CN107034199A - The bacillus pumilus CotA Laccase mutants that a kind of Fixedpoint mutation modified stability and activity are improved - Google Patents

Abstract

The invention discloses the bacillus pumilus CotA Laccase mutants that a kind of substrate specificity is improved, belong to genetic engineering and enzyme engineering field.The present invention is using the single mutant L386W CotA laccase genes of this laboratory early stage structure as template, the 417th Gly of single mutant is further sported into Leu, then using wild type CotA laccases as control, it was found that complex mutation body L386W/G417L is to substrate 2,2 nitrogen two (sulfonic acid of 3 ethyl-benzothiazole 6) di-ammonium salts (ABTS) have higher selectivity, improve the prospects for commercial application of bacillus pumilus CotA laccases.

Description

The bacillus pumilus CotA that a kind of Fixedpoint mutation modified stability and activity are improved Laccase mutant

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, 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.The annual dyeing waste water discharge of China Amount accounts for the 35% of total discharged volume of industrial waste water, and oneself turns into the maximum important pollution sources of harm, most of synthetic dyestuffs be all difficult to by Microbial degradation.These dye compositions are normally used for the colouring agent of weaving, food, plastic cement and biomedicine, annual full generation Boundary's commercial dyes are used up to 7 × 10⁵Ton, species is up to 1 × 10⁴Kind, wherein 5~10% dyestuff is all discharged in trade effluent form Out.Coloured sewage is directly released into environment, and many dyestuffs may be converted into noxious material or carcinogenic substance under anaerobic environment Matter, numerous countries have all promulgated severe regulation to limit the discharge of industrial dye sewage in succession.Because dyestuff itself is special Chemical constitution, using physically or chemically method (condensation, ozone, activated carbon) processing, often effect is not good, and also easily causes secondary dirt Dye.Research shows that bioanalysis (using laccase, manganese peroxidase etc.) has percent of decolourization height, operating cost low, environmental protection Advantage, is the potential effective means for handling dyeing waste water, is the focus of current decolorizing printing and dyeing waste water research.Overwhelming majority discharge Often temperature is very high and pH value meta-alkali for industrial dyeing and printing sewage.In the laccase in all kinds of sources, fungal laccase is due to only in pH meta-acids There is preferable decolorizing effect under environment, be difficult to play a role under the conditions of meta-alkalescence, and heat resisting temperature is less than bacterial laccase, institute With bacterial laccase by its unique advantage dyeing wastewater processing 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 paints that a kind of activity is improved Enzyme mutant (WLF), carries out transformation and obtains one plant of performance stabilization, stability and catalytic activity on this basis (ABTS is substrate) The mutant K317N/WLF of raising.

The content of the invention

The problem to be solved in the present invention is to provide a kind of function admirable, and catalytic activity (ABTS is substrate) and stability are improved Laccase mutant K317N/WLF.The mutant WLF that the catalytic activity that the mutant is built with this laboratory early stage is greatly improved For template, WLF the 317th positively charged lysine of polarity (Lys, K) is further sported into the uncharged day of polarity Winter acid amides (Asn, N), is compared with wild type restructuring B.pumilus W3 CotA (WT) and WLF CotA, finds its stability Increase with catalytic activity, 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), B.pumilus W3 are 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 317th lysine mutation of bacillus pumilus Laccase mutant K317N/WLF is asparagine, 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 317 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 (K317N/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.

The present invention also provides a kind of method for producing the soluble laccase of above-mentioned K317N/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, the bacterium solution inoculation 50mL for taking 1mL to activate shakes Bottle 2-3 hour (OD ≈ 0.5) of culture, shaking table is adjusted to 15 DEG C of static gas wave refrigerator 30min, is subsequently added into final concentration of 0.4mM's IPTG and 0.25mM CuSO₄The induced expression 24h in 15 DEG C of 200rpm shaking table.Collect zymotic fluid, 4 DEG C of 8000rpm centrifugations 10min, abandons supernatant, is resuspended with pH7.0 phosphate buffers, and ultrasonication can obtain laccase supernatant.Due to the CotA of recombination expression Laccase protein carries histidine-tagged (His₆Tag), therefore using nickel ion affinity chromatograph method target protein is separated.Utilize AKTA avant25 protein purification systems are through steps such as overbalance, loading, elutions, the CotA laccases that can be finally purified.

Brief description of the drawings

Fig. 1 Laccase mutant K317N/WLF sequencing results.

Fig. 2 is rite-directed mutagenesis principle schematic.

Fig. 3 for B.pumilus W3 CotA laccases Recombinant protein expression, purifying SDS-PAGE analysis collection of illustrative plates and Native gel electrophoresis collection of illustrative plates (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； 6：WLF after purification；8：K317N/WLF after purification.4th, 5,7,9 be other mutant band.

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 2 kinds of azos, 2 kinds of anthraquinones and a kind of decolorization of triphenylmenthane 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..

