CN113683813B

CN113683813B - Application of dye decoloration peroxidase in degradation of polystyrene

Info

Publication number: CN113683813B
Application number: CN202110947698.8A
Authority: CN
Inventors: 吴敬; 夏伟; 杜妍怡; 姚从禹; 窦明德; 宿玲恰
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-08-18
Filing date: 2021-08-18
Publication date: 2022-09-27
Anticipated expiration: 2041-08-18
Also published as: CN113683813A

Abstract

The invention discloses a dye decolorizing peroxidaseApplication in degradation of polystyrene, belonging to the technical field of enzyme engineering. The invention provides application of dye decolorization peroxidase TcDyP and AnaPX from Thermomonospora curvata and Nostocaceae in degrading Polystyrene (PS). The invention expresses dye decolorizing peroxidase TcDyP and AnaPX in colon bacillus, collects enzyme in fermentation liquor, adds the enzyme into glycine-HCl buffer solution containing PS film, uses H ₂ O ₂ Catalyzing, and reacting in constant-temperature water bath shaking tables with the temperature of 30 ℃ and the temperature of 25 ℃ and the speed of 200rpm for 48 hours to degrade the polystyrene plastic part, thereby having important application value for realizing the biodegradation of the plastic.

Description

Application of dye decoloration peroxidase in degradation of polystyrene

Technical Field

The invention relates to application of dye decoloration peroxidase in polystyrene degradation, belonging to the technical field of enzyme engineering.

Background

Plastics have been widely used in the fields of furniture, automotive parts, packaging, body implants, aviation and aerospace, and have become one of the most commonly used commodities in daily life. The global plastic consumption is over 3.2 hundred million tons per year, and the consumption is increased at a rate of 4-6% per year. Among them, Polystyrene (PS) is a plastic with light performance, heat resistance and low production cost. Until now, styrofoam is sold for free in stores, booths, and even supermarkets. Is widely used in catering industry, electronic industry, medical field and the like, and accounts for about 6.6 percent of the total amount of plastics. Because it is difficult to degrade, this allows the PS plastic waste to continue to accumulate in the environment, which poses a serious ecological threat.

At present, the degradation of Polystyrene (PS) still remains in the stage of chemical physical degradation using acid, base, alcohol, heat, etc. Among them, the chemical degradation method may require a large amount of chemicals, high temperature and high pressure equipment, and may generate a large amount of toxic and harmful substances, which may have a serious influence on the ecological environment. Thus, biodegradation is considered to be the most effective method for controlling plastic contamination.

Because the chemical degradation method used at present has the problems of high cost, serious three-waste pollution and the like, a biological method which is green, pollution-free and low in cost is increasingly used in a plurality of manufacturing fields, and the method gradually becomes a research hotspot in the field of plastic degradation. Currently, reported PS degrading bacteria mainly comprise cephalosporium, acinetobacter, tenebrio molitor intestinal flora and the like, but PS degrading enzymes are not reported yet. Therefore, there is a need to find PS degrading enzymes capable of degrading PS plastics.

Disclosure of Invention

The first object of the present invention is to provide a novel use of a dye decolorizing peroxidase which is (a) or (b) or (c):

(a) NCBI accession numbers of amino acid sequences such as ACY98529.1 or WP _010995754.1 dye decolorizing peroxidase;

(b) the amino acid sequence is shown as SEQ ID NO.1 or SEQ ID NO. 2;

(c) protein derived from (a) or (b) by substituting, deleting or adding one or more amino acids in the amino acid sequence defined by (a) or (b) and having dye decolorizing peroxidase activity.

In one embodiment, the dye decolorizing peroxidase is used in the degradation of polystyrene in the form of a free, whole cell, or physically/chemically immobilized enzyme.

In one embodiment, the degradation is a degradation reaction of dye decolorizing peroxidase with an amino acid sequence shown as SEQ ID No.1 or 2 in a reaction system containing Polystyrene (PS).

In one embodiment, the amount of the dye decolorizing peroxidase added in the reaction system is more than or equal to 1X 10 ³ U/L。

In one embodiment, the dye decolorizing peroxidase represented by SEQ ID NO.1 is added in an amount of 2.6X 10 or more ³ U/L。

In a kind of implementationIn the method, the addition amount of the dye decolorizing peroxidase shown in SEQ ID NO.2 is more than or equal to 3.0 × 10 ⁴ U/L。

In one embodiment, the temperature of the degradation reaction is 20-35 ℃, or 20-25 ℃, or 25-30 ℃, or 25-35 ℃, or 30-35 ℃.

