CN107227302A - The bacillus pumilus CotA Laccase mutants that a kind of amount of soluble expression is improved - Google Patents
The bacillus pumilus CotA Laccase mutants that a kind of amount of soluble expression is improved Download PDFInfo
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- CN107227302A CN107227302A CN201710437791.8A CN201710437791A CN107227302A CN 107227302 A CN107227302 A CN 107227302A CN 201710437791 A CN201710437791 A CN 201710437791A CN 107227302 A CN107227302 A CN 107227302A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- 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)
- C12N9/0061—Laccase (1.10.3.2)
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
- C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
- C12Y110/03002—Laccase (1.10.3.2)
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/308—Dyes; Colorants; Fluorescent agents
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Abstract
The invention discloses bacillus pumilus (Bacillus pumilus W3) CotA Laccase mutants that a kind of amount of soluble expression is improved, belong to bioengineering field.501st Asp of complex mutation body is further sported Gly by complex mutation body L386W/G417L/G57F (WLF) the CotA laccase genes that the catalytic efficiency (ABTS is substrate) that the present invention is built using this laboratory early stage is greatly improved as template.The amount of soluble expression of bacillus pumilus CotA Laccase mutants of the present invention is 3.75 times of WT CotA, is 4.48 times of WLF, and catalytic efficiency is 4.4 times of WT CotA, is 0.993 times of WLF, and keeps good pH, temperature stability.Gained CotA Laccase mutants of the invention will be more suitable for industrial applications, with broader practice prospect.
Description
Technical field
The present invention relates to the bacillus pumilus CotA Laccase mutants that a kind of amount of soluble expression is improved, belong to biological work
Journey technical field.
Background technology
Laccase (Laccase, E.C.1.10.3.2) is a kind of cupric polyphenol oxidase, and the oxidation of energy phenol substance catalytic is also
Original reaction, plays a significant role in the biodegradation of lignin and its precursor analog.The oxidation substrates of laccase are extremely wide
It is general, including phenols and its derivative, arylamine and its derivative, aromatic carboxylic acids and its derivative etc., therefore laccase application potential is huge
Greatly.In wood processing field, laccase can replace chemical adhesive, can not only improve product quality, and can mitigate strong to human body
The injury of health and the pollution to environment；In paper industry, laccase is used for paper bio-bleaching and slurrying, can reduce slurrying and make
The pollution of paper plant, contributes to paper-making industry finally to realize clean manufacturing；In food processing field, laccase can be used for removing in fruit juice
It is muddy caused by phenolic compound, so as to improve the quality of fruit juice.In addition, the also oxidable chlorophenol of laccase and its derivative, reduction
Its toxicity, reduce by the raw material of industry of chlorophenols production dyestuff, preservative, herbicide, agrochemical chemical products and cause
Environmental pollution.
By sources difference can be divided into four major classes to laccase：Plant laccase, insect laccase, fungal laccase and bacterial laccase.Plant
Laccase is mainly isolated by Che Shu lacquer liquid.Fungal laccase is originated more extensive (being present in a variety of basidiomycetes), has single electron
The advantages of oxidation-reduction potential is high, catalytic activity is strong, can be catalyzed the oxidation polymerization of substrate can carry out catalysis drop to lignin again
Solution, is the laccase species of people's primary study all the time.Bacterial laccase then find it is relatively later, be initially 1993 by
Giva μ dan et al. identify what is come in a kind of raw fat azospirillum first.Then, scientific research personnel is again successively in Alteromonas
Bacterium, Escherichia coli, pseudomonad, serratia marcescens, Bacillus sphaericus, Bacillus subtillis, extremely alkaline-resisting brood cell's bar
The functional protein of different types of tool laccase activity, such as withered grass/lichens/short and small bud are found that in the bacteriums such as bacterium, streptomyces griseus
PpoA albumen, C μ eO albumen, the EpoA albumen of streptomyces griseus of Escherichia coli of the CotA albumen of born of the same parents bacillus, extra large monad
Deng these bacterial laccases are similar to fungal laccase protein structure, all with 4 copper ion binding sites.In these laccases,
CotA laccases are most paid close attention to by researcher, wherein the most study of Bacillus subtillis CotA laccases, most deep.
