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Low-temperature catechol 1,2-dioxygenase from animal waste metagenome, and encoding gene and preparing method thereof

A catechol and dioxygenase technology, which is applied in the field of genetic engineering, can solve the problems of unseen and lack of research on animal gastrointestinal environment samples

Active Publication Date: 2016-04-20
YUNNAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In recent years, the research on obtaining aromatic compound metabolic enzymes from the environment by using metagenomic technology has mainly focused on environmental samples such as soil, activated sludge and sewage, and the research on animal gastrointestinal environmental samples is scarce [Beloquietal.JBiolChem, 2006, 281 (32):22933-22942; Fangetal.PLoSOne,2012,7(11):e50312], and there is no report of catechol 1,2-dioxygenase from gastrointestinal tract

Method used

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  • Low-temperature catechol 1,2-dioxygenase from animal waste metagenome, and encoding gene and preparing method thereof
  • Low-temperature catechol 1,2-dioxygenase from animal waste metagenome, and encoding gene and preparing method thereof
  • Low-temperature catechol 1,2-dioxygenase from animal waste metagenome, and encoding gene and preparing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Cloning of catechol 1,2-dioxygenase gene catPL12

[0048] Microbial genomic DNA was extracted from the feces of the loris (the extraction method can refer to the patent "A method for extracting high-molecular-weight genomes from animal feces", publication number 102586234A), and then use the DNA as a template to incorporate primer C 12 OF and C 12 OR (see Table 1 for its nucleotide sequence) was amplified by PCR. The parameters of the PCR reaction are: denaturation at 94°C for 5 min; then denaturation at 94°C for 30 sec, annealing at 48°C for 30 sec, extension at 72°C for 1 min, and after 30 cycles, incubation at 72°C for 7 min to obtain a gene fragment of about 430 bp, which was recovered and combined with pMD19- T carrier connection, sent to Beijing Liuhe Huada Gene Technology Co., Ltd. for sequencing, and obtained catechol 1,2-dioxygenase gene fragment C 12 Nucleotide sequence of O15-d-13.

[0049] with C 12 O15-d-13 was the core sequence, and three ups...

Embodiment 2

[0054] Example 2 Preparation of low temperature catechol 1,2-dioxygenase CatPL12

[0055] The catechol 1,2-dioxygenase gene catPL12 prepared in Example 1 was connected to the plasmid pEasy-E2 to obtain the recombinant expression vector pEasy-E2-catPL12, and then transformed into Escherichia coli BL21 (DE3) to obtain the recombinant Escherichia coli strain BL21 (DE3) / catPL12. The Escherichia coli strain BL21(DE3) / catPL12 containing the recombinant expression vector pEasy-E2-catPL12 was inoculated in LB (containing 100 μg / mL Amp) culture medium at an inoculation amount of 0.1%, and shaken rapidly at 37°C for 16 hours. Then inoculate the activated bacterial solution into fresh LB (containing 100 μg / mL Amp) culture solution with 1% inoculum, and culture it with rapid shaking for about 2–3 hours (OD 600 After reaching 0.6–1.0), add IPTG at a final concentration of 0.7mmol / L for induction, and continue shaking culture at 20°C for about 20h. Centrifuge at 12000rpm for 5min to colle...

Embodiment 3

[0057] Example 3 Determination of properties of low temperature catechol 1,2-dioxygenase CatPL12

[0058]1. Activity analysis of low temperature catechol 1,2-dioxygenase CatPL12

[0059] Enzyme activity was determined by spectrophotometry [Gouetal.BiotechnolLett, 2012,34(1):117-123]: take 10μl of 150mM catechol substrate solution (final concentration is 0.5mM) and 2.94mL of 50mM buffer solution at reaction temperature Preheat for 3 minutes, add 50 μl of appropriately diluted enzyme solution to react for 5 minutes, and measure the increase in absorbance within 5 minutes at the corresponding wavelength. The molar extinction coefficient of the catalyzed product cis,cis-hexadienedioic acid at 260nm is 16800 / Mcm with catechol as the substrate. One enzyme activity unit (U) is defined as the amount of enzyme required to catalyze a substrate to produce 1 μmol of the corresponding product per minute under given conditions.

[0060] 2. The method for determining the optimal pH and pH ...

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Abstract

The invention discloses low-temperature catechol 1,2-dioxygenase from animal waste metagenome. The amino acid sequence of low-temperature catechol 1,2-dioxygenase is shown in SEQ ID NO.2, the total number of amino acids is 309, and the theoretical molecular weight is 33.72 kDa; the nucleotide sequence of an encoding gene is shown in SEQ ID NO.1, and the size of the gene is 930 bp. The optimum action pH value of low-temperature catechol 1,2-dioxygenase is 8.0, and after treatment is carried out for 1 h within the pH value range of 7.0-9.0, remaining enzyme activity is 60% or more; the optimum action temperature is 25 DEG C, and the enzyme activity is about 30% and 60% at the temperature of 0 DEG C and 10 DEG C respectively; low-temperature catechol 1,2-dioxygenase tolerates the temperature of 25 DEG C and 37 DEG C for 1 h, the enzyme activity is not affected, low-temperature catechol 1,2-dioxygenase tolerates the temperature of 25 DEG C, 65% or more of enzyme activity can still be kept, and low-temperature catechol 1,2-dioxygenase can be used for enzymatic synthesis of cis,cis-muconic acid and degradation of aromatic compound in low-temperature environment.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a low-temperature catechol 1,2-dioxygenase derived from animal feces metagenome, its coding gene and its preparation method. Background technique [0002] Aromatic compounds are the second largest organic carbon source after carbohydrates on the earth, and they are widely found in nature. Catechol is an intermediate product of microbial metabolism of phenols and most polycyclic aromatic compounds, and further cracking of catechol plays an important role in whether PAHs can be completely degraded. Catechol 1,2-dioxygenase (EC1.13.11.1) is a key aromatic ring oxygenase that catalyzes the ortho oxidation ring opening of catechol, which catalyzes the cleavage of catechol to form an intermediate— —cis,cis-hexadienedioic acid, which is further degraded into the tricarboxylic acid cycle in the subsequent enzymatic reaction, so catechol 1,2-dioxygenase plays an i...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53
CPCC12N9/0069C12Y113/11001
Inventor 许波熊彩云黄遵锡李俊俊唐湘华杨云娟
Owner YUNNAN NORMAL UNIV
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