In one embodiment, the reaction system has a pH of 3 to 4.

In one embodiment, the reaction system further comprises 50mM glycine-HCl buffer.

In one embodiment, the degradation temperature is 30 ℃ and 25 ℃ and the degradation time is 48 h.

In one embodiment, the degradation system is a glycine-HCl buffer at pH3 and pH3.5, 50 mM.

In one embodiment, the degradation reaction further comprises subjecting the polystyrene to an ultraviolet pretreatment prior to enzymatic hydrolysis; the ultraviolet pretreatment is to expose the polystyrene to ultraviolet rays for a certain time.

In one embodiment, the pre-treatment is performed by irradiating the polystyrene with ultraviolet light at 275-.

The invention also provides a gene for coding the dye decolorizing peroxidase.

In one embodiment, the nucleotide sequence encoding the dye decolorizing peroxidase represented by SEQ ID NO.1 is represented by SEQ ID NO. 3; the nucleotide sequence of the dye decolorizing peroxidase shown in the code SEQ ID NO.2 is shown in the SEQ ID NO. 4.

The invention also provides an expression vector carrying the dye decolorizing peroxidase gene.

In one embodiment, the expression vector is a PET vector.

In one embodiment, the expression vector is a PET-24a (+) plasmid.

The invention also provides a microbial cell containing the expression vector or expressing the dye decolorizing peroxidase.

In one embodiment, the host cell is E.coli.

In one embodiment, the host cell is Escherichia coli BL21

The invention also provides application of the gene or the expression vector or the cell in producing the dye decolorizing peroxidase.

In one embodiment, the production of the dye-decolorizing peroxidase is performed by culturing a microbial cell expressing the dye-decolorizing peroxidase in a culture medium and collecting the dye-decolorizing peroxidase.

Has the advantages that:

(1) the invention provides a new gene for coding heterologous expression dye decolorizing peroxidase, constructs recombinant escherichia coli for heterologous expression dye decolorizing peroxidase, and realizes heterologous expression of dye decolorizing peroxidase from Thermomonospora curvata and Nostocaceae.

(2) The invention provides a new application of dye decolorizing peroxidase in degrading polystyrene. Dye decolorizing peroxidase was added to Polystyrene (PS) -containing films (2 x 2 cm) ² ) The reaction system of (2) can be respectively reacted for 48 hours at 30 ℃, pH3 and 25 ℃, pH3.5, so that the Polystyrene (PS) can be partially degraded, and the surface of the film is obviously corroded. The method has mild reaction conditions and environmental protection, and has extremely high application prospect for realizing the biodegradation of the Polystyrene (PS) plastic.

Drawings

FIG. 1: SDS-PAGE analysis of dye decolorizing peroxidase; wherein (a) AnaPX; (b) TcDyP.

FIG. 2: polystyrene (PS) pre-oxidized by uv and cleaned and changed by ir.

FIG. 3: the dye decolorization peroxidase changes the microstructure and hydrophilic angle of the surface of a Polystyrene (PS) film. Wherein (a) is SEM; (b) a hydrophilic angle.

FIG. 4: dye decolorization peroxidase changes the surface of Polystyrene (PS) thin surface in infrared.

Detailed Description

The invention is further illustrated with reference to specific examples.

The Polystyrene (PS) films referred to in the following examples were obtained from Goodfellow GmbH under product No. ST311030, thickness 0.030 mm; dye decolorization peroxidase tcdyp (thermomonospora curvata) and dye decolorization peroxidase anapx (cyanobacterium) are expressed by way of example.

The media involved in the following examples are as follows:

LB solid Medium (g/L): peptone 10, yeast powder 5, sodium chloride 10, agar 13, pH 7.0.

LB liquid Medium (g/L): peptone 10, yeast powder 5, sodium chloride 10, pH 7.0.

The detection methods referred to in the following examples are as follows:

the detection method of the microstructure on the surface of the Polystyrene (PS) plastic film comprises the following steps:

repeatedly washing the Polystyrene (PS) film treated by dye decolorizing peroxidase with SDS for 2 times, and then washing with deionized water for 1 time; putting the cleaned Polystyrene (PS) film into an oven for drying at 60 ℃; and (3) detecting the microstructure of the surface of the untreated Polystyrene (PS) film and the microstructure of the surface of the Polystyrene (PS) film after dye decoloration peroxidase treatment by using the untreated Polystyrene (PS) film as a reference through a scanning electron microscope.

