Carbohydrate esterase family 3 (CE3) polypeptides having acetylxylan esterase activity and polynucleotides encoding same

Abstract

The present invention relates to carbohydrate esterase family 3 (CE3) polypeptides having acetylxylan esterase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides. The invention also relates to compositions comprising CE3 polypeptides having acetylxylan esterase activity and to the use of these compositions for solubilizing hemicellulosic fibers and increasing the release of monomeric arabinose and / or xylose.

Description

[0001] Reference to the Sequence Listing

[0002] This application contains a sequence listing in computer-readable form, which is incorporated herein by reference. BACKGROUND OF THE INVENTION TECHNICAL FIELD

[0004] The present invention relates to carbohydrate esterase family 3 (CE3) polypeptides having acetylxylan esterase activity, polynucleotides encoding these polypeptides, nucleic acid constructs, vectors and host cells comprising these polynucleotides, and methods for producing and using these polypeptides. The invention also relates to compositions comprising CE3 family polypeptides having acetylxylan esterase activity and the use of these compositions for solubilizing hemicellulose fibers. BACKGROUND ART

[0005] The conversion of cellulosic feedstocks to biofuels is challenging due to the high recalcitrance of cellulosic feedstocks, which typically involves a combination of thermochemical pretreatment and subsequent addition of cellulases and hemicellulases to release soluble carbohydrates. Stimulated by government sustainable development initiatives, the biofuel industry uses corn fiber to produce ethanol in existing corn ethanol facilities. Corn fiber accounts for 10% of the weight of corn kernels and consists of cellulose and hemicellulose from the aleurone layer and pericarp layer. In ethanol facilities, corn fiber ultimately becomes dried distillers grains with solubles (DDGS). The hemicellulose portion of corn fiber is enzymatically hydrolyzed into monomeric C5 sugars (such as xylose and arabinose), and these C5 sugars are fermented into ethanol by C5-fermenting yeasts, and using existing infrastructure, so that ethanol plants can produce more cellulosic ethanol from the same amount of corn. Additional benefits of corn fiber degradation include: better quality of DDGS feed rich in protein, which can be used for animal feed; and the lower fiber content of DDGS will likely qualify it for the monogastric and aquaculture animal feed markets.

[0006] The backbone of arabinoxylan in corn fiber consists of a xylan backbone of β-(1,4)-linked D-xylopyranosyl residues, which is highly substituted by arabinose side chains and to a lesser extent by glucuronic acid residues. The predominantly substituted arabinose residues are linked to the O-2 or O-3 position on the mono-substituted xylopyranosyl or to both the O-2 and O-3 on the di-substituted xylopyranosyl unit. In addition to arabinose, the xylan backbone can also be substituted by D-galactopyranosyl and D-glucuronic acid residues, and / or by acetyl groups. Acetic acid is directly esterified to the xylan backbone at the O-2 or O-3 position, while hydroxycinnamic acids (such as ferulic acid, p-coumaric acid, and the dehydrodimer of ferulic acid) are esterified to the arabinofuranosyl at the O-5 position. Additionally, it has been reported that the xylan is further substituted by xylopyranosyl via (1-3)-bonds, and the arabinofuranosyl can be further modified by xylopyranosyl or even L-galactopyranosyl. Due to the highly branched substitution by different moieties, the enzymatic degradation of corn fiber arabinoxylan to monomeric C5 sugars requires the synergistic action of a mixture of debranching activity and depolymerization activity. The debranching activity mainly includes α-L-arabinofuranosidase (EC3.2.1.55) (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 the depolymerization depends on endo-1,4-β-xylanase (EC 3.2.1.8) and β-xylosidase (EC 3.2.1.37) (BX) activity.

[0007] WO 2006 / 114095 “D1” describes methods and compositions for hydrolyzing arabinoxylan, which include contacting a substrate containing arabinoxylan with an enzyme active against di-substituted arabinose (e.g., a glycoside hydrolase family 43 (GH43) α-L-arabinofuranosidase) and an enzyme active against mono-substituted arabinose at the C2 or C3 position (e.g., a GH family 51, 54, or 62 α-L-arabinofuranosidase). D1 teaches that when these two arabinofuranosidases are added to an arabinoxylan solution, the resulting products will be high molecular weight linear xylose polymers and arabinose molecules, which allows the linear xylose polymers to be easily separated from arabinose by known techniques. These linear xylose polymers can be further partially digested using enzyme 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 the purified linear xylose polymers, the resulting product will be xylose substantially free of arabinose substituents, and for degrading even more complex substrates, or in cases where more complete degradation is desired, even further enzyme activities such as acetylxylan 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 desirable.

[0008] However, supply chain disruptions and inflation have led to an increase in the raw material input costs for the enzymes required to produce fully hydrolyzed complex arabinoxylan substrates, thereby weakening the economic incentive for ethanol facilities to purchase additional enzymes to produce cellulosic ethanol from corn. Since conventional wisdom holds that all seven enzyme activities are required to obtain the highest cellulosic ethanol yield from corn, improved methods and compositions are needed that can increase cellulosic ethanol production by releasing more monomeric arabinose and xylose, with fewer enzyme activities and lower costs, which are more profitable for corn ethanol facilities to maximize the cellulosic ethanol yield from their existing corn inputs. Summary of the Invention

[0009] The present invention provides polypeptides having esterase activity and polynucleotides encoding these polypeptides. The CE3 family polypeptides of the present invention release more monomeric arabinose and / or xylose when used in combination with polypeptides having arabinofuranosidase activity towards di- and mono-substituted arabinose, polypeptides having xylanase activity, and polypeptides having β-xylosidase activity. The addition of α-xylosidase (e.g., GH31 α-xylosidase) further increases the release of monomeric sugars. Surprisingly and unexpectedly, the compositions of the present invention significantly increase the production of monomeric arabinose and / or xylose without ferulic acid esterase and / or α-glucuronidase, although the addition of α-xylosidase to the compositions further increases the production of monomeric arabinose and / or xylose.

[0010] SEQ ID NO:1 is the nucleotide sequence encoding the wild-type Dinemasporium sp. CE3 polypeptide having acetylxylan esterase activity of the present invention.

[0011] SEQ ID NO:2 is the full-length amino acid sequence of the wild-type Dinemasporium sp. CE3 polypeptide having acetylxylan esterase activity of the present invention.

[0012] SEQ ID NO:3 is the mature polypeptide of the wild-type Dinemasporium sp. CE3 polypeptide having acetylxylan esterase activity of the present invention.

[0013] SEQ ID NO:4 is the nucleotide sequence encoding the wild-type Epicoccum sorghinum CE3 polypeptide having acetylxylan esterase activity of the present invention.

[0014] SEQ ID NO:5 is the full-length amino acid sequence of the wild-type Epicoccum sorghinum CE3 polypeptide having acetylxylan esterase activity of the present invention.

[0015] SEQ ID NO:6 is the mature polypeptide of the wild-type Epicoccum sorghinum CE3 polypeptide having acetylxylan esterase activity of the present invention.

[0016] SEQ ID NO:7 is the nucleotide sequence encoding the wild-type Flammulina velutipes CE3 polypeptide having acetylxylan esterase activity of the present invention.

[0017] SEQ ID NO:8 is the full-length amino acid sequence of the wild-type Flammulina velutipes CE3 polypeptide having acetylxylan esterase activity of the present invention.

[0018] SEQ ID NO:9 is the mature polypeptide of the wild-type Flammulina velutipes CE3 polypeptide having acetylxylan esterase activity of the present invention.

[0019] SEQ ID NO:10 is the nucleotide sequence encoding the wild-type Microsphaeropsis arundinis CE3 polypeptide with acetylxylan esterase activity of the present invention.

[0020] SEQ ID NO:11 is the full-length amino acid sequence of the wild-type Microsphaeropsis arundinis CE3 polypeptide with acetylxylan esterase activity of the present invention.

[0021] SEQ ID NO:12 is the mature polypeptide of the wild-type Microsphaeropsis arundinis CE3 polypeptide with acetylxylan esterase activity of the present invention.

[0022] SEQ ID NO:13 is the nucleotide sequence encoding the wild-type Microsphaeropsis arundinis CE3 polypeptide with acetylxylan esterase activity of the present invention.

[0023] SEQ ID NO:14 is the full-length amino acid sequence of the wild-type Microsphaeropsis arundinis CE3 polypeptide with acetylxylan esterase activity of the present invention.

[0024] SEQ ID NO:15 is the mature polypeptide of the wild-type Microsphaeropsis arundinis CE3 polypeptide with acetylxylan esterase activity of the present invention.

[0025] SEQ ID NO:16 is the nucleotide sequence encoding the wild-type Microsphaeropsis arundinis CE3 polypeptide with acetylxylan esterase activity of the present invention.

[0026] SEQ ID NO:17 is the full-length amino acid sequence of the wild-type Microsphaeropsis arundinis CE3 polypeptide with acetylxylan esterase activity of the present invention.

[0027] SEQ ID NO:18 is the mature polypeptide of the wild-type Microsphaeropsis arundinis CE3 polypeptide with acetylxylan esterase activity of the present invention.

[0028] SEQ ID NO:19 is the nucleotide sequence encoding the wild-type Paraphaeosphaeria neglecta CE3 polypeptide with acetylxylan esterase activity of the present invention.

[0029] SEQ ID NO:20 is the full-length amino acid sequence of the wild-type Paraphaeosphaeria neglecta CE3 polypeptide with acetylxylan esterase activity of the present invention.

[0030] SEQ ID NO:21 is the mature polypeptide of the wild-type Paraphaeosphaeria neglecta CE3 polypeptide with acetylxylan esterase activity of the present invention.

[0031] SEQ ID NO:22 is the nucleotide sequence encoding the wild-type Paraphaeosphaeria verruculosa CE3 polypeptide with acetylxylan esterase activity of the present invention.

[0032] SEQ ID NO:23 is the full-length amino acid sequence of the wild-type Paraphaeosphaeria verruculosa CE3 polypeptide with acetylxylan esterase activity of the present invention.

[0033] SEQ ID NO:24 is the mature polypeptide of the wild-type Paraphaeosphaeria verruculosa CE3 polypeptide with acetylxylan esterase activity of the present invention.

[0034] SEQ ID NO:25 is the nucleotide sequence encoding the wild-type Westerdykella purpurea CE3 polypeptide with acetylxylan esterase activity of the present invention.

[0035] SEQ ID NO:26 is the full-length amino acid sequence of the wild-type Westerdykella purpurea CE3 polypeptide with acetylxylan esterase activity of the present invention.

[0036] SEQ ID NO:27 is the mature polypeptide of the wild-type Westerdykella purpurea CE3 polypeptide with acetylxylan esterase activity of the present invention.

[0037] SEQ ID NO:28 is the nucleotide sequence encoding the wild-type Xepicula leucotricha CE3 polypeptide with acetylxylan esterase activity of the present invention.

[0038] SEQ ID NO:29 is the full-length amino acid sequence of the wild-type Xepicula leucotricha CE3 polypeptide with acetylxylan esterase activity of the present invention.

[0039] SEQ ID NO:30 is the mature polypeptide of the wild-type Xepicula leucotricha CE3 polypeptide with acetylxylan esterase activity of the present invention.

[0040] Accordingly, the present invention relates to polypeptides having acetylxylan esterase activity, which polypeptides are selected from the group consisting of:

[0041] (i)

[0042] a polypeptide having at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:2;

[0043] b) a polypeptide having at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3;

[0044] c) a polypeptide having at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO:2;

[0045] d) a polypeptide encoded by a polynucleotide having at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:1;

[0046] (e) A polypeptide derived from SEQ ID NO:2, the mature polypeptide of SEQ ID NO:2, or SEQ ID NO:3, which differs by having 1 to 30 alterations (such as substitutions, deletions, and / or insertions at one or more positions, for example, 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, particularly substitutions);

[0047] (f) A polypeptide derived from a polypeptide of (a), (b), (c), (d), or (e), wherein the N-terminus and / or C-terminus has been extended by adding one or more amino acids; and

[0048] (g) A fragment of a polypeptide of (a), (b), (c), (d), or (e);

[0049] wherein the polypeptide has acetylxylan esterase activity;

[0050] (ii)

[0051] (a) A polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:5;

[0052] (b) A polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:6;

[0053] (c) a polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO:5;

[0054] (d) a polypeptide encoded by a polynucleotide that has at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide coding sequence of SEQ ID NO:4;

[0055] (e) a polypeptide derived from SEQ ID NO:5, the mature polypeptide of SEQ ID NO:5, or SEQ ID NO:6 and differing by 1 - 30 alterations (such as substitutions, deletions, and / or insertions at one or more positions, for example 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, particularly substitutions);

[0056] (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d), or (e), wherein the N - terminus and / or C - terminus has been extended by adding one or more amino acids; and

[0057] (g) a fragment of the polypeptide of (a), (b), (c), (d), or (e);

[0058] wherein the polypeptide has acetylxylan esterase activity;

[0059] (iii)

[0060] a polypeptide having at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:8;

[0061] b) a polypeptide having at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:9;

[0062] c) a polypeptide having at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO:8;

[0063] d) a polypeptide encoded by a polynucleotide having at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:7;

[0064] (e) A polypeptide derived from SEQ ID NO:8, the mature polypeptide of SEQ ID NO:8, or SEQ ID NO:9, which differs by having 1 - 30 alterations (such as substitutions, deletions, and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, particularly substitutions);

[0065] (f) A polypeptide derived from a polypeptide of (a), (b), (c), (d), or (e), wherein the N - terminus and / or C - terminus has been extended by adding one or more amino acids; and

[0066] (g) A fragment of a polypeptide of (a), (b), (c), (d), or (e);

[0067] wherein the polypeptide has acetylxylan esterase activity;

[0068] (iv)

[0069] (a) A polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:11;

[0070] (b) A polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:12;

[0071] (c) a polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO: 11;

[0072] (d) a polypeptide encoded by a polynucleotide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide coding sequence of SEQ ID NO: 10;

[0073] (e) a polypeptide derived from SEQ ID NO: 11, the mature polypeptide of SEQ ID NO: 11, or SEQ ID NO: 12 and differing therefrom by having 1 - 30 alterations (e.g., substitutions, deletions, and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, particularly substitutions);

[0074] (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d), or (e), wherein the N - terminus and / or C - terminus has been extended by adding one or more amino acids; and

[0075] (g) a fragment of the polypeptide of (a), (b), (c), (d), or (e);

[0076] wherein the polypeptide has acetylxylan esterase activity; and

[0077] (v)

[0078] a polypeptide having at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:14;

[0079] b polypeptide having at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:15;

[0080] c polypeptide having at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO:14;

[0081] d polypeptide encoded by a polynucleotide having at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:13;

[0082] (e) A polypeptide derived from SEQ ID NO:14, the mature polypeptide of SEQ ID NO:14, or SEQ ID NO:15, which differs by having 1 - 30 alterations (such as substitutions, deletions, and / or insertions at one or more positions, for example, 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, particularly substitutions);

[0083] (f) A polypeptide derived from a polypeptide of (a), (b), (c), (d), or (e), wherein the N - terminus and / or C - terminus has been extended by adding one or more amino acids; and

[0084] (g) A fragment of a polypeptide of (a), (b), (c), (d), or (e);

[0085] wherein the polypeptide has acetylxylan esterase activity; and

[0086] (vi)

[0087] (a) A polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:17;

[0088] (b) A polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:18;

[0089] (c) a polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO:17;

[0090] (d) a polypeptide encoded by a polynucleotide that has at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide coding sequence of SEQ ID NO:16 or its cDNA sequence;

[0091] (e) a polypeptide derived from SEQ ID NO:17, the mature polypeptide of SEQ ID NO:17, or SEQ ID NO:18 and differing by having 1 - 30 alterations (e.g., substitutions, deletions, and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, particularly substitutions);

[0092] (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d), or (e), wherein the N - terminus and / or C - terminus has been extended by adding one or more amino acids; and

[0093] (g) a fragment of the polypeptide of (a), (b), (c), (d), or (e);

[0094] wherein the polypeptide has acetylxylan esterase activity;

[0095] (vii)

[0096] (a) a polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:20;

[0097] (b) a polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:21;

[0098] (c) a polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO:20;

[0099] (d) a polypeptide encoded by a polynucleotide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:19;

[0100] (e) A polypeptide derived from SEQ ID NO:20, the mature polypeptide of SEQ ID NO:20, or SEQ ID NO:21, which differs by having 1 to 30 alterations (e.g., substitutions, deletions, and / or insertions at one or more positions, such as 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, particularly substitutions);

[0101] (f) A polypeptide derived from a polypeptide of (a), (b), (c), (d), or (e), wherein the N-terminus and / or C-terminus has been extended by adding one or more amino acids; and

[0102] (g) A fragment of a polypeptide of (a), (b), (c), (d), or (e);

[0103] wherein the polypeptide has acetylxylan esterase activity;

[0104] (viii)

[0105] (a) A polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:23;

[0106] (b) A polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:24;

[0107] (c) a polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO:23;

[0108] (d) a polypeptide encoded by a polynucleotide that has at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide coding sequence of SEQ ID NO:22;

[0109] (e) a polypeptide derived from SEQ ID NO:23, the mature polypeptide of SEQ ID NO:23, or SEQ ID NO:24 and differing by having 1 - 30 alterations (such as substitutions, deletions, and / or insertions at one or more positions, for example 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, particularly substitutions);

[0110] (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d), or (e), wherein the N - terminus and / or C - terminus has been extended by adding one or more amino acids; and

[0111] (g) a fragment of the polypeptide of (a), (b), (c), (d) or (e);

[0112] wherein the polypeptide has acetylxylan esterase activity;

[0113] (ix)

[0114] (a) A polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:26;

[0115] (b) A polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:27;

[0116] (c) A polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO:26;

[0117] (d) A polypeptide encoded by a polynucleotide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide coding sequence of SEQ ID NO:25;

[0118] (e) A polypeptide derived from SEQ ID NO:26, the mature polypeptide of SEQ ID NO:26, or SEQ ID NO:27, which differs by having 1 to 30 alterations (such as substitutions, deletions, and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, especially substitutions);

[0119] (f) A polypeptide derived from a polypeptide of (a), (b), (c), (d), or (e), wherein the N-terminus and / or C-terminus has been extended by adding one or more amino acids; and

[0120] (g) A fragment of a polypeptide of (a), (b), (c), (d), or (e);

[0121] wherein the polypeptide has acetylxylan esterase activity; and

[0122] (x)

[0123] (a) A polypeptide having at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:29;

[0124] (b) A polypeptide having at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:30;

[0125] (c) A polypeptide having at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO:29;

[0126] (d) A polypeptide encoded by a polynucleotide that has at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:28;

[0127] (e) A polypeptide derived from SEQ ID NO:29, the mature polypeptide of SEQ ID NO:29, or SEQ ID NO:30 and differing therefrom by having 1 - 30 alterations (e.g., substitutions, deletions, and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, particularly substitutions);

[0128] (f) A polypeptide derived from the polypeptide of (a), (b), (c), (d), or (e), wherein the N - terminus and / or C - terminus has been extended by the addition of one or more amino acids; and

[0129] (g) A fragment of the polypeptide of (a), (b), (c), (d), or (e);

[0130] wherein the polypeptide has acetylxylan esterase activity.

[0131] The present invention also relates to polynucleotides encoding the polypeptides of the present invention; nucleic acid constructs; recombinant expression vectors; recombinant host cells comprising these polynucleotides; and methods for producing these polypeptides.

[0132] The present invention also relates to compositions comprising CE3 family polypeptides and the use of these compositions for solubilizing hemicellulose fibers and increasing the release of monomeric arabinose and / or xylose. BRIEF DESCRIPTION OF THE DRAWINGS

[0133] The drawing is an alignment of exemplary CE3 polypeptides of the present invention, showing that they share conserved active site serine, histidine, and aspartate residues that form the catalytic triad characteristic of the SGNH hydrolase family, a conserved classical GxSxT pentapeptide consensus sequence, and Gly residue in Region II and Asn residue in Region III that form the oxyanion hole.

[0134] DEFINITIONS

[0135] For the purposes of this detailed description, the following definitions apply. Note that the singular forms "a / an" and "the" include plural referents unless the context clearly dictates otherwise.

[0136] Unless otherwise defined or clearly 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.

[0137] Acetylxylan esterase: The term "acetylxylan esterase" means a polypeptide having acetylxylan esterase activity (EC 3.1.1.72) that catalyzes the hydrolysis of acetyl groups from polymeric xylan, acetylated xylose, acetylated glucose, α-naphthyl acetate, and p-nitrophenyl acetate, but not from triacylglycerol.

[0138] Acetylxylan esterase activity: One unit of acetylxylan esterase activity is defined as the amount of enzyme required to release 1 micromole of p-nitrophenol per minute from p-nitrophenyl acetate in 100 mM sodium citrate buffer (pH 5) at 40°C. The 100 mM pNP-acetate is dissolved in DMSO as a substrate stock solution. The stock solution is diluted 50-fold in 100 mM sodium citrate to make a 2 mM pNP-acetate substrate solution. 175 μl of the substrate solution and 25 μl of the diluted enzyme are mixed in a 96-well plate and incubated at 37°C. The released p-nitrophenol is monitored at 410 nm by a spectrophotometer.

[0139] α-L-Arabinofuranosidase: "α-L-Arabinofuranosidase" means an α-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55) that catalyzes the hydrolysis of terminal non-reducing α-L-arabinofuranoside residues in α-L-arabinosides. This enzyme acts on α-L-arabinofuranosides, α-L-arabans containing (1,3)- and / or (1,5)-linkages, arabinogalactan, and arabinoxylan. α-L-Arabinofuranosidase is also known as arabinofuranosidase, α-arabinofuranosidase, α-L-arabinofuranosidase, α-arabinofuranosidase, polysaccharide α-L-arabinofuranosidase, α-L-arabinofuranohydrolase, L-arabinofuranosidase, or α-L-arabanase.

[0140] α-L-arabinofuranosidase activity: For the purposes of the present invention, α-L-arabinofuranosidase activity was determined by using 5 mg of medium-viscosity wheat arabinoxylan (Megazyme International Ireland, Ltd., Bray, Co., Wicklow, Ireland) in 100 mM sodium acetate (pH 5) per ml in a total volume of 200 μl at 40 °C for 30 min, followed by arabinose analysis by AMINEX® HPX-87H column chromatography (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

[0141] α-xylosidase: "α-xylosidase" means α-D-xyloside xylohydrolase (EC 3.2.1.177), which catalyzes the hydrolysis of terminal non-substituted xylosides at the non-reducing ends of xylo-oligosaccharides.

[0142] α-xylosidase activity: For the purposes of the present invention, one unit of α-xylosidase is defined as the production of 1.0 micromole of p-nitrophenolate anion per minute from 1 mM p-nitrophenyl-α-D-xyloside as substrate in 100 mM sodium citrate containing 0.01% TWEEN® 20 at 40 °C, pH 5, in a total volume of 200 μl.

[0143] β-xylosidase: "β-xylosidase" means β-D-xyloside xylohydrolase (E.C. 3.2.1.37), which catalyzes the exo-hydrolysis of short β(1-4)-xylo-oligosaccharides to remove successive D-xylose residues from the non-reducing ends.

[0144] β-xylosidase activity: For the purposes of the present invention, one unit of β-xylosidase is defined as the production of 1.0 micromole of p-nitrophenolate anion per minute from 1 mM p-nitrophenyl-β-D-xyloside as substrate in 100 mM sodium citrate containing 0.01% TWEEN® 20 at 40 °C, pH 5.

[0145] Carbohydrate esterase family 3 (CE3): Carbohydrate esterase family 3 is abbreviated herein as "CE3". The CE3 polypeptides of the present invention have acetylxylan esterase activity (EC 3.1.1.72).

[0146] cDNA: The term "cDNA" means 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 may 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, and then presented as mature, spliced mRNA.

[0147] Coding sequence: The term "coding sequence" means a polynucleotide that directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are typically determined by an open reading frame that begins with a start codon (such as ATG, GTG, or TTG) and ends with a stop codon (such as TAA, TAG, or TGA). The coding sequence can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0148] Control sequence: The term "control sequence" means a nucleic acid sequence involved in regulating the expression of a polynucleotide in a particular organism or in vitro. Each control sequence can be native (i.e., from the same gene) or heterologous (i.e., from a different gene) to the polynucleotide encoding the polypeptide, and native or heterologous to each other. Such control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptides, prepeptides, signal peptides, promoters, terminators, enhancers, and transcription or translation initiation and termination sequences. At a minimum, the control sequence includes a promoter and transcription and translation termination signals. For the purpose of introducing specific restriction sites that facilitate the ligation of the control sequence to the coding region of the polynucleotide encoding the polypeptide, these control sequences can be provided with multiple linkers.

[0149] Expression: The term "expression" means any step involved in the production of a polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0150] Expression vector: An "expression vector" refers to a linear or circular DNA construct that contains a DNA sequence encoding a polypeptide, and the coding sequence is operably linked to appropriate control sequences capable of affecting the expression of the DNA in a suitable host. Such control sequences can include a promoter that affects transcription, optional operator sequences that control transcription, sequences encoding appropriate ribosome binding sites on the mRNA, enhancers, and sequences that control the termination of transcription and translation.

