CN101568640B - Novel laccase, composition and use method - Google Patents
Novel laccase, composition and use method Download PDFInfo
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- CN101568640B CN101568640B CN2007800470185A CN200780047018A CN101568640B CN 101568640 B CN101568640 B CN 101568640B CN 2007800470185 A CN2007800470185 A CN 2007800470185A CN 200780047018 A CN200780047018 A CN 200780047018A CN 101568640 B CN101568640 B CN 101568640B
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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
The cross reference of related application
The name that the application requires to submit on December 18th, 2006 is called the U.S. Provisional Patent Application series number No.60/875 of " Novel Laceases; 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
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
Laccase is a copper enzyme, and known its is good oxygenant in the presence of oxygen.In mikrobe, 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.
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 (like food stains).
Most of laccases have represented best pH and passivation under neutrality or alkaline pH in the acid pH scope.
Known to multiple fungi generation laccase; This fungi comprises the kind of following genus (genii), and Aspergillus (Aspergillus), Neurospora (Neurospora), handle spore shell belong 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), shell monospore belong to (Amerosporium) and Lentinus (Lentinus).Yet, the laccase that still need in multiple application, have the different performance spectrum.
For many application, can render a service through the oxidation of using amboceptor (also being known as toughener) to improve laccase.Known in the artly comprise that the system of laccase and amboceptor is laccase-mediator systems (LMS).Identical compound also can be used for the effect of activation or initial laccase.
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.
Functional group and substituting group are renderd a service for amboceptor has very big influence.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 possibly effectively be inappropriate 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 type of this kind 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.
Therefore, the active amboceptor that needs to identify other activation laccase and/or strengthen the enzyme that represents laccase activity.
Summary of the invention
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
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.
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.
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.
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.
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.
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.
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.
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.
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.
Figure 10 be at 60 ℃ when the 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.
Figure 11 be at 60 ℃ when the 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
Only if this paper has definition in addition, otherwise all technology that this paper uses and scientific terminology have with those skilled in the art are common and understand identical implication.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, HarperPerennial, N.Y. (1991) provides the general dictionary of the many terms that use among the present invention to the technician.Although can be used for practice or check the present invention with those similar or equivalent any methods described herein and material, describe preferable methods and material.Numerical range contains the numerical value that limits this scope.Should understand the present invention and be not limited to described concrete grammar, scheme and reagent, because these can change.
The exercise question that this paper provides does not constitute the restriction to multiple aspect of the present invention or embodiment, and they can obtain with reference to specification sheets through integral body.Therefore, more fully limit with reference to specification sheets through integral body below back to back term.
From the compsn and the methodological purpose of describing and openly can unite with the present invention use, all publications that this paper quotes are through quote as a reference clearly.I. laccase and laccase involved enzyme
In the context of the present invention, laccase and laccase involved enzyme contain any laccase that is included in the enzyme classification (EC1.10.3.2).Known laccase is from mikrobe and plant-sourced.The mikrobe laccase can be derived from the laccase that bacterium or fungi (comprising filamentous fungus and yeast) and suitable instance comprise the bacterial strain that is derived from following genus: Aspergillus, Neurospora (for example Neuraspora crassa (N.crassa.)), handle spore shell genus, Staphlosporonites, gold thread Pseudomonas (Collybia), the black hole of hypodermis genus, grape ear mould, leather ear belong to (Panus; For example remove from office ear (Panus rudis)), fusarium globosum shuttle (Theilava), shelf fungus belong to (Fomes), Lentinus, pleurotus, trametes; For example long wool hair bolt bacterium (T.villosa) and variable color bolt bacterium (T.versicolor), Rhizoctonia; For example R.solani, Coprinus (Coprinus); For example pleat line ghost umbrella (C.plicatilis) and Coprinus cinereus (C.cinereus), Psatyrella, myceliophthora; For example thermophilic ruin the silk mould (M.thermonhila), a 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.
In addition; Can produce laccase or laccase involved enzyme through 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 said host cell transforms to be had the dna sequence dna that carries the said laccase of coding and allow this laccase of coding is expressed.
Can expression vector 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 black mold (Aspergillus niger), and Fusarium (Fusarium) kind, the thermophilic hyphomycete of most preferably cotton shape (Fusarium venenatum).Can form and this protoplast transformation through 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.
Perhaps, 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, like people such as T.Maniatis, Molecular Cloning:A Laboratory Manual, ColdSpring Harbor, the 1982 said conversions of carrying out bacterial cell.Also can be through standard method, the dna sequence dna that is fit to referring to people such as T.Maniatis (document that quote the front) screens and vector construction.
The substratum that is used for the host cell after the culture transformation 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 through known method from wherein reclaiming this enzyme; This method comprises through centrifugal or filtration isolated cell from substratum; Proteinaceous component in the salt deposition substratum of utilization such as ammonium sulfate reclaims through chromatography method such as ion exchange chromatography, affinity chromatography etc. subsequently.
In embodiment, expressive host can be the Trichodermareesei (Trichoderma reesei) (referring to for example United States Patent(USP) No. 5,861,271) with the laccase coding region under CBH1 promotor and terminator regulation and control.Expression vector can be pTrex3g, like the U.S. Patent application No.11/245 that submitted on October 7th, 2005, and 628 disclosed (the file number GC886 of agency).
When the 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.
For example, as used herein, confirm 80% sequence identity by algorithm, and therefore the homology of given sequence has the 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.
Can be used for confirming 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.
During 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 all reading frames all by the nucleotide sequence after the translation to GenBank protein sequence and the aminoacid sequence in 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).
