Method and composition for the synthesis of purine nucleosides by enzyme catalyst
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Patents
- Current Assignee / Owner
- ASYMCHEM LAB TIANJIN
- Filing Date
- 2022-08-30
- Publication Date
- 2026-08-05
Smart Images
Figure 0007901189000021 
Figure 0007901189000022 
Figure 0007901189000023
Abstract
Description
Technical Field
[0001] This application is based on a Chinese application with CN application number 202211021644.X and a filing date of August 24, 2022, and claims its priority. The disclosure content of the CN application is incorporated into this application as a whole again.
[0002] The present invention relates to the field of enzyme catalysis, and specifically to a method for synthesizing purine nucleosides by enzyme catalysis and a composition.
Background Art
[0003] Purine nucleosides and their analogs can be widely applied as precursors or final products in the pharmaceutical field of drugs such as antiviral and antitumor drugs. Currently, purine nucleosides and their analogs are mainly synthesized by chemical methods (WO2021186328A1, CN113173961A, CN111592542A, CN111253455A, CN110627729A, CN102127135A, WO2010113937A1, WO2009058800A2, CN101250210A, US2005171126A1, EP1108724A2). However, their synthesis process is complicated, requires strict reaction conditions, and the purification steps of the products synthesized by chemical methods are complex. It is difficult to separate them from their isomers, and problems such as low yield and high cost are caused. In addition, because toxic reagents are used in chemical synthesis, it always causes great damage to the environment.
[0004] Furthermore, purine nucleosides can also be produced by microbial fermentation (CN112553135A, CN112574934A, CN113151238A, CN113278596A, CN112143751A, CN112126666A, CN113373100A, CN102171346, CN101120090A, CN1831115A, CN1270631A). Currently, the production of purine nucleosides by microbial fermentation is often limited to the production of natural nucleosides such as adenosine, guanosine, and inosine, and is time-consuming, yields low, and is still far from industrial production.
[0005] In recent years, the enzymatic synthesis of purine nucleosides has attracted considerable attention due to its advantages, including fewer steps, high yield, high optical purity, and environmental friendliness, which can circumvent the aforementioned problems. The enzymatic synthesis of purine nucleosides can be achieved through various routes using different enzymes. For example, patent EP1457568A1 discloses a biological enzyme-catalyzed method that uses N-deoxyribosyltransferase II and nucleosidase to react thymidine and guanine as substrates for 32 hours to synthesize 31 mM 2'-deoxyguanosine. Furthermore, this patent describes a two-step method that combines the above two enzymes with adenosine deaminase for approximately 30 hours to ultimately synthesize 2'-deoxyguanosine exceeding 40 mM. Furthermore, pyrimidine nucleoside phosphorylase and purine nucleoside phosphorylase can also be used in the synthesis of purine nucleosides. Patent CN102770532A discloses a method for biologically converting pyrimidine nucleosides and purine bases using pyrimidine nucleoside phosphorylase and purine nucleoside phosphorylase, which can convert uridine or 2'-deoxyuridine and purine bases to 2,6-diaminopurine nucleoside and 2,6-diaminopurine-2'-deoxynucleoside, with a conversion rate exceeding 90% but a yield of less than 5 mM. Patent CN1207417A describes how to use pyrimidine nucleoside phosphorylase and purine nucleoside phosphorylase derived from Bacillus stearothermophilus JTS859, along with uracil and thymine-degrading enzyme solutions derived from the genus Arthrobacter, to produce 14.9 mM 2'-deoxyguanosine by reacting thymidine and a corresponding purine base for 110 hours with a yield of 75%, and how to obtain 17.7 mM 2'-deoxyadenosine by reacting for 90 hours with a yield of 88.5%.However, the enzymatic synthesis methods disclosed above all have problems such as low conversion rates, low yields, or long reaction times. [Overview of the project] [Problems that the invention aims to solve]
[0006] The main objective of the present invention is to provide a method and composition for the synthesis of purine nucleosides using an enzyme catalyst, in order to solve the problem of low yield in conventional enzymatic purine nucleoside synthesis. [Means for solving the problem]
[0007] To achieve the above objective, according to a first aspect of the present invention, a method for synthesizing purine nucleosides by enzyme catalyst is provided, the method comprising using purine nucleoside phosphorylase and either pyrimidine nucleoside phosphorylase or thymidine phosphorylase to catalyze a substrate and synthesize a purine nucleoside, wherein the substrate comprises a substrate nucleoside and a substrate base, the pyrimidine nucleoside phosphorylase comprises PyNP, a protein indicated by SEQ ID NO:1, or a protein having 80% or more homology to PyNP and having the same function, or a protein having 80% or more homology to TP and having the same function, the thymidine phosphorylase comprises TP, a protein indicated by SEQ ID NO:2, or a protein having 80% or more homology to TP and having the same function, and the purine nucleoside phosphorylase comprises PNP, a protein indicated by SEQ ID NO:3, or a protein having 80% or more homology to PNP and having the same function.
