CN109295017B

CN109295017B - Fungal laccase mutant PIE5, and expression strain and application thereof

Info

Publication number: CN109295017B
Application number: CN201810817998.2A
Authority: CN
Inventors: 肖亚中; 尹强; 方泽民; 张学成; 房伟
Original assignee: Anhui University
Current assignee: Anhui University
Priority date: 2018-07-24
Filing date: 2018-07-24
Publication date: 2021-08-31
Anticipated expiration: 2038-07-24
Also published as: CN109295017A

Abstract

The invention discloses a fungal laccase mutant PIE5, an expression strain and application thereof, wherein the fungal laccase mutant PIE5 is a laccase mutant obtained by modifying Coprinus cinereus laccase Lcc9 by means of directed evolution, constructing a mutation library and combining a three-wheel screening method so as to improve the application capability of laccase. The optimum catalysis pH of the fungal laccase mutant PIE5 is 8.5 (guaiacol is used as a substrate), and the fungal laccase mutant PIE5 can still exert good activity when the pH is 9.5, so that the fungal laccase PIE5 has the application advantage of alkaline environment compared with the alkaline environment, the application field of the fungal laccase is widened, the fungal laccase mutant PIE can be directly applied to a textile dyeing process to replace stone mill washing, the pH of waste water does not need to be adjusted in the degradation of indigo blue in the waste water, the improvement of the textile dyeing process of the fungal laccase is facilitated, the operation cost is reduced, and the ecological environment is protected.

Description

Fungal laccase mutant PIE5, and expression strain and application thereof

Technical Field

The invention relates to a fungal laccase mutant PIE5, and an expression strain and application thereof.

Background

Laccase is a copper-containing polyphenol oxidase, can catalyze the oxidation of various substrates such as phenols, arylamines and the like, and can further widen the oxidation substrates of some small molecular substances when the small molecular substances exist as mediators and simultaneously lead O₂Is reduced into water. Laccases are widely distributed, and are distributed in plants, animals, microorganisms and the like. Among them, microorganisms are the most major source species of laccases. In general, fungal laccases have higher redox potentials (470-790mV) and higher specific activities (100-1,000U/mg). In comparison, the bacterial laccase redox potential is lower (340-490mV), and the specific activity is lower than that of the fungal laccase (0.1-10U/mg). Thus, fungal laccases have a broader spectrumHas wide application value.

As the fungal laccases have wide substrate specificity, the fungal laccases have application in various fields of paper pulp bleaching, bioremediation, dye degradation, drug synthesis, biodegradation and the like, and have important economic value. Fungal laccases are currently most widely used in textile finishing and especially indigo bleaching. In the denim finishing and dyeing process, the fungal laccase can be used in the pumice stone washing process and also can be used in the subsequent indigo degradation process. The fungal laccase is only stable but is almost non-active under the neutral or alkaline condition, because the fungal laccase can only play the best activity (pH value is 3-5) under the acidic condition. Therefore, in the denim finishing and dyeing process, the pH value of washing needs to be controlled to be about 5, and after the reaction is finished, the pH value of the wastewater needs to be adjusted to be neutral, so that the washing cost is increased, the concentration of salt ions in water is increased, and the post-treatment and environmental protection are not facilitated. The fungal laccase protein which can exert good activity under neutral or alkaline conditions is obtained through protein engineering modification, which is beneficial to improving the textile finishing and dyeing process of fungal laccase, reducing the operation cost and protecting the ecological environment.

Disclosure of Invention

The invention is based on the defects of the prior art and aims to provide a fungal laccase mutant PIE5, and an expression strain and application thereof.

The fungal laccase mutant PIE5 is obtained by modifying Coprinus cinereus laccase Lcc9 by means of directed evolution, constructing a mutation library and combining a three-wheel screening method, so that the application capability of laccase is improved.

The amino acid sequence of the fungal laccase mutant PIE5 is shown as SEQ ID No: 1 is shown.

