Carboxylesterase as well as coding gene and application thereof

A carboxylesterase and gene technology, applied in the field of genetic engineering, can solve problems such as difficult mass production, limited enzyme production capacity of wild strains, long fermentation cycle, etc., achieve good activity and stability, easy to obtain pure protein, and short cultivation cycle short effect

Inactive Publication Date: 2014-10-22
YUNNAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The use of microorganisms to degrade phthalates mostly relies on intracellular enzymatic hydrolysis, but wild strains have limited enzyme production capaci

Method used

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  • Carboxylesterase as well as coding gene and application thereof
  • Carboxylesterase as well as coding gene and application thereof
  • Carboxylesterase as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Cloning of CareW of carboxylesterase gene

[0042] The genomic DNA of Bacillus K91 was extracted with the Genome Extraction Kit (DP302) of Bacillus sp.

[0043] According to Bacillus K91 whole genome sequencing and gene annotation, analyze the carboxylesterase gene CareEW and design expression primers CareEW F and CareEW R:

[0044] Upstream primer CareW F: 5’-ACTCATCAAATAGTAACGAC-3’

[0045] Downstream primer CareW R: 5’-TTCTCCTTTT GAAGGGAATA G-3’

[0046] Upstream primer T7: 5’-TAATACGACTCACTATAGGG-3’

[0047] Downstream primer T7ter: 5’-TGCTAGTTATTGCTCAGCGG-3’

[0048] T7 and T7ter are primers for PCR detection of positive clones.

[0049] PCR amplification was carried out with the genomic DNA of Bacillus thermophilus K91 as a template. The PCR reaction parameters were: 94°C pre-denaturation for 1min30sec; then 94°C denaturation for 30sec; 58°C annealing (0.5°C per cycle) 30sec; 72°C extension for 1min40sec; After 28 cycles, denaturation at 94°C for 30sec; annealing a...

Embodiment 2

[0051] Example 2: Preparation of Recombinant Carboxyesterase CareEW

[0052] Take the recombinant plasmid pEASY TM -E2-CarEW's BL21(DE3) strain and only contains pEASY TM -E2 empty plasmid BL21(DE3) strain, inoculated in LB (containing 100ug / mL Amp r ) In the culture solution, shake rapidly at 37°C for 16 hours. Then the activated bacterial solution was inoculated into fresh LB (containing 100ug / mL Amp r ) In the culture medium, fast shaking culture for about 2–3h (OD 600 After reaching 0.4-0.7), IPTG with a final concentration of 0.7mM was added for induction, and shaking culture was continued for about 20h at 20°C. Centrifuge at 12000 rpm for 10 min to collect the bacteria. Use appropriate amount of pH7.0 citric acid-Na 2 HPO 4 After suspending the bacteria in the buffer, the bacteria are broken by ultrasonic in a low temperature water bath. After centrifugation of the above concentrated crude enzyme solution at 12,000 rpm for 10 minutes, the supernatant was aspirated and the ...

Embodiment 3

[0053] Example 3: Determination of the properties of purified recombinant carboxylesterase CarEW

[0054] 1. Activity analysis of the recombinant carboxylesterase CarEW

[0055] The purified recombinant esterase CarEW uses p-nitrophenol method for activity determination: Take 4 1.5mL centrifuge tubes, numbered 1, 2, 3, 4, of which 1, 2, 3 test tubes are the experimental group (3 replicates), No. 4 is the control group. Add 420 μL of 50 mM citric acid-Na pH 7.5 to the four centrifuge tubes. 2 HPO 4 Buffer, 0.4% Trion-100, 0.1% gum arabic emulsion and 30μL 10mM substrate pNPC 4 After preheating at 37°C for 5 minutes, add 50μL of enzyme solution diluted by an appropriate multiple to tubes 1, 2 and 3 every 10s, add the same amount of water to the control tube No. 4, react at 37°C for 5 minutes and then turn to 1 every 10s. 2, 3 Add 50μL of 0.1M Na to the experimental tube 2 CO 3 To stop the reaction, add an equal volume of stop solution to the No. 4 control tube and immediately insert...

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Abstract

The invention provides a carboxylesterase, a gene encoding the carboxylestearase, a recombination carrier comprising the gene, a recombination strain comprising the gene, and application of the carboxylesterase in degrading diisobutyl phthalate, wherein the amino acid sequence of the carboxylesterase is shown as SEQ ID NO.1. The carboxylesterase gene CarEW comes from a hot spring environment, is proven to have good activity and stability under the condition of 45DEG C, and can be used for degrading phthalate ester compounds (phthalate) such as diisobutyl phthalate in the environment, thus having important significance on the aspect of environment pollution treatment.

Description

Technical field [0001] The invention belongs to the technical field of genetic engineering, and particularly relates to a carboxylesterase, its coding gene and its application. Background technique [0002] Diisobutyl phthalate (DIBP) is a phthalate compound and is a plasticizer and softener widely used in the plastics industry. Its role is to increase the plasticity and toughness of plastics and improve the strength of plastics (Zhao Zhenhua , Environmental Chemistry, 1991, 10(3): 64-68). In addition, DIBP is also widely used in the production process of coatings, printing and dyeing, cosmetics and fragrances. Recent studies have shown that DIBP only relies on intermolecular interactions to combine with polymer plastics. Over time, such substances can migrate directly into food through food packaging materials, or into the environment through plastic products containing the substance. This causes pollution to air, water, soil and food, and can enter the human body through brea...

Claims

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Application Information

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IPC IPC(8): C12N9/18C12N15/55C12N15/70C12N1/21A62D3/02C12R1/07A62D101/28
CPCA62D3/02A62D2101/28C12N9/18C12Y301/01001
Inventor 黄遵锡丁俊美王超凡李俊俊慕跃林杨云娟
Owner YUNNAN NORMAL UNIV
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