CN104145016B - Polynucleotides with the active polypeptide of xylobiase and the coding polypeptide - Google Patents

Polynucleotides with the active polypeptide of xylobiase and the coding polypeptide Download PDF

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CN104145016B
CN104145016B CN201280067784.9A CN201280067784A CN104145016B CN 104145016 B CN104145016 B CN 104145016B CN 201280067784 A CN201280067784 A CN 201280067784A CN 104145016 B CN104145016 B CN 104145016B
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polypeptide
seq
enzyme
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acid
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CN104145016A (en
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张羽
刘晔
段俊欣
汤岚
B.麦克布拉耶
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Novozymes Inc
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01037—Xylan 1,4-beta-xylosidase (3.2.1.37)

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Abstract

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

Description

Polynucleotides with the active polypeptide of xylobiase and the coding polypeptide
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 with governmental support under 08GO18080.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 carrying 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 including nucleic acid construct, carrier and the host cell of the polynucleotides, and the method for generating and using the polypeptide.
Background technology
Ligno-ccllulose, maximum renewable biomass resources in the world, mainly by lignin, cellulose and hemicellulose It constitutes, the major part of wherein hemicellulose is xylan.Zytase (in such as-and Isosorbide-5-Nitrae-beta-xylanase, EC3.2.1.8) Inside β -1,4- xyloses glycosidic bonds in hydrolyzed xylan are to generate the xylose and wood oligose (xylo- of lower molecular weight oligomer).Xylan is the D- xyloses 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 is keyed by β -1,4-.The Portugal that many microorganisms generate hydrolysis β-connection is poly- 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- connections Aggressiveness.Cellobiose is hydrolyzed into glucose by β-glucosyl enzym.
Lignocellulose-containing raw material (lignocellulosic feedstock) is converted to ethyl alcohol to have the advantage that： Big content of starting materials is readily available, avoids burning or the desirability of 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 produced for ethyl alcohol.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.
Invention content
The present invention relates to the polypeptides with the active separation of xylobiase, are selected from the group：
(a) polypeptide, with SEQ ID NO:6 or SEQ ID NO:8 mature polypeptide has at least 60% sequence identity； With SEQ ID NO:2 mature polypeptide has at least 65% sequence identity；Or with SEQ ID NO:4 or SEQ ID NO:10 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：(i)SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:9 mature polypeptide Coded sequence, (ii) SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7 cDNA sequence, 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 NO:9 mature polypeptide encoded sequence 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 substitution, missing and/or the variant 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 the polynucleotides of the separation of the polypeptide of the coding present invention, includes the nucleic acid structure of 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 through degrading or converting or material containing xylan.
The present invention is also related to the technique for generating tunning comprising：(a) there is xylobiase activity in the present 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 saccharification or material containing xylan to generate tunning；(c) from fermenting back Receive the tunning.
The present invention is also related to fermentable fiber cellulosic material or the technique of 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) 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 amino acid 1 is to 21 or SEQ ID NO:10 amino acid 1 is operably connected to coding albumen to 20 Gene, the albumen is external source for the signal peptide；Including the nucleic acid construct of the polynucleotides, expression vector and Recombinant host cell；With the method for generating albumen.
Description of the drawings
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 xylobiases (P24GP2) are 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 xylobiases (P24GP2) are 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 (EC3.1.1.72), 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%TWEENTM0.5mM in the 50mM sodium acetates pH5.0 of 20 (polyoxyethylenesorbitan monolaurates) Acetic acid p-nitrophenyl acetate is determined as substrate.The acetyl xylan esterase of one unit is defined as can be at pH5,25 DEG C every point Clock discharges the enzyme amount of 1 micromole's p-nitrophenol anion (p-nitrophenolate anion).
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 be caused more in population State property.Gene mutation can be silence (unchanged in the polypeptide of coding) 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 (EC3.2.1.55) is catalyzed Arabic to the end irreducibility α-L- in α-L-arabinose glycosides The hydrolysis of furanoside residue.α-L- of the enzyme to α-L- arabinofuranosidases glucosides, containing (1,3)-and/or (1,5)-key I Primary glycan, araboxylan and arabogalactan work.α-l-arabfuranglycosidase is also referred to as arabinose Glycosides enzyme, α-arabinosidase, α-L-arabinose glycosides enzyme, α-arabinofuranosidase, polysaccharide α-L- arabinofuranoses Glycosides enzyme, α-L- arabinofuranosidase glucosides hydrolase, L-arabinose glycosides enzyme or α-L- arabanases.For the present invention, α-l-arabfuranglycosidase activity is the medium of 5mg in the 100mM sodium acetates pH5 using every ml in 200 μ l of total volume 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 chromatographies (Bio-Rad Laboratories, Inc., Hercules, CA, USA) arabinose analyze to determine.
Alpha-glucuronidase：Term " alpha-glucuronidase " means α-D- glucosiduronic acid glucuronic acid hydrolases (alpha-D-glucosiduronate glucuronohydrolase) (EC3.2.1.139) is catalyzed α-D- glucuronic acids Glycoside hydrolysis is D- glucuronic acids and alcohol.For the present invention, alpha-glucuronidase activity be according to de Vries, 1998,J.Bacteriol.180:What 243-249 was determined.The alpha-glucuronidase of one unit be equal to can in pH5,40 The enzyme amount of 1 micromole's glucuronic acid of release DEG C per minute or 4-O- methylglucuronic acids.
β-glucosyl enzym：Term " β-glucosyl enzym " means β-D- glucoside glucohydralases (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, purification and some biochemical properties,J.Basic Microbiol.42:The method of 55-66 It is determined using p-nitrophenyl-β-D- glucose pyranosides as substrate.The β-glucosyl enzym of one unit is defined as at 25 DEG C, PH4.8 is containing 0.01%From the 1mM p-nitrophenyl-β-D- as substrate in 20 50mM sodium citrates Glucose pyranoside 1.0 micromole's p-nitrophenol anion of generation per minute.
Xylobiase：Term " xylobiase " means β-D- xyloside xylose hydrolases (β-D-xyloside Xylohydrolase) (E.C.3.2.1.37) is catalyzed the outer water of short β (1 → 4) wood oligose (xylooligosaccharide) Solution from non-reducing end to remove continuous D- xylose residues.For the present invention, the xylobiase of a unit is defined as 40 DEG C, pH5 is containing 0.01%From the 1mM p-nitrophenyls-as substrate in 20 100mM sodium citrates β-D- xylosides 1.0 micromole's p-nitrophenol anion of generation per minute.
The polypeptide of the present invention has SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:The xylobiase active at least 20% of 10 mature polypeptide, 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 molecules.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 ripe montage.
Cellobiohydrolase：Term " cellobiohydrolase " means 1,4- callose cellobiohydrolases (Isosorbide-5-Nitrae-beta-D-glucan cellobiohydrolase) (E.C.3.2.1.91 and E.C.3.2.1.176), catalysis fibre The hydrolysis of Isosorbide-5-Nitrae-β-D- glycosidic bonds in the polymer of element, cell-oligosaccharide or any glucose comprising β-Isosorbide-5-Nitrae-connection, 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 Biotechnology15:160-167；Teeri etc., 1998, Trichoderma reesei cellobiohydrolases:why so efficient on crystalline cellulose, Biochem.Soc.Trans.26:173-178).According to Lever etc., 1972, Anal.Biochem.47:273-279；van Tilbeurgh etc., 1982, FEBS Letters149:152-156；Van Tilbeurgh and Claeyssens, 1985, FEBS Letters187:283-288；And Tomme etc., 1988, Eur.J.Biochem.170:The method of 575-581 descriptions determines fine Tie up disaccharide-hydrolysing enzymes 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.Measure cellulolytic activity two kinds of basic skills include：(1) total fiber element degrading activity is measured, 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 Advances24:What 452-481 was summarized.Total fiber element degrading activity typically uses insoluble substrate Come what is measured, the substrate includes Whatman1 filter paper, microcrystalline cellulose, bacteria cellulose, algae cellulose, cotton, warp Pretreated ligno-ccllulose etc..Most common total fiber element degrading activity measuring method is to use Whatman1 filter paper the bottom of as The filter paper measuring method of object.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 by measuring under the following conditions by cellulolytic enzyme progress 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 the PCS of zymoprotein/g cellulose (or other pretreated cellulosic materials) in suitable temperature, such as 50 DEG C, 55 DEG C or 60 DEG C carry out 3-7 days.Usual conditions are：1ml reaction solutions, washed or unwashed PCS, 5% insoluble solid, 50mM sodium acetates pH5,1mM MnSO4, 50 DEG C, 55 DEG C or 60 DEG C, 72 hours, pass throughHPX-87H columns (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 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- glucans, 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 typically 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 hydrogen bond with cellulose and other hemicelluloses, 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 are compiled), pp.105-118, Taylor＆Francis, Washington D.C.； Wyman,1994,Bioresource Technology50:3-16；Lynd,1990,Applied Biochemistry and Biotechnology24/25:695-719；Mosier etc., 1999, Recent Progress in Bioconversion of Lignocellulosics, in Advances in Biochemical Engineering/Biotechnology, T.Scheper is edited, Volume65, pp.23-40, Springer-Verlag, New York).It should be understood herein that Cellulose can be the form of ligno-ccllulose, and ligno-ccllulose is a kind of Plant cell wall material, including lignin, cellulose With the mixed-matrix of hemicellulose.Cellulosic material is any biological material in a preferred aspect,.It is preferred at another 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 maize straw.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 willow.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 aquatile matter.In as used in this article, " aquatile matter " means in water The biomass generated by photosynthesis in raw environment.Aquatile matter 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 row 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 to the mature polypeptide of the coding present invention Polynucleotides expression is required nucleic acid sequence.Each regulating and controlling sequence can be for the polynucleotides for encoding 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 are for that can be each other It is natural or external source.These regulating and controlling sequences include but not limited to targeting sequencing, polyadenylation sequence, propeptide sequence, startup Son, signal peptide sequence and transcription terminator.At least, regulating and controlling sequence includes the termination signal of promoter and transcription and translation.Regulation and control Sequence can match the connector for being ready for use on and introducing specific restriction sites, 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)-Portugals callose 4- Endohydrolase (endo-1,4- β-D-glucan4-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 glucans such as cereal beta-D-glucans or xyloglucan of mixing and other plants containing cellulosic component The interior hydrolysis (endohydrolysis) of β -1,4 keys in material.Endoglucanase activity can be by measuring substrate viscosity It reduces or by reducing sugar test method (Zhang etc., 2006, Biotechnology Advances24:452-481) the reduction determined End increases to determine.It for the present invention, can be according to Ghose, 1987, Pure and Appl.Chem.59:The side of 257-268 Method, in pH5,40 DEG C use carboxymethyl cellulose (CMC) as substrate to determine endoglucanase activity.
Expression：Term " expression " includes any step for being related to polypeptide generation comprising but repaiied 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 multinuclears 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 activities that family member measures and be classified as glycoside hydrolase Families.These enzymes Structurally and functionally pattern 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 Chinese cassia trees Acyl-glycosylhydrolase (EC3.1.1.73) is catalyzed sugar (its of 4- hydroxy-3-methoxies cinnamoyl (asafoetide acyl) group from esterification In " natural biomass " substrate be usually arabinose) hydrolysis, to generate ferulic acid (4- hydroxy-3-methoxy Chinese cassia trees 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 acids p-nitrophenyl ester in 50mM sodium acetates pH5.0 is used to be determined as substrate.The feruloyl esterase of one unit Equal to can be in pH5, the enzyme amount of 25 DEG C of 1 micromole's p-nitrophenol anion of release per minute.
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；The wherein described 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 SEQ ID NO:4 at least 690 amino acid residues, for example, at least 730 amino acid residues or at least 770 amino acid residues.On the other hand, segment contains SEQ ID NO:6 at least 710 amino acid residues, 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 Duan Hanyou SEQ ID NO:10 at least 660 amino acid residues, 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 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 branched and straight-chain polysaccharide mixes 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 It measures.
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 blottings 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, 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 offspring different from parental cell.
