Amalonic acid-free Citrobacter and a method for catalyzing the production of 2'-deoxyguanosine

Abstract

The invention discloses a Citrobacter amalonaticus DHE005, with a deposit number of CGMCC NO.24523 and a deposit date of March 11, 2022; and discloses a method for catalytically producing 2'-deoxyguanosine by fermenting and culturing Citrobacter amalonaticus DHE005 as an enzyme source. The catalytic method of the present invention has the advantages that the catalytic process is simple and easy to operate, the cell does not need to be centrifuged and enriched, the cell does not need to be wall-broken, the catalytic process is significantly reduced, and the maximum concentration of the product 2'-deoxyguanosine can reach 33.5g / L, and the catalytic activity is strong, the product concentration is high, the whole catalytic process cost is low, and it is convenient for industrial production.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a method for producing 2'-deoxyguanosine by utilizing amalonic acid-free Citrobacter and catalysis thereof. Background Art

[0002] 2'-Deoxyguanosine is a structural fragment of the biological macromolecule deoxyribonucleic acid (DNA) that is essential for the development and normal functioning of organisms. Its chemical structure is shown in Formula 1.

[0003]

[0004] As an important component of genetic material, 2'-deoxyguanosine, together with other natural nucleosides, participates in the transmission of genetic information of biological cells and plays a very important role in the growth regulation of biological cells. 2'-deoxyguanosine can be used as a chemical reagent for biochemical research. In the field of drug development, it has been found that 2'-deoxyguanosine itself has good physiological activity. It is not only an important raw material in the field of gene drugs, but also an intermediate to produce antiviral, anti-tumor and anti-AIDS drugs. For example, 6-disulfide-substituted-2'-deoxyguanosine compounds prepared with 2'-deoxyguanosine as raw material show significant tumor inhibition effects on various types of tumors and can be used in drugs for tumors related to human cell proliferation. In addition, with the rapid spread of COVID-19, the demand for 2'-deoxyguanosine, as a relatively important precursor raw material in virus detection kits, is also increasing sharply.

[0005] The traditional preparation of 2'-deoxyguanosine is to use DNA as raw material, hydrolyze it into deoxynucleotides by nuclease, and then use enzymes to remove phosphate, generate deoxynucleosides, and then separate 2'-deoxyguanosine from them. This method has high production costs, and the problem of DNA source also limits the industrial production of 2'-deoxyguanosine. At present, the methods that can be used to prepare 2'-deoxyguanosine mainly include chemical synthesis and bio-enzyme catalysis. Regarding the preparation of 2'-deoxyguanosine by chemical synthesis, there are long reaction steps and harsh reaction conditions, resulting in a low final yield. In addition, various organic reagents are also required in the reaction process, which has obvious environmental pollution. Therefore, the preparation of 2'-deoxyguanosine by chemical synthesis is not suitable for large-scale industrial production. There are many patent reports on the preparation of 2'-deoxyguanosine by bioenzyme catalysis. For example, patent CN 104830930B discloses a method for producing deoxyguanosine by bioconversion, which uses fermented lactobacillus cell slurry as a biocatalyst and performs a biocatalytic reaction in a polyethylene glycol 10000 / potassium hydrogen phosphate two-phase aqueous system to obtain a 2'-deoxyguanosine product. Under the optimal conditions, the thymidine conversion rate can reach 89%; patent CN 102174618A also discloses a method for producing 2'-deoxyguanosine by using acetyl Brevibacterium acetyltransferase cells, which uses acetyl Brevibacterium acetyltransferase cells with nucleoside phosphorylase activity as an enzyme source, mixes it with thymidine and guanine donors as conversion reaction substrates, and performs a conversion reaction to synthesize 2'-deoxyguanosine; in addition, patent CN 108018252A also discloses a method for preparing 2'-deoxyguanosine using enzyme catalysis, which uses recombinant Escherichia coli to obtain N-deoxyribosyltransferase to directly catalyze the synthesis of thymidine and guanine into 2'-deoxyguanosine. The above patents all disclose methods for preparing 2'-deoxyguanosine using biological enzyme catalysis, but the bacteria used in patent CN 104830930B are Lactobacillus, which is a facultative anaerobic bacteria. It is difficult to obtain bacteria under conventional culture conditions, and the reaction system is a two-phase aqueous system. The process is relatively complicated and the manufacturing cost is high, which is not conducive to industrial application; the substrate concentration in the catalytic reaction in patent CN 102174618A is low, and the reaction conversion rate is only 60%, which is not suitable for industrial application; patent CN The recombinant E. coli strain in 108018252A needs to add expensive inducers during the culture process to induce the synthesis of N-deoxyribosyltransferase. In addition, as a heterologous expression protein, the recombinant bacteria have unsuccessful protein folding and easy generation of inclusion bodies when expressing N-deoxyribosyltransferase. Moreover, the enzyme catalysis patents disclosed above all require centrifugation of the bacteria, and most of them require cell wall breaking to obtain the corresponding catalytic enzyme for subsequent catalytic reactions. This process significantly increases the process steps and production costs, which is not conducive to industrial application. Summary of the invention