Due to originated from fungus laccase, in working environment pH meta-alkalescences, activity is very low or almost do not have, heat endurance also compared with
Difference, and filamentous fungi growth cycle is long, culture medium requires high, and mycelia is vulnerable to the damage of high shear force in fermentation tank, and this is big
Limit the application of fungal laccase industrially greatly.Research is disclosed, though bacterial origin laccase oxidation activity is generally slightly below fungi
Source laccase, but they often have the advantages that some itself uniquenesses：As without glycosylation modified, heat endurance is good, enzyme activity
Property optimal pH scope it is wide etc., the exactly current laccase commercial Application of these properties is badly in need of.But the table of bacterial laccase in itself
It is low compared with fungal laccase up to measuring, and inclusion body is easily formed in Escherichia coli, the renaturation of inclusion body is relatively difficult, expends material resources
Financial resources.In recent years, site-directed mutagenesis technique was applied on the solubility expression for improving albumen, and obtained good result, and laccase can
The increase of dissolubility expression quantity, is beneficial to its application industrially.
The annual dyeing waste water discharge capacity of China accounts for the 35% of total discharged volume of industrial waste water, and oneself turns into the maximum important dirt of harm
Dye source, most of synthetic dyestuffs are all difficult to be degraded by microorganisms.These dye compositions are normally used for weaving, food, plastic cement
With biomedical colouring agent, annual whole world commercial dyes are used up to 7 × 105Ton, species is up to 1 × 104Kind, wherein 5~
10% dyestuff is all emitted in trade effluent form.Coloured sewage is directly released into environment, and many dyestuffs are in anaerobism ring
Noxious material or carcinogen may be converted under border, numerous countries have all promulgated severe regulation to limit industrial dye in succession
Expect the discharge of sewage.Due to the special chemical constitution of dyestuff itself, physically or chemically method (condensation, ozone, activated carbon) place is used
Often effect is not good for reason, and also easily causes secondary pollution.Research shows that bioanalysis (using laccase, manganese peroxidase etc.) has
Have that percent of decolourization is high, operating cost is low, environmental protection advantage, be the potential effective means for handling dyeing waste water, be current printing and dyeing
The focus of waste water decoloring research.Often temperature is very high and pH value meta-alkali for the industrial dyeing and printing sewages of overwhelming majority discharge.All kinds of next
In the laccase in source, fungal laccase is difficult to due to only having preferable decolorizing effect under pH meta-acid environment under the conditions of meta-alkalescence
Play a role, and heat resisting temperature is less than bacterial laccase, so bacterial laccase is handled by its unique advantage in dyeing wastewater
In it is more with potential applications.
Therefore, resistant against high temperatures and the bacterial laccase gene and tool of accomplishing scale production of high ph-values (alkaline-resisting) condition are excavated
There are important research meaning and application value.This laboratory research simultaneously reports the bacillus pumilus CotA that a kind of activity is improved
Laccase mutant (WLF), carries out transformation and obtains one plant of performance stabilization, the restructuring that amount of soluble expression is improved is dashed forward on this basis
Modification D 501G/WLF.
The content of the invention
The problem to be solved in the present invention is to provide a kind of function admirable, the Laccase mutant that amount of soluble expression is improved
D501G/WLF.The mutant WLF that the catalytic activity that the mutant is built using this laboratory early stage is greatly improved enters one as template
Step by WLF the 501st negatively charged aspartic acid of polarity (Asp, D) sport the uncharged glycine of polarity (Gly,
G), compared with wild type B.pumilus W3 CotA (WT) and WLF CotA, find the increase of its amount of soluble expression, and
Zymologic property is stable.
In the original parent amino acid sequence and ncbi database of the B.pumilus W3 CotA laccases
B.pumilus W3 CotA laccases consensus amino acid sequence (submit, GenBank accession number by this laboratory：KF040050).
