CN101568640A - Danisco us inc genencor div - Google Patents

Danisco us inc genencor div Download PDF

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CN101568640A
CN101568640A CNA2007800470185A CN200780047018A CN101568640A CN 101568640 A CN101568640 A CN 101568640A CN A2007800470185 A CNA2007800470185 A CN A2007800470185A CN 200780047018 A CN200780047018 A CN 200780047018A CN 101568640 A CN101568640 A CN 101568640A
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laccase
seq
gene
pcr
primer
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CN101568640B (en
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J·C·麦考利夫
王华明
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Danisco USA Inc
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Danisco USA Inc
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Priority claimed from PCT/US2007/025533 external-priority patent/WO2008076322A2/en
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Abstract

Novel laccases, nucleic acid sequences encoding such laccases, and vectors and host cells for expressing the laccases are described. The novel laccase enzymes may be employed in conjunction with mediators to provide an improved method for bleaching denim fabrics.

Description

Novel laccase enzyme, composition and using method
The cross reference of related application
[01] the application's name of requiring to submit on December 18th, 2006 is called the U.S. Provisional Patent Application series number No.60/875 of " Novel Laccases; Compositions and Methods of Use ", the name of submitting on December 18th, 518 and 2006 is called the U.S. Provisional Patent Application series number No.60/875 of " Laccase Mediatorsand Methods of Use ", 454 right of priority.
Invention field
[02] the present invention relates to the nucleotide sequence of laccase and this laccase of coding, and relate to the enzymatic means that is used for bleaching material.
Background of invention
[03] laccase is a copper enzyme, and known its is good oxygenant in the presence of oxygen.In microorganism, fungi and more high biology, found laccase.Laccase is used for multiple application, comprises that paper pulp and textile bleaching, paper pulp wastewater processing, deinking, industry decolouring, bleaching laundry detergent, oral cavity health teeth whitening and conduct are used for the catalyzer or the promotor of polymerization and oxidizing reaction.
[04] laccase can be widely used in many industry, comprises detergent industry, paper industry, industrial textile and foodstuffs industry.In the application, with phenol oxidase as when washing composition cleans from the clothes the auxiliary agent of decontamination (as food stains).
[05] most of laccases have represented best pH and passivation under neutrality or alkaline pH in the acid pH scope.
[06] known to multiple fungi generation laccase, this fungi comprises the kind of following genus (genii), Aspergillus (Aspergillus), Neurospora (Neurospora), handle spore shell belongs to (Podospora), Staphlosporonites (Botrytis), pleurotus (Pleurotus), Fornes, penetrate arteries and veins Pseudomonas (Phlebia), trametes (Trametes), Polyporus (Polyporus), grape ear mould (Stachybotrys), Rhizoctonia (Rhizoctonia), Bipolaris (Bipolaris), Curvularia (Curvularia), the shell monospore belongs to (Amerosporium), and Lentinus (Lentinus).Yet, the laccase that still need in multiple application, have the different performance spectrum.
[07], can render a service by the oxidation of using amboceptor (also being known as toughener) to improve laccase for many application.Known in the artly comprise that the system of laccase and amboceptor is laccase-mediator systems (LMS).Identical compound also can be used to activate or the effect of initial laccase.
[08] there are some known amboceptors that are used for laccase-mediator systems.These comprise HBT (I-hydroxybenzotriazole), ABTS[2; 2 '-azine-two (3-ethyl benzo thiazole phenanthrolines-6-sulfonic acid)], NHA (N-hydroxyacetanilide), NEIAA (N-ethanoyl-N-Phenylhydroxylamine), HBTO (3-hydroxyl 1; 2,3-phentriazine-4 (3H)-ketone) and VIO (violuric acid).In addition, some compounds that contain NH-OH or N-O that can be used as amboceptor have been found to exist.
[09] functional group and substituting group have a significant impact for amboceptor effectiveness tool.Even in similar compound, substituting group can change the specificity of laccase to substrate, thereby increases greatly or reduce amboceptor and render a service.In addition, amboceptor may effectively be unsuitable for Another Application for a kind of concrete application.Another shortcoming of present amboceptor is that they are tending towards polymerization in use.Thereby, need exploitation to be used for effective amboceptor of application-specific.One this kind of class application is the bleaching of textiles, its mesosome costliness within reason or harmful also be very important.Other application of laccase-mediator systems have been provided below.
[10] therefore, the active amboceptor that needs to identify other activation laccase and/or strengthen the enzyme that represents laccase activity.
Summary of the invention
[11] described herein is novel laccase enzyme, the nucleotide sequence of this laccase of encoding and the carrier and the host cell of expressing this laccase.
Brief Description Of Drawings
[12] Fig. 1 is the synoptic diagram of Trichoderma (Trichoderma) the expression plasmid pTrex3g-laccase A of use among the embodiment 7.Available other laccase genes described herein replace laccase A gene.
[13] Fig. 2 is the synoptic diagram of the Aspergillus expression plasmid pKB401 that uses among the embodiment 8a.Available other laccase genes described herein replace laccase B gene.
[14] Fig. 3 is the synoptic diagram of the Aspergillus expression plasmid pKB403 that uses among the embodiment 8b.Laccase B gene fusion is in the gene of the catalyst structure domain of coding glucoamylase.Available other laccase genes described herein replace laccase B gene.
[15] Fig. 4 is the synoptic diagram of the Trichoderma expression plasmid pTrex4-laccase B that uses among the embodiment 8d.The gene fusion of the catalyst structure domain of laccase B gene and coding CBH1.Available other laccase genes described herein replace laccase B gene.
[16] Fig. 5 is the synoptic diagram of streptomyces (Streptomyces) the expression plasmid pKB251 that is used for codon optimized laccase B gene of use among the embodiment 9.
[17] Fig. 6 is the synoptic diagram of bacillus (Bacillus) the expression plasmid p2JMagk1031nk2E-laccase that is used for codon optimized laccase D gene of use among the embodiment 13, the gene fusion of laccase D gene and coding BCE103.
[18] Fig. 7 is a histogram, and the result of the laccase and the multiple amboceptor bleaching indigo blue,soluble that concentration is 50 and 500 μ M of use Thielavia species (Thielavia sp.) is shown.
[19] Fig. 8 is a histogram, and the laccase of the species that use the black hole of Thielavia (Thielavia), myceliophthora (Myceliophthora) and hypodermis to belong to (Cerrena) and multiple amboceptor is bleached indigo blue,soluble at pH5 result are shown.
[20] Fig. 9 is a histogram, and the laccase of the species that use the black hole of Thielavia, myceliophthora and hypodermis genus and multiple amboceptor is bleached indigo blue,soluble at pH7 result are shown.
[21] Figure 10 is when 60 ℃ and pH6, as the reorganization laccase D of the function of mediator concentration and enzyme concn and the total color difference figure of cloves acid amides amboceptor.
[22] Figure 11 is when 60 ℃ and pH6, as the reorganization laccase D of the function of mediator concentration and enzyme concn and the total color difference figure of cloves nitrile (syringonitrile) amboceptor.
Invention specifies
[23] unless this paper has definition in addition, otherwise all technology used herein and scientific terminology have with those skilled in the art and usually understand identical implication. The people such as Singleton, DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY second edition, John Wiley and Sons, New York (1994), and Hale﹠Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, N.Y. (1991) provides the general dictionary of the many terms that use among the present invention to the technical staff. Although can be used for practice or check the present invention with those similar or equivalent any methods described herein and material, describe preferred method and material. Number range contains the numerical value that limits this scope. Should understand the present invention and be not limited to described concrete grammar, scheme and reactant, because these can change.
[24] exercise question provided herein does not constitute the restriction to multiple aspect of the present invention or embodiment, and they can obtain with reference to specification sheets by integral body.Therefore, more fully limit following back to back term with reference to specification sheets by integral body.
[25] for the composition and the methodological purpose of describing and openly can unite with the present invention use, all publications that this paper quotes are by quoting as a reference clearly.
I. laccase and laccase involved enzyme
[26] in the context of the invention, laccase and laccase involved enzyme contain any laccase that is included in the enzyme classification (EC1.10.3.2).Known laccase is from microorganism and plant-sourced.The microorganism laccase can be derived from the laccase that bacterium or fungi (comprising filamentous fungus and yeast) and suitable example comprise the bacterial strain that is derived from following genus: Aspergillus, Neurospora (for example Neuraspora crassa (N.crassa.)), handle spore shell belongs to, Staphlosporonites, gold thread Pseudomonas (Collybia), the black hole of hypodermis belongs to, grape ear mould, the leather ear belongs to (Panus, for example remove from office ear (Panus rudis)), fusarium globosum shuttle (Theilava), shelf fungus belongs to (Fomes), Lentinus, pleurotus, trametes, for example long wool hair bolt bacterium (T.villosa) and variable color bolt bacterium (T.versicolor), Rhizoctonia, R.solani for example, Coprinus (Coprinus), for example pleat line ghost umbrella (C.plicatilis) and Coprinus cinereus (C.cinereus), Psatyrella, myceliophthora, the for example thermophilic silk mould (M.thermonhila) of ruining, Schytalidium, penetrate the arteries and veins Pseudomonas, for example penetrate arteries and veins bacterium (P.radita) (WO 92/01046), or Coriolus Qu61 (Coriolus), for example hairy fungus (C.hirsutus) (JP 2-238885), continuous hole skin Pseudomonas species (Spongipellis Sp.), Polyporus, whiterot fungi (Ceriporiopsis subvermispora), tertia glossy ganoderma (Ganodermatsunodae) and Trichoderma.
[27] in addition, can produce laccase or laccase involved enzyme by such method, this method is included under the condition that allows the laccase expression, in substratum, cultivate host cell, and from culture, reclaim laccase, the recombinant DNA carrier of the dna sequence dna that the dna sequence dna that described host cell transforms to be had the dna sequence dna that carries the described laccase of coding and allow this laccase of coding is expressed.
[28] expression vector can be transformed in the suitable host cell, fungal cell for example, its preferred examples is the Aspergillus species, most preferably aspergillus oryzae (Aspergillus oryzae) and aspergillus niger (Aspergillus niger), and Fusarium (Fusarium) kind, the thermophilic hyphomycete of most preferably cotton shape (Fusarium venenatum).Can form and this protoplast transformation by comprising protoplastis, carry out cell walls regenerated method transformed eukaryotic mycetocyte subsequently in a manner known way.EP238,023 has described the application of Aspergillus as host microorganism.WO 96/00787 and WO97/08325 have described the application of Fusarium as host microorganism.
[29] or, host cell can be bacterium, especially the bacterial classification or the intestinal bacteria of bacillus, Rhodopseudomonas (Pseudomonas), streptomyces.Can be according to conventional methods, for example, as people such as T.Maniatis, Molecular Cloning:A Laboratory Manual, ColdSpring Harbor, the 1982 described conversions of carrying out bacterial cell.Also can be by standard method, the dna sequence dna that is fit to referring to people such as T.Maniatis (document of quoting previously) screens and vector construction.
[30] substratum that is used to cultivate the host cell after the conversion can be the conventional substratum of any host cell of being discussed of being suitable for growing.Can be easily with the enzyme secretion of having expressed in substratum and can be by known method from wherein reclaiming this enzyme, this method comprises by centrifugal or filtration isolated cell from substratum, proteinaceous component in the salt precipitation substratum of utilization such as ammonium sulfate reclaims by chromatography method such as ion exchange chromatography, affinity chromatography etc. subsequently.
