CN103562382A - Method for degrading or converting cellulosic material - Google Patents

Abstract

The present invention relates to a method for degrading or converting a cellulosic material comprising: treating the cellulosic material with an enzyme composition in the presence of a polypeptide having catalase activity; and to an enzyme for degrading or converting a cellulosic material A composition comprising one or more (eg, several) enzymes having cellulolytic and/or hemicellulolytic activity, and a polypeptide having catalase activity.

Description

Method for degrading or converting cellulosic material

References to Sequence Listings

This application contains the Sequence Listing in computer readable form, which is hereby incorporated by reference.

Background of the invention

technical field

The present invention relates to methods for degrading or converting cellulosic materials, and enzyme compositions for degrading or converting cellulosic materials.

Background technique

Catalase [hydrogen peroxide: catalase (EC 1.11.1.6)] is an enzyme that catalyzes the conversion of hydrogen peroxide ( _{H2O2 )} into oxygen ( _O2 ) and water ( _H2O ) . These ubiquitous enzymes have been purified from a variety of animal tissues, plants and microorganisms (Chance and Maehly, 1955, Methods Enzymol. 2:764-791).

Catalase preparations are used commercially in enzyme kits for diagnostics, for the enzymatic production _of sodium gluconate from glucose, for the neutralization of _H2O2 waste, for the removal of _H2O2 from textile fabrics, and for use in food _and Removal of H ₂ O ₂ and/or generation of O ₂ in beverages.

Cellulose is a polymer of monosaccharides covalently linked by β-1,4-bonds. Many microorganisms produce enzymes that hydrolyze β-linked glucans. These enzymes include endoglucanases, cellobiohydrolases and beta-glucosidases. Endoglucanases digest cellulosic polymers at random locations, exposing them to cellobiohydrolase attack. Cellobiohydrolases sequentially release molecules of cellobiose from the ends of cellulose polymers. Cellobiose is a water-soluble β-1,4-linked dimer of glucose. β-glucosidase hydrolyzes cellobiose to glucose.

The conversion of lignocellulosic materials has the advantage that large quantities of feedstock are readily available and ideally avoids burning or landfilling the material. Wood, agricultural residues, herbaceous crops and municipal solid waste are considered as raw materials. These materials are mainly composed of cellulose, hemicellulose and lignin. Once lignocellulose is converted into simple sugars such as glucose, the simple sugars can be further converted into many useful substances such as fuels, drinking ethanol, fermentation products and/or chemicals (e.g. acids, alcohols, ketones, gases, etc.) .

It would be advantageous in the art to improve methods for degrading or converting cellulosic materials.

Contents of the invention

The present invention relates to a method for degrading or converting cellulosic material comprising: treating the cellulosic material with an enzyme composition in the presence of a polypeptide having catalase activity.

The present invention also relates to a method for producing a fermentation product comprising:

(a) saccharifying a cellulosic material with an enzyme composition in the presence of a polypeptide having catalase activity;

(b) fermenting the saccharified cellulosic material with one or more (eg, several) fermenting microorganisms to produce a fermentation product; and

(c) recovering the fermentation product from the fermentation.

The present invention further relates to a method of fermenting a cellulosic material, comprising: fermenting said cellulosic material with one or more (e.g. several) fermenting microorganisms, wherein said cellulosic material is in the presence of a polypeptide having catalase activity Hydrolyzed in the presence of an enzyme composition.

The present invention also relates to an enzyme composition for degrading or converting cellulosic material, comprising an enzyme having cellulolytic activity and/or lignin degrading activity and a polypeptide having catalase activity; and uses of the composition.

Description of drawings

Figure 1 shows the genomic DNA sequence (SEQ ID NO:3) and amino acid sequence (SEQ ID NO:4) of a Talaromyces stipitatus catalase gene.

Figure 2 shows a genomic DNA sequence (SEQ ID NO:5) and amino acid sequence (SEQ ID NO:6) of a Humicola insolens catalase gene.

Figure 3 shows the genomic DNA sequence (SEQ ID NO:7) and amino acid sequence (SEQ ID NO:8) of a Penicillium emersonii catalase gene.

definition

Catalase activity: The term "catalase activity" is defined herein as hydrogen _peroxide : hydrogen peroxide redoxase activity (EC 1.11.1.6), which catalyzes _2H2O2 to _O2 + _2H2 O conversion. For purposes of the present invention, catalase activity is determined according to US Patent No. 5,646,025. One unit of catalase activity is equal to the amount of enzyme that catalyzes the oxidation of 1 μmol of hydrogen peroxide under the conditions of the assay.

In one aspect, the catalase used in the present invention has the mature polypeptide of SEQ ID NO:2, the mature polypeptide of SEQ ID NO:4, the mature polypeptide of SEQ ID NO:6, or the mature polypeptide of SEQ ID NO:8 At least 20%, such as at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the catalase activity.

Acetylxylan esterase: The term "acetylxylan esterase" means catalyzing the synthesis of xylan from polymerized xylan, acetylated xylose, acetylated glucose, alpha-naptyl acetate and p-nitroacetic acid Carboxyl esterase (EC3.1.1.72) that hydrolyzes acetyl groups of p-nitrophenyl acetate. For the purposes of the present invention, acetylxylan esterase activity was obtained using 0.5 mM p-nitrophenyl acetate in 50 mM sodium acetate pH 5.0 containing 0.01% TWEEN ^™ 20 (polyoxyethylene sorbitan monolaurate) determined as a substrate. One unit of acetylxylan esterase is defined as the amount of enzyme capable of releasing 1 micromole of p-nitrophenolate anion (p-nitrophenolate anion) per minute at pH 5, 25°C.

HPX-87H柱层析(Bio-RadLaboratories,Inc.,Hercules,CA,USA)进行阿拉伯糖分析来确定的。α-L-arabinofuranosidase: The term "α-L-arabinofuranosidase" means α-L-arabinofuranoside arabinofuranohydrolase (EC3.2.1.55), which catalyzes the reaction of α-L-arabinofuranoside Hydrolysis of terminal non-reducing α-L-arabinofuranoside residues in The enzyme acts on α-L-arabinofuranosides, α-L-arabinan containing (1,3)- and/or (1,5)-linkages, arabinoxylan and arabinogalactan. α-L-arabinofuranosidase is also known as arabinosidase, α-arabinosidase, α-L-arabinosidase, α-arabinofuranosidase, polysaccharide α-L-arabinofuranosidase, α-L- Arabinofuranoside hydrolase, L-arabinosidase or α-L-arabinanase. For the purposes of the present invention, α-L-arabinofuranosidase activity was measured using 5 mg of medium viscosity wheat arabinoxylan (Megazyme International Ireland, Ltd., Bray, Co. Wicklow, Ireland) per ml of 100 mM sodium acetate pH 5 , a total volume of 200 μl, at 40 °C for 30 min, followed by

Arabinose was determined by HPX-87H column chromatography (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Alpha-glucuronidase: The term "alpha-glucuronidase" means alpha-D-glucosiduronate glucuronohydrolase (EC3.2.1.139) , which catalyzes the hydrolysis of α-D-glucuronoside to D-glucuronic acid and alcohol. For the purposes of the present invention, α-glucuronidase activity is 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, 40°C.

20的50mM柠檬酸钠中每分钟从作为底物的1mM对硝基苯基-β-D-葡糖吡喃糖苷产生1.0微摩尔对硝基苯酚阴离子。β-glucosidase: The term "β-glucosidase" means β-D-glucoside glucohydrolase (beta-D-glucoside glucohydrolase) (ECNo. 3.2.1.21), the catalytic terminal non-reducing β-D - Hydrolysis of glucose residues with release of β-D-glucose. For the purposes of the present invention, β-glucosidase is used according to the method of Venturi et al., 2002, Extracellular beta-D-glucosidase from Chaetomium thermophilum var. coprophilum: production, purification and some biochemical properties, J.BasicMicrobiol.42:55-66 p-Nitrophenyl-β-D-glucopyranoside was assayed as a substrate. One unit of β-glucosidase is defined as at 25°C, pH4.8, containing 0.01%

1.0 micromole of p-nitrophenol anion was generated per minute from 1 mM p-nitrophenyl-β-D-glucopyranoside as substrate in 50 mM sodium citrate at 20.

20的100mM柠檬酸钠中每分钟从作为底物的1mM对硝基苯基-β-D-木糖苷产生1.0微摩尔对硝基苯酚阴离子。β-xylosidase: The term "β-xylosidase" means β-D-xylosidexylohydrolase (EC 3.2.1.37), which catalyzes the short β(1→4) Exohydrolysis of xylooligosaccharides to remove consecutive D-xylose residues from the non-reducing end. For the purposes of the present invention, one unit of β-xylosidase is defined as containing 0.01%

1.0 micromole of p-nitrophenol anion per minute in 100 mM sodium citrate at 20 was generated from 1 mM p-nitrophenyl-β-D-xyloside as substrate.

cDNA: The term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a mature, spliced mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intronic sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps including splicing and then emerges as mature spliced mRNA.

Cellobiohydrolase: The term "cellobiohydrolase" means 1,4-beta-D-glucan cellobiohydrolase (1,4-beta-D-glucan cellobiohydrolase) (E.C.3.2.1.91 and E.C.3.2.1.176), which catalyzes the hydrolysis of 1,4-β-D-glycosidic linkages in cellulose, cellooligosaccharides, or any polymer containing β-1,4-linked glucose, from chain reduction or Non-reducing end releases cellobiose (Teeri, 1997, Crystallinecellulose degradation: New insight into the function of cellobiohydrolases, Trends in Biotechnology 15: 160-167; Teeri et al., 1998, Trichoderma reesei cellobiohydrolases: why so efficient on crystalline? cellobiohydrolases, . Trans. 26:173-178). Cellobiohydrolase activity according to 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 determined by the method described by Tomme et al., 1988, Eur. J. Biochem. 170:575-581. In the present invention, the method of Tomme et al. can be used to determine cellobiohydrolase activity.

Cellulosic material: The term "cellulosic material" means any material comprising cellulose. The most dominant polysaccharide in the primary cell wall of biomass is cellulose, the second most abundant is hemicellulose, and the third is pectin. The secondary cell wall produced after cell growth ceases also contains polysaccharides and is reinforced by polymerized lignin covalently cross-linked to hemicellulose. Cellulose is a homopolymer of anhydrocellobiose and is therefore a linear β-(1-4)-D-glucan, while hemicellulose includes compounds such as xylan, xyloglucan (xyloglucan), arabinoxylan and mannan, forming complex branched structures with a wide variety of substituents. Although cellulose is generally polymorphic, cellulose in plant tissues occurs predominantly as an insoluble crystalline matrix of parallel glucan chains. Hemicelluloses are often hydrogen bonded to cellulose and other hemicelluloses, helping to stabilize the cell wall matrix.

Cellulose is commonly found, for example, in the stems, leaves, shells, barks and cobs of plants, or the leaves, branches and wood of trees. Cellulosic materials can be, but are not limited to, agricultural residues, herbaceous material (including energy crops), municipal solid waste, pulp and paper mill residues, waste paper, and wood (including forestry residues) (see, e.g., Wiselogel et al. , 1995, in Handbook on Bioethanol (edited by Charles E.Wyman), pp.105-118, Taylor&Francis, Washington D.C.; Wyman, 1994, Bioresource Technology50:3-16; Lynd, 1990, Applied Biochemistry and Biotechnology24/25:695- 719; Mosier et al., 1999, Recent Progress in Bioconversion of Lignocellulosics, in Advances in Biochemical Engineering/Biotechnology, edited by T. Scheper, Volume 65, pp.23-40, Springer-Verlag, New York). It is understood herein that the cellulose may be in the form of lignocellulose, a plant cell wall material comprising a mixed matrix of lignin, cellulose and hemicellulose. In a preferred aspect, the cellulosic material is any biomass material. In another preferred aspect, the cellulosic material is lignocellulose comprising cellulose, hemicellulose and lignin.

In one aspect, the cellulosic material is agricultural residues. 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 residues).

In another aspect, the cellulosic material is arundo. In another aspect, the cellulosic material is bagasse. In another aspect, the cellulosic material is bamboo. In another aspect, the cellulosic material is corn cob. In another aspect, the cellulosic material is corn fiber. In another aspect, the cellulosic material is corn stover. In another aspect, the cellulosic material is miscanthus. In another aspect, the cellulosic material is orange peel. In another aspect, the cellulosic material is rice straw. In another aspect, the cellulosic material is switch grass. In another aspect, the cellulosic material is straw.

In another aspect, the cellulosic material is aspen. In another aspect, the cellulosic material is eucalyptus. In another aspect, the cellulosic material is fir. In another aspect, the cellulosic material is pine. In another aspect, the cellulosic material is poplar. In another aspect, the cellulosic material is spruce. In another aspect, the cellulosic material is willow.

In another aspect, the cellulosic material is algal cellulose. In another aspect, the cellulosic material is bacterial cellulose. In another aspect, the cellulosic material is cotton linter. In another aspect, the cellulosic material is filter paper. In another aspect, the cellulosic material is microcrystalline cellulose. In another aspect, the cellulosic material is phosphoric acid treated cellulose.

In another aspect, the cellulosic material is aquatic biomass. As used herein, "aquatic biomass" means biomass produced in an aquatic environment by the process of photosynthesis. The aquatic biomass can be algae, emergent plants, floating-leaf plants or submerged plants.

The cellulosic material can be used as is or pretreated using conventional methods known in the art, as described herein. In a preferred aspect, the cellulosic material is pretreated.

Cellulolytic enzyme or cellulase: The term "cellulolytic enzyme" or "cellulase" means one or more (eg, several) enzymes that hydrolyze cellulosic material. Such enzymes include endoglucanases, cellobiohydrolases, beta-glucosidases, or combinations thereof. Two basic methods of measuring cellulolytic activity include: (1) measuring total cellulolytic activity, and (2) measuring individual cellulolytic activities (endoglucanase, cellobiohydrolase, and β-glucolytic glycosidase), as reviewed by Zhang et al., Outlook for cellulase improvement: Screening and selection strategies, 2006, Biotechnology Advances 24:452-481. Total cellulolytic activity is typically determined using insoluble substrates including Whatman No. 1 filter paper, microcrystalline cellulose, bacterial cellulose, algal cellulose, cotton, pretreated lignocellulose, and the like. The most common assay for total cellulolytic activity is the filter paper assay using Whatman No. 1 filter paper as the substrate. This assay was established by the International Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987, Measurement of cellulase activities, Pure Appl. Chem. 59:257-68).

HPX-87H柱(Bio-RadLaboratories,Inc.,Hercules,CA,USA)进行糖分析。For the purposes of the present invention, cellulolytic enzyme activity is determined by measuring the increase in hydrolysis of cellulosic material by cellulolytic enzymes under the following conditions: 1-50 mg of cellulolytic enzyme protein/g of cellulose in PCS ( or other pretreated cellulosic material) at a suitable temperature, such as 50°C, 55°C or 60°C, for 3-7 days, compared with the control hydrolysis without adding cellulolytic enzyme protein. Typical conditions are: 1ml reaction solution, washed or unwashed PCS, 5% insoluble solids, 50mM sodium acetate pH5, 1mM MnSO ₄ , 50°C, 55°C or 60°C, 72 hours, by

An HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used for sugar analysis.

Coding sequence: The term "coding sequence" means a polynucleotide that directly specifies the amino acid sequence of a polypeptide. The boundaries of the 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 combinations thereof.

Control sequence: The term "control sequence" refers to a nucleic acid sequence necessary for the expression of a polynucleotide encoding a mature polypeptide of the present invention. Each regulatory sequence may be native (i.e., from the same gene) or foreign (i.e., from a different gene) to the polynucleotide encoding the mature polypeptide, or each regulatory sequence may be native to each other or exogenous. These regulatory sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, regulatory sequences include a promoter and termination signals for transcription and translation. The control sequences may be provided with linkers for the introduction of specific restriction sites to facilitate ligation of the control sequences with the coding region of the polynucleotide encoding a polypeptide.

Endoglucanase: The term "endoglucanase" means endo-1,4-(1,3;1,4)-β-D-glucan 4-glucanohydrolase ( endo-1,4-β-D-glucan4-glucanohydrolase) (E.C.3.2.1.4), which catalyzes cellulose, cellulose derivatives (such as carboxymethylcellulose and hydroxyethylcellulose), lichenin 1,4-β-D-glycosidic linkages in β-glucans, mixed β-1,3 glucans such as cereal β-D-glucans or xyloglucans, and β-glucans in other plant materials containing cellulosic components Endohydrolysis of -1,4 bonds. Endoglucanase activity can be determined by measuring a decrease in substrate viscosity or an increase in reducing ends as determined by a reducing sugar assay (Zhang et al., 2006, Biotechnology Advances 24:452-481). In terms of the present invention, according to the method of Ghose, 1987, Pure and Appl.Chem.59:257-268, use carboxymethylcellulose (CMC) as substrate to determine endoglucanase at pH5, 40 ℃ active.

Expression: The term "expression" includes any step involved in the production of a polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

Expression vector: The term "expression vector" means a linear or circular DNA molecule comprising a polynucleotide encoding a polypeptide operably linked to regulatory sequences providing for its expression.

Family 61 glycoside hydrolases: The term "family 61 glycoside hydrolases" or "family GH61" or "GH61" is defined herein as according to Henrissat B., 1991, A classification of glycosyl hydrolases based on amino-acid sequence similarities, Biochem.J 280:309-316, and Henrissat B. and Bairoch A., 1996, Updating the sequence-based classification of glycosylhydrolases, Biochem. J. 316:695-696 Polypeptides belonging to glycoside hydrolase family 61. Enzymes in this family were originally classified as glycoside hydrolases on the basis of very weak endo-1,4-β-D glucanase activity measured in one family member. The structure and mode of action of these enzymes are non-classical and they cannot be considered bona fide glycosidases. However, they were retained in the CAZy classification based on their ability to enhance lignocellulosic breakdown when used with cellulases or mixtures of cellulases.

Ferulic acid esterase: The term "feruloyl esterase" means 4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73), which catalyzes 4-hydroxy-3 - Hydrolysis of methoxycinnamoyl (feruloyl) groups from esterified sugars (which in "natural" substrates is usually arabinose) to produce ferulic acid (4-hydroxy-3-methoxy cinnamic acid). Ferulic acid esterase is also known as ferulic acid esterase, hydroxycinnamoyl esterase, FAE-III, cinnamate hydrolase, FAEA, cinnAE, FAE-I or FAE-II. For the purposes of the present invention, ferulic acid esterase activity is determined using 0.5 mM p-nitrophenyl ferulate in 50 mM sodium acetate pH 5.0 as substrate. One unit of ferulic acid esterase is equal to the amount of enzyme capable of releasing 1 micromole p-nitrophenol anion per minute at pH 5, 25°C.

Fragment: The term "fragment" means a polypeptide having one or more (eg, several) amino acids deleted from the amino and/or carboxyl termini of the mature polypeptide main; wherein the fragment has catalase activity. In one aspect, the fragment comprises at least 632 amino acid residues, such as at least 670 amino acid residues, or at least 608 amino acid residues of SEQ ID NO:2. In another aspect, the fragment contains at least 622 amino acid residues, such as at least 659 amino acid residues, or at least 696 amino acid residues of SEQ ID NO:4. In another aspect, the fragment contains at least 652 amino acid residues, such as at least 689 amino acid residues, or at least 727 amino acid residues of SEQ ID NO:6. In another aspect, the fragment contains at least 614 amino acid residues, such as at least 650 amino acid residues, or at least 686 amino acid residues of SEQ ID NO:8.

Hemicellulolytic enzyme or hemicellulase: The term "hemicellulolytic enzyme" or "hemicellulase" means one or more (eg, several) enzymes that hydrolyze hemicellulosic material. See, eg, Shallom D. and Shoham Y. Microbial hemicellulases. Current Opinion In Microbiology, 2003, 6(3):219-228). Hemicellulases are key components in plant biomass degradation. Examples of hemicellulases include, but are not limited to, acetylmannan esterase, acetylxylan esterase, arabinanase, arabinofuranosidase, coumaryl esterase, ferulic acid esterase, galactosidase , glucuronidase, glucuronyl esterase, mannanase, mannosidase, xylanase and xylosidase. The substrate of these enzymes, hemicellulose, is a heterogeneous group of branched and linear polysaccharides that hydrogen bond to the cellulose microfibrils in plant cell walls, cross-linking them into robust network of. Hemicellulose is also covalently attached to lignin, forming highly complex structures together with cellulose. The variable structure and organization of hemicellulose requires the concerted action of many enzymes for its complete degradation. The catalytic modules of hemicellulases are glycoside hydrolase (GH), which hydrolyzes glycosidic bonds, or ester-linked sugar esterase (CE), which hydrolyzes acetic or ferulic acid side groups. These catalytic modules, based on their primary structural homology, can be assigned to the GH and CE families. Some families have an overall similar fold and can be further grouped into clans, labeled with letters (eg, GH-A). The most informative and up-to-date classification of these and other carbohydrate-active enzymes is available at the Carbohydrate-Active Enzymes (CAZy) database. Hemicellulolytic enzyme activity can be measured according to Ghose and Bisaria, 1987, Pure & Appl. Chem. 59: 1739-1752 at a suitable temperature, such as 50°C, 55°C or 60°C, and pH, such as 5.0 or 5.5.

High stringency conditions: The term "high stringency conditions" means that for probes of at least 100 nucleotides in length, at 42°C, in 5X SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA and Prehybridization and hybridization were performed according to standard Southern blotting in 50% formamide for 12 to 24 hr. The support material was finally washed three times with 2X SSC, 0.2% SDS at 65°C for 15 minutes each.

Host cell: The term "host cell" means any cell type amenable to transformation, transfection, transduction, etc., using a nucleic acid construct or expression vector comprising a catalase of the present invention. The term "host cell" encompasses any progeny of a parent cell that differs from the parent cell due to mutations that occur during replication.

Low stringency conditions: The term "low stringency conditions" means that for probes of at least 100 nucleotides in length, at 42°C, in 5X SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA and Prehybridization and hybridization were performed according to standard Southern blotting in 25% formamide for 12 to 24 hr. The support material was finally washed three times with 2X SSC, 0.2% SDS at 50°C for 15 minutes each.

Mature polypeptide: The term "mature polypeptide" means a polypeptide in its final form after translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, and the like. In one embodiment, the mature polypeptide is amino acids 1 to 746 of SEQ ID NO:2. In another embodiment, amino acids 1 to 19 of SEQ ID NO: 4 are predicted to be a signal peptide according to the SignalP program and the mature polypeptide is amino acids 20 to 733 of SEQ ID NO: 4. In another embodiment, amino acids 1 to 19 of SEQ ID NO: 6 are predicted to be a signal peptide according to the SignalP program, and the mature polypeptide is amino acids 20 to 765 of SEQ ID NO: 6. In another embodiment, amino acids 1 to 19 of SEQ ID NO: 8 are predicted to be a signal peptide according to the SignalP program, and the mature polypeptide is amino acids 20 to 741 of SEQ ID NO: 8. It is known in the art that a host cell can produce a mixture of two or more different mature polypeptides (ie, having different C-terminal and/or N-terminal amino acids) expressed from the same polynucleotide.

Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" means a polynucleotide that encodes a mature polypeptide having catalase activity. In one embodiment, the mature polypeptide coding sequence is nucleotides 1 to 2351 of SEQ ID NO: 1 or a cDNA sequence thereof. In another embodiment, the signal peptide encoded by nucleotides 1 to 57 of SEQ ID NO:3 is predicted according to the SignalP program, and the mature polypeptide coding sequence is nucleotides 58 to 2418 of SEQ ID NO:3 or its cDNA sequence. In another embodiment, the mature polypeptide coding sequence is nucleotides 58 to 3040 of SEQ ID NO: 5, or a cDNA sequence thereof, in which nucleotides 1 to 57 of SEQ ID NO: 5 encode a signal peptide according to the prediction of the SignalP program. In another embodiment, the signal peptide encoded by nucleotides 1 to 57 of SEQ ID NO:7 is predicted according to the SignalP program, and the mature polypeptide coding sequence is nucleotides 58 to 2476 of SEQ ID NO:7 or its cDNA sequence.

Moderately stringent conditions: The term "moderately stringent conditions" means that for probes of at least 100 nucleotides in length, at 42°C, in 5X SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA and Prehybridization and hybridization were performed according to standard Southern blotting in 35% formamide for 12 to 24 hr. The carrier material was finally washed three times with 2X SSC, 0.2% SDS at 55°C for 15 minutes each.

Medium-high stringency conditions: The term "medium-high stringency conditions" means that for probes of at least 100 nucleotides in length, sheared and denatured probes in 5X SSPE, 0.3% SDS, 200 micrograms/ml at 42°C Salmon sperm DNA and 35% formamide were prehybridized and hybridized for 12 to 24 hours according to standard Southern blotting. The support material was finally washed three times with 2X SSC, 0.2% SDS at 60°C for 15 minutes each.

Nucleic acid construct: The term "nucleic acid construct" means a nucleic acid molecule, single- or double-stranded, isolated from a naturally occurring gene, or modified to contain it in a manner that would not otherwise exist in nature. A segment of nucleic acid, or synthetic, comprising one or more regulatory sequences.

Operably linked: The term "operably linked" means a configuration in which a regulatory sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the regulatory sequence directs the expression of the coding sequence.

1.5L(Novozymes A/S,

Denmark)的混合物作为纤维素分解活性的来源。Polypeptide having cellulolytic enhancing activity: The term "polypeptide having cellulolytic enhancing" means a GH61 polypeptide that catalyzes the enhanced hydrolysis of cellulosic material by an enzyme having cellulolytic activity. For the purposes of the present invention, cellulolytic enhancing activity is determined by measuring the increase in reducing sugars or the total amount of cellobiose and glucose due to the hydrolysis of cellulosic material by cellulolytic enzymes under the following conditions: 1-50 mg total protein /g cellulose in PCS, wherein the total protein comprises 50-99.5% w/w cellulolytic enzyme protein, and 0.5-50% w/w protein with cellulolytic enhancing activity of GH61 polypeptide, at a suitable temperature , e.g. 50°C, 55°C or 60°C and pH, e.g. 5.0 or 5.5 for 1-7 days, compared with the same amount of total protein loading without cellulolytic enhancing activity (1-50 mg cellulolytic protein/g PCS Cellulose) compared to the control hydrolysis performed. In a preferred aspect, Aspergillus oryzae beta-glucosidase (recombinantly produced in Aspergillus oryzae according to WO02/095014) at 2-3% of the total protein weight or Aspergillus fumigatus beta-glucosidase at 2-3% of the total protein weight is used. In the presence of a cellulase protein loading of glucosidase (recombinantly produced in Aspergillus oryzae as described in WO2002/095014)

1.5L (Novozymes A/S,

Denmark) as a source of cellulolytic activity.

A GH61 polypeptide having cellulolytic enhancing activity enhances the hydrolysis of cellulosic material catalyzed by an enzyme having cellulolytic activity by reducing the amount of cellulolytic enzyme required to achieve the same level of hydrolysis, preferably by a factor of at least 1.01, such as at least 1.05 times, at least 1.10 times, at least 1.25 times, at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, or at least 20 times.

Pretreated corn stover: The term "PCS" or "pretreated corn stover" means a cellulosic material obtained from corn stover by treatment with heat and dilute sulfuric acid, alkaline pretreatment or neutral pretreatment.

Sequence identity: The parameter "sequence identity" describes the relatedness between two amino acid sequences or between two nucleotide sequences.

