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Method for preparing steroid drug intermediate by mixed bacterial fermentation transformation of phytosterol

A technology of phytosterols and steroidal drugs, applied in the direction of microorganism-based methods, biochemical equipment and methods, fermentation, etc., can solve the problems of difficult temperature and high enzyme activity, and achieve shortened conversion cycle, high product formation rate, and reduced The effect of production costs

Active Publication Date: 2019-03-29
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The mixed-bacteria fermentation also has certain technical difficulties, mainly because there are great differences in the growth and fermentation conditions of different strains, such as temperature, medium, pH, etc. Maximize enzyme activity

Method used

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  • Method for preparing steroid drug intermediate by mixed bacterial fermentation transformation of phytosterol
  • Method for preparing steroid drug intermediate by mixed bacterial fermentation transformation of phytosterol
  • Method for preparing steroid drug intermediate by mixed bacterial fermentation transformation of phytosterol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Effect of Ochraus inoculum Size on Sequential Transformation of Phytosterols to Produce 11α-OH AD

[0025] (1) Mycobacterium (M.neoaurum) TCCC 11028M3 (MNR M3) was used as the production strain, the strain taken from the glycerol tube was streaked on the slant medium, cultured at 29°C for 3-4d, and the slant was activated twice;

[0026] (2) Mycobacterium TCCC 11028M3 (MNR M3) seed culture: pour a certain amount of 0.5% Tween 80 sterile aqueous solution into the solid medium of step (1) under aseptic operation to wash the slant seeds, so that the thalline OD 600The value is controlled at 1.0±0.2, inoculated into 30mL seed medium with 3% inoculum size, and cultured at 28°C and 200r / min until the logarithmic growth phase (36-48h);

[0027] (3) Mycobacterium TCCC 11028M3 (MNR M3) fermentation culture: Accurately weigh an appropriate amount of phytosterols so that the final concentration in the fermentation medium is 3g / L, and inoculum the seeds in step (2) by 8...

Embodiment 2

[0041] Example 2: Effect of Ochrae inoculation time on sequential transformation of phytosterol production (11α-OH AD)

[0042] Except the following content, other is the same as embodiment 1.

[0043] In the sequential transformation system, considering that molds have obvious advantages over the growth and reproduction of mycobacteria, the mycobacteria grow to a certain stage and the side chain degrades phytosterols to generate a certain concentration of androst-4-ene-3 ,17-diketone (AD), it is beneficial to the whole flora system to insert A.

[0044] (1) Biotransformation: After the side chain of mycobacteria degrades phytosterols to generate a certain concentration of androst-4-ene-3,17-dione, it is the 1d, 2d, 3d, and 4d of mycobacterial transformation, respectively. 50% of the inoculum was added to Ochrae sp. CICC41473 cells for subsequent transformation experiments, the culture temperature was 28°C, and the transformation was carried out at a speed of 200r / min for 2 d...

Embodiment 3

[0051] Example 3: Research on mixed culture transformation of phytosterols to generate 11α-OH AD

[0052] Except the following content, other is the same as embodiment 1.

[0053] (1) Biotransformation: Mycobacterium TCCC 11028M3 seed culture solution was inserted in 50mL fermentation medium by 8% inoculum, and the spore suspension of prepared Ochraus ochraci CICC 41473 was 10% at the final concentration of spores. 6 The inoculation amount of each / mL was inserted into the fermentation medium, and then phytosterols with a final concentration of 3g / L were added at 24h, 36h, and 48h respectively, the culture temperature was 28°C, and the transformation was carried out at a speed of 200r / min for 5d;

[0054] Described fermentation medium consists of: glucose 30g / L, citric acid 2g / L, ferric ammonium citrate 0.05g / L, magnesium sulfate 0.5g / L, dipotassium hydrogen phosphate 0.5g / L, diammonium hydrogen phosphate 3.5 g / L, corn steep liquor 40g / L, silkworm chrysalis powder 2g / L, cyclod...

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Abstract

The invention belongs to the technical field of sterol biotransformation, and in particular, relates to a method for preparing a steroid drug intermediate 11[alpha]-hydroxyandrost-4-en-3,17-dione by mixed bacterial fermentation biotransformation of phytosterol. A fermentation culture medium containing phytosterol and a transformation medium cyclodextrin is inoculated with a mycobacterium TCCC 11028 M3, culture is performed for 24 h, then the fermentation culture medium is inoculated with an aspergillus ochraceus CICC 41473 spore solution, and transformation continues to be carried out. One-step transformation from the phytosterol to 11[alpha]-hydroxyandrost-4-en-3,17-dione is achieved by mixed bacterial fermentation. The method improves the production rate of the product, shortens the transformation period, simplifies the operation, saves the cost, and is suitable for production of steroid compound biotransformation.

Description

Technical field: [0001] The invention belongs to the technical field of sterol biotransformation, and in particular relates to a method for preparing steroid drug intermediate 11α-hydroxy-androst-4-ene-3,17-dione through mixed bacteria fermentation and biotransformation of phytosterols. Background technique: [0002] Steroids are terpenoids containing four cyclopentane polyhydrophenanthrenes, widely present in animal and plant tissues and some microbial cells. Steroidal drugs produced from steroidal compounds play a very important role in regulating the body, and are widely used in disease treatment and health care. At present, the market demand for steroidal drugs is second only to antibiotics, such as aldosterone antagonists and eplerenone, which have been clinically used to treat various diseases such as heart failure, hypertension, edema, liver ascites, and respiratory failure. 11α-hydroxy-androst-4-ene-3,17-dione (11α-hydroxyandrost-4-ene-3,17-dione, 11α-OH AD) is the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P39/00C12P33/02C12R1/32C12R1/66
CPCC12P33/02C12P39/00
Inventor 王敏申雁冰杨妍骆健美夏梦雷王喜波
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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