Method for synthesizing purine nucleosides by enzymatic catalysis and composition

Abstract

A method and composition for synthesizing purine nucleosides using enzyme catalysts. Here, the method utilizes a purine nucleoside phosphorylase and an enzyme selected from either a pyrimidine nucleoside phosphorylase or a thymidine phosphorylase to catalyze a substrate to synthesize purine nucleosides. The substrate includes a substrate nucleoside and a substrate base. The pyrimidine nucleoside phosphorylase contains PyNP, a protein represented by SEQ ID NO:1. The thymidine phosphorylase contains TP, a protein represented by SEQ ID NO:2. The purine nucleoside phosphorylase contains PNP, a protein represented by SEQ ID NO:3. It can solve the problem of low yield in the synthesis of purine nucleosides by the enzyme method in the prior art and is applicable to the field of enzyme catalysis.

Description

Technical Field

[0001] This application is based on and claims the priority of a Chinese application with CN application number 202211021644.X and a filing date of August 24, 2022. 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 and composition for synthesizing purine nucleosides by enzyme catalysis.

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 methods (CN112553135A, CN112574934A, CN113151238A, CN113278596A, CN112143751A, CN112126666A, CN113373100A, CN102171346, CN101120090A, CN1831115A, CN1270631A). Currently, the production of purine nucleosides by microbial fermentation methods is often limited to the production of natural nucleosides such as adenosine, guanosine, and inosine. Moreover, it takes a long time, the yield is low, and it is still far from industrial production.

[0005] In recent years, the synthesis of purine nucleosides by the enzymatic method has attracted much attention because it can avoid the above problems due to advantages such as fewer steps, high yield, high optical purity, and environmental friendliness. The synthesis of purine nucleosides by the enzymatic method can be realized through various routes using different enzymes. For example, Patent EP1457568A1 discloses a biological enzyme-catalyzed method that can synthesize 31 mM of 2'-deoxyguanosine by reacting thymidine and guanine as substrates for 32 h using nucleoside deoxyribosyl transferase II and nucleosidase. In addition, this patent combines the above two enzymes with adenosine deaminase and reacts them in a two-step method for about 30 h to finally synthesize more than 40 mM of 2'-deoxyguanosine. 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 into 2,6-diaminopurine nucleosides and 2,6-diaminopurine-2'-deoxynucleosides, with a conversion rate exceeding 90%, but the yield is less than 5 mM. Patent CN1207417A uses pyrimidine nucleoside phosphorylase and purine nucleoside phosphorylase derived from Bacillus stearothermophilus JTS859 and uracil and thymine degrading enzyme solution derived from the genus Arthrobacter, uses thymidine and the corresponding purine base as substrates, reacts for 110 h to produce 14.9 mM of 2'-deoxyguanosine with a yield of 75%, and reacts for 90 h to obtain 17.7 mM of 2'-deoxyadenosine with a yield of 88.5%.However, all of the enzyme-catalyzed syntheses disclosed above have problems such as low conversion rates, low yields, or long reaction times.

SUMMARY OF THE INVENTION

PROBLEMS TO BE SOLVED BY THE INVENTION

[0006] The main object of the present invention is to provide a method and composition for synthesizing purine nucleosides by enzyme catalysis in order to solve the problem of low yields in the synthesis of purine nucleosides by the enzyme method in the prior art.

MEANS FOR SOLVING THE PROBLEMS

[0007] To achieve the above object, according to a first aspect of the present invention, there is provided a method for synthesizing purine nucleosides by enzyme catalysis, the method comprising using purine nucleoside phosphorylase and either pyrimidine nucleoside phosphorylase or thymidine phosphorylase to catalyze a substrate to synthesize purine nucleosides, the substrate comprising a substrate nucleoside and a substrate base, the pyrimidine nucleoside phosphorylase comprising PyNP, a protein represented by SEQ ID NO:1, or a protein having 80% or more homology with PyNP and having the same function, or a protein having 80% or more homology with TP and having the same function, the thymidine phosphorylase comprising TP, a protein represented by SEQ ID NO:2, or a protein having 80% or more homology with TP and having the same function, and the purine nucleoside phosphorylase comprising PNP, a protein represented by SEQ ID NO:3, or a protein having 80% or more homology with PNP and having the same function.

[0008] Furthermore, the substrate nucleoside is a nucleoside represented by Formula I, R1 is selected from -H or -OH, R2 is selected from -H or -CH3, and preferably, the substrate nucleoside comprises thymidine, uridine, or 2'-deoxyuridine.

