CN113717886B - Bacillus coagulans and method for producing 2' -deoxyadenosine by catalysis thereof - Google Patents
Bacillus coagulans and method for producing 2' -deoxyadenosine by catalysis thereof Download PDFInfo
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- CN113717886B CN113717886B CN202110995758.3A CN202110995758A CN113717886B CN 113717886 B CN113717886 B CN 113717886B CN 202110995758 A CN202110995758 A CN 202110995758A CN 113717886 B CN113717886 B CN 113717886B
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- Prior art keywords
- bacillus coagulans
- deoxyadenosine
- seed
- fermentation
- culture
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1077—Pentosyltransferases (2.4.2)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/38—Nucleosides
- C12P19/40—Nucleosides having a condensed ring system containing a six-membered ring having two nitrogen atoms in the same ring, e.g. purine nucleosides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/02—Pentosyltransferases (2.4.2)
- C12Y204/02001—Purine-nucleoside phosphorylase (2.4.2.1)
Abstract
The invention discloses bacillus coagulans (Bacillus coagulans) DHE002, the preservation number of which is CGMCC NO.23118; also disclosed is a method for the catalytic production of 2' -deoxyadenosine by fermenting and culturing bacillus coagulans (Bacillus coagulans) DHE002 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 using bacillus coagulans and catalysis thereof.
Background
2' -Deoxyadenosine (CAS No. 958-09-8), named 2' -deoxydenosine, 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 2' -Deoxyadenosine is shown in the formula 1.
2' -deoxyadenosine, which is an important genetic material component, is involved in the transmission of genetic information of biological cells together with other natural nucleosides, and plays a very important role in the growth control of the biological cells. In the field of medicine research and development, researches show that 2' -deoxyadenosine has good physiological activity, and can be used as an important raw material in the field of gene medicine and also as an intermediate to generate antiviral, antitumor, anti-AIDS and other medicines. 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, a novel coronavirus pneumonitis covd-19, the demand for 2' -deoxyadenosine as a precursor material important in a virus detection kit is also rapidly increasing.
At present, the methods available for preparing 2' -deoxyadenosine mainly comprise a chemical synthesis method and a biological enzyme catalysis method. In the synthesis method for preparing 2' -deoxyadenosine by chemical synthesis, as disclosed in patent CN105884846B, adenosine is esterified, an acylating agent and an acid binding agent are acylated to obtain an acylate, and the obtained acylate is reduced and purified to obtain 2' -deoxyadenosine, so that the purity of the 2' -deoxyadenosine can reach more than 99% finally, however, the preparation of 2' -deoxyadenosine by chemical synthesis has long steps, the reaction condition is severe, the final yield is low, various organic reagents are needed in the reaction process, and environmental pollution is obvious, so that the preparation of 2' -deoxyadenosine by chemical synthesis is not suitable for large-scale industrial production. While a plurality of patents report on the preparation of 2'-deoxyadenosine by a biological enzyme catalysis method, for example, patent CN101575630A discloses a method for producing deoxyadenosine by a biological conversion method, which comprises the steps of culturing lactobacillus helveticus as an enzyme source to catalyze the synthesis of 2' -deoxyadenosine, wherein the highest conversion rate can reach more than 80%; also disclosed in patent CN104178541B is a method for producing 2'-deoxyadenosine by conversion using escherichia coli, which synthesizes 2' -deoxyadenosine by directly using escherichia coli cells having high nucleoside phosphorylase activity as an enzyme source, mixing it with deoxythymidine and adenine as conversion substrates, and performing a conversion reaction; in addition, patent CN 111500659B also discloses a method for preparing 2'-deoxyadenosine pure product by utilizing enzyme catalysis, which utilizes 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 a method for preparing 2' -deoxyadenosine by biological enzyme catalysis, however, the bacterium used in the patent CN101575630A is Lactobacillus helveticus, belongs to a facultative anaerobic bacterium, is difficult to obtain thalli under the conventional culture condition, and requires centrifugation thalli and ultrasonic crushing treatment on the thalli before catalytic reaction, so that the process is complex and is not beneficial to industrial application; in the patent CN104178541B, the substrate concentration in the catalytic reaction is only 3-5mmol, the adenine conversion rate is only 72.5% or more, and the substrate concentration and the substrate conversion rate in the method are low and are not suitable for industrial application; in the patent CN 111500659B, the recombinant escherichia coli strain needs to be added with an expensive inducer in the culture process to induce the synthesis of the N-deoxyribose transferase, in addition, as a heterologous expression protein, recombinant bacteria have the problems of unsuccessful protein folding and easy inclusion body generation when expressing the N-deoxyribose transferase, and the above disclosed enzyme catalysis patents all need to carry out centrifugal treatment on thalli, and most of the thalli need to carry out wall breaking treatment to obtain corresponding catalytic enzyme for carrying out subsequent catalytic reaction.
