CN109022394A

CN109022394A - Polynucleotides with the active polypeptide of xylobiase and the coding polypeptide

Info

Publication number: CN109022394A
Application number: CN201810842943.7A
Authority: CN
Inventors: 张羽; 刘晔; 段俊欣; 汤岚; B.麦克布拉耶
Original assignee: Novozymes Biotech Inc
Current assignee: Novozymes Inc
Priority date: 2011-11-22
Filing date: 2012-11-22
Publication date: 2018-12-18
Also published as: CN104145016A; CN104145016B

Abstract

The polynucleotides with xylobiase active isolated polypeptide and coding said polypeptide of separation are provided.Also provide include the polynucleotides nucleic acid construct, carrier and host cell, and for generate and using the polypeptide method.

Description

Polynucleotides with the active polypeptide of xylobiase and the coding polypeptide

The application is to be based on the applying date on November 22nd, 2012, and priority date is on November 22nd, 2011, application No. is 201280067784.9, denomination of invention are as follows: " polynucleotides with the active polypeptide of xylobiase and the coding polypeptide " The divisional application of patent application.

Statement for the right for the invention completed under the research and development of federal funding

The present invention is in cooperation agreement (Cooperative Agreement) DE-FC36- authorized by U.S. Department of Energy It is completed under 08GO18080 with governmental support.Government has certain right in the present invention.

It is related to sequence table

The application includes the sequence table of computer-reader form, is incorporated herein by mentioning stating.

Background of invention

Technical field

The present invention relates to the polynucleotides with xylobiase active polypeptide and coding said polypeptide.The present invention is also It is related to nucleic acid construct, carrier and host cell comprising the polynucleotides, and the method for generating and using the polypeptide.

Background technique

Ligno-ccllulose, maximum renewable biomass resources in the world, mainly by lignin, cellulose and hemicellulose It constitutes, wherein the major part of hemicellulose is xylan.Zytase (in such as-and Isosorbide-5-Nitrae-beta-xylanase, EC 3.2.1.8) the inside β -1,4- xylose glycosidic bond in hydrolyzed xylan is to generate the xylose and wood oligose (xylo- of lower molecular weight oligomer).Xylan is the D- xylose pyranose (1,4- β-glycoside-linked D- connected from 1,4- β-glucoside Xylopyranose) the polysaccharide formed.Xylobiase is catalyzed the outer hydrolysis of short β (1 → 4)-wood oligose to connect from non-reducing end Continuous removal D- xylose residues.

Cellulose is the polymer that glucose passes through β -1,4- key connection.It is poly- that many microorganisms generate hydrolysis β-connection Portugal The enzyme of sugar.These enzymes include endoglucanase, cellobiohydrolase and β-glucosyl enzym.Endoglucanase is in random order Digestion cellulosic polymer is set, cellobiohydrolase attack (attack) is exposed to.Cellobiohydrolase is from fiber Discharge to the terminal order of plain polymer the molecule of cellobiose.Cellobiose is the glucose two of water-soluble β -1,4- connection Aggressiveness.Cellobiose is hydrolyzed into glucose by β-glucosyl enzym.

Ethyl alcohol is converted by lignocellulose-containing raw material (lignocellulosic feedstock) to have the advantage that Big content of starting materials is readily available, avoids the desirability of burning or embedding material and the spatter property of alcohol fuel.Timber, agricultural residues Object, herbaceous crops and municipal solid waste are considered as the raw material for ethyl alcohol production.These materials are mainly by cellulose, half fiber Dimension element and lignin composition.Once ligno-ccllulose is converted into fermentable sugar such as glucose, fermentable sugar and can be easy Ground is ethyl alcohol by yeast fermentation.

Exist in this field and is used for by supplementing other enzymes improvement cellulose decomposition enzymatic compositions with increasing efficiency and offer The demand of the cost-effective enzyme solutions of xylogen degradation cellulose.

The present invention provides the polynucleotides with xylobiase active polypeptide and coding said polypeptide.

Summary of the invention

The present invention relates to the active isolated polypeptide of xylobiase, it is selected from the group:

(a) polypeptide has at least 60% sequence identity with the mature polypeptide of SEQ ID NO:6 or SEQ ID NO:8； There is at least 65% sequence identity with the mature polypeptide of SEQ ID NO:2；Or with SEQ ID NO:4 or SEQ ID NO:10's Mature polypeptide has at least 75% sequence identity；

(b) polypeptide, by polynucleotide encoding, the polynucleotides under at least medium-high stringency conditions with it is following miscellaneous It hands over: the mature polypeptide of (i) SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:9 Coded sequence, the cDNA sequence of (ii) SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7, or (iii) the overall length complement of (i) or (ii)；

(c) polypeptide, by polynucleotide encoding, the polynucleotides and SEQ ID NO:5 or SEQ ID NO:7 or its The mature polypeptide encoded sequence of cDNA sequence has at least 60% sequence identity；With SEQ ID NO:1 or its cDNA sequence Mature polypeptide encoded sequence has at least 65% sequence identity；Or with SEQ ID NO:3 or its cDNA sequence or SEQ ID The mature polypeptide encoded sequence of NO:9 has at least 75% sequence identity；

(d) SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10 at Ripe polypeptide includes the variant for replacing, lacking and/or being inserted into one or more (such as several) positions；With

(e) (a), (b), (c) or polypeptide (d) has the active segment of xylobiase.

The present invention is also related to encode the isolated polynucleotides of polypeptide of the invention, the nucleic acid structure comprising the polynucleotides Build body, recombinant expression carrier and recombinant host cell；With the method for generating the polypeptide.

The present invention is also related to the technique degraded or convert cellulosic material or the material containing xylan comprising: in the present invention Have and handle cellulosic material or material containing xylan with enzymatic compositions in the presence of the active polypeptide of xylobiase.One A aspect, the technique further include recycling the cellulosic material degraded or converted or material containing xylan.

The present invention is also related to the technique for generating tunning comprising: (a) there is xylobiase activity in of the invention Polypeptide in the presence of use enzymatic compositions saccharified cellulosic material or material containing xylan；(b) with one or more (such as several Kind) fermentative microorganism fermentation the cellulosic material through being saccharified or material containing xylan to generate tunning；(c) from fermenting back Receive the tunning.

The technique that the present invention is also related to fermentable fiber cellulosic material or the material containing xylan comprising with one or more (examples As several) fermentative microorganism ferments the cellulosic material or material containing xylan, wherein the cellulosic material or gathering containing wood Sugared material is saccharified in the presence of present invention polypeptide active with xylobiase with enzymatic compositions.In one aspect, described The fermentation of cellulosic material or the material containing xylan generates tunning.On the other hand, above-mentioned technique further comprise from Fermentation recycling tunning.

The present invention is also related to the polynucleotides of encoded signal peptide, and the signal peptide includes or group becomes (consist of) The amino acid 1 of SEQ ID NO:2 to the amino acid 1 of 19, SEQ ID NO:4 to the amino acid 1 of 19, SEQ ID NO:6 to 19, The amino acid 1 of SEQ ID NO:8 is operably connected to coding albumen to the amino acid 1 of 21 or SEQ ID NO:10 to 20 Gene, the albumen is external source for the signal peptide；Nucleic acid construct, expression vector comprising the polynucleotides and Recombinant host cell；With the method for generating albumen.

The present invention includes following embodiments:

Embodiment 1. is a kind of with the active isolated polypeptide of xylobiase, is selected from the group:

(e) (a), (b), (c) or polypeptide (d) has the active segment of xylobiase.

The polypeptide of 2. embodiment 1 of embodiment, it includes or group become SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10 or SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ The mature polypeptide of ID NO:8 or SEQ ID NO:10.

A kind of isolated polynucleotides of embodiment 3. encode the polypeptide of embodiment 1 or 2.

A kind of recombinant host cell of embodiment 4., it includes the polynucleotides of embodiment 3, the polynucleotides can be grasped It is connected to the regulating and controlling sequence of one or more generations for instructing polypeptide with making.

A kind of method for the polypeptide for generating embodiment 1 or 2 of embodiment 5. comprising:

(a) cell is cultivated under conditions of facilitating the polypeptide and generating, the cell generates institute with its wild-type form State polypeptide；Optionally

(b) polypeptide is recycled.

Embodiment 6. is a kind of to generate the method with the active polypeptide of xylobiase comprising:

(a) host cell of embodiment 4 is cultivated under conditions of facilitating the polypeptide and generating；Optionally

(b) polypeptide is recycled.

A kind of genetically modified plants of embodiment 7., plant part or plant cell use the more of coding embodiment 1 or 2 The polynucleotides of peptide are transformed.

Embodiment 8. is a kind of to generate the method with the active polypeptide of xylobiase comprising:

(a) genetically modified plants of culture embodiment 7 or plant cell under conditions of facilitating the polypeptide and generating；With Optionally

(b) polypeptide is recycled.

A kind of method for the mutant for generating parental cell of embodiment 9., the method includes making to encode embodiment 1 Or 2 polypeptide polynucleotides inactivation, lead to parental cell compared with the mutant of the less polypeptide of generation.

A kind of double-stranded inhibitory RNA (dsRNA) of the subsequence of the polynucleotides comprising embodiment 10 of embodiment 10. Molecule, wherein the optionally described dsRNA is siRNA or miRNA molecule.

A kind of method for inhibiting the expression in cell with the active polypeptide of xylobiase of embodiment 11., including it is right The cell application or double-stranded inhibitory RNA (dsRNA) molecule that embodiment 10 is expressed in the cell.

A kind of isolated polynucleotides of embodiment 12., encoded signal peptide, the signal peptide includes or group becomes SEQ The amino acid 1 of ID NO:2 is to the amino acid 1 of 19, SEQ ID NO:4 to the amino acid 1 of 19, SEQ ID NO:6 to 19, SEQ ID The amino acid 1 of NO:8 is to the amino acid 1 of 21 or SEQ ID NO:10 to 20.

Embodiment 13. is a kind of to produce protedogenous method comprising:

(a) recombinant host cell, the recombinant host cell packet are cultivated under conditions of facilitating the protein and generating Gene containing the coding protein that the polynucleotides with embodiment 12 are operatively connected, wherein the gene pairs is in encoded signal It is external source for the polynucleotides of peptide；Optionally

(b) protein is recycled.

The technique of a kind of degradation of embodiment 14. or conversion cellulosic material or the material containing xylan comprising: implementing Scheme 12 has and handles cellulosic material or material containing xylan with enzymatic compositions in the presence of the active polypeptide of xylobiase Material.

A kind of technique for generating tunning of embodiment 15. comprising:

(a) in the presence of the polypeptide active with xylobiase of embodiment 1 or 2, with enzymatic compositions diastatic fiber Cellulosic material or material containing xylan；

(b) with one or more fermentative microorganisms cellulosic material of the fermentation through being saccharified or material containing xylan to generate hair Ferment product；With

(c) tunning is recycled from the fermentation.

The technique of a kind of fermentable fiber cellulosic material of embodiment 16. or the material containing xylan comprising: with one or more Fermentative microorganism fermentable fiber cellulosic material or material containing xylan, wherein the cellulosic material or material containing xylan are in reality Apply scheme 1 or 2 with being saccharified in the presence of the active polypeptide of xylobiase with enzymatic compositions.

A kind of full nutrient solution formulation of embodiment 17. or cell culture compositions, it includes the more of embodiment 1 or 2 Peptide.

Detailed description of the invention

Fig. 1 shows the restricted figure of pGH3_ZY577211_92.

Fig. 2 shows the restricted figure of pGH3_ZY577202_22.

Fig. 3 shows the restricted figure of pGH3_ZY569167_685.

Fig. 4 shows the restricted figure of pGH3_ZY654890_6424.

Fig. 5 shows the restricted figure of pGH3_PE04100001596.

Fig. 6 shows that tangerine orange thermophilic ascomycete (Thermoascus aurantiacus) GH3 xylobiase (P24GP2) is right In the effect of the pretreated corncob of 50 DEG C of Penicillium kinds (Penicillium sp.) GH10 xylanase hydrolysis.

Fig. 7 shows that tangerine orange thermophilic ascomycete (Thermoascus aurantiacus) GH3 xylobiase (P24GP2) is right In the effect of the pretreated corncob of 60 DEG C of Penicillium kinds (Penicillium sp.) GH10 xylanase hydrolysis.

Definition

Acetyl xylan esterase: term " acetyl xylan esterase " means Carboxylesterase (EC 3.1.1.72), is catalyzed second Acyl group from polymeric xylans, acetylation xylose, acetyl glucose, acetic acid α-naphthylacetate (alpha-napthyl acetate) and The hydrolysis of acetic acid p-nitrophenyl acetate (p-nitrophenyl acetate).For the present invention, acetyl xylan esterase activity is Using containing 0.01%TWEEN^TMIn the 50mM sodium acetate pH 5.0 of 20 (polyoxyethylenesorbitan monolaurates) 0.5mM acetic acid p-nitrophenyl acetate is determined as substrate.The acetyl xylan esterase of one unit is defined as can be in pH 5,25 DEG C per minute release 1 micromole's p-nitrophenol anion (p-nitrophenolate anion) enzyme amount.

Allelic variant (allelic variant): term " allelic variant " means to occupy the base of identical chromosomal loci Any two of cause or more optional form.Allelic variation is natively occurred by mutation, and can lead to more in population State property.Gene mutation can be (unchanged in the polypeptide of coding) of silencing or can encode with the amino acid sequence changed Polypeptide.The allelic variant of polypeptide is the polypeptide encoded by the allelic variant of gene.

α-l-arabfuranglycosidase: term " α-l-arabfuranglycosidase " means α-L- arabinofuranosidase glucosides Arabinofuranosidase hydrolase (EC 3.2.1.55), catalysis to the end irreducibility α-L- in α-L-arabinose glycosides I The hydrolysis of primary furanoside residue.The enzyme is to α-L- arabinofuranosidase glucosides, the α-L- Ah containing (1,3)-and/or (1,5)-key Primary glycan, araboxylan and arabogalactan is drawn to work.α-l-arabfuranglycosidase is also referred to as Arab Glycosidase, α-arabinosidase, α-L-arabinose glycosides enzyme, α-arabinofuranosidase, polysaccharide α-L- arabinofuranosidase Glycosidase, α-L- arabinofuranosidase glucosides hydrolase, L-arabinose glycosides enzyme or α-L- arabanase.With regard to the present invention Speech, α-l-arabfuranglycosidase activity is 5mg in the 100mM sodium acetate pH 5 using every ml in 200 μ l of total volume Medium-viscosity Wheat Arabinoxylan (Megazyme International Ireland, Ltd., Bray, Co.Wicklow, Ireland it) carries out 30 minutes at 40 DEG C, then passes throughHPX-87H column chromatographs (Bio-Rad Laboratories, Inc., Hercules, CA, USA) arabinose analysis determine.

Alpha-glucuronidase: term " alpha-glucuronidase " means α-D- glucosiduronic acid glucuronic acid hydrolase (alpha-D-glucosiduronate glucuronohydrolase) (EC 3.2.1.139) is catalyzed α-D- glucuronic acid Glycoside hydrolysis is D- glucuronic acid and alcohol.For the present invention, alpha-glucuronidase activity be according to de Vries, 1998, J.Bacteriol.180:243-249 determinations.The alpha-glucuronidase of one unit be equal to can in pH 5, The enzyme amount of 40 DEG C of 1 micromole's glucuronic acids of release per minute or 4-O- methylglucuronic acid.

β-glucosyl enzym: term " β-glucosyl enzym " means β-D- glucoside glucohydralase (beta-D-glucoside Glucohydrolase) (E.C.No.3.2.1.21) is catalyzed the hydrolysis of the non-reduced β in end-D-Glucose residue, and discharges β-D-Glucose.For the present invention, β-glucosyl enzym 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 P-nitrophenyl-β-D- glucose pyranoside is used to determine as substrate.The β-glucosyl enzym of one unit is defined as at 25 DEG C, PH 4.8 is containing 0.01%From the 1mM p-nitrophenyl-β-D- as substrate in 20 50mM sodium citrate Glucose pyranoside generates 1.0 micromole's p-nitrophenol anion per minute.

Xylobiase: term " xylobiase " means β-D- xyloside xylose hydrolase (β-D-xyloside Xylohydrolase) (E.C.3.2.1.37) is catalyzed the outer water of short β (1 → 4) wood oligose (xylooligosaccharide) Solution is to remove continuous D- xylose residues from non-reducing end.For the present invention, the xylobiase of a unit is defined as 40 DEG C, pH 5 is containing 0.01%From the 1mM p-nitrophenyl-as substrate in 20 100mM sodium citrate β-D- xyloside generates 1.0 micromole's p-nitrophenol anion per minute.

Polypeptide of the invention has SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ The xylobiase of the mature polypeptide of ID NO:10 active at least 20%, for example, 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%.

CDNA: term " cDNA " means can be by reverse transcription from the maturation derived from eukaryon or prokaryotic cell, montage The DNA molecular that is prepared of mRNA molecule.CDNA lacks the intron sequences being typically found in corresponding gene group DNA.Most First (initial) primary RNA transcript object is the precursor of mRNA, includes montage by the processing of a series of step, then makees Occur for the mRNA of mature montage.

Cellobiohydrolase: term " cellobiohydrolase " means 1,4- callose cellobiohydrolase (Isosorbide-5-Nitrae-beta-D-glucan cellobiohydrolase) (E.C.3.2.1.91 and E.C.3.2.1.176), catalysis fibre Element, cell-oligosaccharide or any hydrolysis comprising the Isosorbide-5-Nitrae-β-D- glycosidic bond in β-Isosorbide-5-Nitrae-connection glucose polymer, from chain Reducing end (cellobiohydrolase I) or non-reducing end (cellobiohydrolase II) release cellobiose (Teeri, 1997, Crystalline cellulose degradation:New insight into the function of cellobiohydrolases,Trends in Biotechnology 15:160-167；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 Letters 149:152-156；Van Tilbeurgh and Claeyssens,1985,FEBS Letters 187:283-288；And Tomme etc., 1988, Eur.J.Biochem.170: The method of 575-581 description determines cellobiohydrolase activity.

Cellulolytic enzyme or cellulase: term " cellulolytic enzyme " or " cellulase " mean one or more The enzyme of (such as several) hydrolysis fiber cellulosic material.This fermentoid includes endoglucanase, cellobiohydrolase, β-glucoside Enzyme, or combinations thereof.Two kinds of basic skills of measurement cellulolytic activity include: (1) measurement total fiber element degrading activity, and (2) individual cellulolytic activity (endoglucanase, cellobiohydrolase and β-glucosyl enzym) is measured, such as Zhang Deng, Outlook for cellulase improvement:Screening and selection strategies, 2006, Biotechnology Advances 24:452-481 is summarized.Total fiber element degrading activity usually uses insoluble bottom Object measures, the substrate include Whatman1 filter paper, microcrystalline cellulose, bacteria cellulose, algae cellulose, cotton, Pretreated ligno-ccllulose etc..The most common total fiber element degrading activity measuring method is made using No. 1 filter paper of Whatman For the filter paper measuring method of substrate.The measuring method is by International Union of Pure and Applied Chemistry(IUPAC)(Ghose,1987,Measurement of cellulase activities,Pure Appl.Chem.59:257-68 it) establishes.

For the present invention, cellulose decomposition enzymatic activity is carried out by cellulolytic enzyme under the following conditions by measurement Cellulosic material hydrolysis is determined compared to the increase for the control hydrolysis for being not added with cellulose decomposition zymoprotein: the fiber of 1-50mg Element decomposes in zymoprotein/g PCS cellulose (or other pretreated cellulosic materials) in suitable temperature, such as 50 DEG C, 55 DEG C or 60 DEG C progress 3-7 days.Usual conditions are as follows: 1ml reaction solution, washed or unwashed PCS, 5% insoluble solid, 50mM sodium acetate pH 5,1mM MnSO₄, 50 DEG C, 55 DEG C or 60 DEG C, 72 hours, pass throughHPX-87H column (Bio- Rad Laboratories, Inc., Hercules, CA, USA) carry out glycan analysis.

Cellulosic material: term " cellulosic material " means to wrap cellulose-containing any material.The blastema of biomass Main polysaccharide in wall (primary cell wall) is cellulose, and secondly the most abundant is hemicellulose, and third is fruit Glue.Secondary cell wall (secondary cell wall) generates after cell stops growing, and equally contains polysaccharide and by altogether Valence is cross-linked to the polymeric lignin of hemicellulose and reinforces.Cellulose is the homopolymer of anhydro cellobiose, therefore is straight chain β- (1-4)-D- glucan, and hemicellulose includes multiple compounds, for example, xylan, xyloglucan (xyloglucan), I Primary xylan and mannosan form the complex branches structure with diversified substituent group.Although cellulose is usually more Shape, but the cellulose being present in plant tissue is mainly the insoluble crystal substrate of parallel dextran chain.Hemicellulose is usual It is connected with cellulose and other hemicelluloses with hydrogen bond, helps to stablize cell wall matrix.

Cellulose is commonly found in stem, leaf, shell, skin and the cob of such as plant, or leaf, branch and the timber of tree.Fiber material Material may be, but not limited to, agricultural residue, herbaceous material (including energy crop), municipal solid waste, paper pulp and paper mill Residue, waste paper and timber (including forestry residue) (see, e.g., Wiselogel etc., 1995, in Handbook on Bioethanol (Charles E.Wyman volume), pp.105-118, Taylor&Francis, Washington D.C.； Wyman,1994,Bioresource Technology 50:3-16；Lynd,1990,Applied Biochemistry and Biotechnology 24/25:695-719；Mosier etc., 1999, Recent Progress in Bioconversion of Lignocellulosics, in Advances in Biochemical Engineering/Biotechnology, T.Scheper chief editor, Volume 65, pp.23-40, Springer-Verlag, New York).It should be understood herein that It is that cellulose can be the form of ligno-ccllulose, ligno-ccllulose is a kind of Plant cell wall material, includes lignin, fibre The mixed-matrix of dimension element and hemicellulose.Cellulosic material is any biological material in a preferred aspect,.At another Preferred aspect, the cellulosic material is ligno-ccllulose, and it includes cellulose, hemicellulose and lignin.

In one aspect, cellulosic material is agricultural residue.On the other hand, cellulosic material is herbaceous material (including energy crop).On the other hand, cellulosic material is municipal solid waste.On the other hand, cellulosic material It is paper pulp and paper mill residue.On the other hand, cellulosic material is waste paper.On the other hand, cellulosic material is Timber (including forestry residue).

On the other hand, cellulosic material is giantreed (arundo).On the other hand, cellulosic material is bagasse. On the other hand, cellulosic material is bamboo.On the other hand, cellulosic material is corncob.On the other hand, Cellulosic material is zein fiber.On the other hand, cellulosic material is corn stover.On the other hand, fiber material Material is Chinese silvergrass category.On the other hand, cellulosic material is orange peel.On the other hand, cellulosic material is rice straw.Another A aspect, cellulosic material are switchgrass (switch grass).On the other hand, cellulosic material is straw.

On the other hand, cellulosic material is white poplar.On the other hand, cellulosic material is eucalyptus.At another Aspect, cellulosic material are fir tree (fir).On the other hand, cellulosic material is pine tree.On the other hand, cellulose Material is poplar.On the other hand, cellulosic material is dragon spruce.On the other hand, cellulosic material is willow.

On the other hand, cellulosic material is algae cellulose.On the other hand, cellulosic material is bacterial fibers Element.On the other hand, cellulosic material is velveteen (cotton linter).On the other hand, cellulosic material is filter Paper.On the other hand, cellulosic material is microcrystalline cellulose.On the other hand, cellulosic material is handled through phosphoric acid Cellulose.

On the other hand, cellulosic material is aquatic biomass.In as used in this article, " aquatic biomass " means in water The biomass generated in raw environment by photosynthesis.Aquatic biomass can be algae, emergent aquactic plant (emergent Plant), floatingleaved plant (floating-leaf plant) or submerged plant (submerged plant).

Cellulosic material (as is) can be used or be pre-processed as it is, and pretreatment uses known in the art normal Rule method, as described herein.Pre-treating cellulosic material in a preferred aspect,.

Coded sequence: term " coded sequence " means the polynucleotides of the amino acid sequence of directly specified polypeptide.Code sequence The boundary of column is usually determined that the open reading frame is started with initiation codon such as ATG, GTG or TTG by open reading frame, and And terminated with terminator codon such as TAA, TAG or TGA.Coded sequence can be genomic DNA, cDNA, synthetic DNA or its group It closes.

Regulating and controlling sequence (control sequence): term " regulating and controlling sequence " means the mature polypeptide of the invention to coding Polynucleotides expression is required nucleic acid sequence.Each regulating and controlling sequence can be for encoding the polynucleotides of the mature polypeptide (that is, coming from different genes) of natural (that is, coming from same gene) or external source or each regulating and controlling sequence for can be each other It is natural or external source.These regulating and controlling sequences include but is not limited to leader sequence, polyadenylation sequence, propeptide sequence, starting Son, signal peptide sequence and transcription terminator.At least, regulating and controlling sequence includes the termination signal of promoter and transcription and translation.Regulation Sequence can be with the connector for introducing specific restriction sites be ready for use on, and the specific restriction sites promote regulating and controlling sequence and coding The connection of the polynucleotide encoding district of polypeptide.

Endoglucanase: term " endoglucanase " means inscribe -1,4- (1,3；1,4)-Portugal callose 4- Endohydrolase (endo-1,4- β-D-glucan 4-glucanohydrolase) (E.C.3.2.1.4), catalytic cellulose, 1,4- β-D- sugar in cellulose derivative (such as carboxymethyl cellulose and hydroxyethyl cellulose), lichenin (lichenin) Glycosidic bond, β -1,3 the glucan such as cereal beta-D-glucans or xyloglucan of mixing and other plants containing cellulosic component The interior hydrolysis (endohydrolysis) of β -1,4 key in material.Endoglucanase activity can pass through measurement substrate viscosity It reduces or is gone back by what reducing sugar test method (Zhang etc., 2006, Biotechnology Advances 24:452-481) were determined Former end increases to determine.For the present invention, can according to Ghose, 1987, Pure and Appl.Chem.59:257-268's Method uses carboxymethyl cellulose (CMC) as substrate at 5,40 DEG C of pH to determine endoglucanase activity.

Expression: term " expression " includes being related to any step of polypeptide generation comprising but repaired after being not limited to transcription, transcription Decorations, translation, posttranslational modification and secretion.

Expression vector: term " expression vector " means linear or cricoid DNA molecular, and it includes the multicores of coding polypeptide Thuja acid, and the polynucleotides are operably connected with the regulating and controlling sequence for being used for its expression is provided.

61 glycoside hydrolase of family: term " 61 glycoside hydrolase of family " or " family GH61 " or " GH61 " mean basis 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, Biochem.J.316:695-696 belongs to the polypeptide of glycoside hydrolase Families 61.Proenzyme in the family is first based in a family Very weak inscribe -1,4- β-D dextranase activity that family member measures and be classified as glycoside hydrolase Families.These enzymes Structurally and functionally mode is non-classical, and they can not be considered as real (bona fide) glycosidase.However, based on when with When the mixture of cellulase or cellulase is used together, the ability that enhancing ligno-ccllulose decomposes, they are retained in In CAZy classification.

Feruloyl esterase: term " feruloyl esterase (feruloyl esterase) " means 4- hydroxy-3-methoxy cortex cinnamomi Acyl-glycosylhydrolase (EC 3.1.1.73) is catalyzed sugar (its of 4- hydroxy-3-methoxy cinnamoyl (asafoetide acyl) group from esterification In " natural biomass " substrate be usually arabinose) hydrolysis, to generate ferulic acid (4- hydroxy-3-methoxy cortex cinnamomi Acid).Feruloyl esterase is also referred to as feruloyl esterase (ferulic acid esterase), hydroxy cinnamate acyl ester enzyme, FAE- III, cinnamate hydrolase, FAEA, cinnAE, FAE-I or FAE-II.For the present invention, ferulaic acid esterase activity is to make The 0.5mM ferulic acid p-nitrophenyl ester in 50mM sodium acetate pH 5.0 is used to determine as substrate.The ferulic acid ester of one unit Enzyme is equal to the enzyme amount that 1 micromole's p-nitrophenol anion can be discharged per minute at 5,25 DEG C of pH.

Segment: term " segment " means one or more (such as several from the amino and/or carboxyl-terminal deletion of mature polypeptide It is a) polypeptide of amino acid；Wherein the segment has xylobiase activity.In one aspect, segment contains SEQ ID NO:2 At least 630 amino acid residues, for example, at least 670 amino acid residues, or at least 710 amino acid residues.At another Aspect, segment contain at least 690 amino acid residues of SEQ ID NO:4, for example, at least 730 amino acid residues or at least 770 amino acid residues.On the other hand, segment contains at least 710 amino acid residues of SEQ ID NO:6, such as extremely Few 750 amino acid residues or at least 790 amino acid residues.On the other hand, segment contains SEQ ID NO:8 at least 630 amino acid residues, for example, at least 670 amino acid residues or at least 710 amino acid residues.On the other hand, piece At least 660 amino acid residues of Duan Hanyou SEQ ID NO:10, for example, at least 700 amino acid residues or at least 740 ammonia Base acid residue.

Hemicellulose catabolic enzyme or hemicellulase: term " hemicellulose catabolic enzyme " or " hemicellulase " mean one kind Or the enzyme of a variety of (such as several) hydrolyzed hemicellulose materials.See, e.g. Shallom D. and Shoham Y.Microbial hemicellulases.Current Opinion In Microbiology,2003,6(3):219-228).Hemicellulase It is the key component in Degrading plant biomass.The example of hemicellulase includes but is not limited to acetyl mannan esterase, second Acyl xylan esterase, arabanase, arabinofuranosidase, coumaric acid esterase, feruloyl esterase, galactosidase, Glucuronidase, glucuronic acid esterase, mannonase mannosidase, zytase and xylosidase.These enzymes Substrate, hemicellulose are that branching and straight-chain polysaccharide mix group, these polysaccharide are by hydrogen bonding in plant cell wall Cellulose microfibers, cross-linking are the network of robust (robust).Hemicellulose also covalently invests lignin, with cellulose It is formed together highly complex structure.The changeable structure and organizational form of hemicellulose needs the synergistic effect of many enzymes to make it It is degradable.The catalytic module of hemicellulase is the glycoside hydrolase (GH) of hydrolyzing glucosidic bonds, or hydrolysis acetic acid or ferulic acid The sugar ester enzyme (CE) of the ester connection of side group.These catalytic modules, the homology based on its primary structure can be assigned as GH and CE family Race.Some families have generally similar folding, can further be classified as clan (clan), with alphabetic flag (for example, GH- A).The classification of most informedness and these and other newest sugared organized enzymes can be in Carbohydrate-Active Enzymes (CAZy) database obtains.Hemicellulose decompose enzymatic activity can according to Ghose and Bisaria, 1987, Pure& Appl.Chem.59:1739-1752 is in suitable temperature, such as 50 DEG C, 55 DEG C or 60 DEG C and pH, such as 5.0 or 5.5 progress Measurement.

High stringency conditions: term " high stringency conditions " means the probe at least 100 nucleotide of length, at 42 DEG C, The salmon sperm DNA that 5X SSPE, 0.3%SDS, 200 micrograms/ml have been sheared and are denaturalized and 50% formamide in, according to standard Southern blotting carry out prehybridization and hybridization 12 to 24 hours.Using 2X SSC, 0.2%SDS at 65 DEG C by carrier material Material is final to be washed three times, and 15 minutes every time.

Host cell: term " host cell " mean to be adapted for use with the nucleic acid construct comprising polynucleotides of the present invention or The cell type that expression vector is converted, transfected, transduceed etc..Term " host cell " cover parental cell it is any due to The mutation that occurs in duplication and the offspring for being different from parental cell.

Separation: term " separation " means substance existing for form or environment not occur in nature.Separation The non-limiting example of substance include (1) any non-naturally occurring substance, (2) it is any at least partly with it is one or more or The substance being all detached from its natural adjoint naturally occurring ingredient, including but not limited to any enzyme, variant, nucleic acid, albumen Matter, peptide or co-factor；(3) it have passed through manually modified substance for any substance seen in the nature；Or (4) It is any by increasing the amount of the substance (for example, the recombination in host cell produces relative to its natural adjoint other components It is raw；Encode the multicopy of the gene of the substance；And using more stronger than with the natural adjoint promoter of the gene that encodes the substance Promoter) and modify substance.

Low stringency condition: term " low stringency condition " means the probe at least 100 nucleotide of length, at 42 DEG C, The salmon sperm DNA that 5X SSPE, 0.3%SDS, 200 micrograms/ml have been sheared and are denaturalized and 25% formamide in, according to standard Southern blotting carry out prehybridization and hybridization 12 to 24 hours.Using 2X SSC, 0.2%SDS at 50 DEG C by carrier material Material is final to be washed three times, and 15 minutes every time.

Mature polypeptide: term " mature polypeptide " means to deposit with its final form after translation and any posttranslational modification Polypeptide, the modification truncates such as the processing of the end N-, the end C-, glycosylation, phosphorylation.In one aspect, according to pre- Survey SEQ ID NO:2 (P244Y5) amino acid 1 to 19 be signal peptide SignalP program (Nielsen etc., 1997, Protein Engineering 10:1-6), mature polypeptide is the amino acid 20 to 777 of SEQ ID NO:2.In another side Face, is the SignalP program of signal peptide according to the amino acid 1 of prediction SEQ ID NO:4 (P244Y4) to 19, and mature polypeptide is The amino acid 20 to 825 of SEQ ID NO:4.On the other hand, according to the amino acid 1 of prediction SEQ ID NO:6 (P241KM) It is the SignalP program of signal peptide to 19, mature polypeptide is the amino acid 20 to 851 of SEQ ID NO:6.On the other hand, It is the SignalP program of signal peptide according to the amino acid 1 of prediction SEQ ID NO:8 (P24QRU) to 21, mature polypeptide is SEQ The amino acid 22 to 767 of ID NO:8.On the other hand, according to the amino acid 1 of prediction SEQ ID NO:10 (P24GP2) to 20 It is the SignalP program of signal peptide, mature polypeptide is the amino acid 21 to 800 of SEQ ID NO:10.It is known in the art place Chief cell can produce the different mature polypeptides of two or more expressed by identical polynucleotides (i.e. with different C-terminal and/or N-terminal amino acid) mixture.

Mature polypeptide encoded sequence: term " mature polypeptide encoded sequence " mean coding have xylobiase it is active at The polynucleotides of ripe polypeptide.In one aspect, according to 1 to 57 encoded signal peptide of nucleotide of prediction SEQ ID NO:1 SignalP program (Nielsen etc., 1997, see on), mature polypeptide encoded sequence is SEQ ID NO:1 or its cDNA sequence Nucleotide 58 to 2399 (D822K1).On the other hand, according to 1 to 57 encoded signal of nucleotide of prediction SEQ ID NO:3 The SignalP program of peptide, mature polypeptide encoded sequence are SEQ ID NO:3 or the nucleotide 58 to 2668 of its cDNA sequence (D822JZ).On the other hand, according to the SignalP journey of 1 to 57 encoded signal peptide of nucleotide of prediction SEQ ID NO:5 Sequence, mature polypeptide encoded sequence are SEQ ID NO:5 or the nucleotide 58 to 2829 (D72UE7) of its cDNA sequence.At another Aspect, according to the SignalP program of 1 to 63 encoded signal peptide of nucleotide of prediction SEQ ID NO:7, mature polypeptide encoded sequence It is SEQ ID NO:7 or the nucleotide 64 to 2634 (D13874) of its cDNA sequence.On the other hand, according to prediction SEQ ID The SignalP program of 1 to 60 encoded signal peptide of nucleotide of NO:9, mature polypeptide encoded sequence be SEQ ID NO:9 or its The nucleotide 61 to 2400 (D82RN1) of cDNA sequence.

Medium stringency condition: term " medium stringency condition " means the probe at least 100 nucleotide of length, 42 DEG C, the salmon sperm DNA that 5X SSPE, 0.3%SDS, 200 micrograms/ml have been sheared and are denaturalized and 35% formamide in, according to The Southern blotting of standard carries out prehybridization and hybridization 12 to 24 hours.It will be carried using 2X SSC, 0.2%SDS at 55 DEG C Body material finally washs three times, and 15 minutes every time.

Medium-high stringency conditions: term " medium-high stringency conditions " means the spy at least 100 nucleotide of length Needle, at 42 DEG C, in the salmon sperm DNA that 5X SSPE, 0.3%SDS, 200 micrograms/ml have been sheared and are denaturalized and 35% formamide In, prehybridization and hybridization 12 to 24 hours are carried out according to the Southern blotting of standard.Using 2X SSC, 0.2%SDS 60 DEG C carrier material is finally washed three times, 15 minutes every time.

Nucleic acid construct: term " nucleic acid construct " means single-stranded or double-stranded nucleic acid molecules, is isolated from naturally occurring Gene or its mode being not present in originally through modifying in (not otherwise exist) nature contain nucleic acid Section or its be synthesis, it includes one or more regulating and controlling sequences.

Be operably connected: term " being operably connected " means such configuration, wherein regulating and controlling sequence is placed in relatively In the appropriate location of the coded sequence of polynucleotides, so that regulating and controlling sequence instructs the expression of coded sequence.

Polypeptide with cellulolytic enhancing activity: term " polypeptide with cellulose decomposition enhancing " means catalysis tool There is the GH61 polypeptide of the enhancing of hydrolysis of the enzyme of cellulolytic activity to cellulosic material.For the present invention, pass through measurement Come free cellulolytic enzyme the reduced sugar increase of hydrolysis fiber cellulosic material or fibre compared with control hydrolysis under the following conditions The total amount increase of disaccharides and glucose is tieed up to determine cellulolytic enhancing activity: the 1-50mg total protein/pretreated corn of g Cellulose in stalk (PCS), wherein total protein includes the cellulose decomposition zymoprotein and 0.5-50%w/ of 50-99.5%w/w The protein of the GH61 polypeptide with cellulolytic enhancing activity of w, in suitable temperature, such as 50 DEG C, 55 DEG C or 60 DEG C And pH, such as 5.0 or 5.5 last 1-7 days, control hydrolysis using the total protein loading capacity of equivalent and cellulose-less decomposes enhancing and lives Property (cellulose in 1-50mg cellulolytic protein/g PCS) carry out.It is used in a preferred aspect, in total protein by weight 2-3% aspergillus oryzae β-glucosyl enzym (recombinating generation in aspergillus oryzae according to WO 02/095014) or total protein quality The cellulase protein of the aspergillus fumigatus β-glucosyl enzym (recombinate and generate in aspergillus oryzae as described in WO 2002/095014) of 2-3% In the presence of loading capacityThe mixture of 1.5L (Novozymes A/S, Bagsvaerd, Denmark) Source as cellulolytic activity.

GH61 polypeptide with cellulolytic enhancing activity reaches the horizontal required cellulose of same hydrolysis by reducing The amount of catabolic enzyme and the hydrolysis for enhancing the cellulosic material by the enzymatic with cellulolytic activity are preferably decreased to few 1.01 times, for example, 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 corn stover: term " PCS " or " pretreated corn stover " mean by at heat and dilute sulfuric acid Reason, oxygenation pretreatment or the neutral pretreated cellulosic material from corn stover.

Sequence identity: parameter " sequence identity " describes between two amino acid sequences or between two nucleotide sequences Correlation.