[0009]

Chemical formula

[0010] Furthermore, 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, and preferably, the substrate base includes guanine, adenine, 2,6-diaminopurine, or 6-methoxyguanine.

[0011]

Chemical formula

[0012] Furthermore, one or more of PyNP, TP, or PNP is a purified protein, a crude enzyme solution, or an immobilized enzyme.

[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 catalytic time of the enzyme catalysis is 2 to 18 h.

[0015] Furthermore, the catalytic temperature of the enzyme catalysis is 60 to 70 °C.

[0016] To achieve the above object, according to a second aspect of the present invention, a composition is provided, which includes a purine nucleoside phosphorylase and an enzyme selected from either a pyrimidine nucleoside phosphorylase or a thymidine phosphorylase, where the pyrimidine nucleoside phosphorylase is PyNP, a protein represented by SEQ ID NO:1, the thymidine phosphorylase is TP, a protein represented by SEQ ID NO:2, and the purine nucleoside phosphorylase is PNP, a protein represented 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, where 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 - 400 mM, and the concentration of the substrate base is 1 - 200 mM.

[0019]

Chemical formula

Advantages of the Invention

[0020] Using the technical solution of the present invention, 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 amplified production can be realized, thereby efficiently synthesizing within a short reaction time and improving the yield of purine nucleosides.

Brief Description of the Drawings

[0021] The accompanying 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 of the present invention and their descriptions are used to interpret the present invention and do not constitute an undue limitation of the present invention. In the drawings,

Figure 1

Figure 2

Figure 3

Figure 4

Mode for Carrying Out the Invention

[0022] In addition, the examples in the present application and the features in the examples 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] The synthetic route of nucleoside phosphorylase by the conventional enzymatic method has many reaction steps, complicated operations, takes a long time, has low yields and rates, and high production costs. For example, the conversion by the enzymatic method according to Patent EP1457568A1 requires a two-step reaction and three kinds of enzymes, takes 30 h, and the yield of 2'-deoxyguanosine is only 40 mM. The conversion by the enzymatic method disclosed in CN1207417A takes more than 90 hours, and moreover, the yield of purine nucleoside is low, and it is necessary to separately produce an enzyme solution having uracil and thymine degrading activities, increasing the production cost. Without adding it, the yield and rate will significantly decrease, and it is only 13% - 50% when added. In the method disclosed in Patent CN102770532A, only 2,6-diaminopurine nucleoside and 2,6-diaminopurine-2'-deoxynucleoside less than 5 mM can be obtained. However, there are problems such as low solubility of the substrate purine base, instability of the enzyme, and easy destruction of the intermediate product (ribose-1-phosphate or 2'-deoxyribose-1-phosphate). Therefore, simply amplifying the reaction or extending the time may disrupt the reaction balance or inactivate the enzyme, resulting in low conversion rates and yields finally obtained, increased production costs, and difficulty in applying to actual commercial production.

[0025] To improve the above situation, in the present application, the inventors attempted to explore a new method for purine nucleoside synthesis by enzyme catalysis, and found the catalytic activity of the combination of PyNP (pyrimidine nucleoside phosphorylase, a protein shown by SEQ ID NO:1), PNP (purine nucleoside phosphorylase, a protein shown by SEQ ID NO:3), TP (thymidine phosphorylase, a protein shown by SEQ ID NO:2) and PNP from among a number of related proteins with potential activity. It was found that the enzyme solution of PyNP, TP and PNP can be used to catalyze the substrate nucleoside and substrate base in a one-step method to produce the target product purine nucleoside. The enzyme catalysis method has a short reaction time and high synthesis efficiency, so the yield of the obtained product is high. Moreover, the applicant proposed a series of protection plans for the present application.

[0026] In the first typical embodiment of the present application, a method for synthesizing purine nucleosides by enzymatic catalysis is provided. The method uses a purine nucleoside phosphorylase and one of the enzymes of pyrimidine nucleoside phosphorylase or thymidine phosphorylase to catalyze a substrate to synthesize purine nucleosides. The substrate includes a substrate nucleoside and a substrate base. The pyrimidine nucleoside phosphorylase includes PyNP, which is a protein represented by SEQ ID NO:1. The thymidine phosphorylase includes TP, which is a protein represented by SEQ ID NO:2. The purine nucleoside phosphorylase includes PNP, which is a protein represented by SEQ ID NO:3. Any of the above pyrimidine nucleoside phosphorylase, thymidine phosphorylase or purine nucleoside phosphorylase includes a protein having a homology of 80%, 85%, 90%, 95%, 98%, 99%, 99.5% or 99.9% or more with PyNP, TP or PNP respectively and having the same function.

