Method for preparing inosine 5 '-triphosphate through enzyme catalysis

Abstract

The invention discloses a preparation method for preparing inosine 5 '-triphosphate through enzyme catalysis, and relates to the technical field of biological catalytic synthesis. According to the method, one-step enzyme catalysis reaction is adopted, adenosine 5 '-triphosphate is used as a raw material, adenosine 5'-triphosphate deaminase is used for direct deamination to generate inosine 5 '-triphosphate, the complex process of a traditional chemical synthesis method or a yeast conversion method is simplified, the production efficiency is remarkably improved, ATP and adenosine 5'-triphosphate deaminase are low in cost, the enzyme can be recycled, and the method is suitable for industrial production. The production cost is reduced; inosine 5 '-triphosphate is synthesized through enzyme catalytic reaction, no harmful chemical substance is needed, and environmental pollution is reduced; through purification, filtration and concentration processes, the reaction conditions are mild, the product is prevented from being damaged by high temperature, strong acid and strong alkali, the purity of the obtained product is high, the product quality is ensured, and the burden of subsequent treatment is reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological catalytic synthesis, and particularly relates to a preparation method of enzyme catalytic preparation of inosine 5'-triphosphate. BACKGROUND

[0002] Inosine 5'-triphosphate (ITP, Inosine 5'-triphosphate) is an important nucleotide, belongs to the purine nucleotide family, has multiple physiological functions in the organism, is formed by connecting three phosphate groups (beta, gamma, alpha) to inosine through ester bonds, the inosine is composed of hypoxanthine and ribose, and the three phosphate groups are sequentially connected to the 5' hydroxyl of the ribose. Can be used as a substrate or inhibitor of enzyme reaction, treatment of diseases of inosine metabolic defects.

[0003] The existing preparation method of inosine 5'-triphosphate mainly includes chemical synthesis method and yeast transformation method. The chemical synthesis method is to protect the hydroxyl groups at positions 2 and 3 of the active group of 5'-inosinic acid, and then use a phosphorylation reagent (POC13) to synthesize inosine 5'-triphosphate. In the preparation process, the yield is high, but the production safety is poor, pollution is easy to cause and impurities are more.

[0004] The yeast transformation method is to use inosinic acid as raw material, and inosine 5'-triphosphate is synthesized under the fermentation of beer yeast enzyme system. Compared with the chemical synthesis method, the method is simpler, has the advantages of low raw material cost, safe reaction and mild reaction conditions, but in the preparation process, a large amount of phosphate and glucose and other raw materials are needed, which causes difficulty in subsequent separation and purification process, needs ion exchange resin purification, causes a large amount of wastewater, increases processing cost, reduces product quality, and in addition, inosine 5'-triphosphate is unstable, in other enzyme catalytic systems, it is easy to be hydrolyzed by cell phosphatase to produce inosine 5'-diphosphate and inosine 5'-phosphate, which is difficult to meet the industrial production. Therefore, the present application proposes a preparation method of enzyme catalytic preparation of inosine 5'-triphosphate to solve the above technical problems. SUMMARY

[0005] The purpose of the present application is to provide a preparation method of enzyme catalytic preparation of inosine 5'-triphosphate to solve the problems in the prior art.

[0006] In order to achieve the above-mentioned purpose, the present application provides the following technical scheme: The preparation method of enzyme catalytic preparation of inosine 5'-triphosphate comprises the following steps: taking adenosine 5'-triphosphate as a raw material, adding catalytic enzyme adenosine 5'-triphosphate deaminase, and obtaining inosine 5'-triphosphate through one-step enzyme catalytic reaction.

[0007] Further, the adenosine 5'-triphosphate deaminase is fused with the elastin-like polypeptide tag after the start codon to form the adenosine 5'-triphosphate deaminase with the elastin-like polypeptide tag, which is purified and then reacted with adenosine 5'-triphosphate through one-step enzyme catalysis.

