Preparation method of inosine 5 '-triphosphoric acid and inosine 5'-triphosphoric acid
By purifying and catalyzing adenosine 5′-triphosphate deaminase, the safety and pollution problems in the production of inosine 5′-triphosphate were solved, and high-purity, low-cost preparation of inosine 5′-triphosphate was achieved.
Patent Information
- Application Number
- CN202510962815.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-11
- Publication Date
- 2025-11-14
AI Technical Summary
Existing methods for producing inosine 5′-triphosphate suffer from poor safety, severe pollution, numerous impurities, and high processing costs. In particular, inosine 5′-triphosphate is unstable in enzyme catalysis systems, making industrial-scale production difficult.
After purification, adenosine 5′-triphosphate deaminase is used to synthesize inosine 5′-triphosphate through a one-step catalytic reaction with adenosine 5′-triphosphate. The purification and catalysis are carried out using adenosine 5′-triphosphate deaminase with an elastin-like tag, which avoids complex purification processes and wastewater generation.
This has enabled the production of inosine 5′-triphosphate with high safety, no pollution, and low cost, resulting in high product purity and avoiding environmental pollution and increased processing costs.
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Figure CN120944989A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the fields of biopharmaceutical and biochemical technology, and relates to a method for preparing inosine 5′-triphosphate and inosine 5′-triphosphate. Background Technology
[0002] Inosine 5′-triphosphate (ITP) is a nucleotide composed of three phosphate groups, a pentose sugar, and the base hypoxanthine. It is an important intermediate derivative of purine nucleotides in the human body and a metabolite of high-energy phosphate bonds such as adenosine triphosphate in cells.
[0003] Sodium salt of inosine 5′-triphosphate can be used as an important intermediate in the production of nucleic acid drugs, health food and biochemical reagent, and is used to manufacture drugs such as inosine diphosphate and polyinosinic acid, playing an important role in the treatment of a variety of major diseases.
[0004] Existing methods for producing inosine 5′-triphosphate mainly include chemical synthesis and yeast conversion. The chemical method involves protecting the hydroxyl groups at the 2 and 3 positions of the active group of 5′-inosine acid, and then using the toxic POC13 reagent as a phosphorylation agent for synthesis. The overall process has a high yield, but poor production safety, is prone to pollution, and produces many impurities.
[0005] The yeast conversion method synthesizes inosine 5′-triphosphate by fermenting inosine acid with brewer's yeast enzyme system. Compared with the chemical method, this method is simple and has the advantages of low raw material cost, safe reaction, and mild reaction conditions. However, it has its drawbacks. The process uses a large amount of raw materials such as phosphate and glucose, which makes subsequent separation and purification processes difficult. Ion exchange resin purification is required, resulting in a large amount of wastewater, increasing processing costs and reducing product quality. In addition, because inosine 5′-triphosphate is unstable, it is easily hydrolyzed by cellular phosphatases in other enzyme catalytic systems to produce inosine 5′-diphosphate and inosine 5′-phosphate. Therefore, conventional enzyme catalytic reaction processes are difficult to scale up for industrial production without adding complex purification processes. Summary of the Invention
[0006] In view of this, this application provides a method for preparing inosine 5′-triphosphate by enzyme catalysis, comprising: raw material: adenosine 5′-triphosphate; catalytic enzyme: adenosine 5′-triphosphate deaminase; using adenosine 5′-triphosphate as raw material, the adenosine 5′-triphosphate deaminase is purified and then synthesized into inosine 5′-triphosphate through a one-step catalytic reaction.
[0007] According to one aspect of this application, the adenosine 5′-triphosphate deaminase start codon is fused with an elastin tag to form an adenosine 5′-triphosphate deaminase with an elastin-like tag. After purification, the adenosine 5′-triphosphate deaminase with the elastin-like tag is reacted with adenosine 5′-triphosphate in a one-step catalytic reaction to synthesize inosine 5′-triphosphate.
[0008] According to one aspect of this application, the DNA sequence of the adenosine 5′-triphosphate deaminase is shown in SEQ ID NO:1, and the DNA sequence of the fused elastin tag is shown in SEQ ID NO:3.
