CN115975841A - Citrobacter malonate free and method for producing 2' -deoxyguanosine by catalyzing with Citrobacter malonate free - Google Patents

Abstract

The invention discloses a malonic acid-free Citrobacter citrate (DHE) 005, wherein the preservation number is CGMCC NO. 2457, and the preservation date is 03 and 11 months in 2022; also discloses a method for catalytically producing 2' -deoxyguanosine by taking Citrobacter alamonafaciens (DHE 005) as an enzyme source through fermentation culture. The catalytic method provided by the invention has the advantages that the catalytic process is simple and easy to operate, the thalli do not need to be subjected to centrifugal enrichment, the wall breaking treatment is not needed, the catalytic process is obviously reduced, the highest concentration of the product 2' -deoxyguanosine can reach 33.5g/L, the catalytic activity is strong, the product concentration is high, the cost of the whole catalytic process is low, and the industrial production is facilitated.

Description

Citrobacter malonate free and method for producing 2' -deoxyguanosine by catalyzing with Citrobacter malonate free

Technical Field

The invention relates to the technical field of bioengineering, in particular to a method for producing 2' -deoxyguanosine by utilizing citric acid bacillus without malonate and catalysis thereof.

Background

2' -Deoxyguanosine (2 ' -Deoxyguanosine) is a structural fragment of biomacromolecule deoxyribonucleic acid (DNA) essential for the development and normal operation of organisms, and the chemical structural formula of the 2' -Deoxyguanosine is shown as a formula 1.

2' -deoxyguanosine is an important genetic material component, is involved in the transmission of genetic information of biological cells together with other natural nucleosides, and plays an important role in the growth regulation of the biological cells. 2'-deoxyguanosine can be used as a chemical reagent for biochemical research, and in the field of drug research and development, the research finds that 2' -deoxyguanosine has good physiological activity, is an important raw material in the field of gene drugs, and can also be used as an intermediate to generate drugs for resisting viruses, tumors, AIDS and the like. For example, the 6-disulfur-substituted-2 '-deoxyguanosine compound prepared by taking 2' -deoxyguanosine as a raw material has obvious tumor inhibition effect on various tumors, and can be applied to medicines for tumors related to human cell proliferation. In addition, with the rapid spread of 2019, the novel coronavirus pneumonia COVID-19, the demand of 2' -deoxyguanosine, which is an important precursor material in a virus detection kit, is also rapidly increasing.

The traditional preparation of 2'-deoxyguanosine uses DNA as raw material, and is hydrolyzed into deoxynucleotide by nuclease, then phosphoric acid is removed by enzyme, and 2' -deoxyguanosine is separated from the deoxynucleotide after the deoxynucleotide is generated. At present, the methods available for the preparation of 2' -deoxyguanosine are mainly chemical synthesis and bio-enzyme catalysis. The chemical synthesis method for preparing 2'-deoxyguanosine has the defects of long reaction steps, harsh reaction conditions, low final yield, and obvious environmental pollution due to the fact that various organic reagents are required in the reaction process, so that the chemical synthesis method for preparing 2' -deoxyguanosine is not suitable for large-scale industrial production. A plurality of patents are reported for preparing 2'-deoxyguanosine by a bio-enzyme catalysis method, for example, CN 104830930B discloses a method for producing deoxyguanosine by a biotransformation method, lactobacillus fermentum thallus pulp is used as a biocatalyst, a biocatalytic reaction is carried out in a polyethylene glycol 10000/dipotassium hydrogen phosphate double aqueous phase system to obtain a 2' -deoxyguanosine product, and the thymidine conversion rate can reach 89% under the optimal condition; patent CN 102174618a also discloses a method for producing 2'-deoxyguanosine by conversion using brevibacterium acetylicum, which uses a strain of brevibacterium acetylicum having nucleoside phosphorylase activity as an enzyme source, and mixes it with thymidine and a guanine donor as conversion reaction substrates to carry out conversion reaction to synthesize 2' -deoxyguanosine; in addition, patent CN 108018252A also discloses a method for preparing 2'-deoxyguanosine by enzyme catalysis, which utilizes recombinant Escherichia coli to obtain N-deoxyribotransferase to directly catalyze thymidine and guanine to synthesize 2' -deoxyguanosine. The above patents all disclose methods for preparing 2' -deoxyguanosine by using biological enzyme catalysis, but the bacterium used in the patent CN 104830930B is lactobacillus, belongs to a facultative anaerobe, is difficult to obtain thallus under conventional culture conditions, and the reaction system is a two-aqueous phase system, so that the process is complex, the manufacturing cost is high, and the industrial application is not facilitated; in patent CN 102174618A, the concentration of a substrate in a catalytic reaction is low, the reaction conversion rate is only 60%, and the method is not suitable for industrial application; in patent CN 108018252A, the recombination colibacillus strain can induce the synthesis of N-deoxyribose transferase only by adding expensive inducer in the culture process, in addition, as heterologous expression protein, the recombination bacteria have the problem of unsuccessful protein folding when expressing the N-deoxyribose transferase, and are easy to generate inclusion bodies, and the enzyme catalysis patents disclosed above all need to carry out centrifugal treatment on the bacteria, most of the enzyme catalysis patents need to carry out wall breaking treatment on the bacteria to obtain corresponding catalytic enzyme for subsequent catalytic reaction, the process obviously increases the process steps, increases the production cost, and is not favorable for industrial application.

