GH61 polypeptide variants and polynucleotides encoding same

Abstract

The present invention relates to GH61 polypeptide variants. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells including the polynucleotides; and methods of using the variants.

Description

[0001] This application is a divisional application of the invention patent application filed on April 26, 2013, with application number 201380021502.6 and entitled "GH61 polypeptide variant and polynucleotide encoding the same".

[0002] References to sequence lists

[0003] This application includes a sequence list in a computer-readable form, which is incorporated herein by reference. Background of the Invention Invention Field

[0004] This invention relates to GH61 polypeptide variants, polynucleotides encoding these variants, methods for generating these variants, and methods for using these variants.

[0005] Related technical specifications

[0006] Cellulose is a polymer of glucose monosaccharide covalently linked by β-1,4-bonds. Many microorganisms produce enzymes that hydrolyze β-linked glucans. These enzymes include endoglucanases, cellobiases, and β-glucosidases. Endoglucanases digest the cellulose polymer at any position, opening it for attack by cellobiases. Cellobiases sequentially release cellobiose molecules from the ends of the cellulose polymer. Cellobiose is a water-soluble β-1,4-linked dimer of glucose. β-glucosidases hydrolyze cellobiose into glucose.

[0007] Converting lignocellulosic feedstocks into ethanol offers several advantages: readily available large quantities of raw materials, avoidance of the desire to burn or landfill materials, and the cleanliness of ethanol fuel. Wood, agricultural residues, herbaceous crops, and municipal solid waste are currently considered suitable feedstocks for ethanol production. These materials are primarily composed of cellulose, hemicellulose, and lignin. Once the lignocellulosic material is converted into fermentable sugars, such as glucose, these sugars can be readily fermented into ethanol by yeast.

[0008] WO 2005 / 074647, WO 2008 / 148131, and WO 2011 / 035027 disclose isolated GH61 polypeptides and their polynucleotides with enhanced cellulose-degrading activity from *Thielavia terrestris*. WO 2005 / 074656 and WO 2010 / 065830 disclose isolated GH61 polypeptides and their polynucleotides with enhanced cellulose-degrading activity from *Thermoascus aurantiacus*. WO 2007 / 089290 and WO 2012 / 149344 disclose isolated GH61 polypeptides and their polynucleotides with enhanced cellulose-degrading activity from *Trichoderma reesei*. WO 2009 / 085935, WO 2009 / 085859, WO 2009 / 085864, and WO 2009 / 085868 disclose isolated GH61 polypeptides and their polynucleotides with enhanced cellulose-degrading activity from *Myceliophthora thermophila*. WO 2010 / 138754 discloses isolated GH61 polypeptides and their polynucleotides with enhanced cellulose-degrading activity from *Aspergillus fumigatus*. WO 2011 / 005867 discloses isolated GH61 polypeptides and their polynucleotides with enhanced cellulose-degrading activity from *Penicillium pinophilum*. WO 2011 / 039319 discloses isolated GH61 polypeptides and their polynucleotides with enhanced cellulose-degrading activity from species of the genus *Thermoascus*. WO 2011 / 041397 discloses isolated GH61 polypeptides and their polynucleotides with enhanced cellulose-degrading activity from *Penicillium* sp. species. WO 2011 / 041504 discloses isolated GH61 polypeptides and their polynucleotides with enhanced cellulose-degrading activity from *Thermoascus crustaceus*. WO 2012 / 030799 discloses isolated GH61 polypeptides and their polynucleotides with enhanced cellulose-degrading activity from *Aspergillus aculeatus*. WO 2012 / 113340 discloses isolated GH61 polypeptides and their polynucleotides with enhanced cellulose-degrading activity from *Thermomyces lanuginosus*.WO 2012 / 122477 discloses isolated GH61 polypeptides and their polynucleotides with enhanced cellulose-degrading activity from *Aurantiporus alborubescens*, *Trichophaea saccata*, and *Penicillium thomii*. WO 2012 / 135659 discloses isolated GH61 polypeptides and their polynucleotides with enhanced cellulose-degrading activity from *Talaromyces stipitatus*. WO 2012 / 146171 discloses isolated GH61 polypeptides and their polynucleotides with enhanced cellulose-degrading activity from *Humicola insolens*. WO2012 / 101206 discloses isolated GH61 polypeptides and their polynucleotides with enhanced cellulose-degrading activity from *Malbranchea cinnamomea*, *Talaromyces leycettanus*, and *Chaetomium thermophilum*. WO 2013 / 043910 discloses isolated GH61 polypeptides and their polynucleotides with enhanced cellulose-degrading activity from *Acrophialophora fusispora* and *Corynascus sepedonium*.

[0009] WO 2012 / 044835 and WO 2012 / 044836 disclose GH61 peptide variants with enhanced cellulose degradation activity along with improved thermal activity and thermal stability.

[0010] There is a need in the art for GH61 peptides with enhanced cellulose decomposition activity and increased thermal stability, as components of enzyme compositions used in the degradation of lignocellulose at high temperatures.

[0011] This invention provides a GH61 peptide variant with increased thermal stability. Invention Overview

[0012] The present invention relates to isolated GH61 polypeptide variants comprising substitutions at one or more (e.g., several) positions corresponding to positions 26, 32, 34, 40, 41, 42, 47, 56, 72, 102, 123, 138, 149, 152, 163, 164, 166, 169, 186, 200, 207, 213, 219, 222, 234, 246, 249, and 250 of the mature polypeptide of SEQ ID NO:30, wherein these variants have cellulose-enhancing activity.

[0013] The present invention also relates to isolated polynucleotides encoding these variants; nucleic acid constructs, vectors, and host cells containing these polynucleotides; and methods for generating these variants.

[0014] The present invention also relates to methods for degrading or converting cellulose materials, comprising: treating the cellulose material with an enzymatic composition in the presence of a GH61 polypeptide variant of the present invention. In one aspect, these methods further comprise recovering the degraded or converted cellulose material.

[0015] The present invention also relates to a method for producing a fermentation product, comprising: (a) saccharifying a cellulose material with an enzyme composition in the presence of a GH61 polypeptide variant of the present invention; (b) fermenting the saccharified cellulose material with one or more (e.g., several) fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.

[0016] The present invention also relates to a method for fermenting cellulose materials, comprising: fermenting the cellulose material with one or more (e.g., several) fermenting microorganisms, wherein the cellulose material is saccharified with an enzyme composition in the presence of the GH61 polypeptide variant of the present invention. In one aspect, the fermentation of the cellulose material produces a fermentation product. In another aspect, these methods further comprise recovering the fermentation product from the fermentation.

[0017] This invention also relates to the following items:

[0018] 1. A GH61 polypeptide variant comprising substitutions at one or more positions corresponding to positions 138, 219, 26, 32, 34, 40, 41, 42, 47, 56, 72, 102, 123, 149, 152, 163, 164, 166, 169, 186, 200, 207, 213, 222, 234, 246, 249, and 250 of the mature polypeptide of SEQ ID NO: 30, wherein the variant has cellulose-enhancing activity, and wherein This variant 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 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence similarity to the parental GH61 polypeptide.

[0019] 2. A variant as described in any one of Item 1, wherein the variant is a variant of the parental GH61 polypeptide selected from the group consisting of the following:

[0020] (a) with SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, A polypeptide having at least 60% sequence identity among the mature polypeptides of 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 56, 158, 160, 162, 164, 166, 168, 409, or 411;

[0021] (b) A polypeptide encoded by a polynucleotide that hybridizes to at least the following under low-rigor conditions: (i) SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 10 5, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 408, or 410, or the full-length complement of (ii)(i);

[0022] (c) Composed of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, A polypeptide encoded by a polynucleotide with at least 60% sequence identity of the mature polypeptide coding sequence of 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 408, or 410; and

[0023] (d)SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54 ,56,58,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100,102,104, A fragment of a mature polypeptide of 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 56, 158, 160, 162, 164, 166, 168, 409, or 411, which has cellulolytic enhancement activity.

[0024] 3. The variant as described in Item 2, wherein the parental GH61 polypeptide comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 10 2. Mature polypeptides of or composed of 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 56, 158, 160, 162, 164, 166, 168, 409, or 411.

[0025] 4. A variant as described in any one of items 1-3, wherein the variant is identical to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134 Mature polypeptides of 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 56, 158, 160, 162, 164, 166, 168, 409, or 411 have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity.

[0026] 5. A variant as described in any one of items 1-4, comprising one or more substitutions selected from the group consisting of: S26I; G32E,S; Y34F; V40A; N41T; Q42I,E,V; S47E,L,R; S56C,E,T; S72Q,T; T102K,P; A123R; Q138C,E,G,K,L,M; V149I; D152S; T163E,F,V; V164C,L; I166L; S169R,C; S186F,K,T,Y; F200I,V; G207P; S213E; S219E,M,Q,C; K222R; S234G,K; A246P; N249Q,R,C; and A250C.

[0027] 6. A variant as described in any one of items 1 to 5, the variant further comprising substitutions at one or more positions corresponding to positions 111, 152, 155, 162, 96, 98, 200, 202, 204, 105, 154, 188, 189, 216, and 229 of the mature polypeptide of SEQ ID NO:30, wherein the variant has cellulose-degrading-enhancing activity.

[0028] 7. A variant as described in item 6, comprising one or more substitutions selected from the group consisting of: L111V, D152S, M155L, A162W, I96V, F98L, F200I, I202L, I204V, E105P,K; E154L; G188A,W; N189K; A216L,Y; and A229W,H,I,Y.

[0029] 8. A variant as described in any one of items 1-7, wherein the variant has an increase in thermal stability of at least 1.01 times, for example at least 1.05 times, at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.8 times, at least 2 times, at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 50 times, at least 75 times, or at least 100 times compared to the parent.

[0030] 9. A separate polynucleotide encoding a variant as described in any one of items 1-8.

[0031] 10. A recombinant host cell comprising the polynucleotides described in item 9.

[0032] 11. A method for generating a GH61 polypeptide variant, the method comprising: culturing recombinant host cells as described in item 10 under conditions suitable for expression of the variant.

[0033] 12. A transgenic plant, plant part or plant cell transformed with the polynucleotides described in Project 9.

[0034] 13. A method for producing a variant as described in any one of items 1-8, the method comprising: culturing a transgenic plant or plant cell containing a polynucleotide encoding the variant under conditions conducive to the production of the variant.

[0035] 14. A method for obtaining a GH61 polypeptide variant, the method comprising: substituting the parental GH61 polypeptide at one or more positions corresponding to positions 138, 219, 26, 32, 34, 40, 41, 42, 47, 56, 72, 102, 123, 149, 152, 163, 164, 166, 169, 186, 200, 207, 213, 222, 234, 246, 249, and 250 of the mature polypeptide of SEQ ID NO:30, wherein the variant has cellulose-degrading-enhancing activity; and recovering the variant.

[0036] 15. A method for degrading or converting a cellulose material, the method comprising: treating the cellulose material with an enzyme composition in the presence of a GH61 polypeptide variant having cellulose-degrading-enhancing activity as described in any one of items 1-8.

[0037] 16. A method for producing a fermentation product, the method comprising: (a) saccharifying a cellulose material with an enzyme composition in the presence of a GH61 polypeptide variant having cellulose-degrading-enhancing activity as described in any one of items 1-8; (b) fermenting the saccharified cellulose material with one or more fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.

[0038] 17. A method for fermenting a cellulose material, the method comprising: fermenting the cellulose material with one or more fermenting microorganisms, wherein the cellulose material is saccharified with an enzyme composition in the presence of a GH61 polypeptide variant having cellulose-degrading-enhancing activity as described in any one of items 1-8.

[0039] 18. A composition comprising any of the variants described in any one of items 1-8.

[0040] 19. A whole culture medium preparation or cell culture composition comprising any of the variants described in any one of items 1-8.

[0041] 20. A detergent composition comprising a surfactant and a variant as described in any one of items 1-8. Brief description of the attached figures

[0042] Figure 1The genomic DNA sequence (SEQ ID NO:29) and the inferred amino acid sequence (SEQ ID NO:30) of the Aspergillus fumigatus gene encoding the GH61B polypeptide with enhanced cellulose degradation activity are shown.

[0043] Figure 2 The restrictive spectrum of pMMar44 is shown.

[0044] Figure 3 The restrictive spectrum of pMMar49 is shown.

[0045] Figure 4 The restrictive spectrum of pMMar45 is shown.

[0046] Figure 5 The restriction spectrum of pDFng113 is shown.

[0047] Figure 6 The restriction spectrum of pDFng153-4 is shown.

[0048] Figure 7 The restriction spectrum of pDFng154-17 is shown.

[0049] Figure 8 The restriction spectrum of pDFng155-33 is shown.

[0050] Figure 9 The restriction spectrum of pDFng156-37 is shown.

[0051] Figure 10 The restriction spectrum of pDFng157-51 is shown.

[0052] Figure 11 The restriction spectrum of pBGMH16 is shown.

[0053] definition

[0054] Acetylxylan esterase: The term "acetylxylan esterase" refers to a carboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of acetyl-self-polymerized xylan, acetylated xylose, acetylated glucose, α-naphthyl acetate, and p-nitrophenyl acetate. For the purposes of this invention, 0.5 mM p-nitrophenyl acetate was used as a substrate in a solution containing 0.01% TWEEN. TM The activity of acetylxylan esterase was determined in 50 mM sodium acetate (pH 5.0) of 20 (polyoxyethylene sorbitan laurate). One unit of acetylxylan esterase was defined as the amount of enzyme capable of releasing 1 μmol of p-nitrophenol anion per minute at pH 5 and 25°C.

[0055] Allelic variants: The term "allelic variant" refers to any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variations arise naturally from mutations and can lead to polymorphism within a population. Gene mutations can be silent (without alteration in the encoded polypeptide) or can encode a polypeptide with a modified amino acid sequence. Allelic variants of a polypeptide are polypeptides encoded by allelic variants of a gene.

[0056] α-L-Arabofuranosaccharidase: The term "α-L-Arabofuranosaccharidase" refers to an α-L-arabinofuranoside arabinofuranoside hydrolase (EC 3.2.1.55) that catalyzes the hydrolysis of terminal non-reducing α-L-arabinofuranoside residues in α-L-arabinoside. This enzyme acts on α-L-arabinofuranoside, α-L-arabinanan containing (1,3)- and / or (1,5)- bonds, arabinosylxylan, and arabinogalactan. α-L-Arabofuranosaccharidase is also known as arabinosaccharidase, α-arabinosaccharidase, α-L-arabinosaccharidase, α-arabinofuranosaccharidase, polysaccharide α-L-arabinofuranosaccharidase, α-L-arabinofuranoside hydrolase, L-arabinosaccharidase, or α-L-arabinananase. For the purposes of this invention, 5 mg of medium-viscosity wheat arabinosyl xylan (Megazyme International Ireland, Ltd., Bray, Co., Wicklow, Ireland) per ml of 100 mM sodium acetate (pH 5) was used at a total volume of 200 μl and incubated at 40°C for 30 minutes, followed by... HPX-87H column chromatography (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to analyze arabinose to determine α-L-arabinofuranosidase activity.

[0057] α-Glucuronidase: The term "α-glucuronidase" refers to an α-D-glucuronide hydrolase (EC 3.2.1.139) that catalyzes the hydrolysis of α-D-glucuronide to D-glucuronide and alcohol. For the purposes of this invention, α-glucuronidase activity was determined according to de Vries, 1998, J. Bacteriol. 180:243-249. One unit of α-glucuronidase is equal to the amount of enzyme capable of releasing 1 micromole of glucuronic acid or 4-O-methylglucuronic acid per minute at pH 5 and 40°C.

[0058] β-Glucosidase: The term "β-glucosidase" refers to a β-D-glucosidase (EC 3.2.1.21) that catalyzes the hydrolysis of terminal non-reducing β-D-glucose residues, releasing β-D-glucose. For the purposes of this invention, β-glucosidase activity was determined using p-nitrophenyl-β-D-glucopyranoside as a substrate, according to the procedure described by Venturi et al., 2002, J. Basic Microbiol. 42:55-66. One unit of β-glucosidase is defined as the activity of β-glucosidase at 25°C and pH 4.8, containing 0.01% glucosidase. 1.0 μmol of p-nitrophenol anion is generated per minute from 1 mM p-nitrophenyl-β-D-glucopyranoside as a substrate in 50 mM sodium citrate.

[0059] β-Xylosidase: The term "β-xylosidase" refers to a β-D-xyloside xylose hydrolase (EC 3.2.1.37) that catalyzes the external hydrolysis of short β(1→4)-xylooligosaccharides to remove consecutive D-xylose residues from their non-reducing ends. For the purposes of this invention, 1 mM p-nitrophenyl-β-D-xyloside was used as a substrate in a solution containing 0.01% TWEEN. TM The activity of β-xylosidase was preferably determined in 20% sodium citrate (pH 5, 40°C) at 100 mM (pH 5, 40°C). One unit of β-xylosidase was defined as the activity of β-xylosidase at 40°C, pH 5, in a solution containing 0.01% sodium citrate. 1.0 μmol of p-nitrophenol anion is generated per minute from 1 mM p-nitrophenyl-β-D-xyloside as a substrate in 20 mM sodium citrate.

[0060] cDNA: The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription of mature, spliced ​​mRNA molecules obtained from eukaryotic or prokaryotic cells. cDNA lacks the intron sequences that can be present in the corresponding genomic DNA. Early initial RNA transcripts are precursors to mRNA, undergoing a series of processing steps, including splicing, before becoming mature, spliced ​​mRNA.

[0061] Cellobiose hydrolase: The term "cellobiose hydrolase" refers to a 1,4-β-D-glucan cellobiose hydrolase (EC3.2.1.91 and EC3.2.1.176) that catalyzes the hydrolysis of 1,4-β-D-glycosidic bonds in cellulose, cellooligosaccharides, or any polymer containing β-1,4-linked glucose, thereby releasing cellobiose from either the reducing end (cellobiose hydrolase I) or the non-reducing end (cellobiose hydrolase II) of the chain (Teeri, 1997, Trends in Biotechnology 15:160-167; Teeri et al., 1998, Biochem.Soc.Trans. 26:173-178). Cellobiose hydrolase activity was preferably determined according to procedures described by Lever et al., 1972, Anal. Biochem. 47:273-279; van Tilbeurgh et al., 1982, FEBS Letters, 149:152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters, 187:283-288; and Tomme et al., 1988, Eur. J. Biochem. 170:575-581.

[0062] Cellulose-degrading enzymes or cellulases: The term “cellulose-degrading enzyme” (or “cellulase”) refers to one or more (e.g., several) enzymes capable of hydrolyzing cellulose materials. These enzymes include one or more endoglucanases, one or more cellobiases, one or more β-glucosidases, or combinations thereof. Two basic methods for measuring cellulose-degrading enzyme activity include: (1) determining total cellulose-degrading enzyme activity, and (2) determining individual cellulose-degrading enzyme activity (endoglucanase, cellobiase, and β-glucosidase), as described in Zhang et al., 2006, Biotechnology. As described in Advances (Biotechnology Progress) 24:452-481, total cellulose degrading activity is typically determined using insoluble substrates, including Whatman No. 1 filter paper, microcrystalline cellulose, bacterial cellulose, algal cellulose, cotton, and pretreated lignocellulose. The most commonly used method for determining total cellulose degrading activity is the filter paper assay using Whatman No. 1 filter paper as the substrate. This method was established by the International Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987, Pure Appl. Chem. 59:257-68).

[0063] For the purposes of this invention, cellulase activity is determined by measuring the increase in hydrolysis of cellulose material by one or more cellulases under the following conditions: 1-50 mg of cellulase protein / g of cellulose (or other pretreated cellulose material) in PCS, for 3-7 days at a suitable temperature (e.g., 40°C-80°C, e.g., 50°C, 55°C, 60°C, 65°C, or 70°C) and a suitable pH (e.g., 4-9, e.g., 5.0, 5.5, 6.0, 6.5, or 7.0), compared to control hydrolysis without added cellulase protein. Typical conditions are: 1 ml reaction, washed or unwashed pretreated corn stalks (PCS), 5% insoluble solids, 50 mM sodium acetate (pH 5), 1 mM MnSO4, 50°C, 55°C, or 60°C, 72 hours, by... Sugar analysis was performed using an HPX-87H column (Bio-Ray Laboratories, Hercules, California, USA).

[0064] Cellulose Material: The term "cellulose material" refers to any material containing cellulose. The primary polysaccharide in the primary cell walls of biomass is cellulose, followed by hemicellulose, and then pectin. Secondary cell walls, formed after cell cessation of growth, also contain polysaccharides and are reinforced by polymeric lignin covalently cross-linked with hemicellulose. Cellulose is a homopolymer of dehydrated cellobiose and is therefore a linear β-(l-4)-D-glucan, while hemicellulose comprises a variety of compounds, such as xylan, xyloglucan, arabinoylxylan, and mannan, which exist with a series of substituents in complex branched structures. Although cellulose is generally polymorphic, it is found in plant tissues primarily as an insoluble crystalline matrix of parallel glucan chains. Hemicellulose is often hydrogen-bonded to cellulose and other hemicelluloses, which helps stabilize the cell wall matrix.

[0065] Cellulose is commonly found in, for example, the stems, leaves, shells, bark, and rachis of plants, or the leaves, branches, and wood of trees. Cellulose materials can be, but are not limited to, agricultural residues, herbaceous materials (including energy crops), municipal solid waste, pulp and paper mill residues, waste paper, and wood (including forestry waste) (see, for example, Wiselogel et al., 1995, Handbook on Bioethanol (Charles E. Wyman, ed.), pp. 105-118, Taylor & Francis, Washington; Wyman, 1994, Bioresource Technology 50:3-16; Lynd, 1990, Applied Biochemistry and Biotechnology 24 / 25:695-719; Mosier et al., 1999, Recent Progress in Bioconversion of Lignocellulosics, Advances in Biochemical *Engineering / Biotechnology*, T. Scheper, Editor-in-Chief, Vol. 65, pp. 23-40, Springer-Verlag, New York. It should be understood here that cellulose can be in the form of lignin cellulose, a plant cell wall material comprising lignin, cellulose, and hemicellulose in a mixed matrix. In one preferred aspect, the cellulose material is any biomass material. In another preferred aspect, the cellulose material is lignocellulose, which comprises cellulose, hemicellulose, and lignin.

[0066] In one aspect, the cellulosic material is agricultural residue. In another aspect, the cellulosic material is herbaceous material (including energy crops). In another aspect, the cellulosic material is municipal solid waste. In another aspect, the cellulosic material is pulp and paper mill residue. In another aspect, the cellulosic material is waste paper. In another aspect, the cellulosic material is wood (including forestry waste).

[0067] In another aspect, the cellulose material is reed. In another aspect, the cellulose material is sugarcane bagasse. In another aspect, the cellulose material is bamboo. In another aspect, the cellulose material is corn cob. In another aspect, the cellulose material is corn fiber. In another aspect, the cellulose material is corn stalk. In another aspect, the cellulose material is miscanthus. In another aspect, the cellulose material is orange peel. In another aspect, the cellulose material is rice straw. In another aspect, the cellulose material is switchgrass. In another aspect, the cellulose material is wheat stalk.

[0068] In another aspect, the cellulose material is aspen. In another aspect, the cellulose material is eucalyptus. In another aspect, the cellulose material is fir. In another aspect, the cellulose material is pine. In another aspect, the cellulose material is poplar. In another aspect, the cellulose material is spruce. In another aspect, the cellulose material is willow.

[0069] In another aspect, the cellulose material is seaweed cellulose. In another aspect, the cellulose material is bacterial cellulose. In another aspect, the cellulose material is cotton linter. In another aspect, the cellulose material is filter paper. In another aspect, the cellulose material is microcrystalline cellulose. In another aspect, the cellulose material is phosphoric acid-treated cellulose.

[0070] In another respect, cellulose material is a type of aquatic biomass. As used herein, the term "aquatic biomass" refers to biomass produced in an aquatic environment through the process of photosynthesis. Aquatic biomass can be algae, emergent plants, floating-leaved plants, or submerged plants.

[0071] Cellulose materials can be used as is or pretreated using conventional methods known in the art, as described herein. In a preferred aspect, the cellulose material has been pretreated.

[0072] Coding sequence: The term "coding sequence" refers to a polynucleotide that directly defines the amino acid sequence of a variant. The boundaries of a coding sequence are generally determined by an open reading frame that begins with a start codon (such as ATG, GTG, or TTG) and ends with a stop codon (such as TAA, TAG, or TGA). A coding sequence can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0073] Control Sequences: The term "control sequence" refers to the nucleic acid sequence required for the expression of a polynucleotide encoding a variant of the present invention. Each control sequence may be native (i.e., from the same gene) or exogenous (i.e., from a different gene) for the polynucleotide encoding that variant, or native or exogenous relative to each other. Such control sequences include, but are not limited to, pre-leaders, polyadenylated sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, control sequences include promoters, as well as transcription and translation termination signals. These control sequences may be provided with multiple linkers for the purpose of introducing specific restriction enzyme sites that facilitate the linking of these control sequences to the coding region encoding a polynucleotide of a variant.

[0074] Endoglucanase: The term "endoglucanase" refers to an endo-1,4-(1,3;1,4)-β-D-glucan-4-glucan hydrolase (EC 3.2.1.4) that catalyzes the endo-hydrolysis of β-1,4-β-D-glycosidic bonds in cellulose, cellulose derivatives (such as carboxymethyl cellulose and hydroxyethyl cellulose), lichen polysaccharides, and mixed β-1,3-glucans such as cereal β-D-glucan or xyloglucan, as well as other plant materials containing cellulose components. Endoglucanase activity can be determined by measuring the decrease in substrate viscosity or by determining the increase in the reducing end through reducing sugar assays (Zhang et al., 2006, Progress in Biotechnology 24:452-481). For the purposes of this invention, endoglucanase activity was determined using carboxymethyl cellulose (CMC) as a substrate at pH 5 and 40°C, according to the procedure described by Gauss, 1987, Pure and Applied Chemistry 59:257-268.

[0075] Expression: The term “expression” includes any step involved in the production of a variant, including (but not limited to) transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0076] Expression vector: The term “expression vector” refers to a straight or circular DNA molecule that includes a polynucleotide encoding a variant and is operatively linked to a control sequence that provides for its expression.

[0077] Family 61 Glycoside Hydrolase: The term "Family 61 Glycoside Hydrolase" or "GH61" refers to a polypeptide belonging to the glycoside hydrolase family 61, according to Henrissat, 1991, Biochem.J. 280:309-316, and Henrissat and Bairoch, 1996, Biochem.J. 316:695-696. Enzymes in this family were initially classified as glycoside hydrolases based on the very weak endo-1,4-β-D-glucanase activity measured in one family member. Recently, GH61 has been classified as a lytic polysaccharide monooxygenase (Quinlan et al., 2011, Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci. USA) 208:15079-15084; Phillips et al., 2011, ACS Chem. Biol. 6:1399-1406; Lin et al., 2012, Structure 20:1051-1061).

[0078] Ferulic acid esterase: The term "ferulic acid esterase" refers to 4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC3.1.1.73), which catalyzes the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyl) group from esterified sugars (which are typically arabinose in natural biomass substrates) to produce ferulic esters (4-hydroxy-3-methoxycinnamoyl esterase). Ferulic acid esterase (FAE) is also known as ferulic acid esterase, hydroxycinnamoyl esterase, FAE-III, cinnamoyl esterase, FAEA, cinnAE, FAE-I, or FAE-II. For the purposes of this invention, the activity of ferulic acid esterase was determined using 0.5 mM p-nitrophenylferulate as a substrate in 50 mM sodium acetate at pH 5.0. One unit of ferulic acid esterase is equal to the amount of enzyme that can release 1 μmol of p-nitrophenol anion per minute at pH 5 and 25°C.

[0079] Fragment: The term "fragment" refers to a polypeptide that lacks one or more (e.g., several) amino acids at the amino and / or carboxyl termini of its mature polypeptide, wherein the fragment has cellulolytic-enhancing activity. In one aspect, the fragment contains at least 85% of the amino acid residues, for example at least 90% or at least 95% of the amino acid residues of the mature polypeptide of the GH61 polypeptide.

[0080] Hemicellulase or hemicellulase: The term “hemicellulase” or “hemicellulase” refers to one or more (e.g., several) enzymes that hydrolyze hemicellulose materials. See, for example, Sharlom and Shoham, Current Opinion in Microbiology, 2003, 6(3):219-228). Hemicellulases are key components in the degradation of plant biomass. Examples of hemicellulases include, but are not limited to, acetylmannan esterase, acetylxylan esterase, arabinogalactanase, arabinofuranase, coumarin esterase, ferulic esterase, galactosidase, glucuronidase, glucuronidase, mannanase, mannosidase, xylanase, and xylosidase. The substrate of these enzymes—hemicellulose—is a heterogeneous group of branched and linear polysaccharides that can be cross-linked into a robust network by hydrogen bonds to cellulose microfibers in the plant cell wall. Hemicellulose is also covalently attached to lignin, thus forming a highly complex structure together with cellulose. The variable structure and organization of hemicellulose require the synergistic action of many enzymes to achieve its complete degradation. The catalytic modules of hemicellulases are glycosidases (GH) that hydrolyze glycosidic bonds, or carbohydrate esterases (CE) that hydrolyze ester bonds on the side groups of acetic acid or ferulic acid. These catalytic modules can be assigned to the GH and CE families based on their primary sequence homology. Certain families with generally similar folds can be grouped into subgroups and labeled alphabetically (e.g., GH-A). The most comprehensive and up-to-date classification of these enzymes, as well as carbohydrate-active enzymes, is available in the Carbohydrate Active Enzymes (CAZY) database. According to Ghose and Bisaria, 1987, Pure & Applied Chemistry 59:1739-1752, hemicellulose-degrading enzyme activity can be measured at suitable temperatures, such as 40°C-80°C, or 50°C, 55°C, 60°C, 65°C, or 70°C, and suitable pH values, such as 4-9, or 5.0, 5.5, 6.0, 6.5, or 7.0.

[0081] High-rigor conditions: The term "high-rigor conditions" refers to the standard Southern blotting procedure followed for probes at least 100 nucleotides in length, involving pre-hybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours. Vector material is finally washed three times at 65°C for 15 minutes each time with 0.2X SSC and 0.2% SDS.

[0082] Host cell: The term "host cell" refers to any cell type that is readily transformed, transfected, transduced, etc., using a nucleic acid construct or expression vector including a polynucleotide of the present invention. The term "host cell" also encompasses any offspring of a parent cell that differs from the parent cell due to mutations occurring during replication.

[0083] Improved properties: The term "improved properties" refers to characteristics that are improved relative to the parent strain and are associated with a variant. Such improved properties include, but are not limited to, increased thermal stability.

[0084] Increased thermal stability: The term "increased thermal stability" refers to the greater retention of cellulose-degrading-enhancing activity of the GH61 peptide variant after incubation at a certain temperature for a period of time relative to the parent. The increased thermal stability of the variant relative to the parent can be assessed, for example, under one or more (e.g., several) temperature conditions. For example, the one or more temperatures can be any one or more temperatures in the range of 45°C to 95°C, such as 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, or 95°C (or in between, such as 62°C, 68°C, 72°C, etc.), at one or more pH values ​​in the range of 3 to 9, such as 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0 (or in between), for a sustained incubation period (time), such as 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 45 minutes, or 60 minutes (or in between, such as 23 minutes, 37 minutes, etc.), so that the variant retains residual activity. However, longer incubation periods can also be used. The terms "increased thermal stability" and "improved thermal stability" can be used interchangeably.