[31] in embodiment, expressive host can be the Trichodermareesei (Trichoderma reesei) (referring to for example U.S. Patent No. 5,861,271) with the laccase coding region under CBH1 promotor and terminator regulation and control.Expression vector can be pTrex3g, as the U.S. Patent application No.11/245 that submitted on October 7th, 2005, and 628 disclosed (the file number GC886 of agency).
[32] prepare following novel gene and laccase in this way:
A. from the black hole of the hypodermis laccase A1 gene of CBS 115.075 bacterial strains, it has sequence (SEQ ID No.1):
ATGAGCTCAA AGCTACTTGC TCTTATCACT GTCGCTCTCG TCTTGCCACT 50
AGGCACCGAC GCCGGCATCG GTCCTGTTAC CGACTTGCGC ATCACCAACC 100
AGGATATCGC TCCAGATGGC TTCACCCGAC CAGCGGTACT AGCTGGGGGC 150
ACATTCCCTG GAGCACTTAT TACCGGTCAG AAGGTATGGG AGATCAACTT 200
GGTTGAATAG AGAAATAAAA GTGACAACAA ATCCTTATAG GGAGACAGCT 250
TCCAAATCAA TGTCATCGAC GAGCTTACCG ATGCCAGCAT GTTGACCCAG 300
ACATCCATTG TGAGTATAAT TTAGGTCCGC TCTTCTGGCT ATCCTTTCTA 350
ACTCTTACCG TCTAGCATTG GCACGGCTTC TTTCAGAAGG GATCTGCGTG 400
GGCCGATGGT CCTGCCTTCG TTACTCAATG CCCTATCGTC ACCGGAAATT 450
CCTTCCTGTA CGACTTTGAT GTTCCCGACC AACCTGGTAC TTTCTGGTAC 500
CATAGTCACT TGTCTACTCA ATATTGCGAT GGTCTTCGTG GCCCGTTCGT 550
TGTATACGAT CCAAAGGATC CTAATAAACG GTTGTACGAC ATTGACAATG 600
GTATGTGCAT CATCATAGAG ATATAATTCA TGCAGCTACT GACCGTGACT 650
GATGCTGCCA GATCATACGG TTATTACCCT GGCAGACTGG TACCACGTTC 700
TCGCAAGAAC TGTTGTCGGA GTCGCGTAAG TACAGTCTCA CTTATAGTGG 750
TCTTCTTACT CATTTTGACA TAGGACACCC GACGCAACCT TGATCAACGG 800
TTTGGGCCGT TCTCCAGACG GGCCAGCAGA TGCTGAGTTG GCTGTCATCA 850
ACGTTAAACG CGGCAAACGG TATGTTATTG AACTCCCGAT TTCTCCATAC 900
ACAGTGAAAT GACTGTCTGG TCTAGTTATC GATTTCGTCT GGTCTCCATC 950
TCATGTGACC CTAATTACAT CTTTTCTATC GACAACCATT CTATGACTGT 1000
CATCGAAGTC GATGGTGTCA ACACCCAATC CCTGACCGTC GATTCTATTC 1050
AAATCTTCGC AGGCCAACGA TACTCGTTCG TCGTAAGTCT CTTTGCACGA 1100
TTACTGCTTC TTTGTCCATT CTCTGACCTG TTTAAACAGC TCCATGCCAA 1150
CCGTCCTGAA AACAACTATT GGATCAGGGC CAAACCTAAT ATCGGTACGG 1200
ATACTACCAC AGACAACGGC ATGAACTCTG CCATTCTGCG ATACAACGGC 1250
GCACCTGTTG CGGAACCGCA AACTGTTCAA TCTCCCAGTC TCACCCCTTT 1300
GCTCGAACAG AACCTTCGCC CTCTCGTGTA CACTCCTGTG GTATGTTTCA 1350
AAGCGTTGTA ATTTGATTGT GGTCATTCTA ACGTTACTGC GTTTGCATAG 1400
CCTGGAAACC CTACGCCTGG CGGCGCCGAT ATTGTCCATA CTCTTGACTT 1450
GAGTTTTGTG CGGAGTCAAC ATTCGTAAAG ATAAGAGTGT TTCTAATTTC 1500
TTCAATAATA GGATGCTGGT CGCTTCAGTA TCAACGGTGC CTCGTTCCTT 1550
GATCCTACCG TCCCCGTTCT CCTGCAAATT CTCAGCGGCA CGCAGAATGC 1600
ACAAGATCTA CTCCCTCCTG GAAGTGTGAT TCCTCTCGAA TTAGGCAAGG 1650
TCGTCGAATT AGTCATACCT GCAGGTGTCG TCGGTGGACC TCATCCGTTC 1700
CATCTCCATG GGGTACGTAA CCCGAACTTA TAACAGTCTT GGACTTACCC 1750
GCTGACAAGT GCATAGCATA ACTTCTGGGT CGTGCGAAGT GCCGGAACCG 1800
ACCAGTACAA CTTTAACGAT GCCATTCTCC GAGACGTCGT CAGTATAGGA 1850
GGAACCGGGG ATCAAGTCAC CATTCGTTTC GTGGTATGTT TCATTCTTGT 1900
GGATGTATGT GCTCTAGGAT ACTAACCGGC TTGCGCGTAT AGACCGATAA 1950
CCCCGGACCG TGGTTCCTCC ATTGCCATAT CGACTGGCAC TTGGAAGCGG 2000
GTCTCGCTAT CGTATTTGCA GAGGGAATTG AAAATACTGC TGCGTCTAAT 2050
TTAACCCCCC GTACGCGGTT TCCCTCACAT CCTGGAGCTA AGCAGCTTAC 2100
TAACATACAT TTGCAGAGGC TTGGGATGAG CTTTGCCCGA AGTATAACGC 2150
GCTCAGCGCA CAAAAGAAGG TTGCATCTAA GAAAGGCACT GCCATCTAAT 2200
TTTTGTAACA AACAAGGAGG GTCTCTTGTA CTTTTATTGG GATTTCTTTC 2250
TTGGGGTTTA TTGTTAAACT TGACTCTACT ATGTTTGGAA GACGAAAGGG 2300
GCTCGCGCAT TTATATACTA TCTCTCTTGG CATCACCTGC AGCTCAATCC 2350
TTCAACCACC TAA 2363
And coding has the laccase A1 of the protein sequence (SEQ ID No.2) after the translation
MSSKLLALIT VALVLPLGTD AGIGPVTDLR ITNQDIAPDG FTRPAVLAGG 50
TFPGALITGQ KGDSFQINVI DELTDASMLT QTSIHWHGFF QKGSAWADGP 100
AFVTQCPIVT GNSFLYDFDV PDQPGTFWYH SHLSTQYCDG LRGPFVVYDP 150
KDPNKRLYDI DNDHTVITIA DWYHVLARTV VGVATPDATL INGLGRSPDG 200
PADAELAVIN VKRGKRYRFR LVSISCDPNY IFSIDNHSMT VIEVDGVNTQ 250
SLTVDSIQIF AGQRYSFVLH ANRPENNYWI RAKPNIGTDT TTDSGMNSAI 300
LRYNGAPVAE PQTVQSPSLT PLLEQNLRPL VYTPVPGNPT PGGADIVHTL 350
DLSFDAGRFS INGASFLDPT VPVLLQILSG TQNAQDLLPP GSVIPLELGK 400
VVELVIPAGV VGGPHPFHLH GHNFWVVRSA GTDQYNFNDA ILRDVVSIGG 450
TGDQVTIRFV TDNPGPWFLH CHIDWHLEAG LAIVFAEGIE NTAASNLTPQ 500
AWDELCPKYN ALSAQKKLNP STT 523
B. deceive hole laccase A2 gene (SEQ ID No.3) from the hypodermis of CBS 154.29 bacterial strains
ATGAGCTCAA AGCTACTTGC TCTTATTACT GTCGCTCTCG TCTTGCCACT 50
AGGCACTGAC GCCGGCATCG GTCCTGTTAC CGACTTGCGC ATCACCAACC 100
AGGATATCGC TCCAGATGGC TTCACCCGAC CAGCTGTACT GGCTGGGGGC 150
ACATTCCCCG GAGCACTGAT TACCGGTCAG AAGGTATGGG AGATCGATTT 200
CGTTGAATAG AGAAATACAA CTGAAAACAA ATTCTTATAG GGAGACAGCT 250
TCCAAATCAA TGTCATCGAC GAGCTTACCG ATGCCAGCAT GTTGACCCAG 300
ACATCCATTG TGAGTATAAT ATGGGTCCGC TCTTCTAGCT ATCCTTTCTA 350
ACTCTTACCC TCTAGCATTG GCACGGCTTC TTTCAGAAGG GATCTGCGTG 400
GGCCGATGGT CCTGCCTTCG TTACTCAATG TCCTATCGTC ACCGGAAATT 450
CCTTCCTGTA CGACTTTGAT GTCCCCGACC AACCTGGTAC TTTCTGGTAC 500
CATAGTCACT TGTCTACTCA ATATTGCGAT GGTCTTCGGG GCCCGTTCGT 550
TGTATACGAT CCAAAGGATC CTAATAAACG GTTGTACGAC ATTGACAATG 600
GTATGTGCAT CATCATAAAA ATATAATTCA TGCAGCTACT GACCGCGACT 650
GATGCTGCCA GATCATACGG TTATTACCCT GGCAGACTGG TACCACGTTC 700
TCGCACGAAC TGTTGTCGGA GTCGCGTAAG TACAGTCTGA CTTATAGTGG 750
TCTTCTTACT CATTTTGACA TAGGACACCC GACGCAACCT TGATCAACGG 800
TTTGGGCCGT TCTCCAGACG GGCCAGCAGA TGCTGAGTTG GCTGTCATCA 850
ACGTTAAACG CGGCAAACGG TATGTCATTG AACTCCCGAT TTCTCCATTC 900
ACATTGAAAT GACTGTCTGG TCTAGTTATC GATTCCGTCT GGTCTCCATC 950
TCATGTGACC CTAATTACAT CTTTTCTATC GACAACCATT CTATGACTGT 1000
CATCGAAGTC GATGGTGTCA ACACCCAATC CCTGACCGTC GATTCTATCC 1050
AAATCTTCGC AGGCCAACGC TACTCGTTCG TCGTAAGTCT CTTTGAATGG 1100
TTGGTGCTTT TTCTGTCCAT TCTCTAACCT GTTTATACAG CTCCATGCCA 1150
ACCGTCCTGA AAACAACTAT TGGATCAGGG CCAAACCTAA TATCGGTACG 1200
GATACTACCA CAGACAACGG CATGAACTCT GCCATTCTGC GATACAACGG 1250
CGCACCTGTT GCGGAACCGC AAACTGTTCA ATCTCCCAGT CTCACCCCTT 1300
TGCTCGAACA GAACCTTCGC CCTCTCGTGT ACACTCCTGT GGTATGTTTC 1350
AAAGCGTTGT AATTTGATTG TGGTCATTCT AACGTTACTG CCTTTGCACA 1400
GCCTGGAAAT CCTACGCCTG GCGGGGCCGA TATTGTCCAT ACTCTTGACT 1450
TGAGTTTTGT GCGGAGTCAA CATTCGTAAA GATAAGAGTG TTTCTAATTT 1500
CTTCAATAAT AGGATGCTGG TCGCTTCAGT ATCAACGGTG CCTCGTTCCT 1550
TGATCCTACC GTCCCTGTTC TCCTGCAAAT TCTCAGCGGC ACGCAGAATG 1600
CACAAGATCT ACTCCCTCCT GGAAGTGTGA TTCCTCTCGA ATTAGGCAAG 1650
GTCGTCGAAT TAGTCATACC TGCAGGTGTT GTCGGTGGAC CTCATCCGTT 1700
CCATCTCCAT GGGGTACGTA ACCCGAACTT ATAACAGTCT TGGACTTACC 1750
CGCTGACAAG TGTATAGCAT AACTTCTGGG TCGTGCGAAG TGCCGGAACC 1800
GACCAGTACA ACTTTAACGA TGCCATTCTC CGAGACGTCG TCAGTATAGG 1850
AGGAACCGAG GATCAAGTCA CCATTCGATT CGTGGTATAT ACTTCATTCT 1900
TGTGGATGTA TGTGCTCTAG GATACTAACT GGCTTGCGCG TATAGACCGA 1950
TAACCCCGGA CCGTGGTTCC TCCATTGCCA TATCGACTGG CACTTGGAAG 2000
CGGGTCTCGC TATCGTATTT GCAGAGGGAA TTGAAAATAC TGCTGCGTCT 2050
AATCCAACCC CCCGTATGCG GTTTCCCACA CATTCTGAAT CTAAGCAGCT 2100
TACTAATATA CATTTGCAGA GGCTTGGGAT GAGCTTTGCC CGAAGTATAA 2150
CGCGCTCAAC GCACAAAAGA AGGTTGCATC TAAGAAAGGC ACTGCCATCT 2200
AATCCTTGTA ACAAACAAGG AGGGTCTCTT GTACTTTTAT TGGGATTTAT 2250
TTCTTGGGGT TTATTGTTCA ACTTGATTCT ACTATGTTTG GAAGTAGCGA 2300
TTACGAAAGG GGCTTGCGCA TTTATATACC ATCTTTCTTG GCACCACCTG 2350
CAGCTCAATC CTTCAACCAC CTAA 2374
Its coding has the laccase A2 of the posttranslational protein matter sequence shown in SEQ ID No.4,
MSSKLLALIT VALVLPLGTD AGIGPVTDLR ITNQDIAPDG FTRPAVLAGG 50
TFPGALITGQ KGDSFQINVI DELTDASMLT QTSIHWHGFF QKGSAWADGP 100
AFVTQCPIVT GNSFLYDFDV PDQPGTFWYH SHLSTQYCDG LRGPFVVYDP 150
KDPNKRLYDI DNDHTVITLA DWYHVLARTV VGVATPDATL INGLGRSPDG 200
PADAELAVIN VKRGKRYRFR LVSISCDPNY IFSIDNHSMT VIEVDGVNTQ 250
SLTVDSIQIF AGQRYSFVLH ANRPENNYWI RAKPNIGTDT TTDNGMNSAI 300
LRYNGAPVAE PQTVQSPSLT PLLEQNLRPL VYTPVPGNPT PGGADIVHTL 350
DLSFDAGRFS INGASFLDPT VPVLLQILSG TQNAQDLLPP GSVIPLELGK 400
VVELVIPAGV VGGPHPFHLH GHNFWVVRSA GTDQYNFNDA ILRDVVSIGG 450
TEDQVTIRFV TDNPGPWFLH CHIDWHLEAG LAIVFAEGIE NTAASNPTPQ 500
AWDELCPKYN ALNAQKKLNP STT 523
C. deceive hole laccase B1 gene (SEQ ID No.5) from the hypodermis of CBS 115.075 bacterial strains
ATGTCTCTTC TTCGTAGCTT GACCTCCCTC ATCGTACTAG TCATTGGTGC 50
ATTTGCTGCA ATCGGTCCAG TCACTGACCT ACATATAGTG AACCAGAATC 100
TCGACCCAGA TGGTTTCAAC CGCCCCACTG TACTCGCAGG TGGTACTTTC 150
CCCGGTCCTC TGATTCGTGG TAACAAGGTA CGCTTCATAA CCGCCCTCCG 200
TAGACGTAGG CTTCGGCTGA CATGACCATC ATCTGTAGGG AGATAACTTT 250
AAAATTAATG TGATTGACGA CTTGACAGAG CACAGTATGC TCAAGGCTAC 300
GTCCATCGTA AGTCCCTGAT TAACGTTTCA CCTGGTCATA TCGCTCAACG 350
TCTCGAAGCA CTGGCATGGG TTCTTCCAGA AGGGAACCAA CTGGGCCGAT 400
GGCCCCGCCT TTGTCACCCA ATGTCCTATC ACATCAGGAA ACGCCTTCCT 450
GTATGATTTC AACGTTCCGG ACCAAGCTGG TACTTTCTGG TACCACAGCC 500
ATCTCTCTAC ACAGTATTGT GACGGTCTTC GTGGTGCCTT TGTCGTCTAT 550
GATCCTAATG ATCCCAACAA GCAACTCTAT GATGTTGATA ACGGCAAGTT 600
CCTTGCATAT TTCATTTCTA TCATATCCTC ACCTGTATTG GCACAGAAAG 650
CACCGTGATT ACCTTGGCTG ATTGGTATCA TGCCCTTGCT CAGACTGTCA 700
CTGGTGTCGC GTGAGTGACA AATGGCCCTC AATTGTTCAC ATATTTTCCT 750
GATTATCATA TGATAGAGTA TCTGATGCAA CGTTGATCAA CGGATTGGGA 800
CGTTCGGCCA CCGGCCCCGC AAATGCCCCT CTGGCGGTCA TCAGTGTCGA 850
GCGGAATAAG AGGTCAGTTC CATAATTATG ATTATTTCCC GCGTTACTTC 900
CTAACAATTA TTTTTGTATC CCTCCACAGA TATCGTTTCC GATTGGTTTC 950
TATTTCTTGC GACCCTAACT TTATTTTCTC AATTGACCAC CACCCAATGA 1000
CCGTAATTGA GATGGACGGT GTTAATACCC AATCTATGAC CGTAGATTCG 1050
ATCCAAATAT TCGCAGGTCA ACGATATTCA TTTGTCGTAG GTTATTATAA 1100
ACTGCCCACC GATCATCTCT CACGTAACTG TTATAGATGC AAGCCAACCA 1150
ACCAGTTGGA AATTATTGGA TCCGCGCTAA ACCTAATGTT GGGAACACAA 1200
CTTTCCTTGG AGGCCTGAAC TCCGCTATAT TACGATATGT GGGAGCCCCT 1250
GACCAAGAAC CGACCACTGA CCAAACACCC AACTCTACAC CGCTCGTTGA 1300
GGCGAACCTA CGACCCCTCG TCTATACTCC TGTGGTATGT TGTTCTCGTT 1350
ACATATACCA AACCTAATAT GAAGACTGAA CGGATCTACT AGCCGGGACA 1400
GCCATTCCCT GGCGGTGCTG ATATCGTCAA GAACTTAGCT TTGGGTTTCG 1450
TACGTGTATT TCACTTCCCT TTTGGCAGTA ACTGAGGTGG AATGTATATA 1500
GAATGCCGGG CGTTTCACAA TCAATGGAGC GTCCCTCACA CCTCCTACAG 1550
TCCCTGTACT ACTCCAGATC CTCAGTGGTA CTCACAATGC ACAGGATCTT 1600
CTCCCAGCAG GAAGCGTGAT CGAACTTGAA CAGAATAAAG TTGTCGAAAT 1650
CGTTTTGCCC GCTGCGGGCG CCGTTGGCGG TCCTCATCCT TTTCACTTAC 1700
ATGGTGTAAG TATCAGACGT CCTCATGCCC ATATTGCTCC GAACCTTACA 1750
CACCTGATTT CAGCACAATT TCTGGGTGGT TCGTAGCGCC GGTCAAACCA 1800
CATACAATTT CAATGATGCT CCTATCCGTG ATGTTGTCAG TATTGGCGGT 1850
GCAAACGATC AAGTCACGAT CCGATTTGTG GTATGTATCT CGTGCCTTGC 1900
ATTCATTCCA CGAGTAATGA TCCTTACACT TCGGGTTCTC AGACCGATAA 1950
CCCTGGCCCA TGGTTCCTTC ACTGTCACAT TGACTGGCAT TTGGAGGCTG 2000
GGTTCGCTGT AGTCTTTGCG GAGGGAATCA ATGGTACTGC AGCTGCTAAT 2050
CCAGTCCCAG GTAAGACTCT CGCTGCTTTG CGTAATATCT ATGAATTTAA 2100
ATCATATCAA TTTGCAGCGG CTTGGAATCA ATTGTGCCCA TTGTATGATG 2150
CCTTGAGCCC AGGTGATACA TGA 2173
Its coding has the laccase B1 of posttranslational protein matter sequence (SEQ ID No.6)
MSLLRSLTSL IVLVIGAFAA IGPVTDLHIV NQNLDPDGFN RPTVLAGGTF 50
PGPLIRGNKG DNFKINVIDD LTEHSMLKAT SIHWHGFFQK GTNWADGPAF 100
VTQCPITSGN AFLYDFNVPD QAGTFWYHSH LSTQYCDGLR GAFVVYDPND 150
PNKQLYDVDN GNTVITLADW YHALAQTVTG VAVSDATLIN GLGRSATGPA 200
NAPLAVISVE RNKRYRFRLV SISCDPNFIF SIDHHPMTVI EMDGVNTQSM 250
TVDSIQIFAG QRYSFVMQAN QPVGNYWIRA KPNVGNTTFL GGLNSAILRY 300
VGAPDQEPTT DQTPNSTPLV EANLRPLVYT PVPGQPFPGG ADIVKNLALG 350
FNAGRFTING ASLTPPTVPV LLQILSGTHN AQDLLPAGSV IELEQNKVVE 400
IVLPAAGAVG GPHPFHLHGH NFWVVRSAGQ TTYNFNDAPI RDVVSIGGAN 450
DQVTIRFVTD NPGPWFLHCH IDWHLEAGFA VVFAEGINGT AAANPVPAAW 500
NQLCPLYDAL SPGDT 515
D. deceive hole laccase B2 gene (SEQ ID No.7) from the hypodermis of CBS 154.29 bacterial strains
CACCGCGATG TCTCTTCTTC GTAGCTTGAC CTCCCTCATC GTACTAGCCA 50
CTGGTGCATT TGCTGCAATC GGTCCAGTCA CCGACCTACA TATAGTGAAC 100
CAGAATCTCG CCCCAGATGG TTTAAACCGC CCCACTGTAC TCGCAGGTGG 150
TACTTTCCCC GGTCCTCTGA TTCGTGGTAA CAAGGTACGC TTCATAACCG 200
CCCTCCGTAG ACGTAGGCTT CGGCTGACAT GACCATCATC TGTAGGGAGA 250
TAACTTTAAA ATTAATGTGA TTGACGACTT GACAGAACAC AGTATGCTCA 300
AGGCTACGTC CATTGTAAGT CCCTGATTAA CGTTTCACCT GGTCATATCG 350
CTCAACGTCT CGAAGCACTG GCATGGGTTC TTCCAGAAGG GAACCAACTG 400
GGCCGATGGC CCCGCCTTTG TCACCCAATG TCCTATCACA TCAGGAAACG 450
CCTTCTTGTA TGATTTCAAC GTTCCGGACC AAGCTGGTAC TTTCTGGTAC 500
CACAGCCATC TCTCYACACA GTATTGTGAC GGTCTTCGTG GTGCCTTTGT 550
CGTCTATGAT CCTAATGATC CCAACAAGCA ACTCTATGAT GTTGATAACG 600
GCAAGTCCCT TGCATATTTC AGTTCTATCA TATCCTCACC TGTATTGGCA 650
CAGAAAGCAC CGTGATTACC TTGGCTGATT GGTATCATGC CCTTGCTCAG 700
ACTGTCACTG GTGTCGCGTG AGTGACAAAT GGCCCTTAAT TGTTCACATA 750
TTTTCCTGAT TATCATATGA TAGAGTATCT GATGCAACGT TGATCAACGG 800
ATTGGGACGT TCGGCCACCG GCCCCGCAAA TGCCCCTCTG GCGGTCATCA 850
GTGTCGAGCG GAATAAGAGG TCAGTTCCAT AATTATGATT ATTTCCCGCG 900
TTACTTCCTA ACGATTATTT TTGTATCCCT CCACAGATAT CGTTTCCGAT 950
TGGTTTCTAT TTCTTGCGAC CCTAACTTTA TTTTCTCAAT TGACCACCAC 1000
CCAATGACCG TAATTGAGAT GGACGGTGTT AATACCCAAT CTATGACCGT 1050
AGATTCGATC CAAATATTCG CAGGTCAACG ATATTCATTT GTCGTAGGTT 1100
ATTATAAACT GCCCACCGAT CATCTCTCAC GTAACTGTTA TAGATGCAAG 1150
CCAACCAACC AGTTGGAAAT TATTGGATCC GYGCTAAACC TAATGTTGGG 1200
AACACAACTT TCCTTGGAGG CCTGAACTCC GCTATATTAC GATATGTGGG 1250
AGCCCCTGAC CAAGAACCGA CCACTGACCA AACACCCAAC TCTACACCGC 1300
TCGTCGAGGC GAACCTACGT CCCCTCGTCT ATACTCCTGT GGTATGTTGT 1350