For the present invention, the degree of sequence identity between two amino acid sequences uses such as EMBOSS software package (EMBOSS:The European Molecular Biology Open Software Suite, Rice etc., 2000, Trends Genet.16:276-277), Needleman-Wunsch performed in the Needle program of preferably 5.0.0 editions or more highest version Algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453) measures.The parameter used is notch opening Point penalty (gap open penalty) 10, gap extension penalty (gap extension penalty) 0.5 and EBLOSUM62 (the EMBOSS version of BLOSUM62) substitution matrix.Using Needle labeled as " highest identity (longest identity) " Output result (- nobrief option is used to obtain) calculates as follows as homogeneity percentage:

(same residue × 100)/(sum for comparing notch in length-comparison)

For the present invention, the degree of sequence identity between two nucleotide sequences uses such as EMBOSS software package (EMBOSS:The European Molecular Biology Open Software Suite, Rice etc., 2000, see on Text), in the Needle program of preferably 5.0.0 edition or more highest version performed by Needleman-Wunsch algorithm (Needleman And Wunsch, 1970, see above) it measures.The parameter used is that notch opens point penalty 10,0.5 He of gap extension penalty EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix.The output knot of " highest identity " is labeled as using Needle Fruit (- nobrief option is used to obtain) calculates as follows as homogeneity percentage:

(same deoxyribonucleotide × 100)/(sum for comparing notch in length-comparison)

Subsequence: term " subsequence (subsequence) " means to lack from the 5 ' of mature polypeptide encoded sequence and/or 3 ' ends Lose the polynucleotides of one or more (such as several) nucleotide；Wherein the subsequence coding has xylobiase active Segment.In one aspect, subsequence contains at least 1890 nucleotide of SEQ ID NO:1, for example, at least 2010 nucleotide, Or at least 2130 nucleotide.On the other hand, subsequence contains at least 2070 nucleotide of SEQ ID NO:3, such as At least 2190 nucleotide or at least 2310 nucleotide.On the other hand, subsequence contains SEQ ID NO:5 at least 2130 nucleotide, for example, at least 2250 nucleotide or at least 2370 nucleotide.On the other hand, subsequence contains At least 1890 nucleotide of SEQ ID NO:7, for example, at least 2010 nucleotide or at least 2370 nucleotide.At another Aspect, subsequence contain at least 1980 nucleotide of SEQ ID NO:9, for example, at least 2100 nucleotide or at least 2220 Nucleotide.

Variant: term " variant " means in one or more (such as several) positions to include to change, that is, replace, be inserted into and/ Or missing has the active polypeptide of xylobiase.Substitution means to replace the amino acid for occupying certain position with different amino acid Generation；Missing means that removal occupies the amino acid of certain position；And it is inserted into the amino acid for meaning in adjoining and and then to occupy certain position Amino acid is added later.

Unusual high stringency conditions: term " very high stringency conditions " means the probe at least 100 nucleotide of length, At 42 DEG C, the salmon sperm DNA that 5X SSPE, 0.3%SDS, 200 micrograms/ml have been sheared and are denaturalized and 50% formamide in, Prehybridization and hybridization 12 to 24 hours are carried out according to the Southern blotting of standard.Using 2X SSC, 0.2%SDS at 70 DEG C Carrier material is finally washed three times, 15 minutes every time.

Unusual low stringency condition: term " very low stringency condition " means the probe at least 100 nucleotide of length, At 42 DEG C, the salmon sperm DNA that 5X SSPE, 0.3%SDS, 200 micrograms/ml have been sheared and are denaturalized and 25% formamide in, Prehybridization and hybridization 12 to 24 hours are carried out according to the Southern blotting of standard.Using 2X SSC, 0.2%SDS at 45 DEG C Carrier material is finally washed three times, 15 minutes every time.

Material containing xylan: term " material containing xylan " means any comprising the xylose residues containing β-(1-4) connection The material of the plant cell wall polysaccharides of skeleton.The xylan of terrestrial plant is that have β-(1-4)-xylopyranose skeleton heteromeric Object, with short sugar chain branches.They include D- glucuronic acid or its 4-O- methyl ether, L-arabinose and/or a variety of packets Xylose containing D-, L-arabinose, D- or L- galactolipin and D-Glucose oligosaccharides.It is poly- that the polysaccharide of xylan type can be divided into wood Sugared (homoxylan) and Heteroxylan (heteroxylan), the latter include glucuronoxylan, (Arab) glucuronic acid Xylan, (glucuronic acid) araboxylan, araboxylan and compound Heteroxylan.See, e.g. Ebringerova Deng 2005, Adv.Polym.Sci.186:1-67.

In the process of the invention, any material containing xylan can be used.It is described containing wood in a preferred aspect, Chitosan material is ligno-ccllulose.

Xylanolytic activities or xylanolytic activity: term " xylanolytic activities " or " xylanolytic activity " Mean to hydrolyze the biological activity of the material containing xylan.The basic methods of two kinds of measurement xylanolytic activities include: (1) measurement Total pentosan degrading activity, and (2) measure individual xylanolytic activity (such as endo-xylanase, xylobiase, Ah Draw primary furanoside enzyme, alpha-glucuronidase, acetyl xylan esterase, feruloyl esterase and α-glucuronic acid esterase (α- glucuronyl esterase)).Recently in the in-progress summary of xylanolitic enzyme assay in several open source literatures, including Biely and Puchard, Recent progress in the assays of xylanolytic enzymes, 2006, Journal of the Science of Food and Agriculture 86 (11): 1636-1647；Spanikova and Biely,2006,Glucuronoyl esterase-Novel carbohydrate esterase produced by Schizophyllum commune,FEBS Letters 580(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 Journal 321:375-381。

Total pentosan degrading activity can be measured by determining the reduced sugar formed from a plurality of types of xylans, the wood Glycan includes that such as oat wheat (oat spelt), beech wood (beechwood) and Larch (larchwood) wood are poly- Sugar, or the xylan fragments of the dyeing released from a variety of xylans covalently dyed can be determined by photometry to measure. The most common total pentosan degrading activity measuring method is based on reduced sugar is generated from the 4-O- methylglucuronic acid xylan of poly, such as Bailey,Biely,Poutanen,1992,Interlaboratory testing of methods for assay of Xylanase activity, Journal of Biotechnology 23 (3): described in 257-270.Xylanase activity is also Can use 0.2%AZCL- araboxylan as substrate at 37 DEG C 0.01%X-100 (4- (1,1,3,3- tetra- Methyl butyl) phenyl-polyethylene glycol) and 200mM sodium phosphate buffer pH 6 in determine.The xylanase activity of one unit It is defined as at 37 DEG C, pH 6 is in 6 buffer of 200mM sodium phosphate pH from the 0.2%AZCL- araboxylan as substrate 1.0 micromole's Bazurins (azurine) are generated per minute.

For the present invention, xylanolytic activities are to be made under following usual conditions by measuring by xylanolytic enzyme At the increase of birch xylan (Sigma Chemical Co., Inc., St.Louis, MO, USA) hydrolysis determine: 1ml Reaction, 5mg/ml substrate (total solid), 5mg xylanolitic protein/g substrate, 50mM sodium acetate, 5,50 DEG C of pH, 24 is small When, such as Lever, 1972, A new reaction for colorimetric determination of Described in carbohydrates, Anal.Biochem 47:273-279 using P-hydroxybenzoic acid hydrazides (PHBAH) measuring method into Row glycan analysis.

Zytase: term " zytase " means 1,4- β-D- xylan-xylose hydrolase (1,4- β-D-xylan- Xylohydrolase) (E.C.3.2.1.8) is catalyzed the interior hydrolysis of Isosorbide-5-Nitrae-β-D- xylose glycosidic bond in xylan.With regard to the present invention Speech, xylanase activity use 0.2%AZCL- araboxylan as substrate at 37 DEG C, and pH 6 is 0.01%It is determined in X-100 and 200mM sodium acetate buffer.The xylanase activity of one unit is defined as at 37 DEG C, PH 6 generates 1.0 from the 0.2%AZCL- araboxylan as substrate in 6 buffer of 200mM sodium phosphate pH per minute Micromole's Bazurin.

Detailed description of the invention

With the active polypeptide of xylobiase

In one embodiment, the present invention relates to isolated polypeptides, with SEQ ID NO:6 or SEQ ID NO:8's Mature polypeptide is at least 60%, for example, at least 65%, at least 70%, 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 100% sequence identity；Have at least 65%, for example, at least 70% with the mature polypeptide of SEQ ID NO:2, until Few 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 100% sequence identity；Or with SEQ ID NO:4 or SEQ The mature polypeptide of ID NO:10 is at least 75%, for example, 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 100% sequence Identity；The polypeptide has xylobiase activity.In one aspect, the polypeptide and SEQ ID NO:2, SEQ ID NO: The mature polypeptide of 4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10 differs up to 10 amino acid, for example, 1,2, 3,4,5,6,7,8,9 or 10 amino acid.

Polypeptide of the invention, which is preferably comprised or organized, becomes SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ The amino acid sequence or its allelic variant of ID NO:8 or SEQ ID NO:10；It or is it with xylobiase active Section.On the other hand, the polypeptide includes or group becomes SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ The mature polypeptide of ID NO:8 or SEQ ID NO:10.On the other hand, the polypeptide includes or group becomes SEQ ID NO:2 Amino acid 20 to 777, the amino acid 20 to 825 of SEQ ID NO:4, the amino acid 20 to 851 of SEQ ID NO:6, SEQ ID The amino acid 22 to 767 of NO:8 or the amino acid 21 to 800 of SEQ ID NO:10.

In another embodiment, the present invention relates to the active isolated polypeptide of xylobiase, by multicore Thuja acid coding, the polynucleotides are in very low stringency condition, low stringency condition, medium stringency condition, medium-high stringency item Part, high stringency conditions, or hybridize under high stringency conditions with following very much: (i) SEQ ID NO:1, SEQ ID NO:3, SEQ ID The mature polypeptide encoded sequence of NO:5, SEQ ID NO:7 or SEQ ID NO:9, (ii) SEQ ID NO:1, SEQ ID NO:3, The cDNA sequence of SEQ ID NO:5 or SEQ ID NO:7, or (iii) (i) or (ii) overall length complement (Sambrook etc., 1989, Molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor, New York)。

Using SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:9's Polynucleotides or its subsequence and SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ The polypeptide of ID NO:10 or its mature polypeptide or its segment design nucleic acid probe, with according to method well known in the art never The bacterial strain identification and clone's coding that belong to or plant have the DNA of the active polypeptide of xylobiase.Specifically, according to standard These probes can be used to hybridize with the genomic DNA of interested cell or cDNA by Southern immunoblot method, with identification and From wherein separating corresponding gene.These probes can be significantly shorter than complete sequence, but should be at least 15 in length, for example, at least 25, at least 35, or at least 70 nucleotide.Preferably, the nucleic acid probe is the length of at least 100 nucleotide, for example, extremely Few 200 nucleotide, at least 300 nucleotide, at least 400 nucleotide, at least 500 nucleotide, at least 600 nucleosides Acid, at least 700 nucleotide, at least 800 nucleotide, or the length of at least 900 nucleotide.Both DNA and rna probe are equal It can be used.Usually by probe mark with detect corresponding gene (for example, with³²P、³H、³⁵S, biotin or avidin (avidin) it marks).These probes are covered by the present invention.

Can from the genomic DNA or cDNA library prepared by such other bacterial strains screening hybridize with above-mentioned probe and Encode the DNA with the active polypeptide of xylobiase.Can be by agarose or polyacrylamide gel electrophoresis, or pass through it Its isolation technics separates genome or other DNA from these other bacterial strains.Can by from library DNA or separation DNA is transferred to nitrocellulose (nitrocellulose) or other suitable carrier materials and is fixed thereon.In order to reflect Fixed and SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID its mature polypeptide of NO:7 or SEQ ID NO:9 is compiled The clone or DNA of code sequence or the hybridization of its subsequence, the carrier material is used in Sounthern trace.

For the present invention, hybridization indicates polynucleotides very down to the nucleic acid under very high stringent condition with label Probe hybridization, the nucleic acid probe correspond to: (i) SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO: 7 or SEQ ID NO:9, (ii) SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID The mature polypeptide encoded sequence of NO:9, (iii) SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7 CDNA sequence, (iv) their overall length complement, or (v) their subsequence.Such as X-ray film (X-ray can be used Film) or other any detection means as known in the art detections under these conditions with the molecule of nucleic acid probe hybridization.

In one aspect, the nucleic acid probe is coding SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ The polypeptide of ID NO:8 or SEQ ID NO:10 or the polynucleotides of its mature polypeptide or their segment.On the other hand, The nucleic acid probe is SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:9, The cDNA sequence of its mature polypeptide encoded sequence or SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7 Column or its mature polypeptide encoded sequence.

In another embodiment, the present invention relates to the active isolated polypeptide of xylobiase, by multicore Thuja acid coding, the mature polypeptide encoded sequence of the polynucleotides and SEQ ID NO:5 or SEQ ID NO:7 or its cDNA sequence With at least 60%, for example, at least 65%, at least 70%, 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 100% sequence identity；Have at least 65% with the mature polypeptide encoded sequence of SEQ ID NO:1 or its cDNA sequence, example Such as at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, until Few 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 100% sequence identity；With SEQ ID NO:3 or its cDNA sequence or the mature polypeptide encoded sequence of SEQ ID NO:9 have at least 75%, for example, 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 100% sequence identity.

In another embodiment, the present invention relates to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ The mature polypeptide of ID NO:8 or SEQ ID NO:10 one or more (such as several) positions include replace, missing and/or The variant of insertion.In one embodiment, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID are imported Amino acid substitution, missing and/or the quantity of insertion of the mature polypeptide of NO:8 or SEQ ID NO:10 are up to 10, such as 1,2,3,4,5,6,7,8,9 or 10.Amino acid change can be secondary property, i.e. conservative amino acid substitution or insertion, The folding and/or activity of protein are not significantly affected；The usually small missing of 1 to about 30 amino acid；Small amino or carboxylic Base end extends, such as amino terminal methionine residues；The small joint peptide of up to 20-25 residue；Or by changing net electricity Lotus or other functions promote the small extension of purifying, such as polyhistidine sequence (poly histidine tract), epitope (antigenic epitope) or binding domain (binding domain).

The example of conservative substitution is with the following group: basic amino acid group (arginine, lysine and histidine), acidity Amino acid group (glutamic acid and aspartic acid), polar amino acid group (glutamine and asparagine), hydrophobic amino acid group (bright ammonia Acid, isoleucine and valine), aromatic amino acid group (phenylalanine, tryptophan and tyrosine) and p1 amino acid group (sweet ammonia Acid, alanine, serine, threonine and methionine).The amino of specific activity (specific activity) is not changed usually It is known in the art that acid, which replaces, and by such as H.Neurath and R.L.Hill, 1979, in The Proteins, It is described in Academic Press, New York.The exchange 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.

Alternatively, amino acid change has the property for causing the physicochemical characteristics of polypeptide to change.For example, amino acid change can Improve the thermal stability of polypeptide, change substrate specificity, changes optimal pH etc..

Can according to methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis method (Cunningham and Wells, 1989, Science 244:1081-1085) identify the essential amino acid in parental polypeptide.It, will in latter technique Single alanine mutation is introduced into each residue in molecule, and tests whether obtained mutating molecule has β-xyloside Enzymatic activity, to identify the crucial amino acid residue of the activity for the molecule.It see also Hilton etc., 1996, J.Biol.Chem.271:4699-4708.The active site of enzyme or other biological interactions can also be by structures Physical analysis determines that structure is measured by these following technologies in conjunction with the mutation of the contact site amino acids for presumption: Such as nuclear magnetic resonance, crystallography, electronic diffraction or photoaffinity labeling.See, for example, de Vos etc., 1992, Science255:306- 312；Smith etc., 1992, J.Mol.Biol.224:899-904；Wlodaver etc., 1992, FEBS Lett.309:59-64. The identity of essential amino acid can also be inferred from the homogeneity analysis with related polypeptide.

Known mutagenesis, recombination and/or Shuffling Method can be used, then carry out relevant screening process, such as by Reidhaar-Olson and Sauer, 1988, Science 241:53-57；Bowie and Sauer, 1989, Proc.Natl.Acad.Sci.USA 86:2152-2156；WO 95/17413；Or those of disclosed in WO 95/22625, It carries out one or more amino acid substitutions, missing and/or insertion and is tested.Other workable methods include fallibility PCR, Phage display (such as Lowman etc., 1991, Biochemistry 30:10832-10837；U.S. Patent number 5,223,409； WO 92/06204) and regiondirected mutagenesis (region-directed mutagenesis) (Derbyshire etc., 1986, Gene 46:145；Deng 1988, DNA 7:127).

Mutagenesis/Shuffling Method can combine with high-throughput, auto-screening method with detect by being cloned of host cell expression, The activity (Ness etc., 1999, Nature Biotechnology 17:893-896) of the polypeptide of mutagenesis.Encoding active polypeptide DNA molecular through mutagenesis can be recycled from host cell and is sequenced rapidly using this field standard method.These methods allow quick Determine the importance of single amino acids residue in polypeptide.

The polypeptide can be hybrid polypeptide, and the region of one of polypeptide is blended in the N-terminal or C in the region of another polypeptide End.

The polypeptide can be fused polypeptide or the fused polypeptide that can cut, and another one peptide fusion is in of the invention more The N-terminal or C-terminal of peptide.It is more that fusion is generated by the way that the polynucleotides for encoding another polypeptide are blended in polynucleotides of the invention Peptide.It is known in the art for generating the technology of fused polypeptide, and the coded sequence including connection coding polypeptide is so that they meet Frame (in frame), and make the expression of fused polypeptide under the control of identical promoters and terminator.Fusion protein also may be used Using interior albumen (intein) technology construct, wherein fusions generate upon translation (Cooper etc., 1993, EMBO J.12: 2575-2583；Dawson etc., 1994, Science 266:776-779).

Fused polypeptide can also include cleavage site between two polypeptides.When secreting fused polypeptide, the site is just It is cut open, discharges described two polypeptides.The example for cutting site includes, but are not limited to be disclosed in Martin etc., and 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, Biotechnology 13:498-503；With Contreras etc., 1991, Biotechnology 9:378-381；Eaton etc., 1986,Biochem.25:505-512)；Collins-Racie etc., 1995, Biotechnology 13:982-987；Carter Deng 1989, Proteins:Structure, Function, and Genetics 6:240-248；And Stevens, 2003, Site in Drug Discovery World4:35-48.

Source with the active polypeptide of xylobiase

Of the invention has the active polypeptide of xylobiase can be obtained from the microorganism of any category.With regard to the present invention Speech, for this paper term " obtained from " related with given source, the meaning should be the polypeptide by polynucleotide encoding by described Source generates, or the bacterial strain by wherein inserting the polynucleotides from the source generates.In one aspect, from given source The polypeptide of acquisition is exocytosis.

In one aspect, the polypeptide is capital spore category (Scytalidium) polypeptide.On the other hand, the polypeptide It is thermophilic capital spore (Scytalidium thermophilum) polypeptide.On the other hand, the polypeptide is Penicillium (Penicillium) polypeptide.On the other hand, the polypeptide is penicillium oxalicum (Penicillium oxalicum) polypeptide. On the other hand, the polypeptide is Rhizomucor (Rhizomucor) polypeptide.On the other hand, the polypeptide is Rhizomucor pumillus polypeptide.On the other hand, the polypeptide is thermophilic ascomycete category (Thermoascus) polypeptide. On the other hand, the polypeptide is tangerine orange thermophilic ascomycete (Thermoascus aurantiacus) polypeptide.

It will be understood that the present invention includes complete and imperfect stage (perfect and for above-mentioned kind Imperfect states) and other taxonomic equivalents (equivalent), such as phorozoon (anamorph), and nothing By kind of name known to them.Those skilled in the art will readily recognize the identity of suitable equivalent.

The bacterial strain of these kinds can easily obtain the public in many culture collections, and the collection is all As American type culture collection (the American Type Culture Collection) (ATCC), Germany are micro- Biology and Cell Culture Collection (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) (DSMZ), fungi strain collection (Centraalbureau Voor Schimmelcultures) (CBS) and agricultural research institute's Patent Culture Collection North research center (Agricultural Research Service Patent Culture Collection,Northern Regional Research Center)(NRRL)。

Above-mentioned probe be can use from other sources, including what is separated from nature (for example, soil, compost, water etc.) Microorganism directly obtains DNA sample from nature material (for example, soil, compost, water etc.), identifies and obtains the polypeptide. For from the technology of Natural habitat (habitat) separate microorganism and DNA being directly well known in the art.Class can then be passed through As screen genomic DNA or cDNA library or the mixed DNA sample of another microorganism to obtain coding said polypeptide Polynucleotides.Once with probe in detecting to the polynucleotides of coding polypeptide, so that it may using known to those of ordinary skill in the art Technology the polynucleotides are separated or are cloned (see, e.g., Sambrook etc., 1989, see above).

Polynucleotides

The present invention is also related to the isolated polynucleotides of coding polypeptide of the invention as described herein.

Technology for separating or cloning polynucleotides is well known in the art, and including from genomic DNA or CDNA, or combinations thereof separation.It can be by using well known polymerase chain reaction (PCR) or the antibody screening of expression library The cloned DNA fragments with apokoinou construction characteristic are detected, to realize from this genomic dna cloning polynucleotides.Referring to, For example, Innis etc., 1990, PCR:A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification methods can be used, such as ligase chain reaction (LCR), connection activated transcription (ligated activated transcription；) and the amplification based on polynucleotides (NASBA) LAT.It can be from capital spore category, mould The bacterial strain or related organisms that category, Rhizomucor or thermophilic ascomycete belong to clone the polynucleotides, thus, for example can be institute State the allelic variant or inter-species variant (species variant) of the polypeptid coding area of polynucleotides.

The polynucleotides that modification encodes polypeptide of the present invention can for synthesizing with the essentially similar polypeptide of the polypeptide It can be required.Term and the polypeptide " essentially similar " refer to the non-naturally occurring form of polypeptide.These polypeptides may be with Some engineered modes and be different from the polypeptide that separates from its natural origin, for example, specific activity, thermal stability, optimal pH Etc. different variants.Can as SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or The mature polypeptide sequence or SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7 of SEQ ID NO:9 Mature polypeptide sequence the polynucleotides that present of cDNA sequence on the basis of and/or replaced by introducing following nucleotide: institute The change for replacing and not leading to polypeptid acid sequence is stated, but meets the codon usage for being intended to generate the host organisms of enzyme； Or replaced by the nucleotide that importing can produce different amino acid sequences to construct variant.Replace about nucleotide general It states, see, e.g., Ford etc., 1991, Protein Expression and Purification 2:95-107.

Nucleic acid construct

The invention further relates to the nucleic acid construct comprising polynucleotides of the invention, the polynucleotides and one or more (such as several) regulating and controlling sequence is operably connected, the regulating and controlling sequence in a suitable host cell with the regulating and controlling sequence phase The expression of coded sequence is instructed under conditions of appearance.

It can use and be permitted polynucleotides described in multi-mode operation in order to the expression of polypeptide.Depending on expression vector, will be more It may be ideal or required for operating on it before nucleotides inserted carrier.Multicore glycosides is modified using recombinant DNA method The technology of acid is well known in the art.

Regulating and controlling sequence can be promoter, by for expressing the host cell institute for encoding the polynucleotides of polypeptide of the invention The polynucleotides of identification.Promoter contains the transcription regulating nucleotide sequence of the expression of direct polypeptide.Promoter can be in host cell It is middle display transcriptional activity any polynucleotides, including mutation, truncated and heterozygosis promoter, and can from coding with The gene of the homologous or heterologous extracellular or intracellular polypeptide of host cell obtains.

The example of suitable promoter for instructing nucleic acid construct of the invention to transcribe in bacterial host cell be from The promoter of following acquisitions: bacillus amyloliquefaciens (Bacillus amyloliquefaciens) alpha-amylase gene (amyQ), bacillus licheniformis (Bacillus amyloliquefaciens) alpha-amylase gene (amyL), lichens gemma bar Bacterium penicillinase gene (penP), bacillus stearothermophilus (Bacillus stearothermophilus) produce malt starch Enzyme gene (amyM), bacillus subtilis (Bacillus subtilis) type froctosan saccharase gene (sacB), bacillus subtilis Bacterium xylA and xylB gene, bacillus thuringiensis (Bacillus thuringiensis) cryIIIA gene (Agaisse and Lereclus, 1994, Molecular Microbiology 13:97-107), E. coli lac operon, Escherichia coli trc Promoter (Egon etc., 1988, Gene 69:301-315), streptomyces coelicolor (Streptomyces coelicolor) agarose Enzyme gene (dagA) and protokaryon beta-lactam enzyme gene (Villa-Kamaroff etc., 1978, Proceedings of the National Academy of Sciences USA 75:3727-3731) and tac promoter (DeBoer etc., 1983, Proc.Natl.Acad.Sci.USA 80:21-25).Other promoter exists " Useful proteins from Recombinant bacteria " is in Gilbert etc., 1980, Scientific American, 242:74-94；With Sambrook etc., 1989, see above middle description.The example of Gene expression is disclosed in WO 99/43835.

Example for the suitable promoter for instructing nucleic acid construct of the invention to transcribe in filamentous fungal host cell It is the promoter obtained from the gene of following enzyme: aspergillus nidulans (Aspergillus nidulans) acetamidase, aspergillus niger (Aspergillus niger) neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, aspergillus niger or aspergillus awamori (Aspergillus awamori) glucoamylase (glaA), aspergillus oryzae (Aspergillus oryzae) TAKA amylase, rice Aspergillus alkali protease, aspergillus oryzae triose-phosphate isomerase, sharp fusarium (Fusarium oxysporum) trypsin-like protease Enzyme (WO 96/00787), empiecement fusarium (Fusarium venenatum) amyloglucosidase (WO 00/56900), empiecement sickle Spore Daria (WO 00/56900), empiecement fusarium Quinn (WO 00/56900), Man Hegen Mucor (Rhizomucor miehei) Lipase, Man Hegen Mucor aspartic protease, trichoderma reesei (Trichoderma reesei) β-glucosyl enzym, Richter scale wood Mould cellobiohydrolase I, trichoderma reesei cellobiohydrolase II, trichoderma reesei endoglucanase I, trichoderma reesei inscribe It is dextranase II, trichoderma reesei endoglucanase III, trichoderma reesei endoglucanase V, Xylanase from Trichoderma reesei I, inner Family name's reesei xylanase II, Xylanase from Trichoderma reesei III, trichoderma reesei xylobiase, trichoderma reesei translation elongation factor, And (a kind of promoter of modification comes from aspergillus neutral alpha-amylase enzyme gene, wherein untranslated NA2-tpi promoter Leader sequence substituted by the untranslated leader sequence of the gene of aspergillus triose-phosphate isomerase；Non-limiting example packet The promoter for including modification, the gene from Aspergillus ni ger neutral alpha-amylase, wherein untranslated leader sequence is by aspergillus nidulans Or the untranslated leader sequence of the gene of aspergillus oryzae triose-phosphate isomerase is substituted)；With it is their mutation, truncated and The promoter of heterozygosis.Other promoters are described in U.S. Patent number 6,011,147.

In yeast host, useful promoter is obtained from following gene: saccharomyces cerevisiae (Saccharomyces Cerevisiae) enolase (ENO-1), saccharomyces cerevisiae galactokinase (GAL1), saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde- 3- phosphate dehydrogenase (ADH1, ADH2/GAP), saccharomyces cerevisiae triose-phosphate isomerase (TPI), brewing yeast metallothionein (CUP1) and saccharomyces cerevisiae glycerol 3-phosphate acid kinase.Useful promoters other for yeast host cell by Romanos etc., 1992, Yeast 8:423-488 description.

Regulating and controlling sequence is also possible to transcription terminator, is identified by host cell to terminate transcription.The terminator and volume 3 ' ends of the polynucleotides of the code polypeptide are operably connected.In the present invention, it may be used at functional in host cell Any terminator.

Terminator preferred for bacterial host cell is obtained from following gene: Bacillus clausii (Bacillus Clausii) alkali protease (aprH), bacillus licheniformis alpha-amylase (amyL) and Escherichia coli rRNA (rrnB).

Terminator preferred for 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, Sharp fusarium trypsin like proteases, trichoderma reesei β-glucosyl enzym, trichoderma reesei cellobiohydrolase I, trichoderma reesei are fine Dimension disaccharide-hydrolysing enzymes II, trichoderma reesei endoglucanase I, trichoderma reesei endoglucanase II, trichoderma reesei inscribe Portugal are poly- Carbohydrase III, trichoderma reesei endoglucanase V, Xylanase from Trichoderma reesei I, Xylanase from Trichoderma reesei II, trichoderma reesei wood Dextranase III, trichoderma reesei xylobiase and trichoderma reesei translation elongation factor.

Terminator preferred for yeast host cell is obtained from the gene of following enzyme: saccharomyces cerevisiae enolase, wine brewing Yeast cells pigment C (CYC1) and S. cerevisiae glyceraldehyde -3- phosphate dehydrogenase.Useful ends other for yeast host cell Only son is by Romanos etc., and 1992, it sees above.

Regulating and controlling sequence can also be that the mRNA of the upstream of coding sequence of promoter downstream and gene stabilizes area, increase institute State the expression of gene.

The example that suitable mRNA stabilizes area is obtained from following gene: bacillus thuringiensis cryIIIA gene (WO And bacillus subtilis SP82 gene (Hue etc., 1995, Journal of Bacteriology 177:3465- 94/25612) 3471)。

Regulating and controlling sequence can also be suitable leader sequence, for the important mRNA untranslated of the translation for host cell Area.Leader sequence is operably connected to 5 '-ends of the polynucleotides of coding polypeptide.It may be used at functional in host cell Any leader sequence.

Leader sequence preferred for filamentous fungal host cell is obtained from the gene of following enzyme: oryzae TAKA amylase With aspergillus nidulans triose-phosphate isomerase.

Leader sequence suitable for yeast host cell is obtained from the gene of following enzyme: saccharomyces cerevisiae enolase (ENO-1), saccharomyces cerevisiae glycerol 3-phosphate acid kinase, cerevisiae alpha-factor and saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde -3- phosphorus Acidohydrogenase (ADH2/GAP).

Regulating and controlling sequence is also possible to polyadenylation sequence, is the sequence being operably connected with 3 ' ends of polynucleotides Column, and in transcription, host cell is identified as poly- adenosine residue being added to the signal of the mRNA of transcription.It may be used at Functional any polyadenylation sequence in host cell.

Polyadenylation sequence preferred for filamentous fungal host cell is obtained from the gene of following enzyme: aspergillus nidulans are adjacent Anthranilate synthase, aspergillus niger glucoamylase, aspergillus niger alpha-Glucosidase, oryzae TAKA amylase and sharp fusarium pancreas egg White enzyme sample protease.

The polyadenylation sequence useful for yeast host cell is by Guo and Sherman, and 1995, Mol.Cellular Biol.15:5983-5990 description.

Regulating and controlling sequence can also be signal peptide coding region, encode the signal peptide being connected with the N-terminal of polypeptide, and guide described Polypeptide enters cell secretory pathway.The end of coded sequence 5 ' of polynucleotides can include inherently signal coding sequence, with volume The section of the coded sequence of the code polypeptide is natively connected to together in translation reading frame.Alternatively, the end of coded sequence 5 ' can contain There is the signal coding sequence for the coded sequence external source.When coded sequence does not naturally contain signal coding sequence, Foreign signal peptide coding sequence may be necessary.Alternatively, directly natural signals peptide can be replaced with foreign signal peptide coding sequence Coded sequence is to enhance the secretion of polypeptide.However, the polypeptide that guidance can be used to express enters appointing for the secretory pathway of host cell What signal coding sequence.

The signal peptide coding that the gene that signal coding sequence effective for bacterial host cell follows from enzyme obtains Sequence: bacillus NCIB 11837 produces maltogenic amylase, bacillus licheniformis subtilopeptidase A (subtilisin), bacillus licheniformis beta-lactamase, bacillus stearothermophilus alpha-amylase, bacillus stearothermophilus Neutral proteinase (nprT, nprS, nprM) and bacillus subtilis prsA.Other signal peptide by Simonen and Palva, 1993, Microbiological Reviews 57:109-137 description.

Signal coding sequence effective for filamentous fungal host cell follows from the signal peptide that the gene of enzyme obtains Coded sequence: Aspergillus ni ger neutral amylase, aspergillus niger glucoamylase, oryzae TAKA amylase, Humicola insolens cellulose Enzyme, dredges cotton like humicola lanuginosa lipase and Man Hegen Mucor aspartic protease at Humicola insolens endoglucanase V.

The signal peptide useful for yeast host cell is obtained from the gene of cerevisiae alpha-factor and Saccharomyces cerevisiae invertase ?.Other useful signal coding sequences are by Romanos etc., and 1992, it sees above.

Regulating and controlling sequence can also be that propeptide code sequence, coding are located at the propetide of polypeptide N-terminal.Gained polypeptide is known as proenzyme (proenzyme) or preceding polypeptide (propolypeptide) (or in some cases be known as proenzyme (zymogen)).Preceding polypeptide is logical It is often inactive, and can be cut by the catalysis or self-catalysis of propetide from preceding polypeptide and be converted into active peptides.It can be from Bacillus subtilis alkali proteinase (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95/33836), the gene of Man Hegen Mucor aspartic protease and cerevisiae alpha-factor obtains propeptide code sequence.

In the presence of both signal peptide and propeptide sequence are equal, propeptide sequence is placed in the N of and then (next to) polypeptide It holds, and signal peptide sequence is placed in the N-terminal of and then propeptide sequence.

It is also desirable that addition adjusts sequence, the expression of polypeptide is adjusted relative to the growth of host cell.It adjusts The example of sequence be cause gene expression respond chemical or physical stimulus object, the presence including modulating compound and open or close Those of system.Adjusting sequence in prokaryotic system includes lac, tac and trp operator system.In yeast, it can be used ADH2 system or GAL1 system.In filamentous fungi, aspergillus niger glucose starch enzyme promoters, aspergillus oryzae TAKA α-can be used Amylase promoter and aspergillus oryzae glucose starch enzyme promoters, trichoderma reesei cellobiohydrolase I promoter and trichoderma reesei Cellobiohydrolase II promoter.The other examples for adjusting sequence are that those allow the sequence of gene magnification.In eukaryotic system In, it includes the dihydrofolate reductase gene expanded in the presence of amethopterin (methotrexate) that these, which adjust sequences, and The metallothionein gene expanded with heavy metal (with heavy metal).In these cases, the multicore glycosides of polypeptide is encoded Acid will be operably connected with sequence is adjusted.

Expression vector

The invention further relates to recombinant expression carrier, the recombinant expression carrier includes polynucleotides of the invention, promoter With transcription and translation termination signal.A variety of nucleotide and regulating and controlling sequence can have joined together to create recombinant expression carrier, institute Stating expression vector may include one or more (such as several) convenient restriction site to allow to be inserted into or take in these sites The polynucleotides of generation coding polypeptide.Alternatively, can include the multicore glycosides by being inserted into the carrier appropriate for expression The nucleic acid construct or polynucleotides of acid express the polynucleotides.During preparing expression vector, by coded sequence It is placed in carrier, to being operably connected to the coded sequence and regulating and controlling sequence appropriate for expression.

Recombinant expression carrier can be it is any can easily carry out recombinant DNA step, and polynucleotides can be generated Expression carrier (for example, plasmid or virus).The selection of carrier will generally depend on carrier and will introduce the host of the carrier The compatibility of cell.Carrier can be linear or closed hoop plasmid.

Carrier can be autonomously replicationg vector, that is, as carrier existing for extrachromosomal entity (entity), duplication is only Chromosome replication is stood on, for example, plasmid, extra-chromosomal element, minichromosome (minichromosome) or artificial chromosome. Carrier can be containing any for ensuring the means (means) of self-replacation.Alternatively, carrier, which can be one kind, to be introduced into host When in cell, the carrier that is integrated into genome and is replicated together with the chromosome for incorporating the carrier.In addition it is possible to use Individual carrier or plasmid or two or more carriers or plasmid, contain the complete of host cell gene group to be introduced jointly DNA (total DNA), or transposons (transposon) can be used.

The carrier preferably contains one or more selected markers, in order to be readily selected inverted, transfection, turn The cell led etc..Selected marker is such gene, and product provides biocide or virus resistance, resists to heavy metal Property, to auxotrophic prototrophy (prototrophy to auxotrophs) etc..

The example of bacterial selectable marker is bacillus licheniformis or bacillus subtilis dal gene, or assigns antibiotic The label of resistance, the antibiotic resistance such as ampicillin, chloramphenicol, kanamycins, neomycin, spectinomycin or Fourth Ring Plain resistance.For yeast host cell suitably mark include but is not limited to ADE2, HIS3, LEU2, LYS2, MET3, TRP1 and URA3.Selected marker for filamentous fungal host cell includes but is not limited to adeA (ribose phosphate aminooimidazole amber carboxylic Amide synthase, phosphoribosylaminoimidazole-succinocarboxamide synthase), adeB (phosphoric acid Ribose aminooimidazole synthase, phosphoribosyl-aminoimidazole synthase), amdS (acetamidase), argB (ornithine transcarbamylase), bar (glufosinate-ammonium (phosphinothricin) transacetylase), hph (hygromycin phosphoric acid Transferase), niaD (nitrate reductase) (nitrate reductase), pyrG (Orotidine-5 ' '-phosphate decarboxylase) (orotidine-5 '-phosphate decarboxylase), sC (sulfate adenylyl transferase) and trpC (o-amino benzoyl Acid synthase (anthranilate synthase)) and their equivalent.Being preferably used in Aspergillus cell is that structure nest is bent Mould or aspergillus oryzae amdS and pyrG gene and streptomyces hygroscopicus (Streptomyces hygroscopicus) bar gene.It is preferred that What it is for trichoderma cell is adeA, adeB, amdS, hph and pyrG gene.

Selected marker can be double selectivity tagging system described in WO 2010/039889.In one aspect, institute Stating double selectivity label is hph-tk double selectivity tagging system.

The carrier, which preferably comprises, allows vector integration to enter host cell gene group or carrier in cell independently of gene The element of group independently replicated.

In order to be integrated into host cell gene group, carrier can rely on the sequence of the polynucleotides of coding polypeptide or for passing through Homologous or non-homologous re-combination enters any other carrier element of genome.Alternatively, carrier can be containing for instructing to pass through Homologous recombination is integrated into the additional polynucleotides of the exact position in host cell gene group chromosome.In order to increase accurate A possibility that position is integrated, integrated element should have high degree of sequence identity with corresponding target sequence containing sufficient amount of Nucleic acid, such as 100 to 10,000 base-pair, 400 to 10,000 base-pair and 800 to 10,000 base-pair, to improve homologous recombination Probability.Integrated element can be any sequence homologous with target sequence in host cell gene group.In addition, integrated element can For non-coding or the polynucleotides of coding.On the other hand, carrier can be passed through into the base of non-homologous re-combination to host cell Because in group.

In order to independently replicate, carrier can also include replication orgin, enable carrier in the host cell from It replicates mainly.Replication orgin can be any plasmid replicon that the mediation functioned in cell independently replicates (replicator).Term " replication orgin " or " plasmid replicon " mean the multicore glycosides that can make to replicate in plasmid or carrier body Acid.

The example of bacterial origin of replication be the pBR322 plasmid for allowing to replicate in Escherichia coli, pUC19, pACYC177 and The replication orgin of pACYC184, and plasmid pUB110, pE194, pTA1060 and pAM β 1 for allowing to replicate in bacillus Replication orgin.

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

In filamentous fungal cells the example of useful replication orgin be AMA1 and ANS1 (Gems etc., 1991, Gene 98: 61-67；Cullen etc., 1987, Nucleic Acids Res.15:9163-9175；WO 00/24883).Separate AMA1 gene Plasmid or carrier with building comprising the gene can be completed according to the method being disclosed in WO 00/24883.

