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Mycobacterium sp. strain for biosynthesis of 20-hydroxy-23,24-bisnorchol-4-ene-3-one and synthetic method thereof

A technology of biosynthesis and mycobacteria, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of long transformation time, many by-products, low transformation efficiency, etc., and achieve easy separation and purification, reduce Pollution, the effect of reducing production costs

Active Publication Date: 2017-06-16
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In summary, the current methods for preparing 20-hydroxyl-23,24-dinorchol-1,4-dien-3-one from sterols still have problems such as low conversion efficiency, many by-products, and long conversion time. It is still necessary to explore the use of new strains to convert sterols to prepare 20-hydroxyl-23,24-dinorchol-4-en-3-one, so as to increase the concentration of feed, yield and product purity, so as to further reduce production costs. while reducing environmental pollution

Method used

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  • Mycobacterium sp. strain for biosynthesis of 20-hydroxy-23,24-bisnorchol-4-ene-3-one and synthetic method thereof
  • Mycobacterium sp. strain for biosynthesis of 20-hydroxy-23,24-bisnorchol-4-ene-3-one and synthetic method thereof
  • Mycobacterium sp. strain for biosynthesis of 20-hydroxy-23,24-bisnorchol-4-ene-3-one and synthetic method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Obtaining strain Mycobacterium sp.FJH0105-16 by mutagenesis

[0022] 1.1 Protoplast preparation

[0023] The original strain is Mycobacterium sp. 1229-3, which transforms phytosterols into the main product androst-4-ene-3,17-dione, and the by-product 20-hydroxy-23,24-dicholesterol -4-en-3-one, the ratio of the two is 2:1. The strain was screened from the soil of Tianjin Yihai Kerry Oil Company. Melt the glycerol tube of the preserved original bacterial strain and inoculate it in the seed medium with an inoculum of 2%, cultivate it at 30°C and 200rpm for 36h, transfer the seed solution to the seed medium containing 12.5g / l glycine with a 5% inoculum , cultivated for 24h, 6000rpm, and collected the bacteria for 10min. Wash the cells twice with hypertonic buffer (plus 0.2% Tween 80). Resuspend the bacteria in hyperosmotic enzymatic hydrolysis solution, the concentration of lysozyme is 50mg / ml, enzymatic hydrolysis for 3h, 8000rpm, 20min centrifugation to rem...

Embodiment 2

[0038] Embodiment 2: The method that the mycobacterium provided by the invention transforms phytosterols into 20-hydroxyl-23,24-dinorchol-4-en-3-one

[0039] 2.1 Inclined seed culture

[0040] Mycobacterium sp. FJH0105-16 (Mycobacterium sp. FJH0105-16), the deposit number is CGMCCNO.13234, was cultured at 30° C. for 3 days. The formula of the slant medium is 200g / l potato, 20g / l glucose, 15g / l agar, and natural pH.

[0041] 2.2 Primary seed cultivation

[0042] The bacterial colonies cultivated by the above slant seeds were inserted into the sterilized liquid seed medium, and cultured at 30° C. and 200 rpm for 2 days to obtain seed liquid. The formula of described liquid seed culture medium is, glucose 6g / l, yeast extract 10g / l, Na 2 HPO 4 4.5g / l, KH 2 PO 4 3.4 g / l, 0.2% Tween 80, pH 7.0.

[0043] 2.3 Fermentation and transformation in fermenter to produce 20-hydroxy-23,24-dinorchol-4-en-3-one

[0044] Inoculate the seed liquid obtained in step 2.2 into the sterilized ...

Embodiment 3

[0047] Embodiment 3: The method that the mycobacterium provided by the present invention transforms phytosterols into 20-hydroxyl-23,24-dinorchol-4-en-3-one

[0048] 3.1 Inclined seed culture

[0049] Mycobacterium sp. FJH0105-16 (Mycobacterium sp. FJH0105-16), the deposit number is CGMCCNO.13234, was cultured at 30° C. for 3 days. The medium components are as shown in Example 2.

[0050] 3.2 Primary seed cultivation

[0051] The bacterial colonies cultivated by the above slant seeds were inserted into the sterilized liquid seed medium, and cultured at 30° C. and 200 rpm for 2 days to obtain seed liquid. The components of the liquid seed medium are as shown in Example 2.

[0052] 3.3 Fermentation and transformation in fermenter to produce 20-hydroxy-23,24-dinorchol-4-en-3-one

[0053] Inoculate the seed liquid obtained in step 3.2 into the sterilized transformation medium, the inoculum size is 10%, the transformation temperature is 30°C, the rotation speed is 400rpm, the a...

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Abstract

The invention discloses a mycobacterium sp. strain FJH0105-16 and a method for producing 20-hydroxy-23,24-bisnorchol-4-ene-3-one through fermentation of phytosterol with the strain. The stain is preserved in China General Microbiological Culture Collection Center on November 3, 2016, with an accession number of CGMCC No. 13234. The production method comprises the following steps: with the mycobacterium sp. strain FJH0105-16 as a bacterial strain, carrying out slant seed culture, primary seed culture and fermentation and transformation in a fermentation tank; then adding ethyl acetate for extraction; carrying out drying with anhydrous sodium sulfate; then removing a solvent under reduced pressure so as to obtain a crude 20-hydroxy-23,24-bisnorchol-4-ene-3-one product, wherein the mol yield of the crude product is no less than 71%; and subjecting the crude product to recrystallization so as to obtain a finished 20-hydroxy-23,24-bisnorchol-4-ene-3-one product, wherein the finished product has a purity of greater than 95%.

Description

technical field [0001] The invention relates to a strain and a preparation method for producing steroid hormones, in particular to a strain that biosynthesizes 20-hydroxyl-23,24-bisnorchol-4-en-3-one (22-hydroxy-23,24-bisnorchol -4-ene-3-one) mycobacterium and its synthetic method. Background technique [0002] [0003] 20-hydroxy-23,24-bisnorchol-4-ene-3-one, the English name is 22-hydroxy-23,24-bisnorchol-4-ene-3-one, the chemical formula is C 22 h 34 o 2 , with a molecular weight of 330.25. 20-Hydroxy-23,24-Dinorchol-4-en-3-one can be used as an intermediate in the synthesis of many steroid hormones, such as adrenocortical hormone, progesterone, anabolic hormone, etc. [0004] In the 1970s, researchers were investigating the use of mycobacteria to transform sterols into C19 steroids (androst-4-ene-3,17-dione, androst-1,4-diene-3,17- diketone), 20-hydroxyl-23,24-dinorchol-4-en-3-one was found and identified as a by-product, and the yield was only 40 μg / ml, which was...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P33/02C12P33/00C12R1/32
CPCC12N1/20C12P33/005C12P33/02C12N1/205C12R2001/32
Inventor 王玉李雪梅冯进辉吴洽庆朱敦明马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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