TCTCGTTACA TATACCAAAC CTAATATGAG GACTGAACGG ATCTACTAGC 1400
CGGGACAGCC ATTCCCTGGC GGTGCTGATA TCGTCAAGAA CTTAGCTTTG 1450
GGTTTCGTAC GTGTATTTCA CTTCCCTTTT GGCAGTAACT GAGGTGGAAT 1500
GTATATAGAA TGCCGGGCGT TTCACAATCA ATGGAACATC CTTCACACCT 1550
CCTACAGTCC CTGTACTACT CCAGATCCTC AGTGGTACTC ACAATGCACA 1600
GGATCTTCTT CCAGCAGGAA GCGTGATCGA ACTTGAACAG AATAAAGTTG 1650
TCGAAATCGT TCTGCCCGCT GCGGGCGCCG TTGGCGGTCC TCATCCTTTC 1700
CACTTACATG GTGTAAGTAT CAGACGTCCT CATGCCTATA TTGCTCCGAA 1750
CCTTACACAC CTGATTTCAG CACAATTTCT GGGTGGTTCG TAGCGCCGGT 1800
CAAACCACAT ACAATTTCAA TGATGCTCCT ATCCGTGATG TTGTCAGTAT 1850
TGGCGGTGCA AACGATCAAG TCACGATCCG ATTTGTGGTA TGTATCTCGT 1900
GCCTTGCATT CATTCCACGA GTAATGATCC TTACACTTCG GGTTCTCAGA 1950
CCGATAACCC TGGCCCATGG TTCCTTCACT GTCACATTGA CTGGCATTTG 2000
GAGGCTGGGT TCGCTGTAGT CTTTGCGGAG GGAATCAATG GCACTGCAGC 2050
TGCTAATCCA GTCCCAGGTA AGACTCTCGC TGCTTTGCGT AATATCTATG 2100
AATTTAAAGC ATATCAATTT GCAGCGGCTT GGAATCAATT GTGCCCGTTG 2150
TATGATGCCT TGAGCCCAGG tGATACATGA TTACTCGTAG CTGTGCTTTC 2200
TTATACATAT TCTATGGGTA TATCGGAGTA GCTGTACTAT AGTATGTACT 2250
ATACTAGGTG GGATATGYTG ATGTTGATTT ATATAATTTT GTTTGAAGAG 2300
TGACTTTATC GACTTGGGAT TTAGCCGAGT ACATACTGAT CTCTCACTAC 2350
AGGCTTGTTT TGTCTTTGGG CGCTTACTCA ACAGTTGACT GTTTTTGCTA 2400
TTACGCATTG AACCGCATTC CGGTCYGACT CGTGTCCTCT ACTGTGACTT 2450
GTATTGGCAT TCTAGCACAT ATGTCTCTTA CCTATAGGAA CAATATGTCT 2500
CAACACTGTT CCAAAACCTG CGTAAACCAA ATATCGTCCA TCAGATCAGA 2550
TCATTAACAG TGCCGCACTA ACCTAATACA CTGGCARGGA CTGTGGAAAT 2600
CCCTATAAAT GACCTCTAGA CCGTGAGGTC ATTGCAAGGT CGCTCTCCTT 2650
GTCAAGATGA CCC 2663
Its coding has the laccase B2 of posttranslational protein matter sequence (SEQ ID No.8)
MSLLRSLTSL IVLATGAFAA IGPVTDLHIV NQNLAPDGLN RPTVLAGGTF 50
PGPLIRGNKG DNFKINVIDD LTEHSMLKAT SIHWHGFFQK GTNWADGPAF 100
VTQCPITSGN AFLYDFNVPD QAGTFWYHSH LSTQYCDGLR GAFVVYDPND 150
PNKQLYDVDN GNTVITLADW YHALAQTVTG VAVSDATLIN GLGRSATGPA 200
NAPLAVISVE RNKRYRFRLV SISCDPNFIF SIDHHPMTVI EMDGVNTQSM 250
TVDSIQIFAG QRYSFVMQAN QPVGNYWIRA KPNVGNTTFL GGLNSAILRY 300
VGAPDQEPTT DQTPNSTPLV EANLRPLVYT PVPGQPFPGG ADIVKNLALG 350
FNAGRFTING TSFTPPTVPV LLQILSGTHN AQDLLPAGSV IELEQNKVVE 400
IVLPAAGAVG GPHPFHLHGH NFWVVRSAGQ TTYNFNDAPI RDVVSIGGAN 450
DQVTIRFVTD NPGPWFLHCH IDWHLEAGFA VVFAEGINGT AAANPVPAAW 500
NQLCPLYDAL SPGDT 515
E. deceive hole laccase B3 gene (part) (SEQ IDNo.9) from the hypodermis of ATCC20013 bacterial strain
GTGGGGGCGG ATCCCTAACT GTTTCGAATC GGCACCGAAG TATGCAGGTG 50
TGACGGAGAT GAGGCGTTTT TTCATCTTCC ACTGCAGTAT AAAATGTCTC 100
AGGTAACGTC CAGCTTTTTG TACCAGAGCT ACCTCCAAAT ACCTTTACTC 150
GCAAAGGTTT CGCGATGTCT CTTCTTCGTA GCTTGACCTC CCTCATCGTA 200
CTAGCCACTG GTGCATTTGC TGCAATCGGT CCAGTCACTG ACCTACATAT 250
AGTGAACCAG AATCTCGCCC CAGATGGTTT CAACCGCCCC ACTGTACTCG 300
CAGGTGGTAC TTTCCCCGGT CCTCTGATTC GTGGTAACAA GGTACGCTTC 350
ATAACCGCCC TCCGTAGACG TAGGCTTCGG CTGACATGAC CATCATCTGT 400
AGGGAGATAA CTTTAAAATT AATGTGATTG ACGACTTGAC AGAACACAGT 450
ATGCTCAAGG CCACGTCCAT TGTAAGTCCC TGATTAACGT TTCACCTGGT 500
CATATCGCTC AACGTCTCGA AGCACTGGCA TGGGTTCTTC CAGAAGGGAA 550
CCAACTGGGC CGATGGCCCC GCCTTTGTCA CCCAATGTCC TATCACATCA 600
GGAAACTCCT TCCTGTATGA TTTCAACGTT CCGGACCAAG CTGGTACTTT 650
CTGGTACCAC AGCCATCTCT CTACACAGTA TTGTGACGGT CTTCGTGGTG 700
CCTTTGTCGT CTATGATCCT AATGATCCCA ACAAGCAACT CTATGATGTT 750
GATAACGGCA AGTCCCTTGC ATATTTCATT TCTATCATAT CCTCACCTGT 800
ATTGGCACAG AAAGCACCGT GATTACCTTG GCTGATTGGT ATCATGCCCT 850
TGCTCAGACT GTCACTGGTG TCGCGTGAGT GACAAATGGC CCTCAATTGT 900
TCACATATTT TCCTGATTAT CATATGATAG AGTATCTGAT GCAACGTTGA 950
TCAACGGATT GGGACGTTCG GCCACCGGCC CCGCAAATGC CCCTCTGGCG 1000
GTCATCAGTG TCGAGCGGAA TAAGAGGTCA GTTCCATAAT TATGATTATT 1050
TCCCGCGTTA CTTCCTAACA ATTATTCTTG TATCCCTCCA CAGATATCGC 1100
TTCCGATTGG TGTCTATTTC TTGCGACCCT AACTTTATTT TCTCAATTGA 1150
TCACCACCCA ATGACCGTAA TTGAGATGGA CGGTGTTAAT ACCCAATCTA 1200
TGACCGTAGA TTCGATCCAA ATATTCGCAG GTCAACGATA TTCATTTGTC 1250
GTAGGTTATT ATAAACTGCC CACCGATCAT CTCTCACGTA ACTGTTATAG 1300
ATGCAAGCCA ACCAACCRGT TGGAAATTAT TGGATCC 1337
Its coding has the laccase B3 of part posttranslational protein matter sequence (SEQ ID No.10)
MSLLRSLTSL IVLATGAFAA IGPVTDLHIV NQNLAPDGFN RPTVLAGGTF 50
PGPLIRGNKG DNFKINVIDD LTEHSMLKAT SIHWHGFFQK GTNWADGPAF 100
VTQCPITSGN SFLYDFNVPD QAGTFWYHSH LSTQYCDGLR GAFVVYDPND 150
PNKQLYDVDN GKTVITLADW YHALAQTVTG VAVSDATLIN GLGRSATGPA 200
NAPLAVISVE RNKRYRFRLV SISCDPNFIF SIDHHPMTVI EMDGVNTQSM 250
TVDSIQIFAG QRYSFVMQAN QPVGNYWI 278
F. deceive hole laccase C gene (part) (SEQ IDNo.11) from the hypodermis of CBS 154.29 bacterial strains
TGCAATCGGA CCGGTBGCTG ACCTTCACAT TACGGACGAT ACCATTGCCC 50
CCGATGGTTT CTCTCGTCCT GCTGTTCTCG CTGGCGGGGG TTTCCCTGGC 100
CCTCTCATCA CCGGAAACAA GGTAATGCCT AATGGTTGCG TCTTTGTTGG 150
TGCTCTCATT CATCCACGAC ATTTTGTACC AGGGCGACGC CTTTAAACTC 200
AATGTCATCG ATGAACTAAC GGACGCATCC ATGCTGAAGY CGACTTCCAT 250
CGTAAGTCTC GCTGTATTGC TCCTTGAGCC ATTTCATTGA CTATAACTAC 300
AACCAGCACT GGCATGGATT CTTCCAAAAG GGTACTAATT GGGCAGATGG 350
TCCCGCTTTT GTGAACCAAT GCCCCATCAC CACGGGAAAC TCCTTCTTGT 400
ACGACTTCCA GGTTCCTGAT CAAGCTGGTA AGCATGAGAT TACACTAGGA 450
AAGTTTAATT TAATAACTAT TCAATCAGGA ACCTACTGGT ATCATAGTCA 500
TTTGTCTACG CAATACTGTG ATGGTCTCAG AGGTGCATTC GTTGTCTACG 550
ACCCTTCAGA TCCTCACAAG GATCTCTACG ACGTCGACGA CGGTGAGCTT 600
TGCTTTTTTC ATTGGTATCC ATTATCGCTC ACGTGTCATT ACTGCGCCAC 650
AGAAAGTACC GTCATCACTT TGGCTGATTG GTATCATACT TTGGCTCGTC 700
AGATTGTTGG CGTTGCGTGA GTAGTCTTGT ACCGACTGAA ACATATTCCA 750
GTTGCTGACT TCCCCACAGC ATTTCTGATA CTACCTTGAT AAACGGTTTG 800
GGCCGCAATA CCAATGGTCC GGCTGATGCT GCTCTTGCTG TGATCAATGT 850
TGACGCTGGC AAACGGTGTG TCCAGATTAC TATACTCCCC ATGACGTCTC 900
AATGCTGATG TGTACTACTT CCAGGTACCG TTTCCGTCTT GTTTCCATAT 950
CCTGTGACCC CAATTGGGTA TTCTCGATTG ACAACCATGA CTTTACGGTC 1000
ATTGAAGTCG ATGGTGTTAA CAGTCAACCT CTCAACGTCG ATTCTGTTCA 1050
GATCTTCGCC GGACAACGTT ACTCGTTCGT 1080
Coding has the laccase C of part posttranslational protein matter sequence (SEQ ID No.12)
AIGPVADLHI TDDTIAPDGF SRPAVLAGGG FPGPLITGNK GDAFKLNVID 50
ELTDASMLKX TSIHWHGFFQ KGTNWADGPA FVNQCPITTG NSFLYDFQVP 100
DQAGTYWYHS HLSTQYCDGL RGAFVVYDPS DPHKDLYDVD DESTVITLAD 150
WYHTLARQIV GVAISDTTLI NGLGRNTNGP ADAALAVINV DAGKRYRFRL 200
VSISCDPNWV FSIDNHDFTV IEVDGVNSQP LNVDSVQIFA GQRYSF 246
G. deceive hole laccase D1 gene (SEQ ID No.13) from the hypodermis of CBS 154.29 bacterial strains
GATTCTAATA GACCAGGCAT ACCAAGAGAT CTACAGGTTG ACAGACCATT 50
CTTCTAGGCG GCATTTATGC TGTAGCGTCA GAAATTATCT CTCCATTTGT 100
ATCCCACAGG TCCTGTAATA ACACGGAGAC AGTCCAAACT GGGATGCCTT 150
TTTTCTCAAC TATGGGCGCA CATAGTCTGG ACGATGGTAT ATAAGACGAT 200
GGTATGAGAC CCATGAAGTC AGAACACTTT TGCTCTCTGA CATTTCATGG 250
TTCACACTCT CGAGATGGGA TTGAACTCGG CTATTACATC GCTTGCTATC 300
TTAGCTCTGT CAGTCGGAAG CTATGCTGCA ATTGGGCCCG TGGCCGACAT 350
ACACATTGTC AACAAAGACC TTGCTCCAGA TGGCGTACAA CGTCCAACCG 400
TGCTTGCCGG AGGCACTTTT CCTGGGACGT TGATCACCGG TCAGAAAGTA 450
AGGGATATTA GTTTGCGTCA AAGAGCCAAC CAAAACTAAC CGTCCCGTAC 500
TATAGGGTGA CAACTTCCAG CTCAATGTCA TCGATGATCT TACCGACGAT 550
CGGATGTTGA CGCCAACTTC CATTGTGAGC CTATTATTGT ATGATTTATC 600
CGAATAGTTT CGCAGTCTGA TCATTGGATC TCTATCGCTA GCATTGGCAC 650
GGTTTCTTCC AGAAGGGAAC CGCTTGGGCC GACGGTCCCG CCTTCGTAAC 700
TCAGTGCCCT ATAATAGCAG ATAACTCTTT TCTGTATGAC TTCGACGTCC 750
CAGACCAAGC TGGTACTTTC TGGTATCATA GTCATCTATC CACTCAGTAC 800
TGTGACGGTT TACGTGGTGC CTTCGTTGTG TACGATCCTA ACGATCCTCA 850
CAAAGACCTA TACGATGTTG ATGACGGTGG GTTCCAAATA TTTGTTCTGC 900
AGACATTGTA TTGACGGTGT TCATTATAAT TTCAGAGAGC ACCGTGATTA 950
CCCTTGCGGA TTGGTACCAT GTTCTCGCCC AGACCGTTGT CGGCGCTGCG 1000
TGAGTAACAC ATACACGCGC TCCGGCACAC TGATACTAAT TTTTTTTTAT 1050
TGTAGCACTC CTGATTCTAC CTTGATCAAC GGGTTAGGCC GTTCACAGAC 1100
CGGACCCGCT GATGCTGAGC TGGCTGTTAT CAGCGTTGAA CATAACAAAC 1150
GGTATGTCAT CTCTACCCAG TATCTTCTCT CCTGCTCTAA TTCGCTGTTT 1200
CACCATAGAT ACCGTTTCCG TTTGGTTTCG ATTTCGTGCG ACCCCAACTT 1250
TACCTTCTCC GTTGATGGTC ATAATATGAC TGTCATCGAA GTCGATGGTG 1300
TCAACACACG ACCCCTGACC GTTGACTCTA TTCAAATCTT CGCCGGACAG 1350
AGGTATTCCT TTGTCGTAAG TTAATCGATA TATTCTCCTT ATTACCCCTG 1400
TGTAATTGAT GTCAATAGCT CAATGCTAAC CAACCCGAAG ACAATTACTG 1450
GATCCGTGCT ATGCCAAACA TCGGTAGAAA TACAACAACA CTGGACGGAA 1500
AGAATGCCGC TATCCTTCGA TACAAGAATG CTTCTGTAGA AGAGCCCAAG 1550
ACCGTTGGGG GCCCCGCTCA ATCCCCGTTG AATGAAGCGG ACCTGCGTCC 1600
ACTCGTACCT GCTCCTGTGG TATGTCTTGT CGCGCTGTTC CATCGCTATT 1650
TCATATTAAC GTTTTGTTTT TGTCAAGCCT GGAAACGCTG TTCCAGGTGG 1700
CGCAGACATC AATCACAGGC TTAACTTAAC TTTCGTACGT ACACCTGGTT 1750
GAAACATTAT ATTTCCAGTC TAACCTCTCT TGTAGAGTAA CGGCCTCTTC 1800
AGCATCAACA ACGCCTCCTT CACTaATCCT TCGGTCCCCG CCTTATTACA 1850
AATTCTGAGC GGTGCTCAGA ACGCTCAAGA TTTACTTCCA ACGGGTAGTT 1900
ACATTGGCCT TGAACTAGGC AAGGTTGTGG AGCTCGTTAT ACCTCCTCTG 1950
GCAGTTGGAG GACCGCACCC TTTCCATCTT CATGGCGTAA GCATACCACA 2000
CTCCCGCAGC CAGAATGACG CAAACTAATC ATGATATGCA GCACAATTTC 2050
TGGGTCGTCC GTAGTGCAGG TAGCGATGAG TATAACTTTG ACGATGCTAT 2100
CCTCAGGGAC GTCGTRAGCA TTGGAGCGGG GACTGATGAA GTCACAATCC 2150
GTTTCGTGGT ATGTCTCACC CCTCGCATTT TGAGACGCAA GAGCTGATAT 2200
ATTTTAACAT AGACCGACAA TCCGGGCCCG TGGTTCCTCC ATTGCCATAT 2250
TGATTGGCAT TTGGAGGCAG GCCTTGCCAT CGTCTTCGCT GAGGGCATCA 2300
ATCAGACCGC TGCAGCCAAC CCAACACCCC GTACGTGACA CTGAGGGTTT 2350
CTTTATAGTG CTGGATTACT GAATCGAGAT TTCTCCACAG AAGCATGGGA 2400
TGAGCTTTGC CCCAAATATA ACGGGTTGAG TGCGAGCCAG AAGGTCAAGC 2450
CTAAGAAAGG AACTGCTATT TAAACGTGGT CCTAGACTAC GGGCATATAA 2500
GTATTCGGGT AGCGCGTGTG AGCAATGTTC CGATACACGT AGATTCATCA 2550
CCGGACACGC TGGGACAATT TGTGTATAAT GGCTAGTAAC GTATCTGAGT 2600
TCTGGTGTGT AGTTCAAAGA GACAGCCCTT CCTGAGACAG CCCTTCCTGA 2650
GACAGCCCTT CCTGAGACGT GACCTCCGTA GTCTGCACAC GATACTYCTA 2700
AATACGTATG GCAAGATGAC AAAGAGGAGG ATGTGAGTTA CTACGAACAG 2750
AAATAGTGCC CGGCCTCGGA GAGATGTTCT TGAATATGGG ACTGGGACCA 2800
ACATCCGGA 2809
Its coding has the laccase D1 of posttranslational protein matter sequence (SEQ ID No.14)
MGLNSAITSL AILALSVGSY AAIGPVADIH IVNKDLAPDG VQRPTVLAGG 50
TFPGTLITGQ KGDNFQLNVI DDLTDDRMLT PTSIHWHGFF QKGTAWADGP 100
AFVTQCPIIA DNSFLYDFDV PDQAGTFWYH SHLSTQYCDG LRGAFVVYDP 150
NDPHKDLYDV DDGGTVITLA DWYHVLAQTV VGAATPDSTL INGLGRSQTG 200
PADAELAVIS VEHNKRYRFR LVSISCDPNF TFSVDGHNMT VIEVDGVNTR 250
PLTVDSIQIF AGQRYSFVLN ANQPEDNYWI RAMPNIGRNT TTLDGKNAAI 300
LRYKNASVEE PKTVGGPAQS PLNEADLRPL VPAPVPGNAV PGGADINHRL 350
NLTFSNGLFS INNASFTNPS VPALLQILSG AQNAQDLLPT GSYIGLELGK 400
VVELVIPPLA VGGPHPFHLH GHNFWVVRSA GSDEYNFDDA ILRDVVSIGA 450
GTDEVTIRFV TDNPGPWFLH CHIDWHLEAG LAIVFAEGIN QTAAANPTPQ 500
AWDELCPKYN GLSASQKVKP KKGTAI 526
H. deceive hole laccase D2 gene (SEQ ID No.15) from the hypodermis of CBS 115.075 bacterial strains
GATCTGGACG ATGGTATATA AGACGATGGT ATGAGACCCA TGAAGTCTGA 50
ACACTTTTGC TCTCTGACAT TTCATGGTTC ATACTCTCGA GATGGGATTG 100
AACTCGGCTA TTACATCGCT TGCTATCTTA GCTCTGTCAG TCGGAAGCTA 150
TGCTGCAATT GGGCCCGTGG CCGACATACA CATTGTCAAC AAAGACCTTG 200
CTCCAGATGG TGTACAACGT CCAACCGTGC TCGCCGGAGG CACTTTTCCT 250
GGGACGTTGA TCACCGGTCA GAAAGTAAGG AATATTAGTT TGCGTCAAAG 300
AGCCAACCAA AATTAACCGT CCCGTCCCAT AGGGTGACAA CTTCCAGCTC 350
AATGTCATTG ATGATCTTAC CGACGATCGG ATGTTGACAC CAACTTCCAT 400
TGTGAGCCTA TTATTGTATG ATTTATCCGT ATAGTTTCTC AGTCTGATCA 450
TTGGCTCTCT ATCGCTAGCA TTGGCACGGT TTCTTCCAGA AGGGAACCGC 500
TTGGGCCGAC GGTCCCGCCT TCGTAACTCA GTGCCCTATA ATAGCAGATA 550
ACTCTTTTCT GTATGACTTC GACGTCCCCG ACCAAGCTGG TACTTTCTGG 600
TATCATAGTC ATCTATCCAC TCAGTACTGT GACGGTTTAC GTGGTGCCTT 650
CGTTGTGTAC GATCCTAACG ATCCTCACAA AGACCTATAC GATGTTGATG 700
ACGGTGGGTT CCAAATACTT GACCAAGAAA CATTATATTG ATAGTATCCA 750
CTCTGATTTT CAGAGAGCAC CGTGATTACC CTTGCGGATT GGTACCATGT 800
TCTCGCCCAG ACCGTTGTCG GCGCTGCGTG AGTAACACAT ACACGCGCTC 850
CGGCACACTG ATACTAATTT TTTATTGTAG CACTCCTGAT TCTACCTTGA 900
TCAACGGGTT AGGCCGTTCA CAGACCGGAC CCGCTGATGC TGAGCTGGCT 950
GTTATCAGCG TTGAACATAA CAAACGGTAT GTCATCTCTA CCCATTATCT 1000
TCTCTCCTGC TTTAATTCGC TGTTTCACCA TAGATACCGA TTCCGTTTGG 1050
TTTCGATTTC GTGCGACCCC AACTTTACCT TCTCCGTTGA TGGTCATAAT 1100
ATGACTGTCA TCGAAGTCGA CGGTGTCAAC ACACGACCCC TGACCGTTGA 1150
CTCTATTCAA ATCTTCGCCG GACAGAGGTA TTCCTTTGTC GTAAGTTAAT 1200
CGATATATTC TCCCTATTAC CCCTGTGTAA TTGATGTCAA CAGCTCAATG 1250
CTAACCAACC CGACGACAAT TACTGGATCC GTGCTATGCC AAACATCGGT 1300
AGAAATACAA CAACACTGGA CGGAAAGAAT GCCGCTATCC TTCGATACAA 1350
GAATGCTTCT GTAGAAGAGC CCAAGACCGT TGGGGGCCCC GCTCAATCCC 1400
CGTTGAATGA AGCGGACCTG CGTCCACTCG TACCTGCTCC TGTGGTATGT 1450
CTTGTCGTGC TGTTCCATCG CTATTTCATA TTAACGTTTT GTTTTTGTCA 1500
AGCCTGGAAA CGCTGTTCCA GGTGGCGCAG ACATCAATCA CAGGCTTAAC 1550
TTAACTTTCG TACGTACACC TGGTTGAAAC ATTATATTTC CAGTCTAACC 1600
TCTTGTAGAG TAACGGCCTT TTCAGCATCA ACAACGCCTC CTTCACTAAT 1650
CCTTCGGTCC CCGCCTTATT ACAAATTCTG AGCGGTGCTC AGAACGCTCA 1700
AGATTTACTT CCAACGGGTA GTTACATTGG CCTTGAACTA GGCAAGGTTG 1750
TGGAGCTCGT TATACCTCCT CTGGCAGTTG GAGGACCGCA CCCTTTCCAT 1800
CTTCATGGCG TAAGCATACC ACACTCCCGC AGCCAGAATG ACGCAAACTA 1850
ATCATGATAT GCAGCACAAT TTCTGGGTCG TCCGTAGTGC AGGTAGCGAT 1900
GAGTATAACT TTGACGATGC TATCCTCAGG GACGTCGTGA GCATTGGAGC 1950
GGGGACTGAT GAAGTCACAA TCCGTTTCGT GGTATGTCTC ACCCCTCGCA 2000
TTTTGAGACG CAAGAGCTGA TATATTTTAA CATAGACCGA CAATCCGGGC 2050
CCGTGGTTCC TCCATTGCCA TATTGATTGG CATTTGGAGG CAGGCCTTGC 2100
CATCGTCTTC GCTGAGGGCA TCAATCAGAC CGCTGCAGCC AACCCAACAC 2150
CCCGTACGTG ACACTGAGGG TTTCTTTATA GTGCTGGATT ACTGAATCGA 2200