In terms of the present invention, the degree of sequence identity between two amino acid sequences is preferably 5.0 using the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16:276-277). The Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) implemented in the Needle program version .0 or later. The parameters used were gap open penalty (gap open penalty) 10, gap extension penalty (gap extension penalty) 0.5 and EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. Use Needle's output marked as "longest identity" (obtained with the "-nobrief" option) as percent identity and calculate as follows:

(identical residues x 100)/(alignment length - total number of gaps in the alignment)

In terms of the present invention, the degree of sequence identity between two nucleotide sequences is determined using a software package such as EMBOSS (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 5.0.0 or later version of the Needle program implemented in the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra). The parameters used were gap opening penalty of 10, gap extension penalty of 0.5 and EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. Use Needle's output marked as "highest identity" (obtained with the -nobrief option) as percent identity and calculate as follows:

(identical deoxyribonucleotides × 100)/(alignment length - total number of gaps in the alignment)

Variant: The term "variant" means a polypeptide having catalase activity comprising alterations, ie substitutions, insertions and/or deletions, at one or more (eg several) positions. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; and insertion means adding an amino acid adjacent and immediately after the amino acid occupying a position.

Very high stringency conditions: The term "very high stringency conditions" means that for probes of at least 100 nucleotides in length, at 42°C, in 5X SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA was prehybridized and hybridized for 12 to 24 hours according to standard Southern blotting in 50% formamide. The support material was finally washed three times for 15 minutes each in 2X SSC, 0.2% SDS at 70°C.

Very low stringency conditions: The term "very low stringency conditions" means that for probes of at least 100 nucleotides in length, at 42°C, in 5X SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA was prehybridized and hybridized for 12 to 24 hours according to standard Southern blotting in 25% formamide. The support material was finally washed three times with 2X SSC, 0.2% SDS at 45°C for 15 minutes each.

Xylan-containing material: The term "xylan-containing material" means any material comprising a plant cell wall polysaccharide comprising a backbone of β-(1-4) linked xylose residues. Xylans of land plants are heteropolymers with a β-(1-4)-xylopyranose backbone branched by short sugar chains. They contain D-glucuronic acid or its 4-O-methyl ether, L-arabinose and/or various compounds containing D-xylose, L-arabinose, D- or L-galactose and D-glucose oligosaccharides. Polysaccharides of the xylan type can be divided into homoxylan (homoxylan) and heteroxylan (heteroxylan), the latter including glucuronoxylan, (arabino) glucuronoxylan, (glucose aldehyde) arabinoxylans, arabinoxylans and complex heteroxylans. See, eg, Ebringerova et al., 2005, Adv. Polym. Sci. 186:1-67.

In the process of the present invention, any xylan-containing material can be used. In a preferred aspect, the xylan-containing material is lignocellulose.

Xylan degrading activity or xylanolytic activity: The term "xylan degrading activity" or "xylanolytic activity" means a biological activity that hydrolyzes xylan-containing material. Two basic methods for measuring xylanolytic activity include: (1) measuring total xylanolytic activity, and (2) measuring individual xylanolytic activities (e.g. endoxylanase, β-xylosidase , arabinofuranosidase, α-glucuronidase, acetylxylan esterase, feruloesterase and α-glucuronyl esterase (α-glucuronyl esterase)). Recent progress in xylanolytic enzyme assays is summarized in several publications, including Biely and Puchard, Recent progress in the assays of xylanolytic enzymes, 2006, Journal of the Science of Food and Agriculture 86(11):1636-1647; Spanikova and Biely, 2006, Glucuronoyl esterase-Novel carbohydrate esterase produced by Schizophyllum commune, FEBS Letters 580(19):4597-4601; Herrmann, Vrsanska, Jurickova, Hirsch, Biely, and Kubicek, 1997, The Trider of D-xyloidase reesei is a multifunctional beta-D-xylanxylohydrolase, Biochemical Journal 321:375-381.

X-100(4-(1,1,3,3-四甲基丁基)苯基-聚乙二醇)和200mM磷酸钠缓冲液pH6中来确定。一个单位的木聚糖酶活性定义为在37℃，pH6在200mM磷酸钠pH6缓冲液中从作为底物的0.2%AZCL-阿拉伯木聚糖每分钟产生1.0微摩尔天青蛋白(azurine)。Total xylan degrading activity can be measured by determining reducing sugars formed from various types of xylans including, for example, oat spelled, beechwood and larchwood wood Glycans, or can be measured by photometrically determining the release of dyed xylan fragments from a variety of covalently dyed xylans. The most common assay of total xylanase activity is based on the production of reducing sugars from polymeric 4-O-methylglucuronoxylan, as in Bailey, Biely, Poutanen, 1992, Interlaboratory testing of methods for assay of xylanase activity, described in Journal of Biotechnology 23(3):257-270. Xylanase activity can also use 0.2% AZCL-arabinoxylan as a substrate at 37°C in 0.01%

X-100 (4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol) and 200mM sodium phosphate buffer pH6 to determine. One unit of xylanase activity was defined as the production of 1.0 micromole azurine per minute at 37°C, pH 6 in 200 mM sodium phosphate pH 6 buffer from 0.2% AZCL-arabinoxylan as substrate.

For the purposes of the present invention, xylan-degrading activity is measured by measuring the hydrolysis of birch wood xylan (Sigma Chemical Co., Inc., St. Louis, MO, USA) by xylan-degrading enzymes under the following general conditions: Determined by increasing: 1ml reaction system, 5mg/ml substrate (total solids), 5mg xylan decomposing protein/g substrate, 50mM sodium acetate, pH5, 50°C, 24 hours, such as Lever, 1972, A new The reaction for colorimetric determination of carbohydrates, Anal. Biochem 47:273-279, used the p-hydroxybenzoic acid hydrazide (PHBAH) assay for sugar analysis.

Xylanase: The term "xylanase" means 1,4-β-D-xylan-xylohydrolase (1,4-β-D-xylan-xylohydrolase) (EC3.2.1.8) , which catalyzes the endohydrolysis of 1,4-β-D-xylosidic linkages in xylan. For the purposes of the present invention, xylanase activity is obtained using 0.2% AZCL-arabinoxylan as a substrate in X-100 and 200mM sodium phosphate pH6 buffer determined at 37°C. One unit of xylanase activity was defined as the production of 1.0 micromole of azurin per minute at 37°C, pH 6 in 200 mM sodium phosphate pH 6 buffer from 0.2% AZCL-arabinoxylan as substrate.

Detailed description of the invention

Process for processing cellulosic materials

The present invention relates to a method for degrading or converting cellulosic material comprising: treating the cellulosic material with an enzyme composition in the presence of a polypeptide having catalase activity. In one aspect, the method further includes recovering the degraded or converted cellulosic material.

The present invention also relates to a method for producing a fermentation product comprising:

(a) saccharifying a cellulosic material with an enzyme composition in the presence of a polypeptide having catalase activity;

(b) fermenting the saccharified cellulosic material with one or more (eg, several) fermenting microorganisms to produce a fermentation product; and

(c) recovering the fermentation product from the fermentation.

The present invention further relates to a method of fermenting a cellulosic material, comprising: fermenting said cellulosic material with one or more (e.g. several) fermenting microorganisms, wherein said cellulosic material is in the presence of a polypeptide having catalase activity Hydrolyzed in the presence of an enzyme composition. In one aspect, the fermentation of the cellulosic material produces a fermentation product. In another aspect, the method further comprises recovering the fermentation product from the fermentation.

In the above method, the presence of the polypeptide having catalase activity increases the hydrolysis of the cellulosic material compared to the absence of the polypeptide having catalase activity.

The methods of the invention can be used to saccharify cellulosic material into fermentable sugars and convert the fermentable sugars into a number of useful fermentation products such as fuels, drinking ethanol and/or platform chemicals (e.g. acids, alcohols, ketones, gases, etc.). Producing a desired fermentation product from cellulosic material typically involves pretreatment, enzymatic hydrolysis (saccharification) and fermentation.

Treatment of cellulosic material according to the invention can be accomplished using conventional methods in the art. Furthermore, the process of the invention may be carried out using any conventional biomass processing equipment configured to operate in accordance with the invention.

Hydrolysis (saccharification) and fermentation, separate or simultaneous, including, but not limited to, separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), simultaneous saccharification and co-fermentation (SSCF), mixed hydrolysis and fermentation ( HHF), separated hydrolysis and co-fermentation (SHCF), hybrid hydrolysis and co-fermentation (HHCF), and direct microbial conversion (DMC), sometimes referred to as consolidated bioprocessing (CBP). SHF uses separate processing steps to first enzymatically hydrolyze the cellulosic material into fermentable sugars, eg, glucose, cellobiose, cellotriose, and pentose monomers, and then ferment the fermentable sugars into ethanol. In SSF, enzymatic hydrolysis of cellulosic material and fermentation of sugars to ethanol are combined in one step (Philippidis, G.P., 1996, Cellulose bioconversion technology, in Handbook on Bioethanol: Production and Utilization, edited by Wyman, C.E, Taylor & Francis, Washington, DC, 179-212). SSCF involves co-fermentation of multiple sugars (Sheehan, J. and Himmel, M., 1999, Enzymes, energy and the environment: A strategic perspective on the U.S. Department of Energy's research and development activities for bioethanol, Biotechnol. Prog. 15: 817-827). HHF consists of a separate hydrolysis step and also includes simultaneous saccharification and hydrolysis steps, which can be performed in the same reactor. The steps in the HHF process can be performed at different temperatures, ie high temperature enzymatic saccharification followed by SSF at a lower temperature that the fermenting strain can tolerate. DMC combines all three processes (enzyme production, hydrolysis, and fermentation) in one or more (eg, several) steps, where the same organisms are used to produce both the conversion of cellulosic material into fermentable sugars and the conversion of fermentable Enzymes that convert sugars into end products (Lynd, L.R., Weimer, P.J., van Zyl, W.H., and Pretorius, I.S., 2002, Microbialcellulose utilization: Fundamentals and biotechnology, Microbiol. Mol. Biol. Reviews 66:506-577). It is understood herein that any method known in the art, including pretreatment, enzymatic hydrolysis (saccharification), fermentation, or combinations thereof, can be used to practice the methods of the invention.

Conventional equipment includes fed batch stirred reactors, batch stirred reactors, continuous flow stirred reactors with ultrafiltration and/or continuous plug flow column reactors (Fernanda de Castilhos Corazza, Flávio Faria de Moraes, Gisella Maria Zanin and Ivo Neitzel, 2003, Optimal control infed-batch reactor for the cellobiose hydrolysis, Acta Scientiarum. Technology 25:33-38; Gusakov, A.V. and Sinitsyn, A.P., 1985, Kinetics of the enzymatic hydrolysis for the model of cellulose: 1. batch reactor process, Enz.Microb.Technol.7:346-352), grinding reactor (Ryu, S.K. and Lee, J.M., 1983, Bioconversion of wastecellulose by using an attrition bioreactor, Biotechnol.Bioeng.25:53-65) , or a reactor with intense stirring induced by an electromagnetic field (Gusakov, A.V., Sinitsyn, A.P., Davydkin, I.Y., Davydkin, V.Y., Protas, O.V., 1996, Enhancement of enzyme cellulose hydrolysis using a novel by type of bioreactor with intensive stirring induced electromagnetic field, Appl. Biochem. Biotechnol. 56:141-153). Other reactor types include: fluidized bed, upflow blanket, immobilized and extrusion type reactors for hydrolysis and/or fermentation.

preprocessing . In the practice of the methods of the present invention, any pretreatment process known in the art may be used to disrupt the cellulosic material components of plant cell walls (Chandra et al., 2007, Substrate pretreatment: The key to effective enzymatic hydrolysis of lignocellulars? Adv. Biochem .Engin./Biotechnol.108:67-93; Galbe and Zacchi,2007,Pretreatment of lignocellularmaterials for efficient bioethanol production,Adv.Biochem.Engin./Biotechnol.108:41-65;Hendriks and Zeeman,2009,Pretreatments to enhance the digestibility of lignocellular biomass, Bioresource Technol.100:10-18; Mosier et al., 2005, Features of promising technologies for pretreatment of lignocellular biomass, Bioresource Technol.96:673-686; Taherzadeh and Karimi, 2008, Pretreatment of lignocellulosic biomass production: A review, Int.J.of Mol.Sci.9:1621-1651; Yang and Wyman, 2008, Pretreatment: the key to unlocking low-costcellular ethanol, Biofuels Bioproducts and Biorefining-Biofpr.2:26-40) .

The cellulosic material may also be subjected to particle size reduction, presoaking, wetting, washing and/or conditioning prior to pretreatment using methods known in the art.

Conventional pretreatments include, but are not limited to, steam pretreatment (with or without blasting), dilute acid pretreatment, hot water pretreatment, alkaline pretreatment, lime pretreatment, wet oxidation, wet blasting, ammonia fiber blasting, organic Solvent pretreatment and biological pretreatment. Other pretreatments include ammonia percolation, sonication, electroporation, microwave, supercritical _CO2 , supercritical _H2O , ozone, ionic liquid, and gamma radiation pretreatments.

Cellulosic material may be pretreated prior to hydrolysis and/or fermentation. Pretreatment is preferably carried out prior to hydrolysis. Alternatively, pretreatment can be performed concurrently with enzymatic hydrolysis to release fermentable sugars such as glucose, xylose and/or cellobiose. In most cases, the pretreatment step itself converts some biomass to fermentable sugars (even in the absence of enzymes).

Steam pretreatment. In steam pretreatment, cellulosic material is heated to break down plant cell wall components, including lignin, hemicellulose, and cellulose, making the cellulose and other fractions, eg, hemicellulose, accessible to enzymes. The cellulosic material is passed or passed through a reaction vessel where steam is injected to raise the temperature to the desired temperature and pressure and maintained therein for the desired reaction time. Steam pretreatment is preferably carried out at 140-230°C, more preferably 160-200°C, and most preferably 170-190°C, with the optimum temperature range depending on the addition of any chemical catalysts. The residence time for steam pretreatment is preferably 1-30 minutes, more preferably 1-15 minutes, even more preferably 3-12 minutes, most preferably 4-10 minutes, where the optimal residence time depends on the temperature range and the addition of any chemical catalysts . Steam pretreatment allows relatively high solids loadings, so that the cellulosic material is usually only slightly wet during pretreatment. Steam pretreatment is often combined with explosive discharge of the pretreated material, known as steam explosion, i.e., a rapid change of material to atmospheric pressure and turbulent flow to increase the accessible surface area by fragmentation (Duff and Murray, 1996, Bioresource Technology 855:1-33; Galbe and Zacchi, 2002, Appl. Microbiol. Biotechnol. 59:618-628; US Patent Application No. 20020164730). During steam pretreatment, the hemicellulose acetyl groups are cleaved and the resulting acid autocatalyzes the partial hydrolysis of hemicellulose into monosaccharides and oligosaccharides. The extent of lignin removal is limited.

Often adding a catalyst such _{as H2SO4} or _SO2 (typically 0.3 to 5% w/w) prior to steam pretreatment reduces time, lowers temperature, increases recovery, and improves enzymatic hydrolysis (Ballesteros et al., 2006, Appl. Biochem. Biotechnol. 129-132:496-508; Varga et al., 2004, Appl. Biochem. Biotechnol. 113-116:509-523; Sassner et al., 2006, Enzyme Microb. Technol. 39:756-762).

Chemical pretreatment: The term "chemical treatment" refers to any chemical treatment that promotes the separation and/or release of cellulose, hemicellulose and/or lignin. Examples of suitable chemical pretreatment processes include, for example, dilute acid pretreatment, lime pretreatment, wet oxidation, ammonia fiber/freeze explosion (AFEX), ammonia percolation (APR), ionic liquid and organic solvent pretreatment.

In dilute acid pretreatment, cellulosic material is mixed with dilute acid (usually _H2SO4 ) and water to form _a slurry, heated by steam to the desired temperature, and rapidly changed to atmospheric pressure after a dwell time. Dilute acid pretreatment can be performed with many reactor types, for example, plug flow reactors, countercurrent reactors, or continuous countercurrent contracted bed reactors (Duff and Murray, 1996, supra; Schell et al., 2004, BioresourceTechnol. 91: 179-188 ; Lee et al., 1999, Adv. Biochem. Eng. Biotechnol. 65:93-115).

Several pretreatment methods under alkaline conditions are also available. These alkaline pretreatments include, but are not limited to, lime pretreatment, wet oxidation, ammonia percolation (APR), and ammonia fiber/freeze explosion (AFEX).

Lime pretreatment is performed with calcium carbonate, sodium hydroxide or ammonia at a low temperature of 85-150°C, with residence times ranging from 1 hour to several days (Wyman et al., 2005, Bioresource Technol. 96:1959-1966; Mosier et al., 2005, Bioresource Technol. 96:673-686). WO2006/110891, WO2006/110899, WO2006/110900 and WO2006/110901 disclose pretreatment methods using ammonia.

Wet oxidation is thermal pretreatment, usually at 180-200°C for 5-15 minutes, with the addition of an oxidizing agent such as hydrogen peroxide or overpressured oxygen (Schmidt and Thomsen, 1998, Bioresource Technol. 64:139-151; Palonen et al., 2004 , Appl. Biochem. Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88: 567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81: 1669-1677). Pretreatment is performed at preferably 1-40% dry matter, more preferably 2-30% dry matter, and most preferably 5-20% dry matter, and often the initial pH will increase due to the addition of a base such as sodium carbonate.

A modification of the wet oxidation pretreatment method, called wet explosion (combination of wet oxidation and steam explosion), is capable of treating up to 30% dry matter. In wet blasting, the oxidizing agent is introduced after a certain residence time during pretreatment. Pretreatment was then terminated by a rapid change to atmospheric pressure (WO2006/032282).

Ammonia fiber explosion (AFEX) involves the treatment of cellulosic material, where the dry matter content can be as high as 60%, with liquid or gaseous ammonia at mild temperatures such as 90-100 °C and high pressures such as 17-20 bar for 5-10 minutes (Gollapalli et al., 2002 , Appl.Biochem.Biotechnol.98:23-35; Chundawat et al., 2007, Biotechnol.Bioeng.96:219-231; Alizadeh et al., 2005, Appl.Biochem.Biotechnol.121:1133-1141; Teymouri et al., 2005, Bioresource Technol. 96:2014-2018). AFEX pretreatment leads to depolymerization of cellulose and partial hydrolysis of hemicellulose. Lignin-sugar complexes are cleaved.

Organic solvent pretreatment delignifies cellulosic material by extraction with aqueous ethanol (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 often added as a catalyst. In organic solvent pretreatment, most of the hemicelluloses are removed.

Other examples of suitable pretreatment methods are Schell et al., 2003, Appl. Biochem and Biotechn. Vol. 105-108:69-85, and Mosier et al., 2005, Bioresource Technology 96:673-686, and U.S. Published Application 2002/0164730 mentioned.

In one aspect, the chemical pretreatment is preferably performed as an acid treatment, and more preferably as a continuous dilute and/or mild acid treatment. The acid is usually 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. The mild acid treatment is carried out at a pH range of preferably 1-5, more preferably 1-4, and most preferably 1-3. In one aspect, the acid concentration is in the range of preferably 0.01 to 20 wt% acid, more preferably 0.05 to 10 wt% acid, even more preferably 0.1 to 5 wt% acid, and most preferably 0.2 to 2.0 wt% acid. The acid is contacted with the cellulosic material and maintained at a temperature preferably in the range of 160-220°C, more preferably 165-195°C, for a period of seconds to minutes, eg 1 second to 60 minutes.

In another aspect, pretreatment is performed as an ammonia fiber explosion step (AFEX pretreatment step).

In another aspect, pretreatment occurs in an aqueous slurry. In a preferred aspect, cellulosic material is present during pretreatment in an amount of preferably 10-80 wt%, more preferably 20-70 wt%, and most preferably 30-60 wt%, such as about 50 wt%. The pretreated cellulosic material can be left unwashed, or washed using any method known in the art, for example, with water.

Mechanical pretreatment: The term "mechanical pretreatment" refers to various types of grinding or milling (eg, dry grinding, wet grinding, or vibratory ball milling).

Physical pretreatment: The term "physical pretreatment" refers to any pretreatment that promotes the separation and/or release of cellulose, hemicellulose and/or lignin from cellulosic material. For example, physical pretreatment can involve radiation (eg, microwave radiation), steaming/steam explosion, hydrothermolysis, and combinations thereof.

Physical pretreatment may involve high pressure and/or high temperature (steam explosion). In one aspect, high pressure refers to pressures in the range of preferably about 300 to about 600 psi, more preferably about 350 to about 550 psi, and most preferably about 400 to about 500 psi, such as about 450 psi. In another aspect, elevated temperature refers to temperatures in the range of about 100 to 300°C, preferably about 140 to about 235°C. In a preferred aspect, the mechanical pretreatment is carried out in a batch process steam gun hydrolyzer system (e.g. Sunds Hydrolyzer from Sunds Defibrator AB, Sweden) using high temperature and pressure as defined above.

Combined physical and chemical pretreatment. Cellulosic materials can be subjected to both physical and chemical pretreatments. For example, the pretreatment step may involve dilute or mild acid pretreatment and high temperature and/or high pressure pretreatment. The physical and chemical pretreatments may be performed sequentially or simultaneously as desired. Mechanical pretreatment may also be included if desired.

Thus, in a preferred aspect, the cellulosic material is mechanically, chemically or physically pretreated, or any combination thereof, to facilitate the separation and/or release of cellulose, hemicellulose and/or lignin.

Biological pretreatment: The term "biological pretreatment" refers to any biological pretreatment that can promote the separation and/or release of cellulose, hemicellulose and/or lignin from cellulosic material. Biological pretreatment techniques may include the application of lignin-dissolving microorganisms (see, e.g., Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol: Production and Utilization, Wyman, C.E, eds., Taylor & Francis, Washington, DC , 179-212; Ghosh and Singh, 1993, Physicochemical and biological treatments for enzymatic/microbial conversion of lignocellulosic biomass, Adv.Appl.Microbiol.39:295-333; McMillan, J.D., 1994, Pretreating lignocellular biomass: a review of Biomass for Fuels Production, Himmel, M.E., Baker, J.O., and Overend, R.P., eds., ACS Symposium Series 566, American Chemical Society, Washington, DC, Chapter 15; Gong, C.S., Cao, N.J., Du, J., and Tsao, G.T., 1999, Ethanol production from renewable resources, in Advances in Biochemical Engineering/Biotechnology, Scheper, T., eds., Springer-Verlag Berlin Heidelberg, Germany, 65:207-241; Olsson and Hahn-Hagerdal, 1996, Fignermentation of os fl. Hydrolysates for ethanol production, Enz.Microb.Tech.18:312-331; and Vallander and Eriksson, 1990, Production of ethanol from lignocellular materials: State of the art, Adv.Biochem.Eng./Biotechnol.42:63-95 ).

saccharification . In the hydrolysis step (also called saccharification), cellulosic material (eg, pretreated cellulosic material) is hydrolyzed to break down cellulose and/or hemicellulose into sugars, such as glucose, cellobiose, xylose, Xylulose, arabinose, mannose, galactose and/or soluble oligosaccharides. The sugars, and/or soluble oligosaccharides can be further used to produce alcohols (e.g., arabic alcohol, n-butanol, isobutanol, ethanol, glycerol, methanol, ethylene glycol, 1,3-propanediol (propylene glycol) , butanediol, glycerin, sorbitol and xylitol); alkanes (such as pentane, hexane, heptane, octane, nonane, decane, undecane and dodecane); cycloalkanes (such as cyclopentane, cyclohexane, cycloheptane, and cyclooctane); alkenes (e.g., pentene, hexene, heptene, and octene); amino acids (e.g., aspartic acid, glutamic acid, glycine, lysine amino acids, serine, and threonine); gases (e.g., methane, hydrogen ( _H2 ), carbon dioxide ( _CO2 ), and carbon monoxide (CO)); isoprene; ketones (e.g., acetone); organic acids (e.g., , 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, oxaloacetic acid, propionic acid, succinic acid, and xylonic acid); and polyketides.

The enzyme composition for hydrolysis is carried out enzymatically in the presence of a polypeptide having catalase activity of the present invention. The enzyme and the polypeptide having catalase activity of the composition may also be added simultaneously or sequentially.

Enzymatic hydrolysis is preferably carried out in a suitable aqueous environment under conditions readily determined by those skilled in the art. In one aspect, hydrolysis is performed under conditions suitable for the activity of the enzyme, ie optimal for the enzyme. The hydrolysis can be carried out as a fed-batch process or as a continuous process in which the cellulosic material is gradually fed, for example, into the enzyme-containing hydrolysis solution.

Saccharification is typically carried out in stirred tank reactors or fermenters under controlled conditions of pH, temperature and mixing. Appropriate treatment time, temperature 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 performed preferably for about 12 to about 120 hours, such as about 16 to about 72 hours, or about 24 to about 48 hours. The temperature preferably ranges from about 25°C to about 70°C, such as from about 30°C to about 65°C, from about 40°C to about 60°C, or from about 50°C to 55°C. The pH is preferably in the range of about 3 to about 8, such as about 3.5 to about 7, about 4 to about 6, or about 5.0 to about 5.5. The dry solids content is preferably in the range of about 5 to about 50 wt%, such as about 10 to about 40 wt%, or about 20 to about 30 wt%.

enzyme composition

Enzyme compositions may comprise any protein useful for degrading or converting cellulosic material. The composition may comprise one enzyme as the main enzyme component, such as a one-component composition, or may comprise multiple enzymes. The compositions may be prepared according to methods known in the art and may be in the form of liquid or dry compositions. The compositions can be stabilized according to methods known in the art.

In one aspect, an enzyme composition for degrading or converting a cellulosic material comprises one or more (eg several) enzymes having cellulolytic and/or hemicellulolytic activity, and an enzyme having catalase activity peptide.

In one embodiment, the enzyme composition comprises, or further comprises, one or more (eg several) proteins selected from the group consisting of cellulase, GH61 polypeptide with cellulolytic enhancing activity, hemicellulose Enzymes, esterases, patulins, laccases, ligninolytic enzymes, pectinases, peroxidases, proteases and swellins. In another aspect, the cellulase is preferably one or more (eg several) enzymes selected from the group consisting of endoglucanases, cellobiohydrolases and beta-glucosidases. In another aspect, the hemicellulase is preferably one or more (eg, several) enzymes selected from the group consisting of acetylmannan esterase, acetylxylan esterase, arabinase, arabinase Furanosidase, coumaric esterase, ferulic acid esterase, galactosidase, glucuronidase, glucuronidase, mannanase, mannosidase, xylanase and xylanase Glycosidase.