[0085] The increased thermal stability of the variant relative to the parent can be determined by differential scanning calorimetry (DSC) using standard methods in the art (see, for example, Sturtevant, 1987, Annual Review of Physical Chemistry 38:463-488; Example 9). The increased thermal stability of the variant relative to the parent can also be determined using protein pyrolysis and folding analysis (see, for example, Example 10 herein). The increased thermal stability of the variant relative to the parent can also be determined by any enzyme assay known in the art for GH61 peptides with enhanced cellulose-degrading activity to measure residual activity after temperature treatment. See, for example, WO 2005 / 074647, WO 2008 / 148131, WO 2005 / 074656, WO 2010 / 065830, WO 2007 / 089290, WO 2009 / 085935, WO 2009 / 085859, WO 2009 / 085864, WO 2009 / 085868, and WO 2008 / 151043, which are incorporated herein by reference. Alternatively, the increase in thermal stability of the variant relative to the parent can be determined using any application determination for that variant in which the performance of the variant is compared to that of the parent. For example, the application determination described in Example 5 can be used.

[0086] Separate: The term “separate” means a substance in a form or environment that is not naturally occurring. Non-limiting examples of separated substances include (1) any substance that is not naturally occurring, (2) any substance, including but not limited to any enzyme, variant, nucleic acid, protein, peptide or cofactor that has been at least partially removed from one or more of the naturally occurring components associated with it; (3) any substance that has been artificially modified by humans relative to a substance found in nature; or (4) any substance that has been modified by increasing the amount of the substance relative to other components naturally associated with it (e.g., recombinant generation in a host cell; multiple copies of the gene encoding the substance; and the use of a promoter stronger than that naturally associated with the gene encoding the substance).

[0087] Low-stringency conditions: The term "low-stringency conditions" refers to pre-hybridization and hybridization for probes of at least 100 nucleotides in length, following standard DNA blotting procedures at 42°C in 5X SSPE, 0.3% SDS, 200 μg / mL cleaved and denatured salmon sperm DNA, and 25% formamide for 12 to 24 hours. Vector material is finally washed three times at 50°C for 15 minutes each time with 0.2X SSC and 0.2% SDS.

[0088] Mature polypeptide: The term "mature polypeptide" refers to a polypeptide that has reached its final form after translation and any post-translational modifications (such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.). In one aspect, based on the SignalP 3.0 program predicting that amino acids 1 to 19 of SEQ ID NO:2 are a signal peptide (Bendtsen et al., 2004, J. Mol. Biol. 340:783-795), the mature polypeptide is amino acids 20 to 326 of SEQ ID NO:2. In another aspect, based on the SignalP program predicting that amino acids 1 to 17 of SEQ ID NO:4 are a signal peptide, the mature polypeptide is amino acids 18 to 239 of SEQ ID NO:4. In yet another aspect, based on the SignalP program predicting that amino acids 1 to 19 of SEQ ID NO:6 are a signal peptide, the mature polypeptide is amino acids 20 to 258 of SEQ ID NO:6. In another aspect, based on the SignalP program predicting that amino acids 1 to 18 of SEQ ID NO:8 are signal peptides, the mature polypeptide is amino acids 19 to 226 of SEQ ID NO:8. In another aspect, based on the SignalP program predicting that amino acids 1 to 19 of SEQ ID NO:10 are signal peptides, the mature polypeptide is amino acids 20 to 304 of SEQ ID NO:10. In another aspect, based on the SignalP program predicting that amino acids 1 to 15 of SEQ ID NO:12 are signal peptides, the mature polypeptide is amino acids 16 to 317 of SEQ ID NO:12. In another aspect, based on the SignalP program predicting that amino acids 1 to 21 of SEQ ID NO:14 are signal peptides, the mature polypeptide is amino acids 22 to 249 of SEQ ID NO:14. In another aspect, based on the SignalP program predicting that amino acids 1 to 19 of SEQ ID NO:16 are signal peptides, the mature polypeptide is amino acids 20 to 249 of SEQ ID NO:16. In another aspect, based on the SignalP program predicting that amino acids 1 to 17 of SEQ ID NO:18 are signal peptides, the mature polypeptide is amino acids 18 to 232 of SEQ ID NO:18. In another aspect, based on the SignalP program predicting that amino acids 1 to 15 of SEQ ID NO:20 are signal peptides, the mature polypeptide is amino acids 16 to 235 of SEQ ID NO:20. In another aspect, based on the SignalP program predicting that amino acids 1 to 18 of SEQ ID NO:22 are signal peptides, the mature polypeptide is amino acids 19 to 323 of SEQ ID NO:22. In another aspect, based on the SignalP program predicting that amino acids 1 to 15 of SEQ ID NO:24 are signal peptides, the mature polypeptide is amino acids 16 to 310 of SEQ ID NO:24.In another aspect, based on the SignalP program predicting that amino acids 1 to 19 of SEQ ID NO:26 are signal peptides, the mature polypeptide is amino acids 20 to 246 of SEQ ID NO:26. In another aspect, based on the SignalP program predicting that amino acids 1 to 21 of SEQ ID NO:28 are signal peptides, the mature polypeptide is amino acids 22 to 354 of SEQ ID NO:28. In another aspect, based on the SignalP program predicting that amino acids 1 to 21 of SEQ ID NO:30 are signal peptides, the mature polypeptide is amino acids 22 to 250 of SEQ ID NO:30. In another aspect, based on the SignalP program predicting that amino acids 1 to 21 of SEQ ID NO:32 are signal peptides, the mature polypeptide is amino acids 22 to 322 of SEQ ID NO:32. In another aspect, based on the SignalP program predicting that amino acids 1 to 23 of SEQ ID NO:34 are signal peptides, the mature polypeptide is amino acids 24 to 444 of SEQ ID NO:34. In another aspect, based on the SignalP program predicting that amino acids 1 to 25 of SEQ ID NO:36 are signal peptides, the mature polypeptide is amino acids 26 to 253 of SEQ ID NO:36. In another aspect, based on the SignalP program predicting that amino acids 1 to 17 of SEQ ID NO:38 are signal peptides, the mature polypeptide is amino acids 18 to 246 of SEQ ID NO:38. In another aspect, based on the SignalP program predicting that amino acids 1 to 19 of SEQ ID NO:40 are signal peptides, the mature polypeptide is amino acids 20 to 334 of SEQ ID NO:40. In another aspect, based on the SignalP program predicting that amino acids 1 to 17 of SEQ ID NO:42 are signal peptides, the mature polypeptide is amino acids 18 to 227 of SEQ ID NO:42. In another aspect, based on the SignalP program predicting that amino acids 1 to 19 of SEQ ID NO:44 are signal peptides, the mature polypeptide is amino acids 20 to 223 of SEQ ID NO:44. In another aspect, based on the SignalP program predicting that amino acids 1 to 21 of SEQ ID NO:46 are signal peptides, the mature polypeptide is amino acids 22 to 368 of SEQ ID NO:46. In another aspect, based on the SignalP program predicting that amino acids 1 to 24 of SEQ ID NO:48 are signal peptides, the mature polypeptide is amino acids 25 to 330 of SEQ ID NO:48. In another aspect, based on the SignalP program predicting that amino acids 1 to 16 of SEQ ID NO:50 are signal peptides, the mature polypeptide is amino acids 17 to 236 of SEQ ID NO:50.In another aspect, based on the SignalP program predicting that amino acids 1 to 18 of SEQ ID NO:52 are signal peptides, the mature polypeptide is amino acids 19 to 250 of SEQ ID NO:52. In another aspect, based on the SignalP program predicting that amino acids 1 to 22 of SEQ ID NO:54 are signal peptides, the mature polypeptide is amino acids 23 to 478 of SEQ ID NO:54. In another aspect, based on the SignalP program predicting that amino acids 1 to 16 of SEQ ID NO:56 are signal peptides, the mature polypeptide is amino acids 17 to 230 of SEQ ID NO:56. In another aspect, based on the SignalP program predicting that amino acids 1 to 19 of SEQ ID NO:58 are signal peptides, the mature polypeptide is amino acids 20 to 257 of SEQ ID NO:58. In another aspect, based on the SignalP program predicting that amino acids 1 to 22 of SEQ ID NO:60 are signal peptides, the mature polypeptide is amino acids 23 to 251 of SEQ ID NO:60. In another aspect, based on the SignalP program predicting that amino acids 1 to 18 of SEQ ID NO:62 are signal peptides, the mature polypeptide is amino acids 19 to 349 of SEQ ID NO:62. In another aspect, based on the SignalP program predicting that amino acids 1 to 23 of SEQ ID NO:64 are signal peptides, the mature polypeptide is amino acids 24 to 436 of SEQ ID NO:64. In another aspect, based on the SignalP program predicting that amino acids 1 to 23 of SEQ ID NO:66 are signal peptides, the mature polypeptide is amino acids 21 to 344 of SEQ ID NO:66. In another aspect, based on the SignalP program predicting that amino acids 1 to 25 of SEQ ID NO:68 are signal peptides, the mature polypeptide is amino acids 26 to 400 of SEQ ID NO:68. In another aspect, based on the SignalP program predicting that amino acids 1 to 20 of SEQ ID NO:70 are signal peptides, the mature polypeptide is amino acids 21 to 389 of SEQ ID NO:70. In another aspect, based on the SignalP program predicting that amino acids 1 to 21 of SEQ ID NO:72 are signal peptides, the mature polypeptide is amino acids 22 to 406 of SEQ ID NO:72. In another aspect, based on the SignalP program predicting that amino acids 1 to 19 of SEQ ID NO:74 are signal peptides, the mature polypeptide is amino acids 20 to 427 of SEQ ID NO:74. In yet another aspect, based on the SignalP program predicting that amino acids 1 to 17 of SEQ ID NO:76 are signal peptides, the mature polypeptide is amino acids 18 to 267 of SEQ ID NO:76.In another aspect, based on the SignalP program predicting that amino acids 1 to 20 of SEQ ID NO:78 are signal peptides, the mature polypeptide is amino acids 21 to 273 of SEQ ID NO:78. In another aspect, based on the SignalP program predicting that amino acids 1 to 20 of SEQ ID NO:80 are signal peptides, the mature polypeptide is amino acids 21 to 322 of SEQ ID NO:80. In another aspect, based on the SignalP program predicting that amino acids 1 to 17 of SEQ ID NO:82 are signal peptides, the mature polypeptide is amino acids 18 to 234 of SEQ ID NO:82. In another aspect, based on the SignalP program predicting that amino acids 1 to 23 of SEQ ID NO:84 are signal peptides, the mature polypeptide is amino acids 24 to 233 of SEQ ID NO:84. In another aspect, based on the SignalP program predicting that amino acids 1 to 16 of SEQ ID NO:86 are signal peptides, the mature polypeptide is amino acids 17 to 237 of SEQ ID NO:86. In another aspect, based on the SignalP program predicting that amino acids 1 to 19 of SEQ ID NO:88 are signal peptides, the mature polypeptide is amino acids 20 to 484 of SEQ ID NO:88. In another aspect, based on the SignalP program predicting that amino acids 1 to 21 of SEQ ID NO:90 are signal peptides, the mature polypeptide is amino acids 22 to 320 of SEQ ID NO:90. In another aspect, based on the SignalP program predicting that amino acids 1 to 22 of SEQ ID NO:92 are signal peptides, the mature polypeptide is amino acids 23 to 272 of SEQ ID NO:92. In another aspect, based on the SignalP program predicting that amino acids 1 to 21 of SEQ ID NO:94 are signal peptides, the mature polypeptide is amino acids 22 to 327 of SEQ ID NO:94. In another aspect, based on the SignalP program predicting that amino acids 1 to 22 of SEQ ID NO:96 are signal peptides, the mature polypeptide is amino acids 23 to 274 of SEQ ID NO:96. In another aspect, based on the SignalP program predicting that amino acids 1 to 17 of SEQ ID NO:98 are signal peptides, the mature polypeptide is amino acids 18 to 227 of SEQ ID NO:98. In another aspect, based on the SignalP program predicting that amino acids 1 to 16 of SEQ ID NO:100 are signal peptides, the mature polypeptide is amino acids 17 to 257 of SEQ ID NO:100. In yet another aspect, based on the SignalP program predicting that amino acids 1 to 19 of SEQ ID NO:102 are signal peptides, the mature polypeptide is amino acids 20 to 246 of SEQ ID NO:102.In another aspect, based on the SignalP program predicting that amino acids 1 to 27 of SEQ ID NO:104 are signal peptides, the mature polypeptide is amino acids 28 to 265 of SEQ ID NO:104. In another aspect, based on the SignalP program predicting that amino acids 1 to 15 of SEQ ID NO:106 are signal peptides, the mature polypeptide is amino acids 16 to 310 of SEQ ID NO:106. In another aspect, based on the SignalP program predicting that amino acids 1 to 20 of SEQ ID NO:108 are signal peptides, the mature polypeptide is amino acids 21 to 354 of SEQ ID NO:108. In another aspect, based on the SignalP program predicting that amino acids 1 to 21 of SEQ ID NO:110 are signal peptides, the mature polypeptide is amino acids 22 to 267 of SEQ ID NO:110. In another aspect, based on the SignalP program predicting that amino acids 1 to 15 of SEQ ID NO:112 are signal peptides, the mature polypeptide is amino acids 16 to 237 of SEQ ID NO:112. In another aspect, based on the SignalP program predicting that amino acids 1 to 19 of SEQ ID NO:114 are signal peptides, the mature polypeptide is amino acids 20 to 234 of SEQ ID NO:114. In another aspect, based on the SignalP program predicting that amino acids 1 to 17 of SEQ ID NO:116 are signal peptides, the mature polypeptide is amino acids 18 to 226 of SEQ ID NO:116. In another aspect, based on the SignalP program predicting that amino acids 1 to 16 of SEQ ID NO:118 are signal peptides, the mature polypeptide is amino acids 17 to 231 of SEQ ID NO:118. In another aspect, based on the SignalP program predicting that amino acids 1 to 21 of SEQ ID NO:120 are signal peptides, the mature polypeptide is amino acids 22 to 248 of SEQ ID NO:120. In another aspect, based on the SignalP program predicting that amino acids 1 to 17 of SEQ ID NO:122 are signal peptides, the mature polypeptide is amino acids 18 to 233 of SEQ ID NO:122. In another aspect, based on the SignalP program predicting that amino acids 1 to 20 of SEQ ID NO:124 are signal peptides, the mature polypeptide is amino acids 21 to 243 of SEQ ID NO:124. In another aspect, based on the SignalP program predicting that amino acids 1 to 20 of SEQ ID NO:126 are signal peptides, the mature polypeptide is amino acids 21 to 363 of SEQ ID NO:126. In yet another aspect, based on the SignalP program predicting that amino acids 1 to 19 of SEQ ID NO:128 are signal peptides, the mature polypeptide is amino acids 20 to 296 of SEQ ID NO:128.In another aspect, based on the SignalP program predicting that amino acids 1 to 15 of SEQ ID NO:130 are signal peptides, the mature polypeptide is amino acids 16 to 318 of SEQ ID NO:130. In another aspect, based on the SignalP program predicting that amino acids 1 to 18 of SEQ ID NO:132 are signal peptides, the mature polypeptide is amino acids 19 to 259 of SEQ ID NO:132. In another aspect, based on the SignalP program predicting that amino acids 1 to 19 of SEQ ID NO:134 are signal peptides, the mature polypeptide is amino acids 20 to 325 of SEQ ID NO:134. In another aspect, based on the SignalP program predicting that amino acids 1 to 18 of SEQ ID NO:136 are signal peptides, the mature polypeptide is amino acids 19 to 298 of SEQ ID NO:136. In another aspect, based on the SignalP program predicting that amino acids 1 to 19 of SEQ ID NO:138 are signal peptides, the mature polypeptide is amino acids 20 to 298 of SEQ ID NO:138. In another aspect, based on the SignalP program predicting that amino acids 1 to 21 of SEQ ID NO:140 are signal peptides, the mature polypeptide is amino acids 22 to 344 of SEQ ID NO:140. In another aspect, based on the SignalP program predicting that amino acids 1 to 19 of SEQ ID NO:142 are signal peptides, the mature polypeptide is amino acids 20 to 330 of SEQ ID NO:142. In another aspect, based on the SignalP program predicting that amino acids 1 to 18 of SEQ ID NO:144 are signal peptides, the mature polypeptide is amino acids 19 to 216 of SEQ ID NO:144. In another aspect, based on the SignalP program predicting that amino acids 1 to 17 of SEQ ID NO:146 are signal peptides, the mature polypeptide is amino acids 18 to 490 of SEQ ID NO:146. In another aspect, based on the SignalP program predicting that amino acids 1 to 20 of SEQ ID NO:148 are signal peptides, the mature polypeptide is amino acids 21 to 306 of SEQ ID NO:148. In another aspect, based on the SignalP program predicting that amino acids 1 to 21 of SEQ ID NO:150 are a signal peptide, the mature polypeptide is amino acids 22 to 339 of SEQ ID NO:150. In another aspect, based on the SignalP program predicting that amino acids 1 to 22 of SEQ ID NO:152 are a signal peptide, the mature polypeptide is amino acids 23 to 334 of SEQ ID NO:152. In yet another aspect, based on the SignalP program predicting that amino acids 1 to 23 of SEQ ID NO:154 are a signal peptide, the mature polypeptide is amino acids 24 to 366 of SEQ ID NO:154.In another aspect, based on the SignalP program predicting that amino acids 1 to 20 of SEQ ID NO:156 are signal peptides, the mature polypeptide is amino acids 21 to 364 of SEQ ID NO:156. In another aspect, based on the SignalP program predicting that amino acids 1 to 21 of SEQ ID NO:158 are signal peptides, the mature polypeptide is amino acids 22 to 344 of SEQ ID NO:158. In another aspect, based on the SignalP program predicting that amino acids 1 to 19 of SEQ ID NO:160 are signal peptides, the mature polypeptide is amino acids 20 to 252 of SEQ ID NO:160. In another aspect, based on the SignalP program predicting that amino acids 1 to 19 of SEQ ID NO:162 are signal peptides, the mature polypeptide is amino acids 20 to 344 of SEQ ID NO:162. In another aspect, based on the SignalP program predicting that amino acids 1 to 21 of SEQ ID NO:164 are signal peptides, the mature polypeptide is amino acids 22 to 347 of SEQ ID NO:164. In another aspect, based on the SignalP program predicting that amino acids 1 to 19 of SEQ ID NO:166 are signal peptides, the mature polypeptide is amino acids 20 to 342 of SEQ ID NO:166. In another aspect, based on the SignalP program predicting that amino acids 1 to 26 of SEQ ID NO:168 are signal peptides, the mature polypeptide is amino acids 27 to 254 of SEQ ID NO:168. In another aspect, based on the SignalP program predicting that amino acids 1 to 22 of SEQ ID NO:409 are signal peptides, the mature polypeptide is amino acids 23 to 272 of SEQ ID NO:409. In another aspect, based on the SignalP program predicting that amino acids 1 to 22 of SEQ ID NO:411 are signal peptides, the mature polypeptide is amino acids 23 to 272 of SEQ ID NO:411. It is known in the art that host cells can produce mixtures of two or more different mature polypeptides (i.e., having a different C-terminal and / or N-terminal amino acid) expressed by the same polynucleotide.

[0089] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" refers to a polynucleotide encoding a mature polypeptide with cellulolytic-enhancing activity. In one aspect, based on the SignalP 3.0 program (Bentson et al., 2004, ibid.) predicting nucleotides 330 to 387 of SEQ ID NO:1 encoding a signal peptide, the mature polypeptide coding sequence is nucleotides 388 to 1332 of SEQ ID NO:1. In another aspect, based on the SignalP program predicting nucleotides 47 to 97 of SEQ ID NO:3 encoding a signal peptide, the mature polypeptide coding sequence is nucleotides 98 to 821 of SEQ ID NO:3. In another aspect, based on the SignalP program predicting nucleotides 69 to 125 of SEQ ID NO:5 encoding a signal peptide, the mature polypeptide coding sequence is nucleotides 126 to 978 of SEQ ID NO:5. In yet another aspect, based on the SignalP program predicting nucleotides 1 to 54 of SEQ ID NO:7 encoding a signal peptide, the mature polypeptide coding sequence is nucleotides 55 to 678 of SEQ ID NO:7 or its genomic DNA sequence. In another aspect, based on the SignalP program predicting that nucleotides 1 to 57 of SEQ ID NO:9 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 58 to 912 of SEQ ID NO:9 or their genomic DNA sequence. In another aspect, based on the SignalP program predicting that nucleotides 1 to 45 of SEQ ID NO:11 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 46 to 951 of SEQ ID NO:11 or their genomic DNA sequence. In another aspect, based on the SignalP program predicting that nucleotides 1 to 63 of SEQ ID NO:13 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 64 to 796 of SEQ ID NO:13. In another aspect, based on the SignalP program predicting that nucleotides 20 to 76 of SEQ ID NO:15 or their genomic DNA sequence encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 77 to 766 of SEQ ID NO:15. In another aspect, based on the SignalP program predicting that nucleotides 1 to 51 of SEQ ID NO:17 encode a signal peptide, the mature polypeptide coding sequence is nucleotides 52 to 921 of SEQ ID NO:17. In another aspect, based on the SignalP program predicting that nucleotides 1 to 45 of SEQ ID NO:19 encode a signal peptide, the mature polypeptide coding sequence is nucleotides 46 to 851 of SEQ ID NO:19. In yet another aspect, based on the SignalP program predicting that nucleotides 1 to 54 of SEQ ID NO:21 encode a signal peptide, the mature polypeptide coding sequence is nucleotides 55 to 1239 of SEQ ID NO:21.In another aspect, based on the SignalP program predicting that nucleotides 1 to 45 of SEQ ID NO:23 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 46 to 1250 of SEQ ID NO:23. In another aspect, based on the SignalP program predicting that nucleotides 1 to 57 of SEQ ID NO:25 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 58 to 811 of SEQ ID NO:25. In another aspect, based on the SignalP program predicting that nucleotides 1 to 63 of SEQ ID NO:27 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 64 to 1112 of SEQ ID NO:27. In another aspect, based on the SignalP program predicting that nucleotides 1 to 63 of SEQ ID NO:29 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 64 to 859 of SEQ ID NO:29. In another aspect, based on the SignalP program predicting that nucleotides 1 to 63 of SEQ ID NO:31 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 64 to 1018 of SEQ ID NO:31. In another aspect, based on the SignalP program predicting that nucleotides 1 to 69 of SEQ ID NO:33 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 70 to 1483 of SEQ ID NO:33. In another aspect, based on the SignalP program predicting that nucleotides 1 to 75 of SEQ ID NO:35 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 76 to 832 of SEQ ID NO:35. In another aspect, based on the SignalP program predicting that nucleotides 1 to 51 of SEQ ID NO:37 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 52 to 875 of SEQ ID NO:37. In another aspect, based on the SignalP program predicting that nucleotides 1 to 57 of SEQ ID NO:39 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 58 to 1250 of SEQ ID NO:39. In another aspect, based on the SignalP program predicting that nucleotides 1 to 51 of SEQ ID NO:41 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 52 to 795 of SEQ ID NO:41. In another aspect, based on the SignalP program predicting that nucleotides 1 to 57 of SEQ ID NO:43 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 58 to 974 of SEQ ID NO:43. In another aspect, based on the SignalP program predicting that nucleotides 1 to 63 of SEQ ID NO:45 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 64 to 1104 of SEQ ID NO:45.In another aspect, based on the SignalP program predicting that nucleotides 1 to 72 of SEQ ID NO:47 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 73 to 990 of SEQ ID NO:47. In another aspect, based on the SignalP program predicting that nucleotides 1 to 48 of SEQ ID NO:49 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 49 to 1218 of SEQ ID NO:49. In another aspect, based on the SignalP program predicting that nucleotides 1 to 54 of SEQ ID NO:51 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 55 to 930 of SEQ ID NO:51. In another aspect, based on the SignalP program predicting that nucleotides 1 to 66 of SEQ ID NO:53 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 67 to 1581 of SEQ ID NO:53. In another aspect, based on the SignalP program predicting that nucleotides 1 to 48 of SEQ ID NO:55 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 49 to 865 of SEQ ID NO:55. In another aspect, based on the SignalP program predicting that nucleotides 1 to 57 of SEQ ID NO:57 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 58 to 1065 of SEQ ID NO:57. In another aspect, based on the SignalP program predicting that nucleotides 1 to 66 of SEQ ID NO:59 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 67 to 868 of SEQ ID NO:59. In another aspect, based on the SignalP program predicting that nucleotides 1 to 54 of SEQ ID NO:61 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 55 to 1099 of SEQ ID NO:61. In another aspect, based on the SignalP program predicting that nucleotides 1 to 69 of SEQ ID NO:63 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 70 to 1483 of SEQ ID NO:63. In another aspect, based on the SignalP program predicting that nucleotides 1 to 60 of SEQ ID NO:65 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 61 to 1032 of SEQ ID NO:65. In another aspect, based on the SignalP program predicting that nucleotides 1 to 75 of SEQ ID NO:67 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 76 to 1200 of SEQ ID NO:67. In another aspect, based on the SignalP program predicting that nucleotides 1 to 60 of SEQ ID NO:69 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 61 to 1167 of SEQ ID NO:69.In another aspect, based on the SignalP program predicting that nucleotides 1 to 63 of SEQ ID NO:71 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 64 to 1218 of SEQ ID NO:71. In another aspect, based on the SignalP program predicting that nucleotides 1 to 57 of SEQ ID NO:73 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 58 to 1281 of SEQ ID NO:73. In another aspect, based on the SignalP program predicting that nucleotides 1 to 51 of SEQ ID NO:75 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 52 to 801 of SEQ ID NO:75. In another aspect, based on the SignalP program predicting that nucleotides 1 to 60 of SEQ ID NO:77 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 61 to 819 of SEQ ID NO:77. In another aspect, based on the SignalP program predicting that nucleotides 1 to 60 of SEQ ID NO:79 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 61 to 966 of SEQ ID NO:79. In another aspect, based on the SignalP program predicting that nucleotides 1 to 51 of SEQ ID NO:81 encode a signal peptide, the mature polypeptide coding sequence is nucleotides 52 to 702 of SEQ ID NO:81 or its genomic DNA sequence. In another aspect, based on the SignalP program predicting that nucleotides 1 to 69 of SEQ ID NO:83 encode a signal peptide, the mature polypeptide coding sequence is nucleotides 70 to 699 of SEQ ID NO:83 or its genomic DNA sequence. In another aspect, based on the SignalP program predicting that nucleotides 1 to 48 of SEQ ID NO:85 encode a signal peptide, the mature polypeptide coding sequence is nucleotides 49 to 711 of SEQ ID NO:85 or its genomic DNA sequence. In another aspect, based on the SignalP program predicting that nucleotides 1 to 75 of SEQ ID NO:87 encode a signal peptide, the mature polypeptide coding sequence is nucleotides 76 to 1452 of SEQ ID NO:87. Alternatively, based on the SignalP program predicting that nucleotides 1 to 63 of SEQ ID NO:89 encode a signal peptide, the mature polypeptide coding sequence is nucleotides 64 to 1018 of SEQ ID NO:89. In another aspect, based on the SignalP program predicting that nucleotides 1 to 66 of SEQ ID NO:91 encode a signal peptide, the mature polypeptide coding sequence is nucleotides 67 to 869 of SEQ ID NO:91. In yet another aspect, based on the SignalP program predicting that nucleotides 1 to 63 of SEQ ID NO:93 encode a signal peptide, the mature polypeptide coding sequence is nucleotides 64 to 1036 of SEQ ID NO:93.In another aspect, based on the SignalP program predicting that nucleotides 1 to 66 of SEQ ID NO:95 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 67 to 878 of SEQ ID NO:95. In another aspect, based on the SignalP program predicting that nucleotides 1 to 51 of SEQ ID NO:97 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 52 to 818 of SEQ ID NO:97. In another aspect, based on the SignalP program predicting that nucleotides 1 to 48 of SEQ ID NO:99 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 49 to 1117 of SEQ ID NO:99. In another aspect, based on the SignalP program predicting that nucleotides 1 to 57 of SEQ ID NO:101 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 58 to 875 of SEQ ID NO:101. In another aspect, based on the SignalP program predicting that nucleotides 1 to 81 of SEQ ID NO:103 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 82 to 1064 of SEQ ID NO:103. In another aspect, based on the SignalP program predicting that nucleotides 1 to 45 of SEQ ID NO:105 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 46 to 1032 of SEQ ID NO:105. In another aspect, based on the SignalP program predicting that nucleotides 1 to 60 of SEQ ID NO:107 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 61 to 1062 of SEQ ID NO:107. In another aspect, based on the SignalP program predicting that nucleotides 1 to 63 of SEQ ID NO:109 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 64 to 801 of SEQ ID NO:109. In another aspect, based on the SignalP program predicting that nucleotides 1 to 45 of SEQ ID NO:111 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 46 to 840 of SEQ ID NO:111. In another aspect, based on the SignalP program predicting that nucleotides 1 to 57 of SEQ ID NO:113 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 58 to 702 of SEQ ID NO:113. In another aspect, based on the SignalP program predicting that nucleotides 1 to 51 of SEQ ID NO:115 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 52 to 750 of SEQ ID NO:115. In yet another aspect, based on the SignalP program predicting that nucleotides 1 to 48 of SEQ ID NO:117 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 49 to 851 of SEQ ID NO:117.In another aspect, based on the SignalP program predicting that nucleotides 1 to 63 of SEQ ID NO:119 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 64 to 860 of SEQ ID NO:119. In another aspect, based on the SignalP program predicting that nucleotides 1 to 51 of SEQ ID NO:121 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 52 to 830 of SEQ ID NO:121. In another aspect, based on the SignalP program predicting that nucleotides 1 to 60 of SEQ ID NO:123 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 61 to 925 of SEQ ID NO:123. In another aspect, based on the SignalP program predicting that nucleotides 1 to 60 of SEQ ID NO:125 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 61 to 1089 of SEQ ID NO:125. In another aspect, based on the SignalP program predicting that nucleotides 1 to 57 of SEQ ID NO:127 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 58 to 1083 of SEQ ID NO:127. In another aspect, based on the SignalP program predicting that nucleotides 1 to 45 of SEQ ID NO:129 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 46 to 1029 of SEQ ID NO:129. In another aspect, based on the SignalP program predicting that nucleotides 1 to 54 of SEQ ID NO:131 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 55 to 1110 of SEQ ID NO:131. In another aspect, based on the SignalP program predicting that nucleotides 1 to 57 of SEQ ID NO:133 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 58 to 1100 of SEQ ID NO:133. In another aspect, based on the SignalP program predicting that nucleotides 1 to 54 of SEQ ID NO:135 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 55 to 1036 of SEQ ID NO:135. In another aspect, based on the SignalP program predicting that nucleotides 1 to 57 of SEQ ID NO:137 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 58 to 1022 of SEQ ID NO:137. In another aspect, based on the SignalP program predicting that nucleotides 1 to 63 of SEQ ID NO:139 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 64 to 1032 of SEQ ID NO:139. In another aspect, based on the SignalP program predicting that nucleotides 1 to 57 of SEQ ID NO:141 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 58 to 1054 of SEQ ID NO:141.In another aspect, based on the SignalP program predicting that nucleotides 1 to 54 of SEQ ID NO:143 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 55 to 769 of SEQ ID NO:143. In another aspect, based on the SignalP program predicting that nucleotides 1 to 51 of SEQ ID NO:145 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 52 to 1533 of SEQ ID NO:145. In another aspect, based on the SignalP program predicting that nucleotides 1 to 60 of SEQ ID NO:147 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 61 to 918 of SEQ ID NO:147. In another aspect, based on the SignalP program predicting that nucleotides 1 to 63 of SEQ ID NO:149 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 64 to 1089 of SEQ ID NO:149. In another aspect, based on the SignalP program predicting that nucleotides 1 to 66 of SEQ ID NO:151 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 67 to 1002 of SEQ ID NO:151. In another aspect, based on the SignalP program predicting that nucleotides 1 to 69 of SEQ ID NO:153 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 70 to 1098 of SEQ ID NO:153. In another aspect, based on the SignalP program predicting that nucleotides 1 to 60 of SEQ ID NO:155 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 61 to 1088 of SEQ ID NO:155. In another aspect, based on the SignalP program predicting that nucleotides 1 to 63 of SEQ ID NO:157 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 64 to 1086 of SEQ ID NO:157. In another aspect, based on the SignalP program predicting that nucleotides 1 to 57 of SEQ ID NO:159 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 58 to 756 of SEQ ID NO:159. In another aspect, based on the SignalP program predicting that nucleotides 1 to 57 of SEQ ID NO:161 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 58 to 1032 of SEQ ID NO:161. In another aspect, based on the SignalP program predicting that nucleotides 1 to 63 of SEQ ID NO:163 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 64 to 1041 of SEQ ID NO:163. In another aspect, based on the SignalP program predicting that nucleotides 1 to 57 of SEQ ID NO:165 encode a signal peptide, the mature polypeptide encoding sequence is nucleotides 58 to 1026 of SEQ ID NO:165.In another aspect, based on the SignalP program predicting that nucleotides 1 to 78 of SEQ ID NO:167 encode a signal peptide, the mature polypeptide coding sequence is nucleotides 79 to 762 of SEQ ID NO:167. In another aspect, based on the SignalP program predicting that nucleotides 1 to 66 of SEQ ID NO:408 encode a signal peptide, the mature polypeptide coding sequence is nucleotides 67 to 881 of SEQ ID NO:408. In another aspect, based on the SignalP program predicting that nucleotides 1 to 66 of SEQ ID NO:410 encode a signal peptide, the mature polypeptide coding sequence is nucleotides 67 to 882 of SEQ ID NO:410. The term "mature polypeptide coding sequence" should be understood herein as a cDNA sequence comprising a genomic DNA sequence or a genomic DNA sequence comprising a cDNA sequence.

