CN109295017A - A kind of fungal laccase mutant PIE5 and its expression bacterial strain and application - Google Patents
A kind of fungal laccase mutant PIE5 and its expression bacterial strain and application Download PDFInfo
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- CN109295017A CN109295017A CN201810817998.2A CN201810817998A CN109295017A CN 109295017 A CN109295017 A CN 109295017A CN 201810817998 A CN201810817998 A CN 201810817998A CN 109295017 A CN109295017 A CN 109295017A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
- C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
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- C12Y110/03002—Laccase (1.10.3.2)
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- D—TEXTILES; PAPER
- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
- D06P—DYEING OR PRINTING TEXTILES; DYEING LEATHER, FURS OR SOLID MACROMOLECULAR SUBSTANCES IN ANY FORM
- D06P5/00—Other features in dyeing or printing textiles, or dyeing leather, furs, or solid macromolecular substances in any form
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- D06P5/132—Fugitive dyeing or stripping dyes with oxidants
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Abstract
The invention discloses a kind of fungal laccase mutant PIE5 and its expression bacterial strain and applications, wherein fungal laccase mutant PIE5, it is to be transformed with directed evolution means to Coprinus cinereus laccase Lcc9, construct mutated library, in conjunction with three-wheel screening technique, the Laccase mutant of acquisition, to improve the application power of laccase.The most suitable catalytic pH of fungal laccase mutant PIE5 of the present invention is 8.5 (guaiacol is substrate); and it still is able to play excellent activity in pH to 9.5; therefore fungal laccase PIE5 has the application advantage of alkaline environment in contrast; the application field of fungal laccase is widened; it may be directly applied to weave whole dyeing technique instead of stone mill washing, without adjustment wastewater pH in the indigo degradation of waste water, advantageously improve the whole dyeing technique of weaving of fungal laccase; operating cost is reduced, is preserved the ecological environment.
Description
Technical field
The present invention relates to a kind of fungal laccase mutant PIE5 and its expression bacterial strain and applications.
Background technique
Laccase is a kind of polyphenol oxidase of cupric, the oxidation such as a variety of substrates such as phenols, arylamine class can be catalyzed, when some
In the presence of small-molecule substance is as mediator, laccase can further widen its oxidation substrates, and simultaneously by O2It is reduced into water.Laccase
It is widely distributed, it is distributed in plant, animal and microorganism etc..Wherein, microorganism is the main source species of laccase.
Under normal circumstances, fungal laccase redox potential with higher (470-790mV) and higher Rate activity (100-1,
000 U/mg).It compares, bacterial laccase redox potential is lower (340-490mV), and Rate activity is also below fungal laccase
(0.1-10 U/mg).Therefore, fungal laccase has wider application value.
Since fungal laccase has wide in range substrate specificity, they are in paper pulp bleaching, biological prosthetic, dyestuff degradation, medicine
The multiple fields such as object synthesis and biodegrade have application, have important economic value.Currently, fungal laccase is in whole dye of weaving
It is most widely used in especially indigo decoloration.During the whole dye of denim, fungal laccase can be used for float stone washing process,
It can be used for the indigo degradation process in later period.Since fungal laccase is only capable of playing optimum activity (pH value 3- in acid condition
5), and under neutral or basic conditions, fungal laccase is although more stable, but almost debility.Therefore, in the whole of denim
During dye, needs the pH value that will be washed control 5 or so, and after the completion of reaction, needs for the pH of waste water to be adjusted to neutral,
This process increases the costs of washing, while improving the concentration of the salt ion in water, are unfavorable for post-processing and environment is protected
Shield.It is transformed by protein engineering, obtains the fungal laccase albumen that can play excellent activity under neutral or basic conditions, will have
Conducive to the whole dyeing technique of weaving for improving fungal laccase, operating cost is reduced, is preserved the ecological environment.
Summary of the invention
The present invention is based on the defects of the prior art, it is desirable to provide a kind of fungal laccase mutant PIE5 and its expression bacterial strain and
Using.
Fungal laccase mutant PIE5 of the present invention is to be transformed with directed evolution means to Coprinus cinereus laccase Lcc9,
Mutated library is constructed, in conjunction with three-wheel screening technique, the Laccase mutant of acquisition, to improve the application power of laccase.
