WO2011150724A1 - Method of preparing cyclic adenosine monophosphate - Google Patents
Method of preparing cyclic adenosine monophosphate Download PDFInfo
- Publication number
- WO2011150724A1 WO2011150724A1 PCT/CN2011/073119 CN2011073119W WO2011150724A1 WO 2011150724 A1 WO2011150724 A1 WO 2011150724A1 CN 2011073119 W CN2011073119 W CN 2011073119W WO 2011150724 A1 WO2011150724 A1 WO 2011150724A1
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- WIPO (PCT)
- Prior art keywords
- adenosine monophosphate
- cyclic adenosine
- tricarboxylic acid
- acid cycle
- fermentation
- Prior art date
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- 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 claims abstract description 39
- 238000000034 method Methods 0.000 title claims abstract description 11
- 230000004151 fermentation Effects 0.000 claims abstract description 33
- 238000000855 fermentation Methods 0.000 claims abstract description 33
- 230000034659 glycolysis Effects 0.000 claims abstract description 25
- 230000004102 tricarboxylic acid cycle Effects 0.000 claims abstract description 20
- 239000002243 precursor Substances 0.000 claims abstract description 19
- 239000012190 activator Substances 0.000 claims abstract description 16
- 239000003112 inhibitor Substances 0.000 claims abstract description 16
- 241000894006 Bacteria Species 0.000 claims abstract 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 21
- 238000004519 manufacturing process Methods 0.000 claims description 15
- 239000000126 substance Substances 0.000 claims description 13
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 claims description 12
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 10
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 9
- 241000186063 Arthrobacter Species 0.000 claims description 8
- 239000002126 C01EB10 - Adenosine Substances 0.000 claims description 6
- 229930010555 Inosine Natural products 0.000 claims description 6
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 claims description 6
- 229960005305 adenosine Drugs 0.000 claims description 6
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 claims description 6
- 229960003786 inosine Drugs 0.000 claims description 6
- AUHDWARTFSKSAC-HEIFUQTGSA-N (2S,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)-2-(6-oxo-1H-purin-9-yl)oxolane-2-carboxylic acid Chemical compound [C@]1([C@H](O)[C@H](O)[C@@H](CO)O1)(N1C=NC=2C(O)=NC=NC12)C(=O)O AUHDWARTFSKSAC-HEIFUQTGSA-N 0.000 claims description 5
- 229930024421 Adenine Natural products 0.000 claims description 5
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims description 5
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 claims description 5
- UDMBCSSLTHHNCD-UHFFFAOYSA-N Coenzym Q(11) Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(O)=O)C(O)C1O UDMBCSSLTHHNCD-UHFFFAOYSA-N 0.000 claims description 5
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 claims description 5
- GRSZFWQUAKGDAV-UHFFFAOYSA-N Inosinic acid Natural products OC1C(O)C(COP(O)(O)=O)OC1N1C(NC=NC2=O)=C2N=C1 GRSZFWQUAKGDAV-UHFFFAOYSA-N 0.000 claims description 5
- 229960000643 adenine Drugs 0.000 claims description 5
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 claims description 5
- LNQVTSROQXJCDD-UHFFFAOYSA-N adenosine monophosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(CO)C(OP(O)(O)=O)C1O LNQVTSROQXJCDD-UHFFFAOYSA-N 0.000 claims description 5
- 229960002685 biotin Drugs 0.000 claims description 5
- 235000020958 biotin Nutrition 0.000 claims description 5
- 239000011616 biotin Substances 0.000 claims description 5
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 claims description 5
- 229940028843 inosinic acid Drugs 0.000 claims description 5
- 235000013902 inosinic acid Nutrition 0.000 claims description 5
- 239000004245 inosinic acid Substances 0.000 claims description 5
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) 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-J 0.000 claims description 4
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 claims description 4
- KRHYYFGTRYWZRS-UHFFFAOYSA-M Fluoride anion Chemical compound [F-] KRHYYFGTRYWZRS-UHFFFAOYSA-M 0.000 claims description 4
- 229960003190 adenosine monophosphate Drugs 0.000 claims description 4
- 229960001456 adenosine triphosphate Drugs 0.000 claims description 4
- 238000004321 preservation Methods 0.000 claims description 4
- 239000003617 indole-3-acetic acid Substances 0.000 claims description 3
- JDNTWHVOXJZDSN-UHFFFAOYSA-N iodoacetic acid Chemical compound OC(=O)CI JDNTWHVOXJZDSN-UHFFFAOYSA-N 0.000 claims description 3
- 241001674044 Blattodea Species 0.000 claims 2
- 230000004907 flux Effects 0.000 abstract description 9
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 6
- 229910052799 carbon Inorganic materials 0.000 abstract description 6
- 230000002503 metabolic effect Effects 0.000 abstract description 6
- 230000004108 pentose phosphate pathway Effects 0.000 abstract description 6
- 241000186073 Arthrobacter sp. Species 0.000 abstract description 5
- 230000037353 metabolic pathway Effects 0.000 abstract description 3
- 238000012136 culture method Methods 0.000 description 8
- 230000015572 biosynthetic process Effects 0.000 description 7
- PUZPDOWCWNUUKD-UHFFFAOYSA-M sodium fluoride Chemical compound [F-].[Na+] PUZPDOWCWNUUKD-UHFFFAOYSA-M 0.000 description 6
- 150000007524 organic acids Chemical class 0.000 description 5
- 230000037361 pathway Effects 0.000 description 5
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 239000011775 sodium fluoride Substances 0.000 description 3
- 235000013024 sodium fluoride Nutrition 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 206010000830 Acute leukaemia Diseases 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 229940009098 aspartate Drugs 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 244000144972 livestock Species 0.000 description 2
- 238000009629 microbiological culture Methods 0.000 description 2
- 235000005985 organic acids Nutrition 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 244000144977 poultry Species 0.000 description 2
- 235000013594 poultry meat Nutrition 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 150000003628 tricarboxylic acids Chemical class 0.000 description 2
- QZFUJXGLCPJZFN-UHFFFAOYSA-N 1,1-dibutylguanidine Chemical compound CCCCN(C(N)=N)CCCC QZFUJXGLCPJZFN-UHFFFAOYSA-N 0.000 description 1