[0090] Medium-toughness conditions: The term "medium-toughness conditions" refers to pre-hybridization and hybridization at 42°C for 12 to 24 hours in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 35% formamide, following a standard DNA blotting procedure. Vector material is finally washed three times at 55°C for 15 minutes each time with 0.2X SSC and 0.2% SDS.

[0091] Medium-high stringency conditions: The term "high stringency conditions" refers to pre-hybridization and hybridization for probes at least 100 nucleotides in length, following a standard Southern blotting procedure at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 35% formamide for 12 to 24 hours. Vector material is finally washed three times at 60°C for 15 minutes each time with 0.2X SSC and 0.2% SDS.

[0092] Mutant: The term "mutant" refers to a polynucleotide that encodes a variant.

[0093] Nucleic acid constructs: The term “nucleic acid construct” refers to single-stranded or double-stranded nucleic acid molecules that are isolated from naturally occurring genes, or that are modified to contain segments of nucleic acid in a manner not normally found in nature, or that are synthetic and include one or more control sequences.

[0094] Operable ligation: The term “operable ligation” refers to a configuration in which a control sequence is placed in an appropriate position relative to the coding sequence of a polynucleotide so that the control sequence directs the expression of the coding sequence.

[0095] Parent or parental GH61 polypeptide: The term "parent" or "parental GH61 polypeptide" refers to a GH61 polypeptide modified at one or more (e.g., several) sites, i.e., substitution, insertion, and / or deletion, to produce the GH61 polypeptide variant of the present invention. The parent can be a naturally occurring (wild-type) polypeptide or a variant or fragment thereof.

[0096] Polypeptides with enhanced cellulose degradation activity: The term “polypeptide with enhanced cellulose degradation activity” refers to GH61 polypeptides or variants thereof that are enhanced by the hydrolysis of cellulose materials catalyzed by enzymes with cellulose degradation activity, namely cellulases. For the purposes of this invention, cellulose-enhancing activity is determined by measuring the increase in reducing sugars or the total amount of cellobiose and glucose from cellulase-hydrolyzed cellulose material under the following conditions: 1-50 mg total protein / g of pretreated corn stalks (PCS) containing cellulose, wherein the total protein comprises 50-99.5% w / w cellulase protein and 0.5-50% w / w GH61 polypeptide or a variant thereof, for 1-7 days at a suitable temperature (e.g., 40°C-80°C, such as 50°C, 55°C, 60°C, 65°C, or 70°C) and a suitable pH (e.g., 4-9, such as 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, or 8.5), compared with a control hydrolysis of an equivalent total protein load (1-50 mg cellulase protein / g of cellulose in PCS) without cellulose-enhancing activity. In one respect, the GH61 peptide enhancement activity was measured using the following method: in the presence of a cellulase protein loading of 2%–3% by weight of Aspergillus oryzae β-glucosidase (recombinantly produced in Aspergillus oryzae according to WO 02 / 095014) or 2%–3% by weight of Aspergillus fumigatus β-glucosidase (recombinantly produced in Aspergillus oryzae as described in WO 02 / 095014), CELLUCLAST 1.5L (Novozymes) A mixture from Denmark was used as a source of cellulose-degrading activity.

[0097] Another assay for determining the cellulose-degrading-enhancing activity of GH61 peptide or its variants involves reacting GH61 peptide or variants with 0.5% phosphate-swellable cellulose (PASC), 100 mM sodium acetate (pH 5), 1 mM MnSO4, 0.1% gallic acid, 0.025 mg / ml Aspergillus fumigatus β-glucosidase, and 0.01% [unclear - possibly a specific chemical or ingredient] at 40 °C. Incubate with X100 for 24–96 hours, then measure the glucose released from PASC.

[0098] GH61 polypeptides or variants thereof with cellulose-degrading enhancement activity enhance the hydrolysis of cellulose materials catalyzed by enzymes with cellulose-degrading activity by reducing the amount of cellulose-degrading enzyme required to achieve the same degree of hydrolysis by preferably at least 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.

[0099] Pretreated corn stalks: The term “PCS” or “pretreated corn stalks” means cellulose material obtained from corn stalks by heat and dilute sulfuric acid treatment, alkali pretreatment, neutral pretreatment, or any pretreatment known in the art.

[0100] Sequence Consistency The correlation between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence consistency".

[0101] For the purposes of this invention, the Needleman-Wunsch algorithm (Niedelman and Wunsch, 1970, J.Mol.Biol. 48:443-453) implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (preferably version 5.0.0 or later) was used to determine sequence identity between two amino acid sequences. The parameters used were a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 substitution matrix (the EMBOSS version of BLOSUM62). The output of the "longest consistency" annotated by Needle (obtained using the -non-simplification option) was used as the percentage consistency and calculated as follows:

[0102] (Consistent residues x 100) / (Alignment length - Total number of vacancies in the alignment)

[0103] For the purposes of this invention, the Niedermann-Onsch algorithm (Niedermann and Onsch, 1970, ibid.) was used to determine sequence consistency between two deoxyribonucleotide sequences, implemented as the Nieder program in the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, ibid.) (preferably version 5.0.0 or later). The parameters used were a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The Nieder-annotated output of “longest consistency” (obtained using the -non-simplified option) was used as the percentage consistency and calculated as follows:

[0104] (Consistent deoxyribonucleotides x 100) / (Alignment length - Total number of vacancies in the alignment)

[0105] Subsequence: The term "subsequence" refers to a polynucleotide in which one or more (e.g., several) nucleotides are deleted from the 5' and / or 3' end of the mature polypeptide coding sequence, wherein the subsequence encodes a fragment having cellulolytic-enhancing activity. In one aspect, a subsequence contains at least 85%, for example, at least 90% or at least 95% of the nucleotides of the mature polypeptide coding sequence of the GH61 polypeptide.

[0106] Variants: The term "variant" means a polypeptide having cellulolytic-enhancing activity that contains alterations, i.e., substitutions, insertions, and / or deletions, at one or more (e.g., several) positions. Substitution means that an amino acid occupying a position is replaced with a different amino acid; deletion means that an amino acid occupying a position is removed; and insertion means that an amino acid is added adjacent to and immediately following an amino acid occupying a position. The variants of the present invention have at least 20%, for example at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the cellulolytic-enhancing activity of its parent GH61 polypeptide.

[0107] Very high stringency conditions: The term "very high stringency conditions" refers to pre-hybridization and hybridization for probes of at least 100 nucleotides in length, following standard DNA blotting procedures at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours. Vector material is finally washed three times at 70°C for 15 minutes each time with 0.2X SSC and 0.2% SDS.

[0108] Very low stringency conditions: The term "very low stringency conditions" refers to pre-hybridization and hybridization for probes of at least 100 nucleotides in length, following standard DNA blotting procedures, at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 25% formamide for 12 to 24 hours. Vector material is finally washed three times at 45°C for 15 minutes each time with 0.2X SSC and 0.2% SDS.

[0109] Wild-type GH61 peptide: The term "wild-type" GH61 peptide refers to GH61 peptides that are naturally produced by microorganisms such as bacteria, yeast, or filamentous fungi found in nature.

[0110] Materials containing xylan: The term "materials containing xylan" refers to any material containing a plant cell wall polysaccharide with a backbone of β-(1-4)-linked xylose residues. Terrestrial plant xylans are heteropolymers with a β-(1-4)-D-xylanose backbone branched by short carbohydrate chains. They include D-glucuronic acid or its 4-O-methyl ether, L-arabinose, and / or various oligosaccharides composed of D-xylose, L-arabinose, D- or L-galactose, and D-glucose. Xylan-type polysaccharides can be classified into homoxylans and heteroxylans, including glucuronide xylan, (arabinose)glucuronide xylan, (glucuronide)arasylxylan, arasylxylan, and complex heteroxylans. See, for example, Ebringerova et al., 2005, Adv. Polym. Sci. 186:1-67.

[0111] In the method of the present invention, any material containing xylan can be used. In a preferred aspect, the material containing xylan is lignocellulose.

[0112] Xylan degradation activity or xylan decomposition activity: The term “xylan degradation activity” or “xylan decomposition activity” refers to the biological activity of hydrolyzing materials containing xylan. Two basic methods for measuring xylan decomposition activity include: (1) measuring total xylan decomposition activity, and (2) measuring individual xylan decomposition activities (e.g., xylan endopeptidase, β-xylosidase, arabinofuranylase, α-glucuronylase, acetylxylan esterase, ferulic acid esterase, and α-glucuronylase). Recent advances in the determination of xylanases have been summarized in several publications, including Biely and Puchard, 2006, Journal of the Science of Food and Agriculture 86(11):1636-1647; Spanikova and Biely, 2006, FEBS Letters 580(19):4597-4601; Herrmann et al., 1997, Biochemical Journal 321:375-381.

[0113] Total xylan degradation activity can be measured by determining reducing sugars formed from different types of xylans (including, for example, oat xylan, beech xylan, and larch xylan), or by spectrophotometric determination of stained xylan fragments released from different covalently stained xylans. The most common assay of total xylan degradation activity is based on reducing sugars produced from polymerized 4-O-methylglucuronic acid xylan, as described in Bele, Bele, Potanen, 1992, Interlaboratory testing of methods for assay of xylanase activity, Journal of Biotechnology 23(3):257-270. Xylanase activity can also be measured at 37°C at 0.01%. X-100 (4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol) and 200 mM sodium phosphate (pH 6) were used with 0.2% AZCL-arabinosylxylan as a substrate. One unit of xylanase activity was defined as the production of 1.0 μmol of azurin per minute from 0.2% AZCL-arabinosylxylan as a substrate at 37 °C and pH 6 in 200 mM sodium phosphate (pH 6).

[0114] For the purposes of this invention, xylan degradation activity was determined by measuring the increase in hydrolysis of birch xylan (Sigma Chemical Co., Inc., St. Louis, MO, USA) induced by one or more xylan-degrading enzymes under the following typical conditions: 1 ml reactant, 5 mg / ml substrate (total solids), 5 mg xylan-degrading protein / g substrate, 50 mM sodium acetate (pH 5), 50 °C, 24 h, and sugar analysis using the p-hydroxybenzoic acid hydrazide (PHBAH) assay, as described in Lever, 1972, Analytical Biochemistry 47: 273-279.

[0115] Xylanase: The term "xylanase" refers to 1,4-β-D-xylan-xylose hydrolase (EC 3.2.1.8), which catalyzes the endo-hydrolysis of the 1,4-β-D-xylosidic bonds in xylan. For the purposes of this invention, at 37°C, at 0.01%... Xylanase activity was determined using 0.2% AZCL-arabinosylxylan as a substrate in X-100 and 200 mM sodium phosphate (pH 6). One unit of xylanase activity was defined as the production of 1.0 μmol of azurin per minute from 0.2% AZCL-arabinosylxylan as a substrate in 200 mM sodium phosphate (pH 6) at 37 °C and pH 6.

[0116] Variant Naming Conventions

[0117] For the purposes of this invention, the mature polypeptide disclosed in SEQ ID NO:30 is used to determine the corresponding amino acid residues in another GH61 polypeptide. The amino acid sequence of the other GH61 polypeptide is aligned with the mature polypeptide disclosed in SEQ ID NO:30, and based on this alignment, the Niederman-Wensch algorithm (Niederman and Wensch, 1970, Journal of Molecular Biology 48:443-453) implemented in the Nieder program, such as the EMBOSS package (EMBOSS: European Open Software Suite for Molecular Biology, Rice et al., 2000, Trends in Genetics 16:276-277) (preferably version 5.0.0 or higher), is used to determine the amino acid position number corresponding to any amino acid residue in the mature polypeptide disclosed in SEQ ID NO:30. The parameters used are a vacancy opening penalty of 10, a vacancy expansion penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The amino acid positions are numbered based on the full-length polypeptide (e.g., including the signal peptide) of SEQ ID NO:30, where position 1 is the first amino acid of the signal peptide (i.e., Met) and position 22 is His of SEQ ID NO:30.

[0118] For example, the position corresponding to position 105 in the Aspergillus fumigatus GH61 polypeptide (SEQ ID NO:30) is position 109 in the Penicillium emersonii GH61 polypeptide (SEQ ID NO:36), position 105 in the Ascomycetes aureus GH61 polypeptide (SEQ ID NO:14), and position 103 in the Aspergillus echinospora GH61 polypeptide (SEQ ID NO:68); the position corresponding to position 188 in the Aspergillus fumigatus GH61 polypeptide is position 192 in the Penicillium emersonii GH61 polypeptide, position 188 in the Ascomycetes aureus GH61 polypeptide, and position 186 in the Aspergillus echinospora GH61 polypeptide; the position corresponding to position 154 in the Aspergillus fumigatus GH61 polypeptide is position 152 in the Aspergillus echinospora GH61 polypeptide; and the position corresponding to position 189 in the Aspergillus fumigatus GH61 polypeptide is position 193 in the Penicillium emersonii GH61 polypeptide and position 187 in the Aspergillus echinospora GH61 polypeptide.

[0119] Several computer programs can be used to identify the corresponding amino acid residues in another GH61 peptide by comparing multiple peptide sequences. These computer programs include, but are not limited to: MUSCLE (multiple sequence comparison by log-expectation; version 3.5 or later; Edgar, 2004, Nucleic Acids Research 32:1792-1797); MAFFT (version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research 30:3059-3066; Katoh et al., 2005, Nucleic Acids Research 33:511-518; Katoh and Toh, 2007, Bioinformatics 23:372-374; Katoh et al., 2009, Methods in Molecular Biology). Biology) 537:39-64; Katoch and Du, 2010, Bioinformatics, 26:1899-1900), and using ClustalW's EMBOSS EMMA (1.83 or later; Thompson et al., 1994, Nucleic Acid Research) 22:4673-4680), using their respective default parameters.

[0120] When another GH61 peptide diverges from the mature peptide of SEQ ID NO:30, making their relationship undetectable by conventional sequence-based comparisons (Lindahl and Elofsson, 2000, Journal of Molecular Biology 295:613-615), alternative paired sequence comparison algorithms can be used. Greater sensitivity in sequence-based searches can be achieved using search procedures that utilize probabilistic representations (characteristic curves) of peptide families to search a database. For example, the PSI-BLAST procedure generates multiple characteristic curves through an iterative database search process and is capable of detecting distant homologs (Atschul et al., 1997, Nucleic Acid Research 25:3389-3402). Even greater sensitivity can be achieved if the peptide family or superfamily has one or more representatives in a protein structure database. Programs (e.g., GenTHREADER) (Jones, 1999, Journal of Molecular Biology 287:797-815; McGuffi and Jones, 2003, Bioinformatics 19:874-881) utilize information from multiple sources (PSI-BLAST, secondary structure prediction, structural alignment feature curves, and solvation probability) as input to neural networks that predict the structural folding of query sequences. Similarly, the method of Gough et al., 2000, Journal of Molecular Biology 313:903-919 can be used to align sequences of unknown structures with superfamily models existing in the SCOP database. These alignments can then be used to generate homology models of peptides, and the accuracy of such models can be evaluated using various tools developed for this purpose.

[0121] For proteins with known structures, several tools and resources are available for retrieving and generating structural alignments. For example, the SCOP superfamily of proteins has already been structurally aligned, and those alignments are accessible and downloadable. Various algorithms (e.g., distance alignment matrices (Holm and Sander, 1998, Proteins 33:88-96) or combined extensions (Shindyalov and Bourne, 1998, Protein Engineering 11:739-747)) can be used to align two or more protein structures, and implementations of these algorithms can be further utilized to query structural databases along with the structure of interest to discover possible structural homologs (e.g., Helm and Park, 2000, Bioinformatics 16:566-567).

[0122] In describing the GH61 polypeptide variants of the present invention, the following nomenclature is used for ease of reference. Recognized IUPAC single-letter or three-letter amino acid abbreviations are used.

[0123] replace. For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Therefore, the substitution of threonine at position 226 with alanine is represented as "Thr226Ala" or "T226A". Multiple mutations are separated by a plus sign ("+"), for example, "Gly205Arg+Ser411Phe" or "G205R+S411F" represent the substitution of glycine (G) with arginine (R) and serine (S) with phenylalanine (F) at positions 205 and 411, respectively.

[0124] Missing. For amino acid deletions, use the following nomenclature: original amino acid, position, *. Therefore, a glycine deletion at position 195 is represented as "Gly195*" or "G195*". Multiple deletions are separated by a plus sign ("+"), for example, "Gly195*+Ser411*" or "G195*+S411*".

[0125] insert. For amino acid insertions, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. Therefore, the insertion of lysine after glycine at position 195 is represented as "Gly195GlyLys" or "G195GK". Multiple amino acid insertions are represented as [original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2; etc.]. For example, the insertion of lysine and alanine after glycine at position 195 is represented as "Glyl95GlyLysAla" or "G195GKA".

[0126] In such cases, the inserted amino acid residues are numbered by adding lowercase letters to the position numbers of the amino acid residues preceding them. In the example above, the sequence would therefore be:

[0127] <![CDATA[ Parent: ]]> <![CDATA[ Variants: ]]> 195 195 195a 195b G GKA

[0128] Multiple replacements. Variants with multiple substitutions are separated by a plus sign (“+”), such as “Arg170Tyr+Gly195Glu” or “R170Y+G195E”, which represent arginine and glycine being replaced by tyrosine and glutamic acid at positions 170 and 195, respectively.

[0129] Different substitutions.When different substitutions can be introduced at a single position, the different substitutions are separated by a comma, for example, "Arg170Tyr,Glu" represents the substitution of arginine at position 170 with either tyrosine or glutamic acid. Therefore, "Tyr167Gly,Ala+Arg170Gly,Ala" represents the following variant:

[0130] “Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and “Tyr167Ala+Arg170Ala”.

[0131] Detailed Description of the Invention

[0132] The present invention relates to isolated GH61 polypeptide variants comprising substitutions at one or more (e.g., several) positions corresponding to positions 26, 32, 34, 40, 41, 42, 47, 56, 72, 102, 123, 138, 149, 152, 163, 164, 166, 169, 186, 200, 207, 213, 219, 222, 234, 246, 249, and 250 of the mature polypeptide of SEQ ID NO:30, wherein these variants have cellulose-enhancing activity.

[0133] variants

[0134] In one embodiment, the variant has at least 60%, such as 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%, or at least 99%, but less than 100%, sequence similarity to the parental GH61 polypeptide.

[0135] In another embodiment, this variant is related to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, The mature polypeptides of 142, 144, 146, 148, 150, 152, 154, 56, 158, 160, 162, 164, 166, 168, 409, or 411 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%, or at least 99%, but less than 100%, sequence identity.

[0136] In one aspect, the number of substitutions in the variants of the invention is 1 to 28, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 and 28 substitutions.

[0137] In another aspect, the variant includes substitutions at one or more (e.g., several) positions corresponding to positions 26, 32, 34, 40, 41, 42, 47, 56, 72, 102, 123, 138, 149, 152, 163, 164, 166, 169, 186, 200, 207, 213, 219, 222, 234, 246, 249, and 250. In another aspect, the variant includes substitutions at two positions corresponding to any of positions 26, 32, 34, 40, 41, 42, 47, 56, 72, 102, 123, 138, 149, 152, 163, 164, 166, 169, 186, 200, 207, 213, 219, 222, 234, 246, 249, and 250. In another aspect, the variant includes substitutions at three positions corresponding to any of the following positions: 26, 32, 34, 40, 41, 42, 47, 56, 72, 102, 123, 138, 149, 152, 163, 164, 166, 169, 186, 200, 207, 213, 219, 222, 234, 246, 249, and 250. In another aspect, the variant includes substitutions at four positions corresponding to any of the following positions: 26, 32, 34, 40, 41, 42, 47, 56, 72, 102, 123, 138, 149, 152, 163, 164, 166, 169, 186, 200, 207, 213, 219, 222, 234, 246, 249, and 250. In another aspect, the variant includes substitutions at five positions corresponding to any of the following positions: 26, 32, 34, 40, 41, 42, 47, 56, 72, 102, 123, 138, 149, 152, 163, 164, 166, 169, 186, 200, 207, 213, 219, 222, 234, 246, 249, and 250. In another aspect, the variant includes substitutions at six positions corresponding to any of the following positions: 26, 32, 34, 40, 41, 42, 47, 56, 72, 102, 123, 138, 149, 152, 163, 164, 166, 169, 186, 200, 207, 213, 219, 222, 234, 246, 249, and 250. In another aspect, the variant includes substitutions at seven positions corresponding to any of the following positions: 26, 32, 34, 40, 41, 42, 47, 56, 72, 102, 123, 138, 149, 152, 163, 164, 166, 169, 186, 200, 207, 213, 219, 222, 234, 246, 249, and 250.In another aspect, the variant includes substitutions at eight positions corresponding to any of the following positions: 26, 32, 34, 40, 41, 42, 47, 56, 72, 102, 123, 138, 149, 152, 163, 164, 166, 169, 186, 200, 207, 213, 219, 222, 234, 246, 249, and 250. In another aspect, the variant includes substitutions at nine positions corresponding to any of the following positions: 26, 32, 34, 40, 41, 42, 47, 56, 72, 102, 123, 138, 149, 152, 163, 164, 166, 169, 186, 200, 207, 213, 219, 222, 234, 246, 249, and 250. In another aspect, the variant includes substitutions at ten positions corresponding to any of the following positions: 26, 32, 34, 40, 41, 42, 47, 56, 72, 102, 123, 138, 149, 152, 163, 164, 166, 169, 186, 200, 207, 213, 219, 222, 234, 246, 249, and 250. In another aspect, the variant includes substitutions at eleven positions corresponding to any of the following positions: 26, 32, 34, 40, 41, 42, 47, 56, 72, 102, 123, 138, 149, 152, 163, 164, 166, 169, 186, 200, 207, 213, 219, 222, 234, 246, 249, and 250. In another aspect, the variant includes substitutions at twelve positions corresponding to any of the following positions: 26, 32, 34, 40, 41, 42, 47, 56, 72, 102, 123, 138, 149, 152, 163, 164, 166, 169, 186, 200, 207, 213, 219, 222, 234, 246, 249, and 250. In another aspect, the variant includes substitutions at thirteen positions corresponding to any of the following positions: 26, 32, 34, 40, 41, 42, 47, 56, 72, 102, 123, 138, 149, 152, 163, 164, 166, 169, 186, 200, 207, 213, 219, 222, 234, 246, 249, and 250. In another aspect, the variant includes substitutions at fourteen positions corresponding to any of the following positions: 26, 32, 34, 40, 41, 42, 47, 56, 72, 102, 123, 138, 149, 152, 163, 164, 166, 169, 186, 200, 207, 213, 219, 222, 234, 246, 249, and 250.In another aspect, the variant includes substitutions at fifteen positions corresponding to any of the following positions: 26, 32, 34, 40, 41, 42, 47, 56, 72, 102, 123, 138, 149, 152, 163, 164, 166, 169, 186, 200, 207, 213, 219, 222, 234, 246, 249, and 250. In another aspect, the variant includes substitutions at sixteen positions corresponding to any of the following positions: 26, 32, 34, 40, 41, 42, 47, 56, 72, 102, 123, 138, 149, 152, 163, 164, 166, 169, 186, 200, 207, 213, 219, 222, 234, 246, 249, and 250. In another aspect, the variant includes substitutions at seventeen positions corresponding to any of the following positions: 26, 32, 34, 40, 41, 42, 47, 56, 72, 102, 123, 138, 149, 152, 163, 164, 166, 169, 186, 200, 207, 213, 219, 222, 234, 246, 249, and 250. In another aspect, the variant includes substitutions at eighteen positions corresponding to any of the following positions: 26, 32, 34, 40, 41, 42, 47, 56, 72, 102, 123, 138, 149, 152, 163, 164, 166, 169, 186, 200, 207, 213, 219, 222, 234, 246, 249, and 250. In another aspect, the variant includes substitutions at nineteen positions corresponding to any of the following positions: 26, 32, 34, 40, 41, 42, 47, 56, 72, 102, 123, 138, 149, 152, 163, 164, 166, 169, 186, 200, 207, 213, 219, 222, 234, 246, 249, and 250. In another aspect, the variant includes substitutions at twenty positions corresponding to any of the following positions: 26, 32, 34, 40, 41, 42, 47, 56, 72, 102, 123, 138, 149, 152, 163, 164, 166, 169, 186, 200, 207, 213, 219, 222, 234, 246, 249, and 250. In another aspect, the variant includes substitutions at twenty-one positions corresponding to any of the following positions: 26, 32, 34, 40, 41, 42, 47, 56, 72, 102, 123, 138, 149, 152, 163, 164, 166, 169, 186, 200, 207, 213, 219, 222, 234, 246, 249, and 250.In another aspect, the variant includes substitutions at twenty-two positions corresponding to any of the following positions: 26, 32, 34, 40, 41, 42, 47, 56, 72, 102, 123, 138, 149, 152, 163, 164, 166, 169, 186, 200, 207, 213, 219, 222, 234, 246, 249, and 250. In another aspect, the variant includes substitutions at twenty-three positions corresponding to any of the following positions: 26, 32, 34, 40, 41, 42, 47, 56, 72, 102, 123, 138, 149, 152, 163, 164, 166, 169, 186, 200, 207, 213, 219, 222, 234, 246, 249, and 250. In another aspect, the variant includes substitutions at twenty-four positions corresponding to any of the following positions: 26, 32, 34, 40, 41, 42, 47, 56, 72, 102, 123, 138, 149, 152, 163, 164, 166, 169, 186, 200, 207, 213, 219, 222, 234, 246, 249, and 250. In another aspect, the variant includes substitutions at twenty-five positions corresponding to any of the following positions: 26, 32, 34, 40, 41, 42, 47, 56, 72, 102, 123, 138, 149, 152, 163, 164, 166, 169, 186, 200, 207, 213, 219, 222, 234, 246, 249, and 250. In another aspect, the variant includes substitutions at twenty-six positions corresponding to any of the following positions: 26, 32, 34, 40, 41, 42, 47, 56, 72, 102, 123, 138, 149, 152, 163, 164, 166, 169, 186, 200, 207, 213, 219, 222, 234, 246, 249, and 250. In another aspect, the variant includes substitutions at twenty-seven positions corresponding to any of the following positions: 26, 32, 34, 40, 41, 42, 47, 56, 72, 102, 123, 138, 149, 152, 163, 164, 166, 169, 186, 200, 207, 213, 219, 222, 234, 246, 249, and 250. In another respect, the variant includes substitutions at each of the positions corresponding to positions 26, 32, 34, 40, 41, 42, 47, 56, 72, 102, 123, 138, 149, 152, 163, 164, 166, 169, 186, 200, 207, 213, 219, 222, 234, 246, 249, and 250.

[0138] In another aspect, variants include or consist of a substitution at position 26. In another aspect, the amino acid at position 26 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Ile. In another aspect, variants include or consist of a substitution S26I of the mature polypeptide of SEQ ID NO:30.

[0139] In another aspect, variants include substitutions or components thereof at the position corresponding to position 32. In another aspect, the amino acid at the position corresponding to position 32 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Glu or Ser. In another aspect, variants include or consist of substituted G32E or G32S of the mature polypeptide of SEQ ID NO:30.

[0140] In another aspect, variants include substitutions or components thereof at the position corresponding to position 34. In another aspect, the amino acid at position 34 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Phe. In another aspect, variants include substituted Y34F or components thereof of the mature polypeptide of SEQ ID NO:30.

[0141] In another aspect, variants include or consist of a substitution at the position corresponding to position 40. In another aspect, the amino acid at position 40 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Ala. In another aspect, variants include or consist of a substitution V40A of the mature polypeptide of SEQ ID NO:30.

[0142] In another aspect, variants include substitutions or components thereof at the position corresponding to position 41. In another aspect, the amino acid at position 41 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Thr. In another aspect, variants include substituted N41T or components thereof of the mature polypeptide of SEQ ID NO:30.

[0143] In another aspect, variants include substitutions or components thereof at the position corresponding to position 42. In another aspect, the amino acid at the position corresponding to position 42 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Ile, Glu, or Val. In another aspect, variants include or consist of substitutions Q42I, Q42E, or Q42V of the mature polypeptide of SEQ ID NO:30.

[0144] In another aspect, variants include substitutions or components thereof at the position corresponding to position 47. In another aspect, the amino acid at the position corresponding to position 47 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Glu, Leu, or Arg. In another aspect, variants include or consist of substitutions S47E, S47L, or S47R of the mature polypeptide of SEQ ID NO:30.

[0145] In another aspect, variants include substitutions or components thereof at the position corresponding to position 56. In another aspect, the amino acid at the position corresponding to position 56 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Cys, Glu, or Thr. In another aspect, variants include or consist of substitutions S56C, S56E, or S56T of the mature polypeptide of SEQ ID NO:30.

[0146] In another aspect, variants include substitutions or components thereof at the position corresponding to position 72. In another aspect, the amino acid at the position corresponding to position 72 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Gln or Thr. In another aspect, variants include substitutions S72Q or S72T or components thereof of the mature polypeptide of SEQ ID NO:30.

[0147] In another aspect, variants include substitutions or components thereof at the position corresponding to position 102. In another aspect, the amino acid at the position corresponding to position 102 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Lys or Pro. In another aspect, variants include substitutions of T102K or T102P for or consist of the mature polypeptide of SEQ ID NO:30.

[0148] In another aspect, variants include or consist of substitutions at positions corresponding to position 123. In another aspect, the amino acid at position 123 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Arg. In another aspect, variants include or consist of the substituted A123R of the mature polypeptide of SEQ ID NO:30.

[0149] In another aspect, variants include substitutions or components thereof at the position corresponding to position 138. In another aspect, the amino acid at the position corresponding to position 138 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Cys, Glu, Gly, Lys, Leu, or Met. In another aspect, variants include or consist of substitutions of Q138C, Q138E, Q138G, Q138K, Q138L, or Q138M of the mature polypeptide of SEQ ID NO:30.

[0150] In another aspect, variants include or consist of a substitution at the position corresponding to position 149. In another aspect, the amino acid at position 149 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Ile. In another aspect, variants include or consist of a substituted V149I of the mature polypeptide of SEQ ID NO:30.