Embodiment 1 recombinates the structure of B.pumilus W3 Laccase mutants

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 317th lysine (Lys, K) sport asparagine (Asn, N).The reverse primer of the related forward primer of design is as follows：

- the AACGATTGTTTTA of leading F 5 'AACAATAAGGCAGGC-3’

After draw R 5 '-GTTTAAAACAATCGTTTGGTTTTCGTAA-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 SuperMix 25 μ L, ddH₂O is mended to 50 μ L, PCR amplification condition 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.

Embodiment 2 recombinates the expression and purification of B.pumilus W3 laccases

Expression：WLF and K317N/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 into 50mL respectively by 2% inoculum concentration LB liquid fermentation mediums (L containing 100mg^-1Ampicillin) 37 DEG C of 200rpm shaking table cultures are to OD₆₀₀Reach 0.5, Ran Houyao Bed tempertaure 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 200rpm culture 24h, removes supernatants in 4 DEG C of 8000rpm centrifugations 10min by zymotic fluid, collects thalline.The thalline of collection is used Phosphate buffer is resuspended, and bacterial cell disruption is discharged into intracellular protein with ultrasonic cell disruption instrument after resuspension, after the completion of crushing, will Broken liquid centrifugation 20min (4 DEG C, 8000rpm), then by supernatant 4 DEG C of 70 DEG C of heating water baths 15min, 10000rpm from Heart 10min goes precipitation, collects 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.

Embodiment 3 recombinates the enzyme activity determination of B.pumilus W3 laccases

Enzyme-activity unit is defined：When determining laccase activity using ABTS methods, define 1 μm of ol substrate of catalysis per minute and be converted into Enzyme amount needed for product is a unit of activity.

(1) enzyme activity determination step：1 preheating：2.4mL pH4.0 citrate buffer solution is taken in test tube, is added in test tube Enter 0.5mLABTS solution (the final concentration of 0.5mM of ABTS) and be placed in 50 DEG C of water-baths to preheat 5min；2 reactions：Add what is diluted 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.

(2) 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, pH 3.6), the pure enzyme liquids of 0.1mL, 0.5mL ABTS solution (are made into 10 μM of final concentration, 20 μM, 40 μM, 60 μ respectively M、80μM、100μM、200μM、300μM、400μM、500μM、1000μM).Reaction system is placed in 50 DEG C of water-baths and preheated, Then the 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 Calculate enzyme activity.

Enzyme activity formula：Enzyme activityRate activity

In formula：The poor V of △ OD- reaction time internal absorbance values_AlwaysThe volume (L) of-reaction system

N- enzyme liquid extension rate △ t- reaction time (min)

Volume (L) m of Vo- enzyme liquids_EnzymeThe 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 K317N/WLF laccases is determined by substrate of ABTS V_max、K_m、k_cat、k_cat/K_m, Rate activity, total activity and protein content.As a result it is as follows：

The kinetic parameter of the wild type of table 1 (WT) and mutant laccase

As it can be seen from table 1 mutant K317N/WLF catalytic efficiency is higher than WT, slightly above WLF.

B.pumilus W3 restructuring laccases and the most suitable action pH of Laccase mutant and pH analysis of stability that embodiment 4 is purified Analysis

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 (as shown in Figure 4).

B.pumilus W3 restructuring laccases and Laccase mutant optimum temperature and temperature stabilization that embodiment 5 is purified Property 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 advantage of warm environment, K317N/WLF shows more preferable temperature stability (as shown in Figure 5).

B.pumilus W3 restructuring laccases and Laccase mutant Salt Tolerance Analysis that embodiment 6 is purified

WT, WLF, K317N/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 pH is 10, and buffer solution is carbonic acid buffer.Experimental result is shown in alkali Property environment pH 10, after 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 96.7%, respectively reach 80.9% to a kind of anthraquinone dyes Acid blue129 percent of decolourization, 85.4% and 88.3%, 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 mutants that a kind of Fixedpoint mutation modified stability and activity are 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 GACATGATGC GGCCGATGGA TATCATCCAG TAA

<210> SEQ ID No.2

<211> 510

<212> PRT

<213>Bacillus pumilus W3 mutant K317N/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 D M M R P M D I I Q *

Claims (6)

1. bacillus pumilus (Bacillus pumilus W3) CotA laccases mutation that a kind of stability and catalytic activity are 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 lysine (Lys) of WLF CotA laccases the 317th is sported into asparagine (Asn)；It is to lead WLF laccase genes Enter 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 (- AACGATTGTTTTA of leading F 5 'AACAATAAGGCAGGC-3 ', after draw R 5 '-GTTTAAAACAATCGTTTGGTTTTCGTAA-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 K317N/WLF CotA laccases stability and catalytic activity recombinate B.pumilus compared with wild type W3 CotA (WT) and WLF CotA increase.

6. application of the genetic engineering bacterium in decolorizing printing and dyeing waste water described in a kind of claim 1.