WO2024082546A1 - 阿糖类核苷一锅法生物合成的方法及组合物 - Google Patents

阿糖类核苷一锅法生物合成的方法及组合物 Download PDF

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WO2024082546A1
WO2024082546A1 PCT/CN2023/083184 CN2023083184W WO2024082546A1 WO 2024082546 A1 WO2024082546 A1 WO 2024082546A1 CN 2023083184 W CN2023083184 W CN 2023083184W WO 2024082546 A1 WO2024082546 A1 WO 2024082546A1
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nucleoside phosphorylase
phosphate
substrate
uridine
protein
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洪浩
詹姆斯•盖吉
肖毅
张娜
焦学成
李明记
李�瑞
丁是卯
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Asymchem Life Science Tianjin Co Ltd
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Priority to AU2023364566A priority patent/AU2023364566A1/en
Priority to KR1020257016020A priority patent/KR20250079035A/ko
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1077—Pentosyltransferases (2.4.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/38—Nucleosides
    • C12P19/40—Nucleosides having a condensed ring system containing a six-membered ring having two nitrogen atoms in the same ring, e.g. purine nucleosides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/38—Nucleosides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/02—Pentosyltransferases (2.4.2)
    • C12Y204/02001—Purine-nucleoside phosphorylase (2.4.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/02—Pentosyltransferases (2.4.2)
    • C12Y204/02002—Pyrimidine-nucleoside phosphorylase (2.4.2.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/02—Pentosyltransferases (2.4.2)
    • C12Y204/02003—Uridine phosphorylase (2.4.2.3)

Definitions

  • the invention relates to the field of enzyme catalysis, and in particular to a one-pot biosynthesis method and composition for arabinose nucleosides.
  • Arabinose nucleosides are nucleosides that replace deoxyribose with arabinose. After being incorporated into DNA, they can prevent DNA replication and thus affect cell division and proliferation. Therefore, they can be used as antiviral and anti-tumor drugs for the treatment of related diseases, such as herpes, tumors, AIDS, hepatitis B, etc.
  • arabinose nucleosides are mainly synthesized by chemical methods (CN1042939C, CN107556356A, CN1128270A, CN103467468A, CN107892707A) and biological methods (CN106929553A, JPH10286097A, CN105237602A).
  • the biological method uses enzymes expressed by microorganisms as catalysts to convert substrates into related products under mild conditions.
  • the operation steps are simple and the reaction conditions are relatively mild. It does not require the use of heavy metals, organic reagents and other substances that are harmful to the human body and the environment.
  • Patent CN106929553A discloses a two-step method for synthesizing adenosine, which can convert uridine arabinoside into arabinose-1-phosphate using uridine phosphorylase, and then synthesize adenosine with adenine nucleoside phosphorylase after separation, with a conversion rate of more than 90%.
  • Wei Xiaokun et al. published a two-step method for synthesizing arabinoguanosine.
  • Enterobacter aerogenes which can express purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase, was used to synthesize 2,6-diaminopurine arabinoside from arabinoguanosine and 2,6-diaminopurine. Then, adenosine deaminase from Aspergillus oryzae was used to deaminize 2,6-diaminopurine arabinoside to further generate arabinoguanosine. When the substrate concentration was lower than 10 mM, the conversion rate of 2,6-diaminopurine arabinoside was 80%. However, when the substrate concentration was higher than 10 mM, the conversion rate of 2,6-diaminopurine arabinoside was significantly reduced to below 50%.
  • the main purpose of the present invention is to provide a one-pot biosynthesis method and composition for arabinose nucleosides to solve the problem of complex operation of preparing arabinose nucleosides by two-step biosynthesis in the prior art.
  • a one-pot biosynthesis method of arabinose nucleosides comprising: a substrate, a uridine nucleoside phosphorylase or a pyrimidine nucleoside phosphorylase, and a purine nucleoside phosphorylase are mixed together and then biosynthesized to directly prepare arabinose nucleosides by a one-pot method; wherein the uridine nucleoside phosphorylase comprises UP, or a protein having more than 80% homology with UP and having the same function, UP is the protein shown in SEQ ID NO: 1; the pyrimidine nucleoside phosphorylase comprises PyNP, or a protein having more than 80% homology with PyNP and having the same function
  • the invention relates to a protein having the function of phosphorylating nucleotides, wherein PyNP is a protein shown in SEQ ID NO: 2; the purine nucleoside phosphory
  • the method includes: uridine arabinoside and substrate phosphate are catalyzed by uridine nucleoside phosphorylase or pyrimidine nucleoside phosphorylase to generate arabinose-1-phosphate and free base; arabinose-1-phosphate is catalyzed by purine nucleoside phosphorylase to replace the phosphate group with the substrate base to obtain arabinose nucleoside.
  • one or more of the purine nucleoside phosphorylase, uridine nucleoside phosphorylase or pyrimidine nucleoside phosphorylase is a purified protein, a crude enzyme solution or an immobilized enzyme.
  • the catalytic time of the one-pot biosynthesis is 4 to 20 h; preferably, the catalytic temperature of the one-pot biosynthesis is 50 to 80° C., more preferably 60 to 70° C.; preferably, the concentration of uridine arabinoside is 2 to 320 mM, the concentration of the substrate base is 1 to 200 mM, and the concentration of the substrate phosphate is 1 to 100 mM.
  • the arabinose nucleoside includes 2,6-diaminopurine arabinoside, nelarabine, 2-chloroadenine arabinoside or fludarabine;
  • the substrate phosphate includes one or more of sodium monohydrogen phosphate, sodium dihydrogen phosphate, potassium monohydrogen phosphate or potassium dihydrogen phosphate, or phosphate buffer, or phosphate buffer.
  • a composition which comprises uridine nucleoside phosphorylase or pyrimidine nucleoside phosphorylase, and purine nucleoside phosphorylase;
  • the uridine nucleoside phosphorylase comprises UP, or a protein having more than 80% homology with UP and having the same function, UP is the protein shown in SEQ ID NO: 1;
  • the pyrimidine nucleoside phosphorylase comprises PyNP, or a protein having more than 80% homology with PyNP and having the same function, PyNP is the protein shown in SEQ ID NO: 2;
  • the purine nucleoside phosphorylase comprises PNP, or a protein having more than 80% homology with PNP and having the same function, PNP is the protein shown in SEQ ID NO: 3;
  • one or more of the purine nucleoside phosphorylase, uridine nucleoside phosphorylase or pyrimidine nucleoside phosphorylase is a purified protein, a crude enzyme solution or an immobilized enzyme.
  • the composition also includes uridine arabinoside, a substrate base and a substrate phosphate; preferably, the substrate phosphate includes one or more of sodium monohydrogen phosphate, sodium dihydrogen phosphate, potassium monohydrogen phosphate or potassium dihydrogen phosphate, or a phosphate buffer, or a phosphate buffer.
