A method for enzymatic synthesis of hypoxanthine

Abstract

This invention provides an enzymatic method for synthesizing hypoxanthine, comprising the following steps: dissolving adenine in an appropriate amount of water, adding an effective amount of deaminase, incubating the reaction at 30-50°C, adjusting the pH to 6.5-7 with acid after the reaction, and filtering and washing with water to obtain hypoxanthine. This invention uses adenine as a raw material and employs a deaminase method to synthesize hypoxanthine, overcoming the shortcomings of natural raw material extraction methods, such as complex extraction processes, low yields, and high costs. Compared with other chemical synthesis methods, it produces no byproducts and has higher yields and purity, making it highly valuable for widespread application.

Description

Technical Field

[0001] This invention relates to the field of hypoxanthine preparation technology, and in particular to a method for synthesizing hypoxanthine using an enzymatic method. Background Technology

[0002] Hypoxanthine, also known as 6-hydroxypurine, has a relative molecular weight of 136.11 and the molecular formula C5H4N4O. It is a non-essential biological purine alkaloid widely distributed in the human body. It participates in regulating some important physiological functions, possessing pharmacological activities such as lowering blood pressure, relieving asthma, and treating gout. It can also be used to treat leukopenia and thrombocytopenia caused by various reasons. Furthermore, hypoxanthine is an important intermediate in the synthesis of the anti-tumor drugs 6-mercaptopurine and azathioprine. In addition, hypoxanthine is a pearlescent biological pigment, safe and non-toxic, and can be used not only as a food coloring, cosmetic pigment, and decorative additive, but also for preservation. In agricultural production, hypoxanthine has bactericidal properties and can be used as a pesticide intermediate.

[0003] Currently, the main methods for synthesizing hypoxanthine include natural extraction, chemical synthesis, chemical acid hydrolysis, and diazotization hydrolysis of adenine. Among these:

[0004] (1) The natural raw material extraction method is based on the fact that hypoxanthine is widely distributed in various animal tissues, such as brain tissue, muscle tissue and soft tissue, and is obtained by directly extracting it from animal tissues. The advantages of this method are: wide availability of raw materials, but the extraction process is complicated, the yield is low and the cost is high.

[0005] (2) The chemical synthesis method involves cyclizing ethyl cyanoacetate with sodium ethoxide and thiourea to obtain 2-mercapto-4-amino-6-hydroxypyrimidine, followed by nitrosation, reduction, elimination, and cyclization to obtain hypoxanthine. Although the raw materials are readily available, this method has problems such as complex reaction process, low reaction yield, and high equipment requirements. This invention uses water as a reaction solvent, which is green, mild, non-toxic, and harmless.

[0006] (3) The chemical acid hydrolysis process for obtaining hypoxanthine from inosine requires a large amount of acid and is difficult to treat wastewater. Furthermore, byproducts are easily generated during the reaction process, and the separation of byproducts is difficult, making it unsuitable for large-scale industrial production.

[0007] (4) Adenine is diazotized and hydrolyzed to synthesize hypoxanthine, which requires a large amount of acid, produces a large amount of salt wastewater that is difficult to treat, and the product has a poor appearance.

[0008] Therefore, developing novel preparation methods that are simple, have high yields, and have few side reactions remains an important research topic in this field. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to provide a method for the enzymatic synthesis of hypoxanthine, so as to solve the above-mentioned technical problems existing in the prior art.

[0010] This invention is achieved using the following technical solution:

[0011] A method for the enzymatic synthesis of hypoxanthine includes the following steps: dissolving adenine in an appropriate amount of water, adding an effective amount of deaminase, incubating the reaction at 30-50°C, adjusting the pH to 6.5-7 with acid after the reaction is complete, and washing with water to obtain hypoxanthine.

[0012] The specific reaction formula is as follows:

[0013]

[0014] Preferably, the deaminase is derived from adenine deaminase of Methanosarcina acetivorans C2A, which can catalyze the conversion of adenine to hypoxanthine.

[0015] Preferably, the recombinant plasmid includes a nucleotide sequence of the expression vector linked to the adenine deaminase.

[0016] Preferably, the host cell contains a recombinant plasmid, the host cell is *Escherichia coli*, the gene sequence of the adenine deaminase is SEQ ID NO:1, and the gene sequence of the adenine deaminase is used for expression in *Escherichia coli*.

[0017] The deaminase was prepared by mining the nucleotide sequence of a protein from a database (NCBI Biodatabase), ligating it into a plasmid to obtain a recombinant plasmid, transforming it to obtain recombinant bacteria, culturing the recombinant bacteria, inducing the expression of the recombinant protein, homogenizing and centrifuging to obtain the deaminase. The specific preparation method is as follows: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] Methanosarcina acetivorans The adenine deaminase gene (NC_003552.1:2843256-2844917) of C2A was synthesized by Sangon Biotech (Shanghai) Co., Ltd. and integrated into plasmid pET-28a(+) ZB04293. It was then transformed into *E. coli* BL21 DE3 for expression. The adenine deaminase gene sequence SEQ ID NO:1 is as follows:

[0018]

[0019] The obtained recombinant strain was inoculated into LB medium and cultured with shaking at 37°C for 7 hours. The culture medium after shaking was transferred to TB medium at an inoculation rate of 1-2% and cultured with shaking at 35-40°C until the OD600 reached 0.6. IPTG was added to a final concentration of 1 mmol / L and cultured at 20-30°C for 5-15 hours. After the culture was completed, the cells were collected by centrifugation at 7000 rpm for 15 minutes. The cells were resuspended in phosphate buffer and sonicated in an ice bath. The supernatant was collected and freeze-dried to obtain the deaminase lyophilized powder.