In another embodiment, the enzyme composition comprises one or more (eg several) cellulolytic enzymes. In another aspect, the enzyme composition comprises or further comprises one or more (eg several) hemicellulolytic enzymes. In another aspect, the enzyme composition comprises one or more (eg, several) cellulolytic enzymes and one or more (eg, several) hemicellulolytic enzymes. In another aspect, the enzyme composition comprises one or more (eg, several) enzymes selected from the group consisting of cellulolytic enzymes and hemicellulolytic enzymes. In another aspect, the enzyme composition comprises an endoglucanase. In another aspect, the enzyme composition comprises a cellobiohydrolase. In another aspect, the enzyme composition comprises beta-glucosidase. In another aspect, the enzyme composition comprises a polypeptide having cellulolytic enhancing activity. In another aspect, the enzyme composition comprises an endoglucanase and a polypeptide having cellulolytic enhancing activity. In another aspect, the enzyme composition comprises a cellobiohydrolase and a polypeptide having cellulolytic enhancing activity. In another aspect, the enzyme composition comprises a beta-glucosidase and a polypeptide having cellulolytic enhancing activity. In another aspect, the enzyme composition comprises an endoglucanase and a cellobiohydrolase. In another aspect, the enzyme composition comprises an endoglucanase and a β-glucosidase. In another aspect, the enzyme composition comprises a cellobiohydrolase and a β-glucosidase. In another aspect, the enzyme composition comprises an endoglucanase, a cellobiohydrolase, and a polypeptide having cellulolytic enhancing activity. In another aspect, the enzyme composition comprises an endoglucanase, a β-glucosidase, and a polypeptide having cellulolytic enhancing activity. In another aspect, the enzyme composition comprises a cellobiohydrolase, a beta-glucosidase, and a polypeptide having cellulolytic enhancing activity. In another aspect, the enzyme composition comprises an endoglucanase, a cellobiohydrolase, and a β-glucosidase. In another aspect, the enzyme composition comprises an endoglucanase, a cellobiohydrolase, a beta-glucosidase, and a polypeptide having cellulolytic enhancing activity.

In another embodiment, the enzyme composition comprises acetylmannan esterase. In another aspect, the enzyme composition comprises acetylxylan esterase. In another aspect, the enzyme composition comprises an arabinanase (eg, alpha-L-arabinanase). In another aspect, the enzyme composition comprises an arabinofuranosidase (eg, alpha-L-arabinofuranosidase). In another aspect, the enzyme composition comprises coumaryl esterase. In another aspect, the enzyme composition comprises ferulic acid esterase. In another aspect, the enzyme composition comprises a galactosidase (eg, alpha-galactosidase and/or beta-galactosidase). In another aspect, the enzyme composition comprises a glucuronidase (eg, alpha-D-glucuronidase). In another aspect, the enzyme composition comprises glucuronyl esterase. In another aspect, the enzyme composition comprises a mannanase. In another aspect, the enzyme composition comprises a mannosidase (eg, β-mannosidase). In another aspect, the enzyme composition comprises xylanase. In a preferred aspect, the xylanase is a Family 10 xylanase. In another aspect, the enzyme composition comprises a xylosidase (eg, β-xylosidase).

In another embodiment, the enzyme composition comprises an esterase. In another aspect, the enzyme composition comprises patulin. In another aspect, the enzyme composition comprises laccase. In another aspect, the enzyme composition comprises a ligninolytic enzyme. In another preferred aspect, the ligninolytic enzyme is manganese peroxidase. In another preferred aspect, the ligninolytic enzyme is lignin peroxidase. In another preferred aspect, the ligninolytic enzyme is an H ₂ O ₂ producing enzyme. In another aspect, the enzyme composition comprises pectinase. In another aspect, the enzyme composition comprises peroxidase. In another aspect, the enzyme composition comprises a protease. In another aspect, the enzyme composition comprises swollenin.

In the method of the present invention, the enzyme may be added before or during saccharification, saccharification and fermentation, or fermentation. The enzyme having cellulolytic and/or hemicellulolytic activity and the polypeptide having catalase activity may be added simultaneously or sequentially.

One or more (eg, several) components of the enzyme composition may be wild-type protein, recombinant protein, or a combination of wild-type and recombinant protein. For example, one or more (eg, several) components can be a protein native to the cell, which is used as a host cell to recombinantly express one or more (eg, several) other components of the enzyme composition. One or more (eg, several) components of the enzyme composition can be produced as single components, which are then combined to form the enzyme composition. The enzyme composition may be a combination of multi-component and single-component protein preparations.

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

The optimal amount of enzyme and polypeptide having catalase activity depends on several factors including, but not limited to, the mixture of component cellulolytic enzymes, the cellulosic material, the concentration of the cellulosic material, pretreatment of the cellulosic material, Temperature, time, pH and inclusion of fermenting organisms (eg, yeast for simultaneous saccharification and fermentation).

In a preferred aspect, the effective amount of cellulolytic enzyme or hemicellulolytic enzyme is about 0.5 to about 50 mg, more preferably about 0.5 to about 40 mg, more preferably about 0.5 to about 25 mg, more preferably about 0.75 mg to cellulosic material. to about 20 mg, more preferably about 0.75 to about 15 mg, even more preferably about 0.5 to about 10 mg, most preferably about 1.0 to about 10 mg per g of cellulosic material.

In another preferred aspect, the effective amount of the polypeptide having catalase activity is about 0.001 to about 100.0 mg, preferably about 0.01 to about 50 mg, more preferably about 0.01 to about 40 mg, more preferably about 0.01 to about 40 mg for the cellulosic material. About 30 mg, more preferably about 0.01 to about 20 mg, more preferably about 0.01 to about 10 mg, more preferably about 0.025 to about 8 mg, more preferably about 0.05 to about 6 mg, more preferably about 0.075 to about 5 mg, more preferably about 0.1 to about 4 mg , even more preferably from about 0.15 to about 3 mg, most preferably from about 0.25 to about 1.0 mg per g of cellulosic material.

In another preferred aspect, the effective amount of the polypeptide having catalase activity for cellulolytic or hemicellulolytic enzymes is from about 0.005 to about 1.0 g, preferably from about 0.01 to about 1.0 g, more preferably from about 0.15 to About 0.75 g, more preferably about 0.15 to about 0.5 g, more preferably about 0.1 to about 0.5 g, even more preferably about 0.1 to about 0.5 g, and most preferably about 0.05 to about 0.2 g per g of cellulolytic enzyme or hemifiber prime enzymes.

In another aspect, the effective amount of the GH61 polypeptide having cellulolytic enhancing activity to cellulosic material is about 0.01 to about 50.0 mg, preferably about 0.01 to about 40 mg, more preferably about 0.01 to about 30 mg, more preferably about 0.01 to about 20 mg, more preferably about 0.01 to about 10 mg, more preferably about 0.01 to about 5 mg, more preferably about 0.025 to about 1.5 mg, more preferably about 0.05 to about 1.25 mg, more preferably about 0.075 to about 1.25 mg, more preferably about 0.1 to About 1.25 mg, even more preferably about 0.15 to about 1.25 mg, and most preferably about 0.25 to about 1.0 mg per gram of cellulosic material.

In another aspect, the effective amount of GH61 polypeptide having cellulolytic enhancing activity to cellulolytic enzyme protein is about 0.005 to about 1.0 g, preferably about 0.01 to about 1.0 g, more preferably about 0.15 to about 0.75 g, more preferably From about 0.15 to about 0.5 g, more preferably from about 0.1 to about 0.5 g, even more preferably from about 0.1 to about 0.5 g, and most preferably from about 0.05 to about 0.2 g per gram of cellulolytic enzyme protein.

Polypeptides having cellulolytic enzyme activity or hemicellulolytic enzyme activity, and other proteins/polypeptides useful for the degradation of cellulosic materials, such as polypeptides having cellulolytic enhancing activity (hereinafter collectively referred to as polypeptides having enzymatic activity) May be derived or obtained from any suitable source, including bacterial, fungal, yeast, plant or mammalian sources. The term "obtained" herein also means that the enzyme can be recombinantly produced in a host organism using the methods described herein, wherein the recombinantly produced enzyme is native or foreign to the host organism, or has a modified amino acid sequence , for example, with one or more (eg several) deletions, insertions and/or substitutions of amino acids, i.e. recombinantly produced enzymes which are fragments and/or mutants of the native amino acid sequence or by amino acid shuffling known in the art Enzymes produced by the method. Natural variants are encompassed within the meaning of native enzyme, while variants obtained recombinantly (eg by site-directed mutagenesis or rearrangement) are encompassed within the meaning of foreign enzyme.

A polypeptide having enzymatic activity may be a bacterial polypeptide. For example, the polypeptide may be a Gram-positive bacterial polypeptide such as Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus, milk Lactobacillus, Lactococcus, Clostridium, Geobacillus, Caldicellulosiruptor, Acidothermus, Thermobifidia, or marine spores Polypeptides of the genus Oceanobacillus, which have enzymatic activity; or polypeptides of Gram-negative bacteria, such as Escherichia coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter A Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter, Neisseria or Ureaplasma polypeptide having enzymatic activity .

In one aspect, the polypeptide is an enzymatically active Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Claus Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium ( Bacillus megaterium), Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis or Bacillus thuringiensis polypeptide.

In another preferred aspect, the polypeptide is Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis or Streptococcus equi subsp. . Zooepidemicus) polypeptide.

In another preferred aspect, the polypeptide is Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus or Streptomyces lividans polypeptide.

The polypeptide having enzymatic activity may also be a fungal polypeptide, and more preferably a yeast polypeptide having enzymatic activity such as Candida, Kluyveromyces, Pichia, Saccharomyces ), Schizosaccharomyces or Yarrowia polypeptides; or more preferably filamentous fungal polypeptides having enzymatic activity such as Acremonium, Agaricus, Alternaria Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Ghost Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Pyrospora (Magnaporthe), Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces (Paecilomyces), Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectunia, Pseudotrichonympha, Rhizomucor, Schizophyllum Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma ( Trichoderma), Trichophaea, Verticillium (V erticillium), Volvariella or Xylaria polypeptides.

In one aspect, the polypeptide is Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces skluyveri, Nordisiae having enzymatic activity. Saccharomyces norbensis or Saccharomyces soviformis polypeptides.

In one aspect, the polypeptide is Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus having enzymatic activity , Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum , Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, machete Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, multiple Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum ), Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicolagrisea, Humicola insolens ), Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthorathermophila, Neurospora crassa, rope Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis ), Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia spededonium, Thielavia setosa, Thielavia subthermophila, native Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma viride or Trichophaea saccata polypeptide.

Chemically modified or protein engineered mutants of polypeptides having enzymatic activity can also be used.

One or more (eg, several) components of the cellulolytic enzyme composition may be recombinant components, that is, obtained by cloning the DNA sequences encoding the individual components and subsequently transforming cells with the DNA sequences and Expression in hosts (see, eg, WO91/17243 and WO91/17244) produces. The host is preferably a heterologous host (enzyme is foreign to the host), but the host can also be a homologous host (enzyme is native to the host) under certain conditions. Monocomponent cellulolytic proteins can also be prepared by purifying such proteins from fermentation broth.

NL(DSM)、

S/L100(DSM)，ROHAMENT^TM7069W(

GmbH)，

LDI(Dyadic International,Inc.)、

LBR(Dyadic International,Inc.)或150L(Dyadic International,Inc.)。所述纤维素酶酶以固体的约0.001至约5.0wt%，例如固体的约0.025至约4.0wt%，或固体的约0.005至约2.0wt%的有效量添加。In one aspect, the one or more (eg, several) cellulolytic enzymes comprise a commercial cellulolytic enzyme preparation. Examples of commercial cellulolytic enzyme preparations suitable for use in the present invention include, for example, CELLIC ^™ CTec Ctec3 (Novozymes A/S), CELLIC ^™ Ctec CTec2 (Novozymes A/S), CTec (Novozymes A/S), CELLUCLAST ^TM (Novozymes A/S), NOVOZYM ^TM 188 (Novozymes A/S), CELLUZYME ^TM (Novozymes A/S), CEREFLO ^TM (Novozymes A/S), and ULTRAFLO ^TM (Novozymes A/S) /S), ACCELERASE ^TM (Genencor Int.), LAMINEX ^TM (Genencor Int.), SPEZYME ^TM CP (Genencor Int.),

NL(DSM),

S/L100(DSM), ^ROHAMENTTM 7069W(

GmbH),

LDI (Dyadic International, Inc.),

LBR (Dyadic International, Inc.) or 150L (Dyadic International, Inc.). The cellulase enzyme is added in an effective amount of about 0.001 to about 5.0 wt% solids, such as about 0.025 to about 4.0 wt% solids, or about 0.005 to about 2.0 wt% solids.

Examples of bacterial endoglucanases that can be used in the methods of the present invention include, but are not limited to, Acidothermus cellulolyticus endoglucanase (WO91/05039; WO93/15186; U.S. Patent 5,275,944; WO96/02551; US Patent 5,536,655, WO00/70031, WO05/093050); Thermobifida fusca endoglucanase III (WO05/093050); and Thermobifida fusca endoglucanase V (WO05/093050).

Examples of fungal endoglucanases that can be used in the present invention include, but are not limited to, Trichoderma reesei endoglucanase I (Penttila et al., 1986, Gene45:253-263, Trichoderma reesei Cel7B endoglucanase Glucanase I (GENBANK ^TM accession number M15665); Trichoderma reesei endoglucanase II (Saloheimo et al., 1988, Gene63:11-22), Trichoderma reesei Cel5A endoglucanase II ( GENBANK ^TM Accession No. M19373); Trichoderma reesei endoglucanase III (Okada et al., 1988, Appl. Environ. Microbiol. 64:555-563; GENBANK ^TM Accession No. AB003694); Trichoderma reesei endoglucanase III Glycanase V (Saloheimo et al., 1994, Molecular Microbiology 13:219-228; GENBANK ^TM accession number Z33381); Aspergillus aculeatus endoglucanase (Ooi et al., 1990, Nucleic Acids Research 18:5884); Aspergillus kawachii ( Aspergillus kawachii) endoglucanase (Sakamoto et al., 1995, Current Genetics27:435-439); Carrot soft rot Erwinia (Erwinia carotovara) endoglucanase (Saarilahti et al., 1990, Gene90:9-14) ; Fusarium oxysporum endoglucanase (GENBANK ^TM accession number L29381); Humicola grisea varietal thermoidea endoglucanase (GENBANK ^TM accession number AB003107); Melanocarpus albomyces endoglucanase (GENBANK ^TM accession number No. MAL515703); Neurospora crassa endoglucanase (GENBANK ^TM Accession No. XM_324477); Humicola insolens endoglucanase V; Myceliophthora thermophila CBS117.65 endoglucanase; Basidiomycetes Basidiomycete CBS495.95 endoglucanase; Basidiomycete CBS494.95 endoglucanase; Thielavia terrestris NRRL8126CEL6B endoglucanase; Thielavia terrestris NRRL8126CEL6C endoglucan Thielavia terrestris NRRL8126CEL7C endoglucanase; Thielavia terrestris NRRL8126CEL7E endoglucanase; Thielavia terrestris NRRL8126CEL7F endoglucanase; Cladorrhinum foecundissimum ATCC62373CEL7A endoglucanase; Trichoderma strain No.VTT-D-80133 cut Dextranase (GENBANK ^™ Accession No. M15665).

Examples of cellobiohydrolase that can be used in the present invention include, but are not limited to, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Humicola insolens cellobiohydrolase I, Myceliophthora thermophilum cellobiohydrolase II, Thielavia terrestris cellobiohydrolase II (CEL6A), Chaetomium thermophilum cellobiohydrolase I, and Chaetomium thermophilum cellobiohydrolase II Glycohydrolase II.

Examples of β-glucosidases that can be used in the present invention include, but are not limited to, Aspergillus oryzae β-glucosidase, Aspergillus fumigatus β-glucosidase, Penicillium brasiliensis IBT20888 β-glucosidase, Aspergillus niger β-glucosidase, and Acanthus Aspergillus sporogenes β-glucosidase.

Aspergillus oryzae beta-glucosidase is available according to WO2002/095014. Aspergillus fumigatus beta-glucosidase is available according to WO2005/047499. Penicillium brasiliensis beta-glucosidase is available according to WO2007/019442. Aspergillus niger beta-glucosidase can be obtained according to Dan et al., 2000, J. Biol. Chem. 275:4973-4980. Aspergillus aculeatus beta-glucosidase is available according to Kawaguchi et al., 1996, Gene 173:287-288.

The β-glucosidase may be a fusion protein. In one aspect, the β-glucosidase is an Aspergillus oryzae β-glucosidase variant BG fusion protein or an Aspergillus oryzae β-glucosidase fusion protein (obtained according to WO2008/057637).

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

Other cellulolytic enzymes useful in the present invention are described in EP495,257, EP531,315, EP531,372, WO89/09259, WO94/07998, WO95/24471, WO96/11262, WO96/29397, WO96/034108, WO97/ 14804, WO98/08940, WO98/012307, WO98/13465, WO98/015619, WO98/015633, WO98/028411, WO99/06574, WO99/10481, WO99/025846, WO99/025847, WO99/0317055, WO09 WO2002/050245，WO2002/0076792，WO2002/101078，WO2003/027306，WO2003/052054，WO2003/052055，WO2003/052056，WO2003/052057，WO2003/052118，WO2004/016760，WO2004/043980，WO2004/048592，WO2005/ 001065，WO2005/028636，WO2005/093050，WO2005/093073，WO2006/074005，WO2006/117432，WO2007/071818，WO2007/071820，WO2008/008070，WO2008/008793，美国专利号4,435,307，美国专利号5,457,046，美国专利No. 5,648,263, U.S. Patent No. 5,686,593, U.S. Patent No. 5,691,178, U.S. Patent No. 5,763,254, and U.S. Patent No. 5,776,757.

In the methods of the invention, any GH61 polypeptide having cellulolytic enhancing activity can be used.

In a first aspect, the GH61 polypeptide having cellulolytic enhancing activity comprises the following motif:

[ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(4)-[HNQ] and [FW]-[TF]-K-[AIV],

Wherein X is any amino acid, X(4,5) is any amino acid at 4 or 5 consecutive positions, and X(4) is any amino acid at 4 consecutive positions.

A GH61 polypeptide comprising the motifs shown above may further comprise:

H-X(1,2)-G-P-X(3)-[YW]-[AILMV],

[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV], or

H-X(1,2)-G-P-X(3)-[YW]-[AILMV] and [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV],

Where X is any amino acid, X(1,2) is any amino acid at 1 position or 2 consecutive positions, X(3) is any amino acid at 3 consecutive positions, and X(2) is 2 consecutive amino acids Any amino acid in position. In the above motifs, the accepted IUPAC one-letter amino acid abbreviations are used.

In a preferred aspect, the GH61 polypeptide having cellulolytic enhancing activity further comprises H-X(1,2)-G-P-X(3)-[YW]-[AILMV]. In another preferred aspect, the GH61 polypeptide having cellulolytic enhancing activity further comprises [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV]. In another preferred aspect, the GH61 polypeptide having cellulolytic enhancing activity further comprises H-X(1,2)-G-P-X(3)-[YW]-[AILMV] and

[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV].

In a second aspect, the GH61 polypeptide having cellulolytic enhancing activity comprises the following motif:

[ILMV]-P-x(4,5)-G-x-Y-[ILMV]-x-R-x-[EQ]-x(3)-A-[HNQ],

Wherein x is any amino acid, x(4,5) is any amino acid at 4 or 5 consecutive positions, and x(3) is any amino acid at 3 consecutive positions. In the above motifs, the accepted IUPAC one-letter amino acid abbreviations are used.

Examples of GH61 polypeptides having cellulolytic enhancing activity that may be used in the methods of the invention include, but are not limited to, polypeptides having cellulolytic enhancing activity from Thielavia terrestris (WO2005/074647, WO2008/148131 and WO2011/035027); Polypeptides with cellulolytic enhancing activity from Thermoascus aurantiacus (WO2005/074656 and WO2010/065830), polypeptides with cellulolytic enhancing activity from Trichoderma reesei (WO2007/089290), from Thermoascus Polypeptides having cellulolytic enhancing activity from Myceliophthora (WO2009/085935, WO2009/085859, WO2009/085864, WO2009/085868), polypeptides having cellulolytic enhancing activity from Aspergillus fumigatus (WO2010/138754), and from Cellulolytic enhancing activity of Penicillium pinophilum (WO2011/005867), Thermoascus sp. (WO2011/039319), Penicillium sp. (WO2011/041397), and Thermoascus crustaceous (WO2011/041504) of polypeptides.

In one aspect, said GH61 polypeptide having cellulolytic enhancing activity is used according to WO2008/151043 in the presence of a soluble activatable divalent metal cation, such as manganese sulfate.

In one aspect, the GH61 polypeptide having cellulolytic enhancing activity is present in dioxygen compounds, bicyclic compounds, heterocyclic compounds, nitrogen-containing compounds, quinone compounds, sulfur-containing compounds or from pretreated cellulosic materials (such as by Pretreated corn stover (PCS)) was used in the presence of liquor obtained.

The dioxygen compound may include any suitable compound containing two or more oxygen atoms. In some aspects, the dioxygen compound contains a substituted aryl moiety as described herein. The dioxygen compound may comprise one or more (eg several) hydroxyl groups and/or hydroxyl derivatives, but also includes substituted aryl modules lacking hydroxyl groups and hydroxyl derivatives. Non-limiting examples of dioxygen compounds include catechol or catechol; caffeic acid; 3,4-dihydroxybenzoic acid; 4-tert-butyl-5-methoxy-1,2-benzenediol; pyrogallol; gallic acid; methyl-3,4,5-trihydroxybenzoic acid; 2,3,4-trihydroxybenzophenone; 2,6-dimethoxyphenol; sinapinic acid; 3, 5-dihydroxybenzoic acid; 4-chloro-1,2-benzenediol; 4-nitro-1,2-benzenediol; tannic acid; ethyl gallate; methyl glycolate; dihydroxyfumaric acid; 2 -butyne-1,4-diol; croconic acid; 1,3-propanediol; tartaric acid; 2,4-pentanediol; 3-ethoxy-1,2-propanediol; 2,4,4'- Trihydroxybenzophenone; cis-2-butene-1,4-diol; 3,4-dihydroxy-3-cyclobutene-1,2-dione; dihydroxyacetone; acroleinacetal ); methyl-4-hydroxybenzoic acid; 4-hydroxybenzoic acid; and methyl-3,5-dimethoxy-4-hydroxybenzoic acid; or a salt or solvate thereof.

离子(flavylium ion)，如任选取代的花色素或任选取代的花色苷，或其衍生物。二环化合物的非限定性实例包括表儿茶素(epicatechin)；槲皮素(quercetin)；杨梅黄酮(myricetin)；黄杉素(taxifolin)；山奈酚(kaempferol)；桑素(morin)；金合欢素(acacetin)；柚皮素(naringenin)；异鼠李黄素(isorhamnetin)；芹菜苷配基(apigenin)；花青素(cyanidin)；花色素苷(cyanin)；kuromanin；花青素鼠李葡糖苷(keracyanin)；或它们的盐或溶剂合物。The bicyclic compound may comprise any suitable substituted fused ring system as described herein. The compounds may contain one or more (eg, several) additional rings, and are not limited to a specific number of rings unless otherwise stated. In one aspect, the bicyclic compound is a flavonoid. In another aspect, the bicyclic compound is an optionally substituted isoflavonoid. In another aspect, the bicyclic compound is an optionally substituted flower

A flavylium ion, such as an optionally substituted anthocyanin or an optionally substituted anthocyanin, or a derivative thereof. Non-limiting examples of bicyclic compounds include epicatechin; quercetin; myricetin; taxifolin; acetin; naringenin; isorhamnetin; apigenin; cyanidin; cyanin; kuromanin; anthocyanin rhamnoside glycosides (keracyanin); or their salts or solvates.

The heterocyclic compound can be any suitable compound, such as an optionally substituted heteroatom-containing aromatic or non-aromatic ring, as described herein. In one aspect, the heterocycle is a compound comprising an optionally substituted heterocycloalkyl moiety or an optionally substituted heteroaryl moiety. In another aspect, the optionally substituted heterocycloalkyl moiety or optionally substituted heteroaryl moiety is an optionally substituted five membered heterocycloalkyl moiety or an optionally substituted five membered heteroaryl moiety. In another aspect, the optionally substituted heterocycloalkyl or optionally substituted heteroaryl moiety is an optionally substituted moiety selected from the group consisting of pyrazolyl, furyl, imidazolyl, isoxazolyl, oxadi Azolyl, oxazolyl, pyrrolyl, pyridyl, pyrimidinyl, pyridazinyl, thiazolyl, triazolyl, thienyl, dihydrothieno-pyrazolyl, thioindenyl, Carbazolyl, benzoimidazolyl, benzothienyl, benzofuryl, indolyl, quinolinyl, benzotriazolyl, benzothiazolyl, benzoxazolyl, Benzimidazolyl, isoquinolyl, isoindolyl, acridinyl, benzisoxazolyl (benzoisazolyl), dimethylhydantoin, pyrazinyl, tetrahydrofuranyl, pyrrolinyl, pyrrolidine morpholinyl, indolyl, diazepinyl, azepinyl, thiepinyl, piperidinyl and oxa Cycloheptatrienyl (oxepinyl). In another aspect, the optionally substituted heterocycloalkyl moiety or optionally substituted heteroaryl moiety is optionally substituted furyl. Non-limiting examples of heterocyclic compounds include (1,2-dihydroxyethyl)-3,4-dihydrofuran-2(5H)-one; 4-hydroxy-5-methyl-3-furanone; 5 -Hydroxy-2(5H)-furanone; [1,2-dihydroxyethyl]furan-2,3,4(5H)-trione; α-hydroxy-γ-butyrolactone; Ribonic acid γ-endo Esters; aldohexuronicaldohexuronic acid gamma-lactone; glucono delta-lactone; 4-hydroxycoumarin; dihydrobenzofuran; 5-(hydroxymethyl)furfural; furfuralin (furoin); 2(5H)-furanone; 5,6-dihydro-2H-pyran-2-one; and 5,6-dihydro-4-hydroxy-6-methyl-2H-pyran- 2-ketones; or their salts or solvates.

The nitrogen-containing compound can be any suitable compound having one or more nitrogen atoms. In one aspect, the nitrogen-containing compound comprises an amine, imine, hydroxylamine or nitroxide moiety. Non-limiting examples of nitrogen-containing compounds include acetone oxime; violuric acid; pyridine-2-aldoxime; 2-aminophenol; 1,2-phenylenediamine; piperidinyloxy; 5,6,7,8-tetrahydrobiopterin; 6,7-dimethyl-5,6,7,8-tetrahydropterin; and maleamic acid; or salts or solvates.

The quinone compound may be any suitable compound comprising a quinone moiety described herein. Non-limiting examples of quinone compounds include 1,4-benzoquinone; 1,4-naphthoquinone; 2-hydroxy-1,4-naphthoquinone; 2,3-dimethoxy-5-methyl-1,4 - Benzoquinone or coenzyme Q ₀ ; 2,3,5,6-tetramethyl-1,4-benzoquinone or tetramethyl-p-benzoquinone; 1,4-dihydroxyanthraquinone; 3-hydroxy-1-methanol 4-tert-butyl-5-methoxy-1,2-benzoquinone; pyrroloquinoline quinone (pyrroloquinoline quinone); or their salts or solvates.

The sulfur-containing compound may be any suitable compound containing one or more sulfur atoms. In one aspect, the sulfur-containing compound comprises a moiety selected from the group consisting of thionyl, thioether, sulfinyl, sulfonyl, sulfamide, sulfonamide, sulfonic acid and sulfonate. Non-limiting examples of sulfur-containing compounds include ethanethiol; 2-propanethiol; 2-propene-1-thiol; 2-mercaptoethanesulfonic acid; benzenethiol; Cystine; Methionine; Glutathione; Cystine; or a salt or solvate thereof.