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
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 as redox mediators term " chemical mediator " " amboceptor " of the interchangeable use of this paper (or) between enzyme that shows oxidase activity and dyestuff, to make the chemical cpd of electron shuttle effectively.Chemical mediator also known in the art is toughener and promotor.
Chemical mediator can be phenoloid, and for example NSC 611398 and relevant compound are of 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, NSC 611398, 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-cyanic acid-2,6-syringol, 4-formamido--2; 6-syringol or the substituted verivate of its N-; 4-(N-NMF base)-2 for example, 6-syringol, 4-[N-(2-hydroxyethyl) formamido-]-2,6-syringol or 4-(N; The dinethylformamide base)-2,6-syringol.
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.
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.
In the above-mentioned formula A can be placed on hydroxyl between the position but not be placed on as in the contraposition that illustrates.
In the embodiment, amboceptor can be Syringylethanone, NSC 611398, 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-cyanic acid-2,6-syringol, 4-formamido--2; 6-syringol or the substituted verivate of its N-; 4-(N-NMF base)-2 for example, 6-syringol, 4-[N-(2-hydroxyethyl) formamido-]-2,6-syringol or 4-(N; The dinethylformamide base)-2,6-syringol.
Amboceptor of the present invention can 0.005-1000 μ mole/g denim concentration have preferred 0.05-500 μ mole/g denim, more preferably 0.5-100 μ mole/g denim.
Can prepare amboceptor through 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
The 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.
Laccase as herein described finds can be used for the dyeing and the bleaching of textiles, fiber, yarn etc.Find that polymerization and crosslinking reaction (for example pigment, coating and biomaterial) and dyestuff activation that this laccase also can be used for the polymerization of the depolymerization of wastewater treatment, paper pulp lignification removal, high molecular weight aggregates, deinking, aromatic compound, free radical mediated are to be coupled with organic compounds.This laccase can be used in cleaning compsns or its component, or is used for washing composition.
As used herein, use oxygen as electron acceptor(EA), the multiple coloring compound that laccase can oxidation has 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.
The laccase that this paper shows 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.
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.
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 property and/or non-phenol property fragrance redox compound, the phenol property of xylogen and non-phenol property unit by the effect institute direct oxidation of these phenol property and/or non-phenol property aromatic compound perhaps this xylogen by other phenol property that oxygenizement produced of these compounds and/or the oxidation of non-phenoloid institute.
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; The characteristic that is used for reinforced paper; Ink removing; Paper dyeing; Sticker (the glue that for example is used for particle board or wad) or the like based on xylogen.
Laccase described herein can be used for field of fodder.For example, the laccase of this paper displaying can 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 separately as fodder additives or as the part of fodder additives; And/or be intended to generation as the processing aid of processing vegetable material and byproduct of food industry and be suitable as the raw-material material/product of feed.
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.
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 (Vadex) 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, isomerizing and Tuo Zhi.
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, in food preservation, is used as Oxygen Scavenger etc.
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.
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 measures etc.
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 prosthetic etc.
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 XC 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.
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 (like meat) or feed as the biocide in the cleaning compsns.
The laccase amboceptor can be used as sanitizer and biocide (like wood protection, washing composition).This amboceptor can be independent of enzyme and use or be used in combination with enzyme.
As used herein, " cleaning compsns " and " cleaning formulation " refers to can be used for remove undesired compound compositions from project to be cleaned (like fabric etc.).This term comprises any material/compound that is selected for the particular type cleaning compsns wanted and this product form (like liquid, gel, particle or spray composite), as long as other enzymes 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 compsns material in use to the desired composition forms of clean conditions.
This term also refers to any compsn 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 preparatory stain remover of clothing (pre-spotter), and dish ware washing composition).
In fact, unless otherwise, otherwise comprise general or heavy scale washing agent, the especially cleaning detergent of particle or powder type like the term " cleaning compsns " that this paper uses; 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; Machine is used 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.
Use like this paper, 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 (like " dishwashing detergents ").The compsn of current consideration is not intended to be limited to any specific detergent formulations or compsn.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 optical dye, caking inhibitor, sequestering agent, enzymic activity agent, inhibitor and solubilizing agent.
Use like this paper, term " hard-surface cleaning compositions " refers to be used for the for example detergent composition of floor, wall, brick, stainless steel vessel (like 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.The amino acid sequence analysis of EXAMPLE Example 1, black pore fungi (Cerrena unicolor) laccase of monochromatic hypodermis
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.Through 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 2a. is from the clone of the black pore fungi laccase A gene of monochromatic hypodermis of ATCC20013 bacterial strain
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
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 Hereulase 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.
II enzyme (Stratagene) is added in this test tube and carries out the PCR reaction for 30 times in following program loop: 98 ℃ continue 30 seconds, 50 ℃ continue 30 seconds with 72 ℃ lasting 2 minutes.Accomplish last extension 5 minutes and with reaction 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
To 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 representes 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.
In order to clone 3 ' end of laccase A gene; In lowT PCR reaction, use primer ACCGTGGTTCCTCCATTGCC (SEQ ID No.40) and primer SEQ ID No.31, in first round lowT PCR reaction with extract deceive pore fungi bacterial strain (CBS 115.075) from monochromatic hypodermis genomic dna as template.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.
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 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, with extract deceive pore fungi CBS 115.075 bacterial strains from monochromatic hypodermis genomic dna as template.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 3a. is from the clone and the order-checking of the black pore fungi laccase B gene of monochromatic hypodermis of ATCC20013 bacterial strain
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).