Described the 501st Aspartic acid mutations of bacillus pumilus Laccase mutant D501G/WLF are glycine, nucleotides
Sequence such as SEQ ID NO.1, its amino acid sequence such as SEQ ID NO.2.
The present invention also provides a kind of method for obtaining the Laccase mutant, it is characterised in that pass through sequence alignment first
501 mutational sites are determined, then using-cotA (WLF) plasmids of pCold II as template, primer is designed, is pinpointed by PCR
Mutation, obtains, containing-the cotA of recombinant plasmid pCold II (D501G/WLF) after mutation, turning DMT competence and (being purchased from
TransGen companies), bacterium colony PCR checkings preserve glycerol tube and are sequenced.By the correct bacterium activation of sequencing result, extract plasmid turn
E.coli BL21 (DE3) competence, and bacterium colony PCR checkings are carried out, by -80 DEG C of preservation glycerine of the correct bacterial strain of gene size
Pipe.
The present invention also provides a kind of method for producing the soluble laccase of above-mentioned D501G/WLF, and being deposited in of building is sweet
Purpose bacterial strain in oil pipe connects the activation of 3mL LB test tubes, and 37 DEG C of 200rpm incubated overnights take the bacterium solution of 1mL activation to be inoculated into and contained
There is the shaking flask of 50mL culture mediums, cultivate 2-3 hour (OD ≈ 0.5), shaking table is adjusted to 15 DEG C of static gas wave refrigerator 30min, is subsequently added into
Final concentration of 0.4mM IPTG and 0.25mM CuSO4 induced expression 24h in 15 DEG C of 200rpm shaking table.Collect fermentation
Liquid, 4 DEG C of 8000rpm centrifuge 10min, abandon supernatant, are resuspended with the phosphate buffers of pH 7.0, and ultrasonication can obtain laccase supernatant
Liquid.Because the CotA laccase proteins of recombination expression carry histidine-tagged (His6Tag), thus use the affine layer of nickel ion
Analysis method separates target protein.Using AKTA avant25 protein purification systems through steps such as overbalance, loading, elutions, finally may be used
The CotA laccases purified.
Brief description of the drawings
Fig. 1 is bacterial laccase and fungal laccase C- end sequence comparison charts.
Fig. 2 is rite-directed mutagenesis principle schematic.
Fig. 3 is the Recombinant protein expression of B.pumilus W3 CotA laccases, the SDS-PAGE collection of illustrative plates of purifying；Wherein
M：Protein molecular weight standard (kDa)；1：WLF supernatants；2：D501G/WLF supernatants；3：WT is purified；4：WLF is purified；5：
D501G/WLF is purified.
Fig. 4 is that B.pumilus W3 CotA recombinate laccase and the optimal reaction pH (A) and pH stability of Laccase mutant
(B) analyze.
Fig. 5 is that B.pumilus W3 CotA recombinate laccase and the optimal reactive temperature (A) and temperature stabilization of Laccase mutant
Property (B, C, D) analysis.
Fig. 6 is that B.pumilus W3 CotA recombinate laccase and the analysis of Laccase mutant high salt concentration stability inferior.
Fig. 7 is that B.pumilus W3 CotA recombinate laccase and Laccase mutant by amboceptor of acetosyringone in alkaline bar
To a kind of azo, a kind of anthraquinone, 2 kinds of triphenylmenthanes and a kind of decolorization of heteroaromatic dyestuff under part.
Embodiment
Used term, unless otherwise specified, typically has those of ordinary skill in the art usual in the present invention
The implication of understanding.
Embodiment and Application Example are prepared with reference to specific, and this hair is described in further detail with reference to data
It is bright.It should be understood that these embodiments are of the invention solely for the purpose of illustration, rather than the scope of the present invention is limited in any way.
Below in an example, the various processes and method not being described in detail are conventional methods as known in the art.