In one aspect, the effective amount of a compound as described above to the cellulosic material is from about 10 −6 to about 10, such as from about ^{10 −6 to} about 7.5, from about 10 ⁻⁶ to about 5, about 10 ^-6 to about 2.5, about 10 ^-6 to about 1, about 10 ^-5 to about 1, about 10 ^-5 to about 10 ^-1 , about 10 ^-4 to about 10 ^-1 , about 10 ^{- 3} to about 10 ^-1 , and about 10 ^-3 to about 10 ^-2 . In another aspect, the effective amount of the compound as described above is about 0.1 μM to about 1M, such as about 0.5 μM to about 0.75M, about 0.75 μM to about 0.5M, about 1 μM to about 0.25M, about 1 μM to about 0.1 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.

The term "liquor" means that the lignocellulosic and/or hemicellulosic material, or its monosaccharides such as xylose, arabinose, mannose etc., the resulting solution phase, ie, aqueous phase, organic phase, or a combination thereof, and its soluble contents. Cellulolytically enhanced liquid formulations for GH61 polypeptides can be produced by applying heat and/or pressure to a cellulosic or hemicellulosic material (or feedstock) and then separating the solution from the residual solids, optionally at This is done in the presence of a catalyst such as an acid, optionally in the presence of an organic solvent, and optionally in combination with physical disruption of the material. Such conditions determine the degree of cellulolytic enhancement achievable by the combination of the solution and the GH61 polypeptide during the hydrolysis of cellulosic material with the cellulase preparation. The liquor can be separated from the treated material using standard methods in the art such as filtration, sedimentation or centrifugation.

In one aspect, the effective amount of the liquid for cellulose is from about 10 ⁻⁶ to about 10 g per gram of cellulose, for example from about 10 ⁻⁶ to about 7.5 g, from about 10 ⁻⁶ to about 5, from about 10 ⁻⁶ to about 2.5 g, about 10 ^-6 to about 1 g, about 10 ^-5 to about 1 g, about 10 ^-5 to about 10 ^-1 g, about 10 ^-4 to about 10 ^-1 g, about 10 ^-3 to about 10 ^{- 1} g, and about 10 ^-3 to about 10 ^-2 g per g of cellulose.

HTec(NovozymesA/S)、

Htec2(Novozymes A/S)、(Novozymes A/S)、(Novozymes A/S)、

HC(Novozymes A/S)、

Xylanase(Genencor)、XY(Genencor)、

XC(Genencor)、

TX-200A(AB Enzymes)、HSP6000Xylanase(DSM)、DEPOL^TM333P(Biocatalysts Limit,Wales,UK)、DEPOL^TM740L(Biocatalysts Limit,Wales,UK)和DEPOL^TM762P(Biocatalysts Limit,Wales,UK)。In one embodiment, the one or more (eg, several) hemicellulolytic enzymes comprise a commercial hemicellulolytic enzyme preparation. Examples of commercial hemicellulolytic enzyme preparations suitable for use in the present invention include, for example, SHEARZYME ^™ (Novozymes A/S),

HTec (Novozymes A/S),

Htec2 (Novozymes A/S), (Novozymes A/S), (Novozymes A/S),

HC (Novozymes A/S),

Xylanase (Genencor), XY (Genencor),

XC (Genencor),

TX-200A (AB Enzymes), HSP6000Xylanase (DSM), DEPOL ^™ 333P (Biocatalysts Limit, Wales, UK), DEPOL ^™ 740L (Biocatalysts Limit, Wales, UK) and DEPOL ^™ 762P (Biocatalysts Limit, Wales, UK).

Examples of xylanases useful in the methods of the invention include, but are not limited to, Aspergillus aculeatus xylanase (GeneSeqP: AAR63790; WO94/21785), Aspergillus fumigatus xylanase (WO2006/078256) , Penicillium pinophilum (WO2011/041405), Penicillium sp. (WO2010/126772), Thielavia terrestris NRRL8126 (WO2009/079210) and Trichophyllum saccharomyces GH10 (WO2011/057083).

Examples of beta-xylosidases useful in the methods of the invention include, but are not limited to, Trichoderma reesei beta-xylosidase (UniProtKB/TrEMBL Accession No. Q92458), Talaromyces emersonii (SwissProt accession number Q8X212), and Neurospora crassa (SwissProt accession number Q7SOW4).

Examples of acetylxylan esterases that can be used in the methods of the invention include, but are not limited to, those from Aspergillus aculeatus (WO2010/108918), Chaetomium globosum (Uniprot accession number Q2GWX4), Chaetomium gracile) (GeneSeqP accession number AAB82124), Humicola insolens DSM1800 (WO2009/073709), Hypocrea jecorina (WO2005/001036), Myceliophthora thermophila (Wo2010/014880), Acetyl xylan esterases of Neurospora crassa (UniProt accession number q7s259), Phaeosphaeria nodorum (Uniprot accession number QOUHJ1 ) and Thielavia terrestris NRRL8126 (WO2009/042846).

Examples of ferulic esterases useful in the methods of the invention include, but are not limited to, Humicola insolens DSM1800 (WO2009/076122) ferulic esterase, Neurospora crassa ferulic esterase (UniProt accession number Q9HGR3), and Neosartorya fischer (UniProt accession number A1D9T4) ferulic acid esterase.

Examples of arabinofuranosidases useful in the methods of the invention include, but are not limited to, enzymes from Aspergillus niger (GeneSeqP Accession No. AAR94170), Humicola insolens DSM1800 (WO2006/114094 and WO2009/073383) and M. . Arabinofuranosidase of giganteus (WO2006/114094).

Examples of alpha-glucuronidases that can be used in the methods of the invention include, but are not limited to, enzymes from Aspergillus clavatus (UniProt accession number alcc12), Aspergillus fumigatus (SwissProt accession number Q4WW45), Aspergillus niger (Uniprot accession number Q96WX9 ), Aspergillus terreus (SwissProt Accession No. Q0CJP9), Humicola insolens (WO2010/014706), Penicillium citricolor (WO2009/068565), Talonomyces emersonii (UniProt Accession No. Q8X211) and Rees α-Glucuronidase from Trichoderma (Uniprot Accession No. Q99024).

Polypeptides having enzymatic activity used in the methods of the present invention can be prepared by using methods known in the art (see, for example, Bennett, J.W. and LaSure, L. (eds.) on a nutrient medium containing suitable carbon and nitrogen sources and inorganic salts. ), More Gene Manipulations in Fungi, Academic Press, CA, 1991) were produced by fermenting the microbial strains indicated above. Suitable media are available from commercial suppliers or may be prepared according to published compositions (eg in catalogs of the American Type Culture Collection). Temperature ranges and other conditions suitable for growth and enzyme production are known in the art (see, e.g., Bailey, J.E. and Ollis, D.F., Biochemical Engineering Fundamentals, McGraw-Hill Book Company, NY, 1986).

The fermentation can be any method of culturing cells that results in the expression or isolation of enzymes or proteins. Thus, fermentation may be understood to include shake flask culture in a suitable medium and under conditions allowing the expression or isolation of said enzyme, or small- or large-scale fermentation in laboratory or industrial fermenters (including continuous, batch, fed-batch or solid state fermentation). The resulting enzymes produced by the methods described above can be recovered from the fermentation medium and purified by conventional methods.

The composition may be a fermentation broth formulation or a cell composition, as described herein. In some embodiments, the composition is a cell-killed whole culture solution comprising an organic acid, killed cells and/or cell debris, and culture medium.

In one aspect, the invention relates to a whole broth formulation or cell culture composition comprising one or more (eg several) enzymes having cellulolytic and/or hemicellulolytic activity and catalase active peptides.

The term "fermentation broth" is used herein to refer to a preparation resulting from the fermentation of cells that has undergone no or only minimal recovery and/or purification. For example, a fermentation broth is produced when a microbial culture is grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis (eg, enzyme expression by the host cell), and secretion into the cell culture medium. The fermentation broth may contain unfractionated or fractionated contents of the fermented material obtained at the end of the fermentation. Typically, the fermentation broth is unfractionated and comprises spent medium and cell debris present after removal of microbial cells (eg, filamentous fungal cells), eg, by centrifugation. In some embodiments, the fermentation broth contains spent cell culture medium, extracellular enzymes, and viable and/or nonviable microbial cells.

In one embodiment, the fermentation broth formulation and cell composition comprise a first organic acid component and a second organic acid component, the first organic acid component comprising at least one organic acid of 1-5 carbons and/or salts thereof, and the second organic acid component comprises at least one organic acid of 6 or more carbons and/or salts thereof. In a specific embodiment, the first organic acid component is acetic acid, formic acid, propionic acid, their salts, or a mixture of two or more of the foregoing, and the second organic acid component is benzoic acid , cyclohexanecarboxylic acid, 4-methylpentanoic acid, phenylacetic acid, their salts, or a mixture of two or more of the foregoing.

In one aspect, the composition contains an organic acid, and optionally also contains killed cells and/or cell debris. In one embodiment, the killed cells and/or cell debris are removed from the whole culture of killed cells to provide a composition free of these components.

The fermentation broth formulation or cell composition may further comprise preservatives and/or antimicrobial (eg bacteriostatic) agents, including but not limited to sorbitol, sodium chloride, potassium sorbate and others known in the art.

The whole broth or composition of killed cells may contain the unfractionated content of the fermented material obtained at the termination of the fermentation. Typically, the killed cell whole broth or composition contains spent medium that exists after microbial cells (e.g., filamentous fungal cells) are grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis and cell debris. In some embodiments, the cell-killed whole broth or composition contains spent cell culture medium, extracellular enzymes, and killed filamentous fungal cells. In some embodiments, microbial cells present in a cell-killing whole broth or composition can be permeabilized and/or lysed using methods known in the art.

Whole medium or cell compositions as described herein are typically liquid, but may contain insoluble components, such as killed cells, cell debris, media components and/or insoluble enzymes. In some embodiments, insoluble components can be removed to provide a clear liquid composition.

The whole broth formulations and cell compositions of the invention can be produced by the methods described in WO90/15861 or WO2010/096673.

In one aspect, the invention relates to the use of an enzyme composition of the invention for degrading or converting cellulosic material.

fermentation . Fermentable sugars obtained from hydrolyzed cellulosic material may be fermented by one or more (eg, 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 comprising a fermentation step. Fermentation processes also include fermentation processes used in the consumer alcohol industry (eg, beer and wine), dairy industry (eg, fermented milk products), leather industry, and tobacco industry. Fermentation conditions depend on the desired fermentation product and fermenting organism, and can be readily determined by those skilled in the art.

In the fermentation step, sugars released from the cellulosic material as a result of the pretreatment and enzymatic hydrolysis steps are fermented by a fermenting organism, such as yeast, into a product, eg, ethanol. As described herein, hydrolysis (saccharification) and fermentation can be separate or simultaneous.

Any suitable hydrolyzed cellulosic material may be used in the fermentation step of practicing the present invention. The materials are generally selected according to the desired fermentation product (ie, material to be obtained from the fermentation) and the method to be used, as is known in the art.

The term "fermentation medium" is understood herein to mean the medium prior to the addition of fermenting microorganisms, eg, the medium resulting from a saccharification process, and the medium used in a simultaneous saccharification and fermentation process (SSF).

"Fermenting microorganism" refers to any microorganism, including bacterial and fungal organisms, suitable for use in a desired fermentation process to produce a fermentation product. The fermenting organism can be a hexose and/or pentose sugar fermenting organism, or a combination thereof. Both hexose and pentose sugar fermenting organisms are well known in the art. Suitable fermenting microorganisms are capable of directly or indirectly fermenting (i.e., converting) sugars (e.g., glucose, xylose, xylulose, arabinose, maltose, mannose, galactose, and/or oligosaccharides) into the desired fermentation product . Examples of bacterial and fungal fermenting organisms that can produce ethanol are described in Lin et al., 2006, Appl. Microbiol. Biotechnol. 69:627-642.

Examples of fermenting microorganisms capable of fermenting hexose sugars include bacterial and fungal organisms, such as yeast. Preferred yeasts include strains of the genera Candida, Kluyveromyces, and Saccharomyces, such as Candida sonorensis, Kluyveromyces marx and Saccharomyces cerevisiae.

Examples of fermenting organisms capable of fermenting pentose sugars in their native state include bacterial and fungal organisms, such as some yeasts. Preferred xylose fermenting yeasts include strains of the genus Candida, preferably Candidasheatae or Candida sonorensis; and strains of the genus Pichia, preferably Pichiastipitis, such as Pichia stipitis Strains of CBS5773. Preferred pentose-fermenting yeasts include strains of the genus Pachysolen, preferably Pachysolen tannophilus. Organisms that are not capable of fermenting pentose sugars such as xylose and arabinose can be genetically modified to ferment pentose sugars by methods known in the art.

Bacteria that efficiently ferment hexose and pentose sugars to ethanol include, for example, Bacillus coagulans, Clostridium acetobutylicum, Clostridium thermocellum, Clostridium phytofermentans, Geobacillus Bacillus species, Thermoanaerobacter saccharolyticum and Zymomonas mobilis (Philippidis, 1996, supra).

Other fermenting organisms include Bacillus species such as Bacillus coagulans; Candida species such as Candidasonorensis, C. methanosorbosa, Candida diddensii, Candida parapsilosis, C. naedodendra, C. blankii, C. entomophilia, C. brassicae, Candida pseudotropicalis, Candida boidinii, Candida utilis and Candida scehatae (C. scehatae); Clostridium species, such as Clostridium acetobutylicum, Clostridium thermocellum, and C. phytofermentans; Escherichia coli, especially strains of E. coli that have been genetically modified to increase ethanol production; Geobacillus sp. Bacillus species; Hansenula, such as Hansenula anomala; Klebsiella, such as Klebsiella oxytoca; Kluyveromyces, such as Kluyveromyces marx, K. lactis, K. thermotolerans, and K. fragilis; Schizosaccharomyces, such as S. pombe; Thermoanaerobacter (Thermoanaerobacter), such as Thermoanaerobacter saccharolyticus, and Zymomonas, such as strains of Zymomonas mobilis.

In a preferred aspect, the yeast is Bretannomyces. In a more preferred aspect, the yeast is Bretannomyces clausenii. In another more preferred aspect, the yeast is Candida. In another more preferred aspect, the yeast is Candida sonorensis. In another more preferred aspect, the yeast is Candida boidinii. In another more preferred aspect, the yeast is Candida blankii. In another more preferred aspect, the yeast is Candida brassicae. In another more preferred aspect, the yeast is Candida didansis. In another more preferred aspect, the yeast is Candida entomophilia. In another more preferred aspect, the yeast is Candida pseudotropicalis. In another more preferred aspect, the yeast is Candida shehata. In another more preferred aspect, the yeast is Candida utilis. In another preferred aspect, the yeast is Clavispora. In another more preferred aspect, the yeast is Clavispora lusitaniae. In another more preferred aspect, the yeast is Clavispora opuntiae. In another preferred aspect, the yeast is Kluyveromyces. In another more preferred aspect, the yeast is Kluyveromyces fragilis. In another more preferred aspect, the yeast is Kluyveromyces marxense. In another more preferred aspect, the yeast is Kluyveromyces thermotolerans. In another preferred aspect, the yeast is Pachysolen. In another more preferred aspect, the yeast is Pachysomyces tannophilus. In another preferred aspect, the yeast is Pichia pastoris. In another more preferred aspect, the yeast is Pichia stipitis. In another preferred aspect, the yeast is a Saccharomyces species. In another preferred aspect, the yeast is Saccharomyces cerevisiae. In another more preferred aspect, the yeast is Saccharomyces distaticus. In another more preferred aspect, the yeast is Saccharomyces uvarum.

In a preferred aspect, the bacteria is a Bacillus species. In a more preferred aspect, the bacterium is Bacillus coagulans. In another more preferred aspect, the bacterium is Clostridium. In another more preferred aspect, the bacterium is Clostridium acetobutylicum. In another more preferred aspect, the bacterium is Clostridium phytofermentans. In another more preferred aspect, the bacterium is Clostridium thermocellum. In another more preferred aspect, the bacterium is a Geobacillus species. In another more preferred aspect, the bacterium is a Thermoanaerobacter species. In another more preferred aspect, the bacterium is Thermoanaerobacter saccharolyticus. In another more preferred aspect, the bacterium is a Zymomonas species. In another more preferred aspect, the bacterium is Zymomonas mobilis.

Commercially available yeast suitable for ethanol production include, for example, BIOFERM ^™ AFT and XR (NABC-North American Bioproducts Corporation, GA, USA), ETHANOL RED ^™ Yeast (Red Star/Lesaffre, USA), FALI ^™ (Fleischmann's Yeast, Burns Philp Food Inc., USA), FERMIOL ^™ (DSM Specialties), GERT STRAND ^™ (Gert Strand AB, Sweden) and SUPERSTART ^™ and THERMOSACC ^™ fresh yeast (Ethanol Technology, WI, USA).

In a preferred aspect, the fermenting microorganism has been genetically modified to provide the ability to ferment pentose sugars, such as xylose-utilizing, arabinose-utilizing, and xylose-arabinose-utilizing microorganisms.

Organisms capable of converting hexose and pentose sugars into ethanol (co-fermentation) have been constructed by cloning heterologous genes into a variety of fermenting microorganisms (Chen and Ho, 1993, Cloning and improving the expression of Pichia stipitis xylose reductase gene in Saccharomyces cerevisiae, Appl.Biochem.Biotechnol.39-40:135-147; Ho et al., 1998, Genetically engineered Saccharomyces yeast capable of effectively cofermenting glucose and xylose, Appl.Environ.Microbiol.64:1852-1859; Kotter and Ciriacy, 1993, Xylose fermentation bySaccharomyces cerevisiae,Appl.Microbiol.Biotechnol.38:776-783;Walfridsson等,1995,Xylose-metabolizing Saccharomyces cerevisiae strains overexpressing theTKL1and TAL1genes encoding the pentose phosphate pathway enzymestransketolase and transaldolase,Appl.Environ.Microbiol.61:4184- 4190;Kuyper等,2004,Minimal metabolic engineering of Saccharomyces cerevisiae for efficientanaerobic xylose fermentation:a proof of principle,FEMS Yeast Research4:655-664;Beall等,1991,Parametric studies of ethanol production from xylose and other sugarsby recombinant Escherichia coli, Biotech.Bioeng.38:296-303; Ingram et al., 1998, Metabolic engine ring of bacteria for ethanol production, Biotechnol.Bioeng.58:204-214; Zhang et al., 1995, Metabolic engineering of a pentose metabolism pathway inethanologenic Zymomonas mobilis, Science267:240-243; Deanda et al., 1996, Development of arabinose Zymomonas mobilis strain by metabolic pathway engineering, Appl. Environ. Microbiol. 62:4465-4470; WO2003/062430, xylose isomerase).

In a preferred aspect, the genetically modified fermenting microorganism is Candida sonorensi. In another preferred aspect, the genetically modified fermenting microorganism is Escherichia coli. In another preferred aspect, the genetically modified fermenting microorganism is Klebsiella oxytoca. In another preferred aspect, the genetically modified fermenting microorganism is Kluyveromyces marxii. In another preferred aspect, the genetically modified fermenting microorganism is Saccharomyces cerevisiae. In another preferred aspect, the genetically modified fermenting microorganism is Zymomonas mobilis.

It is well known in the art that the organisms described above can also be used to produce other substances, as described herein.

Typically the fermenting microorganisms are added to the degraded cellulosic material or hydrolyzate and fermentation is carried out for about 8 to about 96 hours, for example about 24 to about 60 hours. The temperature is typically from about 26°C to about 60°C, eg about 32°C or 50°C, and at about pH 3 to about pH 8, eg about pH 4-5, 6 or 7.

In one aspect, yeast and/or another microorganism is applied to the degraded cellulosic material and fermentation is carried out for about 12 to about 96 hours, such as typically 24-60 hours. In another aspect, the temperature is preferably from about 20°C to about 60°C, such as from about 25°C to about 50°C, and from about 32°C to about 50°C, from about 32°C to about 50°C, and the pH is typically from about pH 3 to about pH 7, such as about pH 4 to about pH 7. However, some fermenting organisms, such as bacteria, have higher optimum fermentation temperatures. Yeast or another microorganism is preferably applied in an amount of about 10 ⁵ -10 ¹² , preferably about 10 ⁷ -10 ¹⁰ , especially about 2×10 ⁸ viable cell counts per ml of fermentation broth. Further guidance on fermentation with yeast can be found, for example, in "The Alcohol Textbook" (eds. K. Jacques, TP Lyons and DR Kelsall, Nottingham University Press, United Kingdom 1999), which is incorporated herein by reference.

For ethanol production, after fermentation, the fermented slurry is distilled to extract ethanol. The ethanol obtained according to the method of the present invention can be used, for example, as fuel ethanol, drinking ethanol, ie drinkable neutral wine, or industrial ethanol.

Fermentation stimulators can be used in combination with any of the methods described herein to further improve the fermentation process, particularly to improve the performance of the fermenting microorganism, eg, rate increase and ethanol yield. "Fermentation stimulant" refers to a stimulant for the growth of fermenting microorganisms, particularly yeast. Preferred fermentation stimulators for growth include vitamins and minerals. Examples of vitamins include multivitamins, biotin, pantothenic acid (salts), niacin, meso-inositol, thiamine, pyridoxine, p-aminobenzoic acid, folic acid, riboflavin, and Vitamins A, B, C, D and E. See, e.g., Alfenore et al., Improving ethanol production and viability of Saccharomyces cerevisiae 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 capable of providing nutrients including P, K, Mg, S, Ca, Fe, Zn, Mn, and Cu.

Fermentation product : A fermentation product can be any substance derived from fermentation. The fermentation product can be, without limitation, an alcohol (e.g., arabic alcohol, n-butanol, isobutanol, ethanol, glycerol, methanol, ethylene glycol, 1,3-propanediol [propylene glycol], butanediol, glycerol, sorbitol and xylitol); alkanes (such as pentane, hexane, heptane, octane, nonane, decane, undecane and dodecane); cycloalkanes (such as cyclopentane, cyclohexane, cyclo heptane, and cyclooctane); alkenes (e.g., pentene, hexene, heptene, and octene); amino acids (e.g., aspartic acid, glutamic acid, glycine, lysine, serine, and threonine) ; gases (e.g., methane, hydrogen (H ₂ ), carbon dioxide (CO ₂ ), and carbon monoxide (CO)); isoprene; ketones (e.g., acetone); organic acids (e.g., acetic acid, acetonic acid, hexadiene Acid, Ascorbic Acid, Citric Acid, 2,5-Diketo-D-Gluconic Acid, Formic Acid, Fumaric Acid, Glucaric Acid, Gluconic Acid, Glucuronic Acid, Glutaric Acid, 3-Hydroxypropane acid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid, oxaloacetic acid, propionic acid, succinic acid, and xylonic acid); and polyketides. The fermentation product can also be protein as a high value product.

In a preferred aspect, the fermentation product is alcohol. It will be understood that the term "alcohol" includes substances comprising one or more hydroxyl moieties. In a more preferred aspect, the alcohol is n-butanol. In another more preferred aspect, the alcohol is isobutanol. In another more preferred aspect, the alcohol is ethanol. In another more preferred aspect, the alcohol is methanol. In another more preferred aspect, the alcohol is arabitol. In another more preferred aspect, the alcohol is butanediol. In another more preferred aspect, the alcohol is ethylene glycol. In another more preferred aspect, the alcohol is glycerin. In another more preferred aspect, the alcohol is glycerol. In another more preferred aspect, the alcohol is 1,3-propanediol. In another more preferred aspect, the alcohol is sorbitol. In another more preferred aspect, the alcohol is xylitol. See, e.g., Gong, C.S., Cao, N.J., Du, J., and Tsao, G.T., 1999, Ethanol production from renewable resources, in Advances in Biochemical Engineering/Biotechnology, Scheper, T. eds., Springer-Verlag Berlin Heidelberg, Germany, 65:207-241; Silveira, M.M., and Jonas, R., 2002, The biotechnological production of sorbitol, Appl. Microbiol. Biotechnol.59: 400-408; Nigam, P. and Singh, D., 1995, Processes for fermentative production of xylitol–a sugar substitute, Process Biochemistry30(2):117-124; Ezeji, T.C., Qureshi, N. and Blaschek, H.P., 2003, Production of acetone, butanol and ethanol by Clostridium beijerinckii ping by BA101and over ysitu gas rec , World Journal of Microbiology and Biotechnology 19(6):595-603.

In another preferred aspect, the fermentation product is an alkane. The alkanes may be unbranched or branched alkanes. In another more preferred aspect, the alkane is pentane. In another more preferred aspect, the alkane is hexane. In another more preferred aspect, the alkane is heptane. In another more preferred aspect, the alkane is octane. In another more preferred aspect, the alkane is nonane. In another more preferred aspect, the alkane is decane. In another more preferred aspect, the alkane is undecane. In another more preferred aspect, the alkane is dodecane.

In another preferred aspect, the fermentation product is a naphthene. In another more preferred aspect, the cycloalkane is cyclopentane. In another more preferred aspect, the cycloalkane is cyclohexane. In another more preferred aspect, the cycloalkane is cycloheptane. In another more preferred aspect, the cycloalkane is cyclooctane.

In another preferred aspect, the fermentation product is an alkene. The olefins may be unbranched or branched olefins. In another more preferred aspect, the olefin is pentene. In another more preferred aspect, the olefin is hexene. In another more preferred aspect, the olefin is heptene. In another more preferred aspect, the olefin is octene.

In another preferred aspect, the fermentation product is an amino acid. In another more preferred aspect, the organic acid is aspartic acid. In another more preferred aspect, the amino acid is glutamic acid. In another more preferred aspect, the amino acid is glycine. In another more preferred aspect, the amino acid is lysine. In another more preferred aspect, the amino acid is serine. In another more preferred aspect, the amino acid is threonine. See, eg, Richard, A. and Margaritis, A., 2004, Empirical modeling of batch fermentation kinetics for poly (glutamic acid) production and other microbial biopolymers, Biotechnology and Bioengineering 87(4):501-515.

In another preferred aspect, the substance is a gas. In another more preferred aspect, the gas is methane. In another more preferred aspect, the gas is _H2 . In another more preferred aspect, the gas is _CO2 . In another more preferred aspect, the gas is CO. See, for example, Kataoka, N., A. Miya, and K. Kiriyama, 1997, Studies on hydrogen production by continuous culture system of hydrogen-producing anaerobic bacteria, Water Science and Technology 36(6-7):41-47; and Gunaseelan, VN, 1997, in Biomass and Bioenergy, volume 13 (1-2): 83-114 pages, 1997, Anaerobic digestion of biomass for methane production: A review.

In another preferred aspect, the fermentation product is isoprene.

In another preferred aspect, the fermentation product is a ketone. It should be understood that the term "ketone" covers substances containing one or more ketone moieties. In another more preferred aspect, the ketone is acetone. See, eg, Qureshi and Blaschek, 2003, supra.

In another preferred aspect, the fermentation product is an organic acid. In another more preferred aspect, the organic acid is acetic acid. In another more preferred aspect, the organic acid is acetonic acid. In another more preferred aspect, the organic acid is adipic acid. In another more preferred aspect, the organic acid is ascorbic acid. In another more preferred aspect, the organic acid is citric acid. In another more preferred aspect, the organic acid is 2,5-diketo-D-gluconic acid. In another more preferred aspect, the organic acid is formic acid. In another more preferred aspect, the organic acid is fumaric acid. In another more preferred aspect, the organic acid is glucaric acid. In another more preferred aspect, the organic acid is gluconic acid. In another more preferred aspect, the organic acid is glucuronic acid. In another more preferred aspect, the organic acid is glutaric acid. In another preferred aspect, the organic acid is 3-hydroxypropionic acid. In another more preferred aspect, the organic acid is itaconic acid. In another more preferred aspect, the organic acid is lactic acid. In another more preferred aspect, the organic acid is malic acid. In another more preferred aspect, the organic acid is malonic acid. In another more preferred aspect, the organic acid is oxalic acid. In another more preferred aspect, the organic acid is propionic acid. In another more preferred aspect, the organic acid is succinic acid. In another more preferred aspect, the organic acid is xylonic acid. See, e.g., Chen, R. and Lee, Y.Y., 1997, Membrane-mediated extractive fermentation for lactic acid production from cellulosic biomass, Appl. Biochem. Biotechnol. 63-65:435-448.