  • purine nucleoside phosphorylase, pyrimidine nucleoside phosphorylase or uridine nucleoside phosphorylase can be used to prepare the target product arabinose nucleoside by one-pot biosynthesis using uridine arabinoside and substrate base as substrates, without the need for mid-feeding or intermediate product purification and re-feeding.
  • the operation is simpler and industrial scale-up production can be utilized.
  • FIG. 1 shows the HPLC spectrum of synthesizing 2,6-diaminopurine arabinoside using uridine arabinoside and 2,6-aminopurine as substrates according to Example 2 of the present invention.
  • FIG. 2 shows the HPLC spectrum of the amplified synthesis of 2,6-diaminopurine arabinoside using uridine arabinoside and 2,6-aminopurine as substrates according to Example 3 of the present invention.
  • FIG. 3 shows the HPLC spectrum of synthesizing nelarabine using uridine arabinoside and 2-amino-6-methoxypurine as substrates according to Example 4 of the present invention.
  • FIG. 4 shows the HPLC spectrum of the amplified synthesis of nelarabine using uridine arabinoside and 2-amino-6-methoxypurine as substrates according to Example 5 of the present invention.
  • FIG. 5 shows the HPLC spectrum of the synthesis of 2-chloroadenine arabinoside using uridine arabinoside and 2-chloroadenine as substrates according to Example 6 of the present invention.
  • FIG. 6 shows the HPLC spectrum of fludarabine synthesized using uridine arabinoside and 2-fluoroadenine as substrates according to Example 7 of the present invention.
  • a one-pot biosynthesis method of arabinose nucleosides comprising: a substrate, a uridine nucleoside phosphorylase or a pyrimidine nucleoside phosphorylase, and a purine nucleoside phosphorylase, which are mixed together and then biosynthesized to directly prepare arabinose nucleosides by a one-pot method; wherein the uridine nucleoside phosphorylase comprises UP, or a protein having more than 80% homology with UP and having the same function, and UP is the protein shown in SEQ ID NO: 1; the pyrimidine nucleoside phosphorylase comprises PyNP, or a protein having more than 80% homology with PyNP and having the same function, and PyNP is the protein shown in SEQ ID NO: 2; the purine nucleoside phosphorylase comprises PNP, or a protein having more than 80% homology with PNP and having the same function, and
  • the uridine nucleoside phosphorylase used in the present application is derived from Trypanosoma cruzi, and is the protein shown in SEQ ID NO: 1, named UP.
  • the pyrimidine nucleoside phosphorylase is derived from Thermus thermophiles, and is the protein shown in SEQ ID NO: 2, named PyNP.
  • the purine nucleoside phosphorylase is derived from Geobacillus stearothermophilus, and is the protein shown in SEQ ID NO: 3, named PNP.
  • arabinuridine and substrate base can be used as substrates for biosynthesis through a one-pot method, without the need to purify the intermediate products before conversion, and the target product arabinose nucleoside can be directly obtained.
  • the above-mentioned uridine nucleoside phosphorylase, pyrimidine nucleoside phosphorylase or purine nucleoside phosphorylase respectively include proteins that have 80%, 85%, 90%, 95%, 98%, 99%, 99.5% or more homology with UP, PyNP or PNP and have the same function (i.e., the same catalytic function).
  • 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).
  • Constant amino acid replacements include but are not limited to:
  • Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;
  • hydrophobic amino acids with bulky side chains (Phe, Tyr, Trp) are replaced by other hydrophobic amino acids with bulky side chains;
  • Amino acids with positively charged side chains are replaced by other amino acids with positively charged side chains;
  • Amino acids with polar and uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar and uncharged side chains. generation.
  • a person 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.
  • the method comprises: uridine arabinoside and substrate phosphate are catalyzed by pyrimidine nucleoside phosphorylase or uridine nucleoside phosphorylase to generate arabinose-1-phosphate and free base; arabinose-1-phosphate is catalyzed by purine nucleoside phosphorylase to replace the phosphate group with the substrate base to obtain arabinose nucleoside.
  • the phosphate group derived from the substrate phosphate replaces the base part of uridine arabinoside and connects to the arabinose structure to form phosphorylated arabinose and uracil.
  • the substrate base replaces the phosphate group on the phosphorylated arabinose to obtain arabinose nucleoside.
  • a two-step method is usually used to synthesize arabinose nucleosides, which affects the yield of the one-pot method.
  • a combination of the above enzymes is obtained by screening enzymes with similar functions.
  • the combination of the above enzymes can be used to prepare arabinose nucleosides in a one-pot method, thereby improving the conversion rate of the substrate and the yield of the target product arabinose nucleosides.
  • the above one-pot biosynthesis method has a conversion rate comparable to the two-step method in the prior art, and the reaction operation is simpler, and there is no need to separate and purify the intermediate product, which is easy to prepare in large quantities in industrial production, and can reduce the time and cost required for the reaction.
  • 2,6-diaminopurine i.e., 2-aminoadenine
  • 2-amino-6-methoxypurine 2-chloroadenine or 2-fluoroadenine
  • 2-fluoroadenine 2,6-diaminopurine
  • one or more of purine nucleoside phosphorylase, uridine nucleoside phosphorylase or pyrimidine nucleoside phosphorylase is a purified protein, a crude enzyme solution or an immobilized enzyme.
  • the above three enzymes can exist in various forms such as purified protein, crude enzyme solution or immobilized enzyme, all of which can catalyze the synthesis of nucleosides containing protective groups.
  • the gene expressing PyNP and/or UP and/or PNP is cloned in a host cell, and after inducing protein expression, the host cell is broken to obtain a crude enzyme solution containing the target protein.
  • the crude enzyme solution is simple to prepare, has good catalytic ability, and can reduce the production cost of the catalytic reaction.
  • the catalytic time of enzyme catalysis is 4 to 20 hours; preferably, the catalytic temperature of enzyme catalysis is 50 to 80°C, more preferably 60 to 70°C; preferably, the concentration of uridine arabinoside is 2 to 320 mM, the concentration of substrate base is 1 to 200 mM, and the concentration of substrate phosphate is 1 to 100 mM.
  • the substrate phosphate is not limited to a specific type, including but not limited to phosphates commonly used in the prior art, including but not limited to one or more of sodium monohydrogen phosphate, sodium dihydrogen phosphate, potassium monohydrogen phosphate or potassium dihydrogen phosphate, or a mixture such as commercially available phosphate buffer (PB) or phosphate buffer (PBS) commonly used in the prior art.
  • the phosphate buffer includes a buffer composed of a certain concentration of sodium dihydrogen phosphate and disodium hydrogen phosphate.
  • the phosphate buffer includes a buffer composed of disodium hydrogen phosphate, potassium dihydrogen phosphate, and salts such as sodium chloride and potassium chloride.
