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WO2018085370A1 - Procédés de réduction de la production de primevérose pendant la saccharification enzymatique de matière lignocellulosique - Google Patents

Procédés de réduction de la production de primevérose pendant la saccharification enzymatique de matière lignocellulosique Download PDF

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WO2018085370A1
WO2018085370A1 PCT/US2017/059498 US2017059498W WO2018085370A1 WO 2018085370 A1 WO2018085370 A1 WO 2018085370A1 US 2017059498 W US2017059498 W US 2017059498W WO 2018085370 A1 WO2018085370 A1 WO 2018085370A1
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beta
xylosidase
lignocellulosic material
polypeptide
enzyme composition
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Eric Abbate
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Novozymes AS
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Novozymes AS
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides

Definitions

  • the present invention relates to processes for increasing the yield of fermentable sugars during saccharification of a lignocellulosic material.
  • Biomass feedstocks for the production of ethanol and other chemicals are complex in composition, comprising cellulose, hemicellulose, lignin, and other constituents.
  • Lignocellulose the world's largest renewable biomass resource, is composed mainly of lignin, cellulose, and hemicellulose.
  • Cellulose is a polymer of glucose linked by beta-1 ,4- bonds.
  • Many microorganisms produce enzymes that hydrolyze beta-linked glucans. These enzymes include endoglucanases, cellobiohydrolases, and beta-glucosidases. Endoglucanases digest the cellulose polymer at random locations, opening it to attack by cellobiohydrolases.
  • Cellobiohydrolases sequentially release molecules of cellobiose from the ends of the cellulose polymer.
  • Cellobiose is a water-soluble beta-1 ,4-linked dimer of glucose. Beta-glucosidases hydrolyze cellobiose to glucose.
  • Xylans are polysaccharides formed from 1 ,4- ⁇ - glycoside-linked D-xylopyranoses.
  • Xylanases e.g., endo-1 ,4-beta-xylanase, EC 3.2.1.8
  • Beta-xylosidases catalyze the exo-hydrolysis of short beta (1 ⁇ 4)-xylooligosaccharides to remove successive D-xylose residues from non-reducing termini
  • Beta-xylosidases of glycoside hydrolase family 3 catalyze hydrolysis of (1 ⁇ 4)-beta- D-xylans, e.g., xylobiose, to remove successive D-xylose residues from the non-reducing termini by a retaining mechanism of action. Retaining beta-xylosidases cleave xylobiose via a 2-step mechanism where a glycosyl-enzyme intermediate forms in the first step. Cleavage of this intermediate with water results in hydrolysis of xylobiose to two xylose molecules.
  • Beta-xylosidases of glycoside hydrolase family 43 catalyze hydrolysis of (1 ⁇ 4)-beta- D-xylans, e.g., xylobiose, to remove successive D-xylose residues from the non-reducing termini by an inverting mechanism of action.
  • Inverting beta-xylosidases (GH43 family) cleave via a 1-step mechanism resulting in the hydrolysis of xylobiose to two xylose molecules.
  • the glucose and xylose can then be fermented into biofuel such as ethanol using suitable fermenting microorganisms.
  • biofuel such as ethanol
  • suitable fermenting microorganisms suitable fermenting microorganisms.
  • primeverose 6-O-beta-D-xylopyranosyl-beta-D- glucopyranose
  • the present invention relates to processes for increasing the yield of fermentable sugars during saccharification of a lignocellulosic or hemicellulosic material.
  • the present invention relates to processes for reducing production of primeverose during saccharification of a lignocellulosic material, the process comprising: saccharifying the lignocellulosic material with an enzyme composition comprising a GH43 beta-xylosidase, wherein the enzyme composition comprising the GH43 beta-xylosidase increases the amount of fermentable sugars from saccharification of the lignocellulosic material by reducing the amount of primeverose produced compared to an enzyme composition comprising a GH3 beta-xylosidase in place of the GH43 beta-xylosidase.
  • the present invention also relates to processes for saccharifying a lignocellulosic material, comprising: treating the lignocellulosic material with an enzyme composition comprising a GH43 beta-xylosidase, wherein the enzyme composition comprising the GH43 beta-xylosidase increases the amount of fermentable sugars from saccharification of the lignocellulosic material by reducing the amount of primeverose produced compared to an enzyme composition comprising a GH3 beta-xylosidase in place of the GH43 beta- xylosidase.
  • the present invention also relates to processes for producing a fermentation product, the process comprising: (a) saccharifying a lignocellulosic material with an enzyme composition comprising a GH43 beta-xylosidase, wherein the GH43 beta-xylosidase increases the amount of fermentable sugars from saccharification of the lignocellulosic material by reducing the amount of primeverose produced compared to an enzyme composition comprising a GH3 beta-xylosidase in place of the GH43 beta-xylosidase; (b) fermenting the saccharified lignocellulosic material with one or more fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.
  • the present invention also relates to processes for fermenting a lignocellulosic material, the process comprising: fermenting the lignocellulosic material with one or more fermenting microorganisms, wherein the lignocellulosic material is saccharified with an enzyme composition comprising a GH43 beta-xylosidase, wherein the enzyme composition comprising the GH43 beta-xylosidase increases the amount of fermentable sugars from saccharification of the lignocellulosic material by reducing the amount of primeverose produced compared to an enzyme composition comprising a GH3 beta-xylosidase in place of the GH43 beta-xylosidase.
  • Figure 1 shows a restriction map of plasmid pl_F02.
  • Figure 2 shows a restriction map of plasmid pEbZn58.
  • Figure 3 shows a chromatogram from Dionex ion chromatography analysis using a PA-10 column of reaction mixtures containing different concentrations of Geobacillus stearothermophilus GH43 beta-xylosidase incubated with xylo-oligomers and glucose.
  • the chromatogram depicts the signal in nanocoulombs (nC) and retention time in minutes.
  • Figure 4 shows a chromatogram from Dionex ion chromatography analysis using a
  • the axes are labelled as in Figure 3.
  • Figure 5 shows a chromatogram from Dionex ion chromatography analysis with a PA-10 column of reaction mixtures containing different concentrations of a combination of a Talaromyces emersonii GH3 beta-xylosidase and a Geobacillus stearothermophilus GH43 beta-xylosidase incubated with xylo-oligomers and glucose.
  • concentration shown is the total concentration of beta-xylosidase in the assay which is a 50:50 mix of T. emersonii GH3 beta-xylosidase and G. stearothermophilus GH43 beta-xylosidase.
  • the axes are labelled as in Figure 3.
  • 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-napthyl acetate, and p-nitrophenyl acetate.
  • Acetylxylan esterase activity can be determined using 0.5 mM p-nitrophenylacetate as substrate in 50 mM sodium acetate pH 5.0 containing 0.01 % TWEENTM 20 (polyoxyethylene sorbitan monolaurate).
  • One unit of acetylxylan esterase is defined as the amount of enzyme capable of releasing 1 ⁇ of p-nitrophenolate anion per minute at pH 5, 25°C.
  • allelic variant means any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences.
  • An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.
  • Alpha-L-arabinofuranosidase means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55) that catalyzes the hydrolysis of terminal non-reducing alpha-L-arabinofuranoside residues in alpha-L- arabinosides.
  • the enzyme acts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1 ,3)- and/or (1 ,5)-linkages, arabinoxylans, and arabinogalactans.
  • Alpha-L- arabinofuranosidase is also known as arabinosidase, alpha-arabinosidase, alpha-L- arabinosidase, alpha-arabinofuranosidase, polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranoside hydrolase, L-arabinosidase, or alpha-L-arabinanase.
  • Alpha-L- arabinofuranosidase activity can be determined using 5 mg of medium viscosity wheat arabinoxylan (Megazyme International Ireland, Ltd., Bray, Co.
  • Alpha-glucuronidase means an alpha-D- glucosiduronate glucuronohydrolase (EC 3.2.1.139) that catalyzes the hydrolysis of an alpha-D-glucuronoside to D-glucuronate and an alcohol.
  • Alpha-glucuronidase activity can be determined according to de Vries, 1998, J. Bacteriol. 180: 243-249.
  • One unit of alpha- glucuronidase equals the amount of enzyme capable of releasing 1 ⁇ of glucuronic or 4- O-methylglucuronic acid per minute at pH 5, 40°C.
  • Auxiliary Activity 9 polypeptide means a polypeptide classified as a lytic polysaccharide monooxygenase (Quinlan et ai, 201 1 , Proc. Natl. Acad. Sci. USA 108: 15079-15084; Phillips et ai, 201 1 , ACS Chem. Biol. 6: 1399-1406; Li et ai, 2012, Structure 20: 1051-1061). AA9 polypeptides were formerly classified into the glycoside hydrolase Family 61 (GH61) according to Henrissat, 1991 , Biochem. J. 280: 309-316, and Henrissat and Bairoch, 1996, Biochem. J. 316: 695-696.
  • GH61 glycoside hydrolase Family 61
  • AA9 polypeptides enhance the hydrolysis of a cellulosic material by an enzyme having cellulolytic activity.
  • Cellulolytic enhancing activity can be determined by measuring the increase in reducing sugars or the increase of the total of cellobiose and glucose from the hydrolysis of a cellulosic material by cellulolytic enzyme under the following conditions: 1-50 mg of total protein/g of cellulose in pretreated corn stover (PCS), wherein total protein is comprised of 50-99.5% w/w cellulolytic enzyme protein and 0.5-50% w/w protein of an AA9 polypeptide for 1-7 days at a suitable temperature, such as 40°C-80°C, e.g., 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, or 80°C and a suitable pH, such as 4-9, e.g., 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0,
  • AA9 polypeptide enhancing activity can be determined using a mixture of
  • beta-glucosidase as the source of the cellulolytic activity, wherein the beta-glucosidase is present at a weight of at least 2-5% protein of the cellulase protein loading.
  • the beta-glucosidase is an Aspergillus oryzae beta-glucosidase (e.g., recombinantly produced in Aspergillus oryzae according to WO 02/095014).
  • the beta-glucosidase is an Aspergillus fumigatus beta-glucosidase (e.g., recombinantly produced in Aspergillus oryzae as described in WO 02/095014).
  • AA9 polypeptide enhancing activity can also be determined by incubating an AA9 polypeptide with 0.5% phosphoric acid swollen cellulose (PASC), 100 mM sodium acetate pH 5, 1 mM MnS0 4 , 0.1 % gallic acid, 0.025 mg/ml of Aspergillus fumigatus beta- glucosidase, and 0.01 % TRITON® X-100 (4-(1 , 1 ,3,3-tetramethylbutyl)phenyl-polyethylene glycol) for 24-96 hours at 40°C followed by determination of the glucose released from the PASC.
  • PASC phosphoric acid swollen cellulose
  • AA9 polypeptide enhancing activity can also be determined according to WO 2013/028928 for high temperature compositions.
  • AA9 polypeptides enhance the hydrolysis of a cellulosic material catalyzed by enzyme having cellulolytic activity by reducing the amount of cellulolytic enzyme required to reach the same degree of hydrolysis preferably at least 1.01-fold, e.g., at least 1.05-fold, at least 1.10-fold, at least 1.25-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4- fold, at least 5-fold, at least 10-fold, or at least 20-fold.
  • the AA9 polypeptide can be used in the presence of a soluble activating divalent metal cation according to WO 2008/151043 or WO 2012/122518, e.g., manganese or copper.
  • the AA9 polypeptide can also be used in the presence of a dioxy compound, a bicylic compound, a heterocyclic compound, a nitrogen-containing compound, a quinone compound, a sulfur-containing compound, or a liquor obtained from a pretreated lignocellulosic material such as pretreated corn stover (WO 2012/021394, WO 2012/021395, WO 2012/021396, WO 2012/021399, WO 2012/021400, WO 2012/021401 , WO 2012/021408, and WO 2012/021410).
  • a pretreated lignocellulosic material such as pretreated corn stover
  • Beta-glucosidase means a beta-D-glucoside glucohydrolase (E.C. 3.2.1.21) that catalyzes the hydrolysis of terminal non-reducing beta-D- glucose residues with the release of beta-D-glucose. Beta-glucosidase activity can be determined using p-nitrophenyl-beta-D-glucopyranoside as substrate according to the procedure of Venturi et al., 2002, J. Basic Microbiol. 42: 55-66.
  • beta-glucosidase is defined as 1.0 ⁇ of p-nitrophenolate anion produced per minute at 25°C, pH 4.8 from 1 mM p-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mM sodium citrate containing 0.01 % TWEEN® 20.
  • Beta-xylosidase means a beta-D-xyloside xylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of short beta (1 ⁇ 4)- xylooligosaccharides to remove successive D-xylose residues from non-reducing termini.
  • Beta-xylosidase activity can be determined using 1 mM p-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodium citrate containing 0.01 % TWEEN® 20 at pH 5, 40°C.
  • beta-xylosidase is defined as 1.0 ⁇ of p-nitrophenolate anion produced per minute at 40°C, pH 5 from 1 mM p-nitrophenyl-beta-D-xyloside in 100 mM sodium citrate containing 0.01 % TWEEN® 20.
  • cDNA means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA.
  • the initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.
  • Catalase means a hydrogen-peroxide:hydrogen-peroxide oxidoreductase (E.C. 1.11.1.6 or E.C. 1.11.1.21) that catalyzes the conversion of two hydrogen peroxides to oxygen and two waters.
  • Catalase activity can be determined by monitoring the degradation of hydrogen peroxide at 240 nm based on the following reaction:
  • the reaction is conducted in 50 mM phosphate pH 7 at 25°C with 10.3 mM substrate (H2O2). Absorbance is monitored spectrophotometrically within 16-24 seconds, which should correspond to an absorbance reduction from 0.45 to 0.4.
  • One catalase activity unit can be expressed as one ⁇ of H2O2 degraded per minute at pH 7.0 and 25°C.
  • Cellobiohydrolase means a 1 ,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91 and E.C. 3.2.1.176) that catalyzes the hydrolysis of 1 ,4- beta-D-glucosidic linkages in cellulose, cellooligosaccharides, or any beta-1 ,4-linked glucose containing polymer, releasing cellobiose from the reducing end (cellobiohydrolase I) or non- reducing end (cellobiohydrolase II) of the chain (Teeri, 1997, Trends in Biotechnology 15: 160-167; Teeri et al., 1998, Biochem. Soc. Trans.
  • Cellobiohydrolase activity can be determined according to the procedures described by Lever et al., 1972, Anal. Biochem. 47: 273-279; van Tilbeurgh et ai, 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.
  • Cellulolytic enzyme or cellulase means one or more (e.g., several) enzymes that hydrolyze a cellulosic material. Such enzymes include endoglucanase(s), cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof.
  • the two basic approaches for measuring cellulolytic enzyme activity include: (1) measuring the total cellulolytic enzyme activity, and (2) measuring the individual cellulolytic enzyme activities (endoglucanases, cellobiohydrolases, and beta-glucosidases) as reviewed in Zhang et al., 2006, Biotechnology Advances 24: 452-481.
  • Total cellulolytic enzyme activity can be measured using insoluble substrates, including Whatman N°1 filter paper, microcrystalline cellulose, bacterial cellulose, algal cellulose, cotton, pretreated lignocellulose, etc.
  • the most common total cellulolytic activity assay is the filter paper assay using Whatman N°1 filter paper as the substrate.
  • the assay was established by the International Union of Pure and Applied Chemistry (lUPAC) (Ghose, 1987, Pure Appl. Chem. 59: 257-68).
  • Cellulolytic enzyme activity can be determined by measuring the increase in production/release of sugars during hydrolysis of a cellulosic material by cellulolytic enzyme(s) under the following conditions: 1-50 mg of cellulolytic enzyme protein/g of cellulose in pretreated corn stover (PCS) (or other pretreated cellulosic material) for 3-7 days at a suitable temperature such as 40°C-80°C, e.g., 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, or 80°C, and a suitable pH, such as 4-9, e.g., 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0, compared to a control hydrolysis without addition of cellulolytic enzyme protein.
  • PCS pretreated corn stover
  • Typical conditions are 1 ml reactions, washed or unwashed PCS, 5% insoluble solids (dry weight), 50 mM sodium acetate pH 5, 1 mM MnS0 4 , 50°C, 55°C, or 60°C, 72 hours, sugar analysis by AMINEX® HPX-87H column chromatography (Bio-Rad Laboratories, Inc.).
  • Coding sequence means a polynucleotide, which directly specifies the amino acid sequence of a polypeptide.
  • the boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon, such as ATG, GTG, or TTG, and ends with a stop codon, such as TAA, TAG, or TGA.
  • the coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.
  • control sequences means nucleic acid sequences necessary for expression of a polynucleotide encoding a polypeptide of interest.
  • Each control sequence may be native (i.e., from the same gene) or heterologous or foreign (i.e., from a different gene) to the polynucleotide encoding the polypeptide or native or foreign to each other.
  • control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator.
  • the control sequences include a promoter, and transcriptional and translational stop signals.
  • the control sequences may be provided with linkers for introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a polypeptide.
  • the saturation level of oxygen is determined at the standard partial pressure (0.21 atmosphere) of oxygen.
  • the saturation level at the standard partial pressure of oxygen is dependent on the temperature and solute concentrations. In an embodiment where the temperature during hydrolysis is 50°C, the saturation level would typically be in the range of 5-5.5 mg oxygen per kg slurry, depending on the solute concentrations.
  • a concentration of dissolved oxygen of 0.5 to 10% of the saturation level at 50°C corresponds to an amount of dissolved oxygen in a range from 0.025 ppm (0.5 x 5/100) to 0.55 ppm (10 x 5.5/100), such as, e.g., 0.05 to 0.165 ppm
  • a concentration of dissolved oxygen of 10-70% of the saturation level at 50°C corresponds to an amount of dissolved oxygen in a range from 0.50 ppm (10 x 5/100) to 3.85 ppm (70 x 5.5/100), such as, e.g., 1 to 2 ppm.
  • oxygen is added in an amount in the range of 0.5 to 5 ppm, such as 0.5 to 4.5 ppm, 0.5 to 4 ppm, 0.5 to 3.5 ppm, 0.5 to 3 ppm, 0.5 to 2.5 ppm, or 0.5 to 2 ppm.
  • Endoglucanase means a 4-(1 ,3; 1 ,4)-beta-D-glucan 4- glucanohydrolase (E.C. 3.2.1.4) that catalyzes endohydrolysis of 1 ,4-beta-D-glycosidic linkages in cellulose, cellulose derivatives (such as carboxymethyl cellulose and hydroxyethyl cellulose), lichenin, beta-1 ,4 bonds in mixed beta-1 ,3-1 ,4 glucans such as cereal beta-D-glucans or xyloglucans, and other plant material containing cellulosic components.
  • Endoglucanase activity can be determined by measuring reduction in substrate viscosity or increase in reducing ends determined by a reducing sugar assay (Zhang et al., 2006, Biotechnology Advances 24: 452-481). Endoglucanase activity can also be determined using carboxymethyl cellulose (CMC) as substrate according to the procedure of Ghose, 1987, Pure andAppl. Chem. 59: 257-268, at pH 5, 40°C.
  • CMC carboxymethyl cellulose
  • expression includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
  • Expression vector means a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression.
  • Feruloyl esterase means a 4-hydroxy-3- methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) that catalyzes the hydrolysis of 4-hydroxy- 3-methoxycinnamoyl (feruloyl) groups from esterified sugar, which is usually arabinose in natural biomass substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate).
  • Feruloyl esterase (FAE) is also known as ferulic acid esterase, hydroxycinnamoyl esterase, FAE-III, cinnamoyl ester hydrolase, FAEA, cinnAE, FAE-I, or FAE-II.
  • Feruloyl esterase activity can be determined using 0.5 mM p-nitrophenylferulate as substrate in 50 mM sodium acetate pH 5.0.
  • One unit of feruloyl esterase equals the amount of enzyme capable of releasing 1 ⁇ of p-nitrophenolate anion per minute at pH 5, 25°C.
  • fragment means a polypeptide having one or more (e.g., several) amino acids absent from the amino and/or carboxyl terminus of a polypeptide, wherein the fragment has enzyme activity. In one aspect, a fragment contains at least 85%, at least 90%, or at least 95% of the number of amino acids of the polypeptide.
