Beta-glucuronidase as well as gene and applications thereof

A technology of β-glucuronidase and glucose, applied in the field of genetic engineering, can solve the problems of low enzyme activity, poor substrate specificity, and low enzyme activity of β-glucuronidase

Active Publication Date: 2017-07-25
BEIJING INSTITUTE OF TECHNOLOGYGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Animal-derived β-glucuronidase has poor substrate specificity. When glycyrrhizic acid is hydrolyzed into GAMG, it will be further used as a substrate to hydrolyze a glucuronic acid group (GlcA) to form glycyrrhetinic acid (Glycyrrhetinic acid, GA), such as figure 1 As shown, so the product is a mixture of GA and GAMG, and the preparation process of the enzyme is complicated and costly
The β-glucuronidase produced by most microorganisms also has the problem of poor...

Method used

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  • Beta-glucuronidase as well as gene and applications thereof
  • Beta-glucuronidase as well as gene and applications thereof
  • Beta-glucuronidase as well as gene and applications thereof

Examples

Experimental program
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Effect test

Embodiment 6

[0059] The monoammonium glycyrrhizinate in Example 6 has a purity of 75%, purchased from Xinjiang Tianshan Pharmaceutical Factory.

[0060] The enzyme activity definition of β-glucuronidase in the present invention: the amount of enzyme needed to generate 1 nmol GAMG per minute is an activity unit (U).

[0061] Among the present invention, the productive rate of GAMG is defined as:

[0062]

Embodiment 1

[0063] Example 1: Amplification of the T. pinophilum β-glucuronidase gene

[0064] The total RNA of T. pinophilum Li-93 was extracted, reverse-transcribed into cDNA and used as a template, and the upstream primer Tpgus-F and downstream Tpgus-R were used to amplify the β-glucuronidase gene of T. pinophilum Tpgus, get the Tpgus gene fragment with a length of 1554bp, such as figure 2 shown. The gene fragment obtained above was connected with the cloning vector pMD19-T, transformed into Escherichia coli DH5α strain, the plasmid pMD19-T-Tpgus was extracted, and the Tpgus gene on the plasmid was sequenced and verified. The sequence was shown in SEQ ID No.1, and the results showed that it was correct .

Embodiment 2

[0065] Example 2: Construction of expression vector for T. pinophilum β-glucuronidase gene

[0066] Taking pMD19-T-Tpgus in Example 1 as a template, using upstream primer Tpgus-F-KpnI and downstream primer Tpgus-R-NotI to amplify T. pinophilus beta-glucuronidase gene Tpgus, agarose electrophoresis Verify the amplification result, and cut the gel to recover the target fragment. After recovering the target fragment with a DNA gel recovery kit, perform double digestion with endonucleases KpnI and NotI, and then ligate with the pGAPZα plasmid that was also double-digested with endonucleases KpnI and NotI. The ligation product was transformed into Escherichia coli DH5α strain, and spread on the LB screening plate containing the antibiotic Zeocin. Colony PCR verification was performed using primers Tpgus-F-KpnI and 3'AOX. The positive clones were inoculated in LB liquid medium containing the antibiotic Zeocin, cultured overnight at 37°C, the plasmid was extracted, and the Tpgus ge...

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Abstract

The invention discloses beta-glucuronidase, as well as a gene, an expression vector and a transformant of beta-glucuronidase, and applications in preparing glycyrrhetinic acid monoglucuronide (GAMG). The gene sequence of the beta-glucuronidase is shown as SEQ ID No.1. After the gene is bonded to the expression vector, the obtained product is transformed into pichia pastoris, and thus a pichia pastoris engineering strain is obtained. When the beta-glucuronidase recombinase expressed by the engineering strain is used for preparing GAMG, the result shows that the enzyme has the advantages that the specificity of the substrate is high, no by-product, namely, glycyrrhetinic acid is generated, and the yield of GAMG is about 95%, which proves that beta-glucuronidase and the gene of the beta-glucuronidase have good application prospects in production of GAMG.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a beta-glucuronidase and its gene and application. Background technique [0002] β-glucuronidase (β-glucuronidase) is a kind of glycosidase that can catalyze the hydrolysis of β-glucuronidase, which widely exists in human body, animals, plants and microorganisms. β-glucuronidase was first discovered to exist in various tissues and body fluids of humans and animals, especially in the liver, spleen and adrenal glands. Early people found that this enzyme was related to tumor invasion and metastasis. Now It has become an important means of tumor diagnosis and treatment by detecting the level of this enzyme in a specific part. Bacterial-derived β-glucuronidases have been well utilized since their discovery. For example, β-glucuronidase derived from Escherichia coli can hydrolyze substrates to form fluorescent or colored substances, so this characteristic can ...

Claims

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Application Information

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IPC IPC(8): C12N9/24C12N15/56C12N15/81C12N1/19C12P19/56
CPCC12N9/2402C12P19/56C12Y302/01031
Inventor 李春徐赢华吕波冯旭东
Owner BEIJING INSTITUTE OF TECHNOLOGYGY
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