H2O2Peroxidase mutant with improved stability and application thereof in dye decolorization

文档序号：1932625 发布日期：2021-12-07 浏览：16次中文

阅读说明：本技术 一种h2o2稳定性提高的过氧化物酶突变体及其在染料脱色中的应用 (H2O2Peroxidase mutant with improved stability and application thereof in dye decolorization ) 是由荚荣黄文汉于 2021-10-21 设计创作，主要内容包括：本发明公开了一种H-(2)O-(2)稳定性提高的过氧化物酶突变体及其在染料脱色中的应用。本发明基于真菌IrpexlacteusF17(CCTCC AF 2014020)的染料脱色过氧化物酶Il-DyP4的晶体结构及氨基酸序列,对Il-DyP4的207位Met进行定点突变。实验通过表达菌株的构建、大肠杆菌的异源表达、变性、复性和纯化,得到Il-DyP4的突变体M207V。本发明的突变体M207V对蒽醌染料氧化的酶活力与Il-DyP4相近,并且对H-(2)O-(2)的稳定性明显高于Il-DyP4,为利用生物酶法处理染料工业废水奠定基础。(The invention discloses a method for producing H 2 O 2 Peroxidase mutants with improved stability and their use in dye decolorization. The invention carries out site-directed mutagenesis on the 207-Met of Il-DyP4 based on the crystal structure and amino acid sequence of the dye decolorization peroxidase Il-DyP4 of fungus IrpexlateusF 17(CCTCC AF 2014020). Experiment through constructing expression strain, heterogeneously expressing colibacillus, denaturating, renaturing and purifying, mutant M207V of Il-DyP4 is obtained. The mutant M207V of the invention has enzyme activity on anthraquinone dye oxidation similar to that of Il-DyP4 and on H 2 O 2 The stability of the enzyme is obviously higher than Il-DyP4, and a foundation is laid for treating dye industrial wastewater by using a biological enzyme method.)

1. H₂O₂A peroxidase mutant with improved stability, characterized in that:

site-directed mutagenesis is carried out on Met 207 of dye decolorizing peroxidase Il-DyP4, Met 207 (ATG) is mutated into Val (GTG), and peroxidase mutant M207V is obtained.

2. The peroxidase mutant according to claim 1, characterized in that:

the amino acid sequence of the peroxidase mutant M207V is shown as SEQ ID No. 1.

3. Use of a peroxidase mutant according to claim 1 or 2, characterized in that:

the peroxidase mutant M207V was used for destaining degradation of dyes.

4. Use according to claim 3, characterized in that:

the dye is an anthraquinone dye.

5. Use according to claim 4, characterized in that:

the decolorization and degradation process of the dye is carried out at pH 3.5-5.0, temperature 35 deg.C and H₂O₂The concentration is 0.4-1.0 mmol/L.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to H₂O₂Peroxidase mutants with improved stability and their use in dye decolorization.

Background

Synthetic dyes are widely used in industry, and a large amount of synthetic dyes are produced each year and discharged as industrial wastewater. Anthraquinone dyes are one of the main types of synthetic dyes, are widely applied to industries such as textile, paper making, printing, leather and the like, have the characteristics of toxicity, oxidation resistance, reduction resistance and the like, pollute the environment and threaten the health of human bodies. Therefore, the dye is decolorized by an enzyme method and degraded by oxidation, and has great application prospect in the aspect of dye industrial wastewater treatment.

Dye-decolorizing peroxidases (DyP, EC 1.11.1.19) are a novel class of heme peroxidases distributed in archaea, bacteria and eukaryotic microorganisms. The enzyme has high-efficiency decolorizing and degrading capability on various dyes with complex structures, especially on anthraquinone dyes. Therefore, dye decolorizing peroxidase is a potential, environmentally friendly biological enzyme capable of removing dyes.

The enzymatic reaction of the dye decolorizing peroxidase is H₂O₂As an electron acceptor, the synthetic dyes are decolorized and degraded by proton, electron transfer and radical generation. However, excessive concentration of H₂O₂Easily leads to enzyme inactivation, and prevents the catalytic oxidation reaction from proceeding. Thus, increasing the H of the dye decolorizing peroxidase₂O₂The stability has larger practical application value.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a H₂O₂Peroxidase mutants with improved stability and their use in dye decolorization.

