CN107034200A - The bacillus pumilus CotA laccase complex mutation bodies that a kind of amount of soluble expression is improved - Google Patents

The bacillus pumilus CotA laccase complex mutation bodies that a kind of amount of soluble expression is improved Download PDF

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CN107034200A
CN107034200A CN201710438493.0A CN201710438493A CN107034200A CN 107034200 A CN107034200 A CN 107034200A CN 201710438493 A CN201710438493 A CN 201710438493A CN 107034200 A CN107034200 A CN 107034200A
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laccase
wlf
cota
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管政兵
罗权
夏静
王凯强
廖祥儒
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Jiangnan University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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    • 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)
    • C—CHEMISTRY; METALLURGY
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • 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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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.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 is templates, the 501st Asp of complex mutation body is further sported into Gly, the 317th Lys sports Asn.The amount of soluble expression of bacillus pumilus CotA Laccase mutants of the present invention is 3.04 times of WT CotA, is 3.63 times of WLF, and catalytic efficiency is 4.6 times of WT CotA, is 1.03 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

The bacillus pumilus CotA laccase complex mutation bodies that a kind of amount of soluble expression is improved
Technical field
The present invention relates to the bacillus pumilus CotA laccase complex mutation bodies that a kind of amount of soluble expression is improved, belong to raw Thing field of engineering technology.
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, Including phenols and its derivative, arylamine and its derivative, aromatic carboxylic acids and its derivative etc., therefore laccase application potential is huge. In wood processing field, laccase can replace chemical adhesive, can not only improve product quality, and can mitigate to health Injury and the pollution to environment；In paper industry, laccase is used for paper bio-bleaching and slurrying, can reduce pulp and paper making Pollution, contributes to paper-making industry finally to realize clean manufacturing；In food processing field, laccase can be used for removing phenols chemical combination in fruit juice It is muddy caused by thing, so as to improve the quality of fruit juice.In addition, the also oxidable chlorophenol of laccase and its derivative, reduce its toxicity, subtract It is few that the environmental pollution that dyestuff, preservative, herbicide, agrochemical chemical products are caused is produced by the raw material of industry of chlorophenols.
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 Givaudan et al. identifies 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 bacillus, The functional protein of different types of tool laccase activity, such as withered grass/lichens/short and small brood cell bar are found that in the bacteriums such as streptomyces griseus The CotA albumen of bacterium, PpoA albumen, the CueO albumen of Escherichia coli, the EpoA albumen of streptomyces griseus of extra large monad etc., this A little bacterial laccases are similar to fungal laccase protein structure, all with 4 copper ion binding sites.In these laccases, CotA paints Enzyme is 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 wealth Power.In recent years, site-directed mutagenesis technique was applied on the solubility expression for improving albumen, and obtained good result, and laccase is solvable The increase of property 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 all emitted in trade effluent form.Coloured sewage is directly released into environment, and many dyestuffs are under anaerobic environment Noxious material or carcinogen may be converted into, numerous countries have all promulgated severe regulation to limit industrial dye sewage in succession Discharge.Due to the special chemical constitution of dyestuff itself, handled often using physically or chemically method (condensation, ozone, activated carbon) Effect is not good, and also easily causes secondary pollution.Research shows that bioanalysis (using laccase, manganese peroxidase etc.), which has, to decolourize Rate is high, operating cost is low, environmental protection advantage, is the potential effective means for handling dyeing waste water, is that current dyeing waste water takes off The focus of color research.Often temperature is very high and pH value meta-alkali for the industrial dyeing and printing sewages of overwhelming majority discharge.Paint in all kinds of sources In enzyme, fungal laccase is difficult to play under the conditions of meta-alkalescence and made due to only having preferable decolorizing effect under pH meta-acid environment With, and heat resisting temperature is less than bacterial laccase, so bacterial laccase has more by its unique advantage in dyeing wastewater processing Application potential.
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 paints that a kind of activity is improved Enzyme mutant (WLF), carries out transformation and obtains one plant of performance stabilization, amount of soluble expression is improved, temperature stability on this basis Enhanced complex mutation body K317N/D501G/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 K317N/D501G/WLF.The mutant WLF that the catalytic activity that the mutant is built using this laboratory early stage is greatly improved is mould Plate, further sports the uncharged sweet ammonia of polarity by WLF the 501st negatively charged aspartic acid of polarity (Asp, D) Sour (Gly, G), the 317th positively charged lysine of polarity (Lys, K) sport the uncharged asparagine of polarity (Asn, N), compared with wild type B.pumilus CotA (WT) and WLF CotA, find the increase of its amount of soluble expression, and zymetology Property is stable, and temperature stability is stronger.
