WO2025128568A1

WO2025128568A1 - Composition and use thereof for increasing hemicellulosic fiber solubilization

Info

Publication number: WO2025128568A1
Application number: PCT/US2024/059376
Authority: WO
Inventors: James Lavigne; Chee-Leong Soong; Xinglian Geng; Hui Xu
Original assignee: Novozymes AS
Current assignee: Novozymes AS
Priority date: 2023-12-11
Filing date: 2024-12-10
Publication date: 2025-06-19
Anticipated expiration: 2026-06-11

Abstract

The invention also relates to compositions comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 beta-xylosidase, a GH30 xylanase, and optionally a CE3 acetyl xylan esterase, and use of the compositions for solubilizing hemicellulosic fiber and increasing release of monomeric arabinose and/or xylose.

Description

COMPOSITION 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.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to a composition comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 beta-xylosidase, and a GH30 xylanase, and optionally a carbohydrate esterase family 3 (CE3) acetyl xylan esterase and use thereof for solubilizing hemicellulosic fiber.

Description of the Related Art

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, a GH5 xylanase, a GH3 beta-xylosidase, and a GH30 xylanase, which due to the combined presence of the GH5 and GH30 xylanases unexpectedly increases hemicellulosic fiber solubilization and releases significantly more monomeric arabinose and xylose compared to similar compositions with the GH5 xylanase or GH30 xylanase alone. Surprisingly and unexpectedly, the compositions of the present invention significantly increase yields of monomeric arabinose and xylose without requiring acetyl xylan esterases, feruloyl esterases, and/or alphaglucuronidases, though the addition of CE3 acetyl xylan esterases and/or alpha-xylosidases (e.g., GH31 alpha-xylosidases) to the compositions further increases those yields. The enzyme compositions may be formulated in solid forms (e.g., granules comprising the enzymes) or liquid form (e.g., liquid compositions comprising the enzymes and one or more formulating agents).

Accordingly, an aspect of the present invention relates to a granule, which comprises:(a) a core comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 beta-xylosidase, a GH30 xylanase, and optionally a carbohydrate esterase family 3 (CE3) acetyl xylan esterase, and, optionally (b) a coating consisting of one or more layer(s) surrounding the core.

In an aspect, the present invention relates to a granule, which comprises: (a) a core, and (b) a coating consisting of one or more layer(s) surrounding the core, wherein the coating comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 beta-xylosidase, a GH30 xylanase, and optionally a CE3 acetyl xylan esterase.

In an aspect, the present invention relates to a composition comprising the granule.

In an aspect, the present invention relates to a liquid composition comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 beta-xylosidase, a GH30 xylanase, and optionally a Ce3 acetyl xylan esterase, and an enzyme stabilizer, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, or lactic acid.

In an embodiment, the liquid composition further comprises a filler or carrier material. In an embodiment, the liquid composition further comprises a preservative.

In an aspect, the present invention relates to a composition comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, a GH3 beta-xylosidase, a GH30 xylanase and optionally a CE3 acetyl xylan esterase.

An aspect of the present invention relates to a process for producing a fermentation product from a starch-containing material comprising the steps of: (a) saccharifying a starch-containing material with a glucoamylase and an alpha-amylase at a temperature below the initial gelatinization temperature of the starch to produce a fermentable sugar; (b) fermenting the sugar with a fermenting organism; wherein a composition comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 beta-xylosidase, a GH30 xylanase, and optionally a CE3 acetyl xylan esterase is present or added during saccharifying step (a) and/or fermenting step (b).

In an aspect, the present invention relates to a process for producing a fermentation product from a starch-containing material comprising the steps of: (a) liquefying a starch- containing material at a temperature above the initial gelatinization temperature of the starch with a thermostable alpha-amylase 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, a GH5 xylanase, a GH3 beta-xylosidase, a GH30 xylanase, and optionally a CE3 acetyl xylan esterase is present or added during saccharifying step (b) and/or fermenting step (c).

The GH5 xylanase may be a GH5_21 xylanase.

The GH5 xylanase may be a GH5_35 xylanase.

The GH30 xylanase may be a GH30_7 xylanase.

The GH30 xylanase may be a GH30_8 xylanase.

In certain embodiments, the granule core comprises the CE3 acetyl xylan esterase.

In certain embodiments, the granule coating comprises the CE3 acetyl xylan esterase.

In certain embodiments, the composition (e.g., liquid composition) comprises the CE3 acetyl xylan esterase.

In certain embodiments, the granule core comprises an alpha-xylosidase (e.g., a GH31 alpha-xylosidase).

In certain embodiments, the granule coating comprises an alpha-xylosidase (e.g., a GH31 alpha-xylosidase).

In certain embodiments, the composition (e.g., liquid composition) comprises an alpha- xylosidase (e.g., a GH31 alpha-xylosidase).

BRIEF DESCRIPTION OF THE FIGURE

The Figure is an alignment of exemplary CE3 polypeptides of the present invention showing they share the conserved active site serine, histidine and aspartic acid residues that form the catalytic triad that is characteristic of the SGNH hydrolase enzyme family, the conserved canonical GxSxT pentapeptide consensus sequence, and the Block II Gly and Block III Asn residues comprising the oxyanion hole.

DEFINITIONS

In accordance with this detailed description, the following definitions apply. Note that the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Unless defined otherwise or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Acetyl xylan esterase: The term “acetyl xylan esterase” means an acetyl xylan esterase (EC 3.1.1.72) that catalyzes the hydrolysis of acetyl groups from polymeric xylan, acetylated xylose, acetylated glucose, alpha-napthyl acetate, p-nitrophenly acetate but not from triacylglycerol.

Acetyl xylan esterase activity: One Unit of acetyl xylan esterase activity is defined as the amount of enzyme required to release one pmole of p-nitrophenol per minute from 4- nitrophenyl acetate in 100 mM sodium citrate buffer, pH 5 at 40°C. 100mM pNP-actate is dissolved in DMSO as substrates stock solution. The stock solution is diluted 50x in 100 mM sodium citrate to make 2 mM pNP-acetate substrate solution. 175 pl substrate solution and 25 ul diluted enzyme is mixed in 96-well plate and incubated at 37°C. The released p-nitrophenol is monitored at 410 nm by a spectrophotometer.

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 liter 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).

Alpha-xylosidase: “Alpha-xylosidase” means an alpha-D-xyloside xylohydrolase (EC 3.2.1.177) that catalyzes hydrolysis of a terminal, unsubstituted xyloside at the extreme reducing end of a xylogluco-oligosaccharide.

Alpha-xylosidase Activity: For purposes of the present invention, one unit of alpha- 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-alpha-D-xyloside as substrate in 100 mM sodium citrate containing 0.01 percent TWEEN(R) 20 in a total volume of 200 micro liters.

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.

Carbohydrate Esterease Family 3 (CE3): Carbohydrate Esterase Family 3 is abbreviated herein as “CE3”. The CE3 polypeptides of the present invention have acetyl xylan esterase activity (EC 3.1.1.72). The CE3 family has several enzymes that have been structurally resolved, including TcAE206 from Talaromyces cellulolyticus and CtCes3-1 from Hungateiclostridium thermocellum. Both structures have an (alpha/beta/alpha)-sandwich fold characteristic of SGNH hydrolase family enzymes. The (alpha/beta/alpha)-sandwich has five central parallel beta-strands forming a curved beta-sheet, which is flanked by 5-6 alpha-helices. Both structures also posses a calcium binding loop motif (DXVGXyDXn(D/N)) found above the N- terminal end of the central beta-strand. This binding motif is conserved across previously characterized CE3s.

Carohydrate esterase family 3 (CE3) acetyl xylan esterases possess the classical catalytic triad of Ser-His-Asp, which is a characteristic feature of the SGNH hydrolase family of enzymes. The active site residues are established by four conserved consensus sequences (Blocks l-lll and V) and contain an altered nucleophilic “elbow” turn motif (-GxSxT- rather than the canonical -GxSxG- motif). The catalytic triad together with the Block II Gly and Block III Asn residues that comprise the oxyanion hole, are conserved across all characterized CE3 enzymes. The Block V Asp residue facilitates the amphoteric nature of the Block V His residue, which extracts a proton from the Block I Ser to render it nucleophilic.

The location of the above features in each of the exemplary CE3 acetyl xylan esterases is shown in Table 1 below and an alignment showing conservation of the features in the mature sequences of the exemplary CE3 acetyl xylan esterases of the present invention is shown in the Figure.

Table 1

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

Coding sequence: The term “coding sequence” means a polynucleotide, which directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon, such as ATG, GTG, or TTG, and ends with a stop codon, such as TAA, TAG, or TGA. The coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acid sequences involved in regulation of expression of a polynucleotide in a specific organism or in vitro. Each control sequence may be native (/.e., from the same gene) or heterologous (/.e., from a different gene) to the polynucleotide encoding the polypeptide, and native or heterologous to each other. Such control sequences include, but are not limited to leader, polyadenylation, prepropeptide, propeptide, signal peptide, promoter, terminator, enhancer, and transcription or translation initiator and terminator sequences. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a polypeptide.

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

Expression vector: An "expression vector" refers to a linear or circular DNA construct comprising a DNA sequence encoding a polypeptide, which coding sequence is operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, enhancers and sequences which control termination of transcription and translation.

Extension: The term “extension” means an addition of one or more amino acids to the amino and/or carboxyl terminus of a polypeptide, wherein the “extended” polypeptide has acetyl xylan esterase activity.

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.

Fusion polypeptide: The term “fusion polypeptide” is a polypeptide in which one polypeptide is fused at the N-terminus and/or the C-terminus of a polypeptide of the present invention. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention, or by fusing two or more polynucleotides of the present invention together. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator. Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779). A fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 7Q: 245-251 ; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991 , Biotechnology 9: 378-381 ; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter eta/., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.

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: “GH 10 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.

GH31 alpha-xylosidase: “GH31 arabinofuranosidase” is an abbreviation for Glycoside Hydrolase Family 31 alpha-xylosidases, which is an alpha-D-xyloside xylohydrolase (EC 3.2.1.177) that catalyzes hydrolysis of a terminal, unsubstituted xyloside at the extreme reducing end of a xylogluco-oligosaccharide. Exemplary alpha-xylosidases from the GH31 family utilize a two-step, double-displacement mechanism employing a covalent glycosyl- enzyme intermediate, and produce a product with an anomeric configuration.

GH30 xylanase: “GH30 xylanase” is an abbreviation for Glycoside Hydrolase 30 family xylanases. The GH30 family is defined in the CAZy database (Henrissat, 1991), and has been reviewed in the literature (Puchart et al., 2021). In the present disclosure, “GH30 xylanase” is intended to only include GH30_1 xylanases, GH30_2 xylanases, GH30_3 xylanases, GH30_4 xylanases, GH30_5 xylanases, GH30_7 xylanases, and GH30_8 xylanases. For the avoidance of doubt, GH30_6 xylanases are excluded from the definition of GH30 xylanase in accordance with the disclosure.

GH30_1 xylanase: “GH30_1 xylanase” is an abbreviation for Glycoside Hydrolase 30 subfamily 1 xylanases, which in the context of the present disclosure only includes glucosylceramidases (EC 3.2.145) that catalyze the reaction of a D-glucosyl-N-acylsphingosine + H2O = D-glucose + a ceramide and xylan 1,4-p-xylosidases (EC 3.2.1.37) that catalyze the hydrolysis of (1- 4)- P -D-xylans, to remove successive D-xylose residues from the nonreducing termini. For the avoidance of doubt, "GH30_1 xylanase” does not include glucosidases (EC 3.2.1.-) and (EC3.2.1.21) in the context of the present disclosure.

GH30_2 xylanase: “GH30_2 xylanase” is an abbreviation for Glycoside Hydrolase 30 subfamily 2 xylanases, which includes xylan 1,4-p-xylosidases (EC 3.2.1.37) that catalyze the hydrolysis of (1^4)-p-D-xylans, to remove successive D-xylose residues from the non-reducing termini. GH30_3 xylanase: “GH30_3 xylanase” is an abbreviation for Glycoside Hydrolase 30 subfamily 3 xylanases, which refers to glucan endo-1 ,6-p-glucosidases (EC 3.2.1.75) that catalyze the random hydrolysis of (1 ^6)-linkages in (1-*6)- P -D-glucans. For the avoidance of doubt, “GH30_3 xylanase” does not include glucosidases (EC 3.2.1.-) in the context of the present disclosure.

GH30_4 xylanase: “GH30_4 xylanase” is an abbreviation for Glycoside Hydrolase 30 subfamily 4 xylanases, which refers to p-D-fucosidases (EC 3.2.1.38) that catalyze the hydrolysis of terminal non-reducing p-D-fucose residues in p-D-fucosides.

GH30_5 xylanase” “GH30_5 xylanase” is an abbreviation for Glycoside Hydrolase 30 subfamily 5 xylanases, which includes endo- p-1 ,6-galactanase (EC 3.2.1.164) that catalyze the endohydrolysis of (1-*6)- P -D-galactosidic linkages in arabinogalactan proteins and (1 — 3):(1 — 6)- P -galactans to yield galactose and (1-*6)- P -galactobiose as the final products and alactan exo-1 ,6-p-galactobiohydrolase (EC 3.2.1.213) that catalyze the hydrolysis of (1 ^6)- P -D- galactosidic linkages in arabinogalactan proteins and (1 -*3):(1 —6)- P -galactans to yield (1-*6)- P -galactobiose as the final product.

