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Method for preparing cycloastragenol by double-enzyme compounding conversion of astragaloside

A technology of astragaloside IV and cycloastragenol, which is applied in the field of preparation of cycloastragenol, can solve the problems of unclear mechanism of enzyme action, low tolerance of enzyme substrate, and low efficiency of separation and purification, and achieve high product yield and bottom High drug tolerance and high separation and purification efficiency

Active Publication Date: 2020-11-06
WEIHAI BAIHE BIOTECH +1
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0005] The purpose of the present invention is to provide a method for preparing cycloastragenol by using double enzymes to convert astragaloside IV, which solves the problems of environmental pollution, cumbersome steps, high energy consumption and low tolerance of enzyme substrates in the existing preparation method of cycloastragenol , The mechanism of enzyme action is not clear, the efficiency of the enzyme is low, and the efficiency of separation and purification is low.

Method used

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  • Method for preparing cycloastragenol by double-enzyme compounding conversion of astragaloside
  • Method for preparing cycloastragenol by double-enzyme compounding conversion of astragaloside
  • Method for preparing cycloastragenol by double-enzyme compounding conversion of astragaloside

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Embodiment 1

[0086] The preparation of embodiment 1 xylosidase Xyl-G9

[0087] Insert the recombinant Pichia pastoris GS115-Xyl-G9 with the Xyl-G9 gene SEQ ID NO.3 into 1L of BMMG medium and culture it at 30°C and 220r / min for 12h; then the enriched bacteria The body was washed and inserted into 5LBMMY medium, cultured at 30°C and 220r / min, and 1% volume fraction of methanol was added every 24h to induce enzyme production. After culturing for 5 days, the fermentation broth was filtered to remove bacteria, and the obtained fermentation supernatant was purified by a nickel column to obtain 100 mL of pure recombinant xylosidase Xyl-G9. The product of 150KDa was detected by electrophoresis. Electrophoresis results see figure 1 .

Embodiment 2

[0088] The preparation of embodiment 2 glucosidase Bgcm

[0089] The recombinant Escherichia coli BL21-Bgcm containing the Bgcm gene was inoculated into 1 L of LB medium, and the fermentation was induced for 24 hours at 20°C in IpTG containing 1‰ volume fraction. After the fermentation, the recombinant cells were collected at 6000r / min, and the cells were resuspended using 200mL of 20mM pH 6.0 phosphate buffer, then ultrasonically disrupted and centrifuged to obtain 200mL of glucosidase Bgcm crude enzyme solution, and then purified by a nickel column to obtain 50mL of Pure Glucosidase Bgcm. The product of 78KDa was detected by electrophoresis. Electrophoresis results see image 3 .

Embodiment 3

[0090] The preparation of embodiment 3 composite enzymes

[0091] 100 mL of pure recombinant xylosidase Xyl-G9 and 50 mL of pure glucosidase Bgcm were freeze-dried respectively to obtain 252 mg of powdery recombinant xylosidase Xyl-G9 and 356 mg of powdery recombinant xylosidase Bgcm. Then get 200mg of the above-mentioned powdery xylosidase Xyl-G9 and 100mg of the above-mentioned powdery glucosidase Bgcm and mix to prepare 300mg of compound enzyme.

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Abstract

The invention relates to the technical field of biotransformation, in particular to a method for preparing cycloastragenol through double-enzyme compounding transformation of astragaloside. Accordingto the method, the astragaloside is used as a substrate, xylosidase and glucosidase are subjected to double-enzyme compounding, then xyloside bonds at the C3 position of the substrate and glucoside bonds at the C6 position of the substrate are broken through one-step hydrolysis, and the cycloastragenol is obtained. The purity of the obtained cycloastragenol can reach 98% or above, and the method is easy to operate, free of pollution, milder in reaction temperature, clear in enzyme conversion mechanism, and wider in enzyme substrate adaptability and is suitable for industrial production.

Description

technical field [0001] The invention relates to the technical field of biotransformation, in particular to a method for preparing cycloastragenol by using double enzymes to convert astragaloside IV. Background technique [0002] Astragaloside IV is the main active ingredient of the traditional Chinese medicine Astragalus, and cycloastragenol (CA) is the aglycon of Astragaloside IV. Cycloastragenol can prolong telomeres by activating telomerase, so as to resist aging and improve cell proliferation. [0003] The chemical formula of cycloastragenol is C 30 h 50 o 5 , The relative molecular mass is 490.71, it is a colorless needle crystal, easily soluble in methanol, n-butanol, etc. The content of cycloastragenol in natural plants is very small, so the production of cycloastragenol is mainly through the hydrolysis of astragaloside IV 3 position of xylosidic bond and C 6 positional glucosidic bond implementation. [0004] There are many methods for preparing cycloastrageno...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P33/20C12P33/06C12N9/24C12N9/42C12N15/81C12N15/70C12R1/19C12R1/84
CPCC12P33/20C12P33/06C12N15/815C12N15/70C12N9/2445C12N9/248C12Y302/01021
Inventor 袁其朋程磊雨梁浩魏斌刘旭东石晔张军杰孙志强
Owner WEIHAI BAIHE BIOTECH
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