Method for synthesizing uracil through enzyme catalysis

Abstract

The invention relates to the technical field of organic synthesis, in particular to a method for synthesizing uracil through enzyme catalysis. Comprising the following steps: step 1, adding 2-hydroxypyrimidine, sodium formate and ferrous chloride into deionized water, heating and stirring for 35-50 minutes, and adjusting the pH value to 8-9; step 2, adding NAD (Nicotinamide Adenine Dinucleotide), stirring for 5-8 minutes, then adding No. 118 enzyme and formate dehydrogenase, heating, stirring and reacting for 5-8 hours, and controlling the pH to be 7.9-8.5 during the reaction period; and step 3, after the reaction is finished, separating out a product, filtering, washing and drying to obtain uracil. The whole preparation process adopts an environment-friendly process, no harmful pollutant is discharged, the preparation cost is at a lower level, and meanwhile, high yield and high purity are realized.

Description

Technical Field

[0001] The present invention relates to the technical field of organic synthesis, and particularly to a method for enzymatically catalyzing the synthesis of uracil. Background Art

[0002] As a base unique to RNA, uracil plays a crucial role in the four-base composition system of RNA, and its function is highly equivalent to thymine (T) in DNA. In the core process of DNA transcription, uracil will replace thymine in DNA and then pair with adenine according to the base complementary pairing principle. This process is essential for the accurate transmission and expression of genetic information.

[0003] In the field of traditional chemical synthesis, the preparation of uracil usually uses malonic ester and urea as starting materials. In the initial stage of the reaction, malonic ester and urea undergo a condensation reaction under specific reaction conditions. This step of the reaction requires strict control of parameters such as temperature, pressure, and the molar ratio of reactants. Subsequently, multiple complex cyclization reactions are required, and the entire process is cumbersome and delicate. However, this traditional preparation method has many significant drawbacks. The reaction conditions are extremely harsh, often requiring high temperatures (usually reaching 150°C - 200°C) and high pressures (generally 5 - 10 MPa), and strong corrosive catalysts such as concentrated sulfuric acid must be used to promote the reaction. Such harsh reaction conditions not only pose extremely high requirements on the material and pressure resistance of the reaction equipment, increasing equipment costs and maintenance difficulties, but also pose significant safety hazards. In addition, the selectivity of this reaction is poor, and a large number of complex by-products will inevitably be generated during the reaction; the treatment costs of the waste acid (such as the acidic waste liquid formed after concentrated sulfuric acid participates in the reaction) and waste solvents generated after the reaction are high, and if not treated properly, they will cause serious pollution to the ecological environment such as soil and water bodies.

[0004] In recent years, the emerging biocatalytic method has provided new ideas for the preparation of uracil and alleviated some problems in the traditional method to a certain extent. The biocatalytic method mainly uses specific enzymes to catalyze the reaction process. However, this method itself also has a series of limitations that cannot be ignored. On the one hand, the activity of the enzymes used to catalyze the reaction is generally not high, which directly leads to a slow reaction rate and cannot meet the efficiency requirements of large-scale production. On the other hand, the substrate conversion rate is not ideal, and it is difficult to achieve efficient conversion of the substrate in most cases, resulting in waste of raw materials. In addition, the entire reaction cycle is relatively long, and it may take several days or even weeks to complete the reaction, which greatly limits the large-scale popularization and application of the biocatalytic method in the industrial field. Summary of the Invention

[0005] In view of the above deficiencies in the technology, the present invention provides a method for enzymatically catalyzing the synthesis of uracil.

[0006] To solve the above technical problems, the present invention adopts the following technical solutions: A method for enzymatically synthesizing uracil, characterized by comprising the following steps: Step 1: Add a 2-hydroxypyrimidine solution, a sodium formate solution, and an FeCl 2 solution to deionized water, heat and stir for 35 - 50 min, and adjust the pH to 8 - 9; Step 2: Add an NAD solution, stir for 5 - 8 min, then add an 118# enzyme solution and a formate dehydrogenase solution, heat and stir for 5 - 8 h, and control the pH to 7.9 - 8.5 during the reaction; Step 3: After the reaction is completed, the product precipitates, filter, wash, and dry to obtain uracil.

[0007] The specific reaction is as follows:

[0008] Preferably, it includes the following raw materials in parts by mass: 400 - 600 parts of 2-hydroxypyrimidine solution, 350 - 450 parts of sodium formate solution, 0.5 - 2 parts of NAD solution, FeCl 2 8 - 15 parts of solution, 0.01 - 0.02 parts of formate dehydrogenase solution, 0.02 - 0.03 parts of 118# enzyme solution, 3500 - 4500 parts of deionized water.