[0009] According to one aspect of this application, the amino acid sequence of the adenosine 5′-triphosphate deaminase is shown in SEQ ID NO:2, and the amino acid sequence of the fused elastin tag is shown in SEQ ID NO:4.
[0010] According to one aspect of this application, the adenosine 5′-triphosphate deaminase gene and the fused elastin tag gene are cloned into an exogenous vector having corresponding restriction enzyme sites.
[0011] According to one aspect of this application, the specific steps for purifying the adenosine 5′-triphosphate deaminase with an elastin-like tag include: firstly, heating and holding the adenosine 5′-triphosphate deaminase with an elastin-like tag, then adding the flocculant polyethyleneimine, allowing it to stand, centrifuging, and collecting the supernatant; cooling the supernatant to precipitate micron-sized particles, and performing a second centrifugation; discarding the supernatant obtained from the second centrifugation, dissolving the micron-sized particles in glycerol buffer, cooling, precipitating micron-sized particles II, centrifuging again, and collecting the bottom filter residue to obtain the purified adenosine 5′-triphosphate deaminase with an elastin-like tag.
[0012] According to one aspect of this application, the adenosine 5′-triphosphate deaminase with an elastin-like tag is heated to 28-30 degrees Celsius during the purification process.
[0013] According to one aspect of this application, the specific steps for synthesizing inosine 5′-triphosphate by purifying the elastin-tagged adenosine 5′-triphosphate deaminase and reacting it with adenosine 5′-triphosphate in a one-step catalytic reaction include: adding an appropriate amount of the adenosine 5′-triphosphate to a round-bottom flask, dissolving it in deionized water, adjusting the pH with NaOH solution, and heating; then adding the purified elastin-tagged adenosine 5′-triphosphate deaminase, stirring to initiate dissolution and reaction, adding hydrochloric acid during the dissolution and reaction process to control the pH at 5.5-6.0; and reacting until the pH reaches the settling point. After the change was observed, HPLC analysis of the sample showed complete conversion of adenosine 5′-triphosphate with no residue. The sample was then cooled and stirred to ensure complete precipitation of adenosine 5′-triphosphate deaminase. The enzyme micron particles were filtered out and recycled. The filtrate was ultrafiltered to remove protein, washed three times with nanofiltration water, and concentrated to a concentration of 100-150 g / L for inosine 5′-triphosphate. The pH was adjusted to 1.0-2.0 with 6 mol / L hydrochloric acid, and precipitated with 3-4 times the volume of alcohol. The precipitate was then collected by filtration and vacuum drying to obtain the final inosine 5′-triphosphate.
[0014] According to one aspect of this application, using the adenosine 5′-triphosphate as a raw material, the reaction temperature for the synthesis of inosine 5′-triphosphate catalyzed by the adenosine 5′-triphosphate deaminase is 35-37°C, and the pH of the reaction system is 5.5-6.0.
[0015] According to one aspect of this application, the molecular formula of inosine 5′-triphosphate is: .
[0016] The beneficial effects of this invention are as follows: By using adenosine 5′-triphosphate as a raw material and adenosine 5′-triphosphate deaminase as a catalytic enzyme; using adenosine 5′-triphosphate as a raw material and adenosine 5′-triphosphate deaminase after purification, inosine 5′-triphosphate is synthesized through a one-step catalytic reaction. Firstly, adenosine 5′-triphosphate and adenosine 5′-triphosphate deaminase are low in cost, relatively safe during the reaction process, and avoid generating a large amount of wastewater during the reaction. The synthesized inosine 5′-triphosphate is free of impurities, has high safety, and avoids environmental pollution. Attached Figure Description
[0017] Figure 1 The molecular formula of inosine 5′-triphosphate in this embodiment is shown; Figure 2 The HPLC reaction chromatogram of inosine 5′-triphosphate in this embodiment is shown. Detailed Implementation
[0018] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.
[0019] Unless otherwise specified, the experimental methods used in the following examples are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.
[0020] This application provides a method for preparing inosine 5′-triphosphate by enzyme catalysis, comprising: raw material: adenosine 5′-triphosphate; catalytic enzyme: adenosine 5′-triphosphate deaminase; using adenosine 5′-triphosphate as raw material, the adenosine 5′-triphosphate deaminase is purified and then synthesized into inosine 5′-triphosphate through a one-step catalytic reaction.