  • Hemicellulolytic enzyme or hemicellulase The term "hemicellulolytic enzyme" or
  • hemicellulase means one or more (e.g., several) enzymes that hydrolyze hemicellulose of a lignocellulosic material. See, for example, Shallom and Shoham, 2003, Current Opinion In Microbiology 6(3): 219-228). Hemicellulases are key components in the degradation of plant biomass.
  • 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 xylanase, and a xylosidase.
  • hemicelluloses are a heterogeneous group of branched and linear polysaccharides that are bound via hydrogen bonds to the cellulose microfibrils in the plant cell wall, crosslinking them into a robust network. Hemicelluloses are also covalently attached to lignin, forming together with cellulose a highly complex structure. The variable structure and organization of hemicelluloses require the concerted action of many enzymes for its complete degradation.
  • the catalytic modules of hemicellulases are either glycoside hydrolases (GHs) that hydrolyze glycosidic bonds, or carbohydrate esterases (CEs), which hydrolyze ester linkages of acetate or ferulic acid side groups.
  • GHs glycoside hydrolases
  • CEs carbohydrate esterases
  • catalytic modules based on homology of their primary sequence, can be assigned into GH and CE families. Some families, with an overall similar fold, can be further grouped into clans, marked alphabetically (e.g., GH-A). A most informative and updated classification of these and other carbohydrate active enzymes is available in the Carbohydrate-Active Enzymes (CAZy) database. Hemicellulolytic enzyme activities can be measured according to Ghose and Bisaria, 1987, Pure & Appl. Chem.
  • 59: 1739-1752 at a suitable temperature such as 40°C- 80°C, e.g., 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, or 80°C, and a suitable pH such as 4-9, e.g., 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0.
  • a suitable temperature such as 40°C- 80°C, e.g., 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, or 80°C
  • a suitable pH such as 4-9, e.g., 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0.
  • host cell means any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of interest.
  • host cell encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.
  • Isolated means a substance in a form or environment that does not occur in nature.
  • 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 of the naturally occurring constituents with which it is associated in nature; (3) any substance modified by the hand of man relative to that substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., recombinant production in a host cell; multiple copies of a gene encoding the substance; and use of a stronger promoter than the promoter naturally associated with the gene encoding the substance).
  • Laccase activity can be determined by the oxidation of syringaldazine (4,4 ' -
  • the reaction is conducted in 23 mM MES pH 5.5 at 30°C with 19 ⁇ substrate (syringaldazine) and 1 g/L polyethylene glycol (PEG) 6000.
  • substrate syringaldazine
  • PEG polyethylene glycol
  • the sample is placed in a spectrophotometer and the change in absorbance is measured at 530 nm every 15 seconds up to 90 seconds.
  • One laccase unit is the amount of enzyme that catalyzes the conversion of 1 ⁇ syringaldazine per minute under the specified analytical conditions.
  • Lignocellulosic material means any material containing cellulose, hemicellulose, and lignin.
  • the predominant polysaccharide in the primary cell wall of biomass is cellulose, the second most abundant is hemicellulose, and the third is pectin.
  • the secondary cell wall, produced after the cell has stopped growing, also contains polysaccharides and is strengthened by polymeric lignin covalently cross-linked to hemicellulose.
  • Cellulose is a homopolymer of anhydrocellobiose and thus a linear beta-(1 -4)-D- glucan, while hemicelluloses include a variety of compounds, such as xylans, xyloglucans, arabinoxylans, and mannans in complex branched structures with a spectrum of substituents. Although generally polymorphous, cellulose is found in plant tissue primarily as an insoluble crystalline matrix of parallel glucan chains.
  • Hemicelluloses usually hydrogen bond to cellulose, as well as to other hemicelluloses, which help stabilize the cell wall matrix.
  • Lignocellulose is generally found, for example, in the stems, leaves, hulls, husks, and cobs of plants or leaves, branches, and wood of trees.
  • the lignocellulosic material can be, but is not limited to, agricultural residue, herbaceous material (including energy crops), municipal solid waste, pulp and paper mill residue, waste paper, and wood (including forestry residue) (see, for example, Wiselogel et al., 1995, in Handbook on Bioethanol (Charles E. Wyman, editor), pp.
  • Hemicelluloses include xylan, glucuronoxylan, arabinoxylan, glucomannan, and xyloglucan. These polysaccharides contain many different sugar monomers. Sugar monomers in hemicellulose can include xylose, mannose, galactose, rhamnose, and arabinose. Hemicelluloses contain most of the D-pentose sugars. Xylose is in most cases the sugar monomer present in the largest amount, although in softwoods mannose can be the most abundant sugar. Xylan contains a backbone of beta-(1-4)-linked xylose residues.
  • Xylans of terrestrial plants are heteropolymers possessing a beta-(1-4)-D-xylopyranose backbone, 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-type polysaccharides can be divided into homoxylans and heteroxylans, which include glucuronoxylans, (arabino)glucuronoxylans, (glucurono)arabinoxylans, arabinoxylans, and complex heteroxylans. See, for example, Ebringerova et al., 2005, Adv. Polym. Sci. 186: 1-67. Hemicellulosic material is also known herein as "xylan-containing material".
  • the lignocellulosic material is any biomass material.
  • the lignocellulosic material is agricultural residue, herbaceous material (including energy crops), municipal solid waste, pulp and paper mill residue, waste paper, or wood (including forestry residue).
  • the lignocellulosic material is arundo, bagasse, bamboo, corn cob, corn fiber, corn stover, miscanthus, rice straw, sugar cane straw, switchgrass, or wheat straw.
  • the lignocellulosic material is aspen, eucalyptus, fir, pine, poplar, spruce, or willow.
  • the lignocellulosic material is an aquatic biomass.
  • aquatic biomass means biomass produced in an aquatic environment by a photosynthesis process.
  • the aquatic biomass can be algae, emergent plants, floating-leaf plants, or submerged plants.
  • the lignocellulosic material may be used as is or may be subjected to pretreatment, using conventional methods known in the art, as described herein. In a preferred aspect, the lignocellulosic material is pretreated.
  • Mature polypeptide means a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.
  • the mature polypeptide is amino acids 21 to 541 of SEQ ID NO: 30 based on the SignalP 3.0 (Bendtsen et al., 2004, J. Mol. Biol. 340: 783-795) that predicts amino acids 1 to 20 of SEQ ID NO: 30 are a signal peptide.
  • the mature polypeptide is amino acids 27 to 375 of SEQ ID NO: 31 based on the SignalP 3.0 that predicts amino acids 1 to 26 of SEQ ID NO: 31 are a signal peptide.
  • the mature polypeptide is amino acids 38 to 1347 of SEQ ID NO: 32 based on the SignalP 3.0 that predicts amino acids 1 to 37 of SEQ ID NO: 32 are a signal peptide.
  • the mature polypeptide is amino acids 19 to 340 of SEQ ID NO: 33 based on the SignalP 3.0 that predicts amino acids 1 to 18 of SEQ ID NO: 33 are a signal peptide.
  • the mature polypeptide is amino acids 21 to 348 of SEQ ID NO: 34 based on the SignalP 3.0 that predicts amino acids 1 to 20 of SEQ ID NO: 34 are a signal peptide.
  • the mature polypeptide is amino acids 19 to 340 of SEQ ID NO: 35 based on the SignalP 3.0 that predicts amino acids 1 to 18 of SEQ ID NO: 35 are a signal peptide.
  • the mature polypeptide is amino acids 21 to 350 of SEQ ID NO: 36 based on the SignalP 3.0 that predicts amino acids 1 to 20 of SEQ ID NO: 36 are a signal peptide.
  • the mature polypeptide is amino acids 19 to 350 of SEQ ID NO: 37 based on the SignalP 3.0 that predicts amino acids 1 to 18 of SEQ ID NO: 37 are a signal peptide.
  • the mature polypeptide is amino acids 17 to 574 of SEQ ID NO: 38 based on the SignalP 3.0 that predicts amino acids 1 to 16 of SEQ ID NO: 38 are a signal peptide.
  • the mature polypeptide is amino acids 21 to 445 of SEQ ID NO: 39 based on the SignalP 3.0 that predicts amino acids 1 to 20 of SEQ ID NO: 39 are a signal peptide.
  • nucleic acid construct means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, which comprises one or more control sequences.
  • operably linked means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs expression of the coding sequence.
  • Peroxidase means an enzyme that converts a peroxide, e.g., hydrogen peroxide, to a less oxidative species, e.g., water. It is understood herein that a peroxidase encompasses a peroxide-decomposing enzyme.
  • peroxide- decomposing enzyme is defined herein as a donor: peroxide oxidoreductase (E.C.
  • Peroxidase activity can be determined by measuring the oxidation of 2,2'-azino-bis(3- ethylbenzthiazoline-6-sulfonic acid (ABTS) by a peroxidase in the presence of hydrogen peroxide as shown below.
  • ABTS 2,2'-azino-bis(3- ethylbenzthiazoline-6-sulfonic acid
  • the reaction product ABTS 0X forms a blue-green color which can be quantified at 418 nm.
  • the reaction is conducted in 0.1 M phosphate pH 7 at 30°C with 1.67 mM substrate (ABTS), 1.5 g/L TRITON® X-405, 0.88 mM hydrogen peroxide, and approximately 0.040 unit enzyme per ml.
  • the sample is placed in a spectrophotometer and the change in absorbance is measured at 418 nm from 15 seconds up to 60 seconds.
  • One peroxidase unit can be expressed as the amount of enzyme required to catalyze the conversion of 1 ⁇ of hydrogen peroxide per minute under the specified analytical conditions.
  • Pretreated lignocellulosic material means a lignocellulosic material derived from biomass by treatment with heat and dilute sulfuric acid, alkaline pretreatment, neutral pretreatment, or any pretreatment known in the art.
  • Pretreated corn stover The term "Pretreated Corn Stover" or "PCS" means a lignocellulosic material derived from corn stover by treatment with heat and dilute sulfuric acid, alkaline pretreatment, neutral pretreatment, or any pretreatment known in the art.
  • Primeverose means the compound 6-O-beta-D- xylopyranosyl-beta-D-glucopyranose with the following structure or a derivative thereof:
  • Sequence identity The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity”.
  • 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 5.0.0 or later.
  • the parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
  • the output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
  • variant means a polypeptide having enzyme activity comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (e.g., several) positions.
  • a substitution means replacement of the amino acid occupying a position with a different amino acid;
  • a deletion means removal of the amino acid occupying a position; and
  • an insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position
  • Xylan-containing material means any material comprising a plant cell wall polysaccharide containing a backbone of beta-(1-4)- linked xylose residues.
  • Xylans of terrestrial plants are heteropolymers possessing a beta-
  • (1-4)-D-xylopyranose backbone 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-type polysaccharides can be divided into homoxylans and heteroxylans, which include glucuronoxylans, (arabino)glucuronoxylans, (glucurono)arabinoxylans, arabinoxylans, and complex heteroxylans. See, for example, Ebringerova et al., 2005, Adv. Polym. Sci. 186: 1-
  • any material containing xylan may be used.
  • the xylan-containing material is lignocellulose.
  • xylan degrading activity or xylanolytic activity means a biological activity that hydrolyzes xylan-containing material.
  • the two basic approaches for measuring xylanolytic activity include: (1) measuring the total xylanolytic activity, and (2) measuring the individual xylanolytic activities (e.g., endoxylanases, beta-xylosidases, arabinofuranosidases, alpha-glucuronidases, acetylxylan esterases, feruloyi esterases, and alpha-glucuronyl esterases).
  • Total xylan degrading activity can be measured by determining the reducing sugars formed from various types of xylan, including, for example, oat spelt, beechwood, and larchwood xylans, or by photometric determination of dyed xylan fragments released from various covalently dyed xylans.
  • a common total xylanolytic activity assay is based on production of reducing sugars from polymeric 4-O-methyl glucuronoxylan as described in Bailey et ai, 1992, Interlaboratory testing of methods for assay of xylanase activity, Journal of Biotechnology 23(3): 257-270.
  • Xylanase activity can also be determined with 0.2% AZCL- arabinoxylan as substrate in 0.01 % TRITON® X-100 and 200 mM sodium phosphate pH 6 at 37°C.
  • One unit of xylanase activity is defined as 1.0 ⁇ of azurine produced per minute at 37°C, pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6.
  • Xylan degrading activity can be determined by measuring the increase in hydrolysis of birchwood xylan (Sigma Chemical Co., Inc.) by xylan-degrading enzyme(s) under the following typical conditions: 1 ml reactions, 5 mg/ml substrate (total solids), 5 mg of xylanolytic protein/g of substrate, 50 mM sodium acetate pH 5, 50°C, 24 hours, sugar analysis using p-hydroxybenzoic acid hydrazide (PHBAH) assay as described by Lever, 1972, Anal. Biochem. 47: 273-279.
  • PBAH p-hydroxybenzoic acid hydrazide
  • xylanase means a 1 ,4-beta-D-xylan-xylohydrolase (E.C.
  • Xylanase activity can be determined with 0.2% AZCL-arabinoxylan as substrate in 0.01 % TRITON® X-100 and 200 mM sodium phosphate pH 6 at 37°C.
  • One unit of xylanase activity is defined as 1.0 ⁇ of azurine produced per minute at 37°C, pH 6 from 0.2% AZCL- arabinoxylan as substrate in 200 mM sodium phosphate pH 6.
  • references to "about” a value or parameter herein includes aspects that are directed to that value or parameter per se. For example, description referring to "about X” includes the aspect "X”.
  • the present invention relates to processes for reducing production of primeverose during saccharification of a lignocellulosic material, the process comprising: saccharifying the lignocellulosic material with an enzyme composition comprising a GH43 beta-xylosidase, wherein the enzyme composition comprising the GH43 beta-xylosidase increases the amount of fermentable sugars from saccharification of the lignocellulosic material by reducing the amount of primeverose produced compared to an enzyme composition comprising a GH3 beta-xylosidase in place of the GH43 beta-xylosidase.
  • the processes further comprise recovering the saccharified lignocellulosic material.
  • the present invention also relates to processes for saccharifying a lignocellulosic material, comprising: treating the lignocellulosic material with an enzyme composition comprising a GH43 beta-xylosidase, wherein the enzyme composition comprising the GH43 beta-xylosidase increases the amount of fermentable sugars from saccharification of the lignocellulosic material by reducing the amount of primeverose produced compared to an enzyme composition comprising a GH3 beta-xylosidase in place of the GH43 beta- xylosidase.
  • the processes further comprise recovering the saccharified lignocellulosic material. Soluble products from the degradation of the lignocellulosic material can be separated from insoluble lignocellulosic material using methods known in the art such as, for example, centrifugation, filtration, or gravity settling.
  • the present invention also relates to processes for producing a fermentation product, the process comprising: (a) saccharifying a lignocellulosic material with an enzyme composition comprising a GH43 beta-xylosidase, wherein the GH43 beta-xylosidase increases the amount of fermentable sugars from saccharification of the lignocellulosic material by reducing the amount of primeverose produced compared to an enzyme composition comprising a GH3 beta-xylosidase in place of the GH43 beta-xylosidase; (b) fermenting the saccharified lignocellulosic material with one or more (e.g., several) fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.
  • the present invention also relates to processes for fermenting a lignocellulosic material, the process comprising: fermenting the lignocellulosic material with one or more fermenting microorganisms, wherein the lignocellulosic material is saccharified with an enzyme composition comprising a GH43 beta-xylosidase, wherein the enzyme composition comprising the GH43 beta-xylosidase increases the amount of fermentable sugars from saccharification of the lignocellulosic material by reducing the amount of primeverose produced compared to an enzyme composition comprising a GH3 beta-xylosidase in place of the GH43 beta-xylosidase.
  • the fermenting of the lignocellulosic material produces a fermentation product.
  • the processes further comprise recovering the fermentation product from the fermentation.
  • Primeverose forms as a transglycosylation product when a retaining beta-xylosidase (i.e., GH3 family beta-xylosidase) acts on xylobiose.
  • a retaining beta-xylosidase i.e., GH3 family beta-xylosidase
  • Retaining beta-xylosidases cleave xylobiose via a 2-step mechanism where a glycosyl-enzyme intermediate forms in the first step. Cleavage of this intermediate with water results in hydrolysis of xylobiose to two xylose molecules.
  • cleavage of the glycosyl-enzyme intermediate with another sugar molecule results in the formation of a transglycosylation product (primeverose) where a new sugar linkage is generated.
  • a transglycosylation product i.e., glucose
  • Inverting beta-xylosidases (GH43 family) cleave via a 1-step mechanism and thus transglycosylation products do not form. Consequently, hydrolysis of xylobiose with an enzyme composition comprising a GH43 beta-xylosidase in the presence of other sugars prevents the production of primeverose and only results in the hydrolysis product of two xylose molecules. In contrast, hydrolysis of xylobiose with an enzyme composition comprising a GH3 beta-xylosidase in the presence of other sugars produces primeverose.
  • the amount of primeverose produced in the processes of the present invention is reduced at least 20%, preferably at least 40%, more preferably at least 60%, even more preferably at least 80%, and most preferably at least 100%.
  • the amount of primeverose can be determined by Dionex ion chromatography using a PA- 10 column and pulsed amperometry detection (IC-PAD, Dionex Corporation) using CHROMELEONTM Software (Dionex Corporation) as described in Example 5.
  • the amount of fermentable sugars in the processes of the present invention is increased at least 0.1 %, at least 0.2%, at least 0.5%, at least 1 %, at least 2.5%, at least 5%, or at least 10% from saccharification of the lignocellulosic material.
  • the the amount of fermentable sugars can be determined by AM IN EX® HPX-87H column chromatography (Bio-Rad Laboratories, Inc.).
  • the processes of the present invention can be used to saccharify a lignocellulosic material to fermentable sugars and to convert the fermentable sugars to many useful fermentation products, e.g., fuel (ethanol, n-butanol, isobutanol, biodiesel, jet fuel) and/or platform chemicals (e.g., acids, alcohols, ketones, gases, oils, and the like).
  • fuel ethanol, n-butanol, isobutanol, biodiesel, jet fuel
  • platform chemicals e.g., acids, alcohols, ketones, gases, oils, and the like.
  • 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), also sometimes called consolidated bioprocessing (CBP).
  • SHF separate hydrolysis and fermentation
  • SSF simultaneous saccharification and fermentation
  • SSCF simultaneous saccharification and co-fermentation
  • HHF hybrid hydrolysis and fermentation
  • SHCF separate hydrolysis and co-fermentation
  • HHCF hybrid hydrolysis and co-fermentation
  • DMC direct microbial conversion
  • SHF uses separate process steps to first enzymatically hydrolyze the lignocellulosic material to fermentable sugars, e.g., glucose, cellobiose, and pentose monomers, and then ferment the fermentable sugars to ethanol.
  • fermentable sugars e.g., glucose, cellobiose, and pentose monomers
  • SSCF involves the co-fermentation of multiple sugars (Sheehan and Himmel, 1999, Biotechnol.
  • HHF involves a separate hydrolysis step, and in addition a simultaneous saccharification and hydrolysis step, which can be carried out in the same reactor.
  • the steps in an HHF process can be carried out at different temperatures, i.e., high temperature enzymatic saccharification followed by SSF at a lower temperature that the fermentation strain can tolerate.
  • DMC combines all three processes (enzyme production, hydrolysis, and fermentation) in one or more (e.g., several) steps where the same organism is used to produce the enzymes for conversion of the lignocellulosic material to fermentable sugars and to convert the fermentable sugars into a final product (Lynd et al., 2002, Microbiol. Mol. Biol. Reviews 66: 506-577). It is understood herein that any method known in the art comprising pretreatment, enzymatic hydrolysis (saccharification), fermentation, or a combination thereof, can be used in the practicing the processes of the present invention.
  • a conventional apparatus can include a fed-batch stirred reactor, a batch stirred reactor, a continuous flow stirred reactor with ultrafiltration, and/or a continuous plug-flow column reactor (de Castilhos Corazza et al., 2003, Acta Scientiarum. Technology 25: 33-38; Gusakov and Sinitsyn, 1985, Enz. Microb. Technol. 7: 346-352), an attrition reactor (Ryu and Lee, 1983, Biotechnol. Bioeng. 25: 53-65). Additional reactor types include fluidized bed, upflow blanket, immobilized, and extruder type reactors for hydrolysis and/or fermentation.
