Dehalogenase DhmB, encoding gene and application thereof

A technology for encoding genes and dehalogenases, applied in applications, genetic engineering, plant genetic improvement, etc., can solve the problems of not easy rapid degradation, threats to human and animal health, etc., and achieve broad industrial application prospects, easy purification, and good stability. Effect

Inactive Publication Date: 2015-06-10
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Halogenated organic compounds are generally environmentally toxic, and generally relatively stable, not easy to be rapidly degrade

Method used

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  • Dehalogenase DhmB, encoding gene and application thereof
  • Dehalogenase DhmB, encoding gene and application thereof
  • Dehalogenase DhmB, encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1, the acquisition of DhmB protein and its coding gene

[0051] Using the genomic DNA of Huangia manganoxydans (Huangia manganoxydans) 8047CGMCC No.6697 as a template, PCR amplification was performed with primers DhmBF and DhmBR to obtain PCR amplification products. The primer sequences are as follows (the underlined sequence is the restriction site):

[0052] DhmBF: 5′-G GAATTCCATATGATGCCCGCTGTCTACGTCTTC-3′;

[0053] DhmBR: 5′-CCC AAGCTT TCAGTCCGCGCGCAGGCTGATC-3'.

[0054] The PCR amplification product was recovered and cloned into pGEM-T vector to obtain the recombinant vector, which was transformed into Escherichia coli DH5α and then sent for sequencing.

[0055] Sequencing results show that: the nucleotide sequence of the PCR amplification product is shown in sequence 1 in the sequence listing, the gene shown in the sequence is named DhmB, the protein encoded by the gene is named DhmB, and the amino acid sequence of DhmB is shown in the sequence lis...

Embodiment 2

[0056] Example 2, the acquisition of dehalogenase DhmB and its functional verification

[0057] 1. Dehalogenase acquisition

[0058] 1. Obtaining the recombinant vector

[0059] Use restriction endonucleases to Nde I and Hind III to perform double enzyme digestion on the PCR amplification product obtained in the above example 1 and the pET-28a(+) vector, and connect to obtain the recombinant vector pET-DhmB, and submit it for sequencing .

[0060] Sequencing results show that: pET-DhmB replaces the DNA between the Nde I and Hind III sites of the pET-28a (+) vector with the DhmB gene shown in sequence 1 in the sequence table, and keeps the other elements of the pET-28a (+) vector Recombinant vector obtained with unchanged sequence. The amino acid sequence of the protein encoded by the DhmB gene is shown in sequence 2 in the sequence listing.

[0061] 2. Acquisition of recombinant bacteria

[0062] The recombinant vector pET-DhmB obtained in step 2 is transformed into Esche...

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Abstract

The invention discloses a dehalogenase DhmB, an encoding genes and application thereof. The dehalogenase DhmB is a protein in the following (a) or (b); (a) an amino acid sequence is a protein of a sequence 2 in a sequence table; (b) a protein associated with dehalogenation is obtained by the amino acid sequence, which is shown in the sequence 2 of the sequence table and subjected to replacement and/or missing and/or adding of one or a plurality of amino acid residues. The dehalogenase DhmB is easy in purification, good in stability, resistant to high-medium temperature, applicable to the optimum temperature of 65 DEG C, resistant to organic solvents and capable of degrading organohalogen compounds with low-carbon chains, classic pesticides and chemical toxicant and the like and has wide industrial application prospects.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a dehalogenase DhmB and its coding gene and application. Background technique [0002] There are a large number of halogenated compounds such as halogenated hydrocarbons, halogenated aromatic acids, halogenated alkanoic acids and halogenated alcohols in nature. These halogenated organic compounds can be used as organic solvents, pesticides, pharmaceutical and pesticide intermediates, etc. Halogenated organic compounds are generally environmentally toxic, and generally relatively stable, not easy to be rapidly degraded, and easy to accumulate in human and animal fat tissues through the food chain, posing a great threat to human and animal health. [0003] The mechanism of dehalogenase (Dehalogenase, EC 3.8.1): It can catalyze the cleavage of carbon-halogen bonds in organic halogen compounds, releasing halogen atoms from the compounds. Dehalogenase can eliminate the toxici...

Claims

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Application Information

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IPC IPC(8): C12N9/14C12N15/55C12N15/70C12N1/21A62D3/02A62D101/22
CPCC12N9/14C12Y308/01
Inventor 董志扬董亮戴欣张山王丽张岩峰
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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