- PQGCEDQWHSBAJP-TXICZTDVSA-N 5-O-phosphono-alpha-D-ribofuranosyl diphosphate Chemical compound O[C@H]1[C@@H](O)[C@@H](O[P@](O)(=O)OP(O)(O)=O)O[C@@H]1COP(O)(O)=O PQGCEDQWHSBAJP-TXICZTDVSA-N 0.000 description 1
- 102000006589 Alpha-ketoglutarate dehydrogenase Human genes 0.000 description 1
- 108020004306 Alpha-ketoglutarate dehydrogenase Proteins 0.000 description 1
- 206010002383 Angina Pectoris Diseases 0.000 description 1
- 206010006458 Bronchitis chronic Diseases 0.000 description 1
- 206010008469 Chest discomfort Diseases 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 208000000059 Dyspnea Diseases 0.000 description 1
- 206010013975 Dyspnoeas Diseases 0.000 description 1
- 102100021758 E3 ubiquitin-protein transferase MAEA Human genes 0.000 description 1
- 108010051696 Growth Hormone Proteins 0.000 description 1
- 208000009525 Myocarditis Diseases 0.000 description 1
- 206010033557 Palpitations Diseases 0.000 description 1
- 102000001105 Phosphofructokinases Human genes 0.000 description 1
- 108010069341 Phosphofructokinases Proteins 0.000 description 1
- 102000012288 Phosphopyruvate Hydratase Human genes 0.000 description 1
- 108010022181 Phosphopyruvate Hydratase Proteins 0.000 description 1
- 201000004681 Psoriasis Diseases 0.000 description 1
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 1
- 108010053763 Pyruvate Carboxylase Proteins 0.000 description 1
- 102100039895 Pyruvate carboxylase, mitochondrial Human genes 0.000 description 1
- 102100038803 Somatotropin Human genes 0.000 description 1
- 241000219094 Vitaceae Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 206010006451 bronchitis Diseases 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 238000009924 canning Methods 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 206010007625 cardiogenic shock Diseases 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 208000007451 chronic bronchitis Diseases 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 102000006602 glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 230000002414 glycolytic effect Effects 0.000 description 1
- 235000021021 grapes Nutrition 0.000 description 1
- 239000000122 growth hormone Substances 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000012269 metabolic engineering Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 239000012450 pharmaceutical intermediate Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 235000013613 poultry product Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
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- 230000008844 regulatory mechanism Effects 0.000 description 1
- 208000004124 rheumatic heart disease Diseases 0.000 description 1
- 208000013220 shortness of breath Diseases 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
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
- C12P19/30—Nucleotides
- C12P19/32—Nucleotides having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide
-
- 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
Definitions
- the invention relates to a method for preparing cyclic adenosine monophosphate, in particular to a method for obtaining cyclic adenosine monophosphate by high-yield fermentation by using Arthrobacter as a producing strain and adding a regulating substance to a fermentation medium.
- Cyclic adenosine monophosphate is a physiologically active substance widely present in the human body. As a second messenger in the cell, it plays an important role in the metabolism and synthesis of sugar, fat, nucleic acid, protein, etc. . Clinically, it can be used for the treatment of angina pectoris, myocardial infarction, myocarditis and cardiogenic shock. It also has a certain effect on improving symptoms such as palpitations, shortness of breath and chest tightness in rheumatic heart disease. It can improve the efficacy of acute leukemia combined with chemotherapy, and can also be used for acute leukemia.
- Adenosine monophosphate can also be used as a pharmaceutical intermediate to prepare dibutyl guanidine cyclic adenosine monophosphate and cyclic adenosine monophosphate, to enhance fat solubility, thereby more effectively exerting physiological and pharmacological effects. It can also be used as a food additive for livestock and poultry. It simulates the action of growth hormone under in vitro conditions, promotes the growth of livestock and poultry, and increases the yield of high quality poultry products.
- cyclic adenosine monophosphate by fermentation.
- the synthesis of cyclic adenosine monophosphate involves the glycolytic pathway, the pentose phosphate pathway, and the tricarboxylic acid cycle.
- excess glycolysis flow must be inhibited, and the pentose phosphate pathway flow must be enhanced while the tricarboxylic acid cycle needs to be strengthened.
- the present invention re-distributes the metabolic flux of the central metabolic pathway by adding a precursor substance, a glycolytic pathway inhibitor, and/or a tricarboxylic acid cycle activator, so that the carbon stream flows more efficiently to the target product, a cyclic adenosine monophosphate.
- a method for preparing cyclic adenosine monophosphate which comprises using Arthrobacter as a production strain, and adding a precursor substance, a glycolytic pathway inhibitor and/or a tricarboxylic acid cycle activator to the fermentation medium, and obtaining the fermentation Cyclic adenosine monophosphate.
- the preservation number of the bacillus is CGMCC No. 3584.
- CGMCC Common Microbiology Center
- the precursor substance may be selected from one or more of adenine, adenosine, adenosine monophosphate, adenosine triphosphate, inosine, inosinic acid, and hypoxanthine.
- concentration of the precursor substance added may be from 0.001 to 100 g, preferably from 1 to 15 g, per liter of the fermentation medium.
- the glycolytic pathway inhibitor may be selected from one or more of the group consisting of fluoride, indole acetic acid, citric acid, and citrate.
- the concentration of the glycolytic pathway inhibitor may be 0.001 to 10 g, preferably 0.05 to lg per liter of the fermentation medium.
- the tricarboxylic acid cycle activator may be selected from one or more of biotin, VB1, VB2, VB3, and cobalt chloride.
- the concentration of the tricarboxylic acid cycle activator added may be 0.0001 to lg per liter of the fermentation medium, preferably 0.001 to lg.