  • any pretreatment process known in the art can be used to disrupt plant cell wall components of the lignocellulosic material (Chandra et al., 2007, Adv. Biochem. Engin. /Biotechnol. 108: 67-93; Galbe and Zacchi, 2007, Adv. Biochem. Engin. /Biotechnol. 108: 41-65; Hendriks and Zeeman, 2009, Bioresource Technology 100: 10-18; Mosier et al., 2005, Bioresource Technology 96: 673-686; Taherzadeh and Karimi, 2008, Int. J. Mol. Sci.
  • the lignocellulosic material can also be subjected to particle size reduction, sieving, pre-soaking, wetting, washing, and/or conditioning prior to pretreatment using methods known in the art.
  • Conventional pretreatments include, but are not limited to, steam pretreatment (with or without explosion), dilute acid pretreatment, hot water pretreatment, alkaline pretreatment, lime pretreatment, wet oxidation, wet explosion, ammonia fiber explosion, organosolv pretreatment, and biological pretreatment.
  • Additional pretreatments include ammonia percolation, ultrasound, electroporation, microwave, supercritical CO2, supercritical H2O, ozone, ionic liquid, and gamma irradiation pretreatments.
  • the lignocellulosic material can be pretreated before hydrolysis and/or fermentation.
  • Pretreatment is preferably performed prior to the hydrolysis.
  • the pretreatment can be carried out simultaneously with enzyme hydrolysis to release fermentable sugars, such as glucose, xylose, and/or cellobiose.
  • fermentable sugars such as glucose, xylose, and/or cellobiose.
  • the pretreatment step itself results in some conversion of biomass to fermentable sugars (even in absence of enzymes).
  • the lignocellulosic material is heated to disrupt the plant cell wall components, including lignin, hemicellulose, and cellulose to make the cellulose and other fractions, e.g., hemicellulose, accessible to enzymes.
  • the lignocellulosic material is passed to or through a reaction vessel where steam is injected to increase the temperature to the required temperature and pressure and is retained therein for the desired reaction time.
  • Steam pretreatment is preferably performed at 140-250°C, e.g., 160-200°C or 170-190°C, where the optimal temperature range depends on optional addition of a chemical catalyst.
  • Residence time for the steam pretreatment is preferably 1-60 minutes, e.g., 1-30 minutes, 1-20 minutes, 3-12 minutes, or 4-10 minutes, where the optimal residence time depends on the temperature and optional addition of a chemical catalyst.
  • Steam pretreatment allows for relatively high solids loadings, so that the lignocellulosic material is generally only moist during the pretreatment.
  • the steam pretreatment is often combined with an explosive discharge of the material after the pretreatment, which is known as steam explosion, that is, rapid flashing to atmospheric pressure and turbulent flow of the material to increase the accessible surface area by fragmentation (Duff and Murray, 1996, Bioresource Technology 855: 1-33; Galbe and Zacchi, 2002, Appl. Microbiol. Biotechnol. 59: 618-628; U.S.
  • Patent Application No. 2002/0164730 During steam pretreatment, hemicellulose acetyl groups are cleaved and the resulting acid autocatalyzes partial hydrolysis of the hemicellulose to monosaccharides and oligosaccharides. Lignin is removed to only a limited extent.
  • Chemical Pretreatment 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 to amorphous cellulose.
  • suitable chemical pretreatment processes include, for example, dilute acid pretreatment, lime pretreatment, wet oxidation, ammonia fiber/freeze expansion (AFEX), ammonia percolation (APR), ionic liquid, and organosolv pretreatments.
  • a chemical catalyst such as H2SO4 or SO2 (typically 0.3 to 5% w/w) is sometimes added prior to steam pretreatment, which decreases the time and temperature, increases the recovery, and improves enzymatic hydrolysis (Ballesteros et al., 2006, Appl. Biochem. Biotechnol. 129-132: 496-508; Varga et al., 2004, Appl. Biochem. Biotechnol. 113-1 16: 509- 523; Sassner et ai, 2006, Enzyme Microb. Technol. 39: 756-762).
  • H2SO4 or SO2 typically 0.3 to 5% w/w
  • the lignocellulosic material is mixed with dilute acid, typically H2SO4, and water to form a slurry, heated by steam to the desired temperature, and after a residence time flashed to atmospheric pressure.
  • the dilute acid pretreatment can be performed with a number of reactor designs, e.g., plug-flow reactors, counter-current reactors, or continuous counter-current shrinking bed reactors (Duff and Murray, 1996, Bioresource Technology 855: 1-33; Schell et al., 2004, Bioresource Technology 91 : 179-188; Lee et al., 1999, Adv. Bi ⁇ hem. Eng. Biotechnol. 65: 93-115).
  • alkaline pretreatments include, but are not limited to, sodium hydroxide, lime, wet oxidation, ammonia percolation (APR), and ammonia fiber/freeze expansion (AFEX) pretreatment.
  • Lime pretreatment is performed with calcium oxide or calcium hydroxide at temperatures of 85-150°C and residence times from 1 hour to several days (Wyman et al., 2005, Bioresource Technology 96: 1959-1966; Mosier et al., 2005, Bioresource Technology 96: 673-686).
  • WO 2006/110891 , WO 2006/110899, WO 2006/110900, and WO 2006/110901 disclose pretreatment methods using ammonia.
  • Wet oxidation is a thermal pretreatment performed typically at 180-200°C for 5-15 minutes with addition of an oxidative agent such as hydrogen peroxide or over-pressure of oxygen (Schmidt and Thomsen, 1998, Bioresource Technology 64: 139-151 ; Palonen et al., 2004, Appl. Biochem. Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88: 567- 574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81 : 1669-1677).
  • the pretreatment is performed preferably at 1-40% dry matter, e.g., 2-30% dry matter or 5-20% dry matter, and often the initial pH is increased by the addition of alkali such as sodium carbonate.
  • a modification of the wet oxidation pretreatment method known as wet explosion (combination of wet oxidation and steam explosion) can handle dry matter up to 30%.
  • wet explosion combination of wet oxidation and steam explosion
  • the oxidizing agent is introduced during pretreatment after a certain residence time.
  • the pretreatment is then ended by flashing to atmospheric pressure (WO 2006/032282).
  • Ammonia fiber expansion involves treating the lignocellulosic 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 dry matter content 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 ai, 2005, Appl. Biochem. Biotechnol. 121 : 1133-1141 ; Teymouri et ai, 2005, Bioresource Technology 96: 2014-2018).
  • cellulose and hemicelluloses remain relatively intact. Lignin-carbohydrate complexes are cleaved.
  • Organosolv pretreatment delignifies the lignocellulosic 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 ai., 2006, Biotechnol. Bioeng. 94: 851-861 ; Kurabi et al., 2005, Appl. Biochem. Biotechnol. 121 : 219-230). Sulphuric acid is usually added as a catalyst. In organosolv pretreatment, the majority of hemicellulose and lignin is removed.
  • the chemical pretreatment is preferably carried out as a dilute acid treatment, and more preferably as a continuous dilute acid treatment.
  • the acid is typically sulfuric acid, but other acids can also be used, such as acetic acid, citric acid, nitric acid, phosphoric acid, tartaric acid, succinic acid, hydrogen chloride, or mixtures thereof.
  • Mild acid treatment is conducted in the pH range of preferably 1-5, e.g., 1-4 or 1-2.5.
  • the acid concentration is in the range from preferably 0.01 to 10 wt. % acid, e.g., 0.05 to 5 wt. % acid or 0.1 to 2 wt. % acid.
  • the acid is contacted with the lignocellulosic material and held at a temperature in the range of preferably 140-200°C, e.g., 165-190°C, for periods ranging from 1 to 60 minutes.
  • pretreatment takes place in an aqueous slurry.
  • the lignocellulosic material is present during pretreatment in amounts preferably between 10-80 wt. %, e.g., 20-70 wt. % or 30-60 wt. %, such as around 40 wt. %.
  • the pretreated lignocellulosic material can be unwashed or washed using any method known in the art, e.g., washed with water.
  • mechanical pretreatment or Physical pretreatment refers to any pretreatment that promotes size reduction of particles.
  • pretreatment can involve various types of grinding or milling (e.g., dry milling, wet milling, or vibratory ball milling).
  • the lignocellulosic material can be pretreated both physically (mechanically) and chemically. Mechanical or physical pretreatment can be coupled with steaming/steam explosion, hydrothermolysis, dilute or mild acid treatment, high temperature, high pressure treatment, irradiation (e.g., microwave irradiation), or combinations thereof.
  • high pressure means pressure in the range of preferably about 100 to about 400 psi, e.g., about 150 to about 250 psi.
  • high temperature means temperature in the range of about 100 to about 300°C, e.g., about 140 to about 200°C.
  • mechanical or physical pretreatment is performed in a batch-process using a steam gun hydrolyzer system that uses high pressure and high temperature as defined above, e.g., a Sunds Hydrolyzer available from Sunds Defibrator AB, Sweden.
  • the physical and chemical pretreatments can be carried out sequentially or simultaneously, as desired.
  • the lignocellulosic material is subjected to physical
  • Biopretreatment refers to any biological pretreatment that promotes the separation and/or release of cellulose, hemicellulose, and/or lignin from the lignocellulosic material.
  • Biological pretreatment techniques can involve applying lignin-solubilizing microorganisms and/or enzymes (see, for example, Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol: Production and Utilization, Wyman, C. E., ed., Taylor & Francis, Washington, DC, 179-212; Ghosh and Singh, 1993, Adv. Appl. Microbiol. 39: 295-333; McMillan, J.
  • Saccharification In the hydrolysis step, also known as saccharification, the lignocellulosic material, e.g., pretreated, is hydrolyzed to break down cellulose and/or hemicellulose to fermentable sugars, such as glucose, cellobiose, xylose, xylulose, arabinose, mannose, galactose, and/or soluble oligosaccharides.
  • the hydrolysis is performed enzymatically by one or more enzyme compositions in one or more stages.
  • the hydrolysis can be carried out as a batch process or series of batch processes.
  • the hydrolysis can be carried out as a fed batch or continuous process, or series of fed batch or continuous processes, where the lignocellulosic material is fed gradually to, for example, a hydrolysis solution containing an enzyme composition.
  • the saccharification is a continuous saccharification in which a lignocellulosic material and a cellulolytic enzyme composition are added at different intervals throughout the saccharification and the hydrolysate is removed at different intervals throughout the saccharification. The removal of the hydrolysate may occur prior to, simultaneously with, or after the addition of the lignocellulosic material and the enzyme composition.
  • Enzymatic hydrolysis is preferably carried out in a suitable aqueous environment under conditions that can be readily determined by one skilled in the art. In one aspect, hydrolysis is performed under conditions suitable for the activity of the enzymes(s), i.e., optimal for the enzyme(s).
  • the saccharification is generally performed in stirred-tank reactors or fermentors under controlled pH, temperature, and mixing conditions. Suitable process time, temperature and pH conditions can readily be determined by one skilled in the art.
  • the total saccharification time can last up to 200 hours, but is typically performed for preferably about 4 to about 120 hours, e.g., about 12 to about 96 hours or about 24 to about 72 hours.
  • the temperature is in the range of preferably about 25°C to about 80°C, e.g., about 30°C to about 70°C, about 40°C to about 60°C, or about 50°C to about 55°C.
  • the pH is in the range of preferably about 3 to about 9, e.g., about 3.5 to about 8, about 4 to about 7, about 4.2 to about 6, or about 4.3 to about 5.5.
  • the dry solids content is in the range of preferably about 5 to about 50 wt. %, e.g., about 10 to about 40 wt. % or about 20 to about 30 wt. %.
  • the saccharification is performed in the presence of dissolved oxygen at a concentration of at least 0.5% of the saturation level.
  • the dissolved oxygen concentration during saccharification is in the range of at least 0.5% up to 30% of the saturation level, such as at least 1 % up to 25%, at least 1 % up to 20%, at least 1 % up to 15%, at least 1 % up to 10%, at least 1 % up to 5%, and at least 1 % up to 3% of the saturation level.
  • the dissolved oxygen concentration is maintained at a concentration of at least 0.5% up to 30% of the saturation level, such as at least 1 % up to 25%, at least 1 % up to 20%, at least 1 % up to 15%, at least 1 % up to 10%, at least 1 % up to 5%, and at least 1 % up to 3% of the saturation level during at least 25% of the saccharification period, such as at least 50% or at least 75% of the saccharification period.
  • the enzyme composition comprises an oxidoreductase the dissolved oxygen concentration may be higher up to 70% of the saturation level.
  • Oxygen is added to the vessel to achieve the desired concentration of dissolved oxygen during saccharification. Maintaining the dissolved oxygen level within a desired range can be accomplished by aeration of the vessel, tank or the like by adding compressed air through a diffuser or sparger, or by other known methods of aeration.
  • the aeration rate can be controlled on the basis of feedback from a dissolved oxygen sensor placed in the vessel/tank, or the system can run at a constant rate without feedback control.
  • aeration can be implemented in one or more or all of the vessels/tanks. Oxygen aeration systems are well known in the art.
  • any suitable aeration system may be used.
  • Commercial aeration systems are designed by, e.g., Chemineer, Derby, England, and built by, e.g., Paul Mueller Company. Fermentation.
  • the fermentable sugars obtained from the hydrolyzed lignocellulosic material can be fermented by one or more (e.g., several) fermenting microorganisms capable of fermenting the sugars directly or indirectly into a desired fermentation product.
  • Fermentation or “fermentation process” refers to any fermentation process or any process comprising a fermentation step.
  • Fermentation processes also include fermentation processes used in the consumable alcohol industry (e.g., beer and wine), dairy industry (e.g., fermented dairy products), leather industry, and tobacco industry.
  • the fermentation conditions depend on the desired fermentation product and fermenting organism and can easily be determined by one skilled in the art.
  • sugars released from the lignocellulosic material as a result of the pretreatment and enzymatic hydrolysis steps, are fermented to a product, e.g., ethanol, by a fermenting organism, such as yeast.
  • Hydrolysis (saccharification) and fermentation can be separate or simultaneous.
  • Any suitable hydrolyzed lignocellulosic material can be used in the fermentation step in practicing the present invention.
  • the material is generally selected based on economics, i.e. , costs per equivalent sugar potential, and recalcitrance to enzymatic conversion.
  • fermentation medium is understood herein to refer to a medium before the fermenting microorganism(s) is(are) added, such as, a medium resulting from a saccharification process, as well as a medium used in a simultaneous saccharification and fermentation process (SSF).
  • SSF simultaneous saccharification and fermentation process
  • “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 thereof. Both hexose and pentose fermenting organisms are well known in the art.
  • Suitable fermenting microorganisms can ferment, i.e., convert, sugars, such as glucose, xylose, xylulose, arabinose, maltose, mannose, galactose, and/or oligosaccharides, directly or indirectly into the desired fermentation product. Examples of bacterial and fungal fermenting organisms producing ethanol are described by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69: 627-642.
  • fermenting microorganisms that can ferment hexose sugars include bacterial and fungal organisms, such as yeast.
  • yeast include strains of Candida, Kluyveromyces, and Saccharomyces, e.g., Candida sonorensis, Kluyveromyces marxianus, and Saccharomyces cerevisiae.
  • Xylose fermenting yeast include strains of Candida, preferably C. sheatae or C. sonorensis; and strains of Pichia, e.g., P. stipitis, such as P. stipitis CBS 5773.
  • Pentose fermenting yeast include strains of Pachysolen, preferably P. tannophilus.
  • Organisms not capable of fermenting pentose sugars, such as xylose and arabinose may be genetically modified to do so by methods known in the art.
  • 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. acetobutylicum, C. thermocellum, and C. phytofermentans; E. coli, especially E.
  • Geobacillus sp. Hansenula, such as 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
  • Zymomonas such as Zymomonas mobilis.
  • yeast suitable for ethanol production include, e.g., BIO-FERM® AFT and XR (Lallemand Specialities, Inc., USA), ETHANOL RED® yeast (Lesaffre et Compagnie, France), FALI® (AB Mauri Food Inc., USA), FERMIOL® (Rymco International AG, Denmark), GERT STRANDTM (Gert Strand AB, Sweden), and SUPERSTARTTM and THERMOSACC® fresh yeast (Lallemand Specialities, Inc., USA).
  • the fermenting microorganism has been genetically modified to provide the ability to ferment pentose sugars, such as xylose utilizing, arabinose utilizing, and xylose and arabinose co-utilizing microorganisms.
  • the fermenting organism comprises one or more polynucleotides encoding one or more cellulolytic enzymes, hemicellulolytic enzymes, and accessory enzymes described herein.
  • the fermenting microorganism is typically added to the saccharified lignocellulosic material or hydrolysate and the fermentation is performed for about 8 to about 96 hours, e.g., about 24 to about 60 hours.
  • the temperature is typically between about 26°C to about 60°C, e.g., about 32°C or 50°C, and about pH 3 to about pH 8, e.g., pH 4-5, 6, or 7.
  • the yeast and/or another microorganism are applied to the saccharified lignocellulosic material and the fermentation is performed for about 12 to about 96 hours, such as typically 24-60 hours.
  • the temperature is preferably between about 20°C to about 60°C, e.g., about 25°C to about 50°C, about 32°C to about 50°C, or about 32°C to about 50°C
  • the pH is generally from about pH 3 to about pH 7, e.g., about pH 4 to about pH 7.
  • some fermenting organisms, e.g., bacteria have higher fermentation temperature optima.
  • Yeast or another microorganism is preferably applied in amounts of approximately 10 5 to 10 12 , preferably from approximately 10 7 to 10 10 , especially approximately 2 x 10 8 viable cell count per ml of fermentation broth. Further guidance in respect of using yeast for fermentation can be found in, e.g., "The Alcohol Textbook” (Editors K. Jacques, T.P. Lyons and D.R. Kelsall, Nottingham University Press, United Kingdom 1999), which is hereby incorporated by reference.
  • a fermentation stimulator can be used in combination with any of the processes described herein to further improve the fermentation process, and in particular, the performance of the fermenting microorganism, such as, rate enhancement and ethanol yield.
  • a "fermentation stimulator” refers to stimulators for growth of the fermenting microorganisms, in particular, yeast.
  • Preferred 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.
  • minerals include minerals and mineral salts that can supply nutrients comprising P, K, Mg, S, Ca, Fe, Zn, Mn, and Cu.
  • a fermentation product can be any substance derived from the fermentation.
  • the fermentation product can be, without limitation, an alcohol (e.g., arabinitol, n-butanol, isobutanol, ethanol, glycerol, methanol, ethylene glycol, 1 ,3- propanediol [propylene glycol], butanediol, glycerin, sorbitol, and xylitol); an alkane (e.g.
  • a cycloalkane e.g., cyclopentane, cyclohexane, cycloheptane, and cyclooctane
  • an alkene ⁇ e.g., pentene, hexene, heptene, and octene
  • an amino acid ⁇ e.g., aspartic acid, glutamic acid, glycine, lysine, serine, and threonine
  • a gas ⁇ e.g., methane, hydrogen (H 2 ), carbon dioxide (CO2), and carbon monoxide (CO)
  • isoprene a ketone ⁇ e.g., acetone
  • an organic acid ⁇ e.g., acetic acid, acetonic acid, adipic acid, ascorbic acid, cit
  • the fermentation product is an alcohol.
  • alcohol encompasses a substance that contains one or more hydroxyl moieties.
  • the alcohol can be, but is not limited to, n-butanol, isobutanol, ethanol, methanol, arabinitol, butanediol, ethylene glycol, glycerin, glycerol, 1 ,3-propanediol, sorbitol, xylitol.
  • the fermentation product is an alkane.
  • the alkane may be an unbranched or a branched alkane.
  • the alkane can be, but is not limited to, pentane, hexane, heptane, octane, nonane, decane, undecane, or dodecane.
  • the fermentation product is a cycloalkane.
  • the cycloalkane can be, but is not limited to, cyclopentane, cyclohexane, cycloheptane, or cyclooctane.
  • the fermentation product is an alkene.
  • the alkene may be an unbranched or a branched alkene.
  • the alkene can be, but is not limited to, pentene, hexene, heptene, or octene.
  • the fermentation product is an amino acid.