Disclosure of Invention

Based on the technical defects, one of the objects of the present invention is to provide a Citrobacter alamus-free DHE005, which is deposited in the common microorganism center of china committee for culture collection of microorganisms (CGMCC), with the address: the No. 3 Xilu No. 1 of Beijing, chaoyang, the institute for microbiology, china academy of sciences, with the collection number of CGMCC NO. 2457 and the collection date of 2022 years, 03 months and 11 days, was registered in a book to prove survival.

The invention also aims to provide a new application of the citric acid bacillus without malonate or seed liquid thereof, or fermentation liquid thereof, or bacterial suspension thereof, or culture liquid thereof.

The invention provides application of Citrobacter acidi (Citrobacter ammoniagenes) or seed liquid thereof, fermentation liquid thereof, bacterial suspension thereof or culture liquid thereof in production of purine nucleotide phosphorylase or products containing purine nucleotide phosphorylase.

The invention provides an application of Citrobacter alameanus or seed liquid thereof, fermentation liquid thereof, bacterial suspension thereof or culture liquid thereof in preparing 2'-deoxyguanosine or a pharmaceutical composition containing 2' -deoxyguanosine.

In an embodiment, the Citrobacter malonates-free bacillus (Citrobacter alamo-ionicus) used in the above application is the Citrobacter malonates-free DHE005.

The invention also aims to provide a preparation method of purine nucleotide phosphorylase, which comprises the step of fermenting and preparing by adopting citric acid bacterium without malonate.

Further, the fermentation process comprises aerobic fermentation in an enzyme-producing medium containing an assimilable carbon and/or nitrogen source.

Further, the carbon source is selected from glucose, maltose, sucrose, glycerol or fructose; glucose is preferred.

Further, the nitrogen source is selected from yeast extract powder, yeast extract, soybean lecithin, corn steep liquor dry powder, soybean meal, peptone and urea; preferably yeast extract or yeast extract powder.

Further, the enzyme production culture medium also comprises a surfactant, wherein the surfactant is selected from sodium dodecyl sulfate, tween-20 and tween-80, and is preferably tween-80.

Further, the enzyme production culture medium also comprises inorganic salt, wherein the inorganic salt is selected from ammonium citrate, monopotassium phosphate, dipotassium phosphate, ammonium sulfate, calcium carbonate, ferrous sulfate, zinc sulfate, copper sulfate, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, ferric chloride and manganese sulfate, and preferably ammonium citrate, magnesium sulfate, monopotassium phosphate, potassium chloride or calcium chloride.

Further, the enzyme production culture medium contains 10-50g/L of glucose, 5-20g/L of yeast extract, 5-15g/L of yeast extract powder, 2-10g/L of tween-80, 2-5g/L of ammonium citrate, 1-5g/L of magnesium sulfate, 2-8g/L of monopotassium phosphate, 1-5g/L of potassium chloride and 1-5g/L of calcium chloride.

Further, the fermentation temperature is 25-37 ℃, and the pH value of the culture medium is 5.0-7.0; the culture time is 24-72 hours; the oxygen introduction amount is 0.5-2.0vvm.

Further, more specifically, the citric acid bacillus malonate free is inoculated to the enzyme fermentation culture medium through seed liquid for fermentation culture;

wherein the seed solution is obtained by seed culture of citric acid bacillus malonate free (Citrobacter alamonaticus) in a seed culture medium.