Disclosure of Invention
Based on the shortcomings 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), address: the collection number is CGMCC No.23118, the collection date is 2021, 8 and 5 days, and the collection is registered in a book to prove survival.
The invention also aims at providing a novel 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 or fermentation liquid or bacterial suspension or culture liquid thereof in producing purine nucleotide phosphorylase or products containing the 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 preparing 2'-deoxyadenosine or a pharmaceutical composition containing 2' -deoxyadenosine.
As one embodiment, the Bacillus coagulans (Bacillus coagulans) described in the above application is the Bacillus coagulans (Bacillus coagulans) DHE002 described above.
It is still another object of the present invention to provide a method for producing purine nucleotide phosphorylase, comprising fermentation production using Bacillus coagulans (Bacillus coagulans).
Further, the fermentation process comprises performing aerobic fermentation in an enzyme-producing medium comprising an assimilable carbon source 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 a yeast extract or a yeast extract powder.
Further, the enzyme-producing medium further comprises a surfactant selected from sodium dodecyl sulfate, tween-20 and tween-80, preferably tween-80.
Further, the enzyme-producing medium further comprises an inorganic salt selected from the group consisting of 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, manganese sulfate, preferably ammonium citrate, magnesium sulfate, potassium dihydrogen phosphate, potassium chloride or calcium chloride.
Further, the enzyme-producing culture medium contains 10-50g/L glucose, 5-20g/L yeast extract, 5-15g/L yeast extract powder, 2-10g/L tween-80, 2-5g/L ammonium citrate, 1-5g/L magnesium sulfate, 2-8g/L potassium dihydrogen phosphate, 1-5g/L potassium chloride and 1-5g/L calcium chloride.
Further, the fermentation temperature is 25-37 ℃, and the pH of the culture medium is 5.0-7.0; the culture time is 24-72 hours; the oxygen flow is 0.5-2.0vvm.
Further, more specifically, the bacillus coagulans is inoculated into the enzyme fermentation medium through seed liquid to perform the fermentation culture;
wherein the seed liquid is obtained by seed culturing 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 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 a specific embodiment, the Bacillus coagulans (Bacillus coagulans) used in the method for producing a purine nucleotide phosphorylase is the Bacillus coagulans (Bacillus coagulans) DHE002 described above.
The invention also aims at providing a preparation method of the 2' -deoxyadenosine.
The preparation method of the 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 performing aerobic fermentation in an enzyme-producing medium comprising an assimilable carbon source 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 or urea; preferably a yeast extract or a yeast extract powder.
Further, the enzyme-producing medium further comprises a surfactant selected from sodium dodecyl sulfate, tween-20 and tween-80, preferably tween-80.
Further, the enzyme-producing medium further comprises an inorganic salt selected from the group consisting of 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, manganese sulfate, preferably ammonium citrate, magnesium sulfate, potassium dihydrogen phosphate, potassium chloride or calcium chloride.
Further, the enzyme-producing culture medium contains 10-50g/L glucose, 5-20g/L yeast extract, 5-15g/L yeast extract powder, 2-10g/L tween-80, 2-5g/L ammonium citrate, 1-5g/L magnesium sulfate, 2-8g/L potassium dihydrogen phosphate, 1-5g/L potassium chloride and 1-5g/L calcium chloride.
Further, the fermentation temperature is 25-37 ℃, and the pH of the culture medium is 5.0-7.0; the culture time is 24-72 hours; the oxygen flow is 0.5-2.0vvm.
Further, more specifically, the bacillus coagulans is inoculated into the enzyme fermentation medium through seed liquid to perform the fermentation culture;
wherein the seed liquid is obtained by seed culturing 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 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 one embodiment, the phosphate is disodium hydrogen phosphate and sodium dihydrogen phosphate, and the addition ratio of the disodium hydrogen phosphate and the sodium dihydrogen phosphate is 0.05-0.5mol/L, and the preferable addition amount of the disodium hydrogen phosphate, the sodium dihydrogen phosphate and the like is the addition amount.