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 represent 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 conform to 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 to carry out 30 circulations in lasting 1 minute with 68 ℃ in 1 minute.Accomplish last extension 7 minutes and with reaction 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.
In order to clone 5 ' end of laccase gene, design also obtains primer: GGACGTGGCCTTGAGCATAC (SEQ ID No.29) from Invitrogen.It is used for first round lowT PCR reaction with degenerate oligonucleotide NNNNNNNNNNGGATCC (SEQ ID No.31) (wherein N representes the mixture of all four kinds of Nucleotide (A, T, C and G)).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 with dilution in 1: 100 and in first round highT PCR reacts, 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 ℃ of lasting 1 minute and 72 ℃ carried out 30 circulations in lasting 1 minute.Accomplish last extension 7 minutes and with reaction 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.
In order to clone 3 ' end of laccase B gene, design also obtains primer: CCTCACCTGTATTGGCACAG (SEQ ID No.33) from Invitrogen, and it is used for the lowT PCR reaction of the first round with primer SEQ IDNo.31.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.
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
Design and from Invitrogen obtain two primer: CACCGCGATGTCTCTTCTTCGTAG (SEQ ID NO.46) and TGRAGRTGGAASGGATGWGGTCC (SEQ ID NO.47) wherein R represent the mixture of Nucleotide A and G; S representes the mixture of Nucleotide C and G, and W representes 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.
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.2663bp dna sequence dna and SEQ ID No.8 that SEQ ID No.7 has listed the laccase B of CBS 154.29 bacterial strains have 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
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).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 DNA and to two plasmids.2173 bp dna sequence dnas and SEQ ID No.6 that SEQ ID No.5 has listed the laccase B of CBS 115.075 bacterial strains have 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
According to the design of the peptide sequence GQRYSFV (SEQ ID No.52) after translation primer ACGAACGAGTANCGTTGNCC (SEQ ID No.51), wherein N representes 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.Laccase C gene order and SEQ ID No.12 that SEQ ID No.11 has listed CBS 154.29 bacterial strains of 1080bp have listed the protein sequence after the translation.Embodiment 5a. is from the clone of the black pore fungi laccase D gene of monochromatic hypodermis of CBS 115.075 bacterial strains
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.
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), uses genomic dna to be dna profiling from CBS 115.075 bacterial strains.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
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.
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.
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 with it 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 to take turns PCR fragment and the order-checking that PCR reacts from second.
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 with it 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
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
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.
II enzyme (Stratagene) is added this test tube and continues 30 seconds at 98 ℃, and 50 ℃ continue to continue circulation in 2 minutes and carry out PCR for 30 times and react with 72 ℃ in 30 seconds.Accomplish last extension 5 minutes and with reaction 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 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 through dna sequencing with particle gun.Use is from Bio-Rad (Hercules; CA)
PDS-1000/he particle delivery system accomplishes the conversion of Trichoderma bacterial strain through 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.The expression of embodiment 8a. laccase B gene in Aspergillus
