CN110016468B

CN110016468B - Azo red dye decolorizing enzyme, and preparation method and application thereof

Info

Publication number: CN110016468B
Application number: CN201910284750.9A
Authority: CN
Inventors: 林英武; 张萍; 何博; 高淑琴
Original assignee: Nanhua University
Current assignee: Nanhua University
Priority date: 2019-04-10
Filing date: 2019-04-10
Publication date: 2021-06-08
Anticipated expiration: 2039-04-10
Also published as: CN110016468A

Abstract

The invention belongs to the field of artificial metalloenzymes, and discloses an efficient azored decolorizing enzyme, and a preparation method and application thereof. The azo red decolorizing enzyme provided by the invention is a three-point mutant protein F43Y/F138W/T67R Mb obtained by using myoglobin with oxygen transport function as a protein molecule design skeleton and introducing tyrosine, tryptophan and arginine at positions 43, 67 and 67 near the myoglobin heme active center, and the amino acid sequence of the azo red decolorizing enzyme is shown in SEQ ID No. 1. The azo red decolorizing enzyme can degrade azo red dye molecules under the condition of low hydrogen peroxide concentration and neutrality to achieve the decolorizing effect, can be widely applied to the pollution treatment of industrial dye wastewater, and provides an easy-to-implement, high-efficiency and environment-friendly method for the degradation of azo dyes. The preparation method of the azoic red decolorizing enzyme is simple to operate and is suitable for large-scale industrial production.

Description

Azo red dye decolorizing enzyme, and preparation method and application thereof

Technical Field

The invention belongs to the field of artificial metalloenzymes, and particularly relates to an azored dye decolorizing enzyme, a preparation method and an application thereof, in particular to a myoglobin three-point mutant-based efficient artificial azored dye decolorizing enzyme, and a preparation method and an application thereof.

Background

Over 100 million tons of dye wastewater are produced worldwide every year, with azo species accounting for about 70%. Azo dyes are the most common synthetic colorants in the textile, paint, varnish, paper, plastic, food, pharmaceutical and cosmetic industries. Azo cleavage products (aromatic amines) are carcinogenic.

The molecular design of the artificial decoloration peroxidase is particularly important for understanding the structure and function relationship of natural enzyme and preparing artificial metalloenzyme with potential application value. Dye-decolorizing peroxidases (DyPs) are novel heme peroxidases, widely exist in bacteria, fungi and other microorganisms, and can be applied to degradation of various industrial dyes, so that the Dye-decolorizing peroxidases have a certain application prospect in the fields of water environment pollution treatment and the like. However, natural bleaching peroxidase is relatively difficult to obtain, and the catalysis conditions of these biological enzymes are relatively harsh, and usually have certain biological catalysis function under relatively acidic conditions. For example, decolorized peroxidase extracted from Vibrio cholerae (VcDyP) exhibits high catalytic oxidation activity under acidic (pH 4) conditions. Some artificially designed metalloporphyrins decolorize the azo dye Amaranth (Amaranth) at pH 10.4. Therefore, it is of great significance to design an artificial decolorizing peroxidase for oxidatively decolorizing dyes under neutral conditions.

Previous researches report that a three-point mutant F43Y/F138W/P88W Mb has been designed based on Myoglobin (Mb) molecules, and although the mutant has remarkable dye decolorization peroxidase activity, the mutant has certain limitations on the catalytic breadth, such as poor catalytic effect on azo dyes Amararth under the condition of pH 7. Therefore, the further design and preparation of the high-efficiency artificial decolorizing peroxidase are very important, so that the peroxidase has practical application prospect in the water environment pollution treatment.

Disclosure of Invention

In view of the above, the present invention aims to provide a high-efficiency azo red dye decolorizing enzyme, and a preparation method and an application thereof.

