Preparation method of cytidine-5 '-diphosphate
By using 5’-cytidine, adenosine triphosphate and sodium hexametaphosphate as raw materials, and using 5’-cytidine kinase and polyphosphate kinase for a one-pot catalytic reaction, the problems of poor safety in cytidine-5’-diphosphate production and difficulty in removing by-product impurities in the prior art were solved, and efficient and low-cost cytidine-5’-diphosphate synthesis was achieved.
Patent Information
- Application Number
- CN202411928729.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-24
- Publication Date
- 2025-03-04
AI Technical Summary
The existing cytidine-5’-diphosphate production methods have problems such as poor safety, difficulty in removing by-product impurities, and expensive raw materials.
采用5’-胞苷酸、 三磷酸腺苷和六偏磷酸钠作为原料,通过5’-胞苷酸激酶和多聚磷酸激酶的一锅催化反应,减少副产物的生成,提高产品纯度。
The efficient synthesis of cytidine-5’-diphosphate is achieved, which reduces production costs, simplifies the subsequent purification process, and improves the purity and safety of the product.
Smart Images
Figure CN119552932A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and in particular to a method for preparing cytidine-5'-diphosphate. Background Art
[0002] Cytidine 5'-diphosphate (CDP) is a nucleotide composed of a pyrophosphate group, a pentose, and the base cytosine. It is an important intermediate product of pyrimidine nucleotides in the human body, one of the important nucleotides that make up RNA, and a precursor for the synthesis of deoxyribonucleotides in the human body.
[0003] The sodium salt of cytidine-5'-diphosphate can be used as an important intermediate, health food and biochemical reagent in the production of nucleic acid drugs, and is used to manufacture drugs such as cytidine triphosphate and polyinosinic acid, playing an important role in the treatment of many major diseases.
[0004] The existing methods for producing cytidine-5'-diphosphate mainly include chemical synthesis and cytidine-5'-triphosphate phosphorus reduction. The chemical method is to protect the active group of 5'-cytidylic acid and use the toxic POC13 reagent as a phosphorylation reagent to synthesize cytidine-5'-diphosphate. Toxic reagents are used in the reaction process, which is high temperature and high pressure, explosive, poor in production safety, and easy to cause pollution.
[0005] The cytidine-5'-triphosphate reduction method utilizes cytidine-5'-triphosphate to convert glucose into cytidine-5'-diphosphate under the action of hexokinase. However, due to the high price of cytidine-5'-triphosphate, it lacks practical value.
[0006] The current mainstream method uses 5'-cytidylate kinase and polyphosphate kinase in whole cells to catalyze the synthesis of cytidine-5'-diphosphate. Due to the influence of the cell's own enzymes, it is easy to react to generate impurities such as cytidine-5'-triphosphate, which makes subsequent purification difficult.
[0007] There are also many methods that use 5'-cytidylic acid as a raw material and polyphosphate kinase to catalyze the reaction in one step to synthesize cytidine-5'-diphosphate. This method can avoid the production of impurities such as cytidine-5'-triphosphate. However, due to the low activity of polyphosphate kinase on pyrimidine nucleotides and the low conversion rate, it still needs to be purified by ion exchange column, and the process is complicated. Summary of the invention
[0008] In view of this, the present application provides a method for preparing cytidine-5'-diphosphate, using the raw materials 5'-cytidylic acid and adenosine triphosphate, which are readily available commodities and relatively cheap.
[0009] According to one aspect of the present application, a method for preparing cytidine-5'-diphosphate is provided, comprising raw materials: 5'-cytidylic acid, adenosine triphosphate and sodium hexametaphosphate; free enzymes: 5'-cytidylic acid kinase and polyphosphate kinase; using 5'-cytidine, adenosine triphosphate and sodium hexametaphosphate as raw materials, 5'-cytidylic acid kinase and polyphosphate kinase to carry out a one-pot catalytic reaction to synthesize cytidine-5'-diphosphate.
[0010] In a possible implementation, the DNA sequence of 5'-cytidylate kinase is shown as SEQ ID NO: 1, and the DNA sequence of polyphosphate kinase is shown as SEQ ID NO: 3.
[0011] In a possible implementation, the amino acid sequence of 5'-cytidylate kinase is shown in SEQ ID NO:2, and the amino acid sequence of polyphosphate kinase is shown in SEQ ID NO:4.
[0012] In a possible implementation, the 5'-cytidylate kinase and the polyphosphate kinase are cloned into an exogenous vector respectively, and the exogenous vector has corresponding restriction enzyme cutting sites.
[0013] In a possible implementation, the exogenous vector is introduced into a microbial cell, which is Escherichia coli, yeast and Bacillus subtilis.
[0014] In a possible implementation, the 5'-cytidylate kinase and polyphosphate kinase are in the form of immobilized enzymes.