The amino acid sequence of the fungal laccase mutant PIE5 is SEQ ID No: 1 amino acid sequence which is substituted, deleted or added with one or more amino acid residues and encodes the same functional protein.

The strain for expressing the fungal laccase mutant PIE5 of the invention contains the encoding gene of fungal laccase protein which is tolerant to alkaline pH, is classified and named as Pichia pastoris pPIC9K-PIE5, is deposited in China center for type culture Collection, and has the address: china, Wuhan university, preservation date: 19/7/2018, accession number: CCTCC NO: m2018451.

The preparation method of the fungal laccase mutant PIE5 comprises the following steps:

step 1: inoculating the strain Pichia pastoris/pPIC9K-PIE5 into a seed liquid culture medium for fermentation, and culturing at 15-35 ℃ and 100-₆₀₀2-6; after centrifugation, the cells were resuspended to OD using fermentation broth₆₀₀Adding methanol every 24h to make the methanol concentration be 0.3-2.0%; centrifuging the induced fermentation liquor to obtain supernatant, namely crude fungal laccase enzyme liquid;

step 2: concentrating and ultrafiltering the crude fungal laccase enzyme solution obtained in the step 1 at the temperature of 4-10 ℃ and under the pressure of 0.1-0.5MPa to obtain concentrated crude enzyme solution; subsequently dialyzing with 20mM disodium hydrogen phosphate-citric acid buffer at pH 3.5-6.5, more preferably pH 6.5, at 4-10 deg.C three times to exchange the protein preservation buffer; and then purifying the fungal laccase by using a DEAE-Sepharose Fast Flow anion exchange column to obtain fungal laccase PIE5 protein, and detecting the protein purity by using gel electrophoresis.

The solvent of the seed liquid culture medium is PBS buffer solution with pH value of 3.5-6.5 and 0.1M, and the solutes and the concentrations thereof are as follows: yeast Extract 10g/L, Tryptone 20g/L, YNB 13.4.4 g/L, glycerin 10g/L, biotin 4X 10^-4g/L。

The solvent of the fermentation liquid culture medium is PBS buffer solution with pH value of 3.5-6.5 and 0.1M, and the solutes and the concentrations thereof are as follows: YNB 13.4g/L, methanol 5g/L, copper sulfate 1.5X 10^-2g/L, biotin 4X 10^-4g/L。

The invention discloses application of a fungal laccase mutant PIE5, which is applied as a decoloring oxidant in a dye decoloring process.

The dye decolorization is preferably an indigo decolorization process.

The application of the fungal laccase mutant PIE5 is used as a decoloring oxidant in a dye decoloring process, and specifically comprises the following steps:

the indigo dye is decolorized under the conditions that the pH value of a reaction system is 4.5-9.0, the temperature is 20-80 ℃, the concentration of laccase PIE5 is 40-200U/L and the concentration of a mediator is 20-300 mu M, and the decolorization rate of the indigo reaches more than 83 percent.

The mediator is syringaldehyde (DHB), Syringic Acid (SA), Methyl Syringate (MS), 2-diaza-bis (3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS) or 1-Hydroxybenzotriazole (HBT).

When methyl syringate MS is used as a mediator, PIE5 is used for indigo decoloring, and the optimal reaction system after optimization is as follows: the enzyme amount of the indigo blue with the concentration of 200 mu M, PIE5 is 160U/L, the mediator MS concentration is 120 mu M, the system pH is 7.5, the reaction is carried out for 50min at 60 ℃, and the decolorizing rate of the indigo blue reaches 83.1 +/-1.1% (figure 2).

When ABTS is used as a mediator, PIE5 is used for indigo decoloring, and the optimal reaction system after optimization is as follows: the enzyme amount of the indigo blue with the concentration of 200 mu M, PIE5 is 180U/L, the mediator ABTS concentration is 200 mu M, the system pH is 7.0, the reaction is carried out for 180min at 60 ℃, and the decolorizing rate of the indigo blue reaches 90.9 +/-0.3% (figure 3).