[0021] According to one aspect of this application, the adenosine 5′-triphosphate deaminase start codon is fused with an elastin tag to form an adenosine 5′-triphosphate deaminase with an elastin-like tag. After purification, the adenosine 5′-triphosphate deaminase with the elastin-like tag is reacted with adenosine 5′-triphosphate in a one-step catalytic reaction to synthesize inosine 5′-triphosphate.
[0022] According to one aspect of this application, the DNA sequence of the adenosine 5′-triphosphate deaminase is shown in SEQ ID NO:1, and the DNA sequence of the fused elastin tag is shown in SEQ ID NO:3.
[0023] According to one aspect of this application, the amino acid sequence of the adenosine 5′-triphosphate deaminase is shown in SEQ ID NO:2, and the amino acid sequence of the fused elastin tag is shown in SEQ ID NO:4.
[0024] According to one aspect of this application, the adenosine 5′-triphosphate deaminase gene and the fused elastin tag gene are cloned into an exogenous vector having corresponding restriction enzyme sites.
[0025] According to one aspect of this application, the specific steps for purifying the adenosine 5′-triphosphate deaminase with an elastin-like tag include: firstly, heating and holding the adenosine 5′-triphosphate deaminase with an elastin-like tag, then adding the flocculant polyethyleneimine, allowing it to stand, centrifuging, and collecting the supernatant; cooling the supernatant to precipitate micron-sized particles, and performing a second centrifugation; discarding the supernatant obtained from the second centrifugation, dissolving the micron-sized particles in glycerol buffer, cooling, precipitating micron-sized particles II, centrifuging again, and collecting the bottom filter residue to obtain the purified adenosine 5′-triphosphate deaminase with an elastin-like tag.
[0026] According to one aspect of this application, the adenosine 5′-triphosphate deaminase with an elastin-like tag is heated to 28-30 degrees Celsius during the purification process.
[0027] According to one aspect of this application, the specific steps for synthesizing inosine 5′-triphosphate by purifying the elastin-tagged adenosine 5′-triphosphate deaminase and reacting it with adenosine 5′-triphosphate in a one-step catalytic reaction include: adding an appropriate amount of the adenosine 5′-triphosphate to a round-bottom flask, dissolving it in deionized water, adjusting the pH with NaOH solution, and heating; then adding the purified elastin-tagged adenosine 5′-triphosphate deaminase, stirring to initiate dissolution and reaction, adding hydrochloric acid during the dissolution and reaction process to control the pH at 5.5-6.0; and reacting until the pH reaches the settling point. After the change was observed, HPLC analysis of the sample showed complete conversion of adenosine 5′-triphosphate with no residue. The sample was then cooled and stirred to ensure complete precipitation of adenosine 5′-triphosphate deaminase. The enzyme micron particles were filtered out and recycled. The filtrate was ultrafiltered to remove protein, washed three times with nanofiltration water, and concentrated to a concentration of 100-150 g / L for inosine 5′-triphosphate. The pH was adjusted to 1.0-2.0 with 6 mol / L hydrochloric acid, and precipitated with 3-4 times the volume of alcohol. The precipitate was then collected by filtration and vacuum drying to obtain the final inosine 5′-triphosphate.
[0028] According to one aspect of this application, using the adenosine 5′-triphosphate as a raw material, the reaction temperature for the synthesis of inosine 5′-triphosphate catalyzed by the adenosine 5′-triphosphate deaminase is 35-37°C, and the pH of the reaction system is 5.5-6.0.
[0029] According to one aspect of this application, the molecular formula of inosine 5′-triphosphate is: .
[0030] In the examples, experimental methods without specific conditions are generally performed under conventional conditions, such as those described in Molecular Cloning: A Laboratory Manual (J. Sambrook, DW. Russell, translated by Huang Peitang, Wang Jiaxi, Zhu Houchu, et al., 3rd edition, Beijing: Science Press, 2002).