[0151] Extension: The term "extension" means adding one or more amino acids to the amino and / or carboxyl terminus of a polypeptide, wherein the "extended" polypeptide has acetylxylan esterase activity.

[0152] Fermentation product: "Fermentation product" means a product produced by a process including fermentation using a fermenting organism. Fermentation products include alcohols (e.g., ethanol, methanol, butanol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, succinic acid, gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H2 and CO2); antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B 12 12 , β-carotene); and hormones. In a preferred embodiment, the fermentation product is ethanol, e.g., fuel ethanol; potable ethanol, i.e., a potable neutral alcoholic beverage; or industrial ethanol or product for the consumable alcohol industry (e.g., beer and wine), dairy industry (e.g., fermented dairy products), leather industry, and tobacco industry. Preferred types of beer include ale, stout, porter, lager, bitter, malt liquor, happoushu, high-alcohol beer, low-alcohol beer, low-calorie beer, or light beer. In one embodiment, the fermentation product is ethanol.

[0153] Fermenting organism: "Fermenting organism" refers to any organism suitable for use in a fermentation process and capable of producing a desired fermentation product, including bacterial and fungal organisms, especially yeast.

[0154] Fragment: The term "fragment" means a polypeptide having one or more amino acids deleted from the amino and / or carboxyl terminus of a mature polypeptide, wherein the fragment has acetylxylan esterase activity.

[0155] Fusion polypeptide: The term "fusion polypeptide" refers to a polypeptide in which one polypeptide is fused to the N-terminus and / or C-terminus of the polypeptide of the present invention. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to the polynucleotide of the present invention or by fusing two or more polynucleotides of the present invention together. Techniques for producing fusion polypeptides are known in the art and include ligating the coding sequences encoding the polypeptides such that they are in frame and the expression of the fusion polypeptide is under the control of one or more identical promoters and terminators. Inteins can also be used to construct fusion polypeptides, where the fusion polypeptide is generated after translation (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266:776-779). The fusion polypeptide may further comprise a cleavage site between the two polypeptides. This site is cleaved when the fusion protein is secreted, thereby releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, those disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3:568-576; Svetina et al., 2000, J. Biotechnol. 76:245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63:3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9:378-381; Eaton et al., 1986, Biochemistry 25:505-512; Collins-Racie et al., 1995, Biotechnology 13:982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6:240-248; and Stevens, 2003, Drug Discovery World 4:35-48.

[0156] GH3 β-xylosidase: "GH3 β-xylosidase" is an abbreviation for glycoside hydrolase family 3 β-xylosidase, which is xylan 1,4-β-xylosidase (EC 3.2.1.37), catalyzing the hydrolysis of (1→4)-β-D-xylan to remove successive D-xylose residues from the non-reducing end.

[0157] GH5 xylanase: "GH5 xylanase" is an abbreviation for glycoside hydrolase family 5 xylanase, which mainly consists of endo-1,4-β-xylanase (EC 3.2.1.8) and catalyzes the endohydrolysis of (1→4)-β-D-xylosidic linkages in xylan.

[0158] GH5_21 xylanase: "GH5_21 xylanase" is an abbreviation for glycoside hydrolase family 5 subfamily 21 endo-β-1,4-xylanase, which has a three-dimensional structure characterized by a (β / α)8 barrel and uses a glutamine residue as the catalytic nucleophile / base.

[0159] GH5_35 xylanase: "GH5_35 xylanase" is an abbreviation for glycoside hydrolase family 5 subfamily 35 endo-β-1,4-xylanase, which has a three-dimensional structure characterized by a (β / α)8 barrel and uses a glutamine residue as the catalytic nucleophile / base.

[0160] GH8 xylanase: "GH8 xylanase" is an abbreviation for glycoside hydrolase family 8 xylanase, which consists of endo-1,4-β-xylanase (EC 3.2.1.8), which catalyzes the endohydrolysis of (1→4)-β-D-xylosidic linkages in xylan.

[0161] GH10 xylanase: "GH10 xylanase" is an abbreviation for glycoside hydrolase family 10 xylanase, which consists of endo-1,3-β-xylanase (EC 3.2.1.32), which catalyzes the random endohydrolysis of (1→3)-β-D-glycosidic linkages in (1→3)-β-D-xylan; and endo-1,4-β-xylanase (EC 3.2.1.8), which catalyzes the endohydrolysis of (1→4)-β-D-xylosidic linkages in xylan.

[0162] GH11 xylanase: "GH11 xylanase" is an abbreviation for glycoside hydrolase family 11 xylanase, which is an endo-β-1,4-xylanase (EC 3.2.1.8) and catalyzes the endohydrolysis of (1→4)-β-D-xylosidic linkages in xylan.

[0163] GH31 α-xylosidase: "GH31 arabinofuranosidase" is an abbreviation for glycoside hydrolase family 31 α-xylosidase, which is an α-D-xylosyl-xylose hydrolase (EC 3.2.1.177) and catalyzes the hydrolysis of the terminal unsubstituted xyloside at the non-reducing end of xylo-oligosaccharides. Exemplary GH31 family α-xylosidases utilize a two-step double-displacement mechanism involving a covalent glycosyl-enzyme intermediate and produce products with anomeric configuration.

[0164] GH30_8 xylanase: "GH30_8 xylanase" is an abbreviation for glycoside hydrolase family 30 subfamily 8 xylanase, including the following: endo-β-1,4-xylanase (EC 3.2.1.8), which catalyzes the endohydrolysis of (1→4)-β-D-xylosidic linkages in xylan; and glucuronyl arabinoxylan-specific endo-β-1,4-xylanase (EC 3.2.1.136), which catalyzes the endohydrolysis of (1→4)-β-D-xylosyl linkages in some glucuronyl arabinoxylans. The endohydrolysis of (1→4)-β-D-xylosyl linkages in some glucuronyl arabinoxylans.

[0165] GH43 arabinofuranosidase: "GH43 arabinofuranosidase" is an abbreviation for glycoside hydrolase family 43 arabinofuranosidase, which is an α-L-arabinofuranosidase (EC 3.2.1.55) that catalyzes the hydrolysis of terminal non-reducing α-L-arabinofuranosyl residues in α-L-arabinofuranosides.

[0166] GH51 arabinofuranosidase: "GH51 arabinofuranosidase" is an abbreviation for glycoside hydrolase family 51 arabinofuranosidase, which is an α-L-arabinofuranosidase (EC 3.2.1.55) that catalyzes the hydrolysis of terminal non-reducing α-L-arabinofuranosyl residues in α-L-arabinofuranosides.

[0167] Initial gelatinization temperature: "Initial gelatinization temperature" means the lowest temperature at which starch gelatinization begins. Starch heated in water begins to gelatinize between 50 °C and 75 °C; the exact temperature of gelatinization depends on the specific starch and can be easily determined by a person skilled in the art. Thus, the initial gelatinization temperature can vary depending on the plant species, the specific variety of the plant species, and the growth conditions. In the context of the present disclosure, the initial gelatinization temperature of a given starch-containing cereal is the temperature at which 5% of the starch granules lose birefringence using the method described by Gorinstein.S. and Lii.C, Starch / Starke [Starch], 44(12), 461-466 (1992).

[0168] Heterologous: For a host cell, the term "heterologous" means that a polypeptide or nucleic acid is not naturally present in the host cell. For a polypeptide or nucleic acid, the term "heterologous" means that the control sequence (e.g., promoter) of the polypeptide or nucleic acid is not naturally associated with the polypeptide or nucleic acid, i.e., the control sequence is from a gene other than the gene encoding the mature polypeptide.

[0169] Host strain or host cell: "Host strain" or "host cell" refers to an organism into which an expression vector, phage, virus, or other DNA construct (including a polynucleotide encoding a polypeptide of interest (e.g., amylase)) has been introduced. Exemplary host strains are microbial cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing a polypeptide of interest and / or fermenting sugars. The term "host cell" includes protoplasts produced from the cells.

[0170] Introduce: In the case of inserting a nucleic acid sequence into a cell, the term "introduce" means "transfect", "transform", or "transduce", as known in the art.

[0171] 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.). Thus, an isolated polypeptide, nucleic acid, cell, or other material is in a form that does not exist in nature. Isolated polypeptides include, but are not limited to, culture broths containing secreted polypeptides expressed in host cells.

[0172] Mature polypeptide: The term "mature polypeptide" means a polypeptide in its mature form after N-terminal and / or C-terminal processing (e.g., removal of the signal peptide). In one aspect, the mature polypeptide is amino acids 19 to 297 of SEQ ID NO:2. In one aspect, the mature polypeptide is SEQ ID NO:3. In one aspect, the mature polypeptide is amino acids 26 to 245 of SEQ ID NO:5. In one aspect, the mature polypeptide is SEQ ID NO:6. In one aspect, the mature polypeptide is amino acids 20 to 289 of SEQ ID NO:8. In one aspect, the mature polypeptide is SEQ ID NO:9. In one aspect, the mature polypeptide is amino acids 23 to 253 of SEQ ID NO:11. In one aspect, the mature polypeptide is SEQ ID NO:12. In one aspect, the mature polypeptide is amino acids 19 to 240 of SEQ ID NO:14. In one aspect, the mature polypeptide is SEQ ID NO:15. In one aspect, the mature polypeptide is amino acids 23 to 244 of SEQ ID NO:17. In one aspect, the mature polypeptide is SEQ ID NO:18. In one aspect, the mature polypeptide is amino acids 25 to 247 of SEQ ID NO:20. In one aspect, the mature polypeptide is SEQ ID NO:21. In one aspect, the mature polypeptide is amino acids 25 to 244 of SEQ ID NO:23. In one aspect, the mature polypeptide is SEQ ID NO:24. In one aspect, the mature polypeptide is amino acids 20 to 240 of SEQ ID NO:26. In one aspect, the mature polypeptide is SEQ ID NO:27. In one aspect, the mature polypeptide is amino acids 17 to 286 of SEQ ID NO:29. In one aspect, the mature polypeptide is SEQ ID NO:30.

[0173] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" means a polynucleotide encoding a mature polypeptide having acetylxylan esterase activity. In one aspect, the mature polypeptide coding sequence is nucleotides 57 to 891 of SEQ ID NO:1. In one aspect, the mature polypeptide coding sequence is nucleotides 78 to 735 of SEQ ID NO:4. In one aspect, the mature polypeptide coding sequence is nucleotides 60 to 867 of SEQ ID NO:7. In one aspect, the mature polypeptide coding sequence is nucleotides 69 to 759 of SEQ ID NO:10. In one aspect, the mature polypeptide coding sequence is nucleotides 57 to 720 of SEQ ID NO:13. In one aspect, the mature polypeptide coding sequence is nucleotides 132 to 712 and 798 to 885 of SEQ ID NO:16, or its cDNA sequence. In one aspect, the mature polypeptide coding sequence is nucleotides 75 to 741 of SEQ ID NO:19. In one aspect, the mature polypeptide coding sequence is nucleotides 75 to 732 of SEQ ID NO:22. In one aspect, the mature polypeptide coding sequence is nucleotides 60 to 720 of SEQ ID NO:25. In one aspect, the mature polypeptide coding sequence is nucleotides 51 to 858 of SEQ ID NO:28.

[0174] Native: The term "native" means a nucleic acid or polypeptide that naturally occurs in a host cell.

[0175] Nucleic acid: The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. The nucleic acid 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 particular amino acid, and the compositions and methods of the present invention encompass nucleotide sequences encoding a particular amino acid sequence. Unless otherwise specified, nucleic acid sequences are presented in the 5' to 3' orientation.

[0176] Nucleic acid construct: The term "nucleic acid construct" means a single-stranded or double-stranded nucleic acid molecule isolated from a naturally occurring gene or modified in a manner not otherwise found in nature to contain segments of nucleic acid or synthetic and comprising one or more control sequences operably linked to a nucleic acid sequence.

[0177] Operably linked: The term "operably linked" means that the designated components are in a relationship that permits them to function in the intended manner (including but not limited to juxtaposition). For example, a regulatory sequence is operably linked to a coding sequence such that the expression of the coding sequence is under the control of the regulatory sequence.

[0178] Purified: The term "purified" means a nucleic acid, polypeptide, or cell that is substantially free of other components, as determined by analytical techniques well known in the art (e.g., a purified polypeptide or nucleic acid may form discrete bands in an electrophoretic gel, a chromatographic eluate, and / or a medium subjected to density gradient centrifugation). A purified nucleic acid or polypeptide is at least about 50% pure, 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., by weight percentage or molar percentage). In a related sense, a composition is enriched in a molecule when the concentration of the molecule increases substantially after applying purification or enrichment techniques. The term "enriched" means that a compound, polypeptide, cell, nucleic acid, amino acid, or other specified material or component is present in a composition at a relative or absolute concentration higher than in the starting composition.

[0179] In one aspect, as used herein, the term "purified" means that a polypeptide or cell is substantially free of components from the production organism (especially insoluble components). In other aspects, the term "purified" means that a polypeptide is substantially free of insoluble components (especially insoluble components) from the native organism from which it is obtained. In one aspect, the polypeptide is separated from some soluble components of the organism and the medium from which it is recovered. A polypeptide can be purified (i.e., separated) by one or more of the unit operations of filtration, precipitation, or chromatography.

[0180] Thus, a polypeptide can be purified such that only small amounts of other proteins, especially other polypeptides, are present. As used herein, the term "purified" can refer to the removal of other components, especially other proteins and most especially other enzymes, present in the cells from which the polypeptide is derived. A polypeptide can be "substantially pure", i.e., free of other components from the organism that produces it (e.g., the host organism used for recombinant production of the polypeptide). In one aspect, the polypeptide is at least 40% pure by weight of the total polypeptide material present in the preparation. In one aspect, the polypeptide is at least 50%, 60%, 70%, 80%, or 90% pure by weight of the total polypeptide material present in the preparation. As used herein, a "substantially pure polypeptide" can refer to a polypeptide preparation that contains, 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 polypeptide material associated with it natively or recombinantly.

[0181] Accordingly, preferably, on a weight basis of the total polypeptide material present in the formulation, the substantially pure polypeptide is at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99% pure, and most preferably at least 99.5% pure. The polypeptides of the invention are preferably in substantially pure form (i.e., the formulation is substantially free of other polypeptide materials associated therewith in native or recombinant form). For example, this can be achieved by preparing the polypeptide using well-known recombinant methods or using classical purification methods.

[0182] Recombinant: The term "recombinant" is used in its conventional meaning and refers to the manipulation (e.g., cutting and rejoining) of nucleic acid sequences to form a sequence population different from the sequence populations found in nature. The term recombinant refers to a cell, nucleic acid, polypeptide, or vector that has been modified from its native state. Thus, for example, a recombinant cell expresses a gene not found within a cell in its native (non-recombinant) form, or expresses a native gene at a different level or under different conditions compared to that found in nature. The terms "recombinant" are synonymous with "genetically modified" and "transgenic".

[0183] Recover: The term "recover (recover or recovery)" means the removal of a polypeptide from at least one fermentation broth component selected from the list of cells, nucleic acids, or other specified materials, for example, recovering the polypeptide from the whole fermentation broth or from a cell-free fermentation broth by methods such as polypeptide crystal harvesting, by filtration (e.g., depth filtration (by using a filter aid or packed filter media, cloth filtration in a cartridge filter, rotary drum filtration, drum filtration, rotary vacuum drum filtration, candle filter, horizontal leaf filter, or the like, sheet or pad filtration in a frame or modular device) or membrane filtration (using plate filtration, module filtration, candle filtration, microfiltration, crossflow, dynamic crossflow, or dead-end operation ultrafiltration)) or by centrifugation (using a sedimentation centrifuge, disk stack centrifuge, hydro cyclone, or the like) or by precipitating the polypeptide and using associated solid-liquid separation methods to harvest the polypeptide from the broth medium by using particle size fractionation. Recover encompasses the separation and / or purification of the polypeptide.

[0184] Sequence identity: The degree of relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity". The Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) is used to determine the sequence identity between two amino acid sequences as the output of "longest identity", which is implemented as in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) version 6.6.0. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (the EMBOSS version of BLOSUM62) substitution matrix. In order for the Needle program to report the longest identity, the non-abbreviated (-nobrief) option must be specified on the command line. The output of "longest identity" marked by Needle is calculated as follows:

[0185] (Identical residues × 100) / (Alignment length - Total number of gaps in the alignment)

[0186] 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", which is implemented as in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, ibid.) version 6.6.0. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (the EMBOSS version of NCBINUC4.4) substitution matrix. In order for the Needle program to report the longest identity, the non-abbreviated option must be specified on the command line. The output of "longest identity" marked by Needle is calculated as follows:

[0187] (Identical deoxyribonucleotides × 100) / (Alignment length – Total number of gaps in the alignment)

[0188] Signal peptide: A "signal peptide" is an amino acid sequence attached to the N-terminal portion of a protein that facilitates the secretion of the protein outside the cell. The mature form of the extracellular protein lacks the signal peptide, which is excised during the secretion process.

[0189] Subsequence: The term "subsequence" means a polynucleotide with one or more nucleotides deleted from the 5'-end and / or 3'-end of a mature polypeptide coding sequence; wherein the subsequence encodes a fragment having acetylxylan esterase activity.

[0190] Thermostable: "Thermostable" means that the enzyme does not denature or inactivate 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 retains its activity at this temperature, then the thermostable enzyme is suitable for liquefaction.

[0191] Distillers' solubles: "Distillers' solubles" refers to the centrifuged filtrate separated from whole distillers' grains, which is pumped to an evaporator to be concentrated into a slurry.

[0192] Variant: The term "variant" means a polypeptide having acetylxylan esterase activity and containing a man-made mutation (i.e., substitution, insertion (including extension) and / or deletion (e.g., truncation)) at one or more positions. Substitution means replacing the amino acid occupying a position with a different amino acid; deletion means removing the amino acid occupying a position; and insertion means adding 1-5 amino acids (e.g., 1-3 amino acids, especially 1 amino acid) adjacent to and immediately following the amino acid occupying a position.

[0193] Whole distillers' grains: "Whole distillers' grains" includes the material remaining at the end of the distillation process after the recovery of a fermentation product (such as ethanol).

[0194] Wild type: When referring to an amino acid sequence or a nucleic acid sequence, the term "wild type" means that the amino acid sequence or nucleic acid sequence is a native or natural sequence. As used herein, the term "naturally occurring" refers to any substance found in nature (such as a protein, amino acid or nucleic acid sequence). In contrast, the term "non-naturally occurring" refers to any substance not found in nature (e.g., recombinant nucleic acid and protein sequences produced in the laboratory, or modifications of wild type sequences).

[0195] Xylanase: "Xylanase" encompasses: endo-1,4-β-xylanase (EC 3.2.1.8), which catalyzes the endohydrolysis of (1→4)-β-D-xylosidic bonds in xylan; and glucuronoxylan endo-1,4-β-xylanase (E.C. 3.2.1.136), which catalyzes the endohydrolysis of 1,4-β-D-xylosyl bonds in some glucuronoxylans.

[0196] Xylanase activity: The activity of EC 3.2.1.8 xylanase can be determined using birchwood xylan as a substrate. One unit of xylanase is defined as the amount of enzyme that hydrolyzes 0.01% Producing 1.0 micromole of reducing sugars per minute per liter from 2 g of birchwood xylan as substrate in 50 mM sodium acetate at pH 2 (measured as glucose equivalents as described in Lever, 1972, A new reaction for colorimetric determination of carbohydrates, Anal. Biochem. 47:273-279). EC 3.2.1.136 xylanase activity can be measured at 37 °C in 0.01% X-100 and 200 mM sodium phosphate (pH 6) using 0.2% AZCL-glucuronoxylan as substrate. One unit of xylanase activity is defined as producing 1.0 micromole of azurine per minute from 0.2% AZCL-glucuronoxylan as substrate in 200 mM sodium phosphate (pH 6) at 37 °C and pH 6. Detailed Description

[0197] Carbohydrate esterase family 3 (CE3) polypeptides with acetylxylan esterase activity

[0198] This invention relates to carbohydrate esterase 3 (CE3) family polypeptides with acetylxylan esterase activity. In one aspect, the invention relates to polypeptides with acetylxylan esterase activity selected from the group consisting of:

[0199] (a) polypeptides having at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:2;

[0200] (b) polypeptides having at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3;

[0201] (c) a polypeptide having at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO:2;

[0202] (d) a polypeptide encoded by a polynucleotide having at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:1;

[0203] (e) a polypeptide derived from SEQ ID NO:2, the mature polypeptide of SEQ ID NO:2, or the polypeptide of SEQ ID NO:3 by substitution, deletion, or addition of one or more amino acids;

[0204] (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d), or (e), wherein the N-terminus and / or C-terminus has been extended by addition of one or more amino acids; and

[0205] (g) a fragment of the polypeptide of (a), (b), (c), (d), or (e);

[0206] wherein the polypeptide has acetylxylan esterase activity.

[0207] In one aspect, the polypeptide has at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:2 or the mature polypeptide of SEQ ID NO:2.

[0208] In another aspect, the polypeptide has at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:3.

[0209] The polypeptide preferably comprises, consists essentially of, or consists of: the amino acid sequence of SEQ ID NO:2 or its mature polypeptide.

[0210] The polypeptide preferably comprises, consists essentially of, or consists of: amino acids 19 to 297 of SEQ ID NO:2.

[0211] The polypeptide preferably comprises, consists essentially of, or consists of: the amino acid sequence of SEQ ID NO:3.

[0212] The polypeptide may have an N-terminal and / or C-terminal extension of one or more amino acids (e.g., 1 - 5 amino acids).

[0213] In another aspect, the polypeptide is a fragment that contains at least 237 amino acid residues (e.g., amino acids 1 to 237 of SEQ ID NO:3), at least 251 amino acid residues (e.g., amino acids 1 to 251 of SEQ ID NO:3), or at least 265 amino acid residues (e.g., amino acids 1 to 265 of SEQ ID NO:3).

[0214] In some embodiments, the polypeptide is encoded by a polynucleotide having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:1.

[0215] The polynucleotide encoding the polypeptide preferably comprises, consists essentially of, or consists of: nucleotides 57 to 891 of SEQ ID NO:1.

[0216] In another aspect, the polypeptide is derived from SEQ ID NO:2 by substitution, deletion, or addition of one or several amino acids. In another aspect, the polypeptide is derived from the mature polypeptide of SEQ ID NO:2 by substitution, deletion, or addition of one or several amino acids. In another aspect, the polypeptide is derived from SEQ ID NO:3 by substitution, deletion, or addition of one or more amino acids. In some embodiments, the polypeptide is a variant of SEQ ID NO:3 that contains substitutions, deletions, and / or insertions at one or more positions. In one aspect, the number of amino acid substitutions, deletions, and / or insertions introduced into the polypeptide of SEQ ID NO:3 is up to 15, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid changes can have a conservative nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; typically small deletions of 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as a methionine residue at the amino terminus; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, such as a polyhistidine segment, an epitope, or a binding module.

[0217] In one aspect, the present invention relates to a polypeptide having acetylxylan esterase activity, which polypeptide is selected from the group consisting of:

[0218] (a) a polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:5;

[0219] (b) a polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:6;

[0220] (c) a polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO:5;

[0221] (d) a polypeptide encoded by a polynucleotide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide coding sequence of SEQ ID NO:4;

[0222] (e) a polypeptide derived from SEQ ID NO:5, the mature polypeptide of SEQ ID NO:5, or the polypeptide of SEQ ID NO:6 by substitution, deletion, or addition of one or several amino acids;

[0223] (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d), or (e), wherein the N-terminus and / or C-terminus has been extended by addition of one or more amino acids; and

[0224] (g) a fragment of the polypeptide of (a), (b), (c), (d), or (e);

[0225] wherein the polypeptide has acetylxylan esterase activity.

[0226] In one aspect, the polypeptide has at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:5 or the mature polypeptide of SEQ ID NO:5.

[0227] In another aspect, the polypeptide has at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:6.

[0228] The polypeptide preferably comprises, consists essentially of, or consists of: the amino acid sequence of SEQ ID NO:5 or its mature polypeptide.

[0229] The polypeptide preferably comprises, consists essentially of, or consists of: amino acids 26 to 245 of SEQ ID NO:5.

[0230] The polypeptide preferably comprises, consists essentially of, or consists of: the amino acid sequence of SEQ ID NO:6.

[0231] The polypeptide may have an N-terminal and / or C-terminal extension of one or more amino acids (e.g., 1 - 5 amino acids).

[0232] In another aspect, the polypeptide is a fragment that contains at least 187 amino acid residues (e.g., amino acids 1 to 187 of SEQ ID NO:6), at least 198 amino acid residues (e.g., amino acids 1 to 198 of SEQ ID NO:6), or at least 209 amino acid residues (e.g., amino acids 1 to 209 of SEQ ID NO:6).

[0233] In some embodiments, the polypeptide is encoded by a polynucleotide having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:4.

[0234] The polynucleotide encoding the polypeptide preferably comprises, consists essentially of, or consists of nucleotides 78 to 735 of SEQ ID NO:4.

