WO2024137248A1

WO2024137248A1 - Compositions comprising arabinofuranosidases and a xylanase, and use thereof for increasing hemicellulosic fiber solubilization

Info

Publication number: WO2024137248A1
Application number: PCT/US2023/083345
Authority: WO
Inventors: Chee-Leong Soong
Original assignee: Novozymes A/S
Priority date: 2022-12-19
Filing date: 2023-12-11
Publication date: 2024-06-27

Abstract

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 beta-xylosidase. The present invention also relates to use of the composition for increasing hemicellulosic fiber solubilization and release of monomeric arabinose and xylose.

Description

COMPOSITIONS COMPRISING ARABINOFURANOSIDASES AND A XYLANASE, AND USE THEREOF FOR INCREASING HEMICELLULOSIC FIBER SOLUBILIZATION

REFERENCE TO A SEQUENCE LISTING

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

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Conversion of cellulosic feedstocks into biofuels is challenging due to their high recalcitrance, typically involved combination of thermochemical pretreatment followed by adding cellulase and hemicellulase enzymes to release soluble carbohydrates. With government sustainability initiatives, the biofuels industry is incentivized to produce ethanol from corn fiber at existing corn ethanol facilities. Corn fiber comprises 10% of the weight of corn kernels and consists of cellulose and hemicellulose from the aleurone and pericarp layers. In ethanol facilities, corn fiber ends up in the Distillers Dried Grains with Solubles (DDGS). Enzymatic hydrolysis of the hemicellulose portion of the corn fiber to monomeric C5 sugars such as xylose and arabinose simultaneously with fermentation of the C5 sugars to ethanol by C5 fermenting yeast, and leveraging existing infrastructure, would allow ethanol plants to produce additional cellulosic ethanol yield from the same amount of corn. Additional benefits from corn fiber degradation include better DDGS feed quality from enriched protein content for animal feed and the lower fiber content of DDGS would potentially qualify for access to the monogastric and aquaculture animal feed market.

The arabinoxylan backbone in corn fiber is composed of a xylan backbone of p-(1 ,4)- linked D-xylopyranosyl residues that highly substituted with arabinose side chains and to a lesser extent with glucuronic acid residues. The main substitutions of arabinose residues linked to the 0-2 or 0-3 position on monosubstituted xylopyranosyls or to both 0-2 and 0-3 on doubly substituted xylopyranosyl units. In addition to arabinose, the xylan backbone can be substituted with D-galactopyranosyl and D-glucuronyl residues, and/or with acetyl groups. Acetic acid is esterified directly to the xylan backbone in position 0-2 or 0-3, whereas hydroxycinnamic acids such as ferulic acid, p-coumaric acid, and dehydrodimers of ferulic acid are esterified to arabinofuranosyls in position 0-5. It has also been reported that xylan is further substituted with xylopyranosyls by a (1-3)- linkage and that the arabinofuranosyls can be further decorated with xylopyranosyls or even L-galactopyranosyls. Because of the highly branched substitution by different moieties, enzymatic degradation of corn fiber arabinoxylan to monomeric C5 sugars requires concerted action of a mixture of debranching and depolymerizing activities. Debranching activities mainly include a-L-arabinofuranosidases (EC 3.2.1.55) (a-AraFs), feruloyl esterases (EC 3.1.1.73), a-glucuronidases (EC 3.2.1.139), and/or acetyl xylan esterases (EC 3.1.1.72), while depolymerization relies on endo-1, 4-p- xylanase (EC 3.2.1.8) and p-xylosidase (EC 3.2.1.37) (BX) activities.

WO 2006/114095 “D1” describes a process and composition for hydrolyzing arabinoxylan, which includes contacting an arabinoxylan containing substrate with an enzyme having activity toward di-substituted arabinoses, e.g., such as a Glycoside Hydrolyase Family 43 (GH43) alpha-L-arabinofuranosidase, and an enzyme having activity towards C2- or C3-position mono-substituted arabinoses, e.g., such as a GH Family 51, 54 or 62 alpha-L-arabinofuranosidase. D1 teaches that when the two arabinofuranosidases are added to an arabinoxylan solution the resulting products will be high molecular weight linear xylose polymers and arabinose molecules that allow for an easy separation of the linear xylose polymer by known techniques from arabinose, which may be further partially digested with enzyme activities, such as beta-xylosidase (preferably GH3), and/or endo-1, 4-beta- xylanase (preferably GH10 or GH11), to yield xylo-oligosaccharides. D1 further teaches that when both endo-1, 4-beta-xylanase and a beta-xylosidase are added to purified linear xylose polymers the resulting product will be xylose that is essentially free of arabinose substituents, and that for degradation of even more complex substrates, or where a more complete degradation is required, the presence of even further enzyme activities may be desired, such as acetyl xylan esterase (EC 3.1.1.72) and/or feruloyl esterase (EC 3.1.1.73) and/or alpha-glucuronidase (EC. 3.2.1.139).

However, supply chain disruptions and inflation have driven up the cost of raw material inputs for producing the enzymes needed for completely hydrolyzing complex arabinoxylan substrates, diminishing financial incentives for ethanol facilities to purchase additional enzymes for producing cellulosic ethanol from corn. Because conventional wisdom suggests all seven enzymatic activities are required to maximize cellulosic ethanol yields from corn, there exists a need for improved processes, and compositions capable of increasing cellulosic ethanol yields by releasing more monomeric arabinose and xylose with less enzymatic activities, and at a lower cost that is more profitable for corn ethanol facilities to maximize cellulosic ethanol yields from their existing corn inputs. SUMMARY OF THE INVENTION

The present invention provides a solution to the above problem by providing compositions comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, and a GH5 xylanase or a GH30_8 xylanase, which unexpectedly increase hemicellulosic fiber solubilization and release significantly more monomeric arabinose and xylose compared to compositions combining the GH43 and GH51 arabinofuranosidases alone or with a GH8 xylanase, a GH10 xylanase or a GH11 xylanases. Surprisingly and unexpectedly, the compositions of the present invention significantly increase yields of monomeric arabinose and xylose without requiring beta-xylosidases, acetyl xylan esterases, feruloyl esterases, and/or alpha-glucuronidases, though the addition of GH3 beta-xylosidases to the compositions further increases those yields.

In an aspect, the invention provides a composition comprising:

(a) a GH43 arabinofuranosidase;

(b) a GH51 arabinofuranosidase; and

(c) a GH5 xylanase.

In an embodiment, the composition comprises a beta-xylosidase. In an embodiment, the beta-xylosidase is a GH3 beta-xylosidase. In an embodiment, the GH5 xylanase is a GH5_21 xylanase.

The GH43 and GH51 arabinofuranosidases and the GH5_21 xylanase together release at least 150% more arabinose from liquefied corn mash than the GH43 and GH51 arabinofuranosidases combined when the GH5_21 xylanase is not present in the composition. The GH43 and GH51 arabinofuranosidases and the GH5_21 xylanase together release at least 2 times more arabinose from liquefied corn mash than the GH43 and GH51 arabinofuranosidases combined when the GH5_21 xylanase is not present in the composition. The GH43 and GH51 arabinofuranosidases, the GH3 beta-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 GH43 and GH51 arabinofuranosidases and GH3 beta-xylosidase combined when the GH5_21 xylanase is not present in the composition. The GH43 and GH51 arabinofuranosidases, the GH3 beta-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 GH43 and GH51 arabinofuranosidases and GH3 beta-xylosidase combined when the GH5_21 xylanase is not present in the composition. The GH43 and GH51 arabinofuranosidases, GH3 beta-xylosidase, and the GH5_21 xylanase together release at least 150% more xylose from liquefied corn mash than the GH43 and GH51 arabinofuranosidases combined when the GH3 beta-xylosidase and GH5_21 xylanase are not present in the composition. The GH43 and GH51 arabinofuranosidases, GH3 beta-xylosidase, and the GH5_21 xylanase together release at least 2.5 times more xylose from liquefied corn mash than the GH43 and GH51 arabinofuranosidases combined when the GH3 beta-xylosidase and GH5_21 xylanase are not present in the composition.

In an embodiment, the GH5 xylanase is a GH5_35 xylanase. The GH43 and GH51 arabinofuranosidases, GH3 beta-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 GH43 and GH51 arabinofuranosidases and GH3 beta-xylosidase combined when the GH5_35 xylanase is not present in the composition.

In aspect, the invention provides a composition comprising:

(a) a GH43 arabinofuranosidase;

(b) a GH51 arabinofuranosidase; and

(c) a GH30_8 xylanase.

The GH43 and GH51 arabinofuranosidases and the GH30_8 xylanase together release at least 150% more arabinose from liquefied corn mash than the GH43 and GH51 arabinofuranosidases combined when the GH30_8 xylanase is not present in the composition. The GH43 and GH51 arabinofuranosidases and the GH30_8 xylanase together release at least 2 times more arabinose from liquefied corn mash than the GH43 and GH51 arabinofuranosidases combined when the GH30_8 xylanase is not present in the composition.

In an aspect, the invention provides a process for producing a fermentation product from a starch-containing material, the process comprising:

(a) saccharifying a starch-containing material at a temperature below the initial gelatinization temperature of the starch with an alpha-amylase and a glucoamylase to produce fermentable a sugar; and

(b) fermenting the sugar 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., GH5_21 xylanase) or a GH30_8 xylanase is present or added during step (a) and/or step (b).

(a) liquefying a starch-containing material with a thermostable alpha-amylase at a temperature above the initial gelatinization temperature of the starch to produce a dextrin;

(b) saccharifying the dextrin with a glucoamylase to produce a fermentable sugar; (c) fermenting the sugar 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., GH5_21 xylanase) or a GH30_8 xylanase present or added during step (b) and/or step (c).

In the processes of the invention, solubilization of hemicellulosic fiber is increased to release significantly more monomeric arabinose and monomeric xylose compared to a control process: (i) lacking the composition; (ii) using a composition with only the GH43 and GH51 arabinofuranosidases alone; (iii) using a composition with the GH43 and GH51 arabinofuranosidases and a GH8 xylanase; (iv) using a composition with the GH43 and GH51 arabinofuranosidases and a GH10 xylanase; and/or (v) using a composition with the GH43 and GH51 arabinofuranosidases and a GH11 xylanase.

In the processes of the invention, residual solids are decreased compared to a control process lacking the composition.

A composition of the present invention can be used in saccharification, fermentation, or simultaneous saccharification and fermentation, to solubilize hemicellulosic fiber to monomeric arabinose and xylose in conventional starch-to-ethanol processes and raw- starch hydrolysis (RSH).

DEFINITIONS

Alpha-L-arabinofuranosidase: "Alpha-L-arabinofuranosidase" means an alpha-L- arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55) that catalyzes the hydrolysis of terminal non-reducing alpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzyme acts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)- and/or (1 ,5)-linkages, arabinoxylans, and arabinogalactans. Alpha-L-arabinofuranosidase is also known as arabinofuranosidase, alpha-arabinofuranosidase, alpha-L-arabinofuranosidase, alpha-arabinofuranosidase, polysaccharide alpha-L- arabinofuranosidase, alpha-L- arabinofuranoside hydrolase, L-arabinofuranosidase, or alpha-L- arabinanase.

Alpha-L-arabinofuranosidase Activity: For purposes of the present invention, alpha-L-arabinofuranosidase activity is determined using 5 mg of medium viscosity wheat arabinoxylan (Megazyme International Ireland, Ltd., Bray, Co. Wicklow, Ireland) per ml of 100 mM sodium acetate pH 5 in a total volume of 200 micro liters for 30 minutes at 40 degrees centigrade followed by arabinose analysis by AMINEX(R) HPX-87H column chromatography (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Beta-xylosidase: "Beta-xylosidase" means a beta-D-xyloside xylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of short beta (1-4)-xylooligosaccharides to remove successive D-xylose residues from non-reducing termini. Beta-xylosidase Activity: For purposes of the present invention, one unit of beta- xylosidase is defined as 1.0 pmole of p-nitrophenolate anion produced per minute at 40 degrees centigrade, pH 5 from 1 mM p-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodium citrate containing 0.01 percent TWEEN(R) 20 in a total volume of 200 micro liters.

Fermentation product: “Fermentation product” means a product produced by a process including fermenting 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, B12, beta-carotene); and hormones. In a preferred embodiment the fermentation product is ethanol, e.g., fuel ethanol; drinking ethanol, i.e., potable neutral spirits; or industrial ethanol or products used in the consumable alcohol industry (e.g., beer and wine), dairy industry (e.g., fermented dairy products), leather industry and tobacco industry. Preferred beer types comprise ales, stouts, porters, lagers, bitters, malt liquors, happoushu, high-alcohol beer, low-alcohol beer, low-calorie beer or light beer. In an embodiment the fermentation product is ethanol.

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.

GH3 beta-xylosidase: “GH3 beta-xylosidase” is an abbreviation for Glycoside Hydrolase Family 3 beta-xylosidases, which are xylan 1 ,4-beta-xylosidases (EC 3.2.1.37) that catalyze the hydrolysis (1— >4)-p-D-xylans, to remove successive D-xylose residues from the non-reducing termini.

GH5 xylanase: “GH5 xylanase” is an abbreviation for Glycoside Hydrolase Family 5 xylanase, which consist primarily of endo-1 ,4- p-xylanases (EC 3.2.1.8) that catalyze the endohydrolysis of (1— >4)-p-D-xylosidic linkages in xylans.