[0015] In a possible implementation, the 5'-cytidylate kinase and the polyphosphate kinase are simultaneously purified and immobilized and then subjected to a one-pot catalytic reaction with 5'-cytidine, adenosine triphosphate and sodium hexametaphosphate;
[0016] The structural formula of the cytidine-5'-diphosphate is:
[0017]
[0018] In a possible implementation, the reaction temperature in step 2 is 37° C., the reaction temperature in step 3 is 35° C., the pH of the reaction system in step 3 is α, and the range of α is: 7.0≤α≤8.0.
[0019] The invention has the beneficial effects of: using 5'-cytidylic acid, adenosine triphosphate and sodium hexametaphosphate as raw materials, 5'-cytidylic acid and adenosine triphosphate are easy to purchase and relatively cheap, using 5'-cytidylic acid kinase, polyphosphate kinase and 5'-cytidylic acid, adenosine triphosphate and sodium hexametaphosphate to carry out a one-pot catalytic reaction to synthesize cytidine-5'-diphosphate, and using 5'-cytidylic acid kinase and polyphosphate kinase can reduce the generation of by-products. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 The synthetic route of cytidine-5'-diphosphate in the embodiment of the present application is shown;
[0021] Figure 2 The plasmid map of pet28a(+) in the examples of the present application is shown. DETAILED DESCRIPTION
[0022] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.
[0023] Unless otherwise specified, the experimental methods used in the following examples are generally carried out under conventional conditions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.
[0024] In the examples, the experimental methods without specific conditions are usually carried out according to conventional conditions, such as the methods described in Molecular Cloning Experiment Manual (J. Sambrook, DW Russell, translated by Huang Peitang, Wang Jiaxi, Zhu Houchu, etc., third edition, Beijing: Science Press, 2002).
[0025] Reagents used in upstream genetic engineering: the genome extraction kit, plasmid extraction kit, and DNA purification and recovery kit used in the examples of the present invention were purchased from Nanjing GenScript Biotechnology Co., Ltd.; E. coli TOP10, E. coli BL21 (DE3), plasmid pET-28a (+), etc. were purchased from Qingke Biotechnology Co., Ltd.; DNA marker, low molecular weight standard protein, protein gel, etc. were purchased from Shanghai Sangon Biotechnology Co., Ltd.; primer synthesis and sequence sequencing were completed by Qingke Biotechnology Co., Ltd. The use of the above reagents refers to the product instructions.
[0026] The present invention detects the progress of the reaction by high performance liquid chromatography (HPLC) and analyzes the product. The HPLC analysis method is: chromatographic column / AQ-C18; column temperature 30°C; flow rate 1mL / min; detection wavelength 254nm; mobile phase: 20mM triethylamine phosphate solution, adjusted to pH 6.0.
[0027] Example 1: Construction of genetically engineered bacteria for 5'-cytidylate kinase and polyphosphate kinase.
[0028] 5'-cytidylate kinase, polyphosphate kinase gene fragments were synthesized by Hangzhou Qingke Biotechnology Co., Ltd. (SEQ ID NO: 1, SEQ ID NO: 2) and recombined into the PET28a+ vector ( Figure 2 ). After double digestion with restriction endonucleases NdeI and EcoRI (purchased from Shanghai Sangon Biotechnology Co., Ltd.) at 37°C for 4 hours, the fragments were separated by 1% agarose gel electrophoresis and gel recovery was performed (gel recovery kit purchased from Shanghai Sangon Biotechnology Co., Ltd.). Subsequently, the fragments were connected with the expression vector pET28a(+) (Novage) that had also been double digested with restriction endonucleases, and placed in a low-temperature ligator under the action of T4 DNA ligase (purchased from Takara) for overnight. The ligation solution was transformed into BL21(DE3) competent cells, and colony PCR screening and sequencing verification were performed to obtain positive recombinant plasmids CMPK-pET28a(+) and PPK-pET28a. The positive transformants were recorded as engineered bacteria CMPK and PPK and stored in a -80°C refrigerator.
[0029] Example 2: Cultivation of engineered strains, induced expression, and enzyme solution preparation.
[0030] After the engineered bacteria containing 5'-cytidylate kinase and polyphosphate kinase genes were activated by streaking on a plate, a single colony was picked and inoculated into 10mL LB liquid culture medium containing 50μg / mL kanamycin, and cultured at 37℃ with shaking for 10h. A 2% inoculum was transferred to 50mL LB liquid culture medium also containing 50μg / mL kanamycin, and cultured at 37℃ with shaking until the OD reached about 0.6, and IPTG with a final concentration of 0.5mM was added, and cultured at 25℃ with shaking for 12h. After the culture was completed, the culture solution was centrifuged at 8000rpm for 10min, the supernatant was discarded, and the bacteria were collected for standby use.