[0008] Further, the specific preparation process of the adenosine 5'-triphosphate deaminase with the elastin-like polypeptide tag is as follows: S1: pET-21a(+) is selected as the vector plasmid, double enzyme digestion method is adopted, NdeI (CATATG) and BamHI (GGATCC) are selected as the restriction enzyme cutting sites, the elastin-like polypeptide tag gene and the ATP (adenosine 5'-triphosphate) deaminase gene are inserted into the vector plasmid to obtain a recombinant plasmid; S2: the calcium chloride method is adopted to introduce the recombinant plasmid into the BL21 (DE3) competent cells, inoculate into the LB culture medium containing kanamycin, and screen to obtain the strain containing the recombinant plasmid; S3: the strain containing the recombinant plasmid is inoculated into the LB culture medium containing 50-60 mg / L kanamycin at an inoculation amount of 1-2 wt%, and is cultured overnight at a temperature of 35-37°C, the next day, the inoculation amount is 5-6 wt%, the LB culture medium containing 50-60 mg / L ampicillin is inoculated, and is cultured at a temperature of 35-37°C and a rotation speed of 200-250 r / min for 2-3 h, until the OD600 (cell density parameter) is 0.6-0.8, to obtain the bacterial liquid, the inducer is added, and is induced at a temperature of 30-35°C and a rotation speed of 200 r / min for 4-5 h, after the induction is completed, the bacterial liquid is centrifuged at a temperature of 3-4°C and a rotation speed of 10000-12000 r / min for 2-3 min, the supernatant is removed, and the culture medium is drained by inversion to obtain the E. coli precipitate, the bacteria breaking liquid is added, the cell is broken by using the ultrasonic cell disruptor, after the end, the filter residue is removed by centrifugation at a rotation speed of 10000-12000 r / min for 2-3 min, to obtain the adenosine 5'-triphosphate deaminase with the elastin-like polypeptide tag.

[0009] Further, the bacteria breaking liquid is the bacteria breaking liquid special for E. coli.

[0010] Further, the DNA sequence of the adenosine 5'-triphosphate deaminase is shown in SEQ ID NO: 1, and the DNA sequence of the elastin-like polypeptide tag is shown in SEQ ID NO: 3.

[0011] Further, the amino acid sequence of the adenosine 5'-triphosphate deaminase is shown in SEQ ID NO: 2, and the amino acid sequence of the elastin-like polypeptide tag is shown in SEQ ID NO: 4.

[0012] Further, the adenosine 5'-triphosphate deaminase and the elastin-like polypeptide tag are inserted into a vector plasmid, and the vector plasmid has corresponding NdeI (CATATG) and BamHI (GGATCC) restriction enzyme sites.

[0013] Further, the purification process comprises the following steps: Step 1: heat treatment of the adenosine 5'-triphosphate deaminase with the elastin-like polypeptide tag, addition of a flocculating agent, standing for 10-15 min, centrifugation, and obtaining a clear enzyme solution; Step 2: taking the clear enzyme solution, cooling, centrifugation, removal of the supernatant, and obtaining microparticles; Step 3: dissolving the microparticles in a glycerol buffer solution, cooling, centrifugation, and removal of the supernatant.

[0014] Further, the heat treatment process in Step 1 is heating to 28-30°C and holding for 10-20 min.

[0015] Further, in Step 2, the cooling process conditions are: cooling at a rate of 2-3°C / min to 20-25°C.

[0016] Further, the centrifugation process conditions are: centrifugation at a speed of 3000-10000 r / min for 10-15 min.

[0017] Further, the flocculating agent is polyethyleneimine.