As one embodiment, the initial final concentration of deoxythymidine is 60-100g/L and the initial final concentration of adenine is 30-50g/L.
In one embodiment, the Bacillus coagulans (Bacillus coagulans) used in the method for producing a purine nucleotide phosphorylase is Bacillus coagulans (Bacillus coagulans) DHE002.
The invention performs HPLC detection by the following conditions:
the liquid phase detection method for detecting the 2' -deoxyadenosine comprises the following conditions:
chromatographic column: inertsil ODS-3 (150 x 4.6mm,5 um) (Shimadzu)
The detection wavelength is 254nm; column temperature: 30 ℃; the flow rate is 1.0ml/min; sample injection volume is 5ul; mobile phase a:20mmol of monoammonium phosphate aqueous solution; mobile phase B: methanol;
elution procedure:
the invention discloses a brand new bacillus coagulans (Bacillus coagulans) DHE002, the preservation number is CGMCC NO.23118, 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 the medium containing thalli for catalytic reaction, and compared with other disclosed technologies, the invention can directly feed into the medium containing thalli for catalytic reaction, does not need to carry out centrifugal enrichment on thalli, does not need to carry out wall breaking treatment on thalli, remarkably reduces the catalytic process, and the highest concentration of 2' -deoxyadenosine of a product can reach 75g/L.
In conclusion, compared with the traditional process, the technology has the series of 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 graph showing colony characteristics of strain DHE002 (CGMCC No. 23118) on a solid medium of beef extract peptone.
FIG. 2 shows the 2' -deoxyadenosine profile detected by HPLC after the catalytic reaction of the strain DHE002 (CGMCC No. 23118) on the substrate.
Detailed Description
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents, and the like used in the following examples are commercially available products unless otherwise specified.
The invention is further described below in connection with specific examples, it being understood that the following examples are presented by way of illustration only and are not intended to limit the scope of the invention.
Example 1: bacterial strain origin
Bacillus coagulans (Bacillus coagulans) DHE002 (CGMCC No. 23118) is isolated from soil of Tiantaishan mountain hillside in Taizhou, zhejiang province.
Cross sampling soil in Tiantaishan region, randomly taking 5 sampling points, taking 10g of soil sample from each point, placing into a conical flask, mixing uniformly, adding 10g of sample into a conical flask (with a magnetic stirrer in the flask) filled with 90mL of sterile water, and stirring with vortex for 30 min to obtain suspension 10 -1 A bacterial suspension. The suspension was diluted with sterile water in a volume ratio of 1:9 dilute to 10 -2 ，10 -3 ，10 -4 ，10 -5 The concentration of the bacterial suspension with different dilution factors is 0.1mL, the bacterial suspension is coated on a beef extract peptone agar culture medium plate, the bacterial suspension is lightly coated on the surface of the culture medium by a sterile coating rod, and the bacterial suspension is placed at room temperature for 30 minutes and then placed in a constant temperature incubator at 25 ℃. After the colonies grow out, the color, transparency, surface and edge morphology of the colonies are observed and recorded. Finally 1000 strains are selected and inoculated onBeef extract peptone agar medium is made into inclined plane, and fermentation and purine nucleoside phosphorylase activity analysis are carried out. Picking up one loop of the slant culture thallus with an inoculating loop, inoculating into 250mL conical flasks containing 20mL seed culture medium, shake culturing at 28deg.C for 1 day, then sucking 1mL, shake culturing at 30deg.C for 3 days in 250mL conical flasks containing 20mL fermentation culture medium, centrifuging to obtain wet thallus, detecting with Purine Nucleotide Phosphorylase (PNP) ELISA kit (Shanghai win-win biotechnology Co., ltd.), and selecting Bacillus coagulans (Bacillus coagulans) DHE002 (CGMCC NO. 23118) containing purine nucleotide phosphorylase strain.
Beef extract peptone solid medium (g/L): beef extract 3.0g/L, peptone 10.0g/L, naCl 5.0g/L, distilled water 1000mL, agar 20g/L, pH 7.0.
Seed culture 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 fix the volume to 1000mL, wherein the pH value is 7.0+/-0.1.
Fermentation medium formulation (g/L): glucose 50g/L, yeast extract powder 5g/L, yeast powder 10g/L, yeast extract 10g/L, calcium carbonate 30g/L, and water to 1000mL, pH 7.0+ -0.1.