  • the above method can carry out an amplified reaction.
  • the concentration of uridine arabinoside includes 2mM, 10mM, 20mM, 50mM, 100mM, 200mM, and can even reach 320mM or more
  • the concentration of the substrate base includes 1mM, 2mM, 10mM, 20mM, 50mM, 100mM, and can even reach 200mM or more, thereby carrying out large-scale preparation of arabinose nucleosides.
  • the enzyme catalyzed reaction can be completed, and the conversion rate of the substrate base, that is, the reaction yield is high. There is no need to add enzymes or other reagents in the middle of the reaction, and there is no need to separate and purify the intermediate product before subsequent catalysis.
  • the reaction can be completed by one-pot catalysis, which is suitable for application in industrial scale-up production.
  • the reaction conditions are mild and easy to control, and the production equipment cost, energy cost and danger can also be reduced.
  • the arabinose nucleoside includes but is not limited to 2,6-diaminopurine arabinoside, nelarabine (9-( ⁇ -D-arabinofuranosyl)-6-methoxy-9H-purin-2-amine), 2-chloroadenine arabinoside or fludarabine (9- ⁇ -D-arabinofuranosyl-2-fluoroadenosine).
  • a composition which includes uridine nucleoside phosphorylase or pyrimidine nucleoside phosphorylase, and purine nucleoside phosphorylase;
  • the uridine nucleoside phosphorylase includes UP, or a protein having more than 80% homology with UP and having the same function, UP is the protein shown in SEQ ID NO: 1;
  • the pyrimidine nucleoside phosphorylase includes PyNP, or a protein having more than 80% homology with PyNP and having the same function, PyNP is the protein shown in SEQ ID NO: 2;
  • the purine nucleoside phosphorylase includes PNP, or a protein having more than 80% homology with PNP and having the same function, PNP is the protein shown in SEQ ID NO: 3.
  • one or more of purine nucleoside phosphorylase, uridine nucleoside phosphorylase or pyrimidine nucleoside phosphorylase is a purified protein, a crude enzyme solution or an immobilized enzyme.
  • the composition further comprises uridine arabinoside, a substrate base and a substrate phosphate.
  • the substrate phosphate is not limited to a specific type, including but not limited to phosphates commonly used in the prior art, including but not limited to one or more of sodium monohydrogen phosphate, sodium dihydrogen phosphate, potassium monohydrogen phosphate or potassium dihydrogen phosphate, or a mixture commonly used in the prior art such as a phosphate buffer.
  • the concentration of uridine arabinoside is 2-320 mM
  • the concentration of substrate base is 1-200 mM
  • the concentration of substrate phosphate is 1-100 mM.
  • the concentrations of uridine arabinoside include 2mM, 10mM, 20mM, 50mM, 100mM, 200mM, and can even reach 320mM or more; the concentrations of substrate bases include 1mM, 2mM, 10mM, 20mM, 50mM, 100mM, and can even reach 200mM or more.
  • the above-mentioned uridine nucleoside phosphorylase is derived from Trypanosoma cruzi, and is a protein shown in SEQ ID NO: 1, named UP.
  • the pyrimidine nucleoside phosphorylase is derived from Thermus thermophiles, and is a protein shown in SEQ ID NO: 2, named PyNP.
  • the purine nucleoside phosphorylase is derived from Geobacillus stearothermophilus, and is a protein shown in SEQ ID NO: 3, named PNP.
  • the above-mentioned composition can catalyze the reaction between the substrate base and arabinuridine to produce arabinose nucleosides.
  • the enzymes in the composition can be independently selected from the forms of purified protein, crude enzyme solution or immobilized enzyme, and can all play a catalytic role.
  • composition can also be prepared into product forms such as a kit.
  • the uridine nucleoside phosphorylase used in the present application is derived from Trypanosoma cruzi (named UP, the amino acid sequence is shown in SEQ ID NO: 1).
  • the pyrimidine nucleoside phosphorylase is derived from Thermus thermophiles (named PyNP, the amino acid sequence is shown in SEQ ID NO: 2).
  • the purine nucleoside phosphorylase is derived from Geobacillus stearothermophilus (named PNP, the amino acid sequence is shown in SEQ ID NO: 3).
  • the DNA sequences encoding the three enzymes are obtained, and the DNA sequence encoding UP is SEQ ID NO: 4; the DNA sequence encoding PyNP is SEQ ID NO: 5; and the DNA sequence encoding PNP is SEQ ID NO: 6. They are cloned into the expression vector pET28a (+) respectively. The obtained plasmid is transformed into the competent host of Escherichia coli BL21 (DE3) to obtain a monoclonal strain.
  • the E. coli strains expressing UP, PyNP and PNP were inoculated into test tubes, respectively, and cultured at 37°C for 16 hours, and then inoculated into 2L shake flasks containing 500mL Luria-Bertani medium at a 1% inoculum, and cultured at 37°C until OD600 reached 0.6, and then 0.1M isopropyl- ⁇ -D-thiogalactoside was added to induce protein expression, and cultured at 20°C for 18 hours. The bacterial solution at the end of the culture was centrifuged at 7000rpm for 10 minutes to collect the bacteria for later use.
  • HPLC detection method Chromatographic column: Agilent Eclipse plus C18, 4.6*100mm, 3.5 ⁇ m, mobile phase: water and acetonitrile, flow rate 1.5mL/min, temperature 40°C, UV: 254nm.
  • reaction system 1 mL of reaction system was prepared in 2 mM phosphate buffer (pH 7.5), including 80 mM 1-beta-D-Arabinofuranosyluracil, 20 mM 2,6-aminopurine, 4.88 mg of UP enzyme solution prepared from bacterial sludge and 6.01 mg of PNP enzyme solution prepared from bacterial sludge.
  • the reaction was carried out at 60°C for 16 h. As shown in Figure 1, the conversion rate of 2,6-aminopurine was 98.78%.
  • reaction system In 2 mM phosphate buffer (pH 7.5), 1 mL of the reaction system was prepared, including 320 mM 1-beta-D-Arabinofuranosyluracil, 200 mM 2, 6-aminopurine, 19.54 mg of UP enzyme solution prepared from bacterial sludge, and 60.06 mg of PNP enzyme solution prepared from bacterial sludge.
  • the reaction was carried out at 60 ° C for 6 h, and the conversion rate of 2, 6-aminopurine was 89.74%. After 16 h of reaction, as shown in Figure 2, the conversion rate of 2, 6-aminopurine was 96.74%, and the yield of 2, 6-diaminopurine arabinoside was 54.61 g/L.
  • reaction system 1 mL of reaction system was prepared in 2 mM phosphate buffer (pH 7.5), including 4 mM 1-beta-D-Arabinofuranosyluracil, 1 mM 2-amino-6-methoxypurine, 0.24 mg of UP enzyme solution prepared from bacterial mud and 0.33 mg of PNP enzyme solution prepared from bacterial mud.
