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Strain capable of efficiently converting phytosterol and application thereof

A phytosterol, high-efficiency technology, applied in the biological field, can solve the problems of low solubility, low substrate feeding concentration and conversion efficiency, and high cost

Inactive Publication Date: 2017-01-04
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Both AD and phytosterols are fat-soluble compounds with extremely low solubility in the water phase, resulting in very low substrate concentration and conversion efficiency during the conversion process, and the cost is much higher than that of phytosterols. These problems directly become 9α-OH - Bottlenecks in AD industrial production
Therefore, it is of great significance to screen a microorganism that efficiently transforms phytosterols into 9α-OH-AD, but there is no report on the use of Mycobacterium sp. LY-1 for transforming phytosterols, which is extremely High conversion purity of 9α-OH-AD is a great advantage for industrial production

Method used

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  • Strain capable of efficiently converting phytosterol and application thereof
  • Strain capable of efficiently converting phytosterol and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1 Isolation and Identification of Mycobacterium sp. LY-1 and Preservation of the Strains

[0016] (1) Isolation and screening of Mycobacterium sp. LY-1

[0017] The present invention obtains soil samples from around the chemical factory building of domestic synthetic steroid compounds, weighs 1.0-2.0g of soil samples in a 250mL triangular flask, suspends them with 10-50mL of physiological saline, then places them in a water bath, and treats them at 60°C for 10min , after cooling, perform gradient dilution, take 0.2mL of the diluted solution and spread it on a solid plate with phytosterol as the only carbon source for primary screening. After culturing at 30°C for 120 h, a single colony with better growth was selected for fermentation verification. Pick a single colony and inoculate it into a fresh liquid seed medium, cultivate it at 30°C and 90-150r / min for 72h, then insert the inoculation amount of 1% (w / w) into the fermentation medium with a liquid volume of ...

Embodiment 2

[0020] Example 2 Transformation of phytosterols using mycobacterium CGMCC No.13031 (Mycobacterium sp.) LY-1

[0021] (1) Preparation of cell liquid culture of mycobacterium CGMCC No.13031 (Mycobacterium sp.) LY-1 strain

[0022] Pick a ring of Mycobacterium sp. LY-1 strain on the solid PDA medium, inoculate it in a 500mL Erlenmeyer flask containing 100mL seed medium, place it on a shaker at 120r / Cultivate at a rotational speed of 60-72 hours to the logarithmic phase, that is, to prepare a liquid cell culture of Mycobacterium sp. LY-1 strain.

[0023] (2) The composition and ratio of the fermentation medium are: phytosterol 5-20g / L, NaNO 3 2-10g / L, corn steep liquor 5-25g / L, (NH 4 ) 2 HPO 4 0.2-1.5g / L, pH 7.5-8.5, sterilized under high-pressure steam at 121°C for 20min.

[0024] (3) shake flask fermentation: the cell liquid culture of above-mentioned mycobacterium CGMCC No.13031 (Mycobacterium sp.) LY-1 bacterial strain is inoculated with the inoculation amount of 0.5-1...

Embodiment 3

[0025] Example 3 Under the two-phase fermentation system of soybean oil and water, mycobacterium CGMCC No.13031 (Mycobacterium sp.) LY-1 transforms phytosterols

[0026] (1) Preparation of cell liquid culture of mycobacterium CGMCC No.13031 (Mycobacterium sp.) LY-1 strain

[0027]Pick a ring of mycobacterium (Mycobacterium sp.) LY-1 strain on the solid PDA medium, inoculate it in a 500mL Erlenmeyer flask containing 100mL seed medium, and place it on a shaker at 120r / h at 30°C. Cultivate at a rotational speed of 60-72 hours to the logarithmic phase, that is, to prepare a cell liquid culture of Mycobacterium CGMCC No.13031 (Mycobacterium sp.) LY-1 strain.

[0028] (2) Fermentation system with Tween 80 added: 0.05% Tween 80 was added to the fermentation medium, and ultrasonically oscillated for 30 minutes.

[0029] (3) The composition and ratio of the fermentation medium are: phytosterol 5-20g / L, NaNO 3 2-10g / L, corn steep liquor 5-25g / L, (NH 4 ) 2 HPO 4 0.2-1.5g / L, pH 7.5...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to mycobacterium sp. LY-1 capable of efficiently converting phytosterol and application thereof. The strain is preserved in the common microorganism center of China Committee for Culture Collection of Microorganisms, and the preservation number is CGMCC No.13031. The mycobacterium sp. LY-1 is capable of converting phytosterol into 9 alpha-hydroxy androstane-4-alkenyl-3,17-diketone, and the product yield is 33% under the condition that the substrate feed amount is 15 g / L.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a Mycobacterium sp. LY-1 strain and a method for transforming phytosterols into 9α-hydroxyandrost-4-ene-3,17-dione. Background technique [0002] 9α-Hydroxyandrost-4-ene-3,17-dione (9α-OH-AD) is the precursor of an important class of steroid hormone drugs, and its 9-position is hydroxylated, after a simple Halogenation reaction can introduce halogen substituents such as F or Cl, thereby effectively improving the medicinal efficacy of certain corticosteroids. Due to the serious industrial pollution caused by the traditional chemical synthesis method, the low yield and high cost have greatly restricted the development of its synthesis, so the use of microbial transformation has gradually become the main development trend in recent years. Therefore, the screening and enrichment of strains that can effectively accumulate 9α-OH-AD has become one of the focuses of steroid drug development...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P33/00C12R1/32
CPCC12P33/00C12N1/205C12R2001/32
Inventor 宋宇迪许正宏史劲松李会王萌慧董玉秀张晓娟
Owner JIANGNAN UNIV
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