CN104350148A

CN104350148A - Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

Info

Publication number: CN104350148A
Application number: CN201280067149.0A
Authority: CN
Inventors: 汤岚; 段俊欣; 张羽; 刘晔
Original assignee: Novozymes Biotech Inc
Current assignee: Novozymes Inc
Priority date: 2011-11-15
Filing date: 2012-11-15
Publication date: 2015-02-11

Abstract

Provided are isolated polypeptides having cellobiohydrolase activity, catalytic domains and cellulose binding domains, and polynucleotides encoding the polypeptides, catalytic domains or cellulose binding domains. Also provided are nucleic acid constructs, vectors and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains or cellulose binding domains.

Description

There are the polypeptide of cellobiohydrolase activity and the polynucleotide of this polypeptide of coding

For the statement of the right of the invention completed under the research and development of federal funding

The present invention completes with governmental support under the cooperation agreement of being authorized by USDOE (Cooperative Agreement) DE-FC36-08GO18080.Government has certain right in the present invention.

Relate to sequence table

The application comprises the sequence table of computer-reader form, and it is incorporated to herein by carrying stating.

Background of invention

Background technology

Mierocrystalline cellulose is simple sugars glucose by the covalently bound polymkeric substance of β-Isosorbide-5-Nitrae-key.The enzyme of the dextran of many production by biological unboiled water solution β-connections.These enzymes comprise endoglucanase, cellobiohydrolase and beta-glucosidase enzyme.Endoglucanase, at random site digest cellulose polymkeric substance, makes it be exposed to cellobiohydrolase and attacks (attack).Cellobiohydrolase discharges the molecule of cellobiose from the terminal order of cellulose polymer compound.Cellobiose is the glucose dimer of water miscible β-Isosorbide-5-Nitrae-connection.Cellobiose is hydrolyzed into glucose by beta-glucosidase enzyme.

Lignocellulose-containing raw material (lignocellulosic feedstock) is converted into ethanol there is following advantage: large content of starting materials is ready-made available, avoid burning or the desirability of embedding material and the spatter property of alcohol fuel.Timber, agricultural residue, herbaceous crops and municipal solid waste are considered to the raw material for alcohol production.These materials form primarily of Mierocrystalline cellulose, hemicellulose and xylogen.Once ligno-cellulose is converted into fermentable sugar as glucose, described fermentable sugar can be easily ethanol by yeast fermentation.

UNIPROT:Q5B2Q4 discloses the possible cellobiohydrolase A albumen from Emericella nidulans (Emericella nidulans).WO2008140749 discloses thermophilic fungus destroyed wire (Myceliophthora thermophila) cellobiohydrolase I.WO2003070939-A1 discloses hairy fungus (Coriolus hirsutus) cellobiohydrolase I albumen.UNIPROT:Q692I2 discloses the CBHI exoglucanase from the thermophilic mutation of chaetomium thermophilum (Chaetomium thermophilum var.thermophilum).

Exist the new demand with the polypeptide of cellobiohydrolase activity for the hydrolysis for cellulose materials in this area.

The invention provides and there are the polypeptide of cellobiohydrolase activity and the polynucleotide of coding said polypeptide.

Technical field

The present invention relates to the polypeptide with cellobiohydrolase activity, catalytic domain, and sugared binding domain, and coding said polypeptide, catalytic domain, and the polynucleotide of sugared binding domain.The present invention also relates to the nucleic acid construct, carrier and the host cell that comprise described polynucleotide, and produces and use described polypeptide, catalytic domain, and the method for sugared binding domain.

Summary of the invention

The present invention relates to the isolated polypeptide with cellobiohydrolase activity, it is selected from lower group:

(a) polypeptide, the mature polypeptide of itself and SEQ ID NO:2 has at least 70% sequence iden, polypeptide, the mature polypeptide of itself and SEQ ID NO:4 has at least 88% sequence iden, polypeptide, the mature polypeptide of itself and SEQ ID NO:6 has at least 66% sequence iden, or polypeptide, and the mature polypeptide of itself and SEQ ID NO:8 has at least 81% sequence iden;

(b) polypeptide, it is by polynucleotide encoding, described polynucleotide are at medium stringency condition, medium-Gao stringent condition, high stringent condition, or under very high stringent condition with following hybridization: the mature polypeptide encoded sequence of (i) SEQ ID NO:1, the mature polypeptide encoded sequence of SEQ ID NO:3, the mature polypeptide encoded sequence of SEQ ID NO:5, or the mature polypeptide encoded sequence of SEQ ID NO:7, (ii) cDNA sequence of SEQ ID NO:1, the cDNA sequence of SEQ ID NO:3, the cDNA sequence of SEQ ID NO:5, or the cDNA sequence of SEQ ID NO:7, or the total length complement of (iii) (i) or (ii).

(c) polypeptide, it is by polynucleotide encoding, and the mature polypeptide encoded sequence of described polynucleotide and SEQ ID NO:1 or its cDNA sequence has at least 70% sequence iden; Polypeptide, it is by polynucleotide encoding, and the mature polypeptide encoded sequence of described polynucleotide and SEQ ID NO:3 or its cDNA sequence has at least 88% sequence iden; Polypeptide, it is by polynucleotide encoding, and the mature polypeptide encoded sequence of described polynucleotide and SEQ ID NO:5 or its cDNA sequence has at least 66% sequence iden; Polypeptide, it is by polynucleotide encoding, and the mature polypeptide encoded sequence of described polynucleotide and SEQ ID NO:7 or its cDNA sequence has at least 81% sequence iden;

The mature polypeptide of (d) SEQ ID NO:2, the mature polypeptide of SEQ ID NO:4, the mature polypeptide of SEQ ID NO:6, or the mature polypeptide of SEQ ID NO:8 comprises replacement, disappearance and/or inserts variant in one or more (such as several) position; With

E the polypeptide of () (a), (b), (c) or (d) has the fragment of cellobiohydrolase activity.

The present invention also relates to isolated polypeptide, and described isolated polypeptide comprises catalytic domain, and described catalytic domain is selected from lower group:

(a) catalytic domain, the amino acid/11 8 to 458 of itself and SEQ ID NO:2 has at least 70% sequence iden; Catalytic domain, the amino acid 21 to 450 of itself and SEQ ID NO:4 has at least 88% sequence iden; Catalytic domain, the amino acid 22 to 457 of itself and SEQ ID NO:6 has at least 66% sequence iden; Or catalytic domain, the amino acid 21 to 461 of itself and SEQ ID NO:8 has at least 81% sequence iden;

(b) catalytic domain, it is by polynucleotide encoding, described polynucleotide under medium stringency condition, medium-Gao stringent condition, high stringent condition or very high stringent condition with following hybridization: the Nucleotide 52 to 1454 of (i) SEQ ID NO:1, the Nucleotide 61 to 1733 of SEQ ID NO:3, the Nucleotide 64 to 1782 of SEQ ID NO:5, or the Nucleotide 52 to 1460 of SEQ ID NO:7, (ii) its cDNA sequence, or the total length complement of (iii) (i) or (ii);

(c) catalytic domain, it is by polynucleotide encoding, and the Nucleotide 52 to 1454 of described polynucleotide and SEQ ID NO:1 has at least 70% sequence iden; Catalytic domain, it is by polynucleotide encoding, and the Nucleotide 61 to 1733 of described polynucleotide and SEQ ID NO:3 has at least 88% sequence iden; Catalytic domain, it is by polynucleotide encoding, and the Nucleotide 64 to 1782 of described polynucleotide and SEQ ID NO:5 has at least 66% sequence iden; Or catalytic domain, it is by polynucleotide encoding, and the Nucleotide 52 to 1460 of described polynucleotide and SEQ ID NO:7 has at least 81% sequence iden;

The amino acid/11 8 to 458 of (d) SEQ ID NO:2, the amino acid 21 to 450 of SEQ ID NO:4, the amino acid 22 to 457 of SEQ ID NO:6, or the amino acid 21 to 461 of SEQ ID NO:8 comprises replacement, disappearance and/or inserts variant in one or more (such as several) position; With

E the catalytic domain of () (a), (b), (c) or (d) has the variant of cellobiohydrolase activity.

The present invention also relates to isolated polypeptide, and described isolated polypeptide comprises sugared binding domain, and described sugared binding domain is selected from lower group:

(a) sugared binding domain, the amino acid 486 to 521 of itself and SEQ ID NO:8 has at least 81% sequence iden;

(b) sugared binding domain, it is by polynucleotide encoding, described polynucleotide under medium stringency condition, medium-Gao stringent condition, high stringent condition or very high stringent condition with following hybridization: the Nucleotide 1533 to 1640 of (i) SEQ ID NO:7, (ii) its cDNA sequence, or the total length complement of (iii) (i) or (ii);

(c) sugared binding domain, it is by polynucleotide encoding, and the Nucleotide 1533 to 1640 of described polynucleotide and SEQ ID NO:7 or its cDNA sequence has at least 81% sequence iden;

The d amino acid 486 to 521 of () SEQ ID NO:8 comprises replacement, disappearance and/or inserts variant in one or more (or several) position; With

E the sugared binding domain of () (a), (b), (c) or (d) has the variant of Sugar-binding activitv.

The present invention also relates to the polynucleotide of the separation of polypeptide of the present invention of encoding, and comprises the nucleic acid construct of described polynucleotide, recombinant expression vector and recombinant host cell; With the method producing described polypeptide.

The present invention also relates to degraded or transforms the technique of cellulose materials, and it comprises: have under the existence of the polypeptide of cellobiohydrolase activity with enzyme composition process cellulose materials of the present invention.In one aspect, described method also comprises the cellulose materials reclaimed through degraded or conversion.

The present invention also relates to and produces the technique of tunning, and it comprises: (a) of the present invention there is the existence of the polypeptide of cellobiohydrolase activity under use enzyme composition saccharified cellulosic material; (b) with the fermentation of one or more (such as several) organism of fermentation through the cellulose materials of saccharification to produce tunning; (c) this tunning is reclaimed from fermentation.

The present invention also relates to the technique of fermentable fiber cellulosic material, it comprises and to ferment described cellulose materials with one or more (such as several) organism of fermentation, and wherein said cellulose materials uses enzyme composition saccharification have the existence of the polypeptide of cellobiohydrolase activity in the present invention under.In one aspect, the fermentation of described cellulose materials produces tunning.In yet another aspect, aforesaid method comprises further from fermentation recovery tunning.

The present invention also relates to the polynucleotide of coded signal peptide, described signal peptide comprises or consists of the amino acid/11 to 17 of (consist of) SEQ ID NO:2, the amino acid/11 to 20 of SEQ ID NO:4, the amino acid/11 to 21 of SEQ ID NO:6, or the amino acid/11 to 17 of SEQ ID NO:8, it is operably connected to the gene of proteins encoded; Comprise the nucleic acid construct of described polynucleotide, expression vector and recombinant host cell; With the protedogenous method of product.

Accompanying drawing explanation

The genomic dna sequence (SEQ ID NO:1) that Fig. 1 code displaying has the gene of camphor tree suede branch mould (Malbranchea cinnamomea) polypeptide of cellobiohydrolase activity and the aminoacid sequence (SEQ ID NO:2) of deriving.

The genomic dna sequence (SEQ ID NO:3) that Fig. 2 code displaying has the gene of thermophilic excellent softgel shell (Corynascus thermophilus) polypeptide of cellobiohydrolase activity and the aminoacid sequence (SEQ ID NO:4) of deriving.

The genomic dna sequence (SEQ ID NO:5) that Fig. 3 code displaying has the gene of the thermophilic excellent softgel shell polypeptide of cellobiohydrolase activity and the aminoacid sequence (SEQ ID NO:6) of deriving.

The genomic dna sequence (SEQ ID NO:7) that Fig. 4 code displaying has the gene of the thermophilic excellent softgel shell polypeptide of cellobiohydrolase activity and the aminoacid sequence (SEQ ID NO:8) of deriving.

Fig. 5 shows the restriction map of pGH7_ZY582279_485.

Fig. 6 shows the restriction map of pGH7_Mf7339.

Fig. 7 shows the restriction map of pGH7_Mf6627.

Fig. 8 shows the restriction map of pGH7_Mf0261.

Definition

Acetyl xylan esterase: term " acetyl xylan esterase " means Carboxylesterase (EC3.1.1.72), its catalysis ethanoyl is from the hydrolysis of polymeric xylans, acetylize wood sugar, acetyl glucose, acetic acid α-naphthylacetate (alpha-napthyl acetate) and acetic acid p-nitrophenyl acetate (p-nitrophenyl acetate).For the present invention, acetyl xylan esterase activity uses containing 0.01%TWEEN ^tM0.5mM acetic acid p-nitrophenyl acetate in the 50mM sodium acetate pH5.0 of 20 (polyoxyethylenesorbitan monolaurates) is determined as substrate.The acetyl xylan esterase of a unit is defined as can at pH5, and 25 DEG C of per minutes discharge the enzyme amount of 1 micromole's p-NP negatively charged ion (p-nitrophenolate anion).

Allelic variant (allelic variant): term " allelic variant " means two or more Alternative Form any of the gene occupying phase syntenic genes seat.Allelic variation is occurred natively by sudden change, and can cause the polymorphism in population.Transgenation can be the polypeptide of reticent (unchanged in the polypeptide of the coding) aminoacid sequence with change of maybe can encoding.The allelic variant of polypeptide is the polypeptide of being encoded by the allelic variant of gene.

α-l-arabfuranglycosidase: term " α-l-arabfuranglycosidase " means α-L-arabinofuranosidase glucosides arabinofuranosidase lytic enzyme (EC3.2.1.55), its catalysis is to the hydrolysis of the end irreducibility α-L-arabinofuranosidase glucosides residue in α-L-arabinose glycosides.This enzyme works to α-L-arabinofuranosidase glucosides, the α-L-arabinan containing (1,3)-and/or (1,5)-key, araboxylan and arabogalactan.α-l-arabfuranglycosidase is also referred to as arabinofuranosidase/xylosidase, α-arabinofuranosidase/xylosidase, α-L-arabinose glycosides enzyme, α-arabinofuranosidase, polysaccharide α-l-arabfuranglycosidase, α-L-arabinofuranosidase glucosides lytic enzyme, L-arabinose glycosides enzyme or α-L-arabanase.For the present invention, α-l-arabfuranglycosidase activity is medium-viscosity Wheat Arabinoxylan (the Megazyme International Ireland of 5mg in the 100mM sodium acetate pH5 of the every ml used in cumulative volume 200 μ l, Ltd., Bray, Co.Wicklow, Ireland) carry out 30 minutes at 40 DEG C, then pass through the pectinose analysis of HPX-87H column chromatography (Bio-Rad Laboratories, Inc., Hercules, CA, USA) is determined.

Alpha-glucuronidase: term " alpha-glucuronidase " means α-D-glucuronide glucuronic acid lytic enzyme (alpha-D-glucosiduronate glucuronohydrolase) (EC3.2.1.139), its catalysis α-D-glucuronic acid glycoside hydrolysis is D-glucuronic acid and alcohol.For the present invention, alpha-glucuronidase activity is that 1998, J.Bacteriol.180:243-249 determines according to de Vries.The alpha-glucuronidase of a unit equals can at pH5, and 40 DEG C of per minutes discharge the enzyme amount of 1 micromole's glucuronic acid or 4-O-methylglucuronic acid.

Beta-glucosidase enzyme: term " beta-glucosidase enzyme " means β-D-glucoside glucohydralase (beta-D-glucoside glucohydrolase) (E.C.No.3.2.1.21), the hydrolysis of its catalysis end non-reduced β-D-Glucose residue, and discharge β-D-Glucose.For the present invention, beta-glucosidase enzyme is according to Venturi etc., 2002, Extracellular beta-D-glucosidase from Chaetomium thermophilum var.coprophilum:production, the method of purification and some biochemical properties, J.Basic Microbiol.42:55-66 uses p-nitrophenyl-β-D-glucose pyranoside to determine as substrate.The beta-glucosidase enzyme of a unit is defined as at 25 DEG C, pH4.8, is containing 0.01% 1.0 micromole's p-NP negatively charged ion are produced from the 1mM p-nitrophenyl-β-D-glucose pyranoside per minute as substrate in the 50mM Trisodium Citrate of 20.

Xylobiase: term " xylobiase " means β-D-xyloside wood sugar lytic enzyme (β-D-xyloside xylohydrolase) (E.C.3.2.1.37), the outer hydrolysis of the short β of its catalysis (1 → 4) wood oligose (xylooligosaccharide) is to remove continuous print D-xylose residues from non-reducing end.For the present invention, the xylobiase of a unit is defined as at 40 DEG C, and pH5 is containing 0.01% 1.0 micromole's p-NP negatively charged ion are produced from the 1mM p-nitrophenyl-β-D-xyloside per minute as substrate in the 100mM Trisodium Citrate of 20.

Sugar binding domain: term " sugared binding domain " means mediation enzyme to the region of the enzyme of the combination of the pars amorpha of sugared substrate.Sugar binding domain (CBD), is also called carbohydrate binding modules, usually sees N-terminal or the C-terminal of enzyme.

Catalytic domain: term " catalytic domain " means the region of the enzyme of the catalysis mechanism (catalytic machinery) containing enzyme.

CDNA: term " cDNA " mean can by reverse transcription from derive from eucaryon or prokaryotic cell prokaryocyte maturation, DNA molecular that the mRNA molecule of montage prepares.CDNA lacks the intron sequences be usually present in corresponding gene group DNA.Initial (initial) primary RNA transcript thing is the precursor of mRNA, and it comprises montage by a series of step processing, and the mRNA then as ripe montage occurs.

Cellobiohydrolase: term " cellobiohydrolase " means 1, 4-callose cellobiohydrolase (1, 4-beta-D-glucan cellobiohydrolase) (E.C.3.2.1.91 and E.C.3.2.1.176), its catalyse cellulose, cell-oligosaccharide, or anyly comprise β-1, in the polymkeric substance of the glucose that 4-connects 1, the hydrolysis of 4-β-D-glycosidic link, cellobiose (Teeri is discharged from the reducing end (cellobiohydrolase I) of chain or non-reducing end (cellobiohydrolase II), 1997, Crystalline cellulose degradation:New insight into the function of cellobiohydrolases, Trends in Biotechnology15:160-167, Teeri etc., 1998, Trichoderma reesei cellobiohydrolases:why so efficient on crystalline cellulose?, Biochem.Soc.Trans.26:173-178).According to Lever etc., 1972, Anal.Biochem.47:273-279; Van Tilbeurgh etc., 1982, FEBS Letters149:152-156; Van Tilbeurgh and Claeyssens, 1985, FEBS Letters187:283-288; And Tomme etc., 1988, the Eur.J.Biochem.170:575-581 method determination cellobiohydrolase activity described.In the present invention, the method for Tomme etc. can be used for determining cellobiohydrolase activity.Or cellobiohydrolase activity can use Microcrystalline Cellulose to determine according to the step described in the embodiment of the present invention 15.

Polypeptide of the present invention has the mature polypeptide of SEQ ID NO:2, the mature polypeptide of SEQ ID NO:4, the mature polypeptide of SEQ ID NO:6, or at least 20% of the cellobiohydrolase activity of the mature polypeptide of SEQ ID NO:8, such as at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100%.

Cellulolytic enzyme or cellulase: term " cellulolytic enzyme " or " cellulase " mean the enzyme of one or more (such as several) hydrolysis fiber cellulosic material.This fermentoid comprises endoglucanase, cellobiohydrolase, beta-glucosidase enzyme, or its combination.The two kinds of basic skills measuring cellulolytic activity comprise: (1) measures total fiber element degrading activity, (2) independent cellulolytic activity (endoglucanase, cellobiohydrolase and beta-glucosidase enzyme) is measured, as Zhang etc., Outlook for cellulase improvement:Screening and selection strategies, 2006, Biotechnology Advances24:452-481 summarizes.Total fiber element degrading activity typically uses insoluble substrate to measure, and described substrate comprises Whatman1 filter paper, Microcrystalline Cellulose, bacteria cellulose, algae Mierocrystalline cellulose, cotton, pretreated ligno-cellulose etc.Modal total fiber element degrading activity assay method uses Whatman1 filter paper as the filter paper assay method of substrate.This assay method is by International Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987, Measurement of cellulase activities, Pure Appl.Chem.59:257-68) establish.

For the present invention, cellulose decomposition enzymic activity is determined compared to the increase of the contrast hydrolysis not adding cellulose decomposition zymoprotein by measuring the cellulosic material hydrolysis undertaken by cellulolytic enzyme under the following conditions: in the PCS of the cellulose decomposition zymoprotein/g of 1-50mg, Mierocrystalline cellulose (or other pretreated cellulose materials) is in suitable temperature, and such as 50 DEG C, 55 DEG C or 60 DEG C are carried out 3-7 day.Usual conditions are: 1ml reaction solution, through washing or unwashed PCS, and 5% insoluble solid, 50mM sodium acetate pH5,1mM MnSO ₄, 50 DEG C, 55 DEG C or 60 DEG C, 72 hours, pass through hPX-87H post (Bio-Rad Laboratories, Inc., Hercules, CA, USA) carries out glycan analysis.

Cellulose materials: term " cellulose materials " means to comprise cellulosic any material.Main polysaccharide in the primary cell wall (primary cell wall) of biomass is Mierocrystalline cellulose, and secondly the abundantest is hemicellulose, and the 3rd is pectin.Secondary cell wall (secondary cell wall) produces after cell stops growing, and it is contained polysaccharide equally and is strengthened by the polymeric lignin being covalently cross-linking to hemicellulose.Mierocrystalline cellulose is the homopolymer of anhydro cellobiose, therefore be straight chain β-(1-4)-D-dextran, and hemicellulose comprises multiple compounds, such as xylan, xyloglucan (xyloglucan), araboxylan and mannosans, formed and have diversified substituent complex branches structure.Although Mierocrystalline cellulose normally multiform, be present in the insoluble crystal substrate of Mierocrystalline cellulose in plant tissue mainly parallel dextran chain.Hemicellulose is connected with hydrogen bond with Mierocrystalline cellulose and other hemicellulose usually, and it helps stabilized cell wall matrix.

Mierocrystalline cellulose sees the stem of such as plant, leaf, shell, skin and cob usually, or the leaf of tree, branch and timber.Cellulose materials can be, but be not limited to, agricultural residue, herbaceous material (comprising energy crop), municipal solid waste, paper pulp and paper mill residue, waste paper and timber (comprising forestry residue) (see, such as, Wiselogel etc., 1995, in Handbook on Bioethanol (Charles E.Wyman compiles), pp.105-118, Taylor & Francis, Washington D.C.; Wyman, 1994, Bioresource Technology50:3-16; Lynd, 1990, Applied Biochemistry and Biotechnology24/25:695-719; Mosier etc., 1999, Recent Progress in Bioconversion of Lignocellulosics, in Advances in Biochemical Engineering/Biotechnology, T.Scheper edits, Volume65, pp.23-40, Springer-Verlag, New York).It should be understood that Mierocrystalline cellulose can be the form of ligno-cellulose in this article, ligno-cellulose is a kind of Plant cell wall material, comprises the mixed-matrix of xylogen, Mierocrystalline cellulose and hemicellulose.In preferred at one, cellulose materials is any biological material.In another is preferred, described cellulose materials is ligno-cellulose, and it comprises Mierocrystalline cellulose, hemicellulose and xylogen.

In one aspect, cellulose materials is agricultural residue.In yet another aspect, cellulose materials is herbaceous material (comprising energy crop).In yet another aspect, cellulose materials is municipal solid waste.In yet another aspect, cellulose materials is paper pulp and paper mill residue.In yet another aspect, cellulose materials is waste paper.In yet another aspect, cellulose materials is timber (comprising forestry residue).

In yet another aspect, cellulose materials is giantreed (arundo).In yet another aspect, cellulose materials is bagasse.In yet another aspect, cellulose materials is bamboo.In yet another aspect, cellulose materials is corn cob.In yet another aspect, cellulose materials is zein fiber.In yet another aspect, cellulose materials is maize straw.In yet another aspect, cellulose materials is that Chinese silvergrass belongs to.In yet another aspect, cellulose materials is orange peel.In yet another aspect, cellulose materials is rice straw.In yet another aspect, cellulose materials is switchgrass (switch grass).In yet another aspect, cellulose materials is straw.

In yet another aspect, cellulose materials is white poplar.In yet another aspect, cellulose materials is eucalyptus.In yet another aspect, cellulose materials is fir (fir).In yet another aspect, cellulose materials is pine tree.In yet another aspect, cellulose materials is willow.In yet another aspect, cellulose materials is dragon spruce.In yet another aspect, cellulose materials is willow.

In yet another aspect, cellulose materials is algae Mierocrystalline cellulose.In yet another aspect, cellulose materials is bacteria cellulose.In yet another aspect, cellulose materials is velveteen (cotton linter).In yet another aspect, cellulose materials is filter paper.In yet another aspect, cellulose materials is Microcrystalline Cellulose.In yet another aspect, cellulose materials is the acid-treated Mierocrystalline cellulose of phosphorus.

In yet another aspect, cellulose materials is hydrobiont matter.As for herein, " hydrobiont matter " means the biomass produced by photosynthesis in aquatic environment.Hydrobiont matter can be algae, emergent (emergent plant), floatingleaved plant (floating-leaf plant) or submerged plant (submerged plant).

Cellulose materials can in statu quo (as is) use or carry out pre-treatment, and pre-treatment uses ordinary method known in the art, as described herein.In preferred at one, pre-treating cellulosic material.

Encoding sequence: term " encoding sequence " means the polynucleotide of the aminoacid sequence of directly specifying polypeptide.The border of encoding sequence is determined by open reading frame usually, described open reading frame with initiator codon as ATG, GTG or TTG start, and with terminator codon as TAA, TAG or TGA terminate.Encoding sequence can be genomic dna, cDNA, synthetic DNA or its combination.

Regulating and controlling sequence (control sequence): it is required nucleotide sequence that term " regulating and controlling sequence " means to express the polynucleotide of coding mature polypeptide of the present invention.Each regulating and controlling sequence can be (that is, from different genes) of natural (that is, from same gene) or external source for the polynucleotide of the described mature polypeptide of coding, or each regulating and controlling sequence is for can be natural or external source each other.These regulating and controlling sequences include but not limited to leader sequence, polyadenylation se-quence, propeptide sequence, promotor, signal peptide sequence and transcription terminator.At least, regulating and controlling sequence comprises promotor and the termination signal transcribed and translate.Regulating and controlling sequence can be equipped with the joint for introducing specific restriction sites, and described specific restriction sites promotes the connection in the polynucleotide encoding district of regulating and controlling sequence and coded polypeptide.

Endoglucanase: term " endoglucanase " means inscribe-Isosorbide-5-Nitrae-(1,3; 1,4)-callose 4-glucan hydrolase (endo-1,4-β-D-glucan4-glucanohydrolase) (E.C.3.2.1.4), in its catalyse cellulose, derivatived cellulose (such as carboxymethyl cellulose and Natvosol), moss starch (lichenin) 1, the β-1 of 4-β-D-glycosidic link, mixing, 3 dextran such as cereal beta-D-glucans or xyloglucan and the interior hydrolysis (endohydrolysis) containing the β-Isosorbide-5-Nitrae key in other vegetable material of cellulosic component.Endoglucanase activity is determined by the minimizing of measuring substrate viscosity or the reducing end increase determined by reducing sugar test method (Zhang etc., 2006, Biotechnology Advances24:452-481).For the present invention, according to Ghose, the method for 1987, Pure and Appl.Chem.59:257-268, at pH5,40 DEG C use carboxymethyl cellulose (CMC) to determine endoglucanase activity as substrate.

Express: term " expressions " comprises any step relating to polypeptide generation, it include but not limited to transcribe, post transcriptional modificaiton, translation, posttranslational modification and secretion.

Expression vector: term " expression vector " means DNA molecular that is linear or ring-type, and it comprises the polynucleotide of coded polypeptide, and described polynucleotide be provided for its regulating and controlling sequence of expressing and be operably connected.

Family 61 glycoside hydrolase: term " family 61 glycoside hydrolase " or " family GH61 " or " GH61 " are defined as according to Henrissat B. in this article, 1991, A classification of glycosyl hydrolases based on amino-acid sequence similarities, Biochem.J.280:309-316, and Henrissat B. and Bairoch A., 1996, Updating the sequence-based classification of glycosyl hydrolases, Biochem.J.316:695-696 belongs to the polypeptide of glycoside hydrolase Families 61.Proenzyme in this family first classifies as glycoside hydrolase Families based on the very weak inscribe measured a family member-Isosorbide-5-Nitrae-β-D dextranase activity.The structure of these enzymes and binding mode are non-classical, and they cannot be considered as real (bona fide) Glycosylase.But based on when the mixture with cellulase or cellulase together uses, it strengthens the ability that ligno-cellulose decomposes, they are retained in CAZy classification.

Feruloyl esterase: term " feruloyl esterase (feruloyl esterase) " means 4-hydroxy-3-methoxy cinnyl-glycosylhydrolase (EC3.1.1.73), its catalysis 4-hydroxy-3-methoxy cinnyl (asafoetide acyl) group from the hydrolysis of the sugar (it is generally pectinose " natural biomass " substrate) of esterification, to produce forulic acid (Ferulic acid).Feruloyl esterase is also referred to as feruloyl esterase (ferulic acid esterase), hydroxy cinnamate acyl group esterase, FAE-III, laurate lytic enzyme, FAEA, cinnAE, FAE-I or FAE-II.For the present invention, ferulaic acid esterase activity uses the 0.5mM forulic acid p-nitrophenyl ester in 50mM sodium acetate pH5.0 to determine as substrate.The feruloyl esterase of a unit equals can at pH5, and 25 DEG C of per minutes discharge the enzyme amount of 1 micromole's p-NP negatively charged ion.

Fragment: term " fragment " means from the amino in mature polypeptide or territory and/or carboxyl-terminal deletion one or more (such as several) amino acid whose polypeptide or catalytic domain or sugared binding domain; Wherein said fragment has cellobiohydrolase activity or Sugar-binding activitv.In one aspect, fragment contains at least 375 amino-acid residues of SEQ ID NO:2, such as at least 397 amino-acid residues, or at least 419 amino-acid residues.In yet another aspect, fragment contains at least 364 amino-acid residues of SEQ ID NO:4, such as at least 386 amino-acid residues or at least 408 amino-acid residues.In yet another aspect, fragment contains at least 370 amino-acid residues of SEQ ID NO:6, such as at least 392 amino-acid residues or at least 414 amino-acid residues.In yet another aspect, fragment contains at least 429 amino-acid residues of SEQ ID NO:8, such as at least 454 amino-acid residues or at least 479 amino-acid residues.

Hemicellulose lytic enzyme or hemicellulase: term " hemicellulose lytic enzyme " or " hemicellulase " mean the enzyme of one or more (such as several) hydrolyzed hemicellulose materials.See, such as Shallom D. and Shoham Y.Microbial hemicellulases.Current Opinion In Microbiology, 2003,6 (3): 219-228).Hemicellulase is the key component in Degrading plant biomass.The example of hemicellulase includes but not limited to acetyl mannan esterase, acetyl xylan esterase, arabanase, arabinofuranosidase, coumaric acid esterase, feruloyl esterase, tilactase, glucuronidase, glucuronic acid esterase, mannonase mannosidase, zytase and xylosidase.The substrate of these enzymes, hemicellulose be branching and straight-chain polysaccharide mix group, these polysaccharide are by the cellulose micro-fibers of hydrogen bonding in plant cell wall, and cross-linking is the network of robust (robust).Hemicellulose also covalently invests xylogen, with the structure of Mierocrystalline cellulose together height of formation complexity.The changeable structure of hemicellulose and organizational form need the synergy of many enzymes to make it degradable.The catalytic module of hemicellulase is the glycoside hydrolase (GH) of hydrolyzing glucosidic bonds, or the sugar ester enzyme (CE) that the ester of hydrolysis acetic acid or forulic acid side base connects.These catalytic module, based on the homology of its primary structure, can be assigned as GH and CE family.Some families, have similar folding generally, can classify as clan (clan) further, with alphabetic flag (such as, GH-A).The classification of most informedness and these and other up-to-date sugared organized enzymes can obtain at Carbohydrate-Active Enzymes (CAZy) database.Hemicellulose lytic enzyme activity can according to Ghose and Bisaria, and 1987, Pure & Appl.Chem.59:1739-1752 is in suitable temperature, and such as 50 DEG C, 55 DEG C or 60 DEG C, and pH, such as 5.0 or 5.5 measure.

High stringent condition: term " high stringent condition " means the probe for length at least 100 Nucleotide, at 42 DEG C, 5X SSPE, 0.3%SDS, 200 micrograms/ml sheared and the salmon sperm DNA of sex change and 50% methane amide in, carry out prehybridization and hybridization 12 to 24 hours according to the Southern blotting of standard.2X SSC, 0.2%SDS is used at 65 DEG C, solid support material finally to be washed three times, each 15 minutes.

Host cell: term " host cell " means to be suitable for using the nucleic acid construct that comprises polynucleotide of the present invention or expression vector to carry out transforming, transfection, transduction etc. cell type.Any sudden change owing to occurring in copying that parental cell contained in term " host cell " and be different from the offspring of parental cell.

