Process for biologically synthesizing extracellular cholesterol oxidase from Brevibacterium and its transferred product cholest-4-ene-3-one

A technology of cholesterol oxidase and Brevibacterium, applied in the direction of oxidoreductase, fermentation, etc., can solve the problems that have not been reported in the public literature, so as to reduce the absorption of cholesterol, increase high-density lipoprotein, and reduce low-density lipoprotein. Effect

Inactive Publication Date: 2005-06-29
JIANGNAN UNIV
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Problems solved by technology

Except the published articles and dissertations of this subject research directed by the inventor, there is no public literature report identical with the technical characteristics involved in the present invention

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  • Process for biologically synthesizing extracellular cholesterol oxidase from Brevibacterium and its transferred product cholest-4-ene-3-one

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Shake Flask Fermentation of Cholesterol Oxidase:

[0055] Using the above-mentioned medium in this manual, DGCDC-82 was used as the production bacteria, cultured on a slant, 30°C, 48hr. Seed liquid culture: Take the slant bacteria into a 250ml Erlenmeyer flask containing 30ml of seed medium, 30°C, 220r / min, and cultivate for 12hrs as the seed liquid.

[0056] Fermentation broth culture: Add 3ml of seed solution into a 250ml Erlenmeyer flask containing 30ml of fermentation medium, 30°C, 220r / min, and after 36hrs of cultivation, the cholesterol oxidase production is as high as 700U / L.

Embodiment 2

[0058] 15L tank fermentation of cholesterol oxidase:

[0059] Fermentation medium conditions are the same as above, seed liquid medium conditions are the same as above, 5% inoculum size, culture temperature 33°C, 500r / min, ventilation rate 1vvm, liquid volume 6-7L, culture in 15L automatic fermenter for 28-32hr, fermented liquid Enzyme activity is over 1000U / L.

Embodiment 3

[0061] A two-phase system utilizes cholesterol oxidase to convert free cholesterol to cholestenone:

[0062] With 30ml pH7.5 potassium phosphate buffer as the aqueous phase and 20ml n-octane as the organic phase, add 2g cholesterol and 16U cholesterol oxidase, and react in a 250ml three-necked flask. The reaction conditions are: oxygen flow rate 40L / hr, temperature 40°C, stirring speed 300r / min. After 40 minutes, the conversion rate of cholesterol reaches over 92%.

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Abstract

A process for biologically synthesizing the extracellular cholesterol oxidase from Brevibacterium is disclosed. The high-output bacterium DGCDC-82 is fermented in optimized condition to obtain said enzyme up to 700-1000 U/L. After purified, its specific enzyme activity can reach 30 U/mg. Said enzyme contains coenzyme FAD and cysteine in the active center of enzyme. In the dual-phase (water phase and organic phase) system, said enzyme can be used to uniquely oxidize the cholesterol to becone cholest-4-ene-3-ketone with 92% of transfer rate, which can be used to reduce cholesterol, blood fat and low-density lipoprotein, so having wide application range.

Description

technical field [0001] The present invention relates to the biosynthesis of extracellular cholesterol oxidase by self-selected Brevibacterium sp. A method for enzymatically catalyzing the oxidation of cholesterol to produce a single oxidation product, cholest-4-en-3-one. The toxicity and blood lipid lowering of cholest-4-en-3-one were studied, indicating its application in food and medicine. The invention belongs to the technical field of microbial enzyme engineering. Background technique [0002] Cholesterol is an indispensable organic component in the human body, and the concentration of cholesterol in the blood is an important physiological indicator that can reflect the health status of the human body. Excessive cholesterol content in food will also have a negative impact on people's health. It is precisely because of the discovery of cholesterol oxidase in microbial cells that there are a series of fast, simple and automatic enzymatic methods for determining the conc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12P33/00
Inventor 王武季文明陈毅力吕陈峰陈亮韩振芳
Owner JIANGNAN UNIV
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