Compositions and their use for increasing dissolution of hemicellulose fibers

Abstract

The present invention also relates to compositions comprising GH43 arabinofuranylase, GH51 arabinofuranylase, GH5 xylanase, GH3 β-xylanase, GH30 xylanase and optionally CE3 acetylxylan esterase, and the use of these compositions for dissolving hemicellulose fibers and increasing the release of monomeric arabinose and / or xylose.

Description

[0001] References to sequence lists This application contains a sequence list in computer-readable form, which is incorporated herein by reference. Background of the Invention Technical Field

[0002] This invention relates to compositions comprising GH43 arabinofuranylase, GH51 arabinofuranylase, GH5 xylanase, GH3 β-xylanase and GH30 xylanase, and optionally carbohydrate esterase family 3 (CE3) acetylxylan esterase, and the use of the compositions for dissolving hemicellulose fibers. Background Technology

[0003] The conversion of cellulosic feedstocks to biofuels is challenging due to their high resistance to degradation, typically involving a combination of thermochemical pretreatment and subsequent addition of cellulase and hemicellulase to release soluble carbohydrates. Driven by government sustainability initiatives, the biofuel industry is utilizing existing corn ethanol facilities to produce ethanol from corn fiber. Corn fiber comprises 10% of the corn kernel weight and consists of cellulose and hemicellulose from the aleurone layer and pericarp layer. In ethanol facilities, corn fiber ultimately becomes dry distillers' grains (DDGS) containing solubles. The hemicellulose portion of corn fiber is enzymatically hydrolyzed into monomeric C5 sugars (such as xylose and arabinose), which are then fermented into ethanol by C5 fermentation yeasts. This, combined with existing infrastructure, allows ethanol plants to produce more cellulosic ethanol from the same amount of corn. Additional benefits of corn fiber degradation include: higher protein content in DDGS, making it suitable for animal feed; and the lower fiber content of DDGS potentially qualifying it for use in monogastric and aquaculture animal feed markets.

[0004] The arabinoyl xylan backbone in corn fiber consists of a xylan backbone of β-(1,4)-linked D-xylopyranosyl residues, which is highly substituted with arabinose side chains and to a lesser extent substituted with glucuronic acid residues. The predominantly substituted arabinose residues are linked to the O-2 or O-3 positions of monosubstituted xylopyranosyl units or to both O-2 and O-3 positions of disubstituted xylopyranosyl units. In addition to arabinose, the xylan backbone can also be substituted with D-galactopyranosyl and D-glucuronic acid residues, and / or with acetyl groups. Acetic acid is directly esterified to the xylan backbone at positions O-2 or O-3, while hydroxycinnamic acids (such as ferulic acid, p-coumaric acid, and dehydrogenated dimers of ferulic acid) are esterified to arabinofuranosyl units at position O-5. Furthermore, it has been reported that xylan is further substituted with xylanosyl groups via (1-3)-bonds, and the arabinofuranosyl groups can be further modified with xylanosyl groups or even L-galactopyranosyl groups. Due to the highly branched substitution of different parts, the enzymatic degradation of maize cellulosic arabinosyl xylan into monomeric C5 sugars requires the synergistic action of a mixture of debranching and depolymerization activities. The debranching activities mainly include α-L-arabinofuranosidase (EC 3.2.1.55) (α-AraF), ferulic acid esterase (EC 3.1.1.73), α-glucuronidase (EC 3.2.1.139), and / or acetylxylan esterase (EC 3.1.1.72), while depolymerization depends on the activities of endo-1,4-β-xylanase (EC 3.2.1.8) and β-xylosidase (EC 3.2.1.37) (BX).

[0005] WO 2006 / 114095 “D1” describes a method and composition for hydrolyzing arabinosylxylan, comprising contacting a substrate containing arabinosylxylan with an enzyme active with disubstituted arabinose (e.g., glycoside hydrolase family 43 (GH43) α-L-arabinofuranosidase) and an enzyme active with monosubstituted arabinose at the C2 or C3 position (e.g., GH family 51, 54 or 62 α-L-arabinofuranosidase). D1 teaches that when these two arabinofuranosidases are added to a solution of arabinosylxylan, the resulting products will be high molecular weight linear xylose polymers and arabinose molecules, which allow the linear xylose polymers to be easily separated from arabinose by known techniques. These linear xylose polymers can be further partially digested using enzymatic activities such as β-xylosidase (preferably GH3) and / or endo-1,4-β-xylanase (preferably GH10 or GH11) to produce xylooligosaccharides. D1 further teaches that when both endo-1,4-β-xylanase and β-xylosidase are added to purified linear xylose polymers, the resulting product will be xylose that is essentially free of arabinose substituents, and for the degradation of even more complex substrates, or where more complete degradation is required, the presence of even further enzymatic activities, such as acetylatedxylan esterase (EC 3.1.1.72) and / or ferulic acid esterase (EC 3.1.1.73) and / or α-glucuronidase (EC 3.2.1.139), may be expected.

[0006] However, supply chain disruptions and inflation have increased the cost of raw material inputs for the enzymes required to fully hydrolyze complex arabinoyl xylan substrates, thereby diminishing the economic incentive for ethanol facilities to purchase additional enzymes to produce cellulosic ethanol from corn. Since conventional wisdom holds that achieving the highest cellulosic ethanol yield from corn requires the activity of all seven enzymes, there is a need for improved methods and compositions that can increase cellulosic ethanol yield by releasing more monomeric arabinose and xylose, while requiring less enzyme activity and at a lower cost, making them more profitable for corn ethanol facilities to maximize the cellulosic ethanol yield from their existing corn inputs. Summary of the Invention

[0007] This invention provides a solution to the aforementioned problems by offering compositions comprising GH43 arabinofuranase, GH51 arabinofuranase, GH5 xylanase, GH3 β-xylosidase, and GH30 xylanase. Compared to similar compositions containing only GH5 or GH30 xylanase, these compositions unexpectedly increase hemicellulose dissolution and release significantly more monomeric arabinose and xylose due to the combined presence of GH5 and GH30 xylanase. Surprisingly and unexpectedly, the compositions of this invention significantly increase the yield of monomeric arabinose and xylose without the need for acetylxylan esterase, ferulic acid esterase, and / or α-glucuronidase; however, the addition of CE3 acetylxylan esterase and / or α-xylosidase (e.g., GH31 α-xylosidase) to the compositions further increases the yield of monomeric arabinose and / or xylose. Enzyme compositions can be formulated in solid form (e.g., particles containing enzymes) or in liquid form (e.g., liquid compositions containing enzymes and one or more formulations).

[0008] Therefore, one aspect of the present invention relates to a particle comprising: (a) a core comprising GH43 arabinofuranylase, GH51 arabinofuranylase, GH5 xylanase, GH3 β-xylanase, GH30 xylanase and optionally carbohydrate esterase family 3 (CE3) acetylxylan esterase, and optionally (b) a coating consisting of one or more layers surrounding the core.

[0009] In one aspect, the present invention relates to a particle comprising: (a) a core, and (b) a coating consisting of one or more layers surrounding the core, wherein the coating comprises GH43 arabinofuranylase, GH51 arabinofuranylase, GH5 xylanase, GH3 β-xylosidase, GH30 xylanase and optionally CE3 acetylatedxylan esterase.

[0010] In one aspect, the present invention relates to a composition comprising the particles.

[0011] In one aspect, the present invention relates to a liquid composition comprising GH43 arabinofuranylase, GH51 arabinofuranylase, GH5 xylanase, GH3 β-xylanase, GH30 xylanase and optionally Ce3 acetylatedxylan esterase, and an enzyme stabilizer, such as a polyol like propylene glycol or glycerol, a sugar or sugar alcohol, or lactic acid.

[0012] In some embodiments, the liquid composition further comprises a filler or carrier material. In some embodiments, the liquid composition further comprises a preservative.

[0013] In one aspect, the present invention relates to a composition comprising GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_21 xylanase, GH3 β-xylosidase, GH30 xylanase and optionally CE3 acetylxylan esterase.

[0014] One aspect of the present invention relates to a method for producing a fermentation product from a starch-containing material, the method comprising the steps of: (a) saccharifying the starch-containing material with glucosylamylase and α-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 GH43 arabinofuranylase, GH51 arabinofuranylase, GH5 xylanase, GH3 β-xylanase, GH30 xylanase and optionally CE3 acetylxylan esterase is present or added during the saccharification step (a) and / or the fermentation step (b).

[0015] In one aspect, the present invention relates to a method for producing a fermentation product from a starch-containing material, the method comprising the steps of: (a) liquefying the starch-containing material with a thermostable α-amylase at a temperature above the initial gelatinization temperature of the starch to produce dextrin; (b) saccharifying the dextrin with a glucosylase to produce a fermentable sugar; and (c) fermenting the sugar with a fermenting organism to produce the fermentation product; wherein a composition comprising GH43 arabinofuranylase, GH51 arabinofuranylase, GH5 xylanase, GH3 β-xylanase, GH30 xylanase and optionally CE3 acetylxylan esterase is present or added during the saccharification step (b) and / or the fermentation step (c).

[0016] GH5 xylanase can be GH5_21 xylanase.

[0017] GH5 xylanase can be GH5_35 xylanase.

[0018] GH30 xylanase can be GH30_7 xylanase.

[0019] GH30 xylanase can be GH30_8 xylanase.

[0020] In some embodiments, the particle core contains CE3 acetylxylan esterase.

[0021] In some embodiments, the particle coating contains CE3 acetylxylan esterase.

[0022] In some embodiments, the composition (e.g., a liquid composition) contains CE3 acetylxylan esterase.

[0023] In some embodiments, the particle core contains α-xylosidase (e.g., GH31 α-xylosidase).

[0024] In some embodiments, the particle coating contains α-xylosidase (e.g., GH31 α-xylosidase).

[0025] In some embodiments, the composition (e.g., a liquid composition) contains α-xylosidase (e.g., GH31 α-xylosidase). Attached Figure Description

[0026] The accompanying figure is a comparison of exemplary CE3 peptides of the present invention, showing that they share conserved active sites of serine, histidine, and aspartic acid residues that form the characteristic catalytic triplet of the SGNH hydrolase family, share conserved classical GxSxT pentapeptide common sequences, and share Gly residues in region II and Asn residues in region III that constitute the oxygen anion pore.

[0027] definition Based on this detailed description, the following definitions apply. Note that the singular forms “a / an” and “the” include plural indicators unless the context explicitly indicates otherwise.

[0028] Unless otherwise defined or explicitly indicated by the 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 pertains.

[0029] Acetylxylan esterase: The term "acetylxylan esterase" refers to acetylxylan esterase (EC 3.1.1.72), which catalyzes the hydrolysis of acetyl groups from polyxylan, acetylated xylose, acetylated glucose, α-naphthyl acetate, and p-nitrobenzene acetate, but does not catalyze the hydrolysis of acetyl groups from triglycerides.

[0030] Acetylxylan esterase activity: One unit of acetylxylan esterase activity is defined as the amount of enzyme required to release 1 μmol of p-nitrophenol per minute from 4-nitrophenyl acetate in 100 mM sodium citrate buffer (pH 5) at 40 °C. 100 mM pNP-acetate was dissolved in DMSO as a substrate stock solution. The stock solution was diluted 50-fold in 100 mM sodium citrate to prepare a 2 mM pNP-acetate substrate solution. 175 μl of the substrate solution and 25 μl of diluted enzyme were mixed in a 96-well plate and incubated at 37 °C. The release of p-nitrophenol was monitored at 410 nm using a spectrophotometer.

[0031] α-L-Arabofuranosaccharidase: "α-L-Arabofuranosaccharidase" refers to α-L-arabinofuranoside arabinofuranoside hydrolase (EC 3.2.1.55), which catalyzes the hydrolysis of terminal non-reducing α-L-arabinofuranoside residues in α-L-arabinoside. This enzyme acts on α-L-arabinofuranoside, α-L-arabinanan containing (1,3)- and / or (1,5)- bonds, arabinosylxylan, and arabinogalactan. α-L-arabinofuranosaccharidase is also known as arabinofuranosaccharidase, α-arabinofuranosaccharidase, α-L-arabinofuranosaccharidase, α-arabinofuranosaccharidase, polysaccharide α-L-arabinofuranosaccharidase, α-L-arabinofuranoside hydrolase, L-arabinofuranosaccharidase, or α-L-arabinananase.

[0032] α-L-arabinofuranosaccharide activity: For the purposes of this invention, α-L-arabinofuranosaccharide activity was determined by analyzing arabinose in 200 μL of medium-viscosity wheat arabinosylxylan (Megazyme International Ireland, Ltd., Bray, Co., Wicklow, Ireland) at 40°C for 30 minutes per ml of 100 mM sodium acetate (pH 5) at 40°C for 30 minutes. The arabinose was then analyzed by AMINEX® HPX-87H column chromatography (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

[0033] α-Xylosidase: "α-Xylosidase" refers to α-D-xyloside xylose hydrolase (EC 3.2.1.177), which catalyzes the hydrolysis of unsubstituted xyloside at the terminal end of the reducing end of xylooligosaccharide glucose.

[0034] α-Xylosidase activity: For the purposes of this invention, one unit of α-xylosidase is defined as the production of 1.0 μmol of p-nitrophenol anion per minute from 1 mM p-nitrophenyl-α-D-xyloside as a substrate in 100 mM sodium citrate containing 0.01% TWEEN(R) 20 at 40 °C, pH 5, and a total volume of 200 μL.

[0035] β-xylosidase: "β-xylosidase" refers to β-D-xyloside xylose hydrolase (EC3.2.1.37), which catalyzes the exolytic hydrolysis of short β(1-4)-xylooligosaccharides to remove continuous D-xylose residues from the non-reducing ends.

[0036] β-xylosidase activity: For the purposes of this invention, one unit of β-xylosidase is defined as the production of 1.0 μmol of p-nitrophenol anion per minute from 1 mM p-nitrophenyl-β-D-xyloside as a substrate in 100 mM sodium citrate containing 0.01% TWEEN(R) 20 at 40 °C and pH 5.

[0037] Carbohydrate esterase family 3 (CE3): Carbohydrate esterase family 3 is abbreviated as "CE3" herein. The CE3 polypeptide of this invention has acetylxylan esterase activity (EC 3.1.1.72). The CE3 family includes several structurally resolved enzymes, including those from *Cladosporium fibronectin* (…). Talaromyces cellulolyticus TcAE206 and Clostridium thermophilum ( ) Hungateiclostridium thermocellum The CtCes3-1 structure exhibits the characteristic (α / β / α) sandwich fold of the SGNH hydrolase family. The (α / β / α)-sandwich has five centrally parallel β-chains forming a curved β-sheet with 5-6 α-helices on either side. Both structures also possess the calcium-binding ring motif (DXVGX7DX). n (D / N)), located above the N-terminus of the central β-chain. This binding motif is conserved in the previously characterized CE3.

[0038] Carbohydrate esterase family 3 (CE3) acetylxylan esterases possess the classic Ser-His-Asp catalytic triad, a characteristic feature of the SGNH hydrolase family. The active site residues are established by four conserved common sequences (regions I-III and V) and contain a modified nucleophilic “elbow” turn motif (-GxSxT- instead of the typical -GxSxG- motif). The catalytic triad, along with the Gly residues in region II and the Asn residues in region III that constitute the oxyanion pore, are conserved in all characterized CE3 enzymes. The Asp residues in region V promote the amphiphilic nature of the His residues in region V, which extract a proton from the Ser residue in region I to make it nucleophilic.

[0039] Table 1 below shows the location of the above features in each exemplary CE3 acetylxylan esterase, and the accompanying drawings show a comparison of the conservation of features in the mature sequence of the exemplary CE3 acetylxylan esterase of the present invention.

[0040] Table 1 cDNA: The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from mature, spliced ​​mRNA molecules obtained from eukaryotic or prokaryotic cells. cDNA lacks intron sequences that can be present in the corresponding genomic DNA. The initial primary RNA transcript is the precursor of mRNA, which is processed through a series of steps (including splicing) to become mature, spliced ​​mRNA.

[0041] Coding sequence: The term "coding sequence" refers to a polynucleotide that directly specifies the amino acid sequence of a polypeptide. The boundaries of a coding sequence are typically defined by an open reading frame (ORF), which begins with a start codon (such as ATG, GTG, or TTG) and ends with a stop codon (such as TAA, TAG, or TGA). Coding sequences can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0042] Control Sequences: The term "control sequence" refers to a nucleic acid sequence involved in regulating the expression of polynucleotides in a particular organism, either in vivo or in vitro. Each control sequence can be native (i.e., from the same gene) or heterologous (i.e., from different genes) to the polynucleotide encoding a polypeptide, and is native or heterologous relative to each other. Such control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptides, propeptides, signal peptides, promoters, terminators, enhancers, and transcription or translation initiator and terminator sequences. At a minimum, control sequences include promoters and transcription and translation termination signals. These control sequences may be provided with multiple linkers for the purpose of introducing specific restriction sites that facilitate the linking of control sequences to the coding regions of polynucleotides encoding polypeptides.

[0043] Expression: The term “expression” refers to any step involved in polypeptide production, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0044] Expression vector: An expression vector is a linear or circular DNA construct containing a DNA sequence encoding a polypeptide, with the coding sequence operatively linked to a suitable control sequence that can influence the expression of the DNA in a suitable host. Such control sequences may include promoters that affect transcription, optional operon sequences that control transcription, sequences encoding suitable ribosome binding sites on mRNA, enhancers, and sequences that control the termination of transcription and translation.

[0045] Extension: The term "extension" refers to the addition of one or more amino acids to the amino and / or carboxyl terminus of a polypeptide, wherein the "extended" polypeptide has acetylxylan esterase activity.

[0046] Fermentation products: "Fermentation products" refers to the products produced through a fermentation process involving the use of fermenting organisms. Fermentation products include alcohols (e.g., ethanol, methanol, butanol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, succinic acid, gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H2 and CO2); antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B vitamins). 12 (e.g., β-carotene); and hormones. In preferred embodiments, the fermentation product is ethanol, such as fuel ethanol; drinking ethanol, i.e., neutral alcoholic beverages; or industrial ethanol or products used in the consumer alcohol industry (e.g., beer and wine), the dairy industry (e.g., fermented dairy products), the leather industry, and the tobacco industry. Preferred beer types include ale, stout, porter, lager, bitters, malt liquor, happyshu, high-alcohol beer, low-alcohol beer, low-calorie beer, or light beer. In an embodiment, the fermentation product is ethanol.

[0047] Fermentation organisms: "Fermentation organisms" refers to any organism suitable for use in the fermentation process and capable of producing the desired fermentation product, including bacteria and fungi, especially yeast.

[0048] Fusion polypeptide: The term "fusion polypeptide" is a polypeptide in which one of the polypeptides of the present invention is fused to the N-terminus and / or C-terminus. Fusion polypeptides are generated by fusing a polynucleotide encoding another polypeptide with a polynucleotide of the present invention, or by fusing two or more polynucleotides of the present invention together. Techniques for generating fusion polypeptides are known in the art and include linking the coding sequences of the polypeptides such that they conform to reading frames, and the expression of the fusion polypeptide is under the control of one or more identical promoters and terminators. Fusion polypeptides can also be constructed using intron technology, wherein the fusion polypeptide is generated post-translational (Cooper et al., 1993, ). EMBO J. [Journal of the European Society for Molecular Biology] 12:2575-2583; Dawson et al., 1994, Science [Science] 266: 776-779. The fusion peptide may further include a cleavage site between the two peptides. This site is cleaved upon secretion of the fusion protein, thereby releasing both peptides. Examples of cleavage sites include, but are not limited to, those disclosed in the following literature: Martin et al., 2003. J. Ind. Microbiol. Biotechnol. [Journal of Industrial Microbiology and Biotechnology] 3: 568-576; Svetina et al., 2000, J. Biotechnol [Journal of Biotechnology] 76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. [Applied and Environmental Microbiology] 63: 3488-3493; Ward et al., 1995, Biotechnology [Biotechnology] 13: 498-503; and Contreras et al., 1991, Biotechnology [Biotechnology] 9: 378-381; Eaton et al., 1986, Biochemistry [Biochemistry] 25: 505-512; Collins-Racie et al., 1995, Biotechnology [Biotechnology] 13: 982-987; Carter et al., 1989, Proteins : Structure, Function, and Genetics [Proteins: Structure, Function, and Genetics] 6:240-248; and Stevens, 2003, Drug Discovery World [Drug Discovery World] 4: 35-48.

[0049] GH3 β-xylosidase: "GH3 β-xylosidase" is an abbreviation for 3 β-xylosidase, a family of glycoside hydrolases. It is a xylan 1,4-β-xylosidase (EC 3.2.1.37) that catalyzes the hydrolysis of (1→4)-β-D-xylan to remove consecutive D-xylose residues from the non-reducing end.

[0050] GH5 xylanase: "GH5 xylanase" is an abbreviation for the 5-xylanase family of glycoside hydrolases. It is mainly composed of endo-1,4-β-xylanase (EC 3.2.1.8), which catalyzes the endo-hydrolysis of the (1→4)-β-D-xyloside bond in xylan.

[0051] GH5_21 xylanase: "GH5_21 xylanase" is an abbreviation for 21-endo-β-1,4-xylanase, a subfamily 5 of the glycoside hydrolase family. It has a three-dimensional structure characterized by (β / α) 8 barrels and uses glutamine residues as catalytic nucleophiles / bases.

[0052] GH5_35 xylanase: "GH5_35 xylanase" is an abbreviation for 35-endo-β-1,4-xylanase, a subfamily 5 of the glycoside hydrolase family. It has a three-dimensional structure characterized by (β / α) 8 barrels and uses glutamine residues as catalytic nucleophiles / bases.

[0053] GH8 xylanase: "GH8 xylanase" is an abbreviation for the 8-xylanase family of glycoside hydrolases, which consists of the following: endo-1,4-β-xylanase (EC 3.2.1.8), which catalyzes the endo-hydrolysis of the (1→4)-β-D-xyloside bond in xylan.

[0054] GH10 xylanase: "GH10 xylanase" is an abbreviation for the 10-xylanase family of glycoside hydrolases, which consists of the following: endo-1,3-β-xylanase (EC 3.2.1.32), which catalyzes the random endo-hydrolysis of the (1→3)-β-D-glycosidic bond in (1→3)-β-D-xylan; and endo-1,4-β-xylanase (EC 3.2.1.8), which catalyzes the endo-hydrolysis of the (1→4)-β-D-xylosidic bond in xylan.

[0055] GH11 xylanase: "GH11 xylanase" is an abbreviation for 11 xylanase, a member of the glycoside hydrolase family. It is an endo-β-1,4-xylanase (EC 3.2.1.8) that catalyzes the endo-hydrolysis of the (1→4)-β-D-xyloside bond in xylan.

[0056] GH31 α-xylosidase: "GH31 arabinofuranosidase" is an abbreviation for the 31 α-xylosidase family of glycoside hydrolases. It is an α-D-xyloside xylose hydrolase (EC 3.2.1.177) that catalyzes the hydrolysis of the terminal unsubstituted xyloside at the reducing end of the xylooligosaccharide glucose. Exemplary GH31 family α-xylosidases utilize a two-step double-displacement mechanism employing a covalent glycosyl-enzyme intermediate and produce products with an anomeric conformation.

[0057] GH30 xylanase: "GH30 xylanase" is an abbreviation for the 30-xylanase family of glycoside hydrolases. The GH30 family is defined in the CAZy database (Henrissat, 1991) and has been reviewed in the literature (Puchart et al., 2021). In this disclosure, "GH30 xylanase" is intended to include only GH30_1 xylanase, GH30_2 xylanase, GH30_3 xylanase, GH30_4 xylanase, GH30_5 xylanase, GH30_7 xylanase, and GH30_8 xylanase. For the avoidance of ambiguity, GH30_6 xylanase is excluded from the definition of GH30 xylanase according to this disclosure.

[0058] GH30_1 xylanase: "GH30_1 xylanase" is an abbreviation for glycoside hydrolase subfamily 30 xylanase, which in the context of this disclosure includes only the following: glucosylceramidinase (EC 3.2.145), which catalyzes the reaction D-glucosyl-N-acylsphingosine + H2O = D-glucose + ceramide; and xylan 1,4-β-xylosidase (EC 3.2.1.37), which catalyzes the hydrolysis of (1→4)-β-D-xylan to remove consecutive D-xylose residues from the non-reducing end. For the avoidance of doubt, in the context of this disclosure, "GH30_1 xylanase" does not include glucosidase (EC 3.2.1.-) and (EC 3.2.1.21).

[0059] GH30_2 xylanase: "GH30_2 xylanase" is an abbreviation for glycoside hydrolase subfamily 30 xylanase, which includes xylan 1,4-β-xylosidase (EC 3.2.1.37), which catalyzes the hydrolysis of (1→4)-β-D-xylan to remove consecutive D-xylose residues from the non-reducing end.