The decolorizing capacity of different types of fungal laccase on indigo is compared with that of a commercially available laccase, preferably a Novoxin laccase preparation. The method takes 2, 2-diazo-bis (3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS) as a laccase substrate, and detects the activity change of the laccase in the fermentation process every 24 hours. The enzyme activity detection system is subjected to ice bath for 30S immediately after reacting for 3min at the temperature of 30 ℃, the light absorption value is detected at the position of 420nm, the enzyme activity is calculated, and the enzyme activity (U) of one unit is defined as the enzyme amount required for converting 1 mu M substrate in one minute. The activity of the fungal laccase in the invention is measured by using ABTS as a substrate.

Compared with the prior art, the invention has the beneficial effects that:

the fungal laccase has wider application value, but the catalytic pH of the fungal laccase is generally between acidic 3 and 5, so the application of the fungal laccase is limited in an alkaline environment. The fungal laccase mutant PIE5 obtained by the invention has the most suitable catalytic pH value of 8.5 (guaiacol is taken as a substrate), and can still play good activity when the pH value is 9.5, so the fungal laccase PIE5 has the application advantage of alkaline environment compared with the prior art, and the application field of the fungal laccase is widened, for example, the fungal laccase PIE5 is used for decoloring indigo dye in textile dye finishing process to replace the traditional process. Textile finishing is one of the more widely applied directions of fungal laccase, and the traditional method uses stone mill washing for denim antique treatment in textile finishing and dyeing, and uses the traditional methods of physical adsorption and the like in dye wastewater treatment. However, the methods are not green and environment-friendly enough, the fungal laccase is a green catalyst and can be used for oxidative degradation of indigo, the alkaline environment of the textile dyeing process limits the application of most fungal laccase, and the alkaline fungal laccase PIE5 obtained by the method can be used for the textile dyeing process, especially for indigo decoloration. The alkaline mutant PIE5 can be directly applied to a textile dyeing process to replace stone mill washing, and the pH of the wastewater does not need to be adjusted in the degradation of the indigo blue of the wastewater, so that the improvement of the textile dyeing process of the fungal laccase is facilitated, the operation cost is reduced, and the ecological environment is protected.

Drawings

FIG. 1 shows electrophoresis detection of purified fungal laccase PIE5, wherein M is protein Maker, and PIE5 is mixed laccase pure protein. From FIG. 1, it can be seen that the target band PIE5 is highly pure and has a size of about 61 kDa.

FIG. 2 is the results of the optimization of PIE5 decolorizing indigo system with MS as mediator, where A represents the effect of pH on decolorization rate, B represents the effect of temperature on decolorization rate, C represents the effect of enzyme amount on decolorization rate, and D represents the effect of mediator concentration on decolorization rate. As can be seen from FIG. 2, the reaction parameters have a great influence on the indigo decoloration of PIE5, and pH and temperature influence the catalytic activity of the enzyme, so that the decoloring rate tends to increase and decrease, and tends to increase and stabilize with the increase of the enzyme amount and the mediator concentration.

FIG. 3 is a graph of PIE5 for oxidative decolorization of indigo using MS as a mediator, where A represents the change in the rate of indigo decolorization at different times and B represents the effect of indigo decolorization at different times. From fig. 3, it can be seen that PIE5 has a significant decolorizing effect on indigo under the action of mediator MS, and from fig. a, it is seen that the indigo decolorizing rate increases with the reaction time and becomes stable after 50min of reaction. It is seen from the graph B that the solution color becomes clear and indigo is decolorized by oxidation as the reaction time is prolonged.