[0031] Reagents used in upstream genetic engineering: The genome extraction kit, plasmid extraction kit, and DNA purification and recovery kit used in this embodiment of the invention were purchased from Nanjing Genscript Biotech Co., Ltd.; E. coli TOP10, E. coli BL21(DE3), plasmid pET-21a(+), etc., were purchased from Qingke Biotechnology Co., Ltd.; DNA markers, low molecular weight standard proteins, protein gels, etc., were purchased from Shanghai Sangon Biotech Co., Ltd.; primer synthesis and sequence sequencing were performed by Qingke Biotechnology Co., Ltd. Refer to the product instructions for the usage of the above reagents.
[0032] This invention utilizes high-performance liquid chromatography (HPLC) to detect the reaction and analyze the products. The HPLC analysis method is as follows: chromatographic column: AQ-C18; column temperature: 30℃; flow rate: 1 mL / min; detection wavelength: 254 nm; mobile phase: 20 mM sodium dihydrogen phosphate solution, adjusted to pH 6.0.
[0033] Example 1: Construction of genetically engineered bacteria for adenosine 5′-triphosphate deaminase Based on its amino acid sequence, Nanjing Genscript Biotech Co., Ltd. was commissioned to optimize the nucleotide sequence to facilitate expression in *E. coli*. The gene encoding adenosine 5′-triphosphate deaminase (SEQ ID NO: 1, amino acid sequence: SEQ ID NO: 3) and the gene encoding an elastin-like tag (SEQ ID NO: 2, amino acid sequence: SEQ ID NO: 4) were cloned into the Ndel and Baml restriction enzyme sites of pET21a(+), with the elastin-like gene preceding the ATG start codon of adenosine 5′-triphosphate deaminase. Following standard procedures, the plasmid was introduced into BL21 (DE3) competent cells via calcium chloride transformation. Plates containing carbamate were spread, and single colonies were picked for enzyme digestion verification. Correct single colonies were selected to obtain the glycerol kinase expression strain BL21 (DE3)-ATP deaminase.
[0034] Example 2: Construction of adenosine 5′-triphosphate deaminase genetically engineered bacteria, culture, induction of expression, and preparation of enzyme solution. Inoculate 1% of the bacterial culture into LB medium containing 50 mg / L ampicillin and incubate overnight at 37 °C. The next day, inoculate 5% of the culture into LB medium containing 50 mg / L ampicillin and incubate at 37 °C with shaking at 200 rpm for 2-3 h. When the OD600 reaches approximately 0.6-0.8, add 0.2 mM IPTG and induce at 30 °C with 200 rpm for 4-5 h. After induction, centrifuge the bacterial culture at 12000 rpm for 2 minutes at 4 °C, discard the supernatant, invert the culture to drain, and then resuspend the cells in a 1:4 (w / w) solution of lysis buffer (pH 7.4). Dissociate the bacterial culture using an ultrasonic homogenizer for 2 minutes. Centrifuge again, and collect the crude enzyme solution (adenosine 5′-triphosphate deaminase) using the same method.
[0035] Example 3: Purification of adenosine 5′-triphosphate deaminase with an elastin-like tag The crude adenosine 5′-triphosphate deaminase solution was heated to 37°C and incubated for 10 minutes. Then, 0.1% polyethyleneimine flocculant was added, and the mixture was allowed to stand for 10 minutes. After centrifugation, the supernatant was collected, cooled to 20-25°C, and micron-sized particles precipitated. The mixture was centrifuged again. The supernatant was discarded, and the micron-sized particles were dissolved in 30-35°C 50mm PBS (10% glycerol) buffer. After cooling to 20-25°C, the micron-sized particles precipitated again. The residue at the bottom was collected, yielding purified adenosine 5′-triphosphate deaminase. SDS-PEGE gel electrophoresis showed a band purity of over 95%.
