A method of efficiently catalyzing dehydroepiandrosterone by utilization of a resting cell of colletotrichum linli

A technology of C. flaxensis and dehydroepiandrosterone, which is applied in the field of high-efficiency catalysis of dehydroepiandrosterone, to achieve the effects of improving utilization rate, increasing product yield, and facilitating separation and extraction

Inactive Publication Date: 2014-07-09
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to improve the existing conversion process aiming at the technical difficulties and existing proble

Method used

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  • A method of efficiently catalyzing dehydroepiandrosterone by utilization of a resting cell of colletotrichum linli
  • A method of efficiently catalyzing dehydroepiandrosterone by utilization of a resting cell of colletotrichum linli
  • A method of efficiently catalyzing dehydroepiandrosterone by utilization of a resting cell of colletotrichum linli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1 Craniospora flax ( Colletotrichum linli )ST-1 Transformation of Growing Cells vs Transformation of Resting Cells

[0023] (1) Preparation of liquid cell culture of C. flaxensis

[0024] Pick an appropriate amount of spiny sporospora mycelium from the slant medium, inoculate it into a 250 mL Erlenmeyer flask filled with 30 mL of seed medium, and culture it on a shaker at 220 r / min for 72 h at 30 °C Perform activation, and then inoculate the primary seeds with (8%-10% V / V) inoculum into a new seed medium (in a 500 mL Erlenmeyer flask containing 100 mL of seed medium) and proceed with the shaker conditions above After expansion and cultivation for 24 h, a considerable concentration of cell liquid culture was obtained.

[0025] (2) Preparation of resting cell suspension of C. flaxensis

[0026] Inject the cell liquid culture prepared in step (1) into the fermentation medium with an inoculum size of 10 % (w / w), with a liquid volume of 100 mL / 500 mL, and cult...

Embodiment 2

[0033] Embodiment 2 pH is to flax sporophyllum ( Colletotrichum linli ) ST-1 Effects of Resting Cell Transformation

[0034] (1) Preparation of liquid cell culture of C. flaxensis

[0035] Same as (1) in Example 1

[0036] (2) Preparation of resting cell suspension of C. flaxensis

[0037] Inject the liquid cell culture prepared in step (1) into the fermentation medium with an inoculum size of 8-10% (w / w), with a liquid volume of 100 mL / 500 mL, at 30°C, at a speed of 220 r / min Cultivate at low temperature for 24 h, collect the cells by centrifugation, wash the cells twice with 0.01M sodium phosphate buffer at pH 7.0 (remove the remaining components in the fermentation broth), collect the cells, and suspend them in pH 5.5 / 6.5 / In 7.5 sodium phosphate buffer, the buffer concentration is 0.2M, and a resting cell suspension with a cell concentration of 10-12 g / L is prepared for use in transformation.

[0038] (3) Transformation of resting cells

[0039] Take 30 mL of the...

Embodiment 3

[0042] The concentration of embodiment 3 buffering solution is to the flax sporophyllum ( Colletotrichum linli ) ST-1 Effects of Resting Cell Transformation

[0043] (1) Preparation of liquid cell culture of C. flaxensis

[0044] Same as (1) in Example 1

[0045] (2) Preparation of resting cell suspension of C. flaxensis

[0046] Inject the liquid cell culture prepared in step (1) into the fermentation medium with an inoculum size of 8-10% (w / w), with a liquid volume of 100 mL / 500 mL, at 30°C, at a speed of 220 r / min Cultivate at low temperature for 24 h, collect the cells by centrifugation, wash the cells twice with 0.01M sodium phosphate buffer at pH 7.0 (remove the remaining components in the fermentation broth), collect the cells, and suspend them in 0.02M / In 0.05M / 0.1M / 0.2M sodium phosphate buffer (both pH 6.5), prepare a resting cell suspension with a cell concentration of 10-12 g / L for transformation.

[0047] (3) Transformation of resting cells

[0048] Take 3...

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Abstract

The invention discloses a method of efficiently catalyzing dehydroepiandrosterone by utilization of a resting cell of colletotrichum linli ST-1. The method combines resting cell conversion and substrate assisted solubilization, and increases the utilization rate of a substrate and the product yield. A sodium phosphate buffer provides a stable pH environment for the conversion reaction. Tween 80 can assist solubilization and promote thallus growth. By utilization of a novel conversion process, the molar product yield is increased by 78.3% than that of traditional conversion methods. The conversion rate of the product in the method is 90.4% while the conversion rate of the product of the traditional methods is 67.3%.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a new method for utilizing Spirospora flaxensis ( Colletotrichum lini ) ST-1 A method for efficient catalysis of dehydroepiandrosterone by quiescent cells. Background technique [0002] Trihydroxyandrostenone (7α, 15α-diOH-DHEA) is an important precursor for the synthesis of the new oral contraceptive drospirenone. The traditional chemical synthesis method has complicated steps and complex processes, and the use of a large amount of organic solvents is also harmful to the environment. brought a lot of pollution. Now, the target product trihydroxyandrostenone can be obtained in one step by catalyzing the substrate dehydroepiandrosterone (DHEA) by microbial method. The reaction steps are simplified, and the use of organic solvents is also reduced. [0003] However, the existing strains also have certain limitations in the transformation of substrates. Firstly, the ori...

Claims

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Application Information

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IPC IPC(8): C12P33/06C12R1/645
Inventor 许正宏史劲松李会张汝金尹思淇
Owner JIANGNAN UNIV
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