[0235] In another aspect, the polypeptide is derived from SEQ ID NO:5 by substitution, deletion or addition of one or several amino acids. In another aspect, the polypeptide is derived from the mature polypeptide of SEQ ID NO:5 by substitution, deletion or addition of one or several amino acids. In another aspect, the polypeptide is derived from SEQ ID NO:6 by substitution, deletion or addition of one or more amino acids. In some embodiments, the polypeptide is a variant of SEQ ID NO:6 that contains substitutions, deletions and / or insertions at one or more positions. In one aspect, the number of amino acid substitutions, deletions and / or insertions introduced into the polypeptide of SEQ ID NO:6 is up to 15, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. The amino acid alterations can be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; typically small deletions of 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as a methionine residue at the amino terminus; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, such as a polyhistidine segment, an antigenic epitope or a binding module.

[0236] In one aspect, the present invention relates to a polypeptide having acetylxylan esterase activity, which polypeptide is selected from the group consisting of:

[0237] (a) a polypeptide having at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:8;

[0238] (b) a polypeptide having at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:9;

[0239] (c) a polypeptide having at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO:8;

[0240] (d) a polypeptide encoded by a polynucleotide having at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide coding sequence of SEQ ID NO:7;

[0241] (e) a polypeptide derived from SEQ ID NO:8, the mature polypeptide of SEQ ID NO:8, or the polypeptide of SEQ ID NO:9 by substitution, deletion, or addition of one or more amino acids;

[0242] (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d), or (e), wherein the N-terminus and / or C-terminus has been extended by addition of one or more amino acids; and

[0243] (g) a fragment of the polypeptide of (a), (b), (c), (d), or (e);

[0244] wherein the polypeptide has acetylxylan esterase activity.

[0245] In one aspect, the polypeptide has at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:8 or the mature polypeptide of SEQ ID NO:8.

[0246] In another aspect, the polypeptide has at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:9.

[0247] The polypeptide preferably comprises, consists essentially of, or consists of: the amino acid sequence of SEQ ID NO:8 or its mature polypeptide.

[0248] The polypeptide preferably comprises, consists essentially of, or consists of: amino acids 20 to 289 of SEQ ID NO:8.

[0249] The polypeptide preferably comprises, consists essentially of, or consists of: the amino acid sequence of SEQ ID NO:9.

[0250] The polypeptide may have an N-terminal and / or C-terminal extension of one or more amino acids (e.g., 1 - 5 amino acids).

[0251] In another aspect, the polypeptide is a fragment that contains at least 230 amino acid residues (e.g., amino acids 1 to 230 of SEQ ID NO:9), at least 243 amino acid residues (e.g., amino acids 1 to 243 of SEQ ID NO:9), or at least 257 amino acid residues (e.g., amino acids 1 to 257 of SEQ ID NO:9).

[0252] In some embodiments, the polypeptide is encoded by a polynucleotide having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:7.

[0253] The polynucleotide encoding the polypeptide preferably comprises, consists essentially of, or consists of nucleotides 60 to 867 of SEQ ID NO:7.

[0254] In another aspect, the polypeptide is derived from SEQ ID NO:8 by substitution, deletion or addition of one or several amino acids. In another aspect, the polypeptide is derived from the mature polypeptide of SEQ ID NO:8 by substitution, deletion or addition of one or several amino acids. In another aspect, the polypeptide is derived from SEQ ID NO:9 by substitution, deletion or addition of one or more amino acids. In some embodiments, the polypeptide is a variant of SEQ ID NO:9 that contains substitutions, deletions and / or insertions at one or more positions. In one aspect, the number of amino acid substitutions, deletions and / or insertions introduced into the polypeptide of SEQ ID NO:9 is up to 15, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. The amino acid alterations can have a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; typically small deletions of 1 - 30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as a methionine residue at the amino terminus; small linker peptides of up to 20 - 25 residues; or small extensions that facilitate purification by altering the net charge or another function, such as a polyhistidine segment, an epitope or a binding module.

[0255] In one aspect, the invention relates to a polypeptide having acetylxylan esterase activity, which polypeptide is selected from the group consisting of:

[0256] (a) A polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:11;

[0257] (b) A polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:12;

[0258] (c) A polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO:11;

[0259] (d) A polypeptide encoded by a polynucleotide that has at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide coding sequence of SEQ ID NO:10;

[0260] (e) A polypeptide derived from SEQ ID NO:11, the mature polypeptide of SEQ ID NO:11, or the polypeptide of SEQ ID NO:12 by substitution, deletion, or addition of one or more amino acids;

[0261] (f) A polypeptide derived from the polypeptide of (a), (b), (c), (d), or (e), wherein the N-terminus and / or C-terminus has been extended by addition of one or more amino acids; and

[0262] (g) A fragment of the polypeptide of (a), (b), (c), (d), or (e);

[0263] wherein the polypeptide has acetylxylan esterase and / or acetylxylan esterase activity.

[0264] In one aspect, the polypeptide has at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:11 or the mature polypeptide of SEQ ID NO:11.

[0265] In another aspect, the polypeptide has at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:12.

[0266] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:11 or its mature polypeptide.

[0267] The polypeptide preferably comprises, consists essentially of, or consists of amino acids 23 to 253 of SEQ ID NO:11 or its mature polypeptide.

[0268] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:12.

[0269] The polypeptide may have an N-terminal and / or C-terminal extension of one or more amino acids (e.g., 1-5 amino acids).

[0270] In another aspect, the polypeptide is a fragment that contains at least 196 amino acid residues (e.g., amino acids 1 to 196 of SEQ ID NO:12), at least 208 amino acid residues (e.g., amino acids 1 to 208 of SEQ ID NO:12), or at least 219 amino acid residues (e.g., amino acids 1 to 219 of SEQ ID NO:12).

[0271] In some embodiments, the polypeptide is encoded by a polynucleotide having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:10.

[0272] The polynucleotide encoding the polypeptide preferably comprises, consists essentially of, or consists of nucleotides 69 to 759 of SEQ ID NO:10.

[0273] In another aspect, the polypeptide is derived from SEQ ID NO:11 by substitution, deletion or addition of one or several amino acids. In another aspect, the polypeptide is derived from the mature polypeptide of SEQ ID NO:11 by substitution, deletion or addition of one or several amino acids. In another aspect, the polypeptide is derived from SEQ ID NO:12 by substitution, deletion or addition of one or more amino acids. In some embodiments, the polypeptide is a variant of SEQ ID NO:12 that contains substitutions, deletions and / or insertions at one or more positions. In one aspect, the number of amino acid substitutions, deletions and / or insertions introduced into the polypeptide of SEQ ID NO:12 is up to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. The amino acid changes can have a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; typically small deletions of 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function (such as a polyhistidine segment, an epitope or a binding module).

[0274] In one aspect, the present invention relates to polypeptides having acetylxylan esterase activity, which polypeptides are selected from the group consisting of:

[0275] (a) a polypeptide having at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:14;

[0276] (b) a polypeptide having at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:15;

[0277] (c) a polypeptide having at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO:14;

[0278] (d) a polypeptide encoded by a polynucleotide having at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:13;

[0279] (e) A polypeptide derived from SEQ ID NO:14, the mature polypeptide of SEQ ID NO:14, or the polypeptide of SEQ ID NO:15 by substitution, deletion, or addition of one or more amino acids;

[0280] (f) A polypeptide derived from a polypeptide of (a), (b), (c), (d), or (e), wherein the N-terminus and / or C-terminus has been extended by addition of one or more amino acids; and

[0281] (g) A fragment of a polypeptide of (a), (b), (c), (d), or (e);

[0282] wherein the polypeptide has acetylxylan esterase activity.

[0283] In one aspect, the polypeptide has at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:14 or the mature polypeptide of SEQ ID NO:14.

[0284] In another aspect, the polypeptide has at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:15.

[0285] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:14 or its mature polypeptide.

[0286] The polypeptide preferably comprises, consists essentially of, or consists of amino acids 19 to 240 of SEQ ID NO:14.

[0287] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:15.

[0288] The polypeptide may have an N-terminal and / or C-terminal extension of one or more amino acids (e.g., 1-5 amino acids).

[0289] In another aspect, the polypeptide is a fragment that contains at least 188 amino acid residues (e.g., amino acids 1 to 188 of SEQ ID NO:15), at least 200 amino acid residues (e.g., amino acids 1 to 200 of SEQ ID NO:15), or at least 211 amino acid residues (e.g., amino acids 1 to 211 of SEQ ID NO:15).

[0290] In some embodiments, the polypeptide is encoded by a polynucleotide having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:13.

[0291] The polynucleotide encoding the polypeptide preferably comprises, consists essentially of, or consists of nucleotides 57 to 720 of SEQ ID NO:13.

[0292] In another aspect, the polypeptide is derived from SEQ ID NO:14 by substitution, deletion, or addition of one or a few amino acids. In another aspect, the polypeptide is derived from the mature polypeptide of SEQ ID NO:14 by substitution, deletion, or addition of one or a few amino acids. In another aspect, the polypeptide is derived from SEQ ID NO:15 by substitution, deletion, or addition of one or more amino acids. In some embodiments, the polypeptide is a variant of SEQ ID NO:15 that contains substitutions, deletions, and / or insertions at one or more positions. In one aspect, the number of amino acid substitutions, deletions, and / or insertions introduced into the polypeptide of SEQ ID NO:15 is up to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid changes can have a conservative nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; typically small deletions of 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function (such as a polyhistidine segment, an epitope, or a binding module).

[0293] In one aspect, the present invention relates to a polypeptide having acetylxylan esterase activity, which polypeptide is selected from the group consisting of:

[0294] (a) a polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:17;

[0295] (b) a polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:18;

[0296] (c) a polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO:17;

[0297] (d) A polypeptide encoded by a polynucleotide that has at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:16 or its cDNA sequence;

[0298] (e) A polypeptide derived from SEQ ID NO:17, the mature polypeptide of SEQ ID NO:17, or the polypeptide of SEQ ID NO:18 by substitution, deletion, or addition of one or more amino acids;

[0299] (f) A polypeptide derived from the polypeptide of (a), (b), (c), (d), or (e), wherein the N-terminus and / or C-terminus has been extended by addition of one or more amino acids; and

[0300] (g) A fragment of the polypeptide of (a), (b), (c), (d), or (e);

[0301] wherein the polypeptide has acetylxylan esterase activity.

[0302] In one aspect, the polypeptide has at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:17 or the mature polypeptide of SEQ ID NO:17.

[0303] In another aspect, the polypeptide has at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:18.

[0304] The polypeptide preferably comprises, consists essentially of, or consists of: the amino acid sequence of SEQ ID NO:17 or its mature polypeptide.

[0305] The polypeptide preferably comprises, consists essentially of, or consists of: amino acids 23 to 244 of SEQ ID NO:17.

[0306] The polypeptide preferably comprises, consists essentially of, or consists of: the amino acid sequence of SEQ ID NO:18.

[0307] The polypeptide may have an N-terminal and / or C-terminal extension of one or more amino acids (e.g., 1 - 5 amino acids).

[0308] In another aspect, the polypeptide is a fragment that contains at least 188 amino acid residues (e.g., amino acids 1 to 188 of SEQ ID NO:18), at least 200 amino acid residues (e.g., amino acids 1 to 200 of SEQ ID NO:18), or at least 211 amino acid residues (e.g., amino acids 1 to 211 of SEQ ID NO:18).

[0309] In some embodiments, the polypeptide is encoded by a polynucleotide having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the mature polypeptide coding sequence of SEQ ID NO:16.

[0310] The polynucleotide encoding the polypeptide preferably comprises, consists essentially of, or consists of: nucleotides 135 to 712 and 798 to 885 of SEQ ID NO:16.

[0311] In another aspect, the polypeptide is derived from SEQ ID NO:17 by substitution, deletion, or addition of one or several amino acids. In another aspect, the polypeptide is derived from the mature polypeptide of SEQ ID NO:17 by substitution, deletion, or addition of one or several amino acids. In another aspect, the polypeptide is derived from SEQ ID NO:18 by substitution, deletion, or addition of one or more amino acids. In some embodiments, the polypeptide is a variant of SEQ ID NO:18 that contains substitutions, deletions, and / or insertions at one or more positions. In one aspect, the number of amino acid substitutions, deletions, and / or insertions introduced into the polypeptide of SEQ ID NO:18 is up to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid changes can have a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; typically small deletions of 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as a methionine residue at the amino terminus; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, such as a polyhistidine segment, an epitope, or a binding module.

[0312] In one aspect, the present invention relates to a polypeptide having acetylxylan esterase activity, which polypeptide is selected from the group consisting of:

[0313] (a) a polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:20;

[0314] (b) a polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:21;

[0315] (c) a polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO: 20;

[0316] (d) a polypeptide encoded by a polynucleotide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide coding sequence of SEQ ID NO: 19;

[0317] (e) a polypeptide derived from SEQ ID NO: 20, the mature polypeptide of SEQ ID NO: 20, or the polypeptide of SEQ ID NO: 21 by substitution, deletion, or addition of one or several amino acids;

[0318] (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d), or (e), wherein the N-terminus and / or C-terminus has been extended by addition of one or more amino acids; and

[0319] (g) a fragment of the polypeptide of (a), (b), (c), (d), or (e);

[0320] wherein the polypeptide has acetylxylan esterase activity.

[0321] In one aspect, the polypeptide has at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:20 or the mature polypeptide of SEQ ID NO:20.

[0322] In another aspect, the polypeptide has at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:21.

[0323] The polypeptide preferably comprises, consists essentially of, or consists of: the amino acid sequence of SEQ ID NO:20 or its mature polypeptide.

[0324] The polypeptide preferably comprises, consists essentially of, or consists of: amino acids 25 to 247 of SEQ ID NO:20.

[0325] The polypeptide preferably comprises, consists essentially of, or consists of: the amino acid sequence of SEQ ID NO:21.

[0326] The polypeptide may have an N-terminal and / or C-terminal extension of one or more amino acids (e.g., 1 - 5 amino acids).

[0327] In another aspect, the polypeptide is a fragment that contains at least 190 amino acid residues (e.g., amino acids 1 to 190 of SEQ ID NO:21), at least 200 amino acid residues (e.g., amino acids 1 to 200 of SEQ ID NO:21), or at least 212 amino acid residues (e.g., amino acids 1 to 212 of SEQ ID NO:21).

[0328] In some embodiments, the polypeptide is encoded by a polynucleotide having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:19.

[0329] The polynucleotide encoding the polypeptide preferably comprises, consists essentially of, or consists of nucleotides 75 to 741 of SEQ ID NO:19.

[0330] In another aspect, the polypeptide is derived from SEQ ID NO:20 by substitution, deletion or addition of one or several amino acids. In another aspect, the polypeptide is derived from the mature polypeptide of SEQ ID NO:20 by substitution, deletion or addition of one or several amino acids. In another aspect, the polypeptide is derived from SEQ ID NO:21 by substitution, deletion or addition of one or more amino acids. In some embodiments, the polypeptide is a variant of SEQ ID NO:21 that contains substitutions, deletions and / or insertions at one or more positions. In one aspect, the number of amino acid substitutions, deletions and / or insertions introduced into the polypeptide of SEQ ID NO:21 is up to 15, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. The amino acid changes can have a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; typically small deletions of 1 - 30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as a methionine residue at the amino terminus; small linker peptides of up to 20 - 25 residues; or small extensions that facilitate purification by altering the net charge or another function (such as a polyhistidine segment, an epitope or a binding module).

[0331] In one aspect, the present invention relates to a polypeptide having acetylxylan esterase activity, which polypeptide is selected from the group consisting of:

[0332] (a) A polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 23;

[0333] (b) A polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 24;

[0334] (c) A polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO: 23;

[0335] (d) A polypeptide encoded by a polynucleotide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide coding sequence of SEQ ID NO: 22;

[0336] (e) Derived from the polypeptide of SEQ ID NO:23, the mature polypeptide of SEQ ID NO:23, or the polypeptide of SEQ ID NO:24 by substitution, deletion, or addition of one or more amino acids;

[0337] (f) A polypeptide derived from the polypeptide of (a), (b), (c), (d), or (e), wherein the N-terminus and / or C-terminus has been extended by addition of one or more amino acids; and

[0338] (g) A fragment of the polypeptide of (a), (b), (c), (d), or (e);

[0339] wherein the polypeptide has acetylxylan esterase activity.

[0340] In one aspect, the polypeptide has at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:23 or the mature polypeptide of SEQ ID NO:23.

[0341] In another aspect, the polypeptide has at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:24.

[0342] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:23 or its mature polypeptide.

[0343] The polypeptide preferably comprises, consists essentially of, or consists of amino acids 25 to 244 of SEQ ID NO:23.

[0344] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:24.

[0345] The polypeptide may have an N-terminal and / or C-terminal extension of one or more amino acids (e.g., 1-5 amino acids).

[0346] In another aspect, the polypeptide is a fragment that contains at least 187 amino acid residues (e.g., amino acids 1 to 187 of SEQ ID NO:24), at least 198 amino acid residues (e.g., amino acids 1 to 198 of SEQ ID NO:24), or at least 209 amino acid residues (e.g., amino acids 1 to 209 of SEQ ID NO:24).

[0347] In some embodiments, the polypeptide is encoded by a polynucleotide having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:22.

[0348] The polynucleotide encoding the polypeptide preferably comprises, consists essentially of, or consists of nucleotides 75 to 732 of SEQ ID NO:22.

[0349] In another aspect, the polypeptide is derived from SEQ ID NO:23 by substitution, deletion or addition of one or several amino acids. In another aspect, the polypeptide is derived from the mature polypeptide of SEQ ID NO:23 by substitution, deletion or addition of one or several amino acids. In another aspect, the polypeptide is derived from SEQ ID NO:24 by substitution, deletion or addition of one or more amino acids. In some embodiments, the polypeptide is a variant of SEQ ID NO:24 that contains substitutions, deletions and / or insertions at one or more positions. In one aspect, the number of amino acid substitutions, deletions and / or insertions introduced into the polypeptide of SEQ ID NO:24 is up to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. The amino acid alterations can have a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; typically small deletions of 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function (such as a polyhistidine segment, an epitope or a binding module).

[0350] In one aspect, the present invention relates to a polypeptide having acetylxylan esterase activity, the polypeptide being selected from the group consisting of:

[0351] (a) a polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:26;

[0352] (b) a polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:27;

[0353] (c) a polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO:26;

[0354] (d) a polypeptide encoded by a polynucleotide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide coding sequence of SEQ ID NO:25;

[0355] (e) A polypeptide derived from SEQ ID NO:26, the mature polypeptide of SEQ ID NO:26, or the polypeptide of SEQ ID NO:27 by substitution, deletion, or addition of one or more amino acids;

[0356] (f) A polypeptide derived from the polypeptide of (a), (b), (c), (d), or (e), wherein the N-terminus and / or C-terminus has been extended by addition of one or more amino acids; and

[0357] (g) A fragment of the polypeptide of (a), (b), (c), (d), or (e);

[0358] wherein the polypeptide has acetylxylan esterase activity.

[0359] In one aspect, the polypeptide has at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:26 or the mature polypeptide of SEQ ID NO:26.

[0360] In another aspect, the polypeptide has at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:27.

[0361] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:26 or its mature polypeptide.

[0362] The polypeptide preferably comprises, consists essentially of, or consists of amino acids 20 to 240 of SEQ ID NO:26.

[0363] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:27.

[0364] The polypeptide may have an N-terminal and / or C-terminal extension of one or more amino acids (e.g., 1-5 amino acids).

[0365] In another aspect, the polypeptide is a fragment that contains at least 188 amino acid residues (e.g., amino acids 1 to 188 of SEQ ID NO:27), at least 199 amino acid residues (e.g., amino acids 1 to 199 of SEQ ID NO:27), or at least 210 amino acid residues (e.g., amino acids 1 to 210 of SEQ ID NO:27).

[0366] In some embodiments, the polypeptide is encoded by a polynucleotide having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:25.

[0367] The polynucleotide encoding the polypeptide preferably comprises, consists essentially of, or consists of nucleotides 60 to 720 of SEQ ID NO:25.

[0368] In another aspect, the polypeptide is derived from SEQ ID NO:26 by substitution, deletion or addition of one or several amino acids. In another aspect, the polypeptide is derived from the mature polypeptide of SEQ ID NO:26 by substitution, deletion or addition of one or several amino acids. In another aspect, the polypeptide is derived from SEQ ID NO:27 by substitution, deletion or addition of one or more amino acids. In some embodiments, the polypeptide is a variant of SEQ ID NO:27 that contains substitutions, deletions and / or insertions at one or more positions. In one aspect, the number of amino acid substitutions, deletions and / or insertions introduced into the polypeptide of SEQ ID NO:27 is up to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. The amino acid changes can have a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; typically small deletions of 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as a methionine residue at the amino terminus; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, such as a polyhistidine segment, an epitope or a binding module.

[0369] In one aspect, the invention relates to a polypeptide having acetylxylan esterase activity, which polypeptide is selected from the group consisting of:

[0370] (a) a polypeptide having at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:29;

[0371] (b) a polypeptide having at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:30;

[0372] (c) a polypeptide having at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO:29;

[0373] (d) a polypeptide encoded by a polynucleotide that has at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide coding sequence of SEQ ID NO:28;

[0374] (e) a polypeptide derived from SEQ ID NO:29, the mature polypeptide of SEQ ID NO:29, or the polypeptide of SEQ ID NO:30 by substitution, deletion, or addition of one or more amino acids;

[0375] (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d), or (e), wherein the N-terminus and / or C-terminus has been extended by addition of one or more amino acids; and

[0376] (g) a fragment of the polypeptide of (a), (b), (c), (d), or (e);

[0377] wherein the polypeptide has acetylxylan esterase activity.

[0378] In one aspect, the polypeptide has at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO:29 or the mature polypeptide of SEQ ID NO:29.

[0379] In another aspect, the polypeptide has at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:30.

[0380] The polypeptide preferably comprises, consists essentially of, or consists of: the amino acid sequence of SEQ ID NO:29 or its mature polypeptide.

[0381] The polypeptide preferably comprises, consists essentially of, or consists of: amino acids 17 to 286 of SEQ ID NO:29 or its mature polypeptide.

[0382] The polypeptide preferably comprises, consists essentially of, or consists of: the amino acid sequence of SEQ ID NO:30.

[0383] The polypeptide may have an N-terminal and / or C-terminal extension of one or more amino acids (e.g., 1 - 5 amino acids).

[0384] In another aspect, the polypeptide is a fragment that contains at least 230 amino acid residues (e.g., amino acids 1 to 230 of SEQ ID NO:30), at least 243 amino acid residues (e.g., amino acids 1 to 243 of SEQ ID NO:30), or at least 257 amino acid residues (e.g., amino acids 1 to 257 of SEQ ID NO:30).

[0385] In some embodiments, the polypeptide is encoded by a polynucleotide having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:28.

[0386] The polynucleotide encoding the polypeptide preferably comprises, consists essentially of, or consists of: nucleotides 51 to 858 of SEQ ID NO:28.

[0387] In another aspect, the polypeptide is derived from SEQ ID NO:29 by substitution, deletion, or addition of one or several amino acids. In another aspect, the polypeptide is derived from the mature polypeptide of SEQ ID NO:29 by substitution, deletion, or addition of one or several amino acids. In another aspect, the polypeptide is derived from SEQ ID NO:30 by substitution, deletion, or addition of one or more amino acids. In some embodiments, the polypeptide is a variant of SEQ ID NO:30 that contains substitutions, deletions, and / or insertions at one or more positions. In one aspect, the number of amino acid substitutions, deletions, and / or insertions introduced into the polypeptide of SEQ ID NO:30 is up to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid alterations can be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; typically small deletions of 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as a methionine residue at the amino terminus; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, such as a polyhistidine segment, an epitope, or a binding module.

[0388] Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, a single alanine mutation is introduced at each residue in the molecule, and the acetylxylan esterase activity of the resulting molecule is tested to identify the amino acid residues that are critical for the activity of the molecule. See also Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. The active site of an enzyme or other biological interaction can also be determined by physical analysis of the structure, such as by the following techniques: such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, together with mutagenesis of the putative contact-site amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identity of essential amino acids can also be inferred from alignment with related polypeptides, and / or from sequence homology and conserved catalytic mechanisms with related polypeptides or polypeptides / proteins within a polypeptide or protein family that share a common ancestor (typically having similar three-dimensional structures, functions, and significant sequence similarity). Additionally or alternatively, protein structure prediction tools can be used for protein structure modeling to identify essential amino acids and / or active sites of a polypeptide. See, for example, Jumper et al., 2021, “Highly accurate protein structure prediction with AlphaFold”, Nature 596:583-589.

[0389] Using known mutagenesis, recombination, and / or shuffling methods, followed by the appropriate screening procedures, single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested, such as the screening procedures disclosed by Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86:2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:10832-10837; US 5,223,409; WO 92 / 06204), and site-directed mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0390] The mutagenesis / shuffling methods can be combined with high-throughput, automated screening methods to detect the activity of the cloned, mutagenized polypeptides expressed by the host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). The mutagenized DNA molecules encoding the active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow for the rapid determination of the importance of individual amino acid residues in the polypeptide.

