Carbohydrate esterase family 1 (CE1) polypeptides having feruloyl esterase and / or acetylxylan esterase activity and polynucleotides encoding same

Abstract

The present invention relates to carbohydrate esterase family 1 (CE1) polypeptides having feruloyl esterase and / or 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 CE1 family polypeptides having feruloyl esterase and / or acetylxylan esterase activity and to the use of these compositions for solubilizing hemicellulosic fibers.

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 1 (CE1) polypeptides having ferulic acid esterase and / or 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 CE1 family polypeptides 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. Motivated by government sustainable development initiatives, the biofuel industry is using corn fiber to produce ethanol at existing corn ethanol facilities. Corn fiber accounts for 10% of the weight of corn kernels and consists of cellulose and hemicellulose from the aleurone and pericarp layers. 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), while these C5 sugars are fermented into ethanol by C5-fermenting yeast, 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 the protein-rich DDGS feed, 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 the activities of endo-1,4-β-xylanase (EC 3.2.1.8) and β-xylosidase (EC 3.2.1.37) (BX).

[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 for the easy separation of the linear xylose polymers from arabinose by known techniques. The 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, it may be desirable to have 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).

[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 CE1 family polypeptides having ferulic acid esterase and / or acetylxylan esterase activity and polynucleotides encoding these polypeptides. The CE1 family polypeptides of the present invention release more monomeric arabinose and / or xylose when used in combination with polypeptides having arabinofuranosidase activity towards di-substituted 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 α-glucuronidase, and 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 Microsphaeropsis arundinis CE1 family polypeptide having ferulic acid esterase and / or acetylxylan esterase activity of the present invention.

[0011] SEQ ID NO:2 is the full-length amino acid sequence of the wild-type Microsphaeropsis arundinis CE1 family polypeptide having ferulic acid esterase and / or acetylxylan esterase activity of the present invention.

[0012] SEQ ID NO:3 is the mature polypeptide of the wild-type Microsphaeropsis arundinis CE1 family polypeptide having ferulic acid esterase and / or acetylxylan esterase activity of the present invention.

[0013] SEQ ID NO:4 is the nucleotide sequence encoding the wild-type Microsphaeropsis arundinis CE1 family polypeptide having ferulic acid esterase and / or acetylxylan esterase activity of the present invention.

[0014] SEQ ID NO:5 is the full-length amino acid sequence of the wild-type Microsphaeropsis arundinis CE1 family polypeptide having ferulic acid esterase and / or acetylxylan esterase activity of the present invention.

[0015] SEQ ID NO:6 is the mature polypeptide of the wild-type Microsphaeropsis arundinis CE1 family polypeptide having ferulic acid esterase and / or acetylxylan esterase activity of the present invention.

[0016] SEQ ID NO:7 is the nucleotide sequence encoding the wild-type Microsphaeropsis arundinis CE1 family polypeptide having ferulic acid esterase and / or acetylxylan esterase activity of the present invention.

[0017] SEQ ID NO:8 is the full-length amino acid sequence of the wild-type Microsphaeropsis arundinis CE1 family polypeptide having ferulic acid esterase and / or acetylxylan esterase activity of the present invention.

[0018] SEQ ID NO:9 is the mature polypeptide of the wild-type Sphaeropsis sapinea CE1 family polypeptide with ferulic acid esterase and / or acetylxylan esterase activity of the present invention.

[0019] SEQ ID NO:10 is the nucleotide sequence encoding the wild-type Sphaeropsis sapinea CE1 family polypeptide with ferulic acid esterase and / or acetylxylan esterase activity of the present invention.

[0020] SEQ ID NO:11 is the full-length amino acid sequence of the wild-type Sphaeropsis sapinea CE1 family polypeptide with ferulic acid esterase and / or acetylxylan esterase activity of the present invention.

[0021] SEQ ID NO:12 is the mature polypeptide of the wild-type Sphaeropsis sapinea CE1 family polypeptide with ferulic acid esterase and / or acetylxylan esterase activity of the present invention.

[0022] SEQ ID NO:13 is the nucleotide sequence encoding the wild-type Sphaeropsis sapinea CE1 family polypeptide with ferulic acid esterase activity and / or acetylxylan esterase of the present invention.

[0023] SEQ ID NO:14 is the full-length amino acid sequence of the wild-type Sphaeropsis sapinea CE1 family polypeptide with ferulic acid esterase and / or acetylxylan esterase activity of the present invention.