  • the organic acid can be, but is not limited to, aspartic acid, glutamic acid, glycine, lysine, serine, or threonine. See, for example, Richard and Margaritis, 2004, Biotechnology and Bioengineering 87(4): 501-515.
  • the fermentation product is a gas.
  • the gas can be, but is not limited to, methane, H2, CO2, or CO. See, for example, Kataoka et al., 1997, Water Science and Technology 36(6-7): 41-47; and Gunaseelan, 1997, Biomass and Bioenergy 13(1-2): 83- 114.
  • the fermentation product is isoprene.
  • the fermentation product is a ketone.
  • ketone encompasses a substance that contains one or more ketone moieties.
  • the ketone can be, but is not limited to, acetone.
  • the fermentation product is an organic acid.
  • the organic acid can be, but is not limited to, acetic acid, acetonic acid, adipic acid, ascorbic acid, citric acid, 2,5- diketo-D-gluconic acid, formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid, propionic acid, succinic acid, or xylonic acid. See, for example, Chen and Lee, 1997, Appl. Biochem. Biotechnol. 63-65: 435-448.
  • the fermentation product is polyketide.
  • the fermentation product(s) can be optionally 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.
  • alcohol is separated from the fermented lignocellulosic material and purified by conventional methods of distillation.
  • Ethanol with a purity of up to about 96 vol. % can be obtained, which can be used as, for example, fuel ethanol, drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.
  • any GH43 beta-xylosidase may be used.
  • the GH43 beta-xylosidase can be obtained from any source, especially microorganisms of any genus.
  • the term "obtained from” as used herein in connection with a given source shall mean that the polypeptide encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted.
  • the GH43 beta-xylosidase obtained from a given source is secreted extracellularly.
  • the GH43 beta-xylosidase may be a bacterial beta-xylosidase.
  • the GH43 beta-xylosidase may be a gram positive bacterial beta-xylosidase such as a Bacillus, Clostridium, Corynebacterium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces beta-xylosidase, or a Gram negative bacterial beta-xylosidase such as an E. coli, Campylobacter, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasma beta-xylosidase.
  • the GH43 beta-xylosidase is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus cereus, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus polymyxa, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Geobacillus stearothermophilus, Lactobacillus plantarum, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp.
  • Bacterial GH43 beta-xylosidases can be obtained from Alkaliphilus metalliredigens (UniProt A6TTC7), Bacillus halodurans (UniProt Q9K6P5), Bacillus pumilus (UniProt P07129), Bacillus subtilis (UniProt P94489), Bacteroides ovatus (UniProt P49943), Bifidobacterium adolescentis (UniProt A1A0H6), Bifidobacterium animalis (SwissProt B2ECL1), Butyrivibrio fibrisolvens (UniProt P45982), Caldicellulosiruptor saccharolyticus (UniProt 030426), Caulobacter crescentus (SwissProt Q9A9J1), Cellvibrio japonicus (UniProt B3PD
  • the GH43 beta-xylosidase may also be a fungal beta-xylosidase, and more preferably a yeast beta-xylosidase such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia beta-xylosidase; or more preferably a filamentous fungal beta-xylosidase such as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Lepto
  • the GH43 beta-xylosidase is a Saccharomyces carlsbergensis
  • Saccharomyces cerevisiae Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis beta- xylosidase.
  • the GH43 beta-xylosidase is an Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus fischeri, Aspergillus flavus, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae (Swiss-Prot Accession number P78581), Aspergillus usamii, Aspergillus ustus, Aspergillus versicolor, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium zonat
  • Filamentous fungal GH43 beta-xylosidases can be obtained from Aspergillus fumigatus (SwissProt Q4X0K2 and SwissProt A0A0J5SK50), Aspergillus oryzae (UniProt Q2URT3), Cochliobolus carbonum (UniProt 093912), Fusarium graminearum (UniProt I 1 S3S2), Fusarium oxysporum (UniProt J9NHC4), Fusarium verticillioides (UniProt W7MVU8, SwissProt W7MCN3, and SwissProt W7MN47), Humicola insolens (UniProt V9TT49 and V9TNS0), Paecilomyces thermophila (UniProt F1APW0), Penicillium herquei (UniProt Q870E8), Penicillium purpurogenum (UniProt K7WBC5),
  • the GH43 beta-xylosidase is selected from the group consisting of:
  • SEQ ID NO: 1 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or 29, or the mature polypeptide of SEQ ID NO: 30, 31 , 32, 33, 34, 35, 36, 37, 38, or 39;
  • polypeptide comprising the polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or 29, or the mature polypeptide of SEQ ID NO: 30, 31 , 32, 33, 34, 35, 36, 37, 38, or 39;
  • the GH43 beta-xylosidase has a sequence identity to the polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23,
  • the GH43 beta-xylosidase differs by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or 29, or the mature polypeptide of SEQ ID NO: 30, 31 , 32, 33, 34, 35, 36, 37, 38, or 39.
  • the GH43 beta-xylosidase comprises or consists of the amino acid sequence of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24,
  • the polypeptide comprises or consists of the polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25,
  • the present invention relates to variants of the polypeptide of
  • SEQ ID NO: 1 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or 29, or the mature polypeptide of SEQ ID NO: 30, 31 , 32, 33, 34, 35, 36, 37,
  • the number of amino acid substitutions, deletions and/or insertions introduced into the polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or 29, or the mature polypeptide of SEQ ID NO: 30, 31 , 32, 33, 34, 35, 36, 37, 38, or 39 is up to 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • amino acid changes may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.
  • conservative substitutions are within 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 do not generally 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.
  • amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered.
  • amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.
  • Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant molecules are tested for GH43 beta-xylosidase activity to identify amino acid residues that are critical to the activity of the molecule. 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 structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. 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.
  • the identity of essential amino acids can also be inferred from an alignment with a related polypeptide.
  • Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed 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 ⁇ e.g., Lowman et al., 1991 , Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).
  • Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.
  • ATCC American Type Culture Collection
  • DSM Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
  • CBS Centraalbureau Voor Schimmelcultures
  • NRRL Northern Regional Research Center
  • beta-xylosidases may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms from natural habitats are well known in the art. The polynucleotide may then be obtained by similarly screening a genomic or cDNA library of such a microorganism.
  • the polynucleotide can be isolated or cloned by utilizing techniques that are well known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).
  • Beta-xylosidases also include fused polypeptides or cleavable fusion polypeptides in which another polypeptide is fused at the N-terminus or the C-terminus of the beta- xylosidase or fragment thereof.
  • a fused polypeptide is produced by fusing a nucleotide sequence (or a portion thereof) encoding another polypeptide to a nucleotide sequence (or a portion thereof) encoding a beta-xylosidase.
  • Techniques for producing fusion polypeptides include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fused polypeptide is under control of the same promoter(s) and terminator.
  • a fusion polypeptide can further comprise a cleavage site.
  • the site Upon secretion of the fusion protein, the site is cleaved releasing the beta-xylosidase from the fusion protein.
  • cleavage sites include, but are not limited to, a Kex2 site that encodes the dipeptide Lys-Arg (Martin et al. , 2003, J. Ind. Microbiol. Biotechnol. 3: 568-76; Svetina et al. , 2000, J. Biotechnol. 76: 245-251 ; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol.
  • the enzyme composition may comprise any protein involved in the processing of a lignocellulosic material to fermentable sugars, e.g., glucose and xylose.
  • the enzyme composition comprises or further comprises one or more
  • the cellulase is preferably one or more (e.g., several) enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase.
  • the hemicellulase is preferably one or more (e.g., 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, and a xylanase.
  • the oxidoreductase is preferably one or more (e.g., several) enzymes selected from the group consisting of a catalase, a laccase, and a peroxidase.
  • the enzyme composition comprises or further comprises one or more (e.g., several) cellulolytic enzymes. In another aspect, the enzyme composition comprises or further comprises one or more (e.g., several) hemicellulolytic enzymes. In another aspect, the enzyme composition comprises or further comprises one or more (e.g., several) cellulolytic enzymes and one or more (e.g., several) hemicellulolytic enzymes. In another aspect, the enzyme composition comprises or further comprises one or more (e.g., several) enzymes selected from the group of cellulolytic enzymes and hemicellulolytic enzymes. In another aspect, the enzyme composition comprises or further comprises an endoglucanase.
  • the enzyme composition comprises or further comprises a cellobiohydrolase. In another aspect, the enzyme composition comprises or further comprises a beta-glucosidase. In another aspect, the enzyme composition comprises or further comprises an AA9 polypeptide. In another aspect, the enzyme composition comprises or further comprises an endoglucanase and an AA9 polypeptide. In another aspect, the enzyme composition comprises or further comprises a cellobiohydrolase and an AA9 polypeptide. In another aspect, the enzyme composition comprises or further comprises a beta-glucosidase and an AA9 polypeptide. In another aspect, the enzyme composition comprises or further comprises an endoglucanase and a cellobiohydrolase.
  • the enzyme composition comprises or further comprises an endoglucanase I, an endoglucanase II, or a combination of an endoglucanase I and an endoglucanase II, and a cellobiohydrolase I, a cellobiohydrolase II, or a combination of a cellobiohydrolase I and a cellobiohydrolase II.
  • the enzyme composition comprises or further comprises an endoglucanase and a beta-glucosidase.
  • the enzyme composition comprises or further comprises an endoglucanase I, an endoglucanase II, or a combination of an endoglucanase I and an endoglucanase II, and a beta-glucosidase.
  • the enzyme composition comprises or further comprises a beta-glucosidase and a cellobiohydrolase.
  • the enzyme composition comprises or further comprises a beta-glucosidase and a cellobiohydrolase I, a cellobiohydrolase II, or a combination of a cellobiohydrolase I and a cellobiohydrolase II.
  • the enzyme composition comprises or further comprises an endoglucanase, an AA9 polypeptide, and a cellobiohydrolase.
  • the enzyme composition comprises or further comprises an endoglucanase I, an endoglucanase II, or a combination of an endoglucanase I and an endoglucanase II, an AA9 polypeptide, and a cellobiohydrolase I, a cellobiohydrolase II, or a combination of a cellobiohydrolase I and a cellobiohydrolase II.
  • the enzyme composition comprises or further comprises an endoglucanase, a beta-glucosidase, and an AA9 polypeptide.
  • the enzyme composition comprises or further comprises a beta-glucosidase, an AA9 polypeptide, and a cellobiohydrolase.
  • the enzyme composition comprises or further comprises a beta-glucosidase, an AA9 polypeptide, and a cellobiohydrolase I, a cellobiohydrolase II, or a combination of a cellobiohydrolase I and a cellobiohydrolase II.
  • the enzyme composition comprises or further comprises an endoglucanase, a beta-glucosidase, and a cellobiohydrolase.
  • the enzyme composition comprises or further comprises an endoglucanase I, an endoglucanase II, or a combination of an endoglucanase I and an endoglucanase II, a beta-glucosidase, and a cellobiohydrolase I, a cellobiohydrolase II, or a combination of a cellobiohydrolase I and a cellobiohydrolase II.
  • the enzyme composition comprises or further comprises an endoglucanase, a cellobiohydrolase, a beta-glucosidase, and an AA9 polypeptide.
  • the enzyme composition comprises or further comprises an endoglucanase I, an endoglucanase II, or a combination of an endoglucanase I and an endoglucanase II, a beta-glucosidase, an AA9 polypeptide, and a cellobiohydrolase I, a cellobiohydrolase II, or a combination of a cellobiohydrolase I and a cellobiohydrolase II.
  • the enzyme composition comprises or further comprises an acetylmannan esterase. In another aspect, the enzyme composition comprises or further comprises an acetylxylan esterase. In another aspect, the enzyme composition comprises or further comprises an arabinanase ⁇ e.g., alpha-L-arabinanase). In another aspect, the enzyme composition comprises or further comprises an arabinofuranosidase (e.g., alpha-L- arabinofuranosidase). In another aspect, the enzyme composition comprises or further comprises a coumaric acid esterase. In another aspect, the enzyme composition comprises or further comprises a feruloyl esterase.
  • the enzyme composition comprises or further comprises a galactosidase (e.g., alpha-galactosidase and/or beta- galactosidase).
  • the enzyme composition comprises or further comprises a glucuronidase (e.g., alpha-D-glucuronidase).
  • the enzyme composition comprises or further comprises a glucuronoyl esterase.
  • the enzyme composition comprises or further comprises a mannanase.
  • the enzyme composition comprises or further comprises a mannosidase (e.g., beta-mannosidase).
  • the enzyme composition comprises or further comprises a xylanase.
  • the xylanase is a Family 10 xylanase.
  • the xylanase is a Family 1 1 xylanase.
  • the enzyme composition comprises or further comprises an esterase. In another aspect, the enzyme composition comprises or further comprises an expansin. In another aspect, the enzyme composition comprises or further comprises a ligninolytic enzyme. In an embodiment, the ligninolytic enzyme is a manganese peroxidase. In another embodiment, the ligninolytic enzyme is a lignin peroxidase. In another embodiment, the ligninolytic enzyme is a H 2 02-producing enzyme. In another aspect, the enzyme composition comprises or further comprises a pectinase. In another aspect, the enzyme composition comprises or further comprises an oxidoreductase. In an embodiment, the oxidoreductase is a catalase.
  • the oxidoreductase is a laccase. In another embodiment, the oxidoreductase is a peroxidase. In another aspect, the enzyme composition comprises or further comprises a protease. In another aspect, the enzyme composition comprises or further comprises a swollenin.
  • the enzyme(s) can be added prior to or during saccharification, saccharification and fermentation, or fermentation.
  • One or more (e.g., several) components of the enzyme composition may be native proteins, recombinant proteins, or a combination of native proteins and recombinant proteins.
  • one or more (e.g., several) components may be native proteins of a cell, which is used as a host cell to express recombinantly one or more (e.g., several) other components of the enzyme composition.
  • the recombinant proteins may be heterologous (e.g., foreign) and/or native to the host cell.
  • One or more (e.g., several) components of the enzyme composition may be produced as monocomponents, which are then combined to form the enzyme composition.
  • the enzyme composition may be a combination of multicomponent and monocomponent protein preparations.
  • the enzymes used in the processes of the present invention may be in any form suitable for use, such as, for example, a fermentation broth formulation or a cell composition, a cell lysate with or without cellular debris, a semi-purified or purified enzyme preparation, or a host cell as a source of the enzymes.
  • the enzyme composition may be a dry powder or granulate, a non-dusting granulate, a liquid, a stabilized liquid, or a stabilized protected enzyme.
  • Liquid enzyme preparations may, for instance, 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 established processes.
  • the optimum amounts of the enzymes depend on several factors including, but not limited to, the mixture of cellulolytic enzymes and/or hemicellulolytic enzymes, the lignocellulosic material, the concentration of lignocellulosic material, the pretreatment(s) of the lignocellulosic material, temperature, time, pH, and inclusion of a fermenting organism (e.g., for Simultaneous Saccharification and Fermentation).
  • an effective amount of cellulolytic or hemicellulolytic enzyme to the lignocellulosic material is about 0.5 to about 50 mg, e.g., about 0.5 to about 40 mg, about 0.5 to about 25 mg, about 0.75 to about 20 mg, about 0.75 to about 15 mg, about 0.5 to about 10 mg, or about 2.5 to about 10 mg per g of the lignocellulosic material.
  • an effective amount of a GH43 beta-xylosidase to the lignocellulosic material is about 0.01 to about 50.0 mg, e.g., 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 g of the lignocellulosic material.
  • an effective amount of a GH43 beta-xylosidase to cellulolytic or hemicellulolytic enzyme is about 0.005 to about 1.0 g, e.g., 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 g of cellulolytic or hemicellulolytic enzyme.
  • polypeptides having cellulolytic enzyme activity or hemicellulolytic enzyme activity as well as other proteins/polypeptides useful in the degradation of the lignocellulosic material can be derived or obtained from any suitable origin, including, archaeal, bacterial, fungal, yeast, plant, or animal origin.
  • the term "obtained” also means herein that the enzyme may have been produced recombinantly in a host organism employing methods described herein, wherein the recombinantly produced enzyme is either native or foreign to the host organism or has a modified amino acid sequence, e.g., having one or more (e.g., 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 native amino acid sequence or an enzyme produced by nucleic acid shuffling processes known in the art.
  • a native enzyme are natural variants and within the meaning of a foreign enzyme are variants obtained by, e.g., site-directed mutagenesis or shuffling.
  • Each polypeptide may be a bacterial polypeptide.
  • each polypeptide may be a Gram-positive bacterial polypeptide having enzyme activity, or a Gram-negative bacterial polypeptide having enzyme activity.
  • Each polypeptide may also be a fungal polypeptide, e.g., a yeast polypeptide or a filamentous fungal polypeptide.
  • Chemically modified or protein engineered mutants of polypeptides may also be used.
  • One or more (e.g., several) components of the enzyme composition may be a recombinant component, i.e., produced by cloning of a DNA sequence encoding the single component and subsequent cell transformed with the DNA sequence and expressed in a host (see, for example, WO 91/17243 and WO 91/17244).
  • the host can be a heterologous host (enzyme is foreign to host), but the host may under certain conditions also be a homologous host (enzyme is native to host).
  • Monocomponent cellulolytic proteins may also be prepared by purifying such a protein from a fermentation broth.
  • the one or more (e.g., several) cellulolytic enzymes comprise a commercial cellulolytic enzyme preparation.
  • commercial cellulolytic enzyme preparations suitable for use in the present invention include, for example, CELLIC® CTec (Novozymes A/S), CELLIC® CTec2 (Novozymes A/S), CELLIC® CTec3 (Novozymes A/S), CELLUCLASTTM (Novozymes A/S), NOVOZYMTM 188 (Novozymes A/S), SPEZYMETM CP (Genencor Int.), ACCELLERASETM TRIO (DuPont), FILTRASE® NL (DSM); METHAPLUS® S/L 100 (DSM), ROHAMENTTM 7069 W (Rohm GmbH), or ALTERNAFUEL® CMAX3TM (Dyadic International, Inc.).
  • the cellulolytic enzyme preparation is added in an amount effective from about 0.001 to about 5.0 wt. % of solids, e.g., about 0.025 to about 4.0 wt. % of solids or about 0.005 to about 2.0 wt. % of solids.
  • bacterial endoglucanases examples include, but are not limited to, Acidothermus cellulolyticus endoglucanase (WO 91/05039; WO 93/15186; U.S. Patent No. 5,275,944; WO 96/02551 ; U.S. Patent No.
  • fungal endoglucanases examples include, but are not limited to, Trichoderma reesei endoglucanase I (Penttila et al., 1986, Gene 45: 253-263, Trichoderma reesei Cel7B endoglucanase I (GenBank:M15665), Trichoderma reesei endoglucanase II (Saloheimo et al., 1988, Gene 63: 1 1-22), Trichoderma reesei Cel5A endoglucanase II (Gen Bank: M 19373), Trichoderma reesei endoglucanase III (Okada et al., 1988, Appl.
  • thermoidea endoglucanase (GenBank:AB003107), Melanocarpus albomyces endoglucanase (GenBank:MAL515703), Neurospora crassa endoglucanase (GenBank:XM_324477), Humicola insolens endoglucanase V, Myceliophthora thermophila CBS 117.65 endoglucanase, Thermoascus aurantiacus endoglucanase I (GenBank:AF487830), Trichoderma reesei strain No. VTT-D-80133 endoglucanase (GenBank:M 15665), and Penicillium pinophilum endoglucanase (WO 2012/062220).
  • cellobiohydrolases useful in the present invention include, but are not limited to, Aspergillus aculeatus cellobiohydrolase II (WO 201 1/059740), Aspergillus fumigatus cellobiohydrolase I (WO 2013/028928), Aspergillus fumigatus cellobiohydrolase II (WO 2013/028928), Chaetomium thermophilum cellobiohydrolase I, Chaetomium thermophilum cellobiohydrolase II, Humicola insolens cellobiohydrolase I, Myceliophthora thermophila cellobiohydrolase II (WO 2009/042871), Penicillium occitanis cellobiohydrolase I (GenBank:AY690482), Talaromyces emersonii cellobiohydrolase I (GenBank:AF439936), Thielavia hyrcanie cellobiohydrolase II (WO 2010/141325), Thielavia terrestris cellobio
  • beta-glucosidases useful in the present invention include, but are not limited to, beta-glucosidases from Aspergillus aculeatus (Kawaguchi et al., 1996, Gene 173:
  • any AA9 polypeptide can be used as a component of the enzyme composition.