Material and reagent：ABTS, ampicillin etc. are purchased from Sigma-Aldrich Sigma-Aldrich (Shanghai) trade
Co., Ltd；Plasmid extraction kit, site-directed mutagenesis kit are purchased from Beijing Quan Shi King Companies；Other reagents be it is domestic or
The AR of person's foreign procurement；E. coli bl21 (DE3) bacterial strain is purchased from precious biotech firms of Chinese T akara etc..
The structure of the B.pumilus W3 Laccase mutants of embodiment 1
Rite-directed mutagenesis principle：Point mutation principle introduces mutational site, PCR S μ perMix synthesis mutation chain (figures using primer
2).The restructuring Laccase mutant WLF successfully constructed using early stage CotA laccase genes sequence designs primer, by laccase as template
In the 501st aspartic acid (Asp, D) sport glycine (Gly, D).The related forward primer and reverse primer of design are such as
Under：
- the AACACGAGGATTAT of leading F 5 'GGCATGATGCGGCC-3’
After draw R 5 '-CCATAATCCTCGTGTTCTAATATGTGAC-3’
Wherein underscore part represents the codon corresponding to 501 glycine of mutant gene coding respectively.PCR expands
Increasing system is：The μ L of DNA 3, the μ L of leading (10 μM) 1, after draw (10 μM) 1 μ L, PCR S μ perMix 25 μ L, ddH2O mend to
50 μ L, PCR amplification conditions are 94 DEG C of denaturation 4min, circulate 25 times (94 DEG C of 20s, 55 DEG C of 20s, 72 DEG C of 3min), last 72 DEG C are prolonged
Stretch 10min.5 μ L PCR primers are taken to be detected through 1% agarose gel electrophoresis, and it is correct to detect purpose band.By 1 μ L DMT
Enzyme is added in remaining PCR primer, is mixed, and 37 DEG C are incubated 1 hour, adds 3 μ L DMT enzymic digestions products in 100 μ L competence
In cell, mixing, ice bath 30 minutes are flicked.42 DEG C of water-bath thermal shock 45s, are immediately placed on 2min on ice；Then plus 900 μ L balance
To the LB culture mediums of room temperature, 37 DEG C of 200rpm cultivate 1h, and converted product finally is coated on into L containing 100mg-1Ampicillin
LB flat boards, through 37 DEG C of incubated overnights, 10 single bacterium colonies are selected from flat board and carry out bacterium colony PCR checkings, from the bacterium colony being proved to be successful
In choose 5 single bacterium colonies and be inoculated into after LB fluid nutrient mediums, 10h each single bacterium colony is preserved into 2 glycerol tubes, a -80 DEG C of guarantors
Hide, portion is used to be sequenced.Will the correct mutant of sequencing from glycerol tube be inoculated into LB fluid nutrient mediums in activated overnight, afterwards
Glycerol tube is first preserved, then remaining bacterium solution extracts plasmid, and converts BL21 (DE3) competent cell.
The expression and purification of the B.pumilus W3 laccases of embodiment 2
Expression：WLF and D501G/WLF weights that wild type restructuring laccase expression bacterial strain and early stage are built are inoculated with from glycerol tube
Group expression bacterial strain is activated into LB culture mediums, and 37 DEG C, 200rpm is stayed overnight (10h).Seed is accessed respectively by 2% inoculum concentration
50mL LB liquid fermentation mediums (L containing 100mg-1Ampicillin) 37 DEG C of 200rpm shaking table cultures are to OD600Reach 0.5,
Then shaking table temperature is adjusted to 15 DEG C of static gas wave refrigerator 30min, is subsequently added into final concentration 0.4mM IPTG and 0.25mM CuSO4 progress
Induction, 15 DEG C of 200rpm cultivate 24h, and zymotic fluid is removed into supernatant in 4 DEG C of 8000 rpm centrifugations 10min, thalline is collected.It will collect
Thalline be resuspended with phosphate buffer, bacterial cell disruption is discharged into intracellular protein with ultrasonic cell disruption instrument after resuspension, crush
After the completion of, broken liquid is centrifuged into 20min (4 DEG C, 8000rpm), then by supernatant in 70 DEG C of heating water bath 15min,
4 DEG C of centrifugation 10min of 10000rpm go precipitation, collect supernatant.The supernatant of collection is purified for CotA laccase proteins.