GH5_21 xylanase: “GH5_21 xylanase” is an abbreviation for Glycoside Hydrolase Family 5 subfamily 21 endo-beta-1 , 4-xylanases that possess a three-dimensional structure characterized by a (P / a) 8 barrel and use a glutamine residue as a catalytic nucleophile/base.

GH5_35 xylanase: “GH5_35 xylanase” is an abbreviation for Glycoside Hydrolase Family 5 subfamily 35 endo-beta-1 , 4-xylanases that possess a three-dimensional structure characterized by a (P / a) 8 barrel and use a glutamine residue as a catalytic nucleophile/base. GH8 xylanase: “GH8 xylanase” is an abbreviation for Glycoside Hydrolase Family 8 xylanases, which consists of endo-1, 4-p-xylanases (EC 3.2.1.8) that catalyze the endohydrolysis of (1— >4)-p-D-xylosidic linkages in xylans.

GH10 xylanase: “GH10 xylanase” is an abbreviation for Glycoside Hydrolase Family

10 xylanases, which consists of endo-1 , 3-p-xylanases (EC 3.2.1.32) that catalyze the random endohydrolysis of (1— >3)-p-D-glycosidic linkages in (1^3)-p-D-xylans, and endo-1, 4-p-xylanases (EC 3.2.1.8) that catalyze the endohydrolysis of (1^4)-p-D-xylosidic linkages in xylans.

GH11 xylanase: “GH11 xylanase” is an abbreviation for Glycoside Hydrolase Family

11 xylanase, which is an endo-p-1,4-xylanase (EC 3.2.1.8) that catalyzes the endohydrolysis of (1^4)-p-D-xylosidic linkages in xylans.

GH30_8 xylanase: “GH30_8 xylanase” is an abbreviation for Glycoside Hydrolase 30 subfamily 8 xylanases, which include endo-beta-1, 4-xylanase (EC 3.2.1.8) that catalyze the endohydrolysis of (1^4)-p-D-xylosidic linkages in xylans and glucuronoarabinoxylan- specific endo-p-1,4-xylanases (EC 3.2.1.136) that catalyze the endohydrolysis of (1^4)-p-D- xylosyl links in some glucuronoarabinoxylans. endohydrolysis of (1^4)-p-D-xylosyl links in some glucuronoarabinoxylans.

GH43 arabinofuranosidase: “GH43 arabinofuranosidase” is an abbreviation for Glycoside Hydrolase Family 43 arabinofuranosidase, which is an alpha-L- arabinofuranosidase (EC 3.2.1.55) that catalyzes the hydrolysis of terminal non-reducing alpha-L-arabinofuranoside residues in alpha-L-arabinosides.

GH51 arabinofuranosidase: “GH51 arabinofuranosidase” is an abbreviation for Glycoside Hydrolase Family 51 arabinofuranosidase, which is an alpha-L- arabinofuranosidase (EC 3.2.1.55) that catalyzes the hydrolysis of terminal non-reducing alpha-L-arabinofuranoside residues in alpha-L-arabinosides.

Initial gelatinization temperature: "Initial gelatinization temperature" means the lowest temperature at which gelatinization of the starch commences. Starch heated in water begins to gelatinize between 50 degrees centigrade and 75 degrees C; the exact temperature of gelatinization depends on the specific starch, and can readily be determined by the skilled artisan. Thus, the initial gelatinization temperature may vary according to the plant species, to the particular variety of the plant species as well as with the growth conditions. In the context of this disclosure the initial gelatinization temperature of a given starch-containing material is the temperature at which birefringence is lost in 5 percent of the starch granules using the method described by Gorinstein. S. and Lii. C, Starch/Starke, Vol. 44 (12) pp. 461-466 (1992). Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”. The sequence identity between two amino acid sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276- 277), version 6.6.0. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order for the Needle program to report the longest identity, the -nobrief option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:

(Identical Residues x 100)/(Length of Alignment - T otal Number of Gaps in Alignment)

The sequence identity between two polynucleotide sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), version 6.6.0. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NLIC4.4) substitution matrix. In order for the Needle program to report the longest identity, the nobrief option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows: (Identical Deoxyribonucleotides x 100)/(Length of Alignment - Total Number of Gaps in Alignment)

Thermostable: “Thermostable” means the enzyme is not denatured or deactivated when it is used in a liquefaction step of a process of the 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 that temperature.

Thin Stillage: “Thin stillage” refers to centrate separated from whole stillage that is pumped toward the evaporators to be concentrated into syrup.

Whole Stillage: "Whole stillage" includes the material that remains at the end of the distillation process after recovery of the fermentation product, e.g., ethanol.

Xylanase: “Xylanase” encompasses endo-1,4- p-xylanases (EC 3.2.1.8) that catalyze the endohydrolysis of (1— >4)-p-D-xylosidic linkages in xylans and glucuronoarabinoxylan endo-1 ,4-beta-xylanases (E.C. 3.2.1.136) that catalyze the endohydrolysis of 1,4-beta-D-xylosyl links in some glucuronoarabinoxylans.

Xylanase Activity: Activity of EC 3.2.1.8 xylanases can be determined using birchwood xylan as substrate. One unit of xylanase is defined as 1.0 pmole of reducing sugar (measured in glucose equivalents as described by Lever, 1972, A new reaction for colorimetric determination of carbohydrates, Anal. Biochem 47: 273-279) produced per minute during the initial period of hydrolysis at 50° C., pH 5 from 2 g of birchwood xylan per liter as substrate in 50 mM sodium acetate containing 0.01% TWEEN® 2. Activity of EC 3.2.1.136 xylanases can be determined with 0.2% AZCL-glucuronoxylan as substrate in 0.01% TRITON® X-100 and 200 mM sodium phosphate pH 6 at 37°C. One unit of xylanase activity is defined as 1.0 pmole of azurine produced per minute at 37°C, pH 6 from 0.2% AZCL-glucuronoxylan as substrate in 200 mM sodium phosphate pH 6.

DESCRIPTION OF THE INVENTION

I. COMPOSITIONS

The present invention relates to compositions comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, and a GH5 xylanase or a GH30_8 xylanase.

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 an otherwise identical compositions lacking the GH5 or GH30_8 xylanases. In particular, work described herein demonstrates that compositions comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, and a GH5 xylanase or GH30_8 xylanase unexpectedly released significantly more monomeric arabinose compared to 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. Surprisingly, compositions comprising a GH5_21 xylanase or GH30_8 xylanase and GH43 and GH51 arabinofuranosidases boosted release of monomeric arabinose three times more than a no enzyme control, and over two times more than a composition comprising the GH43 and GH51 arabinofuranosidases alone, whereas compositions comprising the GH43 and GH51 arabinofuranosidases and a GH8 xylanase, a GH10 xylanase, or a GH11 xylanase performed on par or slightly better in terms of releasing arabinose compared to compositions comprising the GH43 and GH51 arabinofuranosidases alone.

Work described herein further unexpectedly demonstrates that the addition of a beta- xylosidase to compositions comprising the GH43 arabinofuranosidase, GH51 arabinofuranosidase and GH5_21 xylanase or GH30_8 xylanase further increases the amount of monomeric arabinose and/or monomeric xylose released from hemicellulosic fiber in liquefied corn mash.

The present invention contemplates using the compositions of the present invention in saccharification, fermentation, or simultaneous saccharification and fermentation, to increase solubilization of hemicellulosic fibers to monomeric sugars, such as arabinose and xylose, in conventional and raw-starch hydrolysis (RSH) ethanol production processes.

A. Compositions comprising a GH5 xylanase

In an aspect, a composition of the present invention comprises:

(a) a GH43 arabinofuranosidase;

(b) a GH51 arabinofuranosidase; and

(c) a GH5 xylanase.

In an embodiment, the composition includes a beta-xylosidase. In an embodiment, the beta-xylosidase is a GH3 beta-xylosidase.

In an embodiment, the GH5 xylanase is a GH5_21 xylanase. The compositions of the present invention comprising the GH5_21 xylanase provide unexpected and surprising benefits compared to similar compositions without the GH5_21 xylanase. The GH43 and GH51 arabinofuranosidases and the GH5_21 xylanase together releases at least 150% more arabinose from liquefied corn mash than the GH43 and GH51 arabinofuranosidases combined when the GH5_21 xylanase is not present in the composition. The GH43 and GH51 arabinofuranosidases and the GH5_21 xylanase together releases at least 2 times more arabinose from liquefied corn mash than the GH43 and GH51 arabinofuranosidases combined when the GH5_21 xylanase is not present in the composition. The GH43 and GH51 arabinofuranosidases, the GH3 beta-xylosidase, and the GH5_21 xylanase together releases at least 10% more xylose and at least 20% more arabinose from liquefied corn mash than the GH43 and GH51 arabinofuranosidases and GH3 beta-xylosidase combined when the GH5_21 xylanase is not present in the composition. The GH43 and GH51 arabinofuranosidases, the GH3 beta-xylosidase, and the GH5_21 xylanase together releases up to about 70% more xylose and up to about 100% more arabinose from liquefied corn mash than the GH43 and GH51 arabinofuranosidases and GH3 beta-xylosidase combined when the GH5_21 xylanase is not present in the composition. The GH43 and GH51 arabinofuranosidases, GH3 beta-xylosidase, and the GH5_21 xylanase together releases at least 150% more xylose from liquefied corn mash than the GH43 and GH51 arabinofuranosidases combined when the GH3 beta-xylosidase and GH5_21 xylanase are not present in the composition. The GH43 and GH51 arabinofuranosidases, GH3 beta- xylosidase, and the GH5_21 xylanase together releases at least 2.5 times more xylose from liquefied corn mash than the GH43 and GH51 arabinofuranosidases combined when the GH3 beta-xylosidase and GH5_21 xylanase are not present in the composition. The composition comprising the GH43 and GH51 arabinofuranosidases and the GH5_21 xylanase together together releases at least 2 times more arabinose from liquefied corn mash than compositions comprising the GH43 and GH51 arabinofuranosidases combined alone or combined with a GH8 xylanase, a GH10 xylanase or a GH11 xylanase.

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, GH3 beta-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 GH43 and GH51 arabinofuranosidases and GH3 beta-xylosidase combined when the GH5_35 xylanase is not present in the composition.

B. Compositions comprising a GH30_8 xylanase

In an aspect, a composition of the present invention comprises:

(a) a GH43 arabinofuranosidase;

(b) a GH51 arabinofuranosidase; and

(c) a GH30_8 xylanase.

The compositions of the present invention comprising the GH30_8 xylanase provide unexpected and surprising benefits compared to similar compositions without the GH30_8 xylanase. The GH43 and GH51 arabinofuranosidases and the GH30_8 xylanase together release at least 150% more arabinose from liquefied corn mash than the GH43 and GH51 arabinofuranosidases combined when the GH30_8 xylanase is not present in the composition. The GH43 and GH51 arabinofuranosidases and the GH30_8 xylanase together release at least 2 times more arabinose from liquefied corn mash than the GH43 and GH51 arabinofuranosidases combined when the GH30_8 xylanase is not present in the composition.

The composition comprising the GH43 and GH51 arabinofuranosidases and the GH30_8 xylanase together releases at least 2 times more arabinose from hemicellulosic fiber in liquefied corn mash than compositions comprising the GH43 and GH51 arabinofuranosidases combined alone or with a GH8 xylanase, a GH10 xylanase or a GH11 xylanase.

In an embodiment, the composition further comprises a beta-xylosidase. In an embodiment, the the beta-xylosidase is a GH3 beta-xylosidase.

In an aspect, a composition of the present invention comprises: (a) a GH43 arabinofuranosidase;

(b) a GH51 arabinofuranosidase;

(c) a GH3 beta-xylosidase; and

(d) a GH5 xylanase.

The GH43 and GH51 arabinofuranosidases, GH3 beta-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 GH43 and GH51 arabinofuranosidases and GH3 beta- xylosidase combined when the GH5_21 xylanase is not present in the composition.

The GH43 and GH51 arabinofuranosidases, GH3 beta-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 GH43 and GH51 arabinofuranosidases and GH3 beta-xylosidase combined when the GH5_21 xylanase is not present in the composition.

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

Surprisingly, the work described herein demonstrates that a composition comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, and a GH8 xylanase, a GH10 xylanase, or a GH11 xylanase did not provide as significant of an increase in the release of monomeric arabinose as compared to a composition comprising the GH43 and GH51 arabinofuranosidase and the GH5_21 or GH30_8 xylanase. In fact, the examples demonstrate that such compositions performed on par with or only slightly better than compositions comprising only the GH43 and GH51 arabinofuranosidases.

Accordingly, the present invention contemplates emodiments in which the compositions lack a GH8, GH10, or GH11 xylanase. In an embodiment, the composition does not include a GH8 xylanase. In an embodiment, the composition does not include a GH10 xylanase. In an embodiment, the composition does not include a GH11 xylanase.

A. Exemplary GH43 arabinofuranosidases

Aspects of the invention relate to compositions comprising a GH43 arabinofuranosidases in combination with other enzymes to increase hemicellulosic fiber solubilization and production of monomeric arabinose and/or xylose. The present invention contemplates any GH43 arabinofuranosidase that, when used in combination with a GH51 arabinofuronasidase, a GH5 xylanase or GH30_8 xylanase, and optionally a beta- xylosidase, increases production of monomeric arabinose and/or xylose compared to compositions comprising the GH43 and GH51 arabinofuranosidases alone. In an embodiment, the GH43 arabinofuranosidase is a GH43 subfamily 36 arabinofuranosidase.

