CN102268385A - Arthrobacter for fermentation production of cyclic adenosine monophosphate and application thereof - Google Patents
Arthrobacter for fermentation production of cyclic adenosine monophosphate and application thereof Download PDFInfo
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- CN102268385A CN102268385A CN2010101915156A CN201010191515A CN102268385A CN 102268385 A CN102268385 A CN 102268385A CN 2010101915156 A CN2010101915156 A CN 2010101915156A CN 201010191515 A CN201010191515 A CN 201010191515A CN 102268385 A CN102268385 A CN 102268385A
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- arthrobacter
- fermentation
- cyclic monophosphate
- carbon source
- inorganic salt
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- 238000000855 fermentation Methods 0.000 title claims abstract description 31
- 230000004151 fermentation Effects 0.000 title claims abstract description 31
- 241000186063 Arthrobacter Species 0.000 title claims abstract description 24
- IVOMOUWHDPKRLL-KQYNXXCUSA-N Cyclic adenosine monophosphate Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=CN=C2N)=C2N=C1 IVOMOUWHDPKRLL-KQYNXXCUSA-N 0.000 title abstract 4
- 238000004519 manufacturing process Methods 0.000 title description 6
- -1 cyclic monophosphate Chemical class 0.000 claims description 31
- 238000000034 method Methods 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 15
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- 239000008103 glucose Substances 0.000 claims description 10
- 229910052799 carbon Inorganic materials 0.000 claims description 9
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 8
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- HYMXALLEHXXORK-HTDNVCFESA-N (4ar,6r,7r,7as)-6-(6-aminopurin-9-yl)-2-hydroxy-2-oxo-4a,6,7,7a-tetrahydro-4h-furo[3,2-d][1,3,2]dioxaphosphinin-7-ol;(2r,3r,4r,5s)-6-(methylamino)hexane-1,2,3,4,5-pentol Chemical compound CNC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO.C([C@H]1O2)OP(O)(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=CN=C2N)=C2N=C1 HYMXALLEHXXORK-HTDNVCFESA-N 0.000 description 1
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- ZKHQWZAMYRWXGA-KQYNXXCUSA-N Adenosine triphosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-N 0.000 description 1
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- 229930195725 Mannitol Natural products 0.000 description 1
- 208000009525 Myocarditis Diseases 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- 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
- 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
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/06—Arthrobacter
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides an arthrobacter for producing cyclic adenosine monophosphate by fermentation and application thereof, wherein the preservation number of the arthrobacter is CGMCC No. 3584. The yield of the Arthrobacter A302 strain with the preservation number of CGMCC No.3584 is improved by 3 times compared with the cyclic adenosine monophosphate yield of the original strain, and the performance of the strain for producing the cyclic adenosine monophosphate is kept stable after more than 10 generations of passage.
Description
Technical field
The invention belongs to microbial technology field, relate to a kind of high yield cyclic monophosphate Arthrobacter and application thereof.
Background technology
Cyclic monophosphate is a kind of important substance with physiologically active that extensively exists in the human body, and as intracellular second messenger, it plays an important role to the metabolism and synthetic adjusting of sugar, fat, nucleic acid, protein etc.Be used for the treatment of stenocardia, myocardial infarction, myocarditis and cardiogenic shock clinically; The palpitaition of improving rheumatic heart disease, symptom such as out of breath, uncomfortable in chest are also had certain effect; Can improve the curative effect of acute leukemia, also can be used for the inducer remission of acute leukemia in conjunction with chemotherapy; In addition, senile chronic bronchitis, various hepatitis and psoriatic also there is certain curative effect.Cyclic monophosphate also can be used as pharmaceutical intermediate and prepares dibutyryl cyclic adenosine monophosphate and Meglumine Cyclic Adenylate, improve fat-soluble, thereby more effectively bring into play physiology and pharmacological action.Cyclic monophosphate also can be used for the animal foods additive, and the effect of simulation tethelin promotes growth of animals or poultry under isolated condition, increases high-quality poultry product output.
