO: 3. Reaction conditions: 100 mM 2′-fluoro-2′-deoxyuridine, 150 mM adenine, 807 μL of crude TtPyNP enzyme, 1291 μL of crude GsPNP mutant enzyme, 2 mM PBS (pH 7.0), 3 mL, 60°C, 200 rpm, 20 h. The conversion rate of 2′-fluoro-2′-deoxyuridine is shown in Table 1.

[0082] Table 1

[0083] Note: In the above table, - represents a conversion rate of 0.1-10% (excluding the endpoint value of 10%), + represents a conversion rate of 10-20% (excluding the endpoint value of 20%), ++ represents a conversion rate of 20-30% (excluding the endpoint value of 30%), +++ represents a conversion rate of 30-40% (excluding the endpoint value of 40%), ++++ represents a conversion rate of 40-50% (excluding the endpoint value of 50%), +++++ represents a conversion rate of 50-70%, and ++++++ represents a conversion rate>70%;

[0084] # represents a conversion rate of 0.1-5% (excluding the endpoint value of 5%), * represents a conversion rate of 5-10% (excluding the endpoint value of 10%), ** represents a conversion rate of 10-20% (excluding the endpoint value of 20%), *** represents a conversion rate of 20-30% (excluding the endpoint value of 30%), **** represents a conversion rate of 30-40% (excluding the endpoint value of 40%), ***** represents a conversion rate of 40-50%, and ****** represents a conversion rate >50%.

[0085] In the above Step 1 + Step 2 reaction, the conversion rate is calculated based on the conversion rate of the substrate with low concentration. Since the amount of 2′-fluoro-2′-deoxyuridine is small, the enzyme activity is evaluated by evaluating the conversion rate of Step 1.

[0086] In the above Step 1+Step 2 reaction, since both reactions are reversible, as the enzyme activity of the GsPNP mutant increases, the reaction conversion rate of Step 2 increases, and the concentration of the reaction intermediate phosphorylated 2′-fluoropentose is low, thereby promoting the reaction of Step 1 to proceed in the forward direction and achieving a corresponding increase in the reaction conversion rate of Step 1.

[0087] Example 5 Reaction Amplification

[0088] A 2 mL reaction system was prepared in 20 mM phosphate buffer (pH 7.5) containing 200 mM 2′-fluoro-2′-deoxyuridine, 250 mM adenine, 430 μL of a 3x concentrated crude TtPyNP enzyme solution, and 258 μL of a 3x concentrated crude L148P+I69M+Q78A+V98L+M107S+S113N+Y125H+N130D+K144A+S152P+F166V+L175G+I205V enzyme solution. The reaction was incubated at 60°C for 159 h. After completion of the reaction, an equal volume of DMSO was added for dissolution, the solution was diluted 50-fold, and then analyzed by HPLC. The results are shown in Figure 3. The conversion rate of 2′-fluoro-2′-deoxyuridine (2′-Deoxy-2′-fluorouridine) in Step 1 was greater than 81%, the conversion rate of adenine in Step 2 was greater than 63%, and the yield of 2′-fluoro-2′-deoxyadenosine was 42.63 g / L.

[0089] From the above description, it can be seen that the above-mentioned embodiment of the present invention achieves the following technical effects: using the above-mentioned purine nucleoside phosphorylase mutant, it is possible to efficiently catalyze the phosphorylation of 2'-fluoro pentose and the combination of substrate bases to prepare 2'-fluoro nucleosides. Further, using nucleoside phosphorylases such as pyrimidine nucleoside phosphorylase, thymidine phosphorylase, uridine phosphorylase or 5'-methylthioadenosine phosphorylase in the prior art, it is possible to catalyze the cleavage of substrate nucleosides with 2'-fluoro pentose into phosphorylated 2'-fluoro pentose and free bases, providing reaction substrates for the catalytic reaction of the above-mentioned purine nucleoside phosphorylase mutant. Therefore, the above-mentioned two-step catalytic reaction is carried out simultaneously at the same time, in the same container, and under the same reaction conditions, and 2'-fluoro nucleosides are prepared using a one-pot method. Compared with the wild-type enzyme, the above-mentioned purine nucleoside phosphorylase mutant has better enzyme activity, and the conversion rate and efficiency of preparing 2'-fluoro nucleoside products are higher, which can significantly reduce the amount of enzyme used, thereby reducing production costs and being suitable for industrial scale-up production.