[0006] Based on the shortcomings of the above-mentioned technology, one of the objects of the present invention is to provide a Citrobacter amalonaticus DHE005, which is deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with a deposit number of CGMCC NO.24523, a deposit date of March 11, 2022, and is registered to prove survival.

[0007] Another object of the present invention is to provide a new use of Citrobacter amalonaticus or its seed solution or fermentation solution or bacterial suspension or culture solution.

[0008] The invention provides application of Citrobacter amalonaticus or its seed solution or fermentation solution or bacterial suspension or culture solution in producing purine nucleotide phosphorylase or products containing purine nucleotide phosphorylase.

[0009] The present invention provides the use of Citrobacter amalonaticus or its seed solution or fermentation solution or bacterial suspension or culture solution in preparing 2'-deoxyguanosine or a pharmaceutical composition containing 2'-deoxyguanosine.

[0010] As an embodiment, the Citrobacter amalonaticus described in the above application is the Citrobacter amalonaticus DHE005 described above.

[0011] Another object of the present invention is to provide a method for preparing purine nucleotide phosphorylase, comprising fermenting and preparing the purine nucleotide phosphorylase using Citrobacter amalonaticus.

[0012] Furthermore, the fermentation process includes aerobic fermentation in an enzyme-producing medium containing assimilable carbon source and / or nitrogen source.

[0013] Furthermore, the carbon source is selected from glucose, maltose, sucrose, glycerol or fructose; preferably glucose.

[0014] Furthermore, the nitrogen source is selected from yeast extract powder, yeast powder, yeast paste, soybean lecithin, corn steep liquor powder, soybean meal, peptone, and urea; preferably yeast paste or yeast extract powder.

[0015] Furthermore, the enzyme production culture medium also includes a surfactant, and the surfactant is selected from sodium dodecyl sulfate, Tween-20, Tween-80, preferably Tween-80.

[0016] Furthermore, the enzyme-producing culture medium also includes an inorganic salt, and the inorganic salt is selected from ammonium citrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium sulfate, calcium carbonate, ferrous sulfate, zinc sulfate, copper sulfate, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, ferric chloride, and manganese sulfate, preferably ammonium citrate, magnesium sulfate, potassium dihydrogen phosphate, potassium chloride or calcium chloride.

[0017] Furthermore, the enzyme production medium contains 10-50 g / L of glucose, 5-20 g / L of yeast extract, 5-15 g / L of yeast extract powder, 2-10 g / L of Tween-80, 2-5 g / L of ammonium citrate, 1-5 g / L of magnesium sulfate, 2-8 g / L of potassium dihydrogen phosphate, 1-5 g / L of potassium chloride, and 1-5 g / L of calcium chloride.

