METHODS FOR DEGRADING AND FERMENTING A CELLULOSIC MATERIAL, AND FOR PRODUCING A FERMENTATION PRODUCT

专利摘要:
methods to degrade and ferment a cellulosic, and to produce a fermentation product. the present invention relates to methods of degrading or converting a pre-treated cellulosic material with a composition comprising one or more gh61 polypeptides.
公开号:BR112013010008B1
申请号:R112013010008-7
申请日:2011-11-02
公开日:2020-09-08
发明作者:Jason Quinlan；Feng Xu
申请人:Novozymes, Inc.；
IPC主号:

专利说明:

[0001] [0001] The present invention relates to methods of degrading or converting a cellulosic material pretreated with a GH61 polypeptide. DESCRIPTION OF RELATED TECHNIQUE
[0002] [0002] Cellulose is a simple sugar polymer covalently linked by beta-1,4 bonds. Many microorganisms produce enzymes that hydrolyze bound beta-glycans. These enzymes include endoglucanases, cellobiohydrolases and beta-glycosidases. Endoglucanases digest the cellulose polymer at random locations, opening it up to attack by cellobiohydrolases. Cellobiohydrolases sequentially release cellobiose molecules from the ends of the cellulose polymer. Cellobiosis is a dimer bound to water-soluble 1,4-beta glucose. Beta-glycosidases hydrolyze cellobiose into glucose.
[0003] [0003] The conversion of lignocellulolytic raw materials into ethanol has the advantages of the immediate availability of large quantities of raw material, the advantage of avoiding burning or grounding the materials, and the cleaning of fuel ethanol. Wood, agricultural residues, herbaceous crops and municipal solid residues are considered raw materials for the production of ethanol. These materials mainly consist of cellulose, hemicellulose, and lignin. Since lignocellulose is converted into fermentable sugars, for example, glucose, fermentable sugars are easily fermented by yeast in fermentation products, for example, ethanol.
[0004] [0004] WO 2005/074647, WO 2008/148131 and WO 2011/035027 disclose isolated GH61 polypeptides with enhanced cellulolytic activity and their polynucleotides from Thielavia terrestris. WO 2005/074656 and WO 2010/065830 disclose isolated GH61 polypeptides with enhanced cellulolytic activity and their polynucleotides from Thermoascus aurantiacus. WO 2007/089290 discloses an isolated GH61 polypeptide with enhanced cellulolytic activity and its polynucleotide from Trichoderma reesei. WO 2009/085935, WO 2009/085859, WO 2009/085864, and WO 2009/085868 disclose isolated GH61 polypeptides with enhanced cellulolytic activity and their polynucleotides from Myceliophthora thermophila. WO 2010/138754 discloses isolated GH61 polypeptides with enhanced cellulolytic activity and their polynucleotides from Aspergillus fumigatus. WO 2011/005867 discloses isolated GH61 polypeptides with enhanced cellulolytic activity and their polynucleotides from Penicillium pinophilum. WO 2011/039319 discloses isolated GH61 polypeptides with enhanced cellulolytic activity and their polynucleotides from Thermoascus sp. WO 2011/041397 discloses isolated GH61 polypeptides with enhanced cellulolytic activity and their polynucleotides from Penicillium sp. WO 2011/041504 discloses isolated GH61 polypeptides with enhanced cellulolytic activity and their polynucleotides from Thermoascus crustaceous. WO 2008/151043 discloses methods of improving the activity of a GH61 polypeptide with enhanced cellulolytic activity, by adding a soluble activating divalent metal cation to a composition comprising the polypeptide.
[0005] [0005] The ability to improve the pretreatment of a cellulosic material for ethanol production could be an advantage in the technique.
[0006] [0006] The present invention relates to methods of degrading or converting a cellulosic material pretreated with GH61 polypeptide. SUMMARY OF THE INVENTION
[0007] (a) pré-tratar o material celulósico com uma composição compreendendo um ou mais (por exemplo, vários) polipeptídeos GH61; e (b) sacarificar o material celulósico pré-tratado com polipeptídeo GH61 com uma composição enzimática. A presente invenção também se refere aos métodos de produzir um produto de fermentação, compreendendo: (a) pré-tratar um material celulósico com uma composição compreendendo um ou mais (por exemplo, vários) polipeptídeos GH61; (b) sacarificar o material celulósico pré-tratado com polipeptídeo GH61 com uma composição enzimática; (c) fermentar o material celulósico sacarificado com um ou mais (por exemplo, vários) microrganismos fermentadores para sintetizar o produto de fermentação; e (d) recuperar o produto de fermentação a partir da fermentação. [0007] The present invention relates to methods of degrading a cellulosic material, comprising: (a) pretreating the cellulosic material with a composition comprising one or more (for example, several) GH61 polypeptides; and (b) saccharifying the cellulosic material pretreated with GH61 polypeptide with an enzymatic composition. The present invention also relates to methods of producing a fermentation product, comprising: (a) pretreating a cellulosic material with a composition comprising one or more (for example, several) GH61 polypeptides; (b) saccharifying the cellulosic material pretreated with GH61 polypeptide with an enzymatic composition; (c) fermenting the saccharified cellulosic material with one or more (for example, several) fermenting microorganisms to synthesize the fermentation product; and (d) recovering the fermentation product from the fermentation.
[0008] (a) sacarificar um material celulósico com uma composição enzimática, em que o material celulósico é pré-tratado com uma composição compreendendo um ou mais (por exemplo, vários) polipeptídeos GH61; (b) fermentar o material celulósico sacarificado com um ou mais (por exemplo, vários) microrganismos fermentadores para sintetizar o produto de fermentação; e (c) recuperar o produto de fermentação a partir da fermentação. [0008] The present invention also relates to methods of producing a fermentation product, comprising: (a) saccharifying a cellulosic material with an enzymatic composition, wherein the cellulosic material is pretreated with a composition comprising one or more (for example, several) GH61 polypeptides; (b) fermenting the saccharified cellulosic material with one or more (for example, several) fermenting microorganisms to synthesize the fermentation product; and (c) recovering the fermentation product from fermentation.
[0009] [0009] The present invention also relates to methods of fermenting a cellulosic material, comprising: fermenting the cellulosic material with one or more (for example, several) fermenting microorganisms, wherein the cellulosic material is pretreated with a composition comprising a or more (for example, several) GH61 polypeptides, and is saccharified with an enzyme composition. BRIEF DESCRIPTION OF THE FIGURES
[0010] [00010] Figure 1 shows the fractional hydrolysis of (A) PCS washed with hot water, ground and pre-treated with enzyme by a T. reesei cellulase composition, in the presence of the indicated T. terrestris GH61E polypeptide concentration; the PCS washed with hot water was pre-treated by the indicated enzymes for 1 day; and (B) PCS washed with hot water, ground and pre-treated with enzyme by a T. reesei cellulase composition, in the presence of the indicated concentration of T. terrestris GH61E polypeptide; the PCS washed with hot water was pre-treated by the indicated enzymes for 3 days.
[0011] [00011] Figure 2 shows a comparison of the fractional hydrolysis of 1 and 3 days of the PCS washed with hot, ground and pretreated water, of the T. terrestris GH61E polypeptide, by a T. reesei cellulase composition in the presence of indicated concentration of T. terrestris GH61E polypeptide. Definitions
[0012] [00012] Acetylxylan esterase: The term "acetylxylan esterase" means a carboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of acetyl groups from polymeric xylan, acetylated xylose, acetylated glucose, alpha-naphthyl acetate and p-acetate nitrophenyl. For purposes of the present invention, acetylxylan esterase activity is determined using 0.5 mM p-nitrophenylacetate as a substrate in 50 mM sodium acetate pH 5.0, containing 0.01% TWEEN ™ 20 (polyoxyethylene monolaurate and sorbitan) . One unit of acetylxylan esterase is defined as the amount of enzyme capable of releasing 1 µmol of p-nitrophenolate anion per minute at pH 5, 25 ° C.
[0013] [00013] Allelic variant: The term "allelic variant" means any of two or more alternative forms of a gene that occupies the same chromosomal locus. Allelic variation naturally increases through mutation, and can result in polymorphism in populations. Genetic mutations can be silent (no change in the encoded polypeptide,) or can encode polypeptides with altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.
[0014] [00014] Alpha-L-arabinofuranosidase: The term "alpha-L-arabinofuranosidase" means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55), which catalyzes the hydrolysis of alpha-L-arabinofuranoside non-reducing residues in alpha -L-arabinosides. The enzyme acts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3) and / or (1,5) bonds, arabinoxylans and arabinogalactans. Alpha-L-arabinofuranosidase is also known as arabinosidase, alpha-arabinosidase, alpha-L-arabinosidase, alpha-arabinofuranosidase, alpha-L-arabinofuranosidase polysaccharide, alpha-L-arabinofuranoside hydrolase, L-arabinosidase or alpha-L-arabinanase. For the purposes of the present invention, the activity of alpha-L-arabinofuranosidase is determined 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, in a total volume of 200 µL for 30 minutes at 40 ° C, followed by arabinose analysis by AMINEX® HPX-87H column chromatography (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
[0015] [00015] Alpha-glucuronidase: The term "alpha-glucuronidase" means an alpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1.139), which catalyzes the hydrolysis of an alpha-D-glucuronoside to D-glucuronate and an alcohol. For the purposes of the present invention, alpha-glucuronidase activity is determined according to de Vries, 1998, J. Bacteriol. 180: 243-249. One unit of alpha-glucuronidase is equal to the amount of enzyme capable of releasing 1 µmol of glucuronic acid or 4-O-methylglucuronic per minute at pH 5, 40 ° C.
[0016] [00016] Beta-glucosidase: The term “beta-glucosidase” means a beta-D-glycoside glycohydrolase (EC 3.2.1.21) that catalyzes the hydrolysis of non-terminal reducing beta-D-glucose residues, with the release of beta- D-glucose. For purposes of the present invention, beta-glycosidase activity is determined using p-nitrophenyl-beta-D-glycopyranoside as a substrate according to the procedure of Venturi et al., 2002, Extracellular beta-D-glucosidase from Chaetomium thermophilum var. coprophilum: production, purification and some biochemical properties, J. Basic Microbiol. 42: 55-66. One unit of beta-glycosidase is defined as 1.0 μmol of p-nitrophenolate anion produced per minute at 25 ° C, pH 4.8, from 1 mM p-nitrophenyl-beta-D-glycopyranoside as a substrate in citrate 50 mM sodium containing 0.01% TWEEN® 20.
[0017] [00017] Beta-xylosidase: The term “beta-xylosidase” means a beta-D-xyloside xylohydrolase (EC 3.2.1.37), which catalyzes the exohydrolysis of short beta (1 ^ 4) -xyl-oligosaccharides, to remove successive residues of D-xylose from the non-reducing terminations. For the purposes of the present invention, a beta-xylosidase unit is defined as 1.0 μmol of p-nitrophenolate anion produced per minute at 40 ° C, pH 5, from 1 mM p-nitrophenyl-beta-D-xyloside as 100 mM sodium citrate substrate containing 0.01% TWEEN® 20.
[0018] [00018] DNAc: The term "DNAc" means a DNA molecule that can be prepared by reverse transcription from a mature and joined mRNA molecule, obtained from a eukaryotic or prokaryotic cell. CDNA does not have intron sequences that may be present in the corresponding genomic DNA. The initial and primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature and spliced mRNA.
[0019] [00019] Cellobiohydrolase: The term "cellobiohydrolase" means a 1,4-beta-D-glycan cellobiohydrolase (EC 3.2.1.91) that catalyzes the hydrolysis of 1,4-beta-D-glycosidic bonds in cellulose, cell-oligosaccharides, or any beta-1,4-linked glucose containing polymer, which releases cellobiose from the reducing or non-reducing ends of the chain (Teeri, 1997, Crystalline cellulose 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 cellullose , Biochem. Soc. Trans. 26: 173-178). For purposes of the present invention, cellobiohydrolase activity is determined according to the procedures described by Lever et al., 1972, Anal. Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBS Letters, 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters, 187: 283-288; and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581. In the present invention, the method of Lever et al. can be used to ensure cellulose hydrolysis in corn straw, while the methods of van Tilbeurgh et al. and Tomme et al. can be used to determine cellobiohydrolase activity in a fluorescent disaccharide derivative, 4-methylumbelliferyl-β-D-lactoside.
[0020] [00020] Cellulolytic enzyme or cellulase: The term "cellulolytic enzyme" or "cellulase" means one or more (for example, several) enzymes that hydrolyze a cellulosic material. Such enzymes include endoglucanase (s), cellobiohydrolase (s), beta-glycosidase (s), or combinations thereof. The two basic approaches to assess cellulolytic activity include: (1) measuring total cellulolytic activity, and (2) measuring individual cellulolytic activities (endoglucanases, cellobiohydrolases and beta-glucosidases) in the manner reviewed in Zhang et al., Outlook for cellullase improvement: Screening and selection strategies, 2006, Biotechnology Advances 24: 452481. Total cellulolytic activity is assessed in general using insoluble substrates, including Whatman No. 1 filter paper, microcrystalline cellulose, bacterial cellulose, algae cellulose, cotton, pre-lignocellulose -treated, etc. The most common total cellulolytic activity assay is the filter paper assay using Whatman No. 1 filter paper as the substrate. The assay was established by the International Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987, Measurement of cellullase activities, Pure Appl. Chem. 59: 257-68).
[0021] [00021] For purposes of the present invention, cellulolytic enzyme activity is determined by measuring the increase in hydrolysis of a cellulosic material by cellulolytic enzyme (s) under the following conditions: 1-50 mg of cellulolytic enzyme protein / g of cellulose in PCS (or other pretreated cellulosic material) for 3-7 days at an appropriate temperature, for example, 50 ° C, 55 ° C, or 60 ° C, compared to a control hydrolysis without the addition of cellulolytic enzymatic protein . Typical conditions are 1 mL reactions, washed or unwashed PCS, 5% insoluble solids, 50 mM sodium acetate pH 5, 1 mM MnSO4, 50 ° C, 55 ° C, or 60 ° C, 72 hours, analysis of sugar per AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
[0022] [00022] Cellulosic material: The term "cellulosic material" means any material containing cellulose. The predominant polysaccharide in the primary cell wall of the biomass is cellulose, the second most abundant is hemicellulose, and the third is pectin. The secondary cell wall, produced after the cell stops growing, also contains polysaccharides and is reinforced by polymeric lignin covalently cross-linked to hemicellulose. Cellulose is an anhydrocelobiose homopolymer and is thus a linear beta- (1-4) -D-glycan, while hemicelluloses include a variety of compounds, such as xylans, xyloglycans, arabinoxylans, and mannans in complex branched structures with a spectrum of substituents. Although it is generally polymorphic, cellulose is found in plant tissue mainly as an insoluble crystalline matrix of parallel glycan chains. Hemicelluloses in general bind hydrogen to cellulose, as well as other hemicelluloses, which help to stabilize the cell wall matrix.
[0023] [00023] Cellulose is generally found, for example, in the stems, leaves, sepals, barks and cobs of plants or leaves, branches and wood of trees. Cellulosic material can be, but is not limited to, agricultural waste, herbaceous material (including crops intended for energy production), municipal solid waste, pulp and ground paper waste, paper and wood waste (including forest waste) (see, for example) example, Wiselogel et al., 1995, in Handbook on Bioethanol (Charles E. Wyman, editor), pp.105-118, Taylor & Francis, Washington DC; Wyman, 1994, Bioresource Technology 50: 3-16; Lynd, 1990 , Applied Biochemistry and Biotechnology 24/25: 695-719; Mosier et al., 1999, Recent Progress in Bioconversion of Lignocellulosics, in Advances in Biochemical Engineering / Biotechnology, T. Scheper, executive editor, Volume 65, pp.23-40 , Springer-Verlag, New York). It is understood here that cellulose may be in the form of lignocellulose, a plant cell wall material containing lignin, cellulose and hemicellulose in a mixed matrix. In a preferred aspect, the cellulosic material is any material from the biomass. In another preferred aspect, the cellulosic material is lignocellulose, which comprises cellulose, hemicelluloses and lignin.
[0024] [00024] In one aspect, cellulosic material is agricultural waste. In another aspect, cellulosic material is herbaceous material (including crops intended for energy production). In another aspect, cellulosic material is municipal solid waste. In another aspect, the cellulosic material is pulp and ground paper residue. In another aspect, the cellulosic material is waste paper. In another aspect, the cellulosic material is wood (including forest waste).
[0025] [00025] 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 ear of corn. In another aspect, the cellulosic material is corn fiber. In another aspect, the cellulosic material is corn straw. 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 yellow millet. In another aspect, the cellulosic material is wheat straw.
[0026] [00026] In another aspect, the cellulosic material is aspen. In another aspect, the cellulosic material is eucalyptus. In another aspect, the cellulosic material is pine. 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.
[0027] [00027] In another aspect, the cellulosic material is algae cellulose. In another aspect, the cellulosic material is bacterial cellulose. In another aspect, the cellulosic material is cotton lint. 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 cellulose treated with phosphoric acid.
[0028] [00028] In another aspect, the cellulosic material is an aquatic biomass. As used herein the term "aquatic biomass" means biomass produced in an aquatic environment by a process of photosynthesis. Aquatic biomass can be algae, emerging plants, floating leaf plants, or submerged plants.
[0029] [00029] The cellulosic material can be used as is, or can be subjected to pretreatment using conventional methods known in the art, in the manner described herein. In a preferred aspect, the cellulosic material is pre-treated.
[0030] [00030] Control sequences: The term "control sequences" means nucleic acid sequences necessary for the expression of a polynucleotide that encodes a mature polypeptide of the present invention. Each control sequence can be natural (i.e., from the same gene) or foreign (i.e., from a different gene) to the polynucleotide encoding the polypeptide, or natural or foreign to each other. Such control sequences include, but are not limited to, a major sequence, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence and transcription terminator. At a minimum, control sequences include a promoter and transcriptional and translational stop signals. Control sequences can be provided with ligands for the purpose of introducing specific restriction sites that facilitate the binding of control sequences to the polynucleotide coding region that encodes a polypeptide.
[0031] [00031] Endoglucanase: The term “endoglucanase” means an endo-1,4- (1,3; 1,4) -beta-D-glycan 4-glycanhydrolase (EC 3.2.1.4) that catalyzes the bonding endohydrolysis 1,4-beta-D-glycosides in cellulose, cellulose derivatives (such as carboxymethyl cellulose and hydroxyethyl cellulose), lichenine, beta-1,4 bonds in mixed beta-1,3 glycans such as beta-D-glycans or xyloglycans cereal, and other plant material containing cellulosic components. Endoglucanase activity can be determined by measuring the reduction in substrate viscosity, or the increase in reducing ends determined by a reducing sugar assay (Zhang et al., 2006, Biotechnology Advances 24: 452-481). For purposes of the present invention, endoglucanase activity is determined using carboxymethyl cellulose (CMC) as a substrate according to the procedure of Ghose, 1987, Pure and Appl. Chem. 59: 257-268, at pH 5, 40 ° C.
[0032] [00032] 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.
[0033] [00033] Expression vector: The term "expression vector" means a linear or circular DNA molecule that comprises a polynucleotide that encodes a polypeptide, and is operably linked to the control sequences that provide its expression.
[0034] [00034] Glycoside hydrolase family 61: The term "Glycoside hydrolase family 61" or "GH61 family" or "GH61" means a polypeptide that belongs to the glycoside hydrolase family 61 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. The enzymes in this family were originally classified as a family of glycoside hydrolase, based on the measurement of very weak endo-1,4-beta-D-glycanase activity in a family member. The structure and mode of action of these enzymes are non-canonical and cannot be considered as authentic glycosidases. However, they are maintained in the CAZy classification, based on their ability to improve the breakdown of lignocellulose when used in conjunction with a cellulase or a mixture of cellulases.
[0035] [00035] Feruloyl esterase: The term "feruloyl esterase" means a hydrolysis of the 4-hydroxy-3-methoxycinnamyl (EC 3.1.1.73) sugar that catalyzes the hydrolysis of the 4-hydroxy-3-methoxycinnamyl (feruloyl) groups from the sugar esterified, which is generally arabinose on “natural” substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate). Feruloyl esterase is also known as feryl acid esterase, hydroxycinnamyl esterase, FAE-III, cinnamoyl ester hydrolase, FAEA, cinnAE, FAE-I, or FAE-II. For purposes of the present invention, feruloyl esterase activity is determined using 0.5 mM p-nitrophenylferulate as a substrate in 50 mM sodium acetate, pH 5.0. One unit of feruloyl esterase is equivalent to the amount of enzyme capable of releasing 1 µmol of p-nitrophenolate anion per minute at pH 5, 25 ° C.
[0036] [00036] Fragment: The term "fragment" means a polypeptide with one or more (for example, several) amino acids missing at the amino and / or carboxyl terminus of a mature polypeptide; where the fragment has biological activity, for example, enzymatic activity.
[0037] [00037] Hemicellulolytic enzyme or hemicellulase: The term "hemicellulolytic enzyme" or "hemicellulase" means one or more (for example, several) enzymes that hydrolyze a hemicellulosic material. See, for example, Shallom, D. and Shoham, Y. Microbial hemicelullases. Current Opinion in Microbiology, 2003, 6 (3): 219-228). Hemicellulases are key components in the degradation of plant biomass. Examples of hemicellulases include, but are not limited to, an acetylmannan esterase, an acetylxylan esterase, an arabinanase, an arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, a galactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, a mannosidase, a mannosidase, and a xylosidase. The substrates of these enzymes, hemicelluloses, are a heterogeneous group of branched and linear polysaccharides that are linked by means of hydrogen bonds to cellulose microfibrils in the plant cell wall, cross-linking them in a strong network. Hemicelluloses are also covalently attached to lignin, forming together with cellulose a very complex structure. The variable structure and organization of hemicelluloses requires the combined action of many enzymes for their complete degradation. The catalytic modules of hemicellulases are both glycoside hydrolases (GHs), which hydrolyze glycosidic bonds, and carbohydrate esterases (CEs), which hydrolyze acetate ester bonds or lateral groups of ferulic acid. These catalytic modes, based on the homology of their primary sequence, can be determined in the GH and CE families. Some families, with a similar full fold, can be further grouped into clans, marked alphabetically (for example, GH-A). A more informative classification and an updated classification of these and other enzymes active in carbohydrates are available in the Carbohydrate-Active Enzymes (CAZy) database. The activities of the hemicellulolytic enzyme can be evaluated according to Ghose and Bisaria, 1987, Pure & AppI. Chem. 59: 1739-1752, at a suitable temperature, for example, 50 ° C, 55 ° C, or 60 ° C, and at pH, for example, 5.0 or 5.5.
[0038] [00038] High severity conditions: The term "high severity conditions" means probes of at least 100 nucleotides in length, prehybridization and hybridization at 42 ° C in 5X SSPE, 0.3% SDS, 200 micrograms / mL of denatured and sheared DNA from salmon sperm, and 50% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 65 ° C.
[0039] [00039] Host cell: The term "host cell" means any type of cell that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" includes any progeny of a parental cell that is not identical to the parental cell because of the mutations that occur during replication.
