Compositions comprising arabinofuranosidase and xylanase and their use for increasing hemicellulosic fiber solubilization

Abstract

The present invention relates to a composition comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, or a GH308 xylanase, and optionally a [beta]-xylosidase. The invention also relates to the use of this composition for increasing the dissolution of hemicellulosic fibers and the release of monomeric arabinose and xylose.

Description

[0001] References to sequence listings

[0002] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference. Technical Field

[0003] The present invention relates to a composition comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase or a GH30-8 xylanase, and optionally a β-xylosidase. The present invention also relates to the use of the composition for increasing the solubility of hemicellulose fibers and the release of monomeric arabinose and xylose. Background Art

[0004] Due to the high resistance of cellulosic feedstock, converting it to biofuel is challenging and typically involves a combination of thermochemical pretreatment followed by the addition of cellulases and hemicellulases to release soluble carbohydrates. Under government sustainability initiatives, the biofuel industry has been incentivized to produce ethanol from corn fiber in existing corn ethanol facilities. Corn fiber makes up 10% of the weight of the corn kernel and is composed of cellulose and hemicellulose from the aleurone and pericarp layers. In ethanol facilities, corn fiber ends up as distillers dried grains with solubles (DDGS). Enzymatic hydrolysis of the hemicellulose portion of corn fiber to monomeric C5 sugars (such as xylose and arabinose) while simultaneously fermenting the C5 sugars to ethanol by C5 fermenting yeasts and utilizing existing infrastructure would allow ethanol plants to produce additional cellulosic ethanol production from the same amount of corn. Additional benefits of corn fiber degradation include better feed quality for animal feed from DDGS due to its enriched protein content, and the lower fiber content of DDGS may qualify it for entry into the monogastric and aquaculture animal feed markets.

[0005] The arabinoxylan backbone in corn fiber consists of a xylan backbone of β-(1,4)-linked D-xylopyranosyl residues that is highly substituted with arabinose side chains and, to a lesser extent, glucuronic acid residues. The primary substitution with the arabinose residues is at the O-2 or O-3 position on monosubstituted xylopyranosyl units, or at both O-2 and O-3 positions on disubstituted xylopyranosyl units. In addition to arabinose, the xylan backbone can also be substituted with D-galactopyranosyl and D-glucuronic acid residues and / or with acetyl groups. Acetic acid is esterified directly to the xylan backbone at the O-2 or O-3 position, while hydroxycinnamic acids (such as ferulic acid, p-coumaric acid, and the dehydrodimer of ferulic acid) are esterified to the arabinofuranosyl unit at the O-5 position. It has also been reported that xylans are further substituted with xylopyranosyl groups via (1-3)-linkages, and that arabinofuranosyl groups can be further modified with xylopyranosyl groups or even L-galactopyranosyl groups. Due to the high degree of branching substitution with different moieties, the enzymatic degradation of corn fiber arabinoxylans into monomeric C5 sugars requires the synergistic action of a mixture of debranching and depolymerization activities. Debranching activities primarily include α-L-arabinofuranosidase (EC 3.2.1.55) (α-AraF), feruloyl esterase (EC 3.1.1.73), α-glucuronidase (EC 3.2.1.139) and / or acetylxylan esterase (EC 3.1.1.72), while depolymerization relies on endo-1,4-β-xylanase (EC 3.2.1.8) and β-xylosidase (EC 3.2.1.37) (BX) activities.

[0006] WO 2006 / 114095 "D1" describes a method and composition for hydrolyzing arabinoxylans, the method comprising contacting a substrate containing arabinoxylans with an enzyme active towards disubstituted arabinoses, such as, for example, a glycoside hydrolase family 43 (GH43) α-L-arabinofuranosidase, and with an enzyme active towards monosubstituted arabinoses at the C2 or C3 position, such as, for example, a GH family 51, 54, or 62 α-L-arabinofuranosidase. D1 teaches that when two arabinofuranosidases are added to an arabinoxylan solution, the resulting products will be high molecular weight linear xylose polymers and arabinose molecules, which allows for easy separation of the linear xylose polymers from the arabinose by known techniques. The linear xylose polymers can be further partially digested with enzyme activities, such as β-xylosidase (preferably GH3) and / or endo-1,4-β-xylanase (preferably GH10 or GH11), to produce xylo-oligosaccharides. D1 further teaches that when endo-1,4-β-xylanase and β-xylosidase are added to purified linear xylose polymers, the resulting product will be xylose essentially free of arabinose substituents, and that for the degradation of even more complex substrates, or where a more complete degradation is desired, the presence of further enzyme activities, such as acetylxylan esterase (EC 3.1.1.72) and / or feruloyl esterase (EC 3.1.1.73) and / or α-glucuronidase (EC.3.2.1.139), may be required.

[0007] However, supply chain disruptions and inflation have increased the cost of raw material inputs required to produce the enzymes needed to fully hydrolyze complex arabinoxylan substrates, reducing the financial incentive for ethanol facilities to purchase additional enzymes to produce cellulosic ethanol from corn. Since conventional wisdom suggests that all seven enzyme activities are required to maximize cellulosic ethanol yield from corn, there is a need for improved methods and compositions that can maximize cellulosic ethanol yield from existing corn inputs by releasing more of the less enzymatically active monomers arabinose and xylose and increasing cellulosic ethanol yield at a lower cost that is more profitable for corn ethanol facilities. Summary of the Invention

[0008] The present invention provides a solution to the above problems by providing compositions comprising GH43 arabinofuranosidase, GH51 arabinofuranosidase and GH5 xylanase or GH30-8 xylanase, which unexpectedly increase hemicellulose fiber solubilization and release significantly more monomeric arabinose and xylose compared to compositions combining GH43 and GH51 arabinofuranosidase alone or both with GH8 xylanase, GH10 xylanase or GH11 xylanase. Surprisingly and unexpectedly, the compositions of the present invention significantly increase the yield of monomeric arabinose and xylose without the need for β-xylosidase, acetylxylan esterase, feruloyl esterase and / or α-glucuronidase, although the addition of GH3 β-xylosidase to the compositions further increases these yields.

[0009] In one aspect, the present invention provides a composition comprising:

[0010] (a) GH43 arabinofuranosidase;

[0011] (b) GH51 arabinofuranosidase; and

[0012] (c) GH5 xylanase.

[0013] In an embodiment, the composition comprises a β-xylosidase. In an embodiment, the β-xylosidase is a GH3 β-xylosidase. In an embodiment, the GH5 xylanase is a GH5_21 xylanase.

[0014] The GH43 and GH51 arabinofuranosidases and the GH5_21 xylanase together released at least 150% more arabinose from liquefied corn mash than the combination of the GH43 and GH51 arabinofuranosidases when the GH5_21 xylanase was not present in the composition. The GH43 and GH51 arabinofuranosidases and the GH5_21 xylanase together released at least 2-fold more arabinose from liquefied corn mash than the combination of the GH43 and GH51 arabinofuranosidases when the GH5_21 xylanase was not present in the composition. The GH43 and GH51 arabinofuranosidases, the GH3 β-xylosidase, and the GH5_21 xylanase together released at least 10% more xylose and at least 20% more arabinose from liquefied corn mash than the combination of the GH43 and GH51 arabinofuranosidases and the GH3 β-xylosidase when the GH5_21 xylanase was not present in the composition. The GH43 and GH51 arabinofuranosidases, GH3 β-xylosidases, and GH5_21 xylanase together release up to about 70% more xylose and up to about 100% more arabinose from liquefied corn mash than the combination of the GH43 and GH51 arabinofuranosidases and GH3 β-xylosidases in the absence of the GH5_21 xylanase in the composition. The GH43 and GH51 arabinofuranosidases, GH3 β-xylosidases, and GH5_21 xylanase together release at least 150% more xylose from liquefied corn mash than the combination of the GH43 and GH51 arabinofuranosidases in the absence of the GH3 β-xylosidase and GH5_21 xylanase in the composition. The GH43 and GH51 arabinofuranosidases, GH3 β-xylosidase, and GH5_21 xylanase together release at least 2.5 times more xylose from liquefied corn mash than the combination of GH43 and GH51 arabinofuranosidases when the GH3 β-xylosidase and GH5_21 xylanase are not present in the composition.

[0015] In embodiments, the GH5 xylanase is a GH5_35 xylanase. The GH43 and GH51 arabinofuranosidases, the GH3 β-xylosidase, and the GH5_35 xylanase together release at least 50% more xylose and at least 60% more arabinose from liquefied corn mash than the combination of the GH43 and GH51 arabinofuranosidases and the GH3 β-xylosidase in the absence of the GH5_35 xylanase in the composition.

[0016] In one aspect, the present invention provides a composition comprising:

[0017] (a) GH43 arabinofuranosidase;

[0018] (b) GH51 arabinofuranosidase; and

[0019] (c)GH30_8 xylanase.

[0020] The GH43 and GH51 arabinofuranosidases, together with the GH30_8 xylanase, release at least 150% more arabinose from liquefied corn mash than the combination of the GH43 and GH51 arabinofuranosidases when the GH30_8 xylanase is not present in the composition. The GH43 and GH51 arabinofuranosidases, together with the GH30_8 xylanase, release at least 2-fold more arabinose from liquefied corn mash than the combination of the GH43 and GH51 arabinofuranosidases when the GH30_8 xylanase is not present in the composition.

[0021] In one aspect, the invention provides a method for producing a fermentation product from starch-containing material, the method comprising:

[0022] (a) saccharifying a starch-containing material with an α-amylase and a glucoamylase at a temperature below the initial gelatinization temperature of the starch to produce fermentable sugars; and

[0023] (b) fermenting the sugars with a fermenting organism to produce the fermentation product, wherein a composition comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, and a GH5 xylanase (e.g., a GH5_21 xylanase) or a GH30_8 xylanase is present or added during step (a) and / or step (b).

[0024] In one aspect, the invention provides a method for producing a fermentation product from starch-containing material, the method comprising:

[0025] (a) liquefying a starch-containing material with a thermostable α-amylase at a temperature above the initial gelatinization temperature of the starch to produce dextrins;

[0026] (b) saccharifying the dextrin with glucoamylase to produce fermentable sugars;

[0027] (c) fermenting the sugars with a fermenting organism to produce the fermentation product, wherein a composition comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, and a GH5 xylanase (e.g., a GH5_21 xylanase) or a GH30_8 xylanase is present or added during step (b) and / or step (c).

[0028] In the methods of the present invention, solubilization of hemicellulose fibers is increased to release significantly more monomeric arabinose and monomeric xylose compared to the following control methods: (i) lacking the composition; (ii) using a composition comprising only individual GH43 and GH51 arabinofuranosidases; (iii) using a composition comprising GH43 and GH51 arabinofuranosidases and a GH8 xylanase; (iv) using a composition comprising GH43 and GH51 arabinofuranosidases and a GH10 xylanase; and / or (v) using a composition comprising GH43 and GH51 arabinofuranosidases and a GH11 xylanase.

[0029] In the process of the present invention, residual solids are reduced compared to a control process lacking the composition.

[0030] The compositions of the present invention can be used for saccharification, fermentation, or simultaneous saccharification and fermentation to solubilize hemicellulose fibers into monomeric arabinose and xylose in conventional starch-to-ethanol processes and raw starch hydrolysis (RSH).

[0031] definition

[0032] α-L-Arabinofuranosidase: "α-L-Arabinofuranosidase" refers to an α-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55), which catalyzes the hydrolysis of terminal non-reducing α-L-arabinofuranoside residues in α-L-arabinosides. This enzyme acts on α-L-arabinofuranosides, α-L-arabinans containing (1,3)- and / or (1,5)-linkages, arabinoxylans, and arabinogalactans. α-L-Arabinofuranosidase is also known as arabinofuranosidase, α-arabinofuranosidase, α-L-arabinofuranosidase, α-arabinofuranosidase, polysaccharide α-L-arabinofuranosidase, α-L-arabinofuranoside hydrolase, L-arabinofuranosidase, or α-L-arabinanase.

[0033] α-L-Arabinofuranosidase activity: For the purposes of the present invention, α-L-arabinofuranosidase activity was determined using 5 mg of medium viscosity wheat arabinoxylan (Megazyme International Ireland, Ltd., Bray, Co., Wicklow, Ireland) per ml in 100 mM sodium acetate (pH 5) in a total volume of 200 μl at 40°C for 30 minutes, followed by arabinose analysis by AMINEX(R) HPX-87H column chromatography (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

[0034] β-Xylosidase: "β-Xylosidase" means a β-D-xyloside xylose hydrolase (EC 3.2.1.37) that catalyzes the exohydrolysis of short β(1-4)-xylooligosaccharides to remove consecutive D-xylose residues from the non-reducing ends.

[0035] β-Xylosidase Activity: For the purposes of the present invention, one unit of β-xylosidase is defined as the production of 1.0 micromole of p-nitrophenolate anion per minute from 1 mM p-nitrophenyl-β-D-xyloside as a substrate in 100 mM sodium citrate containing 0.01% TWEEN(R) 20 at 40°C, pH 5, in a total volume of 200 microliters.

[0036] Fermentation product: "Fermentation product" means a product produced by a process involving fermentation using a fermenting organism. Fermentation products include alcohols (e.g., ethanol, methanol, butanol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, succinic acid, gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H2 and CO2); antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B 12 , β-carotene); and hormones. In a preferred embodiment, the fermentation product is ethanol, for example, fuel ethanol; drinking ethanol, i.e., neutral drinking alcohol; or industrial ethanol or products used in the consumer alcohol industry (e.g., beer and wine), the dairy industry (e.g., fermented dairy products), the leather industry, and the tobacco industry. Preferred beer types include ale, stout, porter, lager, bitter, malt liquor, happy beer, high alcohol beer, low alcohol beer, low calorie beer, or light beer. In an embodiment, the fermentation product is ethanol.

[0037] Fermenting Organism: "Fermenting organism" refers to any organism, including bacterial and fungal organisms, especially yeast, suitable for use in a fermentation process and capable of producing the desired fermentation product.

[0038] GH3 β-xylosidase: "GH3 β-xylosidase" is the abbreviation of glycoside hydrolase family 3 β-xylosidase, which is a xylan 1,4-β-xylosidase (EC 3.2.1.37) that catalyzes the hydrolysis of (1→4)-β-D-xylans to remove consecutive D-xylose residues from the non-reducing ends.

[0039] GH5 xylanase: "GH5 xylanase" is an abbreviation for glycoside hydrolase family 5 xylanases, which mainly consist of endo-1,4-β-xylanases (EC 3.2.1.8), which catalyze the endohydrolysis of (1→4)-β-D-xylosidic linkages in xylans.

[0040] GH5_21 Xylanase: "GH5_21 Xylanase" is an abbreviation for glycoside hydrolase family 5 subfamily 21 endo-β-1,4-xylanase, which has a three-dimensional structure characterized by a (β / α)8 barrel and uses a glutamine residue as a catalytic nucleophile / base.

[0041] GH5_35 Xylanase: "GH5_35 Xylanase" is an abbreviation for glycoside hydrolase family 5 subfamily 35 endo-β-1,4-xylanase, which has a three-dimensional structure characterized by a (β / α)8 barrel and uses a glutamine residue as a catalytic nucleophile / base.

[0042] GH8 Xylanase: "GH8 Xylanase" is an abbreviation for glycoside hydrolase family 8 xylanases, which consists of endo-1,4-β-xylanases (EC 3.2.1.8), which catalyze the endohydrolysis of (1→4)-β-D-xylosidic linkages in xylans.

[0043] GH10 xylanase: "GH10 xylanase" is an abbreviation for glycoside hydrolase family 10 xylanases, which consists of endo-1,3-β-xylanases (EC 3.2.1.32) that catalyze the random endohydrolysis of (1→3)-β-D-glycosidic bonds in (1→3)-β-D-xylans and endo-1,4-β-xylanases (EC 3.2.1.8) that catalyze the endohydrolysis of (1→4)-β-D-xylosidic bonds in xylans.

[0044] GH11 Xylanase: “GH11 Xylanase” is an abbreviation for Glycoside Hydrolase Family 11 Xylanases, which are endo-β-1,4-xylanases (EC 3.2.1.8) that catalyze the endohydrolysis of (1→4)-β-D-xylosidic linkages in xylans.