[0027] The pyrimidine nucleoside phosphorylase used in the present application is derived from the hyperthermophilic bacterium Thermus thermophilus and is the protein represented by SEQ ID NO:1. This pyrimidine nucleoside phosphorylase is named PyNP. The thymidine phosphorylase is derived from Homo sapiens and is the protein represented by SEQ ID NO:2. This thymidine phosphorylase is named TP. The purine nucleoside phosphorylase is derived from Geobacillus stearothermophilus and is the protein represented by SEQ ID NO:3. This purine nucleoside phosphorylase is named PNP. In the above enzymatic catalysis method, by using two types of nucleoside phosphatases from different origins to efficiently synthesize purine nucleosides in a one-step method, the production cost can be reduced and it can be applied to the large-scale industrial production of related products. The above substrate includes a substrate nucleoside and a substrate base, which may be a natural nucleoside or base structure, or an artificially modified similar structure.

[0028]

Chem.

[0029] In the enzyme-catalyzed method, the above A reaction is carried out under the catalysis of PyNP or TP to cleave the C-N bond between the pyrimidine group and the ribose group of the substrate nucleoside, and a phosphate group is bonded to the nucleoside group to form a ribose phosphate intermediate. Under the action of PNP, the above B reaction is carried out on the ribose phosphate intermediate, and a C-N bond is formed between the C-1' atom of the ribose group and the N-9 atom of the purine-based substrate base to produce a purine nucleoside compound. In the above A and B reactions, it is not necessary to add a coenzyme such as NADPH, and the reaction can be completed by using only the substrate and the protein. The reaction is simple, does not need to be carried out in living cells, and greatly reduces the reaction cost and efficiency. The enzyme-catalyzed method carried out by combining these two types of proteins can complete the catalytic reaction within a relatively short catalytic time, has a high substrate conversion rate, can also be similarly applied to the amplification reaction, and enables large-scale industrial production.

[0030] In a preferred embodiment, the substrate nucleoside is a nucleoside represented by Formula I, R1 is selected from -H or -OH, R2 is selected from -H or -CH3, and preferably, the substrate nucleoside includes thymidine, uridine, or 2'-deoxyuridine.

[0031]

Chem.

[0032] In a preferred embodiment, 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, and preferably, the substrate base includes guanine, adenine, 2,6-diaminopurine, or 6-methoxyguanine.

[0033]

Chem.

[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-methyl-guanosine can be produced by the 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, enzyme-catalyzed reactions can be carried out on different substrates using PyNP and PNP, and various purine nucleosides can be produced.

[0036] In a preferred embodiment, one or more of PyNP, TP, or PNP is a purified protein, a crude enzyme solution, or an immobilized enzyme.

[0037] Through the PyNP, TP, and PNP catalytic reactions, the above three types of proteins can exist in various forms such as purified proteins, crude enzyme solutions, or immobilized enzymes, and all can catalyze the synthesis of purine nucleosides. After cloning the gene expressing PyNP and / or TP and / or PNP into a host cell and inducing the expression of the protein, the host cell can be disrupted to obtain a crude enzyme solution containing the target protein. The production of the crude enzyme solution is simple, and it has good catalytic ability, which can reduce the production cost of the catalytic reaction.

[0038] In a preferred embodiment, in the above method, the concentration of the substrate nucleoside is 2 - 400 mM, and the concentration of the substrate base is 1 - 200 mM.

[0039] The above enzyme-catalyzed method can perform an amplification reaction. In the reaction system, the concentration of the substrate nucleoside is up to 400 mM, and the concentration of the substrate base is up to 200 mM, thereby enabling the large-scale production of purine nucleosides.

[0040] In a preferred embodiment, the catalytic time of the enzyme catalyst is 2 to 18 h.

[0041] In a preferred embodiment, the catalytic temperature of the enzyme catalyst is 60 to 70 °C.

[0042] Within the appropriate catalytic temperature and catalytic 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. There is no need to add enzymes or other reagents during the reaction, and the reaction can be completed by one-step catalytic method, which is suitable for industrialized amplification production applications.