[0018] Furthermore, the fermentation temperature is 25-37°C, the culture medium pH is 5.0-7.0; the culture time is 24-72 hours; and the oxygen flow rate is 0.5-2.0 vvm.

[0019] Further, more specifically, the amalonic acid-free Citrobacter is inoculated into the enzyme fermentation medium by seed liquid to carry out the fermentation culture;

[0020] The seed solution is obtained by culturing Citrobacter amalonaticus in a seed culture medium.

[0021] The conditions for seed culture are as follows: the temperature of seed culture is 25° C.-37° C.; the pH of the culture medium is 5.0-7.0; and the culture time is 12-36 hours.

[0022] The seed culture medium contains 5-30 g / L of glucose, 5-20 g / L of peptone, 2-10 g / L of yeast extract, 5-20 g / L of yeast extract powder, 1-10 g / L of calcium chloride, 1-10 g / L of magnesium sulfate, and 1-10 g / L of potassium dihydrogen phosphate.

[0023] As a specific embodiment, the Citrobacter amalonaticus described in the above-mentioned method for preparing purine nucleotide phosphorylase is the above-mentioned Citrobacter amalonaticus DHE005.

[0024] Another object of the present invention is to provide a method for preparing 2'-deoxyguanosine.

[0025] The method for preparing 2'-deoxyguanosine of the present invention comprises fermenting the Citrobacter amalonaticus DHE005 of claim 1 to obtain a fermentation broth containing purine nucleotide phosphorylase, and adding phosphate, deoxythymidine and guanosine to the fermentation broth to obtain 2'-deoxyguanosine.

[0026] Furthermore, the fermentation process includes aerobic fermentation in an enzyme-producing medium containing assimilable carbon source and / or nitrogen source.

[0027] Furthermore, the carbon source is selected from glucose, maltose, sucrose, glycerol or fructose; preferably glucose.

[0028] Furthermore, the nitrogen source is selected from yeast extract powder, yeast powder, yeast paste, soybean lecithin, corn steep liquor powder, soybean meal, peptone or urea; preferably yeast paste or yeast extract powder.

[0029] Furthermore, the enzyme production culture medium also includes a surfactant, and the surfactant is selected from sodium dodecyl sulfate, Tween-20, Tween-80, preferably Tween-80.

[0030] Furthermore, the enzyme-producing culture medium also includes an inorganic salt, and the inorganic salt is selected from ammonium citrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium sulfate, calcium carbonate, ferrous sulfate, zinc sulfate, copper sulfate, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, ferric chloride, and manganese sulfate, preferably ammonium citrate, magnesium sulfate, potassium dihydrogen phosphate, potassium chloride or calcium chloride.

[0031] Furthermore, the enzyme production medium contains 10-50 g / L of glucose, 5-20 g / L of yeast extract, 5-15 g / L of yeast extract powder, 2-10 g / L of Tween-80, 2-5 g / L of ammonium citrate, 1-5 g / L of magnesium sulfate, 2-8 g / L of potassium dihydrogen phosphate, 1-5 g / L of potassium chloride, and 1-5 g / L of calcium chloride.

[0032] Furthermore, the fermentation temperature is 25-37°C, the culture medium pH is 5.0-7.0; the culture time is 24-72 hours; and the oxygen flow rate is 0.5-2.0 vvm.

[0033] Further, more specifically, the amalonic acid-free Citrobacter is inoculated into the enzyme fermentation medium by seed liquid to carry out the fermentation culture;

[0034] The seed solution is obtained by culturing Citrobacter amalonaticus in a seed culture medium.

[0035] The conditions for seed culture are as follows: the temperature of seed culture is 25° C.-37° C.; the pH of the culture medium is 5.0-7.0; and the culture time is 12-36 hours.