Be separated: term " separation " means the material existed with the form do not occurred at nature or environment.The non-limiting example of the material be separated comprises the material of (1) any non-natural existence, (2) any at least in part with one or more or material that all natural adjoint naturally occurring composition departs from it, include but not limited to any enzyme, variant, nucleic acid, protein, peptide or cofactor; (3) anyly for this material seen by occurring in nature, have passed through manually modified material; Or (4) are any, and by increasing this amount of substance relative to other adjoint components natural in it, (restructuring such as, in host cell produces; To encode the multiple copied of gene of this material; And use than the stronger promotor of the natural adjoint promotor of gene with this material of coding) and the material modified.

Low stringency condition: term " low stringency condition " means the probe for length at least 100 Nucleotide, at 42 DEG C, 5X SSPE, 0.3%SDS, 200 micrograms/ml sheared and the salmon sperm DNA of sex change and 25% methane amide in, carry out prehybridization and hybridization 12 to 24 hours according to the Southern blotting of standard.2X SSC, 0.2%SDS is used at 50 DEG C, solid support material finally to be washed three times, each 15 minutes.

Mature polypeptide: term " mature polypeptide " means the polypeptide with its final form existence after translation and any posttranslational modification, described modification such as N-end processing, the brachymemma of C-end, glycosylation, phosphorylation etc.In one aspect, according to the SignalP program (Nielsen etc. that the amino acid/11 to 17 of prediction SEQ ID NO:2 (P249XX) is signal peptide, 1997, Protein Engineering10:1-6), mature polypeptide is the amino acid/11 8 to 458 of SEQ ID NO:2.In yet another aspect, be the SignalP program of signal peptide according to the amino acid/11 to 20 of prediction SEQ ID NO:4 (P24NX2), mature polypeptide is the amino acid 21 to 450 of SEQ ID NO:4.In yet another aspect, according to the SignalP program (Nielsen etc. that the amino acid/11 to 21 of prediction SEQ ID NO:6 (P24FVN) is signal peptide, 1997, Protein Engineering10:1-6), mature polypeptide is the amino acid 22 to 457 of SEQ ID NO:6.In yet another aspect, be the SignalP program of signal peptide according to the amino acid/11 to 17 of prediction SEQ ID NO:8 (P24FUQ), mature polypeptide is the amino acid/11 8 to 521 of SEQ ID NO:8.

In this area, known host cell can produce the mixture of two or more different mature polypeptides (that is, having different C-ends and/or n terminal amino acid) of identical expression of polypeptides.Also known different host cell processing polypeptides by different way in this area, and therefore, an express polypeptide host cell compared with expressing another host cell of phase homopolypeptide, different mature polypeptides (such as there is different C-ends and/or n terminal amino acid) can be produced.

Mature polypeptide encoded sequence: term " mature polypeptide encoded sequence " means to encode the polynucleotide of the mature polypeptide with cellobiohydrolase activity.In one aspect, according to the SignalP program of Nucleotide 1 to the 51 coded signal peptide of prediction SEQ ID NO:1 (Nielsen etc., 1997, on seeing), mature polypeptide encoded sequence is the Nucleotide 52 to 1454 of SEQ ID NO:1 or its cDNA sequence.In yet another aspect, according to the SignalP program of Nucleotide 1 to the 60 coded signal peptide of prediction SEQ ID NO:3, mature polypeptide encoded sequence is the Nucleotide 61 to 1733 of SEQ ID NO:3 or its cDNA sequence.In yet another aspect, according to the SignalP program of Nucleotide 1 to the 63 coded signal peptide of prediction SEQ ID NO:5 (Nielsen etc., 1997, on seeing), mature polypeptide encoded sequence is the Nucleotide 64 to 1782 of SEQ ID NO:5 or its cDNA sequence.In yet another aspect, according to the SignalP program of Nucleotide 1 to the 51 coded signal peptide of prediction SEQ ID NO:7, mature polypeptide encoded sequence is the Nucleotide 52 to 1640 of SEQ ID NO:7 or its cDNA sequence.

Medium stringency condition: term " medium stringency condition " means the probe for length at least 100 Nucleotide, at 42 DEG C, 5X SSPE, 0.3%SDS, 200 micrograms/ml sheared and the salmon sperm DNA of sex change and 35% methane amide in, carry out prehybridization and hybridization 12 to 24 hours according to the Southern blotting of standard.2X SSC, 0.2%SDS is used at 55 DEG C, solid support material finally to be washed three times, each 15 minutes.

Medium-Gao stringent condition: term " medium-Gao stringent condition " means the probe for length at least 100 Nucleotide, at 42 DEG C, 5X SSPE, 0.3%SDS, 200 micrograms/ml sheared and the salmon sperm DNA of sex change and 35% methane amide in, carry out prehybridization and hybridization 12 to 24 hours according to the Southern blotting of standard.2X SSC, 0.2%SDS is used at 60 DEG C, solid support material finally to be washed three times, each 15 minutes.

Nucleic acid construct: term " nucleic acid construct " means the nucleic acid molecule of strand or double-strand, it is separated from naturally occurring gene, or it modifiedly contains the section of nucleic acid in the mode not originally being present in (not otherwise exist) occurring in nature, or it is for what synthesize, it comprises one or more regulating and controlling sequence.

Be operably connected: term " is operably connected " and means such configuration, wherein regulating and controlling sequence is placed in the appropriate location of the encoding sequence relative to polynucleotide, make regulating and controlling sequence instruct the expression of encoding sequence.

There is the polypeptide of cellulolytic enhancing activity: term " has the polypeptide that cellulose decomposition strengthens " and means the GH61 polypeptide of enzyme to the enhancing of the hydrolysis of cellulose materials that catalysis has cellulolytic activity.For the present invention, by measure free cellulolytic enzyme be hydrolyzed with contrast under the following conditions compared with the reducing sugar of hydrolysis fiber cellulosic material to increase or cellulolytic enhancing activity is determined in the total amount increase of cellobiose and glucose: the middle Mierocrystalline cellulose of the pretreated maize straw of 1-50mg total protein/g (PCS), wherein total protein comprises the cellulose decomposition zymoprotein of 50-99.5%w/w, and the protein with the GH61 polypeptide of cellulolytic enhancing activity of 0.5-50%w/w, in suitable temperature, such as 50 DEG C, 55 DEG C or 60 DEG C and suitable pH, as 4-9, such as 5.0 or 5.5 last 1-7 days, contrast hydrolysis uses the total protein heap(ed) capacity of equivalent and cellulose-less decomposition enhanced activity (in 1-50mg cellulolytic protein/g PCS Mierocrystalline cellulose) carries out.In preferred at one, under the cellulase protein heap(ed) capacity being used in the Aspergillus fumigatus beta-glucosidase enzyme (restructuring produces in aspergillus oryzae as described in WO 2002/095014) of the aspergillus oryzae beta-glucosidase enzyme (recombinating in aspergillus oryzae generation according to WO 02/095014) of the 2-3% of total protein by weight or the 2-3% of total protein quality exists 1.5L (Novozymes A/S, denmark) mixture is as the source of cellulolytic activity.

The GH61 polypeptide with cellulolytic enhancing activity strengthens by the hydrolysis of the enzymatic cellulose materials with cellulolytic activity by reducing the amount of the cellulolytic enzyme reached needed for same hydrolysis level, preferred reduction at least 1.01 times, such as at least 1.05 times, at least 1.10 times, at least 1.25 times, at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, or at least 20 times.

Pretreated maize straw: term " PCS " or " pretreated maize straw " mean by with heat and dilute sulphuric acid process, oxygenation pretreatment or neutrality the pretreated cellulose materials being derived from maize straw.

Sequence iden: parameter " sequence iden " describes the dependency between two aminoacid sequences or between two nucleotide sequences.

For the present invention, degree of sequence identity between two aminoacid sequences uses as EMBOSS software package (EMBOSS:The European Molecular Biology Open Software Suite, Rice etc., 2000, Trends Genet.16:276-277), Needleman-Wunsch algorithm (Needleman and Wunsch performed in the Needle program of preferred 3.0.0,5.0.0 version or more highest version, 1970, J.Mol.Biol.48:443-453) measure.The parameter used opens point penalty (gap open penalty) 10, gap extension penalty (gap extension penalty) 0.5 and EBLOSUM62 (the EMBOSS version of BLOSUM62) substitution matrix for breach.Use Needle to be labeled as the Output rusults (using-nobrief option to obtain) of " the highest identity (longest identity) " as identity per-cent, and be calculated as follows:

(same residue × 100)/sum of breach (in the comparison length-comparison)

For the present invention, degree of sequence identity between two nucleotide sequences uses as EMBOSS software package (EMBOSS:The European Molecular Biology Open Software Suite, Rice etc., 2000, see above), Needleman-Wunsch algorithm performed in the Needle program of preferred 5.0.0 version or more highest version (Needleman and Wunsch, 1970, see above) measure.The parameter used opens point penalty 10, gap extension penalty 0.5 and EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix for breach.Use Needle to be labeled as the Output rusults (using-nobrief option to obtain) of " the highest identity " as identity per-cent, and be calculated as follows:

(same deoxyribonucleotide × 100)/sum of breach (in the comparison length-comparison)

Subsequence: term " subsequence (subsequence) " means to hold from 5 ' and/or 3 ' of mature polypeptide encoded sequence or territory the polynucleotide lacking one or more (such as several) Nucleotide; Wherein said subsequence coding has the fragment of cellobiohydrolase activity or Sugar-binding activitv.In one aspect, subsequence contains at least 1125 Nucleotide of SEQ ID NO:1, such as at least 1191 Nucleotide, or at least 1257 Nucleotide.In yet another aspect, subsequence contains at least 1092 Nucleotide of SEQ ID NO:3, such as at least 1158 Nucleotide or at least 1224 Nucleotide.In one aspect, subsequence contains at least 1110 Nucleotide of SEQ ID NO:5, such as at least 1176 Nucleotide or at least 1242 Nucleotide.In yet another aspect, subsequence contains at least 1287 Nucleotide of SEQ ID NO:7, such as at least 1362 Nucleotide or at least 1437 Nucleotide.

Variant: term " variant " means to comprise change, the polypeptide with cellobiohydrolase activity namely replacing, insert and/or lack in one or more (such as several) position.Replace and mean to occupy the different amino acid replacement of amino acid of certain position; Disappearance means to remove the amino acid occupying certain position; And insertion means to add amino acid after adjoining and and then occupy the amino acid of certain position.

Very high stringent condition: term " very high stringent condition " means the probe for length at least 100 Nucleotide, at 42 DEG C, 5X SSPE, 0.3%SDS, 200 micrograms/ml sheared and the salmon sperm DNA of sex change and 50% methane amide in, carry out prehybridization and hybridization 12 to 24 hours according to the Southern blotting of standard.2X SSC, 0.2%SDS is used at 70 DEG C, solid support material finally to be washed three times, each 15 minutes.

Very low stringency condition: term " very low stringency condition " means the probe for length at least 100 Nucleotide, at 42 DEG C, 5X SSPE, 0.3%SDS, 200 micrograms/ml sheared and the salmon sperm DNA of sex change and 25% methane amide in, carry out prehybridization and hybridization 12 to 24 hours according to the Southern blotting of standard.2X SSC, 0.2%SDS is used at 45 DEG C, solid support material finally to be washed three times, each 15 minutes.

Containing xylan material: term " containing xylan material " means any material comprising the plant cell wall polysaccharides of the xylose residues skeleton connected containing β-(1-4).The xylan of terrestrial plant is the heteropolymer with β-(1-4)-xylopyranose skeleton, and it has short sugar chain branches.They comprise D-glucuronic acid or its 4-O-methyl ether, L-arabinose and/or the multiple oligosaccharides comprising D-wood sugar, L-arabinose, D-or L-semi-lactosi and D-Glucose.The polysaccharide of xylan type can be divided into equal xylan (homoxylan) and Heteroxylan (heteroxylan), the latter comprises glucuronoxylan, (Arab) glucuronoxylan, (glucuronic acid) araboxylan, araboxylan and compound Heteroxylan.See, such as Ebringerova etc., 2005, Adv.Polym.Sci.186:1-67.

In technique of the present invention, any material containing xylan can be used.In preferred at one, described is ligno-cellulose containing xylan material.

Xylanolytic activities or xylanolytic activity: term " xylanolytic activities " or " xylanolytic activity " mean to be hydrolyzed the biologic activity containing xylan material.Two kinds of basic methods measuring xylanolytic activity comprise: (1) measures total pentosan degrading activity, and (2) measure independent xylanolytic activity (such as endo-xylanase, xylobiase, arabinofuranosidase, alpha-glucuronidase, acetyl xylan esterase, feruloyl esterase and α-glucuronic acid esterase (α-glucuronyl esterase)).The nearest in-progress summary at xylanolitic enzyme assay is in several open source literature, comprise Biely and Puchard, 2006, Recent progress in the assays of xylanolytic enzymes, Journal of the Science of Food and Agriculture86 (11): 1636-1647; Spanikova and Biely, 2006, Glucuronoyl esterase-Novel carbohydrate esterase produced by Schizophyllum commune, FEBS Letters580 (19): 4597-4601; Herrmann, Vrsanska, Jurickova, Hirsch, Biely, and Kubicek, 1997, The beta-D-xylosidase of Trichoderma reesei is a multifunctional beta-D-xylan xylohydrolase, Biochemical Journal321:375-381.

Total pentosan degrading activity is by determining that the reducing sugar formed from polytype xylan is measured, described xylan comprises such as oat wheat (oat spelt), beech wood (beechwood) and Larch (larchwood) xylan, or determines that the xylan fragments of the dyeing that the xylan dyeed from multiple covalency discharges is measured by light-intensity method.Modal total pentosan degrading activity assay method produces reducing sugar based on the 4-O-methylglucuronic acid xylan from poly, as Bailey, Biely, Poutanen, 1992, Interlaboratory testing of methods for assay of xylanase activity, Journal of Biotechnology23 (3): described in 257-270.Xylanase activity also can with 0.2%AZCL-araboxylan as substrate at 37 DEG C 0.01% determine in X-100 (4-(1,1,3,3-tetramethyl butyl) phenyl-polyoxyethylene glycol) and 200mM sodium phosphate buffer pH6.The xylanase activity of a unit is defined as at 37 DEG C, and pH6 produces 1.0 micromole's azurins (azurine) from the 0.2%AZCL-araboxylan per minute as substrate in 200mM sodium phosphate pH6 damping fluid.

For the present invention, xylanolytic activities is by measuring birch xylan (the Sigma Chemical Co. caused under following usual conditions by xylanolytic enzyme, Inc., St.Louis, MO, USA) increase be hydrolyzed is determined: 1ml reacts, 5mg/ml substrate (total solid), 5mg xylanolitic protein/g substrate, 50mM sodium acetate, pH5, 50 DEG C, 24 hours, as Lever, 1972, A new reaction for colorimetric determination of carbohydrates, P-hydroxybenzoic acid hydrazides (PHBAH) assay method is used to carry out glycan analysis described in Anal.Biochem47:273-279.

Zytase: term " zytase " means Isosorbide-5-Nitrae-β-D-xylan-wood sugar lytic enzyme (Isosorbide-5-Nitrae-β-D-xylan-xylohydrolase) (E.C.3.2.1.8), the interior hydrolysis of Isosorbide-5-Nitrae-β-D-wood sugar glycosidic bond in its catalysis xylan.For the present invention, xylanase activity use 0.2%AZCL-araboxylan as substrate at 37 DEG C 0.01% x-100 and 200mM sodium acetate buffer, determines in pH6.The xylanase activity of a unit is defined as and in 200mM sodium phosphate pH6 damping fluid, produces 1.0 micromole's azurins at 37 DEG C from the 0.2%AZCL-araboxylan per minute as substrate.

Detailed Description Of The Invention

There is the polypeptide of cellobiohydrolase activity

In one embodiment, the present invention relates to isolated polypeptide, the mature polypeptide of itself and SEQ ID NO:2 has at least 70%, such as at least 75%, at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%; Described polypeptide has cellobiohydrolase activity.In one embodiment, the present invention relates to isolated polypeptide, the mature polypeptide of itself and SEQ ID NO:4 has at least 88%, such as at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%; Described polypeptide has cellobiohydrolase activity.In one embodiment, the present invention relates to isolated polypeptide, the mature polypeptide of itself and SEQ ID NO:6 has at least 66%, such as at least 68%, at least 70%, at least 75%, at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%; Described polypeptide has cellobiohydrolase activity.In one embodiment, the present invention relates to isolated polypeptide, the mature polypeptide of itself and SEQ ID NO:8 has at least 81%, such as at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%; Described polypeptide has cellobiohydrolase activity.In one aspect, the mature polypeptide of described polypeptide and SEQ ID NO:2, the mature polypeptide of SEQ ID NO:4, the mature polypeptide of SEQ ID NO:6, or mature polypeptide difference the most nearly 10 amino acid, such as 1,2,3,4,5,6,7,8,9 or 10 amino acid of SEQ ID NO:8.

Polypeptide of the present invention preferably comprises or consists of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or the aminoacid sequence of SEQ ID NO:8 or its allelic variant; Or for it, there is the fragment of cellobiohydrolase activity.In yet another aspect, described polypeptide comprises or consists of the mature polypeptide of SEQ ID NO:2, the mature polypeptide of SEQ ID NO:4, the mature polypeptide of SEQ ID NO:6, or the mature polypeptide of SEQ ID NO:8.In yet another aspect, described polypeptide comprises or consists of the amino acid 22 to 457 of amino acid 21 to 450, SEQ ID NO:6 of amino acid/11 8 to 458, SEQ ID NO:4 of SEQ ID NO:2, or the amino acid/11 8 to 521 of SEQ ID NO:8.

In another embodiment, the present invention relates to the isolated polypeptide with cellobiohydrolase activity, it is by polynucleotide encoding, described polynucleotide are at unusual low stringency condition, low stringency condition, medium stringency condition, medium-Gao stringent condition, high stringent condition, or under very high stringent condition with following hybridization: the mature polypeptide encoded sequence of (i) SEQ ID NO:1, the mature polypeptide encoded sequence of SEQ ID NO:3, the mature polypeptide encoded sequence of SEQ ID NO:5, or the mature polypeptide encoded sequence of SEQ ID NO:7, (ii) cDNA sequence of SEQ ID NO:1, the cDNA sequence of SEQ ID NO:3, the cDNA sequence of SEQ ID NO:5, or the cDNA sequence of SEQ ID NO:7, or the total length complement (Sambrook etc. of (iii) (i) or (ii), 1989, Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor, New York).

SEQ ID NO:1 can be utilized, SEQ ID NO:3, SEQ ID NO:5, or the polynucleotide of SEQ ID NO:7 or its subsequence, and SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or the polypeptide of SEQ ID NO:8 or its fragment designing nucleic acid probe, there is with the identification of strains never belonging to together according to method well known in the art or plant and clones coding the DNA of the polypeptide of cellobiohydrolase activity.Specifically, according to the Southern immunoblot method of standard, these probes can be used for hybridizing with the genomic dna of interested cell or cDNA, with identify with from being wherein separated corresponding gene.These probes can be significantly shorter than complete sequence, but length should be at least 15, such as at least 25, at least 35, or at least 70 Nucleotide.Preferably, described nucleic acid probe is the length of at least 100 Nucleotide, such as, at least 200 Nucleotide, at least 300 Nucleotide, at least 400 Nucleotide, at least 500 Nucleotide, at least 600 Nucleotide, at least 700 Nucleotide, at least 800 Nucleotide, or the length of at least 900 Nucleotide.Both DNA and rna probe all can use.Usually probe mark (such as, is used to detect corresponding gene ³²p, ³h, ³⁵s, vitamin H or avidin (avidin) mark).These probes are covered by the present invention.

Can screen from the genomic dna prepared by other such bacterial strain or cDNA library with above-mentioned probe hybridization and the DNA of the polypeptide with cellobiohydrolase activity of encoding.Can agarose or polyacrylamide gel electrophoresis be passed through, or be separated from the genome of these other bacterial strains or other DNA by other isolation technique.Can will be transferred to soluble cotton (nitrocellulose) or other suitable solid support material from the DNA in library or the DNA of separation and be fixed thereon.In order to identify and SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7, or its mature polypeptide encoded sequence, or the clone of its subsequence hybridization or DNA, described solid support material is used in Sounthern trace.

For the present invention, hybridization represents that polynucleotide are being low to moderate the nucleic acid probe hybridization with mark under very high stringent condition very much, described nucleic acid probe corresponds to: (i) SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7, (ii) the mature polypeptide encoded sequence of SEQ ID NO:1, the mature polypeptide encoded sequence of SEQ ID NO:3, the mature polypeptide encoded sequence of SEQ ID NO:5, or the mature polypeptide encoded sequence of SEQ ID NO:7, (iii) cDNA sequence of SEQ ID NO:1, the cDNA sequence of SEQ ID NO:3, the cDNA sequence of SEQ ID NO:5, or the cDNA sequence of SEQ ID NO:7, (iv) their total length complement, or (v) their subsequence.Can use such as X-ray film (X-ray film) or other any detection meanss as known in the art detect under these conditions with the molecule of nucleic acid probe hybridization.

In one aspect, described nucleic acid probe is coding SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or the polypeptide of SEQ ID NO:8 or its mature polypeptide, or the polynucleotide of their fragment.In yet another aspect, described nucleic acid probe is SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7; Or the mature polypeptide encoded sequence of SEQ ID NO:1, the mature polypeptide encoded sequence of SEQ ID NO:3, the mature polypeptide encoded sequence of SEQ ID NO:5, or the mature polypeptide encoded sequence of SEQ ID NO:7; Or the cDNA sequence of SEQ ID NO:1, the cDNA sequence of SEQ ID NO:3, the cDNA sequence of SEQ ID NO:5, or the cDNA sequence of SEQ ID NO:7.

In another embodiment, the present invention relates to the isolated polypeptide with cellobiohydrolase activity, it is by polynucleotide encoding, the mature polypeptide encoded sequence of described polynucleotide and SEQ ID NO:1 or its cDNA sequence has at least 70%, such as at least 72%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%, have the isolated polypeptide of cellobiohydrolase activity, it is by polynucleotide encoding, and the mature polypeptide encoded sequence of described polynucleotide and SEQ ID NO:3 or its cDNA sequence has at least 88%, such as at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%, there is the isolated polypeptide of cellobiohydrolase activity, it is by polynucleotide encoding, the mature polypeptide encoded sequence of described polynucleotide and SEQ ID NO:5 or its cDNA sequence has at least 66%, such as at least 68%, at least 70%, at least 75%, at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%, have the isolated polypeptide of cellobiohydrolase activity, it is by polynucleotide encoding, described polynucleotide with or the mature polypeptide encoded sequence of SEQ ID NO:7 or its cDNA sequence have at least 81%, such as at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%.

In another embodiment, the present invention relates to the mature polypeptide of SEQ ID NO:2, the mature polypeptide of SEQ ID NO:4, the mature polypeptide of SEQ ID NO:6, or the mature polypeptide of SEQ ID NO:8 comprises replacement, disappearance and/or inserts variant in one or more (such as several) position.In one aspect, import the mature polypeptide of SEQ ID NO:2, the mature polypeptide of SEQ ID NO:4, the mature polypeptide of SEQ ID NO:6, or the aminoacid replacement of the mature polypeptide of SEQ ID NO:8, disappearance and/or insertion quantity be as many as 10, such as 1,2,3,4,5,6,7,8,9 or 10.Amino acid change can be secondary property, namely conservative aminoacid replacement or insertion, its not remarkably influenced protein folding and/or active; Be generally 1 to about 30 amino acid whose little disappearances; Little amino or C-terminal extend, such as N-terminal methionine residues; The little joint peptide of about 20-25 residue at the most; Or promoted the little extension of purifying by change net charge or other function, as polyhistidine sequence (poly histidine tract), epitope (antigenic epitope) or binding domain (binding domain).

The conservative example replaced is within following group: basic aminoacids group (arginine, Methionin and Histidine), acidic amino acid group (L-glutamic acid and aspartic acid), polare Aminosaeren group (glutamine and l-asparagine), hydrophobic amino acid group (leucine, Isoleucine and α-amino-isovaleric acid), aromatic amino acid group (phenylalanine, tryptophane and tyrosine) and p1 amino acid group (glycine, L-Ala, Serine, Threonine and methionine(Met)).Usually the aminoacid replacement not changing specific activity (specific activity) is known in the art, and by such as H.Neurath and R.L.Hill, 1979, in The Proteins, Academic Press, describe in New York.The exchange the most generally occurred is Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly.

Or amino acid change has the character causing the physics-chem characteristic of polypeptide to change.Such as, amino acid change can improve the thermostability of polypeptide, changes substrate specificity, changes optimal pH etc.

Can according to methods known in the art, such as site-directed mutagenesis or L-Ala scanning mutagenesis method (Cunningham and Wells, 1989, Science244:1081-1085) identify the indispensable amino acid in parental polypeptide.In a rear technology, single alanine mutation is incorporated into each residue in molecule, and whether the mutating molecule that test obtains has cellobiohydrolase activity, to identify the amino-acid residue of the activity key for described molecule.See also Hilton etc., 1996, J.Biol.Chem.271:4699-4708.The reactive site of enzyme or other biological interaction also can by the physical analysiss to structure, sudden change in conjunction with the contact site amino acids for presumption is determined, structure is measured by these technology following: as nucleus magnetic resonance, crystallography, electron diffraction or photoaffinity labeling.See such as de Vos etc., 1992, Science255:306-312; Smith etc., 1992, J.Mol.Biol.224:899-904; Wlodaver etc., 1992, FEBS Lett.309:59-64.Also the identity of indispensable amino acid can be inferred from the identity analysis with related polypeptide.

Known mutagenesis, restructuring and/or Shuffling Method can be used, then carry out the screening process of being correlated with, as by Reidhaar-Olson and Sauer, 1988, Science241:53-57; Bowie and Sauer, 1989, Proc.Natl.Acad.Sci.USA86:2152-2156; WO 95/17413; Or those disclosed in WO 95/22625, carry out one or more aminoacid replacement, disappearance and/or insert and tested.Other spendable methods comprise fallibility PCR, phage display (such as Lowman etc., 1991, Biochemistry30:10832-10837; U.S. Patent number 5,223,409; WO 92/06204) and regiondirected mutagenesis (region-directed mutagenesis) (Derbyshire etc., 1986, Gene46:145; Deng, 1988, DNA7:127).

Mutagenesis/Shuffling Method can with high-throughput, auto-screening method combine with detect by host cell expression through cloning, the activity (Ness etc., 1999, Nature Biotechnology17:893-896) of the polypeptide of mutagenesis.The DNA molecular through mutagenesis of encode active polypeptides can reclaim from host cell and use this area standard method to check order rapidly.These methods allow the importance determining single amino acids residue in polypeptide fast.

Described polypeptide can be hybrid polypeptide, and the N of area merges in the region of another polypeptide of one of them polypeptide holds or C end.

Described polypeptide can be the fusion polypeptide that fusion polypeptide maybe can be cut, and wherein another peptide fusion is held or C end in the N of polypeptide of the present invention.Fusion polypeptide is produced by the polynucleotide of another polypeptide of coding are blended in polynucleotide of the present invention.It is known in the art for producing the technology of fusion polypeptide, and comprises the encoding sequence connecting coded polypeptide and meet frame (in frame) to make them, and makes the expression of fusion polypeptide under the control of identical promoters and terminator.Fusion rotein also can use interior albumen (intein) technique construction, and wherein (Cooper etc., 1993, EMBO J.12:2575-2583 fusions produces upon translation; Dawson etc., 1994, Science266:776-779).

Fusion polypeptide can also comprise cleavage site between two polypeptide.When secreting fusion polypeptide, described site is just cut open, and discharges described two polypeptide.The example cutting site includes, but not limited to be disclosed in Martin etc., 2003, J.Ind.Microbiol.Biotechnol.3:568-76; Svetina etc., 2000, J.Biotechnol.76:245-251; Rasmussen-Wilson etc., 1997, Appl.Environ.Microbiol.63:3488-3493; Ward etc., 1995, Biotechnology13:498-503; With Contreras etc., 1991, Biotechnology9:378-381; Eaton etc., 1986, Biochem.25:505-512); Collins-Racie etc., 1995, Biotechnology13:982-987; Carter etc., 1989, Proteins:Structure, Function, and Genetics6:240-248; And the site in Stevens, 2003, Drug Discovery World4:35-48.

There is the source of the polypeptide of cellobiohydrolase activity

The polypeptide with cellobiohydrolase activity of the present invention can obtain the microorganism from any genus.For the present invention, " obtain certainly " for term relevant with given source herein, the meaning should be and produced by described source by the polypeptide of polynucleotide encoding, or is produced by the bacterial strain wherein inserted from the polynucleotide in described source.In one aspect, the polypeptide obtained from given source is exocytosis.

Described polypeptide can be tungal polypeptide.In one aspect, described polypeptide is suede Thamnidium (Malbranchea) polypeptide.In yet another aspect, described polypeptide is camphor tree suede branch mould (Malbranchea cinnamomea) polypeptide.In yet another aspect, described polypeptide is that excellent softgel shell belongs to (Corynascus) polypeptide.In yet another aspect, described polypeptide is thermophilic excellent softgel shell (Corynascus thermophilus) polypeptide.

Will be understood that for aforesaid kind, the present invention comprises completely and the imperfect state (perfect and imperfect states), with other taxonomic equivalent (equivalent), such as anamorph (anamorph), and no matter their known kind names.Those skilled in the art will easily identify the identity of applicable equivalent.

The bacterial strain of these kinds can easily obtain for the public at many culture collection centers, described preservation center is American type culture collection (the American Type Culture Collection) (ATCC) such as, Mikroorganismen and Cell Culture Collection (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) (DSMZ), fungi strain preservation center (Centraalbureau Voor Schimmelcultures) (CBS) and research centre, agricultural research institute's Patent Culture Collection North (Agricultural Research Service Patent Culture Collection, Northern Regional Research Center) (NRRL).

Above-mentioned probe can be utilized to originate from other, comprise from nature (such as, soil, compost, water etc.) microorganism that is separated or the DNA sample directly obtained from nature material (such as, soil, compost, water etc.), identify and obtain described polypeptide.For directly from the technology of Natural habitat (habitat) separate microorganism and DNA be well known in the art.Subsequently by the DNA sample of the genomic dna or cDNA library or mixing that screen another kind of microorganism similarly to obtain the polynucleotide of coding said polypeptide.Once with the polynucleotide of probe in detecting to coded polypeptide, technology known to persons of ordinary skill in the art just can be used to be separated by described polynucleotide or clone (see, such as, Sambrook etc., 1989, see above).

Catalytic domain

In one embodiment, the present invention also relates to catalytic domain, and the amino acid/11 8 to 458 of itself and SEQ ID NO:2 has at least 70%, such as at least 72%, at least 75%, at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%; Relate to catalytic domain, the amino acid 21 to 450 of itself and SEQ ID NO:4 has at least 88%, such as at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%; Relate to catalytic domain, the amino acid 22 to 457 of itself and SEQ ID NO:6 has at least 66%, such as at least 68%, at least 70%, at least 75%, at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%; Relate to catalytic domain, the amino acid 21 to 461 of itself and SEQ ID NO:8 has at least 81%, such as at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%.In one aspect, described catalytic domain comprises aminoacid sequence, the amino acid/11 8 to 458 of described aminoacid sequence and SEQ ID NO:2, the amino acid 21 to 450 of SEQ ID NO:4, the amino acid 22 to 457 of SEQ ID NO:6, the amino acid 21 to 461 of SEQ ID NO:8 differs the most nearly 10, such as, and 1,2,3,4,5,6,7,8,9 or 10 amino acid.

Described catalytic domain preferably comprises or consists of the amino acid 21 to 461 of amino acid 22 to 457, SEQ ID NO:8 of amino acid 21 to 450, SEQ ID NO:6 of amino acid/11 8 to 458, SEQ ID NO:4 of SEQ ID NO:2; Or its allelic variant, or for it, there is the fragment of cellobiohydrolase activity.

In another embodiment, the present invention also relates to catalytic domain, it is by polynucleotide encoding, described polynucleotide are at medium stringency condition, medium-Gao stringent condition, high stringent condition, or very high stringent condition (as above define) under with following hybridization: the Nucleotide 52 to 1454 of (i) SEQ ID NO:1, the Nucleotide 61 to 1733 of SEQ ID NO:3, the Nucleotide 64 to 14782 of SEQ ID NO:5, or the Nucleotide 52 to 1460 of SEQ ID NO:7, (ii) its cDNA sequence, or the total length complement (Sambrook etc. of (iii) (i) or (ii), 1989, see above).

In another embodiment, the present invention also relates to catalytic domain, it is by polynucleotide encoding, the Nucleotide 52 to 1454 of described polynucleotide and SEQ ID NO:1, or its cDNA sequence has at least 70%, such as at least 72%, at least 75%, at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%.In another embodiment, the present invention also relates to catalytic domain, and it is by polynucleotide encoding, the Nucleotide 61 to 1733 of described polynucleotide and SEQ ID NO:3, or its cDNA sequence has at least 88%, such as at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%.In another embodiment, the present invention also relates to catalytic domain, it is by polynucleotide encoding, the Nucleotide 64 to 1782 of described polynucleotide and SEQ ID NO:5, or its cDNA sequence has at least 66%, such as at least 68%, at least 70%, at least 75%, at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%.In another embodiment, the present invention also relates to catalytic domain, and it is by polynucleotide encoding, the Nucleotide 52 to 1460 of described polynucleotide and SEQ ID NO:7, or its cDNA sequence has at least 81%, such as at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%.