In order to realize the purpose of the invention, the invention adopts the following technical scheme:

the invention utilizes myoglobin with oxygen transport function as a protein molecule design skeleton, tyrosine is introduced at the position 43 near the myoglobin heme active center, tryptophan is introduced at the position 138, arginine is introduced at the position 67, the structure of natural decoloration peroxidase is simulated, and artificial metalloenzyme with catalytic function is constructed to obtain three-point mutant protein F43Y/F138W/T67R Mb, namely, the novel high-efficiency artificial azored decoloration enzyme, wherein the amino acid sequence of the novel high-efficiency artificial azored decoloration enzyme is shown as SEQ ID No. 1.

The invention also provides a DNA molecule for coding the azo red decolorizing enzyme.

Due to the degeneracy of the codons, there may be a wide variety of nucleotide sequences capable of encoding the azo red decolorizing enzymes described herein.

The invention also provides a preparation method of the azored decolorizing enzyme, which comprises the steps of introducing tyrosine at the position 43, tryptophan at the position 138 and arginine at the position 67 near the myoglobin heme active center by using a site-directed mutagenesis technology, expressing in host cells, and then separating and purifying.

Preferably, the host cell in the preparation method of the invention is an escherichia coli expression strain, including Rosetta series and BL21 series strains. In one embodiment, the host cell is strain BL21(DE 3).

Preferably, the separation and purification method according to the preparation method of the present invention is separation using an ion exchange column and a gel column in this order.

In some embodiments, the packing for the ion exchange column chromatography is DEAE 52 resin; the filler of the gel column chromatography is Superdex 75.

The three-mutant F43Y/F138W/T67R Mb azored decolorizing enzyme can degrade amaranth azored dye under neutral condition, simultaneously reduces the tolerance to hydrogen peroxide, can degrade dye molecules in a short time under low hydrogen peroxide concentration, and achieves the decolorizing effect. Therefore, the invention also provides the application of the azo red decolorizing enzyme in the dye for degrading industrial wastewater.

The invention also provides a method for decoloring industrial dye wastewater, which is carried out in H₂O₂In the presence of the azo red dye decolorizing enzyme, the azo red dye decolorizing enzyme is added to react with the wastewater containing the industrial dye.

Preferably, in the method for decoloring the industrial dye wastewater, the H₂O₂Is in a concentration of 6-10 mM. In some embodiments, the H₂O₂Was 6 mM.

Preferably, in the method for decoloring the industrial dye wastewater, the concentration of the azored decoloring enzyme is 1 to 10 μ M. In some embodiments, the concentration of the azored decolorizing enzyme is 10 μ M.

Preferably, in the method for decoloring the industrial dye wastewater, the reaction temperature is 25 ℃, and the reaction pH value is 7.0.

Preferably, in the method for decoloring the industrial dye wastewater, the industrial dye is Amaranth (Amaranth).

Further preferably, in the method for decoloring the industrial dye wastewater, the concentration of the industrial dye is 5 to 140 μ M. In some embodiments, the concentration of the industrial dye amaranth is 0.1 mM.

According to the technical scheme, the invention provides the efficient azored decolorizing enzyme and the preparation method and the application thereof. The azo red decolorizing enzyme provided by the invention is a three-point mutant protein F43Y/F138W/T67R Mb obtained by using myoglobin with oxygen transport function as a protein molecule design skeleton and introducing tyrosine, tryptophan and arginine at positions 43, 67 and 67 near the myoglobin heme active center, and the amino acid sequence of the azo red decolorizing enzyme is shown in SEQ ID No. 1. The azo red decolorizing enzyme can degrade azo red dye molecules under the condition of low hydrogen peroxide concentration and neutrality, thereby achieving the decolorizing effect. The resistance to hydrogen peroxide is reduced. The preparation method of the azoic red decolorizing enzyme is simple to operate and is suitable for large-scale industrial production. Experiments show that the azoic red decolorizing enzyme can be used for reducing H₂O₂Amarath molecules are decolorized in about 100s under the concentration, the catalytic efficiency is improved, and in addition, the reaction conditions are mild, so that the method can be widely applied to pollution treatment of industrial dye wastewater, and a method which is easy to implement, high in efficiency and harmless to the environment is provided for degradation of azo dyes.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows that F43Y/F138W/T67R Mb is in H₂O₂Full spectrum change of catalytic Amararth oxidation decoloration in the presence;