[0391] The CE3 family has several enzymes whose structures have been resolved, including TcAE206 from Talaromyces cellulolyticus and CtCes3-1 from Hungateiclostridium thermocellum. Both of these structures have the (α / β / α)-sandwich fold characteristic of enzymes in the SGNH hydrolase family. The (α / β / α)-sandwich has five central parallel β-strands that form a curved β-sheet, which is flanked on either side by 5-6 α-helices. Both of these structures also have a calcium-binding loop motif (DXVGX7DX n (D / N)), which is present above the N-terminus of the central β-strand. This binding motif is conserved in previously characterized CE3s.

[0392] Carbohydrate esterase family 3 (CE3) polypeptides have a classical Ser-His-Asp catalytic triad, which is a characteristic feature of enzymes of the SGNH hydrolase family. The active site residues are established by four conserved consensus sequences (regions I-III and V) and contain an altered nucleophilic "elbow" turn motif (-GxSxT- instead of the typical -GxSxG- motif). The catalytic triad, together with the Gly residue in region II and the Asn residue in region III that form the oxyanion hole, is conserved in all characterized CE3 enzymes. The Asp residue in region V promotes the amphoteric nature of the His residue in region V, which extracts a proton from the Ser in region I to render it nucleophilic.

[0393] The positions of the above features are shown in Table 1 below, and Table 1 shows an alignment demonstrating the conservation of the features in the exemplary CE3 polypeptide mature sequences of the present invention.

[0394] Table 1

[0395]

[0396] The polypeptide can be a fusion polypeptide.

[0397] In one aspect, the polypeptide is isolated.

[0398] In another aspect, the polypeptide is purified.

[0399] Sources of carbohydrate esterase 3 (CE3) family polypeptides having acetylxylan esterase activity

[0400] The polypeptides of the present invention having acetylxylan esterase activity can be obtained from microorganisms of any genus. For the purposes of the present invention, the term "obtained from" as used herein in connection with a given source shall mean that the polypeptide encoded by the polynucleotide is produced by that source or by a strain into which the polynucleotide of the present invention has been inserted. In one aspect, the polypeptide obtained from a given source is secreted extracellularly.

[0401] The polypeptide with acetylxylan esterase activity of the present invention can be obtained from the genus Dinemasporium. In one aspect, the polypeptide is obtained from a Dinemasporium species. In another aspect, the polypeptide is obtained from Dinemasporium ambiguum. In another aspect, the polypeptide is obtained from Dinemasporium americana. In another aspect, the polypeptide is obtained from Dinemasporium bambusicola. In another aspect, the polypeptide is obtained from Dinemasporium cruciferum. In another aspect, the polypeptide is obtained from Dinemasporium decipiens. In another aspect, the polypeptide is obtained from Dinemasporium ipomoeae. In another aspect, the polypeptide is obtained from Dinemasporium iriomotense. In another aspect, the polypeptide is obtained from Dinemasporium japonicum. In another aspect, the polypeptide is obtained from Dinemasporium longicapillatum. In another aspect, the polypeptide is obtained from Dinemasporium morbidum. In another aspect, the polypeptide is obtained from Dinemasporium nelloi. In another aspect, the polypeptide is obtained from Dinemasporium parastrigosum. In another aspect, the polypeptide is obtained from Dinemasporium polygonum. In another aspect, the polypeptide is obtained from Dinemasporium pseudodecipiens. In another aspect, the polypeptide is obtained from Dinemasporium pseudoindicum. In another aspect, the polypeptide is obtained from Dinemasporium pseudostrigosum. In another aspect, the polypeptide is obtained from Dinemasporium rishiriense. In another aspect, the polypeptide is obtained from Dinemasporium sasae. In another aspect, the polypeptide is obtained from Dinemasporium spinificis. In another aspect, the polypeptide is obtained from Dinemasporium strigosum. In another aspect, the polypeptide is obtained from Dinemasporium trichophoricola.

[0402] In one aspect, the polypeptide is obtained from Epicoccum. In another aspect, the polypeptide is obtained from Epicoccum andropogonearum. In another aspect, the polypeptide is obtained from Epicoccum andropogonis. In another aspect, the polypeptide is obtained from Epicoccum brahmansense. In another aspect, the polypeptide is obtained from Epicoccum brasiliense. In another aspect, the polypeptide is obtained from Epicoccum camelliae. In another aspect, the polypeptide is obtained from Epicoccum catenisporum. In another aspect, the polypeptide is obtained from Epicoccum cedri. In another aspect, the polypeptide is obtained from Epicoccum chloridis. In another aspect, the polypeptide is obtained from Epicoccum dendrobii. In another aspect, the polypeptide is obtained from Epicoccum djirangnandiri. In another aspect, the polypeptide is obtained from Epicoccum draconis. In another aspect, the polypeptide is obtained from Epicoccum duchesneae. In another aspect, the polypeptide is obtained from Epicoccum endophytica. In another aspect, the polypeptide is obtained from Epicoccum henningsii. In another aspect, the polypeptide is obtained from Epicoccum hordei. In another aspect, the polypeptide is obtained from Epicoccum huancayense. In another aspect, the polypeptide is obtained from Epicoccum italicum. In another aspect, the polypeptide is obtained from Epicoccum keratinophilum. In another aspect, the polypeptide is obtained from Epicoccum latusicollum. In another aspect, the polypeptide is obtained from Epicoccum layuense. In another aspect, the polypeptide is obtained from Epicoccum longiostiolatum. In another aspect, the polypeptide is obtained from Epicoccum mackenziei. In another aspect, the polypeptide is obtained from Epicoccum mezzettii. In another aspect, the polypeptide is obtained from Epicoccum mnesitheae. In another aspect, the polypeptide is obtained from Epicoccum mutliceps.On the other hand, the polypeptide is obtained from Epicoccum nigrum. On the other hand, the polypeptide is obtained from Epicoccum oryzae. On the other hand, the polypeptide is obtained from Epicoccum ovisporum. On the other hand, the polypeptide is obtained from Epicoccum phragmospora. On the other hand, the polypeptide is obtained from Epicoccum pimprinum. On the other hand, the polypeptide is obtained from Epicoccum plurivorum. On the other hand, the polypeptide is obtained from Epicoccum pneumoniae. On the other hand, the polypeptide is obtained from Epicoccum poaeiocola. On the other hand, the polypeptide is obtained from Epicoccum poae. On the other hand, the polypeptide is obtained from Epicoccum polychromum. On the other hand, the polypeptide is obtained from Epicoccum proteae. On the other hand, the polypeptide is obtained from Epicoccum pruni. On the other hand, the polypeptide is obtained from Epicoccum pseudokeratinophilum. On the other hand, the polypeptide is obtained from Epicoccum rhynchosporae. On the other hand, the polypeptide is obtained from Epicoccum rosae. On the other hand, the polypeptide is obtained from Epicoccum sorghicola. On the other hand, the polypeptide is obtained from Epicoccum sorghi. On the other hand, the polypeptide is obtained from Epicoccum thailandicum. On the other hand, the polypeptide is obtained from Epicoccum tobaicum. On the other hand, the polypeptide is obtained from Epicoccum triodiae. On the other hand, the polypeptide is obtained from Epicoccum tritici. On the other hand, the polypeptide is obtained from Epicoccum variabile. On the other hand, the polypeptide is obtained from Epicoccum viticis.

[0403] In one aspect, the polypeptide is obtained from Flammulina. In another aspect, the polypeptide is obtained from Flammulina cephalariae. In another aspect, the polypeptide is obtained from Flammulina elastica. In another aspect, the polypeptide is obtained from Flammulina fennae. In another aspect, the polypeptide is obtained from Flammulina filiformis. In another aspect, the polypeptide is obtained from Flammulina finlandica. In another aspect, the polypeptide is obtained from Flammulina Mexicana. In another aspect, the polypeptide is obtained from Flammulina ononidis. In another aspect, the polypeptide is obtained from Flammulina populicola. In another aspect, the polypeptide is obtained from Flammulina rossica. In another aspect, the polypeptide is obtained from Flammulina stratosa. In another aspect, the polypeptide is obtained from Flammulina velutipes. In another aspect, the polypeptide is obtained from Flammulina yunanensis.

[0404] In one aspect, the polypeptide is obtained from Microsphaeropsis. In another aspect, the polypeptide is the polypeptide obtained from Microsphaeropsis amaranthi. In another aspect, the polypeptide is the polypeptide obtained from Microsphaeropsis sphaerosperma. In another aspect, the polypeptide is the polypeptide obtained from Microsphaeropsis fusca. In another aspect, the polypeptide is the polypeptide obtained from Microsphaeropsis hellebori. In another aspect, the polypeptide is the polypeptide obtained from Microsphaeropsis olivacea. In another aspect, the polypeptide is the polypeptide obtained from Microsphaeropsis ononidicola. In another aspect, the polypeptide is the polypeptide obtained from Microsphaeropsis proteae. In another aspect, the polypeptide is the polypeptide obtained from Microsphaeropsis spartii-juncei.

[0405] In one aspect, the polypeptide is obtained from Paraphaeosphaeria. In another aspect, the polypeptide is obtained from Paraphaeosphaeria angularis. In another aspect, the polypeptide is obtained from Paraphaeosphaeria arecacearum. In another aspect, the polypeptide is obtained from Paraphaeosphaeria barriae. In another aspect, the polypeptide is obtained from Paraphaeosphaeria camelliae. In another aspect, the polypeptide is obtained from Paraphaeosphaeria graminicola. In another aspect, the polypeptide is obtained from Paraphaeosphaeria hydei. In another aspect, the polypeptide is obtained from Paraphaeosphaeria michotii. In another aspect, the polypeptide is obtained from Paraphaeosphaeria minitans. In another aspect, the polypeptide is obtained from Paraphaeosphaeria neglecta. In another aspect, the polypeptide is obtained from Paraphaeosphaeria parmeliae. In another aspect, the polypeptide is obtained from Paraphaeosphaeria pilleata. In another aspect, the polypeptide is obtained from Paraphaeosphaeria rceurvifoliae. In another aspect, the polypeptide is obtained from Paraphaeosphaeria rosae. In another aspect, the polypeptide is obtained from Paraphaeosphaeria rosicola. In another aspect, the polypeptide is obtained from Paraphaeosphaeria sardoa. In another aspect, the polypeptide is obtained from Paraphaeosphaeria spartii. In another aspect, the polypeptide is obtained from Paraphaeosphaeria sporulosa. In another aspect, the polypeptide is obtained from Paraphaeosphaeria veruuculosa. In another aspect, the polypeptide is obtained from Paraphaeosphaeria viciae. In another aspect, the polypeptide is obtained from Paraphaeosphaeria viridescens. In another aspect, the polypeptide is obtained from Paraphaeosphaeria xanthorrhoeae.

[0406] In one aspect, the polypeptide is obtained from Westerdykella. In another aspect, the polypeptide is obtained from Westerdykella angulata. In another aspect, the polypeptide is obtained from Westerdykella aquatica. In another aspect, the polypeptide is obtained from Westerdykella aurantiaca. In another aspect, the polypeptide is obtained from Westerdykella capitulum. In another aspect, the polypeptide is obtained from Westerdykella centenaria. In another aspect, the polypeptide is obtained from Westerdykella cylindrica. In another aspect, the polypeptide is obtained from Westerdykella dispersa. In another aspect, the polypeptide is obtained from Westerdykella globosa. In another aspect, the polypeptide is obtained from Westerdykella minutispora. In another aspect, the polypeptide is obtained from Westerdykella multispora. In another aspect, the polypeptide is obtained from Westerdykella nigra. In another aspect, the polypeptide is obtained from Westerdykella ornate. In another aspect, the polypeptide is obtained from Westerdykella purpureopunctata. In another aspect, the polypeptide is obtained from Westerdykella reniformis.

[0407] In one aspect, the polypeptide is obtained from strains of the genus Xepicula. In another aspect, the polypeptide is obtained from Xepicula crassiseta. In another aspect, the polypeptide is obtained from Xepicula jollymannii. In another aspect, the polypeptide is obtained from Xylariaceae. In another aspect, the polypeptide is obtained from Xepicula yifeii.

[0408] It should be understood that for the foregoing species, the present invention encompasses the perfect and imperfect stages as well as other taxonomic equivalents, such as anamorphs, regardless of their known species names. Those skilled in the art will readily recognize the identity of the appropriate equivalents.

[0409] The above-mentioned probes can be used to identify and obtain polypeptides from other sources, including microorganisms isolated from natural sources (such as soil, compost, water, etc.) or DNA samples directly obtained from natural materials (such as soil, compost, water, etc.). Techniques for directly isolating microorganisms and DNA from natural habitats are well known in the art. The polynucleotide encoding the polypeptide can then be obtained by similarly screening the genomic DNA or cDNA library of another microorganism or a mixed DNA sample. Once the polynucleotide encoding the polypeptide has been detected with one or more probes, the polynucleotide can be isolated or cloned by using techniques known to those of ordinary skill in the art (see, for example, Davis et al., 2012, Basic Methods in Molecular Biology, Elsevier).

[0410] Polynucleotide

[0411] The invention also relates to a polynucleotide encoding the polypeptide of the invention, as described herein.

[0412] The polynucleotide can be genomic DNA, cDNA, synthetic DNA, synthetic RNA, mRNA, or a combination thereof. The polynucleotide can be cloned from strains of the genus Trichoderma, the genus Epichloe, the genus Flammulina, the genus Chlorella, the genus Paraphaeosphaeria, the genus Westiellopsis, the genus Xepicula, or related organisms, and can thus be, for example, a polynucleotide sequence encoding a variant of the polypeptide of the invention.

[0413] In one embodiment, the polynucleotide is a subsequence encoding a fragment of the present invention having acetylxylan esterase activity. In one aspect, the subsequence contains at least 711 nucleotides (e.g., nucleotides 57 to 768 of SEQ ID NO:1), at least 753 nucleotides (e.g., nucleotides 57 to 810 of SEQ ID NO:1), or at least 795 nucleotides (e.g., nucleotides 57 to 852 of SEQ ID NO:1). In one embodiment, the polynucleotide is a subsequence encoding a fragment of the present invention having acetylxylan esterase activity. In one aspect, the subsequence contains at least 561 nucleotides (e.g., nucleotides 78 to 639 of SEQ ID NO:4), at least 594 nucleotides (e.g., nucleotides 78 to 672 of SEQ ID NO:4), or at least 627 nucleotides (e.g., nucleotides 78 to 705 of SEQ ID NO:4). In one embodiment, the polynucleotide is a subsequence encoding a fragment of the present invention having acetylxylan esterase activity. In one aspect, the subsequence contains at least 690 nucleotides (e.g., nucleotides 60 to 750 of SEQ ID NO:7), at least 729 nucleotides (e.g., nucleotides 60 to 789 of SEQ ID NO:7), or at least 771 nucleotides (e.g., nucleotides 60 to 831 of SEQ ID NO:7). In one embodiment, the polynucleotide is a subsequence encoding a fragment of the present invention having acetylxylan esterase activity. In one aspect, the subsequence contains at least 588 nucleotides (e.g., nucleotides 69 to 657 of SEQ ID NO:10), at least 624 nucleotides (e.g., nucleotides 69 to 693 of SEQ ID NO:10), or at least 633 nucleotides (e.g., nucleotides 69 to 702 of SEQ ID NO:10). In one embodiment, the polynucleotide is a subsequence encoding a fragment of the present invention having acetylxylan esterase activity. In one aspect, the subsequence contains at least 564 nucleotides (e.g., nucleotides 57 to 657 of SEQ ID NO:13), at least 600 nucleotides (e.g., nucleotides 57 to 657 of SEQ ID NO:13), or at least 633 nucleotides (e.g., nucleotides 57 to 690 of SEQ ID NO:13). In one embodiment, the polynucleotide is a subsequence encoding a fragment of the present invention having acetylxylan esterase activity. In one aspect, the subsequence contains at least 564 nucleotides (e.g., nucleotides 69 to 633 of SEQ ID NO:16), at least 600 nucleotides (e.g., nucleotides 69 to 669 of SEQ ID NO:16), or at least 633 nucleotides (e.g., nucleotides 69 to 759 of SEQ ID NO:16). In one embodiment, the polynucleotide is a subsequence encoding a fragment of the present invention having acetylxylan esterase activity.In one aspect, the subsequence contains at least 570 nucleotides (e.g., nucleotides 75 to 645 of SEQ ID NO:19), at least 600 nucleotides (e.g., nucleotides 75 to 675 of SEQ ID NO:19), or at least 636 nucleotides (e.g., nucleotides 75 to 711 of SEQ ID NO:19). In one embodiment, the polynucleotide is a subsequence encoding a fragment of the present invention having acetylxylan esterase activity. In one aspect, the subsequence contains at least 561 nucleotides (e.g., nucleotides 75 to 636 of SEQ ID NO:22), at least 594 nucleotides (e.g., nucleotides 75 to 669 of SEQ ID NO:22), or at least 627 nucleotides (e.g., nucleotides 75 to 702 of SEQ ID NO:22). In one embodiment, the polynucleotide is a subsequence encoding a fragment of the present invention having acetylxylan esterase activity. In one aspect, the subsequence contains at least 564 nucleotides (e.g., nucleotides 60 to 624 of SEQ ID NO:25), at least 597 nucleotides (e.g., nucleotides 60 to 657 of SEQ ID NO:25), or at least 630 nucleotides (e.g., nucleotides 60 to 690 of SEQ ID NO:25). In one embodiment, the polynucleotide is a subsequence encoding a fragment of the present invention having acetylxylan esterase activity. In one aspect, the subsequence contains at least 690 nucleotides (e.g., nucleotides 51 to 741 of SEQ ID NO:28), at least 729 nucleotides (e.g., nucleotides 51 to 780 of SEQ ID NO:28), or at least 771 nucleotides (e.g., nucleotides 51 to 822 of SEQ ID NO:28).

[0414] In one embodiment, the polynucleotide encoding the polypeptide of the present invention is isolated from cells of the genus Chaetomella. In one embodiment, the polynucleotide encoding the polypeptide of the present invention is isolated from cells of the genus Epichloe. In one embodiment, the polynucleotide encoding the polypeptide of the present invention is isolated from cells of the genus Flammulina. In one embodiment, the polynucleotide encoding the polypeptide of the present invention is isolated from cells of the genus Chlorella. In one embodiment, the polynucleotide encoding the polypeptide of the present invention is isolated from cells of the genus Paraphaeosphaeria. In one embodiment, the polynucleotide encoding the polypeptide of the present invention is isolated from cells of the genus Westiellopsis. In one embodiment, the polynucleotide encoding the polypeptide of the present invention is isolated from cells of the genus Xepicula.

[0415] The polynucleotide can also be mutated by introducing nucleotide substitutions that do not result in changes in the amino acid sequence of the polypeptide, but which correspond to the codon usage of the host organism intended for production of the enzyme, or by introducing nucleotide substitutions that may give rise to a different amino acid sequence. For a general description of nucleotide substitutions, see, for example, Ford et al., 1991, Protein Expression and Purification 2:95-107.

[0416] In one aspect, the polynucleotide is isolated.

[0417] In another aspect, the polynucleotide is purified.

[0418] Nucleic acid construct

[0419] The invention also relates to a nucleic acid construct comprising the polynucleotide of the invention, wherein the polynucleotide is operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with these control sequences.

[0420] The polynucleotide can be manipulated in a variety of ways to provide for expression of the polypeptide. Depending on the expression vector, it may be desirable or necessary to manipulate the polynucleotide prior to insertion into the vector. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

[0421] Promoter

[0422] The control sequence may be a promoter, i.e., a polynucleotide that is recognized by the host cell for expression of the polynucleotide encoding the polypeptide of the invention. The promoter contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter can be any polynucleotide that shows transcriptional activity in the host cell, including mutant promoters, truncated promoters, and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides homologous or heterologous to the host cell.

[0423] Examples of suitable promoters for directing transcription of the polynucleotide of the invention in bacterial host cells are described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab., New York; Davis et al., 2012, ibid.; and Song et al., 2016, PLOS One 11(7):e0158447.

[0424] Examples of suitable promoters for directing transcription of the polynucleotides of the present invention in filamentous fungal host cells are promoters obtained from Aspergillus, Fusarium, Rhizomucor, and Trichoderma cells, such as the promoters described in: Mukherjee et al., 2013, “Trichoderma: Biology and Applications” and Schmoll and 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology.

[0425] For expression in yeast hosts, examples of useful promoters are described by: Smolke et al., 2018, “Synthetic Biology: Parts, Devices and Applications” (Chapter 6: Constitutive and Regulated Promoters in Yeast: How to Design and Make Use of Promoters in S. cerevisiae) and Schmoll and 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology.

[0426] Terminator

[0427] The control sequence may also be a transcription terminator recognized by the host cell to terminate transcription. The terminator is operably linked to the 3'-end of the polynucleotide encoding the polypeptide. Any terminator functional in the host cell can be used in the present invention.

[0428] Preferred terminators for bacterial host cells can be obtained from the genes of: Bacillus clausii alkaline protease (aprH), Bacillus licheniformis α-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

[0429] Preferred terminators for filamentous fungal host cells can be obtained from Aspergillus or Trichoderma species, such as the genes obtained from Aspergillus niger glucoamylase, Trichoderma reesei β-glucosidase, Trichoderma reesei cellobiohydrolase I and Trichoderma reesei endoglucanase I, such as the terminators described in the following: Mukherjee et al., 2013, “Trichoderma: Biology and Applications” and Schmoll and 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology.

[0430] Preferred terminators for yeast host cells can be obtained from the genes of Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1) and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, Yeast 8:423-488.

[0431] mRNA stabilizer

[0432] The control sequence can also be an mRNA stabilizer region downstream of the promoter and upstream of the coding sequence of the gene, which increases the expression of the gene.

[0433] Examples of suitable mRNA stabilizer regions are obtained from the Bacillus thuringiensis cryIIIA gene (WO 94 / 25612) and the Bacillus subtilis SP82 gene (Hue et al., 1995, J. Bacteriol. 177:3465-3471).

[0434] Examples of mRNA stabilizer regions for fungal cells are described in Geisberg et al., 2014, Cell 156(4):812-824 and Morozov et al., 2006, Eukaryotic Cell 5(11):1838-1846.

[0435] Leader sequence

[0436] The control sequence can also be a leader sequence, i.e., an mRNA untranslated region that is important for host cell translation. The leader sequence is operably linked to the 5'-end of the polynucleotide encoding the polypeptide. Any leader sequence that is functional in the host cell can be used.

[0437] Suitable leader sequences for bacterial host cells are described by Hambraeus et al., 2000, Microbiology 146(12):3051-3059 and Kaberdin and 2006, FEMS Microbiol. Rev. 30(6):967-979.

[0438] Preferred leader sequences for filamentous fungal host cells can be obtained from the genes of: Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0439] Suitable leader sequences for yeast host cells can be obtained from the genes of: Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0440] Polyadenylation sequence

[0441] The control sequence can also be a polyadenylation sequence, i.e., a sequence operably linked to the 3'-end of the polynucleotide that is recognized by the host cell as a signal to add polyadenylate residues to the transcribed mRNA. Any polyadenylation sequence that is functional in the host cell can be used.

[0442] Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes of: Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0443] Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15:5983-5990.

[0444] Signal peptide

[0445] The control sequence may also be a signal peptide coding region encoding a signal peptide linked to the N-terminus of the polypeptide and directing the polypeptide into the secretory pathway of the cell. The 5'-end of the coding sequence of the polynucleotide may itself contain a signal peptide coding sequence that is naturally linked in the translation reading frame to the coding sequence segment encoding the polypeptide. Alternatively, the 5'-end of the coding sequence may contain a signal peptide coding sequence that is heterologous to the coding sequence. A heterologous signal peptide coding sequence may be required when the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, the heterologous signal peptide coding sequence may simply replace the native signal peptide coding sequence in order to enhance the secretion of the polypeptide. Any signal peptide coding sequence that directs the expressed polypeptide into the secretory pathway of the host cell may be used.

[0446] An effective signal peptide coding sequence for a bacterial host cell is a signal peptide coding sequence obtained from the genes of the following: Bacillus amyloliquefaciens NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus alpha-amylase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM) and Bacillus subtilis prsA. Additional signal peptides are described by Freudl, 2018, Microbial Cell Factories 17:52.

[0447] An effective signal peptide coding sequence for a filamentous fungal host cell is a signal peptide coding sequence obtained from the genes of the following: Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase and Rhizomucor miehei aspartic protease, such as the signal peptides described by Xu et al., 2018, Biotechnology Letters 40:949-955.

[0448] Useful signal peptides for yeast host cells are obtained from the genes of the following: Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992, ibid.

[0449] Propeptide

[0450] The control sequence may also be a propeptide-encoding sequence encoding a propeptide located at the N-terminus of the polypeptide. The resulting polypeptide is referred to as a proenzyme or pro-polypeptide (or in some cases as a zymogen). Pro-polypeptides are generally inactive and can be converted to active polypeptides by catalytic or autocatalytic cleavage of the propeptide from the pro-polypeptide. The propeptide-encoding sequence may be obtained from the genes of Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95 / 33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae α-factor.