The polynucleotide of described catalytic domain of encoding preferably comprise or consist of the Nucleotide 64 to 1782 of Nucleotide 61 to 1733, SEQ ID NO:5 of Nucleotide 52 to 1454, SEQ ID NO:3 of SEQ ID NO:1, or the Nucleotide 52 to 1460 of SEQ ID NO:7.

In another embodiment, the present invention also relates to the amino acid/11 8 to 458 of SEQ ID NO:2, the amino acid 21 to 450 of SEQ ID NO:4, the amino acid 21 to 461 of amino acid 22 to 457, the SEQ ID NO:8 of SEQ ID NO:6 comprises replacement, disappearance and/or inserts catalytic domain variant in one or more (such as several) position.In one aspect, introduce the amino acid/11 8 to 458 of SEQ ID NO:2, the amino acid 21 to 450 of SEQ ID NO:4, the amino acid 22 to 457 of SEQ ID NO:6, the aminoacid replacement of the sequence of the amino acid 21 to 461 of SEQ ID NO:8, disappearance and/or to insert number be as many as 10, such as 1,2,3,4,5,6,7,8,9 or 10.

Sugar binding domain

In one embodiment, the present invention also relates to sugared binding domain, and the amino acid 486 to 521 of itself and SEQ ID NO:8 has at least 81%, such as at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%.In one aspect, described sugared binding domain comprises aminoacid sequence, and described aminoacid sequence differs as many as 10 with the amino acid 486 to 521 of SEQ ID NO:8, such as, and 1,2,3,4,5,6,7,8,9 or 10 amino acid.

Described sugared binding domain preferably comprises or consists of the amino acid 486 to 521 of SEQ ID NO:8; Or its allelic variant, or for it, there is the fragment of Sugar-binding activitv.

In another embodiment, the present invention also relates to sugared binding domain, it is by polynucleotide encoding, described polynucleotide are at medium stringency condition, medium-Gao stringent condition, high stringent condition, or very high stringent condition (as above define) under with following hybridization: the Nucleotide 1533 to 1640 of (i) SEQ ID NO:7, (ii) its cDNA sequence, or the total length complement (Sambrook etc. of (iii) (i) or (ii), 1989, see above).

In another embodiment, the present invention also relates to sugared binding domain, and it is by polynucleotide encoding, the Nucleotide 1533 to 1640 of described polynucleotide and SEQ ID NO:7 has at least 81%, and such as at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%.

The polynucleotide of described sugared binding domain of encoding preferably comprise or consist of the Nucleotide 1533 to 1640 of SEQ ID NO:7.

In another embodiment, the amino acid 494 to 529 that the present invention also relates to SEQ ID NO:6 comprises replacement, disappearance and/or inserts catalytic domain variant in one or more (such as several) position.In one aspect, introducing the aminoacid replacement of the sequence of the amino acid 494 to 529 of SEQ ID NO:6, disappearance and/or inserting number is as many as 10, such as 1,2,3,4,5,6,7,8,9 or 10.

The catalytic domain be operatively connected and sugared binding domain can from lytic enzymes, isomerase, ligase enzyme, lyase, oxygen is enzyme or transferring enzyme also, such as aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, at, Maltose 4-glucosyltransferase, deoxyribonuclease, endoglucanase, esterase, alpha-galactosidase, beta-galactosidase enzymes, glucoamylase, alpha-glucosidase, beta-glucosidase enzyme, saccharase, laccase, lipase, mannosidase, become glycanase, oxydase, pectin decomposing enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic ferment, rnase, trans-glutaminases, zytase or xylobiase.The polypeptide of coding catalytic domain can obtain from any protokaryon, eucaryon or other source.

Polynucleotide

The present invention also relates to coding polypeptide of the present invention as described herein, catalytic domain, or the polynucleotide of the separation of sugared binding domain.

For separating of or the technology of clone's polynucleotide be well known in the art, and to comprise from genomic dna or cDNA, or its combination is separated.By such as using the antibody screening of polymerase chain reaction (PCR) or the expression library known to detect the cloned DNA fragments with apokoinou construction characteristic, thus realize from this genomic dna cloning polynucleotide.See, such as, Innis etc., 1990, PCR:A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification method can be used, as ligase chain reaction (LCR) (LCR), connect activated transcription (ligated activated transcription; LAT) with based on the amplification (NASBA) of polynucleotide.Can from the bacterial strain of Malbrancheaor Corynascus, or related organisms clones described polynucleotide, therefore, such as, can be allele variant or the variant (species variant) between planting of the polypeptid coding area of described polynucleotide.

The polynucleotide modifying code book invention polypeptide may be required for the synthesis polypeptide substantially similar to described polypeptide.Term refers to described polypeptide " substantially similar " form that the non-natural of polypeptide exists.These polypeptide may be different from from its natural origin isolated polypeptide in some engineered modes, such as, and the variant that the aspects such as specific activity, thermostability, optimal pH are different.Can in the mature polypeptide sequence as SEQ ID NO:1, the mature polypeptide sequence of SEQ ID NO:3, the mature polypeptide sequence of SEQ ID NO:5, or the mature polypeptide sequence of SEQ ID NO:7, or its cDNA sequence, or replace on the basis of polynucleotide that presents of its subsequence and/or by introducing following Nucleotide: described replacement does not cause the change of polypeptid acid sequence, but meets the codon usage being intended to the host organisms producing enzyme; Or build variant by importing the Nucleotide replacement that can produce different aminoacid sequences.About the general introduction that Nucleotide replaces, see, such as, Ford etc., 1991, Protein Expression and Purification2:95-107.

Nucleic acid construct

The invention still further relates to the nucleic acid construct comprising polynucleotide of the present invention, described polynucleotide are operably connected with one or more (such as several) regulating and controlling sequence, and described regulating and controlling sequence instructs the expression of encoding sequence in suitable host cell under the condition compatible with this regulating and controlling sequence.

Can with being permitted polynucleotide described in multi-mode operation so that the expression of polypeptide.Depend on expression vector, it may be desirable or required for operating on it before by polynucleotide insertion vector.The technology using recombinant DNA method to modify polynucleotide is well known in the art.

Regulating and controlling sequence can be promotor, its polynucleotide identified by the host cell of the polynucleotide for expressing polypeptide of the present invention of encoding.Promotor contains the transcription regulating nucleotide sequence of the expression of direct polypeptide.Promotor can be any polynucleotide showing transcriptional activity in host cell, comprise sudden change, brachymemma with the promotor of heterozygosis, and can to obtain by the gene of polypeptide from the born of the same parents of coding and host cell homology or allos or in born of the same parents.

The example of the suitable promoter of transcribing for instructing nucleic acid construct of the present invention in bacterial host cell is from the following promotor obtained: bacillus amyloliquefaciens alpha-amylase gene (amyQ), bacillus licheniformis alpha-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), bacstearothermophilus produces maltogenic amylase gene (amyM), subtilis levansucrase gene (sacB), subtilis xylA and xylB gene, bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994, Molecular Microbiology13:97-107), E. coli lac operon, intestinal bacteria trc promotor (Egon etc., 1988, Gene69:301-315), streptomyces coelicolor agarase gene (dagA) and protokaryon β-lactamase gene (Villa-Kamaroff etc., 1978, Proceedings of the National Academy of Sciences USA75:3727-3731), and tac promotor (DeBoer etc., 1983, Proc.Natl.Acad.Sci.USA80:21-25).In Gilbert etc., 1980, Scientific American, in 242:74-94 at " Useful proteins from recombinant bacteria " for other promotor; With at Sambrook etc., 1989, see above middle description.The example of Gene expression is disclosed in WO 99/43835.

Being used to guide nucleic acid construct of the present invention is the promotor obtained from the gene of following enzyme at the example of the suitable promoter of filamentous fungal host cell transcription: Aspergillus nidulans acetamidase, Aspergillus ni ger neutral α-amylase, Aspergillus niger acid stable α-amylase, aspergillus niger or Aspergillus awamori amylase (glaA), oryzae TAKA amylase, line protease, aspergillus oryzae triose-phosphate isomerase, point sickle spore trypsin like proteases (WO 96/00787), empiecement sickle spore amyloglucosidase (WO 00/56900), empiecement sickle spore Daria (WO 00/56900), empiecement sickle spore Quinn (WO 00/56900), Man Hegen Mucor (Rhizomucor miehei) lipase, Man Hegen Mucor aspartate protease, Trichodermareesei beta-glucosidase enzyme, Trichodermareesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, trichoderma reesei endoglucanase I, trichoderma reesei endoglucanase II, trichoderma reesei endoglucanase III, trichoderma reesei endoglucanase V, Xylanase from Trichoderma reesei I, Xylanase from Trichoderma reesei II, Xylanase from Trichoderma reesei III, Trichodermareesei xylobiase, Trichodermareesei translation elongation factor, and NA2-tpi promotor (a kind of promotor of modification, its come comfortable Aspergillus neutral alpha-amylase gene, wherein untranslated leader sequence by the gene of Aspergillus triose-phosphate isomerase untranslated leader sequence substitute, limiting examples comprises the promotor of modification, and it is from the gene of Aspergillus ni ger neutral α-amylase, wherein untranslated leader sequence by the gene of Aspergillus nidulans or aspergillus oryzae triose-phosphate isomerase untranslated leader sequence substitute), with their sudden change, brachymemma with the promotor of heterozygosis.Other promotor is described in U.S. Patent number 6, and 011,147.

In yeast host, useful promotor obtains from following gene: yeast saccharomyces cerevisiae Hydratase, phosphoenolpyruvate (ENO-1), yeast saccharomyces cerevisiae galactokinase (GAL1), yeast saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP), yeast saccharomyces cerevisiae triose-phosphate isomerase (TPI), brewing yeast metallothionein (CUP1) and yeast saccharomyces cerevisiae glycerol 3-phosphate acid kinase.For other useful promotor of yeast host cell by Romanos etc., 1992, Yeast8:423-488 describes.

Regulating and controlling sequence also can be transcription terminator, and it is transcribed with termination by host cell identification.Described terminator is operably connected with 3 ' end of the polynucleotide of coding said polypeptide.In the present invention, any terminator having function can be used in host cell.

The preferred terminator of bacterial host cell is obtained from following gene: Bacillus clausii Sumizyme MP (aprH), bacillus licheniformis alpha-amylase (amyL) and intestinal bacteria ribosome-RNA(rRNA) (rrnB).

The preferred terminator of filamentous fungal host cell is obtained from the gene of following enzyme: Aspergillus nidulans acetamidase, Aspergillus nidulans anthranilate synthase, aspergillus niger glucoamylase, aspergillus niger alpha-glucosidase, oryzae TAKA amylase, point sickle spore trypsin like proteases, Trichodermareesei beta-glucosidase enzyme, Trichodermareesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, trichoderma reesei endoglucanase I, trichoderma reesei endoglucanase II, trichoderma reesei endoglucanase III, trichoderma reesei endoglucanase V, Xylanase from Trichoderma reesei I, Xylanase from Trichoderma reesei II, Xylanase from Trichoderma reesei III, Trichodermareesei xylobiase and Trichodermareesei translation elongation factor.

The preferred terminator of yeast host cell is obtained from the gene of following enzyme: yeast saccharomyces cerevisiae Hydratase, phosphoenolpyruvate, S. cerevisiae cytochrome C (CYC1) and S. cerevisiae glyceraldehyde-3-phosphate dehydrogenase.For other useful terminator of yeast host cell by Romanos etc., 1992, see above description.

Regulating and controlling sequence can also be the mRNA stabilization district of the encoding sequence upstream of promotor downstream and gene, and it increases the expression of described gene.

The example in suitable mRNA stabilization district obtains from following gene: bacillus thuringiensis cryIIIA gene (WO 94/25612) and subtilis SP82 gene (Hue etc., 1995, Journal of Bacteriology177:3465-3471).

Regulating and controlling sequence can also be suitable leader sequence, and it is for the important mRNA non-translational region of the translation of host cell.Leader sequence is operably connected to 5 '-end of the polynucleotide of coded polypeptide.Any leader sequence having function can be used in host cell.

The preferred leader sequence of filamentous fungal host cell is obtained from the gene of following enzyme: oryzae TAKA amylase and Aspergillus nidulans triose-phosphate isomerase.

The leader sequence suitable for yeast host cell obtains from the gene of following enzyme: yeast saccharomyces cerevisiae Hydratase, phosphoenolpyruvate (ENO-1), yeast saccharomyces cerevisiae glycerol 3-phosphate acid kinase, cerevisiae alpha-factor and yeast saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

Regulating and controlling sequence also can be polyadenylation se-quence, and it is the sequence be operably connected with 3 ' end of polynucleotide, and when transcribing, host cell is identified as the signal poly-adenosine residue being added into the mRNA transcribed.Any polyadenylation se-quence having function can be used in host cell.

The preferred polyadenylation se-quence of filamentous fungal host cell is obtained from the gene of following enzyme: Aspergillus nidulans anthranilate synthase, aspergillus niger glucoamylase, aspergillus niger alpha-glucosidase, oryzae TAKA amylase and sharp sickle spore trypsin like proteases.

For the useful polyadenylation se-quence of yeast host cell by Guo and Sherman, 1995, Mol.Cellular Biol.15:5983-5990 describes.

Regulating and controlling sequence can also be signal peptide coding region, and its coding holds with the N of polypeptide the signal peptide be connected, and and guides described polypeptide to enter emiocytosis approach.Encoding sequence 5 ' the end of polynucleotide can comprise signal coding sequence inherently, and it is connected to natively and translates in reading frame together with the section of the encoding sequence of coding said polypeptide.Or encoding sequence 5 ' is held can containing the signal coding sequence for described encoding sequence external source.When encoding sequence natural containing signal coding sequence time, exogenous signals peptide-coding sequence may be required.Or, directly can replace natural signals peptide-coding sequence to strengthen the secretion of polypeptide with exogenous signals peptide-coding sequence.But, any signal coding sequence of the Secretory Pathway of host cell can be entered by the instruction polypeptide of expressing.

The signal coding sequence obtained from the gene of following enzyme for the effective signal coding sequence of bacterial host cell: bacillus NCIB11837 produces maltogenic amylase, Bacillus licheniformis subtilisin (subtilisin), Bacillus licheniformis β-lactamase, bacillus stearothermophilus alpha-amylase, stearothermophilus neutral proteolytic enzyme (nprT, nprS, nprM) and subtilis prsA.Other signal peptide is by Simonen and Palva, and 1993, Microbiological Reviews57:109-137 describes.

The signal coding sequence obtained from the gene of following enzyme for the effective signal coding sequence of filamentous fungal host cell: Aspergillus ni ger neutral amylase, aspergillus niger glucoamylase, oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens EGV, dredge cotton like humicola lanuginosa lipase and Man Hegen Mucor aspartate protease.

The signal peptide useful for yeast host cell obtains from the gene of cerevisiae alpha-factor and Saccharomyces cerevisiae invertase.Other useful signal coding sequence by Romanos etc., 1992, see above description.

Regulating and controlling sequence can also be propeptide code sequence, and its coding is positioned at the propetide that polypeptide N holds.Gained polypeptide is called proenzyme (proenzyme) or front polypeptide (propolypeptide) (or being called proenzyme (zymogen) in some cases).Front polypeptide normally non-activity, and can by the catalysis of propetide or autocatalysis cutting in the past polypeptide be converted into active polypeptide.Propeptide code sequence can be obtained from the gene of bacillus subtilis alkali proteinase (aprE), Bacillus subtilis neutral proteolytic enzyme (nprT), Myceliophthora thermophila laccase (WO 95/33836), Man Hegen Mucor aspartate protease and cerevisiae alpha-factor.

When both signal peptide and propeptide sequence all exist, propeptide sequence is placed in and then the N end of (next to) polypeptide, and signal peptide sequence is placed in the N end of and then propeptide sequence.

Regulate sequence it is also desirable that add, it regulates the expression of polypeptide relative to the growth of host cell.Regulate the example of sequence to be cause genetic expression to respond chemistry or physical stimulation thing, comprise and regulate the existence of compound and those systems of opening or closing.Adjustment sequence in prokaryotic system comprises lac, tac and trp operator gene system.In yeast, ADH2 system or GAL1 system can be used.In filamentous fungus, aspergillus niger glucoamylase promotor, aspergillus oryzae TAKA α-amylase promotor and aspergillus oryzae glucoamylase promotor, Trichodermareesei cellobiohydrolase I promotor and Trichodermareesei cellobiohydrolase II promotor can be used.Other example of sequence is regulated to be that those allow the sequence of gene amplification.In eukaryotic system, these regulate sequence to be included in the dihydrofolate reductase gene of the lower amplification of methotrexate (methotrexate) existence, and with the metallothionein gene that heavy metal (with heavy metal) increases.In these cases, the polynucleotide of coded polypeptide will be operably connected with adjustment sequence.

Expression vector

The invention still further relates to recombinant expression vector, described recombinant expression vector comprises polynucleotide of the present invention, promotor and transcribes and translation termination signal.Multiple Nucleotide and regulating and controlling sequence can combine to produce recombinant expression vector, described expression vector can comprise one or more (such as several) easily restriction site to allow insert in these sites or replace the polynucleotide of coded polypeptide.Or, can by inserting the nucleic acid construct that comprises described polynucleotide or polynucleotide express described polynucleotide in the suitable carrier for expressing.In the process preparing expression vector, encoding sequence is placed in carrier, thus this encoding sequence is operably connected for expression with suitable regulating and controlling sequence.

Recombinant expression vector can be anyly can carry out recombinant DNA step easily, and can produce the carrier (such as, plasmid or virus) of the expression of polynucleotide.The selection of carrier will usually depend on carrier and will introduce the consistency of the host cell of this carrier.Carrier can be wire or closed hoop plasmid.

Carrier can be autonomously replicationg vector, that is, as the carrier that extrachromosomal entity (entity) exists, it copies independent of chromosome duplication, such as, plasmid, extra-chromosomal element, minichromosome (minichromosome) or artificial chromosome.Carrier can contain any means (means) for guaranteeing self-replacation.Or carrier can be a kind of when being introduced in host cell, to be incorporated in genome and the carrier copied together with the karyomit(e) incorporating this carrier.In addition, can use independent carrier or plasmid or two or more carriers or plasmid, its global DNA (total DNA) jointly containing host cell gene group to be introduced, maybe can use transposon (transposon).

Described carrier preferably contains one or more (such as several) selected marker, so that easily select the cell through conversion, transfection, transduction etc.Selected marker is such gene, its product provide biocide or virus resistance, heavy metal resistance, to auxotrophic prototrophy (prototrophy to auxotrophs) etc.

The example of bacterial selectable marker is Bacillus licheniformis or subtilis dal gene, or gives the mark of antibiotics resistance, and described antibiotics resistance is penbritin, paraxin, kantlex, Liu Suanyan NEOMYCIN SULPHATE, spectinomycin or tetracyclin resistance such as.The mark suitable for yeast host cell includes but not limited to ADE2, HIS3, LEU2, LYS2, MET3, TRP1 and URA3.Selected marker for filamentous fungal host cell includes but not limited to adeA (ribose phosphoric acid aminooimidazole amber carboxylic acid amides synthase, phosphoribosylaminoimidazole-succinocarboxamide synthase), adeB (ribose phosphoric acid aminooimidazole synthase, phosphoribosyl-aminoimidazole synthase), amdS (acetamidase), argB (ornithine transcarbamylase), bar (careless ammonium phosphine (phosphinothricin) Transacetylase), hph (hygromix phosphotransferase), niaD (nitrate reductase) (nitrate reductase), pyrG (Orotidine-5 '-'-phosphate decarboxylase) (orotidine-5 '-phosphate decarboxylase), sC (sulfate adenylyl transferase) and trpC (anthranilate synthase (anthranilate synthase)) and their equivalent.Preferably be used in Aspergillus cell is Aspergillus nidulans or aspergillus oryzae amdS and pyrG gene and streptomyces hygroscopicus (Streptomyces hygroscopicus) bar gene.What be preferred for Trichoderma cell is adeA, adeB, amdS, hph and pyrG gene.

Selected marker can be the double selectivity Mk system described in WO 2010/039889.In one aspect, described double selectivity mark is hph-tk double selectivity Mk system.

Described carrier is preferably containing allowing vector integration to enter host cell gene group or carrier in cell independent of the element of genomic self-replicating.

In order to be integrated into host cell gene group, the sequence of the polynucleotide of the responsible coded polypeptide of carrier or for being entered genomic other carrier element any by homology or non-homologous re-combination.Or carrier can containing being used to guide the extra polynucleotide being integrated into the exact position in host cell gene group chromosome by homologous recombination.In order to be increased in the possibility that exact position is integrated, integrated element should have the nucleic acid of high degree of sequence identity, as 100 to 10 containing sufficient amount with corresponding target sequence, 000 base pair, 400 to 10,000 base pair, with 800 to 10,000 base pair, to improve the probability of homologous recombination.Integrated element can be sequence that is any and the target sequence homology in host cell gene group.In addition, integrated element can be the polynucleotide of non-coding or coding.On the other hand, can by carrier by non-homologous re-combination in the genome of host cell.

In order to self-replicating, carrier also can comprise replication orgin, and it enables carrier independently copy in described host cell.Replication orgin can be any plasmid replicon (replicator) of the mediation self-replicating playing function in cell.Term " replication orgin " or " plasmid replicon " mean the polynucleotide that can make to copy in plasmid or carrier body.

The example of bacterial origin of replication be allow to copy in intestinal bacteria pBR322 plasmid, pUC19, pACYC177 and pACYC184 replication orgin, and allow the replication orgin of plasmid pUB110, pE194, pTA1060 and pAM β 1 copied in bacillus.

Example for the replication orgin in yeast host cell is the combination of 2 micron origin of replication, ARS1, ARS4, ARS1 and CEN3, and the combination of ARS4 and CEN6.

The example of replication orgin useful in filamentous fungal cells is AMA1 and ANS1 (Gems etc., 1991, Gene98:61-67; Cullen etc., 1987, Nucleic Acids Res.15:9163-9175; WO 00/24883).Separation of AM A1 gene and build and comprise the plasmid of this gene or carrier can complete according to the method be disclosed in WO 00/24883.

Can by the polynucleotide Insertion Into Host Cell of the present invention of more than one copy to increase the generation of polypeptide.The increase of polynucleotide copies number obtains by following method: the sequence of at least one additional copy is integrated into host cell gene group, or the selected marker that can increase is included in polynucleotide, wherein select the amplification containing selected marker to copy by culturing cell under the existence of suitable selective agent (selectable agent), and contain the cell of the additional copy of polynucleotide thus.

For connect said elements with build the method for recombinant expression vector of the present invention be well known to those skilled in the art (see, such as, Sambrook etc., 1989, see above).

Host cell

The invention still further relates to recombinant host cell, it comprises polynucleotide of the present invention and is operably connected to the regulating and controlling sequence that one or more (such as several) instruct the generation of polypeptide of the present invention.The construct or carrier that comprise polynucleotide are introduced host cell, described construct or carrier are maintained as chromosomal integrant or as the outer carrier of karyomit(e) of self-replacation as previously mentioned.Term " host cell " comprises any offspring being different from parental cell due to the sudden change occurred in reproduction process of parental cell.The selection of host cell will depend on gene and the source thereof of coded polypeptide to a great extent.

Host cell can be any cell useful in the restructuring of polypeptide of the present invention produces, such as, and protokaryon or eukaryotic cell.

Prokaryotic host cell can be any Gram-positive or gram negative bacterium.Gram positive bacterium includes but not limited to, bacillus, fusobacterium, enterococcus spp, ground bacillus genus, lactobacillus, lactococcus, bacillus marinus genus, Staphylococcus, streptococcus and streptomyces.Gram negative bacterium includes but not limited to, campylobacter, intestinal bacteria, Flavobacterium, Fusobacterium, Helicobacterium, mud Bacillaceae, eisseria, Rhodopseudomonas, salmonella and Ureaplasma.

Bacterial host cell can be any bacillus cell, includes but not limited to Alkaliphilic bacillus, bacillus amyloliquefaciens, bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, bacillus firmus, bacillus lautus, bacillus lentus, Bacillus licheniformis, bacillus megaterium, bacillus pumilus, bacstearothermophilus, subtilis and Bacillus thuringiensis cell.

Bacterial host cell can also be any streptococcus cell, includes but not limited to, streptococcus equisimilis, streptococcus pyogenes, streptococcus uberis and zooepidemicus cell.

Bacterial host cell can also be any Streptomyces cell, includes but not limited to, not streptomyces chromogenes, deinsectization streptomycete, streptomyces coelicolor, streptomyces griseus and shallow Streptomyces glaucoviolaceus cell.

Realize DNA to be incorporated into bacillus cell by following method: protoplast transformation (see, such as, Chang and Cohen, 1979, Mol.Gen.Genet.168:111-115), competent cell transforms (see, such as, Young and Spizizen, 1961, J.Bacteriol.81:823-829 or Dubnau and Davidoff-Abelson, 1971, J.Mol.Biol.56:209-221), electroporation (see, such as, Shigekawa and Dower, 1988, Biotechniques6:742-751) or engage (see, such as, Koehler and Thorne, 1987, J.Bacteriol.169:5771-5278).Realize DNA to be incorporated into Bacillus coli cells by following method: protoplast transformation (see, such as, Hanahan, 1983, J.Mol.Biol.166:557-580) or electroporation (see, such as, Dower etc., 1988, Nucleic Acids Res.16:6127-6145).Realize DNA to be incorporated into Streptomyces cell by following method: protoplast transformation, electroporation (see, such as, Gong etc., 2004, Folia Microbiol. (Praha) 49:399-405), engage (see, such as, Mazodier etc., 1989, J.Bacteriol.171:3583-3585), or transduction (see, such as, Burke etc., 2001, Proc.Natl.Acad.Sci.USA98:6289-6294).Realize DNA to be incorporated into Rhodopseudomonas cell by following method: electroporation (see, such as, Choi etc., 2006, J.Microbiol.Methods64:391-397) or engage (see, such as, Pinedo and Smets, 2005, Appl.Environ.Microbiol.71:51-57).Realize DNA to be incorporated into streptococcus cell by following method: natural competence (natural competence) (see, such as, Perry and Kuramitsu, 1981, Infect.Immun.32:1295-1297), protoplast transformation (see, such as, Catt and Jollick, 1991, Microbios.68:189-207), electroporation (see, such as, Buckley etc., 1999, Appl.Environ.Microbiol.65:3800-3804) or engage (see, such as, Clewell, 1981, Microbiol.Rev.45:409-436).But, any method DNA being introduced host cell known in the art can be used.

Host cell also can be eukaryote, as Mammals, insect, plant or fungal cell.

Host cell can be fungal cell." fungi " is used in and comprises with Xiamen herein: Ascomycota (Ascomycota), Basidiomycota (Basidiomycota), chytrid door (Chytridiomycota) and Zygomycota (Zygomycota) and oomycetes door (Oomycota) and all mitosporic fungi (mitosporic fungi) are (as by Hawksworth etc., in Ainsworth and Bisby ' s Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, defined in Cambridge, UK).

Fungal host cells can be yeast cell." yeast " is used in the yeast comprising ascosporogenous yeast (ascosporogenous yeast) (Endomycetale (Endomycetales)), product load yeast (basidiosporogenous yeast) herein and belong to imperfect fungi (Fungi Imperfecti) (gemma guiding principle (Blastomycetes)).Because the future that is sorted in of yeast may change, for the present invention, yeast is defined as Biology and Activities of Yeast (Skinner, Passmore, compile with Davenport, Soc.App.Bacteriol.Symposium Series No.9,1980) described in.

Yeast host cell can be mycocandida, Hansenula (Hansenula), genus kluyveromyces, Pichia, yeast belong, Schizosaccharomyces or the mould genus cell of Western alpine yarrow, as Kluyveromyces lactis (Kluyveromyces lactis), saccharomyces carlsbergensis, yeast saccharomyces cerevisiae, saccharomyces diastaticus, Doug Laplace yeast, Saccharomyces kluyveri, promise ground yeast, ellipsoideus yeast or solution fat the West alpine yarrow mould (Yarrowia lipolytica) cell.

Fungal host cells can be filamentous fungal cells." filamentous fungus " comprises all filamentous form of the subphylum (as by Hawksworth etc., 1995, see above, defined) of Mycophyta (Eumycota) and oomycetes door.Filamentous fungus is common is characterised in that the mycelia body wall be made up of chitin (chitin), Mierocrystalline cellulose, dextran, chitosan (chitosan), mannosans and other complicated polysaccharide.Extend into row by mycelia to nourish and grow, and carbon katabolism is obligate aerobic.On the contrary, nourishing and growing of yeast such as yeast saccharomyces cerevisiae is undertaken by the gemmation (budding) of unicellular thallus, and carbon katabolism can be fermentable.

Filamentous fungal host cell can be the mould genus of branch top spore, Aspergillus, aureobasidium genus, the mould genus of smoke pipe (Bjerkandera), intend wax Pseudomonas, Chrysosporium, Coprinus (Coprinus), Coriolus Qu61 (Coriolus), genera cryptococcus, Filibasidium, fusarium, Humicola, Magnaporthe grisea belongs to, Mucor, myceliophthora, the mould genus of Xin Kaoma fat, Neurospora, paecilomyces, Penicillium, flat lead fungi belongs to (Phanerochaete), penetrate arteries and veins Pseudomonas (Phlebia), cud Chytridium, pleurotus (Pleurotus), Schizophyllum, Talaromyces, thermophilic ascomycete belongs to, the mould genus of shuttle spore, Tolypocladium, trametes (Trametes) or Trichoderma cell.

Such as, filamentous fungal host cell can be Aspergillus awamori, Aspergillus fumigatus, smelly aspergillus, aspergillus japonicus, Aspergillus nidulans, aspergillus niger, aspergillus oryzae, black thorn smoke pipe bacterium (Bjerkandera adusta), dry plan wax bacterium (Ceriporiopsis aneirina), Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, worm intends wax bacterium (Ceriporiopsis subvermispora), Chrysosporium inops, chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, felt gold pityrosporion ovale, Chrysosporium queenslandicum, chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus (Coprinus cinereus), hairy fungus (Coriolus hirsutus), bar spore shape sickle spore, F.graminearum schw, storehouse prestige sickle spore, machete sickle spore, fusarium graminaria, the red sickle spore of standing grain, different spore sickle spore, albizzia sickle spore, point sickle spore, racemosus sickle spore, pink sickle spore, Williams Elder Twig sickle spore, colour of skin sickle spore, intend branch spore sickle spore, sulphur look sickle spore, circle sickle spore, intend silk spore sickle spore, empiecement sickle spore, Humicola insolens, dredge cotton like humicola lanuginosa, rice black wool is mould, thermophilic fungus destroyed wire, Neuraspora crassa, penicillium purpurogenum, the yellow flat lead fungi of spore (Phanerochaete chrysosporium), arteries and veins bacterium (Phlebia radiata) is penetrated in radiation, pleurotus eryngii (Pleurotus eryngii), autochthonal shuttle spore is mould, long wool Trametes trogii (Trametes villosa), Trametes versicolor (Trametes versicolor), trichoderma harziarum, healthy and free from worry wood is mould, long shoot wood is mould, Trichodermareesei or Trichoderma viride cell.

Can by relating to, protoplastis be formed, the method for protoplast transformation and cell wall-deficient mutant transforms in a per se known way by fungal cell.For transforming the appropriate method of Aspergillus and Trichoderma host cell at EP238023 and Yelton etc., 1984, Proc.Natl.Acad.Sci.USA81:1470-1474 and Christensen etc., describe in 1988, Bio/Technology6:1419-1422.For the appropriate method of transforming Fusarium bacterial classification by Malardier etc., 1989, Gene78:147-156 and WO 96/00787 describes.Can use by the method transformed yeast of following document description: Becker and Guarente, in Abelson, J.N. and Simon, M.I. compile, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume194, pp182-187, Academic Press, Inc., New York; Ito etc., 1983, J.Bacteriol.153:163; With Hinnen etc., 1978, Proc.Natl.Acad.Sci.USA75:1920.

Production method

The invention still further relates to the method for generation of polypeptide of the present invention, it comprises: (a) is contributing to culturing cell under the condition producing polypeptide, and described cell produces described polypeptide with its wild-type form; Optionally (b) reclaims described polypeptide.In one aspect, described cell is suede Thamnidium cell.In yet another aspect, described cell is the mould cell of camphor tree suede branch.In yet another aspect, described cell is that excellent softgel shell belongs to cell.In yet another aspect, described cell is thermophilic excellent softgel shell cell.In yet another aspect, described cell is thermophilic excellent softgel shell NN000308 cell.

The invention still further relates to the method for generation of polypeptide of the present invention, it comprises: (a) is contributing to cultivating recombinant host cell of the present invention under the condition producing polypeptide; Optionally (b) reclaims described polypeptide.

Described host cell uses methods known in the art to cultivate in the nutritional medium being suitable for producing described polypeptide.Such as; can by suitable culture medium and allow expression and/or be separated described polypeptide condition under shake-flask culture, or small-scale in laboratory or industrial fermentation tank or large scale fermentation (comprise continuously, in batches, fed-batch or solid state fermentation) carry out culturing cell.Use methods known in the art to cultivate in suitable nutritional medium, described nutritional medium comprises Carbon and nitrogen sources and inorganic salt.Suitable substratum can obtain from commercial supplier or can according to disclosed composition preparation (such as, in the catalogue of American type culture collection).If polypeptide is secreted in nutritional medium, this polypeptide directly can reclaim from described substratum.If polypeptide is not secreted, it can reclaim from cell lysate (lysate).