Fungal laccase mutant PIE5 of the present invention, amino acid sequence is as shown in SEQ ID No:1.
Fungal laccase mutant PIE5 of the present invention, amino acid sequence are that SEQ ID No:1 is substituted, lacks or adds one
A or several amino residues and the amino acid sequence for encoding identical function protein.
The bacterial strain for expressing fungal laccase mutant PIE5 of the present invention, the coding of the fungal laccase albumen containing tolerance alkaline pH
Gene, classification naming are Pichia pastoris pPIC9K-PIE5, are preserved in China typical culture collection center, address:
Wuhan, China Wuhan University, preservation date: on July 19th, 2018, deposit number: CCTCC NO:M 2018451.
The preparation method of fungal laccase mutant PIE5 of the present invention, includes the following steps:
Step 1: bacterial strain Pichia pastoris/pPIC9K-PIE5 being inoculated in seed fluid nutrient mediums of saccharomycete and is sent out
Ferment is cultivated under the conditions of 15-35 DEG C, 100-250rpm/min to thallus OD600=2~6；After thallus is centrifuged, fermentation liquid is used
Cell is resuspended to OD in body culture medium600=0.5-2.0 makes wherein methanol concentration 0.3-2.0% every methanol is added for 24 hours；It will lure
Supernatant, as fungal laccase crude enzyme liquid are obtained after fermentation liquid centrifugation after leading；
Step 2: step 1 gained fungal laccase crude enzyme liquid is subjected to ultrafiltration concentration under the conditions of 4-10 DEG C, 0.1-0.5MPa,
Obtain concentration crude enzyme liquid；Then with pH value 3.5-6.5, more preferably pH value 6.5, disodium hydrogen phosphate-lemon that concentration is 20mM
Acid buffer is dialysed at 4-10 DEG C and saves buffer three times to replace albumen；Again with DEAE-Sepharose Fast
Flow anion-exchange column purifies fungal laccase, obtains fungal laccase PIE5 albumen, and with detected through gel electrophoresis albumen
Purity.
The solvent of the seed fluid nutrient mediums of saccharomycete is the PBS buffer solution of pH value 3.5-6.5,0.1M, and solute and its concentration are such as
Under: Yeast Extract 10g/L, Tryptone 20g/L, YNB 13.4g/L, glycerol 10g/L, biotin 4 × 10-4g/L。
The solvent of the fermentation broth is the PBS buffer solution of pH value 3.5-6.5,0.1M, and solute and its concentration are such as
Under: YNB 13.4g/L, methanol 5g/L, copper sulphate 1.5 × 10-2G/L, biotin 4 × 10-4g/L。
The purposes of fungal laccase mutant PIE5 of the present invention is the answering as decoloration oxidant during dye decolored
With.
The dye decolored preferably indigo decolorization.
The application of fungal laccase mutant PIE5 of the present invention is to use during dye decolored as decoloration oxidant,
Specifically comprise the following steps:
In pH value of reaction system 4.5-9.0,20-80 DEG C of temperature, laccase PIE5 concentration 40-200U/L, mediator concentration 20-
Under conditions of 300 μM, decolorization is carried out to bipseudoindoxyl dye, indigo percent of decolourization reaches 83% or more.
The mediator is syringaldehyde (DHB), syringic acid (SA), NSC 611398 (MS), 2,2- join (the 3- ethyl-benzene of nitrogen-two
And thiazole -6- sulfonic acid) di-ammonium salts (ABTS) or I-hydroxybenzotriazole (HBT).
When using NSC 611398 MS as mediator, PIE5 is used for indigo decoloration, optimal reaction system after optimization are as follows: indigo
200 μM of concentration, PIE5 enzyme amount 160U/L, 120 μM of mediator MS concentration, system pH react 50min under being 7.5 in 60 DEG C, indigo
Percent of decolourization reaches 83.1 ± 1.1% (Fig. 2).