The detection method of the surface of the Polystyrene (PS) film comprises the following steps:

repeatedly washing the Polystyrene (PS) film treated by dye decolorizing peroxidase with SDS for 2 times, and then washing with deionized water for 1 time; putting the cleaned Polystyrene (PS) film into an oven to be dried at 60 DEG C

The dried PS film is subjected to Scanning Electron Microscopy (SEM), hydrophilic angle and infrared spectroscopy (ATR-FT-IR), and the detection method refers to the article Interaction between Styrofoam and Microalgae spiral tissue in Brackish Water System published in 2021.

The method for measuring the enzyme activity of the dye decolorizing peroxidase comprises the following steps: enzyme activity assay is carried out at optimum pH and optimum temperature in 870. mu.L glycine-HCL buffer (100mM), 10. mu.L hydrogen peroxide (1mM), 20. mu.L ABTS (0.2mM), and 100. mu.L enzyme solution; wherein the optimal temperature of the dye decolorizing peroxidase TcDyP is 30 ℃, and the optimal pH is 3; the optimal temperature of dye decolorizing peroxidase AnaPX is 25 ℃, and the optimal pH is 3.5.

Definition of enzyme activity: the amount of enzyme required to oxidize 1. mu. mol of 2, 2' -azo-bis (3-ethylbenzothiazoline-6-sulfonic Acid (ABTS)) per minute in the reaction system.

Technical terms:

dye decolorizing peroxidase: the term "dye-decolorizing peroxidase" (dye-decolorizing peroxidase) is a protein containing heme that degrades various toxic dyes and can participate in the regulation of oxidative stress of bacteria and increase the resistance of bacteria to oxidative stress. Taking the embodiment of the invention as an example, the dye decolorizing peroxidase is derived from Thermomonospora curvata (Thermomonospora curvata) and has an amino acid sequence shown as NCBI accession number ACY98529.1 or shown as SEQ ID NO.1, or the dye decolorizing peroxidase is derived from Nostocaceae (Nostocaceae) and has an amino acid sequence shown as NCBI accession number WP _010995754.1 or shown as SEQ ID NO. 2.

Gene encoding dye decolorizing peroxidase: the term "gene encoding dye decolorizing peroxidase" directly specifies a polynucleotide encoding dye decolorizing peroxidase, which is typically bounded by an open reading frame beginning with a start codon (e.g., ATG, GTG, or TTG) and ending with a stop codon (e.g., TAA, TAG, or TGA).

Expressing: the term "expression" includes any step involved in the production of dye decolorization peroxidase, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

Expression vector(s): the term "expression vector" means a linear or circular DNA molecule comprising a gene encoding the dye decolorizing peroxidase of the present invention and operably linked to control sequences providing for its expression.

Host cell: the term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

Fermentation liquor: the term "fermentation broth" refers to a preparation produced by fermentation of cells, either unretained or recovered and/or purified. For example, a fermentation broth is produced when a microbial culture is grown to saturation by incubation under carbon-limited conditions that allow for protein synthesis (e.g., expression of an enzyme by the host cell) and secretion of the protein into the cell culture medium. The fermentation broth may contain the contents of the fermented material obtained at the end of the fermentation. For example, the fermentation broth comprises the medium components after utilization by the microorganism and cell debris present after removal of the microorganism cells by centrifugation.

Example 1: construction of recombinant plasmid carrying dye decolorization peroxidase gene TcDyP

Synthesizing a gene which has a nucleotide sequence shown as SEQ ID NO.3 and codes dye decolorizing peroxidase on a carrier pET-24a (+) by a chemical method, directly obtaining a recombinant plasmid, and transforming Escherichia coli (Escherichia coli) JM109 with the recombinant plasmid to obtain a transformation product; coating the transformation product on an LB solid culture medium (containing 40 mu g/mL kanamycin), and carrying out inverted culture in a constant-temperature incubator at 37 ℃ for 8-12 h to obtain a transformant; and (3) selecting a transformant, inoculating the transformant to an LB liquid culture medium, performing shake-flask culture for 8-12 h at 37 ℃ and 120-180 rpm, extracting plasmids, performing sequencing verification, and obtaining the recombinant plasmid pET-24a (+) -TcDyP after verification is correct.