GATTTCTCCA CAGAAGCATG GGATGAGCTT TGCCCCAAAT ATAACGGGTT 2250
GAGTGCGAGC CAGAAGGTCA AGCCTAAGAA AGGAACTGCT ATTTAAACG 2299
Coding has the laccase D2 of posttranslational protein matter sequence (SEQ ID No.16)
MGLNSAITSL AILALSVGSY AAIGPVADIH IVNKDLAPDG VQRPTVLAGG 50
TFPGTLITGQ KGDNFQLNVI DDLTDDRMLT PTSIHWHGFF QKGTAWADGP 100
AFVTQCPIIA DNSFLYDFDV PDQAGTFWYH SHLSTQYCDG LRGAFVVYDP 150
NDPHKDLYDV DDGGTVITLA DWYHVLAQTV VGAATPDSTL INGLGRSQTG 200
PADAELAVIS VEHNKRYRFR LVSISCDPNF TFSVDGHNMT VIEVDGVNTR 250
PLTVDSIQIF AGQRYSFVLN ANQPDDNYWI RAMPNIGRNT TTLDGKNAAI 300
LRYKNASVEE PKTVGGPAQS PLNEADLRPL VPAPVPGNAV PGGADINHRL 350
NLTFSNGLFS INNASFTNPS VPALLQILSG AQNAQDLLPT GSYIGLELGK 400
VVELVIPPLA VGGPHPFHLH GHNFWVVRSA GSDEYNFDDA ILRDVVSIGA 450
GTDEVTIRFV TDNPGPWFLH CHIDWHLEAG LAIVFAEGIN QTAAANPTPQ 500
AWDELCPKYN GLSASQKVKP KKGTAI 526
I. deceive hole laccase E gene (part) (SEQ IDNo.17) from the hypodermis of CBS 154.29 bacterial strains
TGCAATCGGA CCGGTGGCCG ACCTCAAGAT CGTAAACCGA GACATTGCAC 50
CTGACGGTTT TATTCGTCCC GCCGTTCTCG CTGGAGGGTC GTTCCCTGGT 100
CCTCTCATTA CAGGGCAGAA AGTACGTTAC GCTATCTCGG TGCTTTGGCT 150
TAATTAAACT ATTTGACTTT GTGTTCTCTT AGGGGAACGA GTTCAAAATC 200
AATGTAGTCA ATCAACTGAC CGATGGTTCT ATGTTAAAAT CCACCTCAAT 250
CGTAAGCAGA ATGAGCCCTT TGCATCTCGT TTTATTGTTA ATGCGCCCAC 300
TATAGCATTG GCATGGATTC TTCCAGAAGG GAACAAACTG GGCAGACGGT 350
CCTGCGTTCG TGAACCAATG TCCAATCGCC ACGAACAATT CGTTCTTGTA 400
TCAGTTTACC TCACAGGAAC AGCCAGGTGA GTATGAGATG GAGTTCATCC 450
GAGCATGAAC TGATTTATTT GGAACCTAGG CACATTTTGG TACCATAGTC 500
ATCTTTCCAC ACAATACTGC GATGGTTTGC GAGGGCCACT CGTGGTGTAT 550
GACCCACAAG ACCCGCATGC TGTTCTCTAC GACGTCGACG ATGGTTCGTA 600
CTTCGCATAT CCACGCTCGC TTTCATACAA TGTAAACTTT GTTCCTCCAG 650
AAAGTACAAT CATCACGCTC GCGGATTGGT ATCATACCTT GGCTCGGCAA 700
GTGAAAGGCC CAGCGTAAGG CACTTTAGTG TTTCCTCATA GTCCAAGAAA 750
TTCTAACACG CCTTCTTCAT CAGGGTTCCT GGTACGACCT TGATCAACGG 800
GTTGGGGCGT CACAACAATG GTCCTCTAGA TGCTGAACTA GCGGTGATCA 850
GTGTTCAAGC CGGCAAACGG CAAGTTCAAT TCACACTTTT CACTCTGTAC 900
CTTCTTCCTG ACATTCTTTT CTTGTAGTTA CCGCTTCCGC CTGATTTCAA 950
TTTCATGCGA TCCCAACTAC GTATTCTCCA TTGATGGCCA TGATATGACT 1000
GTCATCGAAG TGGATAGTGT TAACAGTCAA CCTCTCAAGG TAGATTCTAT 1050
CCAAATATTT GCAGGTCAGA GATATTCGTT CGTGGTGAGT CAGATCAGGG 1100
CATATCCTTT TGTCGATACG TCATTGACCA TATAATGCTA CAAGCTGAAT 1150
GCCAACCAAC CA G 1163
Its coding has the laccase E of part posttranslational protein matter sequence (SEQ ID No.18)
AIGPVADLKI VNRDIAPDGF IRPAVLAGGS FPGPLITGQK GNEFKINVVN 50
QLTDGSMLKS TSIHWHGFFQ KGTNWADGPA FVNQCPIATN NSFLYQFTSQ 100
EQPGTFWYHS HLSTQYCDGL RGPLVVYDPQ DPHAVLYDVD DESTIITLAD 150
WYHTLARQVK GPAVPGTTLI NGLGRHNNGP LDAELAVISV QAGKRQVQFT 200
LFTLYRFRLI SISCDPNYVF SIDGHDMTVI EVDSVNSQPL KVDSIQIFAG 250
QRYSFVLNAN QP 262
When [33] term of this paper " identity per-cent " refers to use the comparison of sequence alignment program, the nucleic acid between the nucleotide sequence of the laccase described herein of encoding or this laccase aminoacid sequence or the identity level of aminoacid sequence.
[34] for example, as used herein, therefore determine 80% sequence identity by algorithm, and the homology of given sequence has sequence identity greater than 80% on the length of this given sequence.For given sequence, for example, the exemplary sequence identity level of the encoding sequence of laccase as described herein includes but not limited to 80,85,90,95,98% or higher sequence identity.
[35] can be used for determining that the illustrative computer program of identity between two sequences includes but not limited to the blast program external member, for example BLASTN, BLASTX and TBLASTX, BLASTP and TBLASTN, the public can be from interconnected network address Www.ncbi.nlm.nih.gov/BLASTObtain.Also referring to people such as Altschul, 1990 and people such as Altschul, 1997.
[36] during the nucleotide sequence in assessment given nucleotide sequence and GenBank dna sequence dna and other public data storehouses, use the BLASTN program to carry out sequence retrieval usually.The BLASTX program is preferably used for retrieving nucleotide sequence after all reading frames all have been translated at the aminoacid sequence in GenBank protein sequence and other public data storehouses.It is 11.0 and to extend gap penalty be 1.0 default parameters operation that BLASTN and BLASTX use gap penalty, and utilizes BLOSUM-62 matrix (referring to people such as for example Altschul, 1997).
[37] can use the CLUSTAL-W program of MacVector for example 6.5 editions, (comprise gap penalty 10.0 with default parameters, extending gap penalty is 0.1, and BLOSUM 30 similar matrixes) operation carries out the comparison of selected sequence, to confirm two or " the identity per-cent " between multisequencing more.
II. amboceptor
[38] in embodiment, the enzymatic oxidn system also comprises one or more chemical mediator reagent, and it strengthens the activity of laccase.This paper is defined as redox mediators term " chemical mediator " (or " amboceptor " that this paper is used interchangeably) to make the chemical compound of electron shuttle effectively between enzyme that shows oxidase activity and dyestuff.Chemical mediator also known in the art is toughener and promotor.
[39] chemical mediator can be phenoloid, and for example methyl syringate and relevant compound are as described in WO 95/01426 and 96/12845.Chemical mediator also can be N-oxy-compound, N-oxime compound or N-oxide compound, for example N-hydroxybenzotriazole, violuric acid or N-hydroxyacetanilide.Chemical mediator also can be phenoxazine/phenothiazine compounds, for example thiodiphenylamine-10-propionic acid.Chemical mediator also can be 2,2 '-azine-two-(3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) is (ABTS).Other chemical mediators are well known in the art.For example, disclosed compound strengthens laccase activity among the known WO 95/01426.In the embodiment, amboceptor can be Syringylethanone, methyl syringate, Ethyl syringate, syringic acid propyl ester, Syringic acid butyl ester, the own ester of syringic acid or syringic acid monooctyl ester.
[40] preferably, amboceptor is a 4-cyano group-2,6-syringol, 4-formamido--2, the derivative that 6-syringol or its N-replace, 4-(N-methylformamide base)-2 for example, 6-syringol, 4-[N-(2-hydroxyethyl) formamido-]-2,6-syringol or 4-(N, the dinethylformamide base)-2,6-syringol.
[41] can the amboceptor that the present invention uses be described by following general formula:
In this formula A be such as-R ,-D ,-CH=CH-D ,-CH=CH-CH=CH-D ,-CH=N-D ,-N=N-D or-group of N=CH-D, wherein D be selected from-CO-E ,-SO 2-E ,-CN ,-NXY and-N +XYZ, wherein E can be-H ,-OH ,-R ,-OR or-NXY, and X and Y and Z can be identical or different and be selected from-H ,-OH ,-OR ' and-R; R is C 1-C 16Alkyl, preferred C 1-C 8That alkyl, this alkyl can be is saturated or unsaturated, ramose or unbranched and randomly by carboxyl, sulfo group or amino the replacement; And B and C can be identical or different and be selected from C mH 2m+11≤m≤5.
[42] in embodiment, the A in the above-mentioned formula is-CN or-CO-E, wherein E can be-H ,-OH ,-R ,-OR or-NXY, its X and Y can be identical or different and be selected from-H ,-OH ,-OR and-R, R is C 1-C 16Alkyl, preferred C 1-C 8That alkyl, this alkyl can be is saturated or unsaturated, ramose or unbranched and randomly by carboxyl, sulfo group or amino the replacement; And B and C can be identical or different and be selected from C mH 2m+11≤m≤5.
[43] in the above-mentioned formula A can be placed on hydroxyl between the position but not be placed in the contraposition as shown.
[44] in the embodiment, amboceptor can be Syringylethanone, methyl syringate, Ethyl syringate, syringic acid propyl ester, Syringic acid butyl ester, the own ester of syringic acid or syringic acid monooctyl ester.Preferably, amboceptor is a 4-cyano group-2,6-syringol, 4-formamido--2, the derivative that 6-syringol or its N-replace, 4-(N-methylformamide base)-2 for example, 6-syringol, 4-[N-(2-hydroxyethyl) formamido-]-2,6-syringol or 4-(N, the dinethylformamide base)-2,6-syringol.
[45] concentration that amboceptor of the present invention can 0.005-1000 μ mole/g denim exists, preferred 0.05-500 μ mole/g denim, more preferably 0.5-100 μ mole/g denim.
[46] can prepare amboceptor by the known method of those of skill in the art such as WO 97/11217, WO 96/12845 and US 5752980 disclosed those.
III. practicality
[47] industrial application of laccase comprises the bleaching of paper pulp and paper and such as the textile bleaching of the denim fabric of indigo dyeing.Found that also laccase is useful (referring to for example WO95/33836 and WO 95/33837) in hair dyeing.European patent No.0504005 discloses laccase can be used to the wool that dyes.
[48] laccase as herein described finds can be used for the dyeing and the bleaching of textiles, fiber, yarn etc.Find that this laccase also can be used for the polymerization of the polymerization of the depolymerization of wastewater treatment, paper pulp lignification removal, high molecular weight aggregates, deinking, aromatic compound, free radical mediated and crosslinking reaction (for example pigment, coating and biomaterial) and dyestuff activation to be coupled with organic compounds.This laccase can be used in cleaning compositions or its component, or is used for washing composition.
[49] as used herein, use oxygen as electron acceptor(EA), the multiple coloring compound that laccase can oxidation has different chemical structures.Therefore, the laccase that this paper shows can be used for the application that needs change the color relevant with coloring compound, for example cleans, as removing the food color on the fabric.Under some situation, can use amboceptor or toughener to obtain desired effects.
[50] laccase of this paper displaying can be used for textile field.For example, laccase as herein described can be used for processing, processing, arrangement, polishing or the production of fiber or other fabrics or goods.The enzyme of this paper can be used for for example denim processing (bleaching aftertreatment technology); The decolouring of indigo waste water; Textile dyeing; Textile bleaching technology; Fibre modification; Realize enhanced fiber or fabric property etc.
[51] laccase as herein described can be used for leather industry.For example, this laccase can be used for the processing of animal skin, includes but not limited to depilation, liming, the softening and/or tanning of skin.
[52] this paper also discloses and has been used for removing delignification from containing lignocellulosic materials, and bleaching contains lignocellulosic materials (being the enzymatic deinking of recycled writing paper) and/or handles by the technology of making the waste water that paper or Mierocrystalline cellulose produced.This technology is used the laccase available from the black hole of hypodermis species, add simultaneously or quantifying adds the non-fragrant redox agent of phenol and/or the fragrant redox compound of non-phenol, the phenol of xylogen and non-phenol unit by effect institute's direct oxidation of these phenol and/or non-phenol aromatic compound or this xylogen by other phenol that oxygenizement produced of these compounds and/or the oxidation of non-phenoloid institute.
[53] laccase described herein can be used for field of papermaking.For example, laccase can be used for making paper pulp and Time of Fluff Slurry from the starting material such as timber, bamboo wood and cereal rice bar; Manufacturing be used for printing and write, encapsulate, health care and other technologies use paper and cardboard; Regeneration is used for the cellulosic fibre of papermaking and cardboard purpose; With handle by paper pulp or paper shredder and other equipment (especially for making paper, paper pulp or Time of Fluff Slurry) produces and by the waste product of its processing.The enzyme that this paper shows can be used for for example wood working; Association with pulp bleaching; The xylon modification; Be used for the xanthan gum (xylogen activation) that MDF makes; Be used to strengthen the characteristic of paper; Ink removing; Paper dyeing; Binding agent (the glue that for example is used for particle board or fiberboard) or the like based on xylogen.
[54] laccase described herein can be used for field of fodder.For example, the laccase showed of this paper can be used alone as fodder additives or be intended to increase the nutritive value of the animal (for example chicken, ox, pig, fish and pet) that is used for any kind of as the part of fodder additives; And/or be intended to produce as the processing aid of processing vegetable material and byproduct of food industry and be suitable as the raw-material material/product of feed.
[55] laccase described herein can be used for the contact lens cleaning applications.For example, this laccase can be used for contact lens cleaning, deposit, sterilize and/or preserve.
