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Method for synthesizing betulic acid by carrying out biocatalysis on betulin

A technology of betulin alcohol and betulinic acid, which is applied in the fields of bioengineering and microbial fermentation, can solve the problems of affecting the catalytic effect of enzymes on substrates, microbial cell toxicity, long conversion reaction time, etc., so as to shorten the catalytic reaction time and conversion time. Short, easy to recycle effect

Inactive Publication Date: 2010-05-19
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Due to the low solubility of the substrate solution in the organic phase in the aqueous phase system, it not only affects the catalysis of the enzyme on the substrate, but also has certain toxicity to the microbial cells, resulting in the synthesis of betulin from betulin by the strains and fermentation conditions obtained in previous experiments. The yield of acid is still low and the conversion reaction time is very long

Method used

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  • Method for synthesizing betulic acid by carrying out biocatalysis on betulin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1～8

[0035] Examples 1-8 Synthesis of betulinic acid from betulin catalyzed by the fungus ZJUQH in different two-phase systems

[0036] (1) Insert Armillaria chrysanthemum ZJUQH into sterilized potato dextrose agar medium (its raw material mass percentage is composed of: potato 20%, glucose 2% and agar 3%), at 28 ℃, after 4 days of activation, prepare Become Armillaria chrysanthemum ZJUQH spore suspension, then insert potato glucose liquid culture medium (its raw material mass percentage is made up of: potato 20%, glucose 2%, surplus is water, pH is natural), at 28 ℃, rotating speed is 120r / min rotary shaker for 3 days to obtain seed culture solution. The concentration of spores in Armillaria chrysanthemum ZJUQH spore suspension was 1×10 6 individual / ml, the volume ratio of the potato dextrose liquid medium to the Armillaria chrysanthemum ZJUQH spore suspension was 30.

[0037] The seed culture solution was refrigerated and centrifuged at 3000 r / min for 30 minutes, washed with st...

Embodiment 9～14

[0046] Examples 9-14 Synthesis of betulinic acid catalyzed by Clostridium chrysogenum ZJUQH in a two-phase system with different ionic liquid concentrations

[0047] (1) The preparation of Armillaria chrysanthemum ZJUQH wet cells is the same as in Example 1.

[0048] (2) Add 5 mL of ionic liquid or a mixed solvent of ionic liquid and n-hexane into a stoppered test tube to form a two-phase system. The volume concentration of the ionic liquid in the two-phase system is 3% to 100%. ZJUQH wet cells, the amount of ZJUQH wet cells of Clostridium chrysogenum chrysogenum ZJUQH is 200 g per liter of the two-phase system, pre-cultured on a rotary shaker at 28° C. with a rotation speed of 200 r / min for 10 minutes to obtain a pre-culture system.

[0049] (3) Add 0.05 mL of dimethyl sulfoxide solution with a concentration of 7.5 mg / mL betulin to the above pre-culture system, and continue to ferment and culture on a rotary shaker at 28° C. with a rotation speed of 200 rpm for 18 hours to ob...

Embodiment 15～21

[0054] Examples 15-21 Synthesis of betulinic acid catalyzed by Cyclobacter chrysogenum ZJUQH in different co-substrates

[0055] (1) The preparation of Armillaria chrysanthemum ZJUQH wet cells is the same as in Example 1.

[0056] (2) Add 5mL of ionic liquid [EMIM]BF to a stoppered test tube 4 A mixed solvent with n-hexane forms a two-phase system, the volume concentration of the ionic liquid in the two-phase system is 50%, and a co-substrate is added to the two-phase system at the same time, and the amount of the co-substrate added per liter of the two-phase system is 0.1mol. Add the above-mentioned C. chrysogenum ZJUQH wet cells under aseptic conditions. The amount of C. chrysogenum ZJUQH wet cells is 200 g per liter of the two-phase system. Pre-cultivate on a rotary shaker at 28 ° C for 10 min at a speed of 200 r / min to obtain pre-cultured system.

[0057] (3) Add 0.05 mL of dimethyl sulfoxide solution with a concentration of 7.5 mg / mL betulin to the above pre-culture sys...

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Abstract

The invention discloses a method for synthesizing betulic acid by carrying out biocatalysis on betulin, comprising the following steps: (1) preparing armillaria luteo-uirens ZJUQH wet cells; (2) adding armillaria luteo-uirens ZJUQH wet cells into ionic liquid or a two-phase system for pre-culture to obtain a pre-culture system; (3) adding betulin solution in the pre-culture system for fermentation culture for 6-24 hours at 25-30 DEG C to obtain transformed culture solution; (4) processing the transformed culture solution to obtain betulic acid. The two-phase system is mixed solvent of ionic liquid and normal hexane, or mixed solvent of ionic liquid and phosphoric acid buffer solution. Compared with a conventional method, the inventive method has higher product yield, shorter catalytic reaction time, simple product separation, lower volatility of ionic liquid system, better biocompatibility and no pollution to the environment.

Description

technical field [0001] The invention relates to the fields of bioengineering and microbial fermentation, in particular to a method for biocatalyzing betulin to synthesize betulinic acid. Background technique [0002] Biocatalysis is the process of using enzymes or microbial cells to catalyze certain chemical reactions. It has the advantages of strong specificity, high catalytic efficiency, environmental friendliness, and mild operating conditions. Compared with biocatalytic reactions in aqueous phase, non-aqueous phase biocatalysis has its unique advantages. However, traditional organic solvents usually limit the activity and selectivity of enzymes, and cause a lot of industrial pollution. [0003] Ionic liquids (ILs), as a new type of green solvent, are salts composed of organic cations and inorganic anions or organic anions, which are liquid at room temperature or lower temperature (<100°C), have negligible vapor pressure, It has the characteristics of stability, high...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P33/00C12R1/645
CPCY02P20/54Y02P20/582
Inventor 陈启和刘婧傅明亮何国庆刘晓杰章海锋
Owner ZHEJIANG UNIV
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