Separation：Term " separation " means substance in the form of not in nature appearance or existing for environment.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) any to have passed through manually modified substance for the substance seen in nature；Or (4) It is any by relative to increasing the amount of the substance with its natural adjoint other components (for example, the recombination in host cell is produced 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 blottings 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, 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 ends N-, the ends C-, glycosylation, phosphorylation.In one aspect, according to pre- Survey SEQ ID NO:The amino acid 1 of 2 (P244Y5) to SignalP programs that 19 be signal peptide (Nielsen etc., 1997, Protein Engineering10:1-6), mature polypeptide is SEQ ID NO:2 amino acid 20 to 777.On the other hand, According to prediction SEQ ID NO:For the amino acid 1 of 4 (P244Y4) to the SignalP programs that 19 be signal peptide, mature polypeptide is SEQ ID NO:4 amino acid 20 to 825.On the other hand, according to prediction SEQ ID NO:The amino acid 1 of 6 (P241KM) is to 19 It is the SignalP programs of signal peptide, mature polypeptide is SEQ ID NO:6 amino acid 20 to 851.On the other hand, according to Predict SEQ ID NO:The amino acid 1 of 8 (P24QRU) to the SignalP programs that 21 be signal peptide, mature polypeptide is SEQ ID NO:8 amino acid 22 to 767.On the other hand, according to prediction SEQ ID NO:The amino acid 1 of 10 (P24GP2) is to 20 The SignalP programs of signal peptide, mature polypeptide are SEQ ID NO:10 amino acid 21 to 800.It is known in the art host Cell, which can generate the different mature polypeptides of two or more expressed by identical polynucleotides, (has 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 prediction SEQ ID NO:11 to 57 encoded signal peptide of nucleotide SignalP programs (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 prediction SEQ ID NO:31 to 57 encoded signal of nucleotide The SignalP programs of peptide, mature polypeptide encoded sequence are SEQ ID NO:The nucleotide 58 to 2668 of 3 or its cDNA sequence (D822JZ).On the other hand, according to prediction SEQ ID NO:The SignalP journeys of 51 to 57 encoded signal peptide of nucleotide Sequence, mature polypeptide encoded sequence are SEQ ID NO:The nucleotide 58 to 2829 (D72UE7) of 5 or its cDNA sequence.At another Aspect, according to prediction SEQ ID NO:The SignalP programs of 71 to 63 encoded signal peptide of nucleotide, mature polypeptide encoded sequence It is SEQ ID NO:The nucleotide 64 to 2634 (D13874) of 7 or its cDNA sequence.On the other hand, according to prediction SEQ ID NO:The SignalP programs of 91 to 60 encoded signal peptide of nucleotide, mature polypeptide encoded sequence are 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 blottings of standard carry out prehybridization and hybridize 12 to 24 hours.It will be carried at 55 DEG C using 2X SSC, 0.2%SDS Body material finally washs three times, 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 is carried out according to the Southern blottings of standard and is hybridized 12 to 24 hours.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 contain nucleic acid in a manner of being not present in (not otherwise exist) nature originally through modifying Section or its be synthesis, it includes one or more regulating and controlling sequences.
It is 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 are the GH61 polypeptides of the enhancing of the 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：1-50mg total proteins/pretreated the corns of g Cellulose in stalk (PCS), wherein total protein include the cellulose decomposition zymoprotein and 0.5-50%w/ of 50-99.5%w/w The protein of the GH61 polypeptides 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 proteins/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 capacity1.5L(Novozymes A/S,Denmark mixture) Source as cellulolytic activity.
GH61 polypeptides with cellulolytic enhancing activity reach 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 maize straw：Term " PCS " or " pretreated maize straw " mean by at heat and dilute sulfuric acid Reason, oxygenation pretreatment or the neutral pretreated cellulosic material from maize straw.
Sequence identity：Between parameter " sequence identity " two amino acid sequences of description 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 packages (EMBOSS:The European Molecular Biology Open Software Suite, Rice etc., 2000, Trends Genet.16:276-277), Needleman-Wunsch performed in the Needle programs of preferably 5.0.0 editions or more highest version Algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453) measure.The parameter used is opened for notch Point penalty (gap open penalty) 10, gap extension penalty (gap extension penalty) 0.5 and EBLOSUM62 (the EMBOSS versions of BLOSUM62) substitution matrix.Using Needle labeled as " highest identity (longest identity) " Output result (- nobrief options is used to obtain) is used as homogeneity percentage, and calculates as follows：
(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 packages (EMBOSS:The European Molecular Biology Open Software Suite, Rice etc., 2000, see on Text), in the Needle programs of preferably 5.0.0 edition or more highest version performed by Needleman-Wunsch algorithms (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 versions of NCBI NUC4.4) substitution matrix.The output knot of " highest identity " is labeled as using Needle Fruit (- nobrief options is used to obtain) is used as homogeneity percentage, and calculates as follows：
(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；The wherein described subsequence coding has xylobiase active Segment.In one aspect, subsequence contains SEQ ID NO:1 at least 1890 nucleotide, for example, at least 2010 nucleotide, Or at least 2130 nucleotide.On the other hand, subsequence contains SEQ ID NO:3 at least 2070 nucleotide, 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 SEQ ID NO:7 at least 1890 nucleotide, for example, at least 2010 nucleotide or at least 2370 nucleotide.At another Aspect, subsequence contain SEQ ID NO:9 at least 1980 nucleotide, 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 occupying certain position Amino acid is added later.
Very 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 is carried out according to the Southern blottings of standard and is hybridized 12 to 24 hours.Using 2X SSC, 0.2%SDS at 70 DEG C Carrier material is finally washed three times, 15 minutes every time.
Very 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 is carried out according to the Southern blottings of standard and is hybridized 12 to 24 hours.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) connections The material of the plant cell wall polysaccharides of skeleton.The xylan of terrestrial plant is have β-(1-4)-xylopyranose skeleton heteromeric Object, with short sugar chain branches.They include D- glucuronic acids or its 4-O- methyl ether, L-arabinose and/or a variety of packets The oligosaccharides of xylose containing D-, L-arabinose, D- or L- galactolipins and D-Glucose.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 technique of the present 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.Two kinds measurement xylanolytic activities basic methods include：(1) it measures 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 Agriculture86 (11):1636-1647；Spanikova and Biely, 2006,Glucuronoyl esterase-Novel carbohydrate esterase produced by Schizophyllum commune,FEBS Letters580(19):4597-4601；Herrmann,Vrsanska, Jurickova, Hirsch, Biely, and Kubicek, 1997, The beta-D-xylosidase of Trichoderma reesei is a multifunctional beta-D-xylan xylohydrolase,Biochemical Journal321:375-381。
Total pentosan degrading activity 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. Most common total pentosan degrading activity measuring method from the 4-O- methylglucuronic acid xylans of poly based on reduced sugar is generated, such as Bailey,Biely,Poutanen,1992,Interlaboratory testing of methods for assay of xylanase activity,Journal of Biotechnology23(3):Described in 257-270.Xylanase activity is also Can use 0.2%AZCL- araboxylans as substrate at 37 DEG C 0.01%X-100(4-(1,1,3,3- Tetramethyl butyl) phenyl-polyethylene glycol) and 200mM sodium phosphate buffers pH6 in determine.The xylanase activity of one unit Property is defined as at 37 DEG C, and pH6 is in 200mM sodium phosphate pH6 buffer solutions from the 0.2%AZCL- araboxylans as substrate 1.0 micromole's Bazurins (azurine) of generation per minute.
For the present invention, xylanolytic activities are made under following usual conditions by xylanolytic enzyme by measurement At the increase of birch xylan (Sigma Chemical Co., Inc., St.Louis, MO, USA) hydrolysis determine：1ml Reaction, 5mg/ml substrates (total solid), 5mg xylanolitics protein/g substrates, 50mM sodium acetates, pH5,50 DEG C, 24 is small When, such as Lever, 1972, A new reaction for colorimetric determination of carbohydrates,Anal.Biochem47:It is carried out using P-hydroxybenzoic acid hydrazides (PHBAH) measuring method described in 273-279 Glycan analysis.
Zytase：Term " zytase " means 1,4- β-D- xylans-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 bonds in xylan.With regard to the present invention Speech, xylanase activity use 0.2%AZCL- araboxylans as substrate at 37 DEG C, and pH6 is 0.01%It is determined in X-100 and 200mM sodium acetate buffers.The xylanase activity of one unit is defined as at 37 DEG C, PH6 is micro- from the generation 1.0 per minute of the 0.2%AZCL- araboxylans as substrate in 200mM sodium phosphate pH6 buffer solutions Mole Bazurin.
Detailed description of the invention
With the active polypeptide of xylobiase
In one embodiment, the present invention relates to the polypeptides of separation, with SEQ ID NO:6 or SEQ ID NO:8 Mature polypeptide have 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；With SEQ ID NO:2 mature polypeptide has at least 65%, for example, at least 70%, 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 ID NO:10 mature polypeptide 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 Homogeneity；The polypeptide has xylobiase activity.In one aspect, the polypeptide and SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10 mature polypeptide differs up to 10 amino acid, for example, 1,2, 3,4,5,6,7,8,9 or 10 amino acid.
The polypeptide of the present invention, which is preferably comprised or organized, becomes SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10 amino acid sequence or its allelic variant；Or it is it with active of xylobiase Section.On the other hand, the polypeptide includes or group becomes SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10 mature polypeptide.On the other hand, the polypeptide includes or group becomes SEQ ID NO:2 Amino acid 20 to 777, SEQ ID NO:4 amino acid 20 to 825, SEQ ID NO:6 amino acid 20 to 851, SEQ ID NO:8 amino acid 22 to 767 or SEQ ID NO:10 amino acid 21 to 800.
In another embodiment, the present invention relates to the polypeptides with the active separation of xylobiase, by multinuclear Thuja acid encodes, and the polynucleotides are in very low stringency condition, low stringency condition, medium stringency condition, medium-high stringency item Part, high stringency conditions, or hybridize with following under high stringency conditions very much：(i)SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:9 mature polypeptide encoded sequence, (ii) SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7 cDNA sequence, 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 Polynucleotides or its subsequence and SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10 polypeptide or its mature polypeptide or its segment design nucleic acid probe, with according to method well known in the art never The DNA of bacterial strain identification and clone's coding with the active polypeptide of xylobiase for belonging to or planting.Specifically, according to standard These probes can be used to hybridize with the genomic DNA of interested cell or cDNA by Southern immunoblot methods, with identification with From wherein detaching 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 at least 900 nucleotide length.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 Genome or other DNA of the separation of its isolation technics 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 NO:7 or SEQ ID NO:9 its mature polypeptide are compiled The clone or DNA of code sequence or the hybridization of its subsequence, the carrier material is used in Sounthern traces.
For the present invention, hybridization indicates polynucleotides very down to the nucleic acid with label under very high stringent condition Probe hybridizes, and the nucleic acid probe corresponds 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 NO:9 mature polypeptide encoded sequence, (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 ID NO:8 or SEQ ID NO:The polynucleotides of 10 polypeptide or 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, Its mature polypeptide encoded sequence or SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7 cDNA sequences Row or its mature polypeptide encoded sequence.
In another embodiment, the present invention relates to the polypeptides with the active separation of xylobiase, by multinuclear Thuja acid encodes, the polynucleotides and SEQ ID NO:5 or SEQ ID NO:The mature polypeptide encoded sequence of 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；With SEQ ID NO:1 or its cDNA sequence mature polypeptide encoded sequence have at least 65%, 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 SEQ ID NO:9 mature polypeptide encoded sequence has 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 ID NO:8 or SEQ ID NO:10 mature polypeptide one or more (such as several) positions include substitution, missing and/or The variant of insertion.In one embodiment, SEQ ID NO are imported:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:Amino acid substitution, missing and/or the quantity of insertion of 10 mature polypeptide 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, Folding and/or the 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 Acid substitution is known in the art, and by such as H.Neurath and R.L.Hill, 1979, in The Proteins, 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, which has, leads to the property that the physicochemical characteristics of polypeptide changes.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,Science244:1081-1085) 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, Science241:53-57；Bowie and Sauer, 1989, Proc.Natl.Acad.Sci.USA86:2152-2156；WO 95/17413；Or those of disclosed in WO 95/22625, into Row one or more amino acid substitution, missing and/or insertion are simultaneously tested.Other workable methods include fallibility PCR, bite Phage display (such as Lowman etc., 1991, Biochemistry30:10832-10837；U.S. Patent number 5,223,409；WO 92/06204) and regiondirected mutagenesis (region-directed mutagenesis) (Derbyshire etc., 1986, Gene46:145；Deng 1988, DNA7:127).
Mutagenesis/Shuffling Method can combine with high-throughput, auto-screening method to detect by host cell expression through cloning, Activity (Ness etc., 1999, Nature Biotechnology17 of the polypeptide of mutagenesis:893-896).The warp of encoding active polypeptide The DNA molecular of mutagenesis can be recycled from host cell and is sequenced rapidly using this field standard method.These methods allow quickly true 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 be cut, and other in which peptide fusion is more in the present invention's 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 to be blended in the polynucleotides of the present invention Peptide.The technology for generating fused polypeptide is known in the art, and includes that connection encodes the coded sequence of polypeptide 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 It is built using interior albumen (intein) technology, wherein fusions generate that (Cooper etc., 1993, EMBO J.12 upon translation: 2575-2583；Dawson etc., 1994, Science266:776-779).
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, Biotechnology13:498-503；With Contreras etc., 1991, Biotechnology9:378-381；Eaton etc., 1986,Biochem.25:505-512)；Collins-Racie etc., 1995, Biotechnology13:982-987；Carter Deng 1989, Proteins:Structure,Function,and Genetics6:240-248；And Stevens, 2003, Drug Discovery World4:Site in 35-48.
Source with the active polypeptide of xylobiase
The present invention's has the active polypeptide of xylobiase can be obtained from the microorganism of any category.With regard to the present invention Speech should be the polypeptide by polynucleotide encoding by described for term " obtained from " related with given source, the meaning herein 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 polypeptides.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 for above-mentioned kind, the present invention includes complete and imperfect stage (perfect and Imperfect states) and other taxonomic equivalents (equivalent), such as phorozoon (anamorph), and nothing By their known kind of names.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 can be utilized from other sources, include being detached 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 another microorganism of screening genomic DNA or cDNA library or mixed DNA sample obtain coding said polypeptide Polynucleotides.Once with probe in detecting to the polynucleotides of coding polypeptide, so that it may to use known to those of ordinary skill in the art Technology the polynucleotides are detached or are cloned (see, e.g., Sambrook etc., 1989, see above).