When using ABTS as mediator, PIE5 is used for indigo decoloration, optimal reaction system after optimization are as follows: indigo 200 μ of concentration
M, PIE5 enzyme amount 180U/L, 200 μM of mediator ABTS concentration, system pH react 180min, indigo decoloration under being 7.0 in 60 DEG C
Rate reaches 90.9 ± 0.3% (Fig. 3).
The present invention, to indigo decoloring ability, while comparing commercially available laccase, preferably Novi to different type fungal laccase
Letter laccase preparation compares indigo decoloring ability.The present invention joins nitrogen-two (3- ethyl-benzothiazole -6- sulfonic acid) two with 2,2-
Ammonium salt (ABTS) is laccase substrates, is changed every detecting laccase activity in fermentation process for 24 hours.Enzyme activity assay system is at 30 DEG C
Ice bath 30S immediately after 3min is reacted, light absorption value is detected 420nm at and calculates enzyme activity, the enzyme activity (U) for defining a unit is one
Enzyme amount needed for 1 μM of substrate of conversion in minute.Fungal laccase vigor is surveyed using ABTS by substrate in the present invention.
Compared with prior art, the beneficial effects of the present invention are embodied in:
Fungal laccase has a more wide application value, but its catalytic pH is generally in acid 3-5, so in alkaline environment
Under limit the application of fungal laccase.The present invention obtains a fungal laccase mutant PIE5, and most suitable catalytic pH (is more created for 8.5
The wooden phenol is substrate), and still be able to play excellent activity in pH to 9.5, therefore fungal laccase PIE5 has alkali in contrast
Property environment application advantage, widened the application field of fungal laccase, for example, the present invention fungal laccase PIE5 is used to weave it is whole
To replace traditional handicraft in bipseudoindoxyl dye decoloration in dyeing technique.Weave it is whole dye be the widely used direction of fungal laccase it
One, tradition is traditional using stone mill washing progress denim old-imitating treatment, and in dye wastewater treatment in whole dye of weaving
Use the means such as physical absorption.But these modes are not environmentally protective enough, and fungal laccase is a kind of green catalyst, can aoxidize drop
Solve indigo, but the alkaline environment for the whole dyeing technique of weaving limits the application of most of fungal laccase, and the present invention obtains alkalinity very
Bacterium laccase PIE5 can be used for weaving in the especially indigo decoloration of whole dyeing technique.Alkaline mutant PIE5 may be directly applied to weave whole
Dyeing technique is washed instead of stone mill, without adjustment wastewater pH in the indigo degradation of waste water, is beneficial to improve the spinning of fungal laccase
Whole dyeing technique is knitted, operating cost is reduced, preserves the ecological environment.
Detailed description of the invention
Fig. 1 is fungal laccase PIE5 electrophoresis detection after purification, and M is albumen Maker, and PIE5 is laccase pure protein after mixing.
As can be seen from Figure 1 target stripe PIE5 purity is higher, and size is about 61kDa.
PIE5 decolourizes indigo system optimization as a result, the influence that wherein A indicates pH to percent of decolourization when Fig. 2 is using MS as mediator,
B indicates influence of the temperature to percent of decolourization, and C indicates influence of the enzyme amount to percent of decolourization, and D indicates influence of the mediator concentration to percent of decolourization.
From figure 2 it can be seen that response parameter decolourizes to PIE5, indigo influence is very big, and the catalytic activity of pH and temperature influence enzyme make
Percent of decolourization is in first to increase the trend to tend to be steady afterwards with the increase percent of decolourization of enzyme amount and mediator concentration in the trend dropped afterwards is first increased.
PIE5 is used for indigo oxidative decoloration when Fig. 3 is using MS as mediator, and wherein A indicates the indigo percent of decolourization under different time
Variation, B indicate indigo decolorizing effect under different time.As can be seen from Figure 3 mediator MS effect under, PIE5 to it is indigo have it is bright
Aobvious decolorizing effect finds out that indigo percent of decolourization extends with the reaction time and increases from figure A, until tending towards stability after reaction 50min.
Find out from figure B and extend with the reaction time, solution colour is gradually clarified, indigo to be oxidized decoloration.
