Preparation method for preparing inosine-5 '-diphosphate through enzyme catalysis
The enzyme catalytic method uses glycerol kinase and adenosine diphosphate deaminase to convert adenosine triphosphate and sodium hexametaphosphate into inosine-5’-diphosphate, which solves the problems of poor safety, many side reactions and difficulty in purification in the prior art, and achieves a safe, efficient and low-cost production effect.
Patent Information
- Application Number
- CN202411997463.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-31
- Publication Date
- 2025-04-11
AI Technical Summary
The existing inosine-5’-diphosphate production methods have problems such as poor safety, many side reactions, and difficulty in purification, especially the use of toxic reagents by chemical synthesis, and the cost is high.
Using enzyme catalytic method, glycerol kinase and adenosine diphosphate deaminase were used as immobilized enzymes to convert adenosine triphosphate and sodium hexametaphosphate into inosine-5’-diphosphate through a one-pot catalytic reaction.
The safe and efficient production of inosine-5’-diphosphate is achieved, reducing the generation of by-products, reducing production costs, and simplifying the subsequent purification process.
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Abstract
Description
Technical Field
[0001] The invention relates to the field of biotechnology, and in particular to a method for preparing inosine-5'-diphosphate by enzyme catalysis. Background Art
[0002] Inosine diphosphate (IDP) 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 and a high-energy phosphate bond metabolite such as adenosine triphosphate in cells.
[0003] The sodium salt of inosine-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 inosine triphosphate and polyinosinic acid, playing an important role in the treatment of many major diseases.
[0004] The existing methods for producing inosine-5'-diphosphate mainly include chemical synthesis and inosine-5'-triphosphate reduction or inosinic acid kinase synthesis. The chemical method is to protect the active group of 5'-inosinic acid and use the toxic POC13 reagent as a phosphorylation reagent to synthesize inosine-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 inosine-5'-triphosphate reduction method is to use inosine-5'-triphosphate to react with glucose under the action of hexokinase to produce inosine-5'-diphosphate, but because inosine-5'-triphosphate is expensive, it lacks practical value. The inosinic acid kinase synthesis method uses inosinic acid kinase and adenosine triphosphate to synthesize inosine-5'-diphosphate, but because inosinic acid kinase usually uses adenylate kinase as an enzyme, it has low activity for inosinic acid, insufficient conversion rate, and more impurities such as side reactions, which makes subsequent purification difficult. Summary of the invention
[0006] In view of this, the present application provides a method for preparing inosine-5'-diphosphate by enzyme catalysis, comprising: raw materials: adenosine triphosphate and sodium hexametaphosphate; immobilized enzymes: glycerol kinase and adenosine diphosphate deaminase; using adenosine triphosphate and sodium hexametaphosphate as raw materials, glycerol kinase and adenosine diphosphate deaminase are used to carry out a one-pot catalytic reaction to synthesize inosine-5'-diphosphate.
[0007] According to one aspect of the present application, the DNA sequence of glycerol kinase is shown in SEQ ID NO:1, and the DNA sequence of adenosine diphosphate deaminase is shown in SEQ ID NO:3.
[0008] According to one aspect of the present application, the amino acid sequence of glycerol kinase is shown in SEQ ID NO:2, and the amino acid sequence of adenosine diphosphate deaminase is shown in SEQ ID NO:4.
[0009] According to one aspect of the present application, the glycerol kinase and the adenosine diphosphate deaminase are cloned into exogenous vectors respectively, and the exogenous vectors have corresponding restriction enzyme cutting sites.
[0010] According to one aspect of the present application, the glycerol kinase and the ATP deaminase are simultaneously purified and immobilized.
[0011] According to one aspect of the present application, the glycerol kinase and the adenosine diphosphate deaminase are simultaneously purified and immobilized and then subjected to a one-pot catalytic reaction with the adenosine triphosphate and the sodium hexametaphosphate.
[0012] According to one aspect of the present application, the reaction temperature of one-pot catalysis is 30-40° C., the pH of the reaction system is α, and the range of α is: 7.0≤α≤8.0.
[0013] According to one aspect of the present application, the glycerol kinase and the adenosine diphosphate deaminase may also be free enzymes.