[0036] The seed culture medium contains 5-30 g / L of glucose, 5-20 g / L of peptone, 2-10 g / L of yeast extract, 5-20 g / L of yeast extract powder, 1-10 g / L of calcium chloride, 1-10 g / L of magnesium sulfate, and 1-10 g / L of potassium dihydrogen phosphate.

[0037] As an embodiment, the phosphate is disodium hydrogen phosphate and sodium dihydrogen phosphate, and the addition ratio of the two is 0.05-0.5 mol / L. Preferably, the amount of the disodium hydrogen phosphate and sodium dihydrogen phosphate and other substances is added.

[0038] As an embodiment, the initial and final concentrations of deoxythymidine are 20-40 g / L, and the initial and final concentrations of guanosine are 25-50 g / L.

[0039] As an embodiment, the Citrobacter amalonaticus described in the above method for preparing purine nucleotide phosphorylase is Citrobacter amalonaticus DHE005.

[0040] The present invention carries out HPLC detection by the following conditions:

[0041] The liquid phase detection method conditions used for 2'-deoxyguanosine detection of the present invention are as follows:

[0042] Chromatographic column: Welch ultimate AQ-C18 (250x 4.6mm, 5um)

[0043] Detection wavelength: 260nm; column temperature: 60℃; flow rate: 1.0ml / min; injection volume: 5ul; mobile phase: isocratic elution with 10% methanol solution.

[0044] The invention discloses a new Citrobacter amalonaticus DHE005, with a deposit number of CGMCC NO.24523. The strain has extremely high nucleoside phosphorylase activity. After the strain is cultured in an enzyme fermentation medium, the strain can be directly fed into a medium containing bacterial cells for catalytic reaction. The invention can directly feed into a medium containing bacterial cells for catalytic reaction. Compared with other disclosed technologies, the strain does not need to be centrifuged and enriched, and the strain does not need to be wall-breaking treated, which significantly reduces the catalytic process. The maximum concentration of the product 2'-deoxyguanosine can reach 33.5 g / L.

[0045] In summary, compared with the traditional process, the technology of the present invention has a series of advantages such as simple and easy operation of the catalytic process, strong catalytic activity, high product concentration, low cost of the entire catalytic process, and convenience for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is the colony characteristic diagram of strain DHE005 (CGMCC NO.24523) on beef extract peptone solid culture medium.

[0047] Figure 2 This is the 2'-deoxyguanosine spectrum detected by HPLC after the strain DHE005 (CGMCC NO.24523) catalyzed the reaction on the substrate. DETAILED DESCRIPTION

[0048] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0049] Unless otherwise specified, the materials and reagents used in the following examples are all common commercial products and can be purchased on the market.

[0050] The present invention is further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention rather than to limit the scope of the present invention.

[0051] Example 1: Strain Source

[0052] Citrobacter amalonaticus DHE005 (CGMCC NO.24523) was isolated from Tiantaishan Farm in Taizhou City, Zhejiang Province, China.

[0053] Cross sampling was carried out in the Tiantai Mountain farm. Five sampling points were randomly selected. 10g of soil sample was taken from each point and put into a conical bottle. After mixing evenly, 10g of sample was taken and added to a conical bottle filled with 90mL sterile water (there was a magnetic stirrer in the bottle). The suspension was vortexed for 30 minutes to fully mix it into a suspension, which was 10 -1 Use the dilution plate method to dilute the suspension with sterile water at a volume ratio of 1:9 to 10 -2 , 10 -3 , 10 -4 , 10 -5Concentration, take 0.1mL of bacterial suspension with different dilution multiples, spread it on beef extract peptone agar medium plate, and gently spread it on the surface of the medium with a sterile coating stick. Let it stand at room temperature for 30 minutes and then place it in a 25℃ constant temperature incubator. After the colonies grow, observe and record the color, transparency, surface and edge morphology of the colonies. Finally, pick 1000 strains and inoculate them on beef extract peptone agar medium to make a slant, and perform fermentation and purine nucleoside phosphorylase activity analysis. A loop of the slant cultured bacteria was picked up with an inoculating loop and inoculated into 250 mL conical flasks containing 20 mL of seed culture medium. After shaking culture at 28°C for 1 day, 1 mL was transferred to a 250 mL conical flask containing 20 mL of fermentation medium. After shaking culture at 30°C for 3 days, wet bacteria were obtained by centrifugation and tested with a purine nucleotide phosphorylase (PNP) ELISA kit (Shanghai Shuangying Biotechnology Co., Ltd.), and the strain containing purine nucleotide phosphorylase, namely Citrobacter amalonaticus DHE005 (CGMCC NO.24523), was selected.