PIE5 decolourizes indigo system optimization as a result, the shadow that wherein A indicates pH to percent of decolourization when Fig. 4 is using ABTS as mediator
It rings, B indicates influence of the temperature to percent of decolourization, and C indicates influence of the enzyme amount to percent of decolourization, and D indicates mediator concentration to the shadow of percent of decolourization
It rings.Figure 4, it is seen that response parameter is very big to PIE5 indigo influence of decolourizing, pH and temperature influence the catalytic activity of enzyme,
Make percent of decolourization in the trend dropped afterwards is first increased, is in first to increase the trend to tend to be steady afterwards with the increase percent of decolourization of enzyme amount and mediator concentration.
PIE5 is used for indigo oxidative decoloration when Fig. 5 is using ABTS as mediator, and wherein A indicates the indigo decoloration under different time
Rate variation, B indicate indigo decolorizing effect under different time.As can be seen from Figure 5 under mediator ABTS effect, PIE5 being capable of oxygen
It is indigo to change decoloration, figure A shows that indigo percent of decolourization extends with the reaction time and increased, until tending towards stability after reaction 180min.It is aobvious to scheme B
Show and extend with the reaction time, solution colour is gradually clarified, indigo to be oxidized decoloration.
Specific embodiment
Term as used in the present invention generally has those of ordinary skill in the art usual unless otherwise specified
The meaning of understanding.Below in an example, the various processes and method being not described in detail are routine as known in the art
Method.
1, the preparation of fungal laccase PIE5
Bacterial strain: E.coli JM109, P.pastoris GS115
Carrier: pPIC9K
Culture medium:
YPD culture medium (1L): 20g Peptone, 10g Yeast Extract is dissolved in 900mL water, after high pressure sterilization,
10 × D of 100mL, the agar of solid medium addition 1.5% is added.
The solvent of seed fluid nutrient mediums of saccharomycete is pH value 6.0, the PBS buffer solution of 0.1M, and solute and its concentration are as follows: Yeast
Extract 10g/L, Tryptone 20g/L, YNB 13.4g/L, glycerol 10g/L, biotin 4 × 10-4g/L。
The solvent of fermentation broth is pH value 6.0, the PBS buffer solution of 0.1M, and solute and its concentration are as follows: YNB
13.4 g/L, methanol 5g/L, copper sulphate 1.5 × 10-2G/L, biotin 4 × 10-4g/L。
The acquisition of 1.1 mutant PIE5 encoding genes and the building of recombinant plasmid
Using the cDNA of Coprinus cinereus laccase Lcc9 as template, using following primer (underscore is mutational site sequence) in amino
Site mutation at E116K, N229D, I393T tri- is introduced in acid sequence.
116F:GCATTGAACCTGTACTTAAACGACTCTC
116R:GAGAGTCGTTTAAGTACAGGTTCAATGC
229F:GAACTTGTAGTCGGGATCGCACG
229R:TCGTGCGATCCCGACTACAAGTTC
393F:GGCAAAGTATGGGTCGCACCAC
393R:AGTGGTGCGACCCATACTTTGCC
PIE5F:CGGAATTCCAAATCCTTGGCCCG (the identification sequence that underscore part is Eco RI)
PIE5R:AAGGAAAAAAGCGGCCGCTTAAGGAGTGG (the identification sequence that underscore part is Not I)
Mutant PIE5 encoding gene is expanded and obtained in a manner of Standard PCR, and introduces Eco in full length sequence amplification
Restriction enzyme site at RI and Not I two.50 μ L of PCR reaction system, reaction condition are as follows: 95 DEG C of initial denaturation 2min then carry out 30
It recycles (95 DEG C of 20S, 57 DEG C of 20S, 72 DEG C of 1.5min), 72 DEG C of extension 10min, last 4 DEG C of heat preservations take 10 μ L PCR to produce after circulation
Object is detected through 1% agarose gel electrophoresis.With AxyPrep DNA gel QIAquick Gel Extraction Kit by cutting the operation such as glue for PIE5 purpose
Segment.
