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Peroxidase DyP35 gene, and expression protein and application thereof

A peroxidase and gene technology, applied in the field of bioengineering, can solve the problems of low enzyme activity, poor cost, unfavorable effect, etc., and achieve the effect of high enzyme activity and high decolorization efficiency

Active Publication Date: 2017-12-12
JIANGSU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to overcome the problems in the prior art, such as dye decolorization peroxidase of fungal origin, long growth cycle of fungi, low enzyme activity, unfavorable for industrialized production and poor effect of traditional physical and chemical methods for treating waste water High problem, and the optimum pH of dye decolorization peroxidase of some bacterial origins is an acidic environment, is not suitable for the situation that the actual wastewater treatment environment is alkaline; and then provides a kind of dye decolorization peroxidase of high activity, Its amino acid sequence is shown in SEQ ID NO.2

Method used

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  • Peroxidase DyP35 gene, and expression protein and application thereof
  • Peroxidase DyP35 gene, and expression protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1: the purification of protein

[0027] (1) Ammonium sulfate precipitation: Cultivate Comamonas serinivorans SP35 in LB medium (with 1 g / L lignin added as an inducer) for 24 hours, then centrifuge at 12000 rpm to collect the cells, and wash with buffer (Tris- Hydrochloric acid, pH 8.0) was washed once and re-centrifuged, then placed on ice for ultrasonic crushing, 15000 rpm high-speed centrifugation for 20 min, the supernatant was extracted and stirred on a magnetic stirrer, and ammonium sulfate was added in sections and then centrifuged to obtain proteins of different stages Precipitate, then detect the activity, collect the protein in the highly active ammonium sulfate precipitation concentration stage, and then dialyze to remove the ammonium sulfate.

[0028] (2) Ion-exchange column chromatography: The protein solution obtained by dialysis was purified by DEAE-SepharoseCL-6B chromatography column, and linearly eluted with 50 mmol / L Tris-HCl of five column ...

Embodiment 2

[0029] Embodiment 2: Determination of amino acid sequence

[0030] The single protein band obtained in Example 1 was subjected to N-terminal sequencing by Edman degradation method (Suzhou Hongxun Biotechnology Co., Ltd.), and the amino acid sequence of the decolorizing enzyme was obtained, as shown in SEQ ID NO.2. The sequence was compared and analyzed in the GENEBANK database, and the protein was determined to belong to the peroxidase class, named DyP35. At the same time, the nucleotide sequence of the enzyme gene was obtained, as shown in SEQ ID NO.1.

Embodiment 3

[0031] Embodiment 3: the construction of the engineering bacterium of dye decolorization peroxidase DyP35

[0032] (1) Synthesis of dye decolorization peroxidase DyP35 gene

[0033] The full-length gene of dye decolorization peroxidase DyP35 was synthesized by in vitro total gene synthesis method (synthesized by Suzhou Hongxun Biotechnology Co., Ltd.).

[0034] (2) Treatment and connection of dye decolorization peroxidase DyP35 gene

[0035] dye decolorization peroxidase DyP35 gene fragment with T 4 After DNA Polymerase treatment, link with plasmid pET-28a(+) (Suzhou Hongxun Biotechnology Co., Ltd.) DNA at room temperature for 20 min.

[0036] (3) Transformation of recombinant plasmids in Escherichia coli BL21

[0037] Add the ligation product to 50 µL E. coliArctic Expression (DE3) competent cells were placed in an ice bath for 30 min, then heat-shocked for 60 s and continued to ice-bath for 2 min, then added 250 µL of LB medium, and incubated at 37°C for 1 h. Then take...

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Abstract

The invention relates to the technical field of biological engineering, in particular to a peroxidase DyP35 gene, and expression protein and application thereof; the amino acid sequence of the peroxidase DyP35 is as shown in SEQ ID NO.2; the invention also discloses a gene for expressing the dye decoloring enzyme, and the DNA sequence is as shown in SEQ ID No.1; the invention also discloses a purification method of the dye decoloring enzyme and application of the dye decoloring enzyme to printing and dyeing dye removal, papermaking black liquid decolorization, lignin processing and fruit juice production phenol polymerization; and the peroxidase DyP35 has efficient decoloring activity on benzene ring dye and lignin decomposition products and has huge application prospect.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a peroxidase DyP35 gene and its expressed protein and application. Background technique [0002] Peroxidase is an oxidoreductase that can oxidize various organic and inorganic substances with hydrogen peroxide, and plays an important role in biosynthesis and degradation processes. Most of the peroxidases with the function of decolorizing dyes were initially found in fungi. For example, the peroxidases containing heme found in 1995 can decolorize 18 different types of dyes, and then they were also found in bacteria. Peroxidase with decolorization function, for example, YcdB is a kind of peroxidase with decolorization function isolated from Escherichia coli. [0003] The existing dye decolorization methods are mainly physical and chemical methods, which are not only complicated but also costly. However, there are some defects in the enzymes with dye decolorization function...

Claims

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Application Information

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IPC IPC(8): C12N9/08C12N15/53A23L2/84A23K20/189D21C5/00D21C9/10D21C11/00C02F3/34C02F101/30
CPCA23K20/189A23L2/84C02F3/342C02F2101/308C12N9/0065D21C5/005D21C9/1063D21C11/00
Inventor 朱道辰张佩佩孙建中
Owner JIANGSU UNIV
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