WO2024040628A1 - 酶催化合成嘌呤核苷的方法及组合物 - Google Patents

酶催化合成嘌呤核苷的方法及组合物 Download PDF

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WO2024040628A1
WO2024040628A1 PCT/CN2022/115944 CN2022115944W WO2024040628A1 WO 2024040628 A1 WO2024040628 A1 WO 2024040628A1 CN 2022115944 W CN2022115944 W CN 2022115944W WO 2024040628 A1 WO2024040628 A1 WO 2024040628A1
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nucleoside
phosphorylase
purine
substrate
protein
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French (fr)
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洪浩
詹姆斯•盖吉
肖毅
张娜
焦学成
李明记
李�瑞
张燕青
丁是卯
杨益明
李娟�
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凯莱英医药集团（天津）股份有限公司
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    • 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
    • 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

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  • This application is based on the Chinese application with CN application number 202211021644.
  • the present invention relates to the field of enzyme catalysis, and specifically to a method and composition for enzyme-catalyzed synthesis of purine nucleosides.
  • Purine nucleosides and their analogs can be widely used in the pharmaceutical field as precursors or final products, such as antiviral, antitumor and other drugs.
  • purine nucleosides and their analogs are mainly synthesized by chemical methods (WO2021186328A1, CN113173961A, CN111592542A, CN111253455A, CN110627729A, CN102127135A, WO2010113937A1, WO2009058800A2, CN101250 210A, US2005171126A1, EP1108724A2), its synthesis process is complicated and requires strict reaction conditions. , and the purification steps of products synthesized by chemical methods are complex, and it is difficult to separate their isomers, which will lead to low yields and higher costs.
  • chemical synthesis uses toxic reagents, which often causes great damage to the environment.
  • purine nucleosides can also be produced by microbial fermentation (CN112553135A, CN112574934A, CN113151238A, CN113278596A, CN112143751A, CN112126666A, CN113373100A, CN102171346, CN101120090A, CN183 1115A, CN1270631A).
  • microbial fermentation CN112553135A, CN112574934A, CN113151238A, CN113278596A, CN112143751A, CN112126666A, CN113373100A, CN102171346, CN101120090A, CN183 1115A, CN1270631A.
  • the production of purine nucleosides by microbial fermentation is mostly limited to the production of natural nucleosides such as adenosine, guanosine and inosine, which is time-consuming and has low yield. It is
  • patent EP1457568A1 discloses a biological enzyme catalytic method that can utilize N-deoxyribosyl transferase II (nucleoside deoxyribosyl transferase II) and nucleosidase ( nucleosidase) reacts with thymidine and guanine as substrates for 32 hours to synthesize 31mM 2'-deoxyguanosine.
  • Patent CN102770532A discloses a method for bioconverting pyrimidine nucleosides and purine bases using pyrimidine nucleoside phosphorylase and purine nucleoside phosphorylase, which can convert uridine or 2'-deoxyuridine into purine bases.
  • Patent CN1207417A utilizes pyrimidine nucleoside phosphorylase and purine nucleoside phosphorylase derived from Bacillus stearothermophilus JTS859 and uracil and thymine decomposing enzyme liquid derived from Arthrobacter to use thymidine and corresponding The purine base is used as the substrate. After 110 hours of reaction, 14.9mM 2'-deoxyguanosine can be produced with a yield of 75%. After 90 hours of reaction, 17.7mM 2'-deoxyadenosine can be produced with a yield of 88.5%.
  • the above disclosed enzymatic synthesis has problems such as low conversion rate, low yield or long reaction time.
  • the main purpose of the present invention is to provide a method and composition for enzymatic synthesis of purine nucleosides, so as to solve the problem of low yield of enzymatic synthesis of purine nucleosides in the prior art.
  • a method for enzymatically synthesizing purine nucleosides which method utilizes purine nucleoside phosphorylase and any one of the following enzymes: pyrimidine nucleoside phosphorylase or Thymidine phosphorylase catalyzes the synthesis of purine nucleosides from substrates; substrates include substrate nucleosides and substrate bases; pyrimidine nucleoside phosphorylase includes PyNP, or has more than 80% homology with PyNP and has the same function Protein, PyNP is the protein shown in SEQ ID NO: 1, or a protein with more than 80% homology with TP and has the same function; thymidine phosphorylase includes TP, or has more than 80% homology with TP For proteins with the same function, TP is the protein shown in SEQ ID NO: 2; purine nucleoside phosphorylase includes PNP, or proteins with more than 80% homology with P
  • the substrate nucleoside is a nucleoside shown in formula I, R 1 is selected from -H or -OH, R 2 is selected from -H or -CH 3 ; preferably, the substrate nucleoside includes thymidine, uridine or 2'-deoxyuridine.
  • the substrate base is a base shown in formula II or formula III, X is selected from -NH 3 or -OCH 3 , Y is selected from -H or -NH 2 ; preferably, the substrate base includes guanine , adenine, 2,6-diaminopurine or 6-methoxyguanine;
  • one or more of PyNP, TP, or PNP is purified protein, crude enzyme solution, or immobilized enzyme.
  • the concentration of the substrate nucleoside is 2-400mM
  • the concentration of the substrate base is 1-200mM
  • the catalytic time of enzyme catalysis is 2 to 18 hours.
  • the catalytic temperature of enzyme catalysis is 60-70°C.
