CN113717886A - Bacillus coagulans and method for producing 2' -deoxyadenosine by catalyzing same - Google Patents
Bacillus coagulans and method for producing 2' -deoxyadenosine by catalyzing same Download PDFInfo
- Publication number
- CN113717886A CN113717886A CN202110995758.3A CN202110995758A CN113717886A CN 113717886 A CN113717886 A CN 113717886A CN 202110995758 A CN202110995758 A CN 202110995758A CN 113717886 A CN113717886 A CN 113717886A
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- China
- Prior art keywords
- bacillus coagulans
- fermentation
- culture
- deoxyadenosine
- culture medium
- Prior art date
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Abstract
The invention discloses Bacillus coagulans (Bacillus coagulans) DHE002 with the preservation number of CGMCC NO. 23118; meanwhile, discloses a method for catalytically producing 2' -deoxyadenosine by fermenting and culturing Bacillus coagulans (DHE 002) as an enzyme source.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a method for producing 2' -deoxyadenosine by utilizing bacillus coagulans and catalysis of the bacillus coagulans.
Background
2 '-Deoxyadenosine (CAS No: 958-09-8), having the name of 2' -Deoxydeinosine, is a structural fragment of biological macromolecular deoxyribonucleic acid (DNA) essential for the development and normal operation of organisms, and the chemical structural formula of the structural fragment is shown as formula 1.
2' -deoxyadenosine is an important genetic material component, participates 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. In the field of drug research and development, research finds that 2' -deoxyadenosine has good physiological activity, is an important raw material in the field of gene drugs, and can be used as an intermediate to generate drugs for resisting viruses, tumors, AIDS and the like. For example, dideoxyadenosine (2 ', 3' -dideoxyadenosine) and dideoxyinosine (2 ', 3' -dideoxyinosine) prepared from 2 '-deoxyadenosine have high activity against HIV virus, and 2' -fluoro-2 '-deoxyadenosine and cladribine (2' -chloro-2 '-deoxyadenosine) prepared from 2' -deoxyadenosine have excellent antitumor effects. In addition, with the rapid spread of 2019 novel coronavirus pneumonia COVID-19, the demand of 2' -deoxyadenosine as a precursor material which is important in a virus detection kit is also rapidly increasing.
At present, the methods available for preparing 2' -deoxyadenosine are mainly chemical synthesis methods and biological enzyme catalysis methods. Regarding the chemical synthesis method for preparing 2 ' -deoxyadenosine, for example, the synthesis method of 2 ' -deoxyadenosine disclosed in patent CN105884846B is to esterify adenosine, acidylates an acylating agent and an acid-binding agent to obtain an acylate, and then reduces and purifies the obtained acylate to obtain 2 ' -deoxyadenosine, and the final HPLC purity can reach more than 99%. A plurality of patents have been reported for preparing 2 '-deoxyadenosine by a biological enzyme catalysis method, for example, patent CN101575630A discloses a method for producing deoxyadenosine by a biotransformation method, the highest transformation rate can reach more than 80 percent by culturing Lactobacillus helveticus as an enzyme source to catalyze the synthesis of 2' -deoxyadenosine; also disclosed in patent CN104178541B is a method for producing 2' -deoxyadenosine by transformation using Escherichia coli, which is synthesized by mixing a bacterial cell of Escherichia coli having a high nucleoside phosphorylase activity as an enzyme source with deoxythymidine and adenine as substrates for the transformation reaction to carry out the transformation reaction; in addition, patent CN 111500659B also discloses a method for preparing 2 '-deoxyadenosine pure product by enzyme catalysis, which uses recombinant escherichia coli to obtain N-deoxyribotransferase to catalyze the synthesis of 2' -deoxyadenosine, and the conversion rate can reach more than 85%. The above patents all disclose methods for preparing 2' -deoxyadenosine by bio-enzyme catalysis, but the bacterium used in patent CN101575630A is lactobacillus helveticus, which belongs to a facultative anaerobic bacterium, and the bacterium is difficult to obtain under conventional culture conditions, and the bacterium needs to be centrifuged and subjected to ultrasonic disruption treatment before catalytic reaction, so the process is complex and not