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Method for synthesizing betulinic acid by transforming betulin into betulin from silvery mildew

The technology of C. japonica and betulin is applied in the fields of bioengineering and microbial fermentation, and can solve the problems of difficulty in further improving the yield of betulinic acid, long conversion reaction time, low yield of betulinic acid, etc. Yield, short conversion time, mild effect

Inactive Publication Date: 2011-12-21
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The yield of betulinic acid in this patented method is still low, the conversion reaction time is longer, and there are many additional products, and because the conversion mechanism is not clear, it is difficult to further increase the yield of betulinic acid in the future

Method used

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  • Method for synthesizing betulinic acid by transforming betulin into betulin from silvery mildew
  • Method for synthesizing betulinic acid by transforming betulin into betulin from silvery mildew
  • Method for synthesizing betulinic acid by transforming betulin into betulin from silvery mildew

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Conversion of betulin to betulin to betulinic acid by direct growth transformation method

[0036] (1) Preparation of Cunninghamella brevisiae AS 3.910 spore suspension: The Cunninghamella brevisiae AS 3.910 (purchased from the General Microbiology Center of the China Microorganism Collection Management Committee) preserved at 4°C was inoculated on potato agar On a slant medium, activate and culture at 28°C for 6 days; on an ultra-clean table, scrape the activated Cunninghamella brevisiae AS 3.910 with an inoculation needle into a cone containing 100 mL sterile water and several glass beads In the bottle, shake to prepare a spore suspension of Cunninghamella brevisiae AS 3.910, in which the concentration of spores of Cunninghamella brevis AS 3.910 is 1×10 6 Pcs / ml.

[0037] (2) Preparation of pre-culture system: Take 20g of glucose, 5g of yeast extract, 5g of peptone, and 5g of dipotassium phosphate, add 1000mL of water, heat and mix, adjust the pH to 6.5 to obtain...

Embodiment 2

[0041] Example 2 Using resting cell transformation method to convert betulin to betulinic acid

[0042] (1) Preparation of Cunninghamella brevisiae AS 3.910 wet cells: Take 1 mL of Cunninghamella brevisiae AS 3.910 spore suspension (the spore concentration is 1×10 6 A / ml) was added to 30mL growth medium, cultured in a shaker at 28°C and rotating speed 180rpm for 2 days, then stopped the culture, centrifuged at 3000rpm for 30 minutes, washed with distilled water 3 times, and removed the culture solution to obtain Cunninghamella brevisiae AS3.910 wet cell.

[0043] Among them, the preparation method of the Cunninghamella brevisiae AS 3.910 spore suspension and the composition of the growth medium are the same as those in Example 1.

[0044] (2) Preparation of the pre-culture system: On the ultra-clean table, 5g of Cunninghamella brevisiae AS 3.910 wet cells were inserted into 30 mL of 2% glucose-containing phosphate buffer solution (pH 6) to obtain the pre-culture system.

[0045] (3) T...

Embodiment 3-5

[0052] Example 3-5 The Influence of Different Glucose Concentrations in Growth Medium on the Synthesis of Betulinic Acid by Direct Growth Transformation

[0053] Referring to the method of Example 1, in step (2), each liter of growth medium does not contain or contains 20g and 40g glucose, that is, the glucose concentration in the growth medium is 0, 2%, 4%, and the rest of the steps are the same ; Determine the content of betulin and betulinic acid in the sample, and the specific results are shown in Table 2.

[0054] Table 2 The effect of different glucose concentrations in growth medium on the synthesis of betulinic acid by direct growth transformation method

[0055]

[0056] It can be seen from the results in Table 2 that the conversion rate of betulin increases first and then decreases when the glucose concentration is in the range of 0-4%, reaching a maximum of 98.6% when the glucose concentration is 2%; the conversion yield of betulinic acid has an increasing trend, but As t...

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Abstract

The invention discloses a method for synthesizing betulinic acid through converting cunninghamella blakesleeana into betulin, which comprises the following steps of introducing cunninghamella blakesleeana AS 3.910 into sterilized culture solution to obtain a pre-culture system; then, adding betulin solution and surfactants so that the concentration of the betulin reaches 0.01 to 0.1g / L; carrying out conversion reaction for 2 to 6 days at 25 to 30 DEG C to obtain conversion liquid; and obtaining the betulinic acid through separation and purification. The method provided by the invention uses the cunninghamella blakesleeana as a carrier and uses the betulin as substrates for synthesizing the betulinic acid through biotransformation, the process is simple, the condition is mild, the transformation period is short, the product yield is high, the environmental-friendly effect is realized, and the betulinic acid with a higher yield can be obtained.

Description

Technical field [0001] The invention belongs to the field of bioengineering and microbial fermentation, and particularly relates to a method for converting betulin into betulin to synthesize betulinic acid by Cunninghamia brevifolia. Background technique [0002] In the past 30 to 50 years, cancer treatment has mainly relied on radiotherapy and chemotherapy. These treatments inhibit cancer cells and damage normal cells. Therefore, it is very important to find cancer treatment drugs that have no side effects on normal cells. Studies have shown that betulin and its derivatives have this potential ability, especially betulinic acid, which has shown great potential in cancer treatment. Experiments have shown that it has a good inhibitory effect on malignant tumors in mice. Normal cells have extremely low toxicity and are the most potential new pharmaceutical preparations. Related research has become a hot spot in natural organic medicine research in recent years. [0003] Microbial tr...

Claims

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Application Information

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IPC IPC(8): C12P33/00C12R1/645
Inventor 陈启和冯宇刘婧何国庆傅明亮董亚晨
Owner ZHEJIANG UNIV
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