The seed culture conditions are as follows: the temperature of seed culture is 25-37 ℃; the pH value of the culture medium is 5.0-7.0; the culture time is 12-36 hours.

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

In one embodiment, the Citrobacter acidilacticum (Citrobacter alginationus) used in the method for producing a purine nucleotide phosphorylase is DHE005 of the Citrobacter acidilacticum (Citrobacter alginationus).

It is still another object of the present invention to provide a method for preparing 2' -deoxyguanosine.

The method for producing 2'-deoxyguanosine according to the present invention comprises fermenting the Citrobacter alamidosus DHE005 according to 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.

Further, the fermentation process comprises conducting aerobic fermentation in an enzyme-producing medium containing assimilable carbon and/or nitrogen sources.

Further, the carbon source is selected from glucose, maltose, sucrose, glycerol or fructose; glucose is preferred.

Further, the nitrogen source is selected from yeast extract powder, yeast extract, soybean lecithin, corn steep liquor dry powder, soybean meal, peptone or urea; preferably yeast extract or yeast extract powder.

Further, the enzyme production culture medium also comprises a surfactant, wherein the surfactant is selected from sodium dodecyl sulfate, tween-20 and tween-80, and is preferably tween-80.

Further, the enzyme production culture medium further comprises inorganic salt, wherein the inorganic salt is selected from ammonium citrate, monopotassium phosphate, dipotassium phosphate, ammonium sulfate, calcium carbonate, ferrous sulfate, zinc sulfate, copper sulfate, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, ferric chloride and manganese sulfate, and preferably ammonium citrate, magnesium sulfate, monopotassium phosphate, potassium chloride or calcium chloride.

Further, the enzyme production culture medium contains 10-50g/L of glucose, 5-20g/L of yeast extract, 5-15g/L of yeast extract powder, 2-10g/L of tween-80, 2-5g/L of ammonium citrate, 1-5g/L of magnesium sulfate, 2-8g/L of monopotassium phosphate, 1-5g/L of potassium chloride and 1-5g/L of calcium chloride.

Further, the fermentation temperature is 25-37 ℃, and the pH value of the culture medium is 5.0-7.0; the culture time is 24-72 hours; the oxygen introduction amount is 0.5-2.0vvm.

Further, more specifically, the citric acid bacillus malonate free is inoculated to the enzyme fermentation culture medium through seed liquid for fermentation culture;

wherein the seed solution is obtained by seed culture of citric acid bacillus without malonate in a seed culture medium.

The seed culture conditions are as follows: the temperature of seed culture is 25-37 ℃; the pH value of the culture medium is 5.0-7.0; the culture time is 12-36 hours.

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

In one embodiment, the phosphate is disodium hydrogen phosphate and sodium dihydrogen phosphate, both of which are added in a proportion of 0.05 to 0.5mol/L, and preferably, the phosphate is added in the amount of the disodium hydrogen phosphate and the sodium dihydrogen phosphate.

In one embodiment, the initial final concentration of deoxythymidine is 20-40g/L and the initial final concentration of guanosine is 25-50g/L.

In one embodiment, the Citrobacter acidilacticum (Citrobacter alginationus) used in the method for producing a purine nucleotide phosphorylase is Citrobacter acidilacticum (Citrobacter alginationus) DHE005.

The invention carries out HPLC detection by the following conditions:

the conditions of the liquid phase detection method used for detecting the 2' -deoxyguanosine are as follows:

a chromatographic column: welch ultmate AQ-C18 (250x 4.6mm, 5um)

The detection wavelength is 260nm; column temperature: 60 ℃; the flow rate is 1.0ml/min; the sample injection volume is 5ul; mobile phase: the 10% methanol solution was eluted isocratically.

The invention discloses a brand-new citric acid bacillus (Citrobacter alamo) DHE005 without malonic acid, wherein the preservation number is CGMCC NO. 2457, the strain has extremely high nucleoside phosphorylase activity, after the strain is cultured in an enzyme fermentation culture medium, the strain can be directly fed into the culture medium containing thalli for catalytic reaction, the strain can be directly fed into the culture medium containing the thalli for catalytic reaction, compared with other disclosed technologies, the strain does not need to be subjected to centrifugal enrichment, the thalli does not need to be subjected to wall breaking treatment, the catalytic process is obviously reduced, and the maximum concentration of a product 2' -deoxyguanosine can reach 33.5g/L.