With two kinds of limitation restriction endonucleases of Eco RI and Not I respectively by PIE5 target fragment after the recovery and pPIC9K carrier in
Double digestion 8h in 37 DEG C of water-baths respectively takes after being recycled two groups of digestion products by AxyPrep DNA gel QIAquick Gel Extraction Kit
100ng recovery product, by the Eco of PIE5 encoding gene forward direction insertion pPIC9K in 16 DEG C of environment under T4 connection enzyme effect
Between RI and Not I restriction enzyme site, recombinant plasmid is obtained.
The building of 1.2 mutant PIE5 recombinant strains
Recombinant plasmid is transferred in E.coli JM109 competent cell in converted product coated plate is in LB (ammonia containing 100mg/L
Parasiticin) plate 37 DEG C cultivate.Random picking part monoclonal, verifies positive colony in a manner of bacterium colony PCR.By positive gram
It is grand to be inoculated in containing in 5mL liquid LB (ampicillin containing 100mg/L) test tube, it is cultivated in 37 DEG C, 200rpm.Training overnight
After supporting, centrifugation abandons supernatant and collects thallus, extracts recombinant plasmid with AxyPrep plasmid QIAquick Gel Extraction Kit.With Sac I restriction enzyme
Enzyme abundant single endonuclease digestion in 37 DEG C by the recombinant plasmid of 6 μ g obtains linearization plasmid, AxyPrep DNA gel QIAquick Gel Extraction Kit
Linearization plasmid is recycled.
The recombinant plasmid linearized after recycling is transferred in P.pastoris GS115 competent cell in such a way that electricity turns, in
It is cultivated in 28 DEG C with YPD plate, positive colony is selected with the BMM detection plate containing ABTS, screening obtains PIE5 recombination
Express bacterial strain.The recombinant bacterial strain of acquisition is chosen into BMGY culture medium and is stored in 15% after 28 DEG C, 200rpm CMC model
In glycerol tube, it is placed in -80 DEG C to protect bacterium.
1.3 fermented and cultured
Under the conditions of 28 DEG C, 200rpm, by the strain inoculated being stored in glycerol tube in the MGY culture medium of liquid B containing 50mL
250mL shaking flask in culture to Fungal biodiversity OD600It is worth to 2.0-6.0.It is centrifuged thallus, with the training of 100mL fermentation liquid BMM liquid
It supports base weight and hangs thallus, until thallus OD600Value is 1.0, continues culture and makes wherein methanol concentration every addition methanol for 24 hours
0.5%, and with laccase PIE5 vigour changes in ABTS detection fermentation liquid.
ABTS detection architecture are as follows: take the 950 μ L of sodium tartrate buffer of pH4.0, it is female that the ABTS that concentration is 0.5mM is added
17 μ L laccase liquid are added after preheating 3min in 30 DEG C of water-baths in 33 μ L of liquid.After reaction system reacts 3 min at 30 DEG C after mixing
Ice bath 30S immediately detects light absorption value at 420nm and calculates enzyme activity, and the enzyme activity (U) for defining a unit is to convert in one minute
Enzyme amount needed for 1 μM of substrate.
1.4 crude enzyme liquids obtain
When continuous detection laccase activity is begun to decline, terminates to ferment and collect fermentation liquid, be centrifuged at 4 DEG C, 8000g
30min abandons precipitating and collects supernatant 1L, is PIE5 crude enzyme liquid.Using 350mL specification ultrafiltration apparatus under the conditions of 4 DEG C, 0.3MPa
Ultrafiltration concentration is carried out to crude enzyme liquid, fungal laccase PIE5 is obtained and crude enzyme liquid 20mL is concentrated.
PH 6.5 is prepared, concentration is disodium hydrogen phosphate-citric acid elution buffer 3L of 20mM, and crude enzyme liquid is concentrated in PIE5
It is placed in bag filter.In 4 DEG C of environment, bag filter is placed in buffer and carries out dialysis replacement laccase buffer system.Every 5h
An elution buffer is replaced, continuous dialysis three times, obtains laccase PIE5 crude enzyme liquid about 21mL.