The production method of cyclic monophosphate has three kinds of chemical synthesis, enzyme process and fermentation methods.Industrialization is both at home and abroad produced and is all adopted chemical synthesis, is raw material with the adenylic acid, adopts the high efficiency separation post to carry out intermediate and separates, and this method solvent loss amount is big, and yield is low, the cost height, and output is little, and environmental pollution is serious.Utilize enzyme process catalysis Triphosaden generation efficiently cyclic monophosphate, but this method only is confined to laboratory stage, also has certain distance from industrialization.
At present, the domestic report that does not also have fermentation method to prepare cyclic monophosphate.Abroad, the graceful grade of mark in 1963 has detected the cyclic monophosphate of 1.1 nanograms/milliliter the earliest in intestinal bacteria Crookes strain cell.1971, certain dialister bacterium No.205 ATCC21376 had been screened in separation such as tor and the number strain belongs to the bacterium of list shape Bacteriaceae, become the resistance mutant strain by chemomorphosis after, can generate cyclic monophosphate more than the 2g/L from glucose direct fermentation.1973, the Suzuki report utilized the rose-colored arthrobacter paraffineus ATCC15584 of hydrocarbon assimilation bacterium bacterial strain, can be carbon source with the n-tetradecane, fermentation accumulation 1.4g/L cyclic monophosphate.
Ion implantation technique has obtained some important research achievements at aspects such as selection by mutation, plant transgene, origin of life and evolution and environmental radiation and human healths as a kind of new technology of biological variety improvement.Wherein, in the research of microorganism mutation breeding, the ion implantation selection by mutation that has been widely used in microbial strains, and obtained good result.Ion implantation and traditional mutation source is compared, except having the energy deposition effect, also have momentum transfer, quality deposition and charge neutralization and exchange effect, be a kind of with physical mutagenesis and chemomorphosis feature set comprehensive mutafacient system, under the situation about can inject at low dosage, cell injury is lighter, influence physiology, the biochemical property of biomass cells consumingly, cause the change of the fundamental unit-base of genetic material, bring out chromosome structure variation (surplus blast, physics, 1997,26 (6): 333-338).Zhao Hongying (Tianjin Science ﹠ Engineering Univ journal, 2001,17 (1): 14-17) N such as grade
+Ion implantation gentamicin produces the ripe spore of bacterium, and the bacterial strain that obtains through screening produces microbiotic ability raising 27.39%.(JOURNAL OF MICROBIOLOGY, 1998,18 (4): 25-28) wait by the ion implantation mutagenesis screening and obtain the stable high yield bacterium of a strain heritability, the yield bacterium that sets out improves 55%～60% to Wang Ji.(the biotechnology journal, 2000 (16): 478-481) etc. the screening of application ion implantation mutagenesis obtains a strain bacterial strain of high-yield peanut tetraenoic acid to Yao Jianming, and is applied to scale operation, and its rate ratio foreign patent is reported high 1 times.
Summary of the invention
Therefore, technical problem to be solved by this invention provides the high yield fermentation of a strain cyclic monophosphate.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
A kind of Arthrobacter that is used for the fermentative production cyclic monophosphate, the deposit number of this Arthrobacter are CGMCC No.3584.
Classification called after Arthrobacter (Arthrobacter sp.) A302 of the Arthrobacter of the high yield cyclic monophosphate of this conduct production bacterial strain, this bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) at present, depositary institution address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number is CGMCC No.3584, and preservation date is on January 18th, 2010.This bacterial strain be by to soil screening to Arthrobacter carry out low energy ion beam and inject, then the bacterial strain after the mutagenesis is transferred and in plate culture medium, cultivate, select single bacterium colony and transfer, thereby after the liquid seed culture medium of carbonaceous sources, nitrogenous source, inorganic salt and water and fermention medium are cultivated the cyclic monophosphate high yield bacterium that filters out in the extractum carnis slant culture.