The method for described polypeptid specificity known in the art can be used to detect polypeptide.These detection methods include but not limited to the use of specific antibody, the formation of enzyme product or the disappearance of enzyme substrates.Such as, enzyme assay (enzyme assay) can be used for the activity determining polypeptide.

Polypeptide can use methods known in the art to reclaim.Such as, polypeptide can be reclaimed from nutritional medium by ordinary method, described ordinary method includes but not limited to collect, centrifugal, filtration, extraction, spraying dry, evaporation or precipitation.In one aspect, the whole beer comprising polypeptide has been reclaimed.

Polypeptide can by multiple methods known in the art purifying to obtain substantially pure polypeptide, described method includes but not limited to that chromatography (such as, ion-exchange, affine, hydrophobic, chromatofocusing and size exclusion), electrophoresis method (such as, preparative (preparative) isoelectrofocusing), differential solubility (such as, ammonium sulfate precipitation), SDS-PAGE or extraction (see, such as, Protein Purification, Janson and Ryden compiles, VCH Publishers, New York, 1989).

In yet another aspect, do not reclaim polypeptide, but use the host cell of the present invention of expressing described polypeptide as the source of described polypeptide.

Plant

The invention still further relates to the plant of separation, such as, transgenic plant, plant part or vegetable cell, it comprises polynucleotide of the present invention, thus reaches with callable scale and produce described polypeptide or territory.Polypeptide or territory can be reclaimed from plant or plant part.Or, can in statu quo (as such) by the plant containing this polypeptide or territory or plant part for improvement of food or quality of the fodder, such as, improve nutritive value, palatability (palatability) and rheological property (rheological properties), or for destroying antinutritional factor.

Transgenic plant can be dicots (dicotyledonss) or monocotyledonous (monocotyledons).Monocotyledonous example is grass (grasses), as English grass (meadow grass) (bluegrass (blue grass), Poa L. (Poa)); Forage grass (forage grass) is as festuca (Festuca), lolium (Lolium); Cold ground type herbage (temperate grass), as Agrostis (Bentgrass); And cereal, such as, wheat, oat, rye, barley, rice (rice), Chinese sorghum and Zea mays (maize) (corn).

The example of dicotyledons is tobacco (tobacco), beans (legumes), as lupine (lupins), potato, sugar beet (sugar beet), pea, (cruciferous) plant (Cruciferae (family Brassicaceae)) of beans (bean) and soybean (soybean) and Cruciferae, as Cauliflower (cauliflower), Semen Brassicae campestris (rape seed) and the model organism Arabidopis thaliana (Arabidopsis thaliana) be closely related.

The example of plant part is stem (stem), callus (callus), leaf (leaf), root (root), fruit (fruit), seed (seed) and stem tuber (tuber), and comprise the independent body of these parts, such as, epidermis (epidermis), mesophyll (mesophyll), parenchyma (parenchyme), vascular tissue (vascular tissue), meristematic tissue (meristem).Concrete palnt cell compartments (compartments), as chloroplast(id) (chloroplast), apoplast (apoplast), plastosome (mitochondria), vacuole (vacuole), peroxysome (peroxisome) and tenuigenin (cytoplasm) are also considered to plant part.In addition, any vegetable cell, whatsoever tissue-derived, be all considered to plant part.Similarly, plant part, be used for as separated promoting that the concrete tissue of application of the present invention and cell are also considered to plant part, such as embryo (embryo), endosperm (endosperm), aleuron (aleurone) and seed coat (seed coat).

Be contained in the offspring also having these plants, plant part and vegetable cell in the scope of the invention equally.

The transgenic plant in express polypeptide or territory or vegetable cell can build according to means known in the art.In brief, build described plant or vegetable cell by the following method: one or more expression construct in coded polypeptide or territory are imported plant host genome or Chloroplast gene, and be transgenic plant or vegetable cell by the modified plant of gained or vegetable cell breeding.

Expression construct is the nucleic acid construct of the polynucleotide comprising coded polypeptide or territory expediently, and described polynucleotide are operably connected with the suitable adjustment sequence expressed in the plant selected or plant part needed for these polynucleotide.In addition, expression construct can comprise for the useful selected marker of plant identification cell, incorporates expression construct and this construct is incorporated into necessary DNA sequence dna in described plant (the latter depends on the DNA introducing method of use) in described vegetable cell.

Regulate the selection of sequence, the selection of such as promotor and terminator sequence and optionally signal or transit sequence, for example, based on when expecting, where and how express polypeptide or territory and determine.Such as, the expression of the gene in coded polypeptide or territory can be composing type or induction type, can be maybe grow, the stage or tissue-specific, and gene product can target specifically organize or plant part as seed or leaf.Regulate sequence by such as Tague etc., described in 1988, Plant Physiology86:506.

For constructive expression, 35S-CaMV, maize ubiquitin 1 or rice Actin muscle 1 promotor (Franck etc., 1980, Cell21:285-294, Christensen etc., 1992, Plant Mo.Biol.18:675-689 can be used; Zhang etc., 1991, Plant Cell3:1155-1165).Organ specific promoters can be such as from storage tissue (storage sink tissue) such as seed, promotor (Edwards and Coruzzi of potato tuber and fruit, 1990, Ann.Rev.Genet.24:275-303), or from metabolic pool tissue (metabolic sink tissue) such as merismatic promotor (Ito etc., 1994, Plant Mol.Biol.24:863-878), seed specific promoters is such as from the gluten (glutelin) of rice, prolamine (prolamin), sphaeroprotein (globulin) or albumin (albumin) promotor (Wu etc., 1998, Plant Cell Physiol.39:885-889), from the broad bean promotor (Conrad etc. of the Seed Storage Protein gene of the unknown of legumin (legumin) B4 and broad bean (Vicia faba), 1998, J.Plant Physiol.152:708-711), from the promotor (Chen etc. of seed oil bodies albumen (oil body protein), 1998, Plant Cell Physiol.39:935-941), from the storage protein napA promotor of colea (Brassica napus), or the promotor of any other seed-specific well-known in the art, such as, described in WO 91/14772.In addition, promotor can be the specific promotor of leaf, as the rbcs promotor (Kyozuka etc. from rice or tomato, 1993, Plant Physiol.102:991-1000), chlorella virus (chlorella virus) adenine methyltransferase (adenine methyltransferase) gene promoter (Mitra and Higgins, 1994, Plant Mol.Biol.26:85-93), from the aldP gene promoter (Kagaya etc. of rice, 1995, Mol.Gen.Genet.248:668-674), or the promotor of wound induction, as potato pin2 promotor (Xu etc., 1993, Plant Mol.Biol.22:573-588).Similarly, described promotor is induced by abiotic process, described abiotic process such as temperature, arid or salinity altercation, or the material induction of the described promotor of activation to be applied by external source, such as ethanol, oestrogenic hormon (oestrogens), plant hormone (plant hormones) are as ethene, dormin (abscisic acid) and gibberic acid (gibberellic acid), and heavy metal.

Promotor enhancer element also may be used for realizing polypeptide or the territory comparatively high expression level in plant.Such as, promotor enhancer element can be intron, and it is placed between the polynucleotide in promotor and coded polypeptide or territory.Such as Xu etc., 1993, on seeing, disclose and use the First Intron of rice Actin muscle 1 gene with Enhanced expressing.

Any other parts of selected marker and expression construct can be selected from available those in this area.

Nucleic acid construct is imported Plant Genome according to routine techniques known in the art, described routine techniques comprises the conversion that Agrobacterium (Agrobacterium) mediates, virus-mediated conversion, microinjection (microinjection), particle bombardment, Biolistic transformation and electroporation (Gasser etc., 1990, Science244:1293; Potrykus, 1990, Bio/Technology8:535; Shimamoto etc., 1989, Nature338:274).

The transgenosis (gene transfer) that Agrobacterium tumefaciens (Agrobacterium tumefaciens) mediate, it is a kind of generation transgenic dicots (its summary, see Hooykas and Schilperoort, 1992, Plant Mol.Biol.19:15-38), with the method for transforming monocots, although other method for transformation can be used for these plants.A kind of method producing transgenic monocot plant is with particle (gold or tungsten particle with the microcosmic of transfering DNA coating) bombardment embryo callus (embryonic calli) or developmental embryo (developing embryos) (Christou, 1992, Plant J.2:275-281; Shimamoto, 1994, Curr.Opin.Biotechnol.5:158-162; Vasil etc., 1992, Bio/Technology10:667-674).A kind of alternative method of transforming monocots is based on protoplast transformation, as by Omirulleh etc., described by 1993, Plant Mol.Biol.21:415-428.Other method for transformation comprises and is described in U.S. Patent number 6,395,966 and 7,151, those (both are all by reference incorporated herein in its entirety) in 204.

After conversion, have according to method choice well known in the art the expression construct of importing transformant and regeneration become full plants.Usual design method for transformation be used for by the following method regeneration period or in subsequent generation selectivity eliminate Select gene: such as, use with two independently T-DNA construct cotransformation or excise Select gene by specific recombinase locus specificity.

Except directly directly transforming except concrete plant genotype with construct of the present invention, also by the plant that will have a construct with lack the second plant hybridization of this construct to prepare transgenic plant.For example, the construct in coded polypeptide or territory can be introduced specified plant kind by hybridizing, and basic without the need to directly transforming the plant of this given kind.Therefore, the present invention is not only contained from the plant according to the cell Direct Regeneration of the present invention through transforming, and also comprises the offspring (progeny) of this type of plant.As for herein, offspring can refer to the descendant (offspring) of mother plant any generation of preparing according to the present invention.This kind of offspring can comprise the DNA construct prepared according to the present invention.Hybridization causes transgenosis to pass through the introduced plant germline by initial germline donor plant germline crossing pollination.The limiting examples of this type of step is described in U.S. Patent number 7,151,204.

Plant is generated by backcross conversion method.For example, this plant comprises the plant of genotype, germline, inbreeding body (inbred) or the crossbred (hybrid) being called backcross conversion.

Genetic marker can be used to assist one or more transgenosiss of the present invention from a genetic background gene transgression (introgression) to another.The selection that mark is assisted provides the advantage relative to conventional breeding, is that it can be used for the mistake avoiding being caused by phenotypic variation.Further, genetic marker can provide the data about breeding kind matter relative extent in the individual offspring of specific cross.For example, when (otherwise) does not have the genetic background needed for non-agronomy but has plant and the breeding parents of required proterties for this, genetic marker can be used select and not only there is objective trait, also there is the offspring of required kind of matter of relatively large ratio.In this way, the generation number making one or more character genes infiltrate needed for specific genetic background is minimized.

The present invention also relates to the method producing polypeptide of the present invention or territory, it comprises: (a) is contributing to cultivating transgenic plant or vegetable cell under the condition producing described polypeptide or territory, and described plant or vegetable cell comprise the polynucleotide in coded polypeptide or territory; Optionally (b) reclaims described polypeptide or territory.

Remove or reduce cellobiohydrolase activity

The invention still further relates to the method for generation of parental cell mutant, it comprises the polynucleotide or its part that destroy or lack coding polypeptide of the present invention, when described method causes cultivating under the same conditions, the cell of sudden change produces less described polypeptide compared with parental cell.

Method well known in the art (such as, insert, destroy, substitute or disappearance) can be used to build mutant cell by the expression reducing or eliminating polynucleotide.In preferred at one, described polynucleotide are inactivations.Polynucleotide that are to be finished or inactivation can be, such as, and coding region or its part crucial to activity, or express the regulatory element needed for coding region.The example of this adjustment or regulating and controlling sequence can be promoter sequence or its funtion part, that is, be enough to the part affecting polynucleotide expression.Other regulating and controlling sequence for possible modification includes but not limited to leader sequence, polyadenylation se-quence, propeptide sequence, signal peptide sequence, transcription terminator and transcription activator.

Can by imposing mutagenesis to parental cell, and select the mutant cell wherein expression of polynucleotide reduced or eliminated to carry out modification or the inactivation of polynucleotide.Mutagenesis may be specific or random, can by such as using suitable physics or chemical mutagen to carry out, by using suitable oligonucleotide to carry out, or by described DNA sequence dna being carried out the mutagenesis of PCR generation.In addition, mutagenesis can be carried out by using any combination of these mutagenic compound.

The example of the physics or chemical mutagen that are suitable for the object of the invention comprises ultraviolet (UV) irradiation, azanol, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine, nitrous acid, ethyl methanesulfonate (ethyl methane sulphonate) (EMS), sodium bisulfite, formic acid and nucleotide analog.

When using such agents, usually carry out described mutagenesis by the following method: the parental cell that when there are selected mutagenic compound under suitable conditions, incubation is to be mutagenic, and screening and/or select that display genetic expression reduces or without the mutant cells of genetic expression.

The modification of polynucleotide or inactivation also can transcribe or translate required controlling element by the one or more Nucleotide in insertion, replacement or missing gene or its and realize.Such as, can insert or remove Nucleotide thus cause introducing terminator codon, remove initiator codon, or frame be opened in change.The mutagenesis produced by site-directed mutagenesis or PCR according to methods known in the art can realize this modification or inactivation.Although described in theory modification can be carried out in vivo, that is, directly carry out on the cell of expressing polynucleotide to be finished, be preferably as follows and carry out described modification in vitro like that exemplified by face.

The example eliminating or reduce the convenient manner that polynucleotide are expressed has based on gene replacement, genetically deficient, or the technology of gene disruption.Such as, in gene disruption method, the nucleotide sequence corresponding to endogenous polynucleotides is carried out in vitro mutagenesis to produce the nucleotide sequence of defective, be then transformed in parental cell to produce dcc gene.By homologous recombination, described defective nucleotide sequence instead of endogenous polynucleotide.May it is desirable to described defective polynucleotide also coded markings, it can be used for the transformant selecting wherein polynucleotide to be modified or destroy.In one aspect, described polynucleotide are destroyed with selectable mark (as those described herein).

The present invention also relates to the method suppressing to have the expression of the polypeptide of cellobiohydrolase activity in cell, it comprises to use to cell or at cells double-stranded RNA (dsRNA) molecule, wherein said dsRNA comprises the subsequence of polynucleotide of the present invention.In preferred at one, described dsRNA length is about 15,16,17,18,19,20,21,22,23,24,25 or more duplex nucleotides.

Described dsRNA is preferably siRNA (siRNA) or microRNA (miRNA).In preferred at one, described dsRNA is for suppressing the siRNA of transcribing.In another is preferred, described dsRNA is for suppressing the microRNA translated.

The present invention also relates to such double-stranded RNA (dsRNA) molecule, it comprises the mature polypeptide encoded sequence of SEQ ID NO:1, the mature polypeptide encoded sequence of SEQ ID NO:3, the mature polypeptide encoded sequence of SEQ ID NO:5, or a part for the mature polypeptide encoded sequence of SEQ ID NO:7, for the expression suppressing described polypeptide in cell.Although the present invention is not by the restriction of any concrete mechanism of action, described dsRNA can enter cell and cause single stranded RNA (ssRNA) that is similar or identical sequence, comprises the degraded of endogenous mRNA.When cell is exposed to dsRNA, from homogenic mRNA by being called that the process of RNA interference (RNAi) is by degradation selectivity.

DsRNA of the present invention can be used for gene silencing.In one aspect, the invention provides the method using dsRNAi degradation selectivity RNA of the present invention.The method can be implemented in vitro, in vitro or in body.In one aspect, described dsRNA molecule is used in the sudden change that in cell, organ or animal, systematic function is lost.For the preparation of with use the method for dsRNA molecular selectivity degradation of rna to be as known in the art, see, such as U.S. Patent number 6,489,127; 6,506,559; 6,511,824; With 6,515,109.

The invention further relates to the mutant cells of parental cell, it comprises the silence of the gene of the polynucleotide of coded polypeptide or the destruction of its regulating and controlling sequence or disappearance or coding said polypeptide, and this causes mutant cells compared with parental cell produce less polypeptide or do not produce polypeptide.

Polypeptide defective type mutant cell is natural particularly useful with host cell that is heterologous polypeptide as expression.So the invention further relates to the method for producing natural or heterologous polypeptide, it comprises: (a) is contributing to cultivating mutant cell under the condition of producing polypeptide; Optionally (b) reclaims described polypeptide.It is not natural polypeptide that term " heterologous polypeptide " means host cell, such as, and the variant of native protein.Host cell can comprise the polynucleotide more than the described natural or heterologous polypeptide of the coding of a copy.

Method for cultivating the interested product with purifying can be undertaken by methods known in the art.

The present invention is make us interesting especially for generation of the method for the product of essentially no cellobiohydrolase activity in the generation of eucaryon polypeptide, particularly Fungal Protein such as enzyme.Cellobiohydrolase activity deficient cells also may be used for expressing significant heterologous protein such as hormone, somatomedin, acceptor etc. in pharmacy.Term " eucaryon polypeptide " not only comprises natural polypeptides, also comprises by aminoacid replacement, disappearance or interpolation or other such modification and is modified to enhance the polypeptide of activity, thermostability, pH tolerance etc., such as enzyme.

In other respects, the present invention relates to the protein of essentially no cellobiohydrolase activity, it is produced by method of the present invention.

Fermented liquid formulation or cell composition

The present invention also relates to fermented liquid formulation and cell composition, and it comprises polypeptide of the present invention.Described fermented liquid product comprises other composition for zymotechnique further, such as cell (comprise the host cell of the gene containing coding polypeptide of the present invention, it is for generation of interested polypeptide), cell debris, biomass, fermention medium and/or tunning.In some embodiments, described composition is the full nutrient solution having killed cell containing organic acid, the cell be killed and/or cell debris, and substratum.

Term " fermented liquid " is produced for this paper middle finger by cell fermentation, is not experienced or only experience the recovery of minimum and/or the prepared product of purifying.For example, saturated when being grown to by microorganisms cultures, under the condition of restriction carbon, incubation synthesizes (such as by host cell expression enzyme) to allow albumen, and when being secreted into cell culture medium, produces fermented liquid.Described fermented liquid can containing the non-classification of fermented material obtained when fermenting and stopping or the inclusion of classification.Typically, fermented liquid is unassorted, and the substratum used existed after comprising removal (such as by centrifugal) microorganism cells (such as filamentous fungal cells) and cell debris.In some embodiments, described fermented liquid containing the cell culture medium of useful mistake, extracellular enzyme, and can survive and/or (the viable and/or nonviable) microorganism cells that can not survive.

In one embodiment, described fermented liquid formulation and cell composition comprise the first organic acid composition and the second organic acid composition, described first organic acid composition comprises organic acid and/or its salt of at least one 1-5 carbon, and described second organic acid composition comprises organic acid and/or its salt of at least one 6 or more carbon.In a specific embodiment, described first organic acid composition is acetic acid, formic acid, propionic acid, their salt, or two or more mixture aforementioned, and described second organic acid composition is phenylformic acid, hexahydrobenzoic acid, 4-methylvaleric acid, toluylic acid, their salt, or two or more mixture aforementioned.

In one aspect, described composition contains organic acid, and optionally further containing the cell be killed and/or cell debris.In one embodiment, remove from the full nutrient solution killing cell described in the cell that has been killed and/or cell debris to provide not containing the composition of these components.

Described fermented liquid formulation or cell composition can comprise sanitas and/or antimicrobial (such as antibacterial) agent further, include but not limited to sorbyl alcohol, sodium-chlor, potassium sorbate and other is as known in the art.

Described fermented liquid formulation or cell composition can comprise multiple enzymic activity further, as one or more (such as several) are selected from the enzyme of lower group: cellulase, the GH61 polypeptide with cellulolytic enhancing activity, hemicellulase, esterase, claviformin, laccase, lignin decomposition enzyme, polygalacturonase, peroxidase, proteolytic enzyme and swollenin.Described fermented liquid formulation or cell composition also can comprise the enzyme that one or more (such as several) are selected from lower group: lytic enzyme, isomerase, ligase enzyme, lyase, oxygen is enzyme or transferring enzyme also, such as alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase, beta-galactosidase enzymes, beta-glucosidase enzyme, xylobiase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, at, Maltose 4-glucosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucoamylase, saccharase, laccase, lipase, mannosidase, become glycanase, oxydase, pectin decomposing enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic ferment, rnase, trans-glutaminases or zytase.

Described killed cell full nutrient solution or composition can non-classification inclusion containing the fermented material obtained when fermenting and stopping.Typically, described killed cell full nutrient solution or composition contain microorganism cells (such as filamentous fungal cells) grown to saturated, and under the condition of restriction carbon incubation with the substratum and the cell debris that allow albumen to synthesize (such as, express cellulase and Polyglucosidase) existence afterwards.In some embodiments, described killed cell full nutrient solution or composition containing the cell culture medium of useful mistake, extracellular enzyme, and the filamentous fungal cells be killed.In some embodiments, the microorganism cells existed in the full nutrient solution killing cell or composition can use method osmotic as known in the art and/or cracking.

Full nutrient solution or cell composition are generally liquid as described herein, but can contain insoluble component, as the cell, cell debris, nutrient media components and/or the insoluble enzyme that have been killed.In some embodiments, insoluble component can be removed to provide the liquid composition of clarification.

Full nutrient solution formulation of the present invention and cell composition produce by the method described in WO 90/15861 or WO 2010/096673.

Set forth below is the embodiment of the preferable use of composition of the present invention.Other condition that the dosage of described composition and composition use can determine based on means known in the art.

Enzyme composition

The invention still further relates to the composition comprising polypeptide of the present invention.Preferably, described composition is enriched this peptide species.Term " be enriched " show the cellobiohydrolase activity of described composition with such as at least 1.1 enrichment factor increase.

Described composition can comprise polypeptide of the present invention as major enzymatic component, such as single-component composition.Or, described composition can comprise multiple enzymic activity, as being selected from one or more (such as several) enzymes of lower group: cellulase, the GH61 polypeptide with cellulolytic enhancing activity, hemicellulase, esterase, claviformin, laccase, lignin decomposition enzyme, polygalacturonase, peroxidase, proteolytic enzyme and swollenin.Described composition also can comprise the enzyme that one or more (such as several) are selected from lower group: lytic enzyme, isomerase, ligase enzyme, lyase, oxygen is enzyme or transferring enzyme also, such as alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase, beta-galactosidase enzymes, beta-glucosidase enzyme, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, at, Maltose 4-glucosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucoamylase, saccharase, laccase, lipase, mannosidase, become glycanase, oxydase, pectin decomposing enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic ferment, rnase, trans-glutaminases, or zytase.Described composition can be prepared according to method as known in the art, and can be the form of liquid or dry composition.Described composition can according to method stabilization as known in the art.

Hereinafter provide the example of the preferable use of composition of the present invention.Other condition that the dosage of composition and composition use can give methods known in the art to determine.

Purposes

The invention still further relates to following use and there is the polypeptide of cellobiohydrolase activity or the technique of its composition.

The invention still further relates to degraded or transform the method for cellulose materials, it comprises: of the present invention there is the existence of the polypeptide of cellobiohydrolase activity under, with enzyme composition process cellulose materials.In one aspect, described technique comprises the cellulose materials that recovery has been degraded or transformed further.The degraded of described cellulose materials or the soluble product of conversion can be separated from insoluble fibrin materials'use methods known in the art, as such as centrifugal, filter or gravity settling.

The invention still further relates to and produce the technique of tunning, it comprises: (a) of the present invention there is the existence of the polypeptide of cellobiohydrolase enzymic activity under, use enzyme composition saccharified cellulosic material; (b) with the fermentation of one or more (such as several) organism of fermentation through the cellulose materials of saccharification to produce tunning; (c) tunning is reclaimed from fermentation.

The invention still further relates to the technique of fermentable fiber cellulosic material, it comprises: by one or more (such as several) organism of fermentation fermentable fiber cellulosic material, and wherein said cellulose materials has with enzyme composition saccharification under the existence of the polypeptide of cellobiohydrolase activity of the present invention.In one aspect, the fermentation of cellulose materials produces tunning.In yet another aspect, described technique also comprises from fermentation recovery tunning.

Technique of the present invention may be used for cellulose materials saccharification to become fermentable sugars, and fermentable sugars is changed into much useful tunning, such as fuel, drinking alcohol and/or platform chemicals (platform chemical) (such as acid, alcohol, ketone, gas etc.).Produce from cellulose materials the tunning expected and be usually directed to pre-treatment, enzymic hydrolysis (saccharification) and fermentation.

The ordinary method of this area can be used to complete according to the process of cellulose materials of the present invention.In addition, technique of the present invention can use any standard biologic matter processing units be configured to according to invention operation to carry out.

Hydrolysis (saccharification) and fermentation, point other or simultaneously, include but not limited to, the hydrolysis be separated and fermentation (SHF), synchronous glycosylation and fermentation (SSF), synchronous glycosylation and common fermentation (SSCF), the hydrolysis mixed and fermentation (HHF), the hydrolysis be separated and common fermentation (SHCF), the hydrolysis mixed and common fermentation (HHCF), with direct microbial transformation (DMC), sometimes also referred to as consolidated bioprocessing (consolidated bioprocessing, CBP).SHF use be separated treatment step with first by enzymatic hydrolysis of cellulosic material for fermentable sugars, such as, glucose, cellobiose and pentose monomers, then by fermentable sugars fermentation become ethanol.In SSF, enzymic hydrolysis and the sugar of cellulose materials become the fementative composition of ethanol (Philippidis, G.P., 1996 in one step, Cellulose bioconversion technology, in Handbook on Bioethanol:Production and Utilization, Wyman, C.E compile, Taylor & Francis, Washington, DC, 179-212).SSCF comprises the common fermentation (Sheehan of multiple sugar, and Himmel J., M., 1999, Enzymes, energy and the environment:A strategic perspective on the U.S.Department of Energy ' s research and development activities for bioethanol, Biotechnol.Prog.15:817-827).HHF is at the same time outside saccharification and hydrolysing step, and also comprise independent hydrolysing step, described each step can be carried out in same reactor.Step in HHF process can be carried out in different temperature, that is, the saccharification of high temperature enzyme process, and the lesser temps that then can tolerate at fermentation strain carries out SSF.DMC is combined with all three processes (enzyme generation, hydrolysis and fermentation) in one or more (such as several) step, identical organism is wherein used to produce for cellulose materials being changed into fermentable sugars and fermentable sugars being changed into enzyme (the Lynd L.R. of end product, Weimer, P.J., van Zyl, W.H., and Pretorius, I.S., 2002, Microbial cellulose utilization:Fundamentals and biotechnology, Microbiol.Mol.Biol.Reviews66:506-577).Be understandable that herein, any method as known in the art, comprise pre-treatment, enzymic hydrolysis (saccharification), fermentation, or their combination, all can be used for implementing technique of the present invention.

Conventional equipment can comprise Fed-batch stirred reactor, batch-type stirred reactor, the Continuous Flow stirred reactor with ultrafiltration and/or continuous piston flow column reactor (Fernanda de Castilhos Corazza, Fl á vio Faria de Moraes, Gisella Maria Zanin and Ivo Neitzel, 2003, Optimal control in fed-batch reactor for the cellobiose hydrolysis, Acta Scientiarum.Technology25:33-38, Gusakov, and Sinitsyn A.V., A.P., 1985, Kinetics of the enzymatic hydrolysis of cellulose:1.A mathematical model for a batch reactor process, Enz.Microb.Technol.7:346-352), griding reaction device (Ryu, and Lee S.K., J.M., 1983, Bioconversion of waste cellulose by using an attrition bioreactor, Biotechnol.Bioeng.25:53-65), or there is the intensively stirred reactor (Gusakov caused by electromagnetic field, A.V., Sinitsyn, A.P., Davydkin, I.Y., Davydkin, V.Y., Protas, O.V., 1996, Enhancement of enzymatic cellulose hydrolysis using a novel type of bioreactor with intensive stirring induced by electromagnetic field, Appl.Biochem.Biotechnol.56:141-153).Other type of reactor comprises: fluidized-bed, up-flow layer (upflow blanket), immobilization and the reactor of extruder type for being hydrolyzed and/or ferment.

pre-treatment.In the enforcement of technique of the present invention, any preprocessing process known in the art can be used to destroy the cellulose materials component (Chandra etc. of plant cell wall, 2007, Substrate pretreatment:The key to effective enzymatic hydrolysis of lignocellulosics? Adv.Biochem.Engin./Biotechnol.108:67-93; Galbe and Zacchi, 2007, Pretreatment of lignocellulosic materials for efficient bioethanol production, Adv.Biochem.Engin./Biotechnol.108:41-65; Hendriks and Zeeman, 2009, Pretreatments to enhance the digestibility of lignocellulosic biomass, Bioresource Technol.100:10-18; Mosier etc., 2005, Features of promising technologies for pretreatment of lignocellulosic biomass, Bioresource Technol.96:673-686; Taherzadeh and Karimi, 2008, Pretreatment of lignocellulosic wastes to improve ethanol and biogas production:A review, Int.J.of Mol.Sci.9:1621-1651; Yang and Wyman, 2008, Pretreatment:the key to unlocking low-cost cellulosic ethanol, Biofuels Bioproducts and Biorefining-Biofpr.2:26-40).

Cellulose materials also can use method as known in the art to carry out particle size reduction, screening, pre-soaking, soak, wash and/or conditioning (conditioning) before pre-processing.

Conventional pre-treatment includes but not limited to, steam pre-treatment (with or not with explosion), dilute acid pretreatment, hot-water pretreatment, oxygenation pretreatment, Calx preconditioning, wet oxidation, wet explosion, the explosion of ammonia fiber, organic solvent pre-treatment and Biological Pretreatment.Other pre-treatment comprises ammonia diafiltration, ultrasonic, electroporation, microwave, supercritical CO ₂, overcritical H ₂o, ozone, ionic liquid and gamma-radiation pre-treatment.

Can be hydrolyzed and/or pre-treating cellulosic material before fermentation.Pre-treatment is preferably carried out before hydrolysis.Or pre-treatment can be carried out discharging fermentable sugars, as glucose, wood sugar and/or cellobiose with enzymic hydrolysis simultaneously.In most of the cases, pre-treatment step itself makes some Wood Adhesives from Biomass become fermentable sugars (even not depositing in the context of enzymes).

Steam pre-treatment.In steam pre-treatment, heating cellulose material, to destroy plant cell wall component, comprises xylogen, hemicellulose and Mierocrystalline cellulose, makes Mierocrystalline cellulose and other fraction, such as hemicellulose, can be touched by enzyme.Passed through by cellulose materials or pass through reaction vessel, wherein injecting steam is to increase temperature to the temperature and pressure needed, and keeps the reaction times of expectation wherein.Steam pre-treatment is preferably at 140-250 DEG C, and such as 160-200 DEG C, or 170-190 DEG C is carried out, wherein optimum temperature range depends on the interpolation of chemical catalyst.The residence time preferred 1-60 minute of steam pre-treatment, such as 1-30 minute, 1-20 minute, 3-12 minute, or 4-10 minute, the wherein optimum residence time depends on the interpolation of temperature range and chemical catalyst.Steam pre-treatment allows relatively high solid substance heap(ed) capacity, to such an extent as to cellulose materials mostly just becomes moist in preprocessing process.Steam pre-treatment often combines with the explosion blowing (explosive discharge) of pretreated material, this is called steam explosion, namely, quick flickering is to the turbulent flow of normal atmosphere and material, to increase surface-area (Duff and Murray that can contact by broken, 1996, Bioresource Technology855:1-33; Galbe and Zacchi, 2002, Appl.Microbiol.Biotechnol.59:618-628; U.S. Patent application No.20020164730).In steam pre-treatment process, hemicellulose acetyl group is cut open, and the sour autocatalysis hemicellulose fraction hydrolysis obtained becomes monose and oligosaccharides.Xylogen is only removed with limited degree.

Chemical Pretreatment: term " chemical treatment " refers to any chemical treatment that can promote Mierocrystalline cellulose, hemicellulose and/or lignin separation and/or release.Crystal fibre element can be converted into amorphous cellulose by this kind of pre-treatment.The example of suitable Chemical Pretreatment technique comprises such as dilute acid pretreatment, Calx preconditioning, wet oxidation, ammonia fiber/freezing explosion (AFEX), ammonia diafiltration (APR), ionic liquid and organic solvent pre-treatment.

Catalyzer was added as H before steam pre-treatment of being everlasting ₂sO ₄or SO ₂(usual 0.3 to 5%w/w), it can reduce the time, reduces temperature, increases the rate of recovery, and improves enzymic hydrolysis (Ballesteros etc., 2006, Appl.Biochem.Biotechnol.129-132:496-508; Varga etc., 2004, Appl.Biochem.Biotechnol.113-116:509-523; Sassner etc., 2006, Enzyme Microb.Technol.39:756-762).In dilute acid pretreatment, by cellulose materials and diluted acid (normally H ₂sO ₄) and water mixing to form slurry, by the temperature being steam heated to expectation, and after one period of residence time flickering to normal atmosphere.Can carry out dilute acid pretreatment by a lot of reactor design form, such as, plug flow reactor, counter-current reactor or continuous countercurrent shrink bed bioreactor (Duff and Murray, 1996, supra; Schell etc., 2004, Bioresource Technol.91:179-188; Lee etc., 1999, Adv.Biochem.Eng.Biotechnol.65:93-115).

Several pretreatment processs under alkaline condition can also be used.These oxygenation pretreatment include, but not limited to sodium hydroxide, lime, wet oxidation, ammonia diafiltration (APR) and ammonia fiber/freezing explosion (AFEX).

