CN112195163A - Manganese peroxidase mutant independent of manganese ions and application thereof - Google Patents

Manganese peroxidase mutant independent of manganese ions and application thereof Download PDF

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CN112195163A
CN112195163A CN202011250108.8A CN202011250108A CN112195163A CN 112195163 A CN112195163 A CN 112195163A CN 202011250108 A CN202011250108 A CN 202011250108A CN 112195163 A CN112195163 A CN 112195163A
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荚荣
杨俊�
李柳清
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Abstract

The invention discloses a manganese peroxidase mutant independent of manganese ions and application thereof. On the basis of an Il-MnP1 amino acid sequence, site-directed mutagenesis is carried out on a 70 th amino acid residue Arg and a 166 th amino acid residue Glu, and a mutant MnP (R70V/E166A) is obtained through constructing an expression strain, induction expression, denaturation and renaturation and purification. The mutants of the invention are Mn-independent2+The pH value is neutral, the anthraquinone dye can be effectively decolorized, and H can be effectively removed2O2The tolerance of the concentration is improved, which has important significance for treating the water body pollution caused by anthraquinone dye.

Description

Manganese peroxidase mutant independent of manganese ions and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a manganese ion (Mn) independent manganese ion2+) The manganese peroxidase mutant and the application thereof in decoloring anthraquinone dyes.
Background
Manganese peroxidase (MnP, ec1.11.1.13) is a heme-containing peroxidase produced mainly by exocytosis of woody white rot fungi. The MnP can degrade refractory aromatic compounds, including synthetic dyes. Therefore, the enzyme has great application prospect in environmental pollutant treatment.
Anthraquinone dye is a widely used synthetic dye with bright color, and has the structural characteristic that two or more carbonyl groups are contained in a condensed ring conjugated system. Because the dye is of a ketone structure, the dye is not easy to oxidize and generate a photo-oxidation reaction, and has high light fastness, so that the dye is extremely poor in biodegradability and not easy to degrade, the wastewater containing the dye is very difficult to decolorize, the chromaticity of the water is high, and the water environment and the normal life activities of aquatic animals and plants are seriously influenced.
Most of the MnP requires Mn for degradation of anthraquinone dyes2+And the reaction must be carried out under the condition that the pH is slightly acidic (pH 3.5-4.0), which can further introduce metal ions into the water body and cause new environmental pollution. However, there are also some MnP, which can be Mn-independent2+Some small molecular substrates and dyes are directly oxidized, but the catalytic oxidation capability is poor, and the oxidation reaction is H2O2Is less tolerant and, therefore, manganese ion-dependent, more acidic pH conditions, lower catalytic oxidation and poorer H2O2The tolerance of MnP limits the practical application of MnP.
Disclosure of Invention
Aiming at the technical problems, the invention provides a manganese peroxidase mutant independent of manganese ions and application thereof. The mutant is obtained by site-directed mutagenesis of amino acid residue Arg at 70 and amino acid residue Glu at 166 of recombinant Il-MnP1, and can be obtained in the absence of Mn2+Effectively decolors anthraquinone dye under the condition of neutral pH and can decolor H2O2The concentration tolerance is improved.
The amino acid sequence (AGO86670.2) of the recombinant manganese peroxidase Il-MnP1 derived from the fungus Irpex lacteus F17(CCTCC AF 2014020) is shown as SEQ ID NO. 1.
The amino acid sequence of the manganese peroxidase mutant MnP (R70V/E166A) independent of manganese ions is shown as SEQ ID NO. 2.
The construction method of the manganese peroxidase mutant MnP (R70V/E166A) comprises the following steps:
step 1: amplifying by adopting a PCR method to obtain linear plasmids of the mutant;
step 2: phosphorylating and connecting the linear plasmid of the mutant to obtain a recombinant vector;
and step 3: transforming the recombinant vector into an expression strain;
and 4, step 4: culturing the expression strain, and inducing the mutant enzyme to excessively express;
and 5: the expressed mutant enzyme (R70V/E166A) was denatured, renatured, recovered and purified.
