WO2020199612A1 - Method for increasing enzyme activity of highly recombinant dye decolorizing peroxidase - Google Patents

Abstract

Provided is a method for increasing the enzyme activity of a highly recombinant dye decolorizing peroxidase, related to the technical field of bioengineering. Comprised are methods (1) and/or (2). For (1), a dye decolorizing peroxidase (DyP) is heterologously expressed in Escherichia coli, a recombinant strain is acquired and fermented, by adding an exogenous substance glutamate and/or FeCl₂ to a fermentation culture medium, when 80 μmol/L of FeCl₂ is added to the culture medium, the enzyme activity is increased to 1.45 times of that of a control. For (2), by means of the co-expression of hemA genes and DyP genes of a heme synthesis pathway key enzyme, combined with exogenously added 40 μmol/L glutamate, the enzyme activity of the DyP is increased to 13.2 U/mg, and dye decolorization rate is increased to 53.3%.

Description

Method for improving decolorization peroxidase enzyme activity of recombinant dye Technical field

The invention relates to a method for improving the enzymatic activity of recombined dye decolorization peroxidase, and belongs to the technical field of bioengineering.

Background technique

Dye decoloring peroxidase (DyP) is a new member of the peroxidase family. It uses heme as a prosthetic group and uses hydrogen peroxide as an electron acceptor to catalyze various organics. It can be used as anthraquinone dyes and lignin model compounds. The substrate has potential application value, which can realize the degradation of dye wastewater by biological method, thereby avoiding the secondary pollution problem of degradation by chemical and physical methods.

At present, DyP is mainly derived from microorganisms, but DyP obtained from primitive bacteria is not only low in content, but also difficult to separate from other intracellular proteins. Therefore, DyP is often produced by the method of constructing genetic engineering bacteria, such as heterologous expression of DyP in E. coli , But the content of recombinant protein is low, generally below 10mg/L. Recent studies have found that DyP and modified protein fusion expression, the amount of recombinant protein can be increased to about 200mg/L, but the highly overexpressed fusion DyP has almost no activity. The analysis of enzymes by ultraviolet-visible absorption spectroscopy and high-resolution mass spectrometry showed that most of the highly overexpressed enzymes only contain the iron-deficient heme precursor protoporphyrin IX (PPIX) instead of heme. In Escherichia coli, heme synthesis starts from glutamic acid (Glu), through the key precursor 5-aminolevulinic acid (5-ALA), and multi-step biochemical reaction to produce protoporphyrin IX (PPIX), PPIX chelate iron After the formation of heme. Modifying heme synthesis genes to increase heme content is a way to increase DyP production. However, because multiple genes are involved in the pathway and there is feedback control, the improvement is limited.

Adding 5-ALA to the culture medium is another way to increase the yield of DyP, but the cost is high and it is not suitable for large-scale promotion and application.

Therefore, providing an economical, simple and effective method for increasing the yield of DyP is of great significance for the further application of DyP.

Summary of the invention

An object of the present invention is to provide a method for improving the enzyme activity of recombinant DyP, the method being the following (1) and/or (2):

(1) Expressing DyP heterologously in Escherichia coli to obtain a recombinant strain, using the recombinant strain for fermentation, and adding Glu and/or Fe ^{2+ to the} fermentation medium;

(2) Co-express the gene encoding DyP and the key enzyme gene hema of the heme synthesis pathway in E. coli. The amino acid sequence of the key enzyme of the heme synthesis pathway is the sequence shown in GenBank numbering CAQ31712.1.

In one embodiment of the present invention, the final concentration of Glu and/or Fe ²⁺ added is 20-100 μmol/L.

In one embodiment of the present invention, the final concentration of added Glu and/or Fe ²⁺ is 40-100 μmol/L.

In one embodiment of the present invention, the final concentration of the added Glu and/or Fe ²⁺ is 60-100 μmol/L.

In one embodiment of the present invention, the amino acid sequence of the DyP is the sequence shown in GenBank numbering AAZ57111.1 and UniProtKB/Swiss-Prot numbering Q47KB1.