1.5 fungal laccase PIE5 purifying
With anion-exchange column: DEAE-Sepharose Fast Flow purifies fungal laccase, prepares purification solution:
Less salt: 20mM disodium hydrogen phosphate-citric acid pH 6.5；
It is with high salt: the ammonium sulfate of 150mM being added in low salt buffer；
NaCl: concentration is the NaCl solution 500mL of 1M；
NaOH: concentration is the NaOH solution 500mL of 1M；
Ethyl alcohol: volume fraction is 20% ethanol solution 1L；
H2O: ultrapure water 1L.
Ultrasonic degasification after the solution completed is filtered will be prepared.Specific step is as follows for protein purification:
1. conditioning instrumentation flow velocity 1.0mL/min installs anion-exchange column, cylinder 40min is rinsed using 20% ethyl alcohol；
2. rinsing cylinder, H is successively replaced2O→NaCl→H2O→NaOH→H2O → with high salt → H2O → less salt is replaced every time
Rinse 40min.
3. adjusting high salt concentration, successively from low to high (0%-100%) by the PIE5 crude enzyme liquid loading after dialysis into column
Elution albumen is carried out, and collects eluent.
4. detection: pure with the albumen that 15% SDS- polyacrylamide gel (SDS-PAGE) detection is collected in eluent
Degree collects the purer eluent of target protein, spare after mixing (Fig. 1).
2, laccase PIE5 is used for indigo decoloration
5.25g indigo, 4g sodium hydroxide, 2g thiourea dioxide are weighed into 1L water, mixed liquor is heated to 50 DEG C of dissolutions
Prepare indigo solution.With dimethyl sulfoxide (DMSO) for solvent, prepares mediator NSC 611398 (MS) and ABTS mother liquid concentration is
10mM。
Construct the indigo initial reaction system 1mL of laccase oxidative decoloration: indigo 200 μM, PIE5 enzyme activity 200U/L, mediator concentration
100 μM, with the citrate-phosphate salt buffer polishing of pH7.0 to 1mL.System detects at 700nm after 40 DEG C of reaction 3h
The light absorption value variation of reaction front and back, and calculate percent of decolourization, optimize using percent of decolourization as evaluation criterion and establish the indigo decoloration of laccase oxidation
System.Percent of decolourization calculation formula is as follows:
Percent of decolourization=(A0-A1)/A0× 100%
A0For the initial light absorption value of reacting precursor system, A1For system light absorption value after reaction.
2.1 are used for indigo decoloration by mediator of MS
When using NSC 611398 MS as mediator, PIE5 is used for indigo decoloration, it is right respectively in the optimization of reaction system
It influences the parameters such as temperature, enzyme amount, pH, the mediator concentration of decolorizing effect to be optimized, using percent of decolourization as evaluation criterion, after optimization
Optimal reaction system are as follows: 200 μM of indigo concentration, PIE5 enzyme amount 160U/L, 120 μM of mediator MS concentration, system pH be 7.5 under
50min is reacted in 60 DEG C, indigo percent of decolourization reaches 83.1 ± 1.1% (Fig. 2-3).
2.2 are used for indigo decoloration by mediator of ABTS
When using ABTS as mediator, PIE5 is used for indigo decoloration, in the optimization of reaction system, is decolourized respectively to influence
The parameters such as temperature, enzyme amount, pH, the mediator concentration of effect are optimized, using percent of decolourization as evaluation criterion, optimal reaction after optimization
System are as follows: 200 μM of indigo concentration, PIE5 enzyme amount 180U/L, 200 μM of mediator ABTS concentration, system pH are anti-in 60 DEG C under being 7.0
180min is answered, indigo percent of decolourization reaches 90.9 ± 0.3% (Fig. 4-5).