[0090] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A purine nucleoside phosphorylase mutant, characterized in that: include: (a) A protein having a mutation based on the wild-type purine nucleoside phosphorylase GsPNP shown in SEQ ID NO: 3, wherein the mutation is selected from any one or more of the following mutations: L148, P19, Q41, T51, R56, T62, I69, S70, I73, T74, Q78, S79, Y80, N81, V82, Q83, A93, I94, K96, V98, K99 , M107, T108, S109, S113, T119, Y125, A126, T128, N130, F131, K135, Y138, L142, K144, V150, S152, V153, F154, A156, F166, K168, A170, Y172, V174, L175, I205 or L206; or (b) A protein having 70% or more homology with the amino acid sequence defined in (a) and having purine nucleoside phosphorylase activity.

2. The purine nucleoside phosphorylase mutant according to claim 1, characterized in that In (a), the mutation is selected from any one or more of the following mutations: L148P, P19M, P19K, Q41D, Q41G, Q41N, Q41T, T51F, T51W, T51Y, R56K, T62C, I69M, S70G, I73A, I73G, I73L, T74Q, Q78 A. Q78K, S79T, Y80A, Y80P, Y80T, N81G, V82A, Q83G, A93S, I94A, I94L, I94N, K96E, K96P, K96S, V98E, V98H, V98L, V98 M, V98Q, V98S, V98T, K99S, M107E, M107G, M107H, M107N, M107S, M107Y, T108I, T108L, T108R, T108V, S109N, S113N, T119C, T119K, Y125F, Y125H, Y125K, Y125M, Y125Y, A126G, A126S, T128C, N130D, F131A, F131I, F131L, F131P, F131S , F131T, F131W, K135E, Y138S, Y138L, K142R, K144A, K144L, L148A, L148G, L148Q, L148S, L148Y, V150E, V150P, S15 2D, S152G, S152H, S152K, S152N, S152P, V153S, F154L, F154K, A156E, F166I, F166P, F166S, F166T, F166V, K168E, A1 70E, A170R, Y172A, Y172C, Y172G, Y172L, Y172N, Y172Q, Y172R, V174D, V174G, V174L, V174M, V174N, V174Q, V174S, V174T, L175A, L175E, L175G, L175K, L175N, L175S, L175T, I205A, I205V or L206I, wherein the letters before the numbers represent the original amino acids and the letters after the numbers represent the mutated amino acids.