beneficial to industrial application; in patent CN104178541B, the substrate concentration in the catalytic reaction is only 3-5mmol, the adenine conversion rate is only above 72.5%, and the substrate concentration and the substrate conversion rate in the method are both low, so that the method is not suitable for industrial application; in patent CN 111500659B, the recombinant Escherichia coli strain needs to add an expensive inducer in the culture process to induce the synthesis of N-deoxyribose transferase, in addition, as heterologous expression protein, recombinant bacteria have protein folding unsuccessfully when expressing N-deoxyribose transferase, the problem of inclusion bodies is easy to generate, and the above disclosed enzyme catalysis patents need to carry out centrifugal treatment on the bacteria, most of the bacteria need to be subjected to wall breaking treatment to obtain corresponding catalytic enzyme for subsequent catalytic reaction, the process obviously increases the process steps, the production cost is increased, and therefore the industrial application is not facilitated.
Disclosure of Invention
Based on the defects of the technology, one of the purposes of the invention is to provide Bacillus coagulans (Bacillus coagulans) DHE002, which is preserved in China general microbiological culture Collection center (CGMCC), and the address: the No. 3 Xilu-1 of Beijing, Chaoyang, the institute for microbiology, China academy of sciences, with the preservation number of CGMCC NO.23118 and the preservation date of 2021 year, 8 months and 5 days, was registered in a book to prove survival.
Another purpose of the invention is to provide a new application of Bacillus coagulans (Bacillus coagulans) or seed liquid thereof, or fermentation liquid thereof, or bacterial suspension thereof, or culture liquid thereof.
The invention provides application of Bacillus coagulans (Bacillus coagulans) or seed liquid thereof, or fermentation liquid thereof, or bacterial suspension thereof, or culture liquid thereof in production of purine nucleotide phosphorylase or products containing purine nucleotide phosphorylase.
The invention provides application of Bacillus coagulans (Bacillus coagulans) or seed liquid thereof, or fermentation liquid thereof, or bacterial suspension thereof, or culture liquid thereof in preparation of 2 '-deoxyadenosine or a pharmaceutical composition containing 2' -deoxyadenosine.
As an embodiment, the Bacillus coagulans in the above application is Bacillus coagulans DHE 002.
The invention also aims to provide a preparation method of purine nucleotide phosphorylase, which comprises the step of preparing by fermenting Bacillus coagulans.
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 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, 802-10 g/L of tween-802, 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 0.5-2.0 vvm.
Further, more specifically, the bacillus coagulans is inoculated to the enzyme fermentation culture medium through a seed solution for the fermentation culture;
wherein the seed solution is obtained by seed culture of Bacillus coagulans (Bacillus coagulans) 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.
As a specific embodiment, the Bacillus coagulans (Bacillus coagulans) in the preparation method of purine nucleotide phosphorylase is Bacillus coagulans (Bacillus coagulans) DHE 002.
Still another object of the present invention is to provide a method for preparing 2' -deoxyadenosine.
The preparation method of 2 '-deoxyadenosine comprises the steps of fermenting Bacillus coagulans (Bacillus coagulans) DHE002 according to claim 1 to obtain fermentation liquor containing purine nucleotide phosphorylase, and adding phosphate, deoxythymidine and adenine into the fermentation liquor to obtain the 2' -deoxyadenosine.
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 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, 802-10 g/L of tween-802, 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 0.5-2.0 vvm.
Further, more specifically, the bacillus coagulans is inoculated to the enzyme fermentation culture medium through a seed solution for the fermentation culture;
wherein the seed solution is obtained by seed culture of Bacillus coagulans (Bacillus coagulans) 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 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 60-100g/L and the initial final concentration of adenine is 30-50 g/L.
