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.
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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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