In one embodiment of the present invention, the recombinant strain uses a pET series vector as a vector.

In one embodiment of the present invention, the fermentation conditions are: after seed culture, transfer to LB medium with an inoculum of 1-5% (v/v), 200-220r/min, 35- Cultivate at 38°C until the OD ₆₀₀ of the bacteria reaches 0.6-0.8, add IPTG to the medium at a final concentration of 0.1-0.5mmol/L, and induce culture at 28-30°C for 12-20 hours.

In one embodiment of the present invention, the seed culture is to inoculate the recombinant strain into the LB medium, and cultivate for 10-15 hours at 200-220 r/min at 35-38°C.

In one embodiment of the present invention, the formula of LB medium is: tryptone 10g/L, yeast extract 5g/L, and sodium chloride 10g/L.

In one embodiment of the present invention, the E. coli is E. coli BL21 (DE3).

In one embodiment of the present invention, the co-expression uses pET-28a as an expression vector.

The second purpose of the present invention is to provide a genetically engineered bacteria that co-expresses the gene encoding DyP and the key enzyme gene heme in the heme synthesis pathway in Escherichia coli. The amino acid sequence of the DyP is as shown in GenBank numbering AAZ57111.1 The sequence shown, the amino acid sequence of the key enzyme of the heme synthesis pathway is the sequence shown in GenBank numbering CAQ31712.1.

The third objective of the present invention is to provide a method for producing DyP, which is to first co-express the gene encoding DyP and the key enzyme gene heme synthesis pathway heme in E. coli, and then use the co-expressed strain to ferment to produce DyP. The amino acid sequence of DyP is the sequence shown in GenBank numbering AAZ57111.1, and the amino acid sequence of the key enzyme in the heme synthesis pathway is the sequence shown in GenBank numbering CAQ31712.1.

In one embodiment of the present invention, the fermentation conditions are to activate the co-expressed strain at 35-38°C, 200-220r/min for 8-14h, and then press 1-5% inoculum (V/V ) Transfer to LB medium, culture until the OD ₆₀₀ value of the bacteria is 0.6-0.8, add IPTG at a final concentration of 0.1-1 mmol/L, and induce 6-10 hours at 35-38°C.

In one embodiment of the present invention, the fermentation medium is LB medium.

The fourth object of the present invention is to provide a method for decolorizing Reactive Blue 19, which uses Reactive Blue 19 as a substrate and adds the protein expressed by the genetically engineered bacteria.

In one embodiment of the present invention, the decolorization reaction system includes 20 mmol/L acetate buffer, 0.1-0.3 mmol/L final concentration of reactive blue 19, and 180-220 μg of the protein expressed by the genetically engineered bacteria. , H ₂ O ₂ with a final concentration of 0.02-0.2 mmol/L.

Another object of the present invention is to provide the application of the above method in the biological, textile or chemical fields.

The beneficial effects of the present invention:

The invention provides a method for improving the enzyme activity of recombinant DyP.

(1) Firstly express DyP heterologously in Escherichia coli to obtain recombinant strains and carry out fermentation. By adding exogenous substances Glu and/or Fe ^{2+ to the} fermentation medium, the heme saturation in recombinant DyP is increased. When adding 100μmol/L of Glu+FeCl ₂ to the medium, the specific enzyme activity can be increased to 1.37 times that of the control; when adding 80μmol/L of FeCl ₂ to the medium, the enzyme activity can be increased to that of the control 1.45 times. The method is simple and easy to operate, and the added foreign substances such as Glu and FeCl _{2 are} cheap and economical.