The manganese peroxidase mutant MnP (R70V/E166A) of the invention has no Mn2+pH 5.5 and H2O2The decolorization rate of the anthraquinone dye Reactive blue 4 is 59.4 percent under the condition that the concentration is 0.4mmol/L, the decolorization rate of the Reactive blue 5 is 80.8 percent, the decolorization rate of the Reactive blue 19 is 90.56 percent, and the method is expected to be used for decolorization treatment of wastewater water containing anthraquinone dye and has important application value.
Drawings
FIG. 1 is an SDS-PAGE electrophoresis of the mutant enzyme of the present invention.
FIG. 2 mutant enzyme in Mn-independent2+Optimum pH for catalytic Reactive blue 19 under conditions with unmutated enzyme as control.
FIG. 3 mutant enzyme in Mn-independent2+Optimal temperature for catalyzing Reactive blue 19 under conditions with unmutated enzyme as control.
FIG. 4 mutation of enzyme in Mn-independent2+Optimum H for catalyzing Reactive blue 19 under conditions2O2Concentrations were compared to unmutated enzyme.
FIG. 5 decolorization of Reactive blue 4, Reactive blue 5 and Reactive blue 19 by mutant enzymes under optimal conditions, with non-mutant enzymes as controls.
Detailed Description
The present invention is described in detail with reference to specific embodiments, which are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operation procedures are provided.
Example 1: preparation of mutant MnP (R70V/E166A)
1. Site-directed mutagenesis was performed by a one-step PCR method using a plasmid of recombinant Il-MnP1 (pET28a-MnP1) as a template to convert amino acid R (arginine CGT) at position 70 to V (valine GTC) and amino acid E (glutamic acid GAG) at position 166 to A (alanine GCA), respectively. The designed primer sequences (mutation sites underlined) are shown in Table 1.
TABLE 1 primer sequences for site-directed mutagenesis
Figure BDA0002771314960000021
PCR reaction system and conditions: the 50. mu.L system included 1. mu.L each of the upstream and downstream primers, 1. mu.L of DNA template, 2 XPrimeSTAR Max Premix 25. mu.L, ddH2And O is supplemented to 50 mu L. Pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10 s; annealing at 55 ℃ for 5 s; extending for 7min at 72 ℃; after 28 cycles, the final extension is carried out for 10min at 72 ℃, and the temperature is reduced to 4 ℃ for storage.
Plasmid (pET28a-MnP1) of Il-MnP1 is used as a template, R70V-F and R70V-R are used as primers, the plasmid containing R70V mutation sites is amplified, and after DNA gel electrophoresis detection is correct, a glue recovery kit is adopted to recover and purify target genes.
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
Figure BDA0002771314960000031
1) The reaction was carried out at 37 ℃ for 10 min.
2) The reaction was carried out at 70 ℃ for 10 min.
3) 5 mu L of the reaction Solution is put into a new micro-centrifuge tube, 5 mu L of the dilution Solution I is added, and the mixture is uniformly mixed and reacted for 1h at the temperature of 16 ℃.
The reaction solution in 3) was transformed into 50. mu.L of LEscherichia coliRosetta (DE3) competent cells.
Selecting and sequencing a single clone, carrying out plasmid extraction on a positive single clone strain with correct sequencing, and carrying out mutation by using a plasmid of the mutant R70V as a template and E166A-F and E166A-R as primers according to the site-specific mutagenesis method. Finally, the mutant strain R70V/E166A containing two mutation sites is obtained.
2. Inducible expression of mutant strains
1) 50 μ L of the mutant strain was inoculated into 5mL of LB medium containing kanamycin sulfate and chloramphenicol: 220rpm, 37 ℃ overnight culture (kanamycin sulfate final concentration of 50 u g/mL, chloramphenicol final concentration of 34 u g/mL); then transferred into 400mL LB culture medium containing kanamycin sulfate and chloramphenicol for amplification culture for 2-3h to OD6000.4-0.6; adding isopropyl-beta-D-thiogalactoside (IPTG, final concentration of 0.5mM) for induction, and culturing at 37 deg.C for 3-4 h; centrifuging at 4 deg.C and 8000rpm for 10min, and removing supernatant; the inclusion body precipitate was resuspended and solubilized with 50mL of Tris-HCl (50mM, pH8.5), 0.1mL of DTT (1M), 0.4mL of EDTA (500mM), 10. mu.L of PMSF (100mM), and sonicated until the liquid was slightly transparent; centrifuge at 12000rpm for 30min at 4 ℃ and discard the supernatant.