  • composition which composition includes a purine nucleoside phosphorylase and any one of the following enzymes: pyrimidine nucleoside phosphorylase or thymidine phosphorylase , among which, pyrimidine nucleoside phosphorylase is PyNP, PyNP is the protein shown in SEQ ID NO: 1; thymidine phosphorylase is TP, TP is the protein shown in SEQ ID NO: 2; purine nucleoside phosphorylase is PNP, and PNP is the protein shown in SEQ ID NO: 3.
  • pyrimidine nucleoside phosphorylase is PyNP
  • PyNP is the protein shown in SEQ ID NO: 1
  • thymidine phosphorylase is TP
  • TP is the protein shown in SEQ ID NO: 2
  • purine nucleoside phosphorylase is PNP
  • PNP is the protein shown in SEQ ID NO: 3.
  • one or more of pyrimidine nucleoside phosphorylase, thymidine phosphorylase TP or purine nucleoside phosphorylase PNP is a purified protein, crude enzyme solution or immobilized enzyme; preferably, the composition includes PyNP
  • TP or PNP is purified protein, crude enzyme solution or immobilized enzyme.
  • the composition also includes a substrate nucleoside and a substrate base;
  • the substrate nucleoside is a nucleoside shown in formula I, R 1 is selected from -H or -OH, and R 2 is selected from -H or -CH 3 ;
  • the substrate base is a base represented by Formula II or Formula III, X is selected from -NH 3 or -OCH 3 , Y is selected from -H or -NH 2 ;
  • the concentration of the substrate nucleoside is 2 ⁇ 400mM, the concentration of substrate base is 1 ⁇ 200mM;
  • Figure 1 shows a schematic diagram of the standard curve of 2'-deoxyadenosine according to Example 5 of the present invention.
  • Figure 2 shows a schematic diagram of the standard curve of 2'-deoxyguanosine according to Embodiment 7 of the present invention.
  • Figure 3 shows a high performance liquid chromatogram of 2'-deoxyadenosine according to Example 5 of the present invention.
  • Figure 4 shows a high performance liquid chromatogram of 2'-deoxyadenosine according to Example 6 of the present invention.
  • the existing enzymatic synthesis route for nucleoside phosphorylase has many reaction steps, is cumbersome to operate, takes a long time, and has low yield and high production cost.
  • the enzymatic conversion involved in patent EP1457568A1 requires a two-step reaction and three enzymes, takes 30 hours, and the yield of 2'-deoxyguanosine is only 40mM; the enzymatic conversion published in CN1207417A takes more than 90 hours, and the yield of purine nucleosides is It is relatively low, and additional enzyme liquid with uracil and thymine decomposition activity needs to be prepared, which increases the production cost. Without addition, the output and yield will be significantly reduced, only 13% to 50% of those when added.
  • a method for enzyme-catalyzed synthesis of purine nucleosides which method utilizes purine nucleoside phosphorylase and any of the following enzymes: pyrimidine nucleoside phosphorylase or thymidine Phosphorylase, catalyzes the synthesis of purine nucleosides from substrates; substrates include substrate nucleosides and substrate bases; pyrimidine nucleoside phosphorylase includes PyNP, PyNP is the protein shown in SEQ ID NO: 1; thymidine phosphate
  • the purine nucleoside phosphorylase includes TP, which is the protein shown in SEQ ID NO: 2; the purine nucleoside phosphorylase includes PNP, which is the protein shown in SEQ ID NO: 3.
  • the above-mentioned pyrimidine nucleoside phosphorylase, thymidine phosphorylase or purine nucleoside phosphorylase all include 80%, 85%, 90%, 95%, 98%, 99%, 99.5 with PyNP, TP or PNP respectively. % or more than 99.9% homology and have the same function.
  • the pyrimidine nucleoside phosphorylase used in this application is derived from Thermus thermophilus and is the protein shown in SEQ ID NO: 1.
  • This pyrimidine nucleoside phosphorylase is named PyNP;
  • the thymidine phosphorylase is derived from Homo sapiens is the protein shown in SEQ ID NO: 2, and this thymidine phosphorylase is named TP;
  • the purine nucleoside phosphorylase is derived from Geobacillus stearothermophilus, and is the protein shown in SEQ ID NO: 3
  • the protein shown is named PNP for this purine nucleoside phosphorylase.
  • nucleoside phosphatases from two different sources are used to efficiently synthesize purine nucleosides in one step, which can reduce production costs and be applied to large-scale industrial production of related products.
  • the above-mentioned substrate includes a substrate nucleoside and a substrate base, and may be a natural nucleoside or base structure, or may be an artificially modified similar structure.
  • the above-mentioned A reaction is carried out under the catalysis of PyNP or TP, the C-N bond between the pyrimidine group and the ribose group of the substrate nucleoside is broken, and a phosphate group is combined with the nucleoside group. , forming the ribose phosphate intermediate.
  • the ribose phosphate intermediate undergoes the above-mentioned B reaction under the action of PNP, causing the C-1' atom of the ribose group to form a C-N bond with the N-9 atom of the purine substrate base to generate a purine nucleoside compound.
  • the reaction can be completed using only substrates and proteins.
  • the reaction is simple and does not need to be performed in living cells, greatly reducing reaction costs and efficiency.
  • the enzymatic catalysis method performed by combining these two proteins can complete the catalytic reaction in a short catalytic time, and has a high substrate conversion rate. It is also suitable for amplification reactions and can be used for large-scale industrial production.
