CN102676550B - Laccase for biological treatment of papermaking wastewater and encoding gene as well as expression and application of encoding gene - Google Patents
Laccase for biological treatment of papermaking wastewater and encoding gene as well as expression and application of encoding gene Download PDFInfo
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- CN102676550B CN102676550B CN 201210153015 CN201210153015A CN102676550B CN 102676550 B CN102676550 B CN 102676550B CN 201210153015 CN201210153015 CN 201210153015 CN 201210153015 A CN201210153015 A CN 201210153015A CN 102676550 B CN102676550 B CN 102676550B
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Abstract
The invention relates to the field of biotechnology, and discloses a laccase for biological treatment of papermaking wastewater as well as an encoding gene and application of the encoding gene in the biological treatment of the papermaking wastewater. The nucleotide sequence of the laccase gene is shown in SEQ IDNO:2, and the amino acid sequence of the laccase which is contains the gene codes is shown in SEQ IDNO:1. According to the laccase and the encoding gene, an efficient expression can be realized in microzyme, and the defects of the conventional pure culture technology and induced expression are overcome; and the laccase gene can improve the expression activity of the laccase, and is of great importance for promoting the application of the laccase in the biological treatment of the papermaking wastewater.
Description
Technical field
The present invention relates to biological technical field, the application that has related in particular to a kind of laccase for the paper waste biological treatment, encoding gene and carried out a biological disposal upon at paper waste.
Background technology
China's paper industry year wastewater discharge is big, and the intractability height adds " pulp and paper industry pollution discharge standard " promulgation and enforcement (GB3544-2008), and the intractability of paper-making industrial waste water strengthens, and processing costs significantly increases.Along with the enhancing of people's environmental consciousness, constantly sound continuous strictness, technological process for the treatment of papermaking and up to standard the having important practical significance that reaches the discharge index of paper waste of laws and regulations.
The treatment process that paper waste is commonly used mainly contains physics method and chemical method at present, but can't thoroughly eliminate more unmanageable material in the waste water, as Mierocrystalline cellulose, hemicellulose, xylogen and phenolic compound etc.Biological process is a kind of the most effective, the safest of Processing Paper Wastewater and method the most completely.
Laccase is a kind of protein in conjunction with a plurality of copper atoms, belongs to the covellite oxydase.Laccase effect substrate is quite extensive, can be to multiple pollutent such as various phenols dyestuff, substituted phenol, chlorophenol, thiophenol, bis-phenol, aromatic amine etc.In the presence of laccase amboceptor (HBT, ABTS etc.), all right catalyzed degradation ditan relevant with xylogen of laccase is Ji dioxin etc.
Laccase is the biological enzyme of finding tool potentiality in paper industry in recent years, has the ability of catalyzed oxidation lignin.Laccase is the catalysis lignin degradation optionally, can not produce toxic substance, and produces and carry out saving equipment and energy consumption under the mild conditions of normal temperature, normal pressure.Therefore laccase has very great application prospect at aspects such as paper-making industrial waste water processing.
Giving birth to fat azospirillum (Azospirillum lipoferum) successively from people in 1983, replace Zymomonas mobilis (Alteromonas sp.MMB-1), intestinal bacteria (Escherichia coli), pseudomonas (Pseudomonas sp. KU03), clayey Serratia (Serratia marcescens), Bacillus sphaericus (Bacillus sphaericus), extreme alkali-fast bacillus cereus (B. halodurans) and dark blue streptomycete (Streptomyces cyaneus), streptomyces coelicolor (S.coelicolor) and husky streptomycete (S.psammoticus) are waited and all find to have laccase activity in some bacterial strains.(Wang Chunlei etc., 2011)
Owing to lack method and the culture medium of reproducing environment condition, caused most microorganisms to be difficult to be called not culturing micro-organisms (uncultured microorganism) by the recovery of the method for standard laboratory and cultivation.Use standard microorganism to learn culture technique to the mensuration of different habitats microorganism cultivation property, microorganism is educable in the seawater is about 0.001%～0.1%, and fresh water is about 0.25%, and soil is about 0.3%, and active sludge is about 1%～15%.That is to say that present most physical environment microorganisms are difficult to maybe can not obtain its pure culture by traditional pure culture separation method.