[0451] In the case where both a signal peptide sequence and a propeptide sequence are present, the propeptide sequence is located immediately adjacent to the N-terminus of the polypeptide and the signal peptide sequence is located immediately adjacent to the N-terminus of the propeptide sequence. Additionally or alternatively, when both a signal peptide sequence and a propeptide sequence are present, the polypeptide may comprise only a portion of the signal peptide sequence and / or only a portion of the propeptide sequence. Alternatively, the final or isolated polypeptide may comprise a mixture of the mature polypeptide and a polypeptide comprising a partial or full-length propeptide sequence and / or signal peptide sequence.

[0452] Regulatory sequence

[0453] It may also be desirable to add regulatory sequences which regulate the expression of polypeptides related to the growth of the host cell. Examples of regulatory sequences are those which cause the gene expression to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory sequences in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1 system can be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA α-amylase promoter, and Aspergillus oryzae glucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter, and Trichoderma reesei cellobiohydrolase II promoter can be used. Other examples of regulatory sequences are those which allow gene amplification. In fungal systems, these regulatory sequences include the dihydrofolate reductase gene amplified in the presence of methotrexate and the metallothionein gene amplified with heavy metals.

[0454] Transcription factor

[0455] A control sequence may also be a transcription factor, i.e., a polynucleotide encoding a polypeptide that specifically binds DNA, which controls the rate of transcription of genetic information from DNA to mRNA by binding to a specific polynucleotide sequence. Transcription factors can act alone and / or together with one or more other polypeptides or transcription factors in a complex to act by promoting or blocking the recruitment of RNA polymerase. Transcription factors are characterized by containing at least one DNA-binding domain, which is typically attached to a specific DNA sequence adjacent to the genetic element regulated by the transcription factor. Transcription factors can regulate the expression of a target protein directly (i.e., by activating the transcription of a gene encoding the target protein by binding to its promoter) or indirectly (i.e., by activating the transcription of another transcription factor by binding to the promoter of another transcription factor that regulates the transcription of the gene encoding the target protein). Suitable transcription factors for fungal host cells are described in WO 2017 / 144177. Suitable transcription factors for prokaryotic host cells are described in Seshasayee et al., 2011, Subcellular Biochemistry 52:7-23 and Balleza et al., 2009, FEMS Microbiol. Rev. 33(1):133-151.

[0456] Expression vector

[0457] The invention also relates to a recombinant expression vector comprising the polynucleotide, promoter, and transcription and translation termination signals of the invention. The polynucleotides and control sequences can be ligated together to produce a recombinant expression vector, which may include one or more convenient restriction sites to allow insertion or substitution of the polynucleotide encoding the polypeptide at such sites. Alternatively, the polynucleotide can be expressed by inserting the polynucleotide or a nucleic acid construct containing the polynucleotide into an appropriate vector for expression. When producing an expression vector, the coding sequence is positioned in the vector such that the coding sequence is operably linked to the appropriate control sequences for expression.

[0458] The recombinant expression vector can be any vector (e.g., plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can cause the expression of the polynucleotide. The choice of vector will typically depend on the compatibility of the vector with the host cell to be introduced into the vector. The vector can be a linear or closed circular plasmid.

[0459] The vector can be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity and whose replication is independent of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector can contain any means for ensuring self-replication. Alternatively, the vector can be a vector that integrates into the genome when introduced into a host cell and replicates with the chromosome into which it has been integrated. Moreover, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell can be used, or a transposon can be used.

[0460] The vector preferably contains one or more selectable markers that allow for convenient selection of cells such as transformed cells, transfected cells, transduced cells, etc. A selectable marker is a gene whose product provides biocide resistance or virus resistance, resistance to heavy metals, prototrophy for auxotrophs, etc.

[0461] The vector preferably contains at least one element that allows the vector to integrate into the genome of the host cell or to replicate autonomously in the cell independently of the genome.

[0462] For integration into the genome of the host cell, the vector can rely on a polynucleotide sequence encoding a polypeptide or any other element of the vector for integration into the genome by homologous recombination (such as homologous directed repair (HDR)) or non-homologous recombination (such as non-homologous end joining (NHEJ)).

[0463] For autonomous replication, the vector can further contain an origin of replication that enables the vector to replicate autonomously in the host cell under discussion. The origin of replication can be any plasmid replicon that functions in the cell to mediate autonomous replication. The term "origin of replication" or "plasmid replicon" refers to a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0464] More than one copy of the polynucleotide of the present invention can be inserted into a host cell to increase the production of the polypeptide. For example, 2 or 3 or 4 or 5 or more copies are inserted into the host cell. An increased copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the genome of the host cell or by including an amplifiable selectable marker gene together with the polynucleotide, where cells containing the amplified copy of the selectable marker gene and thus the additional copy of the polynucleotide can be selected by culturing the cells in the presence of an appropriate selective agent.

[0465] Host cell

[0466] The present invention also relates to recombinant host cells that contain the polynucleotide of the present invention operably linked to one or more control sequences that direct the production of the polypeptide of the present invention.

[0467] A construct or vector comprising a polynucleotide is introduced into a host cell such that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extrachromosomal vector, as described earlier. The choice of host cell will depend to a large extent on the gene encoding the polypeptide and its source. The polypeptide can be native or heterologous to the recombinant host cell. In addition, at least one of the one or more control sequences can be heterologous to the polynucleotide encoding the polypeptide. The recombinant host cell can contain a single copy or at least two copies of the polynucleotide of the present invention, such as three, four, five or more copies.

[0468] The host cell can be any microbial cell useful for the recombinant production of the polypeptide of the present invention, such as a prokaryotic cell or a fungal cell.

[0469] Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to, the genera Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0470] Bacterial host cells can be any Bacillus cells, including but not limited to Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells. In one embodiment, the Bacillus cells are Bacillus amyloliquefaciens, Bacillus licheniformis, and Bacillus subtilis cells.

[0471] For the purposes of the present invention, Bacillus species / genus / species should be defined as described in Patel and Gupta, 2020, Int. J. Syst. Evol. Microbiol. 70: 406 - 438.

[0472] The bacterial host cell can also be any Streptococcus cell, including but not limited to Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. zooepidemicus cells.

[0473] The bacterial host cell can also be any Streptomyces cell, including but not limited to Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

[0474] Methods for introducing DNA into prokaryotic host cells are well known in the art and any suitable method can be used, including but not limited to protoplast transformation, competent cell transformation, electroporation, conjugation, transduction, wherein the DNA is introduced as a linearized or circular polynucleotide. Those skilled in the art will be able to readily determine the suitable method for introducing DNA into a given prokaryotic cell, for example, based on the genus. Methods for introducing DNA into prokaryotic host cells are described, for example, in Heinze et al., 2018, BMC Microbiology 18: 56; Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98: 6289 - 6294; Choi et al., 2006, J. Microbiol. Methods 64: 391 - 397; and Donald et al., 2013, J. Bacteriol. 195(11): 2612 - 2620.

[0475] The host cell can be a fungal cell. As used herein, "fungal" includes Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota, as well as Oomycota and all mitosporic fungi (as defined in Hawksworth et al., Ainsworth and Bisby’s Dictionary of The Fungi, 8th Edition, 1995, CAB International, University Press, Cambridge, UK).

[0476] Fungal cells can be transformed by processes involving protoplast-mediated transformation, Agrobacterium-mediated transformation, electroporation, gene gun methods, and shock wave-mediated transformation (as reviewed in Li et al., 2017, Microbial Cell Factories 16:168) and by the procedures described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474; Christensen et al., 1988, Bio / Technology 6:1419-1422, and Lubertozzi and Keasling, 2009, Biotechn. Advances 27:53-75. However, any method known in the art for introducing DNA into a fungal host cell can be used, and the DNA can be introduced as a linearized or circular polynucleotide.

[0477] The fungal host cell can be a yeast cell. As used herein, "yeast" includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). For the purposes of the present invention, yeast shall be defined as described in Biology and Activities of Yeast (edited by Skinner, Passmore, and Davenport, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0478] The yeast host cell can be a cell of the genus Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces or Yarrowia, such as Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis or Yarrowia lipolytica cell. In a preferred embodiment, the yeast host cell is a Pichia or Komagataella cell, such as Pichia pastoris cell (Komagataella phaffii).

[0479] The fungal host cell can be a filamentous fungal cell. "Filamentous fungi" includes all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). Filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth of yeast (such as Saccharomyces cerevisiae) is by budding of single cell thalli and carbon catabolism can be fermentative.

[0480] The filamentous fungal host cell may be a cell of Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma. In a preferred embodiment, the filamentous fungal host cell is a cell of Aspergillus, Trichoderma or Fusarium. In another preferred embodiment, the filamentous fungal host cell is an Aspergillus niger, Aspergillus oryzae, Trichoderma reesei or Fusarium venenatum cell.

[0481] For example, the filamentous fungal host cell can be Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum), Fusarium torulosum, Fusarium trichothecioides, Fusarium lamellum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Talaromyces emersonii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei or Trichoderma viride cells.

[0482] In one aspect, the host cell is isolated.

[0483] In another aspect, the host cell is purified.

[0484] Production method

[0485] The present invention also relates to methods for producing the polypeptides of the present invention, which methods comprise (a) culturing a cell that produces the polypeptide in its wild-type form under conditions conducive to the production of the polypeptide; and optionally (b) recovering the polypeptide.

[0486] In one aspect, the cell is a cell of a species of Setomelanomma. In another aspect, the cell is a polysphondylium. In another aspect, the cell is a Dematium americanum. In another aspect, the cell is a cell of Microsporidium bambusae. In another aspect, the cell is a cell of Nematospora cruciformis. In another aspect, the cell is a cell of Sporormiella decipiens. In another aspect, the cell is a cell of Myriangium haraeanum. In another aspect, the cell is a cell of Sirodospora microspora. In another aspect, the cell is a cell of Nematospora japonica. In another aspect, the cell is a cell of Dinemasporium longicapillatum. In another aspect, the cell is a cell of Didymosporium morbiferum. In another aspect, the cell is a cell of Dinemasporium nelloi. In another aspect, the cell is a cell of Nematospora parasitica. In another aspect, the cell is a cell of Dinemasporium polygonum. In another aspect, the cell is a cell of Didymosporium pseudocylindrosporum. In another aspect, the cell is a cell of Streptomyces pseudoirregularis. In another aspect, the cell is a cell of Pseudostraminea longispora. In another aspect, the cell is a cell of Streptomyces rishiriensis. In another aspect, the cell is a cell of Gibberella fujikuroi. In another aspect, the cell is a cell of polysphondylium aculeatum. In another aspect, the cell is a cell of Setomelanomma hispidulum. In another aspect, the cell is a cell of Trichonectria crataegicola.

[0487] In one aspect, the cell is an Epicoccum cell. In another aspect, the cell is Epicoccum masculinum. In another aspect, the cell is Epicoccum aristidae. In another aspect, the cell is Epicoccum brahmanticum. In another aspect, the cell is Epicoccum brazilianum. In another aspect, the cell is Epicoccum camelliae. In another aspect, the cell is Epicoccum catenulatum. In another aspect, the cell is Epicoccum cedri. In another aspect, the cell is Epicoccum chloratum. In another aspect, the cell is Epicoccum dendrobii. In another aspect, the cell is Epicoccum geelongense. In another aspect, the cell is Epicoccum draconis. In another aspect, the cell is Epicoccum duthiei. In another aspect, the cell is Epicoccum endophyticum. In another aspect, the cell is Epicoccum henningsii. In another aspect, the cell is Epicoccum hordei. In another aspect, the cell is Epicoccum huancabambae. In another aspect, the cell is Epicoccum italicum. In another aspect, the cell is Epicoccum keratinophilum. In another aspect, the cell is Epicoccum latifolium. In another aspect, the cell is Epicoccum laurenianum. In another aspect, the cell is Epicoccum longipes. In another aspect, the cell is Epicoccum mackenziei. In another aspect, the cell is Epicoccum maydis. In another aspect, the cell is Epicoccum melinii. In another aspect, the cell is Epicoccum myriosporum. In another aspect, the cell is Epicoccum nigrum. In another aspect, the cell is Epicoccum oryzae. In another aspect, the cell is Epicoccum ovatisporum. In another aspect, the cell is Epicoccum polysporum. In another aspect, the cell is Epicoccum piprines. In another aspect, the cell is Epicoccum polyphagum. In another aspect, the cell is Epicoccum pneumoniae. In another aspect, the cell is Epicoccum poaceiocola. In another aspect, the cell is Epicoccum praecox. In another aspect, the cell is Epicoccum purpurascens. In another aspect, the cell is Epicoccum variabile. In another aspect, the cell is Epicoccum pruni. In another aspect, the cell is Epicoccum pseudokeratinophilum. In another aspect, the cell is Epicoccum uncinulatum. In another aspect, the cell is Epicoccum rosae. In another aspect, the cell is Epicoccum sorghicola. In another aspect, the cell is Epicoccum sorghi. In another aspect, the cell is Epicoccum thailandicum. In another aspect, the cell is Epicoccum nicotianae. In another aspect, the cell is Epicoccum triseptatum. In another aspect, the cell is Epicoccum tritici. In another aspect, the cell is Epicoccum varians. In another aspect, the cell is Epicoccum viticis.

[0488] In one aspect, the cell is a Lentinula cell. In another aspect, the cell is a Flammulina cephalariae cell. In another aspect, the cell is a Lentinus elasticus cell. In another aspect, the cell is a Flammulina fennae cell. In another aspect, the cell is a wild Flammulina velutipes cell. In another aspect, the cell is a Flammulina finlandica cell. In another aspect, the cell is a Lentinula mexicana cell. In another aspect, the cell is a Flammulina ononidis cell. In another aspect, the cell is a yellow Flammulina velutipes cell. In another aspect, the cell is a Lentinus pallidus cell. In another aspect, the cell is a Flammulina stratosa cell. In another aspect, the cell is a Flammulina velutipes cell. In another aspect, the cell is a Lentinula yunnanensis cell.

[0489] In one aspect, the cell is a Chlorella cell. In another aspect, the polypeptide is a polypeptide obtained from Chlorella amygdalina cells. In another aspect, the polypeptide is a polypeptide obtained from Microsphaeropsis sphaeroidea cells. In another aspect, the polypeptide is a polypeptide obtained from Chlorella fusca cells. In another aspect, the polypeptide is a polypeptide obtained from Chlorella luteoviridis cells. In another aspect, the polypeptide is a polypeptide obtained from Chlorella olivacea cells. In another aspect, the polypeptide is a polypeptide obtained from Microsphaeropsis ononidicola cells. In another aspect, the polypeptide is a polypeptide obtained from Chlorella variabilis cells. In another aspect, the polypeptide is a polypeptide obtained from Microsphaeropsis spartii-juncei cells.

[0490] In one aspect, the cell is a Paraphaeosphaeria cell. In another aspect, the cell is a Paraphaeosphaeria angulata cell. In another aspect, the cell is a Paraphaeosphaeria arecae cell. In another aspect, the cell is a Paraphaeosphaeria barriae cell. In another aspect, the cell is a Paraphaeosphaeria camelliae cell. In another aspect, the cell is a Paraphaeosphaeria graminicola cell. In another aspect, the cell is a Paraphaeosphaeria hedericola cell. In another aspect, the cell is a Paraphaeosphaeria mihailovicii cell. In another aspect, the cell is a Paraphaeosphaeria minima cell. In another aspect, the cell is a Paraphaeosphaeria neglecta. In another aspect, the cell is a Paraphaeosphaeria pamerana cell. In another aspect, the cell is a Paraphaeosphaeria pilleata cell. In another aspect, the cell is a Paraphaeosphaeria recurvata cell. In another aspect, the cell is a Paraphaeosphaeria rosae cell. In another aspect, the cell is a Paraphaeosphaeria rosicola cell. In another aspect, the cell is a Paraphaeosphaeria saccardoi cell. In another aspect, the cell is a Paraphaeosphaeria spartica cell. In another aspect, the cell is a Paraphaeosphaeria spora cell. In another aspect, the cell is a Paraphaeosphaeria verrucosa cell. In another aspect, the cell is a Paraphaeosphaeria viciae cell. In another aspect, the cell is a Paraphaeosphaeria virens cell. In another aspect, the cell is a Paraphaeosphaeria xanthomatosis cell.

[0491] In one aspect, the cell is a Westerdykella cell. In another aspect, the cell is a Westerdykella angulospora cell. In another aspect, the cell is a Westerdykella aquatica cell. In another aspect, the cell is a Westerdykella aurantiolutea cell. In another aspect, the cell is a Westerdykella capitulum cell. In another aspect, the cell is a Westerdykella centenaria cell. In another aspect, the cell is a Westerdykella cylindrica cell. In another aspect, the cell is a Westerdykella diffusa cell. In another aspect, the cell is a Westerdykella globosa cell. In another aspect, the cell is a Westerdykella microspora cell. In another aspect, the cell is a Westerdykella polyspora cell. In another aspect, the cell is a Westerdykella pulla cell. In another aspect, the cell is a Westerdykella violacea cell. In another aspect, the cell is a Westerdykella reniformis cell.

[0492] In one aspect, the cell is a Xepicula cell. In another aspect, the cell is a Xepicula crassiseta cell. In another aspect, the cell is a Xepicula jollymannii cell. In another aspect, the cell is a Leucocarpus candidus cell. In another aspect, the cell is a Xepicula yifeii cell.

[0493] The present invention also relates to methods for producing the polypeptides of the present invention, which methods include (a) culturing the recombinant host cells of the present invention under conditions conducive to the production of the polypeptide; and optionally (b) recovering the polypeptide.

[0494] The host cells are cultured in a nutrient medium suitable for producing polypeptides using methods known in the art. For example, the cells can be cultured by shake flask cultivation or by small-scale or large-scale fermentation (including continuous, batch, fed-batch or solid-state and / or microcarrier-based fermentations) in a suitable medium and under conditions that allow the expression and / or isolation of the polypeptide in laboratory or industrial fermentors. Suitable media are available from commercial suppliers or can be prepared according to published compositions (e.g., in the catalogs of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from the cell lysate.

[0495] Polypeptides can be detected using methods specific for polypeptides known in the art, which methods include, but are not limited to, using specific antibodies, enzyme product formation, enzyme substrate disappearance, or assays that measure the relative or specific activity of the polypeptide.

[0496] Polypeptides can be recovered from the medium using methods known in the art, which methods include, but are not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. In one aspect, the whole fermentation broth containing the polypeptide is recovered. In another aspect, the cell-free fermentation broth containing the polypeptide is recovered.

[0497] The polypeptide can be purified by a variety of procedures known in the art to obtain a substantially pure polypeptide and / or polypeptide fragment (see, e.g., Wingfield, 2015, Current Protocols in Protein Science; 80(1):6.1.1-6.1.35; Labrou, 2014, Protein Downstream Processing, 1129:3-10).

[0498] In an alternative aspect, the polypeptide is not recovered.

[0499] Acetylxylan esterase particles

[0500] The present invention also relates to enzyme particles / pellets comprising the polypeptides of the present invention. In one embodiment, the particle comprises a core and optionally one or more coatings (outer layers) surrounding the core.

[0501] The diameter of the core (measured as the equivalent spherical diameter (volume-based average particle size)) can be 20 - 2000 μm, particularly 50 - 1500 μm, 100 - 1500 μm or 250 - 1200 μm. The core diameter measured as the equivalent spherical diameter can be determined using laser diffraction such as with a Malvern Mastersizer and / or the method described under ISO 13320 (2020).

[0502] In one embodiment, the core comprises a polypeptide having acetylxylan esterase activity of the present invention.

[0503] The core can 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.

[0504] The core can include binders such as synthetic polymers, waxes, fats, or carbohydrates.

[0505] Typically as a homogeneous blend, the core can include salts of polyvalent cations, reducing agents, antioxidants, peroxide decomposition catalysts, and / or acidic buffer components.

[0506] The core can include inert particles, where the polypeptide is adsorbed within the inert particles or applied (e.g., by fluidized bed coating) to the surface of the inert particles.

[0507] The diameter of the core can be 20 - 2000 μm, particularly 50 - 1500 μm, 100 - 1500 μm or 250 - 1200 μm.

[0508] The core can be surrounded by at least one coating, e.g., to improve storage stability, reduce dust formation during handling, or for coloring the particles. Optional coatings can include salt coatings or other suitable coating materials such as polyethylene glycol (PEG), methylhydroxypropylcellulose (MHPC), and polyvinyl alcohol (PVA).

[0509] The coating can be applied in an amount of at least 0.1% (e.g., at least 0.5%, at least 1%, at least 5%, at least 10% or at least 15%) by weight of the core. The amount can be at most 100%, 70%, 50%, 40% or 30%.

[0510] 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 thickness of the coating is less than 100 μm, such as less than 60 μm or less than 40 μm.

[0511] The coating should seal the core unit by forming a substantially continuous layer. A substantially continuous layer should be understood as a coating having very few or no holes such that the core unit has very few or no uncoated areas. The layer or coating should in particular be uniform in thickness.

[0512] The coating may further contain other materials known in the art, such as fillers, anti-sticking agents, pigments, dyes, plasticizers, and / or binders, such as titanium dioxide, kaolin, calcium carbonate, or talc.

[0513] The salt coating may comprise at least 60% by weight of salt, 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% by weight.

[0514] To provide acceptable protection, the salt coating is preferably at least 0.1 μm thick, such as 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.

[0515] The salt may be added from a salt solution in which the salt is completely dissolved, or from a salt suspension in which the fine particles are less than 50 μm, such as less than 10 μm or less than 5 μm.

[0516] The salt coating may comprise a single salt or a mixture of two or more salts. The salt 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, such as at least 1 g / 100 g of water, such as at least 5 g / 100 g of water.

[0517] The salt may be an inorganic salt, such as a sulfate, sulfite, phosphate, phosphonate, nitrate, chloride, or carbonate, or a salt of a simple organic acid (less than 10 carbon atoms, such as 6 or fewer carbon atoms) such as citrate, malonate, or acetate. Examples of cations in these salts are alkali or alkaline earth metal ions, ammonium ions, or metal ions of the first transition series, such as sodium, potassium, magnesium, calcium, zinc, or aluminum. Examples of anions include chloride, bromide, iodide, sulfate, sulfite, bisulfite, thiosulfate, phosphate, dihydrogen phosphate, dibasic phosphate, hypophosphite, pyrophosphate, tetraborate, borate, carbonate, bicarbonate, metasilicate, citrate, malate, maleate, malonate, succinate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate, or gluconate. In particular, alkali or 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 may be used.

[0518] The salt in the coating may have a constant humidity of more than 60%, especially more than 70%, more than 80% or more than 85% at 20 °C, or it may be another hydrate form of such a salt (for example, the anhydrous form). The salt coating may be as described in WO00 / 01793 or WO 2006 / 034710.

[0519] Specific examples of suitable salts are NaCl (CH 20 °C = 76%), Na2CO3 (CH 20 °C = 92%), NaNO3 (CH 20 °C = 73%), Na2HPO4 (CH 20 °C = 95%), Na3PO4 (CH 25 °C = 92%), NH4Cl (CH 20 °C = 79.5%), (NH4)2HPO4 (CH 20 °C = 93.0%), NH4H2PO4 (CH 20 °C = 93.1%), (NH4)2SO4 (CH 20 °C = 81.1%), KCl (CH 20 °C = 85%), K2HPO4 (CH 20 °C = 92%), KH2PO4 (CH 20 °C = 96.5%), KNO3 (CH 20 °C = 93.5%), Na2SO4 (CH 20 °C = 93%), K2SO4 (CH 20 °C = 98%), KHSO4 (CH 20 °C = 86%), MgSO4 (CH 20 °C = 90%), ZnSO4 (CH 20 °C = 90%) and sodium citrate (CH 25 °C = 86%). Other examples include NaH2PO4, (NH4)H2PO4, CuSO4, Mg(NO3)2 and magnesium acetate.

[0520] The salt may be in anhydrous form, or it may be a hydrated salt, i.e., a crystalline salt hydrate having one or more bound waters of crystallization, as described in WO 99 / 32595. Specific examples include anhydrous sodium sulfate (Na2SO4), anhydrous magnesium sulfate (MgSO4), 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.

[0521] Preferably, the salt is applied as a salt solution, for example, using a fluidized bed.

[0522] The coating material can be a waxy coating material and a film-forming coating material. Examples of waxy coating materials are poly(ethylene oxide) products (polyethylene glycol, PEG) with an average molar mass of 1000 to 20000; ethoxylated nonylphenols having 16 to 50 ethylene oxide units; ethoxylated fatty alcohols, where the alcohol contains 12 to 20 carbon atoms and where there are 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 by fluidized bed techniques are given in GB 1483591.

[0523] The particles may optionally have one or more additional coatings. Examples of suitable coating materials are polyethylene glycol (PEG), methylhydroxypropylcellulose (MHPC), and polyvinyl alcohol (PVA). Examples of enzyme particles with multiple coatings are described in WO 93 / 07263 and WO 97 / 23606.

[0524] The core can be prepared by granulating a blend of the components, for example, by a method including granulation techniques such as crystallization, precipitation, pan-coating, fluidized bed coating, fluidized bed agglomeration, rotary atomization, extrusion, prilling, spheronization, particle size reduction methods, drum granulation, and / or high shear granulation.