  • the substrate nucleoside is a nucleoside shown in formula I, R 1 is selected from -H or -OH, R 2 is selected from -H or -CH 3 ; preferably, the substrate nucleoside Includes thymidine, uridine or 2'-deoxyuridine;
  • the substrate base is a base represented by Formula II or Formula III, X is selected from -NH 3 or -OCH 3 , Y is selected from -H or -NH 2 , preferably, the base base is Chemical bases include guanine, adenine, 2,6-diaminopurine or 6-methoxyguanine,
  • this enzyme catalytic method can be used to prepare 2'-deoxyadenosine, 2'-deoxyguanosine, 2,6-diaminopurine nucleoside, and 2,6-diamino Purine nucleosides such as purine-2'-deoxynucleoside and 6-O-methyl-guanosine.
  • the R1 , R2 , Purine nucleosides are The R1 , R2 , Purine nucleosides.
  • one or more of PyNP, TP or PNP is purified protein, crude enzyme solution or immobilized enzyme.
  • the above three proteins can exist in various forms such as purified protein, crude enzyme solution or immobilized enzyme, and can all catalyze the synthesis of purine nucleosides.
  • the genes expressing PyNP and/or TP and/or PNP are cloned into host cells. After inducing protein expression, the host cells are disrupted 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 catalytic reactions.
  • the concentration of the substrate nucleoside is 2-400mM, and the concentration of the substrate base is 1-200mM.
  • the above-mentioned enzyme-catalyzed method can carry out amplification reactions.
  • the concentration of the substrate nucleoside can be up to 400mM, and the concentration of the substrate base can be up to 200mM, thereby enabling large-scale preparation of purine nucleosides.
  • the catalytic time of enzyme catalysis is 2 to 18 hours.
  • the catalytic temperature of enzyme catalysis is 60-70°C.
  • the enzyme-catalyzed reaction can be completed, and the conversion rate of the substrate base, that is, the reaction yield is relatively high. There is no need to add enzymes or other reagents during the reaction, and the reaction can be completed through one-step catalysis, which is suitable for application in industrial scale-up production.
  • composition which composition includes a purine nucleoside phosphorylase and any one of the following enzymes: pyrimidine nucleoside phosphorylase or thymidine phosphorylase, wherein , pyrimidine nucleoside phosphorylase is PyNP, PyNP is the protein shown in SEQ ID NO: 1; thymidine phosphorylase is TP, TP is the protein shown in SEQ ID NO: 2; purine nucleotide phosphorylase is PNP, PNP is the protein shown in SEQ ID NO: 3.
  • the composition is used to synthesize purine nucleosides.
  • the composition includes purified protein, crude enzyme solution or immobilized enzyme.
  • the composition also includes a reaction between a substrate nucleoside and a substrate base to generate a purine nucleoside;
  • the substrate nucleoside is a nucleoside shown in formula I, and R 1 is selected from -H or - OH, R 2 is selected from -H or -CH 3 ;
  • the substrate base is a base shown in Formula II or Formula III, X is selected from -NH 3 or -OCH 3 , Y is selected from -H or -NH 2 ;
  • the concentration of the substrate nucleoside is 2-400mM, and the concentration of the substrate base is 1-200mM;
  • PyNP is a pyrimidine nucleoside phosphorylase derived from Thermus thermophilus, and its amino acid sequence is shown in SEQ ID NO: 1.
  • TP is thymidine phosphorylase derived from Homo sapiens, and its amino acid sequence is shown in SEQ ID NO: 2.
  • PNP is a purine nucleoside phosphorylase derived from Geobacillus stearothermophilus, and its amino acid sequence is shown in SEQ ID NO: 3.
  • the composition includes PyNP and PNP, or TP and PNP.
  • the composition can catalyze the reaction of nucleosides such as thymidine and uridine with bases such as guanine and adenine to produce purine nucleosides and their analogs.
  • the protein in the composition can be independently selected from purified protein, crude enzyme solution or immobilized enzyme, all of which can exert a catalytic effect.
  • the pyrimidine nucleoside phosphorylase used in this application is derived from Thermus thermophilus, named PyNP, and the amino acid sequence is shown in SEQ ID NO: 1.
  • Thymidine phosphorylase is derived from Homo sapiens, named TP, and its amino acid sequence is shown in SEQ ID NO: 2.
  • Purine nucleoside phosphorylase is derived from Geobacillus stearothermophilus, named PNP, and its amino acid sequence is shown in SEQ ID NO: 3.
  • the DNA sequences encoding the two enzymes were obtained.
  • the DNA sequence encoding PyNP is SEQ ID NO: 4; the DNA sequence encoding TP is SEQ ID NO: 5; and the DNA sequence encoding PNP is SEQ ID NO: 6. They were cloned into the expression vector pET28a(+) respectively. The obtained plasmid was transferred into the competent E. coli BL21 (DE3) host to obtain a single clonal strain.
  • E. coli strains expressing PyNP, TP, and PNP were inoculated into test tubes respectively, cultured at 37°C for 16 hours, and then inoculated into a 2L shake flask containing 500 mL LB liquid medium at an inoculation volume of 1v/v%, and cultured at 37°C until OD 600 was At 0.6, a final concentration of 0.1M isopropyl- ⁇ -D-thiogalactopyranoside was added to induce protein expression and cultured at 20°C for 18 h. Centrifuge the cultured bacterial solution at 7000 rpm for 10 minutes to collect the bacterial cells for later use.
  • flow rate is 1mL/min
  • column temperature is 40°C
  • UV detector detection wavelength is 254nm
  • detection time is 15 minutes.