[0040] [00040] Isolated: The term "isolated" means a substance in a form or environment that does not occur in nature. Non-limiting examples of isolated substances include (1) any non-naturally occurring substance, (2) any substance including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide or cofactor that is at least partially removed from one or more, or all, the naturally occurring constituents with which it is associated in nature; (3) any substance modified by human manipulation related to that substance found in nature; or (4) any substance modified by increasing the amount of the substance related to other components with which it is naturally associated (for example, multiple copies of a gene encoding the substance; use of a stronger promoter than the promoter naturally associated with the gene encoding the substance). The polypeptide can be used in industrial applications, in the form of a fermentation broth product, that is, the polypeptide is a component of a fermentation broth used as a product in industrial applications (for example, ethanol production). The fermentation broth product, in addition to the polypeptide, will comprise additional ingredients used in the fermentation process such as, for example, cells (including, host cells containing the gene encoding the polypeptide, which are used to produce the polypeptide of interest) , cell debris, biomass, fermentation media and / or fermentation products. The fermentation broth can optionally be subjected to one or more purification steps (including filtration), to remove or reduce more components of a fermentation process. In this way, an isolated substance can be present in a fermentation broth product like this.
[0041] [00041] Low severity conditions: The term "low severity conditions" means probes of at least 100 nucleotides in length, prehybridization and hybridization at 42 ° C in 5X SSPE, 0.3% SDS, 200 micrograms / mL of denatured and sheared DNA from salmon sperm, and 25% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each, for 15 minutes, using 2X SSC, 0.2% SDS at 50 ° C.
[0042] [00042] 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, etc. It is known in the art that a host cell can produce a mixture of two or more different mature polypeptides (i.e., with a different C-terminal and / or N-terminal amino acid) expressed by the same polynucleotide. The mature polypeptide can be predicted using the SignalP program (Nielsen et al., 1997, Protein Engineering 10: 1-6). It is also known in the art that different host cells process polypeptides differently, and thus a host cell that expresses a polynucleotide can produce a different mature polypeptide (for example, with a different C-terminal and / or N-terminal amino acid), compared to another host cell that expresses the same polynucleotide.
[0043] [00043] Sequence encoding the mature polypeptide: The term "sequence encoding the mature polypeptide" is defined herein as a nucleotide sequence encoding a mature polypeptide with biological activity, for example, enzymatic activity. The sequence encoding the mature polypeptide can be predicted using the SignalP program (Nielsen et al., 1997, supra). It is known in the art that a host cell can produce a mixture of two or more different mature polypeptides (i.e., with a different C-terminal and / or N-terminal amino acid) expressed by the same polynucleotide. It is also known in the art that different host cells process polypeptides differently, and thus a host cell that expresses a polynucleotide can produce a different mature polypeptide (for example, with a different C-terminal and / or N-terminal amino acid), compared to another host cell that expresses the same polynucleotide.
[0044] [00044] Medium severity conditions: The term "medium severity conditions" means probes of at least 100 nucleotides in length, prehybridization and hybridization at 42 ° C in 5X SSPE, 0.3% SDS, 200 micrograms / mL of denatured and sheared salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 55 ° C.
[0045] [00045] Medium-high severity conditions: The term “medium-high severity conditions” means probes of at least 100 nucleotides in length, prehybridization and hybridization at 42 ° C in 5X SSPE, 0.3% SDS, 200 micrograms / mL of denatured and sheared salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 60 ° C.
[0046] [00046] Nucleic acid construct: The term '' nucleic acid construct 'means a nucleic acid molecule, both single-stranded and double-stranded, which is isolated from a naturally occurring gene, or is modified to contain nucleic acid segments in a way that could not exist in nature, or that is synthetic, that comprises one or more control sequences.
[0047] [00047] Operably linked: The term "operably linked" means a configuration in which a control sequence is placed in an appropriate position with respect to the coding sequence of a polynucleotide, in such a way that the control sequence directs the expression of the coding sequence .
[0048] [00048] Polypeptide with enhanced cellulolytic activity: The term "polypeptide with enhanced cellulolytic activity" means a GH61 polypeptide that catalyzes the intensification of the hydrolysis of a cellulosic material by an enzyme with cellulolytic activity. For purposes of the present invention, the enhanced cellulolytic activity is determined by measuring the increase in reducing sugars, or the increase in the total cellobiose and glucose from the hydrolysis of a cellulosic material by cellulolytic enzyme under the following conditions: 1-50 mg of protein total / g cellulose in PCS, where the total protein is comprised of 50-99.5% w / w of cellulolytic enzyme protein and 0.5-50% w / w of protein of a GH61 polypeptide with cellulolytic activity intensified for 1-7 days at an appropriate temperature, for example, 50 ° C, 55 ° C, or 60 ° C and pH, for example, 5.0 or 5.5, compared to a control hydrolysis with full protein charge without intensified cellulolytic activity equal (1-50 mg cellulolytic protein / g cellulose in PCS). In a preferred aspect, a mixture of CELLUCLAST® 1.5 L (Novozymes A / S, Bagsv ^ rd, Denmark), in the presence of 2-3% total weight of beta-glucosidase protein from Aspergillus oryzae (recombinantly produced in Aspergillus oryzae according to WO 02/095014), or 2-3 wt.% of beta-glucosidase protein from Aspergillus fumigatus (recombinantly produced in Aspergillus oryzae as described in WO 2002/095014), the cellulase protein load is used as the source of cellulolytic activity.
[0049] [00049] GH61 polypeptides with enhanced cellulolytic activity intensify the hydrolysis of a cellulosic material, catalyzed by enzyme with cellulolytic activity, reducing the amount of cellulolytic enzyme required to achieve the same degree of hydrolysis, preferably at least 1.01 times, for example at least 1.05 times, at least 1.10 times, at least 1.25 times, at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least at least 10 times, or at least 20 times.
[0050] [00050] Pretreated corn straw: The term "PCS" or "Pretreated corn straw" means a cellulosic material derived from corn straw r heat treatment and diluted sulfuric acid, alkaline pretreatment or pretreatment neutral.
[0051] [00051] Sequence identity: the relationship between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".
[0052] [00052] For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0, 5.0.0 or higher. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the replacement matrix EBLOSUM62 (EMBOSS version of BLOSUM62). Needle yield marked as “larger identity” (obtained using the non-summarized option) is used as the percentage identity and is calculated as follows: (Identical residues x 100) / (Alignment size -Total number of intervals in alignment )
[0053] [00053] For purposes of the present invention, the sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra), as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 3.0.0, 5.0.0 or higher. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the replacement matrix EDNAFULL (EMBOSS version of NCBI NUC4.4). The Needle yield marked as “major identity” (obtained using the non-summarized option) is used as the percentage identity, and is calculated as follows: (Identical deoxyribonucleotides x 100) / (Alignment size - Total number of intervals in the alignment)
[0054] [00054] Substring: The term "subsequence" means a polynucleotide with one or more (for example, several) nucleotides missing at the 5 'and / or 3' end of a sequence encoding the mature polypeptide; where the subsequence encodes a fragment with biological activity, for example, enzymatic activity.
[0055] [00055] Variant: The term "variant" means a polypeptide with enzymatic activity comprising a change, that is, a substitution, insertion and / or elimination, in one or more (for example, several) positions. A substitution means changing the amino acid that occupies a position with a different amino acid; an elimination means the removal of the amino acid that occupies a position; and an insertion means adding an amino acid adjacent and immediately after the amino acid that occupies a position.
[0056] [00056] Very high severity conditions: The term "very high severity conditions" means probes of at least 100 nucleotides in length, prehybridization and hybridization at 42 ° C in 5X SSPE, 0.3% SDS, 200 micrograms / mL of denatured and sheared DNA from salmon sperm and 50% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 70 ° C.
[0057] [00057] Very low severity conditions: The term "very low severity conditions" means probes of at least 100 nucleotides in length, prehybridization and hybridization at 42 ° C in SSPE 5X, 0.3% SDS, 200 micrograms / mL of denatured and sheared salmon sperm DNA, and 25% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 45 ° C.
[0058] [00058] Xylan-containing material: The term "xylan-containing material" means any material comprising a plant cell wall polysaccharide containing a major part of xylose residues bound to beta- (1-4). Terrestrial plant xylans are heteropolymers that have a major part of beta- (1-4) -D-xylopyranose, which is branched by short carbohydrate chains. They comprise D-glucuronic acid or its 4-O-methyl ether, L-arabinose, and / or various oligosaccharides composed of D-xylose, L-arabinose, D or L-galactose, and D-glucose. Xylan-like polysaccharides can be divided into homoxylans and heteroxylans, which include glucuronoxylans, (arabino) glucuronoxylans, (glucuron) arabinoxylans, arabinoxylans and complex heteroxylans. See, for example, Ebringerova et al., 2005, Adv. Polym. Sci. 186: 1-67.
[0059] [00059] In the methods of the present invention, any material containing xylan can be used. In a preferred aspect, the material containing xylan is lignocellulose.
[0060] [00060] Activity that degrades xylan or xylanolitic activity: The term "activity that degrades xylan" or "xylanolitic activity" means a biological activity that hydrolyzes material containing xylan. The two basic approaches to assess xylanolitic activity include: (1) measuring total xylanolitic activity, and (2) measuring individual xylanolitic activities (for example, endoxylanases, beta-xylosidases, arabinofuranosidases, alpha-glucuronidases, acetylxylan esterases, feruloyl esterases , and alpha-glucuronyl esterases). Recent progress in the testing of xylanolitic enzymes has been summarized in several publications, including Biely and Puchard, Recent progress in the assays of xylanolytics enzymes, 2006, Journal of the Science of Food and Agriculture 86 (11): 1636-1647; Spanikova and Biely, 2006, Glycuronoyl esterase - Novel carbohydrate esterase produced by Schizophyllum commune, FEBS Letters 580 (19): 4597-4601; Herrmann, Vrsanska, Jurickova, Hirsch, Biely, and Kubicek, 1997, The beta-D-xylosidase of Trichoderma reesei is a multifunctional beta-D-xylan xylohydrolase, Biochemical Journal 321: 375-381.
[0061] [00061] The activity that degrades total xylan can be assessed by determining the reducing sugars formed from various types of xylan including, for example, oat spelled xylans, beech wood and larch wood, or by photometric determination of fragments of colored xylan released from several covalently colored xylans. The most common total xylanolitic activity assay is based on the production of reducing sugars from polymeric 4-O-methyl glucuronoxylane, described in Bailey, Biely, Poutanen, 1992, Interlaboratory testing of methods for assay of xylanase activity, Journal of Biotechnology 23 (3): 257-270. Xylanase activity can also be determined with AZCL-arabinoxylan at 0.2% as a substrate in TRITON® X-100 (4- (1,1,3,3-tetramethylbutyl) phenyl-polyethylene glycol) at 0.01%, and 200 mM sodium phosphate buffer pH 6 at 37 ° C. One unit of xylanase activity is defined as 1.0 qmol of azurine produced per minute at 37 ° C, pH 6, from 0.2% AZCL-arabinoxylan as a substrate, in 200 mM sodium phosphate buffer pH 6.
[0062] [00062] For purposes of the present invention, the activity that degrades xylan is determined by measuring the increase in birch xylan hydrolysis (Sigma Chemical Co., Inc., St. Louis, MO, USA) by enzyme (s) that degrades (m) xylan in the following typical conditions: reactions of 1 ml, 5 mg / ml of substrate (total solids), 5 mg of xylanolitic protein / g of substrate, 50 mM sodium acetate at pH 5, 50 ° C, 24 hours, sugar analysis using the p-hydroxybenzoic acid hydrazide (PHBAH) assay as described by Lever, 1972, A new reaction for colorimetric determination of carbohydrates, Anal. Biochem 47: 273279.
[0063] [00063] Xylanase: The term "xylanase" means a 1,4-beta-D-xylan-xylohydrolase (EC 3.2.1,8) that catalyzes the endo-hydrolysis of the 1,4-beta-D-xylosidic bonds in xylans . For purposes of the present invention, xylanase activity is determined with 0.2% AZCL-arabinoxylan as a substrate in 0.01% TRITON® X-100, and 200 mM sodium phosphate buffer pH 6 at 37 ° C. One unit of xylanase activity is defined as 1.0 pmol of azurine produced per minute at 37 ° C, pH 6, from 0.2% AZCL-arabinoxylan as a substrate in 200 mM sodium phosphate buffer pH 6 . DETAILED DESCRIPTION OF THE INVENTION
[0064] (a) pré-tratar o material celulósico com uma composição compreendendo um ou mais (por exemplo, vários) polipeptídeos GH61; e (b) sacarificar o material celulósico pré-tratado com polipeptídeo GH61 com uma composição enzimática. [00064] The present invention relates to methods of degrading a cellulosic material, comprising: (a) pretreating the cellulosic material with a composition comprising one or more (for example, several) GH61 polypeptides; and (b) saccharifying the cellulosic material pretreated with GH61 polypeptide with an enzymatic composition.
[0065] [00065] In one aspect, the foregoing methods additionally comprise recovering the degraded or converted cellulosic material. Soluble products of degradation or conversion of cellulosic material can be separated from insoluble cellulosic material using technology well known in the art, for example, such as centrifugation, filtration and gravity establishment.
[0066] (a) pré-tratar um material celulósico com uma composição compreendendo um ou mais (por exemplo, vários) polipeptídeos GH61; (b) sacarificar o material celulósico pré-tratado com GH61 com uma composição enzimática; (c) fermentar o material celulósico sacarificado com um ou mais (por exemplo, vários) microrganismos fermentadores para sintetizar o produto de fermentação; e (d) recuperar o produto de fermentação a partir da fermentação. [00066] The present invention also relates to methods of producing a fermentation product, comprising: (a) pretreating a cellulosic material with a composition comprising one or more (for example, several) GH61 polypeptides; (b) saccharifying the cellulosic material pretreated with GH61 with an enzymatic composition; (c) fermenting the saccharified cellulosic material with one or more (for example, several) fermenting microorganisms to synthesize the fermentation product; and (d) recovering the fermentation product from the fermentation.
[0067] (a) sacarificar um material celulósico com uma composição enzimática, em que o material celulósico é pré-tratado com uma composição compreendendo um ou mais (por exemplo, vários) polipeptídeos GH61; (b) fermentar o material celulósico sacarificado com um ou mais (por exemplo, vários) microrganismos fermentadores para sintetizar o produto de fermentação; e (c) recuperar o produto de fermentação a partir da fermentação. [00067] The present invention also relates to methods of producing a fermentation product, comprising: (a) saccharifying a cellulosic material with an enzymatic composition, wherein the cellulosic material is pretreated with a composition comprising one or more (for example, several) GH61 polypeptides; (b) fermenting the saccharified cellulosic material with one or more (for example, several) fermenting microorganisms to synthesize the fermentation product; and (c) recovering the fermentation product from fermentation.
[0068] [00068] The present invention also relates to methods of fermenting a cellulosic material, comprising: fermenting the cellulosic material with one or more (for example, several) fermenting microorganisms, wherein the cellulosic material is pretreated with a composition comprising a or more (for example, several) GH61 polypeptides, and is saccharified with an enzyme composition.
[0069] [00069] 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.
[0070] [00070] An advantage of pretreating the cellulosic material with a GH61 polypeptide is that the treatment intensifies the digestibility of the cellulosic material, by an enzymatic composition comprising the cellulolytic enzyme and / or hemicellulolytic enzyme, that is, it decreases the recalcitrance of the cellulosic material for enzymatic hydrolysis.
[0071] [00071] In another aspect, each of the foregoing methods may further comprise treating the cellulosic material with a chemical pretreatment, a physical pretreatment, or a chemical pretreatment and a physical pretreatment. In one embodiment, each of the foregoing methods further comprises treating the cellulosic material with a chemical pretreatment. In another embodiment, each of the foregoing methods additionally comprises treating the cellulosic material with a physical pretreatment. In another embodiment, each of the previous methods further comprises treating the cellulosic material with a chemical pretreatment and a physical pretreatment.
[0072] [00072] In another aspect, pretreatment with one or more (for example, several) GH61 polypeptides is performed before, during or after chemical pretreatment, physical pretreatment, or chemical pretreatment and physical pretreatment. In one embodiment, pretreatment with one or more (for example, several) GH61 polypeptides is performed before chemical pretreatment, physical pretreatment, or chemical pretreatment and physical pretreatment. In another embodiment, pretreatment with one or more (for example, several) GH61 polypeptides is carried out during chemical pretreatment, physical pretreatment, or chemical pretreatment and physical pretreatment. In another embodiment, pretreatment with one or more (for example, several) GH61 polypeptides is performed after chemical pretreatment, physical pretreatment, or chemical pretreatment and physical pretreatment.
[0073] [00073] In another aspect, pretreatment with one or more (for example, several) GH61 polypeptides is performed before saccharification. In another aspect, the additional treatment of the cellulosic material with a chemical pretreatment, a physical pretreatment, or a chemical pretreatment and a physical pretreatment is carried out before saccharification. In another aspect, pretreatment with one or more (for example, several) GH61 polypeptides and additional treatment of the cellulosic material with a chemical pretreatment, a physical pretreatment, or a chemical pretreatment and a Physical pre-treatment is carried out before saccharification.
[0074] [00074] In another aspect, the one or more (for example, several) GH61 polypeptides are inactivated after pretreating the cellulosic material. The one or more (for example, several) GH61 polypeptides can be inactivated by incubation at an elevated temperature, below the melting temperatures of the respective GH61 polypeptides used, for example, 90 ° C, for a sufficient period of time, for example, at least 30-45 minutes.
[0075] [00075] The processing of cellulosic material according to the present invention, can be carried out using methods conventional in the art. Furthermore, the methods of the present invention can be implemented using any conventional biomass processing apparatus configured to operate in accordance with the invention.
[0076] [00076] Hydrolysis (saccharification) and fermentation, separate or simultaneous, include, but are not limited to, separate hydrolysis and fermentation (SHF); simultaneous saccharification and fermentation (SSF); simultaneous saccharification and co-fermentation (SSCF); hybrid hydrolysis and fermentation (HHF); separate hydrolysis and co-fermentation (SHCF); hybrid hydrolysis and co-fermentation (HHCF) and direct microbial conversion (DMC), sometimes also called consolidated bioprocessing (CBP). SHF uses separate process steps to enzymatically hydrolyze cellulosic material into fermentable sugars, for example, glucose, cellobiose and pentose monomers, and then ferment fermentable sugars in ethanol. In SSF, the enzymatic hydrolysis of cellulosic material and the fermentation of sugars in ethanol are combined in one step (Philippidis, GP, 1996, Cellulose bioconversion technology, in Handbook on Bioethanol: Production and Utilization, Wyman, CE, ed., Taylor & Francis, Washington, DC, 179-212). SSCF involves the co-fermentation of multiple sugars (Sheehan, J., and Himmel, M., 1999, Enzymes, energy and the environment: A strategic perspective on the US Department of Energy's research and development activities for bioethanol, Biotechnol. Prog. 15: 817827). HHF involves a separate hydrolysis step and, in addition, a simultaneous saccharification and hydrolysis step that can be performed in the same reactor. The steps in an HHF process can be carried out at different temperatures, that is, enzymatic saccharification at high temperature followed by SSF at a temperature lower than the fermentation strain can tolerate. DMC combines all three processes (production, hydrolysis and enzymatic fermentation) in one or more (for example, several) steps, where the same organism is used to produce the enzymes for converting cellulosic material into fermentable sugars and to convert sugars fermentable in a final product (Lynd, LR, Weimer, PJ, van Zyl, WH, and Pretorius, IS, 2002, Microbial cellulose utilization: Fundamentals and biotechnology, Microbiol. Mol. Biol. Reviews 66: 506-577). It is understood here that any method known in the art which comprises pretreatment, enzymatic hydrolysis (saccharification), fermentation, or a combination of these, can be used in the practice of the methods of the present invention.
[0077] [00077] A conventional apparatus may include a batch reactor agitated, a batch agitated reactor, a continuous flow agitated reactor with ultrafiltration and / or a continuous piston flow column reactor (Fernanda de Castilhos Corazza, Flávio Faria de Moraes, Gisella Maria Zanin and Ivo Neitzel, 2003, Optimal control in fed-batch reactor for the cellobiose hydrolysis, Acta Scientiarum. Technology 25: 33-38; Gusakov, AV, and Sinitsyn, AP, 1985, Kinetics of the enzymatic hydrolysis of cellulose: 1. A mathematical model for a batch reactor process, Enz. Microb. Technol. 7: 346-352), a friction reactor (Ryu, SK, and Lee, JM, 1983, Bioconversion of waste cellulose using an attrition bioreactor , Biotechnol. Bioeng. 25: 53-65), or a reactor with intensive agitation induced by an electromagnetic field (Gusakov, AV, Sinitsyn, AP, Davydkin, IY, Davydkin, VY, Protas, OV, 1996, Enhancement of enzymatic cellulose hydrolysis using a novel type of bioreactor with intensive stirring induced by electromagnetic field, Appl. Biochem. Biotechnol. 56: 141-153). Additional types of reactors include: fluidized bed, upstream flow, immobilized and extruder reactors for hydrolysis and / or fermentation.
[0078] [00078] Pre-treatment with GH61 polypeptide. Pretreating a cellulosic material with a composition comprising one or more (for example, several) GH61 polypeptides can be carried out at a pH in the range of 4 to 10, for example, 4.0; 4.5; 5.0; 5.5; 6.0; 6.5; 7.0; 7.5; 8.0; 8.5; 9.0; 9.5; or 10, (or between them) and at a temperature in the range of 5 ° C to 70 ° C, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 ° C (or between). However, any pH and temperature can be used.
[0079] [00079] In one aspect, pretreatment is carried out at pH 4.5 and 5 ° C. In another aspect, the pretreatment is carried out at pH 4.5 and 10 ° C. In another aspect, the pretreatment is carried out at pH 4.5 and 15 ° C. In another aspect, the pre-treatment is carried out at pH 4.5 and 20 ° C. In another aspect, the pre-treatment is carried out at pH 4.5 and 25 ° C. In another aspect, the pre-treatment is carried out at pH 4.5 and 30 ° C. In another aspect, pretreatment is carried out at pH 4.5 and 35 ° C. In another aspect, the pretreatment is carried out at pH 4.5 and 40 ° C. In another aspect, the pre-treatment is carried out at pH 4.5 and 45 ° C. In another aspect, the pre-treatment is carried out at pH 4.5 and 50 ° C. In another aspect, the pretreatment is carried out at pH 4.5 and 55 ° C. In another aspect, the pretreatment is carried out at pH 4.5 and 60 ° C. In another aspect, the pretreatment is carried out at pH 4.5 and 65 ° C. In another aspect, the pretreatment is carried out at pH 4.5 and 70 ° C.
[0080] [00080] In another aspect, the pre-treatment is carried out at pH 5.0 and 5 ° C. In another aspect, the pretreatment is carried out at pH 5.0 and 10 ° C. In another aspect, the pre-treatment is carried out at pH 5.0 and 15 ° C. In another aspect, the pre-treatment is carried out at pH 5.0 and 20 ° C. In another aspect, the pre-treatment is carried out at pH 5.0 and 25 ° C. In another aspect, the pretreatment is carried out at pH 5.0 and 30 ° C. In another aspect, the pre-treatment is carried out at pH 5.0 and 35 ° C. In another aspect, the pretreatment is carried out at pH 5.0 and 40 ° C. In another aspect, the pretreatment is carried out at pH 5.0 and 45 ° C. In another aspect, the pretreatment is carried out at pH 5.0 and 50 ° C. In another aspect, the pretreatment is carried out at pH 5.0 and 55 ° C. In another aspect, the pre-treatment is carried out at pH 5.0 and 60 ° C. In another aspect, the pretreatment is carried out at pH 5.0 and 65 ° C. In another aspect, the pre-treatment is carried out at pH 5.0 and 70 ° C.