(2) Through the co-expression of hema and DyP genes, the key enzyme genes in the E.coli endogenous heme synthesis pathway, combined with the addition of cheap exogenous heme synthesis precursors, it was found that:

(a) The intracellular hemoglobin content is greatly increased: after the recombinant bacteria are induced at 37℃ and 0.3mmol/L IPTG, the intracellular hemoglobin test is performed. The heme concentration in the strain pD is 3.4μmol/L, and the co-expressing strain pAD The concentration of heme in the medium increased significantly, reaching 9.8 μmol/L; adding 20 μmol/L FeCl ₂ and 40 μmol/L Glu to the culture medium of the strain pAD further increased the concentration of heme to 11.3 μmol/L and 13.5 μmol/L;

(b) DyP binds more heme: the heme and protein content of DyP is expressed by 408nm (Soret peak) and 280nm wavelength. Soret peak is the characteristic absorption peak of heme, which can be used to characterize the level of heme incorporated into DyP. Except for the strains containing empty plasmids, all other strains showed a Soret peak at 408nm; the analysis peak showed that compared with pD strain 0.637, the co-expression strain pAD was 0.794. It can be seen that the DyP gene and the heme synthesis pathway key enzyme gene heMA Co-expression enhances the binding degree of Dyp and cofactor heme. Exogenous addition of 20μmol/L FeCl ₂ and 40μmol/L Glu respectively increases the peak at 408nm to 0.865 and 0.889;

(c) The Dyp enzyme activity was measured with 2,2-diazo-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS) as a substrate: the Dyp enzyme activity of strain pD was 4.5 U /mg, while the pAD enzyme activity of the co-expression strain reached 9.2U/mg, which was 1.1 times higher than the original. Adding 20μmol/L FeCl ₂ and 40μmol/L Glu separately, the enzyme activity increased to 11.8U/mg, 13.2U/mg .

(d) The decolorization effect of Dyp on Reactive Blue 19: The non-degradable anthraquinone dye Reactive Blue 19 was used as the substrate to carry out the decolorization determination of Dyp; the results showed that the recombinant strains all showed decolorization activity on the dye Reactive Blue 19. The decolorization rate of Dyp expressed by the strain pD was 24.4%, and the Dyp of the co-expression strain pAD was 37.4%. When 20μmol/L FeCl ₂ and 40μmol/LGlu were added, the dye decolorization efficiency was enhanced to 42.7% and 53.3%.

Description of the drawings

Figure 1: The effects of metal ions and amino acids on cell mass and recombinant DyP enzyme activity.

Figure 2: The effect of additives on enzyme activity and heme saturation.

Figure 3: The effect of FeCl ₂ concentration on the enzyme activity of recombinant DyP.

Figure 4: The effect of various strains and additives on the content of recombinant DyP heme.

Figure 5: The effects of various strains and additives on the spectral absorption of recombinant DyP.

Figure 6: The effects of strains and additives on the level of recombinant DyP enzyme activity.

Figure 7: Effects of strains and additives on the decolorization efficiency of recombinant DyP dyes.

detailed description

The corresponding relationship between English abbreviations and full names involved in the present invention is as follows:

DyP: dye decolorizing peroxidase;

Glu: glutamic acid;

Fe ²⁺ ：ferrous ion;

FeCl ₂ : Ferrous chloride;

H ₂ O ₂ : hydrogen peroxide;

Strain pD: a strain co-expressing the dye decolorizing peroxidase gene;

Strain pAD: a strain that co-expresses the dye decoloring peroxidase gene and the key enzyme gene heme in the heme synthesis pathway;

Strain pAD (FeCl ₂ ): Co-express the dye decoloring peroxidase gene and the key enzyme gene heme in the heme synthesis pathway, and add FeCl ₂ exogenously;

Strain pAD (Glu): Co-express the dye decoloring peroxidase gene with hema, a key enzyme gene in the heme synthesis pathway, and add Glu exogenously;

OD ₆₀₀ : absorbance value at a wavelength of 600nm;

CoCl ₂ : Cobalt chloride;

MnCl ₂ : Manganese chloride;

His: histidine;

ABTS: 2,2-Diazo-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt;

5-ALA: 5-aminolevulinic acid.

Example 1

The full-length DyP protein sequence (GenBank numbering AAZ57111.1) was obtained using GenBank database, and the target gene encoding the protein was synthesized. Using plasmid pET-28a(+) as a vector, through restriction digestion and ligation, a recombinant plasmid containing the target gene was synthesized. The recombinant plasmid containing the target gene was transformed into E. coli BL21 for heterologous expression, and a recombinant strain was obtained.