[0060] GH30_3 xylanase: "GH30_3 xylanase" is an abbreviation for glycosidase subfamily 30 xylanase, which refers to endoglucanase-1,6-β-glucosidase (EC 3.2.1.75), catalyzing the random hydrolysis of the (1→6)-bond in (1→6)-β-D-glucan. For the avoidance of ambiguity, in the context of this disclosure, "GH30_3 xylanase" does not include glucosidase (EC 3.2.1.-).

[0061] GH30_4 xylanase: "GH30_4 xylanase" is an abbreviation for 4-xylanase of the 30 subfamily of glycoside hydrolases. It refers to β-D-fucosidase (EC 3.2.1.38), which catalyzes the hydrolysis of the terminal non-reducing β-D-fucose residue in β-D-fucoside.

[0062] GH30_5 xylanase: "GH30_5 xylanase" is an abbreviation for 5-xylanases of the 30 subfamily of glycoside hydrolases, including the following: endo-β-1,6-galactanase (EC 3.2.1.164), which catalyzes the endo-hydrolysis of the (1→6)-β-D-galactoside bond in arabinogalactan protein and (1→3):(1→6)-β-galactan, producing galactose and (1→6)-β-galactobiose as the final products; and alactan exo-1,6-β-galactobiohydrolase (EC... 3.2.1.213), which catalyzes the hydrolysis of the (1→6)-β-D-galactoside bond in arabinogalactan protein and (1→3):(1→6)-β-galactan, producing (1→6)-β-galactobiose as the final product.

[0063] GH30_7 xylanase: "GH30_7 xylanase" is an abbreviation for 7 xylanases of the 30 subfamily of glycoside hydrolases, which includes the following: endo-β-1,4-xylanase (EC 3.2.1.8), which catalyzes the endo-hydrolysis of the (1→4)-β-D-xyloside bond in xylan; and oligosaccharide reducing end xylanase (EC 3.2.1.156), which catalyzes the hydrolysis of (1→4)-β-D-xylose residues from the reducing end of oligosaccharides.

[0064] GH30_8 xylanase: "GH30_8 xylanase" is an abbreviation for xylanases of the 30 subfamily of glycoside hydrolases, including the following: endo-β-1,4-xylanase (EC 3.2.1.8), which catalyzes the endo-hydrolysis of the (1→4)-β-D-xylosidic bond in xylan; and glucuronide-arabinoxylan-specific endo-β-1,4-xylanase (EC 3.2.1.136), which catalyzes the endo-hydrolysis of the (1→4)-β-D-xylosyl bond in some glucuronide-arabinoxylans.

[0065] GH30_9 xylanase: "GH30_9 xylanase" is an abbreviation for xylanase 9 of the glycoside hydrolase subfamily 30. It refers to baicalin-β-D-glucuronidase (EC 3.2.1.167), which catalyzes the reaction of baicalin + H2O = baicalin + D-glucuronic acid.

[0066] GH30_10 xylanase: "GH30_10 xylanase" is an abbreviation for 10 xylanase of the 30 subfamily of glycoside hydrolases. It refers to non-reducing end specific xylobiose hydrolases (EC 3.2.1.-) that catalyze the hydrolysis of xylan or xylooligosaccharides to produce xylobiose.

[0067] GH43 arabinofuranosidase: "GH43 arabinofuranosidase" is an abbreviation for 43 arabinofuranosidase, a member of the glycoside hydrolase family. It is an α-L-arabinofuranosidase (EC 3.2.1.55) that catalyzes the hydrolysis of the terminal non-reducing α-L-arabinofuranoside residue in α-L-arabinoside.

[0068] GH51 arabinofuranosidase: "GH51 arabinofuranosidase" is an abbreviation for 51 arabinofuranosidase, a member of the glycoside hydrolase family. It is an α-L-arabinofuranosidase (EC 3.2.1.55) that catalyzes the hydrolysis of the terminal non-reducing α-L-arabinofuranoside residue in α-L-arabinoside.

[0069] Initial gelatinization temperature: "Initial gelatinization temperature" refers to the lowest temperature at which starch gelatinization begins. Starch heated in water begins to gelatinize between 50°C and 75°C; the exact gelatinization temperature depends on the specific starch and can be readily determined by a technician. Therefore, the initial gelatinization temperature can vary depending on the plant species, the specific variety of the plant species, and the growing conditions. In the context of this disclosure, the initial gelatinization temperature of a given starchy grain is the temperature at which 5% of the starch granules lose birefringence, as described by Gorinstein, S. and Lii, C., Starch / Starke [Starch], Vol. 44 (12), pp. 461-466 (1992).

[0070] Heterogeneous: For host cells, the term "heterogeneous" means that the polypeptide or nucleic acid is not naturally present in the host cell. For polypeptides or nucleic acids, the term "heterogeneous" means that the control sequence (e.g., promoter) of the polypeptide or nucleic acid is not naturally associated with that polypeptide or nucleic acid; that is, the control sequence comes from a gene other than the gene encoding the mature polypeptide.

[0071] Host strain or host cell: A “host strain” or “host cell” refers to an organism in which an expression vector, bacteriophage, virus, or other DNA construct (including a polynucleotide encoding a target polypeptide, such as an amylase) has been introduced. An exemplary host strain is a microbial cell (e.g., bacteria, filamentous fungi, and yeast) capable of expressing a target polypeptide and / or fermenting sugars. The term “host cell” includes protoplasts produced by cells.

[0072] Introduction: In the context of inserting a nucleic acid sequence into a cell, the term “introduction” means “transfection,” “conversion,” or “transduction,” as is known in the art.

[0073] Isolated: The term "isolated" means a polypeptide, nucleic acid, cell, or other specific 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.). Therefore, the isolated polypeptide, nucleic acid, cell, or other material exists in a form not found in nature. Isolated polypeptides include, but are not limited to, culture media containing secreted polypeptides expressed in host cells.

[0074] Mature peptide: The term “mature peptide” refers to a peptide that has been processed at its N-terminus and / or C-terminus (e.g., removal of the signal peptide) to be in its mature form.

[0075] Native: The term "native" refers to nucleic acids or polypeptides that are naturally present in the host cell.

[0076] Nucleic acid: The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding polypeptides. Nucleic acids can be single-stranded or double-stranded and can be chemically modified. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon can be used to encode a specific amino acid, and the compositions and methods of the present invention cover nucleotide sequences encoding specific amino acid sequences. Unless otherwise stated, nucleic acid sequences are presented in a 5' to 3' orientation.

[0077] Purified: The term "purified" means nucleic acids, peptides, or cells that are substantially free of other components, as determined by analytical techniques well known in the art (e.g., in electrophoretic gels, chromatographic eluates, and / or media subjected to density gradient centrifugation, where purified peptides or nucleic acids form discrete bands). Purified nucleic acids or peptides are at least about 50% pure, and typically 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., weight percentage or molar percentage). In a relevant sense, a composition is enriched with the molecule when the concentration of the molecule increases significantly after the application of purification or enrichment techniques. The term “enrichment” refers to the presence of compounds, peptides, cells, nucleic acids, amino acids, or other specified materials or components in a composition at a relative or absolute concentration higher than that of the starting composition.

[0078] In one respect, as used herein, the term "purified" means that the polypeptide or cell is substantially free of components (especially insoluble components) from the producing organism. In another respect, the term "purified" means that the polypeptide is substantially free of insoluble components (especially insoluble components) from the protist from which it was obtained. In one respect, the polypeptide is separated from the soluble components of the organism from which it was recovered and the culture medium. The polypeptide can be purified (i.e., separated) by one or more of unit operation filtration, precipitation, or chromatography.

[0079] Therefore, peptides can be purified such that only small amounts of other proteins, particularly other peptides, are present. As used herein, the term "purified" can refer to the removal of other components, particularly other proteins and most particularly other enzymes, present in the cells from which the peptide originates. A peptide can be "substantially pure," meaning it is free from other components from the organism that produced it (e.g., the host organism used to recombinantly produce the peptide). In one aspect, the peptide is at least 40% pure by weight of the total peptide material present in the formulation. In another aspect, the peptide is at least 50%, 60%, 70%, 80%, or 90% pure by weight of the total peptide material present in the formulation. As used herein, "substantially pure peptide" can mean a peptide formulation containing, by weight, 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% of other peptide material associated with the peptide, either natively or recombinantly.

[0080] Therefore, it is preferred that the substantially pure polypeptide, based on the weight of the total polypeptide material present in the formulation, 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, and most preferably at least 99.5% pure. The polypeptides of the present invention are preferably in a substantially pure form (i.e., the formulation is substantially free of other polypeptide materials associated with it, either natively or recombinantly). This can be achieved, for example, by preparing the polypeptide using well-known recombinant methods or classical purification methods.

[0081] Recombination: The term "recombination," used in its conventional sense, refers to the manipulation (e.g., cutting and rejoining) of nucleic acid sequences to form a sequence group different from that found in nature. The term recombination refers to cells, nucleic acids, peptides, or vectors that have been modified from their native state. Thus, for example, recombinant cells express genes not found in the native (non-recombinant) form of the cell, or express native genes at different levels or under different conditions compared to those found in nature. The term "recombination" is synonymous with "genetically modified" and "transgenic."

[0082] Recovery: The term "recovery" refers to the removal of peptides from at least one fermentation broth component selected from a list of cells, nucleic acids, or other specified materials, for example, by methods such as: harvesting peptides by peptide crystallization, by filtration (e.g., deep filtration (using filter aids or packed filter media, cloth filtration in a box filter, rotary drum filtration, drum filtration, rotary vacuum drum filtration, candle filter, horizontal leaf filter, or the like, using sheet or pad filtration in a frame or modular device) or membrane filtration (using plate filtration, modular filtration, candle filtration, microfiltration, crossflow, dynamic crossflow, or ultrafiltration in dead-end operation)), or by centrifugation (using a sedimentation centrifuge, disc stack centrifuge, hyrdo cyclone, or the like), or by precipitation of peptides and using relevant solid-liquid separation methods to harvest peptides from broth media by particle size fractionation. Recovery encompasses the isolation and / or purification of peptides.

[0083] Sequence identity: The degree of association between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence identity". The Needleman-Wunsch algorithm (Needleman and Wunsch, 1970) is used. J. Mol. Biol. [Journal of Molecular Biology] 48: 443-453) determines the sequence identity between two amino acid sequences as the output of "longest identity," an algorithm such as the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, 48: 443-453). Trends Genet. [Trends in Genetics] 16: 276-277) is implemented in version 6.6.0 of the Niedel program. The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (the EMBOSS version of BLOSUM62) substitution matrix. For the Niedel program to report the longest identity, the non-brief (-nobrief) option must be specified on the command line. The Niedel-marked "longest identity" output is calculated as follows: (Identical residues × 100) / (Alignment length - Total number of vacancies in the alignment) The Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, ibid.) is used to determine the sequence identity between two polynucleotide sequences as the output of "longest identity." This algorithm is implemented in the Niedel program of EMBOSS software package version 6.6.0 (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, ibid.). The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. For the Niedel program to report the longest identity, the non-simplified option must be specified on the command line. The Niedel-marked "longest identity" output is calculated as follows: (Identical deoxyribonucleotides × 100) / (Alignment length – Total number of vacancies in the alignment) Signal peptide: A signal peptide is an amino acid sequence that attaches to the N-terminal portion of a protein and promotes its secretion outside the cell. The mature form of extracellular proteins lacks a signal peptide, which is cleaved during the secretion process.

[0084] Thermostable: "Thermostable" means that the enzyme does not denature or become inactive when used in the liquefaction step of the method of the present invention. In other words, if a thermostable enzyme has a denaturation temperature (Td) compatible with the liquefaction temperature and maintains its activity at this temperature, then the thermostable enzyme is suitable for liquefaction.

[0085] Distillers' grains water: "Distillers' grains water" refers to the centrifuged filtrate separated from whole distillers' grains, which is pumped to an evaporator to concentrate into a slurry.

[0086] Whole lees: "Whole lees" includes the material retained at the end of the distillation process after the recovery of fermentation products (such as ethanol).

[0087] Xylanase: "Xylanase" encompasses any of the following definitions of xylanase: endo-1,4-β-xylanase (EC 3.2.1.8), which catalyzes the endo-hydrolysis of (1→4)-β-D-xylosidic bonds in xylan; glucuronide-arabinoxylan endo-1,4-β-xylanase (EC 3.2.1.136), which catalyzes the endo-hydrolysis of 1,4-β-D-xylosyl bonds in some glucuronide-arabinoxylans; glucoceramitinase (EC 3.2.145), which catalyzes the reaction D-glucosyl-N-acylsphingosine + H₂O = D-glucose + ceramide; xylan 1,4-β-xylosidase (EC 3.2.1.136 ... 3.2.1.37), which catalyzes the hydrolysis of (1→4)-β-D-xylan to remove consecutive D-xylose residues from the non-reducing ends; endo-1,6-β-glucanase (EC 3.2.1.75), which catalyzes the random hydrolysis of the (1→6)-bond in (1→6)-β-D-glucan; β-D-fucosidase (EC 3.2.1.38), which catalyzes the hydrolysis of the terminal non-reducing β-D-fucoside in β-D-fucoside; endo-β-1,6-galactanase (EC 3.2.1.164), which catalyzes the endo-hydrolysis of the (1→6)-β-D-galactoside bond in arabinogalactan protein and (1→3):(1→6)-β-galactan, producing galactose and (1→6)-β-galactobiose as the final products; galactan exo-1,6-β-galactobiose hydrolase (EC 3.2.1.213), which catalyzes the hydrolysis of the (1→6)-β-D-galactoside bond in arabinogalactan protein and (1→3):(1→6)-β-galactan, producing (1→6)-β-galactobiose as the final product; oligosaccharide reducing xylanase (EC 3.2.1.156), which catalyzes the hydrolysis of (1→4)-β-D-xylose residues from the reducing end of oligosaccharides; baicalin-β-D-glucuronidase (EC 3.2.1.156), which catalyzes the hydrolysis of (1→4)-β-D-xylose residues from the reducing end of oligosaccharides; baicalin-β-D-glucuronidase (EC 3.2.1.164), which catalyzes the endo-hydrolysis of the (1→6)-β-D-galactoside bond in arabinogalactan protein and (1→3):(1→6)-β-galactan, producing (1→6)-β-galactobiose as the final product; 3.2.1.167), which catalyzes the reaction of baicalin + H2O = baicalein + D-glucuronic acid; and non-reducing end-specific xylobiose hydrolase (EC 3.2.1.-), which catalyzes the hydrolysis of xylan or xylooligosaccharides to produce xylobiose.

[0088] Xylanase activity: EC 3.2.1.8 Xylanase activity can be determined using birch xylan as a substrate. One unit of xylanase is defined as the production of 1.0 μmol of reducing sugar per liter per minute (measured in glucose equivalents as described in Lever, 1972, A new reaction for colorimetric determination of carbohydrates, Anal. Biochem 47: 273-279) from 2 g of birch xylan as a substrate during the initial hydrolysis phase at 50°C and pH 5. EC 3.2.1.136 Xylanase activity can be determined at 37°C using 0.2% AZCL-glucuronide xylan as a substrate in 0.01% TRITON® X-100 and 200 mM sodium phosphate (pH 6). One unit of xylanase activity is defined as the production of 1.0 μmol azuron per minute from 0.2% AZCL-glucuronide xylan as a substrate in 200 mM sodium phosphate (pH 6) at 37 °C and pH 6. It can be produced in a solution containing purified xylanase and 10 mg / ml. -1 The activities of EC 3.2.1.8 and EC 3.2.1.156 xylanases were measured in a reaction mixture of beech xylan (MEGAZYME International Ireland Ltd., County Wicklow, Ireland) at 40°C for 15 minutes in 50 mm sodium acetate (pH 4.0). The DNS method (Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar) was used. Anal. Chem (Analytical Chemistry, 31, 426–428) Measures the reducing sugars obtained from substrate depolymerization. One unit of enzyme activity is defined as the amount of enzyme that catalyzes the formation of 1 μmol of reducing sugar per minute. Detailed Implementation

[0089] Composition This invention relates to compositions comprising GH43 arabinofuranylase, GH51 arabinofuranylase, GH5 xylanase, GH3 β-xylanase and GH30 xylanase and optionally CE3 acetylxylan esterase and / or α-xylanase, and the use of these compositions for dissolving hemicellulose fibers.

[0090] The work described in co-pending PCT international application PCT / US2023 / 083345 (which is incorporated herein by reference in its entirety) demonstrates that compositions containing GH43 arabinofuranosylase, GH51 arabinofuranosylase, and GH5 xylanase or GH30_8 xylanase unexpectedly increase hemicellulose dissolution and release significantly more monomeric arabinose and xylose compared to compositions that combine GH43 arabinofuranosylase and GH51 arabinofuranosylase alone or in combination with GH8 xylanase, GH10 xylanase, or GH11 xylanase.

[0091] Similarly, the work described in co-pending PCT international application PCT / US2023 / 083351 (which is incorporated herein by reference in its entirety) shows that CE3 acetylated xylan esterase releases more monomeric arabinose and / or xylose when used in combination with GH43 arabinofuranosidase and GH51 arabinofuranosidase, GH5 xylanase and GH3 β-xylosidase.

[0092] Surprisingly and unexpectedly, the work described herein shows that compositions containing GH43 arabinofuranase and G51 arabinofuranase, GH5 xylanase, GH3 β-xylosidase, and GH30 xylanase increase hemicellulose dissolution and release significantly more monomeric arabinose and xylose compared to similar compositions that do not contain GH30 xylanase alone. The work described herein demonstrates that the inclusion of both GH5 and GH30 xylanases in compositions containing GH43 arabinofuranase and GH51 arabinofuranase and GH3 β-xylose produces an unexpected synergistic effect. Surprisingly, the compositions of the present invention significantly increase the yield of monomeric arabinose and xylose without the need for acetylated xylan esterase, ferulic acid esterase, and / or α-glucuronidase; however, the addition of CE3 acetylated xylan esterase and / or α-xylosidase (e.g., GH31 α-xylosidase) to the compositions further increases the yield of monomeric arabinose and / or xylose.

[0093] For example, the work described herein shows that compositions containing GH43 arabinofuranase, GH51 arabinofuranase, GH5_21 xylanase, GH3 β-xylanase, and GH30 xylanase release at least 40% to at least 60% more xylose compared to compositions lacking GH30 xylanase but otherwise identical. Similarly, the work described herein shows that compositions containing GH43 arabinofuranase, GH51 arabinofuranase, GH5_21 xylanase, GH3 β-xylanase, GH30 xylanase, and CE3 acetylxylanase release at least 130% to at least 140% more xylose compared to compositions lacking CE3 acetylxylan esterase but otherwise identical. This work further demonstrates that compositions containing GH43 arabinofuranase, GH51 arabinofuranase, GH5 xylanase, GH3 β-xylosidase, GH30 xylanase and CE3 acetylxylanase release at least about 10% to about 15% more arabinose compared to compositions lacking CE3 acetylxylan esterase but otherwise identical.

[0094] Enzyme compositions can be formulated in solid form (e.g., particles containing enzymes) or in liquid form (e.g., liquid compositions containing enzymes and one or more formulations).

[0095] The present invention envisions the use of the compositions of the present invention in saccharification, fermentation, or simultaneous saccharification and fermentation to increase the solubility of hemicellulose fibers in monomeric sugars (such as arabinose and xylose) during conventional and raw starch hydrolysis (RSH) ethanol production processes.

[0096] A. Exemplary GH43 arabinofuranase Aspects of the present invention relate to compositions comprising GH43 arabinofuranase in combination with other enzymes to increase hemicellulose solubility and the production of monomeric arabinose and / or xylose. The present invention contemplates that any GH43 arabinofuranase, when used in combination with GH51 arabinofuranase, GH5 xylanase, GH3 β-xylosidase, GH30 xylanase, and optionally CE3 acetylxylan esterase and / or GH31 α-xylosidase, increases the production of monomeric arabinose and / or xylose, compared to similar compositions without GH43 arabinofuranase.

[0097] In the examples, GH43 arabinofuranase is GH43_36 arabinofuranase.

[0098] Exemplary GH43 arabinofuranase can be derived from the genus *Pythium* (…). Humicol a) *Diplostomum* ( Lasiodiplodia ), or genus *Porphyra* ( Poronia ).

[0099] Exemplary GH43 arabinofuranase can be derived from species-specific humic fungi ( Humicola insolens ), Dioscorea opposita ( Lasiodiplodia theobromae ), or point hole housing ( Poronia punctata ).

[0100] An exemplary GH43 arabinofuranase has the amino acid sequence of SEQ ID NO: 1. In an embodiment, the GH43 arabinofuranase has the amino acid sequence of SEQ ID NO: 1 containing 0 to 10 conserved amino acid substitutions and has arabinofuranase activity. In an embodiment, the GH43 arabinofuranase 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 with the amino acid sequence of SEQ ID NO: 1 and has arabinofuranase activity. An exemplary GH43 arabinofuranase has the amino acid sequence of SEQ ID NO: 2. In an embodiment, the GH43 arabinofuranase has the amino acid sequence of SEQ ID NO: 2 containing 0 to 10 conserved amino acid substitutions and has arabinofuranase activity. In the embodiments, the GH43 arabinofuranase 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 with the amino acid sequence of SEQ ID NO: 2, and has arabinofuranase activity. An exemplary GH43 arabinofuranase has the amino acid sequence of SEQ ID NO: 3. In the embodiments, the GH43 arabinofuranase has the amino acid sequence of SEQ ID NO: 3 containing 0 to 10 conserved amino acid substitutions and has arabinofuranase activity. In the embodiments, the GH43 arabinofuranase 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 with the amino acid sequence of SEQ ID NO: 3, and has arabinofuranase activity.

[0101] GH43 arabinofuranosaccharidase can be added at concentrations between 0.0001 and 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) during presaccharification, saccharification, and / or simultaneous saccharification and fermentation.

[0102] B. Exemplary GH51 arabinofuranase Aspects of the present invention relate to compositions comprising GH51 arabinofuranase in combination with other enzymes to increase hemicellulose solubility and the production of monomeric arabinose and / or xylose. The present invention contemplates that any GH51 arabinofuranase, when used in combination with GH43 arabinofuranase, GH5 xylanase, GH3 β-xylosidase, GH30 xylanase, and optionally CE3 acetylxylan esterase and / or GH31 α-xylosidase, increases the production of monomeric arabinose and / or xylose, compared to similar compositions without GH51 arabinofuranase.

[0103] In this embodiment, GH51 arabinofuranase is GH51_6 arabinofuranase.

[0104] Exemplary GH51 arabinofuranase can be derived from the genus *Grifola frondosa* (…). Meripulus ), *Trichoderma* genus, or *Acidobacterium* genus ( Acidiella) .

[0105] Exemplary GH51 arabinofuranase can be derived from the species *Grifola frondosa* (…). Meripulus giganteus ), *Dioscorea opposita*, or *Bohemiania* ( Aci diella bohemica ).

[0106] An exemplary GH51 arabinofuranylase has the amino acid sequence of SEQ ID NO: 4. In an embodiment, the GH51 arabinofuranylase has the amino acid sequence of SEQ ID NO: 4 containing 0 to 10 conserved amino acid substitutions and has arabinofuranylase activity. In an embodiment, the GH51 arabinofuranylase 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 with the amino acid sequence of SEQ ID NO: 4 and has arabinofuranylase activity. An exemplary GH51 arabinofuranylase has the amino acid sequence of SEQ ID NO: 5. In an embodiment, the GH51 arabinofuranylase has the amino acid sequence of SEQ ID NO: 5 containing 0 to 10 conserved amino acid substitutions and has arabinofuranylase activity. In the embodiments, GH51 arabinofuranylase 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 with the amino acid sequence of SEQ ID NO: 5, and has arabinofuranylase activity. An exemplary GH51 arabinofuranylase has the amino acid sequence of SEQ ID NO: 6. In the embodiments, GH51 arabinofuranylase has the amino acid sequence of SEQ ID NO: 6 containing 0 to 10 conserved amino acid substitutions and has arabinofuranylase activity. In the embodiments, GH51 arabinofuranylase 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 with the amino acid sequence of SEQ ID NO: 6, and has arabinofuranylase activity.

[0107] GH51 arabinofuranosaccharidase can be added at concentrations between 0.0001 and 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) during presaccharification, saccharification, and / or simultaneous saccharification and fermentation.

[0108] C. Exemplary GH5 xylanase Various aspects of the present invention relate to compositions comprising GH5 xylanase in combination with other enzymes to increase hemicellulose solubility and the production of monomeric arabinose and / or xylose. The present invention contemplates the increased production of monomeric arabinose and / or xylose when any GH5 xylanase is used in combination with GH43 arabinofuranase, GH51 arabinofuranase, GH3 β-xylosidase, GH30 xylanase, and optionally CE3 acetylxylan esterase and / or GH31 α-xylosidase, compared to similar compositions without GH5 xylanase.

[0109] In this example, GH5 xylanase is GH5_21 xylanase.