[0151] In another aspect, variants include or consist of a substitution at the position corresponding to position 152. In another aspect, the amino acid at position 152 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ser. In another aspect, variants include or consist of a substituted D152S of the mature polypeptide of SEQ ID NO:30.

[0152] In another aspect, variants include substitutions or components thereof at the position corresponding to position 163. In another aspect, the amino acid at the position corresponding to position 163 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Glu, Phe, or Val. In another aspect, variants include or consist of substitutions T163E, T163F, or T163V of the mature polypeptide of SEQ ID NO:30.

[0153] In another aspect, variants include or consist of substitutions at the position corresponding to position 164. In another aspect, the amino acid at the position corresponding to position 164 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Cys or Leu. In another aspect, variants include or consist of substituted V164C or V164L of the mature polypeptide of SEQ ID NO:30.

[0154] In another aspect, variants include or consist of a substitution at position 166. In another aspect, the amino acid at position 166 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Leu. In another aspect, variants include or consist of a substituted I166L of the mature polypeptide of SEQ ID NO:30.

[0155] In another aspect, variants include substitutions at or consisting of positions corresponding to position 169. In another aspect, the amino acid at the position corresponding to position 169 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Arg or Cys. In another aspect, variants include substitutions S169R or S169C of the mature polypeptide of SEQ ID NO:30, or consisting of positions thereof. In another aspect, variants include substitutions S173C of the mature polypeptide of SEQ ID NO:36, or consisting of positions thereof. Position 173 corresponds to position 169 in the mature polypeptide of Aspergillus fumigatus GH61.

[0156] In another aspect, variants include substitutions or components thereof at the position corresponding to position 186. In another aspect, the amino acid at the position corresponding to position 186 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Phe, Lys, Thr, or Tyr. In another aspect, variants include or consist of substitutions S186F, S186K, S186T, or S186Y of the mature polypeptide of SEQ ID NO:30.

[0157] In another aspect, variants include or consist of substitutions at the position corresponding to position 200. In another aspect, the amino acid at the position corresponding to position 200 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Ile or Val. In another aspect, variants include or consist of substitutions F200I or F200V of the mature polypeptide of SEQ ID NO:30.

[0158] In another aspect, variants include or consist of a substitution at the position corresponding to position 207. In another aspect, the amino acid at position 207 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Pro. In another aspect, variants include or consist of a substituted G207P of the mature polypeptide of SEQ ID NO:30.

[0159] In another aspect, variants include or consist of substitutions at the position corresponding to position 213. In another aspect, the amino acid at position 213 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Glu. In another aspect, variants include or consist of substitutions S213E of the mature polypeptide of SEQ ID NO:30.

[0160] In another aspect, variants include substitutions or components thereof at the position corresponding to position 219. In another aspect, the amino acid at the position corresponding to position 219 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Glu, Met, Gln, or Cys. In another aspect, variants include substitutions S219E, S219M, S219Q, or S219C of the mature polypeptide of SEQ ID NO:30, or components thereof.

[0161] In another aspect, variants include or consist of a substitution at the position corresponding to position 222. In another aspect, the amino acid at position 222 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Arg. In another aspect, variants include or consist of a substituted K222R of the mature polypeptide of SEQ ID NO:30.

[0162] In another aspect, variants include substitutions or are composed of the amino acid at the position corresponding to position 234. In another aspect, the amino acid at the position corresponding to position 234 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Gly or Lys. In yet another aspect, variants include or are composed of the substitution S234G or S234K of the mature polypeptide of SEQ ID NO:30.

[0163] In another aspect, variants include or consist of a substitution at the position corresponding to position 246. In another aspect, the amino acid at position 246 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Pro. In another aspect, variants include or consist of a substitution A246P of the mature polypeptide of SEQ ID NO:30.

[0164] In another aspect, variants include substitutions or components thereof at the position corresponding to position 249. In another aspect, the amino acid at the position corresponding to position 249 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Gln, Arg, or Cys. In another aspect, variants include substitutions N249Q, N249R, or N249C or components thereof in the mature polypeptide of SEQ ID NO:30. In another aspect, variants include substitutions F253C or components thereof in the mature polypeptide of SEQ ID NO:36. Position 253 corresponds to position 249 in the mature polypeptide of Aspergillus fumigatus GH61.

[0165] In another aspect, variants include or consist of a substitution at position 250. In another aspect, the amino acid at position 250 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Cys. In another aspect, variants include or consist of a substituted A250C of the mature polypeptide of SEQ ID NO:30.

[0166] On the other hand, among the other GH61 peptides described herein, variants include those with SEQ ID One or more (e.g., several) substitutions at the corresponding position of the mature polypeptide NO:30, or consisting of such substitutions, are selected from the group consisting of: S26I, G32E,S, Y34F, V40A, N41T, Q42I,E,V, S47E,L,R, S56C,E,T, S72Q,T, T102K,P, A123R, Q138C,E,G,K,L,M, V149I, D152S, T163E,F,V, V164C,L,I166L, S169R,C, S186F,K,T,Y, F200I,V, G207P, S213E, S219E,M,Q,C, K222R, S234G,K, A246P, N249Q,R C, and A250C; or comprising or consisting of one or more (e.g., several) selected from the group consisting of: S26I, G32E,S, Y34F, V40A, N41T, Q42I,E,V, S47E,L,R, S56C,E,T, S72Q,T, T102K,P, A123R, Q138C,E, G,K,L,M,V149I,D152S,T163E,F,V,V164C,L,I166L,S169R,C,S186F,K,T,Y,F20 0I,V, G207P, S213E, S219E,M,Q,C, K222R, S234G,K, A246P, N249Q,R,C, and A250C.

[0167] In the other GH61 peptides described herein, variants include, or consist of, one or more (e.g., several) of the substitutions described below at the position corresponding to SEQ ID NO:30.

[0168] In another aspect, the variant comprises or consists of the substitution S173C+F253C of the mature polypeptide of SEQ ID NO:36. In yet another aspect, the variant comprises or consists of the substitution L111V+D152S+M155L+A162W+Q138K+K229W of the mature polypeptide of SEQ ID NO:30. In yet another aspect, the variant comprises or consists of the substitution L111V+D152S+M155L+A162W+S47E+K229W of the mature polypeptide of SEQ ID NO:30. In yet another aspect, the variant comprises or consists of the substitution L111V+D152S+M155L+A162W+S56A+K229W of the mature polypeptide of SEQ ID NO:30. In another aspect, the variant comprises, or is composed of, the substitution of the mature polypeptide of SEQ ID NO:30 with L111V+D152S+M155L+A162W+T102K+K229W. In another aspect, the variant comprises, or is composed of, the substitution of the mature polypeptide of SEQ ID NO:30 with L111V+D152S+M155L+A162W+S186T+K229W. In another aspect, the variant comprises, or is composed of, the substitution of the mature polypeptide of SEQ ID NO:30 with L111V+D152S+M155L+A162W+K229W+S234G. In another aspect, the variant comprises, or is composed of, the substitution of the mature polypeptide of SEQ ID NO:30 with L111V+D152S+M155L+A162W+T102K+E105K+K229W. In another aspect, the variant comprises, or is composed of, the substitution of the mature polypeptide of SEQ ID NO:30 with L111V+D152S+M155L+A162W+Q138K+G188F+K229W. In another aspect, the variant comprises, or is composed of, the substitution of the mature polypeptide of SEQ ID NO:30 with L111V+D152S+M155L+A162W+Q138K+V149I+G188F+K229W. In another aspect, the variant comprises, or is composed of, the substitution of the mature polypeptide of SEQ ID NO:30 with L111V+D152S+M155L+A162W+S169C+G188F+K229W+A250C. In another aspect, the variant comprises, or is composed of, the substitution of the mature polypeptide of SEQ ID NO:30 with L111V+D152S+M155L+A162W+S72T+Q138K+V149I+G188F+K229W. In another aspect, the variant comprises, or is composed of, the substitution of the mature polypeptide of SEQ ID NO:30 with L111V+D152S+M155L+A162W+Q138K+V149I+G188F+G207P+K229W.

[0169] These variations may further include one or more additional changes at one or more (e.g., several) other locations, such as substitution, insertion, or deletion.

[0170] These amino acid changes can be minor, i.e., conserved amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions typically ranging from 1 to 30 amino acids; small amino or carboxyl terminus extensions, such as N-terminal methionine residues; small linker peptides of up to 20 to 25 residues; or small extensions that facilitate purification by altering net charge or another function, such as a multihistidine tract, an antigenic epitope, or a binding domain.

[0171] Examples of conserved substitutions are found in the following group: basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that do not alter specific activity in general are known in the art and, for example, described by H. Neurath and RL Hill in *The Proteins*, Academic Press, New York, 1979. Common substitutes are 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.

[0172] Alternatively, amino acid alterations have the property of changing the physicochemical properties of peptides. For example, amino acid alterations can improve the thermal stability of peptides, change substrate specificity, change the optimal pH, and so on.

[0173] Variants of the present invention may further include substitutions at one or more (e.g., several) positions corresponding to positions 111, 152, 155, and 162 of the mature polypeptide of SEQ ID NO:30, wherein these variants have cellulase activity (WO 2012 / 044835).

[0174] In one aspect, the number of the additional substitutions described above in the variants of the invention is 1 to 4, such as 1, 2, 3, or 4 substitutions.

[0175] In another aspect, the variant further or even further includes substitution at one or more (e.g., several) positions corresponding to positions 111, 152, 155, and 162. In another aspect, the variant further or even further includes substitution at any two corresponding positions 111, 152, 155, and 162. In another aspect, the variant further or even further includes substitution at any three corresponding positions 111, 152, 155, and 162. In another aspect, the variant further or even further includes substitution at each position corresponding to positions 111, 152, 155, and 162.

[0176] In another aspect, the variant further or even further includes substitutions at the position corresponding to position 111. In another aspect, the amino acid corresponding to position 111 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Val. In another aspect, the variant further or even further includes the substitution L111V of the mature polypeptide of SEQ ID NO:30.

[0177] In another aspect, the variant further or even further includes substitutions at the position corresponding to position 152. In another aspect, the amino acid corresponding to position 152 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ser substitution. In another aspect, the variant further or even further includes the substituted D152S of the mature polypeptide of SEQ ID NO:30.

[0178] In another aspect, the variant further or even further includes substitutions at the position corresponding to position 155. In another aspect, the amino acid corresponding to position 155 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Leu. In another aspect, the variant further or even further includes the substituted M155L of the mature polypeptide of SEQ ID NO:30.

[0179] In another aspect, the variant further or even further includes substitutions at the position corresponding to position 162. In another aspect, the amino acid corresponding to position 162 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Trp. In another aspect, the variant further or even further includes the substitution A162W of the mature polypeptide of SEQ ID NO:30.

[0180] In another aspect, in other GH61 peptides described herein, variants further or even further comprise one or more (e.g., several) substitutions selected from the group consisting of: L111V, D152S, M155L, and A162W of the mature peptide of SEQ ID NO:30; or one or more (e.g., several) substitutions selected from the group consisting of: L111V, D152S, M155L, and A162W at positions corresponding to the mature peptide of SEQ ID NO:30.

[0181] Variants of the present invention may further include substitutions at one or more (e.g., several) positions corresponding to positions 96, 98, 200, 202, and 204 of the mature polypeptide of SEQ ID NO:30, wherein these variants have cellulolytic-enhancing activity (WO 2012 / 044836).

[0182] In one aspect, the number of the additional substitutions described above in the variants of the invention is 1 to 5, such as 1, 2, 3, 4, or 5 substitutions.

[0183] In another aspect, the variant further or even further includes substitution at one or more (e.g., several) positions corresponding to positions 96, 98, 200, 202, and 204. In another aspect, the variant further or even further includes substitution at two positions corresponding to any of positions 96, 98, 200, 202, and 204. In another aspect, the variant further or even further includes substitution at three positions corresponding to any of positions 96, 98, 200, 202, and 204. In another aspect, the variant further or even further includes substitution at four positions corresponding to any of positions 96, 98, 200, 202, and 204. In another aspect, the variant further or even further includes substitution at each position corresponding to positions 96, 98, 200, 202, and 204.

[0184] In another aspect, the variant further or even further includes a substitution at the position corresponding to position 96. In another aspect, the amino acid corresponding to position 96 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Val. In another aspect, the variant further or even further includes the substitution I96V of the mature polypeptide of SEQ ID NO:30.

[0185] In another aspect, the variant further or even further includes substitutions at the position corresponding to position 98. In another aspect, the amino acid corresponding to position 98 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Leu. In another aspect, the variant further or even further includes the substitution F98L of the mature polypeptide of SEQ ID NO:30.

[0186] In another aspect, the variant further or even further includes substitutions at the position corresponding to position 200. In another aspect, the amino acid corresponding to position 200 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Ile. In another aspect, the variant further or even further includes the substitution F200I of the mature polypeptide of SEQ ID NO:30.

[0187] In another aspect, the variant further or even further includes substitutions at the position corresponding to position 202. In another aspect, the amino acid corresponding to position 202 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Leu. In another aspect, the variant further or even further includes the substitution I202L of the mature polypeptide of SEQ ID NO:30.

[0188] In another aspect, the variant further or even further includes substitutions at the position corresponding to position 204. In another aspect, the amino acid corresponding to position 204 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Val. In another aspect, the variant further or even further includes the substitution I204V of the mature polypeptide of SEQ ID NO:30.

[0189] In another aspect, in other GH61 peptides described herein, variants further or even further include one or more (e.g., several) substitutions selected from the group consisting of I96V, F98L, F200I, I202L, and I204V at the position corresponding to SEQ ID NO:30; or one or more (e.g., several) substitutions selected from the group consisting of I96V, F98L, F200I, I202L, and I204V.

[0190] Variants of the present invention may further include substitutions at one or more (e.g., several) positions corresponding to positions 105, 154, 188, 189, 216, and 229 of the mature polypeptide of SEQ ID NO:30, wherein these variants have cellulose-degrading-enhancing activity.

[0191] In one aspect, the number of the additional substitutions described above in variations of the invention is 1 to 6, such as 1, 2, 3, 4, 5, or 6 substitutions.

[0192] In another aspect, the variant further or even further includes substitutions at one or more (e.g., several) positions corresponding to positions 105, 154, 188, 189, 216, and 229. In another aspect, the variant further or even further includes substitutions at two positions corresponding to any of positions 105, 154, 188, 189, 216, and 229. In another aspect, the variant further or even further includes substitutions at three positions corresponding to any of positions 105, 154, 188, 189, 216, and 229. In another aspect, the variant further or even further includes substitutions at four positions corresponding to any of positions 105, 154, 188, 189, 216, and 229. In another aspect, the variant further or even further includes substitutions at five positions corresponding to any of positions 105, 154, 188, 189, 216, and 229. On the other hand, the variant further or even further includes substitutions at each position corresponding to positions 105, 154, 188, 189, 216, and 229.

[0193] In another aspect, the variant further or even further includes substitutions at the position corresponding to position 105. In another aspect, the amino acid at the position corresponding to position 105 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Pro or Lys. In another aspect, the variant further or even further includes the substitution E105P or E105K of the mature polypeptide of SEQ ID NO:30.

[0194] In another aspect, the variant further or even further includes substitutions at the position corresponding to position 154. In another aspect, the amino acid corresponding to position 154 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Leu. In another aspect, the variant further or even further includes the substitution E154L of the mature polypeptide of SEQ ID NO:30.

[0195] In another aspect, the variant further or even further includes substitutions at the position corresponding to position 188. In another aspect, the amino acid at the position corresponding to position 188 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Ala or Trp. In another aspect, the variant further or even further includes the substitution G188A or G188W of the mature polypeptide of SEQ ID NO:30.

[0196] In another aspect, the variant further or even further includes a substitution at the position corresponding to position 189. In another aspect, the amino acid corresponding to position 189 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Lys. In another aspect, the variant further or even further includes the substituted N189K of the mature polypeptide of SEQ ID NO:30.

[0197] In another aspect, the variant further or even further includes substitutions at the position corresponding to position 216. In another aspect, the amino acid at the position corresponding to position 216 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Leu or Tyr. In another aspect, the variant further or even further includes the substitution A216L or A216Y of the mature polypeptide of SEQ ID NO:30.

[0198] In another aspect, the variant further or even further includes substitutions at the position corresponding to position 229. In another aspect, the amino acid at the position corresponding to position 229 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Trp, His, Ile, or Tyr. In another aspect, the variant further or even further includes substitutions of A229W, A229H, A229I, or A229Y for the mature polypeptide of SEQ ID NO:30.

[0199] In another aspect, among the other GH61 peptides described herein, variants further or even further comprise one or more (e.g., several) substitutions selected from the group consisting of: E105P,K, E154L, G188A,W, N189K, A216L,Y, and A229W,H,I,Y of the mature peptide of SEQ ID NO:30, or one or more (e.g., several) substitutions selected from the group consisting of: E105P,K, E154L, G188A,W, N189K, A216L,Y, and A229W,H,I,Y at positions corresponding to the mature peptide of SEQ ID NO:30.

[0200] These variants may consist of at least 85% of the amino acid residues, for example at least 90% or at least 95% of the amino acid residues of the mature polypeptide of the corresponding parental GH61 polypeptide.

[0201] Essential amino acids in peptides can be identified using procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, a single alanine mutation is introduced into each residue in the molecule, and the resulting mutant molecule is tested against enhanced cellulose-degrading activity to identify amino acid residues critical to the molecule's activity. See also Hilton et al., 1996, J. Biol. Chem., 271:4699-4708. Mutations of amino acids at hypothetical contact sites can also be combined with physical analysis of the structure, such as by techniques like NMR, crystallography, electron diffraction, or photoaffinity labeling, to determine the enzyme's active site or other biological interactions. See, for example, de Vos et al., Science 255:306-312; Smith et al., 1992, Journal of Molecular Biology 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. Essential amino acids can also be inferred from comparisons with related peptides. The essential amino acids in the GH61 peptide correspond to positions 22, 107, 194, and / or 196 of the mature peptide in SEQ ID NO:30.

[0202] In one embodiment, the variant exhibits increased thermal stability compared to its parent GH61 peptide.

[0203] In one aspect, the thermal stability of the variant relative to the parent was determined at pH 3.0 and 45°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 3.0 and 50°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 3.0 and 55°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 3.0 and 60°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 3.0 and 62°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 3.0 and 65°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 3.0 and 68°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 3.0 and 70°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 3.0 and 72°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 3.0 and 75°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 3.0 and 80°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 3.0 and 85°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 3.0 and 90°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 3.0 and 95°C.

[0204] In another aspect, the thermal stability of the variant relative to the parent was determined at pH 3.5 and 45°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 3.5 and 50°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 3.5 and 55°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 3.5 and 60°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 3.5 and 62°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 3.5 and 65°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 3.5 and 68°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 3.5 and 70°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 3.5 and 72°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 3.5 and 75°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 3.5 and 80°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 3.5 and 85°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 3.5 and 90°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 3.5 and 95°C.

[0205] In another aspect, the thermal stability of the variant relative to the parent was determined at pH 4.0 and 45°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 4.0 and 50°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 4.0 and 55°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 4.0 and 60°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 4.0 and 62°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 4.0 and 65°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 4.0 and 68°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 4.0 and 70°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 4.0 and 72°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 4.0 and 75°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 4.0 and 80°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 4.0 and 85°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 4.0 and 90°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 4.0 and 95°C.

[0206] In another aspect, the thermal stability of the variant relative to the parent was determined at pH 4.5 and 45°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 4.5 and 50°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 4.5 and 55°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 4.5 and 60°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 4.5 and 62°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 4.5 and 65°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 4.5 and 68°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 4.5 and 70°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 4.5 and 72°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 4.5 and 75°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 4.5 and 80°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 4.5 and 85°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 4.5 and 90°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 4.5 and 95°C.

[0207] In another aspect, the thermal stability of the variant relative to the parent was determined at pH 5.0 and 45°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 5.0 and 50°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 5.0 and 55°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 5.0 and 60°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 5.0 and 62°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 5.0 and 65°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 5.0 and 68°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 5.0 and 70°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 5.0 and 72°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 5.0 and 75°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 5.0 and 80°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 5.0 and 85°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 5.0 and 90°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 5.0 and 95°C.

[0208] In another aspect, the thermal stability of the variant relative to the parent was determined at pH 5.5 and 45°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 5.5 and 50°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 5.5 and 55°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 5.5 and 60°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 5.5 and 62°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 5.5 and 65°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 5.5 and 68°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 5.5 and 70°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 5.5 and 72°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 5.5 and 75°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 5.5 and 80°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 5.5 and 85°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 5.5 and 90°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 5.5 and 95°C.

[0209] In another aspect, the thermal stability of the variant relative to the parent was determined at pH 6.0 and 45°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 6.0 and 50°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 6.0 and 55°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 6.0 and 60°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 6.0 and 62°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 6.0 and 65°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 6.0 and 68°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 6.0 and 70°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 6.0 and 72°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 6.0 and 75°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 6.0 and 80°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 6.0 and 85°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 6.0 and 90°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 6.0 and 95°C.

[0210] In another aspect, the thermal stability of the variant relative to the parent was determined at pH 6.5 and 45°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 6.5 and 50°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 6.5 and 55°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 6.5 and 60°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 6.5 and 62°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 6.5 and 65°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 6.5 and 68°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 6.5 and 70°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 6.5 and 72°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 6.5 and 75°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 6.5 and 80°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 6.5 and 85°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 6.5 and 90°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 6.5 and 95°C.

[0211] In another aspect, the thermal stability of the variant relative to the parent was determined at pH 7.0 and 45°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 7.0 and 50°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 7.0 and 55°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 7.0 and 60°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 7.0 and 62°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 7.0 and 65°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 7.0 and 68°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 7.0 and 70°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 7.0 and 72°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 7.0 and 75°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 7.0 and 80°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 7.0 and 85°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 7.0 and 90°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 7.0 and 95°C.

[0212] In another aspect, the thermal stability of the variant relative to the parent was determined at pH 7.5 and 45°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 7.5 and 50°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 7.5 and 55°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 7.5 and 60°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 7.5 and 62°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 7.5 and 65°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 7.5 and 68°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 7.5 and 70°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 7.5 and 72°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 7.5 and 75°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 7.5 and 80°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 7.5 and 85°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 7.5 and 90°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 7.5 and 95°C.

[0213] In another aspect, the thermal stability of the variant relative to the parent was determined at pH 8.0 and 45°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 8.0 and 50°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 8.0 and 55°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 8.0 and 60°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 8.0 and 62°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 8.0 and 65°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 8.0 and 68°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 8.0 and 70°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 8.0 and 72°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 8.0 and 75°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 8.0 and 80°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 8.0 and 85°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 8.0 and 90°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 8.0 and 95°C.

[0214] In another aspect, the thermal stability of the variant relative to the parent was determined at pH 8.5 and 45°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 8.5 and 50°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 8.5 and 55°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 8.5 and 60°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 8.5 and 62°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 8.5 and 65°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 8.5 and 68°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 8.5 and 70°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 8.5 and 72°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 8.5 and 75°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 8.5 and 80°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 8.5 and 85°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 8.5 and 90°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 8.5 and 95°C.

[0215] In another aspect, the thermal stability of the variant relative to the parent was determined at pH 9.0 and 45°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 9.0 and 50°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 9.0 and 55°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 9.0 and 60°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 9.0 and 62°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 9.0 and 65°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 9.0 and 68°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 9.0 and 70°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 9.0 and 72°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 9.0 and 75°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 9.0 and 80°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 9.0 and 85°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 9.0 and 90°C. In another aspect, the thermal stability of the variant relative to the parent was determined at pH 9.0 and 95°C.

[0216] In all the above aspects, the thermal stability of the variant relative to the parent can be determined by incubating the variant and the parent for 1 minute. In all the above aspects, the thermal stability of the variant relative to the parent can be determined by incubating the variant and the parent for 5 minutes. In all the above aspects, the thermal stability of the variant relative to the parent can be determined by incubating the variant and the parent for 10 minutes. In all the above aspects, the thermal stability of the variant relative to the parent can be determined by incubating the variant and the parent for 15 minutes. In all the above aspects, the thermal stability of the variant relative to the parent can be determined by incubating the variant and the parent for 20 minutes. In all the above aspects, the thermal stability of the variant relative to the parent can be determined by incubating the variant and the parent for 25 minutes. In all the above aspects, the thermal stability of the variant relative to the parent can be determined by incubating the variant and the parent for 30 minutes. In all the above aspects, the thermal stability of the variant relative to the parent can be determined by incubating the variant and the parent for 45 minutes. In all the above aspects, the thermal stability of the variant relative to the parent can be determined by incubating the variant and the parent for 60 minutes. Longer time periods than 60 minutes can also be used.

[0217] In one respect, the variants with enhanced cellulose decomposition activity exhibit at least 1.01 times greater thermal stability compared to the parent, for example at least 1.05 times, at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.8 times, at least 2 times, at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 50 times, at least 75 times, or at least 100 times.

[0218] Parental GH61 polypeptide

[0219] The parental GH61 peptide can be any GH61 peptide with enhanced cellulose degradation activity.

[0220] The parental GH61 polypeptide can be (a) and SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116. (a) A polypeptide having at least 60% sequence identity with a mature polypeptide of SEQ ID NO: 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 56, 158, 160, 162, 164, 166, 168, 409, or 411; (b) A polypeptide encoded by a polynucleotide that hybridizes to at least the following under low-string conditions: (i) SEQ ID NO: 418, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 56, 158, 160, 162, 164, 166, 168, 409, or 411; NO:1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 10 5, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 408, or 410, or the full-length complement of (ii)(i);Or (c) by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107 A polypeptide encoded by a polynucleotide having at least 60% sequence identity with a mature polypeptide coding sequence of 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 408, or 410.

[0221] In one aspect, this parent is associated with SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, which have cellulose-enhancing activity. The mature polypeptides of 142, 144, 146, 148, 150, 152, 154, 56, 158, 160, 162, 164, 166, 168, 409, or 411 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.

[0222] In one embodiment, the amino acid sequence of the parent is identical to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110 Mature polypeptides of 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 56, 158, 160, 162, 164, 166, 168, 409, or 411 have up to 10 amino acids, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different amino acids.

[0223] In another embodiment, the parent contains SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 10 2. The amino acid sequence of 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 56, 158, 160, 162, 164, 166, 168, 409, or 411 or composed of such sequences.

[0224] In another embodiment, the parent contains SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 10 2. Mature polypeptides of or composed of 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 56, 158, 160, 162, 164, 166, 168, 409, or 411.

[0225] In another embodiment, the parent is a fragment of at least 85% of the amino acid residues, such as at least 90% or at least 95% of the amino acid residues, of a mature polypeptide containing the GH61 polypeptide.

[0226] In another embodiment, the parent is SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 10 2. Allelic variants of mature polypeptides of 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 56, 158, 160, 162, 164, 166, 168, 409, or 411.

[0227] In another aspect, the parent is encoded by polynucleotides that hybridize with the following under very low stringency conditions, low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions: (i) SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 1 Mature polypeptide coding sequences of 13, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 408, or 410, or their full-length complements (Sambrook et al., 1989, A Laboratory Manual of Molecular Cloning, 2nd ed., Cold Spring Harbor, New York).

[0228] SEQ ID NO:1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,5 3, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 10 3, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 408, or 410 polynucleotides or their subsequences, and SEQ ID. NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 ,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,108,110,112,11 4. Peptides or fragments thereof of the numbers 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 56, 158, 160, 162, 164, 166, 168, 409, or 411 can be used to design nucleic acid probes to identify and clone DNA encoding parents from strains of different genera or species, according to methods well known in the art. Specifically, such probes can be hybridized with the genomic DNA or cDNA of cells of interest according to standard DNA blotting procedures to identify and isolate the corresponding genes. Such probes can be much shorter than the entire sequence, but should be at least 15 nucleotides long, for example, at least 25, at least 35, or at least 70 nucleotides. Preferably, the nucleic acid probe is at least 100 nucleotides in length, such as at least 200, 300, 400, 500, 600, 700, 800, or 900 nucleotides. Both DNA and RNA probes can be used. Typically, the probe is labeled for detecting the corresponding gene (e.g., using...). 32 P, 3 H. 35 S, biotin or avidin). This invention covers such probes.

[0229] Genomic DNA or cDNA libraries prepared from other strains of this type can be screened by targeting DNA that hybridizes to and encodes a parent using the probes described above. Genomic DNA or other DNA from these other strains can be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the library or separated DNA can be transferred and immobilized on nitrocellulose or other suitable carrier materials. For identification with SEQ ID... NO:1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,5 5, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 1 Clones or DNA hybridized with 07, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 408, or 410, or their subsequences, are used as vector materials in DNA blotting.

[0230] For the purposes of this invention, hybridization indicator polynucleotides are hybridized with labeled nucleic acid probes corresponding to the following under conditions of very low to very high stringency: (i) SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 408, or 410; (ii) its mature polypeptide coding sequence; (iii) its full-length complement; or (iv) its subsequence. Molecules hybridizing with this nucleic acid probe under these conditions can be detected using, for example, X-ray membrane detection or any other detection method known in the art.

[0231] In one respect, the nucleic acid probe is SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101 Mature polypeptide coding sequences of 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 408, or 410.

[0232] In another embodiment, the nucleic acid probe is encoded with SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 10 4. Polypeptides of 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 56, 158, 160, 162, 164, 166, 168, 409, or 411; their mature polypeptides; or polynucleotide fragments thereof.

[0233] In another embodiment, the nucleic acid probe is SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 9 9, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 408, or 410.

[0234] In another aspect, the parent is encoded by a polynucleotide that is associated with SEQ ID NO. NO:1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 14 1. The mature polypeptide coding sequence of 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 408, or 410 has 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.

[0235] The parent can be a hybrid polypeptide in which a region of one polypeptide is fused to the N-terminus or C-terminus of a region of another polypeptide.

[0236] The parent peptide can be a fusion peptide or a cleavable fusion peptide, wherein another peptide is fused to the N-terminus or C-terminus of the peptide of the present invention. Fusion peptides are generated by fusing a polynucleotide encoding another peptide to the polynucleotide of the present invention. Techniques for generating fusion peptides are known in the art and include linking the coding sequences of the peptides such that they are in a frame and that the expression of the fusion peptide is under the control of the same one or more promoters and terminators. Fusion peptides can also be constructed using integrin techniques, wherein the fusion peptide is generated post-translational (Cooper et al., 1993, Journal of the European Society for Molecular Biology 12:2575-2583; Dawson et al., 1994, Science 266:776-779).

[0237] A fusion peptide may further include a cleavage site between the two peptides. This site is cleaved upon secretion of the fusion protein, thereby releasing both peptides. Examples of cleavage sites include, but are not limited to, those disclosed in the following: Martin et al., 2003, Journal of Industrial Microbiology and Biotechnology (J. Ind. Microbiol. Biotechnol.) 3:568-576; Svetina et al., 2000, Journal of Biotechnology (J. Biotechnol.) 76:245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63:3488-3493; Wa... (rd) et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9:378-381; Eaton et al., 1986, Biochemistry 25:505-512; Collins-Racie et al., 1995, Biotechnology 13:982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6:240-248; and Stevens, 2003, Drug Discovery World 4:35-48.

[0238] The parent can be obtained from any genus of microorganisms. For the purposes of this invention, the term "obtained from" as used herein in conjunction with a given source shall mean that the parent encoded by the polynucleotide is produced by that source or by a strain in which a polynucleotide from that source has been inserted. In one embodiment, the parent is extracellularly secreted.

[0239] The parent can be a bacterial GH61 polypeptide. For example, the parent can be a Gram-positive bacterial polypeptide, such as Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces. s) GH61 polypeptide, or Gram-negative bacterial polypeptides, such as Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasma GH61 polypeptide.

[0240] In one embodiment, the parent is Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus scoagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis GH61 polypeptide.