CGMCC No.3584 bacterial strain has following character:
1, colonial morphology feature:
Bacterium colony is rounded on solid medium, and is moistening, low projection, and smooth surface, diameter is about 1.5-2mm; Glossy, it is yellow that bacterium colony is, and children bacterium in age is light yellow, and along with cell age increases, bacterium colony is deep yellow
2, physiology and biochemical characteristic:
Bacterial strain is the obligate aerobic bacteria, and anaerobic condition is not grown; Oxydase reaction is negative, and the catalase reaction is positive, and the nitrate reduction reaction is positive; The optimum growth temp of this bacterium is 30 ℃, and optimum pH is 7.0, can be in 10.0 times growths of pH; The suitableeest NaCl concentration is 1% (mass ratio), also can grow when NaCl concentration is 5% (mass ratio); Thermotolerance (55 ℃ were heated 20 minutes) is arranged.
3,16S rDNA sequential analysis:
In the registration of GenBank database, registration number is GQ141738 to the 16S rDNA sequence of CGMCC No.3584 bacterial strain.Check order row are compared with relevant kind in the GenBank database, make up 16S rDNA total order and classify the growth of basic system as and set.The result shows: strains A 302 reaches 99% with the homology of Arthrobacter (Arthrobacter), therefore assert that the bacterial strain that the present invention obtains is an Arthrobacter, i.e. Arthrobacter A302 (CGMCC No.3584).
4, nutritional character:
This bacterium can utilize glucose, inositol, sorbyl alcohol, wood sugar, N.F,USP MANNITOL, maltose, sucrose, raffinose, lactose, rhamnosyl; Hydrolyze urea does not produce H
2S; Indole reaction is negative; Hydrolyzable starch, not gelatin hydrolysate.
The present invention also provides the application of above-mentioned Arthrobacter in the output that improves the fermentative production cyclic monophosphate.
The present invention provides a kind of fermentation process of producing cyclic monophosphate again, and this method comprises that the above-mentioned Arthrobacter of employing ferments in liquid fermentation medium.
In above-mentioned fermentation process, liquid seed culture medium can comprise: carbon source 1～100g/L is preferably 10～30g/L; Nitrogenous source 1～100g/L is preferably 10～30g/L; Inorganic salt 0.01～100g/L, 1～10g/L; All the other are water.Liquid fermentation medium can comprise: carbon source 1～100g/L is preferably 30～50g/L; Nitrogenous source 1～100g/L is preferably 10～30g/L; Inorganic salt 0.01～100g/L, 10～20g/L; All the other are water.Wherein, carbon source can be selected from one or more in glucose, wood sugar, fructose and the glycerine; Nitrogenous source can be selected from one or more in extractum carnis, peptone, yeast extract paste, corn steep liquor, soybean cake powder, urea and the ammonium sulfate; Inorganic salt can be selected from one or more in sylvite, sodium salt, phosphoric acid salt, hydrochloride and the vitriol.The initial pH of liquid fermentation medium can be 4.5～9.5, is preferably 6～8.
In above-mentioned fermentation process, leavening temperature can be 25～37 ℃, is preferably 30～35 ℃; Fermentation time can be 24～120 hours, is preferably 60～100 hours.Wherein, fermentation is preceding to be 1～30: 100 according to volume ratio, is preferably 8～12: 100 inoculum size inoculation.
Beneficial effect of the present invention comprises:
1. compare with traditional radiation method and chemical mutagen, ion beam mutagenesis has that damage is light, mutation rate is high, mutation spectrum is wide, inheritance stability, be easy to obtain characteristics such as desirable new variety.
2. cyclic monophosphate high yield Arthrobacter A302 of the present invention (CGMCC No.3584) has the stable characteristics of high-yield character, and this bacterial strain is through going down to posterity more than 10 generations, and the property retention of producing cyclic phosphoric acid acid is stable.
3. cyclic monophosphate high yield Arthrobacter A302 of the present invention (CGMCC No.3584) can utilize several kinds of carbon source and nitrogen source fermentation to produce cyclic monophosphate, and is easy to operate simple.
The preservation of biomaterial
Arthrobacter (Arthrobacter sp.) A302 has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on January 18th, 2010, depositary institution address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number is CGMCCNo.3584.