Purifying：Because the CotA laccases of recombination expression carry polyhistidine label (His6Tag), thus using nickel from
Sub- affinity chromatography separates target protein.The step of nickel ion affinity chromatograph purifying protein：(1) balance：Volume is lived with 10 times
20mM buffer solutions (imidazoles containing 5mM) balance HisTrap HP nickel ions posts (1mL)；(2) loading：The sample anticipated
With 1mL min-1Flow velocity loading；(3) elute：Gradient elution is carried out with high concentration imidazoles, peak type correspondence under elution requirement is collected
Pipe number, and do Enzyme activity assay, collect the unimodal corresponding albumen for having an enzyme activity, run SDS-PAGE protein electrophoresises confirmation form one
Band, obtains the enzyme of purifying.
The enzyme activity determination and expression analysis of the B.pumilus W3 laccases of embodiment 3
(1) enzyme-activity unit is defined：When determining laccase activity using ABTS methods, define 1 μm of ol substrate of catalysis per minute and turn
Enzyme amount needed for turning to product is a unit of activity.
(2) enzyme activity determination step：1 preheating：2.4mL pH4.0 citrate buffer solution is taken in test tube, is added in test tube
Enter 0.5mL ABTS solution (the final concentration of 0.5mM of ABTS) and be placed in 50 DEG C of water-baths to preheat 5min；2 reactions：Addition dilutes
0.1mL sample enzyme liquids, concussion is uniform.3 measurements：Kinetic measurement is carried out to shaking uniform sample with spectrophotometer,
The variable quantity (measurement reaction shows straight line) of OD values per minute in 30s is measured under 420nm wavelength and enzyme activity is calculated.
(3) Determination of Kinetic Parameters：The kinetic parameter of pure enzyme is determined using the ABTS of various concentrations as substrate.Reaction
System is 3mL, final concentration of 10-1000 μM of ABTS.3mL reaction system is slow including 2.4mL disodium hydrogen phosphates-citric acid
Fliud flushing (100mM, pH3.6), the pure enzyme liquids of 0.1mL, 0.5mL ABTS solution (be made into respectively 10 μM of final concentration, 20 μM, 40 μM, 60
μM、80μM、100 μM、200μM、300μM、400μM、500μM、1000μM).Reaction system is placed in 50 DEG C of water-baths in advance
Heat, the then enzyme-added variable quantity (reaction rate at the uniform velocity reaction) that OD values in 30s are determined under 420nm wavelength.According to enzyme activity
Formula calculates enzyme activity.
Enzyme activity formula：
In formula：The poor V of △ OD- reaction time internal absorbance valuesAlwaysThe volume (L) of-reaction system
N- enzyme liquid extension rate △ t- reaction time (min)
Volume (L) m of Vo- enzyme liquidsEnzymeThe quality (mg) of-zymoprotein
ε-substrate molar extinction coefficient, ε420=3.6 × 104L·mol-1·cm-1
The kinetic parameter of wild type, mutant WLF and mutant D501G/WLF laccases is determined by substrate of ABTS
Vmax、Km、 kcat、kcat/Km, Rate activity, total activity and protein content.As a result it is as follows：
The kinetic parameter and expression quantity of the wild type of table 1 (WT) and mutant laccase
Note：Total activity (Vol μm of etric Activity) concentrates 5 times.
As it can be seen from table 1 mutant D501G/WLF catalytic efficiency is higher than WT, slightly below WLF, but soluble table
It is significantly improved up to amount D501G/WLF.