FIG. 4 shows the results of optimizing PIE5 decolorizing indigo system with ABTS as mediator, where A shows the effect of pH on decolorization rate, B shows the effect of temperature on decolorization rate, C shows the effect of enzyme amount on decolorization rate, and D shows the effect of mediator concentration on decolorization rate. As can be seen from FIG. 4, the reaction parameters have a great influence on the indigo decoloration of PIE5, and pH and temperature influence the catalytic activity of the enzyme, so that the decoloring rate tends to increase and decrease, and tends to increase and stabilize with the increase of the enzyme amount and the mediator concentration.

FIG. 5 is a graph of PIE5 for oxidative decolorization of indigo using ABTS as mediator, where A represents the change in the rate of indigo decolorization at different times and B represents the effect of indigo decolorization at different times. It can be seen from fig. 5 that PIE5 was able to oxidatively decolorize indigo under the action of mediator ABTS, and graph a shows that the indigo decolorization rate increased with the reaction time and became stable after 180min of reaction. Panel B shows that as the reaction time is extended, the solution becomes increasingly clear in color and the indigo is oxidatively bleached.

Detailed Description

Terms used in the present invention have generally meanings as commonly understood by one of ordinary skill in the art, unless otherwise specified. In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.

1. Preparation of fungal laccase PIE5

The strain is as follows: coli JM109, P.pastoris GS115

Carrier: pPIC9K

Culture medium:

YPD medium (1L): 20g of Peptone and 10g of Yeast Extract were dissolved in 900mL of water, autoclaved, and then 100mL of 10 XD was added, and 1.5% agar was added to the solid medium.

The solvent of the seed liquid culture medium is PBS buffer solution with pH value of 6.0 and 0.1M, and the solutes and the concentrations thereof are as follows: yeast Extract 10g/L, Tryptone 20g/L, YNB 13.4.4 g/L, glycerin 10g/L, biotin 4X 10^-4g/L。

The solvent of the fermentation liquid culture medium is PBS buffer solution with pH value of 6.0 and 0.1M, and the solutes and the concentrations thereof are as follows: YNB 13.4g/L, methanol 5g/L, copper sulfate 1.5X 10^-2g/L, biotin 4X 10^-4g/L。

1.1 obtaining of mutant PIE5 coding gene and construction of recombinant plasmid

The cDNA of Coprinus cinereus laccase Lcc9 is used as a template, and the following primers (underlined are mutation site sequences) are used for introducing three site mutations of E116K, N229D and I393T in an amino acid sequence.

116F：GCATTGAACCTGTACTTAAACGACTCTC

116R：GAGAGTCGTTTAAGTACAGGTTCAATGC

229F：GAACTTGTAGTCGGGATCGCACG

229R：TCGTGCGATCCCGACTACAAGTTC

393F：GGCAAAGTATGGGTCGCACCAC

393R：AGTGGTGCGACCCATACTTTGCC

PIE5F：CGGAATTCCAAATCCTTGGCCCG (recognition sequence of Eco RI is underlined)

PIE5R：AAGGAAAAAAGCGGCCGCTTAAGGAGTGG (Not I recognition sequence underlined)

The gene encoding the mutant PIE5 is amplified and obtained in a conventional PCR mode, and two enzyme cutting sites of Eco RI and Not I are introduced during full-length sequence amplification. The PCR reaction system is 50 mu L, and the reaction conditions are as follows: pre-denaturation at 95 ℃ for 2min, followed by 30 cycles (95 ℃ 20S, 57 ℃ 20S, 72 ℃ 1.5min), extension at 72 ℃ for 10min after cycling, final incubation at 4 ℃ and detection of 10. mu.L of PCR product by 1% agarose gel electrophoresis. The PIE5 target fragment was excised from an AxyPrep DNA gel recovery kit by a procedure such as gel cutting.

The recovered PIE5 target fragment and pPIC9K vector are subjected to double enzyme digestion for 8h in water bath at 37 ℃ by two restriction endonucleases of Eco RI and Not I respectively, after two groups of enzyme digestion products are recovered by an AxyPrep DNA gel recovery kit, 100ng of the recovered products are taken respectively, and the PIE5 encoding gene is inserted between Eco RI and Not I enzyme digestion sites of pPIC9K in the forward direction under the action of T4 ligase at 16 ℃ to obtain recombinant plasmid.