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.Fragment cloning behind this purifying is arrived the carrier pGAMpR2-GV (referring to U.S. Patent application US20050153399) through Nhel and BstEII digestion.Be transformed in the black mold 2445 through 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.The spore and the dull and stereotyped embolism of mycelial CMA that will contain fusion are transferred 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
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).Make
With two primer TTGCTAGCAACGTGATCTCCAAGCGTGCAATCGGTCCAGTCACTGACCTAC (51mer, SEQ ID No.74) and primer SEQ ID NO.73.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.Fragment cloning behind this purifying is arrived the carrier pGAMpR2-GV (referring to U.S. Patent application US20050153399) through Nhel and BstEI digestion.Be transformed in the black mold 2445 through 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.The spore and the dull and stereotyped embolism of mycelial CMA that will contain fusion are transferred 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
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 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 through 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
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 50TATAGTGAAC? CAGAATCTCG? ACCCAGATGG? TTTCAACCGC? CCCACTGTAC 100TCGCAGGTGG? TACTTTCCCC? GGTCCTCTGA? TTCGTGGTAA? CAAGGTACGC 150TTCATAACCG? CCCTCCGTAG? ACGTAGGCTT? CGGCTGACAT? GACCATCATC 200TGTAGGGAGA? TAACTTTAAA? ATTAATGTGA? TTGACGACTT? GACAGAGCAC 250AGTATGCTCA? AGGCTACGTC? CATCGTAAGT? CCCTGATTAA? CGTTTCACCT 300GGTCATATCG? CTCAACGTCT? CGAAGCACTG? GCATGGGTTC? TTCCAGAAGG 350GAACCAACTG? GGCCGATGGC? CCCGCCTTTG? TCACCCAATG? TCCTATCACA 400TCAGGAAACG? CCTTCCTGTA? TGATTTCAAC? GTTCCGGACC? AAGCTGGTAC 450TTTCTGGTAC? CACAGCCATC? TCTCTACACA? GTATTGTGAC? GGTCTTCGTG 500GTGCCTTTGT? CGTCTATGAT? CCTAATGATC? CCAACAAGCA ? ACTCTATGAT 550GTTGATAACG? GCAAGTTCCT? TGCATATTTC? ATTTCTATCA? TATCCTCACC 600TGTATTGGCA? CAGAAAGCAC? CGTGATTACC? TTGGCTGATT? GGTATCATGC 650CCTTGCTCAG? ACTGTCACTG? GTGTCGCGTG? AGTGACAAAT? GGCCCTCAAT 700TGTTCACATA? TTTTCCTGAT? TATCATATGA ? TAGAGTATCT? GATGCAACGT 750TGATCAACGG? ATTGGGACGT? TCGGCCACCG? GCCCCGCAAA? TGCCCCTCTG 800GCGGTCATCA? GTGTCGAGCG? GAATAAGAGG? TCAGTTCCAT? AATTATGATT 850ATTTCCCGCG? TTACTTCCTA? ACAATTATTT? TTGTATCCCT? CCACAGATAT 900CGTTTCCGAT? TGGTTTCTAT ? TTCTTGCGAC? CCTAACTTTA? TTTTCTCAAT 950TGACCACCAC? CCAATGACCG? TAATTGAGAT? GGACGGTGTT? AATACCCAAT ? 1000CTATGACCGT? AGATTCGATC? CAAATATTCG? CAGGTCAACG? ATATTCATTT ? 1050GTCGTAGGTT? ATTATAAACT? GCCCACCGAT? CATCTCTCAC? GTAACTGTTA ? 1100TAGATGCAAG? CCAACCAACC? AGTTGGAAAT? TATTGGATCC? GCGCTAAACC ? 1150TAATGTTGGG? AACACAACTT? TCCTTGGAGG? CCTGAACTCC? GCTATATTAC ? 1200GATATGTGGG? AGCCCCTGAC? CAAGAACCGA? CCACTGACCA? AACACCCAAC ? 1250TCTACACCGC? TCGTTGAGGC? GAACCTACGA? CCCCTCGTCT? ATACTCCTGT ? 1300GGTATGTTGT? TCTCGTTACA? TATACCAAAC? CTAATATGAA? GACTGAACGG ? 1350ATCTACTAGC? CGGGACAGCC? ATTCCCTGGC? GGTGCTGATA? TCGTCAAGAA ? 1400CTTAGCTTTG? GGTTTCGTAC? GTGTATTTCA? CTTCCCTTTT? GGCAGTAACT ? 1450GAGGTGGAAT? GTATATAGAA? TGCCGGGCGT? TTCACAATCA? ATGGAGCGTC ? 1500CCTCACACCT? CCTACAGTCC? CTGTACTACT? CCAGATCCTC? AGTGGTACTC ? 1550ACAATGCACA? GGATCTTCTC? CCAGCAGGAA? GCGTGATCGA? ACTTGAACAG ? 1600AATAAAGTTG? TCGAAATCGT? TTTGCCCGCT? GCGGGCGCCG? TTGGCGGTCC ? 1650TCATCCTTTT? CACTTACATG? GTGTAAGTAT? CAGACGTCCT? CATGCCCATA ? 1700TTGCTCCGAA? CCTTACACAC? CTGATTTCAG? CACAATTTCT? GGGTGGTTCG? 1750TAGCGCCGGT? CAAACCACAT? ACAATTTCAA? TGATGCTCCT? ATCCGTGATG ? 1800TTGTCAGTAT? TGGCGGTGCA? AACGATCAAG? TCACGATCCG? ATTTGTGGTA ? 1850TGTATCTCGT? GCCTTGCATT? CATTCCACGA? GTAATGATCC? TTACACTTCG ? 1900GGTTCTCAGA? CCGATAACCC? TGGCCCATGG? TTCCTTCACT? GTCACATTGA ? 1950CTGGCATTTG? GAGGCTGGGT? TCGCTGTAGT? CTTTGCGGAG? GGAATCAATG ? 2000GTACTGCAGC? TGCTAATCCA? GTCCCAGGTA? AGACTCTCGC? TGCTTTGCGT ? 2050AATATCTATG? AATTTAAATC? ATATCAATTT? GCAGCGGCTT? GGAATCAATT ? 2100GTGCCCATTG? TATGATGCCT? TGAGCCCAGG? TGATACATGA? TTACAAGGGT ? 2150GGGCGCGCC? 2159 using QIAquick spin column purified and digested with restriction enzymes Spel and Ascl digestion.Then with this fragment (SEQ ID No.66) be 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 through 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.The expression of the laccase B gene of embodiment 9a.CBS bacterial strain 115.075 in strepto-belongs to