[0110] Exemplary GH_21 xylanase can be derived from Bacteroides ( Bacteroides ), Belénella ( Belliella ), Chlorella ( Chryseobacterium ), or Sphingosine mononitrate spp. ( Sphingobacterium ).

[0111] Exemplary GH_21 xylanase can be derived from the species Bacteroides fibronectinus ( Bacteroides cellulosilyticus CL02Y12C19, species of the genus *Bell's bacterium* - 64282, species of the genus *Aureobacter*, *Rainbow Aureobacterium* ( Chryseobacterium oncorhynchi ), or species of the genus *Sphingosporobacter*-64162.

[0112] Exemplary GH5_21 xylanase can be derived from bioreactor metagenomics, elephant dung metagenomics, xanthan gum basic community O, xanthan gum basic community S, or xanthan gum basic community T.

[0113] 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 containing 0 to 10 conserved 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 with 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 containing 0 to 10 conserved amino acid substitutions and has xylanase activity. In the embodiments, 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 with 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 the embodiments, GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 9 containing 0 to 10 conserved amino acid substitutions and has xylanase activity. In the embodiments, 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 with 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 containing 0 to 10 conserved 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 with 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 containing 0 to 10 conserved amino acid substitutions and has xylanase activity.In the embodiments, the GH5_21 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with 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 the embodiments, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 12 containing 0 to 10 conserved amino acid substitutions and has xylanase activity. In the embodiments, the GH5_21 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with 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 containing 0 to 10 conserved 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 with 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 containing 0 to 10 conserved amino acid substitutions and has xylanase activity. In the embodiments, the GH5_21 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with 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 the embodiments, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 15 containing 0 to 10 conserved amino acid substitutions and has xylanase activity.In the embodiments, 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 with 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 the embodiments, GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 16 containing 0 to 10 conserved amino acid substitutions and has xylanase activity. In the embodiments, 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 with 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 containing 0 to 10 conserved 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 with 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 containing 0 to 10 conserved amino acid substitutions and has xylanase activity. In the embodiments, the GH5_21 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with 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 the embodiments, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 19 containing 0 to 10 conserved amino acid substitutions and has xylanase activity.In the embodiments, 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 with 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 the embodiments, GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 20 containing 0 to 10 conserved amino acid substitutions and has xylanase activity. In the embodiments, 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 with 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 containing 0 to 10 conserved 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 with the amino acid sequence of SEQ ID NO: 21 and has xylanase activity.

[0114] In the example, GH5 xylanase is GH5_35 xylanase.

[0115] Example GH5_35 xylanase can be derived from Bacillus spp., Cohenella spp. ( Cohnella ), or Bacillus spp. ( Paenibacillus ).

[0116] Exemplary GH5_35 xylanase can be derived from the species *Bacillus hemicellulose* (… Bacillus hemiccellulosilyticus JCM 9152 、 Hyalinella xylanaceae ( Cohnella xylanilytica ), chitinophilic spore-forming bacteria ( Paenibacillus chitinolyticus ), or Bacillus species-62332.

[0117] An example GH5_35 xylanase can be derived from a compost metagenomics.

[0118] 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 containing 0 to 10 conserved 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 with 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 containing 0 to 10 conserved amino acid substitutions and has xylanase activity. In the embodiments, the GH5_35 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with 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 the embodiments, the GH5_35 xylanase has the amino acid sequence of SEQ ID NO: 24 containing 0 to 10 conserved amino acid substitutions and has xylanase activity. In the embodiments, the GH5_35 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with 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 containing 0 to 10 conserved 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 with 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 containing 0 to 10 conserved amino acid substitutions and has xylanase activity.In the examples, 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 with the amino acid sequence of SEQ ID NO: 26, and has xylanase activity.

[0119] GH5 xylanase can be added at concentrations between 0.0001 and 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) during presaccharification, saccharification, and / or simultaneous saccharification and fermentation.

[0120] D. Exemplary GH3 β-xylosidase Aspects of the present invention relate to compositions comprising GH3 β-xylosidase in combination with other enzymes to increase hemicellulose dissolution and the production of monomeric arabinose and / or xylose. The present invention contemplates the increased production of monomeric arabinose and / or xylose when any GH3 β-xylosidase is used in combination with GH43 arabinofuranase, GH51 arabinofuranase, GH5 xylanase, GH30 xylanase, and optionally CE3 acetylxylan esterase and / or GH31 α-xylosidase, compared to similar compositions without GH3 β-xylosidase.

[0121] Exemplary GH3 β-xylosidases include, but are not limited to, those from the genus Alternaria ( Alternaria Aspergillus ( ) Aspergillus ), genus Chaetomium ( Chaetomium Fusarium ( ) Fusarium ), Thermophyton genus ( Mycothermus ), Penicillium genus ( Penicillium ), genus Corydalis ( Sporormia ), genus Bassilia ( Talaromyces ) or Trichoderma ( Trichoderma GH3 β-xylosidase.

[0122] Exemplary GH3 β-xylosidases include, but are not limited to, GH3 β-xylosidases from the following species: Alternaria terrestris (… Alternaria tellustris Aspergillus echinosporum ( ), Aspergillus aculeatus ), Fisher Aspergillus ( Aspergillus fischeri Aspergillus fumigatus ( ) Aspergillus fumigatus Aspergillus nidus ( ) Aspergillus nidulans ), Chaetomium coccidioides ( Chaetomium globosum ), green chrysophagus ( Chaetomium virescens Fusarium longipes ( Fusarium longipes ), thermophilic bacteria ( Mycothermus thermophilus ), Penicillium Emersonii ( Penicillium emersonii ), Penicillium oxalate ( Penicillium oxalicum ), coccidioidomyces ( Sporormia fimetaria Emerson's basket bacteria ( Talaromyces emersonii ), Bassilized bacteria ( Talaromyces stipitatus ) or Trichoderma reesei ( Trichoderma reesei ).

[0123] An exemplary GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 27. In an embodiment, the GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 27 containing 0 to 10 conserved amino acid substitutions and has β-xylosidase activity. In an embodiment, the GH3 β-xylosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 27 and has β-xylosidase activity. An exemplary GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 28. In an embodiment, the GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 28 containing 0 to 10 conserved amino acid substitutions and has β-xylosidase activity. In the embodiments, the GH3 β-xylosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 28, and has β-xylosidase activity. An exemplary GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 29. In the embodiments, the GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 29 containing 0 to 10 conserved amino acid substitutions and has β-xylosidase activity. In the embodiments, the GH3 β-xylosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 29, and has β-xylosidase activity. An exemplary GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 30. In an embodiment, the GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 30 containing 0 to 10 conserved amino acid substitutions and has β-xylosidase activity. In an embodiment, the GH3 β-xylosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 30 and has β-xylosidase activity. An exemplary GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 31.In the embodiments, GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 31 containing 0 to 10 conserved amino acid substitutions and has β-xylosidase activity. In the embodiments, GH3 β-xylosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 31 and has β-xylosidase activity. An exemplary GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 32. In the embodiments, GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 32 containing 0 to 10 conserved amino acid substitutions and has β-xylosidase activity. In the embodiments, the GH3 β-xylosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 32, and has β-xylosidase activity. An exemplary GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 33. In the embodiments, the GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 33 containing 0 to 10 conserved amino acid substitutions and has β-xylosidase activity. In the embodiments, the GH3 β-xylosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 33, and has β-xylosidase activity. An exemplary GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 34. In an embodiment, the GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 34 containing 0 to 10 conserved amino acid substitutions and has β-xylosidase activity. In an embodiment, the GH3 β-xylosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 34 and has β-xylosidase activity. An exemplary GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 35. In an embodiment, the GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 35 containing 0 to 10 conserved amino acid substitutions and has β-xylosidase activity.In the embodiments, the GH3 β-xylosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 35, and has β-xylosidase activity. An exemplary GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 36. In the embodiments, the GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 36 containing 0 to 10 conserved amino acid substitutions and has β-xylosidase activity. In the embodiments, the GH3 β-xylosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 36, and has β-xylosidase activity. An exemplary GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 37. In an embodiment, the GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 377 containing 0 to 10 conserved amino acid substitutions and has β-xylosidase activity. In an embodiment, the GH3 β-xylosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 37 and has β-xylosidase activity. An exemplary GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 38. In an embodiment, the GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 38 containing 0 to 10 conserved amino acid substitutions and has β-xylosidase activity. In the embodiments, the GH3 β-xylosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 38, and has β-xylosidase activity. An exemplary GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 39. In the embodiments, the GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 39 containing 0 to 10 conserved amino acid substitutions and has β-xylosidase activity.In the embodiments, the GH3 β-xylosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 39, and has β-xylosidase activity. An exemplary GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 40. In the embodiments, the GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 40 containing 0 to 10 conserved amino acid substitutions and has β-xylosidase activity. In the embodiments, the GH3 β-xylosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 40, and has β-xylosidase activity. An exemplary GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 41. In an embodiment, the GH3 β-xylosidase has the amino acid sequence of SEQ ID NO: 41 containing 0 to 10 conserved amino acid substitutions and has β-xylosidase activity. In an embodiment, the GH3 β-xylosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 41 and has β-xylosidase activity.

[0124] GH3 β-xylosidase can be added at concentrations between 0.0001 and 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) during presaccharification, saccharification, and / or simultaneous saccharification and fermentation.

[0125] E. Exemplary GH30 xylanase Aspects of the present invention relate to compositions comprising GH30 xylanase in combination with other enzymes to increase hemicellulose solubility and the production of monomeric arabinose and / or xylose. The present invention contemplates the increased production of monomeric arabinose and / or xylose when any GH30 xylanase is used in combination with GH43 arabinofuranase, GH51 arabinofuranase, GH5 xylanase, GH3 β-xylosidase, and optionally CE3 acetylxylan esterase and / or GH31 α-xylosidase, compared to similar compositions without GH30 xylanase.

[0126] In this example, GH30 xylanase is GH30_1 xylanase.

[0127] In the example, GH30 xylanase is GH30_2 xylanase.

[0128] In the examples, GH30 xylanase is GH30_3 xylanase.

[0129] In the example, GH30 xylanase is GH30_4 xylanase.

[0130] In this embodiment, GH30 xylanase is GH30_5 xylanase.

[0131] In this example, GH30 xylanase is GH30_7 xylanase.

[0132] Exemplary GH30_7 xylanase can be derived from Aspergillus or Evanstokertella. Evanstolkia ), Basilaria and Trichoderma.

[0133] Exemplary GH30_7 xylanase can be derived from the species *Aspergillus ferruginosa* and *Aspergillus fumigatus*. Aspergillus smoking-related Aspergillus neonicotinus () Aspergillus novofumigatus ), Aspergillus pseudoterreus ( Aspergillus pseudo-terrestrial Aspergillus terreus ( ) Aspergillus terreus ), Aspergillus turcica ( Aspergillus turcosus Aspergillus urdagawa ( ) Aspergillus udagawae ), Basilella repens ( Evanstolkia leycettana ), *Paederus cibarius* ( Talaromyces verruculosus ) and Trichoderma reesei.

[0134] 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 containing 0 to 10 conserved 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 with 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 containing 0 to 10 conserved amino acid substitutions and has xylanase activity. In the embodiments, 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 with 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 the embodiments, the GH30_7 xylanase has the amino acid sequence of SEQ ID NO: 44 containing 0 to 10 conserved amino acid substitutions and has xylanase activity. In the embodiments, 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 with 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 containing 0 to 10 conserved 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 with 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 containing 0 to 10 conserved amino acid substitutions and has xylanase activity.In the embodiments, 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 with 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 the embodiments, the GH30_7 xylanase has the amino acid sequence of SEQ ID NO: 47 containing 0 to 10 conserved amino acid substitutions and has xylanase activity. In the embodiments, 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 with 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 containing 0 to 10 conserved 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 with 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 containing 0 to 10 conserved amino acid substitutions and has xylanase activity. In the embodiments, 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 with 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 the embodiments, the GH30_7 xylanase has the amino acid sequence of SEQ ID NO: 50 containing 0 to 10 conserved amino acid substitutions and has xylanase activity.In the embodiments, 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 with 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 the embodiments, the GH30_7 xylanase has the amino acid sequence of SEQ ID NO: 51 containing 0 to 10 conserved amino acid substitutions and has xylanase activity. In the embodiments, 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 with 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 containing 0 to 10 conserved 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 with the amino acid sequence of SEQ ID NO: 52 and has xylanase activity.

[0135] In this embodiment, GH30 xylanase is GH30_8 xylanase.

[0136] Example GH30_8 xylanase can be derived from Bacillus spp., Clostridium spp. ( Clostridium ), Bacillus spp. and Vibrio spp. Vibrio ).

[0137] Exemplary GH30_8 xylanase can be derived from species Bacillus species-18423, Clostridium acetonebutanol ( Clostridium acetobutylicum ), Clostridium sacchariformis ( Clostridium saccharobutylicum ), feed-grade Bacillus ( Paenibacillus pabuli ) and rhizosphere Vibrio ( Vibrio rhizosphaerae ).

[0138] 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 containing 0 to 10 conserved 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 with 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 containing 0 to 10 conserved amino acid substitutions and has xylanase activity. In the embodiments, the GH30_8 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with 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 the embodiments, the GH30_8 xylanase has the amino acid sequence of SEQ ID NO: 55 containing 0 to 10 conserved amino acid substitutions and has xylanase activity. In the embodiments, the GH30_8 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with 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 containing 0 to 10 conserved 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 with 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 containing 0 to 10 conserved amino acid substitutions and has xylanase activity.In the examples, 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 with the amino acid sequence of SEQ ID NO: 57, and has xylanase activity.

[0139] In the example, GH30 xylanase is GH30_9 xylanase.

[0140] In the example, GH30 xylanase is GH30_10 xylanase.

[0141] GH30 xylanase can be added at concentrations between 0.0001 and 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) during presaccharification, saccharification, and / or simultaneous saccharification and fermentation.

[0142] F. Exemplary CE3 acetylation esterase Aspects of the present invention relate to compositions comprising CE3 acetylxylan esterase in combination with other enzymes to increase hemicellulose solubility and the production of monomeric arabinose and / or xylose. The present invention contemplates that any CE3 acetylxylan esterase, when used in combination with GH43 arabinofuranase, GH51 arabinofuranase, GH5 xylanase, GH3 β-xylosidase, GH30 xylanase, and optionally GH31 α-xylosidase, increases the production of monomeric arabinose and / or xylose compared to similar compositions without CE3 acetylxylan esterase.

[0143] An exemplary CE3 acetylxylan 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 containing 0 to 10 conserved amino acid substitutions and has acetylxylan 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 with the amino acid sequence of SEQ ID NO: 58 and has acetylxylan esterase activity. An exemplary CE3 acetylxylan 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 containing 0 to 10 conserved amino acid substitutions and has acetylxylan esterase activity. In the embodiments, 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 with the amino acid sequence of SEQ ID NO: 59, and has acetylxylan esterase activity. An exemplary CE3 acetylxylan esterase has the amino acid sequence of SEQ ID NO: 60. In the embodiments, the CE3 polypeptide has the amino acid sequence of SEQ ID NO: 60 containing 0 to 10 conserved amino acid substitutions and has acetylxylan esterase activity. In the embodiments, 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 with the amino acid sequence of SEQ ID NO: 60, and has acetylxylan esterase activity. An exemplary CE3 acetylxylan esterase has the amino acid sequence of SEQ ID NO: 61. In the embodiments, the CE3 polypeptide has the amino acid sequence of SEQ ID NO: 61 containing 0 to 10 conserved amino acid substitutions and has acetylxylan esterase activity. In the examples, 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 with the amino acid sequence of SEQ ID NO:61, and has acetylxylan esterase activity.An exemplary CE3 acetylxylan 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 containing 0 to 10 conserved amino acid substitutions and has acetylxylan 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 with the amino acid sequence of SEQ ID NO: 62 and has acetylxylan esterase activity. An exemplary CE3 acetylxylan 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 containing 0 to 10 conserved amino acid substitutions and has acetylxylan esterase activity. In the embodiments, 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 with the amino acid sequence of SEQ ID NO: 63, and has acetylxylan esterase activity. An exemplary CE3 acetylxylan esterase has the amino acid sequence of SEQ ID NO: 64. In the embodiments, the CE3 polypeptide has the amino acid sequence of SEQ ID NO: 64 containing 0 to 10 conserved amino acid substitutions and has acetylxylan esterase activity. In the embodiments, 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 with the amino acid sequence of SEQ ID NO: 64, and has acetylxylan esterase activity. An exemplary CE3 acetylxylan esterase has the amino acid sequence of SEQ ID NO: 65. In the embodiments, the CE3 polypeptide has the amino acid sequence of SEQ ID NO: 65 containing 0 to 10 conserved amino acid substitutions and has acetylxylan esterase activity. In the examples, 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 with the amino acid sequence of SEQ ID NO: 65, and has acetylxylan esterase activity.An exemplary CE3 acetylxylan 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 containing 0 to 10 conserved amino acid substitutions and has acetylxylan 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 with the amino acid sequence of SEQ ID NO: 66 and has acetylxylan esterase activity. An exemplary CE3 acetylxylan 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 containing 0 to 10 conserved amino acid substitutions and has acetylxylan esterase activity. In the examples, 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 with the amino acid sequence of SEQ ID NO:67, and has acetylxylan esterase activity.

[0144] CE3 acetylated xylan esterase can be added at concentrations between 0.0001 and 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) during presaccharification, saccharification, and / or simultaneous saccharification and fermentation.

[0145] G. Exemplary GH31 α-xylosidase Aspects of the present invention relate to compositions comprising GH31 α-xylosidase in combination with other enzymes to increase hemicellulose solubility and the production of monomeric arabinose and / or xylose. The present invention contemplates the increased production of monomeric arabinose and / or xylose when any GH31 α-xylosidase is used in combination with GH43 arabinofuranase, GH51 arabinofuranase, GH5 xylanase, GH3 β-xylosidase, and GH30 xylanase and optionally CE3 acetylxylan esterase, compared to similar compositions without GH31 α-xylosidase.

[0146] Exemplary GH31 α-xylosidase can be derived from the genus *Acridobacter* (…). Herbaceous plant ).

[0147] Exemplary GH31 α-xylosidase can be derived from the species *Hemicellulosinus hemicellulose* (… Herbaceous plant hemicellulosilytica).

[0148] An exemplary GH31 α-xylosidase has the amino acid sequence of SEQ ID NO: 68. In an embodiment, the GH31 α-xylosidase has the amino acid sequence of SEQ ID NO: 68 containing 0 to 10 conserved amino acid substitutions and has β-xylosidase activity. In an embodiment, the GH31 α-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 with the amino acid sequence of SEQ ID NO: 68 and has α-xylosidase activity.

[0149] GH31 α-xylosidase can be added at concentrations between 0.0001 and 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) during presaccharification, saccharification, and / or simultaneous saccharification and fermentation.

[0150] H. Exemplary fermented organisms Various aspects of this invention relate to the use of fermentation organisms for the production of fermentation products. Particularly suitable fermentation organisms are those capable of directly or indirectly fermenting sugars (such as arabinose, glucose, maltose, and / or arabinose) into (i.e., converting them into) desired fermentation products (such as ethanol). Examples of fermentation organisms include fungal organisms, such as yeast. Preferred yeasts include strains of the genus *Saccharomyces*, particularly *Saccharomyces cerevisiae*.

[0151] Examples of commercially available yeasts include, for example, RED STAR™ and ETHANOL RED™ yeast (available from Fermentisis / Lesaffre, USA), FALI (available from Fleischmann's Yeast, USA), SUPERSTART and THERMOSACC™ fresh yeast (available from Ethanol Technology, Wisconsin, USA), BIOFERM AFT and XR (available from NABC-North American Bioproducts Corporation, Georgia, USA), GERT STRAND (available from Gert Strand AB, Sweden), and FERMIOL (available from DSM Specialties). Other available yeast strains can be obtained from biological collections such as the American Type Culture Collection (ATCC) or the German Microbial Culture Collection (DSMZ), such as BY4741 (e.g., ATCC 201388); Y108-1 (ATCC PTA.10567) and NRRL YB-1952 (ARS Culture Collection). Other suitable Saccharomyces cerevisiae strains as host cells include 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 yeast species 1400, 424A (LNH-ST), 259A (LNH-ST) and their derivatives.

[0152] As used herein, “derivatives” of a strain are derived from a reference strain, such as through mutagenesis, recombinant DNA technology, mating, cell fusion, or cell-directed transfer between yeast strains. Those skilled in the art will understand that genetic alterations (including the metabolic modifications exemplified herein) can be described with reference to a suitable host organism and its corresponding metabolic responses or a suitable source organism for the desired genetic material (such as genes for desired metabolic pathways). However, given the vast diversity of organisms available for whole-genome sequencing and the high level of skill in the field of genomics, those skilled in the art can apply the teachings and guidelines provided herein to other organisms. For example, the metabolic alterations exemplified herein can be readily applied to other species by incorporating similar coding nucleic acids, either from the same species or from a species different from the reference species.

[0153] The fermenting organism can be a yeast strain, such as a *Saccharomyces cerevisiae* strain produced using the methods described and involved in U.S. Patent No. 8,257,959-BB. In one embodiment, the recombinant cell is a derivative of strain *Saccharomyces cerevisiae* CIBTS1260 (deposited at the Agricultural Research Service Culture Collection (NRRL) at NRRL Y-50973, Illinois 61604, USA).

[0154] Fermentation organisms can also be *Saccharomyces cerevisiae* strain NMI V14 / 004037 (see WO 2015 / 143324 and WO2015 / 143317, each of which is incorporated herein by reference), strain numbers V15 / 004035, V15 / 004036 and V15 / 004037 (see WO 2016 / 153924, which is incorporated herein by reference), strain numbers V15 / 001459, V15 / 001460, V15 / 001461 (see WO 2016 / 138437, which is incorporated herein by reference), strain number NRRL Y67342 (see WO2018 / 098381, which is incorporated herein by reference), strain numbers NRRL Y67549 and NRRL Y67700 (see WO... Derivatives of any strain described in WO 2019 / 161227 (which is incorporated herein by reference), or WO 2017 / 087330 (which is incorporated herein by reference).

[0155] Fermentation organisms may contain one or more heteropolynucleotides encoding α-amylase, glucosylamylase, protease, and / or cellulase. Examples of α-amylases, glucosylamylases, proteases, and cellulases suitable for expression in fermentation organisms are known in the art (see WO 2021 / 231623, which is incorporated herein by reference).

[0156] Fermented organisms can be in the form of a composition comprising fermented organisms and naturally occurring and / or non-naturally occurring components.

[0157] Fermentation organisms can be in any viable form, including pulverized, dried (including active dried and instantaneous), compressed, paste (liquid) forms, etc. In one embodiment, the fermentation organism (e.g., a Saccharomyces cerevisiae strain) is dry yeast, such as active dry yeast or instantaneous yeast. In one embodiment, the fermentation organism is pulverized yeast. In one embodiment, the fermentation organism is compressed yeast. In one embodiment, the fermentation organism is paste yeast.

[0158] In one embodiment, a composition comprises a fermentation organism (e.g., a strain of Saccharomyces cerevisiae) as described herein and one or more components selected from the group consisting of surfactants, emulsifiers, gums, swelling agents, antioxidants, and other processing aids.

[0159] The compositions described herein may comprise the fermentation organisms described herein (e.g., Saccharomyces cerevisiae strains) and any suitable surfactants. In one embodiment, one or more surfactants are anionic surfactants, cationic surfactants, and / or nonionic surfactants.

[0160] The compositions described herein may comprise the fermentation organisms described herein (e.g., Saccharomyces cerevisiae strains) and any suitable emulsifier. In one embodiment, the emulsifier is a fatty acid ester of sorbitan. In another embodiment, the emulsifier is selected from the group consisting of: sorbitan monostearate (SMS), citrate of mono- and diglycerides, polyglycerol esters, and fatty acid esters of propylene glycol.

[0161] In one embodiment, the composition comprises the fermentation organism described herein (e.g., a strain of Saccharomyces cerevisiae) and Olindronal SMS, Olindronal SK, or Olindronal SPL, including the compositions described in European Patent No. 1,724,336 (which is incorporated herein by reference). For active dry yeast, these products are commercially available from Bussetti AG of Austria.

[0162] The compositions described herein may comprise the fermentation organisms described herein (e.g., Saccharomyces cerevisiae strains) and any suitable gum. In one embodiment, the gum is selected from the group consisting of: pagoda tree gum, guar gum, astragalus gum, gum arabic, xanthan gum, and gum arabic, particularly for paste, compressed, and dry yeast.

[0163] The compositions described herein may comprise the fermentation organisms described herein (e.g., Saccharomyces cerevisiae strains) and any suitable swelling agent. In one embodiment, the swelling agent is methylcellulose or carboxymethylcellulose.

[0164] The compositions described herein may comprise the fermentation organisms described herein (e.g., Saccharomyces cerevisiae strains) and any suitable antioxidants. In one embodiment, the antioxidant is butylated hydroxyanisole (BHA) and / or butylated hydroxytoluene (BHT), or ascorbic acid (vitamin C), particularly for active dry yeast.

