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Method utilizing bacillus subtilis to produce adenosine cyclophosphate

A technology for cyclic adenosine monophosphate and its production method is applied in the field of constructing high-yielding cyclic adenosine monophosphate strains by gene recombination and its fermentation production, which can solve the problems of toxicity and pollution, expensive industrial production, and low yield, and achieve simple equipment, The effect of good effect and feasible method

Inactive Publication Date: 2015-02-11
JIANSGU INST OF MICROBIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The raw materials of this method are easy to obtain, and the reaction conditions are mild and moderate, but the yield is low (49%)
[0012] However, the above methods are all chemical synthesis methods. The difficulty is that the raw materials required for the reaction are toxic and polluting, and industrial production is too expensive.

Method used

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  • Method utilizing bacillus subtilis to produce adenosine cyclophosphate

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Effect test

Embodiment 1

[0020] The present invention includes extracting a protein gene containing adenylyl cyclase activity, such as the protein with the amino acid sequence shown in SEQ ID NO: 1.

[0021] DNA fragments containing the adenylyl cyclase gene can be obtained from microorganisms capable of producing cyclic adenosine monophosphate. Any microorganism capable of producing cyclic adenosine monophosphate can be used in the present invention. A cruciferous vegetable black rot fungus jsim-3017 is owned and preserved by the inventor.

[0022] The method for obtaining the adenylyl cyclase gene from Brassicaceae vegetable black rot fungus jsim-3017 is described below.

[0023] Chromosomal DNA was prepared from Brassicaceae vegetable black rot fungus jsim-3017 by conventional DNA isolation methods such as the phenol method (Biochem. Biophys. Acta, 72, 619-629).

[0024] The DNA fragment of sequence 1 can be amplified by PCR with primers synthesized by conventional methods, and the PCR product ca...

Embodiment 2

[0037] Inoculate in a 250ml shaker flask containing 30ml of liquid fermentation medium (50g of glucose, 10g of tryptone, 45g of K2HPO, 3g of corn steep liquor, 3g of NaCl dissolved in 1L of tap water, pH7.5), in a shaker at 30°C with a rotation speed of 200rpm After culturing for 72 hours, the yield of cyclic adenosine monophosphate was detected by HPLC, and 4 strains with a fermentation unit of 3 g / L or more were screened. Among them, the fermentation unit of Bacillus.subtilis jsim-1277 reached 5.5 g / L.

Embodiment 3

[0039] Dissolve Bacillus.subtilis jsim-1277 in 250ml shake flask with 30ml liquid fermentation medium (30g glucose, 10g tryptone, 3g corn steep liquor, 3g NaCl dissolved in 1L tap water, pH7.5) at 30°C with a rotation speed of 200rpm Cultivate 18h in the shaker in the shaker, take 3ml and inoculate into the 250ml shaking flask (glucose 50g, tryptone 10g, corn steep liquor 3g, NaCl3g is dissolved in 1L tap water, pH7.5) that 30ml liquid fermentation medium is housed as seed solution, in Cultivate for 72 hours at 30° C. in a shaker with a rotational speed of 200 rpm, and obtain a final fermentation liquid with a cyclic adenosine monophosphate fermentation unit of 6.1 g / L.

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Abstract

The invention discloses an industrial method to produce adenosine cyclophosphate, which is used as a medicine and biochemical reagent. The invention further discloses a gene which codes a protein with adenylate cyclase activity and can be applied to the production method mentioned above, a recombinant DNA containing the gene, a novel bacterium strain containing the recombinant DNA, and a method utilizing the novel bacterium strain to produce adenosine cyclophosphate.

Description

technical field [0001] The invention belongs to the technical field of microbial strain screening, and in particular relates to the technology of using gene recombination to construct a high-yield cyclic adenosine monophosphate strain and its fermentation production. Background technique [0002] The present invention relates to an industrial process for the production of cyclic adenosine monophosphate useful as a drug and a biochemical reagent, a gene encoding a protein having adenylate cyclase activity and which can be used in the production process, a recombinant DNA containing the gene, containing the A transformant of recombinant DNA, and a method for producing cyclic adenosine monophosphate using the transformant. Cyclic adenosine monophosphate is a medicine for the circulatory system and belongs to a kind of energy mixture. It is mainly used clinically for heart failure, myocarditis, sick sinus syndrome, coronary heart disease and cardiomyopathy, and can also be used ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/32C12R1/125
Inventor 朱晓宏朱晓慧魏薇单志萍孟妤陆茂林杨晓伟司竑飞
Owner JIANSGU INST OF MICROBIOLOGY
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