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 also with XbaI and BclI digestion.Use the postdigestive fragment of the centrifugal column purification of QIAquick and it is cloned into the carrier pKB105 (referring to US 20060154843) through Xbal and BamHI digestion.Confirm the exactness of the plasmid pKB251 (Fig. 5) of generation through 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
The synthetic laccase B gene of optimizing (SEQ ID NO:67): ACTAGTGTCG CCGTTTACAA ACGCGCAATC GGTCCCGTCA CTGACCTGCA 50TATTGTGAAC CAGAATCTCG ACCCCGATGG TTTCAACCGC CCCACTGTCC 100TCGCAGGTGG TACTTTCCCC GGTCCTCTGA TTCGTGGTAA CAAGGGAGAT 150AACTTTAAAA TTAATGTGAT TGACGACTTG ACAGAGCACA GCATGCTCAA 200GGCTACGTCC ATCCACTGGC ATGGCTTCTT CCAGAAGGGA ACCAACTGGG 250CCGATGGCCC CGCCTTTGTC ACCCAATGTC CTATCACATC AGGAAACGCC 300TTCCTGTACG ATTTCAACGT TCCGGACCAA GCTGGTACTT TCTGGTACCA 350CAGCCATCTC TCTACACAGT ACTGTGACGG TCTTCGTGGT GCCTTTGTCG 400TCTACGATCC TAATGATCCC AACAAGCAAC TCTACGATGT TGATAACGGC 450AACACCGTGA TTACCTTGGC TGATTGGTAC CATGCCCTTG CTCAGACTGT 500CACTGGTGTC GCAGTCTCTG ATGCAACGTT GATCAACGGA TTGGGACGTT 550CGGCCACCGG CCCCGCAAAT GCCCCTCTGG CGGTCATCAG CGTCGAGCGC 600AATAAGCGCT ATCGTTTCCG ATTGGTTTCT ATTTCTTGCG ACCCTAACTT 650TATTTTCTCA ATTGACCACC ACCCCATGAC CGTCATTGAG ATGGACGGTG 700TTAATACCCA ATCTATGACC GTAGATTCGA TCCAAATCTT CGCAGGTCAA 750CGATACTCAT TTGTCATGCA AGCCAACCAA CCAGTTGGAA ATTACTGGAT 800CCGCGCTAAA CCTAATGTTG GCAACACAAC TTTCCTTGGA GGCCTGAACT 850CCGCTATCTT GCGATACGTG GGAGCCCCTG ACCAAGAACC GACCACTGAC 900CAAACACCCA ACTCTACACC GCTCGTTGAG GCGAACCTGC GACCCCTCGT 950CTACACTCCT GTGCCGGGAC AGCCATTCCC TGGCGGTGCT GATATCGTCA 1000AGAACTTGGC TTTGGGTTTC AATGCCGGGC GTTTCACAAT CAATGGAGCG 1050TCCCTCACAC CTCCTACAGT CCCTGTCCTG CTCCAGATCC TCAGCGGTAC 1100TCACAATGCA CAGGATCTTC TCCCGGCAGG AAGCGTGATC GAACTTGAAC 1150AGAATAAAGT TGTCGAAATC GTTTTGCCCG CTGCGGGCGC CGTTGGCGGT 1200CCTCATCCTT TTCACTTGCA TGGTCACAAT TTCTGGGTGG TTCGTAGCGC 1250CGGTCAAACC ACATACAATT TCAATGATGC TCCTATCCGT GATGTTGTCA 1300GCATTGGCGG TGCAAACGAT CAAGTCACGA TCCGATTTGT GACCGATAAC 1350CCTGGCCCAT GGTTCCTTCA CTGTCACATT GACTGGCATT TGGAGGCTGG 1400ATTCGCTGTC GTCTTTGCGG AGGGAATCAA TGGTACTGCA GCTGCTAATC 1450CCGTCCCGGC GGCTTGGAAT CAATTGTGCC CGTTGTACGA TGCCTTGAGC 1500CCGGGTGATA CATGAGGCGC GCC 1523 its coding laccase B genes; By McLab Inc. (Molecular Cloning Laboratories; 384 Oyster Point Blvd; Suite 15; South San Francisco, CA94080) synthetic.Digest the synthetic 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.The expression of embodiment 11a. laccase D gene in Trichoderma
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 DNAs 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 through 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
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 as dna profiling.Use the centrifugal column purification PCR of QIAquick fragment and digest, and it is cloned into also the pTrex4 carrier that digests through Spel and Ascl (referring to U.S. Patent application 10/590,956 with Restriction Enzyme Spel and Ascl; 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 through 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