Example 2: morphological, cultural, physiological and biochemical characteristics of bacillus coagulans (Bacillus coagulans) DHE002 (CGMCC No. 23118).
The colony characteristics of the strain DHE002 after being cultured for 24 hours in the beef extract peptone solid medium at 30 ℃ are shown in figure 1, and the colony is flat, milky white, convex and opaque, and shows that the color of the front side and the back side is consistent. The gram staining of the bacterial cells was positive, and the bacterial cells were observed under a microscope to be rod-shaped, and some bacterial cells had spores.
The physiological reactions of the strains are shown in Table 1, referring to the identification according to the relevant contents in the books of Bojg's Manual of bacteriology of the system, manual of identification of the common bacterial System, etc.
TABLE 1 physiological reaction of Bacillus coagulans (Bacillus coagulans) DHE002 (CGMCC No. 23118)
Remarks: +: positive; -: negative.
Example 3 identification of species
The 16S rDNA sequence analysis of Bacillus coagulans (Bacillus coagulans) DHE002 (CGMCC No. 23118) was performed with reference to the relevant content in the book of the molecular cloning Experimental guidelines. The bacterial cells were collected and then the total DNA was extracted with a bacterial DNA extraction kit.
Designing a primer: forward primer27F (5'-AGAGTTTGATCCTGGCTCAG-3'), reverse primer 1492R (5'-GGTTACCTTGTTACGACTT-3', PCR amplification, PCR product detection using 0.8% agarose gel electrophoresis, PCR product purification recovery using SanPrep column PCR purification product kit, purified PCR product directly sent to biological engineering (Shanghai) stock company for sequence determination.
The sequence of the 16S rDNA detected by the strain DHE002 (CGMCC No. 23118) is compared with the sequence of related species and genus in the GenBank database by the homologous sequence BLAST so as to determine the classification status of the strain.
The 16S rDNA sequence obtained by the strain DHE002 (CGMCC No. 23118) is submitted to NCBI and BLAST comparison is carried out on related sequences in GenBank, and the result is shown in Table 2 (only the mode strains with higher homology are listed in the table).
TABLE 2 homology of strain DHE002 (CGMCC No. 23118) and typical model strain
By sequencing the region of the strain DHE002 (CGMCC No. 23118) 16S rDNA (SEQ ID No. 1), and comparing with the sequences of related species and genus in GenBank database by homologous sequence BLAST, the homology with bacillus coagulans (Bacillus coagulans DSM1, bacillus coagulans strain NBRC 12583, bacillus coagulans NBRC106567, bacillus coagulans strain C4 and Bacillus coagulansT 3) is found to be up to 99.86% -100%, and meanwhile, the apparent characteristic test is carried out on the strain DHE002 (CGMCC No. 23118), and the classification related parameters of the strain and bacillus coagulans (Bacillus coagulans) are found to be very close, so the strain DHE002 (CGMCC No. 23118) is identified as bacillus coagulans (Bacillus coagulans) strain.
Example 4 catalytic production of 2' -deoxyadenosine
(1) Preparing and culturing inclined plane strains:
slant culture 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, pH 7.2-7.4 before digestion, 30X 200mm of test tube and 15mL of loading, sterilizing at 121 ℃ for 20min, cooling to about 55-60 ℃, placing an inclined plane, inoculating to the inclined plane after cooling and solidifying, and culturing for 3 days at 30+/-1 ℃ to obtain the strain.
(2) Preparing and culturing seed liquid:
the formula of the seed culture medium comprises: glucose 30g/L, peptone 10g/L, yeast extract 5g/L, calcium chloride 2g/L, magnesium sulfate 2g/L, and potassium dihydrogen phosphate 5g/L. Pre-digestion pH 7.0;250mL triangular shake flask, 50mL,121 ℃ sterilization 20min. Inoculating 1-2 slant single colonies into seed culture medium, and shake culturing at 30+ -1deg.C and 250rpm for 24 hr, wherein pH of culture solution is 6.8-7.2, and thallus OD600 is 10-20.
(3) Preparation of enzyme fermentation medium and thallus culture:
the formula of the enzyme fermentation medium comprises: 10g/L of glucose, 5g/L of yeast extract powder, 2g/L of tween-802, 1g/L of magnesium sulfate, 2g/L of monopotassium phosphate, 1g/L of potassium chloride and 1g/L of calcium chloride, and adjusting the pH to 5.0 before sterilization. 250mL triangular shake flask, 20mL,121 ℃ sterilization 20min. The seed solution was inoculated at an inoculum size of 10% (volume ratio). The culture was carried out at 25.+ -. 1 ℃ and 250rpm for 72 hours.