The invention carries out site-directed mutagenesis on the 207-Met of Il-DyP4 based on the crystal structure and amino acid sequence of the dye decolorization peroxidase Il-DyP4 of fungus IrpexlateusF 17(CCTCC AF 2014020). Constructing an expression strain, and carrying out heterologous expression, denaturation, renaturation and purification on escherichia coli to obtain a mutant M207V of Il-DyP 4. The mutant M207V of the invention has enzyme activity on anthraquinone dye oxidation similar to that of Il-DyP4 and on H₂O₂The stability of the enzyme is obviously higher than Il-DyP4, and a foundation is laid for treating dye industrial wastewater by using a biological enzyme method.

II-DyP 4 crystal structure (PDB:7D8M) and Met 207 are shown in FIG. 1.

The cDNA sequence of dye decolorization peroxidase Il-DyP4 is detailed in Il-DyP4(MG209114) in GenBank.

The amino acid sequence of the peroxidase mutant M207V is shown in SEQ ID No. 1.

The construction method of the peroxidase mutant M207V comprises the following steps:

step 1: obtaining M207V linear plasmid by PCR with plasmid pET28a-DyP4 as template;

step 2: phosphorylating and connecting the ends of the linear plasmids to obtain a recombinant vector;

and step 3: transforming the recombinant vector into an expression strain by a heat shock method;

and 4, step 4: culturing an expression strain and performing induced expression on mutant protein;

and 5: and (5) denaturing, renaturing and purifying to obtain the mutant enzyme M207V.

The application of the peroxidase mutant M207V is to decolorize and degrade dyes.

The dye is preferably an anthraquinone dye.

Peroxidase mutant M207V of the present invention and different concentrations of H₂O₂Incubating for 30min at pH 3.5-5.0, temperature 35 deg.C, and H₂O₂The results of dye decolorization using anthraquinone dyes Reactive Blue 4(RB4), Reactive Blue 5(RB5), Reactive Blue 19(RB19) and Reactive Blue 74(RB74) as substrates at a concentration of 0.4 to 1.0mmol/L showed that H of mutant M207V₂O₂The stability is respectively 6.2, 4.8, 6.3 and 6.4 times of that of Il-DyP4 (enzyme pair H)₂O₂The stability results were determined by measuring the residual 50% decolorization H₂O₂Concentration). Meanwhile, the mutant M207V has enzyme activity similar to that of Il-DyP 4. The mutant was incubated at pH 3.5, temperature 35 ℃ and H₂O₂The enzyme activities of the four anthraquinone dyes under the condition of the concentration of 0.1mmol/L reach 81.36, 34.38, 24.9 and 74.07U/mg respectively. In conclusion, the mutant M207V H obtained by the invention₂O₂Compared with the non-mutated Il-DyP4, the stability is obviously improved, and the Il is maintainedDyP4, indicating that the enzyme has greater application value for treating the dye industrial wastewater.

Drawings

FIG. 1 is a schematic diagram showing the crystal structure and mutation site of recombinase Il-DyP4 of the present invention.

FIG. 2 is an SDS-PAGE electrophoresis of recombinant enzyme Il-DyP4 and mutant M207V of the present invention.

FIG. 3 is a UV-visible scanning spectrum of recombinase Il-DyP4 and mutant M207V of the invention.

FIG. 4 shows the enzymatic activities of the mutants on the oxidation of the anthraquinone dyes Reactive Blue 4(4a), Reactive Blue 5(4b), Reactive Blue 19(4c) and Reactive Blue 74(4d), with the unmutated enzyme Il-DyP4 as a control.

FIG. 5 shows the optimum H values of the oxidation substrates Reactive Blue 4(5a), Reactive Blue 5(5b), Reactive Blue 19(5c) and Reactive Blue 74(5d) of the mutants₂O₂Concentrations were controlled with the unmutated enzyme Il-DyP 4.

FIG. 6 shows that the mutant enzymes were used under optimum reaction conditions for H in Reactive Blue 4(6a), Reactive Blue 5(6b), Reactive Blue 19(6c) and Reactive Blue 74(6d)₂O₂Stability, using the unmutated enzyme Il-DyP4 as a control.

Detailed Description

The generation and property studies of the mutants of the present invention are described in detail below, and the results of the experiments are analyzed.

Example 1: preparation of mutant M207V

1. The 207-position Met (ATG) is mutated to Val (GTG) by a one-step PCR method using the plasmid pET28a-DyP4 as a template. As shown in Table 1, the designed primer sequences (mutation sites are drawn in horizontal lines).