DNA (SEQ ID NO:70): ACTAGTGTCG CCGTTTACAA ACGCGCTATT GGACCAGTTG CTGATCTGCA 50CATCGTTAAC AAGGATTTGG CCCCAGACGG CGTCCAGCGC CCAACTGTTC 100TGGCCGGTGG AACTTTTCCG GGCACGCTGA TTACCGGTCA AAAGGGCGAC 150AACTTCCAGC TGAACGTGAT TGATGACCTG ACCGACGATC GCATGTTGAC 200CCCTACTTCG ATCCATTGGC ATGGTTTCTT CCAGAAGGGA ACCGCCTGGG 250CCGACGGTCC GGCTTTCGTT ACACAGTGCC CTATTATCGC AGACAACTCC 300TTCCTCTACG ATTTCGACGT TCCCGACCAG GCGGGCACCT TCTGGTACCA 350CTCACACTTG TCTACACAGT ACTGCGACGG TCTGCGCGGT GCCTTCGTTG 400TTTACGACCC CAACGACCCT CACAAGGACC TTTATGATGT CGATGACGGT 450GGCACAGTTA TCACATTGGC TGACTGGTAT CACGTCCTCG CTCAGACCGT 500TGTCGGAGCT GCTACACCCG ACTCTACGCT GATTAACGGC TTGGGACGCA 550GCCAGACTGG CCCCGCCGAC GCTGAGCTGG CCGTTATCTC TGTTGAACAC 600AACAAGAGAT ACCGTTTCAG ACTCGTCTCC ATCTCGTGCG ATCCCAACTT 650CACTTTTAGC GTCGACGGTC ACAACATGAC GGTTATCGAG GTTGATGGCG 700TGAATACCCG CCCTCTCACC GTCGATTCCA TTCAAATTTT CGCCGGCCAG 750CGATACTCCT TTGTGCTGAA TGCCAATCAG CCCGAGGATA ACTACTGGAT 800CCGCGCTATG CCTAACATCG GACGAAACAC CACTACCCTT GATGGCAAGA 850ATGCCGCTAT CCTGCGATAC AAGAACGCCA GCGTTGAGGA GCCCAAAACC 900GTCGGAGGAC CCGCGCAGAG CCCATTGAAC GAGGCCGACC TGCGACCTCT 950GGTGCCCGCT CCTGTCCCTG GCAACGCAGT TCCTGGTGGT GCGGACATCA 1000ACCACCGCCT GAACCTGACA TTCAGCAACG GCCTCTTCTC TATCAATAAC 1050GCATCATTTA CAAACCCCAG CGTCCCTGCC TTGTTGCAGA TTCTTTCCGG 1100CGCACAAAAC GCTCAGGATC TGCTTCCCAC CGGTTCTTAT ATCGGCTTGG 1150AGTTGGGCAA GGTCGTTGAA CTCGTGATCC CTCCCTTGGC CGTTGGTGGC 1200CCCCATCCAT TCCACTTGCA CGGCCACAAC TTTTGGGTCG TCCGAAGCGC 1250TGGTTCTGAC GAGTATAATT TCGACGATGC AATTTTGCGC GACGTGGTCA 1300GCATTGGCGC GGGAACTGAC GAGGTTACTA TCCGTTTTGT CACTGATAAC 1350CCAGGCCCTT GGTTCCTCCA TTGCCACATC GACTGGCACC TCGAAGCCGG 1400CCTCGCCATT GTTTTCGCCG AAGGCATCAA TCAAACCGCA GCCGCCAACC 1450CGACTCCACA GGCCTGGGAC GAACTCTGCC CCAAGTATAA CGGACTCTCC 1500GCTTCCCAGA AAGTGAAGCC CAAGAAGGGA ACAGCCATCT AAGGCGCGCC 1550 its coding laccase D genes (according to gene) from CBS 115.075; By DNA2.0Inc. (1455Adams Drive; Menlo Park, CA94025) synthetic.With Restriction Enzyme Spel and Ascl digestion synthetic DNA and from gel, separates the dna fragmentation of 1.5kb, it is cloned into the pTrex4 carrier generation expression plasmid (pTrex4-laccase Dopt) that is also digested by Spel and Ascl.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
DNA (SEQ ID NO:71): GGATCCTGAA GCTATCGGTC CGGTTGCAGA TTTACACATC GTAAACAAAG 50ATCTTGCACC TGACGGCGTT CAACGTCCAA CTGTACTTGC TGGTGGAACA 100TTCCCTGGTA CACTTATTAC TGGTCAAAAA GGTGACAACT TCCAATTAAA 150CGTAATTGAC GATCTTACAG ATGACCGTAT GCTTACACCG ACTTCAATTC 200ACTGGCACGG TTTCTTTCAA AAAGGAACAG CATGGGCTGA TGGTCCTGCA 250TTCGTTACAC AATGTCCAAT CATTGCTGAT AACTCTTTCC TTTACGATTT 300TGACGTTCCT GATCAAGCTG GTACATTCTG GTATCACTCA CACTTATCCA 350CACAATACTG CGATGGACTT CGCGGAGCTT TCGTAGTTTA CGACCCAAAC 400GATCCTCATA AAGACCTTTA CGATGTAGAT GATGGTGGAA CAGTTATCAC 450ATTAGCTGAT TGGTACCATG TACTTGCTCA AACAGTTGTA GGTGCAGCTA 500CACCAGATTC AACACTTATC AATGGATTAG GACGTTCTCA AACTGGTCCT 550GCTGACGCAG AACTTGCTGT AATCTCTGTT GAACATAACA AACGTTACAG 600ATTCCGTCTT GTTAGCATTT CTTGCGATCC AAACTTCACA TTTTCAGTTG 650ACGGACATAA CATGACAGTT ATCGAAGTAG ATGGTGTAAA CACACGTCCA 700CTTACTGTAG ACTCTATCCA AATCTTCGCA GGACAACGTT ACTCATTCGT 750ATTAAACGCA AATCAACCAG AAGATAACTA CTGGATTCGT GCAATGCCAA 800ACATCGGACG TAACACTACA ACTCTTGACG GCAAAAACGC AGCTATTCTT 850CGTTACAAAA ACGCTTCTGT TGAAGAACCT AAAACAGTTG GTGGACCAGC 900ACAATCACCA CTTAACGAAG CTGACTTACG TCCACTGGTT CCAGCACCTG 950TACCTGGAAA CGCTGTACCA GGAGGTGCTG ATATTAATCA TAGACTTAAC 100CTTACTTTCT CTAACGGTCT GTTCTCAATC AACAACGCTT CATTCACAAA 1050TCCTTCAGTT CCAGCACTTT TACAAATTCT TAGCGGTGCA CAAAATGCTC 1100AGGATCTTTT ACCAACTGGA TCTTACATTG GTCTTGAACT GGGTAAAGTA 1150GTTGAATTAG TAATTCCTCC GCTTGCTGTA GGTGGACCAC ATCCTTTCCA 1200TCTTCACGGT CATAACTTCT GGGTTGTACG TTCTGCTGGT TCAGATGAAT 1250ACAACTTCGA TGACGCAATT CTTCGTGATG TTGTATCTAT TGGTGCTGGA 1300ACAGATGAAG TAACTATTCG TTTCGTAACA GATAACCCTG GTCCTTGGTT 1350CTTACATTGT CATATCGATT GGCATCTTGA AGCTGGACTT GCTATTGTTT 1400TCGCTGAAGG AATCAATCAA ACAGCTGCAG CTAACCCAAC ACCTCAAGCA 1450TGGGACGAAT TATGTCCAAA ATACAACGCA CTTTCTCCAG GAGATACTTA 1500AAAGCTT 1507 its coding laccase D genes (according to gene) from CBS 115.075; By DNA2.0Inc. (1455 Adams Drive; Menlo Park, CA94025) synthetic.With Restriction Enzyme BamHI and HindIII digestion synthetic 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
In 96 hole titer plate, undertaken by the detection of laccase/amboceptor combination the indigo substrate bleaching of dissolved by following.