- the present invention also provides the use of a precursor substance, a glycolytic pathway inhibitor and/or a tricarboxylic acid cycle activator for the preparation of cyclic adenosine monophosphate by fermentation using Arthrobacter as a producing strain.
- the preservation number of Arthrobacter is CGMCC No. 3584.
- the precursor substance may be selected from one or more of adenine, adenosine, adenosine monophosphate, adenosine triphosphate, inosine, inosinic acid, and hypoxanthine.
- the precursor substance may be used in a concentration of from 0.001 to 100 g, preferably from 1 to 15 g, per liter of the fermentation medium.
- the glycolytic pathway inhibitor may be selected from one or more of the group consisting of fluoride, iodoacetic acid, citric acid, and citrate.
- the glycolytic pathway inhibitor may be used in a concentration of 0.001 to 10 g, preferably 0.05 to lg per liter of the fermentation medium.
- the tricarboxylic acid cycle activator may be selected from one or more of biotin, VB1, VB2, VB3, and cobalt chloride. Adding the tricarboxylic acid cycle activator to a concentration of fermentation per liter of fermentation
- the base is 0.0001 to lg, preferably 0.00 lg.
- the present inventors have realized through metabolic flow and key node analysis that in order to obtain high yield of cyclic adenosine monophosphate and reduce the concentration of by-product organic acid, the following two points must be achieved: 1. Reduce the flux of the glycolysis pathway, on the one hand Metabolic flux can be migrated, allowing more carbon to flow to the pentose phosphate pathway, providing more precursors for cyclic adenosine monophosphate. On the other hand, it can alleviate the "overflow" of the carbon source existing between the glycolytic pathway and the tricarboxylic acid cycle, and reduce the formation of by-product organic acids. 2.
- Increasing the circulating flux of the tricarboxylic acid can provide more precursor aspartate for cyclic adenosine monophosphate; on the other hand, it can match the high-throughput glycolysis pathway, in reducing organic The formation of acid reduces the waste of glucose.
- the addition of the precursor substance allows the bacillus to synthesize cyclic adenosine monophosphate through a salvage synthesis pathway, which consumes a large amount of accumulated phosphoribosyl pyrophosphate, which greatly enhances the pentose phosphate pathway, thereby increasing the production of cyclic adenosine monophosphate.
- glycolytic inhibitors inhibits many key enzymes in this pathway, such as phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase, and enolase, and increases the metabolic flux of the glycolysis pathway.
- the flux of the pentose phosphate pathway increases the production of cyclic adenosine monophosphate.
- a tricarboxylic acid cycle activator activates many key enzymes involved in this pathway, such as pyruvate carboxylase, pyruvate dehydrogenase, ⁇ -ketoglutarate dehydrogenase, etc., and accelerates the tricarboxylic acid.
- the acid cycle provides more precursor aspartate for cAMP and matches the high-throughput EMP pathway to reduce organic acid formation.
- CGMCC General Microbiology Center of China Microbial Culture Collection Management Committee
- the fermentation medium is in a common medium (based on the weight percentage of the medium, including the grapes) Sugar 5%, K 2 HP0 4 1%, KH 2 P0 4 1%, MgS0 4 1%, urea 0.5%, peptone 0.5%) based on the addition of 0.1 g NaF, O.lg VBl and 5 g times per liter of medium Huang Wei.
- the fermentation medium was prepared by adding 6 g of hypoxanth per liter of medium to the ordinary medium (as in Example 1).
- the culture method was the same as in Example 1.
- the yield of cyclic adenosine monophosphate was 4.6 g/L, which was 0.92 times higher than that of the conventional medium alone (2.4 g/L).
- the fermentation medium was prepared by adding 0.2 g of sodium fluoride per liter of the medium on the basis of the ordinary medium (same as in Example 1).
- the culture method was the same as in Example 1.
- the yield of cyclic adenosine monophosphate was 3.2 g/L, which was 0.33 times higher than that of the conventional medium alone (2.4 g/L).
- the fermentation medium was prepared by adding O.lg iodoacetic acid, O.lg VBl and 5 g of hypoxanthine per liter of the medium in the usual medium (as in Example 1).
- the culture method was the same as in Example 1.
- the production of cyclic adenosine monophosphate was 3.2 g/L, which was 0.33 times higher than that of the conventional medium alone (2.4 g/L).
- the fermentation medium was added with 0.5 g/L of indole acetic acid, O. lg biotin and 3 g of inosine per liter of medium on the basis of ordinary medium (same as in Example 1).
- the culture method was the same as in Example 1.
- the production of cyclic adenosine monophosphate was 5.2 g/L, which was 1.17 times higher than that of the conventional medium alone (2.4 g/L).
- Example 6 The fermentation medium was prepared by adding O.lg citric acid, 0.05 g of VB2 and 4 g of adenine per liter of medium on the basis of ordinary medium (same as in Example 1).
- the culture method was the same as in Example 1.
- the production of cyclic adenosine monophosphate was 2.8 g/L, which was 0.06 times higher than the yield (2.4 g/L) when using only ordinary medium.
- the fermentation medium was prepared by adding 0.3 g of sodium fluoride, lg VB3 and 8 g of adenosine per liter of the medium on the basis of the ordinary medium (as in Example 1).
- the culture method was the same as in Example 1.
- the production of cyclic adenosine monophosphate was 9.2 g/L, which was 2.83 times higher than that of the conventional medium alone (2.4 g/L).
- the fermentation medium was prepared by adding 0.6 g of citric acid, O.lg of cobalt chloride and 10 g of inosinic acid per liter of the medium on the basis of the ordinary medium (as in Example 1).
- the culture method was the same as in Example 1.
- the production of cyclic adenosine monophosphate was 7.2 g/L, which was twice as high as that of the conventional medium alone (2.4 g/L).