As an embodiment, the Bacillus coagulans (Bacillus coagulans) described in the above preparation method of purine nucleotide phosphorylase is Bacillus coagulans (Bacillus coagulans) DHE 002.
The invention carries out HPLC detection by the following conditions:
the conditions of the liquid phase detection method used for detecting the 2' -deoxyadenosine are as follows:
a chromatographic column: inertsil ODS-3(150x 4.6mm,5um) (Shimadzu)
The detection wavelength is 254 nm; column temperature: 30 ℃; the flow rate is 1.0 ml/min; the sample injection volume is 5 ul; mobile phase A: 20mmol of ammonium dihydrogen phosphate aqueous solution; mobile phase B: methanol;
elution procedure:
the invention discloses a brand-new Bacillus coagulans (Bacillus coagulans) DHE002 with the preservation number of CGMCC NO.23118, the strain has extremely high nucleoside phosphorylase activity, and after the strain is cultured in an enzyme fermentation culture medium, the strain can be directly fed into the culture medium containing the strain for catalytic reaction.
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 DHE002(CGMCC NO.23118) on beef extract peptone solid medium.
FIG. 2 is a 2' -deoxyadenosine chromatogram detected by HPLC after a substrate is catalyzed by strain DHE002(CGMCC NO. 23118).
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 all common commercially available products and are commercially available unless otherwise specified.
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
Bacillus coagulans DHE002(CGMCC NO.23118) is separated from soil in Tiantaishan mountain slope of Taizhou city of Taizhou province of Zhejiang province.
Cross sampling is carried out on the soil in the Tiantai mountain area, 5 sampling points are randomly selected, 10g of soil sample is taken at each point, the soil samples are put into a conical flask, 10g of soil sample is taken after uniform mixing, the soil samples are added into the conical flask filled with 90mL of sterile water (a magnetic stirrer is arranged in the conical flask), vortex stirring is carried out for 30 minutes, the soil samples are fully and uniformly mixed to prepare suspension, namely 10-1And (4) bacterial suspension. Mixing the suspension with sterile water according to a volume ratio of 1: 9 to 10-2，10-3，10-4，10-5And (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 ring of the slant culture thallus is picked by an inoculating loop, the thallus is respectively inoculated in a 250mL conical flask containing 20mL of seed culture medium, after shaking culture is carried out for 1 day at the temperature of 28 ℃, 1mL of the thallus is further sucked and transplanted in a 250mL conical flask containing 20mL of fermentation culture medium, after shaking culture is carried out for 3 days at the temperature of 30 ℃, wet thallus is obtained by centrifugation, detection is carried out by a Purine Nucleotide Phosphorylase (PNP) ELISA kit (Shanghai double win biotechnology, Inc.), and a strain containing the purine nucleotide phosphorylase, namely Bacillus coagulans DHE002(CGMCC NO.23118) 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, and adding water to a constant volume of 1000mL and pH of 7.0 +/-0.1.
Fermentation medium formula (g/L): 50g/L of glucose, 5g/L of yeast extract powder, 10g/L of yeast extract and 30g/L of calcium carbonate, and adding water to a constant volume of 1000mL, wherein the pH value is 7.0 +/-0.1.
Example 2: morphology, cultural characteristics, physiological and biochemical characteristics of Bacillus coagulans DHE002(CGMCC NO. 23118).
The characteristics of the bacterial colony of the strain DHE002 cultured in a beef extract peptone solid medium at 30 ℃ for 24h are shown in figure 1, the bacterial colony is flat, milky, convex and opaque, shows dryness and has consistent front and back 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 reactions of Bacillus coagulans DHE002(CGMCC NO.23118)
Remarking: +: positive; -: and (4) negativity.