[0018] Further, the enzyme catalysis reaction process comprises the following steps: Step (1): adding adenosine 5'-triphosphate in a three-necked round-bottom flask, dissolving in deionized water, adjusting pH, and heating to obtain an enzyme catalysis substrate; Step (2): adding the adenosine 5'-triphosphate deaminase with the elastin-like polypeptide tag in the enzyme catalysis substrate, stirring until dissolution, starting the reaction, controlling pH 5.5-6.0 with 6-7 mol / L hydrochloric acid in the middle, controlling the temperature at 35-37°C, after the pH no longer changes, detecting that the adenosine 5'-triphosphate is completely converted without residue; cooling to 20-25°C, stirring for 10-15 min, and after the adenosine 5'-triphosphate deaminase is completely precipitated; nanofiltration, washing 3-4 times with deionized water, concentrating to a concentration of 100-150 g / L of inosine 5'-triphosphate, adding 5-7 mol / L hydrochloric acid to adjust pH 1.0-2.0, adding an ethanol solution with a concentration of 70-75 vt%, filtering, and vacuum drying to obtain inosine-5'-triphosphate.

[0019] Further, in Step (1), the pH adjustment process is: using 6-7 mol / L NaOH solution to adjust the pH to 5.5-6.0.

[0020] Further, in step (1), the temperature of heating is 35-37℃.

[0021] Further, in step (2), the mass ratio of enzyme catalytic substrate to adenosine 5'-triphosphate deaminase of the elastin-like polypeptide tag is (8-15):0.1.

[0022] Further, in step (2), the stirring process condition is that the stirring speed is 300-400 r / min and the stirring time is 8-10 min.

[0023] Further, in step (2), the pH adjusting process is that the pH is controlled to be 5.5-6.0 using hydrochloric acid with a concentration of 5-7 mol / L until the pH does not change.

[0024] Further, in step (2), the vacuum drying process condition is that the vacuum degree is 0.3-0.5 MPa and the temperature is 60-70℃, and the drying time is 1-2 h.

[0025] Further, in step (2), the volume of the ethanol solution is 3-4 times of the reaction system.

[0026] Further, the reaction formula of the enzyme catalytic preparation of inosine 5'-triphosphate is: .

[0027] In the above technical solution, the present application adopts one-step enzyme catalytic reaction, uses adenosine 5'-triphosphate (ATP) as raw material, and directly generates inosine 5'-triphosphate (ITP) through adenosine 5'-triphosphate deaminase to simplify the complex process of the traditional chemical synthesis method or the yeast conversion method, significantly improve the production efficiency, the raw material (ATP) and the enzyme (adenosine 5'-triphosphate deaminase) have low cost, and the enzyme can be recycled to reduce the production cost; the traditional chemical method needs to use toxic phosphating reagents such as POC13, and the present application is realized through enzyme catalytic reaction, does not need harmful chemicals, reduces environmental pollution; compared with the yeast conversion method which depends on ion exchange resin purification to produce a large amount of wastewater, the present application greatly reduces the generation of wastewater through the processes of purification, filtration and concentration, meets the green chemistry requirements, and the reaction condition is mild, avoids the damage of high temperature or strong acid and alkali to the product, obtains the product with high purity, guarantees the product quality, and reduces the burden of subsequent treatment. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 The reaction formula of the enzyme catalytic preparation of inosine 5'-triphosphate in the present embodiment is: Figure 2 The high-performance liquid chromatography reaction spectrum of inosine 5'-triphosphate in the present embodiment is: DETAILED DESCRIPTION

[0029] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all of them. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0030] Unless otherwise specified, the experimental methods used in the following embodiments are the experimental methods described in the embodiments without specific conditions. Generally, the materials, reagents and the like used in the following embodiments are commercially available, unless otherwise specified.

[0031] Unless otherwise specified, the experimental methods are generally performed according to the conventional conditions, such as the methods described in the Experimental Guide of Molecular Cloning (J. Sambrook, D. W. Russell, Huang Peitang, Wang Jiaxi, Zhu Houchu, etc. Translated from the third edition, Beijing: Science Press, 2002).