  • the reaction was carried out at 70° C. for 6 h. As shown in FIG3 , the conversion rate of 2-amino-6-methoxypurine was 98.48%.
  • reaction system 1 mL of reaction system was prepared in 2 mM phosphate buffer (pH 7.5), including 320 mM 1-beta-D-Arabinofuranosyluracil, 80 mM 2-Amino-6-methoxypurine, 19.54 mg UP enzyme solution prepared from bacterial sludge and 26.42 mg PNP enzyme solution prepared from bacterial sludge, and reacted at 70 ° C for 16 h.
  • the conversion rate of 6-methoxypurine was 74.82%.
  • the conversion rate of 2-amino-6-methoxypurine was 94.96%, as shown in Figure 4, and the yield of nelarabine was 22.58 g/L.
  • reaction system 1 mL of reaction system was prepared in 2 mM phosphate buffer (pH 7.5), including 2 mM 1-beta-D-Arabinofuranosyluracil, 1 mM purine analog 2-fluoroadenine, 0.12 mg of PyNP enzyme solution prepared from bacterial sludge, and an appropriate amount of PNP enzyme solution prepared from bacterial sludge, and reacted at 70°C for 20 h. As shown in Figure 6, the conversion rate of 2-fluoroadenine was 92.51%.
  • the above purine nucleoside phosphorylase, pyrimidine nucleoside phosphorylase or uridine nucleoside phosphorylase can be used in the present application to prepare the target product arabinose nucleoside by one-pot biosynthesis with uridine arabinoside and substrate base as substrates, without the need for midway feeding or intermediate product purification and re-feeding.
  • the operation is simpler and the reaction time is short; compared with the one-pot biosynthesis in the prior art, the reaction rate is faster, the substrate conversion rate is higher, and a higher substrate concentration can be tolerated, and large-scale industrial production can be achieved in a shorter reaction time; compared with chemical synthesis, the reaction conditions are milder, the process is simple, the cost is low and it is environmentally friendly.

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Abstract

本发明提供了一种阿糖类核苷一锅法生物合成的方法及组合物。其中，阿糖类核苷一锅法生物合成的方法包括：底物、尿嘧啶核苷磷酸化酶或嘧啶核苷磷酸化酶、以及嘌呤核苷磷酸化酶，共同混合后进行生物合成，一锅法直接制备获得阿糖类核苷。尿嘧啶核苷磷酸化酶包括SEQ ID NO：1所示的UP；嘧啶核苷磷酸化酶包括SEQ ID NO：2所示的PyNP；嘌呤核苷磷酸化酶包括SEQ ID NO：3所示的PNP；底物包括阿糖尿苷和底物碱基。能够解决现有技术中生物合成两步法制备阿糖类核苷操作复杂的问题，适用于酶催化领域。

Description

阿糖类核苷一锅法生物合成的方法及组合物
本申请是以CN申请号为202211296616.9，申请日为2022年10月21日的中国申请为基础，并主张其优先权，该CN申请的公开内容再次作为整体引入本申请中。
技术领域
本发明涉及酶催化领域，具体而言，涉及一种阿糖类核苷一锅法生物合成的方法及组合物。
背景技术
阿糖类核苷是将脱氧核糖替换为阿拉伯糖的核苷，在被掺入DNA后可阻止DNA的复制进而影响细胞的分裂和增殖，因而可作为抗病毒和抗肿瘤类药物用于相关疾病的治疗，如疱疹，肿瘤、艾滋病、乙肝等。目前，阿糖核苷类主要以化学法(CN1042939C，CN107556356A，CN1128270A，CN103467468A，CN107892707A)和生物法合成(CN106929553A，JPH10286097A，CN105237602A)。其中，化学合成工艺过程复杂，成本较高，且反应条件苛刻，需使用重金属，有机溶剂等，会对人员健康造成危害并对引起环境污染等问题。而生物法是利用微生物表达的酶作为催化剂，在温和条件下即可将底物转化为相关产品，操作步骤简单，且反应条件则相对温和，不需要使用重金属和有机试剂等对人体和环境有害的物质。