[0081] [00081] In another aspect, the pre-treatment is carried out at pH 5.5 and 5 ° C. In another aspect, the pre-treatment is carried out at pH 5.5 and 10 ° C. In another aspect, the pre-treatment is carried out at pH 5.5 and 15 ° C. In another aspect, the pre-treatment is carried out at pH 5.5 and 20 ° C. In another aspect, the pre-treatment is carried out at pH 5.5 and 25 ° C. In another aspect, the pretreatment is carried out at pH 5.5 and 30 ° C. In another aspect, the pretreatment is carried out at pH 5.5 and 35 ° C. In another aspect, the pre-treatment is carried out at pH 5.5 and 40 ° C. In another aspect, the pre-treatment is carried out at pH 5.5 and 45 ° C. In another aspect, the pre-treatment is carried out at pH 5.5 and 50 ° C. In another aspect, the pre-treatment is carried out at pH 5.5 and 55 ° C. In another aspect, the pretreatment is carried out at pH 5.5 and 60 ° C. In another aspect, the pre-treatment is carried out at pH 5.5 and 65 ° C. In another aspect, the pre-treatment is carried out at pH 5.5 and 70 ° C.
[0082] [00082] In another aspect, the pre-treatment is carried out at pH 6.0 and 5 ° C. In another aspect, the pretreatment is carried out at pH 6.0 and 10 ° C. In another aspect, the pre-treatment is carried out at pH 6.0 and 15 ° C. In another aspect, the pretreatment is carried out at pH 6.0 and 20 ° C. In another aspect, the pretreatment is carried out at pH 6.0 and 25 ° C. In another aspect, the pretreatment is carried out at pH 6.0 and 30 ° C. In another aspect, the pretreatment is carried out at pH 6.0 and 35 ° C. In another aspect, the pretreatment is carried out at pH 6.0 and 40 ° C. In another aspect, the pretreatment is carried out at pH 6.0 and 45 ° C. In another aspect, the pre-treatment is carried out at pH 6.0 and 50 ° C. In another aspect, the pretreatment is carried out at pH 6.0 and 55 ° C. In another aspect, the pre-treatment is carried out at pH 6.0 and 60 ° C. In another aspect, the pretreatment is carried out at pH 6.0 and 65 ° C. In another aspect, the pretreatment is carried out at pH 6.0 and 70 ° C.
[0083] [00083] In another aspect, the pre-treatment is carried out at pH 6.5 and 5 ° C. In another aspect, the pretreatment is carried out at pH 6.5 and 10 ° C. In another aspect, the pretreatment is carried out at pH 6.5 and 15 ° C. In another aspect, the pretreatment is carried out at pH 6.5 and 20 ° C. In another aspect, the pretreatment is carried out at pH 6.5 and 25 ° C. In another aspect, the pretreatment is carried out at pH 6.5 and 30 ° C. In another aspect, the pretreatment is carried out at pH 6.5 and 35 ° C. In another aspect, the pretreatment is carried out at pH 6.5 and 40 ° C. In another aspect, the pretreatment is carried out at pH 6.5 and 45 ° C. In another aspect, the pretreatment is carried out at pH 6.5 and 50 ° C. In another aspect, the pretreatment is carried out at pH 6.5 and 55 ° C. In another aspect, the pretreatment is carried out at pH 6.5 and 60 ° C. In another aspect, pretreatment is carried out at pH 6.5 and 65 ° C. In another aspect, the pre-treatment is carried out at pH 6.5 and 70 ° C.
[0084] [00084] In another aspect, the pre-treatment is carried out at pH 7.0 and 5 ° C. In another aspect, the pretreatment is carried out at pH 7.0 and 10 ° C. In another aspect, the pretreatment is carried out at pH 7.0 and 15 ° C. In another aspect, the pretreatment is carried out at pH 7.0 and 20 ° C. In another aspect, the pretreatment is carried out at pH 7.0 and 25 ° C. In another aspect, the pretreatment is carried out at pH 7.0 and 30 ° C. In another aspect, the pretreatment is carried out at pH 7.0 and 35 ° C. In another aspect, the pretreatment is carried out at pH 7.0 and 40 ° C. In another aspect, the pretreatment is carried out at pH 7.0 and 45 ° C. In another aspect, the pretreatment is carried out at pH 7.0 and 50 ° C. In another aspect, the pretreatment is carried out at pH 7.0 and 55 ° C. In another aspect, the pretreatment is carried out at pH 7.0 and 60 ° C. In another aspect, the pretreatment is carried out at pH 7.0 and 65 ° C. In another aspect, the pretreatment is carried out at pH 7.0 and 70 ° C.
[0085] [00085] In another aspect, the pre-treatment is carried out at pH 7.5 and 5 ° C. In another aspect, the pre-treatment is carried out at pH 7.5 and 10 ° C. In another aspect, the pretreatment is carried out at pH 7.5 and 15 ° C. In another aspect, the pretreatment is carried out at pH 7.5 and 20 ° C. In another aspect, the pretreatment is carried out at pH 7.5 and 25 ° C. In another aspect, the pretreatment is carried out at pH 7.5 and 30 ° C. In another aspect, the pretreatment is carried out at pH 7.5 and 35 ° C. In another aspect, the pretreatment is carried out at pH 7.5 and 40 ° C. In another aspect, the pretreatment is carried out at pH 7.5 and 45 ° C. In another aspect, the pre-treatment is carried out at pH 7.5 and 50 ° C. In another aspect, the pretreatment is carried out at pH 7.5 and 55 ° C. In another aspect, the pretreatment is carried out at pH 7.5 and 60 ° C. In another aspect, the pretreatment is carried out at pH 7.5 and 65 ° C. In another aspect, the pretreatment is carried out at pH 7.5 and 70 ° C.
[0086] [00086] In another aspect, the pre-treatment is carried out at pH 8.0 and 5 ° C. In another aspect, the pre-treatment is carried out at pH 8.0 and 10 ° C. In another aspect, the pre-treatment is carried out at pH 8.0 and 15 ° C. In another aspect, the pretreatment is carried out at pH 8.0 and 20 ° C. In another aspect, the pretreatment is carried out at pH 8.0 and 25 ° C. In another aspect, the pretreatment is carried out at pH 8.0 and 30 ° C. In another aspect, the pretreatment is carried out at pH 8.0 and 35 ° C. In another aspect, the pretreatment is carried out at pH 8.0 and 40 ° C. In another aspect, the pretreatment is carried out at pH 8.0 and 45 ° C. In another aspect, the pretreatment is carried out at pH 8.0 and 50 ° C. In another aspect, the pretreatment is carried out at pH 8.0 and 55 ° C. In another aspect, the pretreatment is carried out at pH 8.0 and 60 ° C. In another aspect, the pretreatment is carried out at pH 8.0 and 65 ° C. In another aspect, the pretreatment is carried out at pH 8.0 and 70 ° C.
[0087] [00087] In another aspect, the pre-treatment is carried out at pH 8.5 and 5 ° C. In another aspect, the pretreatment is carried out at pH 8.5 and 10 ° C. In another aspect, the pre-treatment is carried out at pH 8.5 and 15 ° C. In another aspect, the pre-treatment is carried out at pH 8.5 and 20 ° C. In another aspect, the pretreatment is carried out at pH 8.5 and 25 ° C. In another aspect, the pre-treatment is carried out at pH 8.5 and 30 ° C. In another aspect, the pretreatment is carried out at pH 8.5 and 35 ° C. In another aspect, the pretreatment is carried out at pH 8.5 and 40 ° C. In another aspect, the pretreatment is carried out at pH 8.5 and 45 ° C. In another aspect, the pretreatment is carried out at pH 8.5 and 50 ° C. In another aspect, the pretreatment is carried out at pH 8.5 and 55 ° C. In another aspect, the pretreatment is carried out at pH 8.0 and 60 ° C. In another aspect, the pretreatment is carried out at pH 8.5 and 65 ° C. In another aspect, the pre-treatment is carried out at pH 8.5 and 70 ° C.
[0088] [00088] In another aspect, the pre-treatment is carried out at pH 9.0 and 5 ° C. In another aspect, the pretreatment is carried out at pH 9.0 and 10 ° C. In another aspect, the pretreatment is carried out at pH 9.0 and 15 ° C. In another aspect, the pretreatment is carried out at pH 9.0 and 20 ° C. In another aspect, the pretreatment is carried out at pH 9.0 and 25 ° C. In another aspect, the pretreatment is carried out at pH 9.0 and 30 ° C. In another aspect, the pretreatment is carried out at pH 9.0 and 35 ° C. In another aspect, the pretreatment is carried out at pH 9.0 and 40 ° C. In another aspect, the pretreatment is carried out at pH 9.0 and 45 ° C. In another aspect, the pretreatment is carried out at pH 9.0 and 50 ° C. In another aspect, the pretreatment is carried out at pH 9.0 and 55 ° C. In another aspect, the pretreatment is carried out at pH 9.0 and 60 ° C. In another aspect, the pretreatment is carried out at pH 9.0 and 65 ° C. In another aspect, the pretreatment is carried out at pH 9.0 and 70 ° C.
[0089] [00089] In another aspect, the pre-treatment is carried out at pH 9.5 and 5 ° C. In another aspect, the pretreatment is carried out at pH 9.5 and 10 ° C. In another aspect, the pretreatment is carried out at pH 9.5 and 15 ° C. In another aspect, the pretreatment is carried out at pH 9.5 and 20 ° C. In another aspect, the pretreatment is carried out at pH 9.5 and 25 ° C. In another aspect, the pretreatment is carried out at pH 9.5 and 30 ° C. In another aspect, the pretreatment is carried out at pH 9.5 and 35 ° C. In another aspect, the pretreatment is carried out at pH 9.5 and 40 ° C. In another aspect, the pretreatment is carried out at pH 9.5 and 45 ° C. In another aspect, the pre-treatment is carried out at pH 9.5 and 50 ° C. In another aspect, the pretreatment is carried out at pH 9.5 and 55 ° C. In another aspect, the pretreatment is carried out at pH 9.5 and 60 ° C. In another aspect, the pretreatment is carried out at pH 9.5 and 65 ° C. In another aspect, the pretreatment is carried out at pH 9.5 and 70 ° C.
[0090] [00090] In another aspect, the pre-treatment is carried out at pH 10.0 and 5 ° C. In another aspect, the pretreatment is carried out at pH 10.0 and 10 ° C. In another aspect, the pretreatment is carried out at pH 10.0 and 15 ° C. In another aspect, the pretreatment is carried out at pH 10.0 and 20 ° C. In another aspect, the pretreatment is carried out at pH 10.0 and 25 ° C. In another aspect, the pretreatment is carried out at pH 10.0 and 30 ° C. In another aspect, the pretreatment is carried out at pH 10.0 and 35 ° C. In another aspect, the pretreatment is carried out at pH 10.0 and 40 ° C. In another aspect, the pre-treatment is carried out at pH 10.0 and 45 ° C. In another aspect, the pretreatment is carried out at pH 10.0 and 50 ° C. In another aspect, the pretreatment is carried out at pH 10.0 and 55 ° C. In another aspect, the pretreatment is carried out at pH 8.0 and 60 ° C. In another aspect, the pretreatment is carried out at pH 10.0 and 65 ° C. In another aspect, the pretreatment is carried out at pH 10.0 and 70 ° C.
[0091] [00091] During pretreatment of a cellulosic material, an efficient amount of a GH61 polypeptide with enhanced cellulolytic activity for the cellulosic material is about 0.01 to about 50.0 mg, about 0.01 to about 40 mg, about 0.01 to about 30 mg, about 0.01 to about 20 mg, about 0.01 to about 10 mg, about 0.01 to about 5 mg, about 0.025 to about 1.5 mg, about 0.05 to about 1.25 mg, about 0.075 to about 1.25 mg, about 0.1 to about 1.25 mg, still about from 0.15 to about 1.25 mg, or about 0.25 to about 1.0 mg per g of cellulosic material.
[0092] [00092] Pretreatment with one or more (for example, several) GH61 polypeptides with enhanced cellulolytic activity can be carried out for a period of minutes to hours, to days, to weeks, depending on the amount of an GH61 polypeptide with activity intensified cellulolytic for the cellulosic material used, temperature and pH. In one aspect, the time is 30 minutes to 60 minutes. In another aspect, the time is 1 hour to 24 hours. In another aspect, the time is 1 day to 7 days. In another aspect, the time is 1 week to 4 weeks. For example, in 1 mg of GH61 polypeptide per gram of cellulosic material at 50 ° C and pH 5.0, the pretreatment would be 3 days. However, any suitable time to achieve enhanced digestibility of cellulosic material by pretreatment with a GH61 polypeptide can be used, and is easily determined by those skilled in the art.
[0093] [00093] In another aspect, pretreatment with one or more (for example, several) GH61 polypeptides with enhanced cellulolytic activity is carried out in the presence of a soluble activating divalent metal cation (WO 2008/151043), for example, manganese or copper. [00094] In another aspect, pretreatment with one or more (for example, several) GH61 polypeptides with enhanced cellulolytic activity is carried out in the presence of a dioxy compound, a bicyclic compound, a heterocyclic compound, a compound containing nitrogen, quinone compound, a sulfur-containing compound, or a liquor obtained from a pre-treated cellulosic material, such as pre-treated corn straw (PCS).
[0094] [00094] In another aspect, pretreatment with one or more (for example, several) GH61 polypeptides with enhanced cellulolytic activity is carried out in the presence of a dioxy compound, a bicyclic compound, a heterocyclic compound, a compound containing nitrogen, quinone compound, a sulfur-containing compound, or a liquor obtained from a pre-treated cellulosic material, such as pre-treated corn straw (PCS).
[0095] [00095] The dioxide compound can include any suitable compound containing two or more oxygen atoms. In some respects, the dioxide compounds contain a substituted aryl fraction, as described herein. Dioxy compounds may comprise one or more (for example, several) hydroxyl and / or hydroxyl derivatives, but also include fractions of substituted aryl that do not contain hydroxyl and hydroxyl derivatives. Non-limiting examples of dioxide compounds include pyrocatechol or catechol; caffeic acid; 3,4-dihydroxybenzoic acid; 4-tert-butyl-5-methoxy-1,2-benzenediol; pyrogallol; gallic acid; methyl-3,4,5-trihydroxybenzoate; 2,3,4-trihydroxybenzophenone; 2,6-dimethoxyphenol; synapinic acid; 3,5-dihydroxybenzoic acid; 4-chloro-1,2-benzenediol; 4-nitro-1,2-benzenediol; tannic acid; ethyl gallate; methyl glycolate; dihydroxyfumaric acid; 2-butyn-1,4-diol; (crochonic acid; 1,3-propanediol; tartaric acid; 2,4-pentanediol; 3-ethioxy-1,2-propanediol; 2,4,4'-trihydroxybenzophenone; cis-2-butene-1,4-diol; 3,4-dihydroxy-3-cyclobutene-1,2-dione; dihydroxyacetone; acrolein acetal; methyl-4-hydroxybenzoate; 4-hydroxybenzoic acid; and methyl-3,5-dimethoxy-4-hydroxybenzoate; or a salt or solvate of these.
[0096] [00096] The bicyclic compound can include any suitable substituted fused ring system, as described herein. The compounds may comprise one or more (for example, 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 flavilium ion, such as an optionally substituted anthocyanidin or optionally substituted anthocyanin, or derivatives thereof. Non-limiting examples of bicyclic compounds include epicatechin; quercetin; myricetin; taxifoline; caenferol; morina; acacetin; naringenin; isoramnetine; apigenin; cyanidin; cyanine; curomanine; kerakianin; or a salt or solvate of these.
[0097] [00097] The heterocyclic compound can be any suitable compound, such as an optionally substituted or non-aromatic aromatic ring comprising a heteroatom, in the manner described herein. In one aspect, the heterocyclic is a compound comprising an optionally substituted heterocycloalkyl fraction, or an optionally substituted heteroaryl fraction. In another aspect, the optionally substituted heterocycloalkyl fraction or the optionally substituted heteroaryl fraction is an optionally substituted 5-element heterocycloalkyl, or an optionally substituted 5-element heteroaryl fraction. In another aspect, the optionally substituted heterocycloalkyl or optionally substituted heteroaryl fraction is an optionally substituted fraction selected from pyrazolyl, furanyl, imidazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrrolyl, pyridyl, pyrimidyl, pyridazinyl, thiazolyl, thiazolyl, thiazolyl, thiazolyl, thiazolyl, thiazolyl, thiazolyl, thiazolyl, thiazolyl, thiazolyl, pyrazolyl, tianaftenyl, carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl, indolyl, quinolinyl, benzotriazolila, benzothiazolyl, benzoxazolyl, benzimidazolyl, isoquinolinepyl, isoindolyl, acridinyl, pyridine, pyridine, diazylilinyl, pyridine tiepinila, piperidinila, and oxepinila. In another aspect, the optionally substituted heterocycloalkyl fraction or the optionally substituted heteroaryl fraction is an optionally substituted furanyl. Non-limiting examples of heterocyclic compounds include (1,2-dihydroxyethyl) -3,4-dihydroxyfuran-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-y-butyrolactone; ribonic γ-lactone; aldoexuronicaldohexuronic acid γ-lactone; δ-lactone gluconic acid; 4-hydroxycoumarin; dihydrobenzofuran; 5- (hydroxymethyl) furfural; furoin; 2 (5H) -furanone; 5,6-dihydro-2H-pyran-2-one; and 5,6-dihydro-4-hydroxy-6-methyl-2H-pyran-2-one; or a salt or solvate of these.
[0098] [00098] The nitrogen-containing compound can be any suitable compound with one or more nitrogen atoms. In one aspect, the nitrogen-containing compound comprises an amine, imine, hydroxylamine, or nitroxide fraction. Non-limiting examples of nitrogen-containing compounds include acetone oxime; violuric acid; pyridine-2-aldoxime; 2-aminophenol; 1,2-benzenediamine; 2,2,6,6-tetramethyl-1-piperidinyloxy; 5,6,7,8-tetrahydrobiopterin; 6,7-dimethyl-5,6,7,8-tetrahydropterin and maleamic acid; or a salt or solvate of these.
[0099] [00099] The quinone compound can be any suitable compound comprising a fraction of quinone in the manner 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 Q0; 2,3,5,6-tetramethyl-1,4-benzoquinone or duroquinone; 1,4-dihydroxyanthraquinone; 3-hydroxy-1-methyl-5,6-indolinedione or adrenochrome; 4-tert-butyl-5-methoxy-1,2-benzoquinone; quinone pyrroloquinoline; or a salt or solvate of these.
[0100] The sulfur-containing compound can be any suitable compound comprising one or more sulfur atoms. In one aspect, the sulfur-containing compound comprises a selected fraction of thionyl, thioether, sulfinyl, sulfonyl, sulfamide, sulfonamide, sulfonic acid and sulfonic ester. Non-limiting examples of sulfur-containing compounds include ethanethiol; 2-propanetiol; 2-propene-1-thiol; 2-mercaptoethane sulfonic acid; benzenethiol; benzene-1,2-dithiol; cysteine; methionine; glutathione; cystine or a salt or solvate thereof.
[0101] [000101] In one aspect, an efficient amount of a compound like this described above for cellulosic material, such as a molar ratio for glucosyl cellulose units, is about 10-6 to about 10, for example, about 10 -6 to about 7.5, 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, an efficient amount of a compound like the one described above is about 0.1 μM to about 1 M, for example, about 0.5 μM to about 0.75 M, about 0.75 μM to about 0.5 M, about 1 μM to about 0.25 M, about 1 μM to about 0.1 M, about 5 μM to about 50 mM, about 10 μM to about 25 mM , about 50 μM to about 25 mM, about 10 μM to about 10 mM, about 5 μM to about 5 mM, and about 0.1 mM to about 1 mM.
[0102] [000102] The term "liquor" means the phase of the solution, both aqueous, organic, and a combination thereof, which arises from the treatment of a lignocellulose and / or hemicellulose material in a sludge, or monosaccharides of these, for example, xylose, arabinose, mannose, etc., in conditions as described herein, and their soluble contents. A liquor for the cellulolytic intensification of a GH61 polypeptide can be produced by treating a lignocellulose or hemicellulose material (or raw material), applying heat and / or pressure, optionally in the presence of a catalyst, for example, acid, optionally in the presence of a organic solvent, and optionally in combination with physical interruption of the material, and then separating the solution from the residual solids. Such conditions determine the degree of cellulolytic intensification, obtained by combining liquor and a GH61 polypeptide, during the hydrolysis of a cellulosic substrate in a cellulase preparation. The liquor can be separated from the treated material using standard methods in the art, such as filtration, sedimentation or centrifugation.
[0103] [000103] In one aspect, such an efficient amount of a cellulose liquor is about 10-6 to about 10 g per gram of cellulose, for example, about 10-6 to about 7.5 g, about from 10-6 to about 5, about 106 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 gram of cellulose.
[0104] [000104] Pretreatment with one or more (for example, several) GH61 polypeptides can be performed in situ before chemical / physical pretreatment, during chemical / physical pretreatment, or after chemical / pretreatment / physical, or in a device intended for saccharification before saccharification. Examples of chemical and physical pretreatments are described here.
[0105] [000105] In the methods of the present invention, any GH61 polypeptide with enhanced cellulolytic activity can be used.
[0106] [000106] In a first aspect, the GH61 polypeptide with enhanced cellulolytic activity comprises the following reasons: [ILMV] -PX (4,5) -GXY - [ILMV] -XRX- [EQ] -X (4) - [HNQ] (SEQ ID NO: 157 or SEQ ID NO: 158) and [FW] - [ TF] -K- [AIV], where X is any amino acid, X (4,5) is any amino acid in 4 or 5 contiguous positions, and X (4) is any amino acid in 4 contiguous positions.
[0107] [000107] The GH61 polypeptide comprising the aforementioned motifs may additionally comprise: HX (1,2) -GPX (3) - [YW] - [AILMV] (SEQ ID NO: 159 or SEQ ID NO: 160), [EQ] -XYX (2) -CX- [EHQN] - [FILV] -X- [ILV] (SEQ ID NO: 161), or HX (1,2) -GPX (3) - [YW] - [AILMV] (SEQ ID NO: 162 or SEQ ID NO: 163) and [EQ] -XYX (2) -CX- [EHQN] - [FILV ] -X- [ILV] (SEQ ID NO: 164), where X is any amino acid, X (1,2) is any amino acid in 1 position or 2 contiguous positions, X (3) is any amino acid in 3 contiguous positions, and X (2) is any amino acid in 2 contiguous positions. In the previous reasons, the letter abbreviated amino acid accepted by IUPAC is used.
[0108] [000108] In a preferred embodiment, the GH61 polypeptide with enhanced cellulolytic activity further comprises H-X (1,2) -G-P-X (3) - [YW] - [AILMV] (SEQ ID NO: 165 or SEQ ID NO: 166). In another preferred embodiment, the GH61 polypeptide with enhanced cellulolytic activity further comprises [EQ] -X-Y-X (2) -C-X- [EHQN] - [FILV] -X- [ILV] (SEQ ID NO: 167). In another preferred embodiment, the GH61 polypeptide with enhanced cellulolytic activity further comprises HX (1,2) -GPX (3) - [YW] - [AILMV] (SEQ ID NO: 168 or SEQ ID NO: 169) and [EQ ] -XYX (2) -CX- [EHQN] - [FILV] -X- [ILV] (SEQ ID NO: 170).