[56] laccase described herein can be used for the starch field.For example, this laccase can be used for substrate (comprising starch and/or cereal) is processed as glucose (dextrose) syrup, fructose syrups or any other slurries, alcohols (beverage or fuel) or carbohydrate.This starch processing can comprise the procedure of processing such as substrate liquefaction, saccharification, isomerization and Tuo Zhi.
[57] laccase described herein can be used for field of food.For example, this laccase can be used to prepare, process food or as the activeconstituents of food, for example be used for the human consumption yellow fat, teas beverage, cook product, bakery and frozen product.This laccase can for example be used as bread improver, is used as Oxygen Scavenger etc. in food preservation.
[58] laccase described herein can be used for personal care field.For example, this laccase can be used to prepare individual product such as fragrant atmosphere and the skin care, hair-care, oral hygiene, personal cleanliness and the reodorant that are used for the people and/or the product of antiperspirant that is used for the people.The enzyme that this paper shows for example can be used for, hair dyeing and/or bleaching, nail dyeing and/or bleaching; Skin dyeing and/or bleaching; Finishing (for example as coupling agent); As biocide; Be used for taste removal; Tooth whitening etc.
[59] laccase described herein can be used for cleaning applications.For example, this laccase can be used for cleaning, processing or the nursing of laundry project such as clothes or fabric; The cleaning that is used for the family expenses crust; Be used to coil the ware nursing, comprise dishwasher use; Be used for soap bar and liquid and/or synthetic surfactant rod and liquid.The enzyme that this paper shows can be used for for example decontamination/decolouring, and/or is used for taste removal, and/or is used for sanitary measure etc.
[60] laccase described herein can be used for field of waste water treatment.For example, this laccase can be used for the decolouring of coloring compound; Be used for the detoxifcation of phenolic constituent; Be used for antimicrobial acivity (for example being used for water cycle); Be used for biological restoration etc.
[61] laccase described herein can be used for technical field of biological material.For example, this laccase can be with the biological catalyst that acts on various organic reactions; And/or be used for biological polymer; Be used for encapsulation; Be used for adhesive agent; Be used for finishing (activation and coupling agent); Be used for the production of primary alcohol; Be used for biological inductor and/or organic synthesis etc.
[62] laccase described herein can be used for antimicrobial field.For example, this laccase can perhaps be used for reducing or eliminating the microbial biomass of numerous food (as meat) or feed as the biocide in the cleaning compositions.
[63] the laccase amboceptor can be used as sanitizer and biocide (as wood protection, washing composition).This amboceptor can be independent of enzyme and use or be used in combination with enzyme.
[64] as used herein, " cleaning compositions " and " cleaning formulation " refers to can be used for remove undesired compound compositions from project to be cleaned (as fabric etc.).This term comprises any material/compound that is selected for the particular type cleaning compositions wanted and this product form (as liquid, gel, particle or spray composite), as long as other enzyme compatibilities of using in said composition and laccase and the said composition.Consider surface, project or fabric to be cleaned, and, can easily carry out the specific selection of cleaning compositions material in use at the desired composition forms of clean conditions.
[65] this term also refers to any composition that is suitable for cleaning and/or bleaching any object and/or surface.This term is intended to include but not limited to detergent composition (for example liquid and/or solid laundry detergent and carefully fabric detergent; The hard-surface cleaning preparation for example is used for glass, timber, pottery and metal counter top and window; Carpet cleaner; The baking box sanitising agent; With textiles and the pre-stain remover of clothing (pre-spotter), and dish ware washing composition).
[66] in fact, unless otherwise, otherwise term " cleaning compositions " comprises the general of particle or powder type or heavy scale washing agent, especially cleaning detergent as used herein; The all purpose cleaner of liquid, gel or the form of paste, especially so-called heavy-filth liquid (HDL) type; The thin fabric detergent of liquid; Manual dishwasher detergent or light dirty dishwasher detergent especially enrich those of foam type; The machine dishwasher detergent comprises the multiple tablet, particle, liquid and bright dish agent (rinse-aid) type that are used for family and public organizations' use; Liquid cleaning and sterilizing agent, vehicle or carpet washings, bathroom detergent; Shampoo and hair rinse agent; Bathing agents and body wash and metal detergent; And cleaning additive, for example bleach additive and " super dirt-removing rod (stain stick) " or pre-treatment type.
[67] as used herein, term " detergent composition " and " detergent formulations " are used in reference to the employed mixture of cleaning medium that is intended to be used for clean the object of pollution.In some embodiments, this term is used in reference to the washing composition (for example " laundry detergent ") of laundering of textile fabrics and/or clothes.In the alternate embodiment, this term refers to other washing composition, as is used for cleaning deskitte ware, cutlery etc. those (as " dishwashing detergents ").The composition of current consideration is not intended to be limited to any specific detergent formulations or composition.In fact, except laccase, this term is intended to comprise the washing composition that contains tensio-active agent, transferring enzyme, lytic enzyme, washing assistant (builder), SYNTHETIC OPTICAL WHITNER, bleach-activating agent, chlorinated lime and fluorescence dye, caking inhibitor, sequestering agent, enzymic activity agent, antioxidant and solubilizing agent.
[68] as used herein, term " hard-surface cleaning compositions " refers to be used for for example detergent composition of floor, wall, brick, stainless steel vessel (as fermentor tank), bathroom and kitchen fixture etc. of cleaning of hard surfaces.In any form, include but not limited to that solid, liquid, emulsion etc. provide said composition.
Embodiment
The amino acid sequence analysis of embodiment 1, black pore fungi (Cerrena unicolor) laccase of monochromatic hypodermis
[69] use the commercially available laccase that gets to obtain four peptide sequence: AIGPVADLHI (SEQ IDNO.19), MLTPTSI (SEQ ID No.20), TVGGPA (SEQ ID No.21) and YSFVLNANQP (SEQ ID NO.22).This commercially available laccase that gets of purifying.The N-end sequencing obtains SEQ ID No.19.Carry out proteolytic digestion with the sample of trypsinase after to purifying.By the gel electrophoresis isolated fragment, select 3 bands and manual the collection.Carry out peptide sequencing and obtain SEQ ID No.20,21 and 22 for each band.
Embodiment 2
A. the clone who deceives pore fungi laccase A gene from the monochromatic hypodermis of ATCC20013 bacterial strain
[70] for from ATCC 20013 bacterial strains clones laccase A gene, two primers are respectively according to obtaining available from dna sequence dna (referring to the embodiment 3a) design of the laccase B gene of ATCC20013 bacterial strain and from Invitrogen: TTCGCAGGTCAACGATATTC (SEQ ID No.35) and design and obtain from Invitrogen according to the laccase A gene (referring to embodiment 2c) available from CBS 115.075 bacterial strains: GTTAGGTGGTTGAAGGATTG (SEQ ID NO.36).In the highT PCR reaction of genomic dna that contains available from ATCC 20013 bacterial strains, use this primer (referring to embodiment 3) as template.Use is from the QIAquick centrifugal column purification PCR fragment of Qiagen and use TOPO clone's test kit (Invitrogen) that it is cloned into the pTOPO plasmid.22 of use Ready-To-Go PCR pearl (GE Healthcare) amplifications are cloned and 3 PCR fragments (2-1,2-3 and 2-6) are checked order.SEQ ID No.37 has listed the 1316bp dna sequence dna from the laccase A gene of ATCC20013.
B. the clone who deceives pore fungi laccase A gene from the monochromatic hypodermis of CBS 154.29 bacterial strains
[71] in order to clone laccase A gene from CBS 154.29 bacterial strains, two primers are respectively according to obtaining available from laccase A gene (referring to the embodiment 2c) design of CBS 115.075 bacterial strains and from Invitrogen: CACCAGCATGAGCTCAAAGCTAC (SEQ ID No.45) and acquisition primer SEQ ID No.36.In containing, use this primer in the Herculaase PCR of the 4%DMSO reaction available from genomic dna template, dNTP, primer and the 1x damping fluid of CBS 154.29 bacterial strains.This PCR mixture heating up to 98 ℃ is continued to make in 4 minutes this dna profiling sex change.Will
Figure A20078004701800271
II enzyme (Stratagene) adds in this test tube and carries out the PCR reaction for 30 times in following program loop: 98 ℃ continue 30 seconds, and 50 ℃ continued 30 seconds and 72 ℃ lasting 2 minutes.Finished last extension 5 minutes and reaction is cooled to 4 ℃ at 72 ℃.Use QIAquick centrifugal column purification PCR fragment and it is cloned in the pENTR/D-TOPO carrier (Invitrogen).Use 15 clones of Ready-To-Go PCR pearl amplification and reach dna profiling is checked order from two clones (pENTR15-24 and pENTR15-30) separation quality grain.Acquisition is from the 2374bp dna sequence dna of the laccase A gene of CBS 154.29.SEQ ID No.3 has listed this dna sequence dna and SEQ ID No.4 has listed the protein sequence after the translation.
C. the clone who deceives pore fungi laccase A gene from the monochromatic hypodermis of CBS 115.075 bacterial strains
[72] will be used for lowT PCR reaction (referring to embodiment 3a) according to primer CAATCTATGACCGTAGATTC (SEQ ID No.39) and primer NNNNNNNNNNCGATCG (SEQ ID No.38) (wherein N represents the mixture of all four kinds of Nucleotide (A, T, C and G)) from the laccase B gene (referring to embodiment 3a) of ATCC20013 bacterial strain.Extract genomic dna and in first round lowTPCR reaction, be used as template from the black pore fungi bacterial strain (CBS 115.075) of monochromatic hypodermis.Take turns in the lowT PCR reaction to be used as template second with this PCR fragment of the centrifugal column purification of QIAquick and with it, this second is taken turns lowT PCR reaction and uses according to primer SEQ ID No.35 and primer SEQ ID No.38 from the laccase B gene (referring to embodiment 3a) of ATCC20013 bacterial strain.Use TOPO clone test kit with this PCR fragment cloning to the pTOPO plasmid.Use 16 clones of Ready-To-Go PCR pearl amplification and to 3 clones' PCR fragment (B2#1, B2#4 and B2#11) order-checking.
[73] in order to clone 3 ' end of laccase A gene, in lowT PCR reacts, use primer ACCGTGGTTCCTCCATTGCC (SEQ ID No.40) and primer SEQ ID No.31, in first round lowT PCR reaction, deceive the genomic dna of pore fungi bacterial strain (CBS 115.075) as template with extracting from monochromatic hypodermis.Take turns in the lowT PCR reaction to be used as template second with this PCR fragment of the centrifugal column purification of QIAquick and with it, this second is taken turns lowT PCR reaction and uses primer GACTGGCACTTGGAAGCGGG (SEQ ID No.41) and primer SEQ ID No.31.Use TOPO clone test kit with this PCR fragment cloning to the pTOPO plasmid.Use 22 clones of Ready-To-Go PCR pearl amplification and to 1 clone's PCR fragment (D2#2) order-checking.
[74] in order to clone 5 ' end of laccase A gene, according to laccase B gene order design primer GGACCAAGCTGGTACTTTC (SEQ ID No.42).Itself and primer SEQ ID No.36 are used for amplification of DNA fragments.The genomic dna that will extract from the black pore fungi bacterial strain (CBS 115.075) of monochromatic hypodermis is used as pcr template.Obtain 1.7kb PCR fragment and with it with the centrifugal column purification of QIAquick and use TOPO to clone test kit to be cloned in the pTOPO plasmid.
Use Ready-To-Go PCR pearl to analyze 22 clones.To plasmid DNA order-checking from clone (C5#20).In order further to clone 5 ' of laccase A gene, in lowT PCR reaction, use primer CGTGGTACCAGTCTGCCAGGG (SEQ ID NO.43) and primer SEQID No.31, deceive the genomic dna of pore fungi CBS 115.075 bacterial strains as template with extracting from monochromatic hypodermis.Take turns the lowT PCR reaction to be used as template second from this PCR fragment of first round lowT PCR reaction purification and with it with the centrifugal post of QIAquick, this second is taken turns lowT PCR reaction and uses primer GGCAGCATCAGTCACGGTCAG (SEQ ID No.44) and primer SEQ IDNo.31.This PCR fragment (a3) that increases once more also is used as template with it in third round lowT PCR reaction, primer GGCAGCATCAGTCACGGTCAG (SEQ ID No.44) and primer SEQ IDNo.31 are used in this third round lowT PCR reaction.Use TOPO clone test kit that PCR fragment (a3-2) is cloned into the pTOPO plasmid.Use 11 clones of Ready-To-Go PCR pearl amplification and to 2 clones' PCR fragment (a3-2#10 and a3-2#11) order-checking.SEQ ID No.1 has listed from the dna sequence dna of the laccase A gene of 5 ' and the 3 ' sequence that comprises the coding region of CBS 115.075 bacterial strains and SEQ ID No.2 has listed the protein sequence after the translation.
Embodiment 3
The clone and the order-checking of a. deceiving pore fungi laccase B gene from the monochromatic hypodermis of ATCC20013 bacterial strain
[75] for the dna fragmentation of the black hole of clones coding hypodermis laccase gene, obtain 4 degenerated primers according to peptide sequence AIGPVADLHI (SEQ ID No.19) design and from Invitrogen.With their called after:
Primer A:GCAATCGGACCNGTNGCAGA (SEQ ID No.23);
Primer B:GCAATCGGACCNGTNGCTGA (SEQ ID No.24);
Primer C:GCAATCGGACCNGTNGCGGA (SEQ ID No.25) and
Primer D:GCAATCGGACCNGTNGCCGA (SEQ ID No.26).
[76] obtain 2 degenerated primers according to peptide sequence YSFVLNANQP (SEQ ID No.22) design and from Invitrogen.With their called after:
Primer E:GGTTGATTTGCATTNAGNAC (SEQ ID No.27) and
Primers F: GGTTGATTTGCGTTNAGNAC (SEQ ID No.28)
Wherein N represents the mixture of four kinds of Nucleotide (A, T, C and G).From the ATCC20013 bacterial strain extract genomic dna and it is being contained below the lowT PCR reaction of combination of primers in be used as template: PCR reaction 1 and do not contain DNA and primer; PCR reaction 2 contains primer A and primer E; PCR reaction 3 contains primer B and primer E; PCR reaction 4 contains primer C and primer E; PCR reaction 5 contains primer D and primer E; PCR reaction 6 contains primer A and primers F; PCR reaction 7 contains primer B and primers F; PCR reaction 8 contains primer C and primers F and PCR reaction 9 and contains primer D and primers F.This PCR reaction mixture contains dna profiling, primer, 1 * damping fluid, 0.2mM dNTP and 1 Taq of unit archaeal dna polymerase.This PCR is reflected at 95 ℃ and continues 1 minute, and 45 ℃ continue 1 minute and 68 ℃ and carried out 30 circulations in lasting 1 minute.Finished last extension 7 minutes and reaction is cooled to 4 ℃ at 72 ℃.Scale off the PCR fragment and the collection of autoreaction 4,5 and 8 from 1.2% sepharose.Use the centrifugal post of Qiagen to extract the PCR fragment and use TOPO clone test kit that it is cloned into the pTOPO plasmid from gel.The dna sequence dna of selecting 32 clones' PCR fragment and using the order-checking of Ready-To-Go PCR pearl and will clone #A30 is accredited as laccase B gene.
[77] in order to clone 5 ' end of laccase gene, design also obtains primer: GGACGTGGCCTTGAGCATAC (SEQ ID No.29) from Invitrogen.Itself and degenerate oligonucleotide NNNNNNNNNNGGATCC (SEQ ID No.31) (wherein N represents the mixture of all four kinds of Nucleotide (A, T, C and G)) are used for first round lowT PCR reaction.Take turns in the lowT PCR reaction to be used as template second with this PCR product of the centrifugal column purification of QIAquick and with it, this second is taken turns lowT PCR reaction and contains primer TCTGTCAAGTCGTCAATCAC (SEQ IDNo.30) and primer SEQ ID No.31.Use QIAquick this PCR fragment of centrifugal column purification and with 1: 10 and dilution in 1: 100 and in first round highT PCR reaction, be used as template, this highTPCR reaction continues 1 minute with two primers (SEQ ID No.30 and SEQ ID No.31) at 95 ℃, and 50 ℃ continue 1 minute and 72 ℃ and carried out 30 circulations in lasting 1 minute.Finished last extension 7 minutes and reaction is cooled to 4 ℃ at 72 ℃.Use QIAquick this PCR fragment of centrifugal column purification and it is used for second with primer TTACCACGAATCAGAGGACC (SEQ ID No.32) and SEQ ID No.31 and take turns highT PCR reaction.PCR fragment (D13) is checked order.
[78] in order to clone 3 ' end of laccase B gene, design also obtains primer: CCTCACCTGTATTGGCACAG (SEQ ID No.33) from Invitrogen, and itself and primer SEQ IDNo.31 are used for the lowT PCR reaction of the first round.Take turns in the lowT PCR reaction to be used as template second with this PCR fragment of the centrifugal column purification of QIAquick and with it, primer TTGGTATCATGCCCTTGCTC (SEQ ID NO.34) and primer SEQID No.31 are used in this second lowT PCR reaction of taking turns.Use TOPO clone test kit with this PCR fragment cloning to the pTOPO plasmid.Use 16 clones of Ready-To-Go PCR pearl amplification and to 4 clones' PCR fragment (C3, C4, C5 and C7) order-checking.
[79] obtain the 1337bp dna fragmentation.SEQ ID No.9 has listed dna sequence dna and SEQ IDNo.10 has listed the protein sequence after the translation.
B. the clone who deceives pore fungi laccase B gene from the monochromatic hypodermis of CBS 154.29 bacterial strains
[80] design and obtain two primers from Invitrogen:
CACCGCGATGTCTCTTCTTCGTAG (SEQ ID NO.46) and
TGRAGRTGGAASGGATGWGGTCC(SEQ ID NO.47)
Wherein R represents the mixture of Nucleotide A and G, and S represents the mixture of Nucleotide C and G, and W represents the mixture of Nucleotide A and T.These two primers are used for highT PCR reaction.Use QIAquick centrifugal column purification PCR fragment (A3).Use TOPO clone test kit with this PCR fragment cloning to the pTOPO plasmid.Use 16 of Ready-To-Go PCR pearl amplifications to clone and 2 PCR fragments (A3#1 and A3#5) are checked order.
[81] in order to clone 3 ' end from the laccase B gene of CBS 154.29 bacterial strains, design also obtains primer: GTCCCTGTACTACTCCAGATCC (SEQ IDNo.48) from Invitrogen, and the primer of itself and SEQ ID No.31 is used for the lowT PCR reaction of the first round.Take turns in the lowT PCR reaction to be used as template second with this PCR fragment of the centrifugal column purification of QIAquick and with it, this second is taken turns lowT PCR reaction and uses primer CCAGCAGGAAGCGTGATCGAAC (SEQ ID No.49) and primer SEQ IDNo.31.Use TOPO clone test kit with this PCR fragment cloning to the pTOPO plasmid.Use 16 of Ready-To-Go PCR pearl amplifications to clone and 3 PCR fragments (7#6,7#7 and 7#8) are checked order.SEQ ID No.7 has listed the 2663bp dna sequence dna of laccase B of CBS 154.29 bacterial strains and SEQ ID No.8 has listed the protein sequence after the translation.