[0009] Preferably, in step 1, the heating temperature is 30 - 40 °C, and the pH is adjusted to 8.

[0010] Preferably, the 118# enzyme is prepared by the following method: Add yeast to a fermenter for high-density fermentation, control the temperature at 25 - 30 °C, adjust the pH, add p-hydroxybenzoic acid, ferment for 40 - 50 h, centrifuge, ultrasonically treat, and concentrate and dry to obtain the 118# enzyme.

[0011] Preferably, in step 2, the heating temperature is 35 - 40 °C, and the pH is controlled at 7.9 - 8.1.

[0012] Preferably, step 3 is specifically: after the reaction is completed, adjust the pH to 7, cool down to 10 - 15 °C, filter, wash 3 times with deionized water, and dry at 40 - 45 °C for 36 - 48 h to obtain uracil.

[0013] A uracil obtained by the method for enzymatically synthesizing uracil.

[0014] In summary, the present invention has the following beneficial effects: In the process of preparing uracil, the present invention significantly reduces the severity of reaction conditions. Compared with the extreme reaction environment of high temperature and high pressure in the traditional chemical synthesis method, the reaction conditions of the present invention are mild, and the requirements for equipment are relatively low, greatly improving the safety during the operation process and effectively reducing potential safety risks. During the reaction process, the present invention selects enzyme 118# and formate dehydrogenase for biocatalytic reaction. As a biocatalyst, the enzyme has high specificity and selectivity. Enzyme 118# can accurately recognize and act on specific substrates, work in cooperation with formate dehydrogenase, and efficiently catalyze the reaction towards the direction of generating uracil, greatly reducing the occurrence of side reactions. Compared with the traditional chemical synthesis method, using the biocatalytic method of the present invention, the selectivity of the target product uracil is increased by 30%-40%, making the product purity higher. The high purity characteristic of the product directly simplifies the subsequent purification work, without complex multi-step separation and purification processes, saving a large amount of time and labor costs. The waste generated by the biocatalytic method of the present invention is significantly reduced. The enzyme-catalyzed reaction is usually carried out in a mild aqueous solution environment, without the need for a large amount of chemical reagents, and the small amount of by-products generated after the reaction are mostly environmentally friendly substances and are easy to handle. This greatly reduces the waste treatment cost and effectively reduces the environmental pollution pressure. Detailed implementation mode

[0015] The present invention will be further described below in conjunction with the detailed implementation mode, but the embodiments do not limit the present invention in any form.

[0016] Formate dehydrogenase was purchased from Shandong Jinwanhua Fine Chemical Co., Ltd.

[0017] Example 1 A method for enzyme-catalyzed synthesis of uracil, comprising the following steps: Step 1: Add the 2-hydroxypyrimidine solution, sodium formate solution and FeCl 2 solution to deionized water, heat to 30°C, stir for 35 min, and adjust the pH to 8; wherein, 400 parts of the 2-hydroxypyrimidine solution, with a concentration of 100 g / L; 350 parts of the sodium formate solution, with a concentration of 100 g / L; 8 parts of the FeCl 2 solution, with a concentration of 2 g / L; 3500 parts of deionized water; Step 2: Add the NAD solution, stir for 5 min, then add the 118# enzyme solution and formate dehydrogenase solution, heat to 35°C, and stir for reaction for 5 h. Control the pH at 7.9 during the reaction. Among them, 0.5 part of the NAD solution has a concentration of 0.2 g / L; 0.02 part of the 118# enzyme solution has a concentration of 5 mg / L; 0.01 part of the formate dehydrogenase solution has a concentration of 2 mg / L. Among them, the 118# enzyme is prepared by the following method: Add yeast to a fermenter for high-density fermentation, control the temperature at 25°C, adjust the pH to 5.5, add p-hydroxybenzoic acid, ferment for 40 h, then centrifuge at 6000 rpm for 5 min, perform ultrasonic treatment at 30 kHz for 10 min, and concentrate and dry at 40°C for 15 h to obtain the 118# enzyme; the mass ratio of p-hydroxybenzoic acid to yeast is 1:0.05; Step 3: After the reaction is completed, adjust the pH to 7, cool down to 10°C, filter, wash with deionized water 3 times, and dry at 40°C for 36 h to obtain uracil.