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- 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)
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- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention provides a method of preparing cyclic adenosine monophosphate, which includes the use of Arthrobacter sp. as an engineering bacterium, and the addition of precursors, inhibitors of glycolytic pathway and/or activators of tricarboxylic acid cycle into a fermentation medium, so as to acquire the cyclic adenosine monophosphate by fermentation. Further provided is a use of precursors, inhibitors of glycolytic pathway and/or activators of tricarboxylic acid cycle for preparing cyclic adenosine monophosphate by Arthrobacter sp.. The present invention inhibits the excessive glycolysis, increases the flux of pentose phosphate pathway, and enhances the tricarboxylic acid cycle by the addition of precursors, inhibitors of glycolytic pathway and/or activators of tricarboxylic acid cycle, so as to redistribute rationally the metabolic flux of central metabolic pathways, make the carbon flux more efficiently direct to the target product - cyclic adenosine monophosphate, and synthesize cyclic adenosine monophosphate by using carbon source efficiently.
Description
环磷酸腺苷的制备方法 技术领域 Method for preparing cyclic adenosine monophosphate
本发明涉及一种环磷酸腺苷的制备方法， 具体涉及一种采用节杆菌为 生产菌， 并且向发酵培养基中添加调节物质， 经发酵高产量获得环磷酸腺 苷的方法。 背景技术 The invention relates to a method for preparing cyclic adenosine monophosphate, in particular to a method for obtaining cyclic adenosine monophosphate by high-yield fermentation by using Arthrobacter as a producing strain and adding a regulating substance to a fermentation medium. Background technique
环磷酸腺苷 （cAMP )是人体内广泛存在的一种具有生理活性的重要 物质， 作为细胞内的第二信使， 它对糖、 脂肪、 核酸、 蛋白质等的代谢和 合成调节起着重要的作用。 临床上可用于治疗心绞痛、 心肌梗死、 心肌炎 及心源性休克； 对改善风湿性心脏病的心悸、 气急、 胸闷等症状也有一定 的作用； 可提高急性白血病结合化疗的疗效， 亦可用于急性白血病的诱导 緩解； 此外， 对老年慢性支气管炎、 各种肝炎和银屑病也有一定疗效。 环 磷酸腺苷也可作为药物中间体制备二丁跣环磷酸腺苷和环磷酸腺苷葡甲 胺， 以提高脂溶性， 从而更有效的发挥生理及药理作用。 环碑酸腺苷亦可 用于畜禽食品添加剂，在离体条件下模拟生长激素的作用，促进畜禽生长， 增加优质禽产品的产量。 Cyclic adenosine monophosphate (cAMP) is a physiologically active substance widely present in the human body. As a second messenger in the cell, it plays an important role in the metabolism and synthesis of sugar, fat, nucleic acid, protein, etc. . Clinically, it can be used for the treatment of angina pectoris, myocardial infarction, myocarditis and cardiogenic shock. It also has a certain effect on improving symptoms such as palpitations, shortness of breath and chest tightness in rheumatic heart disease. It can improve the efficacy of acute leukemia combined with chemotherapy, and can also be used for acute leukemia. Induction of remission; In addition, it has a certain effect on chronic bronchitis, various hepatitis and psoriasis in the elderly. Adenosine monophosphate can also be used as a pharmaceutical intermediate to prepare dibutyl guanidine cyclic adenosine monophosphate and cyclic adenosine monophosphate, to enhance fat solubility, thereby more effectively exerting physiological and pharmacological effects. It can also be used as a food additive for livestock and poultry. It simulates the action of growth hormone under in vitro conditions, promotes the growth of livestock and poultry, and increases the yield of high quality poultry products.
在之前的发酵法制备环磷酸腺苷的过程中发现， 有机酸通过糖酵解途 径被大量合成， 大大降低了 cAMP的生产效率。 为了降低有机酸的产生， 重新分配代谢流以提高目的产物的生产效率， Palme通过批发酵过程中的 底物流加策略降低了酸的形成， 并提高了产率。 Cameron和 Tong用代谢 工程的方法改变细胞调控机制和代谢途径来抑制糖酵解途径。 最近， Chen 通过添加柠檬酸来抑制糖酵解途径， 加大了 HMP途径的流量， 提高了肌 苷的产量。 但是， 现有的生产环磷酸腺苷的发酵方法的产量还有待于进一 步提高。 发明内容 In the previous fermentation process for the preparation of cyclic adenosine monophosphate, it was found that the organic acid was synthesized in a large amount by the glycolysis pathway, which greatly reduced the production efficiency of cAMP. In order to reduce the production of organic acids and redistribute the metabolic stream to increase the production efficiency of the target product, Palme reduces the formation of acid and improves the yield by the bottom stream addition strategy in the batch fermentation process. Cameron and Tong use metabolic engineering methods to alter cellular regulatory mechanisms and metabolic pathways to inhibit the glycolytic pathway. Recently, Chen added citric acid to inhibit the glycolysis pathway, increased the flow of the HMP pathway, and increased the production of inosine. However, the yield of the existing fermentation method for producing cyclic adenosine monophosphate has yet to be further improved. Summary of the invention
因此， 本发明的目的是提供一种提高发酵法生产环磷酸腺苷产量的方 法。 环磷酸腺苷的合成涉及到糖酵解途径、 磷酸戊糖途径和三羧酸循环。
为了有效地利用碳源合成环磷酸腺苷， 过量的糖酵解流量必须被抑制， 磷 酸戊糖途径流量须增强， 而同时三羧酸循环也需得到加强。 本发明通过添 加前体物质、糖酵解途径抑制剂和 /或三羧酸循环激活剂， 重新合理地分配 中心代谢途径的代谢流， 使碳流更有效的流向目的产物一环磷酸腺苷。 Accordingly, it is an object of the present invention to provide a method for increasing the production of cyclic adenosine monophosphate by fermentation. The synthesis of cyclic adenosine monophosphate involves the glycolytic pathway, the pentose phosphate pathway, and the tricarboxylic acid cycle. In order to effectively utilize the carbon source to synthesize cyclic adenosine monophosphate, excess glycolysis flow must be inhibited, and the pentose phosphate pathway flow must be enhanced while the tricarboxylic acid cycle needs to be strengthened. The present invention re-distributes the metabolic flux of the central metabolic pathway by adding a precursor substance, a glycolytic pathway inhibitor, and/or a tricarboxylic acid cycle activator, so that the carbon stream flows more efficiently to the target product, a cyclic adenosine monophosphate.