[0036] Example 4: Synthesis of inosine 5′-triphosphate using adenosine 5′-triphosphate deaminase as catalysis Weigh 150g of adenosine 5′-triphosphate into a 2L three-necked round-bottom flask, add 1L of deionized water to dissolve, and adjust the pH to 5.5-6.0 with 6mol / L NaOH solution to ensure complete dissolution. Heat to 35-37℃, add 1.5g of adenosine 5′-triphosphate deaminase, stir to dissolve, and begin the reaction. Maintain the pH at 5.5-6.0 and the temperature at 35-37℃ using 6mol / L hydrochloric acid. After the pH no longer changes, take a sample for HPLC analysis to confirm complete conversion of adenosine 5′-triphosphate with no residue. The solution was cooled to 20-25℃ and stirred for 10 minutes. After complete precipitation of adenosine 5′-triphosphate deaminase, the enzyme micron particles were filtered out and recycled. The filtrate was ultrafiltered to remove protein, washed three times with nanofiltration water, and concentrated to a concentration of 100-150 g / L for inosine 5′-triphosphate. The pH was adjusted to 1.0-2.0 with 6 mol / L hydrochloric acid, and precipitated with 3-4 times the volume of alcohol. After filtration, 186 g of inosine 5′-triphosphate was obtained. After vacuum drying, 140 g of inosine 5′-triphosphate was obtained. The purity was determined to be 99.1% by HPLC. Figure 2 As shown.
[0037] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope disclosed in the present invention, based on the technical solution and concept of the present invention, should be covered within the scope of protection of the present invention.
[0038] sequence list <110> Shanghai Riguan Biotechnology Co., Ltd. <120> Enzyme-catalyzed preparation method of inosine 5′-triphosphate <160> 4 <210> 1 <211> 1563 <212> DNA <213> Artificial synthesis <400> 1 60 atgctagcga tggcggctct agtaggagca tcatttgtgg ctgtatccgc ttattatatg 120 caccgcaaaa ccctaaatca acttctcgag ttcgccaaga cggtagagag agaacgagaa 180 agagacgaca actccgacgg cggcggcgga ggaggaggag gatcgtcgcc gcagcatttg 240 aagaagcgcc ggagtcacgg acggaggaag ggaagtagcg gttatactataa caagcgtggc 300 tcggcttcgt taccggacgt gacggcgata tacggtggtg gaattgatgg agaggagaaa 360 cgtaatggtc aggtggttta tgtcgaaggg attccggctg gattgccgag gcttcatact 420 ttgcctgaag ggaaatcttc tgggcatatc aagaggcctg gaagttttat cagaccaact 480 tctccaaagt ctcctggtgc tagtgctagt gcctttgaca gcgtggaagg atcagatgat 540 gagataaca tgactgacaa ttctaacta gactacat atctgcatgt taatggaat 600 gctgatataa aggatgtttt accacac attaatgca atggagacca agtccctata 660 cctgcttcaa gcatgatcg atcccatagt gtgtctggtg acttgcatgg tgttcagcct 720 gatcctattg ctgctgatat tctaggaaa gaaccagaac aggaacttt tgcacgactt 780 aaaatttctc ctatggaggt gccatcacca gatgagtgg attcctatat agttctcaa 840 gatgtcttg aaatgcgaaa aagatatgta ttcaggaag caatcgcacc atgggaaa 900 gaaattattt ctgaccccag tacaccaaag cctaatcctg accctctc ctttacacca 960 gaggaaaat ctgatcatta ttttgagatg cagatgggg taatccatgt ctatccaaat 1020 aaagactcta aggagaact ttttctgtt gctgatgcga caacgtttt cactgatttg 1080 catcacatac ttcgagttat tgcaattgga atatcagaa ctctatgtca tcatcggttg 1140 atatctcctag aaaaaaatt caacttcat ttgatgctta