Polynucleotides
The present invention is also related to encode the polynucleotides of the separation of the polypeptide of the present invention as described herein.
Technology for detaching or cloning polynucleotides is well known in the art, and include from genomic DNA or CDNA, or combinations thereof separation.It can be by using well known PCR (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 Belong to, the bacterial strain or the related organisms clone polynucleotides of Rhizomucor or thermophilic ascomycete category, 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 synthesis and the essentially similar polypeptide of the polypeptide Can be required.Term refers to the non-naturally occurring form of polypeptide with the polypeptide " essentially similar ".These polypeptides may be with Some engineered modes and different from the polypeptide that is detached from its natural origin, for example, specific activity, thermal stability, optimal pH Etc. different variants.It can be as SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9 mature polypeptide sequence or SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7 Mature polypeptide sequence the polynucleotides that present of cDNA sequence on the basis of and/or pass through and introduce the substitution of following nucleotide：Institute Stating substitution does not lead to the change of polypeptid acid sequence, but meets the codon usage for the host organisms for being intended to generate enzyme； Or the nucleotide substitution of different amino acid sequences can be generated to build variant by importing.About the general of nucleotide substitution It states, see, e.g., Ford etc., 1991, Protein Expression and Purification2:95-107.
Nucleic acid construct
The invention further relates to the nucleic acid construct of the polynucleotides comprising the present invention, the polynucleotides and one or more (such as several) regulating and controlling sequence is operably connected, the regulating and controlling sequence in 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 to be operated on it before nucleotides inserted carrier.Multinuclear 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 the host cell institute for expressing the polynucleotides for encoding the polypeptide of the present invention The polynucleotides of identification.Promoter contains the transcription regulating nucleotide sequence of the expression of direct polypeptide.Promoter can be in host cell Any polynucleotides of middle display transcriptional activity, 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.
For instructed in bacterial host cell the present invention nucleic acid construct transcription suitable promoter example 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 genes (Agaisse and Lereclus,1994,Molecular Microbiology13:97-107), E. coli lac operon, Escherichia coli trc Promoter (Egon etc., 1988, Gene69:301-315), 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 USA75:3727-3731) and tac promoters (DeBoer etc., 1983, Proc.Natl.Acad.Sci.USA80:21-25).Other promoter exists " Useful proteins from Recombinant bacteria " are in Gilbert etc., 1980, Scientific American, 242:In 74-94；With Sambrook etc., 1989, see above described in.The example of Gene expression is disclosed in WO 99/43835.
The example of suitable promoter for instructing the nucleic acid construct of the present invention to be transcribed 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 Mucors (Rhizomucor miehei) Lipase, Man Hegen Mucors 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 promoters Targeting sequencing substituted by the untranslated targeting sequencing of the gene of aspergillus triose-phosphate isomerase；Non-limiting examples packet The promoter for including modification, the gene from Aspergillus ni ger neutral alpha-amylase, wherein untranslated targeting sequencing is by aspergillus nidulans Or the untranslated targeting sequencing 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 dehydrogenases (ADH1, ADH2/GAP), saccharomyces cerevisiae triose-phosphate isomerase (TPI), brewing yeast metallothionein (CUP1) and saccharomyces cerevisiae glycerol 3-phosphate acid kinase.For the other useful promoters of yeast host cell by Romanos etc., 1992,Yeast8:423-488 is described.
Regulating and controlling sequence can also be transcription terminator, be 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.
The preferred terminator of 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).
The preferred terminator of filamentous fungal host cell is obtained from the gene of following enzyme：Aspergillus nidulans acetamidase, Aspergillus nidulans anthranilate synthase, aspergillus niger glucoamylase, aspergillus niger alpha-Glucosidase, oryzae TAKA amylase, 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.
The preferred terminator of 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 dehydrogenases.For yeast host cell other useful ends 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 genes (WO And bacillus subtilis SP82 genes (Hue etc., 1995, Journal of Bacteriology177 94/25612):3465- 3471)。
Regulating and controlling sequence can also be suitable targeting sequencing, for the important mRNA untranslateds of the translation for host cell Area.Targeting sequencing is operably connected to 5 '-ends of the polynucleotides of coding polypeptide.It may be used at functional in host cell Any targeting sequencing.
The preferred targeting sequencing of filamentous fungal host cell is obtained from the gene of following enzyme：Oryzae TAKA amylase With aspergillus nidulans triose-phosphate isomerase.
The suitable targeting sequencing of 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 can also be polyadenylation sequence, be the sequence being operably connected with 3 ' ends of polynucleotides Row, 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.
The preferred polyadenylation sequence of 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.
For the useful polyadenylation sequence of yeast host cell by Guo and Sherman, 1995, Mol.Cellular Biol.15:5983-5990 is described.
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, appointing for the secretory pathway for instructing the polypeptide of expression to enter host cell can be used What signal coding sequence.
The signal peptide coding that the gene of enzyme obtains is followed from for the effective signal coding sequence of bacterial host cell Sequence：Bacillus NCIB11837 productions 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 Reviews57:109-137 is described.
The signal peptide that the gene of enzyme obtains is followed from for the effective signal coding sequence of filamentous fungal host cell 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 proteases at Humicola insolens endoglucanase V.
The useful signal peptide of 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 propeptide code sequence, propetide of the coding positioned at 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 from preceding polypeptide by the catalysis or self-catalysis of propetide 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 Mucors 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 regulatory 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, including modulating compound presence and be turned on and off Those of system.Regulatory sequence in prokaryotic system includes lac, tac and trp operator system.In yeast, it can be used ADH2 systems or GAL1 systems.In filamentous fungi, aspergillus niger glucose starch enzyme promoters, aspergillus oryzae TAKA α-shallow lake can be used Powder enzyme promoters and aspergillus oryzae glucose starch enzyme promoters, trichoderma reesei cellobiohydrolase I promoters and trichoderma reesei are fine Tie up disaccharide-hydrolysing enzymes II promoters.Other examples of regulatory sequence are the sequences of those permission gene magnifications.In eukaryotic system, These regulatory sequences are included in the dihydrofolate reductase gene expanded in the presence of amethopterin (methotrexate), and with weight The metallothionein gene of metal (with heavy metal) amplification.In these cases, the polynucleotides for encoding polypeptide will It is operably connected with regulatory sequence.
Expression vector
The invention further relates to recombinant expression carrier, the recombinant expression carrier includes the polynucleotides of the present 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 It may include one or more (such as several) convenient restriction site to allow to be inserted into or take in these sites to state expression vector The polynucleotides of generation coding polypeptide.Alternatively, can include the multinuclear glycosides by being inserted into the carrier appropriate for expressing The nucleic acid construct or polynucleotides of acid express the polynucleotides.During preparing expression vector, by coded sequence It is placed in carrier, to be 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), replicate only Chromosome replication is stood on, for example, plasmid, extra-chromosomal element, minichromosome (minichromosome) or artificial chromosome. Carrier can contain any means (means) for ensuring self-replacation.Alternatively, carrier, which can be one kind, being 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 genes, 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 not limited to ADE2, HIS3, LEU2, LYS2, MET3, TRP1 and URA3.Selected marker for filamentous fungal host cell includes but not limited to adeA (ribose phosphate aminooimidazole amber carboxylics 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 benzoyls Acid synthase (anthranilate synthase)) and their equivalent.It is that structure nest is bent to be preferably used in Aspergillus cell Mould or aspergillus oryzae amdS and pyrG gene and streptomyces hygroscopicus (Streptomyces hygroscopicus) bar genes.It is preferred that What it is for trichoderma cell is adeA, adeB, amdS, hph and pyrG gene.
Selected marker can be the double selectivity tagging system described in WO 2010/039889.In one aspect, institute It is hph-tk double selectivity tagging systems to state double selectivity label.
The carrier, which preferably comprises, allows vector integration to enter host cell gene group or carrier in cell independently of gene The element of the autonomous replication of group.
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.It is useful for instructing passing through alternatively, carrier can contain Homologous recombination is integrated into the additional polynucleotides of the exact position in host cell gene group chromosome.In order to increase accurate The possibility that position is integrated, integrated element should have high degree of sequence identity containing sufficient amount of with corresponding target sequence 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 the base of non-homologous re-combination to host cell Because in group.
For autonomous replication, carrier can include also replication orgin, enable carrier in the host cell from It replicates mainly.Replication orgin can be any plasmid replicon of the mediation autonomous replication functioned in cell (replicator).Term " replication orgin " or " plasmid replicon " mean the multinuclear 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 allow plasmid pUB110, pE194, pTA1060 and pAM β 1 replicated 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, Gene98: 61-67；Cullen etc., 1987, Nucleic Acids Res.15:9163-9175；WO 00/24883).Detach AMA1 genes It can be completed according to the method being disclosed in WO 00/24883 with plasmid of the structure comprising the gene or carrier.
It can be by the polynucleotides Insertion Into Host Cell of the present invention of more than one copy to increase the generation of polypeptide.Multinuclear 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.
In the method for building the recombinant expression carrier of the present invention it 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 the polynucleotides of the present 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 Carrier maintains outside the chromosome as chromosomal integrant or as self-replacation as previously described for the construct or carrier.Art Language " host cell " includes any offspring for being different from parental cell due to the mutation occurred 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 recombinate any cell useful in generation in polypeptide of the invention, 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 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, Biotechniques6: 742-751) or engagement (see, e.g., Koehler and Thorne, 1987, J.Bacteriol.169:5771-5278).It can lead to It crosses following method realization and DNA is introduced into Bacillus coli cells：Protoplast transformation (see, e.g., Hanahan, 1983, J.Mol.Biol.166:557-580) or electroporation is (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), engage (see, e.g., Mazodier etc., 1989, J.Bacteriol.171:3583-3585), or transduction (see, e.g., Burke etc., 2001, Proc.Natl.Acad.Sci.USA98:6289-6294).It can realize that DNA is introduced into pseudomonas is thin by the following method Born of the same parents：Electroporation (see, e.g., Choi etc., 2006, J.Microbiol.Methods64:391-397) or engagement is (referring to example Such as, 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 herein that " yeast ", which is used in, 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 fat 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 fat, 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, it is 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 EP 238023 and Yelton Deng 1984, Proc.Natl.Acad.Sci.USA81:1470-1474 and Christensen etc., 1988, Bio/ Technology6:Described in 1419-1422.For converting the appropriate method of Fusarium strain by Malardier etc., 1989, Gene78:147-156 and WO 96/00787 is described.The method transformed yeast described by following document can be used：Becker and Guarente, in Abelson, J.N. and Simon, M.I. is compiled, Guide to Yeast Genetics and Molecular Biology,Methods in Enzymology,Volume194,pp182-187,Academic Press,Inc.,New York；Ito etc., 1983, J.Bacteriol.153:163；With Hinnen etc., 1978, Proc.Natl.Acad.Sci.USA75: 1920。
Production method
The invention 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 It is Rhizomucor cell to state cell.On the other hand, the cell is Rhizomucor pumillus cells.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 of the polypeptide for generating the present invention comprising：(a) in the item for helping to create polypeptide The recombinant host cell of the present invention is cultivated under part；Optionally (b) recycles the polypeptide.
The host cell is trained using methods known in the art in the nutrient medium for being suitable for generating the polypeptide It supports.For example, can by the shaking flask culture in suitable culture medium and under conditions of allow to express and/or detach 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).
Polypeptide can be detected using the method known in the art for the polypeptid specificity.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.
Polypeptide can be recycled using methods known in the art.For example, polypeptide can be by conventional method from nutrition culture It is recycled in base, the conventional method includes but not limited to collection, centrifuges, filtering, extraction, spray drying, evaporates or precipitate.One A aspect has recycled the whole beer of the polypeptide comprising the present 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 extraction (see, e.g., Protein Purification, Janson and Ryden is compiled, VCH Publishers, New York, and 1989).
On the other hand, polypeptide is not recycled, but uses the host cell of the present invention of the expression polypeptide as institute State the source of polypeptide.
Plant
The invention further relates to the plants of separation, for example, genetically modified plants, plant part or plant cell, it includes this hairs Bright polynucleotides, to express and generate the polypeptide with recyclable amount.Polypeptide can be recycled from plant or plant part.Or Person (as such) can be used for the plant containing the polypeptide or plant part to improve food or quality of the fodder, example as it is 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 Potato, sugar beet (sugar beet), pea, beans (bean) and (cruciferous) of soybean (soybean) and Cruciferae plant 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 detached for promoting the specific tissue of the application of the present invention and cell also to be recognized 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 also have these plants, plant part and plant cell offspring.
The genetically modified plants or plant cell for expressing polypeptide can build according to means known in the art.In brief, lead to It crosses following method and builds the plant or plant cell：The one or more expression constructs for encoding polypeptide are imported into plant host Genome or Chloroplast gene, and be genetically modified plants or plant by the modified plant of gained or plant cell breeding Cell.
Expression construct advantageously include coding polypeptide polynucleotides nucleic acid construct, the polynucleotides with The regulatory 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 can include that expression structure is incorporated in the plant cell for the useful selected marker of plant identification cell It builds body and the construct is introduced into necessary DNA sequence dna in the plant (the latter depends on the DNA introducing methods used).