本发明的目的通过以下技术方案实现： The object of the invention is achieved by the following technical solutions:
一种环磷酸腺苷的制备方法， 该方法包括采用节杆菌为生产菌， 并且 在发酵培养基中添加前体物质、 糖酵解途径抑制剂和 /或三羧酸循环激活 剂， 经发酵获得环磷酸腺苷。 A method for preparing cyclic adenosine monophosphate, which comprises using Arthrobacter as a production strain, and adding a precursor substance, a glycolytic pathway inhibitor and/or a tricarboxylic acid cycle activator to the fermentation medium, and obtaining the fermentation Cyclic adenosine monophosphate.
在上述制备方法中， 优选地， 节杆菌的保藏编号为 CGMCC No.3584。 即节杆菌 A302 rthrobacter sp 2010年 1月 18日保藏于中国微生物菌 种保藏管理委员会普通微生物中心 （CGMCC ) , 保藏编号为 CGMCC Νο·3584。 In the above preparation method, preferably, the preservation number of the bacillus is CGMCC No. 3584. Arthrobacter sp. A302 rthrobacter sp was deposited on January 18, 2010 at the Common Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee, with the accession number CGMCC Νο·3584.
前体物质可以选自腺嘌呤、 腺苷、 腺苷单磷酸、 腺苷三磷酸、 肌苷、 肌苷酸和次黄嘌呤中的一种或多种。 添加所述前体物质的浓度可以为每升 发酵培养基 0.001~100g, 优选为 l~15g。 The precursor substance may be selected from one or more of adenine, adenosine, adenosine monophosphate, adenosine triphosphate, inosine, inosinic acid, and hypoxanthine. The concentration of the precursor substance added may be from 0.001 to 100 g, preferably from 1 to 15 g, per liter of the fermentation medium.
糖酵解途径抑制剂可以选自氟化物、 坱乙酸、 柠檬酸和柠檬酸盐中的 一种或多种。 添加所述糖酵解途径抑制剂的浓度可以为每升发酵培养基 0.001~10g， 优选为 0.05~lg。 The glycolytic pathway inhibitor may be selected from one or more of the group consisting of fluoride, indole acetic acid, citric acid, and citrate. The concentration of the glycolytic pathway inhibitor may be 0.001 to 10 g, preferably 0.05 to lg per liter of the fermentation medium.
三羧酸循环激活剂可以选自生物素、 VB1、 VB2、 VB3和氯化钴中的 一种或多种。 添加所述三羧酸循环激活剂的浓度可以为每升发酵培养基 0.0001~lg， 优选为 0.001~lg。 The tricarboxylic acid cycle activator may be selected from one or more of biotin, VB1, VB2, VB3, and cobalt chloride. The concentration of the tricarboxylic acid cycle activator added may be 0.0001 to lg per liter of the fermentation medium, preferably 0.001 to lg.
本发明还提供了前体物质、糖酵解途径抑制剂和 /或三羧酸循环激活剂 在采用节杆菌为生产菌经发酵制备环磷酸腺苷中的用途。 The present invention also provides the use of a precursor substance, a glycolytic pathway inhibitor and/or a tricarboxylic acid cycle activator for the preparation of cyclic adenosine monophosphate by fermentation using Arthrobacter as a producing strain.
在上述用途中， 优选地， 节杆菌的保藏编号为 CGMCC No.3584。 前体物质可以选自腺嘌呤、 腺苷、 腺苷单磷酸、 腺苷三磷酸、 肌苷、 肌苷酸和次黄嘌呤中的一种或多种。 所述前体物质的使用浓度可以为每升 发酵培养基 0.001~100g， 优选为 l〜15g。 In the above use, preferably, the preservation number of Arthrobacter is CGMCC No. 3584. The precursor substance may be selected from one or more of adenine, adenosine, adenosine monophosphate, adenosine triphosphate, inosine, inosinic acid, and hypoxanthine. The precursor substance may be used in a concentration of from 0.001 to 100 g, preferably from 1 to 15 g, per liter of the fermentation medium.
糖酵解途径抑制剂可以选自氟化物、 碘乙酸、 柠檬酸和柠檬酸盐中的 一种或多种。 所述糖酵解途径抑制剂的使用浓度可以为每升发酵培养基 0.001~10g， 优选为 0.05~lg。 The glycolytic pathway inhibitor may be selected from one or more of the group consisting of fluoride, iodoacetic acid, citric acid, and citrate. The glycolytic pathway inhibitor may be used in a concentration of 0.001 to 10 g, preferably 0.05 to lg per liter of the fermentation medium.
三羧酸循环激活剂可以选自生物素、 VB1、 VB2、 VB3和氯化钴中的 一种或多种。 添加所述三羧酸循环激活剂的使用浓度可以为每升发酵培养
基 0.0001〜lg， 优选为 0.00卜 lg。 The tricarboxylic acid cycle activator may be selected from one or more of biotin, VB1, VB2, VB3, and cobalt chloride. Adding the tricarboxylic acid cycle activator to a concentration of fermentation per liter of fermentation The base is 0.0001 to lg, preferably 0.00 lg.