Example 2: construction of recombinant plasmid carrying dye decolorizing peroxidase gene AnaPX

Synthesizing a gene which has a nucleotide sequence shown as SEQ ID NO.4 and codes dye decolorizing peroxidase on a carrier pET-24a (+) by a chemical method, directly obtaining a recombinant plasmid, and transforming Escherichia coli (Escherichia coli) JM109 with the recombinant plasmid to obtain a transformation product; coating the transformation product on an LB solid culture medium (containing 40 mu g/mL kanamycin), and carrying out inverted culture in a constant-temperature incubator at 37 ℃ for 8-12 h to obtain a transformant; and (3) selecting a transformant, inoculating the transformant to an LB liquid culture medium, performing shake-flask culture for 8-12 h at 37 ℃ and 120-180 rpm, extracting plasmids, performing sequencing verification, and obtaining the recombinant plasmid pET-24a (+) -AnaPX after verification is correct.

Example 3: construction of recombinant Escherichia coli expressing dye decolorizing peroxidase

Transforming the recombinant plasmid into Escherichia coli (Escherichia coli) BL21 to obtain a transformation product; coating the transformation product on an LB solid culture medium (containing 50 mu g/mL kanamycin), and carrying out inverted culture in a constant-temperature incubator at 37 ℃ for 8-12 h to obtain a transformant; and (3) selecting a transformant, inoculating the transformant to an LB liquid culture medium, performing shake flask culture for 8-12 h at 37 ℃ and 120-180 rpm, extracting plasmids, performing enzyme digestion verification and sequencing verification, and obtaining the recombinant escherichia coli BL21-pET-24a (+) -TcDyP after verification is correct.

Example 4: construction of recombinant Escherichia coli expressing dye decolorizing peroxidase

Transforming the recombinant plasmid into Escherichia coli (Escherichia coli) BL21 to obtain a transformation product; coating the transformation product on an LB solid culture medium (containing 50 mu g/mL kanamycin), and carrying out inverted culture in a constant-temperature incubator at 37 ℃ for 8-12 h to obtain a transformant; and (3) selecting a transformant, inoculating the transformant to an LB liquid culture medium, performing shake flask culture for 8-12 h at 37 ℃ and 120-180 rpm, extracting plasmids, performing enzyme digestion verification and sequencing verification, and obtaining the recombinant escherichia coli BL21-pET-24a (+) -AnaPX after verification is correct.

Example 5: recombinant expression of dye decolorizing peroxidase

Respectively culturing the recombinant Escherichia coli BL21-pET-24a (+) -TcDyP obtained in the construction of the example 3 and the recombinant Escherichia coli BL21-pET-24a (+) -AnaPX obtained in the construction of the example 4 in a shake flask containing an LB culture medium for 8-12 h, respectively transferring the bacterial liquid with the inoculation amount of 5% (v/v) to 100mL of LB liquid culture medium, and shake-culturing at 37 ℃ and 200rpm for 3h to OD ₆₀₀ After the concentration is 0.8, IPTG with a final concentration of 0.4mM and 5-aminolevulinic acid with a final concentration of 2mM are added, and recombinant escherichia coli BL21-pET-24a (+) -TcDyP is subjected to shaking culture at 30 ℃ and 200rpm for 20 hours to obtain TcDyP fermentation liquor (OD is about 7-10); continuously shaking-culturing BL21-pET-24a (+) -AnaPX at 25 ℃ and 200rpm for 20h to obtain AnaPX fermentationLiquid (OD about 7-10).

Respectively centrifuging fermentation liquor obtained by fermentation for 15min at 8000rpm to obtain precipitate; suspending the precipitate with a pH 7.0100 mM potassium phosphate buffer to obtain a resuspension; breaking the wall of the heavy suspension by using a high-pressure homogenizer under the pressure of 800Bar to obtain a wall-broken liquid; centrifuging the wall-broken solution at 8000rpm for 15min, and separating the wall-broken supernatant and the wall-broken precipitate.

The enzyme activity of the dye decolorizing peroxidase was measured separately (as shown in Table 1). SDS-PAGE analysis of the wall-broken supernatant showed that there were crude protein bands at the 41.9kDa and 54.3kDa bands, i.e., TcDyP and AnaPX, which are the dye decolorizing peroxidases (shown in FIG. 1).

