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Method for preparing 7alpha, 15alpha-dihydroxy androstenone by pre-inducing substrate

A technology of hydroxyandrostenone and substrate, which is applied in the field of bioengineering and can solve the problems of limited transformation ability and a large number of toxic reagents

Inactive Publication Date: 2012-10-31
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] 7α, 15α-Dihydroxyandrostenone is produced by the hydroxylation of dehydroepiandrosterone at the 7-position and 15-position. If the reaction is realized by pure chemical methods, nearly ten steps of cumbersome reactions are required, and the reaction process Requires large amounts of toxic reagents, etc.
According to relevant literature reports, there are strains such as Gibberella sp. and C. flaxensis that have the ability to dihydroxylate DHEA 7α, 15α, but their transformation ability is limited, so the further improvement of the transformation process is the research of steroid biotransformation. a big problem

Method used

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  • Method for preparing 7alpha, 15alpha-dihydroxy androstenone by pre-inducing substrate
  • Method for preparing 7alpha, 15alpha-dihydroxy androstenone by pre-inducing substrate

Examples

Experimental program
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Effect test

Embodiment 1

[0020] (1) Place the inoculated slant in a constant temperature incubator at 30°C and culture it for 3 days to obtain mature spores. Inoculate the spores into a 500mL shaker flask containing 100mL seed medium and shake at 28°C and 220rpm. Cultivate on the bed for 4 days to obtain seed culture solution suitable for inoculation.

[0021] (2) Put the seed culture solution into a 250mL shake flask containing 30mL of fermentation medium at an inoculum amount of 10%, and continue culturing for 24h under the same conditions to obtain a cell culture solution.

[0022] (3) Accurately weigh 8 g / L of the substrate and put it into the bacterial cell fermentation liquid obtained in step (2), transform it under the same conditions for 60 hours and put it into a bottle. The extracted product was analyzed by HPLC, and the conversion rate was 83.79%.

Embodiment 2

[0024] (1) Slope culture, seed culture and fermentation culture are the same as in Example 1.

[0025] (2) Put the seed culture solution into a 250mL shake flask containing 30mL fermentation medium at an inoculum amount of 10%, and cultivate it for 12 hours under the culture conditions of 28°C and 220rpm, and then accurately weigh 2g / L (substrate weight / volume of fermentation broth) the substrate was put into the cell fluid and continued to cultivate for 12h.

[0026] (3) Accurately weigh 8 g / L of the substrate dehydroepiandrosterone and add it to the bacterial cell culture solution obtained in step (2), so that the final concentration of the substrate is 8 g / L, continue the transformation for 48 hours and put it in the bottle. Take 1mL of fermentation broth to detect, and the conversion rate is 89.95%.

Embodiment 3

[0028] (1) Slope culture, seed culture and fermentation culture are the same as in Example 1.

[0029] (2) Put 10% inoculum of the seed culture solution into a 250mL shake flask containing 30mL fermentation medium, cultivate for 8h, culture conditions are 28°C, 220rpm, and then accurately weigh 2g / L (substrate weight / Fermentation broth volume) the substrate was put into the cell fluid to continue culturing for 16 hours.

[0030] (3) Accurately weigh 8 g / L of the substrate dehydroepiandrosterone and add it to the bacterial cell culture solution obtained in step (2), so that the final concentration of the substrate is 8 g / L, continue the transformation for 48 hours and put it in the bottle. Take 1mL of fermentation broth to detect, and the conversion rate is 86.95%.

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Abstract

The invention discloses a fermentation process method for biologically synthesizing 7alpha,15alpha-dihydroxy androstenone by taking colletotrichum lini ST-1 as a strain. The method is characterized in that a certain amount of substrate dehydroepiandrosterone is added at the beginning of a fermentation process, and the substrate is fed again after the substrate dehydroepiandrosterone is cultured for a period of time, so that not only a key enzyme can be pre-induced in the transformation process, but also the toxicity to the strain cell caused by feeding the substrate at a time can be reduced. Therefore, the yield of the product can be increased. Through the adoption of the fermentation process, the 7alpha,15alpha-dihydroxy androstenone is synthesized, meanwhile, the feeding concentration of the substrate can reach 8 g / L (weight of substrate / volume of fermentation broth), and the transformation rate can reach about 90%. Compared with a once feeding way, the way of feeding the substrate reduces the toxicity to the cell caused by the substrate, increases the yield of the product to a certain extent, and increases the feeding amount of the substrate.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a kind of C. flaxensis ( Colletotrichum lini ) ST-1 is a method for efficiently preparing 7α, 15α-dihydroxyandrostenone by pre-inducing substrates in the fermentation process of strains. Background technique [0002] Drospirenone (DRSP) is an aldosterone antagonist with anti-mineralcorticosterone activity. It is used in combination with ethinyl estradiol to form Yasmin, a new high-efficiency monophasic oral contraceptive. 7α, 15α-dihydroxyandrostenone is an important pharmaceutical intermediate for the production of drospirenone, and its production methods mainly include traditional chemical synthesis, semi-synthesis, and biotransformation. [0003] 7α, 15α-Dihydroxyandrostenone is produced by the hydroxylation of dehydroepiandrosterone at the 7-position and 15-position. If the reaction is realized by pure chemical methods, nearly ten steps of cumbersome reac...

Claims

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Application Information

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IPC IPC(8): C12P33/06C12R1/645
Inventor 许正宏付珍珍朱晓宏
Owner JIANGNAN UNIV
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