A method for purifying and refining biosynthesized adenine and its application

Abstract

This invention belongs to the field of biochemical preparation technology, specifically relating to a method for refining and purifying adenine from biosynthesis and its application. The method includes the following steps: S1, heating the adenine-containing bio-fermentation filtrate to boiling, then maintaining the temperature, and separating the solid and liquid to obtain the filtrate; S2, heating and evaporating the filtrate to concentrate it, then cooling to crystallize, and separating the solid and liquid to obtain crude adenine; S3, adding water at 2-12°C to the crude adenine for suspension washing, separating the solid to obtain the solid, and drying to obtain pure adenine. This invention, by precisely controlling parameters such as heating, boiling, and evaporation concentration, combined with a targeted crystal washing process, can minimize the loss of the target product and effectively remove impurities, achieving a final product yield of over 95% and a purity of over 98%. Furthermore, the process is simple to operate, does not require acid / alkali adjustment or activated carbon, and reduces waste emissions, possessing significant economic potential and suitable for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of biochemical preparation technology, specifically relating to a method for purifying and refining adenine through biosynthesis and its application. Background Technology

[0002] Adenine is an important purine compound and one of the basic building blocks of nucleic acids (DNA and RNA). It has significant applications in medicine, food, and agriculture, serving as an important precursor for the synthesis of antiviral and anticancer drugs. It can also be used as a nutrient fortifier and for regulating plant growth.

[0003] Traditional methods for producing adenine mainly include chemical synthesis (such as hypoxanthine chlorination and ammoniation) and biological enzymatic methods (such as adenosine cleavage).

[0004] Chemical synthesis method: such as CN102321086A, uses highly polluting reagents such as phosphorus oxychloride, which generates a large amount of phosphorus-containing wastewater. In addition, the purification process requires multiple decolorization and crystallization, and the amount of activated carbon used is large (0.2-0.5 times the crude quality), resulting in high energy consumption.

[0005] Bio-enzymatic method: The raw materials rely on inosine or adenosine fermentation, which limits the source. At the same time, the content of by-products is high, requiring complex post-processing for separation and purification. For example, CN 111378705 A has a high proportion of D-ribose, which requires the separation of D-ribose and adenine, and the steps are relatively complicated.

[0006] The refining methods in patents such as JP56131584A, JP58008083A, JP56131585A, JP57062279A, CN101125854A, CN102127081A, and CN102321086A all require adjusting the acid and alkali levels, using large amounts of water and activated carbon, which generates a large amount of waste and increases environmental protection costs.

[0007] Therefore, developing a purification method that requires no chemical reagents, is easy to operate, and can efficiently remove multiple impurities has become a key requirement for improving the quality and industrialization benefits of synthetic adenine products. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the prior art and provide a purification method for biosynthesized adenine, so as to achieve efficient and green production of adenine, reduce wastewater generation, and lower environmental protection costs.

[0009] To achieve the above objectives, the technical solution adopted by the present invention includes:

[0010] In a first aspect, the present invention provides a method for purifying and refining biosynthetic adenine, comprising the following steps:

[0011] S1. The adenine-containing bio-fermentation filtrate is heated to boiling, then kept at the temperature, and the solid and liquid are separated to obtain the filtrate.

[0012] S2. The filtrate is heated to evaporate and concentrate, then cooled to crystallize, and the solid and liquid are separated to obtain crude adenine.

[0013] S3. Add water at 2-12℃ to the crude adenine product for suspension washing, separate the solid and liquid to obtain the solid, dry it, and then obtain the pure adenine product.

[0014] This invention develops a purification method for adenine obtained through bio-fermentation. In this purification method, step S1 involves heating and boiling followed by heat preservation. Utilizing the high solubility of adenine in hot water and the high-temperature denaturation characteristics of biomacromolecules such as proteins and polysaccharides, most impurities are removed through solid-liquid separation. Step S2 then employs evaporation concentration combined with cooling crystallization. Taking advantage of the high solubility of adenine at high temperatures and the rapid decrease in solubility at low temperatures, the target product precipitates as high-purity crystals. Step S3, cold water suspension washing, further utilizes the low solubility of adenine in cold water, removing sugars and other impurities adhering to the crystal surface while ensuring a low washing loss rate. The physical purification process described in this invention is simple to operate, generates little wastewater, and reduces emissions by eliminating the need for activated carbon. It achieves efficient, energy-saving, and environmentally friendly industrial production while ensuring the quality of the adenine product, demonstrating significant economic potential.