专利CN106929553A公开了一种两步法合成阿糖腺苷的方法，可通过使用尿苷磷酸化酶将阿糖尿苷转化为阿糖-1-磷酸，经分离后再利用腺嘌呤核苷磷酸化酶将其和腺嘌呤合成阿糖腺苷，其转化率达90％以上。魏晓琨等人发表了一种两步法合成阿糖鸟苷的方法，首先利用可表达嘌呤核苷磷酸化酶和嘧啶核苷磷酸化酶的产气肠杆菌(Enterobacter aerogenes)将阿糖尿苷和2，6-二氨基嘌呤合成2，6-二氨基嘌呤阿拉伯糖苷，然后利用来源于米曲霉(Aspergillus oryzae)的腺苷脱氨酶将2，6-二氨基嘌呤阿拉伯糖苷脱去氨基进一步生成阿糖鸟苷，当底物浓度低于10mM时，2，6-二氨基嘌呤阿拉伯糖苷转化率为80％；但底物浓度高于10mM时，2，6-二氨基嘌呤阿拉伯糖苷的转化率则显著降低至50％以下。
发明内容
本发明的主要目的在于提供一种阿糖类核苷一锅法生物合成的方法及组合物，以解决现有技术中生物合成两步法制备阿糖类核苷操作复杂的问题。
为了实现上述目的，根据本发明的第一个方面，提供了一种阿糖类核苷一锅法生物合成的方法，该方法包括：底物、尿嘧啶核苷磷酸化酶或嘧啶核苷磷酸化酶、以及嘌呤核苷磷酸化酶，共同混合后进行生物合成，一锅法直接制备获得阿糖类核苷；其中，尿嘧啶核苷磷酸化酶包括UP、或与UP具有80％以上同源性且具有相同功能的蛋白质，UP为SEQ ID NO：1所示的蛋白质；嘧啶核苷磷酸化酶包括PyNP、或与PyNP具有80％以上同源性且具有相同功 能的蛋白质，PyNP为SEQ ID NO：2所示的蛋白质；嘌呤核苷磷酸化酶包括PNP、或与PNP具有80％以上同源性且具有相同功能的蛋白质，PNP为SEQ ID NO：3所示的蛋白质；底物包括阿糖尿苷、底物碱基和底物磷酸盐。
进一步地，方法包括：阿糖尿苷和底物磷酸盐在尿嘧啶核苷磷酸化酶或嘧啶核苷磷酸化酶的催化下，生成阿拉伯糖-1-磷酸和游离碱基；阿拉伯糖-1-磷酸在嘌呤核苷磷酸化酶的催化下，底物碱基取代磷酸基团，获得阿糖类核苷。
进一步地，底物碱基为式I所示的碱基，R1选自-NH2、＝O、-OMe或-Cl，R2选自-H、-NH2、-Cl或-F；优选地，底物碱基包括2，6-二氨基嘌呤、2-氨基-6-甲氧基嘌呤、2-氯腺嘌呤或2-氟腺嘌呤；
进一步地，嘌呤核苷磷酸化酶、尿嘧啶核苷磷酸化酶或嘧啶核苷磷酸化酶中的一种或多种为纯化蛋白、粗酶液或固定化酶。
进一步地，一锅法生物合成的催化时间为4～20h；优选地，一锅法生物合成的催化温度为50～80℃，更优选为60～70℃；优选地，阿糖尿苷的浓度为2～320mM，底物碱基的浓度为1～200mM，底物磷酸盐的浓度为1～100mM。
进一步地，阿糖类核苷包括2，6-二氨基嘌呤阿拉伯糖苷、奈拉滨、2-氯腺嘌呤阿拉伯糖苷或氟达拉滨；底物磷酸盐包括磷酸一氢钠、磷酸二氢钠、磷酸一氢钾或磷酸二氢钾中的一种或多种，或磷酸缓冲液，或磷酸盐缓冲液。
为了实现上述目的，根据本发明的第二个方面，提供了一种组合物，该组合物包括尿嘧啶核苷磷酸化酶或嘧啶核苷磷酸化酶、以及嘌呤核苷磷酸化酶；尿嘧啶核苷磷酸化酶包括UP、或与UP具有80％以上同源性且具有相同功能的蛋白质，UP为SEQ ID NO：1所示的蛋白质；嘧啶核苷磷酸化酶包括PyNP、或与PyNP具有80％以上同源性且具有相同功能的蛋白质，PyNP为SEQ ID NO：2所示的蛋白质；嘌呤核苷磷酸化酶包括PNP、或与PNP具有80％以上同源性且具有相同功能的蛋白质，PNP为SEQ ID NO：3所示的蛋白质；
进一步地，嘌呤核苷磷酸化酶、尿嘧啶核苷磷酸化酶或嘧啶核苷磷酸化酶中的一种或多种为纯化蛋白、粗酶液或固定化酶。
进一步地，组合物还包括阿糖尿苷、底物碱基和底物磷酸盐；优选地，底物磷酸盐包括磷酸一氢钠、磷酸二氢钠、磷酸一氢钾或磷酸二氢钾中的一种或多种，或磷酸缓冲液，或磷酸盐缓冲液。
进一步地，底物碱基为式I所示的碱基，R1选自-NH2、＝O、-OMe或-Cl，R2选自-H、-NH2、-Cl或-F；优选地，底物碱基包括2，6-二氨基嘌呤、2-氨基-6-甲氧基嘌呤、2-氯腺嘌呤或2-氟腺嘌呤；优选地，阿糖尿苷的浓度为2～320mM，底物碱基的浓度为1～200mM，底物磷酸盐的浓度为1～100mM；
应用本发明的技术方案，利用嘌呤核苷磷酸化酶、以及嘧啶核苷磷酸化酶或尿嘧啶核苷磷酸化酶，能够以阿糖尿苷和底物碱基为底物，一锅法生物合成制备目标产物阿糖类核苷，无需进行中途补料或中间产物纯化再投料等操作，相较于两步法制备，操作更简单，利用工业化放大生产。
附图说明
构成本申请的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1示出了根据本发明实施例2的以阿糖尿苷和2，6-氨基嘌呤为底物合成2，6-二氨基嘌呤阿拉伯糖苷的HPLC图谱。
图2示出了根据本发明实施例3的以阿糖尿苷和2，6-氨基嘌呤为底物放大合成2，6-二氨基嘌呤阿拉伯糖苷的HPLC图谱。
图3示出了根据本发明实施例4的以阿糖尿苷和2-氨基-6-甲氧基嘌呤为底物合成奈拉滨的HPLC图谱。
图4示出了根据本发明实施例5的以阿糖尿苷和2-氨基-6-甲氧基嘌呤为底物放大合成奈拉滨的HPLC图谱。
图5示出了根据本发明实施例6的以阿糖尿苷和2-氯腺嘌呤为底物合成2-氯腺嘌呤阿拉伯糖苷的HPLC图谱。
图6示出了根据本发明实施例7的以阿糖尿苷和2-氟腺嘌呤为底物合成氟达拉滨的HPLC图谱。
具体实施方式
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将结合实施例来详细说明本发明。
如背景技术所提到的，现有技术中生物合成阿糖腺苷多采用两步法，操作繁琐，所需时间长，不利于实现工业化的大量生产。因而，在本申请中发明人尝试探究一种一锅法生物合成制备阿糖类核苷的方法，因而提出了本申请的一系列保护方案。