In another preferred aspect, the substance is a polyketide.

Recovery Fermentation product can optionally be recovered from the fermentation medium using any method known in the art, including, but not limited to, chromatography, electrophoretic methods, differential solubility, distillation, or extraction. For example, the alcohol is separated and purified from the fermented cellulosic material by conventional distillation methods. Ethanol is available in a purity of up to about 96 vol.%, which can be used, for example, as fuel ethanol, potable ethanol (ie, a drinkable neutral alcoholic beverage), or industrial ethanol.

Polypeptides with catalase activity

In the methods of the present invention, the polypeptide having catalase activity can be any polypeptide having catalase activity. A polypeptide having catalase activity may be present as an enzyme in an enzyme composition and/or as one or more protein components added to said composition. In a preferred aspect, the polypeptide having catalase activity is foreign to one or more components of the cellulase composition.

Polypeptides having catalase activity can be obtained from microorganisms of any genus. In one aspect, the polypeptide obtained from a given source is secreted extracellularly.

The polypeptide having catalase activity may be a bacterial polypeptide. For example, the polypeptide can be a Gram-positive bacterial polypeptide such as Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterobacteriaceae having catalase activity. Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus, or Oceanobacillus polypeptides; or Gram-negative bacteria Polypeptides such as E. coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacterium with catalase activity (Flavobacterium), Fusobacterium, Ilyobacter, Neisseria or Ureaplasma polypeptides.

In one aspect, the polypeptide having catalase activity is Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, cyclic Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis or Bacillus thuringiensis polypeptides.

In another aspect, the polypeptide having catalase activity is Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus uberis having catalase activity. Streptococcus equi subsp. Zooepidemicus polypeptide.

In another aspect, the polypeptide having catalase activity is Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, gray Streptomyces griseus or Streptomyces lividans polypeptide.

The polypeptide having catalase activity can also be a fungal polypeptide, and more preferably a yeast polypeptide such as Candida, Kluyveromyces, Pichia, etc. having catalase activity. A polypeptide of the genus Pichia, Saccharomyces, Schizosaccharomyces or Yarrowia; or more preferably a filamentous fungal polypeptide such as Acremonium having catalase activity ), Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Gold Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Chromasporium ( Diplodia), Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichoderma phaea), Verticillium, Volvariella or Xylaria polypeptides.

In another aspect, the polypeptide is Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces skluyveri having catalase activity. ), Saccharomyces norbensis or Saccharomycesoviformis polypeptides.

In another aspect, the polypeptide is Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium tropicalis (Chrysosporium tropicum), Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwell ), Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum oxysporum), Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Sulfur Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicola insens olens), Humicola lanuginosa, Irpex lacteus, Mucormiehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium emersonii, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Talaromyces stipitatus, Thermoascus aurantiacus, Thielavia ( Thielavia achromatica), Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia tumefaciens (Thielaviaspededonium), Thielavia setosa, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichodermakoningii, Trichoderma longibrachiatum, Trichoderma reesei or Trichoderma viride polypeptides.

In a preferred embodiment, the polypeptide having catalase activity is a catalase from Thermoascus, Talaromyces, Humicola or Penicillium. In a more preferred embodiment, the polypeptide having catalase activity is a catalase from Thermoascus aurantiacus, Talaromyces stipitatus, Humicola insolens or Penicillium emersonii.

Non-limiting examples of suitable catalases and their coding sequences are listed in the table below.

SEQ ID NO: 1 and 2: Polynucleotides and polypeptides from catalase from Thermoascus aurantiacus as described in JP2004261137A.

SEQ ID NO: 3 and 4: Polynucleotides and polypeptides of catalase from Talaromyces stipitatus, which can be prepared as described in Examples 9-13.

SEQ ID NOs: 5 and 6: Polynucleotides and polypeptides of catalase from Humicola insolens, which can be prepared as described in Examples 14-20.

SEQ ID NOs: 7 and 8: Polynucleotides and polypeptides of catalase from Penicillium emersonii, which can be prepared as described in Examples 21-27.

SEQ ID NO: 9 and 10: Polynucleotides and polypeptides of catalase from Thermus Brockianus, as described in WO2005/044994.

SEQ ID NO: 11 and 12: Polynucleotides and polypeptides of catalase from Saccharomyces pastorianus as described in WO2007/105350.

SEQ ID NO: 13 and 14: Polynucleotides and polypeptides of catalase from Saccharomyces pastorianus as described in WO2007/105350.

SEQ ID NO: 15 and 16: Polynucleotides and polypeptides of catalase from Penicillium pinophilum as described in WO2009/104622.

SEQ ID NO: 17 and 18: Polynucleotides and polypeptides of catalase from Humicola grisea, as described in WO2009/104622.

SEQ ID NO: 19 and 20: Polynucleotides and polypeptides of catalase from Thielavia terrestris as described in WO2010/074972.

SEQ ID NO: 21 and 22: polynucleotides and polypeptides from catalase of Bacillus thermoglucosidasius as described in JP11243961A.

SEQ ID NO: 23 and 24: Polynucleotides and polypeptides of catalase from Aspergillus oryzae as described in JP2002223772A.

SEQ ID NO: 25 and 26: Polynucleotides and polypeptides of catalase from Thermoascus aurantiacus, as described in JP2007143405A.

SEQ ID NO: 27 and 28: Polynucleotides and polypeptides of catalase from Bacillus thermoglucosidase, as described in US6,022,721.

SEQ ID NO: 29 and 30: Polynucleotides and polypeptides of catalase from Bacillus thermoglucosidase, as described in US6,022,721.

SEQ ID NO: 31 and 32: Polynucleotides and polypeptides from catalase from Alcaligenes aquamarinus as described in WO98/00526.

SEQ ID NO: 33 and 34: polynucleotides and polypeptides from catalase from blackening Microscilla furvescens as described in WO98/00526.

SEQ ID NO: 35 and 36: Polynucleotides and polypeptides of catalase from Aspergillus niger, as described in US5,360,901.

SEQ ID NO37: A polypeptide of Humicola niger heat-tolerant catalase (GENESEQP: AXQ55105, disclosed in WO2009104622).

In one embodiment, the catalase used in the present invention is matured with the mature polypeptide of SEQ ID NO:2, the mature polypeptide of SEQ ID NO:4, the mature polypeptide of SEQ ID NO:6, the mature polypeptide of SEQ ID NO:8 Polypeptide, the mature polypeptide of SEQ ID NO:10, the mature polypeptide of SEQ ID NO:12, the mature polypeptide of SEQ ID NO:14, the mature polypeptide of SEQ ID NO:16, the mature polypeptide of SEQ ID NO:18, SEQ ID NO: The mature polypeptide of 20, the mature polypeptide of SEQ ID NO:22, the mature polypeptide of SEQ ID NO:24, the mature polypeptide of SEQ ID NO:26, the mature polypeptide of SEQ ID NO:28, the mature polypeptide of SEQ ID NO:30, The mature polypeptide of SEQ ID NO:32, the mature polypeptide of SEQ ID NO:34, the mature polypeptide of SEQ ID NO:36, the mature polypeptide of SEQ ID NO:37 have at least 60%, such as at least 65%, at least 70%, at least 75%, at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% , at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, which has catalase active.

The SignalP program predicts that amino acids 1 to 19 of SEQ ID NO:4 are the signal peptide and the mature polypeptide is amino acids 20 to 733 of SEQ ID NO:4. In another aspect, amino acids 1 to 19 of SEQ ID NO: 6 are predicted to be a signal peptide according to the SignalP program, and the mature polypeptide is amino acids 20 to 765 of SEQ ID NO: 6. In another aspect, amino acids 1 to 19 of SEQ ID NO: 8 are predicted to be a signal peptide according to the SignalP program, and the mature polypeptide is amino acids 20 to 741 of SEQ ID NO: 8. It is known in the art that a host cell can produce a mixture of two or more different mature polypeptides (ie, having different C-terminal and/or N-terminal amino acids) expressed from the same polynucleotide.

In another embodiment, the catalase used in the present invention is encoded by a polynucleotide that operates under low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions. Hybridizes under stringent conditions to: (i) the mature polypeptide coding sequence of SEQ ID NO:1, the mature polypeptide coding sequence of SEQ ID NO:3, the mature polypeptide coding sequence of SEQ ID NO:5, or the mature polypeptide coding sequence of SEQ ID NO:7 Polypeptide coding sequences, (ii) their cDNA sequences, or (iii) the full-length complementary strands of (i) or (ii) (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York) .

For purposes of the present invention, hybridization indicates that the polynucleotide hybridizes under very low to very high stringency conditions to nucleic acid probes corresponding to the following labels: (i) SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO :5 or SEQ ID NO:7; (ii) the mature polypeptide coding sequence of SEQ ID NO:1, the mature polypeptide coding sequence of SEQ ID NO:3, the mature polypeptide coding sequence of SEQ ID NO:5, or the mature polypeptide coding sequence of SEQ ID NO: 7; (iii) their cDNA sequences; or (iv) their full-length complementary strands; or their subsequences. Molecules that hybridize to the nucleic acid probe under these conditions can be detected using, for example, x-rays or any other means of detection known in the art.

In one embodiment, the catalase used in the present invention is encoded by a polynucleotide that is identical to the mature polypeptide coding sequence of SEQ ID NO: 1, the mature polypeptide coding sequence of SEQ ID NO: 3, SEQ ID NO: The mature polypeptide coding sequence of ID NO:5, or the mature polypeptide coding sequence of SEQ ID NO:7, or their cDNA sequences have at least 60%, such as at least 65%, at least 70%, at least 75%, at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92% , at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

In one embodiment, the mature polypeptide coding sequence is nucleotides 1 to 2351 of SEQ ID NO: 1 or a cDNA sequence thereof. In another embodiment, the signal peptide encoded by nucleotides 1 to 57 of SEQ ID NO:3 is predicted according to the SignalP program, and the mature polypeptide coding sequence is nucleotides 58 to 2418 of SEQ ID NO:3 or its cDNA sequence. In another embodiment, the signal peptide encoded by nucleotides 1 to 57 of SEQ ID NO:5 is predicted according to the SignalP program, and the mature polypeptide coding sequence is nucleotides 58 to 3040 of SEQ ID NO:5 or its cDNA sequence. In another embodiment, the signal peptide encoded by nucleotides 1 to 57 of SEQ ID NO:7 is predicted according to the SignalP program, and the mature polypeptide coding sequence is nucleotides 58 to 2476 of SEQ ID NO:7 or its cDNA sequence.

In another embodiment, the catalase used in the present invention relates to a variant of the mature polypeptide of SEQ ID NO:2, a variant of the mature polypeptide of SEQ ID NO:4, a variant of the mature polypeptide of SEQ ID NO:6 Variants, or variants of the mature polypeptide of SEQ ID NO: 8, which comprise substitutions, deletions and/or insertions at one or more (eg several) positions. In one embodiment, amino acid substitutions, deletions and/or insertions of the mature polypeptide of SEQ ID NO:2, the mature polypeptide of SEQ ID NO:4, the mature polypeptide of SEQ ID NO:6, the mature polypeptide of SEQ ID NO:8 are introduced The number is up to 10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Amino acid changes may be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; typically small deletions of 1 to about 30 amino acids; small amino- or carboxy-terminal extensions of amino- or carboxyl-terminal extensions, such as amino-terminal methionine residues; small linker peptides of up to approximately 20-25 residues; or small extensions that facilitate purification by altering net charge or other functions, such as polyhistidine acid sequence (poly histidine tract), antigenic epitope (antigenic epitope) or binding domain (binding domain).

Examples of conservative substitutions are within the following groups: the group of basic amino acids (arginine, lysine, and histidine), the group of acidic amino acids (glutamic acid and aspartic acid), the group of polar amino acids (glutamine amides and asparagine), hydrophobic amino acid groups (leucine, isoleucine, and valine), aromatic amino acid groups (phenylalanine, tryptophan, and tyrosine), and small amino acid groups (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions which generally do not alter a particular activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979, in The Proteins, Academic Press, New York. The most commonly occurring exchanges 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.

Alternatively, amino acid changes have the property of resulting in changes in the physicochemical properties of the polypeptide. For example, amino acid changes can improve the thermal stability of the polypeptide, change the substrate specificity, change the optimal pH, etc.

Essential amino acids in the parental polypeptide can be identified according to methods 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, single alanine mutations are introduced at every residue in the molecule, and the resulting mutated molecules are tested for catalase activity to identify amino acid residues critical to the activity of the molecule. See also Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. Enzyme active sites or other biological interactions can also be determined by physical analysis of structures determined by techniques such as NMR, crystallography, electron diffraction or photoaffinity labeling, combined with mutations to putative contact site amino acids. See, eg, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identity of essential amino acids can also be inferred from alignments with related polypeptides.

Known methods of mutagenesis, recombination and/or shuffling can be used, followed by related screening procedures, as described by Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86:2152-2156; WO95/17413; or those disclosed in WO95/22625, one or more amino acid substitutions, deletions and/or insertions are made and tested. Other methods that can be used include error-prone PCR, phage display (e.g. Lowman et al., 1991, Biochemistry 30:10832-10837; U.S. Pat. No. 5,223,409; WO92/06204) and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; et al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect the activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). Mutagenized DNA molecules encoding active polypeptides can be recovered from host cells and rapidly sequenced using methods standard in the art. These methods allow rapid determination of the importance of individual amino acid residues in polypeptides.

The polypeptide may be a hybrid polypeptide in which a region of one polypeptide is fused to the N- or C-terminus of a region of another polypeptide.

The polypeptide may be a fusion polypeptide or a cleavable fusion polypeptide wherein another polypeptide is fused to the N- or C-terminus of the polypeptide of the invention. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the invention. Techniques for producing fusion polypeptides are known in the art and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fused polypeptide is under the control of the same promoter and terminator. Fusion polypeptides can also be constructed using intein technology, where fusions are generated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266:776-779).

Fusion polypeptides can also contain a cleavage site between the two polypeptides. Once the fusion polypeptide is secreted, the site is cleaved, releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, those disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3:568-76; Svetina et al., 2000, J. Biotechnol. etc., 1997, Appl.Environ.Microbiol.63:3488-3493; Ward et al., 1995, Biotechnology 13:498-503; 512); Collins-Racie et al., 1995, Biotechnology 13:982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6:240-248; and sites in Stevens, 2003, Drug Discovery World 4:35-48 .

Examples of commercial catalase preparations suitable for use in the present invention include, for example, Terminox Ultra50L/200L (Novozymes A/S), Catazyme 25L (Novozymes A/S), GC118 (Danisco A/S), Oxygone T100/T400 (Danisco A/S), /S), ASC Super200L (Mitsubishi Chemicals, Japan) and Reyonet200L (Nagase, Japan).

nucleic acid construct

Isolated polynucleotides encoding polypeptides (e.g., cellulolytic enzymes, polypeptides having catalase activity, polypeptides having cellulolytic enhancing activity, etc.) can be manipulated in a variety of ways by constructing nucleic acid constructs to To provide expression of said polypeptide, the nucleic acid construct comprises an isolated polynucleotide encoding said polypeptide operably linked to one or more (eg, several) control sequences, said control sequences being expressed in a suitable host cell directs the expression of a coding sequence under conditions compatible with the regulatory sequences. The polynucleotides can be manipulated in a number of ways to provide expression of the polypeptide. Depending on the expression vector, it may be desirable or necessary to manipulate the polynucleotide prior to its insertion into the vector. Techniques for modifying polynucleotide sequences using recombinant DNA methods are well known in the art.

The control sequence may be a promoter, which is a polynucleotide recognized by a host cell for expression of a polynucleotide encoding a polypeptide. A promoter contains transcriptional regulatory sequences that mediate the expression of a polypeptide. The promoter may be any polynucleotide that exhibits transcriptional activity in the host cell, including mutated, truncated, and hybrid promoters, and may be derived from an extracellular or intracellular polypeptide encoding homologous or heterologous to the host cell gene acquisition.

Examples of suitable promoters for directing transcription of nucleic acid constructs of the invention in bacterial host cells are promoters obtained from: Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus stearothermophilus maltogenic 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 13:97-107), E. coli lac operon, E. coli trc promoter (Egon et al., 1988, Gene69:301-315), Streptomyces coelicolor agarase Gene (dagA) and prokaryotic β-lactamase gene (Villa-Kamaroff et al., 1978, Proceedings of the National Academy of Sciences USA75:3727-3731), and tac promoter (DeBoer et al., 1983, Proc.Natl.Acad.Sci .USA80:21-25). Additional promoters are described in "Useful proteins from recombinant bacteria" in Gilbert et al., 1980, Scientific American, 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promoters are disclosed in WO99/43835.

Examples of suitable promoters for directing transcription of nucleic acid constructs of the invention in filamentous fungal host cells are promoters obtained from the genes of the following enzymes: Aspergillus nidulans acetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid-stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like Protease (WO96/00787), Fusarium venariens amyloglucosidase (WO00/56900), Fusarium venariens Daria (WO00/56900), Fusarium venariens Quinn (WO00/56900), Rhizomucor miehei ) lipase, Rhizomucor mehnheich aspartic protease, Trichoderma reesei β-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei Endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase Glucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei β-xylosidase, and the NA2-tpi promoter (a modified promoter, It is derived from the Aspergillus neutral alpha-amylase gene in which the untranslated leader sequence is replaced by the untranslated leader sequence of the Aspergillus triose phosphate isomerase gene; non-limiting examples include modified promoters whose from the gene of Aspergillus niger neutral alpha-amylase, wherein the untranslated leader sequence is replaced by the untranslated leader sequence of the gene of Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerase); and their mutant, truncated Short and heterozygous promoters.

In yeast hosts, useful promoters are obtained from the following genes: S. cerevisiae enolase (ENO-1), S. cerevisiae galactokinase (GAL1), S. cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase enzymes (ADH1, ADH2/GAP), S. cerevisiae triose phosphate isomerase (TPI), S. cerevisiae metallothionein (CUP1) and S. cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8:423-488.

The regulatory sequence can also be a transcription terminator, which is recognized by a host cell to terminate transcription. The terminator is operably linked to the 3' end of the polynucleotide encoding the polypeptide. In the present invention, any terminator that is functional in the host cell can be used.

Preferred terminators for bacterial host cells are obtained from the following genes: Bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha-amylase (amyL) and E. coli ribosomal RNA (rrnB).

Preferred terminators for filamentous fungal host cells are obtained from the genes for the following enzymes: Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase and Fusarium Spore trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genes for the following enzymes: S. cerevisiae enolase, S. cerevisiae cytochrome C (CYC1 ), and S. cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, supra.

A regulatory sequence may also be an mRNA stabilizing region downstream of a promoter and upstream of the coding sequence of a gene, which increases the expression of said gene.

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

The regulatory sequence may also be a suitable leader sequence, which is an untranslated region of an mRNA important for translation by the host cell. A leader sequence is operably linked to the 5'-terminus of the polynucleotide encoding the polypeptide. Any leader sequence that is functional in the host cell can be used.

Preferred leader sequences for filamentous fungal host cells are obtained from the genes for the following enzymes: Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

Suitable leader sequences for yeast host cells are obtained from the genes for the following enzymes: S. cerevisiae enolase (ENO-1), S. cerevisiae 3-phosphoglycerate kinase, S. cerevisiae alpha factor and S. cerevisiae alcohol dehydrogenase/glyceraldehyde- 3-phosphate dehydrogenase (ADH2/GAP).

The regulatory sequence can also be a polyadenylation sequence, which is a sequence operably linked to the 3' end of a polynucleotide and, when transcribed, is recognized by the host cell as adding polyadenylation residues to the transcribed mRNA signal of. Any polyadenylation sequence that is functional in the host cell can be used.

Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes of the following enzymes: Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase and Fusarium oxysporum trypsin-like proteases.

Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15:5983-5990.

The control sequence may also be a signal peptide coding region, which codes for a signal peptide linked to the N-terminus of the polypeptide and directs the polypeptide into the cell's secretory pathway. The 5' end of the coding sequence of a polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the polypeptide. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding sequence foreign to the coding sequence. An exogenous signal peptide coding sequence may be necessary when the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, an exogenous signal peptide coding sequence can simply replace the native signal peptide coding sequence to enhance secretion of the polypeptide. However, any signal peptide coding sequence that directs the expressed polypeptide into the secretory pathway of the host cell can be used.

Signal peptide coding sequences effective for bacterial host cells are those obtained from the genes of the following enzymes: Bacillus sp. NCIB11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis β-lactamase , Bacillus stearothermophilus α-amylase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM) and Bacillus subtilis prsA. Additional signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Signal peptide coding sequences effective for filamentous fungal host cells are those obtained from the genes of the following enzymes: Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens fiber Sulfase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase and Rhizomucor mehnhee aspartic protease.

Signal peptides useful for yeast host cells are obtained from the genes for S. cerevisiae alpha factor and S. cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence, which codes for a propeptide at the N-terminus of the polypeptide. The resulting polypeptide is called a proenzyme or propolypeptide (or in some cases a zymogen). Propolypeptides are generally inactive and can be converted from the propolypeptide to the active polypeptide by catalytic or autocatalytic cleavage of the propeptide. Can be obtained from Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO95/33836), Rhizomucor mehnhee aspartic protease and Saccharomyces cerevisiae alpha factor The gene obtains the propeptide coding sequence.

When both signal peptide and propeptide sequences are present, the propeptide sequence is placed next to the N-terminus of the polypeptide, and the signal peptide sequence is placed next to the N-terminus of the propeptide sequence.

It is also desirable to add regulatory sequences that regulate the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory sequences are those 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 the lac, tac, and trp operator sequences. In yeast, the ADH2 system or the GAL1 system can be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, the Aspergillus oryzae TAKA alpha-amylase promoter and the Aspergillus oryzae glucoamylase promoter can be used. Other examples of regulatory sequences are those that allow gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene amplified in the presence of methotrexate, and the metallothionein gene amplified with heavy metals. In these cases, the polynucleotide encoding the polypeptide will be operably linked to regulatory sequences.

Expression vector

The various nucleotide and regulatory sequences described above can be combined to produce a recombinant expression vector, which can include one or more (e.g., several) convenient restriction sites to allow insertion or substitution of the encoded polypeptide at these sites Polynucleotides for polypeptides such as cellulolytic enzymes, polypeptides having catalase activity, polypeptides having cellulolytic enhancing activity, and the like. Alternatively, the polynucleotide can be expressed by inserting a nucleic acid construct or polynucleotide comprising the polynucleotide in an appropriate vector for expression. In preparing an expression vector, a coding sequence is placed in the vector such that it is operably linked with appropriate regulatory sequences for expression.

A recombinant expression vector can be any vector (eg, a plasmid or virus) that facilitates recombinant DNA procedures and is capable of producing expression of a polynucleotide. The choice of vector will generally depend on the compatibility of the vector with the host cell into which it will be introduced. Vectors can be linear or closed circular plasmids.

A vector may be an autonomously replicating vector, ie, a vector that exists as an extrachromosomal entity that replicates independently of chromosomal replication, eg, a plasmid, extrachromosomal element, minichromosome or artificial chromosome. A vector may contain any means for ensuring self-replication. Alternatively, the vector may be one that, when introduced into a host cell, integrates into the genome and replicates together with the chromosome into which the vector has been integrated. In addition, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell may be used, or a transposon may be used.

The vector preferably contains one or more (eg, several) selectable markers that allow easy selection of transformed, transfected, transduced, etc. cells. Selectable markers are genes whose products confer biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are the Bacillus licheniformis or Bacillus subtilis dal genes, or markers that confer antibiotic resistance such as 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. Selectable markers for filamentous fungal host cells include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (humidity Mycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (adenylyl sulfate transfer enzyme) and trpC (anthranilate synthase) and their equivalents. Preferred for use in Aspergillus cells are the A. nidulans or A. oryzae amdS and pyrG genes and the Streptomyces hygroscopicus bar gene.

The vector preferably contains elements that allow integration of the vector into the host cell genome or autonomous replication of the vector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on a polynucleotide encoding a polypeptide or any other vector element for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides to direct integration by homologous recombination into the precise chromosomal location of the host cell genome. To increase the probability of integration at precise locations, integration elements should contain sufficient amounts of nucleic acid, such as 100 to 10,000 bp, 400 to 10,000 bp, and 800 to 10,000 bp, that are highly sequenced to the corresponding target sequence identity to enhance the probability of homologous recombination. An integrating element can be any sequence that is homologous to a sequence of interest in the genome of the host cell. Furthermore, integrating elements can be non-coding or coding polynucleotides. Alternatively, the vector can be integrated into the genome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin of replication, which enables the vector to replicate autonomously in said host cell. The origin of replication can be any plasmid replicator that mediates autonomous replication, which functions in the cell. The term "origin of replication" or "plasmid replicator" means a polynucleotide capable of in vivo replication of a plasmid or vector.

Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177 and pACYC184 which permit replication in E. coli, and the origins of replication of plasmids pUB110, pE194, pTA1060 and pAMβ1 which permit replication in Bacillus.

Examples of origins of replication for use in yeast host cells are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful 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; WO00/24883). Isolation of the AMA1 gene and construction of a plasmid or vector containing the gene can be accomplished according to the methods disclosed in WO00/24883.

More than one copy of a polynucleotide can be inserted into a host cell to increase production of the polypeptide. An increase in the copy number of a polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome, or by including an amplifiable selectable marker gene in the polynucleotide, which can be obtained by adding an appropriate selection agent Cells are grown in the presence of a (selectable agent) to select for cells that contain an amplified copy of the selectable marker gene, and thus contain additional copies of the polynucleotide.

Methods for joining the above elements to construct recombinant expression vectors are well known to those skilled in the art (see, eg, Sambrook et al., 1989, supra).

host cell

Recombinant host cells comprising polynucleotides encoding polypeptides (eg, cellulolytic enzymes, polypeptides having catalase activity, polypeptides having cellulolytic enhancing activity) and the like can be advantageously used to recombinantly produce the polypeptides. A construct or vector comprising a polynucleotide is introduced into a host cell and maintained as a chromosomal integrant or as a self-replicating extrachromosomal vector as previously described. The term "host cell" includes any progeny of a parent cell that differs from the parent cell due to mutations that occur during replication. The choice of host cell will depend largely on the gene encoding the polypeptide and its source.

A host cell can be any cell useful in the recombinant production of a polypeptide, eg, a prokaryotic or eukaryotic cell.

Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, marine Bacillus, Staphylococcus, Streptococcus, and Streptomyces belongs to. Gram-negative bacteria include, but are not limited to, Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Gleobacter, Neisseria, Pseudomonas, Salmonella, and urea body.

The bacterial host cell can be any Bacillus genus cell, including but not limited to Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus candidia Bacillus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis and Bacillus thuringiensis cells.

The bacterial host cell can also be any Streptococcus cell including, but not limited to, S. equisimilis, S. pyogenes, S. uberis, and S. equi subsp. zooepidemicus cells.