Embodiment
Below in conjunction with embodiment the present invention is further described in detail, the present invention may be better understood according to following embodiment.Yet what those skilled in the art should be easily understood that is that the described concrete material proportion of embodiment, processing condition and result thereof only are used to illustrate the present invention, and should also can not limit the scope of the present invention that claims are contained.
Embodiment 1
With the bacterial strain that separates the product cyclic monophosphate obtain from soil is starting strain, will be through N
+(vacuum tightness is 2 * 10 to ion beam mutagenesis in the low energy ion implanter target chamber
-4, the injection energy is 20kev, dosage is 5 * 10
13Ionscm
-2.s
-1) after spore suspension after 100 times of sterilized water dilutions, get 0.1ml and coat on the extractum carnis plate culture medium 30 ℃ and cultivated 3 days down, therefrom select single bacterium colony extractum carnis slant culture 2 days.Switching one encircles in the 500mL that 30mL liquid seed culture medium (glucose 10g/L, peptone 10g/L, yeast extract paste 5g/L, extractum carnis 10g/L, NaCl 3g/L) is housed shakes in the bottle, cultivates 20 hours in 30 ℃ of following 250rpm shaking tables.4mL transfer again in 40mL liquid fermentation medium (glucose 50g/L, K are housed
2HPO
410g/L, KH
2PO
410g/L, MgSO
41g/L, urea 5g/L, peptone 5g/L) 500mL shake in the bottle, cultivated 72 hours in 30 ℃ of following 280rpm shaker fermentations.Through primary dcreening operation, obtain the 88 strains mutant strain higher than starting strain output.With carrying out multiple sieve under the same condition, finally obtain the highest strains A of output 302, cyclic phosphoric acid glycosides output production peak reaches 2.4g/L, improves 2 times than the 1.2g/L of original strain.
Embodiment 2
Strains A 302 encircled in the 500mL that 30mL liquid seed culture medium (glucose 20g/L, peptone 8g/L, yeast extract paste 5g/L, extractum carnis 15g/L, NaCl 3g/L) is housed from extractum carnis inclined-plane switching one shake in the bottle, cultivated 20 hours in 30 ℃ of following 250rpm shaking tables.4mL is in 40mL liquid fermentation medium (glucose 40g/L, K are housed in switching
2HPO
410g/L, KH
2PO
45g/L, MgSO
44g/L, urea 8g/L, peptone 5g/L) 500mL shake in the bottle, cultivated 72 hours in 32 ℃ of following 260rpm shaker fermentations.Cyclic phosphoric acid glycosides output reaches 2.0g/L.
Embodiment 3
Strains A 302 encircled in the 500mL that 30mL liquid seed culture medium (glucose 10g/L, peptone 3g/L, yeast extract paste 4g/L, extractum carnis 10g/L, NaCl 3g/L) is housed from extractum carnis inclined-plane switching one shake in the bottle, cultivated 20 hours in 30 ℃ of following 250rpm shaking tables.4mL is in 40mL liquid fermentation medium (glucose 45g/L, K are housed in switching
2HPO
45g/L, KH
2PO
410g/L, MgSO
40.5g/L, urea 3g/L, peptone 5g/L) 500mL shake in the bottle, cultivated 72 hours in 30 ℃ of following 300rpm shaker fermentations.Cyclic phosphoric acid glycosides output reaches 3.6g/L.
Embodiment 4
Strains A 302 was passed for 10 generations continuously, under the culture condition of embodiment 3, carry out fermentation culture, produce cyclic monophosphate.The result shows (seeing Table 1 and 2), and continuous 10 generations heredity is cultivated, the stable performance that strains A 302 is produced cyclic monophosphate.
Table 1 passes the production cyclic monophosphate performance of the strains A 302 in 1～5 generation continuously
Table 2 passes the production cyclic monophosphate performance of the strains A 302 in 6～10 generations continuously
Claims (11)
1. Arthrobacter that is used for the fermentative production cyclic monophosphate, the deposit number of this Arthrobacter is CGMCC No.3584.
2. the application of Arthrobacter according to claim 1 in the output that improves the fermentative production cyclic monophosphate.
3. fermentation process of producing cyclic monophosphate, this method comprise and adopt Arthrobacter according to claim 1 to ferment in liquid fermentation medium.