B.pumilus W3 restructuring laccases and the most suitable action pH of Laccase mutant and pH stability analyses that embodiment 4 is purified
Using conventional determining method (having document to refer to), using ABTS as substrate, reaction temperature is 50 DEG C.1
Individual enzyme-activity unit is defined as the enzyme amount needed for 1 minute internal oxidition, 1 μM of substrate.Experimental result fully shows restructuring laccase and laccase
Mutant all has the advantages that long-time alkali resistance environment is good, although mutant D501G/WLF pH stability is poor compared with WT and WLF
Some, it is but still very stable, belong to stability very strong enzyme (as shown in Figure 4) compared to more other kinds of enzyme.
B.pumilus W3 restructuring laccases and Laccase mutant optimum temperature and temperature stability that embodiment 5 is purified
Analysis
Using conventional determining method, using ABTS as substrate, pH3.6 is reacted.1 enzyme-activity unit was defined as in 1 minute
Aoxidize the enzyme amount needed for 1 μM of substrate A BTS.Experimental result fully shows that restructuring laccase and Laccase mutant have resistance to height for a long time
The good advantage of warm environment (as shown in Figure 5).
B.pumilus W3 restructuring laccases and Laccase mutant Salt Tolerance Analysis that embodiment 6 is purified
WT, WLF, D501G/WLF laccase are all stored in the NaCl solution of three kinds of various concentrations respectively first (100mM,
500mM, 1M), 4 DEG C of refrigerator 10h are stored in, enzyme activity, reaction system is then surveyed by substrate of ABTS under the conditions of pH3.6,50 DEG C
Enzyme activity determination method in 3mL, method be the same as Example 3.As a result experimental result as shown in fig. 6, show that restructuring laccase and laccase are prominent
Variant has good stability under high salt concn.
The B.pumilus W3 of embodiment 7 recombinate laccase and Laccase mutant in the basic conditions to azo, Anthraquinones,
The percent of decolourization of triphenylmethane and heteroaromatic class dyestuff is determined
Using conventional determining method：Reaction system is 5mL, dyestuff 0.25mg, the μ g of laccase 10 after purification, and amboceptor is acetyl
Syringone, mediator concentration 1mM, reaction temperature is 37 DEG C, and pH10, buffer solution is carbonic acid buffer.Experimental result is shown in alkalescence
After environment pH10,10 h of effect, three kinds of restructuring laccases are respectively reached to a kind of azo dyes Acid red 1 percent of decolourization
86.5%th, 93.3% and 92.1%, respectively reach 80.9% to a kind of anthraquinone dyes Acid bl μ e 129 percent of decolourization,
85.4% and 95.6%, also equally there is good decolorizing effect to triphenylmethane and heteroaromatic class dyestuff, to aromatic series
The percent of decolourization of heterocyclic dyestuff will be less than other several types dyestuffs (as shown in Figure 7).Data above shows that the restructuring laccase exists
Good decolorizing effect is respectively provided with to azo dyes and anthraquinone dyes and triphenylmethane dye under alkaline environment, had
Larger application potential.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, can do various changes and modification, therefore the protection of the present invention
What scope should be defined by claims is defined.
Sequence table
<110>Southern Yangtze University
<120>The bacillus pumilus CotA Laccase mutants that a kind of amount of soluble expression is improved
<160> 2
<210> SEQ ID No.1
<211> 1533bp
<212> DNA
<213>Designed according to gene order, for gene expression.
