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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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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 effective contact between steroids and biological enzymes very difficult, seriously affecting the speed and yield of steroid conversion reactions

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

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Embodiment 1

[0042]A method for preparing cholestenone using eutectic as a solubilizing agent by transforming resting cells. In this embodiment, the catalytic conversion effect of different types of DES is detected by changing the mixing ratio of DES, as follows:

[0043] ⑴ Scrape Arthrobacter simplex into sterile water, shake and mix well, and add it into a 50ml / 250ml shake flask for reaction according to the concentration of OD 0.440. The culture time of strain fermentation is 18~-22h, the temperature is 30°C, and the rotation speed of the shaker is 160rpm.

[0044] (2) After the cells are cultured, collect the cells by centrifugation, and resuspend the cells in 20ml of phosphate buffer to make the concentration 0.4g DW / mL. Put 5‰ of substrate cholesterol into the conversion solution, and add eutectic to carry out microbial conversion reaction, wherein the added eutectic is choline chloride / ethylene glycol mixed molar ratio of 1:2, 1:1 , 2:1, or the mixed molar ratio of choline chlori...

Embodiment 2

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

[0051] ⑴ Scrape Arthrobacter simplex into sterile water, shake and mix well, and add it into a 50ml / 250ml shake flask for reaction according to the concentration of OD 0.440. The culture time of strain fermentation is 18~-22h, the temperature is 30°C, and the rotation speed of the shaker is 160rpm.

[0052] (2) After the bacteria are cultured, collect the bacteria by centrifugation to make the concentration 0.4g DW / mL Use 20ml phosphate buffer to resuspend the cells. Put 5‰ of the substrate cholesterol into the conversion liquid, and add the eutectic choline chloride / urea (molar ratio is 1:1) for conversion, wherein the eutectic solvent accounts for 100% of the DES / phosphate buffer system The component cont...

Embodiment 3

[0055] A method for preparing cholestenone by using eutectic as a solubilizing agent by transforming resting cells. In this embodiment, by changing the substrate concentration, the catalytic conversion effect of the substrate concentration change is detected, as follows:

[0056] ⑴ Scrape Arthrobacter simplex into sterile water, shake and mix well, and add it into a 50ml / 250ml shake flask for reaction according to the concentration of OD 0.440. The culture time of strain fermentation is 18-22h, the temperature is 30°C, and the rotation speed of the shaker is 160rpm.

[0057] (2) After the cells are cultured, collect the cells by centrifugation, and resuspend the cells in 20ml of phosphate buffer to make the concentration 0.4g DW / mL. Put the substrate cholesterol into the conversion liquid, wherein the substrate addition amount is 1‰, 5‰, 1%, 2%, 3%, and add 2% choline chloride / urea (molar ratio is 1:1) low The co-melt undergoes microbial transformation reactions. The conve...

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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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