  • Example 5 After performing Example 5, add 1.35g of adenine and 135.0mg of enzyme solution prepared from PNP bacterial mud, and continue the reaction for 24 hours. After the reaction, take 1 mL of the reaction solution and add an equal volume of DMSO to dissolve it, dilute it 20 times and send it to HPLC for detection. As shown in Figure 4, the thymidine conversion rate is 74.97% and the purine conversion rate is 81.30%. According to the results shown in Figure 1 Standard curve, calculated 2'-deoxyadenosine production is 68.3g/L.
  • the substrate base and the substrate nucleoside can be used as substrates in a short catalytic time , synthesize purine nucleoside products, and can scale up production, and efficiently synthesize 2'-deoxyadenosine, 2'-deoxyguanosine, 2,6-diaminopurine nucleoside, 2,6 in a short reaction time -Diaminopurine-2'-deoxynucleoside and 6-O-methyl-guanosine, the yields can reach 68.3g/L, 17.5g/L, 54.3g/L, 59.6g/L and 58.6g/L respectively .

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Abstract

一种酶催化合成嘌呤核苷的方法及组合物。其中，该方法利用嘌呤核苷磷酸化酶以及如下任一种酶：嘧啶核苷磷酸化酶或胸苷磷酸化酶，催化底物合成嘌呤核苷；底物包括底物核苷和底物碱基；嘧啶核苷磷酸化酶包括PyNP，PyNP为SEQ ID NO：1所示的蛋白质；胸苷磷酸化酶包括TP，TP为SEQ ID NO：2所示的蛋白质；嘌呤核苷磷酸化酶包括PNP，PNP为SEQ ID NO：3所示的蛋白质。能够解决现有技术中的利用酶法合成嘌呤核苷产量低的问题，适用于酶催化领域。

Description

酶催化合成嘌呤核苷的方法及组合物
本申请是以CN申请号为202211021644.X，申请日为2022年8月24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，涉及一种酶催化合成嘌呤核苷的方法及组合物。
背景技术
嘌呤核苷及其类似物可作为前体或者终产品广泛应用于医药领域，如抗病毒，抗肿瘤等药物。目前，嘌呤核苷及其类似物主要以化学法合成(WO2021186328A1、CN113173961A、CN111592542A、CN111253455A、CN110627729A、CN102127135A，WO2010113937A1，WO2009058800A2、CN101250210A、US2005171126A1、EP1108724A2)，其合成工艺较为繁琐，需要严苛的反应条件，且化学法合成的产品纯化步骤复杂，与其异构体难以分离，会导致收率不高而引起成本较高等问题。此外，化学合成会用到毒性试剂，往往对环境造成较大的破坏。
此外，嘌呤核苷还可以微生物发酵法生产(CN112553135A、CN112574934A、CN113151238A、CN113278596A、CN112143751A、CN112126666A、CN113373100A、CN102171346、CN101120090A、CN1831115A、CN1270631A)。目前微生物发酵法生产嘌呤类核苷多限于天然核苷如腺苷，鸟苷和肌苷等的生产，且耗时长，产量较低，离工业化生产还有一定的距离。
近些年，嘌呤核苷的酶法合成由于步骤少，收率较高，光学纯度高，环境友好等优点，可以避免上述的问题而受到较多的关注。嘌呤核苷的酶法合成可经多种路线使用不同酶得以实现，如专利EP1457568A1公开了一种生物酶催化方法，可以利用N-脱氧核糖转移酶II(nucleoside deoxyribosyl transferase II)和核苷酶(nucleosidase)以胸苷和鸟嘌呤为底物反应32h合成31mM 2'-脱氧鸟苷。此外，该专利还以上述两种酶并结合腺苷脱氨酶(adenosine deaminase)用两步法反应约30h最终合成超过40mM 2'-脱氧鸟苷。此外，嘧啶核苷磷酸化酶(pyrimidine nucleoside phosphorylase)和嘌呤核苷磷酸化酶(purine nucleoside phosphorylase)也可以被用于合成嘌呤核苷。专利CN102770532A公开了一种可以利用嘧啶核苷磷酸化酶和嘌呤核苷磷酸化酶将嘧啶核苷和嘌呤碱基进行生物转化的方法，可以将尿苷或2'-脱氧尿苷和嘌呤碱基转化成2,6-二氨基嘌呤核苷和2,6-二氨基嘌呤-2'-脱氧核苷，转化率超过90％，但产量低于5mM。专利CN1207417A利用芽孢杆菌属脂肪嗜热杆菌(Bacillus stearothermophilus JTS859)来源的嘧啶核苷磷酸化酶和嘌呤核苷磷酸化酶以及节细菌属来源的尿嘧啶和胸腺嘧啶分解酶液，以胸苷和相应的嘌呤碱基为底物，反应110小时可生产14.9mM 2'-脱氧鸟苷，产率为75％，反应90小时可得到17.7mM的2'-脱氧腺苷，产率为88.5％。但上述公开的酶法合成，均具有转化率低、产量低或反应时间长等的问题。