The polynucleotides Insertion Into Host Cell of the invention of more than one copy can be increased to the generation of polypeptide.Multicore The increase of thuja acid copy number can obtain by the following method: the sequence of at least one additional copy is integrated into host cell gene Group, or amplifiable selected marker is included in polynucleotides, wherein can be by suitable selective agent Cell is cultivated in the presence of (selectable agent) to select the amplification containing selected marker to copy, and is thus contained The cell of the additional copy of polynucleotides.

It in the method for constructing recombinant expression carrier of the invention is that those skilled in the art are known for connecting said elements (see, e.g., Sambrook etc., 1989, see above).

Host cell

The invention further relates to recombinant host cells, and it includes polynucleotides of the invention to be operably connected to one or more The regulating and controlling sequence of the generation of a guidance polypeptide of the present invention.Construct comprising polynucleotides or carrier are introduced into host cell, made The construct or carrier are used as chromosomal integrant as previously described or maintain as carrier outside the chromosome of self-replacation.Art Language " host cell " includes any offspring for being different from parental cell due to the mutation that occurs in reproduction process of parental cell. The selection of host cell will be largely dependent upon gene and its source of coding polypeptide.

Host cell can be useful any cell in the recombination of polypeptide of the invention generates, for example, protokaryon or true Nucleus.

Prokaryotic host cell can be any Gram-positive or gramnegative bacterium.Gram-positive bacterium include but Be not limited to, bacillus (Bacillus), fusobacterium (Clostridium), enterococcus spp (Enterococcus), gemma Bacillus (Geobacillus), lactobacillus (Lactobacillus), lactococcus (Lactococcus), ocean gemma bar Pseudomonas (Oceanobacillus), staphylococcus (Staphylococcus), streptococcus (Streptococcus) and strepto- Pseudomonas (Streptomyces).Gramnegative bacterium includes but is not limited to campylobacter (Campylobacter), large intestine Bacillus, Flavobacterium (Flavobacterium), Fusobacterium (Fusobacterium), Helicobacterium (Helicobacter), Mud Bacillus (lIyobacter), eisseria (Neisseria), pseudomonas (Pseudomonas), Salmonella (Salmonella) and Ureaplasma (Ureaplasma).

Bacterial host cell can be any bacillus cell, including but not limited to Alkaliphilic bacillus (Bacillus alka/ophilus), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), short gemma bar Bacterium (Bacillus brevis), Bacillus circulans (Bacillus circulans), Bacillus clausii, condensation gemma Bacillus (Bacillus coagulans), bacillus firmus (Bacillus firmus), bacillus lautus (Bacillus Lautus), bacillus lentus (Bacillus lentus), bacillus licheniformis, bacillus megaterium (Bacillus Megaterium), bacillus pumilus (Bacillus pumilus), bacillus stearothermophilus, bacillus subtilis and Soviet Union Cloud gold bacillus cell.

Bacterial host cell can also be any streptococcus cell, including but not limited to, streptococcus equisimilis (Streptococcus equisimilis), streptococcus pyogenes (Streptococcus pyogenes), streptococcus uberis (Streptococcus uberis) and zooepidemicus (Streptococcus equi Subsp.Zooepidemicus) cell.

Bacterial host cell can also be any Streptomyces cell, including but not limited to, not streptomyces chromogenes (Streptomyces achromogenes), deinsectization streptomycete (Streptomyces avermitilis), streptomyces coelicolor, Streptomyces griseus (Streptomyces griseus) and shallow Streptomyces glaucoviolaceus (Streptomyces lividans) cell.

It can realize by the following method and DNA is introduced into bacillus cell: protoplast transformation (see, e.g., Chang and Cohen, 1979, Mol.Gen.Genet.168:111-115), competent cell conversion (see, e.g., Young and Spizizen, 1961, J.Bacteriol.81:823-829 or Dubnau and Davidoff-Abelson, 1971, J.Mol.Biol.56:209-221), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6:742-751) or engagement (see, e.g., Koehler and Thorne, 1987, J.Bacteriol.169:5771-5278).It can It realizes by the following method and DNA is introduced into Bacillus coli cells: protoplast transformation (see, e.g., Hanahan, 1983, J.Mol.Biol.166:557-580) or electroporation (see, e.g., Dower etc., 1988, Nucleic Acids Res.16: 6127-6145).It can realize by the following method and DNA is introduced into Streptomyces cell: protoplast transformation, electroporation (ginseng See, for example, Gong etc., 2004, Folia Microbiol. (Praha) 49:399-405), engagement (see, e.g., Mazodier etc., 1989, J.Bacteriol.171:3583-3585), or transduction (see, e.g., Burke etc., 2001, Proc.Natl.Acad.Sci.USA 98:6289-6294).It can realize by the following method and DNA is introduced into pseudomonas Cell: electroporation (see, e.g., Choi etc., 2006, J.Microbiol.Methods 64:391-397) or engagement (referring to, For example, Pinedo and Smets, 2005, Appl.Environ.Microbiol.71:51-57).Can realize by the following method by DNA is introduced into streptococcus cell: natural competence (natural competence) (see, e.g., Perry and Kuramitsu, 1981, Infect.Immun.32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick, 1991, Microbios.68:189-207), electroporation (see, e.g., Buckley etc., 1999, Appl.Environ.Microbiol.65:3800-3804) or engagement (see, e.g., Clewell, 1981, Microbiol.Rev.45:409-436).However, any method known in the art that DNA is introduced to host cell can be used.

Host cell can be also eucaryote, such as mammal, insect, plant or fungal cell.

Host cell can be fungal cell.It includes with Xiamen that " fungi ", which is used in herein: Ascomycota (Ascomycota), load Daughter bacteria door (Basidiomycota), chytridiomycota (Chytridiomycota) and Zygomycota (Zygomycota) and oomycota (Oomycota) and all mitosporic fungis (mitosporic fungi) (such as by Hawksworth, in Ainsworth and Bisby ' s Dictionary of The Fungi, the 8th edition, 1995, CAB International, Defined in University Press, Cambridge, UK).

Fungal host cells can be yeast cells.It includes ascosporogenous yeast (ascosporogenous that " yeast " is used in herein Yeast) (Endomycetale (Endomycetales)), basidiosporogenous yeast (basidiosporogenous yeast) and belong to half The yeast of perfect fungus (Fungi Imperfecti) (gemma guiding principle (Blastomycetes)).Since the future that is sorted in of yeast can Can change, for the present invention, by yeast be defined as Biology and Activities of Yeast (Skinner, Passmore, and Davenport are compiled, Soc.App.Bacteriol.Symposium Series No.9,1980) described in.

Yeast host cell can be candida, Hansenula (Hansenula), Kluyveromyces, finish red ferment Female category, saccharomyces, Schizosaccharomyces or the mould category cell of Western alpine yarrow, such as Kluyveromyces lactis (Kluyveromyces Lactis), saccharomyces carlsbergensis, saccharomyces cerevisiae, saccharomyces diastaticus, Doug Laplace yeast, Saccharomyces kluyveri, promise yeast, ellipsoideus yeast, Or solution mould (Yarrowia lipolytica) cell of rouge the West alpine yarrow.

Fungal host cells can be filamentous fungal cells." filamentous fungi " includes Eumycota (Eumycota) and oomycota All filamentous forms of subphylum (such as by Hawksworth, 1995, see above, defined).The common feature of filamentous fungi exists It is constituted in by chitin (chitin), cellulose, glucan, chitosan (chitosan), mannosan and other complicated polysaccharide Mycelia body wall.Row nutrient growth is extended by mycelia, and carbon catabolism is obligate aerobic.On the contrary, yeast is for example made The nutrient growth of brewer yeast is carried out by the gemmation (budding) of unicellular thallus, and carbon catabolism can be fermentation Property.

Filamentous fungal host cell can be acremonium, aspergillus, Aureobasidium, the mould category of smoke pipe, quasi- wax Pseudomonas, golden spore Daughter bacteria category, Coprinus, Coriolus Qu61, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe grisea category, mucor, The mould category of myceliophthora, Xin Kaoma rouge, Neurospora, paecilomyces, Penicillium, flat lead fungi category, penetrate arteries and veins Pseudomonas, cud Chytridium, The mould category of Pleurotus, Schizophyllum, Talaromyces, thermophilic ascomycete category, shuttle spore, Tolypocladium, Trametes or trichoderma cell (Acremonium,Aspergillus,Aureobasidium,Bjerkandera,Ceriporiopsis, Chrysosporium,Coprinus,Coriolus,Cryptococcus,Filibasidium,Fusarium,Humicola, Magnaporthe,Mucor,Myceliophthora,Neocallimastix,Neurospora,Paecilomyces, Penicillium,Phanerochaete,Phlebia,Piromyces,Pleurotus,Schizophyllum, Talaromyces,Thermoascus,Thielavia,Tolypocladium,Trametes,or Trichoderma cell)。

For example, filamentous fungal host cell can be aspergillus awamori, aspergillus fumigatus, smelly aspergillus, aspergillus japonicus, aspergillus nidulans, black Aspergillus, 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 intend 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 fusarium (Fusarium bactridioides), F.graminearum schw, library prestige fusarium, machete fusarium, grass Section's fusarium, red fusarium of standing grain, different spore fusarium, albizzia fusarium, sharp fusarium, racemosus fusarium, pink fusarium, elder fusarium, the colour of skin Fusarium, sulphur color fusarium, circle fusarium, quasi- silk spore fusarium, empiecement fusarium, Humicola insolens, dredges cotton like detritus at intend branch spore fusarium Mould, rice black wool mould, thermophilic fungus destroyed wire, Neuraspora crassa, penicillium purpurogenum (Fusarium cerealis, Fusarium crookwellense,Fusarium culmorum,Fusarium graminearum,Fusarium graminum, Fusarium heterosporum,Fusarium negundi,Fusarium oxysporum,Fusarium reticulatum,Fusarium roseum,Fusarium sambucinum,Fusarium sarcochroum,Fusarium sporotrichioides,Fusarium sulphureum,Fusarium torulosum,Fusarium trichothecioides,Fusarium venenatum,Humicola insolens,Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila,Neurospora crassa,Penicillium Purpurogenum), the yellow flat lead fungi of spore (Phanerochaete chrysosporium), penetrate arteries and veins bacterium (Phlebia Radiata), pleurotus eryngii (Pleurotus eryngii), autochthonal shuttle spore be mould, long wool Trametes trogii (Trametes villosa), Trametes versicolor (Trametes versicolor), Trichoderma harzianum, trichodermaharzianum, long shoot trichoderma, trichoderma reesei or Trichoderma viride are thin Born of the same parents.

It can be by fungal cell by being related to the method for protoplast formation, protoplast transformation and cell wall-deficient mutant with this Mode well known to body converts.For converting the appropriate method of aspergillus and pyr-trichoderma host cell in EP238023 and Yelton Deng, 1984, Proc.Natl.Acad.Sci.USA 81:1470-1474 and Christensen etc., 1988, Bio/ It is described in Technology 6:1419-1422.For converting the appropriate method of Fusarium strain by Malardier etc., 1989, Gene 78:147-156 and WO 96/00787 is described.The method transformed yeast described by following document: Becker can be used And Guarente, in Abelson, J.N. and Simon, M.I. is compiled, Guide to Yeast Genetics and Molecular Biology,Methods in Enzymology,Volume 194,pp 182-187,Academic Press,Inc.,New York；Ito etc., 1983, J.Bacteriol.153:163；With Hinnen etc., 1978, Proc.Natl.Acad.Sci.USA 75:1920。

Production method

The invention further relates to the methods for generating polypeptide of the present invention comprising: (a) in the condition for helping to create polypeptide Lower culture cell, the cell generate the polypeptide with its wild-type form；Optionally (b) recycles the polypeptide.At one Aspect, the cell are the cells that capital spore belongs to.On the other hand, the cell is thermophilic capital spore cell.At another Aspect, the cell are Penicillium cells.On the other hand, the cell is penicillium oxalicum cell.On the other hand, institute Stating cell is Rhizomucor cell.On the other hand, the cell is Rhizomucor pumillus cell.At another Aspect, the cell are thermophilic ascomycete category cells.On the other hand, the cell is tangerine orange thermophilic ascomycete cell.

The invention further relates to the methods for generating polypeptide of the invention comprising: (a) in the item for helping to create polypeptide Recombinant host cell of the invention is cultivated under part；Optionally (b) recycles the polypeptide.

The host cell is trained in the nutrient medium for being suitable for generating the polypeptide using methods known in the art It supports.For example, can by the shaking flask culture in suitable culture medium and under conditions of allowing to express and/or separate the polypeptide, Or in laboratory or industrial fermentation tank small-scale or large scale fermentation (including it is continuous, in batches, fed-batch or solid state fermentation) To cultivate cell.It is cultivated in suitable nutrient medium using methods known in the art, the nutrient medium packet Carbonaceous sources and nitrogen source and inorganic salts.Suitable culture medium can be obtained from commercial supplier or can be prepared according to disclosed composition (for example, in catalogue of American type culture collection).If polypeptide is secreted into nutrient medium, which can be with It is directly recycled from the culture medium.If polypeptide is not secreted, can be recycled from cell lysate (lysate).

The method known in the art for the polypeptid specificity can be used to detect polypeptide.These detection method packets Include but be not limited to the use of specific antibody, the formation of enzyme product or the disappearance of zymolyte.For example, enzyme assay (enzyme Assay it) can be used for determining the activity of polypeptide.

Methods known in the art recycling can be used in polypeptide.For example, polypeptide can be by conventional method from nutrition culture It is recycled in base, the conventional method includes but is not limited to collection, be centrifuged, filtering, extract, spray drying, evaporates or precipitate.One A aspect has recycled the whole beer comprising polypeptide of the invention.

Polypeptide can by a variety of methods known in the art purify to obtain substantially pure polypeptide, the method includes But chromatography (for example, ion exchange, affine, hydrophobic, chromatofocusing and size exclusion), electrophoresis method are not limited to (for example, preparative (preparative) isoelectric focusing), differential solubility (for example, ammonium sulfate precipitation), SDS-PAGE or extract (see, e.g., Protein Purification, Janson and Ryden are compiled, VCH Publishers, New York, and 1989).

On the other hand, polypeptide is not recycled, but uses the host cell of the invention for expressing the polypeptide as institute State the source of polypeptide.

Plant

The invention further relates to isolated plants, for example, genetically modified plants, plant part or plant cell, it includes this hairs Bright polynucleotides, to recyclable amount expression and generate the polypeptide.Polypeptide can be recycled from plant or plant part.Or Plant containing the polypeptide or plant part (as such) can be used to improve food or quality of the fodder, example as it is by person Such as, nutritive value, palatability (palatability) and the rheological equationm of state (rheological properties) are improved, or is used for Destroy anti-nutritional factors.

Genetically modified plants can be dicots (dicotyledon) or monocotyledonous (monocotyledon).Monocotyledon Example be careless (grasses), such as English grass (meadow grass) (bluegrass (blue grass), Poa L. (Poa))；Forage grass (forage grass) such as Festuca (Festuca), Lolium (Lolium)；Cold ground type herbage (temperate grass), such as Agrostis (Bentgrass)；And cereal, for example, wheat, oat, rye, barley, rice (rice), sorghum and maize (maize) (corn).

The example of dicotyledon is tobacco (tobacco), beans (legumes), such as lupin (lupins), Ma Ling (cruciferous) of potato, sugar beet (sugar beet), pea, beans (bean) and soybean (soybean) and Cruciferae plants Object (Cruciferae (family Brassicaceae)), such as cauliflower (cauliflower), rapeseed (rape seed) and tight Close relevant model organism arabidopsis (Arabidopsis thaliana).

The example of plant part is stem (stem), callus (callus), leaf (leaf), root (root), fruit (fruit), seed (seed) and stem tuber (tuber), and the independent body comprising these parts, for example, epidermis (epidermis), mesophyll (mesophyll), parenchymal tissue (parenchyme), vascular tissue (vascular tissue), point Raw tissue (meristem).Specific palnt cell compartments (compartments), outside chloroplaset (chloroplast), matter Body (apoplast), mitochondria (mitochondria), vacuole (vacuole), peroxisome (peroxisome) and thin Cytoplasm (cytoplasm) is also considered as plant part.In addition, any plant cell, whatsoever tissue-derived, it is considered to It is plant part.Similarly, plant part is such as separated for promoting the specific tissue of application of the invention also to be recognized with cell To be plant part, such as embryo (embryo), endosperm (endosperm), aleuron (aleurone) and kind skin (seed coat).

Be again included in the scope of the invention there are also these plants, plant part and plant cell offspring.

The genetically modified plants or plant cell for expressing polypeptide can construct according to means known in the art.In brief, lead to It crosses following method and constructs the plant or plant cell: the one or more expression constructs for encoding polypeptide are imported into plant host Genome or Chloroplast gene, and resulting modified plant or plant cell are bred as genetically modified plants or plant Cell.

Expression construct advantageously include encode polypeptide polynucleotides nucleic acid construct, the polynucleotides with Adjusting sequence appropriate needed for the polynucleotides is expressed in the plant of selection or plant part to be operably connected.In addition, table Expression constructs may include the selected marker useful for plant identification cell, and expression structure is incorporated in the plant cell It builds body and the construct is introduced into necessary DNA sequence dna in the plant (the latter depends on the DNA introducing method used).

The selection of sequence, such as the selection of promoter and terminator sequence and optional earth signal or transit sequence are adjusted, is lifted For example, based on expectation when, where and how to express polypeptide and determine.For example, the expression of the gene of coding polypeptide can be with Be composing type or induction type, or can be development, stage or tissue specificity, and gene product can target it is specific Tissue or plant part such as seed or leaf.Sequence is adjusted by such as Tague etc., 1988, Plant Physiology 86:506 It is described.

For constructive expression, 1 promoter (Franck of 35S-CaMV, maize ubiquitin 1 or rice actin can be used Deng, 1980, Cell 21:285-294, Christensen etc., 1992, Plant Mo.Biol.18:675-689；Zhang etc., 1991,Plant Cell 3:1155-1165).Organ specific promoters can be for example from storage tissue (storage Sink tissue) such as seed, potato tubers and fruit promoter (Edwards and Coruzzi, 1990, Ann.Rev.Genet.24:275-303), or from metabolic pool tissue (metabolic sink tissue) such as separate living tissue Promoter (Ito etc., 1994, Plant Mol.Biol.24:863-878), seed specific promoters are such as from the paddy of rice Albumen (glutelin), alcohol soluble protein (prolamin), globulin (globulin) or albumin (albumin) promoter (Wu Deng 1998, Plant Cell Physiol.39:885-889), come from legumin (legumin) B4 and semen viciae fabae (Vicia Faba semen viciae fabae promoter (Conrad etc., 1998, J.Plant Physiol.152:708- of unknown Seed Storage Protein gene) 711) promoter (Chen etc., 1998, Plant Cell of seed oil bodies albumen (oil body protein), are come from Physiol.39:935-941), the storage protein napA promoter or this technology of colea (Brassica napus) are come from The promoter of any other seed specific well known to field, for example, described in WO 91/14772.In addition, promoter Can be leaf specificity promoter, such as from rice or tomato rbcs promoter (Kyozuka, 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) come It is opened from what the aldP gene promoter (Kagaya etc., 1995, Mol.Gen.Genet.248:668-674) or wound of rice induced Mover, such as potato pin2 promoter (Xu, 1993, Plant Mol.Biol.22:573-588).Similarly, the starting Son can be induced by abiotic processing, abiotic processing such as temperature, arid or the salinity altercation, or be applied by external source The substance induction of the activation promoter added, such as ethyl alcohol, estrogen (oestrogens), plant hormone (plant Hormones) such as ethylene, abscisic acid (abscisic acid) and gibberellic acid (gibberellic acid) and heavy metal.

Promoter enhancer element can be used for realizing higher expression of the polypeptide in plant.For example, promoter enhances Subcomponent can be introne, be placed between promoter and the polynucleotides for encoding polypeptide.Such as Xu etc., 1993, see on, it is public The First Intron using 1 gene of rice actin has been opened with Enhanced expressing.

It any other part of selected marker and expression construct can be those of available selected from the art.

Nucleic acid construct is imported into Plant Genome according to routine techniques known in the art, the routine techniques includes soil Conversion, the virus-mediated conversion, microinjection (microinjection), grain of earth Bacillus (Agrobacterium) mediation Son bombardment, Biolistic transformation and electroporation (Gasser etc., 1990, Science 244:1293；Potrykus,1990,Bio/ Technology 8:535；Shimamoto etc., 1989, Nature 338:274).

The gene transfer (gene transfer) that Agrobacterium tumefaciens (Agrobacterium tumefaciens) mediate, Be a kind of generation transgenic dicots (it is summarized, referring to Hooykas and Schilperoort, 1992, Plant Mol.Biol.19:15-38), and for transforming monocots method, although other turn can be used for these plants Change method.It is a kind of generate transgenic monocot plant method be with particle (with conversion DNA coating microcosmic gold or tungsten particle Son) 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/Technology 10:667-674).A kind of alternative of transforming monocots is turned based on protoplast Change, such as by Omirulleh, described in 1993, Plant Mol.Biol.21:415-428.Other method for transformation include retouching It those of is set forth in U.S. Patent number 6,395,966 and 7,151,204 (being both by reference incorporated herein in its entirety).

After conversion, there is transformant and the regeneration of the expression construct imported according to method choice well known in the art As full plants.Method for transformation is commonly designed for selectively disappearing during regeneration or in subsequent generation by the following method Except selection gene: for example, special there are two the cotransformation of independent T-DNA construct or by specific recombinase site using band Selection gene is cut off anisotropicly.

It, can also be by that will have building other than directly directly converting specific plant genotype with construct of the invention The plant of body and the second plant hybridization for lacking the construct carry out prepare transgenosis plant.For example, polypeptide will can be encoded Construct introduces specified plant kind by hybridization, and at all without directly converting the plant for giving kind.Therefore, this hair It is bright not only to cover from the plant according to the transformed cell Direct Regeneration of the present invention, it further include the offspring of such plant (progeny).As used in this article, offspring can refer to the descendant according to mother plant prepared by the present invention any generation (offspring).Such offspring may include the DNA construct prepared according to the present invention.Hybridization causes transgenosis by that will originate Germline donor plant germline crossing pollination and introduced plant germline.The non-limiting example of such step is described in U.S. Patent number 7,151,204。

Plant is generated by backcross conversion method.For example, the plant include referred to as the genotype of backcross conversion, kind It is, the plant of inbreeding body (inbred) or hybrid (hybrid).

It can be used genetic marker to assist one or more transgenosis of the invention from a genetic background gene transgression (introgression) to another.The selection that label is assisted provides the advantage relative to conventional breeding, is that it can be used for Avoid the mistake as caused by phenotypic variation.Further, genetic marker can provide related breeding in the individual offspring of specific cross The data of germplasm relative extent.For example, when (otherwise) with genetic background needed for non-agronomy but does not have for this When the plant of required character and breeding parents, genetic marker can be used to select not only to have objective trait, also there is phase To the offspring of the required germplasm of larger proportion.By this method, one or more character genes are penetrated into needed for specific genetic background Generation number minimized.

The present invention is also related to the method for generating polypeptide of the invention comprising: (a) in the item for helping to create the polypeptide Genetically modified plants or plant cell are cultivated under part, the plant or plant cell include the polynucleotides of coding polypeptide；With it is optional Recycle the polypeptide in ground (b).

Remove or reduce xylobiase activity

The invention further relates to the methods for generating parental cell mutant comprising destroys or missing coding is of the invention Polynucleotides of polypeptide or part thereof, when the method causes to cultivate under the same conditions, what is be mutated compared with parental cell is thin Born of the same parents generate the less polypeptide.

Method well known in the art (for example, insertion, destruction, substitution or missing) can be used by reducing or eliminating multicore The expression of thuja acid constructs mutant cell.The polynucleotides are inactivations in a preferred aspect,.It is to be finished or inactivation Polynucleotides can be, for example, regulating element needed for code area or its part to active key, or expression code area.This Kind is adjusted or the example of regulating and controlling sequence can be promoter sequence or its funtion part, that is, is enough to influence polynucleotides expression Part.Other regulating and controlling sequences for possible modification include but is not limited to leader sequence, polyadenylation sequence, propetide sequence Column, signal peptide sequence, transcription terminator and transcription activator.

It can be by imposing mutagenesis to parental cell, and select wherein to have reduced or eliminated the expression of polynucleotides Mutant cell carries out modification or the inactivation of polynucleotides.Mutagenesis may be specificity or it is random, can be for example, by making With suitably physically or chemically mutagens carry out, by using suitable oligonucleotides carry out, or by by the DNA sequence dna into The mutagenesis that row PCR is generated.Furthermore, it is possible to carry out mutagenesis by using any combination of these mutagens.

The example for being suitable for the physically or chemically mutagens of the object of the invention includes ultraviolet light (UV) irradiation, azanol, N- first Base-N'- nitro-N nitrosoguanidine (MNNG), O- methyl hydroxylamine, nitrous acid, ethyl methanesulfonate (ethyl methane Sulphonate) (EMS), sodium hydrogensulfite, formic acid and nucleotide analog.

When using such agents, it usually carries out the mutagenesis by the following method: existing under suitable conditions selected Mutagens when incubate parental cell to be mutagenic, and screen and/or selection display gene expression is reduced or without gene expression Mutant cells.

The modification of polynucleotides or inactivation can by one or more nucleotide in insertion, substitution or missing gene or The controlling element that it is transcribed or translation is required is realized.For example, can be inserted or remove nucleotide so as to cause termination codon is introduced Son removes initiation codon, or changes and open frame.It is lured according to methods known in the art by what site-directed mutagenesis or PCR were generated This modification or inactivation may be implemented in change.Although the theoretically described modification can carry out in vivo, that is, directly to be repaired in expression Carried out on the cell of the polynucleotides of decorations, but it is preferably as illustrated below as carry out the modification in vitro.

The example for eliminating or reducing the convenient manner of polynucleotides expression has to be replaced based on gene, gene delection or gene The technology of destruction.For example, the nucleic acid sequence that will correspond to endogenous polynucleotides carries out mutagenesis in vitro in gene disruption method To generate the nucleic acid sequence of defective, then it is transformed into parental cell to generate dcc gene.Pass through homologous recombination, institute Defective nucleic acid sequence is stated instead of endogenous polynucleotide.It may be desirable that the defective polynucleotides also encode mark Note can be used for selecting the transformant that wherein polynucleotides are modified or destroy.In one aspect, (such as with selectable label Those described herein) destroy the polynucleotides.

The present invention is also related to inhibit the method with the expression of the active polypeptide of xylobiase in cell comprising to Double-stranded RNA (dsRNA) molecule is expressed in cell application in cell, wherein the dsRNA includes polynucleotides of the invention Subsequence.The dsRNA length is about 15,16,17,18,19,20,21,22,23,24,25 or more in a preferred aspect, Multiple duplex nucleotides.

The dsRNA is preferably siRNA (siRNA) or microRNA (miRNA).It is described in a preferred aspect, DsRNA is the siRNA for inhibiting transcription.At another preferred aspect, the dsRNA is for inhibiting the micro- of translation RNA。

The present invention is also related to such double-stranded RNA (dsRNA) molecule, it includes SEQ ID NO:1, SEQ ID NO:3, A part of the mature polypeptide encoded sequence of SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:9, in cell Inhibit the expression of the polypeptide.Although the present invention is not limited by any specific mechanism of action, the dsRNA can enter thin Born of the same parents and the single stranded RNA (ssRNA) for leading to sequence similar or identical, the degradation including endogenous mRNA.When cell is exposed to dsRNA When, the mRNA from homologous gene is by the process of referred to as RNA interference (RNAi) by degradation selectivity.

DsRNA of the invention can be used for gene silencing.In one aspect, the present invention provides use dsRNAi of the invention The method of degradation selectivity RNA.This method can be implemented in vitro, in vitro or in vivo.In one aspect, the dsRNA molecule can For the mutation that systematic function is lost in cell, organ or animal.It is used to prepare and use dsRNA molecular selection to degrade The method of RNA is it is well known in the art that see, e.g. 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, and it includes the polynucleotides of coding polypeptide or its regulations The silencing of the gene of the destruction of sequence or missing or coding said polypeptide, this causes the mutant cells compared with parental cell to generate Less polypeptide does not generate polypeptide.

Polypeptide deficiency mutant cell is particularly useful as the host cell for expressing natural and heterologous polypeptide.So this hair It is bright further to production is natural or the method for heterologous polypeptide comprising: (a) culture is prominent under conditions of helping to produce polypeptide Attenuate born of the same parents；Optionally (b) recycles the polypeptide.Term " heterologous polypeptide " means it is not natural polypeptide, example to host cell Such as, the variant of native protein.Host cell may include coding natural or heterologous polypeptide the multicore glycosides copied more than one Acid.

Method for cultivating and purifying interested product can be carried out by methods known in the art.

The present invention is used to generate the method substantially without the active product of xylobiase in eukaryon polypeptide, especially fungi It is especially to make us interesting in the generation of protein such as enzyme.Xylobiase deficient cells can be used for expression in pharmacy Upper significant heterologous protein such as hormone, growth factor, receptor etc..Term " eukaryon polypeptide " not only includes natural polypeptides, Also include be modified by amino acid substitutions, deletions, or additions or other such modifications with enhance activity, thermal stability, The polypeptide of pH tolerance etc., such as enzyme.

In other respects, the present invention relates to substantially without the active protein product of xylobiase, through the invention Method generate.

Fermentation liquid formulation or cell composition

The present invention is also related to fermentation liquid formulation and cell composition, and it includes polypeptides of the invention.The fermentation liquid produces Object further includes other ingredients for zymotechnique, such as cell (including containing the gene for encoding polypeptide of the invention Host cell is used to generate interested polypeptide), cell fragment, biomass, fermentation medium and/or tunning.One In a little embodiments, the composition is the full nutrient solution for having killed cell containing organic acid, the cell being killed and/or thin Born of the same parents' fragment and culture medium.

Term " fermentation liquid " be used for herein refer to by cell fermentation generate, do not suffer from or only undergo minimum recycling and/ Or the prepared product of purifying.For example, it when culture of microorganism is grown to saturation, incubates under conditions of limiting carbon to allow Albumen synthesizes (such as by host cell expression enzyme), and when being secreted into cell culture medium, generates fermentation liquid.The fermentation liquid can contain There is the content for not being classified or being classified of the fermented material obtained when fermenting and terminating.Typically, fermentation liquid is unassorted, And include removal (such as passing through centrifugation) microbial cell (such as filamentous fungal cells) after existing for used culture medium with And cell fragment.In some embodiments, the fermentation liquid contains used cell culture medium, ectoenzyme, and can survive And/or (viable and/or nonviable) microbial cell that cannot be survived.

In one embodiment, the fermentation liquid formulation and cell composition include the first organic acid composition and second Organic acid composition, first organic acid composition includes the organic acid and/or its salt of at least one 1-5 carbon, and described second has Machine acid constituents includes the 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 aforementioned mixture, and described second Organic acid composition be benzoic acid, cyclohexane-carboxylic acid, 4- methylvaleric acid, phenylacetic acid, they salt or it is aforementioned two or more Mixture.

In one aspect, the composition contains organic acid, and optionally further containing the cell being killed and/or Cell fragment.In one embodiment, removed from the full nutrient solution for killed cell the cell being killed and/or Cell fragment is free of the composition of these components to provide.

The fermentation liquid formulation or cell composition can further include preservative and/or antimicrobial (such as antibacterial) Agent, including but not limited to sorbierite, sodium chloride, potassium sorbate and other as known in the art.

The fermentation liquid formulation or cell composition can further include a variety of enzymatic activitys, such as one or more (such as It is several) enzyme selected from the group below: cellulase, hemicellulase, esterase, clavacin (expansin), laccase, lignin decompose Enzyme, pectase, peroxidase, protease and swollenin (swollenin).The fermentation liquid formulation or cell composition are also May include one or more (such as several) enzymes selected from the group below: hydrolase, isomerase, ligase, lyase, oxygen also enzyme turn Move enzyme, such as alpha-galactosidase, alpha-Glucosidase, aminopeptidase, amylase, beta galactosidase, β-glucosyl enzym, β-xylose Glycosides enzyme, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin sugar Based transferase, deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase, laccase, lipase, sweet dew Glycosidase becomes dextranase, oxidizing ferment, pectin decomposing enzyme, peroxidase, phytase, polyphenol oxidase, proteolytic enzyme, core Ribonuclease T., transglutaminase or zytase.

The full nutrient solution for having killed cell or composition contain the fermented material obtained when fermenting and terminating not It is classified content.Typically, the full nutrient solution for having killed cell or composition contain by microbial cell (such as silk Shape fungal cell) saturation is grown to, and incubate under conditions of limiting carbon to allow albumen synthesis (for example, expression cellulase And glucosidase) there are used culture medium and cell fragments later.In some embodiments, the cell killed Full nutrient solution or composition contain used cell culture medium, ectoenzyme, and the filamentous fungal cells being killed.In some realities It applies in scheme, the microbial cell present in the full nutrient solution or composition for having killed cell can be used as known in the art Method infiltration and/or cracking.

Full nutrient solution as described herein or cell composition are usually liquid, but can contain insoluble component, such as Cell, cell fragment, nutrient media components and/or the insoluble enzyme being killed.In some embodiments, it can remove insoluble group Divide to provide clear liquid composition.

Full nutrient solution formulation of the invention and cell composition can be by WO 90/15861 or WO 2010/096673 The method of description generates.

Set forth below is the embodiments of the preferable use of composition of the invention.The dosage and composition of the composition make Other conditions can be determined based on means known in the art.

Enzymatic compositions

The invention further relates to the compositions comprising polypeptide of the invention.Preferably, the composition be enriched it is such more Peptide.Term " being enriched " shows that the xylobiase activity of the composition is increased with for example, at least 1.1 enrichment factor.

The composition may include polypeptide of the invention as major enzymatic component, such as single-component composition.Alternatively, described Composition may include a variety of enzymatic activitys, such as one or more (such as several) enzymes selected from the group below: cellulase, hemicellulose Enzyme, esterase, clavacin, laccase, lignin decomposition enzyme, pectase, peroxidase, protease and swollenin.The hair group Closing object also may include one or more (such as several) enzymes selected from the group below: hydrolase, isomerase, ligase, lyase, oxygen is also Enzyme or transferase, for example, alpha-galactosidase, alpha-Glucosidase, aminopeptidase, amylase, beta galactosidase, β-glucosyl enzym, Carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyl transfer Enzyme, deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase, laccase, lipase, mannosidase, Become dextranase, oxidizing ferment, pectin decomposing enzyme, peroxidase, phytase, polyphenol oxidase, proteolytic enzyme, ribonucleic acid Enzyme, transglutaminase or zytase.The composition can be prepared according to method as known in the art, and can be liquid Or the form of dry composition.The composition can be stabilized according to method as known in the art.

The example that the preferable use of composition of the invention is given below.The dosage and composition of composition use its Its condition can give methods known in the art to determine.

Purposes

The technique with the active polypeptide of xylobiase or combinations thereof object is used the invention further relates to following.

The invention further relates to the methods degraded or convert cellulosic material or the material containing xylan comprising: in the present invention There is the active polypeptide of xylobiase in the presence of, handle cellulosic material or material containing xylan with enzymatic compositions.? On one side, the technique further comprises the cellulosic material or material containing xylan that recycling has been degraded or converted.The fibre The soluble product for tieing up degradation or the conversion of cellulosic material or the material containing xylan can be from insoluble fibrin material or containing xylan The separation of materials'use methods known in the art, such as such as centrifugation, filtering or gravitational settling.

The invention further relates to the techniques for generating tunning comprising: (a) there is xylobiase activity in of the invention Polypeptide in the presence of, with enzymatic compositions saccharified cellulosic material or material containing xylan；(b) with one or more (such as several Kind) fermentative microorganism fermentation the cellulosic material through being saccharified or material containing xylan to generate tunning；(c) from fermenting back Receive tunning.

The invention further relates to fermentable fiber cellulosic material or the techniques of the material containing xylan comprising: with one or more (examples As several) fermentative microorganism fermentable fiber cellulosic material or material containing xylan, wherein the cellulosic material or material containing xylan Material is to be saccharified in the presence of polypeptide active with xylobiase of the invention with enzymatic compositions.In one aspect, fine The fermentation for tieing up cellulosic material or the material containing xylan generates tunning.On the other hand, the technique further includes from fermenting back Receive tunning.

Technique of the invention can be used for cellulosic material or the saccharification of material containing xylan into fermentable sugars, and can Sugar fermentation is converted to many useful tunnings, such as fuel, drinking alcohol and/or platform chemicals (platform Chemical) (such as acid, alcohol, ketone, gas etc.).It is logical that desired tunning is generated from cellulosic material or material containing xylan Often it is related to pretreatment, enzyme hydrolysis (saccharification) and fermentation.

The conventional method that this field can be used in the processing of cellulosic material according to the present invention or the material containing xylan is complete At.It is configured to carry out according to any standard biologic matter process equipment of invention operation in addition, technique of the invention can be used.

Hydrolyze (saccharification) and ferment, respectively or simultaneously, including but not limited to, isolated hydrolysis and fermentation (SHF), together When be saccharified and fermentation (SSF), synchronous glycosylation and common fermentation (SSCF), the hydrolysis of mixing and fermentation (HHF), the hydrolysis of separation and altogether It ferments (SHCF), the hydrolysis and common fermentation (HHCF) of mixing, and directly microorganism conversion (DMC), otherwise referred to as joint biology It processes (consolidated bioprocessing, CBP).SHF is using isolated processing step first by cellulosic material Enzyme hydrolysis is fermentable sugars, for example, glucose, cellobiose and pentose monomers, then become ethyl alcohol for fermentable sugars fermentation.? In SSF, the enzyme hydrolysis of cellulosic material and sugar become the fementative composition of ethyl alcohol in one step (Philippidis, G.P., 1996, Cellulose bioconversion technology, in Handbook on Bioethanol:Production And Utilization, Wyman, C.E volume, Taylor&Francis, Washington, DC, 179-212).SSCF includes more Common fermentation (Sheehan, J. and Himmel, M., 1999, Enzymes, the energy and the environment:A of kind sugar strategic perspective on the U.S.Department of Energy’s research and development activities for bioethanol,Biotechnol.Prog.15:817-827).HHF is sugared at the same time It further include individual hydrolysing step, each step can carry out in the same reactor except change and hydrolysing step.HHF In the process the step of, can carry out in different temperature, that is, then the saccharification of high temperature enzyme process is resistant to lower in fermentation strain Temperature carries out SSF.DMC is combined with all three processes in one or more (such as several) steps, and (enzyme is generated, hydrolyzes and is sent out Ferment), wherein being generated using identical organism for cellulosic material to be converted to fermentable sugars and is converted to fermentable sugars Final product enzyme (Lynd L.R., Weimer, P.J., van Zyl, W.H., and Pretorius, I.S., 2002, Microbial cellulose utilization:Fundamentals and biotechnology, Microbiol.Mol.Biol.Reviews 66:506-577).Herein it is understood that any as known in the art Method, including pretreatment, enzyme hydrolysis (saccharification), fermentation or their combination, can be used in implementing technique of the invention.

Conventional equipment may include Fed-batch stirred reactor, batch-type stirred reactor, the continuous flow with ultrafiltration Stirred reactor 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.Technology 25: 33-38；Gusakov, A.V. and Sinitsyn, 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, S.K. and Lee, J.M., 1983, Bioconversion of waste cellulose by using an attrition bioreactor, Biotechnol.Bioeng.25:53-65), or have the intensively stirred reactor as caused by electromagnetic field (Gusakov, 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 include: fluidized bed, up-flow layer (upflow Blanket), the reactor of immobilization and the extruder type for hydrolyzing and/or fermenting.

