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Heat-stable catechol 1,2-dioxygenase derived from animal manure metagenome and coding gene thereof, and preparation method of heat-stable catechol 1,2-dioxygenase

A catechol and dioxygenase technology, applied in the field of microorganisms and genetic engineering, can solve the problems of limiting the breadth and effectiveness of screening

Active Publication Date: 2016-05-11
YUNNAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional pure culture technology of microorganisms makes it impossible to isolate non-cultivable microorganisms, which account for more than 99% of microbial species, so the traditional method of screening novel enzymes by isolating and cultivating microorganisms greatly limits the breadth and effectiveness of screening.

Method used

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  • Heat-stable catechol 1,2-dioxygenase derived from animal manure metagenome and coding gene thereof, and preparation method of heat-stable catechol 1,2-dioxygenase
  • Heat-stable catechol 1,2-dioxygenase derived from animal manure metagenome and coding gene thereof, and preparation method of heat-stable catechol 1,2-dioxygenase
  • Heat-stable catechol 1,2-dioxygenase derived from animal manure metagenome and coding gene thereof, and preparation method of heat-stable catechol 1,2-dioxygenase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 The acquisition of catechol 1,2-dioxygenase gene catPLCgl

[0044] 1. Construction of a metagenomic library for the fecal microorganisms of the Japanese slow loris

[0045] Microbial genomic DNA was extracted from the feces of the loris (see the patent "A method for extracting high molecular weight genomes from animal feces" for details on the extraction method, publication number: CN102586234A, application date: 2012-03-12), after fragmentation Separation by pulsed field electrophoresis and agarose gel electrophoresis to recover a DNA fragment with a size of about 40kb. The recovered DNA fragment was ligated with the fosmid vector pCC1FOS and transfected with host bacteria E.coliEPI300, and spread on an LB plate containing 12.5 μg / mL chloramphenicol Transformants were obtained by culturing overnight at 37°C, and the clone library was stored in a 96-well plate at -80°C.

[0046] 2. Extraction and sequencing of fosmid plasmid

[0047] The fosmid mixed plasmid...

Embodiment 2

[0050] Example 2 Preparation of thermostable catechol 1,2-dioxygenase CatPLCgl

[0051] The catechol 1,2-dioxygenase gene catPLCgl prepared in Example 1 was connected to the plasmid pEasy-E2 to obtain the recombinant expression vector pEasy-E2-catPLCgl, and then transformed into Escherichia coli BL21 (DE3) to obtain recombinant Escherichia coli strain BL21 (DE3) / catPLCgl. Take the Escherichia coli strain BL21(DE3) / catPLCgl containing the recombinant expression vector pEasy-E2-catPLCgl, and inoculate it in LB (containing 100 μg / mL Amp) culture medium with a 0.1% inoculum amount, and shake 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 9500rpm for 5min to colle...

Embodiment 3

[0053] Embodiment 3 Thermostable catechol 1,2-dioxygenase CatPLCgl property determination

[0054] 1. Activity analysis of thermostable catechol 1,2-dioxygenase CatPLCgl

[0055]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 catalytic 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.

[0056] 2. Determination of optimum pH and pH stability of thermosta...

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Abstract

The invention discloses a heat-stable catechol 1,2-dioxygenase derived from animal manure metagenome. The amino acid sequence is disclosed as SEQ ID NO.2, the catechol 1,2-dioxygenase has 283 amino acids, and the theoretical molecular weight is 31.91kDa. The nucleotide sequence of the coding gene is disclosed as SEQ ID NO.1, and the gene size is 852bp. The optimal action pH of the heat-stable catechol 1,2-dioxygenase is 7.0; after the catechol 1,2-dioxygenase is used for treatment within the pH range of 7.0-10.0 for 1 hour, the residual enzyme activity is 90% or above. The optimal action temperature is 40 DEG C; after the catechol 1,2-dioxygenase resists the temperatures of 25 DEG C and 40 DEG C for 210 hours, the enzyme activity is basically not influenced, and thus, the catechol 1,2-dioxygenase is a typical heat-stable enzyme; and the catechol 1,2-dioxygenase can be used for enzyme-process synthesis of cis-cis-muconic acid and degradation of polycyclic aromatic hydrocarbons.

Description

technical field [0001] The application belongs to the field of microbiology and genetic engineering, and in particular relates to a thermostable catechol 1,2-dioxygenase derived from animal feces metagenomics, 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-oxidative ring opening of catechol, which catalyzes the cleavage of catechol to form an intermediate— —cis, cis-hexadienate, which is further degraded into succinate and acetyl-CoA in subsequent enzymatic reactions, and enters the tricarboxylic acid cycle, and...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/70
CPCC12N9/0069C12Y113/11001
Inventor 许波邓梦黄遵锡李俊俊唐湘华杨云娟
Owner YUNNAN NORMAL UNIV
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