C. the clone who deceives pore fungi laccase B gene from the monochromatic hypodermis of CBS 115.075 bacterial strains
[82] design and obtain primer: GTAATCATGTATCACCTGGGCTCAAGG (SEQ ID NO.50) from Invitrogen.This primer and primer SEQ ID No.46 are used for Herculase PCR reaction (referring to embodiment 2b).With this PCR fragment of the centrifugal column purification of QIAquick.Use TOPO clone test kit with this PCR fragment cloning to the pTOPO plasmid.Using Ready-To-Go PCR pearl to analyze 17 clones and the PCR fragment (#1, #2, #4 and #5) from 4 clones is checked order.Check order from two clones (pENTR-laccase B CBS 115075#1 and pENTR-laccase B CBS 115075#3) preparation plasmid DNA and to two plasmids.SEQ ID No.5 has listed the 2173bp dna sequence dna of laccase B of CBS 115.075 bacterial strains and SEQ ID No.6 has listed the protein sequence after the translation.
Embodiment 4, from the clone of the black pore fungi laccase C gene of the monochromatic hypodermis of CBS 154.29 bacterial strains
[83] according to the design of the peptide sequence GQRYSFV (SEQ ID No.52) after translation primer ACGAACGAGTANCGTTGNCC (SEQ ID No.51), wherein N represents the mixture of all four kinds of Nucleotide (being A, T, C and G).This peptide is (referring to the embodiment 2 and 3) that guards between laccase A gene and laccase B gene.This primer is used for the lowT reaction available from Invitrogen and with primer SEQ ID No.24.Use this PCR fragment of the centrifugal column purification of QIAquick.Use TOPO clone test kit with this PCR fragment cloning to the pTOPO plasmid.Using Ready-To-Go PCR pearl to analyze 33 clones and the PCR fragment (#12, #5a, #19a and #21a) from 4 clones is checked order.SEQ ID No.11 has listed the laccase C gene order of CBS 154.29 bacterial strains of 1080bp and SEQ ID No.12 has listed the protein sequence after the translation.
Embodiment 5
A. the clone who deceives pore fungi laccase D gene from the monochromatic hypodermis of CBS 115.075 bacterial strains
[84] in order to clone 5 ' end, according to laccase D gene (referring to embodiment 5b) design primer (AACACGGAGACAGTCCAAAC, SEQ ID NO.62) from CBS 154.29 bacterial strains from the laccase D gene of CBS 115.075 bacterial strains.This primer and primer SEQ ID No.56 are used for highT PCR reaction.Use this PCR fragment of the centrifugal column purification of QIAquick and order-checking.
[85] in order to clone laccase D gene from CBS 115.075 bacterial strains, according to two primer CACCTCTCGAGATGGGATTGAAC of laccase D gene (referring to embodiment 5b) design from the CBS154.29 bacterial strain, SEQ ID NO.63 and CGTTTAAATAGCAGTTCCTTTC, SEQ ID No.64.This primer is used for Herculase PCR reaction (referring to embodiment 2b), and using the genomic dna from CBS 115.075 bacterial strains is dna profiling.With this PCR fragment of the centrifugal column purification of QIAquick and be cloned into the pENTR/D-TOPO carrier.Use 16 clones of Ready-To-Go PCR pearl amplification and to producing from 4 clones' PCR sequencing fragment.From clone's #2 (pENTRE-laccase D#2) isolated plasmid dna and to its order-checking.Obtain the dna sequence dna of 2809bp from the laccase D gene of CBS 115.075.SEQ ID No.15 has listed this dna sequence dna and SEQ ID No.16 has listed the protein sequence after the translation.
B. the clone who deceives pore fungi laccase D gene from the monochromatic hypodermis of CBS 154.29 bacterial strains
[86] according to peptide sequence LNANQP (SEQ ID No.54) design primer CTGGTTGGTTNGCATTNAG (SEQ ID No.53).This primer is used for lowT PCR reaction available from Invitrogen and with primer SEQ ID No.26.Use QIAquick this PCR fragment of centrifugal column purification and use TOPO clone test kit with this PCR fragment cloning to the pTOPO plasmid.Use Ready-To-Go PCR pearl to analyze 18 clones and to PCR sequencing fragment from the clone.
[87] in order to clone 3 ' end of laccase D gene, design primer (CACACGACCCCTGACCGTTG, SEQ ID No.55).This primer and primer SEQ ID No.31 are used for lowT PCR reaction.Use QIAquick this PCR fragment of centrifugal column purification and use TOPO clone test kit with this PCR fragment cloning to the pTOPO plasmid.Use Ready-To-Go PCR pearl to analyze 24 clones and to PCR sequencing fragment from the clone.
[88] in order to clone laccase D gene more 3 ' and 5 ' end, use inverse PCR.The genomic dna 1.5 hours that belongs to CBS 154.29 bacterial strains with the EcoRV Restriction Enzyme at 37 ℃ of digestion 0.4 μ g from the black hole of hypodermis.With ethanol sedimentation through postdigestive genomic DNA fragment.In 100 μ l volumes, connect linear DNA fragment more than 5 hours with the T4DNA ligase enzyme.Dna fragmentation after connecting is heated to 100 ℃ of dna profilings that continued 3 minutes and be used as in the first round highT PCR reaction, two primer TGACCGGTGATCAACGTCCC are used in this first round highT PCR reaction, SEQ ID NO.56 and GGCGCAGACATCAATCACAG, SEQ ID No.57.Use QIAquick this PCR fragment of centrifugal column purification and used as second dna profiling of taking turns in the highT PCR reaction, this second is taken turns highT PCR reaction and uses two primer TCTTCAGCATCAACAACGCC, SEQ ID NO.58 and TCCGGCAAGCACGGTTGG, SEQ ID No.59.Use the centrifugal column purification of QIAquick also to check order from the second PCR fragment of taking turns the PCR reaction.
[89] in order to clone the more 3 ' end of laccase D gene, use inverse PCR from CBS 154.29 bacterial strains.The genomic dna 1.5 hours that belongs to CBS 154.29 bacterial strains with the SmaI Restriction Enzyme at 37 ℃ of digestion 0.4 μ g from the black hole of hypodermis.With ethanol sedimentation through postdigestive genomic DNA fragment.In 100 μ l volumes, connect linear DNA fragment more than 5 hours with the T4DNA ligase enzyme.Dna fragmentation after connecting is heated to 100 ℃ of dna profilings that continued 3 minutes and be used as in the first round highT PCR reaction, and primer TCGTCTTCGCTGAGGGCATC, SEQ ID NO.60 and primer SEQ ID No.56 are used in this first round highT PCR reaction.Use QIAquick this PCR fragment of centrifugal column purification and used as second dna profiling of taking turns in the highT PCR reaction, this second is taken turns highT PCR reaction and uses primer CAGACCGCTGCAGCCAACCC, SEQ ID No.61 and primer SEQ ID No.59.Use the centrifugal column purification of QIAquick to take turns the PCR fragment of PCR reaction and use TOPO to clone test kit and be cloned into the pTOPO plasmid with this its from second.Use Ready-To-Go PCR pearl to analyze 21 clones and to PCR sequencing fragment from clone #Ce11 and #Ce14.SEQ ID No.13 listed 2809bp from the laccase D gene order of CBS 154.29.49 bacterial strain and SEQ IDNo.14 has listed the protein sequence after the translation.
Embodiment 6, from the clone of the black pore fungi laccase E gene of the monochromatic hypodermis of CBS 154.29 bacterial strains
[90] primer SEQ ID No.53 and primer SEQ ID No.26 (referring to embodiment 5b) are used for lowT PCR reaction.Use QIAquick this PCR fragment of centrifugal column purification and use TOPO clone test kit with this PCR fragment cloning to the pTOPO plasmid.Use Ready-To-Go PCR pearl to analyze 18 clones and to PCR sequencing fragment from clone #Ae17.SEQ ID No.17 listed 1163bp from the laccase E gene order of CBS 154.29.49 bacterial strain and SEQ IDNo.18 has listed the protein sequence after the translation.
Embodiment 7, the expression of laccase A gene in Trichoderma
[91] in order to make up the laccase A expression of gene plasmid that is used for CBS bacterial strain 115.075, in containing, use two primers (SEQ ID No.45 and SEQ ID No.36) in the Herculase PCR of the 4%DMSO reaction available from genomic dna template, dNTP and the 1 * damping fluid of 115.075 bacterial strains.This PCR mixture heating up to 98 ℃ is continued to make in 4 minutes this dna profiling sex change.Will
Figure A20078004701800351
II enzyme (Stratagene) adds this test tube and continues 30 seconds at 98 ℃, and 50 ℃ continue to continue in 30 seconds and 72 ℃ circulation in 2 minutes and carry out PCR for 30 times and react.Finished last extension 5 minutes and reaction is cooled to 4 ℃ at 72 ℃.Use QIAquick centrifugal column purification PCR fragment and be cloned into the pENTR/D-TOPO carrier.Use 15 clones of Ready-To-Go PCR pearl amplification and from pENTR-laccase A-CBS 115.075#11 clone and separate plasmid DNA.Laccase A Gene Partial is checked order with the fidelity of reproduction of the pcr amplification of confirming laccase A gene.Contain in the LB clonase II reaction (Invitrogen) of 6.5 μ l TE, 1 μ l pTrex3g carrier (0.1mg/ml) and 2 μ l Clonasell at 10 μ l, pENTR-laccase A-CBS 115.075#11 plasmid (50ng) is converted into expression plasmid pTrex3g-laccase A (Fig. 1).Confirm this expression plasmid and it is converted into the Trichoderma bacterial strain by dna sequencing with particle gun.Use from Bio-Rad (Hercules, CA)
Figure A20078004701800352
PDS-1000/he particle delivery system finishes the conversion of Trichoderma bacterial strain by the particle gun conversion method according to manufacturer specification (referring to WO05/001036 and US 2006/0003408).Select 66 transformant and be transferred on the new flat board.At 30 ℃, 15 stable transformant growths continue 2 days altogether in 30ml Proflo substratum.5ml is transferred to the defined medium that 50ml contains 1mM copper from Proflo substratum culture in 2 day age.Make culture growth 5 days at 28 ℃.Centrifugation medium also is used for ABTS with supernatant and detects.
Embodiment 8
A. the expression of laccase B gene in Aspergillus
[92] in order to make up the laccase B expression of gene plasmid that is used for CBS bacterial strain 115.075, use two primer GCAGATCTGCGATGTCTCTTCTTCGTAGCTTGAC (SEQ ID No.72) and GAGGTCACCTCTAGATCATGTATCACCTGGGCTCAAGGCATC (SEQ ID NO.73) in containing available from the Herculase PCR reaction (referring to embodiment 2b) of the genomic dna template of 115.075 bacterial strains.Use the centrifugal column purification PCR of QIAquick fragment and use Restriction Enzyme BglII and XbaI digestion.Be cloned into pGAPT carrier with the centrifugal column purification dna fragmentation of QIAquick and with it once more through BglII and XbaI digestion.Confirm the fidelity of reproduction of plasmid by dna sequencing.The plasmid pKB401 (Fig. 2) that generates is transformed in the aspergillus niger 2445 to detect laccase B expression of gene.Select 34 transformant and be transferred on the MM flat board and to grow 4 days at 30 ℃.The Microembolism (plug) that will comprise spore and mycelial single bacterium colony is inoculated on the CMA flat board and in 30 ℃ of growths 4 days.To contain the spore of fusion and the embolism of mycelial CMA flat board and transfer to the Promosoy special culture medium (pH6.2) that 30ml contains 1mM copper.Culture was grown 5 days in 30 ℃.Centrifugation medium also is used for ABTS with supernatant and detects.
B. the expression of laccase B gene in Aspergillus of merging with the catalyst structure domain of glucoamylase
[93] in order to make up the laccase B gene Fusion expression plasmid that is used for CBS bacterial strain 115.075, use two primer TTGCTAGCAACGTGATCTCCAAGCGTGCAATCGGTCCAGTCACTGACCTAC (51mer, SEQ ID No.74) and primer SEQ ID NO.73 in containing available from the Herculase PCR reaction (referring to embodiment 2b) of the genomic dna template of CBS115.075 bacterial strain.Use the centrifugal column purification PCR of QIAquick fragment and also use the centrifugal column purification of QIAquick once more with Restriction Enzyme Nhel and BstEII digestion.With the fragment cloning behind this purifying to carrier pGAMpR2-GV (referring to U.S. Patent application US20050153399) through Nhel and BstEI digestion.Be transformed in the aspergillus niger 2445 by the plasmid pKB403 (Fig. 3) of sequencing analysis affirmation generation and with it.Select 28 transformant and be transferred on the MM flat board and to grow 4 days at 30 ℃.The fritter that will comprise spore and mycelial single bacterium colony is inoculated on the CMA flat board and in 30 ℃ of growths 4 days.To contain the spore of fusion and the embolism of mycelial CMA flat board and transfer to the Promosoy special culture medium (pH6.2) (referring to U.S. Patent application US20050153399) that 30ml contains 1mM copper.Culture was grown 5 days in 30 ℃.Centrifugation medium also is used for ABTS with supernatant and detects.
C. the expression of laccase B gene in trichoderma
[94] structure is used for the expression plasmid of the laccase B gene (referring to embodiment 2b) of CBS 115.075 bacterial strains.Design also obtains primer: GTAATCATGTATCACCTGGGCTCAAGG (SEQ ID NO.50) from Invitrogen.This primer and primer SEQ ID No.46 are used for Herculase PCR reaction (referring to embodiment 2b).Use QIAquick centrifugal column purification PCR fragment and it is cloned into pENTR/D-TOPO carrier (Invitrogen).Use 17 clones of Ready-To-Go PCR pearl amplification and from pENTR-CBS 115.075#1 clone and separate plasmid DNA (referring to embodiment 3c).Laccase B Gene Partial is checked order to confirm the fidelity of reproduction of pcr amplification.Contain in the LB clonase II reaction (Invitrogen) of 6.5 μ l TE, 1 μ lpTrex3g carrier (0.1mg/ml) and 2 μ l Clonasell at 10 μ l, pENTR-laccase B-CBS 115.075#1 plasmid (50ng) is converted into expression plasmid pTrex3g-laccase B (referring to Fig. 1, just replacing laccase A gene with laccase B gene).Confirm this expression plasmid and it is converted into the Trichoderma bacterial strain by dna sequencing with particle gun.Select 60 transformant and it is transferred on the new plate.At 30 ℃, 20 stable transformant growths continue 2 days altogether in 30ml Proflo substratum.3ml is transferred to 30ml from Proflo substratum culture in 2 day age to be contained in the defined medium (referring to U.S. Patent application 20050153399) of 1mM copper.Make culture growth 4 days at 28 ℃.Centrifugation medium also is used for ABTS with supernatant and detects.
D. laccase B gene and CBH1 merge the expression in Trichoderma
[95] in order to make up the laccase B expression of gene plasmid that is used for CBS bacterial strain 115.075, design also obtains primer: GGACTAGTGTCGCCGTTTACAAACGCGCAATCGGTCCAGTCACTGACC, SEQ ID NO.65 from Invitrogen.This primer and reverse primer (available from New England Biolab) combination is used to contain the Herculase PCR reaction of pENTR-laccase B CBS 115.075#1 (referring to embodiment 3c) as dna profiling.PCR fragment (SEQ ID No.66)
ACTAGTGTCG CCGTTTACAA ACGCGCAATC GGTCCAGTCA CTGACCTACA 50
TATAGTGAAC CAGAATCTCG ACCCAGATGG TTTCAACCGC CCCACTGTAC 100
TCGCAGGTGG TACTTTCCCC GGTCCTCTGA TTCGTGGTAA CAAGGTACGC 150
TTCATAACCG CCCTCCGTAG ACGTAGGCTT CGGCTGACAT GACCATCATC 200
TGTAGGGAGA TAACTTTAAA ATTAATGTGA TTGACGACTT GACAGAGCAC 250
AGTATGCTCA AGGCTACGTC CATCGTAAGT CCCTGATTAA CGTTTCACCT 300
GGTCATATCG CTCAACGTCT CGAAGCACTG GCATGGGTTC TTCCAGAAGG 350
GAACCAACTG GGCCGATGGC CCCGCCTTTG TCACCCAATG TCCTATCACA 400
TCAGGAAACG CCTTCCTGTA TGATTTCAAC GTTCCGGACC AAGCTGGTAC 450
TTTCTGGTAC CACAGCCATC TCTCTACACA GTATTGTGAC GGTCTTCGTG 500
GTGCCTTTGT CGTCTATGAT CCTAATGATC CCAACAAGCA ACTCTATGAT 550
GTTGATAACG GCAAGTTCCT TGCATATTTC ATTTCTATCA TATCCTCACC 600
TGTATTGGCA CAGAAAGCAC CGTGATTACC TTGGCTGATT GGTATCATGC 650
CCTTGCTCAG ACTGTCACTG GTGTCGCGTG AGTGACAAAT GGCCCTCAAT 700
TGTTCACATA TTTTCCTGAT TATCATATGA TAGAGTATCT GATGCAACGT 750
TGATCAACGG ATTGGGACGT TCGGCCACCG GCCCCGCAAA TGCCCCTCTG 800
GCGGTCATCA GTGTCGAGCG GAATAAGAGG TCAGTTCCAT AATTATGATT 850
ATTTCCCGCG TTACTTCCTA ACAATTATTT TTGTATCCCT CCACAGATAT 900
CGTTTCCGAT TGGTTTCTAT TTCTTGCGAC CCTAACTTTA TTTTCTCAAT 950
TGACCACCAC CCAATGACCG TAATTGAGAT GGACGGTGTT AATACCCAAT 1000
CTATGACCGT AGATTCGATC CAAATATTCG CAGGTCAACG ATATTCATTT 1050
GTCGTAGGTT ATTATAAACT GCCCACCGAT CATCTCTCAC GTAACTGTTA 1100
TAGATGCAAG CCAACCAACC AGTTGGAAAT TATTGGATCC GCGCTAAACC 1150
TAATGTTGGG AACACAACTT TCCTTGGAGG CCTGAACTCC GCTATATTAC 1200
GATATGTGGG AGCCCCTGAC CAAGAACCGA CCACTGACCA AACACCCAAC 1250
TCTACACCGC TCGTTGAGGC GAACCTACGA CCCCTCGTCT ATACTCCTGT 1300
GGTATGTTGT TCTCGTTACA TATACCAAAC CTAATATGAA GACTGAACGG 1350
ATCTACTAGC CGGGACAGCC ATTCCCTGGC GGTGCTGATA TCGTCAAGAA 1400
CTTAGCTTTG GGTTTCGTAC GTGTATTTCA CTTCCCTTTT GGCAGTAACT 1450
GAGGTGGAAT GTATATAGAA TGCCGGGCGT TTCACAATCA ATGGAGCGTC 1500
CCTCACACCT CCTACAGTCC CTGTACTACT CCAGATCCTC AGTGGTACTC 1550
ACAATGCACA GGATCTTCTC CCAGCAGGAA GCGTGATCGA ACTTGAACAG 1600
AATAAAGTTG TCGAAATCGT TTTGCCCGCT GCGGGCGCCG TTGGCGGTCC 1650
TCATCCTTTT CACTTACATG GTGTAAGTAT CAGACGTCCT CATGCCCATA 1700
TTGCTCCGAA CCTTACACAC CTGATTTCAG CACAATTTCT GGGTGGTTCG 1750
TAGCGCCGGT CAAACCACAT ACAATTTCAA TGATGCTCCT ATCCGTGATG 1800
TTGTCAGTAT TGGCGGTGCA AACGATCAAG TCACGATCCG ATTTGTGGTA 1850
TGTATCTCGT GCCTTGCATT CATTCCACGA GTAATGATCC TTACACTTCG 1900
GGTTCTCAGA CCGATAACCC TGGCCCATGG TTCCTTCACT GTCACATTGA 1950
CTGGCATTTG GAGGCTGGGT TCGCTGTAGT CTTTGCGGAG GGAATCAATG 2000
GTACTGCAGC TGCTAATCCA GTCCCAGGTA AGACTCTCGC TGCTTTGCGT 2050
AATATCTATG AATTTAAATC ATATCAATTT GCAGCGGCTT GGAATCAATT 2100
GTGCCCATTG TATGATGCCT TGAGCCCAGG TGATACATGA TTACAAGGGT 2150
GGGCGCGCC 2159
Digest with the centrifugal column purification of QIAquick and with Restriction Enzyme Spel and Ascl.Then this fragment (SEQ ID No.66) is cloned into also through the pTrex4 carrier of Spel and Ascl digestion with produce expression vector (pTrex4-laccase B, Fig. 4).Confirm the fidelity of reproduction of expression plasmid and use particle gun that it is transformed in the Trichoderma bacterial strain by dna sequencing.Produce the transformant more than 100 and 60 transformant are transferred to new plate.At 30 ℃, on 30ml Proflo substratum, amount to 20 stable transformant growths 2 days.5ml is transferred to the defined medium that 50ml contains 1mM copper from the culture in 2 day age of Proflo substratum.Culture was grown 4 days in 28 ℃.Centrifugation medium also is used for ABTS with supernatant and detects.