The bacterial host cell can also be any Streptomyces cell, including, but not limited to, S. achromogenes, S. avermitilis, S. coelicolor, S. griseus, and S. lividans cells.

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

The host cell can also be a eukaryote, such as a mammalian, insect, plant or fungal cell.

"Fungi" as used herein includes the following phyla: Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as well as Oomycota and all mitotic spore fungi ( mitosporic 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).

A fungal host cell can be a yeast cell. "Yeast" as used herein includes ascosporogenous yeasts (Endomycetales), basidiosporogenous yeasts, and yeasts belonging to the Fungi Imperfecti (Blastomycetes) . Since the classification of yeast may change in the future, for the purposes of the present invention, yeast is defined as Biology and Activities of Yeast (Skinner F.A.,, Passmore S.M. and Davenport R.R. eds., Soc. App. Bacteriol. Symposium Series No. 9, 1980 ) described in .

The yeast host cell can be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as Kluyveromyces lactis ( Kluyveromyces lactis), Karl's yeast, Saccharomyces cerevisiae, Saccharomyces cerevisiae, Saccharomyces douglasia, Kluyveromyces, Nordica, Saccharomyces ovale, or Yarrowia lipolytica cells.

A fungal host cell can be a filamentous fungal cell. "Filamentous fungi" include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). Filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth occurs by hyphal extension, whereas carbon catabolism is obligately aerobic. In contrast, vegetative growth of yeast such as Saccharomyces cerevisiae proceeds by budding of the unicellular thallus, while carbon catabolism may be fermentative.

Filamentous fungal host cells can be Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceremonas, Chrysosporium, Coprinus, Coriolus (Coriolus), Cryptococcus, Filibasidium, Fusarium, Humicola, Pyrospora, Mucor, Myceliophthora, Neocoma, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Rumenochytrium, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia genus, Curvularia, Trametes or Trichoderma cells.

For example, the filamentous fungal host cell can be Aspergillus awamori, Aspergillus fumigatus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsisaneirina, Ceriporiopsis caregiea、Ceriporiopsis gilvescens、Ceriporiopsis pannocinta、Ceriporiopsis rivulosa、Ceriporiopsis subrufa、虫拟蜡菌(Ceriporiopsissubvermispora)、Chrysosporium inops、嗜角质金孢子菌、Chrysosporiumlucknowense、Chrysosporium merdarium、毡金孢子菌、Chrysosporiumqueenslandicum、热带金孢子菌、Chrysosporium zonatum , Coprinus cinereus, Coriolus hirsutus, Fusarium baculum, Fusarium graminearum, Fusarium kuwei, Fusarium spp., Fusarium gramineae, Fusarium graminearum, Heterospora Fusarium, Fusarium albizia, Fusarium oxysporum, Fusarium multibranchus, Fusarium pink, Fusarium elderberry, Fusarium complexion, Fusarium cladoides, Fusarium thiochrome, Fusarium torus, Fusarium mycelia spores, Fusarium venerae, Humicola insolens, Humicola lanuginosa, Mucor miera, Myceliophthora thermophila, Neurospora crassa, Penicillium purpura, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametesvillosa, Trametes versicolor, Trichoderma harzianum, Trichoderma korningii, Trichoderma longibrachiae , Trichoderma reesei or Trichoderma viride cells.

Fungal cells can be transformed in a manner known per se by methods involving protoplast formation, transformation of the protoplasts and regeneration of the cell wall. Suitable methods for transforming Aspergillus and Trichoderma host cells are described in EP238023 and Yelton et al., 1984, Proc. . Suitable methods for transformation of Fusarium species are described by Malardier et al., 1989, Gene 78:147-156 and WO96/00787. Yeast can be transformed using the methods described by Becker and Guarente, in Abelson, J.N. and Simon, M.I., eds., Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York ; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75:1920.

Generation method

A method for producing a polypeptide (e.g., a cellulolytic enzyme, a polypeptide having catalase activity, a polypeptide having cellulolytic enhancing activity) and the like, comprising (a) culturing cells under conditions conducive to production of the polypeptide, wherein producing the polypeptide in its wild-type form by the cell; and (b) recovering the polypeptide. In a preferred aspect, the cell is an Aspergillus, Thermoascomyces, Talaromyces, Trichoderma, Humicola or Penicillium cell. In a more preferred aspect, the cells are Aspergillus niger, Aspergillus oryzae, Aspergillus fumigatus, Thermoascus aurantiacus, Talaromyces stipitatus, Trichoderma reesei, Humicola insolens or Penicillium emersonii cells.

Alternatively, a method for producing a polypeptide, such as a cellulolytic enzyme, a polypeptide having catalase activity, a polypeptide having cellulolytic enhancing activity, etc., comprising (a) culturing the recombinant host under conditions conducive to production of the polypeptide cells; and (b) recovering the polypeptide.

The host cells are cultured in a nutrient medium suitable for production of the polypeptide using methods known in the art. For example, it can be cultured in shake flasks in a suitable medium and under conditions that allow expression and/or isolation of the polypeptide, or small-scale or large-scale fermentation (including continuous, batch, supplemented) in laboratory or industrial fermenters. Feed batch or solid state fermentation) to cultivate cells. Cultivation is carried out in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts using methods known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (eg, in catalogs of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from said medium. If the polypeptide is not secreted, it can be recovered from cell lysates.

Polypeptides can be detected using methods known in the art that are specific for the polypeptide. 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, enzyme assays can be used to determine the activity of a polypeptide. Polypeptides having cellulolytic enhancing activity are detected using the methods described herein.

Polypeptides can be recovered using methods known in the art. For example, polypeptides can be recovered from the nutrient medium by conventional methods including, but not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. In one aspect, whole fermentation broth is recovered.

Polypeptides can be purified to obtain substantially pure polypeptides by a variety of methods known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromofocusing, and size exclusion), electrophoresis Method (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, Ed. Janson and Ryden, VCH Publishers, New York, 1989) .

In another aspect, the polypeptide is not recovered, but a host cell expressing the polypeptide is used as the source of the polypeptide.

The present invention is further described by the following examples, which should not be construed as limiting the scope of the present invention.

Example

strain

The fungal strain NN70 was obtained from the Centraalbureau voor Schimmelcultures under the designation CBS375.48. Based on both morphological characteristics and the ITS rDNA sequence, strain NN70 was identified as Talaromyces stipitatus.

Fungal strain NN38 was isolated from soil samples collected from China by the dilution plate method with PDA medium at 45°C. It was then purified by transferring one monoconidia to a YG agar plate. Based on both morphological characteristics and the ITS rDNA sequence, strain NN38 was identified as Humicola insolens.

The fungal strain NN051602 was isolated from soil samples collected from China by the dilution plate method with PDA medium at 45°C. It was then purified by transferring one monoconidia to a YG agar plate. Based on both morphological characteristics and the ITS rDNA sequence, strain NN051602 was identified as Penicillium emersonii.

culture medium

PDA medium consisted of 39 grams of Potato Dextrose Agar and deionized water up to 1 liter.

YG agar plates consisted of 5.0 g of yeast extract, 10.0 g of glucose, 20.0 g of agar, and deionized water up to 1 liter.

YPM medium contains 1% yeast extract, 2% peptone, and 2% maltose in deionized water.

YPG medium contains 0.4% yeast extract in deionized water, 0.1% KH ₂ PO ₄ , 0.05% MgSO ₄ ·7H ₂ O, 1.5% glucose.

A minimal medium plate consisted of 342 g of sucrose, 20 ml of a saline solution consisting of 2.6% KCl, 2.6% MgSO ₄ 7H ₂ O, 7.6% KH ₂ , and 1 liter of deionized water, followed by 20 g of agar. PO ₄ , 2 ppm Na ₂ B ₄ O ₇ 10H ₂ O, 20 ppm CuSO ₄ 5H ₂ O, 40 ppm FeSO ₄ 7H ₂ O, 40 ppm MnSO ₄ 2H ₂ O, 40 ppm Na ₂ MoO ₄ 2H ₂ O, and 400 ppm Composed of ZnSO ₄ ·7H ₂ O.

Example 1: Enhancing effect of Thermoascus aurantiacus catalase or Talaromyces stipitatus catalase on the hydrolysis of pretreated corn stover (PCS)

Catalase from Thermoascus aurantiacus (shown in SEQ ID NO: 2) was expressed by Aspergillus niger and purified as described in Japanese Publication No. 2004261137A. Catalase from Talaromyces stipitatus (as shown in SEQ ID NO:4) was cloned, expressed and purified as in Examples 9-13. The corn stover was treated at the U.S. Department of Energy National Renewable Energy Laboratory (NREL) using dilute sulfuric acid in a pretreatment reactor at 190 °C, 1 minute retention time, 0.05 g acid/g dry biomass , and pretreatment at 30% total solids concentration.

CTec2，可从Novozymes A/S,Bagsvaerd,Denmark获得)添加至PCS进行酶水解，其中里氏木霉纤维素酶组合物对纤维素的比例为0.5%(w/w)，即基于蛋白量为5mg/g纤维素。将桔橙嗜热子囊菌过氧化氢酶或Talaromyces stipitatus过氧化氢酶以下表1中所示的剂量添加入水解系统。不添加过氧化氢酶的水解系统用作对照。将烧瓶在50℃，在130rpm振荡下温育72小时。除非另行指明，总水解时间为72小时。在水解完成后，糖通过高效液相色谱(HPLC)进行分析。PCS was hydrolyzed at 10% starting total solids (TS) and 20 g total weight of the hydrolysis system. Trichoderma reesei cellulase composition (

CTec2, available from Novozymes A/S, Bagsvaerd, Denmark) was added to PCS for enzymatic hydrolysis, wherein the ratio of Trichoderma reesei cellulase composition to cellulose was 0.5% (w/w), i.e. based on the amount of protein 5mg/g cellulose. Thermoascus aurantiacus catalase or Talaromyces stipitatus catalase was added to the hydrolysis system at the dosage indicated in Table 1 below. A hydrolysis system without addition of catalase was used as a control. The flasks were incubated at 50°C for 72 hours with shaking at 130 rpm. Total hydrolysis time was 72 hours unless otherwise specified. After hydrolysis was complete, sugars were analyzed by high performance liquid chromatography (HPLC).

HPX-87H柱(Bio-RadLaboratories,Inc.,Hercules,CA,USA)来测量，即用0.005M H₂SO₄在65℃以0.7ml每分钟的流速洗脱，并通过对折射率检测(

1100HPLC,Agilent Technologies,Santa Clara,CA,USA)所得的、经纯糖样品校准的葡萄糖信号积分来定量。使用所得的葡萄糖对于每个反应计算来自葡聚糖的葡萄糖产量的百分比。对测得的糖浓度根据合适的稀释因子进行调整。将测得的糖浓度针对在零时点在未经洗涤的生物质中相应的背景糖浓度进行调整，来确定酶产生的糖的净浓度。所有的HPLC数据处理使用MICROSOFT EXCEL^TM软件(Microsoft,Richland,WA,USA)进行。For HPLC measurements, collected samples were filtered using a 0.22 μm syringe filter (Millipore, Bedford, MA, USA), and the filtrate was analyzed for sugar content as described below. The sugar concentration of the sample diluted in _0.005MH2SO4 used _7.8 x 300mm

HPX-87H column (Bio-RadLaboratories, Inc., Hercules, CA, USA) to measure, that is to use 0.005M H ₂ SO ₄ at 65 ℃ with a flow rate of 0.7ml per minute elution, and by the detection of the refractive index (

1100 HPLC, Agilent Technologies, Santa Clara, CA, USA) obtained by the integration of glucose signals calibrated for pure sugar samples. The resulting glucose was used to calculate the percent glucose yield from dextran for each reaction. Adjust the measured sugar concentration for the appropriate dilution factor. The measured sugar concentration was adjusted for the corresponding background sugar concentration in the unwashed biomass at time zero to determine the net concentration of enzyme-produced sugar. All HPLC data processing was performed using MICROSOFT EXCEL ^™ software (Microsoft, Richland, WA, USA).

The degree of conversion of glucose into glucose was calculated according to the publication of Zhu, Y. et al. 2010, Bioresource Technology.102(3):2897-2903.

The results shown in Table 1 show that the conversion of PCS to glucose can be significantly improved by adding a small amount of catalase.

Table 1: Effect of catalase from T. aurantiacus or from T. stipitatus on glucose conversion of PCS

Example 2: Single-component potentiation of cellulase by Talaromyces stipitatus catalase

PH-101(Fluka11365,Sigma-Aldrich(Shanghai),中国上海)，一种微晶纤维素，以5g/l的终浓度和0.5ml的水解系统的总体积进行水解。将pH通过50mM乙酸钠调整并维持在5.0。此外，抗坏血酸以0.5mM的终浓度存在于水解系统中，或不存在于水解系统中。硫酸锰(II)以1mM的终浓度存在于水解系统中。Will

PH-101 (Fluka 11365, Sigma-Aldrich (Shanghai), Shanghai, China), a microcrystalline cellulose, was hydrolyzed at a final concentration of 5 g/l and a total volume of the hydrolysis system of 0.5 ml. The pH was adjusted and maintained at 5.0 with 50 mM sodium acetate. In addition, ascorbic acid was present in the hydrolysis system at a final concentration of 0.5 mM, or was absent from the hydrolysis system. Manganese(II) sulfate was present in the hydrolysis system at a final concentration of 1 mM.

的水解。使用10mg单组分纤维素酶/g

和5mg过氧化氢酶/g

将试管在50℃在600rpm振荡下温育72小时。所有实验进行一式三次。Cloned, expressed and purified cellobiohydrolase (CBH) I from Aspergillus fumigatus (WO2011/057140), cellobiohydrolase (CBH) II from Aspergillus fumigatus (WO2011/057140), from Trichoderma reesei Endoglucanase (EG) I (WO2011/057140) and β-glucanase (BG) from Aspergillus oryzae (WO02/095014). Apply these single components individually to

of hydrolysis. Use 10mg single-component cellulase/g

and 5mg catalase/g

The tubes were incubated at 50°C for 72 hours with shaking at 600 rpm. All experiments were performed in triplicate.

HPLC analysis for the degree of hydrolysis was performed according to the procedure described in Example 1.

The degree of cellulose conversion is calculated based on the mass ratio of solubilized glycosyl units to the starting mass of insoluble cellulose. For soluble sugars only glucose and cellobiose were measured because cellodextrins longer than cellobiose were only present in negligible concentrations (due to enzymatic hydrolysis). The degree of total cellulose conversion was calculated using the following formula:

(Formula 1)

The factors for glucose and cellobiose are 1.111 and 1.053, respectively, which take into account when glycosyl units in cellulose (average molecular weight 162 Daltons) are converted to glucose (molecular weight 180 Daltons) or cellobiose sugars Unit (average molecular weight 171 Daltons) of the mass increase.

Table 2: Effect of catalase from T. stipitatus on glucose conversion of Avicel.

As shown in Table 2, the hydrolysis of each monocomponent cellulase was enhanced by T. stipitatus catalase in the presence of ascorbic acid.

Example 3: Enhancement of the hydrolysis of PCS by Humicola insolens catalase

The preparation of PCS and the setting of the hydrolysis system were the same as in Example 1. Catalase from Humicola insolens was cloned, expressed and purified as shown in Examples 14-20.

The PCS was hydrolyzed with 10% of the initial TS and 20 g of the total weight of the hydrolysis system. Using Trichoderma reesei cellulase composition (available from Novozymes A/S, Bagsvaerd, Denmark CTec2) for enzymatic hydrolysis. Five percent by weight of Ctec2 based on protein amount was replaced with Humicola insolens catalase and the total enzyme dosage was 4 mg/g cellulose. A hydrolysis system containing 4 mg T. reesei cellulase composition/g cellulose but no catalase was used as a control. The flasks were incubated at 50°C for 72 hours with shaking at 130 rpm. The total hydrolysis time was 72 hours.

The calculation of glucose conversion is the same as in Example 1, and the boosting effect is shown in Table 3.

Table 3: Effect of catalase from Humicola insolens on glucose conversion of PCS

control Humicola insolens catalase Glucose conversion (%) 50.4±1.1 58.4±0.9

Example 4: Enhancement of the hydrolysis of PCS by Humicola insolens catalase

The preparation of PCS and the setting of the hydrolysis system were the same as in Example 1. Catalase from Humicola insolens was cloned, expressed and purified as shown in Examples 14-20.

CTec3)进行酶水解。将按重量计百分之五的Ctec3基于蛋白量用特异腐质霉过氧化氢酶替代，且总酶剂量为4mg/g纤维素。使用含4mg里氏木霉纤维素酶组合物/g纤维素、但不含过氧化氢酶的水解系统作为对照。将烧瓶在50℃，在130rpm振荡下温育72小时。总水解时间为72小时。The PCS was hydrolyzed with 10% of the initial TS and 20 g of the total weight of the hydrolysis system. Using Trichoderma reesei cellulase composition (available from Novozymes A/S, Bagsvaerd, Denmark

CTec3) for enzymatic hydrolysis. Five percent by weight of Ctec3 based on protein amount was replaced with Humicola insolens catalase and the total enzyme dosage was 4 mg/g cellulose. A hydrolysis system containing 4 mg T. reesei cellulase composition/g cellulose but no catalase was used as a control. The flasks were incubated at 50°C for 72 hours with shaking at 130 rpm. The total hydrolysis time was 72 hours.

The calculation of glucose conversion is the same as in Example 1, and the boosting effect is shown in Table 4.

Table 4: Effect of catalase from Humicola insolens on glucose conversion of PCS

control Humicola insolens catalase Glucose conversion (%) 70.9±1.4 80.1±1.2

Example 5: Synergistic effect of Thermoascus aurantiacus catalase and Thermoascus aurantiacus GH61A on the hydrolysis of PCS

可从Novozymes A/S,Bagsvaerd,Denmark获得)添加入PCS进行酶水解，其中里氏木霉纤维素酶组合物对纤维素的比例为0.8%(w/w)。将来自桔橙嗜热子囊菌的过氧化氢酶，来自桔橙嗜热子囊菌的GH61A多肽(WO2005/074656)，或其组合分别添加入水解系统。过氧化氢酶、GH61A多肽、或其组合的剂量基于纤维素的重量计算。含里氏木霉纤维素酶组合物但不含过氧化氢酶和GH61的水解系统用作对照。将烧瓶在50℃，在130rpm振荡下温育72小时。所有实验进行一式三次。对水解程度的HPLC分析根据实施例1中所述的步骤进行。在72小时水解之后PCS至葡萄糖的转化示于下表5。PCS was prepared according to the procedure described in Example 1 and hydrolyzed with 10% of the starting TS and 20 g of the total weight of the hydrolysis system. The pH was adjusted to 5.0 using 10M sodium hydroxide. Trichoderma reesei cellulase composition (in the presence of Aspergillus fumigatus β-glucosidase (WO2005/047499) at 10% total protein weight

available from Novozymes A/S, Bagsvaerd, Denmark) was added to PCS for enzymatic hydrolysis, wherein the ratio of Trichoderma reesei cellulase composition to cellulose was 0.8% (w/w). Catalase from T. aurantiacus, GH61A polypeptide from T. aurantiacus (WO2005/074656), or a combination thereof were added to the hydrolysis system, respectively. Doses of catalase, GH61A polypeptide, or combinations thereof are based on the weight of cellulose. A hydrolysis system containing the T. reesei cellulase composition without catalase and GH61 was used as a control. The flasks were incubated at 50°C for 72 hours with shaking at 130 rpm. All experiments were performed in triplicate. HPLC analysis for the degree of hydrolysis was performed according to the procedure described in Example 1. The conversion of PCS to glucose after 72 hours of hydrolysis is shown in Table 5 below.

Table 5: Synergistic effect of T. aurantiacus catalase and T. aurantiacus GH61A on the hydrolysis of PCS

As shown in Table 5, either catalase alone or GH61A polypeptide alone enhanced the hydrolysis of PCS. Surprisingly, it was found that hydrolysis was significantly improved when catalase and GH61A polypeptide were used together. These results indicated that catalase and GH61A polypeptide had a significant synergistic effect on the hydrolysis of PCS.

Example 6: Enhancement of Penicillium emersonii catalase to PCS hydrolysis

The preparation of PCS and the setting of the hydrolysis system were the same as in Example 1. Catalase from P. emersonii was cloned, expressed and purified as shown in Examples 21-27.

CTec2)添加入PCS进行酶水解。将按重量计百分之五的Ctec2基于蛋白量用P.emersonii过氧化氢酶替代，且总酶剂量为4mg/g纤维素。使用含里氏木霉纤维素酶组合物、但不含过氧化氢酶的水解系统作为对照。将烧瓶在50℃、130rpm振荡下温育72小时。The PCS was hydrolyzed with 10% of the initial TS and 20 g of the total weight of the hydrolysis system. Trichoderma reesei cellulase composition (available from Novozymes A/S, Bagsvaerd, Denmark

CTec2) was added into PCS for enzymatic hydrolysis. Five percent by weight of Ctec2 based on protein amount was replaced with P. emersonii catalase with a total enzyme dosage of 4 mg/g cellulose. A hydrolysis system containing the T. reesei cellulase composition but no catalase was used as a control. The flasks were incubated for 72 hours at 50°C with shaking at 130 rpm.

The calculation of glucose conversion is the same as in Example 1, and the boosting effect is shown in Table 6.

Table 6: Effect of catalase from P. emersonii on glucose conversion of PCS

control P. emersonii catalase Glucose conversion (%) 48.6±0.7 54.3±0.8

Example 7: Enhancement of the hydrolysis of PCS by Thermoascus aurantiacus catalase at relatively high TS

CTec2)添加入PCS进行酶水解。将按重量计百分之五的Ctec2基于蛋白量用桔橙嗜热子囊菌过氧化氢酶替代，总酶剂量为7mg/g纤维素。使用含里氏木霉纤维素酶组合物、但不含过氧化氢酶的水解系统作为对照。将烧瓶在50℃，在130rpm振荡下温育72小时。葡萄糖转化的计算与实施例1相同，且加强作用示于表7。The preparation of PCS and the setting of the hydrolysis system were the same as in Example 1. PCS was hydrolyzed at 20% of the starting TS and 20 g of the total weight of the hydrolysis system. Trichoderma reesei cellulase composition (available from NovozymesA/S, Bagsvaerd, Denmark

CTec2) was added into PCS for enzymatic hydrolysis. Five percent by weight of Ctec2 based on protein amount was replaced with T. aurantiacus catalase for a total enzyme dose of 7 mg/g cellulose. A hydrolysis system containing the T. reesei cellulase composition but no catalase was used as a control. The flasks were incubated at 50°C for 72 hours with shaking at 130 rpm. The calculation of glucose conversion is the same as in Example 1, and the boosting effect is shown in Table 7.

Table 7: Effect of catalase from T. aurantiacus on glucose conversion of PCS

control 5% Thermoascus aurantiacus catalase replacement Glucose conversion (%) 58.6±1.4 64.8±0.8

Example 8: Enhancement of the hydrolysis of PCS by Thermoascus aurantiacus catalase at relatively high TS

CTec3)进行酶水解。将按重量计百分之五的Ctec3基于蛋白量用桔橙嗜热子囊菌过氧化氢酶替代，且总酶剂量为6mg/g纤维素。使用含里氏木霉纤维素酶组合物、但不含过氧化氢酶的水解系统作为对照。将烧瓶在50℃、130rpm振荡下温育72小时。The preparation of PCS and the setting of the hydrolysis system were the same as in Example 1. PCS was hydrolyzed at 20% of the starting TS and 20 g of the total weight of the hydrolysis system. Using Trichoderma reesei cellulase composition (available from Novozymes A/S, Bagsvaerd, Denmark

CTec3) for enzymatic hydrolysis. Five percent by weight of Ctec3 based on protein amount was replaced with T. aurantiacus catalase with a total enzyme dosage of 6 mg/g cellulose. A hydrolysis system containing the T. reesei cellulase composition but no catalase was used as a control. The flasks were incubated for 72 hours at 50°C with shaking at 130 rpm.

The calculation of glucose conversion is the same as in Example 1, and the enhancement effect of catalase is shown in Table 8.

Table 8: Effect of catalase from T. aurantiacus on glucose conversion of PCS

control 5% Thermoascus aurantiacus catalase replacement Glucose conversion (%) 72.0± 80.5±0.8

Example 9: Talaromyces stipitatus Genomic DNA Extraction

植物小提试剂盒(Plant Mini Kit)(QIAGEN Inc.,Valencia,CA,USA)分离基因组DNA。Talaromyces stipitatus strain NN70 was grown on PDA agar plates at 45°C for 3 days. Mycelia were collected directly from the agar plates into sterilized pestles and mortars under liquid nitrogen cooling. Grind the frozen mycelia to a fine powder with a pestle and mortar, and use

Plant Mini Kit (Plant Mini Kit) (QIAGEN Inc., Valencia, CA, USA) was used to isolate genomic DNA.

Example 10: Cloning of the Talaromyces stipitatus catalase gene from genomic DNA

Based on the DNA information of European Molecular Biology Laboratory (EMBL): EQ962660 (ie SEQ ID NO: 3) and the protein sequence SWISSPROT: B8MT74 (ie SEQ ID NO: 4), the following oligo was designed Nucleotide primers to amplify the catalase gene from genomic DNA of Talaromyces stipitatus NN70. Primers were manufactured by Invitrogen (Invitrogen, Beijing, China).

Forward primer: 5'ACACAACTGGGGATCC ACC atgcgaggggcatactctctc3' (SEQ ID NO:38)

Reverse primer: 5'GTCACCCTCTAGATCT aacaagttactcgtgttaatcgtggaa3' (SEQID NO:39)

The lowercase letters represent the sequence of the gene, while the uppercase part is homologous to the insertion site of the pPFJO355 vector described in US2010306879.

The expression vector pPFJO355 contains the TAKA-amylase promoter from Aspergillus oryzae and the Aspergillus niger glucoamylase terminator element. In addition pPFJO355 has pUC18-derived sequences for selection and propagation in E. coli, and the pyrG gene encoding orotidine decarboxylase from Aspergillus nidulans for selection of transformants of pyrG mutant Aspergillus strains.

Twenty pmoles of primer pairs (forward and reverse) were used in PCR reactions consisting of 2 μl of Talaromyces stipitatus NN70 genomic DNA, 10 μl of 5X GC buffer, 1.5 μl of DMSO, 2.5 mM each of dATP, dTTP, dGTP and dCTP, and 0.6 units of PHUSION ^™ high-fidelity DNA polymerase (Finnzymes Oy, Espoo, Finland) were constituted in a final volume of 50 μl. Amplification was performed using a Peltier Thermal Cycler (MJ Research Inc., South San Francisco, CA, USA), programmed as follows: denaturation at 98°C for 40 seconds; 8 cycles of denaturation at 98°C for 15 seconds and annealing at 70°C for each cycle 30 seconds, each cycle of 1°C decrease, and 80 seconds extension at 72°C; and another 23 cycles, each cycle at 98°C for 15 seconds, 62°C for 30 seconds, and 72°C for 80 seconds; The final extension is 5 minutes. The heat block then goes into a 4°C soak cycle.

PCR reaction products were separated by 1.0% agarose gel electrophoresis using 90 mM Tris boronic acid and 1 mM EDTA (TBE) buffer, which showed a single product band of the expected size (approximately 2.4 kb) under UV light, and then analyzed using ILLUSTRA ^™ GFX ^™ PCR DNA and Gel Band Purification Kit (GE Healthcare, Buckinghamshire, UK) was purified from solution according to the manufacturer's instructions.