TABLE 1 dye decolorizing peroxidase enzyme Activity

Example 6: ultraviolet pre-oxidation and cleaning of Polystyrene (PS) film

The method comprises the following specific steps:

polystyrene (PS) films were irradiated for 8 days at UVC (275-200 nm). The film was placed 20 cm from the lamp and turned over each day to allow each side of the samples to be uniformly illuminated. Cutting the UV-irradiated 8-day PS film into 2 × 2cm ² And the size of the membrane is that 2 times of ultrasonic washing is carried out by 2% SDS, then deionized water is used for washing twice, then 75% ethanol is used for 2 times of ultrasonic washing and deionized water is used for washing twice, finally n-hexane is used for standing and washing for 2 hours, then deionized water is used for washing twice, and the residual n-hexane on the membrane is thoroughly removed and then dried to be used as an enzyme degradation substrate for later use. And performing infrared spectrum measurement on the dried PS film.

The infrared spectrum of the Polystyrene (PS) subjected to ultraviolet pre-oxidation and washing is detected, and the result shows that the Polystyrene (PS) film can be oxidized by the ultraviolet pre-oxidation, oxygen-containing functional groups such as C ═ O, -OH and the like are inserted into the Polystyrene (PS) film, so that an action target point is generated for the subsequent dye decolorizing peroxidase to attack the Polystyrene (PS) film, and meanwhile, some small-molecule oxidation products are generated, but the small-molecule oxidation products can be removed after washing, and the influence of the small-molecule oxidation products on the subsequent degradation of the Polystyrene (PS) film by the dye decolorizing peroxidase is avoided (the detection result is shown in fig. 2).

Example 7: degradation effect of dye decolorizing peroxidase on Polystyrene (PS) film

(1) Degradation of polystyrene by dye decoloration peroxidase TcDyP

A Polystyrene (PS) film (2X 2 cm) treated in example 6 was used ² ) After placing in a 50mL centrifuge tube, the reaction system was adjusted to pH3.5 with potassium phosphate buffer (pH 3.5 and pH 2,100mM) and added to a final concentration of 2.6X 10 ³ U/L of the peroxidase solution TcDyP for decolorization of dye obtained in example 5, to which H was added ₂ O ₂ (2mM and 1 mM). Reacting for 48 hours at 30 ℃, pH3 and 200rpm, separating the membrane from the degradation liquid, cleaning and drying the surface of the membrane, and carrying out SEM (scanning Electron microscope), hydrophilic angle and infrared spectrum measurement on the dried PS film.

(2) Degradation of polystyrene by dye decoloration peroxidase AnaPX

A Polystyrene (PS) film (2X 2 cm) treated in example 6 was used ² ) After being placed in a 50mL centrifuge tube, the dye decolorizing peroxidase AnaPX was adjusted to pH3 with potassium phosphate buffer (pH 3.5 and pH 2,100mM) at 3.0X 10 ⁴ U/L addition amount the dye decolorizing peroxidase AnaPX enzyme solution obtained in example 5 was added, and H was added ₂ O ₂ (2mM and 1 mM). Reacting at 25 ℃, pH3.5 and 200rpm for 48 hours, separating the membrane from the degradation liquid, cleaning and drying the surface of the membrane, and carrying out SEM, hydrophilic angle and infrared spectrum measurement on the dried PS film.

The detection of the surface microstructure and the change of the hydrophilic angle of the Polystyrene (PS) treated with the dye decolorization peroxidase solution shows that the dye decolorization peroxidases TcDyP and AnaPX have an etching effect on the surface of the Polystyrene (PS) film and can improve the hydrophilicity of the surface of the Polystyrene (PS) film (see the detection result in fig. 3). The infrared spectrum of the Polystyrene (PS) treated with the dye decolorization peroxidase solution is detected, and the result shows that the dye decolorization peroxidase TcDyP and AnaPX can oxidize the Polystyrene (PS) film to further generate oxygen-containing functional groups such as C ═ O, -OH, C-O-C, and the like, so that the Polystyrene (PS) is depolymerized (see fig. 4).

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> university of south of the Yangtze river

