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High-yield AD/ADD strain and method for high-efficient production of AD/ADD

A technology of strains and microorganisms, applied in the direction of microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve the problem of low single substrate feeding amount of AD and/or ADD, and achieve the effect of increasing the quantity

Active Publication Date: 2013-03-20
广东本科生物工程股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] For this reason, the technical problem to be solved by the present invention is the low problem of AD and / or ADD single substrate feeding amount in the prior art, and then provides a mycobacterium that can transform the AD and / or ADD of high feeding amount strain

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1 Activation and Proliferation Culture of Bacterial Strains

[0031] Prepare activated slant medium (g / L): glucose 20, peptone 10, yeast powder 5, beef extract 5, potassium dihydrogen phosphate 1, ammonium nitrate 1, magnesium sulfate 0.5, agar 20, control pH 7.0. And the culture medium was sterilized at 121°C, 0.1Mpa, and steam for 20min. After sterilization, the culture medium is divided into 250ml eggplant-shaped bottles, and the filling volume of each bottle is 50ml.

[0032] The preserved strain Mycobacterium.sp-BK-1 (preservation number CGMCC No.5707) was taken out from the -80°C refrigerator, inoculated aseptically on the above slant medium, and cultured at 30°C for 5 days.

[0033] Prepare the shake flask seed medium (g / L): glucose 20, peptone 5, yeast powder 2.5, magnesium sulfate 0.5, control pH 7.0, 121°C, 0.1Mpa, steam sterilization for 20min. The seed medium is divided into 500ml Erlenmeyer flasks, and the volume of each bottle is 100ml.

[00...

Embodiment 2

[0035] Example 2 Production of AD and / or ADD by fermentation

[0036] Preparation of fermenter medium (g / L): Potassium dihydrogen phosphate 1, disodium hydrogen phosphate 0.5, refined molasses 60, magnesium sulfate 0.3, ammonium nitrate 3, sterol 25, soybean oil 147 (the average density of soybean oil is 0.92g / ml , about 0.159L, accounting for about 16% of the volume ratio of the fermentation broth), control pH 6.8, prepare a fermenter with a volume of 7.5L, a liquid volume of 4L, sterilize at 121°C, 0.1Mpa, and steam for 20 minutes, and set aside.

[0037] According to the inoculum size of 10%, the culture obtained in the seed shake flask in Example 1 was transferred into a fermenter for biotransformation at 31° C., the stirring speed was 350 rpm, the ventilation flow rate was 1.0 L / L / min, and the fermentation time was cultivated for 168 hours.

[0038] When the fermentor was inoculated, the pH was controlled to be 6.5, and then the pH changed automatically. When the fermenta...

Embodiment 3

[0040] The fermentation medium and operation steps selected in this example are the same as in Example 2, the difference is that the pH is controlled at 7.0 during inoculation, adjusted to pH 7.5 in 24 hours, 8.0 in 48 hours, and above 8.5 after 72 hours. At the end of the fermentation, the pH of the fermentation broth was detected to be 9.1; the detected sterol residue was 7.2%, and the conversion rate was 68.0%; the total amount of AD and / or ADD was 10.41g / L, wherein AD:ADD=96:4.

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PUM

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Abstract

The invention belongs to the field of microbial fermentation and particularly relates to a method for fermentation production of AD and / or ADD by utilizing a high-yield strain. The invention discloses a mycobacterium strain which can realize high-efficient transformation of sterol substances for producing the AD and / or the ADD, the classification name of the mycobacterium strain is Mycobacterium. sp-BK-1, and the mycobacterium strain is collected in China General Microbiological Culture Collection Center with the collection number of CGMCC No. 5707. The invention further discloses the method for producing the AD and / or the ADD by utilizing the strain to transform sterol. The strain disclosed by the invention can completely transform the sterol substances with the concentration of 3.5 percent in a fermentation culture medium to the AD and / or the ADD by relying on an existing process for producing the AD and / or the ADD by microbial transformation of the sterol substances, and the transformation rate is about 70-80 percent, so that the single-time production quantity is increased on the basis of ensuring the single transformation rate.

Description

technical field [0001] The invention belongs to the field of microbial fermentation, and in particular relates to a method for fermenting and producing AD and / or ADD by using high-yield bacterial strains. Background technique [0002] Androst-4-ene-3.17-dione (androst-4-ene-3,17-dione, referred to as androstenedione, AD) and androst-1,-4-diene-3.17-dione (androst- 1,4-diene-3,17-dione, referred to as androstenedione, ADD) are important steroid drug intermediates, androstenedione (AD) can be used as androgen, protein synthesis hormone and other hormones The precursor of substances can also be used to synthesize spironolactone, hydrocortisone, prednisone oxide, dexamethasone and other drugs; while androstenedione (ADD) can be used for the synthesis of estrogen, oral contraceptives and other drugs . Compared with ADD, AD is used in a wider and deeper range in the pharmaceutical industry. More than 90% of steroid hormone drugs are produced with AD as the basic raw material. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P33/02C12R1/01
Inventor 林智德王宏辉毕锡阳张雅娟辜丹锋
Owner 广东本科生物工程股份有限公司
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