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Method for degrading triphenylmethane dye by utilizing recombinational lipoxygenase

A technology of lipoxygenase and triphenylmethane, applied in the biological field, can solve the problems of unsatisfactory treatment effect and difficult degradation of biological treatment system

Inactive Publication Date: 2015-05-06
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

And some of these dyes are difficult to degrade, which often leads to the unsatisfactory treatment effect of conventional biological treatment systems
There is no report on the use of lipoxygenase to catalyze the degradation of triphenylmethane dyes

Method used

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  • Method for degrading triphenylmethane dye by utilizing recombinational lipoxygenase
  • Method for degrading triphenylmethane dye by utilizing recombinational lipoxygenase
  • Method for degrading triphenylmethane dye by utilizing recombinational lipoxygenase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Cloning of Pseudomonas aeruginosa ATCC27853 lipoxygenase gene (LOX-27853)

[0031] The bacterial cells of Pseudomonas aeruginosa ATCC27853 were collected by centrifugation, and the genomic DNA of Pseudomonas aeruginosa ATCC27853 was extracted with the Shanghai Sangon Genomic DNA Extraction Kit.

[0032] According to the registered Pseudomonas lipoxygenase gene (No.CP006832.1) in the Genebank database, two primers were designed:

[0033] Upstream primer F-1: 5'-ATGAAACGCAGGAGTGTGCTCTTG-3' (SEQ ID NO.3);

[0034] Downstream primer R-1: 5'-TCAGATATTGGTGCTCGCCGGGATC-3' (SEQ ID NO.4);

[0035] In a 50 μl system, the final concentration of each primer is 1 μM, the final concentration of dNTPs is 0.2 mM, 10 ng of genomic DNA of Pseudomonas aeruginosa ATCC27853 strain, 2 U of Pfu DNA polymerase. The amplification program was 94°C for 3min; 30×(94°C for 40s, 53°C for 50s, 72°C for 90s); 72°C for 10min. Agarose gel electrophoresis, gel cutting, recovery by Shanghai ...

Embodiment 2

[0036] Embodiment 2: the construction of Pseudomonas aeruginosa ATCC27853 lipoxygenase gene (LOX-27853) prokaryotic expression vector (attached figure 1 )

[0037] According to the obtained laccase gene sequence, two primers were designed, the upstream primer plus the SacI recognition sequence, and the downstream primer plus the XhoI recognition sequence (the underlined part is the restriction enzyme recognition sequence):

[0038] Upstream primer F-2: 5′-CGC GAGCTC ATGAAACGCAGGAGTGTGCTCTTG-3' (SEQ ID NO. 5)

[0039] Downstream primer R-2: 5′-CCG CTCGAG TCAGATATTGGTGCTCGCCGGGATC-3' (SEQ ID NO. 6)

[0040] Add each component according to the following PCR system to amplify the LOX gene:

[0041]

[0042] The PCR program is: 94°C for 3min; 30×(94°C for 40s; 53°C for 50s; 72°C for 90s); 72°C for 10min.

[0043] Purify the PCR product with Shanghai Sangon PCR Product Purification Kit, add SacI, XhoI double enzyme digestion, inactivation, ethanol precipitation, ddH 2 O was...

Embodiment 3

[0044] Embodiment 3: Fermentative production of Pseudomonas aeruginosa ATCC27853 lipoxygenase gene (LOX-27853) in Escherichia coli

[0045] The fermentation production of Pseudomonas aeruginosa ATCC27853 lipoxygenase gene (LOX-27853) in Escherichia coli involves the following three media:

[0046] (1) Solid plate medium: 10g / L tryptone, 5g / L yeast extract, 10g / L sodium chloride, 15g / L agar powder

[0047] (2) Seed liquid medium: 10g / L tryptone, 5g / L yeast extract, 10g / L sodium chloride

[0048] (3) Fermentation medium: 10g / L lactose, 0.5g / L glucose, 5g / La glycerol, 3.4g / L KH 2 PO 4 , 1.2g / L MgSO 4 , 10g / L Enzymatic Sodium Caseinate, 5g / L Yeast Extract, 8.95g / L Na 2 HPO 4 .12H 2 O, 1.42g / L Na 2 SO 4 ,2.67g / L NH 4 Cl and 1ml trace element mixture.

[0049] (4) Trace element mixture: 50μM FeCl 3 .6H 2 O, 20 μM CaCl 2 .2H 2 O, 10 μM MnCl 2 .4H 2 O, 10 μM ZnSO 4 .7H 2 O, 2 μM CoCl 2 .6H2O, 2μM CuCl 2 ,2μM NiCl 2 ,2μM Na 2 SeO 3 ,2μM H 3 B 4 o 7 .

[0050]...

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Abstract

The invention discloses a method for degrading a triphenylmethane dye by utilizing recombinational lipoxygenase and application of the recombinational lipoxygenase rLOX-27853 in degrading the triphenylmethane dye. The amino acid sequence of the recombinational lipoxygenase rLOX-27853 is as shown in SEQ ID NO.2. The method for degrading the triphenylmethane dye by utilizing the recombinational lipoxygenase comprises the following steps: adding the recombinational lipoxygenase rLOX-27853 to a medium which contains sodium linoleate and the triphenylmethane dye to degrade the medium. The method disclosed by the invention obtains a novel prokaryotic lipoxygenase gene LOX-27853 by cloning from a pseudomonas aeruginosa ATCC27853 strain genome and realizes the heterogenetic high-efficiency expression of host bacteria in escherichia coli by utilizing a fermentation culture method, thereby efficiently catalyzing the degradation of the triphenylmethane dyes, namely aniline blue, malachite green and brilliant green.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for degrading triphenylmethane dyes by using recombinant lipoxygenase. Background technique [0002] Triphenylmethane dye is a kind of polyphenyl ring compound, which is widely used and used in a large amount, and its output is listed as the third major dye. Some of the dyes and intermediate degradation products have "three effects". Moreover, some of these dyes are difficult to degrade, which often leads to unsatisfactory treatment effects of conventional biological treatment systems. Effective dye wastewater treatment methods and technologies are an important guarantee for improving the ecological environment, ensuring food safety and maintaining human health. The biological method is to screen or construct specific microbial strains with high performance for the decolorization of dye wastewater. It is an economical, effective and suitable technology for large-scale wastew...

Claims

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Application Information

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IPC IPC(8): C02F3/34C12N9/02C12N15/70C12R1/385C02F101/38
CPCC02F3/342C02F2101/308C02F2301/00C12N9/0004
Inventor 张充陆兆新卢静吕凤霞别小妹赵海珍
Owner NANJING AGRICULTURAL UNIVERSITY
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