Manganese peroxidase MNP-1 and gene and application thereof

A technology of manganese peroxidase and MNP-1, applied in the field of genetic engineering, can solve problems such as low yield and hindering large-scale application

Active Publication Date: 2016-11-23
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

[0006] So far, only dozens of manganese peroxidases have been isolated from

Method used

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  • Manganese peroxidase MNP-1 and gene and application thereof
  • Manganese peroxidase MNP-1 and gene and application thereof
  • Manganese peroxidase MNP-1 and gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Cloning of Irpex lactus manganese peroxidase coding gene MNP-1

[0048] Extraction of total RNA from Irpex lactus:

[0049] 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

[0054] The preparation of embodiment 2 recombinant manganese peroxidase

[0055] The expression vector pET-28a(+) was double-enzymatically digested (BamHI and NotI), and the gene MNP-1 encoding manganese peroxidase was double-enzymatically digested (BamHI and Not I) to cut out the mature manganese peroxidase The gene fragment of the enzyme was connected with the expression vector pET-28a(+), and the recombinant plasmid pET28a-MNP-1 containing the Irpex lacteus manganese peroxidase gene MNP-1 was obtained and transformed into Escherichia coli DE3 to obtain the recombinant Escherichia coli strain BL21(DE3 ) / MNP-1.

[0056] The recombinant expression vector containing the signal peptide sequence was constructed in the same way, and transformed into Escherichia coli strain.

[0057] Take the DE3 strain containing the recombinant plasmid, inoculate it in 40mL LB culture medium, and culture it with shaking at 250rpm at 37°C for 12h, then transfer it to 300mL LB medium at a ratio of...

Embodiment 3

[0058] The activity analysis of embodiment 3 recombinant manganese peroxidase

[0059] 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, 50μl 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 invention relates to the field of gene engineering, in particular to manganese peroxidase MNP-1 and a gene and an application thereof. An amino acid sequence is shown in SEQ ID No.1. The manganese peroxidase has the following properties that the most suitable pH (potential of hydrogen) value is 4.0, and the most suitable temperature is 60 DEG C; the different structural types of dyes can be effectively degraded; the manganese peroxidase is used as a novel enzyme preparation, and can be widely applied to the fields of biological restoration, biological energy source, textiles, papermaking and food industry.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to a manganese peroxidase MNP-1 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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IPC IPC(8): C12N9/08C12N15/53C12N15/70C12N1/21C12R1/19
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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