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Acidic lipoxygenase and preparation method and application thereof

A lipoxygenase, acid technology, applied in the field of acid lipoxygenase and its preparation

Active Publication Date: 2017-06-13
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there are no reports at home and abroad about lipoxygenase derived from recombinant Myxococcus aurantium and its application in dye decolorization

Method used

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  • Acidic lipoxygenase and preparation method and application thereof
  • Acidic lipoxygenase and preparation method and application thereof
  • Acidic lipoxygenase and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Myxococcus xanthus Cloning of DK1622 lipoxygenase gene (rMxLOX)

[0029] centrifuge collection Myxococcus xanthus DK1622 cells, extracted with Shanghai Sangon Genomic DNA Extraction Kit Myxococcus xanthus Genomic DNA of DK1622.

[0030] Two primers were designed according to the Myxococcus aurantiacus lipoxygenase gene (No.CP006832.1) registered in the Genebank database:

[0031] Upstream primer F-1: 5'-ATGAAACGCAGGAGTGTGCTCTTG-3' (SEQ ID NO.3);

[0032] Downstream primer R-1: 5'-TCAGATATTGGTGCTCGCCGGGATC-3' (SEQ ID NO.4);

[0033] obtained by extracting Myxococcus xanthus DK1622 genomic DNA is used as a template, and the above two primers are used for PCR amplification. The PCR reaction system is as follows:

[0034] 2× GC Buffer 25 µL MXLOX-F 2 µL MXLOX-R 2 µL 2.5 mM dNTPs 8 µL genomic DNA 1 µL Pfu DNA polymerase 1 µL wxya 2 o

Make up to 50 µL

[0035] PCR reaction conditions: pre-denaturati...

Embodiment 2

[0037] Embodiment 2: the construction of prokaryotic expression vector of myxococcus aureus DK1622 lipoxygenase gene (rMxLOX) (attachment figure 1 )

[0038] According to the obtained rMxLOX gene sequence, design two primers (SEQ ID NO.3, SEQ ID NO.4), the upstream primer plus Bam H I recognition sequence, downstream primer plus Hind ШRecognition sequence:

[0039] Add components according to the following PCR system to amplify the LOX gene:

[0040] 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.

[0041] Purify the PCR product with Shanghai Sangon PCR Product Purification Kit, add Bam H I. Hind ШDouble restriction enzyme digestion, connection with appropriate amount of vector pET-28a digested with the same restriction enzyme, and transformation into Escherichia coli DH5α. Randomly pick a few colonies from the transformation plate, insert them into LB liquid medium, shake culture, extract plasmids, perform elect...

Embodiment 3

[0043] Method for the production of Myxococcus aureus DK1622 lipoxygenase gene (rMxLOX) by fermentation in Escherichia coli:

[0044] (1) Transform Escherichia coli expression host strain BL21(DE3) containing rMxLOX expression plasmid pET-28a-rMxLOX (product of Novagen, Germany), and spread it on solid plate medium containing kanamycin (50 μg / ml), 37 Cultivate for 12-16 hours at ℃;

[0045] (2) Pick a single colony, insert it into 50ml seed liquid medium containing kanamycin (50μg / ml), and culture overnight at 70-90rpm 30°C;

[0046] (3) Take the seed liquid according to the volume ratio of 1% and add it to 100ml fermentation medium containing kanamycin (50μg / ml), shake and cultivate at 37°C and 180rpm for 2-3 hours until the OD600 is about 0.6;

[0047](4) Adjust the fermentation temperature to 16°C and shake at 180rpm for 16 hours.

[0048] (5) Collect the bacteria by centrifugation, add pH 3.0 citric acid buffer, break the bacteria by ultrasonic, and collect the supernata...

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Abstract

The invention discloses acidic lipoxygenase and a preparation method and application thereof. A novel lipoxygenase gene MxLOX is obtained by cloning from an Myxococcus xanthus DK1622 strain genome, and the amino acid sequence of the novel lipoxygenase gene is shown as SEQ ID NO.2. The lipoxygenase has the highest activity under the conditions that the pH is 3.0 and the temperature is 30 DEG C, and has stable activity under the conditions that the pH is 2.5-5.0 and the temperature is 30 DEG C. Recombinant lipoxygenase rMxLOX can efficiently catalyze degradation of triphenylmethane dyes such as aniline blue and methyl blue, and has an potential application value in treatment of acidic industrial wastewater.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to an acid lipoxygenase and its preparation method and application. Background technique [0002] Triphenylmethane dye is a kind of polyphenyl ring compound, which has "carcinogenic, teratogenic and mutagenic" effects. The treatment of dye wastewater is mainly physical and chemical methods, although effective, but the general cost is high, and easy to produce secondary pollutants. However, biodegradable dyes have become a research hotspot of scholars at home and abroad because of their low cost, less secondary pollution, and good ecological restoration. Biodegradable dyes are mainly decolorized by the extracellular oxidase system secreted by microorganisms, including laccase, lignin peroxidase and manganese peroxidase. Therefore, effective dye wastewater treatment methods and technologies are an important guarantee for improving the ecological environment and maintaining human health. ...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C12N15/11C12N15/70C02F3/34C02F101/38
CPCC02F3/342C02F2101/308C02F2101/40C12N9/0069C12N15/70C12Y113/11012
Inventor 吕凤霞陆兆新钱辉张充别小妹赵海珍
Owner NANJING AGRICULTURAL UNIVERSITY
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