本发明人通过代谢流和关键节点分析认识到， 要使环磷酸腺苷得到高 产， 使副产物有机酸的浓度降低， 必须做到以下两点： 1. 降低糖酵解途 径通量，一方面可使代谢流发生迁移，使更多碳流流向磷酸戊糖途径途径， 为环磷酸腺苷提供更多的前体。 另一方面， 可緩解糖酵解途径与三羧酸循 环之间存在的碳源"溢流"现象， 减少副产物有机酸的形成。 2. 增大三羧 酸循环通量， 一方面可以为环磷酸腺苷提供更多的前体天冬氨酸； 另一方 面， 可与高通量的糖酵解途径相匹配， 在减少有机酸的形成的同时降低葡 萄糖的浪费。 The present inventors have realized through metabolic flow and key node analysis that in order to obtain high yield of cyclic adenosine monophosphate and reduce the concentration of by-product organic acid, the following two points must be achieved: 1. Reduce the flux of the glycolysis pathway, on the one hand Metabolic flux can be migrated, allowing more carbon to flow to the pentose phosphate pathway, providing more precursors for cyclic adenosine monophosphate. On the other hand, it can alleviate the "overflow" of the carbon source existing between the glycolytic pathway and the tricarboxylic acid cycle, and reduce the formation of by-product organic acids. 2. Increasing the circulating flux of the tricarboxylic acid, on the one hand, can provide more precursor aspartate for cyclic adenosine monophosphate; on the other hand, it can match the high-throughput glycolysis pathway, in reducing organic The formation of acid reduces the waste of glucose.
本发明的有益效果如下： The beneficial effects of the present invention are as follows:
1、 前体物质的添加使节杆菌通过补救合成途径合成环磷酸腺苷， 消 耗了大量积累的磷酸核糖焦磷酸， 使磷酸戊糖途径大大增强， 从而提高了 环磷酸腺苷的产量。 1. The addition of the precursor substance allows the bacillus to synthesize cyclic adenosine monophosphate through a salvage synthesis pathway, which consumes a large amount of accumulated phosphoribosyl pyrophosphate, which greatly enhances the pentose phosphate pathway, thereby increasing the production of cyclic adenosine monophosphate.
2、 糖酵解抑制剂的添加， 使该途径的众多关键酶， 如磷酸果糖激酶、 3-磷酸甘油醛脱氢酶、 烯醇化酶受到抑制， 糖酵解途径代谢流量降低的同 时增大了磷酸戊糖途径的通量， 从而提高了环磷酸腺苷的产量。 2. The addition of glycolytic inhibitors inhibits many key enzymes in this pathway, such as phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase, and enolase, and increases the metabolic flux of the glycolysis pathway. The flux of the pentose phosphate pathway increases the production of cyclic adenosine monophosphate.
3、 三羧酸循环激活剂的添加， 使涉及该途径的众多关键酶， 如丙酮 酸羧化酶、 丙酮酸脱氢酶、 α-酮戊二酸脱氢酶等得到激活， 加速了三羧酸 循环， 为 cAMP提供更多的前体天冬氨酸， 同时可与高通量的 EMP途径 相匹配， 减少有机酸的形成。 生物材料的保藏 3. The addition of a tricarboxylic acid cycle activator activates many key enzymes involved in this pathway, such as pyruvate carboxylase, pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, etc., and accelerates the tricarboxylic acid. The acid cycle provides more precursor aspartate for cAMP and matches the high-throughput EMP pathway to reduce organic acid formation. Preservation of biological materials
节杆菌 A302, Arthrobacter sp. , 2010年 1月 18日保藏于中国微生物 菌种保藏管理委员会普通微生物中心 （CGMCC, 地址： 北京市朝阳区大 屯路， 中国科学院微生物研究所）， 保藏编号为 CGMCC No.3584。 Arthrobacter sp. A302, Arthrobacter sp., deposited on January 18, 2010 at the General Microbiology Center of China Microbial Culture Collection Management Committee (CGMCC, Address: Datun Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), with accession number CGMCC No. 3584.
具体实施方式 detailed description
下面结合具体实施方式对本发明进行进一步的详细描述， 给出的实施 例仅为了阐明本发明， 而不是为了限制本发明的范围。 实施例 1 The present invention is further described in detail with reference to the preferred embodiments thereof. Example 1
发酵培养基为在普通培养基（基于培养基的重量百分比计， 包含葡萄
糖 5%， K2HP04 1%, KH2P04 1%, MgS04 1%, 尿素 0.5%， 蛋白胨 0.5% ) 的基础上每升培养基添加 0.1g NaF、 O.lg VBl和 5g次黄嘌呤。 The fermentation medium is in a common medium (based on the weight percentage of the medium, including the grapes) Sugar 5%, K 2 HP0 4 1%, KH 2 P0 4 1%, MgS0 4 1%, urea 0.5%, peptone 0.5%) based on the addition of 0.1 g NaF, O.lg VBl and 5 g times per liter of medium Huang Wei.