The selection of regulatory sequence, for example, promoter and terminator sequence and optional earth signal or transit sequence selection, lift For example, based on it is expected 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 outcome can target it is specific Tissue or plant part such as seed or leaf.Regulatory sequence is by such as Tague etc., 1988, Plant Physiology86:506 It is described.
For constructive expression, 1 promoter (Franck of 35S-CaMV, maize ubiquitin 1 or rice actin can be used Deng 1980, Cell21:285-294, Christensen etc., 1992, Plant Mo.Biol.18:675-689；Zhang etc., 1991,Plant Cell3:1155-1165).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), the seed specific promoters such as paddy from rice Albumen (glutelin), alcohol soluble protein (prolamin), globulin (globulin) or albumin (albumin) promoter (Wu Deng 1998, Plant Cell Physiol.39:885-889), legumin (legumin) B4 and broad bean (Vicia are come from Faba broad bean promoter (Conrad etc., 1998, J.Plant Physiol.152 of unknown Seed Storage Protein gene):708- 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 promoters 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 promoters (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 From the aldP gene promoters of rice (Kagaya etc., 1995, Mol.Gen.Genet.248:668-674) or wound induction open Mover, such as potato pin2 promoters (Xu, 1993, Plant Mol.Biol.22:573-588).Similarly, the startup 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.
Any other part of selected marker and expression construct can be selected from those of available in the art.
Nucleic acid construct is imported into Plant Genome according to routine techniques known in the art, the routine techniques includes soil Earth Bacillus (Agrobacterium) mediate convert, virus-mediated conversion, microinjection (microinjection), grain Son bombardment, Biolistic transformation and electroporation (Gasser etc., 1990, Science244:1293；Potrykus,1990,Bio/ Technology8:535；Shimamoto etc., 1989, Nature338:274).
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/Technology10:667-674).A kind of alternative of transforming monocots is turned based on protoplast Change, such as by Omirulleh, 1993, Plant Mol.Biol.21:Described in 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, using the cotransformation of independent T-DNA constructs with there are two or passing through specific recombinase site spy Selection gene is cut off anisotropicly.
It, can also be by that will have structure other than directly directly converting specific plant genotype with the construct of the present invention The plant of body carrys out prepare transgenosis plant with the second plant hybridization for lacking the construct.For example, polypeptide will can be encoded Construct introduces specified plant kind by hybridization, and is not necessarily to directly convert the plant of the given kind at all.Therefore, this hair It is bright not only to cover from the plant according to inverted cell Direct Regeneration of the invention, further include the offspring of such plant (progeny).As used in this article, offspring can refer to the descendant of the mother plant any generation prepared according to the present invention (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 examples of such step are 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).
Genetic marker can be used 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 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) does not have the genetic background needed for non-agronomy but have for this When the plant of required character is with 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 is made to penetrate into needed for specific genetic background Generation number minimized.
The present invention is also related to the method for generating the polypeptide of the present 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 encodes the present invention's 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, be inserted into, destroy, substitute or lack) can be used by reducing or eliminating multinuclear The expression of thuja acid builds mutant cell.The polynucleotides are inactivations in a preferred aspect,.It is to be finished or inactivation Polynucleotides can be, for example, code area or its part to active key, or the regulating element needed for 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 not limited to targeting sequencing, polyadenylation sequence, propetide sequence Row, 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 combinations 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 nitrosoguanidines (MNNG), O- methyl hydroxylamines, nitrous acid, ethyl methanesulfonate (ethyl methane Sulphonate) (EMS), sodium hydrogensulfite, formic acid and nucleotide analog.
When using such agents, usually the mutagenesis is carried out by the following method：Exist under suitable conditions selected Mutagens when incubate parental cell to be mutagenic, and screen and/or selection show it is that gene expression is reduced or without gene expression Mutant cells.
The modification of polynucleotides or inactivation can by one or more of insertion, substitution or missing gene nucleotide 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 by what site-directed mutagenesis or PCR were generated according to methods known in the art This modification or inactivation may be implemented in change.Although the modification described in theory 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 based on gene substitution, gene delection or gene The technology of destruction.For example, in gene disruption method, the nucleic acid sequence that will correspond to endogenous polynucleotides carries out mutagenesis in vitro 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 for the expression with 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 the polynucleotides of the present 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, and it includes SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:A part for 9 mature polypeptide encoded sequence, 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 similar or identical sequence, include the degradation of endogenous mRNA.When cell is exposed to dsRNA When, the mRNA from homologous gene interferes the process of (RNAi) by degradation selectivity by referred to as RNA.
The dsRNA of the present invention can be used for gene silencing.In one aspect, the present invention provides the dsRNAi for using the present invention The method of degradation selectivity RNA.This method can be implemented in vitro, in vitro or in vivo.In one aspect, the dsRNA molecules can For the mutation that systematic function is lost in cell, organ or animal.It is used to prepare and uses dsRNA molecule degradation selectivities 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 regulation and control The silence 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 Become cell；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 the coding copied more than one natural or heterologous polypeptide the multinuclear glycosides 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 is such as hormone, growth factor, receptor.Term " eukaryon polypeptide " includes not only 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 tolerances 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.
Zymotic fluid formulation or cell composition
The present invention is also related to zymotic fluid formulation and cell composition, and it includes the polypeptides of the present invention.The zymotic fluid production Object further includes other ingredients for zymotechnique, such as cell (includes the gene of the polypeptide containing the coding present 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 " zymotic fluid " 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, is incubated 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 zymotic fluid.The zymotic fluid can contain There is the content for not being classified or being classified of the fermented material obtained when fermenting and terminating.Typically, zymotic fluid is unassorted, And include after removal (such as passing through centrifugation) microbial cell (such as filamentous fungal cells) existing for used culture medium with And cell fragment.In some embodiments, the zymotic fluid 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 zymotic fluid 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 the aforementioned mixture of two or more, and described second Organic acid composition be benzoic acid, cyclohexane-carboxylic acid, 4- methylvaleric acids, phenylacetic acid, they salt or it is aforementioned two or more Mixture.
In one aspect, the composition contains organic acid, and optionally further contain the cell being killed and/or Cell fragment.In one embodiment, the cell that has been killed described in being removed from the full nutrient solution for killed cell and/or Cell fragment is free of the composition of these components to provide.
The zymotic fluid 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 zymotic fluid 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 zymotic fluid formulation or cell composition are also It 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 incubated 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 is permeated 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.
The full nutrient solution formulation and cell composition of the present invention 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 the composition of the present invention.The dosage and composition of the composition make Other conditions can be based on means known in the art and determine.
Enzymatic compositions
The invention further relates to the compositions of the polypeptide comprising the present 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 the polypeptide of the present 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 It closes object and 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 of the preferable use of the composition of the present 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 Materials'use methods known in the art detach, 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 the present 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 saccharification 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 saccharified with enzymatic compositions in the presence of present invention polypeptide active with xylobiase.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.
The technique of the present invention can be used for cellulosic material or material containing xylan being saccharified 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 processing of cellulosic material according to the present invention or the material containing xylan can use the conventional method of this field complete At.It is configured to carry out according to any standard biologic matter process equipment of invention operation in addition, the technique of the present invention can be used.
Hydrolyze (saccharification) and ferment, respectively or simultaneously, including but not limited to, the hydrolysis and fermentation (SHF) of separation, 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 The hydrolysis ferment (SHCF), mixed and common fermentation (HHCF), and directly microorganism conversion (DMC), otherwise referred to as joint biology It processes (consolidated bioprocessing, CBP).SHF is using the processing step of separation with first by cellulosic material Enzyme hydrolysis is fermentable sugars, for example, glucose, cellobiose and pentose monomers, then become ethyl alcohol by 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 volumes, Taylor＆Francis, Washington, DC, 179-212).SSCF includes more Common fermentation (Sheehan, J. and Himmel, M., 1999, Enzymes, the energy and the environment of kind sugar:A 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 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, high temperature enzyme process is saccharified, and is then resistant in fermentation strain relatively low 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.Reviews66:506-577).Herein it is understood that any side as known in the art Method, including pretreatment, enzyme hydrolysis (saccharification), fermentation or combination thereof, the technique that can be used in implementing the present 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.Technology25:33- 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 with the intensively stirred reactor 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：Fluid bed, up-flow layer (upflow Blanket), the reactor of immobilization and the extruder type for hydrolyzing and/or fermenting.
Pretreatment.In the implementation of the technique of the present invention, any preprocessing process known in the art can be used to 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 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 pretreatments.
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 at the same time 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 enzymes 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 Technology855:1-33；Galbe and Zacchi, 2002, Appl.Microbiol.Biotechnol.59:618-628；It is beautiful State patent application No.20020164730).During steam pre-treatment, hemicellulose acetyl group is cut open, and is obtained Sour self-catalysis hemicellulose fraction hydrolysis become 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 crystal fibre element to amorphous cellulose.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 mixing 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, the residence time is from 1 hour to several with calcium oxide or calcium hydroxide 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, is usually carried out 5-15 minutes at 180-200 DEG C, and oxidant such as hydrogen peroxide is added Or over-pressed 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% dries Matter, such as 2-30% dry matters or 5-20% dry matters 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 by being extracted 30-60 minutes at 160-200 DEG C with hydrous ethanol (40-60% ethyl alcohol) 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 Technology96:673-686, 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 typically sulfuric acid, but can also use other acid, 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 ranges 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 is happened 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：It is big that term " mechanical pretreatment " or " physics pretreatment " refer to any promotion particle The pretreatment of small reduction.For example, such pretreatment can relate 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 About 150 to the range of about 250psi pressure.On the other hand, high temperature refers to about 100 to 300 DEG C, and 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 sequentially can be carried out or are carried out at the same time.
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 volumes, 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 Series566, American Chemical Society, Washington, DC, the 15th chapter；Gong, C.S., Cao, N.J., Du, J., and Tsao, G.T., 1999, Ethanol production from renewable resources, in Advances in Biochemical Engineering/Biotechnology, Scheper, T. 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 using enzymatic compositions in the present invention 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, hydrolysis is in the activity 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 in stirred tank reactor or fermentation tank under controlled pH, temperature and mixing condition.Properly Processing time, temperature and pH conditions 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 the range of 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. Ranges of the pH in preferably from about 3 to about 8, for example, about 3.5 to about 7, about 4 to about 6, or 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 convert cellulosic material or the material containing xylan.
In one aspect, the enzymatic compositions include or also include one or more (such as several) eggs selected from the group below In vain：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 It includes endoglucanase and the polypeptide with cellulolytic enhancing activity to state enzymatic compositions.On the other hand, the enzyme Composition includes cellobiohydrolase and the polypeptide with cellulolytic enhancing activity.On the other hand, the enzyme group It includes β-glucosyl enzym and the polypeptide with cellulolytic enhancing activity to close object.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- are 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- glucuronidases). 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 It includes zytase to state enzymatic compositions.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 technique of the present invention, enzyme can be saccharified, and be saccharified and ferment, or be added 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.
Can be any applicable form for the enzyme in present invention process, such as zymotic fluid formulation, cell composition contain Or the cell pyrolysis liquid without cell fragment, half purifies or the enzyme prepared product of purifying, or the source as enzyme host cell.Institute It can be dry powder or particle to state enzymatic compositions, non-dusting particle, and liquid stabilizes liquid or stabilizes shielded enzyme.Liquid Enzyme prepared product can according to established technique, such as by add 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 materials 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, and 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 per g cellulosic materials 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 enzymes 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 polypeptides with cellulolytic enhancing activity are (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 Row recombinate the enzyme of generation for example, being lacked with one or more (such as several), the amino acid of insertion and/or substitution, It is the segment and/or mutant or the enzyme that is generated by amino acid Shuffling Method known in the art of natural acid sequence. What is covered in the meaning of native enzyme is natural variant, and cover in the meaning of external enzyme be recombination (such 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 can also be 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 fat, 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, being encoded by cloning The DNA sequence dna of the independent component simultaneously then with the DNA sequence dna transformed cells and is expressed (see, e.g., WO91/ in host 17243 and WO91/17244) and generate.The host is preferably heterologous host (enzyme is external source to host), but the host Can also be homologous host under certain condition (enzyme is natural to host).Homofil element decomposition of protein can also pass through Such protein is purified from zymotic fluid 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, CELLICTM Ctec (Novozymes A/S)、CELLICTM CTec2(Novozymes A/S)、CELLUCLASTTM(Novozymes A/S)、 NOVOZYMTM188(Novozymes A/S)、CELLUZYMETM(Novozymes A/S)、CEREFLOTM(Novozymes A/S) And ULTRAFLOTM(Novozymes A/S), ACCELERASETM(Genencor Int.)、LAMINEXTM(Genencor Int.)、SPEZYMETMCP (Genencor Int.),NL(DSM)、S/L100 (DSM), ROHAMENTTM7069W(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 the technique of the present 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 IIIs (WO 05/093050)；It is poly- with Thermobifida fusca inscribes Portugal Carbohydrase V (WO 05/093050).