[0165] During fermentation (such as SSF), the appropriate concentration of viable fermentation organisms is well known in the art or can be readily determined by those skilled in the art. In one embodiment, fermentation organisms (such as ethanol fermentation yeast (e.g., Saccharomyces cerevisiae)) are added to the fermentation medium such that the count of viable fermentation organisms (such as yeast) per mL of fermentation medium is 10. 5 Up to 10 12 Within the range of 10, preferably 10 7 Up to 10 10 One, especially about 5 × 10 7 indivual.

[0166] Particles This invention also relates to enzyme particles / granules 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 this invention. In embodiments, the particle comprises a core and optionally one or more coatings (outer layers) surrounding the core. This invention contemplates the use of any one of the exemplary GH43 arabinofuranylase, GH51 arabinofuranylase, exemplary GH5 xylanase, exemplary GH3 β-xylosidase, exemplary GH30 xylanase, exemplary CE3 acetylxylan esterase, and exemplary GH31 α-xylosidase in the enzyme particles / granules described herein.

[0167] The diameter of the core (measured as equivalent sphere diameter (volume-average particle size)) can be 20–2000 µm, particularly 50–1500 µm, 100–1500 µm, or 250–1200 µm. The core diameter as an equivalent sphere diameter can be determined using laser diffraction, such as with the Malvern Mastersizer, and / or the methods described under ISO 13320 (2020).

[0168] In some embodiments, the core comprises the GH43 arabinofuranylase of the present invention. In some embodiments, the core comprises the GH51 arabinofuranylase of the present invention. In some embodiments, the core comprises the GH5 xylanase of the present invention. In some embodiments, the core comprises the GH3 β-xylosidase of the present invention. In some embodiments, the core comprises the GH30 xylanase of the present invention. In some embodiments, the core comprises the acetylxylan esterase of the present invention. In some embodiments, the core comprises the GH31 α-xylosidase of the present invention.

[0169] In the embodiments, 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: GH43 arabinofuranase, GH51 arabinofuranase, GH5 xylanase, GH3 β-xylanase, GH30 xylanase, CE3 acetylxylan esterase, and GH31 α-xylanase of the present invention.

[0170] In one embodiment, the core comprises GH43 arabinofuranylase, GH51 arabinofuranylase, GH5 xylanase, GH3 β-xylanase, and GH30 xylanase. In another embodiment, the core comprises GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_21 xylanase, GH3 β-xylanase, and GH30_7 xylanase. In yet another embodiment, the core comprises GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_21 xylanase, GH3 β-xylanase, and GH30_8 xylanase. In yet another embodiment, the core comprises GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_35 xylanase, GH3 β-xylanase, and GH30_7 xylanase. In the embodiments, the core comprises GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_35 xylanase, GH3 β-xylanase and GH30_8 xylanase.

[0171] In one embodiment, the core comprises GH43 arabinofuranylase, GH51 arabinofuranylase, GH5 xylanase, GH3 β-xylosidase, GH30 xylanase, and CE3 acetylxylan esterase. In another embodiment, the core comprises GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_21 xylanase, GH3 β-xylosidase, GH30_7 xylanase, and CE3 acetylxylan esterase. In yet another embodiment, the core comprises GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_21 xylanase, GH3 β-xylosidase, GH30_8 xylanase, and CE3 acetylxylan esterase. In this embodiment, the core comprises GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_35 xylanase, GH3 β-xylosidase, GH30_7 xylanase, and CE3 acetylxylan esterase. In this embodiment, the core comprises GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_35 xylanase, GH3 β-xylosidase, GH30_8 xylanase, and CE3 acetylxylan esterase.

[0172] In one embodiment, the core comprises GH43 arabinofuranylase, GH51 arabinofuranylase, GH5 xylanase, GH3 β-xylanase, GH30 xylanase, CE3 acetylxylan esterase, and GH31 α-xylanase. In another embodiment, the core comprises GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_21 xylanase, GH3 β-xylanase, GH30_7 xylanase, CE3 acetylxylan esterase, and GH31 α-xylanase. In yet another embodiment, the core comprises GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_21 xylanase, GH3 β-xylanase, GH30_8 xylanase, CE3 acetylxylan esterase, and GH31 α-xylanase. In this embodiment, the core comprises GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_35 xylanase, GH3 β-xylanase, GH30_7 xylanase, CE3 acetylxylan esterase, and GH31 α-xylanase. In this embodiment, the core comprises GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_35 xylanase, GH3 β-xylanase, GH30_8 xylanase, CE3 acetylxylan esterase, and GH31 α-xylanase.

[0173] The core may include additional materials such as fillers, fibrous materials (cellulose or synthetic fibers), stabilizers, solubilizers, suspending agents, viscosity modifiers, light spheres, plasticizers, salts, lubricants, and fragrances.

[0174] The core may include binders such as synthetic polymers, waxes, fats, or carbohydrates.

[0175] The core, typically as a homogeneous blend, may include salts of polyvalent cations, reducing agents, antioxidants, peroxide decomposition catalysts, and / or acidic buffer components.

[0176] The core may include inert particles into which one or more enzymes are adsorbed or applied (e.g., by fluidized bed coating) to the surface of the inert particles.

[0177] The diameter of the core can be 20-2000 µm, especially 50-1500 µm, 100-1500 µm or 250-1200 µm.

[0178] The core may be surrounded by at least one coating, for example, to improve storage stability, reduce dust formation during processing, or to color the particles. Optional coatings may include salt coatings or other suitable coating materials such as polyethylene glycol (PEG), methyl hydroxypropyl cellulose (MHPC), and polyvinyl alcohol (PVA).

[0179] In some embodiments, the coating comprises the GH43 arabinofuranylase of the present invention. In some embodiments, the coating comprises the GH51 arabinofuranylase of the present invention. In some embodiments, the coating comprises the GH5 xylanase of the present invention. In some embodiments, the coating comprises the GH3 β-xylosidase of the present invention. In some embodiments, the coating comprises the GH30 xylanase of the present invention. In some embodiments, the coating comprises the acetylxylan esterase of the present invention. In some embodiments, the coating comprises the GH31 α-xylosidase of the present invention.

[0180] In the embodiments, 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: GH43 arabinofuranase, GH51 arabinofuranase, GH5 xylanase, GH3 β-xylanase, GH30 xylanase, CE3 acetylxylan esterase, and GH31 α-xylanase of the present invention.

[0181] In some examples, the coating contains GH43 arabinofuranylase, GH51 arabinofuranylase, GH5 xylanase, GH3 β-xylanase, and GH30 xylanase. In some examples, the coating contains GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_21 xylanase, GH3 β-xylanase, and GH30_7 xylanase. In some examples, the coating contains GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_21 xylanase, GH3 β-xylanase, and GH30_8 xylanase. In some examples, the coating contains GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_35 xylanase, GH3 β-xylanase, and GH30_7 xylanase. In the embodiments, the coating contains GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_35 xylanase, GH3 β-xylanase and GH30_8 xylanase.

[0182] In some examples, the coating contains GH43 arabinofuranylase, GH51 arabinofuranylase, GH5 xylanase, GH3 β-xylosidase, GH30 xylanase, and CE3 acetylxylan esterase. In some examples, the coating contains GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_21 xylanase, GH3 β-xylosidase, GH30_7 xylanase, and CE3 acetylxylan esterase. In some examples, the coating contains GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_21 xylanase, GH3 β-xylosidase, GH30_8 xylanase, and CE3 acetylxylan esterase. In some examples, the coating contains GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_35 xylanase, GH3 β-xylosidase, GH30_7 xylanase, and CE3 acetylxylan esterase. In other examples, the coating contains GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_35 xylanase, GH3 β-xylosidase, GH30_8 xylanase, and CE3 acetylxylan esterase.

[0183] In some examples, the coating contains GH43 arabinofuranylase, GH51 arabinofuranylase, GH5 xylanase, GH3 β-xylanase, GH30 xylanase, CE3 acetylxylan esterase, and GH31 α-xylanase. In some examples, the coating contains GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_21 xylanase, GH3 β-xylanase, GH30_7 xylanase, CE3 acetylxylan esterase, and GH31 α-xylanase. In some examples, the coating contains GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_21 xylanase, GH3 β-xylanase, GH30_8 xylanase, CE3 acetylxylan esterase, and GH31 α-xylanase. In some examples, the coating contains GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_35 xylanase, GH3 β-xylanase, GH30_7 xylanase, CE3 acetylxylan esterase, and GH31 α-xylanase. In other examples, the coating contains GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_35 xylanase, GH3 β-xylanase, GH30_8 xylanase, CE3 acetylxylan esterase, and GH31 α-xylanase.

[0184] The coating may be applied at a rate of at least 0.1% (e.g., at least 0.5%, at least 1%, at least 5%, at least 10%, or at least 15%) of the core weight. This amount may be at most 100%, 70%, 50%, 40%, or 30%.

[0185] The coating is preferably at least 0.1 µm thick, particularly at least 0.5 µm, at least 1 µm, or at least 5 µm thick. In some embodiments, the coating thickness is less than 100 µm, such as less than 60 µm or less than 40 µm.

[0186] The coating should seal the core unit by forming a substantially continuous layer. A substantially continuous layer should be understood as a coating with very few or no pores, such that the core unit has very few or no uncoated areas. The layer or coating should in particular be uniform in thickness.

[0187] The coating may further contain other materials as known in the art, such as fillers, anti-sticking agents, pigments, dyes, plasticizers and / or adhesives, such as titanium dioxide, kaolin, calcium carbonate or talc.

[0188] Salt coatings may contain at least 60% salt by weight, such as 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% salt by weight.

[0189] To provide acceptable protection, the salt coating is preferably at least 0.1 µm thick, for example at least 0.5 µm, at least 1 µm, at least 2 µm, at least 4 µm, at least 5 µm, or at least 8 µm. In particular embodiments, the thickness of the salt coating is less than 100 µm, such as less than 60 µm or less than 40 µm.

[0190] Salt can be added from a salt solution (where the salt is completely dissolved) or from a salt suspension (where the fine particles are less than 50 µm, such as less than 10 µm or less than 5 μm).

[0191] Salt coatings may contain a single salt or a mixture of two or more salts. The salts may be water-soluble, particularly having a solubility of at least 0.1 g in 100 g of water at 20°C, preferably at least 0.5 g / 100 g of water, for example at least 1 g / 100 g of water, or for example at least 5 g / 100 g of water.

[0192] Salts can be inorganic salts, such as sulfates, sulfites, phosphates, phosphonates, nitrates, chlorides, or carbonates, or salts of simple organic acids (less than 10 carbon atoms, such as 6 or fewer carbon atoms), such as citrates, malonates, or acetates. Examples of cations in these salts are alkali or alkaline earth metal ions, ammonium ions, or first transition metal ions, such as sodium, potassium, magnesium, calcium, zinc, or aluminum. Examples of anions include chloride, bromine, iodine, sulfate, sulfite, bisulfite, thiosulfate, phosphate, dihydrogen phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, metasilicate, citrate, malate, maleate, malonic acid, succinate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate, or gluconate. In particular, alkali or alkaline earth metal salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate, or salts of simple organic acids such as citrate, malonate or acetate can be used.

[0193] The salt in the coating may have a constant humidity of 60% or more, particularly 70%, 80% or more or 85% or more at 20°C, or it may be another hydrated form of such salt (e.g., anhydrous form). Salt coatings may be as described in WO 00 / 01793 or WO 2006 / 034710.

[0194] A specific example of a suitable salt is NaCl (CH4). 20℃ = 76%), Na2CO3 (CH 20℃ = 92%), NaNO3 (CH 20℃ =73%), Na2HPO4 (CH 20℃ = 95%), Na3PO4 (CH 25℃ = 92%), NH4Cl (CH 20℃ = 79.5%), (NH4)2HPO4 (CH 20℃ =93.0%), NH4H2PO4 (CH 20℃ = 93.1%), (NH4)2SO4 (CH 20℃ = 81.1%), KCl (CH 20℃ = 85%), K2HPO4 (CH 20℃ = 92%), KH2PO4 (CH 20℃ = 96.5%), KNO3 (CH 20℃ = 93.5%), Na2SO4 (CH 20℃ = 93%), K2SO4 (CH 20℃ =98%), KHSO4 (CH 20℃= 86%), MgSO4 (CH 20℃ = 90%), ZnSO4 (CH 20℃ = 90%) and sodium citrate (CH 25℃ = 86%). Other examples include NaH2PO4, (NH4)H2PO4, CuSO4, Mg(NO3)2, and magnesium acetate.

[0195] Salts can be in anhydrous form, or they can be hydrated salts, i.e., crystalline salt hydrates with one or more bound water crystals, as described in WO 99 / 32595. Specific examples include anhydrous sodium sulfate (Na₂SO₄), anhydrous magnesium sulfate (MgSO₄), and magnesium sulfate heptahydrate (MgSO₄). . 7H2O), zinc sulfate heptahydrate (ZnSO4) . 7H2O), disodium hydrogen phosphate heptahydrate (Na2HPO4) . 7H2O), magnesium nitrate hexahydrate (Mg(NO3)2(6H2O)), sodium citrate dihydrate and magnesium acetate tetrahydrate.

[0196] Preferably, the salt is used as a salt solution, for example, using a fluidized bed.

[0197] The coating material can be a waxy coating material or a film-forming coating material. Examples of waxy coating materials are poly(ethylene oxide) products (polyethylene glycol, PEG) with an average molar amount of 1,000 to 20,000; ethoxylated nonylphenol having 16 to 50 ethylene oxide units; ethoxylated fatty alcohols containing 12 to 20 carbon atoms and having 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and monoglycerides, diglycerides, and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application via fluidized bed technology are given in GB 1483591.

[0198] The particles may optionally have one or more additional coatings. Examples of suitable coating materials are polyethylene glycol (PEG), methyl hydroxypropyl cellulose (MHPC), and polyvinyl alcohol (PVA). Examples of enzyme particles with multiple coatings are described in WO 93 / 07263 and WO 97 / 23606.

[0199] The core can be prepared by granulating blends of the components, for example by methods including granulation techniques such as crystallization, precipitation, pan-coating, fluidized bed coating, fluidized bed agglomeration, rotary atomization, extrusion, granulation, spheronization, particle size reduction, drum granulation, and / or high-shear granulation.

[0200] Methods for preparing the core can be found in *Handbook of Powder Technology*; CE Capes, *Particle Size Enlargement*; Volume 1; 1980; Elsevier. Preparation methods include known feed and pellet formulation techniques, such as: (a) Spray-dried products, wherein a liquid solution containing one or more enzymes is atomized in a spray drying tower to form small droplets, which are then dried to form particulate material containing one or more enzymes as they descend along the drying tower. Very small particles can be produced in this way (Michael S. Showell (ed.); Powdered detergents [Powdered Detergents]; Surfactant Science Series; 1998; Volume 71; pp. 140-142; Marcel Dekker.

[0201] (b) Layered products in which one or more enzymes are coated in layers around pre-formed inert core particles, wherein the solution containing one or more enzymes is typically atomized in a fluidized bed apparatus, in which the pre-formed core particles are fluidized and the solution containing one or more enzymes adheres to the core particles and is dried until a dried layer of one or more enzymes remains on the surface of the core particles. If useful core particles of the desired size can be found, particles of the desired size can be obtained in this manner. This type of product is described, for example, in WO 97 / 23606.

[0202] (c) Adsorbed core particles, wherein instead of coating one or more enzymes in layers around the core, one or more enzymes are adsorbed onto and / or into the surface of the core. Such a method is described in WO 97 / 39116.

[0203] (d) Extruded or pelletized products, wherein a paste containing one or more enzymes is pressed into pellets or extruded under pressure through a small opening and cut into particles, which are then dried. Such particles typically have a fairly large size because the material with the extrusion opening (usually a flat plate with perforations) limits the permissible pressure drop across the extrusion opening. Furthermore, when using small openings, the very high extrusion pressure increases heat generation in the paste containing one or more enzymes, which is detrimental to the enzymes themselves (Michael S. Showell (edited);). Powdered detergents[Powdered Detergents]; Surfactant Science Series; 1998; Volume 71; pp. 140-142; Marcel Dekker.

[0204] (e) Spray-granulated products, wherein a powder containing one or more enzymes is suspended in molten wax and the suspension is sprayed (e.g., via a rotary atomizer) into a cooling chamber where the droplets rapidly solidify (Michael S. Showell (ed.)). Powdered detergents [Powdered Detergent]; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker. The resulting product is one or more enzymes evenly distributed throughout the inert material rather than concentrated on its surface. This technique is described in US 4,016,040 and US 4,713,245.

[0205] (f) A mixer-granulated product in which a liquid containing one or more enzymes is added to a dry powder composition of conventional granulation components. The liquid and powder are mixed in a suitable ratio, and as moisture from the liquid is absorbed into the dry powder, the components of the dry powder begin to adhere and aggregate, and particles accumulate to form granules containing one or more enzymes. Such methods are described in US 4,106,991, EP 170360, EP 304332, EP 304331, WO 90 / 09440, and WO90 / 09428. In certain aspects of this process, various high-shear mixers can be used as granulators. Granules consisting of one or more enzymes, fillers, and binders are mixed with cellulose fibers to reinforce the granules, producing so-called T-granules. The reinforced granules are more robust and release less enzyme dust.

[0206] (g) Particle size reduction, in which a core is produced by grinding or crushing larger particles, pellets, tablets, briquettes, etc., containing one or more enzymes. The desired core particle fraction is obtained by sieving the ground or crushed product. Oversized and undersized particles can be recovered. Particle size reduction is described in Martin Rhodes (ed.); Principles of Powder Technology; 1990; Chapter 10; John Wiley & Sons.

[0207] (h) Fluidized bed granulation. Fluidized bed granulation involves suspending microparticles in an airflow and spraying liquid through a nozzle onto the fluidized particles. The particles hit by the sprayed droplets become wetted and sticky. The sticky particles collide with and adhere to other particles to form granules.

[0208] (i) These cores can be dried, such as in a fluidized bed dryer. Technicians can use other known methods used in the feed or enzyme industries for drying particles. Drying is preferably carried out at a product temperature of 25°C to 90°C. For some enzymes, it is important that the core containing one or more enzymes contains a small amount of water before salt coating. If one or more water-sensitive enzymes are salt-coated before excess water removal, excess water will be trapped in the core and may negatively affect the activity of one or more enzymes. After drying, these cores preferably contain 0.1%–10% w / w water.

[0209] Dust-free particulate matter can be generated, for example, as disclosed in US 4,106,991 and US 4,661,452, and can optionally be coated by methods known in the art.

[0210] The particles may further contain one or more additional enzymes, such as hydrolases, isomerases, ligases, lyases, oxidoreductases, and transferases. The one or more additional enzymes are preferably selected from the group consisting of: acetylxylan esterase, acylglycerol lipase, amylase, α-amylase, β-amylase, arabinofuranosylase, cellobiase, cellulase, ferulic acid esterase, galactanase, α-galactosidase, β-galactosidase, β-glucanase, β-glucosidase, lysophospholipase, lysozyme, α-mannosidase, β-mannosidase (mannanase), phytase, phospholipase A1, phospholipase A2, phospholipase D, protease, pullulanase, pectin esterase, triacylglycerol lipase, xylanase, β-xylosidase, or any combination thereof. Each enzyme will then be present in more particles, ensuring a more uniform distribution of the enzymes and also reducing the physical separation of different enzymes due to the varying particle sizes. The method for generating multi-enzyme coparticles is disclosed in IP.com disclosure IPCOM000200739D.

[0211] Another example of preparing one or more enzymes using co-particles is disclosed in WO 2013 / 188331.

[0212] The present invention also relates to one or more protected enzymes prepared according to the method disclosed in EP 238216.

[0213] Liquid preparations 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 present invention. The compositions may comprise enzyme stabilizers (examples of which include polyols (such as propylene glycol or glycerol), sugars or sugar alcohols, lactic acid, reversible protease inhibitors, boric acid or boric acid derivatives such as aromatic borate esters, or phenyl boric acid derivatives such as 4-formylphenylboronic acid).

[0214] The present invention contemplates the use of any one of the exemplary GH43 arabinofuranylase, GH51 arabinofuranylase, exemplary GH5 xylanase, exemplary GH3 β-xylosidase, exemplary GH30 xylanase, exemplary CE3 acetylxylan esterase, and exemplary GH31 α-xylosidase in the liquid formulations described herein.

[0215] In some embodiments, one or more fillers or one or more carrier materials are included to increase the volume of such compositions. Suitable fillers or carrier materials include, but are not limited to, various salts of sulfate, carbonate, and silicate, as well as talc, clay, etc. Suitable fillers 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, these compositions contain about 5% to about 90% of such materials.

[0216] In one aspect, the liquid formulation contains 20%-80% w / w polyol. In one embodiment, the liquid formulation contains 0.001%-2% w / w preservative.

[0217] The following exemplary liquid formulations provide %w / w ranges for enzyme proteins in different combinations of enzymes present in the compositions of the present invention. It should be understood that each class of enzymes may be added at different concentrations to achieve the specified total %w / w for each composition.

[0218] In another embodiment, the present invention relates to liquid formulations comprising: (A) 0.01%-25% w / w of the composition of the present invention, the composition comprising GH43 arabinofuranase, GH51 arabinofuranase, GH5 xylanase, GH3 β-xylanase and GH30 xylanase of the present invention; (B) 20%-80% w / w polyols; (C) Optional 0.001%-2% w / w preservative; and (D) Water.

[0219] In another embodiment, the present invention relates to liquid formulations comprising: (A) 0.01%-25% w / w of the composition of the present invention, the composition comprising GH43 arabinofuranase, GH51 arabinofuranase, GH5_21 xylanase, GH3 β-xylanase and GH30_7 xylanase; (B) 20%-80% w / w polyols; (C) Optional 0.001%-2% w / w preservative; and (D) Water.

[0220] In another embodiment, the present invention relates to liquid formulations comprising: (A) 0.01%-25% w / w of the composition of the present invention, the composition comprising GH43 arabinofuranase, GH51 arabinofuranase, GH5_21 xylanase, GH3 β-xylanase and GH30_8 xylanase; (B) 20%-80% w / w polyols; (C) Optional 0.001%-2% w / w preservative; and (D) Water.

[0221] In another embodiment, the present invention relates to liquid formulations comprising: (A) 0.01%-25% w / w of the composition of the present invention, the composition comprising GH43 arabinofuranase, GH51 arabinofuranase, GH5_35 xylanase, GH3 β-xylanase and GH30_7 xylanase; (B) 20%-80% w / w polyols; (C) Optional 0.001%-2% w / w preservative; and (D) Water.

[0222] In another embodiment, the present invention relates to liquid formulations comprising: (A) 0.01%-25% w / w of the composition of the present invention, the composition comprising GH43 arabinofuranase, GH51 arabinofuranase, GH5-35 xylanase, GH3 β-xylanase and GH30-8 xylanase; (B) 20%-80% w / w polyols; (C) Optional 0.001%-2% w / w preservative; and (D) Water.

[0223] In another embodiment, the present invention relates to liquid formulations comprising: (A) 0.01%-25% w / w of the composition of the present invention, comprising GH43 arabinofuranylase, GH51 arabinofuranylase, GH5 xylanase, GH3 β-xylosidase, GH30 xylanase and CE3 acetylatedxylan esterase; (B) 20%-80% w / w polyols; (C) Optional 0.001%-2% w / w preservative; and (D) Water.

[0224] In another embodiment, the present invention relates to liquid formulations comprising: (A) 0.01%-25% w / w of the composition of the present invention, comprising GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_21 xylanase, GH3 β-xylosidase, GH30_7 xylanase and CE3 acetylatedxylan esterase; (B) 20%-80% w / w polyols; (C) Optional 0.001%-2% w / w preservative; and (D) Water.

[0225] In another embodiment, the present invention relates to liquid formulations comprising: (A) 0.01%-25% w / w of the composition of the present invention, comprising GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_21 xylanase, GH3 β-xylosidase, GH30_8 xylanase and CE3 acetylatedxylan esterase; (B) 20%-80% w / w polyols; (C) Optional 0.001%-2% w / w preservative; and (D) Water.

[0226] In another embodiment, the present invention relates to liquid formulations comprising: (A) 0.01%-25% w / w of the composition of the present invention, comprising GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_35 xylanase, GH3 β-xylosidase, GH30_7 xylanase and CE3 acetylatedxylan esterase; (B) 20%-80% w / w polyols; (C) Optional 0.001%-2% w / w preservative; and (D) Water.

[0227] In another embodiment, the present invention relates to liquid formulations comprising: (A) 0.01%-25% w / w of the composition of the present invention, comprising GH43 arabinofuranylase, GH51 arabinofuranylase, GH5-35 xylanase, GH3 β-xylanase, GH30-8 xylanase and CE3 acetylatedxylan esterase; (B) 20%-80% w / w polyols; (C) Optional 0.001%-2% w / w preservative; and (D) Water.