[0014] According to one aspect of the present application, the immobilized enzyme is derived from overexpressed microorganisms, preferably Escherichia coli.
[0015] The beneficial effects of the present invention are as follows: by using adenosine triphosphate and sodium hexametaphosphate as raw materials, adenosine triphosphate and sodium hexametaphosphate are easy to purchase and relatively cheap, using glycerol kinase and adenosine diphosphate deaminase as immobilized enzymes, using glycerol kinase and adenosine diphosphate deaminase to carry out a one-pot catalytic reaction with adenosine triphosphate and sodium hexametaphosphate to synthesize inosine-5'-diphosphate, and using adenosine triphosphate and sodium hexametaphosphate can reduce the generation of by-products. DETAILED DESCRIPTION
[0016] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.
[0017] 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.
[0018] The invention discloses a method for preparing inosine-5'-diphosphate by enzyme catalysis, comprising: raw materials: adenosine triphosphate and sodium hexametaphosphate; immobilized enzymes: glycerol kinase and adenosine diphosphate deaminase; using adenosine triphosphate and sodium hexametaphosphate as raw materials, glycerol kinase and adenosine diphosphate deaminase to carry out a one-pot catalytic reaction to synthesize inosine-5'-diphosphate.
[0019] According to one aspect of the present application, the DNA sequence of glycerol kinase is shown in SEQ ID NO:1, and the DNA sequence of adenosine diphosphate deaminase is shown in SEQ ID NO:3.
[0020] According to one aspect of the present application, the amino acid sequence of glycerol kinase is shown in SEQ ID NO:2, and the amino acid sequence of adenosine diphosphate deaminase is shown in SEQ ID NO:4.
[0021] According to one aspect of the present application, the glycerol kinase and the adenosine diphosphate deaminase are cloned into exogenous vectors respectively, and the exogenous vectors have corresponding restriction enzyme cutting sites.
[0022] According to one aspect of the present application, the glycerol kinase and the ATP deaminase are simultaneously purified and immobilized.
[0023] According to one aspect of the present application, the glycerol kinase and the adenosine diphosphate deaminase are simultaneously purified and immobilized and then subjected to a one-pot catalytic reaction with the adenosine triphosphate and the sodium hexametaphosphate.
[0024] According to one aspect of the present application, the reaction temperature of one-pot catalysis is 30-40° C., the pH of the reaction system is α, and the range of α is: 7.0≤α≤8.0.
[0025] According to one aspect of the present application, the glycerol kinase and the adenosine diphosphate deaminase may also be free enzymes.
[0026] According to one aspect of the present application, the immobilized enzyme is derived from overexpressed microorganisms, preferably Escherichia coli.
[0027] 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).
[0028] 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.
[0029] 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 sodium dihydrogen phosphate solution, adjusted to pH 6.0.
[0030] Example 1: Construction of genetically engineered bacteria for glycerol kinase and adenosine diphosphate deaminase
[0031] According to its amino acid sequence, Nanjing GenScript Biotech Co., Ltd. was commissioned to optimize the nucleotide sequence to facilitate expression in Escherichia coli. The coding genes of glycerol kinase and adenosine diphosphate deaminase are SEQ ID NO: 1 and SEQ ID NO: 3, respectively, and the amino acid sequences are SEQ ID NO: 2 and SEQ ID NO: 4, respectively cloned into the NdeI and Baml restriction enzyme sites of pET21a (+). According to conventional operation methods, the plasmid was introduced into BL21 (DE3) competent cells by calcium chloride method, and a plate containing ampicillin was smeared, and a single colony was picked for enzyme verification, and the correct single colony was selected to obtain the glycerol kinase expression strain BL21 (DE3)-glycerol kinase and the adenosine diphosphate deaminase expression strain BL21 (DE3)-adenosine diphosphate deaminase.