FIG. 2 shows F43Y/F138W/T67R Mb in H₂O₂Catalyzing Amaranth to oxidize and decolor in the presence, wherein the change trend of an absorption value at 521nm of an Amaranth characteristic peak along with the reaction time is shown;

FIG. 3 is a color chart of a solution before and after 5 mu M F43Y/F138W/T67R Mb catalytic Amaranthh oxidation decoloration;

FIG. 4 is a color chart of a solution before and after oxidation and decoloration of Amaranthh catalyzed by 1 mu M F43Y/F138W/T67R Mb;

FIG. 5 is a graph of the steady state rate constant and H for F43Y/F138W/T67R Mb catalyzed Amaranthh oxidation₂O₂The relationship between different concentrations;

FIG. 6 is a graph of the relationship between the steady state rate constant for Amaranthh oxidation catalyzed by F43Y/F138W/T67R Mb and different concentrations of substrate Amaranthh;

FIG. 7 shows WT Mb at H₂O₂Full spectrum change of catalytic Amararth oxidation decoloration in the presence;

FIG. 8 shows WT Mb at H₂O₂Catalyzing Amaranth to oxidize and decolor in the presence, wherein the change trend of an absorption value at 521nm of an Amaranth characteristic peak along with the reaction time is shown;

FIG. 9 is a color chart of the solution before and after decolorization by WT Mb catalyzed Amararth oxidation;

FIG. 10 shows F43Y/F138W/P88W Mb in H₂O₂Full spectrum change of catalytic Amararth oxidation decoloration in the presence;

FIG. 11 shows F43Y/F138W/P88W Mb at H₂O₂Catalyzing Amaranth to oxidize and decolor in the presence, wherein the change trend of an absorption value at 521nm of an Amaranth characteristic peak along with the reaction time is shown;

FIG. 12 is a color chart of a solution before and after F43Y/F138W/P88W Mb catalytic Amaranthh oxidation decoloration.

Detailed Description

The invention discloses an azo red dye decolorizing enzyme, and a preparation method and application thereof. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and products of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.

In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available.

Example 1

Based on gene engineering and protein engineering, site-directed mutagenesis technology is applied, tyrosine is introduced at the 43 th site, tryptophan is introduced at the 138 th site, arginine is introduced at the 67 th site near the myoglobin heme active center, and then the protein is expressed in escherichia coli BL21(DE3), and the protein is separated and purified by an ion exchange column (DEAE 52 resin) and a gel column (Superdex 75) separation method, so that F43Y/F138W/T67R Mb mutant protein is obtained.

The amino acid sequence (SEQ ID NO.1) of the F43Y/F138W/T67R Mb mutant is as follows:

VLSEGEWQLVLHVWAKVEADVAGHGQDILIRLFKSHPETLEKYDRFKHLKTEAEMKASEDLKKHGVRVLTALGAILKKKGHHEAELKPLAQSHATKHKIPIKYLEFISEAIIHVLHSRHPGDFGADAQGAMNKALELWRKDIAAKYKELGYQG。

example 2

10 μ M F43Y/F138W/T67R Mb was prepared in 100mM phosphate buffer (pH 7.0). At the same time, 6mM H was prepared with ionized water₂O₂And 100mM Amaranth (Amarath) mother liquor, the concentration of which is calibrated by ultraviolet spectrum (H)₂O₂Is epsilon_240nm＝39.4M^- ¹cm^-1Amaranth is epsilon_521nm＝13.6mM^-1cm^-1)。

2mL of the above F43Y/F138W/T67R Mb solution was added with 2.4. mu.L of 100mM Amaranthh mother liquor, mixed well and placed in a fast-residence spectrometer C sample injector, and 6mM H was taken₂O₂2mL was placed in the D injector. The catalytic reaction was carried out by mixing samples from the sample injectors C and D, the reaction time was 100s, and a total of 500 spectra were collected (FIG. 1). The Amaranth characteristic absorption 521nm absorption peak was monitored as a function of time (FIG. 2).