在本申请第一种典型的实施方式中，提供了一种阿糖类核苷一锅法生物合成的方法，该方法包括：底物、尿嘧啶核苷磷酸化酶或嘧啶核苷磷酸化酶、以及嘌呤核苷磷酸化酶，共同混合后进行生物合成，一锅法直接制备获得阿糖类核苷；其中，尿嘧啶核苷磷酸化酶包括UP、或与UP具有80％以上同源性且具有相同功能的蛋白质，UP为SEQ ID NO：1所示的蛋白质；嘧啶核苷磷酸化酶包括PyNP、或与PyNP具有80％以上同源性且具有相同功能的蛋白质，PyNP为SEQ ID NO：2所示的蛋白质；嘌呤核苷磷酸化酶包括PNP、或与PNP具有80％以上同源性且具有相同功能的蛋白质，PNP为SEQ ID NO：3所示的蛋白质；底物包括阿糖尿苷和底物碱基；底物还可包括底物磷酸盐，用于提供反应所需的磷酸基团；阿糖类核苷为由阿拉伯糖结构和碱基结构组合的核苷。
本申请中所使用尿嘧啶核苷磷酸化酶来源于克氏锥虫Trypanosoma cruzi，为SEQ ID NO：1所示的蛋白质，命名为UP。嘧啶核苷磷酸化酶来源于嗜热栖热菌Thermus thermophiles，为SEQ ID NO：2所示的蛋白质，命名为PyNP。嘌呤核苷磷酸化酶来源于嗜热脂肪地芽孢杆菌Geobacillus stearothermophilus，为SEQ ID NO：3所示的蛋白质，命名为PNP。利用嘌呤核苷磷酸化酶以及如下任一种酶：尿嘧啶核苷磷酸化酶或嘧啶核苷磷酸化酶，能够以阿糖尿苷和底物碱基为底物，通过一锅法进行生物合成，无需对中间产物进行纯化后再进行转化，能够直接获得目标产物阿糖类核苷。上述尿嘧啶核苷磷酸化酶、嘧啶核苷磷酸化酶或嘌呤核苷磷酸化酶，均分别包括与UP、PyNP或PNP具有80％、85％、90％、95％、98％、99％、99.5％或99.9％以上同源性且具有相同功能(即相同催化功能)的蛋白质。
如本文所用，氨基酸残基缩写如下：丙氨酸(Ala；A)、天冬酰胺(Asn；N)、天冬氨酸(Asp；D)、精氨酸(Arg；R)、半胱氨酸(Cys；C)、谷氨酸(Glu；E)、谷氨酰胺(Gln；Q)、甘氨酸(Gly；G)、组氨酸(His；H)、异亮氨酸(Ile；I)、亮氨酸(Leu；L)、赖氨酸(Lys；K)、蛋氨酸(Met；M)、苯丙氨酸(Phe；F)、脯氨酸(Pro；P)，丝氨酸(Ser；S)、苏氨酸(Thr；T)、色氨酸(Trp；W)、酪氨酸(Tyr；Y)和缬氨酸(Val；V)。
取代、替换等规则，一般情况下，哪些氨基酸性质类似，替换后的效果也类似。例如，在上述同源蛋白中，可发生保守的氨基酸替换。“保守的氨基酸替换”包括但不限于：
疏水性氨基酸(Ala、Cys、Gly、Pro、Met、Val、Ile、Leu)被其他疏水性氨基酸取代；
侧链粗大的疏水性氨基酸(Phe、Tyr、Trp)被其他侧链粗大的疏水性氨基酸取代；
侧链带正电的氨基酸(Arg、His、Lys)被其他侧链带正电的氨基酸取代；
侧链有极性不带电的氨基酸(Ser、Thr、Asn、Gln)被其他侧链有极性不带电的氨基酸取 代。
本领域技术人员也可以根据现有技术中的“blosum62评分矩阵”等本领域技术人员熟知的氨基酸替换规则对氨基酸进行保守替换。
在一种优选的实施例中，方法包括：阿糖尿苷和底物磷酸盐在嘧啶核苷磷酸化酶或尿嘧啶核苷磷酸化酶的催化下，生成阿拉伯糖-1-磷酸和游离碱基；阿拉伯糖-1-磷酸在嘌呤核苷磷酸化酶的催化下，底物碱基取代磷酸基团，获得阿糖类核苷。
上述方法的反应路线如下：
首先底物阿糖尿苷在尿嘧啶核苷磷酸化酶或嘧啶核苷磷酸化酶的催化下，来源于底物磷酸盐的磷酸基团取代阿糖尿苷的碱基部分连接在阿拉伯糖结构上，形成磷酸化阿拉伯糖和尿嘧啶(Uracil)。之后在嘌呤核苷磷酸化酶的催化下，底物碱基取代磷酸化阿拉伯糖上的磷酸基团，获得阿糖类核苷。
在现有技术中，通常使用两步法合成阿糖类核苷，影响一锅法的收率。而本申请中，从功能类似的酶中进行了筛选，获得上述酶的组合，利用上述酶的组合，能够在一锅法制备阿糖类核苷，从而提高底物的转化率和目标产物阿糖类核苷的产率。上述一锅法生物合成的方法，转化率与现有技术中的两步法相当，而反应操作更加简单，也无需对中间产物进行分离纯化，易于工业生产中大批量制备，能够降低反应所需的时间和成本。
在一种优选的实施例中，底物碱基为式I所示的碱基，R1选自-NH2、＝O、-OMe或-Cl，R2选自-H、-NH2、-Cl或-F；优选地，底物碱基包括2，6-二氨基嘌呤(即2-氨基腺嘌呤)、2-氨基-6-甲氧基嘌呤、2-氯腺嘌呤或2-氟腺嘌呤；
在一种优选的实施例中，嘌呤核苷磷酸化酶、尿嘧啶核苷磷酸化酶或嘧啶核苷磷酸化酶中的一种或多种为纯化蛋白、粗酶液或固定化酶。
在催化反应中，上述3种酶可以为纯化蛋白、粗酶液或固定化酶等多种形式存在，均能够催化合成含有保护基的核苷。将表达PyNP和/或UP和/或PNP的基因克隆在宿主细胞中，诱导蛋白表达后，破碎宿主细胞即能够获得含有目标蛋白的粗酶液。粗酶液的制备简单，且有良好的催化能力，且能够降低催化反应的生产成本。
在一种优选的实施例中，酶催化的催化时间为4～20h；优选地，酶催化的催化温度为50～80℃，更优选为60～70℃；优选地，阿糖尿苷的浓度为2～320mM，底物碱基的浓度为1～200mM，底物磷酸盐的浓度为1～100mM。底物磷酸盐不限定特定种类，包括但不限于现有技术中常用的磷酸盐，包括但不限于磷酸一氢钠、磷酸二氢钠、磷酸一氢钾或磷酸二氢钾中的一种或多种，或是现有技术中常见的如市售的磷酸缓冲液(PB)、或磷酸盐缓冲液(PBS)等混合物。其中，磷酸缓冲液包括一定浓度的磷酸二氢钠和磷酸氢二钠组成的缓冲液。磷酸盐缓冲液包括磷酸氢二钠、磷酸二氢钾、和氯化钠、氯化钾等盐组成的缓冲液。
上述方法，能够进行放大反应，在反应体系中，阿糖尿苷的浓度包括2mM、10mM、20mM、50mM、100mM、200mM，乃至能够达到320mM甚至更多，底物碱基的浓度包括1mM、2mM、10mM、20mM、50mM、100mM，乃至能够达到200mM甚至更多，从而进行大规模的阿糖类核苷的制备。
在上述适宜的催化温度和催化时间内，该酶催化反应即能够完成，且底物碱基的转化率，即反应收率较高。在反应中途也无需补加酶或其他试剂，也无需将中间产物进行分离纯化后，再进行后续催化。利用一锅法催化即能够完成反应，适宜在工业化放大生产上的应用。反应条件温和易控制，也能够降低生产的装置成本、能源成本及危险性。