[0109] [000109] In a second aspect, the GH61 polypeptide with enhanced cellulolytic activity comprises the following reason: [ILMV] -PX (4,5) -GXY - [ILMV] -XRX- [EQ] -X (3) -A- [HNQ] (SEQ ID NO: 171 or SEQ ID NO: 172), where X is any amino acid, X (4,5) is any amino acid in 4 or 5 contiguous positions, and X (3) is any amino acid in 3 contiguous positions. In the previous reason, the letter abbreviated amino acid accepted by IUPAC is used.
[0110] [000110] In a third aspect, the GH61 polypeptide with enhanced cellulolytic activity comprises an amino acid sequence that has a sequence identity with the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO : 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40 , SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO : 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, or SEQ ID NO: 156 of at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, by at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100 %.
[0111] [000111] In a preferred embodiment, the mature polypeptide has amino acids 20 to 326 of SEQ ID NO: 2, amino acids 18 to 239 of SEQ ID NO: 4, amino acids 20 to 258 of SEQ ID NO: 6, amino acids 19 to 226 of SEQ ID NO: 8, amino acids 20 to 304 of SEQ ID NO: 10, amino acids 16 to 317 of SEQ ID NO: 12, amino acids 23 to 250 of SEQ ID NO: 14, amino acids 20 to 249 of SEQ ID NO: 16 , amino acids 18 to 232 of SEQ ID NO: 18, amino acids 16 to 235 of SEQ ID NO: 20, amino acids 19 to 323 of SEQ ID NO: 22, amino acids 16 to 310 of SEQ ID NO: 24, amino acids 20 to 246 of SEQ ID NO: 26, amino acids 22 to 354 of SEQ ID NO: 28, amino acids 22 to 250 of SEQ ID NO: 30, or amino acids 22 to 322 of SEQ ID NO: 32, amino acids 24 to 444 of SEQ ID NO: 34 , amino acids 26 to 253 of SEQ ID NO: 36, amino acids 20 to 223 of SEQ ID NO: 38, amino acids 18 to 246 of SEQ ID NO: 40, amino acids 20 to 334 of SEQ ID NO: 42, amino acids 18 to 227 of SEQ ID NO: 44, amino acids 22 to 368 of SEQ ID NO: 46, amino acids 25 to 330 of SEQ ID NO: 48, amino acids 17 to 236 of SEQ ID NO: 50, amino acids 19 to 250 of SEQ ID NO: 52, amino acids 23 to 478 of SEQ ID NO: 54, amino acids 17 to 230 of SEQ ID NO: 56, amino acids 20 to 257 of SEQ ID NO: 58, amino acids 23 to 251 of SEQ ID NO: 60, amino acids 19 to 349 of SEQ ID NO: 62, amino acids 24 to 436 of SEQ ID NO: 64, amino acids 21 to 344 of SEQ ID NO : 134, 21 to 389 of SEQ ID NO: 136, amino acids 22 to 406 of SEQ ID NO: 138, amino acids 20 to 427 of SEQ ID NO: 140, amino acids 18 to 267 of SEQ ID NO: 142, amino acids 21 to 273 of SEQ ID NO: 144, amino acids 21 to 322 of SEQ ID NO: 146, amino acids 18 to 234 of SEQ ID NO: 148, amino acids 24 to 233 of SEQ ID NO: 150, amino acids 17 to 237 of SEQ ID NO: 152 , amino acids 20 to 484 of SEQ ID NO: 154, or amino acids 22 to 320 of SEQ ID NO: 156.
[0112] [000112] In a fourth aspect, the GH61 polypeptide with enhanced cellulolytic activity is encoded by a polynucleotide that hybridizes under at least very low severity conditions, at least low severity conditions, at least medium severity conditions, at least severity conditions medium-high, at least conditions of high severity, or at least conditions of very high severity (i) in the sequence encoding the mature polypeptide of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID N O: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, or SEQ ID NO : 155, (ii) in the genomic DNA sequence of the sequence encoding the mature polypeptide of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 145, SEQ ID NO: 147, or SEQ ID NO: 149, or in the cDNA sequence of the sequence encoding the mature polypeptide of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 151, SEQ ID NO: 153, or SEQ ID NO: 155, (iii) in a subsequence of (i ) or (ii), or (iv) in a complement and total size of (i), (ii) or (iii) (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor, New York). A substring of the sequence encoding the mature polypeptide of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, or SEQ ID NO: 155 contains at least 100 contiguous nucleotides or preferably at least 200 contiguous nucleotides. Furthermore, the subsequence can encode a polypeptide fragment that has enhanced cellulolytic activity.
[0113] [000113] In a preferred embodiment, the sequence encoding the mature polypeptide has nucleotides 388 to 1332 of SEQ ID NO: 1, nucleotides 98 to 821 of SEQ ID NO: 3, nucleotides 126 to 978 of SEQ ID NO: 5, nucleotides 55 to 678 of SEQ ID NO: 7, nucleotides 58 to 912 of SEQ ID NO: 9, nucleotides 46 to 951 of SEQ ID NO: 11, nucleotides 67 to 796 of SEQ ID NO: 13, nucleotides 77 to 766 of SEQ ID NO: 15, nucleotides 52 to 921 of SEQ ID NO: 17, nucleotides 46 to 851 of SEQ ID NO: 19, nucleotides 55 to 1239 of SEQ ID NO: 21, nucleotides 46 to 1250 of SEQ ID NO: 23, nucleotides 58 to 811 of SEQ ID NO: 25, nucleotides 64 to 1112 of SEQ ID NO: 27, nucleotides 64 to 859 of SEQ ID NO: 29, nucleotides 64 to 1018 of SEQ ID NO: 31, nucleotides 70 to 1483 of SEQ ID NO: 33, nucleotides 76 to 832 of SEQ ID NO: 35, nucleotides 58 to 974 of SEQ ID NO: 37, nucleotides 52 to 875 of SEQ ID NO: 39, nucleotides 58 to 1250 of SEQ ID NO: 41, nucleotides 52 to 795 of SEQ ID NO: 43, nucleotides 64 to 1104 of SEQ ID NO: 45, nucleotides 73 to 990 of SEQ ID NO: 47, nucleotides 49 to 1218 of SEQ ID NO: 49, nucleotides 55 to 930 of SEQ ID NO: 51, nucleotides 67 to 1581 of SEQ ID NO: 53, nucleotides 49 to 865 of SEQ ID NO: 55, nucleotides 58 to 1065 of SEQ ID NO: 57, nucleotides 67 to 868 of SEQ ID NO: 59, nucleotides 55 to 1099 of SEQ ID NO: 61, nucleotides 70 to 1483 of SEQ ID NO: 63, nucleotides 61 to 1032 of SEQ ID NO: 133, nucleotides 61 to 1167 of SEQ ID NO: 135, nucleotides 64 to 1218 of SEQ ID NO: 137, nucleotides 58 to 1281 of SEQ ID NO: 139 , nucleotides 52 to 801 of SEQ ID NO: 141, nucleotides 61 to 819 of SEQ ID NO: 143, nucleotides 61 to 966 of SEQ ID NO: 145, nucleotides 52 to 702 of SEQ ID NO: 147, nucleotides 70 to 699 of SEQ ID NO: 149, nucleotides 49 to 711 of SEQ ID NO: 151, nucleotides 76 to 1452 of SEQ ID NO: 153, or nucleotides 64 to 1018 of SEQ ID NO: 155.
[0114] [000114] In a fifth aspect, the GH61 polypeptide with enhanced cellulolytic activity is encoded by a polynucleotide comprising or consisting of a sequence of nucleotides that has a sequence identity with the sequence encoding the mature polypeptide of SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO : 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 , SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO : 153, or SEQ ID NO: 155 of at least 60%, at least 65%, at least at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least least 96%, at least 97%, at least 98%, at least 99%, or at least 100%.
[0115] [000115] In a sixth aspect, the GH61 polypeptide with enhanced cellulolytic activity is an artificial variant comprising a substitution, elimination, and / or insertion in one or more (for example, several) positions of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO : 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52 , SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO : 154, or SEQ ID NO: 156; or a homologous sequence of these.
[0116] [000116] Amino acid changes may be of a minor nature, that is, conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions, typically 1-30 amino acids; small amino or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small binding peptide of up to 20-25 residues; or a small extension that facilitates purification by changing the net load or another function, such as a polyhistidine tract, an antigenic epitope or a binding domain.
[0117] [000117] Examples of conservative substitutions are in the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that generally do not alter specific 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. Common substitutions 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.
[0118] [000118] Alternatively, the amino acid changes are of such a nature that the physical-chemical properties of the polypeptides are altered. For example, amino acid changes can improve the thermal stability of the polypeptide, change the specificity of the substrate, change the ideal pH and the like.
[0119] [000119] The essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scan mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, simple alanine mutations are introduced into each residue in the molecule, and the resulting mutant molecules are tested for enhanced cellulolytic activity to identify amino acid residues that are important for the molecule's activity. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of the structure, in the manner determined by such techniques as nuclear magnetic resonance, crystallography, electronic diffraction, or photo-affinity tagging, along with the mutation of the possible amino acids of the contact site. See, for example, 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.
[0120] [000120] Single or multiple amino acid substitutions, deletions, and / or insertions can be performed and tested using known methods of mutagenesis, recombination and / or shuffling, followed by a relevant selection procedure, such as those revealed by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error prone PCR, phage display (for example, Lowman et al., 1991, Biochemistry 30: 10832-10837; US patent 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).
[0121] [000121] Mutagenesis / scrambling methods can be combined with automated, high-throughput selection methods to detect the activity of cloned, mutagenized and expressed polypeptides 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 be sequenced quickly using standard methods in the art. These methods allow rapid determination of the importance of individual amino acid residues in a polypeptide.
[0122] [000122] The total number of amino acid substitutions, deletions and / or insertions of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO : 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60 , SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, or SEQ ID NO: 156 is up to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
[0123] [000123] Other pre-treatments. In carrying out the methods of the present invention, any pretreatment process known in the art can be used to disrupt the plant cell wall components of the cellulosic material (Chandra et al., 2007, Substrate pretreatment: The key to effective enzymatic hydrolysis of lignocellulosics Adv. Biochem. Engin./Biotechnol. 108: 67-93; Galbe and Zacchi, 2007, Pretreatment of lignocelullosic materials for efficient bioethanol production, Adv. Biochem. Engin./Biotechnol. 108: 41-65; Hendriks and Zeeman, 2009, Pretreatment to enhance the digestibillity of lignocellulosic biomass, Bioresource Technol. 100: 10-18; Mosier et al., 2005, Features of promising technologies for pretreatment of lignocellulosic biomass, Bioresource Technol. 96: 673-686; Taherzadeh and Karimi, 2008, Pretreatment of lignocellulosic wastes to improve ethanol and biogas production: A review, Int. J. de Mol. Sci. 9: 1621-1651; Yang and Wyman, 2008, Pretreatment: the key to unlocking low-cost cellulosic ethano l, Biofuels Bioproducts and Biorefining-Biofpr. 2: 26-40).
[0124] [000124] Conventional pretreatments include, but are not limited to, steam pretreatment (with or without explosion), diluted acid pretreatment, hot water pretreatment, alkaline pretreatment, lime pretreatment , wet oxidation, wet explosion, ammonia fiber explosion, organosolv pretreatment and biological pretreatment. Additional pre-treatments include percolation treatments with ammonia, ultrasound, electroporation, microwave, supercritical CO2, supercritical H2O, ozone, ionic liquid and gamma irradiation.
[0125] [000125] Cellulosic material can be pre-treated before hydrolysis and / or fermentation. Pre-treatment is preferably carried out before hydrolysis. Alternatively, pretreatment can be carried out simultaneously with enzymatic hydrolysis to release fermentable sugars, such as glucose, xylose and / or cellobiose. In many cases, the pre-treatment step itself results in some conversion of biomass into fermentable sugars (even in the absence of enzymes).
[0126] [000126] Pre-treatment with steam. In the steam pretreatment, cellulosic material is heated to break up the components of plant cell walls, including lignin, hemicellulose and cellulose, to make cellulose and other fractions, for example, hemicellulose, accessible to enzymes. The cellulosic material passes through a reaction vessel where the steam is injected to increase the temperature to the required temperature and pressure and is maintained there for the desired reaction time. Pre-treatment with steam is preferably carried out at 140-250 ° C, for example, 160-200 ° C or 170-190 ° C, where the ideal temperature range depends on the addition of a chemical catalyst. The residence time for pre-treatment with steam is preferably 1-60 minutes, for example, 1-30 minutes, 1-20 minutes, 3-12 minutes or 4-10 minutes, where the ideal residence time depends on the range temperature and the addition of a chemical catalyst. Pre-treatment with steam allows relatively high solid loads, in such a way that the cellulosic material is generally moist only during the pre-treatment. Steam pretreatment is often combined with an explosive discharge of material after pretreatment, which is known as a steam explosion, that is, rapid burning at atmospheric pressure and turbulent flow of the material to increase the surface area accessible by fragmentation (Duff and Murray, 1996, Bioresource Technology 855: 1-33; Galbe and Zacchi, 2002, Appl. Microbiol. Biotechnol. 59: 618-628; US patent application 20020164730). During the steam pretreatment, the acetyl hemicellulose groups are cleaved and the resulting acid autocatalyzes the partial hydrolysis of hemicellulose into monosaccharides and oligosaccharides. Lignin is removed only to a limited extent.
[0127] [000127] Chemical pretreatment: The term "chemical treatment" refers to any chemical pretreatment that promotes the separation and / or release of cellulose, hemicellulose and / or lignin. Such a pretreatment can convert crystalline cellulose into amorphous cellulose. Examples of suitable chemical pretreatment processes include, for example, pretreatment with dilute acid, pretreatment with lime, wet oxidation, fiber explosion with ammonia / freezing (AFEX), percolation with ionic liquid ammonia (APR) and pre-treatments with organosolv.
[0128] [000128] A catalyst, such as H2SO4 or SO2 (typically 0.3 to 5% w / w), is generally added to the steam pretreatment which decreases time and 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). In the pre-treatment with diluted acid, the cellulosic material is mixed with the diluted acid, typically H2SO4, and water to form a slurry, heated by steam at the desired temperature, and after a residence time it is burned at atmospheric pressure. Pretreatment with dilute acid can be performed with numerous reactor designs, for example, piston flow reactors, counter current reactors, or agitated bed reactors against direct current (Duff and Murray, 1996, supra; Schell et al ., 2004, Bioresource Technol. 91: 179-188; Lee et al., 1999, Adv. Biochem. Eng. Biotechnol. 65: 93-115).
[0129] [000129] Various pre-treatment methods in alkaline conditions can also be used. These alkaline pretreatments include, but are not limited to, sodium hydroxide, lime, wet oxidation, percolation with ammonia (APR), and fiber burst with ammonia / freezing (AFEX).
[0130] [000130] Pre-treatment with lime is carried out with calcium oxide or calcium hydroxide at temperatures of 85-150 ° C and residence time from 1 hour to several days (Wyman et al., 2005, Bioresource Technol. 96: 19591966; Mosier et al., 2005, Bioresource Technol. 96: 673-686). WO 2006/110891, WO 2006/110899, WO 2006/110900, and WO 2006/110901 disclose pretreatment methods that use ammonia.
[0131] [000131] Wet oxidation is a thermal pretreatment typically carried out at 180-200 ° C for 5-15 minutes with the addition of an oxidizing agent, such as hydrogen peroxide or oxygen overpressure (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: 567574; Martin et al., 2006, J. Chem Technol, Biotechnol 81: 1669-1677). Pre-treatment is preferably carried out on 1-40% dry material, for example, 2-30% dry material or 5-20% dry material, and often the initial pH is increased by the addition of alkali, such as carbonate sodium.
[0132] [000132] A modification of the pre-treatment method with wet oxidation, known as wet explosion (combination of wet oxidation and steam explosion), can handle dry material up to 30%. In the wet explosion, the oxidizing agent is introduced during the pre-treatment after a certain period of residence. The pre-treatment is then finished by burning at atmospheric pressure (WO 2006/032282).
[0133] [000133] The fiber explosion with ammonia (AFEX) involves treating the cellulosic material with liquid or gaseous ammonia at moderate temperatures, such as 90-150 ° C, and high pressure such as 17-20 bar for 5-10 minutes, where the content of the dry material can be as high as 60% (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). During pretreatment with AFEX, cellulose and hemicellulose remain relatively intact. Lignin-carbohydrate complexes are cleaved.
[0134] [000134] Pretreatment with organosolv delignifies cellulosic material by extraction using 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 generally added as a catalyst. In pre-treatment with organosolv, most of the hemicellulose and lignin is removed.
[0135] [000135] Other examples of suitable pretreatment methods are described by Schell et al., 2003, Appl. Biochem. and Biotechnol. 105-108, p. 69-85, and Mosier et al., 2005, Bioresource Technology 96: 673-686, and U.S. published application 2002/0164730.
[0136] [000136] In one aspect, chemical pretreatment is preferably carried out as a diluted acid treatment, and more preferably as a continuous diluted acid treatment. The acid is typically sulfuric acid, but other acids can also be used, such as acetic acid, citric acid, nitric acid, phosphoric acid, tartaric acid, succinic acid, hydrogen chloride or mixtures thereof. Weak acid treatment is conducted in the pH range of preferably 1-5, for example, 1-4 or 1-2.5. In one aspect, the concentration of the acid is in the range of preferably 0.01 to 10% by weight of acid, for example, 0.05 to 5% by weight of acid or 0.1 to 2% by weight of acid. The acid is brought into contact with the cellulosic material and maintained at a temperature in the range of preferably 140-200 ° C, for example, 165-190 ° C, for periods ranging from 1 to 60 minutes.
[0137] [000137] In another aspect, pretreatment takes place in an aqueous sludge. In preferred aspects, the cellulosic material is present during pre-treatment in amounts preferably between 10-80% by weight, for example, 20-70% by weight or 30-60% by weight, such as around 40% by weight. Weight. The pre-treated cellulosic material can be unwashed or washed using any method known in the art, for example, washed with water.
[0138] [000138] Mechanical pretreatment or physical pretreatment: The term "mechanical pretreatment" or "physical pretreatment" refers to any pretreatment that promotes particle size reduction. For example, such pre-treatment can involve various types of crushing or grinding (for example, dry grinding, wet grinding, or vibrating ball grinding) to break and / or reduce the particle size of the plant cell wall components of the material. cellulosic.
[0139] [000139] Cellulosic material can be pretreated both physically (mechanically) and chemically. The mechanical or physical pretreatment can be coupled with vapor / vapor explosion, hydrothermolysis, weak or diluted acid treatment, high temperature, high pressure treatment, irradiation (for example, microwave irradiation), or combinations of these. In one aspect, high pressure means pressure in the range of preferably about 100 to about 400 psi (689.9 kPa to about 2.8 MPa), for example, about 150 to about 250 psi (1.03 MPa at about 1.72 MPa). In another aspect, elevated temperature means temperatures in the range of about 100 to about 300 ° C, for example, about 140 to about 200 ° C. In a preferred aspect, mechanical or physical pretreatment is carried out in a batch process using a steam gun hydrolyzer system that uses high pressure and high temperature in the manner defined above, for example, a Sunds hydrolyzer available from Sunds Defibrator AB , Sweden. Chemical or physical pretreatments can be carried out sequentially or simultaneously, if desired.
[0140] [000140] Thus, in a preferred aspect, the cellulosic material is subjected to physical (mechanical) or chemical pretreatment, or any combination of these, to promote the separation and / or release of cellulose, hemicellulose and / or lignin.
[0141] [000141] Biological pretreatment: The term "biological pretreatment" refers to any biological pretreatment that promotes the separation and / or release of cellulose, hemicellulose and / or lignin from cellulosic material. Biological pretreatment techniques may involve applying microorganisms and / or enzymes that solubilize lignin (see, for example, Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol: Production and Utilization, Wyman, CE , ed., Taylor & Francis, Washington, DC, 179-212; Ghosh and Singh, 1993, Physicochemical and biological treatments for enzymatic / microbial conversion of cellulosic biomass, Adv. Appl. Microbiol. 39: 295-333; McMillan, JD , 1994, Pretreating lignocellulosic biomass: a review, in Enzymatic Conversion of Biomass for Fuels Production, Himmel, ME, Baker, JO, and Overend, RP, eds., ACS Symposium Series 566, American Chemical Society, Washington, DC, chapter 15 ; Gong, CS, Cao, NJ, Du, J., and Tsao, GT, 1999, Ethanol production from renewable resources, in Advances in Biochemical Engineering / Biotechnology, Scheper, T., ed., Springer-Verlag Berlin Heidelberg, Germany , 65: 207-241; Olsson and Hahn-Hagerdal, 1996, Fermentation of lignocellulosic hydrolysates for ethanol production, Enz. Microb. Tech. 18: 312-331; and Vallander and Eriksson, 1990, Production of ethanol from material lignocelullosics: State of art, Adv. Biochem. Eng./Biotechnol. 42: 63-95).
[0142] [000142] Saccharification. In the hydrolysis stage, also known as saccharification, the pre-treated cellulosic material is hydrolyzed to break down cellulose and / or hemicellulose into fermentable sugars, such as glucose, cellobiose, xylose, xylulose, arabinose, mannose, galactose, and / or oligosaccharides soluble. Hydrolysis is carried out enzymatically by an enzymatic composition. The components of the compositions can be added simultaneously or sequentially.
[0143] [000143] Enzymatic hydrolysis is preferably carried out in a suitable aqueous environment, under conditions that can be easily determined by those skilled in the art. In one aspect, hydrolysis is carried out under conditions suitable for the activity of the components of the enzyme (s), that is, ideal for the enzyme (s). Hydrolysis can be carried out as a continuous or batch fed process, where the pre-treated cellulosic material is fed gradually, for example, to an enzyme containing hydrolysis solution.
[0144] [000144] Saccharification is generally carried out in reactors or fermenters in agitated tanks under controlled conditions of pH, temperature and mixture. The suitable conditions of process time, temperature and pH can be easily determined by those skilled in the art. For example, saccharification can last up to 200 hours, but is typically performed for preferably about 12 to about 120 hours, for example, about 16 to about 72 hours or about 24 to about 48 hours. The temperature is preferably in the range of about 25 ° C to about 70 ° C, for example, about 30 ° C to about 65 ° C, about 40 ° C to about 60 ° C, or about 50 ° C to about 55 ° C. The pH is preferably in the range of about 3 to about 8, for example, about 3.5 to about 7, about 4 to about 6, or about 5.0 to about 5.5. The dry solids content is preferably in the range of about 5 to about 50% by weight, for example, about 10 to about 40% by weight or about 20 to about 30% by weight.
[0145] [000145] The ideal amounts of enzymes depend on several factors including, but not limited to, the mixture of cellulolytic enzymes and / or component hemicellulolytic enzymes, the cellulosic material, the concentration of cellulosic material, the pretreatment (s) cellulosic material, temperature, time, pH and inclusion of fermenting organism (for example, yeast for simultaneous saccharification and fermentation).
[0146] [000146] In one aspect, an efficient amount of cellulolytic or hemicellulolytic enzyme for cellulosic material is about 0.5 to about 50 mg, for example, about 0.5 to about 40 mg, about 0.5 about 25 mg, about 0.75 to about 20 mg, about 0.75 to about 15 mg, about 0.5 to about 10 mg, or about 2.5 to about 10 mg per gram of cellulosic material.