SEQ ID No:1
MSRKLFSLAYLAVVLVSVAGQILGPTSTMTVSNIDASPDGFNRPVVAVNGQHPGPLVRANK GDNFRI
NVVNDLNDPTMLRQTSVHWHGVFQHGTAWADGPDGVTQCPIAQNGESFKYRFN AGNEAGTFWYHSHFGTQYCDGL
RGPLVIYDPNDPHRNLYDVDNADTVITLVDWYHLQAPS IEGPALSDATLINGKGRRPGGPETDIAIVNVQRNRRY
RFRLVSMSCDPDYKFSIDGHKLTVIE ADGQLTEPLMVDEIQIFAGQRYSFVLSANRPVGNYWIRAIPNVGSNNLP
NFSSGGINSAILRY AGAPNANPTSTPVTNPVALHESNLHALLNPGAPGGSGPADENIVLQMGLGPAGFEINGVTW
ANPDSPVMVQIMNGVPPADIVPSGATHTLPRNRVVEVSIPGFELAGPHPFHLHGHAFSVVRS AGSSTYNYENPVR
RDVVDVGGASDNVTIRFTTDNPGPWFFHCHIEFHLVLGLAMVFMEAP SDIPSTSPPPPSWSELCPKFESLPASAT
SIQIVPTP。
SEQUENCE LISTING
<110>University of Anhui
<120>a kind of fungal laccase mutant PIE5 and its expression bacterial strain and application
<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 (10)
1. a kind of fungal laccase mutant PIE5, it is characterised in that:
It is to be transformed with directed evolution means to Coprinus cinereus laccase Lcc9, constructs mutated library, in conjunction with three-wheel screening technique,
The Laccase mutant of acquisition, to improve the application power of laccase.
2. fungal laccase mutant PIE5 according to claim 1, it is characterised in that: its amino acid sequence such as SEQ ID
Shown in No:1.
3. fungal laccase mutant PIE5 according to claim 1, it is characterised in that: its amino acid sequence is SEQ ID
No:1 is substituted, lacks or adds one or several amino residues and encodes the amino acid sequence of identical function protein.
4. a kind of bacterial strain for expressing fungal laccase mutant PIE5 described in claim 1,2 or 3, it is characterised in that:
The classification naming of the bacterial strain is Pichia pastoris pPIC9K-PIE5, is preserved in China typical culture collection
Center, the deposit date is on July 19th, 2018, deposit number was CCTCC NO:M 2018451.
5. a kind of purposes of fungal laccase mutant PIE5 described in claim 1,2 or 3, it is characterised in that:
Application of the fungal laccase mutant PIE5 during dye decolored as decoloration oxidant.
6. a kind of application of fungal laccase mutant PIE5 described in claim 1,2 or 3, it is characterised in that:
The fungal laccase mutant PIE5 is used during dye decolored as decoloration oxidant, specifically includes following step
It is rapid:
In pH value of reaction system 4.5-9.0,20-80 DEG C of temperature, laccase PIE5 concentration 40-200U/L, 20-300 μM of mediator concentration
Under conditions of, decolorization is carried out to dyestuff, indigo percent of decolourization reaches 83% or more.
7. application according to claim 6, it is characterised in that:
The mediator is syringaldehyde, syringic acid, NSC 611398,2,2- connection nitrogen-two (3- ethyl-benzothiazole -6- sulfonic acid) two
Ammonium salt or I-hydroxybenzotriazole.
8. application according to claim 6, it is characterised in that:
The dyestuff is bipseudoindoxyl dye.
9. application according to claim 7 or 8, it is characterised in that:
When using NSC 611398 as mediator, PIE5 is used for indigo decoloration, 200 μM of indigo concentration, is situated between PIE5 enzyme amount 160U/L
120 μM of body MS concentration, system pH react 50min under being 7.5 in 60 DEG C, and indigo percent of decolourization reaches 83.1 ± 1.1%.
10. application according to claim 7 or 8, it is characterised in that:
When being mediator with 2,2- connection (3- ethyl-benzothiazole -6- sulfonic acid) di-ammonium salts of nitrogen-two, PIE5 is used for indigo decoloration,
200 μM of indigo concentration, PIE5 enzyme amount 180U/L, 200 μM of mediator ABTS concentration, system pH react in 60 DEG C under being 7.0
180min, indigo percent of decolourization reach 90.9 ± 0.3%.
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| CN113930402A (en) * | 2021-12-21 | 2022-01-14 | 中国农业科学院北京畜牧兽医研究所 | Method for improving laccase catalytic activity, mutant Lcc9-M2, gene and application |
| CN114891757A (en) * | 2022-06-13 | 2022-08-12 | 安徽大学 | Laccase directed evolution method, mutant laccase gene, expression vector, recombinant strain and application of mutant laccase gene in detoxification of aflatoxin |
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