3. The purine nucleoside phosphorylase mutant according to claim 1, characterized in that The mutation includes any of the following amino acid mutations: L148P+P19M、L148P+P19K、L148P+Q41D、L148P+Q41G、L148P+Q41N、L148P+Q41T、L148P+T51F、L148P+T51W、L148P+T51Y、L148P+R56K、L148P+T62C、L148P+I69M、L148P+S70G、L148P+I73A、L148P+I73G、L148P+I73L、L148P+T74Q、L148P+Q78A、L148P+Q78K、L148P+S79T、L148P+Y80A、L148P+Y80P、L148P+Y80T、L148P+N81G、L148P+V82A、L148P+Q83G、L148P+A93S、L148P+I94A、L148P+I94L、L148P+I94N、L148P+K96E、L148P+K96P、L148P+K96S、L148P+V98E、L148P+V98H、L148P+V98L、L148P+V98M、L148P+V98Q、L148P+V98S、L148P+V98T、L148P+K99S、L148P+M107E、L148P+M107G、L148P+M107H、L148P+M107N、L148P+M107S、L148P+M107Y、 L148P+T108I、L148P+T108L、L148P+T108R、L148P+T108V、L148P+S109N、L148P+S113N、L148P+T119C、L148P+T119K、L148P+Y125F、L148P+Y125H、L148P+Y125K、L148P+Y125M、L148P+Y125Y、L148P+A126G、L148P+A126S、L148P+T128C、L148P+N130D、L148P+F131A、L148P+F131I、L148P+F131L、L148P+F131P、L148P+F131S、L148P+F131T、L148P+F131W、L148P+K135E、L148P+Y138S、L148P+Y138L、L148P+K142R、L148P+K144A、L148P+K144L、L148P+V150E、L148P+V150P、L148P+S152D、L148P+S152G、L148P+S152H、L148P+S152K、L148P+S152N、L148P+S152P、L148P+V153S、L148P+F154L、L148P+F154K、L148P+A156E、L148P+F166I、L148P+F166P、L148P+F166S、L148P+F166T、L148P+F166V、L148P+K168E、L148P+A170E、L148P+A170R、L148P+Y172A、L148P+Y172C、L148P+Y172G、L148P+Y172L、L148P+Y172N、L148P+Y172Q、L148P+Y172R、L148P+V174D、L148P+V174G、L148P+V174L、L148P+V174M、L148P+V174N、L148P+V174Q、L148P+V174S、L148P+V174T、L148P+L175A、L148P+L175E、L148P+L175G、L148P+L175K、L148P+L175N、L148P+L175S、L148P+L175T、L148P+I205A、L148P+I205V、L148P+L206I、L148P+P19K+M107G、L148P+P19M+F166P、L148P+Q41G+T119K、L148P+Q41D+A156E、L148P+Q41N+K96E、L148P+Q41T+T51W、L148P+Q41T+N81G、L148P+Q41T+T108R、L148P+Q41T+L175K、L148P+T51F+F166I、L148P+T51W+V174S、L148P+T51Y+F166P、L148P+R56K+A126S、L148P+T62C+M107H、L148P+I69M+K135E、L148P+S70G+Y125H、L148P+I73A+S152N、L148P+I73A+M107Y、L148P+I73G+V98Q、L148P+T74Q+I94N、L148P+Q78A+V174S、L148P+Q78K+L175G、L148P+S79T+A93S、L148P+Y80A+F154L、L148P+Y80P+Y172A、L148P+Y80T+L206I、L148P+Q83G+S152D、L148P+V98E+V153S、L148P+V98L+V174L、L148P+V98M+F131I、L148P+V98H+K168E、L148P+V98S+Y172Q、L148P+M107S+S152K、L148P+T108L+V174D、L148P+T108V+L175K、L148P+S113N+S152H、L148P+T119C+S152G、L148P+Y125M+Y172L、L148P+A126G+Y172G、L148P+T128C+V174N、L148P+N130D+V150E、L148P+F131L+I205V、L148P+F131P+Y172C、L148P+F131W+L175S、L148P+Y138S+V174Q、L148P+F166S+V174M、L148P+F166V+L175E、L148P+P19K+T108V+Y125Y、L148P+P19M+M107N+F166P、L148P+Q41D+M107G+S152P、L148P+Q41N+Y80P+K144A、L148P+Q41T+M107H+V174L、L148P+Q41T+L175K+I73A、L148P+T51W+Y80P+V174S、L148P+T51Y+F131T+V174G、L148P+R56K+Q78A+F166S、L148P+T62C+Y125K+V150P、L148P+I73A+T108L+F131S、L148P+I73G+K96P+S152H、L148P+I73L+V98M+F166P、L148P+V82A+A93S+F131L、L148P+I69M+A156E+L175E、L148P+S70G+S113N+Y172R、L148P+S79T+A126G+S152D、L148P+I94A+F131P+V174M、L148P+I94L+Y138L+V174T、L148P+I94N+N130D+L206I、L148P+K96E+F131W+F166I、L148P+V98H+T119K+F166T、L148P+V98Q+Y138S+A170E、L148P+V98L+K135E+V174L、L148P+M107E+S152N+L175K、L148P+T108R+A126S+K168E、 