在一种优选的实施例中，阿糖类核苷包括但不限于2，6-二氨基嘌呤阿拉伯糖苷、奈拉滨(9-(β-D-阿拉伯呋喃糖基)-6-甲氧基-9H-嘌呤-2-胺)、2-氯腺嘌呤阿拉伯糖苷或氟达拉滨(9-β-D-阿拉伯呋喃糖-2-氟腺苷)。
在本申请第二种典型的实施方式中，提供了一种组合物，该组合物包括尿嘧啶核苷磷酸化酶或嘧啶核苷磷酸化酶、以及嘌呤核苷磷酸化酶；尿嘧啶核苷磷酸化酶包括UP、或与UP具有80％以上同源性且具有相同功能的蛋白质，UP为SEQ ID NO：1所示的蛋白质；嘧啶核苷磷酸化酶包括PyNP、或与PyNP具有80％以上同源性且具有相同功能的蛋白质，PyNP为SEQ ID NO：2所示的蛋白质；嘌呤核苷磷酸化酶包括PNP、或与PNP具有80％以上同源性且具有相同功能的蛋白质，PNP为SEQ ID NO：3所示的蛋白质。
在一种优选的实施例中，嘌呤核苷磷酸化酶、尿嘧啶核苷磷酸化酶或嘧啶核苷磷酸化酶中的一种或多种为纯化蛋白、粗酶液或固定化酶。
在一种优选的实施例中，组合物还包括阿糖尿苷、底物碱基和底物磷酸盐。底物磷酸盐不限定特定种类，包括但不限于现有技术中常用的磷酸盐，包括但不限于磷酸一氢钠、磷酸二氢钠、磷酸一氢钾或磷酸二氢钾中的一种或多种，或是现有技术中常见的如磷酸缓冲液中等混合物。
在一种优选的实施例中，底物碱基为式I所示的碱基，R1选自-NH2，＝O，-OMe或-Cl，R2选自-H，-NH2，-Cl或-F；优选地，底物碱基包括2，6-氨基嘌呤、2-氨基-6-甲氧基嘌呤、2-氯腺嘌呤或2-氟腺嘌呤；
在一种优选的实施例中，阿糖尿苷的浓度为2～320mM，底物碱基的浓度为1～200mM，底物磷酸盐的浓度为1～100mM。
阿糖尿苷的浓度包括2mM、10mM、20mM、50mM、100mM、200mM，乃至能够达到320mM甚至更多，底物碱基的浓度包括1mM、2mM、10mM、20mM、50mM、100mM，乃至能够达到200mM甚至更多。
上述尿嘧啶核苷磷酸化酶来源于克氏锥虫Trypanosoma cruzi，为SEQ ID NO：1所示的蛋白质，命名为UP。嘧啶核苷磷酸化酶来源于嗜热栖热菌Thermus thermophiles，为SEQ ID NO：2所示的蛋白质，命名为PyNP。嘌呤核苷磷酸化酶来源于嗜热脂肪地芽孢杆菌Geobacillus stearothermophilus，为SEQ ID NO：3所示的蛋白质，命名为PNP。上述组合物能够催化底物碱基和阿糖尿苷发生反应，生产阿糖类核苷。组合物中的酶，能够分别独立选自纯化蛋白、粗酶液或固定化酶等形式，均能够发挥催化作用。
上述组合物还可以制备成试剂盒等产品形式。
下面将结合具体的实施例来进一步详细解释本申请的有益效果。
实施例1
1、菌株构建
本申请中所使用尿嘧啶核苷磷酸化酶来源于克氏锥虫Trypanosoma cruzi(命名UP，氨基酸序列如SEQ ID NO：1所示)。嘧啶核苷磷酸化酶来源于嗜热栖热菌Thermus thermophiles(命名PyNP，氨基酸序列如SEQ ID NO：2所示)。嘌呤核苷磷酸化酶来源于嗜热脂肪地芽孢杆菌Geobacillus stearothermophilus(命名PNP，氨基酸序列如SEQ ID NO：3所示)。密码子优化后获得编码三个个酶的DNA序列，编码UP的DNA序列为SEQ ID NO：4；编码PyNP的DNA序列为SEQ ID NO：5；编码PNP的DNA序列为SEQ ID NO：6。将其分别克隆到表达载体pET28a(+)上。将得到的质粒化转至大肠杆菌BL21(DE3)宿主感受态中，获得单克隆菌株。


2、蛋白表达
将表达UP、PyNP和PNP的大肠杆菌菌株分别接种于试管，37℃培养16h后以1％接种量，接种于含500mL Luria-Bertani培养基的2L摇瓶，37℃培养至OD600为0.6时，加入终浓度为0.1M异丙基-β-D-硫代半乳糖苷诱导蛋白的表达，20℃培养18h。将培养结束的菌液7000rpm离心10min收集菌体，备用。
3、酶液制备
称取0.1g菌泥，添加1mL pH为7.5的磷酸钾缓冲液，震荡混匀后，用超声破碎仪，将菌体混悬液破碎，功率30％，时间5min。
4、检测方法
HPLC检测方法：色谱柱：Agilent Eclipse plus C18，4.6*100mm，3.5μm，流动相为水和乙腈，流苏1.5mL/min，温度40℃，UV：254nm。
实施例2 2，6-二氨基嘌呤阿拉伯糖苷(2，6-Diamino-9-(beta-D-arabinofuranosyl)purine)的合成
在2mM磷酸缓冲液(pH 7.5)配制1mL反应体系，其中包括80mM阿糖尿苷(1-beta-D-Arabinofuranosyluracil)，20mM 2，6-氨基嘌呤(2，6-Diaminopurine)，4.88mg菌泥制备的UP酶液和6.01mg菌泥制备的PNP酶液，60℃反应16h，如图1所示，2，6-氨基嘌呤转化率为98.78％。
实施例3 2，6-二氨基嘌呤阿拉伯糖苷合成的放大反应
在2mM磷酸缓冲液(pH 7.5)配制1mL反应体系，其中包括320mM阿糖尿苷(1-beta-D-Arabinofuranosyluracil)，200mM 2，6-氨基嘌呤(2，6-Diaminopurine)，19.54mg菌泥制备的UP酶液和60.06mg菌泥制备的PNP酶液，60℃反应6h，2，6-氨基嘌呤转化率为89.74％；反应16h，如图2所示，2，6-氨基嘌呤转化率为96.74％，2，6-二氨基嘌呤阿拉伯糖苷产量为54.61g/L。
实施例4奈拉滨(Nelarabine，9-(β-D-阿拉伯呋喃糖基)-6-甲氧基-9H-嘌呤-2-胺)的合成
在2mM磷酸缓冲液(pH 7.5)配制1mL反应体系，其中包括4mM阿糖尿苷(1-beta-D-Arabinofuranosyluracil)，1mM 2-氨基-6-甲氧基嘌呤(2-Amino-6-methoxypurine)，0.24mg菌泥制备的UP酶液和0.33mg菌泥制备的PNP酶液，70℃反应6h，如图3所示，2-氨基-6-甲氧基嘌呤转化率为98.48％。
实施例5奈拉滨(9-(β-D-阿拉伯呋喃糖基)-6-甲氧基-9H-嘌呤-2-胺)的放大反应