[0045] GH30_8 Xylanase: "GH30_8 Xylanase" is an abbreviation for glycoside hydrolase 30 subfamily 8 xylanases, which include endo-β-1,4-xylanases (EC 3.2.1.8) that catalyze the endohydrolysis of (1→4)-β-D-xylosidic linkages in xylans and glucuronylarabinoxylan-specific endo-β-1,4-xylanases (EC 3.2.1.136) that catalyze the endohydrolysis of (1→4)-β-D-xylosidic linkages in some glucuronylarabinoxylans.

[0046] GH43 Arabinofuranosidase: "GH43 Arabinofuranosidase" is an abbreviation for glycoside hydrolase family 43 arabinofuranosidase, which is an α-L-arabinofuranosidase (EC 3.2.1.55) that catalyzes the hydrolysis of terminal non-reducing α-L-arabinofuranoside residues in α-L-arabinosides.

[0047] GH51 Arabinofuranosidase: "GH51 Arabinofuranosidase" is an abbreviation for Glycoside Hydrolase Family 51 Arabinofuranosidase, which is an α-L-arabinofuranosidase (EC 3.2.1.55) that catalyzes the hydrolysis of terminal non-reducing α-L-arabinofuranoside residues in α-L-arabinosides.

[0048] Initial gelatinization temperature: "Initial gelatinization temperature" means the lowest temperature at which gelatinization of starch begins. Starch heated in water begins to gelatinize between 50°C and 75°C; the exact temperature of gelatinization depends on the specific starch and can be easily determined by the skilled person. Therefore, the initial gelatinization temperature can vary depending on the plant species, the specific variety of the plant species, and the growing conditions. In the context of the present disclosure, the initial gelatinization temperature of a given starch-containing material is the temperature at which 5% of the starch granules lose their birefringence using the method described by Gorinstein.S. and Lii.C., Starch / Starke [Starch], Vol. 44(12), pp. 461-466 (1992).

[0049] Sequence identity: the degree of association between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence identity". The sequence identity between two amino acid sequences is determined as the output of "longest identity" using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol. [Journal of Molecular Biology] 48: 443-453), as implemented in the Needle program of version 6.6.0 of the EMBOSS software package (EMBOSS:The European Molecular Biology Open Software Suite [EMBOSS:European Molecular Biology Open Software Suite], Rice et al., 2000, Trends Genet. [Genetics Trends] 16: 276-277). The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order for the Needle program to report the longest identity, the non-simplified (-nobrief) option must be specified in the command line. The output of the "longest identity" of the Needle tag is calculated as follows:

[0050] (identical residues x 100) / (alignment length - total number of gaps in the alignment)

[0051] Use Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, the same) to determine the sequence identity between two polynucleotide sequences as the output of " longest identity ", this algorithm is as implemented in the Needle program of EMBOSS software package (EMBOSS:TheEuropean Molecular Biology Open Software Suite [EMBOSS:European Molecular Biology Open Software Suite], Rice et al., 2000, the same) (6.6.0 version).The parameters used are gap opening penalty 10, gap extension penalty 0.5 and EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix.In order to make Needle program report the longest identity, it is necessary to specify non-simplified options in the command line.The output calculation of " longest identity " of Needle mark is as follows:

[0052] (number of identical deoxyribonucleotides x 100) / (length of alignment – ​​total number of gaps in the alignment)

[0053] Thermostable: "Thermostable" means that the enzyme is not denatured or inactivated when used in the liquefaction step of the process of the present invention. In other words, a thermostable enzyme is suitable for liquefaction if it has a denaturation temperature (Td) that is compatible with the liquefaction temperature and retains its activity at this temperature.

[0054] Thin Stillage: "Thin Stillage" refers to the separated liquid from the whole stillage, which is pumped to the evaporator to be concentrated into a slurry.

[0055] Whole stillage: "Whole stillage" includes the material remaining at the end of the distillation process after recovery of fermentation products, such as ethanol.

[0056] Xylanases: "Xylanase" encompasses endo-1,4-β-xylanases (EC 3.2.1.8) that catalyze the endohydrolysis of (1→4)-β-D-xylosidic linkages in xylans and glucuronylarabinoxylan endo-1,4-β-xylanases (EC 3.2.1.136) that catalyze the endohydrolysis of 1,4-β-D-xylosyl linkages in some glucuronylarabinoxylans.

[0057] Xylanase activity: EC 3.2.1.8 xylanase activity can be determined using birchwood xylan as substrate. One unit of xylanase is defined as the activity of the enzyme in the initial hydrolysis at 50°C, pH 5, in a solution containing 0.01% The activity of the EC 3.2.1.136 xylanase can be measured at 37°C in 0.01% HO and 50 mM sodium acetate at 1 liter per minute from 2 g of birchwood xylan as substrate (measured in glucose equivalents as described in Lever, 1972, A new reaction for colorimetric determination of carbohydrates, Anal. Biochem 47:273-279). The activity was determined using 0.2% AZCL-glucuronoxylan as substrate in 200 mM sodium phosphate (pH 6) and X-100. One unit of xylanase activity was defined as the production of 1.0 μmol of azurin per minute from 0.2% AZCL-glucuronoxylan as substrate in 200 mM sodium phosphate (pH 6) at 37°C. DETAILED DESCRIPTION

[0058] I. Composition

[0059] The present invention relates to a composition comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, and a GH5 xylanase or a GH30_8 xylanase.

[0060] The work described herein unexpectedly demonstrates that compositions comprising GH43 and GH51 arabinofuranosidases and a GH5 xylanase or a GH30-8 xylanase release significantly more arabinose from hemicellulosic fiber (e.g., in liquefied corn mash) compared to otherwise identical compositions lacking the GH5 or GH30-8 xylanase. In particular, the work described herein demonstrates that compositions comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, and a GH8 polypeptide having xylanase activity, a GH10 polypeptide having xylanase activity, or a GH11 polypeptide having xylanase activity unexpectedly release significantly more monomeric arabinose than compositions comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, and a GH5 xylanase or a GH30-8 xylanase. Surprisingly, compositions comprising a GH5_21 xylanase or a GH30_8 xylanase and a GH43 and GH51 arabinofuranosidase enhanced the release of monomeric arabinose three-fold over the no enzyme control and more than two-fold over compositions comprising GH43 and GH51 arabinofuranosidases alone, while compositions comprising GH43 and GH51 arabinofuranosidases and a GH8 xylanase, a GH10 xylanase, or a GH11 xylanase performed comparable or slightly better in releasing arabinose than compositions comprising GH43 and GH51 arabinofuranosidases alone.

[0061] The work described herein further unexpectedly demonstrates that the addition of a β-xylosidase to a composition comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, and either a GH5_21 xylanase or a GH30_8 xylanase further increases the amount of monomeric arabinose and / or monomeric xylose released from hemicellulosic fiber in liquefied corn mash.

[0062] The present invention contemplates using the compositions of the present invention in saccharification, fermentation, or simultaneous saccharification and fermentation to increase the solubilization of hemicellulosic fiber to monomeric sugars (eg, arabinose and xylose) in conventional and raw starch hydrolysis (RSH) ethanol production processes.

[0063] A. Compositions Comprising GH5 Xylanases

[0064] In one aspect, the compositions of the invention comprise:

[0065] (a) GH43 arabinofuranosidase;

[0066] (b) GH51 arabinofuranosidase; and

[0067] (c) GH5 xylanase.

[0068] In embodiments, the composition comprises a β-xylosidase. In embodiments, the β-xylosidase is a GH3 β-xylosidase.

[0069] In an embodiment, the GH5 xylanase is a GH5_21 xylanase. Compared to similar compositions without the GH5_21 xylanase, the compositions of the present invention comprising the GH5_21 xylanase provide unexpected and surprising benefits. The arabinose released from liquefied corn mash by the GH43 and GH51 arabinofuranosidases together with the GH5_21 xylanase is at least 150% greater than the combination of the GH43 and GH51 arabinofuranosidases when the GH5_21 xylanase is not present in the composition. The arabinose released from liquefied corn mash by the GH43 and GH51 arabinofuranosidases together with the GH5_21 xylanase is at least 2 times greater than the combination of the GH43 and GH51 arabinofuranosidases when the GH5_21 xylanase is not present in the composition. The GH43 and GH51 arabinofuranosidases, GH3 β-xylosidases, and GH5_21 xylanase together release at least 10% more xylose and at least 20% more arabinose from liquefied corn mash than the combination of the GH43 and GH51 arabinofuranosidases and GH3 β-xylosidases when the GH5_21 xylanase is not present in the composition. The GH43 and GH51 arabinofuranosidases, GH3 β-xylosidases, and GH5_21 xylanase together release up to about 70% more xylose and up to about 100% more arabinose from liquefied corn mash than the combination of the GH43 and GH51 arabinofuranosidases and GH3 β-xylosidases when the GH5_21 xylanase is not present in the composition. The GH43 and GH51 arabinofuranosidases, GH3 β-xylosidase, and GH5_21 xylanase together release at least 150% more xylose from liquefied corn mash than the combination of the GH43 and GH51 arabinofuranosidases when the GH3 β-xylosidase and GH5_21 xylanase are not present in the composition. The GH43 and GH51 arabinofuranosidases, GH3 β-xylosidase, and GH5_21 xylanase together release at least 2.5 times more xylose from liquefied corn mash than the combination of the GH43 and GH51 arabinofuranosidases when the GH3 β-xylosidase and GH5_21 xylanase are not present in the composition. The composition comprising GH43 and GH51 arabinofuranosidases together with a GH5-21 xylanase released at least 2-fold more arabinose from liquefied corn mash than a composition comprising the GH43 and GH51 arabinofuranosidases alone or in combination with a GH8 xylanase, a GH10 xylanase, or a GH11 xylanase.

[0070] In an embodiment, the GH5 xylanase is a GH5_35 xylanase. The compositions of the present invention comprising the GH5_35 xylanase provide unexpected and surprising benefits compared to similar compositions without the GH5_35 xylanase. The GH43 and GH51 arabinofuranosidases, the GH3 β-xylosidase, and the GH5_35 xylanase together release at least 50% more xylose and at least 60% more arabinose from liquefied corn mash than the combination of the GH43 and GH51 arabinofuranosidases and the GH3 β-xylosidase when the GH5_35 xylanase is not present in the composition.

[0071] B. Compositions containing GH30_8 xylanases

[0072] In one aspect, the compositions of the invention comprise:

[0073] (a) GH43 arabinofuranosidase;

[0074] (b) GH51 arabinofuranosidase; and

[0075] (c)GH30_8 xylanase.

[0076] The compositions of the present invention comprising a GH30_8 xylanase provide unexpected and surprising benefits compared to similar compositions without the GH30_8 xylanase. The GH43 and GH51 arabinofuranosidases, together with the GH30_8 xylanase, release at least 150% more arabinose from liquefied corn mash than the combination of the GH43 and GH51 arabinofuranosidases when the GH30_8 xylanase is not present in the composition. The GH43 and GH51 arabinofuranosidases, together with the GH30_8 xylanase, release at least 2-fold more arabinose from liquefied corn mash than the combination of the GH43 and GH51 arabinofuranosidases when the GH30_8 xylanase is not present in the composition.

[0077] Compositions comprising GH43 and GH51 arabinofuranosidases together with a GH30-8 xylanase released at least 2-fold more arabinose from hemicellulosic fiber in liquefied corn mash than compositions comprising the GH43 and GH51 arabinofuranosidases alone or in combination with a GH8 xylanase, a GH10 xylanase, or a GH11 xylanase.

[0078] In embodiments, the composition further comprises a β-xylosidase. In embodiments, the β-xylosidase is a GH3 β-xylosidase.

[0079] In one aspect, the compositions of the invention comprise:

[0080] (a) GH43 arabinofuranosidase;

[0081] (b) GH51 arabinofuranosidase;

[0082] (c) GH3 β-xylosidase; and

[0083] (d) GH5 xylanase.

[0084] The GH43 and GH51 arabinofuranosidases, GH3 β-xylosidase, and GH5_21 xylanase together release at least 10% more xylose and at least 20% more arabinose from liquefied corn mash than the combination of the GH43 and GH51 arabinofuranosidases and GH3 β-xylosidase when the GH5_21 xylanase is not present in the composition.

[0085] The GH43 and GH51 arabinofuranosidases, GH3 β-xylosidase, and GH5_21 xylanase together release up to about 70% more xylose and up to about 100% more arabinose from liquefied corn mash than the combination of the GH43 and GH51 arabinofuranosidases and GH3 β-xylosidase when the GH5_21 xylanase is not present in the composition.

[0086] C. Compositions without GH8, GH10 and / or GH11 xylanases

[0087] Surprisingly, the work described herein demonstrates that compositions comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase and a GH8 xylanase, a GH10 xylanase, or a GH11 xylanase do not provide the same significant increase in monomeric arabinose release compared to compositions comprising GH43 and GH51 arabinofuranosidases and either a GH5-21 or GH30-8 xylanase. In fact, the examples demonstrate that such compositions perform comparable to or only slightly better than compositions comprising only GH43 and GH51 arabinofuranosidases.

[0088] Thus, the present invention contemplates embodiments in which the composition lacks a GH8, GH10, or GH11 xylanase. In embodiments, the composition does not include a GH8 xylanase. In embodiments, the composition does not include a GH10 xylanase. In embodiments, the composition does not include a GH11 xylanase.

[0089] A. Exemplary GH43 arabinofuranosidases

[0090] Aspects of the invention relate to compositions comprising a GH43 arabinofuranosidase in combination with other enzymes to increase hemicellulose fiber solubilization and the production of monomeric arabinose and / or xylose. The invention contemplates any GH43 arabinofuranosidase that, when used in combination with a GH51 arabinofuranosidase, a GH5 xylanase, or a GH30-8 xylanase, and optionally a β-xylosidase, increases the production of monomeric arabinose and / or xylose compared to a composition comprising the GH43 and GH51 arabinofuranosidases alone.

[0091] In embodiments, the GH43 arabinofuranosidase is a GH43 subfamily 36 arabinofuranosidase.

[0092] Exemplary GH43 arabinofuranosidases can be from Humicola, Lasiodiplodia, or Poronia.

[0093] Exemplary GH43 arabinofuranosidases can be from the species Humicola insolens, Lasiodiplodia theobromae, and Poronia punctata.

[0094] Exemplary GH43 arabinofuranosidases are arabinofuranosidases having the amino acid sequence of SEQ ID NO: 1. In embodiments, the GH43 arabinofuranosidase has the amino acid sequence of SEQ ID NO: 1 with 0 to 10 conservative amino acid substitutions and has arabinofuranosidase activity. In embodiments, the GH43 arabinofuranosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 and has arabinofuranosidase activity. Exemplary GH43 arabinofuranosidases are arabinofuranosidases having the amino acid sequence of SEQ ID NO: 2. In embodiments, the GH43 arabinofuranosidase has the amino acid sequence of SEQ ID NO: 2 with 0 to 10 conservative amino acid substitutions and has arabinofuranosidase activity. In embodiments, the GH43 arabinofuranosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 2 and has arabinofuranosidase activity. An exemplary GH43 arabinofuranosidase is an arabinofuranosidase having the amino acid sequence of SEQ ID NO: 3. In embodiments, the GH43 arabinofuranosidase has the amino acid sequence of SEQ ID NO: 3 with 0 to 10 conservative amino acid substitutions and has arabinofuranosidase activity. In embodiments, the GH43 arabinofuranosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 3 and has arabinofuranosidase activity.

[0095] The GH43 arabinofuranosidase can be dosed in pre-saccharification, saccharification, and / or simultaneous saccharification and fermentation at a concentration between 0.0001-1 mg EP (enzyme protein) / g DS, e.g., 0.0005-0.5 mg EP / g DS, such as 0.001-0.1 mg EP / g DS or 0.001-0.01 mg EP / g DS.

[0096] B. Exemplary GH51 Arabinofuranosidases

[0097] Aspects of the invention relate to compositions comprising a GH51 arabinofuranosidase in combination with other enzymes to increase hemicellulose fiber solubilization and the production of monomeric arabinose and / or xylose. The invention contemplates the use of any GH51 arabinofuranosidase that, when used in combination with a GH43 arabinofuranosidase, a GH5 xylanase, or a GH30-8 xylanase, and optionally a beta-xylosidase, increases the production of monomeric arabinose and / or xylose compared to a composition comprising the GH43 and GH51 arabinofuranosidases alone.