TABLE 1 primer sequences for site-directed mutagenesis

PCR reaction system and conditions: total 50. mu.L, plasmid pET28a-DyP4 template 1. mu.L, upstream and downstream primers 1. mu.L each, 2 XPrimeSTAR Max Premix 25. mu.L, ddH₂O supplementTo 50. mu.L. Pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10 s; annealing at 55 ℃ for 5 s; extension at 72 ℃ for 40 s; after 30 cycles, the final extension is carried out for 10min at 72 ℃, and the temperature is reduced to 4 ℃ for storage.

The PCR product is detected by 0.8 percent DNA gel electrophoresis, and the target gene is recovered and purified by adopting a gel recovery kit.

And (3) phosphorylating and connecting the recovered product by using a Blunting hybridization Ligation (BKL) Kit. Table 2 shows the phosphorylation system.

TABLE 2 phosphorylation System

The reaction solution is respectively reacted for 10min at 37 ℃ and 70 ℃ for phosphorylation; add 5. mu. of ligation Solution I and mix, react at 16 ℃ for 1h for ligation.

The ligation products were transformed into a 50. mu.L volume of an Escherichia coli Rosetta (DE3) competent cell solution by heat shock method.

And (4) selecting a single clone for sequencing, and storing the strain with successful sequencing in a refrigerator at the temperature of-80 ℃.

2. Inducible expression of the mutant

1) 50. mu.L of the strain from which sequencing was successful was added to 5mL of LB liquid medium containing kanamycin (final concentration: 50. mu.g/mL) and chloramphenicol (final concentration: 34. mu.g/mL) for activation, followed by overnight culture at 37 ℃ and 220 rpm; the activated cells were transferred to 400mL of LB liquid medium containing kanamycin (final concentration: 50. mu.g/mL) and chloramphenicol (final concentration: 34. mu.g/mL), and cultured at 37 ℃ at 220rpm for 3 hours to OD₆₀₀Adding isopropyl-beta-D-thiogalactoside (IPTG, final concentration of 0.5mM) and inducing at 37 ℃ for 3-4 h; centrifuging at 8000rpm for 10min, and discarding the supernatant; resuspending and dissolving the pellet with 50mL Tris-HCl (50mM, pH 7.5), 0.4mL EDTA (500mM), 10 μ L PMSF (100mM), and ultrasonicating for 25 min; centrifuge at 12000rpm for 20min at 4 ℃ and discard the supernatant.

2) The pellet was resuspended in 5mL of urea (8M), 5. mu.L of EDTA (500mM) and placed at 4 ℃ for 3 h.

3) The denatured solution was added to the reconstituted solution after the preparation to dilute the urea concentration to 0.8M, and the reconstituted solution (45 mL): 44.65 mL sodium acetate (10mM, pH 6.0), 100. mu.L EDTA (500mM), 250. mu.L hemin (1 mM); standing at 4 deg.C for 36 h.

4) 500mL of sodium acetate (10mM, pH 6.0)) was used as a dialysate, and the dialysis membrane was selected to have a pore size of 13-14kDa and dialyzed at 4 ℃ for 24 hours; the dialyzate was centrifuged at 12000rpm for 20min at 4 ℃ and the supernatant was filtered by suction (through a 0.22 μm aqueous membrane) to obtain a crude enzyme solution (to be further purified).

5) And (3) further purifying the crude enzyme solution by using a Ni-NTA affinity chromatography gravity column, eluting the hybrid protein by using 40mM imidazole after loading the protein, washing the target protein by using 150mM imidazole, and detecting by using protein glue.

6) The protein concentration is measured by an extinction coefficient method, and the protein extinction coefficient is 222196M^-1cm^-1(ii) a Diluted with sodium acetate (10mM, pH 6.0) to the corresponding concentration.

The purification results are shown in SDS-PAGE electrophoresis (molecular weight 54kDa) of FIG. 2 and UV-visible spectrum of FIG. 3.

Example 2: determination of the enzymatic Properties of the mutant enzyme M207V

1. Determination of enzymatic Activity of mutant enzyme M207V

Four anthraquinone dyes Reactive Blue 4, Reactive Blue 5, Reactive Blue 19 and Reactive Blue 74 are selected as substrates to measure the activity of Il-DyP4 and mutant enzymes.

The enzyme activity calculation formula is as follows:

wherein A represents a change in absorbance.