[0043] In the second typical embodiment of the present application, a composition is provided, which comprises purine nucleoside phosphorylase and an enzyme selected from either pyrimidine nucleoside phosphorylase or thymidine phosphorylase. Here, the pyrimidine nucleoside phosphorylase is PyNP, which is the protein represented by SEQ ID NO:1, the thymidine phosphorylase is TP, which is the protein represented by SEQ ID NO:2, and the purine nucleoside phosphorylase is PNP, which is the protein represented by SEQ ID NO:3. The composition is used for the synthesis of purine nucleosides.

[0044] In a preferred embodiment, the composition comprises a purified protein, a crude enzyme solution, or an immobilized enzyme.

[0045] In a preferred embodiment, the composition further comprises reacting a substrate nucleoside with a substrate base to produce a purine nucleoside. The substrate nucleoside is a nucleoside represented by Formula I, where 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.

[0046]

Chemical formula

[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 contains PyNP and PNP, or TP and PNP, and by using this composition, the reaction completion between nucleosides such as thymidine and uridine and bases such as guanine and adenine can be catalyzed to produce purine nucleosides and their analogs. The proteins in the composition can each independently be selected from forms such as purified proteins, crude enzyme solutions, or immobilized enzymes, and all can exhibit catalytic activity.

[0048] Hereinafter, the beneficial effects of the present application will be described in more detail with specific examples.

[0049] (Example 1) 1. Strain construction The pyrimidine nucleoside phosphorylase used in the present 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 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. These were each cloned into the expression vector pET28a(+). The resulting plasmids were transformed into competent Escherichia coli BL21(DE3) hosts to obtain monoclonal strains.

[0051] TIFF2025520835000009.tif48161

[0052] TIFF2025520835000010.tif50161

[0053] TIFF2025520835000011.tif32161

[0054] TIFF2025520835000012.tif84161

[0055] TIFF2025520835000013.tif80160

[0056] TIFF2025520835000014.tif53161

[0057] 2. Protein Expression E. coli strains expressing PyNP, TP, and PNP were each inoculated into test tubes and cultured at 37°C for 16 h. Then, they were inoculated at an inoculum size of 1 v / v% into 2 L shaker flasks containing 500 mL of LB liquid medium and cultured at 37°C until the OD 600 reached 0.6. At this time, isopropyl-β-D-thiogalactoside with a final concentration of 0.1 M was added to induce protein expression, and the culture was continued at 20°C for 18 h. After the culture was completed, the bacterial solution was centrifuged at 7000 rpm for 10 min to collect the cells, which were then prepared for use.

[0058] 3. Preparation of Enzyme Solution Weighed 0.1 g of bacterial sludge, added 1 mL of potassium phosphate buffer with a pH of 7.5, mixed and kneaded by shaking, and then disrupted the cell suspension with an ultrasonic crusher, setting the power at 30% and the time at 5 min.

[0059] 4. HPLC Detection Method The chromatography column was Atlantis T3 Column, 4.6 mm x 150 mm. The mobile phase was methanol containing 0.1 v / v% trifluoroacetic acid (TFA), the flow rate was 1 mL / min, the column temperature was 40 °C, the UV detector was used, the detection wavelength was 254 nm, and the detection time was 15 min.

[0060] (Example 2) Enzymatic Catalysis of Purine Nucleosides and Their Analogs Prepared a 1 mL reaction system containing 2 mM thymidine or uridine, and PyNP enzyme solution prepared from different weights of bacterial sludge of 1 mM purine or purine analog and PNP enzyme solution prepared from different weights of bacterial sludge in 2 mM phosphate buffer (pH 7.5), and reacted at 60 °C for 18 h. After the reaction, 1 mL of DMSO was added, and detection was performed by HPLC. The experimental results are shown in Table 1. As a result, the reaction conversion rates of adenine and purine analogs were both higher than 79%. Here, in the synthesis of 2,6-diaminopurine nucleoside and 2,6-diaminopurine-2'-deoxynucleoside, it was shown that the conversion rate of 2,6-diaminopurine could reach 96%.

[0061]

Table 1

[0062] (Example 3) Influence of Substrate Concentration Increase on Enzymatic Catalysis of Purine Nucleosides and Their Analogs

[0063] A 1 mL reaction system containing 2 mM phosphate buffer (pH 7.5), 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 was formulated and reacted at 70 °C for 2 h. After the reaction was completed, 1 mL of DMSO was added and detection was performed by HPLC. The experimental results are shown in Table 2. As a result, it was shown that by increasing the substrate concentration of thymidine and shortening the time to 2 h, the reaction conversion rates of adenine and purine analogs still improved and were both higher than 90%.