[0228] In another embodiment, the present invention relates to liquid formulations comprising: (A) 0.01%-25% w / w of the composition of the present invention, comprising GH43 arabinofuranylase, GH51 arabinofuranylase, GH5-35 xylanase, GH3 β-xylanase, GH30-8 xylanase and CE3 acetylatedxylan esterase; (B) 20%-80% w / w polyols; (C) Optional 0.001%-2% w / w preservative; and (D) Water.

[0229] In each of the above examples of liquid formulations, the composition of the present invention may further include GH31 α-xylosidase.

[0230] In another embodiment, the present invention relates to liquid formulations comprising: (A) 0.01%-25% w / w of the composition of the present invention, the composition comprising GH43 arabinofuranase, GH51 arabinofuranase, GH5 xylanase, GH3 β-xylanase and GH30 xylanase of the present invention; (B) 0.001%-2% w / w preservative; (C) Optional 20%-80% w / w polyols; and (D) Water.

[0231] In another embodiment, the present invention relates to liquid formulations comprising: (A) 0.01%-25% w / w of the composition of the present invention, the composition comprising GH43 arabinofuranase, GH51 arabinofuranase, GH5_21 xylanase, GH3 β-xylanase and GH30_7 xylanase; (B) 0.001%-2% w / w preservative; (C) Optional 20%-80% w / w polyols; and (D) Water.

[0232] In another embodiment, the present invention relates to liquid formulations comprising: (A) 0.01%-25% w / w of the composition of the present invention, the composition comprising GH43 arabinofuranase, GH51 arabinofuranase, GH5_21 xylanase, GH3 β-xylanase and GH30_8 xylanase; (B) 0.001%-2% w / w preservative; (C) Optional 20%-80% w / w polyols; and (D) Water.

[0233] In another embodiment, the present invention relates to liquid formulations comprising: (A) 0.01%-25% w / w of the composition of the present invention, the composition comprising GH43 arabinofuranase, GH51 arabinofuranase, GH5_35 xylanase, GH3 β-xylanase and GH30_7 xylanase; (B) 0.001%-2% w / w preservative; (C) Optional 20%-80% w / w polyols; and (D) Water.

[0234] In another embodiment, the present invention relates to liquid formulations comprising: (A) 0.01%-25% w / w of the composition of the present invention, the composition comprising GH43 arabinofuranase, GH51 arabinofuranase, GH5-35 xylanase, GH3 β-xylanase and GH30-8 xylanase; (B) 0.001%-2% w / w preservative; (C) Optional 20%-80% w / w polyols; and (D) Water.

[0235] In another embodiment, the present invention relates to liquid formulations comprising: (A) 0.01%-25% w / w of the composition of the present invention, comprising GH43 arabinofuranylase, GH51 arabinofuranylase, GH5 xylanase, GH3 β-xylosidase, GH30 xylanase and CE3 acetylatedxylan esterase; (B) 0.001%-2% w / w preservative; (C) Optional 20%-80% w / w polyols; and (D) Water.

[0236] In another embodiment, the present invention relates to liquid formulations comprising: (A) 0.01%-25% w / w of the composition of the present invention, comprising GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_21 xylanase, GH3 β-xylosidase, GH30_7 xylanase and CE3 acetylatedxylan esterase; (B) 0.001%-2% w / w preservative; (C) Optional 20%-80% w / w polyols; and (D) Water.

[0237] In another embodiment, the present invention relates to liquid formulations comprising: (A) 0.01%-25% w / w of the composition of the present invention, comprising GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_21 xylanase, GH3 β-xylosidase, GH30_8 xylanase and CE3 acetylatedxylan esterase; (B) 0.001%-2% w / w preservative; (C) Optional 20%-80% w / w polyols; and (D) Water.

[0238] In another embodiment, the present invention relates to liquid formulations comprising: (A) 0.01%-25% w / w of the composition of the present invention, comprising GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_35 xylanase, GH3 β-xylosidase, GH30_7 xylanase and CE3 acetylatedxylan esterase; (B) 0.001%-2% w / w preservative; (C) Optional 20%-80% w / w polyols; and (D) Water.

[0239] In another embodiment, the present invention relates to liquid formulations comprising: (A) 0.01%-25% w / w of the composition of the present invention, comprising GH43 arabinofuranylase, GH51 arabinofuranylase, GH5-35 xylanase, GH3 β-xylanase, GH30-8 xylanase and CE3 acetylatedxylan esterase; (B) 0.001%-2% w / w preservative; (C) Optional 20%-80% w / w polyols; and (D) Water.

[0240] In another embodiment, the present invention relates to liquid formulations comprising: (A) 0.01%-25% w / w of the composition of the present invention, comprising GH43 arabinofuranylase, GH51 arabinofuranylase, GH5-35 xylanase, GH3 β-xylanase, GH30-8 xylanase and CE3 acetylatedxylan esterase; (B) 0.001%-2% w / w preservative; (C) Optional 20%-80% w / w polyols; and (D) Water.

[0241] In each of the above examples of liquid formulations, the composition of the present invention may further include GH31 α-xylosidase.

[0242] The liquid formulation may further comprise one or more formulations, such as formulations selected from the group consisting of: polyols, 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 formulations selected from the group consisting of: sodium sulfate, dextrin, cellulose, sodium thiosulfate, kaolin, and calcium carbonate. In one embodiment, the polyol is selected from the group consisting of: glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propanediol or 1,3-propanediol, dipropylene glycol, polyethylene glycol (PEG) with an average molecular weight of less than about 600, and polypropylene glycol (PPG) with an average molecular weight of less than about 600, more preferably selected from the group consisting of: glycerol, sorbitol, and propylene glycol (MPG), or any combination thereof.

[0243] The liquid formulation may contain 20%-80% polyols (i.e., the total amount of polyols), such as 25%-75% polyols, 30%-70% polyols, 35%-65% polyols, or 40%-60% polyols. In one embodiment, the liquid formulation contains 20%-80% polyols, such as 25%-75% polyols, 30%-70% polyols, 35%-65% polyols, or 40%-60% polyols, wherein the polyols are selected from the group consisting of: glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propanediol or 1,3-propanediol, dipropylene glycol, polyethylene glycol (PEG) with an average molecular weight of less than about 600, and polypropylene glycol (PPG) with an average molecular weight of less than about 600. Liquid formulations may contain 20%-80% polyols (i.e., the total amount of polyols), such as 25%-75% polyols, 30%-70% polyols, 35%-65% polyols, or 40%-60% polyols, wherein the polyols are selected from the group consisting of glycerol, sorbitol, and propylene glycol (MPG).

[0244] 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 contains 0.02%-1.5% w / w preservative, such as 0.05%-1% w / w or 0.1%-0.5% w / w preservative. In one embodiment, the liquid formulation contains 0.001%-2% w / w preservative (i.e., the total amount of preservative), such as 0.02%-1.5% w / w, 0.05%-1% w / w, 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.

[0245] Liquid formulations may further contain one or more additional enzymes, such as hydrolases, isomerases, ligases, lyases, oxidoreductases, and transferases. The one or more additional enzymes are preferably selected from the group consisting of: acetylxylan esterase, acylglycerol lipase, amylase, α-amylase, β-amylase, arabinofuranylase, cellobiase, cellulase, ferulic acid esterase, galactanase, α-galactosidase, β-galactosidase, β-glucanase, β-glucosidase, lysophospholipase, lysozyme, α-mannosidase, β-mannosidase (mannanase), phytase, phospholipase A1, phospholipase A2, phospholipase D, protease, pullulanase, pectin esterase, triacylglycerol lipase, xylanase, β-xylosidase, or any combination thereof.

[0246] Method for producing fermentation products from starch-containing materials One aspect of the invention relates to a method for producing fermentation products (e.g., fuel ethanol) from materials containing gelatinized starch, wherein a composition comprising GH43 arabinofuranase, GH51 arabinofuranase, GH5 xylanase, GH3 β-xylosidase, GH30 xylanase and optionally CE3 acetylxylan esterase and / or GH31 α-xylosidase is present or added during saccharification and / or fermentation.

[0247] In an embodiment, a method for producing fermentation products from starch-containing materials includes the following steps: (a) Using a heat-stable α-amylase to liquefy starch-containing material at a temperature above the initial gelatinization temperature of starch to produce dextrin; (b) Saccharifying the dextrin using glucosylamylase to produce fermentable sugars; and (c) Fermenting the sugar using a fermentation organism to produce a fermentation product; The composition comprising GH43 arabinofuranase, GH51 arabinofuranase, GH5 xylanase, GH3 β-xylanase, GH30 xylanase and optionally CE3 acetylxylan esterase and / or GH31 α-xylanase is present or added during the saccharification step (b) and / or fermentation step (c).

[0248] The present invention contemplates the use of any one of the following exemplary GH43 arabinofuranylase, GH51 arabinofuranylase, GH5 xylanase, GH3 β-xylanase, GH30 xylanase, CE3 acetylxylan esterase, and GH31 α-xylanase in the compositions of the present invention and in the methods of using the compositions of the present invention (including in the following exemplary compositions used in the methods for producing fermentation products).

[0249] The exemplary compositions used in steps (b) and / or (c) comprise GH43 arabinofuranylase, GH51 arabinofuranylase, GH5 xylanase, GH3 β-xylanase, and GH30 xylanase. In examples, the GH30 xylanase activity is GH30_1 xylanase. In examples, the GH30 xylanase activity is GH30_2 xylanase. In examples, the GH30 xylanase activity is GH30_3 xylanase. In examples, the GH30 xylanase activity is GH30_4 xylanase. In examples, the GH30 xylanase activity is GH30_5 xylanase. In examples, the GH30 xylanase activity is GH30_7 xylanase. In examples, the GH30 xylanase activity is GH30_8 xylanase. In examples, the GH30 xylanase activity is GH30_9 xylanase. In the examples, the GH30 xylanase activity is GH30_10 xylanase.

[0250] In this embodiment, the GH5 family xylanase is GH5_21 xylanase. The exemplary compositions used in step (b) and / or step (c) comprise GH43 arabinofuranase, GH51 arabinofuranase, GH5_21 xylanase, GH3 β-xylosidase, and GH30_7 xylanase. The exemplary compositions used in step (b) and / or step (c) comprise GH43 arabinofuranase, GH51 arabinofuranase, GH5_21 xylanase, GH3 β-xylosidase, and GH30_8 xylanase. In this embodiment, the GH5 family xylanase is GH5_35 xylanase. In this embodiment, the core comprises GH43 arabinofuranase, GH51 arabinofuranase, GH5_35 xylanase, GH3 β-xylosidase, and GH30_7 xylanase. The exemplary compositions used in step (b) and / or step (c) comprise GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_35 xylanase, GH3 β-xylanase and GH30_8 xylanase.

[0251] In the embodiments, the compositions used in step (b) and / or step (c) include CE3 acetylxylan esterase. Exemplary compositions used in step (b) and / or step (c) comprise GH43 arabinofuranase, GH51 arabinofuranase, GH5 xylanase, GH3 β-xylosidase, GH30 xylanase, and CE3 acetylxylan esterase. Exemplary compositions used in step (b) and / or step (c) comprise GH43 arabinofuranase, GH51 arabinofuranase, GH5_21 xylanase, GH3 β-xylosidase, GH30_7 xylanase, and CE3 acetylxylan esterase. The exemplary compositions used in steps (b) and / or (c) comprise GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_21 xylanase, GH3 β-xylosidase, GH30_8 xylanase, and CE3 acetylxylan esterase. The exemplary compositions used in steps (b) and / or (c) comprise GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_35 xylanase, GH3 β-xylosidase, GH30_7 xylanase, and CE3 acetylxylan esterase. The exemplary compositions used in steps (b) and / or (c) comprise GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_35 xylanase, GH3 β-xylosidase, GH30_8 xylanase, and CE3 acetylxylan esterase.

[0252] In the embodiments, the compositions used in steps (b) and / or (c) include GH31 α-xylosidase. In the embodiments, the compositions used in steps (b) and / or (c) include CE3 acetylxylan esterase and GH31 α-xylosidase. Exemplary compositions used in steps (b) and / or (c) comprise GH43 arabinofuranase, GH51 arabinofuranase, GH5 xylanase, GH3 β-xylosidase, GH30 xylanase, CE3 acetylxylan esterase, and GH31 α-xylosidase. Exemplary compositions used in steps (b) and / or (c) comprise GH43 arabinofuranase, GH51 arabinofuranase, GH5_21 xylanase, GH3 β-xylosidase, GH30_7 xylanase, CE3 acetylxylan esterase, and GH31 α-xylosidase. The exemplary compositions used in steps (b) and / or (c) comprise GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_21 xylanase, GH3 β-xylanase, GH30_8 xylanase, CE3 acetylxylan esterase, and GH31 α-xylanase. The exemplary compositions used in steps (b) and / or (c) comprise GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_35 xylanase, GH3 β-xylanase, GH30_7 xylanase, CE3 acetylxylan esterase, and GH31 α-xylanase. The exemplary compositions used in step (b) and / or step (c) comprise GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_35 xylanase, GH3 β-xylosidase, GH30_8 xylanase, CE3 acetylxylan esterase, and GH31 α-xylosidase.

[0253] In this embodiment, GH43 arabinofuranase is GH43_36 arabinofuranase. In this embodiment, GH51 arabinofuranase is GH51_6 arabinofuranase.

[0254] In one embodiment, the composition is added during saccharification step (b). In another embodiment, the composition is added during fermentation step (c). In yet another embodiment, steps (b) and (c) are performed simultaneously in simultaneous saccharification and fermentation (SSF). In yet another embodiment, the composition is added during SSF.

[0255] In some embodiments, a thermostable glucosylamylase is added during liquefaction step (a). In some embodiments, a thermostable endoglucanase is added during liquefaction step (a). In some embodiments, a thermostable lipase is added during liquefaction step (a). In some embodiments, a thermostable phytase is added during liquefaction step (a). In some embodiments, a thermostable protease is added during liquefaction step (a). In some embodiments, a thermostable branched-chain amylase is added during liquefaction step (a). In some embodiments, a thermostable xylanase is added during liquefaction step (a). In a preferred embodiment, a thermostable α-amylase and a thermostable protease are added during liquefaction step (a). In some embodiments, a thermostable α-amylase and a thermostable xylanase are added during liquefaction step (a). In a preferred embodiment, a thermostable α-amylase, a thermostable protease, and a thermostable xylanase are added during liquefaction step (a).

[0256] In some embodiments, α-amylase is added during steps (b) and / or (c). In some embodiments, α-glucosidase is added during steps (b) and / or (c). In some embodiments, β-amylase is added during steps (b) and / or (c). In some embodiments, β-glucanase is added during steps (b) and / or (c). In some embodiments, β-glucosidase is added during steps (b) and / or (c). In some embodiments, cellobiase is added during steps (b) and / or (c). In some embodiments, endoglucanase is added during steps (b) and / or (c). In some embodiments, lipase is added during steps (b) and / or (c). In some embodiments, polysaccharide-lysing monooxygenase (LPMO) is added during steps (b) and / or (c). In some embodiments, maltose-producing α-amylase is added during steps (b) and / or (c). In some embodiments, pectinase is added during steps (b) and / or (c). In some embodiments, peroxidase is added during steps (b) and / or (c). In some embodiments, phytase is added during steps (b) and / or (c). In some embodiments, protease is added during steps (b) and / or (c). In some embodiments, trehalase is added during steps (b) and / or (c).

[0257] In this embodiment, the fermenting organism is yeast. In this embodiment, the yeast expresses α-amylase in situ during step (b) and / or step (c). In this embodiment, the yeast expresses glucosylamylase in situ during step (b) and / or step (c).

[0258] process parameters Process parameters for producing fermentation products (such as ethanol from starch-containing materials (e.g., corn)) are well known in the art. See, for example, WO 2006 / 086792, WO 2013 / 082486, WO 2012 / 088303, WO 2013 / 055676, WO2014 / 209789, WO 2014 / 209800, WO 2015 / 035914, WO 2017 / 112540, WO 2020 / 014407, WO2021 / 126966 (each of these patents is incorporated herein by reference).

[0259] Starch-containing materials Any suitable starch-containing starting material can be used. The material is selected based on the desired fermentation product. Examples of starch-containing materials include, but are not limited to, barley, legumes, cassava, cereals, corn, milo, peas, potatoes, rice, rye, sago, sorghum, sweet potatoes, tapioca, wheat, and whole grains, or any mixture thereof. Starch-containing materials can also be waxy or non-waxy types of corn and barley. Commonly used commercial starch-containing materials include corn, milo, and / or wheat.

[0260] Reduced particle size of starch-containing materials Prior to the liquefaction step (a), the particle size of the starch-containing material can be reduced, for example by dry milling.

[0261] slurry Prior to liquefaction step (a), a slurry comprising starch-containing material (e.g., preferably ground) and water can be formed. α-Amylase and optionally protease can be added to the slurry. During this process, the slurry can be heated above the initial gelatinization temperature of the starch-containing material to initiate starch gelatinization.

[0262] Spray cooking Optionally, the slurry can be spray-cooked before adding α-amylase during liquefaction step (a) to further gelatinize the starch in the slurry. Spray cooking can be carried out at a temperature range of 100°C to 120°C for up to at least 15 minutes.

[0263] liquefaction temperature The temperature range used during liquefaction step (a) can be from 70°C to 110°C, such as 75°C to 105°C, 80°C to 100°C, 85°C to 95°C, or 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.

[0264] liquefied pH The pH range used during liquefaction step (a) can be 4 to 6, 4.5 to 5.5, or 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.

[0265] liquefaction time The time range for performing the liquefaction step (a) can be from 30 minutes to 5 hours, from 1 hour to 3 hours, or from 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.

[0266] Liquefying enzyme This invention considers the use of thermostable enzymes during liquefaction step (a). Various thermostable enzymes are well known in the art to be used during liquefaction step (a), including, for example, thermostable α-amylase, thermostable glucosylase, thermostable endoglucanase, thermostable lipase, thermostable phytase, thermostable protease, thermostable branched-chain amylase, and / or thermostable xylanase. This invention considers the use of any thermostable enzyme in liquefaction step (a). Guidelines for determining the denaturation temperature of candidate thermostable enzymes used in liquefaction step (a) are provided in the Materials and Methods section below. The following published patent applications describe activity assays for determining whether a considered candidate thermostable enzyme used in liquefaction step (a) will be inactivated at the temperature considered in liquefaction step (a).

[0267] Examples of suitable thermostable α-amylases and guidelines for their use in liquefaction step (a) include, but are not limited to, the α-amylases described below: WO 94 / 18314, WO 94 / 02597, WO 96 / 23873, WO 96 / 23874, WO 96 / 39528, WO 97 / 41213, WO 97 / 43424, WO 99 / 19467, WO 00 / 60059, WO 2002 / 010355, WO 2002 / 092797, WO 2009 / 149130, WO 2009 / 61378, WO 2009 / 061379, WO 2009 / 061380, WO 2009 / 061381, WO 2009 / 098229, WO WO 2009 / 100102, WO 2010 / 115021, WO 2010 / 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 these patents is incorporated herein by reference).

[0268] Examples of suitable thermostable glucosylamylases include, but are not limited to, the glucosylamylases described in the following: WO2011 / 127802, WO 2013 / 036526, WO 2013 / 053801, WO 2018 / 164737, WO 2020 / 010101, and WO2022 / 090564 (each of these patents is incorporated herein by reference).

[0269] Examples of suitable thermostable endoglucanases include, but are not limited to, the endoglucanases described in WO 2015 / 035914 (which is incorporated herein by reference).

[0270] Examples of suitable thermostable lipases include, but are not limited to, the lipases described in WO 2017 / 112542 and WO 2020 / 014407 (both of which are incorporated herein by reference).

[0271] Examples of suitable thermostable phytases include, but are not limited to, the phytases described below: 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, WO2003 / 066847, WO 2004 / 085638, WO 2006 / 037327, WO 2006 / 037328, WO 2006 / 038062, WO2006 / 063588, WO WO 2007 / 112739, WO 2008 / 092901, WO 2008 / 116878, WO 2009 / 129489, and WO2010 / 034835 (each of these patents is incorporated herein by reference). Commercially available phytase-containing products include BIO-FEEDPHYTASE™, PHYTASE NOVO™ CT or L, LIQMAX or RONOZYME™ NP, RONOZYME® HIPHOS, RONOZYME® P5000 (CT), and NATUPHOS™ NG 5000.

[0272] Examples of suitable thermostable proteases include, but are not limited to, the proteases described in the following: WO 1992 / 02614, WO 98 / 56926, WO 2001 / 151620, WO 2003 / 048353, WO 2006 / 086792, WO 2010 / 008841, WO 2011 / 076123, WO 2011 / 087836, WO 2012 / 088303, WO 2013 / 082486, WO 2014 / 209789, WO 2014 / 209800, WO 2018 / 098124, WO 2018 / 118815 A1, and WO 2018 / 169780A1 (each of these patents is incorporated herein by reference).

[0273] Suitable commercially available protease-containing products include AVANTEC AMP®, FORTIVA REVO®, and FORTIVA HEMI®.

[0274] Examples of suitable heat-stable amylopectins include, but are not limited to, the amylopectins described in the following patents: WO2015 / 007639, WO 2015 / 110473, WO 2016 / 087327, WO 2017 / 014974, and WO 2020 / 187883 (each of these patents is incorporated herein by reference in its entirety). Suitable commercially available amylopectin products include PROMOZYME 400L, PROMOZYME™ D2 (Novozymes A / S, Denmark), OPTIMAX L-300 (Genencor Int., USA), and AMANO 8 (Amano, Japan).

[0275] Examples of suitable heat-stable xylanases include, but are not limited to, the xylanases described in WO 2017 / 112540 and WO 2021 / 126966 (each of these patents is incorporated herein by reference). Suitable commercially available products containing heat-stable xylanases include FORTIVA HEMI®.

[0276] The one or more enzymes described above are intended to be used in the method of the present invention in an "effective amount". Guidelines for determining the effective amount of the enzyme used in liquefaction step (a) and for performing activity assays to determine the activity of those enzymes can be found in each of the cited published patent applications for the different heat-stable liquefying enzymes.

[0277] Saccharification temperature Saccharification can be carried out in a temperature range of 20°C to 75°C, 30°C to 70°C, or 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.

[0278] Saccharification pH Saccharification can be carried out in a pH range of 4 to 5. Preferably, the pH is about 4.5.

[0279] Saccharification time Glycation can last from about 24 hours to about 72 hours.

[0280] Fermentation time Fermentation can last from 6 to 120 hours, 24 to 96 hours, or 35 to 60 hours.

[0281] Simultaneous saccharification and fermentation SSF can be carried out at temperatures of 25°C to 40°C, 28°C to 35°C, or 30°C to 5°C, and at pH values ​​of 3.5 to 5 or 3.8 to 4.3 for 24 to 96 hours, 36 to 72 hours, or 48 to 60 hours. Preferably, SSF is carried out at about 32°C and at a pH value of 3.8 to 4.5 for 48 to 60 hours.

[0282] Saccharification and / or fermentation enzymes This invention considers the use of enzymes during the saccharification step (b) and / or fermentation step (c). Various enzymes are well known in the art to be used during the saccharification step (b) and / or fermentation step (c), including, for example, α-amylase, α-glucosidase, β-amylase, β-glucanase, β-glucosidase, cellobiase, endoglucanase, glucosylamylase, lipase, polysaccharide lysing monooxygenase (LPMO), maltose-producing α-amylase, pectinase, peroxidase, phytase, protease, and trehalase.

[0283] The enzymes used in the saccharification step (b) and / or fermentation step (c) may be added exogenously as single components or formulated into compositions containing these enzymes. The enzymes used in the saccharification step (b) and / or fermentation step (c) may be added via in situ expression from a fermentation organism (e.g., yeast).

[0284] Suitable examples of α-amylases include, but are not limited to, the α-amylases described in the following: WO 2004 / 055178, WO 2006 / 069290, WO 2013 / 006756, WO 2013 / 034106, WO 2013 / 044867, WO 2021 / 163011, and WO 2021 / 163030 (each of these patents is incorporated herein by reference).

[0285] Suitable examples of glucosylamylases include, but are not limited to, the glucosylamylases described in the following: 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, WO2016 / 062875, WO 2017 / 066255, and WO 2018 / 191215 (each of these patents is incorporated herein by reference).

[0286] Examples of suitable compositions comprising α-amylase and glucosylamylase include, but are not limited to, compositions described in: WO 2006 / 069290, WO 2009 / 052101, WO 2011 / 068803, and WO 2013 / 006756 (each of these patents is incorporated herein by reference). Commercially available compositions containing glucosylamylase 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); OPTIDEX™ 300, GC480, and GC417 (from DuPont-Genencor); AMIGASE™ and AMIGASE™ PLUS (from DSM); and G-ZYME™ G900, G-ZYME™, and G990ZR (from DuPont-Genencor).