[0008] Furthermore, the substrate nucleoside is a nucleoside represented by formula I, where R1 is selected from -H or -OH, and R2 is selected from -H or -CH3. Preferably, the substrate nucleoside includes thymidine, uridine, or 2'-deoxyuridine.
[0009] [ka]
[0010] Furthermore, the substrate base is a base represented by formula II or formula III, where X is selected from -NH3 or -OCH3, and Y is selected from -H or -NH2. Preferably, the substrate base includes guanine, adenine, 2,6-diaminopurine, or 6-methoxyguanine.
[0011] [ka]
[0012] Furthermore, one or more of PyNP, TP, or PNP are purified proteins, crude enzyme solutions, or immobilized enzymes.
[0013] Furthermore, in the above method, the concentration of the substrate nucleoside is 2 to 400 mM, and the concentration of the substrate base is 1 to 200 mM.
[0014] Furthermore, the catalysis time of the enzyme catalyst is 2 to 18 hours.
[0015] Furthermore, the catalytic temperature of the enzyme catalyst is 60-70°C.
[0016] To achieve the above objective, a composition is provided according to a second aspect of the present invention, the composition comprising a purine nucleoside phosphorylase and either a pyrimidine nucleoside phosphorylase or a thymidine phosphorylase, wherein the pyrimidine nucleoside phosphorylase is PyNP, a protein indicated by SEQ ID NO:1, the thymidine phosphorylase is TP, a protein indicated by SEQ ID NO:2, and the purine nucleoside phosphorylase is PNP, a protein indicated by SEQ ID NO:3.
[0017] Furthermore, one or more of pyrimidine nucleoside phosphorylase, thymidine phosphorylase TP or purine nucleoside phosphorylase PNP is a purified protein, a crude enzyme solution or an immobilized enzyme. Preferably, the composition contains one or more of PyNP, TP or PNP and is a purified protein, a crude enzyme solution or an immobilized enzyme.
[0018] Furthermore, the composition further contains a substrate nucleoside and a substrate base. The substrate nucleoside is a nucleoside represented by Formula I, R1 is selected from -H or -OH, R2 is selected from -H or -CH3, the substrate base is a base represented by Formula II or Formula III, X is selected from -NH3 or -OCH3, Y is selected from -H or -NH2. Preferably, the concentration of the substrate nucleoside is 2 to 400 mM, and the concentration of the substrate base is 1 to 200 mM.
[0019]
Chemical formula
Advantages of the Invention
[0020] When the technical solution of the present invention is used, pyrimidine nucleoside phosphorylase and purine nucleoside phosphorylase, or thymidine phosphorylase and purine nucleoside phosphorylase are utilized to perform enzymatic catalysis together, including PyNP and PNP, or TP and PNP. Using a substrate nucleoside and a substrate base as raw materials, multiple types of purine nucleosides can be generated through catalytic reaction in a one-step method, and amplification production can be realized. Thereby, they can be efficiently synthesized within a short reaction time, and the yield of purine nucleosides can be improved.
Brief Description of the Drawings
[0021] The attached drawings of the specification that form a part of this application are used to provide a further understanding of the present invention. The exemplary embodiments and their descriptions of the present invention are used to interpret the present invention and do not constitute an undue limitation of the present invention. In the drawings, [Figure 1]It is a schematic diagram showing a calibration curve of 2'-deoxyadenosine according to Example 5 of the present invention. [Figure 2] It is a schematic diagram showing a calibration curve of 2'-deoxyguanosine according to Example 7 of the present invention. [Figure 3] It shows a high performance liquid chromatogram of 2'-deoxyadenosine according to Example 5 of the present invention. [Figure 4] It shows a high performance liquid chromatogram of 2'-deoxyadenosine according to Example 6 of the present invention.