Embodiment 9
The expression of the laccase B gene of a.CBS bacterial strain 115.075 in strepto-belongs to
[96] use according to muta lead mycillin (Streptomyces lividans) codon, be used for laccase B protein sequence codon optimized.To be used for the synthetic laccase B expression of gene plasmid that strepto-belongs to CBS 115.075 bacterial strains in order making up, in the Herculase of the dna profiling that contains optimization PCR reaction (referring to embodiment 2b), to use two primer ACGCAGCCTGAACTAGTTGCGATCCTCTAGAG (SEQ ID NO.75) and CTCTGATCAAGGTCATCAGGTGTCGCCCGGGGACAGG (SEQ IDNO.76).Use the centrifugal column purification PCR of QIAquick fragment and use XbaI and BclI digestion.Use the postdigestive fragment of the centrifugal column purification of QIAquick and it is cloned into carrier pKB105 (referring to US 20060154843) through Xbal and BamHI digestion.Confirm the exactness of the plasmid pKB251 (Fig. 5) of generation by dna sequencing.The DNA of plasmid pKB251 is transformed into muta lead mycillin g3s3 bacterial strain (referring to US 20060154843).Choose 12 thiostrepton resistance transformant and be transferred to the production of hybrid seeds and shake bottle (20ml TSG substratum contains 50 μ g/ml thiostreptons in DMSO), 30 ℃ of growths 2 days.The culture in 2 day age that 3ml is shaken bottle from the production of hybrid seeds is transferred to the production medium ii that 30ml contains the strepto-genus improvement of 1mM copper.Culture was grown 4 days in 30 ℃.Centrifugation medium also is used for ABTS with supernatant and detects.
Embodiment 10, the laccase B gene synthetic gene and the expression of CBH1 fusion in Trichoderma that son is optimized that access to your password
[97] the synthetic laccase B gene (SEQ ID NO:67) of You Huaing:
ACTAGTGTCG CCGTTTACAA ACGCGCAATC GGTCCCGTCA CTGACCTGCA 50
TATTGTGAAC CAGAATCTCG ACCCCGATGG TTTCAACCGC CCCACTGTCC 100
TCGCAGGTGG TACTTTCCCC GGTCCTCTGA TTCGTGGTAA CAAGGGAGAT 150
AACTTTAAAA TTAATGTGAT TGACGACTTG ACAGAGCACA GCATGCTCAA 200
GGCTACGTCC ATCCACTGGC ATGGCTTCTT CCAGAAGGGA ACCAACTGGG 250
CCGATGGCCC CGCCTTTGTC ACCCAATGTC CTATCACATC AGGAAACGCC 300
TTCCTGTACG ATTTCAACGT TCCGGACCAA GCTGGTACTT TCTGGTACCA 350
CAGCCATCTC TCTACACAGT ACTGTGACGG TCTTCGTGGT GCCTTTGTCG 400
TCTACGATCC TAATGATCCC AACAAGCAAC TCTACGATGT TGATAACGGC 450
AACACCGTGA TTACCTTGGC TGATTGGTAC CATGCCCTTG CTCAGACTGT 500
CACTGGTGTC GCAGTCTCTG ATGCAACGTT GATCAACGGA TTGGGACGTT 550
CGGCCACCGG CCCCGCAAAT GCCCCTCTGG CGGTCATCAG CGTCGAGCGC 600
AATAAGCGCT ATCGTTTCCG ATTGGTTTCT ATTTCTTGCG ACCCTAACTT 650
TATTTTCTCA ATTGACCACC ACCCCATGAC CGTCATTGAG ATGGACGGTG 700
TTAATACCCA ATCTATGACC GTAGATTCGA TCCAAATCTT CGCAGGTCAA 750
CGATACTCAT TTGTCATGCA AGCCAACCAA CCAGTTGGAA ATTACTGGAT 800
CCGCGCTAAA CCTAATGTTG GCAACACAAC TTTCCTTGGA GGCCTGAACT 850
CCGCTATCTT GCGATACGTG GGAGCCCCTG ACCAAGAACC GACCACTGAC 900
CAAACACCCA ACTCTACACC GCTCGTTGAG GCGAACCTGC GACCCCTCGT 950
CTACACTCCT GTGCCGGGAC AGCCATTCCC TGGCGGTGCT GATATCGTCA 1000
AGAACTTGGC TTTGGGTTTC AATGCCGGGC GTTTCACAAT CAATGGAGCG 1050
TCCCTCACAC CTCCTACAGT CCCTGTCCTG CTCCAGATCC TCAGCGGTAC 1100
TCACAATGCA CAGGATCTTC TCCCGGCAGG AAGCGTGATC GAACTTGAAC 1150
AGAATAAAGT TGTCGAAATC GTTTTGCCCG CTGCGGGCGC CGTTGGCGGT 1200
CCTCATCCTT TTCACTTGCA TGGTCACAAT TTCTGGGTGG TTCGTAGCGC 1250
CGGTCAAACC ACATACAATT TCAATGATGC TCCTATCCGT GATGTTGTCA 1300
GCATTGGCGG TGCAAACGAT CAAGTCACGA TCCGATTTGT GACCGATAAC 1350
CCTGGCCCAT GGTTCCTTCA CTGTCACATT GACTGGCATT TGGAGGCTGG 1400
ATTCGCTGTC GTCTTTGCGG AGGGAATCAA TGGTACTGCA GCTGCTAATC 1450
CCGTCCCGGC GGCTTGGAAT CAATTGTGCC CGTTGTACGA TGCCTTGAGC 1500
CCGGGTGATA CATGAGGCGC GCC 1523
Its coding laccase B gene, (Suite 15 for Molecular Cloning Laboratories, 384Oyster Point Blvd, and South San Francisco is CA94080) synthetic by McLab Inc..Digest the synthetic plasmid DNA and from gel, separate the 1.5kb dna fragmentation with Ascl with Restriction Enzyme Spel, it is cloned into the pTrex4 carrier that is also digested by Spel and Ascl produces expression plasmid (pTrex4-laccase Bopt), expression plasmid shown in this plasmid and Fig. 4 is similar, except replace (unoptimizable) laccase B gene with codon optimized laccase B gene.This plasmid is transformed into the Trichoderma bacterial strain with particle gun.Produce the transformant more than 30 and it is transferred to new plate.20 stable transformant of selective summarizing also are transferred to the defined medium that 30ml contains 1mM copper with mycelium.28 ℃ of grown culture 4 days.Centrifugation medium also is used for ABTS with supernatant and detects.
Embodiment 11
A. the expression of laccase D gene in Trichoderma
[98] in order to make up the laccase D expression of gene plasmid that is used for CBS 115.075 bacterial strains, use two primers (SEQ ID No.63 and SEQ ID No.64) in containing available from the Herculase PCR reaction (referring to embodiment 2b) of the genomic dna template of CBS 115.075 bacterial strains.Use QIAquick centrifugal column purification PCR fragment and it is cloned into the pENTR/D-TOPO carrier.Use 16 of Ready-To-Go PCR pearl amplifications to clone and 4 plasmid DNA are checked order.Select pENTR-laccase D CBS 115.075#2 clone.Contain in the LB clonase II reaction of 6.5 μ l TE, 1 μ l pTrex3g carrier (0.1mg/ml) and 2 μ l Clonasell at 10 μ l, pENTR-laccase D CBS 115.075#2 plasmid (50ng) is converted into expression plasmid pTrex3g-laccase D, expression vector shown in this expression plasmid and Fig. 1 is similar, except replace laccase A gene with codon optimized laccase D gene.Reaffirm this expression plasmid and it is converted into the Trichoderma bacterial strain by dna sequencing with particle gun.Select 45 transformant and be transferred to new plate.To be transferred to the defined medium that 30ml contains 0.5mM copper from the mycelium of 28 stable conversion bodies.Make culture growth 4 days at 28 ℃.Centrifugation medium also is used for ABTS with supernatant and detects.
B. laccase D gene and CBH1 merge the expression in Trichoderma
[99] in order to make up the laccase D expression of gene plasmid that is used for CBS 115.075 bacterial strains, design also obtains two primer GGACTAGTGTCGCCGTTTACAAACGCGCAATTGGGCCCGTGGCCGAC from Invitrogen, SEQ ID No.68 and AAGGCGCGCCTTAAATAGCAGTTCCTTTCTTAG, SEQ ID No.69.This primer is used for containing with the Herculase PCR reaction of CBS 115.075 strain gene group DNAs dna profiling.Use the centrifugal column purification PCR of QIAquick fragment and with Restriction Enzyme Spel and Ascl digestion, and it is cloned into also pTrex4 carrier through Spel and Ascl digestion (referring to U.S. Patent application 10/590,956; WO 05/093050) to produce expression plasmid pTrex4-laccase D.Confirm the fidelity of reproduction of this expression plasmid and it is converted into the Trichoderma bacterial strain by dna sequencing with particle gun.Produce the transformant more than 300 and 60 transformant are transferred to new plate.To be transferred to the defined medium that 30ml contains 0.5mM copper from the mycelium of 25 stable conversion bodies.Make culture growth 4 days at 28 ℃.Centrifugation medium also is used for ABTS with supernatant and detects.
Embodiment 12, the laccase D gene synthetic gene and the expression of CBH1 fusion in Trichoderma that son is optimized that access to your password
[100]DNA(SEQ ID NO：70)：
ACTAGTGTCG CCGTTTACAA ACGCGCTATT GGACCAGTTG CTGATCTGCA 50
CATCGTTAAC AAGGATTTGG CCCCAGACGG CGTCCAGCGC CCAACTGTTC 100
TGGCCGGTGG AACTTTTCCG GGCACGCTGA TTACCGGTCA AAAGGGCGAC 150
AACTTCCAGC TGAACGTGAT TGATGACCTG ACCGACGATC GCATGTTGAC 200
CCCTACTTCG ATCCATTGGC ATGGTTTCTT CCAGAAGGGA ACCGCCTGGG 250
CCGACGGTCC GGCTTTCGTT ACACAGTGCC CTATTATCGC AGACAACTCC 300
TTCCTCTACG ATTTCGACGT TCCCGACCAG GCGGGCACCT TCTGGTACCA 350
CTCACACTTG TCTACACAGT ACTGCGACGG TCTGCGCGGT GCCTTCGTTG 400
TTTACGACCC CAACGACCCT CACAAGGACC TTTATGATGT CGATGACGGT 450
GGCACAGTTA TCACATTGGC TGACTGGTAT CACGTCCTCG CTCAGACCGT 500
TGTCGGAGCT GCTACACCCG ACTCTACGCT GATTAACGGC TTGGGACGCA 550
GCCAGACTGG CCCCGCCGAC GCTGAGCTGG CCGTTATCTC TGTTGAACAC 600
AACAAGAGAT ACCGTTTCAG ACTCGTCTCC ATCTCGTGCG ATCCCAACTT 650
CACTTTTAGC GTCGACGGTC ACAACATGAC GGTTATCGAG GTTGATGGCG 700
TGAATACCCG CCCTCTCACC GTCGATTCCA TTCAAATTTT CGCCGGCCAG 750
CGATACTCCT TTGTGCTGAA TGCCAATCAG CCCGAGGATA ACTACTGGAT 800
CCGCGCTATG CCTAACATCG GACGAAACAC CACTACCCTT GATGGCAAGA 850
ATGCCGCTAT CCTGCGATAC AAGAACGCCA GCGTTGAGGA GCCCAAAACC 900
GTCGGAGGAC CCGCGCAGAG CCCATTGAAC GAGGCCGACC TGCGACCTCT 950
GGTGCCCGCT CCTGTCCCTG GCAACGCAGT TCCTGGTGGT GCGGACATCA 1000
ACCACCGCCT GAACCTGACA TTCAGCAACG GCCTCTTCTC TATCAATAAC 1050
GCATCATTTA CAAACCCCAG CGTCCCTGCC TTGTTGCAGA TTCTTTCCGG 1100
CGCACAAAAC GCTCAGGATC TGCTTCCCAC CGGTTCTTAT ATCGGCTTGG 1150
AGTTGGGCAA GGTCGTTGAA CTCGTGATCC CTCCCTTGGC CGTTGGTGGC 1200
CCCCATCCAT TCCACTTGCA CGGCCACAAC TTTTGGGTCG TCCGAAGCGC 1250
TGGTTCTGAC GAGTATAATT TCGACGATGC AATTTTGCGC GACGTGGTCA 1300
GCATTGGCGC GGGAACTGAC GAGGTTACTA TCCGTTTTGT CACTGATAAC 1350
CCAGGCCCTT GGTTCCTCCA TTGCCACATC GACTGGCACC TCGAAGCCGG 1400
CCTCGCCATT GTTTTCGCCG AAGGCATCAA TCAAACCGCA GCCGCCAACC 1450
CGACTCCACA GGCCTGGGAC GAACTCTGCC CCAAGTATAA CGGACTCTCC 1500
GCTTCCCAGA AAGTGAAGCC CAAGAAGGGA ACAGCCATCT AAGGCGCGCC 1550
Its laccase D gene (according to the gene from CBS 115.075) of encoding, (1455Adams Drive, Menlo Park CA94025) synthesize by DNA2.0Inc..With Restriction Enzyme Spel and Ascl digestion synthetic plasmid DNA and from gel, separates the dna fragmentation of 1.5kb, it is cloned into also pTrex4 carrier generation expression plasmid (pTrex4-laccase Dopt) by Spel and Ascl digestion.This plasmid is transformed into the Trichoderma bacterial strain with particle gun.40 transformant are transferred to new plate.24 stable transformant of selective summarizing also are transferred to the defined medium that 30ml contains 0.5mM copper with mycelium.At 28 ℃, grown culture 4 days.Centrifugation medium also is used for ABTS with supernatant and detects.
Embodiment 13, the laccase D gene synthetic gene and the expression of BCE103 fusion in bacillus that son is optimized that access to your password
[101]DNA(SEQ ID NO：71)：
GGATCCTGAA GCTATCGGTC CGGTTGCAGA TTTACACATC GTAAACAAAG 50
ATCTTGCACC TGACGGCGTT CAACGTCCAA CTGTACTTGC TGGTGGAACA 100
TTCCCTGGTA CACTTATTAC TGGTCAAAAA GGTGACAACT TCCAATTAAA 150
CGTAATTGAC GATCTTACAG ATGACCGTAT GCTTACACCG ACTTCAATTC 200
ACTGGCACGG TTTCTTTCAA AAAGGAACAG CATGGGCTGA TGGTCCTGCA 250
TTCGTTACAC AATGTCCAAT CATTGCTGAT AACTCTTTCC TTTACGATTT 300
TGACGTTCCT GATCAAGCTG GTACATTCTG GTATCACTCA CACTTATCCA 350
CACAATACTG CGATGGACTT CGCGGAGCTT TCGTAGTTTA CGACCCAAAC 400
GATCCTCATA AAGACCTTTA CGATGTAGAT GATGGTGGAA CAGTTATCAC 450
ATTAGCTGAT TGGTACCATG TACTTGCTCA AACAGTTGTA GGTGCAGCTA 500
CACCAGATTC AACACTTATC AATGGATTAG GACGTTCTCA AACTGGTCCT 550
GCTGACGCAG AACTTGCTGT AATCTCTGTT GAACATAACA AACGTTACAG 600
ATTCCGTCTT GTTAGCATTT CTTGCGATCC AAACTTCACA TTTTCAGTTG 650
ACGGACATAA CATGACAGTT ATCGAAGTAG ATGGTGTAAA CACACGTCCA 700
CTTACTGTAG ACTCTATCCA AATCTTCGCA GGACAACGTT ACTCATTCGT 750
ATTAAACGCA AATCAACCAG AAGATAACTA CTGGATTCGT GCAATGCCAA 800
ACATCGGACG TAACACTACA ACTCTTGACG GCAAAAACGC AGCTATTCTT 850
CGTTACAAAA ACGCTTCTGT TGAAGAACCT AAAACAGTTG GTGGACCAGC 900
ACAATCACCA CTTAACGAAG CTGACTTACG TCCACTGGTT CCAGCACCTG 950
TACCTGGAAA CGCTGTACCA GGAGGTGCTG ATATTAATCA TAGACTTAAC 100
CTTACTTTCT CTAACGGTCT GTTCTCAATC AACAACGCTT CATTCACAAA 1050
TCCTTCAGTT CCAGCACTTT TACAAATTCT TAGCGGTGCA CAAAATGCTC 1100
AGGATCTTTT ACCAACTGGA TCTTACATTG GTCTTGAACT GGGTAAAGTA 1150
GTTGAATTAG TAATTCCTCC GCTTGCTGTA GGTGGACCAC ATCCTTTCCA 1200
TCTTCACGGT CATAACTTCT GGGTTGTACG TTCTGCTGGT TCAGATGAAT 1250
ACAACTTCGA TGACGCAATT CTTCGTGATG TTGTATCTAT TGGTGCTGGA 1300
ACAGATGAAG TAACTATTCG TTTCGTAACA GATAACCCTG GTCCTTGGTT 1350
CTTACATTGT CATATCGATT GGCATCTTGA AGCTGGACTT GCTATTGTTT 1400
TCGCTGAAGG AATCAATCAA ACAGCTGCAG CTAACCCAAC ACCTCAAGCA 1450
TGGGACGAAT TATGTCCAAA ATACAACGCA CTTTCTCCAG GAGATACTTA 1500
AAAGCTT 1507
Its laccase D gene (according to the gene from CBS 115.075) of encoding, (1455Adams Drive, Menlo Park CA94025) synthesize by DNA2.0Inc..With Restriction Enzyme BamHI and HindIII digestion synthetic plasmid DNA and from gel, separates the dna fragmentation of 1.5kb, be connected to p2JMagk1031nk2 carrier (referring to US20050202535A1) by two kinds of identical Restriction Enzymes digestion with generation expression plasmid p2JMagk1031nk2E-laccase (Fig. 6).This plasmid is transformed into Bacillus subtilus (B.subtilis) bacterial strain (degUHy32, oppA, DspoIIE, DaprE, DnprE, Depr, DispA, Dbpr, Dvpr, DwprA, Dmpr-ybfJ, DnprB, amyE::xylRPxylAcomK-ermC) (referring to US20050202535A1).Have two transformant of selection on the Luria Broth agar plate of 5mg/ml paraxin, screening has the clone of higher gene copy number then, will clone line continuously on the Luria Broth agar plate of 25mg/ml paraxin up to the quick colony growth of acquisition having.The transformant that increases is inoculated into 30ml to be contained in the MBD substratum (referring to US20050202535A1) of 0.5mM copper.Make culture growth 60 hours at 37 ℃.Centrifugation medium also is used for ABTS with supernatant and detects.