[0054] Beef extract peptone solid medium (g / L): beef extract 3.0 g / L, peptone 10.0 g / L, NaCl 5.0 g / L, distilled water 1000 mL, agar 20 g / L, pH 7.0.

[0055] Seed culture medium formula (g / L): glucose 10g / L, peptone 10g / L, yeast extract powder 5g / L, calcium carbonate 15g / L, magnesium sulfate 1.5g / L, potassium dihydrogen sulfate 1g / L, add water to make up to 1000mL, pH 7.0±0.1.

[0056] Fermentation medium formula (g / L): glucose 50 g / L, yeast extract powder 5 g / L, yeast powder 10 g / L, yeast paste 10 g / L, calcium carbonate 30 g / L, add water to make up to 1000 mL, pH 7.0±0.1.

[0057] Example 2: Morphological, cultural, physiological and biochemical characteristics of Citrobacter amalonaticus DHE005 (CGMCCNO.24523).

[0058] The colony characteristics of strain DHE005 after being cultured in beef extract peptone solid medium at 30℃ for 24h are as follows Figure 1 As shown, the colonies are flat, milky white, convex, opaque, dry, and have the same color on both sides. The bacteria are Gram-positive, and are rod-shaped under a microscope, with spores in some bacteria.

[0059] The identification was carried out according to the relevant contents in books such as Bergey's Manual of Systematic Bacteriology and Manual of Identification of Common Bacterial Systems. The physiological responses of the strains are shown in Table 1.

[0060] Table 1. Physiological responses of Citrobacter amalonaticus DHE005 (CGMCCNO.24523)

[0061]

[0062]

[0063] Remarks: +: positive; -: negative.

[0064] Example 3 Bacterial species identification

[0065] The 16S rDNA sequence analysis of Citrobacter amalonaticus DHE005 (CGMCC NO.24523) was carried out according to the relevant contents in the Molecular Cloning Experiment Guide. The bacteria were collected and then the total DNA was extracted using a bacterial DNA extraction kit.

[0066] Primers: Forward primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 2)), Revese primer 1492R (5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 3)) were designed for PCR amplification. The PCR product was detected by 0.8% agarose gel electrophoresis. The PCR product was purified and recovered using the SanPrep column PCR purification product kit. The purified PCR product was directly sent to Sangon Biotech (Shanghai) Co., Ltd. for sequence determination.

[0067] The 16S rDNA sequence of strain DHE005 (CGMCC NO.24523) was proofread and compared with the sequences of related species and genera in the GenBank database by BLAST to determine the taxonomic status of the strain.

[0068] The 16S rDNA sequence of strain DHE005 (CGMCC NO.24523) was submitted to NCBI for BLAST comparison with the relevant sequences in GenBank. The results are shown in Table 2 (only the model strains with higher homology are listed in the table).