[0147] [000147] In another aspect, an efficient amount of a GH61 polypeptide with enhanced cellulolytic activity for the cellulosic material is about 0.01 to about 50.0 mg, for example, about 0.01 to about 40 mg , about 0.01 to about 30 mg, about 0.01 to about 20 mg, about 0.01 to about 10 mg, about 0.01 to about 5 mg, about 0.025 to about 1.5 mg, about 0.05 to about 1.25 mg, about 0.075 to about 1.25 mg, about 0.1 to about 1.25 mg, about 0.15 to about 1.25 mg, or about 0.25 to about 1.0 mg per gram of cellulosic material.
[0148] [000148] In another aspect, an efficient amount of a GH61 polypeptide with enhanced cellulolytic activity for the cellulolytic or hemicellulolytic enzyme is about 0.005 to about 1.0 g, for example, about 0.01 to about 1, 0 g, about 0.15 to about 0.75 g, about 0.15 to about 0.5 g, about 0.1 to about 0.5 g, about 0.1 to about 0.25 g, or about 0.05 to about 0.2 g per gram of cellulolytic or hemicellulolytic enzyme.
[0149] [000149] Fermentation. Fermentable sugars obtained from hydrolyzed cellulosic material can be fermented by one or more (for example, several) fermenting microorganisms capable of fermenting sugars directly or indirectly in a desired fermentation product. "Fermentation" or "fermentation process" refers to any fermentation process or any process that comprises a fermentation step. Fermentation processes also include fermentation processes used in the alcohol consumption industry (for example, beer and wine), the bakery industry (for example, fermented bakery products), the leather industry and the tobacco industry. The fermentation conditions depend on the desired fermentation product and the fermenting organism, and can be easily determined by those skilled in the art.
[0150] [000150] In the fermentation step, the sugars, released from the cellulosic material as a result of the pre-treatment and enzymatic hydrolysis steps, are fermented in a product, for example, ethanol, by a fermenting organism, such as yeast. Hydrolysis (saccharification) and fermentation can be separated or simultaneous, as described here.
[0151] [000151] Any suitable hydrolyzed cellulosic material can be used in the fermentation step in the practice of the present invention. The material is in general selected on the basis of the desired fermentation product, that is, the substance to be obtained from the fermentation, and in the process employed, in a manner well known in the art.
[0152] [000152] It is understood here that the term "fermentation medium" refers to a medium before the fermenting microorganism (s) is (are) added, such as a medium that results from a saccharification process, as well as a medium used in a simultaneous saccharification and fermentation process (SSF).
[0153] [000153] "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 hexose and / or pentose fermenting organisms, or a combination of these. Both hexose and pentose fermenting organisms are well known in the art. Suitable fermenting microorganisms are capable of fermenting, that is, converts, sugars, such as glucose, xylose, xylulose, arabinose, maltose, mannose, galactose and / or oligosaccharides, directly or indirectly into the desired fermentation product.
[0154] [000154] Examples of bacterial and fungal fermenting organisms that produce ethanol are described by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69: 627-642.
[0155] [000155] Examples of fermenting microorganisms that can ferment hexose sugars include bacterial and fungal organisms, such as yeast. Preferred yeasts include strains of Candida, Kluyveromyces and Saccharomyces, for example, Candida sonorensis, Kluyveromyces marxianus and Saccharomyces cerevisiae.
[0156] [000156] Examples of fermenting organisms that can ferment pentose sugars in their natural state include bacterial and fungal organisms, such as some yeasts. Preferred xylose fermenting yeasts include strains of Candida, preferably C. sheatae or C. sonorensis; and Pichia strains, preferably P. stipitis, such as P. stipitis CBS 5773. Preferred pentose fermenting yeasts include Pachysolen strains, preferably P. tannophilus. Organisms that are not able to ferment pentose sugars, such as xylose and arabinose, can be genetically modified to accomplish this by methods known in the art.
[0157] [000157] Examples of bacteria that can efficiently ferment hexose and pentose in ethanol include, for example, Bacillus coagulans, Clostridium acetobutilicum, Clostridium thermocellum, Clostridium phytofermentans, Geobacillus sp., Thermoanaerobacter saccharolyticum and Zymomonas mobilis (Philippid.,).
[0158] [000158] Other fermenting organisms include strains of Bacillus, such as Bacillus coagulans; Candida, such as C. sonorensis, C. methanosorbosa, C. diddensiae, C. parapsilosis, C. naedodendra, C. blankii, C. entomophilia, C. brassicae, C. pseudotropicalis, C. boidinii, C. utilis and C. scehatae; Clostridium, such as C. acetobutilicum, C. thermocellum and C. phytofermentans; E. coli, especially strains of E. coli that have been genetically modified to improve ethanol yield; Geobacillus sp .; Hansenula, like Hansenula anomala; Klebsiella, such as K. oxytoca; Kluyveromyces, such as K. marxianus, K. lactis, K. thermotolerans and K. fragilis; Schizosaccharomyces, such as S. pombe; Thermoanaerobacter, such as Thermoanaerobacter saccharolyticum and Zymomonas, such as Zymomonas mobilis.
[0159] [000159] In a preferred aspect, yeast is a Bretannomyces. In a more preferred aspect, the yeast is Bretannomyces clausenii. In another preferred aspect, the yeast is a 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 diddensii. In another more preferred aspect, the yeast is Candida entomophiliia. In another more preferred aspect, the yeast is Candida pseudotropicalis. In another more preferred aspect, the yeast is Candida scehatae. In another more preferred aspect, the yeast is Candida utilis. In another preferred aspect, the yeast is a 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 a Kluyveromyces. In another more preferred aspect, the yeast is Kluyveromyces fragilis. In another more preferred aspect, the yeast is Kluyveromyces marxianus. In another more preferred aspect, the yeast is Kluyveromyces thermotolerans. In another preferred aspect, the yeast is a Pachysolen. In another more preferred aspect, the yeast is Pachysolen tannophilus. In another preferred aspect, the yeast is a Pichia. In another more preferred aspect, the yeast is a Pichia stipitis. In another preferred aspect, the yeast is Saccharomyces spp. In another more 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.
[0160] [000160] In a preferred aspect, the bacterium is a Bacillus. In a more preferred aspect, the bacterium is Bacillus coagulans. In another preferred aspect, the bacterium is a Clostridium. In another more preferred aspect, the bacterium is Clostridium acetobutilicum. 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 Geobacilus sp. In another more preferred aspect, the bacterium is a Thermoanaerobacter. In another more preferred aspect, the bacterium is Thermoanaerobacter saccharolyticum. In another preferred aspect, the bacterium is a Zymomonas. In another more preferred aspect, the bacterium is Zymomonas mobilis.
[0161] [000161] Yeasts commercially available and suitable for ethanol production include, for example, BIOFERM ™ AFT and XR (NABC -North American Bioproducts Corporation, GA, USA), yeast ETANOL RED ™ (Fermentis / Lesaffre, USA), FALI ™ (Fleischmann's Yeast, USA), FERMIOL ™ (DSM Specialties), GERT STRAND ™ (Gert Strand AB, Sweden), and SUPERSTART ™ and THERMOSACC ™ fresh yeast (Ethanol Technology, WI, USA).
[0162] [000162] In a preferred aspect, the fermenting microorganism has been genetically modified to provide the ability to ferment pentose sugars, such as microorganisms that use xylose, which use arabinose, and which co-use xylose and arabinose.
[0163] [000163] The cloning of heterologous genes in various fermenting microorganisms led to the construction of organisms capable of converting hexoses and pentoses into ethanol (co-fermentation) (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 by Saccharomyces cerevisiae, Appl. Microbiol. Biotechnol. 38: 776-783; Walfridsson et al., 1995, Xylose-metabolizing Saccharomyces cerevisiae strains overexpressing the TKL1 and TAL1 genes encoding the pentose phosphate pathway enzymes transketolase and transketolase and Appl. Environ. Microbiol. 61: 4184-4190; Kuyper et al., 2004, Minimal metabolic engineering of Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: a proof of principl and, FEMS Yeast Research 4: 655-664; Beall et al., 1991, Parametric studies of ethanol production from xylose and other sugars by recombinant Escherichia coli, Biotech. Bioeng. 38: 296-303; Ingram et al., 1998, Metabolic engineering of bacteria for ethanol production, Biotechnol. Bioeng. 58: 204-214; Zhang et al., 1995, Metabolic engineering of a pentose metabolism pathway in ethanologenic Zymomonas mobilis, Science 267: 240-243; Deanda et al., 1996, Development of an arabinose-fermenting Zymomonas mobilis strain by metabolic pathway engineering, Appl. Environ. Microbiol. 62: 4465-4470; WO 2003/062430, xylose isomerase).
[0164] [000164] In a preferred aspect, the genetically modified fermenting microorganism is Candida sonorensis. 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 marxianus. In another preferred aspect, the genetically modified fermenting microorganism is Saccharomyces cerevisiae. In another preferred aspect, the genetically modified fermenting microorganism is Zymomonas mobilis.
[0165] [000165] It is well known in the art that the organisms described above can also be used to produce other substances, in the manner described herein.
[0166] [000166] The fermenting microorganism is typically added to the degraded or hydrolyzed cellulosic material, and fermentation is carried out for about 8 to about 96 hours, for example, about 24 to about 60 hours. The temperature is typically between about 26 ° C to about 60 ° C, for example, about 32 ° C or 50 ° C, and about pH 3 to about pH 8, for example, pH 4-5, 6 , or 7.
[0167] [000167] 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 between about 20 ° C to about 60 ° C, for example, about 25 ° C to about 50 ° C, about 32 ° C to about 50 ° C, or about 32 ° C to about 50 ° C, and the pH is generally about pH 3 to about pH 7, for example, about pH 4 to about pH 7. However, some fermenting organisms, for example, bacteria, have a higher ideal fermentation temperature. Yeast or another microorganism is preferably applied in amounts of approximately 105 to 1012, preferably in approximately 107 to 1010, essentially approximately counts of 2 x 108 viable cells per ml of fermentation broth. Additional guidance regarding the use of yeast for fermentation can be found in, for example, “The Alcohol Textbook” (Editors K. Jacques, TP Lyons and DR Kelsall, Nottingham University Press, UK 1999), which is incorporated by reference.
[0168] [000168] A fermentation stimulator can be used in combination with any of the processes described here to further improve the fermentation process and, in particular, the performance of the fermenting microorganism, such as intensifying the ethanol rate and yield. A "fermentation stimulator" refers to stimulators for the growth of fermenting microorganisms, in particular, yeasts. Preferable fermentation stimulators for growth include vitamins and minerals. Examples of vitamins include multivitamins, biotin, pantothenate, nicotinic acid, meso-inositol, thiamine, pyridoxine, para-aminobenzoic acid, folic acid, riboflavin and vitamins A, B, C, D, and E. See, for example, 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 by reference. Examples of minerals include minerals and mineral salts that can supply nutrients that comprise P, K, Mg, S, Ca, Fe, Zn, Mn and Cu.
[0169] [000169] Fermentation products: A fermentation product can be any substance derived from fermentation. The fermentation product can be, without limitation, an alcohol (for example, arabinitol, n-butanol, isobutanol, ethanol, glycerol, methanol, ethylene glycol, 1,3-propanediol [ropylene glycol], butanediol, glycerin, sorbitol and xylitol ); an alkane (for example, pentane, hexane, heptane, octane, nonane, decane, undecane, and dodecane), a cycloalkane (for example, cyclopentane, cyclohexane, cycloheptane and cyclooctane), an alkene (for example pentene, hexene, heptene and octene); an amino acid (for example, aspartic acid, glutamic acid, glycine, lysine, serine and threonine); a gas (for example, methane, hydrogen (H2), carbon dioxide (CO2) and carbon monoxide (CO)); isoprene; a ketone (for example, acetone); an organic acid (for example, acetic acid, acetonic acid, adipic acid, ascorbic acid, citric acid, 2,5-diceto-D-gluconic acid, formic acid, fumaric acid, gluconic 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 polyketide. The fermentation product can also be protein as a high-value product.
[0170] [000170] In a preferred aspect, the fermentation product is an alcohol. It will be well understood that the term "alcohol" includes a substance that contains one or more hydroxyl fractions. 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 arabinitol. 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, for example, Gong, CS, Cao, NJ, Du, J., and Tsao, GT, 1999, Ethanol production from renewable resources, in Advances in Biochemical Engineering / Biotechnology, Scheper, T., ed., Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Silveira, M. M., and Jonas, R., 2002, The biotechnological production of sorbitol, Appl. Microbiol. Biotechnol. 59: 400408; Nigam, P., and Singh, D., 1995, Processs for fermentative production of xylitol - a sugar substitute, Process Biochemistry 30 (2): 117-124; Ezeji, T. C., Qureshi, N. and Blaschek, H. P., 2003, Production of acetone, butanol and ethanol by Clostridium beijerinckii BA101 and in situ recovery by gas stripping, World Journal of Microbiology and Biotechnology 19 (6): 595-603.
[0171] [000171] In another preferred aspect, the fermentation product is an alkane. The alkane can be an unbranched or branched alkane. 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 dean. In another more preferred aspect, the alkane is undecane. In another more preferred aspect, the alkane is dodecane.
[0172] [000172] In another preferred aspect, the fermentation product is a cycloalkane. In another more preferred aspect, cycloalkane is cyclopentane. In another more preferred aspect, cycloalkane is cyclohexane. In another more preferred aspect, the cycloalkane is cycloeptane. In another more preferred aspect, cycloalkane is cycloctane.
[0173] [000173] In another preferred aspect, the fermentation product is an alkene. The alkene can be an unbranched or branched alkene. In another more preferred aspect, the alkene is pentene. In another more preferred aspect, the alkene is hexene. In another more preferred aspect, the alkene is heptene. In another more preferred aspect, the alkene is octene.
[0174] [000174] 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, for example, 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.
[0175] [000175] In another preferred aspect, the fermentation product 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 V.N. in Biomass and Bioenergy, Vol. 13 (1-2), pp. 83-114, 1997, Anaerobic digestion of biomass for methane production: A review.
[0176] [000176] In another preferred aspect, the fermentation product is isoprene.
[0177] [000177] In another preferred aspect, the fermentation product is a ketone. It will be well understood that the term "ketone" includes a substance that contains one or more fractions of ketone. In another more preferred aspect, the ketone is acetone. See, for example, Qureshi and Blaschek, 2003, supra.
[0178] [000178] 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-diceto-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, 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, for example, 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.
[0179] [000179] In another preferred aspect, the fermentation product is polyketide.
[0180] [000180] Recovery. The fermentation product (s) can optionally be recovered from the fermentation medium using any method known in the art including, but not limited to, chromatography, electrophoretic procedures, differential solubility, distillation or extraction. For example, the alcohol is separated from the fermented cellulosic material and purified by conventional distillation methods. Ethanol with a purity of up to about 96 vol. % can be obtained, which can be used, for example, as ethanol fuel, ethanol for drinks, that is, neutral potable spirits or industrial ethanol. Enzyme compositions
[0181] [000181] Enzyme compositions can comprise any protein that is used in the degradation or conversion of cellulosic material. The compositions can comprise an enzyme as the main enzyme component, for example, a one-component, or multiple enzyme composition. The compositions can be prepared according to methods known in the art, and can be in the form of a liquid or dry composition. The compositions can be stabilized according to methods known in the art.
[0182] [000182] The compositions can be a fermentation broth formulation or a cell composition in the manner described herein. In some embodiments, the composition is a complete broth of dead cells containing organic acid (s), dead cells and / or cell debris, and culture medium.
[0183] [000183] The term "fermentation broth", as used here, refers to a preparation produced by cell fermentation that undergoes minimal or no purification and / or recovery. For example, fermentation broths are produced when the Microbial cultures are grown in saturation, incubated under conditions of carbon limitation, to allow protein synthesis (eg, expression of enzymes by host cells) and secretion in a cell culture medium.The fermentation broth may contain unfractionated contents or fractionated, from the fermentation materials derived at the end of fermentation. Typically, the fermentation broth is unfractionated and comprises the culture medium used and the cell debris present after the microbial cells (eg, filamentous fungus cells) are removed, for example, by centrifugation. In some embodiments, the fermentation broth contains used cell culture medium, extracellular enzymes and cells viable and / or non-viable microbial bacteria.
[0184] [000184] In one embodiment, the fermentation broth formulation and cellular compositions comprise a first organic acid component, comprising at least one 1-5 carbon organic acid and / or a salt thereof, and a second organic acid component comprising at least an organic acid of 6 or more carbons and / or a salt thereof. In a specific embodiment, the first component of organic acid is acetic acid, formic acid, propionic acid, a salt thereof, or a mixture of two or more of the above, and the second component of organic acid is benzoic acid, cyclohexanecarboxylic acid, 4-methylvaleric acid, phenylacetic acid, a salt thereof, or a mixture of two or more of the foregoing.
[0185] [000185] In one aspect, the composition contains organic acid (s) and, optionally, contains dead cells and / or additional cell debris. In one embodiment, dead cells and / or cell debris are removed from a broth complete with dead cells to provide a composition that is free of these components.
[0186] [000186] Fermentation broth formulations or cellular compositions may additionally comprise a preservative and / or antimicrobial agent (for example, bacteriostatic), including, but not limited to, sorbitol, sodium chloride, potassium sorbate and others known in the art. technical.
[0187] [000187] The broth or complete composition of dead cells may contain the unfractionated contents of fermentation materials derived from the end of fermentation. Typically, the broth or complete composition of dead cells contains the culture medium used and the cell debris present after the microbial cells (for example, filamentous fungus cells) grow by saturation, are incubated under conditions of carbon limitation to allow synthesis protein (for example, expression of the cellulase enzyme (s) and / or glycosidase). In some embodiments, the broth or complete composition of dead cells contains the culture medium used, extracellular enzymes and dead cells of filamentous fungus. In some embodiments, microbial cells present in the broth or complete composition of dead cells can be permeabilized and / or lysed using methods known in the art.
[0188] [000188] A complete cell composition or broth, as described herein, is typically a liquid, but may contain insoluble components, such as dead cells, cellular debris, components of the culture medium, and / or insoluble enzyme (s) ( s). In some embodiments, insoluble components can be removed to provide a clear liquid composition.
[0189] [000189] The full-broth formulations and cellular compositions of the present invention can be produced by a method described in WO 90/15861 or WO 2010/096673.
[0190] [000190] In one aspect, the enzyme composition further comprises or comprises one or more (for example, several) proteins selected from the group consisting of a cellulase, a GH61 polypeptide with enhanced cellulolytic activity, a hemicellulase, an esterase, an expandin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease and a swolenin. In another aspect, cellulase is preferably one or more (for example, several) enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase and a beta-glucosidase. In another aspect, hemicellulase is preferably one or more (for example, several) enzymes selected from the group consisting of an acetylmannan esterase, an acetylxylan esterase, an arabinanase, an arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, a galactosidase , a glucuronidase, a glucuronoyl esterase, a mannanase, a mannosidase, a xylanase and a xylosidase.
[0191] [000191] In another aspect, the enzyme composition comprises one or more (for example, several) cellulolytic enzyme. In another aspect, the enzyme composition further comprises or comprises one or more (for example, several) hemicellulolytic enzyme. In another aspect, the enzyme composition comprises one or more (for example, several) cellulolytic enzyme and one or more (for example, several) hemicellulolytic enzyme. In another aspect, the enzyme composition comprises one or more (for example, several) enzymes selected from the group 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 a beta-glycosidase. In another aspect, the enzyme composition comprises a polypeptide with enhanced cellulolytic activity. In another aspect, the enzyme composition comprises an endoglucanase and a polypeptide with enhanced cellulolytic activity. In another aspect, the enzyme composition comprises a cellobiohydrolase and a polypeptide with enhanced cellulolytic activity. In another aspect, the enzyme composition comprises a beta-glycosidase and a polypeptide with enhanced cellulolytic activity. In another aspect, the enzyme composition comprises an endoglucanase and a cellobiohydrolase. In another aspect, the enzyme composition comprises an endoglucanase and a beta-glycosidase. In another aspect, the enzyme composition comprises a cellobiohydrolase and a beta-glycosidase. In another aspect, the enzyme composition comprises an endoglucanase, a cellobiohydrolase, and a polypeptide with enhanced cellulolytic activity. In another aspect, the enzyme composition comprises an endoglucanase, a beta-glycosidase, and a polypeptide with enhanced cellulolytic activity. In another aspect, the enzyme composition comprises a cellobiohydrolase, a beta-glycosidase, and a polypeptide with enhanced cellulolytic activity. In another aspect, the enzyme composition comprises an endoglucanase, a cellobiohydrolase, and a beta-glycosidase. In another aspect, the enzyme composition comprises an endoglucanase, a cellobiohydrolase, a beta-glycosidase, and a polypeptide with enhanced cellulolytic activity.
[0192] [000192] In another aspect, the enzyme composition comprises an acetylmannan esterase. In another aspect, the enzyme composition comprises an acetylxylane esterase. In another aspect, the enzyme composition comprises an arabinanase (for example, alpha-L-arabinanase). In another aspect, the enzyme composition comprises an arabinofuranosidase (for example, alpha-L-arabinofuranosidase). In another aspect, the enzyme composition comprises coumaric acid esterase. In another aspect, the enzyme composition comprises feruloyl esterase. In another aspect, the enzyme composition comprises a galactosidase (for example, alpha-galactosidase and / or beta-galactosidase). In another aspect, the enzyme composition comprises a glucuronidase (for example, alpha-D-glucuronidase). In another aspect, the enzyme composition comprises a glucuronoyl esterase. In another aspect, the enzyme composition comprises mannanase. In another aspect, the enzyme composition comprises a mannosidase (for example, beta-mannosidase). In another aspect, the enzyme composition comprises a xylanase. In a preferred aspect, xylanase is a family 10 xylanase. In another aspect, the enzyme composition comprises a xylosidase (e.g., beta-xylosidase).
[0193] [000193] In another aspect, the enzyme composition comprises an esterase. In another aspect, the enzyme composition comprises an expansin. In another aspect, the enzyme composition comprises a laccase. In another aspect, the enzyme composition comprises a lignolytic enzyme. In a preferred aspect, the lignolytic enzyme is a manganese peroxidase. In another preferred aspect, the lignolytic enzyme is a lignin peroxidase. In another preferred aspect, the lignolytic enzyme is an H2O2-producing enzyme. In another aspect, the enzyme composition comprises a pectinase. In another aspect, the enzyme composition comprises a peroxidase. In another aspect, the enzyme composition comprises a protease. In another aspect, the enzyme composition comprises a swolenin.
[0194] [000194] In the methods of the present invention, the enzyme (s) can (s) be added before or during saccharification, saccharification and fermentation, or fermentation.
[0195] [000195] One or more (for example, several) components of the enzyme composition can be wild type proteins, recombinant proteins, or a combination of wild type proteins and recombinant proteins. For example, one or more (for example, several) components can be natural proteins of a cell, which are used as a host cell to recombinantly express one or more (for example, several) other components of the enzyme composition. One or more (for example, several) components of the enzyme composition can be produced as monocomponents, which are then combined to form the enzyme composition. The enzyme composition can be a combination of multicomponent and monocomponent protein preparations.
[0196] [000196] The enzymes used in the methods of the present invention can be in any form suitable for use, such as, for example, a fermentation broth formulation or cell composition, a cell lysate with or without cell debris, a preparation of semi-purified or purified enzyme, or a host cell as a source of the enzymes. The enzyme composition can be a dry powder or granulate, a non-powdered granulate, a liquid, a stabilized liquid, or a protected stabilized enzyme. Liquid enzyme preparations, for example, can be stabilized by adding stabilizers such as a sugar, a sugar alcohol or another polyol, and / or lactic acid or another organic acid according to the established processes.