Example 3 species identification
The 16S rDNA sequence analysis of Bacillus coagulans DHE002(CGMCC NO.23118) is tested according to the contents in the book 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') and Revese primer 1492R (5'-GGTTACCTTGTTACGACTT-3') are subjected to PCR amplification, 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 biological engineering (Shanghai) corporation for sequence determination.
The 16S rDNA sequence measured by strain DHE002(CGMCC NO.23118) is collated and then is compared with homologous sequence BLAST in related species and genus sequences in GenBank database to determine the classification status of the strain.
The 16S rDNA sequence of strain DHE002(CGMCC NO.23118) was subjected to BLAST comparison with related sequences in GenBank, and the results are shown in Table 2 (only the model strains with higher homology are listed in the table).
TABLE 2 homology of the Strain DHE002(CGMCC NO.23118) with the model Strain
Through sequencing the 16S rDNA (SEQ ID NO:1) region of the strain DHE002(CGMCC NO.23118) and carrying out BLAST comparison with homologous sequences of related species and genera in a GenBank database, the strain is found to have homology as high as 99.86-100% with Bacillus coagulans (Bacillus coagulans DSM1, Bacillus coagulans strain NBRC 12583, Bacillus coagulans NBRC106567, Bacillus coagulans strain C4 and Bacillus coagulans T3), and meanwhile, the strain DHE002(CGMCC NO.23118) is subjected to an apparent characteristic test, so that the strain is very close to classification related parameters of the Bacillus coagulans (CGMCC NO.23118), and the strain DHE002(CGMCC NO.23118) is identified as the Bacillus coagulans strain.
Example 4 catalytic production of 2' -deoxyadenosine
(1) Preparing and culturing slant strains:
slant culture medium formula (g/L): 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 with the specification is filled with 50mL and sterilized at 121 ℃ for 20 min. 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) Preparation of enzyme fermentation medium and thallus culture:
the formula of the enzyme fermentation culture medium is as follows: 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 with the specification is filled with 20mL and sterilized at 121 ℃ for 20 min. The seed liquid was inoculated at an inoculum size of 10% (by volume). The culture was carried out at 25. + -. 1 ℃ for 72 hours with shaking at 250 rpm.
(4) Catalytic reaction of 2' -deoxyadenosine:
after the fermentation of the bacillus coagulans is finished, 0.05mol of each of disodium hydrogen phosphate and sodium dihydrogen phosphate is added into the fermentation liquor, the temperature is raised to 60 ℃, then 60g/L of deoxythymidine and 30g/L of adenine are added, the reaction is finished after stirring for 3h, and the content of 2' -deoxyadenosine in the catalytic reaction liquor is detected by an HPLC method and is measured to be 52 g/L.
Example 5 catalytic production of 2' -deoxyadenosine
(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 formula of the enzyme fermentation culture medium is as follows: 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 with the specification is filled with 20mL and sterilized at 121 ℃ for 20 min. The seed liquid was inoculated at an inoculum size of 10% (by volume). The cells were cultured at 37. + -. 1 ℃ for 24 hours with shaking at 250 rpm.
(4) Catalytic reaction of 2' -deoxyadenosine:
after the fermentation of the bacillus coagulans is finished, 0.1mol of each of disodium hydrogen phosphate and sodium dihydrogen phosphate is added into the fermentation liquor, the temperature is raised to 55 ℃, then 80g/L of deoxythymidine and 40g/L of adenine are added, the reaction is finished after 2 hours of stirring, the content of 2' -deoxyadenosine in the catalytic reaction liquor is detected by an HPLC method, and the content is measured to be 67 g/L.
Example 6 catalytic production of 2' -deoxyadenosine
(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 primary seed culture medium are the same as those in step (2) of 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 by steam at 121 ℃ for 20min, cooling to 30 ℃, inoculating 200mL of first-stage shake flask seed solution, stirring at 200rpm, ventilating at 1.0vvm, 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 seeding 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 is carried out for 24 hours at the temperature of 30 ℃.
