Method for preparing glycyrrhetinic acid through biological catalysis of novel glucuronidase

A technology of glucuronidase and immobilized enzyme catalyzing licorice, applied in biochemical equipment and methods, glycosylase, enzyme and other directions, can solve problems such as unfavorable industrial application, impure final product, cleavage of glycyrrhizic acid, etc. The effect of increasing difficulty and workload, reducing production costs and economical savings

Active Publication Date: 2021-01-15
BEIJING UNIV OF CHEM TECH
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Problems solved by technology

[0004]Most of the existing β-glucuronidases will break the two sugar chains of glycyrrhizic acid, resulting in the formation of the intermediate product monoglucuronyl glycyrrhetinic acid, so that the final The product is impure
Moreover, the large-scale use of free enzy

Method used

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  • Method for preparing glycyrrhetinic acid through biological catalysis of novel glucuronidase
  • Method for preparing glycyrrhetinic acid through biological catalysis of novel glucuronidase
  • Method for preparing glycyrrhetinic acid through biological catalysis of novel glucuronidase

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0031]Example 1

[0032]This example provides the screening and expression of β-glucuronidase, and the specific method is as follows: firstly, through the gene database NCBI, sequence comparison of the existing β-glucuronidase, which is widely used, is screened out. Β-glucuronidase from Aspergillus spp (85% amino acid sequence homology), gene number KAE8374379.1, the target gene (such asfigure 1 Shown), the primers are:

[0033]Primer KAE-F: 5'-GGGAAAGGATCCATTCCGACCGATGCGCAGA-3', restriction site is BamHI;

[0034]Primer KAE-R: 5'-GGGAAAGTCGACCTGGCACGCCTGGCCTTC-3', restriction site is SalI.

[0035]Then the target gene is connected with the expression vector plasmid peT28a by means of restriction enzyme digestion (such asfigure 2 ,image 3 As shown), introduce the expression bacteria-Escherichia coli BL21 (DE3), culture in 100 mL LB liquid medium, add the inducer IPTG at OD 0.6, induce at 16°C for 24 hours, centrifuge, and use 20 mL, 20 mM, pH 7 for the bacteria .0 phosphate buffer solution resu...

Example Embodiment

[0036]Example 2

[0037]This example constructed an immobilized enzyme Fe3O4@Zr-2MIm@KAE, the specific method is:

[0038]1) Expression vector Fe3O4@Zr-2MIm's construction

[0039]Will Fe3O4The microspheres (100 mg) were added to the methanol solution (50 ml, 1 mg / mL) dissolved in PVP, sonicated in an sonicator for 60 min, and the supernatant was removed by magnet adsorption. Under ultrasound, the precipitate was collected and dispersed in a methanol (25 ml, 40 mm) solution of 2-methylimidazole (2-MIm). Then, Zr(NO3)4·5H2A methanol solution of O (25 ml, 40 mm) was added to the solution. After standing for 10 minutes, collect with a magnet and rinse thoroughly with methanol solution. The precipitate was dried at 80°C for 8h.

[0040]2) Immobilized enzyme Fe3O4@Zr-2MIm@KAE's construction

[0041]Take the β-glucuronidase crude enzyme solution prepared in Example 1, 2 mL per tube, and divide them into centrifuge tubes. Add 3 mg of the constructed carrier Fe to each tube.3O4@Zr-2MIm, at 25 degrees Cels...

Example Embodiment

[0043]Example 3

[0044]This example uses the immobilized enzyme Fe constructed in Example 23O4@Zr-2MIm@KAE catalyzes the preparation of glycyrrhetinic acid from glycyrrhizic acid.Figure 5 As shown, the specific method is:

[0045]Weigh 10 mg of glycyrrhizic acid, dissolve it in 5ml pH5.0 20mM acetic acid-sodium acetate buffer, take 1ml of glycyrrhizic acid solution, and add the immobilized enzyme Fe to remove the supernatant phosphate buffer3O4In @Zr-2MIm@KAE, the reaction was carried out in a shaker at 60°C for 2h, and after 2h, the supernatant was separated by centrifugation at 12000rpm, and the precipitate was the product glycyrrhetinic acid.

[0046]Dissolve the obtained precipitate in 1mL methanol, such asFigure 6As shown, HPLC (methanol: 0.2% phosphoric acid 90:10, wavelength 254nm, flow rate 1mL / min, column temperature 35°C) detects the content of glycyrrhizic acid and glycyrrhetinic acid in the supernatant and precipitate, and the final conversion rate is 95%.

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Abstract

The invention provides a method for preparing glycyrrhetinic acid through biological catalysis of novel glucuronidase. The method comprises the following steps of screening and expressing novel recombinant glucuronidase through a genetic engineering means; by taking glycyrrhizic acid as a substrate, performing a catalytic reaction to obtain the glycyrrhetinic acid with higher purity; incubating beta-glucuronidase and Fe3O4@Zr-2MIm in a phosphate buffer solution environment to obtain an immobilized enzyme; and carrying out a catalytic reaction on the immobilized enzyme and the glycyrrhizic acidto obtain the glycyrrhetinic acid. The beta-glucuronidase screened out in the method is high in catalytic activity; the glycyrrhizic acid can be rapidly and completely converted within 2 hours, and an intermediate product glycyrrhetinic acid monoglucuronide cannot be generated; and the high-purity glycyrrhetinic acid can be conveniently obtained. Meanwhile, the immobilized enzyme can be recycled,so that the production cost is reduced; and the process is simple, economic and very suitable for large-scale industrial production. The conversion rate of the glycyrrhizic acid by the immobilized enzyme reaches 95% or above, and the substrate glycyrrhizic acid is basically and completely converted into the glycyrrhetinic acid.

Description

technical field [0001] The invention belongs to the field of catalysts, in particular to a method for preparing glycyrrhetinic acid by biocatalysis of novel glucuronidase. Background technique [0002] β-glucuronidase (KAE), present in various tissues and body fluids of the human body, is an acid hydrolase that can hydrolyze glucuronide. It is most abundant in liver tissue and plays an important role in promoting cell proliferation. The activity of this enzyme is the lowest in normal human serum, and its activity gradually increases with the change of cell proliferation, and the activity increases significantly when canceration occurs. β-glucuronidase, as an enzyme marker in vivo, is an active enzyme in vivo related to the occurrence and development of liver cancer, and may become an auxiliary indicator for the diagnosis of liver cancer. Although human β-glucuronidases play important roles in disease, the majority of β-glucuronidases present in humans are located in the mi...

Claims

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Application Information

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IPC IPC(8): C12P33/06C12P33/00C12N11/14C12N9/24
CPCC12P33/06C12P33/00C12N11/14C12N9/2402C12Y302/01031
Inventor 梁浩魏斌高惠玲徐海畅刘晓洁
Owner BEIJING UNIV OF CHEM TECH
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