L148P+S109N+F131I+F154L、L148P+T119C+Y125H+K135E、L148P+F131A+K142R+V174S、L148P+S152G+F166V+I205V、L148P+P19K+K96P+V150P+A126S、L148P+P19M+M107N+F166P+V174T、L148P+Q41D+T108L+K144A+V153S、L148P+Q41G+T51Y+S113N+F166I、L148P+Q41N+T51F+A93S+F131A、L148P+Q41N+Y80A+S152G+L175E、L148P+Q41T+Y80A+V150E+Y172A、L148P+Q41T+I73A+S113N+L175K、L148P+T51W+Y80P+Y125M+V174S、L148P+T51Y+I73A+Y125K+L142R、L148P+R56K+M107H+A126G+L206I、L148P+T62C+V98Q+M107G+S152P、L148P+I69M+F131W+L175K+L206I、L148P+I73G+F131L+S152K+Y172C、L148P+I73L+I94A+T119C+S152D、L148P+T74Q+V98S+T108I+I205V、L148P+Q78A+S152P+F166V+L175G、L148P+Q78K+I94L+L175E+L206I、L148P+S79T+K96E+Y138L+V174M、L148P+Y80T+M107S+S152H+Y172R、L148P+N81G+Y125Y+F131T+A156E、L148P+Q83G+M107Y+A126S+K168E、L148P+V98L+T119K+K135E+V174L、L148P+V98T+M107E+Y138S+I205A、L148P+Y125H+F131I+S152H+L175T、L148P+P19M+T51Y+M107N+F166P+V174T、L148P+Q41T+I73A+A126S+L175K+S113N、L148P+T51W+Y80P+Y125M+S152P+V174S、L148P+I73L+Y80T+M107S+S152H+Y172R、L148P+Q78A+S152P+F166V+L175G+I205V、L148P+V98L+T119K+K135E+F166P+V174L、L148P+P19M+T51Y+M107N+S152H+F166P+V174T、L148P+Q41D+V98L+T119K+K135E+F166P+V174L、L148P+Q41T+I73A+A126S+K144A+L175K+S113N、L148P+T51W+Y80P+M107N+S113N+F166V+I205A、L148P+T51W+Y80P+Y125M+S152P+V174S+L206I、L148P+R56K+I73L+Y80T+M107S+S152H+Y172R、L148P+Q78A+M107S+S152P+F166V+L175G+I205V、L148P+P19M+T51Y+I73A+M107N+S152H+F166P+V174T、L148P+Q41D+V98L+T119K+K135E+S152H+F166P+V174L、L148P+Q41T+I73A+S113N+A126S+K144A+L175K+I205V、L148P+T51W+Y80P+M107N+S113N+F166V+L175G+I205A、L148P+T51W+Y80P+V98L+Y125M+S152P+V174S+L206I、L148P+R56K+I73L+Y80T+M107S+T119K+S152H+Y172R、L148P+Q78A+M107S+Y125H+S152P+F166V+L175G+I205V、L148P+P19M+Q41T+I73A+S113N+A126S+K144A+L175K+I205V、L148P+P19M+T51Y+I73A+V98T+M107N+S152H+F166P+V174T、L148P+Q41D+V98L+T119K+K135E+S152H+F166P+V174L+I205V、L148P+T51W+Y80P+V98T+M107N+S113N+F166V+L175G+I205A、L148P+T51W+Y80P+V98L+Y125M+S152P+F166V+V174S+L206I、L148P+R56K+I73L+Y80T+M107S+T119K+S152H+Y172R+L206I、L148P+Q78A+M107S+S113N+Y125H+S152P+F166V+L175G+I205V、L148P+P19M+Q41T+I69M+I73A+S113N+A126S+K144L+L175K+I205V、 L148P+P19M+T51Y+I73A+V98T+M107N+S152H+F166P+V174T+L206I, L148P+Q41D+S70G+V98L+T119K+K135E+S152H+F166P+V174L+ I205V, L148P+T51W+I69M+Y80P+V98T+M107N+S113N+F166V+L175G+I205A, L148P+T51W+Y80P+V98L+Y125M+K144L+S152P+F166V+V 174S+L206I, L148P+R56K+I73L+Y80T+M107S+T119K+K144A+S152H+Y172R+L206I, L148P+Q78A+V98L+M107S+S113N+Y125H+S152P +F166V+L175G+I205V, L148P+P19M+Q41T+I69M+I73A+S113N+Y125F+A126S+T128C+N130D+K144L+L175K+I205V, L148P+P19M+Q41D +T51Y+I73A+V98T+M107N+A126G+K135E+S152H+F166P+V174T+L206I, L148P+Q41D+S70G+V98L+T108L+T119K+Y125F+K135E+K144 A+S152H+F166P+V174L+I205V, L148P+T51W+I69M+Y80P+V98T+M107N+S113N+N130D+K142R+F154L+F166V+L175G+I205A, L148P+T5 1W+T74Q+Y80P+V98L+T108I+S113N+Y125M+K144L+S152P+F166V+V174S+L206I, L148P+R56K+I73L+Y80T+V98M+M107S+T119K+Y125F+F131S+K144A+S152H+Y172R+L206I, or L148P+I69M+Q78A+V98L+M107S+S113N+Y125H+N130D+K144A+S152P+F166V+L175G+I205V.