Pretreatment.In the implementation of technique of the invention, any preprocessing process known in the art can be used and destroy The cellulosic material of plant cell wall or material component containing xylan (Chandra etc., 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)。

Cellulosic material or material containing xylan can also be carried out using method as known in the art before pre-processing Granularity reduction, screening, pre-soaking, wetting, washing and/or conditioning (conditioning).

Conventional pretreatment includes but is not limited to, steam pre-treatment (adjoint or be not accompanied by explosion), dilute acid pretreatment, hot water Pretreatment, oxygenation pretreatment, Lime Pretreatment, wet oxidation, wet explosion, the explosion of ammonia fiber, organic solvent pretreatment and the pre- place of biology Reason.Other pretreatments include ammonia diafiltration, ultrasound, electroporation, microwave, supercritical CO₂, overcritical H₂O, ozone, ionic liquid and γ radiation pretreatment.

Can before hydrolysis and/or fermentation pre-treating cellulosic material or material containing xylan.Pretreatment is preferably in water It is carried out before solution.Alternatively, pretreatment can be carried out simultaneously with enzyme hydrolysis to discharge fermentable sugars, such as glucose, xylose and/or fiber Disaccharides.In most cases, pre-treatment step itself makes some biomass be converted to fermentable sugars (or even there is no enzyme In the case of).

Steam pre-treatment.In steam pre-treatment, heating cellulose material or material containing xylan are to destroy plant cell Wall ingredient, including lignin, hemicellulose and cellulose, make cellulose and other fractions, such as hemicellulose, can be touched by enzyme And.Cellulosic material or material containing xylan are passed over or through into reaction vessel, wherein injection steam is to increase temperature to needs Temperature and pressure, and keep the desired reaction time wherein.Steam pre-treatment is preferably at 140-250 DEG C, such as 160- 200 DEG C or 170-190 DEG C progress, wherein optimal temperature range depends on the addition of chemical catalyst.Steam pre-treatment is stopped Stay the time 1-60 minutes preferred, such as 1-30 minutes, 1-20 minutes, 3-12 minutes or 4-10 minutes, wherein when optimal stop Between depend on temperature range and chemical catalyst addition.Steam pre-treatment allows relatively high solid content loading capacity, so that It is mostly just become moist through in preprocessing process in cellulosic material or material containing xylan.Steam pre-treatment is often located with pre- The explosion blowing (explosive discharge) of substance after reason combines, this is known as steam blasting, that is, quick flickering is to big The turbulent flow of air pressure and substance, with by it is broken increase accessible surface area (Duff and Murray, 1996, Bioresource Technology 855:1-33；Galbe and Zacchi, 2002, Appl.Microbiol.Biotechnol.59:618-628； U.S. Patent application No.20020164730).During steam pre-treatment, hemicellulose acetyl group is cut open, and The sour self-catalysis hemicellulose fraction hydrolysis arrived becomes monosaccharide and oligosaccharides.Lignin is only removed to a limited degree.

Chemical pretreatment: term " chemical treatment " refer to can promote cellulose, hemicellulose and/or lignin separation and/or Any chemical treatment of release.Such pretreatment can convert amorphous cellulose for crystal fibre element.The suitable pre- place of chemistry The example of science and engineering skill includes such as dilute acid pretreatment, Lime Pretreatment, wet oxidation, ammonia fiber/freezing explosion (AFEX), ammonia diafiltration (APR), ionic liquid and organic solvent pretreatment.

Catalyst such as H is often added before steam pre-treatment₂SO₄Or SO₂(usual 0.3 to 5%w/w), when can reduce Between, reduce temperature, increase the rate of recovery, and improve enzyme hydrolysis (Ballesteros etc., 2006, Appl.Biochem.Biotechnol.129-132:496-508；Varga etc., 2004, Appl.Biochem.Biotechnol.113-116:509-523；The such as Sassner, 2006, Enzyme Microb.Technol.39:756-762).It is in dilute acid pretreatment, cellulosic material or material containing xylan and diluted acid is (logical It is often H₂SO₄) and water be mixed to form slurry, by being steam heated to desired temperature, and after one section of residence time flickering to big Air pressure.Dilute acid pretreatment can be carried out with many reactor design forms, for example, plug flow reactor, counter-current reactor or company Continuous adverse current shrink bed reactor (Duff and Murray, 1996, supra；Schell etc., 2004, Bioresource Technol.91:179-188；Lee etc., 1999, Adv.Biochem.Eng.Biotechnol.65:93-115).

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

Lime Pretreatment is carried out in 85-150 DEG C of temperature with calcium oxide or calcium hydroxide, and the residence time is from 1 hour to several Its (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 preprocess method using ammonia.

Wet oxidation is a kind of Grape berry, usually 180-200 DEG C progress 5-15 minutes, be added oxidant such as hydrogen peroxide Or over-voltage 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).Pretreatment is with preferred 1-40% dry Matter, such as 2-30% dry matter or 5-20% dry matter carry out, and increase initially frequently by alkali such as sodium carbonate is added pH。

The amending method of wet oxidation preprocess method, referred to as wet explosion (combination of wet oxidation and steam blasting), can locate The dry matter of reason up to 30%.In wet explosion, in preprocessing process, oxidant is introduced after certain residence time.So Terminate to pre-process (WO 2006/032282) by flickering to atmospheric pressure afterwards.

Ammonia fiber explosion (AFEX) is related in such as 90-150 DEG C of moderate temperature and high pressure such as 17-20bar, with liquefied ammonia or ammonia By cellulosic material or material processing containing xylan 5-10 minutes, wherein dry matter content can be up to 60% (Gollapalli Deng 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 In, cellulose and hemicellulose keep relatively complete.Lignin-saccharide complex is cut open.

Organic solvent pretreatment is incited somebody to action and with hydrous ethanol (40-60% ethyl alcohol) at 160-200 DEG C extraction 30-60 minutes Cellulosic material or the delignification of material containing xylan (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).Sulfuric acid is frequently added as catalyst.In organic solvent pretreatment In, most of hemicellulose and lignin are removed.

Other examples such as Schell etc., 2003, Appl.Biochem and of suitable preprocess method Biotechn.Vol.105-108:69-85, and Mosier etc., 2005, Bioresource Technology 96:673-686, With described in U.S. Published Application 2002/0164730.

In one aspect, chemical pretreatment is carried out preferably as dilute acid pretreatment, and more preferably as continuous dilute acid pretreatment. Acid is usually sulfuric acid, but other acid also can be used, such as acetic acid, citric acid, nitric acid, phosphoric acid, tartaric acid, succinic acid, hydrogen chloride Or mixtures thereof.Weak acid (mild acid) processing is carried out in preferred 1-5, such as the pH range of 1-4 or 1-2.5.A side Face, acid concentration is in preferably 0.01 to 10wt% acid, such as the range of 0.05 to 5wt% acid or 0.1 to 2wt% acid.By acid and fibre Cellulosic material or material containing xylan are tieed up, and at preferred 140-200 DEG C, such as the temperature of 165-190 DEG C of range keeps 1 to 60 The time of minute.

On the other hand, pretreatment occurs in aqueous slurry.The cellulose in preprocessing process in a preferred aspect, With preferred 10-80wt%, such as 20-70wt% or 30-60wt%, the such as from about amount of 40wt% is deposited for material or material containing xylan ?.Pretreated cellulosic material or material containing xylan can not be washed or be washed using any of method in this field It washs, for example, being washed with water.

Mechanical pretreatment or physics pretreatment: term " mechanical pretreatment " or " physics pretreatment " refer to that any promotion particle is big The pretreatment of small reduction.For example, such pretreatment can be related to various types of grindings (grinding) or grind (milling) (for example, dry grinding, wet-milling or vibratory milling).

Cellulosic material or material containing xylan can be through both physics (machinery) and chemical pretreatment.Mechanically or physically pre- place Reason can be with following couplings: decatize/steam blasting, aquathermolysis (hydrothermolysis), diluted acid or weak acid treatment, high temperature, height Pressure processing, radiation (such as microwave radiation), or combinations thereof.In one aspect, high pressure refers to preferably from about 100 to about 400psi, such as The pressure of about 150 to about 250psi range.On the other hand, high temperature refers to about 100 to 300 DEG C, for example, about 140 to about 200 The temperature of DEG C range.It mechanically or physically pre-processes in a preferred aspect, and utilizes high temperature as defined above and height in use The steam gun hydrolyzer system (such as Sunds Hydrolyzer from Sunds Defibrator AB, Sweden) of pressure It is carried out in batch process.The physics and chemical pretreatment optionally can be carried out sequentially or be carried out simultaneously.

Therefore, physics (machinery) or chemistry are carried out to cellulosic material or material containing xylan in a preferred aspect, Pretreatment or any combination of them, to promote the separation and/or release of cellulose, hemicellulose and/or lignin.

Biological Pretreatment: term " Biological Pretreatment ", which refers to, can promote cellulose, hemicellulose and/or lignin from fiber Cellulosic material or any Biological Pretreatment of the separation of material containing xylan and/or release.Biological Pretreatment Techniques may include application The microorganism of dissolved lignin and/or enzyme (see, e.g., Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol:Production and Utilization, Wyman, C.E volume, 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. are compiled, ACS Symposium Series 566, 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. are compiled, 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 cellulosic material or material containing xylan, such as pretreated cellulosic material Or material containing xylan hydrolysis cellulose and hemicellulose are resolved into fermentable sugars, as glucose, cellobiose, xylose, Xylulose, arabinose, mannose, galactolipin and/or soluble oligosaccharides.Hydrolysis has β-wood in the present invention using enzymatic compositions Enzymatic progress as described herein in the presence of the polypeptide of glycosidase activity.The enzyme component of composition can also simultaneously or sequentially add Enter.

Enzyme hydrolysis preferably under conditions of being easy to be determined by those skilled in the art, carries out in suitable aqueous environment. In one aspect, it hydrolyzes in the activity for being suitable for enzyme component, i.e., for being carried out under enzyme component optimal conditions.Hydrolysis can be with feed supplement Partial or continuous process carries out, and gradually fills into cellulosic material or material containing xylan in continuous process, for example, filling into In hydrating solution containing enzyme.

Saccharification usually carries out under controlled pH, temperature and mixing condition in stirred tank reactor or fermentor.Properly Processing time, temperature and pH condition can be readily determined by those skilled in the art.For example, saccharification is sustainable to be up to 200 Hour, but preferably from about 12 to about 120 hours are usually carried out, for example, about 16 to about 72 hours, or about 24 to about 48 hours.Temperature In preferably from about 25 DEG C to about 70 DEG C, for example, about 30 DEG C to about 65 DEG C, about 40 DEG C to about 60 DEG C, or about 50 DEG C to 55 DEG C of range. PH is in preferably from about 3 to about 8, for example, about 3.5 to about 7, about 4 to about 6, or the range of about 5.0 to about 5.5.Dry solid content exists Preferably from about 5 to about 50wt%, for example, about 10 to about 40wt%, or about 20 to about 30wt% range.

Enzymatic compositions may include any albumen that can be used for degrading or converting cellulosic material or the material containing xylan.

In one aspect, the enzymatic compositions include or also comprising one or more (such as several) eggs selected from the group below It is white: cellulase, the polypeptide with cellulolytic enhancing activity, hemicellulase, esterase, clavacin, laccase, lignin Catabolic enzyme, pectase, peroxidase, protease and swollenin.On the other hand, the cellulase be it is preferred a kind of or A variety of (such as several) enzymes selected from the group below: endoglucanase, cellobiohydrolase and β-glucosyl enzym.In another side Face, the hemicellulase are preferred one or more (such as several) enzymes selected from the group below: acetyl mannan esterase, acetyl Xylan esterase, arabanase, arabinofuranosidase, coumaric acid esterase, feruloyl esterase, galactosidase, Portugal Uronic acid glycosidase, glucuronic acid esterase, mannonase mannosidase, zytase and xylosidase.

On the other hand, the enzymatic compositions include one or more (such as several) cellulolytic enzymes.Another A aspect, the enzymatic compositions include or further include one or more (such as several) hemicellulose catabolic enzymes.Another A aspect, the enzymatic compositions include one or more (such as several) cellulolytic enzymes and one or more (such as several) Hemicellulose catabolic enzyme.On the other hand, the enzymatic compositions include one or more (such as several) enzymes selected from the group below: Cellulolytic enzyme and hemicellulose catabolic enzyme.On the other hand, the enzymatic compositions include endoglucanase.Another A aspect, the enzymatic compositions include cellobiohydrolase.On the other hand, the enzymatic compositions include β-glucoside Enzyme.On the other hand, the enzymatic compositions include the polypeptide with cellulolytic enhancing activity.On the other hand, institute Stating enzymatic compositions includes endoglucanase and the polypeptide with cellulolytic enhancing activity.On the other hand, the enzyme Composition includes cellobiohydrolase and the polypeptide with cellulolytic enhancing activity.On the other hand, the enzyme group Closing object includes β-glucosyl enzym and the polypeptide with cellulolytic enhancing activity.On the other hand, the enzymatic compositions packet Containing endoglucanase and cellobiohydrolase.On the other hand, the enzymatic compositions include endoglucanase and β- Glucosidase.On the other hand, the enzymatic compositions include cellobiohydrolase and β-glucosyl enzym.In another side Face, the enzymatic compositions include endoglucanase, cellobiohydrolase and the polypeptide with cellulolytic enhancing activity. On the other hand, the enzymatic compositions comprising endoglucanase, β-glucosyl enzym and have cellulolytic enhancing activity Polypeptide.On the other hand, the enzymatic compositions comprising cellobiohydrolase, β-glucosyl enzym and have cellulose decomposition Enhance active polypeptide.On the other hand, the enzymatic compositions include endoglucanase, cellobiohydrolase and β-Portugal Glycosidase.On the other hand, the enzymatic compositions include endoglucanase, cellobiohydrolase, β-glucosyl enzym and Polypeptide with cellulolytic enhancing activity.

On the other hand, the enzymatic compositions include acetyl mannan esterase.On the other hand, the enzyme combination Object includes acetyl xylan esterase.On the other hand, the enzymatic compositions include that (such as α-L- is Arabic for arabanase Dextranase).On the other hand, the enzymatic compositions include arabinofuranosidase (such as α-L- arabinofuranosidase glucosides Enzyme).On the other hand, the enzymatic compositions include coumaric acid esterase.On the other hand, the enzymatic compositions include asafoetide Acid esters enzyme.On the other hand, the enzymatic compositions include galactosidase (such as alpha-galactosidase and/or beta galactose Glycosides enzyme).On the other hand, the enzymatic compositions include glucuronidase (such as α-D- glucuronidase).? On the other hand, the enzymatic compositions include glucuronic acid esterase.On the other hand, the enzymatic compositions include mannosan Enzyme.On the other hand, the enzymatic compositions include mannosidase (such as beta-Mannosidase).On the other hand, institute Stating enzymatic compositions includes zytase.The zytase is 10 zytase of family in a preferred aspect,.At another Aspect, the enzymatic compositions include xylosidase (such as xylobiase).

On the other hand, the enzymatic compositions include esterase.On the other hand, the enzymatic compositions include Aspergillusclavatus Element.On the other hand, the enzymatic compositions include laccase.On the other hand, the enzymatic compositions are decomposed comprising lignin Enzyme.At another preferred aspect, the lignin decomposition enzyme is manganese peroxidase.It is described at another preferred aspect Lignin decomposition enzyme is lignin peroxidase.At another preferred aspect, the lignin decomposition enzyme is to generate H₂O₂'s Enzyme.On the other hand, the enzymatic compositions include pectase.On the other hand, the enzymatic compositions include peroxide Enzyme.On the other hand, the enzymatic compositions include protease.On the other hand, the enzymatic compositions include swollenin.

In the process of the invention, enzyme can be saccharified, and be saccharified and ferment, or add before or during fermentation.

One or more (such as several) components of the enzymatic compositions can be wild-type protein, recombinant protein or wild type The combination of albumen and recombinant protein.For example, one or more (such as several) components can be the native protein of cell, use Make host cell to recombinantly express one or more (such as several) other components of enzymatic compositions.It can be by one kind of enzymatic compositions Or a variety of (such as several) components are generated as independent component, are then combined to form enzymatic compositions.The enzymatic compositions It can be the combination of multicomponent and one pack system protein preparation.

It can be any applicable form for the enzyme in present invention process, such as fermentation liquid formulation, cell composition contain Or the cell pyrolysis liquid without cell fragment, the half enzyme prepared product for purifying or purifying, or the host cell in the source as enzyme.Institute Stating enzymatic compositions can be for dry powder or particle, non-dusting particle, liquid, stabilizes liquid or stabilizes shielded enzyme.Liquid Enzyme prepared product can according to established technique, such as by addition stabilizer such as sugar, sugar alcohol or other polyalcohols and/or lactic acid or Other organic acids stabilize.

Optimal dose with the active enzyme of xylobiase and polypeptide depends on several factors comprising but be not limited to, it is fine Dimension element decomposes and/or mixture, cellulosic material or the material containing xylan of hemicellulose catabolic enzyme component, cellulosic material or The concentration of the material containing xylan, the pretreatment of cellulosic material or the material containing xylan, temperature, the time, pH and including fermentation give birth to Object (for example, yeast of synchronous glycosylation and fermentation).

In one aspect, cellulolytic enzyme or hemicellulose catabolic enzyme are for cellulosic material or the material containing xylan Effective quantity be about 0.5 to about 50mg, for example, about 0.5 to about 40mg, about 0.5 to about 25mg, about 0.75 to about 20mg, about 0.75 to About 15mg, about 0.5 to about 10mg, or about 2.5 to about 10mg every g cellulosic materials or material containing xylan.

On the other hand, the having for cellulosic material or the material containing xylan with the active polypeptide of xylobiase Effect amount is about 0.01 to about 50.0mg, for example, about 0.01 to about 40mg, about 0.01 to about 30mg, about 0.01 to about 20mg, about 0.01 to about 10mg, about 0.01 to about 5mg, about 0.025 to about 1.5mg, about 0.05 to about 1.25mg, about 0.075 to about 1.25mg, about 0.1 to about 1.25mg, about 0.15 to about 1.25mg, or about 0.25 to about 1.0mg every g cellulosic material or containing wood Chitosan material.

On the other hand, have the active polypeptide of xylobiase for cellulolytic enzyme or hemicellulose catabolic enzyme Effective quantity be about 0.005 to about 1.0g, for example, 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, about 0.1 to about 0.25g, or about 0.05 to about 0.2g every g cellulolytic enzyme or hemicellulose catabolic enzyme.

With cellulose decomposition enzymatic activity or the active polypeptide of hemicellulose catabolic enzyme and other it can be used for fiber material The protein/polypeptide of the degradation of material or the material containing xylan, such as the GH61 polypeptide with cellulolytic enhancing activity is (herein In be referred to as the polypeptide with enzymatic activity) may originate from or obtained from any suitable source, including bacterium, fungi, yeast, plant Or mammal source.Term " acquisition " still means that the enzyme can use method described herein in host organism herein Recombination generates, wherein the enzyme generated through recombination is natural or external source for host organism, or the amino acid sequence with modification Column recombinate the enzyme of generation for example, having one or more (such as several) missing, insertion and/or the amino acid replaced, Be natural acid sequence segment and/or mutant or by amino acid Shuffling Method known in the art generate enzyme. What is covered in the meaning of native enzyme is natural variant, and cover in the meaning of external enzyme be recombination (as by site-directed mutagenesis or Reset) obtain variant.

Polypeptide with enzymatic activity can be bacterial peptide.For example, the polypeptide can be gram-positive bacterium polypeptide Such as bacillus (Bacillus), streptococcus (Streptococcus), streptomyces (Streptomyces), grape ball Pseudomonas (Staphylococcus), enterococcus spp (Enterococcus), lactobacillus (Lactobacillus), lactococcus (Lactococcus), fusobacterium (Clostridium), ground bacillus category (Geobacillus), pyrolysis cellulose Pseudomonas (Caldicellulosiruptor), hot acid Pseudomonas (Acidothermus), Thermobifidia or bacillus marinus category (Oceanobacillus) polypeptide, the polypeptide have enzymatic activity；Or gramnegative bacterium polypeptide, such as Escherichia coli, false list Born of the same parents Pseudomonas (Pseudomonas), Salmonella (Salmonella), campylobacter (Campylobacter), Helicobacterium (Helicobacter), Flavobacterium (Flavobacterium), Fusobacterium (Fusobacterium), mud Bacillus (Ilyobacter), eisseria (Neisseria) or Ureaplasma (Ureaplasma) polypeptide, the polypeptide have enzyme activity Property.

In one aspect, the polypeptide is the Alkaliphilic bacillus with enzymatic activity, bacillus amyloliquefaciens, short gemma bar Bacterium, Bacillus circulans, Bacillus clausii, bacillus coagulans, bacillus firmus, bacillus lautus, slow bud Spore bacillus, bacillus licheniformis, bacillus megaterium, bacillus pumilus, bacillus stearothermophilus, bacillus subtilis or Bacillus thuringiensis polypeptide.

At another preferred aspect, the polypeptide is the streptococcus equisimilis with enzymatic activity, streptococcus pyogenes, breast chain Coccus or zooepidemicus polypeptide.

At another preferred aspect, the polypeptide is the not streptomyces chromogenes with enzymatic activity, deinsectization streptomycete, sky blue Streptomycete, streptomyces griseus or shallow Streptomyces glaucoviolaceus polypeptide.

Polypeptide with enzymatic activity is also possible to tungal polypeptide, and more preferably yeast polypeptides such as candida, Crewe Saccharomyces, pichia, saccharomyces, Schizosaccharomyces or the mould category polypeptide of Western alpine yarrow are tieed up, with enzymatic activity；Or more preferable silk Shape tungal polypeptide such as acremonium, Agaricus, Alternaria, aspergillus, Aureobasidium, Botryospaeria, quasi- wax bacterium Category, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinus, Coptotermes, stick softgel shell Category, the red shell Pseudomonas of hidden clump, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, full flagellate Category, Humicola, rake teeth Pseudomonas, Agaricus, Leptospaeria, Magnaporthe grisea category, Melanocarpus, Polyporus, Mucor The mould category of category, myceliophthora, Xin Kaoma rouge, Neurospora, paecilomyces, Penicillium, flat lead fungi category, cud Chytridium, Poitrasia, false black Peziza, Pseudotrichonympha, Rhizomucor, Schizophyllum, capital spore category, Talaromyces, The mould category of thermophilic ascomycete category, shuttle spore, Tolypocladium, trichoderma, Trichophaea, Verticillium, Volvaria or Xylaria Polypeptide, with enzymatic activity.

In one aspect, the polypeptide is the saccharomyces carlsbergensis with enzymatic activity, saccharomyces cerevisiae, saccharomyces diastaticus, Doug Laplace Yeast, Saccharomyces kluyveri, promise ground yeast or ellipsoideus yeast polypeptide.

On the other hand, the polypeptide be the solution fiber branch acremonium with enzymatic activity, 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 spore Bacterium, Chrysosporium queenslandicum, Chrysosporium zonatum, bar spore shape fusarium, F.graminearum schw, library It is prestige fusarium, machete fusarium, fusarium graminaria, red fusarium of standing grain, different spore fusarium, albizzia fusarium, sharp fusarium, racemosus fusarium, pink Fusarium, elder fusarium, colour of skin fusarium, intend branch spore fusarium, sulphur color fusarium, circle fusarium, quasi- silk spore fusarium, empiecement fusarium, ash Humicola lanuginosa, Humicola insolens dredge cotton like humicola lanuginosa, white rake teeth bacterium, rice black wool mould, thermophilic fungus destroyed wire, Neuraspora crassa, rope form blueness Mould, penicillium purpurogenum, the flat lead fungi of yellow spore, Thielavia achromatica, Thielavia albomyces, Thielavia Albopilosa, Australia shuttle spore are mould, Thielavia fimeti, small spore shuttle spore is mould, ovum spore shuttle spore is mould, Thielavia Peruviana, tumor spore shuttle spore is mould, hair shuttle spore is mould, Thielavia subthermophila, autochthonal shuttle spore are mould, Trichoderma harzianum, health Peaceful trichoderma, long shoot trichoderma, trichoderma reesei, Trichoderma viride or brown spore become mildewed cup fungi (Trichophaea saccata) polypeptide.

The mutant of the polypeptide with enzymatic activity being transformed through chemical modification or protein engineering can also be used.

One or more (such as several) components of the composition can be recombination component, also that is, passing through clone's coding The DNA sequence dna of the independent component and then with the DNA sequence dna transformed cells and in host expression (see, e.g., WO91/ 17243 and WO91/17244) and generate.The host is preferably heterologous host (enzyme is external source to host), but the host It is also possible to homologous host under certain condition (enzyme is natural to host).Homofil element decomposition of protein can also pass through Such protein is purified from fermentation liquid to prepare.

In one aspect, one or more (such as several) cellulolytic enzymes include commercial cellulolytic enzyme Prepared product.The example for being suitable for the invention the cellulolytic enzyme prepared product of business includes, for example, CELLIC^TM Ctec (Novozymes A/S)、CELLIC^TM CTec2(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/L 100 (DSM), ROHAMENT^TM7069 W(GmbH),LDI(Dyadic International, Inc.)、LBR (Dyadic International, Inc.) or150L (Dyadic International,Inc.).The cellulose enzyme is such as solid with about the 0.001 to about 5.0wt% of solid content About 0.005 to about 2.0wt% effective quantity of about 0.025 to about 4.0wt% or solid of shape object adds.

The example that can be used for the bacterial endo glucanases of technique of the invention includes but are not limited to, and solves 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)；It is poly- with the fusca inscribe Portugal Thermobifida Carbohydrase V (WO 05/093050).

The example that can be used for fungal endoglucanase of the invention includes but are not limited to, and trichoderma reesei inscribe Portugal is poly- Carbohydrase I (Penttila etc., 1986, Gene 45:253-263, trichoderma reesei Cel7B endoglucanase i (GENBANK^TMIt logs in Number M15665)；Trichoderma reesei endoglucanase II (Saloheimo etc., 1988, Gene 63:11-22), trichoderma reesei 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 inscribe Portugal Dextranase V (Saloheimo etc., 1994, Molecular Microbiology 13:219-228；GENBANK^TMAccession number Z33381)；Microorganism Aspergillus aculeatus endoglucanase (Ooi etc., 1990, Nucleic Acids Research18:5884)；Valley is bent Mould (Aspergillus kawachii) endoglucanase (Sakamoto etc., 1995, Current Genetics 27:435- 439)；Carrot soft rot Erwinia (Erwinia carotovara) endoglucanase (Saarilahti etc., 1990, Gene 90:9-14)；Sharp fusarium endoglucanase (GENBANK^TMAccession number L29381)；Grey humicola lanuginosa thermoidea mutation inscribe Dextranase (GENBANK^TMAccession number AB003107)；Melanocarpus albomyces endoglucanase (GENBANK^TM Accession number MAL515703)；Neuraspora crassa endoglucanase (GENBANK^TMAccession number XM_324477)；In Humicola insolens Cut dextranase V；117.65 endoglucanase of thermophilic fungus destroyed wire CBS；Basidiomycetes (basidiomycete) CBS 495.95 endoglucanase；494.95 endoglucanase of Basidiomycetes CBS；In autochthonal mould 8126 CEL6B of NRRL of shuttle spore Cut dextranase；The autochthonal mould 8126 CEL6C endoglucanase of NRRL of shuttle spore；In autochthonal mould 8126 CEL7C of NRRL of shuttle spore Cut dextranase；The autochthonal mould 8126 CEL7E endoglucanase of NRRL of shuttle spore；In autochthonal mould 8126 CEL7F of NRRL of shuttle spore Cut dextranase；62373 CEL7A endoglucanase of Cladorrhinum foecundissimum ATCC；And Richter scale Trichoderma strain No.VTT-D-80133 endoglucanase (GENBANK^TMAccession number M15665).

The example of cellobiohydrolase for use in the present invention includes but are not limited to, the hydrolysis of microorganism Aspergillus aculeatus cellobiose Enzyme II (WO 2011/059740), chaetomium thermophilum (Chaetomium thermophilum) cellobiohydrolase I, it is thermophilic Cupreum cellobiohydrolase II, Humicola insolens cellobiohydrolase I, thermophilic fungus destroyed wire cellobiohydrolase II, (WO2009/042871), Thielavia hyrcanie cellobiohydrolase II (WO 2010/141325), autochthonal shuttle spore are mould Cellobiohydrolase II (CEL6A, WO 2006/074435), trichoderma reesei cellobiohydrolase I, trichoderma reesei fiber two Glycosylhydrolase II and brown spore become mildewed cup fungi cellobiohydrolase II (WO 2010/057086).

The example of β-glucosyl enzym for use in the present invention include but are not limited to from microorganism Aspergillus aculeatus (Kawaguchi etc., 1996, Gene 173:287-288), aspergillus fumigatus (WO 2005/047499), aspergillus niger (Dan etc., 2000, J.Biol.Chem.275:4973-4980), aspergillus oryzae (WO 2002/095014), Brazil mould IBT20888 (WO 2007/ 019442 and WO 2010/088387), autochthonal shuttle spore mould (WO 2011/035029) and brown spore become mildewed cup fungi (WO 2007/ 019442) β-glucosyl enzym.

The β-glucosyl enzym can be fusion protein.In one aspect, the β-glucosyl enzym is WO aspergillus oryzae β-Portugal Glucosides enzyme variants BG fusion protein (WO 2008/057637) or aspergillus oryzae β-glucosyl enzym fusion protein (2008/057637).

Other available endoglucanases, cellobiohydrolase and β-glucosyl enzym are disclosed in using basis 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 enzymes for use in the present invention are 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, United States Patent (USP) No.5,457, 046, United States Patent (USP) No.5,648,263 and United States Patent (USP) No.5,686,593.

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

The example of the GH61 polypeptide with cellulolytic enhancing activity of technique for use in the present invention includes but unlimited In mould (WO 2005/074647, WO 2008/148131 and WO 2011/035027) from autochthonal shuttle spore；The hot sac fungus of tangerine orange (WO 2005/074656 and WO 2010/065830), trichoderma reesei (WO 2007/089290), thermophilic fungus destroyed wire (WO 2009/ 085935, WO 2009/085859, WO 2009/085864, WO 2009/085868), aspergillus fumigatus (WO 2010/138754) GH61 polypeptide, from thermophilic loose mould (WO 2011/005867), thermophilic ascomycete category strain (WO 2011/039319), Penicillium The GH61 polypeptide of strain (WO 2011/041397) and Thermoascus crustaceous (WO 2011/041504).

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

On the other hand, the GH61 polypeptide with cellulolytic enhancing activity is in dioxy compound, Fused bicyclic Close object, heterocyclic compound, nitrogenous compound, naphtoquinone compounds, sulfur-containing compound or pre- from pretreated cellulosic material such as warp It is used in the presence of the liquor that the corn stover (PCS) of processing obtains.

The dioxy compound may include any the suitable compound containing two or more oxygen atoms.In some respects, The dioxy compound contains the aryl module (moiety) replaced as described herein.The dioxy compound may include one A or multiple (such as several) hydroxyl and/or hydroxy derivatives, but also include the substituted virtue for lacking hydroxyl and hydroxy derivatives Basic mode block.The non-limiting example of dioxy compound includes catechol or catechol；Caffeic acid；3,4- dihydroxy-benzoic acid； 4- tert-butyl -5- methoxyl group -1,2- benzenediol；Pyrogallol；Gallic acid；Methyl -3,4,5-trihydroxy benzoic acid；2,3,4- Trihydroxybenzophenone；2,6- syringol；Sinapic acid；3,5- dihydroxy-benzoic acid；The chloro- 1,2- benzenediol of 4-；4- nitre Base -1,2- benzenediol；Tannic acid；Progallin A；Glycolic acid methyl esters；Dihydroxy fumaric acid；2- butine -1,4- glycol；Gram Ketone acid；1,3- propylene glycol；Tartaric acid；2,4-PD；3- ethyoxyl -1,2- propylene glycol；2,4,4 '-trihydroxybenzophenones； Cis-2-butene -1,4- glycol；3,4- dihydroxy -3- cyclobutane -1,2- diketone；Dihydroxyacetone (DHA)；Acetyl acrolein (acrolein acetal)；Methyl -4-HBA；4-HBA；With methyl -3,5- dimethoxy-4 '-hydroxy benzenes Formic acid；Or their salt or solvate (solvate).

The bicyclic compound may include any substitution fused ring system suitable as described herein.The compound can Comprising one or more (such as several) other ring, and that, unless otherwise stated, it is not limited to specific number of rings.In one aspect, The bicyclic compound is flavonoids.On the other hand, the bicyclic compound is the isoflavonoid optionally replaced (isoflavonoid).On the other hand, the bicyclic compound is the pattern optionally replacedIon (flavylium Ion), the anthocyanidin such as optionally replaced or the anthocyanin optionally replaced, or derivatives thereof.The non-limiting example of bicyclic compound Including epicatechin (epicatechin)；Quercetin (quercetin)；Myricetin (myricetin)；Taxifolin (taxifolin)；Kaempferol (kaempferol)；Mulberrin (morin)；Acacetin (acacetin)；Naringenin (naringenin)；Isorhamnetin (isorhamnetin)；Apigenin (apigenin)；Anthocyanidin (cyanidin)； Anthocyanin (cyanin)；kuromanin；Keracyanin (keracyanin)；Or their salt or solvate.

The heterocyclic compound can be any suitable compound, and what is optionally replaced as described herein includes hetero atom Aromatic ring or non-aromatic ring.In one aspect, the heterocycle is the Heterocyclylalkyl module comprising optionally replacing or optionally replaces miscellaneous The compound of aryl module.On the other hand, the Heterocyclylalkyl module optionally replaced or the heteroaryl basic mode optionally replaced Block is the five-ring heterocycles alkyl optionally replaced or the quinary heteroaryl module optionally replaced.On the other hand, optionally replace Heterocyclylalkyl or the heteroaryl module optionally replaced are the modules chosen from the followings optionally replaced: pyrazolyl, furyl, imidazoles Base, isoxazolyl, oxadiazoles base, oxazolyl, pyrrole radicals, pyridyl group, pyrimidine radicals, pyridazinyl, thiazolyl, triazolyl, thienyl (thienyl), dihydro-thiophene-pyrazolyl (dihydrothieno-pyrazolyl), thianaphthenyl, carbazyl, benzimidazolyl, Benzothienyl (benzothienyl), benzofuranyl, indyl, quinolyl, benzotriazole base, benzothiazolyl, benzo Oxazolyl (benzooxazolyl), benzimidazolyl, isoquinolyl, isoindolyl, acridinyl, benzo isoxazolyl (benzoisazolyl), dimethyl hydantoin, pyrazinyl, tetrahydrofuran base, pyrrolinyl, pyrrolidinyl, morpholinyl, Yin Diindyl base, diaza cycloheptatriene base (diazepinyl), azepine cycloheptatriene base (azepinyl), thia cycloheptatriene base (thiepinyl), piperidyl and oxepin base (oxepinyl).The heterocycle alkane optionally replaced on the other hand Basic mode block or the heteroaryl module optionally replaced are the furyls optionally replaced.The non-limiting example of heterocyclic compound includes (1,2- dihydroxy ethyl) -3,4- dihydrofuran -2 (5H) -one；4- hydroxy-5-methyl base -3- furanone；5- hydroxyl -2 (5H)-furans Ketone；[1,2- dihydroxy ethyl] furans -2,3,4 (5H)-triketone；Alpha-hydroxy-gamma-butyrolacton；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- pyrans -2- Ketone；With 5,6- dihydro -4- hydroxyl -6- methyl -2H- pyran-2-one；Or their salt or solvate.

The nitrogenous compound can be any the suitable compound with one or more nitrogen-atoms.In one aspect, institute Stating nitrogenous compound includes amine, imines, azanol or nitrous oxide (nitroxide) module.The non-limiting reality of nitrogenous compound Example includes acetoxime；Violuric acid；Pyridine -2- aldoxime；Ortho-Aminophenol；1,2- phenylenediamine；2,2,6,6- tetramethyl -1- piperidyl Oxygen (piperidinyloxy)；5,6,7,8- tetrahydrobiopterin；6,7- dimethyl -5,6,7,8- tetrahydro pterin；With Malaysia acyl Amino acid；Or their salt or solvate.

The naphtoquinone compounds can be any suitable compound comprising quinone module described herein.Naphtoquinone compounds it is non- Limited example includes 1,4- benzoquinones；1,4- naphthoquinones；2 hydroxy 1,4 naphthoquinone (lawsone)；2,3- dimethoxy -5- methyl-1,4- benzoquinones Or ubiquinone₀；2,3,5,6- tetramethyl -1,4- benzoquinones or duroquinone；1,4- dihydroxy anthraquinone；3- hydroxyl -1- methyl - 5,6- indoline diketone or adrenochrome；4- tert-butyl -5- methoxyl group -1,2- benzoquinones；Pyrroloquinoline quinone (pyrroloquinoline quinone)；Or their salt or solvate.

The sulfur-containing compound can be any suitable compound comprising one or more sulphur atoms.In one aspect, The sulfur-containing compound includes module chosen from the followings: thionyl, thioether, sulfenyl, sulphonyl, sulfonamide (sulfamide), sulphur Amide (sulfonamide), sulfonic acid and sulphonic acid ester.The non-limiting example of sulfur-containing compound includes ethyl mercaptan；2- propanethiol；2- Propylene -1- mercaptan；Mistabrom；Benzenethiol；Two mercaptan of benzene -1,2-；Cysteine；Methionine；Glutathione；Guang ammonia Acid；Or their salt or solvate.

In one aspect, such compound as described above is to the effective quantity of cellulosic material, to cellulose sugar unit Molar ratio meter, be about 10^-6To about 10, for example, about 10^-6To about 7.5, about 10^-6To about 5, about 10^-6To about 2.5, about 10^-6Extremely 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.Another On one side, the effective quantity of compound as described above is about 0.1 μM to about 1M, for example, about 0.5 μM to about 0.75M, about 0.75 μ M to about 0.5M, about 1 μM to about 0.25M, about 1 μM to about 0.1M, about 5 μM to about 50mM, about 10 μM to about 25mM, about 50 μM extremely About 25mM, about 10 μM to about 10mM, about 5 μM to about 5mM, or about 0.1mM to about 1mM.

Term " liquor (liquor) " mean it is described herein under conditions of, pass through the ligno-ccllulose in processing slurry And/or hemicellulosic materials or its monosaccharide such as xylose, arabinose, mannose etc., generated solution phase, i.e. water phase have Machine phase or combinations thereof, and its soluble content.The liquor that cellulose decomposition for GH61 polypeptide enhances can pass through, and optionally exist Catalyst for example acid in the presence of, optionally in presence of organic solvent, and optionally with the physical damage phase group to the material Close come by heat is applied and/or pressure handles cellulosic material or hemicellulosic materials (or raw material), then by solution with it is residual Remaining solid separation is to generate.Such conditional decision passes through during by Cellulase preparation hydrolysis fiber cellulosic material The degree of the enhancing of cellulose decomposition obtained by the combination of liquor and GH61 polypeptide.The standard in this field can be used in the liquor Method such as filtering, deposition are centrifuged from the separation of processed material.

In one aspect, the liquor is about 10 to the effective quantity of cellulose^-6To the every g cellulose of about 10g, for example, about 10^-6 To 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 cellulose of g.