Carry out Calx preconditioning with calcium oxide or calcium hydroxide the temperature of 85-150 DEG C, the residence time was from 1 hour to several days (Wyman etc., 2005, Bioresource Technol.96:1959-1966; Mosier etc., 2005, Bioresource Technol.96:673-686).WO 2006/110891, WO 2006/110899, WO 2006/110900 and WO 2006/110901 disclose the pretreatment process using ammonia.

Wet oxidation is a kind of Grape berry, usually carries out 5-15 minute at 180-200 DEG C, adds oxygenant as hydrogen peroxide or overvoltage oxygen (Schmidt and Thomsen, 1998, Bioresource Technol.64:139-151; Palonen etc., 2004, Appl.Biochem.Biotechnol.117:1-17; Varga etc., 2004, Biotechnol.Bioeng.88:567-574; Martin etc., 2006, J.Chem.Technol.Biotechnol.81:1669-1677).Pre-treatment is with preferred 1-40% dry-matter, and such as 2-30% dry-matter, or 5-20% dry-matter carries out, and frequently by adding alkali if sodium carbonate is to increase initial pH.

The amending method of wet oxidation pretreatment process, is called wet explosion (combination of wet oxidation and steam explosion), can processes the dry-matter up to 30%.In wet explosion, in preprocessing process, after certain residence time, introduce oxygenant.Then pre-treatment (WO 2006/032282) is terminated by flickering to normal atmosphere.

Ammonia fiber explosion (AFEX) relates in moderate temperature if 90-150 DEG C and high pressure are as 17-20bar, with liquefied ammonia or ammonia by cellulose materials process 5-10 minute, wherein dry matter content can up to 60% (Gollapalli etc., 2002, Appl.Biochem.Biotechnol.98:23-35; Chundawat etc., 2007, Biotechnol.Bioeng.96:219-231; Alizadeh etc., 2005, Appl.Biochem.Biotechnol.121:1133-1141; Teymouri etc., 2005, Bioresource Technol.96:2014-2018).In AFEX preprocessing process, Mierocrystalline cellulose and hemicellulose keep relative complete.Xylogen-saccharide complex is cut open.

Organic solvent pre-treatment is by using aqueous ethanol (40-60% ethanol) 160-200 DEG C of extraction 30-60 minute by cellulose materials delignification (Pan etc., 2005, Biotechnol.Bioeng.90:473-481; Pan etc., 2006, Biotechnol.Bioeng.94:851-861; Kurabi etc., 2005, Appl.Biochem.Biotechnol.121:219-230).Often add sulfuric acid as catalyzer.In organic solvent pre-treatment, most of hemicellulose and xylogen are removed.

Other examples of suitable pretreatment process as Schell etc., 2003, Appl.Biochem and Biotechn.Vol.105-108:69-85, with Mosier etc., 2005, Bioresource Technology96:673-686, and the U.S. openly applies for described in 2002/0164730.

In one aspect, Chemical Pretreatment preferably as dilute acid pretreatment, and is more preferably carried out as continuous dilute acid pretreatment.Acid is sulfuric acid normally, but also can use other acid, as acetic acid, citric acid, nitric acid, phosphoric acid, tartrate, succsinic acid, hydrogenchloride or its mixture.Weak acid (mild acid) process such as, at preferred 1-5,1-4, or the pH scope of 1-2.5 is carried out.In one aspect, acid concentration in preferred 0.01 to 10wt% acid, the scope of such as 0.05 to 5wt% acid or 0.1 to 2wt% acid.Acid is contacted with cellulose materials, and at preferred 140-200 DEG C, the temperature of such as 165-190 DEG C of scope keeps the time of 1 to 60 minute.

In yet another aspect, pre-treatment occurs in aqueous slurry.In preferred, in preprocessing process, cellulose materials such as, with preferred 10-80wt%, 20-70wt% or 30-60wt%, and the amount of 40wt% exists according to appointment.Pretreated cellulose materials can not wash or use any known method washing in this area, such as, washes with water.

Mechanical pretreatment or physics pre-treatment: term " mechanical pretreatment " or " physics pre-treatment " refer to the pre-treatment that any promotion granular size reduces.For example, this kind of pre-treatment can relate to various types of grinding (grinding) or grind (milling) (such as, dry grinding, wet-milling or vibratory milling).

Cellulose materials can through both physics (machinery) and Chemical Pretreatment.Machinery or physics pre-treatment can with following coupling: decatize/steam explosion, aquathermolysis (hydrothermolysis), diluted acid or weak acid treatment, high temperature, autoclaving, radiation (such as microwave radiation), or its combination.In one aspect, high end finger is preferably about 100 to about 400psi, and such as about 150 to the pressure of the scope of about 250psi.In yet another aspect, high temperature refers to about 100 to 300 DEG C, the temperature of such as about 140 to about 200 DEG C of scopes.In preferred at one, machinery or physics pre-treatment utilize in the batchwise process of the vapor gun hydrolyzer system of high temperature and high pressure as defined above (such as from the Sunds Hydrolyzer of Sunds Defibrator AB, Sweden) in use to be carried out.Described physics and chemistry pre-treatment can optionally sequentially be carried out or carry out simultaneously.

Therefore, in preferred at one, physics (machinery) or Chemical Pretreatment are carried out to cellulose materials, or their any combination, to promote separation and/or the release of Mierocrystalline cellulose, hemicellulose and/or xylogen.

Biological Pretreatment: term " Biological Pretreatment " refers to any Biological Pretreatment that can promote that Mierocrystalline cellulose, hemicellulose and/or xylogen are separated from cellulose materials and/or discharge.Biological Pretreatment Techniques can comprise the application microorganism of dissolved lignin and/or enzyme (see, such as, Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol:Production and Utilization, Wyman, C.E compiles, Taylor & Francis, Washington, DC, 179-212; Ghosh and Singh, 1993, Physicochemical and biological treatments for enzymatic/microbial conversion of lignocellulosic biomass, Adv.Appl.Microbiol.39:295-333; McMillan, J.D., 1994, Pretreating lignocellulosic biomass:a review, in Enzymatic Conversion of Biomass for Fuels Production, Himmel, M.E., Baker, J.O., and Overend, R.P., compile, ACS Symposium Series566, American Chemical Society, Washington, DC, the 15th chapter; Gong, C.S., Cao, N.J., Du, J., and Tsao, G.T., 1999, Ethanol production from renewable resources, in Advances in Biochemical Engineering/Biotechnology, Scheper, T., compile, Springer-Verlag Berlin Heidelberg, Germany, 65:207-241; Olsson and Hahn-Hagerdal, 1996, Fermentation of lignocellulosic hydrolysates for ethanol production, Enz.Microb.Tech.18:312-331; With Vallander and Eriksson, 1990, Production of ethanol from lignocellulosic materials:State of the art, Adv.Biochem.Eng./Biotechnol.42:63-95).

saccharification.In hydrolysing step, by cellulose materials, such as pretreated cellulosic material hydrolysis so that Mierocrystalline cellulose and hemicellulose are resolved into fermentable sugars, as glucose, cellobiose, wood sugar, xylulose, pectinose, seminose, semi-lactosi and/or solvable oligosaccharides.Hydrolysis utilizes enzyme composition to have under the existence of the polypeptide of cellobiohydrolase activity enzymatic as described herein in the present invention to carry out.The enzyme component of composition can also add simultaneously or sequentially.

Enzymic hydrolysis preferably, under the easy condition determined by those skilled in the art, is carried out in suitable aqueous environment.In one aspect, be hydrolyzed in the activity being suitable for enzyme component, namely carry out under enzyme component optimal conditions.Hydrolysis can be carried out with fed-batch or continuous print process, is filled into gradually by cellulose materials in successive processes, such as, fills in the hydrating solution containing enzyme.

Saccharification is carried out usually in stirred-tank reactor or fermentor tank under controlled pH, temperature and mixing condition.Suitable treatment time, temperature and pH condition easily can be determined by those skilled in the art.Such as, saccharification is sustainable reaches 200 hours, but usually carries out preferably about 12 to about 120 hours, such as about 16 to about 72 hours, or about 24 to about 48 hours.Temperature at preferably about 25 DEG C to about 70 DEG C, such as about 30 DEG C to about 65 DEG C, about 40 DEG C to about 60 DEG C, or the scope of about 50 DEG C to 55 DEG C.PH such as, preferred about 3 to about 8, about 3.5 to about 7, about 4 to about 6, or the scope of about 5.0 to about 5.5.Dry solid content is preferred about 5 to about 50wt%, and such as about 10 to about 40wt%, or the scope of about 20 to about 30wt%.

Enzyme composition can comprise any albumen that can be used for degradation of fibers cellulosic material.

In one aspect, described enzyme composition comprises or also comprises the protein/polypeptide that one or more (such as several) are selected from lower group: cellulase, have the GH61 polypeptide of cellulolytic enhancing activity, hemicellulase, esterase, claviformin, laccase, lignin decomposition enzyme, polygalacturonase, peroxidase, proteolytic enzyme and swollenin.In yet another aspect, described cellulase is that preferably one or more (such as several) are selected from the enzyme of lower group: endoglucanase, cellobiohydrolase and beta-glucosidase enzyme.In yet another aspect, described hemicellulase is that preferably one or more (such as several) are selected from the enzyme of lower group: acetyl mannan esterase, acetyl xylan esterase, arabanase, arabinofuranosidase, coumaric acid esterase, feruloyl esterase, tilactase, glucuronidase, glucuronic acid esterase, mannonase mannosidase, zytase and xylosidase.

In yet another aspect, described enzyme composition comprises one or more (such as several) cellulolytic enzymes.In yet another aspect, described enzyme composition comprises or comprises further one or more (such as several) hemicellulose lytic enzymes.In yet another aspect, described enzyme composition comprises one or more (such as several) cellulolytic enzymes and one or more (such as several) hemicellulose lytic enzymes.In yet another aspect, described enzyme composition comprises the enzyme that one or more (such as several) are selected from lower group: cellulolytic enzyme and hemicellulose lytic enzyme.In yet another aspect, described enzyme composition comprises endoglucanase.In yet another aspect, described enzyme composition comprises cellobiohydrolase.In yet another aspect, described enzyme composition comprises beta-glucosidase enzyme.In yet another aspect, described enzyme composition comprises the polypeptide with cellulolytic enhancing activity.In yet another aspect, described enzyme composition comprises endoglucanase and has the polypeptide of cellulolytic enhancing activity.In yet another aspect, described enzyme composition comprises cellobiohydrolase and has the polypeptide of cellulolytic enhancing activity.In yet another aspect, described enzyme composition comprises beta-glucosidase enzyme and has the polypeptide of cellulolytic enhancing activity.In yet another aspect, described enzyme composition comprises endoglucanase and cellobiohydrolase.In yet another aspect, described enzyme composition comprises endoglucanase and beta-glucosidase enzyme.In yet another aspect, described enzyme composition comprises cellobiohydrolase and beta-glucosidase enzyme.In yet another aspect, described enzyme composition comprises endoglucanase, cellobiohydrolase and has the polypeptide of cellulolytic enhancing activity.In yet another aspect, described enzyme composition comprises endoglucanase, beta-glucosidase enzyme and has the polypeptide of cellulolytic enhancing activity.In yet another aspect, described enzyme composition comprises cellobiohydrolase, beta-glucosidase enzyme and has the polypeptide of cellulolytic enhancing activity.In yet another aspect, described enzyme composition comprises endoglucanase, cellobiohydrolase and beta-glucosidase enzyme.In yet another aspect, described enzyme composition comprises endoglucanase, cellobiohydrolase, beta-glucosidase enzyme and has the polypeptide of cellulolytic enhancing activity.

In yet another aspect, described enzyme composition comprises acetyl mannan esterase.In yet another aspect, described enzyme composition comprises acetyl xylan esterase.In yet another aspect, described enzyme composition comprises arabanase (such as α-L-arabanase).In yet another aspect, described enzyme composition comprises arabinofuranosidase (such as α-l-arabfuranglycosidase).In yet another aspect, described enzyme composition comprises coumaric acid esterase.In yet another aspect, described enzyme composition comprises feruloyl esterase.In yet another aspect, described enzyme composition comprises tilactase (such as alpha-galactosidase and/or beta-galactosidase enzymes).In yet another aspect, described enzyme composition comprises glucuronidase (such as α-D-glucuronidase).In yet another aspect, described enzyme composition comprises glucuronic acid esterase.In yet another aspect, described enzyme composition comprises mannase.In yet another aspect, described enzyme composition comprises mannosidase (such as beta-Mannosidase).In yet another aspect, described enzyme composition comprises zytase.In preferred at one, described zytase is family 10 zytase.In yet another aspect, described enzyme composition comprises xylosidase (such as xylobiase).

In yet another aspect, described enzyme composition comprises esterase.In yet another aspect, described enzyme composition comprises claviformin.In yet another aspect, described enzyme composition comprises laccase.In yet another aspect, described enzyme composition comprises lignin decomposition enzyme.In another is preferred, described lignin decomposition enzyme is manganese peroxidase.In another is preferred, described lignin decomposition enzyme is lignin peroxidase.In another is preferred, described lignin decomposition enzyme produces H ₂o ₂enzyme.In yet another aspect, described enzyme composition comprises polygalacturonase.In yet another aspect, described enzyme composition comprises peroxidase.In yet another aspect, described enzyme composition comprises proteolytic enzyme.In yet another aspect, described enzyme composition comprises swollenin.

In technique of the present invention, enzyme can in saccharification, saccharification and fermentation, or fermentation before or add in process.

One or more (such as several) components of described enzyme composition can be the combination of wild-type protein, recombinant protein or wild-type protein and recombinant protein.For example, one or more (such as several) components can be the native protein of cell, and it is used as host cell with one or more (such as several) other components of recombinant expressed enzyme composition.One or more (such as several) components of enzyme composition can be produced as independent component, then be combined to form enzyme composition.Described enzyme composition can be the combination of polycomponent and single component protein preparation.

Can be any applicable form for the enzyme in present invention process, such as fermented liquid formulation, cell composition, containing or not containing the cell pyrolysis liquid of cell debris, the enzyme prepared product of half purifying or purifying, or the host cell in source as enzyme.Described enzyme composition can be dry powder or particle, non-dusting particle, liquid, stabilization liquid or the shielded enzyme of stabilization.Liquid enzyme preparations can according to the technique of establishing, and such as, by adding stablizer as sugar, sugar alcohol or other polyvalent alcohols, and/or lactic acid or other organic acids carry out stabilization.

The optimal dose of the enzyme and polypeptide with cellobiohydrolase activity depends on several factor, it includes but not limited to, mixture, cellulose materials, the concentration of cellulose materials, pre-treatment, temperature, time, the pH of cellulose materials of cellulose decomposition and/or hemicellulose lytic enzyme component and comprise fermenting organisms (such as, the yeast of synchronous glycosylation and fermentation).

In one aspect, cellulolytic enzyme or hemicellulose lytic enzyme are about 0.5 to about 50mg for the significant quantity of cellulose materials, such as about 0.5 to about 40mg, about 0.5 to about 25mg, about 0.75 to about 20mg, about 0.75 to about 15mg, and about 0.5 to about 10mg, or about 2.5 to about 10mg every g cellulose materialss.

In yet another aspect, the polypeptide with cellobiohydrolase activity is about 0.01 to about 50.0mg for the significant quantity of cellulose materials, and such as about 0.01 to about 40mg, about 0.01 to about 30mg, about 0.01 to about 20mg, and about 0.01 to about 10mg, and about 0.01 to about 5mg, about 0.025 to about 1.5mg, about 0.05 to about 1.25mg, and about 0.075 to about 1.25mg, and about 0.1 to about 1.25mg, about 0.15 to about 1.25mg, or about 0.25 to about 1.0mg every g cellulose materials.

In yet another aspect, the polypeptide with cellobiohydrolase activity is about 0.005 to about 1.0g for the significant quantity of cellulolytic enzyme or hemicellulose lytic enzyme, such as about 0.01 to about 1.0g, about 0.15 to about 0.75g, about 0.15 to about 0.5g, about 0.1 to about 0.5g, and about 0.1 to about 0.25g, or about 0.05 to about 0.2g every g cellulolytic enzyme or hemicellulose lytic enzyme.

There is the polypeptide of cellulose decomposition enzymic activity or hemicellulose lytic enzyme activity, and other can be used for the protein/polypeptide of the degraded of cellulose materials, the GH61 polypeptide (being referred to as the polypeptide with enzymic activity in this article) such as with cellulolytic enhancing activity can be derived from or obtain from any suitable source, comprises bacterium, fungi, yeast, plant or Mammals source.Term " acquisition " also means this enzyme in this article and described method restructuring can be used in host living beings herein to produce, the enzyme wherein produced through recombinating is natural or external source for host living beings, or there is the aminoacid sequence of modification, such as, the amino acid there is one or more (such as several) disappearance, inserting and/or replace, namely recombinate the enzyme produced, it is the fragment of natural acid sequence and/or mutant or the enzyme produced by amino acid Shuffling Method known in the art.What contain in the implication of natural enzyme is natural variant, and what contain in the implication of external enzyme is the variant that restructuring (as by site-directed mutagenesis or rearrangement) obtains.

The polypeptide with enzymic activity can be bacterial peptide.Such as, described polypeptide can be that gram positive bacterium polypeptide is as bacillus (Bacillus), streptococcus (Streptococcus), streptomyces (Streptomyces), Staphylococcus (Staphylococcus), enterococcus spp (Enterococcus), lactobacillus (Lactobacillus), lactococcus (Lactococcus), fusobacterium (Clostridium), ground bacillus belongs to (Geobacillus), pyrolysis Mierocrystalline cellulose Pseudomonas (Caldicellulosiruptor), hot acid Pseudomonas (Acidothermus), Thermobifidia or bacillus marinus belong to (Oceanobacillus) polypeptide, described polypeptide has enzymic activity, or gram negative bacterium polypeptide, as intestinal bacteria, Rhodopseudomonas (Pseudomonas), salmonella (Salmonella), campylobacter (Campylobacter), Helicobacterium (Helicobacter), Flavobacterium (Flavobacterium), Fusobacterium (Fusobacterium), mud Bacillaceae (Ilyobacter), eisseria (Neisseria) or Ureaplasma (Ureaplasma) polypeptide, described polypeptide has enzymic activity.

In one aspect, described polypeptide has the Alkaliphilic bacillus of enzymic activity, bacillus amyloliquefaciens, bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, bacillus firmus, bacillus lautus, bacillus lentus, Bacillus licheniformis, bacillus megaterium, bacillus pumilus, bacstearothermophilus, subtilis or bacillus thuringiensis polypeptide.

In another is preferred, described polypeptide has the streptococcus equisimilis of enzymic activity, streptococcus pyogenes, streptococcus uberis or zooepidemicus polypeptide.

In another is preferred, described polypeptide has the not streptomyces chromogenes of enzymic activity, deinsectization streptomycete, streptomyces coelicolor, streptomyces griseus or shallow Streptomyces glaucoviolaceus polypeptide.

The polypeptide with enzymic activity also can be tungal polypeptide, and more preferably yeast polypeptides genus polypeptide as mould in mycocandida, genus kluyveromyces, Pichia, yeast belong, Schizosaccharomyces or Western alpine yarrow, it has enzymic activity, or more preferably filamentous fungal polypeptide as the mould genus of branch top spore, Agaricus, Alternaria, Aspergillus, aureobasidium genus, Botryospaeria, intend wax Pseudomonas, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinus, Coptotermes, rod softgel shell belongs to, the red shell Pseudomonas of hidden clump, genera cryptococcus, Diplodia, Exidia, Filibasidium, fusarium, Gibberella, full flagellum Eimeria, Humicola, rake teeth Pseudomonas, Agaricus, Leptospaeria, Magnaporthe grisea belongs to, Melanocarpus, Polyporus, Mucor, myceliophthora, the mould genus of Xin Kaoma fat, Neurospora, paecilomyces, Penicillium, flat lead fungi belongs to, cud Chytridium, Poitrasia, false black Peziza, Pseudotrichonympha, Rhizomucor, Schizophyllum, capital spore belongs to, Talaromyces, thermophilic ascomycete belongs to, the mould genus of shuttle spore, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvaria or Xylaria polypeptide, it has enzymic activity.

In one aspect, described polypeptide has the saccharomyces carlsbergensis of enzymic activity, yeast saccharomyces cerevisiae, saccharomyces diastaticus, Doug Laplace yeast, Saccharomyces kluyveri, promise ground yeast or ellipsoideus yeast polypeptide.

In yet another aspect, described polypeptide is that to have the solution fiber branch top spore of enzymic activity mould, microorganism Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus, smelly aspergillus, aspergillus japonicus, Aspergillus nidulans, aspergillus niger, aspergillus oryzae, chrysosporium keratinophilum, Chrysosporium lucknowense, chrysosporium tropicum, Chrysosporium merdarium, Chrysosporium inops, felt gold pityrosporion ovale, Chrysosporium queenslandicum, Chrysosporium zonatum, bar spore shape sickle spore, F.graminearum schw, storehouse prestige sickle spore, machete sickle spore, fusarium graminaria, the red sickle spore of standing grain, different spore sickle spore, albizzia sickle spore, point sickle spore, racemosus sickle spore, pink sickle spore, Williams Elder Twig sickle spore, colour of skin sickle spore, intend branch spore sickle spore, sulphur look sickle spore, circle sickle spore, intend silk spore sickle spore, empiecement sickle spore, ash humicola lanuginosa, Humicola insolens, dredge cotton like humicola lanuginosa, white rake teeth bacterium, rice black wool is mould, thermophilic fungus destroyed wire, Neuraspora crassa, penicillium funiculosum, penicillium purpurogenum, the flat lead fungi of yellow spore, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Australia shuttle spore is mould, Thielavia fimeti, little spore shuttle spore is mould, ovum spore shuttle spore is mould, Thielavia peruviana, knurl spore shuttle spore is mould, hair shuttle spore is mould, Thielavia subthermophila, autochthonal shuttle spore is mould, trichoderma harziarum, healthy and free from worry wood is mould, long shoot wood is mould, Trichodermareesei, viride or brown spore become mildewed cup fungi (Trichophaea saccata) polypeptide.

The mutant transformed through chemically modified or protein engineering of the polypeptide with enzymic activity can also be used.

One or more (such as several) components of described composition can be restructuring components, that is, by the DNA sequence dna of component independent described in clones coding and subsequently with this DNA sequence dna transformant and express in host (see, such as, WO91/17243 and WO91/17244) and produce.Described host is preferably heterologous host (enzyme is external source to host), but this host also can be homology host (enzyme is natural to host) under certain condition.Monocomponent fibre element decomposition of protein can also be prepared by such protein of purifying from fermented liquid.

In one aspect, one or more (such as several) cellulolytic enzymes described comprise commercial cellulolytic enzyme prepared product.The example being applicable to the cellulolytic enzyme prepared product of business of the present invention comprises, such as, and CELLIC ^tMctec (Novozymes A/S), CELLIC ^tMcTec2 (Novozymes A/S), cTec3 (Novozymes A/S), CELLUCLAST ^tM(Novozymes A/S), NOVOZYM ^tM188 (Novozymes A/S), CELLUZYME ^tM(Novozymes A/S), CEREFLO ^tM(Novozymes A/S) and ULTRAFLO ^tM(Novozymes A/S), ACCELERASE ^tM(Genencor Int.), LAMINEX ^tM(Genencor Int.), SPEZYME ^tMcP (Genencor Int.), nL (DSM), s/L100 (DSM), ROHAMENT ^tM7069W lDI (Dyadic International, Inc.), lBR (Dyadic International, Inc.) or 150L (Dyadic International, Inc.).Described cellulose enzyme with about 0.001 of solid substance to about 5.0wt%, such as solid substance about 0.025 to about 4.0wt%, or the significant quantity of about 0.005 of solid to about 2.0wt% is added.

The example that may be used for the bacterial endo glucanases of technique of the present invention includes but are not limited to, and separates fiber hot acid bacterium (Acidothermus cellulolyticus) endoglucanase (WO 91/05039; WO 93/15186; United States Patent (USP) 5,275,944; WO 96/02551; United States Patent (USP) 5,536,655, WO 00/70031, WO 05/093050); Thermobifida fusca EG III (WO 05/093050); With Thermobifida fusca EGV (WO 05/093050).

The example that may be used for fungal endoglucanase of the present invention includes but are not limited to, trichoderma reesei endoglucanase I (Penttila etc., 1986, Gene45:253-263, Trichodermareesei Cel7B endoglucanase i (GENBANK ^tMaccession number M15665); Trichoderma reesei endoglucanase II (Saloheimo etc., 1988, Gene63:11-22), Trichodermareesei Cel5A EG II (GENBANK ^tMaccession number M19373); Trichoderma reesei endoglucanase III (Okada etc., 1988, Appl.Environ.Microbiol.64:555-563; GENBANK ^tMaccession number AB003694); Trichoderma reesei endoglucanase V (Saloheimo etc., 1994, Molecular Microbiology13:219-228; GENBANK ^tMaccession number Z33381); Microorganism Aspergillus aculeatus endoglucanase (Ooi etc., 1990, Nucleic Acids Research18:5884); Valley aspergillus (Aspergillus kawachii) endoglucanase (Sakamoto etc., 1995, Current Genetics27:435-439); Carrot soft rot Erwinia (Erwinia carotovara) endoglucanase (Saarilahti etc., 1990, Gene90:9-14); Point sickle spore endoglucanase (GENBANK ^tMaccession number L29381); Ash humicola lanuginosa thermoidea mutation endoglucanase (GENBANK ^tMaccession number AB003107); Melanocarpus albomyces endoglucanase (GENBANK ^tMaccession number MAL515703); Neuraspora crassa endoglucanase (GENBANK ^tMaccession number XM_324477); Humicola insolens EGV; Thermophilic fungus destroyed wire CBS117.65 endoglucanase; Basidiomycetes (basidiomycete) CBS495.95 endoglucanase; Basidiomycetes CBS494.95 endoglucanase; The mould NRRL8126CEL6B endoglucanase of autochthonal shuttle spore; The mould NRRL8126CEL6C endoglucanase of autochthonal shuttle spore; The mould NRRL8126CEL7C endoglucanase of autochthonal shuttle spore; The mould NRRL8126CEL7E endoglucanase of autochthonal shuttle spore; The mould NRRL8126CEL7F endoglucanase of autochthonal shuttle spore; Cladorrhinum foecundissimum ATCC62373CEL7A endoglucanase; And Li's Trichoderma strains No.VTT-D-80133 endoglucanase (GENBANK ^tMaccession number M15665).

The example of cellobiohydrolase used in the present invention includes but are not limited to, microorganism Aspergillus aculeatus cellobiohydrolase II (WO 2011/059740), chaetomium thermophilum (Chaetomium thermophilum) cellobiohydrolase I, chaetomium thermophilum cellobiohydrolase II, Humicola insolens cellobiohydrolase I, thermophilic fungus destroyed wire cellobiohydrolase II, (WO2009/042871), Thielavia hyrcanie cellobiohydrolase II (WO 2010/141325), the mould cellobiohydrolase II of autochthonal shuttle spore (CEL6A, WO 2006/074435), Trichodermareesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, and brown spore becomes mildewed cup fungi cellobiohydrolase II (WO 2010/057086).

The example of beta-glucosidase enzyme used in the present invention includes but are not limited to from microorganism Aspergillus aculeatus (Kawaguchi etc., 1996, Gene173:287-288), Aspergillus fumigatus (WO 2005/047499), aspergillus niger (Dan etc., 2000, J.Biol.Chem.275:4973-4980), aspergillus oryzae (WO 2002/095014), Brazilian mould IBT20888 (WO 2007/019442 and WO 2010/088387), mould (WO 2011/035029) of autochthonal shuttle spore and brown spore become mildewed the beta-glucosidase enzyme of cup fungi (WO 2007/019442).

Described beta-glucosidase enzyme can be fusion rotein.In one aspect, described beta-glucosidase enzyme is WO aspergillus oryzae beta-glucosidase enzyme variant BG fusion rotein (WO 2008/057637) or aspergillus oryzae beta-glucosidase enzyme fusion rotein (2008/057637).

Other available endoglucanase, cellobiohydrolase and beta-glucosidase enzyme are disclosed in and use according to Henrissat B., 1991, A classification of glycosyl hydrolases based on amino-acid sequence similarities, Biochem.J.280:309-316 and Henrissat B. and Bairoch A., 1996, Updating the sequence-based classification of glycosyl hydrolases, in many glycosyl hydrolase families of the classification of Biochem.J.316:695-696.

Other cellulolytic enzyme used in the present invention is described in WO 98/13465, WO 98/015619, WO 98/015633, WO 99/06574, WO 99/10481, WO 99/025847, WO 99/031255, WO 2002/101078, WO 2003/027306, WO 2003/052054, WO 2003/052055, WO 2003/052056, WO 2003/052057, WO 2003/052118, WO 2004/016760, WO 2004/043980, WO 2004/048592, WO 2005/001065, WO 2005/028636, WO 2005/093050, WO 2005/093073, WO 2006/074005, WO 2006/117432, WO 2007/071818, WO 2007/071820, WO 2008/008070, WO 2008/008793, U.S. Patent No. 5, 457, 046, U.S. Patent No. 5, 648, 263 and U.S. Patent No. 5, 686, 593.

In the method for the invention, any component of GH61 polypeptide as enzyme composition with cellulolytic enhancing activity can be used.

In first, described in there is cellulolytic enhancing activity GH61 polypeptide comprise following motif:

[ILMV]-P-X (4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X (4)-[HNQ] (SEQ ID NO:29 or SEQ ID NO:30) and [FW]-[TF]-K-[AIV],

Wherein X is arbitrary amino acid, and X (4,5) is the arbitrary amino acid on 4 or 5 continuous positions, and X (4) is the arbitrary amino acid on 4 continuous positions.

The isolated polypeptide comprising above-mentioned shown motif can also comprise:

H-X (1,2)-G-P-X (3)-[YW]-[AILMV] (SEQ ID NO:31 or SEQ ID NO:32),

[EQ]-X-Y-X (2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO:33), or

H-X (1,2)-G-P-X (3)-[YW]-[AILMV] (SEQ ID NO:31 or SEQ ID NO:32) and [EQ]-X-Y-X (2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO:33)

Wherein X is arbitrary amino acid, and X (1,2) is the arbitrary amino acid on 1 position or 2 continuous positions, and X (3) is the arbitrary amino acid on 3 continuous positions, and X (2) is the arbitrary amino acid on 2 continuous positions.In above-mentioned motif, adopt the IUPAC one letter amino abbreviation of generally acknowledging.

In preferred at one, the described GH61 polypeptide with the separation of cellulolytic enhancing activity also comprises H-X (1,2)-G-P-X (3)-[YW]-[AILMV] (SEQ ID NO:31 or SEQ ID NO:32).In another is preferred, the GH61 polypeptide with the separation of cellulolytic enhancing activity also comprises [EQ]-X-Y-X (2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO:33).In another is preferred, the GH61 polypeptide with the separation of cellulolytic enhancing activity also comprises H-X (1,2)-G-P-X (3)-[YW]-[AILMV] (SEQ ID NO:31 or SEQ ID NO:32) and [EQ]-X-Y-X (2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO:33).

In second, described in there is cellulolytic enhancing activity isolated polypeptide comprise following motif:

[ILMV]-P-x (4,5)-G-x-Y-[ILMV]-x-R-x-[EQ]-x (3)-A-[HNQ] (SEQ ID NO:34 or SEQ ID NO:35),

Wherein x is arbitrary amino acid, and x (4,5) is the arbitrary amino acid on 4 or 5 continuous positions, and x (3) is the arbitrary amino acid on 3 continuous positions.In above-mentioned motif, adopt the IUPAC one letter amino abbreviation of generally acknowledging.

The example with the GH61 polypeptide of cellulolytic enhancing activity of technique used in the present invention includes but not limited to from autochthonal shuttle spore mould (WO 2005/074647, WO 2008/148131 and WO 2011/035027), the tangerine hot ascomycetes of orange (WO 2005/074656 and WO 2010/065830), Trichodermareesei (WO 2007/089290), thermophilic fungus destroyed wire (WO 2009/085935, WO 2009/085859, WO 2009/085864, WO 2009/085868), the GH61 polypeptide of Aspergillus fumigatus (WO 2010/138754), from addicted to loose mould (WO 2011/005867), thermophilic ascomycete bacterial classification (WO 2011/039319), Penicillium species (WO 2011/041397), with the GH61 polypeptide (WO 2011/041504) of crust thermophilic ascomycete (Thermoascus crustaceous).

In one aspect, described in there is cellulolytic enhancing activity the solubility activation divalent metal of GH61 polypeptide described in WO 2008/151043, such as, use under the existence of manganese or copper.

In yet another aspect, the GH61 polypeptide described in cellulolytic enhancing activity uses under titanium dioxide compound, bicyclic compound, heterogeneous ring compound, nitrogenous compound, naphtoquinone compounds, sulfocompound or the existence of liquor that obtains as pretreated maize straw (PCS) from pretreated cellulose materials.