[0031] The collected bacteria after the culture was completed were washed twice with pH 8 phosphate buffer (50mM), then the bacteria were added to pH = 7.5 PBS (50mM) to resuspend the cells, and ultrasonically disrupted 30 times under the following conditions: power 400W, disruption for 2s, and rest for 5s. The cell disruption solution was centrifuged at 4℃ 8000rpm for 10min to remove the precipitate. The supernatant obtained was the crude enzyme solution.
[0032] Example 3: One-step purification and immobilization of 5'-cytidylate kinase, polyphosphate kinase
[0033] After acid-base regeneration, the metal chelate resin is immersed in a 10 mm nickel sulfate solution at a ratio of 50 g of the resin per liter of nickel sulfate solution. After immersion for 2 hours, the green nickel sulfate solution becomes colorless, and the resin turns green after absorbing nickel ions. It is washed three times with pure water, and 100 mL of the crude enzyme solution prepared above is added with 10 g of the resin chelating nickel ions. The mixture is slowly stirred on a shaking table for 30 minutes, taken out, washed three times with a 20 mm imidazole solution, washed three times with pure water, and stored in a 50 mm pH 7.0 Tris-HCl buffer.
[0034] Example 4: Preparation of cytidine-5'-diphosphate from 5'-cytidylic acid using immobilized enzyme.
[0035] Weigh 50g of 5'-cytidylic acid (CMP) in a 1L glass reactor, add appropriate amount of deionized water to dissolve, adjust pH to 6.5 with 6mol / L NaOH solution to completely dissolve, and obtain 5'-cytidylic acid solution. Add 0.1g of disodium adenosine triphosphate, 25g of sodium hexametaphosphate, and 5g of magnesium chloride, adjust pH to 7.0-7.5, and the temperature to 35°C. Add immobilized 5'-cytidylic acid kinase and polyphosphate kinase, stir slowly for 2h, and detect 5'-cytidylic acid conversion rate of more than 97% by HPLC. Filter and remove the immobilized enzyme, wash the immobilized enzyme with pure water, and store it at 4°C, 50mm pH7.0 Tris-HCl buffer. After the enzyme was used for repeated catalytic reaction 10 times, the reaction time was extended to 3h, and the 5'-cytidylic acid conversion rate reached 95%.
[0036] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical solutions and concepts of the present invention within the scope disclosed by the present invention, which should be covered by the protection scope of the present invention.
[0037]
[0038]
[0039]
[0040]
Claims
1. A method for preparing cytidine-5'-diphosphate, characterized in that: include: Raw materials: 5'-cytidylic acid, adenosine triphosphate and sodium hexametaphosphate; Immobilized enzymes: 5'-cytidylate kinase, polyphosphate kinase; Cytidine, adenosine triphosphate and sodium hexametaphosphate are used as raw materials, 5'-cytidylate kinase and polyphosphate kinase are used to carry out a one-pot catalytic reaction to synthesize cytidine-5'-diphosphate.
2. The method for preparing cytidine-5'-diphosphate according to claim 1, characterized in that: The DNA sequence of 5'-cytidylate kinase is shown in SEQ ID NO:1, and the DNA sequence of polyphosphate kinase is shown in SEQ ID NO:
3.
3. The method for preparing cytidine-5'-diphosphate according to claim 1, characterized in that: The amino acid sequence of 5'-cytidylate kinase is shown in SEQ ID NO:2, and the amino acid sequence of polyphosphate kinase is shown in SEQ ID NO:
4.
4. The method for preparing cytidine-5'-diphosphate according to claim 2 or 3, characterized in that: The 5'-cytidylate kinase and the polyphosphate kinase are cloned into an exogenous vector respectively, and the exogenous vector has corresponding restriction enzyme cutting sites.
5. The method for preparing cytidine-5'-diphosphate according to claim 1, characterized in that: The 5'-cytidylate kinase and polyphosphate kinase are immobilized enzymes.
6. The method for preparing cytidine-5'-diphosphate according to claim 5, characterized in that: The immobilized enzyme is subjected to one-step affinity purification.
7. The method for preparing cytidine-5'-diphosphate according to claim 6, characterized in that: The immobilized enzyme is derived from overexpressed microorganisms, preferably Escherichia coli, yeast and Bacillus subtilis.
8. The method for preparing cytidine-5'-diphosphate according to claim 7, characterized in that: The 5'-cytidylate kinase and the polyphosphate kinase are simultaneously purified and immobilized and then subjected to a one-pot catalytic reaction with 5'-cytidine, adenosine triphosphate and sodium hexametaphosphate; The structural formula of the cytidine-5'-diphosphate is:
9. The method for preparing cytidine-5'-diphosphate according to claim 8, characterized in that: The reaction temperature in step 2 is 37° C., the reaction temperature in step 3 is 35° C., the pH of the reaction system in step 3 is α, and the range of α is: 7.0≤α≤8.0.