In one aspect, one or more (such as several) hemicellulose catabolic enzymes include commercial hemicellulose point Solve enzyme prepared product.The example for being suitable for the invention commercial hemicellulose catabolic enzyme prepared product includes, such as SHEARZYME^TM (Novozymes A/S)、HTec(Novozymes A/S)、Htec2(Novozymes A/S)、(Novozymes A/S)、(Novozymes A/S)、HC (Novozymes A/S)、Xylanase(Genencor)、XY (Genencor)、XC(Genencor)、TX-200A(AB Enzymes)、HSP 6000 Xylanase(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 is not limited to come from microorganism Aspergillus aculeatus (Aspergillus aculeatus)(GeneSeqP:AAR63790；WO 94/21785), aspergillus fumigatus (Aspergillus fumigatus) (WO 2006/078256), thermophilic loose mould (WO 2011/041405), Penicillium species (WO 2010/126772), autochthonal shuttle spore are mould (Thielavia terrestris) NRRL 8126 (WO 2009/079210) and brown spore become mildewed cup fungi GH10 (WO 2011/ 057083) zytase.

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

The example that can be used for the acetyl xylan esterase of present invention process includes but is not limited to come from microorganism Aspergillus aculeatus (WO 2010/108918), chaetomium globosum (Chaetomium globosum) (Uniprot accession number Q2GWX4), thin beautiful cupreum (Chaetomium gracile) (GeneSeqP accession number AAB82124), Humicola insolens (Humicola insolens) DSM 1800 (WO 2009/073709), Hypocrea jecorina (Hypocrea jecorina) (WO 2005/001036), thermophilic fungus destroyed wire (Wo 2010/014880), Neuraspora crassa (UniProt accession number q7s259), phaeosphaeria nodorum (Phaeosphaeria Nodorum) (Uniprot accession number Q0UHJ1) and the acetyl wood of the mould NRRL 8126 (WO 2009/042846) of autochthonal shuttle spore are poly- Sugar ester enzyme.

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

The example that can be used for the arabinofuranosidase of present invention process includes but is not limited to come from aspergillus niger (Aspergillus niger) (GeneSeqP accession number AAR94170), Humicola insolens (Humicola insolens) DSM The arabinofuranosidase of 1800 (WO 2006/114094 and WO 2009/073383) and M.giganteus (WO 2006/114094) Glycosidase.

The example that can be used for the alpha-glucuronidase of the method for the present invention includes but is not limited to come from Aspergillusclavatus It is (Aspergillus clavatus) (UniProt accession number alcc12), aspergillus fumigatus (SwissProt accession number Q4WW45), black Aspergillus (Uniprot accession number Q96WX9), Aspergillus terreus (Aspergillus terreus) (SwissProt accession number Q0CJP9), (UniProt is logged in for Humicola insolens (WO 2010/014706), tangerine ash mould (WO 2009/068565), Ai Mosen ankle section bacterium Number Q8X211) and trichoderma reesei (Uniprot accession number Q99024) alpha-glucuronidase.

The polypeptide with enzymatic activity for present invention process can be by containing suitable carbon source and nitrogen source and inorganic salts On nutrient medium, using means known in the art (see, e.g. Bennett, J.W. and LaSure, L. (eds.), More Gene Manipulations in Fungi, Academic Press, CA, 1991) the above-mentioned microbial strains pointed out of fermentation It generates.Suitable culture medium can be obtained from supplier, or can be prepared according to published composition (such as American Type culture is protected The catalogue at hiding center).Be known in the art suitable for growing the temperature range generated with enzyme and other conditions (see, e.g. Bailey, J.E. and Ollis, D.F., Biochemical Engineering Fundamentals, McGraw-Hill Book Company,NY,1986)。

The method that the fermentation can be any culture cell for leading to enzyme or protein expression or separation.Therefore, fermentation can With the shaking flask training for being to be understood as included in suitable culture medium and being carried out under conditions of allowing the enzyme to be able to express or separate Support, or in laboratory or industrial fermentation tank it is small-or large scale fermentation (including it is continuous, in batches, fed-batch or solid-state hair Ferment).The resulting enzyme generated by the above method can be recycled from fermentation medium and be purified by conventional method.

Fermentation.Sugar can be directly or indirectly fermented into the hair of required tunning by one or more (such as several) The fermentable sugars that ferment microbial fermentation is obtained from the cellulosic material through hydrolyzing or material containing xylan." fermentation " or " fermentation side Method " refers to any fermentation process or any method comprising fermentation step.Fermentation process further includes for the industrial (example of consumer goods alcohol Such as, beer and grape wine), dairy husbandry (for example, fermented dairy product), leather industry and tobacco fermentation process.Fermentation condition according to The desired tunning of Lai Yu and fermenting organisms, and can be readily determined by those skilled in the art.

In fermentation step, the result as pretreatment and enzyme hydrolysis step is released from cellulosic material or material containing xylan The sugar put becomes product by fermenting organisms (such as yeast) fermentation, for example, ethyl alcohol.As described herein, hydrolyze (saccharification) and Fermentation can be separately or simultaneously.

Any suitable cellulosic material through hydrolyzing or poly- containing wood can be used in implementing fermentation step of the invention Sugared material.The material is selected generally according to required fermented product (that is, the substance to obtain from fermentation) and the method used, As known in the art.

Term " fermentation medium " can be regarded as referring to herein the culture medium before fermentative microorganism is added, e.g., by sugar Culture medium used in the culture medium and synchronous glycosylation and fermentation process (SSF) that change process generates.

" fermentative microorganism " refer to suitable for ideal fermentation process generate tunning any microorganism, including bacterium and Fungal organism.Fermenting organisms can be hexose and/or pentose fermentation biology body or their combination.Hexose and pentose hair Ferment organism is well known in this field.Suitable fermentative microorganism can be by sugared (such as glucose, xylose, xylulose, Arab Sugar, maltose, mannose, galactolipin and/or oligosaccharides) (that is, conversion) is directly or indirectly fermented into required fermented product.It can The bacterium of generation ethyl alcohol and the example such as Lin of fungi fermentation organism etc., 2006, Described in Appl.Microbiol.Biotechnol.69:627-642.

The example of the fermentative microorganism of energy zymohexose includes bacterium and fungal organism, such as yeast.Preferred yeast packet Include candida, Kluyveromyces and saccharomyces, such as Candida sonorensis, kluyveromyces marxianus and wine The bacterial strain of brewer yeast.

Example with the fermenting organisms of its native state energy ferment pentoses includes bacterium and fungal organism, such as some ferment It is female.Preferred wood-sugar fermentation yeast includes candida, preferably shehatae candida (Candida sheatae) or Candida sonorensis；And pichia, the preferably bacterial strain of pichia stipitis (Pichia stipitis) are such as set The bacterial strain of dry Pichia pastoris CBS 5773.Preferred pentose fermentation yeast includes pipe capsule saccharomyces (Pachysolen), preferably thermophilic The bacterial strain of tan pipe capsule yeast (Pachysolen tannophilus).Can not ferment pentoses such as xylose and arabinose biology Means known in the art genetic modification ferment pentoses can be passed through.

Can effectively include at the bacterium of ethyl alcohol by hexose and pentose fermentation, for example, bacillus coagulans (Bacillus Coagulans), clostridium acetobutylicum (Clostridium acetobutylicum), Clostridium thermocellum (Clostridium Thermocellum), Clostridium phytofermentans, ground bacillus category strain, Thermoanaerobactersaccharolyticum (Thermoanaerobacter saccharolyticum) and zymomonas mobilis (Zymomonas mobilis) (Philippidis, 1996, see above).

Other fermenting organisms include bacillus, such as bacillus coagulans；Candida, such as Candida Sonorensis, C.methanosorbosa, Di Dansi Candida (Candida diddensii), Candida parapsilosis (Candida parapsilosis), C.naedodendra, C.blankii, C.entomophilia, rape Candida (C.brassicae), candida pseudotropicalis (Candida pseudotropicalis), Candida boidinii (Candida Boidinii), candida utili (Candida utilis) and shehatae candida (C.scehatae)；Fusobacterium, such as Clostridium acetobutylicum, Clostridium thermocellum and C.phytofermentans；Escherichia coli, especially genetically modified promotion ethyl alcohol The coli strain of generation；Ground bacillus category strain；Hansenula, such as Hansenula anomala (Hansenula anomala)；Klebsiella (Klebsiella), such as acid-producing Klebsiella bacterium (Klebsiella oxytoca)；Crewe dimension Saccharomyces, such as kluyveromyces marxianus, Kluyveromyces lactis (K.lactis), K.thermotolerans and crisp wall Crewe Tie up yeast；Schizosaccharomyces, such as schizosaccharomyces pombe (S.pombe)；Hot anaerobic bacillus(cillus anaerobicus) category (Thermoanaerobacter), such as Thermoanaerobactersaccharolyticum and zymomonas (Zymomonas), such as the bacterial strain of zymomonas mobilis.

Yeast is brettanomyce category (Bretannomyces) in a preferred aspect,.In terms of one is preferred, Yeast is gram Lawson's brettanomyce (Bretannomyces clausenii).In another more preferred aspect, yeast is false silk Yeast.In another more preferred aspect, yeast is Candida sonorensis.In another more preferred aspect, yeast It is Candida boidinii.In another more preferred aspect, yeast is Candida blankii.It is preferred at another Aspect, yeast are rape Candidas.In another more preferred aspect, yeast is Di Dansi Candida.Another more Preferred aspect, yeast is Candida entomophiliia.In another more preferred aspect, yeast is pseudo-heat band vacation silk Yeast.In another more preferred aspect, yeast is shehatae candida.In another more preferred aspect, yeast is to produce Protein Candida.At another preferred aspect, yeast is stick spore saccharomyces (Clavispora).In another preferred side Face, yeast are Clavisporalusitaniae (Clavispora lusitaniae).In another more preferred aspect, yeast is celestial People slaps stick spore yeast (Clavispora opuntiae).At another preferred aspect, yeast is kluyveromyces.Another A preferred aspect, yeast are Kluyveromyces fragilis.In another more preferred aspect, yeast is Marx's Crewe dimension ferment It is female.In another more preferred aspect, yeast is Kluyveromyces thermotolerans.In another preferred side Face, yeast are pipe capsule saccharomyces (Pachysolen).In another more preferred aspect, yeast is pachysolen tannophilus.Another One preferred aspect, yeast is Pichia pastoris.In another more preferred aspect, yeast is pichia stipitis.Another A preferred aspect, yeast are Saccharomyces sps.At another preferred aspect, yeast is saccharomyces cerevisiae.It is more excellent at another The aspect of choosing, yeast are saccharomyces diastaticus (Saccharomyces distaticus).In another more preferred aspect, yeast is Saccharomyces uvarum (Saccharomyces uvarum).

Bacterium is bacillus in a preferred aspect,.At a preferred aspect, bacterium is condensation gemma bar Bacterium.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 ground bacillus category strain.It is preferred at another Aspect, bacterium are hot anaerobic bacillus(cillus anaerobicus) categories.In another more preferred aspect, bacterium is Thermoanaerobactersaccharolyticum.Another more Preferred aspect, bacterium is zymomonas.In another more preferred aspect, bacterium is zymomonas mobilis.

The yeast that commercially available suitable ethyl alcohol generates includes, 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^TM Fresh yeast (Ethanol Technology, WI, USA).

Fermentative microorganism has already passed through genetic modification to provide the ability of ferment pentoses, such as in a preferred aspect, Using xylose, utilize arabinose and the common microorganism for utilizing xylose and arabinose.

Constructs by the way that heterologous gene is cloned into a variety of fermentative microorganisms and hexose and pentose can be converted to ethyl alcohol Organism (Chen and Ho, 1993, the Cloning and improving the expression of Pichia of (common fermentation) stipitis xylose reductase gene in Saccharomyces cerevisiae, Appl.Biochem.Biotechnol.39-40:135-147；Ho etc., 1998, Genetically engineeredSaccharomyces yeast capable of effectively cofermenting glucose and xylose,Appl.Environ.Microbiol.64:1852-1859；Kotter and Ciriacy, 1993, Xylose fermentation bySaccharomyces 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 Research 4:655-664；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,Science 267: 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).

It is in a preferred aspect, Candida sonorensi by the fermentative microorganism of genetic modification.At another Preferred aspect, the fermentative microorganism by genetic modification is Escherichia coli.At another preferred aspect, by genetic modification Fermentative microorganism be acid-producing Klebsiella bacterium.At another preferred aspect, the genetically modified fermentative microorganism is Kluyveromyces marxianus.At another preferred aspect, the genetically modified fermentative microorganism is saccharomyces cerevisiae.Another One preferred aspect, the fermentative microorganism by genetic modification is zymomonas mobilis.

It is well known in the art that above-mentioned organism can be used for generating other materials, as described herein.

Fermentative microorganism usually is added to the cellulosic material of degradation or material containing xylan or hydrolysate, and carries out about 8 To about 96 hours, ferment within for example, about 24 to about 60 hours.Temperature is typically about 26 DEG C to about 60 DEG C, and for example, about 32 DEG C or 50 DEG C, And in about pH 3 to about pH 8, for example, about pH 4-5,6 or 7.

In one aspect, yeast and/or another microorganism are applied to the cellulosic material of degradation or material containing xylan, And carry out about 12 to about 96 hours, it ferments within such as usually 24-60 hours.On the other hand, temperature is preferably from about 20 DEG C to about 60 DEG C, for example, 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 typically about pH 3 To about pH 7, for example, about pH 4 to about pH 7.However, some fermenting organisms such as bacterium, has higher most suitable fermentation temperature Degree.Yeast or another microorganism are preferably with about 10⁵-10¹², preferably from about 10⁷-10¹⁰, particularly from about 2x10⁸Viable count is every The amount of ml fermentation liquid is applied.Such as " The Alcohol is found in about the further guidance for using yeast to ferment Textbook " (K.Jacques, T.P.Lyons and D.R.Kelsall are compiled, Nottingham University Press, United Kingdom 1999), it is incorporated herein by mentioning stating.

Fermentation stimulating substance can be applied in combination with any method as described herein, to be further improved zymotechnique, especially It is the performance for improving fermentative microorganism, e.g., rate increases and alcohol getting rate." fermentation stimulating substance " refers to (special for fermentative microorganism Not yeast) growth stimulant.The fermentation stimulating substance for being preferably used in growth includes vitamin and mineral.The reality of vitamin Example includes multivitamin, biotin, pantothenic acid (salt), niacin, meso inositol (meso-inositol), thiamine, pyridoxol (pyridoxine), p-aminobenzoic acid, folic acid, riboflavin and vitamin A, B, C, D and E.See, e.g., Alfenore etc., Improving ethanol production and viability ofSaccharomyces cerevisiae by a Vitamin feeding strategy during fed-batch process, Springer-Verlag (2002) lead to It crosses to mention stating and be incorporated herein.The example of minerals includes the minerals and mineral salt for being capable of providing nutrients, the nutrients packet Include P, K, Mg, S, Ca, Fe, Zn, Mn and Cu.

Tunning: tunning can be any substance from fermentation.Tunning can be, and be not limited to, alcohol (example Such as, arabite, n-butanol, isobutanol, ethyl alcohol, glycerol, methanol, ethylene glycol, 1,3-PD (propylene glycol), butanediol, third Triol, sorbierite and xylitol)；Alkane (such as pentane, hexane, heptane, octane, nonane, decane, hendecane and dodecane)； Cycloalkane (such as pentamethylene, hexamethylene, cycloheptane and cyclooctane)；Alkene (such as amylene, hexene, heptene and octene)；Amino Sour (for example, aspartic acid, glutamic acid, glycine, lysine, serine and threonine)；Gas is (for example, methane, hydrogen (H₂), carbon dioxide (CO₂) and carbon monoxide (CO))；Isoprene；Ketone (for example, acetone)；Organic acid is (for example, acetic acid, methylacetal Acid, adipic acid, ascorbic acid, citric acid, 2,5- diketo-D gluconate, formic acid, fumaric acid, glucosaccharic acid, gluconic acid, Glucuronic acid, glutaric acid, 3- hydracrylic acid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid, oxaloacetic acid, propionic acid, amber Acid and xylonic)；And polyketide.Tunning can also be the protein as high-value product.

Tunning is alcohol in a preferred aspect,.It will be appreciated that term " alcohol " includes comprising one or more hydroxyls The substance of base group.At preferred aspect, the alcohol is n-butanol.In another more preferred aspect, the alcohol is isobutyl Alcohol.In another more preferred aspect, the alcohol is ethyl alcohol.In another more preferred aspect, the alcohol is methanol.Another A preferred aspect, the alcohol are arabites.In another more preferred aspect, the alcohol is butanediol.Another A preferred aspect, the alcohol are ethylene glycol.In another more preferred aspect, the alcohol is glycerine (glycerin). In another more preferred aspect, the alcohol is glycerol (glycerol).In another more preferred aspect, the alcohol is 1,3- Propylene glycol.In another more preferred aspect, the alcohol is sorbierite.In another more preferred aspect, the alcohol is xylose Alcohol.See, e.g., 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. are compiled, Springer-Verlag Berlin Heidelberg, Germany, 65:207-241；Silveira, And Jonas, R., 2002, M.M., 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 Biochemistry 30 (2):117-124；Ezeji, T.C., Qureshi, N. and Blaschek, H.P., 2003, Production of acetone, butanol and ethanol by Clostridium beijerinckii BA101and in situ recovery by gas stripping,World Journal of Microbiology and Biotechnology 19(6):595-603。

At another preferred aspect, the tunning is alkane.The alkane is unbranched or branching alkane.? Another preferred aspect, the alkane is pentane.In another more preferred aspect, the alkane is hexane.Another A preferred aspect, the alkane are heptane.In another more preferred aspect, the alkane is octane.Another more Preferred aspect, the alkane is nonane.In another more preferred aspect, the alkane is decane.It is more preferable at another Aspect, the alkane is hendecane.In another more preferred aspect, the alkane is dodecane.

At another preferred aspect, the tunning is cycloalkane.In another more preferred aspect, the cycloalkanes Hydrocarbon is pentamethylene.In another more preferred aspect, the cycloalkane is hexamethylene.In another more preferred aspect, described Cycloalkane is cycloheptane.In another more preferred aspect, the cycloalkane is cyclooctane.

At another preferred aspect, the tunning is alkene.The alkene can be unbranched or branching alkene. In another more preferred aspect, the alkene is amylene.In another more preferred aspect, the alkene is hexene.Another One preferred aspect, the alkene is heptene.In another more preferred aspect, the alkene is octene.

At another preferred aspect, the tunning is amino acid.In another more preferred aspect, described organic Acid is aspartic acid.In another more preferred aspect, the amino acid is glutamic acid.In another more preferred aspect, institute Stating amino acid is glycine.In another more preferred aspect, the amino acid is lysine.In another preferred side Face, the amino acid are serines.In another more preferred aspect, the amino acid is threonine.See, e.g., Richard, A. and Margaritis, A., 2004, Empirical modeling of batch fermentation kinetics for poly(glutamic acid)production and other microbial biopolymers, Biotechnology and Bioengineering 87(4):501-515。

At another preferred aspect, the substance is gas.In another more preferred aspect, the gas is first Alkane.In another more preferred aspect, the gas is H₂.In another more preferred aspect, the gas is CO₂.Another A preferred aspect, the gas are CO.See, e.g., Kataoka, N., A.Miya and K.Kiriyama, 1997, Studies on hydrogen production by continuous culture system of hydrogen- producing anaerobic bacteria,Water Science and Technology 36(6-7):41-47；With Gunaseelan, V.N., in Biomass and Bioenergy, Vol.13 (1-2), pp83-114,1997, Anaerobic digestion of biomass for methane production:A review。

At another preferred aspect, the tunning is isoprene.

At another preferred aspect, the tunning is ketone.It should be understood that term " ketone " is covered containing one Or the ketone of multiple ketone modules.In another more preferred aspect, the ketone is acetone.See, e.g. Qureshi and Blaschek, 2003, it sees above.

At another preferred aspect, the tunning is organic acid.In another more preferred aspect, described organic Acid is acetic acid.In another more preferred aspect, the organic acid is acetone acid.In another more preferred aspect, described to have Machine acid is adipic acid.In another more preferred aspect, the organic acid is ascorbic acid.In another more preferred aspect, The organic acid is citric acid.In another more preferred aspect, the organic acid is 2,5- diketo-D gluconate.Another A preferred aspect, the organic acid are formic acid.In another more preferred aspect, the organic acid is fumaric acid. In another more preferred aspect, the organic acid is glucosaccharic acid.In another more preferred aspect, the organic acid is Portugal Saccharic acid.In another more preferred aspect, the organic acid is glucuronic acid.In another more preferred aspect, described organic Acid is glutaric acid.At another preferred aspect, the organic acid is 3- hydracrylic acid.In another more preferred aspect, institute Stating organic acid is itaconic acid.In another more preferred aspect, the organic acid is lactic acid.In another more preferred aspect, The organic acid is malic acid.In another more preferred aspect, the organic acid is malonic acid.In another preferred side Face, the organic acid are oxalic acid.In another more preferred aspect, the organic acid is propionic acid.In another preferred side Face, the organic acid are succinic acids.In another more preferred aspect, the organic acid is xylonic.See, e.g., Chen, And Lee, Y.Y., 1997, R. Membrane-mediated extractive fermentation for lactic acid production from cellulosic biomass,Appl.Biochem.Biotechnol.63-65:435-448。

At another preferred aspect, the substance is polyketide.

RecycleAny method known in the art can be used, optionally recycle tunning from fermentation medium, it is described Method includes, but are not limited to chromatography, electrophoresis method, differential solubility, distillation or extraction.For example, by conventional distil-lation method from The cellulosic material of fermentation or the separation of material containing xylan and purified alcohols.The ethyl alcohol with high purity of about 96vol.% can be obtained, It can serve as, for example, alcohol fuel, drinking alcohol (that is, drinkable neutrality pick-me-up) or industrial alcohol.

Signal peptide

The invention further relates to the isolated polynucleotides of encoded signal peptide, the signal peptide includes or group becomes SEQ ID The amino acid 1 of NO:2 is to the amino acid 1 of 19, SEQ ID NO:4 to the amino acid 1 of 19, SEQ ID NO:6 to 19, SEQ ID The amino acid 1 of NO:8 is to the amino acid 1 of 21 or SEQ ID NO:10 to 17.The polynucleotides can further include coding egg White gene, is operably connected to signal peptide.The albumen is external source preferably for the signal peptide.A side Face, the polynucleotides for encoding the signal peptide are the nucleotide 1 to 57 of SEQ ID NO:1.On the other hand, the letter is encoded The polynucleotides of number peptide are the nucleotide 1 to 57 of SEQ ID NO:3.On the other hand, the multicore glycosides of the signal peptide is encoded Acid is the nucleotide 1 to 57 of SEQ ID NO:5.On the other hand, the polynucleotides for encoding the signal peptide are SEQ ID The nucleotide 1 to 63 of NO:7.On the other hand, the polynucleotides for encoding the signal peptide are the nucleotide 1 of SEQ ID NO:9 To 60.

The invention further relates to the nucleic acid construct comprising such polynucleotides, expression vector and recombinant host cells.

The invention further relates to for producing protedogenous method, comprising: (a) culture includes the recombination place of such polynucleotides Chief cell, the polynucleotides are operably connected to the gene of coding albumen；Optionally (b) recycles the protein.

The protein can be host cell natural or heterologous.Term " protein " this paper the meaning not Refer to the coded product of specific length, and therefore covers peptide, oligopeptides and polypeptide.Term " protein " also covers combined with shape At the two or more polypeptides of coded product.The protein further includes hybrid polypeptide and fused polypeptide.

Preferred protein is hormone, enzyme, receptor or part thereof, antibody or part thereof or reporter protein (reporter).Example Such as, the protein can be hydrolase, isomerase, ligase, lyase (lyase), oxidoreducing enzyme or transferase, such as α- Galactosidase, alpha-Glucosidase, aminopeptidase, amylase, beta galactosidase, β-glucosyl enzym, xylobiase, carbohydrase, Carboxypeptidase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyl transferases, takes off catalase Oxygen ribalgilase, esterase, glucoamylase, invertase, laccase, lipase, mannosidase, becomes poly- at endoglucanase Carbohydrase (mutanase), oxidizing ferment, pectin decomposing enzyme, peroxidase, phytase, polyphenol oxidase, proteolysis Enzyme, ribalgilase, transglutaminase or zytase.

Gene can be obtained from any protokaryon, eukaryon or other sources.

By following embodiment, the present invention is further described, but should not be construed as the limit to the scope of the invention System.

Embodiment

Bacterial strain

The fungal bacterial strain of NN047338 is named as by diluting on PDA plate at 45 DEG C and then single mitogenetic by shifting It purifies on spore to YG agar plate and is separated from the pedotheque collected is saved from Hunan China.NN047338 bacterial strain is based on form Feature and ITS rDNA Sequence Identification are thermophilic capital spore.

The fungal bacterial strain of NN051380 is named as by diluting on PDA plate at 25 DEG C and then single mitogenetic by shifting It purifies on spore to PDA plate and is separated from the pedotheque collected in China.Bacterial strain NN051380 is based on morphological feature and ITS RDNA Sequence Identification is penicillium oxalicum.

The fungal bacterial strain of NN046782 is named as by diluting on PDA plate at 45 DEG C and then single mitogenetic by shifting It purifies on spore to YG agar plate and is separated from the pedotheque collected in China.Bacterial strain NN046782 is based on morphological feature It is Rhizomucor pusillus (Rhizomucor pusillus) with ITS rDNA Sequence Identification.

The fungal bacterial strain of NN044936 is named as by diluting on PDA plate at 45 DEG C and then single mitogenetic by shifting It purifies on spore to YG agar plate and is separated from the pedotheque collected is saved in Chinese yunnan.Bacterial strain NN044936 is based on form Feature and ITS rDNA Sequence Identification are tangerine orange thermophilic ascomycete.

Culture medium

PDA plate adds to 1 liter by 39 grams of potato dextrose agar and deionized water and constitutes.

Yeast extract of the YG agar plate by 5g, the glucose of 10g, the agar and deionized water of 20g add to 1 liter of structure At.

YPG culture medium is by 0.4% yeast extract in deionized water, 0.1% KH₂PO₄, 0.05% MgSO₄· 7H₂O and 1.5% glucose are constituted.

By 1% yeast extract in deionized water, 2% peptone and 2% maltose are constituted YPM culture medium.

Sucrose of Czapek ' the s culture medium by 30g, the NaNO of 3g₃, the MgSO of 0.5g₄·7H₂The FeSO of O, 0.01g₄· 7H₂The K of O, 1g₂HPO₄, the KCl and deionized water of 0.5g add to 1 liter of composition.PH is adjusted with 1M HCl to pH 4.

Soy meal of the FG4 culture medium by 30g, the maltose of 15g, the Bacto peptone and deionized water of 5g add to 1 liter It constitutes.

Sucrose of the minimal medium plate by 342g, the salting liquid of 20ml, the agar and deionized water of 20g add to 1 liter of structure At.Salting liquid is by the 2.6%KCl in deionized water, 2.6%MgSO₄7H2O, 7.6%KH₂PO₄, 2ppm Na₂B₄O₇· 10H₂O, 20ppm CuSO₄·5H₂O, 40ppm FeSO₄·7H₂O, 40ppm MnSO₄·2H₂O, 40ppm Na₂MoO₄·2H₂O, With 400ppm ZnSO₄·7H₂O is constituted.

Embodiment 1: extracting genome DNA

By thermophilic capital spore bacterial strain NN047338 be inoculated on PDA plate and 45 DEG C Incubation in dark 3 days.By several mycelia Body-PDA bolt (plug) is inoculated with the 500ml shaking flask into the YPG culture medium containing 100ml.By bottle at 45 DEG C under 160rpm oscillation It incubates 3.Mycelium by via(Calbiochem, La Jolla, CA, USA) filters to collect And freeze in liquid nitrogen.The mycelium freezed is milled to fine-powder by mortar and pestle, and is used Plant Maxi kit (QIAGEN Inc., Valencia, CA, USA) follows the instruction separation genomic DNA of manufacturer.

By penicillium oxalicum bacterial strain NN051380 be inoculated on PDA plate and 25 DEG C Incubation in dark 5 days.By several mycelia Body-PDA bolt kind enters the 500ml shaking flask of Czapek ' the s culture medium containing 100ml.By bottle in 30 DEG C of temperature under 160rpm oscillation It educates 3.Mycelium by viaFiltering is to collect and freeze in liquid nitrogen.The mycelium freezed is led to It crosses mortar and pestle is milled to fine-powder, and usePlant Maxi kit follows the instruction point of manufacturer From genomic DNA.

By Rhizomucor pusillus bacterial strain NN046782 be inoculated on PDA plate and 45 DEG C Incubation in dark 3 days.By several mycelia Body-PDA bolt kind enters the 500ml shaking flask of the FG4 culture medium containing 100ml.Bottle is incubated 3 under 160rpm oscillation at 45 DEG C Day.Mycelium by viaFiltering is to collect and freeze in liquid nitrogen.The mycelium freezed is passed through Mortar and pestle are milled to fine-powder, and usePlant Maxi kit follows the instruction separation of manufacturer Genomic DNA.

By tangerine orange thermophilic ascomycete bacterial strain NN044936 be inoculated on PDA plate and 45 DEG C Incubation in dark 3 days.It will be several Mycelium-PDA bolt kind enters the 500ml shaking flask of the YPG culture medium containing 100ml.By bottle in 45 DEG C of temperature under 160rpm oscillation It educates 3.Mycelium by viaFiltering is to collect and freeze in liquid nitrogen.The mycelium freezed is led to It crosses mortar and pestle is milled to fine-powder, and usePlant Maxi kit follows the instruction point of manufacturer From genomic DNA.

Embodiment 2: thermophilic capital spore bacterial strain NN047338, penicillium oxalicum bacterial strain NN051380, Rhizomucor pusillus Gene order-checking, compilation and the annotation of NN046782 and Thermoascus aurantiacus NN044936

By the genome DNA sample of extraction be delivered to Beijing Genome Institute (BGI, Shenzhen, China) for usingThe genome of GA2System (Illumina, Inc., San Diego, CA, USA) Sequencing.Rough read is used into program SOAPdenovo (Li et al., 2010, Genome Research 20 (2): 265-72) in BGI It collects.The sequence of compilation is analyzed using standard bioinformatic methods for gene search (gene finding) And function prediction.It is pre- that gene is carried out using geneID (Parra etc., 2000, Genome Research 10 (4): 511-515) It surveys.Use Blastall version 2 .2.10 (Altschul etc., 1990, J.Mol.Biol.215 (3): 403-410, National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) and HMMER version 2 .1.1 (National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) is based on structure Homology forecast function.Go out β-glucosyl enzym by the analysis Direct Identification of Blast result.Using Agene program (Munch and Krogh, 2006, BMC Bioinformatics 7:263) and SignalP program (Nielsen etc., 1997, Protein Engineering 10:1-6) identification initiation codon.Further use SignalP program predicted signal peptide.It uses The isoelectric point for the amino acid sequence that Pepstats (Rice etc., 2000, Trends Genet.16 (6): 276-277) prediction derives And molecular weight.

Embodiment 3: from the thermophilic capital spore GH3 xylobiase coded sequence of genomic dna cloning

Based on the DNA information (SEQ ID NO:1 and 3) obtained from the gene order-checking in embodiment 2, devise shown below Oligonucleolide primers are with from the genomic DNA amplification GH3 xylobiase gene of thermophilic capital spore NN047338, GH3_ ZY577211_92 and GH3_ZY577202_22.Primer is synthesized by Invitrogen, Beijing, China.

SEQID1_ forward primer:

5’-ACACAACTGGGGATCCACCatgaccaggctgaccagcatc-3’(SEQ ID NO:11)

SEQID1_ reverse primer:

5’-GTCACCCTCTAGATCTcgtaccccactgccgttattg-3’(SEQ ID NO:12)

SEQID3_ forward primer:

5’-ACACAACTGGGGATCCACCatgaaggccctgactagaagg-3’(SEQ ID NO:13)

SEQID3_ reverse primer:

5’-GTCACCCTCTAGATCTtaccggacatgaacatgacagtagg-3’(SEQ ID NO:14)

Lowercase represents the coded sequence of gene in forward primer, and the flank of gene is represented in reverse primer Area, and capitalization represents the area (WO2011/005867) with the insertion point derived from plasmid pPFJO355.

For each gene, each forward and reverse primer pair of 20 picomoles is used for PCR reaction, the reaction is by 2 μ The thermophilic capital spore NN047338 genomic DNA of l, 10 μ l 5X GC Buffer (Finnzymes Oy, Espoo, Finland), the DMSO of 1.5 μ l, the PHUSION of dATP, dTTP, dGTP and dCTP of each 2.5mM and 0.6 unit^TM High- Fidelity archaeal dna polymerase (Finnzymes Oy, Espoo, Finland) is constituted, and final volume is 50 μ l.Amplification uses Peltier Thermal Cycler (MJ Research Inc., South San Francisco, CA, USA) is carried out, journey Sequence is as follows: being denaturalized 1 minute at 98 DEG C；6 circulations, are each denaturalized 15 seconds at 98 DEG C, anneal 30 seconds at 65 DEG C, and every circulation reduces by 1 DEG C, and extend 3 minutes at 72 DEG C；23 circulations, each carry out 15 seconds at 98 DEG C, carry out 30 seconds and 72 DEG C carrying out 3 points at 62 DEG C Clock；And finally extend 5 minutes at 72 DEG C.Then heat block enters 4 DEG C of infusions (soak cycle).

PCR product is by using 1.0% Ago-Gel of 90mM Tris- boric acid and 1mM EDTA (TBE) buffer electricity Swimming separation, wherein visible under w light for the independent product band of the 3kb of each reaction.Then it usesPCR and Gel Band Purification kit (GE Healthcare, Buckinghamshire, UK) according to the instruction of manufacturer from solution purification PCR product.

Plasmid pPFJO355 Bam HI and Bgl II is digested, by using 1.0% Ago-Gel of tbe buffer liquid Electrophoretic separation, and usePCR and Gel Band Purification kit is according to life The instruction of business men purifies.

Table 1: plasmid

Gene	Plasmid	DNA spectrum
			GH3_ZY577211_92	pGH3_ZY577211_92	Fig. 1
GH3_ZY577202_22	pGH3_ZY577202_22	Fig. 2

Every kind of PCR product and the carrier of digestion are used into IN-CF Dry-down Cloning kit (Clontech Laboratories, Inc., Mountain View, CA, USA) links together, and obtains matter shown in table 1 Grain: pGH3_ZY577211_92 (Fig. 1) and pGH3_ZY577202_22 (Fig. 2), wherein thermophilic capital spore GH3 xylobiase is compiled The transcription of code sequence is under the regulation of oryzae alpha-amylase gene promoter.In short, 30ng is used Bam HI and Bgl It is small that the various thermophilic capital spore GH3 xylobiase PCR products of the purifying of the pPFJO355 and 60ng of II digestion are added to reaction Bottle, and the final volume for being resuspended in 10 μ l by adding deionized water.Reaction is incubated 15 minutes at 37 DEG C then in 50 DEG C of temperature It educates 15 minutes.Escherichia coli TOP10 competent cell (TIANGEN Biotech (Beijing) is converted using the reactant of three μ l Co.Ltd., Beijing, China).Escherichia coli transformant containing expression construct is detected by bacterium colony PCR.Bacterium colony PCR It is a kind of method being inserted into for directly quickly screening plasmid from E. coli clones.In short, the premix in each PCR pipe The PCR solution aliquot of conjunction (includes PCR buffer, MgCl₂, dNTP, and the primer pair from its generation PCR fragment) in, lead to The liquid relief point picking with sterilizing is crossed, and the liquid relief point is rotated in reaction solution to add single bacterium colony.Usually screening 7-10 bacterium colony.After PCR, analyzed 1.0% agarose gel electrophoresis by using tbe buffer liquid is reacted. It usesSpin Miniprep kit (QIAGEN GmbH, Hilden, Germany) has from display to be expected The bacterium colony of the insert of size prepares Plasmid DNA.That is inserted into pGH3_ZY577211_92 and pGH3_ZY577202_22 is thermophilic Capital spore GH3 xylobiase coded sequence is by using 3730XL DNA Analyzer (Applied Biosystems Inc., Foster City, CA, USA) DNA sequencing confirm.

Embodiment 4: thermophilic capital spore GH3 xylobiase coded sequence is expressed in aspergillus oryzae

It will be according to Christensen etc., the aspergillus oryzae of the method preparation of 1988, Bio/Technology 6:1419-1422 HowB101 (WO95/35385) protoplast is converted with the pGH3_ZY577211_92 or pGH3_ZY577202_22 of 3 μ g.Each Conversion generates about 50 transformant.Eight transformant from each conversion are separated to individual minimal medium plate.

Four transformant from each conversion are inoculated with to the YPM culture medium of the 3ml in 24 orifice plates respectively, and at 30 DEG C It is incubated under 150rpm stirring.After 3 incubate, by the supernatant of the 20 μ l from each culture by using 2- containing 50mM (N- morpholino) ethanesulfonic acid (MES) 4-12%Bis-Tris Gel (Invitrogen Corporation, Carlsbad, CA, USA) SDS-PAGE analyzed according to the instruction of manufacturer.Resulting gel is used(Expedeon Ltd., Babraham Cambridge, UK) dyeing.The SDS-PAGE of culture General picture shows that the transformant of pGH3_ZY577211_92 and pGH3_ZY577202_22 is respectively provided with the master in 90kDa and 95kDa Want protein band (table 2).A transformant from each conversion is elected to be expression strain, and is respectively designated as aspergillus oryzae O5JAC With aspergillus oryzae O5JA9.

Table 2: expression

Plasmid	Express strain	The size (KD) of recombinant protein
			pGH3_ZY577211_92	O5JAC	90
pGH3_ZY577202_22	O5JA9	95

By each expression strain, the inclined-plane (slant) of aspergillus oryzae O5JAC and aspergillus oryzae O5JA9 are washed with the YPM of 10ml, and are connect Kind enters 2 liters of flasks of the YPM culture medium containing 400ml.In harvest culture on the 3rd, and use 0.45 μmMembrane (Millipore, Bedford, MA, USA) filtering.

Embodiment 5: thermophilic capital spore GH3 xylobiase is recombinated from aspergillus oryzae O5JAC purifying

By the filtered culture solution of the aspergillus oryzae O5JAC (embodiment 4) of 3200ml volume with ammonium sulfate (80% saturation) Precipitating, is redissolved in the 20mM sodium acetate pH 5.0 of 50ml, dialyses for same buffer, and passes through 0.45 μm of filter mistake Filter.Final volume is 80ml.Solution is imposed on to the 40ml Q balanced with 20mM sodium acetate pH 5.0Fast Flow column (GE Healthcare, Buckinghamshire, UK).By the linear 0-0.5M NaCl gradient elution of albumen.By grade Divide and collects, collects and impose on the 40ml SP balanced in 20mM sodium acetate pH 5.0Fast Flow column (GE Healthcare,Buckinghamshire,UK).By albumen with linear 0.2-0.5M NaCl gradient elution.Fraction passes through Using with 50mM MES'sThe SDS-PAGE of 4-12%Bis-Tris Gel is divided Analysis.Collect the fraction of the band containing about 90kDa, and is concentrated by ultrafiltration.

Embodiment 6: from genomic dna cloning penicillium oxalicum GH3 xylosidase coded sequence

Based on the DNA information (SEQ ID NO:5) obtained from the gene order-checking in embodiment 2, widow shown below is devised Nucleotide primer is with from the genomic DNA amplification GH3 xylosidase gene of penicillium oxalicum, GH3_ZY569167_685.

Forward primer:

5’-ACACAACTGGGGATCCACCatgctggccctggcatc-3’(SEQ ID NO:15)

Reverse primer:

5’-GTCACCCTCTAGATCTtcaaaatcctcttgtgctacctctcaagaa-3’(SEQ ID NO:16)

Lowercase represents the DNA sequence dna of gene in forward primer, and the flanking region of gene is represented in reverse primer, And capitalization represents the area with the insertion point derived from plasmid pPFJO355.