atgcggatag agagttctt 1200 gctcagaaaa gtgctccaca ccgtgacttt tataatgtca ggaagttga tacccatgtt 1260 catcattctg catgcatgaa ccagaaacat cttttaaggt ttataaagtc aaaactgagg 1320 aaagagcctg atgaggttgt aatttttcga gatggaacat acttgacatt gaaagaagtg 1380 tttgagagtt tggatttgac tgggtatgat ctcaatgttg accttttaga tgttcacgcg 1440 gacaagagca catttcatcg ctttgataag ttcaacctga agtacaatcc ttgtggtcag 1500 agtagactta gggagatttt ccttaaacaa gacaatctta tccaaggccg tttccttggt 1560 gaactgacaa aacaagtctt ttctgatctt tctgcaagta aatatcagat ggctgaatac 1563 taa <210> 2 <211> 540 <212> AA <213> Synthetic <400> 2 60 MEAYSLHLAM AALVGASFVA VSAYYMHRKT LNQLLEFAKT VERERERGDN SDGGGGGGGG 120 SSPQHLKKRR SHGRRKGSSG YYNKRGSASL PDVTAIYGGG IDGEEKRNGQ VVYVDGIPAG 180 LPRLHTLPEG KSSGHIKRPG SFIRPTSPKS PGASASAFDS VEGSDDEDNM TDNSKLDTTY 240 LHVNGNADIK DVLPQHINAN GDQVPIPASS MIRSHSVSGD LHGVQPDPIA ADILRKEPEQ 300 ETFARLKISP MEVPSPDEVD SYIVLQECLE MRKRYVFKEA IAPWEKEIIS DPSTPKPNPD 360 PFSFTPEGKS DHYFEMQDGV IHVYPNKDSK EELFPVADAT TFFTDLHHIL RVIAIGNIRT 420 LCHHRLNLLE QKFNLHLMLN ADREFLAQKS APHRDFYNVR KVDTHVHHSA CMNQKHLLRF 480 IKSKLRKEPD EVVIFRDGTY LTLKEVFESL DLTGYDLNVD LLDVHADKST FHRFDKFNLK 540 YNPCGQSRLR EIFLKQDNLI QGRFLGELTK QVFCDLSASK YQMAEYRISI YGRKQSEWDQ <210> 3 <211> 909 <212> DNA <213> Synthetic <400> 3 60 atgtgggttc caggcattgg agtgccaggc attggcgtac caggtattgg agttccaggt 120 attggggtac cgggcatcgg agttcctggg atcggagttc cgggaattgg tgtgccgggt 180 atcggtgtgc ctgggatcgg tgttccaggt atcggggttc cgggtatcgg cgttcccggc 240 attggtgttc caggcatcgg tgtgccggga attggggttc cggggattgg tgtacctggc 300 attggggtac ctggaatcgg cgtgcctggt attggcgtgc ctggcatcgg cgttcctggc 360 attggtgttc caggcattgg agtgccaggc attggcgtac caggtattgg agttccaggt 420 attggggtac cgggcatcgg agttcctggg atcggagttc cgggaattgg tgtgccgggt 480 atcggtgtgc ctgggatcgg tgttccaggt atcggggttc cgggtatcgg cgttcccggc 540 attggtgttc caggcatcgg tgtgccggga attggggttc cggggattgg tgtacctggc 600 attggggtac ctggaatcgg cgtgcctggt attggcgtgc ctggcatcgg cgttcctggc 660 attggtgttc caggcattgg agtgccaggc attggcgtac caggtattgg agttccaggt 720 attggggtac cgggcatcgg agttcctggg atcggagttc cgggaattgg tgtgccgggt 780 atcggtgtgc ctgggatcgg tgttccaggt atcggggttc cgggtatcgg cgttcccggc 840 attggtgttc caggcatcgg tgtgccggga attggggttc cggggattgg tgtacctggc 900 attggggtac ctggaatcgg cgtgcctggt attggcgtgc ctggcatcgg cgttcctggc 909 attggttgc <210> 4 <211> 315 <212> AA <213> Synthetic <400> 4 60 MWVPGIGVPG IGVPGIGVPG IGVPGIGVPG IGVPGIGVPG IGVPGIGVPG IGVPGIGVPG 120 IGVPGIGVPG IGVPGIGVPG IGVPGIGVPG IGVPGIGVPG IGVPGIGVPG 180 IGVPGIGVPG IGVPGIGVPG IGVPGIGVPG IGVPGIGVPG IGVPGIGVPG 240 IGVPGIGVPG IGVPGIGVPG IGVPGIGVPG IGVPGIGVPG IGVPGIGVPG 300 IGVPGIGVPG IGVPGIGVPG IGVPGIGVPG IGVPGIGVPG IGVPGIGVPG 303 IGC.
Claims
1. A method for preparing inosine 5′-triphosphate via enzyme catalysis, characterized in that, include: Raw material: adenosine 5′-triphosphate; Catalytic enzyme: adenosine 5′-triphosphate deaminase; Using adenosine 5′-triphosphate as a raw material, adenosine 5′-triphosphate deaminase was purified and then synthesized into inosine 5′-triphosphate through a one-step catalytic reaction.