The results show that F43Y/F138W/T67R Mb can substantially eliminate the absorption peak of the dye Amaranth at 521nm in about a few seconds (100 s).

Example 3

Mu. M F43Y/F138W/T67R Mb was prepared in 100mM phosphate buffer (pH 7.0). At the same time, 200mM H was prepared in ionized water₂O₂And 100mM Amaranth stock solution (Amaranthh) at a concentration calibrated by ultraviolet spectroscopy (H)₂O₂Is epsilon_240nm＝39.4M^- ¹cm^-1Amaranth is epsilon_521nm＝13.6mM^-1cm^-1)。

Two cuvettes (clean and dry) of the same specification were taken, 2mL of the above F43Y/F138W/T67R Mb solution was added, and 1.2. mu.L of 100mM Amaranthh mother liquor was added to each cuvette and mixed well. 30 μ L of 200mM H was added to the cuvette₂O₂This operation requires to be performed simultaneously. The reaction was carried out for 100s and photographed, and the color of the solution before and after the reaction is shown in FIG. 3.

The results show that F43Y/F138W/T67R Mb can decolorize the dye Amaranthh completely in about 100s (note: the solution color after the reaction is F43Y/F138W/T67R Mb protein color, non-dye color).

Examples 4,

Two cuvettes of the same dimensions (clean and dry) were taken and added separately2mL of the F43Y/F138W/T67R Mb solution was added, and 4. mu.L of 10mM Amaranthh mother solution was added and mixed well. 30 μ L of 200mM H was added to the cuvette₂O₂This operation requires to be performed simultaneously. The reaction was carried out for 100s and photographed, and the color of the solution before and after the reaction is shown in FIG. 4.

The results show that F43Y/F138W/T67R Mb can substantially decolorize the dye Amarath in about 100 s.

Examples 5,

10 μ M F43Y/F138W/T67R Mb was prepared in 100mM phosphate buffer (pH 7.0). At the same time, 200mM H was prepared in ionized water₂O₂And 100mM Amaranth stock solution (Amaranthh) at a concentration calibrated by ultraviolet spectroscopy (H)₂O₂Is epsilon_240nm＝39.4M^- ¹cm^-1Amaranth is epsilon_521nm＝13.6mM^-1cm^-1)。

2mL of the above F43Y/F138W/T67R Mb solution was added with 1.2. mu.L of 100mM Amararth H₂O₂And an amount of 200mM H₂O₂Mother liquor of H₂O₂The final concentration was 600. mu.M, 800. mu.M, 1mM, 2mM, 3mM, 5mM, 10mM, and placed in the sample feeder of fast-dwell spectrometer D, 2mL of the above F43Y/F138W/T67R Mb solution was added with 2.4. mu.L of 100mM Amaranthh stock solution, mixed well and placed in the sample feeder of fast-dwell spectrometer C. And (3) carrying out catalytic reaction after sample injection and mixing by using a C sample injector and a D sample injector, wherein the reaction time is 100s, collecting 500 spectra in total, monitoring the change of an absorption peak at 521nm along with the time, and fitting an initial linear part of a curve to obtain the initial reaction rate of Amarath oxidation. The initial reaction rate was plotted against the amantath concentration, as shown in fig. 5.

