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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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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 enzymes leads to high production costs, which is not conducive to industrial applications. Therefore, it is of high industrial application value to find a new type of β-glucuronidase that can directly convert glycyrrhizic acid into glycyrrhetinic acid efficiently and quickly.

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

Embodiment 1

[0032] This example provides the screening and expression of β-glucuronidase. The specific method is as follows: firstly, through the gene database NCBI, compare the sequences of the existing β-glucuronidases that are widely used, and screen out a β-glucuronidase from primary The β-glucuronidase of Aspergillus fungi (85% amino acid sequence homology), gene number KAE8374379.1, the target gene was obtained by PCR amplification (such as figure 1 shown), the primers are:

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

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

[0035] Then, the target gene was connected with the expression vector plasmid peT28a by restriction enzyme ligation (such as figure 2 , image 3 shown), introduced the expression bacteria-Escherichia coli BL21(DE3), cultured in 100mL LB liquid medium, added the inducer IPTG at OD0.6, induced at 16°C for 24h, centrifuged,...

Embodiment 2

[0037] This example constructed the immobilized enzyme Fe 3 o 4 @Zr-2MIm@KAE, the specific method is:

[0038] 1) Expression vector Fe 3 o 4 Build by @Zr-2MIm

[0039] Will Fe 3 o 4Microspheres (100 mg) were added into a methanol solution (50 ml, 1 mg / mL) of PVP, sonicated in an ultrasonic instrument for 60 min, and the supernatant was removed by adsorption with a magnet. The precipitate was collected and dispersed in a solution of 2-methylimidazole (2-MIm) in methanol (25 ml, 40 mm) under sonication. Then, Zr(NO 3 ) 4 ·5H 2 O in methanol (25 ml, 40 mm) was added to the solution. After standing for 10 minutes, collect it with a magnet and rinse it thoroughly with methanol solution. The precipitate was dried at 80 °C for 8 h.

[0040] 2) Immobilized enzyme Fe 3 o 4 Build of @Zr-2MIm @KAE

[0041] Get the crude enzyme solution of β-glucuronidase prepared in Example 1, 2mL per tube, subpackage in centrifuge tubes, add 3mg of carrier Fe constructed in each tube 3 o...

Embodiment 3

[0044] This embodiment utilizes the immobilized enzyme Fc constructed in Example 2 3 o 4 @Zr-2MIm@KAE catalyzes the preparation of glycyrrhetinic acid from glycyrrhizic acid, and the reaction schematic diagram is as follows Figure 5 As shown, the specific method is:

[0045] Weigh 10mg of glycyrrhizic acid, dissolve in 5ml pH5.0 20mM acetic acid-sodium acetate buffer, take 1ml of glycyrrhizic acid solution, add the immobilized enzyme Fe that absorbs the supernatant phosphate buffer 3 o 4 In @Zr-2MIm@KAE, react in a shaking table at 60°C for 2 hours. After 2 hours, centrifuge at 12000rpm to separate the supernatant, and the precipitate is the product glycyrrhetinic acid.

[0046] Dissolve the resulting precipitate in 1 mL of methanol as Figure 6 As 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...

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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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