[0015] Preferably, in step S1, the heat preservation treatment time is 1-2 hours.

[0016] The core function of the heat treatment after boiling is to increase the solubility of adenine in water and promote the full denaturation and precipitation of thermally unstable impurities in the fermentation filtrate, thereby removing impurities through solid-liquid separation. If the heat treatment time is too short, impurities cannot be fully denatured or coagulated and precipitated, leading to co-precipitation with adenine in subsequent steps, directly reducing the purity of the crude product. Simultaneously, residual colloidal impurities may adsorb some adenine, increasing adenine loss during subsequent separation and indirectly reducing yield. Conversely, if the heat treatment time is too long, some adenine will undergo trace degradation, resulting in the loss of the target product, and may even promote secondary reactions of some soluble impurities in the solution, leading to a decrease in the purity of the final product. Experimental research has shown that a heat treatment of 1-2 hours can achieve a balance between sufficient impurity precipitation and adenine stability, thus ensuring the removal of most impurities while effectively improving the yield and purity of the final product.

[0017] Preferably, in step S2, the volume concentration factor of the heating evaporation concentration step is 10-30 times.

[0018] Experimental studies have revealed that the evaporation concentration factor significantly impacts the yield and purity of the final adenine product. If the concentration factor is too low, the adenine concentration cannot reach supersaturation, resulting in insufficient crystallization during cooling and leaving a large amount of adenine residue in the mother liquor, thus significantly reducing yield. Conversely, if the concentration factor is too high, the supersaturation increases rapidly, leading to rapid precipitation of adenine and the formation of irregular crystals. These crystals are prone to encapsulating impurities, resulting in a decrease in the purity of the crude product. Therefore, employing the optimized concentration factor range described above ensures efficient crystallization of adenine without co-precipitation of impurities, thereby further improving product yield and purity.

[0019] Preferably, in step S2, the specific method for cooling and crystallization is: stirring at 2-12℃ for 1-3 hours.

[0020] Adenine solubility decreases with decreasing temperature, making cooling a key method to promote its precipitation. Experimental studies have shown that using the optimized cooling and crystallization conditions described above ensures sufficient adenine precipitation while minimizing impurity contamination, thus guaranteeing yield and purity. However, excessively low temperatures lead to a sharp drop in adenine solubility, resulting in excessively rapid crystallization and increased impurity adsorption. Simultaneously, the solubility of some impurities may decrease significantly with low temperatures, reaching supersaturation and co-precipitating with adenine, leading to decreased product purity. Conversely, excessively high temperatures result in high adenine solubility, insufficient crystallization, and reduced product yield.

[0021] Preferably, in step S3, the mass ratio of crude adenine to water is 1:(10-30), and the temperature of the water is 2-12°C.

[0022] This invention further removes impurities adsorbed on the surface of crude adenine by washing with cold water, while simultaneously reducing the solubility of adenine at low temperatures (2-12°C) to minimize dissolution loss. Furthermore, with a crude product to water ratio of 1:(10-30), the water volume is sufficient to adequately wet the crude product particles and disperse the solid, allowing impurities to fully dissolve in the water, while avoiding excessive adenine dissolution due to excessive water volume. At this point, impurities are effectively washed away, and because adenine has low solubility at low temperatures, dissolution loss is controlled within a reasonable range, thus achieving a balance between product purity and yield.

[0023] More preferably, the mass ratio of crude adenine to water is 1:(10-15).

[0024] Preferably, in step S3, the drying temperature is 115-125℃ and the drying time is 2-3 hours.

[0025] Preferably, in steps S1-S3, solid-liquid separation is performed by centrifugation, and the centrifugation speed is 1500-2500 rpm.

[0026] This invention effectively regulates the dissolution and crystallization rate of adenine and the behavior of impurities by precisely controlling various key parameters in the purification method, thereby collectively influencing and improving the purity and yield of the product. Only by controlling the above key conditions within the range defined by this invention can the orderly precipitation of adenine and the minimization of impurities be achieved, thus balancing high purity and high yield; deviation from this range will significantly lead to a decrease in yield or purity.