[0032] The genomic extraction kit, plasmid extraction kit and DNA purification and recovery kit used in the embodiments of the present application are purchased from Nanjing Kingsriver Biological Technology Co., Ltd. E. coli TOP10, E. coli BL21 (DE3), plasmid pET-21a (+) and the like are purchased from GenScript Biotech Co., Ltd. DNA marker, low molecular weight standard protein, protein gel and the like are purchased from Shanghai Sangon Biological Engineering Co., Ltd. The primer synthesis and sequence determination are completed by GenScript Biotech Co., Ltd. The inducer is IPTG (isopropyl thiogalactoside), and the concentration is 0.2 mmol / L. The flocculating agent is polyethyleneimine, and the concentration is 0.1 wt%; Glycerol buffer, 50 mmol / L PBS (phosphate buffer), 10 wt% glycerol; Bacterium breaking liquid, model PR0202, bacterium breaking liquid special for E. coli; The DNA sequence of adenosine 5'-triphosphate deaminase is shown in SEQ ID NO: 1; The amino acid sequence of adenosine 5'-triphosphate deaminase is shown in SEQ ID NO: 2; The DNA sequence of elastin-like tag is shown in SEQ ID NO: 3; The amino acid sequence of elastin-like tag is shown in SEQ ID NO: 4; According to the amino acid sequence, the nucleotide sequence optimization is entrusted to Nanjing Kingsriver Biological Technology Co., Ltd. to facilitate the expression in E. coli. Embodiment The preparation method of inosine 5'-triphosphate prepared by enzyme catalysis comprises the following steps: taking adenosine 5'-triphosphate as raw material, adding catalytic enzyme adenosine 5'-triphosphate deaminase, and obtaining inosine 5'-triphosphate through one-step enzyme catalysis reaction.

[0033] The adenosine 5'-triphosphate deaminase is fused with the elastin-like polypeptide tag after the start codon to form the adenosine 5'-triphosphate deaminase with the elastin-like polypeptide tag.

[0034] The specific preparation process of the adenosine 5'-triphosphate deaminase with the elastin-like polypeptide tag is as follows: S1: pET-21a(+) is selected as a carrier plasmid, a double enzyme digestion method is adopted, NdeI (CATATG) and BamHI (GGATCC) are selected as restriction enzyme cutting sites, the elastin-like polypeptide tag gene and the ATP deaminase gene are inserted into the carrier plasmid to obtain a recombinant plasmid; S2: the calcium chloride method is adopted to introduce the recombinant plasmid into BL21 (DE3) competent cells, inoculate into LB culture medium containing kanamycin, and screen to obtain a strain containing the recombinant plasmid; S3: the strain containing the recombinant plasmid is inoculated into LB culture medium containing 50 mg / L kanamycin at an inoculation amount of 1%, and is cultured overnight at a temperature of 37 DEG C; the next day, the inoculation amount is 5%, the LB culture medium contains 50 mg / L ampicillin, the culture is carried out at a temperature of 37 DEG C, the rotation speed is 200 r / min, the culture is carried out for 2.5 h, the OD600 (cell density parameter) is 0.6, the bacterial liquid is obtained, the inducer is added, the culture is carried out at a temperature of 30 DEG C, the rotation speed is 200 r / min, the induction is carried out for 4 h, after the induction is completed, the bacterial liquid is centrifuged at a temperature of 4 DEG C and a rotation speed of 12000 r / min for 2 min, the supernatant is removed, the culture medium is drained by inversion, the E. coli precipitate is obtained, the bacterium breaking liquid is added, the cell is broken by using an ultrasonic cell disruptor, after the operation is completed, the filter residue is removed by centrifugation at a rotation speed of 12000 r / min for 2 min, and the adenosine 5'-triphosphate deaminase with the elastin-like polypeptide tag is obtained; the mass ratio of the E. coli precipitate to the bacterium breaking liquid is 1:4; the pH is 7.4.