Described titanium dioxide compound can comprise any suitable combination thing containing two or more Sauerstoffatoms.In some respects, described titanium dioxide compound contains the aryl module (moiety) replaced as described herein.Described titanium dioxide compound can comprise one or more (such as several) hydroxyl and/or hydroxy derivatives, but also comprises the aryl module of the replacement lacking hydroxyl and hydroxy derivatives.The non-limiting example of titanium dioxide compound comprises pyrocatechol or catechol; Coffic acid; PCA; The 4-tertiary butyl-5-methoxyl group-1,2-dihydroxy-benzene; Pyrogallol; Gallic acid; Methyl-Gallic Acid; 2,3,4-trihydroxybenzophenone; 2,6-syringol; Sinapinic acid; 3,5-resorcylic acid; Chloro-1, the 2-dihydroxy-benzene of 4-; 4-nitro-1,2-dihydroxy-benzene; Tannic acid; Progallin A; Hydroxyethanoic acid methyl esters; Dihydroxyl fumaric acid; 2-butyne-Isosorbide-5-Nitrae-glycol; Croconic acid; 1,3-PD; Tartrate; 2,4-pentanediol; 3-oxyethyl group-1,2-PD; 2,4,4 '-trihydroxybenzophenone; Cis-2-butene-Isosorbide-5-Nitrae-glycol; Squaric acid; Otan; Acetyl acrolein (acrolein acetal); Methyl-4-HBA; 4-HBA; With methyl-3,5-dimethoxy-4 's-hydroxy-benzoic acid; Or their salt or solvate (solvate).

Described bicyclic compound can comprise any replacement carbocyclic fused ring system suitable as described herein.Described compound can comprise one or more (such as several) other ring, and unless otherwise specified, is not limited to concrete number of rings.In one aspect, described bicyclic compound is flavonoid.In yet another aspect, described bicyclic compound is the optional isoflavonoid (isoflavonoid) replaced.In yet another aspect, described bicyclic compound is the optional pattern replaced ion (flavylium ion), as the cyanidin(e) optionally replaced or the anthocyanogen optionally replaced, or derivatives thereof.The non-limiting example of bicyclic compound comprises l-Epicatechol (epicatechin); Quercetin (quercetin); Myricetin (myricetin); Taxifolin (taxifolin); Kaempferol (kaempferol); Sang Su (morin); Acacetin (acacetin); Naringenin (naringenin); Isorhamnetin (isorhamnetin); Apigenin (apigenin); Anthocyanidin (cyanidin); Anthocyanin (cyanin); Kuromanin; Keracyanin (keracyanin); Or their salt or solvate.

Described heterogeneous ring compound can be any suitable compound, and what optionally replace as described herein comprises heteroatomic aromatic ring or non-aromatic ring.In one aspect, described heterocycle is the compound comprising the optional Heterocyclylalkyl module replaced or the heteroaryl module optionally replaced.In yet another aspect, the Heterocyclylalkyl module of described optional replacement or the heteroaryl module of optional replacement are the optional five-membered ring alkyl replaced or the quinary heteroaryl module optionally replaced.In yet another aspect, the optional Heterocyclylalkyl replaced or the heteroaryl module optionally replaced are the modules being selected from following optional replacement: pyrazolyl, furyl, imidazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrryl, pyridyl, pyrimidyl, pyridazinyl, thiazolyl, triazolyl, thienyl (thienyl), dihydro-thiophene-pyrazolyl (dihydrothieno-pyrazolyl), thianaphthenyl, carbazyl, benzimidazolyl-, benzothienyl (benzothienyl), benzofuryl, indyl, quinolyl, benzotriazole base, benzothiazolyl, benzoxazolyl (benzooxazolyl), benzimidazolyl-, isoquinolyl, pseudoindoyl, acridyl, benzoisoxazole base (benzoisazolyl), T10, pyrazinyl, tetrahydrofuran base, pyrrolinyl, pyrrolidyl, morpholinyl, indyl, diazacyclo heptantriene base (diazepinyl), nitrogen heterocyclic heptantriene base (azepinyl), thia cycloheptatriene base (thiepinyl), piperidyl and oxepin base (oxepinyl).The Heterocyclylalkyl module of described optional replacement or the heteroaryl module of optional replacement are the optional furyls replaced in yet another aspect.The non-limiting example of heterogeneous ring compound comprises (1,2-dihydroxy ethyl)-3,4-dihydrofuran-2 (5H)-one; 4-hydroxy-5-methyl base-3-furanone; 5-hydroxyl-2 (5H)-furanone; [1,2-dihydroxy ethyl] furans-2,3,4 (5H)-triketone; Alpha-hydroxy-gamma-butyrolactone; Ribonic acid gamma lactone; Hexanal saccharic acid gamma lactone (aldohexuronicaldohexuronic acid γ-lactone); Glucopyrone; 4 hydroxy coumarin; Dihydrobenzofuranes; 5-(methylol) furfural; Furoin (furoin); 2 (5H)-furanones; 5,6-dihydro-2H-pyran-2-one; With 5,6-dihydro-4-hydroxyl-6-methyl-2H-pyran-2-one; Or their salt or solvate.

Described nitrogenous compound can be any suitable combination thing with one or more nitrogen-atoms.In one aspect, described nitrogenous compound comprises amine, imines, azanol or Nitrous Oxide (nitroxide) module.The non-limiting example of nitrogenous compound comprises acetoxime; Violuric acid; Pyridine-2-aldoxime; Ortho-Aminophenol; 1,2-phenylenediamine; 2,2,6,6-tetramethyl--piperidino oxygen (piperidinyloxy); 5,6,7,8-tetrahydrobiopterin; 6,7-dimethyl-5,6,7,8-tetrahydrochysene pterin; And maleinamic acid; Or their salt or solvate.

Described naphtoquinone compounds can be any described suitable compound comprising quinone module herein.The non-limiting example of naphtoquinone compounds comprises Isosorbide-5-Nitrae-benzoquinones; 1,4-naphthoquinone; 2 hydroxy 1,4 naphthoquinone (lawsone); 2,3-dimethoxy-5-methyl isophthalic acid, 4-benzoquinones or ubiquinone ₀; 2,3,5,6-tetramethyl--Isosorbide-5-Nitrae-benzoquinones or duroquinone; Isosorbide-5-Nitrae-dihydroxyanthraquinone; 3-hydroxyl-1-methyl-5,6-indoline diketone or carbazochrome; The 4-tertiary butyl-5-methoxyl group-1,2-benzoquinones; Pyrroloquinoline quinone (pyrroloquinoline quinone); Or their salt or solvate.

Described sulfocompound can be any suitable compound comprising one or more sulphur atom.In one aspect, described sulfocompound comprises and is selected from following module: thionyl, thioether, sulfinyl, sulphonyl, sulphamide (sulfamide), sulphonamide (sulfonamide), sulfonic acid and sulphonate.The non-limiting example of sulfocompound comprises sulfur alcohol; 2-propylmercaptan; 2-propylene-1-mercaptan; Mistabrom; Benzenethiol; Benzene-1,2-bis-mercaptan; Halfcystine; Methionine(Met); Gsh; Gelucystine; Or their salt or solvate.

In one aspect, this kind of compound as above, to the significant quantity of cellulose materials, in the molar ratio to cellulose sugar unit, is about 10 ^-6such as, to about 10, about 10 ^-6to about 7.5, about 10 ^-6to about 5, about 10 ^-6to about 2.5, about 10 ^-6to about 1, about 10 ^-5to about 1, about 10 ^-5to about 10 ^-1, about 10 ^-4to about 10 ^-1, about 10 ^-3to about 10 ^-1, or about 10 ^-3to about 10 ^-2.In yet another aspect, the significant quantity of compound as above be about 0.1 μM to about 1M, such as about 0.5 μM to about 0.75M, about 0.75 μM to about 0.5M, about 1 μM to about 0.25M, about 1 μM to about 0.1M, about 5 μMs to about 50mM, about 10 μMs to about 25mM, about 50 μMs to about 25mM, about 10 μMs to about 10mM, about 5 μMs to about 5mM, or about 0.1mM to about 1mM.

Under term " liquor (liquor) " means condition described in this article, by the ligno-cellulose in process slurry and/or hemicellulosic materials, or its monose such as wood sugar, pectinose, seminose etc., the solution phase produced, i.e. aqueous phase, organic phase or its combination, and solubility inclusion.The liquor strengthened for the cellulose decomposition of GH61 polypeptide by, optionally under the existence of catalyzer such as acid, optionally in presence of organic solvent, and optional process cellulose materials or hemicellulosic materials (or raw material) with to the physical damage of described material is combined by applying hot and/or pressure, then solution is separated with residual solid and produces.This type of conditional decision in the process by Cellulase preparation hydrolysis fiber cellulosic material by degree that the obtainable cellulose decomposition of combination of liquor and GH61 polypeptide strengthens.Described liquor can use the standard method in this area as filtered, deposition or centrifugal from treated material separation.

In one aspect, described liquor is about 10 to cellulosic significant quantity ^-6to the every g Mierocrystalline cellulose of about 10g, such as about 10 ^-6to about 7.5g, about 10 ^-6to about 5, about 10 ^-6to about 2.5g, about 10 ^-6to about 1g, about 10 ^-5to about 1g, about 10 ^-5to about 10 ^-1g, about 10 ^-4to about 10 ^-1g, about 10 ^-3to about 10 ^-1g, or about 10 ^-3to about 10 ^-2the every g Mierocrystalline cellulose of g.

In one aspect, one or more (such as several) hemicellulose lytic enzymes described comprise commercial hemicellulose lytic enzyme prepared product.The example being applicable to commercial hemicellulose lytic enzyme prepared product of the present invention comprises, such as SHEARZYME ^tM(Novozymes A/S), hTec (Novozymes A/S), hTec2 (Novozymes A/S), hTec3 (Novozymes A/S), (Novozymes A/S), (Novozymes A/S), hC (Novozymes A/S), xylanase (Genencor), xY (Genencor), xC (Genencor), tX-200A (AB Enzymes), HSP6000Xylanase (DSM), DEPOL ^tM333P (Biocatalysts Limit, Wales, UK), DEPOL ^tM740L (Biocatalysts Limit, Wales, UK) and DEPOL ^tM762P (Biocatalysts Limit, Wales, UK).

The example that can be used for the zytase of present invention process includes but not limited to from microorganism Aspergillus aculeatus (Aspergillus aculeatus) (GeneSeqP:AAR63790; WO 94/21785), Aspergillus fumigatus (Aspergillus fumigatus) (WO 2006/078256), cup fungi GH10 (WO 2011/057083) that to become mildewed addicted to loose mould (WO 2011/041405), Penicillium species (WO 2010/126772), autochthonal shuttle spore mould (Thielavia terrestris) NRRL8126 (WO 2009/079210) and brown spore zytase.

The example that can be used for the xylobiase of present invention process includes but not limited to the xylobiase saving bacterium (Talaromyces emersonii) (SwissProt accession number Q8X212) from Neuraspora crassa (Neurospora crassa) (SwissProt accession number Q7SOW4), Trichodermareesei (Trichoderma reesei) (UniProtKB/TrEMBL accession number Q92458) and Ai Mosen ankle.

The example that can be used for the acetyl xylan esterase of present invention process includes but not limited to from microorganism Aspergillus aculeatus (WO 2010/108918), chaetomium globosum (Chaetomium globosum) (Uniprot accession number Q2GWX4), thin beautiful chaetomium (Chaetomium gracile) (GeneSeqP accession number AAB82124), Humicola insolens (Humicola insolens) DSM1800 (WO 2009/073709), Hypocrea jecorina (Hypocrea jecorina) (WO 2005/001036), thermophilic fungus destroyed wire (Wo2010/014880), Neuraspora crassa (UniProt accession number q7s259), the acetyl xylan esterase of grain husk withered septoria musiva (Phaeosphaeria nodorum) (Uniprot accession number Q0UHJ1) and the mould NRRL8126 of autochthonal shuttle spore (WO 2009/042846).

The example that can be used for the feruloyl esterase of present invention process includes but not limited to the feruloyl esterase from Humicola insolens DSM1800 (WO 2009/076122), Fei Xixinsatuo bacterium (Neosartorya fischer) (UniProt accession number A1D9T4), Neuraspora crassa (UniProt accession number Q9HGR3), tangerine ash mould (WO 2009/127729) and autochthonal shuttle spore mould (WO 2010/053838 and WO 2010/065448).

The example that can be used for the arabinofuranosidase of present invention process includes but not limited to the arabinofuranosidase from aspergillus niger (Aspergillus niger) (GeneSeqP accession number AAR94170), Humicola insolens (Humicola insolens) DSM1800 (WO 2006/114094 and WO 2009/073383) and M.giganteus (WO 2006/114094).

The example that can be used for the alpha-glucuronidase of the inventive method includes but not limited to from excellent aspergillus (Aspergillus clavatus) (UniProt accession number alcc12), Aspergillus fumigatus (SwissProt accession number Q4WW45), aspergillus niger (Uniprot accession number Q96WX9), terreus (Aspergillus terreus) (SwissProt accession number Q0CJP9), Humicola insolens (WO 2010/014706), tangerine ash mould (WO 2009/068565), the alpha-glucuronidase of Ai Mosen ankle joint bacterium (UniProt accession number Q8X211) and Trichodermareesei (Uniprot accession number Q99024).

For the polypeptide with enzymic activity of present invention process by the nutritional medium containing suitable Carbon and nitrogen sources and inorganic salt, use means known in the art (see, such as Bennett, and LaSure J.W., L. (volume), More Gene Manipulations in Fungi, Academic Press, CA, 1991) the above-mentioned microorganism strains pointed out that ferments produces.Suitable substratum can obtain from supplier, or can according to published composition preparation (catalogue of such as American type culture collection).The temperature range being suitable for growing and enzyme produces is known in the art with other conditions (see, such as Bailey, J.E. and Ollis, D.F., Biochemical Engineering Fundamentals, McGraw-Hill Book Company, NY, 1986).

Described fermentation can be any method causing the culturing cell of enzyme or protein expression or separation.Therefore, fermentation can be understood as and to be included in suitable substratum and at the shake-flask culture allowing to carry out under the described enzyme condition being expressed or be separated, or in laboratory or industrial fermentation tank little-or large scale fermentation (comprise continuously, in batches, fed-batch or solid state fermentation).The enzyme of the gained produced by aforesaid method can from fermention medium recovery and by ordinary method purifying.

Fermentation.The fermentable sugars that the cellulose materials that hydrolysis is hung oneself in the organism of fermentation fermentation that sugar directly or indirectly can be fermented into required tunning by one or more (such as several) obtains." fermentation " or " fermentation process " refers to any fermentation process or any method comprising fermentation step.Fermentation process also comprises the fermentation process for consumer's goods alcohol industry (such as, beer and grape wine), Dairy industry (such as, fermented milk prod), leather industry and tobacco.Fermentation condition depends on tunning and the fermenting organisms of expectation, and easily can be determined by those skilled in the art.

In fermentation step, the sugar that the result as pre-treatment and enzyme hydrolysis step discharges from cellulose materials, becomes product by fermenting organisms (as yeast) fermentation, such as, and ethanol.As described herein, it can be separately or simultaneously for being hydrolyzed (saccharification) and fermenting.

Implementing in fermentation step of the present invention, to use any suitable cellulose materials through hydrolysis.Leavened prod needed for usual basis (that is, the material that will obtain from fermentation) and the method used select described material, as known in the art.

Term " fermention medium " can be regarded as the substratum before referring to add organism of fermentation in this article, e.g., and the substratum produced by saccharifying, and the substratum of synchronous glycosylation and the middle use of fermentation process (SSF).

" organism of fermentation " refers to be applicable to any microorganism that desirable fermentation process produces tunning, comprises bacterium and fungal organism.Fermenting organisms can be hexose and/or pentose fermentation organism, or their combination.Hexose and pentose fermentation organism are all known in this area.The leavened prod that sugar (as glucose, wood sugar, xylulose, pectinose, maltose, seminose, semi-lactosi and/or oligosaccharides) can ferment needed for (that is, conversion) one-tenth by suitable organism of fermentation directly or indirectly.The example of the bacterium and fungi fermentation organism that can produce ethanol as Lin etc., described in 2006, Appl.Microbiol.Biotechnol.69:627-642.

The example of the organism of fermentation of energy zymohexose comprises bacterium and fungal organism, as yeast.Preferred yeast comprises mycocandida, genus kluyveromyces and yeast belong, such as the bacterial strain of Candida sonorensis, kluyveromyces marxianus and yeast saccharomyces cerevisiae.

Bacterium and fungal organism is comprised, as some yeast with the example of the fermenting organisms of its native state energy ferment pentoses.Preferred wood-sugar fermentation yeast comprises mycocandida, preferred shehatae candida (Candida sheatae) or Candida sonorensis; And Pichia, the bacterial strain of preferred pichia stipitis (Pichia stipitis), as the bacterial strain of pichia stipitis CBS5773.Preferred pentose fermentation yeast comprises pipe capsule yeast belong (Pachysolen), the bacterial strain of preferred pachysolen tannophilus (Pachysolen tannophilus).Can not ferment pentoses as the biology of wood sugar and pectinose by means known in the art genetic modification ferment pentoses.

Can the bacterium of ethanol be effectively become by hexose to comprise with pentose fermentation, such as, Bacillus coagulans (Bacillus coagulans), clostridium acetobutylicum (Clostridium acetobutylicum), Clostridium thermocellum (Clostridium thermocellum), Clostridium phytofermentans, ground bacillus belong to bacterial classification, separate sugared hot anaerobic bacillus(cillus anaerobicus) (Thermoanaerobacter saccharolyticum) and zymomonas mobilis (Zymomonas mobilis) (Philippidis, 1996, see above).

Other fermenting organism comprises bacillus, as Bacillus coagulans, mycocandida, as Candida sonorensis, C.methanosorbosa, Di Dansi candiyeast (Candida diddensii), Candida parapsilosis (Candida parapsilosis), C.naedodendra, C.blankii, C.entomophilia, rape candiyeast (C.brassicae), candida pseudotropicalis (Candida pseudotropicalis), Candida boidinii (Candida boidinii), Candida utilis (Candida utilis) and shehatae candida (C.scehatae), fusobacterium, as clostridium acetobutylicum, Clostridium thermocellum and C.phytofermentans, the coli strain that intestinal bacteria, particularly genetically modified promotion ethanol produce, ground bacillus belongs to bacterial classification, Hansenula, as Hansenula anomala (Hansenula anomala), Klebsiella (Klebsiella), as acid-producing Klebsiella bacterium (Klebsiella oxytoca), genus kluyveromyces, as kluyveromyces marxianus, Kluyveromyces lactis (K.lactis), K.thermotolerans and Kluyveromyces fragilis, Schizosaccharomyces, as schizosaccharomyces pombe (S.pombe), hot anaerobic bacillus(cillus anaerobicus) belongs to (Thermoanaerobacter), as separated sugared hot anaerobic bacillus(cillus anaerobicus), and zymomonas (Zymomonas), as the bacterial strain of zymomonas mobilis.

In preferred at one, yeast is that Brettanomyces belongs to (Bretannomyces).In preferred at one, yeast is Ke Laosen Brettanomyces (Bretannomyces clausenii).In another more preferred aspect, yeast is candiyeast.In another more preferred aspect, yeast is Candida sonorensis.In another more preferred aspect, yeast is Candida boidinii.In another more preferred aspect, yeast is Candida blankii.In another more preferred aspect, yeast is rape candiyeast.In another more preferred aspect, yeast is Di Dansi candiyeast.In another more preferred aspect, yeast is Candida entomophiliia.In another more preferred aspect, yeast is candida pseudotropicalis.In another more preferred aspect, yeast is shehatae candida.In another more preferred aspect, yeast is Candida utilis.In another is preferred, yeast is excellent spore yeast belong (Clavispora).In another more preferred aspect, yeast is Clavispora lusitaniae yeast (Clavispora lusitaniae).In another more preferred aspect, yeast is Root and stem of Cholla rod spore yeast (Clavispora opuntiae).In another is preferred, yeast is kluyveromyces.In another more preferred aspect, yeast is Kluyveromyces fragilis.In another more preferred aspect, yeast is kluyveromyces marxianus.In another more preferred aspect, yeast is Kluyveromyces thermotolerans.In another is preferred, yeast is pipe capsule yeast belong (Pachysolen).In another more preferred aspect, yeast is pachysolen tannophilus.In another is preferred, yeast is pichia spp.In another more preferred aspect, yeast is pichia stipitis.In another is preferred, yeast is Saccharomyces sp.In another is preferred, yeast is yeast saccharomyces cerevisiae.In another more preferred aspect, yeast is saccharomyces diastaticus (Saccharomyces distaticus).In another more preferred aspect, yeast is saccharomyces uvarum (Saccharomyces uvarum).

In preferred at one, bacterium is bacillus.In preferred at one, bacterium is Bacillus coagulans.In another more preferred aspect, bacterium is fusobacterium.In another more preferred aspect, bacterium is clostridium acetobutylicum.In another more preferred aspect, bacterium is Clostridium phytofermentans.In another more preferred aspect, bacterium is Clostridium thermocellum.In another more preferred aspect, bacterium is that ground bacillus belongs to bacterial classification.In another more preferred aspect, bacterium is that hot anaerobic bacillus(cillus anaerobicus) belongs to.In another more preferred aspect, bacterium separates sugared hot anaerobic bacillus(cillus anaerobicus).In another more preferred aspect, bacterium is zymomonas.In another more preferred aspect, bacterium is zymomonas mobilis.

The yeast that commercially available applicable ethanol produces comprises, such as BIOFERM ^tMaFT and XR (NABC-North American Bioproducts Corporation, GA, USA), ETHANOL RED ^tMyeast (Red Star/Lesaffre, USA), FALI ^tM(Fleischmann ' s Yeast, Burns Philp Food Inc., USA), FERMIOL ^tM(DSM Specialties), GERT STRAND ^tM(Gert Strand AB, Sweden) and SUPERSTART ^tMand THERMOSACC ^tMfresh yeast (Ethanol Technology, WI, USA).

In preferred at one, organism of fermentation provides the ability of ferment pentoses through genetic modification, as utilized wood sugar, utilizes pectinose and jointly utilizes the microorganism of wood sugar and pectinose.

Construct organism (Chen and Ho that hexose and pentose can be changed into ethanol (altogether fermentation) by heterologous gene being cloned into multiple organism of fermentation, 1993, Cloning and improving the expression of Pichia stipitis xylose reductase gene in Saccharomyces cerevisiae, Appl.Biochem.Biotechnol.39-40:135-147; Ho etc., 1998, Genetically engineered Saccharomyces yeast capable of effectively cofermenting glucose and xylose, Appl.Environ.Microbiol.64:1852-1859; Kotter and Ciriacy, 1993, Xylose fermentation by Saccharomyces cerevisiae, Appl.Microbiol.Biotechnol.38:776-783; Walfridsson etc., 1995, Xylose-metabolizing Saccharomyces cerevisiae strains overexpressing the TKL1and TAL1genes encoding the pentose phosphate pathway enzymes transketolase and transaldolase, Appl.Environ.Microbiol.61:4184-4190; Kuyper etc., 2004, Minimal metabolic engineering of Saccharomyces cerevisiae for efficient anaerobic xylose fermentation:a proof of principle, FEMS Yeast Research4:655-664; Beall etc., 1991, Parametric studies of ethanol production from xylose and other sugars by recombinant Escherichia coli, Biotech.Bioeng.38:296-303; Ingram etc., 1998, Metabolic engineering of bacteria for ethanol production, Biotechnol.Bioeng.58:204-214; Zhang etc., 1995, Metabolic engineering of a pentose metabolism pathway in ethanologenic Zymomonas mobilis, Science267:240-243; Deanda etc., 1996, Development of an arabinose-fermenting Zymomonas mobilis strain by metabolic pathway engineering, Appl.Environ.Microbiol.62:4465-4470; WO 2003/062430, xylose isomerase).

In preferred at one, the organism of fermentation through genetic modification is Candida sonorensi.In another is preferred, the organism of fermentation through genetic modification is intestinal bacteria.In another is preferred, the organism of fermentation through genetic modification is acid-producing Klebsiella bacterium.In another is preferred, described genetically modified organism of fermentation is kluyveromyces marxianus.In another is preferred, described genetically modified organism of fermentation is yeast saccharomyces cerevisiae.In another is preferred, the organism of fermentation through genetic modification is zymomonas mobilis.

As known in the art, above-mentioned organism can also for generation of other material, as described herein.

Usually add organism of fermentation to the cellulose materials of degrading or hydrolyzate, and carry out about 8 to about 96 hours, such as about 24 to about 60 hours fermentation.Temperature is generally about 26 DEG C to about 60 DEG C, such as about 32 DEG C or 50 DEG C, and at about pH3 such as, to about pH8, about pH4-5,6 or 7.

In one aspect, yeast and/or another kind of microorganism are used to the cellulose materials of degraded, and carries out about 12 to about 96 hours, as being generally 24-60 hours fermentation.In yet another aspect, temperature is preferably about 20 DEG C to about 60 DEG C, such as about 25 DEG C to about 50 DEG C, and about 32 DEG C to about 50 DEG C, about 32 DEG C to about 50 DEG C, and pH is generally about pH3 to about pH7, such as about pH4 to about pH7.But some fermenting organisms such as bacterium, has the suitableeest higher leavening temperature.Yeast or another kind of microorganism are preferably with about 10 ⁵-10 ¹², preferably about 10 ⁷-10 ¹⁰, particularly about 2x10 ⁸the amount of the every ml fermented liquid of viable count is used." The Alcohol Textbook " (K.Jacques is found in such as about the further guidance using yeast to carry out fermenting, T.P.Lyons and D.R.Kelsall compiles, Nottingham University Press, United Kingdom1999), it is incorporated to herein by carrying stating.

Fermentation stimulating substance can use with any Combination of Methods as herein described, to improve zymotechnique further, particularly improves the performance of organism of fermentation, and e.g., speed increases and alcohol getting rate." fermentation stimulating substance " refers to the stimulant grown for organism of fermentation (particularly yeast).Preferably comprise VITAMIN and mineral substance for the fermentation stimulating substance grown.The example of VITAMIN comprises multivitamin, vitamin H, pantothenic acid (salt), nicotinic acid, meso-inositol (meso-inositol), VitB1, pyridoxol (pyridoxine), para-amino benzoic acid, folic acid, riboflavin and vitamin A, B, C, D and E.See, such as, Alfenore etc., Improving ethanol production and viability of Saccharomyces cerevisiae by a vitamin feeding strategy during fed-batch process, Springer-Verlag (2002), it is incorporated to herein by carrying stating.The example of mineral substance comprises can provide nutraceutical mineral substance and mineral salt, and described nutrition comprises P, K, Mg, S, Ca, Fe, Zn, Mn and Cu.

tunning: tunning can be any material being derived from fermentation.Tunning can be, be not limited to, alcohol (such as, arabitol, propyl carbinol, isopropylcarbinol, ethanol, glycerine, methyl alcohol, ethylene glycol, 1,3-PD (propylene glycol), butyleneglycol, glycerol, sorbyl alcohol and Xylitol); Alkane (such as pentane, hexane, heptane, octane, nonane, decane, undecane and dodecane); Naphthenic hydrocarbon (such as pentamethylene, hexanaphthene, suberane and cyclooctane); Alkene (such as amylene, hexene, heptene and octene); Amino acid (such as, aspartic acid, L-glutamic acid, glycine, Methionin, Serine and Threonine); Gas (such as, methane, hydrogen (H ₂), carbonic acid gas (CO ₂) and carbon monoxide (CO)); Isoprene; Ketone (such as, acetone); Organic acid (such as, acetic acid, acetonic acid, hexanodioic acid, xitix, citric acid, 2,5-diketo-D gluconate, formic acid, FUMARIC ACID TECH GRADE, saccharic acid, glyconic acid, glucuronic acid, pentanedioic acid, 3-hydroxy-propionic acid, methylene-succinic acid, lactic acid, oxysuccinic acid, propanedioic acid, oxalic acid, oxaloacetic acid, propionic acid, succsinic acid and xylosic acid); And polyketide.Tunning can also be the protein as high-value product.

In preferred at one, tunning is alcohol.Will be understood that, term " alcohol " comprises the material comprising one or more oh group.In preferred, described alcohol is propyl carbinol.In another more preferred aspect, described alcohol is isopropylcarbinol.In another more preferred aspect, described alcohol is ethanol.In another more preferred aspect, described alcohol is methyl alcohol.In another more preferred aspect, described alcohol is arabitol.In another more preferred aspect, described alcohol is butyleneglycol.In another more preferred aspect, described alcohol is ethylene glycol.In another more preferred aspect, described alcohol is glycerol (glycerin).In another more preferred aspect, described alcohol is glycerine (glycerol).In another more preferred aspect, described alcohol is 1,3-PD.In another more preferred aspect, described alcohol is sorbyl alcohol.In another more preferred aspect, described alcohol is Xylitol.See, such as, Gong, C.S., Cao, N.J., Du, J., and Tsao, G.T., 1999, Ethanol production from renewable resources, in Advances in Biochemical Engineering/Biotechnology, Scheper, T. compile, Springer-Verlag Berlin Heidelberg, Germany, 65:207-241; Silveira, M.M., and Jonas, R., 2002, The biotechnological production of sorbitol, Appl.Microbiol.Biotechnol.59:400-408; Nigam, P. and Singh, D., 1995, Processes for fermentative production of xylitol – a sugar substitute, Process Biochemistry30 (2): 117-124; Ezeji, T.C., Qureshi, and Blaschek N., H.P., 2003, Production of acetone, butanol and ethanol by Clostridium beijerinckii BA101and in situ recovery by gas stripping, World Journal of Microbiology and Biotechnology19 (6): 595-603.

In another is preferred, described tunning is alkane.Described alkane is the alkane of non-branching or branching.In another more preferred aspect, described alkane is pentane.In another more preferred aspect, described alkane is hexane.In another more preferred aspect, described alkane is heptane.In another more preferred aspect, described alkane is octane.In another more preferred aspect, described alkane is nonane.In another more preferred aspect, described alkane is decane.In another more preferred aspect, described alkane is undecane.In another more preferred aspect, described alkane is dodecane.

In another is preferred, described tunning is naphthenic hydrocarbon.In another more preferred aspect, described naphthenic hydrocarbon is pentamethylene.In another more preferred aspect, described naphthenic hydrocarbon is hexanaphthene.In another more preferred aspect, described naphthenic hydrocarbon is suberane.In another more preferred aspect, described naphthenic hydrocarbon is cyclooctane.

In another is preferred, described tunning is alkene.Described alkene can be the alkene of non-branching or branching.In another more preferred aspect, described alkene is amylene.In another more preferred aspect, described alkene is hexene.In another more preferred aspect, described alkene is heptene.In another more preferred aspect, described alkene is octene.

In another is preferred, described tunning is amino acid.In another more preferred aspect, described organic acid is aspartic acid.In another more preferred aspect, described amino acid is L-glutamic acid.In another more preferred aspect, described amino acid is glycine.In another more preferred aspect, described amino acid is Methionin.In another more preferred aspect, described amino acid is Serine.In another more preferred aspect, described amino acid is Threonine.See, such as, Richard, and Margaritis A., A., 2004, Empirical modeling of batch fermentation kinetics for poly (glutamic acid) production and other microbial biopolymers, Biotechnology and Bioengineering87 (4): 501-515.

In another is preferred, described material is gas.In another more preferred aspect, described gas is methane.In another more preferred aspect, described gas is H ₂.In another more preferred aspect, described gas is CO ₂.In another more preferred aspect, described gas is CO.See, such as, Kataoka, N., A.Miya, and K.Kiriyama, 1997, Studies on hydrogen production by continuous culture system of hydrogen-producing anaerobic bacteria, Water Science and Technology36 (6-7): 41-47; And Gunaseelan, V.N., in Biomass and Bioenergy, Vol.13 (1-2), pp83-114,1997, Anaerobic digestion of biomass for methane production:A review.

In another is preferred, described tunning is isoprene.

In another is preferred, described tunning is ketone.It should be understood that term " ketone " covers the ketone containing one or more ketone module.In another more preferred aspect, described ketone is acetone.See, such as Qureshi and Blaschek, 2003, see above.

In another is preferred, described tunning is organic acid.In another more preferred aspect, described organic acid is acetic acid.In another more preferred aspect, described organic acid is acetonic acid.In another more preferred aspect, described organic acid is hexanodioic acid.In another more preferred aspect, described organic acid is xitix.In another more preferred aspect, described organic acid is citric acid.In another more preferred aspect, described organic acid is 2,5-diketo-D gluconate.In another more preferred aspect, described organic acid is formic acid.In another more preferred aspect, described organic acid is FUMARIC ACID TECH GRADE.In another more preferred aspect, described organic acid is saccharic acid.In another more preferred aspect, described organic acid is glyconic acid.In another more preferred aspect, described organic acid is glucuronic acid.In another more preferred aspect, described organic acid is pentanedioic acid.In another is preferred, described organic acid is 3-hydroxy-propionic acid.In another more preferred aspect, described organic acid is methylene-succinic acid.In another more preferred aspect, described organic acid is lactic acid.In another more preferred aspect, described organic acid is oxysuccinic acid.In another more preferred aspect, described organic acid is propanedioic acid.In another more preferred aspect, described organic acid is oxalic acid.In another more preferred aspect, described organic acid is propionic acid.In another more preferred aspect, described organic acid is succsinic acid.In another more preferred aspect, described organic acid is xylosic acid.See, such as, Chen, R. and Lee, Y.Y., 1997, Membrane-mediated extractive fermentation for lactic acid production from cellulosic biomass, Appl.Biochem.Biotechnol.63-65:435-448.

In another is preferred, described material is polyketide.

reclaimcan use any method known in the art, optionally reclaim tunning from fermention medium, described method includes, but not limited to chromatography, electrophoresis method, differential solubility, distillation or extraction.Such as, be separated and purified alcohols by the cellulose materials of conventional distil-lation method from fermentation.Can obtain the ethanol that high purity is about 96vol.%, it can be used as, such as, and alcohol fuel, drinking alcohol (that is, drinkable neutral alcoholic drinks), or industrial alcohol.