Exemplary GH43 arabinfuranosidases may be from the genus Humicola, Lasiodiplodia, or Poronia.

Exemplary GH43 arabinofuranosidase may be from the species Humicola insolens, Lasiodiplodia theobromae, and Poronia punctata.

An exemplary GH43 arabinofuranosidase is an arabinofuranosidase having the amino acid sequence of SEQ ID NO: 1. In an embodiment, the GH43 arabinofuranosidase has the amino acid sequence of SEQ ID NO: 1 with from 0 to 10 conservative amino acid substitutions and has arabinofuranosidase activity. In an embodiment, 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. An exemplary GH43 arabinofuranosidase is an arabinofuranosidase having the amino acid sequence of SEQ ID NO: 2. In an embodiment, the GH43 arabinofuranosidase has the amino acid sequence of SEQ ID NO: 2 with from 0 to 10 conservative amino acid substitutions and has arabinofuranosidase activity. In an embodiment, 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 an embodiment, the GH43 arabinofuranosidase has the amino acid sequence of SEQ ID NO: 3 with from 0 to 10 conservative amino acid substitutions and has arabinofuranosidase activity. In an embodiment, 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.

The GH43 arabinofuranosidase may be dosed in pre-saccharification, saccharification, and/or simultaneous saccharification and fermentation in 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.

B. Exemplary GH51 arabinofuranosidases

Aspects of the invention relate to compositions comprising GH51 arabinofuranosidases in combination with other enzymes to increase hemicellulosic fiber solubilization and production of monomeric arabinose and/or xylose. The present invention contemplates using any GH51 arabinofuranosidase that, when used in combination with a GH43 arabinofuronasidase, a GH5 xylanase or GH30_8 xylanase, and optionally a beta- xylosidase, increases production of monomeric arabinose and/or xylose compared to compositions comprising the GH43 and GH51 arabinofuranosidases alone.

In an embodiment, the GH51 arabinofuranosidase is a GH51 subfamily 6 arabinofuranosidase.

Exemplary GH51 arabinfuranosidases include, without limitation, ones from the genus Meripulus, Lasiodiplodia, or Acidiella.

Exemplary GH51 arabinfuranosidases include, without limitation, ones from the species Meripulus giganteus, Lasiodiplodia theobromae, or Acidiella bohemica.

An exemplary GH51 arabinofuranosidase has the amino acid sequence of SEQ ID NO: 4. In an embodiment, the GH51 arabinofuranosidase has the amino acid sequence of SEQ ID NO: 4 with from 0 to 10 conservative amino acid substitutions and has arabinofuranosidase activity. In an embodiment, 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. An exemplary GH51 arabinofuranosidase has the amino acid sequence of SEQ ID NO: 5. In an embodiment, the GH51 arabinofuranosidase has the amino acid sequence of SEQ ID NO: 5 with from 0 to 10 conservative amino acid substitutions and has arabinofuranosidase activity. In an embodiment, 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. An exemplary GH51 arabinofuranosidase has the amino acid sequence of SEQ ID NO: 6. In an embodiment, the GH51 arabinofuranosidase has the amino acid sequence of SEQ ID NO: 6 with from 0 to 10 conservative amino acid substitutions and has arabinofuranosidase activity. In an embodiment, 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.

The GH51 arabinofuranosidase may be dosed in pre-saccharification, saccharification, and/or simultaneous saccharification and fermentation in a concentration of betweenO.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. C. Exemplary GH5 xylanases

Aspects of the invention relate to compositions comprising GH5 family xylanases arabinofuranosidases in combination with other enzymes to increase hemicellulosic fiber solubilization and production of monomeric arabinose and/or xylose. The present invention contemplates using any GH5 xylanase that, when used in combination with GH43 and GH51 arabinofuronasidases, and optionally a beta-xylosidase, increases production of monomeric arabinose and/or xylose compared to compositions comprising the GH43 and GH51 arabinofuranosidases alone.

In an embodiment, the xylanase is a GH5 family xylanase.

In an embodiment, the xylanase is a GH5_21 xylanase.

Exemplary GH5_21 xylanases include, without limitation, ones from the genus Bacteroides, Belliella, Chryseobacterium, or Sphingobacterium.

Exemplary GH5_21 xylanases include, without limitation, ones from the species Bacteroides cellulosilyticus CL02Y12C19, Belliella sp-64282, Chryseobacterium sp., Chryseobacterium oncorhynchi, or Sphingobacterium sp-64162.

Exemplary GH5_21 xylanases include, without limitation, ones from bioreactor metagenome, Elephant dung metagenome, Xanthan alkaline community O, Xanthan alkaline community S, or Xanthan alkaline community T.

An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 7. In an embodiment, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 7 with from 0 to 10 conservative amino acid substitutions and has 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: 7 and has xylanase activity. An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 8. In an embodiment, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 8 with from 0 to 10 conservative amino acid substitutions and has 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 from 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. An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 10. In an embodiment, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 10 with from 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: 10 and has xylanase activity. An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 11. In an embodiment, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 11 with from 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: 11 and has xylanase activity. An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO:12. In an embodiment, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 12 with from 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: 12 and has xylanase activity. An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 13. In an embodiment, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 13 with from 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 of SEQ ID NO: 13 and has xylanase activity. An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 14. In an embodiment, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 14 with from 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: 14 and has xylanase activity. An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 15. In an embodiment, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 15 with from 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 of SEQ ID NO: 15 and has xylanase activity. An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 16. In an embodiment, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 16 with from 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: 16 and has xylanase activity. An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 17. In an embodiment, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 17 with from 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: 17 and has xylanase activity. An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 18. In an embodiment, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 18 with from 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: 18 and has xylanase activity. An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 19. In an embodiment, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 19 with from 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: 19 and has xylanase activity. An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 20. In an embodiment, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 20 with from 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 to the amino acid sequence of SEQ ID NO: 20 and has xylanase activity. An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 21. In an embodiment, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 21 with from 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: 21 and has xylanase activity.

In an embodiment, the GH5 xylanase is a GH5_35 xylanase.

Exemplary GH5_35 xylanases include, without limitation, ones from the genus Bacillus, Cohnella, or Paenibacillus.

Exemplary GH5_35 xylanases include, without limitation, ones from the species Bacillus hemiccellulosilyticus JCM 9152, Cohnella xylanilytica, Paenibacillus chitinolyticus, or Paenibacillus sp-62332.

Exemplary GH5_35 xylanases include, without limitation, ones from compost metagenome.

An exemplary GH5_35 xylanase has the amino acid sequence of SEQ ID NO: 22. In an embodiment, the GH5_35 xylanase has the amino acid sequence of SEQ ID NO: 22 with from 0 to 10 conservative amino acid substitutions and has xylanase activity. In an embodiment, the GH_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: 22 and has xylanase activity. An exemplary GH5_35 xylanase has the amino acid sequence of SEQ ID NO: 23. In an embodiment, the GH5_35 xylanase has the amino acid sequence of SEQ ID NO: 23 with from 0 to 10 conservative amino acid substitutions and has xylanase activity. In an embodiment, the GH_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 an embodiment, the GH5_35 xylanase has the amino acid sequence of SEQ ID NO: 24 with from 0 to 10 conservative amino acid substitutions and has xylanase activity. In an embodiment, 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. An exemplary GH5_35 xylanase has the amino acid sequence of SEQ ID NO: 25. In an embodiment, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 25 with from 0 to 10 conservative amino acid substitutions and has xylanase activity. In an embodiment, 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. An exemplary GH5_35 xylanase has the amino acid sequence of SEQ ID NO: 26. In an embodiment, the GH5_35 xylanase has the amino acid sequence of SEQ ID NO: 26 with from 0 to 10 conservative amino acid substitutions and has xylanase activity. In an embodiment, 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.

The GH5 xylanase may be dosed in pre-saccharification, saccharification, and/or simultaneous saccharification and fermentation in 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

Aspects of the invention relate to compositions comprising a GH30_8 xylanase in combination with other enzymes to increase hemicellulosic fiber solubilization and production of monomeric arabinose and/or xylose. The present invention contemplates using any GH30_8 xylanase that, when used in combination with GH43 and GH51 arabinofuronasidases, and optionally a beta-xylosidase, increases production of monomeric arabinose and/or xylose compared to compositions comprising the GH43 and GH51 arabinofuranosidases alone.

Exemplary GH30_8 xylanases include, without limitation, ones from the genus Bacillus.

Exemplary GH30_8 xylanases include, without limitation, ones from the species Bacillus sp- 18423.

An exemplary GH30_8 xylanase has the amino acid sequence of SEQ ID NO: 27. In an embodiment, the GH30_8 xylanase has the amino acid sequence of SEQ ID NO: 27 with from 0 to 10 conservative amino acid substitutions and has xylanase activity. In an embodiment, 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.

Additional exemplary GH30_8 xylanases suitable for use in the compositions of the present invention are described in WO 2019/055455 (which is incorporated herein by reference in its entirety). The GH30_8 xylanase may be dosed in pre-saccharification, saccharification, and/or simultaneous saccharification and fermentation in 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.

E. Exemplary Beta-xylosidases

Aspects of the invention relate to compositions comprising a beta-xylosidase (e.g., a GH3 beta-xylosidase) in combination with other enzymes to increase hemicellulosic fiber solubilization and production of monomeric arabinose and/or xylose. The present invention contemplates using any beta-xylosidase (e.g., GH3 beta-xylosidase) that, when used in combination with GH43 and GH51 arabinofuronasidases, and a GH5 xylanase or GH30_8 xylanase, further increases production of monomeric arabinose and/or xylose compared to compositions comprising the GH43, GH51 and GH5 xylanase or GH30_8 xylanase alone.

In an embodiment, the beta-xylosidase is a GH3 beta-xylosidase.

Exemplary GH3 beta-xylosidases include, without limitation, ones from the genus Alternaria, Aspergillus, Chaetomium, Fusarium, Mycothermus, Penicillium, Sporormia, Talaromyces, or Trichoderma.

Exemplary GH3 beta-xylosidases include, without limitation, ones from the species Alternaria tellustris, Aspergillus aculeatus, Aspergillus fischeri, Aspergillus fumigatus, Aspergillus nidulans, Chaetomium globosum, Chaetomium virescens, Fusarium longipes, Mycothermus thermophilus, Penicillium emersonii, Penicillium oxalicum, Sporormia fi meta ria, Talaromyces emersonii, Talaromyces stipitatus, or Trichoderma reesei.

An exemplary GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 28. In an embodiment, the GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 28 with from 0 to 10 conservative amino acid substitutions and has beta-xylosidase activity. In an embodiment, the GH3 beta-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 beta-xylosidase activity.

An exemplary GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 29. In an embodiment, the GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 29 with from 0 to 10 conservative amino acid substitutions and has beta-xylosidase activity. In an embodiment, the GH3 beta-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 beta-xylosidase activity. An exemplary GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 30. In an embodiment, the GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 30 with from 0 to 10 conservative amino acid substitutions and has beta-xylosidase activity. In an embodiment, the GH3 beta-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.

An exemplary GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 44. In an embodiment, the GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 44 with from 0 to 10 conservative amino acid substitutions and has beta-xylosidase activity. In an embodiment, the GH3 beta-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 beta-xylosidase activity.

An exemplary GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 45. In an embodiment, the GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 45 with from 0 to 10 conservative amino acid substitutions and has beta-xylosidase activity. In an embodiment, the GH3 beta-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 beta-xylosidase activity.

An exemplary GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 46. In an embodiment, the GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 46 with from 0 to 10 conservative amino acid substitutions and has beta-xylosidase activity. In an embodiment, the GH3 beta-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 beta-xylosidase activity.

An exemplary GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 47. In an embodiment, the GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 47 with from 0 to 10 conservative amino acid substitutions and has beta-xylosidase activity. In an embodiment, the GH3 beta-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 beta-xylosidase activity. An exemplary GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 48. In an embodiment, the GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 48 with from 0 to 10 conservative amino acid substitutions and has beta-xylosidase activity. In an embodiment, the GH3 beta-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 beta-xylosidase activity.

An exemplary GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 49. In an embodiment, the GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 49 with from 0 to 10 conservative amino acid substitutions and has beta-xylosidase activity. In an embodiment, the GH3 beta-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 beta-xylosidase activity.

An exemplary GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 50. In an embodiment, the GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 50 with from 0 to 10 conservative amino acid substitutions and has beta-xylosidase activity. In an embodiment, the GH3 beta-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 beta-xylosidase activity.

An exemplary GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 51. In an embodiment, the GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 51 with from 0 to 10 conservative amino acid substitutions and has beta-xylosidase activity. In an embodiment, the GH3 beta-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 beta-xylosidase activity.

An exemplary GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 52. In an embodiment, the GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 52 with from 0 to 10 conservative amino acid substitutions and has beta-xylosidase activity. In an embodiment, the GH3 beta-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 beta-xylosidase activity. An exemplary GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 53. In an embodiment, the GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 53 with from 0 to 10 conservative amino acid substitutions and has beta-xylosidase activity. In an embodiment, the GH3 beta-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 beta-xylosidase activity.

An exemplary GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 54. In an embodiment, the GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 54 with from 0 to 10 conservative amino acid substitutions and has beta-xylosidase activity. In an embodiment, the GH3 beta-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 beta-xylosidase activity.

