A method for converting cholesterol into cholest-4-en-3-one by using whole cells of recombinant bacillus subtilis

A cholesterol and recombinant vector technology, applied in the fields of genetic engineering and enzyme engineering, can solve problems such as potential safety hazards, and achieve the effects of easy control, high utilization rate of raw materials, and high product purity

Active Publication Date: 2016-11-23
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When ChOx from different sources is expressed in E. coli, since the E. coli strain itself is an opportunistic pathogen, there are certain safety risks in the heterologous expression of ChOx in E. coli

Method used

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  • A method for converting cholesterol into cholest-4-en-3-one by using whole cells of recombinant bacillus subtilis
  • A method for converting cholesterol into cholest-4-en-3-one by using whole cells of recombinant bacillus subtilis
  • A method for converting cholesterol into cholest-4-en-3-one by using whole cells of recombinant bacillus subtilis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1: Construction of recombinant B.subtilis168 bacterial strain

[0039] The new Mycobacterium aureus with cholesterol oxidase activity in the laboratory was used as the starting strain, and its chromosome was used as a template to obtain the gene of the enzyme by means of PCR, and then connected to the cloning vector to achieve a large amount of amplification of the gene. A large number of amplified KSDD gene fragments were purified and connected to the pMA5 vector. After successful verification, the model strain Bacillus subtilis 168 was transformed. Positive transformants were screened on the resistance plate, inoculated in shake flasks for fermentation, and the product cholest-4-en-3-one in the fermentation broth was detected by TLC and HPLC. The product cholest-4-en-3-one was detected, indicating that the recombinant strain capable of converting cholesterol into cholest-4-en-3-one was successfully constructed. The present invention finally obtains the B. ...

Embodiment 2

[0040] Embodiment 2: the enzyme activity assay of recombinant bacterial strain

[0041] The strain was cultured overnight in LB medium, centrifuged at 8000 rpm for 10 min, washed 3 times with 50 mM Tris-HCl buffer solution of pH 7.0, and resuspended in 5 ml of the buffer solution. Ultrasonic crushing with 40% power in an ice bath, working for 2 seconds and resting for 5 seconds, and working time for 10 minutes. Centrifuge at 10,000r / min for 30min to obtain the supernatant, which contains the target protein. The enzyme activity determination method is as follows: 3mL solution A (4-amino-antipyrine 1mmol / L; phenol 6mmol / L; sodium azide 0.2g / L; peroxidase 5000U / L; potassium phosphate buffer 25mmol / L , pH 7.5), 150 μL solution B (cholesterol 8.26g / L; Triton X-100 4.26%; isopropanol as solvent), 50 μL enzyme solution, reacted at 37°C for 5min, boiled water bath for 3min, and measured the absorbance value OD at 500nm 500 . Definition of enzyme activity unit: at 37°C, the amount o...

Embodiment 3

[0042] Example 3: Detection of whole cell transformation performance of recombinant strains

[0043] The bacterial strain was inoculated in 100mL LB medium with 1% inoculum, cultivated for 24h, recovered the thalli by centrifugation, washed twice, resuspended with an appropriate 50mM Tris-HCl (pH7.0) buffer, added 0.1% ( w / v) cholesterol and 0.1% (v / v) Tween-80, continue to culture at 37°C, 160rpm for 24h. After detection and analysis by HPLC, such as image 3 As shown, the final substrate molar conversion rates reached 67% and 83%, respectively, which were about 20 and 25 times higher than that of the original bacteria.

[0044]

[0045]

[0046]

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Abstract

The invention relates to a method for producing cholest-4-en-3-one through whole-cell transformation of cholesterol by using recombinant Bacillus subtilis and belongs to the field of genetic engineering and enzyme engineering. According to the method, the gene amplification of cholesterol oxidase (ChoM) in mycobacterium neoaurum is achieved firstly, and then, the overexpression of the gene in a type strain bacillus subtilis 168 is realized by using plasmid pMA5. According to the method, a bacillus subtilis engineering strain, which takes cholesterol as a substrate, takes whole-cell transformation as a method, is capable of highly yielding cholest-4-en-3-one is constructed, and researches on the enzyme activity and fermentation performance of the strain show that the activity of the cholesterol oxidase is obviously improved as compared with the activity of a starting strain, and the molar conversion rate of the substrate (0.1% (w / v) cholesterol) reaches 67% and 83% by carrying out whole-cell transformation for 24 hours and is 20 and 25 times the relative transformation rate of the starting strain. The method provides a beneficial guide for the industrialization of microbial one-step fermentation production of cholest-4-en-3-one.

Description

technical field [0001] The invention discloses a method for transforming cholesterol into cholest-4-en-3-one by using whole cells of recombinant Bacillus subtilis, belonging to the fields of genetic engineering and enzyme engineering. technical background [0002] Steroidal drugs can be obtained through total synthesis or degradation of natural steroidal compounds and transformation of their functional groups. Steroidal drugs have strong pharmacological effects such as anti-infection, just allergy, anti-virus and anti-shock. With the continuous development of the times, steroid drugs have become the second largest class of drugs after antibiotics. In 2000, the sales of steroid drugs in the global drug market have exceeded 20 billion US dollars, accounting for about 66% of the world's total pharmaceutical sales. %. [0003] Classification of steroid hormone drugs: adrenocortical hormones, including hydrocortisone, prednisone, etc. It can treat Addison's disease, anti-inflam...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/75C12N1/21C12P33/00C12R1/125
Inventor 饶志明许正宏邵明龙张显徐美娟杨套伟李会
Owner JIANGNAN UNIV
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