[0064]

Table 2

[0065] (Example 4) Optimization of the amount of enzyme for 2'-deoxyadenosine synthesis reaction A 1 mL reaction system containing 2 mM phosphate buffer (pH 7.5), 400 mM thymidine, 200 mM adenine, PyNP enzyme solution prepared from 9.7 - 48.4 mg of bacterial sludge, and PNP enzyme solution prepared from 2.7 - 13.5 mg of bacterial sludge was formulated and reacted at 60 °C for 16 h. After the reaction was completed, 1 mL of DMSO was added and detection was performed by HPLC. The experimental results are shown in Table 3. As a result, it was shown that when a low amount of enzyme was used, the conversion rate of purine could still be maintained above 90%. For example, when the PyNP enzyme solution prepared from 9.7 mg of bacterial sludge and the PNP enzyme solution prepared from 2.7 mg of bacterial sludge were used, the conversion rate was 93.17%.

[0066]

Table 3

[0067] (Example 5) Amplification reaction of 2'-deoxyadenosine synthesis A 50 mL reaction system containing 400 mM thymidine, 200 mM adenine, the PyNP enzyme solution prepared from 484.0 mg of bacterial sludge, and the PNP enzyme solution prepared from 135.0 mg of bacterial sludge was prepared in 2 mM phosphate buffer (pH 7.5), reacted at 60 °C for 16 h to produce thymine and 2'-deoxyadenosine. After the reaction was completed, an equal volume of DMSO was added to 1 mL of the reaction solution to dissolve it, diluted 20-fold, and then detected by HPLC. As shown in Figure 3, the conversion rate of thymidine was 39.20%, and the conversion rate of purine was 94.72%. From the calibration curve shown in Figure 1, the yield of 2'-deoxyadenosine was calculated to be 51.1 g / L.

[0068] (Example 6) Influence of the amount of added enzyme on the 2'-deoxyadenosine synthesis amplification reaction After Example 5 was carried out, 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 h. After the reaction was completed, an equal volume of DMSO was added to 1 mL of the reaction solution to dissolve it, diluted 20-fold, and then detected 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) Enzymatic catalytic reaction of 2'-deoxyguanosine 1. Guanine with a concentration of 1 M was prepared and dissolved with NaOH. 120 mM thymidine, 40 mM guanine, the PyNP enzyme solution prepared from 58 mg of bacterial sludge, and the PNP enzyme solution prepared from 120 mg of bacterial sludge were added to a 1 mL reaction system of 2 mM phosphate buffer (pH 7.5), reacted at 60 °C for 16 h, then sampled, an equal volume of DMSO was added to dissolve it, 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 highest yield of 2'-deoxyguanosine was calculated to be 9.8 g / L.

[0070] 2. Prepare guanine with a concentration of 1 M, dissolve it with NaOH, add 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 to a reaction system of 1 mL of 2 mM phosphate buffer (pH 7.5), react at 60 °C for 16 h, then sample, add an equal volume of DMSO to dissolve, dilute 20-fold, detect by HPLC. The conversion rate of thymidine is 31.79%, and the conversion rate of purine is 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 above experimental results are shown in Table 4.

[0072] [Table 4]

[0073] (Example 8) Reaction for the synthesis of 2,6-diaminopurine nucleoside Prepare a 10 mL reaction system 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 in 2 mM phosphate buffer (pH 7.5), and react at 60 °C for 16 h. After the reaction is completed, add an equal volume of DMSO to 1 mL of the reaction solution to dissolve, dilute 20-fold, and then detect by HPLC. The conversion rate of uridine is 54.92%, the conversion rate of purine is 96.18%, and the yield of 2,6-diaminopurine nucleoside is 54.3 g / L.

[0074] (Example 9) Reaction for the synthesis of 2,6-diaminopurine-2'-deoxynucleoside A 10 mL reaction system 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 was prepared in 2 mM phosphate buffer (pH 7.5), and reacted at 50 °C for 16 h. After the reaction was completed, an equal volume of DMSO was added to 1 mL of the reaction solution to dissolve it, and after 20-fold dilution, detection was carried out 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) Enzymatic catalytic reaction for 6-O-methyl-guanosine synthesis A 10 mL reaction system 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 was prepared in 2 mM phosphate buffer (pH 7.5), and reacted at 60 °C for 24 h. After the reaction was completed, an equal volume of DMSO was added to 1 mL of the reaction solution to dissolve it, and after 20-fold dilution, detection was carried out by HPLC. The conversion rate of uridine was 51.27%, the conversion rate of purine was 98.67%, and the yield of 6-O-methyl-guanosine was 58.6 g / L.