[0069] Table 2 Homology of strain DHE005 (CGMCC NO.24523) and typical model strains

[0070]

[0071]

[0072] By sequencing the 16S rDNA (SEQ ID NO: 1) region of strain DHE005 (CGMCC NO. 24523) and comparing the homologous sequences with the sequences of related species and genera in the GenBank database by BLAST, it was found that the homology between strain DHE005 and Citrobacter amalonaticus (Citrobacter amalonaticus strain CECT 863, Citrobacter amalonaticus strain HAMBI1296, Citrobacter amalonaticus strain 133355-SW-C4-Cam, Citrobacter amalonaticus strain SWTPB14 and Citrobacter amalonaticus JCM 1661) was as high as 99.93%-100%. At the same time, the apparent characteristics test of strain DHE005 (CGMCC NO. 24523) was performed, and it was found that the strain had a high homology with Citrobacter amalonaticus (Citrobacter amalonaticus strain CECT 863, Citrobacter amalonaticus strain HAMBI1296, Citrobacter amalonaticus strain 133355-SW-C4-Cam, Citrobacter amalonaticus strain SWTPB14 and Citrobacter amalonaticus JCM 1661). amalonaticus, the strain DHE005 (CGMCC NO.24523) was identified as a Citrobacter amalonaticus strain.

[0073] Example 4 Catalytic production of 2'-deoxyguanosine

[0074] (1) Preparation and cultivation of slant bacteria:

[0075] Slant culture medium formula (g / L): yeast extract powder 4.0g / L, malt extract 10.0g / L, glucose 4.0g / L, agar 20.0g / L, pH before sterilization 7.2-7.4, test tube 30×200mm, filling volume 15mL, sterilize at 121℃ for 20min, cool to about 55-60℃ and swing on the slant, after cooling and solidifying, inoculate on the slant, and culture at 30±1℃ for 3 days until the strain matures.

[0076] (2) Preparation and cultivation of seed solution:

[0077] Seed culture medium formula: glucose 30g / L, peptone 10g / L, yeast extract 10g / L, yeast extract powder 5g / L, calcium chloride 2g / L, magnesium sulfate 2g / L, potassium dihydrogen phosphate 5g / L. pH 7.0 before sterilization; 250mL triangular shake flask, 50mL, sterilized at 121℃ for 20min. Inoculate 1-2 single colonies on the slant into the seed culture medium, 30±1℃, 250rpm shaking culture for 24 hours, at which time the culture solution pH is 6.8-7.2, and the bacterial OD600 is 10-20.

[0078] (3) Preparation of enzyme fermentation medium and bacterial culture:

[0079] Enzyme fermentation medium formula: glucose 10g / L, yeast extract 5g / L, yeast extract powder 5g / L, Tween-802g / L, ammonium citrate 2g / L, magnesium sulfate 1g / L, potassium dihydrogen phosphate 2g / L, potassium chloride 1g / L, calcium chloride 1g / L, pH adjusted to 5.0 before sterilization. 250mL triangular shake flask, filled with 20mL, sterilized at 121℃ for 20min. Inoculate the seed liquid at a 10% (volume ratio) inoculation amount. Culture at 25±1℃, 250rpm shaking for 72 hours.

[0080] (4) Catalytic reaction of 2'-deoxyguanosine:

[0081] After the fermentation of Citrobacter malonic acidus was completed, 0.05 mol of disodium hydrogen phosphate and sodium dihydrogen phosphate were added to the fermentation broth, and the temperature was raised to 40°C. Subsequently, 20 g / L of deoxythymidine and 25 g / L of guanosine were added. The reaction was stirred for 0.5 h and ended. The content of 2'-deoxyguanosine in the catalytic reaction liquid was detected by HPLC and was found to be 18.4 g / L.

[0082] Example 5 Catalytic production of 2'-deoxyguanosine

[0083] (1) The slant culture medium formulation and culture conditions are the same as step (1) in Example 4;

[0084] (2) The seed culture medium formulation and culture conditions are the same as step (2) in Example 4;

[0085] (3) Preparation of enzyme fermentation medium and bacterial culture:

[0086] Enzyme fermentation medium formula: glucose 30g / L, yeast extract 10g / L, yeast extract powder 10g / L, Tween-806g / L, ammonium citrate 4g / L, magnesium sulfate 3g / L, potassium dihydrogen phosphate 6g / L, potassium chloride 3g / L, calcium chloride 3g / L, pH adjusted to 6.0 before sterilization. 250mL triangular shake flask, filled with 20mL, sterilized at 121℃ for 20min. Inoculate the seed liquid at a 10% (volume ratio) inoculation amount. Culture at 37±1℃, 250rpm shaking for 24 hours.