Mode for Carrying Out the Invention
[0022] In addition, the examples and features in the examples in the present application can be combined with each other as long as they do not conflict. Hereinafter, the present invention will be described in detail with reference to examples.
[0023] As mentioned in the background art, all of the conventional methods for synthesizing purine nucleosides have problems such as high cost, long time consumption, and low yield, which are disadvantageous for the production and subsequent utilization of purine nucleosides and their analogs.
[0024] Conventional enzymatic synthesis routes for nucleoside phosphorylases involve numerous reaction steps, complicated procedures, long processing times, low yields, and high production costs. For example, the enzymatic conversion described in patent EP1457568A1 requires two reaction steps and three types of enzymes, takes 30 hours, and yields only 40 mM of 2'-deoxyguanosine. The enzymatic conversion described in CN1207417A takes over 90 hours, yields low purine nucleosides, requires the separate preparation of enzyme solutions with uracil and thymine-degrading activity, increases production costs, and significantly reduces yields without the addition, reaching only 13% to 50% of the yield with the addition. The method described in patent CN102770532A yields only less than 5 mM of 2,6-diaminopurine nucleoside and 2,6-diaminopurine-2'-deoxynucleoside. However, due to problems such as the low solubility of the substrate purine base, the instability of the enzyme, and the easy destruction of intermediate products (ribose-1-phosphate or 2'-deoxyribose-1-phosphate), simply amplifying the reaction or extending the reaction time can disrupt the reaction balance or inactivate the enzyme, resulting in low conversion rates and yields, increased production costs, and making it difficult to apply to actual commercial production.
[0025] To improve the situation described above, in this application, the inventors attempted to explore a new method for enzyme-catalyzed purine nucleoside synthesis. From among numerous related proteins with latent activity, they discovered catalytic activity in combinations of PyNP (pyrimidine nucleoside phosphorylase, indicated by SEQ ID NO: 1), PNP (purine nucleoside phosphorylase, indicated by SEQ ID NO: 3), and TP (thymidine phosphorylase, indicated by SEQ ID NO: 2). They found that substrate nucleosides and substrate bases can be catalyzed in a single step using enzyme solutions of PyNP, TP, and PNP to produce the target product, purine nucleoside. This enzyme-catalyzed method has a short reaction time and high synthesis efficiency, resulting in a high yield of the obtained product. Based on this, the applicant proposed the series of protection proposals in this application.
[0026] A first typical embodiment of the present application provides an enzyme-catalyzed method for synthesizing purine nucleosides, which utilizes a purine nucleoside phosphorylase and either a pyrimidine nucleoside phosphorylase or a thymidine phosphorylase to catalyze a substrate and synthesize a purine nucleoside, wherein the substrate comprises a substrate nucleoside and a substrate base, the pyrimidine nucleoside phosphorylase comprises PyNP, a protein indicated by SEQ ID NO:1, the thymidine phosphorylase comprises TP, a protein indicated by SEQ ID NO:2, and the purine nucleoside phosphorylase comprises PNP, a protein indicated by SEQ ID NO:3. The pyrimidine nucleoside phosphorylase, thymidine phosphorylase, or purine nucleoside phosphorylase described above all contain proteins that have 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, or 99.9% or more homology with PyNP, TP, or PNP, respectively, and that have the same function.
[0027] The pyrimidine nucleoside phosphorylase used in this invention is derived from the hyperthermophilic bacterium Thermus thermophilus and is a protein indicated by SEQ ID NO:1. This pyrimidine nucleoside phosphorylase is named PyNP. The thymidine phosphorylase is derived from Homo sapiens and is a protein indicated by SEQ ID NO:2. This thymidine phosphorylase is named TP. The purine nucleoside phosphorylase is derived from Geobacillus stearothermophilus and is a protein indicated by SEQ ID NO:3. This purine nucleoside phosphorylase is named PNP. In the above enzyme-catalyzed method, by utilizing two types of nucleoside phosphatases from different origins, purine nucleosides can be efficiently synthesized in a single step, thereby reducing production costs and enabling application to large-scale industrial production of related products. The above-mentioned substrate comprises a substrate nucleoside and a substrate base, and may be a natural nucleoside or base structure, or an artificially modified similar structure.
