Gene contained in A.xylosoxidans LHB21 strain and used for coding catechol 2,3-dioxygenase

A catechol and dioxygenase technology, applied in genetic engineering, oxidoreductase, plant gene improvement, etc., can solve problems such as not suitable for cold regions, lack of cold resistance mechanism, etc.

Inactive Publication Date: 2011-06-15
DALIAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in these studies, the C23O genes are all derived from mesophilic microorganisms, and the optimum temperature is under mesophilic conditions, which lacks the mechanism of cold resistance and is not suitable for functioning in cold regions.

Method used

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  • Gene contained in A.xylosoxidans LHB21 strain and used for coding catechol 2,3-dioxygenase
  • Gene contained in A.xylosoxidans LHB21 strain and used for coding catechol 2,3-dioxygenase
  • Gene contained in A.xylosoxidans LHB21 strain and used for coding catechol 2,3-dioxygenase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Genomic DNA Extraction of Strain A.xylosoxidans LHB21

[0022] Use TaKaRa MiniBEST Bacterial Genomic DNA Extraction Kit Ver.2.0 (Code No.DV810A) to extract the genomic DNA of strain A.xylosoxidans LHB21, the specific operation is as follows:

[0023] 1. Bacterial culture: Pick a single colony and inoculate it into 1-4ml of LB (component concentration (W / V): peptone 1%, yeast extract 0.5%, NaCl 1%) liquid medium for overnight cultivation at 30°C.

[0024] 2. Take 1-4ml of supernatant, centrifuge at 10,000rpm for 2 minutes, and discard the supernatant.

[0025] 3. Fully suspend the bacterial pellet with 150ul of SP Buffer (containing RNase A1).

[0026] 4. Add 20ul of Lysozyme solution, mix well and let stand at room temperature for 5 minutes.

[0027] 5. Add 30ul of EDTA Buffer, mix well and let stand at room temperature for 5 minutes.

[0028] 6. Add 200ul of Solution A, shake vigorously and keep warm at 65°C for 10 minutes.

[0029] 7. Add 400ul of Solut...

Embodiment 2

[0042] Embodiment 2: C23O gene PCR clone

[0043] 1. Cloning of C23O gene by PCR method

[0044] 1.1 Design and synthesis of primers

[0045] According to the C23O gene of A.xylosoxidans kf701 strain (as shown in SEQ ID NO: 3), the oligonucleotide primers used for C23O gene amplification were designed and synthesized, and the Nco I restriction enzyme was designed at the 5' end of the upstream primer F0 site, a Hind III restriction site was designed at the 5' end of the downstream primer R0.

[0046] PCR primers:

[0047] F 5'-ATGAACAAAGGTGTAATGCGAC-3' (SEQ ID NO: 4)

[0048] R 5'-TCAGGTCAGCACGGTCATGAA-3' (SEQ ID NO: 5)

[0049] YF2 5'-TGTTCACCAAGGTGCTCGG-3' (SEQ ID NO: 6)

[0050] YR2 5'-GGTCGAAGAAGTAGATGGTC-3' (SEQ ID NO: 7)

[0051] YF3 5'-GCCTCCATCATGTGTCCTTC-3' (SEQ ID NO: 8)

[0052] YR3 5'-TGTGTCGGTCATGGAGATCA-3' (SEQ ID NO: 9)

[0053] R02 5'-TCAGGTCAGCACGGTCATGAATCGTTCGTTGAGAAT-3' (SEQ ID NO: 10)

[0054] F0 5′- CCATGG CTATGAACAAAGGTGTAATGCGAC-3' (SEQ ID NO:...

Embodiment 3

[0079] Example 3: C23O gene expression

[0080] With pET-32a (+) (structure see Figure 4 ), construct the fusion gene expression vector pDSFL01 (see the structure Figure 5 ). Sequencing results showed that the C23O gene on the pDSFLO1 expression vector was a full-length C23O gene.

[0081] Pick a single colony of the positive expresser and inoculate it in 2ml liquid medium containing 100lg / ml ampicillin at 37°C, 200rpm and culture overnight. In 6ml LB medium, insert 100μl of the culture of Escherichia coli JM109 containing pDSFLO1 and grow to OD 600 0.55-0.7, add IPTG to make the final concentration reach 1.0mM, and induce expression at 30°C for 3 hours. Collect bacteria for electrophoresis analysis. SDS-PAGE electrophoresis results (see Image 6 ), Image 6 Middle M: ​​Protein MW marker (Broad) (kDa), A: pET-32a(+) whole cell, B: pET-32a(+) supernatant, C: pET-32a(+) pellet, D: pDSFL01 whole cell , E: pDSFLO1 supernatant, F: pDSFLO1 pellet.

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Abstract

The invention provides a separated gene for coding catechol 2,3-dioxygenase (C230). The C230 gene is derived from an achromobacter xylosoxidans LHB21 (A.xylosoxidans LHB21 for short) strain, has the overall length of 924bp, starts from an ATG initiation codon, ends with a TGA termination codon, and codes 307 amino acid residues; and the molecular weight of the expressed protein is 35.16kDa, and the enzyme activity of the expressed protein is 32U/mg.

Description

technical field [0001] The present invention relates to the fields of genetic engineering, enzyme engineering, microbiology, biochemistry and the like, in particular to a strain of Achromobacter xylosoxidans LHB21 (abbreviated as A. xylosoxidans LHB21) The gene (C23O) encoding catechol 2,3-dioxygenase (catechol 2,3-dioxygenase, C23O) in the depository center, numbered CGMCC 1.2679). Background technique [0002] Catechol 2,3-dioxygenase (C23O) catalyzes the ortho-cleavage of catechol, exists in a variety of aromatic hydrocarbon degrading microorganisms, and is the key enzyme for the degradation of aromatic compounds. Since Kojima first reported catechol 2,3-dioxygenase from Pseudomonas putida mt-2 in 1961, it has been reported to contain catechol 2,3-dioxygenase Microorganisms with enzyme and dioxygenase region genes include: Pseudomonas, Comamonas, A.eutrophus335, A.xylosoxidansKF 701, RhodococcusV 49, Balillus stearothermophilus. The C23O produced by these microorganisms ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10
Inventor 张庆芳迟乃玉李兵
Owner DALIAN UNIV
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