[0525] Methods for preparing the core can be found in Handbook of Powder Technology; Particle size enlargement by C.E. Capes; Volume 1; 1980; Elsevier. The preparation methods include known feed and particle formulation techniques, such as:

[0526] (a) Spray-dried products, where a liquid polypeptide-containing solution is atomized in a spray-drying tower to form small droplets, which are dried during their descent through the drying tower to form polypeptide-containing particulate material. In this way, very small particles can be produced (Michael S. Showell (ed.); Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140 - 142; Marcel Dekker).

[0527] (b) Layered products, where the polypeptide is coated in layers around pre-formed inert core particles. Usually, a polypeptide-containing solution is atomized in a fluidized bed apparatus, where the pre-formed core particles are fluidized and the polypeptide-containing solution adheres to the core particles and is dried until a dry polypeptide layer 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 way. This type of product is described, for example, in WO 97 / 23606.

[0528] (c) Absorbed core particles, where instead of coating the polypeptide in layers around the core, the polypeptide is absorbed onto and / or into the surface of the core. Such a method is described in WO 97 / 39116.

[0529] (d) Extruded or pelletized products, where a polypeptide-containing paste is pressed into pellets or extruded under pressure through small openings and cut into particles, which are then dried. Such particles usually have a rather large size because the material with the extrusion openings (usually a flat plate with drilled holes) sets a limit on the allowable pressure drop across the extrusion openings. In addition, when using small openings, the very high extrusion pressure increases the heat generation in the polypeptide paste, which is harmful to the polypeptide (Michael S. Showell (ed.); Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140 - 142; Marcel Dekker).

[0530] (e) Prilled products, where the polypeptide-containing powder is suspended in molten wax and the suspension is sprayed (e.g., through a rotary atomizer) into a cooling chamber where the droplets solidify rapidly (Michael S. Showell (ed.); Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker). The resulting product is one where the polypeptide is uniformly distributed throughout the inert material rather than concentrated on its surface. US 4,016,040 and US 4,713,245 describe this technique.

[0531] (f) Mixer granulation products, where the polypeptide-containing liquid is added to a dry powder composition of conventional granulation components. The liquid and powder are mixed in a suitable ratio, and as the moisture of the liquid is absorbed in the dry powder, the components of the dry powder will start to adhere and agglomerate, and the particles will accumulate to form particulate matter containing the polypeptide. Such methods are described in US4,106,991, EP 170360, EP 304332, EP 304331, WO 90 / 09440 and WO 90 / 09428. In a particular aspect of this process, various high-shear mixers can be used as granulators. The particulate matter composed of polypeptide, filler, binder, etc. is mixed with cellulose fibers to strengthen the particles, thus producing the so-called T-particulate matter. The strengthened particles are more robust and release less enzyme dust.

[0532] (g) Particle size reduction, where cores are produced by milling or crushing larger particles, pellets, tablets, briquettes, etc. containing the polypeptide. The desired core particle fraction is obtained by sieving the milled or crushed product. Oversize and undersize particles can be recycled. Particle size reduction is described in Martin Rhodes (ed.); Principles of Powder Technology; 1990; Chapter 10; John Wiley & Sons.

[0533] (h) Fluidized bed granulation. Fluidized bed granulation involves suspending fine particles in an air stream and spraying a liquid through a nozzle onto the fluidized particles. The particles hit by the sprayed droplets become wet and sticky. The sticky particles collide with other particles and attach to them to form granules.

[0534] (i) These cores can withstand drying, such as in a fluidized bed dryer. A person skilled in the art can use other known methods for drying granules in the feed or enzyme industry. Drying is preferably carried out at a product temperature of 25 °C to 90 °C. For some polypeptides, it is important that the cores containing the polypeptide contain a small amount of water before coating with salt. If a water-sensitive polypeptide is coated with salt before removing the excess water, the excess water will be trapped in the core and may have a negative impact on the activity of the polypeptide. After drying, these cores preferably contain 0.1 - 10% w / w water.

[0535] Dust-free particulate matter can be produced, 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.

[0536] The particulate matter can 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, arabinofuranosidase, cellobiohydrolase, 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. Then, each enzyme will be present in more particles, ensuring a more uniform distribution of the enzymes and also reducing the physical separation of different enzymes due to different particle sizes. The method for producing multi-enzyme co-particulate matter is disclosed in the ip.com disclosure IPCOM000200739D.

[0537] Another example of formulating a polypeptide by using co-particulate matter is disclosed in WO 2013 / 188331.

[0538] The present invention also relates to a protected polypeptide prepared by the method disclosed in EP 238216.

[0539] Liquid formulations

[0540] The present invention also relates to a liquid composition comprising the polypeptide of the present invention. The composition can contain an enzyme stabilizer (examples of the enzyme stabilizer 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 phenylboric acid derivatives such as 4-formylphenylboric acid).

[0541] In some embodiments, one or more fillers or one or more carrier materials are included to increase the volume of such a composition. Suitable fillers or carrier materials include, but are not limited to, various salts of sulfate, carbonate, and silicate radicals, as well as talc, clay, and the like. 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 from about 5% to about 90% of such materials.

[0542] 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.

[0543] In another embodiment, the present invention relates to liquid formulations that comprise:

[0544] (A) 0.001%-25% w / w of the polypeptide having acetylxylan esterase activity of the present invention;

[0545] (B) 20%-80% w / w polyol;

[0546] (C) Optionally 0.001%-2% w / w preservative; and

[0547] (D) Water.

[0548] In another embodiment, the present invention relates to liquid formulations that comprise:

[0549] (A) 0.001%-25% w / w of the polypeptide having acetylxylan esterase activity of the present invention;

[0550] (B) 0.001%-2% w / w preservative;

[0551] (C) Optionally 20%-80% w / w polyol; and

[0552] (D) Water.

[0553] In another embodiment, the liquid formulation comprises 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 polyols are selected from the group consisting of: glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1,2 - propylene glycol or 1,3 - propylene glycol, dipropylene glycol, polyethylene glycol (PEG) 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.

[0554] In another embodiment, the liquid formulation comprises 20% - 80% polyol (i.e., the total amount of polyol), such as 25% - 75% polyol, 30% - 70% polyol, 35% - 65% polyol or 40% - 60% polyol. In one embodiment, the liquid formulation comprises 20% - 80% polyol, such as 25% - 75% polyol, 30% - 70% polyol, 35% - 65% polyol or 40% - 60% polyol, wherein the polyols are selected from the group consisting of: glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1,2 - propylene glycol or 1,3 - propylene glycol, dipropylene glycol, polyethylene glycol (PEG) with an average molecular weight of less than about 600 and polypropylene glycol (PPG) with an average molecular weight of less than about 600. In one embodiment, the liquid formulation comprises 20% - 80% polyol (i.e., the total amount of polyol), such as 25% - 75% polyol, 30% - 70% polyol, 35% - 65% polyol or 40% - 60% polyol, wherein the polyols are selected from the group consisting of: glycerol, sorbitol and propylene glycol (MPG).

[0555] In another embodiment, the preservatives are selected from the group consisting of: sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof. In one embodiment, the liquid formulation comprises 0.02% - 1.5% w / w preservative, such as 0.05% - 1% w / w preservative or 0.1% - 0.5% w / w preservative. In one embodiment, the liquid formulation comprises 0.001% - 2% w / w preservative (i.e., the total amount of preservative), such as 0.02% - 1.5% w / w preservative, 0.05% - 1% w / w preservative or 0.1% - 0.5% w / w preservative, wherein the preservatives are selected from the group consisting of: sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof.

[0556] In another embodiment, the liquid formulation further comprises 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, arabinofuranosidase, cellobiohydrolase, 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.

[0557] Composition

[0558] The present invention relates to compositions that comprise a carbohydrate esterase family 3 (CE3) polypeptide having acetylxylan esterase activity, a polypeptide having arabinofuranosidase activity against di - substituted arabinose, a polypeptide having arabinofuranosidase activity against mono - substituted arabinose, xylanase, β - xylosidase, and optionally α - xylosidase.

[0559] The present invention contemplates using the compositions of the present invention in saccharification, fermentation, or simultaneous saccharification and fermentation to increase the solubilization of hemicellulose fibers to monomeric sugars, such as arabinose and xylose, in conventional and raw starch hydrolysis (RSH) ethanol production processes.

[0560] A. Exemplary CE3 polypeptides having acetylxylan esterase activity

[0561] Aspects of the present invention relate to compositions that comprise a combination of a CE3 polypeptide having acetylxylan esterase activity with other enzymes to increase hemicellulose fiber solubilization and the production of monomeric arabinose and / or xylose. The present invention contemplates that any CE3 polypeptide having acetylxylan esterase activity, when used in combination with a polypeptide having arabinofuranosidase activity against di - substituted and mono - substituted arabinose, xylanase, β - xylosidase, and optionally α - xylosidase, increases the production of monomeric arabinose and / or xylose as compared to compositions comprising only a polypeptide having arabinofuranosidase activity against di - substituted and mono - substituted arabinose, xylanase, β - xylosidase, and optionally α - xylosidase.

[0562] Exemplary CE3 polypeptides having acetylxylan esterase activity have the amino acid sequence of SEQ ID NO:3. In one embodiment, the CE3 polypeptide has the amino acid sequence of SEQ ID NO:3 with 0 to 10 conservative amino acid substitutions and has acetylxylan esterase activity. In one 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:3 and has acetylxylan esterase activity. Exemplary CE3 polypeptides having acetylxylan esterase activity have the amino acid sequence of SEQ ID NO:6. In one embodiment, the CE3 polypeptide has the amino acid sequence of SEQ ID NO:6 with 0 to 10 conservative amino acid substitutions and has acetylxylan esterase activity. In one 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:6 and has acetylxylan esterase activity. Exemplary CE3 polypeptides having acetylxylan esterase activity have the amino acid sequence of SEQ ID NO:9. In one embodiment, the CE3 polypeptide has the amino acid sequence of SEQ ID NO:9 with 0 to 10 conservative amino acid substitutions and has acetylxylan esterase activity. In one 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:9 and has acetylxylan esterase activity. Exemplary CE3 polypeptides having acetylxylan esterase activity have the amino acid sequence of SEQ ID NO:12. In one embodiment, the CE3 polypeptide has the amino acid sequence of SEQ ID NO:12 with 0 to 10 conservative amino acid substitutions and has acetylxylan esterase activity. In one 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:12 and has acetylxylan esterase activity.Exemplary CE3 polypeptides having acetylxylan esterase activity have the amino acid sequence of SEQ ID NO:15. In one embodiment, the CE3 polypeptide has the amino acid sequence of SEQ ID NO:15 with 0 to 10 conservative amino acid substitutions and has acetylxylan esterase activity. In one 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:15 and has acetylxylan esterase activity. Exemplary CE3 polypeptides having acetylxylan esterase activity have the amino acid sequence of SEQ ID NO:18. In one embodiment, the CE3 polypeptide has the amino acid sequence of SEQ ID NO:18 with 0 to 10 conservative amino acid substitutions and has acetylxylan esterase activity. In one 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:18 and has acetylxylan esterase activity. Exemplary CE3 polypeptides having acetylxylan esterase activity have the amino acid sequence of SEQ ID NO:21. In one embodiment, the CE3 polypeptide has the amino acid sequence of SEQ ID NO:21 with 0 to 10 conservative amino acid substitutions and has acetylxylan esterase activity. In one 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:21 and has acetylxylan esterase activity. Exemplary CE3 polypeptides having acetylxylan esterase activity have the amino acid sequence of SEQ ID NO:24. In one embodiment, the CE3 polypeptide has the amino acid sequence of SEQ ID NO:24 with 0 to 10 conservative amino acid substitutions and has acetylxylan esterase activity.In one 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:24 and has acetylxylan esterase activity. An exemplary CE3 polypeptide having acetylxylan esterase activity has the amino acid sequence of SEQ ID NO:27. In one embodiment, the CE3 polypeptide has the amino acid sequence of SEQ ID NO:27 with 0 to 10 conservative amino acid substitutions and has acetylxylan esterase activity. In one 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:27 and has acetylxylan esterase activity. An exemplary CE3 polypeptide having acetylxylan esterase activity has the amino acid sequence of SEQ ID NO:30. In one embodiment, the CE3 polypeptide has the amino acid sequence of SEQ ID NO:30 with 0 to 10 conservative amino acid substitutions and has acetylxylan esterase activity. In one 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:30 and has acetylxylan esterase activity.

[0563] The CE3 polypeptide having acetylxylan esterase activity can be added at a concentration between 0.0001 - 1 mg EP (enzyme protein) / g DS (e.g., 0.0005 - 0.5 mg EP / g DS, such as 0.001 - 0.1 mg EP / g DS or 0.001 - 0.01 mg EP / g DS) in pre-saccharification, saccharification, and / or simultaneous saccharification and fermentation.

[0564] B. Exemplary polypeptides having arabinofuranosidase activity towards di-substituted arabinose

[0565] Aspects of the present invention relate to compositions that comprise a combination of an arabinofuranosidase active against di-substituted arabinose with other enzymes to increase hemicellulose fiber solubilization and the production of monomeric arabinose and / or xylose. The present invention contemplates that any polypeptide having arabinofuranosidase activity against di-substituted arabinose, when used in combination with a CE3 polypeptide having acetylxylan esterase activity, a polypeptide having arabinofuranosidase activity against mono-substituted arabinose, a xylanase, a β-xylosidase, and optionally an α-xylosidase, increases the production of monomeric arabinose and / or xylose as compared to compositions comprising the CE3 polypeptide having acetylxylan esterase activity, the polypeptide having arabinofuranosidase activity against mono-substituted arabinose, the xylanase, the β-xylosidase, and optionally the α-xylosidase alone.

[0566] In one embodiment, the polypeptide having arabinofuranosidase activity against di-substituted arabinose is a GH43 arabinofuranosidase. In one embodiment, the GH43 arabinofuranosidase is a GH43_36 arabinofuranosidase.

[0567] Exemplary GH43 arabinofuranosidases can be from the genus Humicola, Lasiodiplodia, or Poronia.

[0568] Exemplary GH43 arabinofuranosidases can be from Humicola insolens, Lasiodiplodia theobromae, or Poronia punctata.

[0569] Exemplary GH43 arabinofuranosidases have the amino acid sequence of SEQ ID NO:31. In one embodiment, the GH43 arabinofuranosidase has the amino acid sequence of SEQ ID NO:31 with 0 to 10 conservative amino acid substitutions and has arabinofuranosidase activity. In one embodiment, the GH43 arabinofuranosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:31 and has arabinofuranosidase activity. Exemplary GH43 arabinofuranosidases have the amino acid sequence of SEQ ID NO:32. In one embodiment, the GH43 arabinofuranosidase has the amino acid sequence of SEQ ID NO:32 with 0 to 10 conservative amino acid substitutions and has arabinofuranosidase activity. In one embodiment, the GH43 arabinofuranosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:32 and has arabinofuranosidase activity. Exemplary GH43 arabinofuranosidases have the amino acid sequence of SEQ ID NO:33. In one embodiment, the GH43 arabinofuranosidase has the amino acid sequence of SEQ ID NO:33 with 0 to 10 conservative amino acid substitutions and has arabinofuranosidase activity. In one embodiment, the GH43 arabinofuranosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:33 and has arabinofuranosidase activity.

[0570] Polypeptides having arabinofuranosidase activity against di-substituted arabinose can be added at a concentration between 0.0001 - 1 mg EP (enzyme protein) / g DS (e.g., 0.0005 - 0.5 mg EP / g DS, such as 0.001 - 0.1 mg EP / g DS or 0.001 - 0.01 mg EP / g DS) in pre-saccharification, saccharification, and / or simultaneous saccharification and fermentation.

[0571] C. Exemplary polypeptides having arabinofuranosidase activity against mono-substituted arabinose

[0572] Aspects of the invention relate to compositions that comprise a combination of an arabinofuranosidase that is active against mono-substituted arabinose and other enzymes to increase hemicellulose fiber solubilization and the production of monomeric arabinose and / or xylose. The invention contemplates that any polypeptide having arabinofuranosidase activity against mono-substituted arabinose, when used in combination with a CE3 polypeptide having acetylxylan esterase activity, a polypeptide having arabinofuranosidase activity against di-substituted arabinose, a xylanase, a β-xylosidase, and optionally an α-xylosidase, increases the production of monomeric arabinose and / or xylose compared to compositions comprising the CE3 polypeptide having acetylxylan esterase activity, the polypeptide having arabinofuranosidase activity against di-substituted arabinose, the xylanase, the β-xylosidase, and optionally the α-xylosidase alone.

[0573] In one embodiment, the polypeptide having arabinofuranosidase activity against mono-substituted arabinose is a GH51 arabinofuranosidase. In one embodiment, the GH51 arabinofuranosidase is a GH51_6 arabinofuranosidase.

[0574] Exemplary GH51 arabinofuranosidases can be from the genus Meripulus, the genus Lasiodiplodia, or the genus Acidiella.

[0575] Exemplary GH51 arabinofuranosidases can be from the species Meripulus giganteus, Lasiodiplodia theobromae, or Acidiella bohemica.

[0576] Exemplary GH51 arabinofuranosidases have the amino acid sequence of SEQ ID NO:34. In one embodiment, the GH51 arabinofuranosidase has an amino acid sequence of SEQ ID NO:34 with 0 to 10 conservative amino acid substitutions and has arabinofuranosidase activity. In one embodiment, the GH51 arabinofuranosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:34 and has arabinofuranosidase activity. Exemplary GH51 arabinofuranosidases have the amino acid sequence of SEQ ID NO:35. In one embodiment, the GH51 arabinofuranosidase has an amino acid sequence of SEQ ID NO:35 with 0 to 10 conservative amino acid substitutions and has arabinofuranosidase activity. In one embodiment, the GH51 arabinofuranosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:35 and has arabinofuranosidase activity. Exemplary GH51 arabinofuranosidases have the amino acid sequence of SEQ ID NO:36. In one embodiment, the GH51 arabinofuranosidase has an amino acid sequence of SEQ ID NO:36 with 0 to 10 conservative amino acid substitutions and has arabinofuranosidase activity. In one embodiment, the GH51 arabinofuranosidase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:36 and has arabinofuranosidase activity.

[0577] Polypeptides having arabinofuranosidase activity towards mono-substituted arabinose can be dosed at a concentration between 0.0001 - 1 mg EP (enzyme protein) / g DS (e.g., 0.0005 - 0.5 mg EP / g DS, such as 0.001 - 0.1 mg EP / g DS or 0.001 - 0.01 mg EP / g DS) in pre-saccharification, saccharification, and / or simultaneous saccharification and fermentation.

[0578] D. Exemplary polypeptides having xylanase activity

[0579] Aspects of the invention relate to compositions that comprise a combination of a polypeptide having xylanase activity with other enzymes to increase hemicellulose fiber solubilization and the production of monomeric arabinose and / or xylose. The invention contemplates that any polypeptide having xylanase activity, when used in combination with a CE3 polypeptide having acetylxylan esterase activity, a polypeptide having arabinofuranosidase activity towards di- and mono-substituted arabinose, a β-xylosidase, and optionally an α-xylosidase, increases the production of monomeric arabinose and / or xylose compared to compositions comprising a CE3 polypeptide alone, a polypeptide having arabinofuranosidase activity towards di- and mono-substituted arabinose, a β-xylosidase, and optionally an α-xylosidase.

[0580] In one embodiment, the polypeptide having xylanase activity is a GH5_21 xylanase.

[0581] Exemplary GH_21 xylanases can be from the genus Bacteroides, Belliella, Chryseobacterium, or Sphingobacterium.

[0582] Exemplary GH_21 xylanases can be from the species Bacteroides cellulosilyticus CL02Y12C19, Belliella sp.-64282, Chryseobacterium sp., Chryseobacterium oncorhynchi, or Sphingobacterium sp.-64162.

[0583] Exemplary GH5_21 xylanases can be from a bioreactor metagenome, an elephant feces metagenome, the xanthan gum alkaline community O, the xanthan gum alkaline community S, or the xanthan gum alkaline community T.

[0584] Exemplary GH5_21 xylanases have the amino acid sequence of SEQ ID NO:37. In one embodiment, the GH5_21 xylanase has an amino acid sequence of SEQ ID NO:37 with 0 to 10 conservative amino acid substitutions and has xylanase activity. In one 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:37 and has xylanase activity. Exemplary GH5_21 xylanases have the amino acid sequence of SEQ ID NO:38. In one embodiment, the GH5_21 xylanase has an amino acid sequence of SEQ ID NO:38 with 0 to 10 conservative amino acid substitutions and has xylanase activity. In one 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:38 and has xylanase activity. Exemplary GH5_21 xylanases have the amino acid sequence of SEQ ID NO:39. In one embodiment, the GH5_21 xylanase has an amino acid sequence of SEQ ID NO:39 with 0 to 10 conservative amino acid substitutions and has xylanase activity. In one 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:39 and has xylanase activity. Exemplary GH5_21 xylanases have the amino acid sequence of SEQ ID NO:40. In one embodiment, the GH5_21 xylanase has an amino acid sequence of SEQ ID NO:40 with 0 to 10 conservative amino acid substitutions and has xylanase activity. In one 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:40 and has xylanase activity. Exemplary GH5_21 xylanases have the amino acid sequence of SEQ ID NO:41.In one embodiment, the GH5_21 xylanase has an amino acid sequence of SEQ ID NO:41 with 0 to 10 conservative amino acid substitutions and has xylanase activity. In one 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:41 and has xylanase activity. An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO:42. In one embodiment, the GH5_21 xylanase has an amino acid sequence of SEQ ID NO:42 with 0 to 10 conservative amino acid substitutions and has xylanase activity. In one 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:42 and has xylanase activity. An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO:43. In one embodiment, the GH5_21 xylanase has an amino acid sequence of SEQ ID NO:43 with 0 to 10 conservative amino acid substitutions and has xylanase activity. In one 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:43 and has xylanase activity. An exemplary GH5_21 xylanase has the amino acid sequence of SEQ IDNO:44. In one embodiment, the GH5_21 xylanase has an amino acid sequence of SEQ ID NO:44 with 0 to 10 conservative amino acid substitutions and has xylanase activity. In one 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:44 and has xylanase activity. An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO:45. In one embodiment, the GH5_21 xylanase has an amino acid sequence of SEQ ID NO:45 with 0 to 10 conservative amino acid substitutions and has xylanase activity.In one 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: 45 and has xylanase activity. An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 46. In one embodiment, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 46 with 0 to 10 conservative amino acid substitutions and has xylanase activity. In one 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: 46 and has xylanase activity. An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 47. In one embodiment, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 47 with 0 to 10 conservative amino acid substitutions and has xylanase activity. In one 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: 47 and has xylanase activity. An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 48. In one embodiment, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 48 with 0 to 10 conservative amino acid substitutions and has xylanase activity. In one 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: 48 and has xylanase activity. An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 49. In one embodiment, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 49 with 0 to 10 conservative amino acid substitutions and has xylanase activity.In one 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:49 and has xylanase activity. An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO:50. In one embodiment, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO:50 with 0 to 10 conservative amino acid substitutions and has xylanase activity. In one 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:50 and has xylanase activity. An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO:51. In one embodiment, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO:51 with 0 to 10 conservative amino acid substitutions and has xylanase activity. In one 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:51 and has xylanase activity.

[0585] In one embodiment, the polypeptide having xylanase activity is a GH5_35 xylanase.

[0586] Exemplary GH5_35 xylanases can be from the genus Bacillus, Cohnella, or Paenibacillus.

[0587] Exemplary GH5_35 xylanases can be from the species Bacillus hemiccellulosilyticus JCM 9152, Cohnella xylanilytica, Paenibacillus chitinolyticus, or Paenibacillus sp.-62332.

[0588] Exemplary GH5_35 xylanases can be from a compost metagenome.

[0589] Exemplary GH5_35 xylanases have the amino acid sequence of SEQ ID NO:52. In one embodiment, the GH5_35 xylanase has the amino acid sequence of SEQ ID NO:52 with 0 to 10 conservative amino acid substitutions and has xylanase activity. In one 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:52 and has xylanase activity. Exemplary GH5_35 xylanases have the amino acid sequence of SEQ ID NO:53. In one embodiment, the GH5_35 xylanase has the amino acid sequence of SEQ ID NO:53 with 0 to 10 conservative amino acid substitutions and has xylanase activity. In one 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:53 and has xylanase activity. Exemplary GH5_35 xylanases have the amino acid sequence of SEQ ID NO:54. In one embodiment, the GH5_35 xylanase has the amino acid sequence of SEQ ID NO:54 with 0 to 10 conservative amino acid substitutions and has xylanase activity. In one 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:54 and has xylanase activity. Exemplary GH5_35 xylanases have the amino acid sequence of SEQ ID NO:55. In one embodiment, the GH5_35 xylanase has the amino acid sequence of SEQ ID NO:55 with 0 to 10 conservative amino acid substitutions and has xylanase activity. In one 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:55 and has xylanase activity. Exemplary GH5_35 xylanases have the amino acid sequence of SEQ ID NO:56.In one embodiment, the GH5_35 xylanase has an amino acid sequence of SEQ ID NO:56 with 0 to 10 conservative amino acid substitutions and has xylanase activity. In one 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:56 and has xylanase activity.