[0098] In embodiments, the GH51 arabinofuranosidase is a GH51 subfamily 6 arabinofuranosidase.

[0099] Exemplary GH51 arabinofuranosidases include, but are not limited to, those from Meripilus, Chromodispora, or Acidiella.

[0100] Exemplary GH51 arabinofuranosidases include, but are not limited to, those from the species Meripilus giganteus, Lactodiplodia theobrominum, or Acidiella bohemica.

[0101] Exemplary GH51 arabinofuranosidases have the amino acid sequence of SEQ ID NO: 4. In embodiments, the GH51 arabinofuranosidase has the amino acid sequence of SEQ ID NO: 4 with 0 to 10 conservative amino acid substitutions and has arabinofuranosidase activity. In embodiments, the GH51 arabinofuranosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 4 and has arabinofuranosidase activity. Exemplary GH51 arabinofuranosidases have the amino acid sequence of SEQ ID NO: 5. In embodiments, the GH51 arabinofuranosidase has the amino acid sequence of SEQ ID NO: 5 with 0 to 10 conservative amino acid substitutions and has arabinofuranosidase activity. In embodiments, the GH51 arabinofuranosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 5 and has arabinofuranosidase activity. Exemplary GH51 arabinofuranosidases have the amino acid sequence of SEQ ID NO: 6. In embodiments, the GH51 arabinofuranosidase has the amino acid sequence of SEQ ID NO: 6 with 0 to 10 conservative amino acid substitutions and has arabinofuranosidase activity. In embodiments, the GH51 arabinofuranosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 6 and has arabinofuranosidase activity.

[0102] The GH51 arabinofuranosidase can be dosed in presaccharification, saccharification, and / or simultaneous saccharification and fermentation at a concentration between 0.0001-1 mg EP (enzyme protein) / g DS, e.g., 0.0005-0.5 mg EP / g DS, such as 0.001-0.1 mg EP / g DS or 0.001-0.01 mg EP / g DS.

[0103] C. Exemplary GH5 Xylanases

[0104] Aspects of the invention relate to compositions comprising a GH5 family xylanase, an arabinofuranosidase, in combination with other enzymes to increase hemicellulose fiber solubilization and the production of monomeric arabinose and / or xylose. The invention contemplates the use of any GH5 xylanase that, when used in combination with a GH43 and GH51 arabinofuranosidase, and optionally a β-xylosidase, increases the production of monomeric arabinose and / or xylose compared to a composition comprising the GH43 and GH51 arabinofuranosidases alone.

[0105] In embodiments, the xylanase is a GH5 family xylanase.

[0106] In embodiments, the xylanase is a GH5-21 xylanase.

[0107] Exemplary GH5-21 xylanases include, but are not limited to, those from Bacteroides, Belliella, Chryseobacterium, or Sphingobacterium.

[0108] Exemplary GH5-21 xylanases include, but are not limited to, those from species Bacteroides cellulosilyticus CL02Y12C19, Bell's sp.-64282, Chryseobacterium sp., Chryseobacterium on corhynchi, or Sphingobacterium sp.-64162.

[0109] Exemplary GH5_21 xylanases include, but are not limited to, those from a bioreactor metagenome, an elephant dung metagenome, a xanthan alkaline community O, a xanthan alkaline community S, or a xanthan alkaline community T.

[0110] Exemplary GH5_21 xylanases have the amino acid sequence of SEQ ID NO: 7. In embodiments, the GH5_21 xylanases have the amino acid sequence of SEQ ID NO: 7 with 0 to 10 conservative amino acid substitutions and have xylanase activity. In embodiments, the GH_21 xylanases have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 7 and have xylanase activity. Exemplary GH5_21 xylanases have the amino acid sequence of SEQ ID NO: 8. In embodiments, the GH5_21 xylanases have the amino acid sequence of SEQ ID NO: 8 with 0 to 10 conservative amino acid substitutions and have xylanase activity. In an embodiment, the GH_21 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 8 and has xylanase activity. An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 9. In an embodiment, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 9 with 0 to 10 conservative amino acid substitutions and has xylanase activity. In an embodiment, the GH5_21 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 9 and has xylanase activity. Exemplary GH5_21 xylanases have the amino acid sequence of SEQ ID NO: 10. In embodiments, the GH5_21 xylanases have the amino acid sequence of SEQ ID NO: 10 with 0 to 10 conservative amino acid substitutions and have xylanase activity. In embodiments, the GH5_21 xylanases have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 10 and have xylanase activity. Exemplary GH5_21 xylanases have the amino acid sequence of SEQ ID NO: 11. In embodiments, the GH5_21 xylanases have the amino acid sequence of SEQ ID NO: 11 with 0 to 10 conservative amino acid substitutions and have xylanase activity.In embodiments, the GH5_21 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 11 and has xylanase activity. Exemplary GH5_21 xylanases have the amino acid sequence of SEQ ID NO: 12. In embodiments, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 12 with 0 to 10 conservative amino acid substitutions and has xylanase activity. In embodiments, the GH5_21 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 12 and has xylanase activity. Exemplary GH5_21 xylanases have the amino acid sequence of SEQ ID NO: 13. In embodiments, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 13 with 0 to 10 conservative amino acid substitutions and has xylanase activity. In embodiments, the GH5_21 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 13 and has xylanase activity. Exemplary GH5_21 xylanases have the amino acid sequence of SEQ ID NO: 14. In embodiments, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 14 with 0 to 10 conservative amino acid substitutions and has xylanase activity. In embodiments, the GH5_21 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 14 and has xylanase activity. Exemplary GH5_21 xylanases have the amino acid sequence of SEQ ID NO: 15. In embodiments, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 15 with 0 to 10 conservative amino acid substitutions and has xylanase activity.In embodiments, the GH5_21 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 15 and has xylanase activity. Exemplary GH5_21 xylanases have the amino acid sequence of SEQ ID NO: 16. In embodiments, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 16 with 0 to 10 conservative amino acid substitutions and has xylanase activity. In embodiments, the GH5_21 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 16 and has xylanase activity. Exemplary GH5_21 xylanases have the amino acid sequence of SEQ ID NO: 17. In embodiments, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 17 with 0 to 10 conservative amino acid substitutions and has xylanase activity. In embodiments, the GH5_21 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 17 and has xylanase activity. Exemplary GH5_21 xylanases have the amino acid sequence of SEQ ID NO: 18. In embodiments, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 18 with 0 to 10 conservative amino acid substitutions and has xylanase activity. In embodiments, the GH5_21 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 18 and has xylanase activity. Exemplary GH5_21 xylanases have the amino acid sequence of SEQ ID NO: 19. In embodiments, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 19 with 0 to 10 conservative amino acid substitutions and has xylanase activity.In embodiments, the GH5_21 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 19 and has xylanase activity. Exemplary GH5_21 xylanases have the amino acid sequence of SEQ ID NO: 20. In embodiments, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 20 with 0 to 10 conservative amino acid substitutions and has xylanase activity. In embodiments, the GH5_21 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 20 and has xylanase activity. Exemplary GH5_21 xylanases have the amino acid sequence of SEQ ID NO: 21. In embodiments, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 21 with 0 to 10 conservative amino acid substitutions and has xylanase activity. In embodiments, the GH5_21 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 21 and has xylanase activity.

[0111] In embodiments, the GH5 xylanase is a GH5_35 xylanase.

[0112] Exemplary GH5-35 xylanases include, but are not limited to, those from Bacillus, Cohnella, or Paenibacillus.

[0113] Exemplary GH5-35 xylanases include, but are not limited to, those from the species Bacillus hemiccellulosilyticus JCM 9152, Cohnella xylanilytica, Paenibacillus chitinolyticus, or Paenibacillus sp.-62332.

[0114] Exemplary GH5_35 xylanases include, but are not limited to, those from compost metagenomes.

[0115] Exemplary GH5_35 xylanases have the amino acid sequence of SEQ ID NO: 22. In embodiments, the GH5_35 xylanases have the amino acid sequence of SEQ ID NO: 22 with 0 to 10 conservative amino acid substitutions and have xylanase activity. In embodiments, the GH_35 xylanases have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 22 and have xylanase activity. Exemplary GH5_35 xylanases have the amino acid sequence of SEQ ID NO: 23. In embodiments, the GH5_35 xylanases have the amino acid sequence of SEQ ID NO: 23 with 0 to 10 conservative amino acid substitutions and have xylanase activity. In embodiments, the GH5_35 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 23 and has xylanase activity. An exemplary GH5_35 xylanase has the amino acid sequence of SEQ ID NO: 24. In embodiments, the GH5_35 xylanase has the amino acid sequence of SEQ ID NO: 24 with 0 to 10 conservative amino acid substitutions and has xylanase activity. In embodiments, the GH5_35 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 24 and has xylanase activity. Exemplary GH5_35 xylanases have the amino acid sequence of SEQ ID NO: 25. In embodiments, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 25 with 0 to 10 conservative amino acid substitutions and has xylanase activity. In embodiments, the GH5_35 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 25 and has xylanase activity. Exemplary GH5_35 xylanases have the amino acid sequence of SEQ ID NO: 26. In embodiments, the GH5_35 xylanase has the amino acid sequence of SEQ ID NO: 26 with 0 to 10 conservative amino acid substitutions and has xylanase activity.In embodiments, the GH5_35 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 26 and has xylanase activity.

[0116] GH5 xylanases can be dosed in pre-saccharification, saccharification, and / or simultaneous saccharification and fermentation at a concentration of between 0.0001-1 mg EP (enzyme protein) / g DS, e.g., 0.0005-0.5 mg EP / g DS, such as 0.001-0.1 mg EP / g DS or 0.001-0.01 mg EP / g DS. D. Exemplary GH30-8 xylanases

[0117] Aspects of the invention relate to compositions comprising a GH30_8 xylanase in combination with other enzymes to increase hemicellulose fiber solubilization and the production of monomeric arabinose and / or xylose. The invention contemplates the use of any GH30_8 xylanase, when used in combination with a GH43 and GH51 arabinofuranosidase, and optionally a β-xylosidase, to increase the production of monomeric arabinose and / or xylose compared to a composition comprising the GH43 and GH51 arabinofuranosidases alone.

[0118] Exemplary GH30-8 xylanases include, but are not limited to, those from Bacillus.

[0119] Exemplary GH30-8 xylanases include, but are not limited to, those from species Bacillus sp.-18423.

[0120] Exemplary GH30_8 xylanases have the amino acid sequence of SEQ ID NO: 27. In embodiments, the GH30_8 xylanases have the amino acid sequence of SEQ ID NO: 27 with 0 to 10 conservative amino acid substitutions and have xylanase activity. In embodiments, the GH30_8 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 27 and has xylanase activity.

[0121] Additional exemplary GH30-8 xylanases suitable for use in the compositions of the invention are described in WO 2019 / 055455 (which is incorporated herein by reference in its entirety).

[0122] The GH30_8 xylanase can be dosed in pre-saccharification, saccharification, and / or simultaneous saccharification and fermentation at a concentration of between 0.0001-1 mg EP (enzyme protein) / g DS, e.g., 0.0005-0.5 mg EP / g DS, such as 0.001-0.1 mg EP / g DS or 0.001-0.01 mg EP / g DS.

[0123] E. Exemplary β-xylosidases

[0124] Aspects of the invention relate to compositions comprising β-xylosidases (e.g., GH3 β-xylosidases) in combination with other enzymes to increase hemicellulose fiber solubilization and the production of monomeric arabinose and / or xylose. The invention contemplates the use of any β-xylosidase (e.g., GH3 β-xylosidases) that, when used in combination with GH43 and GH51 arabinofuranosidases and GH5 xylanases or GH30-8 xylanases, further increase the production of monomeric arabinose and / or xylose compared to compositions comprising GH43, GH51, and GH5 xylanases or GH30-8 xylanases alone.

[0125] In embodiments, the β-xylosidase is a GH3 β-xylosidase.

[0126] Exemplary GH3 β-xylosidases include, but are not limited to, those from Alternaria, Aspergillus, Chaetomium, Fusarium, Mycothermus, Penicillium, Sporormia, Talaromyces, or Trichoderma.

[0127] Exemplary GH3 β-xylosidases include, but are not limited to, those from the following species: Alternaria tellustris, Aspergillus aculeatus, Aspergillus fischeri, Aspergillus fumigatus, Aspergillus nidulans, Chaetomium globosum, Chaetomium virescens, Fusarium longipes, Mycothermus thermophilus, Penicillium emersonii, Penicillium oxalicum, Sporormia fimetaria, Talaromyces emersonii, Talaromyces stipitatus, or Trichoderma reesei.

[0128] Exemplary GH3 β-xylosidases have the amino acid sequence of SEQ ID NO: 28. In embodiments, the GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 28 with 0 to 10 conservative amino acid substitutions and has β-xylosidase activity. In embodiments, the GH3 β-xylosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 28 and has β-xylosidase activity.

[0129] Exemplary GH3 β-xylosidases have the amino acid sequence of SEQ ID NO: 29. In embodiments, the GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 29 with 0 to 10 conservative amino acid substitutions and has β-xylosidase activity. In embodiments, the GH3 β-xylosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 29 and has β-xylosidase activity.

[0130] Exemplary GH3 β-xylosidases have the amino acid sequence of SEQ ID NO: 30. In embodiments, the GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 30 with 0 to 10 conservative amino acid substitutions and has β-xylosidase activity. In embodiments, the GH3 β-xylosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 30.

[0131] Exemplary GH3 β-xylosidases have the amino acid sequence of SEQ ID NO: 44. In embodiments, the GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 44 with 0 to 10 conservative amino acid substitutions and has β-xylosidase activity. In embodiments, the GH3 β-xylosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 44 and has β-xylosidase activity.

[0132] Exemplary GH3 β-xylosidases have the amino acid sequence of SEQ ID NO: 45. In embodiments, the GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 45 with 0 to 10 conservative amino acid substitutions and has β-xylosidase activity. In embodiments, the GH3 β-xylosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 45 and has β-xylosidase activity.

[0133] Exemplary GH3 β-xylosidases have the amino acid sequence of SEQ ID NO: 46. In embodiments, the GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 46 with 0 to 10 conservative amino acid substitutions and has β-xylosidase activity. In embodiments, the GH3 β-xylosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 46 and has β-xylosidase activity.

[0134] Exemplary GH3 β-xylosidases have the amino acid sequence of SEQ ID NO: 47. In embodiments, the GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 47 with 0 to 10 conservative amino acid substitutions and has β-xylosidase activity. In embodiments, the GH3 β-xylosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 47 and has β-xylosidase activity.

[0135] Exemplary GH3 β-xylosidases have the amino acid sequence of SEQ ID NO: 48. In embodiments, the GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 48 with 0 to 10 conservative amino acid substitutions and has β-xylosidase activity. In embodiments, the GH3 β-xylosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 48 and has β-xylosidase activity.

[0136] Exemplary GH3 β-xylosidases have the amino acid sequence of SEQ ID NO: 49. In embodiments, the GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 49 with 0 to 10 conservative amino acid substitutions and has β-xylosidase activity. In embodiments, the GH3 β-xylosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 49 and has β-xylosidase activity.

[0137] Exemplary GH3 β-xylosidases have the amino acid sequence of SEQ ID NO: 50. In embodiments, the GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 50 with 0 to 10 conservative amino acid substitutions and has β-xylosidase activity. In embodiments, the GH3 β-xylosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 50 and has β-xylosidase activity.

[0138] Exemplary GH3 β-xylosidases have the amino acid sequence of SEQ ID NO: 51. In embodiments, the GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 51 with 0 to 10 conservative amino acid substitutions and has β-xylosidase activity. In embodiments, the GH3 β-xylosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 51 and has β-xylosidase activity.

[0139] Exemplary GH3 β-xylosidases have the amino acid sequence of SEQ ID NO: 52. In embodiments, the GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 52 with 0 to 10 conservative amino acid substitutions and has β-xylosidase activity. In embodiments, the GH3 β-xylosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 52 and has β-xylosidase activity.

[0140] Exemplary GH3 β-xylosidases have the amino acid sequence of SEQ ID NO: 53. In embodiments, the GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 53 with 0 to 10 conservative amino acid substitutions and has β-xylosidase activity. In embodiments, the GH3 β-xylosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 53 and has β-xylosidase activity.