An exemplary GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 55. In an embodiment, the GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 55 with from 0 to 10 conservative amino acid substitutions and has beta-xylosidase activity. In an embodiment, the GH3 beta-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 beta-xylosidase activity.

The beta-xylosidase, e.g., GH3 beta-xylosidase may be dosed in presaccharification, saccharification, and/or simultaneous saccharification and fermentation in 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.

F. Exemplary Fermenting Organisms

Aspects of the invention relate to the use of a fermenting organism for producing a fermentation product. Especially suitable fermenting organisms are able to ferment, i.e. , convert, sugars, such as arabinose, glucose, maltose, and/or xylose, directly or indirectly into the desired fermentation product, such as ethanol. Examples of fermenting organisms include fungal organisms, such as yeast. Preferred yeast includes strains of Saccharomyces spp., in particular, Saccharomyces cerevisiae.

Examples of commercially available yeast includes, e.g., RED STAR™ and ETHANOL RED™ yeast (available from Fermentis/Lesaffre, USA), FALI (available from Fleischmann’s Yeast, USA), SUPERSTART and THERMOSACC™ fresh yeast (available from Ethanol Technology, Wl, USA), BIOFERM AFT and XR (available from NABC - North American Bioproducts Corporation, GA, 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 Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), such as, e.g., BY4741 (e.g., ATCC 201388); Y108-1 (ATCC PTA.10567) and NRRL YB- 1952 (ARS Culture Collection). Still other S. cerevisiae strains suitable as host cells DBY746, [Alpha][Eta]22, S150-2B, GPY55-15Ba, CEN.PK, USM21, TMB3500, TMB3400, VTT-A-63015, VTT-A-85068, VTT-c-79093 and their derivatives as well as Saccharomyces sp. 1400, 424A (LNH-ST), 259A (LNH-ST) and derivatives thereof.

As used herein, a “derivative” of strain is derived from a referenced strain, such as through mutagenesis, recombinant DNA technology, mating, cell fusion, or cytoduction between yeast strains. Those skilled in the art will understand that the genetic alterations, including metabolic modifications exemplified herein, may be described with reference to a suitable host organism and their corresponding metabolic reactions or a suitable source organism for desired genetic material such as genes for a desired metabolic pathway. However, given the complete genome sequencing of a wide variety of organisms and the high level of skill in the area of genomics, those skilled in the art can apply the teachings and guidance provided herein to other organisms. For example, the metabolic alterations exemplified herein can readily be applied to other species by incorporating the same or analogous encoding nucleic acid from species other than the referenced species.

The fermenting organism may be Saccharomyces strain, e.g., Saccharomyces cerevisiae strain produced using the method described and concerned in US patent no. 8,257,959-BB. In one embodiment, the recombinant cell is a derivative of a strain Saccharomyces cerevisiae CIBTS1260 (deposited under Accession No. NRRL Y-50973 at the Agricultural Research Service Culture Collection (NRRL), Illinois 61604 U.S.A.).

The fermenting organism may also be a derivative of Saccharomyces cerevisiae strain NMI V14/004037 (See, WO2015/143324 and WO2015/143317 each incorporated herein by reference), strain nos. V15/004035, V15/004036, and V15/004037 (See, WO 2016/153924 incorporated herein by reference), strain nos. V15/001459, V15/001460, V15/001461 (See, WO2016/138437 incorporated herein by reference), strain no. NRRL Y67342 (See, WO2018/098381 incorporated herein by reference), strain nos. NRRL Y67549 and NRRL Y67700 (See, WO 2019/161227 incorporated herein by reference), or any strain described in WO2017/087330 (incorporated herein by reference).

The fermenting organisms may comprise one or more heterologous polynucleotides encoding an alpha-amylase, glucoamylase, protease and/or cellulase. Examples of alpha- amylase, glucoamylase, protease and cellulases suitable for expression in the fermenting organism are known in the art (See, WO2021/231623 incorporated herein by reference), The fermenting organism may be in the form of a composition comprising a fermenting organism and a naturally occurring and/or a non-naturally occurring component. The fermenting organism may be in any viable form, including crumbled, dry, including active dry and instant, compressed, cream (liquid) form etc. In one embodiment, the fermenting organism (e.g., a Saccharomyces cerevisiae yeast strain) is dry yeast, such as active dry yeast or instant yeast. In one embodiment, the fermenting organism is crumbled yeast. In one embodiment, the fermenting organism is a compressed yeast. In one embodiment, the fermenting organism is cream yeast.

In one embodiment is a composition comprising a fermenting organism described herein (e.g., a Saccharomyces cerevisiae yeast strain), and one or more of the components selected from the group consisting of: surfactants, emulsifiers, gums, swelling agent, and antioxidants and other processing aids.

The compositions described herein may comprise a fermenting organism described herein (e.g., a Saccharomyces cerevisiae yeast strain) and any suitable surfactants. In one embodiment, the surfactant(s) is/are an anionic surfactant, cationic surfactant, and/or nonionic surfactant.

The compositions described herein may comprise a fermenting organism described herein (e.g., a Saccharomyces cerevisiae yeast 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 of sorbitan monostearate (SMS), citric acid esters of monodiglycerides, polyglycerolester, fatty acid esters of propylene glycol.

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

The compositions described herein may comprise a fermenting organism described herein (e.g., a Saccharomyces cerevisiae yeast strain) and any suitable gum. In one embodiment, the gum is selected from the group of carob, guar, tragacanth, arabic, xanthan and acacia gum, in particular for cream, compressed and dry yeast.

The compositions described herein may comprise a fermenting organism described herein (e.g., a Saccharomyces cerevisiae yeast strain) and any suitable swelling agent. In one embodiment, the swelling agent is methyl cellulose or carboxymethyl cellulose. The compositions described herein may comprise a fermenting organism described herein (e.g., a Saccharomyces cerevisiae yeast strain) and any suitable anti-oxidant. In one embodiment, the antioxidant is butylated hydroxyanisol (BHA) and/or butylated hydroxytoluene (BHT), or ascorbic acid (vitamin C), particular for active dry yeast.

Suitable concentrations of the viable fermenting organism during fermentation, such as SSF, are well known in the art or can easily be determined by the skilled person in the art. In one embodiment the fermenting organism, such as ethanol fermenting yeast, (e.g., Saccharomyces cerevisiae) is added to the fermentation medium so that the viable fermenting organism, such as yeast, count per mL of fermentation medium is in the range from 105 to 1012, preferably from 107 to 1010, especially about 5x107.

II. Process for producing a fermentation product from a gelatinized starch -containing material

An aspect of the invention relates to a process for producing a fermentation product, (e.g., fuel ethanol), from a gelatinized starch-containing material, wherein a composition comprising a GH5 xylanase or GH30_8 xylanase of the present invention, or a GH5 xylanase of the present invention, is present or added during saccharification or fermentation. This process of the invention contemplates any of the compositions described in Section I above, including any combination of the enzymes described in Section II, especially the compositions demonstrated in the examples below.

In an embodiment, a process for producing a fermentation product from a starch- containing material, comprises the steps of:

(a) liquefying a starch-containing material at a temperature above the initial gelatinization temperature of the starch using a thermostable alpha-amylase to produce a dextrin;

(b) saccharifying the dextrin using a glucoamylase to produce a fermentable sugar; and

(c) fermenting the sugar using a fermenting organism to produce the fermentation product; 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).

In an embodiment, the composition used in step (b) and/or step (c) includes a beta- xylosidase. In an embodiment, the composition used in step (b) and/or step (c) includes a GH3 beta-xylosidase. In an embodiment, the composition is added during saccharifying step (b). In an embodiment, the composition is added during fermenting step (c). In an embodiment, steps (b) and (c) are performed simultaneously in a simultaneous saccharification and fermentation (SSF). In an embodiment, the composition is added during SSF.

In an embodiment, a thermostable glucoamylase is added during liquefying step (a). In an embodiment, a thermostable endoglucanase is added during liquefying step (a). In an embodiment, a thermostable lipase is added during liquefying step (a). In an embodiment, a thermostable phytase is added during liquefying step (a). In an embodiment, a thermostable protease is added during liquefying step (a). In an embodiment, a thermostable pullulanase is added during liquefying step (a). In an embodiment, a thermostable xylanase is added during liquefying step (a). In a preferred embodiment, a thermostable alpha-amylase and a thermostable protease are added during liquefying step (a). In an embodiment, a thermostable alpha-amylase and a thermostable xylanase are added during liquefying step

(a). In a preferred embodiment, a thermostable alpha-amylase, a thermostable protease and a thermostable xylanase are added during liquefying step (a).

In an embodiment, an alpha-amylase is added during step (b) and/or step (c). In an embodiment, an alpha-glucosidase is added during step (b) and/or step (c). In an embodiment, a beta-amylase is added during step (b) and/or step (c). In an embodiment, a beta-glucanase is added during step (b) and/or step (c). In an embodiment, a betaglucosidase is added during step (b) and/or step (c). In an embodiment, a cellobiohydrolase is added during step (b) and/or step (c). In an embodiment, an endoglucanase is added during step (b) and/or step (c). In an embodiment a lipase is added during step (b) and/or step (c). In an embodiment, a lytic polysaccharide monooxygenase (LPMO) is added during step (b) and/or step (c). In an embodiment, a maltogenic alpha-amylsae is added during step

(b) and/or step (c). In an embodiment, a pectinase is added during step (b) and/or step (c). In an embodiment, a peroxidase is added during step (b) and/or step (c). In an embodiment, a phytase is added during step (b) and/or step (c). In an embodiment, a protease is added during step (b) and/or step (c). In an embodiment, a trehalase is added during step (b) and/or step (c).

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).

Process Parameters

The parameters for processes for producing fermentation products, such as the production of ethanol from starch-containing material (e.g., corn) are well known in the art. See, e.g., 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).

Starch-containing material

Any suitable starch-containing starting material may be used. The material is selected based on the desired fermentation product. Examples of starch-containing materials, include without limitation, barley, beets, beans, cassava, cereals, corn, milo, oats peas, potatoes, rice, rye, sago, sorghum, sweet potatoes, tapioca, wheat, and whole grains, or any mixture thereof. The starch-containing material may also be a waxy or non-waxy type of corn and barley. Commonly used commercial starch-containing materials include corn, milo and/or wheat.

Starch-Containing Material Particle Size Reduction

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

Slurry

Prior to liquefying step (a), a slurry comprising the starch-containing material (e.g., preferably milled) and water may be formed. Alpha-amylase and optionally protease may be added to the slurry. The slurry may be heated to between to above the initial gelatinization temperature of the starch-containing material to begin gelatinization of the starch.

Jet Cook

The slurry may optionally be jet-cooked to further gelatinize the starch in the slurry before adding alpha-amylase during liquefying step (a). Jet cooking can be performed at temperatures ranging from 100 °C to 120 °C for up to at least 15 minutes.

Liquefaction Temperature

The temperature used during liquefying step (a) may range 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.

Liquefaction pH

The pH used during liquefying step (a) may range 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. Liquefaction Time

The time for performing liquefying step (a) may range 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.

Liquefaction Enzymes

The present invention contemplates the use of thermostable enzymes during liquefying step (a). It is well known in the art to use various thermostable enzymes during liquefying step (a), including, for example, thermostable alpha-amylases, thermostable glucoamylases, thermostable endoglucanases, thermostable lipases, thermostable phytase, thermostable proteases, thermostable pullulanases, and/or thermostable xylanases. The present invention contemplates the use of any thermostable enzyme in liquefying step (a). Guidance for determining the denaturation temperature of a candidate thermostable enzyme for use in liquefying step (a) is provided in the Materials & Methods section below. The published patent applications listed below describe activity assays for determining whether a candidate thermostable enzyme contemplated for use in liquefying step (a) will be deactivated at a temperature contemplated for liquefying step (a).

Examples of suitable thermostable alpha-amylases and guidance for using them in liquefying step (a) include, without limitation, the alpha-amylases described in WO94/18314, WO94/02597, 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 2009/098229, WO 2009/100102, WO 2010/115021 , WO2010/115028, WO 2010/036515, WO 2011/082425, WO 2013/096305, WO 2013/184577, WO 2014/007921 , WO 2014/164777, WO 2014/164800, WO 2014/164834, WO 2019/113413, WO 2019/113415, WO 2019/197318 (each of which is incorporated herein by reference).

Examples of suitable thermostable glucoamylases include, without limitation, 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).

Examples of suitable thermostable endoglucanases include, without limitation, the endoglucanases described in WO 2015/035914 (which is incorporated herein by reference)

Examples of suitable thermostable lipases include, without limitation, the lipases described in WO 2017/112542 and WO 2020/014407 (which are both incorporated herein by reference). Examples of suitable thermostable phytases include, without limitation, 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-FEED PHYTASE™, PHYTASE NOVO™ CT or L, LIQMAX or RONOZYME™ NP, RONOZYME® HIPHOS, RONOZYME® P5000 (CT), NATUPHOS™ NG 5000.

Examples of suitable thermostable proteases include, without limitation, 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, WO2018/118815 A1, and WO2018/169780A1 (each of which is incorporated herein by reference).

Suitable commercially available protease containing products include AVANTEC AMP®, FORTIVA REVO®, FORTIVA HEMI®.

Examples of suitable thermostable pullulanases include, without limitation, the 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 pullulanase products include PROMOZYME 400L, PROMOZYME™ D2 (Novozymes A/S, Denmark), OPTIMAX L-300 (Genencor Int. , USA), and AMANO 8 (Amano, Japan).

