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Microbial strain for preparing androstenedione and application thereof

A technology of androstenedione and microorganisms, applied in the direction of microorganism-based methods, microorganisms, biochemical equipment and methods, etc., can solve the problems of 4AD fermentation strain instability, low feeding concentration, low conversion rate, etc.

Active Publication Date: 2013-11-06
HUBEI GONGTONG BIOLOGICAL SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The technical problem to be solved in the present invention is to provide a high-efficiency fermentation strain of mycobacteria for the problems of unstable 4AD fermentation strains, low feed concentration and low conversion rate in the fermentation process, and simultaneously utilize surfactants, soybean oil and The fermentation broth forms a two-way system, improves the fermentation process, and greatly improves the amount of raw materials and the conversion rate of microorganisms

Method used

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  • Microbial strain for preparing androstenedione and application thereof
  • Microbial strain for preparing androstenedione and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1. Composite mutagenesis to obtain 9-hydroxylase-deficient mutant strains

[0056] 1.1 Starting strain: Mycobacteriumsp.NRRLB-3683

[0057] 1.2 Screening route: original strain→isolation and purification→starting bacteria (Mycobacterium sp.NRRLB-3683)→fresh slant strain→spore suspension→UV irradiation→ 60 Co irradiation→nitrosoguanidine treatment→YAG double frequency pulsed laser→plate separation→single colony→primary screening, secondary screening→positive mutant mutant strains

[0058] 1.3 Preparation of spore suspension: Wash the fresh slant of the starting strain (Mycobacterium sp.NRRL B-3683) with sterile water, and transfer it to a sterilized Erlenmeyer flask containing glass beads, at 28°C, 200r / min Oscillate on a rotating shaker for 30 minutes to make a concentration of about 10 7 cells / ml of bacterial suspension.

[0059] 1.4 UV irradiation: put the bacterial suspension under a 15W ultraviolet lamp (distance 30cm), irradiate for 1, 2, 3, 4, and 5 min...

Embodiment 2

[0064] Example 2. Microorganisms convert phytosterols into androstenedione fermentation regulation process in a biphasic system

[0065] 2.1 Primary seed tank (700L) fermentation

[0066] 2.1.1 Medium composition

[0067] Element Concentration, g / L The effective volume is 400L, Kg corn syrup 50.0 20.00 glucose / brown sugar 6.0 2.40 NaNO 3 5.4 2.16 (NH 4 ) 2 HPO 4 0.6 0.24 Bubble enemy 1.0 0.40

[0068] 2.1.2 Ingredients: Accurately weigh each ingredient according to the above formula, put all the medium ingredients except Paodi into the fermenter, and set the volume to 360L. After constant volume, the pH of the medium is about 3.5-4.3, adjust it to 8.3-8.5 with NaOH (40%), then add foam enemy, and sterilize at 124 degrees for 30 minutes.

[0069] 2.1.3 Culture conditions: inoculum size 1%, 31°C, 180rpm, 0.05MPa, 30-42h

[0070] 2.2 Secondary seed tank (4000L) fermentation

[0071] 2.2.1 Medium composition

[0072] ...

Embodiment 3

[0084] Example 3. Microorganisms convert phytosterols into androstenedione in a biphasic system

[0085] 3.1 HPLC detection method:

[0086] 3.1.1 Detection conditions: liquid chromatography pump; purple visible detector; water: distilled water, and then filtered through a 0.45μm filter membrane; methanol: HPLC reagent.

[0087] 3.1.2 Chromatographic conditions: chromatographic column: C18 (4.6*1505um); mobile phase: methanol: water = 65:35; flow rate: 1ml / min; solvent: methanol; detection wavelength: 240nm.

[0088] 3.1.3 Steps:

[0089] ① Precisely weigh 20mg of working standard, place it in a 100ml volumetric flask, dissolve it with a small amount of methanol, dilute to the mark with mobile phase, shake well, and use it as the reference solution.

[0090] ② Take a certain volume of oily phase of fermentation liquid (weigh its weight W 1 ), add an equal volume of chloroform (weigh its weight W 2 ). Mix well, centrifuge for 15min, weigh the weight of the upper layer wate...

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Abstract

The invention discloses a microbial strain for preparing androstenedione. The collection number of the microbial strain is CCTCC NO:M2012522. The preparation of androstenedione by the utilization of the strain comprises the following steps: A, primary seeding tank fermentation: inoculation amount of the strain is 0.5-1.5%, and cultivation is carried out at 160-200rpm and at 0.03-0.07MPa for 30-42h; B, secondary seeding tank fermentation: inoculation amount of the strain is 8-12%, and cultivation is carried out under the same conditions for 20-30h; C, fermentation tank fermentation for conversion to generate androstenedione: inoculation amount of the strain is 10-14%, and cultivation is carried out under the conditions of 27-35 DEG C and 0.03-0.07MPa; and D, termination of conversion: sterol is lower than 0.5%, pH is 8.9-9.0, discharging, heating to 90-100 DEG C and keeping for 30-50min, cooling to 30-50 DEG C, stopping stirring and standing for 4-6h. The invention provides a high-efficiency fermentation strain of mycobacterium. Meanwhile, a surfactant, soya-bean oil and a broth are used to form a two-way system. The fermentation technology is improved, and feeding amount of raw materials and conversion rate of microorganisms are greatly raised.

Description

technical field [0001] The invention relates to the technical field of biological fermentation. Background technique [0002] At present, there are two main pathways for the preparation of steroidal drug intermediate androstenedione (androst 4-ene-3,20-dione, AD), one is the diosgenin pathway, and the other is the microbial transformation pathway. Diosgenin is generally extracted from wild Chinese medicinal materials such as "Chuandilong" and other plants, and then chemically synthesized to prepare androstenedione. The process is complicated, the cost is high, and the environmental pollution is serious. The work of microbial degradation of sterols began in the 1960s. It has been found that a variety of microorganisms can degrade sterols, and its products androstenedione (AD) and androstene 1,4-diene-3,17-dione (ADD) can As an intermediate in the synthesis of many steroid hormones such as testosterone, methyltestosterone, estrone and adrenocortical hormone. In the early 197...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P33/16C12R1/32
Inventor 系组斌卢蕾卢方欣
Owner HUBEI GONGTONG BIOLOGICAL SCI & TECH
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