[0141] Exemplary GH3 β-xylosidases have the amino acid sequence of SEQ ID NO: 54. In embodiments, the GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 54 with 0 to 10 conservative amino acid substitutions and has β-xylosidase activity. In embodiments, the GH3 β-xylosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 54 and has β-xylosidase activity.

[0142] Exemplary GH3 β-xylosidases have the amino acid sequence of SEQ ID NO: 55. In embodiments, the GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 55 with 0 to 10 conservative amino acid substitutions and has β-xylosidase activity. In embodiments, the GH3 β-xylosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 55 and has β-xylosidase activity.

[0143] The β-xylosidase (e.g., GH3 β-xylosidase) can be dosed in pre-saccharification, saccharification, and / or simultaneous saccharification and fermentation at a concentration of between 0.0001-1 mg EP (enzyme protein) / g DS, e.g., 0.0005-0.5 mg EP / g DS, such as 0.001-0.1 mg EP / g DS or 0.001-0.01 mg EP / g DS.

[0144] F. Exemplary Fermenting Organisms

[0145] Aspects of the present invention relate to the use of fermenting organisms for producing fermentation products. Particularly suitable fermenting organisms are capable of fermenting sugars (such as arabinose, glucose, maltose and / or xylose) directly or indirectly into (i.e., converting into) a desired fermentation product (such as ethanol). Examples of fermenting organisms include fungal organisms, such as yeast. Preferred yeasts include strains of Saccharomyces species, particularly Saccharomyces cerevisiae.

[0146] Examples of commercially available yeast include, for example, RED STAR TM and ETHANOL RED TM Yeast (available from Fermentisis / Lesaffre, USA), FALI (available from Fleischmann's Yeast, USA), SUPERSTART and THERMOSACC TMFresh yeast (available from Ethanol Technology, Wisconsin, USA), BIOFERM AFT and XR (available from NABC-North American Bioproducts Corporation, Georgia, USA), GERT STRAND (available from Gert Strand AB, Sweden), and FERMIOL (available from DSM Specialties). Other useful yeast strains are available from biological depositories such as the American Type Culture Collection (ATCC) or the German Collection of Microorganisms (DSMZ), such as, for example, BY4741 (e.g., ATCC 201388); Y108-1 (ATCC PTA.10567), and NRRL YB-1952 (Agricultural Research Culture Collection). Other suitable host cells include Saccharomyces cerevisiae strains DBY746, [Alpha][Eta]22, S150-2B, GPY55-15Ba, CEN.PK, USM21, TMB3500, TMB3400, VTT-A-63015, VTT-A-85068, VTT-c-79093 and derivatives thereof, and Saccharomyces species 1400, 424A (LNH-ST), 259A (LNH-ST) and derivatives thereof.

[0147] As used herein, " derivatives " of bacterial strains are derived from reference strains, such as by mutagenesis, recombinant DNA technology, mating, cell fusion, or cell conduction between yeast strains. It will be understood by those skilled in the art that genetic changes (including metabolic modifications exemplified herein) can be described with reference to suitable host organisms and their corresponding metabolic reactions or suitable source organisms for desired genetic material (such as genes of desired metabolic pathways). However, in view of the high-level skills in the whole genome sequencing and genomics fields of various organisms, those skilled in the art can apply the teachings and guidance provided herein to other organisms. For example, the metabolic changes exemplified herein can be easily applied to other species by incorporating the same or similar encoding nucleic acids from species different from the reference species.

[0148] The fermenting organism can be a strain of Saccharomyces, such as a strain of Saccharomyces cerevisiae produced using the methods described and referred to in U.S. Patent No. 8,257,959-BB. In one embodiment, the recombinant cell is a derivative of strain Saccharomyces cerevisiae CIBTS1260 (deposited with the Agricultural Research Service Type Culture Collection (NRRL) in Illinois, USA under accession number NRRL Y-50973).

[0149] The fermenting organism may also be Saccharomyces cerevisiae strain NMI V14 / 004037 (see, WO 2015 / 143324 and WO 2015 / 143317, each of which is incorporated herein by reference), strain numbers V15 / 004035, V15 / 004036, and V15 / 004037 (see, WO 2016 / 153924, incorporated herein by reference), strain numbers V15 / 001459, V15 / 001460, V15 / 001461 (see, WO 2016 / 138437, incorporated herein by reference), strain number NRRL Y67342 (see, WO 2018 / 098381, incorporated herein by reference), strain numbers NRRL Y67549 and NRRL Y67700 (see, WO 2016 / 153924, incorporated herein by reference), 2019 / 161227, incorporated herein by reference), or derivatives of any strain described in WO 2017 / 087330 (incorporated herein by reference).

[0150] The fermenting organism may comprise one or more heterologous polynucleotides encoding alpha-amylases, glucoamylases, proteases and / or cellulases. Examples of alpha-amylases, glucoamylases, proteases and cellulases suitable for expression in fermenting organisms are known in the art (see WO 2021 / 231623, which is incorporated herein by reference).

[0151] The fermenting organism can be in the form of a composition comprising the fermenting organism and naturally occurring and / or non-naturally occurring components. The fermenting organism can be in any active form, including crushing, drying, including active dry and instant, compressed, paste (liquid) forms, etc. In one embodiment, the fermenting organism (e.g., a saccharomyces cerevisiae strain) is a dry yeast, such as active dry yeast or instant yeast. In one embodiment, the fermenting organism is a crushed yeast. In one embodiment, the fermenting organism is a compressed yeast. In one embodiment, the fermenting organism is a paste yeast.

[0152] In one embodiment is a composition comprising a fermenting organism as described herein (eg, a strain of Saccharomyces cerevisiae) and one or more components selected from the group consisting of surfactants, emulsifiers, gums, swelling agents, and antioxidants and other processing aids.

[0153] The compositions described herein can comprise a fermenting organism described herein (e.g., a Saccharomyces cerevisiae strain) and any suitable surfactant. In one embodiment, the one or more surfactants are anionic surfactants, cationic surfactants, and / or nonionic surfactants.

[0154] The compositions described herein can comprise a fermenting organism as described herein (e.g., a Saccharomyces cerevisiae strain) and any suitable emulsifier. In one embodiment, the emulsifier is a fatty acid ester of sorbitan. In one embodiment, the emulsifier is selected from the group consisting of sorbitan monostearate (SMS), citric acid mono- and diglycerides, polyglycerol esters, and propylene glycol fatty acid esters.

[0155] In one embodiment, the composition comprises a fermenting organism as described herein (e.g., a Saccharomyces cerevisiae strain) and Olindronal SMS, Olindronal SK, or Olindronal SPL, including compositions described in European Patent No. 1,724,336 (incorporated herein by reference). For active dry yeast, these products are commercially available from Bussetti, Austria.

[0156] The compositions described herein can comprise a fermenting organism as described herein (e.g., a Saccharomyces cerevisiae strain) and any suitable gum. In one embodiment, the gum is selected from the group consisting of locust bean gum, guar gum, tragacanth gum, gum arabic, xanthan gum, and gum arabic, particularly for paste, compressed, and dry yeast.

[0157] The compositions described herein can comprise a fermenting organism described herein (eg, a Saccharomyces cerevisiae strain) and any suitable swelling agent. In one embodiment, the swelling agent is methylcellulose or carboxymethylcellulose.

[0158] The compositions described herein can comprise a fermenting organism as described herein (e.g., a Saccharomyces cerevisiae strain) and any suitable antioxidant. In one embodiment, the antioxidant is butylated hydroxyanisole (BHA) and / or butylated hydroxytoluene (BHT), or ascorbic acid (vitamin C), particularly for active dry yeast.

[0159] Suitable concentrations of viable fermenting organisms during fermentation (e.g., SSF) are well known in the art or can be readily determined by one skilled in the art. In one embodiment, the fermenting organism (e.g., ethanol fermenting yeast (e.g., Saccharomyces cerevisiae)) is added to the fermentation medium so that the viable fermenting organism (e.g., yeast) counts per mL of fermentation medium are between 10 and 20. 5 to 10 12 , preferably from 10 7 to 10 10 range, especially about 5x10 7 indivual.

[0160] II. Methods for producing fermentation products from gelatinized starch-containing material

[0161] One aspect of the invention relates to a method for producing a fermentation product (e.g., fuel ethanol) from gelatinized starch-containing material, wherein a GH5 xylanase or GH30-8 xylanase of the invention, or a composition comprising a GH5 xylanase of the invention, is present or added during saccharification or fermentation. This method of the invention contemplates any of the compositions described in Section I above, including any combination of the enzymes described in Section II, and particularly the compositions demonstrated in the Examples below.

[0162] In an embodiment, a method for producing a fermentation product from starch-containing material comprises the following steps:

[0163] (a) liquefying a starch-containing material using a thermostable α-amylase at a temperature above the initial gelatinization temperature of the starch to produce dextrins;

[0164] (b) saccharifying the dextrin using glucoamylase to produce fermentable sugars; and

[0165] (c) fermenting the sugar using a fermenting organism to produce the fermentation product;

[0166] wherein a composition comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase and a GH5 xylanase or a GH30-8 xylanase is present or added during step (b) and / or step (c).

[0167] In embodiments, the composition used in step (b) and / or step (c) comprises a β-xylosidase. In embodiments, the composition used in step (b) and / or step (c) comprises a GH3 β-xylosidase. In embodiments, the composition is added during saccharification step (b). In embodiments, the composition is added during fermentation step (c). In embodiments, steps (b) and (c) are performed simultaneously in simultaneous saccharification and fermentation (SSF). In embodiments, the composition is added during SSF.

[0168] In an embodiment, a thermostable glucoamylase is added during the liquefaction step (a). In an embodiment, a thermostable endoglucanase is added during the liquefaction step (a). In an embodiment, a thermostable lipase is added during the liquefaction step (a). In an embodiment, a thermostable phytase is added during the liquefaction step (a). In an embodiment, a thermostable protease is added during the liquefaction step (a). In an embodiment, a thermostable pullulanase is added during the liquefaction step (a). In an embodiment, a thermostable xylanase is added during the liquefaction step (a). In a preferred embodiment, a thermostable α-amylase and a thermostable protease are added during the liquefaction step (a). In an embodiment, a thermostable α-amylase and a thermostable xylanase are added during the liquefaction step (a). In a preferred embodiment, a thermostable α-amylase, a thermostable protease, and a thermostable xylanase are added during the liquefaction step (a).

[0169] In embodiments, an α-amylase is added during step (b) and / or step (c). In embodiments, an α-glucosidase is added during step (b) and / or step (c). In embodiments, a β-amylase is added during step (b) and / or step (c). In embodiments, a β-glucanase is added during step (b) and / or step (c). In embodiments, a β-glucosidase is added during step (b) and / or step (c). In embodiments, a cellobiohydrolase is added during step (b) and / or step (c). In embodiments, an endoglucanase is added during step (b) and / or step (c). In embodiments, a lipase is added during step (b) and / or step (c). In embodiments, a lytic polysaccharide monooxygenase (LPMO) is added during step (b) and / or step (c). In embodiments, a maltose-generating α-amylase is added during step (b) and / or step (c). In an embodiment, pectinase is added during step (b) and / or step (c). In an embodiment, peroxidase is added during step (b) and / or step (c). In an embodiment, phytase is added during step (b) and / or step (c). In an embodiment, protease is added during step (b) and / or step (c). In an embodiment, trehalase is added during step (b) and / or step (c).

[0170] In an embodiment, the fermenting organism is yeast. In an embodiment, the yeast expresses an alpha-amylase in situ during step (b) and / or step (c). In an embodiment, the yeast expresses a glucoamylase in situ during step (b) and / or step (c).

[0171] Process parameters

[0172] Parameters for producing fermentation products, such as ethanol from starch-containing materials (e.g., corn), are well known in the art. See, for example, WO 2006 / 086792, WO 2013 / 082486, WO 2012 / 088303, WO 2013 / 055676, WO 2014 / 209789, WO 2014 / 209800, WO 2015 / 035914, WO 2017 / 112540, WO 2020 / 014407, WO 2021 / 126966 (each of which is incorporated herein by reference).

[0173] starch-containing materials

[0174] Any suitable starch-containing starting material can be used. Material is selected based on the desired fermentation product. Examples of starch-containing materials include, but are not limited to, barley, beets, beans, cassava, cereals, corn, milo, oats, peas, potatoes, rice, rye, sago, sorghum, sweet potatoes, cassava, wheat and whole grains or any mixture thereof. Starch-containing materials can also be waxy or non-waxy corn and barley. Common commercial starch-containing materials include corn, milo and / or wheat.

[0175] Particle size reduction of starch-containing materials

[0176] Prior to the liquefaction step (a), the particle size of the starch-containing material may be reduced, for example by dry milling.

[0177] slurry

[0178] Prior to the liquefaction step (a), a slurry comprising starch-containing material (e.g., preferably ground) and water may be formed. An alpha-amylase and preferably a protease may be added to the slurry. The slurry may be heated to between or above the initial gelatinization temperature of the starch-containing material to initiate gelatinization of the starch.

[0179] Jet cooking

[0180] Prior to adding the alpha-amylase during the liquefaction step (a), the slurry may optionally be jet cooked to further gelatinize the starch in the slurry.Jet cooking may be performed at a temperature ranging from 100°C to 120°C for up to at least 15 minutes.

[0181] liquefaction temperature

[0182] The temperature used during the liquefaction step (a) may be in the range of from 70° C. to 110° C., such as from 75° C. to 105° C., from 80° C. to 100° C., from 85° C. to 95° C., or from 88° C. to 92° C. Preferably, the temperature is at least 70° C., at least 80° C., at least 85° C., at least 88° C. or at least 90° C.

[0183] Liquefaction pH

[0184] The pH used during liquefaction step (a) may be in the range of from 4 to 6, from 4.5 to 5.5, or from 4.8 to 5.2. Preferably, the pH is at least 4.5, at least 4.6, at least 4.7, at least 4.8, at least 4.9, at least 5.0, or at least 5.1.

[0185] liquefaction time

[0186] The time for carrying out liquefaction step (a) can be in the range of from 30 minutes to 5 hours, from 1 hour to 3 hours, or 90 minutes to 150 minutes. Preferably, the time is at least 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 90 minutes, or at least about 2 hours.

[0187] Liquefaction enzyme

[0188] The present invention contemplates the use of a thermostable enzyme during the liquefaction step (a). It is well known in the art to use various thermostable enzymes during the liquefaction step (a), including, for example, thermostable α-amylases, thermostable glucoamylases, thermostable endoglucanases, thermostable lipases, thermostable phytases, thermostable proteases, thermostable pullulanases, and / or thermostable xylanases. The present invention contemplates the use of any thermostable enzyme in the liquefaction step (a). Guidance for determining the denaturation temperature of a candidate thermostable enzyme for the liquefaction step (a) is provided in the following materials and methods section. The following published patent applications describe activity assays for determining whether a candidate thermostable enzyme for the liquefaction step (a) will be inactivated at the temperature considered for the liquefaction step (a).

[0189] Examples of suitable thermostable α-amylases and guidance for their use in the liquefaction step (a) include, but are not limited to, the α-amylases described in WO 94 / 18314, WO 94 / 02597, WO 96 / 23873, WO 96 / 23874, WO 96 / 39528, WO 97 / 41213, WO 97 / 43424, WO 99 / 19467, WO 00 / 60059, WO 2002 / 010355, WO 2002 / 092797, WO 2009 / 149130, WO 2009 / 61378, WO 2009 / 061379, WO 2009 / 061380, WO 2009 / 061381, WO 2014 / 164800, WO 2014 / 164834, WO 2019 / 113413, WO 2019 / 113415, WO 2019 / 197318 (each of which is incorporated herein by reference).

[0190] Examples of suitable thermostable glucoamylases include, but are not limited to, the glucoamylases described in WO 2011 / 127802, WO 2013 / 036526, WO 2013 / 053801, WO 2018 / 164737, WO 2020 / 010101, and WO 2022 / 090564 (each of which is incorporated herein by reference).

[0191] Examples of suitable thermostable endoglucanases include, but are not limited to, those described in WO 2015 / 035914 (which is incorporated herein by reference).