4. method according to claim 3 is characterized in that, the liquid seed culture medium of described fermentation comprises: carbon source 1～100g/L is preferably 10～30g/L; Nitrogenous source 1～100g/L is preferably 10～30g/L; Inorganic salt 0.01～100g/L is preferably 1～10g/L; All the other are water.
5. method according to claim 4 is characterized in that, the liquid fermentation medium of described fermentation comprises: carbon source 1～100g/L is preferably 30～50g/L; Nitrogenous source 1～100g/L is preferably 10～30g/L; Inorganic salt 0.01～100g/L, 10～20g/L; All the other are water.
6. according to claim 4 or 5 described methods, it is characterized in that described carbon source is selected from one or more in glucose, wood sugar, fructose and the glycerine
7. according to each described method in the claim 4 to 6, it is characterized in that described nitrogenous source is selected from one or more in extractum carnis, peptone, yeast extract paste, corn steep liquor, soybean cake powder, urea and the ammonium sulfate.
8. according to each described method in the claim 3 to 7, it is characterized in that described inorganic salt are selected from one or more in sylvite, sodium salt, phosphoric acid salt, hydrochloride and the vitriol.
9. according to each described method in the claim 3 to 8, it is characterized in that the initial pH of described liquid fermentation medium is 4.5～9.5, is preferably 6～8.
10. according to each described method in the claim 3 to 9, it is characterized in that described leavening temperature is 25～37 ℃, be preferably 30～35 ℃; Fermentation time is 24～120 hours, is preferably 60～100 hours.
11., it is characterized in that described fermentation is preceding to be 1～30: 100 according to volume ratio, is preferably 8～12 according to each described method in the claim 3 to 10: 100 inoculum size inoculation.
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| CN102433292A (en) * | 2011-12-19 | 2012-05-02 | 南京工业大学 | Recombinant escherichia coli for high yield of cyclic adenosine monophosphate and application thereof |
| CN103320373A (en) * | 2013-06-20 | 2013-09-25 | 南京工业大学 | Arthrobacter for overexpression of hypoxanthine phosphoribosyltransferase gene and construction method and application thereof |
| CN103571779A (en) * | 2013-11-04 | 2014-02-12 | 河南科技学院 | Fermentation medium, bacterial strain and production method for fermentation production of adenosine cyclophosphate |
| CN104962574A (en) * | 2015-06-10 | 2015-10-07 | 南京工业大学 | Arthrobacter expression plasmid and application |
| CN105505933A (en) * | 2016-01-07 | 2016-04-20 | 南京工业大学 | Novel arthrobacterium promoter sequence and application thereof |
| CN108795958A (en) * | 2018-07-11 | 2018-11-13 | 南京工业大学 | Recombinant bacterium for expressing polyphosphate kinase and application thereof |
| CN112300968A (en) * | 2020-11-19 | 2021-02-02 | 河南巨龙生物工程股份有限公司 | Arthrobacter producing adenosine cyclophosphate and application thereof |
| CN115843936A (en) * | 2022-11-23 | 2023-03-28 | 南京工业大学 | Preparation and application of adenosine cyclophosphate-containing pig feed additive |
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| CN102433292A (en) * | 2011-12-19 | 2012-05-02 | 南京工业大学 | Recombinant escherichia coli for high yield of cyclic adenosine monophosphate and application thereof |
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| CN103571779A (en) * | 2013-11-04 | 2014-02-12 | 河南科技学院 | Fermentation medium, bacterial strain and production method for fermentation production of adenosine cyclophosphate |
| CN103571779B (en) * | 2013-11-04 | 2016-10-26 | 河南科技学院 | Fermentation medium, bacterial strain and the production method of a kind of fermenting and producing adenosine cyclophosphate |
| CN104962574B (en) * | 2015-06-10 | 2017-11-24 | 南京工业大学 | Arthrobacter expression plasmid and application |
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| CN115843936A (en) * | 2022-11-23 | 2023-03-28 | 南京工业大学 | Preparation and application of adenosine cyclophosphate-containing pig feed additive |
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