Measuring enzyme activities of Il-DyP4 and mutant M207V; experimental reaction system (1 mL): 875. mu.L of 0.10mol/L sodium tartrate buffer (pH 3.5), 50. mu.L of enzyme (10. mu.g/mL concentration), 50. mu.L of 0.1mmol/L dye, 0.1mmol/LH₂O₂25 μL，No enzyme was added as a control, and H was added₂O₂The reaction was started, reacted at 35 ℃ for 2min and absorbance was measured on a spectrophotometer.

As shown in FIG. 4 and Table 3, the enzyme activities of Il-DyP4 on Reactive Blue 4, Reactive Blue 5, Reactive Blue 19 and Reactive Blue 74 were 79.5, 31.5, 26.6 and 67.1U/mg, respectively, while the enzyme activities of M207V mutant on the four anthraquinone dyes were 81.4, 34.4, 24.9 and 74.1U/mg, respectively; from the results, the enzyme activity of M207V is not obviously different from that of Il-DyP4, which preliminarily shows that the catalytic oxidation capability of the enzyme is not changed by the 207-Met mutation of Il-DyP 4.

TABLE 3 enzyme activities of Il-DyP4 and mutant M207V on oxidation of different dyes

2. Optimum H of mutant M207V for decoloring four anthraquinone dyes₂O₂Concentration of

The optimum H of the mutant for decoloring different anthraquinone dyes is determined by taking the unmutated enzyme as a control₂O₂And (4) concentration. H₂O₂The concentration was set at 0.1, 0.4, 1.0, 4.0, 10.0, 20.0, 60.0, 100.0, 150.0, 200.0, 250.0 mM. 1mL of the reaction system was: 0.10mol/L sodium tartrate buffer (pH 3.5) 900. mu.L, 10. mu.g/mL enzyme 25. mu.L, 40mmol/L dye 50. mu.L (final concentration of 0.1mmol/L), 25. mu.L of H at various concentrations was added₂O₂Starting the reaction, adding no enzyme in the experimental control group, carrying out water bath reaction at 35 ℃ for 4min, then measuring the light absorption value, and calculating the decolorization rate.

Decolorization ratio calculation formula:

TABLE 4 optimum H for decolourization of the mutant by four anthraquinone dyes₂O₂Concentration and decolorization rate

As shown in FIG. 5 and Table 4, Il-DyP4 was found to be 1.0, 0.6mM H₂O₂The mutant M207V has the highest decolorization rate for Reactive Blue 4, Reactive Blue 5, Reactive Blue 19 and Reactive Blue 74, and the most suitable H for decolorizing four anthraquinone dyes₂O₂The optimal H of M207V for decoloring four anthraquinone dyes with the concentrations of 0.6, 0.4 and 1.0mM respectively₂O₂The concentrations were all similar to Il-DyP4, which indicated that the M207 mutation to Il-DyP4 did not significantly alter the optimum H for the enzyme reaction₂O₂And (4) concentration.

3. H of mutant₂O₂Stability of

1) Mutants and H₂O₂Decolorization rate of incubation for different time

Reactive Blue 19 as substrate, H₂O₂Selecting 15mM of concentration, incubating with enzyme for 10min, 20min, 30min, 40 min, 50 min and 60min, and respectively measuring the decolorization rate of the mutant to Reactive Blue 19; reaction system: 0.10mol/L sodium tartrate (pH 3.5) 912. mu.L, incubated enzyme protein 13. mu.L (40. mu.g/mL), 2mmol/LReactive blue 1950. mu.L (final concentration 0.1mmol/L), 16mmol/LH₂O₂(final concentration 0.4mmol/L) 25. mu.L; reacting in water bath at 35 ℃ for 4min, and calculating the decolorization rate.

TABLE 5M 207V mutant with H₂O₂Destaining rate of RB19 by incubation for different periods of time

As shown in Table 5, the Il-DyP4 and M207V mutants were at 15mM H₂O₂The decolorization rate of Il-DyP4 on Reactive Blue 19 is gradually reduced along with the extension of the incubation time, while the decolorization rate of M207V mutant on Reactive Blue 19 is always kept at about 90% along with the extension of the incubation time, which indicates that the decolorization rate of mutant M207V on H is reduced₂O₂The stability is obviously excellentIn Il-DyP 4.

