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Manganese peroxidase MNP-2, gene and applications thereof

A technology of manganese peroxidase and enzyme manganese peroxide, applied in the field of genetic engineering, can solve problems such as hindering large-scale application, low yield and the like

Active Publication Date: 2017-02-22
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, only dozens of manganese peroxidases have been isolated from white-rot fungi and their production is low, which seriously hinders large-scale application.

Method used

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  • Manganese peroxidase MNP-2, gene and applications thereof
  • Manganese peroxidase MNP-2, gene and applications thereof
  • Manganese peroxidase MNP-2, gene and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Cloning of Irpex lactus manganese peroxidase coding gene MNP-1

[0046] Extraction of total RNA from Irpex lactus:

[0047] Collect the mycelia cultured in liquid for 5 days, press dry on filter paper, and thoroughly grind into powder in liquid nitrogen; add 800 μl Trizol reagent to 100 mg mycelia, blow and mix, and place at room temperature for 5 minutes; add 200 μl chloroform, and shake vigorously for 15 seconds , placed at room temperature for 3 minutes, centrifuged at 4°C, 12,000 rpm for 15 minutes; aspirated the supernatant, added an equal volume of isopropanol, mixed well, placed at room temperature for 10 minutes, and centrifuged at 4°C, 12,000 rpm for 10 minutes; discarded the supernatant, and washed the precipitate with 70% ethanol for 1 The second time, the precipitate was dried in the air for 5 minutes; an appropriate amount of RNase-Free deionized water was added to dissolve the RNA, and the RNA concentration was measured on the NanoDrop micronucle...

Embodiment 2

[0052] The preparation of embodiment 2 recombinant manganese peroxidase

[0053] The expression vector pET-28a(+) was double digested (BamHI and XhoI), and the gene MNP-2 encoding manganese peroxidase was double digested (BamHI and XhoI) to cut out the mature manganese peroxidase The gene fragment of the gene was connected with the expression vector pET-28a(+), and the recombinant plasmid pET28a-MNP-2 containing the Irpex lacteus manganese peroxidase gene MNP-2 was obtained and transformed into Escherichia coli DE3 to obtain the recombinant Escherichia coli strain BL21(DE3) / MNP-2.

[0054] In the same way, a recombinant expression vector containing the complete cDNA sequence of the signal peptide gene sequence was constructed and transformed into Escherichia coli.

[0055] Take the DE3 strain containing the recombinant plasmid, inoculate it in 40mL LB culture medium, shake it at 250rpm at 37°C for 12h, transfer it to 300mLLB medium at a ratio of 2%, and culture it at 250rpm ...

Embodiment 3

[0056] The activity analysis of embodiment 3 recombinant manganese peroxidase

[0057] ABTS method (absorption coefficient 36000M -1 cm -1 ): The specific method is as follows: at pH 4.5 and 25°C, a 200μl reaction system includes 20μl of appropriately diluted enzyme solution, 20μl of 10mM ABTS, 50ul of 4mM manganese sulfate, and 50μl of 0.4mM hydrogen peroxide to start the reaction, and measure at 420nm Absorbance value, record a data every 20s, read 3min. One enzyme activity unit (U) is defined as the amount of enzyme required to oxidize 1 μmol ABTS per minute under given conditions.

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Abstract

The present invention relates to the field of genetic engineering, particularly to a manganese peroxidase MNP-2, a gene and applications thereof. The present invention provides a manganese peroxidase MNP-2 derived from lignocellulose degradation bacteria, wherein the amino acid sequence is represented by SEQ ID NO.1. According to the present invention, the manganese peroxidase has the following properties that the optimal pH value is 4.0, the optimal temperature is 60 DEG C, the manganese peroxidase MNP-2 can effectively degrade dyes having different structure types, and can be used as the novel enzyme preparation so as to be widely used in the fields of bioremediation, biological energy sources, textile, papermaking, food industry, and the like.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to a manganese peroxidase MNP-2 and its gene and application. Background technique [0002] With the development of dye production and printing and dyeing industry, the wastewater produced has become one of the most important sources of water pollution. In the production and use of dyes, about 10%-15% of dyes are released into the environment. Due to the complex structure and strong stability of dyes, it is difficult to be effectively degraded, causing serious environmental pollution problems. Therefore, it is urgent to find an efficient and environmentally friendly method for dye degradation or dye wastewater treatment. [0003] Currently, dye wastewater treatment methods include physical, chemical and biological methods. Although the research on the treatment of dye wastewater by physical and chemical methods has become mature and widely used in practice...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/08C12N15/53C12N15/70C12N1/21
CPCC12N9/0065C12Y111/01013
Inventor 姚斌苏小运秦星罗会颖黄火清柏映国王苑王亚茹涂涛
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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