发明内容
本发明的主要目的在于提供一种酶催化合成嘌呤核苷的方法及组合物，以解决现有技术中的酶法合成嘌呤核苷产量低的问题。
为了实现上述目的，根据本发明的第一个方面，提供了一种酶催化合成嘌呤核苷的方法，该方法利用嘌呤核苷磷酸化酶以及如下任一种酶：嘧啶核苷磷酸化酶或胸苷磷酸化酶，催化底物合成嘌呤核苷；底物包括底物核苷和底物碱基；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：1所示的蛋白质、或与TP具有80％以上同源性且具有相同功能的蛋白质；胸苷磷酸化酶包括TP、或与TP具有80％以上同源性且具有相同功能的蛋白质，TP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，底物核苷为式I所示的核苷，R 1选自-H或-OH，R 2选自-H或-CH 3；优选地，底物核苷包括胸苷，尿苷或2'-脱氧尿苷。
Figure PCTCN2022115944-appb-000001
进一步地，底物碱基为式II或式III所示的碱基，X选自-NH 3或-OCH 3，Y选自-H或-NH 2；优选地，底物碱基包括鸟嘌呤，腺嘌呤、2,6-二氨基嘌呤或6-甲氧基鸟嘌呤；
Figure PCTCN2022115944-appb-000002
进一步地，PyNP、TP、或PNP中的一种或多种为纯化蛋白、粗酶液或固定化酶。
进一步地，上述方法中：底物核苷的浓度为2～400mM，底物碱基的浓度为1～200mM。
进一步地，酶催化的催化时间为2～18h。
进一步地，酶催化的催化温度为60～70℃。
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，该组合物包括嘌呤核苷磷酸化酶以及如下任一种酶：嘧啶核苷磷酸化酶或胸苷磷酸化酶，其中，嘧啶核苷磷酸化酶为PyNP，PyNP为SEQ ID NO：1所示的蛋白质；胸苷磷酸化酶为TP，TP为SEQ ID NO：2所示的蛋白质；嘌呤核苷磷酸化酶为PNP，PNP为SEQ ID NO：3所示的蛋白质。
进一步地，嘧啶核苷磷酸化酶、胸苷磷酸化酶TP或嘌呤核苷磷酸化酶PNP中的一种或多种为纯化蛋白、粗酶液或固定化酶；优选地，组合物包括PyNP、TP或PNP中的一种或多种为纯化蛋白、粗酶液或固定化酶。
进一步地，组合物还包括底物核苷和底物碱基；底物核苷为式I所示的核苷，R 1选自-H或-OH，R 2选自-H或-CH 3；底物碱基为式II或式III所示的碱基，X选自-NH 3或-OCH 3，Y选自-H或-NH 2；优选地，底物核苷的浓度为2～400mM，底物碱基的浓度为1～200mM；
Figure PCTCN2022115944-appb-000003
应用本发明的技术方案，利用嘧啶核苷磷酸化酶和嘌呤核苷磷酸化酶，或胸苷磷酸化酶和嘌呤核苷磷酸化酶，包括PyNP和PNP、或TP和PNP，共同进行酶催化，以底物核苷和底物碱基为原料，一步法催化生成多种嘌呤核苷，并能够实现放大生产，从而在较短的反应时间内高效合成，提高嘌呤核苷的产量。
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5的2'-脱氧腺苷的标准曲线示意图。
图2示出了根据本发明实施例7的2'-脱氧鸟苷的标准曲线示意图。
图3示出了根据本发明实施例5的2'-脱氧腺苷的高效液相色谱图。
图4示出了根据本发明实施例6的2'-脱氧腺苷的高效液相色谱图。
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，现有技术中合成嘌呤核苷的方法，均具有成本高、耗时长、产量低等问题，不利于嘌呤核苷及其类似物的制备和后续利用。
现有的酶法合成核苷磷酸化酶路线反应步骤多，操作繁琐，耗时较长，产量产率较低造成生产成本较高。如专利EP1457568A1涉及的酶法转化需要两步反应和三种酶，耗时30h，2'-脱氧鸟苷产量仅为40mM；CN1207417A公布的酶法转化耗时90小时以上，而且嘌呤核苷的产量较低，还需要额外制备具有尿嘧啶和胸腺嘧啶分解活性的酶液，增加了生产成本，如无添加则产量和产率显著降低，仅为添加时的13％～50％。专利CN102770532A公布的方法仅能得到低于5mM的2,6-二氨基嘌呤核苷和2,6-二氨基嘌呤-2'-脱氧核苷。而由于底物嘌呤碱基溶解度较低，酶不稳定和中间产物(核糖-1-磷酸或2’-脱氧核糖-1-磷酸)易被破坏等问题，单纯的对反应进行放大或延长时间可能使反应平衡被破坏，酶失活等，最终得到的转化率和收率偏低，使得生产成本增加，因而难以被应用到实际的商业生产中。
为改善上述现状，在本申请中，发明人尝试探究新的酶催化合成嘌呤核苷的方法，从大量具有潜在活性的相关蛋白中，发现了PyNP(嘧啶核苷磷酸化酶，如SEQ ID NO：1所示的蛋白质)和PNP(嘌呤核苷磷酸化酶，如SEQ ID NO：3所示的蛋白质)及TP(胸苷磷酸化酶包括，如SEQ ID NO：2所示的蛋白质)和PNP组合作用的催化活性，发现利用PyNP，TP和PNP的酶液能够一步法催化底物核苷和底物碱基，生成目标产物嘌呤核苷。该酶催化方法反应时间短，合成效率高，因而得到的产品产量高。在此基础上，申请人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，提供了一种酶催化合成嘌呤核苷的方法，该方法利用嘌呤核苷磷酸化酶以及如下任一种酶：嘧啶核苷磷酸化酶或胸苷磷酸化酶，，催化底物合成嘌呤核苷；底物包括底物核苷和底物碱基；嘧啶核苷磷酸化酶包括PyNP，PyNP为SEQ ID NO：1所示的蛋白质；胸苷磷酸化酶包括TP，TP为SEQ ID NO：2所示的蛋白质；嘌呤核苷磷酸化酶包括PNP，PNP为SEQ ID NO：3所示的蛋白质。上述嘧啶核苷磷酸化酶、胸苷磷酸化酶或嘌呤核苷磷酸化酶，均分别包括与PyNP、TP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的蛋白质。