[0027] Preferably, in step S1, the adenine-containing bio-fermentation filtrate is obtained by fermentation with engineered bacteria of Corynebacterium glutamicum;

[0028] The engineered *Corynebacterium glutamicum* strain uses *Corynebacterium glutamicum* as the chassis strain, knocking out the serine / threonine protein kinase gene PknG and replacing the PknG knockout site with the glutamine synthase gene glnA; knocking out the purine nucleotide repressor protein gene purR and replacing the purR knockout site with the 5-phosphoribose-1-pyrophosphate synthase gene PRPP; knocking out the phosphorylacetyltransferase gene pta and replacing the pta knockout site with the glucose-6-phosphate dehydrogenase gene zwf; knocking out the adenine phosphoribosyltransferase gene apt and replacing the apt knockout site with the polyphosphokinase gene ppk; the chassis strain also overexpresses the adenylate succinate synthase gene purA and the adenylate succinate lyase gene purB.

[0029] The adenine purification method described in this invention is not only applicable to low-concentration conventional fermentation systems such as *E. coli*, but can also be directly applied to the purification of high-density fermentation broths from genetically engineered bacteria with higher impurity (such as protein and metabolic byproduct) content and more complex systems. The *Corynebacterium glutamicum* engineered bacteria constructed in the early stages of this invention can effectively improve the adenine synthesis efficiency, resulting in an adenine concentration of 6.7 g / L in the fermentation filtrate. Combined with the purification method described in this invention, the adenine recovery rate and purity can be maximized, achieving an adenine yield ≥95% and a product content ≥98%.

[0030] Therefore, the adenine purification method described in this invention provides a practical solution for the efficient preparation of adenine by balancing yield and purity as well as meeting the requirements of efficient fermentation, and has important application value in the fields of bio-fermentation and fine chemicals.

[0031] Preferably, the method for preparing the adenine-containing bio-fermentation filtrate includes the following steps:

[0032] (1) The engineered strain of Corynebacterium glutamicum was fermented and cultured, and then a synthase inducer was added to induce protein expression. The culture was continued, and the cells were collected by centrifugation to obtain the whole cell catalyst.

[0033] (2) The whole-cell catalyst was added to a Tris-HCl buffer containing ATP, magnesium sulfate heptahydrate, polyP, glycine, aspartic acid and glucose for catalytic culture to obtain the adenine-containing bio-fermentation filtrate.

[0034] Preferably, in step (2), the temperature of the catalytic culture is 25-37°C; and the final concentration of magnesium ions in the Tris-HCl buffer is 1-5 mM.

[0035] Secondly, the present invention provides the application of the purification method described above in the preparation of pure adenine.

[0036] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0037] (1) In terms of improving product yield and purity, this invention can minimize the loss of target product and effectively remove impurities by precisely controlling parameters such as heating, boiling and heat preservation treatment, evaporation and concentration, and combining them with targeted crystal washing process. The final product yield reaches more than 95% and the purity can reach more than 98%. Moreover, the process is simple to operate and suitable for industrial production.

[0038] (2) In terms of environmental protection and economy, the present invention uses water as a reaction solvent, which avoids the use of organic solvents. It is not only safe and non-toxic, but also reduces solvent recycling costs and environmental risks. At the same time, the entire process does not require acid and alkali adjustment and does not introduce additional inorganic salt impurities, thus reducing the generation of waste acid and waste alkali. In addition, the process does not use activated carbon, thus avoiding solid waste pollution. The overall emissions of waste gas, wastewater, and solid waste are low, which is highly environmentally friendly and meets the requirements of green chemistry and clean production. Attached Figure Description

[0039] Figure 1 This is a flowchart illustrating the purification method described in this invention.

[0040] Figure 2 This is a high-performance liquid chromatography (HPLC) chromatogram of the initial bio-fermentation filtrate.

[0041] Figure 3 The high-performance liquid chromatography (HPLC) chromatogram shows the adenine purified by the purification method described in Example 1.

[0042] Figure 4 Images of the initial fermentation broth (A) and the adenine dry powder prepared in Example 1 (B). Detailed Implementation

[0043] To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0044] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; and the materials and reagents used are commercially available unless otherwise specified.

[0045] The fermentation strain, *Corynebacterium glutamicum*, is an engineered strain derived from the HG-VB4-01 strain in patent application (application number 202511049390.6). The construction method of the strain is as follows:

[0046] Using Corynebacterium glutamicum ATCC 13032 (purchased from Shanghai Beinuo Biotechnology Co., Ltd.) as the chassis strain, the serine / threonine protein kinase gene PknG was knocked out, and the PknG knockout site was replaced with the glutamine synthase gene glnA; the purine nucleotide repressor protein gene purR was knocked out, and the purR knockout site was replaced with the 5-phosphoribose-1-pyrophosphate synthase gene PRPP; the phosphorylacetyltransferase gene pta was knocked out, and the pta knockout site was replaced with the glucose-6-phosphate dehydrogenase gene zwf; the adenine phosphoribosyltransferase gene apt was knocked out, and the apt knockout site was replaced with the polyphosphokinase gene ppk; the chassis strain also overexpressed the adenylate succinate synthase gene purA and the adenylate succinate lyase gene purB.

