Method for producing dehydroepiandrosterone through microbial fermentation

A technology for dehydroepiandrosterone and microbial fermentation, applied in the direction of microorganism-based methods, microorganisms, biochemical equipment and methods, etc., can solve the problems of high toxicity of organic solvents, expensive raw materials, cumbersome preparation steps, etc., and achieve improved product quality The effect of quality yield, reduction of production cost and simplification of extraction process

Inactive Publication Date: 2014-12-24
宋浩雷
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] 1. Most of them use benzene or halogenated alkanes as solvents, and organic solvents are highly toxic, which leads to potential safety hazards in drugs;
[0007] 2. Raw materials are expensive and production costs are high;
[0008] 3. The preparation steps are cumbersome, and the use of organic solvents in the preparation process leads to low product purity, cumbersome purification operations in the later stage, and low production efficiency
Chinese patent application 201210038094.2 proposes a method for preparing dehydroepiandrosterone using 4-AD, that is, phytosterols are first fermented to produce 4-AD, and then DHEA is obtained by chemical methods. Although the above problems have been improved, there are still problems caused by multi-step reactions. Problems such as high production cost, low total yield, and high pressure on environmental protection

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  • Method for producing dehydroepiandrosterone through microbial fermentation

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preparation example Construction

[0040] 2.1 Preparation of Competent Cells

[0041] will concentration 10 7 -10 9 Individuals / ml of Mycobacterium smegmatis was inoculated in LB liquid medium with 10% inoculum size, containing 0.05% Tween 80 by weight percentage in the LB liquid medium, and cultivated to OD at 30°C 600 =0.4-0.5, add glycine with a final concentration of 1% to continue culturing, when OD 600 When it reaches 0.8-1.0, start to prepare competent state;

[0042] First, the bacterial suspension was centrifuged at 11000g at 4°C for 20 min, and the supernatant was discarded; then, the bacteria were resuspended in a glycerol Tween solution containing 0.05% Tween 80 by weight in an amount of 1 / 6 of the medium volume, The mass percentage of the glycerin solution is 10%, centrifuged at 4000g, 4°C for 10min, discard the supernatant after the centrifugation is completed; wash the bacteria twice again with the above method, and the volume of the glycerol Tween solution used is 1 / 15 of the medium volume re...

Embodiment 1

[0046] Propagate ZS3-△MSMEG_5228 in the seed medium first, and wait until the seed medium OD 600 When ≈15, insert it with 30% inoculum in the fermenter that fermentation medium and phytosterol are housed and carry out fermentation transformation, and the weight percent of phytosterol and fermentation medium is 1%, and the condition of fermentation transformation is: temperature 29-31°C, tank pressure 0.05MPa, stirring speed 150-200rpm, culture period 4-5day;

[0047] The components of the seed medium are: glucose 15g / L, K 2 HPO 4 0.3g / L, MgSO 4 ·7H 2 O0.8g / L, (NH 4 ) 2 Fe(SO 4 ) 2 ·6H 2 O0.02g / L, CaCO 3 5.5g / L and citric acid 1.0g / L, the pH of the seed medium is 6.0;

[0048] The components of the fermentation medium are: glucose 15g / L, peptone 30g / L, bean cake powder 25g / L, K 2 HPO 4 0.5g / L, MgSO 4 ·7H 2 O1.0g / L, (NH 4 ) 2 Fe(SO 4 )2·6H 2 O0.06g / L, CaCO 3 4.5g / L, citric acid 3.0g / L and NH 4 NO 3 1.5g / L, the pH of the fermentation medium is 6.0.

[0049] ...

Embodiment 2

[0052] Change the inoculum amount in Example 1 to 40%, and the rest of the conditions remain unchanged.

[0053] The test result is: the final yield of DHEA is 88.75%.

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Abstract

The invention provides a method for producing dehydroepiandrosterone through microbial fermentation. The method can be used for producing dehydroepiandrosterone by transforming phytosterol by utilizing the single water phase fermentation of recombinant mycobacterium smegmatis and comprises the following steps of: constructing knockout original 5228up-kan-5228down; constructing a recombinant strain; propagating the strain, and fermenting and transforming. According to the method provided by the invention, the recombinant mycobacterium smegmatis is obtained by knocking out a 3 beta-hydroxysteroid dehydrogenase encoding gene MSMEG_5228 by utilizing a molecular biology method and dehydroepiandrosterone is produced in a single water phase fermentation system by taking phytosterin as a raw material. The method reduces the production cost, achieves the mass yield of dehydroepiandrosterone more than 90%, simplifies the extraction process, reduces the extraction cost and has the advantages of little pollution, low environmental-friendly pressure and great industrial production superiority.

Description

technical field [0001] The invention relates to a production method of a steroid drug intermediate, in particular to a method for producing dehydroepiandrosterone by microbial fermentation of recombinant mycobacterium smegmatis. Background technique [0002] Dehydroepiandrosterone (DHEA) is a C 19 Steroid carrier compound, chemical name 3β-hydroxyandrost-5-en-17-one, molecular formula C 19 h 28 o 2 , the structural formula is: [0003] [0004] DHEA is a large number of adrenal steroid compounds that coexist in human plasma. It has anti-inflammatory, anti-proliferation and anti-depressant activities. Its derivatives can promote immune response in animal experiments and have good neuroprotective effects. In addition, DHEA is also an important intermediate in the synthesis of steroidal drugs, such as the steroidal contraceptive drospirenone. [0005] Most of the existing DHEA preparation techniques are synthesized by traditional chemical techniques, for example, pregna...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N15/66C12P33/06C12R1/32
Inventor 宋浩雷刘倩孟祥雷张亚朋
Owner 宋浩雷
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