[0028] [ka]
[0029] In this enzyme-catalyzed method, reaction A is carried out under the catalysis of PyNP or TP, cleaving the CN bond between the pyrimidine group and the ribose group of the substrate nucleoside and attaching a phosphate group to the nucleoside group to form a ribose phosphate intermediate. This ribose phosphate intermediate then undergoes reaction B under the action of PNP, where the C-1' atom of the ribose group and the N-9 atom of the purine substrate base form a CN bond, producing a purine nucleoside compound. Reactions A and B do not require the addition of coenzymes such as NADPH; the reaction can be completed using only the substrate and protein, making the reaction simple, eliminating the need to perform it in living cells, and significantly reducing reaction costs and efficiency. This enzyme-catalyzed method, which combines these two types of proteins, can complete the catalytic reaction within a relatively short catalytic time, has a high substrate conversion rate, can be similarly applied to amplification reactions, and enables large-scale industrial production.
[0030] In preferred embodiments, the substrate nucleoside is a nucleoside represented by formula I, where R1 is selected from -H or -OH, and R2 is selected from -H or -CH3, and preferably the substrate nucleoside comprises thymidine, uridine, or 2'-deoxyuridine.
[0031] [ka]
[0032] In preferred embodiments, the substrate base is a base represented by formula II or formula III, where X is selected from -NH3 or -OCH3, and Y is selected from -H or -NH2. Preferably, the substrate base includes guanine, adenine, 2,6-diaminopurine, or 6-methoxyguanine.
[0033] [ka]
[0034] Using the above-mentioned substrate nucleoside and substrate base, purine nucleosides such as 2'-deoxyadenosine, 2'-deoxyguanosine, 2,6-diaminopurine nucleoside, 2,6-diaminopurine-2'-deoxynucleoside, and 6-O-methylguanosine can be produced by this enzyme-catalyzed method.
[0035] Since the R1, R2, X, and Y substituents in the above-mentioned substrate nucleoside and substrate base are all far from the reaction site, PyNP and PNP can be used to carry out enzyme-catalyzed reactions with different substrates and produce a variety of purine nucleosides.
[0036] In preferred embodiments, one or more of PyNP, TP, or PNP are purified protein, crude enzyme solution, or immobilized enzyme.
[0037] Through the catalytic reactions of PyNP, TP, and PNP, the three proteins described above can exist in various forms, such as purified proteins, crude enzyme solutions, or immobilized enzymes, and all can catalyze the synthesis of purine nucleosides. By cloning genes expressing PyNP and / or TP and / or PNP into host cells, inducing protein expression, and then disrupting the host cells, a crude enzyme solution containing the target protein can be obtained. The crude enzyme solution is easy to produce, has good catalytic activity, and can reduce the production cost of catalytic reactions.
[0038] In preferred examples, the above method involves a substrate nucleoside concentration of 2 to 400 mM and a substrate base concentration of 1 to 200 mM.
[0039] The above enzyme-catalyzed method allows for amplification reactions, with substrate nucleoside concentrations reaching a maximum of 400 mM and substrate base concentrations reaching a maximum of 200 mM in the reaction system, thereby enabling large-scale production of purine nucleosides.
[0040] In preferred embodiments, the catalysis time of the enzyme catalyst is 2 to 18 hours.
[0041] In preferred embodiments, the catalytic temperature of the enzyme catalyst is 60-70°C.
[0042] Within the appropriate catalytic temperature and time described above, the enzyme-catalyzed reaction can be completed, and the conversion rate of the substrate base, i.e., the reaction yield, is high. Since there is no need to add enzymes or other reagents during the reaction, the reaction can be completed with a one-step catalyst, making it suitable for industrial amplification production applications.