Plasmid pPFJO355 was digested with Bam HI and Bgl II, separated by 1.0% agarose gel electrophoresis using TBE buffer, and purified using the ILLUSTRA ^™ GFX ^™ PCR DNA and Gel Band Purification Kit according to the manufacturer's instructions.

The fragment was directly cloned into expression vector pPFJO355 without restriction digestion and ligation using IN-FUSION ^™ CF Dry-down Cloning kit (Clontech Laboratories, Inc., Mountain View, CA, USA).

The PCR reaction product and the digested vector were ligated together using IN-FUSION ^TM CF dry-type PCR cloning (Dry-down PCR Cloning) to obtain plasmid pTs, in which the transcription of the Talaromyces stipitatus catalase gene was from Aspergillus oryzae α-starch under the regulation of the promoter of the enzyme gene. Cloning was performed according to the manufacturer's instructions. Briefly, 30 ng of Bam HI and Bgl II digested pPFJO355, and 60 ng of purified Talaromyces stipitatus catalase PCR reaction product were added to a reaction vial, and deionized water was added to resuspend the powder in a final volume of 10 μl. Reactions were incubated at 37°C for 15 minutes, then at 50°C for 15 minutes. 3 μl of the reaction product was used to transform Escherichia coli TOP10 competent cells (TIANGEN Biotech (Beijing) Co. Ltd., Beijing, China). E. coli transformants harboring the expression construct were detected by colony PCR, a method for rapid screening of plasmid insertions directly from E. coli colonies. Briefly, in each PCR tube aliquots of premixed PCR solution (containing PCR buffer, MgCl ₂ , dNTPs, and primer pairs for PCR fragment generation) were Single colonies were added by tip picking and twirl the pipette tip in the reaction solution. Typically 7 to 10 colonies are screened. After the PCR procedure, the reaction products were checked on an agarose gel. Amplified colonies giving the expected size likely contain the correct insertion. Plasmid DNA was prepared using QIAprep Spin Miniprep Kit (QIAGEN Inc., Valencia, CA, USA). The Talaromyces stipitatus catalase gene inserted in the plasmid pTs was confirmed by DNA sequencing using a 3730XL DNA Analyzer (Applied Biosystems Inc, Foster City, CA, USA).

Example 11: Expression of Talaromyces stipitatus catalase gene in Aspergillus oryzae

Aspergillus oryzae HowB101 (described in patent WO9535385 Example 1) protoplasts were prepared according to the method of Christensen et al., 1988, Bio/Technology 6:1419-1422. Aspergillus oryzae HowB101 was transformed with 3 μg of plasmid pTs.

Aspergillus oryzae HowB101 was transformed with plasmid pTs, yielding approximately 50 transformants per transformation. Eight transformants were isolated to a single minimal medium plate.

Four transformants from each transformation were individually inoculated into 3 ml of YPM in a 24-well plate and incubated at 30°C, 150 rpm. After 3 days of incubation, 20 μl of the supernatant from each culture was run on a NuPAGE Novex 4-12% Bis-Tris gel containing (2-N-morpholino)ethanesulfonic acid (MES) (Invitrogen Corporation, Carlsbad, CA, USA) were analyzed according to the manufacturer's instructions. The resulting gel was stained with INSTANT BLUE ^™ (Expedeon Ltd., Babraham Cambridge, UK). The SDS-PAGE profile of the culture showed the expression of protein bands detected. The size of the main band of the gene is approximately 92KD. The expression strain was named EXP84.

Example 12: Fermentation of expression strain EXP84

One slant expressing strain EXP84 was washed with 10 ml of YPM and inoculated into eight 2-liter flasks containing 400 ml of YPM medium to generate broth for enzyme identification. Cultures were harvested on day 3 and filtered using a 0.45 μm DURAPORE membrane (Millipore, Bedford, MA, USA).

Embodiment 13: Purify recombinant Talaromyces stipitatus catalase from Aspergillus oryzae EXP84

Fast Flow柱(GE Healthcare,Buckinghamshire,UK)。收集用0.08-0.2M NaCl洗脱的级分，并进一步在40ml Q

Fast Flow柱(GE Healthcare,Buckinghamshire,UK)上用线性NaCl梯度(0.14–0.2M)纯化。级分通过SDS-PAGE(NP0336BOX,NUPAGE4-12%BT GEL1.5MM15W)评估。汇集含有大约92kDa的条带的级分。然后将汇集的溶液通过超滤浓缩。3200 ml of the supernatant of recombinant strain EXP84 was precipitated with ammonium sulfate (80% saturated) and resuspended in 50 ml of 20 mM Tris-HCl buffer, pH 7.5, then dialyzed against the same buffer and filtered through a 0.45 mm filter to a final volume of 100ml. The solution was applied to 40 ml of Q equilibrated in 20 mM Tris-HCl buffer pH 7.5

Fast Flow columns (GE Healthcare, Buckinghamshire, UK). Collect the fractions eluted with 0.08-0.2M NaCl, and further in 40ml Q

Purification was performed on a Fast Flow column (GE Healthcare, Buckinghamshire, UK) with a linear NaCl gradient (0.14-0.2M). Fractions were evaluated by SDS-PAGE (NP0336BOX, NUPAGE4-12%BT GEL1.5MM15W). Fractions containing a band at approximately 92 kDa were pooled. The pooled solutions were then concentrated by ultrafiltration.

Example 14: Extraction of Humicola insolens Genomic DNA

(Calbiochem,La Jolla,CA,USA)的过滤来收集，并冻结在液氮中。将冻结的菌丝体通过杵和研钵磨碎为精细粉末，而基因组DNA使用

植物大提试剂盒(Plant MaxiKit)(QIAGEN Inc.,Valencia,CA,USA)根据生产商的指示分离。Humicola insolens strain NN38 was inoculated on a PDA plate, and incubated at 45° C. in the dark for 3 days. Several mycelium-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml YPG medium. The flasks were incubated at 45°C for 3 days with shaking at 160 rpm. Mycelium passes through

(Calbiochem, La Jolla, CA, USA) and frozen in liquid nitrogen. Frozen mycelium was ground into a fine powder by pestle and mortar, while genomic DNA was used

Plant MaxiKit (QIAGEN Inc., Valencia, CA, USA) was isolated according to the manufacturer's instructions.

Example 15: Genome sequencing, assembly and annotation of Humicola insolens strain NN38

GA2系统(Illumina,Inc.,San Diego,CA,USA)的基因组测序。在BGI将原始读取(raw read)使用程序SOAPdenovo(Li等,2010,GenomeResearch20(2):265-72)进行组装。将组装后的序列使用标准的用于基因查找(gene finding)和功能预测的生物信息学手段进行分析。简言之，使用geneID(Parra等,2000,Genome Research10(4):511-515)进行基因预测。使用Blastallversion2.2.10((Altschul等,1990,J.Mol.Biol.215(3):403–410;National Centerfor Biotechnology Information(NCBI),Bethesda,MD,USA)和HMMER version2.1.1(National Center for Biotechnology Information(NCBI),Bethesda,MD,USA)基于结构同源性预测功能。通过对Blast结果的分析直接鉴定出过氧化氢酶基因(对于DNA序列为SEQ ID NO:5，对于蛋白序列为SEQ ID NO:6)。使用Agene程序(Munch和Krogh,2006,BMC Bioinformatics7:263)和SignalP程序(Nielsen等,1997,Protein Engineering10:1-6)鉴定起始密码子。还用SignalP预测信号肽。使用Pepstats(European Bioinformatics Institute,Hinxton,Cambridge CB101SD,UK)预测蛋白的等电点和分子量。The extracted genomic DNA samples were sent to Beijing Genome Institute (BGI, Shenzhen, China) for use

Genome sequencing by the GA2 system (Illumina, Inc., San Diego, CA, USA). Raw reads were assembled at BGI using the program SOAPdenovo (Li et al., 2010, GenomeResearch 20(2):265-72). The assembled sequences were analyzed using standard bioinformatics tools for gene finding and function prediction. Briefly, gene prediction was performed using geneID (Parra et al., 2000, Genome Research 10(4):511-515). Using Blastallversion2.2.10 ((Altschul et al., 1990, J.Mol.Biol.215(3):403-410; National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) and HMMER version2.1.1 (National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) based on structural homology prediction function. The catalase gene (for DNA sequence is SEQ ID NO:5 for DNA sequence is SEQ ID NO:5 for protein sequence by the analysis of Blast result directly NO: 6). Use the Agene program (Munch and Krogh, 2006, BMC Bioinformatics 7: 263) and the SignalP program (Nielsen et al., 1997, Protein Engineering 10: 1-6) to identify the initiation codon. Also use SignalP to predict the signal peptide. Use Pepstats (European Bioinformatics Institute, Hinxton, Cambridge CB101SD, UK) predicted the isoelectric point and molecular weight of the protein.

Example 16: Cloning of Humicola insolens catalase from genomic DNA

Based on the DNA information of Humicola insolens catalase, the oligonucleotide primers shown below were designed to amplify the catalase gene from the genomic DNA of Humicola insolens NN38. Primers were manufactured by Invitrogen (Invitrogen, Beijing, China).

Forward primer: 5'ACACAACTGGGGATCC ACC atgaacagagtcacgaatctcctcg3' (SEQ ID NO: 40)

Reverse primer: 5'GTCACCCTCTAGATCT ggtacaactcccaccctattccttctc3' (SEQ ID NO: 41)

The lowercase letters represent the gene sequence in the forward primer, and the flanking region at the 3' end of the gene in the reverse primer, while the uppercase part is homologous to the insertion site of the pPFJO355 vector described in US2010306879.

The expression vector pPFJO355 contains the TAKA-amylase promoter from Aspergillus oryzae and the Aspergillus niger glucoamylase terminator element. In addition pPFJO355 has pUC18-derived sequences for selection and propagation in E. coli, and the pyrG gene encoding orotidine decarboxylase from Aspergillus nidulans for selection of transformants of pyrG mutant Aspergillus strains.

20 pmoles of primer pairs (forward and reverse) were used in a PCR reaction consisting of 2 μl of Humicola insolens NN38 genomic DNA, 10 μl of 5X GC buffer, 1.5 μl of DMSO, 2.5 mM each of dATP , dTTP, dGTP and dCTP, and 0.6 units of PHUSION ^™ high-fidelity DNA polymerase (Finnzymes Oy, Espoo, Finland) in a final volume of 50 μl. Amplification was performed using a PeltierThermal Cycler (MJ Research Inc., South San Francisco, CA, USA), programmed as follows: denaturation at 98°C for 1 minute; 6 cycles of denaturation at 98°C for 15 seconds and annealing at 63°C 30 seconds, each cycle of 1°C lowering, and 3 minutes extension at 72°C; and another 22 cycles, each cycle of 98°C for 15 seconds, 62°C for 30 seconds, and 72°C for 3 minutes; The final stretch was 7 minutes. The heat block then goes into a 4°C soak cycle.

PCR reaction products were separated by 1.0% agarose gel electrophoresis using 90 mM Tris boric acid and 1 mM EDTA (TBE) buffer, where a single product band of the expected size (approximately 3.1 kb) was visualized under UV light, which was then used in ILLUSTRA ^™ GFX ^™ PCR DNA and Gel Band Purification Kit (GE Healthcare, Buckinghamshire, UK) was purified from solution according to the manufacturer's instructions.

Plasmid pPFJO355 was digested with Bam HI and Bgl II, separated by 1.0% agarose gel electrophoresis using TBE buffer, and purified using the ILLUSTRA ^™ GFX ^™ PCR DNA and Gel Band Purification Kit according to the manufacturer's instructions.

The fragment was cloned directly into expression vector pPFJO355 using IN-FUSION ^™ CF Dry Cloning Kit (Clontech Laboratories, Inc., Mountain View, CA, USA) without restriction digestion and ligation.

The PCR reaction product and the digested vector were ligated together using IN-FUSION ^TM CF dry-type PCR cloning (Dry-down PCR Cloning) to obtain the plasmid pHi, in which the transcription of the Humicola insolens catalase gene was in the Under the regulation of the promoter of the α-amylase gene. Cloning was performed according to the manufacturer's instructions. Briefly, 30 ng of Bam HI and Bgl II digested pPFJO355, and 60 ng of purified Humicola insolens catalase PCR reaction product were added to a reaction vial, and deionized water was added to resuspend the powder in 10 μl of the final volume. Reactions were incubated at 37°C for 15 minutes, then at 50°C for 15 minutes. 3 μl of the reaction product was used to transform Escherichia coli TOP10 competent cells (TIANGEN Biotech (Beijing) Co. Ltd., Beijing, China). E. coli transformants containing the reporter construct were detected by colony PCR, a method for rapid screening of plasmid insertions directly from E. coli colonies. Briefly, in each PCR tube aliquots of premixed PCR solution (containing PCR buffer, MgCl ₂ , dNTPs, and primer pairs for PCR fragment generation) were Pick and twirl the pipette tip in the reaction solution to add single colonies. Typically 7 to 10 colonies are screened. After the PCR procedure, the reaction products were checked on an agarose gel. Amplified colonies giving the expected size likely contain the correct insertion. Plasmid DNA was prepared using QIAprep Spin Miniprep Kit (QIAGEN Inc., Valencia, CA, USA). The inserted Humicola insolens catalase gene in plasmid pHi was confirmed by DNA sequencing using a 3730XL DNA Analyzer (Applied Biosystems Inc, Foster City, CA, USA).

Example 17: Expression of the Humicola insolens catalase gene in Aspergillus oryzae

Aspergillus oryzae HowB101 (described in patent WO9535385 Example 1) protoplasts were prepared according to the method of Christensen et al., 1988, Bio/Technology 6:1419-1422. Aspergillus oryzae HowB101 was transformed with 3 μg of plasmid pHi.

Transformation of Aspergillus oryzae HowB101 with plasmid pHi yielded approximately 50 transformants per transformation. Eight transformants were isolated to individual minimal media plates.

Four transformants from each transformation were individually inoculated into 3 ml of YPM in a 24-well plate and incubated at 30 °C, 150 rpm. After 3 days of incubation, 20 μl of supernatant from each culture was analyzed on NuPAGE Novex 4-12% Bis-Tris gels (Invitrogen Corporation, Carlsbad, CA, USA) with MES according to the manufacturer's instructions. The resulting gel was stained with INSTANT BLUE ^™ (Expedeon Ltd., Babraham Cambridge, UK). The SDS-PAGE profile of the culture showed the expression of protein bands detected. The size of the major band of the gene is approximately 80KD. The expression strain was named O5.

Example 18: Fermentation of expression strain O5

One slant expressing strain O5 was washed with 10 ml of YPM and inoculated into 12 2-liter flasks containing 400 ml of YPM medium to generate broth for enzyme identification. Cultures were harvested on day 3 and filtered using a 0.45 μm DURAPORE membrane (Millipore, Bedford, MA, USA).

Example 19: Purification of recombinant Humicola insolens catalase from Aspergillus oryzae O5

Fast Flow柱(GE Healthcare,Buckinghamshire,UK)，并用线性NaCl梯度(0–0.25M)洗脱蛋白。收集用0.2-0.5M NaCl洗脱的级分，并进一步在平衡于20mM Bis-Tris缓冲液pH6.0中的40ml Q

Fast Flow柱(GE Healthcare,Buckinghamshire,UK)上纯化，并用线性NaCl梯度(0.2–0.5M)洗脱蛋白。级分通过SDS-PAGE(NP0336BOX,NUPAGE4-12%BTGEL1.5MM15W)评估。汇集含有大约80kDa的条带的级分。然后将汇集的溶液通过超滤浓缩。4000 ml of the recombinant strain O5 supernatant was precipitated with ammonium sulfate (80% saturated) and resuspended in 50 ml of 20 mM Tris-HCl buffer, pH 6.0, then dialyzed against the same buffer and filtered through a 0.45 mm filter to a final volume of 140ml. The solution was applied to 40 ml of Q equilibrated in 20 mM Bris-Tris buffer, pH 6.0

Fast Flow column (GE Healthcare, Buckinghamshire, UK), and the protein was eluted with a linear NaCl gradient (0-0.25M). Fractions eluted with 0.2-0.5M NaCl were collected and further in 40ml Q

Purification was performed on a Fast Flow column (GE Healthcare, Buckinghamshire, UK), and the protein was eluted with a linear NaCl gradient (0.2–0.5M). Fractions were evaluated by SDS-PAGE (NP0336BOX, NUPAGE4-12%BTGEL1.5MM15W). Fractions containing a band of approximately 80 kDa were pooled. The pooled solutions were then concentrated by ultrafiltration.

The mature polypeptide of Humicola insolens catalase has 99.25% identity with Humicola grisea heat-tolerant catalase protein (WO2009104622-A1).

Embodiment 20: Determination of catalase activity

Purified Humicola insolens catalase was examined for catalase activity using the following protocol.

The substrate was prepared by diluting 30% H ₂ O ₂ (from Xilong Chemical, Guangdong, China) 1000 times with double distilled water (ddH ₂ O) to a final concentration of 10.3 mM. Reactions were initiated by adding 1 μl of a sample of purified Humicola insolens catalase to 1000 μl of substrate. The optical density (OD) at 240 nm was read at 0 and 16 seconds with an Ultrospec 3300 (GE Healthcare, Buckinghamshire, UK), respectively, and the decrease in OD (from 0.400 to 0.102) indicates the relative activity of Humicola insolens catalase.

Example 21: Penicillium emersonii Genomic DNA Extraction

(Calbiochem,La Jolla,CA,USA)过滤来收集菌丝体，并在液氮中冷冻。将冷冻的菌丝体用杵和研钵磨碎至精细粉末，并使用真菌基因组DNA大提试剂盒-柱式(Large-Scale Column Fungal DNAout)(Baoman Biotechnology,中国上海)根据生产商的指示分离基因组DNA。Penicillium emersonii strain NN051602 was inoculated on a PDA plate and incubated at 45° C. in the dark for 3 days. Several mycelium-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated at 45°C for 3 days with shaking at 160 rpm. pass

(Calbiochem, La Jolla, CA, USA) to collect mycelium by filtration and freeze in liquid nitrogen. Frozen mycelium was ground to a fine powder with a pestle and mortar, and isolated using the Fungal Genomic DNA Large-Scale Column Fungal DNAout (Baoman Biotechnology, Shanghai, China) according to the manufacturer's instructions Genomic DNA.

Example 22: Genome Sequencing, Assembly and Annotation

GA2系统(Illumina,Inc.,San Diego,CA,USA)的基因组测序。在BGI将原始读取(raw read)使用程序SOAPdenovo(Li等,2010,GenomeResearch20(2):265-72)进行组装。将组装的序列使用标准的供基因鉴定和功能预测的生物信息学手段进行分析。简言之，使用geneID(Parra等,2000,GenomeResearch10(4):511-515)进行基因预测。使用Blastall2.2.10版本(Altschul等,1990,J.Mol.Biol.215(3):403–410,http://blast.ncbi.nlm.nih.gov/Blast.cgi)和HMMER2.1.1版本(National Center for Biotechnology Information(NCBI),Bethesda,MD,USA,http://hmmer.janelia.org)基于结构同源性预测功能。通过对Blast结果的分析直接鉴定出了过氧化氢酶。使用Agene程序(Munch和Krogh,2006,BMC Bioinformatics7:263)和SignalP程序(Nielsen等,1997,ProteinEngineering10:1-6)鉴定起始密码子。进一步使用SignalP预测信号肽。使用Pepstats(Rice等,2000,Trends Genet.16(6):276-277)估计蛋白的等电点和分子量。The extracted genomic DNA samples were delivered to Beijing Genome Institute (BGI, Shenzhen, China) for use

Genome sequencing by the GA2 system (Illumina, Inc., San Diego, CA, USA). Raw reads were assembled at BGI using the program SOAPdenovo (Li et al., 2010, GenomeResearch 20(2):265-72). The assembled sequences were analyzed using standard bioinformatics tools for gene identification and function prediction. Briefly, gene prediction was performed using geneID (Parra et al., 2000, Genome Research 10(4):511-515). Using Blastall2.2.10 version (Altschul et al., 1990, J.Mol.Biol.215(3):403–410, http://blast.ncbi.nlm.nih.gov/Blast.cgi) and HMMER2.1.1 version ( National Center for Biotechnology Information (NCBI), Bethesda, MD, USA, http://hmmer.janelia.org) based on structural homology prediction functions. Catalase was directly identified by analysis of Blast results. Initiation codons were identified using the Agene program (Munch and Krogh, 2006, BMC Bioinformatics 7:263) and the SignalP program (Nielsen et al., 1997, Protein Engineering 10:1-6). Signal peptides were further predicted using SignalP. Isoelectric points and molecular weights of proteins were estimated using Pepstats (Rice et al., 2000, Trends Genet. 16(6):276-277).

Example 23: Cloning of Penicillium emersonii catalase from genomic DNA

A catalase gene PE04230007241 (SEQ ID NO: 7) was selected for expression cloning.

Based on the gene information obtained by genome sequencing, the oligonucleotide primers shown below were designed to amplify the catalase gene PE04230007241 from the genomic DNA of Penicillium emersonii. Primers were manufactured by Invitrogen (Invitrogen, Beijing, China).

forward primer 5'ACACAACTGGGGATCC ACC atgcgcgcagtgcagct3' SEQ ID NO:42 reverse primer 5'GTCACCCCTCTAGATCTgtcgactattccaaccttcctatatggacac3' SEQ ID NO:43

The lowercase letters of the forward primer represent the coding sequence of the gene, the lowercase letters of the reverse primer represent the flanking region of the gene, and the uppercase part is homologous to the insertion site of the pPFJO355 vector described in US2010306879.

The fragment was cloned directly into the expression vector pPFJO355 described in US2010306879 using IN-FUSION ^™ CF Dry Cloning Kit (Clontech Laboratories, Inc., Mountain View, CA, USA) without restriction digestion and ligation.

The expression vector pPFJO355 contains the TAKA-amylase promoter from Aspergillus oryzae and the Aspergillus niger glucoamylase terminator element. In addition pPFJO355 has pUC18-derived sequences for selection and propagation in E. coli, and the pyrG gene encoding orotidine decarboxylase from Aspergillus nidulans, a transformant for selection of pyrG mutant Aspergillus strains.

Twenty pmoles of each of the above were used in a PCR reaction consisting of 2 μl of Penicilliumemersonii genomic DNA, 10 μl of 5X GC buffer, 1.5 μl of DMSO, 2.5 mM each of dATP, dTTP, dGTP, and dCTP, and 0.6 units PHUSION ^™ high-fidelity DNA polymerase (Finnzymes Oy, Espoo, Finland) in a final volume of 50 μl. Amplification was performed using a Peltier ThermalCycler (MJ Research Inc., South San Francisco, CA, USA), and the program was as follows: denaturation at 98°C for 1 minute; 8 cycles, each cycle denaturation at 98°C for 15 seconds, annealing at 65°C 30 seconds, each cycle of 1 °C decrease and 3 min extension at 72 °C; and another 22 cycles, each cycle of 98 °C for 15 seconds, 58 °C for 30 seconds, and 72 °C for 3 minutes 15 seconds; Final extension at 72°C for 10 minutes. The heat block then goes into a 4°C soak cycle.

The reaction products were separated by 1.0% agarose gel electrophoresis using 90 mM Tris boronic acid and 1 mM EDTA (TBE) buffer, in which a product band of approximately 2.5 kb was excised from the gel, and then separated using ILLUSTRA ^™ GFX ^™ PCRDNA and gel Band purification kit (GE Healthcare, Buckinghamshire, UK) was purified from solution according to the manufacturer's instructions.

Plasmid pPFJO355 was digested with Bam HI and Bgl II, separated by 1.0% agarose gel electrophoresis using TBE buffer, and purified using the ILLUSTRA ^™ GFX ^™ PCR DNA and Gel Band Purification Kit according to the manufacturer's instructions.

The PCR reaction product and the digested vector were ligated together using IN-FUSION ^™ CF dry-type PCR cloning (Dry-down PCR Cloning) to obtain plasmid pCat_PE04230007241, in which the transcription of the Penicillium emersonii catalase gene was from Aspergillus oryzae α-starch under the regulation of the promoter of the enzyme gene. Cloning was performed according to the manufacturer's instructions. Briefly, 30 ng of Bam HI and Bgl II digested pPFJO355, and 60 ng of purified Penicillium emersonii catalase PCR reaction product were added to a reaction vial, and deionized water was added to resuspend the powder in a final volume of 10 μl. Reactions were incubated at 37°C for 15 minutes, then at 50°C for 15 minutes. 3 μl of the reaction product was used to transform Escherichia coli TOP10 competent cells (TIANGEN Biotech (Beijing) Co. Ltd., Beijing, China). One E. coli transformant containing pCat_PE04230007241 was detected by colony PCR. Colony PCR is a method for rapid screening of plasmid insertions directly from E. coli colonies. Briefly, in each PCR tube aliquots of premixed PCR solution (containing PCR buffer, MgCl ₂ , dNTPs, and primer pairs to generate PCR fragments) were Pick and twirl the pipette tip in the reaction solution to add single colonies. Typically 7 to 10 colonies are screened. After PCR, reaction products were analyzed by 1.0% agarose gel electrophoresis using TBE buffer. Plasmid DNA was prepared using QIAprep Spin Miniprep Kit (QIAGEN Inc., Valencia, CA, USA). The Penicillium emersonii catalase gene inserted in pCat_PE04230007241 was confirmed by DNA sequencing using a 3730XL DNA analyzer (Applied Biosystems Inc, Foster City, CA, USA).

Example 24: Expression of Penicillium emersonii catalase gene in Aspergillus oryzae

Protoplasts of Aspergillus oryzae HowB101 (described in Example 1 of patent WO9535385) were prepared according to the method of Christensen et al., 1988, Bio/Technology 6:1419-1422. Aspergillus oryzae HowB101 was transformed with 3 μg of pCat_PE04230007241.

Transformation of Aspergillus oryzae HowB101 with pCat_PE04230007241 resulted in approximately 50 transformants. The four transformants were segregated to individual minimal media plates.

Four transformants were each inoculated into 3 ml of YPM in a 24-well plate and incubated at 30°C, 150 rpm. After 3 days of incubation, 20 μl of supernatant from each culture was run on a NuPAGE Novex 4-12% Bis-Tris gel (Invitrogen Corporation, Carlsbad) containing 2-(N-morpholino)ethanesulfonic acid (MES). , CA, USA) were analyzed according to the manufacturer's instructions. The resulting gel was stained with INSTANT BLUE ^™ (Expedeon Ltd., Babraham Cambridge, UK). SDS-PAGE overview of the cultures showed a band of approximately 80 KDa in all transformants. The expression strain was named O6YTS.

Embodiment 25: Fermentation of Aspergillus oryzae expression strain O6YTS

One slant expressing strain O6YTS was washed with 10 ml of YPM and inoculated into seven 2 liter flasks containing 400 ml of YPM medium to generate broth for enzyme identification. Cultures were harvested on day 3 and filtered using a 0.45 μm DURAPORE membrane (Millipore, Bedford, MA, USA).