[0241] The parent can be a fungal GH61 polypeptide. For example, the parent can be a yeast GH61 polypeptide, such as those from the genera *Candida*, *Kluyveromyces*, *Pichia pastoris*, *Saccharomyces*, *Schizosaccharomyces*, or *Yarrowia*; or a filamentous fungal GH61 polypeptide, such as those from the genera *Cladosporium*, *Agaricus*, *Alternaria*, *Aspergillus*, *Bacillus*, *Botryospaeria*, *Ceriporiopsis*, *Chaetomidium*, *Aureobasidium*, *Cyperus*, *Cyperus*, *Cryptotermes*, *Cryphonectria*, *Cryptococcus*, *Diplosporium*, *Auricularia*, *Filibasidium*, *Fusarium*, *Gibberellinia*, or *Pteranophora*. GH61 polypeptide from the genera *Pyrophyllus*, *Irpex*, *Lentinula*, *Leptospaeria*, *Blastomyces*, *Melanocarpus*, *Meripilus*, *Mucor*, *Hypericum*, *Neocallimastix*, *Neurospora*, *Penicillium*, *Phanerochaete*, *Ruminocyticum*, *Poitrasia*, *Pseudotrichonympha*, *Rhizopus*, *Scytalidium*, *Arthrophyllus*, *Thermoascus*, *Clostridium*, *Trichoderma*, *Pterocarya*, *Verticillium*, *Smallopsis*, or *Carya*.

[0242] In another embodiment, the parent is a *Saccharomyces carlsbergensis*, *Saccharomyces cerevisiae*, *Saccharomyces sacchariformis*, *Saccharomyces douglasii*, *Saccharomyces kluyveri*, *Nordijanthi*, or *Saccharomyces ovalis* GH61 polypeptide.

[0243] In another embodiment, the parent is one of the following fungi: *Acremonium cellulolyticus*, *Aspergillus echinosporum*, *Aspergillus avocado*, *Aspergillus odorata*, *Aspergillus fumigatus*, *Aspergillus niger*, *Aspergillus niger*, *Aspergillus oryzae*, *Aspergillus terreus*, *Chrysosporium inops*, *Chrysosporium keratinophilum*, *Chrysosporium lucknowense*, *Chrysosporium medarium*, *Chrysosporium pannicola*, *Chrysosporium queenslandicum*, *Chrysosporium tropicum*, *Chrysosporium zonatum*, and *Fennellia*. Fusarium bactridioides, Fusarium crookwellense, Fusarium graminum, Fusarium heterosporum, Fusarium albinosum, Fusarium oxysporum, Fusarium spp., Fusarium oxysporum, Fusarium rosenbergii, Fusarium oxysporum, Fusarium rosenbergii, Fusarium rosenbergii, Fusarium scabra, Fusarium scabra, Fusarium sulfideum, Fusarium torulosum, Fusarium trichothecioides, Fusarium variegatum, Fusarium spp. ... leycettanus, thermophilic ascomycetes, Thievora achromatica, Thievora albomyces, Thievora albopilosa, Thievora australeinsis, Thievora fimeti, Thievora microspora, Thievora ovispora, Thievora peruvianaThe fungi include peruviana, Thievia setosa, Thievia spededonium, Thievia subthermophila, Thievia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibranchii, Trichoderma reesei, or Trichoderma viride GH61 polypeptide.

[0244] It will be understood that, for the purposes described above, the invention encompasses both the complete and incomplete states, as well as other taxonomic equivalents, such as asexual forms, regardless of their known species names. Those skilled in the art will readily identify the appropriate equivalents.

[0245] Strains of these species are readily available to the public at many culture collections, such as the American Type Culture Collection (ATCC), the German Microbial Culture Collection (DSMZ), the Dutch Culture Collection (CentraalbureauVoor Schimmelcultures, CBS), and the Northern Research Center (NRRL) of the Agricultural Research Culture Collection (ARC).

[0246] Parents can be identified and obtained from other sources, including microorganisms isolated from nature (e.g., soil, compost, water, etc.) using the probes described above, or DNA samples obtained directly from natural materials (e.g., soil, compost, water, etc.). Techniques for directly isolating microorganisms and DNA from the natural living environment are well known in the art. The polynucleotide encoding the parent can then be obtained by similarly screening in a library of genomic DNA or cDNA from another microorganism or a mixed DNA sample. Once a polynucleotide encoding a parent has been detected with one or more probes, the polynucleotide can be isolated or cloned using techniques known to those skilled in the art (see, for example, Sambrook et al., 1989, ibid.).

[0247] Preparation of variants

[0248] The present invention also relates to methods for obtaining GH61 polypeptide variants having cellulolytic-enhancing activity, the methods comprising: (a) replacing the introduced parental GH61 polypeptide at one or more (e.g., several) positions corresponding to positions 26, 32, 34, 40, 41, 42, 47, 56, 72, 102, 123, 138, 149, 152, 163, 164, 166, 169, 186, 200, 207, 213, 219, 222, 234, 246, 249, and 250 of the mature polypeptide of SEQ ID NO: 30, wherein the variant has cellulolytic-enhancing activity; and (b) recovering the variant. In one aspect, the methods further or even more further comprise replacing the introduced parental GH61 polypeptide at one or more (e.g., several) positions corresponding to positions 111, 152, 155, and 162 of the mature polypeptide of SEQ ID NO: 30, wherein the variant has cellulolytic-enhancing activity. In another aspect, these methods further or even further include replacing the introduced parental GH61 polypeptide at one or more (e.g., several) positions corresponding to positions 96, 98, 200, 202, and 204 of the mature polypeptide of SEQ ID NO:30, wherein the variant has cellulolytic-enhancing activity. In another aspect, these methods further or even further include replacing the introduced parental GH61 polypeptide at one or more (e.g., several) positions corresponding to positions 105, 154, 188, 189, 216, and 229 of the mature polypeptide of SEQ ID NO:30, wherein the variant has cellulolytic-enhancing activity.

[0249] These variants can be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc.

[0250] Site-directed mutagenesis is a technique in which one or more (e.g., several) mutations are introduced at one or more defined sites in a polynucleotide encoding a parent. Any site-directed mutagenesis procedure can be used in this invention. Many commercially available kits are available for preparing variants.

[0251] Site-directed mutagenesis can be achieved in vitro via PCR using primers containing oligonucleotides with the desired mutation. Site-directed mutagenesis can also be performed in vitro via cassette mutagenesis involving cleavage by a restriction endonuclease at a site containing a polynucleotide encoding the parent, followed by ligation of an oligonucleotide containing the mutation in that polynucleotide. Typically, the restriction endonuclease used to digest the plasmid and the oligonucleotide is the same to allow the sticky ends of the plasmid and the insert to ligate to each other. See, for example, Scherer and Davis, 1979, Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA) 76:4949-4955; and Barton et al., 1990, Nucleic Acid Research, 18:7349-4966.

[0252] Site-directed mutagenesis can also be achieved in vivo using methods known in the art. See, for example, U.S. Patent Application Publication No. 2004 / 0171154; Storici et al., 2001, Nature Biotechnology 19:773-776; Kren et al., 1998, Nat. Med. 4:285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43:15-16.

[0253] Site saturation mutagenesis systematically replaces the polypeptide coding sequence with a sequence encoding all 19 amino acids at one or more (e.g., several) specific sites (Parikh and Matsumura, 2005, Journal of Molecular Biology 352:621-628).

[0254] Synthetic gene construction requires the in vitro synthesis of a designed polynucleotide molecule to encode a polypeptide of interest. Gene synthesis can be performed using a variety of techniques, such as the multi-microchip-based technique described by Tian et al. (2004, Nature 432:1050-1054), and similar techniques in which oligonucleotides are synthesized and assembled on optically programmable microfluidic chips.

[0255] Using known mutagenesis, recombination, and / or tampering methods, followed by an associated screening procedure, one or more amino acid substitutions, deletions, and / or insertions can be made and tested. These associated screening procedures are, for example, those described by Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA (Proceedings of the National Academy of Sciences) 86:2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include: error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:10832-10837; US Patent No. 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0256] The activity of cloned, mutagenic peptides expressed by host cells can be detected by combining mutagenesis / reorganization methods with high-throughput automated screening methods (Ness et al., 1999, Nature Biotechnology 17:893-896). Mutagenic DNA molecules encoding active peptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow for the rapid determination of the importance of individual amino acid residues within the peptide.

[0257] Semi-synthetic gene construction is achieved through a combination of various methods, including synthetic gene construction, and / or site-directed mutagenesis, and / or random mutagenesis, and / or shuffling. Semi-synthetic construction typically involves combining a process of synthesizing polynucleotide fragments with PCR technology. Therefore, specific regions of the gene can be synthesized de novo, while other regions can be amplified using site-specific mutagenesis primers, and still others can undergo error-prone or non-error-prone PCR amplification. The polynucleotide subsequence can then be shuffled.

[0258] polynucleotides

[0259] The present invention also relates to isolated polynucleotides encoding the GH61 polypeptide variant of the present invention.

[0260] Nucleic acid constructs

[0261] The present invention also relates to a nucleic acid construct comprising a polynucleotide encoding a GH61 polypeptide variant of the present invention, the polynucleotide being operatively linked to one or more control sequences that, in a suitable host cell, guide the expression of the encoding sequence under conditions compatible with the control sequences.

[0262] This polynucleotide can be manipulated in a variety of ways to provide expression of the GH61 polypeptide variant. Depending on the expression vector, manipulation of the polynucleotide prior to its insertion into the vector may be desired or necessary. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

[0263] The control sequence can be a promoter, i.e., a polynucleotide recognized by the host cell to express a polynucleotide encoding a variant of the present invention. The promoter contains a transcriptional control sequence mediating the expression of the GH61 polypeptide variant. The promoter can be any polynucleotide exhibiting transcriptional activity in the host cell, including mutant, truncated, and heterozygous promoters, and can be derived from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to that of the host cell.

[0264] In bacterial host cells, examples of promoters suitable for guiding the transcription of the nucleic acid constructs of this invention are promoters obtained from the following: Bacillus amyloliquefaciens α-amylase gene (amyQ), Bacillus licheniformis α-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus thermophilus malt amylase gene (amyM), Bacillus subtilis fructan sucrase gene (sacB), Bacillus subtilis xylA and xylB genes, Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994, Molecular Microbiology). Molecular Microbiology 13:97-107), E. coli lac operon, E. coli trc promoter (Egon et al., 1988, Genes 69:301-315), Streptomyces agarase gene (dagA), and prokaryotic β-lactamase gene (Villa-Kamaroff et al., 1978, Proceedings of the National Academy of Sciences 75:3727-3731), along with the tac promoter (DeBoer et al., 1983, Proceedings of the National Academy of Sciences 80:21-25). Other promoters are described in “Useful proteins from recombinant bacteria”, Gilbert et al., 1980, Scientific American, 242:74-94; and Sambrook et al., 1989, ibid. Examples of tandem promoters are disclosed in WO 99 / 43835.

[0265] In filamentous fungal host cells, examples of suitable promoters for guiding the transcription of the nucleic acid constructs of this invention are promoters derived from the following genes: Aspergillus nidulans acetamase, Aspergillus niger neutral α-amylase, Aspergillus niger acid-stable α-amylase, Aspergillus niger or Aspergillus avocado glucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO 96 / 00787), Fusarium variegatum amyglucosidase (WO 00 / 56900), Fusarium variegatum Daria (WO 00 / 56900), Fusarium variegatum Quinn (WO 00 / 56900), etc. 00 / 56900), *Rhizopus oryzae* lipase, *Rhizopus oryzae* aspartic protease, *Trichoderma reesei* β-glucosidase, *Trichoderma reesei* cellobiose hydrolase I, *Trichoderma reesei* cellobiose hydrolase II, *Trichoderma reesei* glucan endonuclease I, *Trichoderma reesei* glucan endonuclease II, *Trichoderma reesei* glucan endonuclease III, *Trichoderma reesei* glucan endonuclease V, *Trichoderma reesei* xylanase I, *Trichoderma reesei* xylanase II, *Trichoderma reesei* xylanase III, *Trichoderma reesei* β-xylosidase, *Trichoderma reesei* Mold translation elongation factors, together with the NA2-tpi promoter (a modified promoter from a neutral α-amylase gene of Aspergillus, wherein the untranslated pre-promoter has been replaced with a untranslated pre-promoter from a triose phosphate isomerase gene of one of the Aspergillus species; non-limiting examples include a modified promoter from a neutral α-amylase gene of Aspergillus niger, wherein the untranslated pre-promoter has been replaced with a untranslated pre-promoter from a triose phosphate isomerase gene of Aspergillus nidulans or Aspergillus oryzae); and mutant, truncated, and hybrid promoters thereof. Other promoters are described in U.S. Patent No. 6,011,147.

[0266] In a yeast host, useful promoters are derived from the following genes: *Saccharomyces cerevisiae* enolase (ENO-1), *Saccharomyces cerevisiae* galactokinase (GAL1), *Saccharomyces cerevisiae* alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP), *Saccharomyces cerevisiae* triose phosphate isomerase (TPI), *Saccharomyces cerevisiae* metallothionein (CUP1), and *Saccharomyces cerevisiae* 3-phosphate glycerate kinase. Romanos et al., 1992, *Yeast* 8:423-488, describe other useful promoters in yeast host cells.

[0267] The control sequence can also be a transcription terminator recognized by the host cell to terminate transcription. This terminator is operatively linked to the 3' end of the polynucleotide encoding the GH61 polypeptide variant. Any terminator that is functional in the host cell can be used.

[0268] Preferred terminators for bacterial host cells are derived from genes of Bacillus clausti alkaline protease (aprH), Bacillus licheniformis α-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

[0269] Preferred terminators for filamentous fungal host cells are derived from the genes of: Aspergillus nidulans acetamase, Aspergillus nidulans o-aminobenzoate synthase, Aspergillus niger glucosylamylase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, Fusarium oxysporum trypsin-like protease, Trichoderma reesei β-glucosidase, Trichoderma reesei cellobiose hydrolase I, Trichoderma reesei cellobiose hydrolase II, Trichoderma reesei glucan endonuclease I, Trichoderma reesei glucan endonuclease II, Trichoderma reesei glucan endonuclease III, Trichoderma reesei glucan endonuclease V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei xylanase III, Trichoderma reesei β-xylosidase, and Trichoderma reesei translation elongation factor.

[0270] Preferred terminators for yeast host cells are derived from the genes of *Saccharomyces cerevisiae* enolase, *Saccharomyces cerevisiae* cytochrome C (CYC1), and *Saccharomyces cerevisiae* glyceraldehyde-3-phosphate dehydrogenase. Romanos et al., 1992, ibid., describe other useful terminators for yeast host cells.

[0271] The control sequence can also be a stabilizer region of mRNA downstream of the promoter and upstream of the gene coding sequence, which increases the expression of the gene.

[0272] Examples of suitable mRNA stable regions were obtained from the following: Bacillus thuringiensis cryIIIA gene (WO94 / 25612) and Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

[0273] The control sequence can also be a pre-signal, a non-translated mRNA region important for translation in the host cell. This pre-signal is operatively linked to the 5' end of the polynucleotide encoding the GH61 polypeptide variant. Any pre-signal that is functional in the host cell can be used.

[0274] The preferred precursors for filamentous fungal host cells are obtained from the genes of Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0275] Suitable precursors for yeast host cells are derived from the genes of the following: Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphoglycerate dehydrogenase (ADH2 / GAP).

[0276] The control sequence can also be a polyadenylated sequence, which is a sequence operatively linked to the 3' end of the coding sequence of the GH61 polypeptide variant and, during transcription, is recognized by the host cell as a signal to add polyadenylated residues to the transcribed mRNA. Any polyadenylated sequence that functions in the host cell can be used.

[0277] Preferred polyadenylated sequences for use in filamentous fungal host cells are derived from the genes of Aspergillus nidulans anthranilate synthase, Aspergillus niger glucosidase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0278] Polyadenylation sequences that can be used in yeast host cells have been described in Guo and Sherman, 1995, Molecular Cellular Biology, 15:5983-5990.

[0279] The control sequence can also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of the GH61 polypeptide variant and directs the variant into the cellular secretion pathway. The 5' end of the polynucleotide coding sequence may inherently contain a signal peptide coding sequence, which is naturally linked in the translation reading frame along with a segment encoding the variant's coding sequence. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding sequence that is exogenous to the coding sequence. In cases where the coding sequence does not naturally contain a signal peptide coding sequence, an exogenous signal peptide coding sequence may be required. Alternatively, an exogenous signal peptide coding sequence can simply replace the native signal peptide coding sequence to increase the variant's secretion. However, any signal peptide coding sequence that directs the expressed variant into the host cell's secretion pathway can be used.

[0280] The effective signal peptide coding sequences for bacterial host cells are derived from the following genes: Bacillus NCIB 11837 malt amylase, Bacillus subtilis protease, Bacillus subtilis β-lactamase, Bacillus thermophilus α-amylase, Bacillus thermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Additional signal peptides are described in Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

[0281] The effective signal peptide coding sequences for filamentous fungal host cells are obtained from the following genes: Aspergillus niger neutral amylase, Aspergillus niger glucosylase, Aspergillus oryzae TAKA amylase, Aspergillus oryzae cellulase, Aspergillus oryzae endoglucanase V, Aspergillus sparseis lipase, and Rhizopus oryzae aspartic protease.

[0282] Signal peptides useful to yeast host cells are obtained from the genes of *Saccharomyces cerevisiae* α-factor and *Saccharomyces cerevisiae* invertase. Romanos et al., 1992, ibid., described the coding sequences of other useful signal peptides.

[0283] The control sequence can also be a propeptide-coding sequence, encoding a propeptide located at the N-terminus of the GH61 polypeptide variant. The resulting polypeptide is called a proenzyme or propeptide progenitor (or, in some cases, a zymogen). The propeptide progenitor is usually inactive and can be converted into an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propeptide progenitor. The propeptide-coding sequence can be obtained from the genes of Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Thermophilus laccase (WO 95 / 33836), Rhizopus oryzae aspartic protease, and Saccharomyces cerevisiae α-factor.

[0284] In the presence of both the signal peptide and the propeptide sequence, the propeptide sequence is located immediately after the N-terminus of the GH61 polypeptide variant, and the signal peptide sequence is located immediately after the N-terminus of the propeptide sequence.

[0285] It may also be desirable to add regulatory sequences that modulate the expression of the GH61 polypeptide variant in relation to the growth of the host cell. Examples of regulatory sequences are those sequences that cause gene expression to be turned on or off in response to chemical or physical stimuli, including the presence of regulatory compounds. Regulatory sequences in prokaryotic systems include lac, tac, and trp-operated gene systems. In yeast, the ADH2 or GAL1 system can be used. In filamentous fungi, the *Aspergillus niger* glucosylamylase promoter, the *Aspergillus oryzae* TAKAα-amylase promoter, and the *Aspergillus oryzae* glucosylamylase promoter, the *Trichoderma reesei* cellobiose hydrolase I promoter, and the *Trichoderma reesei* cellobiose hydrolase II promoter can be used. Other examples of regulatory sequences are those that allow gene amplification. In eukaryotic systems, these regulatory sequences include dihydrofolate reductase genes amplified in the presence of methotrexate and metallothionein genes amplified with heavy metals. In these cases, the polynucleotide encoding the variant will be operatively linked to the regulatory sequence.

[0286] expression vector

[0287] The present invention also relates to a recombinant expression vector comprising a polynucleotide encoding a GH61 polypeptide variant of the present invention, a promoter, and transcription and translation termination signals. Different nucleotides and control sequences can be linked together to produce a recombinant expression vector, which may include one or more convenient restriction enzyme sites to allow insertion or substitution of the polynucleotide encoding the variant at these sites. Alternatively, the polynucleotide can be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into a suitable vector for expression. In producing the expression vector, the coding sequence is located within the vector such that the coding sequence is operatively linked to a suitable control sequence for expression.

[0288] The recombinant expression vector can be any vector (e.g., plasmid or virus) that can readily undergo recombinant DNA procedures and induce polynucleotide expression. The choice of vector will typically depend on its compatibility with the host cell to which it will be introduced. The vector can be a linear or closed circular plasmid.

[0289] The vector can be a self-replicating vector, i.e., a vector existing as an extrachromosomal entity whose replication is independent of chromosome replication, such as a plasmid, extrachromosomal element, mini-chromosome, or artificial chromosome. The vector can contain any means to ensure self-replication. Alternatively, the vector can be one that, when introduced into the host cell, is integrated into the genome and replicates along with one or more chromosomes in which it has been integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids (which together contain the total DNA of the genome to be introduced into the host cell), or transposons can be used.

[0290] The vector preferably contains one or more selective markers that allow easy selection of transformed, transfected, transduced, or similar cells. The selective marker is a gene whose product provides resistance to biocides or viruses, heavy metals, auxotrophic prototrophs, etc.

[0291] Examples of selective bacterial markers include the dal gene in Bacillus licheniformis or Bacillus subtilis, or markers that confer antibiotic resistance (e.g., ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline resistance). Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selective markers for use in filamentous fungal host cells include, but are not limited to, adeA (phosphogluconoylaminoimidazolium-succinate carboxylamine synthase), adeB (phosphogluconoylaminoimidazolium synthase), amdS (acetamipase), argB (ornithine carbamoyltransferase), bar (glufosinate acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotate nucleoside-5'-phosphate decarboxylase), sC (adenosyl sulfate transtransferase), and trpC (o-aminobenzoic acid synthase), along with their equivalents. The preferred genes used in Aspergillus cells are *Aspergillus nidus* or *Aspergillus oryzae* amdS and pyrG genes, and *Streptomyces hygroscopicus* bar genes. The preferred genes used in *Trichoderma* cells are adeA, adeB, amdS, hph, and pyrG genes.

[0292] The selective marker can be a biselective marker system as described in WO 2010 / 039889. In one aspect, the biselective marker is the hph-tk biselective marker system. The vector preferably contains one or more elements that allow the vector to integrate into the host cell's genome or to replicate autonomously in the cell independently of the genome.

[0293] To integrate into the host cell genome, the vector may rely on a polynucleotide sequence encoding a GH61 polypeptide variant or any other element of the vector for integration into the genome via homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides to guide integration into one or more precise locations on one or more chromosomes within the host cell genome via homologous recombination. To increase the likelihood of integration at precise locations, these integrating elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, that have high sequence identity with the corresponding target sequence to enhance the likelihood of homologous recombination. These integrating elements can be any sequence homologous to the target sequence within the host cell genome. Furthermore, these integrating elements can be non-coding or coding polynucleotides. On the other hand, the vector can integrate into the host cell genome via non-homologous recombination.

[0294] For autonomous replication, the vector may further include an origin of replication that enables the vector to replicate autonomously in the host cell in question. The origin of replication can be any plasmid replicon that mediates autonomous replication and functions within the cell. The terms "origin of replication" or "plasmid replicator" refer to a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0295] Examples of bacterial origins of replication are plasmids pBR322, pUC19, pACYC177, and pACYC184, which allow replication in Escherichia coli, and pUB110, pE194, pTA1060, and pAMβ1, which allow replication in Bacillus.

[0296] Examples of replication origins used in yeast host cells are 2-micron replication origins, ARS1, ARS4, a combination of ARS1 and CEN3, and a combination of ARS4 and CEN6.

[0297] Examples of useful origins of replication in filamentous fungal cells are AMA1 and ANS1 (Gems et al., 1991, Gene 98:61-67; Cullen et al., 1987, Nucleic Acids Res. 15:9163-9175; WO 00 / 24883). The isolation of the AMA1 gene and the construction of plasmids or vectors containing this gene can be performed according to the methods disclosed in WO00 / 24883.

[0298] One or more copies of the polynucleotide of the present invention can be inserted into host cells to increase the production of GH61 polypeptide variants. An increased number of polynucleotide copies can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selective marker gene associated with the polynucleotide, wherein cells containing an amplified copy of the selective marker gene, and thus additional copies of the polynucleotide, can be selected by culturing cells in the presence of a suitable selective reagent.

[0299] The procedures for connecting the above-described elements to construct the recombinant expression vector of the present invention are well known to those skilled in the art (see, for example, Sarabrook et al., 1989, ibid.).

[0300] host cells

[0301] This invention also relates to recombinant host cells containing a polynucleotide encoding a GH61 polypeptide variant of the invention, operably linked to one or more control sequences that direct the generation of variants of the invention. The construct or vector containing the polynucleotide is introduced into the host cell such that the construct or vector is maintained as a chromosomal integrator or as an autonomously replicating extrachromosomal vector, as previously described. The term "host cell" encompasses any progeny of a parent cell that differs from the parent cell due to mutations occurring during replication. The selection of the host cell will depend largely on the gene encoding the variant and its origin.

[0302] The host cell can be any cell that is useful in the recombinant generation of the GH61 peptide variant, such as a prokaryotic or eukaryotic cell.

[0303] Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Agrobacterium, Lactobacillus, Lactococcus, Marinebacterium, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Sludge, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0304] The bacterial host cell can be any Bacillus cell, including but not limited to Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus croceus, Bacillus coagulans, Bacillus sturdier, Bacillus splendidus, Bacillus stagnation, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus thermophilus, Bacillus subtilis, and Bacillus thuringiensis.

[0305] The bacterial host cell can also be a cell of any streptococcus genus, including but not limited to cells of Streptococcus equi, Streptococcus pyogenes, Streptococcus lactis, and Streptococcus equi subsp. vespermiae.

[0306] The bacterial host cell can also be any Streptomyces cell, including but not limited to non-chromogenic Streptomyces, Streptomyces avermectin, Streptomyces cerevisiae, Streptomyces grayi, and Streptomyces cerevisiae.

[0307] DNA can be introduced into Bacillus cells via the following methods: protoplast transformation (see, for example, Chang and Cohen, 1979, Molecular Genetics and Genomics, Gen. Genet., 168:111-115), competent cell transformation (see, for example, Young and Spizizen, 1961, Journal of Bacteriology, 81:823-829; or Dubainu...). DNA can be introduced into E. coli cells via protoplast transformation (see, for example, Dubnau et al., Davidoff-Abelson, 1971, Journal of Molecular Biology 56:209-221), electroporation (see, for example, Shigekawa and Dower, 1988, Biotechnology 6:742-751), or conjugation (see, for example, Koehler and Thorne, 1987, Journal of Bacteriology 169:5271-5278). DNA can be introduced into Streptomyces cells via protoplast transformation, electroporation (see, for example, Gong et al., 2004, Folia Microbiol. (Praha) 49:399-405), conjugation (see, for example, Mazodier et al., 1989, Journal of Bacteriology 171:3583-3585), or transduction (see, for example, Burke et al., 2001, Proceedings of the National Academy of Sciences 98:6289-6294). DNA can be introduced into Pseudomonas cells via electroporation (see, for example, Choi et al., 2006, Journal of Microbiological Methods 64:391-397) or conjugation (see, for example, Pinedo and Smets, 2005, Applied Environmental Microbiology 71:51-57).DNA can be introduced into Streptococcus cells via the following methods: native competent cells (see, for example, Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, for example, Catt and Jollick, 1991, Microbios 68:189-207), electroporation (see, for example, Buckley et al., 1999, Applied Environmental Microbiology 65:3800-3804), or conjugation (see, for example, Clewell, 1981, Microbiol. Rev. 45:409-436). However, any method known in the art for introducing DNA into host cells can be used.

[0308] The host cell can also be a eukaryotic cell, such as a mammalian, insect, plant, or fungal cell.

[0309] The host cell can be a fungal cell. As used herein, “fungus” includes Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota together with Oomycota and all mitotic fungi (as defined by Hawksworth et al. in Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).

[0310] The host cell of this fungus can be a yeast cell. As used herein, "yeast" includes Ascomycota (Endosporales), Basidiomycota, and yeasts belonging to the Deuteromycetes (budders). Since the classification of yeast may change in the future, for the purposes of this invention, yeast should be defined as described in *Biology and Activities of Yeast* (edited by Skinner, Passmore, and Davenport, Soc. App. Bacteriol. Symposium, Vol. 9, 1980).

[0311] Yeast host cells can be Candida, Hansenula, Kluyveromyces, Pichia pastoris, yeasts, fission yeasts, or Yarrowia cells, such as Kluyveromyces lactis, Kalmarosacchari, Saccharomyces cerevisiae, Saccharomyces sacchari, Douglas yeast, Kluyveromyces, Nodi yeast, Ovoyces, or Yarrowia lipolytica cells.

[0312] Fungal host cells can be filamentous fungal cells. "Filamentous fungi" includes all filamentous forms of the phylum Eumycota and subphyla of Oomycetes (as defined by Hawkesworth et al., 1995, see above). Filamentous fungi are typically characterized by a hyphal wall composed of chitosan, cellulose, glucan, mannan, and other complex polysaccharides. Vegetative growth occurs through hyphal elongation, and carbon metabolism is obligate aerobic. In contrast, yeast (such as Saccharomyces cerevisiae) grows vegetatively through budding of single-celled cells, and carbon metabolism can be fermentative.

[0313] The host cells of filamentous fungi can be cells of the genera *Cladosporium*, *Aspergillus*, *Briefomus*, *Fungiella*, *Aureosporium*, *Coprinus*, *Gynostemma*, *Cryptococcus*, *Smuts*, *Fusarium*, *Pyrophyllus*, *Orychophragmus*, *Mucor*, *Hydrophyllus*, *Neococcus*, *Penicillium*, *Penicillium*, *Pleurotus*, *White Rot*, *Ruminocyticum*, *Pleurotus*, *Schizophyllum*, *Arthrophyllus*, *Thermophilus*, *Clostridium*, *Cytotrichum*, or *Trichoderma*.

[0314] For example, the host cells of filamentous fungi can be *Aspergillus bubomori*, *Aspergillus sulphureus*, *Aspergillus fumigatus*, *Aspergillus japonicus*, *Aspergillus nidus*, *Aspergillus oryzae*, *Ceriporiopsis caregiea*, *Ceriporiopsis pannocinta*, *Ceriporiopsis rivulosa*, *Ceriporiopsis subrufa*, *Ceriporiopsis larvae*, *Chrysosporium inopsinatus*, *Chrysosporium lucknowense*, *Chrysosporium lucknowense*, and *Chrysosporium lucknowense*. *Chrysosporium queenslandicum*, *Chrysosporium queenslandicum*, *Chrysosporium tropicum*, *Chrysosporium brunneoincarnatum*, *Coprinus comatus*, *Tricholoma matsutake*, *Fusarium graminearum ... cells of Trichoderma harzianum, Trichoderma cornigerum, Trichoderma longifolium, Trichoderma reesei, or Trichoderma viride.

[0315] Fungal cells can be transformed in a manner known per se through methods involving protoplast formation, protoplast transformation, and cell wall regeneration. Suitable procedures for transforming Aspergillus and Trichoderma host cells are described in EP 238023, Yelton et al., 1984, Proceedings of the National Academy of Sciences 81:1470-1474, and Christensen et al., 1988, Bio / Technology 6:1419-1422. Suitable methods for transforming Fusarium species are described in Malardier et al., 1989, Gene 78:147-156 and WO 96 / 00787. Yeast can be transformed using procedures described in the following literature: Becker and Guarente, in Abelson, JN and Simon, MI eds., Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Vol. 194, pp. 182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal of Bacteriology 153:163; and Hinnen et al., 1978, Proceedings of the National Academy of Sciences 75:1920.

[0316] Generation method

[0317] The present invention also relates to a method for generating a GH61 peptide variant, the method comprising: (a) culturing the recombinant host cell of the present invention under conditions favorable for generating the variant; and optionally (b) recovering the variant.

[0318] The host cells are cultured in a nutrient medium suitable for producing the GH61 peptide variant using methods known in the art. For example, cells can be cultured in multi-well plates (e.g., 24, 48, or 96-well plates), shake flasks, or in a suitable medium under conditions allowing for the expression and / or isolation of the variant, either on a small-scale or large-scale basis in a laboratory or industrial fermenter (including continuous, batch, fed-batch, or solid-state fermentation). This culture occurs using procedures known in the art in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts. Suitable media are available from commercial suppliers or can be prepared according to publicly available compositions (e.g., in the U.S. Type Culture Collection). If the variant is secreted into the nutrient medium, it can be recovered directly from the medium. If the variant is not secreted, it can be recovered from cell lysates.