[0590] In one embodiment, the polypeptide having xylanase activity is a GH30_8 xylanase.

[0591] Exemplary GH30_8 xylanases can be from the genus Bacillus.

[0592] Exemplary GH30_8 xylanases can be from the species Bacillus sp.-18423.

[0593] Exemplary GH30_8 xylanases have the amino acid sequence of SEQ ID NO:57. In one embodiment, the GH30_8 xylanase has an amino acid sequence of SEQ ID NO:57 with 0 to 10 conservative amino acid substitutions and has xylanase activity. In one 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:57 and has xylanase activity.

[0594] The polypeptide having xylanase activity can be added at a concentration between 0.0001 - 1 mg EP (enzyme protein) / g DS (e.g., 0.0005 - 0.5 mg EP / g DS, such as 0.001 - 0.1 mg EP / g DS or 0.001 - 0.01 mg EP / g DS) in pre-saccharification, saccharification, and / or simultaneous saccharification and fermentation.

[0595] E. Exemplary polypeptides having β-xylosidase activity

[0596] Aspects of the present invention relate to compositions that comprise a combination of a polypeptide having β-xylosidase activity with other enzymes to increase hemicellulose fiber solubilization and the production of monomeric arabinose and / or xylose. The present invention contemplates that any polypeptide having β-xylosidase activity, when used in combination with a CE3 polypeptide having acetylxylan esterase activity, a polypeptide having arabinofuranosidase activity towards di- and mono-substituted arabinose, a polypeptide having xylanase activity, and optionally a polypeptide having α-xylosidase activity, increases the production of monomeric arabinose and / or xylose as compared to compositions comprising a CE3 polypeptide alone, a polypeptide having arabinofuranosidase activity towards di- and mono-substituted arabinose, a xylanase, and optionally an α-xylosidase.

[0597] In one embodiment, the β-xylosidase is a GH3 β-xylosidase.

[0598] Exemplary GH3 β-xylosidases can be from the genus Aspergillus or Talaromyces.

[0599] Exemplary GH3 β-xylosidases can be from the species Aspergillus fumigatus, Aspergillus nidulans, or Talaromyces emersonii.

[0600] Exemplary GH3β-xylosidases have the amino acid sequence of SEQ ID NO:58. In one embodiment, the GH3β-xylosidase has the amino acid sequence of SEQ ID NO:58 with 0 to 10 conservative amino acid substitutions and has β-xylosidase activity. In one embodiment, the GH3β-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:58 and has β-xylosidase activity. Exemplary GH3β-xylosidases have the amino acid sequence of SEQ ID NO:59. In one embodiment, the GH3β-xylosidase has the amino acid sequence of SEQ ID NO:59 with 0 to 10 conservative amino acid substitutions and has β-xylosidase activity. In one embodiment, the GH3β-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:59 and has β-xylosidase activity. Exemplary GH3β-xylosidases have the amino acid sequence of SEQ ID NO:60. In one embodiment, the GH3β-xylosidase has the amino acid sequence of SEQ ID NO:60 with 0 to 10 conservative amino acid substitutions and has β-xylosidase activity. In one embodiment, the GH3β-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:60 and has β-xylosidase activity.

[0601] Polypeptides having β-xylosidase can be added at a concentration between 0.0001 - 1 mg EP (enzyme protein) / g DS (e.g., 0.0005 - 0.5 mg EP / g DS, such as 0.001 - 0.1 mg EP / g DS or 0.001 - 0.01 mg EP / g DS) in pre-saccharification, saccharification, and / or simultaneous saccharification and fermentation.

[0602] F. Exemplary polypeptides having α-xylosidase activity

[0603] Aspects of the present invention relate to compositions that comprise a combination of a polypeptide having α-xylosidase activity with other enzymes to increase hemicellulose fiber solubilization and the production of monomeric arabinose and / or xylose. The present invention contemplates that any polypeptide having α-xylosidase activity, when used in combination with a CE polypeptide having acetylxylan esterase activity, a polypeptide having arabinofuranosidase activity towards di- and mono-substituted arabinose, a polypeptide having xylanase activity, and a polypeptide having β-xylosidase activity, increases the production of monomeric arabinose and / or xylose compared to compositions comprising a CE3 polypeptide alone, a polypeptide having arabinofuranosidase activity towards di- and mono-substituted arabinose, a xylanase, and a β-xylosidase.

[0604] In one embodiment, the α-xylosidase comprises a GH31 α-xylosidase.

[0605] Exemplary GH31 α-xylosidases can be from the genus Herbinix.

[0606] Exemplary GH31 α-xylosidases can be from the species Herbinix hemicellulosilytica.

[0607] Exemplary GH31 α-xylosidases have the amino acid sequence of SEQ ID NO:61. In one embodiment, the GH31 α-xylosidase has an amino acid sequence of SEQ ID NO:61 that contains 0 to 10 conservative amino acid substitutions and has β-xylosidase activity. In one 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 to the amino acid sequence of SEQ ID NO:61 and has α-xylosidase activity.

[0608] The polypeptide having α-xylosidase can be dosed at a concentration between 0.0001 - 1 mg EP (enzyme protein) / g DS (e.g., 0.0005 - 0.5 mg EP / g DS, such as 0.001 - 0.1 mg EP / g DS or 0.001 - 0.01 mg EP / g DS) in pre-saccharification, saccharification, and / or simultaneous saccharification and fermentation.

[0609] G. Exemplary fermenting organisms

[0610] Aspects of the present invention relate to the use of fermenting organisms for the production of fermentation products. Particularly suitable fermenting organisms are capable of directly or indirectly fermenting sugars (such as arabinose, glucose, maltose, and / or xylose) into (i.e., converting them into) a desired fermentation product (such as ethanol). Examples of fermenting organisms include fungal organisms such as yeast. Preferred yeasts include strains of the species Saccharomyces, particularly Saccharomyces cerevisiae.

[0611] Examples of commercially available yeasts include, for example, RED STAR TM and ETHANOL RED TM yeast (available from Fermentis / Lesaffre, USA), FALI (available from Fleischmann’s Yeast, USA), SUPERSTART and THERMOSACC TM fresh yeast (available from Ethanol Technology, Wisconsin, USA), BIOFERM AFT and XR (available from North American Bioproducts Corporation, Georgia, USA), GERT STRAND (available from Gert Strand AB, Sweden), and FERMIOL (available from DSM Specialties). Other available yeast strains are available from biological depositories such as the American Type Culture Collection (ATCC) or the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), such as, for example, BY4741 (e.g., ATCC 201388); Y108-1 (ATCC PTA.10567) and NRRL YB-1952 (ARS Culture Collection). Still other Saccharomyces cerevisiae strains suitable as host cells are DBY746, [Alpha][Eta]22, S150-2B, GPY55-15Ba, CEN.PK, USM21, TMB3500, TMB3400, VTT-A-63015, VTT-A-85068, VTT-c-79093 and their derivatives, as well as Saccharomyces species 1400, 424A (LNH-ST), 259A (LNH-ST) and their derivatives.

[0612] As used herein, a "derivative" of a strain is derived from a reference strain, such as by mutagenesis, recombinant DNA technology, mating, cell fusion, or cytoduction 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 reactions or a suitable source organism for the desired genetic material (such as genes of a desired metabolic pathway). However, given the whole-genome sequencing of a wide variety of organisms 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 the same or similar coding nucleic acids from a species different from the reference species.

[0613] The fermenting organism can be a strain of the genus Saccharomyces, 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 the strain Saccharomyces cerevisiae CIBTS1260 (deposited under accession number NRRL Y-50973 at the Agricultural Research Service Culture Collection (NRRL), 61604, Illinois, USA).

[0614] The fermenting organism can also be a derivative of the Saccharomyces cerevisiae strain NMI V14 / 004037 (see WO 2015 / 143324 and WO2015 / 143317, each incorporated herein by reference), strain numbers V15 / 004035, V15 / 004036, and V15 / 004037 (see WO 2016 / 153924, incorporated herein by reference), strain numbers V15 / 001459, V15 / 001460, V15 / 001461 (see WO 2016 / 138437, incorporated herein by reference), strain number NRRL Y67342 (see WO2018 / 098381, incorporated herein by reference), strain numbers NRRL Y67549 and NRRL Y67700 (see WO 2019 / 161227, incorporated herein by reference), or any strain described in WO 2017 / 087330 (incorporated herein by reference).

[0615] The fermenting organism can contain one or more heterologous polynucleotides encoding α-amylase, glucoamylase, protease, and / or cellulase. Examples of α-amylase, glucoamylase, protease, and cellulase suitable for expression in a fermenting organism are known in the art (see WO 2021 / 231623, incorporated herein by reference).

[0616] The fermenting organism can be in the form of a composition that includes the fermenting organism and naturally occurring and / or non-naturally occurring components.

[0617] The fermenting organism can be in any viable form, including crushed, dried, including active dry and instant, compressed, paste (liquid) form, etc. In one embodiment, the fermenting organism (e.g., a Saccharomyces cerevisiae strain) is dry yeast, such as active dry yeast or instant yeast. In one embodiment, the fermenting organism is crushed yeast. In one embodiment, the fermenting organism is compressed yeast. In one embodiment, the fermenting organism is paste yeast.

[0618] In one embodiment, it is a composition that includes the fermenting organism (e.g., a Saccharomyces cerevisiae strain) described herein and one or more components selected from the group consisting of: surfactants, emulsifiers, gums, swelling agents, and antioxidants and other processing aids.

[0619] The composition described herein can include the fermenting organism (e.g., a Saccharomyces cerevisiae strain) described herein and any suitable surfactant. In one embodiment, one or more surfactants are anionic surfactants, cationic surfactants, and / or nonionic surfactants.

[0620] The composition described herein can include the fermenting organism (e.g., a Saccharomyces cerevisiae strain) described herein and any suitable emulsifier. In one embodiment, the emulsifier is a fatty acid ester of sorbitan. In one embodiment, the emulsifier is selected from the group consisting of: sorbitan monostearate (SMS), citric acid esters of mono- and diglycerides, polyglycerol esters, and fatty acid esters of propylene glycol.

[0621] In one embodiment, the composition includes the fermenting organism (e.g., a Saccharomyces cerevisiae strain) described herein and Olindronal SMS, Olindronal SK, or Olindronal SPL, including the compositions described in European Patent No. 1,724,336 (which is hereby incorporated by reference). For active dry yeast, these products are commercially available from Bussetti of Austria.

[0622] The composition described herein can include the fermenting organism (e.g., a Saccharomyces cerevisiae strain) described herein and any suitable gum. In one embodiment, the gum is selected from the group consisting of: locust bean gum, guar gum, tragacanth gum, gum arabic, xanthan gum, and gum acacia, particularly for paste, compressed, and dry yeast.

[0623] The composition described herein can include the fermenting organism (e.g., a Saccharomyces cerevisiae strain) described herein and any suitable swelling agent. In one embodiment, the swelling agent is methylcellulose or carboxymethylcellulose.

[0624] The compositions described herein may comprise a fermenting organism (e.g., a Saccharomyces cerevisiae strain) described herein and any suitable antioxidant. In one embodiment, the antioxidant is butylated hydroxyanisole (BHA) and / or butylated hydroxytoluene (BHT), or ascorbic acid (vitamin C), particularly for active dry yeast.

[0625] During fermentation (such as SSF), suitable concentrations of viable fermenting organisms are well known in the art or can be readily determined by those skilled in the art. In one embodiment, a fermenting organism (such as an ethanol-fermenting yeast (e.g., Saccharomyces cerevisiae)) is added to the fermentation medium such that the viable fermenting organism (such as yeast) count per mL of the fermentation medium is in the range of 10 5 to 10 12 , preferably 10 7 to 10 10 , particularly about 5×10 7 .

[0626] Method for producing a fermentation product from a gelatinized starch material

[0627] One aspect of the present invention relates to a method for producing a fermentation product (e.g., fuel ethanol) from a gelatinized starch material, wherein a composition comprising a CE3 polypeptide having acetylxylan esterase or a CE3 polypeptide having acetylxylan esterase is present or added during saccharification and / or fermentation.

[0628] In one embodiment, the method for producing a fermentation product from a starch-containing material comprises the following steps:

[0629] (a) Liquefying the starch-containing material using a thermostable α-amylase at a temperature above the initial gelatinization temperature of the starch to produce dextrin;

[0630] (b) Saccharifying the dextrin using glucoamylase to produce fermentable sugars; and

[0631] (c) Fermenting the sugars using a fermenting organism to produce a fermentation product;

[0632] wherein a CE3 polypeptide having acetylxylan esterase activity or a composition comprising a CE3 polypeptide having acetylxylan esterase activity is present or added during saccharification step (b) and / or fermentation step (c).

[0633] The present invention contemplates any CE3 polypeptide or composition described herein for use in a method for producing a fermentation product. In one embodiment, the composition used in step (b) and / or step (c) comprises a polypeptide having arabinofuranosidase activity towards di-substituted arabinose. In one embodiment, the polypeptide having arabinofuranosidase activity towards di-substituted arabinose is a GH43 arabinofuranosidase. In one embodiment, the GH43 arabinofuranosidase is a GH43_36 arabinofuranosidase. In one embodiment, the composition used in step (b) and / or step (c) comprises a polypeptide having arabinofuranosidase activity towards mono-substituted arabinose. In an embodiment, the polypeptide having arabinofuranosidase activity towards mono-substituted arabinose is a GH51 arabinofuranosidase. In one embodiment, the GH51 arabinofuranosidase is a GH51_6 arabinofuranosidase. In one embodiment, the composition used in step (b) and / or step (c) comprises a polypeptide having xylanase activity. In one embodiment, the polypeptide having xylanase activity is a GH5 family xylanase. In one embodiment, the GH5 family xylanase is a GH5_21 xylanase. In one embodiment, the GH5 family xylanase is a GH5_35 xylanase. In one embodiment, the polypeptide having xylanase activity is a GH30_8 xylanase. In one embodiment, the composition used in step (b) and / or step (c) comprises a β-xylosidase. In one embodiment, the β-xylosidase is a GH3 β-xylosidase. In one embodiment, the composition used in step (b) and / or step (c) comprises an α-xylosidase. In one embodiment, the α-xylosidase is a GH31 α-xylosidase.

[0634] The present invention contemplates the use of any of the exemplary CE3 polypeptides, exemplary polypeptides having arabinofuranosidase activity towards di- and mono-substituted arabinose, exemplary polypeptides having xylanase activity, exemplary polypeptides having β-xylosidase activity, and exemplary polypeptides having α-xylosidase activity in a composition and in a method of using the composition of the present invention, including in the following exemplary compositions for use in a method for producing a fermentation product.

[0635] The exemplary composition used in step (b) and / or step (c) comprises a CE3 polypeptide having acetylxylan esterase activity, a polypeptide having arabinofuranosidase activity towards di-substituted arabinose, a polypeptide having arabinofuranosidase activity towards mono-substituted arabinofuranose, a polypeptide having xylanase activity, and a polypeptide having β-xylosidase activity.

[0636] The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a polypeptide having xylanase activity, and a polypeptide having β-xylosidase activity. The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, and a polypeptide having β-xylosidase activity. The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, and a GH3 β-xylosidase. The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, and a GH3 β-xylosidase. The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_35 xylanase, and a GH3 β-xylosidase. The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH30_8 xylanase, and a GH3 β-xylosidase.

[0637] The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a GH43_36 arabinofuranosidase, a GH51_6 arabinofuranosidase, a polypeptide having xylanase activity, and a polypeptide having β-xylosidase activity. The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a GH43_36 arabinofuranosidase, a GH51_6 arabinofuranosidase, a GH5 xylanase, and a polypeptide having β-xylosidase activity. The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a GH43_36 arabinofuranosidase, a GH51_6 arabinofuranosidase, a GH5 xylanase, and a GH3 β-xylosidase. The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a GH43_36 arabinofuranosidase, a GH51_6 arabinofuranosidase, a GH5_21 xylanase, and a GH3 β-xylosidase. The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a GH43_36 arabinofuranosidase, a GH51_6 arabinofuranosidase, a GH5_35 xylanase, and a GH3 β-xylosidase. The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a GH43_36 arabinofuranosidase, a GH51_6 arabinofuranosidase, a GH30_8 xylanase, and a GH3 β-xylosidase.

[0638] The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a polypeptide having arabinofuranosidase activity towards di-substituted arabinose, a polypeptide having arabinofuranosidase activity towards mono-substituted arabinofuranoside, a polypeptide having xylanase activity, a polypeptide having β-xylosidase activity, and a polypeptide having α-xylosidase activity.

[0639] The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a polypeptide having xylanase activity, a polypeptide having β-xylosidase activity, and a polypeptide having α-xylosidase activity. The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a polypeptide having β-xylosidase activity, and a polypeptide having α-xylosidase activity. The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 β-xylosidase, and a polypeptide having α-xylosidase activity. The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, a GH3 β-xylosidase, and a polypeptide having α-xylosidase activity. The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_35 xylanase, a GH3 β-xylosidase, and a polypeptide having α-xylosidase activity. The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH30_8 xylanase, and a GH3 β-xylosidase, and a polypeptide having α-xylosidase activity. The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 β-xylosidase, and a GH31 α-xylosidase. The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, a GH3 β-xylosidase, and a GH31 α-xylosidase. The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_35 xylanase, a GH3 β-xylosidase, and a GH31 α-xylosidase.The exemplary composition used in step (b) and / or step (c) comprises a CE3 polypeptide having acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH30_8 xylanase, and a GH3 β-xylosidase, and a GH31 α-xylosidase.

[0640] In one embodiment, the composition is added during the saccharification step (b). In one embodiment, the composition is added during the fermentation step (c). In one embodiment, steps (b) and (c) are carried out simultaneously in simultaneous saccharification and fermentation (SSF). In one embodiment, the composition is added during SSF.

[0641] In one embodiment, a thermostable glucoamylase is added during the liquefaction step (a). In one embodiment, a thermostable endoglucanase is added during the liquefaction step (a). In one embodiment, a thermostable lipase is added during the liquefaction step (a). In one embodiment, a thermostable phytase is added during the liquefaction step (a). In one embodiment, a thermostable protease is added during the liquefaction step (a). In one embodiment, a thermostable pullulanase is added during the liquefaction step (a). In one embodiment, a thermostable xylanase is added during the liquefaction step (a). In a preferred embodiment, a thermostable α-amylase and a thermostable protease are added during the liquefaction step (a). In one embodiment, a thermostable α-amylase and a thermostable xylanase are added during the liquefaction step (a). In a preferred embodiment, a thermostable α-amylase, a thermostable protease, and a thermostable xylanase are added during the liquefaction step (a).

[0642] In one embodiment, α-amylase is added during step (b) and / or step (c). In one embodiment, α-glucosidase is added during step (b) and / or step (c). In one embodiment, β-amylase is added during step (b) and / or step (c). In one embodiment, β-glucanase is added during step (b) and / or step (c). In one embodiment, β-glucosidase is added during step (b) and / or step (c). In one embodiment, cellobiohydrolase is added during step (b) and / or step (c). In one embodiment, endoglucanase is added during step (b) and / or step (c). In one embodiment, lipase is added during step (b) and / or step (c). In one embodiment, lytic polysaccharide monooxygenase (LPMO) is added during step (b) and / or step (c). In one embodiment, maltogenic α-amylase is added during step (b) and / or step (c). In one embodiment, pectinase is added during step (b) and / or step (c). In one embodiment, peroxidase is added during step (b) and / or step (c). In one embodiment, phytase is added during step (b) and / or step (c). In one embodiment, protease is added during step (b) and / or step (c). In one embodiment, trehalase is added during step (b) and / or step (c).

[0643] In one embodiment, the fermenting organism is yeast. In one embodiment, the yeast in situ expresses α-amylase during step (b) and / or step (c). In one embodiment, the yeast in situ expresses glucoamylase during step (b) and / or step (c).

[0644] Process parameters

[0645] Process parameters for producing a fermentation product (such as ethanol from a starch-containing material (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).

[0646] Starch-containing material

[0647] 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, beans, cassava, cereal grains, corn, milo, peas, potatoes, rice, rye, sago, sorghum, sweet potatoes, tapioca, wheat, and whole grains, or any mixture thereof. The starch-containing material can also be waxy or non-waxy types of corn and barley. Commonly used commercial starch-containing materials include corn, milo, and / or wheat.

[0648] Reduction in the particle size of the starch-containing material

[0649] Before the liquefaction step (a), the particle size of the starch-containing material can be reduced, for example, by dry milling.

[0650] Slurry

[0651] Before the liquefaction step (a), a slurry can be formed comprising the starch-containing material (e.g., preferably ground) and water. α-Amylase and optionally protease can be added to the slurry. The slurry can be heated therebetween to above the initial gelatinization temperature of the starch-containing material to initiate gelatinization of the starch.

[0652] Jet cooking

[0653] Before adding α-amylase during the liquefaction step (a), the slurry can optionally be jet-cooked to further gelatinize the starch in the slurry. Jet cooking can be carried out in the temperature range of 100 °C to 120 °C for up to at least 15 minutes.

[0654] Liquefaction temperature

[0655] The temperature range used during the liquefaction step (a) can be 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.

[0656] Liquefaction pH

[0657] The pH range used during the 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.

[0658] Liquefaction time

[0659] 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.

[0660] Liquefying enzyme

[0661] The present invention contemplates the use of thermostable enzymes during the liquefaction step (a). It is well known in the art to use a variety of thermostable enzymes during the liquefaction step (a), including, for example, thermostable α-amylase, thermostable glucoamylase, thermostable endoglucanase, thermostable lipase, thermostable phytase, thermostable protease, thermostable pullulanase, and / or thermostable xylanase. The present invention contemplates the use of any thermostable enzyme in the liquefaction step (a). Guidelines for determining the denaturation temperature of candidate thermostable enzymes used in the liquefaction step (a) are provided in the following Materials and Methods section. The published patent applications listed below describe activity assays for determining whether a candidate thermostable enzyme under consideration for use in the liquefaction step (a) will be inactivated at the temperatures contemplated for the liquefaction step (a).

[0662] Examples of suitable thermostable α-amylases and guidelines for their use in the liquefaction step (a) include, but are not limited to, the α-amylases described in the following: 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 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).

[0663] Examples of suitable thermostable glucoamylases include, but are not limited to, glucoamylases 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).

[0664] Examples of suitable thermostable endoglucanases include, but are not limited to, the endoglucanases described in WO 2015 / 035914 (which is incorporated herein by reference).

[0665] Examples of suitable thermostable lipases include, but are not limited to, the lipases described in WO 2017 / 112542 and WO 2020 / 014407 (both of these patents are incorporated herein by reference).

[0666] Examples of suitable thermostable phytases include, but are not limited to, phytases described in the following: 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 2007 / 112739, WO 2008 / 092901, WO 2008 / 116878, WO 2009 / 129489, and WO2010 / 034835 (each of these patents is incorporated by reference). Commercially available phytase products include BIO-FEEDPHYTASE TM 、PHYTASE NOVO TM CT or L、LIQMAX or RONOZYME TM NP、 HIPHOS、 P5000(CT)、NATUPHOS TM NG 5000。

[0667] Examples of suitable thermostable proteases include, but are not limited to, 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, WO2018 / 118815A1, and WO2018 / 169780A1 (each of these patents is incorporated herein by reference).

[0668] Suitable commercially available protease-containing products include AVANTEC FORTIVA FORTIVA

[0669] Examples of suitable thermostable pullulanases include, but are not limited to, pullulanases described in the following: 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 pullulanase products include PROMOZYME 400L, PROMOZYME TM D2 (Novozymes A / S, Denmark), OPTIMAX L-300 (Genencor Int., USA), and AMANO 8 (Amano, Japan).

[0670] Examples of suitable thermostable xylanases include, but are not limited to, xylanases described in WO 2017 / 112540 and WO 2021 / 126966 (each of these patents is incorporated herein by reference). Suitable commercially available thermostable xylanase-containing products include FORTIVA

[0671] One or more of the enzymes described above are to be used in "effective amounts" in the methods of the present invention. Guidelines for determining the effective amounts of the enzymes used in the liquefaction step (a) and guidelines for performing activity assays to determine the activities of those enzymes can be found in the published patent applications cited for each of the different thermostable liquefying enzymes.

[0672] Saccharification temperature

[0673] The saccharification can be carried out within 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.

[0674] Saccharification pH

[0675] The saccharification can be carried out within a pH range of 4 to 5. Preferably, the pH is about 4.5.

[0676] Saccharification time

[0677] The saccharification can last for about 24 hours to about 72 hours.

[0678] Fermentation time

[0679] The fermentation can last for 6 to 120 hours, 24 hours to 96 hours, or 35 hours to 60 hours.

[0680] Simultaneous saccharification and fermentation

[0681] SSF can be carried out at a temperature of 25 °C to 40 °C, 28 °C to 35 °C, or 30 °C to [temperature value not provided] °C, and a pH 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 a pH of 3.8 to 4.5 for 48 to 60 hours.