<120> application of dye decolorizing peroxidase in degradation of polystyrene

<130> GBAA211060B

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 381

<212> PRT

<213> Artificial sequence

<400> 1

Met Ser Glu Glu Arg Ala Ala Gly Ala Arg Pro Ser Ala Thr Gln Thr

1 5 10 15

Thr Gly Thr Ala Thr Glu Pro Phe His Gly Pro His Gln Ala Gly Ile

20 25 30

Ala Thr Pro Pro Gln Ala His Ala Val Phe Leu Gly Leu Asp Leu Arg

35 40 45

Lys Gly Thr Gly Arg Lys Glu Leu Gly Arg Leu Met Arg Leu Leu Thr

50 55 60

Asp Asp Ala Arg Arg Leu Thr Gln Gly Arg Pro Ala Leu Ala Asp Pro

65 70 75 80

Glu Pro Asp Leu Ala Pro Leu Pro Ser Arg Leu Thr Phe Thr Phe Gly

85 90 95

Phe Gly Pro Gly Leu Phe Lys Ala Ala Gly Leu Glu Lys Gln Arg Pro

100 105 110

Glu Gly Leu Arg Pro Leu Pro Pro Phe Lys Val Asp Arg Leu Glu Asp

115 120 125

Arg Trp Ser Gly Gly Asp Leu Leu Val Gln Ile Cys Cys Asp Asp Pro

130 135 140

Ile Thr Leu Ala His Ala Leu Arg Met Thr Val Lys Asp Ala Arg Ala

145 150 155 160

Phe Thr Arg Val Arg Trp Val Gln Arg Gly Phe Arg Arg Ser Pro Gly

165 170 175

Val Gln Ser Ser Gly Ala Thr Gln Arg Asn Leu Met Gly Gln Leu Asp

180 185 190

Gly Thr Val Asn Pro Val Pro Gly Thr Ala Asp Phe Asp Gln Ala Val

195 200 205

Trp Val Gln Asp Gly Pro Glu Trp Leu Arg Gly Gly Thr Thr Leu Val

210 215 220

Leu Arg Arg Ile Arg Met Glu Leu Glu Lys Trp Asp Glu Ala Asp Pro

225 230 235 240

Ala Gly Lys Glu Phe Ala Val Gly Arg Arg Leu Thr Ser Gly Ala Pro

245 250 255

Leu Thr Gly Arg His Glu His Asp Glu Pro Asp Phe Asp Ala Val Asp

260 265 270

Ser Ala Gly Phe Pro Val Ile Ala Glu Asn Ala His Ile Arg Leu Ala

275 280 285

His Val Asp Ser Pro Arg Leu Arg Met Leu Arg Arg Pro Tyr Asn Tyr

290 295 300

Asp Glu Gly Leu Thr Ala Asp Gly Arg Ser Asp Ala Gly Leu Leu Phe

305 310 315 320

Ala Ala Tyr Gln Ala Asp Ile Asp Arg Gln Phe Ile Pro Val Gln Arg

325 330 335

Arg Leu Asp Glu Gly Gly Asp Leu Leu Asn Leu Trp Thr Thr Pro Ile

340 345 350

Gly Ser Ala Val Phe Ala Ile Pro Pro Gly Cys Asp Glu Asn Gly Trp

355 360 365

Ile Gly Gln Gly Leu Leu Gly His His His His His His

370 375 380

<210> 2

<211> 475

<212> PRT

<213> Artificial sequence

<400> 2

Met Ala Leu Thr Glu Lys Asp Leu Lys Asn Leu Pro Glu Asp Gly Ile

1 5 10 15

Asp Ser Glu Asn Pro Gly Lys Tyr Arg Asn Leu Leu Asn Asp Leu Gln

20 25 30

Gly Asn Ile Leu Lys Gly His Gly Arg Asp His Ser Val His Leu Phe

35 40 45

Leu Gln Phe Lys Pro Glu Gln Val Glu Val Val Lys Gln Trp Ile Gln

50 55 60

Ser Phe Ala Gln Thr Tyr Ile Thr Ser Ala Lys Lys Gln Ala Asp Glu

65 70 75 80

Ala Phe Lys Tyr Arg Gln Lys Gly Val Ser Gly Asp Val Phe Ala Asn

85 90 95

Phe Phe Leu Ser Arg His Gly Tyr Glu Tyr Leu Glu Ile Glu Pro Phe

100 105 110

Gln Ile Pro Gly Asp Lys Pro Phe Arg Met Gly Met Lys Asn Glu Glu

115 120 125

Ile Arg Ser Ser Leu Gly Asp Pro Lys Ile Ala Thr Trp Glu Leu Gly

130 135 140

Phe Gln Ser Glu Ile His Ala Leu Val Leu Ile Ala Asp Asp Asp Ile

145 150 155 160

Val Asp Leu Leu Gln Ile Val Asn Gln Ile Thr Gln Lys Leu Arg Gln

165 170 175

Ile Ala Glu Ile Val His Arg Glu Asp Gly Phe Ile Leu Arg Asn Gln

180 185 190