GH30_7 xylanase: “GH30_7 xylanase” is an abbreviation for Glycoside Hydrolase 30 subfamily 7 xylanases, which includes endo-p-1 ,4-xylanases (EC 3.2.1.8) that catalzye the endohydrolysis of (1^4)-p-D-xylosidic linkages in xylans and oligosasccharide reducing-end xylanases (EC 3.2.1.156) that catalyze the hydrolysis of (1^4)-p-D-xylose residues from the reducing end of oligosaccharides.

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.

GH30_9 xylanase: “GH30_9 xylanase” is an abbreviation for Glycoside Hydrolase 30 subfamily 9 xylanases, which refers to baicalin-p-D-glucuronidases (EC 3.2.1.167) that catalyze the reaction of baicalin + H2O = baicalein + D-glucuronate.

GH30_10 xylanase: “GH30_10 xylanase” is an abbreviation for Glycoside Hydrolase 30 subfamily 10 xylanases, which refers to non-reducing end-specific xylobiohydrolases (EC 3.2.1.-) that catalyzes the hydrolysis of xylan or xylo-oligosaccharides to produce xylobiose.

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 grain 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).

Heterologous: The term "heterologous" means, with respect to a host cell, that a polypeptide or nucleic acid does not naturally occur in the host cell. The term "heterologous" means, with respect to a polypeptide or nucleic acid, that a control sequence, e.g., promoter, of a polypeptide or nucleic acid is not naturally associated with the polypeptide or nucleic acid, i.e., the control sequence is from a gene other than the gene encoding the mature polypeptide.

Host Strain or Host Cell: A "host strain" or "host cell" is an organism into which an expression vector, phage, virus, or other DNA construct, including a polynucleotide encoding a polypeptide of interest (e.g., an amylase) has been introduced. Exemplary host strains are microorganism cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing the polypeptide of interest and/or fermenting saccharides. The term "host cell" includes protoplasts created from cells.

Introduced: The term "introduced" in the context of inserting a nucleic acid sequence into a cell, means "transfection", "transformation" or "transduction," as known in the art.

Isolated: The term “isolated” means a polypeptide, nucleic acid, cell, or other specified material or component that has been separated from at least one other material or component, including but not limited to, other proteins, nucleic acids, cells, etc. An isolated polypeptide, nucleic acid, cell or other material is thus in a form that does not occur in nature. An isolated polypeptide includes, but is not limited to, a culture broth containing the secreted polypeptide expressed in a host cell.

Mature polypeptide: The term “mature polypeptide” means a polypeptide in its mature form following N-terminal and/or C-terminal processing (e.g., removal of signal peptide).

Native: The term "native" means a nucleic acid or polypeptide naturally occurring in a host cell.

Nucleic acid: The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids may be single stranded or double stranded, and may be chemical modifications. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in 5'-to-3' orientation.

Purified: The term “purified” means a nucleic acid, polypeptide or cell that is substantially free from other components as determined by analytical techniques well known in the art (e.g., a purified polypeptide or nucleic acid may form a discrete band in an electrophoretic gel, chromatographic eluate, and/or a media subjected to density gradient centrifugation). A purified nucleic acid or polypeptide is at least about 50% pure, usually at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8% or more pure (e.g., percent by weight or on a molar basis). In a related sense, a composition is enriched for a molecule when there is a substantial increase in the concentration of the molecule after application of a purification or enrichment technique. The term "enriched" refers to a compound, polypeptide, cell, nucleic acid, amino acid, or other specified material or component that is present in a composition at a relative or absolute concentration that is higher than a starting composition.

In one aspect, the term "purified" as used herein refers to the polypeptide or cell being essentially free from components (especially insoluble components) from the production organism. In other aspects, the term "purified" refers to the polypeptide being essentially free of insoluble components (especially insoluble components) from the native organism from which it is obtained. In one aspect, the polypeptide is separated from some of the soluble components of the organism and culture medium from which it is recovered. The polypeptide may be purified (/.e., separated) by one or more of the unit operations filtration, precipitation, or chromatography.

Accordingly, the polypeptide may be purified such that only minor amounts of other proteins, in particular, other polypeptides, are present. The term "purified" as used herein may refer to removal of other components, particularly other proteins and most particularly other enzymes present in the cell of origin of the polypeptide. The polypeptide may be "substantially pure", i.e., free from other components from the organism in which it is produced, e.g., a host organism for recombinantly produced polypeptide. In one aspect, the polypeptide is at least 40% pure by weight of the total polypeptide material present in the preparation. In one aspect, the polypeptide is at least 50%, 60%, 70%, 80% or 90% pure by weight of the total polypeptide material present in the preparation. As used herein, a "substantially pure polypeptide" may denote a polypeptide preparation that contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0.5% by weight of other polypeptide material with which the polypeptide is natively or recombinantly associated.

It is, therefore, preferred that the substantially pure polypeptide is at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99% pure, most preferably at least 99.5% pure by weight of the total polypeptide material present in the preparation. The polypeptide of the present invention is preferably in a substantially pure form (/.e., the preparation is essentially free of other polypeptide material with which it is natively or recombinantly associated). This can be accomplished, for example by preparing the polypeptide by well-known recombinant methods or by classical purification methods.

Recombinant: The term "recombinant" is used in its conventional meaning to refer to the manipulation, e.g., cutting and rejoining, of nucleic acid sequences to form constellations different from those found in nature. The term recombinant refers to a cell, nucleic acid, polypeptide or vector that has been modified from its native state. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell, or express native genes at different levels or under different conditions than found in nature. The term “recombinant” is synonymous with “genetically modified” and “transgenic”.

Recover: The terms "recover" or “recovery” means the removal of a polypeptide from at least one fermentation broth component selected from the list of a cell, a nucleic acid, or other specified material, e.g., recovery of the polypeptide from the whole fermentation broth, or from the cell-free fermentation broth, by polypeptide crystal harvest, by filtration, e.g. depth filtration (by use of filter aids or packed filter medias, cloth filtration in chamber filters, rotary-drum filtration, drum filtration, rotary vacuum-drum filters, candle filters, horizontal leaf filters or similar, using sheed or pad filtration in framed or modular setups) or membrane filtration (using sheet filtration, module filtration, candle filtration, microfiltration, ultrafiltration in either cross flow, dynamic cross flow or dead end operation), or by centrifugation (using decanter centrifuges, disc stack centrifuges, hyrdo cyclones or similar), or by precipitating the polypeptide and using relevant solid-liquid separation methods to harvest the polypeptide from the broth media by use of classification separation by particle sizes. Recovery encompasses isolation and/or purification of the polypeptide.

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 - Total 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)

Signal Peptide: A "signal peptide" is a sequence of amino acids attached to the N- terminal portion of a protein, which facilitates the secretion of the protein outside the cell. The mature form of an extracellular protein lacks the signal peptide, which is cleaved off during the secretion process.

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 any one of the following definitions of xylanases: endo-1 ,4- p-xylanases (EC 3.2.1.8) that catalyze the endohydrolysis of (1— >4)-p-D-xylosidic linkages in xylans; 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; glucosylceramidases (EC 3.2.145) that catalyze the reaction of a D-glucosyl-N-acylsphingosine + H2O = D-glucose + a ceramide; xylan 1 ,4-p-xylosidases (EC 3.2.1.37) that catalyze the hydrolysis of (1- 4)- P -D-xylans, to remove successive D-xylose residues from the nonreducing termini; glucan endo-1 ,6-p-glucosidases (EC 3.2.1.75) that catalyze the random hydrolysis of (1 ^6)-linkages in (1 ^6)- P -D-glucans; p-D-fucosidases (EC 3.2.1.38) that catalyze the hydrolysis of terminal non-reducing p-D-fucose residues in p-D-fucosides; endo- p- 1 ,6-galactanase (EC 3.2.1.164) that catalyze the endohydrolysis of (1 ^6)- P -D-galactosidic linkages in arabinogalactan proteins and (1 -*3):(1 —6)- P -galactans to yield galactose and (1 — 6)- 13 -galactobiose as the final products; galactan exo-1 ,6-p-galactobiohydrolase (EC 3.2.1.213) that catalyze the hydrolysis of (1-*6)- P -D-galactosidic linkages in arabinogalactan proteins and (1 -*3):(1 —6)- P -galactans to yield (1 —6)- P -galactobiose as the final product; oligosasccharide reducing-end xylanases (EC 3.2.1.156) that catalyze the hydrolysis of (1— >4)-p-D-xylose residues from the reducing end of oligosaccharides; baicalin-p-D-glucuronidases (EC 3.2.1.167) that catalyze the reaction of baicalin + H2O = baicalein + D-glucuronate; and nonreducing end-specific xylobiohydrolases (EC 3.2.1.-) that catalyzes the hydrolysis of xylan or xylo-oligosaccharides to produce xylobiose;

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. Activity of EC 3.2.1 .8 and EC 3.2.1 .156 xylanases can be measured in a reaction mixture containing purified xylanase and 10 mg ml-1 beechwood xylan (MEGAZYME, Wicklow, Ireland) in 50 mm sodium acetate (pH 4.0) at 40 °C for 15 min. The reducing sugars from depolymerization of the substrate are measured using the DNS method (Miller G. L. (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31 , 426-428). One unit of enzyme activity is defined as the amount of enzyme that catalyzes the formation of 1 pmol of reducing sugar/min.

DETAILED DESCRIPTION OF THE INVENTION

Compositions

The present invention relates to compositions comprising a GH43 arabinofuranosidase, a GH51 arabinforuanosidase, a GH5 xylanase, a GH3 beta-xylosidase, and a GH30 xylanase, and optionally a CE3 acetyl xylan esterase and/or an alpha-xylosidase and use thereof for solubilizing hemicellulosic fiber.

The work described in co-pending PCT International Application No. PCT/US2023/083345 (incorporated herein by reference in its entirety) demonstrated that a composition a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, and either a GH5 xylanase or a GH30_8 xylanase unexpectedly increased hemicellulosic fiber solubilization and released 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.

Similarly, the work described in co-pending PCT International Application No. PCT/US2023/083351 (incorporated herein by reference in its entirety) demonstrated that CE3 acetyl xylan esterases released more monomeric arabinose and/or xylose when used in combination with GH43 and GH51 arabinofuranosidases, GH5 xylanases, and GH3 beta- xylosidases.

Surprisingly and unexpectedly, the work described herein demonstrates that compositions comprising GH43 and G51 arabinofuranosidases, GH5 xylanases, GH3 beta- xylosidases and GH30 xylanases increases hemicellulosic fiber solubilization and releases significantly more monomeric arabinose and xylose compared to a similar composition without GH30 xylanase alone. That is the work described herein demonstrates an unexpected synergistic effect of including both the GH5 and GH30 xylanases in the composition with the GH43 and GH51 arabinofuranosidases and the GH3 beta-xylosidase. Surprisingly, the compositions of the present invention significantly increase yields of monomeric arabinose and xylose without requiring acetyl xylan esterases, feruloyl esterases, and/or alphaglucuronidases, though the addition of CE3 acetyl xylan esterases and/or alpha-xylosidases (e.g., GH31 alpha-xylosidases) to the compositions further increases those yields.

For instance, work described herein demonstrates that compositions comprising a GH43 arabinofuranosiase, a GH51 arabinofuranosidase, a GH5_21 xylanase, a GH3 beta- xylosidase and GH30 xylanase release from at least 40% to at least 60% more xylose compared to an otherwise identical composition that lacks the GH30 xylanase. Similarly, work described herein demonstrates that compositions comprising a GH43 arabinofuranosiase, a GH51 arabinofuranosidase, a GH5_21 xylanase, a GH3 beta-xylosidase, GH30 xylanase and a CE3 acetyl xylan esterase releases at least 130% to at least 140% more xylose compared to an otherwise identical composition that lacks the CE3 acetyl xylan esterase. The work further demonstrates that the composition comprising the GH43 arabinofuranosidase, GH51 arabinofuranosidase, GH5 xylanase, GH3 beta-xylosidase, GH30 xylanase and CE3 acetyl xylan esterase also releases at least about 10% to about 15% more arabinose compared to an otherwise identical composition that lacks the CE3 acetyl xylan esterase.

The enzyme compositions may be formulated in solid forms (e.g., granules comprising the enzymes) or liquid form (e.g., liquid compositions comprising the enzymes and one or more formulating agents).

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. Exemplary GH43 arabinofuranosidases

Aspects of the invention relate to compositions comprising a GH43 arabinofuranosidase 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 arabinofuranosidase, a GH5 xylanase, a GH3 beta-xylosidase, a GH30 xylanase and optionally a CE3 acetyl xylan esterase and/or GH31 alpha-xylosidase, increases production of monomeric arabinose and/or xylose compared to similar compositions without the GH43 arabinofuranosidase.

In an embodiment, the GH43 arabinofuranosidase is a GH43_36 arabinofuranosidase.

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

Exemplary GH43 arabinofuranosidases may be from the species Humicola insolens, Lasiodiplodia theobromae, or Poronia punctata.

An exemplary GH43 arabinofuranosidase has 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 has 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 has 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 any GH51 arabinofuranosidase that, when used in combination with a GH43 arabinofuranosidase, a GH5 xylanase, a GH3 beta-xylosidase, a GH30 xylanase and optionally a CE3 acetyl xylan esterase and/or a GH31 alpha-xylosidase, increases production of monomeric arabinose and/or xylose compared to similar compositions without the GH51 arabinofuranosidase.