1.2 construction of mutant PIE5 recombinant expression Strain

The recombinant plasmid was transferred into E.coli JM109 competent cells, and the transformant was plated on LB (100 mg/L ampicillin-containing) plates and cultured at 37 ℃. Some single clones were randomly picked and positive clones were verified by colony PCR. Positive clones were inoculated into a tube containing 5mL of liquid LB (containing 100mg/L of ampicillin), and cultured at 37 ℃ and 200 rpm. After overnight culture, the supernatant was centrifuged and collected, and recombinant plasmids were extracted using AxyPrep plasmid recovery kit. 6 mu.g of recombinant plasmid is completely and singly digested at 37 ℃ by Sac I restriction enzyme to obtain linearized plasmid, and the linearized plasmid is recovered by an AxyPrep DNA gel recovery kit.

Transferring the recovered linearized recombinant plasmid into a P.pastoris GS115 competent cell in an electrotransfer mode, culturing the competent cell at 28 ℃ by using a YPD plate, selecting a positive clone by using a BMM detection plate containing ABTS, and screening to obtain a PIE5 recombinant expression strain. The obtained recombinant strain was picked up to BMGY medium, cultured at 28 ℃ and 200rpm, stored in a 15% glycerol tube, and placed at-80 ℃ for bacterial conservation.

1.3 fermentation culture

The strain preserved in the glycerin tube is inoculated into a 250mL shaking flask containing 50mL liquid BMGY culture medium and cultured at 28 ℃ and 200rpm until the biomass OD of the strain is obtained₆₀₀The value is 2.0-6.0. Centrifuging the cells, suspending the cells in 100mL BMM broth medium until the OD of the cells₆₀₀The value is 1.0, the culture is continued, methanol is added every 24 hours to make the methanol concentration be 0.5%, and the change of the laccase PIE5 activity in the fermentation liquor is detected by ABTS.

The ABTS detection system is as follows: taking 950 μ L of sodium tartrate buffer solution with pH of 4.0, adding 33 μ L of ABTS mother liquor with concentration of 0.5mM, preheating in water bath at 30 deg.C for 3min, and adding 17 μ L of laccase solution. After uniform mixing, the reaction system reacts for 3min at 30 ℃, ice bath is carried out for 30S immediately, the light absorption value is detected at 420nm, the enzyme activity is calculated, and the enzyme activity (U) of one unit is defined as the enzyme quantity required for converting 1 mu M substrate in one minute.

1.4 obtaining crude enzyme solution

And (3) when the activity of the laccase begins to decrease by continuous detection, ending fermentation, collecting fermentation liquor, centrifuging at 4 ℃ and 8000g for 30min, discarding the precipitate, and collecting 1L of supernatant as crude enzyme liquid of PIE 5. And (3) concentrating and ultrafiltering the crude enzyme solution by using an ultrafiltration device with the specification of 350mL at the temperature of 4 ℃ and under the pressure of 0.3MPa to obtain 20mL of the fungal laccase PIE5 concentrated crude enzyme solution.

3L of 20mM disodium hydrogen phosphate-citric acid dialysis buffer, pH 6.5, was prepared, and the concentrated crude enzyme solution of PIE5 was placed in a dialysis bag. And (3) placing the dialysis bag in a buffer solution to dialyze and replace the laccase buffer system in the environment of 4 ℃. The dialysis buffer was changed every 5h and dialysis was continued three times to obtain about 21mL of crude laccase PIE5 enzyme solution.

1.5 fungal laccase PIE5 purification

Using an anion exchange column: DEAE-Sepharose Fast Flow purified fungal laccase, preparing a purified solution:

low salt content: 20mM disodium phosphate-citric acid pH 6.5;

high salt content: adding 150mM ammonium sulfate into a low-salt buffer;

NaCl: 500mL of NaCl solution with the concentration of 1M;

NaOH: 500mL of NaOH solution with the concentration of 1M;

ethanol: 1L of 20% ethanol solution by volume fraction;

H₂o: 1L of ultrapure water.