  • AA9 polypeptides useful in the processes of the present invention include, but are not limited to, AA9 polypeptides from Thielavia terrestris (WO 2005/074647, WO 2008/148131 , and WO 201 1/035027), Thermoascus aurantiacus (WO 2005/074656 and WO 2010/065830), Trichoderma reesei (WO 2007/089290 and WO 2012/149344), Myceliophthora thermophila (WO 2009/085935, WO 2009/085859, WO 2009/085864, WO 2009/085868, and WO 2009/033071), Aspergillus fumigatus (WO 2010/138754), Penicillium pinophilum (WO 201 1/005867), Thermoascus sp.
  • the AA9 polypeptide is used in the presence of a soluble activating divalent metal cation according to WO 2008/151043 or WO 2012/122518, e.g., manganese or copper.
  • the AA9 polypeptide is used in the presence of a dioxy compound, a bicylic compound, a heterocyclic compound, a nitrogen-containing compound, a quinone compound, a sulfur-containing compound, or a liquor obtained from a pretreated lignocellulosic material such as pretreated corn stover (WO 2012/021394, WO 2012/021395, WO 2012/021396, WO 2012/021399, WO 2012/021400, WO 2012/021401 , WO 2012/021408, and WO 2012/021410).
  • a pretreated lignocellulosic material such as pretreated corn stover
  • such a compound is added at a molar ratio of the compound to glucosyl units of cellulose of about 10 "6 to about 10, e.g., 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 , or about 10 "3 to about 10 “2 .
  • an effective amount of such a compound is about 0.1 ⁇ to about 1 M, e.g., about 0.5 ⁇ to about 0.75 M, about 0.75 ⁇ to about 0.5 M, about 1 ⁇ to about 0.25 M, about 1 ⁇ to about 0.1 M, about 5 ⁇ to about 50 mM, about 10 ⁇ to about 25 mM, about 50 ⁇ to about 25 mM, about 10 ⁇ to about 10 mM, about 5 ⁇ to about 5 mM, or about 0.1 mM to about 1 mM.
  • liquid means the solution phase, either aqueous, organic, or a combination thereof, arising from treatment of a lignocellulose and/or hemicellulose material in a slurry, or monosaccharides thereof, e.g., xylose, arabinose, mannose, etc., under conditions as described in WO 2012/021401 , and the soluble contents thereof.
  • a liquor for cellulolytic enhancement of an AA9 polypeptide can be produced by treating a lignocellulose or hemicellulose material (or feedstock) by applying heat and/or pressure, optionally in the presence of a catalyst, e.g., acid, optionally in the presence of an organic solvent, and optionally in combination with physical disruption of the material, and then separating the solution from the residual solids.
  • a catalyst e.g., acid
  • organic solvent optionally in the presence of an organic solvent
  • the liquor can be separated from the treated material using a method standard in the art, such as filtration, sedimentation, or centrifugation.
  • an effective amount of the liquor to cellulose is about 10 "6 to about 10 g per g of cellulose, e.g. , about 10 "6 to about 7.5 g, about 10 "6 to about 5 g, about 10 "6 to about 2.5 g, about 10 "6 to about 1 g, about 10 "5 to about 1 g, about 10 "5 to about 10 "1 g, about 10 “4 to about 10 "1 g, about 10 "3 to about 10 "1 g, or about 10 "3 to about 10 "2 g per g of cellulose.
  • the one or more (e.g., several) hemicellulolytic enzymes comprise a commercial hemicellulolytic enzyme preparation.
  • commercial hemicellulolytic enzyme preparations suitable for use in the present invention include, for example, SHEARZYMETM (Novozymes A/S), CELLIC® HTec (Novozymes A/S), CELLIC® HTec2 (Novozymes A/S), CELLIC® HTec3 (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), DEPOLTM
  • Aspergillus aculeatus GeneSeqP:AAR63790; WO 94/21785
  • Aspergillus fumigatus WO 2006/078256
  • Penicillium pinophilum WO 2011/04140
  • the hemicellulase may be added in an amount effective to hydrolyze hemicellulose, such as, in amounts from about 0.001 to 0.5 wt. % of total solids (TS), more preferably from about 0.05 to 0.5 wt. % of TS.
  • TS total solids
  • Xylanases may be added in amounts of 0.001-1.0 g/kg DM (dry matter) substrate, preferably in the amount of 0.005-0.5 g/kg DM substrate, and most preferably from 0.05-0.10 g/kg DM substrate.
  • acetylxylan esterases useful in the processes of the present invention include, but are not limited to, acetylxylan esterases from Aspergillus aculeatus (WO 2010/108918), Chaetomium globosum (UniProt:Q2GWX4), Chaetomium gracile (GeneSeqP:AAB82124), Humicola insolens DSM 1800 (WO 2009/073709), Hypocrea jecorina (WO 2005/001036), Myceliophtera thermophila (WO 2010/014880), Neurospora crassa (UniProt:q7s259), Phaeosphaeria nodorum (UniProt:Q0UHJ1), and Thielavia terrestris NRRL 8126 (WO 2009/042846).
  • feruloyi esterases form Humicola insolens DSM 1800 (WO 2009/076122), Neosartorya fischeri (UniProt:A1 D9T4), Neurospora crassa (UniProt:Q9HGR3), Penicillium aurantiogriseum (WO 2009/127729), and Thielavia terrestris (WO 2010/053838 and WO 2010/065448).
  • arabinofuranosidases useful in the processes of the present invention include, but are not limited to, arabinofuranosidases from Aspergillus niger (GeneSeqP:AAR94170), Humicola insolens DSM 1800 (WO 2006/1 14094 and WO 2009/073383), and M. giganteus (WO 2006/1 14094).
  • alpha-glucuronidases useful in the processes of the present invention include, but are not limited to, alpha-glucuronidases from Aspergillus clavatus (UniProt:alcc12), Aspergillus fumigatus (SwissProt:Q4WW45), Aspergillus niger (UniProt:Q96WX9), Aspergillus terreus (SwissProt:Q0CJP9), Humicola insolens (WO 2010/014706), Penicillium aurantiogriseum (WO 2009/068565), Talaromyces emersonii (UniProt:Q8X211), and Trichoderma reesei (UniProt:Q99024).
  • alpha-glucuronidases from Aspergillus clavatus (UniProt:alcc12), Aspergillus fumigatus (SwissProt:Q4WW45), Asperg
  • oxidoreductases useful in the processes of the present invention include, but are not limited to, Aspergillus lentilus catalase, Aspergillus fumigatus catalase, Aspergillus niger catalase, Aspergillus oryzae catalase, Humicola insolens catalase, Neurospora crassa catalase, Penicillium emersonii catalase, Scytalidium thermophilum catalase, Talaromyces stipitatus catalase, Thermoascus aurantiacus catalase, Coprinus cinereus laccase, Myceliophthora thermophila laccase, Polyporus pinsitus laccase, Pycnoporus cinnabarinus laccase, Rhizoctonia solani laccase, Streptomyces coelicolor laccase, Coprinus cinereus peroxidase, Soy peroxidase, and Royal palm peroxidas
  • polypeptides having enzyme activity used in the processes of the present invention may be produced by fermentation of the above-noted microbial strains on a nutrient medium containing suitable carbon and nitrogen sources and inorganic salts, using procedures known in the art (see, e.g., Bennett, J.W. and LaSure, L. (eds.), More Gene Manipulations in Fungi, Academic Press, CA, 1991). Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). Temperature ranges and other conditions suitable for growth and enzyme production are known in the art (see, e.g., Bailey, J.E., and Ollis, D.F., Biochemical Engineering Fundamentals, McGraw-Hill Book Company, NY, 1986).
  • the fermentation can be any method of cultivation of a cell resulting in the expression or isolation of an enzyme or protein. Fermentation may, therefore, be understood as comprising shake flask cultivation, or small- or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the enzyme to be expressed or isolated.
  • the resulting enzymes produced by the methods described above may be recovered from the fermentation medium and purified by conventional procedures.
  • the enzyme composition may comprise an enzyme as the major enzymatic component, e.g., a mono-component composition.
  • the enzyme composition may comprise multiple enzymatic activities, such as one or more (e.g., several) enzymes selected from the group consisting of a cellulase, a hemicellulase, an AA9 polypeptide, a cellulose inducible protein (CIP), a catalase, an esterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin.
  • a cellulase e.g., a hemicellulase, an AA9 polypeptide, a cellulose inducible protein (CIP), a catalase, an esterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase,
  • compositions may also comprise or further comprise one or more (e.g., several) enzymes selected from the group consisting of a hydrolase, an isomerase, a ligase, a lyase, an oxidoreductase, or a transferase, e.g., an alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase, beta-galactosidase, beta-glucosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pect
  • the enzyme composition may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition.
  • the compositions may be stabilized in accordance with methods known in the art.
  • the enzyme composition may be a fermentation broth formulation or a cell composition.
  • the fermentation broth product further comprises additional ingredients used in the fermentation process, such as, for example, cells (including, the host cells containing the gene encoding a polypeptide of interest which are used to produce the polypeptide), cell debris, biomass, fermentation media and/or fermentation products.
  • the composition is a cell-killed whole broth containing organic acid(s), killed cells and/or cell debris, and culture medium.
  • fermentation broth refers to a preparation produced by cellular fermentation that undergoes no or minimal recovery and/or purification.
  • fermentation broths are produced when microbial cultures are grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis ⁇ e.g., expression of enzymes by host cells) and secretion into cell culture medium.
  • the fermentation broth can contain unfractionated or fractionated contents of the fermentation materials derived at the end of the fermentation.
  • the fermentation broth is unfractionated and comprises the spent culture medium and cell debris present after the microbial cells (e.g. , filamentous fungal cells) are removed, e.g., by centrifugation.
  • the fermentation broth contains spent cell culture medium, extracellular enzymes, and viable and/or nonviable microbial cells.
  • the fermentation broth formulation and cell 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 one 6 or more carbon organic acid and/or a salt thereof.
  • the first organic acid component is acetic acid, formic acid, propionic acid, a salt thereof, or a mixture of two or more of the foregoing and the second organic acid component is benzoic acid, cyclohexanecarboxylic acid, 4-methylvaleric acid, phenylacetic acid, a salt thereof, or a mixture of two or more of the foregoing.
  • the composition contains an organic acid(s), and optionally further contains killed cells and/or cell debris.
  • the killed cells and/or cell debris are removed from a cell-killed whole broth to provide a composition that is free of these components.
  • the fermentation broth formulations or cell compositions may further comprise a preservative and/or anti-microbial (e.g., bacteriostatic) agent, including, but not limited to, sorbitol, sodium chloride, potassium sorbate, and others known in the art.
  • a preservative and/or anti-microbial agent including, but not limited to, sorbitol, sodium chloride, potassium sorbate, and others known in the art.
  • the fermentation broth formulations or cell compositions may further comprise multiple enzymatic activities, such as one or more (e.g., several) enzymes selected from the group consisting of a cellulase, a hemicellulase, an AA9 polypeptide, a cellulose inducible protein (CIP), a catalase, an esterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin.
  • a cellulase e.g., several enzymes selected from the group consisting of a cellulase, a hemicellulase, an AA9 polypeptide, a cellulose inducible protein (CIP), a catalase, an esterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase,
  • the fermentation broth formulations or cell compositions may also comprise or further comprise one or more (e.g., several) enzymes selected from the group consisting of a hydrolase, an isomerase, a ligase, a lyase, an oxidoreductase, or a transferase, e.g., an alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase, beta-galactosidase, beta-glucosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase, laccase, lipase, mannosidase, mutanase, oxid
  • the cell-killed whole broth or composition may contain the unfractionated contents of the fermentation materials derived at the end of the fermentation.
  • the cell-killed whole broth or composition contains the spent culture medium and cell debris present after the microbial cells (e.g., filamentous fungal cells) are grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis (e.g., expression of cellulase and/or glucosidase enzyme(s)).
  • the cell-killed whole broth or composition contains the spent cell culture medium, extracellular enzymes, and killed filamentous fungal cells.
  • the microbial cells present in the cell-killed whole broth or composition can be permeabilized and/or lysed using methods known in the art.
  • a whole broth or cell composition as described herein is typically a liquid, but may contain insoluble components, such as killed cells, cell debris, culture media components, and/or insoluble enzyme(s). In some embodiments, insoluble components may be removed to provide a clarified liquid composition.
  • the whole broth formulations and cell compositions may be produced by a method described in WO 90/15861 or WO 2010/096673.
  • Methods of producing a polypeptide having enzyme activity comprise (a) cultivating a cell, which in its wild-type form is capable of producing the polypeptide, under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.
  • methods of producing a polypeptide having enzyme activity or cellulolytic enhancing activity comprise (a) cultivating a recombinant host cell under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.
  • the cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods well known in the art.
  • the cell may be cultivated by shake flask cultivation, and small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated.
  • the 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 may be prepared according to published compositions (e.g., in catalogues 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 into the medium, it can be recovered from cell lysates.
  • the polypeptides having enzyme activity can be detected using the methods described herein or methods known in the art.
  • the resulting broth may be used as is with or without cellular debris or the polypeptide may be recovered using methods known in the art.
  • the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.
  • polypeptides may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS- PAGE, or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure polypeptides.
  • chromatography e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion
  • electrophoretic procedures e.g., preparative isoelectric focusing
  • differential solubility e.g., ammonium sulfate precipitation
  • SDS- PAGE or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden,
  • Bacillus subtilis strain IH14 was used as an expression host for the Geobacillus stearothermophilus GH43 polypeptide.
  • LB medium was composed of 10 g of tryptone, 5 g of yeast extract, 5 g of NaCI, and deionized water to 1 liter.
  • MRS medium was composed of 55 g of DIFCOTM Lactobacilli MRS powder and deionized water to 1 liter.
  • Spizizen II medium was composed of 6 g of KH2PO4, 14 g of K2HPO4, 2 g of (NH 4 )2S0 4 , 1 g of NasCeHsOy, 0.2 g of MgS0 -7H 2 0, 5 g of glucose, 0.2 g of casein hydrolysate, 1 g of yeast extract, 50 mg of tryptophan, 0.055 g of CaC , 0.24 g of MgC , 0.761 g of EGTA, and deionized water to 1 liter.
  • TBAB + CM plates were composed of 33 g of Tryptose blood agar base (TBAB), 0.5 ml of 5 mg of chloramphenicol, and deionized water to 1 liter.
  • 2XYT + Amp plates were composed of 16 g of tryptone, 10 g of yeast extract, 5 g of NaCI, 15 g of Bacto agar, 1 ml of 100 mg/ml ampicillin stock solution, and deionized water to 1 liter.
  • Talaromyces emersonii GH3 beta-xylosidase (SEQ ID NO: 40 [DNA sequence] and SEQ ID NO: 41 [deduced amino acid sequence]; P4UE) was prepared recombinantly according to Rasmussen et al., 2006, Biotechnology and Bioengineering 94: 869-876 using Aspergillus oryzae Jal_355 as a host (WO 2003/070956).
  • the harvested broth was sterile filtered using a 0.22 ⁇ polyethersulfone membrane (Millipore).
  • the filtered broth was concentrated and buffer exchanged with 50 mM sodium acetate pH 5.0 using a tangential flow concentrator (Pall Filtron) equipped with a 10 kDa polyethersulfone membrane at approximately 20 psi.
  • Desalted material was examined on 8- 16% CRITERIONTM SDS-PAGE gels (Bio-Rad Laboratories, Inc.) stained with GELCODE® Blue Stain Reagent (Thermo Fisher Scientific).
  • the protein was >90% pure as judged by SDS-PAGE. Protein concentration was determined using a BCA Protein Assay Kit (Thermo Fisher Scientific) in which bovine serum albumin was used as a protein standard.
  • Plasmid pEbZn58 was constructed as described below for expression of the Geobacillus stearothermophilus GH43 polypeptide (SEQ ID NO: 1 [deduced amino acid sequence]).
  • Plasmid pLF02 Figure 1 ; SEQ ID NO: 42 [vector DNA sequence]
  • Plasmid pLF02 is a shuttle vector for transformation into B. subtilis via double cross-over of the crylllA stabilizer sequence and pelB locus.
  • the digested plasmid was purified using a PCR Purification Kit (QIAGEN Inc.) according to the manufacturer's instructions.
  • the Geobacillus stearothermophilus GH43 beta-xylosidase coding sequence (SEQ ID NO: 43 [genomic DNA sequence]) was optimized for expression in Bacillus subtilis using GeneArt® GeneOptimzer® software (SEQ ID NO: 44 [B. subtilis optimized DNA sequence]) and synthesized by Life Technologies.
  • the optimized sequence was amplified by PCR using the primers shown in Table 1. Bold letters represent coding sequence. The remaining sequences are homologous to insertion sites of pLF02.
  • the amplification reaction was performed in a thermocycler programmed for 1 cycle at 98°C for 30 seconds; 30 cycles each at 98°C for 5 seconds, 62°C for 30 seconds, and 72°C for 45 seconds; and a final elongation at 72°C for 10 minutes.
  • the heat block then went to a 4°C soak cycle.
  • reaction product was isolated by 1.0% agarose gel electrophoresis using 40 mM Tris base-20 mM sodium acetate-1 mM disodium EDTA (TAE) buffer where a 1602 bp band was excised from the gel and extracted using an Agarose Gel Purification Kit (Clontech Laboratories, Inc.) according to the manufacturer's instructions.
  • TAE Tris base-20 mM sodium acetate-1 mM disodium EDTA
  • the homologous ends of the 1602 bp PCR product and the digested pLF02 were joined together using an IN-FUSIONTM HD Cloning Kit (Clontech Laboratories, Inc.).
  • a total of 100 ng of the 1602 bp PCR product and 100 ng of the Kpn ⁇ IXba I digested plasmid pLF02 were used in a reaction containing 2 ⁇ of 5X IN-FUSIONTM reaction buffer (Clontech Laboratories, Inc.) and 1 ⁇ of IN-FUSIONTM enzyme (Clontech Laboratories, Inc.) in a final volume of 10 ⁇ .
  • the reaction was incubated for 15 minutes at 50°C and then placed on ice.
  • E. coli STELLAR® Cells (Stratagene) according to the manufacturer's instructions. E. coli transformants were selected on 2XYT + Amp plates. Plasmid DNA from several of the resulting E. coli transformants was prepared using a BIOROBOT® 9600 (QIAGEN Inc.). The Geobacillus stearothermophilus GH43 polypeptide coding sequence insert was confirmed by DNA sequencing with a Model 377 XL Automated DNA Sequencer (Applied Biosystems Inc.) using dye-terminator chemistry (Giesecke et al., 1992, J. Virol. Methods 38: 47-60). Sequencing primers used for verification of the gene insert and sequence are shown in Table 2.
  • a plasmid containing the correct Geobacillus stearothermophilus GH43 polypeptide coding sequence was selected and designated pEbZn58 ( Figure 2).
  • Example 3 Expression of the Geobacillus stearothermophilus GH43 beta-xylosidase in Bacillus subtil is strain IH14
  • Bacillus subtilis strain SM025 (U.S. Patent No. 8,580,536) was modified to generate Bacillus subtilis strain IH14 via the deletion of the native GH11 xylanase (xynA) gene, insertion of a spectinomycin resistance marker from transposon Tn554 (Murphy et ai, 1985, EMBO Journal 4 (12): 3357-3365), replacement of the pectate lyase (pelB) gene with a triple promoter (Pam_4i99/Phort consensus amyo/PcryiiiA /crylllA stabilizer sequence triple promoter) (U.S. Patent No.
  • B. subtilis strain I H 14 was made competent using the method described by Anagnostopoulos and Spizizen, 1961 , Journal of Bacteriology 81 : 741-746. Once cells were deemed competent by measuring their log-phase growth the entire culture was centrifuged at 3836 x g for 10 minutes. The supernatant was removed and 18 ml of the removed supernatant was mixed with 2 ml of glycerol. The cell pellet was resuspended in this supernatant/glycerol mixture, distributed in 0.5 ml aliquots, and stored at -80°C.