Therefore, the molecular biology method that utilize to make up gene library screens new efficient laccase gene, utilizes the pichia spp constitutive expression to improve the expression vigor of laccase, and is significant for the application that promotes laccase in the paper waste biological treatment.
Summary of the invention
The invention provides a kind of new laccase gene that from the active sludge of papermaking wastewater treatment plant, filters out, this gene can be in yeast cell constitutive expression.The yeast expression bacterium that contains new laccase gene can breed and express laccase in paper waste, handle thereby paper waste is carried out bio-synergistic.
In order to solve the problems of the technologies described above, the present invention is solved by following technical proposals:
A kind of laccase for the treatment of paper waste, its aminoacid sequence shown in SEQ ID NO:1, totally 544 amino acid.Concrete, described gene nucleotide series is shown in SEQ ID NO:2, and total length is 1662bp, and GC content is 40.07%.
The laccase gene that the present invention relates to is a kind of new gene, does not have homology with isoformgene.The laccase of its coding is a kind of new laccase, compares with the laccase aminoacid sequence that other have been reported, and similarity is less than 40%.Should be pointed out that the coded aminoacid sequence of laccase gene of the present invention is carried out one or more amino acid to be replaced, insert or lack resulting functional analogue and all belong to protection scope of the present invention.Under the situation of known laccase gene of the present invention, those of ordinary skill of the present invention can obtain described laccase by this area ordinary method carrier construction, conversion, expression.
The invention still further relates to the host bacterium that contains described laccase gene.
The invention still further relates to the recombinant vectors that contains described gene, the recombinant vectors that will contain described gene is converted in the host bacterium (being generally yeast), expresses, and can obtain laccase of the present invention.
The invention still further relates to the application of described gene in preparation reorganization laccase.
The invention still further relates to the application of described laccase in the paper waste biological treatment, the optimum temperuture of laccase of the present invention is 30-40 ℃, and optimal pH is 5-6.After genetic engineering bacterium carried out enlarged culturing, the addition by 0.05% adds in the aeration tank to be handled.
The present invention has significant technique effect owing to adopted above technical scheme:
The invention provides a kind of new laccase gene, can in yeast, efficiently express by composing type, overcome the deficiency of traditional pure culture technigne and abduction delivering, can improve the expression vigor of laccase, significant for the application that promotes laccase in the paper waste biological treatment.
Description of drawings
Fig. 1 is the optimal pH curve of laccase;
Fig. 2 is the optimum temperuture curve of laccase.
Embodiment
Describe in further detail with the present invention of embodiment below in conjunction with accompanying drawing 1 to accompanying drawing 2:
Embodiment 1: the structure in activated sludge DNA library
1, the extraction of activated sludge DNA
Gather mud from certain paper waste sewage work, carry out the extraction of DNA through suitably diluting as sample.12000 rmin
-1Centrifugal 5 min collect sample, and after the resuspended washing once of TE, adding N,O-Diacetylmuramidase to final concentration is 100 μ gml
-1, 37 ℃ of water-baths 2 hours.Add 5 M NaCl and adjust final concentration to 0.5 M, add 40 μ l Proteinase Ks behind the mixing and 20 % SDS(final concentrations are 0.5 ~ 1 %), 37 ℃ are spent the night or 50 ℃ of water-bath 3 h clarify to the solution thickness.After adding saturated NaCl thermal agitation 15 s of 1/3 volume, 12000 rmin
-1Centrifugal 5 min collect in supernatant to the sterilization centrifuge tube, add 0.6 V Virahol mixing postprecipitation, 12000 rmin
-1Centrifugal collecting precipitation with 70 % washing with alcohol, is dissolved in after drying up in the 400 μ l sterilization deionized water.
