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A kind of carboxylesterase and application thereof resistant to metal ions and organic solvents

A technology of organic solvents and metal ions, which is applied in the field of enzyme engineering, can solve the problems of low carboxylesterase expression, low tolerance, and limit the application of carboxylesterase, and achieve the effect of good organic solvent tolerance

Active Publication Date: 2022-05-17
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the expression level of carboxylesterase in wild bacteria is low, which cannot meet the needs of industrial applications; with the application of genetic engineering in the study of carboxylesterase, many carboxylesterase genes have been expressed heterologously
[0004] However, most carboxylesterases have low tolerance to metal ions and organic solvents, which severely limits the application of carboxylesterases.

Method used

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  • A kind of carboxylesterase and application thereof resistant to metal ions and organic solvents
  • A kind of carboxylesterase and application thereof resistant to metal ions and organic solvents
  • A kind of carboxylesterase and application thereof resistant to metal ions and organic solvents

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Construction of engineered strains

[0044] Synthetic nucleotide sequence as shown in SEQ ID NO.2 (amino acid sequence as shown in SEQ ID NO.1) carboxylate enzyme BaCEs04 gene sequence. BaCEs04 gene sequence and plasmid vector pColdII were cleaved with restriction enzymes SacI. and XbaI. and then ligation into E.coli BL21 (DE3) competent cells to obtain recombinant bacteria E.coli BL21-pColdII-BaCEs04.

Embodiment 2

[0045] Example 2: Expression and purification of carboxylate enzyme (BaCEs04).

[0046] LB medium g / L: sodium chloride 10, tryptone 10, Yeast Extract 5, pH 7.

[0047]Recombinant E. coli BL21-pColdII-BaCEs04 was inoculated with 100 mg·mL -1 In LB liquid medium for ampicilum, transfer to pClodII plasmid in the original strain E.coli BL21 (DE3) and no-load strain (E.coli BL21 (DE3)) as a control, 37 °C, 200rmp culture for 12 h, and then inoculate 500 μL of the above seed solution in 50 mL LB culture medium containing 50 μL ampicle, 37 °C for 2.5 h, to OD 600 For 0.6, cool the shaker to 15 °C and let stand for 30 min. Add 40 μL of IPTG with a final concentration of 0.4 mol / L per bottle as the inducer, take no inducer as the control group, and incubate at 15 °C200rmp for 24 h.

[0048] Collect the bacterial fluid, 4 °C, 8000rmp centrifugation for 10 min to obtain the bacterial body, add 5mL phosphate buffer (0.02mol / L, pH 7.0) to resuspend the bacterial body, ultrasonic crusher crush...

Embodiment 3

[0049] Example 3: Enzyme viability assay of carboxylate enzyme (BaCEs04).

[0050] In the disodium hydrogen phosphate- potassium dihydrogen phosphate buffer (pH 7), with 2-naphthyl acetate as the substrate, in the range of 15 to 75 °C, every 5 °C, the carboxylate enzyme BaCEs04 enzyme activity was determined, and the optimal temperature of the carboxylate enzyme BaCEs04 was 60 °C. Under the optimal reaction temperature of 60 °C, the pH range of 5.0 to 8.0, every 0.5, the enzyme activity was determined, and the optimal reaction pH was determined to be 7.5.

[0051] Under the optimal reaction conditions, i.e., disodium hydrogen phosphate - potassium dihydrogen phosphate buffer (pH 7.5), at 60 °C, 0.6M1-naphthalene acetate and 2-naphthalene acetate as substrates were determined in Example 2 the purified carboxylate enzyme BaCEs04 specific enzyme activity, purified carboxylate baCEs04 specific enzyme activity reached 0.28U / mg and 0.13U / mg, respectively.

[0052] Temperature stabil...

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Abstract

The invention discloses a carboxylesterase resistant to metal ions and organic solvents and an application thereof, belonging to the field of enzyme engineering. In the present invention, the carboxylesterase BaCEs04 whose amino acid sequence is shown in SEQ ID NO.1 is heterologously expressed in Escherichia coli, and the obtained carboxylesterase BaCEs04 is expressed in Zn 2+ , NH 4+ In the presence of , the enzyme activities were increased by 24.3% and 23.5%, respectively, and the remaining metal ions Na + , K + , Mg 2+ , Ca 2+ , Cu 2+ , Fe 3+ There is almost no effect on the enzyme activity or even a slight increase, indicating that the carboxylesterase is relatively stable in the presence of metal ions. After the enzyme is placed in acetone, n-hexane, and isopropanol for 1 hour, the enzyme activity increases by 22%, 30%, and 26% respectively, indicating that the enzyme has good tolerance to organic solvents.

Description

Technical field [0001] The present invention relates to a metal ion-resistant and organic solvent carboxylate enzyme and its applications, belonging to the field of enzyme engineering. Background [0002] Carboxylesterase (EC 3.1.1.1) refers to non-specific esterases capable of catalyzing hydrolyzed carboxylates to produce carboxylic acids and alcohols. Carboxylates are widely used in production and practical life, and have been favored by more and more modern pharmaceutical industries, chiral compound synthesis, fine chemicals and other related industries. Carboxylates are used as catalysts to catalyze chiral molecular synthesis, such as the production of naproxen and 2-aryl naphthalene, while carboxylates are also used as green catalysts to degrade pesticide residues in soil and plasticizer phthalates in soil and water. Due to the defects of low enzyme activity, low yield, complex extraction, poor thermal stability and other defects of carboxylate enzymes produced by automatic...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70A62D3/02B09B3/70C12R1/19
CPCC12N9/18C12Y301/01001C12N15/70A62D3/02B09B3/00A62D2101/47
Inventor 廖祥儒黄琳杨邵岚孟迪蔡宇杰管政兵
Owner JIANGNAN UNIV
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