  • Plasmid pEbZn58 was linearized with Sal I. A 0.5 ml volume of Spizizen II medium
  • the re-streaked plates were used to inoculate 3 ml of LB medium, which was incubated at 37°C for 6 hours with shaking at 300 rpm.
  • Five hundred ⁇ volumes of the culture were inoculated into 125 ml plastic non-baffled shake flasks containing 50 ml of MRS medium.
  • the shake flasks were incubated at 37°C with shaking at 250 rpm.
  • the cultures were transferred to centrifuge bottles and the cells were pelleted at 3,234 ⁇ g for 20 minutes at 4°C.
  • the supernatants were then filtered through a 0.22 ⁇ EMD Millipore STERICUPTM Sterile Vacuum Filter Unit (EMD Millipore) and stored at -20°C until purification.
  • EMD Millipore STERICUPTM Sterile Vacuum Filter Unit EMD Millipore
  • the sterile filtered supernatant from Example 3 was adjusted to pH 8 with 50% NaOH and applied to a NUVIATM IMAC column (Bio-Rad Laboratories, Inc.) equilibrated with 50 mM Tris-HCI pH 8.0, 150 mM NaCI, 20 mM CaCI 2 , 10 mM imidazole. The column was washed extensively with equilibration buffer and then washed with 20 mM Tris-HCI pH 8.0. Bound proteins were eluted with an imidazole gradient (20 column volumes) of 0 M imidazole to 250 mM imidazole in 20 mM Tris-HCI pH 8.0.
  • Eluted protein was pooled and applied to a CAPTOTM Q column (GE Healthcare) equilibrated with 20 mM Tris-HCI pH 8. Bound proteins were eluted with a salt gradient (15 column volumes) of 0 M NaCI to 1 M NaCI in 20 mM Tris-HCI pH 8.0. Fractions were examined on 8-16% CRITERIONTM STAIN- FREETM SDS-PAGE gels. GH43 beta-xylosidase containing fractions were pooled and judged to be >90% pure by SDS-PAGE. Protein concentration was determined using a BCA Protein Assay Kit in which bovine serum albumin was used as a protein standard.
  • Example 5 Assay of the Talaromyces emersonii GH3 beta-xylosidase and Geobacillus stearothermophilus GH43 beta-xylosidase with glucose and xylo-oligomers
  • the Talaromyces emersonii GH3 beta-xylosidase and Geobacillus stearothermophilus GH43 beta-xylosidase were assayed for the degradation of xylo- oligomers in the presence of glucose. Assays were conducted in 0.3 ml 96 well round bottom polypropylene plates (Corning). Reactions were initiated by the addition of 10 ⁇ of enzyme to 90 ⁇ of substrate (100 mg/ml glucose, 50 mg/ml xylo-oligosaccharides (Cascade Analytical Reagents and Biochemicals), 50 mM sodium acetate pH 5, 0.01 % TWEEN® 20 (Sigma-Aldrich).
  • GH3 beta-xylosidase and GH43 beta-xylosidase were dosed at 0.0750, 0.0188, and 0.0023 mg/ml final concentration in the assay. No enzyme controls were also run by the addition of 10 ⁇ of water to 90 ⁇ of substrate. The reactions were incubated at 50°C for 24 hours. Following hydrolysis, samples were filtered with a 0.45 ⁇ Multiscreen 96-well filter plate (Millipore) and filtrates analyzed for carbohydrate content as described below.
  • Hydrolysate samples were diluted 1 :50 with 10 mM NaOH prior to analysis.
  • the hydrolysis samples were analyzed by Dionex ion chromatography with pulsed amperometry detection (IC-PAD, Dionex Corporation) using CHROMELEONTM Software (Dionex Corporation). Chromatographic separation was obtained using a PA-10 column and elution was achieved with an isocratic gradient of 13 mM NaOH, 2.5 mM sodium acetate for 20 minutes, followed by a linear gradient from 13 to 50 mM NaOH for 10 minutes, and then a linear gradient for 20 minutes from 0.5 to 62.5 mM sodium acetate and 75 mM NaOH.
  • GH3 beta-xylosidase was incubated with xylo-oligomers and glucose. As shown in Figure 4, the GH3 beta-xylosidase degraded xylo-oligomers and xylobiose and also generated xylose. However, it also generated two other products with retention times of 33 minutes and 39.5 minutes.
  • the 33 minute product was identified as primeverose (6- ⁇ - ⁇ - D-xylopyranosyl-D-glucose) by 2D NMR spectroscopy. The identity of the 39.5 minute product is unknown but has a retention time suggesting it is oligomeric in nature.
  • a mixture (50:50) of the G. stearothermophilus GH43 beta-xylosidase and T. emersonii GH3 beta-xylosidase did not prevent production of primeverose.
  • the mixture of the GH43 beta-xylosidase and GH3 beta-xylosidase can degrade xylo-oligomers and xylobiose and generate xylose.
  • the mixture of the two beta- xylosidases failed to prevent production of primeverose and the unknown product.
  • a process for reducing production of primeverose during saccharification of a lignocellulosic material comprising: saccharifying the lignocellulosic material with an enzyme composition comprising a GH43 beta-xylosidase, wherein the enzyme composition comprising the GH43 beta-xylosidase increases the amount of fermentable sugars from saccharification of the lignocellulosic material by reducing the amount of primeverose produced compared to an enzyme composition comprising a GH3 beta-xylosidase in place of the GH43 beta-xylosidase.
  • Paragraph 2 The process of paragraph 1 , wherein the lignocellulosic material is pretreated before saccharification.
  • Paragraph 3 The process of paragraph 1 or 2, wherein the enzyme composition further comprises one or more enzymes selected from the group consisting of a cellulase, an AA9 polypeptide, a hemicellulase, a cellulose inducible protein, an esterase, an expansin, a ligninolytic enzyme, an oxidoreductase, a pectinase, a protease, and a swollenin.
  • enzymes selected from the group consisting of a cellulase, an AA9 polypeptide, a hemicellulase, a cellulose inducible protein, an esterase, an expansin, a ligninolytic enzyme, an oxidoreductase, a pectinase, a protease, and a swollenin.
  • Paragraph 4 The process of paragraph 3, wherein the cellulase is one or more enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase.
  • Paragraph 5 The process of paragraph 3, wherein the hemicellulase is one or more enzymes selected from the group consisting of a xylanase, an acetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, and a glucuronidase.
  • Paragraph 6 The process of any one of paragraphs 1-5, further comprising recovering the saccharified lignocellulosic material.
  • a process for saccharifying a lignocellulosic material comprising: treating the lignocellulosic material with an enzyme composition comprising a GH43 beta- xylosidase, wherein the enzyme composition comprising the GH43 beta-xylosidase increases the amount of fermentable sugars from saccharification of the lignocellulosic material by reducing the amount of primeverose produced compared to an enzyme composition comprising a GH3 beta-xylosidase in place of the GH43 beta-xylosidase.
  • Paragraph 8 The process of paragraph 7, wherein the lignocellulosic material is pretreated.
  • Paragraph 9 The process of paragraph 7 or 8, wherein the enzyme composition further comprises one or more enzymes selected from the group consisting of a cellulase, an AA9 polypeptide, a hemicellulase, a cellulose inducible protein, an esterase, an expansin, a ligninolytic enzyme, an oxidoreductase, a pectinase, a protease, and a swollenin.
  • Paragraph 10 The process of paragraph 9, wherein the cellulase is one or more enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase.
  • Paragraph 1 1. The process of paragraph 9, wherein the hemicellulase is one or more enzymes selected from the group consisting of a xylanase, an acetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, and a glucuronidase.
  • Paragraph 12 The process of any one of paragraphs 7-11 , further comprising recovering the saccharified lignocellulosic material.
  • Paragraph 13 The process of paragraph 12, wherein the saccharified lignocellulosic material is a sugar.
  • Paragraph 14 The process of paragraph 13, wherein the sugar is selected from the group consisting of glucose, xylose, mannose, galactose, and arabinose.
  • a process for producing a fermentation product comprising: (a) saccharifying a lignocellulosic material with an enzyme composition comprising a GH43 beta-xylosidase, wherein the GH43 beta-xylosidase increases the amount of fermentable sugars from saccharification of the lignocellulosic material by reducing the amount of primeverose produced compared to an enzyme composition comprising a GH3 beta-xylosidase in place of the GH43 beta-xylosidase; (b) fermenting the saccharified lignocellulosic material with one or more fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.
  • Paragraph 16 The process of paragraph 15, wherein the lignocellulosic material is pretreated.
  • Paragraph 17 The process of paragraph 15 or 16, wherein the enzyme composition further comprises one or more enzymes selected from the group consisting of a cellulase, an AA9 polypeptide, a hemicellulase, a cellulose inducible protein, an esterase, an expansin, a ligninolytic enzyme, an oxidoreductase, a pectinase, a protease, and a swollenin.
  • Paragraph 18 The process of paragraph 17, wherein the cellulase is one or more enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase.
  • Paragraph 19 The process of paragraph 17, wherein the hemicellulase is one or more enzymes selected from the group consisting of a xylanase, an acetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, and a glucuronidase.
  • Paragraph 20 The process of any one of paragraphs 15-19, wherein steps (a) and (b) are performed simultaneously in a simultaneous saccharification and fermentation.
  • Paragraph 21 The process of any one of paragraphs 15-20, 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.
  • Paragraph 22 A process for fermenting a lignocellulosic material, the process comprising: fermenting the lignocellulosic material with one or more fermenting microorganisms, wherein the lignocellulosic material is saccharified with an enzyme composition comprising a GH43 beta-xylosidase, wherein the enzyme composition comprising the GH43 beta-xylosidase increases the amount of fermentable sugars from saccharification of the lignocellulosic material by reducing the amount of primeverose produced compared to an enzyme composition comprising a GH3 beta-xylosidase in place of the GH43 beta-xylosidase.
  • Paragraph 23 The process of paragraph 22, wherein the fermenting of the lignocellulosic material produces a fermentation product.
  • Paragraph 24 The process of paragraph 23, further comprising recovering the fermentation product from the fermentation.
  • Paragraph 25 The process of paragraph 23 or 24, 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.
  • Paragraph 26 The process of any one of paragraphs 22-25, wherein the lignocellulosic material is pretreated before saccharification.
  • Paragraph 27 The process of any one of paragraphs 22-26, wherein the enzyme composition further comprises one or more enzymes selected from the group consisting of a cellulase, an AA9 polypeptide, a hemicellulase, a cellulose inducible protein, an esterase, an expansin, a ligninolytic enzyme, an oxidoreductase, a pectinase, a protease, and a swollenin.
  • Paragraph 28 The process of paragraph 27, wherein the cellulase is one or more enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase.
  • Paragraph 29 The process of paragraph 27, wherein the hemicellulase is one or more enzymes selected from the group consisting of a xylanase, an acetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, and a glucuronidase.
  • Paragraph 30 The process of any one of paragraphs 1-29, wherein the GH43 beta- xylosidase is selected from the group consisting of.
  • polypeptide comprising the polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or 29, or the mature polypeptide of SEQ ID NO: 30, 31 , 32, 33, 34, 35, 36, 37, 38, or 39;
  • Paragraph 31 The process of paragraph 30, wherein the GH43 beta-xylosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the beta-xylosidase of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or 29, or the mature polypeptide of SEQ ID NO: 30, 31 , 32, 33, 34, 35, 36, 37, 38, or 39.
  • Paragraph 32 The process of paragraph 30, wherein the GH43 beta-xylosidase comprises or consists of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or 29, or the mature polypeptide of SEQ ID NO: 30, 31 , 32, 33, 34, 35, 36, 37, 38, or 39.
  • Paragraph 33 The process of paragraph 30, wherein the GH43 beta-xylosidase is a variant of the polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or 29, or the mature polypeptide of SEQ ID NO: 30, 31 , 32, 33, 34, 35, 36, 37, 38, or 39 comprising a substitution, deletion, and/or insertion at one or more positions.
  • Paragraph 34 The process of paragraph 30, wherein the GH43 beta-xylosidase is a fragment of the polypeptide the GH43 beta-xylosidase of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or 29, or the mature polypeptide of SEQ ID NO: 30, 31 , 32, 33, 34, 35, 36, 37, 38, or 39 that has GH43 beta- xylosidase activity.
  • Paragraph 35 The process of any one of paragraphs 1-34, wherein the amount of primeverose produced is reduced at least 20%, preferably at least 40%, more preferably at least 60%, even more preferably at least 80%, and most preferably at least 100%.
  • Paragraph 36 The process of any one of paragraphs 1-34, wherein the amount of fermentable sugars is increased at least 0.1 %, at least 0.2%, at least 0.5%, at least 1 %, at least 2.5%, at least 5%, or at least 10% from saccharification of the lignocellulosic material.

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Abstract

La présente invention concerne des procédés pour augmenter le rendement de sucres fermentables pendant la saccharification d'un matière lignocellulosique par réduction de la quantité de primevérose produite.
PCT/US2017/059498 2016-11-02 2017-11-01 Procédés de réduction de la production de primevérose pendant la saccharification enzymatique de matière lignocellulosique Ceased WO2018085370A1 (fr)

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Citations (83)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990015861A1 (fr) 1989-06-13 1990-12-27 Genencor International, Inc. Procede pour la neutralisation de cellules sans lyse cellulaire
WO1991005039A1 (fr) 1989-09-26 1991-04-18 Midwest Research Institute Endoglucanases thermostables purifiees tirees de la bacterie thermophile acidothermus cellulolyticus
WO1991017243A1 (fr) 1990-05-09 1991-11-14 Novo Nordisk A/S Preparation de cellulase comprenant un enzyme d'endoglucanase
WO1991017244A1 (fr) 1990-05-09 1991-11-14 Novo Nordisk A/S Enzyme capable de degrader la cellulose ou l'hemicellulose
WO1992006204A1 (fr) 1990-09-28 1992-04-16 Ixsys, Inc. Banques de recepteurs heteromeres a expression en surface
US5223409A (en) 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
WO1993015186A1 (fr) 1992-01-27 1993-08-05 Midwest Research Institute Endoglucanases thermostables purifiees obtenues a partir de la bacterie thermophile acidothermus cellulolyticus
WO1994021785A1 (fr) 1993-03-10 1994-09-29 Novo Nordisk A/S Enzymes derivees d'aspergillus aculeatus presentant une activite de xylanase
WO1995017413A1 (fr) 1993-12-21 1995-06-29 Evotec Biosystems Gmbh Procede permettant une conception et une synthese evolutives de polymeres fonctionnels sur la base d'elements et de codes de remodelage
WO1995022625A1 (fr) 1994-02-17 1995-08-24 Affymax Technologies N.V. Mutagenese d'adn par fragmentation aleatoire et reassemblage
WO1996002551A1 (fr) 1994-07-15 1996-02-01 Midwest Research Institute Gene codant l'endoglucanase e1
EP1038878A1 (fr) * 1999-03-19 2000-09-27 Amano Pharmaceutical Co., Ltd. Méthode pour séparer un saccharide d'un glycoside
WO2000070031A1 (fr) 1999-05-19 2000-11-23 Midwest Research Institute Variants d'endoglucanase e1: y245g, y82r et w42r
US20020164730A1 (en) 2000-02-24 2002-11-07 Centro De Investigaciones Energeticas, Medioambientales Y Tecnologicas (C.I.E.M.A.T.) Procedure for the production of ethanol from lignocellulosic biomass using a new heat-tolerant yeast
WO2002095014A2 (fr) 2001-05-18 2002-11-28 Novozymes A/S Polypeptides presentant une activite de cellobiase et polynucleotides codant pour de tels polypeptides
WO2003062430A1 (fr) 2002-01-23 2003-07-31 Royal Nedalco B.V. Fermentation de sucres pentose
WO2003070956A1 (fr) 2002-02-19 2003-08-28 Novozymes A/S Procedes de clonage d'expression dans des champignons filamenteux
WO2005001036A2 (fr) 2003-05-29 2005-01-06 Genencor International, Inc. Nouveaux genes de trichoderma
WO2005047499A1 (fr) 2003-10-28 2005-05-26 Novozymes Inc. Polypeptides presentant une activite beta-glucosidase et polynucleotides codant pour ceux-ci
WO2005074656A2 (fr) 2004-02-06 2005-08-18 Novozymes, Inc. Polypeptides presentant une amelioration de l'activite cellulolytique et polynucleotides codant pour de tels polypeptides
WO2005074647A2 (fr) 2004-01-30 2005-08-18 Novozymes Inc. Polypeptides presentant une activite favorisant l'activite cellulolytique, et polynucleotides codant lesdits polypeptides
WO2005093050A2 (fr) 2004-03-25 2005-10-06 Genencor International, Inc. Proteine de fusion cellulase et construction de fusion cellulase heterologue codant ladite proteine