[0043] A second typical embodiment of the present application provides a composition comprising a purine nucleoside phosphorylase and either a pyrimidine nucleoside phosphorylase or a thymidine phosphorylase, wherein the pyrimidine nucleoside phosphorylase is PyNP, a protein indicated by SEQ ID NO:1; the thymidine phosphorylase is TP, a protein indicated by SEQ ID NO:2; and the purine nucleoside phosphorylase is PNP, a protein indicated by SEQ ID NO:3. The composition is used for the synthesis of purine nucleosides.
[0044] In preferred embodiments, the composition comprises purified protein, crude enzyme solution, or immobilized enzyme.
[0045] In preferred embodiments, the composition further comprises reacting a substrate nucleoside with a substrate base to produce a purine nucleoside, wherein the substrate nucleoside is a nucleoside represented by formula I, where R1 is selected from -H or -OH, and R2 is selected from -H or -CH3; the substrate base is a base represented by formula II or formula III, where X is selected from -NH3 or -OCH3, and Y is selected from -H or -NH2; preferably, the concentration of the substrate nucleoside is 2 to 400 mM, and the concentration of the substrate base is 1 to 200 mM.
[0046] [ka]
[0047] PyNP is a pyrimidine nucleoside phosphorylase derived from the hyperthermophilic bacterium Thermus thermophilus, and its amino acid sequence is shown in SEQ ID NO:1. TP is a 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 comprises PyNP and PNP, or TP and PNP, and this composition can be used to catalyze the completion of the reaction between nucleosides such as thymidine and uridine and bases such as guanine and adenine, thereby producing purine nucleosides and their analogues. The proteins in the composition can each be independently selected from the form of purified protein, crude enzyme solution, or immobilized enzyme, and all can exert catalytic activity.
[0048] The beneficial effects of this invention will be explained in more detail below, with reference to specific examples.
[0049] (Example 1) 1. Strain construction The pyrimidine nucleoside phosphorylase used in this application is derived from the hyperthermophilic bacterium Thermus thermophilus, named PyNP, and its amino acid sequence is shown in SEQ ID NO:1. The thymidine phosphorylase is derived from Homo sapiens, named TP, and its amino acid sequence is shown in SEQ ID NO:2. The purine nucleoside phosphorylase is derived from Geobacillus stearothermophilus, named PNP, and its amino acid sequence is shown in SEQ ID NO:3.
[0050] After codon optimization, DNA sequences encoding two enzymes were obtained: the DNA sequence encoding PyNP was SEQ ID NO:4, the DNA sequence encoding TP was SEQ ID NO:5, and the DNA sequence encoding PNP was SEQ ID NO:6. These were cloned into the expression vector pET28a(+). The resulting plasmids were transformed into competent E. coli BL21(DE3) hosts to obtain monoclonal strains.
[0051] TIFF0007901189000008.tif48161
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[0054] TIFF0007901189000011.tif84161
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[0056] TIFF0007901189000013.tif53161
[0057] 2. Protein expression E. coli strains expressing PyNP, TP, and PNP were inoculated into test tubes and incubated at 37°C for 16 hours. Afterward, they were inoculated at a 1v / v% dose into a 2L shaker flask containing 500mL LB liquid medium and incubated at 37°C. 600 When the culture was maintained until the ratio reached 0.6, isopropyl-β-D-thiogalactoside at a final concentration of 0.1 M was added to induce protein expression, and the culture was maintained at 20°C for 18 hours. The cultured bacterial suspension was centrifuged at 7000 rpm for 10 minutes to collect the cells, and the cells were prepared for use.
[0058] 3. Production of enzyme solution 0.1 g of bacterial sludge was weighed, 1 mL of potassium phosphate buffer with a pH of 7.5 was added, and after shaking and kneading, the bacterial suspension was broken up using an ultrasonic disruptor at a power of 30% for 5 minutes.
[0059] 4. HPLC detection method The chromatography column used was an Atlantis T3 Column, 4.6 mm x 150 mm. The mobile phase was methanol containing 0.1 v / v% trifluoroacetic acid (TFA), with a flow rate of 1 mL / min, a column temperature of 40°C, a UV detector, a detection wavelength of 254 nm, and a detection time of 15 min.