2, the structure in DNA library
(1) enzyme of DNA is cut
With the total DNA of the partially digested active sludge of Sau3A I, carry out enzyme with detection type reaction system earlier and cut, the suitableeest definite enzyme is cut concentration and time, and the type that finally is prepared is partially digested.Detection type endonuclease reaction system (10 μ L): total DNA 2 μ L, Sau3AI is an amount of, and damping fluid 1 μ L adds ddH
2O is to final volume 10 μ L.Preparation type endonuclease reaction system (100 μ L): total DNA 20 μ L, Sau3AI is an amount of, and damping fluid 10 μ L add ddH
2O is to final volume 100 μ L.Add wherein that Sau3AI enzyme amount is cut concentration according to enzyme and the time gropes, cut the dna fragmentation that glue reclaims 2 ~ 4 kb bands, as the external source fragment.
(2) preparation of dephosphorization carrier
Extract the puc18 plasmid, carry out single endonuclease digestion with BamHI, the linear fragment that reclaims the 1.8kb size carries out the dephosphorylation reaction.Reaction system below setting up in Eppendorf tube: linearizing puc18 carrier 28ul, 10 * alkaline phosphatase damping fluid, 5 μ l, CIAP(16 U/ μ l TAKARA) 0.5ul adds ddH
2O is to final volume 50 μ l.50 ℃ are reacted 30 min, add the EDTA termination reaction of 1/10 volume 0.5M; Merge four effective ddH
2O complements to 500ul; Add 400ul phenol/chloroform extracting 1 time, the sucking-off supernatant; The 3M NaAc that adds 1/10V, the 2V precooled ethanol is at-20 degree precipitation 60min; Centrifugal collecting precipitation with drying up after 200 μ l, the 70 % washing with alcohol, dissolves with 20 μ lTE.Electrophoresis is quantitative, and it is in 50% the sterile glycerol that the packing of 50 ~ 100ng/ pipe is stored in final concentration, standby.
(3) transform
External source fragment and carrier are spent the night enzyme even in proportion.Get
E.coliThe efficient competent cell of DH5 α (the about 200 μ l of every pipe) melts in ice bath, and every pipe adds 2 μ l enzymes and connects product, rotates gently with the mixing content, place 30 min in the ice bath, centrifuge tube is put into 42 ℃ circulator bath, and heat shock 90 s do not shake centrifuge tube.Fast centrifuge tube is moved on to make cell cooling 1 ~ 2min in the ice bath after every pipe add the LB substratum that 800 μ l are preheated to 37 ℃, behind the mixing in 37 ℃ of shaking tables, 150 rmin
-1Incubation 45 min make bacteria resuscitation, and the antibiotics resistance gene of expression plasmid coding.Conversion fluid after the coating recovery is to adding penbritin (100 μ gml
-1) fresh LB flat board on, treat that bacterium liquid is absorbed fully after, be inverted dull and stereotypedly in 37 ℃ of cultivations, bacterium colony appears behind 12～16 h.
(4) inspection in library and preservation
150 positive colonies of picking are inoculated on the microbiotic flat board, 37 ℃ be cultured to bacterium colony and grow after, picking part transformant carries out plasmid and extracts at random, checks that the external source fragment inserts Qing Condition, and cultured flat board is standby in 4 ℃ of preservations.Flat board after the detection washes the back glycerol adding to final concentration 15% with sterilized water with bacterium colony, and is standby in-70 ℃ of preservations.
Embodiment 2: the screening of laccase gene and sequential analysis
Clone's in the DNA library is cultivated preservation respectively, carry out positive colony and efficient screening of producing the laccase transformant.
The primary dcreening operation substratum
The BavendammShi reaction culture medium: adding tannic acid in the PDA solid medium, to make its final concentration be 0. 4 mmo l/ L;
Zone of oxidation is measured substratum: adding methyl catechol in the PDA solid medium, to make its final concentration be 0. 04%.