Examples 6,

10 μ M F43Y/F138W/T67R Mb was prepared in 100mM phosphate buffer (pH 7.0). At the same time, 6mM H was prepared with ionized water₂O₂And 100mM Amaranth stock solution (Amaranthh) at a concentration calibrated by ultraviolet spectroscopy (H)₂O₂Is epsilon_240nm＝39.4M^-1cm^-1Amaranth is epsilon_521nm＝13.6mM^-1cm^-1)。

2mL of the F43Y/F138W/T67R Mb solution was added with a certain amount of 100mM Amaranth mother liquor, the final concentration of Amaranth is 5. mu.M, 10. mu.M, 20. mu.M, 30. mu.M, 40. mu.M, 60. mu.M, 80. mu.M, 100. mu.M, 120. mu.M, 140. mu.M, mixed well and placed in a fast-stop spectrometer C sample injector, and 6mM H is taken₂O₂2mL was placed in the D injector. And (3) carrying out catalytic reaction after sample injection and mixing by using a C sample injector and a D sample injector, wherein the reaction time is 100s, collecting 500 spectra in total, monitoring the change of an absorption peak at 521nm along with the time, and fitting an initial linear part of a curve to obtain the initial reaction rate of Amarath oxidation. Initial reaction rates were plotted against Amaranthh concentrations (FIG. 6), and then fitted with the Michaelis-Menten enzymatic kinetics equation, from which the kinetic parameter k of the catalyzed reaction was derived_cat(0.75s^-1) And K_m(27. mu.M), it follows that the catalytic efficiency is k_cat/K_m＝27.8×10³M^-1s^-1。

Comparative examples 1,

10 μ M wild-type myoglobin (WT Mb) was prepared with 100mM phosphate buffer (pH 7.0). At the same time, 6mM H was prepared with ionized water₂O₂And 100mM Amaranth stock solution (Amaranthh) at a concentration calibrated by ultraviolet spectroscopy (H)₂O₂Is epsilon_240nm＝39.4M^- ¹cm^-1Amaranth is epsilon_521nm＝13.6mM^-1cm^-1)。

2mL of the WT Mb solution was taken, 2.4. mu.L of 100mM Amaranthh stock solution was added, mixed well and placed in a fast-residence spectrometer C sample injector, and 6mM H was taken₂O₂2mL was placed in the D injector. The catalytic reaction was carried out by mixing the samples from the sample injectors C and D, the reaction time was 100 seconds, and a total of 500 spectra were collected (FIG. 7). The Amaranth characteristic absorption 521nm absorption peak was monitored as a function of time (FIG. 8).

The results show that WT Mb does not allow the extinction of the dye Amaranth at 521 nm.

Comparative examples 2,

5 μ M WT Mb was prepared with 100mM phosphate buffer (pH 7.0). At the same time, 1.2mM H is prepared by using ionized water₂O₂And 100mM Amaranth stock solution (Amaranthh) at a concentration calibrated by ultraviolet spectroscopy (H)₂O₂Is epsilon_240nm＝39.4M^-1cm^-1Amaranth is epsilon_521nm＝13.6mM^-1cm^-1)。

Two cuvettes of the same size (clean and dry) were added with 1mL of the WT Mb solution and 1.2. mu.L of 100mM Amararth stock solution, respectively, and mixed well. Then 30. mu.L of 200mM H was added to the cuvette₂O₂This operation requires to be performed simultaneously. The reaction was carried out for 100 seconds and photographed, and the color of the solution before and after the reaction is shown in FIG. 9.

The results show that WT Mb cannot decolorize the dye Amaranthh.

Comparative examples 3,

F43Y/F138W/P88W Mb was prepared according to the Chinese patent publication No. CN108676783A, example 1.

10 μ M F43Y/F138W/P88W Mb was prepared with 100mM phosphate buffer (pH 7.0). At the same time, 6mM H was prepared with ionized water₂O₂And 100mM Amaranth stock solution (Amaranthh) at a concentration calibrated by ultraviolet spectroscopy (H)₂O₂Is epsilon_240nm＝39.4M^-1cm^-1Amaranth is epsilon_521nm＝13.6mM^-1cm^-1)。

A cuvette (clean and dry) was added with 1mL of the above F43Y/F138W/P88W Mb solution, 1.2. mu.L of 100mM Amaranth mother liquor and mixed well. 1mL of 6mM H was added to the cuvette₂O₂. The reaction time was 100s and a total of 200 spectra were collected (FIG. 10). The Amaranth characteristic absorption 521nm absorption peak was monitored as a function of time (FIG. 11).