[0047] For details on the specific construction method of the HG-VB4-01 strain, please refer to the patent application number 202511049390.6.

[0048] The method for preparing the adenine-containing bio-fermentation filtrate includes the following steps:

[0049] 1. Fermentation of engineered bacteria

[0050] (1) Take out the frozen glycerol bacterial suspension of engineered Bacillus glutamate HG-VB4-01, and after thawing, use the streak plate method to streak on solid medium containing chloramphenicol BHIG with a final concentration of 50 mg / L. Incubate the plates in a constant temperature incubator at 34℃ for 12 h.

[0051] (2) Select single colonies with a diameter of 0.12-0.15 mm, inoculate them into 10 mL of BHIG medium, add chloramphenicol to a final concentration of 50 mg / L and culture in a shaker. The culture conditions are: 34℃, 220 rpm, and the OD600 of the culture solution is between 3.0 and 4.0 after 8 h of culture to obtain the activated primary seed solution.

[0052] (3) Take 10 mL of the activated primary seed culture and inoculate it into a 5 L fermenter containing 2.4 L of BHIG medium. Add chloramphenicol to the fermenter at a final concentration of 50 mg / L and culture under the following conditions: pH 7.0, temperature 34 °C, initial aeration ratio of 1 vvm, initial rotation speed of 220 rpm, tank pressure of 0.05 MPa, stirring and contacting dissolved oxygen (DO) at 30%, and culture for 12 h. Add the synthase inducer at 8 h of fermentation and control the temperature at 25 °C to induce the correct expression of purA and purB.

[0053] The BHIG culture medium comprises the following components at the following concentrations: brain and heart extract 37 g / L, glucose 10 g / L;

[0054] The BHIG solid culture medium comprises the following components at the following concentrations: brain heart extract 37 g / L, glucose 10 g / L, and agar powder 15 g / L.

[0055] The synthase inducer is IPTG, with an optimal concentration of 0.1 mmol / L.

[0056] 2. Whole-cell catalysis to produce adenine

[0057] (1) Collection of recombinant strains: The collection method is to centrifuge at 4℃, 5000rpm for 10min, and wash the cells twice with 50mM Tris-HCl at pH=7.4 to remove culture medium residue;

[0058] (2) Prepare the whole cell catalytic system: Use 50mM Tris-HCl as buffer and add 5mM ATP, 5mM magnesium sulfate heptahydrate, 2mM polyP, 50mM glycine, 50mM aspartic acid, 10g / L glucose. Adjust the pH to 7.4 and then add the cells obtained in step (1) to a final concentration of OD600 = 40.

[0059] (3) Whole-cell catalysis: The whole-cell catalysis system obtained in step (2) was placed at 34℃ and catalyzed at 500 rpm for 24 h; after the reaction was completed, the adenine yield was determined by high performance liquid chromatography.

[0060] The 50mM Tris-HCl comprises the following components at the following concentrations: Tris 6.06 g / L, and the pH is adjusted to 7.4 using HCl;

[0061] The high-performance liquid chromatography (HPLC) detection method is as follows: wavelength 260 nm, flow rate 1.0 mL / min, sample solution: DMSO, injection volume: 10 μL, column temperature 35℃, run time 20 min, and column usage. C18 (5μm) HPLC columns 4.6mm×25cm were used for isocratic elution with methanol:pure water (v / v) = 30:70 as the mobile phase.

[0062] The concentration of adenine in the fermentation filtrate obtained by the above-mentioned bio-fermentation method was found to be 6.7 g / L. This filtrate was used as the bio-fermentation filtrate for purification in the following examples and comparative examples.

[0063] Example 1

[0064] This embodiment provides a method for purifying biosynthesized adenine, including the following steps:

[0065] S1. Take 5L of adenine-containing bio-fermentation filtrate catalyzed by HG-VB4-01 strain, heat to boiling and keep warm for 2 hours; while hot, perform solid-liquid separation using a plate centrifuge at 1500rpm to obtain the filtrate.

[0066] S2. The filtrate was heated and evaporated to concentrate it to a volume concentration of 20 times. It was then cooled to 2°C in an ice bath and stirred for 2 hours. After crystallization was complete, the solid and liquid were separated using a plate centrifuge at 1500 rpm to obtain crude adenine.