[0018] Example 2 A method for enzyme-catalyzed synthesis of uracil, comprising the following steps: Step 1: Add the 2-hydroxypyrimidine solution, sodium formate solution and FeCl 2 solution to deionized water, heat to 40°C, stir for 50 min, and adjust the pH to 9. Among them, 600 parts of the 2-hydroxypyrimidine solution has a concentration of 100 g / L; 450 parts of the sodium formate solution has a concentration of 100 g / L; FeCl 2 solution 15 parts, with a concentration of 2 g / L; 4500 parts of deionized water; Step 2: Add the NAD solution, stir for 8 min, then add the 118# enzyme solution and formate dehydrogenase solution, heat to 40°C, and stir for reaction for 8 h. Control the pH at 8.1 during the reaction. Among them, 2 parts of the NAD solution has a concentration of 0.2 g / L; 0.03 part of the 118# enzyme solution has a concentration of 5 mg / L; 0.02 part of the formate dehydrogenase solution has a concentration of 2 mg / L. Among them, the 118# enzyme is prepared by the following method: Add yeast to a fermenter for high-density fermentation, control the temperature at 30°C, adjust the pH to 6.5, add p-hydroxybenzoic acid, ferment for 50 h, then centrifuge at 8000 rpm for 8 min, perform ultrasonic treatment at 40 kHz for 15 min, and concentrate and dry at 45°C for 24 h to obtain the 118# enzyme; the mass ratio of p-hydroxybenzoic acid to yeast is 1:0.1; Step 3: After the reaction is completed, adjust the pH to 7, cool down to 15°C, filter, wash with deionized water 3 times, and dry at 45°C for 48 h to obtain uracil.

[0019] Example 3 A method for enzymatically synthesizing uracil, comprising the following steps: Step 1: Add a 2-hydroxypyrimidine solution, a sodium formate solution, and an FeCl 2 solution to deionized water, heat to 37 °C, stir for 35 min, and adjust the pH to 8; wherein, 500 parts of the 2-hydroxypyrimidine solution, with a concentration of 100 g / L; 400 parts of the sodium formate solution, with a concentration of 100 g / L; FeCl 2 solution 10 parts, with a concentration of 2 g / L; 4000 parts of deionized water; Step 2: Add an NAD solution, stir for 7 min, then add a 118# enzyme solution and a formate dehydrogenase solution, heat to 37 °C, and stir and react for 6 h, controlling the pH to be 8 during the reaction; wherein, 1 part of the NAD solution, with a concentration of 0.2 g / L; 0.025 parts of the 118# enzyme solution, with a concentration of 5 mg / L; 0.01 parts of the formate dehydrogenase solution, with a concentration of 2 mg / L; wherein, the 118# enzyme is prepared by the following method: Add yeast to a fermentation tank for high-density fermentation, control the temperature at 28 °C, adjust the pH to 6, add p-hydroxybenzoic acid, ferment for 45 h, then centrifuge at 7000 rpm for 6 min, perform ultrasonic treatment at 40 kHz for 13 min, and concentrate and dry at 45 °C for 20 h to obtain the 118# enzyme; the mass ratio of p-hydroxybenzoic acid to yeast is 1.5:0.08; Step 3: After the reaction is completed, adjust the pH to 7, cool down to 13 °C, filter, wash 3 times with deionized water, and dry at 43 °C for 40 h to obtain uracil.

[0020] Comparative Example 1 A method for enzymatically synthesizing uracil, comprising the following steps: Step 1: Add a 2-hydroxypyrimidine solution, a sodium formate solution, and an FeCl 2 solution to deionized water, heat to 30 °C, stir for 35 min, and adjust the pH to 8; wherein, 400 parts of the 2-hydroxypyrimidine solution, with a concentration of 100 g / L; 350 parts of the sodium formate solution, with a concentration of 100 g / L; FeCl 2 solution 8 parts, with a concentration of 2 g / L; 3500 parts of deionized water; Step 2: Add the NAD solution, stir for 5 min, then add the 118# enzyme solution, heat to 35°C, and stir and react for 5 h. Control the pH at 7.9 during the reaction. Among them, the NAD solution is 0.5 parts with a concentration of 0.2 g / L; the 118# enzyme solution is 0.02 parts with a concentration of 5 mg / L. Among them, the 118# enzyme is prepared by the following method: Add yeast to a fermenter for high-density fermentation, control the temperature at 25°C, adjust the pH to 5.5, add p-hydroxybenzoic acid, ferment for 40 h, then centrifuge at 6000 rpm for 5 min, perform ultrasonic treatment at 30 kHz for 10 min, and concentrate and dry at 40°C for 15 h to obtain the 118# enzyme; the mass ratio of p-hydroxybenzoic acid to yeast is 1:0.05. Step 3: After the reaction is completed, adjust the pH to 7, cool down to 10°C, filter, wash 3 times with deionized water, and dry at 40°C for 36 h to obtain uracil.

