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Carboxylesterase resistant to metal ions and organic solvent and application of carboxylesterase

A technology of organic solvents and metal ions, 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: 2019-07-30
JIANGNAN UNIV
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  • 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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  • Carboxylesterase resistant to metal ions and organic solvent and application of carboxylesterase
  • Carboxylesterase resistant to metal ions and organic solvent and application of carboxylesterase
  • Carboxylesterase resistant to metal ions and organic solvent and application of carboxylesterase

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Experimental program
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Embodiment 1

[0043] Embodiment 1: Construction of engineering strains

[0044] The artificially synthesized carboxylesterase BaCEs04 gene sequence whose nucleotide sequence is shown in SEQ ID NO.2 (amino acid sequence is shown in SEQ ID NO.1). The BaCEs04 gene sequence and the plasmid vector pColdII were digested with restriction endonucleases SacI and XbaI, then ligated and transformed into E.coli BL21(DE3) competent cells to obtain recombinant E.coli BL21-pColdII-BaCEs04.

Embodiment 2

[0045] Embodiment 2: expression and purification of carboxylesterase (BaCEs04)

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

[0047] Inoculate the recombinant Escherichia coli E.coli BL21-pColdII-BaCEs04 in a solution containing 100mg·mL -1 In 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.

[0048] Collect the bacterial liquid, centrifuge at 4°C, 8000rmp for 10min to obtain the...

Embodiment 3

[0049] Embodiment 3: the enzyme activity assay of carboxylesterase (BaCEs04)

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

[0051] Under optimum reaction conditions, i.e. in disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution (pH 7.5), at 60°C, 0.6M 1-naphthyl acetate and 2-naphthyl acetate were used as substrates to determine Example 2 The specific enzyme activity of the carboxylesterase BaCEs04 purified in the middle, the specific enzyme activity of the purified carboxylesterase BaCEs04 reached 0.28U / mg and 0.13U / mg respectively. ...

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Abstract

The invention discloses carboxylesterase resistant to metal ions and an organic solvent and application of the carboxylesterase, and belongs to the field of enzyme engineering. The carboxylesterase BaCEs04 with the amino acid sequence shown in SEQ ID NO.1 is subjected to heterologous expression in escherichia coli, the enzyme activity of the obtained carboxylesterase BaCEs04 is improved by 24.3% and 23.5% separately in the presence of Zn2+ and NH4+, remaining metal ions of Na+, K+, Mg2+, Ca2+, Cu2+ and Fe3+ nearly have no influence on the enzyme activity or even slightly improve the enzyme activity, which shows that the carboxylesterase is stable in an environment with metal ions. After the carboxylesterase is placed in acetone, n-hexane and isopropanol for 1 hour, the enzyme activity is improved by 22%, 30% and 26% separately, which shows that the carboxylesterase has high organic solvent tolerance.

Description

technical field [0001] The invention relates to a carboxylesterase resistant to metal ions and organic solvents and an 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 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70A62D3/02B09B3/00C12R1/19
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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