[0197] [000197] Enzymes (collectively "polypeptides with enzymatic activity"), can be derived from or obtained from any suitable source, including bacterial, fungal, yeast, plant or mammal origin. The term "obtained" also means here that the enzyme may have been produced recombinantly in a host organism, employing methods described herein, in which the enzyme produced recombinantly is either natural or foreign to the host organism, or has a modified sequence of amino acids, for example. example, with one or more (for example, several) amino acids that are deleted, inserted and / or substituted, i.e., a recombinantly produced enzyme that is a mutant and / or a fragment of a natural amino acid sequence, or an enzyme produced by nucleic acid scrambling processes known in the art. Natural variants are included in the meaning of a natural enzyme, and variants are recombinantly obtained in the meaning of a foreign enzyme, such as by site-directed mutagenesis or shuffling.
[0198] [000198] A polypeptide with enzymatic activity can be a bacterial polypeptide. For example, the polypeptide can be a polypeptide of Gram positive bacteria, such as a polypeptide with enzymatic activity from Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus, Caldicellulosiria, Therdicellothermidid, Therdacellusidid, Therdacellusidid, Thermo, a polypeptide from Gram negative bacteria such as a polypeptide with enzymatic activity from E. coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter Neisseria or Ureaplasma.
[0199] [000199] In one respect, the polypeptide is a polypeptide with enzymatic activity from Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus lentus, Bacillus lentus, Bacillus lentus, Bacillus lentus, Bacillus lentus, Bacillus lentus, Bacillus lentus, Bacillus lentus, Bacillus lentus, Bacillus lentus, Bacillus lentus, Bacillus lentus, Bacillus lentus, Bacillus lentus. pumilus, Bacillus stearothermophilus, Bacillus subtilis or Bacillus thuringiensis.
[0200] [000200] In another aspect, the polypeptide is a polypeptide with enzymatic activity from Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberi, or Streptococcus equi subsp. Zooepidemicus.
[0201] [000201] In another aspect, the polypeptide is a polypeptide with enzymatic activity from Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus or Streptomyces lividans.
[0202] [000202] The polypeptide with enzymatic activity can also be a fungal polypeptide, and more preferably a yeast polypeptide such as a polypeptide with enzymatic activity from Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces or Yarrowia; or more preferably a filamentous fungus polypeptide such as a polypeptide with enzymatic activity of Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Cryptography, Cryptography, Cryptography, Cryptography Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillis, Picherichia, Picherich, Picherich, Pony, Pony Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella or Xylaria.
[0203] [000203] In one aspect, the polypeptide is a polypeptide with enzymatic activity from Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomyces oviformis.
[0204] [000204] In another aspect, the polypeptide is a polypeptide with enzymatic activity from Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulus, Aspergillus nerisillis, kermisillispor, Gillispor Chrysosporium tropicum, Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminearum, Fusarium graminumum, Fusarium graminumum, Fusarium graminum, Fusarium graminum, Fusarium graminumum , Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthoram, Myceliophthoram spora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia ovispora, Thielavia ovispora, Thielavia triandis, Thielavia terruviana, Thielavia terruviana , Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma viride or Trichophaea saccata.
[0205] [000205] Chemically modified or protein engineered mutants can also be used.
[0206] [000206] One or more (several) components of the enzyme composition can be a recombinant component, that is, produced by cloning a DNA sequence that encodes the single component and the subsequent cell transformed with the DNA sequence and expressed in a host (see, for example, WO 91/17243 and WO 91/17244). The host is preferably a heterologous host (the enzyme is foreign to the host), but the host can also, under certain conditions, be a homologous host (the enzyme is natural to the host). Mono-component cellulolytic proteins can also be prepared by purifying a protein like this from a fermentation broth.
[0207] [000207] In one aspect, the one or more (for example, 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 (Novozymes AS), CELLIC® CTec2 (Novozymes AS), CELLUCLAST ™ (Novozymes AS), NOVOZYM ™ 188 (Novozymes AS), CELLUZYME ™ (Novozymes AS), CEREFLO ™ (Novozymes AS), and ULTRAFLO ™ (Novozymes AS), ACCELERASE ™ (Genencor Int.), LAMINEX ™ (Genencor Int.), SPEZYME ™ CP (Genencor Int.), FILTRASE® NL (DSM); METHAPLUS® S / L 100 (DSM), ROHAMENT ™ 7069 W (Rohm GmbH), FIBREZYME® LDI (Dyadic International, Inc.), FIBREZYME® LBR (Dyadic International, Inc.), or VISCOSTAR® 150L (Dyadic International, Inc .). Cellulase enzymes are added in efficient amounts of about 0.001 to about 5.0% by weight of solids, for example, about 0.025 to about 4.0% by weight of solids or about 0.005 to about 2, 0% by weight of solids.
[0208] [000208] Examples of bacterial endoglucanases that can be used in the methods of the present invention include, but are not limited to, an Acidothermus cellulolyticus endoglucanase (WO 91/05039; WO 93/15186; US patent 5,275,944; WO 96/02551; US patent 5,536,655, WO 00/70031, WO 05/093050); endoglucanase III d e Thermobifida fusca (WO 05/093050) and endoglucanase V from Thermobifida fusca (WO 05/093050).
[0209] [000209] Examples of fungal endoglucanases that can be used in the present invention include, but are not limited to, an endoglucanase I from Trichoderma reesei (Penttila et al., 1986, Gene 45: 253-263; endoglucanase I from Trichoderma reesei Cel7B; number access to GENBANK ™ M15665; SEQ ID NO: 66); endoglucanase II from Trichoderma reesei (Saloheimo, et al., 1988, Gene 63: 11-22; endoglucanase II from Trichoderma reesei Cel5A; GENBANK ™ accession number M19373; SEQ ID NO: 68); endoglucanase III from Trichoderma reesei (Okada et al., 1988, Appl. Environ. Microbiol. 64: 555-563; GENBANK ™ AB003694 accession number; SEQ ID NO: 70); endoglucanase V from Trichoderma reesei (Saloheimo et al., 1994, Molecular Microbiology 13: 219-228; GENBANK ™ accession number Z33381; SEQ ID NO: 72); endoglucanase from Aspergillus aculeatus (Ooi et al., 1990, Nucleic Acids Research 18: 5884); endoglucanase from Aspergillus kawachii (Sakamoto et al., 1995, Current Genetics 27: 435-439); endoglucanase from Erwinia carotovara (Saarilahti et al., 1990, Gene 90: 9-14); Fusarium oxysporum endoglucanase (GENBANK ™ L29381 accession number); endoglucanase from Humicola grisea var. thermoidea (GENBANK ™ AB003107 accession number); Melanocarpus albomyces endoglucanase (GENBANK ™ MAL515703 accession number); Neurospora crassa endoglucanase (GENBANK ™ accession number XM_324477); endoglucanase V from Humicola insolens (SEQ ID NO: 74); Myceliophthora thermophila CBS 117.65 endoglucanase (SEQ ID NO: 76); basiodiomycete endoglucanase CBS 495.95 (SEQ ID NO: 78); basiodiomycete endoglucanase CBS 494.95 (SEQ ID NO: 80); endoglucanase from Tielavia terrestris NRRL 8126 CEL6B (SEQ ID NO: 82); endoglucanase from Tielavia terrestris NRRL 8126 CEL6C (SEQ ID NO: 84); endoglucanase from Tielavia terrestris NRRL 8126 CEL7C (SEQ ID NO: 86); endoglucanase from Tielavia terrestris NRRL 8126 CEL7E (SEQ ID NO: 88); endoglucanase from Tielavia terrestris NRRL 8126 CEL7F (SEQ ID NO: 90); Cladorrhinum foecundissimum endoglucanase ATCC 62373 CEL7A (SEQ ID NO: 92) and Trichoderma reesei endoglucanase strain No. VTT-D-80133 (SEQ ID NO: 94; GENBANK ™ accession number M15665). The endoglucanases of SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80 , SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, and SEQ ID NO: 94 described above are encoded by the sequence that encodes the mature polypeptide of SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO : 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, and SEQ ID NO: 93, respectively.
[0210] [000210] Examples of cellobiohydrolases used in the present invention include, but are not limited to, cellobiohydrolase I of Trichoderma reesei (SEQ ID NO: 96); cellobiohydrolase II from Trichoderma reesei (SEQ ID NO: 98); cellobiohydrolase I from Humicola insolens (SEQ ID NO: 100); cellobiohydrolase II from Myceliophthora thermophila (SEQ ID NO: 102 and SEQ ID NO: 104); cellobiohydrolase II of Tielavia terrestris (CEL6A) (SEQ ID NO: 106); cellobiohydrolase I from Chaetomium thermophiluma (SEQ ID NO: 108); cellobiohydrolase II from Chaetomium thermophiluma (SEQ ID NO: 110); cellobiohydrolase I from Aspergillus fumigatus (SEQ ID NO: 112) and cellobiohydrolase II from Aspergillus fumigatus (SEQ ID NO: 114).
[0211] [000211] The cellobiohydrolases of SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, and SEQ ID NO: 114 described above are encoded by the sequence encoding the mature polypeptide of SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, and SEQ ID NO: 113, respectively.
[0212] [000212] Examples of beta-glucosidases used in the present invention include, but are not limited to, beta-glucosidase from Aspergillus oryzae (SEQ ID NO: 116); beta-glucosidase from Aspergillus fumigatus (SEQ ID NO: 118); beta-glucosidase from Penicillium brasilianum IBT 20888 (SEQ ID NO: 120); beta-glucosidase from Aspergillus niger (SEQ ID NO: 122) and beta-glucosidase from Aspergillus aculeatus (SEQ ID NO: 124). The beta-glucosidases of SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, and SEQ ID NO: 124 described above are encoded by the sequence encoding the mature polypeptide of SEQ ID NO : 115, SEQ ID NO: 117, and SEQ ID NO: 119, SEQ ID NO: 121, and SEQ ID NO: 123, respectively.
[0213] [000213] Examples of other beta-glucosidases used in the present invention include a beta-glucosidase fusion variant of Aspergillus oryzae of SEQ ID NO: 126 or the beta-glucosidase fusion protein of Aspergillus oryzae of SEQ ID NO: 128 The beta-glucosidase fusion proteins of SEQ ID NO: 126 and SEQ ID NO: 128 are encoded by SEQ ID NO: 125 and SEQ ID NO: 127, respectively.
[0214] [000214] Aspergillus oryzae beta-glycosidase can be obtained according to WO 2002/095014. Beta-glycosidase from Aspergillus fumigatus can be obtained according to WO 2005/047499. Beta-glycosidase from Penicillium brasilianuma can be obtained according to WO 2007/019442. Beta-glycosidase from Aspergillus niger can be obtained according to Dan et al., 2000, J. Biol. Chem. 275: 4973-4980. Beta-glycosidase from Aspergillus aculeatus can be obtained according to Kawaguchi et al., 1996, Gene 173: 287-288.
[0215] [000215] Other endoglucanases, cellobiohydrolases and beta-glucosidases used are disclosed in several families of glycosyl hydrolase using the classification according to Henrissat B., 1991, The classification of glycosyl hydrolases based on amino-acid sequence similarities, Biochem. J. 280: 309316, and Henrissat B., and Bairoch A., 1996, Updating the sequence-based classification of glycosyl hydrolases, Biochem. J. 316: 695-696.
[0216] [000216] Other cellulolytic enzymes that can be used in the present invention are described in WO 98/13465, WO 98/015619, WO 98/015633, WO 99/06574, WO 99/10481, WO 99/025847, WO 99/031255 , WO 2002/101078, WO 2003/027306, WO 2003/052054, WO 2003/052055, WO 2003/052056, WO 2003/052057, WO 2003/052118, WO 2004/016760, WO 2004/043980, WO 2004/048592 , WO 2005/001065, WO 2005/028636, WO 2005/093050, WO 2005/093073, WO 2006/074005, WO 2006/117432, WO 2007/071818, WO 2007/071820, WO 2008/008070, WO 2008/008793 , US patent 5,457,046, US patent 5,648,263 and US patent 5,686,593.
[0217] [000217] In one aspect, one or more (for example, 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), CELLIC® HTec (Novozymes A / S), CELLIC® HTec2 (Novozymes A / S), VISCOZYME® (Novozymes A / S), ULTRAFLO® (Novozymes A / S), PULPZYME® HC (Novozymes A / S), MULTIFECT® Xylanase (Genencor), ACCELLERASE® XY (Genencor), ACCELLERASE® XC (Genencor), ECOPULP® TX -200A (AB Enzymes), HSP 6000 Xylanase (DSM), DEPOL ™ 333P (Biocatalysts Limit, Wales, UK), DEPOL ™ 740L. (Biocatalysts Limit, Wales, UK), and DEPOL ™ 762P (Biocatalysts Limit, Wales, UK).
[0218] [000218] Examples of xylanases used in the methods of the present invention include, but are not limited to, Aspergillus aculeatus xylanases (GeneSeqP: AAR63790; WO 94/21785), Aspergillus fumigatus xylanases (WO 2006/078256; xyl 3 SEQ ID NO: 130 [DNA sequence] and SEQ ID NO: 68 [deduced amino acid sequence]), Penicillium pinophilum (WO 2011/041405), Penicillium sp. (WO 2010/126772), Thielavia terrestris NRRL 8126 (WO 2009/079210), and Trichophaea saccata GH10 (WO 2011/057083).
[0219] [000219] Examples of beta-xylosidases used in the methods of the present invention include, but are not limited to, Trichoderma reesei beta-xylosidase (accession number UniProtKB / TrEMBL Q92458; SEQ ID NO: 131 [DNA sequence] and SEQ ID NO : 132 [deduced amino acid sequence]), Talaromyces emersonii (SwissProt accession number Q8X212), and Neurospora crassa (SwissProt accession number Q7SOW4).
[0220] [000220] Examples of acetylxylan esterases used in the methods of the present invention include, but are not limited to, acetylxylan esterases from Aspergillus aculeatus (WO 2010/108918), Chaetomium globosum (Uniprot accession number Q2GWX4), Chaetomium gracile (GeneSeqP accession number AAB82124 ), Humicola insolens DSM 1800 (WO 2009/073709), Hypocrea jecorina (WO 2005/001036), Myceliophtera thermophila (WO 2010/014880), Neurospora crassa (UniProt accession number q7s259), Phaeosphaeria nodorum (accession number Uniprot QUU, Q0 and Thielavia terrestris NRRL 8126 (WO 2009/042846).
[0221] [000221] Examples of feruloyl esterases (ferulic acid esterases) used in the methods of the present invention include, but are not limited to, feruloyl esterases of Humicola insolens DSM 1800 (WO 2009/076122), Neosartorya fischeri (UniProt access number A1D9T4), Neurospora crassa (accession number UniProt Q9HGR3), Penicillium aurantiogriseum (WO 2009/127729), and Thielavia terrestris (WO 2010/053838 and WO 2010/065448).
[0222] [000222] Examples of arabinofuranosidases used in the methods of the present invention include, but are not limited to, Aspergillus niger arabinofuranosidases (GeneSeqP accession number AAR94170), Humicola insolens DSM 1800 (WO 2006/114094 and WO 2009/073383), and M. giganteus (WO 2006/114094).
[0223] [000223] Examples of alpha-glucuronidases used in the methods of the present invention include, but are not limited to, alpha-glucuronidases of Aspergillus clavatus (UniProt access number alcc12), Aspergillus fumigatus (SwissProt access number Q4WW45), Aspergillus niger (number of access Uniprot Q96WX9), Aspergillus terreus (SwissProt access number Q0CJP9), Humicola insolens (WO 2010/014706), Penicillium aurantiogriseum (WO 2009/068565), Talaromyces emersonii (UniProt access number Q8X211), and Trichoderma access number Uniprot Q99024).
[0224] [000224] Polypeptides with enzymatic activity, used in the methods of the present invention, can be produced by fermenting the aforementioned microbial strains in a nutrient medium containing suitable sources of carbon and nitrogen and inorganic salts, using procedures known in the art (see, for example, example, Bennett, JW and LaSure, L. (eds.), More Gene Manipulations in Fungi, Academic Press, CA, 1991). Suitable media are available from commercial suppliers, or can be prepared according to published compositions (for example, in catalogs of the American Type Culture Collection). Temperature ranges and other conditions suitable for enzyme growth and production are known in the art (see, for example, Bailey, J.E., and Ollis, D.F., Biochemical Engineering Fundamentals, McGraw-Hill Book Company, NY, 1986).
[0225] [000225] Fermentation can be any method of culturing a cell that results in the expression or isolation of an enzyme or protein. Therefore, fermentation can be understood as that comprising shaking bottle cultivation, or small or large scale fermentation (including continuous, batch, fed batch or solid fermentation) in laboratory or industrial fermenters, carried out in a appropriate medium and under conditions that allow the enzyme to be expressed or isolated. The resulting enzymes produced by the methods described above can be recovered from the fermentation medium and purified by conventional procedures. Nucleic acid constructs
[0226] [000226] An isolated polypeptide encoding a polypeptide, for example, a GH61 polypeptide with enhanced cellulolytic activity, a cellulolytic enzyme, a hemicellulolytic enzyme, etc., can be manipulated in a variety of ways to provide polypeptide expression by constructing a construct nucleic acid comprising an isolated polypeptide, which encodes the polypeptide operably linked to one or more (for example, several) control sequences that direct expression of the coding sequence in a suitable host cell, under conditions compatible with the control sequences . The manipulation of the polynucleotide sequences before insertion into a vector may be desirable or necessary, depending on the expression vector. Techniques for modifying polynucleotide sequences using recombinant DNA methods are well known in the art.
[0227] [000227] The control sequence can be a promoter, a polynucleotide that is recognized by a host cell for the expression of a polynucleotide that encodes the polypeptide. The promoter contains transcriptional control sequences that mediate polypeptide expression. The promoter can be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides, both homologous and heterologous to the host cell.
[0228] [000228] Examples of promoters suitable for directing the transcription of nucleic acid constructs in a bacterial host cell are promoters obtained from the 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 levansucrase (sacB) gene, Bacillus subtilis xylA and xylB genes, Bacillus subtilis gene 1994 , Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trc promoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicolor (dagA) agarase gene and beta gene prokaryote lactamase (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci USA 80: 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 WO 99/43835.
[0229] [000229] Examples of promoters suitable for directing transcription of the nucleic acid constructs in a filamentous fungus host cell are promoters obtained from the genes for Aspergillus nidulans acetamidase, neutral alpha-amylase from Aspergillus niger, alpha-amylase stable to Aspergillus niger acid, Aspergillus niger glycoamylase (glaA) or Aspergillus awamori, Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, oxide protease, and type 7 protease amyloglycosidase from Fusarium venenatum (WO 00/56900), Fusarium venenatum Daria (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor miehei lipase, Rhizomucor miehei aspartic proteinase, beta-glucosidase from Trichoderma reesei, cellulose I of Trichoderma reesei, cellobiohydrolase II of Trichoderma reesei, endoglucanase I of Trichoderma reesei, endoglucanase II of Trichoderma reesei, endogluc anase III of Trichoderma reesei, endoglucanase V of Trichoderma reesei, xylanase I of Trichoderma reesei, xylanase II of Trichoderma reesei, xylanase III of Trichoderma reesei, beta-xylidasidase of Trichoderma reesei and lengthening factor of the translation as promoter of Trichoderma reesei NA2-tpi (a modified promoter from a neutral Aspergillus alpha-amylase gene, in which the untranslated main sequence was replaced by a major region from an Aspergillus triose phosphate isomerase gene; non-limiting examples include promoters modified from a neutral Aspergillus niger alpha-amylase gene, where the untranslated main region has been replaced by an untranslated main sequence from an Aspergillus nidulans or Aspergillus oryzae isomerase triose gene) and mutant, truncated and hybrid promoters thereof. Other promoters are described in U.S. patent 6,011,147.
[0230] [000230] In a host yeast, the promoters used are obtained from Saccharomyces cerevisiae enolase (ENO-1) genes, Saccharomyces cerevisiae galactokinase (GAL1), alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP) Saccharomyces cerevisiae, triose phosphate isomerase (TPI) from Saccharomyces cerevisiae, metallothionein (CUP1) from Saccharomyces cerevisiae and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other promoters used for yeast host cells are described by Romanos et al., 1992, Yeast 8: 423-488.
[0231] [000231] The control sequence can also be a transcription terminator, which is recognized by a host cell to terminate transcription. The finisher is operably linked at the 3 'end of the polynucleotide encoding the polypeptide. Any finisher that is functional in the host cell can be used in the present invention.
[0232] [000232] Preferred finalizers for bacterial host cells are obtained from Bacillus clausii alkaline protease (aprH) genes, Bacillus licheniformis alpha-amylase (amyL) and Escherichia coli ribosomal RNA (rrnB).
[0233] [000233] The preferred finishers for filamentous fungi host cells are obtained from Aspergillus nidulans acetamidase genes, Aspergillus nidulans anthranilate synthase, Aspergillus niger glycoamylase, Aspergillus niger alpha-glycosidase, TAKA amylase from Aspergillus type of aspergine asease from Fusarium oxysporum, beta-glucosidase from Trichoderma reesei, cellobiohydrolase I from Trichoderma reesei, cellobiohydrolase II from Trichoderma reesei, endoglucanase I from Trichoderma reesei, endoglucanase II from Trichoderma reesei, endoglucanase III from Trichoderma reesei, endoglucanase III from Trichoderma reese Trichoderma reesei, Trichoderma reesei xylanase II, Trichoderma reesei xylanase III, Trichoderma reesei beta-xylosidase and translation lengthening factor of Trichoderma reesei.
[0234] [000234] Preferred finalizers for yeast host cells are obtained from Saccharomyces cerevisiae enolase genes, Saccharomyces cerevisiae cytochrome C (CYC1) and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other finalizers used for yeast host cells are described by Romanos et al., 1992, supra.
[0235] [000235] The control sequence can also be a stabilizing region of mRNA, downstream of a promoter and upstream of the coding sequence of a gene that increases the expression of the variant.
[0236] [000236] Examples of suitable mRNA stabilizing regions are obtained from a Bacillus thuringiensis crylIIA gene (WO 94/25612) and a Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177: 3465-3471) .
[0237] [000237] The control sequence can also be a main sequence, an untranslated region of an mRNA that is important for translation by the host cell. The main sequence is operably linked at the 5 'end of the polynucleotide that encodes the polypeptide. Any major sequence that is functional in the host cell can be used.
[0238] [000238] The preferred major sequences for the host cells of filamentous fungi are obtained from the genes for TAKA amylase from Aspergillus oryzae and triose phosphate isomerase from Aspergillus nidulans.
[0239] [000239] The appropriate leader sequences for yeast host cells are obtained from Saccharomyces cerevisiae enolase (ENO-1) genes, Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha factor and dehydrogenase / glyceraldehyde-3-phosphate alcohol dehydrogenase (ADH2 / GAP) from Saccharomyces cerevisiae.
[0240] [000240] The control sequence can also be a polyadenylation sequence, a sequence operably linked at the 3 'end of the polynucleotide and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to the transcribed mRNA. Any polyadenylation sequence that is functional in the host cell can be used.
[0241] [000241] The preferred polyadenylation sequences for filamentous fungus host cells are obtained from Aspergillus nidulans anthranilate synthase genes, Aspergillus niger glycoamylase, Aspergillus niger alpha glycosidase, TAKA Aspergillus oryzae amylase and protease type of tyrosine oxide and tyrosine protease .