[0319] The GH61 peptide variant can be detected using methods known in the art that are specific to this variant. These detection methods include, but are not limited to, the use of specific antibodies, the formation of enzyme products, or the disappearance of enzyme substrates. For example, an enzyme assay can be used to determine the activity of this variant. One specific assay for determining GH61 protein involves reacting the GH61 peptide variant with 0.5% phosphate-swellable cellulose (PASC), 100 mM sodium acetate (pH 5), 1 mM MnSO4, 0.1% gallic acid, 0.025 mg / ml Aspergillus fumigatus β-glucosidase, and 0.01% [unclear - possibly a specific enzyme or solution] at 40°C. Incubate with X100 for 24–96 hours, then determine the glucose released from the PASC by measuring this reaction. See the determination described in Example 5.

[0320] The GH61 peptide variant can be recovered using methods known in the art. For example, the variant can be recovered from the nutrient medium using a variety of conventional procedures, including, but not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. In one aspect, the entire fermentation broth containing the variant of the present invention was recovered.

[0321] GH61 peptide variants can be purified to obtain substantially pure variants using a variety of procedures known in the art, including, but not limited to, chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatographic focusing, and size exclusion chromatography), electrophoresis procedures (e.g., preparative isoelectric point focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, for example, Protein Purification, edited by Janson and Ryden, VCH Publishers, New York, 1989).

[0322] In an alternative, instead of recycling the GH61 peptide variant, the present invention’s host cells expressing the variant are used as the source of the variant.

[0323] Fermentation broth preparations or cell compositions

[0324] The present invention also relates to a fermentation broth formulation or a cell composition comprising a variant of the invention. The fermentation broth product further comprises additional components used in the fermentation process, such as cells (including host cells containing genes encoding a variant of the invention, which are used to produce the variant), cell debris, biomass, fermentation medium, and / or fermentation products. In some embodiments, the composition is a cell-killed whole culture containing one or more organic acids, killed cells and / or cell debris, and a culture medium.

[0325] As used herein, the term "fermentation broth" refers to a preparation produced by cell fermentation that undergoes little or no recovery and / or purification. For example, fermentation broth is produced when a microbial culture is incubated to saturation under carbon-limited conditions that allow protein synthesis (e.g., expression by enzymes of the host cell) and secretion of proteins into the cell culture medium. Fermentation broth may contain unfractionated or fractionated contents of the fermentation material obtained at the end of fermentation. Typically, fermentation broth is unfractionated and includes depleted culture medium and cell debris present, for example, after removal of microbial cells (e.g., filamentous fungal cells) by centrifugation. In some embodiments, fermentation broth comprises depleted cell culture medium, extracellular enzymes, and viable and / or non-viable microbial cells.

[0326] In one embodiment, the fermentation broth formulation and cell composition comprise a first organic acid component (comprising at least one 1-5 carbon organic acid and / or its salt) and a second organic acid component (comprising at least one 6- or more carbon organic acid and / or its salt). In one specific embodiment, the first organic acid component is acetic acid, formic acid, propionic acid, its salt, or a mixture of two or more of the aforementioned acids; and the second organic acid component is benzoic acid, cyclohexanecarboxylic acid, 4-methylvaleric acid, phenylacetic acid, its salt, or a mixture of two or more of the aforementioned acids.

[0327] In one aspect, the composition comprises one or more organic acids and optionally further comprises killed cells and / or cell debris. In one embodiment, these killed cells and / or cell debris are removed from the cell-killing whole culture medium to provide a composition free of these components.

[0328] These fermentation broth formulations or cell compositions may further contain a preservative and / or antimicrobial (e.g., bacteriostatic) agent, including but not limited to sorbitol, sodium chloride, potassium sorbate, and other reagents known in the art.

[0329] Fermentation broth formulations or cell compositions may further contain a variety of enzyme activities, such as one or more (e.g., several) enzymes selected from the group consisting of: cellulase, hemicellulase, esterase, dilatin, laccase, lignin-degrading enzyme, pectinase, peroxidase, protease, and swollenin. Fermentation broth formulations or cell compositions may also contain one or more (e.g., several) enzymes selected from the group consisting of: hydrolases, isomerases, ligases, lyases, oxidoreductases, or a transferase, such as α-galactosidase, α-glucosidase, aminopeptidase, amylase, β-galactosidase, β-glucosidase, β-xylosidase, glycosylase, carboxypeptidase, catalase, cellobiase, cellulase, chitinase, keratinase, cyclodextrin glycosyltransferase, deoxyribonuclease, dextran endonuclease, esterase, glucosylamylase, invertase, laccase, lipase, mannosidase, polysaccharidase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenol oxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

[0330] Cell-killing whole cultures or compositions may contain ungraded contents of fermentation material derived at the end of fermentation. Typically, cell-killing whole cultures or compositions contain depleted culture medium from which microbial cells (e.g., filamentous fungal cells) have grown to saturation or are present, incubated under carbon-limited conditions to allow protein synthesis (e.g., expression of cellulase and / or one or more glucosidases). In some embodiments, cell-killing whole cultures or compositions contain depleted cell culture medium, extracellular enzymes, and killed filamentous fungal cells. In some embodiments, methods known in the art can be used to permeate and / or lyse the microbial cells present in the cell-killing whole cultures or compositions.

[0331] The whole culture medium or cell composition described herein is typically a liquid, but may contain insoluble components, such as killed cells, cell debris, culture medium components, and / or one or more insoluble enzymes. In some embodiments, insoluble components may be removed to provide a clear liquid composition.

[0332] The whole culture medium formulations and cell compositions of the present invention can be produced by the methods described in WO 90 / 15861 or WO 2010 / 096673.

[0333] Examples of preferred uses of the compositions of the present invention are given below. The dosage of the composition and other conditions for using the composition can be determined based on methods known in the art.

[0334] Enzyme composition

[0335] The present invention also relates to compositions comprising a variant of the invention. Preferably, these compositions are enriched with such a variant. The term "enrichment" indicates that the composition exhibits enhanced cellulose-degrading activity, for example, an increase in enrichment factor of at least 1.1.

[0336] These compositions may contain a variant of the invention as the main enzyme component, such as a single-component composition. Alternatively, these compositions may contain a variety of enzyme activities, such as one or more (e.g., several) enzymes selected from the group consisting of: cellulase, hemicellulase, GH61 polypeptide, esterase, dilatin, laccase, lignin-degrading enzyme, pectinase, peroxidase, protease, and swollenin. These compositions may also include one or more (e.g., several) enzymes selected from the group consisting of: hydrolases, isomerases, ligases, lyases, oxidoreductases, or transferases, such as α-galactosidase, α-glucosidase, aminopeptidase, amylase, β-galactosidase, β-glucosidase, β-xylosidase, glycosylase, carboxypeptidase, catalase, cellobiase, cellulase, chitosanase, keratinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucosylamylase, invertase, laccase, lipase, mannosidase, polysaccharidase, oxidase, pectinase, peroxidase, phytase, polyphenol oxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

[0337] These compositions can be prepared according to methods known in the art and can be in the form of liquid or dry compositions. These compositions can be stabilized according to methods known in the art.

[0338] Examples of preferred uses of the compositions of the present invention are given below. The dosage of the composition and other conditions for using the composition can be determined based on methods known in the art.

[0339] use

[0340] The present invention also relates to the following methods using GH61 polypeptide variants or combinations thereof that have cellulolytic-enhancing activity.

[0341] The present invention also relates to methods for degrading or converting cellulose materials, comprising treating the cellulose material with an enzymatic composition in the presence of a GH61 polypeptide variant of the present invention. In one aspect, these methods further comprise recovering the degraded or converted cellulose material. The soluble products of the degradation or conversion of the cellulose material can be separated from the insoluble cellulose material using methods known in the art, such as centrifugation, filtration, or gravity sedimentation.

[0342] The present invention also relates to a method for producing a fermentation product, comprising: (a) saccharifying a cellulose material with an enzyme composition in the presence of a GH61 polypeptide variant of the present invention; (b) fermenting the saccharified cellulose material with one or more (e.g., several) fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.

[0343] The present invention also relates to a method for fermenting cellulose materials, comprising: fermenting the cellulose material with one or more (e.g., several) fermenting microorganisms, wherein the cellulose material is saccharified with an enzyme composition in the presence of the GH61 polypeptide variant of the present invention. In one aspect, the fermentation of the cellulose material produces a fermentation product. In another aspect, these methods further comprise recovering the fermentation product from the fermentation.

[0344] The method of the present invention can be used to saccharify cellulosic materials into fermentable sugars and convert the fermentable sugars into a variety of useful fermentation products, such as fuels (ethanol, n-butanol, isobutanol, biodiesel, jet fuel) and / or platform compounds (e.g., acids, alcohols, ketones, gases, oils, etc.). The production of desired fermentation products from cellulosic materials typically involves pretreatment, enzymatic hydrolysis (saccharification), and fermentation.

[0345] The processing of the cellulose material according to the present invention can be accomplished using methods conventional in the art. Furthermore, the methods of the present invention can be implemented using any conventional biomass processing equipment configured to operate according to the present invention.

[0346] Separate or simultaneous hydrolysis (saccharification) and fermentation include, but are not limited to: separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), simultaneous saccharification and co-fermentation (SSCF), hybrid hydrolysis and fermentation (HHF), separate hydrolysis and co-fermentation (SHCF), hybrid hydrolysis and co-fermentation (HHCF), and direct microbial transformation (DMC), sometimes referred to as combined bioprocessing (CBP). SHF uses separate processing steps to first enzymatically hydrolyze cellulosic material into fermentable sugars (e.g., glucose, cellobiose, and pentose monomers), and then ferment the fermentable sugars into ethanol. In SSF, the enzymatic hydrolysis of cellulose material and the fermentation of sugars to ethanol are combined in a single step (Philippidis, GP, 1996, Cellulose bioconversion technology, in Handbook on Bioethanol: Production and Utilization, Wyman, CE, editor, Taylor & Francis, Washington, D.C., 179-212). SSCF involves the co-fermentation of multiple sugars (Sheehan and Himmel, 1999, Biotechnol. Prog. 15:817-827). HHF involves a separate hydrolysis step and an additional simultaneous saccharification and hydrolysis step, which can be carried out in the same reactor. The steps in the HHF process can be carried out at different temperatures, i.e., high-temperature enzymatic saccharification followed by SSF at a lower temperature tolerable by the fermentation strain. DMC combines all three processes (enzyme production, hydrolysis, and fermentation) in one or more (e.g., several) steps, using the same bio-production enzymes for converting cellulosic material into fermentable sugars and for converting fermentable sugars into the final product (Lynd et al., 2002, Microbiol. Mol. Biol. Reviews 66: 506-577). It should be understood here that any method known in the art, including pretreatment, enzymatic hydrolysis (saccharification), fermentation, or combinations thereof, can be used to implement the method of the present invention.

[0347] Conventional apparatus may include a fed-batch stirred reactor, a batch stirred reactor, a continuous flow stirred reactor with ultrafiltration, and / or a continuous plug-flow column reactor (de Castilhos Corazza et al., 2003, Acta Scientiarum. Technology 25:33-38; Gusakov and Sinitsyn, 1985, Enzymology and Microbiology Technol. 7:346-352), and a grinding reactor (Ryu and Lee, 1983, Biotechnol. Bioeng. 25:53-65). Other reactor types include fluidized bed reactors for hydrolysis and / or fermentation, upflow blanket reactors, immobilized reactors, and extruder-type reactors.

[0348] Preprocessing. In practicing the process of this invention, any pretreatment process known in the art can be used to disrupt the plant cell wall components of the cellulose material (Chandra et al., 2007, Adv. Biochem. Engin. / Biotechnol. 108:67-93; Galbe and Zacchi, 2007, Adv. Biochem. Engin. / Biotechnol. 108:41-65; Hendriks and Zeeman, 2009, Bioresource Technology). Technol. 100:10-18; Mosier et al., Bioresource Technology, 2005, 96:673-686; Taherzadeh and Karimi, 2008, International Journal of Molecular Sciences, 9:1621-1651; Yang and Wyman, 2008, Biofuels, Bioproducts and Biorefining, 2:26-40.

[0349] Cellulose materials may also undergo particle size reduction, sieving, pre-soaking, wetting, washing and / or conditioning before being pretreated using methods known in the art.

[0350] Conventional pretreatment includes, but is not limited to, steam pretreatment (with or without bursting), dilute acid pretreatment, hot water pretreatment, alkali pretreatment, lime pretreatment, wet oxidation, wet bursting, ammonia cellulose bursting, organic solvent pretreatment, and biological pretreatment. Other pretreatments include ammonia permeation, ultrasonication, electroporation, microwave, supercritical CO2, supercritical H2O, ozone, ionic liquids, and gamma radiation pretreatment.

[0351] Cellulose materials can be pretreated before hydrolysis and / or fermentation. Pretreatment is preferred before hydrolysis. Alternatively, pretreatment can be performed simultaneously with enzymatic hydrolysis to release fermentable sugars, such as glucose, xylose, and / or cellobiose. In most cases, the pretreatment step itself results in the conversion of biomass into fermentable sugars (even in the absence of enzymes).

[0352] Steam pretreatment. In steam pretreatment, the cellulose material is heated to break down plant cell wall components, including lignin, hemicellulose, and cellulose, so that cellulose and other fractions, such as hemicellulose, become accessible to enzymes. The cellulose material is passed through or through a reaction vessel, into which steam is injected to increase the temperature to the desired temperature and pressure, and the steam is maintained therein for the desired reaction time. Steam pretreatment is preferably carried out at 140°C–250°C, for example 160°C–200°C or 170°C–190°C, where the optimal temperature range depends on the optional addition of a chemical catalyst. The residence time for steam pretreatment is preferably 1–60 minutes, for example 1–30 minutes, 1–20 minutes, 3–12 minutes, or 4–10 minutes, where the optimal residence time depends on the temperature and the optional addition of a chemical catalyst. Steam pretreatment allows for a relatively high solids loading, so that the cellulose material typically only becomes moist during pretreatment. Steam pretreatment is often combined with an explosive discharge of the pretreated material, known as a steam burst, which involves rapid evaporation to atmospheric pressure and turbulence of the material to increase the accessible surface area through breakup (Duff and Murray, 1996, Bioresource Technology 855:1-33; Gelbe and Sage, 2002, Appl. Microbiol. Biotechnol. 59:618-628; US Patent Application No. 2002 / 0164730). During steam pretreatment, hemicellulose acetyl groups are cleaved, and the resulting acid autocatalytically hydrolyzes the hemicellulose into monosaccharides and oligosaccharides. Lignin is removed only to a limited extent.

[0353] Chemical pretreatment: The term "chemical treatment" refers to any chemical pretreatment that promotes the separation and / or release of cellulose, hemicellulose, and / or lignin. Such pretreatment can convert crystalline cellulose into amorphous cellulose. Examples of suitable chemical pretreatment processes include, for example, dilute acid pretreatment, lime pretreatment, wet oxidation, ammonia cellulose / freeze expansion (AFEX), ammonia permeation (APR), ionic liquids, and organic solvent pretreatment.

[0354] A chemical catalyst (e.g., H₂SO₄ or SO₂) (typically 0.3 to 5% w / w) is often added prior to steam pretreatment. This catalyst reduces time and temperature, increases recovery, and improves enzymatic hydrolysis (Ballesteros et al., 2006, Applied Biochemistry and Biotechnology 129-132:496-508; Varga et al., 2004, Applied Biochemistry and Biotechnology 113-116:509-523; Sassner et al., 2006, Enzyme Microb. Technol. 39:756-762). In dilute acid pretreatment, cellulose material is mixed with dilute acid (typically H₂SO₄) and water to form a slurry, heated by steam to the desired temperature, and rapidly evaporated to atmospheric pressure after a residence time. Various reactor designs can be used for dilute acid pretreatment, such as plug flow reactors, countercurrent reactors, or continuous countercurrent shrink-bed reactors (Duff and Murray, 1996, ibid.; Schell et al., 2004, Bioresource Technology 91: 179-188; Lee et al., 1999, Adv. Biochem. Engin. / Biotechnol. 65: 93-115).

[0355] Several pretreatment methods under alkaline conditions can also be used. These alkaline pretreatments include, but are not limited to: sodium hydroxide, lime, wet oxidation, ammonia permeation (APR), and ammonia cellulose / freeze expansion (AFEX) pretreatment.

[0356] Lime pretreatment using calcium oxide or calcium hydroxide at temperatures of 85°C–150°C, with residence times ranging from one hour to several days (Wyman et al., 2005, Bioresource Technol. 96: 1959–1966; Mosier et al., 2005, Bioresource Technol. 96: 673–686). WO 2006 / 110891, WO 2006 / 110899, WO 2006 / 110900, and WO 2006 / 110901 disclose pretreatment methods using ammonia.

[0357] Wet oxidation typically involves adding an oxidizing agent such as hydrogen peroxide or overpressurized oxygen and performing a thermal pretreatment at 180°C–200°C for 5–15 minutes (Schmidt and Thomsen, 1998, Bioresource Technology, 64: 139–151; Palonen et al., 2004, Applied Biochemistry and Biotechnology, 117: 1–17; Varga et al., 2004, Biotechnology and Bioengineering, 88: 567–574; Martin et al., 2006, Journal of Chemical Technology and Biotechnology, 81: 1669–1677). Pretreatment is preferably performed on 1%-40% dry matter, such as 2%-30% or 5%-20% dry matter, and the initial pH is usually increased by adding an alkali, such as sodium carbonate.

[0358] A modified wet oxidation pretreatment method, termed wet burst (a combination of wet oxidation and steam burst), can handle up to 30% dry matter. In wet burst, an oxidizing agent is introduced during pretreatment after a certain residence time. The pretreatment is then terminated by rapid evaporation to atmospheric pressure (WO 2006 / 03228).

[0359] Ammonium cellulose expansion (AFEX) involves treating cellulosic materials with liquid or gaseous ammonia at moderate temperatures (e.g., 90°C–150°C) and high pressures (e.g., 17–20 bar) for 5–10 minutes, where the dry matter content can be as high as 60% (Gollapalli et al., 2002, Appl. Biochem. Biotechnol. 98:23–35; Chundawat et al., 2007, Biotechnology and Bioengineering (Biotechnol. Bioeng. 96:219–231; Alizadeh et al., 2005, Appl. Biochem. Biotechnol. 121:1133–1141; Teymouri et al., 2005, Bioresource Technol. 96:2014–2018). During the AFEX pretreatment process, cellulose and hemicellulose remain relatively intact. The lignin-carbohydrate complex is cleaved.

[0360] Organic solvent pretreatment can delignify cellulosic materials by extraction with an aqueous ethanol solution (40%-60% ethanol) at 160-200°C for 30-60 minutes (Pan et al., 2005, Biotechnol. Bioeng. 90: 473-481; Pan et al., 2006, Biotechnol. Bioeng. 94: 851-861; Kurabi et al., 2005, Appl. Biochem. Biotechnol. 121: 219-230). Sulfuric acid is usually added as a catalyst. During organic solvent pretreatment, most of the hemicellulose and lignin are removed.

[0361] Other examples of suitable pretreatment methods are described by Sher et al., 2003, Applied Biochemistry and Biotechnology 105-108:69-85, and Mosier et al., 2005, Bioresource Technology 96:673-686, and U.S. Patent Application 2002 / 0164730.

[0362] In one aspect, the chemical pretreatment is preferably carried out as a dilute acid treatment, and more preferably as a continuous dilute acid treatment. The acid is typically sulfuric acid, but other acids such as acetic acid, citric acid, nitric acid, phosphoric acid, tartaric acid, succinic acid, hydrogen chloride, or mixtures thereof may also be used. A mild acid treatment is preferably carried out in a pH range of 1-5, for example 1-4 or 1-2.5. In one aspect, the acid concentration is preferably in the range of 0.01 to 10 wt.% of acid, for example 0.05 to 5 wt.% or 0.1 to 2 wt.% of acid. The acid is contacted with the cellulose material, and preferably at a temperature in the range of 140°C to 200°C, for example 165°C to 190°C, for a time ranging from 1 to 60 minutes.

[0363] On the other hand, pretreatment is carried out in an aqueous slurry. In a preferred aspect, the cellulose material present during pretreatment is in an amount preferably 10-80 wt%, for example 20-70 wt% or 30-60 wt%, such as about 40 wt%. The pretreated cellulose material can be treated without washing or by washing using any method known in the art, for example, washing with water.

[0364] Mechanical or physical pretreatment: The terms “mechanical pretreatment” or “physical pretreatment” refer to any pretreatment that promotes particle size reduction. For example, such pretreatment can involve different types of grinding or milling (e.g., dry grinding, wet grinding, or vibratory ball milling).

[0365] The cellulose material can be subjected to physical (mechanical) and chemical pretreatments. Mechanical or physical pretreatments can be combined with steam / steam bursts, hydrothermal hydrolysis, dilute or weak acid treatment, high temperature, high pressure treatment, irradiation (e.g., microwave irradiation), or combinations thereof. In one aspect, high pressure refers to a pressure preferably in the range of about 100 to about 400 psi, for example, about 150 to about 250 psi. In another aspect, high temperature means a temperature in the range of about 100°C to about 300°C, for example, about 140°C to about 200°C. In a preferred aspect, a steam gun hydrolyzer system is used for batch mechanical or physical pretreatment using high pressure and high temperature as defined above, for example, the Sunds hydrolyzers available from Sunds Defibrator AB, Sweden. These physical and chemical pretreatments can be performed sequentially or simultaneously as needed.

[0366] Therefore, in a preferred aspect, the cellulose material is subjected to physical (mechanical) or chemical pretreatment, or any combination thereof, to promote the separation and / or release of cellulose, hemicellulose, and / or lignin.

[0367] Biological pretreatment: The term “biological pretreatment” refers to any biological pretreatment that promotes the separation and / or release of cellulose, hemicellulose, and / or lignin from cellulosic materials. Biological pretreatment techniques can involve the application of lignin-dissolving microorganisms and / or enzymes (see, for example, Hsu, T.-A., 1996, Pretreatment of biomass, in Bioethanol: A Handbook of Production and Utilization, Wyman, CE, ed., Taylor & Francis, Washington, D.C., 179-212; Ghosh and Singh, 1993, Adv. Appl. Microbiol. 39:295-333; McMillan, JD, 1994, Pretreating lignocellulosic biomass: a review, in Enzymatic Conversion of Biomass for Fuels Production, Himmel, ME, Baker, JO, and Overend, RP, ed., ACS Symposium). Series 566, American Chemical Society, Washington, D.C., Chapter 15; Gong, CS, Cao, NJ, Du, J., and Tsao, GT, 1999, Ethanol production from renewable resources, in Advances in Biochemical In Engineering / Biotechnology, Scheper, T., editor, Springer-Verlag, Berlin-Heidelberg, Germany, 65:207-241; Olsson and Hahn-Hagerdal, 1996, Enz. Microb. Tech. 18:312-331; and Vallander and Eriksson, 1990, Adv. Biochem. Eng. / Biotechnol. 42:63-95).

[0368] GlycationIn the hydrolysis step, also known as saccharification, cellulose material (e.g., pretreated) is hydrolyzed to break down cellulose and / or hemicellulose into fermentable sugars, such as glucose, cellobiose, xylose, xylulose, arabinose, mannose, galactose, and / or soluble oligosaccharides. Hydrolysis is enzymatically propelled by an enzyme composition in the presence of the GH61 polypeptide variant of the present invention. The enzymes in these compositions may be added simultaneously or sequentially.

[0369] Enzymatic hydrolysis is preferably performed under conditions readily determined by those skilled in the art, in a suitable aqueous environment. In one aspect, hydrolysis is carried out under conditions suitable for the activity of one or more enzymes, i.e., optimal for those enzymes. Hydrolysis can be carried out as a batch or continuous process, wherein cellulose material is gradually fed into, for example, an enzyme-containing hydrolysis solution.

[0370] Saccharification is typically carried out in a stirred tank reactor or fermenter under controlled pH, temperature, and mixing conditions. Suitable processing times, temperatures, and pH conditions can be readily determined by those skilled in the art. For example, saccharification can last up to 200 hours, but is typically preferred to be carried out for about 12 to about 120 hours, for example, about 16 to about 72 hours or about 24 to about 48 hours. Preferred temperatures are in the range of about 25°C to about 70°C, for example, about 30°C to about 65°C, about 40°C to about 60°C, or about 50°C to about 55°C. Preferred pH is in the range of about 3 to about 8, for example, about 3.5 to about 7, about 4 to about 6, or about 5 to about 5.5. Preferred dry solids content is in the range of about 5 to about 50 wt%, for example, about 10 to about 40 wt% or about 20 to about 30 wt%.

[0371] These enzyme compositions can contain any protein used to degrade cellulose materials.

[0372] In one aspect, the enzyme composition comprises or further comprises one or more proteins selected from the group consisting of: cellulase, GH61 polypeptide with cellulolytic-enhancing activity, hemicellulase, esterase, expansin, lignin-degrading enzyme, oxidoreductase, pectinase, protease, and swelling agent. In another aspect, the cellulase is preferably one or more enzymes selected from the group consisting of: endoglucanase, cellobiase, and β-glucosidase. In yet another aspect, the hemicellulase is preferably one or more enzymes selected from the group consisting of: acetylmannan esterase, acetylxylan esterase, arabinonanase, arabinofuranase, coumarin esterase, ferulic acid esterase, galactosidase, glucuronidase, glucuronidase, mannanase, mannosidase, xylanase, and xylosidase. In another aspect, the oxidoreductase is preferably one or more (e.g., several) enzymes selected from the group consisting of catalase, laccase, and peroxidase.

[0373] In another aspect, the enzyme composition comprises one or more (e.g., several) cellulases. In another aspect, the enzyme composition comprises or further comprises one or more (e.g., several) hemicellulases. In another aspect, the enzyme composition comprises one or more (e.g., several) cellulases and one or more (e.g., several) hemicellulases. In another aspect, the enzyme composition comprises one or more (e.g., several) enzymes selected from the group consisting of cellulases and hemicellulases. In another aspect, the enzyme composition comprises an endoglucanase. In another aspect, the enzyme composition comprises a cellobiose hydrolase. In another aspect, the enzyme composition comprises a β-glucosidase. In another aspect, the enzyme composition comprises a GH61 polypeptide with cellulolytic enhancement activity. In another aspect, the enzyme composition comprises an endoglucanase and a GH61 polypeptide with cellulolytic enhancement activity. In another aspect, the enzyme composition comprises a cellobiose hydrolase and a GH61 polypeptide with cellulolytic enhancement activity. In another aspect, the enzyme composition comprises a β-glucosidase and a GH61 polypeptide with cellulolytic enhancement activity. In another aspect, the enzyme composition comprises an endoglucanase and a cellobiase. In another aspect, the enzyme composition comprises an endoglucanase and a cellobiase I, a cellobiase II, or a combination of cellobiase I and cellobiase II. In another aspect, the enzyme composition comprises an endoglucanase and a β-glucosidase. In another aspect, the enzyme composition comprises a β-glucosidase and a cellobiase. In another aspect, the enzyme composition comprises a β-glucosidase and a cellobiase I, a cellobiase II, or a combination of cellobiase I and cellobiase II. In another aspect, the enzyme composition comprises an endoglucanase, a GH61 polypeptide with enhanced cellulose-degrading activity, and a cellobiase. In another aspect, the enzyme composition comprises an endoglucanase, a GH61 polypeptide with enhanced cellulolytic activity, and a cellobiase I, a cellobiase II, or a combination of cellobiase I and cellobiase II. In another aspect, the enzyme composition comprises an endoglucanase, a β-glucosidase, and a GH61 polypeptide with enhanced cellulolytic activity. In another aspect, the enzyme composition comprises a β-glucosidase, a GH61 polypeptide with enhanced cellulolytic activity, and a cellobiase. In another aspect, the enzyme composition comprises a β-glucosidase, a GH61 polypeptide with enhanced cellulolytic activity, and a cellobiase I, a cellobiase II, or a combination of cellobiase I and cellobiase II.In another aspect, the enzyme composition comprises an endoglucanase, a β-glucosidase, and a cellobiase. In yet another aspect, the enzyme composition comprises an endoglucanase, a β-glucosidase, and a cellobiase I, a cellobiase II, or a combination of cellobiase I and cellobiase II. In yet another aspect, the enzyme composition comprises an endoglucanase, a cellobiase, a β-glucosidase, and a GH61 polypeptide with enhanced cellulose-degrading activity. In yet another aspect, the enzyme composition comprises an endoglucanase, a β-glucosidase, a GH61 polypeptide with enhanced cellulose-degrading activity, and a cellobiase I, a cellobiase II, or a combination of cellobiase I and cellobiase II.

[0374] In another aspect, the enzyme composition comprises an acetylmannan esterase. In another aspect, the enzyme composition comprises an acetylxylan esterase. In yet another aspect, the enzyme composition comprises an arabinogalactanase (e.g., α-L-arabinogalactanase). In yet another aspect, the enzyme composition comprises an arabinofuranase (e.g., α-L-arabinofuranase). In yet another aspect, the enzyme composition comprises a coumarin esterase. In yet another aspect, the enzyme composition comprises a ferulic esterase. In yet another aspect, the enzyme composition comprises a galactosidase (e.g., α-galactosidase and / or β-galactosidase). In yet another aspect, the enzyme composition comprises a glucuronidase (e.g., α-D-glucuronidase). In yet another aspect, the enzyme composition comprises a glucuronidase. In yet another aspect, the enzyme composition comprises a mannanase. In yet another aspect, the enzyme composition comprises a mannosidase (e.g., β-mannosidase). In another aspect, the enzyme composition comprises a xylanase. In a preferred aspect, the xylanase is a family 10 xylanase. In another preferred aspect, the xylanase is a family 11 xylanase. In yet another aspect, the enzyme composition comprises a xylosidase (e.g., β-xylosidase).

[0375] In another aspect, the enzyme composition comprises an esterase. In another aspect, the enzyme composition comprises an expanding protein. In another aspect, the enzyme composition comprises a lignin-degrading enzyme. In a preferred aspect, the lignin-degrading enzyme is a manganese peroxidase. In another preferred aspect, the lignin-degrading enzyme is a lignin peroxidase. In another preferred aspect, the lignin-degrading enzyme is an H₂O₂-producing enzyme. In another aspect, the enzyme composition comprises a pectinase. In another aspect, the enzyme composition comprises a redox enzyme. In another preferred aspect, the redox enzyme is a catalase. In another preferred aspect, the redox enzyme is a laccase. In another preferred aspect, the redox enzyme is a peroxidase. In another aspect, the enzyme composition comprises a protease. In another aspect, the enzyme composition comprises an expanding protein.

[0376] In the method of the present invention, one or more enzymes may be added before or during saccharification, saccharification and fermentation, or fermentation.

[0377] One or more (e.g., several) components of an enzyme composition may be native proteins, recombinant proteins, or combinations of native and recombinant proteins. For example, one or more (e.g., several) components may be native proteins of a cell that can be used as a host cell to recombinantly express one or more (e.g., several) other components of the enzyme composition. It should be understood that the recombinant protein may be heterologous (e.g., exogenous) and native to the host cell. One or more (e.g., several) components of an enzyme composition may be generated as single components and then combined to form the enzyme composition. The enzyme composition may be a combination of multi-component and single-component protein formulations.

[0378] The enzymes used in the methods of this invention can be in any suitable form, such as fermentation broth formulations or cell compositions, cell lysates with or without cell debris, semi-purified or purified enzyme preparations, or host cells as the source of the enzyme. The enzyme composition can be a dry powder or granules, dust-free particles, a liquid, a stabilized liquid, or a stabilized protected enzyme. Liquid enzyme preparations can be stabilized according to established methods, for example, by adding stabilizers such as sugars, sugar alcohols, or other polyols and / or lactic acid.