The recombinant strain was inoculated into LB medium containing kanamycin, cultured at 200r/min, 37°C for 12h, and transferred to a 500mL shake flask containing 100mL of fresh LB medium at 4% (v/v) inoculum , And cultivate under the same conditions until the OD ₆₀₀ of the bacteria reaches 0.6-0.8. Add isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.3mmol/L to the culture medium, induce culture at 30°C for 14h, and centrifuge at 8000rpm for 20min to obtain the bacteria. Cells were resuspended in 50mmol/L pH 7.4 potassium phosphate buffer solution, sonicated for 30min, centrifuged at 10000rpm for 30min to collect the supernatant, passed through a 0.22μm water filter membrane to obtain the crude enzyme solution, separated and purified by 1mL His Trap ^TM HP affinity nickel column , Collect the target protein and perform SDS-PAGE electrophoresis to identify the purity of the recombinant DyP.

100μmol/L Glu, FeCl ₂ , CoCl ₂ , MnCl ₂ , Glu+FeCl _{2 were} added to the culture medium, the final concentration of IPTG added was 0.3 mmol/L, and the culture was induced at 30°C for 14 hours. The cells were collected and centrifuged. Suspend, break, centrifuge, collect the supernatant and pass through a 0.22μm aqueous membrane to obtain the crude enzyme solution, which is separated and purified by a 1mL His Trap ^TM HP affinity nickel column, and linearly eluted with 10 column volumes of elution buffer. The target protein performs full-wavelength scanning and hemoglobin saturation calculation.

Heme saturation is defined as the ratio of absorbance at 408nm to 280nm, that is, A ₄₀₈ /A ₂₈₀ .

The results showed that the addition of CoCl ₂ reduced the heme saturation, the addition of MnCl ₂ had no significant effect (Table 1), while the addition of Glu+FeCl ₂ significantly increased the heme saturation in the recombinant DyP.

Table 1 The effect of different exogenous additives on heme saturation in recombinant DyP

Note: B is the control group without any additives.

Add His, Glu, FeCl ₂ , CoCl ₂ , MnCl ₂ , Glu+FeCl ₂ to the medium with a final concentration of 100 μmol/L, and investigate the effects of amino acids and metal ions on the enzyme activity and bacterial growth of recombinant DyP. Add any additives as the control group (B). Add IPTG to a final concentration of 0.3mmol/L, induce culture at 30℃ for 14h, collect the bacteria by centrifugation, suspension, breakage, centrifugation, collect the supernatant and pass through 0.22μm water filter membrane to obtain the crude enzyme solution, pass 1mL His Trap ^TM HP affinity nickel column separation and purification, linear elution with 10 column volumes of elution buffer, full wavelength scanning of the target protein and calculation of specific enzyme activity.

DyP enzyme activity determination method one:

Add 100mmol/L sodium acetate buffer solution (pH 4.5), 100μmol/L Reactive Blue 19 and an appropriate amount of enzyme solution to the reaction system, and finally add a final concentration of 0.1mMH ₂ O _{2 to} start the reaction, enzymatic reaction for 10 minutes, and then pass Add 200 μL 2% SDS to stop the reaction. At room temperature, the change in absorbance was detected at a wavelength of 595nm. The protein concentration was determined using the BCA method, with bovine serum albumin as the standard protein.

Definition of DyP enzyme activity: at 25°C and pH 4.5, the amount of enzyme required to oxidize 1 μmol of Reactive Blue 19 per minute is 1 U.

Specific enzyme activity (U/mg) = enzyme activity (U/mL) × [protein concentration (mg/mL)] ^-1 .