And (4) carrying out suction filtration on the prepared solutions, and then carrying out ultrasonic degassing. The specific steps of protein purification are as follows:

adjusting the flow rate of the instrument to be 1.0mL/min, installing an anion exchange column, and washing the column body for 40min by using 20% ethanol;

flushing the column, replacing H in sequence₂O→NaCl→H₂O→NaOH→H₂O → high salt → H₂O → Low salt, rinse for 40min for each change.

Thirdly, loading the dialyzed crude PIE5 enzyme solution into a column, adjusting the high salt concentration, eluting protein from low to high (0-100 percent) in sequence, and collecting the eluent.

And fourthly, detection: the protein purity in the eluate was checked by 15% SDS-polyacrylamide gel (SDS-PAGE), and the eluate containing the target protein in a relatively pure form was collected and mixed (FIG. 1).

2. Laccase PIE5 for indigo blue decolouring

Weighing 5.25g of indigo, 4g of sodium hydroxide and 2g of thiourea dioxide into 1L of water, and heating the mixed solution to 50 ℃ to dissolve the mixture to prepare an indigo solution. Dimethyl sulfoxide (DMSO) was used as a solvent to prepare a stock solution containing mediator Methyl Syringate (MS) and ABTS at a concentration of 10 mM.

Constructing 1mL of initial reaction system for oxidizing and decoloring indigo by laccase: indigo blue 200U M, PIE5 enzyme activity 200U/L, mediator concentration 100U M, pH7.0 citric acid-phosphate buffer solution to make up to 1 mL. After the system reacts for 3 hours at 40 ℃, the change of light absorption values before and after the reaction is detected at 700nm, the decolorization rate is calculated, and the decolorization rate is used as an evaluation standard to optimize and establish a laccase oxidation indigo blue decolorization system. The decolorization ratio calculation formula is as follows:

decolorization ratio ═ A₀-A₁)/A₀×100％

A₀For the initial absorbance of the reaction precursor system, A₁The absorbance of the system after reaction.

2.1 use of MS as mediator for indigo blue decolorization

When methyl syringate MS is used as a mediator, PIE5 is used for indigo blue decolorization, parameters such as temperature, enzyme amount, pH, mediator concentration and the like which influence the decolorization effect are respectively optimized in the optimization of a reaction system, the decolorization rate is used as an evaluation standard, and the optimized reaction system is as follows: the enzyme amount of the indigo blue with the concentration of 200 mu M, PIE5 is 160U/L, the mediator MS concentration is 120 mu M, the system pH is 7.5, the reaction is carried out for 50min at 60 ℃, and the decolorizing rate of the indigo blue reaches 83.1 +/-1.1% (figure 2-3).

2.2 use of ABTS as mediator for indigo decoloring

When ABTS is used as a mediator, PIE5 is used for indigo blue decolorization, parameters such as temperature, enzyme amount, pH, mediator concentration and the like which influence the decolorization effect are respectively optimized in the optimization of a reaction system, and the optimized reaction system is as follows by taking the decolorization rate as an evaluation standard: the enzyme amount of the indigo blue with the concentration of 200 mu M, PIE5 is 180U/L, the mediator ABTS concentration is 200 mu M, the system pH is 7.0, the reaction is carried out for 180min at 60 ℃, and the decolorizing rate of the indigo blue reaches 90.9 +/-0.3% (figure 4-5).