[0379] The optimal amount of enzyme and GH61 polypeptide variant depends on several factors, including but not limited to: the mixture of cellulase and / or hemicellulose-degrading enzyme, the cellulose material, the concentration of the cellulose material, one or more pretreatments of the cellulose material, temperature, time, pH, and the inclusion of fermentation organisms (e.g., for simultaneous saccharification and fermentation).

[0380] In one aspect, the effective amount of cellulase or hemicellulase for cellulosic material is about 0.5 to about 50 mg per gram of cellulosic material, for example, about 0.5 to about 40 mg, about 0.5 to about 25 mg, about 0.75 to about 20 mg, about 0.75 to about 15 mg, about 0.5 to about 10 mg, or about 2.5 to about 10 mg.

[0381] In another aspect, the effective amount of the GH61 peptide variant for cellulose material is about 0.01 to about 50.0 mg per gram of cellulose material, for example, about 0.01 to about 40 mg, about 0.01 to about 30 mg, about 0.01 to about 20 mg, about 0.01 to about 10 mg, about 0.01 to about 5 mg, about 0.025 to about 1.5 mg, about 0.05 to about 1.25 mg, about 0.075 to about 1.25 mg, about 0.1 to about 1.25 mg, about 0.15 to about 1.25 mg, or about 0.25 to about 1.0 mg.

[0382] In another aspect, the effective amount of the GH61 polypeptide variant against cellulase or hemicellulase is about 0.005 to about 1.0 g per gram of cellulase or hemicellulase, for example, about 0.01 to about 1.0 g, about 0.15 to about 0.75 g, about 0.15 to about 0.5 g, about 0.1 to about 0.5 g, about 0.1 to about 0.25 g, or about 0.05 to about 0.2 g.

[0383] Polypeptides with cellulase or hemicellulase activity, as well as other proteins / peptides with the ability to degrade cellulose materials (collectively referred to below as "enzymatically active polypeptides"), can be derived or obtained from any suitable source, including archaea, bacteria, fungi, yeast, plants, or mammals. The term "obtained" herein also means that the enzyme has been recombined in a host organism using the methods described herein, wherein the recombinant enzyme may be native or exogenous to the host organism, or have a modified amino acid sequence, such as having one or more (e.g., several) deleted, inserted, and / or substituted amino acids, i.e., a recombinant enzyme that is a mutant and / or fragment of the native amino acid sequence, or an enzyme generated by nucleic acid shuffling processes known in the art. The term "native enzyme" includes naturally occurring variants, while the term "exogenous enzyme" includes variants such as those obtained through site-directed mutagenesis or shuffling.

[0384] The enzymatic polypeptide can be a bacterial polypeptide. For example, the polypeptide can be a Gram-positive bacterial polypeptide with enzymatic activity, such as polypeptides from Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium, Bacillus terrestris, Caldicellulosiruptor, Acidothermus, Thermobifidia, or Oceanobacillus; or a Gram-negative bacterial polypeptide with enzymatic activity, such as polypeptides from Escherichia coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, or Ureaplasma.

[0385] The enzymatically active polypeptide can also be a fungal polypeptide, and more preferably a yeast polypeptide, such as polypeptides from the genera *Candida*, *Kluyveromyces*, *Pichia pastoris*, *Saccharomyces*, *Schizosaccharomyces*, and *Yarlonia*; or more preferably, polypeptides from filamentous fungi, such as polypeptides from the genera *Apocytosporum*, *Larix*, *Cladosporium*, *Aspergillus*, *Briefomus*, *Botryospaeria*, *Pseudomonas*, *Trichoderma*, *Aureospora*, *Ceratophyllum*, *Ceratophyllum*, *Ceratophyllum*, *Coprinus*, *Termites*, *Ceratophyllum*, *Pseudomonas*, *Cryptococcus*, *Monosporium*, *Auricularia*, *Solanum*, *Fusarium*, and *Gibberellinia*. The following genera are listed: *Heterobacterium*, *Pyrophyllus*, *Phyllostachys*, *Lentinula*, *Schlumbergera*, *Melanocarpus*, *Grey Tree Fungus*, *Mucor*, *Hydrophyllus*, *Neurospora*, *Penicillium*, *Penicillium*, *Pyrophyllus*, *Poitrasia*, *Pseudotrichonympha*, *Rhizopus*, *Schizophyllum*, *Cyclophorus*, *Heterophyllus*, *Clostridium*, *Clostridium*, *Clostridium*, *Pleurotus*, or *Carya* polypeptides.

[0386] In one respect, the polypeptide is a polypeptide from *Karlsbergia latifolia*, *Saccharomyces cerevisiae*, *Saccharomyces sacchariformis*, *Saccharomyces douglas*, *Klufers*, *Nordimium*, or *Ovozymes*.

[0387] On another front, polypeptides are enzymatically active fungi such as *Apertiliomyces fibronectinus*, *Aspergillus echinosporus*, *Aspergillus avocado*, *Aspergillus fumigatus*, *Aspergillus sulphureus*, *Aspergillus japonicus*, *Aspergillus nidus*, *Aspergillus niger*, *Aspergillus oryzae*, *Chrysosporium lucknowense*, *Chrysosporium merdarium*, *Chrysosporium inops*, *Chrysosporium queenslandicum*, and *Chrysosporium moniliforme*. zonatum), Fusarium moniliforme, Fusarium graminearum, Fusarium kuwai, Fusarium chrysogenum, Fusarium graminearum, Fusarium graminearum, Fusarium graminearum, Fusarium moniliforme ... peruviana), fusarium spores, fusarium spores, heat-resistant fusarium spores, fusarium terrestris, Trichoderma harzianum, Trichoderma corningensis, Trichoderma longibranchii, Trichoderma reesei, Trichoderma viride, or cystic long hairy disc polypeptide.

[0388] Chemically modified or protein-engineered mutants of peptides with enzymatic activity can also be used.

[0389] One or more (e.g., several) components of the enzyme composition can be recombinant components, i.e., produced by cloning a DNA sequence encoding a single component, subsequently transforming cells with that DNA sequence, and then expressing it in a host (see, for example, WO 91 / 17243 and WO 91 / 17244). The preferred host is a heterologous host (the enzyme is heterologous to the host), but under certain conditions, the host can also be a homologous host (the enzyme is native to the host). Single-component cellulose-degrading proteins can also be prepared by purifying such a protein from fermentation broth.

[0390] In one aspect, one or more (e.g., several) cellulases include commercial cellulase formulations. Examples of commercial cellulase formulations suitable for use in this invention include, for example: CTec (Novozymes A / S) CTec2 (Novozymes A / S) CTec3 (Novozymes A / S), CELLUCLAST TM (Novozymes A / S Company), NOVOZYM TM 188 (Novozymes A / S), SPEZYME TM CP (Genencor Int.), ACCELERASE TM TRIO (DuPont) NL (DSM Company); S / L 100 (DSM Company), ROHAMENT TM 7069W (ROHM Company) GmbH), or CMAX3 TM (Dyadic International, Inc.) The effective amount of cellulase added is from about 0.001 to about 5.0 wt.% in solids, for example, from about 0.025 to about 4.0 wt.% in solids, or from about 0.005 to about 2.0 wt.% in solids.

[0391] Examples of bacterial endoglucanases that can be used in the methods of this invention include, but are not limited to: Acidothermus cellulolyticus endoglucanase (WO 91 / 05039; WO 93 / 15186; U.S. Patent Application No. 5,275,944; WO 96 / 02551; U.S. Patent Application No. 5,536,655, WO 00 / 70031, WO 05 / 093050), Erwinia carotovara endoglucanase (Saarilahti et al., 1990, Gene 90:9-14), Thermobifida fusca endoglucanase III (WO 05 / 093050), and Thermobifida Fusca) Endoglucanase V (WO05 / 093050).

[0392] Examples of fungal endoglucanases that can be used in this invention include, but are not limited to: *Trichoderma reesei* endoglucanase I (Penttila et al., 1986, gene 45:253-263, *Trichoderma reesei* Cel7B endoglucanase I (GenBank: M15665); *Trichoderma reesei* endoglucanase II (Saloheimo et al., 1988, gene 63:11-22, *Trichoderma reesei* Cel5B endoglucanase I (GenBank: M15665); *Trichoderma reesei* Cel5B endoglucanase II (Saloheimo et al., 1988, gene 63:11-22, *Trichoderma reesei* Cel5B endoglucanase I (GenBank: M15665); Endoglucanase II (GenBank: M19373); *Trichoderma reesei* endoglucanase III (Okada et al., 1988, Applied and Environmental Microbiology, 64:555-563, GenBank: AB003694); *Trichoderma reesei* endoglucanase V (Salohemo et al., 1994, Molecular Microbiology). Microbiology (l3:219-228, GenBank: Z33381); Aspergillus echinosporum endoglucanase (Huang et al., 1990, Nucleic Acid Research l8:5884); Aspergillus kawachii endoglucanase (Sakamoto et al., 1995, Current Genetics 27:435-439); Fusarium oxysporum endoglucanase (GenBank: L29381); Humicola grisea var. thermoidea endoglucanase (GenBank: AB003107); Melanocarpus thermophilus Endoglucanases from *Albomyces* (GenBank: MAL515703); *Neurospora crassa* (GenBank: XM_324477); *Potassium Specificum* endoglucanase V; *Tricholoma thermophilum* CBS117.65 endoglucanase; *Thermophilum aureum* endoglucanase I (GenBank: AF487830); and *Trichoderma reesei* strain VTT-D-80133 endoglucanase (GenBank: M15665).

[0393] Examples of cellobiose hydrolases that can be used in this invention include, but are not limited to: *Aspergillus echinosporum* cellobiose hydrolase II (WO 2011 / 059740), *Chaetomium thermophilum* cellobiose hydrolase I, *Chaetomium thermophilum* cellobiose hydrolase II, *Humicola insolens* cellobiose hydrolase I, *Myceliophthora thermophila* cellobiose hydrolase II (WO2009 / 042871), *Penicillium occitanis* cellobiose hydrolase I (GenBank: AY690482), *Emersonia spp.* cellobiose hydrolase I (GenBank: AF439936), *Thielavia hyrcanie* cellobiose hydrolase II (WO2010 / 141325), and *Thielavia hyrcanie*. The following are cellobiase II (CEL6A, WO2006 / 074435) from *Trichoderma reesei*, cellobiase I from *Trichoderma reesei*, cellobiase II from *Trichodermareesei*, and cellobiase II from *Trichophaea saccata* (WO 2010 / 057086).

[0394] Examples of β-glucosidases that can be used in this invention include, but are not limited to, β-glucosidases derived from: *Aspergillus echinosporum* (Kawaguchi et al., 1996, gene 173:287-288), *Aspergillus fumigatus* (WO 2005 / 047499), *Aspergillus niger* (Dan et al., 2000, Journal of Biochemistry 275:4973-4980), *Aspergillus oryzae* (WO 02 / 095014), *Penicillium brasilianum* IBT 20888 (WO 2007 / 019442 and WO 2010 / 088387), *Clostridium terrestris* (WO 2011 / 035029), and *Clostridium sacchariformis* (WO 2007 / 019442).

[0395] β-glucosidase can be a fusion protein. In one aspect, β-glucosidase is a BG fusion protein of an Aspergillus oryzae β-glucosidase variant (WO 2008 / 057637) or an Aspergillus oryzae β-glucosidase fusion protein (WO 2008 / 057637).

[0396] Other useful endoglucanases, cellobiases, and β-glucosidases are disclosed in many families of glycosyl hydrolases classified according to the following literature: Henrissat, 1991, Biochem.J. 280: 309-316, and Henrissat and Bairoch, 1996, Biochem.J. 316: 695-696.

[0397] Other cellulases that can be used in this invention are described in the following references: WO 98 / 13465, WO 98 / 015619, WO 98 / 015633, WO 99 / 06574, WO 99 / 10481, WO 99 / 025847, WO 99 / 031255, WO2002 / 101078, WO 2003 / 027306, WO 2003 / 052054, WO 2003 / 052055, WO 2003 / 052056, WO2003 / 052057, WO 2003 / 052118, WO 2004 / 016760, WO 2004 / 043980, WO WO 2004 / 048592, WO 2005 / 001065, WO 2005 / 028636, WO 2005 / 093050, WO 2005 / 093073, WO 2006 / 074005, WO 2006 / 117432, WO 2007 / 071818, WO 2007 / 071820, WO 2008 / 008070, WO 2008 / 008793, U.S. Patent No. 5,457,046, U.S. Patent No. 5,648,263, and U.S. Patent No. 5,686,593.

[0398] In the method of the present invention, any GH61 polypeptide having cellulolytic-enhancing activity can be used as a component of the enzyme composition.

[0399] Examples of GH61 peptides that can be used in the methods of this invention include, but are not limited to, GH61 peptides derived from the following: *Thielavia terrestris* (WO 2005 / 074647, WO 2008 / 148131 and WO2011 / 035027), *Thermophila aurea* (WO 2005 / 074656 and WO 2010 / 065830), *Trichoderma reesei* (WO 2007 / 089290), *Myceliophthora thermophila* (WO 2009 / 085935, WO 2009 / 085859, WO 2009 / 085864 and WO 2009 / 085868), and *Aspergillus fumigatus* (WO 2009 / 085935, WO 2009 / 085859, WO 2009 / 085864 and WO 2009 / 085868). 2010 / 138754), *Penicillium pinophilum* (WO2011 / 005867), *Thermoascus sp.* (WO 2011 / 039319), *Penicillium sp.* (WO 2011 / 041397), *Thermoascus crustaceous* (WO 2011 / 041504), *Aspergillus echinosporum* (WO 2012 / 125925), *Thermoascus crustaceous* (WO 2012 / 113340, WO 12 / 129699, and WO2012 / 130964), *Aurantiporus alborubescens* (WO 2012 / 122477), *Discocele cystis* (WO 2012 / 122477), *Penicillium tommex* (WO 2010 / 138754), *Penicillium pinophilum* (WO 2011 / 005867), *Thermoascus sp.* (WO 2011 / 039319), *Penicillium sp.* (WO 2011 / 041397), *Thermoascus crustaceous* (WO 2011 / 041504), *Aspergillus echinosporum* (WO 2012 / 122477), *Aurantiporus alborubescens* (WO 2010 / 138754), *Aurantiporus alborubescens* (WO 2010 / 122477), *Penicillium tommex* (WO 2010 / 122477), *Aurantiporus alborubescens* (WO 2010 / 122477), *Aurantiporus alborubescens* ( 2012 / 122477), *Talaromyces leycettanus* (WO 2012 / 135659), *Pseudomonas aeruginosa* (WO 2012 / 146171), *Talaromyces leycettanus* (WO 2012 / 101206), and *Talaromyces leycettanus* (WO 2012 / 101206).

[0400] In one aspect, GH61 peptide variants and GH61 peptides with cellulose-enhancing activity are used in the presence of soluble activated divalent metal cations (e.g., manganese or copper) according to WO2008 / 151043.

[0401] In another aspect, GH61 peptide variants and GH61 peptides with cellulose-degrading-enhancing activity are used in the presence of dioxins, bicyclic compounds, heterocyclic compounds, nitrogen-containing compounds, quinone compounds, sulfur-containing compounds, or liquids obtained from pretreated cellulose materials (such as pretreated corn stalks) (WO 2012 / 021394, WO 2012 / 021395, WO2012 / 021396, WO 2012 / 021399, WO2012 / 021400, WO 2012 / 021401, WO 2012 / 021408, and WO2012 / 021410).

[0402] In one aspect, such a compound is added at the following molar ratio of the compound to glucosyl units of cellulose: approximately 10 -6 Up to approximately 10, for example, approximately 10 -6 Up to approximately 7.5, approximately 10 -6 Up to approximately 5, approximately 10 -6 Up to approximately 2.5, approximately 10 -6 To approximately 1, approximately 10 -5 To approximately 1, approximately 10 -5 Up to approximately 10 -1 Approximately 10 -4 Up to approximately 10 -1 Approximately 10 -3 Up to approximately 10 -1 or about 10 -3 Up to approximately 10 -2 In another aspect, the effective amount of such a compound is about 0.1 μM to about 1 M, for example about 0.5 μM to about 0.75 M, about 0.75 μM to about 0.5 M, about 1 μM to about 0.25 M, about 1 μM to about 0.1 M, about 5 μM to about 50 mM, about 10 μM to about 25 mM, about 50 μM to about 25 mM, about 10 μM to about 10 mM, about 5 μM to about 5 mM, or about 0.1 mM to about 1 mM.

[0403] The term "liquor" refers to the solution phase (aqueous phase, organic phase, or a combination thereof) and its soluble contents resulting from the treatment of lignocellulose and / or hemicellulose material, or its monosaccharides (e.g., xylose, arabinose, mannose, etc.) in a pulp, under the conditions described herein. A liquid for enhancing the decomposition of GH61 polypeptide fibers can be generated by treating the lignocellulose or hemicellulose material (raw material) with heat and / or pressure, optionally in the presence of a catalyst such as an acid, optionally in the presence of an organic solvent, and optionally in combination with physical degradation of the material, followed by separation of the solution from the residual solids. The degree of enhanced cellulose decomposition resulting from the combination of the liquid and the GH61 polypeptide during the hydrolysis of cellulose substrates by cellulase preparations is determined by these conditions. The liquid can be separated from the treated material using standard methods in the art, such as filtration, precipitation, or centrifugation.

[0404] In one respect, the effective amount of liquid for cellulose is approximately 10 gram of cellulose per gram of cellulose. -6 Up to approximately 10g, for example, approximately 10g of cellulose per gram. -6 Up to approximately 7.5g, approximately 10 -6 Up to approximately 5g, approximately 10 -6 Up to approximately 2.5g, approximately 10 -6 Up to about 1g, about 10 -5 Up to about 1g, about 10 -5 Up to approximately 10 -1 g, approximately 10 -4 Up to approximately 10 -1 g, approximately 10 -3 Up to approximately 10 -1 g, or about 10 g -3 Up to approximately 10 -2 g.

[0405] In one aspect, one or more (e.g., several) hemicellulose-degrading enzymes comprise commercial hemicellulose-degrading enzyme formulations. Examples of commercial hemicellulose-degrading enzyme formulations suitable for use in this invention include, for example, SHEARZYME. TM (Novozymes A / S) HTec (Novozymes A / S) HTec2 (Novozymes A / S) HTec3 (Novozymes A / S) (Novozymes A / S) (Novozymes A / S) HC (Novozymes A / S), Xylanase (Genergen) XY (Genentech) XC (Genetronics) TX-200A (AB Enzymes), HSP 6000 xylanase (DSM), DEPOL TM 333P (Biocatalysts Limit, Wales, UK) and DEPOL TM 740L. (Biocatalyst Ltd, Wales, UK) and DEPOL TM 762P (Biocatalyst Ltd, Wales, UK), ALTERNA FUEL100P (Dyadic), and ALTERNA FUEL 200P (Dyadic).

[0406] Examples of xylanases useful in the methods of the present invention include, but are not limited to, xylanases derived from: *Aspergillus aculeatus* (gene SeqP: AAR63790; WO 94 / 21785), *Aspergillus fumigatus* (WO 2006 / 078256), *Penicillium pinophilum* (WO2011 / 041405), *Penicillium sp.* (WO 2010 / 126772), *Talaromyces thermophilus* GH11 (WO2012 / 130965), *Talaromyces thermophilus* GH11 (WO 2012 / 13095), *Thielavia terrestris* NRRL 8126 (WO 2009 / 079210), and *Trichophaea*. saccata)GH10 (WO 2011 / 057083).

[0407] The β-xylosidases useful in the methods of the present invention include, but are not limited to, β-xylosidases from: Neurospora crassa (SwissProt:Q7SOW4), Trichoderma reesei (UniProtKB / TrEMBL:Q92458), Talaromyces emersonii (SwissProt:Q8X212), and Talaromyces GH11 (WO 2012 / 13095).

[0408] Examples of acetylated xylan esterases useful in the methods of the present invention include, but are not limited to, acetylated xylan esterases from: *Aspergillus aculeatus* (WO 2010 / 108918), *Chaetomium globosum* (UniProt:Q2GWX4), *Chaetomium gracile* (GeneSeqP:AAB82124), *Humicola insolens* DSM 1800 (WO 2009 / 073709), *Hypocrea jecorina* (WO 2005 / 001036), *Myceliophtera thermophila* (WO2010 / 014880), *Neurospora crassa* (UniProt:q7s259), and *Phaeosphaeria*. nodorum (UniProt:Q0UHJ1) and Thievaria terrestris NRRL 8126 (WO 2009 / 042846).

[0409] Examples of ferulic esterases used in the methods of the present invention include, but are not limited to, ferulic esterases derived from: Humicola insolens DSM 1800 (WO 2009 / 076122), Neosartorya fischeri (UniProt:A1D9T4), Neurospora crassa (UniProt:Q9HGR3), Penicillium aurantiogriseum (WO 2009 / 127729), and Thievaria terrestris (WO 2010 / 053838 and WO 2010 / 065448).

[0410] Examples of arabinofuranosidases useful in the methods of the present invention include, but are not limited to, arabinofuranosidases derived from: Aspergillus niger (GeneSeqP:AAR94170), Humicola insolens DSM 1800 (WO 2006 / 114094 and WO 2009 / 073383), and M. giganteus (WO 2006 / 114094).

[0411] Examples of α-glucuronidases useful in the methods of the present invention include, but are not limited to, α-glucuronidases derived from: *Aspergillus clavatus* (UniProt:alcc12), *Aspergillus fumigatus* (SwissProt:Q4WW45), *Aspergillus niger* (UniProt:Q96WX9), *Aspergillus terreus* (SwissProt:Q0CJP9), *Humicolainsolens* (WO 2010 / 014706), *Penicillium chrysogenum* (WO 2009 / 068565), *Emersonia spp.* (UniProt:Q8X211), and *Trichoderma reesei* (UniProt:Q99024).

[0412] Examples of oxidoreductases useful in the methods of this invention include, but are not limited to: catalase from *Aspergillus fumigatus*, catalase from *Aspergillus niger*, catalase from *Aspergillus oryzae*, catalase from *Potassium spp.*, catalase from *Neurospora crassa*, catalase from *Penicillium emersonii*, catalase from *Scytalidium thermophilum*, catalase from *Stenocystis aegyptiacus*, catalase from *Thermophilus aureus*, laccase from *Coprinus cinereus*, laccase from *Streptomyces coelicolor*, peroxidase from *Coprinus cinereus*, soybean peroxidase, and *Royal palmatum*. palm peroxidase.

[0413] The enzymatically active polypeptides used in the process of this invention can be produced by fermentation of the microbial strains described above using a procedure known in the art on a nutrient medium containing suitable carbon and nitrogen sources and inorganic salts (see, for example, Bennett, JW, and LaSure, L. (ed.), More Gene Manipulations in Fungi, Academic Press, California, 1991). Suitable culture media are available from commercial suppliers or can be prepared according to publicly available compositions (e.g., in the U.S. Type Culture Collection catalogue). Suitable temperature ranges and other conditions for growth and enzyme production are known in the art (see, for example, Bailey, JE, and Ollis, DF, Biochemical Engineering Fundamentals, McGraw-Hill Book Company, New York, 1986).

[0414] Fermentation can be any method of cultured cells that results in the expression or isolation of an enzyme or protein. Therefore, fermentation can be understood as including shake-flask culture, or small-scale or large-scale fermentation (including continuous fermentation, batch fermentation, fed-batch fermentation, or solid-state fermentation) in a suitable culture medium and under conditions that allow for the expression or isolation of the enzyme in a laboratory or industrial fermenter. The resulting enzymes can be recovered from the fermentation medium and purified using standard procedures.

[0415] Fermentation. Fermentable sugars obtained from hydrolyzed cellulose material can be fermented using one or more (e.g., several) fermenting microorganisms capable of directly or indirectly fermenting sugars into desired fermentation products. "Fermentation" or "fermentation process" refers to any fermentation process or any process that includes a fermentation step. Fermentation processes also include those used in the consumer alcohol industry (e.g., beer and wine), the dairy industry (e.g., fermented dairy products), the leather industry, and the tobacco industry. Fermentation conditions depend on the desired fermentation product and the fermenting organism, and can be readily determined by those skilled in the art.

[0416] In the fermentation step, sugars released from cellulose material are fermented into products such as ethanol by a fermenting organism, such as yeast, as a result of pretreatment and enzymatic hydrolysis. Hydrolysis (saccharification) and fermentation can be separate or simultaneous.

[0417] In the fermentation steps of this invention, any suitable hydrolyzed cellulose material can be used. The material is generally selected based on economics, i.e., the cost per unit sugar potential, and the recalcitrantness to enzymatic conversion.

[0418] The term “fermentation medium” can be understood here as a medium prior to the addition of one or more fermenting microorganisms, such as a medium produced by a saccharification process, and a medium used in a simultaneous saccharification and fermentation (SSF) process.

[0419] "Fermentation microorganism" refers to any microorganism, including bacterial and fungal organisms, suitable for a desired fermentation process to produce a fermentation product. Fermentation organisms can be hexose and / or pentose fermentation organisms, or combinations thereof. Both hexose and pentose fermentation organisms are well known in the art. Suitable fermentation microorganisms are capable of fermenting (i.e., converting) sugars (e.g., glucose, xylose, xylulose, arabinose, maltose, mannose, galactose, and / or oligosaccharides) directly or indirectly into the desired fermentation product. Examples of bacterial and fungal fermentation organisms for ethanol production are described by Lin et al., 2006, Applied Microbiology and Biotechnology 69:627-642.

[0420] Examples of fermenting microorganisms capable of fermenting hexoses include bacterial and fungal organisms, such as yeast. Yeasts include strains of the genera *Candida*, *Kluyveromyces*, and *Saccharomyces*, such as *Candida sonorensis*, *Kluyveromyces martensii*, and *Saccharomyces cerevisiae*.

[0421] Examples of fermentative microorganisms capable of fermenting pentoses in their native state include bacterial and fungal organisms, such as some yeasts. Yeasts that ferment xylose include strains of the genus *Candida*, preferably *Candida sheatae* or *Candida sonorensis*; and strains of the genus *Pichia*, such as *Pichia stylosa*, like *Pichia stylosa* CBS 5773. Yeasts that ferment pentoses include strains of the genus *Pichia*, preferably *Pichia tannophilus*. Organisms that cannot ferment pentoses (such as xylose and arabinose) can be genetically modified to ferment pentoses using methods known in the art.

[0422] Examples of bacteria that can effectively ferment hexoses and pentoses into ethanol include, for example, Bacillus coagulans, Clostridium acetobutyricum, Clostridium thermophilum, Clostridium phytofermentans, species of Bacillus terrestris, Thermoanaerobacter saccharolyticum, and motile fermentation monoclonal bacteria (Philippidis, 1996, ibid.).

[0423] Other fermenting organisms include strains of the genus *Bacillus*, such as *Bacillus coagulans*; strains of the genus *Candida*, such as *Candida sonorensis*, *Candida methanosa*, *Candida didensia*, *Candida parapsilosis*, *Candida naedodendra*, *Candida bronkii*, *Candida entomophilia*, *Candida brassicae*, *Candida tropicalis*, *Candida booidinii*, *Candida utilis*, and *Candida shurata*; and strains of the genus *Clostridium*, such as *Clostridium acetone-butanol*, *Candida thermocellum*, and *Clostridium difficile*. *Phytofermentans*; *Escherichia coli*, especially genetically modified strains to improve ethanol production; *Bacillus* species; *Hansenula*, such as *Hansenula aberrant*; *Klebsiella*, such as *Klebsiella acidogenetica*; *Kluyveromyces*, such as *Kluyveromyces martensii*, *Kluyveromyces lactis*, *Kluyveromyces thermostableis*, and *Kluyveromyces brittleis*; *Schizosaccharizoa*, such as *Schizosaccharizoa pombe*; *Thermoanaerobacter accharolyticum*; and fermentation monopores, such as *Fermentomonas motiformis*.

[0424] Commercially available yeasts suitable for ethanol production include, for example, BIOFERM. TM AFT and XR (NABC - North American Bioproducts Corporation, Georgia, USA), Ethanol Red Yeast (ETHANOLRED) TM yeast (Fermentis / Lesaffre, USA), FALI TM (Fleischmann's Yeast Company, USA), FERMIOL TM (DSM Specialties), GERT STRAND TM (Gert Strand AB, Sweden), and SUPERSTARTTM and THERMOSACC TM Fresh yeast (Ethanol Technology, Wisconsin, USA).

[0425] In one respect, fermenting microorganisms have been genetically modified to provide the ability to ferment pentoses, such as microorganisms that utilize xylose, microorganisms that utilize arabinose, and microorganisms that utilize both xylose and arabinose.

[0426] Heterologous genes have been cloned into various fermenting microorganisms to construct organisms capable of converting hexoses and pentoses into ethanol (co-fermentation) (Chen and Ho, 1993, Applied Biochemistry and Biotechnology, 39-40:135-147; Ho et al., 1998, Applied and Environmental Microbiology, 64:1852-1859; Kotter and Ciriacy, 1993, Applied Microbiology and Biotechnology, 38:776-783; Walfridsson et al., 1995, Applied and Environmental Microbiology, 61:4184-4190; Kuyper et al., 2004, FEMS Yeast Research). Research) 4:655-664; Beall et al., 1991, Biotechnology and Bioengineering (Biotech.Bioeng.) 38:296-303; Ingram et al., 1998, Biotechnology and Bioengineering 58:204-214; Zhang et al., 1995, Science 267:240-243; Deanda et al., 1996, Applied and Environmental Microbiology 62:4465-4470; WO 2003 / 062430).

[0427] As is well known in the art, the organisms described above can also be used to produce other substances, as described herein.

[0428] Typically, fermentative microorganisms are added to degraded cellulose materials or hydrolysates and fermentation is carried out for approximately 8 to approximately 96 hours, for example, approximately 24 to approximately 60 hours. The temperature is typically between approximately 26°C and approximately 60°C, for example, approximately 32°C or 50°C, and the pH is between approximately 3 and approximately 8, for example, pH 4-5, 6, or 7.

[0429] In one aspect, yeast and / or another microorganism are applied to degraded cellulose material and fermentation is carried out for approximately 12 to approximately 96 hours, typically 24-60 hours. In another aspect, the temperature is preferably between approximately 20°C and approximately 60°C, for example, approximately 25°C to approximately 50°C, approximately 32°C to approximately 50°C, or approximately 32°C to approximately 50°C, and the pH is generally from approximately pH 3 to approximately pH 7, for example, approximately pH 4 to approximately pH 7. However, some fermenting organisms, such as bacteria, have optimal fermentation temperatures. A temperature of approximately 10... 5 Up to 10 12 The amount is preferably from about 10 7 Up to 10 10 Especially about 2 x 10 8 Live cell count / ml fermentation broth using yeast or another microorganism. Further guidance on the use of yeast for fermentation can be found, for example, in "The Alcohol Textbook" (edited by T.P. Lyons and D.R. Kelsall, Nottingham University Press, UK), which is hereby incorporated by reference.

[0430] Fermentation stimulants can be used in combination with any of the methods described herein to further improve fermentation methods, particularly the performance of fermenting microorganisms, such as increasing speed and ethanol yield. "Fermentation stimulant" refers to an agent used to stimulate the growth of fermenting microorganisms (especially yeast). Preferred fermentation stimulants for growth include vitamins and minerals. Examples of vitamins include multivitamins, biotin, pantothenate (esters), niacin, meso-inositol, thiamine, pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and vitamins A, B, C, D, and E, for example, see Alfenore et al., "Improving ethanol production and viability of Saccharomyces cerevisia by a vitamin feeding strategy during fed-batch process," Springer-Verlag (2002), which is incorporated herein by reference. Examples of minerals include minerals and mineral salts that can supply nutrients containing P, K, Mg, S, Ca, Fe, Zn, Mn and Cu.