4. The purine nucleoside phosphorylase mutant according to claim 1, characterized in that: The purine nucleoside phosphorylase mutant includes: a protein shown in SEQ ID NO: 5, or a protein having more than 80% homology with the protein shown in SEQ ID NO: 5 and having purine nucleoside phosphorylase activity.

5. A DNA molecule, characterized in that The DNA molecule encodes the purine nucleoside phosphorylase mutant according to any one of claims 1 to 4.

6. A recombinant plasmid, characterized in that: The recombinant plasmid is connected to the DNA molecule according to claim 5.

7. A host cell, characterized in that The host cell contains the DNA molecule according to claim 5 or the recombinant plasmid according to claim 6.

8. A method for preparing 2′-fluoronucleoside, characterized in that: The preparation method comprises: using the purine nucleoside phosphorylase according to any one of claims 1 to 4 to catalyze the phosphorylation of 2′-fluoropentose and the combination with the substrate base to prepare the 2′-fluoronucleoside.

9. The preparation method according to claim 8, characterized in that: The preparation method of the phosphorylated 2'-fluoropentose comprises: utilizing nucleoside phosphorylase to catalyze the cleavage of substrate nucleosides having a 2'-fluoropentose structure into the phosphorylated 2'-fluoropentose and free bases.

10. The preparation method according to claim 9, characterized in that: The nucleoside phosphorylase includes pyrimidine nucleoside phosphorylase, thymidine phosphorylase, uridine phosphorylase or 5'-methylthioadenosine phosphorylase.

11. The preparation method according to claim 9, characterized in that: The substrate nucleoside includes 2'-fluoro-2'-deoxyuridine, 2'-fluoro-2'-deoxycytidine or 2'-deoxy-2'-fluorothymidine.

12. The preparation method according to claim 8, characterized in that: The substrate bases include modified or unmodified adenine, guanine, thymine, cytosine, or modified uracil.

13. The preparation method according to claim 12, characterized in that: The substrate base includes adenine, guanine or 2-aminoadenine.

14. The preparation method according to any one of claims 8 to 13, characterized in that: The 2'-fluoro nucleosides include nucleosides containing a 2'-fluoro-2'-deoxypentose structure.

15. The preparation method according to claim 14, characterized in that: The 2′-fluoro nucleosides include 2′-fluoro-2′-deoxyadenosine, 2′-fluoro-2′-deoxy-2-aminoadenosine, 2′-fluoro-2′-deoxyguanosine or 2′-fluoro-2′-deoxycytidine; Alternatively, the 2′-fluoro nucleoside is selected from 2′-fluoro-2′-deoxynucleosides having a substituent on the base structure.

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