Ala Gly Gln Ile Ile Glu His Phe Gly Phe Val Asp Gly Val Ser Gln

195 200 205

Pro Leu Phe Met Lys Arg Asp Val Val Arg Glu Arg Val Asn Asn Cys

210 215 220

Asp Phe Asp Lys Trp Asp Pro Lys Ala Pro Leu Asp Ser Ile Leu Val

225 230 235 240

Glu Asp Pro Asn Gly Asn Thr Lys Asp Ser Tyr Gly Ser Tyr Leu Val

245 250 255

Tyr Arg Lys Leu Glu Gln Asn Val Lys Ala Phe Arg Glu Asp Gln Arg

260 265 270

Lys Leu Ala Gln Lys Leu Asn Ile Gln Glu Asn Leu Ala Gly Ala Leu

275 280 285

Ile Val Gly Arg Phe Ala Asp Gly Thr Pro Val Thr Leu Ser Asp Ile

290 295 300

Pro Thr Tyr Ala Val Thr Pro Thr Asn Asn Phe Asn Tyr Asp Gly Asp

305 310 315 320

Leu Ala Ala Thr Lys Cys Pro Phe His Ser His Thr Arg Lys Thr Asn

325 330 335

Pro Arg Gly Asp Thr Ala Arg Leu Leu Thr Thr Asp Gly His Phe Asp

340 345 350

Glu Ala Phe Lys Glu Glu Arg Gly His Arg Ile Thr Arg Arg Ala Val

355 360 365

Ser Tyr Gly Glu Asn Asn Pro Ser Lys Glu Pro Val Ser Gly Ser Gly

370 375 380

Leu Leu Phe Leu Cys Phe Gln Ser Asn Ile Glu Asn Gln Phe Asn Phe

385 390 395 400

Met Gln Ser Arg Trp Ala Asn Pro Gln Asn Phe Val Gln Val Asn Thr

405 410 415

Gly Pro Asp Pro Leu Ile Gly Gln Pro Ser Gly Thr Gln Lys Trp Pro

420 425 430

Lys Lys Trp Gly Glu Pro Glu Thr Glu Glu Tyr Asn Phe Gln Leu Trp

435 440 445

Ile Asn Met Lys Gly Gly Glu Tyr Phe Phe Ala Pro Ser Ile Ser Phe

450 455 460

Leu Lys Thr Leu Ala His His His His His His

465 470 475

<210> 3

<211> 1195

<212> DNA

<213> Artificial sequence

<400> 3

aactttaaga aggagatata catatgtcag aagagcgtgc agcaggagca cgtcccagtg 60

ccacccaaac aactggaacg gcaacggaac ccttccacgg gccacatcag gcggggattg 120

ctacgccccc tcaggcacat gccgtattcc ttggattaga cttacgtaaa gggacgggac 180

gtaaggagct tggacgcttg atgcgtttac tgactgacga tgctcgccgc ttaacacagg 240

gtcgtcccgc actggccgac ccggaaccgg acttggctcc tttaccctct cgccttactt 300

tcacttttgg tttcggcccc gggcttttta aagctgctgg tttagaaaaa cagcgtccgg 360

agggtctgcg tccattgccc ccttttaagg ttgatcgcct ggaagaccgc tggtccggcg 420

gagacttact ggtacagatc tgctgtgatg acccaatcac tcttgcacat gctcttcgca 480

tgacggtaaa ggacgcgcgc gcttttactc gtgtccgctg ggtccagcgc ggcttccgcc 540

gttcgcctgg agtgcaaagt tccggcgcaa cgcaacgtaa tttgatgggg cagctggacg 600

ggactgttaa ccccgtgcct ggcacagctg attttgacca ggcggtgtgg gttcaagatg 660

gacctgagtg gctgcgtggg ggtacaactt tagtacttcg ccgtattcgc atggagctgg 720

agaaatggga tgaagcggac cctgctggaa aagagttcgc ggtgggtcgc cgtctgacat 780

caggggcccc tttgactggt cgtcacgagc atgacgaacc agactttgac gccgttgatt 840

cggccggttt tcctgtaatt gcagaaaacg cgcatatccg cttggcacat gtcgacagtc 900

cacgcttacg catgttacgc cgtccataca attacgatga aggacttact gcggatggac 960

gctctgacgc aggattgctg tttgctgcgt accaagcaga cattgatcgt cagttcattc 1020

cagttcagcg ccgcttggat gagggtgggg atctgttgaa cttatggacg acaccaatcg 1080

gctccgctgt tttcgctatt cctcccggtt gtgatgaaaa tggctggatt ggtcaggggt 1140

tattagggca tcatcaccat catcattaag aattcgagct ccgtcgacaa gcttg 1195

<210> 4

<211> 1428

<212> DNA

<213> Artificial sequence

<400> 4

atggccctga ccgagaagga cctgaagaac ctgcccgagg acggcatcga cagcgagaac 60

cccggcaagt acaggaacct gctgaacgac ctgcagggca acatcctgaa gggccacggc 120