Signal peptide

The invention still further relates to the polynucleotide of the separation of coded signal peptide, described signal peptide comprises or consists of the amino acid/11 to 17 of SEQ ID NO:2, the amino acid/11 to 20 of SEQ ID NO:4, the amino acid/11 to 21 of SEQ ID NO:6, or the amino acid/11 to 17 of SEQ ID NO:8.Described polynucleotide can comprise the gene of proteins encoded further, and it is operably connected to signal peptide.Described albumen is preferably external source for described signal peptide.In one aspect, the polynucleotide of described signal peptide of encoding are the Nucleotide 1 to 51 of SEQ ID NO:1.In yet another aspect, the polynucleotide of described signal peptide of encoding are the Nucleotide 1 to 60 of SEQ ID NO:3.In yet another aspect, the polynucleotide of described signal peptide of encoding are the Nucleotide 1 to 63 of SEQ ID NO:5.In yet another aspect, the polynucleotide of described signal peptide of encoding are the Nucleotide 1 to 51 of SEQ ID NO:7.

The invention still further relates to the nucleic acid construct, expression vector and the recombinant host cell that comprise this kind of polynucleotide.

The invention still further relates to for generation of method of protein, comprising: (a) cultivates the recombinant host cell comprising the gene of this kind of polynucleotide and the encoding said proteins be operatively connected; Optionally (b) reclaims described protein.

Described protein can be natural or allos for host cell.Term " protein " does not refer to the coded product of length-specific in the meaning herein, and therefore contains peptide, oligopeptides and polypeptide.Term " protein " is also contained through combination with the two or more polypeptide forming coded product.Described protein also comprises hybrid polypeptide and fusion polypeptide.

Preferred protein is hormone, enzyme, acceptor or its part, antibody or its part, or reporter protein (reporter).Such as, described protein can be lytic enzyme, isomerase, ligase enzyme, lyase (lyase), oxydo-reductase or transferring enzyme, such as alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase, beta-galactosidase enzymes, beta-glucosidase enzyme, xylobiase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, at, Maltose 4-glucosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucoamylase, saccharase, laccase, lipase, mannosidase, become glycanase (mutanase), oxydase, pectin decomposing enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic ferment, rnase, trans-glutaminases, or zytase.

Gene can obtain from any protokaryon, eucaryon or other source.

By following examples, the present invention is further described, but should not be understood as limitation of the scope of the invention.

Embodiment

Bacterial strain

Fungal bacterial strain NN044758 is by being separated by the dilution plate method that PDA substratum carries out the pedotheque collected since China at 45 DEG C.Then by it by single conidium is transferred to purifying on YG agar plate.Bacterial strain NN044758 is accredited as camphor tree suede branch based on morphological feature and ITS rDNA sequences mould.

Fungal bacterial strain NN000308 purchased from fungi strain preservation center (Centraalbureau Voor Schimmelcultures), called after CBS174.70.Based on morphological feature and ITS rDNA sequences, bacterial strain NN000308 is accredited as thermophilic excellent softgel shell (being before accredited as thermophilic shuttle spore shell (Thielavia thermophila), homonymus or synonymum Myceliophthora fergusii (Myceliophthora fergusii)).

Substratum

PDA substratum adds to 1 liter by the potato dextrose agar of 39 grams and deionized water and forms.

YG agar plate by the yeast extract of 5.0g, the glucose of 10.0g, the agar of 20.0g, and deionized water adds to 1 liter of formation.

YPG substratum by the yeast extract of 0.4% in deionized water, the KH of 0.1% ₂pO ₄, the MgSO of 0.05% ₄7H ₂o, and 1.5% glucose is formed.

YPM substratum by the yeast extract of 1% in deionized water, the peptone of 2%, and 2% maltose form.

The dull and stereotyped sucrose by 342g of minimum medium, the salts solution of 20ml, the agar of 20g, and deionized water adds to 1 liter of formation.Salts solution is by 2.6%KCl, 2.6%MgSO ₄7H ₂o, 7.6%KH ₂pO ₄, 2ppm Na ₂b ₄o ₇10H ₂o, 20ppm CuSO ₄5H ₂o, 40ppm FeSO ₄7H ₂o, 40ppm MnSO ₄2H ₂o, 40ppm Na ₂moO ₄2H ₂o, and 400ppm ZnSO ₄7H ₂o is formed.

Embodiment 1: the mould extracting genome DNA of camphor tree suede branch

Camphor tree suede branch trichoderma strain NN044758 to be inoculated on PDA flat board and 45 DEG C of Incubation in dark 3 days.Several mycelium-PDA bolt kind is entered the 500ml shaking flask of the YPG substratum containing 100ml.By bottle at 45 DEG C of incubations 3 days under 160rpm vibration.Mycelium by via (Calbiochem, La Jolla, CA, USA) filters and collects and freeze under liquid nitrogen.The mycelium freezed is milled to fine-powder by mortar and pestle, and uses Large-Scale Column Fungal DNAout (BAOMAN BIOTECHNOLOGY, Shanghai, China) according to the instruction isolation of genomic DNA of manufacturer.

Embodiment 2: gene order-checking, compilation and annotation

The genome DNA sample of extraction is delivered to Beijing Genome Institute (BGI, Shenzhen, China) for use the gene order-checking of GA2System (Illumina, Inc., San Diego, CA, USA).Skimming reading being taken at BGI uses SOAPdenovo program (Li etc., 2010, Genome Research20 (2): 265-72) to collect.Standard bioinformatic methods is used to carry out analyzing for gene identification and function prediction the sequence of compilation.GeneID (Parra etc., 2000, Genome Research10 (4): 511-515) is used to carry out predictive genes.Use Blastall version 2 .2.10 (Altschul etc., 1990, J.Mol.Biol.215 (3): 403-410, National Center for Biotechnology Information (NCBI), Bethesda, MD, and HMMER version 2 .1.1 (National Center for Biotechnology Information (NCBI) USA), Bethesda, MD, USA) structure based homology forecast function.GH7 family cellobiohydrolase polypeptide is gone out by the analysis Direct Identification of Blast result.Agene program (Munch and Krogh, 2006, BMC Bioinformatics7:263) and SignalP program (Nielsen etc., 1997, Protein Engineering10:1-6) is used to identify initiator codon.Further use SignalP program predicted signal peptide.The iso-electric point of the aminoacid sequence using Pepstats (Rice etc., 2000, Trends Genet.16 (6): 276-277) prediction to derive and molecular weight.

Embodiment 3: coding has the sign of the mould genome sequence of camphor tree suede branch of the active polypeptide of cellobiohydrolase (CBH)

The genomic dna sequence (SEQ ID NO:1) of the mould polypeptid coding sequence of camphor tree suede branch and the aminoacid sequence (SEQ ID NO:2) of derivation are shown in Fig. 1.Encoding sequence is 1457bp, comprises terminator codon, and it is interrupted by an intron of 80bp (Nucleotide 613 to 692).The G+C content not containing the mature polypeptide encoded sequence of intron and terminator codon is 52.38%.The albumen of the prediction of coding is 458 amino acid.Use SignalP program (Nielsen etc., 1997, Protein Engineering10:1-6), predict the signal peptide of 17 residues.The maturation protein of prediction contains 441 amino acid, has the iso-electric point of the molecular weight of 47890.24 daltonian predictions and the prediction of 4.12.The subfamily module subsequence comparison (Cantarel etc. defined by aminoacid sequence being used BLAST and all CAZY, 2009, Nucleic Acids Res.37:D233-238), wherein use the most significant single comparison in subfamily to predict GH7 territory, doping cellobiohydrolase catalytic domain is amino acid/11 8 to 458.

Use Needleman and Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453) with the gap open penalty of 10, the gap extension penalty of 0.5 and EBLOSUM62 matrix determine aminoacid sequence comparative by overall comparison.The maturing part of the aminoacid sequence of the mould encoding sequence of camphor tree suede branch of comparison code displaying cellobiohydrolase polypeptide has 69.23% identity with the aminoacid sequence (accession number: UNIPROT:Q5B2Q4) from the derivation of the cellobiohydrolase A of Emericella nidulans.

Embodiment 4: from genomic dna cloning camphor tree suede branch mould GH7 cellobiohydrolase (CBH) gene

CH7CBH gene GH7_ZY582279_485 is selected to clone.

The Oligonucleolide primers shown in following table 1 is devised with the genomic DNA amplification CBH gene from the mould NN044758 of camphor tree suede branch based on the DNA information obtained from gene order-checking (SEQ ID NO:1).Primer is synthesized by Invitrogen, Beijing, China.

Table 1: primer

Forward primer	ACACAACTGGGGATCCACCatgcatcgccaactcgctc	SEQ ID NO:9
			Reverse primer	GTCACCCTCTAGATCTgacacgcagcatgctaggagac	SEQ ID NO:10

Lowercase represents the coding region of gene in forward primer, and in reverse primer, represent the flanking region of gene, and upper-case portion is with the insertion point coming from the pPFJO355 carrier described in WO 2011/005867.

For each gene, each primer pair of 20 picomole is used for PCR reaction, described reaction by the mould NN044758 genomic dna of camphor tree suede branch of 2 μ l, 5X GC Buffer (the Finnzymes Oy of 10 μ l, Espoo, Finland), the methyl-sulphoxide (DMSO) of 1.5 μ l, the dATP of each 2.5mM, dTTP, dGTP, and dCTP, and the PHUSION of 0.6 unit ^tMhigh-Fidelity DNA Polymerase (Finnzymes Oy, Espoo, Finland) is formed, and final volume is 50 μ l.Amplification uses Peltier Thermal Cycler (MJ Research Inc., South San Francisco, CA, USA) carry out, its program was as follows: 94 DEG C of sex change 1 minute, 6 circulations, each 94 DEG C of sex change 15 seconds, 68 DEG C of annealing 30 seconds, often circulation reduction by 1 DEG C, and extend 100 seconds at 72 DEG C; 23 circulations, eachly carry out 15 seconds at 94 DEG C, carry out 30 seconds, and 72 DEG C are carried out 100 seconds at 63 DEG C; And finally extend 5 minutes at 72 DEG C.Then heat block enters 4 DEG C of infusion.

PCR primer is separated with 1.0% agarose gel electrophoresis of 1mM EDTA (TBE) damping fluid by using 90mM Tris-boric acid, wherein expects that the single product band of about size 1.5kb develops under w light.Then PCR primer is passed through to use from solution pCR DNA and Gel Band Purification Kit (GE Healthcare, Buckinghamshire, UK) carries out purifying according to the instruction of manufacturer.

Plasmid pPFJO355 Bam HI and Bgl II being digested, by using 1.0% agarose gel electrophoresis of tbe buffer liquid to be separated, and using ILLUSTRA ^tMgFX ^tMpCR DNA And Gel Band Purification Kit is according to the instruction purifying of manufacturer.

The carrier of PCR primer and digestion is used cF Dry-down PCR Cloning Kit (Clontech Laboratories, Inc., Mountain View, CA, USA) link together, obtain plasmid pGH7_ZY582279_485 (Fig. 1), wherein the mould GH7CBH gene of camphor tree suede branch transcribe the regulation and control be in from the promotor of oryzae alpha-amylase gene under.Clone operations is according to the instruction of manufacturer.In brief, for each ligation, by the pPFJO355 digested with Bam HI and Bgl II of 30ng, and the mould GH7CBH PCR primer of camphor tree suede branch of the purifying of 60ng is added into reaction bottle, and is resuspended in the final volume of 10 μ l by adding deionized water.To react 37 DEG C of incubations 15 minutes then 50 DEG C of incubations 15 minutes.Use the reaction product transformation of E. coli TOP10 competent cell (TIANGEN Biotech (Beijing) Co.Ltd., Beijing, China) of three microlitres.Intestinal bacteria transformant containing expression construct is detected by bacterium colony PCR.Bacterium colony PCR is the method for inserting from the direct rapid screening plasmid of E. coli clones.In brief, the PCR solution aliquots containig of the pre-mixing in each PCR pipe (comprises PCR damping fluid, MgCl ₂, dNTP, and PCR fragment generates primer pair used) in, by moving liquid point picking with sterilizing, and the described liquid point that moves is rotated and adds single bacterium colony in reaction soln.Usually, screened 7-10 bacterium colony.After PCR, reaction is analyzed by using 1.0% agarose gel electrophoresis of tbe buffer liquid.Use the bacterium colony that Spin Miniprep Kit (QIAGEN GmbH, Hilden, Germany) has the inset of the size of expectation from display prepares plasmid DNA.The mould GH7CBH gene of camphor tree suede branch inserted in pGH7_ZY582279_485 confirms by using the DNA sequencing of 3730XL DNA Analyzer (Applied Biosystems Inc., Foster City, CA, USA).

Embodiment 5: express the mould GH7CBH gene of camphor tree suede branch in aspergillus oryzae

Aspergillus oryzae HowB101 (WO9535385 embodiment 1) protoplastis is according to Christensen etc., and the method preparation of 1988, Bio/Technology6:1419-1422, transforms with the pGH7_ZY582279_485 of 3 μ g.Transform aspergillus oryzae HowB101 with pGH7_ZY582279_485 and produce about 50 transformant.Eight transformant are separated to independent minimum medium dull and stereotyped.

Four transformant are inoculated respectively the YPM substratum of the 3ml in 24 orifice plates, and at 30 DEG C of incubations under 150rpm stirs.After incubation on the 3rd, by the supernatant of the 20 μ l from each cultivation by using containing 50mM MES's the SDS-PAGE of 4-12%Bis-Tris Gel (Invitrogen Corporation, Carlsbad, CA, USA) analyzes according to the instruction of manufacturer.By the gel INSTANTBLUE of gained ^tM(Expedeon Ltd., Babraham Cambridge, UK) dyes.The SDS-PAGE general picture of culture shows the main band of all 4 clonal expressions at about 45kDa.Express strain called after aspergillus oryzae O5XGY.

Embodiment 6: the fermentation expressing strain

The YPM substratum of the inclined-plane 10ml of aspergillus oryzae O5XGY is washed, and the 2 liters of flasks inoculated into six each YPM substratum containing 400ml are to generate fermented liquid.By culture results on the 3rd, and use 0.45 μm membrane (Millipore, Bedford, MA, USA) filters.

Embodiment 7: from the mould CBH of aspergillus oryzae O5XGY purification of Recombinant camphor tree suede branch

By filtered for the aspergillus oryzae O5XGY (embodiment 6) of 2400ml volume supernatant ammonium sulfate (80% is saturated) precipitation, and be again dissolved in 50ml20mM sodium acetate pH5.5, for same buffer dialysis, and through 0.45 μm of frit.Final volume is 80ml.Solution is imposed on the 40ml Q balanced in 20mM sodium acetate pH5.5 fast Flow post (GE Healthcare, Buckinghamshire, UK).By albumen linear NaCI gradient (0-0.5M) wash-out.Level part from post has 50mM MES's by using the SDS-PAGE of 4-12%Bis-Tris Gel analyzes.The fraction of the band containing approximately 45 kda is collected and is concentrated by ultrafiltration.

Embodiment 8: thermophilic excellent softgel shell extracting genome DNA

Thermophilic excellent softgel shell bacterial strain NN000308 to be inoculated on PDA flat board and 45 DEG C of Incubation in dark 3 days.Several mycelium-PDA bolt kind is entered the 500ml shaking flask of the YPG substratum containing 100ml.By bottle at 45 DEG C of incubations 4 days under 160rpm vibration.Mycelium by via (Calbiochem, La Jolla, CA, USA) filters and collects and freeze in liquid nitrogen.The mycelium freezed is milled to fine-powder by mortar and pestle, and uses plant Maxi Kit (QIAGEN GmbH, Hilden, Germany) isolation of genomic DNA.

Embodiment 9: gene order-checking, compilation and annotation

The genome DNA sample of extraction is delivered to Beijing Genome Institute (BGI, Shenzhen, China) for use the gene order-checking of GA2System (Illumina, Inc., San Diego, CA, USA).Skimming reading being taken at BGI uses SOAPdenovo program (Li etc., 2010, Genome Research20 (2): 265-72) to collect.Standard bioinformatic methods is used to carry out analyzing for gene identification and function prediction the sequence of compilation.GeneID (Parra etc., 2000, Genome Research10 (4): 511-515) is used to carry out predictive genes.Use Blastall version 2 .2.10 (Altschul etc., 1990, J.Mol.Biol.215 (3): 403-410, National Center for Biotechnology Information (NCBI), Bethesda, MD, and HMMER version 2 .1.1 (National Center for Biotechnology Information (NCBI) USA), Bethesda, MD, USA) structure based homology forecast function.GH7 family cellobiohydrolase polypeptide is gone out by the analysis Direct Identification of Blast result.Agene program (Munch and Krogh, 2006, BMC Bioinformatics 7:263) and SignalP program (Nielsen etc., 1997, Protein Engineering10:1-6) is used to identify initiator codon.Further use SignalP program predicted signal peptide.The iso-electric point of the aminoacid sequence using Pepstats (Rice etc., 2000, Trends Genet.16 (6): 276-277) prediction to derive and molecular weight.

Embodiment 10: coding has the sign of the thermophilic excellent softgel shell genome sequence of the active polypeptide of cellobiohydrolase (CBH)

The genomic dna sequence (SEQ ID NO:3) of thermophilic excellent softgel shell polypeptid coding sequence and the aminoacid sequence (SEQ ID NO:4) of derivation are shown in Fig. 2.Encoding sequence is 1736bp, comprise terminator codon, it is by 76bp (Nucleotide 568 to 643), 68bp (Nucleotide 781 to 848), 75bp (Nucleotide 1062 to 1136), 94bp (Nucleotide 1384 to 1477), and 5 introns of 70bp (Nucleotide 1609 to 1678) interrupt.The G+C content not containing the mature polypeptide encoded sequence of intron and terminator codon is 65.9%.The albumen of the prediction of coding is 450 amino acid.Use SignalP program (Nielsen etc., 1997, see above), predict the signal peptide of 20 residues.The maturation protein of prediction contains 430 amino acid, has the iso-electric point of the molecular weight of 46517.43 daltonian predictions and the prediction of 4.88.The subfamily module subsequence comparison (Cantarel etc. defined by aminoacid sequence being used BLAST and all CAZY, 2009, Nucleic Acids Res.37:D233-238), wherein use the most significant single comparison in subfamily to predict GH7 territory, doping cellobiohydrolase catalytic domain is amino acid 21 to 450.

Use Needleman and Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453) with the gap open penalty of 10, the gap extension penalty of 0.5 and EBLOSUM62 matrix determine aminoacid sequence comparative by overall comparison.The maturing part of the aminoacid sequence of the thermophilic excellent softgel shell encoding sequence of comparison code displaying cellobiohydrolase polypeptide has 86.98% identity with the aminoacid sequence (accession number: GENESEQP:ATS95014 and WO2008140749) from the cellobiohydrolase I of thermophilic fungus destroyed wire (Myceliophthora thermophila).

The genomic dna sequence (SEQ ID NO:5) of thermophilic excellent softgel shell polypeptid coding sequence and the aminoacid sequence (SEQ ID NO:6) of derivation are shown in Fig. 3.Encoding sequence is 1785bp, comprise terminator codon, it is by 131bp (Nucleotide 160 to 290), 94bp (Nucleotide 750 to 843), 94bp (Nucleotide 1052 to 1145), and 4 introns of 92bp (Nucleotide 1501 to 1592) interrupt.The G+C content not containing the mature polypeptide encoded sequence of intron and terminator codon is 63.38%.The albumen of the prediction of coding is 457 amino acid.Use SignalP program (Nielsen etc., 1997, see above), predict the signal peptide of 21 residues.The maturation protein of prediction contains 436 amino acid, has the iso-electric point of the molecular weight of 47254.04 daltonian predictions and the prediction of 4.35.The subfamily module subsequence comparison (Cantarel etc. defined by aminoacid sequence being used BLAST and all CAZY, 2009, Nucleic Acids Res.37:D233-238), wherein use the most significant single comparison in subfamily to predict GH7 territory, doping cellobiohydrolase catalytic domain is amino acid 22 to 457.

Use Needleman and Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453) with the gap open penalty of 10, the gap extension penalty of 0.5 and EBLOSUM62 matrix determine aminoacid sequence comparative by overall comparison.The maturing part of the aminoacid sequence of the thermophilic excellent softgel shell encoding sequence of comparison code displaying cellobiohydrolase polypeptide and the aminoacid sequence (WO2003070939-A1) of hairy fungus (Coriolus hirsutus) cellobiohydrolase I albumen have 65.51% identity.

The genomic dna sequence (SEQ ID NO:7) of thermophilic excellent softgel shell polypeptid coding sequence and the aminoacid sequence (SEQ ID NO:8) of derivation are shown in Fig. 4.Encoding sequence is 1643bp, comprises terminator codon, and it is interrupted by 1 intron of 77bp (Nucleotide 410 to 486).The G+C content not containing the mature polypeptide encoded sequence of intron and terminator codon is 65.61%.The albumen of the prediction of coding is 521 amino acid.Use SignalP program (Nielsen etc., 1997, see above), predict the signal peptide of 17 residues.The maturation protein of prediction contains 504 amino acid, has the iso-electric point of the molecular weight of 53756.07 daltonian predictions and the prediction of 4.67.The subfamily module subsequence comparison (Cantarel etc. defined by aminoacid sequence being used BLAST and all CAZY, 2009, Nucleic Acids Res.37:D233-238), wherein use the most significant single comparison in subfamily to predict GH7 territory and CBM territory, dope cellobiohydrolase catalytic domain and CBM territory is amino acid 21 to 461 and amino acid 486 to 521.

Use Needleman and Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453) with the gap open penalty of 10, the gap extension penalty of 0.5 and EBLOSUM62 matrix determine aminoacid sequence comparative by overall comparison.The maturing part of the aminoacid sequence of the thermophilic excellent softgel shell encoding sequence of comparison code displaying cellobiohydrolase polypeptide has 80.61% identity with the aminoacid sequence (accession number: UNIPROT:Q692I2) from the derivation of the CBHI endoglucanase of the mould thermophilic mutation of Chaetomium thermophile (Chaetomium thermophilum var.thermophilum).

Embodiment 11: from the thermophilic excellent softgel shell GH7 cellobiose hydrolase gene of genomic dna cloning

As shown in table 2, have selected three CBH genes for cloning by expression.

Table 2:CBH gene

Work name	DNA sequence dna	Protein sequence
			GH7_Mf7339	SEQ ID NO:3	SEQ ID NO:4
GH7_Mf6627	SEQ ID NO:5	SEQ ID NO:6
			GH7_Mf0261	SEQ ID NO:7	SEQ ID NO:8

Based on the DNA information design obtained from gene order-checking, the Oligonucleolide primers shown in following table 3 is with the genomic DNA amplification gene from thermophilic excellent softgel shell NN000308.Primer is synthesized by Invitrogen, Beijing, China.

Table 3: primer

Lowercase represents the coding region of gene in forward primer, and in reverse primer, represent the flanking region of gene, and upper-case portion represents the insertion point of plasmid pPFJO355.

For each gene, each primer pair of 20 picomole is used for PCR reaction, described reaction by the thermophilic excellent softgel shell NN000308 genomic dna of 2 μ l, the 5X GC Buffer of 10 μ l, 1.5 the DMSO of μ l, the dATP of each 2.5mM, dTTP, dGTP, and dCTP, and the PHUSION of 0.6 unit ^tMhigh-Fidelity DNA Polymerase is formed, and final volume is 50 μ l.Amplification uses Peltier Thermal Cycler to carry out, and its program was as follows: 98 DEG C of sex change 1 minute, 6 circulations, each 98 DEG C of sex change 15 seconds, 67 DEG C of annealing 30 seconds, and often circulation reduction by 1 DEG C, and extend 3 minutes at 72 DEG C; 23 circulations, eachly carry out 15 seconds at 94 DEG C, carry out 30 seconds, and 72 DEG C are carried out 3 minutes at 63 DEG C; And finally extend 5 minutes at 72 DEG C.Then heat block enters 4 DEG C of infusion.

PCR primer is separated by using 1.0% agarose gel electrophoresis of tbe buffer liquid, wherein GH7_Mf7339, GH7_Mf6627, and the single product band that the expection size of GH7_Mf0261 is respectively about 1.8kb, 1.8kb, 1.7kb develops under w light.Then PCR primer is passed through to use from solution pCR DNA and Gel Band Purification Kit carries out purifying according to the instruction of manufacturer.

Table 4: plasmid

Gene	Plasmid	DNA schemes
			GH7_Mf7339	pGH7_Mf7339	Fig. 6
GH7_Mf6627	pGH7_Mf6627	Fig. 7
			GH7_Mf0261	pGH7_Mf0261	Fig. 8

The carrier of PCR primer and digestion is used cF Dry-down PCR Cloning Kit (Clontech Laboratories, Inc., Mountain View, CA, USA) link together, obtain plasmid (table 4) pGH7_Mf7339 (Fig. 6) respectively, pGH7_Mf6627 (Fig. 7) and pGH7_Mf0261 (Fig. 8), wherein thermophilic excellent softgel shell CBH gene transcribe the regulation and control be in from the promotor of oryzae alpha-amylase gene under.In brief, for each ligation, by the pPFJO355 digested with Bam HI and Bgl II of 30ng, and the thermophilic excellent softgel shell GH7CBH PCR primer of the purifying of 60ng is added into reaction bottle, and is resuspended in the final volume of 10 μ l by adding deionized water.To react 37 DEG C of incubations 15 minutes then 50 DEG C of incubations 15 minutes.Use the reaction product transformation of E. coli TOP10 competent cell (TIANGEN Biotech (Beijing) Co.Ltd., Beijing, China) of three microlitres.Intestinal bacteria transformant containing expression construct is detected by bacterium colony PCR as described in Example 4.PGH7_Mf7339, pGH7_Mf6627, and the thermophilic excellent softgel shell GH7CBH gene inserted in pGH7_Mf0261 confirms by using the DNA sequencing of 3730XL DNA Analyzer.

Embodiment 12: express thermophilic excellent softgel shell GH7CBH gene in aspergillus oryzae

Aspergillus oryzae HowB101 protoplastis is according to Christensen etc., and the method preparation of 1988, Bio/Technology6:1419-1422, transforms with the pGH7_Mf7339 of each 3 μ g, pGH7_Mf6627 and pGH7_Mf0261.

Transform aspergillus oryzae HowB101 with pGH7_Mf6627 and pGH7_Mf0261 and about 50 transformant are produced for each conversion.Eight of each conversion transformant are separated to independent minimum medium dull and stereotyped.

Four of each conversion transformant are inoculated respectively the YPM substratum of the 3ml in 24 orifice plates, and at 30 DEG C of incubations under 150rpm stirs.After incubation on the 3rd, the supernatant of the 20 μ l from each cultivation is being contained MES's 4-12%Bis-Tris Gel (Invitrogen Corporation, Carlsbad, CA, USA) analyzes according to the instruction of manufacturer.By the gel INSTANTBLUE of gained ^tM(Expedeon Ltd., Babraham Cambridge, UK) dyes.The expression of the SDS-PAGE general picture display GH7 cellobiohydrolase polypeptide of culture.The size of the main band of GH7 cellobiohydrolase polypeptide is shown in following table 5.Express strain to name as shown in the second hurdle.

Table 5: express

Express	Express strain	The size of recombinant protein
			pGH7_Mf6627	O7J2B	50kDa
pGH7_Mf0261	O7J19	60kDa

Embodiment 6: the fermentation expressing strain O7J2B and O7J19

The YPM substratum washing of the inclined-plane 10ml of transformant O7J2B, and the 2 liters of flasks inoculated into eight each YPM substratum containing 400ml are to generate fermented liquid.Then by bottle at 80rpm, 30 DEG C vibration 3 days.By culture results on the 3rd, and use 0.45 μm membrane (Millipore, Bedford, MA, USA) filters.

The YPM substratum washing of the inclined-plane 10ml of transformant O7J19, and the 2 liters of flasks inoculated into four each YPM substratum containing 400ml are to generate fermented liquid.Then by bottle at 80rpm, 30 DEG C vibration 3 days.By culture results on the 3rd, and use 0.45 μm membrane (Millipore, Bedford, MA, USA) filters.

Embodiment 14: from aspergillus oryzae O7J2B and the mould CBH of O7J19 purification of Recombinant camphor tree suede branch

By filtered for the aspergillus oryzae O7J2B (embodiment 13) of 3200ml volume supernatant ammonium sulfate (80% is saturated) precipitation, and be again dissolved in 50ml20mM Bis-Tris pH6.0, for same buffer dialysis, and through 0.45 μm of frit.Final volume is 100ml.Solution is imposed on the 40ml Q by 20mM Bis-Tris pH6.0 balance fast Flow post (GE Healthcare, Buckinghamshire, UK).By albumen linear NaCI gradient (0-0.25M) wash-out.Level part of collection 0.15-0.25M NaCl wash-out at identical Q fast Flow post (GE Healthcare, Buckinghamshire, UK) is further purified with NaCl gradient (0.0-0.2M).Level part has 50mM MES's by using the SDS-PAGE of 4-12%Bis-Tris Gel analyzes.The fraction of the band containing about 50kDa is collected and is concentrated by ultrafiltration.

By filtered for the aspergillus oryzae O7J19 (embodiment 13) of 1600ml volume supernatant ammonium sulfate (80% is saturated) precipitation, and be again dissolved in 50ml20mM Bis-Tris pH6.0, for same buffer dialysis, and through 0.45 μm of frit.Final volume is 60ml.Solution is imposed on the 40ml Q by 20mM Bis-TrispH6.0 balance fast Flow post.By albumen linear NaCI gradient (0 – 0.5M) wash-out.Level part of collection 0.1-0.3M NaCl wash-out also uses 40ml Phenyl 6Fast Flow post (GE Healthcare, Buckinghamshire, UK) is further purified with linear 1.2-0M (NH4) 2SO4 gradient.Level part has 50mM MES's by using the SDS-PAGE of 4-12%Bis-Tris Gel analyzes.The gel INSTANTBLUE of gained ^tMdyeing.The fraction of the band containing about 60kDa is collected and is concentrated by ultrafiltration.

Embodiment 15: for GH7 cellobiohydrolase (CBH) determination of activity of the Mierocrystalline cellulose (PASC) of phosphoric acid swollen

Preparation PASC lay in pulp solution, by 5g Microcrystalline Cellulose ( jRS Pharma, Holzm ü hle1, Rosenberg, Germany) with water-wet, then add 85% ice-cold ortho-phosphoric acid of 150ml.Suspension is stirred 1 hour lentamente in ice bath.Then the acetone that interpolation 500ml is ice-cold is simultaneously being stirred.Slurry is used (EMD Millipore Bioscience, Billerica, MA, USA) filters, and then uses the washing with acetone three times (draining as far as possible after each washing) that 100ml is ice-cold.Finally, by filtered slurry 500ml water washing twice, then again drain as far as possible after each washing.PASC and deionized water are mixed to the cumulative volume of 500ml, there is the concentration that 10g/ rises, be mixed to homogeneous (use homogenizer, Cole-Parmer, Vernon Hills, IL, USA), and be stored in refrigerator and reach one month most.

PASC liquid storage 50mM sodium acetate pH5.0 damping fluid is diluted to the concentration that 2g/ rises, and is used as substrate.To the PASC liquid storage of 150 μ l, add 20 μ l enzyme samples, and by reaction mixture incubation 60 minutes under 850rpm vibration.At the end of incubation, add the 2%NaOH of 50 μ l with termination reaction.Reaction mixture is centrifugal at 1,000x g.The sugar discharged by by the reaction mixture of 10 μ l first with the 0.4%NaOH of 90 μ l, then with 1.5% P-hydroxybenzoic acid hydrazides (PHBAH, Sigma Chemical Co., the St.Loius in the 2%NaOH of 50 μ l, MO, USA) mix and measure.Mixture is boiled 5 minutes at 100 DEG C, then 100 μ l is transferred to titer plate for the absorbance reading (Spectra Max M2, Molecular devices Sunnyvale, CA, USA) at 410nm.Blank by ignoring PASC and by substituting hydrolyzate to prepare with damping fluid in sugared determining step in hydrolysing step.Cellobiohydrolase activity calculates based on the absorbancy of sample and the difference of blank absorbancy.

Its result, comprise the O5XGY of camphor tree suede branch mould cellobiohydrolase I (P249XX) and comprise the O7J2B display section cellobiohydrolase of thermophilic excellent softgel shell cellobiohydrolase I (P24FVN), and the O7J19 comprising thermophilic excellent softgel shell cellobiohydrolase I (P24FUQ) is presented at the cellobiohydrolase activity that 410nm has the absorbancy of 1.1407.

Embodiment 16: pretreated maize straw hydrolysis measures

Use 1.4wt% sulfuric acid 165 DEG C and 107psi pre-treatment 8 minutes at U.S.Department of Energy National Renewable Energy Laboratory (USDOE National Renewable Energy Laboratory) (NREL) maize straw.Water-fast solid substance in pretreated maize straw (PCS) contains 56.5% Mierocrystalline cellulose, 4.6% hemicellulose and 28.4% xylogen.By two benches sulphuric acid hydrolysis, then by using the efficient liquid phase chromatographic analysis sugar of NREL standard analyzer (Standard Analytical Procedure) #002 to measure Mierocrystalline cellulose and hemicellulose.Xylogen uses NREL standard analyzer #003 to determine with gravimetry after with sulphuric acid hydrolysis Mierocrystalline cellulose and hemicellulose fraction.