[0087] (4) Catalytic reaction of 2'-deoxyguanosine:

[0088] After the fermentation of Citrobacter malonic acidus was completed, 0.1 mol each of disodium hydrogen phosphate and sodium dihydrogen phosphate was added to the fermentation broth, the temperature was raised to 60°C, and then 30 g / L of deoxythymidine and 35 g / L of guanosine were added. The reaction was stirred for 3 hours and ended. The content of 2'-deoxyguanosine in the catalytic reaction liquid was detected by HPLC and was found to be 27.6 g / L.

[0089] Example 6 Catalytic production of 2'-deoxyguanosine

[0090] (1) The slant culture medium formulation and culture conditions are the same as step (1) in Example 4; the primary seed culture medium formulation and culture conditions are the same as step (2) in Example 4;

[0091] (2) Preparation of seed solution in seed tank:

[0092] The formula of the seed liquid culture medium in the seed tank is the same as the seed culture medium in step (2) of Example 4;

[0093] 10 L of seed culture medium was added to a 15 L seed tank, and sterilized with steam at 121° C. for 20 min. After cooling to 30° C., 200 mL of seed solution from a primary shake flask was added, the stirring speed was 200 rpm, the ventilation volume was 1.0 vvm, and the culture was carried out at 30±1° C. for 16 hours, at which time the pH of the seed solution was 7.0-7.4, and the bacterial concentration was 12-15% (volume ratio);

[0094] (3) Preparation of enzyme fermentation medium and bacterial culture:

[0095] Enzyme fermentation medium formula: glucose 50g / L, yeast extract 20g / L, yeast extract powder 15g / L, Tween-8010g / L, ammonium citrate 5g / L, magnesium sulfate 5g / L, potassium dihydrogen phosphate 8g / L, potassium chloride 5g / L, calcium chloride 5g / L, adjust the pH to 7.0 before sterilization.

[0096] The fermentation tank has a volume of 50 L and a feed volume of 30 L. It is sterilized with steam at 121°C for 20 min. After cooling to 30°C, 3 L of seed liquid in the seed tank is added. The stirring speed is 300-600 rpm (the speed gradually increases from 300 rpm to 600 rpm in the first 3 days), the ventilation volume is 2.0 vvm, and the culture is carried out at 30°C for 24 hours.

[0097] (4) Catalytic reaction of 2'-deoxyguanosine:

[0098] After the fermentation of Citrobacter malonic acidus was completed, 0.5 mol of disodium hydrogen phosphate and sodium dihydrogen phosphate were directly added to the fermentation liquid in the tank, the temperature was raised to 70°C, and then 40 g / L of deoxythymidine and 50 g / L of guanosine were added. The reaction was stirred for 5 hours and ended. The content of 2'-deoxyguanosine in the catalytic reaction liquid was detected by HPLC and was measured to be 33.5 g / L.

Claims

1. A non-malonic acid Citrobacter ( Citrobacter amalonaticus )DHE005, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC), with the deposit number CGMCC NO.24523 and the deposit date of March 11, 2022.

2. A method comprising the non-malonic acid Citrobacter ( Citrobacter amalonaticus ) Fermentation broth of DHE005.

3. A non-malonic acid Citrobacter as claimed in claim 1 ( Citrobacter amalonaticus )Application of DHE005 or its seed solution or fermentation solution or bacterial suspension or culture solution in the production of purine nucleotide phosphorylase or products containing purine nucleotide phosphorylase; Or, a non-malonic acid Citrobacter as claimed in claim 1 ( Citrobacter amalonaticus ) Use of DHE005 or its seed solution or fermentation solution or bacterial suspension or culture solution in the preparation of 2'-deoxyguanosine or a pharmaceutical composition containing 2'-deoxyguanosine.