agggaccaca gcgtgcacct gttcctgcag ttcaagcccg agcaggtgga ggtggtgaag 180

cagtggatcc agagcttcgc ccagacctac atcaccagcg ccaagaagca ggccgacgag 240

gccttcaagt acaggcagaa gggcgtgagc ggcgacgtgt tcgccaactt cttcctgagc 300

aggcacggct acgagtacct ggagatcgag cccttccaga tccccggcga caagcccttc 360

aggatgggca tgaagaacga ggagatcagg agcagcctgg gcgaccccaa gatcgccacc 420

tgggagctgg gcttccagag cgagatccac gccctggtgc tgatcgccga cgacgacatc 480

gtggacctgc tgcagatcgt gaaccagatc acccagaagc tgaggcagat cgccgagatc 540

gtgcacaggg aggacggctt catcctgagg aaccaggccg gccagatcat cgagcacttc 600

ggcttcgtgg acggcgtgag ccagcccctg ttcatgaaga gggacgtggt gagggagagg 660

gtgaacaact gcgacttcga caagtgggac cccaaggccc ccctggacag catcctggtg 720

gaggacccca acggcaacac caaggacagc tacggcagct acctggtgta caggaagctg 780

gagcagaacg tgaaggcctt cagggaggac cagaggaagc tggcccagaa gctgaacatc 840

caggagaacc tggccggcgc cctgatcgtg ggcaggttcg ccgacggcac ccccgtgacc 900

ctgagcgaca tccccaccta cgccgtgacc cccaccaaca acttcaacta cgacggcgac 960

ctggccgcca ccaagtgccc cttccacagc cacaccagga agaccaaccc caggggcgac 1020

accgccaggc tgctgaccac cgacggccac ttcgacgagg ccttcaagga ggagaggggc 1080

cacaggatca ccaggagggc cgtgagctac ggcgagaaca accccagcaa ggagcccgtg 1140

agcggcagcg gcctgctgtt cctgtgcttc cagagcaaca tcgagaacca gttcaacttc 1200

atgcagagca ggtgggccaa cccccagaac ttcgtgcagg tgaacaccgg ccccgacccc 1260

ctgatcggcc agcccagcgg cacccagaag tggcccaaga agtggggcga gcccgagacc 1320

gaggagtaca acttccagct gtggatcaac atgaagggcg gcgagtactt cttcgccccc 1380

agcatcagct tcctgaagac cctggcccac caccaccacc accactga 1428

Claims

1. The application of the dye decolorizing peroxidase in degrading polystyrene or polystyrene-containing products is characterized in that the amino acid sequence of the dye decolorizing peroxidase is shown as SEQ ID No.1 or SEQ ID No. 2.

2. The use according to claim 1, wherein the dye decolorizing peroxidase is used in the degradation of polystyrene in the form of a free, whole cell, or physically/chemically immobilized enzyme.

3. A method for degrading polystyrene is characterized in that dye decoloration peroxidase is reacted in a reaction system containing polystyrene; the addition amount of the dye decolorizing peroxidase in the reaction system is more than or equal to 1 multiplied by 10 ³ U/L；

The amino acid sequence of the dye decolorizing peroxidase is shown in SEQ ID NO.1 or SEQ ID NO. 2.

4. The method according to claim 3, wherein the temperature of the degradation reaction is 20 to 35 ℃.

5. The method according to claim 3 or 4, wherein the reaction system has a pH of 3 to 4.

6. The method according to claim 3 or 4, characterized in that before the enzymatic hydrolysis, the polystyrene is also subjected to a UV pre-treatment; the ultraviolet pretreatment is to expose the polystyrene to ultraviolet rays for a certain time.

7. The method of claim 5, wherein prior to enzymatic hydrolysis, the polystyrene is further subjected to an ultraviolet pretreatment; the ultraviolet pretreatment is to expose the polystyrene to ultraviolet rays for a certain time.