2. The method for preparing inosine 5′-triphosphate by enzyme catalysis according to claim 1, characterized in that, The adenosine 5′-triphosphate deaminase start codon is fused with an elastin tag to form an adenosine 5′-triphosphate deaminase with an elastin-like tag. After purification, the adenosine 5′-triphosphate deaminase with an elastin-like tag is reacted with adenosine 5′-triphosphate in a one-step catalytic reaction to synthesize inosine 5′-triphosphate.
3. The method for preparing inosine 5′-triphosphate by enzyme catalysis according to claim 2, characterized in that, The DNA sequence of the adenosine 5′-triphosphate deaminase is shown in SEQ ID NO:1, and the DNA sequence of the fused elastin tag is shown in SEQ ID NO:
3.
4. The method for preparing inosine 5′-triphosphate by enzyme catalysis according to claim 2, characterized in that, The amino acid sequence of the adenosine 5′-triphosphate deaminase is shown in SEQ ID NO:2, and the amino acid sequence of the fused elastin tag is shown in SEQ ID NO:
4.
5. The method for preparing inosine 5′-triphosphate by enzyme catalysis according to any one of claims 2-4, characterized in that, The adenosine 5′-triphosphate deaminase gene and the fused elastin tag gene were cloned into an exogenous vector, which has corresponding restriction enzyme sites.
6. The method for preparing inosine 5′-triphosphate by enzyme catalysis according to claim 5, characterized in that, The specific steps for purifying the adenosine 5′-triphosphate deaminase with the elastin-like tag include: First, adenosine 5′-triphosphate deaminase with an elastin-like tag is heated and kept warm. Then, the flocculant polyethyleneimine is added, and the mixture is allowed to stand, centrifuged, and the supernatant is collected. The supernatant was cooled to precipitate micron-sized particles, and then centrifuged a second time. Discard the supernatant obtained from the second centrifugation, dissolve the micron-sized particles in glycerol buffer, cool down, precipitate micron-sized particles II, centrifuge again, collect the bottom filter residue, and obtain the purified adenosine 5′-triphosphate deaminase with an elastin-like tag.
7. The method for preparing inosine 5′-triphosphate by enzyme catalysis according to claim 6, characterized in that, The adenosine 5′-triphosphate deaminase with an elastin-like tag is heated to 28-30 degrees Celsius during purification.
8. The method for preparing inosine 5′-triphosphate by enzyme catalysis according to claim 5, characterized in that, The specific steps for synthesizing inosine 5′-triphosphate by purifying the elastin-like tagged adenosine 5′-triphosphate and reacting it with adenosine 5′-triphosphate in a one-step catalytic reaction include: Add an appropriate amount of the adenosine 5′-triphosphate to a round-bottom flask, dissolve it in deionized water, adjust the pH with NaOH solution, and heat. Then, the purified adenosine 5′-triphosphate deaminase with the elastin-like tag is added, and the mixture is stirred to allow it to begin dissolving and reacting. During the dissolution and reaction process, hydrochloric acid is added to control the pH at 5.5-6.
0. After the reaction continued until the pH no longer changed, a sample was taken for HPLC analysis to confirm that adenosine 5′-triphosphate was completely converted and there was no residue. Begin cooling and stirring to allow adenosine 5′-triphosphate deaminase to precipitate completely; The enzyme micron particles were recycled, and the filtrate was ultrafiltered to remove proteins. It was then washed three times with nanofiltration water and concentrated to a concentration of 100-150 g / L for inosine 5′-triphosphate. The pH was adjusted to 1.0-2.0 with 6 mol / L hydrochloric acid, and 3-4 times the volume of alcohol was added for alcohol precipitation. The inosine 5′-triphosphate was collected by filtration and then vacuum dried to obtain the final inosine 5′-triphosphate.
9. The method for preparing inosine 5′-triphosphate by enzyme catalysis according to claim 8, characterized in that, Using the aforementioned adenosine 5′-triphosphate as a raw material, the reaction temperature for the synthesis of inosine 5′-triphosphate catalyzed by the adenosine 5′-triphosphate deaminase is 35-37℃, and the pH of the reaction system is 5.5-6.
0.
10. An inosine 5′-triphosphate, characterized in that, Prepared by the preparation method of any one of claims 1-9, the molecular formula of inosine 5′-triphosphate is: 。