[0060] (Example 2) Enzyme catalysts of purine nucleosides and their analogues A 1 mL reaction system was prepared in 2 mM phosphate buffer (pH 7.5) containing PyNP enzyme solution prepared from different weights of bacterial sludge containing 2 mM thymidine or uridine and 1 mM purine or purine analog, and PNP enzyme solution prepared from different weights of bacterial sludge. The reaction was carried out at 60°C for 18 hours. After the reaction was complete, 1 mL of DMSO was added, and the reaction was detected by HPLC. The experimental results are shown in Table 1. As a result, the reaction conversion rates of both adenine and purine analog were higher than 79%, demonstrating that the conversion rate of 2,6-diaminopurine can reach 96% in the synthesis of 2,6-diaminopurine nucleoside and 2,6-diaminopurine-2'-deoxynucleoside.
[0061] [Table 1]
[0062] (Example 3) Effect of increased substrate concentration on enzyme catalysts of purine nucleosides and their analogues
[0063] A 1 mL reaction system was prepared in 2 mM phosphate buffer (pH 7.5) containing 4 mM thymidine or uridine, 1 mM purine or purine analog, PyNP enzyme solution prepared from different weights of bacterial sludge, and PNP enzyme solution prepared from different weights of bacterial sludge. The reaction was carried out at 70°C for 2 hours. After the reaction was complete, 1 mL of DMSO was added, and the results were detected by HPLC. The experimental results are shown in Table 2. The results showed that increasing the substrate concentration of thymidine and shortening the reaction time to 2 hours still improved the reaction conversion rates of adenine and purine analog, both of which were higher than 90%.
[0064] [Table 2]
[0065] (Example 4) Optimization of the amount of 2'-deoxyadenosine synthase. A 1 mL reaction system was prepared in 2 mM phosphate buffer (pH 7.5) containing 400 mM thymidine, 200 mM adenine, and PyNP enzyme solution prepared from 9.7–48.4 mg of bacterial sludge, as well as PNP enzyme solution prepared from 2.7–13.5 mg of bacterial sludge. The reaction was carried out at 60°C for 16 hours. After the reaction was complete, 1 mL of DMSO was added, and the results were detected by HPLC. The experimental results are shown in Table 3. The results showed that even with low enzyme amounts, the purine conversion rate could still be maintained at over 90%. For example, when using PyNP enzyme solution prepared from 9.7 mg of bacterial sludge and PNP enzyme solution prepared from 2.7 mg of bacterial sludge, the conversion rate was 93.17%.
[0066] [Table 3]
[0067] (Example 5) Amplification reaction of 2'-deoxyadenosine synthesis A 50 mL reaction system was prepared in 2 mM phosphate buffer (pH 7.5) containing 400 mM thymidine, 200 mM adenine, PyNP enzyme solution prepared from 484.0 mg of bacterial sludge, and PNP enzyme solution prepared from 135.0 mg of bacterial sludge. The reaction was carried out at 60°C for 16 hours to produce thymine and 2'-deoxyadenosine. After the reaction was complete, an equal volume of DMSO was added to 1 mL of the reaction solution to dissolve it, and after dilution 20-fold, detection was performed by HPLC. As shown in Figure 3, the conversion rate of thymidine was 39.20%, and the conversion rate of purine was 94.72%. Based on the calibration curve shown in Figure 1, the yield of 2'-deoxyadenosine was calculated to be 51.1 g / L.
[0068] (Example 6) Effect of additional enzyme amount on the 2'-deoxyadenosine synthesis amplification reaction After carrying out Example 5, an enzyme solution prepared from 1.35 g of adenine and 135.0 mg of PNP bacterial sludge was added, and the reaction was continued for 24 hours. After the reaction was complete, an equal volume of DMSO was added to 1 mL of the reaction solution to dissolve it, and after dilution 20-fold, detection was performed by HPLC. As shown in Figure 4, the conversion rate of thymidine was 74.97%, and the conversion rate of purine was 81.30%. From the calibration curve shown in Figure 1, the yield of 2'-deoxyadenosine was calculated to be 68.3 g / L.