Through 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.
In 96 hole titer plate, carry out the 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 TV in every hole is transferred to 200 μ L.Only contain twice of the contrast repetition of laccase.Seal this plate and (Thermomixer Eppendorf) goes up and to hatch 2 hours with 800rpm at 50 ℃ of whisking appliances 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 through using the titer plate reader to confirm 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.
Fig. 7 shows the result from Thielavia species laccase (Ecostone LCC10, AB enzymes, 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), NSC 611398 (MS), 4-(N-NMF 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 mediator concentration was 500 μ M, the most effectively amboceptor that is used for indigo bleaching was ABTS, follows 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; Using Thielavia species laccase and concentration is that the multiple amboceptor of 500 and 50 μ M carries out after the bleaching of soluble indigo blue at absorbancy variation (n=2)
at 600nm place embodiment 15; Soluble indigo blue bleaching with different laccases under two kinds of pH values detects
Two kinds of pH values; Assessment is ruined Hyphomyces (Myceliophtora) (
II with being derived from of lower molecular weight amboceptor combination; Novozymes; Bagsvaerd, Denmark), Thielavia (Ecostone LCC10, AB enzymes; Darmstadt, Germany) and the indigo ability of its bleaching dissolved of laccase of the black hole of hypodermis species.
In the pH value is 5 and 7 times, uses 3 kinds of different laccases, in 96 hole titer plate, carries out the indigo bleaching of dissolved by embodiment 14 is said.Used amboceptor is sinapinic acid, 4-formamido--2,6-syringol (SA), methyl 4-acetyl cloves acid esters (AMS), NSC 611398 (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; After use is carried out the soluble indigo blue bleaching from Thielavia, the laccase of ruining the black hole of Hyphomyces and hypodermis species; Change
table 3, be under the 250 μ M with respect to the absorbancy of contrast at the 600nm place in pH 7, mediator concentration; Use changes the definite of
embodiment 16, purifying and specific activity at 600nm place with respect to the absorbancy of contrast after carrying out the soluble indigo blue bleaching from Thielavia, the laccase of ruining the black hole of Hyphomyces and hypodermis species
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 through 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 on the UFC sample after the 500ml diafiltration appearance to contain Poros HS-20 resin (Applied Biosystems, 20X275mm 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 level branch of laccase activity and uses the Amicon 400mL agitation elements with 10K film further to concentrate.Use BSA as standard, measuring gross protein through 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 through 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 copper of causing owing to high specific acitivity in environment, discharging is lower than other laccases (like the Stachybotrys atra laccase).Embodiment 17, be used for the program of denim bleaching
Amboceptor
4-hydroxyl-3, (the cloves acid amides is SA) available from PunjabChemicals & Crop Protection Limited (Mumbai, India) for 5-syringol methane amide.4-hydroxyl-3, the 5-dimethoxy-benzyl nitrile (the cloves nitrile, SN) available from Stereochemical, Inc., (Newark, DE) or PunjabChemicals & Crop Protection Limited (Mumbai, India).
Enzyme
(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
Under the different condition that relates to pH, temperature, enzyme concn and mediator concentration, accomplish enzyme in ATLASLP 2Launder-O-meter and hatch.
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 under the program of cotton dry and assess with Elna pressure electriciron in AEG IPX4 whizzer.
The granite-wash of denim
Under following condition; Denim destarch in UnimacUF 50 washing machines with the 12 car pin (leg) of heavily about 3kg: with 10: 1 liquor ratios; In 50 ℃ with 0.5g/l (15g) Optisize160 glycase (Genencor) and 0.5g/l (15g) nonionogenic tenside (Rucogen BFA for example; (Rudolf Chemie) or Ultravon GPN, (Huntsman)) destarch 15 minutes was with twice cold rinsing of 30: 1 liquor ratios 5 minutes.
After the destarch; Under following condition; This denim of granite-wash in Unimac UF 50 washing machines: with 55 ℃ of the cold rinsings of liquor ratio in 10: 15 minutes; 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.
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
Measure the color of five denim samples in the CIE Lab color space with D65 light source with Minolta Chromameter CR 310.Before with laccase treatment with after handling, measure and the result of 5 samples is averaged.Calculate total color difference (TCD).Can use equality: TCD=√ (Δ L)
2+ (Δ is a)
2+ (Δ b)
2Calculate total color difference.
DenimThe assessment of car pin
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
The laccase of granite-wash denim bleaching: will heavily about 3kg 12
DenimThe car pin is pressed embodiment 17 said destarch and granite-wash.After the granite-wash; In Unimac UF 50 washing machines, carry out laccase treatment according to following technology: carry out 30 minutes pH 6 (21g SODIUM PHOSPHATE, MONOBASIC and 5g hexanodioic acids with 10: 1 liquor ratios; Laccase D) or pH 4.8 (8.6g SODIUM PHOSPHATE, MONOBASIC and 16.8g hexanodioic acid; Novoprime Base 268 laccases) after laccase (laccase D or Novoprime Base 268) amboceptor (cloves acid amides (SA) and cloves nitrile (the SN)) laccase treatment, with 30: 1 liquor ratios to rinsing in twice cold water of denim 5 minutes.
Carry out laccase experiment and result and be 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 |
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 between 40 ℃ and 60 ℃, bleaching action is provided.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
The influence (with sodium hydroxide 4N solution adjustment 50mM phosphate sodium dihydrogen buffer solution pH) of laccase and mediator concentration is assessed in the experiment of carrying out following table pH6 and 60 ℃.
