Method for preparing recombinant heat-proof beta-glucuronic acid enzyme

A glucuronidase, heat-resistant technology, applied in biochemical equipment and methods, recombinant DNA technology, enzymes and other directions, can solve problems such as low expression level, achieve the effect of improved expression level, high yield, and beneficial to industrial fermentation

Inactive Publication Date: 2008-08-27
南京仙奕基因科技有限公司
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AI Technical Summary

Problems solved by technology

The β-glucuronidase (GenBank NO.NC_000853) produced by Thermotoga maritima has good thermostability (Rober H, Thomas L, Helmut K, et al.Thermotoga maritima sp.nov.represents a new genus of unique extremelythermophilic eubacteria growing up 90℃. Arch Microbiol, 1986, 144: 324-333), but the expression level of genes derived from thermophilic microorganisms is usually low when expressed in normal temperature bacteria such as Escherichia coli
Hamzah M. Salleh et al. used the pET28a vector to express the Thermotoga maritima β-glucuronidase gene in Escherichia coli, and only about 5 mg / L protein could be obtained.

Method used

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  • Method for preparing recombinant heat-proof beta-glucuronic acid enzyme
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  • Method for preparing recombinant heat-proof beta-glucuronic acid enzyme

Examples

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Effect test

Embodiment 1

[0028] Example 1: High-efficiency expression and application of Thermotoga maritima β-glucuronidase gene

[0029](1) Cultivate Thermotoga maritima according to conventional methods, and extract genomic DNA from Thermotoga maritima; design primers according to the known heat-resistant β-glucuronidase gene (GenBank NO.NC_000853), and use Thermotoga maritima Genomic DNA of Togamota was used as a template, and PCR amplification was carried out with synthetic primers to obtain the original gene of heat-resistant β-glucuronidase; the extraction of genomic DNA and PCR amplification and other gene operations were carried out according to the third chapter of "Molecular Cloning Manual". (Sambrook and Russell, 2001, CSHL press, Cold Spring Harbor, New York), primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.; tool enzymes used for gene manipulation were purchased from Bao Bioengineering (Dalian ) Ltd. After digestion and purification of the PCR ampl...

Embodiment 2

[0041] Embodiment 2: the application of recombinant heat-resistant β-glucuronidase in the preparation of glycyrrhetinic acid, the steps are as follows:

[0042] Purified recombinant thermostable β-glucuronidase was used to catalyze the hydrolysis reaction from glycyrrhizic acid to glycyrrhetinic acid, the reaction condition was 60°C, and the buffer used in the reaction was pH4.6 phosphate buffer.

Embodiment 3

[0043] Embodiment 3: the application of recombinant heat-resistant β-glucuronidase in the preparation of glycyrrhetinic acid, the steps are as follows:

[0044] The hydrolysis reaction from glycyrrhizic acid to glycyrrhetinic acid was catalyzed by unpurified recombinant thermostable β-glucuronidase, the reaction condition was 80°C, and the buffer used in the reaction was imidazole potassium hydrogen phthalate buffer at pH 7.4.

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Abstract

The invention relates to a method for preparing recombinant extremely heat-resistant beta-glucuronidase, which comprises flowing steps: inserting heat-resistant beta-glucuronidase genes into an expression vector pHsh, transforming escherichia coli to obtain expression plasmids of heat-resistant beta-glucuronidase, then, mutating genes, changing a secondary structure of mRNA, obtaining optimized expression plasmids of the heat-resistant beta-glucuronidase, then, transforming the expression plasmids of the heat-resistant beta-glucuronidase or the optimized expression plasmids of the heat-resistant beta-glucuronidase into the escherichia coli, obtaining genetic engineering bacteria, enabling the beta-glucuronidase to obtain over-expression through hot-shock inducement, finally, collecting cells, breaking wall, centrifuging, then, obtaining crude enzyme, further purifying, then, and obtaining pure enzyme. The method of the invention uses the expression vector pHsh to express the genes of the heat-resistant beta-glucuronidase on high level for the first time and applies the recombinant heat-resistant beta-glucuronidase in preparing glycyrrhetinic acid for the first time.

Description

technical field [0001] The invention relates to the fields of molecular biology, enzymology, bioinformatics, genetic engineering and the like. Specifically, it relates to preparation of recombinant thermostable β-glucuronidase by using a pHsh expression system, and a method for preparing glycyrrhetinic acid by using the recombinant thermostable β-glucuronidase. Background technique [0002] β-Glucuronidase degrades glycyrrhizinate to produce glycyrrhetinic acid or monoglucuronoglycyrrhetinic acid (Takashi K.Microbial production of glycyrrhetic acid 3-0-mono-β-D-glucuronide from glycyrrhizin by cryptococcus magnus MG-27.Biosci Biotech Biochem, 1994, 58:455-458). Domestic and foreign scholars' research on β-glucuronidase focuses on (1) the research on the conversion pathway of glycyrrhizic acid to glycyrrhetinic acid or monoglucuronyl glycyrrhetinic acid, as well as the pharmacology and physiological functions of glycyrrhizic acid and glycyrrhetinic acid (2) Research on the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N15/70C12N9/00C12P7/42
Inventor 邵蔚蓝裴建军王卓
Owner 南京仙奕基因科技有限公司
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