[0242] [000242] The polyadenylation sequences used for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.
[0243] [000243] The control sequence can also be a signal peptide coding region that encodes a signal peptide attached at the N-terminus of a polypeptide, and directs the polypeptide in the cell's secretory pathway. The 5 'ends of the polynucleotide coding sequence may inherently contain a coding signal peptide sequence, naturally linked to the reading frame, with the segment of the coding sequence encoding the polypeptide. Alternatively, the 5 'end of the coding sequence may contain a coding signal peptide sequence that is foreign to the coding sequence. A foreign coding signal peptide sequence may be required where the coding sequence does not naturally contain a coding signal peptide sequence. Alternatively, a foreign signal peptide coding sequence can simply replace the natural signal peptide coding sequence in order to enhance secretion of the polypeptide. However, any signal encoding peptide sequence, which directs the expressed polypeptide into the secretory pathway of a host cell, can be used.
[0244] [000244] The efficient signal peptide sequences that encode bacterial host cells are the coding signal peptide sequences obtained from the genes for Bacillus maltogenic amylase NCIB 11837, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus alpha-amylase stearothermophilus, neutral proteases from Bacillus stearothermophilus (nprT, nprS, nprM) and prsA from Bacillus subtilis. Additional signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.
[0245] [000245] The efficient signal peptide sequences encoding filamentous fungus host cells are the signal peptide sequences encoding obtained from the genes for Aspergillus niger amylase, Aspergillus niger glycoamylase, TAKA Aspergillus oryzae amylase, Humicola insolens cellulase, endoglucanase, endoglucan Humicola insolens, Humicola lanuginosa lipase and Rhizomucor miehei aspartic proteinase.
[0246] [000246] The signal peptides used for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha factor and Saccharomyces cerevisiae invertase. Other coding signal peptide sequences used are described by Romanos et al., 1992, supra.
[0247] [000247] The control sequence can also be a coding propeptide sequence that encodes a pro-peptide positioned at the N-terminus of a polypeptide. The resulting polypeptide is known as a pro-enzyme or pro-polypeptide (or a zymogen in some cases). A pro-polypeptide is generally inactive, and can be converted to an active polypeptide by catalytic or auto-catalytic cleavage of the pro-peptide from the pro-polypeptide. The coding propeptide sequence can be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase and alpha factor Saccharomyces cerevisiae.
[0248] [000248] Where both the signal peptide and the pro-peptide sequence are present, the pro-peptide sequence is positioned close to the N-terminus of the polypeptide, and the signal peptide sequence is positioned close to the N-terminus of the pro-peptide sequence. peptide.
[0249] [000249] It may also be desirable to add regulatory sequences that regulate the expression of the polypeptide with respect to host cell growth. Examples of regulatory sequences are those that cause gene expression to be triggered and deactivated in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory sequences in prokaryotic systems include lac, tac and trp operator systems. In yeast, the ADH2 system or the GAL1 system can be used. In filamentous fungi, the Aspergillus niger glycoamylase promoter, Aspergillus oryzae TAKA alpha-amylase promoter and the Aspergillus oryzae glycoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter and Troboderma reesei cellobiohydrolase promoter. Other examples of regulatory sequences are those that allow the amplification of the gene. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals. In these cases, the polynucleotide encoding the polypeptide can be operably linked to the regulatory sequence. Expression Vectors
[0250] [000250] The various nucleotide and control sequences described above can be brought together to produce a recombinant expression vector, which may include one or more (for example, several) convenient restriction sites to allow insertion or replacement of a polynucleotide that encodes a polypeptide, for example, a GH61 polypeptide with enhanced cellulolytic activity, a cellulolytic enzyme, a hemicellulolytic enzyme, etc., at such sites. Alternatively, the polynucleotide can be expressed by inserting the polynucleotide or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector, such that the coding sequence is operably linked in the appropriate control sequences for expression.
[0251] [000251] The recombinant expression vector can be any vector (for example, a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures, and can result in the expression of the polynucleotide. The choice of the vector will typically depend on the vector's compatibility with the host cell, into which the vector will be introduced. The vector can be a closed linear or circular plasmid.
[0252] [000252] The vector can be a vector that replicates autonomously, that is, a vector that exists as an extrachromosomal entity, whose replication is independent of chromosomal replication, for example, a plasmid, an extrachromosomal element, a minichromosome, or a chromosome artificial. The vector can contain any means to guarantee self-replication. Alternatively, the vector can be one that, when introduced into the host cell, is integrated into the genome and replicated along with the chromosome (s) into which it has been integrated. In addition, a single vector, or plasmid, or two or more vectors or plasmids that together contain the total DNA to be introduced into the host cell's genome, or a transposon, can be used.
[0253] [000253] The vector preferably contains one or more (for example, several) selectable markers that allow easy selection of transformed, transfected, transduced or similar cells. A selectable marker is a product of the gene that provides biocidal or viral resistance, resistance to heavy metals, prototrophy to auxotrophic and the like.
[0254] [000254] Examples of selectable bacterial markers are the genes of Bacillus licheniformis or Bacillus subtilis, or markers that confer resistance to antibiotics, such as resistance to ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin or tetracycline. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1 and URA3. Selectable markers for use in a filamentous fungus host cell include, but are not limited to, adeA (phosphoribosylaminoimidazole-succinocarboxamide synthase), adeB (phosphoribosyl-aminoimidazole synthase), amdS (acetamidase), argB (ornithine carbamoyltransferase), bar , hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (adenyl transferase sulfate), and trpC (anthranylate synthase), as well as equivalents thereof. Preferred for use in an Aspergillus cell are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae, and a bar gene of Streptomyces hygroscopicus. The genes adeA, adeB, amdS, hph and pyrG are preferred for use in a Trichoderma cell.
[0255] [000255] The selectable marker can be a double selectable marker system, as described in WO 2010/039889. In one aspect, the double selectable marker is a hph-tk double selectable marker system.
[0256] [000256] The vector preferably contains an element (s) that allows integration of the vector into the genome of the host cell or autonomous replication of the vector in the cell independent of the genome.
[0257] [000257] For integration into the host cell genome, the vector may depend on the polynucleotide sequence encoding the polypeptide, or any other element of the vector 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 host cell genome at an exact location (s) on the chromosome (s). To increase the likelihood of integration in a precise location, the integrational elements may contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs and 800 to 10,000 base pairs, which have a high degree of sequence identity with the corresponding target sequence to enhance the likelihood of homologous recombination. The integrational elements can be any sequence that is homologous to the target sequence in the host cell genome. Furthermore, the integrating elements can be non-coding or coding polynucleotides. On the other hand, the vector can be integrated into the host cell genome by non-homologous recombination.
[0258] [000258] For autonomous replication, the vector may additionally comprise an origin of replication that enables the vector to replicate autonomously in the host cell in question. The origin of replication can be any replicator plasmid that mediates autonomous replication that functions in a cell. The term "origin of replication" or "replicator plasmid" means a polynucleotide that enables a plasmid or vector to replicate in vivo.
[0259] [000259] Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 that allow replication in E. coli, and pUB110, pE194, pTA1060, and pAMBl that allow replication in Bacillus.
[0260] [000260] Examples of origins of replication for use in a yeast as a host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.
[0261] [000261] Examples of origins of replication used in a filamentary cell are AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al., 1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). The isolation of the AMA1 gene and the construction of plasmids or vectors comprising the gene can be carried out according to the methods disclosed in WO 00/24883.
[0262] [000262] More than one copy of a polynucleotide can be inserted into a host cell to increase production of a polypeptide. An increase in the number of copies of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the genome of the host cell, or by including a selectable marker gene amplifiable with the polynucleotide, where cells containing amplified copies of the selectable marker gene and, through in addition, additional copies of the polynucleotide can be selected by culturing the cells in the presence of the appropriate selectable agent.
[0263] [000263] The procedures used to link the elements described above to construct the recombinant expression vectors are well known to those skilled in the art (see, for example, Sambrook et al., 1989, supra). Host cells
[0264] [000264] Recombinant host cells comprising a polynucleotide encoding a polypeptide, for example, a GH61 polypeptide with enhanced cellulolytic activity, a cellulolytic enzyme, a hemicellulolytic enzyme, etc., can be advantageously used in the recombinant production of the polypeptide. A construct or vector comprising a polynucleotide such as this is introduced into a host cell, in such a way that the vector is maintained as a chromosomal integral, or as an extra-chromosomal self-replicating vector in the manner described above. The term "host cell" includes any progeny of a parental cell that is not identical to the parental cell, due to mutations that occur during replication. The choice of a host cell will depend to some degree on the gene encoding the polypeptide and on your source.
[0265] [000265] The host cell can be any cell used in the production of a recombinant polypeptide, for example, a prokaryote or a eukaryote.
[0266] [000266] The prokaryotic host cell can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella and Ureaplasma.
[0267] [000267] The bacterial host cell can be qq. Bacillus cells including, but not limited to, Bacillus alkalophilus cells, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus michus, Bacillus licheniformis, Bacillus megillus Bacillus subtilis and Bacillus thuringiensis.
[0268] [000268] The bacterial host cell can also be any Streptococcus cell including, but not limited to, Streptococcus equisimilis cells, Streptococcus pyogenes, Streptococcus uberis and Streptococcus equi subsp. Zooepidemicus.
[0269] [000269] The bacterial host cell can also be any Streptomyces cell including, but not limited to, Streptomyces achromogenes cells, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus and Streptomyces lividans.
[0270] [000270] The introduction of DNA into a Bacillus cell can be carried out by protoplast transformation (see, for example, Chang and Cohen, 1979, Mol. Gen. Genet. 168: 111-115), competent cell transformation (see , for example, Young and Spizizen, 1961, J. Bacteriol. 81: 823829, 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, for example, Koehler and Thorne, 1987, J. Bacteriol. 169: 5271-5278). The introduction of DNA into an E. coli cell can be carried out by transformation of protoplasts (see, for example, Hanahan, 1983, J. Mol. Biol. 166: 557-580) or electroporation (see, for example, Dower et al., 1988, Nucleic Acids Res. 16: 6127-6145). The introduction of DNA into a Streptomyces cell can be carried out by protoplast transformation, electroporation (see, for example, Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405), conjugation (see, for example , Mazodier et al., 1989, J. Bacteriol. 171: 3583-3585), or transduction (see, for example, Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98: 6289-6294). The introduction of DNA into a Pseudomonas cell can be performed by electroporation (see, for example, Choi et al., 2006, J. Microbiol. Methods 64: 391-397), or conjugation (see, for example, Pinedo and Smets , 2005, Appl. Environ. Microbiol. 71: 51-57). The introduction of DNA into a Streptococcus cell can be accomplished by natural competence (see, for example, Perry and Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), transformation of protoplasts (see, for example, Catt and Jollick , 1991, Microbes 68: 189-207), electroporation (see, for example, Buckley et al., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or conjugation (see, for example, Clewell, 1981, Microbiol Rev. 45: 409-436). However, any method known in the art to introduce DNA into a host cell can be used.
[0271] [000271] The host cell can also be from a eukaryote, such as a mammalian cell, insect, plant or fungus.
[0272] [000272] The host cell can be a fungal cell. “Fungi”, as used herein, includes the phyla Ascomycota, Basidiomycota, Chytridiomycota and Zygomycota, as well as Oomycota and all mythosporic 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).
[0273] [000273] The fungal host cell can be a yeast cell. “Yeast”, as used here, includes ascosporogenic yeasts (Endomycetales), basidiosporogenic yeasts and yeasts that belong to imperfect fungi (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeasts can be defined in the manner described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, RR, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).
[0274] [000274] Yeast as a host cell can be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as a Kluyveromyces lactis cell, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyyces, Saccharomyy , Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica.
[0275] [000275] The fungal host cell can be a filamentous fungus cell. "Filamentous fungi" include all filamentous forms in the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). Filamentous fungi are generally characterized by a mycelium wall composed of chitin, cellulose, glycan, chitosan, mannan and other complex polysaccharides. Vegetative growth is by stretching hyphae and carbon catabolism is mandatory aerobic. On the contrary, the vegetative growth of yeasts, such as Saccharomyces cerevisiae, is by budding from a single-celled stem and the carbon catabolism can be fermentative.
[0276] [000276] The host cells of the filamentous fungus may be an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Neocor, Mycelio, Mycelio, Mycelio Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma.
[0277] [000277] For example, the host cells of the filamentous fungus can be a cell of Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Ceriororisis, Cerjorisisipisis, Ceri Ceriporiopsis pannocinta, rivulose Ceriporiopsis, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, lucknowense Chrysosporium, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum cinereus Coprinus, Coriolus hirsutus, Fusarium bactridioides, cerealis Fusarium, Fusarium crookwellense , Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium arium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotielusyon, terrestrial, terrestrial, terrarium , Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.
[0278] [000278] Fungal cells can be transformed by a process involving formation of protoplasts, transformation of protoplasts and regeneration of the cell wall in a manner known per se. Suitable procedures for transforming Aspergillus and Trichoderma host cells are described in EP 238023 and Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81: 1470-1474, and Christensen ET. AL., 1988, Bio / Technology 6: 1419-1422. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147156, and WO 96/00787. Yeast can be transformed using the procedures described by Becker and Guarente, In Abelson, JN and Simon, MI, editors, 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. Production Methods
[0279] [000279] The methods for producing a polypeptide, for example, a GH61 polypeptide with enhanced cellulolytic activity, a cellulolytic enzyme, a hemicellulolytic enzyme, etc., comprise (a) cultivating a cell, which in its wild type is capable of producing the polypeptide, under conductive conditions for the production of the polypeptide; and (b) recovering the polypeptide. In a preferred aspect, the cell is of the Aspergillus genus. In a more preferred aspect, the cell is Aspergillus fumigatus.
[0280] [000280] Alternatively, methods for producing a polypeptide, for example, a GH61 polypeptide with enhanced cellulolytic activity, a cellulolytic enzyme, a hemicellulolytic enzyme, etc., comprise (a) culturing a recombinant host cell under conductive conditions for the production of the polypeptide ; and (b) recovering the polypeptide.
[0281] [000281] The cells are grown in a nutrient medium suitable for the production of the polypeptide using methods known in the art. For example, cells can be grown by shaking flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed batch or solid fermentation) in laboratory or industrial fermenters, in a medium and under suitable conditions that allow the polypeptide to be expressed and / or isolated. Cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers, or can be prepared according to published compositions (for example, in catalogs of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.
[0282] [000282] The polypeptide can be detected using methods known in the art that are specific to the polypeptides. These detection methods include, but are not limited to, the use of specific antibodies, the formation of an enzyme product, or the disappearance of an enzyme substrate. For example, an enzyme assay can be used to determine the activity of the polypeptide. Polypeptides with enhanced cellulolytic activity are detected using the methods described here.
[0283] [000283] The resulting broth can be used as such, or the polypeptide can be recovered using methods known in the art. For example, the variant can be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation or precipitation. In one aspect, the entire fermentation broth is recovered.
[0284] [000284] The polypeptide can be purified by a variety of procedures known in the art including, but not limited to, chromatography (for example, ion exchange, affinity, hydrophobic, isoelectric focusing and size exclusion), electrophoretic procedures (for example , preparative isoelectric focus), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, for example, Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, Nova York, 1989) to obtain substantially pure polypeptides.
[0285] [000285] In an alternative aspect, the polypeptide is not recovered, but certainly a host cell that expresses a polypeptide is used as a source of the polypeptide.
[0286] [000286] The present invention is further described by the following examples, which are not to be construed as limiting the scope of the invention. Examples Example 1: Pretreatment of corn straw
[0287] [000287] Corn straw was pretreated at the US Department of Energy National Renewable Energy Laboratory (NREL) using 1.4% (w / v) sulfuric acid, for 8 minutes at 165 ° C and 107 psi (737, 7 kPa). The water-insoluble solids in the pre-treated corn straw contained 57.5% cellulose, 4.6% hemicelluloses, and 28.4% lignin. Cellulose and hemicellulose were determined by hydrolysis with sulfuric acid in two stages, with subsequent analysis of sugars by high performance liquid chromatography using the standard analytical procedure NREL # 002. Lignin was determined gravimetrically after hydrolyzing the cellulose and hemicellulose fractions with sulfuric acid, using the standard analytical procedure NREL # 003.
[0288] [000288] The pretreated corn straw was adjusted to pH 5.0 by repeatedly adding 10 N NaOH in aliquots of a few millimeters, followed by complete mixing and incubation at room temperature for approximately 1 hour. The pH was confirmed after overnight incubation at 4 ° C, and the pH-adjusted corn straw was autoclaved for 20 minutes at approximately 120 ° C, and then stored at 4 ° C to reduce the risk of contamination microbial. The dry weight of the pre-treated corn straw was 33% TS (total solids), which was confirmed before each use.
[0289] [000289] The pre-treated corn straw was ground before use. Pre-treated milled corn straw (initial dry weight 32.35% TS) was prepared by grinding in a wet multi-utility grinder Cosmos ICMG 40 (EssEmm Corporation, Tamil Nadu, India). The pre-treated ground corn straw, in some cases, was also subsequently washed with deionized water by repeated dilution after decanting the supernatant fraction. The dry weight of pre-treated, ground corn and washed with water was 7.11% TS.
[0290] [000290] Alternatively, the pre-treated, ground and washed corn straw was washed extensively with water at 50 ° C. Approximately 600 mL of pre-treated corn straw washed with water was diluted with approximately 500 mL of distilled and deionized water, and were incubated at 50 ° C with shaking for 7 days. The pretreated corn straw was allowed to settle three to four times a day, and the water in the supernatant decanted and was replaced by 500 ml of fresh deionized water. The dry weight of pre-treated corn straw, ground and washed with hot water was 6.74% TS. Example 2: Preparation of Tielavia terrestris GH61E polypeptide with enhanced cellulolytic activity
[0291] [000291] Tielavia terrestris GH61E polypeptide with enhanced cellulolytic activity was produced recombinantly in Aspergillus oryzae JaL250, as described in U.S. patent 7,361,495. Example 3: Evaluation of the saccharification of cellulosic material
[0292] [000292] The pre-treated corn straw, ground and washed with hot water was prepared in the manner described in example 1, and was used as the source of the cellulosic material.
[0293] [000293] A cellulase preparation of Trichoderma reesei (CELLUCLAST® supplemented with beta-glucosidase from Aspergillus oryzae, available from Novozymes A / S, Bagsvaerd, Denmark) was used in the hydrolysis reactions, and is determined here in the examples as “composition of Trichoderma reesei cellulase ".
[0294] [000294] The hydrolysis of PCS ground and washed with hot water was conducted using 96-well, 2.0-ml deep plates (Axygen, Union City, CA, USA), in a total reaction volume of 1.0 ml. Each hydrolysis was performed with 50 mg of PCS (29.5 mg of cellulose) per mL of 50 mM sodium acetate buffer, pH 5.0, containing 1 mM manganese sulfate and the T. reesei cellulase composition in 4 mg of protein per gram of cellulose, with and without the GH61 polypeptide with enhanced cellulolytic activity, in various concentrations between 0 and 1 mg per gram of cellulose (unless otherwise specified). The plate was then sealed using an ALPS-300 ™ (Abgene, Epsom, UK), completely mixed and incubated at 50 ° C for 7 days, shaking at 150 rpm in an Innova 4080 shaking incubator (New Brunswick Scientific, Edison , NJ, USA). All experiments were carried out in triplicate.
[0295] [000295] At various time points, between 24 and 168 hours of incubation, aliquots of 100 pL were removed and the degree of hydrolysis was assessed by high performance liquid chromatography (HPLC), using the protocol described below.
[0296] [000296] For HPLC analysis, samples were filtered using a 96-well plate with 0.45 pm MULTISCREEN® filter (Millipore, Bedford, MA, USA), and the filtrates were analyzed for the sugar content of the described below. Sugar concentrations of the samples diluted in 0.005 M H2SO4 were measured using a 4.6 x 250 mm AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, CA, USA) by elution with 0.5% w / w benzoic acid - 5 mM H2SO4, at a flow rate of 0.6 mL per minute at 65 ° C for 11 minutes, and quantification by integrating glucose and cellobiose signals from the detection of the refractive index (CHEMSTATION® , AGILENT® 1100 HPLC, Agilent Technologies, Santa Clara, CA, USA) was calibrated by pure sugar samples. The resulting equivalents were used to calculate the percentage of cellulose conversion for each reaction. The degree of each hydrolysis was determined as the fraction of total cellulose converted to cellobiose + glucose, and was not corrected for the soluble sugars present in the pretreated corn straw liqueur, or was corrected for the soluble sugars present in the liquor, from indicated way.
[0297] [000297] All HPLC data processing was performed using KALEIDAGRAPH® software (Synergy software, Reading, PA, USA) or MICROSOFT EXCEL® (Microsoft, Seattle, WA, USA). The evaluated sugar concentrations were adjusted to the appropriate dilution factor. Glucose and cellobiose were separated and chromatographically integrated, and their respective concentrations were determined independently. To calculate the total conversion, the glucose and cellobiose values were combined. Fractional hydrolysis is reported as the ratio of the corrected concentrations of glucose and cellobiose mass to the initial cellulose concentration according to equation 1. The triplicate data points were averaged and the standard deviation was calculated.
[0298] [000298] The dependence of the concentration of the dependent enhancement of the GH61 polypeptide on cellulose hydrolysis by the T. reesei cellulase composition was determined by titration of the GH61 polypeptide between 0 and 24% (w / w) of total protein added in a constant concentration of T. reesei cellulase of 4 mg per gram cellulose, graphically representing fractional hydrolysis against the concentration of GH61 polypeptide. Example 4: Effect of treating pre-treated, ground and washed corn straw with hot water with Tielavia terrestris GH61E polypeptide, or Tielavia terrestris GH61E polypeptide with Triehoderma reesei cellulase composition
[0299] [000299] PCS ground and washed with hot water was generated in the manner described in example 1. Fifty ml of PCS ground and washed with hot water were incubated with 5% of the total solids in 50 mM sodium acetate, MnSO4 1 mM pH 5 , 0 to 50 ° C, either for 1 or 3 days, with 1 mg of T. terrestris GH61E polypeptide per gram of cellulose, 4 mg of the cellulosease composition of Triehoderma reesei per gram of cellulose, or 4 mg of the cellulose composition T. reesei cellulase per gram of cellulose with 1 mg of the GH61E polypeptide of Thelavia terrestris per gram of cellulose. After incubation, the enzyme activity was inactivated by incubation at 90 ° C for more than 30 minutes. The complete inactivation of the enzymatic activity (saccharification) was confirmed by further incubating the aliquots of these samples at 50 ° C for 5 days, and was evaluated by hydrolysis in the manner described in example 3.
[0300] [000300] Treatment with T. terrestris polypeptide GH61E alone yielded 0.0 fractional hydrolysis after both 1 and 3 days. The treatment for 1 or 3 days with the cellulase composition of T. reesei yielded fractional hydrolysis of 0.37 or 0.47, respectively. The treatment for 1 or 3 days with the cellulase composition of T. reesei and with the polypeptide GH61E of T. terrestris yielded fractional hydrolysis of 0.36 or 0.46, respectively.
[0301] [000301] The dry weight of the residual PCS was determined using an IR120 moisture analyzer (Denver Instrument, Bohemia, NY, USA), and then adjusted to 5% by the addition of deionized water. The PCSs treated in various ways were then hydrolyzed on a 1 mL scale, as described in example 3, using 4 mg of the T. reesei cellulase composition per gram of cellulose, with various concentrations of T. terrestris GH61E polypeptide between 0 and 1 mg per gram of cellulose. The degree of hydrolysis was determined in 1 and 5 days of hydrolysis, as described in example 3.
