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Method for synthesizing 7,21-dyhydroxyl-20-methyl pregnane-4-alkene-3-ketone by microorganisms

A technology for microbial synthesis and methylpregnant, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of low substrate conversion rate, long conversion cycle, single hydroxylation product, etc.

Active Publication Date: 2019-04-26
北京泛球生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, Mycobacterium is mainly capable of transforming steroid hormone drug intermediates, which not only has a long transformation cycle, low substrate conversion rate, but also easily forms other by-products.
In addition, the hydroxylation products of steroid hormone drug intermediates are single, only the hydroxylation of 9α, 14α, and 15α has been reported, and there are few studies on the hydroxylation of 7α or 7β

Method used

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  • Method for synthesizing 7,21-dyhydroxyl-20-methyl pregnane-4-alkene-3-ketone by microorganisms
  • Method for synthesizing 7,21-dyhydroxyl-20-methyl pregnane-4-alkene-3-ketone by microorganisms
  • Method for synthesizing 7,21-dyhydroxyl-20-methyl pregnane-4-alkene-3-ketone by microorganisms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Method for synthesizing 7α- and 7β-hydroxyl-4-HBC by Mucor circinelloides lusitanicus bacteria GB1

[0040] 1. Preparation of cell liquid culture of Mucor circiniferus strain GB1

[0041] Pick a ring of Mucor circiniferii strain GB1 on the solid medium, inoculate it in a Erlenmeyer flask with seed medium, and culture it on a shaker at 200r / min at 28°C until the logarithmic growth phase. That is, the cell liquid culture of Mucor circinifolia was obtained.

[0042] 2. Shake flask fermentation of Mucor circinelloides lusitanicus strain

[0043] The above-mentioned Mucor circinelloides lusitanicus (Mucor circinelloides lusitanicus) cell culture solution is inserted into the fermentation medium with an inoculum size of 8% (v / v), and the filling volume is 30ml fermentation medium in a 250ml Erlenmeyer flask, and the culture temperature is 25- At 30°C, the bacteria were cultured at a rotational speed of 220r / min to enter the logarithmic growth phase.

[0044] 3. B...

Embodiment 2

[0056] Embodiment 2 Acremonium strictum (Acremonium strictum) GB2 synthesizes the method for 7α- and 7β-hydroxyl-4-HBC

[0057] 1. Preparation of cell liquid culture of Acremonium erectus strain GB2

[0058] Pick a ring of Acremonium erecta strain GB2 on the solid medium, inoculate it in a Erlenmeyer flask with seed medium, and culture it on a shaker at 200r / min at 28°C until logarithmic phase. That is, the cell liquid culture of Acremonium erectum was obtained.

[0059] 2. Shake flask fermentation of Acremonium strictum (Acremonium strictum) bacterial strain

[0060] The above-mentioned upright Acremonium strictum (Acremonium strictum) cell culture solution is inserted into the fermentation medium with an inoculum size of 8% (v / v), and the filling volume is 30ml of the fermentation medium in a 250ml Erlenmeyer flask, and the culture temperature is 25- The bacteria were cultured at 30°C at a rotational speed of 200r / min to enter the logarithmic growth phase. 3. Biotransform...

Embodiment 3

[0072] Example 3 Method for Synthesizing 7α- and 7β-Hydroxyl-4-HBC from Mucor.racemosus GB3

[0073] 1. Prepare the cell liquid culture of Mucor.racemosus GB3

[0074] Pick a ring of Mucor racemosus strain GB3 on the solid medium, inoculate it in a Erlenmeyer flask with seed medium, and culture it on a shaker at 200r / min at 28°C until the logarithmic phase, namely A liquid culture of cells of Mucor racemosum was prepared.

[0075] 2. Shake flask fermentation of Mucor.racemosus strain

[0076] The above-mentioned Mucor.racemosus (Mucor.racemosus) cell culture solution was inserted into the fermentation medium with an inoculum size of 8% (v / v). At 30°C, the bacteria were cultured at a rotational speed of 200r / min to enter the logarithmic growth phase.

[0077] 3. Biotransformation of 4-HBC by Mucor.racemosus strain

[0078] Accurately weigh an appropriate amount of substrate 4-HBC, put it into the cell fermentation broth in step 2, make the final concentration 20g / l, transfo...

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Abstract

The invention provides a method for synthesizing 7,21-dyhydroxyl-20-methyl pregnane-4-alkene-3-ketone by microorganisms. Through a two-step fermentation method, one or more kinds of fermentation liquor including mucor circinelloides lusitanicus, acremonium strictum, mucor.racemosus and aspergillus fumigatus can be used for efficiently converting 4-HBC into 7 alpha-OH-4-HBC and 7beta-OH-4-HBC. Under a condition that the inventory of substrate 4-HBC is 10-20 g / L, a conversion rate is high, two products, including 7alpha and 7beta-hydroxy-4-HBC are separated from the product, and the total yieldof the product is 46.5% to 85.32%. The method disclosed by the invention is convenient in operation, low in cost and high in yield, and has good economic value, and a new method is provided for the development and the synthesis of steroid drugs.

Description

technical field [0001] The invention belongs to the technical field of microbial fermentation technology and biosynthesis of steroidal compounds, and in particular relates to a method for synthesizing 7,21-dihydroxy-20-methylpregn-4-en-3-one by using microorganisms. Background technique [0002] Steroidal compounds are widely distributed in nature and living organisms, and are an important class of bioactive molecules. They are known as the "golden eggs" in the field of pharmacy. With the development of microbial transformation technology, steroidal drugs have become the second largest sales volume in the world. The largest drug, second only to antibiotics, has a global annual production value of nearly 10 billion US dollars. 21-Hydroxy-20-methylpregn-4-en-3-one (4-HBC), which can be biotransformed from phytosterols, is an important intermediate in the synthesis of progesterone and some other adrenocortical hormones. At present, the country has taken the development of new ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P39/00C12P33/06C12N1/14C12R1/785C12R1/68
CPCC12N1/14C12P33/06C12P39/00
Inventor 王福聚董华强李小连孙强
Owner 北京泛球生物科技有限公司
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