In an embodiment, the GH51 arabinofuranosidase is a GH51_6 arabinofuranosidase. Exemplary GH51 arabinofuranosidases may be from the genus Meripulus, Lasiodiplodia, or Acidiella.

Exemplary GH51 arabinofuranosidases may be from the species Meripulus giganteus, Lasiodiplodia theobromae, or Ac/diella 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 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, which 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 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. C. Exemplary GH5 xylanases

Aspects of the invention relate to compositions comprising a GH5 xylanase in combination with other enzymes to increase hemicellulosic fiber solubilization and production of monomeric arabinose and/or xylose. The present invention contemplates any GH5 xylanase that, when used in combination with a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH3 beta-xylosidase, a GH30 xylanase, and optionally a CE3 acetyl xylan esterase and/or a GH31 alpha-xylosidase, increases production of monomeric arabinose and/or xylose compared to similar compositions without the GH5 xylanase.

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

Exemplary GH_21 xylanases may be from the genus Bacteroides, Belliella, Chryseobacterium, or Sphingobacterium.

Exemplary GH_21 xylanases may be from the species Bacteroides cellulosilyticus CL02Y12C19, Belliella sp-64282, Chryseobacterium sp., Chryseobacterium oncorhynchi, or Sphingobacterium sp-64162.

Exemplary GH5_21 xylanases may be 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 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: 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 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: 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 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 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 and 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 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 may be from the genus Bacillus, Cohnella, or Paenibacillus.

Exemplary GH5_35 xylanase may be from the species Bacillus hemiccellulosilyticus JCM 9152, Cohnella xylanilytica, Paenibacillus chitinolyticus, or Paenibacillus sp-62332.

Exemplary GH5_35 xylanases may be 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 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: 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 GH5_35 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 23, and has xylanase activity. An exemplary GH5_35 xylanase has the amino acid sequence of SEQ ID NO: 24. In 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_35 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 to the amino acid sequence of SEQ ID NO: 26, and has xylanase activity.

The GH5 xylanases 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 GH3 beta-xylosidases

Aspects of the invention relate to compositions comprising GH3 beta-xylosidases in combination with other enzymes to increase hemicellulosic fiber solubilization and production of monomeric arabinose and/or xylose. The present invention contemplates any GH3 beta- xylosidase that, when used in combination with a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH30 xylanase, and optionally a a CE3 acetyl xylan esterase and/or a GH31 alpha-xylosidase, increases production of monomeric arabinose and/or xylose compared to similar compositions without the 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 fimetaria, Talaromyces emersonii, Talaromyces stipitatus, or Trichoderma reesei.

An exemplary GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 27. In an embodiment, the GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 27 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: 27, and has beta-xylosidase activity. 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 and has beta-xylosidase activity. An exemplary GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 31. In an embodiment, the GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 31 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: 31 and has beta-xylosidase activity. An exemplary GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 32. In an embodiment, the GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 32 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: 32 and has beta-xylosidase activity. An exemplary GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 33. In an embodiment, the GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 33 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: 33 and has beta-xylosidase activity. An exemplary GH3 beta- xylosidase has the amino acid sequence of SEQ ID NO: 34. In an embodiment, the GH3 beta- xylosidase has the amino acid sequence of SEQ ID NO: 34 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: 34 and has beta-xylosidase activity. An exemplary GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 35. In an embodiment, the GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 35 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: 35 and has beta-xylosidase activity. An exemplary GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 36. In an embodiment, the GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 36 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: 36 and has beta-xylosidase activity. An exemplary GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 37. In an embodiment, the GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 377 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: 37 and has beta-xylosidase activity. An exemplary GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 38. In an embodiment, the GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 38 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: 38 and has beta-xylosidase activity. An exemplary GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 39. In an embodiment, the GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 39 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: 39 and has beta-xylosidase activity. An exemplary GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 40. In an embodiment, the GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 40 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: 40 and has beta-xylosidase activity. An exemplary GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 41. In an embodiment, the GH3 beta-xylosidase has the amino acid sequence of SEQ ID NO: 41 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: 41 and has beta-xylosidase activity.

The GH3 beta-xylosidase 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 GH30 xylanases

Aspects of the invention relate to compositions comprising GH30 xylanases in combination with other enzymes to increase hemicellulosic fiber solubilization and production of monomeric arabinose and/or xylose. The present invention contemplates any GH30 xylanase that, when used in combination with a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 beta-xylosidase, and optionally a CE3 acetyl xylan esterase and/or a GH31 alpha-xylosidase, increases production of monomeric arabinose and/or xylose compared to similar compositions without the GH30 xylanase.

In an embodiment, the GH30 xylanase is a GH30_1 xylanase.

In an embodiment, the GH30 xylanase is a GH30_2 xylanase.

In an embodiment, the GH30 xylanase is a GH30_3 xylanase.

In an embodiment, the GH30 xylanase is a GH30_4 xylanase.

In an embodiment, the GH30 xylanase is a GH30_5 xylanase.

In an embodiment, the GH30 xylanase is a GH30_7 xylanase.

Exemplary GH30_7 xylanases may be from the genera Aspergillus, Evansstolkia, Talaromyces and Trichoderma.

Exemplary GH30_7 xylanases may be from the species Aspergillus fischeri, Aspergillus fumigatiaffinis, Aspergillus novofumigatus, Aspergillus pseudoterreus, Aspergillus terreus, Aspergillus turcosus, Aspergillus udagawae, Evansstolkia leycettana, Talaromyces verruculosus, and Trichoderma reesei.

An exemplary GH30_7 xylanase has the amino acid sequence of SEQ ID NO: 42. In an embodiment, the GH30_7 xylanase has the amino acid sequence of SEQ ID NO: 42 with from 0 to 10 conservative amino acid substitutions and has xylanase activity. In an embodiment, the GH30_7 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: 42, and has xylanase activity. An exemplary GH30_7 xylanase has the amino acid sequence of SEQ ID NO: 43. In an embodiment, the GH30_7 xylanase has the amino acid sequence of SEQ ID NO: 43 with from 0 to 10 conservative amino acid substitutions and has xylanase activity. In an embodiment, the GH30_7 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: 43, and has xylanase activity. An exemplary GH30_7 xylanase has the amino acid sequence of SEQ ID NO: 44. In an embodiment, the GH30_7 xylanase has the amino acid sequence of SEQ ID NO: 44 with from 0 to 10 conservative amino acid substitutions and has xylanase activity. In an embodiment, the GH30_7 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: 44, and has xylanase activity. An exemplary GH30_7 xylanase has the amino acid sequence of SEQ ID NO: 45. In an embodiment, the GH30_7 xylanase has the amino acid sequence of SEQ ID NO: 45 with from 0 to 10 conservative amino acid substitutions and has xylanase activity. In an embodiment, the GH30_7 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: 45, and has xylanase activity. An exemplary GH30_7 xylanase has the amino acid sequence of SEQ ID NO: 46. In an embodiment, the GH30_7 xylanase has the amino acid sequence of SEQ ID NO: 46 with from 0 to 10 conservative amino acid substitutions and has xylanase activity. In an embodiment, the GH30_7 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: 46, and has xylanase activity. An exemplary GH30_7 xylanase has the amino acid sequence of SEQ ID NO: 47. In an embodiment, the GH30_7 xylanase has the amino acid sequence of SEQ ID NO: 47 with from 0 to 10 conservative amino acid substitutions and has xylanase activity. In an embodiment, the GH30_7 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: 47, and has xylanase activity. An exemplary GH30_7 xylanase has the amino acid sequence of SEQ ID NO: 48. In an embodiment, the GH30_7 xylanase has the amino acid sequence of SEQ ID NO: 48 with from 0 to 10 conservative amino acid substitutions and has xylanase activity. In an embodiment, the GH30_7 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: 48, and has xylanase activity. An exemplary GH30_7 xylanase has the amino acid sequence of SEQ ID NO: 49. In an embodiment, the GH30_7 xylanase has the amino acid sequence of SEQ ID NO: 49 with from 0 to 10 conservative amino acid substitutions and has xylanase activity. In an embodiment, the GH30_7 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: 49, and has xylanase activity. An exemplary GH30_7 xylanase has the amino acid sequence of SEQ ID NO: 50. In an embodiment, the GH30_7 xylanase has the amino acid sequence of SEQ ID NO: 50 with from 0 to 10 conservative amino acid substitutions and has xylanase activity. In an embodiment, the GH30_7 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: 50, and has xylanase activity. An exemplary GH30_7 xylanase has the amino acid sequence of SEQ ID NO: 51. In an embodiment, the GH30_7 xylanase has the amino acid sequence of SEQ ID NO: 51 with from 0 to 10 conservative amino acid substitutions and has xylanase activity. In an embodiment, the GH30_7 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: 51, and has xylanase activity. An exemplary GH30_7 xylanase has the amino acid sequence of SEQ ID NO: 52. In an embodiment, the GH30_7 xylanase has the amino acid sequence of SEQ ID NO: 52 with from 0 to 10 conservative amino acid substitutions and has xylanase activity. In an embodiment, the GH30_7 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: 52, and has xylanase activity.

In an embodiment, the GH30 xylanase is a GH30_8 xylanase.

Exemplary GH30_8 xylanases may be from the genus Bacillus, Clostridium, Paenibacillus, and Vibrio.

Exemplary GH30_8 xylanases may be from the species Bacillus sp-18423, Clostridium acetobutylicum, Clostridium saccharobutylicum, Paenibacillus pabuli, and Vibrio rhizosphaerae.

An exemplary GH30_8 xylanase has the amino acid sequence of SEQ ID NO: 53. In an embodiment, the GH30_8 xylanase has the amino acid sequence of SEQ ID NO: 53 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: 53, and has xylanase activity. An exemplary GH30_8 xylanase has the amino acid sequence of SEQ ID NO: 54. In an embodiment, the GH30_8 xylanase has the amino acid sequence of SEQ ID NO: 54 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: 54, and has xylanase activity. An exemplary GH30_8 xylanase has the amino acid sequence of SEQ ID NO: 55. In an embodiment, the GH30_8 xylanase has the amino acid sequence of SEQ ID NO: 55 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: 55, and has xylanase activity. An exemplary GH30_8 xylanase has the amino acid sequence of SEQ ID NO: 56. In an embodiment, the GH30_8 xylanase has the amino acid sequence of SEQ ID NO: 56 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: 56, and has xylanase activity. An exemplary GH30_8 xylanase has the amino acid sequence of SEQ ID NO: 57. In an embodiment, the GH30_8 xylanase has the amino acid sequence of SEQ ID NO: 57 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: 57, and has xylanase activity.

In an embodiment, the GH30 xylanase is a GH30_9 xylanase.

In an embodiment, the GH30 xylanase is a GH30_10 xylanase.

The GH30 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.

F. Exemplary CE3 acetyl xylan esterases

Aspects of the invention relate to compositions comprising a CE3 acetyl xylan esterase in combination with other enzymes to increase hemicellulosic fiber solubilization and production of monomeric arabinose and/or xylose. The present invention contemplates any CE3 acetyl xylan esterase that, when used in combination with a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 beta-xylosidase, a GH30 xylanase and optionally a GH31 alpha-xylosidase, increases production of monomeric arabinose and/or xylose compared to similar compositions without the CE3 acetyl xylan esterase.

An exemplary CE3 acetyl xylan esterase has the amino acid sequence of SEQ ID NO: 3. In an embodiment, the CE3 polypeptide has the amino acid sequence of SEQ ID NO: 58 with from 0 to 10 conservative amino acid substitutions and has acetyl xylan esterase activity. In an embodiment, the CE3 polypeptide has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 58, and has acetyl xylan esterase activity. An exemplary CE3 acetyl xylan esterase has the amino acid sequence of SEQ ID NO: 59. In an embodiment, the CE3 polypeptide has the amino acid sequence of SEQ ID NO: 59 with from 0 to 10 conservative amino acid substitutions and has acetyl xylan esterase activity. In an embodiment, the CE3 polypeptide has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 59, and has acetyl xylan esterase activity. An exemplary CE3 acetyl xylan esterase has the amino acid sequence of SEQ ID NO: 60. In an embodiment, the CE3 polypeptide has the amino acid sequence of SEQ ID NO: 60 with from 0 to 10 conservative amino acid substitutions and has acetyl xylan esterase activity. In an embodiment, the CE3 polypeptide has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 60, and has acetyl xylan esterase activity. An exemplary CE3 acetyl xylan esterase has the amino acid sequence of SEQ ID NO: 61. In an embodiment, the CE3 polypeptide has the amino acid sequence of SEQ ID NO: 61 with from 0 to 10 conservative amino acid substitutions and has acetyl xylan esterase activity. In an embodiment, the CE3 polypeptide has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 61, and has acetyl xylan esterase activity. An exemplary CE3 acetyl xylan esterase has the amino acid sequence of SEQ ID NO: 62. In an embodiment, the CE3 polypeptide has the amino acid sequence of SEQ ID NO: 62 with from 0 to 10 conservative amino acid substitutions and has acetyl xylan esterase activity. In an embodiment, the CE3 polypeptide has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 62, and has acetyl xylan esterase activity. An exemplary CE3 acetyl xylan esterase has the amino acid sequence of SEQ ID NO: 63. In an embodiment, the CE3 polypeptide has the amino acid sequence of SEQ ID NO: 63 with from 0 to 10 conservative amino acid substitutions and has acetyl xylan esterase activity. In an embodiment, the CE3 polypeptide has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 63, and has acetyl xylan esterase activity. An exemplary CE3 acetyl xylan esterase has the amino acid sequence of SEQ ID NO: 64. In an embodiment, the CE3 polypeptide has the amino acid sequence of SEQ ID NO: 64 with from 0 to 10 conservative amino acid substitutions and has acetyl xylan esterase activity. In an embodiment, the CE3 polypeptide has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 64, and has acetyl xylan esterase activity. An exemplary CE3 acetyl xylan esterase has the amino acid sequence of SEQ ID NO: 65. In an embodiment, the CE3 polypeptide has the amino acid sequence of SEQ ID NO: 65 with from 0 to 10 conservative amino acid substitutions and has acetyl xylan esterase activity. In an embodiment, the CE3 polypeptide has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 65, and has acetyl xylan esterase activity. An exemplary CE3 acetyl xylan esterase has the amino acid sequence of SEQ ID NO: 66. In an embodiment, the CE3 polypeptide has the amino acid sequence of SEQ ID NO: 66 with from 0 to 10 conservative amino acid substitutions and has acetyl xylan esterase activity. In an embodiment, the CE3 polypeptide has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 66, and has acetyl xylan esterase activity. An exemplary CE3 acetyl xylan esterase has the amino acid sequence of SEQ ID NO: 67. In an embodiment, the CE3 polypeptide has the amino acid sequence of SEQ ID NO: 67 with from 0 to 10 conservative amino acid substitutions and has acetyl xylan esterase activity. In an embodiment, the CE3 polypeptide has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 67, and has acetyl xylan esterase activity.