[0035] The purification process comprises the following steps: Step 1: the adenosine 5'-triphosphate deaminase with the elastin-like polypeptide tag is heated to 37 DEG C, after 10 min of incubation, the flocculating agent is added, the liquid is placed for 10 min, and the liquid is centrifuged to obtain the clear enzyme liquid; Step 2: the clear enzyme liquid is taken, cooled to 20 DEG C, centrifuged, and the supernatant is removed to obtain the microparticles; Step 3: The microparticles are added into the glycerol buffer solution, cooled, centrifuged, and the supernatant is removed to obtain the purified adenosine 5'-triphosphate deaminase with the elastin-like polypeptide tag.

[0036] In the above step, the centrifugation process conditions are as follows: a rotation speed of 12000 r / min and centrifugation for 2 min.

[0037] The enzyme catalysis reaction process comprises the following steps: Step (1): 150 g of adenosine 5'-triphosphate is added into a 2 L three-necked round-bottom flask, dissolved in 1 L of deionized water, the pH is adjusted to 5.5 by using a 6 mol / L NaOH solution, and the enzyme catalysis substrate is obtained by heating to 35℃. Step (2): 1.5 g of the adenosine 5'-triphosphate deaminase with the elastin-like polypeptide tag is added into the enzyme catalysis substrate, stirred until dissolved, and the reaction is started. During the reaction, the pH is controlled to be 5.5 by using a 6 mol / L hydrochloric acid, and the temperature is controlled to be 35℃. After the pH no longer changes, the complete conversion of the adenosine 5'-triphosphate is detected, and after the residual adenosine 5'-triphosphate is removed, the temperature is lowered to 20℃, and the adenosine 5'-triphosphate deaminase is completely precipitated after stirring for 10 min. The obtained solution is subjected to nanofiltration, washed with deionized water for three times, concentrated to a concentration of 150 g / L, adjusted to a pH of 1.0 by adding a 6 mol / L hydrochloric acid, and then added with a 70 vt% ethanol solution, filtered, and 186 g of inosine 5'-triphosphate is obtained. The inosine 5'-triphosphate is dried at a vacuum degree of 0.3 MPa and a temperature of 60℃ for 1 h to obtain 140 g of inosine-5'-triphosphate.

[0038] Experiment: The reaction is detected by high performance liquid chromatography (HPLC), and the product is analyzed. The HPLC analysis method is as follows: a chromatographic column / AQ-C18; a column temperature of 30℃; a flow rate of 1 mL / min; a detection wavelength of 254 nm; and a mobile phase of 20 mmol / L sodium dihydrogen phosphate solution, adjusted to a pH of 6.0. The purity detected by HPLC is 99.1%, as shown in Figure 2 .

[0039] Conclusion: The one-step enzyme catalysis reaction of the present application directly deaminates adenosine 5'-triphosphate (ATP) to generate inosine 5'-triphosphate (ITP) by using adenosine 5'-triphosphate deaminase, which simplifies the complex process of the traditional chemical synthesis method or the yeast conversion method, significantly improves the production efficiency, obtains a product with high purity, guarantees the product quality, and reduces the burden of subsequent processing.

[0040] It will be apparent to those skilled in the art that the application is not limited to the details of the above-exemplified embodiments and that the present application can be implemented in other particular forms without departing from the spirit or essential characteristics of the present application. The embodiments should therefore be considered in all respects as illustrative and not restrictive, the scope of the application being indicated by the appended claims rather than by the above description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims

1. A process for the enzymatic production of inosine 5'-triphosphate characterized in that: It comprises the following steps: The adenosine 5'-triphosphate is used as raw material, and the catalytic enzyme adenosine 5'-triphosphate deaminase is added to obtain inosine 5'-triphosphate through one-step enzyme catalytic reaction.