In conclusion, compared with the traditional process, the technology of the invention has the series advantages of simple and easy operation of the catalytic process, strong catalytic activity, high product concentration, lower cost of the whole catalytic process and convenience for industrial production.

Drawings

FIG. 1 is a characteristic diagram of bacterial colony of strain DHE005 (CGMCC NO. 2457) on beef extract peptone solid medium.

FIG. 2 shows the HPLC-detected 2' -deoxyguanosine chromatogram after the catalytic reaction of the strain DHE005 (CGMCC NO. 2457) on the substrate.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

The materials, reagents and the like used in the following examples are ordinary and commercially available products unless otherwise specified, and are commercially available.

The present invention is further illustrated by the following examples, which are intended to be purely exemplary of the invention and are not intended to limit its scope.

Example 1: the source of the strain

The Citrobacter malononitrile-free DHE005 (CGMCC NO. 2457) is isolated from a Tiantaishan farm in Taizhou city, zhejiang province, china.

Cross sampling is carried out in a Tiantai mountain farm, 5 sampling points are randomly taken, 10g of soil sample is taken at each point and put into a conical flask, and the soil samples are uniformly mixedThen 10g of sample is taken and added into a conical flask filled with 90mL of sterile water (a magnetic stirrer is arranged in the flask), vortex stirring is carried out for 30 minutes, and the mixture is fully and uniformly mixed to prepare suspension, namely 10g of suspension ^-1 And (4) bacterial suspension. Mixing the suspension with sterile water according to a volume ratio of 1:9 to 10 ^-2 ，10 ^-3 ，10 ^-4 ，10 ^-5 And (3) taking 0.1mL of bacterial suspension with different dilution times, coating the bacterial suspension on a beef extract peptone agar medium plate, slightly coating the bacterial suspension on the surface of a medium by using a sterile coating rod, standing at room temperature for 30 minutes, and then placing in a constant-temperature incubator at 25 ℃. And after the bacterial colony grows out, observing and recording the color, the transparency, the surface and the edge form of the bacterial colony. Finally, 1000 strains are selected and inoculated in a beef extract peptone agar culture medium to be made into a slant, and fermentation and purine nucleoside phosphorylase activity analysis are carried out. A loop of the strain cultured on the inclined plane is picked by an inoculating loop, the loop is respectively inoculated in a 250mL conical flask containing 20mL of seed culture medium, after shaking culture is carried out for 1 day under the condition of 28 ℃, 1mL of the strain is further absorbed and inoculated in a 250mL conical flask containing 20mL of fermentation culture medium, after shaking culture is carried out for 3 days under the condition of 30 ℃, wet strain is obtained by centrifugation, and the wet strain is detected by a Purine Nucleotide Phosphorylase (PNP) ELISA kit (Shanghai double win Biotech Co., ltd.), and a strain containing purine nucleotide phosphorylase, namely Citrobacter alamonacina oracina-free DHE005 (CGMCC NO. 2457) is selected.

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

Seed culture medium formula (g/L): 10g/L of glucose, 10g/L of peptone, 5g/L of yeast extract powder, 15g/L of calcium carbonate, 1.5g/L of magnesium sulfate and 1g/L of potassium dihydrogen sulfate, adding water to fix the volume to 1000mL, and adjusting the pH to 7.0 +/-0.1.

Fermentation medium formula (g/L): 50g/L glucose, 5g/L yeast extract powder, 10g/L yeast extract, 30g/L calcium carbonate, water to 1000mL, pH7.0 +/-0.1.

Example 2: the morphology, the cultural characteristics and the physiological and biochemical characteristics of the Citrobacter malonodiacremophilus DHE005 (CGMCC NO. 2457) are avoided.

The characteristics of the bacterial colony of the strain DHE005 cultured in a beef extract peptone solid medium at 30 ℃ for 24 hours are shown in figure 1, the bacterial colony is flat, milky, convex and opaque, shows dryness and has consistent front and back side colors. The gram staining of the thallus is positive, and the thallus is observed to be rod-shaped under a microscope, and spores exist in partial thallus.

The identification was carried out according to the contents of the book "Bergey's Manual of bacteriology of the System" and "Manual of identification of common bacteria System", etc., and the physiological reactions of the strains are shown in Table 1.

