Dehalogenase HadD14 as well as coding gene and application thereof

A dehalogenase and encoding technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve serious, environmental and health consequences, microbial toxicity, etc.

Active Publication Date: 2021-04-20
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, because the stability of halogenated organic substances is generally high, and they are even toxic to microorganisms that may degrade them, the biodegradability

Method used

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  • Dehalogenase HadD14 as well as coding gene and application thereof
  • Dehalogenase HadD14 as well as coding gene and application thereof
  • Dehalogenase HadD14 as well as coding gene and application thereof

Examples

Experimental program
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Embodiment 1

[0059] Embodiment 1, expression and activity determination of HadD14 protein

[0060] 1. Acquisition of the gene encoding HadD14 protein

[0061] Using the genomic DNA of the new strain D14 isolated from deep-sea sediment samples in the Indian Ocean as a template, PCR amplification was performed with D14F and D14R primers:

[0062] D14F: 5'-AAGAAGGAGATATACATATGACCGTGATCGCCGGCATCAAG-3'

[0063] D14R: 5'-TCGAGTGCGGCCGCAAGCTTCGCCACCAGGCGGGGAAGTTC-3'

[0064] The PCR amplification product was recovered and cloned into the pET21a(+) vector (Novagen) to obtain the recombinant vector pET-D14, which was transformed into Escherichia coli BL21(DE3) and then submitted for sequencing.

[0065] Sequencing results show that: the vector contains the nucleotides shown in sequence 1 (without GDDDGDDDGDDD solubilizing tag), the gene contained in the sequence is named HadD14, and the coding region of the gene is the 1st-675th position of sequence 1, the The protein encoded by the gene is name...

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Abstract

The invention discloses a dehalogenase HadD14 as well as a coding gene and application thereof. The protein provided by the invention is a protein shown as a sequence 2 in a sequence table. Experiments prove that the soluble expression quantity of the dehalogenase HadD14 (sequence 2) can be remarkably increased through prokaryotic expression of escherichia coli by adopting the modified gene sequence (sequence 1), and the obtained dehalogenase HadD14 has high activity, has thermal stability under the treatment condition of not higher than 70 DEG C, and has pH stability under the condition of pH 5.0-8.2; and under the treatment conditions of not higher than 10 percent of methanol, 10 percent of ethanol and 30 percent of DMSO (Dimethyl Sulfoxide), relatively strong organic solvent stability is achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a dehalogenase HadD14 and its coding gene and application. Background technique [0002] Halogenated organic compounds are widely used in many fields. Among them, due to their extensive use in industrial enterprises and daily necessities, they have become an important class of environmental pollutants. These halogenated organics have continued to cause widespread environmental problems in the biosphere for many years due to their very stable chemical bonds. Halogenated organics are the most used and studied class of organics, mainly due to their relatively simple synthetic routes. However, due to the generally high stability of halogenated organic substances and their toxicity to microorganisms that may degrade them, the biodegradability of halogenated organic substances has been poor for many years. Excessive exposure to these halogenated pollutants can lead to seriou...

Claims

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Application Information

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IPC IPC(8): C12N9/14C12N15/55C12P7/54A62D3/02A62D101/22
CPCY02A50/20
Inventor 董志扬张重雄王丽戴欣
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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