Use DyP specific enzyme activity determination method-determine DyP enzyme activity. The results are as follows:

(1) As shown in Figure 1, CoCl ₂ has a certain inhibitory effect on the recombinant DyP enzyme activity. Adding His can basically keep the recombinant DyP enzyme activity and bacterial growth unchanged. Exogenous addition of FeCl ₂ , MnCl ₂ and Glu can all Improve recombinant DyP enzyme activity and bacterial growth. When 100μmol/L FeCl ₂ was added to the culture medium, the enzyme activity of recombinant DyP increased from 22.34U/L to 26.20U/L, and the bacterial growth increased from 5.45g/L to 6.17g/L; When 100μmol/L of Glu was added to the mixture, the enzyme activity of recombinant DyP increased from 22.34U/L to 23.33U/L, and the cell growth increased from 5.45g/L to 5.78g/L;

(2) As shown in Figure 2, when 100μmol/L of Glu+FeCl ₂ is added to the medium, the specific enzyme activity increases to 1.37 times that of the control (101U/g);

(3) Add FeCl ₂ with final concentrations of 40, 60, 80, 100, 120 μmol/L to the culture medium, as shown in Figure 3. As the concentration of FeCl ₂ increases, the enzyme activity first increases and then decreases. When the concentration is At 80μmol/L, the enzyme activity reached 31.31U/L, which was 1.45 times that of the control.

Example 2

(1) Construction of recombinant plasmid

Refer to NCBI (https://www.ncbi.nlm.nih.gov/nuccore/AM946981.2:1251856..1253112) for the nucleotide sequence of the heMA gene encoding glutamine tRNA reductase from Escherichia coli.

Refer to NCBI (https://www.ncbi.nlm.nih.gov/nuccore/CP000088.1:3600559..3601851) for the nucleotide sequence of the gene encoding DyP from Thermobifida fusca. The Dyp gene was cloned into the pET-28a vector to construct the recombinant plasmid pDyP. The hema and Dyp genes were co-expressed on the pET-28a vector to construct the recombinant plasmid phemA-DyP. The plasmid pDyP and phemA-DyP were introduced into E. coli BL21 (DE3) to form recombinant bacteria pD and pAD respectively.

(2) Expression and purification identification of recombinant protein

After the recombinant bacteria were activated and cultured overnight at 37°C and 200 r/min, they were transferred to the LB liquid medium containing 50 μg/mL Kan antibiotic according to the 4% inoculum. When the OD ₆₀₀ value of the bacteria is 0.6-0.8, add IPTG at a final concentration of 0.3 mmol/L and induce 8 hours at 37°C. After IPTG induction, intracellular hemoglobin was detected. The heme concentration of pD strain was 3.4 μmol/L, while the heme concentration of co-expression strain pAD increased significantly to 9.8 μmol/L (Figure 4).

The cells were suspended in phosphate buffer (20mmol/L, pH 7.4), the cells were disrupted by ultrasound for 20 minutes, and centrifuged at 4°C for 30 minutes at 10,000 r/min. The crude enzyme solution is filtered with a 0.45μm filter membrane to remove impurities. After equilibration with phosphate buffer containing 20mmol/L imidazole, load the sample, and finally linear elution with phosphate buffer containing 500mmol/L imidazole. According to the elution profile, collect the enzyme solution corresponding to the elution peak. The purified protein was analyzed by 10% SDS-PAGE.

(3) Add heme synthesis precursor to the recombinant culture medium

Exogenously add 20μmol/L FeCl ₂ and 40μmol/L Glu to the culture medium of strain pAD.

(4) Determination of heme concentration

Fluorescence method is used to detect the heme concentration. The heme is heated in an oxalic acid reagent to remove ferrous ions and reduced to porphyrin with fluorescent characteristics. The fluorescence value is detected by a microplate reader to calculate the hemoglobin concentration. According to the formula V=8/OD, take the induced bacterial solution, centrifuge at 8000g at 4°C for 5min, and discard the supernatant. After washing with ultrapure water, resuspend the bacteria and transfer to a 1.5mL amber centrifuge tube. Add 500 μL of 20 mM oxalic acid solution to each centrifuge tube, and place in a dark room at 4°C for 16 hours. Then, 500μL, 2mol/L oxalic acid solution was added, and the control group and the experimental group were treated in a water bath at room temperature and 95°C for 30 minutes. After the sample is naturally cooled, centrifuge at 12000g at 4°C for 5min, and take 200μL of supernatant on a black 96-well fluorescent plate for fluorescence detection (excitation wavelength and emission wavelength are set to 400nm and 620nm, respectively).