TABLE 1 physiological response of Citrobacter malononitrile-free DHE005 (CGMCC NO. 2457)

Remarking: +: positive; -: and (4) negativity.

EXAMPLE 3 species identification

16S rDNA sequence analysis of DHE005 (CGMCC NO. 2457) of Citrobacter malononitrile (Citrobacter alamionicus) was performed according to the contents of the "molecular cloning instruction". The cells were collected and total DNA was extracted using a bacterial DNA extraction kit.

Designing a primer: forward primer27F (5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 2)), revese primer1492R (5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 3)), PCR amplification is carried out, 0.8% agarose gel electrophoresis is adopted for PCR product detection, a SanPrep column type PCR purified product kit is adopted for PCR product purification and recovery, and the purified PCR product is directly sent to Shanghai Kongkun company of biological engineering (Shanghai) for sequence determination.

The 16S rDNA sequence measured by strain DHE005 (CGMCC NO. 2457) is checked and compared with homologous sequence BLAST in GenBank database to determine the classification status of the strain.

The result of BLAST comparison of the 16S rDNA sequence measured by the strain DHE005 (CGMCC NO. 245923) with the related sequence in GenBank is shown in Table 2 (only the model strains with higher homology are listed in the table).

TABLE 2 homology of the Strain DHE005 (CGMCC NO. 245123) with the model Strain

/>

By sequencing the region of strain DHE005 (CGMCC NO. 245123) 16S rDNA (SEQ ID NO: 1), comparing with homologous sequences BLAST of related species and genera in GenBank database, it was found that homology was up to 99.93% -100% with that of Citrobacter malonate-free (Citrobacter alaminationicus strain CECT 863, citrobacter alaminationicus MBI1296, citrobacter alaminaticus strain 133355-SW-C4-Cam, citrobacter alaminationicus strain SWTPB14 and Citrobacter alaminaticus JCM 1661), and performing apparent feature test on strain DHE (CGMCC NO. 245123), it was found that the strain and the Citrobacter malonate-free (Citrobacter) strain were identified as being very close to the classification related parameters of the strain DHE005 (CGMCC NO. 23), and the strain DHE005 (CGMCC NO. 23) was identified as the strain NO malonate.

Example 4 catalytic production of 2' -deoxyguanosine

(1) Preparing and culturing slant strains:

formula (g/L) of slant culture medium: 4.0g/L of yeast extract powder, 10.0g/L of malt extract, 4.0g/L of glucose, 20.0g/L of agar, 7.2-7.4 of pH before digestion, 30 x 200mm of test tube and 15mL of package, sterilizing at 121 ℃ for 20min, cooling to 55-60 ℃, placing an inclined plane, inoculating to the inclined plane after cooling and solidification, and culturing at 30 +/-1 ℃ for 3 days to ensure that the strain is mature.

(2) Preparing and culturing a seed solution:

the seed culture medium formula comprises: 30g/L of glucose, 10g/L of peptone, 10g/L of yeast extract, 5g/L of yeast extract powder, 2g/L of calcium chloride, 2g/L of magnesium sulfate and 5g/L of monopotassium phosphate. pH7.0 before digestion; a250 mL triangular shake flask was filled with 50mL medium and sterilized at 121 ℃ for 20min. Inoculating 1-2 single colonies on the inclined plane into a seed culture medium, performing shake culture at 30 + -1 deg.C and 250rpm for 24 hr, wherein the pH of the culture solution is 6.8-7.2, and the thallus OD600 is 10-20.

(3) Preparing an enzyme fermentation culture medium and culturing thalli:

the enzyme fermentation culture medium formula comprises: 10g/L glucose, 5g/L yeast extract powder, 802g/L tween-2, 2g/L ammonium citrate, 1g/L magnesium sulfate, 2g/L potassium dihydrogen phosphate, 1g/L potassium chloride and 1g/L calcium chloride, and adjusting the pH value to 5.0 before sterilization. A250 mL triangular shake flask is filled with 20mL and sterilized at 121 ℃ for 20min. The seed liquid is inoculated with an inoculum size of 10% (volume ratio). The culture was carried out at 25. + -. 1 ℃ for 72 hours with shaking at 250 rpm.