<400> SEQ ID No.1
1 ATGAACCTAG AAAAATTTGT TGACGAGCTG CCAATTCCAG AAGTCGCAGA GCCCGTCAAA
61 AAAAACCCAA GACAAACGTA TTATGAAATC GCTATGGAGG AGGTGTTCCT AAAAGTTCAT
121 AGTGATCTGC CCCCAACCAA GTTATGGACC TATAATGGCG GTTTGCCTTT TCCAACCATT
181 AAAGCGAATC GAAATGAAAA GGTCAAAGTG AAATGGATGA ACAAATTGCC GCTGAAACAC
241 TTCCTGCCTG TCGATCATAC CATTCACGCT GGACACCATG ATGAACCCGA AGTCAAAACA
301 GTCGTTCACC TGCATGGGGG CGTGACACCT GCAAGCAGTG ACGGTTATCC AGAGGCTTGG
361 TTTTCGCGAG ACTTTGAAGC GACCGGCCCC TTCTTTGAAC GAGAGGTTTA CGAATACCCT
421 AATCACCAGC AAGCATGCAC ATTGTGGTAT CACGATCATG CCATGGCATT AACACGATTA
481 AATGTATACG CCGGATTAGC TGGCTTTTAT TTGATCTCAG ATGCGTTTGA AAAATCACTC
541 GAATTACCGA AAGATGAATA TGATATCCCT TTAATGATCA TGGACCGGAC ATTCCAGGAG
601 GATGGCGCAC TATTTTATCC AAGTAGACCA AATAACACAC CAGAAGATAG TGACCTTCCA
661 GATCCCTCTA TCGTGCCATT TTTTTGCGGG GAAACGATTT TAGTCAATGG AAAAGTATGG
721 CCATATTTAG AAGTAGAGCC TCGAAAATAT CGTTTTCGTA TCTTAAACGC GTCCAATACA
781 AGAACTTACG AGCTTCATCT AGATAACGAC GCCACAATCT TACAAATTGG ATCTGATGGC
841 GGCTTTTTGC CAAGACCTGT TCATCACCAA TCTTTTAGCA TTGCACCTGC TGAACGTTTT
901 GATGTCATCA TCGACTTCTC AGCTTACGAA AACCAAACGA TTGTTTTAAA AAATAAGGCA
961 GGCTGCGGTC AGGAAGTCAA TCCAGAAACA GATGCGAACA TTATGCAATT TAAAGTCACT
1021 CGACCGCTCA AAGGAAGAGC AGCTAAAACA TTACGTCCGA TCTTCAAACC ACTTCCACCA
1081 CTCCGGCCGA GCCGAGCTGA TAACGAGCGA ACGCTGACCC TTACTGGCAC ACAAGATAAA
1141 TATGGGCGCC CTATTTGGTT ACTAGATAAC CAGTTTTGGA ATGATCCTGT TACGGAAAAT
1201 CCTCGACTTG GCAGTGTAGA GGTATGGAAC ATCGTTAACC CAACAAGGCT CACACACCCT
1261 ATTCATTTAC ATCTTGTTCA ATTTCGGGTG ATTGATAGAA GACCATTCGA TACAGACATC
1321 TATCAATCAA CAGGTGAAAT CGTGTACACG GGACCAAATG AAGCGCCTCC TTTGCATGAA
1381 CAAGGATACA AGGATACAAT TCAGGCGCAT GCCGGTGAAG TCATTCGCAT CATCGCTCGG
1441 TTTGTCCCAT ATAGCGGACG GTATGTGTGG CATTGTCACA TATTAGAACA CGAGGATTAT
1501 GGCATGATGC GGCCGATGGA TATCATCCAG TAA
<210> SEQ ID No.2
<211> 510
<212> PRT
<213>Bacillus pumilus W3 mutant D501G/WLF (B. pumilus W3 D501G/WLF)
1 M N L E K F V D E L P I P E V A E P V K
21 K N P R Q T Y Y E I A M E E V F L K V H
41 S D L P P T K L W T Y N G G L P F P T I
61 K A N R N E K V K V K W M N K L P L K H
81 F L P V D H T I H A G H H D E P E V K T
101 V V H L H G G V T P A S S D G Y P E A W
121 F S R D F E A T G P F F E R E V Y E Y P
141 N H Q Q A C T L W Y H D H A M A L T R L
161 N V Y A G L A G F Y L I S D A F E K S L
181 E L P K D E Y D I P L M I M D R T F Q E
201 D G A L F Y P S R P N N T P E D S D L P
221 D P S I V P F F C G E T I L V N G K V W
241 P Y L E V E P R K Y R F R I L N A S N T
261 R T Y E L H L D N D A T I L Q I G S D G
281 G F L P R P V H H Q S F S I A P A E R F
301 D V I I D F S A Y E N Q T I V L K N K A
321 G C G Q E V N P E T D A N I M Q F K V T
341 R P L K G R A A K T L R P I F K P L P P
361 L R P S R A D N E R T L T L T G T Q D K
381 Y G R P I W L L D N Q F W N D P V T E N
401 P R L G S V E V W N I V N P T R L T H P
421 I H L H L V Q F R V I D R R P F D T D I
441 Y Q S T G E I V Y T G P N E A P P L H E
461 Q G Y K D T I Q A H A G E V I R I I A R
481 F V P Y S G R Y V W H C H I L E H E D Y
501 G M M R P M D I I Q *
Claims (6)
1. bacillus pumilus (Bacillus pumilus W3) CotA Laccase mutants that a kind of amount of soluble expression is improved,
Characterized in that, being on the basis of mutant L386W/G417L/G57F (WLF) laccase gene, to be obtained by rite-directed mutagenesis；
It is that the aspartic acid Asp of WLF CotA laccases the 501st is sported into glycine Gly；It is to import WLF laccase genes
The genetic engineering bacterium that Escherichia coli BL21 (DE3) are obtained.