Sieve substratum again
Potato 200 g, glucose 20 g, potassium primary phosphate 3 g, magnesium sulfate heptahydrate 1. 5 g, yeast extract 1 g, distilled water 1 000 mL boil filtration, and cooling back is divided and is filled to 250 mL triangular flasks, every bottled 50 mL substratum of going into.
Produce the enzyme basic medium
Glucose 30 g, SODIUMNITRATE 2 g, magnesium sulfate heptahydrate 0. 5 g, three water dipotassium hydrogen phosphates, 1 g, iron vitriol 0. 01 g, Repone K 0. 5 g, distilled water 1 000 mL, natural pH.
The primary dcreening operation of superior strain:
The conventional sterilization of BavendammShi reaction culture medium is fallen dull and stereotyped, get 2 of cultured diameter 12 mm, 7 d cell ages bacterium plugs to be measured and be inoculated in the primary dcreening operation flat board, 28 ℃ of biochemical incubators are cultivated, it is good to select mycelial growth, make tannic acid, strong, the fireballing flat board of variable color of the two metachrosis of methyl catechol, and be inoculated in the PDA inclined-plane, in the lump in 4 ℃ of preservations.
The multiple sieve of superior strain:
Under aseptic condition, punch on the flat board of primary dcreening operation gained bacterium with punch tool, make diameter 12 mm bacterium plug, the 250 mL triangular flasks that 50 mL produce the enzyme substratum are equipped with in access, 2 of every bottle graft kinds, and 3 are parallel, 28 ℃, 120 r/ min shaking culture, every day, the sampling and measuring laccase activity continued 10 d, selected the high bacterial strain of laccase vigor.
The laccase vigor is measured with guaiacol method, and an enzyme unit definition alive makes the required enzyme amount of substrate conversion of 1 μ mol for per minute under 25 ℃ of conditions.
Choose the laccase vigor the highest clone's, extract plasmid insertion sequence checked order.Laccase gene called after LacN, carrier called after puc-LacN.
Sequencing result carries out sequence alignment at NCBI Genbank database, and the result shows there is not the sequence of discovery and this gene order homology in database, and it should be a new sequence.Through to the analysis of laccase gene LacN, predicted the possible encoding sequence of this enzyme, remove signal peptide after, comprise initiation codon and termination codon, totally 1662 Nucleotide (SEQ ID NO:2), 544 amino acid (SEQ ID NO:1) of encoding.
Embodiment 3: the expression of laccase gene in yeast cell
1, the structure of expression vector
Puc-LacN with EcoR I/Not I double digestion, is reclaimed the external source fragment, is inserted into the pichia spp constitutive expression carrier pGAPZaA(U.S. invitrogen company of same double digestion with correct reading frame) connect, transform
E.coliDH5 α obtains recombinant plasmid pGAPZaA-LacN.This recombinant plasmid is through EcoR I/Not I double digestion checking.
2, the preparation of electric transformed competence colibacillus
In containing the 50 ml centrifuge tubes of 5 ml YPD, the culturing yeast cell, 30 spend night; Get 30 ~ 50 ul overnight culture, 250 ml that inoculation contains 50 ml fresh cultures shake bottle, and overnight growth is to OD600=1.3 ~ 1.5; At 4 degree, the centrifugal 5 min collecting cells of 1500 g are with the aqua sterilisa suspension cell of 50 ml precoolings; As above centrifugal, with the aqua sterilisa suspension cell of 25 ml precoolings; As above centrifugal, with the 1M sorbyl alcohol suspension cell of 2 ml precoolings; As above centrifugal, with the 1M sorbyl alcohol suspension cell of 100 ul precoolings.