[0287] Examples of suitable β-glucanases include, but are not limited to, the β-glucanases described in WO 2021 / 055395 (which is incorporated herein by reference).

[0288] Examples of suitable β-glucosidases include, but are not limited to, the β-glucosidases described in the following: WO 2005 / 047499, WO 2013 / 148993, WO 2014 / 085439 and WO 2012 / 044915 (each of these patents is incorporated herein by reference).

[0289] Examples of suitable cellobiases include, but are not limited to, cellobiases described in the following: WO2013 / 148993, WO 2014 / 085439, WO 2014 / 138672, and WO 2016 / 040265 (each of these patents is incorporated herein by reference).

[0290] Examples of suitable endoglucanases include, but are not limited to, those described in WO 2013 / 148993 and WO 2014 / 085439 (both of which are incorporated herein by reference).

[0291] Suitable examples of maltose-producing α-amylases are described in U.S. Patent Nos. 4,598,048, 4,604,355, and 6,162,628, which are hereby incorporated by reference.

[0292] Examples of suitable lipases include, but are not limited to, the lipases described in WO 2017 / 112533, WO2017 / 112539, and WO 2020 / 076697 (each of these patents is incorporated herein by reference).

[0293] Suitable examples of LPMOs include, but are not limited to, those described in the following: WO 2013 / 148993, WO2014 / 085439, and WO 2019 / 083831 (each of these patents is incorporated herein by reference).

[0294] Examples of suitable phytases include, but are not limited to, the phytases described in WO 2001 / 62947 (which is incorporated herein by reference).

[0295] Examples of suitable pectinases include, but are not limited to, the pectinases described in WO 2022 / 173694 (which is incorporated herein by reference).

[0296] Examples of suitable peroxidases include, but are not limited to, the peroxidases described in WO 2019 / 231944 (which is incorporated herein by reference).

[0297] Examples of suitable proteases include, but are not limited to, the proteases described in the following: WO 2017 / 050291, WO2017 / 148389, WO 2018 / 015303, and WO 2018 / 015304 (each of these patents is incorporated herein by reference).

[0298] Suitable examples of trehalases include, but are not limited to, the trehalases described in the following: WO 2016 / 205127, WO 2019 / 005755, WO 2019 / 030165, and WO 2020 / 023411 (each of these patents is incorporated herein by reference).

[0299] Method for producing fermentation products from materials containing ungelatinized starch One aspect of the invention relates to a method for producing fermentation products from materials containing ungelatinized starch (i.e., granular starch – commonly referred to as “raw starch hydrolysis”), wherein a composition comprising GH43 arabinofuranase, GH51 arabinofuranase, GH5 xylanase, GH3 β-xylosidase, GH30 xylanase and optionally CE3 acetylxylan esterase and / or GH31 α-xylosidase is present or added during saccharification and / or fermentation.

[0300] In an embodiment, a method for producing fermentation products from materials containing ungelatinized starch includes the following steps: (a) Saccharifying starch-containing materials using α-amylase and glucosylase at a temperature below the initial gelatinization temperature of starch to produce fermentable sugars; and (b) Fermenting the sugar using a fermentation organism to produce a fermentation product; The composition comprising GH43 arabinofuranylase, GH51 arabinofuranylase, GH5 xylanase, GH3 β-xylosidase, GH30 xylanase and optionally CE3 acetylxylan esterase and / or GH31 α-xylosidase is present in or added to the saccharification step (a) and / or fermentation step (b).

[0301] The present invention contemplates the use of any one of the following exemplary GH43 arabinofuranylase, GH51 arabinofuranylase, GH5 xylanase, GH3 β-xylanase, GH30 xylanase, CE3 acetylxylan esterase, and GH31 α-xylanase in the compositions of the present invention and in the methods of using the compositions of the present invention (including in the following exemplary compositions used in the methods for producing fermentation products).

[0302] The exemplary compositions used in step (a) and / or step (b) comprise GH43 arabinofuranylase, GH51 arabinofuranylase, GH5 xylanase, GH3 β-xylanase, and GH30 xylanase. In an example, the GH30 xylanase activity is GH30_1 xylanase. In an example, the GH30 xylanase activity is GH30_2 xylanase. In an example, the GH30 xylanase activity is GH30_3 xylanase. In an example, the GH30 xylanase activity is GH30_4 xylanase. In an example, the GH30 xylanase activity is GH30_5 xylanase. In an example, the GH30 xylanase activity is GH30_7 xylanase. In an example, the GH30 xylanase activity is GH30_8 xylanase. In an example, the GH30 xylanase activity is GH30_9 xylanase. In the examples, the GH30 xylanase activity is GH30_10 xylanase.

[0303] In this embodiment, the GH5 family xylanase is GH5_21 xylanase. The exemplary compositions used in step (a) and / or step (b) comprise GH43 arabinofuranase, GH51 arabinofuranase, GH5_21 xylanase, GH3 β-xylosidase, and GH30_7 xylanase. The exemplary compositions used in step (a) and / or step (b) comprise GH43 arabinofuranase, GH51 arabinofuranase, GH5_21 xylanase, GH3 β-xylosidase, and GH30_8 xylanase. In this embodiment, the GH5 family xylanase is GH5_35 xylanase. In this embodiment, the core comprises GH43 arabinofuranase, GH51 arabinofuranase, GH5_35 xylanase, GH3 β-xylosidase, and GH30_7 xylanase. The exemplary compositions used in step (a) and / or step (b) comprise GH43 arabinofuranase, GH51 arabinofuranase, GH5_35 xylanase, GH3 β-xylanase and GH30_8 xylanase.

[0304] In the embodiments, the compositions used in step (a) and / or step (b) include CE3 acetylxylan esterase. Exemplary compositions used in step (a) and / or step (b) comprise GH43 arabinofuranylase, GH51 arabinofuranylase, GH5 xylanase, GH3 β-xylosidase, GH30 xylanase, and CE3 acetylxylan esterase. Exemplary compositions used in step (a) and / or step (b) comprise GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_21 xylanase, GH3 β-xylosidase, GH30_7 xylanase, and CE3 acetylxylan esterase. The exemplary compositions used in step (a) and / or step (b) comprise GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_21 xylanase, GH3 β-xylosidase, GH30_8 xylanase, and CE3 acetylxylan esterase. The exemplary compositions used in step (a) and / or step (b) comprise GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_35 xylanase, GH3 β-xylosidase, GH30_7 xylanase, and CE3 acetylxylan esterase. The exemplary compositions used in step (a) and / or step (b) comprise GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_35 xylanase, GH3 β-xylosidase, GH30_8 xylanase, and CE3 acetylxylan esterase.

[0305] In the embodiments, the compositions used in step (a) and / or step (b) include GH31 α-xylosidase. In the embodiments, the compositions used in step (a) and / or step (b) include CE3 acetylxylan esterase and GH31 α-xylosidase. Exemplary compositions used in step (a) and / or step (b) comprise GH43 arabinofuranase, GH51 arabinofuranase, GH5 xylanase, GH3 β-xylosidase, GH30 xylanase, CE3 acetylxylan esterase, and GH31 α-xylosidase. Exemplary compositions used in step (a) and / or step (b) comprise GH43 arabinofuranase, GH51 arabinofuranase, GH5_21 xylanase, GH3 β-xylosidase, GH30_7 xylanase, CE3 acetylxylan esterase, and GH31 α-xylosidase. The exemplary compositions used in step (a) and / or step (b) comprise GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_21 xylanase, GH3 β-xylanase, GH30_8 xylanase, CE3 acetylxylan esterase, and GH31 α-xylanase. The exemplary compositions used in step (a) and / or step (b) comprise GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_35 xylanase, GH3 β-xylanase, GH30_7 xylanase, CE3 acetylxylan esterase, and GH31 α-xylanase. The exemplary compositions used in step (a) and / or step (b) comprise GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_35 xylanase, GH3 β-xylosidase, GH30_8 xylanase, CE3 acetylxylan esterase, and GH31 α-xylosidase.

[0306] In this embodiment, GH43 arabinofuranase is GH43_36 arabinofuranase. In this embodiment, GH51 arabinofuranase is GH51_6 arabinofuranase.

[0307] In one embodiment, the composition is added during saccharification step (b). In another embodiment, the composition is added during fermentation step (c). In yet another embodiment, steps (b) and (c) are performed simultaneously in simultaneous saccharification and fermentation (SSF). In yet another embodiment, the composition is added during SSF.

[0308] Raw starch hydrolysis (RSH) is well known in the art. Those skilled in the art will understand that, except for the process parameters related to the liquefaction step (a) which is not performed in the RSH process, the process parameters described in Part II above apply to the methods described in this part, including the selection of starch-containing materials, reduction of grain particle size, saccharification temperature, time, and pH, conditions for simultaneous saccharification and fermentation, and saccharifying enzymes. Process parameters for the exemplary raw starch hydrolysis method are further detailed in WO 2004 / 106533 (which is incorporated herein by reference).

[0309] Examples of α-amylases preferably used in step (a) and / or step (b) include, but are not limited to, α-amylases described in the following: WO 2004 / 055178, WO 2005 / 003311, WO 2006 / 069290, WO 2013 / 006756, WO 2013 / 034106, WO 2021 / 163015, and WO 2021 / 163036 (each of these patents is incorporated herein by reference).

[0310] Examples of glucosylamylase preferably used in step (a) and / or step (b) include, but are not limited to, WO1999 / 28448, WO 2005 / 045018, WO2005 / 069840, and WO 2006 / 069289 (each of these patents is incorporated herein by reference).

[0311] Examples of compositions comprising α-amylase and glucosylamylase preferably used in step (a) and / or step (b) include, but are not limited to, the compositions described in WO 2015 / 031477 (which is incorporated herein by reference).

[0312] Back-end or downstream processing A. Recovery of fermentation products and production of whole distiller's grains Following fermentation or SSF, the fermentation products can be separated from the fermentation medium. Fermentation products (e.g., ethanol) can optionally be recovered from the fermentation medium using any method known in the art, including but not limited to chromatography, electrophoresis, differential solubility, distillation, or extraction. For example, alcohols can be separated and purified from fermented starch-containing materials using conventional distillation methods.

[0313] Therefore, in one embodiment, the method of the present invention further includes distillation to obtain a fermentation product, such as ethanol. Fermentation and distillation may be carried out simultaneously and / or separately / sequentially; optionally, this is followed by one or more process steps for further refining the fermentation product. After the distillation process is completed, the remaining material is considered as whole lees.

[0314] As another example, the desired fermentation product can be extracted from the fermentation medium using microfiltration or membrane filtration techniques. Ethanol with a purity of up to approximately 96 vol.% can be obtained, which can be used as, for example, fuel ethanol, drinking ethanol (i.e., a drinkable neutral alcoholic beverage), or industrial ethanol.

[0315] In some embodiments of these methods, the recovered fermentation product is substantially pure. With respect to these methods herein, "substantially pure" means that the recovered formulation contains no more than 15% impurities, where impurities refer to compounds other than the fermentation product (e.g., ethanol). In one variant, a substantially pure formulation is provided, wherein the formulation contains no more than 25% impurities, or no more than 20% impurities, or no more than 10% impurities, or no more than 5% impurities, or no more than 3% impurities, or no more than 1% impurities, or no more than 0.5% impurities.

[0316] Suitable assays for the production of ethanol and contaminants, as well as sugar consumption, can be performed using methods known in the art. For example, ethanol products and other organic compounds can be analyzed by methods such as HPLC (High Performance Liquid Chromatography), GC-MS (Gas Chromatography-Mass Spectrometry), and LC-MS (Liquid Chromatography-Mass Spectrometry) or other suitable analytical methods using conventional procedures well known in the art. The release of ethanol from fermentation broth can also be tested using culture supernatants. HPLC (Lin et al.) can be used, for example, with refractive index detectors for glucose and alcohols, and UV detectors for organic acids. Biotechnol. Bioeng. [Biotechnology and Bioengineering] 90:775-779 (2005) or quantify byproducts and residual sugars (e.g., glucose or xylose) in the fermentation medium using other suitable assays and detection methods known in the art.

[0317] B. Process the entire lees into lees water and wet cake. In one embodiment, the whole grains are processed into two streams—a wet cake and a centrifugal filtrate. The whole grains are separated or partitioned into solid and liquid phases by one or more methods that separate the centrifugal filtrate from the wet cake. The centrifugal filtrate is split into two streams—grains water, which enters an evaporator; and a countercurrent stream, which is recycled back to the front of the plant. Any suitable separation technique, including centrifugation, pressing, and filtration, can be used to separate the whole grains into a centrifugal filtrate (e.g., grains water, when pumped to an evaporator rather than the front of the plant) and a wet cake to remove most of the liquid / water. In a preferred embodiment, separation / dehydration is performed by centrifugation. In industry, the preferred centrifuge is a sedimentation centrifuge, preferably a high-speed sedimentation centrifuge. An example of a suitable centrifuge is the NX 400 steep cone series from Alfa Laval, a high-performance decanter. Similar sedimentation centrifuges are also available from FLOTTWEG. In another preferred embodiment, separation is performed using other conventional separation equipment (such as plate / frame filter presses, belt filter presses, screw presses, gravity thickeners, and dewatering machines) or similar equipment.

[0318] C. Processing of distiller's grains Distillers' grains water is the term for the supernatant used in whole-grain centrifugation. Typically, distillers' grains water contains 4%–8% dry solids (DS) (primarily proteins, soluble fiber, fats, fine fibers, and cell wall components) and is at a temperature of approximately 60°C–90°C. The distillers' grains water stream can be condensed by evaporation to provide two process streams: (i) an evaporator condensate stream, which contains the condensate removed from the distillers' grains water during evaporation; and (ii) a slurry stream, which contains a more concentrated stream of non-volatile dissolved and undissolved solids, such as non-fermentable sugars and oils remaining in the distillers' grains water due to the removal of the evaporated water.

[0319] Optionally, oil can be removed from the lees water, or it can be removed as an intermediate step in an evaporation process that typically uses a series of evaporation stages.

[0320] The slurry and / or deoiled slurry can be introduced into a dryer together with the wet cake (from the whole lees separation step) to provide a product known as soluble dry lees, which can also be used as animal feed. In an embodiment, the slurry and / or deoiled slurry is sprayed into one or more dryers to combine the slurry and / or deoiled slurry with the whole lees to produce soluble dry lees.

[0321] Distiller's grains water (e.g., optionally hydrolyzed) of between 5-90 vol.%, such as between 10%-80%, between 15%-70%, or between 20%-60%, can be recycled (as a countercurrent) to step (a). The recycled distiller's grains water (i.e., countercurrent) can account for about 1-70 vol.%, preferably 15-60 vol.%, and especially about 30-50 vol.%. In embodiments, the process further includes, optionally, recycling at least a portion of the distiller's grains water stream into the slurry after oil has been extracted from the distiller's grains water stream.

[0322] D. Drying of wet cakes and production of dried lees and dried lees containing solubles. After the wet cake containing about 25-40 wt.%, preferably 30-38 wt.%, of dry solids has been separated from the distillers' grains water (e.g., dehydrated), it can be dried in a drum dryer, spray dryer, ring dryer, fluidized bed dryer, etc., to produce "dried distillers' grains" (DDG). DDG is a valuable feed ingredient for animals such as livestock, poultry, and fish. DDG is preferably provided with a moisture content of less than about 10-12 wt.% to avoid mold and microbial decomposition and to increase shelf life. Additionally, a high moisture content makes transporting DDG more expensive. The wet cake is preferably dried under conditions that do not denature the proteins in it. The wet cake can be blended with a slurry separated from the distillers' grains water and dried to DDG containing solubles (DDGS). Partially dried intermediates, such as sometimes referred to as modified wet distillers' grains, can be produced by partially drying the wet cake, optionally adding slurry before, during, or after the drying process.

[0323] Example The enzymes used in the example: GH43A: An exemplary GH43 arabinofuranase from *Hymenopsporum bisporum* disclosed in SEQ ID NO: 1; GH43B: An exemplary GH43 arabinofuranase from *Dioscorea thecogene* disclosed in SEQ ID NO: 2; GH43C: An exemplary GH43 arabinofuranase from a perforated shell disclosed in SEQ ID NO: 3; GH51A: An exemplary GH51 arabinofuranase from *Grifola frondosa* disclosed in SEQ ID NO: 4; GH51B: An exemplary GH51 arabinofuranase from *Trichoderma cocovenenans* disclosed in SEQ ID NO: 5; GH51C: An exemplary GH51 arabinofuranase from *Bohemiania* disclosed in SEQ ID NO: 6; GH5_21A: Exemplary GH5_21 xylanase from Bacteroides CL02T12C19 disclosed in SEQ ID NO: 7; GH5_21B: An exemplary GH5_21 xylanase from xanthan gum basic community S disclosed in SEQ ID NO: 8; GH5_21C: An exemplary GH5_21 xylanase from genus Sphingosporobacter-64162 disclosed in SEQ ID NO: 9; GH5_21D: An exemplary GH5_21 xylanase from genus Sphingosporobacter-64162 disclosed in SEQ ID NO: 10; GH5_21E: Exemplary GH5_21 xylanase from xanthan gum basic community O disclosed in SEQ ID NO: 11; GH5_21F: An exemplary GH5_21 xylanase from a bioreactor metagenomics disclosed in SEQ ID NO: 12; GH5_21G: Exemplary GH5_21 xylanase from xanthan gum basic community T disclosed in SEQ ID NO: 13; GH5_21H: Exemplary GH5_21 xylanase from xanthan gum basic community S disclosed in SEQ ID NO: 14; GH5_21I: An exemplary GH5_21 xylanase from species-64282 of the genus *Beleria* disclosed in SEQ ID NO: 15; GH5_21J: Exemplary GH5_21 xylanase from *Iridaceae iridozoae* disclosed in SEQ ID NO: 16; GH5_21K: Exemplary GH5_21 xylanase from xanthan gum basic community T disclosed in SEQ ID NO: 17; GH5_21L: Exemplary GH5_21 xylanase from the genus *Sphingosporobacter* disclosed in SEQ ID NO: 18; GH5_21M: An exemplary GH5_21 xylanase from elephant feces metagenomics disclosed in SEQ ID NO: 19; GH5_21N: An exemplary GH5_21 xylanase from elephant feces metagenomics disclosed in SEQ ID NO: 20; GH5_21O: An exemplary GH5_21 xylanase from a species of the genus *Chlorella* disclosed in SEQ ID NO: 21; GH5_35A: Exemplary GH5_35 xylanase from *Hydroxylum hygrophytes* disclosed in SEQ ID NO: 22; GH5_35B: Exemplary GH5_35 xylanase from Bacillus hemicellulose JCM 9152 disclosed in SEQ ID NO: 23; GH5_35C: An exemplary GH5_35 xylanase from Bacillus species-62332 disclosed in SEQ ID NO: 24; GH5_35D: An exemplary GH5_35 xylanase from a compost metagenomics disclosed in SEQ ID NO: 25; GH5_35E: Exemplary GH5_35 xylanase from Bacillus chrysogenus disclosed in SEQ ID NO: 26; GH3A: An exemplary GH3 β-xylosidase from Aspergillus fumigatus disclosed in SEQ ID NO: 27; GH3B: An exemplary GH3 β-xylosidase from Aspergillus nidulans disclosed in SEQ ID NO: 28; GH3C: An exemplary GH3 β-xylosidase from *Eimersoniana* disclosed in SEQ ID NO: 29; GH3D: Aspergillus terreus disclosed in SEQ ID NO: 30 Aspergillus terrestris Exemplary GH3 β-xylosidase; GH3E: An exemplary GH3 β-xylosidase from Aspergillus echinospora disclosed in SEQ ID NO: 31; GH3F: An exemplary GH3 β-xylosidase from Aspergillus Fischer disclosed in SEQ ID NO: 32; GH3G: An exemplary GH3 β-xylosidase from Chaetomium globosa disclosed in SEQ ID NO: 33; GH3H: An exemplary GH3 β-xylosidase from Chaetoceros spp. disclosed in SEQ ID NO: 34; GH3I: An exemplary GH3 β-xylosidase from Fusarium spp. disclosed in SEQ ID NO: 35; GH3J: An exemplary GH3 β-xylosidase from thermophilic bacteria disclosed in SEQ ID NO: 36; GH3K: An exemplary GH3 β-xylosidase from *Penicillium emersonii* disclosed in SEQ ID NO: 37; GH3L: An exemplary GH3 β-xylosidase from Penicillium oxalate disclosed in SEQ ID NO: 38; GH3M: An exemplary GH3 β-xylosidase from Coleoptera faecalis disclosed in SEQ ID NO: 39; GH3N: An exemplary GH3 β-xylosidase from *Basilella spicata* disclosed in SEQ ID NO: 40; GH3O: An exemplary GH3 β-xylosidase from Trichoderma reesei disclosed in SEQ ID NO: 41; GH30_7A: Exemplary GH30_7 xylanase from *Basilella repens* disclosed in SEQ ID NO: 42; GH30_8A: A species of Bacillus disclosed in SEQ ID NO: 53 - Exemplary GH30_8 xylanase of 18423; CE3A: An exemplary CE3 polypeptide containing acetylated xylan esterase from a species of the genus Trichoderma, disclosed as SEQ ID NO: 58; CE3B: Disclosed as SEQ ID NO: 59 from *Pseudomonas sorghum* ( Epicoccum sorghum Exemplary CE3 acetylxylan esterase; CE3C: Disclosed as SEQ ID NO: 60 from enoki mushrooms ( Flammulina velutipes Exemplary CE3 acetylxylan esterase; CE3D: Disclosed as SEQ ID NO: 61 from Microsporum ( Microsphaeropsis reundinis Exemplary CE3 acetylxylan esterase; CE3E: Exemplary CE3 acetylatedlanase from Micrococcus pluvialis, disclosed as SEQ ID NO: 62; CE3F: Exemplary CE3 acetylatedlanase from Micrococcus pluvialis as SEQ ID NO: 63; CE3G: Disclosed as SEQ ID NO: 64 from *Parasitic glomerulosa* ( Paraphaeospheres neglected Exemplary CE3 acetylxylan esterase; CE3H: Disclosed as SEQ ID NO: 65, from *Paranoia verrucous* ( Paraphaeospheres warty Exemplary CE3 acetylxylan esterase; CE3I: Disclosed as SEQ ID NO: 66 from *Vibrio purpureus* ( Westerdykella purpurea Exemplary CE3 acetylxylan esterase; CE3J: Disclosed as SEQ ID NO: 67 from *Hylocereus undatus* ( White-tailed eagle Exemplary CE3 acetylxylan esterase; GH31A: An exemplary GH31 α-xylosidase from *Hymenopterinella hemicellulose* disclosed in SEQ ID NO: 68; GH8: An exemplary GH8 xylanase from Bacillus species KK-1 disclosed in SEQ ID NO: 69; GH10: An exemplary GH10 xylanase from *Aspergillus echinococcosis* disclosed in SEQ ID NO: 70; and GH11: From *Thermophilus spp.* disclosed in SEQ ID NO: 71 ( Thermomyces downy An example of GH11 xylanase.

[0324] Liquefying enzyme blend 1: from *Bacillus stearothermophilus* disclosed in SEQ ID NO: 72 ( Bacillus stearothermophilus Exemplary thermostable α-amylases; those from *Streptococcus virens* disclosed in SEQ ID NO: 74. Pyrococcus furiosus Examples of thermostable proteases.

[0325] Liquefying enzyme blend 2: an exemplary heat-stable α-amylase from *Bacillus stearothermophilus* disclosed in SEQ ID NO: 73; an exemplary heat-stable protease from *Cyclophorus virescens* disclosed in SEQ ID NO: 74; and an exemplary heat-stable protease from *Thermophyton floccosum* disclosed in SEQ ID NO: 75. Sea-dwelling thermotoga An example of a thermostable xylanase.

[0326] Saccharifying enzyme blend: from *Entoloma viminalis* disclosed in SEQ ID NO: 76 ( Gloeophyllum hedgerow Exemplary glucosyl amylases; those from *Rhizopus microphylla* disclosed in SEQ ID NO: 77. Rhizomucor little one Exemplary α-amylases; those from *Basilella fragilis* disclosed in SEQ ID NO: 78. Talaromyces rope-like Exemplary trehalases; an exemplary β-glucosidase from Aspergillus fumigatus disclosed in SEQ ID NO: 79; an exemplary cellobiase from Aspergillus fumigatus disclosed in SEQ ID NO: 80; and an exemplary endoglucanase from Trichoderma reesei disclosed in SEQ ID NO: 81.

[0327] Hemicellulase blends: GH43 arabinofuranase (GH43A), GH51 arabinofuranase (GH51A), GH5_21 xylanase (GH5_21O) and GH3 β-xylosidase (GH3A).

[0328] Td of liquefying enzyme was determined by differential scanning calorimetry. The thermal stability of the enzyme was determined by differential scanning calorimetry (DSC) using a VP-capillary differential scanning calorimeter (MicroCal Inc., Piscataway, NJ, USA). The thermal analysis chromatogram (Cp vs. T) obtained after heating the enzyme solution (approximately 0.5 mg / ml) in buffer (50 mM acetate, pH 5.0) at a constant programmed heating rate of 200 K / h was used, with the denaturation temperature Td (°C) considered as the apex of the denaturation peak (the main endothermic peak).