[0069] (Example 7) Enzyme-catalyzed reaction of 2'-deoxyguanosine 1. Guanine at a concentration of 1 M was prepared and dissolved in NaOH. 120 mM thymidine, 40 mM guanine, PyNP enzyme solution prepared from 58 mg of bacterial sludge, and PNP enzyme solution prepared from 120 mg of bacterial sludge were added to a reaction system of 1 mL of 2 mM phosphate buffer (pH 7.5). The reaction was carried out at 60°C for 16 hours, after which a sample was taken, dissolved in an equal volume of DMSO, diluted 20-fold, and detected by HPLC. The conversion rate of thymidine was 37.77%, and the conversion rate of purine was 86.42%. From the calibration curve shown in Figure 2, the maximum yield of 2'-deoxyguanosine was calculated to be 9.8 g / L.
[0070] 2. Guanine at a concentration of 1 M was prepared and dissolved in NaOH. 240 mM thymidine, 80 mM guanine, PyNP enzyme solution prepared from 58 mg of bacterial sludge, and PNP enzyme solution prepared from 120 mg of bacterial sludge were added to a reaction system of 1 mL of 2 mM phosphate buffer (pH 7.5). The reaction was carried out at 60°C for 16 hours, after which a sample was taken, dissolved in an equal volume of DMSO, diluted 20-fold, and detected by HPLC. The conversion rate of thymidine was 31.79%, and the conversion rate of purine was 77.07%. From the calibration curve shown in Figure 2, the maximum yield of 2'-deoxyguanosine was calculated to be 17.5 g / L.
[0071] The results of the above experiment are shown in Table 4.
[0072] [Table 4]
[0073] (Example 8) Reaction for the synthesis of 2,6-diaminopurine nucleoside A 10 mL reaction system was prepared in 2 mM phosphate buffer (pH 7.5) containing 320 mM uridine, 200 mM 2,6-diaminopurine, PyNP enzyme solution prepared from 195.4 mg of bacterial sludge, and PNP enzyme solution prepared from 150.1 mg of bacterial sludge. The reaction was carried out at 60°C for 16 hours. After the reaction was complete, an equal volume of DMSO was added to 1 mL of the reaction solution to dissolve it, and after dilution 20-fold, detection was performed by HPLC. The conversion rate of uridine was 54.92%, the conversion rate of purine was 96.18%, and the yield of 2,6-diaminopurine nucleoside was 54.3 g / L.
[0074] (Example 9) Reaction for the synthesis of 2,6-diaminopurine-2'-deoxynucleoside A 10 mL reaction system was prepared in 2 mM phosphate buffer (pH 7.5) containing 400 mM 2'-deoxyuridine, 200 mM 2,6-diaminopurine, TP enzyme solution prepared from 230.0 mg of bacterial sludge, and PNP enzyme solution prepared from 75.0 mg of bacterial sludge. The reaction was carried out at 50°C for 16 hours. After the reaction was complete, an equal volume of DMSO was added to 1 mL of the reaction solution to dissolve it, and after dilution 20-fold, detection was performed by HPLC. The conversion rate of 2'-deoxyuridine was 43.89%, the conversion rate of purine was 97.75%, and the yield of 2,6-diaminopurine-2'-deoxynucleoside was 59.6 g / L.
[0075] (Example 10) Enzyme-catalyzed reaction of 6-O-methylguanosine synthesis A 10 mL reaction system was prepared in 2 mM phosphate buffer (pH 7.5) containing 320 mM uridine, 200 mM 6-O-methylguanine, PyNP enzyme solution prepared from 195.4 mg of bacterial sludge, and PNP enzyme solution prepared from 165.0 mg of bacterial sludge. The reaction was carried out at 60°C for 24 hours. After the reaction was complete, an equal volume of DMSO was added to 1 mL of the reaction solution to dissolve it, and after dilution 20-fold, detection was performed by HPLC. The conversion rate of uridine was 51.27%, the conversion rate of purine was 98.67%, and the yield of 6-O-methylguanosine was 58.6 g / L.
[0076] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects. The above enzyme-catalyzed method allows for the synthesis and amplification of purine nucleoside products using substrate bases and substrate nucleosides as substrates within a short catalytic time. Within a short reaction time, 2'-deoxyadenosine, 2'-deoxyguanosine, 2,6-diaminopurine nucleoside, 2,6-diaminopurine-2'-deoxynucleoside, and 6-O-methylguanosine can be efficiently synthesized, with yields reaching 68.3 g / L, 17.5 g / L, 54.3 g / L, 59.6 g / L, and 58.6 g / L, respectively.