[0192] Examples of suitable thermostable lipases include, but are not limited to, the lipases described in WO 2017 / 112542 and WO 2020 / 014407 (both of which are incorporated herein by reference).

[0193] Examples of suitable thermostable phytases include, but are not limited to, the phytases described in WO 1996 / 28567, WO 1997 / 33976, WO 1997 / 38096, WO 1997 / 48812, WO 1998 / 05785, WO 1998 / 06856, WO 1998 / 13480, WO 1998 / 20139, WO 1998 / 028408, WO 1999 / 48330, WO 1999 / 49022, WO 2003 / 066847, WO 2004 / 085638, WO 2006 / 037327, WO 2006 / 037328, WO 2006 / 038062, WO 2006 / 063588, WO 2007 / 112739, WO 2008 / 092901, WO 2008 / 116878, WO 2009 / 129489 and WO 2010 / 034835 (each of which is incorporated by reference). Commercially available phytase-containing products include BIO-FEEDPHYTASE TM 、PHYTASENOVO TM CT or L, LIQMAX or RONOZYME TM NP, HIPHOS, P5000(CT), NATUPHOS TM NG 5000.

[0194] Examples of suitable thermostable proteases include, but are not limited to, the proteases described in WO 1992 / 02614, WO 98 / 56926, WO 2001 / 151620, WO 2003 / 048353, WO 2006 / 086792, WO 2010 / 008841, WO 2011 / 076123, WO 2011 / 087836, WO 2012 / 088303, WO 2013 / 082486, WO 2014 / 209789, WO 2014 / 209800, WO 2018 / 098124, WO 2018 / 118815A1, and WO 2018 / 169780A1 (each of which is incorporated herein by reference).

[0195] Suitable commercially available protease-containing products include AVANTEC FORTIVA FORTIVA

[0196] Examples of suitable thermostable pullulanases include, but are not limited to, pullulanases described in WO 2015 / 007639, WO 2015 / 110473, WO 2016 / 087327, WO 2017 / 014974, and WO 2020 / 187883 (each of which is incorporated herein by reference in its entirety). Suitable commercially available pullulanases include PROMOZYME 400L, PROMOZYME TM D2 (Novozymes A / S, Denmark), OPTIMAX L-300 (Genencor Int., USA), and AMANO 8 (Amano, Japan).

[0197] Examples of suitable thermostable xylanases include, but are not limited to, xylanases described in WO 2017 / 112540 and WO 2021 / 126966 (each of which is incorporated herein by reference). Suitable commercially available products containing thermostable xylanases include FORTIVA

[0198] One or more of the enzymes described above will be used in the methods of the invention in an effective amount. Guidance for determining effective amounts of enzymes to be used in the liquefaction step (a), as well as guidance for conducting activity assays to determine the activity of those enzymes, can be found in the cited published patent applications for each of the different thermostable liquefaction enzymes.

[0199] saccharification temperature

[0200] Saccharification can be performed at a temperature ranging from 20 to 75° C., 30 to 70° C., or 40 to 65° C. Preferably, the saccharification temperature is at least about 50° C., at least about 55° C., or at least about 60° C.

[0201] Saccharification pH

[0202] Saccharification can occur at a pH ranging from 4 to 5. Preferably, the pH is about 4.5.

[0203] Saccharification time

[0204] Saccharification can last from about 24 hours to about 72 hours.

[0205] Fermentation time

[0206] Fermentation can last from 6 to 120 hours, from 24 to 96 hours, or from 35 to 60 hours.

[0207] Simultaneous saccharification and fermentation

[0208] SSF can be carried out at a temperature of from 25° C. to 40° C., from 28° C. to 35° C., or from 30° C. to 5° C., at a pH of from 3.5 to 5 or from 3.8 to 4.3, for 24 to 96 hours, 36 to 72 hours, or 48 to 60 hours. Preferably, SSF is carried out at about 32° C. at a pH of 3.8 to 4.5 for 48 to 60 hours.

[0209] Saccharification and / or fermentation enzymes

[0210] The present invention contemplates the use of enzymes during the saccharification step (b) and / or the fermentation step (c). The use of various enzymes during the saccharification step (b) and / or the fermentation step (c) is well known in the art and includes, for example, α-amylases, α-glucosidases, β-amylases, β-glucanases, β-glucosidases, cellobiohydrolases, endoglucanases, glucoamylases, lipases, lytic polysaccharide monooxygenases (LPMOs), maltose-generating α-amylases, pectinases, peroxidases, phytases, proteases, and trehalases.

[0211] The enzymes used in the saccharification step (b) and / or the fermentation step (c) can be added exogenously as a single component or formulated as a composition comprising the enzyme. The enzymes used in the saccharification step (b) and / or the fermentation step (c) can also be added via in situ expression from a fermenting organism (e.g., yeast).

[0212] Examples of suitable alpha-amylases include, but are not limited to, the alpha-amylases described in WO 2004 / 055178, WO 2006 / 069290, WO 2013 / 006756, WO 2013 / 034106, WO 2013 / 044867, WO 2021 / 163011, and WO 2021 / 163030 (each of which is herein incorporated by reference).

[0213] Examples of suitable glucoamylases include, but are not limited to, the glucoamylases described in WO 1984 / 02921, WO 1992 / 00381, WO 1999 / 28448, WO 2000 / 04136, WO 2001 / 04273, WO 2006 / 069289, WO 2011 / 066560, WO 2011 / 066576, WO 2011 / 068803, WO 2011 / 127802, WO 2012 / 064351, WO 2013 / 036526, WO 2013 / 053801, WO 2014 / 039773, WO 2014 / 177541, WO 2014 / 177546, WO 2016 / 062875, WO 2017 / 066255 and WO 2018 / 191215 (each of which is incorporated by reference).

[0214] Examples of suitable compositions comprising α-amylase and glucoamylase include, but are not limited to, compositions described in WO 2006 / 069290, WO 2009 / 052101, WO 2011 / 068803, and WO 2013 / 006756, each of which is incorporated herein by reference. Commercially available compositions comprising glucoamylase include AMG 200L; AMG 300L; SAN TM SUPER、SAN TM EXTRA L、SPIRIZYME TM PLUS, SPIRIZYME TM FUEL、SPIRIZYME TM B4U、SPIRIZYME TM ULTRA, SPIRIZYME TM EXCEL, SPIRIZYME ACHIEVE, and AMG TM E (from Novozymes); OPTIDEX TM 300, GC480, GC417 (from DuPont-Genencor); AMIGASE TM and AMIGASE TM PLUS (from DSM); G-ZYME TM G900, G-ZYME TM and G990 ZR (from DuPont Genencor).

[0215] Examples of suitable β-glucanases include, but are not limited to, the β-glucanases described in WO 2021 / 055395 (which is incorporated herein by reference).

[0216] Examples of suitable beta-glucosidases include, but are not limited to, the beta-glucosidases described in WO 2005 / 047499, WO 2013 / 148993, WO 2014 / 085439, and WO 2012 / 044915 (each of which is incorporated herein by reference).

[0217] Examples of suitable cellobiohydrolases include, but are not limited to, those described in WO 2013 / 148993, WO 2014 / 085439, WO 2014 / 138672, and WO 2016 / 040265 (each of which is herein incorporated by reference).

[0218] Examples of suitable endoglucanases include, but are not limited to, those described in WO 2013 / 148993 and WO 2014 / 085439 (both of which are incorporated herein by reference).

[0219] Examples of suitable maltose-generating alpha-amylases are described in US Pat. Nos. 4,598,048, 4,604,355, and 6,162,628, which are hereby incorporated by reference.

[0220] Examples of suitable lipases include, but are not limited to, those described in WO 2017 / 112533, WO 2017 / 112539, and WO 2020 / 076697 (each of which is herein incorporated by reference).

[0221] Examples of suitable LPMOs include, but are not limited to, those described in WO 2013 / 148993, WO 2014 / 085439, and WO 2019 / 083831 (each of which is incorporated herein by reference).

[0222] Examples of suitable phytases include, but are not limited to, the phytases described in WO 2001 / 62947 (which is incorporated herein by reference).

[0223] Examples of suitable pectinases include, but are not limited to, the pectinases described in WO 2022 / 173694 (which is incorporated herein by reference).

[0224] Examples of suitable peroxidases include, but are not limited to, the peroxidases described in WO 2019 / 231944 (which is incorporated herein by reference).

[0225] Examples of suitable proteases include, but are not limited to, those described in WO 2017 / 050291, WO 2017 / 148389, WO 2018 / 015303, and WO 2018 / 015304 (each of which is incorporated herein by reference).

[0226] Examples of suitable trehalases include, but are not limited to, those described in WO 2016 / 205127, WO 2019 / 005755, WO 2019 / 030165, and WO 2020 / 023411 (each of which is herein incorporated by reference).

[0227] III. Methods for producing fermentation products from ungelatinized starch-containing materials

[0228] One aspect of the invention relates to a method for producing a fermentation product from ungelatinized starch-containing material (i.e., granulated starch - often referred to as a "raw starch hydrolysis" process), wherein a GH5 xylanase or GH30-8 xylanase of the invention, or a composition comprising a GH5 xylanase of the invention, is present or added during saccharification or fermentation. This method of the invention contemplates any of the compositions described in Section I above, including any combination of the enzymes described in Section II, and particularly the compositions demonstrated in the Examples below.

[0229] In an embodiment, a method for producing a fermentation product from ungelatinized starch-containing material comprises the following steps:

[0230] (a) saccharifying a starch-containing material using an α-amylase and a glucoamylase at a temperature below the initial gelatinization temperature of the starch to produce fermentable sugars; and

[0231] (b) fermenting the sugar using a fermenting organism to produce a fermentation product;

[0232] wherein a composition comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase and a GH5 xylanase or a GH30-8 xylanase is present or added during step (a) and / or step (b).

[0233] In an embodiment, the GH5 xylanase is a GH5_21 xylanase. In an embodiment, the GH5 xylanase is a GH5_35 xylanase.

[0234] In embodiments, the composition used in step (a) and / or step (b) comprises a β-xylosidase. In embodiments, the composition used in step (a) and / or step (b) comprises a GH3 β-xylosidase. In embodiments, the composition is added during saccharification step (a). In embodiments, the composition is added during fermentation step (b). In embodiments, steps (a) and (b) are performed simultaneously in simultaneous saccharification and fermentation (SSF). In embodiments, the composition is added during SSF.

[0235] Raw starch hydrolysis (RSH) processes are well known in the art. Those skilled in the art will appreciate that, with the exception of process parameters associated with the liquefaction step (a) which is not performed in the RSH process, the process parameters described in Section II above are applicable to the methods described in this section, including the selection of starch-containing material, reduction of grain particle size, saccharification temperature, time, and pH, conditions for simultaneous saccharification and fermentation, and saccharifying enzymes. Process parameters for an exemplary raw starch hydrolysis process are described in further detail in WO 2004 / 106533 (incorporated herein by reference).

[0236] Examples of α-amylases preferably used in step (a) and / or step (b) include, but are not limited to, the α-amylases described in WO 2004 / 055178, WO 2005 / 003311, WO 2006 / 069290, WO 2013 / 006756, WO 2013 / 034106, WO 2021 / 163015, and WO 2021 / 163036 (each of which is incorporated herein by reference).

[0237] Examples of glucoamylases preferably used in step (a) and / or step (b) include, but are not limited to, WO 1999 / 28448, WO 2005 / 045018, WO 2005 / 069840, WO 2006 / 069289 (each of which is incorporated herein by reference).

[0238] Examples of compositions comprising an α-amylase and a glucoamylase preferably used in step (a) and / or step (b) include, but are not limited to, the compositions described in WO 2015 / 031477 (which is incorporated herein by reference).

[0239] Back-end or downstream processing

[0240] A. Fermentation product recovery and whole grain production

[0241] After fermentation or SSF, the fermentation product can be separated from the fermentation medium. The fermentation product (e.g., ethanol) can be optionally recovered from the fermentation medium using any method known in the art, including but not limited to chromatography, electrophoresis, differential solubility, distillation, or extraction. For example, alcohol can be separated and purified from the fermented starch-containing material by conventional distillation methods.

[0242] Thus, in one embodiment, the method of the present invention further comprises distillation to obtain a fermentation product, such as ethanol. Fermentation and distillation can be performed simultaneously and / or separately / sequentially; optionally, this can be followed by one or more process steps for further refining the fermentation product. After the distillation process is complete, the remaining material is considered whole stillage.

[0243] As another example, the desired fermentation product can be extracted from the fermentation medium by microfiltration or membrane filtration techniques. 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., a neutral drinking alcoholic beverage), or industrial ethanol.

[0244] In some embodiments of these methods, the fermentation product after recovery is substantially pure. With respect to these methods herein, "substantially pure" means that the preparation recovered comprises no more than 15% impurities, wherein impurities mean compounds other than the fermentation product (e.g., ethanol). In a variant, a substantially pure preparation is provided, wherein the preparation comprises no more than 25% impurities, or no more than 20% impurities, or no more than 10% impurities, or no more than 5% impurities, or no more than 3% impurities, or no more than 1% impurities, or no more than 0.5% impurities.

[0245] Can use methods known in the art to carry out suitable mensuration to test the generation and sugar consumption of ethanol and pollutant.For example, can by method (such as HPLC (high performance liquid chromatography), GC-MS (gas chromatography-mass spectrometry) and LC-MS (liquid chromatography-mass spectrometry)) or use other suitable analytical methods of routine procedures well known in the art to analyze ethanol product and other organic compounds.Can also use the release of the ethanol in culture supernatant test fermentation liquid.Can use for example the refractive index detector for glucose and alcohols and for the UV detector of organic acid by HPLC (people such as Lin, Biotechnol.Bioeng.[biotechnology and bioengineering] 90:775-779 (2005)) or use other suitable mensuration well known in the art and detection method quantization by product and residual sugar (for example, glucose or xylose) in fermentation medium.

[0246] B. Processing of whole lees

[0247] In one embodiment, whole stillage is processed into two streams: wet cake and separated liquid. The whole stillage is separated or distributed into a solid phase and a liquid phase by one or more methods for separating the separated liquid from the wet cake. The separated liquid is divided into two streams: stillage, which enters the evaporator, and a countercurrent, which is recycled to the front of the plant. The whole stillage can be separated into separated liquid (e.g., stillage when pumped to the evaporator rather than the front of the plant) and wet cake using any suitable separation technique, including centrifugation, pressing, and filtration, to remove a significant portion of the liquid / water. In a preferred embodiment, separation / dehydration is performed by centrifugation. In industry, the preferred centrifuge is a decanter centrifuge, preferably a high-speed decanter centrifuge. An example of a suitable centrifuge is the NX 400 steep cone series from Alfa Laval, which is a high-performance decanter centrifuge. Similar decanter centrifuges are also available from Flottweg. In another preferred embodiment, the separation is performed using other conventional separation equipment such as plate / frame filter press, belt filter press, screw press, gravity thickener and dewatering machine or similar equipment.

[0248] C. Processing of lees water

[0249] Thins water is the term used for the supernatant from whole stillage centrifugation. Typically, thins water contains 4% to 8% dry solids (DS) (primarily protein, soluble fiber, fat, fine fiber, and cell wall components) and has a temperature of approximately 60°C to 90°C. The thins water stream can be condensed by evaporation to provide two process streams: (i) an evaporator condensate stream, which contains the condensed water removed from the thins water during evaporation; and (ii) a slurry stream, which contains a more concentrated stream of non-volatile dissolved and undissolved solids, such as non-fermentable sugars and oils, remaining from the thins water as a result of the removal of the evaporated water.

[0250] Optionally, oil may be removed from the thin stillage, or the oil may be removed as an intermediate step in an evaporation process, which is typically performed using a series of several evaporation stages.

[0251] The slurry and / or de-oiled slurry can be introduced into a dryer along with the wet cake (from the whole stillage separation step) to provide a product referred to as dried stillage with solubles, which can also be used as animal feed. In an embodiment, the slurry and / or de-oiled slurry is sprayed into one or more dryers to combine the slurry and / or de-oiled slurry with the whole stillage to produce dried stillage with solubles.