Examples of suitable thermostable xylanases include, without limitation, the xylanases described in WO 2017/112540 and WO 2021/126966 (each of which is incorporated herein by reference). Suitable commercially available thermostable xylanase containing products include FORTIVA HEMI®

The enzyme(s) described above are to be used in effective amounts in the processes of the present invention. Guidance for determining effective amounts of enzymes to be used in liquefying step (a) can be found in the published patent applications cited for each of the different thermostable liquefaction enzymes, along with guidance for performing activity assays for determining the activity of those enzymes.

Saccharification Temperature

Saccharification may be performed at temperatures ranging from 20 °C to 75 °C, from 30 °C to 70 °C, or from 40 °C to 65 °C. Preferably, the saccharification temperature is at least about 50 °C, at least about 55 °C, or at least about 60 °C. Saccharification pH

Saccharification may occur at a ph ranging from 4 to 5. Preferably, the pH is about

4.5.

Saccharification Time

Saccharification may last from about 24 hours to about 72 hours.

Fermentation Time

Fermentation may last from 6 to 120 hours, from 24 hours to 96 hours, or from 35 hours to 60 hours.

Simultaneous Saccharification and Fermentation

SSF may be performed at a temperature from 25 °C to 40 °C, from 28 °C to 35 °C, or from 30 °C to °C, at a pH from 3.5 to 5 or from 3.8 to 4.3., for 24 to 96 hours, 36 to 72 hours, or from 48 to 60 hours. Preferably, SSF is performed at about 32 °C, at a pH from 3.8 to 4.5 for from 48 to 60 hours.

Saccharification and/or Fermentation Enzymes

The present invention contemplates the use of enzymes during saccharifying step (b) and/or fermenting step (c). It is well known in the art to use various enzymes during saccharifying step (b) and/or fermenting step (c), including, for example, alpha-amylases, alpha-glucosidases, beta-amylases, beta-glucanases, beta-glucosidases, cellobiohydrolases, endoglucanases, glucoamylases, lipases, lytic polysaccharide monooxygenases (LPMOs), maltogenic alpha-amylases, pectinases, peroxidases, phytases, proteases, and trehalases.

The enzymes used in saccharifying step (b) and/or fermenting step (c) may be added exogenously as mono-components or formulated as compositions comprising the enzymes. The enzymes used in saccharifying step (b) and/or fermenting step (c) may also be added via in situ expression from the fermenting organism (e.g., yeast).

Examples of suitable alpha-amylases include, without limitation, 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 incorporated herein by reference).

Examples of suitable glucoamylases include, without limitation, 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 herein by reference.

Examples of suitable compositions comprising alpha-amylases and glucoamylases include, without limitation, the compositons described in WO 2006/069290, WO 2009/052101, WO 2011/068803, and WO 2013/006756 (each of which is incorporated by reference herein). Commercially available compositions comprising glucoamylase include AMG 200L; AMG 300 L; SAN™ SUPER, SAN™ EXTRA L, SPIRIZYME™ PLUS, SPIRIZYME™ FUEL, SPIRIZYME™ B4U, SPIRIZYME™ ULTRA, SPIRIZYME™ EXCEL, SPIRIZYME ACHIEVE and AMG™ E (from Novozymes A/S); OPTIDEX™ 300, GC480, GC417 (from DuPont-Genencor); AMIGASE™ and AMIGASE™ PLUS (from DSM); G- ZYME™ G900, G-ZYME™ and G990 ZR (from DuPont-Genencor).

Examples of suitable beta-glucanases include, without limitation, the beta-glucanases described in WO 2021/055395 (which is incorporated herein by reference).

Examples of suitable beta-glucosidases include, without limitation, the betaglucosidases described in WO 2005/047499, WO 2013/148993, WO 2014/085439 and WO 2012/044915 (each of which is incorporated herein by reference).

Examples of suitable cellobiohydrolases include, without limitation, the cellobiohydrolases described in WO 2013/148993, WO 2014/085439, WO 2014/138672, and WO 2016/040265 (each of which is incorporated herein by reference).

Examples of suitable endoglucanases include, without limitation, the endoglucanases described in WO 2013/148993 and WO 2014/085439 (both of which are incorporated herein by reference).

Examples of suitable maltogenic alpha-amylases are described in US Patent nos. 4,598,048, 4,604,355 and 6,162,628, which are hereby incorporated by reference.

Examples of suitable lipases include, without limitation, the lipases described in WO 2017/112533, WO 2017/112539, and WO 2020/076697 (each of which is incorporated herein by reference).

Examples of suitable LPMOs include, without limitation, the LPMOs described in WO 2013/148993, WO 2014/085439, and WO 2019/083831 (each of which is incorporated herein by reference).

Examples of suitable phytases include, without limitation, the phytases described in WO 2001/62947 (which is incorporated herein by reference).

Examples of suitable pectinases include, without limitation, the pectinases described in WO 2022/173694 (which is incorporated herein by reference).

Examples of suitable peroxidases include, without limitation, the peroxidases described in WO 2019/231944 (which is incorporated herein by reference). Examples of suitable proteases include, without limitation, the proteases described in WO 2017/050291, WO 2017/148389, WO 2018/015303, and WO 2018/015304 (each of which is incorporated herein by reference).

Examples of suitable trehalases include, without limitation, the trehalases described in WO 2016/205127, WO 2019/005755, WO 2019/030165, and WO 2020/023411 (each of which is incorporated herein by reference).

III. Process for producing a fermentation product from ungelatinized starch -containing material

An aspect of the invention relates to a process for producing a fermentation product from an ungelatinized starch-containing material (i.e., granularized starch--often referred to as a “raw starch hydrolysis” process), wherein a composition comprising a GH5 xylanase or GH30_8 xylanase of the present invention, or a GH5 xylanase of the present invention, is present or added during saccharification or fermentation. This process of the invention contemplates any of the compositions described in Section I above, including any combination of the enzymes described in Section II, especially the compositions demonstrated in the examples below.

In an embodiment, a process for producing a fermentation product from an ungelatinized starch-containging material comprises the following steps:

(a) saccharifying a starch-containing material at a temperature below the initial gelatinization temperature of the starch using an alpha-amylase and a glucoamylase to produce a fermentable sugar; and

(b) fermenting the sugar using a fermentation organism to produce a fermentation product; 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).

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

In an embodiment, the composition used in step (a) and/or step (b) includes a beta- xylosidase. In an embodiment, the composition used in step (a) and/or step (b) includes a GH3 beta-xylosidase. In an embodiment, the composition is added during saccharifying step (a). In an embodiment, the composition is added during fermenting step (b). In an embodiment, steps (a) and (b) are performed simultaneously in a simultaneous saccharification and fermentation (SSF). In an embodiment, the composition is added during SSF. Raw starch hydrolysis (RSH) processes are well-known in the art. The skilled artisan will appreciate that, except for the process parameters relating to liquefying step (a) which is not done in a RSH process, the process parameters described in Section II above are applicable to the process described in this section, including selection of the starch- containing material, reducing the grain particle size, saccharification temperature, time and pH, conditions for simultaneous saccharification and fermentation, and saccharification enzymes. The process parameters for an exemplary raw-starch hydrolysis process are described in further detail in WO 2004/106533 (which is incorporated herein by reference).

Examples of alpha-amylases that are preferably used in step (a) and/or step (b) include, without limitation, the alpha-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 by reference herein).

Examples of glucoamylases that are preferably used in step (a) and/or step (b) include, without limitation, WO 1999/28448, WO 2005/045018, W02005/069840, WO 2006/069289 (each of which is incorporated by reference herein).

Examples of compositions comprising alpha-amylases and glucoamylase that are preferably used in step (a) and/or step (b) include, without limitation, the compositions described in WO 2015/031477 (which is incorporated by reference herein).

Backend or downstream processing

A. Recovery of the fermentation product and production of whole stillage

Subsequent to fermentation or SSF, the fermentation product may be separated from the fermentation medium. The fermentation product, e.g., ethanol, can optionally be recovered from the fermentation medium using any method known in the art including, but not limited to, chromatography, electrophoretic procedures, differential solubility, distillation, or extraction. For example, alcohol is separated from the fermented starch-containing material and purified by conventional methods of distillation.

Thus, in one embodiment, the method of the invention further comprises distillation to obtain the fermentation product, e.g., ethanol. The fermentation and the distillation may be carried out simultaneously and/or separately/sequentially; optionally followed by one or more process steps for further refinement of the fermentation product. Following the completion of the distillation process, the material remaining is considered the whole stillage.

As another example, the desired fermentation product may be extracted from the fermentation medium by micro 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. , potable neutral spirits, or industrial ethanol. In some embodiments of the methods, the fermentation product after being recovered is substantially pure. With respect to the methods herein, "substantially pure" intends a recovered preparation that contains no more than 15% impurity, wherein impurity intends compounds other than the fermentation product (e.g., ethanol). In one variation, a substantially pure preparation is provided wherein the preparation contains no more than 25% impurity, or no more than 20% impurity, or no more than 10% impurity, or no more than 5% impurity, or no more than 3% impurity, or no more than 1% impurity, or no more than 0.5% impurity.

Suitable assays to test for the production of ethanol and contaminants, and sugar consumption can be performed using methods known in the art. For example, ethanol product, as well as other organic compounds, can be analyzed by methods such as HPLC (High Performance Liquid Chromatography), GC-MS (Gas Chromatography Mass Spectroscopy) and LC-MS (Liquid Chromatography-Mass Spectroscopy) or other suitable analytical methods using routine procedures well known in the art. The release of ethanol in the fermentation broth can also be tested with the culture supernatant. Byproducts and residual sugar in the fermentation medium (e.g., glucose or xylose) can be quantified by HPLC using, for example, a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 90:775 -779 (2005)), or using other suitable assay and detection methods well known in the art.

B. Processing of Whole Stillage

In one embodiment, the whole stillage is processed into two streams — wet cake and centrate. The whole stillage is separated or partitioned into a solid and liquid phase by one or more methods for separating the centrate from the wet cake. The centrate is split into two flows--thin stillage, which goes to the evaporators, and backset, which is recycled to the front of the plant. Separating whole stillage into centrate (e.g., thin stillage when pumped toward the evaporators rather than the front end of the plant) and wet cake to remove a significant portion of the liquid/water, may be done using any suitable separation technique, including centrifugation, pressing and filtration. In a preferred embodiment, the separation/dewatering is carried out by centrifugation. Preferred centrifuges in industry are decanter type centrifuges, preferably high speed decanter type centrifuges. An example of a suitable centrifuge is the NX 400 steep cone series from ALFA LAVAL which is a high-performance decanter. A similar decanter centrifuge can also be purchased from FLOTTWEG. In another preferred embodiment, the separation is carried out using other conventional separation equipment such as a plate/frame filter presses, belt filter presses, screw presses, gravity thickeners and deckers, or similar equipment. C. Processing of Thin Stillage

Thin stillage is the term used for the supernatant of the centrifugation of the whole stillage. Typically, the thin stillage contains 4-8 percent dry solids (DS) (mainly proteins, soluble fiber, fats, fine fibers, and cell wall components) and has a temperature of about 60- 90 degrees centigrade. The thin stillage stream may be condensed by evaporation to provide two process streams including: (i) an evaporator condensate stream comprising condensed water removed from the thin stillage during evaporation, and (ii) a syrup stream, comprising a more concentrated stream of the non-volatile dissolved and non-dissolved solids, such as non-fermentable sugars and oil, remaining present from the thin stillage as the result of removing the evaporated water.

Optionally, oil can be removed from the thin stillage or can be removed as an intermediate step to the evaporation process, which is typically carried out using a series of several evaporation stages.

Syrup and/or de-oiled syrup may be introduced into a dryer together with the wet cake (from the whole stillage separation step) to provide a product referred to as distillers dried grain with solubles, which also can be used as animal feed. In an embodiment, syrup and/or de-oiled syrup is sprayed into one or more dryers to combine the syrup and/or deoiled syrup with the whole stillage to produce distillers dried grain with solubles.

Between 5-90 vol-%, such as between 10-80%, such as between 15-70%, such as between 20-60% of thin stillage (e.g., optionally hydrolyzed) may be recycled (as backset) to step (a). The recycled thin stillage (i.e. , backset) may constitute from about 1-70 vol.-%, preferably 15-60% vol.-%, especially from about 30 to 50 vol.-% of the slurry formed in step (a). In an embodiment, the process further comprises recycling at least a portion of the thin stillage stream to the slurry, optionally after oil has been extracted from the thin stillage stream.

D. Drying of Wet Cake and Producing Distillers Dried Grains and Distillers Dried Grains with Solubles

After the wet cake, containing about 25-40 wt-%, preferably 30-38 wt-% dry solids, has been separated from the thin stillage (e.g., dewatered) it may be dried in a drum dryer, spray dryer, ring drier, fluid bed drier or the like in order to produce “Distillers Dried Grains” (DDG). DDG is a valuable feed ingredient for animals, such as livestock, poultry and fish. It is preferred to provide DDG with a content of less than about 10-12 wt.-% moisture to avoid mold and microbial breakdown and increase the shelf life. Further, high moisture content also makes it more expensive to transport DDG. The wet cake is preferably dried under conditions that do not denature proteins in the wet cake. The wet cake may be blended with syrup separated from the thin stillage and dried into DDG with Solubles (DDGS). Partially dried intermediate products, such as are sometimes referred to as modified wet distillers grains, may be produced by partially drying wet cake, optionally with the addition of syrup before, during or after the drying process.