2) The pellet was resuspended in 5mL of urea (8M), 200. mu.L of EDTA (500mM), 10. mu.L of DTT (1M), and allowed to stand under a closed condition at 4 ℃ for 2h 40 min.
3) Adding the inclusion body denaturation resuspension into a renaturation solution (53.366mL), wherein the renaturation solution comprises the following components: 42.074mL Tris-HCl (50mM, pH8.5), 10% glycerol, 3.2mLCaCl2(2.5M), 1.326mL hemin (1mM), 1.06mL KCl (1M), 380. mu.L GSSG (0.07M), 26. mu.L MnSO4(0.1M); incubate 4 ℃ under sealed and dark conditions for 36 h.
4) Dialyzed against 1L of sodium acetate (10mmol/L, pH 6.0) and magnetically stirred at 4 ℃ for 24 h; after the dialysis was completed, the mixture was centrifuged at 12000rpm for 30min at 4 ℃ and the supernatant was filtered (through a 0.22 μm aqueous membrane) to obtain an enzyme solution (to be further purified).
5) Loading the enzyme solution to a Ni-NTA affinity chromatography gravity column, eluting with imidazole (30mM, 100mM and 200mM, and pH 7.4) with different concentrations, collecting 200mM imidazole eluate, concentrating the eluate, and replacing buffer solution (sodium acetate) to obtain pure mutant enzyme.
6) The concentration of the above enzyme was determined using a modified Brandford method protein concentration assay kit and diluted to 100. mu.g/mL with sodium acetate (10mmol/L, pH 6.0). Each 1mL of diluted enzyme solution (100. mu.g/mL) was supplemented with 25. mu.L of CaCl2(2.5M), and left to stand at 4 ℃ for 6 days.
As a result of the purification, the protein size was about 43kDa as shown by SDS-PAGE in FIG. 1.
Example 2: determination of the enzymatic Properties of the mutant enzyme R70V/E166A
1. Mutant enzyme R70V/E166A is Mn-independent2+Optimum pH for catalytic Oxidation of Reactive blue 19 under conditions
With Reactive blue 19 as a model dye and an unmutated enzyme as a control, the mutated enzyme R70V/E166A was tested in the presence of Mn independence2+Optimum pH for catalytic oxidation of Reactive blue 19 under conditions: as a buffer for measuring enzyme activity, sodium lactate buffers (0.11mol/L) having different pH values (pH values of 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.53, and 8.0) were used. 1mL of the reaction system was: 0.11mol/L sodium lactate buffer solution with different pH values, 100. mu.g/mL MnP (protein concentration is the same, final concentration is 10. mu.g/mL), 2mmol/LReactive blue 19 (final concentration is 0.1mmol/L), 4mmol/L H is added2O2(final concentration is 0.2mmol/L) are mixed evenly to start reaction, no enzyme is added in a control group of an experiment, after 30min of water bath reaction at 30 ℃, the light absorption value is measured at 595nm, and then the decolorization rate is calculated.
Decolorization ratio calculation formula:
Figure BDA0002771314960000041
in the formula: a. the0Is the absorbance at 595nm of the control group, AtIs the absorbance at 595nm after the experimental reaction t (min).
The decolorization rates of the mutant enzymes R70V/E166A on the anthraquinone dyes Reactive blue 4(598nm) and Reactive blue 5(600nm) were calculated as in the above equation.
The results are shown in FIG. 2. As can be seen from FIG. 2, the optimum pH of the unmutated enzyme was 3.5, while that of the mutated enzyme R70V/E166A was 5.5. Independent of Mn2+At this time, the decolourization rate of Reactive blue 19 by the mutant enzyme R70V/E166A was significantly higher than that of the non-mutant enzyme, the decolourization rate at the optimum pH (91.94%) was 5.12 times that of the non-mutant enzyme at the optimum pH (17.94%), and the mutant enzyme R70V/E166A still had 84% decolourization rate of Reactive blue 19 at pH 6.5And (4) rate. The optimum pH of the mutant enzyme R70V/E166A is improved, and the catalytic oxidation capability of the enzyme in a neutral environment is also enhanced.