2. The process for the enzymatic production of inosine 5'-triphosphate according to claim 1, characterized in that: The adenosine 5'-triphosphate deaminase is fused with the elastin-like polypeptide tag after the start codon to form the adenosine 5'-triphosphate deaminase with the elastin-like polypeptide tag, and the adenosine 5'-triphosphate deaminase with the elastin-like polypeptide tag is purified and then reacted with the adenosine 5'-triphosphate through one-step enzyme catalytic reaction.

3. The method of enzymatic catalysis production of inosine 5'-triphosphate according to claim 1, characterized by that: The DNA sequence of the adenosine 5'-triphosphate deaminase is SEQ ID NO:1, the DNA sequence of the elastin-like polypeptide tag is SEQ ID NO:3, the amino acid sequence of the adenosine 5'-triphosphate deaminase is SEQ ID NO:2, and the amino acid sequence of the elastin-like polypeptide tag is SEQ ID NO:

4.

4. The method of enzymatic catalyzed production of inosine 5'-triphosphate according to claim 2, characterized in that: The purification process comprises the following steps: Step 1: The adenosine 5'-triphosphate deaminase with the elastin-like polypeptide tag is heat-treated, a flocculating agent is added, and then the mixture is left to stand for 10-15 min, centrifuged to obtain a clear enzyme solution; Step 2: The clear enzyme solution is cooled, centrifuged, and the supernatant is removed to obtain microparticles; Step 3: The microparticles are dissolved in a glycerol buffer solution, cooled, centrifuged, and the supernatant is removed.

5. The method of enzymatic catalysis production of inosine 5'-triphosphate according to claim 1, characterized by that: The enzyme catalytic reaction process comprises the following steps: Step (1): adenosine 5'-triphosphate is added to a three-necked round-bottom flask, dissolved in deionized water, adjusted in pH, and heated to obtain an enzyme catalytic substrate; Step (2): the adenosine 5'-triphosphate deaminase with the elastin-like polypeptide tag is added to the enzyme catalytic substrate, stirred until dissolved, and then the reaction is started; during the reaction, hydrochloric acid is used to control the pH to be 5.5-6.0 and the temperature to be 35-37℃; after the pH no longer changes, it is detected that the adenosine 5'-triphosphate is completely converted without any residue; then the temperature is lowered to 20-25℃, the mixture is stirred for 10-15 min, and then the adenosine 5'-triphosphate deaminase is completely precipitated; the mixture is subjected to nanofiltration, washed with deionized water for 3-4 times, concentrated to a concentration of inosine 5'-triphosphate to be 100-150 g / L, adjusted in pH to be 1.0-2.0 by adding hydrochloric acid, and then filtered and vacuum dried to obtain inosine-5'-triphosphate.

6. The method of enzymatic catalysis production of inosine 5'-triphosphate according to claim 4, characterized by that: In Step 1, the heat treatment process is heating to 28-30℃ and maintaining for 10-20 min; in Step 2, the cooling process is cooling at a rate of 2-3℃ / min to 20-25℃.

7. The method of enzymatic catalysis production of inosine 5'-triphosphate according to claim 2, characterized by that: The genes of the adenosine 5'-triphosphate deaminase and the elastin-like polypeptide tag are inserted into a vector plasmid, and the vector plasmid has corresponding NdeI (CATATG) and BamHI (GGATCC) restriction enzyme sites.

8. The method of enzymatic catalysis production of inosine 5'-triphosphate according to claim 5, characterized by that: In Step (1), the pH is adjusted by using 6-7 mol / L NaOH solution to be 5.5-6.0; and the heating temperature is 35-37℃.

9. The method of enzymatic catalysis production of inosine 5'-triphosphate according to claim 5, characterized by that: In Step (2), the mass ratio of the enzyme catalytic substrate to the adenosine 5'-triphosphate deaminase with the elastin-like polypeptide tag is (8-15):0.

1.

10. The method for preparing inosine 5′-triphosphate by enzyme catalysis according to claim 1, characterized in that: The reaction formula of the enzyme catalytic preparation of inosine 5'-triphosphate is as follows: 。

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