[0252] Between 5 vol-% and 90 vol-%, such as between 10% and 80%, such as between 15% and 70%, such as between 20% and 60% of the thin-skin water (e.g., optionally hydrolyzed) can be recycled (as a countercurrent) to step (a). The recycled thin-skin water (i.e., the countercurrent) can comprise about 1 vol.-% to 70 vol.-%, preferably 15 vol.-% to 60 vol.-%, in particular from about 30 vol.-% to 50 vol.-% of the slurry formed in step (a). In embodiments, the method further comprises recycling at least a portion of the thin-skin water stream to the slurry, optionally after oil has been extracted from the thin-skin water stream.

[0253] D. Drying of wet cake and production of Dried Distillers Grains and Dried Distillers Grains with Solubles

[0254] After the wet cake containing about 25 wt% to 40 wt%, preferably 30 wt% to 38 wt%, of dry solids has been separated from the stillage (e.g., dewatered), it can be dried in a drum dryer, spray dryer, ring dryer, fluidized bed dryer, or the like to produce "distiller's dried grains (DDG)." DDG is a valuable feed ingredient for animals such as livestock, poultry, and fish. DDG is preferably provided at a moisture content of less than about 10 wt% to 12 wt% to avoid mold and microbial decomposition and to increase shelf life. Furthermore, a high moisture content makes transporting DDG more expensive. The wet cake is preferably dried under conditions that do not denature the proteins in the wet cake. The wet cake can be blended with the slurry separated from the stillage and dried to produce DDG with solubles (DDGS). A partially dried intermediate product, sometimes referred to as modified wet distiller's grains, can be produced by partially drying the wet cake, optionally adding a slurry before, during, or after the drying process.

[0255] The invention is further summarized in the following paragraphs:

[0256] 1. A composition comprising:

[0257] (a) GH43 arabinofuranosidase;

[0258] (b) GH51 arabinofuranosidase; and

[0259] (c) GH5_21 xylanase or GH30_8 xylanase.

[0260] 2. The composition of paragraph 1, wherein:

[0261] (a) the GH43 and GH51 arabinofuranosidases and the GH5_21 or GH30_8 xylanase together release at least 150% more arabinose from liquefied corn mash than the combination of the GH43 and GH51 arabinofuranosidases in the absence of the GH5_21 or GH30_8 xylanase in the composition; and / or

[0262] (b) the GH43 and GH51 arabinofuranosidases and the GH5_21 or GH30_8 xylanase together release at least 2-fold more arabinose from liquefied corn mash than the combination of the GH43 and GH51 arabinofuranosidases in the absence of the GH5_21 or GH30_8 xylanase in the composition.

[0263] 3. The composition of paragraph 1 or 2, further comprising β-xylosidase.

[0264] 4. The composition of any of paragraphs 1-3, wherein the β-xylosidase is a GH3 β-xylosidase.

[0265] 5. The composition of paragraph 4, wherein:

[0266] (a) the GH43 and GH51 arabinofuranosidases, the GH3 β-xylosidase, and the GH5_21 xylanase together release at least 150% more xylose from liquefied corn mash than the combination of the GH43 and GH51 arabinofuranosidases in the absence of the GH3 β-xylosidase and the GH5_21 xylanase in the composition; and / or

[0267] (b) the GH43 and GH51 arabinofuranosidases, the GH3 β-xylosidase, and the GH5_21 xylanase together release at least 2.5 times more xylose from liquefied corn mash than the combination of the GH43 and GH51 arabinofuranosidases in the absence of the GH3 β-xylosidase and the GH5_21 xylanase in the composition.

[0268] 6. A composition comprising:

[0269] (a) GH43 arabinofuranosidase;

[0270] (b) GH51 arabinofuranosidase;

[0271] (c) GH3 β-xylosidase; and

[0272] (d) GH5 xylanase.

[0273] 7. The composition of paragraph 6, wherein the GH5 xylanase is a GH5_21 xylanase.

[0274] 8. The composition of paragraph 7, wherein:

[0275] (a) the GH43 and GH51 arabinofuranosidases, GH3 β-xylosidase, and the GH5_21 xylanase together release at least 10% more xylose and at least 20% more arabinose from liquefied corn mash than the combination of the GH43 and GH51 arabinofuranosidases and GH3 β-xylosidase in the absence of the GH5_21 xylanase in the composition; and / or

[0276] (b) the GH43 and GH51 arabinofuranosidases, GH3 β-xylosidase, and the GH5_21 xylanase together release up to about 70% more xylose and up to about 100% more arabinose from liquefied corn mash than the combination of the GH43 and GH51 arabinofuranosidases and GH3 β-xylosidase when the GH5_21 xylanase is not present in the composition.

[0277] 9. The composition of paragraph 6, wherein the GH5 xylanase is a GH5_35 xylanase.

[0278] 10. The composition of paragraph 9, wherein:

[0279] (a) the GH43 and GH51 arabinofuranosidases, GH3 β-xylosidase, and the GH5_35 xylanase together release at least 50% more xylose and at least 60% more arabinose from liquefied corn mash than the combination of the GH43 and GH51 arabinofuranosidases and GH3 β-xylosidase in the absence of the GH5_35 xylanase in the composition.

[0280] 11. The composition of any of paragraphs 1-10, wherein the composition does not comprise a GH8 xylanase, a GH10 xylanase, or a GH11 xylanase.

[0281] 12. The composition of any of paragraphs 1-11, wherein the GH43 arabinofuranosidase is GH43_36.

[0282] 13. The composition of any of paragraphs 1-12, wherein the GH43 arabinofuranosidase is from Humicola, Chromobacterium, or Podophyllum.

[0283] 14. The composition of any of paragraphs 1-13, wherein the GH43 arabinofuranosidase is from the species Humicola insolens, Lactodiplodia theobrominata, or Aspergillus niger.

[0284] 15. The composition of any of paragraphs 1-14, wherein the GH43 arabinofuranosidase has an amino acid sequence selected from the group consisting of:

[0285] (i) an amino acid sequence of SEQ ID NO: 1 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1, which has arabinofuranosidase activity;

[0286] (ii) an amino acid sequence of SEQ ID NO: 2 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to an amino acid sequence of SEQ ID NO: 2, which has arabinofuranosidase activity; and

[0287] (iii) an amino acid sequence of SEQ ID NO: 3 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 3, which has arabinofuranosidase activity.

[0288] 16. The composition of any of paragraphs 1-15, wherein the GH51 arabinofuranosidase is from the genus Melilotrichia, Chromodispora, or Acidobacterium.

[0289] 17. The composition of any of paragraphs 1-16, wherein the GH51 arabinofuranosidase is from the species Subgrossinus giganteus, Diplodia theobrominus, or Acidobacterium bohemiensis.

[0290] 18. The composition of any of paragraphs 1-17, wherein the GH51 arabinofuranosidase has an amino acid sequence selected from the group consisting of:

[0291] (i) an amino acid sequence of SEQ ID NO: 4 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to an amino acid sequence of SEQ ID NO: 4, which has arabinofuranosidase activity;

[0292] (ii) an amino acid sequence of SEQ ID NO: 5 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to an amino acid sequence of SEQ ID NO: 5, which has arabinofuranosidase activity;

[0293] (iii) an amino acid sequence of SEQ ID NO: 6 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 6, which has arabinofuranosidase activity.

[0294] 19. The composition of any of paragraphs 1-18, wherein the GH5_21 xylanase is from Bacteroides, Bell's, Chryseobacterium, or Sphingobacterium.

[0295] 20. The composition of any of paragraphs 1-19, wherein the GH5-21 xylanase is from the species Bacteroides cellulosus CL02Y12C19, Bell's species-64282, Chryseobacterium species, Chryseobacterium oncorhynchus, or Sphingobacterium species-64162.

[0296] 21. The composition of any of paragraphs 1-20, wherein the GH5_21 xylanase is from a bioreactor metagenome, an elephant dung metagenome, a xanthan alkaline community O, a xanthan alkaline community S, or a xanthan alkaline community T.

[0297] 22. The composition of any of paragraphs 1-21, wherein the GH5-21 xylanase has an amino acid sequence selected from the group consisting of:

[0298] (i) an amino acid sequence of SEQ ID NO: 7 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to an amino acid sequence of SEQ ID NO: 7, which has xylanase activity;

[0299] (ii) an amino acid sequence of SEQ ID NO: 8 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to an amino acid sequence of SEQ ID NO: 8, which has xylanase activity;

[0300] (iii) an amino acid sequence of SEQ ID NO: 9 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to an amino acid sequence of SEQ ID NO: 9, which has xylanase activity;

[0301] (iv) an amino acid sequence of SEQ ID NO: 10 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to an amino acid sequence of SEQ ID NO: 10, which has xylanase activity;

[0302] (v) an amino acid sequence of SEQ ID NO: 11 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to an amino acid sequence of SEQ ID NO: 11, which has xylanase activity;

[0303] (vi) an amino acid sequence of SEQ ID NO: 12 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to an amino acid sequence of SEQ ID NO: 12, which has xylanase activity;

[0304] (vii) an amino acid sequence of SEQ ID NO: 13 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to an amino acid sequence of SEQ ID NO: 13, which has xylanase activity;

[0305] (viii) an amino acid sequence of SEQ ID NO: 14 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to an amino acid sequence of SEQ ID NO: 14, which has xylanase activity;

[0306] (ix) an amino acid sequence of SEQ ID NO: 15 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to an amino acid sequence of SEQ ID NO: 15, which has xylanase activity;

[0307] (x) an amino acid sequence of SEQ ID NO: 16 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to an amino acid sequence of SEQ ID NO: 16;

[0308] (xi) an amino acid sequence of SEQ ID NO: 17 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to an amino acid sequence of SEQ ID NO: 17, which has xylanase activity;

[0309] (xii) an amino acid sequence of SEQ ID NO: 18 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to an amino acid sequence of SEQ ID NO: 18, which has xylanase activity;

[0310] (xiii) an amino acid sequence of SEQ ID NO: 19 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to an amino acid sequence of SEQ ID NO: 19, which has xylanase activity;

[0311] (xiv) an amino acid sequence of SEQ ID NO:20 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to an amino acid sequence of SEQ ID NO:20, which has xylanase activity; and

[0312] (xv) an amino acid sequence of SEQ ID NO: 21 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to an amino acid sequence of SEQ ID NO: 21, which has xylanase activity.

[0313] 23. The composition of any of paragraphs 1-22, wherein the GH5_35 xylanase is from Bacillus, Coenella, or Paenibacillus.

[0314] 24. The composition of any of paragraphs 1-23, wherein the GH5-35 xylanase is from the species Bacillus hemicellulolyticus JCM 9152, Coenella xylanolyticus, Paenibacillus chitininolyticus, or Paenibacillus sp.-62332.

[0315] 25. The composition of any of paragraphs 1-24, wherein the GH5_35 xylanase is from a compost metagenome.

[0316] 26. The composition of any of paragraphs 1-25, wherein the GH5_35 xylanase has an amino acid sequence selected from the group consisting of:

[0317] (i) an amino acid sequence of SEQ ID NO: 22 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to an amino acid sequence of SEQ ID NO: 22, which has xylanase activity;

[0318] (ii) an amino acid sequence of SEQ ID NO:23 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to an amino acid sequence of SEQ ID NO:23, which has xylanase activity;

[0319] (iii) an amino acid sequence of SEQ ID NO: 24 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to an amino acid sequence of SEQ ID NO: 24, which has xylanase activity;

[0320] (iv) an amino acid sequence of SEQ ID NO: 25 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to an amino acid sequence of SEQ ID NO: 25, which has xylanase activity; and

[0321] (v) an amino acid sequence of SEQ ID NO: 26 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to an amino acid sequence of SEQ ID NO: 26, which has xylanase activity.

[0322] 27. The composition of any of paragraphs 1-26, wherein the GH30-8 xylanase is from Bacillus.

[0323] 28. The composition of any of paragraphs 1-27, wherein the GH30-8 xylanase is from species Bacillus sp.-18423.

[0324] 29. The composition of any of paragraphs 1-28, wherein the GH30-8 xylanase has the amino acid sequence of SEQ ID NO: 27 with 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 27, which has xylanase activity.

[0325] 30. The composition of any of paragraphs 1-29, wherein the GH3 β-xylosidase is from Aspergillus or Talaromyces.

[0326] 31. The composition of any of paragraphs 1-30, wherein the GH3 β-xylosidase is from the species Aspergillus fumigatus, Aspergillus nidulans, or Talaromyces emersonii.

[0327] 32. The composition of any of paragraphs 1-31, wherein the GH3 β-xylosidase has an amino acid sequence selected from the group consisting of:

[0328] (i) an amino acid sequence of SEQ ID NO: 28 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to an amino acid sequence of SEQ ID NO: 28, which has β-xylosidase activity;

[0329] (ii) an amino acid sequence of SEQ ID NO: 29 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to an amino acid sequence of SEQ ID NO: 29, which has β-xylosidase activity;

[0330] (iii) an amino acid sequence of SEQ ID NO: 30 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to an amino acid sequence of SEQ ID NO: 30, which has β-xylosidase activity;

[0331] (iv) an amino acid sequence of SEQ ID NO: 44 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to an amino acid sequence of SEQ ID NO: 44, which has β-xylosidase activity;

[0332] (v) an amino acid sequence of SEQ ID NO: 45 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to an amino acid sequence of SEQ ID NO: 45, which has β-xylosidase activity;

[0333] (vi) an amino acid sequence of SEQ ID NO: 46 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to an amino acid sequence of SEQ ID NO: 46, which has β-xylosidase activity;

[0334] (vii) an amino acid sequence of SEQ ID NO: 47 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to an amino acid sequence of SEQ ID NO: 47, which has β-xylosidase activity;

[0335] (viii) an amino acid sequence of SEQ ID NO: 48 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to an amino acid sequence of SEQ ID NO: 48, which has β-xylosidase activity;

[0336] (ix) an amino acid sequence of SEQ ID NO: 49 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 49, which has β-xylosidase activity;

[0337] (x) an amino acid sequence of SEQ ID NO:50 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to an amino acid sequence of SEQ ID NO:50, which has β-xylosidase activity;

[0338] (xi) an amino acid sequence of SEQ ID NO:51 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to an amino acid sequence of SEQ ID NO:51, which has β-xylosidase activity;

[0339] (x) an amino acid sequence of SEQ ID NO:52 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to an amino acid sequence of SEQ ID NO:52, which has β-xylosidase activity;

[0340] (xi) an amino acid sequence of SEQ ID NO:53 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to an amino acid sequence of SEQ ID NO:53, which has β-xylosidase activity;

[0341] (xii) an amino acid sequence of SEQ ID NO: 54 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to an amino acid sequence of SEQ ID NO: 54, which has β-xylosidase activity; and

[0342] (xiii) an amino acid sequence of SEQ ID NO: 55 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 55, which has β-xylosidase activity.

[0343] 33. A method for producing a fermentation product from starch-containing material, the method comprising:

[0344] (a) saccharifying a starch-containing material with an α-amylase and a glucoamylase at a temperature below the initial gelatinization temperature of the starch to produce fermentable sugars; and

[0345] (b) fermenting the sugar using a fermenting organism to produce the fermentation product, wherein:

[0346] (i) a xylanase according to any one of claims 19 to 29 is present or added during step (a) and / or step (b); or

[0347] (j) the presence or addition of a composition as claimed in any one of claims 1 to 32 during step (a) and / or step (b).

[0348] 34. The method of paragraph 33, wherein steps (a) and (b) are performed simultaneously.

[0349] 35. A method for producing a fermentation product from starch-containing material, the method comprising:

[0350] (d) liquefying the starch-containing material with a thermostable α-amylase at a temperature above the initial gelatinization temperature of the starch to produce dextrins;

[0351] (e) saccharifying the dextrin with glucoamylase to produce fermentable sugars;

[0352] (f) fermenting the sugar using a fermenting organism to produce the fermentation product, wherein:

[0353] (i) a xylanase according to any one of claims 19 to 29 is present or added during step (b) and / or step (c); or

[0354] (ii) the composition of any one of claims 1 to 32 is present or added during step (b) and / or step (c).

[0355] 36. The method of paragraph 35, wherein steps (b) and (c) are performed simultaneously.

[0356] 37. The method of paragraph 35 or 36, wherein the thermostable α-amylase has the amino acid sequence of SEQ ID NO: 34 with 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 34, which has α-amylase activity.

[0357] 38. The method of paragraph 35 or 36, wherein the thermostable alpha-amylase has the amino acid sequence of SEQ ID NO: 35 with 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 35, which has alpha-amylase activity.