[0032] Example 2: Construction of genetically engineered bacteria for glycerol kinase and adenosine diphosphate deaminase, cultivation of genetically engineered strains, induced expression, and enzyme solution preparation
[0033] Inoculate the glycerol tube of the strain at a 1% inoculation amount into LB medium containing 50mg / L ampicillin and culture overnight at 37°C. The next day, inoculate the tube with a 5% inoculation amount into LB medium containing 50mg / L ampicillin and culture at 37°C and 200rpm for 2-3h. When the OD600 is about 0.6-0.8, add 0.2mM IPTG at 30°C and 200rmp for induction for 4-5h. After induction, centrifuge the bacterial solution at 12000rpm at 4°C for 2 minutes, pour off the supernatant, invert to drain the culture medium, and then resuspend the bacteria in a 1:4 (w / w) bacterial lysis solution (pH 7.4) buffer. Use an ultrasonic disruptor to break the bacterial solution for 2 minutes. Centrifuge, and the supernatant obtained is the crude enzyme solution. Glycerol kinase and adenosine diphosphate deaminase crude enzyme solutions are collected in this way.
[0034] Example 3: Preparation of inosine-5'-diphosphate from 5'-inosinic acid using glycerol kinase and adenosine diphosphate deaminase
[0035] Weigh 200g of ATP in a 2L three-necked round-bottom flask, add 1L of deionized water to dissolve, adjust the pH to 6.5 with 6mol / L NaOH solution to make it completely dissolved, add 10g of magnesium chloride and 50g of glycerol, adjust the pH to 7.0-7.5, and the temperature to 35°C. Add glycerol kinase and crude enzyme solution of adenosine diphosphate deaminase, stir slowly for 2h, adjust the pH to 7.0 with 6mol / L NaOH solution in the middle, and the ATP conversion rate is more than 99.9% by HPLC detection. Remove the enzyme solution by ultrafiltration, add 3 times the volume of alcohol for crystallization, and filter to obtain 130g of crude inosine-5'-diphosphate. Dissolve the crude product in 500mL of pure water, decolorize with activated carbon, filter, add 3 times the volume of alcohol to the filtrate for recrystallization, filter and collect the filter cake, and vacuum dry to obtain 100g of inosine-5'-diphosphate, with a purity of 99.5% as detected by HPLC.
[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 inosine-5'-diphosphate by enzyme catalysis, characterized in that: include: Raw materials: adenosine triphosphate and sodium hexametaphosphate; Immobilized enzymes: glycerol kinase and adenosine diphosphate deaminase; Inosine-5'-diphosphate was synthesized by using adenosine triphosphate and sodium hexametaphosphate as raw materials and glycerol kinase and adenosine diphosphate deaminase in a one-pot catalytic reaction.
2. The method for preparing inosine-5'-diphosphate by enzyme catalysis according to claim 1, characterized in that: The DNA sequence of glycerol kinase is shown in SEQ ID NO:1, and the DNA sequence of adenosine diphosphate deaminase is shown in SEQ ID NO:
3.
3. The method for preparing inosine-5'-diphosphate by enzyme catalysis according to claim 1, characterized in that: The amino acid sequence of glycerol kinase is shown in SEQ ID NO:2, and the amino acid sequence of adenosine diphosphate deaminase is shown in SEQ ID NO:
4.
4. The method for preparing inosine-5'-diphosphate by enzyme catalysis according to claim 2 or 3, characterized in that: The glycerol kinase and the adenosine diphosphate deaminase are cloned into an exogenous vector respectively, and the exogenous vector has corresponding restriction enzyme cutting sites.
5. The method for preparing inosine-5'-diphosphate by enzyme catalysis according to claim 1, characterized in that: The glycerol kinase and the adenosine diphosphate deaminase are simultaneously purified and immobilized.
6. The method for preparing inosine-5'-diphosphate by enzyme catalysis according to claim 5, characterized in that: The glycerol kinase and the adenosine diphosphate deaminase are simultaneously purified and immobilized and then subjected to a one-pot catalytic reaction with the adenosine triphosphate and the sodium hexametaphosphate.
7. The method for preparing inosine-5'-diphosphate by enzyme catalysis according to claim 6, characterized in that: The reaction temperature of one-pot catalysis is 30-40° C., the pH of the reaction system is α, and the range of α is: 7.0≤α≤8.
0.
8. The method for preparing inosine-5'-diphosphate by enzyme catalysis according to claim 7, characterized in that: The glycerol kinase and the adenosine diphosphate deaminase may also be free enzymes.
9. The method for preparing inosine-5'-diphosphate by enzyme catalysis according to claim 8, characterized in that: The immobilized enzyme is derived from overexpressed microorganisms, preferably Escherichia coli.