The invention is further summarized in the following paragraphs:

1. A composition comprising:

(a) a GH43 arabinofuranosidase;

(b) a GH51 arabinofuranosidase; and

(c) a GH5_21 xylanase or a GH30_8 xylanase.

2. The composition of paragraph 1, wherein:

(a) the GH43 and GH51 arabinofuranosidases and GH5_21 or GH30_8 xylanase together releases at least150% more arabinose from liquefied corn mash than the GH43 and GH51 arabinofuranosidases combined when the GH5_21 or GH30_8 xylanase is not present in the composition; and/or

(b) the GH43 and GH51 arabinofuranosidases and GH5_21 or GH30_8 xylanase together releases at least 2 times more arabinose from liquefied corn mash than the GH43 and GH51 arabinofuranosidases combined when the GH5_21 or GH30_8 xylanase is not present in the composition.

3. The composition of paragraph 1 or 2, further comprising a beta-xylosidase.

4. The composition of any one of paragraphs 1-3, wherein the beta-xylosidase is a GH3 beta-xylosidase.

5. The composition of paragraph 4, wherein:

(a) the GH43 and GH51 arabinofuranosidases, GH3 beta-xylosidase, and the GH5_21 xylanase together release at least 150% more xylose from liquefied corn mash than the GH43 and GH51 arabinofuranosidases combined when the GH3 beta-xylosidase and GH5_21 xylanase are not present in the composition; and/or

(b) the GH43 and GH51 arabinofuranosidases, GH3 beta-xylosidase, and the GH5_21 xylanase together release at least 2.5 times more xylose from liquefied corn mash than the GH43 and GH51 arabinofuranosidases combined when the GH3 beta-xylosidase and GH5_21 xylanase are not present in the composition.

6. A composition comprising:

(a) a GH43 arabinofuranosidase;

(b) a GH51 arabinofuranosidase; (c) a GH3 beta-xylosidase; and

(d) a GH5 xylanase.

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

8. The composition of paragraph 7, wherein:

(a) the GH43 and GH51 arabinofuranosidases, GH3 beta-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 GH43 and GH51 arabinofuranosidases and GH3 beta-xylosidase combined when the GH5_21 xylanase is not present in the composition; and/or

(b) the GH43 and GH51 arabinofuranosidases, GH3 beta-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 GH43 and GH51 arabinofuranosidases and GH3 beta-xylosidase combined when the GH5_21 xylanase is not present in the composition.

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

10. The composition of paragraph 9, wherein:

(a) the GH43 and GH51 arabinofuranosidases, GH3 beta-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 GH43 and GH51 arabinofuranosidases and GH3 beta-xylosidase combined when the GH5_35 xylanase is not present in the composition.

11. The composition of any one of paragraphs 1-10, wherein the composition does not include a GH8 xylanase, a GH10 xylanase, or a GH11 xylanase.

12. The composition of any one of paragraphs 1-11 , wherein the GH43 arabinofuranosidase is a GH43_36.

13. The composition of any one of paragraphs 1-12, wherein the GH43 arabinofuranosidase is from the genus Humicola, Lasiodiplodia, or Poronia. 14. The composition of any one of paragraphs 1-13, wherein the GH43 arabinofuranosidase is from the species Humicola insolens, Lasiodiplodia theobromae, or Poronia punctata.

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

(i) the amino acid sequence of SEQ ID NO: 1 with from 0 to 10 conservative amino acid substitutions or one 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;

(ii) the amino acid sequence of SEQ ID NO: 2 with from 0 to 10 conservative amino acid substitutions or one 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: 2, which has arabinofuranosidase activity; and

(iii) the amino acid sequence of SEQ ID NO: 3 with from 0 to 10 conservative amino acid substitutions or one 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.

16. The composition of any one of paragraphs 1-15, wherein the GH51 arabinofuranosidase is from the genus Meripilus, Lasiodiplodia, or Acidiella.

17. The composition of any one of paragraphs 1-16, wherein the GH51 arabinofuranosidase is from the species Meripilus giganteus, Lasiodiplodia theobromae, or Acidiella bo he mica.

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

(i) the amino acid sequence of SEQ ID NO: 4 with from 0 to 10 conservative amino acid substitutions or one 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 to the amino acid sequence of SEQ ID NO: 4, which has arabinofuranosidase activity; (ii) the amino acid sequence of SEQ ID NO: 5 with from 0 to 10 conservative amino acid substitutions or one 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: 5, which has arabinofuranosidase activity;

(iii) the amino acid sequence of SEQ ID NO: 6 with from 0 to 10 conservative amino acid substitutions or one 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.

19. The composition of any one of paragraphs 1-18, wherein the GH5_21 xylanase is from the genus Bacteroides, Belliella, Chryseobacterium, or Sphingobacterium.

20. The composition of any one of paragraphs 1-19, wherein the GH5_21 xylanase is from the species Bacteroides cellulosilyticus CL02Y12C19, Belliella sp-64282, Chryseobacterium sp., Chryseobacterium oncorhynchi, or Sphingobacterium sp-64162.

21. The composition of any one of paragraphs 1-20, wherein the GH5_21 xylanase is from bioreactor metagenome, Elephant dung metagenome, Xanthan alkaline community O, Xanthan alkaline community S, or Xanthan alkaline community T.

22. The composition of any one of paragraphs 1-21 , wherein the GH5_21 xylanase has an amino acid sequence selected from the group consisting of:

(i) the amino acid sequence of SEQ ID NO: 7 with from 0 to 10 conservative amino acid substitutions or one 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: 7, which has xylanase activity;

(ii) the amino acid sequence of SEQ ID NO: 8 with from 0 to 10 conservative amino acid substitutions or one 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: 8, which has xylanase activity;

(iii) the amino acid sequence of SEQ ID NO: 9 with from 0 to 10 conservative amino acid substitutions or one 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: 9, which has xylanase activity;

(iv) the amino acid sequence of SEQ ID NO: 10 with from 0 to 10 conservative amino acid substitutions or one 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: 10, which has xylanase activity;

(v) the amino acid sequence of SEQ ID NO: 11 with from 0 to 10 conservative amino acid substitutions or one 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: 11 , which has xylanase activity;

(vi) the amino acid sequence of SEQ ID NO: 12 with from 0 to 10 conservative amino acid substitutions or one 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: 12, which has xylanase activity;

(vii) the amino acid sequence of SEQ ID NO: 13 with from 0 to 10 conservative amino acid substitutions or one 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: 13, which has xylanase activity;

(viii) the amino acid sequence of SEQ ID NO: 14 with from 0 to 10 conservative amino acid substitutions or one 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: 14, which has xylanase activity;

(ix) the amino acid sequence of SEQ ID NO: 15 with from 0 to 10 conservative amino acid substitutions or one 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: 15, which has xylanase activity;

(x) the amino acid sequence of SEQ ID NO: 16 with from 0 to 10 conservative amino acid substitutions or one 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: 16; (xi) the amino acid sequence of SEQ ID NO: 17 with from 0 to 10 conservative amino acid substitutions or one 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: 17, which has xylanase activity;

(xii) the amino acid sequence of SEQ ID NO: 18 with from 0 to 10 conservative amino acid substitutions or one 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: 18, which has xylanase activity;

(xiii) the amino acid sequence of SEQ ID NO: 19 with from 0 to 10 conservative amino acid substitutions or one 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: 19, which has xylanase activity;

(xiv) the amino acid sequence of SEQ ID NO: 20 with from 0 to 10 conservative amino acid substitutions or one 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: 20, which has xylanase activity; and

(xv) the amino acid sequence of SEQ ID NO: 21 with from 0 to 10 conservative amino acid substitutions or one 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: 21 , which has xylanase activity.

23. The composition of any one of paragraphs 1-22, wherein the GH5_35 xylanase is from the genus Bacillus, Cohnella, or Paenibacillus.

24. The composition of any one of paragraphs 1-23, wherein the GH5_35 xylanase is from the species Bacillus hemiccellulosilyticus JCM 9152, Cohnella xylanilytica, Paenibacillus chitinolyticus, or Paenibacillus sp-62332.

25. The composition of any one of paragraphs 1-24, wherein the GH5_35 xylanase is from compost metagenome. 26. The composition of any one of paragraphs 1-25, wherein the GH5_35 xylanase has an amino acid sequence selected from the group consisting of:

(i) the amino acid sequence of SEQ ID NO: 22 with from 0 to 10 conservative amino acid substitutions or one 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: 22, which has xylanase activity;

(ii) the amino acid sequence of SEQ ID NO: 23 with from 0 to 10 conservative amino acid substitutions or one 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: 23, which has xylanase activity;

(iii) the amino acid sequence of SEQ ID NO: 24 with from 0 to 10 conservative amino acid substitutions or one 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: 24, which has xylanase activity;

(iv) the amino acid sequence of SEQ ID NO: 25 with from 0 to 10 conservative amino acid substitutions or one 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: 25, which has xylanase activity; and

(v) the amino acid sequence of SEQ ID NO: 26 with from 0 to 10 conservative amino acid substitutions or one 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 to the amino acid sequence of SEQ ID NO: 26, which has xylanase activity.

27. The composition of any one of paragraphs 1-26 wherein the GH30_8 xylanase is from the genus Bacillus.

28. The composition of any one of paragraphs 1-27, wherein the GH30_8 xylanase is from the species Bacillus sp- 8423.

29. The composition of any one of paragraphs 1-28, wherein the GH30_8 xylanase has the amino acid sequence of SEQ ID NO: 27 with from 0 to 10 conservative amino acid substitutions or one 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.

30. The composition of any one of paragraphs 1-29, wherein the GH3 beta- xylosidase is from the genus Aspergiluus or Talaromyces.

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

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

(i) the amino acid sequence of SEQ ID NO: 28 with from 0 to 10 conservative amino acid substitutions or one 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: 28, which has beta-xylosidase activity;

(ii) the amino acid sequence of SEQ ID NO: 29 with from 0 to 10 conservative amino acid substitutions or one 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: 29, which has beta-xylosidase activity;

(iii) the amino acid sequence of SEQ ID NO: 30 with from 0 to 10 conservative amino acid substitutions or one 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: 30, which has beta-xylosidase activity;

(iv) the amino acid sequence of SEQ ID NO: 44 with from 0 to 10 conservative amino acid substitutions or one 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: 44, which has beta-xylosidase activity;

(v) the amino acid sequence of SEQ ID NO: 45 with from 0 to 10 conservative amino acid substitutions or one 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: 45, which has beta-xylosidase activity;

(vi) the amino acid sequence of SEQ ID NO: 46 with from 0 to 10 conservative amino acid substitutions or one 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: 46, which has beta-xylosidase activity;

(vii) the amino acid sequence of SEQ ID NO: 47 with from 0 to 10 conservative amino acid substitutions or one 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: 47, which has beta-xylosidase activity;

(viii) the amino acid sequence of SEQ ID NO: 48 with from 0 to 10 conservative amino acid substitutions or one 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: 48, which has beta-xylosidase activity;

(ix) the amino acid sequence of SEQ ID NO: 49 with from 0 to 10 conservative amino acid substitutions or one 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 beta-xylosidase activity;

(x) the amino acid sequence of SEQ ID NO: 50 with from 0 to 10 conservative amino acid substitutions or one 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: 50, which has beta-xylosidase activity;

(xi) the amino acid sequence of SEQ ID NO: 51 with from 0 to 10 conservative amino acid substitutions or one 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: 51 , which has beta-xylosidase activity;

(x) the amino acid sequence of SEQ ID NO: 52 with from 0 to 10 conservative amino acid substitutions or one 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: 52, which has beta-xylosidase activity; (xi) the amino acid sequence of SEQ ID NO: 53 with from 0 to 10 conservative amino acid substitutions or one 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: 53, which has beta-xylosidase activity;

(xii) the amino acid sequence of SEQ ID NO: 54 with from 0 to 10 conservative amino acid substitutions or one 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: 54, which has beta-xylosidase activity; and

(xiii) the amino acid sequence of SEQ ID NO: 55 with from 0 to 10 conservative amino acid substitutions or one 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 beta-xylosidase activity.

33. A process for producing a fermentation product from a starch-containing material, the process comprising:

(a) saccharifying a starch-containing material at a temperature below the initial gelatinization temperature of the starch- with an alpha-amylase and a glucoamylase to produce fermentable a sugar; and

(b) fermenting the sugar with a fermenting organism to produce the fermentation product, wherein:

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

(j) the composition of any one of claims 1-32 is present or added during step (a) and/or step (b).

34. The process of paragraph 33, wherein steps (a) and (b) are performed simultaneously.

35. A process for producing a fermentation product from a starch-containing material, the process comprising:

(d) liquefying a starch-containing material with a thermostable alpha-amylase at a temperature above the initial gelatinization temperature of the starch to produce a dextrin;

(e) saccharifying the dextrin with a glucoamylase to produce a fermentable sugar; (f) fermenting the sugar with a fermenting organism to produce the fermentation product, wherein:

(i) the xylanase of any one of claims 19 to 29 is present or added during step (b) and/or step (c); or

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

36. The process of paragraph 35, wherein steps (b) and (c) are performed simultaneously.

37. The process of paragraph 35 or 36, wherein the thermostable alpha-amylase has the amino acid sequence of SEQ ID NO: 34 with from 0 to 10 conservative amino acid substitutions or an amino acid sequence that is 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 alpha-amylase activity.