SEQ ID No：1

MSRKLFSLAYLAVVLVSVAGQILGPTSTMTVSNIDASPDGFNRPVVAVNGQHPGPLVRANK GDNFRINVVNDLNDPTMLRQTSVHWHGVFQHGTAWADGPDGVTQCPIAQNGESFKYRFN AGNEAGTFWYHSHFGTQYCDGLRGPLVIYDPNDPHRNLYDVDNADTVITLVDWYHLQAPS IEGPALSDATLINGKGRRPGGPETDIAIVNVQRNRRYRFRLVSMSCDPDYKFSIDGHKLTVIE ADGQLTEPLMVDEIQIFAGQRYSFVLSANRPVGNYWIRAIPNVGSNNLPNFSSGGINSAILRY AGAPNANPTSTPVTNPVALHESNLHALLNPGAPGGSGPADENIVLQMGLGPAGFEINGVTW ANPDSPVMVQIMNGVPPADIVPSGATHTLPRNRVVEVSIPGFELAGPHPFHLHGHAFSVVRS AGSSTYNYENPVRRDVVDVGGASDNVTIRFTTDNPGPWFFHCHIEFHLVLGLAMVFMEAP SDIPSTSPPPPSWSELCPKFESLPASATSIQIVPTP。

SEQUENCE LISTING

<110> university of Anhui

<120> fungal laccase mutant PIE5, and expression strain and application thereof

<130>

<160> 1

<170> PatentIn version 3.1

<210> 1

<211> 525

<212> PRT

<213> Coprinopsis cinerea

<220>

<221> SEQ ID No：1

<222> (1)..(525)