The example that can be used for the fungal endoglucanase of the present invention includes but are not limited to, and trichoderma reesei inscribe Portugal is poly- Carbohydrase I (Penttila etc., 1986, Gene45:253-263, trichoderma reesei Cel7B endoglucanase is (GENBANKTMIt logs in Number M15665)；Trichoderma reesei endoglucanase II (Saloheimo etc., 1988, Gene63:11-22), trichoderma reesei Cel5A EG II (GENBANKTMAccession number M19373)；Trichoderma reesei endoglucanase III (Okada etc., 1988, Appl.Environ.Microbiol.64:555-563；GENBANKTMAccession number AB003694)；Trichoderma reesei endo-glucanase Enzyme V (Saloheimo etc., 1994, Molecular Microbiology13:219-228；GENBANKTMAccession number Z33381)； Microorganism Aspergillus aculeatus endoglucanase (Ooi etc., 1990, Nucleic Acids Research18:5884)；Valley aspergillus (Aspergillus kawachii) endoglucanase (Sakamoto etc., 1995, Current Genetics27:435- 439)；Carrot soft rot Erwinia (Erwinia carotovara) endoglucanase (Saarilahti etc., 1990, Gene90:9-14)；Sharp fusarium endoglucanase (GENBANKTMAccession number L29381)；Grey humicola lanuginosa thermoidea mutation Endoglucanase (GENBANKTMAccession number AB003107)；Melanocarpus albomyces endoglucanases (GENBANKTMAccession number MAL515703)；Neuraspora crassa endoglucanase (GENBANKTMAccession number XM_324477)；It is special Different humicola lanuginosa endoglucanase V；Thermophilic fungus destroyed wire CBS117.65 endoglucanases；Basidiomycetes (basidiomycete) CBS495.95 endoglucanases；Basidiomycetes CBS494.95 endoglucanases；In the autochthonal mould NRRL8126CEL6B of shuttle spore Cut dextranase；The autochthonal mould NRRL8126CEL6C endoglucanases of shuttle spore；Autochthonal shuttle spore mould NRRL8126CEL7C inscribes Portugal Dextranase；The autochthonal mould NRRL8126CEL7E endoglucanases of shuttle spore；The autochthonal mould NRRL8126CEL7F endo-glucanases of shuttle spore Enzyme；Cladorrhinum foecundissimum ATCC62373CEL7A endoglucanases；And Li's Trichoderma strains No.VTT-D-80133 endoglucanases (GENBANKTMAccession 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 cellobiohydrolases 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,Gene173:287-288), 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 oryzaes β-Portugal Glucosides enzyme variants BG fusion proteins (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, Biochem.J.316:In many glycosyl hydrolase families of the classification of 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 polypeptides with cellulolytic enhancing activity can be used to be combined as enzyme The component of object.
The example of the GH61 polypeptides 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 polypeptides, from thermophilic loose mould (WO 2011/005867), thermophilic ascomycete category strain (WO 2011/039319), Penicillium The GH61 polypeptides of strain (WO 2011/041397) and Thermoascus crustaceous (WO 2011/041504).
In one aspect, the GH61 polypeptides with cellulolytic enhancing activity are 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 polypeptides with cellulolytic enhancing activity are 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 maize straw (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 acids； 4- tertiary butyl -5- methoxyl group -1,2- benzenediols；Pyrogallol；Gallic acid；Methyl -3,4,5-trihydroxy benzoic acid；2,3,4- Trihydroxybenzophenone；2,6- syringol；Sinapic acid；3,5- dihydroxy-benzoic acids；The chloro- 1,2- benzenediols of 4-；4- nitre Base -1,2- benzenediols；Tannic acid；Progallin A；Oxyacetic 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 's-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 Including the ring that one or more (such as several) are other, 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 to replace comprising the Heterocyclylalkyl module optionally replaced or optionally 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, oxadiazolyl, oxazolyls, 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, benzoxazine (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 ethyls) -3,4- dihydrofuran -2 (5H) -one；4- hydroxy-5-methyl base -3- furanones；5- hydroxyls -2 (5H)-furans Ketone；[1,2- dihydroxy ethyls] 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-ones；Or their salt or solvate.
The nitrogenous compound can be any the suitable compound with one or more nitrogen-atoms.In one aspect, institute It includes amine, imines, azanol or nitrous oxide (nitroxide) module to state nitrogenous compound.The non-limiting reality of nitrogenous compound Example includes acetoxime；Violuric acid；Pyridine -2- aldoximes；Ortho-Aminophenol；1,2- phenylenediamines；2,2,6,6- tetramethyl -1- piperidyls Oxygen (piperidinyloxy)；5,6,7,8- tetrahydrobiopterins；6,7- dimethyl -5,6,7,8- tetrahydrochysene pterins；With Malaysia acyl Amino acid；Or their salt or solvate.
The naphtoquinone compounds can be any suitable compound for including 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-1s, 4- benzoquinones Or ubiquinone0；2,3,5,6- tetramethyl -1,4- benzoquinones or duroquinone；1,4- dihydroxy anthraquinones；3- hydroxyl -1- methyl - 5,6- indoline diketone or adrenochrome；4- tertiary 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 for including 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- propanethiols；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, with 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 handled in slurry And/or hemicellulosic materials or its monosaccharide, such as xylose, arabinose, mannose, generated solution phase, i.e. water phase have Machine phase or combinations thereof, and its soluble content.The liquor that cellulose decomposition for GH61 polypeptides 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 detaches to generate.Such conditional decision passes through during by Cellulase preparation hydrolysis fiber cellulosic material The degree that cellulose decomposition obtained by the combination of liquor and GH61 polypeptides enhances.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 about 10g per g celluloses, 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 10-2G is per g celluloses.
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 SHEARZYMETM (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)、HSP6000Xylanase(DSM)、DEPOLTM333P(Biocatalysts Limit,Wales, UK)、DEPOLTM740L (Biocatalysts Limit, Wales, UK) and DEPOLTM762P(Biocatalysts Limit, Wales,UK)。
The example that can be used for the zytase of present invention process includes but 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) NRRL8126 (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 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 sections 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 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) DSM1800 (WO 2009/073709), it Hypocrea jecorina (Hypocrea jecorina) (WO 2005/001036), thermophilic ruins Silk mould (Wo 2010/014880), Neuraspora crassa (UniProt accession number q7s259), phaeosphaeria nodorum (Phaeosphaeria Nodorum) the acetyl xylan of (Uniprot accession number Q0UHJ1) and the autochthonal mould NRRL8126 of shuttle spore (WO 2009/042846) Esterase.
The example that can be used for the feruloyl esterase of present invention process includes but 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 not limited to come from aspergillus niger (Aspergillus niger) (GeneSeqP accession number AAR94170), Humicola insolens (Humicola insolens) The Arab of DSM1800 (WO 2006/114094 and WO 2009/073383) and M.giganteus (WO 2006/114094) Furanoside enzyme.
The example that can be used for the alpha-glucuronidase of the method for the present invention includes but 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 sections 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 Tibetan 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 fermentation can be the method for 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 detach 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).Enzyme as obtained by above method generation 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 hydrolysis or material containing xylan." fermentation " or " fermentation side Method " refers to any fermentation process or any method for including fermentation step.Fermentation process further includes for the industrial (example of consumer goods alcohol Such as, beer and grape wine), the fermentation process of dairy husbandry (for example, fermented dairy product), leather industry and tobacco.Fermentation condition according to The desired tunnings 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, for example, ethyl alcohol by fermenting organisms (such as yeast) fermentation.As described herein, hydrolyze (saccharification) and Fermentation can be separately or simultaneously.
Any suitable cellulosic material through hydrolysis or poly- containing wood can be used in the fermentation step for implementing the present invention Sugared material.The material is selected generally according to required fermented product (that is, the substance to be obtained 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 The culture medium used in 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 combination thereof.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 example such as Lin etc. of the bacterium and fungi fermentation organism of generation ethyl alcohol, 2006, Appl.Microbiol.Biotechnol.69:Described in 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 CBS5773.Preferred pentose fermentation yeast includes pipe capsule saccharomyces (Pachysolen), preferably thermophilic tan The bacterial strain of pipe capsule yeast (Pachysolen tannophilus).Can not the biology of ferment pentoses such as xylose and arabinose can Ferment pentoses by means known in the art genetic modification.
Can include at the bacterium of ethyl alcohol by hexose and pentose fermentation effectively, 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 is tieed up 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 Candidas.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 production 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 BIOFERMTMAFT and XR (NABC-North American Bioproducts Corporation, GA, USA), ETHANOL REDTMYeast (Red Star/Lesaffre, USA)、FALITM(Fleischmann ' s Yeast, Burns Philp Food Inc., USA), FERMIOLTM(DSM Specialties), GERT STRANDTM(Gert Strand AB, Sweden) and SUPERSTARTTMAnd THERMOSACCTM 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 engineered Saccharomyces yeast capable of effectively cofermenting glucose and xylose, Appl.Environ.Microbiol.64:1852-1859；Kotter and Ciriacy, 1993, Xylose fermentation by Saccharomyces cerevisiae,Appl.Microbiol.Biotechnol.38:776-783；Walfridsson Deng 1995, Xylose-metabolizing Saccharomyces cerevisiae strains overexpressing the TKL1and TAL1genes encoding the pentose phosphate pathway enzymes transketolase and transaldolase,Appl.Environ.Microbiol.61:4184-4190；Kuyper etc., 2004,Minimal metabolic engineering of Saccharomyces cerevisiae for efficient anaerobic xylose fermentation:a proof of principle,FEMS Yeast Research4:655- 664；Beall etc., 1991, Parametric studies of ethanol production from xylose and other sugars by recombinant Escherichia coli,Biotech.Bioeng.38:296-303；Ingram Deng, 1998, Metabolic engineering of bacteria for ethanol production, Biotechnol.Bioeng.58:204-214；Zhang etc., 1995, Metabolic engineering of a pentose metabolism pathway in ethanologenic Zymomonas mobilis,Science267:240-243； Deanda etc., 1996, Development of an arabinose-fermenting Zymomonas mobilis strain by metabolic pathway engineering,Appl.Environ.Microbiol.62:4465-4470；WO 2003/ 062430,xylose isomerase)。
It is Candida sonorensi to pass through the fermentative microorganism of genetic modification in a preferred aspect,.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, for example, about 32 DEG C or 50 DEG C, And in about pH3 to about pH8, for example, about pH4-5,6 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 pH3 extremely About pH7, for example, about pH4 are to about pH7.However, some fermenting organisms such as bacterium, has higher most suitable fermentation temperature.Ferment Female or another microorganism is preferably with about 105-1012, preferably from about 107-1010, particularly from about 2x108Viable count ferments per ml The amount of liquid is applied.It is found in such as " The Alcohol Textbook " about the further guidance for using yeast to ferment (K.Jacques, T.P.Lyons and D.R.Kelsall are compiled, Nottingham University Press, United Kingdom1999), it is incorporated herein by carrying 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 of Saccharomyces cerevisiae by a Vitamin feeding strategy during fed-batch process, Springer-Verlag (2002) lead to It crosses to carry 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 derived from any substance of fermentation.Tunning can be not limited to, alcohol (example Such as, arabite, n-butanol, isobutanol, ethyl alcohol, glycerine, 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 gluconates, formic acid, fumaric acid, glucosaccharic acid, gluconic acid, Glucuronic acid, glutaric acid, 3- hydracrylic acids, 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 glycerine (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 Biochemistry30 (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 Biotechnology19(6):595-603。
At another preferred aspect, the tunning is alkane.The alkane is unbranched or branched 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 branched 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 It is glycine to state amino acid.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 Bioengineering87(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 Technology36(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 there are one term " ketone " covers and contains 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 gluconates.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 acids.In another more preferred aspect, institute It is itaconic acid to state organic 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 recycles 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 that purity is up to 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 polynucleotides of the separation of 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 amino acid 1 is to 21 or SEQ ID NO:10 amino acid 1 is 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 SEQ ID NO:1 nucleotide 1 to 57.On the other hand, the letter is encoded The polynucleotides of number peptide are SEQ ID NO:3 nucleotide 1 to 57.On the other hand, the multinuclear glycosides of the signal peptide is encoded Acid is SEQ ID NO:5 nucleotide 1 to 57.On the other hand, the polynucleotides for encoding the signal peptide are SEQ ID NO:7 nucleotide 1 to 63.On the other hand, the polynucleotides for encoding the signal peptide are SEQ ID NO:9 nucleotide 1 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, including：(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 natural or heterologous for host cell.Term " protein " this paper the meaning not Refer to the coded product of specific length, and therefore covers peptide, oligopeptides and polypeptide.Term " protein " is also contemplated by 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 being diluted on PDA plate at 45 DEG C and then single mitogenetic by shifting It purifies on spore to YG agar plates and is detached from the pedotheque collected is saved from Hunan China.NN047338 bacterial strains are based on form Feature and ITS rDNA Sequence Identifications are thermophilic capital spore.
The fungal bacterial strain of NN051380 is named as by being diluted on PDA plate at 25 DEG C and then single mitogenetic by shifting It purifies on spore to PDA plate and is detached from the pedotheque collected in China.Bacterial strain NN051380 is based on morphological feature and ITS RDNA Sequence Identifications are penicillium oxalicum.
The fungal bacterial strain of NN046782 is named as by being diluted on PDA plate at 45 DEG C and then single mitogenetic by shifting It purifies on spore to YG agar plates and is detached from the pedotheque collected in China.Bacterial strain NN046782 is based on morphological feature It is Rhizomucor pusillus (Rhizomucor pusillus) with ITS rDNA Sequence Identifications.