Experimentize with cloves acid amides (SA) and cloves nitrile (SN) amboceptor.
(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 |
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 said 60 ℃ of processing by embodiment 17.Press embodiment 17 these samples of said assessment.Table 8?
TCD = total color table 9?
TCD = total color
Last table and Figure 10 and 11 show needs enzyme and amboceptor to bleach.Its also be illustrated in realize certain javelle at ordinary times enzyme/amboceptor ratio certain handiness is arranged.Embodiment 20, the dose response effect (Unimac) of laccase D of recombinating for bleachability
Through 12 of the laccase bleaching of the denim of granite-wash-will heavily about 3kg
DenimThe car pin is pressed embodiment 17 said 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.
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 |
On expressed the result.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.
Should be understood that embodiment described herein and embodiment only be used for illustration purpose and based on this multiple modification or to change be predictable to those skilled in the art, and it is included in the application's the scope of spirit and scope and accompanying claims.All publications, patent and patented claim that this paper quotes are quoted as a reference through integral body at this.
Claims (11)
1. laccase, its nucleotide sequence by SEQ ID NO.70 is coded.
2. nucleotide sequence, its sequence is the SEQ ID NO.70 and the laccase as claimed in claim 1 of encoding.
3. expression vector comprises the nucleotide sequence of claim 2.
4. host cell comprises the carrier of claim 3.
5. the method for the dyestuff in the liquid lime chloride, this method comprise with the laccase of claim 1 and effectively amboceptor handle the dyestuff in solution.
6. according to the method for claim 5, its mesosome is selected from Syringylethanone, syringic aldehyde, cloves acid amides, methyl cloves acid amides, 2-hydroxyethyl cloves acid amides, NSC 611398, cloves nitrile, dimethyl-cloves acid amides and syringic acid.
7. method of using the laccase bleached woven fabric, its improvement comprises the laccase that uses claim 1.
8. the method for claim 7 also comprises the amboceptor that is selected from Syringylethanone, syringic aldehyde, cloves acid amides, methyl cloves acid amides, 2-hydroxyethyl cloves acid amides, NSC 611398, cloves nitrile, dimethyl-cloves acid amides and syringic acid.
9. according to the method for claim 7, wherein with vat dyes to textile dyeing.
10. according to the method for claim 7, wherein this fabric is the mixture of cellulosic fabric, cellulosic fibre or the mixture of cellulose fiber peacekeeping synthon.
11. according to the method for claim 7, wherein this fabric is a denim.
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| US87551806P | 2006-12-18 | 2006-12-18 | |
| US60/875,454 | 2006-12-18 | ||
| US60/875,518 | 2006-12-18 | ||
| PCT/US2007/025533 WO2008076322A2 (en) | 2006-12-18 | 2007-12-12 | Novel laccases, compositions and methods of use |
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| CN102115724B (en) * | 2010-12-02 | 2013-05-22 | 东北林业大学 | Bacillus amyloliquefaciens LS01 laccase and application thereof |
| CN102115722B (en) * | 2010-12-02 | 2013-05-22 | 东北林业大学 | Bacillus subtilis ls02 laccase and application thereof |
| CN102115723B (en) * | 2010-12-02 | 2013-05-22 | 东北林业大学 | Bacillus amyloliquefaciens LC02 and application thereof |
| CN102154147B (en) * | 2010-12-22 | 2013-05-08 | 东北林业大学 | Bacillus pumilus LC01 laccase and application thereof |
| CN103444431B (en) * | 2013-08-06 | 2015-03-11 | 中南林业科技大学 | Panus rudis strain and application method thereof |
| CN108570459B (en) * | 2018-04-10 | 2022-10-04 | 南京农业大学 | Method for producing recombinant bacterial laccase by high-efficiency fermentation |
| CN111689585B (en) * | 2020-04-16 | 2022-09-13 | 安徽工程大学宣城产业技术研究院有限公司 | Method for degrading and decoloring rhodamine B by adopting white rot fungus coculture |
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| CN1430668A (en) * | 2000-05-23 | 2003-07-16 | 芬兰技术研究中心 | Novel laccase enzyme and gene encoding the enzyme |
| CN1594543A (en) * | 2003-09-11 | 2005-03-16 | 中国科学院微生物研究所 | Laccase and gene encoding same and engineering bacterium |
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| KR100371433B1 (en) * | 1994-10-20 | 2003-03-15 | 노보자임스 에이/에스 | Bleaching process comprising use of a phenol oxidizing enzyme, a hydrogen peroxide source and an enhancing agent |
| WO1997011217A1 (en) * | 1995-09-19 | 1997-03-27 | Novo Nordisk A/S | Stain bleaching |
| US5908472A (en) * | 1996-01-12 | 1999-06-01 | Novo Nordisk A/S | Fabric treated with cellulase and oxidoreductase |
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2007
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|---|---|---|---|---|
| CN1430668A (en) * | 2000-05-23 | 2003-07-16 | 芬兰技术研究中心 | Novel laccase enzyme and gene encoding the enzyme |
| CN1594543A (en) * | 2003-09-11 | 2005-03-16 | 中国科学院微生物研究所 | Laccase and gene encoding same and engineering bacterium |
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| zhang min et al.Characterization and decolorization ability of a laccase from Panus rudis.《ENZYME AND MICROBIAL TECHNOLOGY》.2006,第39卷(第1期),92-97. * |
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