Embodiment 14, with the bleaching indigo of different laccases to dissolved
[102] in 96 hole titer plate, undertaken by the detection of laccase/amboceptor combination the indigo substrate bleaching of dissolved by following.
[103] by at room temperature stirring the indigo saturated solution among indigo (30mg) of N-Methyl pyrrolidone (NMP) in (10ml) preparation in the 5 hours NMP.This nmp solution of dilution obtains blue solution for 10 times in aqueous buffer.For example, be diluted in the 50mM sodium-acetate buffer of pH 5, perhaps in the 50mM sodium phosphate buffer of pH 7.Solution is fully vibration before use.
[104] in 96 hole titer plate, carry out detection to the bleaching of the indigo substrate of dissolved, wherein every hole is received in the soluble indigo blue solution (180 μ L) in the 50mM sodium-acetate buffer of pH5, laccase (10ppm enzyme) and amboceptor solution (from the 20mM mother liquor in the methyl alcohol).With deionized water the cumulative volume in every hole is transferred to 200 μ L.The contrast that only contains laccase repeats twice.Seal this plate and (Thermomixer Eppendorf) goes up and to hatch 2 hours with 800rpm at 50 ℃ of agitators in heating.After this stage, open this plate and add ascorbic acid solution (10% aqueous solutions of 20 μ L) so that reduce the oxidised form of amboceptor to every hole.Then by using the titer plate reader to determine that every hole assesses the degree of indigo bleaching in the absorbancy of 600nm.The absorbancy reading is low more, and indigo bleaching degree is high more.
[105] Fig. 7 shows the result from Thielavia species laccase (Ecostone LCC10, ABenzymes, Darmstadt, Germany).Used amboceptor is 2,2 '-azine-two (3-ethyl benzo thiazole phenanthrolines-6-sulfonic acid) (ABTS), syringic acid, 4-formamido--2,6-syringol (SA), methyl syringate (MS), 4-(N-methylformamide base)-2,6-syringol (MSA), 10-(carboxylic propyl group)-thiodiphenylamine (PTP) and syringic aldehyde.Table 1 has been listed the absorbancy variation of 600nm place with respect to contrast, and wherein the maximum of absorbancy changes corresponding to maximum indigo bleaching.
When [106] mediator concentration was 500 μ M, the most effective amboceptor that is used for indigo bleaching was ABTS, followed by N-methane amide (MSA) and unsubstituted acid amides 4-formamido--2,6-syringol (SA).When the low mediator concentration of 50 μ M, ABTS still is the most effective amboceptor, and remaining amboceptor is about the same.Exception is a syringic acid, its with collating condition under do not have an effect to the bleaching of soluble indigo blue is the same.
Table 1, to use Thielavia species laccase and concentration be that the multiple amboceptor of 500 and 50 μ M carries out after the bleaching of soluble indigo blue the absorbancy variation (n=2) at 600nm place
Figure A20078004701800451
Embodiment 15, the soluble indigo blue bleaching with different laccases under two kinds of pH values detect
[107] two kinds of pH values, assessment and being derived from of lower molecular weight amboceptor combination ruin Hyphomyces (Myceliophtora) (
Figure A20078004701800452
II, Novozymes, Bagsvaerd, Denmark), the indigo ability of its bleaching dissolved of laccase of Thielavia (Ecostone LCC10, AB enzymes, Darmstadt, Germany) and the black hole of hypodermis species.
[108] be 5 and 7 times in the pH value, use 3 kinds of different laccases, in 96 hole titer plate, carry out the indigo bleaching of dissolved by embodiment 14 is described.Used amboceptor is sinapinic acid, 4-formamido--2,6-syringol (SA), methyl 4-acetyl cloves acid esters (AMS), methyl syringate (MS) and 2,2 '-azine-two (3-ethyl benzo thiazole phenanthrolines-6-sulfonic acid) are (ABTS).It is 5 and 7 o'clock that Fig. 8 and 9 shows the pH value, uses respectively to be derived from three kinds of laccases ruining the black hole of Hyphomyces, Thielavia and hypodermis species result for the soluble indigo blue bleaching.These data lists are in table 2 and 3.
Table 2, at pH5, mediator concentration is under the 250 μ M, use carry out the soluble indigo blue bleaching from Thielavia, the laccase of ruining Hyphomyces and the black hole of hypodermis species after, at 600nm place with respect to the absorbancy variation of contrast
Figure A20078004701800461
Table 3, be under the 250 μ M in pH 7, mediator concentration, use carry out the soluble indigo blue bleaching from Thielavia, the laccase of ruining Hyphomyces and the black hole of hypodermis species after, at 600nm place with respect to the absorbancy variation of contrast
Figure A20078004701800462
Figure A20078004701800471
Determining of embodiment 16, purifying and specific activity
[109] in 14 liters of fermentor tanks, use is called the application US60/984 common co-pending of " Signal Sequences andco-expressed chaperones for improved heterologous protein production ina host cell " in name, expression system described in 430 (the file number No.GC993P of agency, on November 1st, 2007 submitted) is expressed the gene (SEQID NO:70) that laccase D optimizes.Collected fermented liquid and concentrated by ultrafiltration process (UFC 20070245) at 184 hours.The 25mM sodium-acetate is arrived in enriched material diafiltration (diafilter), in the pH4.0 damping fluid.Then with sample on the UFC sample after the 500ml diafiltration to contain Poros HS-20 resin (Applied Biosystems, 20X 275mm column), with the 25mM sodium-acetate buffer, in the pH4.0 equilibrated ion exchange column.With the 25mM sodium-acetate buffer of 10 times of column volumes, pH4.0 washes post.Use is at the 25mM sodium-acetate buffer, among the pH4.0 from the salt gradient (12 times of column volumes) of 40mM to 80mM sodium-chlor from this post wash-out laccase D protein.Collection contains the fraction of laccase activity and uses the Amicon 400mL agitation elements with 10K film further to concentrate.Use BSA as standard, measuring gross protein by the SDS protein gel is 4mg/ml (＞90% purity).Water was also at room temperature deposited the laccase diluted sample 18 hours and was deposited greater than 24 hours at 4 ℃ for 10,000 times.Measuring the ABTS activity is 8570 units/ml.Then by obtaining 2140 units/ml protein divided by the specific activity that 4mg/ml calculates reorganization laccase D with 8570 units/ml, this is active higher 100 times than Stachybotrys atra laccase (16U/mg), referring to people such as Mander, Appl.Environ.Microbiol. (2006) 72:5020-5026).Therefore, this enzyme causes the copper that discharges in environment lower than other laccases (as the Stachybotrys atra laccase) owing to high specific acitivity.
Embodiment 17, be used for the program of denim bleaching
Amboceptor
[110] 4-hydroxyl-3, (the cloves acid amides is SA) available from PunjabChemicals﹠amp for 5-syringol methane amide; Crop Protection Limited (Mumbai, India).4-hydroxyl-3, the 5-dimethoxy-benzyl nitrile (the cloves nitrile, SN) available from Stereochemical, Inc., (Newark, DE) or PunjabChemicals﹠amp; Crop Protection Limited (Mumbai, India).
Enzyme
[111] (embodiment 16,8570U/ml, 4mg protein/ml) to use the laccase be derived from the black pore fungi of monochromatic hypodermis in the experiment.
Program
[112] under the different condition that relates to pH, temperature, enzyme concn and mediator concentration, finish enzyme in ATLASLP 2Launder-O-meter and hatch.
[113] in containing the 500ml stainless steel reaction container of 100ml liquid, react.For each container, add 5 parts of (Stainless Steel Balls of the denim sample of 7 * 7cm) granite-wash (ACG denim 80270 types) and 6 6mm diameters.The off-response container is also put into the launder-O-meter that preheats to preferred temperature.Carry out hatching in 30 minutes, clean this sample with " mobile " tap water after this, Rotary drying and be suitable for Elna pressure electric iron dryly under the program of cotton also assessing in AEG IPX4 whizzer.
The granite-wash of denim
[114] under the following conditions, with denim destarch in UnimacUF 50 washing machines of the 12 car pin (leg) of heavily about 3kg:
With 10: 1 liquor ratios, in 50 ℃ with 0.5g/l (15g) Optisize160 amylase (Genencor) and 0.5g/l (15g) nonionogenic tenside (Rucogen BFA for example, (Rudolf Chemie) or Ultravon GPN, (Huntsman)) destarch is 15 minutes
With twice cold rinsing of 30: 1 liquor ratios 5 minutes.
[115] after the destarch, under the following conditions, this denim of granite-wash in Unimac UF 50 washing machines:
With the cold rinsing of liquor ratio in 10: 15 minutes
55 ℃, use the 1kg float stone, citrate buffer (30g citrate trisodium dihydrate and 30g Citric acid monohydrate Food grade) and 35g IndiAge 2XL cellulase (Genencor) were with liquor ratio granite-wash in 10: 1 60 minutes.
With twice cold rinsing of 30: 1 liquor ratios 5 minutes.
[116] this denim is dry in Miele Novotronic T494C family expenses fabric dryer.Cut the sample of 7 * 7cm from this denim car pin.
The assessment of denim sample
[117] with the color of Minolta Chromameter CR 310 at five denim samples of CIE Lab color space measurement with D65 light source.Measure before with laccase treatment and after handling and the result of 5 samples is averaged.Calculate total color difference (TCD).Can use equation: TCD=√ (Δ L) 2+ (Δ is a) 2+ (Δ b) 2Calculate total color difference.
DenimThe assessment of car pin
[118] has the CIE Lab color space assessment of D 65 light sources with Minolta Chromameter CR 310 DenimThe car pin.Only after laccase treatment, measure.For every DenimThe car pin carry out measuring for 8 times and to the result of 12 car pin (96 times measure) average.According to following formula Δ E=(Δ L 2+ Δ a 2+ Δ b 2) 1/2From beginning CIE L *a *b *Value and final CIE L *a *b *Difference between the value is calculated total color difference (Δ E).
Embodiment 18, temperature are for the influence (Unimac) of reorganization laccase D bleachability
[119] laccase of granite-wash denim bleaching: will heavily about 3kg 12 DenimThe car pin is pressed embodiment 17 described destarch and granite-wash.After the granite-wash, in Unimac UF 50 washing machines, carry out laccase treatment according to following technology:
Carried out 30 minutes with 10: 1 liquor ratios
PH 6 (21g SODIUM PHOSPHATE, MONOBASIC and 5g hexanodioic acid, laccase D) or pH 4.8 (8.6g SODIUM PHOSPHATE, MONOBASIC and 16.8g hexanodioic acid, Novoprime Base 268 laccases)
Laccase (laccase D or Novoprime Base 268)
Amboceptor (cloves acid amides (SA) and cloves nitrile (SN))
After the laccase treatment, with 30: 1 liquor ratios to rinsing in twice cold water of denim 5 minutes.
[120] carry out laccase and test and the results are shown in table 4 and 5.
Table 4
Laccase D concentration Amboceptor Mediator concentration Temperature (℃) Bleaching level (CIE L)
0.05g/l/0.4U/ml SA 0.33mM 60 35.6
0.05g/l/0.4U/ml SN 0.47mM 60 35.9
0.05g/l/0.4U/ml SA 0.33mM 40 35.6
0.05g/l/0.4U/ml SN 0.47mM 40 35.7
Table 5
Novoprime base 268 concentration Mediator concentration Temperature (℃) Bleaching level (CIE L)
0.05g/l 0.023g/l 60 35.9
0.05g/l 0.023g/l 40 33.7
[121] at a lower temperature, reorganization laccase D has better properties than the present commercially available laccase that gets.Laccase D (when having amboceptor) is lower than 60 ℃ in temperature, preferably provides bleaching action between 40 ℃ and 60 ℃.Therefore, this laccase can provide the energy benefit to the textiles processor.
Embodiment 19, reorganization laccase and mediator concentration are for the influence (Launder-O-meter) of bleachability
[122] influence (adjusting 50mM phosphate sodium dihydrogen buffer solution pH with sodium hydroxide 4N solution) of laccase and mediator concentration is assessed in the experiment of carrying out following table pH6 and 60 ℃.
[123] experimentize with cloves acid amides (SA) and cloves nitrile (SN) amboceptor.
[124] (7 * 7cm) beaker adds the 100ml damping fluid to having 5 duplicate samples.Gross weight is a 12g (denim: liquor ratio=1: 8).Use laccase and mediator concentration shown in the according to the form below.
Table 6
Laccase concentration (μ l/l) Corresponding activity (laccase unit/g denim)
10 0.67
33 2.17
55 3.67
78 5.17
100 6.67
Table 7
Mediator concentration (mM)
0.10
0.33
0.55
0.78
1.00
[125] will be as cloves acid amides or each beaker of cloves nitrile amboceptor adding as shown in the table of the diluent of 275mM SA in 98% methyl alcohol or SN mother liquor.To add each beaker as shown in the laccase according to the form below of the diluent of 400 units/ml laccase mother liquor.Close beaker and described 60 ℃ of processing by embodiment 17.Press embodiment 17 these samples of described assessment.
Table 8
Figure A20078004701800511
Figure A20078004701800521
The TCD=total color difference
Table 9
Figure A20078004701800522
Figure A20078004701800531
The TCD=total color difference
[126] last table and Figure 10 and 11 show and need enzyme and amboceptor to bleach.Its also show realize certain Javelle water at ordinary times enzyme/amboceptor ratio certain handiness is arranged.
Embodiment 20, reorganization laccase D are for the dose response effect (Unimac) of bleachability
[127] through 12 of the laccase bleaching of the denim of granite-wash-will heavily about 3kg DenimThe car pin is pressed embodiment 17 described destarch and granite-wash.After the granite-wash, carry out laccase treatment: handled 30 minutes with laccase and amboceptor with 10: 1 liquor ratios and pH 6 (21g SODIUM PHOSPHATE, MONOBASIC and 5g hexanodioic acid) and 60 ℃ according to following technology.After the laccase treatment, with 30: 1 liquor ratios to twice cold rinse of this denim 5 minutes.
[128] carry out following experiment.
Cloves acid amides 0.33mM:
The black pore fungi laccase concentration (g/l) of monochromatic hypodermis Bleaching level (CIE L)
0.010 34.6
0.05 36.2
0.25 36.2
Cloves nitrile 0.39mM:
Laccase D concentration (g/l) Bleaching level (CIE L)
0.25 37.7
0.4 39.5
0.53 38.8
[129] expressed the result on.This has shown utilization reorganization laccase D and acid amides amboceptor, and the bleaching level very rapidly flattens.Enzyme concn is 0.05 and 0.25 o'clock, obtains identical bleaching level.For reorganization laccase D and nitrile amboceptor, the bleaching level increases to 0.4g/l at the most, and this appears as the best.
[130] should understand, embodiment described herein and embodiment only be used for illustration purpose and based on this multiple modification or to change be predictable for those skilled in the art, and it is included in the scope of the application's spirit and scope and claims.All publications, patent and patent application that this paper quotes are quoted as a reference by integral body at this.

Claims (14)

1, a kind of SEQ of being selected from ID NO.2,4,6,8,10,12,14,16,18 laccase and a kind of and any SEQ ID NO.2,4,6,8,10,12,14,16 or 18 laccases with at least 90% identity.
2, a kind of nucleotide sequence of the laccase of encoding, wherein said laccase are selected from SEQ ID NOS.2,4,6,8,10,12,14,16,18 and be and any SEQ ID NOS.2,4,6,8,10,12,14,16 or 18 laccases with at least 90% identity.
3, a kind of nucleotide sequence of the laccase of encoding, wherein said nucleotide sequence are selected from SEQ ID NOS.1,3,5,7,9,11,13,15 and 17.
4, a kind of expression vector comprises the nucleotide sequence of claim 2.
5, a kind of expression vector comprises the nucleotide sequence of claim 3.
6, a kind of host cell comprises the carrier of claim 4.
7, a kind of host cell comprises the carrier of claim 5.
8, the method for the dyestuff in a kind of liquid lime chloride, this method comprise the dyestuff in solution with the laccase of claim 1 and effective amboceptor processing.
9, method according to Claim 8, its mesosome are selected from Syringylethanone, syringic aldehyde, cloves acid amides, methyl cloves acid amides, 2-hydroxyethyl cloves acid amides, methyl syringate, cloves nitrile, dimethyl cloves acid amides and syringic acid.
10, a kind of method of using the laccase bleached woven fabric, its improvement comprises the laccase that uses claim 1.
11, the method for claim 10 also comprises the amboceptor that is selected from Syringylethanone, syringic aldehyde, cloves acid amides, methyl cloves acid amides, 2-hydroxyethyl cloves acid amides, methyl syringate, cloves nitrile, dimethyl cloves acid amides and syringic acid.
12, according to the method for claim 10, wherein with vat dyes to textile dyeing.
13, according to the method for claim 10, wherein this fabric is the mixture of cellulosic fabric, cellulosic fibre or the mixture of cellulose fiber peacekeeping synthon.
14, according to the method for claim 10, wherein this fabric is a denim.
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