[0067] S3. The obtained crude adenine product was washed with 2℃ cold pure water (solid-liquid mass ratio 1:15) and stirred for 2 hours. Then, solid-liquid separation was performed using a plate centrifuge at 1500 rpm. The solid was dried at 120℃ for 3 hours to obtain pure adenine product.

[0068] Example 2

[0069] This embodiment provides a method for purifying biosynthesized adenine, including the following steps:

[0070] S1. Take 5L of adenine-containing bio-fermentation filtrate catalyzed by HG-VB4-01 strain, heat to boiling and keep warm for 1 hour; while hot, perform solid-liquid separation using a plate centrifuge at 1500rpm to obtain the filtrate.

[0071] S2. The filtrate was heated and evaporated to concentrate it to a volume concentration of 30 times. It was then cooled to 12°C in an ice bath and stirred for 3 hours. After crystallization was complete, the solid and liquid were separated using a plate centrifuge at 1500 rpm to obtain crude adenine.

[0072] S3. The obtained crude adenine was washed with 12°C cold pure water (solid-liquid mass ratio 1:30) and stirred for 2 hours. Then, solid-liquid separation was performed using a plate centrifuge at 1500 rpm. The solid was dried at 120°C for 3 hours to obtain pure adenine.

[0073] Example 3

[0074] This embodiment provides a method for purifying biosynthesized adenine, including the following steps:

[0075] S1. Take 5L of adenine-containing bio-fermentation filtrate catalyzed by HG-VB4-01 strain, heat to boiling and keep warm for 2 hours; while hot, perform solid-liquid separation using a plate centrifuge at 1500rpm to obtain the filtrate.

[0076] S2. The filtrate was heated and evaporated to concentrate it to a volume concentration of 10 times. It was then cooled to 6°C in an ice bath and stirred for 1 hour. After crystallization was complete, the solid and liquid were separated using a plate centrifuge at 1500 rpm to obtain crude adenine.

[0077] S3. The obtained crude adenine was washed with 6°C cold pure water (solid-liquid mass ratio 1:10) and stirred for 2 hours. Then, solid-liquid separation was performed using a plate centrifuge at 1500 rpm. The solid was dried at 120°C for 3 hours to obtain pure adenine.

[0078] Comparative Example 1

[0079] This comparative example provides a method for purifying biosynthesized adenine. The only difference between this method and Example 1 is that in step S2, the filtrate is heated and evaporated to concentrate it to a volume concentration of 50 times. The rest of the preparation process is the same as in Example 1.

[0080] Comparative Example 2

[0081] This comparative example provides a method for purifying biosynthesized adenine. The only difference between this method and Example 1 is that in step S2, the filtrate is heated and evaporated to concentrate it to a volume concentration of 5 times. The rest of the preparation process is the same as in Example 1.

[0082] Comparative Example 3

[0083] This comparative example provides a method for purifying biosynthesized adenine, which differs from Example 1 only in that: in step S2, the temperature is lowered to 25°C in an ice bath and stirred for 2 hours to induce crystallization; in step S3, pure water at 25°C is added to wash the crystals; the rest of the preparation process is the same as in Example 1.

[0084] Comparative Example 4

[0085] This comparative example provides a purification method for biosynthesized adenine, which differs from Example 1 only in that: in step S3, the mass ratio of crude adenine to pure water is 1:50, and the rest of the preparation process is the same as in Example 1.

[0086] Comparative Example 5

[0087] This comparative example provides a method for purifying biosynthesized adenine. The only difference between this method and Example 1 is that in step S3, the mass ratio of crude adenine to pure water is 1:5, and the rest of the preparation process is the same as in Example 1.

[0088] Example of effect

[0089] The adenine obtained by the purification methods described in Examples 1-3 and Comparative Examples 1-5 was used as the test sample, and its yield and purity were tested. The specific test results are shown in Table 1.