The CE3 acetyl xylan esterase 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.

G. Exemplary GH31 alpha-xylosidases

Aspects of the invention relate to compositions comprising GH31 alpha-xylosidases in combination with other enzymes to increase hemicellulosic fiber solubilization and production of monomeric arabinose and/or xylose. The present invention contemplates any GH31 alpha- xylosidase that, when used in combination with a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 beta-xylosidase, and a GH30 xylanase, and optionally a CE3 acetyl xylan esterase, increases production of monomeric arabinose and/or xylose compared to similar compositions without the GH31 alpha-xylosidase.

Exemplary GH31 alpha-xylosidases may be from the genus Herbinix. Exemplary GH31 alpha-xylosidases may be from the species Herbinix hemicellulosilytica.

An exemplary GH31 alpha-xylosidase has the amino acid sequence of SEQ ID NO: 68. In an embodiment, the GH31 alpha-xylosidase has the amino acid sequence of SEQ ID NO: 68 with from 0 to 10 conservative amino acid substitutions and has beta-xylosidase activity. In an embodiment, the GH31 alpha-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: 68, and has alpha-xylosidase activity.

The GH31 alpha-xylosidase 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.

H. 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 arabinose, 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 alphaamylase, 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.

Granules

The present invention also relates to enzyme granules/particles comprising at least one, at least two, at least three, at least four, at least five, at least six, or at least seven enzymes of the invention. In an embodiment, the granule comprises a core, and optionally one or more coatings (outer layers) surrounding the core. The present invention contemplates using any of the exemplary GH43 arabinofuranosidases, GH51 arabinofuranosidases, exemplary GH5 xylanses, exemplary GH3 beta-xylosidases, exemplary GH30 xylanases, exemplary CE3 acetyl xylan esterases, and exemplary GH31 alpha-xylosidases in the enzyme granules/particles described herein.

The core may have a diameter, measured as equivalent spherical diameter (volume based average particle size), of 20-2000 pm, particularly 50-1500 pm, 100-1500 pm or 250-1200 pm. The core diameter, measured as equivalent spherical diameter, can be determined using laser diffraction, such as using a Malvern Mastersizer and/or the method described under I S013320 (2020). In an embodiment, the core comprises a GH43 arabinofuranosidase of the present invention. In an embodiment, the core comprises a GH51 arabinofuranosidase of the present invention. In an embodiment, the core comprises a GH5 xylanase of the present invention. In an embodiment, the core comprises a GH3 beta-xylosidase of the present invention. In an embodiment, the core comprises a GH30 xylanase of the present invention. In an embodiment, the core comprises a acetyl xylan esterase of the present invention. In an embodiment, the core comprises a GH31 alpha-xylosidase of the present invention.

In an embodiment, the core comprises at least one, at least two, at least three, at least four, at least five, at least six, or at least seven enzymes selected from the group consisting of a GH43 arabinofuranosidase of the present invention, a GH51 arabinofuranosidase of the present invention, a GH5 xylanase of the present invention, a GH3 beta-xylosidase of the present invention, a GH30 xylanase of the present invention, a CE3 acetyl xylan esterase of the present invention, and a GH31 alpha-xylosidase of the present invention.

In an embodiment, the core comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 beta-xylosidase, and a GH30 xylanase. In an embodiment, the core comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, a GH3 beta-xylosidase, and a GH30_7 xylanase. In an embodiment, the core comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, a GH3 beta-xylosidase, and a GH30_8 xylanase. In an embodiment, the core comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_35 xylanase, a GH3 beta-xylosidase, and a GH30_7 xylanase. In an embodiment, the core comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_35 xylanase, a GH3 beta-xylosidase, and a GH30_8 xylanase.

In an embodiment, the core comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 beta-xylosidase, a GH30 xylanase, and a CE3 acetyl xylan esterase. In an embodiment, the core comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, a GH3 beta-xylosidase, a GH30_7 xylanase, and a CE3 acetyl xylan esterase. In an embodiment, the core comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, a GH3 beta-xylosidase, a GH30_8 xylanase, and a CE3 acetyl xylan esterase. In an embodiment, the core comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_35 xylanase, a GH3 beta- xylosidase, a GH30_7 xylanase, and a CE3 acetyl xylan esterase. In an embodiment, the core comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_35 xylanase, a GH3 beta-xylosidase, a GH30_8 xylanase, and a CE3 acetyl xylan esterase.

In an embodiment, the core comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 beta-xylosidase, a GH30 xylanase, a CE3 acetyl xylan esterase, and a GH31 alpha-xylosidase. In an embodiment, the core comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, a GH3 beta-xylosidase, a GH30_7 xylanase, a CE3 acetyl xylan esterase, and a GH31 alpha-xylosidase. In an embodiment, the core comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, a GH3 beta-xylosidase, a GH30_8 xylanase, a CE3 acetyl xylan esterase, and a GH31 alpha-xylosidase. In an embodiment, the core comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_35 xylanase, a GH3 beta-xylosidase, a GH30_7 xylanase, a CE3 acetyl xylan esterase, and a GH31 alpha-xylosidase. In an embodiment, the core comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_35 xylanase, a GH3 beta-xylosidase, a GH30_8 xylanase, a CE3 acetyl xylan esterase, and a GH31 alpha-xylosidase.

The core may include additional materials such as fillers, fiber materials (cellulose or synthetic fibers), stabilizing agents, solubilizing agents, suspension agents, viscosity regulating agents, light spheres, plasticizers, salts, lubricants and fragrances.

The core may include a binder, such as synthetic polymer, wax, fat, or carbohydrate.

The core may include a salt of a multivalent cation, a reducing agent, an antioxidant, a peroxide decomposing catalyst and/or an acidic buffer component, typically as a homogenous blend.

The core may include an inert particle with the enzyme(s) absorbed into it, or applied onto the surface, e.g., by fluid bed coating.

The core may have a diameter of 20-2000 pm, particularly 50-1500 pm, 100-1500 pm or 250-1200 pm.

The core may be surrounded by at least one coating, e.g., to improve the storage stability, to reduce dust formation during handling, or for coloring the granule. The optional coating(s) may include a salt coating, or other suitable coating materials, such as polyethylene glycol (PEG), methyl hydroxy-propyl cellulose (MHPC) and polyvinyl alcohol (PVA).

In an embodiment, the coating comprises a GH43 arabinofuranosidase of the present invention. In an embodiment, the coating comprises a GH51 arabinofuranosidase of the present invention. In an embodiment, the coating comprises a GH5 xylanase of the present invention. In an embodiment, the coating comprises a GH3 beta-xylosidase of the present invention. In an embodiment, the coating comprises a GH30 xylanase of the present invention. In an embodiment, the coating comprises a acetyl xylan esterase of the present invention. In an embodiment, the coating comprises a GH31 alpha-xylosidase of the present invention.

In an embodiment, the coating comprises at least one, at least two, at least three, at least four, at least five, at least six, or at least seven enzymes selected from the group consisting of a GH43 arabinofuranosidase of the present invention, a GH51 arabinofuranosidase of the present invention, a GH5 xylanase of the present invention, a GH3 beta-xylosidase of the present invention, a GH30 xylanase of the present invention, a CE3 acetyl xylan esterase of the present invention, and a GH31 alpha-xylosidase of the present invention. In an embodiment, the coating comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 beta-xylosidase, and a GH30 xylanase. In an embodiment, the coating comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, a GH3 beta-xylosidase, and a GH30_7 xylanase. In an embodiment, the coating comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, a GH3 beta-xylosidase, and a GH30_8 xylanase. In an embodiment, the coating comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_35 xylanase, a GH3 beta-xylosidase, and a GH30_7 xylanase. In an embodiment, the coating comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_35 xylanase, a GH3 beta-xylosidase, and a GH30_8 xylanase.

In an embodiment, the coating comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 beta-xylosidase, a GH30 xylanase, and a CE3 acetyl xylan esterase. In an embodiment, the coating comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, a GH3 beta-xylosidase, a GH30_7 xylanase, and a CE3 acetyl xylan esterase. In an embodiment, the coating comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, a GH3 beta-xylosidase, a GH30_8 xylanase, and a CE3 acetyl xylan esterase. In an embodiment, the coating comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_35 xylanase, a GH3 beta- xylosidase, a GH30_7 xylanase, and a CE3 acetyl xylan esterase. In an embodiment, the coating comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_35 xylanase, a GH3 beta-xylosidase, a GH30_8 xylanase, and a CE3 acetyl xylan esterase.

In an embodiment, the coating comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 beta-xylosidase, a GH30 xylanase, a CE3 acetyl xylan esterase, and a GH31 alpha-xylosidase. In an embodiment, the coating comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, a GH3 beta-xylosidase, a GH30_7 xylanase, a CE3 acetyl xylan esterase, and a GH31 alpha-xylosidase. In an embodiment, the coating comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, a GH3 beta-xylosidase, a GH30_8 xylanase, a CE3 acetyl xylan esterase, and a GH31 alpha-xylosidase. In an embodiment, the coating comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_35 xylanase, a GH3 beta-xylosidase, a GH30_7 xylanase, a CE3 acetyl xylan esterase, and a GH31 alpha-xylosidase. In an embodiment, the coating comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_35 xylanase, a GH3 beta-xylosidase, a GH30_8 xylanase, a CE3 acetyl xylan esterase, and a GH31 alpha-xylosidase.

The coating may be applied in an amount of at least 0.1% by weight of the core, e.g., at least 0.5%, at least 1%, at least 5%, at least 10%, or at least 15%. The amount may be at most 100%, 70%, 50%, 40% or 30%. The coating is preferably at least 0.1 pm thick, particularly at least 0.5 pm, at least 1 pm or at least 5 pm. In some embodiments, the thickness of the coating is below 100 pm, such as below 60 pm, or below 40 pm.

The coating should encapsulate the core unit by forming a substantially continuous layer. A substantially continuous layer is to be understood as a coating having few or no holes, so that the core unit has few or no uncoated areas. The layer or coating should, in particular, be homogeneous in thickness.

The coating can further contain other materials as known in the art, e.g., fillers, antisticking agents, pigments, dyes, plasticizers and/or binders, such as titanium dioxide, kaolin, calcium carbonate or talc.

A salt coating may comprise at least 60% by weight of a salt, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% by weight.

To provide acceptable protection, the salt coating is preferably at least 0.1 pm thick, e.g., at least 0.5 pm, at least 1 pm, at least 2 pm, at least 4 pm, at least 5 pm, or at least 8 pm. In a particular embodiment, the thickness of the salt coating is below 100 pm, such as below 60 pm, or below 40 pm.

The salt may be added from a salt solution where the salt is completely dissolved or from a salt suspension wherein the fine particles are less than 50 pm, such as less than 10 pm or less than 5 pm.

The salt coating may comprise a single salt or a mixture of two or more salts. The salt may be water soluble, in particular, having a solubility at least 0.1 g in 100 g of water at 20°C, preferably at least 0.5 g per 100 g water, e.g., at least 1 g per 100 g water, e.g., at least 5 g per 100 g water.

The salt may be an inorganic salt, e.g., salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids (less than 10 carbon atoms, e.g., 6 or less carbon atoms) such as citrate, malonate or acetate. Examples of cations in these salts are alkali or earth alkali metal ions, the ammonium ion or metal ions of the first transition series, such as sodium, potassium, magnesium, calcium, zinc or aluminum. Examples of anions include chloride, bromide, iodide, sulfate, sulfite, bisulfite, thiosulfate, phosphate, monobasic phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, metasilicate, citrate, malate, maleate, malonate, succinate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate or gluconate. In particular, alkali- or earth alkali metal salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids such as citrate, malonate or acetate may be used.