The fungal bacterial strain of NN044936 is named as by being diluted on PDA plate at 45 DEG C and then single mitogenetic by shifting It purifies on spore to YG agar plates and is detached from the pedotheque collected is saved in Chinese yunnan.Bacterial strain NN044936 is based on form Feature and ITS rDNA Sequence Identifications 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.
YG agar plates add to 1 liter of structure by the yeast extract of 5g, the glucose of 10g, the agar and deionized water of 20g At.
YPG culture mediums are 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 mediums.
Czapek ' s culture mediums are by the sucrose of 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 pH4.
FG4 culture mediums add to 1 liter by the soy meal of 30g, the maltose of 15g, the Bacto peptones and deionized water of 5g It constitutes.
Minimal medium tablet adds to 1 liter of structure by the sucrose of 342g, the salting liquid of 20ml, the agar and deionized water of 20g 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
Thermophilic capital spore bacterial strain NN047338 is inoculated on PDA plate and in 45 DEG C of Incubation in dark 3 days.By several mycelia Body-PDA bolts (plug) are inoculated with the 500ml shaking flasks into the YPG culture mediums containing 100ml.By bottle at 45 DEG C under 160rpm oscillations 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 kits (QIAGEN Inc., Valencia, CA, USA) follow the instruction separation genomic DNA of manufacturer.
Penicillium oxalicum bacterial strain NN051380 is inoculated on PDA plate and in 25 DEG C of Incubation in dark 5 days.By several mycelia Body-PDA bolts kind enter the 500ml shaking flasks of Czapek ' the s culture mediums containing 100ml.By bottle in 30 DEG C of temperature under 160rpm oscillations It educates 3.Mycelium by viaIt filters 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 kits follow the instruction point of manufacturer From genomic DNA.
Rhizomucor pusillus bacterial strain NN046782 is inoculated on PDA plate and in 45 DEG C of Incubation in dark 3 days.By several mycelia Body-PDA bolts kind enter the 500ml shaking flasks of the FG4 culture mediums containing 100ml.Bottle is incubated 3 at 45 DEG C under 160rpm oscillations Day.Mycelium by viaIt filters to collect and freeze in liquid nitrogen.The mycelium freezed is passed through Mortar and pestle are milled to fine-powder, and usePlant Maxi kits follow the instruction separation of manufacturer Genomic DNA.
Tangerine orange thermophilic ascomycete bacterial strain NN044936 is inoculated on PDA plate and in 45 DEG C of Incubation in dark 3 days.It will be several Mycelium-PDA bolts kind enter the 500ml shaking flasks of the YPG culture mediums containing 100ml.By bottle in 45 DEG C of temperature under 160rpm oscillations It educates 3.Mycelium by viaIt filters 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 kits follow 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 Research20 (2) in BGI：265-72) into Row compilation.The sequence of compilation is analyzed using standard bioinformatic methods for gene search (gene finding) and Function prediction.Use geneID (Parra etc., 2000, Genome Research10 (4)：511-515) carry out predictive genes.Make With Blastall version 2s .2.10 (Altschul etc., 1990, J.Mol.Biol.215 (3)：403-410, National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) and HMMER version 2s .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 results.Using Agene programs (Munch and Krogh, 2006, BMC Bioinformatics7：263) and SignalP programs (Nielsen etc., 1997, Protein Engineering10：1-6) identify initiation codon.Further use SignalP program predicted signal peptides.Use Pepstats (Rice etc., 2000, Trends Genet.16 (6)：276-277) the isoelectric point and molecular weight for the amino acid sequence that prediction derives.
Embodiment 3：From the thermophilic capital spore GH3 xylobiase coded sequences of genomic dna cloning
Based on DNA information (the SEQ ID NO obtained from the gene order-checking in embodiment 2:1 and 3), it devises shown below Oligonucleolide primers are with from the genomic DNA amplification GH3 xylobiase genes of thermophilic capital spore NN047338, GH3_ ZY577211_92 and GH3_ZY577202_22.Primer is synthesized by Invitrogen, Beijing, China.
SEQID1_ forward primers：
5’-ACACAACTGGGGATCCACCatgaccaggctgaccagcatc-3’(SEQ ID NO:11)
SEQID1_ reverse primers：
5’-GTCACCCTCTAGATCTcgtaccccactgccgttattg-3’(SEQ ID NO:12)
SEQID3_ forward primers：
5’-ACACAACTGGGGATCCACCatgaaggccctgactagaagg-3’(SEQ ID NO:13)
SEQID3_ reverse primers：
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 (WO 2011/005867) with the insertion point derived from plasmid pPFJO355.
For each gene, each forward and reverse primer pair of 20 picomoles is reacted for PCR, the reaction is by 2 μ The thermophilic capital spore NN047338 genomic DNAs 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 unitTM High- Fidelity archaeal dna polymerases (Finnzymes Oy, Espoo, Finland) are constituted, and final volume is 50 μ l.Amplification uses Peltier Thermal Cycler (MJ Research Inc., South San Francisco, CA, USA) are carried out, journey Sequence is as follows：It is denaturalized 1 minute at 98 DEG C；6 cycles, are each denaturalized 15 seconds at 98 DEG C, anneal 30 seconds at 65 DEG C, and often cycle reduces by 1 DEG C, and extend 3 minutes at 72 DEG C；23 cycles, 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 solution 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 kits (GE Healthcare, Buckinghamshire, UK) according to the instruction of manufacturer from solution purification PCR product.
Plasmid pPFJO355 Bam HI and Bgl II are digested, by using 1.0% Ago-Gel of tbe buffer liquid Electrophoretic separation, and usePCR and Gel Band Purification kits according to The instruction of manufacturer purifies.
Table 1：Plasmid
Gene Plasmid DNA is composed
GH3_ZY577211_92 pGH3_ZY577211_92 Fig. 1
GH3_ZY577202_22 pGH3_ZY577202_22 Fig. 2
Each PCR product and the carrier of digestion are usedCF Dry-down Cloning kits (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 xylobiases are compiled Under regulation and control of the transcription in oryzae alpha-amylase gene promoter of code sequence.In short, by 30ng 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 it is resuspended in the final volume of 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.Use reactant conversion Escherichia coli TOP10 competent cells (the TIANGEN Biotech (Beijing) 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 In the PCR solution aliquot (including PCR buffer solutions, MgCl2, dNTP, and the primer pair from its generation PCR fragment) of conjunction, 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, 1.0% agarose gel electrophoresis of the reaction by using tbe buffer liquid is analyzed. It usesSpin Miniprep kits (QIAGEN GmbH, Hilden, Germany) have 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 xylobiases 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 sequences are expressed in aspergillus oryzae
Will according to Christensen etc., 1988, Bio/Technology6:Aspergillus oryzae prepared by the method for 1419-1422 HowB101 (WO95/35385) protoplasts are 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 detached to individual minimal medium tablet.
Four transformant from each conversion are inoculated with to the YPM culture mediums of the 3ml in 24 orifice plates respectively, and at 30 DEG C It is incubated under 150rpm stirrings.After being incubated on 3rd, by the supernatant of the 20 μ l from each culture by using containing 50mM2- (N- morpholinoes) ethanesulfonic acid (MES) 4-12%Bis-Tris Gel (Invitrogen Corporation, Carlsbad, CA, USA) SDS-PAGE analyzed according to the instruction of manufacturer.The gel of gained is used(Expedeon Ltd., Babraham Cambridge, UK) is dyed.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 mediums containing 400ml.Culture was harvested on 3rd, and used 0.45 μmMembrane (Millipore, Bedford, MA, USA) is filtered.
Embodiment 5：Thermophilic capital spore GH3 xylobiases are recombinated from aspergillus oryzae O5JAC purifying
By the filtered culture solution of the aspergillus oryzae O5JAC (embodiment 4) of 3200ml volumes with ammonium sulfate (80% saturation) Precipitation, is redissolved in the 20mM sodium acetate pH5.0 of 50ml, dialyses for same buffer, and is filtered by 0.45 μm of filter. Final volume is 80ml.Solution is imposed on into the 40ml Q with 20mM sodium acetates pH5.0 balancesFast Flow columns (GE Healthcare, Buckinghamshire, UK).By the linear 0-0.5M NaCl gradient elutions of albumen.By grade Divide and collects, collects and impose on the 40ml SP balanced in 20mM sodium acetates pH5.0Fast Flow columns (GE Healthcare,Buckinghamshire,UK).By albumen with linear 0.2-0.5M NaCl gradient elutions.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 sequences
Based on DNA information (the SEQ ID NO obtained from the gene order-checking in embodiment 2:5) widow shown below, is devised Nucleotide primer is with from the genomic DNA amplification GH3 xylosidase genes 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.
Each above-mentioned primer of 20 picomoles is reacted for PCR, 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 PHUSIONTMHigh-Fidelity archaeal dna polymerases are constituted, and final volume is 50 μ l.Amplification uses Peltier Thermal Cycler is carried out, and program is as follows：It is denaturalized 1 minute at 98 DEG C；6 cycles, are each denaturalized 15 seconds at 98 DEG C, the annealing 30 at 65 DEG C Second, often cycle reduces by 1 DEG C, and extends 3 minutes at 72 DEG C；25 cycles, each carry out 15 seconds at 98 DEG C, and 30 are carried out 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 detached 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 kits It is purified according to the instruction of manufacturer.
Plasmid pPFJO355 Bam HI and Bgl II are digested, by using 1.0% Ago-Gel of tbe buffer liquid Electrophoretic separation, and usePCR and Gel Band Purification kits according to The instruction of manufacturer purifies.
The 3kb PCR products and the carrier of digestion are usedCF Dry-down Cloning examinations Agent box links together, and obtains pGH3_ZY569167_685 (Fig. 3), wherein turn of penicillium oxalicum GH3 xylosidases coded sequence Under regulation and control of the record in oryzae alpha-amylase gene promoter.In short, by 30ng Bam HI and Bgl II digestion The penicillium oxalicum GH3 xylobiase PCR products of the purifying of pPFJO355 and 60ng are added to reaction bottle, and pass through addition Deionized water is resuspended in the final volume of 10 μ l.Reaction is incubated 15 minutes at 37 DEG C and then incubated 15 minutes at 50 DEG C.It uses The reactant of three μ l converts Escherichia coli TOP10 competent cells.Escherichia coli transformant containing pGH3_ZY569167_685 Pass through bacterium colony PCR detections as described in example 3 above.It usesSpin Miniprep kits (QIAGEN GmbH, Hilden, Germany) prepare 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 sequences are 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 tablet.
Four transformant are inoculated with to the YPM culture mediums of the 3ml in 24 orifice plates respectively, and are stirred in 150rpm at 30 DEG C Mix lower incubation.After being incubated on 3rd, by the supernatant of the 20 μ l from each culture by using the MES's containing 50mM4-12%Bis-Tris Gel are analyzed.The gel of gained is usedDyeing.The most of transformant of SDS-PAGE general pictures display of culture 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 mediums of 10ml, and is inoculated with into the YPM culture mediums containing 400ml 2 liters of flasks.Culture was harvested on 3rd, and used 0.45 μmMembrane(Millipore, Bedford, MA, USA) filtering.
Embodiment 8：From genomic dna cloning Rhizomucor pusillus GH3 xylobiase coded sequences
Based on DNA information (the SEQ ID NO obtained from gene order-checking:7), devise Oligonucleolide primers shown below with From the genomic DNA amplification GH3 xylobiase genes of Rhizomucor pusillus NN046782, GH3_ZY654890_6424.Primer by Invitrogen, Beijing, China are synthesized.
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.
Each above-mentioned primer of 20 picomoles is reacted for PCR, 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 PHUSIONTMHigh-Fidelity archaeal dna polymerases are constituted, and final volume is 50 μ l.Amplification uses Peltier Thermal Cycler is carried out, and program is as follows：It is denaturalized 1 minute at 98 DEG C；6 cycles, are each denaturalized 30 seconds at 98 DEG C, the annealing 30 at 63 DEG C Second, often cycle reduces by 1 DEG C, and extends 2.5 minutes at 72 DEG C；24 cycles, each carry out 15 seconds at 94 DEG C, and 30 are carried out 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 detached 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 examinations Agent box is purified according to the instruction of manufacturer.
Plasmid pPFJO355 Bam HI and Bgl II are digested, by using 1.0% Ago-Gel of tbe buffer liquid Electrophoretic separation, and usePCR and Gel Band Purification kits are according to life The instruction of business men purifies.
It usesCF Dry-down Cloning kits are by direct gram of each 2.6kb PCR fragments It is grand enter expression vector pPFJO355, without restrictive digestion and connection.
PCR product and the carrier of digestion are usedCF Dry-down Cloning kits connect Together, pGH3_ZY654890_6424 (Fig. 4) is obtained, wherein at the transcription of Rhizomucor pusillus GH3 xylosidases coded sequence Under the regulation and control of oryzae alpha-amylase gene promoter.In short, by 30ng Bam HI and Bgl II digestion The Rhizomucor pusillus GH3 xylobiase PCR products of the purifying of pPFJO355 and 60ng are 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 then incubated 15 minutes at 50 DEG C.Make Escherichia coli TOP10 competent cells are converted with the reactant of three μ l.Escherichia coli containing pGH3_ZY654890_6424 turn Change body and passes through bacterium colony PCR detections as described in example 3 above.It usesIt is prepared by Spin Miniprep kits Plasmid DNA.The Rhizomucor pusillus GH3 xylobiases 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 sequences
Based on DNA information (the SEQ ID NO obtained from the gene order-checking in embodiment 2:9) widow shown below, is devised Nucleotide primer is with from the genomic DNA amplification GH3 xylobiase genes 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)
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.