38. The process of paragraph 35 or 36, wherein the thermostable alpha-amylase has the amino acid sequence of SEQ ID NO: 35 with from 0 to 10 conservative amino acid substitutions or an amino acid sequence that is 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.

39. The process of any one of paragraphs 35 to 38, wherein a thermostable protease and/or a thermostable xylanase are added in liquefying step (a).

40. The process of any one of paragraphs 35 to 39, wherein the thermostable protease has the amino acid sequence of SEQ ID NO: 36 with from 0 to 10 conservative amino acid substitutions or an amino acid sequence that is 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.

41. The process of any one of paragraphs 35 to 40, wherein the thermostable xylanase has an amino acid sequence of SEQ ID NO: 37 with from 0 to 10 conservative amino acid substitutions or an amino acid sequence that is 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.

42. The process of any one of paragraphs 33 to 41, wherein the glucoamylase has an amino acid sequence of SEQ ID NO: 38 with from 0 to 10 conservative amino acid substitutions or an amino acid sequence that is 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.

43. The process of any one of paragraphs 35 to 42, further comprising adding an alpha-amylase during saccharifying step (b) and/or fermenting step (c).

44. The process of any one of paragraphs 33 to 43, wherein the alpha-amylase has an amino acid sequence of SEQ ID NO: 39 with from 0 to 10 conservative amino acid substitutions or an amino acid sequence that is 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.

45. The process of any one of paragraphs 33 to 44, wherein a trehalase is added during the saccharifying step and/or the fermenting step.

46. The process of any one of paragraph 45, wherein the trehalase has an amino acid sequence of SEQ ID NO: 40 with from 0 to 10 conservative amino acid substitutions or an amino acid sequence that is 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.

47. The process of any one of paragraphs 33 to 47, wherein a composition comprising a beta-glucosidase, a cellobiohydrolase, and an endoglucanase are added during the saccharifying step and/or the fermenting step.

48. The process of paragraph 47, wherein the beta-glucosidase has an amino acid sequence of SEQ ID NO: 41 with from 0 to 10 conservative amino acid substitutions or an amino acid sequence that is 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 beta-glucosidase activity.

49. The process of paragraphs 47-48, wherein the cellobiohydrolase has an amino acid sequence of SEQ ID NO: 42 with from 0 to 10 conservative amino acid substitutions or an amino acid sequence that is 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.

50. The process of any one of paragraphs 47-49, wherein the endoglucanase has an amino acid sequence of SEQ ID NO: 43 with from 0 to 10 conservative amino acid substitutions or an amino acid sequence that is 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.

51. The process of any one of paragraphs 33 to 50, wherein the starch-containing material comprises beets, maize, corn, wheat, rye, barley, oats, triticale, rice, sorghum, sweet potatoes, millet, pearl millet, and/or foxtail millet.

52. The process of any one of paragraphs 33 to 51, wherein the starch-containing material comprises corn.

53. The process of any one of paragraphs 33 to 52, wherein the fermentation product is ethanol, preferably fuel ethanol.

54. The process of any one of paragraphs 33 to 53, wherein the fermenting organism is yeast.

55. The process of any one of paragraphs 33 to 54, wherein solubilization of hemicellulosic fiber is increased to release significantly more monomeric arabinose and monomeric xylose compared to a control process:

(i) lacking the composition; (ii) using a composition with only the GH43 and GH51 arabinofuranosidases alone;

(iii) using a composition with the GH43 and GH51 arabinofuranosidases and a GH8 xylanase;

(iv) using a composition with the GH43 and GH51 arabinofuranosidases and a GH10 xylanase; and/or

(v) using a composition with the GH43 and GH51 arabinofuranosidases and a GH11 xylanase.

56. The process of any one of paragraphs 33-55, wherein residual solids are decreased compared to a control process lacking the composition.

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

EXAMPLES

Materials & Methods

Enzymes used in the examples

GH43A: exemplary GH43 arabinofuranosidase from Humicola insolens disclosed in SEQ ID NO: 1.

GH43B: exemplary GH43 arabinofuranosidase from Lasiodiplodia theobromane disclosed in SEQ ID NO: 2.

GH43C: exemplary GH43 arabinofuranosidase from Poronia punctata disclosed in SEQ ID NO: 3.

GH51A: exemplary GH51 arabinofuranosidase from Meripilus giganteus disclosed in SEQ ID NO: 4.

GH51B: exemplary GH51 arabinofuranosidase from Lasiodiplodia theobromae disclosed in SEQ ID NO: 5. GH51C: exemplary GH51 arabinofuranosidase from Acidiella bohemica disclosed in SEQ ID NO: 6.

GH5_21A: exemplary GH5_21 xylanase from Bacteroides cellulosilyticus CL02T12C19 disclosed in SEQ ID NO: 7.

GH5_21B: exemplary GH5_21 xylanase from Xanthan alkaline community S disclosed in SEQ ID NO: 8.

GH5_21C: exemplary GH5_21 xylanase from Sphingobacterium sp-64162 disclosed in SEQ ID NO: 9.

GH5_21D: exemplary GH5_21 xylanase from Sphingobacterium sp-64162 disclosed in SEQ ID NO: 10.

GH5_21E: exemplary GH5_21 xylanase from Xanthan alkaline community O disclosed in SEQ ID NO: 11.

GH5_21F: exemplary GH5_21 xylanase from bioreactor metagenome disclosed in SEQ ID NO: 12.

GH5_21G: exemplary GH5_21 xylanase from Xanthan alkaline community T disclosed in SEQ ID NO: 13.

GH5_21H: exemplary GH5_21 xylanase from Xanthan alkaline community S disclosed in SEQ ID NO: 14.

GH5_21I: exemplary GH5_21 xylanase from Belliella sp-64282 disclosed in SEQ ID NO: 15.

GH5_21J: exemplary GH5_21 xylanase from Chryseobacterium oncorhynchi disclosed in SEQ ID NO: 16.

GH5_21K: exemplary GH5_21 xylanase from Xanthan alkaline community T disclosed in SEQ ID NO: 17.

GH5_21L: exemplary GH5_21 xylanase from Sphingobacterium disclosed in SEQ ID NO: 18.

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

GH5_21N: exemplary GH5_21 xylanase from elephant dung metagenome disclosed in SEQ ID NO: 20.

GH5_21O: exemplary GH5_21 xylanase from Chryseobacterium sp disclosed in SEQ ID NO: 21.

GH5_35A: exemplary GH5_35 xylanase from Cohnella xylanilytica disclosed in SEQ ID NO: 22.

GH5_35B: exemplary GH5_35 xylanase from Bacillus hemicellulosilyticus JCM 9152 disclosed in SEQ ID NO: 23. GH5_35C: exemplary GH5_35 xylanase from Paenibacillus sp-62332 disclosed in SEQ ID NO: 24.

GH5_35D: exemplary GH5_35 xylanase from compost metagenome disclosed in SEQ ID NO: 25.

GH5_35E: exemplary GH5_35 xylanase from Paenibacillus chitinolyticus disclosed in SEQ ID NO: 26.

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

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

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

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

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

GH10: exemplary GH10 xylanase from Aspergillus aculeatus disclosed in SEQ ID NO: 32.

GH11: exemplary GH11 xylanase from Thermomyces lanuginosus disclosed in SEQ ID NO: 33.

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

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

Saccharification Enzyme Blend: exemplary glucoamylase from Gloeophyllum sepiarium disclosed in SEQ ID NO: 38; exemplary alpha-amylase from Rhizomucor pusillus disclosed in SEQ ID NO: 39; exemplary trehalase from Talaromyces funiculosus disclosed in SEQ ID NO: 40; exemplary beta-glucosidase from Aspergillus fumigatus disclosed in SEQ ID NO: 41; exemplary celliobiohydrolase from Aspergillus fumigatus disclosed in SEQ ID NO: 42; exemplary endoglucanase from Trichoderma reesei disclosed in SEQ ID NO: 43.

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

Aa BX: exemplary GH3 beta-xylosidase from Aspergillus aculeatus disclosed in SEQ ID NO: 45. Af BX: exemplary GH3 beta-xylosidase beta-xylosidase from Aspergillus fischeri disclosed in SEQ ID NO: 46.

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

Cv BX: exemplary GH3 beta-xylosidase beta-xylosidase from Chaetomium virescens disclosed in SEQ ID NO: 48.

Fl BX: exemplary GH3 beta-xylosidase beta-xylosidase from Fusarium longipes disclosed in SEQ ID NO: 49.

Mt BX: exemplary GH3 beta-xylosidase beta-xylosidase from Mycothermus thermophilus disclosed in SEQ ID NO: 50.

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

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

Sf BX: exemplary GH3 beta-xylosidase beta-xylosidase from Sporormia fimetaria disclosed in SEQ ID NO: 53.

Ts BX: exemplary GH3 beta-xylosidase beta-xylosidase from Talaromyces stipitatus disclosed in SEQ ID NO: 54.

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

Determination of Td by Differential Scanning Calorimetry for Liquefaction Enzymes

The thermostability of an enzyme is determined by Differential Scanning Calorimetry (DSC) using a VP-Capillary Differential Scanning Calorimeter (MicroCai Inc., Piscataway, NJ, USA). The thermal denaturation temperature, Td (°C), is taken as the top of denaturation peak (major endothermic peak) in thermograms (Cp vs. T) obtained after heating enzyme solutions (approx. 0.5 mg/ml) in buffer (50 mM acetate, pH 5.0) at a constant programmed heating rate of 200 K/hr.

Sample- and reference-solutions (approx. 0.2 ml) are loaded into the calorimeter (reference: buffer without enzyme) from storage conditions at 10°C and thermally preequilibrated for 20 minutes at 20°C prior to DSC scan from 20°C to 120°C. Denaturation temperatures are determined at an accuracy of approximately +/- 1°C.

Strains

Yeast strain MEJI797 is MBG5012 of WO2019/161227 further expressing a Pycnopous sanguineus glucoamylase (SEQ ID NO: 4 of WO2011/066576) and a hybrid Rhizomucor pusillus alpha amylase expression cassette (as described in WO2013/006756).

Example 1 - Effect of xylanases from GH families 5, 8, 10, 11 and 30 in combination with arabinofuranosidases from GH families 43 and 51 for increasing xylose and arabinose in simultaneous saccharification and fermentation process

An industrial liquefied mash prepared using Liquefaction Enzyme Blend 1 was used for the experiment. The dry solid determined by moisture balance was about 34%DS and pH was adjusted to pH 5.0 following by supplemented with 3 ppm of penicillin and 500 ppm of urea. Simultaneous saccharification and fermentation (SSF) was performed via mini-scale fermentations. Approximately 4.0 g of the industrial liquefied corn mash was added to 15 ml tube vials. Each vial was dosed with 0.42 AGU/gDS of Saccharification Enzyme Blend and appropriate amount of respective xylanase and arabinofuranosidase with the dosing scheme treatments as shown in Table 1. Saccharification Enzyme blend was used as a control, without addition of xylanase or arabinofuranosidase. Actual enzymes dosages were based on the exact weight of corn slurry in each vial. After enzymes addition, fermentation was initiated by addition of 50 pL of propagated yeast strain MEJI797. Tubes were incubated at 32°C with three replicates for each treatment. After 65 hours SSF, tubes were taken out from incubator and added 50 pL of 34% H2SO4 and then subjected to centrifugation 3500 rpm for 10 min follow by filtering through a 0.45 micrometer filter. Sugars concentrations were determined using HPLC system equipped with H column (Benson Polymeric, BP-700 H, 300 x 7.8 mm).

Table 1 : Dosing scheme of arabinofuranosidase with or without xylanase

Table 2: Results

% Boost arabinose = [(Mean arabinose experimental ppm - mean arabinose control) I mean arabinose control] x 100

Table 2 shows that GH5_21 or GH30_8 xylanases combined with GH43 and GH51 arabinofuranosidases release the highest concentration of arabinose compared to GH43 or GH51 arabinofuranosidase alone or their combination without xylanase.

Example 2 - Effect of GH3 family beta-xylosidase combination with arabinofuranosidase from GH 43 and 51 families and xylanase from GH5_21 for increasing xylose in simultaneous saccharification and fermentation process

An industrial liquefied mash prepared using Liquefaction Enzyme Blend 2 was used for the experiment. The dry solid determined by moisture balance was about 35.9%DS and pH was adjusted to pH 5.0 following by supplemented with 3 ppm of penicillin and 500 ppm of urea. Simultaneous saccharification and fermentation (SSF) was performed via mini-scale fermentations. Approximately 4.2 g of the industrial liquefied corn mash was added to 15 ml tube vials. Each vial was dosed with 0.42 AGU/gDS of Saccharification Enzyme Blend and appropriate amount of respective xylanase, arabinofuranosidase and beta-xylosidase as listed in Table 3. The dosing scheme followed the fixed amount of GH5_21 xylanase, GH43 arabinofuranosidase and GH51 arabinofuranosidase of each 10 ug/g dry solids, respectively, with or without beta-xylosidase GH3A, GH3B or GH3C at a dosage of 25, 50, 100 or 200 ug/g dry solids. Saccharification Enzyme Blend was used as a control, without addition of xylanases or beta-xylosidases. Actual enzymes dosages were based on the exact weight of corn slurry in each vial. After enzymes addition, fermentation was initiated by addition of 50 pL of propagated yeast strain MEJI797. Tubes were incubated at 32°C with three replicates for each treatment. After 65 hours SSF, tubes were taken out from incubator and then subjected to centrifugation 3500 rpm for 10 min follow by filtering through a 0.45 micrometer filter. Sugars concentrations were determined using HPLC system equipped with lead column (Benson Polymeric, BP-800 Pb, 300 x 7.8 mm).