(4) Catalytic reaction of 2' -deoxyadenosine:
after the Bacillus coagulans fermentation is finished, adding 0.05mol of disodium hydrogen phosphate and 0.05mol of sodium dihydrogen phosphate into the fermentation liquid, heating to 60 ℃, then adding 60g/L of deoxythymidine and 30g/L of adenine, stirring and reacting for 3 hours, and detecting the content of 2' -deoxyadenosine in the catalytic reaction liquid by an HPLC method to obtain 52g/L.
Example 5 catalytic production of 2' -deoxyadenosine
(1) The slant culture medium formulation and culture conditions were the same as in step (1) of example 4;
(2) The formula and culture conditions of the seed culture medium are the same as in the step (2) in the example 4;
(3) Preparation of enzyme fermentation medium and thallus culture:
the formula of the enzyme fermentation medium comprises: 30g/L of glucose, 10g/L of yeast extract powder, 806g/L of tween-4 g/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 adjusting the pH to 6.0 before sterilization. 250mL triangular shake flask, 20mL,121 ℃ sterilization 20min. The seed solution was inoculated at an inoculum size of 10% (volume ratio). The culture was carried out at 37.+ -. 1 ℃ and 250rpm for 24 hours.
(4) Catalytic reaction of 2' -deoxyadenosine:
after the Bacillus coagulans fermentation is finished, adding 0.1mol of disodium hydrogen phosphate and 0.1mol of sodium dihydrogen phosphate into the fermentation liquid, heating to 55 ℃, then adding 80g/L of deoxythymidine and 40g/L of adenine, stirring and reacting for 2 hours, and detecting the content of 2' -deoxyadenosine in the catalytic reaction liquid by an HPLC method to obtain 67g/L.
Example 6 catalytic production of 2' -deoxyadenosine
(1) The slant culture medium formulation and culture conditions were the same as in step (1) of example 4; the formula and culture conditions of the primary seed culture medium are the same as in the step (2) in the example 4;
(2) Preparing seed tank seed liquid:
the formula of the seed liquid culture medium in the seed tank is the same as that of the seed culture medium in the step (2) in the example 4;
putting 10L of seed culture medium into a 15L seed tank, sterilizing with steam, sterilizing at 121deg.C for 20min, cooling to 30deg.C, inoculating 200mL of first-stage shake flask seed solution, stirring at 200rpm, introducing air at 1.0vvm, and culturing at 30+ -1deg.C for 16 hr, wherein the seed solution has pH of 7.0-7.4 and thallus concentration of 12-15% (volume ratio);
(3) Preparation of enzyme fermentation medium and thallus culture:
the formula of the enzyme fermentation medium comprises: 50g/L of glucose, 20g/L of yeast extract, 15g/L of yeast extract powder, 8010g/L of tween-8010, 5g/L of ammonium citrate, 5g/L of magnesium sulfate, 8g/L of monopotassium phosphate, 5g/L of potassium chloride and 5g/L of calcium chloride, and regulating the pH to 7.0 before sterilization.
The volume of the fermentation tank is 50L, the feeding volume is 30L, steam sterilization is carried out, the temperature is 121 ℃ and 20min, 3L of seed tank seed liquid is introduced after the fermentation tank is cooled to 30 ℃, the stirring speed is 300-600rpm (the rotation speed gradually increases from 300rpm to 600rpm in the previous 3 days), the ventilation rate is 2.0vvm, and the fermentation tank is cultured for 24 hours at 30 ℃.
(4) Catalytic reaction of 2' -deoxyadenosine:
after fermentation on the Bacillus coagulans tank is finished, directly adding 0.5mol of disodium hydrogen phosphate and 0.5mol of sodium dihydrogen phosphate into fermentation liquid in the tank, heating to 45 ℃, then adding 100g/L of deoxythymidine and 50g/L of adenine, stirring and reacting for 1h, and detecting the content of 2' -deoxyadenosine in the catalytic reaction liquid by an HPLC method to obtain 75g/L.