[0358] 39. The method of any of paragraphs 35 to 38, wherein a thermostable protease and / or a thermostable xylanase is added in the liquefaction step (a).

[0359] 40. The method of any of paragraphs 35 to 39, wherein the thermostable protease has the amino acid sequence of SEQ ID NO: 36 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 36, which has protease activity.

[0360] 41. The method of any of paragraphs 35 to 40, wherein the thermostable xylanase has the amino acid sequence of SEQ ID NO: 37 with 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 37, which has xylanase activity.

[0361] 42. The method of any of paragraphs 33 to 41, wherein the glucoamylase has the amino acid sequence of SEQ ID NO: 38 with 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 37, which has glucoamylase activity.

[0362] 43. The method of any of paragraphs 35 to 42, further comprising adding an alpha-amylase during saccharification step (b) and / or fermentation step (c).

[0363] 44. The method of any of paragraphs 33 to 43, wherein the alpha-amylase has the amino acid sequence of SEQ ID NO: 39 with 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 39, which has alpha-amylase activity.

[0364] 45. The method of any of paragraphs 33 to 44, wherein trehalase is added during the saccharification step and / or the fermentation step.

[0365] 46. ​​The method of any of paragraph 45, wherein the trehalase has the amino acid sequence of SEQ ID NO: 40 with 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 40, which has trehalase activity.

[0366] 47. The method of any of paragraphs 33 to 47, wherein a composition comprising β-glucosidase, cellobiohydrolase, and endoglucanase is added during the saccharification step and / or the fermentation step.

[0367] 48. The method of paragraph 47, wherein the β-glucosidase has the amino acid sequence of SEQ ID NO: 41 with 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 41, which has β-glucosidase activity.

[0368] 49. The method of paragraphs 47-48, wherein the cellobiohydrolase has the amino acid sequence of SEQ ID NO: 42 with 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 42, which has cellobiohydrolase activity.

[0369] 50. The method of any of paragraphs 47-49, wherein the endoglucanase has an amino acid sequence of SEQ ID NO: 43 carrying 0 to 10 conservative amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 43, which has endoglucanase activity.

[0370] 51. The method of any of paragraphs 33 to 50, wherein the starch-containing material comprises sugar beets, maize, corn, wheat, rye, barley, oats, triticale, rice, sorghum, sweet potato, millet, pearl millet and / or foxtail millet.

[0371] 52. The method of any of paragraphs 33 to 51, wherein the starch-containing material comprises corn.

[0372] 53. The method of any of paragraphs 33 to 52, wherein the fermentation product is ethanol, preferably fuel ethanol.

[0373] 54. The method of any of paragraphs 33 to 53, wherein the fermenting organism is yeast.

[0374] 55. The method of any of paragraphs 33 to 54, wherein compared to a control method:

[0375] (i) lack of the composition;

[0376] (ii) using a composition comprising only individual GH43 and GH51 arabinofuranosidases; (iii) using a composition comprising GH43 and GH51 arabinofuranosidases and a GH8 xylanase;

[0377] (iv) using a composition comprising GH43 and GH51 arabinofuranosidases and a GH10 xylanase; and / or

[0378] (v) Using the composition containing the GH43 and GH51 arabinofuranosidases and the GH11 xylanase, solubilization of hemicellulose fibers is increased to release significantly more monomeric arabinose and monomeric xylose.

[0379] 56. The method of any of paragraphs 33-55, wherein residual solids are reduced compared to a control method lacking the composition.

[0380] The invention described and claimed herein is not limited to the scope of the specific embodiments disclosed herein, as these embodiments are intended to serve as illustrative illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of the invention. Indeed, various modifications of the invention, in addition to those shown and described herein, will become clear to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the event of a conflict, the present disclosure, including definitions, will prevail. Various references are cited herein, the disclosures of which are incorporated herein by reference in their entirety. The invention is further described by the following examples, which should not be construed as limiting the scope of the invention.

[0381] Examples

[0382] Materials and Methods

[0383] Enzymes used in the examples

[0384] GH43A: An exemplary GH43 arabinofuranosidase from Humicola insolens disclosed in SEQ ID NO: 1.

[0385] GH43B: An exemplary GH43 arabinofuranosidase from Lactodiplodia theobromin disclosed in SEQ ID NO:2.

[0386] GH43C: An exemplary GH43 arabinofuranosidase from Aspergillus niger disclosed in SEQ ID NO:3.

[0387] GH51A: An exemplary GH51 arabinofuranosidase from G. gigantea disclosed in SEQ ID NO:4.

[0388] GH51B: An exemplary GH51 arabinofuranosidase from Lactodiplodia theobromin disclosed in SEQ ID NO:5.

[0389] GH51C: An exemplary GH51 arabinofuranosidase from Acidobacterium bohemiensis disclosed in SEQ ID NO:6.

[0390] GH5_21A: Exemplary GH5_21 xylanase from Bacteroides cellulosus CL02T12C19 disclosed in SEQ ID NO:7.

[0391] GH5_21B: Exemplary GH5_21 xylanase from Xanthan Alkaline Cluster S disclosed in SEQ ID NO:8.

[0392] GH5_21C: Exemplary GH5_21 xylanase from Sphingobacterium sp.-64162 disclosed in SEQ ID NO:9.

[0393] GH5_21D: Exemplary GH5_21 xylanase from Sphingobacterium sp.-64162 disclosed in SEQ ID NO: 10.

[0394] GH5_21E: Exemplary GH5_21 xylanase from Xanthan Alkaline Community O disclosed in SEQ ID NO: 11.

[0395] GH5_21F: an exemplary GH5_21 xylanase from a bioreactor metagenome disclosed in SEQ ID NO: 12.

[0396] GH5_21G: Exemplary GH5_21 xylanase from Xanthan Alkaline Cluster T disclosed in SEQ ID NO: 13.

[0397] GH5_21H: Exemplary GH5_21 xylanase from Xanthan Alkaline Cluster S disclosed in SEQ ID NO: 14.

[0398] GH5_21I: Exemplary GH5_21 xylanase from Belleriana sp.-64282 disclosed in SEQ ID NO: 15.

[0399] GH5_21J: Exemplary GH5_21 xylanase from Chryseobacterium oncorhynchus disclosed in SEQ ID NO:16.

[0400] GH5_21K: An exemplary GH5_21 xylanase from Xanthan Alkaline Cluster T disclosed in SEQ ID NO: 17.

[0401] GH5_21L: Exemplary GH5_21 xylanase from Sphingobacterium disclosed in SEQ ID NO: 18.

[0402] GH5_21M: an exemplary GH5_21 xylanase from an elephant dung metagenome disclosed in SEQ ID NO: 19.

[0403] GH5_21N: An exemplary GH5_21 xylanase from an elephant dung metagenome disclosed in SEQ ID NO: 20.

[0404] GH5_210: An exemplary GH5_21 xylanase from Chryseobacterium sp. disclosed in SEQ ID NO:21.

[0405] GH5_35A: Exemplary GH5_35 xylanase from Coenella xylanicum disclosed in SEQ ID NO:22.

[0406] GH5_35B: Exemplary GH5_35 xylanase from Bacillus hemicellulolyticus JCM 9152 disclosed in SEQ ID NO:23.

[0407] GH5_35C: Exemplary GH5_35 xylanase from Paenibacillus sp.-62332 disclosed in SEQ ID NO:24.

[0408] GH5_35D: an exemplary GH5_35 xylanase from a compost metagenome disclosed in SEQ ID NO: 25.

[0409] GH5_35E: Exemplary GH5_35 xylanase from Paenibacillus chitinoilyticus disclosed in SEQ ID NO:26.

[0410] GH30_8: An exemplary GH30_8 xylanase from Bacillus sp.-18423 disclosed in SEQ ID NO:27.

[0411] GH3A: An exemplary GH3 beta-xylosidase from Aspergillus fumigatus disclosed in SEQ ID NO:28.

[0412] GH3B / An BX: an exemplary GH3 beta-xylosidase from Aspergillus nidulans disclosed in SEQ ID NO:29.

[0413] GH3C: An exemplary GH3 beta-xylosidase from Talaromyces emersonii disclosed in SEQ ID NO:30.

[0414] GH8: An exemplary GH8 xylanase from Bacillus sp. KK-1 disclosed in SEQ ID NO:31.

[0415] GH10: An exemplary GH10 xylanase from Aspergillus aculeatus disclosed in SEQ ID NO:32.

[0416] GH11: An exemplary GH11 xylanase from Thermomyces lanuginosus disclosed in SEQ ID NO:33.

[0417] Liquefaction Enzyme Blend 1: an exemplary thermostable alpha-amylase from Bacillus stearothermophilus disclosed in SEQ ID NO:34; an exemplary thermostable protease from Pyrococcus furiosus disclosed in SEQ ID NO:36.

[0418] Liquefaction Enzyme Blend 2: an exemplary thermostable alpha-amylase from Bacillus stearothermophilus disclosed in SEQ ID NO:35; an exemplary thermostable protease from Pyrococcus furiosus disclosed in SEQ ID NO:36; an exemplary thermostable xylanase from Thermotoga maritima disclosed in SEQ ID NO:37.

[0419] Saccharifying enzyme blend: an exemplary glucoamylase from Gloeophyllum sepiarium disclosed in SEQ ID NO:38; an exemplary alpha-amylase from Rhizomucorpus illus disclosed in SEQ ID NO:39; an exemplary trehalase from Talaromyces funiculosus disclosed in SEQ ID NO:40; an exemplary beta-glucosidase from Aspergillus fumigatus disclosed in SEQ ID NO:41; an exemplary cellobiohydrolase from Aspergillus fumigatus disclosed in SEQ ID NO:42; an exemplary endoglucanase from Trichoderma reesei disclosed in SEQ ID NO:43.

[0420] At BX: an exemplary GH3 beta-xylosidase from Aspergillus tellustris disclosed in SEQ ID NO:44.

[0421] Aa BX: The exemplary GH3 beta-xylosidase from Aspergillus aculeatus disclosed in SEQ ID NO:45.

[0422] Af BX: The exemplary GH3 beta-xylosidase from Aspergillus fischerii disclosed in SEQ ID NO:46.

[0423] Cg BX: The exemplary GH3 beta-xylosidase from Chaetomium globosum disclosed in SEQ ID NO:47.

[0424] Cv BX: The exemplary GH3 beta-xylosidase from Chaetomium chlororosum disclosed in SEQ ID NO:48.

[0425] F1BX: an exemplary GH3 beta-xylosidase from Fusarium oleiferum disclosed in SEQ ID NO:49.

[0426] Mt BX: The exemplary GH3 beta-xylosidase from Thermus thermophilus disclosed in SEQ ID NO:50.

[0427] Pe BX: The exemplary GH3 beta-xylosidase from Penicillium emersonii disclosed in SEQ ID NO:51.

[0428] Po BX: an exemplary GH3 beta-xylosidase from Penicillium oxalicum disclosed in SEQ ID NO:52.

[0429] Sf BX: The exemplary GH3 beta-xylosidase from Sphaerotheca septicum disclosed in SEQ ID NO:53.

[0430] Ts BX: The exemplary GH3 beta-xylosidase from Talaromyces stipulosa disclosed in SEQ ID NO:54.

[0431] Tr BX: The exemplary GH3 beta-xylosidase from Trichoderma reesei disclosed in SEQ ID NO:55.

[0432] Determination of Td of liquefaction enzyme by differential scanning calorimetry

[0433] The thermal stability of the enzyme was determined by differential scanning calorimetry (DSC) using a VP-capillary differential scanning calorimeter (MicroCal Inc., Piscataway, NJ, USA). The thermal denaturation temperature, Td (° C.), was taken as the top of the denaturation peak (major endothermic peak) in the thermogram (Cp vs. T) obtained after heating an enzyme solution (approximately 0.5 mg / ml) in buffer (50 mM acetate, pH 5.0) at a constant programmed heating rate of 200 K / h.

[0434] The sample solution and the reference solution (approximately 0.2 ml) were loaded into the calorimeter from storage conditions at 10° C. (reference solution: buffer without enzyme) and thermally pre-equilibrated at 20° C. for 20 minutes, followed by a DSC scan from 20° C. to 120° C. The denaturation temperature was determined with an accuracy of approximately + / - 1° C.

[0435] strain

[0436] Yeast strain MEJI797 is MBG5012 of WO 2019 / 161227, further expressing a Pycnopous sanguineus glucoamylase (SEQ ID NO: 4 of WO 2011 / 066576) and a hybrid Rhizomucor pusillus alpha amylase expression cassette (as described in WO 2013 / 006756).

[0437] Example 1 - Effect of xylanases from GH families 5, 8, 10, 11 and 30 in combination with arabinofuranosidases from GH families 43 and 51 to increase xylose and arabinose in a simultaneous saccharification and fermentation process

[0438] The experiment was conducted using industrial liquefied mash prepared using liquefaction enzyme blend 1. The dry solids determined by moisture balance were approximately 34% DS, and the pH was adjusted to pH 5.0, followed by supplementation with 3 ppm penicillin and 500 ppm urea. Simultaneous saccharification and fermentation (SSF) was performed via small-scale fermentation. Approximately 4.0 g of industrial liquefied corn mash was added to a 15 ml tube vial. Each vial was given 0.42 AGU / g DS of the saccharifying enzyme blend and appropriate amounts of the corresponding xylanase and arabinofuranosidase, with the treatment regimen shown in Table 1. The saccharifying enzyme blend was used as a control, without the addition of xylanase or arabinofuranosidase. The actual enzyme dosage was based on the exact weight of the corn slurry in each vial. After the enzymes were added, fermentation was started by adding 50 μL of the propagated yeast strain MEJI797. The test tubes were incubated at 32°C, and each treatment was repeated three times. After 65 hours of SSF, the tubes were removed from the incubator, 50 μL of 34% H2SO4 was added, and the tubes were centrifuged at 3500 rpm for 10 min and then filtered through a 0.45 μm filter. Sugar concentrations were determined using an HPLC system equipped with an H column (Benson Polymeric, BP-700H, 300×7.8 mm).

[0439] Table 1: Dosing regimen of arabinofuranosidase in the presence or absence of xylanase

[0440]

[0441] Table 2: Results

[0442]

[0443]

[0444] Increased arabinose % = [(arabinose experimental mean ppm - arabinose control mean) / arabinose control mean] x 100

[0445] Table 2 shows that the highest concentration of arabinose was released by the combination of GH5_21 or GH30_8 xylanase with GH43 and GH51 arabinofuranosidase compared to either GH43 or GH51 arabinofuranosidase alone or their combination without xylanase.

[0446] Example 2 - Effect of GH3 family β-xylosidase in combination with arabinofuranosidases from GH 43 and 51 families and xylanase from GH5_21 to increase xylose in a simultaneous saccharification and fermentation process

[0447] The experiment was conducted using industrial liquefied mash prepared using liquefaction enzyme blend 2. The dry solids determined by moisture balance were approximately 35.9% DS, and the pH was adjusted to pH 5.0, followed by supplementation with 3 ppm penicillin and 500 ppm urea. Simultaneous saccharification and fermentation (SSF) was performed via small-scale fermentation. Approximately 4.2 g of industrial liquefied corn mash was added to 15 ml vials. Each vial was dosed with 0.42 AGU / g DS of the saccharifying enzyme blend and appropriate amounts of the corresponding xylanase, arabinofuranosidase, and β-xylosidase, as listed in Table 3. The dosing regimen followed a fixed amount of GH5_21 xylanase, GH43 arabinofuranosidase, and GH51 arabinofuranosidase (each at 10 ug / g dry solids), with or without β-xylosidase GH3A, GH3B, or GH3C, at doses of 25, 50, 100, or 200 ug / g dry solids. The saccharifying enzyme blend was used as a control without the addition of xylanase or β-xylosidase. The actual enzyme dosage was based on the exact weight of the corn slurry in each vial. After the enzymes were added, fermentation was started by adding 50 μL of the propagated yeast strain MEJI797. The test tubes were incubated at 32°C, with each treatment repeated three times. After 65 hours of SSF, the test tubes were removed from the incubator, centrifuged at 3500 rpm for 10 min, and then filtered through a 0.45 micron filter. Sugar concentration was determined using an HPLC system equipped with a lead column (Benson Polymers, BP-800Pb, 300x7.8 mm).