本申请中所使用嘧啶核苷磷酸化酶来源于嗜热栖热菌Thermus thermophilus，为SEQ ID NO：1所示的蛋白质，将此嘧啶核苷磷酸化酶命名PyNP；胸苷磷酸化酶来源于智人Homo sapiens，为SEQ ID NO：2所示的蛋白质，将此胸苷磷酸化酶命名TP；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，将此嘌呤核苷磷酸化酶命名PNP。在上述酶催化方法中，利用两种不同来源的核苷磷酸酶，一步法高效合成嘌呤核苷，能够降低生产成本，并应用于大规模工业生产相关产品。上述底物包括底物核苷和底物碱基，可以为天然的核苷或碱基结构，也可以为人为改造的类似结构。
Figure PCTCN2022115944-appb-000004
在该酶催化方法中，在PyNP或TP的催化下进行上述A反应，将底物核苷的嘧啶基团和核糖基团间的C-N键断开，并在核苷基团上结合磷酸基团，形成磷酸核糖中间体。该磷酸核糖中间体在PNP的作用下，进行上述B反应，使核糖基团的C-1'原子与嘌呤类底物碱基的N-9原子形成C-N键，生成嘌呤核苷类化合物。在上述A和B反应中，不需要额外添加NADPH等辅酶，仅利用底物和蛋白即可完成反应，反应简单，无需在活细胞中进行，大大降低反应成本和效率。此2种蛋白组合进行的该酶催化方法，能够在较短的催化时间内完成催化反应，且底物的转化率高，在放大反应中同样适用，能够大规模的进行工业生产。
在一种优选的实施例中，底物核苷为式I所示的核苷，R 1选自-H或-OH，R 2选自-H或-CH 3；优选地，底物核苷包括胸苷，尿苷或2'-脱氧尿苷；
Figure PCTCN2022115944-appb-000005
在一种优选的实施例中，底物碱基为式II或式III所示的碱基，X选自-NH 3或-OCH 3，Y选自-H或-NH 2，优选地，底物碱基包括鸟嘌呤，腺嘌呤、2,6-二氨基嘌呤或6-甲氧基鸟嘌呤，
Figure PCTCN2022115944-appb-000006
利用上述底物核苷和底物碱基，能够利用该酶催化方法，制备2'-脱氧腺苷，2'-脱氧鸟苷，2,6-二氨基嘌呤核苷，2,6-二氨基嘌呤-2'-脱氧核苷和6-O-甲基-鸟苷等嘌呤核苷。
上述底物核苷和底物碱基中的R 1、R 2、X和Y取代基均距离反应位点较远，因此可以利 用PyNP和PNP对不同的底物进行酶催化反应，制备多种嘌呤核苷。
在一种优选的实施例中，PyNP、TP或PNP中的一种或多种为纯化蛋白、粗酶液或固定化酶。
利用PyNP，TP和PNP催化反应，上述3种蛋白可以以纯化蛋白、粗酶液或固定化酶等多种形式存在，均能够催化合成嘌呤核苷。将表达PyNP和/或TP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，在上述方法中，底物核苷的浓度为2～400mM，底物碱基的浓度为1～200mM。
上述酶催化方法，能够进行放大反应，在反应体系中，底物核苷的浓度最大为400mM，底物碱基的浓度最大为200mM，从而进行大规模的嘌呤核苷的制备。
在一种优选的实施例中，酶催化的催化时间为2～18h。
在一种优选的实施例中，酶催化的催化温度为60～70℃。
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，一步法催化即能够完成反应，适宜在工业化放大生产上的应用。
在本申请第二种典型的实施方式中，提供了一种组合物，该组合物包括嘌呤核苷磷酸化酶以及如下任一种酶：嘧啶核苷磷酸化酶或胸苷磷酸化酶，其中，嘧啶核苷磷酸化酶为PyNP，PyNP为SEQ ID NO：1所示的蛋白质；胸苷磷酸化酶为TP，TP为SEQ ID NO：2所示的蛋白质；嘌呤核苷酸磷酸化酶为PNP，PNP为SEQ ID NO：3所示的蛋白质。该组合物用于合成嘌呤核苷。
在一种优选的实施例中，组合物包括纯化蛋白、粗酶液或固定化酶。
在一种优选的实施例中，组合物还包括底物核苷和底物碱基反应，生成嘌呤核苷；底物核苷为式I所示的核苷，R 1选自-H或-OH，R 2选自-H或-CH 3；底物碱基为式II或式III所示的碱基，X选自-NH 3或-OCH 3，Y选自-H或-NH 2；优选地，底物核苷的浓度为2～400mM，底物碱基的浓度为1～200mM；
Figure PCTCN2022115944-appb-000007
PyNP为来源于嗜热栖热菌Thermus thermophilus的嘧啶核苷磷酸化酶，氨基酸序列如SEQ ID NO：1所示。TP为来源于智人Homo sapiens的胸苷磷酸化酶，氨基酸序列如SEQ ID NO：2所示。PNP为来源于嗜热脂肪地芽孢杆菌Geobacillus stearothermophilus的嘌呤核苷磷酸化酶，氨基酸序列如SEQ ID NO：3所示。组合物包括PyNP和PNP、或TP和PNP，利用该组合物，能够催化胸苷、尿苷等核苷与鸟嘌呤、腺嘌呤等碱基完成反应，生产嘌呤核苷及其类似物。组合物中的蛋白，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。
