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Catechol 2,3-dioxygenase gene and application thereof

A catechol and dioxygenase technology, applied in the field of genetic engineering, can solve the problems of low enzyme activity and high temperature, and achieve the effect of solving environmental pollution problems and considerable economic benefits

Inactive Publication Date: 2018-01-19
HENAN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, with the development of molecular biology techniques, although there have been many studies on the gene expression of catechol 2,3-dioxygenase, the expression of these catechol 2,3-dioxygenase The enzyme activity is often relatively low, and the temperature required for the catalytic reaction is relatively high, which is not suitable for use under low temperature conditions. Therefore, it is very necessary to construct a low-temperature and efficient catechol 2,3-dioxygenase genetically engineered strain

Method used

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  • Catechol 2,3-dioxygenase gene and application thereof
  • Catechol 2,3-dioxygenase gene and application thereof
  • Catechol 2,3-dioxygenase gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Cloning of catechol 2,3-dioxygenase gene

[0044] (1) Extraction of total bacterial DNA

[0045] After the bacterial strain Sphingbium sp.MEA3-1 was cultured in LB liquid medium for 18 hours, the bacterial cells were collected by centrifugation at 5000r / min, and the total DNA of the bacterial cells was extracted using a genomic DNA extraction kit (Azanno Biotech), and the mass fraction was 0.75% agar Glycogel electrophoresis was used to detect the quality of the total DNA extracted.

[0046] (2) Primer design and synthesis

[0047] The upstream and downstream primers of catechol 2,3-dioxygenase were designed according to the genome information of Sphingbium bacteria, and the specific sequences are as follows:

[0048] Primer C23O-F: 5'- CATATG GCGATTCGTGGATTGCTC-3';

[0049] Primer C23O-R: 5'- CTCGAG GGTCAGAACGTTCAGAAAAGCT-3';

[0050] The underlined parts are NdeI and XhoI restriction sites respectively;

[0051] (3) PCR amplification of catechol dio...

Embodiment 2

[0064] Example 2 High-efficiency expression of recombinant catechol 2,3-dioxygenase protein

[0065] (1) Digest the positive clone plasmid successfully sequenced in Example 1 with NdeI and XhoI double enzyme digestion system (50 μL): NdeI 1 μL, Xho I 1 μL, 10×H buffer 5 μL, positive clone plasmid 20 μL, wxya 2 O 1 μL; react in a water bath at 37°C for more than 3 hours. After the digested product was detected by agarose gel electrophoresis with a mass fraction of 0.75%, a fragment of about 900 bp was recovered using a DNA gel recovery kit.

[0066](2) Referring to step (1), the vector pET-29a(+) (Merck-Novagen, Cat No. 69871) was double digested with NdeI and XhoI.

[0067] (3) Ligate the target fragment recovered in step (1) with the vector pET-29a(+) digested in step (2) to obtain the pET-29a(+) containing catechol 2,3-dioxygenase gene 29a(+) recombinant plasmid; the pET-29a(+) recombinant plasmid containing the catechol 2,3-dioxygenase gene linked by the enzyme was trans...

Embodiment 3

[0070] Example 3 Purification and concentration of recombinant catechol 2,3-dioxygenase

[0071] Using the six histidine tags introduced during primer design, the crude enzyme solution (prepared in step (4) of Example 2) that induces expression of catechol 2,3-dioxygenase was passed through Ni-NTAResin (Sangon Biotech) Purify the affinity chromatography column, the steps are as follows: first equilibrate the column with 10mL buffer solution A (20mM PBS pH 8.0, 300mM NaCl, 0mM imidazole), take 2.5mL crude enzyme solution and load it on the Ni-NTA affinity chromatography column, Elute with 5mL buffer solution A, and then use 5mL buffer B (20mM PBS pH 8.0, 300mM NaCl, 20mM imidazole), buffer C (20mM PBS pH 8.0, 300mM NaCl, 50mM imidazole), buffer D (20mM PBS Gradient elution was performed with buffer E (20 mM PBS pH 8.0, 300 mM NaCl, 100 mM imidazole), buffer E (20 mM PBS pH 8.0, 300 mM NaCl, 250 mM imidazole) and buffer F (20 mM PBS pH 8.0, 300 mM NaCl, 500 mM imidazole). Use S...

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Abstract

The invention belongs to the field of genetic engineering and specifically relates to a catechol 2,3-dioxygenase gene and an application thereof. The nucleotide sequence of the catechol 2,3-dioxygenase gene is shown as SEQ ID NO.1. The gene expression product catechol 2,3-dioxygenase is capable of effectively hydrolyzing aromatic compound catechol, 4-methyl catechol, catechol, 1,2,4-pyrogallol and2,6-dimethyl-1,3,4-pyrogallol. The catechol 2,3-dioxygenase gene can be applied to the industries of biological remediation for environmental pollution, industrial wastewater treatment and 2-hydroxylfurfural production, not only can solve the environmental pollution of catechol aromatic compounds, but also can acquire great economic benefit.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular to a catechol 2,3-dioxygenase gene and its application. Background technique [0002] Catechol 2,3-dioxygenase, also called mutacatecholase (MPC), exists in many bacteria that degrade aromatic compounds. It can catalyze the ortho-position cleavage of the benzene ring, turning colorless or slightly brown The catechol is transformed into yellow 2-hydroxy mucofuroic acid semialdehyde. The C23O produced by different bacteria has different degrees of difference in physical and chemical properties, catalytic activity, enzyme substrate, etc., and the gene encoding C23O also varies with the bacterial species. Searching for C23O genes and expression products from different bacterial sources is of great significance for solving environmental pollution problems caused by different environmental conditions and different types of aromatic compounds. [0003] The strain Sphingbium sp.MEA3-1 c...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N15/70C12N15/66C12N1/21C12N9/02C02F3/34A62D3/02C12R1/19C02F101/34A62D101/28
Inventor 侯颖徐建强邵嫄尤晓颜马丽苹秦翠丽牛明福宫强李阳裴韬王维宇
Owner HENAN UNIV OF SCI & TECH
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