The original parent amino acid sequence of the B.pumilus CotA laccases and the B.pumilus in ncbi database CotA laccases consensus amino acid sequence (submit, GenBank accession number by this laboratory：KF040050), bacillus pumilus (B.pumilus W3) is obtained by this laboratory separation screening, and bacterial strain is stored in China typical culture collection center, and numbering is CCTCC No：M2015018.
Described the 501st Aspartic acid mutations of bacillus pumilus Laccase mutant K317N/D501G/WLF are glycine, 317th lysine mutation is asparagine, nucleotide 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 Company), bacterium colony PCR checkings preserve glycerol tube and are sequenced.The correct bacterium activation of sequencing result, extraction plasmid are turned into E.coli BL21 (DE3) competence, and bacterium colony PCR checkings are carried out, by -80 DEG C of preservation glycerol tubes of the correct bacterial strain of gene size.Then use Identical method, with-the cotA of recombinant plasmid pCold II (D501G/WLF) for template, enters performing PCR with 317 mutant primers and expands Increase, finally can obtain complex mutation body K317N/D501G/WLF.
The present invention also provides a kind of method for producing the soluble laccases of above-mentioned K317N/D501G/WLF, by the guarantor built The purpose bacterial strain ensconced in glycerol tube connects the activation of 3mL LB test tubes, 37 DEG C of 200rpm incubated overnights, the bacterium solution inoculation for taking 1mL to activate To the shaking flask containing 50mL culture mediums, 2-3 hour (OD ≈ 0.5) is cultivated, shaking table is adjusted to 15 DEG C of static gas wave refrigerator 30min, then Add final concentration of 0.4mM IPTG and 0.25mM CuSO4The induced expression 24h in 15 DEG C of 200rpm shaking table.Collect hair Zymotic fluid, 4 DEG C of 8000rpm centrifuge 10min, abandon supernatant, are resuspended with pH7.0 phosphate buffers, and ultrasonication can obtain laccase supernatant Liquid.Because the CotA laccase proteins of recombination expression carry histidine-tagged (His6Tag), therefore nickel ion affinity chromatograph is used Method separates target protein.Using AKTA avant25 protein purification systems through steps such as overbalance, loading, elutions, it can finally obtain To the CotA laccases of purifying.
Brief description of the drawings
Fig. 1 is mutated 317 surveys to be mutated into D501G/WLF sequencing results by template of WLF, by template of D501G/WLF Sequence result.
Fig. 2 is rite-directed mutagenesis principle schematic.
Fig. 3 is the Recombinant protein expression, the SDS-PAGE collection of illustrative plates of purifying and non-change of B.pumilus W3 CotA laccases Property gel electrophoresis spectrum (green)；Wherein M：Protein molecular weight standard (kDa)；
Band is respectively 1 in A：Unloaded supernatant；2：WLF supernatants；3：WT supernatants；4 and 6：WLF is purified；5 and 7：WT is purified. Wherein 6 and 7 bands are non denatured electrophoretograms, using ABTS as substrate staining.
Band M in B：Protein molecular weight standard (kDa)；1：Unloaded supernatant；2：K317N/WLF supernatants；3：WLF supernatants； 4：D501G/WLF supernatants；5：K317N/D501G/WLF supernatants；6：WLF after purification；7：K317N/D501G/ after purification WLF；8：K317N/WLF after purification；9 D501G/WLF after purification.
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 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 SuperMix 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), be mutated after being mutated successfully with D501G/WLF templates 317.The related forward primer and reverse primer of design are as follows：
501 F5 '-AACACGAGGATTATGGCATGATGCGGCC-3’
R5’-CCATAATCCTCGTGTTCTAATATGTGAC-3’
317 F5 '-AACGATTGTTTTAAACAATAAGGCAGGC-3’
R5’-GTTTAAAACAATCGTTTGGTTTTCGTAA-3’
Wherein underscore part represents the codon corresponding to the amino acid of mutant gene coding respectively.PCR expands body It is to be：The μ L of DNA 3, the μ L of leading (10 μM) 1, after draw (10 μM) 1 μ L, PCR SuperMix 25 μ L, ddH2O is mended 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 of extensions 10min.5 μ L PCR primers are taken to be detected through 1% agarose gel electrophoresis, and it is correct to detect purpose band.1 μ L DMT enzymes are added Enter in remaining PCR primer, mix, 37 DEG C are incubated 1 hour, add 3 μ L DMT enzymic digestions products in 100 μ L competent cells In, flick mixing, ice bath 30 minutes.42 DEG C of water-bath thermal shock 45s, are immediately placed on 2min on ice；Plus 900 μ L are balanced to room temperature then LB culture mediums, 37 DEG C of 200rpm cultivate 1h, converted product finally are coated on into L containing 100mg-1The LB flat boards of ampicillin, Through 37 DEG C of incubated overnights, 10 single bacterium colonies are selected from flat board and carry out bacterium colony PCR checkings, 5 are chosen from the bacterium colony being proved to be successful Single bacterium colony is inoculated into after LB fluid nutrient mediums, 10h preserves 2 glycerol tubes, a -80 DEG C of preservations, Yi Fenyong by each single bacterium colony In sequencing.Will the correct mutant of sequencing from glycerol tube be inoculated into LB fluid nutrient mediums in activated overnight, glycerine is first preserved afterwards Pipe, 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 K317N/ that wild type restructuring laccase expression bacterial strain and early stage are built are