[0431] Fermentation products:Fermentation products can be any substance obtained from fermentation. Fermentation products can be, but are not limited to, an alcohol (e.g., arabinitol, n-butanol, isobutanol, ethanol, glycerol, methanol, ethylene glycol, 1,3-propanediol [propylene glycol], butanediol, glycerin, sorbitol, and xylitol); an alkane (e.g., pentane, hexane, heptane, octane, nonane, decane, undecane, and dodecane); a cycloalkane (e.g., cyclopentane, cyclohexane, cycloheptane, and cyclooctane); an alkene (e.g., pentene, hexene, heptene, and octene); or an amino acid (e.g., aspartic acid, glutamic acid, etc.). Fermentation products include amino acids (glycine, lysine, serine, and threonine); a gas (e.g., methane, hydrogen (H2), carbon dioxide (CO2), and carbon monoxide (CO)); isoprene; a ketone (e.g., acetone); an organic acid (e.g., acetic acid, acetoic acid, adipic acid, ascorbic acid, citric acid, 2,5-diketo-D-gluconic acid, formic acid, fumaric acid, gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid, oxaloacetic acid, propionic acid, succinic acid, and xylic acid); and polyketide compounds. Fermentation products can also be proteins that are high-value products.

[0432] In a preferred aspect, the fermentation product is an alcohol. It will be understood that the term "alcohol" encompasses substances containing one or more hydroxyl moieties. Alcohols can be, but are not limited to: n-butanol, isobutanol, ethanol, methanol, arabinitol, butanediol, ethylene glycol, glycerin, glycerol, 1,3-propanediol, sorbitol, and xylitol. See, for example, Gong et al., 1999, Ethanol production from renewable resources, in Advances in Biochemical Engineering / Biotechnology, Skapier, T., ed., Springer Publishers Berlin-Heidelberg, Germany, 65:207-241; Silveira and Jonas, 2002, Appl. Microbiol. Biotechnol. 59:400-408; Nigam and Singh, 1995, Process Biochemistry 30(2):117-124; Ezeji et al., 2003, World Journal of Microbiology and Biotechnology 19(6):595-603.

[0433] In another preferred aspect, the fermentation product is an alkane. The alkane can be unbranched or branched. The alkane can be, but is not limited to: pentane, hexane, heptane, octane, nonane, decane, undecane, or dodecane.

[0434] In another preferred aspect, the fermentation product is a cycloalkane. The cycloalkane may be, but is not limited to, cyclopentane, cyclohexane, cycloheptane, or cyclooctane.

[0435] In another preferred aspect, the fermentation product is an olefin. This olefin can be unbranched or branched. The olefin can be, but is not limited to, pentene, hexene, hepten, or octene.

[0436] In another preferred aspect, the fermentation product is an amino acid. The organic acid may be, but is not limited to, aspartic acid, glutamic acid, glycine, lysine, serine, or threonine. See, for example, Richard and Margaritis, 2004, Biotechnology and Bioengineering 87(4):501-515.

[0437] In another preferred aspect, the fermentation product is a gas. The gas can be, but is not limited to, methane, H2, CO2, or CO. See, for example, Kataoka et al., 1997, Water Science and Technology 36(6-7):41-47; and Gunaseelan, 1997, Biomass and Bioenergy 13(1-2):83-114.

[0438] In another preferred aspect, the fermentation product is isoprene.

[0439] In another preferred aspect, the fermentation product is a ketone. It should be understood that the term "ketone" encompasses substances containing one or more ketone moieties. A ketone can be, but is not limited to, acetone.

[0440] In another preferred aspect, the fermentation product is an organic acid. The organic acid may be, but is not limited to: acetic acid, acetonic acid, adipic acid, ascorbic acid, citric acid, 2,5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid, propionic acid, succinic acid, or xylonic acid. See, for example, Chen and Lee, 1997, Biochem. Biotechnol. 63-65:435-448.

[0441] In another preferred aspect, the fermentation product is a polyketide compound.

[0442] Recycle One or more fermentation products can optionally be recovered from the fermentation medium using any method known in the art, including but not limited to chromatography, electrophoresis, differential solubility, distillation, or extraction. For example, alcohols can be separated and purified from fermented cellulose material by conventional distillation methods. Ethanol with a purity of up to about 96 vol.% can be obtained, which can be used as, for example, fuel ethanol, drinking ethanol (i.e., neutral drinking ethanol), or industrial ethanol.

[0443] Detergent composition

[0444] The present invention also relates to detergent compositions comprising the GH61 peptide variant of the present invention and a surfactant. The GH61 peptide variant of the present invention can be added to detergent compositions and thus become a component of the detergent composition.

[0445] The detergent compositions of the present invention can be formulated as, for example, hand-wash or machine-wash laundry detergent compositions, including a laundry additive composition suitable for pre-treating stained fabrics and a fabric softener composition added during rinsing, or formulated as detergent compositions for general household hard surface cleaning operations, or formulated for hand-wash or machine-wash dishwashing operations. In one aspect, the present invention also relates to a method for cleaning or washing hard surfaces or clothing, the method comprising contacting the hard surface or clothing with the detergent composition of the present invention.

[0446] In one particular aspect, the present invention provides a detergent additive comprising a GH61 polypeptide variant of the present invention. The detergent additive and the detergent composition may comprise one or more (e.g., several) enzymes selected from the group consisting of: amylase, arabinase, keratinase, carbohydrate enzyme, cellulase, galactanase, laccase, lipase, mannanase, oxidase, pectinase, peroxidase, protease, and xylanase.

[0447] Generally speaking, the characteristics of the selected one or more enzymes should be compatible with the selected detergent (i.e., optimal pH, compatibility with other enzymes and non-enzyme components, etc.), and the one or more enzymes should be present in an effective amount.

[0448] Cellulase Suitable cellulases include those of bacterial or fungal origin. This includes chemically modified or protein-engineered mutants. Suitable cellulases include those from the genera *Bacillus*, *Pseudomonas*, *Pyrophyllus*, *Fusarium*, *Clostridium*, and *Cladosporium*, such as fungal cellulases produced by *Pyrophyllus*, *Thermophyllus*, and *Fusarium* as disclosed in US 4,435,307, US 5,648,263, US 5,691,178, US 5,776,757, and WO 89 / 09259.

[0449] Particularly suitable cellulases are alkaline or neutral cellulases that offer color protection benefits. Examples of such cellulases are those described in EP 0 495 257, EP 0 531 372, WO 96 / 11262, WO 96 / 29397, and WO 98 / 08940. Other examples are cellulase variants, such as those described in WO 94 / 07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95 / 24471, WO 98 / 12307, and PCT / DK 98 / 00299.

[0450] Commercially available cellulases include CELLUZYME TM and CAREZYME TM (Novozymes), CLAZINASE TM and PURADAX HA TM (Genetronics International Limited), and KAC-500(B) TM (Kao Corporation).

[0451] Protease:Suitable proteases include those of animal, plant, or microbial origin. Microbial origin is preferred. This includes chemically modified or protein-engineered mutants. The protease can be a serine protease or a metalloproteinase, preferably an alkaline microbial protease or a trypsin-like protease. Examples of alkaline proteases are subtilisin proteases, especially those derived from Bacillus, such as subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147, and subtilisin 168 (described in WO 89 / 06279). Examples of trypsin-like proteases are trypsins (e.g., of porcine or bovine origin) and Fusarium proteases described in WO 89 / 06270 and WO 94 / 25583.

[0452] Examples of useful proteases are variants as described in WO 92 / 19729, WO 98 / 20115, WO 98 / 20116 and WO 98 / 34946, particularly variants in which substitution occurs at one or more of the following positions: 27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235, and 274.

[0453] Preferred commercially available proteases include ALCALASE TM SAVINASE TM PRIMASE TM DURALASE TM ESPERASE TM , and KANNASE TM (Novozymes), MAXATASE TM MAXACAL TM MAXAPEM TM PROPERASE TM PURAFECT TM PURAFECT OXP TM FN2 TM and FN3 TM (Genetronics International)

[0454] lipaseSuitable lipases include those of bacterial or fungal origin. This includes chemically modified or protein-engineered mutants. Examples of useful lipases include those from the following: *Pseudomonas* (synonymous with *Thermophilus*), such as those from *Pseudomonas latifolia* (*Thermophilus filamentosa*) as described in EP 258 068 and EP 305 216, or those from specific *Pseudomonas* species as described in WO 96 / 13580; *Pseudomonas* lipases, such as those from *Pseudomonas alkaloids* or *Pseudomonas alkaloids* (EP 218 272), *Pseudomonas cepacia* (EP 331 376), *Pseudomonas schrenckii* (GB 1,372,034), *Pseudomonas fluorescens*, *Pseudomonas* species strain SD 705 (WO 95 / 06720 and WO 96 / 27002), *Pseudomonas wisconsinii* (WO 13580). 96 / 12012); Bacillus lipases, such as those from Bacillus subtilis (Dartois et al., 1993, Biochemica et Biophysica Acta, 1131:253-360), thermophilic steatobacterium (JP 64 / 744992) or Bacillus pumilus (WO 91 / 16422).

[0455] Other examples of lipase variants are those described in WO 92 / 05249, WO 94 / 01541, EP 407 225, EP 260105, WO 95 / 35381, WO 96 / 00292, WO 95 / 30744, WO 94 / 25578, WO 95 / 14783, WO 95 / 22615, WO 97 / 04079, and WO 97 / 07202.

[0456] Preferred commercially available lipases include: LIPOLASE TM And LIPOLASE ULTRA TM (Novozymes)

[0457] amylase: Suitable amylases (α and / or β) include those of bacterial or fungal origin. This includes chemically modified or protein-engineered mutants. Amylases include, for example, α-amylases obtained from specific strains of Bacillus, such as Bacillus licheniformis, described in more detail in GB 1,296,839.

[0458] Examples of useful amylases are the variants described in WO 94 / 02597, WO 94 / 18314, WO 96 / 23873, and WO 97 / 43424, especially variants with substitutions at one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391, 408, and 444.

[0459] Commercially available amylase is DURAMYL TM TERMAMYL TM FUNGAMYL TM and BAN TM (Novozymes), Rapidase TM and PURASTAR TM (From Genentech International).

[0460] Peroxidase / oxidase: Suitable peroxidases / oxidases include those of plant, bacterial, or fungal origin. This includes chemically modified or protein-engineered mutants. Examples of useful peroxidases include peroxidases from *Coprinus comatus*, such as those from *Coprinus gracilistylus* and their variants, as described in WO 93 / 24618, WO 95 / 10602, and WO 98 / 15257.

[0461] Commercially available peroxidases include GUARDZYME TM (Novozymes)

[0462] One or more detergent enzymes can be included in a detergent composition by adding a single additive containing one or more (e.g., several) enzymes, or by adding an additive containing a combination of all these enzymes. The detergent additives of the present invention, i.e., individual additives or combined additives, can be formulated as, for example, granules, liquids, slurries, etc. Preferred detergent additive formulations are granules, especially non-dust granules; liquids, especially stabilized liquids; or slurries.

[0463] Non-dust-forming particles can be produced, such as those disclosed in US 4,106,991 and 4,661,452, and can optionally be coated using methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products (polyethylene glycol, PEG) having an average molar weight of 1,000 to 20,000; ethoxylated nonylphenol having 16 to 50 ethylene oxide units; ethoxylated fatty alcohols containing 12 to 20 carbon atoms and having 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and monoglycerides, diglycerides, and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application via fluidized bed technology are given in GB 1483591. Liquid enzyme preparations can be stabilized, for example, by adding polyols (such as propylene glycol), sugars or sugar alcohols, lactic acid, or boric acid according to established methods. Protected enzymes can be prepared according to the methods disclosed in EP238,216.

[0464] The detergent compositions of the present invention can be in any convenient form, such as strips, tablets, powders, granules, pastes, or liquids. Liquid detergents can be aqueous, typically containing up to 70% water and 0-30% organic solvents, or they can be non-aqueous.

[0465] The detergent composition contains one or more (e.g., several) surfactants, which may be nonionic (including semi-polar) and / or anionic and / or cationic and / or zwitterionic. These surfactants are typically present at levels of 0.1% to 60% by weight.

[0466] When it includes anionic surfactants, the detergent will typically contain about 1% to about 40% of anionic surfactants, such as linear alkylbenzene sulfonates, α-alkenyl sulfonates, alkyl sulfates (fatty alcohol sulfates), alcohol ethoxy sulfates, secondary alkyl sulfonates, α-sulfonyl fatty acid methyl esters, alkyl or alkenyl succinic acids, or soaps.

[0467] When it includes a nonionic surfactant, the detergent will typically contain about 0.2% to about 40% of a nonionic surfactant, such as alcohol ethoxide, nonylphenol ethoxide, alkyl polyglycoside, alkyl dimethylamine oxide, ethoxylated fatty acid monoethanolamine, fatty acid monoethanolamine, polyhydroxyalkyl fatty acid amide, or N-acyl N-alkyl derivatives of glucosamine (“glucosamide”).

[0468] The detergent may contain 0-65% detergent builders or complexing agents, such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitric acid triacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl or alkenyl succinic acid, soluble silicate, or layered silicate (e.g., SKS-6 from Hoechst).

[0469] The detergent may contain one or more (e.g., several) polymers. Examples include carboxymethyl cellulose, poly(vinylpyrrolidone), poly(ethylene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazolium), polycarboxylate (such as polyacrylate), maleic acid / acrylic acid copolymer, and lauryl methacrylate / acrylic acid copolymer.

[0470] Detergents may contain a bleaching system, which may include an H2O2 source, such as perboric acid or percarbonate, which may be combined with a bleaching activator that forms a peracid (such as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate). Alternatively, the bleaching system may include, for example, amides, imides, or sulfone-type peroxy acids.

[0471] One or more enzymes in the detergent compositions of the present invention can be stabilized using conventional stabilizers, such as polyols (e.g., propylene glycol or glycerol), sugars or sugar alcohols, lactic acid, boric acid, or boric acid derivatives (e.g., aromatic borate esters, or phenyl boric acid derivatives (e.g., 4-formylphenyl boric acid)), and the compositions can be formulated as described in, for example, WO 92 / 19709 and WO 92 / 19708.

[0472] The detergent may also contain other conventional detergent ingredients, such as fabric conditioners including clay, foam promoters, defoamers, corrosion inhibitors, suspending agents, anti-redeposition agents, dyes, bactericides, optical brighteners, water-soluble agents, dulling inhibitors, or fragrances.

[0473] In detergent compositions, the amount of any enzyme added can correspond to 0.01-100 mg of enzyme protein per liter of detergent solution, preferably 0.05-5 mg of enzyme protein per liter of detergent solution, and especially 0.1-1 mg of enzyme protein per liter of detergent solution.

[0474] In the detergent composition, the amount of the GH61 polypeptide variant of the present invention having cellulose-degrading and enhancing activity can correspond to 0.001-100 mg of protein per liter of detergent solution, preferably 0.005-50 mg of protein, more preferably 0.01-25 mg of protein, even more preferably 0.05-10 mg of protein, most preferably 0.05-5 mg of protein, and even most preferably 0.01-1 mg of protein.

[0475] The GH61 polypeptide variant of the present invention, which has enhanced cellulose decomposition activity, can also be incorporated into detergent formulations disclosed in WO 97 / 07202 (linked hereto by reference).

[0476] plant

[0477] The present ...

Claims

1. a GH61 polypeptide variant, which is substituted at one or more positions corresponding to position 26, 32, 34, 40, 41, 42, 47, 56, 72, 102, 123, 138, 149, 152, 163, 164, 166, 169, 186, 200, 207, 213, 219, 222, 234, 246, 249, and 250 of the mature polypeptide of SEQ ID NO:30, wherein the The variant has cellulolytic enhancing activity, and wherein the variant has at least 80% but less than 100% sequence identity with the amino acid sequence of the parent GH61 polypeptide; wherein the substitution corresponding to position 26 is Ile, the substitution corresponding to position 32 is Glu or Ser; the substitution corresponding to position 34 is Phe; the substitution corresponding to position 40 is Ala; the substitution corresponding to position 41 is Thr; the substitution corresponding to position 42 is Ile, Glu or Val; the substitution corresponding to position 47 is Glu, Leu or Arg; the substitution corresponding to position 56 is Cys, Glu or T hr; substitution at position 72 with Gln or Thr; substitution at position 102 with Lys or Pro; substitution at position 123 with Arg; substitution at position 138 with Cys, Glu, Gly, Lys, Leu or Met; substitution at position 149 with Ile; substitution at position 152 with Ser; substitution at position 163 with Glu, Phe or Val; substitution at position 164 with Cys or Leu; substitution at position 166 with Leu; substitution at position 169 with Arg or C ys; the substitution at position 186 is Phe, Lys, Thr or Tyr; the substitution at position 200 is Ile or Val; the substitution at position 207 is Pro; the substitution at position 213 is Glu; the substitution at position 219 is Glu, Met, Gln or Cys; the substitution at position 222 is Arg; the substitution at position 234 is Gly or Lys; the substitution at position 246 is Pro; the substitution at position 249 is Gln, Arg or Cys; the substitution at position 250 is Cys.

2. variant as claimed in claim 1, wherein the variant of parent GH61 polypeptide is selected from the group consisting of: (a) and SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 56, 158, 160, 162, 164, 166, 168, 409, or 411; (b) a polypeptide encoded by a polynucleotide that hybridizes under high stringency conditions to the full-length complementary strand of: (i) SEQ ID NO:1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63, 65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,1 21, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 408, or 410 mature polypeptide coding sequence, wherein the high stringency conditions refer to prehybridization and hybridization at 42°C in 5XSSPE, 0.3% SDS, 200 micrograms / ml sheared and denatured salmon sperm DNA and 50% formamide, and using 0.2X SSC, 0.2% SDS, washed three times at 65°C for 15 minutes each time; (c) by the same sequence as SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107 , 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 408, or 410. A polypeptide encoded by a polynucleotide having at least 80% sequence identity to the mature polypeptide coding sequence of 3. variant as claimed in claim 2, wherein the parent GH61 polypeptide and SEQ ID NO:2,4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,108,110,112,114,116,118,120,122,124,126,128,130,132,133 4, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 56, 158, 160, 162, 164, 166, 168, 409, or 411 has a mature polypeptide of 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.

4. variant as claimed in claim 2, wherein this parent GH61 polypeptide is by the polynucleotide encoding of hybridizing with the complete complementary strand of the following under high stringency conditions: SEQ ID NO:1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,120 21, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 408, or 410 of the mature polypeptide coding sequence, wherein the high stringency conditions refer to prehybridization and hybridization at 42°C in 5XSSPE, 0.3% SDS, 200 micrograms / ml sheared and denatured salmon sperm DNA and 50% formamide, and washing three times at 65°C with 0.2X SSC, 0.2% SDS for 15 minutes each.

5. variant as claimed in claim 2, wherein the parent GH61 polypeptide is composed of SEQ ID NO:1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,13 1, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 408, or 410 has a mature polypeptide coding sequence having 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 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a polynucleotide.

6. variant as claimed in claim 2, wherein the parent GH61 polypeptide comprises SEQ ID NO:2,4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100,10 2, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 56, 158, 160, 162, 164, 166, 168, 409, or 411 of the mature polypeptide, or consist of the same.

7. The variant of any one of claims 1-6, which is different from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134 , 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 56, 158, 160, 162, 164, 166, 168, 409, or 411 has a mature polypeptide of 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%, or at least 99%, but less than 100% sequence identity.

8. The variant of any one of claims 1-7, further comprising 1, 2, 3 or 4 substitutions at positions corresponding to positions 111, 152, 155, and 162 of the mature polypeptide of SEQ ID NO:30, wherein the variant has cellulolytic enhancing activity; wherein the substitution corresponding to position 111 is Val; the substitution corresponding to position 152 is Ser; the substitution corresponding to position 155 is Leu; the substitution corresponding to position 162 is Trp.

9. The variant of any one of claims 1-8, further comprising 1, 2, 3, 4, 5 or 6 substitutions at positions corresponding to positions 96, 98, 200, 202, and 204 of the mature polypeptide of SEQ ID NO:30, wherein the variant has cellulolytic enhancing activity; wherein the substitution corresponding to position 96 is Val; the substitution corresponding to position 98 is Leu; the substitution corresponding to position 200 is Ile; the substitution corresponding to position 202 is Leu; the substitution corresponding to position 204 is Val.

10. The variant of any one of claims 1-9, further comprising 1, 2, 3, 4, 5 or 6 substitutions at positions corresponding to positions 105, 154, 188, 189, 216, and 229 of the mature polypeptide of SEQ ID NO:30, wherein the variant has cellulolytic enhancing activity; wherein the substitution corresponding to position 105 is Pro or Lys; the substitution corresponding to position 154 is Leu; the substitution corresponding to position 188 is Ala or Trp; the substitution corresponding to position 189 is Lys; the substitution corresponding to position 216 is Leu or Tyr; the substitution corresponding to position 229 is Trp, His, lie or Tyr.

11. The variant of any one of claims 1-10, having at least a 1.01-fold increased thermal stability compared to the parent.

12. The variant of any one of claims 11, having at least a 1.05-fold increased thermal stability compared to the parent.

13. The variant of any one of claims 11, having at least 1.1-fold increased thermal stability compared to the parent.

14. The variant of any one of claims 11, having at least a 1.2-fold increased thermal stability compared to the parent.

15. The variant of any one of claims 11, having at least 1.4-fold increased thermal stability compared to the parent.

16. The variant of any one of claims 11, having at least 1.5-fold increased thermal stability compared to the parent.

17. The variant of any one of claims 11, having at least a 1.8-fold increased thermal stability compared to the parent.

18. The variant of any one of claims 11, having at least a 2-fold increased thermal stability compared to the parent.

19. The variant of any one of claims 11, having at least 5-fold increased thermal stability compared to the parent.

20. The variant of any one of claims 11, having at least a 10-fold increased thermal stability compared to the parent.

21. The variant of any one of claims 11, having at least a 20-fold increased thermal stability compared to the parent.

22. The variant of any one of claims 11, having at least 25-fold increased thermal stability compared to the parent.

23. The variant of any one of claims 11, having at least a 30-fold increased thermal stability compared to the parent.

24. The variant of any one of claims 11, having at least a 50-fold increased thermal stability compared to the parent.

25. The variant of any one of claims 11, having at least 75-fold increased thermal stability compared to the parent.

26. The variant of any one of claims 11, having at least a 100-fold increased thermal stability compared to the parent.

27. An isolated polynucleotide encoding the variant of any one of claims 1-26.

28. A nucleic acid construct comprising the polynucleotide of claim 27.

29. An expression vector comprising the polynucleotide of claim 27.

30. A recombinant host cell transformed with the polynucleotide of claim 27.

31. A method for producing a GH61 polypeptide variant, comprising: The recombinant host cell of claim 30 is cultured under conditions suitable for expression of the variant.

32. The method of claim 31 , further comprising recovering the variant.

33. A method of producing a variant as described in any one of claims 1 to 26, comprising: The transgenic plant or plant cell comprising the polynucleotide encoding the variant is cultured under conditions conducive for production of the variant.

34. The method of claim 33, further comprising recovering the variant.

35. A method for obtaining a GH61 polypeptide variant, the method comprising: A variant is introduced into a parent GH61 polypeptide at one or more positions corresponding to positions 26, 32, 34, 40, 41, 42, 47, 56, 72, 102, 123, 138, 149, 152, 163, 164, 166, 169, 186, 200, 207, 213, 219, 222, 234, 246, 249, and 250 of the mature polypeptide of SEQ ID NO: 30, wherein the variant has cellulolytic enhancing activity; and wherein the variant has at least 80%, but less than 100%, of the amino acid sequence of the parent GH61 polypeptide. sequence identity; wherein the substitution at position 26 is Ile, the substitution at position 32 is Glu or Ser; the substitution at position 34 is Phe; the substitution at position 40 is Ala; the substitution at position 41 is Thr; the substitution at position 42 is Ile, Glu or Val; the substitution at position 47 is Glu, Leu or Arg; the substitution at position 56 is Cys, Glu or Thr; the substitution at position 72 is Gln or Thr; the substitution at position 102 is L ys or Pro; the substitution at position 123 is Arg; the substitution at position 138 is Cys, Glu, Gly, Lys, Leu or Met; the substitution at position 149 is Ile; the substitution at position 152 is Ser; the substitution at position 163 is Glu, Phe or Val; the substitution at position 164 is Cys or Leu; the substitution at position 166 is Leu; the substitution at position 169 is Arg or Cys; the substitution at position 186 is Phe, Lys or Val. s, Thr or Tyr; substitution corresponding to position 200 is Ile or Val; substitution corresponding to position 207 is Pro; substitution corresponding to position 213 is Glu; substitution corresponding to position 219 is Glu, Met, Gln or Cys; substitution corresponding to position 222 is Arg; substitution corresponding to position 234 is Gly or Lys; substitution corresponding to position 246 is Pro; substitution corresponding to position 249 is Gln, Arg or Cys; substitution corresponding to position 250 is Cys and the variant is recovered.

36. The method of claim 35, further comprising introducing 1, 2, 3 or 4 substitutions into a parent GH61 polypeptide at one or more positions corresponding to positions 111, 152, 155, and 162 of the mature polypeptide of SEQ ID NO:30, wherein the variant has cellulolytic enhancing activity; wherein the substitution corresponding to position 111 is Val; the substitution corresponding to position 152 is Ser; the substitution corresponding to position 155 is Leu; and the substitution corresponding to position 162 is Trp.

37. The method of claim 35 or 36, further comprising introducing 1, 2, 3, 4, 5 or 6 substitutions into a parent GH61 polypeptide at one or more positions corresponding to positions 96, 98, 200, 202, and 204 of the mature polypeptide of SEQ ID NO:30, wherein the variant has cellulolytic enhancing activity; wherein the substitution corresponding to position 96 is Val; the substitution corresponding to position 98 is Leu; the substitution corresponding to position 200 is Ile; the substitution corresponding to position 202 is Leu; the substitution corresponding to position 204 is Val.

38. The method of any one of claims 35-37, further comprising introducing 1, 2, 3, 4, 5 or 6 substitutions into a parent GH61 polypeptide at one or more positions corresponding to positions 105, 154, 188, 189, 216, and 229 of the mature polypeptide of SEQ ID NO:30, wherein the variant has cellulolytic enhancing activity; wherein the substitution corresponding to position 105 is Pro or Lys; the substitution corresponding to position 154 is Leu; the substitution corresponding to position 188 is Ala or Trp; the substitution corresponding to position 189 is Lys; the substitution corresponding to position 216 is Leu or Tyr; the substitution corresponding to position 229 is Trp, His, lie or Tyr.

39. A method for degrading or converting a cellulosic material, comprising: The cellulosic material is treated with an enzyme composition containing the GH61 polypeptide variant having cellulolytic enhancing activity as described in any one of claims 1-26.

40. The method of claim 39, wherein the cellulosic material has been pretreated.

41. The method of claim 39 or 40, further comprising recovering the degraded or converted cellulosic material.

42. The method of any one of claims 39-41, wherein the enzyme composition further comprises one or more enzymes selected from the group consisting of: Cellulases, polypeptides having cellulolytic enhancing activity, hemicellulases, esterases, ligninases, pectinases, peroxidases, and proteases.

43. The method of any one of claims 39-42, wherein the enzyme composition further comprises an expansin and / or an swollenin.

44. The method of any one of claims 42, wherein the ligninolytic enzyme is a laccase.

45. The method of claim 42, wherein the cellulase is one or more enzymes selected from the group consisting of: Endoglucanases, cellobiohydrolases, and β-glucosidases.

46. ​​The method of claim 42, wherein the hemicellulase is one or more enzymes selected from the group consisting of: Xylanases, acetylxylan esterases, ferulic acid esterases, arabinofuranosidases, xylosidases, and glucuronidases.

47. The method of any one of claims 39-46, wherein the degraded or converted cellulosic material is a sugar.

48. The method of claim 47, wherein the sugar is selected from the group consisting of: Glucose, xylose, mannose, galactose, and arabinose.

49. A method for producing a fermentation product, the method comprising: (a) saccharifying a cellulosic material using an enzyme composition containing a GH61 polypeptide variant having cellulolytic enhancing activity as described in any one of claims 1-26; (b) fermenting the saccharified cellulosic material with one or more fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.

50. The method of claim 49, wherein the cellulosic material is pretreated.

51. The method of claim 49 or 50, wherein the enzyme composition comprises one or more enzymes selected from the group consisting of: Cellulases, polypeptides having cellulolytic enhancing activity, hemicellulases, esterases, ligninases, pectinases, peroxidases, and proteases.

52. The method of any one of claims 49-51, wherein the enzyme composition further comprises an expansin and / or an swollenin.

53. The method of any one of claims 51, wherein the ligninolytic enzyme is a laccase.

54. The method of claim 51, wherein the cellulase is one or more enzymes selected from the group consisting of: Endoglucanases, cellobiohydrolases, and β-glucosidases.

55. The method of claim 51, wherein the hemicellulase is one or more enzymes selected from the group consisting of: Xylanases, acetylxylan esterases, ferulic acid esterases, arabinofuranosidases, xylosidases, and glucuronidases.

56. The method of any one of claims 49-55, wherein steps (a) and (b) are performed simultaneously in a simultaneous saccharification and fermentation.

57. The method of any one of claims 49-56, wherein the fermentation product is an alcohol, an alkane, a cycloalkane, an alkene, a gas, a ketone, an organic acid, or a polyketide.

58. The method of claim 57, wherein the organic acid is an amino acid.

59. The method of claim 57, wherein the olefin is isoprene.

60. A method of fermenting a cellulosic material, comprising: The cellulosic material is fermented with one or more fermenting microorganisms, wherein the cellulosic material is saccharified with an enzyme composition containing a GH61 polypeptide variant having cellulolytic enhancing activity as described in any one of claims 1-26.

61. The method of claim 60, wherein the cellulosic material is pretreated prior to saccharification.

62. The method of claim 60 or 61, wherein the enzyme composition comprises one or more enzymes selected from the group consisting of: Cellulases, polypeptides having cellulolytic enhancing activity, hemicellulases, esterases, ligninases, pectinases, peroxidases, and proteases.

63. The method of any one of claims 60-62, wherein the enzyme composition further comprises an expansin and / or an swollenin.

64. The method of any one of claims 62, wherein the ligninolytic enzyme is a laccase.

65. The method of claim 62, wherein the cellulase is one or more enzymes selected from the group consisting of: Endoglucanases, cellobiohydrolases, and β-glucosidases.

66. The method of claim 62, wherein the hemicellulase is one or more enzymes selected from the group consisting of: Xylanases, acetylxylan esterases, ferulic acid esterases, arabinofuranosidases, xylosidases, and glucuronidases.

67. The method of any one of claims 60-66, wherein the fermentation of the cellulosic material produces a fermentation product.

68. The method of claim 67, further comprising recovering the fermentation product from the fermentation.

69. The method of claim 67 or 68, wherein the fermentation product is an alcohol, an alkane, a cycloalkane, an alkene, a gas, a ketone, an organic acid, or a polyketide.

70. The method of claim 69, wherein the organic acid is an amino acid.

71. The method of claim 69, wherein the olefin is isoprene.

72. A composition comprising the variant of any one of claims 1-26.

73. A whole culture broth formulation or cell culture composition comprising the variant of any one of claims 1-26.

74. A detergent composition comprising a surfactant and a variant according to any one of claims 1-26.

75. The composition of claim 74, further comprising one or more enzymes selected from the group consisting of: Amylases, arabinases, cutinases, carbohydrases, cellulases, galactanases, laccases, lipases, mannanases, oxidases, pectinases, peroxidases, proteases, and xylanases.

76. The composition of claim 74 or 75, formulated as a bar, tablet, powder, granules, paste, or liquid.

77. A method for cleaning or washing a hard surface or laundry, the method comprising contacting the hard surface or the laundry with a composition as claimed in any one of claims 74 to 76.

Images