(4) Catalytic reaction of 2' -deoxyguanosine:

after fermentation of the citric acid bacterium without malonic acid was completed, disodium hydrogenphosphate and sodium dihydrogenphosphate were added to the fermentation broth in an amount of 0.05mol each, the temperature was raised to 40 ℃ and then 20g/L deoxythymidine and 25g/L guanosine were added thereto, the reaction was completed after stirring for 0.5 hour, and the content of 2' -deoxyguanosine in the catalytic reaction solution was measured by HPLC and found to be 18.4g/L.

Example 5 catalytic production of 2' -deoxyguanosine

(1) The formulation of the slant culture medium and the culture conditions were the same as those in step (1) of example 4;

(2) The formulation of the seed culture medium and the culture conditions were the same as those in step (2) of example 4;

(3) Preparation of enzyme fermentation medium and thallus culture:

the enzyme fermentation culture medium formula comprises: 30g/L of glucose, 10g/L of yeast extract powder, 806g/L of tween-citrate, 4g/L of ammonium citrate, 3g/L of magnesium sulfate, 6g/L of monopotassium phosphate, 3g/L of potassium chloride and 3g/L of calcium chloride, and the pH value is adjusted to 6.0 before sterilization. A250 mL triangular shake flask was filled with 20mL and sterilized at 121 ℃ for 20min. The seed liquid is inoculated with an inoculum size of 10% (volume ratio). The culture was performed at 37. + -. 1 ℃ for 24 hours with shaking at 250 rpm.

(4) Catalytic reaction of 2' -deoxyguanosine:

after fermentation of the citric acid bacterium without malonic acid was completed, disodium hydrogenphosphate and sodium dihydrogenphosphate were added to the fermentation broth in an amount of 0.1mol each, the temperature was raised to 60 ℃ and then 30g/L deoxythymidine and 35g/L guanosine were added thereto, the reaction was stirred for 3 hours and then ended, and the content of 2' -deoxyguanosine in the catalytic reaction solution was measured by HPLC and found to be 27.6g/L.

Example 6 catalytic production of 2' -deoxyguanosine

(1) The formulation of the slant culture medium and the culture conditions were the same as those in step (1) of example 4; the formula and culture conditions of the first-stage seed culture medium are the same as those in the step (2) in example 4;

(2) Preparing seed liquid in a seeding tank:

the formula of the seed liquid culture medium in the seeding tank is the same as the seed culture medium in the step (2) in the example 4;

putting 10L of seed culture medium into a 15L seeding tank, sterilizing with steam at 121 ℃ for 20min, cooling to 30 ℃, inoculating 200mL of first-stage shake flask seed solution, stirring at 200rpm, ventilating at 1.0vvm, and culturing at 30 +/-1 ℃ for 16 hours, wherein the pH of the seed solution is 7.0-7.4, and the thallus concentration is 12-15% (volume ratio);

(3) Preparation of enzyme fermentation medium and thallus culture:

the formula of the enzyme fermentation culture medium is as follows: 50g/L glucose, 20g/L yeast extract, 15g/L yeast extract powder, 8010g/L tween-tween, 5g/L ammonium citrate, 5g/L magnesium sulfate, 8g/L potassium dihydrogen phosphate, 5g/L potassium chloride and 5g/L calcium chloride, and the pH value is adjusted to 7.0 before sterilization.

The volume of the fermentation tank is 50L, the feeding volume is 30L, the fermentation tank is sterilized by steam, the temperature is 121 ℃, the fermentation tank is used for 20min, 3L seed solution in the seed tank is inoculated after the fermentation tank is cooled to 30 ℃, the stirring speed is 300-600rpm (the rotation speed is gradually increased from 300rpm to 600rpm in the first 3 days), the ventilation volume is 2.0vvm, and the fermentation tank is cultured for 24 hours at the temperature of 30 ℃.

(4) Catalytic reaction of 2' -deoxyguanosine:

after the fermentation of the citric acid bacillus without malonic acid on a tank is finished, 0.5mol of each of disodium hydrogen phosphate and sodium dihydrogen phosphate is directly added into fermentation liquor in the tank, the temperature is raised to 70 ℃, then 40g/L of deoxythymidine and 50g/L of guanosine are added, the stirring reaction is finished after 5 hours, the content of 2' -deoxyguanosine in the catalytic reaction liquid is detected by an HPLC method, and the content is 33.5g/L.

Claims (19)

1. A Citrobacter alamocarpus-free DHE005 is preserved in China general microbiological culture collection center (CGMCC), with the preservation number of CGMCC NO. 245123 and the preservation date of 2022 years, 03 months and 11 days.