[0329] The sample solution and reference solution (approximately 0.2 ml) were loaded into the calorimeter from storage conditions at 10°C (reference: enzyme-free buffer) and pre-equilibrated at 20°C for 20 minutes, followed by DSC scanning from 20°C to 120°C. The denaturation temperature was determined with an accuracy of approximately + / -1°C.

[0330] strain Yeast strain MEJI797 is MBG5012 of WO 2019 / 161227, which further expresses *Hemiberlesia javanica* (…). Pycnopous sanguineus ) glucosyl amylase (SEQ ID NO: 4 of WO 2011 / 066576) and a heterozygous Rhizopus microsporus α-amylase expression cassette (as described in WO 2013 / 006756).

[0331] Culture media and solutions The PDA plate consists of 39 grams of potato dextrose agar and 1 liter of deionized water.

[0332] The skim milk culture medium consists of a 10% skim milk powder solution and is autoclaved.

[0333] Example 1: The combination of xylanases from GH family 5, 8, 10, 11 and 30 with arabinofuranases from GH family 43 and 51 enhances the xylose and arabinose effects in simultaneous saccharification and fermentation methods. Industrial liquefied mash prepared with liquefying enzyme blend 1 was used in the experiment. The dry solids (DS) was determined by a moisture balance to be approximately 34%, and the pH was adjusted to 5.0, followed by the addition of 3 ppm penicillin and 500 ppm urea. Simultaneous saccharification and fermentation (SSF) was performed via small-scale fermentation. Approximately 4.0 g of industrially liquefied corn mash was added to 15 ml vials. 0.42 AGU / gDS of the saccharifying enzyme blend and appropriate amounts of the corresponding xylanase and arabinofuranosaccharidase were added to each vial, as shown in Table 2. The saccharifying enzyme blend served as a control without the addition of xylanase or arabinofuranosaccharidase. The actual enzyme dosage was based on the precise weight of the corn mash in each vial. After enzyme addition, fermentation was initiated by adding 50 µL of propagated yeast strain MEJI797. The tubes were incubated at 32°C, and each treatment was repeated three times. After 65 hours of SSF, the tubes were removed from the incubator, 50 µL of 34% H2SO4 was added, and the tubes were centrifuged at 3500 rpm for 10 minutes, followed by filtration through a 0.45 μm filter. Sugar concentrations were determined using an HPLC system equipped with an H column (Benson Polymeric, BP-700 H, 300 × 7.8 mm).

[0334] Table 2: Dosage regimens of arabinofuranosylase with or without xylanase Table 3: Results Increase in arabinose (%) = [(Average arabinose experimental ppm - Average arabinose control) / Average arabinose control] × 100 Table 3 shows that GH5_21 or GH30_8 xylanase, in combination with GH43 and GH51 arabinofuranase, releases the highest concentration of arabinose compared to GH43 or GH51 arabinofuranase alone or their combination without xylanase.

[0335] Example 2: Combining GH3 family β-xylosidases with arabinofuranases from GH 43 and 51 families and xylanases from GH5_21 enhances the xylose effect in simultaneous saccharification and fermentation methods. Industrial liquefied mash prepared with liquefying enzyme blend 2 was used in the experiment. The dry solids content (DS) was determined by a moisture balance to be approximately 35.9%, and the pH was adjusted to 5.0, followed by the addition of 3 ppm penicillin and 500 ppm urea. Simultaneous saccharification and fermentation (SSF) were carried out via small-scale fermentation. Approximately 4.2 g of industrial liquefied corn mash was added to 15 ml pipettes. 0.42 AGU / gDS of the saccharifying enzyme blend and appropriate amounts of the corresponding xylanase, arabinofuranase, and β-xylosidase (as listed in Table 4) were added to each vial. The dosing protocol followed a fixed amount of GH5_21 xylanase, GH43 arabinofuranase, and GH51 arabinofuranase, each at 10 μg / g dry solids, with or without 25, 50, 100, or 200 μg / g dry solids of β-xylosidase GH3A, GH3B, or GH3C. A saccharifying enzyme blend was used as a control without the addition of xylanase or β-xylosidase. Actual enzyme dosages were based on the precise weight of corn steep liquor in each vial. After enzyme addition, fermentation was initiated by adding 50 µL of propagated yeast strain MEJI797. Tubes were incubated at 32°C, with each treatment repeated three times. After 65 hours of SSF, tubes were removed from the incubator and centrifuged at 3500 rpm for 10 minutes, followed by filtration through a 0.45 μm filter. Sugar concentrations were determined using an HPLC system equipped with a lead column (Benson Polymeric, BP-800 Pb, 300 × 7.8 mm).

[0336] result Table 4 shows that the combination of β-xylosidase with GH43, GH51 arabinofuranase and GH5_21 xylanase significantly increases xylose release, and higher enzyme doses correspond to higher xylose release.

[0337] Table 4 Example 3: Combining GH 5 xylanase subfamily 21 and 35 with Hi GH 43 and Mg GH51 arabinofuranosidase and AfGH3 β-xylosidase to enhance the xylose and arabinose effects in simultaneous saccharification and fermentation methods. Industrial liquefied mash prepared with liquefying enzyme blend 1 was used in the experiments. The dry solids (DS) was determined by a moisture balance to be approximately 33.7%, and the pH was adjusted to 5.0, followed by the addition of 3 ppm penicillin and 500 ppm urea. Simultaneous saccharification and fermentation (SSF) was performed via small-scale fermentation. Approximately 4.2 g of industrially liquefied corn mash was added to 15 ml vials. Each vial was added with 0.42 AGU / gDS of the saccharifying enzyme blend, 10 μg / gDS of GH43A arabinofuranylase, 10 μg / gDS of GH51A arabinofuranylase, 25 μg / gDS of GH3A β-xylosidase, and 10 μg / gDS of the corresponding xylanase (as listed in Table 5). The saccharifying enzyme blend served as a control without the addition of arabinofuranylase, xylanase, or β-xylosidase. The actual enzyme dosage was based on the precise weight of corn steep liquor in each vial. After enzyme addition, fermentation was initiated by adding 50 µL of propagated yeast strain MEJI797. The tubes were incubated at 32°C, with each treatment repeated three times. After 65 hours of SSF, the tubes were removed from the incubator, 50 µL of 34% H₂SO₄ was added, and the tubes were centrifuged at 3500 rpm for 10 minutes, followed by filtration through a 0.45 μm filter. Sugar concentrations were determined using an HPLC system equipped with a lead column (Benson Polymeric, BP-800 Pb, 300 × 7.8 mm).

[0338] result Table 5 shows that, compared with the control or the treatment consisting of GH43, GH51 arabinofuranosidase and GH3 β-xylosidase without xylanase, the addition of xylanase from GH5_21 and GH5_35 significantly increased the release of xylose and arabinose.

[0339] Table 5 Example 4: Single, dual, triple, or quadruple combinations of hemicellulases such as GH5_21 xylanase, GH43 and GH51 arabinofuranase, and GH3 β-xylosidase enhance the xylose and arabinose effects in simultaneous saccharification and fermentation methods. Industrial liquefied mash prepared with liquefying enzyme blend 2 was used in the experiments. The dry solids content (DS) was determined by a moisture balance to be approximately 33.4%, and the pH was adjusted to 5.0, followed by the addition of 3 ppm penicillin and 500 ppm urea. Simultaneous saccharification and fermentation (SSF) was performed via small-scale fermentation. Approximately 4.2 g of industrially liquefied corn mash was added to 15 ml vials. 0.6 AGU / gDS of the saccharifying enzyme blend was added to each vial, following a dosing regimen of 10 μg / gDS GH5_21O xylanase, 10 μg / gDS GH43A arabinofuranase, 10 μg / gDS GH51A arabinofuranase, and / or 25 μg / gDS GH3A β-xylosidase. As a control, only the saccharifying enzyme blend was added without arabinofuranase, xylanase, or β-xylosidase. The actual enzyme dosage was based on the precise weight of corn mash in each vial. After enzyme addition, fermentation was initiated by adding 50 µL of hydrated yeast strain MEJI797. Tubes were incubated at 32°C, with each treatment repeated three times. After 65 hours of SSF, the tubes were removed from the incubator, 50 µL of 34% H2SO4 was added, and the tubes were centrifuged at 3500 rpm for 10 minutes, followed by filtration through a 0.45 μm filter. Sugar concentrations were determined using an HPLC system equipped with an H column (Benson Polymeric, BP-700 H, 300 × 7.8 mm). Decanters containing wet corn mash at the end of fermentation were freeze-dried under vacuum for 3 days. The weight of the dried solids in each tube was determined, and the residual solids were calculated as the ratio of the final solid weight to the initial solid weight.

[0340] result Table 6 Table 6 shows that, compared with the control or the treatment consisting of GH43, GH51 arabinofuranosidase and GH3 β-xylosidase without xylanase, the addition of GH5_21 xylanase, along with GH43, GH51 arabinofuranosidase and GH3 β-xylosidase, increased the release of xylose and arabinose.

[0341] Example 5: Combining arabinofuranases from GH family 43 and 51 with xylanases from GH family subfamily 5 21 enhances the effect of arabinose in simultaneous saccharification and fermentation methods. Industrial liquefied mash prepared with liquefying enzyme blend 2 was used in the experiments. The dry solids (DS) was determined by a moisture balance to be approximately 36%, and the pH was adjusted to 5.0, followed by the addition of 3 ppm penicillin and 500 ppm urea. Simultaneous saccharification and fermentation (SSF) was performed via small-scale fermentation. Approximately 4.0 g of industrial liquefied corn mash was added to 15 ml vials. 0.42 AGU / g DS of the saccharifying enzyme blend and appropriate amounts of arabinofuranylase combinations from families GH43 and GH51 (as listed in Table 7) were added to each vial. The addition protocol followed a fixed amount of GH5_21O xylanase, GH43 arabinofuranylase, and GH51 arabinofuranylase, each at 10 μg / g DS. As a control, only the saccharifying enzyme blend was used without the addition of xylanase or arabinofuranylase. Actual enzyme dosages were based on the precise weight of the corn mash in each vial. After enzyme addition, fermentation was initiated by adding 50 µL of proliferated yeast strain MEJI797. The tubes were incubated at 32 °C, with each treatment repeated three times. After 65 hours of SSF, the tubes were removed from the incubator, 50 µL of 34% H₂SO₄ was added, and the tubes were centrifuged at 3500 rpm for 10 minutes, followed by filtration through a 0.45 μm filter. Sugar concentrations were determined using an HPLC system equipped with an H column (Benson Polymeric, BP-700 H, 300 × 7.8 mm).

[0342] result Table 7 Increase in arabinose (%) = [(Average arabinose experimental ppm - Average arabinose control) / Average arabinose control] × 100 Table 7 shows that, compared with the control without arabinofuranase and xylanase, the combination of GH43 and GH51 arabinofuranase with GH5_21 xylanase increased arabinose.

[0343] Example 6: Combining arabinofuranases from GH family 43 and 51 with xylanases from GH family subfamily 5 21 enhances the effect of arabinose in simultaneous saccharification and fermentation methods. Industrial liquefied mash prepared with liquefying enzyme blend 2 was used in the experiments. The dry solids (DS) was determined by a moisture balance to be approximately 33.8%, and the pH was adjusted to 5.0, followed by the addition of 3 ppm penicillin and 500 ppm urea. Simultaneous saccharification and fermentation (SSF) was performed via small-scale fermentation. Approximately 4.2 g of industrially liquefied corn mash was added to 15 ml vials. 0.42 AGU / g DS of the saccharifying enzyme blend and appropriate amounts of arabinofuranylase combinations from families GH43 and GH51 (as listed in Table 8) were added to each vial. The addition protocol followed a fixed amount of GH5_21 xylanase, GH43 arabinofuranylase, and GH51 arabinofuranylase, each at 10 μg / g DS. As a control, only the saccharifying enzyme blend was used without the addition of xylanase or arabinofuranylase. Actual enzyme dosages were based on the precise weight of the corn mash in each vial. After enzyme addition, fermentation was initiated by adding 50 µL of proliferated yeast strain MEJI797. The tubes were incubated at 32 °C, with each treatment repeated three times. After 65 hours of SSF, the tubes were removed from the incubator, 50 µL of 34% H₂SO₄ was added, and the tubes were centrifuged at 3500 rpm for 10 minutes, followed by filtration through a 0.45 μm filter. Sugar concentrations were determined using an HPLC system equipped with an H column (Benson Polymeric, BP-700 H, 300 × 7.8 mm).

[0344] result Table 8 Increase in arabinose (%) = [(Average arabinose experimental ppm - Average arabinose control) / Average arabinose control] × 100 Table 8 shows that, compared with the absence of xylanase and arabinofuranase, the combination of GH43 and GH1 arabinofuranase with GH5_21 xylanase increased arabinose release.

[0345] Example 7: An exemplary CE3 polypeptide with acetylated xylan esterase activity, with or without the exemplary GH31 α-xylosidase, combined with exemplary GH43 arabinofuranase, exemplary GH51 arabinofuranase, exemplary GH5_21 xylanase, and exemplary GH3 β-xylosidase, to enhance the xylose and arabinose effects in a simultaneous saccharification and fermentation process. Industrial liquefied mash prepared with liquefying enzyme blend 1 was used in the experiments. The dry solids (DS) were determined by a moisture balance to be approximately 34%, and the pH was adjusted to 5.0, followed by the addition of 3 ppm penicillin and 500 ppm urea. Simultaneous saccharification and fermentation (SSF) was performed via small-scale fermentation. Approximately 4.2 g of industrially liquefied corn mash was added to 10 ml vials. 0.42 AGU / gDS of the saccharifying enzyme blend, along with the amounts of hemicellulase blend shown in Table 9 and the corresponding CE3 peptide at 10 μg / gDS (Table 10), were added to each vial, followed by 50 µL of proliferated yeast strain MEJI797 per 4.2 g mash. As a control, only the saccharifying enzyme blend was used without the addition of the hemicellulase blend or CE3 peptide. The actual enzyme dosage was based on the precise weight of the corn mash in each vial. The vials were incubated at 32°C, with each treatment repeated three times. After 65 hours of SSF, the tubes were removed from the incubator and centrifuged at 3500 rpm for 10 minutes, followed by filtration through a 0.45-micron filter. Sugar concentrations were determined using an HPLC system equipped with an H column (Benson Polymeric, BP-700 H, 300 × 7.8 mm). Decanters containing wet corn mash at the end of fermentation were freeze-dried under vacuum for 3 days. The weight of dried solids in each tube was determined, and the residual solids were calculated as the ratio of final solid weight to initial solid weight.

[0346] Table 9: Enzyme dosage in hemicellulase blends result As shown in Table 10 below, the combination of CE3 peptide and hemicellulase blend increased xylose and arabinose release compared to the control or the hemicellulase blend without CE3 peptide. Compared to the control, the hemicellulase blend significantly reduced residual solids, and the addition of CE3 peptide further reduced residual solids, indicating that the hemicellulase blend and its combination with CE3 peptide increased maize fiber degradation.

[0347] Table 10 Example 8: An exemplary CE3 polypeptide with acetylated xylan esterase activity, with or without the exemplary GH31 α-xylosidase, combined with exemplary GH43 arabinofuranase, exemplary GH51 arabinofuranase, exemplary GH5_21 xylanase, and exemplary GH3 β-xylosidase, to enhance the xylose and arabinose effects in a simultaneous saccharification and fermentation process. Industrial liquefied mash prepared with liquefying enzyme blend 1 was used in the experiment. The dry solids (DS) was determined by a moisture balance to be approximately 35%, and the pH was adjusted to pH 5.0, followed by the addition of 3 ppm penicillin and 500 ppm urea. Simultaneous saccharification and fermentation (SSF) was carried out via small-scale fermentation. Approximately 4.2 g of industrial liquefied corn mash was added to 10 ml vials. With or without the use of the exemplary GH31 α-xylosidase (GH31A) at 10 ug / g DS, 0.42 AGU / g DS of the saccharifying enzyme blend, along with the amount of hemicellulase blend shown in Table 11 and the corresponding CE3 peptide shown in Table 9 above at 10 ug / g DS, were added to each vial, followed by 50 µL of proliferated yeast strain MEJI797 per 4.2 g mash. As a control, only the saccharifying enzyme blend was used without the addition of hemicellulase blend, CE3 peptide, or GH31 α-xylosidase. The actual enzyme dosage was based on the precise weight of corn mash in each vial. The vials were incubated at 32°C, with each treatment repeated three times. After 65 hours of SSF, the tubes were removed from the incubator and centrifuged at 3500 rpm for 10 minutes, followed by filtration through a 0.45 μm filter. Sugar concentrations were determined using an HPLC system equipped with a lead column (Benson Polymeric, BP-800 Pb, 300 × 7.8 mm). Decanters containing wet corn mash at the end of fermentation were freeze-dried under vacuum for 3 days. The weight of the dried solids in each tube was determined, and the residual solids were calculated as the ratio of the final solid weight to the initial solid weight.

[0348] result As shown in Table 11 below, the combination of CE3 peptide and hemicellulase blend increased xylose and arabinose release compared to the control or the hemicellulase blend without CE3 peptide. The addition of the exemplary α-xylosidase (GH31A) further increased xylose and arabinose release when mixed with the CE3 peptide and hemicellulase blend. Compared to the control, the hemicellulase blend significantly reduced residual solids, and the addition of CE3 peptide and GH31 further reduced residual solids, indicating that the hemicellulase blend and its combination with CE3 peptide and GH31 α-xylosidase increased maize fiber degradation.

[0349] Table 11 Example 9: The effect of combining β-xylosidase (BX) with hemicellulase blends (basic C5) from different sources to enhance xylose in simultaneous saccharification and fermentation processes, with or without Aspergillus fumigatus BX. Industrially prepared liquefied mash from the liquefaction product of liquefying enzyme blend 1 was used in the experiments. The dry solids (DS) was determined by a moisture balance to be approximately 34.1%, and the pH was adjusted to 5.0, followed by the addition of 3 ppm penicillin and 1000 ppm urea. Simultaneous saccharification and fermentation (SSF) were performed via small-scale fermentation. Approximately 4.2 g of industrially liquefied corn mash was added to 10 ml vials. 0.6 AGU / gDS of the saccharifying enzyme blend, along with the hemicellulase blend (basic C5, with or without GH3A) as shown in Table 12, and appropriate amounts of the corresponding β-xylosidase from various sources (Table 9), were added to each vial, followed by 50 μL of hydrated ETHANOL RED yeast per 4.2 g mash. As a control, only glucoamylase was used without the addition of basic C5 or GH3 enzyme. Actual enzyme dosages were based on the precise weight of the corn mash in each vial. The vials were incubated at 32°C, with each treatment repeated three times. After 65 hours of SSF, the tubes were removed from the incubator and centrifuged at 3500 rpm for 10 minutes, followed by filtration through a 0.45 μm filter. Sugar concentrations were determined using an HPLC system equipped with an H column (Benson Polymeric, BP-700 H, 300 × 7.8 mm).

[0350] Table 12: Hemicellulase blends (basal C5) result As shown in Table 13 below, with or without GH3A, the combination of BX in the basic C5 hemicellulase blend (especially with Po BX) significantly increased xylose release.

[0351] Table 13 Example 10: The effect of combining GH30 xylanase subfamily 7 or 8 with hemicellulase (basic C5) in simultaneous saccharification and fermentation methods, with or without CE3 enzyme, to enhance the xylose and arabinose effects. Industrially prepared liquefied mash from the liquefaction product of liquefying enzyme blend 1 was used in the experiment. The dry solids (DS) were determined by a moisture balance to be approximately 35%, and the pH was adjusted to 5.0, followed by the addition of 3 ppm penicillin and 1000 ppm urea. Simultaneous saccharification and fermentation (SSF) was performed via small-scale fermentation. Approximately 4.0 g of industrially liquefied corn mash was added to 10 ml vials. 0.6 AGU / gDS of the saccharifying enzyme blend, along with the hemicellulase blend (basic C5) as shown in Table 12 of Example 9, and appropriate amounts of the corresponding GH30_7, GH30_8, and CE3 enzymes (Table 14), were added to each vial, followed by 50 µL of propagated Ethanol Red yeast per 4.0 g mash. As a control, only glucoamylase was used without the addition of basic C5, GH30 xylanase, or CE3 enzyme. Actual enzyme dosages were based on the precise weight of the corn mash in each vial. The vials were incubated at 32°C, with each treatment repeated three times. After 65 hours of SSF, the tubes were removed from the incubator and centrifuged at 3500 rpm for 10 minutes, followed by filtration through a 0.20 μm filter. Sugar concentrations were determined using an HPLC system equipped with an H column (Benson Polymeric, BP-700 H, 300 × 7.8 mm).

[0352] result As shown in Table 14 below, the combination of GH30 subfamily 7 or 8 xylanases with basal C5 hemicellulase increased xylose and arabinose release compared to the control or basal C5 alone. Adding CE3 enzyme to the combination of basal C5 and GH30 xylanase further increased xylose and arabinose release.

[0353] Table 14 The invention described and claimed herein is not limited to the specific aspects disclosed herein, as these aspects are intended to illustrate several aspects of the invention. Any equivalent aspects are intended to be within the scope of the invention. In fact, various modifications to 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 case of conflict, the disclosure including the definition shall prevail.

[0354] The invention is further defined by the following numbered paragraphs: 1. A particle, said particle comprising: (a) A core comprising GH43 arabinofuranylase, GH51 arabinofuranylase, GH5 xylanase, GH3 β-xylanase and GH30 xylanase, and optionally... (b) A coating consisting of one or more layers surrounding the core.

[0355] 2. A particle, said particle comprising: (a) A core comprising GH43 arabinofuranylase, GH51 arabinofuranylase, GH5 xylanase, GH3 β-xylosidase, and GH30 xylanase and CE3 acetylxylan esterase, and optionally... (b) A coating consisting of one or more layers surrounding the core.

[0356] 3. A particle, said particle comprising: (a) core, and (b) A coating consisting of one or more layers surrounding the core, wherein the coating comprises GH43 arabinofuranylase, GH51 arabinofuranylase, GH5 xylanase, GH3 β-xylanase, and GH30 xylanase.

[0357] 4. A particle, said particle comprising: (a) core, and (b) A coating consisting of one or more layers surrounding the core, wherein the coating comprises GH43 arabinofuranylase, GH51 arabinofuranylase, GH5 xylanase, GH3 β-xylosidase, GH30 xylanase and CE3 acetylxylan esterase.

[0358] 5. A liquid composition comprising GH43 arabinofuranylase, GH51 arabinofuranylase, GH5 xylanase, GH3 β-xylanase, GH30 xylanase, and an enzyme stabilizer, such as a polyol like propylene glycol or glycerol, a sugar or sugar alcohol, or lactic acid.

[0359] 6. A liquid composition comprising GH43 arabinofuranylase, GH51 arabinofuranylase, GH5 xylanase, GH3 β-xylanase, GH30 xylanase, CE3 acetylxylan esterase, and an enzyme stabilizer, such as a polyol like propylene glycol or glycerol, a sugar or sugar alcohol, or lactic acid.

[0360] 7. The liquid composition as described in paragraph 5 or 6, wherein the liquid composition further comprises a filler or carrier material.

[0361] 8. The liquid composition as described in any one of paragraphs 5 to 7, wherein the liquid composition further comprises a preservative.

[0362] 9. A composition comprising particles as described in any one of paragraphs 1 to 4, or a liquid composition as described in any one of paragraphs 5 to 8.

[0363] 10. The composition as described in paragraph 9, wherein the composition is a liquid composition, a solid composition, a solution, a dispersion, a paste, a powder, granules, particulate matter, coated particulate matter, a tablet, a cake, a crystal, a crystal slurry, a gel, or a pellet.

[0364] 11. A composition comprising GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_21 xylanase, GH3 β-xylanase, and GH30 xylanase.

[0365] 12. A composition comprising GH43 arabinofuranylase, GH51 arabinofuranylase, GH5_21 xylanase, GH3 β-xylosidase, GH30 xylanase and CE3 acetylatedxylan esterase.

[0366] 13. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, wherein the GH43 arabinofuranase is GH43_36 arabinofuranase.

[0367] 14. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition or composition as described in paragraph 13, wherein the GH43 arabinofuranoside is derived from the genus *Pyrophyllus*, *Trichophyllus*, or *Porphyra*.

[0368] 15. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition, or composition as described in paragraphs 13 or 14, wherein the GH43 arabinofuranoside is derived from species-specific humic molds, cocoa spp., or punctate seat shells.