WO2006032282A1 (fr) 2004-09-24 2006-03-30 Cambi Bioethanol Aps Procede de traitement de biomasse et de dechets organiques pour la generation de produits a base biologique desires
WO2006074435A2 (fr) 2005-01-06 2006-07-13 Novozymes, Inc. Polypeptides possedant une activite de cellobiohydrlase et des polynucleotides codant ceux-ci
WO2006078256A2 (fr) 2004-02-12 2006-07-27 Novozymes, Inc. Polypeptides presentant une activite xylanase et polynucleotides codant pour ceux-ci
WO2006110900A2 (fr) 2005-04-12 2006-10-19 E. I. Du Pont De Nemours And Company Traitement de biomasse en vue d'obtenir de l'ethanol
WO2006114094A1 (fr) 2005-04-26 2006-11-02 Novozymes A/S Arabinofuranosidases
WO2007019442A2 (fr) 2005-08-04 2007-02-15 Novozymes, Inc. Polypeptides presentant une activite beta-glucosidase et polynucleotides codant pour ceux-ci
WO2007089290A2 (fr) 2005-09-30 2007-08-09 Novozymes, Inc. Procédés d'amélioration de la dégradation ou de la conversion de matière cellulosique
WO2008148131A1 (fr) 2007-05-31 2008-12-04 Novozymes, Inc. Polypeptides ayant une activité cellulolytique améliorée et les polynucléotides codant pour ceux-ci
WO2008151043A1 (fr) 2007-05-31 2008-12-11 Novozymes, Inc. Procédés d'augmentation de l'activité favorisant l'activité cellulolytique d'un polypeptide
WO2009033071A2 (fr) 2007-09-07 2009-03-12 Dyadic International, Inc. Enzymes fongiques inédites
WO2009042846A1 (fr) 2007-09-28 2009-04-02 Novozymes A/S Polypeptides à activité acétylxylane estérase et polynucléotides codant ces polypeptides
WO2009042871A1 (fr) 2007-09-28 2009-04-02 Novozymes A/S Polypeptides à activité de cellobiohydrolase ii et polynucléotides les codant
WO2009068565A1 (fr) 2007-11-27 2009-06-04 Novozymes A/S Polypeptides ayant une activité d'alpha-glucuronidase et polynucléotides codant pour ceux-ci
WO2009073383A1 (fr) 2007-11-30 2009-06-11 Novozymes A/S Polypeptides ayant une activité d'arabinofuranosidase et polynucléotides les encodant
WO2009073709A1 (fr) 2007-12-06 2009-06-11 Novozymes A/S Polypeptides ayant une activité d'acétylxylane estérase et polynucléotides les codant
WO2009076122A1 (fr) 2007-12-07 2009-06-18 Novozymes A/S Polypeptides ayant une activité féruloyl estérase et polynucléotides codant pour ceux-ci
WO2009079210A2 (fr) 2007-12-05 2009-06-25 Novozymes A/S Polypeptides ayant une activité de xylanase et polynucléotides codant pour ceux-ci
WO2009085935A2 (fr) 2007-12-19 2009-07-09 Novozymes A/S Polypeptides présentant une activité cellulolytique et polynucléotides codant pour ceux-ci
WO2009085864A2 (fr) 2007-12-19 2009-07-09 Novozymes A/S Polypeptides présentant une activité d'activation cellulolytique et polynucléotides codant pour ceux-ci
WO2009085868A1 (fr) 2007-12-19 2009-07-09 Novozymes A/S Polypeptides présentant une activité d'activation cellulolytique et polynucléotides codant pour ceux-ci
WO2009085859A2 (fr) 2007-12-19 2009-07-09 Novozymes A/S Polypeptides présentant une activité d'activation cellulolytique et polynucléotides codant pour ceux-ci
WO2009127729A1 (fr) 2008-04-17 2009-10-22 Novozymes A/S Polypeptides à activité acide férulique estérase et polynucléotides codant pour ceux-ci
WO2010014706A1 (fr) 2008-07-29 2010-02-04 Novozymes A/S Polypeptides présentant une activité alpha-glucuronidase et polynucléotides codant pour ceux-ci
WO2010014880A1 (fr) 2008-07-31 2010-02-04 Novozymes A/S Polypeptides ayant une activité d'acétylxylane estérase et polynucléotides codant ceux-ci
WO2010053838A1 (fr) 2008-11-10 2010-05-14 Novozymes, Inc Polypeptides ayant une activité feruloyl estérase et polynucléotides les codant
WO2010057086A2 (fr) 2008-11-14 2010-05-20 Microsoft Corporation Réutilisation de canal avec des signaux cognitifs de faible interférence
WO2010065830A1 (fr) 2008-12-04 2010-06-10 Novozymes, Inc. Polypeptides ayant une activité d’activation cellulolytique et polynucléotides codant pour ceux-ci
WO2010065448A1 (fr) 2008-12-04 2010-06-10 Novozymes, Inc. Polypeptides présentant une activité féruloylestérase et polynucléotides codant lesdits polypeptides
WO2010088387A1 (fr) 2009-01-28 2010-08-05 Novozymes, Inc. Polypeptides à activité bêta-glucosidase, et polynucléotides les codant
WO2010096673A1 (fr) 2009-02-20 2010-08-26 Danisco Us Inc. Préparations de bouillon de fermentation
WO2010108918A1 (fr) 2009-03-24 2010-09-30 Novozymes A/S Polypeptides exerçant une activité acétyl xylane estérase et polynucléotides les codant
WO2010126772A1 (fr) 2009-04-30 2010-11-04 Novozymes, Inc. Polypeptides ayant une activité xylanase et poly-nucléotides codant pour eux
WO2010138754A1 (fr) 2009-05-29 2010-12-02 Novozymes, Inc. Procédés d'amélioration de la dégradation ou de la conversion de matière cellulosique
WO2010141325A1 (fr) 2009-06-02 2010-12-09 Novozymes, Inc. Polypeptides ayant une activité cellobiohydrolase et polynucléotides les codant
WO2011005867A1 (fr) 2009-07-07 2011-01-13 Novozymes, Inc. Polypeptides ayant une activité cellulolytique améliorée et polynucléotides codant pour ceux-ci
WO2011035029A1 (fr) 2009-09-18 2011-03-24 Novozymes, Inc. Polypeptides à activité bêta-glucosidase et polynucléotides codant pour lesdits polypeptides
WO2011035027A2 (fr) 2009-09-17 2011-03-24 Novozymes, Inc. Polypeptides ayant une activité cellulolytique améliorée et polynucléotides codant pour ceux-ci
WO2011041504A1 (fr) 2009-09-30 2011-04-07 Novozymes, Inc. Polypeptides ayant une activité cellulolytique renforcée et polynucléotides codant pour ces polypeptides
WO2011041405A1 (fr) 2009-09-29 2011-04-07 Novozymes, Inc. Polypeptides présentant une activité xylanase et polynucléotides codant pour ceux-ci
WO2011041397A1 (fr) 2009-09-29 2011-04-07 Novozymes, Inc. Polypeptides présentant une activité favorisant l'activité cellulolytique et polynucléotides codant pour ceux-ci
WO2011039319A1 (fr) 2009-09-30 2011-04-07 Novozymes A/S Polypeptides ayant une activité cellulolytique amplifiée et polynucléotides codant pour ceux-ci
WO2011057083A1 (fr) 2009-11-06 2011-05-12 Novozymes, Inc. Polypeptides présentant une activité xylanase et polynucléotides codant pour ceux-ci
WO2011059740A1 (fr) 2009-10-29 2011-05-19 Novozymes, Inc. Polypeptides ayant une activité cellobiohydrolase et polynucléotides codant pour ceux-ci
WO2012000892A1 (fr) 2010-06-29 2012-01-05 Dsm Ip Assets B.V. Polypeptide présentant une activité de dégradation de glucides ou facilitant cette activité, et ses applications
WO2012021400A1 (fr) 2010-08-12 2012-02-16 Novozymes, Inc. Compositions comprenant un polypeptide présentant une activité augmentant la cellulolyse et un composé hétérocyclique, et leurs utilisations
WO2012030799A1 (fr) 2010-08-30 2012-03-08 Novozymes A/S Polypeptides ayant une activité d'amplification de la cellulolyse, et polynucléotides codant pour ceux-ci
WO2012062220A1 (fr) 2010-11-12 2012-05-18 Novozymes A/S Polypeptides ayant une activité endoglucanase et polynucléotides codant pour ceux-ci
WO2012101206A2 (fr) 2011-01-26 2012-08-02 Novozymes A/S Nouvelles glycosides hydrolases de champignons thermophiles
WO2012113340A1 (fr) 2011-02-23 2012-08-30 Novozymes Inc. Polypeptides permettant de faciliter l'activité cellulolytique et polynucléotides codant pour de tels polypeptides
WO2012122477A1 (fr) 2011-03-10 2012-09-13 Novozymes A/S Polypeptides ayant une activité améliorant l'activité cellulolytique et polynucléotides codant pour ceux-ci
WO2012122518A1 (fr) 2011-03-09 2012-09-13 Novozymes A/S Procédés permettant d'accroître l'activité de renforcement de la cellulolyse d'un polypeptide
US8268586B2 (en) 2006-12-21 2012-09-18 Novozymes, Inc. Modified messenger RNA stabilizing sequences for expressing genes in bacterial cells
WO2012129697A1 (fr) 2011-04-01 2012-10-04 Adrian Tsang Nouvelles enzymes de déconstruction de parois cellulaires de talaromyces thermophilus et leurs utilisations
WO2012135659A2 (fr) 2011-03-31 2012-10-04 Novozymes A/S Procédés d'augmentation de dégradation ou de conversion de matière cellulosique
WO2012130964A1 (fr) 2011-04-01 2012-10-04 Dsm Ip Assets B.V. Nouvelles enzymes de déconstruction des parois cellulaires de thermomyces lanuginosus et leurs utilisations
WO2012146171A1 (fr) 2011-04-25 2012-11-01 Novozymes, Inc. Polypeptides capables de favoriser l'activité cellulolytique et polynucléotides codant pour ceux-ci
WO2012149344A1 (fr) 2011-04-29 2012-11-01 Novozymes, Inc. Procédés pour améliorer la dégradation ou la conversion de matériau cellulosique
WO2013028928A1 (fr) 2011-08-24 2013-02-28 Novozymes, Inc. Compositions d'enzymes cellulolytiques et leurs utilisations
WO2013043910A1 (fr) 2011-09-20 2013-03-28 Novozymes A/S Polypeptides ayant une activité d'amélioration cellulolytique et polynucléotides les codant
US8580536B2 (en) 2009-11-06 2013-11-12 Novozymes, Inc. Compositions for saccharification of cellulosic material
WO2015175308A1 (fr) * 2014-05-13 2015-11-19 Danisco Us Inc. Hydrolyse enzymatique améliorée de biomasse

Patent Citations (97)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5223409A (en) 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
WO1990015861A1 (fr) 1989-06-13 1990-12-27 Genencor International, Inc. Procede pour la neutralisation de cellules sans lyse cellulaire
WO1991005039A1 (fr) 1989-09-26 1991-04-18 Midwest Research Institute Endoglucanases thermostables purifiees tirees de la bacterie thermophile acidothermus cellulolyticus
US5275944A (en) 1989-09-26 1994-01-04 Midwest Research Institute Thermostable purified endoglucanas from acidothermus cellulolyticus ATCC 43068
US5536655A (en) 1989-09-26 1996-07-16 Midwest Research Institute Gene coding for the E1 endoglucanase
WO1991017243A1 (fr) 1990-05-09 1991-11-14 Novo Nordisk A/S Preparation de cellulase comprenant un enzyme d'endoglucanase
WO1991017244A1 (fr) 1990-05-09 1991-11-14 Novo Nordisk A/S Enzyme capable de degrader la cellulose ou l'hemicellulose
WO1992006204A1 (fr) 1990-09-28 1992-04-16 Ixsys, Inc. Banques de recepteurs heteromeres a expression en surface
WO1993015186A1 (fr) 1992-01-27 1993-08-05 Midwest Research Institute Endoglucanases thermostables purifiees obtenues a partir de la bacterie thermophile acidothermus cellulolyticus
WO1994021785A1 (fr) 1993-03-10 1994-09-29 Novo Nordisk A/S Enzymes derivees d'aspergillus aculeatus presentant une activite de xylanase
WO1995017413A1 (fr) 1993-12-21 1995-06-29 Evotec Biosystems Gmbh Procede permettant une conception et une synthese evolutives de polymeres fonctionnels sur la base d'elements et de codes de remodelage
WO1995022625A1 (fr) 1994-02-17 1995-08-24 Affymax Technologies N.V. Mutagenese d'adn par fragmentation aleatoire et reassemblage
WO1996002551A1 (fr) 1994-07-15 1996-02-01 Midwest Research Institute Gene codant l'endoglucanase e1
EP1038878A1 (fr) * 1999-03-19 2000-09-27 Amano Pharmaceutical Co., Ltd. Méthode pour séparer un saccharide d'un glycoside
WO2000070031A1 (fr) 1999-05-19 2000-11-23 Midwest Research Institute Variants d'endoglucanase e1: y245g, y82r et w42r
US20020164730A1 (en) 2000-02-24 2002-11-07 Centro De Investigaciones Energeticas, Medioambientales Y Tecnologicas (C.I.E.M.A.T.) Procedure for the production of ethanol from lignocellulosic biomass using a new heat-tolerant yeast
WO2002095014A2 (fr) 2001-05-18 2002-11-28 Novozymes A/S Polypeptides presentant une activite de cellobiase et polynucleotides codant pour de tels polypeptides
WO2003062430A1 (fr) 2002-01-23 2003-07-31 Royal Nedalco B.V. Fermentation de sucres pentose
WO2003070956A1 (fr) 2002-02-19 2003-08-28 Novozymes A/S Procedes de clonage d'expression dans des champignons filamenteux
WO2005001036A2 (fr) 2003-05-29 2005-01-06 Genencor International, Inc. Nouveaux genes de trichoderma
WO2005047499A1 (fr) 2003-10-28 2005-05-26 Novozymes Inc. Polypeptides presentant une activite beta-glucosidase et polynucleotides codant pour ceux-ci
WO2005074647A2 (fr) 2004-01-30 2005-08-18 Novozymes Inc. Polypeptides presentant une activite favorisant l'activite cellulolytique, et polynucleotides codant lesdits polypeptides
WO2005074656A2 (fr) 2004-02-06 2005-08-18 Novozymes, Inc. Polypeptides presentant une amelioration de l'activite cellulolytique et polynucleotides codant pour de tels polypeptides
WO2006078256A2 (fr) 2004-02-12 2006-07-27 Novozymes, Inc. Polypeptides presentant une activite xylanase et polynucleotides codant pour ceux-ci
WO2005093050A2 (fr) 2004-03-25 2005-10-06 Genencor International, Inc. Proteine de fusion cellulase et construction de fusion cellulase heterologue codant ladite proteine
WO2006032282A1 (fr) 2004-09-24 2006-03-30 Cambi Bioethanol Aps Procede de traitement de biomasse et de dechets organiques pour la generation de produits a base biologique desires
WO2006074435A2 (fr) 2005-01-06 2006-07-13 Novozymes, Inc. Polypeptides possedant une activite de cellobiohydrlase et des polynucleotides codant ceux-ci
WO2006110900A2 (fr) 2005-04-12 2006-10-19 E. I. Du Pont De Nemours And Company Traitement de biomasse en vue d'obtenir de l'ethanol
WO2006110891A2 (fr) 2005-04-12 2006-10-19 E. I. Du Pont De Nemours And Company Obtention de produit chimique cible par traitement de biomasse
WO2006110901A2 (fr) 2005-04-12 2006-10-19 E. I. Du Pont De Nemours And Company Traitement de biomasse en vue d'obtenir des sucres fermentescibles
WO2006110899A2 (fr) 2005-04-12 2006-10-19 E. I. Du Pont De Nemours And Company Procede d'integration d'autres charges d'alimentation dans le traitement d'une biomasse et utilisation du procede
WO2006114094A1 (fr) 2005-04-26 2006-11-02 Novozymes A/S Arabinofuranosidases
WO2007019442A2 (fr) 2005-08-04 2007-02-15 Novozymes, Inc. Polypeptides presentant une activite beta-glucosidase et polynucleotides codant pour ceux-ci
WO2007089290A2 (fr) 2005-09-30 2007-08-09 Novozymes, Inc. Procédés d'amélioration de la dégradation ou de la conversion de matière cellulosique
US8268586B2 (en) 2006-12-21 2012-09-18 Novozymes, Inc. Modified messenger RNA stabilizing sequences for expressing genes in bacterial cells
WO2008148131A1 (fr) 2007-05-31 2008-12-04 Novozymes, Inc. Polypeptides ayant une activité cellulolytique améliorée et les polynucléotides codant pour ceux-ci
WO2008151043A1 (fr) 2007-05-31 2008-12-11 Novozymes, Inc. Procédés d'augmentation de l'activité favorisant l'activité cellulolytique d'un polypeptide
WO2009033071A2 (fr) 2007-09-07 2009-03-12 Dyadic International, Inc. Enzymes fongiques inédites
WO2009042846A1 (fr) 2007-09-28 2009-04-02 Novozymes A/S Polypeptides à activité acétylxylane estérase et polynucléotides codant ces polypeptides
WO2009042871A1 (fr) 2007-09-28 2009-04-02 Novozymes A/S Polypeptides à activité de cellobiohydrolase ii et polynucléotides les codant
WO2009068565A1 (fr) 2007-11-27 2009-06-04 Novozymes A/S Polypeptides ayant une activité d'alpha-glucuronidase et polynucléotides codant pour ceux-ci
WO2009073383A1 (fr) 2007-11-30 2009-06-11 Novozymes A/S Polypeptides ayant une activité d'arabinofuranosidase et polynucléotides les encodant
WO2009079210A2 (fr) 2007-12-05 2009-06-25 Novozymes A/S Polypeptides ayant une activité de xylanase et polynucléotides codant pour ceux-ci
WO2009073709A1 (fr) 2007-12-06 2009-06-11 Novozymes A/S Polypeptides ayant une activité d'acétylxylane estérase et polynucléotides les codant
WO2009076122A1 (fr) 2007-12-07 2009-06-18 Novozymes A/S Polypeptides ayant une activité féruloyl estérase et polynucléotides codant pour ceux-ci
WO2009085935A2 (fr) 2007-12-19 2009-07-09 Novozymes A/S Polypeptides présentant une activité cellulolytique et polynucléotides codant pour ceux-ci
WO2009085864A2 (fr) 2007-12-19 2009-07-09 Novozymes A/S Polypeptides présentant une activité d'activation cellulolytique et polynucléotides codant pour ceux-ci
WO2009085868A1 (fr) 2007-12-19 2009-07-09 Novozymes A/S Polypeptides présentant une activité d'activation cellulolytique et polynucléotides codant pour ceux-ci
WO2009085859A2 (fr) 2007-12-19 2009-07-09 Novozymes A/S Polypeptides présentant une activité d'activation cellulolytique et polynucléotides codant pour ceux-ci
WO2009127729A1 (fr) 2008-04-17 2009-10-22 Novozymes A/S Polypeptides à activité acide férulique estérase et polynucléotides codant pour ceux-ci
WO2010014706A1 (fr) 2008-07-29 2010-02-04 Novozymes A/S Polypeptides présentant une activité alpha-glucuronidase et polynucléotides codant pour ceux-ci
WO2010014880A1 (fr) 2008-07-31 2010-02-04 Novozymes A/S Polypeptides ayant une activité d'acétylxylane estérase et polynucléotides codant ceux-ci
WO2010053838A1 (fr) 2008-11-10 2010-05-14 Novozymes, Inc Polypeptides ayant une activité feruloyl estérase et polynucléotides les codant
WO2010057086A2 (fr) 2008-11-14 2010-05-20 Microsoft Corporation Réutilisation de canal avec des signaux cognitifs de faible interférence
WO2010065830A1 (fr) 2008-12-04 2010-06-10 Novozymes, Inc. Polypeptides ayant une activité d’activation cellulolytique et polynucléotides codant pour ceux-ci
WO2010065448A1 (fr) 2008-12-04 2010-06-10 Novozymes, Inc. Polypeptides présentant une activité féruloylestérase et polynucléotides codant lesdits polypeptides