2. encode the gene of mutant described in claim 1.
3. plasmid or cell containing gene described in claim 1.
4. obtain the method for mutant described in claim 1, it is characterised in that with-the cotA of plasmid pCold II (WLF) for template,
Design the primer (- AACACGAGGATTAT of leading F 5 'GGCATGATGCGGCC-3 ', after draw R 5 '-CCATAATCCTCGTGTTCTAATATGTGAC-3 '), mutational site is introduced in overlapping region, encoding mutant body is obtained by PCR
Gene and plasmid, the plasmid Transformed E .coli BL21 (DE3) after mutation are then expressed into bacterial strain, it is flat by ammonia benzyl antibiotic
Screen selects positive transformant and carries out sequence verification.
5. a kind of method that laccase is produced with genetic engineering bacterium described in claim 1, its step is as follows：By recombinant expression plasmid
PCold II-cotA (WLF) Transformed E .coli BL21 (DE3), the engineering bacteria containing recombinant plasmid is inoculated in containing 100 μ g mL-1Ammonia
In the 3mL LB test tubes of parasiticin, 37 DEG C of 200rpm activate 10h, then take 1mL bacterium solutions to be inoculated in containing the μ g of ampicillin 100
mL-150mL LB culture mediums in, 200rpm shaking table cultures are to OD ≈ 0.5 under the conditions of 37 DEG C, then 15 DEG C of static gas wave refrigerator 30min,
It is subsequently added into final concentration of 0.4mM IPTG and 0.25mM CuSO4The induced expression 24h in 15 DEG C of 200rpm shaking table.Receive
Collect thalline, supernatant is collected in ultrasonication, and SDS-PAGE analyses can obtain obvious specific band, band molecular weight and expected size point
Sub- amount~65KD is consistent.Mutant D501G/WLF CotA laccases amount of soluble expression is compared with wild type B.pumilus W3CotA
(WT) it is substantially higher with WLF CotA.
6. application of the genetic engineering bacterium in decolorizing printing and dyeing waste water described in a kind of claim 1.
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| CN109294936A (en) * | 2018-10-29 | 2019-02-01 | 江南大学 | A kind of heterologous recombination Pichia yeast engineering GS115-pPIC9K-LacGWLF and its application |
| CN109439635A (en) * | 2018-10-23 | 2019-03-08 | 江南大学 | The CotA laccase and its application that a kind of catalytic efficiency improves |
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| CN105200020A (en) * | 2015-10-29 | 2015-12-30 | 江南大学 | High-substrate-specificity Bacillus pumilus CotA laccase modified with composite point mutation |
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| CN109439635A (en) * | 2018-10-23 | 2019-03-08 | 江南大学 | The CotA laccase and its application that a kind of catalytic efficiency improves |
| CN109294936A (en) * | 2018-10-29 | 2019-02-01 | 江南大学 | A kind of heterologous recombination Pichia yeast engineering GS115-pPIC9K-LacGWLF and its application |
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