Sequence listing
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gacgaacgct ggcggcgtgc ctaatacatg caagtcgtgc ggacctttta aaagcttgct 60
tttaaaaggt tagcggcgga cgggtgagta acacgtgggc aacctgcctg taagactggg 120
ataacgccgg gaaaccgggg ctaataccng atagtttttt cctccgcatg gaggaaaaag 180
gaaaggcggc ttcggctgcc acttacagat gggcccgcgg cgcattagct agttggcggg 240
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 (15)
1. Bacillus coagulans @Bacillus coagulans) DHE002 is preserved in China general microbiological culture Collection center (CGMCC), with preservation number of CGMCC No.23118 and preservation date of 2021, 8 and 5.
2. A method of producing a bacterium comprising the Bacillus coagulans strain of claim 1Bacillus coagulans) Fermentation broth of DHE002.
3. Bacillus coagulans @Bacillus coagulans) Or seed solution or bacterial suspension thereof in the production of purine nucleotide phosphorylase or products containing the same;
or bacillus coagulans @Bacillus coagulans) Or seed solution or bacterial suspension thereof in preparing 2'-deoxyadenosine or pharmaceutical composition containing 2' -deoxyadenosine;
the bacillus coagulans is%Bacillus coagulans) The bacillus coagulans strain according to claim 1Bacillus coagulans）DHE002。
4. A method for preparing purine nucleotide phosphorylase, which is characterized in that: comprising the use of the bacillus coagulans according to claim 1Bacillus coagulans) DHE002 was prepared by fermentation.
5. A preparation method of 2' -deoxyadenosine is characterized in that:
comprising the use of the bacillus coagulans according to claim 1Bacillus coagulans) Fermenting DHE002 to obtain the product containing purineAnd adding phosphate, deoxythymidine and adenine into the fermentation broth of the nucleotide phosphorylase to obtain 2' -deoxyadenosine.
6. The method of claim 4 or 5, wherein: the fermentation process involves aerobic fermentation in an enzyme-producing medium containing an assimilable carbon source and/or nitrogen source.
7. The method of manufacturing according to claim 6, wherein: the carbon source is selected from glucose, maltose, sucrose, glycerol or fructose;
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.
8. The method of manufacturing according to claim 6, wherein: the enzyme-producing culture medium also comprises a surfactant, wherein the surfactant is selected from sodium dodecyl sulfate, tween-20 and tween-80.
9. The method of manufacturing according to claim 6, wherein: the enzyme-producing 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.
10. The method of manufacturing according to claim 6, wherein: the enzyme-producing culture medium contains 10-50g/L glucose, 5-20g/L yeast extract, 5-15g/L yeast extract powder, 2-10g/L tween-80, 2-5g/L ammonium citrate, 1-5g/L magnesium sulfate, 2-8g/L potassium dihydrogen phosphate, 1-5g/L potassium chloride and 1-5g/L calcium chloride.
11. The method of claim 4 or 5, wherein: the fermentation temperature is 25-37 ℃, and the pH of the culture medium is 5.0-7.0; the culture time is 24-72 hours; the oxygen flow is 0.5-2.0vvm.
12. The method of manufacturing according to claim 6, wherein: the bacillus coagulans is inoculated into an enzyme-producing fermentation medium through seed liquid for fermentation culture;
wherein the seed liquid is prepared from bacillus coagulansBacillus coagulans) Seed culture is carried out in a seed culture medium to obtain the seed;
and/or the seed culture conditions are: the temperature of seed culture is 25-37 ℃; the pH 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 glucose, 5-20g/L peptone, 2-10g/L yeast extract, 5-20g/L yeast extract powder, 1-g/L calcium chloride, 1-10g/L magnesium sulfate and 1-10g/L potassium dihydrogen phosphate.
13. The method of manufacturing according to claim 5, wherein: the phosphate is disodium hydrogen phosphate and sodium dihydrogen phosphate, and the adding proportion of the disodium hydrogen phosphate and the sodium dihydrogen phosphate is 0.05-0.5 mol/L.
14. The method according to claim 5, wherein: the initial final concentration of deoxythymidine is 60-100g/L, and the initial final concentration of adenine is 30-50g/L.
15. The method of manufacturing according to claim 5, wherein: the reaction temperature is 45-60 ℃ and the reaction time is 1-3 h.
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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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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (1)
| Title |
|---|
| Synthesis of 2′-deoxyibosylnucleosides using new 2′-deoxyribosyltransferase microorganism producers;J. Fernández-Lucas等;《Enzyme and Microbial Technology》;20071231;第40卷(第5期);第1147-1155页 * |
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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 |