Each above-mentioned primer of 20 picomoles is reacted for PCR, 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 PHUSIONTMHigh-Fidelity archaeal dna polymerases are constituted, and final volume is 50 μ l.Amplification uses Peltier Thermal Cycler is carried out, and program is as follows：It is denaturalized 1 minute at 98 DEG C；8 cycles, are each denaturalized 15 seconds at 98 DEG C, the annealing 30 at 65 DEG C Second, often cycle reduces by 1 DEG C, and extends 3.25 minutes at 72 DEG C；22 cycles, each carry out 15 seconds at 98 DEG C, are carried 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 detached 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 examinations Agent box purifies.
Plasmid pPFJO355 Bam HI and Bgl II are digested, by using 1.0% Ago-Gel of tbe buffer liquid Electrophoretic separation, and usePCR and Gel Band Purification kits.
The 2.4kb PCR products and the carrier of digestion are usedCF Dry-down Cloning Kit links together, and obtains pGH3_PE04100001596 (Fig. 5), wherein tangerine orange thermophilic ascomycete GH3 xylobiases Under regulation and control of the transcription in oryzae alpha-amylase gene promoter of coded sequence.In short, by 30ng with Bam HI and The tangerine orange thermophilic ascomycete GH3 xylobiase PCR products of the purifying of the pPFJO355 and 60ng of Bgl II digestion are added to instead Bottle is answered, and is resuspended in the final volume of 10 μ l by adding deionized water.Reaction is incubated 15 minutes at 37 DEG C then 50 DEG C incubate 15 minutes.Escherichia coli TOP10 competent cells are converted using the reactant of three μ l.Contain pGH3_ The Escherichia coli transformant of PE04100001596 passes through bacterium colony PCR detections as described in example 3 above.It usesSpin Miniprep kits prepare Plasmid DNA.The tangerine orange being inserted into pGH3_PE04100001596 is thermophilic Sac fungus GH3 xylobiases coded sequence is confirmed by using the DNA sequencing of 3730XL DNA Analyzer.
Embodiment 10：Tangerine orange thermophilic ascomycete GH3 xylobiase coded sequences are 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 tablet.
Four transformant are inoculated with to the YPM culture mediums of the 3ml in 24 orifice plates respectively, and are stirred in 150rpm at 30 DEG C Mix lower incubation.After being incubated on 3rd, 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.The gel of gained is usedDyeing.The most of transformant of SDS-PAGE general pictures display of culture 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 mediums of 10ml, and is inoculated with into the YPM culture mediums containing 400ml 2 liters of flasks.Culture was harvested on 3rd, and used 0.45 μmMembrane(Millipore, Bedford, MA, USA) filtering.
It is again molten by the aspergillus oryzae O6YKQ filtered culture solutions of 2400ml volumes ammonium sulfate (80% saturation) precipitation Solution is dialysed in the 20mM Tris-HCl pH6.5 of 50ml for same buffer, and is filtered by 0.45 μm of filter.Final body Product is 75ml.Solution is imposed on to the 40ml Q balanced in 20mM Tris-HCl pH6.5Fast Flow columns.The fraction with 0.08-0.1M NaCl elutions is collected, and is further existed with linear NaCI gradient (0.03-0.11M) 40ml QIt is purified on Fast Flow columns.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：Encode the characterization of the genomic DNA of GH3 xylobiases
The genomic dna sequence of thermophilic capital spore GH3 xylobiase coded sequences 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 programs (Nielsen etc., 1997, Protein Engineering10：1-6), the signal peptide of 19 residues is predicted. The maturation protein of prediction contains 758 amino acid, with 83.06kDa prediction molecular weight and 6.15 prediction etc. it is electric Point.
Using Needleman and Wunsch algorithms (Needleman and Wunsch, 1970, J.Mol.Biol.48：443- 453) with 10 gap open penalty, 0.5 gap extension penalty and EBLOSUM62 matrixes the comparison of amino acid sequence is 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 xylobiases Amino acid sequence (the UNIPROT of row and the derivation of the GH3 xylobiases from Pyrenophora tritici-repentis B2W9Y0) there is 62.96% sequence identity (excluding notch).
The genomic dna sequence of thermophilic capital spore GH3 xylobiase coded sequences 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, including terminator codon, It is by 68bp (nucleotide 192 to 259), 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 programs (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 with 10 notch using Needleman and Wunsch algorithms (Needleman and Wunsch, 1970, see above) Put point penalty, 0.5 gap extension penalty and EBLOSUM62 matrixes the comparative by overall comparison of amino acid sequence is determined. Compare amino acid sequence and the chaetomium globosum of the derivation of the thermophilic capital spore genomic DNA of code displaying GH3 xylobiases The amino acid sequence (UNIPROT Q2HEP1) of the derivation of (Chaetomium globosum) GH3 xylobiases has 70.39% homogeneity (excludes notch).
The genomic dna sequence of penicillium oxalicum GH3 xylobiase coded sequences 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, including 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 It is 851 amino acid in vain.Using SignalP programs (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 with 10 notch using Needleman and Wunsch algorithms (Needleman and Wunsch, 1970, see above) Put point penalty, 0.5 gap extension penalty and EBLOSUM62 matrixes the comparative by overall comparison of amino acid sequence is determined. Compare the amino acid sequence of the derivation of the penicillium oxalicum genomic DNA of code displaying GH3 xylobiases and from wheel branch fusarium The amino acid sequence (GENESEQP AZG45438) of the derivation of the GH3 enzymes of bacterium (Fusarium verticillioides) has 47.51% homogeneity (excludes notch).
The genomic dna sequence of Rhizomucor pusillus GH3 xylobiase coded sequences 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, including 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 programs (Nielsen etc., 1997, see above), the signal peptide of 21 residues is predicted.Prediction at Soft-boiled eggs contain 746 amino acid in vain, the isoelectric point of the molecular weight of the prediction with 82.03kDa and 5.02 prediction.
It is opened with 10 notch using Needleman and Wunsch algorithms (Needleman and Wunsch, 1970, see above) Put point penalty, 0.5 gap extension penalty and EBLOSUM62 matrixes the comparative by overall comparison of amino acid sequence is determined. It compares the amino acid sequence of the derivation of the Rhizomucor pusillus genomic DNA of code displaying GH3 xylobiases 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% homogeneity (excluding notch).
The amino acid sequence of the genomic dna sequence and derivation of tangerine orange thermophilic ascomycete GH3 xylobiase coded sequences It is shown in SEQ ID NO:9 (D82RN1) and SEQ ID NO:10(P24GP2).Coded sequence is 2403bp, including terminating close Numeral is free of any introne.The albumen of the prediction of coding is 800 amino acid.Using SignalP programs (Nielsen etc., 1997, see above), predict the signal peptide of 20 residues.The maturation protein of prediction contains 780 amino acid, has The isoelectric point of the molecular weight of the prediction of 84.58kDa and 5.03 prediction.
It is opened with 10 notch using Needleman and Wunsch algorithms (Needleman and Wunsch, 1970, see above) Put point penalty, 0.5 gap extension penalty and EBLOSUM62 matrixes the comparative by overall comparison of amino acid sequence is determined. Compare code displaying GH3 xylobiases 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% homogeneity (to exclude Notch).
Embodiment 12：Pretreated corncob hydrolysis measures
Corncob is located at 120 DEG C in 15% gross dry weight amount solid (TS) in advance with NaOH (0.08g/g dry weights cob) Reason 60 minutes.The material with water of gained is washed, until it is pH8.2, obtains washed, oxygenation pretreatment corncob (APCC).Through grinding, screening, 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 Cosmos ICMG40 wet type multipurpose bevellers (wet multi-utility Grinder it) grinds in (EssEmm Corporation, Tamil Nadu, India), and comes within 45 minutes in 121 DEG C of steam sterilizings It prepares, final TS is 3.33%.The hydrolysis of GS-APCC uses 2.2ml deep-well plates (Axygen, Union City, CA, USA) It is carried out with the total reaction volume of 1.0ml.
50mM sodium acetate pH5.0 buffer solution of the hydrolysis with the GS-APCC total solids of 10mg per ml manganese sulfates containing 1mM and a variety of The multiple protein load (being expressed as every gram of cellulose of mg albumen) of enzymatic compositions.Prepare enzymatic compositions, then by it with 50 μ l extremely The volume of 200 μ l ranges is added in all holes simultaneously so that final volume is 1ml in each reaction.Then tablet is made Use ALPS-300TMTablet heats seal instrument (Abgene, Epsom, United Kingdom) and seals, and is sufficiently mixed, and in specific temperature Degree incubates 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 columns (Bio-Rad Laboratories, Inc., Hercules, CA, USA) pass through following measurement：Use 0.05%w/w Benzoic acid -0.005M H2SO4It is eluted with 0.6ml flow velocitys per minute at 65 DEG C, and by will be from being corrected with pure sugar-like product Refractive index detection (1100HPLC,Agilent Technologies, Santa Clara, CA, USA) glucose, cellobiose and xylose signal integration quantified.The glucose equivalent of gained For calculating the cellulose conversion percentages each reacted.The xylose equivalents of gained are used to calculate the wood oligose conversion each reacted 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 EXCELTMSoftware (Microsoft, Richland, WA, USA) carries out.
The degree that wood oligose is converted into xylose uses following equation calculations：% xyloses=xylose concentration/limitation digestion In xylose concentration.In order to calculate % conversions, based on cellulase control (trichoderma reesei cellulase of 100mg, supplement P.emersonii GH61A polypeptides (WO 2011/041397)), aspergillus fumigatus GH10 zytases (xyn3) (WO 2006/ 078256) and Ai Mosen ankle section bacterium GH3 xylobiases (WO 2003/070956) every gram of cellulose) setting 100% conversion Point, and then all values divided by the numerical value are multiplied by 100.Triplicate data point is averaged and calculates standard deviation.
Embodiment 13：The preparation of Penicillium kind bacterial strain NN51602GH10 zytases
Penicillium kind bacterial strain NN51602GH10 zytases (SEQ ID NO:21 [DNA sequence dnas] 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 20mM Tris pH8.0 buffer-exchangeds.Permeate through desalination is loaded on The Q balanced in 20mM Tris pH8.0High Performance columns (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 FREETMSDS-PAGE the and CRITERION STAIN FREE of gelTM Imaging System SDS-PAGE (Bio-Rad Laboratories, Inc., Hercules, CA, USA) are analyzed.It converges The fraction of band of the collection containing about 50kDa.Albumen concentration uses Microplate BCATMProtein Assay kits (Thermo Fisher Scientific, Waltham, MA, USA) is determined, wherein bovine serum albumin(BSA) is used as protein standard.
Embodiment 14：Ai Mosen ankle sections bacterium (Talaromyces emersonii) CBS393.64GH3 xylobiases (P4UE) preparation
Ai Mosen ankle section bacterium CBS393.64 xylobiases (SEQ ID NO:23 [DNA sequence dnas] and SEQ ID NO:24 [amino acid sequence of derivation]) according to Rasmussen etc., 2006, Biotechnology and Bioengineering94: 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) films concentrates and uses 50mM sodium acetate pH5.0 desalinations.Albumen concentration uses Microplate BCATMProtein Assay kits determine that wherein bovine serum albumin(BSA) is used as protein standard.
Embodiment 15：The quantitative gel of tangerine orange thermophilic ascomycete GH3 xylobiases (P24GP2)
The total protein content of tangerine orange thermophilic ascomycete GH3 xylobiases is determined by quantitative gel.Albumen concentration passes through Use 8-16%Tris HCl CRITERION STAIN FREETMSDS-PAGE the and CRITERION STAIN of gel FREETMImaging System SDS-PAGE (Bio-Rad Laboratories, Inc., Hercules, CA, USA) are determining, Wherein Ai Mosen ankles section bacterium GH3 xylobiases are used as protein standard.
Embodiment 16：When the thermophilic ascus of tangerine orange when pH4.0 to 7.0 supplements Penicillium kind GH10 zytases using APCC The effect of bacterium GH3 xylobiases (P24GP2)
Washed, oxygenation pretreatment corncob (APCC) is used to be commented as substrate from pH4.0 to 7.0 at 50 DEG C and 60 DEG C Estimate the tangerine orange thermophilic ascomycete xylobiase (P24GP2) of supplement Penicillium kind GH10 zytases (embodiment 13).As than Compared with, by supplement Penicillium kind GH10 zytases Ai Mosen ankle section bacterium GH3 xylobiases (P4UE) be added to APCC.By β- Xylosidase is added to APCC per g cellulose supplement 4.0mg total proteins with 0.025mg total proteins per the zytase of g celluloses Hydrolysis, and results of hydrolysis is compared with the result of the zytase only containing the every g celluloses of 4.0mg total proteins.
