Method for degrading phthalic acid esters (PAEs) containing metal ions or organic solvents
A technology of phthalic acid and metal ions, applied in the field of enzyme engineering, can solve the problems of low tolerance of metal ions and organic solvents, limited application of carboxylesterase, etc., and achieve the effect of good temperature stability
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Embodiment 1
[0042] Embodiment 1: Construction of engineering strains
[0043] The artificially synthesized carboxylesterase BaCEs01 gene sequence whose nucleotide sequence is shown in SEQ ID NO.2 (amino acid sequence is shown in SEQ ID NO.1). Carboxylesterase BaCEs01 gene sequence and 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 strain E.coliBL21-pColdII-BaCEs01 .
Embodiment 2
[0044] Embodiment 2: Expression and purification of carboxylesterase (BaCEs01)
[0045] LB medium g / L: sodium chloride 10, tryptone 10, Yeast Extract 5, pH 7.
[0046] Inoculate the recombinant Escherichia coli E.coli BL21-pColdII-BaCEs01 in a solution containing 100 mg·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.
[0047] Collect the bacterial liquid, centrifuge at 4°C, 8000rmp for 10min to obtain the...
Embodiment 3
[0048] Embodiment 3: the enzyme activity assay of carboxylesterase (BaCEs01)
[0049] In disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution (pH 7), with 2-naphthyl acetate as substrate, in the range of 20-50 °C, every 5 °C, measure the enzyme activity of carboxylesterase BaCEs01, it can be known The optimum temperature of carboxylesterase BaCEs01 was 40℃. Under the optimum reaction temperature of 40°C, within the range of pH 5.0-9.0, the enzyme activity was measured every 0.5, and the optimum reaction pH was determined to be 6.5.
[0050] Under optimum reaction conditions, i.e. in disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution (pH 6.5), at 40°C, respectively use 0.6M 1-naphthyl acetate and 2-naphthyl acetate as substrates to measure Example 2 The specific enzyme activities of the purified carboxylesterase BaCEs01 reached 5.20U / mg and 1.20U / mg respectively.
[0051] Temperature stability: Store 250 μL of carboxylesterase (Ba...
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