(4) Catalytic reaction of 2' -deoxyadenosine:
after fermentation on a bacillus coagulans 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 45 ℃, then 100g/L of deoxythymidine and 50g/L of adenine are added, stirring reaction is finished after 1h, and the content of 2' -deoxyadenosine in catalytic reaction liquid is detected by an HPLC method to obtain 75 g/L.
Sequence listing
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gtaacggccc accaaggcaa cgatgcgtag ccgacctgag agggtgatcg gccacattgg 300
gactgagaca cggcccaaac tcctacggga ggcagcagta gggaatcttc cgcaatggac 360
gaaagtctga cggagcaacg ccgcgtgagt gaagaaggcc ttcgggtcgt aaaactctgt 420
tgccggggaa gaacaagtgc cgttcgaaca gggcggcgcc ttgacggtac ccggccagaa 480
agccacggct aactacgtgc cagcagccgc ggtaatacgt aggtggcaag cgttgtccgg 540
aattattggg cgtaaagcgc gcgcaggcgg cttcttaagt ctgatgtgaa atcttgcggc 600
tcaaccgcaa gcggtcattg gaaactggga ggcttgagtg cagaagagga gagtggaatt 660
ccacgtgtag cggtgaaatg cgtagagatg tggaggaaca ccagtggcga aggcggctct 720
ctggtctgta actgacgctg aggcgcgaaa gcgtggggag caaacaggat tagataccct 780
ggtagtccac gccgtaaacg atgagtgcta agtgttagag ggtttccgcc ctttagtgct 840
gcagctaacg cattaagcac tccgcctggg gagtacggcc gcaaggctga aactcaaagg 900
aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgaagc aacgcgaaga 960
accttaccag gtcttgacat cctctgacct ccctggagac agggccttcc ccttcggggg 1020
acagagtgac aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt 1080
cccgcaacga gcgcaaccct tgaccttagt tgccagcatt gagttgggca ctctaaggtg 1140
actgccggtg acaaaccgga ggaaggtggg gatgacgtca aatcatcatg ccccttatga 1200
cctgggctac acacgtgcta caatggatgg tacaaagggc tgcgagaccg cgaggttaag 1260
ccaatcccag aaaaccattc ccagttcgga ttgcaggctg caacccgcct gcatgaagcc 1320
ggaatcgcta gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg gccttgtaca 1380
caccgcccgt cacaccacga gagtttgtaa cacccgaagt cggtgaggta acctttacgg 1440
agccagccgc cgaaggtggg acagatgatt ggggtgaag 1479
Claims (17)
1. Bacillus coagulans (DHE 002) is preserved in China general microbiological culture Collection center (CGMCC), with the preservation number of CGMCC NO.23118 and the preservation date of 2021, 8 and 5 days.
2. A fermentation broth comprising Bacillus coagulans DHE002 of claim 1.
3. The application of Bacillus coagulans 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 Bacillus coagulans or seed liquid or fermentation liquid or bacterial suspension or culture liquid thereof in preparing 2 '-deoxyadenosine or the pharmaceutical composition containing the 2' -deoxyadenosine.
4. Use according to claim 3, characterized in that: the Bacillus coagulans (Bacillus coagulans) DHE002 is the Bacillus coagulans (Bacillus coagulans) according to claim 1.
5. A method for producing a purine nucleotide phosphorylase, characterized by comprising: comprises the preparation by fermenting Bacillus coagulans (Bacillus coagulans).
6. A method for preparing 2' -deoxyadenosine is characterized in that:
comprises the steps of fermenting by using Bacillus coagulans (Bacillus coagulans) to obtain fermentation liquor containing purine nucleotide phosphorylase, and adding phosphate, deoxythymidine and adenine into the fermentation liquor to obtain 2' -deoxyadenosine.
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 of claim 7, wherein: 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 is 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, 2-5g/L of tween-802-10 g/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 0.5-2.0 vvm.