Result Table 3 shows beta-xylosidase combined with GH43, GH51 arabinofuranosidases and GH5_21 xylanase significantly increases xylose release and higher enzyme dosages corresponded to higher xylose release.

Table 3

Example 3 - Effect of GH 5 xylanase subfamilies 21 and 35 combination with Hi GH 43 and Mg GH51 arabinofuranosidases and Af GH3 beta-xylosidase for increasing xylose and arabinose in simultaneous saccharification and fermentation process

An industrial liquefied mash prepared using Liquefaction Enzyme Blend 1 was used for the experiment. The dry solid determined by moisture balance was about 33.7%DS and pH was adjusted to pH 5.0 following by supplemented with 3 ppm of penicillin and 500 ppm of urea. Simultaneous saccharification and fermentation (SSF) was performed via mini-scale fermentations. Approximately 4.2 g of the industrial liquefied corn mash was added to 15 ml tube vials. Each vial was dosed with 0.42 AGU/gDS of Saccharification Enzyme Blend, 10 ug/gDS of GH43A arabinofuranosidase, 10 ug/gDS of GH51A arabinofuranosidase, 25 ug/gDS of GH3A beta-xylosidase and 10 ug/gDS of respective xylanase as listed in Table 4. Saccharification Enzyme Blend was used as a control, without addition of arabinofuranosidases, xylanases or beta-xylosidases. Actual enzymes dosages were based on the exact weight of corn slurry in each vial. After enzymes addition, fermentation was initiated by addition of 50 pL of propagated yeast strain MEJI797. Tubes were incubated at 32°C with three replicates for each treatment. After 65 hours SSF, tubes were taken out from incubator and added 50 pL of 34% H2SO4 and then subjected to centrifugation 3500 rpm for 10 min follow by filtering through a 0.45 micrometer filter. Sugars concentrations were determined using HPLC system equipped with lead column (Benson Polymeric, BP-800 Pb, 300 x 7.8 mm). Result

Table 4 shows that the addition of xylanases from GH5_21 and GH5_35 significantly increase xylose and arabinose release compared to control or treatment consist of GH43, GH51 arabinofuranosidase and GH3 beta-xylosidase, without xylanase. Table 4

Example 4 - Effect of single, double, triple or quadruple combination of hemicellulases of GH5_21 xylanase, GH43 and GH51 arabinofuranosidase and GH3 beta-xylosidase for increasing xylose and arabinose in simultaneous saccharification and fermentation process

An industrial liquefied mash prepared using Liquefaction Enzyme Blend 2 was used for the experiment. The dry solid determined by moisture balance was about 33.4%DS and pH was adjusted to pH 5.0 following by supplemented with 3 ppm of penicillin and 500 ppm of urea. Simultaneous saccharification and fermentation (SSF) was performed via mini-scale fermentations. Approximately 4.2 g of the industrial liquefied corn mash was added to 15 ml tube vials. Each vial was dosed with 0.6 AGU/gDS of Saccharification Enzyme Blend and followed the dosing scheme of 10 ug/gDS GH5_21O xylanase, 10 ug/gDS of GH43A arabinofuranosidase, 10 ug/gDS of GH51A arabinofuranosidase and/or 25 ug/gDS of GH3A beta-xylosidase. As control, only Saccharification Enzyme Blend was added without arabinofuranosidases, xylanases or beta-xylosidases. Actual enzymes dosages were based on the exact weight of corn slurry in each vial. After enzymes addition, fermentation was initiated by addition of 50 pL of hydrated yeast strain MEJI797. Tubes were incubated at 32°C with three replicates for each treatment. After 65 hours SSF, tubes were taken out from incubator and added 50 pL of 34% H2SO4 and then subjected to centrifugation 3500 rpm for 10 min follow by filtering through a 0.45 micrometer filter. Sugars concentrations were determined using HPLC system equipped with H column (Benson Polymeric, BP-700 H, 300 x 7.8 mm). The decanted tubes containing wet corn mash at the end of fermentation were taken to vacuum freeze drying for 3 days. The dried solids weight of each tube was determined, and residual solids were calculated as ratio of final solid weight over the initial solid weight.

Result

Table 5

Table 5 shows that the addition of GH5_21 xylanase together with GH43, GH51 arabinofuranosidase and GH3 beta-xylosidase increase xylose and arabinose release compared to control or treatment consist of GH43, GH51 arabinofuranosidase and GH3 beta-xylosidase, without xylanase. Example 5 - Effect of arabinofuranosidase from GH families 43, and 51 combinations with xylanase from GH family 5 subfamily 21 for increasing arabinose in simultaneous saccharification and fermentation process

An industrial liquefied mash prepared using Liquefaction Enzyme Blend 2 was used for the experiment. The dry solid determined by moisture balance was about 36%DS and pH was adjusted to pH 5.0 following by supplemented with 3 ppm of penicillin and 500 ppm of urea. Simultaneous saccharification and fermentation (SSF) was performed via mini-scale fermentations. Approximately 4.0 g of the industrial liquefied corn mash was added to 15 ml tube vials. Each vial was dosed with 0.42 AGU/gDS of Saccharification Enzyme Blend and appropriate amount of arabinofuranosidase combinations from families GH43 and GH51, as listed in Table 6. The dosing scheme followed the fixed amount of GH5_21O xylanase, GH43 arabinofuranosidase and GH51 arabinofuranosidase of each 10 ug/g dry solids, respectively. As control, only Saccharification Enzyme Blend was used with no addition of xylanase or arabinofuranosidase. Actual enzymes dosages were based on the exact weight of corn slurry in each vial. After enzymes addition, fermentation was initiated by addition of 50 pL of propagated yeast strain MEJI797. Tubes were incubated at 32°C with three replicates for each treatment. After 65 hours SSF, tubes were taken out from incubator and added 50 pL of 34% H2SO4 and then subjected to centrifugation 3500 rpm for 10 min follow by filtering through a 0.45 micrometer filter. Sugars concentrations were determined using HPLC system equipped with H column (Benson Polymeric, BP-700 H, 300 x 7.8 mm).

Result

Table 6

Table 6 shows that GH43 and GH51 arabinofuranosidases in combination with GH5_21 xylanase increase arabinose compared to control without arabinofuranosidases and xylanase. Example 6 - Effect of arabinofuranosidase from GH families 43, and 51 combination with xylanase from GH family 5 subfamily 21 for increasing arabinose in simultaneous saccharification and fermentation process

An industrial liquefied mash prepared using Liquefaction Enzyme Blend 2 was used for the experiment. The dry solid determined by moisture balance was about 33.8%DS and pH was adjusted to pH 5.0 following by supplemented with 3 ppm of penicillin and 500 ppm of urea. Simultaneous saccharification and fermentation (SSF) was performed via mini-scale fermentations. Approximately 4.2 g of the industrial liquefied corn mash was added to 15 ml tube vials. Each vial was dosed with 0.42 AGU/gDS of Saccharification Enzyme Blend and appropriate amount of arabinofuranosidase combination from families GH43 and GH51, as listed in Table 8. The dosing scheme followed the fixed amount of GH5_21 xylanase, GH43 arabinofuranosidase and GH51 arabinofuranosidase of each 10 ug/g dry solids, respectively. As control, only Saccharification Enzyme Blend with no addition of xylanase or arabinofuranosidase. Actual enzymes dosages were based on the exact weight of corn slurry in each vial. After enzymes addition, fermentation was initiated by addition of 50 pL of propagated yeast strain MEJI797. Tubes were incubated at 32°C with three replicates for each treatment. After 65 hours SSF, tubes were taken out from incubator and added 50 pL of 34% H2SO4 and then subjected to centrifugation 3500 rpm for 10 min follow by filtering through a 0.45 micrometer filter. Sugars concentrations were determined using HPLC system equipped with H column (Benson Polymeric, BP-700 H, 300 x 7.8 mm).

Result

Table 7

Table 7 shows that GH43 and GH51 arabinofuranosidases in combination with GH5_21 xylanase increase arabinose release compared to control without arabinofuranosidases and xylanase.

Example 7 - Effect of beta-xylosidase (BX) from different sources combination with hemicellulases blend (Base C5), in the presence or absence of A. fumigatus BX, for increasing xylose in simultaneous saccharification and fermentation process An industrial prepared liquefied mash using liquefaction product of Liquefaction Enzyme Blend 1 was used for the experiment. The dry solid determined by moisture balance was about 34.1%DS and pH was adjusted to pH 5.0 following by supplemented with 3 ppm of penicillin and 1000 ppm of urea. Simultaneous saccharification and fermentation (SSF) was performed via mini-scale fermentations. Approximately 4.2 g of the industrial liquefied corn mash was added to 10 ml tube vials. Each vial was dosed with 0.6 AGU/gDS of Saccharification Enzyme Blend together with hemicellulases blend (Base 05) as shown in Table 8, with or without the addition of GH3A, and appropriate amount of respective beta- xylosidase from various sources (Table 9) followed by addition of 50 pL of hydrated ETHANOL RED yeast per 4.2 g slurry. As control, only glucoamylase with no addition of Base 05 or GH3 enzyme. Actual enzymes dosages were based on the exact weight of corn slurry in each vial. Vials were incubated at 32°C with three replicates for each treatment. After 65 hours SSF, tubes were taken out from incubator and subjected to centrifugation 3500 rpm for 10 min follow by filtering through a 0.45 micrometer filter. Sugars concentrations were determined using HPLC system equipped with H column (Benson Polymeric, BP-700 H, 300 x 7.8 mm).

Table 8: Hemicellulases blend (Base 05)

Result

As shown in results Table 9 below, combination of BX particularly with Po BX, with or without Aspergillus fumigatus BX in Base C5 hemicellulases blend significantly increases xylose release.

Table 9

Claims

CLAIMS What is claimed is:

1. A composition comprising:

(a) a GH43 arabinofuranosidase;

(b) a GH51 arabinofuranosidase; and

(c) a GH5 xylanase.

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

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

4. The composition of any one of claims 1-3, wherein the GH5 xylanase is a GH5_21 xylanase.

5. The composition of any one of claims 1-4, 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 GH43 and GH51 arabinofuranosidases combined when the GH5_21 xylanase is not present in the composition;

(b) the GH43 and GH51 arabinofuranosidases and the GH5_21 xylanase together release at least 2 times more arabinose from liquefied corn mash than the GH43 and GH51 arabinofuranosidases combined when the GH5_21 xylanase is not present in the composition;

(c) the GH43 and GH51 arabinofuranosidases, the GH3 beta-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 GH43 and GH51 arabinofuranosidases and GH3 beta-xylosidase combined when the GH5_21 xylanase is not present in the composition;

(d) the GH43 and GH51 arabinofuranosidases, the GH3 beta-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 GH43 and GH51 arabinofuranosidases and GH3 beta-xylosidase combined when the GH5_21 xylanase is not present in the composition; (e) the GH43 and GH51 arabinofuranosidases, GH3 beta-xylosidase, and the GH5_21 xylanase together release at least 150% more xylose from liquefied corn mash than the GH43 and GH51 arabinofuranosidases combined when the GH3 beta-xylosidase and GH5_21 xylanase are not present in the composition; and/or

(f) the GH43 and GH51 arabinofuranosidases, GH3 beta-xylosidase, and the GH5_21 xylanase together release at least 2.5 times more xylose from liquefied corn mash than the GH43 and GH51 arabinofuranosidases combined when the GH3 beta-xylosidase and GH5_21 xylanase are not present 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 beta-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 GH43 and GH51 arabinofuranosidases and GH3 beta-xylosidase combined when the GH5_35 xylanase is not present in the composition.

8. A composition comprising:

(a) a GH43 arabinofuranosidase;

(b) a GH51 arabinofuranosidase; and

(c) a 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 GH43 and GH51 arabinofuranosidases combined when the GH30_8 xylanase is not present in the composition; and/or

(b) the GH43 and GH51 arabinofuranosidases and the GH30_8 xylanase together release at least 2 times more arabinose from liquefied corn mash than the GH43 and GH51 arabinofuranosidases combined when the GH30_8 xylanase is not present in the composition.

10. A process for producing a fermentation product from a starch-containing material, the process comprising: (a) saccharifying a starch-containing material at a temperature below the initial gelatinization temperature of the starch with an alpha-amylase and a glucoamylase to produce fermentable a sugar; and

(b) fermenting the sugar with a fermenting organism to produce the fermentation product, wherein the composition of any one of claims 1-9 is present or added during step

(a) and/or step (b).

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

12. A process for producing a fermentation product from a starch-containing material, the process comprising:

(b) saccharifying the dextrin with a glucoamylase to produce a fermentable sugar;

(c) fermenting the sugar with a fermenting organism to produce the fermentation product, wherein the composition of any one of claims 1-9 is present or added during step

(b) and/or step (c).

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

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

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

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

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

18. The process of any one of claims 10-15, wherein solubilization of hemicellulosic fiber is increased to release significantly more monomeric arabinose and monomeric xylose compared to a control process:

(i) lacking the composition;

(ii) using a composition with only the GH43 and GH51 arabinofuranosidases alone;

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