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Catechol 1, 2-dioxygenase as well as coding group, preparation method and application thereof

A dioxygenase and catechol technology, applied in the biological field, can solve the problems of high enzyme production and limited application of enzyme preparations, and achieve the effect of overcoming poor environmental adaptability and improving microbial degradation rate

Pending Publication Date: 2022-08-05
ENVIRONMENTAL PROTECTION RES INST OF LIGHT IND +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the protein expression process of wild strains is regulated by many factors, it is often difficult to achieve a high level of enzyme production, which limits the application of enzyme preparations in the field of pollution remediation

Method used

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  • Catechol 1, 2-dioxygenase as well as coding group, preparation method and application thereof
  • Catechol 1, 2-dioxygenase as well as coding group, preparation method and application thereof
  • Catechol 1, 2-dioxygenase as well as coding group, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] This embodiment provides the extraction of the genomic DNA of Arthrobacter sp.FB24 and the specific amplification of the catechol 1,2-dioxygenase encoding gene sequence, as follows:

[0052] (1) Extraction of genomic DNA from Arthrobacter sp.FB24

[0053] Arthrobacter sp. FB24 strain was inoculated into liquid LB medium, cultured overnight at 37°C with shaking, and the cells were collected by centrifugation at 8000 r / min. The genome was extracted using the Omega genome kit E.Z.N.A. Bacterial DNA Kit, and the specific extraction steps were carried out according to the instructions of the kit.

[0054] (2) PCR amplification of the gene encoding catechol 1,2-dioxygenase

[0055] According to the coding gene sequence of catechol 1,2-dioxygenase (the nucleotide sequence of which is shown in SEQ ID NO. 1 of the sequence table), a gene amplification carrying restriction enzyme sites EcoR I and Hind III was designed and synthesized. Increase primer, upstream primer ArcatF: 5'...

Embodiment 2

[0058] This embodiment provides a method for constructing a cloned strain containing a gene encoding catechol 1,2-dioxygenase, specifically including:

[0059] (1) Enzyme cleavage reaction

[0060] The PCR product and plasmid pMD19T amplified in Example 1 were double digested with EcoR I and Hind III. The digestion system was as follows: 1 μL EcoR I, 1 μL Hind III, 5 μL 10×buffer, vector or PCR product 500 ng; ddH 2 Make up to 50 μL with O. The above system was reacted at 37 °C for 6 h to obtain the corresponding enzyme cleavage product.

[0061] (2) Construction of recombinant cloning plasmid pMD19T-Arcat

[0062] The PCR product that was double digested with EcoR I and Hind III was ligated to the pM D19T vector that was also double digested. The 10 μL ligation system was as follows: 1 μL pMD19T, 5 μL PCR product, 1 μL 10×Ligase buffer, 1 μL T4 DNALigase, 2 μL ddH 2 O, mix well, take out in a warm bath at 16°C for 16-20h, directly transform or store at -20°C to obtain the ...

Embodiment 3

[0066] This embodiment provides a method for constructing a catechol 1,2-dioxygenase-encoding gene expression strain, which specifically includes:

[0067] (1) Enzyme cleavage reaction

[0068] The recombinant cloning plasmid pMD19T-Arcat was extracted from the cloned strain, and the recombinant cloning plasmid pMD19T-Arcat and plasmid pET28a were double digested with EcoR I and Hind III. The digestion system was as follows: 1 μL Eco R I, 1 μL Hind III, 5 μL 10×buffer , vector or recombinant cloning plasmid pMD19T-Arcat 500ng; ddH 2 Make up to 50 μL with O. The above system was reacted at 37°C for 6 h to obtain the Arcat gene fragment.

[0069] (2) Construction of recombinant expression plasmid pET28a-Arcat

[0070] The Arcat gene fragment that had been double digested with EcoR I and Hind III was ligated to the pET28a vector that had also been double digested. The 10 μL ligation system was as follows: 1 μL pET28a, 5 μL Arcat gene fragment, 1 μL 10×Ligasebuffer, 1 μL T4 DNA...

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Abstract

The invention is applicable to the technical field of biology, and provides catechol 1, 2-dioxygenase as well as a coding group, a preparation method and application thereof, the amino acid sequence of the catechol 1, 2-dioxygenase is as shown in SEQ ID NO.2 in a sequence table, and the preparation method comprises the following steps: carrying out double enzyme digestion on an amplification product of the coding gene of the catechol 1, 2-dioxygenase, and carrying out enzyme digestion on the amplification product of the coding gene of the catechol 1, 2-dioxygenase to obtain the catechol 1, 2-dioxygenase. Connecting to a cloning plasmid to obtain a recombinant cloning plasmid; electrically transferring the recombinant clone plasmid to the first strain to obtain a clone strain; carrying out double enzyme digestion treatment on the recombinant clone plasmid and the expression plasmid, and connecting enzyme digestion producing areas of the recombinant clone plasmid and the expression plasmid to obtain a recombinant expression plasmid; electrically transforming the recombinant expression plasmid onto a second strain to obtain an expression strain; and carrying out induced expression culture on the expression strain to obtain the catechol 1, 2-dioxygenase. The catechol 1, 2-dioxygenase can be used for improving the microbial degradation rate of heavy oil.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a catechol 1,2-dioxygenase and its coding group, preparation method and application. Background technique [0002] With the increase in the use of crude oil and its processed products, the continuous consumption of conventional oil and gas resources, the proportion of heavy oil in my country's industry is increasing, and the environmental pollution incidents caused by heavy oil are also increasing year by year. Microbial remediation technology is regarded as an ideal technical means for heavy oil pollution control due to its advantages of greenness, high efficiency, economy, and no secondary pollution. The application of microbial remediation technology in heavy oil pollution remediation is limited because heavy oil is rich in refractory and highly toxic polycyclic aromatic hydrocarbons. Therefore, how to efficiently remove PAH components in heavy oil is the key to impro...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N1/21C12R1/19
CPCC12N9/0069C12N15/70C12Y113/11001Y02A50/20
Inventor 代小丽吕静
Owner ENVIRONMENTAL PROTECTION RES INST OF LIGHT IND
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