2. Mutant enzyme R70V/E166A is Mn-independent2+Optimum temperature of catalytic oxidation Reactive blue 19 under the conditions
With Reactive blue 19 as a model dye and an unmutated enzyme as a control, the mutated enzyme R70V/E166A was tested in the presence of Mn independence2+The optimum temperature of the catalytic Reactive blue 19 under the conditions is 10, 15, 20, 25, 30, 35 and 40 ℃. The same procedure as in (1) was repeated except that 0.11mol/L sodium lactate buffer solution (pH 4.0) was used.
The results are shown in FIG. 3. As can be seen from FIG. 3, the dependence on Mn is not observed2+The optimum temperatures for the unmutated enzyme and the mutated enzyme R70V/E166A were 30 ℃ and 25 ℃, respectively. The optimal temperature difference between the unmutated enzyme and the mutated enzyme R70V/E166A is 5 ℃, however, more importantly, the decolouring rate of the mutated enzyme R70V/E166A to Reactive blue 19 is 4-6 times that of the unmutated enzyme, and the catalytic oxidation capability of the enzyme is greatly improved.
3. Mutant enzyme R70V/E166A is Mn-independent2+Optimum H of catalytic oxidation Reactive blue 19 under the condition2O2Concentration of
With Reactive blue 19 as a mode dye and unmutated enzyme as a control, the mutated enzyme R70V/E166A is tested in the presence of Mn independence2+Optimum H of catalytic oxidation Reactive blue 19 under the condition2O2Concentration of H2O2The concentration ranges are 0.01, 0.02, 0.03, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 1.2 mmol/L. The same procedure as in (1) was repeated except that 0.11mol/L sodium lactate buffer solution (pH 4.0) was used.
The results are shown in FIG. 4. As can be seen from FIG. 4, the H-most sites of the unmutated and mutated enzymes R70V/E166A2O2Optimum H of mutant enzyme R70V/E166A at concentrations of 0.02mmol/L and 0.4mmol/L, respectively2O2The concentration is greatly improved, and the enzyme is used for H2O2The tolerance of (2) is enhanced. Independent of Mn2+And H2O2When the concentration is more than 0.05mmol/L, the decolourization rate of the mutant enzyme R70V/E166A to Reactive blue 19 is higher than that of the non-mutant enzyme, and when H is higher than H, the mutant enzyme R70V/E166A to Reactive blue 19 is used2O2The maximum decolorization rate is reached at a concentration of 0.4mmol/L, which is 84.02%.
4. Mutant enzyme R70V/E166A is Mn-independent2+And decolorization of Reactive blue 4, Reactive blue 5 and Reactive blue 19 under optimum conditions
The mutant enzyme R70V/E166A was prepared by adding H at a final concentration of 0.4mmol/L to a buffer of sodium lactate (0.11mol/L) at pH 5.5, as substrates of 2mmol/L Reactive blue 4 (final concentration of 0.2mmol/L), 2mmol/L Reactive blue 5 (final concentration of 0.1mmol/L) and 2mmol/L Reactive blue 19 (final concentration of 0.1mmol/L), respectively2O2Mixing uniformly, starting reaction, and carrying out water bath reaction at 25 ℃ for 30 min. The unmutated enzyme was prepared by adding H to a final concentration of 0.02mmol/L using sodium lactate (0.11mol/L) at pH 3.5 as a buffer and the above dyes as substrates, respectively2O2Mixing uniformly, starting reaction, and carrying out water bath reaction at 30 ℃ for 30 min. The measurement and calculation methods are the same as those in (1).