[0076] From the above description, it can be seen that the above examples of the present invention achieve the following technical effects. By the above enzymatic catalytic method, in a short catalytic time, using substrate bases and substrate nucleosides as substrates, purine nucleoside-based products can be synthesized, and amplification production is possible. In a short reaction time, 2'-deoxyadenosine, 2'-deoxyguanosine, 2,6-diaminopurine nucleoside, 2,6-diaminopurine-2'-deoxynucleoside, and 6-O-methyl-guanosine can be efficiently synthesized, and the yields can reach 68.3 g / L, 17.5 g / L, 54.3 g / L, 59.6 g / L, and 58.6 g / L respectively.

[0077] The above are only preferred embodiments of the present invention and do not limit the present invention. For those skilled in the art, various changes and modifications are possible to the present invention. Any changes, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for synthesizing purine nucleoside by enzymatic catalysis, wherein the method uses purine nucleoside phosphorylase and one of pyrimidine nucleoside phosphorylase or thymidine phosphorylase to catalyze a substrate to synthesize the purine nucleoside, the substrate includes a substrate nucleoside and a substrate base, the pyrimidine nucleoside phosphorylase includes PyNP which is a protein represented by SEQ ID NO: 1, or a protein having 80% or more homology with the PyNP and having the same function, the thymidine phosphorylase includes TP which is a protein represented by SEQ ID NO: 2, or a protein having 80% or more homology with the TP and having the same function, the purine nucleoside phosphorylase includes PNP which is a protein represented by SEQ ID NO: 3, or a protein having 80% or more homology with the PNP and having the same function, and a method for synthesizing purine nucleoside by enzymatic catalysis is characterized by this.

2. The substrate nucleoside is a nucleoside represented by Formula I, and R １ is selected from -H or -OH, and R ２ is selected from -H or -CH ３ and is selected from 【Chemical 1】 Preferably, the substrate nucleoside includes thymidine, uridine or 2'-deoxyuridine, and the method according to claim 1 is characterized by this.

3. The base of the substrate is a base represented by Formula II or Formula III, and X is -NH ３ or -OCH ３ selected from, and Y is selected from -H or -NH ２ selected from, 【Chemical Formula 2】 Preferably, the substrate base includes guanine, adenine, 2,6-diaminopurine or 6-methoxyguanine, and the method according to claim 1 is characterized by this.

4. One or more of the pyrimidine nucleoside phosphorylase, the thymidine phosphorylase or the purine nucleoside phosphorylase is a purified protein, a crude enzyme solution or an immobilized enzyme, and the method according to claim 1 is characterized by this.

5. In the method, the concentration of the substrate nucleoside is 2 to 400 mM, and the concentration of the substrate base is 1 to 200 mM, and the method according to claim 1 is characterized by this.

6. The catalytic time of the enzymatic catalysis is 2 to 18 h, and the method according to claim 1 is characterized by this.

7. The catalytic temperature of the enzymatic catalysis is 60 to 70 °C, and the method according to claim 1 is characterized by this.

8. A composition comprising purine nucleoside phosphorylase and one of pyrimidine nucleoside phosphorylase or thymidine phosphorylase, wherein The pyrimidine nucleoside phosphorylase is PyNP, a protein represented by SEQ ID NO: 1, or a protein having 80% or more homology with the PyNP and having the same function. The thymidine phosphorylase is TP, a protein represented by SEQ ID NO: 2, or a protein having 80% or more homology with the TP and having the same function. The purine nucleoside phosphorylase is PNP, a protein represented by SEQ ID NO: 3, or a protein having 80% or more homology with the PNP and having the same function, and the composition is characterized by this. Claim 9 One or more of the pyrimidine nucleoside phosphorylase, the thymidine phosphorylase, or the purine nucleoside phosphorylase is a purified protein, a crude enzyme solution, or an immobilized enzyme, and the composition according to claim 8 is characterized by this. Claim 10 The composition further contains a substrate nucleoside and a substrate base. The substrate nucleoside is a nucleoside represented by Formula I, and R １ is selected from -H or -OH, and R ２ is selected from -H or -CH ３ and is selected from The base of the substrate is a base represented by Formula II or Formula III, and X is -NH ３ or -OCH ３ selected from, and Y is selected from -H or -NH ２ selected from, [Chemical Formula 3] Preferably, the concentration of the substrate nucleoside is 2 to 400 mM, and the concentration of the substrate base is 1 to 200 mM, and the composition according to claim 8 or 9 is characterized by this.

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