The salt in the coating may have a constant humidity at 20°C above 60%, particularly above 70%, above 80% or above 85%, or it may be another hydrate form of such a salt (e.g., anhydrate). The salt coating may be as described in WO 00/01793 or WO 2006/034710. Specific examples of suitable salts are NaCI (CH₂₀°c=76%), Na₂CO₃ (CH₂o°c=92%), NaNO₃ (CH₂₀°C=73%), Na₂HPO₄ (CH₂₀°c=95%), Na₃PO₄ (CH₂₅°c=92%), NH₄CI (CH₂₀°c = 79.5%), (NH₄)₂HPO₄ (CH₂₀°C = 93,0%), NH₄H₂PO₄ (CH₂₀°C = 93.1%), (NH₄)₂SO₄ (CH₂₀°c=81 .1%), KCI (CH₂₀°C=85%), K₂HPO₄ (CH₂₀°C=92%), KH₂PO₄ (CH₂₀°C=96.5%), KNO₃ (CH₂₀°C=93.5%), Na₂SO₄ (CH₂₀°C=93%), K₂SO₄ (CH₂₀°C=98%), KHSO₄ (CH₂₀°C=86%), MgSO₄ (CH₂₀°c=90%), ZnSO₄ (CH₂O°C=9O%) and sodium citrate (CH₂s°c=86%). Other examples include NaH₂PO₄, (NH₄)H₂PO₄, CuSO₄, Mg(NO₃)₂ and magnesium acetate.

The salt may be in anhydrous form, or it may be a hydrated salt, i.e., a crystalline salt hydrate with bound water(s) of crystallization, such as described in WO 99/32595. Specific examples include anhydrous sodium sulfate (Na₂SO₄), anhydrous magnesium sulfate (MgSO₄), magnesium sulfate heptahydrate (MgSO₄7H₂O), zinc sulfate heptahydrate (ZnSO₄7H₂O), sodium phosphate dibasic heptahydrate (Na₂HPO₄7H₂O), magnesium nitrate hexahydrate (Mg(NO₃)₂(6H₂O)), sodium citrate dihydrate and magnesium acetate tetrahydrate.

Preferably the salt is applied as a solution of the salt, e.g., using a fluid bed.

The coating materials can be waxy coating materials and film-forming coating materials. Examples of waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591.

The granule may optionally have one or more additional coatings. Examples of suitable coating materials are polyethylene glycol (PEG), methyl hydroxy-propyl cellulose (MHPC) and polyvinyl alcohol (PVA). Examples of enzyme granules with multiple coatings are described in WO 93/07263 and WO 97/23606.

The core can be prepared by granulating a blend of the ingredients, e.g., by a method comprising granulation techniques such as crystallization, precipitation, pan-coating, fluid bed coating, fluid bed agglomeration, rotary atomization, extrusion, prilling, spheronization, size reduction methods, drum granulation, and/or high shear granulation.

Methods for preparing the core can be found in the Handbook of Powder Technology; Particle size enlargement by C. E. Capes; Vol. 1 ; 1980; Elsevier. Preparation methods include known feed and granule formulation technologies, e.g.,

(a) Spray dried products, wherein a liquid enzyme(s)-containing solution is atomized in a spray drying tower to form small droplets which during their way down the drying tower dry to form an enzyme(s)-containing particulate material. Very small particles can be produced this way (Michael S. Showell (editor); Powdered detergents, Surfactant Science Series; 1998; Vol. 71 ; pages 140-142; Marcel Dekker). (b) Layered products, wherein the enzyme(s) is coated as a layer around a pre-formed inert core particle, wherein an enzyme(s)-containing solution is atomized, typically in a fluid bed apparatus wherein the pre-formed core particles are fluidized, and the enzyme(s)-containing solution adheres to the core particles and dries up to leave a layer of dry enzyme(s) on the surface of the core particle. Particles of a desired size can be obtained this way if a useful core particle of the desired size can be found. This type of product is described in, e.g., WO 97/23606.

(c) Absorbed core particles, wherein rather than coating the enzyme(s) as a layer around the core, the enzyme(s) is absorbed onto and/or into the surface of the core. Such a process is described in WO 97/39116.

(d) Extrusion or pelletized products, wherein a enzyme(s)-containing paste is pressed to pellets or under pressure is extruded through a small opening and cut into particles which are subsequently dried. Such particles usually have a considerable size because of the material in which the extrusion opening is made (usually a plate with bore holes) sets a limit on the allowable pressure drop over the extrusion opening. Also, very high extrusion pressures when using a small opening increase heat generation in the enzyme(s) paste, which is harmful to the enzyme(s) (Michael S. Showell (editor); Powdered detergents’, Surfactant Science Series; 1998; Vol. 71 ; pages 140-142; Marcel Dekker).

(e) Prilled products, wherein a enzyme(s)-containing powder is suspended in molten wax and the suspension is sprayed, e.g., through a rotating disk atomizer, into a cooling chamber where the droplets quickly solidify (Michael S. Showell (editor); Powdered detergents’, Surfactant Science Series; 1998; Vol. 71 ; pages 140-142; Marcel Dekker). The product obtained is one wherein the enzyme(s) is uniformly distributed throughout an inert material instead of being concentrated on its surface. US 4,016,040 and US 4,713,245 describe this technique.

(f) Mixer granulation products, wherein a enzyme(s)-containing liquid is added to a dry powder composition of conventional granulating components. The liquid and the powder in a suitable proportion are mixed and as the moisture of the liquid is absorbed in the dry powder, the components of the dry powder will start to adhere and agglomerate and particles will build up, forming granulates comprising the enzyme(s). Such a process is described in US 4,106,991 , EP 170360, EP 304332, EP 304331 , WO 90/09440 and WO 90/09428. In a particular aspect of this process, various high-shear mixers can be used as granulators. Granulates consisting of enzyme(s), fillers and binders etc. are mixed with cellulose fibers to reinforce the particles to produce a so-called T-granulate. Reinforced particles, are more robust, and release less enzymatic dust.

(g) Size reduction, wherein the cores are produced by milling or crushing of larger particles, pellets, tablets, briquettes etc. containing the enzyme(s). The wanted core particle fraction is obtained by sieving the milled or crushed product. Over and undersized particles can be recycled. Size reduction is described in Martin Rhodes (editor); Principles of Powder Technology; 1990; Chapter 10; John Wiley & Sons. (h) Fluid bed granulation. Fluid bed granulation involves suspending particulates in an air stream and spraying a liquid onto the fluidized particles via nozzles. Particles hit by spray droplets get wetted and become tacky. The tacky particles collide with other particles and adhere to them to form a granule.

(i) The cores may be subjected to drying, such as in a fluid bed drier. Other known methods for drying granules in the feed or enzyme industry can be used by the skilled person. The drying preferably takes place at a product temperature of from 25 to 90°C. For some enzyme(s), it is important the cores comprising the enzyme(s) contain a low amount of water before coating with the salt. If water sensitive enzyme(s) are coated with a salt before excessive water is removed, the excessive water will be trapped within the core and may affect the activity of the enzyme(s) negatively. After drying, the cores preferably contain 0.1-10% w/w water.

Non-dusting granulates may be produced, e.g., as disclosed in US 4,106,991 and US 4,661 ,452 and may optionally be coated by methods known in the art.

The granulate may further comprise one or more additional enzymes, e.g., hydrolase, isomerase, ligase, lyase, oxidoreductase, and transferase. The one or more additional enzymes are preferably selected from the group consisting of acetylxylan esterase, acylglycerol lipase, amylase, alpha-amylase, beta-amylase, arabinofuranosidase, cellobiohydrolases, cellulase, feruloyl esterase, galactanase, alpha-galactosidase, beta-galactosidase, beta-glucanase, betaglucosidase, lysophospholipase, lysozyme, alpha-mannosidase, beta-mannosidase (mannanase), phytase, phospholipase A1 , phospholipase A2, phospholipase D, protease, pullulanase, pectin esterase, triacylglycerol lipase, xylanase, beta-xylosidase or any combination thereof. Each enzyme will then be present in more granules securing a more uniform distribution of the enzymes, and also reduces the physical segregation of different enzymes due to different particle sizes. Methods for producing multi-enzyme co-granulates is disclosed in the ip.com disclosure IPCOM000200739D.

Another example of formulation of enzyme(s) by the use of co-granulates is disclosed in WO 2013/188331.

The present invention also relates to protected enzyme(s) prepared according to the method disclosed in EP 238216.

Liquid Formulations

The present invention also relates to liquid compositions comprising at least one, at least two, at least three, at least four, at least five, at least six, or at least seven enzymes of the invention. The composition may comprise an enzyme stabilizer (examples of which include polyols such as propylene glycol or glycerol, sugar or sugar alcohol, lactic acid, reversible protease inhibitor, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid). The present invention contemplates using any of the exemplary GH43 arabinofuranosidases, GH51 arabinofuranosidases, exemplary GH5 xylanses, exemplary GH3 beta-xylosidases, exemplary GH30 xylanases, exemplary CE3 acetyl xylan esterases, and exemplary GH31 alpha-xylosidases in the liquid formulations described herein.

In some embodiments, filler(s) or carrier material(s) are included to increase the volume of such compositions. Suitable filler or carrier materials include, but are not limited to, various salts of sulfate, carbonate and silicate as well as talc, clay and the like. Suitable filler or carrier materials for liquid compositions include, but are not limited to, water or low molecular weight primary and secondary alcohols including polyols and diols. Examples of such alcohols include, but are not limited to, methanol, ethanol, propanol and isopropanol. In some embodiments, the compositions contain from about 5% to about 90% of such materials.

In an aspect, the liquid formulation comprises 20-80% w/w of polyol. In one embodiment, the liquid formulation comprises 0.001-2% w/w preservative.

The following exemplary liquid formulations provide ranges in %w/w of enzyme protein for different combinations of enzymes present in the compositions of the present invention. It is to be understood that enzymes from each class may be added in different concentrations to achieve the total % w/w indicated for each composition.

In another embodiment, the invention relates to liquid formulations comprising:

(A) 0.01-25% w/w of a composition of the present invention comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 beta-xylosidase, and a GH30 xylanase of the present invention;

(B) 20-80% w/w of polyol;

(C) optionally 0.001-2% w/w preservative; and

(D) water.

In another embodiment, the invention relates to liquid formulations comprising:

(A) 0.01-25% w/w of composition of the present invention comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, a GH3 beta-xylosidase, and a GH30_7 xylanase;

(B) 20-80% w/w of polyol;

(C) optionally 0.001-2% w/w preservative; and

(D) water.

In another embodiment, the invention relates to liquid formulations comprising:

(A) 0.01-25% w/w of a composition of the present invention comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, a GH3 beta-xylosidase, and a GH30_8 xylanase;

(B) 20-80% w/w of polyol;

(C) optionally 0.001-2% w/w preservative; and

(D) water. In another embodiment, the invention relates to liquid formulations comprising:

(A) 0.01-25% w/w of a composition of the present invention comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_35 xylanase, a GH3 beta-xylosidase, and a GH30_7 xylanase;

(B) 20-80% w/w of polyol;

(C) optionally 0.001-2% w/w preservative; and

(D) water.

In another embodiment, the invention relates to liquid formulations comprising:

(A) 0.01-25% w/w of a composition of the present invention comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_35 xylanase, a GH3 beta-xylosidase, and a GH30_8 xylanase;

(B) 20-80% w/w of polyol;

(C) optionally 0.001-2% w/w preservative; and

(D) water.

In another embodiment, the invention relates to liquid formulations comprising:

(A) 0.01-25% w/w of a composition of the present invention comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 beta-xylosidase, a GH30 xylanase, and a CE3 acetyl xylan esterase;

(B) 20-80% w/w of polyol;

(C) optionally 0.001-2% w/w preservative; and

(D) water.

In another embodiment, the invention relates to liquid formulations comprising:

(A) 0.01-25% w/w of a composition of the present invention comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, a GH3 beta-xylosidase, a GH30_7 xylanase, and a CE3 acetyl xylan esterase;

(B) 20-80% w/w of polyol;

(C) optionally 0.001-2% w/w preservative; and

(D) water.

In another embodiment, the invention relates to liquid formulations comprising:

(A) 0.01-25% w/w of a composition of the present invention comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, a GH3 beta-xylosidase, a GH30_8 xylanase, and a CE3 acetyl xylan esterase;

(B) 20-80% w/w of polyol;

(C) optionally 0.001-2% w/w preservative; and

(A) 0.01-25% w/w of a composition of the present invention comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_35 xylanase, a GH3 beta-xylosidase, a GH30_7 xylanase, and a CE3 acetyl xylan esterase;

(B) 20-80% w/w of polyol;

(C) optionally 0.001-2% w/w preservative; and

(D) water.

In another embodiment, the invention relates to liquid formulations comprising:

(A) 0.01-25% w/w of a composition of the present invention comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_35 xylanase, a GH3 beta-xylosidase, a GH30_8 xylanase, and a CE3 acetyl xylan esterase;

(B) 20-80% w/w of polyol;

(C) optionally 0.001-2% w/w preservative; and

(D) water.

In another embodiment, the invention relates to liquid formulations comprising:

(B) 20-80% w/w of polyol;

(C) optionally 0.001-2% w/w preservative; and

(D) water.

In each of the above liquid formulation embodiments, the composition of the present invention may further include a GH31 alpha-xylosidase.

In another embodiment, the invention relates to liquid formulations comprising:

(B) 0.001-2% w/w preservative

(C) optionally 20-80% w/w of polyol;; and

(D) water.

In another embodiment, the invention relates to liquid formulations comprising:

(B) 0.001-2% w/w preservative ;

(C) optionally 20-80% w/w of polyol; and

(B) 0.001-2% w/w preservative ;

(C) optionally 20-80% w/w of polyol; and

(D) water.