The results are shown in FIG. 5. As can be seen from FIG. 5, the dependence on Mn is not observed2+The decolorization rate of the 3 anthraquinone dyes by the mutant enzyme R70V/E166A was greater than that of the unmutated enzyme. Specifically, the decolourization rates of the mutant enzyme R70V/E166A on Reactive blue 4, Reactive blue 5 and Reactive blue 19 reach 59.4%, 80.8% and 90.56% respectively, and are 3.47 times, 5.91 times and 6.62 times of the decolourization rate of the non-mutant enzyme. The catalytic oxidation capacity of the mutant enzyme R70V/E166A to anthraquinone dye is obviously enhanced.
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<110> organization name:universityof Anhui
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<120> Title, manganese peroxidase mutant independent of manganese ions and application thereof
<130> AppFileReference :
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Sequence
--------
<213> OrganismName : Irpex lacteus
<400> PreSequenceString :
MAFKHLIAAL SIVLSFGIAQ AAITKRVACP DGKNTATNAA CCSLFAIRDD IQANLFDGGE 60
CGEEVHESFR LTFHDAIGTG SFGGGGADGS IIVFDDIETN FHANNGVDEI IDEQKPFIAR 120
HNITPGDFIQ FAGAVGVSNC PGAPRLDFFL GRPNPVAAAP DKTVPEPFDT VDSILARFKD 180
AGGFTPAEVV ALLGSHTIAA ADHVDPTIPG TPFDSTPEVF DTQVFVEVQL RGTLFPGTGG 240
NQGEVQSPLR GEIRLQSDHD LARDSRTACE WQSFVNNQAK LQSAFKAAFK KLSVLGHNIN 300
NLIDCSEVIP EPPNVKVKPA TFPAGITHAD VEQACATTPF PTLATDPGPA TSVAPVPPS 359
<212> Type : PRT
<211> Length : 359
SequenceName : SEQ ID NO.1
SequenceDescription :
Sequence
--------
<213> OrganismName : Irpex lacteus
<400> PreSequenceString :
MAFKHLIAAL SIVLSFGIAQ AAITKRVACP DGKNTATNAA CCSLFAIRDD IQANLFDGGE 60
CGEEVHESFV LTFHDAIGTG SFGGGGADGS IIVFDDIETN FHANNGVDEI IDEQKPFIAR 120
HNITPGDFIQ FAGAVGVSNC PGAPRLDFFL GRPNPVAAAP DKTVPAPFDT VDSILARFKD 180
AGGFTPAEVV ALLGSHTIAA ADHVDPTIPG TPFDSTPEVF DTQVFVEVQL RGTLFPGTGG 240
NQGEVQSPLR GEIRLQSDHD LARDSRTACE WQSFVNNQAK LQSAFKAAFK KLSVLGHNIN 300
NLIDCSEVIP EPPNVKVKPA TFPAGITHAD VEQACATTPF PTLATDPGPA TSVAPVPPS 359
<212> Type : PRT
<211> Length : 359
SequenceName : SEQ ID NO.2
SequenceDescription :

Claims (5)

1. A manganese peroxidase mutant independent of manganese ions, characterized in that:
the manganese peroxidase mutant is obtained by carrying out site-directed mutagenesis on 70 th amino acid residue Arg and 166 th amino acid residue Glu of recombinant Il-MnP 1.
2. The manganese peroxidase mutant according to claim 1, characterized in that:
the amino acid sequence of the manganese peroxidase mutant is shown as SEQ ID NO. 2.
3. Use of a manganese peroxidase mutant according to claim 1 or 2, wherein:
the manganese peroxidase mutant is used for carrying out decoloration treatment on waste water containing anthraquinone dye.
4. Use according to claim 3, characterized in that:
the manganese peroxidase mutant does not contain Mn2+The anthraquinone dye can be effectively decolored under the condition of neutral pH environment, and H can be effectively decolored2O2The concentration tolerance is improved.
5. Use according to claim 4, characterized in that:
the manganese peroxidase mutant does not contain Mn2+pH 5.5 and H2O2The decolorization ratio of anthraquinone dye Reactive blue 4 under the condition of concentration of 0.4mmol/L is 59.4%, and the decolorization ratio of anthraquinone dye Reactive blue 5 isThe color ratio was 80.8%, and the decolorization ratio for Reactive blue 19 was 90.56%.
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