inoculated with from glycerol tube D501G/WLF recombinant strains are activated into LB culture mediums, 37 DEG C, and 200rpm is stayed overnight (10h).Respectively will by 2% inoculum concentration Seed access 50mL LB liquid fermentation mediums (L containing 100mg-1Ampicillin) 37 DEG C of 200rpm shaking table cultures are to OD600Reach To 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 CuSO4Induced, 15 DEG C of 200rpm cultivate 24h, zymotic fluid is removed into supernatant in 4 DEG C of 8000rpm centrifugations 10min, bacterium is collected Body.The thalline of collection is resuspended with phosphate buffer, bacterial cell disruption is discharged into intracellular with ultrasonic cell disruption instrument after resuspension Albumen, after the completion of crushing, centrifuges 20min (4 DEG C, 8000rpm), then by supernatant in 70 DEG C of heating water baths by broken liquid 4 DEG C of centrifugation 10min of 15min, 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 under elution requirement is collected corresponding Guan Hao, and do Enzyme activity assay, collects the unimodal corresponding albumen for having an enzyme activity, runs the band of SDS-PAGE protein electrophoresises confirmation form one, Obtain 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 pH 4.0 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 includes 2.4mL disodium hydrogen phosphates-lemon acid buffering Liquid (100mM, pH3.6), the pure enzyme liquids of 0.1mL, 0.5mLABTS solution (be made into respectively 10 μM of final concentration, 20 μM, 40 μM, 60 μM, 80μM、100μM、200μM、300μM、400μM、500μM、1000μM).Reaction system is placed in 50 DEG C of water-baths and preheated, so The enzyme-added variable quantity (reaction rate at the uniform velocity reaction) that OD values in 30s are determined under 420nm wavelength afterwards.According to enzyme activity formula meter Calculate 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 (Volumetric Activity) concentrates 5 times.
As it can be seen from table 1 mutant K317N/D501G/WLF catalytic efficiency is higher than WT, and WLF is quite, but can Dissolubility expression quantity complex mutation body is significantly improved.
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 that restructuring laccase and laccase are prominent Variant all has the advantages that long-time alkali resistance environment is good, and mutant K317N/D501G/WLF pH stability is poor compared with WT and WLF Seldom (as shown in Figure 4), stability very strong enzyme is belonged to 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, complex mutation body is more more stable than WT (as shown in Figure 5).
B.pumilus W3 restructuring laccases and Laccase mutant Salt Tolerance Analysis that embodiment 6 is purified
WT, WLF, K317N/D501G/WLF laccase are all stored in the NaCl solution of three kinds of various concentrations respectively first (100mM, 500mM, 1M), is stored in 4 DEG C of refrigerator 10h, then surveys enzyme activity by substrate of ABTS under the conditions of pH3.6,50 DEG C, instead Answer the enzyme activity determination method in system 3mL, method be the same as Example 3.Experimental result as shown in fig. 6, as a result show restructuring laccase and Laccase mutant 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, effect 10h, 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 blue129 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 model of the present invention Enclose being defined of being defined by claims.
Sequence table
<110>Southern Yangtze University
<120>The bacillus pumilus CotA laccase complex mutation bodies 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 CAATAAGGCA
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 K317N/D501G/WLF (B. pumilus W3 K317N/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 N 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 complex mutations that a kind of amount of soluble expression is improved Body, it is characterised in that be on the basis of mutant L386W/G417L/G57F (WLF) laccase gene, to be obtained by rite-directed mutagenesis 's；It is that the aspartic acid Asp of WLF CotA laccases the 501st is sported into glycine Gly, the 317th lysine Lys is sported Asparagine Asn, is that WLF laccase genes are imported into the genetic engineering bacterium that E.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 primer as follows：
501-AACACGAGGATTAT of F 5 'GGCATGATGCGGCC-3’
R 5’-CCATAATCCTCGTGTTCTAATATGTGAC-3’
317-AACGATTGTTTTA of F 5 'AACAATAAGGCAGGC-3
R 5’-GTTTAAAACAATCGTTTGGTTTTCGTAA-3’
Mutational site is introduced in overlapping region, the gene and plasmid of encoding mutant body are obtained by PCR, then by the matter after mutation Grain Transformed E .coli BL21 (DE3) expression bacterial strains, by ammonia benzyl antibiotic plate screening positive transformant and carry out sequencing and test Card.
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.Complex mutation body K317N/D501G/WLF CotA laccase amount of soluble expression is compared with wild type B.pumilus CotA (WT) and WLF CotA are substantially higher, and temperature stability is more stable compared with wild type.
6. application of the genetic engineering bacterium in decolorizing printing and dyeing waste water described in a kind of claim 1.
CN201710438493.0A 2017-06-12 2017-06-12 The bacillus pumilus CotA laccase complex mutation bodies that a kind of amount of soluble expression is improved Pending CN107034200A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108570459A (en) * 2018-04-10 2018-09-25 南京农业大学 A kind of method of high-efficiency fermenting production recombinant bacteria laccase
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
CN114107364A (en) * 2021-12-22 2022-03-01 河北省微生物研究所有限公司 Construction method of laccase-producing recombinant pichia pastoris engineering strain