2) Determination of mutant H with four anthraquinone dyes as substrates₂O₂Stability of

(ii) H of the enzyme at different concentrations₂O₂In (c) incubation, H₂O₂Concentrations were set at 0, 0.2, 0.6, 1.0, 4.0, 10.0, 15.0, 20.0, 30.0, 40.0mM, incubation system (150 μ L): h was washed with 0.10mol/L sodium tartrate buffer (pH 5.5)₂O₂(400 mmol/L) diluted to different concentrations and mixed with 60. mu.L of enzyme (100. mu.g/mL) mixed with different concentrations of H₂O₂Incubate at 25 ℃ for 30 min.

② with H₂O₂The incubated enzyme decolorizes four anthraquinone dyes, decolorizing the reaction system (1 mL): 0.10mol/L sodium tartrate buffer (reaction pH was adjusted to optimum pH for different substrates by enzyme: Reactive Blue 4(pH 5.0), Reactive Blue 5(pH 3.5), Reactive Blue 19(pH 4.5), Reactive Blue 74(pH 5.0)) 912. mu.L, enzyme after incubation (40. mu.g/mL) 13. mu.L, 2mmol/L dye 50. mu.L (final concentration of 0.1mmol/L), 25. mu.L LH₂O₂(the concentration is shown in optimum H in Table 4₂O₂Concentration), adding enzyme to start reaction, adding no enzyme to a control group of an experiment, carrying out water bath reaction at 35 ℃ for 4min, then measuring the light absorption value, and calculating the decolorization rate.

TABLE 6 mutant H oxidation of four anthraquinone dyes₂O₂Stability of

a incubation of 30min after Il-DyP4 residual H with 50% destaining rate₂O₂Concentration;

b residual 50% destaining H of M207V after 30min incubation₂O₂Concentration;

c enzyme incubation 30min after destaining anthraquinone dye, M207V residual H with 50% destaining rate₂O₂Concentration divided by H of Il-DyP4 residual 50% decolorization₂O₂And (4) concentration.

As shown in Table 6, the M207V mutant had H compared to Il-DyP4₂O₂The stability is obviously improved, and the concrete expression is as follows: measurement H with Reactive Blue 4, Reactive Blue 5, Reactive Blue 19 and Reactive Blue 74 as substrates, respectively₂O₂Stability, M207V is 6.2, 4.8, 6.3, 6.4 times greater than Il-DyP4 respectively; as shown in FIG. 6, H at 15mM₂O₂After 30min of medium incubation, the decoloring rates of M207V on Reactive Blue 4, Reactive Blue 5, Reactive Blue 19 and Reactive Blue 74 reach 48.9, 77.6, 92.7 and 37.6 percent respectively, and the decoloring rates of Il-DyP4 on four anthraquinone dyes reach 13.1, 29.8, 19.5 and 6.8 percent respectively. Thus, H of mutant M207V₂O₂The stability is obviously improved, and the practical application value of the enzyme is enhanced.

1, the amino acid sequence of SEQ ID NO:

MHVKRARSTP LIGSFPGQPP LPTIAQVQST SAGNDSLPFE NIQGDILVGM KKDKEKFVFF 60

HINNATAFKS VLKTYAPANI TSVATIIGPV ANQPLAFVNL AFSHAGFGAL NVTDDLQDTA 120

FSDGQFKDSP NLGDDTSTWE EAFKGTNVDG VFLIGSNDES ITAQYRDDLN AKFGDAWTIV 180

YDLDSAARPG NEKGHEHFGY LDGISNPTIP GFGTPHPGQA VVDPGIIFTG RSKDPVVNRP 240

SWALDGSFLV FRKLKQLVPE FNKYVLDNAL QNQAGNLTVE EGAELLGSRM FGRWKSGAPI 300

DLSPDFDDPA LGNDIERNNN FNYSHPGSDL ATDQTRCPFT AHIRKTNPRD LEGQGLFGDT 360

FHAIRAGTPY GPEVTDYEAS SNTTTIDRGL AFVEYQSVIG NGFRFQQQAW ANNPRFPFSK 420

GPSIQLGLDP VIGQGSPRET FGLDPRNASE SFTVPQVIIS NGGEYFFSPS ITAIVEKFAA 480

<110> organization name:universityof Anhui

Application Project

-------------------

<120> Title, a peroxidase mutant with improved H2O2 stability and application thereof in dye decolorization

<130> AppFileReference :

<140> CurrentAppNumber :

<141> CurrentFilingDate : ____-__-__

Sequence

--------

<213> OrganismName : Irpex lacteus

<400> PreSequenceString :