[0369] 16. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition, or composition as described in any one of paragraphs 13 to 15, wherein the GH43 arabinofuranase has an amino acid sequence selected from the group consisting of: (i) An amino acid sequence of SEQ ID NO: 1 containing 0 to 10 conserved amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 1, having arabinofuranyl glycosidase activity; (ii) An amino acid sequence of SEQ ID NO: 2 containing 0 to 10 conserved amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 2, having arabinofuranosylase activity; and (iii) An amino acid sequence of SEQ ID NO: 3 containing 0 to 10 conserved amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 3, having arabinofuranyl glycosidase activity.

[0370] 17. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition, or composition as described in any one of paragraphs 13 to 16, wherein the GH51 arabinofuranase is GH51_6 arabinofuranase.

[0371] 18. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition, or composition as described in any one of paragraphs 13 to 17, wherein the GH51 arabinofuranase has an amino acid sequence selected from the group consisting of: (i) An amino acid sequence of SEQ ID NO: 4 containing 0 to 10 conserved amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 4, having arabinofuranyl glycosidase activity; (ii) An amino acid sequence of SEQ ID NO: 5 containing 0 to 10 conserved amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 5, having arabinofuranosylase activity; and (iii) An amino acid sequence of SEQ ID NO: 6 containing 0 to 10 conserved amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 6, having arabinofuranyl glycosidase activity.

[0372] 19. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition, or composition as described in any one of paragraphs 13 to 18, wherein the GH5 xylanase is GH5_21 xylanase.

[0373] 20. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition, or composition as described in any one of paragraphs 13 to 19, wherein the GH5_21 xylanase is derived from Bacteroides, Beleuth, Chrysobacterium, or Sphingosporobacter.

[0374] 21. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition, or composition as described in any one of paragraphs 13 to 20, wherein the GH5_21 xylanase is derived from species Bacteroides filamentosa CL02Y12C19, Belylella spp. species-64282, Chlorella spp. species, Iridobacterium iridocycline, or Sphingomyelinobacterium spp. species-64162.

[0375] 22. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition, or composition as described in any one of paragraphs 13 to 21, wherein the GH5_21 xylanase is derived from a bioreactor metagenomics, an elephant dung metagenomics, a xanthan gum basic community O, a xanthan gum basic community S, or a xanthan gum basic community T.

[0376] 23. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition, or composition as described in any one of paragraphs 13 to 22, wherein the GH5_21 xylanase has an amino acid sequence selected from the group consisting of: (i) An amino acid sequence of SEQ ID NO: 7 containing 0 to 10 conserved amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 7, having xylanase activity; (ii) An amino acid sequence of SEQ ID NO: 8 containing 0 to 10 conserved amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 8, having xylanase activity; (iii) An amino acid sequence of SEQ ID NO: 9 containing 0 to 10 conserved amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 9, having xylanase activity; (iv) An amino acid sequence of SEQ ID NO: 10 containing 0 to 10 conserved amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 10, having xylanase activity; (v) An amino acid sequence of SEQ ID NO: 11 containing 0 to 10 conserved amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 11, having xylanase activity; (vi) An amino acid sequence of SEQ ID NO: 12 containing 0 to 10 conserved amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 12, having xylanase activity; (vii) An amino acid sequence of SEQ ID NO: 13 containing 0 to 10 conserved amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 13, having xylanase activity; (viii) An amino acid sequence of SEQ ID NO: 14 containing 0 to 10 conserved amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 14, having xylanase activity; (ix) An amino acid sequence containing 0 to 10 conserved amino acid substitutions of SEQ ID NO: 15, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 15, having xylanase activity; (x) An amino acid sequence of SEQ ID NO: 16 containing 0 to 10 conserved amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 16, having xylanase activity; (xi) An amino acid sequence containing 0 to 10 conserved amino acid substitutions of SEQ ID NO: 17, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 17, having xylanase activity; (xii) An amino acid sequence containing 0 to 10 conserved amino acid substitutions of SEQ ID NO: 18, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 18, having xylanase activity; (xiii) An amino acid sequence containing 0 to 10 conserved amino acid substitutions of SEQ ID NO: 19, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 19, having xylanase activity; (xiv) An amino acid sequence of SEQ ID NO: 20 containing 0 to 10 conserved amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 20, having xylanase activity; and (xv) An amino acid sequence containing 0 to 10 conserved amino acid substitutions of SEQ ID NO: 21, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 21, having xylanase activity.

[0377] 24. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition, or composition as described in any one of paragraphs 13 to 23, wherein the GH5 xylanase is GH5_35 xylanase.

[0378] 25. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition, or composition as described in any one of paragraphs 13 to 24, wherein the GH5_35 xylanase is derived from Bacillus, Coenella, or Bacillus-like organisms.

[0379] 26. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition, or composition as described in any one of paragraphs 13 to 25, wherein the GH5_35 xylanase is derived from species Bacillus hemicellulosee JCM 9152, Heterocystis tumefaciens, Bacillus tachyzoa, or Bacillus species-62332.

[0380] 27. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition, or composition as described in any one of paragraphs 13 to 26, wherein the GH5_35 xylanase is derived from a compost metagenomics.

[0381] 28. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition, or composition as described in 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 containing 0 to 10 conserved amino acid substitutions, or the amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 22, having xylanase activity; (ii) An amino acid sequence of SEQ ID NO: 23 containing 0 to 10 conserved amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 23, having xylanase activity; (iii) An amino acid sequence of SEQ ID NO: 24 containing 0 to 10 conserved amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 24, having xylanase activity; (iv) An amino acid sequence of SEQ ID NO: 25 containing 0 to 10 conserved amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 25, having xylanase activity; and (v) An amino acid sequence of SEQ ID NO: 26 containing 0 to 10 conserved amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 26, having xylanase activity.

[0382] 29. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition, or composition as described in any one of paragraphs 13 to 28, wherein the GH3 β-xylosidase is derived from Aspergillus or Bassilago farfara.

[0383] 30. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition, or composition as described in any one of paragraphs 13 to 29, wherein the GH3 β-xylosidase is derived from the species Aspergillus fumigatus, Aspergillus nidus, or Aspergillus emersonii.

[0384] 31. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition, or composition as described in any one of paragraphs 13 to 30, wherein the GH3 β-xylosidase has an amino acid sequence selected from the group consisting of: (i) The amino acid sequence of SEQ ID NO: 27 containing 0 to 10 conserved amino acid substitutions, or the amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 27, having β-xylosidase activity; (ii) An amino acid sequence containing 0 to 10 conserved amino acid substitutions of SEQ ID NO: 28, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 28, having β-xylosidase activity; (iii) An amino acid sequence of SEQ ID NO: 29 containing 0 to 10 conserved amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 29, having β-xylosidase activity; (iv) An amino acid sequence of SEQ ID NO: 30 containing 0 to 10 conserved amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 30, having β-xylosidase activity; (v) An amino acid sequence of SEQ ID NO: 31 containing 0 to 10 conserved amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 31, having β-xylosidase activity; (vi) An amino acid sequence containing 0 to 10 conserved amino acid substitutions of SEQ ID NO: 32, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 32, having β-xylosidase activity; (vii) An amino acid sequence containing 0 to 10 conserved amino acid substitutions of SEQ ID NO: 33, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 33, having β-xylosidase activity; (viii) An amino acid sequence of SEQ ID NO: 34 containing 0 to 10 conserved amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 34, having β-xylosidase activity; (ix) An amino acid sequence containing 0 to 10 conserved amino acid substitutions of SEQ ID NO: 35, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 35, having β-xylosidase activity; (x) An amino acid sequence of SEQ ID NO: 36 containing 0 to 10 conserved amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 36, having β-xylosidase activity; (xi) An amino acid sequence containing 0 to 10 conserved amino acid substitutions of SEQ ID NO: 37, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 37, having β-xylosidase activity; (xii) An amino acid sequence containing 0 to 10 conserved amino acid substitutions of SEQ ID NO: 38, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 38, having β-xylosidase activity; (xiii) An amino acid sequence containing 0 to 10 conserved amino acid substitutions of SEQ ID NO: 39, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 39, having β-xylosidase activity; (xiv) An amino acid sequence containing 0 to 10 conserved amino acid substitutions of SEQ ID NO: 40, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 40, having β-xylosidase activity; and (xv) An amino acid sequence containing 0 to 10 conserved amino acid substitutions of SEQ ID NO: 41, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 41, having β-xylosidase activity.

[0385] 32. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition, or composition as described in any one of paragraphs 13 to 31, wherein the GH30 xylanase is GH30_1 xylanase.

[0386] 33. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition, or composition as described in any one of paragraphs 13 to 32, wherein the GH30 xylanase is GH30_2 xylanase.

[0387] 34. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition, or composition as described in any one of paragraphs 13 to 33, wherein the GH30 xylanase is GH30_3 xylanase.

[0388] 35. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition, or composition as described in any one of paragraphs 13 to 34, wherein the GH30 xylanase is GH30_4 xylanase.

[0389] 36. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition, or composition as described in any one of paragraphs 13 to 35, wherein the GH30 xylanase is GH30_5 xylanase.

[0390] 37. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition, or composition as described in any one of paragraphs 13 to 36, wherein the GH30 xylanase is GH30_7 xylanase.

[0391] 38. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition, or composition as described in any one of paragraphs 13 to 37, wherein the GH30_7 xylanase is derived from the genera selected from Aspergillus, Evenstoc, Basilaria, and Trichoderma.

[0392] 39. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition, or composition as described in any one of paragraphs 13 to 38, wherein the GH30_7 xylanase is derived from a species selected from the group consisting of Aspergillus Fischer, Aspergillus fumigatus, Aspergillus neonicotinus, Aspergillus pseudoterreus, Aspergillus terreus, Aspergillus turcica, Aspergillus urdagawa, Aspergillus resemblingus, Aspergillus verrucosum, and Trichoderma reesei.

[0393] 40. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition, or composition as described in any one of paragraphs 13 to 39, wherein the GH30_7 xylanase is selected from the group consisting of: (i) An amino acid sequence containing 0 to 10 conserved amino acid substitutions of SEQ ID NO: 42, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 42, having xylanase activity; (ii) An amino acid sequence of SEQ ID NO: 43 containing 0 to 10 conserved amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 43, having xylanase activity; (iii) An amino acid sequence containing 0 to 10 conserved amino acid substitutions of SEQ ID NO: 44, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 44, having xylanase activity; (iv) An amino acid sequence of SEQ ID NO: 45 containing 0 to 10 conserved amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 45, having xylanase activity; (v) An amino acid sequence of SEQ ID NO: 46 containing 0 to 10 conserved amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 46, having xylanase activity; (vi) An amino acid sequence containing 0 to 10 conserved amino acid substitutions of SEQ ID NO: 47, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 47, having xylanase activity; (vii) An amino acid sequence containing 0 to 10 conserved amino acid substitutions of SEQ ID NO: 48, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 48, having xylanase activity; (viii) An amino acid sequence of SEQ ID NO: 49 containing 0 to 10 conserved amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 49, having xylanase activity; (ix) An amino acid sequence of SEQ ID NO: 50 containing 0 to 10 conserved amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 50, having xylanase activity; (x) An amino acid sequence containing 0 to 10 conserved amino acid substitutions of SEQ ID NO: 51, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 51, having xylanase activity; and (xi) An amino acid sequence containing 0 to 10 conserved amino acid substitutions of SEQ ID NO: 52, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 52, having xylanase activity.

[0394] 41. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition, or composition as described in any one of paragraphs 13 to 40, wherein the GH30 xylanase is GH30_8 xylanase.

[0395] 42. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition, or composition as described in any one of paragraphs 13 to 41, wherein the GH30_8 xylanase is derived from Bacillus, Clostridium, Bacillus-like organisms, or Vibrio.

[0396] 43. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition, or composition as described in any one of paragraphs 13 to 42, wherein the GH30_8 xylanase is derived from species Bacillus species-18423, Clostridium acetobutanol, Clostridium sacchaributanol, Bacillus foragerella, and Vibrio rhizosphereus.

[0397] 44. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition, or composition as described in any one of paragraphs 13 to 43, wherein the GH30_8 xylanase is selected from the group consisting of: (i) An amino acid sequence containing 0 to 10 conserved amino acid substitutions of SEQ ID NO: 53, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 53, having xylanase activity; (ii) An amino acid sequence of SEQ ID NO: 54 containing 0 to 10 conserved amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 54, having xylanase activity; (iii) An amino acid sequence containing 0 to 10 conserved amino acid substitutions of SEQ ID NO: 55, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 55, having xylanase activity; (iv) An amino acid sequence of SEQ ID NO: 56 containing 0 to 10 conserved amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 56, having xylanase activity; and (v) An amino acid sequence of SEQ ID NO: 57 containing 0 to 10 conserved amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 57, having xylanase activity.

[0398] 45. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition, or composition as described in any one of paragraphs 13 to 44, wherein the GH30 xylanase is GH30_9 xylanase.

[0399] 46. ​​The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition, or composition as described in any one of paragraphs 13 to 45, wherein the GH30 xylanase is GH30_10 xylanase.

[0400] 47. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition, or composition as described in any one of paragraphs 13 to 46, wherein the CE3 acetylxylan esterase is selected from the group consisting of: (i) The amino acid sequence of SEQ ID NO: 58 containing 0 to 10 conserved amino acid substitutions, or the amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 58, having acetylxylan esterase activity; (ii) An amino acid sequence containing 0 to 10 conserved amino acid substitutions of SEQ ID NO: 59, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 59, having acetyloxylan esterase activity; (iii) An amino acid sequence containing 0 to 10 conserved amino acid substitutions of SEQ ID NO: 60, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 60, having acetyloxylan esterase activity; (iv) An amino acid sequence containing 0 to 10 conserved amino acid substitutions of SEQ ID NO: 61, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 61, having acetyloxylan esterase activity; (v) An amino acid sequence of SEQ ID NO: 62 containing 0 to 10 conserved amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 62, having acetyloxylan esterase activity; (vi) An amino acid sequence containing 0 to 10 conserved amino acid substitutions of SEQ ID NO: 63, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 63, having acetyloxylan esterase activity; (vii) An amino acid sequence containing 0 to 10 conserved amino acid substitutions of SEQ ID NO: 64, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 64, having acetyloxylan esterase activity; (viii) An amino acid sequence containing 0 to 10 conserved amino acid substitutions of SEQ ID NO: 65, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 65, having acetyloxylan esterase activity; (ix) An amino acid sequence containing 0 to 10 conserved amino acid substitutions of SEQ ID NO: 66, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 66, having acetyloxylan esterase activity; (x) An amino acid sequence containing 0 to 10 conserved amino acid substitutions of SEQ ID NO: 67, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 67, having acetylxylan esterase activity.

[0401] 48. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition or composition as described in any one of paragraphs 13 to 47, further comprising GH31 α-xylosidase.

[0402] 49. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition or composition as described in any one of paragraphs 13 to 48, wherein the GH31 α-xylosidase is derived from the genus *Actinidia*.

[0403] 50. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition, or composition as described in any one of paragraphs 13 to 49, wherein the GH31 α-xylosidase is derived from the species Hemicellulosinus hemicellulosinus.

[0404] 51. The particles as described in any one of paragraphs 1 to 4, the liquid composition as described in any one of paragraphs 5 to 8, or the composition as described in any one of paragraphs 9 to 12, or the particles, liquid composition, or composition as described in any one of paragraphs 13 to 50, wherein the GH31 α-xylosidase has the amino acid sequence of SEQ ID NO: 68 containing 0 to 10 conserved amino acid substitutions, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 68, and has α-xylosidase activity.

[0405] 52. A method for producing fermentation products from starch-containing materials, the method comprising the following steps: (a) Saccharifying starch-containing material with α-amylase and glucosylase at a temperature below the initial gelatinization temperature of the starch to produce fermentable sugars; (b) Fermenting the sugar with a fermenting organism to produce the fermentation product; During the saccharification step (a) and / or fermentation step (b), particles as described in any one of paragraphs 1 to 4, liquid compositions as described in any one of paragraphs 5 to 8, compositions as described in any one of paragraphs 9 to 12, or particles, liquid compositions, or compositions as described in any one of paragraphs 13 to 50 are present or added.

[0406] 53. The method as described in paragraph 52, wherein particles as described in any one of paragraphs 1 to 4 and 13 to 51 are present or added during the saccharification step (a) and / or fermentation step (b).

[0407] 54. The method as described in paragraph 52, wherein a liquid composition as described in any one of paragraphs 5 to 8 and 13 to 51 is present or added during the saccharification step (a) and / or fermentation step (b).

[0408] 55. The method as described in paragraph 52, wherein the composition as described in any one of paragraphs 9 to 51 is present or added during the saccharification step (a) and / or fermentation step (b).

[0409] 56. The method as described in any one of paragraphs 52 to 55, wherein the saccharification step (a) and the fermentation step (b) are carried out simultaneously.

[0410] 57. A method for producing fermentation products from starch-containing materials, the method comprising the following steps: (a) The starch-containing material is liquefied with a heat-stable α-amylase at a temperature above the initial gelatinization temperature of the starch to produce dextrin; (b) Saccharifying the dextrin with glucosylamylase to produce fermentable sugars; (c) Fermenting the sugar with a fermenting organism to produce the fermentation product; During the saccharification step (b) and / or fermentation step (c), particles as described in any one of paragraphs 1 to 4, liquid compositions as described in any one of paragraphs 5 to 8, compositions as described in any one of paragraphs 9 to 12, or particles, liquid compositions, or compositions as described in any one of paragraphs 13 to 51 are present or added.

[0411] 58. The method as described in paragraph 57, wherein particles as described in any one of paragraphs 1 to 4 and 13 to 51 are present or added during the saccharification step (a) and / or fermentation step (b).

[0412] 59. The method as described in paragraph 57, wherein a liquid composition as described in any one of paragraphs 5 to 8 and 13 to 51 is present or added during the saccharification step (a) and / or fermentation step (b).

[0413] 60. The method as described in paragraph 57, wherein the composition as described in any one of paragraphs 9 to 51 is present or added during the saccharification step (a) and / or fermentation step (b).

[0414] 61. The method as described in any one of paragraphs 57 to 60, wherein the saccharification step (a) and the fermentation step (b) are carried out simultaneously.

[0415] 62. The method of any one of paragraphs 57 to 61, wherein the thermostable α-amylase has an amino acid sequence of SEQ ID NO: 72 containing 0 to 10 conserved amino acid substitutions or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 72, and has α-amylase activity.

[0416] 63. The method of any one of paragraphs 57 to 61, wherein the thermostable α-amylase has an amino acid sequence of SEQ ID NO: 73 containing 0 to 10 conserved amino acid substitutions or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 73, and has α-amylase activity.

[0417] 64. The method of any one of paragraphs 57 to 63, wherein a thermostable protease and / or a thermostable xylanase are added in the liquefaction step (a).

[0418] 65. The method of any one of paragraphs 57 to 64, wherein the thermostable protease has an amino acid sequence of SEQ ID NO: 74 containing 0 to 10 conserved amino acid substitutions or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 74, and has protease activity.

[0419] 66. The method of any one of paragraphs 57 to 65, wherein the thermostable xylanase has an amino acid sequence of SEQ ID NO: 75 containing 0 to 10 conserved amino acid substitutions or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 75, and has xylanase activity.

[0420] 67. The method of any one of paragraphs 57 to 66, wherein the glucosylamylase has an amino acid sequence of SEQ ID NO: 76 containing 0 to 10 conserved amino acid substitutions or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 76, and has glucosylamylase activity.

[0421] 68. The method as described in any one of paragraphs 57 to 67, the method further comprising adding α-amylase during the saccharification step (b) and / or fermentation step (c).

[0422] 69. The method of any one of paragraphs 52 to 68, wherein the α-amylase has an amino acid sequence of SEQ ID NO: 77 containing 0 to 10 conserved amino acid substitutions or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 77, and has α-amylase activity.

[0423] 70. The method of any one of paragraphs 57 to 69, wherein trehalase is added during the saccharification step and / or the fermentation step.

[0424] 71. The method as described in paragraph 70, wherein the trehalase has an amino acid sequence of SEQ ID NO: 78 containing 0 to 10 conserved amino acid substitutions or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 78, and has trehalase activity.

[0425] 72. The method of any one of paragraphs 57 to 71, wherein a composition comprising β-glucosidase, cellobiase, and endoglucanase is added during the saccharification step and / or the fermentation step.

[0426] 73. The method as described in paragraph 72, wherein the β-glucosidase has an amino acid sequence of SEQ ID NO: 79 containing 0 to 10 conserved amino acid substitutions or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 79, and has β-glucosidase activity.

[0427] 74. The method as described in paragraph 72 or 73, wherein the cellobiose hydrolase has an amino acid sequence of SEQ ID NO: 80 containing 0 to 10 conserved amino acid substitutions or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 80, and has cellobiose hydrolase activity.

[0428] 75. The method of any one of paragraphs 72 to 74, wherein the endoglucanase has an amino acid sequence of SEQ ID NO: 81 containing 0 to 10 conserved amino acid substitutions or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 81, and has endoglucanase activity.

[0429] 76. The method of any one of paragraphs 52 to 75, wherein the starch-containing material comprises beet, corn, maize, wheat, rye, oats, barley, black wheat, rice, sweet potato, sorghum, millet, pearl millet and / or foxtail millet.

[0430] 77. The method of any one of paragraphs 52 to 76, wherein the starch-containing material comprises corn.

[0431] 78. The method of any one of paragraphs 52 to 77, wherein the fermentation product is ethanol, preferably fuel ethanol.

[0432] 79. The method as described in any one of paragraphs 52 to 78, wherein the fermenting organism is yeast.

Claims

1. A particle, said particle comprising: (a) A core comprising GH43 arabinofuranylase, GH51 arabinofuranylase, GH5 xylanase, GH3 β-xylosidase, GH30 xylanase, and optionally carbohydrate esterase family 3 (CE3) acetylxylan esterase, and optionally... (b) A coating consisting of one or more layers surrounding the core.

2. A particle, said particle comprising: (a) core, and (b) A coating consisting of one or more layers surrounding the core, wherein the coating comprises GH43 arabinofuranylase, GH51 arabinofuranylase, GH5 xylanase, GH3 β-xylanase, GH30 xylanase and optionally CE3 acetylxylan esterase.

3. A composition comprising the particles as described in claim 1 or 2.

4. A liquid composition comprising GH43 arabinofuranylase, GH51 arabinofuranylase, GH5 xylanase, GH3 β-xylosidase, GH30 xylanase and optionally Ce3 acetylatedxylan esterase, and an enzyme stabilizer, such as a polyol like propylene glycol or glycerol, a sugar or sugar alcohol, or lactic acid.

5. The liquid composition of claim 4, wherein the liquid composition further comprises a filler or carrier material and / or a preservative.

6. A composition comprising GH43 arabinofuranylase, GH51 arabinofuranylase, GH521 xylanase, GH3 β-xylosidase, GH30 xylanase, and optionally CE3 acetylatedxylan esterase.

7. The particles as claimed in claim 1 or 2, or the composition as claimed in any one of claims 3 to 6, wherein the GH5 xylanase is GH5_21 xylanase or GH5_35 xylanase.

8. The particles as claimed in claim 1 or 2, or the composition as claimed in any one of claims 3 to 6, wherein the GH30 xylanase is GH30_7 xylanase or GH30_8 xylanase.

9. The particles or composition according to any one of claims 1 to 8, wherein the particles or composition further comprise α-xylosidase (e.g., GH31 α-xylosidase).

10. A method for producing fermentation products from starch-containing materials, the method comprising the following steps: (a) Saccharifying starch-containing material with glucosylamylase and α-amylase at a temperature below the initial gelatinization temperature of the starch to produce fermentable sugars; (b) Fermenting the sugar using a fermenting organism; The composition comprising GH43 arabinofuranase, GH51 arabinofuranase, GH5 xylanase, GH3 β-xylanase, GH30 xylanase and optionally CE3 acetylxylan esterase is present or added during the saccharification step (a) and / or fermentation step (b).

11. A method for producing fermentation products from starch-containing materials, the method comprising the following steps: (a) The starch-containing material is liquefied with a heat-stable α-amylase at a temperature above the initial gelatinization temperature of the starch to produce dextrin; (b) Saccharifying the dextrin with glucosylamylase to produce fermentable sugars; (c) Fermenting the sugar with a fermenting organism to produce the fermentation product; The composition comprising GH43 arabinofuranase, GH51 arabinofuranase, GH5 xylanase, GH3 β-xylanase, GH30 xylanase and optionally CE3 acetylxylan esterase is present or added during the saccharification step (b) and / or fermentation step (c).

12. The method of claim 10 or 11, wherein the composition comprises the CE3 acetylxylan esterase.

13. The method of any one of claims 10 to 12, wherein the composition further comprises α-xylosidase (e.g., GH31 α-xylosidase).

14. The method of any one of claims 10 to 13, wherein the GH5 xylanase is GH5_21 xylanase or GH5_35 xylanase.

15. The method of any one of claims 10 to 14, wherein the GH30 xylanase is GH30_7 xylanase or GH30_8 xylanase.

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