By adding 10M NaOH and fully the pH of PCS is adjusted to 5.0 by mixing, then 120 DEG C of steam sterilizings 20 minutes, prepare without grinding, without the PCS (full slurry PCS) of washing.The dry weight of full slurry PCS is 29%.Prepare by grinding full slurry PCS in Cosmos ICMG40 wet type multi-usage shredder (EssEmm Corporation, Tamil Nadu, India) through grinding, without the PCS (dry weight 32.35%) washed.

The hydrolysis of PCS uses 2.2ml deep-well plates (Axygen, Union City, CA, USA) to carry out in the total reaction volume of 1.0ml.The every ml of insoluble PCS solid of hydrolysis 50mg carries out containing the 50mM sodium acetate pH5.0 damping fluid of 1mM manganous sulfate and the multiple enzyme composition (being expressed as mg albumen every gram of Mierocrystalline cellulose) of multiple protein heap(ed) capacity.Prepare enzyme composition, be then added into institute with the volume of 50 μ l to 200 μ l scopes porose simultaneously, the final volume of 1ml to each reaction.Then ALPS-300 is used ^tMdull and stereotyped heat sealer (Abgene, Epsom, United Kingdom) seal plate, fully mixes, and specified temp incubation 72 hours.The reaction of all reports is carried out in triplicate.

After hydrolysis, 0.45 μm is used 96 hole screen plate (Millipore, Bedford, MA, USA) filtered sample, then analyze the sugared content of permeate as mentioned below.When not using immediately, the aliquots containig of filtration is frozen in-20 DEG C.Following measurement is diluted in 0.005M H ₂sO ₄the sugared concentration of sample: use 4.6x250mm hPX-87H post (Bio-Rad Laboratories, Inc., Hercules, CA, USA) by 65 DEG C with 0.05%w/w phenylformic acid-0.005M H ₂sO ₄with the flow velocity wash-out of 0.6ml per minute, and by use the refractive index corrected by pure sugared sample detect ( 1100HPLC, Agilent Technologies, Santa Clara, CA, USA) integration of glucose, cellobiose and wood sugar signal is carried out quantitatively.Use the glucose of gained and cellobiose equivalent for the per-cent of each Response calculation cellulose conversion.

Measure glucose and cellobiose respectively.According to the sugared concentration that the adjustment of suitable dilution factor records.Hang oneself grind, the net concentration of sugar that the enzyme process of unwashed PCS produces by just in the unwashed PCS of zero time point the sugared concentration of corresponding background adjust the sugared concentration recorded and determine.All HPLC data processing all use MICROSOFT EXCEL ^tMsoftware (Microsoft, Richland, WA, USA) carries out.

Following formula is used to calculate the degree that cellulose conversion is glucose: % transforms=(glucose concn in glucose concn/restriction digestion) x100.Transforming to calculate %, setting 100% point of inversion based on cellulase contrast (the trichoderma reesei cellulase every gram Mierocrystalline cellulose of 100mg), and all values is also then multiplied by 100 divided by this numerical value.Are averaged in three repeat number strong points, and calculate standard deviation.

Embodiment 17: the preparation of enzyme composition

Aspergillus fumigatus GH6A cellobiohydrolase II (SEQ ID NO:17 [DNA sequence dna] and SEQ ID NO:18 [aminoacid sequence of derivation]) is restructuring preparation in aspergillus oryzae as described in WO 2011/057140.The filtered nutrient solution of Aspergillus fumigatus cellobiohydrolase II is used 400ml SEPHADEX ^tMg-25 post (GE Healthcare, United Kingdom) buffer-exchanged enters 20mM Tris pH8.0.Fraction is collected, and is adjusted to 1.2M ammonium sulfate-20mM Tris pH8.0.Counter-balanced albumen is loaded on the PHENYL SEPHAROSE be equilibrated at containing in the 20mM Tris pH8.0 of 1.2M ammonium sulfate ^tMon 6Fast Flow post (high sub) (GE Healthcare, Piscataway, NJ, USA), and the albumen the combined 20mM Tris pH8.0 wash-out of not liquid containing ammonium sulfate.Fraction is collected.

Penicillium species (emersonii) GH61A polypeptide (SEQ ID NO:19 [DNA sequence dna] and SEQ ID NO:20 [aminoacid sequence of derivation]) is recombinated according to WO 2011/041397 and is prepared.Penicillium species (emersonii) GH61A polypeptide gene is according to WO 2011/041397 purifying.

Trichodermareesei GH5 EG II (SEQ ID NO:21 [DNA sequence dna] and SEQ ID NO:22 [aminoacid sequence of derivation]) uses aspergillus oryzae to recombinate preparation as host according to WO 2011/057140.Filtered for trichoderma reesei endoglucanase II nutrient solution is used and is configured with 10kDa poly (ether sulfone) film (Pall Filtron, Northborough, MA, USA) tangent line stream thickener (Pall Filtron, Northborough, MA, USA) desalination buffer-exchanged enters 20mM Tris pH8.0.Protein concentration uses Microplate BCA ^tMprotein Assay Kit (Thermo Fischer Scientific, Waltham, MA, USA) determines, wherein bovine serum albumin is used as standard protein sample.

Aspergillus fumigatus GH10 zytase (xyn3) (SEQ ID NO:23 [DNA sequence dna] and SEQ ID NO:24 [aminoacid sequence of derivation]) uses aspergillus oryzae BECh2 (WO 2000/39322) to recombinate preparation as host according to WO 2006/078256.The filtered nutrient solution of Aspergillus fumigatus zytase is used 26/10Desalting Column (GE Healthcare, Piscataway, NJ, USA) desalination and buffer-exchanged enter 50mM Trisodium Citrate pH5.0.

Aspergillus fumigatus NN055679Cel3A beta-glucosidase enzyme (SEQ ID NO:25 [DNA sequence dna] and SEQ ID NO:26 [aminoacid sequence of derivation]) uses aspergillus oryzae to recombinate preparation as host according to WO 2005/047499.Filtered nutrient solution 20% Trisodium Citrate is adjusted to pH8.0, and this makes solution muddy.In order to remove muddiness, by solution at 20,000x g centrifugal 20 minutes, and supernatant is filtered through 0.2 μm of filtering unit (Nalgene, Rochester, NY, USA).Permeate deionized water is diluted to the specific conductivity identical with 50mM Tris/HCl, pH8.0.Enzyme solution through adjustment is imposed at 50mM Tris-HCl, the Q balanced in pH8.0 fast Flow post (GE Healthcare, Piscataway, NJ, USA), and with the linear gradient elution of 0 to 500mM sodium-chlor.Fraction is collected, and by 1% (w/v) activated carbon treatment to collect removal color from beta-glucosidase enzyme.Described charcoal removes by being filtered through 0.2 μm of filtering unit by suspension.Permeate 20% acetic acid is adjusted to pH5.0, and dilutes 10 times with deionized water.Permeate through adjustment is imposed on the SP balanced in 10mM succsinic acid pH5.0 fast Flow post (GE Healthcare, Piscataway, NJ, USA), and with the linear gradient elution of 0 to 500mM sodium-chlor.Collect level part, and use p-nitrophenyl-β-glucopyranoside as its beta-glucosidase activity of substrate assay.P-nitrophenyl-β-glucopyranoside liquid storage is prepared by the DMSO substrate of 50mg being dissolved in 1.0ml.Just before the use, substrate solution passes through the 100mM succsinic acid by the liquid storage of 100 μ l and 4900 μ l, 100mM HEPES, 100mM CHES, 100mM CABS, 1mM CaCl ₂, 150mM KCl, 0.01% prepared by X-100, pH5.0 (mensuration damping fluid) mixing.The substrate solution of 200 μ l volumes is dispensed into test tube, and is placed on ice, then add 20 μ l enzyme samples and (be diluted in 0.01% x-100).Measure and come initial by test tube being transferred to hot mixed instrument, described hot mixed instrument is set to the mensuration temperature of 37 DEG C.By test tube on hot mixed instrument with its maximum oscillation speed (1400rpm) incubation 15 minutes.Measure by test tube being transferred back to ice bath and adding stop bath (the 500mM H of 600 μ l ₃bO ₃/ NaOH pH9.7) stop.Then mix test tube, and allow it to reach room temperature.200 μ l supernatants are transferred to titer plate, and read in absorbancy the measuring as beta-glucosidase activity of 405nm.Buffer control (substituting enzyme) is comprised in mensuration.The level part with beta-glucosidase activity is analyzed by SDS-PAGE.Wherein will only find that the fraction of a band is collected as purified product on the SDS-PAGE gel of coomassie dyeing.Protein concentration uses Microplate BCA ^tMprotein Assay Kit determines, wherein bovine serum albumin is used as standard protein sample.

Aspergillus fumigatus bacterial strain NN051616GH3 xylobiase (SEQ ID NO:27 [DNA sequence dna] and SEQ ID NO:28 [aminoacid sequence of derivation]) is restructuring preparation in aspergillus oryzae as described in WO 2011/057140.The filtered nutrient solution of Aspergillus fumigatus xylobiase is used 26/10Desalting Column desalination buffer-exchanged enters 50mM sodium acetate pH5.0.

Protein concentration for above-mentioned each single component uses Microplate BCA ^tMprotein Assay Kit determines, wherein bovine serum albumin is used as standard protein sample.Enzyme composition is prepared as and is made up of each single component as described below: 25% Aspergillus fumigatus Cel6A cellobiohydrolase II, 15%Penicillium emersonii GH61A polypeptide, 10% Trichodermareesei GH5 EG II, 5% Aspergillus fumigatus GH10 zytase, 5% Aspergillus fumigatus beta-glucosidase enzyme, and 3% Aspergillus fumigatus xylobiase.Described Aspergillus fumigatus enzyme composition is called after " cellulose decomposition enzyme composition " in this article.

Embodiment 18: thermophilic excellent softgel shell cellobiohydrolase I to the hydrolysis of cellulose hydrolysis enzyme composition through grinding, without the effect of PCS of washing

Two kinds of thermophilic excellent softgel shell cellobiohydrolase I (P24FVN and P24FUQ) all be have evaluated it at 50 DEG C, 55 DEG C, 60 DEG C and 65 DEG C at 1.89mg total protein every g Mierocrystalline cellulose fortifying fibre element lytic enzyme composition (embodiment 17) to through grinding, without the ability of the hydrolysis of the PCS (embodiment 16) of washing.Thermophilic excellent softgel shell cellobiohydrolase adds with the every g Mierocrystalline cellulose of 1.11mg albumen.Also described cellulose decomposition enzyme composition is not run with not adding cellobiohydrolase I at 1.89mg albumen every g Mierocrystalline cellulose or 3mg albumen every g Mierocrystalline cellulose.

Measure and carry out as described in example 16 above.With through grinding, without washing PCS 1ml reaction (5% insoluble solid) in the 50mM sodium acetate pH5.0 containing 1mM manganous sulfate, carry out 72 hours.Institute responds and carries out the same form three times, and relates to the single mixing when being hydrolyzed beginning.

As shown in table 6, the cellulose decomposition enzyme composition comprising arbitrary thermophilic excellent softgel shell cellobiohydrolase I (P24FVN or P24FUQ) performance compared with the cellulose decomposition enzyme composition (1.89mg albumen every g Mierocrystalline cellulose) of not fibre-bearing disaccharide-hydrolysing enzymes I is better.For the thermophilic excellent softgel shell cellobiohydrolase I (P24FVN or P24FUQ) being added into cellulose decomposition enzyme composition, cellulose conversion is that the degree of glucose is at 50 DEG C, 55 DEG C, 60 DEG C higher compared with the cellulose decomposition enzyme composition not adding cellobiohydrolase I with 65 DEG C.

Table 6: the effect of thermophilic excellent softgel shell cellobiohydrolase I

The present invention is further described by following numbering paragraph:

[1] have an isolated polypeptide for cellobiohydrolase activity, it is selected from lower group:

(a) polypeptide, the mature polypeptide of itself and SEQ ID NO:2 has at least 70%, such as at least 72%, at least 74%, at least 75%, at least 77%, at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%; Polypeptide, the mature polypeptide of itself and SEQ ID NO:4 has at least 88%, such as at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%; Polypeptide, the mature polypeptide of itself and SEQ ID NO:6 has at least 66%, such as at least 68%, at least 70%, at least 75%, at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%; Polypeptide, the mature polypeptide of itself and SEQ ID NO:8 has at least 81%, such as at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%;

(b) polypeptide, it is by polynucleotide encoding, described polynucleotide are at medium stringency condition, medium-Gao stringent condition, high stringent condition, or under very high stringent condition with following hybridization: the mature polypeptide encoded sequence of (i) SEQ ID NO:1, the mature polypeptide encoded sequence of SEQ ID NO:3, the mature polypeptide encoded sequence of SEQ ID NO:5, or the mature polypeptide encoded sequence of SEQ ID NO:7, (ii) cDNA sequence of SEQ ID NO:1, the cDNA sequence of SEQ ID NO:3, the cDNA sequence of SEQ ID NO:5, or the cDNA sequence of SEQ ID NO:7, or the total length complement of (iii) (i) or (ii),

(c) polypeptide, it is by polynucleotide encoding, the mature polypeptide encoded sequence of described polynucleotide and SEQ ID NO:1 or its cDNA sequence has at least 70%, such as at least 72%, at least 74%, at least 75%, at least 77%, at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%, polypeptide, it is by polynucleotide encoding, and the mature polypeptide encoded sequence of described polynucleotide and SEQ ID NO:3 or its cDNA sequence has at least 88%, such as at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%, polypeptide, it is by polynucleotide encoding, the mature polypeptide encoded sequence of described polynucleotide and SEQ ID NO:5 or its cDNA sequence has at least 66%, such as at least 68%, at least 70%, at least 75%, at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%, polypeptide, it is by polynucleotide encoding, described polynucleotide with or the mature polypeptide encoded sequence of SEQ ID NO:7 or its cDNA sequence have at least 81%, such as at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%,

[2] polypeptide of section 1, the mature polypeptide of itself and SEQ ID NO:2 has at least 70%, at least 72%, at least 74%, at least 75%, at least 77%, at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%; At least 88%, at least 89% is had, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% with the mature polypeptide of SEQ ID NO:4, or the sequence iden of 100%; At least 66% is had, such as at least 68%, at least 70%, at least 75%, at least 78% with the mature polypeptide of SEQ ID NO:6, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%; Or have at least 81% with the mature polypeptide of SEQ ID NO:8, such as at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%.

[3] polypeptide of section 1 or 2, it is by polynucleotide encoding, described polynucleotide are at medium-Gao, with following hybridization under high or very high stringent condition: the mature polypeptide encoded sequence of (i) SEQ ID NO:1, the mature polypeptide encoded sequence of SEQ ID NO:3, the mature polypeptide encoded sequence of SEQ ID NO:5, or the mature polypeptide encoded sequence of SEQ ID NO:7, (ii) cDNA sequence of SEQ ID NO:1, the cDNA sequence of SEQ ID NO:3, the cDNA sequence of SEQ ID NO:5, or the cDNA sequence of SEQ ID NO:7, or the total length complement of (iii) (i) or (ii).

[4] polypeptide of section 1, it comprises or consists of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8; Or comprise or consist of the mature polypeptide of SEQ ID NO:2, the mature polypeptide of SEQ ID NO:4, the mature polypeptide of SEQ ID NO:6, or the mature polypeptide of SEQ ID NO:8.

[5] polypeptide of section 4, wherein said mature polypeptide is the amino acid 22 to 457 of amino acid 21 to 450, the SEQ ID NO:6 of amino acid/11 8 to 458, the SEQ ID NO:4 of SEQ ID NO:2, or the amino acid/11 8 to 521 of SEQ ID NO:8.

[6] polypeptide of any one of section 1-5, it is by polynucleotide encoding, the mature polypeptide encoded sequence of described polynucleotide and SEQ ID NO:1 or its cDNA sequence has at least 70%, at least 72%, at least 74%, at least 75%, at least 77%, at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%, it is by polynucleotide encoding, and the mature polypeptide encoded sequence of described polynucleotide and SEQ ID NO:3 or its cDNA sequence has at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%, it is by polynucleotide encoding, and the mature polypeptide encoded sequence of described polynucleotide and SEQ ID NO:5 or its cDNA sequence has at least 66%, at least 68%, at least 70%, at least 75%, at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%, or it is by polynucleotide encoding, described polynucleotide with or the mature polypeptide encoded sequence of SEQ ID NO:7 or its cDNA sequence have at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%,

[7] polypeptide described in any one of section 1-6, it is SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or the mature polypeptide of SEQ ID NO:8 comprises replacement, disappearance and/or inserts variant in one or more position, wherein said variant has cellobiohydrolase activity.

[8] polypeptide of section 1, it is SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or the fragment of SEQ ID NO:8, and wherein said fragment has cellobiohydrolase activity.

[9] isolated polypeptide, it comprises catalytic domain, and described catalytic domain is selected from lower group:

(a) catalytic domain, the amino acid/11 8 to 458 of itself and SEQ ID NO:2 has at least 70%, such as at least 72%, at least 74%, at least 75%, at least 77%, at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%; Catalytic domain, the amino acid 21 to 450 of itself and SEQ ID NO:4 has at least 88%, such as at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%; Catalytic domain, the amino acid 22 to 457 of itself and SEQ ID NO:6 has at least 66%, such as at least 68%, at least 70%, at least 75%, at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%; Catalytic domain, the amino acid 21 to 461 of itself and SEQ ID NO:8 has at least 81%, such as at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%;

(b) catalytic domain, it is by polynucleotide encoding, described polynucleotide under medium, medium-Gao, height or very high stringent condition with following hybridization: the Nucleotide 52 to 1454 of (i) SEQ ID NO:1, the Nucleotide 61 to 1733 of SEQ ID NO:3, the Nucleotide 64 to 1782 of SEQ ID NO:5, or the Nucleotide 52 to 1460 of SEQ ID NO:7, (ii) its cDNA sequence, or the total length complement of (iii) (i) or (ii);

(c) catalytic domain, it is by polynucleotide encoding, the Nucleotide 52 to 1454 of described polynucleotide and SEQ ID NO:1, or its cDNA sequence has at least 70%, such as at least 72%, at least 74%, at least 75%, at least 77%, at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%, or catalytic domain, it is by polynucleotide encoding, the Nucleotide 61 to 1733 of described polynucleotide and SEQ ID NO:3, or its cDNA sequence has at least 88%, such as at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%, catalytic domain, it is by polynucleotide encoding, the Nucleotide 64 to 1782 of described polynucleotide and SEQ ID NO:5, or its cDNA sequence has at least 66%, such as at least 68%, at least 70%, at least 75%, at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%, or catalytic domain, it is by polynucleotide encoding, the Nucleotide 52 to 1460 of described polynucleotide and SEQ ID NO:7, or its cDNA sequence has at least 81%, such as at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%,

The amino acid/11 8 to 458 of (d) SEQ ID NO:2, the amino acid 21 to 450 of SEQ ID NO:4, the amino acid 22 to 457 of SEQ ID NO:6, or the amino acid 21 to 461 of SEQ ID NO:8 comprises replacement, disappearance and/or inserts variant in one or more position; With

[10] polypeptide of section 9, it comprises sugared binding domain further.

[11] isolated polypeptide, it comprises sugared binding domain, and described sugared binding domain is selected from lower group:

(a) sugared binding domain, the amino acid 486 to 521 of itself and SEQ ID NO:8 has at least 81%, such as at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%;

(b) sugared binding domain, it is by polynucleotide encoding, described polynucleotide under medium, medium-Gao, height or very high stringent condition with following hybridization: the Nucleotide 1533 to 1640 of (i) SEQ ID NO:7, (ii) its cDNA sequence, or the total length complement of (iii) (i) or (ii);

(c) sugared binding domain, it is by polynucleotide encoding, and the Nucleotide 1533 to 1640 of described polynucleotide and SEQ ID NO:7 has at least 81%, and such as at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%;

The d amino acid 486 to 521 of () SEQ ID NO:8 comprises replacement, disappearance and/or inserts variant in one or more position; With

E () (a), (b), (c) or (d) have the fragment of Sugar-binding activitv.

[12] polypeptide of section 11, wherein sugared binding domain is operably connected to catalytic domain, preferred described catalytic domain obtains from following: lytic enzyme, isomerase, ligase enzyme, lyase, oxygen is enzyme or transferring enzyme also, such as aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, at, Maltose 4-glucosyltransferase, deoxyribonuclease, endoglucanase, esterase, alpha-galactosidase, beta-galactosidase enzymes, glucoamylase, alpha-glucosidase, beta-glucosidase enzyme, saccharase, laccase, lipase, mannosidase, become glycanase, oxydase, pectin decomposing enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic ferment, rnase, trans-glutaminases, zytase or xylobiase.

[13] composition, the polypeptide of any one of its section of comprising 1-12.

[14] polynucleotide for separation, the polypeptide of any one of its coding section 1-12.

[15] nucleic acid construct or an expression vector, the polynucleotide of its section of comprising 14, described polynucleotide are operably connected to one or more regulating and controlling sequence, and described regulating and controlling sequence instructs the generation of described polypeptide in expressive host.

[16] recombinant host cell, the polynucleotide of its section of comprising 14, described polynucleotide are operably connected to one or more regulating and controlling sequence, and described regulating and controlling sequence instructs the generation of polypeptide.

[17] method for the polypeptide any one of the section of generation 1-12, it comprises:

A () be culturing cell under the condition contributing to the generation of described polypeptide, described cell produces described polypeptide with its wild-type form; Optionally

B () reclaims described polypeptide.

[18] generation has a method for the polypeptide of cellobiohydrolase activity, and it comprises:

A () is contributing to the host cell of the section of cultivation 16 under the condition that described polypeptide produces; Optionally

B () reclaims described polypeptide.

[19] transgenic plant, plant part or a vegetable cell, the polynucleotide of the polypeptide of its encoded section of any one of 1-12 transform.

[20] generation has a method for the polypeptide of cellobiohydrolase activity, and it comprises:

A () is contributing to the transgenic plant of the section of cultivation 19 or vegetable cell under the condition that described polypeptide produces; Optionally

B () reclaims described polypeptide.

[21] produce a method for the mutant of parental cell, described method comprises the polynucleotide inactivation of the polypeptide made any one of coding section 1-12, causes mutant to produce less described polypeptide compared with parental cell.

[22] mutant cell produced by the method for section 21.

[23] mutant cell of section 22, it comprises the gene of encode natural or heterologous protein further.

[24] a kind of protedogenous method of product, it comprises:

A () is contributing to the mutant cell of the section of cultivation 22 or 23 under the condition that described albumen produces; Optionally

B () reclaims described albumen.

[25] double-stranded inhibitory RNA (dsRNA) molecule, the subsequence of the polynucleotide of its section of comprising 14, wherein optionally this dsRNA is siRNA or miRNA molecule.

[26] double-stranded inhibitory RNA (dsRNA) molecule of section 25, its length is about 15,16,17,18,19,20,21,22,23,24,25 or more duplex nucleotides.

[27] suppression has a method for the expression of polypeptide in cell of cellobiohydrolase, and it comprises to use cell or at double-stranded inhibitory RNA (dsRNA) molecule of cells section 25 or 26.

[28] cell produced by the method for section 27.

[29] cell of section 28, it comprises the gene of encode natural or heterologous protein further.

[30] a kind of protedogenous method of product, it comprises:

A () is contributing to the cell of the section of cultivation 28 or 29 under the condition that described albumen produces; Optionally

B () reclaims described albumen.

[31] a kind of polynucleotide of separation, its coded signal peptide, described signal peptide comprises or consists of the amino acid/11 to 17 of SEQ ID NO:2, the amino acid/11 to 20 of SEQ ID NO:4, the amino acid/11 to 21 of SEQ ID NO:6, or the amino acid/11 to 17 of SEQ ID NO:8.

[32] nucleic acid construct or an expression vector, it comprises the gene of the proteins encoded of the polynucleotide of the section of being operably connected to 31, and wherein said gene is external source for the polynucleotide of the described signal peptide of coding.

[33] recombinant host cell, it comprises the gene of the proteins encoded of the polynucleotide of the section of being operably connected to 31, and wherein said gene is external source for the polynucleotide of the described signal peptide of coding.

[34] a kind of protedogenous method of product, it comprises:

A () cultivates recombinant host cell under the condition contributing to the generation of described albumen, described recombinant host cell comprises the gene of the proteins encoded of the polynucleotide of the section of being operably connected to 31, and wherein said gene is external source for the polynucleotide of the described signal peptide of coding; Optionally

B () reclaims described albumen.

[35] a kind of full nutrient solution formulation or cell culture compositions, the polypeptide of any one of its section of comprising 1-12.

[36] technique for degraded or conversion cellulose materials, it comprises: with cellulose materials described in enzyme composition process under the polypeptide with cellobiohydrolase activity any one of section 1-12 exists.

[37] technique of section 36, wherein said cellulose materials is through pre-treatment.

[38] technique of any one of section 36 or 37, wherein said enzyme composition comprises the enzyme that one or more are selected from lower group: cellulase, the GH61 polypeptide with cellulolytic enhancing activity, hemicellulase, esterase, claviformin, laccase, lignin decomposition enzyme, polygalacturonase, peroxidase, proteolytic enzyme and swollenin.

[39] technique of section 38, wherein said cellulase is that one or more are selected from the enzyme of lower group: endoglucanase, cellobiohydrolase and beta-glucosidase enzyme.

[40] technique of section 38, wherein said hemicellulase is that one or more are selected from the enzyme of lower group: zytase, acetyl xylan esterase, feruloyl esterase, arabinofuranosidase, xylosidase and glucuronidase.

[41] technique any one of section 36-40, also comprises the cellulose materials reclaiming or transform through degraded.

[42] technique of section 41, the cellulose materials wherein through degraded or conversion is sugar.

[43] technique of section 42, wherein said sugar is selected from lower group: glucose, wood sugar, seminose, semi-lactosi, and pectinose.

[44] produce a technique for tunning, it comprises:

A (), under the polypeptide with cellobiohydrolase activity any one of section 1-12 exists, uses enzyme composition saccharified cellulosic material;

(b) with the fermentation of one or more organism of fermentation through the cellulose materials of saccharification to produce tunning; With

C () reclaims tunning from fermentation.

[45] technique of section 44, wherein said cellulose materials is pretreated.

[46] technique of section 44 or 45, wherein said enzyme composition comprises the enzyme that one or more (such as several) are selected from lower group: cellulase, the GH61 polypeptide with cellulolytic enhancing activity, hemicellulase, esterase, claviformin, laccase, lignin decomposition enzyme, polygalacturonase, peroxidase, proteolytic enzyme and swollenin.

[47] technique of section 46, wherein said cellulase is that one or more are selected from the enzyme of lower group: endoglucanase, cellobiohydrolase and beta-glucosidase enzyme.

[48] technique of section 46, wherein said hemicellulase is that one or more are selected from the enzyme of lower group: zytase, acetyl xylan esterase, feruloyl esterase, arabinofuranosidase, xylosidase and glucuronidase.

[49] technique any one of section 44-48, wherein step (a) and optionally (b) at the same time saccharification and fermentation in carry out simultaneously.

[50] technique any one of section 44-49, wherein tunning is alcohol, alkane, naphthenic hydrocarbon, alkene, amino acid, gas, isoprene, ketone, organic acid or polyketide.

[51] a kind of technique of fermentable fiber cellulosic material, it comprises: by one or more organism of fermentation fermentable fiber cellulosic material, and wherein said cellulose materials is having with enzyme composition saccharification under the existence of the polypeptide of cellobiohydrolase activity any one of section 1-12.

[52] technique of section 51, the fermentation of wherein said cellulose materials produces tunning.

[53] technique of section 52, also comprises and reclaims tunning from fermentation.

[54] technique of any one of section 51-53, wherein said cellulose materials before saccharification through pre-treatment.

[55] technique of any one of section 51-54, wherein said enzyme composition comprises the enzyme that one or more are selected from lower group: cellulase, the GH61 polypeptide with cellulolytic enhancing activity, hemicellulase, esterase, claviformin, laccase, lignin decomposition enzyme, polygalacturonase, peroxidase, proteolytic enzyme and swollenin.

[56] technique of section 55, wherein said cellulase is that one or more are selected from the enzyme of lower group: endoglucanase, cellobiohydrolase and beta-glucosidase enzyme.

[57] technique of section 55, wherein said hemicellulase is that one or more are selected from the enzyme of lower group: zytase, acetyl xylan esterase, feruloyl esterase, arabinofuranosidase, xylosidase and glucuronidase.

[58] technique of any one of section 52-57, wherein said tunning is alcohol, alkane, naphthenic hydrocarbon, alkene, amino acid, gas, isoprene, ketone, organic acid or polyketide.

Description and claimed the present invention is herein not limited in the scope of concrete aspect disclosed herein, because these aspects are intended to the explanation as the several aspect of the present invention.Any equivalent aspect is intended to be included within the scope of the present invention.In fact, from the foregoing description, except herein shown and described, multiple amendment of the present invention is apparent for a person skilled in the art.These amendments are also intended to fall in the scope of appended section.In the case of a conflict, be as the criterion with the disclosure comprising definitional part.

Claims

1. have an isolated polypeptide for cellobiohydrolase activity, it is selected from lower group:

(b) polypeptide, it is by polynucleotide encoding, described polynucleotide under at least medium stringency condition with following hybridization: the mature polypeptide encoded sequence of (i) SEQ ID NO:1, the mature polypeptide encoded sequence of SEQ ID NO:3, the mature polypeptide encoded sequence of SEQ ID NO:5, or the mature polypeptide encoded sequence of SEQ ID NO:7, (ii) cDNA sequence of SEQ ID NO:1, the cDNA sequence of SEQ ID NO:3, the cDNA sequence of SEQ ID NO:5, or the cDNA sequence of SEQ ID NO:7, or the total length complement of (iii) (i) or (ii),

2. the polypeptide of claim 1, it comprises or consists of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8; Or comprise or consist of the mature polypeptide of SEQ ID NO:2, the mature polypeptide of SEQ ID NO:4, the mature polypeptide of SEQ ID NO:6, or the mature polypeptide of SEQ ID NO:8.

3. an isolated polypeptide, it comprises catalytic domain, and described catalytic domain is selected from lower group:

4. an isolated polypeptide, it comprises sugared binding domain, and described sugared binding domain is selected from lower group:

(c) sugared binding domain, it is by polynucleotide encoding, and Nucleotide 1533 to 1640 or its cDNA sequence of described polynucleotide and SEQ ID NO:7 have at least 81%, such as at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the sequence iden of 100%;

The d amino acid 486 to 521 of () SEQ ID NO:8 comprises replacement, disappearance and/or inserts variant in one or more position;

(e) (a), (b), (c) or (d) have the fragment of Sugar-binding activitv.

5. a composition, it comprises the polypeptide of any one of claim 1-4.

6. the polynucleotide be separated, the polypeptide of any one of its coding claim 1-4.

7. a recombinant host cell, it comprises the polynucleotide of claim 6, and described polynucleotide are operably connected to the regulating and controlling sequence of the generation of the described polypeptide of one or more guidance.

8. produce a method for the polypeptide of any one of claim 1-4, it comprises:

B () reclaims described polypeptide.

9. generation has a method for the polypeptide of cellobiohydrolase activity, and it comprises:

A () cultivates the host cell of claim 7 under the condition contributing to the generation of described polypeptide; Optionally

B () reclaims described polypeptide.

10. transgenic plant, plant part or a vegetable cell, it transforms with the polynucleotide of the polypeptide of any one of coding claim 1-4.

11. 1 kinds of generations have the method for the polypeptide of cellobiohydrolase activity, and it comprises:

A () cultivates transgenic plant or the vegetable cell of claim 10 under the condition contributing to the generation of described polypeptide; Optionally

B () reclaims described polypeptide.

12. produce a method for the mutant of parental cell, described method comprises the polynucleotide inactivation of the polypeptide making any one of coding claim 1-4, causes mutant to produce less described polypeptide compared with parental cell.

13. 1 kinds of polynucleotide be separated, its coded signal peptide, described signal peptide comprises or consists of the amino acid/11 to 17 of SEQ ID NO:2, the amino acid/11 to 20 of SEQ ID NO:4, the amino acid/11 to 21 of SEQ ID NO:6, or the amino acid/11 to 17 of SEQ ID NO:8.

14. 1 kinds produce method of protein, and it comprises:

A () cultivates recombinant host cell under the condition contributing to the generation of described protein, described recombinant host cell comprises the gene of the coded protein be operatively connected with the polynucleotide of claim 13, and wherein said gene is external source for the polynucleotide of coded signal peptide; Optionally

B () reclaims described polypeptide.

15. 1 kinds of full nutrient solution formulations or cell culture compositions, it comprises the polypeptide of any one of claim 1-4.

The method of 16. 1 kinds of degradeds or conversion cellulose materials, it comprises: with enzyme composition process cellulose materials under the polypeptide with cellobiohydrolase activity of any one of claim 1-4 exists.

17. 1 kinds of methods producing tunning, it comprises:

A (), under the polypeptide with cellobiohydrolase activity of any one of claim 1-4 exists, uses enzyme composition saccharified cellulosic material;

C () reclaims described tunning from described fermentation.

The method of 18. 1 kinds of fermentable fiber cellulosic material, it comprises: by one or more organism of fermentation fermentable fiber cellulosic material, and wherein said cellulose materials is having with enzyme composition saccharification under the existence of the polypeptide of cellobiohydrolase activity in any one of claim 1-4.