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Allophanic acid hydrolase and preparation method thereof

A technology of allophanate and hydrolase, which is applied in the field of molecular biology, can solve the problems of high temperature requirements, poor tolerance of allophanate hydrolase, inability to exert activity, etc., and achieves easy operation, simple method, strong The effect of enzymatic activity

Active Publication Date: 2018-05-04
HUBEI UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The allophanate hydrolase produced at present has poor tolerance to low temperature, and can hardly exert its activity at low temperature. In industrial production, the temperature requirement is relatively high.

Method used

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  • Allophanic acid hydrolase and preparation method thereof
  • Allophanic acid hydrolase and preparation method thereof
  • Allophanic acid hydrolase and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] The acquisition of embodiment 1 allophanate hydrolase gene

[0025] 1) Obtain the strain containing the target gene

[0026] The present invention utilizes strains of the genus Cryobacterium, which are screened from the soil of Changbai Mountain. The specific screening process is like the Chinese invention patent with the patent number of 201710034491.5. The strain is named: Cryobacteriumbaishanse 02, and it is preserved in the China Type Culture Collection Center , deposit number CCTCC NO: M2016604. The date of deposit is October 31, 2016, and the deposit address is Wuhan University, Wuhan, China.

[0027] 2) Genome extraction

[0028] The genome of Cryobacteriumbaishanse 02 strain was extracted using a DNA extraction kit (TAKARA Dalian) as a template, and the extracted genome samples were stored at -20°C until use.

[0029] 3) PCR amplification of the target gene

[0030] Design primers: the forward primer is shown in SEQ ID NO.3, and the reverse primer is shown i...

Embodiment 2

[0036] Example 2 Constructing the expression vector of allophanate hydrolase

[0037] The PCR amplified product obtained in Example 1 was carried out to gel recovery, and the PCR amplified product was carried out double digestion reaction with restriction endonuclease EcoRI and NdeI; Carrier pET21a (+ ) for double enzyme digestion reaction, and then catalyzed by high-efficiency DNA ligase High Ligation (TOYOBO) to connect pET21a(+) and PCR amplification product after double enzyme digestion reaction; construct the expression vector pET21a-APH for allophanate hydrolase , the expression vector pET21a-APH was constructed as figure 2 shown.

Embodiment 3

[0038] Example 3 Expression and Purification of Bacterial Allophanate Hydrolase

[0039] The expression vector pET21a-APH obtained in Example 2 is transformed into the competent cells of Escherichia coli BL21, and the preparation of the competent cells of Escherichia coli BL21 adopts the CaCl2 method, and the preparation of the competent cells of Escherichia coli BL21 by the CaCl2 method is a routine experimental method in the laboratory , which will not be repeated here. The obtained expression vector pET21a-APH was transformed into Escherichia coli BL21 to obtain the recombinant strain Ecoli BL21-pET21a-APH. Expand the culture of Ecoli BL21-pET21a-APH to OD600=0.6, add 1mM IPTG, and induce the expression of allophanate hydrolase protein by delayed induction at 18°C. After purification with chromatographic column, the purified allophanate hydrolase protein is obtained, such as image 3 As shown, the protein molecular weight of the obtained allophanate hydrolase protein is b...

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Abstract

The invention relates to the field of molecular biology, and specifically relates to an allophanic acid hydrolase and a preparation method thereof. The preparation method comprises following steps: (1) utilizing a PCR technology to carry out amplification to obtain clones of allophanic acid hydrolase; (2) constructing an expression carrier of allophanic acid hydrolase; (3) transferring the expression carrier into a competent escherichia coli to obtain recombinant escherichia coli, and finally carrying out induced expression and protein purification to obtain allophanic acid hydrolase. The method is simple, the operation is easy, allophanic acid hydrolase with a novel gene sequence is found out, and the allophanic acid hydrolase still has strong enzymatic activity at a low temperature. Furthermore, the invention also provides a polyclonal antibody of the allophanic acid hydrolase and a detection method thereof. A novel evidence is provided for the identification of a low temperature bacterium Cryobacterium, and a new research direction is provided for the research on allophanic acid hydrolase.

Description

technical field [0001] The invention belongs to the field of molecular biology, and mainly relates to an allophanate hydrolase and its application. Background technique [0002] Allophanate hydrolase, English name: Allophanate hydrolase. Allophanate hydrolase plays an important role in the degradation process of triazine herbicides. It catalyzes the degradation of allophanate to generate ammonia and carbon dioxide. It can degrade triazine-containing pollutants such as alatzine herbicide, and has broad application prospects in the field of environmental protection. [0003] The use of allophanate hydrolase to degrade the intermediate allophanate of herbicide pollutants has great application prospects. Under low temperature conditions, allophanate is used as the substrate to degrade and generate ammonia gas, which has a good effect in purifying the environment. [0004] The currently produced allophanate hydrolase has poor tolerance to low temperature and can hardly exert it...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/80C12N15/55C12N15/70C12N15/11C07K16/40C07K16/06
CPCC07K16/06C07K16/40C12N9/80C12N15/70C12Y305/01054
Inventor 宫春杰薛栋升陶冶胡征杨波
Owner HUBEI UNIV OF TECH
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