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
本申请中所使用嘧啶核苷磷酸化酶来源于嗜热栖热菌Thermus thermophilus，命名PyNP，氨基酸序列如SEQ ID NO：1所示。胸苷磷酸化酶来源于智人Homo sapiens，命名TP，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两个酶的DNA序列，编码PyNP的DNA序列为SEQ ID NO：4；编码TP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。
Figure PCTCN2022115944-appb-000008
Figure PCTCN2022115944-appb-000009
Figure PCTCN2022115944-appb-000010
2.蛋白表达
将表达PyNP、TP、和PNP的大肠杆菌菌株分别接种于试管，37℃培养16h后以1v/v％接种量，接种于含500mL LB液体培养基的2L摇瓶，37℃培养至OD 600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.HPLC检测方法
色谱柱Atlantis T3Column，4.6mm x 150mm，流动相为含有0.1v/v％三氟乙酸(TFA)的甲醇，流速为1mL/min，柱温40℃，UV检测器，检测波长254nm，检测时长为15min。
实施例2嘌呤核苷及其类似物的酶催化
在2mM磷酸缓冲液(pH 7.5)配制1mL反应体系，其中包括2mM胸苷或尿苷，以及1mM嘌呤或嘌呤类似物不同重量菌泥制备的PyNP酶液和不同重量菌泥制备的PNP酶液，60℃反应18h。反应结束后，加入1mL DMSO，送HPLC检测，实验结果参见表1。结果显示，腺嘌呤和嘌呤类似物反应转化率均高于79％，其中在2,6-二氨基嘌呤核苷和2,6-二氨基嘌呤-2'-脱氧核苷的合成中，2,6-二氨基嘌呤的转化率可达96％。
表1.嘌呤核苷及其类似物的酶催化
Figure PCTCN2022115944-appb-000011
Figure PCTCN2022115944-appb-000012
实施例3提高底物浓度对嘌呤核苷及其类似物酶催化的影响
在2mM磷酸缓冲液(pH 7.5)配制1mL反应体系，其中包括4mM胸苷或尿苷，以及1mM嘌呤或嘌呤类似物，不同重量菌泥制备的PyNP酶液和不同重量菌泥制备的PNP酶液，70℃反应2h。反应结束后，加入1mL DMSO，送HPLC检测，实验结果参见表2。结果显示，提高胸苷的底物浓度并缩短时间至2h，腺嘌呤和嘌呤类似物反应转化率仍有提高，均高于90％。
表2.酶量对嘌呤核苷及其类似物酶催化的影响
Figure PCTCN2022115944-appb-000013
实施例4 2'-脱氧腺苷合成反应酶量的优化
在2mM磷酸缓冲液(pH 7.5)配制1mL反应体系，其中包括400mM胸苷，200mM腺嘌呤，9.7～48.4mg菌泥制备的PyNP酶液和2.7～13.5mg菌泥制备的PNP酶液，60℃反应16h。反应结束后，加入1mL DMSO，送HPLC检测，实验结果参见表3。结果显示，当使用较低酶量时，嘌呤转化率仍可维持90％以上。例如，使用9.7mg菌泥制备的PyNP酶液和2.7mg菌泥制备的PNP酶液时，转化率为93.17％。
表3. 2'-脱氧腺苷合成反应酶量的优化
Figure PCTCN2022115944-appb-000014
Figure PCTCN2022115944-appb-000015
实施例5 2'-脱氧腺苷合成的放大反应
在2mM磷酸缓冲液(pH 7.5)配制50mL反应体系，其中包括400mM胸苷(Thymidine)，200mM腺嘌呤(Adenine)，484.0mg菌泥制备的PyNP酶液和135.0mg菌泥制备的PNP酶液，60℃反应16h，生成胸腺嘧啶(Thymine)和2'-脱氧腺苷(2'-Deoxyadenosine)。反应结束后，取1mL反应液加入等体积的DMSO溶解，稀释20倍后送HPLC检测，如图3所示，胸苷转化率为39.20％，嘌呤转化率为94.72％，根据图1所示的标准曲线，计算2'-脱氧腺苷(2'-Deoxyadenosine)产量为51.1g/L。
实施例6补加酶量对2'-脱氧腺苷合成放大反应的影响
在进行实施例5后，补加1.35g腺嘌呤和PNP菌泥135.0mg制备的酶液，继续反应24h。反应结束后，取1mL反应液加入等体积的DMSO溶解，稀释20倍后送HPLC检测，如图4所示，胸苷转化率为74.97％，嘌呤转化率为81.30％，根据图1所示的标准曲线，计算2'-脱氧腺苷产量为68.3g/L。
实施例7 2'-脱氧鸟苷的酶催化反应
1.配制浓度为1M的鸟嘌呤用NaOH溶解，向1mL 2mM磷酸缓冲液(pH 7.5)的反应体系种加入含120mM的胸苷，40mM的鸟嘌呤，58mg菌泥制备的PyNP酶液和120mg菌泥制备的PNP酶液，60℃反应16h后取样加入等体积的DMSO溶解，稀释20倍，送HPLC检测，胸苷转化率为37.77％，嘌呤转化率为86.42％，根据图2所示的标准曲线，计算2'-脱氧鸟苷最高产量为9.8g/L。
2.配制浓度为1M的鸟嘌呤用NaOH溶解，向1mL 2mM磷酸缓冲液(pH 7.5)的反应体系种加入含240mM的胸苷，80mM的鸟嘌呤，58mg菌泥制备的PyNP酶液和120mg菌泥制备的PNP酶液，60℃反应16h后取样加入等体积的DMSO溶解，稀释20倍，送HPLC检测，胸苷转化率为31.79％，嘌呤转化率为77.07％，根据图2所示的标准曲线，计算2'-脱氧鸟苷最高产量为17.5g/L。