Every kind of 20 picomoles above-mentioned primer is used to PCR to react, the penicillium oxalicum genomic DNA reacted by 2 μ l, 5X the GC Buffer, the DMSO of 1.5 μ l, dATP, dTTP, dGTP and dCTP of each 2.5mM of 10 μ l and 0.6 unit PHUSION^TMHigh-Fidelity archaeal dna polymerase is constituted, and final volume is 50 μ l.Amplification uses Peltier Thermal Cycler is carried out, and program is as follows: being denaturalized 1 minute at 98 DEG C；6 circulations, are each denaturalized 15 seconds at 98 DEG C, in 65 DEG C of annealing 30 Second, every circulation reduces by 1 DEG C, and extends 3 minutes at 72 DEG C；25 circulations, each carry out 15 seconds at 98 DEG C, carry out 30 at 62 DEG C Second and 72 DEG C carry out 3 minutes；And finally extend 5 minutes at 72 DEG C.Then heat block enters 4 DEG C of infusions.

PCR product is separated by using 1.0% agarose gel electrophoresis of tbe buffer liquid, wherein by the product of about 3kb Band is cut out from gel, and is usedPCR and Gel Band Purification kit It is purified according to the instruction of manufacturer.

The 3kb PCR product and the carrier of digestion are used into IN-CF Dry-down Cloning reagent Box links together, and obtains pGH3_ZY569167_685 (Fig. 3), wherein the transcription of penicillium oxalicum GH3 xylosidase coded sequence Under regulation in oryzae alpha-amylase gene promoter.In short, by 30ng Bam HI and Bgl II digestion The penicillium oxalicum GH3 xylobiase PCR product of the purifying of pPFJO355 and 60ng is added to reaction bottle, and passes through addition Deionized water is resuspended in the final volume of 10 μ l.Reaction is incubated 15 minutes at 37 DEG C and is then incubated 15 minutes at 50 DEG C.It uses The reactant of three μ l converts Escherichia coli TOP10 competent cell.Escherichia coli transformant containing pGH3_ZY569167_685 It is detected by bacterium colony PCR as described in example 3 above.It usesSpin Miniprep kit (QIAGEN GmbH, Hilden, Germany) preparation Plasmid DNA.The penicillium oxalicum GH3 β-xyloside being inserted into pGH3_ZY569167_685 Enzyme coded sequence is confirmed by using the DNA sequencing of 3730XL DNA Analyzer.

Embodiment 7: penicillium oxalicum GH3 xylobiase coded sequence is expressed in aspergillus oryzae

Will be according to Christensen etc., 1988, the aspergillus oryzae HowB101 (WO95/35385) of the method preparation seen above Protoplast is converted with the pGH3_ZY569167_685 of 3 μ g.The conversion generates about 50 transformant.By four transformant point From to individual minimal medium plate.

Four transformant are inoculated with to the YPM culture medium of the 3ml in 24 orifice plates respectively, and are stirred at 30 DEG C in 150rpm Mix lower incubation.After 3 incubate, by the supernatant of the 20 μ l from each culture by using the MES's containing 50mM4-12%Bis-Tris Gel is analyzed.Resulting gel is usedDyeing.The SDS-PAGE general picture of culture shows that most of transformant has about 98kDa's Band.One transformant is elected to be expression strain, and is named as aspergillus oryzae O4S4Q.

The inclined-plane of aspergillus oryzae O4S4Q is washed with the YPM culture medium of 10ml, and is inoculated with into the YPM culture medium containing 400ml 2 liters of flasks.In harvest culture on the 3rd, and use 0.45 μmMembrane(Millipore, Bedford, MA, USA) filtering.

Embodiment 8: from genomic dna cloning Rhizomucor pusillus GH3 xylobiase coded sequence

Based on the DNA information (SEQ ID NO:7) obtained from gene order-checking, devise Oligonucleolide primers shown below with From the genomic DNA amplification GH3 xylobiase gene of Rhizomucor pusillus NN046782, GH3_ZY654890_6424.Primer by Invitrogen, Beijing, China synthesis.

Forward primer:

5’-ACACAACTGGGGATCCACCatggcgtttatcaagcagagc-3’(SEQ ID NO:17)

Reverse primer:

5’-GTCACCCTCTAGATCTaccgtggaaacagcagcag-3’(SEQ ID NO:18)

Lowercase represents the code area of gene in forward primer, and the flanking region of gene is represented in reverse primer, And capitalization represents the area with the insertion point derived from plasmid pPFJO355.

Every kind of 20 picomoles above-mentioned primer is used for PCR reaction, the Rhizomucor pusillus genome reacted by 2 μ l 5X the GC Buffer, the DMSO of 1.5 μ l, dATP, dTTP, dGTP and dCTP of each 2.5mM of DNA, 10 μ l and 0.6 unit PHUSION^TMHigh-Fidelity archaeal dna polymerase is constituted, and final volume is 50 μ l.Amplification uses Peltier Thermal Cycler is carried out, and program is as follows: being denaturalized 1 minute at 98 DEG C；6 circulations, are each denaturalized 30 seconds at 98 DEG C, in 63 DEG C of annealing 30 Second, every circulation reduces by 1 DEG C, and extends 2.5 minutes at 72 DEG C；24 circulations, each carry out 15 seconds at 94 DEG C, carry out 30 at 58 DEG C Second and 72 DEG C carry out 2.5 minutes；And finally extend 5 minutes at 72 DEG C.Then heat block enters 4 DEG C of infusions.

Reaction product is separated by using 1.0% agarose gel electrophoresis of tbe buffer liquid, wherein by about 2.6kb's Product band is cut out from gel, and is usedPCR and Gel Band Purification examination Agent box is purified according to the instruction of manufacturer.

Use IN-CF Dry-down Cloning kit is by each 2.6kb PCR fragment Direct Cloning Enter expression vector pPFJO355, without restrictive digestion and connection.

PCR product and the carrier of digestion are used into IN-CF Dry-down Cloning kit is connected to Together, pGH3_ZY654890_6424 (Fig. 4) is obtained, wherein the transcription of Rhizomucor pusillus GH3 xylosidase coded sequence is in Under the regulation of oryzae alpha-amylase gene promoter.In short, by 30ng Bam HI and Bgl II digestion The Rhizomucor pusillus GH3 xylobiase PCR product of the purifying of pPFJO355 and 60ng is added to reaction bottle, and by adding Deionized water is added to be resuspended in the final volume of 10 μ l.Reaction is incubated 15 minutes at 37 DEG C and is then incubated 15 minutes at 50 DEG C.Make Escherichia coli TOP10 competent cell is converted with the reactant of three μ l.Escherichia coli containing pGH3_ZY654890_6424 turn Change body to detect by bacterium colony PCR as described in example 3 above.It usesThe preparation of Spin Miniprep kit Plasmid DNA.The Rhizomucor pusillus GH3 xylobiase coded sequence being inserted into pGH3_ZY654890_6424 by using The DNA sequencing of 3730XL DNA Analyzer confirms.

Embodiment 9: from genomic dna cloning tangerine orange thermophilic ascomycete GH3 xylobiase coded sequence

Based on the DNA information (SEQ ID NO:9) obtained from the gene order-checking in embodiment 2, widow shown below is devised Nucleotide primer is with from the genomic DNA amplification GH3 xylobiase gene of tangerine orange thermophilic ascomycete, PE04100001596.Draw Object is synthesized by Invitrogen, Beijing, China.

Forward primer:

5’-ACACAACTGGGGATCCACCatggccaccctcaagtcagttct-3’(SEQ ID NO:19)

Reverse primer:

5’-GTCACCCTCTAGATCTtcgctcactcactcactgagaagc-3’(SEQ ID NO:20)

Every kind of 20 picomoles above-mentioned primer is used for PCR reaction, the tangerine orange thermophilic ascomycete gene reacted by 2 μ l Group DNA, 5X the GC Buffer, the DMSO of 1.5 μ l, dATP, dTTP, dGTP and dCTP of each 2.5mM of 10 μ l and 0.6 unit PHUSION^TMHigh-Fidelity archaeal dna polymerase is constituted, and final volume is 50 μ l.Amplification uses Peltier Thermal Cycler is carried out, and program is as follows: being denaturalized 1 minute at 98 DEG C；8 circulations, are each denaturalized 15 seconds at 98 DEG C, in 65 DEG C of annealing 30 Second, every circulation reduces by 1 DEG C, and extends 3.25 minutes at 72 DEG C；22 circulations, each carry out 15 seconds at 98 DEG C, carry out at 58 DEG C It carries out 3.25 minutes within 30 seconds and 72 DEG C；And finally extend 10 minutes at 72 DEG C.Then heat block enters 4 DEG C of infusions.

Reaction product is separated by using 1.0% agarose gel electrophoresis of tbe buffer liquid, wherein by about 2.4kb's Product band is cut out from gel, and is usedPCR and Gel Band Purification examination The purifying of agent box.

Plasmid pPFJO355 Bam HI and Bgl II is digested, by using 1.0% Ago-Gel of tbe buffer liquid Electrophoretic separation, and usePCR and Gel Band Purification kits.

The 2.4kb PCR product and the carrier of digestion are used into IN-CF Dry-down Cloning examination Agent box links together, and obtains pGH3_PE04100001596 (Fig. 5), and wherein tangerine orange thermophilic ascomycete GH3 xylobiase is compiled The transcription of code sequence is under the regulation of oryzae alpha-amylase gene promoter.In short, 30ng is used Bam HI and Bgl It is small that the tangerine orange thermophilic ascomycete GH3 xylobiase PCR product of the purifying of the pPFJO355 and 60ng of II digestion is added to reaction Bottle, and the final volume for being resuspended in 10 μ l by adding deionized water.Reaction is incubated 15 minutes at 37 DEG C then in 50 DEG C of temperature It educates 15 minutes.Escherichia coli TOP10 competent cell is converted using the reactant of three μ l.Contain pGH3_PE04100001596's Escherichia coli transformant is detected by bacterium colony PCR as described in example 3 above.It usesSpin Miniprep Kit prepares Plasmid DNA.The tangerine orange thermophilic ascomycete GH3 xylobiase code sequence being inserted into pGH3_PE04100001596 Column are confirmed by using the DNA sequencing of 3730XL DNA Analyzer.

Embodiment 10: tangerine orange thermophilic ascomycete GH3 xylobiase coded sequence is expressed in aspergillus oryzae

Will be according to Christensen etc., 1988, the aspergillus oryzae HowB101 (WO95/35385) of the method preparation seen above Protoplast is converted with the pGH3_PE04100001596 of 3 μ g.The conversion generates about 50 transformant.By four transformant point From to individual minimal medium plate.

Four transformant are inoculated with to the YPM culture medium of the 3ml in 24 orifice plates respectively, and are stirred at 30 DEG C in 150rpm Mix lower incubation.After 3 incubate, by the supernatant of the 20 μ l from each culture by using the MES's containing 50mMThe SDS-PAGE of 4-12%Bis-Tris Gel is analyzed.Resulting gel is usedDyeing.The SDS-PAGE general picture of culture shows that most of transformant has about 90kDa's Band.One transformant is elected to be expression strain, and is named as aspergillus oryzae O6YKQ.

The inclined-plane of aspergillus oryzae O6YKQ is washed with the YPM culture medium of 10ml, and is inoculated with into the YPM culture medium containing 400ml 2 liters of flasks.In harvest culture on the 3rd, and use 0.45 μmMembrane(Millipore, Bedford, MA, USA) filtering.

It is again molten by the aspergillus oryzae O6YKQ filtered culture solution of 2400ml volume ammonium sulfate (80% saturation) precipitating Solution is dialysed in the 20mM Tris-HCl pH 6.5 of 50ml for same buffer, and by 0.45 μm of filter filtering.Final body Product is 75ml.Solution is imposed on to the 40ml Q balanced in 20mM Tris-HCl pH 6.5Fast Flow column.The fraction with 0.08-0.1M NaCl elution is collected, and is further existed with linear NaCI gradient (0.03-0.11M) 40ml QIt is purified on Fast Flow column.Fraction is by using with 50mM MES'sThe SDS-PAGE of 4-12%Bis-Tris Gel is assessed.Collect containing about 84kDa Band fraction.Then the solution collected is concentrated by ultrafiltration.

Embodiment 11: the characterization of the genomic DNA of coding GH3 xylobiase

The genomic dna sequence of thermophilic capital spore GH3 xylobiase coded sequence and the amino acid sequence difference of derivation It is shown in SEQ ID NO:1 (D822K1) and SEQ ID NO:2 (P244Y5).Coded sequence is 2402bp, including terminator codon, It is interrupted by the introne (nucleotide 192 to 259) of a 68bp.The albumen of the prediction of coding is 777 amino acid.It uses SignalP program (Nielsen etc., 1997, Protein Engineering 10:1-6), predicts the signal of 19 residues Peptide.The maturation protein of prediction contains 758 amino acid, with 83.06kDa prediction molecular weight and 6.15 prediction etc. Electric point.

Using Needleman and Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443- 453) with 10 gap open penalty, 0.5 gap extension penalty and EBLOSUM62 matrix the comparison of amino acid sequence has been determined Property is by overall comparison.Compare the amino acid sequence of the derivation of the thermophilic capital spore genomic DNA of code displaying GH3 xylobiase Amino acid sequence (the UNIPROT of column and the derivation of the GH3 xylobiase from Pyrenophora tritici-repentis B2W9Y0) there is 62.96% sequence identity (excluding notch).

The genomic dna sequence of thermophilic capital spore GH3 xylobiase coded sequence and the amino acid sequence difference of derivation It is shown in SEQ ID NO:3 (D822JZ) and SEQ ID NO:4 (P244Y4).Coded sequence is 2671bp, includes terminator codon, It is by 68bp (nucleotide 192 to 259), and three of 62bp (nucleotide 564 to 625) and 63bp (nucleotide 1001 to 1063) Introne interrupts.The albumen of the prediction of coding is 825 amino acid.Using SignalP program (Nielsen etc., 1997, see on Text), predict the signal peptide of 19 residues.The maturation protein of prediction contains 806 amino acid, the prediction with 86.94kDa Molecular weight and 5.35 prediction isoelectric point.

It is opened using Needleman and Wunsch algorithm (Needleman and Wunsch, 1970, see above) with 10 notch Put point penalty, 0.5 gap extension penalty and EBLOSUM62 matrix the comparative by overall comparison of amino acid sequence has been determined. Compare amino acid sequence and the chaetomium globosum of the derivation of the thermophilic capital spore genomic DNA of code displaying GH3 xylobiase The amino acid sequence (UNIPROT Q2HEP1) of the derivation of (Chaetomium globosum) GH3 xylobiase has 70.39% identity (excludes notch).

The genomic dna sequence of penicillium oxalicum GH3 xylobiase coded sequence and the amino acid sequence of derivation show respectively In SEQ ID NO:5 (D72UE7) and SEQ ID NO:6 (P241KM).Coded sequence is 2832bp, includes terminator codon, It is interrupted by two intrones of 82bp (nucleotide 222 to 303) and 194bp (nucleotide 418 to 611).The egg of the prediction of coding White is 851 amino acid.Using SignalP program (Nielsen etc., 1997, see above), the signal of 19 residues is predicted Peptide.The maturation protein of prediction contains 832 amino acid, with 90.45kDa prediction molecular weight and 4.83 prediction etc. it is electric Point.

It is opened using Needleman and Wunsch algorithm (Needleman and Wunsch, 1970, see above) with 10 notch Put point penalty, 0.5 gap extension penalty and EBLOSUM62 matrix the comparative by overall comparison of amino acid sequence has been determined. Compare the amino acid sequence of the derivation of the penicillium oxalicum genomic DNA of code displaying GH3 xylobiase and from wheel branch fusarium The amino acid sequence (GENESEQP AZG45438) of the derivation of the GH3 enzyme of bacterium (Fusarium verticillioides) has 47.51% identity (excludes notch).

The genomic dna sequence of Rhizomucor pusillus GH3 xylobiase coded sequence and the amino acid sequence difference of derivation It is shown in SEQ ID NO:7 (D13874) and SEQ ID NO:8 (P24QRU).Coded sequence is 2637bp, includes terminator codon, It is by 51bp (nucleotide 288 to 338), 58bp (nucleotide 444 to 501), 58bp (nucleotide 540 to 597), 59bp (nucleosides Sour 707 to 765) it is interrupted with five intrones of 107bp (nucleotide 1618 to 1724).The albumen of the prediction of coding is 767 Amino acid.Using SignalP program (Nielsen etc., 1997, see above), the signal peptide of 21 residues is predicted.Prediction at Soft-boiled eggs are white containing 746 amino acid, have the isoelectric point of the molecular weight of the prediction of 82.03kDa and 5.02 prediction.

It is opened using Needleman and Wunsch algorithm (Needleman and Wunsch, 1970, see above) with 10 notch Put point penalty, 0.5 gap extension penalty and EBLOSUM62 matrix the comparative by overall comparison of amino acid sequence has been determined. It compares the amino acid sequence of the derivation of the Rhizomucor pusillus genomic DNA of code displaying GH3 xylobiase and comes from disk base net Amino acid sequence (the UNIPROT of the derivation of the xylobiase of handle bacterium (Dictyostelium discoideum) AYM76588) there is 43.44% identity (excluding notch).

The genomic dna sequence of tangerine orange thermophilic ascomycete GH3 xylobiase coded sequence and the amino acid sequence of derivation It is shown in SEQ ID NO:9 (D82RN1) and SEQ ID NO:10 (P24GP2).Coded sequence is 2403bp, close comprising terminating Numeral is free of any introne.The albumen of the prediction of coding is 800 amino acid.Using SignalP program (Nielsen etc., 1997, see above), predict the signal peptide of 20 residues.The maturation protein of prediction contains 780 amino acid, has The molecular weight of the prediction of 84.58kDa and 5.03 prediction isoelectric point.

It is opened using Needleman and Wunsch algorithm (Needleman and Wunsch, 1970, see above) with 10 notch Put point penalty, 0.5 gap extension penalty and EBLOSUM62 matrix the comparative by overall comparison of amino acid sequence has been determined. Compare code displaying GH3 xylobiase tangerine orange thermophilic ascomycete genomic DNA derivation amino acid sequence with come from it is inner There is the amino acid sequence (GENESEQP ARZ21779) of the derivation of the xylobiase of family name's trichoderma 70.5% identity (to exclude Notch).

Embodiment 12: pretreated corncob hydrolysis measurement

Corncob is located at 120 DEG C in 15% gross dry weight amount solid (TS) in advance with NaOH (0.08g/g dry weight cob) Reason 60 minutes.Resulting material with water is washed, until it is pH 8.2, obtains washed, oxygenation pretreatment corncob (APCC).Ground, screened, the corncob of oxygenation pretreatment (GS-APCC) by by the pH of APCC by addition 6M HCl and Water and being sufficiently mixed is adjusted to 5.0, by APCC in 40 wet type multipurpose beveller (wet multi- of Cosmos ICMG Utility grinder) it grinds in (EssEmm Corporation, Tamil Nadu, India), and in 121 DEG C of steam sterilizings It prepares within 45 minutes, final TS is 3.33%.The hydrolysis of GS-APCC using 2.2ml deep-well plates (Axygen, Union City, CA, USA) it is carried out with the total reaction volume of 1.0ml.

5.0 buffer of 50mM sodium acetate pH of the hydrolysis every ml manganese sulfate containing 1mM of GS-APCC total solid of 10mg and more The multiple protein of kind enzymatic compositions loads (being expressed as every gram of cellulose of mg albumen).Enzymatic compositions are prepared, then by it with 50 μ l Volume to 200 μ l ranges is added in all holes simultaneously, so that final volume is 1ml in each reaction.Then by plate Use ALPS-300^TMPlate heats seal instrument (Abgene, Epsom, United Kingdom) sealing, is sufficiently mixed, and specific Incubation at temperature 72 hours.The experiment being had been reported that carries out a formula three times.

After hydrolysis, sample is used 0.45 μm96 hole filters (Millipore, Bedford, MA, USA) filtering, and sugared content is analyzed as described below to permeate.When not immediately in use, by filtered etc. Sample is divided to freeze in -20 DEG C.In 0.005M H₂SO₄In diluted sample sugared concentration use 4.6x250mm HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, CA, USA) passes through following measurement: using 0.05%w/w Benzoic acid -0.005M H₂SO₄In 65 DEG C of flow velocity elutions per minute with 0.6ml, and by will be from being corrected with pure sugar-like product Refractive index detection (1100 HPLC,Agilent Technologies, Santa Clara, CA, USA) glucose, cellobiose and xylose signal integration quantified.Resulting glucose equivalent For calculating the cellulose conversion percentages of each reaction.Resulting xylose equivalents are used to calculate the wood oligose conversion of each reaction Percentage.

Glucose, cellobiose and xylose are measured respectively.The sugared concentration measured is carried out for suitable dilution gfactor Adjustment.All HPLC data mart modelings use MICROSOFT EXCEL^TMSoftware (Microsoft, Richland, WA, USA) carries out.

The degree that wood oligose is converted into xylose uses following equation calculations: % xylose=xylose concentration/limitation digests In xylose concentration.In order to calculate % conversion, based on cellulase control (trichoderma reesei cellulase of 100mg, supplement P.emersonii GH61A polypeptide (WO 2011/041397)), aspergillus fumigatus GH10 zytase (xyn3) (WO 2006/ 078256) and Ai Mosen ankle section bacterium GH3 xylobiase (WO 2003/070956) every gram of cellulose) setting 100% conversion Point, and by all values divided by the numerical value and then multiplied by 100.Triplicate data point is averaged and calculates standard deviation.

Embodiment 13: the preparation of Penicillium kind bacterial strain NN51602GH10 zytase

Penicillium kind bacterial strain NN51602GH10 zytase (SEQ ID NO:21 [DNA sequence dna] and SEQ ID NO:22 [amino acid sequence of derivation]) it is prepared by recombinant according to WO 2010/126772.The culture solution use of filtering is gathered configured with 10kDa Ether sulfone film (Pall Filtron, Northborough, MA, USA) tangent flow inspissator (Pall Filtron, Northborough, MA, USA) concentration and with 8.0 buffer-exchanged of 20mM Tris pH.Permeate through desalination is loaded on The Q balanced in 20mM Tris pH 8.0High Performance column (GE Healthcare, Piscataway, NJ, USA) on, and the linear gradient elution of the albumen 0-1000mM sodium chloride combined.Fraction by using 8-16%Tris HCl CRITERION STAIN FREE^TMSDS-PAGE the and CRITERION STAIN FREE of gel^TM Imaging System SDS-PAGE (Bio-Rad Laboratories, Inc., Hercules, CA, USA) is analyzed.It converges The fraction of band of the collection containing about 50kDa.Protein concentration uses Microplate BCA^TMProtein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA) is determined, wherein bovine serum albumin(BSA) is used as protein standard.

Embodiment 14: Ai Mosen ankle section bacterium (Talaromyces emersonii) CBS 393.64GH3 xylobiase (P4UE) preparation

Ai Mosen ankle section bacterium CBS 393.64 xylobiase (SEQ ID NO:23 [DNA sequence dna] and SEQ ID NO:24 [amino acid sequence of derivation]) according to Rasmussen etc., 2006, Biotechnology and Bioengineering 94: 869-876 uses aspergillus oryzae JaL355 to be prepared by recombinant as host (WO 2003/070956).The culture solution use of filtering is matched The tangent flow inspissator for being equipped with 10kDa poly (ether sulfone) film is concentrated and uses 5.0 desalination of 50mM sodium acetate pH.Protein concentration uses Microplate BCA^TMProtein Assay kit determines that wherein bovine serum albumin(BSA) is used as protein standard.

Embodiment 15: the quantitative gel of tangerine orange thermophilic ascomycete GH3 xylobiase (P24GP2)

The total protein content of tangerine orange thermophilic ascomycete GH3 xylobiase is determined by quantitative gel.Protein concentration passes through Use 8-16%Tris HCl CRITERION STAIN FREE^TMSDS-PAGE the and CRITERION STAIN of gel FREE^TMImaging System SDS-PAGE (Bio-Rad Laboratories, Inc., Hercules, CA, USA) is determining, Wherein Ai Mosen ankle section bacterium GH3 xylobiase is used as protein standard.

Embodiment 16: when the thermophilic son of tangerine orange when pH 4.0 to 7.0 is using APCC supplement Penicillium kind GH10 zytase The effect of capsule bacterium GH3 xylobiase (P24GP2)

Use washed, oxygenation pretreatment corncob (APCC) as substrate from pH 4.0 to 7.0 at 50 DEG C and 60 DEG C The tangerine orange thermophilic ascomycete xylobiase (P24GP2) of assessment supplement Penicillium kind GH10 zytase (embodiment 13).As Compare, the Ai Mosen ankle section bacterium GH3 xylobiase (P4UE) for supplementing Penicillium kind GH10 zytase is added to APCC.It will Xylobiase is added to the zytase of the every g cellulose supplement every g cellulose of 4.0mg total protein of 0.025mg total protein APCC hydrolysis, and results of hydrolysis is compared with the result of the zytase only containing the every g cellulose of 4.0mg total protein.

Measurement carries out as described in example 12 above.Containing 1mM manganese sulfate with the 1ml reaction of APCC (1% total solid) It is carried out 72 hours in 50mM Tris (pH 6.0 to 7.0) or 50mM sodium acetate (pH 4.0 to 5.5).All reactions are triplicate It carries out, and is related to the single mixing when hydrolyzing starting.

It is shown in Fig. 6 in 50 DEG C of results, and is shown in Fig. 7 in 60 DEG C of result.As shown in fig. 1, tangerine orange thermophilic ascomycete GH3 xylobiase compared with only Penicillium kind GH10 xylosidase, increases xylan at 50 DEG C and pH 4.0 to 7.0 significantly To the hydrolysis of xylose.At 50 DEG C, tangerine orange thermophilic ascomycete GH3 xylobiase has optimal activity in pH 4.0 to 5.5.This Outside, tangerine orange thermophilic ascomycete GH3 beta-xylanase is in pH 5.0 to 7.0 and 50 DEG C and Ai Mosen ankle section bacterium GH3 xylobiase It is hydrolyzed compared to increasing.The tangerine orange thermophilic ascomycete GH3 beta-xylanase of zytase is supplemented in pH 7.0 and 50 DEG C and supplement wood The Ai Mosen ankle section bacterium GH3 xylobiase of dextranase is compared to increase xylan to the conversion of xylose up to 3.19 times.

As shown in Figure 7, tangerine orange thermophilic ascomycete GH3 beta-xylanase is in 60 DEG C and pH 4.0 to 6.0 and only Penicillium Kind GH10 zytase is compared, and increases the hydrolysis of xylan to xylose significantly.At 60 DEG C, tangerine orange thermophilic ascomycete GH3 β-wood Dextranase has optimal activity in pH 4.0 to 5.0.In addition, tangerine orange thermophilic ascomycete GH3 beta-xylanase is in pH 5.0 to 6.0 Increase hydrolysis compared with Ai Mosen ankle section bacterium GH3 xylobiase with 60 DEG C.Supplement the tangerine orange thermophilic ascomycete GH3 of zytase Beta-xylanase increases wood compared with the Ai Mosen ankle section bacterium GH3 xylobiase of supplement zytase at pH 6.0 and 60 DEG C and gathers Sugar is to the conversion of xylose up to 1.95 times.

The present invention is further described by following number paragraphs:

[1] a kind of with the active isolated polypeptide of xylobiase, it is selected from the group:

(a) polypeptide has at least 60% sequence identity with the mature polypeptide of SEQ ID NO:6 or SEQ ID NO:8； There is at least 65% sequence identity with the mature polypeptide of SEQ ID NO:2；Or with SEQ ID NO:4 or SEQ ID NO:10's Mature polypeptide has at least 75% sequence identity；(b) polypeptide, by polynucleotide encoding, the polynucleotides are at least Hybridize Deng under-high stringency conditions with following: (i) SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, Or the mature polypeptide encoded sequence of SEQ ID NO:9, (ii) SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ The cDNA sequence of ID NO:7, or the overall length complement of (iii) (i) or (ii)；(c) polypeptide, it is described by polynucleotide encoding The mature polypeptide encoded sequence of polynucleotides and SEQ ID NO:5 or SEQ ID NO:7 or its cDNA sequence has at least 60% Sequence identity；There is at least 65% sequence identity with the mature polypeptide encoded sequence of SEQ ID NO:1 or its cDNA sequence； Or have at least 75% sequence same with SEQ ID NO:3 or its cDNA sequence or the mature polypeptide encoded sequence of SEQ ID NO:9 One property；(d) maturation of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10 Polypeptide includes the variant for replacing, lacking and/or being inserted into one or more (such as several) positions；(e) (a), (b), (c) or (d) polypeptide has the active segment of xylobiase.

[2] polypeptide of paragraph 1 has at least 60% with the mature polypeptide of SEQ ID NO:6 or SEQ ID NO:8, until Few 65%, at least 70%, 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 100% sequence identity；With The mature polypeptide of SEQ ID NO:2 is at least 65%, at least 70%, 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 100% sequence identity；Have at least 75% with the mature polypeptide of SEQ ID NO:4 or SEQ ID NO:10, until Few 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 100% sequence identity.

[3] polypeptide of paragraph 1, by polynucleotide encoding, the polynucleotides are in medium-high, high or unusual high stringency Under the conditions of hybridize with following: (i) SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID The mature polypeptide encoded sequence of NO:9, (ii) SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7 CDNA sequence, or the overall length complement of (iii) (i) or (ii).

[4] polypeptide of paragraph 1, by polynucleotide encoding, the polynucleotides and SEQ ID NO:5 or SEQ ID NO: 7 or its cDNA sequence mature polypeptide encoded sequence have at least 60%, at least 65%, at least 70%, 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 100% sequence identity；It is more with the maturation of SEQ ID NO:1 or its cDNA sequence Peptide-coding sequence has at least 65%, at least 70%, 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 100% Sequence identity；Or have at least with SEQ ID NO:3 or its cDNA sequence or the mature polypeptide encoded sequence of SEQ ID NO:9 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 100% sequence identity.

[5] polypeptide of section 1, it includes or group become SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10 or SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ The mature polypeptide of ID NO:10.

[6] polypeptide of section 5, wherein the mature polypeptide is the amino acid 20 to 777 of SEQ ID NO:2, SEQ ID NO:4 Amino acid 20 to 850, the amino acid 20 to 851 of SEQ ID NO:6, the amino acid 22 to 767 or SEQ of SEQ ID NO:8 The amino acid 21 to 800 of ID NO:10.

[7] polypeptide of section 1 is SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or The mature polypeptide of SEQ ID NO:10 includes the variant for replacing, lacking and/or being inserted into one or more (such as several) positions.

[8] polypeptide of any one of section 1-7 is SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID The segment of NO:8 or SEQ ID NO:10, wherein the segment has xylobiase activity.

[9] a kind of composition, it includes the polypeptides of any one of section 1-8.

[10] a kind of isolated polynucleotides, the polypeptide of any one of coding section 1-8.

[11] a kind of nucleic acid construct or expression vector, it includes the polynucleotides of section 10, the polynucleotides can be operated Ground is connected to one or more regulating and controlling sequences, and the regulating and controlling sequence instructs generation of the polypeptide in expressive host.

[12] a kind of recombinant host cell, it includes the polynucleotides of section 10, the polynucleotides are operably connected to One or more regulating and controlling sequences, the regulating and controlling sequence instruct the generation of polypeptide.

[13] a kind of method for the polypeptide for generating any one of section 1-8 comprising: in the item for facilitating the polypeptide generation Cell is cultivated under part, the cell generates the polypeptide with its wild-type form.

[14] method of section 13 further includes recycling the polypeptide.

It is [15] a kind of to generate the method with the polypeptide of xylanase activity comprising: it is generated facilitating the polypeptide Under conditions of cultivate section 12 host cell.

[16] method of section 15 further includes recycling the polypeptide.

[17] a kind of genetically modified plants, plant part or plant cell, the multicore of the polypeptide of any one of encoded section of 1-8 Thuja acid conversion.

It is [18] a kind of to generate the method with the polypeptide of xylanase activity comprising: it is generated facilitating the polypeptide Under conditions of cultivate section 17 genetically modified plants or plant cell.

[19] method of section 18 further includes recycling the polypeptide.

[20] a kind of method for the mutant for generating parental cell, the method includes making any one of coding section 1-8's The polynucleotides of polypeptide inactivate, and mutant is caused to generate the less polypeptide compared with parental cell.

[21] mutant cell generated by the method for section 20.

[22] mutant cell of section 21 further includes the gene for encoding natural or heterologous protein.

[23] it is a kind of generate albumen method comprising: facilitate the albumen generate under conditions of culture section 21 or 22 mutant cell.

[24] method of section 23 further includes recycling the albumen.

[25] a kind of double-stranded inhibitory RNA (dsRNA) molecule, it includes the subsequences of the polynucleotides of section 10, wherein appointing The selection of land dsRNA is siRNA or miRNA molecule.

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

[27] a kind of method for inhibiting that there is expression of the polypeptide of xylanase activity in cell comprising cell is applied With or in cell express section 25 or 26 double-stranded inhibitory RNA (dsRNA) molecule.

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

[29] cell of section 28 further includes the gene for encoding natural or heterologous protein.

[30] it is a kind of generate albumen method comprising: facilitate the albumen generate under conditions of culture section 28 or 29 cell.

[31] method of section 30 further includes recycling the polypeptide.

[32] a kind of isolated polynucleotides, encoded signal peptide, the signal peptide includes or group becomes SEQ ID NO:2 Amino acid 1 to 19, SEQ ID NO:4 amino acid 1 to 19, SEQ ID NO:6 amino acid 1 to 19, SEQ ID NO:8's Amino acid 1 is to the amino acid 1 of 21 or SEQ ID NO:10 to 20.

[33] a kind of nucleic acid construct or expression vector, it includes the codings for the polynucleotides for being operably connected to section 32 The gene of albumen, wherein the gene pairs is external source for the polynucleotides for encoding the signal peptide.

[34] a kind of recombinant host cell, it includes the bases of the coding albumen for the polynucleotides for being operably connected to section 32 Cause, wherein the gene pairs is external source for the polynucleotides for encoding the signal peptide.

[35] a kind of method for generating albumen comprising: culture recombination place under conditions of facilitating the albumen and generating Chief cell, the recombinant host cell include the gene for being operably connected to the coding albumen of the polynucleotides of section 32, wherein The gene is external source for encoding the polynucleotides of the signal peptide.

[36] method of section 35 further includes recycling the albumen.

[37] a kind of degradation of fibers cellulosic material or the technique of the material containing xylan comprising: in the tool of any one of section 1-8 The cellulosic material or material containing xylan are handled with enzymatic compositions in the presence of the polypeptide of xylanase activity.

[38] technique of section 37, wherein the cellulosic material or material containing xylan are by pretreatment.

[39] technique of any one of section 37 or 38, wherein the enzymatic compositions include that one or more (such as several) are selected from The enzyme of the following group: cellulase, the polypeptide with cellulolytic enhancing activity, hemicellulase, esterase, clavacin, laccase, Lignin decomposition enzyme, pectase, peroxidase, protease and swollenin.

[40] technique of section 39, wherein the cellulase is one or more enzymes selected from the group below: endoglucanase, Cellobiohydrolase and β-glucosyl enzym.

[41] technique of section 39, wherein the hemicellulase is one or more enzymes selected from the group below: zytase, second Acyl xylan esterase, feruloyl esterase, arabinofuranosidase, xylosidase and glucuronidase.

[42] technique of any one of section 37-41 further includes recycling the cellulosic material through degrading or material containing xylan.

[43] technique of section 42, wherein the cellulosic material through degrading or material containing xylan are sugar.

[44] technique of section 43, wherein the sugar is selected from the group: glucose, xylose, mannose, galactolipin, and it is Arabic Sugar.

[45] a kind of technique for generating tunning comprising: (a) there is xylanase activity in any one of section 1-8 In the presence of the polypeptide of property, with enzymatic compositions saccharified cellulosic material or material containing xylan；(b) with one or more micro- lifes of fermentation Object cellulosic material of the fermentation through being saccharified or material containing xylan are to generate tunning；(c) tunning is recycled from fermentation.

[46] technique of section 45, wherein the cellulosic material or material containing xylan are pretreated.

[47] technique of section 45 or 46, wherein the enzymatic compositions include that one or more (such as several) are selected from the group below Enzyme: cellulase, the polypeptide with cellulolytic enhancing activity, hemicellulase, esterase, clavacin, laccase, lignin Catabolic enzyme, pectase, peroxidase, protease and swollenin.

[48] technique of section 47, wherein the cellulase is one or more enzymes selected from the group below: endoglucanase, Cellobiohydrolase and β-glucosyl enzym.

[49] technique of section 47, wherein the hemicellulase is one or more enzymes selected from the group below: zytase, second Acyl xylan esterase, feruloyl esterase, arabinofuranosidase, xylosidase and glucuronidase.

[50] technique of any one of section 45-49, wherein step (a) and (b) are saccharified at the same time and carry out simultaneously in fermenting.

[51] technique of any one of section 45-50, wherein tunning is alcohol, alkane, cycloalkane, alkene, amino acid, gas Body, isoprene, ketone, organic acid or polyketide.

[52] a kind of fermentable fiber cellulosic material or the technique of the material containing xylan comprising: with one or more (such as several Kind) fermentative microorganism fermentable fiber cellulosic material or material containing xylan, wherein the cellulosic material or material containing xylan are It is saccharified in the presence of the polypeptide with xylanase activity of any one of section 1-8 with enzymatic compositions.

[53] technique of section 52, wherein the fermentation of the cellulosic material or the material containing xylan generates tunning.

[54] technique of section 53 further includes recycling tunning from fermentation.

[55] technique of section 53 or 54, wherein the tunning be alcohol, alkane, cycloalkane, alkene, amino acid, gas, Isoprene, ketone, organic acid or polyketide.

[56] technique of any one of section 52-55, wherein the cellulosic material or material containing xylan pass through before saccharification Pretreatment.

[57] technique of any one of section 52-56, wherein the enzymatic compositions include that one or more (such as several) are selected from The enzyme of the following group: cellulase, the polypeptide with cellulolytic enhancing activity, hemicellulase, esterase, clavacin, laccase, Lignin decomposition enzyme, pectase, peroxidase, protease and swollenin.

[58] technique of section 57, wherein the cellulase is one or more enzymes selected from the group below: endoglucanase, Cellobiohydrolase and β-glucosyl enzym.

[59] technique of section 57, wherein the hemicellulase is one or more enzymes selected from the group below: zytase, second Acyl xylan esterase, feruloyl esterase, arabinofuranosidase, xylosidase and glucuronidase.

[60] a kind of full nutrient solution formulation or cell culture compositions, it includes the polypeptides of any one of section 1-8.

Invention described and claimed herein is not limited in the range of specific aspect disclosed herein, because this A little aspects are intended as the explanation of the several aspects of the present invention.It is intended to for any equivalent aspect being included within the scope of the present invention. In fact, from the foregoing description, except herein shown and described, the skill of a variety of modifications of the invention for this field It is obvious for art personnel.These modifications, which are also intended to, to be fallen into the range of appended section.It in the case of a conflict, will be with Subject to the disclosure including defining part.

Sequence table

<110>Novozymes Biotech Inc.