3, electricity transforms
PGAPZaA-LacN with Bgl II linearization for enzyme restriction, with 5 ~ 10ug linearizing DNA and 80 uL electricity transformant mixing, is transferred to 0.1 cm electricity and transforms cup, ice bath 5 min.Electricity transforms parameter: 0.75kV, 25uF, 200 Ω.The 1M sorbyl alcohol that adds the 1mL precooling after the electric shock immediately.Get behind the recovery 1h on the YPD flat board that 150ul coating contains the Zeocin resistance and cultivate.Hatch dull and stereotyped to clone's generation at 30 degree.
4, produce the screening of laccase transformant
Single bacterium colony on the transformant flat board is cultivated preservation respectively, carry out positive colony and efficient screening of producing the laccase transformant.Screening method is with embodiment 2.
Embodiment 4: the characteristic research of laccase
1, the pH value is to the influence of laccase activity
Laccase is added in the buffer solution system of different pH values and measure enzyme activity, pH arranges from 2.0 to 10.0, gets a mensuration every 0.5.Enzyme assay is reflected at 25 ℃ to be carried out, and the reaction times is 15 min.The result as shown in Figure 1, the optimum pH of laccase is 5 ~ 6, in the pH4.0-6.5 scope, laccase work can remain on more than 50%.
2, temperature is to the influence of laccase activity
The optimal reactive temperature of laccase is determined under the optimum pH and carries out, and the reaction times is 15 min, and the temperature setting is got a mensuration from 15 ℃ to 75 ℃ every 5 ℃.Result such as Fig. 2 show that the optimum temperuture of laccase is 30 ~ 40 ℃, and the enzyme work in the time of 20 ~ 50 ℃ can keep more than 60%.
Embodiment 5:The application of laccase gene engineering bacteria in paper waste is handled
Because laccase is constitutive expression in the genetic engineering bacterium, so after genetic engineering bacterium can being carried out enlarged culturing, the addition by 0.05% adds in certain SBR of paper waste treatment plant technology (sequence intermittent activated sludge process) aeration tank to be handled.Compare with control group, the laccase gene engineering bacteria can improve about 15% with the paper waste processing efficiency, in the paper waste in future is handled the important use meaning is arranged.
In a word, the above only is preferred embodiment of the present invention, and all equalizations of doing according to the present patent application claim change and modify, and all should belong to the covering scope of patent of the present invention.
SEQUENCE LISTING
＜110〉Zhejiang Prov Sunda Environmental Protection Co., Ltd
＜120〉a kind of laccase, encoding gene and expression thereof and application for the paper waste biological treatment
<130>
<160> 2
<170> PatentIn version 3.4
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<211> 554
<212> PRT
<213> Unknown
<220>
＜223〉artificial sequence
<400> 1
1 Met Ile Ala Glu Gly Asn Val Ser Gly Leu Glu Met Ile Asp Leu 15
16 Leu Lys Pro Arg Gln Gln Tyr Ile Glu Ser Thr Tyr His Asp Leu 30