2. A fermentation broth comprising Citrobacter malonate free (Citrobacter alamonaticus) DHE005 of claim 1.

3. The application of Citrobacter acidi (Citrobacter ammoniagenes) or seed liquid thereof, or fermentation liquid thereof, or bacterial suspension thereof, or culture liquid thereof in the production of purine nucleotide phosphorylase or products containing purine nucleotide phosphorylase;

or the application of the Citrobacter acidi alatus (Citrobacter alamionicus) or the seed liquid thereof, the fermentation liquid thereof, the bacterial suspension thereof or the culture liquid thereof in the preparation of 2'-deoxyguanosine or the pharmaceutical composition containing 2' -deoxyguanosine.

4. Use according to claim 3, characterized in that: the Citrobacter malonodicus (Citrobacter alamionicus) DHE005 according to claim 1.

5. A method for preparing purine nucleotide phosphorylase, which is characterized in that: comprises adopting citric acid bacillus without malonate (Citrobacter ammonianatrii) to carry out fermentation preparation.

6. A preparation method of 2' -deoxyguanosine is characterized by comprising the following steps:

comprises fermenting with citric acid-free Bacillus acidi-citri (Citrobacter alamionicus) to obtain fermentation liquid containing purine nucleotide phosphorylase, and adding phosphate, deoxythymidine and guanosine to obtain 2' -deoxyguanosine.

7. The production method according to claim 5 or 6, characterized in that: the fermentation process comprises aerobic fermentation in an enzyme-producing medium containing assimilable carbon and/or nitrogen sources.

8. The method for producing according to claim 7, characterized in that: the carbon source is selected from glucose, maltose, sucrose, glycerol or fructose; preferably glucose;

and/or the nitrogen source is selected from yeast extract powder, yeast extract, soybean lecithin, corn steep liquor dry powder, soybean meal, peptone and urea; preferably yeast extract or yeast extract powder.

9. The method of claim 7, wherein: the enzyme production culture medium also comprises a surfactant, wherein the surfactant is selected from sodium dodecyl sulfate, tween-20 and tween-80, and preferably tween-80.

10. The method of claim 7, wherein: the enzyme production culture medium also comprises inorganic salt, wherein the inorganic salt is selected from ammonium citrate, monopotassium 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, and preferably ammonium citrate, magnesium sulfate, monopotassium phosphate, potassium chloride or calcium chloride.

11. The method of claim 7, wherein: the enzyme production culture medium contains 10-50g/L of glucose, 5-20g/L of yeast extract, 5-15g/L of yeast extract powder, 2-10g/L of tween-80, 2-5g/L of ammonium citrate, 1-5g/L of magnesium sulfate, 2-8g/L of monopotassium phosphate, 1-5g/L of potassium chloride and 1-5g/L of calcium chloride.

12. The production method according to claim 5 or 6, characterized in that: the fermentation temperature is 25-37 ℃, and the pH value of the culture medium is 5.0-7.0; the culture time is 24-72 hours; the oxygen introduction amount is 0.5-2.0vvm.

13. The method for producing according to claim 7, characterized in that: the citric acid bacillus malonate free is inoculated to the enzyme fermentation medium through seed liquid to carry out fermentation culture;

wherein the seed solution is obtained by seed culture of citric acid bacillus without malonate in a seed culture medium;

and/or the conditions of the seed culture are as follows: the temperature of seed culture is 25-37 ℃; the pH value of the culture medium is 5.0-7.0; the culture time is 12-36 hours;

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

14. The method of claim 6, wherein: the phosphate is disodium hydrogen phosphate and sodium dihydrogen phosphate, the addition ratio of the two is 0.05-0.5mol/L, and the addition amount of the substances such as the disodium hydrogen phosphate and the sodium dihydrogen phosphate is preferably selected.

15. The method of claim 6, wherein: the initial final concentration of the deoxythymidine is 20-40g/L, and the initial final concentration of the guanosine is 25-50g/L.

16. The method of claim 6, wherein: the reaction temperature is 40-70 ℃, and the reaction time is 0.5-5 h.

17. The production method according to any one of claims 5 to 16, characterized in that: the Citrobacter malonodicus (Citrobacter alamionicus) DHE005 according to claim 1.

18. A purine nucleotide phosphorylase prepared by the method of claim 5.

19. 2' -deoxyguanosine prepared by the method of claim 6.

Images