[0020] The inventors made an unexpected discovery: hypoxanthine can be synthesized in one step using adenine as a raw material via deaminase. The reaction conditions are mild, there are no byproducts, and the product yield is high with high purity, specifically 96.0–99.0% yield and 99.0–99.8% purity. Based on the above discovery, the inventors completed this invention.

[0021] Preferably, the amount of deaminase added is 0.3-0.5% of the mass of adenine.

[0022] Preferably, the heat preservation reaction time is 8-18 hours.

[0023] Preferably, adenine is dissolved in water to prepare a solution with a mass percentage concentration of 2%-15%.

[0024] Preferably, the acid is an inorganic acid, specifically selected from at least one of hydrochloric acid and dilute sulfuric acid.

[0025] This invention uses adenine as a raw material and employs a deaminase method to synthesize hypoxanthine, overcoming the shortcomings of natural raw material extraction methods, such as complex extraction processes, low yields, and high costs. Compared with other chemical synthesis methods, it produces no byproducts and has higher yields and purity, making it highly valuable for widespread application. Detailed Implementation

[0026] The following description is provided to disclose the invention and enable those skilled in the art to implement it. The preferred embodiments described below are merely examples and are not intended to limit the scope of the invention; other obvious variations will occur to those skilled in the art. The basic principles of the invention defined in the following description can be applied to other embodiments, modifications, improvements, equivalents, and other technical solutions that do not depart from the spirit and scope of the invention.

[0027] Unless otherwise specified, all instruments and raw materials used in this invention are conventional commercial instruments or materials. The deaminase is prepared using the following method:

[0028] will come from Methanosarcina acetivoransThe adenine deaminase gene of C2A (NC_003552.1:2843256-2844917) was synthesized by Sangon Biotech (Shanghai) Co., Ltd. and integrated into plasmid pET-28a(+) ZB04293. It was then transformed into *E. coli* BL21 DE3 for expression. The gene sequence is as follows:

[0029]

[0030] The obtained recombinant strain was inoculated into LB medium and cultured with shaking at 37°C for 7 hours. The culture medium after shaking was transferred to TB medium at an inoculation rate of 1-2% and cultured with shaking at 35-40°C until the OD600 reached 0.6. IPTG was added to a final concentration of 1 mmol / L and cultured at 20-30°C for 5-15 hours. After the culture was completed, the cells were collected by centrifugation at 7000 rpm for 15 minutes. The cells were resuspended in phosphate buffer and sonicated in an ice bath. The supernatant was collected and freeze-dried to obtain the deaminase lyophilized powder.

[0031] Unless otherwise mentioned, all detection indicators involved in the embodiments of this invention were detected using conventional detection methods in the art. The purity of hypoxanthine was detected using high-performance liquid chromatography (HPLC). The specific chromatographic conditions were as follows: column: C18 250*4.6mm, 5µm; column temperature: 30℃; detection wavelength: 256 nm; flow rate: 1 ml / min; injection volume: 20 µL; mobile phase: 0.02 mol / L KH₂PO₄ solution (pH adjusted to 2.5 with dilute phosphoric acid): methanol = 97.5:2.5.

[0032] Example 1

[0033] 50g of adenine, 0.25g of deaminase, and 500g of water were added to a 1000ml three-necked flask. The mixture was stirred and kept at 40℃ for 8 hours. The pH was adjusted to 6.5-7 with industrial hydrochloric acid. After the reaction was complete, the mixture was filtered and washed with water to obtain 49.5g of dried hypoxanthine. The yield was 99.0%, and the purity was 99.8%.

[0034] Example 2

[0035] 50g of adenine, 0.15g of deaminase, and 500g of water were added to a 1000ml three-necked flask. The mixture was stirred and kept at 35℃ for 12 hours. The pH was adjusted to 6.5-7 with industrial hydrochloric acid. After the reaction was complete, the mixture was filtered and washed with water to obtain 49g of dried hypoxanthine. The yield was 98.0%, and the purity was 99.0%.

[0036] Those skilled in the art should understand that the above embodiments are merely examples and do not limit the invention. The objectives of the invention have been fully and effectively achieved. The functions and structural principles of the invention have been shown and explained in the embodiments, and any variations or modifications can be made to the implementation of the invention without departing from the stated principles.

Claims

1. A method for the enzymatic synthesis of hypoxanthine comprising the steps of: Adenine is dissolved in an appropriate amount of water, an effective amount of deaminase is added, and the reaction is incubated at 30-50℃ for 8-18 hours. The deaminase is derived from Methanosarcina acetivorans an adenine deaminase of C2A that is capable of catalyzing the conversion of adenine to hypoxanthine; The gene sequence of the adenine deaminase is SEQ ID NO. 1, and the gene sequence of the adenine deaminase is used for expression in Escherichia coli.

2. The method of claim 1, wherein, The recombinant plasmid comprises the nucleotide sequence of the adenine deaminase.

3. The method of claim 2, wherein, The host cell comprises the recombinant plasmid, and the host cell is Escherichia coli.

4. The method of claim 1, wherein, The amount of deaminase added is 0.3-0.5% of the mass of adenine.

5. The method of claim 1, wherein, Adenine is dissolved in water to prepare a solution with a mass percentage concentration of 2%-15%.

6. The method of claim 1, wherein, The acid is an inorganic acid.

7. The method of claim 6, wherein, The acid is selected from at least one of hydrochloric acid and dilute sulfuric acid.

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