[0302] [000302] Figure 1 shows the fractionated hydrolysis of PCS pretreated with enzyme, ground and washed with hot water. PCS was pretreated with only T. terrestris GH61E polypeptide, T. reesei cellulase composition, or T. reesei cellulase composition with T. terrestris GH61E polypeptide, in the manner indicated for 1 day ( Figure 1A) or 3 days (Figure 1B). Figure 2 shows a comparison of the fractionated hydrolysis of the pre-treated PCS, ground and washed with hot water with the T. terrestris polypeptide GH61E for 1 or 3 days.
[0303] [000303] PCS pretreated for 3 days showed much more digestibility when the GH61 polypeptide was included in the pretreatment (Figure 1B). The PCS pretreated with GH61 polypeptide only for 3 days was hydrolyzed almost in an equivalent manner to the PCS pretreated with the T. reesei cellulase composition. This indicated that pretreatments of PCS with the GH61 polypeptide increased the digestibility of cellulose. In 5 days of saccharification, the complete conversion to pre-treated PCS, both with the T. reesei cellulase composition and with the T. reesei cellulase composition and the GH61 polypeptide, was almost equivalent in each concentration of GH61 polypeptide. Although the hydrolysis that occurred during pretreatments with the T. reesei cellulase composition or the T. reesei cellulase composition and the GH61 polypeptide was equivalent, the first day of additional saccharification with increasing concentrations of GH61 polypeptide showed more conversion at all concentrations of the GH61 polypeptide. This result occurred regardless of an additional 3 days of saccharification time for PCS that was pretreated with cellulase. Additionally, fractional hydrolysis in 1 day for PCS pretreated with GH61 polypeptide for 3 days was substantially better than both for untreated PCS and for PCS pretreated with GH61 polypeptide for 1 day, 0.350 ± 0 , 00159 compared to 0.315 ± 0.00267 or 0.306 ± 0.00105 in the absence of the GH61 polypeptide.
[0304] [1] Um método de degradar um material celulósico, compreendendo: (a) pré-tratar o material celulósico com uma composição compreendendo um ou mais (por exemplo, vários) polipeptídeos GH61; e (b) sacarificar o material celulósico pré-tratado com polipeptídeo GH61 com uma composição enzimática. [2] O método do parágrafo 1, que compreende adicionalmente tratar o material celulósico com um pré-tratamento químico, um pré-tratamento físico, ou um pré-tratamento químico e um pré-tratamento físico. [3] O método do parágrafo 2, em que o pré-tratamento com o um ou mais (por exemplo, vários) polipeptídeos GH61 é realizado antes, durante ou após o pré-tratamento químico, o pré-tratamento físico, ou o pré-tratamento químico e o pré-tratamento físico. [4] O método do parágrafo 2 ou 3, em que o tratamento adicional do material celulósico com um pré-tratamento químico, um pré-tratamento físico, ou um pré-tratamento químico e um pré-tratamento físico é realizado antes da sacarificação. [5] O método de quaisquer dos parágrafos 1-4, em que o pré-tratamento com o um ou mais (por exemplo, vários) polipeptídeos GH61 é realizado antes da sacarificação. [6] O método de quaisquer dos parágrafos 1-5, em que o um ou mais (por exemplo, vários) polipeptídeos GH61 são inativados após pré-tratamento do material celulósico. [7] O método de quaisquer dos parágrafos 1-6, em que a composição enzimática compreende uma ou mais (por exemplo, várias) enzimas selecionadas do grupo que consiste em uma celulase, um polipeptídeo GH61 com atividade celulolítica intensificada, uma hemicelulase, uma esterase, uma expansina, uma laccase, uma enzima ligninolítica, uma pectinase, uma peroxidase, uma protease e uma swolenina. [8] O método do parágrafo 7, em que a celulase é uma ou mais (por exemplo, várias) enzimas selecionadas do grupo que consiste em uma endoglucanase, uma celobioidrolase, e uma beta-glucosidase. [9] O método do parágrafo 7, em que a hemicelulase é uma ou mais (por exemplo, várias) enzimas selecionadas do grupo que consiste em uma xilanase, uma acetixilano esterase, uma feruloil esterase, uma arabinofuranosidase, uma xilosidase e uma glucuronidase. [10] O método de quaisquer dos parágrafos 1-9, compreendendo adicionalmente recuperar o material celulósico degradado. [11] O método do parágrafo 10, em que o material celulósico degradado é um açúcar. [12] O método do parágrafo 11, em que o açúcar é selecionado do grupo que consiste em glicose, xilose, manose, galactose e arabinose. [13] Um método de produzir um produto de fermentação, compreendendo: (a) pré-tratar um material celulósico com uma composição compreendendo um ou mais (por exemplo, vários) polipeptídeos GH61; (b) sacarificar o material celulósico pré-tratado com GH61 com uma composição enzimática; (c) fermentar o material celulósico sacarificado com um ou mais (por exemplo, vários) microrganismos fermentadores para sintetizar o produto de fermentação; e (d) recuperar o produto de fermentação a partir da fermentação. [14] O método do parágrafo 13, que compreende adicionalmente tratar o material celulósico com um pré-tratamento químico, um pré-tratamento físico, ou um pré-tratamento químico e um pré-tratamento físico. [15] O método do parágrafo 14, em que o pré-tratamento com o um ou mais (por exemplo, vários) polipeptídeos GH61 é realizado antes, durante ou após o pré-tratamento químico, o pré-tratamento físico, ou o pré-tratamento químico e o pré-tratamento físico. [16] O método do parágrafo 14 ou 15, em que o tratamento adicional do material celulósico com um pré-tratamento químico, um pré-tratamento físico, ou um pré-tratamento químico e um pré-tratamento físico é realizado antes da sacarificação. [17] O método de quaisquer dos parágrafos 13-16, em que o pré-tratamento com o um ou mais (por exemplo, vários) polipeptídeos GH61 é realizado antes da sacarificação. [18] O método de quaisquer dos parágrafos 13-17, em que o um ou mais (por exemplo, vários) polipeptídeos GH61 são inativados após pré-tratamento do material celulósico. [19] O método de quaisquer dos parágrafos 13-18, em que a composição enzimática compreende uma ou mais (por exemplo, várias) enzimas selecionadas do grupo que consiste em uma celulase, um polipeptídeo GH61 com atividade celulolítica intensificada, uma hemicelulase, uma esterase, uma expansina, uma laccase, uma enzima ligninolítica, uma pectinase, uma peroxidase, uma protease e uma swolenina. [20] O método do parágrafo 19, em que a celulase é uma ou mais (por exemplo, várias) enzimas selecionadas do grupo que consiste em uma endoglucanase, uma celobioidrolase, e uma beta-glucosidase. [21] O método do parágrafo 19, em que a hemicelulase é uma ou mais (por exemplo, várias) enzimas selecionadas do grupo que consiste em uma xilanase, uma acetixilano esterase, uma feruloil esterase, uma arabinofuranosidase, uma xilosidase e uma glucuronidase. [22] O método de quaisquer dos parágrafos 13-21, em que as etapas (c) e (d) são realizadas simultaneamente em uma sacarificação e fermentação simultânea. [23] O método de quaisquer dos parágrafos 13-22, em que o produto de fermentação é um álcool, um alcano, um cicloalcano, um alceno, um aminoácido, um gás, isopreno, uma cetona, um ácido orgânico, ou policetídeo. [24] Um método de produzir um produto de fermentação, compreendendo: (a) sacarificar um material celulósico com uma composição enzimática, em que o material celulósico é pré-tratado com uma composição compreendendo um ou mais (por exemplo, vários) polipeptídeos GH61; (b) fermentar o material celulósico sacarificado com um ou mais (por exemplo, vários) microrganismos fermentadores para sintetizar o produto de fermentação; e (c) recuperar o produto de fermentação a partir da fermentação. [25] O método do parágrafo 24, que compreende adicionalmente tratar o material celulósico com um pré-tratamento químico, um pré-tratamento físico, ou um pré-tratamento químico e um pré-tratamento físico. [26] O método do parágrafo 25, em que o pré-tratamento com o um ou mais (por exemplo, vários) polipeptídeos GH61 é realizado antes, durante ou após o pré-tratamento químico, o pré-tratamento físico, ou o pré-tratamento químico e o pré-tratamento físico. [27] O método do parágrafo 25 ou 26, em que o tratamento adicional do material celulósico com um pré-tratamento químico, um pré-tratamento físico, ou um pré-tratamento químico e um pré-tratamento físico é realizado antes da sacarificação. [28] O método de quaisquer dos parágrafos 24-27, em que o pré-tratamento com o um ou mais (por exemplo, vários) polipeptídeos GH61 é realizado antes da sacarificação. [29] O método de quaisquer dos parágrafos 24-28, em que o um ou mais (por exemplo, vários) polipeptídeos GH61 são inativados após pré-tratamento do material celulósico. [30] O método de quaisquer dos parágrafos 24-29, em que a composição enzimática compreende uma ou mais (por exemplo, várias) enzimas selecionadas do grupo que consiste em uma celulase, um polipeptídeo GH61 com atividade celulolítica intensificada, uma hemicelulase, uma esterase, uma expansina, uma laccase, uma enzima ligninolítica, uma pectinase, uma peroxidase, uma protease e uma swolenina. [31] O método do parágrafo 30, em que a celulase é uma ou mais (por exemplo, várias) enzimas selecionadas do grupo que consiste em uma endoglucanase, uma celobioidrolase, e uma beta-glucosidase. [32] O método do parágrafo 30, em que a hemicelulase é uma ou mais (por exemplo, várias) enzimas selecionadas do grupo que consiste em uma xilanase, uma acetixilano esterase, uma feruloil esterase, uma arabinofuranosidase, uma xilosidase e uma glucuronidase. [33] O método de quaisquer dos parágrafos 24-32, em que as etapas (a) e (b) são realizadas simultaneamente em uma sacarificação e fermentação simultânea. [34] O método de quaisquer dos parágrafos 24-33, em que o produto de fermentação é um álcool, um alcano, um cicloalcano, um alceno, um aminoácido, um gás, isopreno, uma cetona, um ácido orgânico, ou policetídeo. [35] Um método para fermentar um material celulósico, compreendendo: fermentar o material celulósico com um ou mais (por exemplo, vários) microrganismos fermentadores, em que o material celulósico é pré-tratado com uma composição compreendendo um ou mais (por exemplo, vários) polipeptídeos GH61, e é sacarificado com uma composição enzimática. [36] O método do parágrafo 35, que compreende adicionalmente tratar o material celulósico com um pré-tratamento químico, um pré-tratamento físico, ou um pré-tratamento químico e um pré-tratamento físico. [37] O método do parágrafo 36, em que o pré-tratamento com o um ou mais (por exemplo, vários) polipeptídeos GH61 é realizado antes, durante ou após o pré-tratamento químico, o pré-tratamento físico, ou o pré-tratamento químico e o pré-tratamento físico. [38] O método do parágrafo 36 ou 37, em que o tratamento adicional do material celulósico com um pré-tratamento químico, um pré-tratamento físico, ou um pré-tratamento químico e um pré-tratamento físico é realizado antes da sacarificação. [39] O método de quaisquer dos parágrafos 35-38, em que o pré-tratamento com o um ou mais (por exemplo, vários) polipeptídeos GH61 é realizado antes da sacarificação. [40] O método de quaisquer dos parágrafos 35-39, em que o um ou mais (por exemplo, vários) polipeptídeos GH61 são inativados após pré-tratamento do material celulósico. [41] O método de quaisquer dos parágrafos 35-40, em que a composição enzimática compreende uma ou mais (por exemplo, várias) enzimas selecionadas do grupo que consiste em uma celulase, um polipeptídeo GH61 com atividade celulolítica intensificada, uma hemicelulase, uma esterase, uma expansina, uma laccase, uma enzima ligninolítica, uma pectinase, uma peroxidase, uma protease e uma swolenina. [42] O método do parágrafo 41, em que a celulase é uma ou mais (por exemplo, várias) enzimas selecionadas do grupo que consiste em uma endoglucanase, uma celobioidrolase, e uma beta-glucosidase. [43] O método do parágrafo 41, em que a hemicelulase é uma ou mais (por exemplo, várias) enzimas selecionadas do grupo que consiste em uma xilanase, uma acetixilano esterase, uma feruloil esterase, uma arabinofuranosidase, uma xilosidase e uma glucuronidase. [44] O método de quaisquer dos parágrafos 35-43, em que a fermentação do material celulósico produz um produto de fermentação. [45] O método do parágrafo 44, compreendendo adicionalmente recuperar o produto de fermentação a partir da fermentação. [46] O método do parágrafo 44 ou 45, em que o produto de fermentação é um álcool, um alcano, um cicloalcano, um alceno, um aminoácido, um gás, isopreno, uma cetona, um ácido orgânico, ou policetídeo. [000304] The present invention is further described by the following numbered paragraphs: [1] A method of degrading a cellulosic material, comprising: (a) pretreating the cellulosic material with a composition comprising one or more (for example, several) GH61 polypeptides; and (b) saccharifying the cellulosic material pretreated with GH61 polypeptide with an enzymatic composition. [2] The method of paragraph 1, which further comprises treating the cellulosic material with a chemical pretreatment, a physical pretreatment, or a chemical pretreatment and a physical pretreatment. [3] The method of paragraph 2, where pretreatment with one or more (for example, several) GH61 polypeptides is performed before, during or after chemical pretreatment, physical pretreatment, or pretreatment -chemical treatment and physical pre-treatment. [4] The method of paragraph 2 or 3, in which the additional treatment of the cellulosic material with a chemical pretreatment, a physical pretreatment, or a chemical pretreatment and a physical pretreatment is carried out before saccharification. [5] The method of any of paragraphs 1-4, in which pretreatment with the one or more (for example, several) GH61 polypeptides is carried out before saccharification. [6] The method of any of paragraphs 1-5, wherein the one or more (for example, several) GH61 polypeptides are inactivated after pretreatment of the cellulosic material. [7] The method of any of paragraphs 1-6, wherein the enzyme composition comprises one or more (for example, several) enzymes selected from the group consisting of a cellulase, a GH61 polypeptide with enhanced cellulolytic activity, a hemicellulase, a esterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease and a swolenin. [8] The method of paragraph 7, in which cellulase is one or more (for example, several) enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase. [9] The method of paragraph 7, in which hemicellulase is one or more (for example, several) enzymes selected from the group consisting of a xylanase, an acetixylan esterase, a feruloyl esterase, an arabinofuranosidase, a xylosidase and a glucuronidase. [10] The method of any of paragraphs 1-9, further comprising recovering the degraded cellulosic material. [11] The method of paragraph 10, in which the degraded cellulosic material is sugar. [12] The method of paragraph 11, in which sugar is selected from the group consisting of glucose, xylose, mannose, galactose and arabinose. [13] A method of producing a fermentation product, comprising: (a) pretreating a cellulosic material with a composition comprising one or more (for example, several) GH61 polypeptides; (b) saccharifying the cellulosic material pretreated with GH61 with an enzymatic composition; (c) fermenting the saccharified cellulosic material with one or more (for example, several) fermenting microorganisms to synthesize the fermentation product; and (d) recovering the fermentation product from fermentation. [14] The method of paragraph 13, which further comprises treating the cellulosic material with a chemical pretreatment, a physical pretreatment, or a chemical pretreatment and a physical pretreatment. [15] The method of paragraph 14, in which pretreatment with one or more (for example, several) GH61 polypeptides is performed before, during or after chemical pretreatment, physical pretreatment, or pretreatment -chemical treatment and physical pre-treatment. [16] The method of paragraph 14 or 15, in which additional treatment of the cellulosic material with a chemical pretreatment, a physical pretreatment, or a chemical pretreatment and a physical pretreatment is carried out before saccharification. [17] The method of any of paragraphs 13-16, in which pretreatment with the one or more (for example, several) GH61 polypeptides is performed before saccharification. [18] The method of any of paragraphs 13-17, wherein the one or more (for example, several) GH61 polypeptides are inactivated after pretreatment of the cellulosic material. [19] The method of any of paragraphs 13-18, wherein the enzyme composition comprises one or more (for example, several) enzymes selected from the group consisting of a cellulase, a GH61 polypeptide with enhanced cellulolytic activity, a hemicellulase, a esterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease and a swolenin. [20] The method of paragraph 19, in which cellulase is one or more (for example, several) enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase. [21] The method of paragraph 19, in which hemicellulase is one or more (for example, several) enzymes selected from the group consisting of a xylanase, an acetixylan esterase, a feruloyl esterase, an arabinofuranosidase, a xylosidase and a glucuronidase. [22] The method of any of paragraphs 13-21, in which steps (c) and (d) are carried out simultaneously in simultaneous saccharification and fermentation. [23] The method of any of paragraphs 13-22, 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 polyketide. [24] A method of producing a fermentation product, comprising: (a) saccharifying a cellulosic material with an enzymatic composition, wherein the cellulosic material is pretreated with a composition comprising one or more (for example, several) GH61 polypeptides ; (b) fermenting the saccharified cellulosic material with one or more (for example, several) fermenting microorganisms to synthesize the fermentation product; and (c) recovering the fermentation product from the fermentation. [25] The method of paragraph 24, which further comprises treating the cellulosic material with a chemical pretreatment, a physical pretreatment, or a chemical pretreatment and a physical pretreatment. [26] The method of paragraph 25, in which pretreatment with the one or more (for example, several) GH61 polypeptides is performed before, during or after chemical pretreatment, physical pretreatment, or pretreatment -chemical treatment and physical pre-treatment. [27] The method of paragraph 25 or 26, in which additional treatment of cellulosic material with chemical pretreatment, physical pretreatment, or chemical pretreatment and physical pretreatment is carried out before saccharification. [28] The method of any of paragraphs 24-27, in which pretreatment with the one or more (for example, several) GH61 polypeptides is performed before saccharification. [29] The method of any of paragraphs 24-28, in which the one or more (for example, several) GH61 polypeptides are inactivated after pretreatment of the cellulosic material. [30] The method of any of paragraphs 24-29, wherein the enzyme composition comprises one or more (for example, several) enzymes selected from the group consisting of a cellulase, a GH61 polypeptide with enhanced cellulolytic activity, a hemicellulase, a esterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease and a swolenin. [31] The method of paragraph 30, in which cellulase is one or more (for example, several) enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase. [32] The method of paragraph 30, wherein hemicellulase is one or more (for example, several) enzymes selected from the group consisting of a xylanase, an acetixylan esterase, a feruloyl esterase, an arabinofuranosidase, a xylosidase and a glucuronidase. [33] The method of any of paragraphs 24-32, in which steps (a) and (b) are performed simultaneously in simultaneous saccharification and fermentation. [34] The method of any of paragraphs 24-33, 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 polyketide. [35] 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 pretreated with a composition comprising one or more (for example, several) GH61 polypeptides, and is saccharified with an enzymatic composition. [36] The method of paragraph 35, which further comprises treating the cellulosic material with a chemical pretreatment, a physical pretreatment, or a chemical pretreatment and a physical pretreatment. [37] The method of paragraph 36, where pretreatment with one or more (for example, several) GH61 polypeptides is performed before, during or after chemical pretreatment, physical pretreatment, or pretreatment -chemical treatment and physical pre-treatment. [38] The method of paragraph 36 or 37, in which additional treatment of cellulosic material with chemical pretreatment, physical pretreatment, or chemical pretreatment and physical pretreatment is carried out before saccharification. [39] The method of any of paragraphs 35-38, in which pretreatment with one or more (for example, several) GH61 polypeptides is performed before saccharification. [40] The method of any of paragraphs 35-39, wherein the one or more (for example, several) GH61 polypeptides are inactivated after pretreatment of the cellulosic material. [41] The method of any of paragraphs 35-40, wherein the enzyme composition comprises one or more (for example, several) enzymes selected from the group consisting of a cellulase, a GH61 polypeptide with enhanced cellulolytic activity, a hemicellulase, a esterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease and a swolenin. [42] The method of paragraph 41, in which cellulase is one or more (for example, several) enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase. [43] The method of paragraph 41, in which hemicellulase is one or more (for example, several) enzymes selected from the group consisting of a xylanase, an acetixylan esterase, a feruloyl esterase, an arabinofuranosidase, a xylosidase and a glucuronidase. [44] The method of any of paragraphs 35-43, in which the fermentation of the cellulosic material produces a fermentation product. [45] The method of paragraph 44, further comprising recovering the fermentation product from fermentation. [46] The method of paragraph 44 or 45, 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 polyketide.
[0305] [000305] The invention described and claimed herein should not be limited in scope by the specific aspects disclosed herein, since these aspects are intended as illustrations of various aspects of the invention. Any equivalent aspect is intended to be within the scope of this invention. In fact, various modifications of the invention, in addition to those shown and described here, will become apparent to those skilled in the art from the preceding description. Such modifications are also intended to be within the scope of the appended claims. In the event of a conflict, the present disclosure, including definitions, will prevail.

权利要求:
Claims (7)
[0001]
Method for degrading a cellulosic material, characterized by the fact that it comprises: (a) pretreating the cellulosic material with a composition comprising one or more polypeptides glycoside hydrolase of the family 61 (GH61); and (b) saccharifying the cellulosic material pretreated with GH61 polypeptide with an enzymatic composition.
[0002]
Method according to claim 1, characterized in that it further comprises treating the cellulosic material with a chemical pretreatment, a physical pretreatment, or a chemical pretreatment and a physical pretreatment.
[0003]
Method according to claim 2, characterized by the fact that the pretreatment with one or more polypeptides glycoside hydrolase of the family 61 (GH61) is carried out before, during or after the chemical pretreatment, the physical pretreatment, or chemical pretreatment and physical pretreatment.
[0004]
Method according to any one of claims 1 to 3, characterized by the fact that the one or more polypeptides glycoside hydrolase of the family 61 (GH61) are inactivated after the pretreatment of the cellulosic material.
[0005]
Method according to any one of claims 1 to 4, characterized in that the enzyme composition comprises one or more enzymes selected from the group consisting of a cellulase, a glycoside hydrolase polypeptide of the family 61 (GH61) with enhanced cellulolytic activity, an hemicellulase, an esterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease and a swolenin.
[0006]
Method for producing a fermentation product, characterized by the fact that it comprises: (a) pretreating a cellulosic material with a composition comprising one or more polypeptides glycoside hydrolase of the family 61 (GH61); (b) saccharifying the cellulosic material pretreated with glycoside hydrolase of the family 61 (GH61) with an enzymatic composition; (c) fermenting the saccharified cellulosic material with one or more fermenting microorganisms to synthesize the fermentation product; and (d) recovering the fermentation product from the fermentation.
[0007]
Method according to claim 6, characterized in that it further comprises treating the material with a chemical pretreatment, a physical pretreatment, or a chemical pretreatment and a physical pretreatment.

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法律状态:
2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according art. 34 industrial property law|

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2019-06-04| B06T| Formal requirements before examination|

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2019-11-26| B07A| Technical examination (opinion): publication of technical examination (opinion)|

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2020-07-21| B09A| Decision: intention to grant|

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2020-09-08| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 02/11/2011, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US40946910P| true| 2010-11-02|2010-11-02|

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US61/409469|2010-11-02|

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PCT/US2011/058995|WO2012061517A1|2010-11-02|2011-11-02|Methods of pretreating cellulosic material with a gh61 polypeptide|

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