In another embodiment, the invention relates to liquid formulations comprising:

(B) .001-2% w/w preservative ;

(C) optionally 20-80% w/w of polyol 0; and

(D) water.

In another embodiment, the invention relates to liquid formulations comprising:

(B) 0.001-2% w/w preservative ;

(C) optionally 20-80% w/w of polyol; and

(D) water.

In another embodiment, the invention relates to liquid formulations comprising:

(B) 0.001-2% w/w preservative ;

(C) optionally 20-80% w/w of polyol; and

(D) water.

In another embodiment, the invention relates to liquid formulations comprising:

(B) 0.001-2% w/w preservative ;

(C) optionally 20-80% w/w of polyol; and

(B) 0.001-2% w/w preservative;

(C) optionally 20-80% w/w of polyol; and

(D) water.

In another embodiment, the invention relates to liquid formulations comprising:

(B) 0.001-2% w/w preservative ;

(C) optionally 20-80% w/w of polyol; and

(D) water.

In another embodiment, the invention relates to liquid formulations comprising:

(B) 0.001-2% w/w preservative ;

(C) optionally 20-80% w/w of polyol; and

(D) water.

In another embodiment, the invention relates to liquid formulations comprising:

(B) 0.001-2% w/w preservative;

(C) optionally 20-80% w/w of polyol; and

(D) water.

The liquid formulation may further comprise one or more formulating agents, such as a formulating agent selected from the group consisting of polyol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, PVA, acetate and phosphate, preferably selected from the group consisting of sodium sulfate, dextrin, cellulose, sodium thiosulfate, kaolin and calcium carbonate. In one embodiment, the polyols is selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1 ,2-propylene glycol or 1 ,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight below about 600 and polypropylene glycol (PPG) having an average molecular weight below about 600, more preferably selected from the group consisting of glycerol, sorbitol and propylene glycol (MPG) or any combination thereof.

The liquid formulation may comprises 20-80% polyol (/.e., total amount of polyol), e.g., 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40-60% polyol. In one embodiment, the liquid formulation comprises 20-80% polyol, e.g., 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40- 60% polyol, wherein the polyol is selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1 ,2-propylene glycol or 1 ,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight below about 600 and polypropylene glycol (PPG) having an average molecular weight below about 600. The liquid formulation may comprise 20-80% polyol (/.e., total amount of polyol), e.g., 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40-60% polyol, wherein the polyol is selected from the group consisting of glycerol, sorbitol and propylene glycol (MPG).

The preservative may be selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof. In one embodiment, the liquid formulation comprises 0.02-1.5% w/w preservative, e.g., 0.05-1% w/w preservative or 0.1-0.5% w/w preservative. In one embodiment, the liquid formulation comprises 0.001-2% w/w preservative (/.e., total amount of preservative), e.g., 0.02-1.5% w/w preservative, 0.05-1% w/w preservative, or 0.1-0.5% w/w preservative, wherein the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof.

The liquid formulation may further comprise one or more additional enzymes, e.g., hydrolase, isomerase, ligase, lyase, oxidoreductase, and transferase. The one or more additional enzymes are preferably selected from the group consisting of acetylxylan esterase, acylglycerol lipase, amylase, alpha-amylase, beta-amylase, arabinofuranosidase, cellobiohydrolases, cellulase, feruloyl esterase, galactanase, alpha-galactosidase, beta-galactosidase, beta- glucanase, beta-glucosidase, lysophospholipase, lysozyme, alpha-mannosidase, beta- mannosidase (mannanase), phytase, phospholipase A1 , phospholipase A2, phospholipase D, protease, pullulanase, pectin esterase, triacylglycerol lipase, xylanase, beta-xylosidase or any combination thereof.

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 GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 beta-xylosidase, a GH30 xylanase, and optionally a CE3 acetyl xylan esterase and/or a GH31 alpha-xylosidase, is present or added during saccharification and/or fermentation.

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, GH5 xylanase, a GH3 beta-xylosidase, a GH30 xylanase, and optionally a CE3 acetyl xylan esterase and/or GH31 alpha-xylosidase, is present or added during saccharifying step (b) and/or fermenting step (c)

The present invention contemplates using any of the exemplary GH43 arabinofuranosidases, GH51 arabinofuranosidases, exemplary GH5 xylanses, exemplary GH3 beta-xylosidases, exemplary GH30 xylanases, exemplary CE3 acetyl xylan esterases, and exemplary GH31 alpha-xylosidases in the compositions and processes of using the compositions of the invention, including in the following exemplary compostions used in the process for producing a fermentation product.

An exemplary composition used in step (b) and/or step (c) comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 beta-xylosidase, and a GH30 xylanase. In an embodiment, the GH30 xylanase activity is a GH30_1 xylanase. In an embodiment, the GH30 xylanase activity is a GH30_2 xylanase. In an embodiment, the GH30 xylanase activity is a GH30_3 xylanase. In an embodiment, the GH30 xylanase activity is a GH30_4 xylanase. In an embodiment, the GH30 xylanase activity is a GH30_5 xylanase. In an embodiment, the GH30 xylanase activity is a GH30_7 xylanase. In an embodiment, the GH30 xylanase activity is a GH30_8 xylanase. In an embodiment, the GH30 xylanase activity is a GH30_9 xylanase. In an embodiment, the GH30 xylanase activity is a GH30_10 xylanase.

In an embodiment, the GH5 family xylanase is a GH5_21 xylanase. An exemplary composition used in step (b) and/or step (c) comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, a GH3 beta-xylosidase, and a GH30_7 xylanase. An exemplary composition used in step (b) and/or step (c) comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, a GH3 beta-xylosidase, and a GH30_8 xylanase. In an embodiment, the GH5 family xylanase is a GH5_35 xylanase. In an embodiment, the core comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_35 xylanase, a GH3 beta-xylosidase, and a GH30_7 xylanase. An exemplary composition used in step (b) and/or step (c) comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_35 xylanase, a GH3 beta-xylosidase, and a GH30_8 xylanase.

In an embodiment, the composition used in step (b) and/or step (c) includes a CE3 acetyl xylan esterase. An exemplary composition used in step (b) and/or step (c) comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 beta-xylosidase, a GH30 xylanase, and a CE3 acetyl xylan esterase. An exemplary composition used in step (b) and/or step (c) comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, a GH3 beta-xylosidase, a GH30_7 xylanase, and a CE3 acetyl xylan esterase. An exemplary composition used in step (b) and/or step (c) comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, a GH3 beta-xylosidase, a GH30_8 xylanase, and a CE3 acetyl xylan esterase. An exemplary composition used in step (b) and/or step (c) comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_35 xylanase, a GH3 beta-xylosidase, a GH30_7 xylanase, and a CE3 acetyl xylan esterase. An exemplary composition used in step (b) and/or step (c) comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_35 xylanase, a GH3 beta-xylosidase, a GH30_8 xylanase, and a CE3 acetyl xylan esterase.

In an embodiment, the composition used in step (b) and/or step (c) includes a GH31 alpha-xylosidase. In an embodiment, the composition used in step (b) and/or (c) includes a CE3 acetyl xylan esterase and a GH31 alpha-xylosidase. An exemplary composition used in step (b) and/or step (c) comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 beta-xylosidase, a GH30 xylanase, a CE3 acetyl xylan esterase, and a GH31 alpha-xylosidase. An exemplary composition used in step (b) and/or step (c) comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, a GH3 beta-xylosidase, a GH30_7 xylanase, a CE3 acetyl xylan esterase, and a GH31 alpha-xylosidase. An exemplary composition used in step (b) and/or step (c) comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, a GH3 beta-xylosidase, a GH30_8 xylanase, a CE3 acetyl xylan esterase, and a GH31 alpha-xylosidase. An exemplary composition used in step (b) and/or step (c) comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_35 xylanase, a GH3 beta-xylosidase, a GH30_7 xylanase, a CE3 acetyl xylan esterase, and a GH31 alpha-xylosidase. An exemplary composition used in step (b) and/or step (c) comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_35 xylanase, a GH3 beta-xylosidase, a GH30_8 xylanase, a CE3 acetyl xylan esterase, and a GH31 alpha-xylosidase.

In an embodiment, the GH43 arabinofuranosidase is a GH43_36 arabinofuranosidase. In an embodiment, the GH51 arabinofuranosidase is a GH51_6 arabinofuranosidase.

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 beta-glucosidase 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, beans, cassava, cereals, corn, milo, 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 beta-glucosidases described in WO 2005/047499, WO 2013/148993, WO 2014/085439 and WO 2012/044915 (each of which is incorporated herein by reference).

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).

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 GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 beta-xylosidase, a GH30 xylanase, and optionally a CE3 acetyl xylan esterase and/or a GH31 alpha-xylosidase, is present or added during saccharification and/or fermentation.

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, GH5 xylanase, a GH3 beta-xylosidase, a GH30 xylanase, and optionally a CE3 acetyl xylan esterase and/or GH31 alpha-xylosidase, is present or added to saccharifying step (a) and/or fermenting step (b)

An exemplary composition used in step (a) and/or step (b) comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 beta-xylosidase, and a GH30 xylanase. In an embodiment, the GH30 xylanase activity is a GH30_1 xylanase. In an embodiment, the GH30 xylanase activity is a GH30_2 xylanase. In an embodiment, the GH30 xylanase activity is a GH30_3 xylanase. In an embodiment, the GH30 xylanase activity is a GH30_4 xylanase. In an embodiment, the GH30 xylanase activity is a GH30_5 xylanase. In an embodiment, the GH30 xylanase activity is a GH30_7 xylanase. In an embodiment, the GH30 xylanase activity is a GH30_8 xylanase. In an embodiment, the GH30 xylanase activity is a GH30_9 xylanase. In an embodiment, the GH30 xylanase activity is a GH30_10 xylanase.

In an embodiment, the GH5 family xylanase is a GH5_21 xylanase. An exemplary composition used in step (a) and/or step (b) comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, a GH3 beta-xylosidase, and a GH30_7 xylanase. An exemplary composition used in step (a) and/or step (b) comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, a GH3 beta-xylosidase, and a GH30_8 xylanase. In an embodiment, the GH5 family xylanase is a GH5_35 xylanase. In an embodiment, the core comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_35 xylanase, a GH3 beta-xylosidase, and a GH30_7 xylanase. An exemplary composition used in step (a) and/or step (b) comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_35 xylanase, a GH3 beta-xylosidase, and a GH30_8 xylanase.

In an embodiment, the composition used in step (a) and/or step (b) includes a CE3 acetyl xylan esterase. An exemplary composition used in step (a) and/or step (b) comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 beta-xylosidase, a GH30 xylanase, and a CE3 acetyl xylan esterase. An exemplary composition used in step (a) and/or step (b) comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, a GH3 beta-xylosidase, a GH30_7 xylanase, and a CE3 acetyl xylan esterase. An exemplary composition used in step (a) and/or step (b) comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, a GH3 beta-xylosidase, a GH30_8 xylanase, and a CE3 acetyl xylan esterase. An exemplary composition used in step (a) and/or step (b) comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_35 xylanase, a GH3 beta-xylosidase, a GH30_7 xylanase, and a CE3 acetyl xylan esterase. An exemplary composition used in step (a) and/or step (b) comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_35 xylanase, a GH3 beta-xylosidase, a GH30_8 xylanase, and a CE3 acetyl xylan esterase.

In an embodiment, the composition used in step (a) and/or step (b) includes a GH31 alpha-xylosidase. In an embodiment, the composition used in step (a) and/or (b) includes a CE3 acetyl xylan esterase and a GH31 alpha-xylosidase. An exemplary composition used in step (a) and/or step (b) comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 beta-xylosidase, a GH30 xylanase, a CE3 acetyl xylan esterase, and a GH31 alpha-xylosidase. An exemplary composition used in step (a) and/or step (b) comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, a GH3 beta-xylosidase, a GH30_7 xylanase, a CE3 acetyl xylan esterase, and a GH31 alpha-xylosidase. An exemplary composition used in step (a) and/or step (b) comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, a GH3 beta-xylosidase, a GH30_8 xylanase, a CE3 acetyl xylan esterase, and a GH31 alpha-xylosidase. An exemplary composition used in step (a) and/or step (b) comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_35 xylanase, a GH3 beta-xylosidase, a GH30_7 xylanase, a CE3 acetyl xylan esterase, and a GH31 alpha-xylosidase. An exemplary composition used in step (a) and/or step (b) comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_35 xylanase, a GH3 beta-xylosidase, a GH30_8 xylanase, a CE3 acetyl xylan esterase, and a GH31 alpha-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.

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 into Thin Stillage and Wet Cake

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 de-oiled 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.