[0448] result

[0449] Table 3 shows that the combination of β-xylosidase with GH43, GH51 arabinofuranosidase and GH5_21 xylanase significantly increased xylose release, with higher enzyme dosages corresponding to higher xylose release.

[0450] Table 3

[0451]

[0452] Example 3 - Effect of Combination of GH5 Xylanase Subfamilies 21 and 35 with HiGH43 and Mg GH51 Arabinofuranosidases and AfGH3 β-Xylosidases to Increase Xylose and Arabinose in a Simultaneous Saccharification and Fermentation Process

[0453] The experiment was conducted using industrial liquefied mash prepared using liquefaction enzyme blend 1. The dry solids determined by moisture balance were approximately 33.7% DS, and the pH was adjusted to pH 5.0, followed by supplementation with 3 ppm penicillin and 500 ppm urea. Simultaneous saccharification and fermentation (SSF) was performed via small-scale fermentation. Approximately 4.2 g of industrial liquefied corn mash was added to 15 ml vials. Each vial was dosed with 0.42 AGU / g DS of the saccharifying enzyme blend, 10 ug / g DS of the GH43A arabinofuranosidase, 10 ug / g DS of the GH51A arabinofuranosidase, 25 ug / g DS of the GH3A β-xylosidase, and 10 ug / g DS of the corresponding xylanase, as listed in Table 4. The saccharifying enzyme blend was used as a control, without the addition of arabinofuranosidase, xylanase, or β-xylosidase. The actual enzyme dosage was based on the exact weight of the corn slurry in each vial. After adding the enzyme, fermentation was started by adding 50 μL of the yeast strain MEJI797. The test tubes were incubated at 32°C, with three replicates per treatment. After 65 hours of SSF, the test tubes were removed from the incubator, 50 μL of 34% HSO were added, and the mixture was centrifuged at 3500 rpm for 10 min and then filtered through a 0.45 micron filter. Sugar concentration was determined using an HPLC system equipped with a lead column (Benson Polymers, BP-800Pb, 300 x 7.8 mm).

[0454] result

[0455] Table 4 shows that addition of xylanases from GH5_21 and GH5_35 significantly increased xylose and arabinose release compared to the control or treatment consisting of GH43, GH51 arabinofuranosidase and GH3 β-xylosidase (xylanase absent).

[0456] Table 4

[0457]

[0458]

[0459] Example 4 - Effect of single, double, triple or quadruple combinations of hemicellulases of GH5_21 xylanase, GH43 and GH51 arabinofuranosidases and GH3 β-xylosidase to increase xylose and arabinose in a simultaneous saccharification and fermentation process

[0460] The experiment was conducted using industrial liquefied mash prepared using liquefaction enzyme blend 2. The dry solids determined by moisture balance were approximately 33.4% DS, and the pH was adjusted to pH 5.0, followed by supplementation with 3 ppm penicillin and 500 ppm urea. Simultaneous saccharification and fermentation (SSF) was performed via small-scale fermentation. Approximately 4.2 g of industrial liquefied corn mash was added to 15 ml vials. Each vial was dosed with 0.6 AGU / gDS of the saccharifying enzyme blend and followed a dosing regimen of 10 ug / gDS of GH5_210 xylanase, 10 ug / gDS of GH43A arabinofuranosidase, 10 ug / gDS of GH51A arabinofuranosidase, and / or 25 ug / gDS of GH3A β-xylosidase. As a control, only the saccharifying enzyme blend was added without arabinofuranosidase, xylanase, or β-xylosidase. The actual enzyme dosage is based on the exact weight of the corn slurry in each vial. After adding the enzyme, fermentation was started by adding 50 μL of hydrated yeast strain MEJI797. The test tubes were incubated at 32°C, and each treatment was repeated three times. After 65 hours of SSF, the test tubes were removed from the incubator, 50 μL of 34% H2SO4 were added, and then centrifuged at 3500 rpm for 10 min and then filtered through a 0.45 micron filter. Sugar concentration was determined using an HPLC system equipped with an H column (Benson Polymers, BP-700H, 300x7.8 mm). The decanters containing the wet corn mash at the end of the fermentation were vacuum freeze-dried for 3 days. The dry solid weight of each test tube was measured, and the residual solids were calculated as the ratio of the final solid weight to the initial solid weight.

[0461] result

[0462] Table 5

[0463]

[0464] Table 5 shows that the addition of GH5_21 xylanase along with GH43, GH51 arabinofuranosidase and GH3 β-xylosidase increased xylose and arabinose release compared to the control or treatment consisting of GH43, GH51 arabinofuranosidase and GH3 β-xylosidase (without xylanase).

[0465] Example 5 - Effect of a combination of arabinofuranosidases from GH families 43 and 51 with a xylanase from GH family 5 subfamily 21 to increase arabinose in a simultaneous saccharification and fermentation process

[0466] The experiment was carried out using industrial liquefied mash prepared using liquefaction enzyme blend 2. The dry solids determined by moisture balance were approximately 36% DS, and the pH was adjusted to pH 5.0, followed by supplementation with 3ppm penicillin and 500ppm urea. Simultaneous saccharification and fermentation (SSF) was performed via small-scale fermentation. Approximately 4.0g of industrial liquefied corn mash was added to a 15ml tube bottle. Each vial was given a saccharifying enzyme blend of 0.42AGU / gDS and an appropriate combination of arabinofuranosidases from families GH43 and GH51, as listed in Table 6. The administration regimen followed a fixed amount of GH5_210 xylanase, GH43 arabinofuranosidase, and GH51 arabinofuranosidase (each at 10ug / g dry solids). As a control, only the saccharifying enzyme blend was used without the addition of xylanase or arabinofuranosidase. The actual enzyme dosage was based on the exact weight of the corn slurry in each vial. After enzyme addition, fermentation was initiated by adding 50 μL of propagated yeast strain MEJI797. The test tubes were incubated at 32°C, with three replicates per treatment. After 65 hours of SSF, the tubes were removed from the incubator, 50 μL of 34% H2SO4 was added, and the tubes were centrifuged at 3500 rpm for 10 min and then filtered through a 0.45 micron filter. Sugar concentration was determined using an HPLC system equipped with an H column (Benson Polymers, BP-700H, 300 x 7.8 mm).

[0467] result

[0468] Table 6

[0469] deal with Average value of arabinose, ppm Increased arabinose% comparison 165 0 GH43A+GH51A 2744 1563％ GH43B+GH51A 2559 1451％ GH43A+GH51C 1842 1016％ GH43A+GH51B 2198 1232％

[0470] Increased arabinose % = [(arabinose experimental mean ppm - arabinose control mean) / arabinose control mean] x 100

[0471] Table 6 shows that the combination of GH43 and GH51 arabinofuranosidases with GH5_21 xylanase increased arabinose compared to the control without arabinofuranosidase and xylanase.

[0472] Example 6 - Effect of a combination of arabinofuranosidases from GH families 43 and 51 with a xylanase from GH family 5 subfamily 21 to increase arabinose in a simultaneous saccharification and fermentation process

[0473] The experiment was conducted using industrial liquefied mash prepared using liquefaction enzyme blend 2. The dry solids determined by moisture balance were approximately 33.8% DS, and the pH was adjusted to pH 5.0, followed by supplementation with 3ppm penicillin and 500ppm urea. Simultaneous saccharification and fermentation (SSF) was performed via small-scale fermentation. Approximately 4.2g of industrial liquefied corn mash was added to a 15ml vial. Each vial was given a saccharifying enzyme blend of 0.42AGU / gDS and an appropriate combination of arabinofuranosidases from families GH43 and GH51, as listed in Table 8. The administration regimen followed a fixed amount of GH5_21 xylanase, GH43 arabinofuranosidase, and GH51 arabinofuranosidase (each at 10ug / g dry solids). As a control, only the saccharifying enzyme blend was used without the addition of xylanase or arabinofuranosidase. The actual enzyme dosage was based on the exact weight of the corn slurry in each vial. After enzyme addition, fermentation was initiated by adding 50 μL of propagated yeast strain MEJI797. The test tubes were incubated at 32°C, with three replicates per treatment. After 65 hours of SSF, the tubes were removed from the incubator, 50 μL of 34% H2SO4 was added, and the tubes were centrifuged at 3500 rpm for 10 min and then filtered through a 0.45 micron filter. Sugar concentration was determined using an HPLC system equipped with an H column (Benson Polymers, BP-700H, 300 x 7.8 mm).

[0474] result

[0475] Table 7

[0476] deal with Average value of arabinose, ppm Increased arabinose% comparison 415 0％ GH43A+GH51A 2120 410％ GH43C+GH51A 1902 358％

[0477] Increased arabinose % = [(arabinose experimental mean ppm - arabinose control mean) / arabinose control mean] x 100

[0478] Table 7 shows that the combination of GH43 and GH51 arabinofuranosidases with GH5_21 xylanase increased arabinose release compared to the control without arabinofuranosidase and xylanase.

[0479] Example 7 - Effect of β-xylosidases (BX) from different sources in combination with a hemicellulase blend (base C5) in increasing xylose in a simultaneous saccharification and fermentation process in the presence or absence of Aspergillus fumigatus BX

[0480] The experiment was conducted using industrially prepared liquefied mash of the liquefaction product of liquefaction enzyme blend 1. The dry solids determined by moisture balance were approximately 34.1% DS, and the pH was adjusted to pH 5.0, followed by supplementation with 3 ppm penicillin and 1000 ppm urea. Simultaneous saccharification and fermentation (SSF) was performed via small-scale fermentation. Approximately 4.2 g of industrially liquefied corn mash was added to 10 ml vials. Each vial was dosed with 0.6 AGU / g DS of a saccharifying enzyme blend and a hemicellulase blend (basic C5), as shown in Table 8 (with or without GH3A), as well as appropriate amounts of the corresponding β-xylosidase from various sources (Table 9), followed by addition of 50 μL of hydrated ETHANOL RED yeast per 4.2 g of slurry. As a control, only glucoamylase was used without adding basic C5 or GH3 enzymes. The actual enzyme dosage was based on the exact weight of the corn slurry in each vial. The vials were incubated at 32°C, with each treatment replicated three times. After 65 hours of SSF, the tubes were removed from the incubator, centrifuged at 3500 rpm for 10 min, and then filtered through a 0.45 μm filter. Sugar concentrations were determined using an HPLC system equipped with an H column (Benson Polymers, BP-700H, 300×7.8 mm).

[0481] Table 8: Hemicellulase blend (Base C5)

[0482] enzymes GH5_21 GH43A GH51A GH3A

[0483] result

[0484] As shown in the results of Table 9 below, the combination of BX (especially with Po BX), in the presence or absence of A. fumigatus BX, significantly increased xylose release in the base C5 hemicellulase blend.

[0485] Table 9

[0486]

[0487]

Claims

1. A composition comprising: (a) GH43 arabinofuranosidase; (b) GH51 arabinofuranosidase; and (c) GH5 xylanase.

2. The composition of claim 1, further comprising β-xylosidase.

3. The composition of claim 2, wherein the β-xylosidase is a GH3 β-xylosidase.

4. The composition of any one of claims 1 to 3, wherein the GH5 xylanase is a GH5-21 xylanase.

5. The composition of any one of claims 1 to 4, wherein: (a) the GH43 and GH51 arabinofuranosidases and the GH5_21 xylanase together release at least 150% more arabinose from liquefied corn mash than the combination of the GH43 and GH51 arabinofuranosidases in the absence of the GH5_21 xylanase in the composition; (b) the GH43 and GH51 arabinofuranosidases and the GH5-21 xylanase together release at least 2-fold more arabinose from liquefied corn mash than the combination of the GH43 and GH51 arabinofuranosidases in the absence of the GH5-21 xylanase in the composition; (c) the GH43 and GH51 arabinofuranosidases, the GH3 β-xylosidase, and the GH5_21 xylanase together release at least 10% more xylose and at least 20% more arabinose from liquefied corn mash than the combination of the GH43 and GH51 arabinofuranosidases and the GH3 β-xylosidase in the absence of the GH5_21 xylanase in the composition; (d) the GH43 and GH51 arabinofuranosidases, the GH3 β-xylosidase, and the GH5_21 xylanase together release up to about 70% more xylose and up to about 100% more arabinose from liquefied corn mash than the combination of the GH43 and GH51 arabinofuranosidases and the GH3 β-xylosidase in the absence of the GH5_21 xylanase in the composition; (e) the GH43 and GH51 arabinofuranosidases, GH3 β-xylosidase, and GH5_21 xylanase together release at least 150% more xylose from liquefied corn mash than the combination of the GH43 and GH51 arabinofuranosidases in the absence of the GH3 β-xylosidase and GH5_21 xylanase in the composition; and / or (f) the GH43 and GH51 arabinofuranosidases, GH3 β-xylosidase, and GH5_21 xylanase together release at least 2.5 times more xylose from liquefied corn mash than the combination of the GH43 and GH51 arabinofuranosidases in the absence of the GH3 β-xylosidase and GH5_21 xylanase in the composition.

6. The composition of any one of claims 1-3, wherein the GH5 xylanase is a GH5-35 xylanase.

7. The composition of any one of claims 1-3 and 6, wherein the GH43 and GH51 arabinofuranosidases, GH3 β-xylosidase, and the GH5_35 xylanase together release at least 50% more xylose and at least 60% more arabinose from liquefied corn mash than the combination of the GH43 and GH51 arabinofuranosidases and GH3 β-xylosidase in the absence of the GH5_35 xylanase in the composition.

8. A composition comprising: (a) GH43 arabinofuranosidase; (b) GH51 arabinofuranosidase; and (c)GH30_8 xylanase.

9. The composition of claim 8, wherein: (a) the GH43 and GH51 arabinofuranosidases and the GH30-8 xylanase together release at least 150% more arabinose from liquefied corn mash than the combination of the GH43 and GH51 arabinofuranosidases in the absence of the GH30-8 xylanase in the composition; and / or (b) the GH43 and GH51 arabinofuranosidases and the GH30_8 xylanase together release at least 2-fold more arabinose from liquefied corn mash than the combination of the GH43 and GH51 arabinofuranosidases in the absence of the GH30_8 xylanase in the composition.

10. A method for producing a fermentation product from starch-containing material, the method comprising: (a) saccharifying a starch-containing material with an α-amylase and a glucoamylase at a temperature below the initial gelatinization temperature of the starch to produce fermentable sugars; as well as (b) fermenting the sugars using a fermenting organism to produce the fermentation product, wherein the composition of any one of claims 1 to 9 is present or added during step (a) and / or step (b).

11. The method of claim 10, wherein steps (a) and (b) are performed simultaneously.

12. A method for producing a fermentation product from starch-containing material, the method comprising: (a) liquefying a starch-containing material with a thermostable α-amylase at a temperature above the initial gelatinization temperature of the starch to produce dextrins; (b) saccharifying the dextrin with glucoamylase to produce fermentable sugars; (c) fermenting the sugars using a fermenting organism to produce the fermentation product, wherein the composition of any one of claims 1 to 9 is present or added during step (b) and / or step (c).

13. The method of claim 12, wherein steps (b) and (c) are performed simultaneously.

14. The method of any one of claims 10-13, wherein the starch-containing material comprises sugar beet, maize, corn, wheat, rye, barley, oats, triticale, sorghum, sweet potato, rice, millet, pearl millet and / or foxtail millet.

15. The method of any one of claims 10-14, wherein the starch-containing material comprises corn.

16. The method of any one of claims 10-15, wherein the fermentation product is ethanol.

17. The method of any one of claims 10-16, wherein the fermenting organism is yeast.

18. The method of any one of claims 10-15, wherein compared to a control method: (i) lacking the composition; (ii) using a composition containing only individual GH43 and GH51 arabinofuranosidases; (iii) using a combination comprising GH43 and GH51 arabinofuranosidases and a GH8 xylanase; (iv) using a composition comprising GH43 and GH51 arabinofuranosidases and a GH10 xylanase; and / or (v) Using the composition containing the GH43 and GH51 arabinofuranosidases and the GH11 xylanase, the solubilization of hemicellulose fibers is increased to release significantly more monomeric arabinose and monomeric xylose.

19. The method of any one of claims 10-16, wherein residual solids are reduced compared to a control method lacking the composition.

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