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Method for producing adenosine by fermentation

A technology of adenosine and fermentation liquid, which is applied in the field of microbial fermentation, can solve the problems of not reaching the stage of large-scale production, and achieve the effects of wide sugar tolerance concentration range, high color purity, and simple control process

Pending Publication Date: 2022-07-05
HANGZHOU ZHONGMEI HUADONG PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The fourth is that the high-level adenosine-producing bacteria that can be reported only exist at the laboratory level, and none of them have reached the stage of large-scale production. Industrial scale-up is a very difficult point for genetically engineered bacteria. Therefore, it is necessary to develop a fermentation that can stably produce adenosine. method and be able to stabilize industrial scale-up is very necessary

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] 1. Bacterial recovery and activation: Take the working strain glycerol tube (CGMCC NO.23448), thaw it at room temperature, inoculate 0.1 ml of the bacterial suspension onto the LB solid plate, spread evenly, and incubate it in a 35°C incubator for 24 hours , to obtain a single colony that was activated and resuscitated.

[0022] 2. Liquid seed preparation: Take the activated single colony, pick a single colony with an inoculation shovel, inoculate it into a 500ml Erlenmeyer flask containing 100ml LB liquid medium, wrap it and place it on a shaker at 35°C and 250rpm for shaking Cultivate for 20h, and control the OD value of the seed solution to be ≥0.5. The pH of the LB liquid medium was adjusted to 7.0 before disinfection, and the disinfection conditions were 121-123 °C for 30 min.

[0023] 3. Seed cultivation in seed tank: take qualified liquid seeds, inoculate them into a 50L automatic control fermenter containing 20L liquid fermentation medium at a ratio of 0.5% (V / ...

Embodiment 2

[0027] 1. The preparation of seed liquid is the same as in Example 1;

[0028]2. Fermentation tank culture: Take the qualified seed tank seeds and insert them into a 50L self-controlled fermenter equipped with 25L liquid fermentation medium at a ratio of 10% (V / V), the temperature is controlled at 36°C, and the dissolved oxygen is controlled. ≥20%, adjust the air flow and stirring speed during the process. The glucose concentration of the fermentation broth was controlled to be 5-10g / L, 10-20g / L, 20-30g / L, 30-40g / L, 40-50g / L, 50-60g / L, and the ammonia flow was added to automatically control pH6 .3-6.4. Cultured for 48h and put in the tank.

[0029] 3. The liquid fermentation medium formula is composed of: glucose monohydrate 180g / L, yeast paste 20g / L, yeast powder 30g / L ammonium sulfate 10g / L, potassium dihydrogen phosphate 3.5g / L, potassium chloride 2g / L, Manganese sulfate 0.02g / L, urea 7g / L, magnesium sulfate 2g / L, sodium chloride 0.5g / L. After disinfection, the pH of am...

Embodiment 3

[0033] 1. The preparation of seed liquid is the same as in Example 1;

[0034] 2. Fermentation tank culture: Take the qualified seed tank seeds and insert them into a 50L self-controlled fermenter equipped with 25L liquid fermentation medium at a ratio of 10% (V / V), the temperature is controlled at 36°C, and the dissolved oxygen is controlled. ≥20%, adjust the air flow and stirring speed during the process. The glucose concentration of the fermentation broth is controlled to be 30-40g / L in the process, and the ammonia water flow is added to automatically control pH 5.5, 5.8, 6.1, 6.4, 6.7, 7.0, 7.3, and the culture is put in the tank for 48 hours; the liquid fermentation medium formula is composed of: 180g of glucose monohydrate / L, yeast extract 20g / L, yeast powder 30g / L, ammonium sulfate 10g / L, potassium dihydrogen phosphate 3.5g / L, potassium chloride 2g / L, manganese sulfate 0.02g / L, urea 7g / L, magnesium sulfate 2g / L, sodium chloride 0.5g / L. After disinfection, the pH of t...

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Abstract

The invention discloses a method for producing adenosine by fermentation, and relates to a deep liquid fermentation culture medium formula, a fermentation control method, a material supplementing method and the like for producing adenosine by using the strain, the color purity level of the finally obtained fermentation liquor is close to 90%, and an adenosine finished product meeting the pharmacopoeia standard can be obtained through extraction and purification. According to the method, deep liquid fermentation is carried out under the formula of using high-concentration glucose as a carbon source and yeast extract, urea, ammonium sulfate and the like as nitrogen sources, the level of a 50L fermentation tank can reach 11.2 g / L, and the level of a 1T fermentation tank can reach 10.5 g / L.

Description

technical field [0001] The invention belongs to the field of microbial fermentation, and in particular relates to a method for producing adenosine by fermentation. Background technique [0002] Adenosine, chemical name 6-amino-9-β-D-ribofuranosyl-9-hydropurine, adenosine is an important nucleotide derivative and is the product of adenine nucleotide dephosphorylation. As an endogenous nucleoside throughout human cells, it can directly enter the myocardium to be phosphorylated to generate adenylic acid, and can directly enter the myocardium to be phosphorylated to generate adenylic acid, which participates in myocardial energy metabolism, and also participates in the expansion of coronary blood vessels. Increase blood flow. It can be used for the treatment of supraventricular tachycardia, for the supraventricular arrhythmia related to the atrioventricular and the treatment of angina pectoris, myocardial infarction, coronary insufficiency, arteriosclerosis, essential hypertens...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/40C12N1/20C12R1/125
CPCC12P19/40C12N1/20
Inventor 高祥张璇郑玲辉澎湘屏陈世敏朱进伟
Owner HANGZHOU ZHONGMEI HUADONG PHARMA
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