在2mM磷酸缓冲液(pH 7.5)配制1mL反应体系，其中包括320mM阿糖尿苷(1-beta-D-Arabinofuranosyluracil)，80mM 2-氨基-6-甲氧基嘌呤(2-Amino-6-methoxypurine)，19.54mg菌泥制备的UP酶液和26.42mg菌泥制备的PNP酶液，70℃反应16h，6-甲氧基嘌呤转化率为74.82％。反应40h，2-氨基-6-甲氧基嘌呤转化率为94.96％，如图4所示，奈拉滨的产量为22.58g/L。
实施例6 2-氯腺嘌呤阿拉伯糖苷(2-chloroadeninearabinoside)的合成
在2mM磷酸缓冲液(pH 7.5)配制1mL反应体系，其中包括2mM阿糖尿苷 (1-beta-D-Arabinofuranosyluracil)，1mM 2-氯腺嘌呤(2-Chloroadenine)，0.12mg菌泥制备的PyNP酶液和适量菌泥制备的PNP酶液，70℃反应20h。如图5所示，2-氯腺嘌呤转化率为93.97％。
实施例7氟达拉滨(Fludarabine，9-β-D-阿拉伯呋喃糖-2-氟腺苷)的合成
在2mM磷酸缓冲液(pH 7.5)配制1mL反应体系，其中包括2mM阿糖尿苷(1-beta-D-Arabinofuranosyluracil)，1mM嘌呤类似物2-氟腺嘌呤(2-Fluoroadenine)，0.12mg菌泥制备的PyNP酶液和适量菌泥制备的PNP酶液，70℃反应20h。如图6所示，2-氟腺嘌呤转化率为92.51％。
从以上的描述中，可以看出，本发明上述的实施例实现了如下技术效果：本申请中利用上述嘌呤核苷磷酸化酶、以及嘧啶核苷磷酸化酶或尿嘧啶核苷磷酸化酶，能够以阿糖尿苷和底物碱基为底物，一锅法生物合成制备目标产物阿糖类核苷，无需进行中途补料或中间产物纯化再投料等操作。相较于现有技术中两步法生物合成的操作更简单，反应所需时间短；相较于现有技术中的一锅法生物合成，反应速率更快，底物转化率更高，能够耐受较高的底物浓度，在较短的反应时间内实现大规模的工业化生产；相较于化学合成，反应所需条件更温和，工艺过程简单，成本低且对环境友好。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种阿糖类核苷一锅法生物合成的方法，其特征在于，所述方法包括：
    底物、尿嘧啶核苷磷酸化酶或嘧啶核苷磷酸化酶、以及嘌呤核苷磷酸化酶，共同混合后进行生物合成，一锅法直接制备获得所述阿糖类核苷；
    其中，所述尿嘧啶核苷磷酸化酶包括UP、或与所述UP具有80％以上同源性且具有相同功能的蛋白质，所述UP为SEQ ID NO：1所示的蛋白质；
    所述嘧啶核苷磷酸化酶包括PyNP、或与所述PyNP具有80％以上同源性且具有相同功能的蛋白质，所述PyNP为SEQ ID NO：2所示的蛋白质；
    所述嘌呤核苷磷酸化酶包括PNP、或与所述PNP具有80％以上同源性且具有相同功能的蛋白质，所述PNP为SEQ ID NO：3所示的蛋白质；
    所述底物包括阿糖尿苷、底物碱基和底物磷酸盐。
  2. 根据权利要求1所述的方法，其特征在于，所述方法包括：
    所述阿糖尿苷和所述底物磷酸盐在所述尿嘧啶核苷磷酸化酶或所述嘧啶核苷磷酸化酶的催化下，生成阿拉伯糖-1-磷酸和游离碱基；
    所述阿拉伯糖-1-磷酸在所述嘌呤核苷磷酸化酶的催化下，所述底物碱基取代所述阿拉伯糖-1-磷酸上的磷酸基团，获得所述阿糖类核苷。
  3. 根据权利要求1所述的方法，其特征在于，所述底物碱基为式I所示的碱基，R1选自-NH2、＝O、-OMe或-Cl，R2选自-H、-NH2、-Cl或-F；
    优选地，所述底物碱基包括2，6-二氨基嘌呤、2-氨基-6-甲氧基嘌呤、2-氯腺嘌呤或2-氟腺嘌呤。
  4. 根据权利要求1所述的方法，其特征在于，所述嘌呤核苷磷酸化酶、所述尿嘧啶核苷磷酸化酶或所述嘧啶核苷磷酸化酶中的一种或多种为纯化蛋白、粗酶液或固定化酶。
  5. 根据权利要求1所述的方法，其特征在于，所述一锅法生物合成的催化时间为4～20h；
    优选地，所述一锅法生物合成的催化温度为50～80℃，更优选为60～70℃；
    优选地，所述阿糖尿苷的浓度为2～320mM，所述底物碱基的浓度为1～200mM，所述底物磷酸盐的浓度为1～100mM。
  6. 根据权利要求1所述的方法，其特征在于，所述阿糖类核苷包括2，6-二氨基嘌呤阿拉伯糖苷、奈拉滨、2-氯腺嘌呤阿拉伯糖苷或氟达拉滨；
    优选地，所述底物磷酸盐包括磷酸一氢钠、磷酸二氢钠、磷酸一氢钾或磷酸二氢钾中的一种或多种。
  7. 一种组合物，其特征在于，所述组合物包括尿嘧啶核苷磷酸化酶或嘧啶核苷磷酸化酶、以及嘌呤核苷磷酸化酶；
    所述尿嘧啶核苷磷酸化酶包括UP、或与所述UP具有80％以上同源性且具有相同功能的蛋白质，所述UP为SEQ ID NO：1所示的蛋白质；
    所述嘧啶核苷磷酸化酶包括PyNP、或与所述PyNP具有80％以上同源性且具有相同功能的蛋白质，所述PyNP为SEQ ID NO：2所示的蛋白质；
    所述嘌呤核苷磷酸化酶包括PNP、或与所述PNP具有80％以上同源性且具有相同功能的蛋白质，所述PNP为SEQ ID NO：3所示的蛋白质。
  8. 根据权利要求7所述的组合物，其特征在于，所述嘌呤核苷磷酸化酶、所述尿嘧啶核苷磷酸化酶或所述嘧啶核苷磷酸化酶中的一种或多种为纯化蛋白、粗酶液或固定化酶。
  9. 根据权利要求7所述的组合物，其特征在于，所述组合物还包括阿糖尿苷、底物碱基和底物磷酸盐。
  10. 根据权利要求9所述的组合物，其特征在于，所述底物碱基为式I所示的碱基，R1选自-NH2、＝O、-OMe或-Cl，R2选自-H、-NH2、-Cl或-F；
    优选地，所述底物碱基包括2，6-二氨基嘌呤、2-氨基-6-甲氧基嘌呤、2-氯腺嘌呤或2-氟腺嘌呤；
    优选地，所述底物磷酸盐包括磷酸一氢钠、磷酸二氢钠、磷酸一氢钾或磷酸二氢钾中的一种或多种；
    优选地，所述阿糖尿苷的浓度为2～320mM，所述底物碱基的浓度为1～200mM，所述底物磷酸盐的浓度为1～100mM。
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