[0090] Table 1

[0091] Example 1 32.85 98.06 99.2 Example 2 31.89 95.19 98.6 Example 3 30.94 97.52 98.2 Comparative Example 1 29.56 88.24 95.7 Comparative Example 2 31.11 92.86 93.1 Comparative Example 3 29.90 89.25 97.8 Comparative Example 4 31.01 92.57 99.5 Comparative Example 5 31.21 93.15 97.4

[0092] Table 1 shows that key parameters such as the concentration factor, cooling / crystallization / washing temperature, and the mass ratio of crude product to washing water have a significant impact on the yield and purity of adenine in the adenine purification method described in this invention. In Examples 1-3, when adenine was purified using the preferred parameter range of this invention, the yield reached over 95% and the purity reached over 98%. In particular, the adenine prepared by the purification method described in Example 1 achieved a yield of 98.06% and a purity of 99.2%, fully realizing high yield and high purity of adenine. The high-performance liquid chromatography (HPLC) analysis chromatogram of the initial bio-fermentation filtrate is shown below. Figure 2 As shown, the high-performance liquid chromatography (HPLC) chromatogram of the pure adenine obtained by the purification method described in Example 1 is as follows. Figure 3 As shown, images of the initial fermentation broth and the adenine dry powder prepared in Example 1 are as follows. Figure 4 As shown.

[0093] When the concentration factor involved in the purification methods described in Comparative Examples 1-2 exceeds the preferred range of this invention, the yield and purity of adenine are significantly lower than those in the examples. Similarly, in Comparative Example 3, when the temperatures during cooling crystallization and washing exceed the preferred range of this invention, the yield of adenine also decreases significantly, and the purity also decreases to some extent. This indicates that the preferred low-temperature conditions of 2-12℃ in this invention can promote the complete crystallization of adenine while reducing the dissolution and residue of impurities, and are the key temperature range for ensuring yield and purity. In Comparative Example 4, when the water volume during washing is too high, although the purity is not significantly affected, the excessive water volume leads to the dissolution and loss of some adenine, resulting in a significant decrease in yield. In Comparative Example 5, when the water volume during washing is too low, impurities cannot be fully washed away, leading to a decrease in adenine purity. Although the loss of adenine is small, the low purity also does not meet the purification requirements. Therefore, the preferred mass ratio range of this invention can effectively remove impurities while reducing the dissolution and loss of adenine, achieving a sufficient balance between yield and purity.

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for purifying and refining biosynthesized adenine, characterized in that, Includes the following steps: S1. The adenine-containing bio-fermentation filtrate is heated to boiling, then kept at the temperature, and the solid and liquid are separated to obtain the filtrate. S2. The filtrate is heated to evaporate and concentrate, then cooled to crystallize, and the solid and liquid are separated to obtain crude adenine. S3. Add water at 2-12℃ to the crude adenine product for suspension washing, separate the solid and liquid to obtain the solid, dry it, and then obtain the pure adenine product. In step S1, the adenine-containing bio-fermentation filtrate is obtained by fermentation with engineered *Corynebacterium glutamicum*. The engineered *Corynebacterium glutamicum* strain uses *Corynebacterium glutamicum* as the chassis strain, knocking out the serine / threonine protein kinase gene PknG and replacing the PknG knockout site with the glutamine synthase gene glnA; knocking out the purine nucleotide repressor protein gene purR and replacing the purR knockout site with the 5-phosphoribose-1-pyrophosphate synthase gene PRPP; knocking out the phosphorylacetyltransferase gene pta and replacing the pta knockout site with the glucose-6-phosphate dehydrogenase gene zwf; knocking out the adenine phosphoribosyltransferase gene apt and replacing the apt knockout site with the polyphosphokinase gene ppk; the chassis strain also overexpresses the adenylate succinate synthase gene purA and the adenylate succinate lyase gene purB. The method for preparing the adenine-containing bio-fermentation filtrate includes the following steps: (1) The engineered strain of Corynebacterium glutamicum was fermented and cultured, and then a synthase inducer was added to induce protein expression. After further culture, the cells were collected by centrifugation to obtain whole-cell catalyst. (2) The whole cell catalyst was added to a Tris-HCl buffer containing ATP, magnesium sulfate heptahydrate, polyP, glycine, aspartic acid and glucose for catalytic culture to obtain the adenine-containing bio-fermentation filtrate. In step S2, the volume concentration factor of the heating evaporation concentration step is 10-30 times; In step S3, the mass ratio of crude adenine to water is 1:(10-30).

2. The purification method as described in claim 1, characterized in that, In step S1, the heat preservation treatment time is 1-2 hours.

3. The purification method as described in claim 1, characterized in that, In step S2, the specific method for cooling and crystallization is as follows: stirring at 2-12℃ for 1-3 hours.

4. The purification method as described in claim 1, characterized in that, In step S3, the drying temperature is 115-125℃ and the drying time is 2-3 hours.

5. The application of the purification method according to any one of claims 1-4 in the preparation of pure adenine.

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