31 Leu Met Arg Glu Tyr Ile Gln Leu Ser Tyr Arg Asn Leu Leu Pro 45
46 Thr Glu Cys Trp Ser Gln Asp Gly Leu Phe Leu Gly Pro Tyr Leu 60
61 Glu Met Pro Lys Asp Val Asn Leu Tyr Val Arg Val Met Asn Ser 75
76 Met Pro Leu Pro His Leu Asn Pro Ile Val His Thr Thr Tyr Ile 90
91 Ser Ala Tyr Gln Gln Gly Glu Pro Lys Val Lys Tyr Val Val Lys 105
106 Ser His Gly Gly Asp Thr Pro Asp Asp Ala Ser Ile Ser Pro Ser 120
121 Ala Gln Phe Ser Met Asp Phe Gln Glu Thr Asp Pro Tyr Phe Arg 135
136 Arg Glu Val Ile His Tyr Ala Ile Gln Leu Arg Gly Ala Lys Leu 150
151 Trp Asp Asp Asp Gln Ser Met Ala Leu Thr Arg Met Asn Val Thr 165
166 Ala Gly Tyr Val Ser Ala Asn Ile Ile Gln Tyr Pro Lys Ser Lys 180
181 Arg Cys Arg Leu His Pro Asp Val Asp Asp Val Ser Ser Leu Tyr 195
196 Thr Asp Asn Ser Ile Ser Thr Asp Gly Ile Leu Leu Val Pro Thr 210
211 Ala Lys Ala Asn Thr Ser Pro Ser Asp Pro Arg Leu Ala Ile Ser 225
226 Gln Ala Phe Ile Gly Asp Ser Ile Leu Val Asp Gly Lys Cys Trp 240
241 Pro Thr Leu Glu Ala Ile Pro Asn Glu Tyr Ser Thr Arg Val Thr 255
256 Asn Ala Cys His Thr Lys His Tyr Asn Tyr Ala Leu Glu Leu Gly 270
271 Glu Asp Phe Ile Leu Leu Gly Ala Tyr Gly Gly Trp Leu Pro Ile 285
286 Ser Val Asn Lys Asn Ser Lys Ser Leu Gly Ser Ala Asp Ser Tyr 300
301 Asp Ile Ile Ile Asn Phe Thr Gly Tyr Glu Gly Ser Ser Ile Leu 315
316 Thr Ala Ile Ser Ala Glu Ser Gly Gly Arg Val Asn Leu Gln Thr 330
331 Asp Asp His Ile Leu Lys Phe Arg Glu Pro Lys Leu Met Ala Gln 345
346 Gly Asp Asp Tyr Arg Lys His Lys Ala Pro Ala Ser Ala Pro Ser 360
361 Gly Gln His Glu Leu Phe Gln Asn Leu Arg Thr Ser Lys Leu Asp 375
376 Arg Thr Gln Asp Glu Tyr Gly Arg Pro Val Leu Leu Leu Asn Asn 390
391 Lys Arg Trp His Asp Pro Val Thr Thr Thr Pro Phe Val Gly Thr 405
406 Thr Gly Met Ala Ser Ile Asn Tyr Pro Thr Thr Gly Ala His Pro 420
421 Ile Leu Leu His Phe Ile Ser Phe Ser Val Leu Ser Leu Arg Thr 435
436 Ser Asp Ile Ile Arg Thr Tyr Glu Ser Asp Gln Leu Tyr Val Thr 450
451 Val Lys Ala Val Pro Trp Pro Leu Ser Glu Asn Thr Trp Lys Arg 465
466 Thr Ile Gln Ala His Ala Glu Glu Val Tyr Arg Ile Ala Phe Thr 480
481 Phe Glu Thr Tyr Ser His Arg Gly Gly Trp His Thr His Ile Ser 495
496 Gln His Glu Ile Tyr Asp Ser Met Arg Gln Arg Asp Met Ala Asp 510
511 Pro Asn Lys Leu Pro Leu Ile Pro Ser Pro Glu Phe His Tyr Gly 525
526 Lys Cys Tyr Leu Ser Thr Ala Asp Ser Tyr Tyr Gln Glu Asn Tyr 540
541 Leu Gly Pro Cys Leu Gly Ser Gln His Cys Ser Thr Pro End 554
<210> 2
<211> 1662
<212> DNA
<213> Unknown
<220>
＜223〉artificial sequence
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atgattgctg aaggtaatgt ttcgggattg gagatgatag atttactgaa gcctagacaa 60
cagtatatag aaagcactta ccatgatctt ctaatgaggg agtatataca actatcgtat 120
aggaatttgc tcccaactga atgttggtcc caggacggtt tgttcctagg tccatacctt 180
gaaatgccta aagacgtcaa cttatacgtt agggtaatga actcaatgcc attacctcat 240
ttgaatccaa ttgtacatac tacttatatc tctgcttatc aacaaggaga acctaaagtt 300
aagtacgttg ttaaatcgca tggtggtgat acacctgatg atgcttccat ttcaccaagt 360
gctcaatttt caatggattt tcaagaaact gatccatact ttagaagaga agtcattcat 420
tacgcaatcc aacttagagg tgctaaattg tgggatgatg atcagtctat ggcactgact 480