EXAMPLES

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;

GH51 B: 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_21 B: 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_21 H: exemplary GH5_21 xylanase from Xanthan alkaline community S disclosed in SEQ ID NO: 14;

GH5_21 I: exemplary GH5_21 xylanase from Belliella sp-64282 disclosed in SEQ ID NO: 15;

GH5_21 J: exemplary GH5_21 xylanase from Chryseobacterium oncorhynchi disclosed in SEQ ID NO: 16;

GH5_21 K: exemplary GH5_21 xylanase from Xanthan alkaline community T disclosed in SEQ ID NO: 17;

GH5_21 L: 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_21 N: 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;

GH3A: exemplary GH3 beta-xylosidase from Aspergillus fumigatus disclosed in SEQ ID NO: 27;

GH3B: exemplary GH3 beta-xylosidase from Aspergillus nidulans disclosed in SEQ ID NO: 28;

GH3C: exemplary GH3 beta-xylosidase from Talaromyces emersonii disclosed in SEQ ID NO: 29;

GH3D: exemplary GH3 beta-xylosidase from Aspergillus tellustris disclosed in SEQ ID NO: 30;

GH3E: exemplary GH3 beta-xylosidase from Aspergillus aculeatus disclosed in SEQ ID NO: 31;

GH3F: exemplary GH3 beta-xylosidase beta-xylosidase from Aspergillus fischeri disclosed in SEQ ID NO: 32;

GH3G: exemplary GH3 beta-xylosidase beta-xylosidase from Chaetomium globosum disclosed in SEQ ID NO: 33;

GH3H: exemplary GH3 beta-xylosidase beta-xylosidase from Chaetomium virescens disclosed in SEQ ID NO: 34;

GH3I: exemplary GH3 beta-xylosidase beta-xylosidase from Fusarium longipes disclosed in SEQ ID NO: 35;

GH3J: exemplary GH3 beta-xylosidase beta-xylosidase from Mycothermus thermophilus disclosed in SEQ ID NO: 36;

GH3K: exemplary GH3 beta-xylosidase beta-xylosidase from Penicillium emersonii disclosed in SEQ ID NO: 37;

GH3L: exemplary GH3 beta-xylosidase beta-xylosidase from Penicillium oxalicum disclosed in SEQ ID NO: 38;

GH3M: exemplary GH3 beta-xylosidase beta-xylosidase from Sporormia fimetaria disclosed in SEQ ID NO: 39;

GH3N: exemplary GH3 beta-xylosidase beta-xylosidase from Talaromyces stipitatus disclosed in SEQ ID NO: 40; GH30: exemplary GH3 beta-xylosidase beta-xylosidase from Trichoderma reesei disclosed in SEQ ID NO: 41 ;

GH30_7A: exemplary GH30_7 xylanase from Evansstolkia leycettana disclosed in SEQ ID NO: 42;

GH30_8A: exemplary GH30_8 xylanase from Bacillus sp-18423 disclosed in SEQ ID NO: 53;

CE3A: exemplary CE3 polypeptide having acetyl xylan esterase from Dinemasporium sp. Disclosed as SEQ ID NO: 58;

CE3B: exemplary CE3 acetyl xylan esterase from Epicoccum sorghinum disclosed as SEQ ID NO: 59;

CE3C: exemplary CE3 acetyl xylan esterase Flammulina velutipes disclosed as SEQ ID NO: 60;

CE3D: exemplary CE3 acetyl xylan esterase from Microsphaeropsis arundinis disclosed as SEQ ID NO: 61;

CE3E: exemplary CE3 acetyl xylan esterase from Microsphaeropsis arundinis disclosed as SEQ ID NO: 62;

CE3F: exemplary CE3 acetyl xylan esterase from Microsphaeropsis arundinis disclosed as SEQ ID NO: 63;

CE3G: exemplary CE3 acetyl xylan esterase from Paraphaeosphaeria neglecta disclosed as SEQ ID NO: 64;

CE3H: exemplary CE3 acetyl xylan esterase from Paraphaeosphaeria verruculosa disclosed as SEQ ID NO: 65;

CE3I: exemplary CE3 acetyl xylan esterase from Westerdykella purpurea disclosed as SEQ ID NO: 66;

CE3J: exemplary CE3 acetyl xylan esterase from Xepicula leucotricha disclosed as SEQ ID NO: 67;

GH31A: exemplary GH31 alpha-xylosidase from Herbinix hemicellulosilytica disclosed in SEQ ID NO: 68;

GH8: exemplary GH8 xylanase from Bacillus sp. KK-1 disclosed in SEQ ID NO: 69;

GH10: exemplary GH 10 xylanase from Aspergillus aculeatus disclosed in SEQ ID NO: 70; and

GH11: exemplary GH11 xylanase from Thermomyces lanuginosus disclosed in SEQ ID NO: 71.

Liquefaction Enzyme Blend 1 : exemplary thermostable alpha-amylase from Bacillus stearothermophilus disclosed in SEQ ID NO: 72; exemplary thermostable protease from Pyrococcus furiosus disclosed in SEQ ID NO: 74.

Liquefaction Enzyme Blend 2: exemplary thermostable alpha-amylase from Bacillus stearothermophilus disclosed in SEQ ID NO: 73; exemplary thermostable protease from Pyrococcus furiosus disclosed in SEQ ID NO: 74; exemplary thermostable xylanase from Thermotoga maritima disclosed in SEQ ID NO: 75.

Saccharification Enzyme Blend: exemplary glucoamylase from Gloeophyllum sepiarium disclosed in SEQ ID NO: 76; exemplary alpha-amylase from Rhizomucor pusillus disclosed in SEQ ID NO: 77; exemplary trehalase from Talaromyces funiculosus disclosed in SEQ ID NO: 78; exemplary beta-glucosidase from Aspergillus fumigatus disclosed in SEQ ID NO: 79; exemplary celliobiohydrolase from Aspergillus fumigatus disclosed in SEQ ID NO: 80; exemplary endoglucanase from Trichoderma reesei disclosed in SEQ ID NO: 81.

Hemicellulase Blend: GH43 arabinofuranosidase (GH43A), GH51 arabinofuranosidase (GH51A), GH5_21 xylanase (GH5_21O), and GH3 beta-xylosidase (GH3A).

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 pre-equilibrated 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 WQ2011/066576) and a hybrid Rhizomucor pusillus alpha amylase expression cassette (as described in WQ2013/006756).

Media and Solutions

PDA plates were composed of 39 grams of potato dextrose agar and deionized water to 1 liter.

Skimmed milk media was composed of a 10% solution of skimmed milk powder and autoclaved. 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 2. 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 2: Dosing scheme of arabinofuranosidase with or without xylanase

Table 3: Results

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

Table 3 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 4. 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 4 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 4

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 5. 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 5 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 5

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 6

Table 6 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 7. 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 7

Table 7 shows that GH43 and GH51 arabinofuranosidases in combination with GH5_21 xylanases 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 8

Table 8 shows that GH43 and GH1 arabinofuranosidases in combination with GH5_21 xylanases increase arabinose release compared to without xylanase and arabinofuranosidases.

Example 7: Effect of exemplary CE3 polypeptides having acetyl xylan esterase activity in combination with exemplary GH43 arabinofuranosidase, exemplary GH51 arabinofuranosidase, exemplary GH5_21 xylanase and exemplary GH3 beta-xylosidase with or without an exemplary GH31 alpha-xylosidase for increasing xylose and arabinose in simultaneous saccharification and fermentation process

An industrial liquefied mash using 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 4.2 g of the industrial liquefied corn mash was added to 10 ml tube vials. Each vial was dosed with 0.42 AGU/gDS of Saccharification Enzyme Blend together with amounts of a Hemicellulase blend shown in Table 9 below and 10 ug/gDS of respective CE3 polypeptide (Table 10) followed by addition of 50 pL of propagated yeast strain MEJI797 per 4.2 g slurry. As control, only Saccharification Enzyme Blend without addition of the Hemicellulase Blend or the CE3 polypeptide. 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). 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.

Table 9: Dose of enzymes in Hemicellulase Blend

Result

As shown in Table 10 below, the combination of the CE3 polypeptide with the Hemicellulase Blend increases xylose and arabinose release compared to control or the Hemicellulase Blend without the CE3 polyeptide. The Hemicellulase Blend significantly reduced residual solids compared to control and the addition of the CE3 polypeptide further decreased the residual solids, indicating increased corn fiber degradation by the Hemicellulase Blend and its combination with the CE3 polypeptide.

Table 10

Example 8: Effect of exemplary CE3 polypeptides having acetyl xylan esterase activity in combination with exemplary GH43 arabinofuranosidase, exemplary GH51 arabinofuranosidase, exemplary GH5_21 xylanase and a exemplary GH3 beta-xylosidase with or without an exemplary GH31 alpha-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 35%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 10 ml tube vials. Each vial was dosed with 0.42 AGU/gDS of Saccharification Enzyme Blend together with amounts of a Hemicellulase Blend shown in Table 11 and 10 ug/gDS of respective CE3 polypeptide shown in Table 9 above with or without 10 ug/gDS of an exemplary GH31 alpha- xylosidase (GH31A), followed by addition of 50 pL of propagated yeast strain MEJI797 per 4.2 g slurry. As control, only Saccharification Enzyme Blend without addition of Hemicellulase Blend, CE3 polypeptide or GH31 alpha-xylosidase. 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 systems equipped with lead column (Benson Polymeric, BP-800 Pb, 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

As shown in Table 11 below, the combination of CE3 polypeptide with the Hemicellulase Blend increases release of xylose and arabinose compared to control or Hemicellulase Blend without the CE3 polypeptide. Addition of an exemplary alpha-xylosidase (GH31A) further boost xylose and arabinose release when mixed with the CE3 polypeptide and Hemicellulase Blend. The Hemicellulase Blend significantly reduced residual solids compared to control and addition of the CE3 polypeptide and the GH31 further decreased residual solids, indicating increased corn fiber degradation by the Hemicellulase Blend and its combination with the CE3 polypeptide and the GH31 alpha-xylosidase.

Table 11

Example 9: 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 12, 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 C5 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 12: Hemicellulases blend (Base C5)

Result

As shown in results Table 13 below, combination of BX particularly with Po BX, with or without GH3A in Base C5 hemicellulases blend significantly increases xylose release.

Table 13

Example 10: Effect of GH30 xylanase subfamily 7 or 8 with or without CE3 enzyme combination with hemicellulases blend (Base C5) for increasing xylose and arabinose 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 35%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 4.0 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 12 in Example 9, and appropriate amount of respective GH30_7, GH30_8 and CE3 enzymes (Table 14) followed by addition of 50 pL of propagated Ethanol Red yeast per 4.0 g slurry. As control, only glucoamylase with no addition of Base 05, GH30 xylanases or CE3 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 0.20 micrometer filter. Sugars concentrations were determined using HPLC system equipped with H column (Benson Polymeric, BP-700 H, 300 x 7.8 mm).

Result

As shown in Table 14 below, combination of GH30 subfamily 7 or 8 xylanases with Base 05 hemicellulases blend increase xylose and arabinose release compared to control or Base 05 alone. Addition of CE3 enzyme to the combination of Base 05 with GH30 xylanase further boost xylose and arabinose release.

Table 14

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

The invention is further defined by the following numbered paragraphs:

1. A granule, which comprises:

(a) a core comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 beta-xylosidase, and a GH30 xylanase, and, optionally

(b) a coating consisting of one or more layer(s) surrounding the core.

2. A granule, which comprises:

(a) a core comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 beta-xylosidase, and a GH30 xylanase, and a CE3 acetyl xylan esterase, and, optionally

(b) a coating consisting of one or more layer(s) surrounding the core.

3. A granule, which comprises:

(a) a core, and

(b) a coating consisting of one or more layer(s) surrounding the core, wherein the coating comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 beta-xylosidase, a GH30 xylanase.

4. A granule, which comprises:

(a) a core, and

(b) a coating consisting of one or more layer(s) surrounding the core, wherein the coating comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 beta-xylosidase, a GH30 xylanase, and a CE3 acetyl xylan esterase. 5. A liquid composition comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 beta-xylosidase, a GH30 xylanase, and an enzyme stabilizer, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, or lactic acid.

6. A liquid composition comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 beta-xylosidase, a GH30 xylanase, a CE3 acetyl xylan esterase, and an enzyme stabilizer, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, or lactic acid.

7. The liquid composition of paragraphs 5 or 6, further comprising a filler or carrier material.

8. The liquid composition of any one of paragraphs 5 to 7, further comprising a preservative.

9. A composition comprising the granule of any one of paragraphs 1 to 4, or the liquid composition of any one of paragraphs 5 to 8.

10. The composition of paragraph 9, which is a liquid composition, solid composition, solution, dispersion, paste, powder, granule, granulate, coated granulate, tablet, cake, crystal, crystal slurry, gel or pellet.

11. A composition comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, a GH3 beta-xylosidase, a GH30 xylanase.

12. A composition comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, a GH3 beta-xylosidase, a GH30 xylanase, and a CE3 acetyl xylan esterase.

13. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, wherein the GH43 arabinofuranosidase is a GH43_36 arabinofuranosidase.

14. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of paragraph 13, wherein the GH43 arabinofuranosidase is from the genus Humicola, Lasiodiplodia, or Poronia. 15. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of paragraph 13 or 14, wherein the GH43 arabinofuranosidase is from the species Humicola insolens, Lasiodiplodia theobromae, or Poronia punctata.

16. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 15, 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.

17. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 16, wherein the GH51 arabinofuranosidase is a GH51_6 arabinofuranosidase.

18. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 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; and

(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 granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 18, wherein the GH5 xylanase is a GH5_21 xylanase.

20. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 19, wherein the GH5_21 xylanase is from the genus Bacteroides, Belliella, Chryseobacterium, or Sphingobacterium.

21. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 20, wherein the GH5_21 xylanase is from the species Bacteroides cellulosilyticus CL02Y12C19, Belliella sp-64282, Chryseobacterium sp., Chryseobacterium oncorhynchi, or Sphingobacterium sp-64162.

22. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 21 , 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.

23. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 22, 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, which has xylanase activity;

(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.

24. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 23, where in the GH5 xylanase is a GH5_35 xylanase.

25. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 24, wherein the GH5_35 xylanase is from the genus Bacillus, Cohnella, or Paenibacillus.

26. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 25, wherein the GH5_35 xylanase is from the species Bacillus hemiccellulosilyticus JCM 9152, Cohnella xylanilytica, Paenibacillus chitinolyticus, or Paenibacillus sp-62332.

27. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 26, wherein the GH5_35 xylanase is from compost metagenome.

28. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 27, 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.

29. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 28, wherein the GH3 beta- xylosidase is from the genus Aspergillus or Talaromyces.

30. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 29, wherein the GH3 beta- xylosidase is from the species Aspergillus fumigatus, Aspergillus nidulans, or Talaromyces emersonii.

31. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 30, 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: 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 beta- xylosidase activity;

(ii) 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;

(iii) 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

(iv) 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;

(v) the amino acid sequence of SEQ ID NO: 31 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: 31 , which has beta- xylosidase activity;

(vi) the amino acid sequence of SEQ ID NO: 32 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: 32, which has beta- xylosidase activity;

(vii) the amino acid sequence of SEQ ID NO: 33 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: 33, which has beta- xylosidase activity;

(viii) the amino acid sequence of SEQ ID NO: 34 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: 34, which has beta- xylosidase activity;

(ix) the amino acid sequence of SEQ ID NO: 35 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: 35, which has beta- xylosidase activity; (x) the amino acid sequence of SEQ ID NO: 36 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: 36, which has beta- xylosidase activity;

(xi) the amino acid sequence of SEQ ID NO: 37 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: 37, which has beta- xylosidase activity;

(xii) the amino acid sequence of SEQ ID NO: 38 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: 38, which has beta- xylosidase activity;

(xiii) the amino acid sequence of SEQ ID NO: 39 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: 39, which has beta- xylosidase activity;

(xiv) the amino acid sequence of SEQ ID NO: 40 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: 40, which has beta- xylosidase activity; and

(xv) the amino acid sequence of SEQ ID NO: 41 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: 41, which has beta- xylosidase activity.

32. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 31, wherein the GH30 xylanase is a GH30_1 xylanase.

33. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 32, wherein the GH30 xylanase is a GH30_2 xylanase.

34. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 33, wherein the GH30 xylanase is a GH30_3 xylanase.

35. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 34 wherein the GH30 xylanase is a GH30_4 xylanase.

36. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 35, wherein the GH30 xylanase is a GH30_5 xylanase.

37. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 36, wherein the GH30 xylanase is a GH30_7 xylanase.

38. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 37, wherein the GH30_7 xylanase is from a genus selected from Aspergillus, Evansstolkia, Talaromyces, and Trichoderma.

39. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 38, wherein the GH30_7 xylanase is from a species selected from Aspergillus fischeri, Aspergillus fumigatiaffinis, Aspergillus novofumigatus, Aspergillus pseudoterreus, Aspergillus terreus, Aspergillus turcosus, Aspergillus udagawae, Evansstolkia leycettana, Talaromyces verruculosus, and Trichoderma reesei.

40. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 39, wherein the GH30_7 xylanase is selected from the group consisting of:

(i) the amino acid sequence of SEQ ID NO: 42 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: 42, which has xylanase activity;

(ii) the amino acid sequence of SEQ ID NO: 43 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: 43, which has xylanase activity;

(iii) 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 xylanase activity;

(iv) 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 xylanase activity;

(v) 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 xylanase activity;

(vi) 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 xylanase activity;

(vii) 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 xylanase activity; (viii) 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 xylanase activity;

(ix) 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 xylanase activity;

(x) 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 xylanase activity; and

(xi) 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 xylanase activity.

41. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 40, wherein the GH30 xylanase is a GH30_8 xylanase.

42. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 41, wherein the GH30_8 xylanase is from the genus Bacillus, Clostridium, Paenibacillus, or Vibrio.

43. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 42, wherein the GH30_8 xylanase is from the species Bacillus sp- 18423, Clostridium acetobutylicum, Clostridium saccharobutylicum, Paenibacillus pabuli, and Vibrio rhizosphaerae. 44. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 43, wherein the GH30_8 xylanase is selected from the group consisting of:

(i) 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 xylanase activity;

(ii) 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 xylanase activity;

(iii) 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 xylanase activity;

(iv) the amino acid sequence of SEQ ID NO: 56 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: 56, which has xylanase activity; and

(v) the amino acid sequence of SEQ ID NO: 57 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: 57, which has xylanase activity.

45. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 44, wherein the GH30 xylanase is a GH30_9 xylanase.

46. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 45, wherein the GH30 xylanase is a GH30_10 xylanase.

47. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 46, wherein the CE3 acetyl xylan esterase is selected from the group consisting of:

(i) the amino acid sequence of SEQ ID NO: 58 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: 58, which has acetyl xylan esterase activity;

(ii) the amino acid sequence of SEQ ID NO: 59 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: 59, which has acetyl xylan esterase activity;

(iii) the amino acid sequence of SEQ ID NO: 60 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: 60, which has acetyl xylan esterase activity;

(iv) the amino acid sequence of SEQ ID NO: 61 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: 61 , which has acetyl xylan esterase activity;

(v) the amino acid sequence of SEQ ID NO: 62 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: 62, which has acetyl xylan esterase activity;

(vi) the amino acid sequence of SEQ ID NO: 63 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: 63, which has acetyl xylan esterase activity; (vii) the amino acid sequence of SEQ ID NO: 64 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: 64, which has acetyl xylan esterase activity;

(viii) the amino acid sequence of SEQ ID NO: 65 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: 65, which has acetyl xylan esterase activity;

(ix) the amino acid sequence of SEQ ID NO: 66 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: 66, which has acetyl xylan esterase activity;

(x) the amino acid sequence of SEQ ID NO: 67 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: 67, which has acetyl xylan esterase activity;

48. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 47, further comprising a GH31 alpha-xylosidase.

49. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 48, wherein GH31 alpha- xylosidase is from the genus Herbinix.

50. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 49, wherein the GH31 alpha- xylosidase is from the species Herbinix hemicellulosilytica.

51. The granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, or the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 50, wherein the GH31 alpha- xylosidase has the amino acid sequence of SEQ ID NO: 68 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: 68, which has alpha-xylosidase activity.

52. A process for producing a fermentation product from a starch-containing material comprising the steps of:

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

(b) fermenting the sugar with a fermenting organism to produce the fermentation product; wherein the granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 50, is present or added during saccharifying step (a) and/or fermenting step (b).

53. The process of praragraph 52, wherein the granule of any one of paragraphs 1 to 4 and 13 to 51 is present or added during saccharifying step (a) and/or fermenting step (b).

54. The process of praragraph 52, wherein the liquid composition of any one of paragraphs 5 to 8 and 13 to 51 is present or added during saccharifying step (a) and/or fermenting step (b).

55. The process of praragraph 52, wherein the composition of any one of paragraphs 9 to 51 is present or added during saccharifying step (a) and/or fermenting step (b).

56. The process of any one of paragraphs 52 to 55, wherein saccharifying step (a) and fermenting step (b) are performed simultaneously.

57. A process for producing a fermentation product from a starch-containing material comprising the steps of:

(a) liquefying a starch-containing material at a temperature above the initial gelatinization temperature of the starch with a thermostable alpha-amylase 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 the granule of any one of paragraphs 1 to 4, the liquid composition of any one of paragraphs 5 to 8, the composition of any one of paragraphs 9 to 12, or the granule, liquid composition, or composition of any one of paragraphs 13 to 51, is present or added during saccharifying step (b) and/or fermenting step (c).

58. The process of praragraph 57, wherein the granule of any one of paragraphs 1 to 4 and 13 to 51 is present or added during saccharifying step (a) and/or fermenting step (b).

59. The process of praragraph 57, wherein the liquid composition of any one of paragraphs 5 to 8 and 13 to 51 is present or added during saccharifying step (a) and/or fermenting step (b).

60. The process of praragraph 57, wherein the composition of any one of paragraphs 9 to 51 is present or added during saccharifying step (a) and/or fermenting step (b).

61. The process of any one of paragraphs 57 to 60, wherein saccharifying step (a) and fermenting step (b) are performed simultaneously.

62. The process of any one of paragraphs 57 to 61 , wherein the thermostable alpha-amylase has the amino acid sequence of SEQ ID NO: 72 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: 72, which has alpha-amylase activity.

63. The process of any one of paragraphs 57 to 61 , wherein the thermostable alpha-amylase has the amino acid sequence of SEQ ID NO: 73 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: 73, which has alpha-amylase activity.

64. The process of any one of paragraphs 57 to 63, wherein a thermostable protease and/or a thermostable xylanase are added in liquefying step (a).

65. The process of any one of paragraphs 57 to 64, wherein the thermostable protease has the amino acid sequence of SEQ ID NO: 74 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: 74, which has protease activity.

66. The process of any one of paragraphs 57 to 65, wherein the thermostable xylanase has an amino acid sequence of SEQ ID NO: 75 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: 75, which has xylanase activity.

67. The process of any one of paragraphs 57 to 66, wherein the glucoamylase has an amino acid sequence of SEQ ID NO: 76 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: 76, which has glucoamylase activity.

68. The process of any one of paragraphs 57 to 67, further comprising adding an alphaamylase during saccharifying step (b) and/or fermenting step (c).

69. The process of any one of paragraphs 52 to 68, wherein the alpha-amylase has an amino acid sequence of SEQ ID NO: 77 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: 77, which has alpha-amylase activity.

70. The process of any one of paragraphs 57 to 69, wherein a trehalase is added during the saccharifying step and/or the fermenting step.

71. The process of paragraph 70, wherein the trehalase has an amino acid sequence of SEQ ID NO: 78 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: 78, which has trehalase activity. 72. The process of any one of paragraphs 57 to 71, wherein a composition comprising a beta-glucosidase, a cellobiohydrolase, and an endoglucanase are added during the saccharifying step and/or the fermenting step.

73. The process of paragraph 72, wherein the beta-glucosidase has an amino acid sequence of SEQ ID NO: 79 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: 79, which has betaglucosidase activity.

74. The process of paragraphs 72 or 73, wherein the cellobiohydrolase has an amino acid sequence of SEQ ID NO: 80 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: 80, which has cellobiohydrolase activity.

75. The process of any one of paragraphs 72 to 74, wherein the endoglucanase has an amino acid sequence of SEQ ID NO: 81 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: 81, which has endoglucanase activity.

76. The process of any one of paragraphs 52 to 75, wherein the starch-containing material comprises beets, maize, corn, wheat, rye, oats, barley, triticale, rice, sweet potatoes, sorghum, millet, pearl millet, and/or foxtail millet.

77. The process of any one of paragraphs 52 to 76, wherein the starch-containing material comprises corn.

78. The process of any one of paragraphs 52 to 77, wherein the fermentation product is ethanol, preferably fuel ethanol.

79. The process of any one of paragraphs 52 to 78, wherein the fermenting organism is yeast.

Claims

CLAIMS What is claimed is:

1. A granule, which comprises:

(a) a core comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 beta-xylosidase, a GH30 xylanase, and optionally a carbohydrate esterase family 3 (CE3) acetyl xylan esterase, and, optionally

(b) a coating consisting of one or more layer(s) surrounding the core.

2. A granule, which comprises:

(a) a core, and

(b) a coating consisting of one or more layer(s) surrounding the core, wherein the coating comprises a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 beta- xylosidase, a GH30 xylanase, and optionally a CE3 acetyl xylan esterase.

3. A composition comprising the granule of claim 1 or 2.

4. A liquid composition comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 beta-xylosidase, a GH30 xylanase, and optionally a Ce3 acetyl xylan esterase, and an enzyme stabilizer, e.g. , a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, or lactic acid.

5. The liquid composition of claim 4, further comprising a fdler or carrier material and/or a preservative.

6. A composition comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 21 xylanase, a GH3 beta-xylosidase, a GH30 xylanase and, optionally, a CE3 acetyl xylan esterase.

7. The granule of claims 1 or 2 or the composition of any one of claims 3 to 6, wherein the GH5 xylanase is a GH5 21 xylanase or a GH5 35 xylanase.

8. The granule of claims 1 or 2 or the composition of any one of claims 3 to 6, wherein the GH30 xylanase is a GH30 7 xylanase or a GH30 8 xylanase.

9. The granule or composition of any one of claims 1 to 8, further comprising an alpha-xylosidase (e.g., a GH31 alpha-xylosidase).

10. A process for producing a fermentation product from a starch-containing material comprising the steps of:

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

(b) fermenting the sugar with a fermenting organism; wherein a composition comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 beta-xylosidase, a GH30 xylanase, and optionally a CE3 acetyl xylan esterase is present or added during saccharifying step (a) and/or fermenting step (b).

11. A process for producing a fermentation product from a starch-containing material comprising the steps of:

(c) fermenting the sugar with a fermenting organism to produce the fermentation product; wherein a composition comprising a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 beta-xylosidase, a GH30 xylanase, and optionally a CE3 acetyl xylan esterase is present or added during saccharifying step (b) and/or fermenting step (c).

12. The process of claim 10 or 11, wherein the composition comprises the CE3 acetyl xylan esterase.

13. The process of any one of claims 10 to 12, wherein the composition further comprises an alpha- xylosidase (e.g., a GH31 alpha-xylosidase).

14. The process of any one of claims 10 to 13, wherein the GH5 xylanase is a GH5 21 xylanase or a GH5 35 xylanase.

15. The process of any one of claims 10 to 14, wherein the GH30 xylanase is a GH30 7 xylanase or a GH30 8 xylanase.