Method for preparing cholestenone by using eutectic mixture as solubilization promoting agent through resting cell transformation

A technology of resting cell transformation and eutectic matter, applied in fermentation and other directions, can solve the problems of difficult contact between steroids and biological enzymes, affecting the speed and yield of steroid transformation reactions, low solubility, etc. It is of great significance to achieve industrialization , Mild operating conditions, the effect of improving the dispersion rate

Inactive Publication Date: 2015-02-18
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Claims
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Problems solved by technology

The solubility of this class of substrates in water is very low, in the range of 10 -5 ~10 -6 mol/L, this poor solubility makes the effec

Method used

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  • Method for preparing cholestenone by using eutectic mixture as solubilization promoting agent through resting cell transformation
  • Method for preparing cholestenone by using eutectic mixture as solubilization promoting agent through resting cell transformation
  • Method for preparing cholestenone by using eutectic mixture as solubilization promoting agent through resting cell transformation

Examples

Experimental program
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Example Embodiment

[0041] Example 1:

[0042] A method for preparing cholestenone by transforming resting cells using eutectic as a solvent. In this example, the catalytic conversion effects of different types of DES are tested by changing the mixing ratio of DES, as follows:

[0043] ⑴ Scrape a loop of simple Arthrobacter into sterile water, shake and mix, and add it to a 50ml / 250ml shake flask at a concentration of 0.440 for the reaction. The fermentation time of the strain is 18-22h, the temperature is 30℃, and the rotating speed of the shaker is 160rpm.

[0044] ⑵After culturing the bacteria, collect the bacteria by centrifugation, and resuspend the bacteria with 20ml phosphate buffer to make the concentration 0.4g DW / mL. Put 5‰ of the substrate cholesterol into the conversion solution, and add the eutectic to carry out the microbial conversion reaction. The added eutectic is choline chloride / ethylene glycol mixed with a molar ratio of 1:2, 1:1 , 2:1, or choline chloride / glycerin mixed molar rat...

Example Embodiment

[0049] Example 2:

[0050] A method for preparing cholestenone by transforming resting cells using eutectic as a solvent. In this example, the catalytic conversion effect of different concentrations of DES is tested by changing the percentage of DES, as follows:

[0051] ⑴ Scrape a loop of simple Arthrobacter into sterile water, shake and mix, and add it to a 50ml / 250ml shake flask at a concentration of 0.440 for the reaction. The fermentation time of the strain is 18-22h, the temperature is 30℃, and the rotating speed of the shaker is 160rpm.

[0052] ⑵After culturing the bacteria, collect the bacteria by centrifugation to make the concentration of 0.4g DW / mL Use 20ml phosphate buffer to resuspend the bacteria. Put 5‰ of the substrate cholesterol into the conversion solution, and add the eutectic choline chloride / urea (molar ratio 1:1) for conversion. The eutectic solvent accounts for 100% of the DES / phosphate buffer system. The sub-contents are 0%, 2%, 4%, 6%, 8%. The conversio...

Example Embodiment

[0054] Example 3:

[0055] A method for preparing cholestenone by using a eutectic as a solvent to transform resting cells. In this embodiment, the substrate concentration is changed to detect the catalytic conversion effect of the substrate concentration change, as follows:

[0056] ⑴ Scrape a loop of simple Arthrobacter into sterile water, shake and mix, and add it to a 50ml / 250ml shake flask at a concentration of 0.440 for the reaction. The fermentation time of the strain is 18-22h, the temperature is 30℃, and the rotating speed of the shaker is 160rpm.

[0057] ⑵After culturing the bacteria, collect the bacteria by centrifugation, and resuspend the bacteria with 20ml phosphate buffer to make the concentration 0.4g DW / mL. Put the substrate cholesterol into the conversion solution, where the substrate addition amount is 1‰, 5‰, 1%, 2%, 3%, and 2% choline chloride / urea (molar ratio 1:1) is added. The eutectic undergoes microbial transformation reactions. The conversion time is 4...

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Abstract

The invention relates to a method for preparing cholestenone by using a eutectic mixture as a solubilization promoting agent through resting cell transformation. The method comprises the steps of (1) performing centrifugation after resting cells are cultivated in a fermentation medium, and adding a sterile phosphate buffer solution for re-suspension so as to be used as transformation liquid, wherein the resting cells have the capacity of preparing cholestenone by oxidizing cholesterol; (2) feeding cholesterol into the transformation liquid, adding the eutectic mixture serving as the solubilization promoting agent, extracting the transformation liquid by ethyl acetate, and detecting the yield of a product 4-cholesten-3-one by an HPLC (high performance liquid chromatography) method; (3) separating the substrate cholesterol from the product cholestenone through a column chromatography separation method, and purifying to obtain a pure cholestenone product. According to the method, the eutectic mixture (DES) replaces an organic solvent such as methyl alcohol and serves as a green additive; after the eutectic mixture is added, the dispersion rate of the substrate is increased, contact of the substrate cholesterol and bacteria is effectively promoted, and the transformation efficiency of the resting cells is effectively improved; in adding and transforming processes, the operation conditions are mild, and the steps are simple.

Description

technical field [0001] The invention belongs to the technical field of microbial transformation, in particular to a method for preparing cholestenone by transformation of resting cells by using eutectic as a solubilizing agent. technical background [0002] Cholester-4-en-3-one (4-Cholesten-3-one), also known as cholestenone, molecular formula C 27 h 44 O, is one of the important products of cholesterol obtained by means of chemical synthesis and microbial transformation, and is an oxidation product of cholesterol. Cholesterenone can be used to treat liver disease, can be used as a weight loss accelerator, and has the functions of preventing skin keratinization and the like. In addition, the specific structure of cholestenone can be used as an intermediate of steroidal drugs and is widely used in the pharmaceutical industry. [0003] It has been reported in the literature that Fusarium spp. screened from soil samples optimized the culture medium and fermentation condition...

Claims

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Application Information

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IPC IPC(8): C12P33/02
Inventor 路福平毛淑红于璐吉少娴张会图刘晓光
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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