培养方法： 将节杆菌 A302接入种子培养基（基于培养基的重量百分 比计， 包含葡萄糖 1%，蛋白胨 1%，酵母膏 0.5%,牛肉膏 1%， NaCl 0.3% ) 中， 初始 pH为 7.0， 在 30 、 240rpm下培养 18小时。 10%的接种量接入 5L发酵罐中的发酵培养基中， 以 NaOH控制 pH为 7.0， 溶氧控制为 30%, 400rpm、 30 下发酵 72小时。 放罐时， 环嶙酸腺苷的产量是 10.2g/L， 比 仅利用普通培养基时的产量（2.4g/L )提高了 3.25倍。 实施例 2 Culture method: Connect the Arthrobacter A302 into the seed medium (based on the weight percentage of the medium, including glucose 1%, peptone 1%, yeast extract 0.5%, beef extract 1%, NaCl 0.3%), the initial pH is 7.0. , Incubate for 18 hours at 30, 240 rpm. 10% of the inoculum was introduced into the fermentation medium in a 5 L fermenter, and the pH was 7.0 with NaOH, 30% with dissolved oxygen, and 72 hours at 400 rpm and 30 hours. When put in a can, the yield of adenosine citrate was 10.2 g/L, which was 3.25 times higher than that when using only normal medium (2.4 g/L). Example 2
发酵培养基为在普通培养基（同实施例 1 ) 的基础上每升培养基添加 6g次黄嘌呤。培养方法同实施例 1。放罐时， 环磷酸腺苷的产量是 4.6g/L， 比仅利用普通培养基时的产量（2.4g/L )提高了 0.92倍。 实施例 3 The fermentation medium was prepared by adding 6 g of hypoxanth per liter of medium to the ordinary medium (as in Example 1). The culture method was the same as in Example 1. At the time of canning, the yield of cyclic adenosine monophosphate was 4.6 g/L, which was 0.92 times higher than that of the conventional medium alone (2.4 g/L). Example 3
发酵培养基为在普通培养基（同实施例 1 ) 的基础上每升培养基添加 0.2g氟化钠。 培养方法同实施例 1。 放罐时， 环磷酸腺苷的产量是 3.2g/L， 比仅利用普通培养基时的产量（2.4g/L )提高了 0.33倍。 实施例 4 The fermentation medium was prepared by adding 0.2 g of sodium fluoride per liter of the medium on the basis of the ordinary medium (same as in Example 1). The culture method was the same as in Example 1. When the can was placed, the yield of cyclic adenosine monophosphate was 3.2 g/L, which was 0.33 times higher than that of the conventional medium alone (2.4 g/L). Example 4
发酵培养基为在普通培养基（同实施例 1 ) 的基础上每升培养基添加 O.lg碘乙酸、 O.lg VBl和 5g次黄嘌呤。 培养方法同实施例 1。放罐时， 环 磷酸腺苷的产量是 3.2g/L, 比仅利用普通培养基时的产量（2.4g/L )提高 了 0.33倍。 实施例 5 The fermentation medium was prepared by adding O.lg iodoacetic acid, O.lg VBl and 5 g of hypoxanthine per liter of the medium in the usual medium (as in Example 1). The culture method was the same as in Example 1. When the can was placed, the production of cyclic adenosine monophosphate was 3.2 g/L, which was 0.33 times higher than that of the conventional medium alone (2.4 g/L). Example 5
发酵培养基为在普通培养基（同实施例 1 ) 的基础上每升培养基添加 0.5g/L坱乙酸、 O.lg生物素和 3g肌苷。 培养方法同实施例 1。放罐时， 环 磷酸腺苷的产量是 5.2g/L， 比仅利用普通培养基时的产量（2.4g/L )提高 了 1.17倍。 实施例 6
发酵培养基为在普通培养基（同实施例 1 ) 的基础上每升培养基添加 O.lg柠檬酸、 0.05g VB2和 4g腺嘌呤。 培养方法同实施例 1。 放罐时， 环 磷酸腺苷的产量是 2.8g/L， 比仅利用普通培养基时的产量（2.4g/L )提高 了 0.06倍。 实施例 7 The fermentation medium was added with 0.5 g/L of indole acetic acid, O. lg biotin and 3 g of inosine per liter of medium on the basis of ordinary medium (same as in Example 1). The culture method was the same as in Example 1. When the can was placed, the production of cyclic adenosine monophosphate was 5.2 g/L, which was 1.17 times higher than that of the conventional medium alone (2.4 g/L). Example 6 The fermentation medium was prepared by adding O.lg citric acid, 0.05 g of VB2 and 4 g of adenine per liter of medium on the basis of ordinary medium (same as in Example 1). The culture method was the same as in Example 1. When the can was placed, the production of cyclic adenosine monophosphate was 2.8 g/L, which was 0.06 times higher than the yield (2.4 g/L) when using only ordinary medium. Example 7
发酵培养基为在普通培养基（同实施例 1 ) 的基础上每升培养基添加 0.3g氟化钠， lg VB3和 8g腺苷。 培养方法同实施例 1。 放罐时， 环磷酸 腺苷的产量是 9.2g/L， 比仅利用普通培养基时的产量（2.4g/L )提高了 2.83 倍。 实施例 8 The fermentation medium was prepared by adding 0.3 g of sodium fluoride, lg VB3 and 8 g of adenosine per liter of the medium on the basis of the ordinary medium (as in Example 1). The culture method was the same as in Example 1. When the can was placed, the production of cyclic adenosine monophosphate was 9.2 g/L, which was 2.83 times higher than that of the conventional medium alone (2.4 g/L). Example 8
发酵培养基为在普通培养基（同实施例 1 ) 的基础上每升培养基添加 0.6g柠檬酸， O.lg氯化钴和 10g肌苷酸。 培养方法同实施例 1。 放罐时， 环磷酸腺苷的产量是 7.2g/L， 比仅利用普通培养基时的产量（2.4g/L )提 高了 2倍。
The fermentation medium was prepared by adding 0.6 g of citric acid, O.lg of cobalt chloride and 10 g of inosinic acid per liter of the medium on the basis of the ordinary medium (as in Example 1). The culture method was the same as in Example 1. When the can was placed, the production of cyclic adenosine monophosphate was 7.2 g/L, which was twice as high as that of the conventional medium alone (2.4 g/L).
Claims
1. 一种环磷酸腺苷的制备方法， 该方法包括采用节杆菌为生产菌， 并 且在发酵培养基中添加前体物质、糖酵解途径抑制剂和 /或三羧酸循环激活 剂， 经发酵获得环磷酸腺苷。 A method for preparing cyclic adenosine monophosphate, which comprises using Arthrobacter as a producing strain, and adding a precursor substance, a glycolytic pathway inhibitor and/or a tricarboxylic acid cycle activator to the fermentation medium, Fermentation obtained cyclic adenosine monophosphate.
2. 根据权利要求 1所述的制备方法，其特征在于， 所述节杆菌的保藏 编号为 CGMCC No.3584。 The method according to claim 1, wherein the Arthrobacter has a deposit number of CGMCC No. 3584.
3. 根据权利要求 1或 2所述的制备方法，其特征在于， 所述前体物质 选自腺嘌呤、 腺苷、 腺苷单磷酸、 腺苷三磷酸、 肌苷、 肌苷酸和次黄嘌呤 中的一种或多种。 The preparation method according to claim 1 or 2, wherein the precursor substance is selected from the group consisting of adenine, adenosine, adenosine monophosphate, adenosine triphosphate, inosine, inosinic acid, and hypoxanthine One or more of the cockroaches.
