A method for preparing cycloastragaloside by compound transformation of astragaloside IV with double enzymes

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

Active Publication Date: 2022-07-22
WEIHAI BAIHE BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • A method for preparing cycloastragaloside by compound transformation of astragaloside IV with double enzymes
  • A method for preparing cycloastragaloside by compound transformation of astragaloside IV with double enzymes
  • A method for preparing cycloastragaloside by compound transformation of astragaloside IV with double enzymes

Examples

Experimental program
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Effect test

Embodiment 1

[0086] Example 1 Preparation of xylosidase Xyl-G9

[0087] The recombinant Pichia GS115-Xyl-G9 integrated with the Xyl-G9 gene SEQ ID NO.3 was inserted into 1L of BMMG medium, and cultured at 30°C and 220r / min for 12h; The body was washed and transferred to 5LBMMY medium and 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 bacterial cells, and the obtained fermentation supernatant was purified by nickel column to obtain 100 mL of pure recombinant xylosidase Xyl-G9. The product of 150KDa was obtained by electrophoresis. For 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 in IpTG containing 1‰ volume fraction at 20 °C for 24 h. After the fermentation, the recombinant cells were collected at 6000 r / min, and the cells were resuspended using 200 mL of 20 mM pH 6.0 phosphate buffer, then sonicated and centrifuged to obtain 200 mL of glucosidase Bgcm crude enzyme liquid, and then purified by nickel column to obtain 50 mL of glucosidase Bgcm. Pure Glucosidase Bgcm. The product of 78KDa was obtained by electrophoresis. For electrophoresis results see image 3 .

Embodiment 3

[0090] Example 3 Preparation of complex enzyme

[0091] 100 mL of pure recombinant xylosidase Xyl-G9 and 50 mL of pure glucosidase Bgcm were freeze-dried to obtain 252 mg of powdered recombinant xylosidase Xyl-G9 and 356 mg of powdered recombinant xylosidase Bgcm. Then, 200 mg of the above powdered xylosidase Xyl-G9 and 100 mg of the above powdered glucosidase Bgcm were mixed to prepare 300 mg of a composite enzyme.

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Abstract

The invention relates to the technical field of biotransformation, in particular to a method for preparing cycloastragalol by compound transformation of astragaloside IV with double enzymes. The method of the present invention takes astragaloside IV as a substrate, utilizes xylosidase and glucosidase double-enzyme complex, and then undergoes one-step hydrolysis to cleave the substrate C. 3 position of the xylosidic bond and C 6 The method for obtaining cycloastragalus by the position of the glucosidic bond. The purity of cycloastragalol obtained by the invention can reach more than 98%, and the method has simple operation, no pollution, milder reaction temperature, clear enzymatic transformation mechanism and wider 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 cycloastragaloside by utilizing double-enzyme compound transformation of astragaloside IV. Background technique [0002] Astragaloside IV is the main active component of traditional Chinese medicine Astragalus, and cycloastragaloside (CA) is the aglycone of astragaloside IV. Cyclo astragalus can prolong telomeres by activating telomerase, so as to achieve the effect of resisting aging and improving the ability of cell proliferation. [0003] The chemical formula of cycloastragalus is C 30 H 50 O 5 , The relative molecular mass is 490.71, it is a colorless needle-like crystal, easily soluble in methanol, n-butanol, etc. The content of cycloastragalus in natural plants is very small, so the production of cycloastragalus is mainly through the hydrolysis of astragaloside C 3 position of the xylosidic bond and C 6 position of the glucosidic bond ...

Claims

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Application Information

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Patent Type & Authority Patents(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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