4. A method for preparing purine nucleotide phosphorylase, characterized in that: comprising using the non-malonic acid Citrobacter as claimed in claim 1 ( Citrobacter amalonaticus )DHE005 was prepared by fermentation.

5. A method for preparing 2'-deoxyguanosine, characterized in that: comprising using the non-malonic acid Citrobacter as claimed in claim 1 ( Citrobacter amalonaticus ) DHE005 is fermented to obtain a fermentation broth containing purine nucleotide phosphorylase, and phosphate, deoxythymidine and guanosine are added to the fermentation broth to obtain 2'-deoxyguanosine.

6. The preparation method according to claim 4 or 5, characterized in that: The fermentation process involves aerobic fermentation in an enzyme-producing medium containing an assimilable carbon source and / or nitrogen source.

7. The preparation method according to claim 6, characterized in that: The carbon source is selected from glucose, maltose, sucrose, glycerol or fructose; And / or the nitrogen source is selected from yeast extract powder, yeast powder, yeast paste, soybean lecithin, corn steep liquor powder, soybean meal, peptone and urea.

8. The preparation method according to claim 6, characterized in that: The enzyme production culture medium also includes a surfactant, and the surfactant is selected from sodium dodecyl sulfate, Tween-20 and Tween-80.

9. The preparation method according to claim 6, characterized in that: The enzyme production culture medium also includes inorganic salts, which are selected from ammonium citrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium sulfate, calcium carbonate, ferrous sulfate, zinc sulfate, copper sulfate, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, ferric chloride, and manganese sulfate.

10. The preparation method according to claim 6, characterized in that: The enzyme production culture medium contains 10-50 g / L of glucose, 5-20 g / L of yeast extract, 5-15 g / L of yeast extract powder, 2-10 g / L of Tween-80, 2-5 g / L of ammonium citrate, 1-5 g / L of magnesium sulfate, 2-8 g / L of potassium dihydrogen phosphate, 1-5 g / L of potassium chloride, and 1-5 g / L of calcium chloride.

11. The preparation method according to claim 4 or 5, characterized in that: The fermentation temperature is 25-37°C, the culture medium pH is 5.0-7.0; the culture time is 24-72 hours; and the oxygen flow rate is 0.5-2.0 vvm.

12. The preparation method according to claim 6, characterized in that: The nonmalonic acid Citrobacter ( Citrobacter amalonaticus ) DHE005 is fermented by inoculating seed liquid into the enzyme production medium; Wherein, the seed liquid is a non-malonic acid Citrobacter ( Citrobacter amalonaticus )DHE005 was obtained by seed culture in seed medium; The seed culture conditions are as follows: the seed culture temperature is 25°C-37°C; the seed culture medium pH is 5.0-7.0; the culture time is 12-36 hours; The seed culture medium contains 5-30 g / L of glucose, 5-20 g / L of peptone, 2-10 g / L of yeast extract, 5-20 g / L of yeast extract powder, 1-10 g / L of calcium chloride, 1-10 g / L of magnesium sulfate, and 1-10 g / L of potassium dihydrogen phosphate.

13. The preparation method according to claim 5, characterized in that: The phosphates are disodium hydrogen phosphate and sodium dihydrogen phosphate, and the addition ratio of the two is 0.05-0.5 mol / L.

14. The preparation method according to claim 13, characterized in that: The amount of substances such as disodium hydrogen phosphate and sodium dihydrogen phosphate is added.

15. The method according to claim 5, characterized in that: The initial and final concentrations of the deoxythymidine are 20-40 g / L, and the initial and final concentrations of the guanosine are 25-50 g / L.

16. The preparation method according to claim 5, characterized in that: The reaction temperature is 40~70℃ and the reaction time is 0.5~5 h.

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