The results show that F43Y/F138W/P88W Mb does not enable the absorption peak of the dye Amaranth at 521nm to disappear.

Comparative examples 4,

Mu. M F43Y/F138W/P88W Mb was prepared with 100mM phosphate buffer (pH 7.0). At the same time, 1.2mM H was prepared in ionized water₂O₂And 100mM Amaranth stock solution (Amaranthh) at a concentration calibrated by ultraviolet spectroscopy (H)₂O₂Is epsilon_240nm＝39.4M^- ¹cm^-1Amaranth is epsilon_521nm＝13.6mM^-1cm^-1)。

Taking two cuvettes with same specification(clean and dry), 2mL of the above F43Y/F138W/P88W Mb solution was added, and 1.2. mu.L of 100mM Amararth mother liquor was added and mixed well. 30 μ L of 200mM H was added to the cuvette₂O₂This operation requires to be performed simultaneously. The reaction was carried out for 100 seconds and photographed, and the color of the solution before and after the reaction is shown in FIG. 12.

The results show that F43Y/F138W/P88W Mb does not decolorize the dye Amarath completely.

Sequence listing

<110> university of southern China

<120> azo red dye decolorizing enzyme, preparation method and application thereof

<130> MP1903239

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 153

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Val Leu Ser Glu Gly Glu Trp Gln Leu Val Leu His Val Trp Ala Lys

1 5 10 15

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

20 25 30

Phe Lys Ser His Pro Glu Thr Leu Glu Lys Tyr Asp Arg Phe Lys His

35 40 45

Leu Lys Thr Glu Ala Glu Met Lys Ala Ser Glu Asp Leu Lys Lys His

50 55 60

Gly Val Arg Val Leu Thr Ala Leu Gly Ala Ile Leu Lys Lys Lys Gly

65 70 75 80

His His Glu Ala Glu Leu Lys Pro Leu Ala Gln Ser His Ala Thr Lys

85 90 95

His Lys Ile Pro Ile Lys Tyr Leu Glu Phe Ile Ser Glu Ala Ile Ile

100 105 110

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

115 120 125

Gly Ala Met Asn Lys Ala Leu Glu Leu Trp Arg Lys Asp Ile Ala Ala

130 135 140

Lys Tyr Lys Glu Leu Gly Tyr Gln Gly

145 150

Claims

1. An azo red dye decolorizing enzyme, whose amino acid sequence is shown in SEQ ID NO. 1.

2. A DNA molecule encoding the azo red dye decolorizing enzyme of claim 1.

3. A process for preparing azored dye decolorizing enzyme, as claimed in claim 1, wherein site-directed mutagenesis is used to introduce tyrosine at position 43, tryptophan at position 138 and arginine at position 67 near the myoglobin heme active center, which is then expressed in host cells and separated and purified.

4. The method according to claim 3, wherein the host cell is Escherichia coli BL21(DE 3); the separation and purification method sequentially uses an ion exchange column and a gel column for separation.

5. A process for decoloring waste water of industrial dye in H₂O₂Adding the azo red dye decolorizing enzyme of claim 1 to react with amaranth-containing wastewater in the presence of the enzyme.

6. The method of claim 5, wherein H is₂O₂The concentration of (B) is 0.6-10 mM.

7. The method according to claim 5, wherein the concentration of azo red dye decolorizer azo red decolorizer is 1 to 10 μ M.

8. The process according to claim 5, wherein the temperature of the reaction is 25 ℃ and the pH of the reaction is 7.0.

9. The method of claim 5, wherein the amaranth is at a concentration of 5-140 μ M.