[0077] The foregoing are merely preferred embodiments of the present invention and do not limit it, and various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention shall be within the scope of protection of the present invention.
Claims
1. A method for synthesizing purine nucleosides by enzyme catalyst, wherein the method utilizes purine nucleoside phosphorylase and either pyrimidine nucleoside phosphorylase or thymidine phosphorylase to catalyze a substrate and synthesize the purine nucleoside, The substrate comprises a substrate nucleoside and a substrate base. The pyrimidine nucleoside phosphorylase comprises PyNP, which is the protein indicated by SEQ ID NO: 1, or a protein that has 90% or more identity with PyNP and has the same function. The thymidine phosphorylase comprises TP, which is a protein indicated by SEQ ID NO: 2, or a protein that is 90% or more identical to TP and has the same function. A method for synthesizing purine nucleosides by enzyme catalyst, characterized in that the purine nucleoside phosphorylase contains PNP, which is a protein indicated by SEQ ID NO: 3, or a protein that has 90% or more identity with and the same function as PNP. The substrate nucleoside is a nucleoside represented by formula I, where R1 is selected from -H or -OH, and R2 is selected from -H or -CH3. 【Chemistry 1】 The substrate base is a base represented by formula II or formula III, where X is selected from -NH₂ or -OCH₃, and Y is selected from -H or -NH₂. 【Chemistry 2】 A method for synthesizing purine nucleosides by enzyme catalyst, characterized in that the purine nucleoside comprises 2'-deoxyadenosine, 2'-deoxyguanosine, 2,6-diaminopurine nucleoside, 2,6-diaminopurine-2'-deoxynucleoside, or 6-O-methylguanosine.
2. The method according to claim 1, characterized in that the substrate nucleoside comprises thymidine, uridine, or 2'-deoxyuridine.
3. The method according to claim 1, characterized in that the substrate base comprises guanine, adenine, 2,6-diaminopurine, or 6-methoxyguanine.
4. The method according to claim 1, characterized in that one or more of the pyrimidine nucleoside phosphorylase, thymidine phosphorylase, or purine nucleoside phosphorylase are purified proteins, crude enzyme solutions, or immobilized enzymes.
5. The method according to claim 1, characterized in that the concentration of the substrate nucleoside is 2 to 400 mM and the concentration of the substrate base is 1 to 200 mM.
6. The method according to claim 1, characterized in that the catalytic time of the enzyme catalyst is 2 to 18 hours.
7. The method according to claim 1, characterized in that the catalytic temperature of the enzyme catalyst is 60 to 70°C.
8. A composition used for the synthesis of purine nucleosides, comprising purine nucleoside phosphorylase and either pyrimidine nucleoside phosphorylase or thymidine phosphorylase, wherein The pyrimidine nucleoside phosphorylase is PyNP, which is the protein indicated by SEQ ID NO: 1, or a protein that has 90% or more identity with PyNP and has the same function. The thymidine phosphorylase is TP, which is the protein indicated by SEQ ID NO: 2, or a protein that has 90% or more identity with TP and has the same function. The purine nucleoside phosphorylase is PNP, which is the protein indicated by SEQ ID NO: 3, or a protein that has 90% or more identity with PNP and has the same function. The composition further comprises a substrate nucleoside and a substrate base, The substrate nucleoside is a nucleoside represented by formula I, where R1 is selected from -H or -OH, and R2 is selected from -H or -CH3. A composition used for the synthesis of purine nucleosides, characterized in that the substrate base is a base represented by formula II or formula III, where X is selected from -NH₂ or -OCH₃, and Y is selected from -H or -NH₂. 【Transformation 3】
9. The composition according to claim 8, characterized in that one or more of the pyrimidine nucleoside phosphorylase, thymidine phosphorylase, or purine nucleoside phosphorylase are purified proteins, crude enzyme solutions, or immobilized enzymes.
10. The composition according to claim 8 or 9, characterized in that the concentration of the substrate nucleoside is 2 to 400 mM, and the concentration of the substrate base is 1 to 200 mM.