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

AI Technical Summary

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, et...

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

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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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