WO2010088387A1 (fr) 2009-01-28 2010-08-05 Novozymes, Inc. Polypeptides à activité bêta-glucosidase, et polynucléotides les codant
WO2010096673A1 (fr) 2009-02-20 2010-08-26 Danisco Us Inc. Préparations de bouillon de fermentation
WO2010108918A1 (fr) 2009-03-24 2010-09-30 Novozymes A/S Polypeptides exerçant une activité acétyl xylane estérase et polynucléotides les codant
WO2010126772A1 (fr) 2009-04-30 2010-11-04 Novozymes, Inc. Polypeptides ayant une activité xylanase et poly-nucléotides codant pour eux
WO2010138754A1 (fr) 2009-05-29 2010-12-02 Novozymes, Inc. Procédés d'amélioration de la dégradation ou de la conversion de matière cellulosique
WO2010141325A1 (fr) 2009-06-02 2010-12-09 Novozymes, Inc. Polypeptides ayant une activité cellobiohydrolase et polynucléotides les codant
WO2011005867A1 (fr) 2009-07-07 2011-01-13 Novozymes, Inc. Polypeptides ayant une activité cellulolytique améliorée et polynucléotides codant pour ceux-ci
WO2011035027A2 (fr) 2009-09-17 2011-03-24 Novozymes, Inc. Polypeptides ayant une activité cellulolytique améliorée et polynucléotides codant pour ceux-ci
WO2011035029A1 (fr) 2009-09-18 2011-03-24 Novozymes, Inc. Polypeptides à activité bêta-glucosidase et polynucléotides codant pour lesdits polypeptides
WO2011041405A1 (fr) 2009-09-29 2011-04-07 Novozymes, Inc. Polypeptides présentant une activité xylanase et polynucléotides codant pour ceux-ci
WO2011041397A1 (fr) 2009-09-29 2011-04-07 Novozymes, Inc. Polypeptides présentant une activité favorisant l'activité cellulolytique et polynucléotides codant pour ceux-ci
WO2011041504A1 (fr) 2009-09-30 2011-04-07 Novozymes, Inc. Polypeptides ayant une activité cellulolytique renforcée et polynucléotides codant pour ces polypeptides
WO2011039319A1 (fr) 2009-09-30 2011-04-07 Novozymes A/S Polypeptides ayant une activité cellulolytique amplifiée et polynucléotides codant pour ceux-ci
WO2011059740A1 (fr) 2009-10-29 2011-05-19 Novozymes, Inc. Polypeptides ayant une activité cellobiohydrolase et polynucléotides codant pour ceux-ci
WO2011057083A1 (fr) 2009-11-06 2011-05-12 Novozymes, Inc. Polypeptides présentant une activité xylanase et polynucléotides codant pour ceux-ci
US8580536B2 (en) 2009-11-06 2013-11-12 Novozymes, Inc. Compositions for saccharification of cellulosic material
WO2012000892A1 (fr) 2010-06-29 2012-01-05 Dsm Ip Assets B.V. Polypeptide présentant une activité de dégradation de glucides ou facilitant cette activité, et ses applications
WO2012021394A1 (fr) 2010-08-12 2012-02-16 Novozymes, Inc. Compositions comprenant un polypeptide présentant une activité augmentant la cellulolyse et un composé de quinone, et leurs utilisations
WO2012021401A1 (fr) 2010-08-12 2012-02-16 Novozymes, Inc. Compositions comprenant un polypeptide ayant une activité améliorant la cellulolyse et un composé bicyclique, et utilisations correspondantes
WO2012021395A1 (fr) 2010-08-12 2012-02-16 Novozymes, Inc. Compositions comprenant un polypeptide à activité renforçant l'activité cellulolytique, composé contenant du soufre et utilisations correspondantes
WO2012021399A1 (fr) 2010-08-12 2012-02-16 Novozymes, Inc. Compositions contenant un polypeptide à activité cellulolytique renforcée et un composé à base d'azote, et leurs utilisations
WO2012021410A1 (fr) 2010-08-12 2012-02-16 Novozymes, Inc. Compositions contenant un polypeptide à activité cellulolytique renforcée et une solution aqueuse et leurs utilisations
WO2012021408A1 (fr) 2010-08-12 2012-02-16 Novozymes, Inc. Compositions comprenant un polypeptide ayant une activité cellulolytique accrue et un composé dioxy et les utilisations de celles-ci
WO2012021400A1 (fr) 2010-08-12 2012-02-16 Novozymes, Inc. Compositions comprenant un polypeptide présentant une activité augmentant la cellulolyse et un composé hétérocyclique, et leurs utilisations
WO2012021396A1 (fr) 2010-08-12 2012-02-16 Novozymes, Inc. Compositions comprenant un polypeptide présentant une activité augmentant la cellulolyse et un composé organique, et leurs utilisations
WO2012030799A1 (fr) 2010-08-30 2012-03-08 Novozymes A/S Polypeptides ayant une activité d'amplification de la cellulolyse, et polynucléotides codant pour ceux-ci
WO2012062220A1 (fr) 2010-11-12 2012-05-18 Novozymes A/S Polypeptides ayant une activité endoglucanase et polynucléotides codant pour ceux-ci
WO2012101206A2 (fr) 2011-01-26 2012-08-02 Novozymes A/S Nouvelles glycosides hydrolases de champignons thermophiles
WO2012113340A1 (fr) 2011-02-23 2012-08-30 Novozymes Inc. Polypeptides permettant de faciliter l'activité cellulolytique et polynucléotides codant pour de tels polypeptides
WO2012122518A1 (fr) 2011-03-09 2012-09-13 Novozymes A/S Procédés permettant d'accroître l'activité de renforcement de la cellulolyse d'un polypeptide
WO2012122477A1 (fr) 2011-03-10 2012-09-13 Novozymes A/S Polypeptides ayant une activité améliorant l'activité cellulolytique et polynucléotides codant pour ceux-ci
WO2012135659A2 (fr) 2011-03-31 2012-10-04 Novozymes A/S Procédés d'augmentation de dégradation ou de conversion de matière cellulosique
WO2012129697A1 (fr) 2011-04-01 2012-10-04 Adrian Tsang Nouvelles enzymes de déconstruction de parois cellulaires de talaromyces thermophilus et leurs utilisations
WO2012130950A1 (fr) 2011-04-01 2012-10-04 Dsm Ip Assets B.V. Nouvelles enzymes de déconstruction de la paroi des cellules de talaromyces thermophilus et utilisations de celles-ci
WO2012130964A1 (fr) 2011-04-01 2012-10-04 Dsm Ip Assets B.V. Nouvelles enzymes de déconstruction des parois cellulaires de thermomyces lanuginosus et leurs utilisations
WO2012129699A1 (fr) 2011-04-01 2012-10-04 Adrian Tsang Enzymes de déconstruction de parois cellulaires de thermomyces lanuginosus et leurs utilisations
WO2012146171A1 (fr) 2011-04-25 2012-11-01 Novozymes, Inc. Polypeptides capables de favoriser l'activité cellulolytique et polynucléotides codant pour ceux-ci
WO2012149344A1 (fr) 2011-04-29 2012-11-01 Novozymes, Inc. Procédés pour améliorer la dégradation ou la conversion de matériau cellulosique
WO2013028928A1 (fr) 2011-08-24 2013-02-28 Novozymes, Inc. Compositions d'enzymes cellulolytiques et leurs utilisations
WO2013043910A1 (fr) 2011-09-20 2013-03-28 Novozymes A/S Polypeptides ayant une activité d'amélioration cellulolytique et polynucléotides les codant
WO2015175308A1 (fr) * 2014-05-13 2015-11-19 Danisco Us Inc. Hydrolyse enzymatique améliorée de biomasse

Non-Patent Citations (141)

* Cited by examiner, † Cited by third party
Title
"GenBank", Database accession no. AB003107
"GenBank", Database accession no. AB003694
"GenBank", Database accession no. AF439936
"GenBank", Database accession no. AF487830
"GenBank", Database accession no. AY690482
"GenBank", Database accession no. L29381
"GenBank", Database accession no. M15665
"GenBank", Database accession no. M19373
"GenBank", Database accession no. MAL515703
"GenBank", Database accession no. XM_324477
"GenBank", Database accession no. Z33381
"More Gene Manipulations in Fungi", 1991, ACADEMIC PRESS
"Protein Purification", 1989, VCH PUBLISHERS
"The Alcohol Textbook", 1999, NOTTINGHAM UNIVERSITY PRESS
ALFENORE ET AL.: "Improving ethanol production and viability of Saccharomyces cerevisiae by a vitamin feeding strategy during fed-batch process", 2002, SPRINGER-VERLAG
ALIZADEH ET AL., APPL. BIOCHEM. BIOTECHNOL., vol. 121, 2005, pages 1133 - 1141
ANAGNOSTOPOULOS; SPIZIZEN, JOURNAL OF BACTERIOLOGY, vol. 81, 1961, pages 741 - 746
BAILEY ET AL.: "Interlaboratory testing of methods for assay of xylanase activity", JOURNAL OF BIOTECHNOLOGY, vol. 23, no. 3, 1992, pages 257 - 270, XP023704921, DOI: doi:10.1016/0168-1656(92)90074-J
BAILEY, J.E.; OLLIS, D.F.: "Biochemical Engineering Fundamentals", 1986, MCGRAW-HILL BOOK COMPANY
BALLESTEROS ET AL., APPL. BIOCHEM. BIOTECHNOL., vol. 129-132, 2006, pages 496 - 508
BEALL ET AL., BIOTECH. BIOENG., vol. 38, 1991, pages 296 - 303
BENDTSEN ET AL., J. MOL. BIOL., vol. 340, 2004, pages 783 - 795
BIELY; PUCHARD, JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, vol. 86, no. 11, 2006, pages 1636 - 1647
BOWIE; SAUER, PROC. NATL. ACAD. SCI., vol. 86, 1989, pages 2152 - 2156
CARTER ET AL., PROTEINS: STRUCTURE, FUNCTION, AND GENETICS, vol. 6, 1989, pages 240 - 248
CHANDRA ET AL., ADV. BIOCHEM. ENGIN./BIOTECHNOL., vol. 108, 2007, pages 67 - 93
CHEN; HO, APPL. BIOCHEM. BIOTECHNOL., vol. 39-40, 1993, pages 135 - 147
CHEN; LEE, APPL. BIOCHEM. BIOTECHNOL., vol. 63-65, 1997, pages 435 - 448
CHUNDAWAT, BIOTECHNOL. BIOENG., vol. 96, 2007, pages 219 - 231
COLLINS-RACIE ET AL., BIOTECHNOLOGY, vol. 13, 1995, pages 982 - 987
CONTRERAS ET AL., BIOTECHNOLOGY, vol. 9, 1991, pages 378 - 381
CUNNINGHAM; WELLS, SCIENCE, vol. 244, 1989, pages 1081 - 1085
DAN ET AL., J. BIOL. CHEM., vol. 275, 2000, pages 4973 - 4980
DE CASTILHOS CORAZZA ET AL., ACTA SCIENTIARUM. TECHNOLOGY, vol. 25, 2003, pages 33 - 38
DE VOS, SCIENCE, vol. 255, 1992, pages 306 - 312
DE VRIES, J. BACTERIOL., vol. 180, 1998, pages 243 - 249
DEANDA ET AL., APPL. ENVIRON. MICROBIOL., vol. 62, 1996, pages 4465 - 4470
DERBYSHIRE, GENE, vol. 46, 1986, pages 145
DUFF; MURRAY, BIORESOURCE TECHNOLOGY, vol. 855, 1996, pages 1 - 33
EATON ET AL., BIOCHEM., vol. 25, 1986, pages 505 - 512
EBRINGEROVA ET AL., ADV. POLYM. SCI., vol. 186, 2005, pages 1 - 67
EBRINGEROVA, ADV. POLYM. SCI., vol. 186, 2005, pages 1 - 67
EZEJI, WORLD JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY, vol. 19, no. 6, 2003, pages 595 - 603
GALBE; ZACCHI, ADV. BIOCHEM. ENGIN./BIOTECHNOL., vol. 108, 2007, pages 41 - 65
GALBE; ZACCHI, APPL. MICROBIOL. BIOTECHNOL., vol. 59, 2002, pages 618 - 628
GHOSE, PURE AND APPL. CHEM., vol. 59, 1987, pages 257 - 268
GHOSE, PURE APPL. CHEM., vol. 59, 1987, pages 257 - 68
GHOSE; BISARIA, PURE & APPL. CHEM., vol. 59, 1987, pages 1739 - 1752
GHOSH; SINGH, ADV. APPL. MICROBIOL., vol. 39, 1993, pages 295 - 333
GIESECKE ET AL., J. VIROL. METHODS, vol. 38, 1992, pages 47 - 60
GOLLAPALLI, APPL. BIOCHEM. BIOTECHNOL., vol. 98, 2002, pages 23 - 35
GONG, C. S.; CAO, N. J.; DU, J.; TSAO, G. T.: "Advances in Biochemical Engineering/Biotechnology", vol. 65, 1999, SPRINGER-VERLAG BERLIN HEIDELBERG, article "Ethanol production from renewable resources", pages: 207 - 241
GONG: "Advances in Biochemical Engineering/Biotechnology", vol. 65, 1999, SPRINGER-VERLAG, article "Ethanol production from renewable resources", pages: 207 - 241
GUNASEELAN, BIOMASS AND BIOENERGY, vol. 13, no. 1-2, 1997, pages 83 - 114
GUSAKOV; SINITSYN, ENZ. MICROB. TECHNOL., vol. 7, 1985, pages 346 - 352
H. NEURATH; R.L. HILL: "The Proteins", 1979, ACADEMIC PRESS
HENDRIKS; ZEEMAN, BIORESOURCE TECHNOLOGY, vol. 100, 2009, pages 10 - 18
HENRISSAT, BIOCHEM. J., vol. 280, 1991, pages 309 - 316
HENRISSAT; BAIROCH, BIOCHEM. J., vol. 316, 1996, pages 695 - 696
HERRMANN ET AL., BIOCHEMICAL JOURNAL, vol. 321, 1997, pages 375 - 381
HILTON, J. BIOL. CHEM., vol. 271, 1996, pages 4699 - 4708
HO ET AL., APPL. ENVIRON. MICROBIOL., vol. 64, 1998, pages 1852 - 1859
HSU, T.-A.: "Handbook on Bioethanol: Production and Utilization", 1996, TAYLOR & FRANCIS, article "Pretreatment of biomass", pages: 179 - 212
INGRAM ET AL., BIOTECHNOL. BIOENG., vol. 58, 1998, pages 204 - 214
KATAOKA, WATER SCIENCE AND TECHNOLOGY, vol. 36, no. 6-7, 1997, pages 41 - 47
KAWAGUCHI ET AL., GENE, vol. 173, 1996, pages 287 - 288
KOTTER; CIRIACY, APPL. MICROBIOL. BIOTECHNOL., vol. 38, 1993, pages 776 - 783
KURABI, APPL. BIOCHEM. BIOTECHNOL., vol. 121, 2005, pages 219 - 230
KUYPER ET AL., FEMS YEAST RESEARCH, vol. 4, 2004, pages 655 - 664
LEE, ADV. BIOCHEM. ENG. BIOTECHNOL., vol. 65, 1999, pages 93 - 115
LEVER ET AL., ANAL. BIOCHEM., vol. 47, 1972, pages 273 - 279
LEVER, ANAL. BIOCHEM., vol. 47, 1972, pages 273 - 279
LI ET AL., STRUCTURE, vol. 20, 2012, pages 1051 - 1061
LIN, APPL. MICROBIOL. BIOTECHNOL., vol. 69, 2006, pages 627 - 642
LOWMAN ET AL., BIOCHEMISTRY, vol. 30, 1991, pages 10832 - 10837
LYND, APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY, vol. 24/25, 1990, pages 695 - 719
LYND, MICROBIOL. MOL. BIOL. REVIEWS, vol. 66, 2002, pages 506 - 577
MARTIN ET AL., J. CHEM. TECHNOL. BIOTECHNOL., vol. 81, 2006, pages 1669 - 1677
MARTIN ET AL., J. IND. MICROBIOL. BIOTECHNOL., vol. 3, 2003, pages 568 - 76
MCKENZIE ET AL., PLASMID, vol. 15, no. 2, 1986, pages 93 - 103
MCMILLAN, J. D.: "Enzymatic Conversion of Biomass for Fuels Production", 1994, AMERICAN CHEMICAL SOCIETY, article "Pretreating lignocellulosic biomass: a review"
MOSIER ET AL., BIORESOURCE TECHNOLOGY, vol. 96, 2005, pages 673 - 686
MOSIER ET AL.: "Advances in Biochemical Engineering/Biotechnology", vol. 65, 1999, SPRINGER-VERLAG, article "Recent Progress in Bioconversion of Lignocellulosics", pages: 23 - 40
MOSIER, BIORESOURCE TECHNOLOGY, vol. 96, 2005, pages 673 - 686
MURPHY ET AL., EMBO JOURNAL, vol. 4, no. 12, 1985, pages 3357 - 3365
NEEDLEMAN; WUNSCH, J. MOL. BIOL., vol. 48, 1970, pages 443 - 453
NER, DNA, vol. 7, 1988, pages 127
NESS ET AL., NATURE BIOTECHNOLOGY, vol. 17, 1999, pages 893 - 896
NIGAM; SINGH, PROCESS BIOCHEMISTRY, vol. 30, no. 2, 1995, pages 117 - 124
OKADA ET AL., APPL. ENVIRON. MICROBIOL., vol. 64, 1988, pages 555 - 563
OLSSON; HAHN-HAGERDAL, ENZ. MICROB. TECH., vol. 18, 1996, pages 312 - 331
OOI ET AL., NUCLEIC ACIDS RESEARCH, vol. 18, 1990, pages 5884
PALONEN ET AL., APPL. BIOCHEM. BIOTECHNOL., vol. 117, 2004, pages 1 - 17
PAN ET AL., BIOTECHNOL. BIOENG., vol. 94, 2006, pages 851 - 861
PAN, BIOTECHNOL. BIOENG., vol. 90, 2005, pages 473 - 481
PENTTILA ET AL., GENE, vol. 45, 1986, pages 253 - 263
PHILIPPIDIS, G. P.: "Handbook on Bioethanol: Production and Utilization", 1996, TAYLOR & FRANCIS, article "Cellulose bioconversion technology", pages: 179 - 212
PHILLIPS ET AL., ACS CHEM. BIOL., vol. 6, 2011, pages 1399 - 1406
QUINLAN ET AL., PROC. NATL. ACAD. SCI., vol. 108, 2011, pages 15079 - 15084
RASMUSSEN ET AL., BIOTECHNOLOGY AND BIOENGINEERING, vol. 94, 2006, pages 869 - 876
RASMUSSEN-WILSON ET AL., APPL. ENVIRON. MICROBIOL., vol. 63, 1997, pages 3488 - 3493
REIDHAAR-OLSON; SAUER, SCIENCE, vol. 241, 1988, pages 53 - 57
RICE ET AL., TRENDS GENET., vol. 16, 2000, pages 276 - 277
RICHARD; MARGARITIS, BIOTECHNOLOGY AND BIOENGINEERING, vol. 87, no. 4, 2004, pages 501 - 515
RYU; LEE, BIOTECHNOL. BIOENG., vol. 25, 1983, pages 53 - 65
SAARILAHTI ET AL., GENE, vol. 90, 1990, pages 9 - 14
SAKAMOTO ET AL., CURRENT GENETICS, vol. 27, 1995, pages 435 - 439
SALOHEIMO ET AL., GENE, vol. 63, 1988, pages 11 - 22
SALOHEIMO ET AL., MOLECULAR MICROBIOLOGY, vol. 13, 1994, pages 219 - 228
SASSNER ET AL., ENZYME MICROB. TECHNOL., vol. 39, 2006, pages 756 - 762
SCHELL, APPL. BIOCHEM. BIOTECHNOL., vol. 105-108, 2003, pages 69 - 85
SCHELL, BIORESOURCE TECHNOLOGY, vol. 91, 2004, pages 179 - 188
SCHMIDT; THOMSEN, BIORESOURCE TECHNOLOGY, vol. 64, 1998, pages 139 - 151
SHALLOM; SHOHAM, CURRENT OPINION IN MICROBIOLOGY, vol. 6, no. 3, 2003, pages 219 - 228
SHEEHAN; HIMMEL, BIOTECHNOL. PROG., vol. 15, 1999, pages 817 - 827
SILVEIRA; JONAS, APPL. MICROBIOL. BIOTECHNOL., vol. 59, 2002, pages 400 - 408
SMITH ET AL., J. MOL. BIOL., vol. 224, 1992, pages 899 - 904
SPANIKOVA; BIELY, FEBS LETTERS, vol. 580, no. 19, 2006, pages 4597 - 4601
STEVENS, DRUG DISCOVERY WORLD, vol. 4, 2003, pages 35 - 48
SVETINA, J. BIOTECHNOL., vol. 76, 2000, pages 245 - 251
TAHERZADEH; KARIMI, INT. J. MOL. SCI., vol. 9, 2008, pages 1621 - 1651
TEERI ET AL., BIOCHEM. SOC. TRANS., vol. 26, 1998, pages 173 - 178
TEERI, TRENDS IN BIOTECHNOLOGY, vol. 15, 1997, pages 160 - 167
TEYMOURI ET AL., BIORESOURCE TECHNOLOGY, vol. 96, 2005, pages 2014 - 2018
TOMME ET AL., EUR. J. BIOCHEM., vol. 170, 1988, pages 575 - 581
VALLANDER; ERIKSSON, ADV. BIOCHEM. ENG./BIOTECHNOL., vol. 42, 1990, pages 63 - 95
VAN TILBEURGH ET AL., FEBS LETTERS, vol. 149, 1982, pages 152 - 156
VAN TILBEURGH; CLAEYSSENS, FEBS LETTERS, vol. 187, 1985, pages 283 - 288
VARGA ET AL., APPL. BIOCHEM. BIOTECHNOL., vol. 113-116, 2004, pages 509 - 523
VARGA ET AL., BIOTECHNOL. BIOENG., vol. 88, 2004, pages 567 - 574
VENTURI ET AL., J. BASIC MICROBIOL., vol. 42, 2002, pages 55 - 66
VETERE A ET AL: "Enzymatic synthesis and characterization of 6-O-beta-d-xylopyranosyl-2-acetamido-2-deoxy-d-glucopyranose, a structural analog of primeverose", CARBOHYDRATE RESEARCH, PERGAMON, GB, vol. 311, no. 1-2, 1 September 1998 (1998-09-01), pages 79 - 83, XP004142809, ISSN: 0008-6215, DOI: 10.1016/S0008-6215(98)00184-0 *
WALFRIDSSON ET AL., APPL. ENVIRON. MICROBIOL., vol. 61, 1995, pages 4184 - 4190
WARD ET AL., BIOTECHNOLOGY, vol. 13, 1995, pages 498 - 503
WISELOGEL ET AL.: "Handbook on Bioethanol", 1995, TAYLOR & FRANCIS, pages: 105 - 118
WLODAVER, FEBS LETT., vol. 309, 1992, pages 59 - 64
WYMAN, BIORESOURCE TECHNOLOGY, vol. 50, 1994, pages 3 - 16
WYMAN, BIORESOURCE TECHNOLOGY, vol. 96, 2005, pages 1959 - 1966
YANG; WYMAN, BIOFUELS BIOPRODUCTS AND BIOREFINING-BIOFPR., vol. 2, 2008, pages 26 - 40
ZHANG ET AL., SCIENCE, vol. 267, 1995, pages 240 - 243
ZHANG, BIOTECHNOLOGY ADVANCES, vol. 24, 2006, pages 452 - 481

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