13. The method of claim 7, wherein: the bacillus coagulans is inoculated into the enzyme fermentation culture medium through seed liquid for the fermentation culture;
wherein the seed solution is obtained by seed culture of Bacillus coagulans (Bacillus coagulans) in a seed culture medium;
and/or the conditions of the seed culture are: 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 preferable amount of the substances such as the disodium hydrogen phosphate and the sodium dihydrogen phosphate is added.
15. The method of claim 6, wherein: the initial final concentration of the deoxythymidine is 60-100g/L, and the initial final concentration of the adenine is 30-50 g/L.
16. The method of claim 6, wherein: the reaction temperature is 45-60 ℃, and the reaction time is 1-3 h.
17. The method according to any one of claims 5 to 16, wherein: the Bacillus coagulans (Bacillus coagulans) DHE002 is the Bacillus coagulans (Bacillus coagulans) according to claim 1.
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| CN202110995758.3A CN113717886B (en) | 2021-08-27 | 2021-08-27 | Bacillus coagulans and method for producing 2' -deoxyadenosine by catalysis thereof |
| PCT/CN2022/115185 WO2023025292A1 (en) | 2021-08-27 | 2022-08-26 | Bacillus coagulans and method for catalytic production of 2'-deoxyadenosine by using bacillus coagulans |
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| WO2023025292A1 (en) * | 2021-08-27 | 2023-03-02 | 浙江珲达生物科技有限公司 | Bacillus coagulans and method for catalytic production of 2'-deoxyadenosine by using bacillus coagulans |
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| JP2003070494A (en) * | 2001-08-31 | 2003-03-11 | Yuki Gosei Kogyo Co Ltd | Method of producing 2'-deoxynucleoside compound |
| CN1670207A (en) * | 2005-03-04 | 2005-09-21 | 上海秋之友生物科技有限公司 | Immobilized nucleoside phosphorylase microbe and its application in synthesizing purine nucleosidase |
| CN104178541A (en) * | 2013-11-06 | 2014-12-03 | 西藏天虹科技股份有限责任公司 | Method for converting escherichia coli to produce 2'-deoxyadenosine |
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| JP3864357B2 (en) * | 1997-08-04 | 2006-12-27 | 有機合成薬品工業株式会社 | Method for producing purine nucleoside compound |
| JP4058663B2 (en) * | 2000-09-22 | 2008-03-12 | 有機合成薬品工業株式会社 | Method for producing ribose 1-phosphates and nucleoside compounds |
| CN113717886B (en) * | 2021-08-27 | 2023-08-18 | 浙江珲达生物科技有限公司 | Bacillus coagulans and method for producing 2' -deoxyadenosine by catalysis thereof |
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| JP2003070494A (en) * | 2001-08-31 | 2003-03-11 | Yuki Gosei Kogyo Co Ltd | Method of producing 2'-deoxynucleoside compound |
| CN1670207A (en) * | 2005-03-04 | 2005-09-21 | 上海秋之友生物科技有限公司 | Immobilized nucleoside phosphorylase microbe and its application in synthesizing purine nucleosidase |
| CN104178541A (en) * | 2013-11-06 | 2014-12-03 | 西藏天虹科技股份有限责任公司 | Method for converting escherichia coli to produce 2'-deoxyadenosine |
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| WO2023025292A1 (en) * | 2021-08-27 | 2023-03-02 | 浙江珲达生物科技有限公司 | Bacillus coagulans and method for catalytic production of 2'-deoxyadenosine by using bacillus coagulans |
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Denomination of invention: A method for producing 2 '- deoxyadenosine catalyzed by Bacillus coagulans Effective date of registration: 20231202 Granted publication date: 20230818 Pledgee: Hangzhou Bank Co.,Ltd. Huanbei Branch Pledgor: Zhejiang Hunda Biotechnology Co.,Ltd. Registration number: Y2023330002894 |