Embodiment 26: Purify recombinant Penicillium emersonii catalase from Aspergillus oryzae O6YTS

Fast Flow柱(GE Healthcare,Buckinghamshire,UK)。用0.18-0.25M NaCl洗脱的级分通过SDS-PAGE(NP0336BOX,NUPAGE4-12%BT GEL1.5MM15W)评估。汇集含有大约80kDa的条带的级分。然后将汇集的溶液通过超滤浓缩。2800 ml of the supernatant of the recombinant strain O6YTS was precipitated with ammonium sulfate (80% saturated) and resuspended in 50 ml of 20 mM Tris-HCl buffer, pH 8.0, then dialyzed against the same buffer and filtered through a 0.45 mm filter to a final volume of 80ml. The solution was applied to 40 ml of Q equilibrated in 20 mM Tris-HCl buffer pH 8.0

Fast Flow columns (GE Healthcare, Buckinghamshire, UK). Fractions eluted with 0.18-0.25M NaCl were evaluated by SDS-PAGE (NP0336BOX, NUPAGE4-12%BT GEL1.5MM15W). Fractions containing a band of approximately 80 kDa were pooled. The pooled solutions were then concentrated by ultrafiltration.

Example 27: Determination of Catalase Activity

Purified Penicillium emersonii catalase was examined for catalase activity using the following protocol.

The substrate was prepared by diluting 30% H ₂ O ₂ (from Xilong Chemical, Guangdong, China) 1000 times with double distilled water (ddH ₂ O) to a final concentration of 10.3 mM. Reactions were initiated by adding 1 μl of a sample of purified Penicillium emersonii catalase to 1000 μl of substrate. The relative activity of Penicillium emersonii catalase was indicated by the decrease in OD (from 0.505 to 0.284) at 0 and 16 s with an Ultrospec 3300 (GE Healthcare, Buckinghamshire, UK) to read the optical density (OD) at 240 nm.

The invention is further described by the following numbered paragraphs:

[1] A method for degrading or converting a cellulosic material, comprising: treating the cellulosic material with an enzyme composition in the presence of a polypeptide having catalase activity.

[2] The method of paragraph 1, wherein the enzyme composition comprises one or more (for example several) enzymes selected from the group consisting of cellulase, GH61 polypeptide having cellulolytic enhancing activity, hemicellulase , esterase, patulin, laccase, ligninolytic enzyme, pectinase, peroxidase, protease and swellin.

[3] The method of paragraph 2, wherein the cellulase is one or more (eg, several) enzymes selected from the group consisting of endoglucanase, cellobiohydrolase, and β-glucosidase .

[4] The method of paragraph 2, wherein the hemicellulase is one or more (for example, several) enzymes selected from the group consisting of xylanase, acetylxylan esterase, ferulic acid esterase , arabinofuranosidase, xylosidase and glucuronidase.

[5] The method of any of paragraphs 1-4, wherein the cellulosic material is selected from the group consisting of agricultural residues, herbaceous materials, municipal solid waste, pulp and paper mill residues, waste paper, and wood; preferably Arundis , bagasse, bamboo, corn cobs, corn fiber, corn stover, miscanthus, orange peel, rice straw, switchgrass, straw, eucalyptus, fir, pine, poplar, spruce, willow, algal cellulose, Bacterial cellulose, cotton linters, filter paper, microcrystalline cellulose, or phosphoric acid-treated cellulose.

[6] The method of any of paragraphs 1-5, wherein the cellulosic material is pretreated, in particular by chemical, physical or biochemical pretreatment.

[7] The method of any of paragraphs 1-6, further comprising recovering the degraded cellulosic material.

[8] The method of paragraph 7, wherein the degraded cellulosic material is sugar.

[9] The method of paragraph 8, wherein the sugar is selected from the group consisting of glucose, xylose, mannose, galactose and arabinose.

[10] The method of any of paragraphs 1-9, wherein the presence of the polypeptide having catalase activity increases hydrolysis of the cellulosic material compared to the absence of the polypeptide having catalase activity.

[11] The method of any one of paragraphs 1-10, wherein the polypeptide having catalase activity is selected from the group consisting of:

(a) a polypeptide having at least 60% sequence identity to the mature polypeptide of SEQ ID NO:2, the mature polypeptide of SEQ ID NO:4, the mature polypeptide of SEQ ID NO:6, or the mature polypeptide of SEQ ID NO:8;

(b) a polypeptide encoded by a polynucleotide that hybridizes under low, medium, medium-high, high or very high stringency conditions to: (i) SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO :5, or the mature polypeptide coding sequence of SEQ ID NO:7, (ii) their cDNA sequence, or (iii) the full-length complementary strand of (i) or (ii);

(c) a polypeptide encoded by a polynucleotide having at least 60% sequence identity to: the mature polypeptide coding sequence of SEQ ID NO:1, the mature polypeptide coding sequence of SEQ ID NO:3, the mature polypeptide coding sequence of SEQ ID NO:5 The polypeptide coding sequence, or the mature polypeptide coding sequence of SEQ ID NO: 7, or its cDNA sequence;

(d) a variant of the mature polypeptide of SEQ ID NO: 2, a variant of the mature polypeptide of SEQ ID NO: 4, a variant of the mature polypeptide of SEQ ID NO: 6, or a variant of the mature polypeptide of SEQ ID NO: 8 variants comprising substitutions, deletions and/or insertions at one or more (eg several) positions; and

(e) A fragment of the polypeptide of (a), (b), (c) or (d) having catalase activity.

[12] The method of any one of paragraphs 1-11, wherein the polypeptide having catalase activity is a catalase from Thermoascus, Talaromyces, Humicola, or Penicillium .

[13] The method of paragraph 12, wherein the polypeptide having catalase activity is a catalase from Thermoascus aurantiacus, Talaromyces stipitatus, Humicola insolens, or Penicillium emersonii.

[14] A method for producing a fermentation product, comprising:

(a) saccharifying a cellulosic material with an enzyme composition in the presence of a polypeptide having catalase activity;

(b) fermenting the saccharified cellulosic material with one or more (eg, several) fermenting microorganisms to produce a fermentation product; and

(c) recovering the fermentation product from the fermentation.

[15] The method of paragraph 14, wherein the enzyme composition comprises one or more (eg several) enzymes selected from the group consisting of cellulase, GH61 polypeptide having cellulolytic enhancing activity, hemicellulase , esterase, patulin, laccase, ligninolytic enzyme, pectinase, peroxidase, protease and swellin.

[16] The method of paragraph 15, wherein the cellulase is one or more (eg, several) enzymes selected from the group consisting of endoglucanase, cellobiohydrolase and β-glucosidase .

[17] The method of paragraph 15, wherein the hemicellulase is one or more (eg, several) enzymes selected from the group consisting of xylanase, acetylxylan esterase, ferulic acid esterase , arabinofuranosidase, xylosidase and glucuronidase.

[18] The method of any of paragraphs 14-17, wherein the cellulosic material is selected from the group consisting of agricultural residues, herbaceous materials, municipal solid waste, pulp and paper mill residues, waste paper, and wood; preferably Arundis , bagasse, bamboo, corn cobs, corn fiber, corn stover, miscanthus, orange peel, rice straw, switchgrass, straw, eucalyptus, fir, pine, poplar, spruce, willow, algal cellulose, Bacterial cellulose, cotton linters, filter paper, microcrystalline cellulose, or phosphoric acid-treated cellulose.

[19] The method of any of paragraphs 14-18, wherein the cellulosic material is pretreated, in particular by chemical pretreatment, physical pretreatment or biochemical pretreatment; or wherein (a) and (b ) simultaneously in simultaneous saccharification and fermentation.

[20] The method of any of paragraphs 14-19, wherein the fermentation product is an alcohol, an alkane, a cycloalkane, an alkene, an amino acid, a gas, isoprene, a ketone, an organic acid, or a polyketide.

[21] The method of any of paragraphs 14-20, wherein the presence of the polypeptide having catalase activity increases hydrolysis of the cellulosic material compared to the absence of the polypeptide having catalase activity.

[22] The method of any one of paragraphs 14-21, wherein the polypeptide having catalase activity is selected from the group consisting of:

(a) a polypeptide having at least 60% sequence identity to the mature polypeptide of SEQ ID NO:2, the mature polypeptide of SEQ ID NO:4, the mature polypeptide of SEQ ID NO:6, or the mature polypeptide of SEQ ID NO:8;

(b) a polypeptide encoded by a polynucleotide that hybridizes under low, medium, medium-high, high or very high stringency conditions to: (i) SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO :5, or the mature polypeptide coding sequence of SEQ ID NO:7, (ii) their cDNA sequence, or (iii) the full-length complementary strand of (i) or (ii);

(c) a polypeptide encoded by a polynucleotide having at least 60% sequence identity to the following: the mature polypeptide coding sequence of SEQ ID NO:1, the mature polypeptide coding sequence of SEQ ID NO:3, the mature polypeptide coding sequence of SEQ ID NO:5 The mature polypeptide coding sequence of, or the mature polypeptide coding sequence of SEQ ID NO: 7, or its cDNA sequence;

(d) a variant of the mature polypeptide of SEQ ID NO: 2, a variant of the mature polypeptide of SEQ ID NO: 4, a variant of the mature polypeptide of SEQ ID NO: 6, or a variant of the mature polypeptide of SEQ ID NO: 8 variants comprising substitutions, deletions and/or insertions at one or more (eg several) positions; and

(e) A fragment of the polypeptide of (a), (b), (c) or (d) having catalase activity.

[23] The method of any one of paragraphs 14-22, wherein the polypeptide having catalase activity is a catalase from Thermoascus, Talaromyces, Humicola, or Penicillium .

[24] The method of paragraph 23, wherein the polypeptide having catalase activity is a catalase from Thermoascus aurantiacus, Talaromyces stipitatus, Humicola insolens, or Penicillium emersonii.

[25] A method for fermenting a cellulosic material, comprising: fermenting the cellulosic material with one or more (for example, several) fermenting microorganisms, wherein the cellulosic material is expressed in a polypeptide having catalase activity Hydrolyzed by the enzyme composition in the presence of

[26] The method of paragraph 25, wherein the enzyme composition comprises one or more (eg several) enzymes selected from the group consisting of cellulase, GH61 polypeptide having cellulolytic enhancing activity, hemicellulase , esterase, patulin, laccase, ligninolytic enzyme, pectinase, peroxidase, protease and swellin.

[27] The method of paragraph 26, wherein the cellulase is one or more (eg several) enzymes selected from the group consisting of endoglucanase, cellobiohydrolase and β-glucosidase .

[28] The method of paragraph 26, wherein the hemicellulase is one or more enzymes selected from the group consisting of xylanase, acetylxylan esterase, feruloesterase, arabinofuranosidase , xylosidase and glucuronidase.

[29] The method of any one of paragraphs 25-28, wherein the cellulosic material is selected from the group consisting of agricultural residues, herbaceous materials, municipal solid waste, pulp and paper mill residues, waste paper, and wood; preferably Arundis , bagasse, bamboo, corn cobs, corn fiber, corn stover, miscanthus, orange peel, rice straw, switchgrass, straw, eucalyptus, fir, pine, poplar, spruce, willow, algal cellulose, Bacterial cellulose, cotton linters, filter paper, microcrystalline cellulose, or phosphoric acid-treated cellulose.

[30] The method of any of paragraphs 25-29, wherein the cellulosic material is pretreated, in particular by chemical, physical or biochemical pretreatment.

[31] The method of any of paragraphs 25-30, wherein the fermentation produces a fermentation product.

[32] The method of paragraph 31, further comprising recovering the fermentation product.

[33] The method of paragraph 32, wherein the fermentation product is an alcohol, an alkane, a cycloalkane, an alkene, an amino acid, a gas, isoprene, a ketone, an organic acid, or a polyketide.

[34] The method of any of paragraphs 25-33, wherein the presence of the polypeptide having catalase activity increases hydrolysis of the cellulosic material compared to the absence of the polypeptide having catalase activity.

[35] The method of any one of paragraphs 25-34, wherein the polypeptide having catalase activity is selected from the group consisting of:

(a) a polypeptide having at least 60% sequence identity to the mature polypeptide of SEQ ID NO:2, the mature polypeptide of SEQ ID NO:4, the mature polypeptide of SEQ ID NO:6, or the mature polypeptide of SEQ ID NO:8;

(b) a polypeptide encoded by a polynucleotide that hybridizes under low, medium, medium-high, high or very high stringency conditions to: (i) SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO :5, or the mature polypeptide coding sequence of SEQ ID NO:7, (ii) their cDNA sequence, or (iii) the full-length complementary strand of (i) or (ii);

(c) a polypeptide encoded by a polynucleotide having at least 60% sequence identity to: the mature polypeptide coding sequence of SEQ ID NO:1, the mature polypeptide coding sequence of SEQ ID NO:3, the mature polypeptide coding sequence of SEQ ID NO:5 The polypeptide coding sequence, or the mature polypeptide coding sequence of SEQ ID NO: 7, or its cDNA sequence;

(d) a variant of the mature polypeptide of SEQ ID NO: 2, a variant of the mature polypeptide of SEQ ID NO: 4, a variant of the mature polypeptide of SEQ ID NO: 6, or a variant of the mature polypeptide of SEQ ID NO: 8 variants comprising substitutions, deletions and/or insertions at one or more (eg several) positions; and

(e) A fragment of the polypeptide of (a), (b), (c) or (d) having catalase activity.

[36] The method of any one of paragraphs 25-35, wherein the polypeptide having catalase activity is a catalase from Thermoascus, Talaromyces, Humicola, or Penicillium .

[37] The method of paragraph 36, wherein the polypeptide having catalase activity is a catalase from Thermoascus aurantiacus, Talaromyces stipitatus, Humicola insolens, or Penicillium emersonii.

[38] An enzyme composition for degrading or converting a cellulosic material, comprising one or more (for example, several) enzymes having cellulolytic and/or hemicellulolytic activity, and hydrogen peroxide Enzymatically active polypeptides.

[39] The enzyme composition of paragraph 38, which further comprises one or more (eg, several) enzymes selected from the group consisting of GH61 polypeptides having cellulolytic enhancing activity, esterases, patulins, laccases , ligninases, pectinases, peroxidases, proteases and swellins.

[40] The enzyme composition of paragraph 38 or 39, wherein the enzyme having cellulolytic activity is an enzyme selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase.

[41] The enzyme composition of any one of paragraphs 38-40, wherein the enzyme having hemicellulolytic activity is an enzyme selected from the group consisting of xylanase, acetylxylan esterase, ferulate enzymes, arabinofuranosidase, xylosidase, and glucuronidase.

[42] The enzyme composition of any one of paragraphs 38-41, wherein the polypeptide having catalase activity is selected from the group consisting of:

(a) a polypeptide having at least 60% sequence identity to the mature polypeptide of SEQ ID NO:2, the mature polypeptide of SEQ ID NO:4, the mature polypeptide of SEQ ID NO:6, or the mature polypeptide of SEQ ID NO:8;

(b) a polypeptide encoded by a polynucleotide that hybridizes under low, medium, medium-high, high or very high stringency conditions to: (i) SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO :5, or the mature polypeptide coding sequence of SEQ ID NO:7, (ii) their cDNA sequence, or (iii) the full-length complementary strand of (i) or (ii);

(c) a polypeptide encoded by a polynucleotide having at least 60% sequence identity to: the mature polypeptide coding sequence of SEQ ID NO:1, the mature polypeptide coding sequence of SEQ ID NO:3, the mature polypeptide coding sequence of SEQ ID NO:5 The polypeptide coding sequence, or the mature polypeptide coding sequence of SEQ ID NO: 7, or its cDNA sequence;

(d) a variant of the mature polypeptide of SEQ ID NO: 2, a variant of the mature polypeptide of SEQ ID NO: 4, a variant of the mature polypeptide of SEQ ID NO: 6, or a variant of the mature polypeptide of SEQ ID NO: 8 variants comprising substitutions, deletions and/or insertions at one or more (eg several) positions; and

(e) A fragment of the polypeptide of (a), (b), (c) or (d) having catalase activity.

[43] The enzyme composition of any one of paragraphs 38-42, wherein the polypeptide having catalase activity is a peroxidase from Thermoascus, Talaromyces, Humicola, or Penicillium. Hydrogenase.

[44] The enzyme composition of paragraph 43, wherein the polypeptide having catalase activity is a catalase from Thermoascus aurantiacus, Talaromyces stipitatus, Humicola insolens, or Penicillium emersonii.

[45] Use of the enzyme composition of any of paragraphs 38-44 for degrading or converting cellulosic material.

[46] The use of paragraph 45, wherein the cellulosic material is selected from the group consisting of agricultural residues, herbaceous materials, municipal solid waste, pulp and paper mill residues, waste paper, and wood; preferably Arundis, bagasse, bamboo , corn cob, corn fiber, corn stover, Miscanthus, orange peel, rice straw, switchgrass, straw, eucalyptus, fir, pine, poplar, spruce, willow, algae cellulose, bacterial cellulose, cotton fleece, filter paper, microcrystalline cellulose, or phosphoric acid-treated cellulose.

[47] The use of paragraph 45 or 46, wherein the cellulosic material is pretreated, in particular by chemical, physical or biochemical pretreatment.

[48] A whole culture broth formulation or cell culture composition comprising one or more (for example, several) enzymes having cellulolytic and/or hemicellulolytic activity, and enzymes having catalase activity of polypeptides.

The invention described and claimed herein is not to be limited in scope to the specific aspects disclosed herein, as these are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In case of conflict, the present disclosure including definitions will control.

Claims (25)

1. for degrading or transforming the method for cellulose materials, it comprises: under the existence of polypeptide with catalase activity, with enzyme composition, process cellulose materials.

2. the method for claim 1, wherein said enzyme composition comprises one or more (for example several) and is selected from the enzyme of lower group: cellulase, have the GH61 polypeptide of cellulolytic enhancing activity, hemicellulase, esterase, claviformin, laccase, lignin decomposition enzyme, polygalacturonase, peroxidase, proteolytic enzyme and swollenin.

3. claim 1 or 2 method, wherein said cellulose materials is selected from lower group: agricultural residue, draft material, municipal solid waste, paper pulp and paper mill resistates, waste paper and timber; Preferred giantreed, bagasse, bamboo, corn cob, zein fiber, maize straw, Chinese silvergrass platymiscium, orange peel, rice straw, switchgrass, straw, eucalyptus, fir, pine tree, willow, dragon spruce, willow, algae Mierocrystalline cellulose, bacteria cellulose, velveteen, filter paper, Microcrystalline Cellulose or the acid-treated Mierocrystalline cellulose of phosphorus.

4. the method for claim 1-3 any one, wherein said cellulose materials is pretreated, particularly pretreated by Chemical Pretreatment, physics pre-treatment or biological chemistry pre-treatment.

5. the method for claim 1-4 any one, the wherein said polypeptide with catalase activity is selected from lower group:

(a) polypeptide, itself and the mature polypeptide of SEQ ID NO:2 are, the mature polypeptide of the mature polypeptide of SEQ ID NO:4, SEQ ID NO:6 or the mature polypeptide of SEQ ID NO:8 have at least 60% sequence identity;

(b) polypeptide, its by under low, medium, medium-Gao, height or very high stringent condition with the polynucleotide encoding of following hybridization: (i) the mature polypeptide encoded sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7, (ii) their cDNA sequence, or (iii) (i) or total length complementary strand (ii);

(c) polypeptide, it is by the polynucleotide encoding with having below at least 60% sequence identity: the mature polypeptide encoded sequence of the mature polypeptide encoded sequence of the mature polypeptide encoded sequence of SEQ ID NO:1, SEQ ID NO:3, the mature polypeptide encoded sequence of SEQ ID NO:5 or SEQ ID NO:7, or its cDNA sequence;

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

(e) (a), the fragment with catalase activity of (b), (c) or polypeptide (d).

6. the method for claim 1-5 any one, the wherein said polypeptide with catalase activity is from thermophilic ascomycete, to belong to the catalase of (Thermoascus), Talaromyces (Talaromyces), Humicola (Humicola) or Penicillium (Penicillium).

7. for generation of a method for tunning, it comprises:

(a) under the existence of polypeptide with catalase activity, use enzyme composition diastatic fiber cellulosic material;

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

(c) from fermentation, reclaim tunning.

8. the method for claim 7, wherein said enzyme composition comprises one or more (for example several) and is selected from the enzyme of lower group: cellulase, have the GH61 polypeptide of cellulolytic enhancing activity, hemicellulase, esterase, claviformin, laccase, lignin decomposition enzyme, polygalacturonase, peroxidase, proteolytic enzyme and swollenin.

9. claim 7 or 8 method, wherein said cellulose materials is selected from lower group: agricultural residue, draft material, municipal solid waste, paper pulp and paper mill resistates, waste paper and timber; Preferred giantreed, bagasse, bamboo, corn cob, zein fiber, maize straw, Chinese silvergrass platymiscium, orange peel, rice straw, switchgrass, straw, eucalyptus, fir, pine tree, willow, dragon spruce, willow, algae Mierocrystalline cellulose, bacteria cellulose, velveteen, filter paper, Microcrystalline Cellulose or the acid-treated Mierocrystalline cellulose of phosphorus.

10. the method for claim 7-9 any one, wherein said tunning is alcohol, alkane, naphthenic hydrocarbon, alkene, amino acid, gas, isoprene, ketone, organic acid or polyketide.

The method of 11. claim 7-10 any one, wherein compares with not there is not the polypeptide with catalase activity, and the existence with the polypeptide of catalase activity increases the hydrolysis of cellulose materials.

12. the method for claim 7-11 any one, the wherein said polypeptide with catalase activity is selected from lower group:

(a) polypeptide, itself and the mature polypeptide of SEQ ID NO:2 are, the mature polypeptide of the mature polypeptide of SEQ ID NO:4, SEQ ID NO:6 or the mature polypeptide of SEQ ID NO:8 have at least 60% sequence identity;

(b) polypeptide, its by under low, medium, medium-Gao, height or very high stringent condition with the polynucleotide encoding of following hybridization: (i) the mature polypeptide encoded sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7, (ii) their cDNA sequence, or (iii) (i) or total length complementary strand (ii);

(c) polypeptide, it is by the polynucleotide encoding with having below at least 60% sequence identity: the mature polypeptide encoded sequence of the mature polypeptide encoded sequence of the mature polypeptide encoded sequence of SEQ ID NO:1, SEQ ID NO:3, the mature polypeptide encoded sequence of SEQ ID NO:5 or SEQ ID NO:7, or its cDNA sequence;

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

(e) (a), (b), (c) or polypeptide (d) have the fragment of catalase activity.

The method of 13. claim 7-12 any one, the wherein said polypeptide with catalase activity is the catalase from thermophilic ascomycete genus, Talaromyces, Humicola or Penicillium.

14. a method for fermented cellulose material, it comprises that for example, wherein said cellulose materials is hydrolyzed with enzyme composition under the existence of polypeptide with catalase activity with one or more (several) organism of fermentation described cellulose materials that ferments.

The method of 15. claims 14, wherein said enzyme composition comprises one or more (for example several) and is selected from the enzyme of lower group: cellulase, have the GH61 polypeptide of cellulolytic enhancing activity, hemicellulase, esterase, claviformin, laccase, lignin decomposition enzyme, polygalacturonase, peroxidase, proteolytic enzyme and swollenin.

The method of 16. claims 14 or 15, wherein said cellulose materials is selected from lower group: agricultural residue, draft material, municipal solid waste, paper pulp and paper mill resistates, waste paper and timber; Preferred giantreed, bagasse, bamboo, corn cob, zein fiber, maize straw, Chinese silvergrass platymiscium, orange peel, rice straw, switchgrass, straw, eucalyptus, fir, pine tree, willow, dragon spruce, willow, algae Mierocrystalline cellulose, bacteria cellulose, velveteen, filter paper, Microcrystalline Cellulose or the acid-treated Mierocrystalline cellulose of phosphorus.

17. the method for claim 14-16 any one, the polypeptide wherein with catalase activity is selected from lower group:

(a) polypeptide, itself and the mature polypeptide of SEQ ID NO:2 are, the mature polypeptide of the mature polypeptide of SEQ ID NO:4, SEQ ID NO:6 or the mature polypeptide of SEQ ID NO:8 have at least 60% sequence identity;

(b) polypeptide, it is by polynucleotide encoding, described polynucleotide are hybridized with following under low, medium, medium-Gao, height or very high stringent condition: (i) the mature polypeptide encoded sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7, (ii) their cDNA sequence, or (iii) (i) or total length complementary strand (ii);

(c) polypeptide, it is by the polynucleotide encoding with having below at least 60% sequence identity: the mature polypeptide encoded sequence of the mature polypeptide encoded sequence of the mature polypeptide encoded sequence of SEQ ID NO:1, SEQ ID NO:3, the mature polypeptide encoded sequence of SEQ ID NO:5 or SEQ ID NO:7, or its cDNA sequence;

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

(e) (a), the fragment with catalase activity of (b), (c) or polypeptide (d).

The method of 18. claim 14-17 any one, the wherein said polypeptide with catalase activity is the catalase from thermophilic ascomycete genus, Talaromyces, Humicola or Penicillium.

For example, 19. for degrading or transforming the enzyme composition of cellulose materials, it comprises one or more (several) and has the enzyme of cellulose decomposition and/or hemicellulose degrading activity, and has the polypeptide of catalase activity.

The enzyme composition of 20. claims 19, it further comprises one or more (for example several) and is selected from the enzyme of lower group: GH61 polypeptide, esterase, claviformin, laccase, lignin decomposition enzyme, polygalacturonase, peroxidase, proteolytic enzyme and the swollenin with cellulolytic enhancing activity.

21. the enzyme composition of claim 19 or 20, the wherein said polypeptide with catalase activity is selected from lower group:

(a) polypeptide, itself and the mature polypeptide of SEQ ID NO:2 are, the mature polypeptide of the mature polypeptide of SEQ ID NO:4, SEQ ID NO:6 or the mature polypeptide of SEQ ID NO:8 have at least 60% sequence identity;

(b) polypeptide, its by under low, medium, medium-Gao, height or very high stringent condition with the polynucleotide encoding of following hybridization: (i) the mature polypeptide encoded sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7, (ii) their cDNA sequence, or (iii) (i) or total length complementary strand (ii);

(c) polypeptide, its by with the following polynucleotide encoding with at least 60% sequence identity: the mature polypeptide encoded sequence of the mature polypeptide encoded sequence of the mature polypeptide encoded sequence of SEQ ID NO:1, SEQ ID NO:3, the mature polypeptide encoded sequence of SEQ ID NO:5 or SEQ ID NO:7, or its cDNA sequence;

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

(e) (a), the fragment with catalase activity of (b), (c) or polypeptide (d).

The enzyme composition of 22. claim 19-21 any one, the wherein said polypeptide with catalase activity is the catalase from thermophilic ascomycete genus, Talaromyces, Humicola or Penicillium.

The enzyme composition of 23. claim 19-22 any one is in degraded or transform the purposes in cellulose materials.

The purposes of 24. claims 23, wherein said cellulose materials is selected from lower group: agricultural residue, draft material, municipal solid waste, paper pulp and paper mill resistates, waste paper and timber; Preferred giantreed, bagasse, bamboo, corn cob, zein fiber, maize straw, Chinese silvergrass platymiscium, orange peel, rice straw, switchgrass, straw, eucalyptus, fir, pine tree, willow, dragon spruce, willow, algae Mierocrystalline cellulose, bacteria cellulose, velveteen, filter paper, Microcrystalline Cellulose or the acid-treated Mierocrystalline cellulose of phosphorus.

25. full nutrient solution formulation or a cell culture compositions, it comprises one or more (for example several) and has the enzyme of cellulose decomposition and/or hemicellulose degrading activity, and has the polypeptide of catalase activity.

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