上述实验结果参见表4。
表4. 2'-脱氧鸟苷的催化反应
Figure PCTCN2022115944-appb-000016
实施例8 2,6-二氨基嘌呤核苷合成的反应
在2mM磷酸缓冲液(pH 7.5)配制10mL反应体系，其中包括320mM尿苷，200mM2,6-二氨基嘌呤，195.4mg菌泥制备的PyNP酶液和150.1mg菌泥制备的PNP酶液，60℃反应16h。反应结束后，取1mL反应液加入等体积的DMSO溶解，稀释20倍后送HPLC检测，尿苷转化率为54.92％，嘌呤转化率为96.18％，2,6-二氨基嘌呤核苷产量为54.3g/L。
实施例9 2,6-二氨基嘌呤-2'-脱氧核苷合成的反应
在2mM磷酸缓冲液(pH 7.5)配制10mL反应体系，其中包括400mM 2'-脱氧尿苷，200mM 2,6-二氨基嘌呤，230.0mg菌泥制备的TP酶液和75.0mg菌泥制备的PNP酶液，50℃反应16h。反应结束后，取1mL反应液加入等体积的DMSO溶解，稀释20倍后送HPLC检测，2'-脱氧尿苷转化率为43.89％，嘌呤转化率为97.75％，2,6-二氨基嘌呤-2'-脱氧核苷产量为59.6g/L。
实施例10 6-O-甲基-鸟苷合成的酶催化反应
在2mM磷酸缓冲液(pH 7.5)配制10mL反应体系，其中包括320mM尿苷，200mM 6-O-甲基鸟嘌呤，195.4mg菌泥制备的PyNP酶液和165.0mg菌泥制备的PNP酶液，60℃反应24h。反应结束后，取1mL反应液加入等体积的DMSO溶解，稀释20倍后送HPLC检测，尿苷转化率为51.27％，嘌呤转化率为98.67％，6-O-甲基鸟苷产量为58.6g/L。
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：利用上述酶催化方法，能够在较短的催化时间内，以底物碱基和底物核苷为底物，合成嘌呤核苷类产物，并能够放大生产，在较短的反应时间内，高效合成2'-脱氧腺苷，2'-脱氧鸟苷，2,6-二氨基嘌呤核苷，2,6-二氨基嘌呤-2'-脱氧核苷和6-O-甲基-鸟苷，产量分别可达68.3g/L，17.5g/L，54.3g/L，59.6g/L和58.6g/L。
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. 一种酶催化合成嘌呤核苷的方法，其特征在于，所述方法利用嘌呤核苷磷酸化酶以及如下任一种酶：嘧啶核苷磷酸化酶或胸苷磷酸化酶，催化底物合成所述嘌呤核苷；
    所述底物包括底物核苷和底物碱基；
    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：1所示的蛋白质；
    所述胸苷磷酸化酶包括TP、或与所述TP具有80％以上同源性且具有相同功能的蛋白质，所述TP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。
  2. 根据权利要求1所述的方法，其特征在于，所述底物核苷为式I所示的核苷，R 1选自-H或-OH，R 2选自-H或-CH 3，
    Figure PCTCN2022115944-appb-100001
    优选地，所述底物核苷包括胸苷，尿苷或2'-脱氧尿苷。
  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II或式III所示的碱基，X选自-NH 3或-OCH 3，Y选自-H或-NH 2，
    Figure PCTCN2022115944-appb-100002
    优选地，所述底物碱基包括鸟嘌呤、腺嘌呤、2,6-二氨基嘌呤或6-甲氧基鸟嘌呤。
  4. 根据权利要求1所述的方法，其特征在于，所述嘧啶核苷磷酸化酶、所述胸苷磷酸化酶或所述嘌呤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，在所述方法中，所述底物核苷的浓度为2～400mM，所述底物碱基的浓度为1～200mM。
  6. 根据权利要求1所述的方法，其特征在于，所述酶催化的催化时间为2～18h。
  7. 根据权利要求1所述的方法，其特征在于，所述酶催化的催化温度为60～70℃。
  8. 一种组合物，其特征在于，所述组合物包括嘌呤核苷磷酸化酶以及如下任一种酶：嘧啶核苷磷酸化酶或胸苷磷酸化酶，其中，
    所述嘧啶核苷磷酸化酶为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：1所示的蛋白质；
    所述胸苷磷酸化酶为TP、或与所述TP具有80％以上同源性且具有相同功能的蛋白质，所述TP为SEQ ID NO：2所示的蛋白质；
    所述嘌呤核苷磷酸化酶为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。
  9. 根据权利要求8所述的组合物，其特征在于，所述嘧啶核苷磷酸化酶、所述胸苷磷酸化酶或所述嘌呤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。
  10. 根据权利要求8或9所述的组合物，其特征在于，所述组合物还包括底物核苷和底物碱基；
    所述底物核苷为式I所示的核苷，R 1选自-H或-OH，R 2选自-H或-CH 3；
    所述底物碱基为式II或式III所示的碱基，X选自-NH 3或-OCH 3，Y选自-H或-NH 2；
    Figure PCTCN2022115944-appb-100003
    优选地，所述底物核苷的浓度为2～400mM，所述底物碱基的浓度为1～200mM。
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