It measures and carries out as described in example 12 above.Containing 1mM manganese sulfates with the 1ml reactions of APCC (1% total solid) It is carried out 72 hours in 50mM Tris (pH6.0 to 7.0) or 50mM sodium acetates (pH4.0 to 5.5).It is all reaction in triplicate into Row, and be related to hydrolyzing single mixing when originating.
It is shown in Fig. 6 in 50 DEG C of results, and Fig. 7 is shown in 60 DEG C of result.As shown in fig. 1, tangerine orange thermophilic ascomycete GH3 xylobiases compared with only Penicillium kind GH10 xylosidases, significantly increase xylan at 50 DEG C and pH4.0 to 7.0 To the hydrolysis of xylose.At 50 DEG C, tangerine orange thermophilic ascomycete GH3 xylobiases have optimal activity in pH4.0 to 5.5.In addition, Tangerine orange thermophilic ascomycete GH3 beta-xylanases are at pH5.0 to 7.0 and 50 DEG C compared with Ai Mosen ankle section bacterium GH3 xylobiases Increase hydrolysis.The tangerine orange thermophilic ascomycete GH3 beta-xylanases of zytase are supplemented in pH7.0 and 50 DEG C and supplement zytase Ai Mosen ankle section bacterium GH3 xylobiases 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-xylanases are in 60 DEG C and pH4.0 to 6.0 and only Penicillium kind GH10 zytases are compared, and hydrolysis of the xylan to xylose is significantly increased.At 60 DEG C, tangerine orange thermophilic ascomycete GH3 β-wood is poly- Carbohydrase has optimal activity in pH4.0 to 5.0.In addition, tangerine orange thermophilic ascomycete GH3 beta-xylanases are in pH5.0 to 6.0 and 60 Increase hydrolysis DEG C compared with Ai Mosen ankle section bacterium GH3 xylobiases.Supplement tangerine orange thermophilic ascomycete GH3 β-wood of zytase Dextranase increases xylan extremely at pH6.0 and 60 DEG C compared with the Ai Mosen ankle section bacterium GH3 xylobiases of supplement zytase The conversion of xylose is up to 1.95 times.
The present invention is further described by following number paragraphs：
[1] a kind of polypeptide with the active separation of xylobiase, is selected from the group：
(a) polypeptide, with SEQ ID NO:6 or SEQ ID NO:8 mature polypeptide has at least 60% sequence identity； With SEQ ID NO:2 mature polypeptide has at least 65% sequence identity；Or with SEQ ID NO:4 or SEQ ID NO:10 Mature polypeptide has at least 75% sequence identity；(b) polypeptide, by polynucleotide encoding, the polynucleotides are at least Hybridize with following Deng under-high stringency conditions：(i)SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, Or SEQ ID NO:9 mature polypeptide encoded sequence, (ii) SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7 cDNA sequence, or (iii) (i) or (ii) overall length complement；(c) polypeptide, it is described by polynucleotide encoding Polynucleotides and SEQ ID NO:5 or SEQ ID NO:7 or its cDNA sequence mature polypeptide encoded sequence have at least 60% Sequence identity；With SEQ ID NO:1 or its cDNA sequence mature polypeptide encoded sequence have at least 65% sequence identity； Or with SEQ ID NO:3 or its cDNA sequence or SEQ ID NO:9 mature polypeptide encoded sequence has at least 75% sequence same One property；(d)SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10 maturation Polypeptide includes substitution, missing and/or the variant 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 claim 1, with SEQ ID NO:6 or SEQ ID NO:8 mature polypeptide has 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 are same Property；With SEQ ID NO:2 mature polypeptide have at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, until Few 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 SEQ ID NO:4 or SEQ ID NO:10 mature polypeptide has 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.
[3] polypeptide of claim 1, by polynucleotide encoding, the polynucleotides are medium-high, high or very high Hybridize with following under stringent condition：(i)SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:9 mature polypeptide encoded sequence, (ii) SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7 cDNA sequence, or (iii) (i) or (ii) overall length complement.
[4] polypeptide of claim 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%, 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；With SEQ ID NO:1 or its cDNA sequence at Ripe polypeptid coding sequence have 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 with SEQ ID NO:3 or its cDNA sequence or SEQ ID NO:9 mature polypeptide encoded sequence With 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.
[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 ID NO:10 mature polypeptide.
[6] polypeptide of section 5, wherein the mature polypeptide is SEQ ID NO:2 amino acid 20 to 777, SEQ ID NO:4 Amino acid 20 to 850, SEQ ID NO:6 amino acid 20 to 851, SEQ ID NO:8 amino acid 22 to 767 or SEQ ID NO:10 amino acid 21 to 800.
[7] polypeptide of section 1 is SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10 mature polypeptide includes substitution, missing and/or the variant 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 NO:8 or SEQ ID NO:10 segment, 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 polynucleotides of separation, 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 are operable 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 generating any one of section 1-8 comprising：In the item for contributing to the polypeptide to generate Cell is cultivated under part, the cell generates the polypeptide with its wild-type form.
[14] method of section 13 further includes the recycling polypeptide.
[15] a kind of method generating the polypeptide with xylanase activity comprising：It is generated contributing to the polypeptide Under conditions of cultivate section 12 host cell.
[16] method of section 15 further includes the recycling polypeptide.
[17] a kind of genetically modified plants, plant part or plant cell, the multinuclear of the polypeptide of any one of encoded section of 1-8 Thuja acid converts.
[18] a kind of method generating the polypeptide with xylanase activity comprising：It is generated contributing to the polypeptide Under conditions of culture section 17 genetically modified plants or plant cell.
[19] method of section 18 further includes the recycling polypeptide.
[20] a kind of method for the mutant 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] a kind of method generating albumen comprising：Under conditions of contributing to the albumen to generate culture section 21 or 22 mutant cell.
[24] method of section 23 further includes the recycling 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 of about 15,16,17,18,19,20,21,22, 23,24,25 or more duplex nucleotides.
[27] method of expression of a kind of polypeptide of the inhibition with 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] a kind of method generating albumen comprising：Under conditions of contributing to the albumen to generate culture section 28 or 29 cell.
[31] method of section 30 further includes the recycling polypeptide.
[32] a kind of polynucleotides of separation, 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 Amino acid 1 is to 21 or SEQ ID NO:10 amino acid 1 is 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 generating albumen comprising：Culture recombination place under conditions of contributing to the albumen to generate Chief cell, the recombinant host cell include the gene of the coding albumen for the polynucleotides for being operably connected to section 32, wherein The gene is external source for encoding the polynucleotides of the signal peptide.
[36] method of section 35 further includes the recycling 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 In the presence of the polypeptide of xylanase activity the cellulosic material or material containing xylan are handled with enzymatic compositions.
[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 are selected from comprising one or more (such as several) 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 degradation or material containing xylan.
[43] technique of section 42, wherein the cellulosic material through degradation 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 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 saccharification 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 carried out at the same time in being saccharified and ferment at the same time.
[51] technique of any one of section 45-50, wherein tunning are 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 with enzymatic compositions in the presence of the polypeptide with xylanase activity of any one of section 1-8.
[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 are selected from comprising one or more (such as several) 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 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.

Claims (37)

1. a kind of polypeptide with the active separation of xylobiase, is selected from the group：
(a) polypeptide, with SEQ ID NO:10 have 100% sequence identity；
(b) polypeptide, by SEQ ID NO:9 polynucleotide encoding；
The polypeptide is thermophilic ascomycete category (Thermoascus) polypeptide.
2. the polypeptide of claim 1, wherein the polypeptide is tangerine orange thermophilic ascomycete polypeptide.
3. the polypeptide of claim 1, consisting of SEQ ID NO:10 or SEQ ID NO:10 mature polypeptide.
4. the polypeptide of claim 3, wherein the mature polypeptide is SEQ ID NO:10 amino acid 21 to 800.
5. a kind of composition, it includes the polypeptides of any one of claim 1-4.
6. a kind of polynucleotides of separation, the polypeptide of any one of coding claim 1-4.
7. a kind of nucleic acid construct or expression vector, it includes the polynucleotides of claim 6, the polynucleotides are operationally One or more regulating and controlling sequences are connected to, the regulating and controlling sequence instructs generation of the polypeptide in expressive host.
8. a kind of recombinant host cell, it includes the polynucleotides of claim 6, the polynucleotides are operably connected to one A or multiple regulating and controlling sequences, the regulating and controlling sequence instruct the generation of polypeptide.
9. a kind of method for the polypeptide generating any one of claim 1-4 comprising：In the item for contributing to the polypeptide to generate Cell is cultivated under part, the cell generates the polypeptide with its wild-type form.
10. the method for claim 9 further includes the recycling polypeptide.
11. a kind of generating the method with the active polypeptide of xylobiase comprising：In the item for contributing to the polypeptide to generate The host cell of claim 8 is cultivated under part.
12. the method for claim 11 further includes the recycling polypeptide.
13. the technique of a kind of degradation of fibers cellulosic material or the material containing xylan comprising：Any one of claim 1-4's With handling the cellulosic material or material containing xylan with enzymatic compositions in the presence of the active polypeptide of xylobiase.
14. the technique of claim 13, wherein the cellulosic material or material containing xylan are by pretreatment.
15. the technique of any one of claim 13 or 14, wherein the enzymatic compositions include one or more enzymes selected from the group below： Cellulase, the polypeptide with cellulolytic enhancing activity, hemicellulase, esterase, laccase, lignin decomposition enzyme, pectin Enzyme, peroxidase, protease.
16. the technique of claim 15, wherein the cellulase is one or more enzymes selected from the group below：Endo-glucanase Enzyme, cellobiohydrolase and β-glucosyl enzym.
17. the technique of claim 15, wherein the hemicellulase is one or more enzymes selected from the group below：Zytase, Acetyl xylan esterase, feruloyl esterase, arabinofuranosidase, xylosidase and glucuronidase.
18. the technique of any one of claim 16-17 further includes recycling the cellulosic material through degradation or material containing xylan Material.
19. the technique of claim 18, wherein the cellulosic material through degradation or material containing xylan are sugar.
20. the technique of claim 19, wherein the sugar is selected from the group：Glucose, xylose, mannose, galactolipin, and it is Arabic Sugar.
21. a kind of technique generating tunning comprising：(a) there is xylobiase in any one of claim 1-4 In the presence of active polypeptide, with enzymatic compositions saccharified cellulosic material or material containing xylan；(b) micro- with one or more fermentations Cellulosic material of the biofermentation through saccharification or material containing xylan are to generate tunning；(c) fermentation production is recycled from fermentation Object.
22. the technique of claim 21, wherein the cellulosic material or material containing xylan are pretreated.
23. the technique of claim 21 or 22, wherein the enzymatic compositions include one or more enzymes selected from the group below：Cellulose Enzyme, the polypeptide with cellulolytic enhancing activity, hemicellulase, esterase, laccase, lignin decomposition enzyme, pectase, peroxide Compound enzyme, protease.
24. the technique of claim 23, wherein the cellulase is one or more enzymes selected from the group below：Endo-glucanase Enzyme, cellobiohydrolase and β-glucosyl enzym.
25. the technique of claim 23, wherein the hemicellulase is one or more enzymes selected from the group below：Zytase, Acetyl xylan esterase, feruloyl esterase, arabinofuranosidase, xylosidase and glucuronidase.
26. the technique in claim 21, wherein step (a) and (b) are carried out at the same time in being saccharified and ferment at the same time.
27. the technique in claim 21, wherein tunning be alcohol, alkane, cycloalkane, alkene, gas, ketone, organic acid or Polyketide.
28. the technique of a kind of fermentable fiber cellulosic material or the material containing xylan comprising：It is sent out with one or more fermentative microorganisms Ferment cellulosic material or material containing xylan, wherein the cellulosic material or material containing xylan are in claim 1-4 Any one with being saccharified with enzymatic compositions in the presence of the active polypeptide of xylobiase.
29. the technique of claim 28, wherein the fermentation of the cellulosic material or the material containing xylan generates tunning.
30. the technique of claim 28 further includes recycling tunning from fermentation.
31. the technique of claim 29 or 30, wherein the tunning is alcohol, alkane, cycloalkane, alkene, gas, ketone, has Machine acid or polyketide.
32. the technique of claim 28, wherein the cellulosic material or material containing xylan pass through pretreatment before saccharification.
33. the technique of claim 28, wherein the enzymatic compositions include one or more enzymes selected from the group below：Cellulase, Polypeptide, hemicellulase with cellulolytic enhancing activity, esterase, laccase, lignin decomposition enzyme, pectase, peroxidating Object enzyme, protease.
34. the technique of claim 33, wherein the cellulase is one or more enzymes selected from the group below：Endo-glucanase Enzyme, cellobiohydrolase and β-glucosyl enzym.
35. the technique of claim 33, wherein the hemicellulase is one or more enzymes selected from the group below：Zytase, Acetyl xylan esterase, feruloyl esterase, arabinofuranosidase, xylosidase and glucuronidase.
36. the technique of claim 27 or 31, wherein organic acid are amino acid, alkene is isoprene.
37. a kind of full nutrient solution formulation or cell culture compositions, it includes the polypeptides of any one of claim 1-4.
CN201280067784.9A 2011-11-22 2012-11-22 Polynucleotides with the active polypeptide of xylobiase and the coding polypeptide Active CN104145016B (en)

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