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Endophytic fungus strain for efficient biotransformation of betulinic acid and application of endophytic fungus strain

A technology of biotransformation and betulin alcohol, applied in the field of bioengineering, can solve the problems of high price, high price of betulinic acid, low yield and low purity of betulinic acid, etc.

Active Publication Date: 2019-12-03
NORTHEAST FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the complexity of the triterpenoid synthesis pathway and the low activity of plant-derived enzymes in microorganisms, there are still many challenges in the microbial synthesis of plant triterpenoids.
[0004]At this stage, the synthesis of betulinic acid all depends on the redox preparation of betulin. This method is expensive and the yield and purity of betulinic acid are low. lower, resulting in high prices of betulinic acid in the market

Method used

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  • Endophytic fungus strain for efficient biotransformation of betulinic acid and application of endophytic fungus strain
  • Endophytic fungus strain for efficient biotransformation of betulinic acid and application of endophytic fungus strain
  • Endophytic fungus strain for efficient biotransformation of betulinic acid and application of endophytic fungus strain

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Experimental program
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Effect test

Embodiment 1

[0051] Example 1, Isolation, Screening and Identification of Phomopsis WDS2

[0052] 1. Isolation and screening of endophytic fungi

[0053] Collect pea plants in Acheng District, Heilongjiang Province, wash the collected materials continuously with tap water, carry out explant disinfection and endophytic fungus isolation and culture, the specific method refers to Gao Jian (Gao Jian. Diversity and ecological distribution of endophytic fungi in mangroves[D] . Guangdong Ocean University, 2013.). Endophytic fungi were isolated and cultivated using the method of Yuan Zhilin from Zhejiang University (Yuan Zhilin. Endophytic fungal resource mining, phylogenetic analysis and preliminary research on the function of wild rice verruca [D]. Zhejiang University, 2010.) to obtain endophytic fungi WDS2. The inventor of the present invention has also isolated other 111 strains of endophytic fungi from soybeans, tamarisk branches, bark and birch branches, leaves, and bark. These 112 strains...

Embodiment 2

[0070] Example 2 Highly efficient transformation of betulinic acid endophytic fungus culture time optimization

[0071] Using Phomopsis WDS2 as the material, the mycelium was picked and cultured in 500ml PDB medium for 2 days to prepare a first-grade seed solution. 2 ml of 5 mg / ml betulin was added to 98 ml of PDB medium to obtain a substrate medium with a final concentration of betulin of 0.1 mg / ml. Draw 10ml of seed liquid into the substrate medium, place it on a shaker at a speed of 120r / min, and ferment in dark culture (wrapped in tin foil) at 25°C. Take 10ml of fermentation broth and freeze-dry it in a freeze dryer, add 5ml of absolute ethanol for ultrasonic extraction for 6h, repeat twice, combine the organic phase, evaporate to dryness in a water bath at 40°C, and redissolve in 2ml of methanol to obtain a sample solution. The HPLC method detects the content of betulinic acid in the sample liquid. Repeat 3 times for each group.

[0072] The results showed that Phomops...

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Abstract

The invention discloses an endophytic fungus strain for efficient biotransformation of betulinic acid and application of the endophytic fungus strain. The invention firstly provides a Phomopsis sp. WDS2 strain, and the preservation number of the Phomopsis sp. WDS2 in China Center for Type Culture Collection is CCTCC NO: M 20199394. The invention further provides a microbial inoculum containing thePhomopsis sp. WDS2 and application of the microbial inoculum. The Phomopsis sp. WDS2 disclosed by the invention has the capability of efficiently and biologically converting betulin into betulinic acid; the betulinic acid conversion amount reaches 23.5 mg / L, so that the Phomopsis sp. WDS2 provides an excellent bacterial strain material for the development of anticancer and antiviral products in the future, and has an important value.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to an endophytic fungal strain for highly efficient biotransformation of betulinic acid and its application. Background technique [0002] The main source of plant triterpenoids is to extract from plants, the use of a large amount of acid and alkali and organic solvents brings serious environmental problems, and the cultivation of plants takes a long time, in addition the content of triterpenoids in plants is low, and There are more structural analogs, resulting in high extraction costs. Betulin (Betulin) and betulinic acid (Betulinic acid, BA) are important anticancer and antiviral pentacyclic triterpenoid natural compounds, which have important value and significance in the treatment of human diseases, and betulin and betulin The drug effect of acid is different from other anti-tumor drugs, and its strong targeting and non-toxicity to normal cells make it more ...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12P33/00A61K31/56A61P35/00A61P31/12C12R1/66
CPCC12N1/14C12P33/00A61K31/56A61P35/00A61P31/12C12R2001/66C12N1/145
Inventor 尹静詹亚光肖佳雷徐志强孙建广张玉琦王宇李影
Owner NORTHEAST FORESTRY UNIVERSITY
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