4. 根据权利要求 1至 3中任一项所述的制备方法，其特征在于， 添加 所述前体物质的浓度为每升发酵培养基 0.001~100g, 优选为 l~15g。 The preparation method according to any one of claims 1 to 3, wherein the concentration of the precursor substance added is 0.001 to 100 g, preferably 1 to 15 g per liter of the fermentation medium.
5. 根据权利要求 1至 4中任一项所述的制备方法，其特征在于， 所述 糖酵解途径抑制剂选自氟化物、 捵乙酸、 柠檬酸和柠檬酸盐中的一种或多 种。 The preparation method according to any one of claims 1 to 4, wherein the glycolytic pathway inhibitor is one or more selected from the group consisting of fluoride, indole acetic acid, citric acid, and citrate Kind.
6. 根据权利要求 1至 5中任一项所述的制备方法，其特征在于， 添加 所述糖酵解途径抑制剂的浓度为每升发酵培养基 0.001〜10g， 优选为 0.05〜lg。 The preparation method according to any one of claims 1 to 5, wherein the concentration of the glycolytic pathway inhibitor is 0.001 to 10 g, preferably 0.05 to lg, per liter of the fermentation medium.
7. 根据权利要求 1至 6中任一项所述的制备方法，其特征在于， 所述 三羧酸循环激活剂选自生物素、 VB1、 VB2、 VB3和氯化钴中的一种或多 种。 The preparation method according to any one of claims 1 to 6, wherein the tricarboxylic acid cycle activator is one or more selected from the group consisting of biotin, VB1, VB2, VB3, and cobalt chloride. Kind.
8. 根据权利要求 1至 6中任一项所述的制备方法，其特征在于， 添加 所述三羧酸循环激活剂的浓度为每升发酵培养基 0.0001~lg， 优选为 0.001〜lg。 The preparation method according to any one of claims 1 to 6, wherein the concentration of the tricarboxylic acid cycle activator added is 0.0001 to lg per liter of the fermentation medium, preferably 0.001 to lg.
9. 前体物质、 糖酵解途径抑制剂和 /或三羧酸循环激活剂在采用节杆 菌为生产菌经发酵制备环磷酸腺苷中的用途。 9. Use of a precursor substance, a glycolytic pathway inhibitor and/or a tricarboxylic acid cycle activator for the production of cyclic adenosine monophosphate by fermentation of a bacterium.
10. 根据权利要求 9所述的用途， 其特征在于， 所述节杆菌的保藏编 号为 CGMCC No.3584。 10. Use according to claim 9, characterized in that the preservation number of the Arthrobacter is CGMCC No. 3584.
11. 根据权利要求 9或 10所述的用途， 其特征在于， 所述前体物盾选 自腺嘌呤、 腺苷、 腺苷单磷酸、 腺苷三磷酸、 肌苷、 肌苷酸和次黄嘌呤中 的一种或多种。 The use according to claim 9 or 10, wherein the precursor shield is selected from the group consisting of adenine, adenosine, adenosine monophosphate, adenosine triphosphate, inosine, inosinic acid and hypoxanthine One or more of the cockroaches.
12. 根据权利要求 9至 11中任一项所述的用途， 其特征在于， 所述糖 酵解途径抑制剂为氟化物、 碘乙酸、 柠檬酸和柠檬酸盐中的一种或多种。The use according to any one of claims 9 to 11, characterized in that the sugar The glycolysis inhibitor is one or more of fluoride, iodoacetic acid, citric acid, and citrate.
13. 根据权利要求 9至 12中任一项所述的用途，其特征在于， 所述三 羧酸循环激活剂选自生物素、 VB1、 VB2、 VB3和氯化钴中的一种或多种。 The use according to any one of claims 9 to 12, wherein the tricarboxylic acid cycle activator is selected from one or more of biotin, VB1, VB2, VB3 and cobalt chloride. .
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| 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 |
| CN104151385B (en) * | 2014-08-15 | 2016-12-07 | 刘琬一 | A kind of method extracting cyclic adenosine monophosphate and application |
| CN104745616B (en) * | 2015-04-17 | 2018-10-23 | 南京工业大学 | The arthrobacterium and its construction method of a kind of inosine dehydrogenase gene deficiency and application |
| CN105343119B (en) * | 2015-11-19 | 2018-08-07 | 广东赛法洛医药科研有限公司 | Application of cyclic adenosine monophosphate or derivative thereof in postoperative subretinal hemorrhage |
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| CN111088207A (en) * | 2020-01-07 | 2020-05-01 | 南京工业大学 | Biocatalysis method for improving yield of synthesized glutaric acid in escherichia coli |
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| US3630842A (en) * | 1968-08-09 | 1971-12-28 | Kikkoman Shoyu Co Ltd | Production of 3{40 ,5{40 -cyclic adenylic acid with micro-organisms |
| US3816251A (en) * | 1971-05-07 | 1974-06-11 | Kyowa Hakko Kogyo Kk | Process for producing adenosine-3,5 -cyclic monophosphoric acid by fermentation |
| CN101805770A (en) * | 2010-03-12 | 2010-08-18 | 南京工业大学 | Method for producing cyclic adenosine monophosphate by using whole cell biocatalysis |
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| US3630842A (en) * | 1968-08-09 | 1971-12-28 | Kikkoman Shoyu Co Ltd | Production of 3{40 ,5{40 -cyclic adenylic acid with micro-organisms |
| US3816251A (en) * | 1971-05-07 | 1974-06-11 | Kyowa Hakko Kogyo Kk | Process for producing adenosine-3,5 -cyclic monophosphoric acid by fermentation |
| CN101805770A (en) * | 2010-03-12 | 2010-08-18 | 南京工业大学 | Method for producing cyclic adenosine monophosphate by using whole cell biocatalysis |
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