[0021] Comparative Example 2 A method for enzymatically synthesizing uracil includes the following steps: Step 1: Add the 2-hydroxypyrimidine solution, sodium formate solution, and FeCl 2 solution to deionized water, heat to 30°C, stir for 35 min, and adjust the pH to 8. Among them, the 2-hydroxypyrimidine solution is 400 parts with a concentration of 100 g / L; the sodium formate solution is 350 parts with a concentration of 100 g / L; the FeCl 2 solution is 8 parts with a concentration of 2 g / L; deionized water is 3500 parts; Step 2: Add the NAD solution, stir for 5 min, then add the formate dehydrogenase solution, heat to 35°C, and stir and react for 5 h. Control the pH at 7.9 during the reaction. Among them, the NAD solution is 0.5 parts with a concentration of 0.2 g / L; the formate dehydrogenase is 0.01 parts with a concentration of 2 mg / L; Step 3: After the reaction is completed, adjust the pH to 7, cool down to 10°C, filter, wash 3 times with deionized water, and dry at 40°C for 36 h to obtain uracil.

[0022] Performance Test Test the purity and yield of the uracil prepared in Examples 1-3 and Comparative Examples 1-2; the test results are as follows.

[0023] Table 1 Test Results of Yield and Purity

[0024] As can be seen from the results in Table 1, enzyme 118# can achieve precise recognition and specific binding to the target substrate based on the specific structural features of the substrate molecule by virtue of its unique molecular structure and active site. During the process of participating in the uracil synthesis reaction in cooperation with formate dehydrogenase, after enzyme 118# binds to the substrate, it promotes specific conformational changes in the substrate molecule, thereby significantly reducing the activation energy of the reaction. Formate dehydrogenase, in the reaction system, efficiently regulates the redox process of the reaction through its own catalytic active center, forming a tight co-catalytic network with enzyme 118#. Compared with the traditional reaction system, the dual-enzyme co-catalytic system of the present invention greatly improves the quality of the product and the production efficiency.

[0025] This specific embodiment is only an explanation of the present invention and does not limit the present invention. After reading this specification, those skilled in the art can make modifications to this embodiment without creative contributions as needed, but as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A method for synthesizing uracil by enzyme catalysis, characterized in that: The following steps are involved: Step 1, add 2-hydroxypyrimidine solution, sodium formate solution and FeCl2 solution into deionized water, heat and stir for 35-50 minutes, and adjust the pH to 8-9; Step 2, add NAD solution, stir for 5-8 minutes, then add 118# enzyme solution and formate dehydrogenase solution, heat and stir to react for 5-8 hours, and control the pH at 7.9-8.5 during the reaction; Step 3: After the reaction is completed, the product is precipitated, filtered, washed, and dried to obtain uracil.

2. The method for enzymatically synthesizing uracil according to claim 1, characterized in that: The invention comprises the following raw materials in parts by weight: 400-600 parts of 2-hydroxypyrimidine solution, 350-450 parts of sodium formate solution, 0.5-2 parts of NAD, 8-15 parts of FeCl2 solution, 0.01-0.02 parts of formate dehydrogenase solution, 0.02-0.03 parts of 118# enzyme solution, and 3500-4500 parts of deionized water.

3. The method for enzymatically synthesizing uracil according to claim 1, characterized in that: In the step 1, the heating temperature is 30-40° C., and the pH is adjusted to 8.

4. The method for enzymatically synthesizing uracil according to claim 1, characterized in that: The 118# enzyme is prepared by the following method: The yeast was added into the fermentation tank for high-density fermentation, the temperature was controlled at 25-30°C, the pH was adjusted, p-hydroxybenzoic acid was added, the fermentation was carried out for 40-50 hours, centrifuged, ultrasonically treated, concentrated and dried to obtain 118# enzyme.

5. The method for enzymatically synthesizing uracil according to claim 1, characterized in that: In the step 2, the heating temperature is 35-40° C., and the pH is controlled to be 7.9-8.

1.

6. The method for enzymatically synthesizing uracil according to claim 1, characterized in that: The step 3 is specifically as follows: after the reaction is completed, the pH is adjusted to 7, the temperature is lowered to 10-15° C., filtered, washed with deionized water for 3 times, and dried at 40-45° C. for 36-48 hours to obtain uracil.

7. A uracil, characterized in that: The product is prepared by the method for synthesizing uracil by enzyme catalysis according to any one of claims 1 to 6.

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