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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AI-Extracted 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 achiev...

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.

Application Domain

BacteriaMicroorganism based processes +5

Technology Topic

Enzyme digestionMolecular biology +10

Image

Examples

Experimental program(7)

Example Embodiment

[0050] Example 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'-CCG GAATTC ATGACCAAGG TTCCGGAG-3' (as shown in SEQ ID NO.3 of the sequence table), the underline indicates the EcoRI restriction site; the downstream primer ArcatR: 5'-GGG A AGCTT T TAGTCCTGGGGGTCGAG-3' (as shown in SEQ ID NO. 4 of the sequence listing), the underline indicates the Hind III restriction site. The gene sequence Arcat encoding catechol 1,2-dioxygenase was amplified by PCR. A 20μL system was used for PCR amplification, including E×TaqDNA polymerase 0.3μL, 10×PCR buffer 2μL, dNTP 0.5μL, PCR template Arthrobacter sp.Z5T genomic DNA 1μL, upstream primer ArcatF 1μL, downstream primer ArcatR 1μL, ddH 2 O 14.2 μL. PCR amplification conditions were pre-denaturation at 95 °C for 3 min, followed by 30 cycles, each cycle included denaturation at 95 °C for 45 s, annealing at 57 °C for 45 s, and extension at 72 °C for 1 min.

[0056] The PCR products were further detected by agarose gel electrophoresis. 1% agarose, 5 μL PCR product loading volume, 1×TAE electrophoresis buffer, 100V electrophoresis for 30-40min, EB staining and observation under UV light, PCR products were electrophoresis bands of about 841bp. The experimental results are as figure 1 shown.

Example Embodiment

[0057] Example 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 first ligation product.

[0063] (3) Conversion reaction

[0064] The above-mentioned first ligation product was electro-transformed into E.coli DH5α, and an E.coli DH5α cloned strain of the C at recombinant cloning plasmid pMD19T-Arcat was obtained by LB-Amp plate screening. The experimental results are as figure 2 and image 3 shown.

Example Embodiment

[0065] Example 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 Ligase, 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 second ligation product.

[0071] (3) Conversion reaction

[0072] The above-mentioned second ligation product was electrotransformed into E. coli BL21 (DE3), and an E. coli BL21 expression strain (DE3) of the ArCat recombinant expression plasmid pET28a-Arcat was obtained by LB-Kan plate screening. The experimental results are as figure 2 and image 3 shown.

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