aggatgaacg ttactgctgg ttatgttagt gcgaacatta ttcaatatcc aaagtcaaag 540
aggtgtagat tgcaccctga tgtcgacgat gtatcatctt tgtatactga taacagtatt 600
agcaccgatg gtattttact tgtacctact gctaaagcca acacttctcc atctgatcca 660
agacttgcta ttagtcaggc ttttatcggt gattcaattt tggtcgacgg taaatgctgg 720
ccgaccttgg aagcgatccc aaacgagtac agtactagag ttaccaacgc ttgtcacact 780
aagcattaca actatgcttt ggaactcggt gaggatttta ttctacttgg tgcttatggt 840
ggatggttgc caatttctgt taacaagaac tctaaatctt tgggatcagc tgattcatac 900
gatatcatta ttaattttac tggatacgaa ggatcctcta ttttgacggc tatctcggct 960
gaaagtggtg gccgagttaa cctgcaaact gatgatcaca ttttgaaatt tagagaacct 1020
aagcttatgg ctcaaggcga tgattataga aagcacaagg caccggcttc tgctccatct 1080
ggccaacatg aattattcca aaaccttaga acgtccaagt tggatcgtac tcaagatgaa 1140
tacggtagac cagttttgtt gttgaacaac aagagatggc atgatccagt tactacaact 1200
ccattcgttg gtactacagg aatggcgtct attaattacc caactactgg tgcccatcca 1260
atacttttgc atttcatttc tttttctgtt ttgagtctca gaacatctga tataattaga 1320
acatacgaat ctgatcaatt gtatgtcact gttaaagctg ttccttggcc actgtctgaa 1380
aatacttgga agaggactat tcaggctcat gctgaggaag tttacagaat tgcttttact 1440
tttgaaacat actcacatag agggggttgg catactcata tttcccaaca tgagatttac 1500
gattccatga gacagaggga tatggccgat ccaaacaagt taccacttat accttcgcca 1560
gaatttcact acggaaaatg ttatttgagc acggccgact cttactacca agaaaactat 1620
ctgggaccat gcttgggatc tcaacactgt agcactccct aa 1662
Claims (7)
1. laccase gene, it is characterized in that: its nucleotide sequence is shown in SEQ ID NO:2.
2. the laccase of genes encoding according to claim 1, it is characterized in that: its aminoacid sequence is shown in SEQ ID NO:1.
3. contain the recombinant expression vector that right requires 1 described laccase gene.
4. contain the host bacterium that right requires 1 described laccase gene.
5. the application of laccase gene as claimed in claim 1 in preparation reorganization laccase.
6. contain the application of engineering bacteria in paper waste is handled that right requires 1 described laccase gene, it is characterized in that: the optimum temperuture of described laccase is 30-40 ℃, and optimal pH is 5-6.
7. contain the application of engineering bacteria in paper waste is handled that right requires 1 described laccase gene, it is characterized in that: after genetic engineering bacterium was carried out enlarged culturing, the addition by 0.05% adds in the aeration tank to be handled.
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| FI113879B (en) * | 2000-05-23 | 2004-06-30 | Valtion Teknillinen | A new coating enzyme |
| CN1308435C (en) * | 2004-04-21 | 2007-04-04 | 中国科学院微生物研究所 | Laccase, preparing method thereof and strain special for same |
| CN101023165B (en) * | 2004-09-21 | 2011-08-24 | 生化酶股份有限公司 | Novel laccase enzyme and use thereof |
| CN102080046A (en) * | 2010-09-25 | 2011-06-01 | 江南大学 | High-yield laccase strain and method for producing laccase through fermentation |
| CN102115724B (en) * | 2010-12-02 | 2013-05-22 | 东北林业大学 | Bacillus amyloliquefaciens LS01 laccase and application thereof |
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