Method for producing 9-alpha-hydroxyandrostenedione by microbial fermentation

A technology for hydroxyandrostenedione and microbial fermentation, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve problems such as pollution and unsuitability for industrial production of 9α-OHAD, and achieve simple extraction and high yield High, low environmental pressure effect

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

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Problems solved by technology

[0005] No matter from the environmental pollution of the fermentation process, or from the yield and p...

Method used

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  • Method for producing 9-alpha-hydroxyandrostenedione by microbial fermentation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Strain propagation: ZS3-9α was prepared into a bacterial concentration of 10 7 -10 9 Bacterial suspension of individual / ml, insert in the seed tank of seed culture medium with 5%-10% inoculum size afterwards and cultivate, and seed culture medium comprises: glucose 8g / L, MgSO 4 ·7H 2 O 0.8g / L, (NH 4 ) 2 Fe(SO 4 ) 2 ·6H 2 O 0.02g / L, CaCO 3 5.5g / L and citric acid 1.0g / L, the pH of the seed medium is 7.0;

[0038] Fermentation transformation: OD of seed liquid in the seed tank 600 When it is 15-20, insert it into the transformation medium with 20% inoculum size for cultivation, and the fermentation medium includes: glucose 30g / L, peptone 15g / L, bean cake powder 25g / L, MgSO 4 ·7H 2 O 0.5g / L, (NH 4 ) 2 Fe(SO 4 )2·6H 2 O 0.06g / L, CaCO 3 4.5g / L, 3.0g / L citric acid, pH of the fermentation medium is 7.0; wherein, the substrate phytosterol is added to the prepared basic fermentation medium with a mass fraction of 2.0%. The transformation culture conditions are as ...

Embodiment 2

[0042] In Example 1, the inoculum amount of strains during fermentation transformation was changed to 30%, and the transformation culture conditions were changed to: temperature 32°C, tank pressure 0.05MPa, stirring 150-200rpm, dissolved oxygen 30%, cycle 100h.

[0043] The test result is: the final conversion rate of 9α-OH AD is 93.2%.

Embodiment 3

[0045] Change the inoculum amount of the bacterial classification to 50% during fermentation transformation in Example 1, change the mass fraction of phytosterols to 3.0%, and change the transformation culture conditions to: temperature 32°C, tank pressure 0.05MPa, stirring 150-200rpm, cycle 120h .

[0046] The test result is: the final conversion rate of 9α-OH AD is 95.8%.

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Abstract

The invention provides a method for producing 9-alpha-hydroxyandrostenedione by microbial fermentation. Recombinant Mycobacterium smegmatis is fermented to convert phytosterin to produce the 9-alpha-hydroxyandrostenedione. The method comprises the following steps: constructing a recombinant expression vector pMV261-ksh; constructing a recombinant strain; and carrying out culture propagation and fermentation conversion. Gene engineering means are utilized to obtain the gene engineering strain for overexpressing 3-sterone-9-alpha-hydroxylase gene (ksh); after the strain is fermented for 120 hours, the conversion rate of the phytosterin is up to 95% above; and the method has the characteristics of high yield, fewer byproducts, short fermentation time simple extraction, small pollution, low pressure for environmental protection and the like, and has huge advantages in industrial production.

Description

technical field [0001] The invention relates to a production method of a steroid drug intermediate, in particular to a method for producing 9α-hydroxyandrostenedione by microbial fermentation. Background technique [0002] 9α-hydroxy androstenedione (9α-hydroxy androstenedione) is abbreviated as 9α-OH AD (the structural formula is shown below), which was first isolated from the fermentation broth of Nocardia as a steroid substance. 9α-OH AD can be used to synthesize corticosteroids, antiandrogen, antiestrogens and contraceptive drugs, and is a very important steroid hormone intermediate. [0003] [0004] At present, there are two main methods for the substrate of 9α-OH AD produced by microbial fermentation: the first method is to use Nocardia, Rhodococcus, Corynebacterium and Cylindrocarpon radicicola to substrate Androstenedione (AD) undergoes 9α-hydroxylation to generate 9α-hydroxyandrostenedione (9α-OH AD). For example, the patent "Method for preparing 9α-hydroxy-an...

Claims

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Application Information

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IPC IPC(8): C12P33/00C12N15/74C12N1/21C12R1/34
Inventor 宋浩雷张敏然张耀勋周利坤
Owner 宋浩雷
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