<223>

<400> 1

Met Ser Arg Lys Leu Phe Ser Leu Ala Tyr Leu Ala Val Val Leu Val

1 5 10 15

Ser Val Ala Gly Gln Ile Leu Gly Pro Thr Ser Thr Met Thr Val Ser

20 25 30

Asn Ile Asp Ala Ser Pro Asp Gly Phe Asn Arg Pro Val Val Ala Val

35 40 45

Asn Gly Gln His Pro Gly Pro Leu Val Arg Ala Asn Lys Gly Asp Asn

50 55 60

Phe Arg Ile Asn Val Val Asn Asp Leu Asn Asp Pro Thr Met Leu Arg

65 70 75 80

Gln Thr Ser Val His Trp His Gly Val Phe Gln His Gly Thr Ala Trp

85 90 95

Ala Asp Gly Pro Asp Gly Val Thr Gln Cys Pro Ile Ala Gln Asn Gly

100 105 110

Glu Ser Phe Lys Tyr Arg Phe Asn Ala Gly Asn Glu Ala Gly Thr Phe

115 120 125

Trp Tyr His Ser His Phe Gly Thr Gln Tyr Cys Asp Gly Leu Arg Gly

130 135 140

Pro Leu Val Ile Tyr Asp Pro Asn Asp Pro His Arg Asn Leu Tyr Asp

145 150 155 160

Val Asp Asn Ala Asp Thr Val Ile Thr Leu Val Asp Trp Tyr His Leu

165 170 175

Gln Ala Pro Ser Ile Glu Gly Pro Ala Leu Ser Asp Ala Thr Leu Ile

180 185 190

Asn Gly Lys Gly Arg Arg Pro Gly Gly Pro Glu Thr Asp Ile Ala Ile

195 200 205

Val Asn Val Gln Arg Asn Arg Arg Tyr Arg Phe Arg Leu Val Ser Met

210 215 220

Ser Cys Asp Pro Asp Tyr Lys Phe Ser Ile Asp Gly His Lys Leu Thr

225 230 235 240

Val Ile Glu Ala Asp Gly Gln Leu Thr Glu Pro Leu Met Val Asp Glu

245 250 255

Ile Gln Ile Phe Ala Gly Gln Arg Tyr Ser Phe Val Leu Ser Ala Asn

260 265 270

Arg Pro Val Gly Asn Tyr Trp Ile Arg Ala Ile Pro Asn Val Gly Ser

275 280 285

Asn Asn Leu Pro Asn Phe Ser Ser Gly Gly Ile Asn Ser Ala Ile Leu

290 295 300

Arg Tyr Ala Gly Ala Pro Asn Ala Asn Pro Thr Ser Thr Pro Val Thr

305 310 315 320

Asn Pro Val Ala Leu His Glu Ser Asn Leu His Ala Leu Leu Asn Pro

325 330 335

Gly Ala Pro Gly Gly Ser Gly Pro Ala Asp Glu Asn Ile Val Leu Gln

340 345 350

Met Gly Leu Gly Pro Ala Gly Phe Glu Ile Asn Gly Val Thr Trp Ala

355 360 365

Asn Pro Asp Ser Pro Val Met Val Gln Ile Met Asn Gly Val Pro Pro

370 375 380

Ala Asp Ile Val Pro Ser Gly Ala Thr His Thr Leu Pro Arg Asn Arg

385 390 395 400

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

405 410 415

Phe His Leu His Gly His Ala Phe Ser Val Val Arg Ser Ala Gly Ser

420 425 430

Ser Thr Tyr Asn Tyr Glu Asn Pro Val Arg Arg Asp Val Val Asp Val

435 440 445

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

450 455 460

Gly Pro Trp Phe Phe His Cys His Ile Glu Phe His Leu Val Leu Gly

465 470 475 480

Leu Ala Met Val Phe Met Glu Ala Pro Ser Asp Ile Pro Ser Thr Ser

485 490 495

Pro Pro Pro Pro Ser Trp Ser Glu Leu Cys Pro Lys Phe Glu Ser Leu

500 505 510

Pro Ala Ser Ala Thr Ser Ile Gln Ile Val Pro Thr Pro

515 520 525

Claims

1. A fungal laccase mutant PIE5, characterized in that:

the method is characterized in that coprinus cinereus laccase Lcc9 is transformed by an oriented evolution means to construct a mutation library, and a three-wheel screening method is combined to obtain a laccase mutant so as to improve the application capability of laccase;

2. A strain expressing the fungal laccase mutant PIE5 of claim 1, characterized in that:

the classification of the strains is namedPichia pastoris pPIC9K-PIE5And the culture is preserved in China center for type culture Collection with the preservation date of 2018, 7 and 19 months and the preservation number of CCTCC NO: m2018451.

3. Use of the fungal laccase mutant PIE5 of claim 1, characterized in that:

the fungal laccase mutant PIE5 is applied as a decoloring oxidant in a dye decoloring process.

4. Use of the fungal laccase mutant PIE5 according to claim 1, wherein the fungal laccase mutant PIE5 comprises:

the fungal laccase mutant PIE5 is used as a decoloring oxidant in a dye decoloring process, and specifically comprises the following steps:

dye is decolorized under the conditions that the pH value of a reaction system is 4.5-9.0, the temperature is 20-80 ℃, the concentration of laccase PIE5 is 40-200U/L and the concentration of a mediator is 20-300 mu M, and the decolorization rate of indigo reaches more than 83 percent.

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

the mediator is syringaldehyde, syringic acid, methyl syringate, 2-diaza-bis (3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt or 1-hydroxybenzotriazole.

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

the dye is an indigo dye.

7. Use according to claim 5 or 6, characterized in that:

when methyl syringate is used as a mediator, PIE5 is used for indigo decoloration, the enzyme amount of 160U/L with the indigo concentration of 200 mu M, PIE5, the MS concentration of the mediator of 120 mu M and the pH value of the system are reacted for 50min at 60 ℃, and the indigo decoloration rate reaches 83.1 +/-1.1%.

8. Use according to claim 5 or 6, characterized in that:

when 2, 2-dinitrogen-di (3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt is used as a mediator, PIE5 is used for indigo decoloring, the indigo decoloring rate reaches 90.9 +/-0.3 percent, the enzyme amount is 180U/L at the indigo concentration of 200 mu M, PIE5, the ABTS concentration is 200 mu M at the mediator concentration, and the system pH is 7.0, and the reaction is carried out for 180min at 60 ℃.