<120>polynucleotides with the active polypeptide of xylobiase and the coding polypeptide

<130> 12211-CN-PCD[2]

<160> 24

<170> PatentIn version 3.5

<210> 1

<211> 2402

<212> DNA

<213>thermophilic capital spore (Scytalidium thermophilum)

<400> 1

atgaccaggc tgaccagcat cggagtcctc gcggcctatg cggccggtgc gctcgccggg 60

gtcctccccg actgcagcaa gccgccgctg agcctcaaca agatctgcga ccagaatgcg 120

agcccgcggg agcgcgctga ggcgctcgtg gcagctatga cggttgaaga gaagctggcc 180

aaccttgtca ggtaaggggt ttgactggca acaccagctt ccagtctgca tctgctccgg 240

actaacacca aatcattagc aaagccgctg gcgtcccccg tctgggcttc cctgcgtaca 300

actggtggtc tgaggcgctg catggcgtcg ccggtgcccc tggcatctca ttcgcgcccc 360

ccttcgacta tgccacttca ttccccatgc ccttgatgat ggcggcggcc ttcgacgacg 420

atctcatcga gaagattgcc gtcgtcattg gcaacgagtc cagggcgttt ggtaacaacg 480

accgttctgg catcgactac tggacccccg atgtcaaccc gtacagggac ccgcgttggg 540

gccgcggatc cgagacgccg ggtgaggatg tcctcgtcat caagagatac accaagcacc 600

tgctgcgtgg cttggagggg actagcaaga cgcgccgcat catcgccacc tgcaagcact 660

atgccggcta cgacctcgag tcgtggcagg gaacgacccg ccatgacttc gacgcgcgca 720

tcacacccca ggagcttgcc gagtactaca tgcagccgtt ccagcagtgc gctcgtgact 780

ctaaagtcgg cagcatcatg tgctcttaca accgggtgaa cggagtcccg gcctgcgctt 840

cggactacct cctcaacaag gtcctgaggg agcactggaa ctggactgag agcaacaact 900

acatcaccag cgactgcgag gccgttgccg acgtctctct caaccacaag tatgctccta 960

ccctggccgc tggcactgcg atgtgcttca acaacggcat ggacttgagc tgcgagtatg 1020

agggctcgtc cgatattccg ggcgcgtgga acagcggagc cctcaacgag accatcgttg 1080

accgggccct cgtccgcctc ttccagggtc tcgtccacgg tggctacttc gacggcccta 1140

cgaacgaatg ggcatccctt ggccgtgctg acgtcgacac cccgtacgct cgtcagctgg 1200

ctctgcagtc ggccattgac ggccttgtgc tgctcaagaa cgaccgcaac accctgccct 1260

tgcctctcag gaagggctcc aaggtggcca tgatcggctt ctgggccgat gacgggccga 1320

agctcaaggg catctacagc ggcccttcgc ccttcatcca cacccctgtc tgggctgccc 1380

gtgaagccgg ctacgaggtt gccgttgctc cgggccctat cctgcagaca aatgctgccc 1440

aggacaactg gaccgaggct gccctcgctg cggcgaagaa gtctgattac atcctctact 1500

tcggtggaat cgatacgaat gctgtcaacg agggtgttga ccgtctcacc atcgcttggc 1560

ccgaggccca gctgactctc atcaacaagc ttgccgcctt gcgcaagccg ctcgtcatca 1620

tccagcttgg cgaccagctt gacaacaccc ctctgctcaa gctccagggc gccccgtcga 1680

ttctctgggc caactggccg ggtcaggacg gcggtccggc catcatctcc gtcatcaacg 1740

gcactcacgc tcccgcgggc cgtctccctg tgacccaata cccggccaac tacaccgaga 1800

tcgtccccat gaccgacatg aaccttcgtc ccaacccgag caccggtaac cctggccgga 1860

cctacaagtg gtacccgcac gccgtccagc cgtttggcac tggtctgcac tacaccactt 1920

tcgacgctca cttcgaccgt ccgctgccag cccctggaaa gccgggcacg ccgggcacga 1980

agccggtcgt cctgaagctc aacatccaat ccctcctcgc ggattgcaag aacacttaca 2040

aggacacttg cgcccttccc ccgctcgagg tgcaagtcac caaccgcggg ccgcgccaca 2100

cctcggatta cgtcgccttg gtcttcatta ccgaaacccc aggccccaag cctcaccctc 2160

tcaagagcct ggccacgtat ggcaggttga ggaacatcaa gccgggtcgc accgagcggg 2220

ccaagctcga gtggactgtg gcttcgctgg cgaggcatga caaagatgga aactcgatct 2280

tgtaccctgg tcgttacact ctggttctgg atgtgccgca gaaggatgag gtcgttgtgg 2340

agttgagcgg caaggaagag gtgctggatg tctggcctgt tgaccccaac ctcaagaatt 2400

ga 2402

<210> 2

<211> 777

<212> PRT

<213>thermophilic capital spore (Scytalidium thermophilum)

<400> 2

Met Thr Arg Leu Thr Ser Ile Gly Val Leu Ala Ala Tyr Ala Ala Gly

1 5 10 15

Ala Leu Ala Gly Val Leu Pro Asp Cys Ser Lys Pro Pro Leu Ser Leu

20 25 30

Asn Lys Ile Cys Asp Gln Asn Ala Ser Pro Arg Glu Arg Ala Glu Ala

35 40 45

Leu Val Ala Ala Met Thr Val Glu Glu Lys Leu Ala Asn Leu Val Ser

50 55 60

Lys Ala Ala Gly Val Pro Arg Leu Gly Phe Pro Ala Tyr Asn Trp Trp

65 70 75 80

Ser Glu Ala Leu His Gly Val Ala Gly Ala Pro Gly Ile Ser Phe Ala

85 90 95

Pro Pro Phe Asp Tyr Ala Thr Ser Phe Pro Met Pro Leu Met Met Ala

100 105 110

Ala Ala Phe Asp Asp Asp Leu Ile Glu Lys Ile Ala Val Val Ile Gly

115 120 125

Asn Glu Ser Arg Ala Phe Gly Asn Asn Asp Arg Ser Gly Ile Asp Tyr

130 135 140

Trp Thr Pro Asp Val Asn Pro Tyr Arg Asp Pro Arg Trp Gly Arg Gly

145 150 155 160

Ser Glu Thr Pro Gly Glu Asp Val Leu Val Ile Lys Arg Tyr Thr Lys

165 170 175

His Leu Leu Arg Gly Leu Glu Gly Thr Ser Lys Thr Arg Arg Ile Ile

180 185 190

Ala Thr Cys Lys His Tyr Ala Gly Tyr Asp Leu Glu Ser Trp Gln Gly

195 200 205

Thr Thr Arg His Asp Phe Asp Ala Arg Ile Thr Pro Gln Glu Leu Ala

210 215 220

Glu Tyr Tyr Met Gln Pro Phe Gln Gln Cys Ala Arg Asp Ser Lys Val

225 230 235 240

Gly Ser Ile Met Cys Ser Tyr Asn Arg Val Asn Gly Val Pro Ala Cys

245 250 255

Ala Ser Asp Tyr Leu Leu Asn Lys Val Leu Arg Glu His Trp Asn Trp

260 265 270

Thr Glu Ser Asn Asn Tyr Ile Thr Ser Asp Cys Glu Ala Val Ala Asp

275 280 285

Val Ser Leu Asn His Lys Tyr Ala Pro Thr Leu Ala Ala Gly Thr Ala

290 295 300

Met Cys Phe Asn Asn Gly Met Asp Leu Ser Cys Glu Tyr Glu Gly Ser

305 310 315 320

Ser Asp Ile Pro Gly Ala Trp Asn Ser Gly Ala Leu Asn Glu Thr Ile

325 330 335

Val Asp Arg Ala Leu Val Arg Leu Phe Gln Gly Leu Val His Gly Gly

340 345 350

Tyr Phe Asp Gly Pro Thr Asn Glu Trp Ala Ser Leu Gly Arg Ala Asp

355 360 365

Val Asp Thr Pro Tyr Ala Arg Gln Leu Ala Leu Gln Ser Ala Ile Asp

370 375 380

Gly Leu Val Leu Leu Lys Asn Asp Arg Asn Thr Leu Pro Leu Pro Leu

385 390 395 400

Arg Lys Gly Ser Lys Val Ala Met Ile Gly Phe Trp Ala Asp Asp Gly

405 410 415

Pro Lys Leu Lys Gly Ile Tyr Ser Gly Pro Ser Pro Phe Ile His Thr

420 425 430

Pro Val Trp Ala Ala Arg Glu Ala Gly Tyr Glu Val Ala Val Ala Pro

435 440 445

Gly Pro Ile Leu Gln Thr Asn Ala Ala Gln Asp Asn Trp Thr Glu Ala

450 455 460

Ala Leu Ala Ala Ala Lys Lys Ser Asp Tyr Ile Leu Tyr Phe Gly Gly

465 470 475 480

Ile Asp Thr Asn Ala Val Asn Glu Gly Val Asp Arg Leu Thr Ile Ala

485 490 495

Trp Pro Glu Ala Gln Leu Thr Leu Ile Asn Lys Leu Ala Ala Leu Arg

500 505 510

Lys Pro Leu Val Ile Ile Gln Leu Gly Asp Gln Leu Asp Asn Thr Pro

515 520 525

Leu Leu Lys Leu Gln Gly Ala Pro Ser Ile Leu Trp Ala Asn Trp Pro

530 535 540

Gly Gln Asp Gly Gly Pro Ala Ile Ile Ser Val Ile Asn Gly Thr His

545 550 555 560

Ala Pro Ala Gly Arg Leu Pro Val Thr Gln Tyr Pro Ala Asn Tyr Thr

565 570 575

Glu Ile Val Pro Met Thr Asp Met Asn Leu Arg Pro Asn Pro Ser Thr

580 585 590

Gly Asn Pro Gly Arg Thr Tyr Lys Trp Tyr Pro His Ala Val Gln Pro

595 600 605

Phe Gly Thr Gly Leu His Tyr Thr Thr Phe Asp Ala His Phe Asp Arg

610 615 620

Pro Leu Pro Ala Pro Gly Lys Pro Gly Thr Pro Gly Thr Lys Pro Val

625 630 635 640

Val Leu Lys Leu Asn Ile Gln Ser Leu Leu Ala Asp Cys Lys Asn Thr

645 650 655

Tyr Lys Asp Thr Cys Ala Leu Pro Pro Leu Glu Val Gln Val Thr Asn

660 665 670

Arg Gly Pro Arg His Thr Ser Asp Tyr Val Ala Leu Val Phe Ile Thr

675 680 685

Glu Thr Pro Gly Pro Lys Pro His Pro Leu Lys Ser Leu Ala Thr Tyr

690 695 700

Gly Arg Leu Arg Asn Ile Lys Pro Gly Arg Thr Glu Arg Ala Lys Leu

705 710 715 720

Glu Trp Thr Val Ala Ser Leu Ala Arg His Asp Lys Asp Gly Asn Ser

725 730 735

Ile Leu Tyr Pro Gly Arg Tyr Thr Leu Val Leu Asp Val Pro Gln Lys

740 745 750

Asp Glu Val Val Val Glu Leu Ser Gly Lys Glu Glu Val Leu Asp Val

755 760 765

Trp Pro Val Asp Pro Asn Leu Lys Asn

770 775

<210> 3

<211> 2671

<212> DNA

<213>thermophilic capital spore (Scytalidium thermophilum)

<400> 3

atgaaggccc tgactagaag gctggcgagc tttctcgcca tcaccggggt tgtcggcttg 60

gagtatccga attgcatcaa cggacctttg gccagtaaca cggtatgcga tgtgaccgcg 120

gcgccggcgg atcgtgccgc ggccttggtc aaggctatga cggtggcgga gaagctggac 180

aacctcgttg agtatgcttt aatattgcat catccttgga gtttttggtg gcttgtctga 240

catggattga cacgacaagc atgagcaaag gagctccccg actgggcctc ccgccgtatg 300

cctggtggaa cgaagcactt catggcgttg ccctatctcc tggggtcact ttcaacccat 360

tagggtctga cttctccaat gcgacctcgt ttgcaaacac catcaccctg gcagccgcct 420

tcgatgacca cctggtgtac caggtggcca gcgcgatcag caccgaggct cgggcttttg 480

ccaacgcagg gcttgccgga ctcgactact ggtccccgaa cattaacccg tacaaggacc 540

cgagatgggg aagagggcat gaggtgacac ctcgtcagga gtccttgtat actgttgaac 600

gaggcaggct aacaattcca actagacccc aggcgaagac cctgttcgca tcaagggcta 660

tgtccgggcg ttcctcgccg gcttggaggg cgacggtccc gtccggaagg tcatagccac 720

atgcaagcat tacgccgcct acgatctgga acggtggcag ggcctcacac gacaccagtt 780

caacgccatc gtgtcgctcc aagacctgtc cgagtactac atgcctccgt tccagcaatg 840

tgccagggac tccaatgtcg gctccatcat gtgctcatac aacgctgtca acggcactcc 900

tgcctgtgcc aatacctatt tgatggacga catcctgcgg aagcattgga actggacagg 960

ccataacaac tacatcacca gcgattgcta cgccattcag gtaacacgcg gtccatgagt 1020

cccattactc ttctctggca gctagactta ttctccctat tagaacttcc tccccagttg 1080

gcgcaactac tcccaatctc ccgcggaggc ggtcgctgct gctctcaatg ctggcaccga 1140

caccatctgc gaagttgccg ggtggctacc ctacgccgac gtcgttggcg cctacgacca 1200

aggtctcctc tccgaagccg tcatcgaccg agccctgcgc cgtctctacg aggggctcgt 1260

ccgggtaggc tactttgacc ctccaacctc ctcctcccca gcggccgcct accgctccct 1320

gtccgccgcc aacgtaagta ccaccgagca ccagctcctc gctctccagt ccgctctgga 1380

cgggatggtc ctcctcaaga acctcaacca aaccctcccc ttacgcgacg acgcaatccc 1440

ggtcccccct ttcaccacca ccgctgccca agtagcaatc atcggccact gggctgctcc 1500

caacgcccac atgctcggcg gcttcagcgg cattccgccc tacctcctct ccccactcca 1560

cgccgctgag ttgctccaac tcaactacac ctacgccccc ggtgcacccg tcgtgatcac 1620

caacaccagc cccgacacac ccgacacctg gaccacccca gccctcgccg ccgcctcttc 1680

ggcctcgtac atcctctact tcggcggctc cgacctctcc ctcgcgcgcg aagatctcga 1740

cagggacagc atctcctggc cgcgcgccga gctcgagctc atcaccaccc tctcctccct 1800

cgggaagcct gtcatcgtga tccagctcgg cgaccagctc gacaccgcgc cgctgctctc 1860

caacccgaat atctccgcca ttctctgggc cggatacccc ggccaagcgg gcgggttggc 1920

cgccatgtac accctgctcg gcatctcctc ccctgccggg cggctgccgg taacccaata 1980

cgccgaggag tacaccaagc gggtagcact gacagacatg cgcctgcgcc cggacgcgca 2040

aaaccctttc gatctcagca cgccggtcca tctccggccc aacactacca gctcgttccc 2100

tggcaggaca taccgctggc tcccgcaccc ctcctcctcc tcttcctcat catcatcatc 2160

cctcgtaacc ctccccttcg gtcacggcct ccactacgcc ccccttcgcg ccaaattcgg 2220

catcttcacc accctctccc tcaccaccgc cgacctcctc tcctcctgca acttgactct 2280

ccacaacaac caccccgacc tctgcccttt ccccctccaa gtctccgtct ggacgaccaa 2340

cctctccccg tcaaacggag gtttcacgac cgactatgtc gccctcgttt ttgtcaccgg 2400

cgaatatggc cccagaccct accccgtcaa aactctcgtg gggtacactc gcctgcgagc 2460

catcgggccg ggggagacca aggcggcctt ggtggacatc aagctcgggg atttggcgcg 2520

gatggacgag gccgggaatc gcgtcttgta tcctgggcgg tacaagttta tgttggatgt 2580

cggggaagac ggaggcgggg tggacgaggt ggagattgaa gtgacgggga aggaggtggt 2640

gttggcgttt tggcctcagc caaaggggtg a 2671

<210> 4

<211> 825

<212> PRT

<213>thermophilic capital spore (Scytalidium thermophilum)