(5) Spectral analysis of DyP

DyP has an obvious characteristic absorption peak of heme at 408nm, so the binding degree of DyP and heme can be analyzed by spectrum. Take 200μL of pure enzyme sample, use the multifunctional microplate reader biotek to scan the wavelength at 280-700nm.

The heme and protein content of DyP is expressed at 408nm (Soret peak) and 280nm wavelength. Soret peak is the characteristic absorption peak of heme, which can be used to characterize the level of heme incorporated into Dyp. Except for the strains containing empty plasmids, all other strains showed a Soret peak at 408nm. The peak analysis showed that, compared with pD strain 0.637, the co-expression strain pAD was 0.794. It can be seen that co-expression of Dyp gene and heMA gene enhances the binding degree of Dyp and cofactor heme (see Figure 5).

(6) Dyp enzyme activity detection method two

Use ABTS as a substrate to detect the activity of DyP. The reaction system includes 20mmol/L acetate buffer, 0.2mmol/L ABTS, 100μL pure enzyme solution after proper dilution, H ₂ O ₂ with final concentration 0.2mmol/L to start the reaction, reaction time is 30S, terminator It is 200 μL, 2% SDS. An ultraviolet-visible spectrophotometer was used to detect the light absorption value of the reaction product at 420 nm (ε=36,000M ^-1 cm ^-1 ).

An enzyme activity unit is defined as the amount of enzyme required to oxidize 1 μmol ABTS under the conditions of 25°C and pH 4.5.

Specific enzyme activity (U·mg ^-1 ) = enzyme activity (U·mL ^-1 )×[protein concentration (mg·mL ^-1 )] ^-1

Using Dyp's enzyme activity detection method two, the Dyp enzyme activity was measured with ABTS as a substrate. The Dyp enzyme activity of the overexpression strain pD alone was 4.5 U/mg, while the pAD enzyme activity of the co-expression strain reached 9.2 U/mg, which was 1.1 times higher than the original (see Figure 6).

(7) Decolorization of DyP on Reactive Blue 19

Taking Reactive Blue 19 as the research object, the reaction system includes 20mmol/L acetate buffer, 0.1mmol/L final concentration of Reactive Blue 19, and appropriate dilution of 100μL pure enzyme solution (about 200μg of protein), final concentration 0.2mmol/L L H ₂ O ₂ initiates the reaction, and the terminator is 200 μL, 2% SDS. Under the conditions of 25°C and pH 4.5, the light absorption value of the solution at 595nm was detected after 15 minutes of reaction, and it was recorded as A. Decolorization rate (%) of Reactive Blue 19=(A ₀ -A)×100%/A ₀ . Where A ₀ is the light absorption value of the control group.

The results showed that the recombinant strains all showed decolorizing activity to the dye reactive blue 19. The pD decolorization rate of the single expression strain was 24.4%, and the co-expression strain pAD was 37.4% (see Figure 7).

Example 3

Exogenously added 20 μmol/L FeCl _{2 to the} strain pAD medium, and other conditions were the same as in Example 2. The heme concentration was further increased to 11.3μmol/L, the peak at 408nm was increased to 0.865, the enzyme activity of Dyp was increased to 11.8U/mg (see Dyp's enzyme activity detection method 2), and the dye decolorization rate was increased to 42.7%.

Add 40 μmol/L Glu exogenously to the strain pAD medium, and other conditions are the same as in Example 2. The heme concentration increased to 13.5μmol/L, the peak at 408nm increased to 0.889, the enzyme activity of Dyp increased to 13.2U/mg (see Dyp's enzyme activity detection method 2 for the determination method), and the dye decolorization rate increased to 53.3%.

Although the present invention has been disclosed as above in preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. The protection scope of the present invention should be defined by the claims.

