Recombinant bacteria for producing carboxylesterase and application thereof

A carboxylesterase and carboxylester-producing technology, which is applied in the field of enzyme engineering, can solve the problem that the enzyme activity of carboxylesterase cannot meet industrial needs and other problems, and achieves the effect of efficient degradation

Active Publication Date: 2019-06-28
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But these carboxylesterase enzymatic activity still can't reach industrial demand

Method used

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  • Recombinant bacteria for producing carboxylesterase and application thereof
  • Recombinant bacteria for producing carboxylesterase and application thereof
  • Recombinant bacteria for producing carboxylesterase and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Embodiment 1: Construction of engineering strains

[0040] The artificially synthesized carboxylesterase BaCEs02 gene sequence whose nucleotide sequence is shown in SEQ ID NO.2 (amino acid sequence is shown in SEQ ID NO.1). The BaCEs02 gene sequence and the plasmid vector pColdII were digested with restriction endonucleases SacI and XbaI and then ligated and transformed into E.coli BL21 (DE3) competent cells to obtain the recombinant strain E.coli BL21-pColdII-BaCEs02.

Embodiment 2

[0041] Embodiment 2: Expression and purification of carboxylesterase (BaCEs02)

[0042] LB medium g / L: sodium chloride 10, tryptone 10, Yeast Extract 5, pH 7.

[0043] Inoculate the recombinant Escherichia coli E.coli BL21-pColdII-BaCEs02 in a solution containing 100mg·mL -1In the LB liquid medium of ampicillin, the original strain E.coli BL21(DE3) and the empty strain (E.coli BL21(DE3) was transformed into the pClodII plasmid) were used as controls, cultured at 37°C, 200rmp for 12h, and then 500μL of the above The seed solution was inoculated in 50 mL LB medium containing 50 μL ampicillin, and cultured at 37 ° C for 2.5 h until the OD 600 to 0.6, cool the shaker to 15°C and let it stand for 30 minutes. 40 μL of IPTG with a final concentration of 0.4 mol / L was added to each bottle as an inducer, and no inducer was used as a control group, and cultured at 15°C and 200 rpm for 24 hours.

[0044] Collect the bacterial liquid, centrifuge at 4°C, 8000rmp for 10min to obtain the ...

Embodiment 3

[0045] Embodiment 3: the enzyme activity assay of carboxylesterase (BaCEs02)

[0046] In disodium hydrogen phosphate-potassium dihydrogen phosphate buffer (pH 7), with 2-naphthyl acetate as substrate, in the range of 15 to 60°C, every 5°C, measure the enzyme activity of carboxylesterase BaCEs02, it can be known The optimum temperature for carboxylesterase BaCEs02 is 45°C (see figure 1 ). Under the optimum reaction temperature of 45°C, within the range of pH 5.0 to 8.0, measure the enzyme activity every 0.5 to determine the optimum reaction pH as 6.5 (see figure 2 ).

[0047] Under optimum reaction conditions, i.e. in disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution (pH 6.5), at 45°C, 0.6M 1-naphthyl acetate and 2-naphthyl acetate were used as substrates to determine the examples The enzyme activity of the crude enzyme liquid obtained in 2 and the specific enzyme activity of the carboxylesterase BaCEs02 obtained by purification, wherein the enzyme ...

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Abstract

The invention discloses recombinant bacteria for producing carboxylesterase and application thereof, and belongs to the field of enzyme engineering. The carboxylesterase BaCEs02 with the amino acid sequence shown in SEQ ID NO.1 is heterologously expressed in escherichia coli, wherein the enzyme activity of 1-naphthyl acetate and 2-naphthyl acetate being substrates in crude enzyme liquid is 13831.8U / L and 8135.9 U / L. The degradation rates of carboxylesterase BaCEs02 pure enzyme liquid obtained after purification for phthalic acid ester (diethyl phthalate or dibutyl phthalate or diisobutyl phthalate) reach up to 92.2%, 95.6% and 87.3% respectively. A method for efficiently degrading a plasticizer phthalic acid ester is provided.

Description

technical field [0001] The invention relates to a recombinant bacterium producing carboxylesterase and application thereof, belonging to the field of enzyme engineering. Background technique [0002] Carboxylesterase (EC 3.1.1.1) refers to a non-specific esterase that can catalyze the hydrolysis of carboxylic esters to generate carboxylic acids and alcohols. Carboxylesterase is widely used in production and practical life, and has been favored by more and more modern pharmaceutical industry, chiral compound synthesis, fine chemical industry and other related industries. Use carboxylesterase as a catalyst to catalyze the synthesis of chiral molecules, such as the production of naproxen and 2-aryl naphthoic acid; at the same time, carboxylesterase is also used as a green catalyst to degrade pesticide residues in soil and plasticization in soil and water phthalates. Carboxylesterases produced in animals and plants have defects such as low enzyme activity, low yield, complicat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/55C12N9/18C02F3/34C12R1/19C02F101/34
Inventor 廖祥儒黄琳杨邵岚李静蔡宇杰管政兵
Owner JIANGNAN UNIV
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