<400> 4

Met Lys Ala Leu Thr Arg Arg Leu Ala Ser Phe Leu Ala Ile Thr Gly

1 5 10 15

Val Val Gly Leu Glu Tyr Pro Asn Cys Ile Asn Gly Pro Leu Ala Ser

20 25 30

Asn Thr Val Cys Asp Val Thr Ala Ala Pro Ala Asp Arg Ala Ala Ala

35 40 45

Leu Val Lys Ala Met Thr Val Ala Glu Lys Leu Asp Asn Leu Val Asp

50 55 60

Met Ser Lys Gly Ala Pro Arg Leu Gly Leu Pro Pro Tyr Ala Trp Trp

65 70 75 80

Asn Glu Ala Leu His Gly Val Ala Leu Ser Pro Gly Val Thr Phe Asn

85 90 95

Pro Leu Gly Ser Asp Phe Ser Asn Ala Thr Ser Phe Ala Asn Thr Ile

100 105 110

Thr Leu Ala Ala Ala Phe Asp Asp His Leu Val Tyr Gln Val Ala Ser

115 120 125

Ala Ile Ser Thr Glu Ala Arg Ala Phe Ala Asn Ala Gly Leu Ala Gly

130 135 140

Leu Asp Tyr Trp Ser Pro Asn Ile Asn Pro Tyr Lys Asp Pro Arg Trp

145 150 155 160

Gly Arg Gly His Glu Thr Pro Gly Glu Asp Pro Val Arg Ile Lys Gly

165 170 175

Tyr Val Arg Ala Phe Leu Ala Gly Leu Glu Gly Asp Gly Pro Val Arg

180 185 190

Lys Val Ile Ala Thr Cys Lys His Tyr Ala Ala Tyr Asp Leu Glu Arg

195 200 205

Trp Gln Gly Leu Thr Arg His Gln Phe Asn Ala Ile Val Ser Leu Gln

210 215 220

Asp Leu Ser Glu Tyr Tyr Met Pro Pro Phe Gln Gln Cys Ala Arg Asp

225 230 235 240

Ser Asn Val Gly Ser Ile Met Cys Ser Tyr Asn Ala Val Asn Gly Thr

245 250 255

Pro Ala Cys Ala Asn Thr Tyr Leu Met Asp Asp Ile Leu Arg Lys His

260 265 270

Trp Asn Trp Thr Gly His Asn Asn Tyr Ile Thr Ser Asp Cys Tyr Ala

275 280 285

Ile Gln Asn Phe Leu Pro Ser Trp Arg Asn Tyr Ser Gln Ser Pro Ala

290 295 300

Glu Ala Val Ala Ala Ala Leu Asn Ala Gly Thr Asp Thr Ile Cys Glu

305 310 315 320

Val Ala Gly Trp Leu Pro Tyr Ala Asp Val Val Gly Ala Tyr Asp Gln

325 330 335

Gly Leu Leu Ser Glu Ala Val Ile Asp Arg Ala Leu Arg Arg Leu Tyr

340 345 350

Glu Gly Leu Val Arg Val Gly Tyr Phe Asp Pro Pro Thr Ser Ser Ser

355 360 365

Pro Ala Ala Ala Tyr Arg Ser Leu Ser Ala Ala Asn Val Ser Thr Thr

370 375 380

Glu His Gln Leu Leu Ala Leu Gln Ser Ala Leu Asp Gly Met Val Leu

385 390 395 400

Leu Lys Asn Leu Asn Gln Thr Leu Pro Leu Arg Asp Asp Ala Ile Pro

405 410 415

Val Pro Pro Phe Thr Thr Thr Ala Ala Gln Val Ala Ile Ile Gly His

420 425 430

Trp Ala Ala Pro Asn Ala His Met Leu Gly Gly Phe Ser Gly Ile Pro

435 440 445

Pro Tyr Leu Leu Ser Pro Leu His Ala Ala Glu Leu Leu Gln Leu Asn

450 455 460

Tyr Thr Tyr Ala Pro Gly Ala Pro Val Val Ile Thr Asn Thr Ser Pro

465 470 475 480

Asp Thr Pro Asp Thr Trp Thr Thr Pro Ala Leu Ala Ala Ala Ser Ser

485 490 495

Ala Ser Tyr Ile Leu Tyr Phe Gly Gly Ser Asp Leu Ser Leu Ala Arg

500 505 510

Glu Asp Leu Asp Arg Asp Ser Ile Ser Trp Pro Arg Ala Glu Leu Glu

515 520 525

Leu Ile Thr Thr Leu Ser Ser Leu Gly Lys Pro Val Ile Val Ile Gln

530 535 540

Leu Gly Asp Gln Leu Asp Thr Ala Pro Leu Leu Ser Asn Pro Asn Ile

545 550 555 560

Ser Ala Ile Leu Trp Ala Gly Tyr Pro Gly Gln Ala Gly Gly Leu Ala

565 570 575

Ala Met Tyr Thr Leu Leu Gly Ile Ser Ser Pro Ala Gly Arg Leu Pro

580 585 590

Val Thr Gln Tyr Ala Glu Glu Tyr Thr Lys Arg Val Ala Leu Thr Asp

595 600 605

Met Arg Leu Arg Pro Asp Ala Gln Asn Pro Phe Asp Leu Ser Thr Pro

610 615 620

Val His Leu Arg Pro Asn Thr Thr Ser Ser Phe Pro Gly Arg Thr Tyr

625 630 635 640

Arg Trp Leu Pro His Pro Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser

645 650 655

Leu Val Thr Leu Pro Phe Gly His Gly Leu His Tyr Ala Pro Leu Arg

660 665 670

Ala Lys Phe Gly Ile Phe Thr Thr Leu Ser Leu Thr Thr Ala Asp Leu

675 680 685

Leu Ser Ser Cys Asn Leu Thr Leu His Asn Asn His Pro Asp Leu Cys

690 695 700

Pro Phe Pro Leu Gln Val Ser Val Trp Thr Thr Asn Leu Ser Pro Ser

705 710 715 720

Asn Gly Gly Phe Thr Thr Asp Tyr Val Ala Leu Val Phe Val Thr Gly

725 730 735

Glu Tyr Gly Pro Arg Pro Tyr Pro Val Lys Thr Leu Val Gly Tyr Thr

740 745 750

Arg Leu Arg Ala Ile Gly Pro Gly Glu Thr Lys Ala Ala Leu Val Asp

755 760 765

Ile Lys Leu Gly Asp Leu Ala Arg Met Asp Glu Ala Gly Asn Arg Val

770 775 780

Leu Tyr Pro Gly Arg Tyr Lys Phe Met Leu Asp Val Gly Glu Asp Gly

785 790 795 800

Gly Gly Val Asp Glu Val Glu Ile Glu Val Thr Gly Lys Glu Val Val

805 810 815

Leu Ala Phe Trp Pro Gln Pro Lys Gly

820 825

<210> 5

<211> 2832

<212> DNA

<213>penicillium oxalicum

<400> 5

atgctggccc tggcatcaat ggcaatgctc accagcgttt acgcaagaag tgaagctgcc 60

accagctgtg aagcttccac aaagtaccta gggtgctatt ccgatccgaa ggtcacgatt 120

cttacctcgg ccaagctgtc cacaatcgct atgacgccgc agttctgcgc tgactggtgc 180

ggccagcgag ggttctcaca cagcggcatc gaatttggga cgtgagtgtt cccctgcgcc 240

acgctaacaa tatatcgtaa ctcacccctg gtaaatgaag aattgtttct cacacttgtc 300

taggcaatgc ttctgtggtg cagaacctaa cttgagtgat gccactcgaa cagacgacgg 360

cgattgcaac acgccctgcc ccttggaacc gtccagttcg tgcggcgcaa cctacgtgta 420

cgtcaccgaa tcacttgccc ctcccttgct ataggagcca acaatcgcta acctatgccg 480

tcttcaccgt tgtgcagtat gtcgttatat caaattataa atccccaagg agggaacccc 540

gacacgcgct ttgtgcctgc ctgccaacgg caacctttga gcagccaccc agtgtgcaat 600

actgccctta gtattcccga gagagtcaag tctctggttg gttcactgac ccaggaagaa 660

aaaatcttga acttggtgga tgctgcagcg ggatcagaac gtcttggttt gccatcttat 720

gaatggtgga gtgaggcaac tcatggtgtt gggtccgcgc ctggcgtgca atttacacgt 780

gcccccgcca atttcagttc ggccacgagc tttcctgccc cgattctcac agcagcttcc 840

tttgacgatg cgctgtttca cgatattggc gaagttacag gaaaagaggg acgagctttt 900

gccaacaacg gcttctccgg attcgacttc tgggctccca acattaacgc cttcagggac 960

ccccgctggg gaagaggcca ggagacgccc ggcgaagatg tgctggtggc ccaaaactac 1020

gtccgtaagt tcgtccaggg gctccagggt gatgacccca aggagaagca agtgatcgcc 1080

acatgcaaac attttgcggt ctatgacatc gagactgatc gatatggcaa caatttcaac 1140

cccacacaac aagaactcgg ggaatacttt ttgccaccat tcaagacatg tgctcgagat 1200

agcggcgtgg gaagtgtgat gtgcgcctat aatgccgtgt ttggtgtccc cgcctgtgca 1260

agcgaatatc tgctcggcga tgttctgaga gatcattgga acttcacggc cgattacaac 1320

tatgtcgtct cggattgcac tgcggtgacg gaaatttggc agagccacaa ctttaccaat 1380

tctgctgagg aggcggcttc ggtcgctctc aattccgggg tggatttgga atgtggaaac 1440

tcatacctga aactcaatga atcgctggcc tccaaccaca catctatcga aactttggac 1500

cgatccttgc aaaggctgta ctcggccctt ttcacggttg gtttcttcga tggaggaaag 1560

tacacggacc ttgactacgc ggatgtttcc acgccaagtg cgcaaatctt ggcctatgcc 1620

gccgcggtgg aaggaatgac attgctcaaa aatgacggtc tgcttcctct tggtacaaag 1680

caccatttca agactgtcgc tgtcattgga ccttatggca atgccacgac tcagatgcaa 1740

ggagattact cgggcatggc ttctcacatt gtaagtcctc tagaggcgtt tcagagcgca 1800

agtcagtggg aagtcaatta cgcgcaaggt accactatca ccaacgagac gagtactgga 1860

tttggcgaag ccctgcgcgc ggcggaaaag agcgatttga tcgtgtttct cggaggcatt 1920

gacaattctc tcgagaatga gggtcttgat cgcaaatctc tcgcttggcc ccagaatcaa 1980

atggacctca tgacagagct ggcaaagacc aagaagccaa tgatcgtggt ccagttcggt 2040

gggggtcaag tcgacgacag cgcgcttctt caaaacgatc atgtgaatgc gatcgtttgg 2100

gcgggatacc ccagtcaaag cggtggcact gctcttatgg atattcttca gggcaaagtg 2160

tcgattgcgg gtcgcctacc tgtcacccag tatccagcca gctatgcgga tcaagttggt 2220

ctttgggatc tcagtcttcg gcccaacgcg aatacttcat atccaggacg gacatataga 2280

tggtatactg gcgagccggt ctttccattc ggctatgggc tgcactacac caaatttgaa 2340

tatgagtggg aagagggcct gcacaagcaa tacaatattc aagagcttgt tggatcatgc 2400

aaaagagagt cgggtggctc tattaatgat gtcacgccct ttgcttctgt caaagtacgc 2460

gtccgaaacg tgggtcacga gaattctgac tatgtcagcc tgcttttcct ctcgagtacc 2520

gatgcaggac ccgcacctca tccctccaag acactggtcg cctactctcg ccttcatggc 2580

atcaaaaaga accatgcgca gactaccacc ctaaatttga gcctgggctc tctggccaga 2640

gctgatgaga aaggaagtct agtaatctac ccgggccatt acaagcttgt cctggacgtc 2700

gatgaaagtc ttgcgcttga attctcatta cacggagacc cagaagtgat tgagactctt 2760

cccgagccgc aggagcagta tgactacacg gtcccggttc atattcagcc gccaagtacc 2820

gggccactgt ga 2832

<210> 6

<211> 851

<212> PRT

<213>penicillium oxalicum

<400> 6

Met Leu Ala Leu Ala Ser Met Ala Met Leu Thr Ser Val Tyr Ala Arg

1 5 10 15

Ser Glu Ala Ala Thr Ser Cys Glu Ala Ser Thr Lys Tyr Leu Gly Cys

20 25 30

Tyr Ser Asp Pro Lys Val Thr Ile Leu Thr Ser Ala Lys Leu Ser Thr

35 40 45

Ile Ala Met Thr Pro Gln Phe Cys Ala Asp Trp Cys Gly Gln Arg Gly

50 55 60

Phe Ser His Ser Gly Ile Glu Phe Gly Thr Gln Cys Phe Cys Gly Ala

65 70 75 80

Glu Pro Asn Leu Ser Asp Ala Thr Arg Thr Asp Asp Gly Asp Cys Asn

85 90 95

Thr Pro Cys Pro Leu Glu Pro Ser Ser Ser Cys Gly Ala Thr Tyr Val

100 105 110

Ile Pro Glu Arg Val Lys Ser Leu Val Gly Ser Leu Thr Gln Glu Glu

115 120 125

Lys Ile Leu Asn Leu Val Asp Ala Ala Ala Gly Ser Glu Arg Leu Gly

130 135 140

Leu Pro Ser Tyr Glu Trp Trp Ser Glu Ala Thr His Gly Val Gly Ser

145 150 155 160

Ala Pro Gly Val Gln Phe Thr Arg Ala Pro Ala Asn Phe Ser Ser Ala

165 170 175

Thr Ser Phe Pro Ala Pro Ile Leu Thr Ala Ala Ser Phe Asp Asp Ala

180 185 190

Leu Phe His Asp Ile Gly Glu Val Thr Gly Lys Glu Gly Arg Ala Phe

195 200 205

Ala Asn Asn Gly Phe Ser Gly Phe Asp Phe Trp Ala Pro Asn Ile Asn

210 215 220

Ala Phe Arg Asp Pro Arg Trp Gly Arg Gly Gln Glu Thr Pro Gly Glu

225 230 235 240

Asp Val Leu Val Ala Gln Asn Tyr Val Arg Lys Phe Val Gln Gly Leu

245 250 255

Gln Gly Asp Asp Pro Lys Glu Lys Gln Val Ile Ala Thr Cys Lys His

260 265 270

Phe Ala Val Tyr Asp Ile Glu Thr Asp Arg Tyr Gly Asn Asn Phe Asn

275 280 285

Pro Thr Gln Gln Glu Leu Gly Glu Tyr Phe Leu Pro Pro Phe Lys Thr

290 295 300

Cys Ala Arg Asp Ser Gly Val Gly Ser Val Met Cys Ala Tyr Asn Ala

305 310 315 320

Val Phe Gly Val Pro Ala Cys Ala Ser Glu Tyr Leu Leu Gly Asp Val

325 330 335

Leu Arg Asp His Trp Asn Phe Thr Ala Asp Tyr Asn Tyr Val Val Ser

340 345 350

Asp Cys Thr Ala Val Thr Glu Ile Trp Gln Ser His Asn Phe Thr Asn

355 360 365

Ser Ala Glu Glu Ala Ala Ser Val Ala Leu Asn Ser Gly Val Asp Leu

370 375 380

Glu Cys Gly Asn Ser Tyr Leu Lys Leu Asn Glu Ser Leu Ala Ser Asn

385 390 395 400

His Thr Ser Ile Glu Thr Leu Asp Arg Ser Leu Gln Arg Leu Tyr Ser

405 410 415

Ala Leu Phe Thr Val Gly Phe Phe Asp Gly Gly Lys Tyr Thr Asp Leu

420 425 430

Asp Tyr Ala Asp Val Ser Thr Pro Ser Ala Gln Ile Leu Ala Tyr Ala

435 440 445

Ala Ala Val Glu Gly Met Thr Leu Leu Lys Asn Asp Gly Leu Leu Pro

450 455 460

Leu Gly Thr Lys His His Phe Lys Thr Val Ala Val Ile Gly Pro Tyr

465 470 475 480

Gly Asn Ala Thr Thr Gln Met Gln Gly Asp Tyr Ser Gly Met Ala Ser

485 490 495

His Ile Val Ser Pro Leu Glu Ala Phe Gln Ser Ala Ser Gln Trp Glu

500 505 510

Val Asn Tyr Ala Gln Gly Thr Thr Ile Thr Asn Glu Thr Ser Thr Gly

515 520 525

Phe Gly Glu Ala Leu Arg Ala Ala Glu Lys Ser Asp Leu Ile Val Phe

530 535 540

Leu Gly Gly Ile Asp Asn Ser Leu Glu Asn Glu Gly Leu Asp Arg Lys

545 550 555 560

Ser Leu Ala Trp Pro Gln Asn Gln Met Asp Leu Met Thr Glu Leu Ala

565 570 575

Lys Thr Lys Lys Pro Met Ile Val Val Gln Phe Gly Gly Gly Gln Val

580 585 590

Asp Asp Ser Ala Leu Leu Gln Asn Asp His Val Asn Ala Ile Val Trp

595 600 605

Ala Gly Tyr Pro Ser Gln Ser Gly Gly Thr Ala Leu Met Asp Ile Leu

610 615 620

Gln Gly Lys Val Ser Ile Ala Gly Arg Leu Pro Val Thr Gln Tyr Pro

625 630 635 640

Ala Ser Tyr Ala Asp Gln Val Gly Leu Trp Asp Leu Ser Leu Arg Pro

645 650 655

Asn Ala Asn Thr Ser Tyr Pro Gly Arg Thr Tyr Arg Trp Tyr Thr Gly

660 665 670

Glu Pro Val Phe Pro Phe Gly Tyr Gly Leu His Tyr Thr Lys Phe Glu

675 680 685

Tyr Glu Trp Glu Glu Gly Leu His Lys Gln Tyr Asn Ile Gln Glu Leu

690 695 700

Val Gly Ser Cys Lys Arg Glu Ser Gly Gly Ser Ile Asn Asp Val Thr

705 710 715 720

Pro Phe Ala Ser Val Lys Val Arg Val Arg Asn Val Gly His Glu Asn

725 730 735

Ser Asp Tyr Val Ser Leu Leu Phe Leu Ser Ser Thr Asp Ala Gly Pro

740 745 750

Ala Pro His Pro Ser Lys Thr Leu Val Ala Tyr Ser Arg Leu His Gly

755 760 765

Ile Lys Lys Asn His Ala Gln Thr Thr Thr Leu Asn Leu Ser Leu Gly

770 775 780

Ser Leu Ala Arg Ala Asp Glu Lys Gly Ser Leu Val Ile Tyr Pro Gly

785 790 795 800

His Tyr Lys Leu Val Leu Asp Val Asp Glu Ser Leu Ala Leu Glu Phe

805 810 815

Ser Leu His Gly Asp Pro Glu Val Ile Glu Thr Leu Pro Glu Pro Gln

820 825 830

Glu Gln Tyr Asp Tyr Thr Val Pro Val His Ile Gln Pro Pro Ser Thr

835 840 845

Gly Pro Leu

850

<210> 7

<211> 2624

<212> DNA

<213>Rhizomucor pusillus

<400> 7

atggcgttta tcaagcagag cgttctactc tgcttgcttg gtctgaatgc attgatgcaa 60

gctcaagaat acgggacact ccgtcctgaa gaatacgatc ggcctggagc cattgaccct 120

gatatcaagg aaatggtttc acgcatgaca ttgcctgaga aaattggtca aatgacacaa 180

ttggatcaag ccatggtgct gcagcctgac ggtactctga acaagactgc agttgagtac 240

tatgcgcaga aatactatgt cggatcctat ctcaaccaac tggcccggta agttggggtt 300

cggattatgc aagtcaaacg ttcttacttg aagattagcg atggccgcaa ccttgatcac 360

aaggagtacg cggacaggat cgaagagata cagcagataa caatggctgc aaactctaca 420

tttaaaatac caattattta cgggtacgtg tctcaagcga agataacatt ttccgcgcta 480

atactctctc ttttgtatca ggttggatca cattcacggt gcgcattatg tagcaaagtg 540

taaggttcct ttttactttt tttcttgacc tctttgaata cttagactgg agaacagcta 600

ccttgttccc gcagggtatc aacattgctg caacatttaa tcccaagctg gcatacgaag 660

cagcttccat tacagccaga gacactcgtg cggcgaatgt acactggtag gcaaaggaaa 720

agaaggccag ggtatccttt tttgacggat tcgaaatgtg tttaggactt ttgctcccgt 780

gctcgatatt cccgttacaa agcaatgggc gcgtgtgtac gagaactttg gagaagatcc 840

ttacctttcc agtgtcatgg gagtcgctgc cattcgaggc taccagggca agtacaagtc 900

agacagaacc aaagtggctg cctcgatgaa gcactttatt gcttacggtg caccgtacag 960

cggtcaggac cgtgacacaa cggtagcctc cgaccgcatg atttacgata cttttgtgcc 1020

tggtttcaag gctgcaattg atgctggtgt ggcgacagct atggaaagct acattgatgt 1080

caatggtgaa cctgtagttg catcccacaa gtatctgcag cagctcttgc gcgagcagct 1140

gggattccaa ggcatgcttg tgacggattg ggctgaaatt gagaatttgt acactacaca 1200

caaggtcgct gccactcaca aggatgcagt ccggctatct atcagcgaca cgagtgtaga 1260

catgtccatg gtaccaagtg acgttatttt tgccgactcg ttgcacgacc ttgtcaagga 1320

gggcaccatt ccagagtctc gcgtcaatga gtcgactgag cgtctgttgc agcttaaaaa 1380

agatcttggt ttgctagagc ccgatggctg gaaagcaaac cgtgccctgc aagaaatggt 1440

cggacggccc gaggatgtgg aggtttctag acaagcggca cgcgagtcac ttgtgctact 1500

caagaatgac aatggtgtgc taccatttaa tgagtctgtg cgccgcatcc ttattgttgg 1560

gccgactgct aatgacctta gtcacctggc tggcggctgg actataaact ggcaagggtg 1620

agttgcaggc agctcgtgga gcgagtgaga aggagagaga gagtgaaaga taaaagggaa 1680

gcaaacagcg agaaaagaca ataactcatt tattgaatat ttagagctac cgaagatcga 1740

tggcaaggcc gcatcagcga cgaccaattt tatgcaaacg gtgtgaccat tgctaacgga 1800

cttcgttcgg ctgcccctca gggcacacag attgactaca ttgaaggatt tgacgtctat 1860

ggcaatgaca cgggcctgga caaggtgttg caagctgcaa acaattacga tgtcattgtg 1920

gcggctgtcg gcgaacacgt gtacgcggaa gcaccgggtg atatccacga tattagactt 1980

gcacagggcc agattgacgg tgtcaaggcc ttggcagcga caaacaaacc tgtcgtgaca 2040

gtgcttgtcg agggcagacc gcgcgtgcta gatagtatcc ctgatcactc acaagctatc 2100

cttcacgcgc ttttgcctgg accttggggt ggtcaagcta tcggcgaagt gctttttggt 2160

ctcgttaatc cctctggcaa gctgccatac acatatccaa agaatgcagg tgacatggca 2220

ctcaattatt ggcgtcaagc caacgatgtc tgggaccctc tctacgagtt tggccacggc 2280

ttgagctatt cgcaattcaa ctatagccaa ctgacagcag acgacaagac tatctctagc 2340

gacaagccag ttaccgtatc tgtccaagta acaaacaatg gtcccatgga cggcatggaa 2400

agcgtcttga tgtttatcca gcagcctgtc cgacgagtga caccgcctgc caagctgcta 2460

aaggggttca aaaagctcca gcttgcaaat ggagagacgg ccacagtcaa cttcgaagtt 2520

agcgcagacg cgttcaaata tactggtttg gatggcgtcc ctggtggctc cctggatgca 2580

ggcccagtca aggtgatgat tggcgaccag gaaattgacc ttga 2624

<210> 8

<211> 767

<212> PRT

<213>Rhizomucor pusillus

<400> 8

Met Ala Phe Ile Lys Gln Ser Val Leu Leu Cys Leu Leu Gly Leu Asn

1 5 10 15

Ala Leu Met Gln Ala Gln Glu Tyr Gly Thr Leu Arg Pro Glu Glu Tyr

20 25 30

Asp Arg Pro Gly Ala Ile Asp Pro Asp Ile Lys Glu Met Val Ser Arg

35 40 45

Met Thr Leu Pro Glu Lys Ile Gly Gln Met Thr Gln Leu Asp Gln Ala

50 55 60

Met Val Leu Gln Pro Asp Gly Thr Leu Asn Lys Thr Ala Val Glu Tyr

65 70 75 80

Tyr Ala Gln Lys Tyr Tyr Val Gly Ser Tyr Leu Asn Gln Leu Ala Arg

85 90 95

Asp Gly Arg Asn Leu Asp His Lys Glu Tyr Ala Asp Arg Ile Glu Glu

100 105 110

Ile Gln Gln Ile Thr Met Ala Ala Asn Ser Thr Phe Lys Ile Pro Ile

115 120 125

Ile Tyr Gly Leu Asp His Ile His Gly Ala His Tyr Val Ala Lys Ser

130 135 140

Thr Leu Phe Pro Gln Gly Ile Asn Ile Ala Ala Thr Phe Asn Pro Lys

145 150 155 160

Leu Ala Tyr Glu Ala Ala Ser Ile Thr Ala Arg Asp Thr Arg Ala Ala

165 170 175

Asn Val His Trp Thr Phe Ala Pro Val Leu Asp Ile Pro Val Thr Lys

180 185 190

Gln Trp Ala Arg Val Tyr Glu Asn Phe Gly Glu Asp Pro Tyr Leu Ser

195 200 205

Ser Val Met Gly Val Ala Ala Ile Arg Gly Tyr Gln Gly Lys Tyr Lys

210 215 220

Ser Asp Arg Thr Lys Val Ala Ala Ser Met Lys His Phe Ile Ala Tyr

225 230 235 240

Gly Ala Pro Tyr Ser Gly Gln Asp Arg Asp Thr Thr Val Ala Ser Asp

245 250 255

Arg Met Ile Tyr Asp Thr Phe Val Pro Gly Phe Lys Ala Ala Ile Asp

260 265 270

Ala Gly Val Ala Thr Ala Met Glu Ser Tyr Ile Asp Val Asn Gly Glu

275 280 285

Pro Val Val Ala Ser His Lys Tyr Leu Gln Gln Leu Leu Arg Glu Gln

290 295 300

Leu Gly Phe Gln Gly Met Leu Val Thr Asp Trp Ala Glu Ile Glu Asn

305 310 315 320

Leu Tyr Thr Thr His Lys Val Ala Ala Thr His Lys Asp Ala Val Arg

325 330 335

Leu Ser Ile Ser Asp Thr Ser Val Asp Met Ser Met Val Pro Ser Asp

340 345 350

Val Ile Phe Ala Asp Ser Leu His Asp Leu Val Lys Glu Gly Thr Ile

355 360 365

Pro Glu Ser Arg Val Asn Glu Ser Thr Glu Arg Leu Leu Gln Leu Lys

370 375 380

Lys Asp Leu Gly Leu Leu Glu Pro Asp Gly Trp Lys Ala Asn Arg Ala

385 390 395 400

Leu Gln Glu Met Val Gly Arg Pro Glu Asp Val Glu Val Ser Arg Gln

405 410 415

Ala Ala Arg Glu Ser Leu Val Leu Leu Lys Asn Asp Asn Gly Val Leu

420 425 430

Pro Phe Asn Glu Ser Val Arg Arg Ile Leu Ile Val Gly Pro Thr Ala

435 440 445

Asn Asp Leu Ser His Leu Ala Gly Gly Trp Thr Ile Asn Trp Gln Gly

450 455 460

Ala Thr Glu Asp Arg Trp Gln Gly Arg Ile Ser Asp Asp Gln Phe Tyr

465 470 475 480

Ala Asn Gly Val Thr Ile Ala Asn Gly Leu Arg Ser Ala Ala Pro Gln

485 490 495

Gly Thr Gln Ile Asp Tyr Ile Glu Gly Phe Asp Val Tyr Gly Asn Asp

500 505 510

Thr Gly Leu Asp Lys Val Leu Gln Ala Ala Asn Asn Tyr Asp Val Ile

515 520 525

Val Ala Ala Val Gly Glu His Val Tyr Ala Glu Ala Pro Gly Asp Ile

530 535 540

His Asp Ile Arg Leu Ala Gln Gly Gln Ile Asp Gly Val Lys Ala Leu

545 550 555 560

Ala Ala Thr Asn Lys Pro Val Val Thr Val Leu Val Glu Gly Arg Pro

565 570 575

Arg Val Leu Asp Ser Ile Pro Asp His Ser Gln Ala Ile Leu His Ala

580 585 590

Leu Leu Pro Gly Pro Trp Gly Gly Gln Ala Ile Gly Glu Val Leu Phe

595 600 605

Gly Leu Val Asn Pro Ser Gly Lys Leu Pro Tyr Thr Tyr Pro Lys Asn

610 615 620

Ala Gly Asp Met Ala Leu Asn Tyr Trp Arg Gln Ala Asn Asp Val Trp

625 630 635 640

Asp Pro Leu Tyr Glu Phe Gly His Gly Leu Ser Tyr Ser Gln Phe Asn

645 650 655

Tyr Ser Gln Leu Thr Ala Asp Asp Lys Thr Ile Ser Ser Asp Lys Pro

660 665 670

Val Thr Val Ser Val Gln Val Thr Asn Asn Gly Pro Met Asp Gly Met

675 680 685

Glu Ser Val Leu Met Phe Ile Gln Gln Pro Val Arg Arg Val Thr Pro

690 695 700

Pro Ala Lys Leu Leu Lys Gly Phe Lys Lys Leu Gln Leu Ala Asn Gly

705 710 715 720

Glu Thr Ala Thr Val Asn Phe Glu Val Ser Ala Asp Ala Phe Lys Tyr

725 730 735

Thr Gly Leu Asp Gly Val Pro Gly Gly Ser Leu Asp Ala Gly Pro Val

740 745 750

Lys Val Met Ile Gly Asp Gln Glu Ile Asp Leu Asp Leu Gln Pro

755 760 765

<210> 9

<211> 2403

<212> DNA

<213>tangerine orange thermophilic ascomycete

<400> 9

atggccaccc tcaagtcagt tctcgccctc gtggcggcct tggtgccaac caccttggcc 60

caggccaaca cgacatacgc gaactactct gtcaagtccc agcccgacct gacgcctcag 120

acggtggcca ccatcgatct gtccttccca gactgcgtca atggaccgct cagctcgaat 180

ctcgtgtgca acacgtcggc ggacccccag gctcgagcag cctccctcgt ctcgctcttc 240

accctggagg agttgatcaa caacacgggg aacacggccc cgggggttcc ccgactgggt 300

ctccccagct atcaagtgtg gagtgagtcc ctgcatggat tggaccgtgc caatttcacg 360

ccggaagggg agtacagctg gtcgacctcc ttccccatgc cgatcctgtc gatggcgtcg 420

ttgaaccgca ccctgatcaa ccagatcgca tccatcattt cgacccaggg ccgtgcgttc 480

aacaacgccg gaagatacgg cctggatgtc tacgccccca acatcaacgg tttcaggcac 540

ccgctctggg gccgtggaca ggagacgcca ggcgaggacg cgttctatct gacctcggtc 600

tatgcgtacg agtacatcac cggcatccaa ggcggagttg atccgcagcc tctgaagttg 660

gccgccacgg cgaagcactt tgccggctac gacctggaga actggggagg ccattctcgc 720

ctgggcaacg atctcagcat cacgcagcaa gatctcgccg agtactacac cccgcagttc 780

ttcgtggcca cgcggtacgc caaggtgcgc agcatcatgt gctcgtacaa cgcggtcaac 840

ggggtgccga gctgctccaa ttccttcttc ctgcagaccc tgctccgcga cacgtggaac 900

ttcgtcgagg acggatacgt ctcgtccgac tgcgatgccg tgtacaacgt cttcaaccct 960

cacatgtacg ccctgaacca gtccgcggcc gcggccgact cgctcagggc aggcaccgac 1020

atcgactgcg gcacgaccta ccagtactac ctgaacgagt cctttgccga cggatatgtg 1080

tcccgcgccg acatcgaact cggcgtcaag cgcctctact cgacgctggt tcgcgctggc 1140

tacttcgacg gcaacggcag cgcataccgg gacctcacct ggaacgacgt ggtgaccacc 1200

gacgcgtgga acatctcgta cgaggccgcg gtggagggaa tcaccctgct caagaacgac 1260

ggaaccctgc cgctgtccaa gtccgtccgc agcgtcgcgc tcatcggacc ctgggcgaac 1320

gccacgaccc agatgcaggg caactacttc ggcccggccc cgtacctgat cagccccctg 1380

gcggccttcg aggcgtccga cctgaaggtg aactacgcgc ccggcaccgg catctcatcc 1440

gactccacgg agggcttcgc ggaggccctc gccgcggcga agaagtccga cgcgatcatc 1500

ttcgccggcg gcatcgacaa caccatcgag gccgagggca tggaccgcat gaacatcacc 1560

tggcccggca accagctcga cctgatccac cagctgagcg agctgcgcaa gccgctggtc 1620

gtcctccaga tgggcggcgg gcaggtcgac tcgtcgtcgc tcaaggccaa cccgcacgtc 1680

aactcgctga tctggggcgg ctacccgggc cagtcgggcg gacaggccct gttcgacatc 1740

atcaccggca agcgcgcgcc cgccggccgc ctcgtcacga cgcagtatcc cgctgaatac 1800

gcgacgcagt tcccggccac ggacatgagc ctgcggccga gcgggaagaa cccgggccag 1860

acgtacatgt ggtacacggg caagcccgtg tacgagttcg gccacggcct cttctacacc 1920

accttccaca tctccctcga cagcagtcac atcaagaaga actccgcagg agcgacatac 1980

aacatcgccg ccctcctctc ccaaccgcac ccggaccacg agttcattga acaggtcccc 2040

ctcctcaact tcaccgtcaa ggtgaccaac accggccacc gcgcgtcccc gtactcggcc 2100

atgctcttcg ccagcaccag ggacgccggc cccgcgccct acccgaacaa gtggctcggc 2160

gggttcgacc gcctgccgac gctggcaccc ggcgagtccg cgacgctgac gatccccgtg 2220

gccatcggca gcgtcacccg cgtggatgag cagggtaatc gcgtgctgta cccggggcgg 2280

tacgagctgg cgctgaacaa cgagcgcgat gccgtcctgt cgtttacgct gacgggcgac 2340

gaggccgttg tcgcgaagtg gccgctggag gcgcagttga ttccgggggc ggcttctcag 2400

tga 2403

<210> 10

<211> 800

<212> PRT

<213>tangerine orange thermophilic ascomycete

<400> 10

Met Ala Thr Leu Lys Ser Val Leu Ala Leu Val Ala Ala Leu Val Pro

1 5 10 15

Thr Thr Leu Ala Gln Ala Asn Thr Thr Tyr Ala Asn Tyr Ser Val Lys

20 25 30

Ser Gln Pro Asp Leu Thr Pro Gln Thr Val Ala Thr Ile Asp Leu Ser

35 40 45

Phe Pro Asp Cys Val Asn Gly Pro Leu Ser Ser Asn Leu Val Cys Asn

50 55 60

Thr Ser Ala Asp Pro Gln Ala Arg Ala Ala Ser Leu Val Ser Leu Phe

65 70 75 80

Thr Leu Glu Glu Leu Ile Asn Asn Thr Gly Asn Thr Ala Pro Gly Val

85 90 95

Pro Arg Leu Gly Leu Pro Ser Tyr Gln Val Trp Ser Glu Ser Leu His

100 105 110

Gly Leu Asp Arg Ala Asn Phe Thr Pro Glu Gly Glu Tyr Ser Trp Ser

115 120 125

Thr Ser Phe Pro Met Pro Ile Leu Ser Met Ala Ser Leu Asn Arg Thr

130 135 140

Leu Ile Asn Gln Ile Ala Ser Ile Ile Ser Thr Gln Gly Arg Ala Phe

145 150 155 160

Asn Asn Ala Gly Arg Tyr Gly Leu Asp Val Tyr Ala Pro Asn Ile Asn

165 170 175

Gly Phe Arg His Pro Leu Trp Gly Arg Gly Gln Glu Thr Pro Gly Glu

180 185 190

Asp Ala Phe Tyr Leu Thr Ser Val Tyr Ala Tyr Glu Tyr Ile Thr Gly

195 200 205

Ile Gln Gly Gly Val Asp Pro Gln Pro Leu Lys Leu Ala Ala Thr Ala

210 215 220

Lys His Phe Ala Gly Tyr Asp Leu Glu Asn Trp Gly Gly His Ser Arg

225 230 235 240

Leu Gly Asn Asp Leu Ser Ile Thr Gln Gln Asp Leu Ala Glu Tyr Tyr

245 250 255

Thr Pro Gln Phe Phe Val Ala Thr Arg Tyr Ala Lys Val Arg Ser Ile

260 265 270

Met Cys Ser Tyr Asn Ala Val Asn Gly Val Pro Ser Cys Ser Asn Ser

275 280 285

Phe Phe Leu Gln Thr Leu Leu Arg Asp Thr Trp Asn Phe Val Glu Asp

290 295 300

Gly Tyr Val Ser Ser Asp Cys Asp Ala Val Tyr Asn Val Phe Asn Pro

305 310 315 320

His Met Tyr Ala Leu Asn Gln Ser Ala Ala Ala Ala Asp Ser Leu Arg

325 330 335

Ala Gly Thr Asp Ile Asp Cys Gly Thr Thr Tyr Gln Tyr Tyr Leu Asn

340 345 350

Glu Ser Phe Ala Asp Gly Tyr Val Ser Arg Ala Asp Ile Glu Leu Gly

355 360 365

Val Lys Arg Leu Tyr Ser Thr Leu Val Arg Ala Gly Tyr Phe Asp Gly

370 375 380

Asn Gly Ser Ala Tyr Arg Asp Leu Thr Trp Asn Asp Val Val Thr Thr

385 390 395 400

Asp Ala Trp Asn Ile Ser Tyr Glu Ala Ala Val Glu Gly Ile Thr Leu

405 410 415

Leu Lys Asn Asp Gly Thr Leu Pro Leu Ser Lys Ser Val Arg Ser Val

420 425 430

Ala Leu Ile Gly Pro Trp Ala Asn Ala Thr Thr Gln Met Gln Gly Asn

435 440 445

Tyr Phe Gly Pro Ala Pro Tyr Leu Ile Ser Pro Leu Ala Ala Phe Glu

450 455 460

Ala Ser Asp Leu Lys Val Asn Tyr Ala Pro Gly Thr Gly Ile Ser Ser

465 470 475 480

Asp Ser Thr Glu Gly Phe Ala Glu Ala Leu Ala Ala Ala Lys Lys Ser

485 490 495

Asp Ala Ile Ile Phe Ala Gly Gly Ile Asp Asn Thr Ile Glu Ala Glu

500 505 510

Gly Met Asp Arg Met Asn Ile Thr Trp Pro Gly Asn Gln Leu Asp Leu

515 520 525

Ile His Gln Leu Ser Glu Leu Arg Lys Pro Leu Val Val Leu Gln Met

530 535 540

Gly Gly Gly Gln Val Asp Ser Ser Ser Leu Lys Ala Asn Pro His Val

545 550 555 560

Asn Ser Leu Ile Trp Gly Gly Tyr Pro Gly Gln Ser Gly Gly Gln Ala

565 570 575

Leu Phe Asp Ile Ile Thr Gly Lys Arg Ala Pro Ala Gly Arg Leu Val

580 585 590

Thr Thr Gln Tyr Pro Ala Glu Tyr Ala Thr Gln Phe Pro Ala Thr Asp

595 600 605

Met Ser Leu Arg Pro Ser Gly Lys Asn Pro Gly Gln Thr Tyr Met Trp

610 615 620

Tyr Thr Gly Lys Pro Val Tyr Glu Phe Gly His Gly Leu Phe Tyr Thr

625 630 635 640

Thr Phe His Ile Ser Leu Asp Ser Ser His Ile Lys Lys Asn Ser Ala

645 650 655

Gly Ala Thr Tyr Asn Ile Ala Ala Leu Leu Ser Gln Pro His Pro Asp

660 665 670

His Glu Phe Ile Glu Gln Val Pro Leu Leu Asn Phe Thr Val Lys Val

675 680 685

Thr Asn Thr Gly His Arg Ala Ser Pro Tyr Ser Ala Met Leu Phe Ala

690 695 700

Ser Thr Arg Asp Ala Gly Pro Ala Pro Tyr Pro Asn Lys Trp Leu Gly

705 710 715 720

Gly Phe Asp Arg Leu Pro Thr Leu Ala Pro Gly Glu Ser Ala Thr Leu

725 730 735

Thr Ile Pro Val Ala Ile Gly Ser Val Thr Arg Val Asp Glu Gln Gly

740 745 750

Asn Arg Val Leu Tyr Pro Gly Arg Tyr Glu Leu Ala Leu Asn Asn Glu

755 760 765

Arg Asp Ala Val Leu Ser Phe Thr Leu Thr Gly Asp Glu Ala Val Val

770 775 780

Ala Lys Trp Pro Leu Glu Ala Gln Leu Ile Pro Gly Ala Ala Ser Gln

785 790 795 800

<210> 11

<211> 40

<212> DNA

<213>artificial sequence

<220>

<223>construct is synthesized

<400> 11

acacaactgg ggatccacca tgaccaggct gaccagcatc 40

<210> 12

<211> 37

<212> DNA

<213>artificial sequence

<220>

<223>construct is synthesized

<400> 12

gtcaccctct agatctcgta ccccactgcc gttattg 37

<210> 13

<211> 40

<212> DNA

<213>artificial sequence

<220>

<223>construct is synthesized

<400> 13

acacaactgg ggatccacca tgaaggccct gactagaagg 40

<210> 14

<211> 41

<212> DNA

<213>artificial sequence

<220>

<223>construct is synthesized

<400> 14

gtcaccctct agatcttacc ggacatgaac atgacagtag g 41

<210> 15

<211> 36

<212> DNA

<213>artificial sequence

<220>

<223>construct is synthesized

<400> 15

acacaactgg ggatccacca tgctggccct ggcatc 36

<210> 16

<211> 46

<212> DNA

<213>artificial sequence

<220>

<223>construct is synthesized

<400> 16

gtcaccctct agatcttcaa aatcctcttg tgctacctct caagaa 46

<210> 17

<211> 40

<212> DNA

<213>artificial sequence

<220>

<223>construct is synthesized

<400> 17

acacaactgg ggatccacca tggcgtttat caagcagagc 40

<210> 18

<211> 35

<212> DNA

<213>artificial sequence

<220>

<223>construct is synthesized

<400> 18

gtcaccctct agatctaccg tggaaacagc agcag 35

<210> 19

<211> 42

<212> DNA

<213>artificial sequence

<220>

<223>construct is synthesized

<400> 19

acacaactgg ggatccacca tggccaccct caagtcagtt ct 42

<210> 20

<211> 40

<212> DNA

<213>artificial sequence

<220>

<223>construct is synthesized

<400> 20

gtcaccctct agatcttcgc tcactcactc actgagaagc 40

<210> 21

<211> 1387

<212> DNA

<213>Penicillium species

<400> 21

atggttcgcc tcagtccagt cctgctggca tcgatcgcag gctctggcct gcctctgtac 60

gcacaagcag ccggcctcaa caccgccgcc aaagccatcg gcctgaaata cttcggcacg 120

gcgaccgaca accccgaact gagcgacacc gcgtacgaga cggaactgaa caacacgcag 180

gatttcgggc agttgacacc tgcgaattcg atgaaggtga gtctgacagc tcccccccct 240

cctggggtga gtgagtgagt tcgacgctaa tggtttttgc agtgggacgc aaccgagccc 300

cagcaaaaca ctttcacgtt cagcggcggc gatcagatcg ctaacctggc caaggcgaat 360

ggccagatgt tgaggtgcca taatcttgtt tggtataatc agttgccgtc gtggggtatg 420

tatagtacct gcgtacttgt ttgtaatgat tgtcttggct gatttgtgaa gtcaccggtg 480

gatcctggac caacgagacg ctgcttgctg ccatgaagaa tcacatcacc aacgtcgtta 540

cccattacaa gggccagtgc tatgcatggg atgtcgtgaa tgagggtacg tccatataat 600

tgctgttact atcgagagga atcagctaat gacgacagcc ctcaacgacg acggcaccta 660

ccgcagcaac gtcttctacc agtatatcgg ggaggcgtac atccccatcg ccttcgcgac 720

ggccgccgcc gccgaccccg acgccaagct gtactacaac gactacaaca tcgagtaccc 780

cggcgccaag gccacggcgg cgcagaacat cgtcaagctg gtgcagtcgt acggggcgcg 840

catcgacggc gtcggcctgc agtcgcactt catcgtgggc cagacgccca gcacgagcgc 900

ccagcagcag aacatggccg ccttcaccgc gctgggcgtc gaggtcgcca tcaccgagct 960

cgacatccgc atgcagctgc ccgagacgtc cgcgcagctg acgcagcagg cgaccgacta 1020

ccagagcacg gtccaggcct gcgtcaacac cgacagctgc gtcggcatta ccctctggga 1080

ctggaccgac aagtactcgt gggtgcccag caccttctca ggctggggcg acgcctgtcc 1140

ctgggacgac aactaccaga agaaacccgc gtacaacggc atcctcactg ctctgggagg 1200

cacgccctcc tccagtacca gctacaccct cacgccgacg acgacctcaa gcggcggcag 1260

tggcagcccg actgacgtgg cccagcattg ggagcagtgc ggtggcctgg gctggactgg 1320

gccgacggtt tgcgccagtg gcttcacttg cactgtcatc aacgagtatt actcgcagtg 1380

tctgtaa 1387

<210> 22

<211> 403

<212> PRT

<213>Penicillium species

<400> 22

Met Val Arg Leu Ser Pro Val Leu Leu Ala Ser Ile Ala Gly Ser Gly

1 5 10 15

Leu Pro Leu Tyr Ala Gln Ala Ala Gly Leu Asn Thr Ala Ala Lys Ala

20 25 30

Ile Gly Leu Lys Tyr Phe Gly Thr Ala Thr Asp Asn Pro Glu Leu Ser

35 40 45

Asp Thr Ala Tyr Glu Thr Glu Leu Asn Asn Thr Gln Asp Phe Gly Gln

50 55 60

Leu Thr Pro Ala Asn Ser Met Lys Trp Asp Ala Thr Glu Pro Gln Gln

65 70 75 80

Asn Thr Phe Thr Phe Ser Gly Gly Asp Gln Ile Ala Asn Leu Ala Lys

85 90 95

Ala Asn Gly Gln Met Leu Arg Cys His Asn Leu Val Trp Tyr Asn Gln

100 105 110

Leu Pro Ser Trp Val Thr Gly Gly Ser Trp Thr Asn Glu Thr Leu Leu

115 120 125

Ala Ala Met Lys Asn His Ile Thr Asn Val Val Thr His Tyr Lys Gly

130 135 140

Gln Cys Tyr Ala Trp Asp Val Val Asn Glu Ala Leu Asn Asp Asp Gly

145 150 155 160

Thr Tyr Arg Ser Asn Val Phe Tyr Gln Tyr Ile Gly Glu Ala Tyr Ile

165 170 175

Pro Ile Ala Phe Ala Thr Ala Ala Ala Ala Asp Pro Asp Ala Lys Leu

180 185 190

Tyr Tyr Asn Asp Tyr Asn Ile Glu Tyr Pro Gly Ala Lys Ala Thr Ala

195 200 205

Ala Gln Asn Ile Val Lys Leu Val Gln Ser Tyr Gly Ala Arg Ile Asp

210 215 220

Gly Val Gly Leu Gln Ser His Phe Ile Val Gly Gln Thr Pro Ser Thr

225 230 235 240

Ser Ala Gln Gln Gln Asn Met Ala Ala Phe Thr Ala Leu Gly Val Glu

245 250 255

Val Ala Ile Thr Glu Leu Asp Ile Arg Met Gln Leu Pro Glu Thr Ser

260 265 270

Ala Gln Leu Thr Gln Gln Ala Thr Asp Tyr Gln Ser Thr Val Gln Ala

275 280 285

Cys Val Asn Thr Asp Ser Cys Val Gly Ile Thr Leu Trp Asp Trp Thr

290 295 300

Asp Lys Tyr Ser Trp Val Pro Ser Thr Phe Ser Gly Trp Gly Asp Ala

305 310 315 320

Cys Pro Trp Asp Asp Asn Tyr Gln Lys Lys Pro Ala Tyr Asn Gly Ile

325 330 335

Leu Thr Ala Leu Gly Gly Thr Pro Ser Ser Ser Thr Ser Tyr Thr Leu

340 345 350

Thr Pro Thr Thr Thr Ser Ser Gly Gly Ser Gly Ser Pro Thr Asp Val

355 360 365

Ala Gln His Trp Glu Gln Cys Gly Gly Leu Gly Trp Thr Gly Pro Thr

370 375 380

Val Cys Ala Ser Gly Phe Thr Cys Thr Val Ile Asn Glu Tyr Tyr Ser

385 390 395 400

Gln Cys Leu

<210> 23

<211> 2388

<212> DNA

<213>Ai Mosen ankle section bacterium

<400> 23

atgatgactc ccacggcgat tctcaccgca gtggcggcgc tcctgcccac cgcgacatgg 60

gcacaggata accaaaccta tgccaattac tcgtcgcagt ctcagccgga cctgtttccc 120

cggaccgtcg cgaccatcga cctgtccttc cccgactgtg agaatggccc gctcagcacg 180

aacctggtgt gcaacaaatc ggccgatccc tgggcccgag ctgaggccct catctcgctc 240

tttaccctcg aagagctgat taacaacacc cagaacaccg ctcctggcgt gccccgtttg 300

ggtctgcccc agtatcaggt gtggaatgaa gctctgcacg gactggaccg cgccaatttc 360

tcccattcgg gcgaatacag ctgggccacg tccttcccca tgcccatcct gtcgatggcg 420

tccttcaacc ggaccctcat caaccagatt gcctccatca ttgcaacgca agcccgtgcc 480

ttcaacaacg ccggccgtta cggccttgac agctatgcgc ccaacatcaa tggcttccgc 540

agtcccctct ggggccgtgg acaggagacg cctggtgagg atgcgttctt cttgagttcc 600

acctatgcgt acgagtacat cacaggcctg cagggcggtg tcgacccaga gcatgtcaag 660

atcgtcgcga cggcgaagca cttcgccggc tatgatctgg agaactgggg caacgtctct 720

cggctggggt tcaatgctat catcacgcag caggatctct ccgagtacta cacccctcag 780

ttcctggcgt ctgctcgata cgccaagacg cgcagcatca tgtgctccta caatgcagtg 840

aatggagtcc caagctgtgc caactccttc ttcctccaga cgcttctccg agaaaacttt 900

gacttcgttg acgacgggta cgtctcgtcg gattgcgacg ccgtctacaa cgtcttcaac 960

ccacacggtt acgcccttaa ccagtcggga gccgctgcgg actcgctcct agcaggtacc 1020

gatatcgact gtggtcagac cttgccgtgg cacctgaatg agtccttcgt agaaggatac 1080

gtctcccgcg gtgatatcga gaaatccctc acccgtctct actcaaacct ggtgcgtctc 1140

ggctactttg acggcaacaa cagcgagtac cgcaacctca actggaacga cgtcgtgact 1200

acggacgcct ggaacatctc gtacgaggcc gcggtggaag gtatcaccct gctcaagaac 1260

gacggaacgc tgccgctgtc caagaaggtc cgcagcattg cgctcatcgg tccttgggcc 1320

aatgccacgg tgcagatgca gggtaactac tatggaacgc caccgtatct gatcagtccg 1380

ctggaagccg ccaaggccag tgggttcacg gtcaactatg cattcggtac caacatctcg 1440

accgattcta cccagtggtt cgcggaagcc atcgcggcgg cgaagaagtc ggacgtgatc 1500

atctacgccg gtggtattga caacacgatc gaggcagagg gacaggaccg cacggatctc 1560

aagtggccgg ggaaccagct ggatctgatc gagcagctca gccaggtggg caagcccttg 1620

gtcgtcctgc agatgggcgg tggccaggtg gattcgtcgt cactcaaggc caacaagaat 1680

gtcaacgctc tggtgtgggg tggctatccc ggacagtcgg gtggtgcggc cctgtttgac 1740

atccttacgg gcaagcgtgc gccggccggt cgtctggtga gcacgcagta cccggccgag 1800

tatgcgacgc agttcccggc caacgacatg aacctgcgtc cgaacggcag caacccggga 1860

cagacataca tctggtacac gggcacgccc gtgtatgagt tcggccacgg tctgttctac 1920

acggagttcc aggagtcggc tgcggcgggc acgaacaaga cgtcgacttt cgacattctg 1980

gaccttttct ccacccctca tccgggatac gagtacatcg agcaggttcc gttcatcaac 2040

gtgactgtgg acgtgaagaa cgtcggccac acgccatcgc cgtacacggg tctgttgttc 2100

gcgaacacga cagccgggcc caagccgtac ccgaacaaat ggctcgtcgg gttcgactgg 2160

ctgccgacga tccagccggg cgagactgcc aagttgacga tcccggtgcc gttgggcgcg 2220

attgcgtggg cggacgagaa cggcaacaag gtggtcttcc cgggcaacta cgaattggca 2280

ctgaacaatg agcgatcggt agtggtgtcg ttcacgctga cgggcgatgc ggcgactcta 2340

gagaaatggc ctttgtggga gcaggcggtt ccgggggtgc tgcagcaa 2388

<210> 24

<211> 796

<212> PRT

<213>Ai Mosen ankle section bacterium

<400> 24

Met Met Thr Pro Thr Ala Ile Leu Thr Ala Val Ala Ala Leu Leu Pro

1 5 10 15

Thr Ala Thr Trp Ala Gln Asp Asn Gln Thr Tyr Ala Asn Tyr Ser Ser

20 25 30

Gln Ser Gln Pro Asp Leu Phe Pro Arg Thr Val Ala Thr Ile Asp Leu

35 40 45

Ser Phe Pro Asp Cys Glu Asn Gly Pro Leu Ser Thr Asn Leu Val Cys

50 55 60

Asn Lys Ser Ala Asp Pro Trp Ala Arg Ala Glu Ala Leu Ile Ser Leu

65 70 75 80

Phe Thr Leu Glu Glu Leu Ile Asn Asn Thr Gln Asn Thr Ala Pro Gly

85 90 95

Val Pro Arg Leu Gly Leu Pro Gln Tyr Gln Val Trp Asn Glu Ala Leu

100 105 110

His Gly Leu Asp Arg Ala Asn Phe Ser His Ser Gly Glu Tyr Ser Trp

115 120 125

Ala Thr Ser Phe Pro Met Pro Ile Leu Ser Met Ala Ser Phe Asn Arg

130 135 140

Thr Leu Ile Asn Gln Ile Ala Ser Ile Ile Ala Thr Gln Ala Arg Ala

145 150 155 160

Phe Asn Asn Ala Gly Arg Tyr Gly Leu Asp Ser Tyr Ala Pro Asn Ile

165 170 175

Asn Gly Phe Arg Ser Pro Leu Trp Gly Arg Gly Gln Glu Thr Pro Gly

180 185 190

Glu Asp Ala Phe Phe Leu Ser Ser Thr Tyr Ala Tyr Glu Tyr Ile Thr

195 200 205

Gly Leu Gln Gly Gly Val Asp Pro Glu His Val Lys Ile Val Ala Thr

210 215 220

Ala Lys His Phe Ala Gly Tyr Asp Leu Glu Asn Trp Gly Asn Val Ser

225 230 235 240

Arg Leu Gly Phe Asn Ala Ile Ile Thr Gln Gln Asp Leu Ser Glu Tyr

245 250 255

Tyr Thr Pro Gln Phe Leu Ala Ser Ala Arg Tyr Ala Lys Thr Arg Ser

260 265 270

Ile Met Cys Ser Tyr Asn Ala Val Asn Gly Val Pro Ser Cys Ala Asn

275 280 285

Ser Phe Phe Leu Gln Thr Leu Leu Arg Glu Asn Phe Asp Phe Val Asp

290 295 300

Asp Gly Tyr Val Ser Ser Asp Cys Asp Ala Val Tyr Asn Val Phe Asn

305 310 315 320

Pro His Gly Tyr Ala Leu Asn Gln Ser Gly Ala Ala Ala Asp Ser Leu

325 330 335

Leu Ala Gly Thr Asp Ile Asp Cys Gly Gln Thr Leu Pro Trp His Leu

340 345 350

Asn Glu Ser Phe Val Glu Gly Tyr Val Ser Arg Gly Asp Ile Glu Lys

355 360 365

Ser Leu Thr Arg Leu Tyr Ser Asn Leu Val Arg Leu Gly Tyr Phe Asp

370 375 380

Gly Asn Asn Ser Glu Tyr Arg Asn Leu Asn Trp Asn Asp Val Val Thr

385 390 395 400

Thr Asp Ala Trp Asn Ile Ser Tyr Glu Ala Ala Val Glu Gly Ile Thr

405 410 415

Leu Leu Lys Asn Asp Gly Thr Leu Pro Leu Ser Lys Lys Val Arg Ser

420 425 430

Ile Ala Leu Ile Gly Pro Trp Ala Asn Ala Thr Val Gln Met Gln Gly

435 440 445

Asn Tyr Tyr Gly Thr Pro Pro Tyr Leu Ile Ser Pro Leu Glu Ala Ala

450 455 460

Lys Ala Ser Gly Phe Thr Val Asn Tyr Ala Phe Gly Thr Asn Ile Ser

465 470 475 480

Thr Asp Ser Thr Gln Trp Phe Ala Glu Ala Ile Ala Ala Ala Lys Lys

485 490 495

Ser Asp Val Ile Ile Tyr Ala Gly Gly Ile Asp Asn Thr Ile Glu Ala

500 505 510

Glu Gly Gln Asp Arg Thr Asp Leu Lys Trp Pro Gly Asn Gln Leu Asp

515 520 525

Leu Ile Glu Gln Leu Ser Gln Val Gly Lys Pro Leu Val Val Leu Gln

530 535 540

Met Gly Gly Gly Gln Val Asp Ser Ser Ser Leu Lys Ala Asn Lys Asn

545 550 555 560

Val Asn Ala Leu Val Trp Gly Gly Tyr Pro Gly Gln Ser Gly Gly Ala

565 570 575

Ala Leu Phe Asp Ile Leu Thr Gly Lys Arg Ala Pro Ala Gly Arg Leu

580 585 590

Val Ser Thr Gln Tyr Pro Ala Glu Tyr Ala Thr Gln Phe Pro Ala Asn

595 600 605

Asp Met Asn Leu Arg Pro Asn Gly Ser Asn Pro Gly Gln Thr Tyr Ile

610 615 620

Trp Tyr Thr Gly Thr Pro Val Tyr Glu Phe Gly His Gly Leu Phe Tyr

625 630 635 640

Thr Glu Phe Gln Glu Ser Ala Ala Ala Gly Thr Asn Lys Thr Ser Thr

645 650 655

Phe Asp Ile Leu Asp Leu Phe Ser Thr Pro His Pro Gly Tyr Glu Tyr

660 665 670

Ile Glu Gln Val Pro Phe Ile Asn Val Thr Val Asp Val Lys Asn Val

675 680 685

Gly His Thr Pro Ser Pro Tyr Thr Gly Leu Leu Phe Ala Asn Thr Thr

690 695 700

Ala Gly Pro Lys Pro Tyr Pro Asn Lys Trp Leu Val Gly Phe Asp Trp

705 710 715 720

Leu Pro Thr Ile Gln Pro Gly Glu Thr Ala Lys Leu Thr Ile Pro Val

725 730 735

Pro Leu Gly Ala Ile Ala Trp Ala Asp Glu Asn Gly Asn Lys Val Val

740 745 750

Phe Pro Gly Asn Tyr Glu Leu Ala Leu Asn Asn Glu Arg Ser Val Val

755 760 765

Val Ser Phe Thr Leu Thr Gly Asp Ala Ala Thr Leu Glu Lys Trp Pro

770 775 780

Leu Trp Glu Gln Ala Val Pro Gly Val Leu Gln Gln

785 790 795

Claims

1. it is a kind of with the active isolated polypeptide of xylobiase, it is selected from the group:

(a) polypeptide has at least 60% sequence identity with the mature polypeptide of SEQ ID NO:6 or SEQ ID NO:8；With The mature polypeptide of SEQ ID NO:2 has at least 65% sequence identity；Or with SEQ ID NO:4 or SEQ ID NO:10 at Ripe polypeptide has at least 75% sequence identity；

(b) polypeptide, by polynucleotide encoding, the polynucleotides hybridize under at least medium-high stringency conditions with following: (i) mature polypeptide of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:9 are compiled Code sequence, the cDNA sequence of (ii) SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7, or (iii) the overall length complement of (i) or (ii)；

(c) polypeptide, by polynucleotide encoding, the polynucleotides and SEQ ID NO:5 or SEQ ID NO:7 or its cDNA sequence The mature polypeptide encoded sequence of column has at least 60% sequence identity；It is more with the maturation of SEQ ID NO:1 or its cDNA sequence Peptide-coding sequence has at least 65% sequence identity；Or with SEQ ID NO:3 or its cDNA sequence or SEQ ID NO:9 at Ripe polypeptid coding sequence has at least 75% sequence identity；

(d) maturation of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10 are more Peptide includes the variant for replacing, lacking and/or being inserted into one or more (such as several) positions；With

(e) (a), (b), (c) or polypeptide (d) has the active segment of xylobiase.

2. the polypeptide of claim 1, it includes or group become SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10 or SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or The mature polypeptide of SEQ ID NO:10.

3. a kind of isolated polynucleotides encode the polypeptide of claims 1 or 2.

4. a kind of recombinant host cell, it includes the polynucleotides of claim 3, the polynucleotides are operably connected to one The regulating and controlling sequence of a or multiple generations for instructing polypeptide.

5. a kind of method for the polypeptide for generating claims 1 or 2 comprising:

(a) cell is cultivated under conditions of facilitating the polypeptide and generating, the cell generates described more with its wild-type form Peptide；Optionally

(b) polypeptide is recycled.

6. a kind of generate the method with the active polypeptide of xylobiase comprising:

(a) host cell of claim 4 is cultivated under conditions of facilitating the polypeptide and generating；Optionally

(b) polypeptide is recycled.

7. a kind of genetically modified plants, plant part or plant cell, with the polynucleotides of the polypeptide of coding claims 1 or 2 It is transformed.

8. a kind of generate the method with the active polypeptide of xylobiase comprising:

(a) genetically modified plants of culture claim 7 or plant cell under conditions of facilitating the polypeptide and generating；With it is optional Ground

(b) polypeptide is recycled.

9. a kind of method for the mutant for generating parental cell, the method includes making the more of the polypeptide for encoding claims 1 or 2 Nucleotide inactivation leads to the mutant that the less polypeptide is generated compared with parental cell.

10. double-stranded inhibitory RNA (dsRNA) molecule of a kind of subsequence of the polynucleotides comprising claim 10, wherein appointing DsRNA described in selection of land is siRNA or miRNA molecule.