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105200021A (en) * 2015-10-29 2015-12-30 江南大学 High-activity Bacillus pumilus CotA laccase modified with site-directed mutagenesis
CN105200020A (en) * 2015-10-29 2015-12-30 江南大学 High-substrate-specificity Bacillus pumilus CotA laccase modified with composite point mutation
CN105219744A (en) * 2015-10-29 2016-01-06 江南大学 Bacillus pumilus CotA Laccase mutant that a kind of catalytic activity improves and preparation method thereof
CN105296442A (en) * 2015-10-29 2016-02-03 江南大学 High-catalytic-activity bacillus pumilus CotA laccase mutant

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105200021A (en) * 2015-10-29 2015-12-30 江南大学 High-activity Bacillus pumilus CotA laccase modified with site-directed mutagenesis
CN105200020A (en) * 2015-10-29 2015-12-30 江南大学 High-substrate-specificity Bacillus pumilus CotA laccase modified with composite point mutation
CN105219744A (en) * 2015-10-29 2016-01-06 江南大学 Bacillus pumilus CotA Laccase mutant that a kind of catalytic activity improves and preparation method thereof
CN105296442A (en) * 2015-10-29 2016-02-03 江南大学 High-catalytic-activity bacillus pumilus CotA laccase mutant

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KOSCHORRECK, K等: "Improving the functional expression of a Bacillus licheniformis laccase by random and site-directed mutagenesis", 《BMC BIOTECHNOLOGY》 *
NIRUPAMA GUPTA等: "Laboratory evolution of laccase for substrate specificity", 《JOURNAL OF MOLECULAR CATALYSIS B:ENZYMATIC》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108570459A (en) * 2018-04-10 2018-09-25 南京农业大学 A kind of method of high-efficiency fermenting production recombinant bacteria laccase
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
CN114107364A (en) * 2021-12-22 2022-03-01 河北省微生物研究所有限公司 Construction method of laccase-producing recombinant pichia pastoris engineering strain
CN114107364B (en) * 2021-12-22 2023-07-28 河北省微生物研究所有限公司 Construction method of laccase-producing recombinant pichia pastoris engineering strain

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