Claims (18)

1. A method for improving the enzymatic activity of recombinant dye decoloring peroxidase, characterized in that the method is the following (1) and/or (2):

   (1) Expressing dye decoloring peroxidase heterologously in E. coli to obtain a recombinant strain, using the recombinant strain for fermentation, and adding Glu and/or Fe ^{2+ to the} fermentation medium;

   (2) Co-express the gene encoding dye decoloring peroxidase and the key enzyme gene heme in the heme synthesis pathway in Escherichia coli. The amino acid sequence of the key enzyme in the heme synthesis pathway is the sequence shown in GenBank numbering CAQ31712.1 .
2. The method of claim 1, wherein the final concentration of the added Glu and/or Fe ²⁺ is 20-100 μmol/L.
3. The method according to claim 1 or 2, characterized in that the final concentration of added Glu and/or Fe ²⁺ is 40-100 μmol/L.
4. The method of claim 1, wherein the amino acid sequence of the dye decoloring peroxidase is the sequence shown in GenBank numbering AAZ57111.1.
5. The method of claim 1, wherein the E. coli is E. coli BL21 (DE3).
6. The method of claim 1 or 5, wherein the recombinant strain uses a pET series vector as a vector.
7. The method according to claim 1, characterized in that the fermentation conditions are: after seed culture, transfer to LB medium with an inoculum of 1-5% (v/v), 200-220r/min, 35- Cultivate at 38°C until the OD ₆₀₀ of the bacteria reaches 0.6-0.8, add IPTG to the medium at a final concentration of 0.1-0.5mmol/L, and induce culture at 28-30°C for 12-20 hours.
8. The method according to claim 7, wherein the seed culture is by inoculating the recombinant strain into LB medium, and culturing at 200-220r/min at 35-38°C for 10-15h.
9. The method according to claim 7, wherein the formula of the LB medium is as follows: tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L.
10. The method of claim 1, wherein the co-expression uses pET-28a as an expression vector.
11. A genetically engineered bacteria that co-expresses the gene encoding dye decoloring peroxidase and the key enzyme gene heme synthesis pathway hema in E. coli. The amino acid sequence of the dye decoloring peroxidase is as GenBank numbered AA257111.1 The sequence shown, the amino acid sequence of the key enzyme of the heme synthesis pathway is the sequence shown in GenBank numbering CAQ31712.1.
12. A method of producing dye decoloring peroxidase is to first co-express the gene encoding dye decoloring peroxidase and the key enzyme gene heme synthesis pathway in E. coli, and then use the co-expressed strains to ferment to produce dye decolorization. Oxidase; the amino acid sequence of the dye decoloring peroxidase is as shown in GenBank numbering AA257111.1, and the amino acid sequence of the key enzyme in the heme synthesis pathway is as shown in GenBank numbering CAQ31712.1 sequence.
13. The method according to claim 12, characterized in that, the conditions of the fermentation are that the co-expressed strain is activated and cultured at 35-38°C, 200-220r/min for 8-14h, and then the inoculum amount is 1-5% ( V/V) was transferred to LB medium, cultured until the OD ₆₀₀ value of the bacteria was 0.6-0.8, and IPTG was added at a final concentration of 0.1-1 mmol/L, and induced at 35-38°C for 6-10 hours.
14. The method according to claim 12 or 13, characterized in that the fermentation medium is LB medium.
15. A method for decolorizing Reactive Blue 19, which is characterized in that the Reactive Blue 19 is used as a substrate and the protein expressed by the genetically engineered bacteria of claim 11 is added.
16. The method according to claim 15, wherein the decolorization reaction system comprises 20 mmol/L acetate buffer, a final concentration of 0.1-0.3 mmol/L reactive blue 19, and 180-220 μg of the claim 11 The protein expressed by genetically engineered bacteria has a final concentration of 0.02-0.2mmol/L H ₂ O ₂ .
17. Application of the method of any one of claims 1-10 in the biological, textile or chemical fields.
18. The application of the method according to any one of claims 12-16 in the biological, textile or chemical fields.

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