[0682] Saccharification and / or fermentation enzymes

[0683] The present invention contemplates the use of enzymes during the saccharification step (b) and / or the fermentation step (c). It is well known in the art to use a variety of enzymes during the saccharification step (b) and / or the fermentation step (c), including, for example, α-amylase, α-glucosidase, β-amylase, β-glucanase, β-glucosidase, cellobiohydrolase, endoglucanase, glucoamylase, lipase, lytic polysaccharide monooxygenase (LPMO), maltogenic α-amylase, pectinase, peroxidase, phytase, protease, and trehalase.

[0684] The enzymes used in the saccharification step (b) and / or the fermentation step (c) can be added exogenously as a single component, or formulated into a composition containing these enzymes. The enzymes used in the saccharification step (b) and / or the fermentation step (c) can be added via in situ expression from a fermenting organism (e.g., yeast).

[0685] Examples of suitable α - amylases include, but are not limited to, 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).

[0686] Examples of suitable glucoamylases include, but are not limited to, glucoamylases 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, WO2013 / 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).

[0687] Examples of suitable compositions containing α - amylase and glucoamylase include, but are not limited to, compositions described in the following: 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 glucoamylase include AMG 200L; AMG300L; SAN TM SUPER, SAN TM EXTRA L, SPIRIZYME TM PLUS, SPIRIZYME TM FUEL, SPIRIZYME TM B4U, SPIRIZYME TM ULTRA, SPIRIZYME TM EXCEL, SPIRIZYME ACHIEVE and AMG TM E (from Novozymes); OPTIDEX TM 300, GC480, GC417 (from DuPont - Genencor); AMIGASETM and AMIGASE TM PLUS (from DSM); G-ZYME TM G900, G-ZYME TM and G990 ZR (from DuPont - Genencor).

[0688] Examples of suitable β - glucanases include, but are not limited to, the β - glucanases described in WO 2021 / 055395 (incorporated herein by reference).

[0689] Examples of suitable β - glucosidases include, but are not limited to, the β - glucosidases described in: WO 2005 / 047499, WO 2013 / 148993, WO 2014 / 085439, and WO 2012 / 044915 (each of these patents is incorporated herein by reference).

[0690] Examples of suitable cellobiohydrolases include, but are not limited to, the cellobiohydrolases described in: WO2013 / 148993, WO 2014 / 085439, WO 2014 / 138672, and WO 2016 / 040265 (each of these patents is incorporated herein by reference).

[0691] Examples of suitable endoglucanases include, but are not limited to, the endoglucanases described in WO 2013 / 148993 and WO 2014 / 085439 (both of these patents are incorporated herein by reference).

[0692] Examples of suitable maltogenic 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.

[0693] 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).

[0694] Examples of suitable LPMOs include, but are not limited to, the LPMOs described in: WO 2013 / 148993, WO2014 / 085439, and WO 2019 / 083831 (each of these patents is incorporated herein by reference).

[0695] Examples of suitable phytases include, but are not limited to, the phytases described in WO 2001 / 62947 (incorporated herein by reference).

[0696] Examples of suitable pectinases include, but are not limited to, the pectinases described in WO 2022 / 173694 (which is incorporated herein by reference).

[0697] Examples of suitable peroxidases include, but are not limited to, the peroxidases described in WO 2019 / 231944 (which is incorporated herein by reference).

[0698] Examples of suitable proteases include, but are not limited to, the proteases described in: WO 2017 / 050291, WO2017 / 148389, WO 2018 / 015303, and WO 2018 / 015304 (each of these patents is incorporated herein by reference).

[0699] Examples of suitable trehalases include, but are not limited to, the trehalases described in: WO 2016 / 205127, WO 2019 / 005755, WO 2019 / 030165, and WO 2020 / 023411 (each of these patents is incorporated herein by reference).

[0700] Method for producing a fermentation product from a material containing ungelatinized starch

[0701] One aspect of the present invention relates to a method for producing a fermentation product from a material containing ungelatinized starch (i.e., granular starch - commonly referred to as the "raw starch hydrolysis" method), wherein a composition comprising a CE3 polypeptide having acetylxylan esterase or a CE3 polypeptide having acetylxylan esterase is present or added during saccharification and / or fermentation.

[0702] In one embodiment, the method for producing a fermentation product from a material containing ungelatinized starch comprises the steps of:

[0703] (a) saccharifying the starch-containing material using α-amylase and glucoamylase at a temperature below the initial gelatinization temperature of the starch to produce fermentable sugars; and

[0704] (b) fermenting the sugar using a fermenting organism to produce a fermentation product;

[0705] wherein a CE3 polypeptide having acetylxylan esterase activity or a composition comprising a CE3 polypeptide having acetylxylan esterase activity is present or added to the saccharification step (a) and / or the fermentation step (b).

[0706] The present invention contemplates any CE3 polypeptide or composition described herein for use in a method for producing a fermentation product. In one embodiment, the composition used in step (b) and / or step (c) comprises a polypeptide having arabinofuranosidase activity towards di-substituted arabinose. In one embodiment, the composition used in step (a) and / or step (b) comprises a polypeptide having arabinofuranosidase activity towards di-substituted arabinose. In one embodiment, the polypeptide having arabinofuranosidase activity towards di-substituted arabinose is a GH43 arabinofuranosidase. In one embodiment, the GH43 arabinofuranosidase is a GH43_36 arabinofuranosidase. In one embodiment, the composition used in step (a) and / or step (b) comprises a polypeptide having arabinofuranosidase activity towards mono-substituted arabinose. In an embodiment, the polypeptide having arabinofuranosidase activity towards mono-substituted arabinose is a GH51 arabinofuranosidase. In one embodiment, the GH51 arabinofuranosidase is a GH51_6 arabinofuranosidase. In one embodiment, the composition used in step (a) and / or step (b) comprises a polypeptide having xylanase activity. In one embodiment, the polypeptide having xylanase activity is a GH5 family xylanase. In one embodiment, the GH5 family xylanase is a GH5_21 xylanase. In one embodiment, the GH5 family xylanase is a GH5_35 xylanase. In one embodiment, the polypeptide having xylanase activity is a GH30_8 xylanase. In one embodiment, the composition used in step (a) and / or step (b) comprises a β-xylosidase. In one embodiment, the β-xylosidase is a GH3 β-xylosidase. In one embodiment, the composition used in step (a) and / or step (b) comprises an α-xylosidase. In one embodiment, the α-xylosidase is a GH31 α-xylosidase.

[0707] The present invention contemplates the use of any of the exemplary CE3 polypeptides, exemplary polypeptides having arabinofuranosidase activity towards di-substituted and mono-substituted arabinose, exemplary polypeptides having xylanase activity, exemplary polypeptides having β-xylosidase activity, and exemplary polypeptides having α-xylosidase activity in a composition and in a method of using the composition of the present invention, including in the following exemplary compositions for use in a method for producing a fermentation product.

[0708] The exemplary composition used in step (b) and / or step (c) comprises a CE3 polypeptide having acetylxylan esterase activity, a polypeptide having arabinofuranosidase activity towards di-substituted arabinose, a polypeptide having arabinofuranosidase activity towards mono-substituted arabinofuranose, a polypeptide having xylanase activity, and a polypeptide having β-xylosidase activity.

[0709] The exemplary composition used in step (b) and / or step (c) comprises a CE3 polypeptide having acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a polypeptide having xylanase activity, and a polypeptide having β-xylosidase activity. The exemplary composition used in step (b) and / or step (c) comprises a CE3 polypeptide having acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, and a polypeptide having β-xylosidase activity. The exemplary composition used in step (b) and / or step (c) comprises a CE3 polypeptide having acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, and a GH3 β-xylosidase. The exemplary composition used in step (b) and / or step (c) comprises a CE3 polypeptide having acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, and a GH3 β-xylosidase. The exemplary composition used in step (b) and / or step (c) comprises a CE3 polypeptide having acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_35 xylanase, and a GH3 β-xylosidase. The exemplary composition used in step (b) and / or step (c) comprises a CE3 polypeptide having acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH30_8 xylanase, and a GH3 β-xylosidase.

[0710] The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a GH43_36 arabinofuranosidase, a GH51_6 arabinofuranosidase, a polypeptide having xylanase activity, and a polypeptide having β-xylosidase activity. The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a GH43_36 arabinofuranosidase, a GH51_6 arabinofuranosidase, a GH5 xylanase, and a polypeptide having β-xylosidase activity. The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a GH43_36 arabinofuranosidase, a GH51_6 arabinofuranosidase, a GH5 xylanase, and a GH3 β-xylosidase. The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a GH43_36 arabinofuranosidase, a GH51_6 arabinofuranosidase, a GH5_21 xylanase, and a GH3 β-xylosidase. The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a GH43_36 arabinofuranosidase, a GH51_6 arabinofuranosidase, a GH5_35 xylanase, and a GH3 β-xylosidase. The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a GH43_36 arabinofuranosidase, a GH51_6 arabinofuranosidase, a GH30_8 xylanase, and a GH3 β-xylosidase.

[0711] The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a polypeptide having arabinofuranosidase activity towards di-substituted arabinose, a polypeptide having arabinofuranosidase activity towards mono-substituted arabinofuranoside, a polypeptide having xylanase activity, a polypeptide having β-xylosidase activity, and a polypeptide having α-xylosidase activity.

[0712] The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a polypeptide having xylanase activity, a polypeptide having β-xylosidase activity, and a polypeptide having α-xylosidase activity. The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a polypeptide having β-xylosidase activity, and a polypeptide having α-xylosidase activity. The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 β-xylosidase, and a polypeptide having α-xylosidase activity. The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, a GH3 β-xylosidase, and a polypeptide having α-xylosidase activity. The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_35 xylanase, a GH3 β-xylosidase, and a polypeptide having α-xylosidase activity. The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH30_8 xylanase, and a GH3 β-xylosidase, and a polypeptide having α-xylosidase activity. The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, a GH3 β-xylosidase, and a GH31 α-xylosidase. The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, a GH3 β-xylosidase, and a GH31 α-xylosidase. The exemplary compositions used in step (b) and / or step (c) comprise a CE3 polypeptide having acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_35 xylanase, a GH3 β-xylosidase, and a GH31 α-xylosidase.The exemplary composition used in step (b) and / or step (c) comprises a CE3 polypeptide having acetylxylan esterase activity, GH43 arabinofuranosidase, GH51 arabinofuranosidase, GH30_8 xylanase, and GH3 β-xylosidase, and GH31 α-xylosidase.

[0713] In one embodiment, the composition is added during the saccharification step (b). In one embodiment, the composition is added during the fermentation step (c). In one embodiment, steps (b) and (c) are carried out simultaneously in simultaneous saccharification and fermentation (SSF). In one embodiment, the composition is added during SSF.

[0714] Raw starch hydrolysis (RSH) methods are well known in the art. Those skilled in the art will understand that the process parameters described in Part II above apply to the methods described in this part, except for the process parameters related to the liquefaction step (a) not carried out in the RSH method, including the selection of starch-containing materials, reducing the grain size, saccharification temperature, time and pH, conditions for simultaneous saccharification and fermentation, and saccharifying enzymes. The process parameters for the exemplary raw starch hydrolysis method are further detailed in WO 2004 / 106533 (which is incorporated herein by reference).

[0715] 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, WO2013 / 034106, WO 2021 / 163015, and WO 2021 / 163036 (each of these patents is incorporated herein by reference).

[0716] Examples of glucoamylases preferably used in step (a) and / or step (b) include, but are not limited to, WO1999 / 28448, WO 2005 / 045018, WO2005 / 069840, WO 2006 / 069289 (each of these patents is incorporated herein by reference).

[0717] Examples of compositions comprising α-amylase and glucoamylase preferably used in step (a) and / or step (b) include, but are not limited to, the compositions described in WO 2015 / 031477 (which is incorporated herein by reference).

[0718] Back-end or downstream processing

[0719] A. Recovery of fermentation products and production of whole distillers grains

[0720] After fermentation or SSF, the fermentation product can be separated from the fermentation medium. Any method known in the art can be used to optionally recover the fermentation product (e.g., ethanol) from the fermentation medium, and the method includes but is not limited to chromatography, electrophoresis procedures, differential solubility, distillation, or extraction. For example, alcohols are separated and purified from fermented starch-containing materials by conventional distillation methods.

[0721] Thus, in one embodiment, the method of the present invention further includes distillation to obtain the fermentation product, e.g., ethanol. Fermentation and distillation can be carried out simultaneously and / or separately / sequentially; optionally, followed by one or more process steps for further refining the fermentation product. After the distillation process is completed, the remaining material is regarded as whole stillage.

[0722] As another example, the desired fermentation product can be extracted from the fermentation medium by microfiltration or membrane filtration techniques. Ethanol with a purity of up to about 96 vol.% can be obtained, which can be used as, for example, fuel ethanol, drinking ethanol (i.e., potable neutral alcoholic beverages), or industrial ethanol.

[0723] 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 preparation contains no more than 15% impurities, where impurities mean compounds other than the fermentation product (e.g., ethanol). In one variant, a substantially pure preparation is provided, wherein the preparation 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.

[0724] ...

Claims

1. A polypeptide having acetylxylan esterase activity, said polypeptide selected from the group consisting of: (i) (a) a polypeptide having at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:2; (b) a polypeptide having at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; (c) a polypeptide having at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO:2; (d) a polypeptide encoded by a polynucleotide having at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:1; (e) A polypeptide derived from SEQ ID NO:2, the mature polypeptide of SEQ ID NO:2, or SEQ ID NO:3, which differs by having 1 - 30 alterations (e.g., substitutions, deletions, and / or insertions at one or more positions, such as 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, especially substitutions); (f) A polypeptide derived from the polypeptide of (a), (b), (c), (d), or (e), wherein the N - terminus and / or C - terminus has been extended by adding one or more amino acids; and (g) A fragment of the polypeptide of (a), (b), (c), (d), or (e); wherein the polypeptide has acetylxylan esterase activity; (ii) (a) A polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:5; (b) A polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:6; (c) A polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO:5; (d) A polypeptide encoded by a polynucleotide that has at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide coding sequence of SEQ ID NO:4; (e) A polypeptide derived from SEQ ID NO:5, the mature polypeptide of SEQ ID NO:5, or SEQ ID NO:6 and differing by 1 - 30 alterations (e.g., substitutions, deletions, and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, particularly substitutions); (f) A polypeptide derived from the polypeptide of (a), (b), (c), (d), or (e) wherein the N - terminus and / or C - terminus has been extended by the addition of one or more amino acids; and (g) A fragment of the polypeptide of (a), (b), (c), (d), or (e); wherein the polypeptide has acetylxylan esterase activity; (iii) (a) A polypeptide having at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:8; (b) A polypeptide having at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:9; (c) a polypeptide having at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO:8; (d) a polypeptide encoded by a polynucleotide having at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:7; (e) a polypeptide derived from SEQ ID NO:8, the mature polypeptide of SEQ ID NO:8, or SEQ ID NO:9 and differing by having 1 - 30 alterations (e.g., substitutions, deletions, and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, particularly substitutions); (f) a polypeptide of the polypeptide derived from (a), (b), (c), (d), or (e) wherein the N - terminus and / or C - terminus has been extended by the addition of one or more amino acids; and (g) a fragment of the polypeptide of (a), (b), (c), (d), or (e); wherein the polypeptide has acetylxylan esterase activity; (iv) (a) a polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:11; (b) a polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:12; (c) a polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO:11; (d) a polypeptide encoded by a polynucleotide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:10; (e) a polypeptide derived from SEQ ID NO:11, the mature polypeptide of SEQ ID NO:11, or SEQ ID NO:12 and differing by 1 - 30 alterations (such as substitutions, deletions, and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, particularly substitutions); (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d), or (e) wherein the N - terminus and / or C - terminus has been extended by adding one or more amino acids; and (g) a fragment of the polypeptide of (a), (b), (c), (d), or (e); wherein the polypeptide has acetylxylan esterase activity; and (v) (a) A polypeptide having at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:14; (b) A polypeptide having at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:15; (c) A polypeptide having at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO:14; (d) A polypeptide encoded by a polynucleotide having at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:13; (e) A polypeptide derived from SEQ ID NO:14, the mature polypeptide of SEQ ID NO:14, or SEQ ID NO:15, which differs by having 1 to 30 alterations (such as substitutions, deletions, and / or insertions at one or more positions, such as 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, particularly substitutions); (f) A polypeptide derived from the polypeptide of (a), (b), (c), (d), or (e), wherein the N-terminus and / or C-terminus has been extended by adding one or more amino acids; and (g) A fragment of the polypeptide of (a), (b), (c), (d), or (e); wherein the polypeptide has acetylxylan esterase activity; and (vi) (a) A polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:17; (b) A polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:18; (c) A polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO:17; (d) A polypeptide encoded by a polynucleotide that has at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:16 or its cDNA sequence; (e) A polypeptide derived from SEQ ID NO:17, the mature polypeptide of SEQ ID NO:17, or SEQ ID NO:18 and differing by 1 - 30 alterations (such as substitutions, deletions, and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, particularly substitutions); (f) A polypeptide derived from the polypeptide of (a), (b), (c), (d), or (e) wherein the N - terminus and / or C - terminus has been extended by the addition of one or more amino acids; and (g) A fragment of the polypeptide of (a), (b), (c), (d), or (e); wherein the polypeptide has acetylxylan esterase activity; (vii) (a) A polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:20; (b) A polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:21; (c) a polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO:20; (d) a polypeptide encoded by a polynucleotide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide coding sequence of SEQ ID NO:19; (e) a polypeptide derived from SEQ ID NO:20, the mature polypeptide of SEQ ID NO:20, or SEQ ID NO:21 and differing by having 1 - 30 alterations (e.g., substitutions, deletions, and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, especially substitutions); (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d), or (e) wherein the N - terminus and / or C - terminus has been extended by adding one or more amino acids; and (g) a fragment of the polypeptide of (a), (b), (c), (d), or (e); wherein the polypeptide has acetylxylan esterase activity; (viii) (a) a polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:23; (b) a polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:24; (c) a polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO:23; (d) a polypeptide encoded by a polynucleotide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:22; (e) a polypeptide derived from SEQ ID NO:23, the mature polypeptide of SEQ ID NO:23, or SEQ ID NO:24 and differing by 1 - 30 alterations (e.g., substitutions, deletions, and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, particularly substitutions); (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d), or (e) wherein the N - terminus and / or C - terminus has been extended by the addition of one or more amino acids; and (g) a fragment of the polypeptide of (a), (b), (c), (d), or (e); wherein the polypeptide has acetylxylan esterase activity; (ix) (a) A polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 26; (b) A polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 27; (c) A polypeptide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 26; (d) A polypeptide encoded by a polynucleotide having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 25; (e) A polypeptide derived from SEQ ID NO:26, the mature polypeptide of SEQ ID NO:26, or SEQ ID NO:27, which differs by having 1 - 30 alterations (e.g., substitutions, deletions, and / or insertions at one or more positions, such as 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, particularly substitutions); (f) A polypeptide derived from the polypeptide of (a), (b), (c), (d), or (e), wherein the N - terminus and / or C - terminus has been extended by adding one or more amino acids; and (g) A fragment of the polypeptide of (a), (b), (c), (d), or (e); wherein the polypeptide has acetylxylan esterase activity; and (x) (a) A polypeptide having at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:29; (b) A polypeptide having at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:30; (c) A polypeptide having at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO:29; (d) A polypeptide encoded by a polynucleotide that has at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:28; (e) A polypeptide derived from SEQ ID NO:29, the mature polypeptide of SEQ ID NO:29, or SEQ ID NO:30 and differing by 1 - 30 alterations (e.g., substitutions, deletions, and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, particularly substitutions); (f) A polypeptide derived from the polypeptide of (a), (b), (c), (d), or (e) wherein the N - terminus and / or C - terminus has been extended by the addition of one or more amino acids; and (g) A fragment of the polypeptide of (a), (b), (c), (d), or (e); wherein the polypeptide has acetylxylan esterase activity.

2. The polypeptide according to claim 1, wherein the polypeptide comprises the following, consists essentially of the following, or consists of the following: SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:

29.

3. The polypeptide according to claim 1, wherein the polypeptide comprises, consists essentially of, or consists of the following: SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:

30.

4. The polypeptide according to claim 1, wherein the polypeptide comprises, consists essentially of, or consists of the following: The mature polypeptides of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:

29.

5. A particle, comprising: (a) A core comprising the polypeptide according to any one of claims 1 - 4, and optionally (b) A coating consisting of one or more layers surrounding the core.

6. A particle, comprising: (a) A core, and (b) A coating consisting of one or more layers surrounding the core, wherein the coating comprises the polypeptide as described in any one of claims 1-4.

7. A composition, the composition comprising the polypeptide as described in any one of claims 1-4 or the particles as described in claim 5 or 6.

8. A composition, the composition comprising a polypeptide having arabinofuranosidase activity against di-substituted arabinose, a polypeptide having arabinofuranosidase activity against mono-substituted arabinose, xylanase, β-xylosidase and a carbohydrate esterase family 3 (CE3) polypeptide having acetylxylan esterase activity, and optionally α-xylosidase.

9. The composition as described in claim 8, wherein the polypeptide having arabinofuranosidase activity against di-substituted arabinose is a GH43 arabinofuranosidase.

10. The composition as described in claim 8 or 9, wherein the polypeptide having arabinofuranosidase activity against mono-substituted arabinose is a GH51 arabinofuranosidase.

11. The composition as described in any one of claims 8-10, wherein the polypeptide having xylanase activity is a GH5 xylanase.

12. The composition as described in claim 11, wherein the GH5 xylanase is a GH5_21 xylanase.

13. The composition as described in claim 11, wherein the GH5 xylanase is a GH5_35 xylanase.

14. The composition as described in any one of claims 8-13, wherein the polypeptide having xylanase activity is a GH30_8 xylanase.

15. The composition as described in any one of claims 8-14, wherein the polypeptide having β-xylosidase activity is a GH3 β-xylosidase.

16. The composition as described in any one of claims 8-15, the composition comprising the α-xylosidase.

17. The composition as described in any one of claims 8-16, wherein the α-xylosidase is a GH31 α-xylosidase.

18. The composition as described in any one of claims 7-17, wherein the CE3 polypeptide having acetylxylan esterase activity is the polypeptide as described in any one of claims 1-4.

19. A method for producing a fermentation product from a starch-containing material, the method comprising the following steps: (a) saccharifying the starch-containing material with glucoamylase and α-amylase at a temperature below the initial gelatinization temperature of the starch to produce fermentable sugars; (b) fermenting the sugars with a fermenting organism; wherein a CE3 polypeptide having acetylxylan esterase activity or a composition comprising a CE3 polypeptide having acetylxylan esterase activity is present or added during the saccharification step (a) and / or the fermentation step (b).

20. The method as described in claim 19, wherein the CE3 polypeptide is the CE3 polypeptide as described in any one of claims 1-4, or is formulated into particles as described in claims 5-6.

21. The method as described in claim 19 or 20, wherein the composition comprising the CE3 polypeptide is the composition as described in any one of claims 7-18.

22. 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 glucoamylase to produce fermentable sugars; (c) fermenting the sugars with a fermenting organism to produce the fermentation product; wherein a CE3 polypeptide having acetylxylan esterase activity or a composition comprising the CE3 polypeptide having acetylxylan esterase activity is present or added during the saccharification step (b) and / or the fermentation step (c).

23. The method according to claim 22, wherein the CE3 polypeptide is the CE3 polypeptide according to any one of claims 1-4, or is formulated into a particle according to claims 5-6.

24. The method according to claim 22 or 23, wherein the composition comprising the CE3 polypeptide is the composition according to any one of claims 7-18.

25. A polynucleotide encoding a polypeptide according to any one of claims 1-4.

26. The polynucleotide according to claim 25, the polynucleotide comprising: (i) SEQ ID NO:1 or nucleotides 57 to 891 of SEQ ID NO:1; (ii) SEQ ID NO:4 or nucleotides 72 to 735 of SEQ ID NO:4; (iii) SEQ ID NO:7 or nucleotides 60 to 867 of SEQ ID NO:7; (iv) SEQ ID NO:10 or nucleotides 69 to 759 of SEQ ID NO:10; (v) SEQ ID NO:13 or nucleotides 57 to 720 of SEQ ID NO:13; (vi) SEQ ID NO:16 or nucleotides 131 to 712 and 798 to 882 of SEQ ID NO:16 or its cDNA; (vii) SEQ ID NO:19 or nucleotides 75 to 741 of SEQ ID NO:19; (viii) SEQ ID NO:22 or nucleotides 75 to 732 of SEQ ID NO:22; (ix) SEQ ID NO:25 or nucleotides 60 to 720 of SEQ ID NO:25; or (x) SEQ ID NO:28 or nucleotides 51 to 858 of SEQ ID NO:

28.

27. A nucleic acid construct or expression vector comprising the polynucleotide according to claim 25 or 26, the polynucleotide being operably linked to one or more control sequences directing the production of the polypeptide in an expression host.

28. A recombinant host cell comprising the nucleic acid construct or expression vector according to claim 27.

29. A method for producing a polypeptide having acetylxylan esterase activity, the method comprising culturing the recombinant host cell according to claim 28 under conditions conducive to the production of the polypeptide.

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