[0024] SEQ ID NO:15 is the mature polypeptide of the wild-type Sphaeropsis sapinea CE1 family polypeptide with ferulic acid esterase and / or acetylxylan esterase activity of the present invention.

[0025] Accordingly, the present invention relates to a polypeptide having ferulic acid esterase and / or acetylxylan esterase activity, which polypeptide is selected from the group consisting of:

[0026] (i)

[0027] (a) a polypeptide having 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:2;

[0028] (b) a polypeptide having 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:3;

[0029] (c) a polypeptide having 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:2;

[0030] (d) a polypeptide encoded by a polynucleotide having 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:1;

[0031] (e) a polypeptide derived from SEQ ID NO:2, the mature polypeptide of SEQ ID NO:2, or SEQ ID NO:3 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);

[0032] (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

[0033] (g) a fragment of the polypeptide of (a), (b), (c), (d), or (e);

[0034] wherein the polypeptide has feruloyl esterase and / or acetylxylan esterase activity;

[0035] (ii)

[0036] (a) a polypeptide having at least 70% identity, at least 71% identity, at least 72% identity, at least 73% identity, at least 74% identity, at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity with SEQ ID NO:5;

[0037] (b) a polypeptide having at least 70% identity, at least 71% identity, at least 72% identity, at least 73% identity, at least 74% identity, at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity with SEQ ID NO:6;

[0038] (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;

[0039] (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;

[0040] (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 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);

[0041] (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

[0042] (g) a fragment of the polypeptide of (a), (b), (c), (d), or (e);

[0043] wherein the polypeptide has feruloyl esterase and / or acetylxylan esterase activity;

[0044] (iii)

[0045] (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:8;

[0046] (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:9;

[0047] (c) a polypeptide having at least 70% identity, at least 71% identity, at least 72% identity, at least 73% identity, at least 74% identity, at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO:8;

[0048] (d) a polypeptide encoded by a polynucleotide having at least 70% identity, at least 71% identity, at least 72% identity, at least 73% identity, at least 74% identity, at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity with the mature polypeptide coding sequence of SEQ ID NO:7;

[0049] (e) a polypeptide derived from SEQ ID NO:8, the mature polypeptide of SEQ ID NO:8, or SEQ ID NO:9, 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);

[0050] (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

[0051] (g) a fragment of the polypeptide of (a), (b), (c), (d), or (e);

[0052] wherein the polypeptide has feruloyl esterase and / or acetylxylan esterase activity;

[0053] (iv)

[0054] (a) a polypeptide having at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity with SEQ ID NO:11;

[0055] (b) a polypeptide having at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity with SEQ ID NO:12;

[0056] (c) a polypeptide having at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO:11;

[0057] (d) A polypeptide encoded by a polynucleotide that has at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity with the mature polypeptide coding sequence of SEQ ID NO:10;

[0058] (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 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);

[0059] (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

[0060] (g) A fragment of the polypeptide of (a), (b), (c), (d), or (e);

[0061] wherein the polypeptide has ferulic acid esterase and / or acetylxylan esterase activity; and

[0062] (v)

[0063] A polypeptide having at least 70% identity, at least 71% identity, at least 72% identity, at least 73% identity, at least 74% identity, at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity to SEQ ID NO:14;

[0064] A polypeptide having at least 70% identity, at least 71% identity, at least 72% identity, at least 73% identity, at least 74% identity, at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity to SEQ ID NO:15;

[0065] A polypeptide having at least 70% identity, at least 71% identity, at least 72% identity, at least 73% identity, at least 74% identity, at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO:14;

[0066] (d) A polypeptide encoded by a polynucleotide that has at least 70% identity, at least 71% identity, at least 72% identity, at least 73% identity, at least 74% identity, at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity with the mature polypeptide coding sequence of SEQ ID NO:13;

[0067] (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, 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);

[0068] (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

[0069] (g) A fragment of the polypeptide of (a), (b), (c), (d), or (e);

[0070] wherein the polypeptide has ferulic acid esterase and / or acetylxylan esterase activity.

[0071] 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.

[0072] The present invention also relates to compositions comprising CE1 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

[0073] The accompanying drawings are alignments of exemplary CE1 polypeptides of the present invention, showing that they share conserved active site serine, histidine, and aspartic acid residues that form the catalytic triad responsible for the classical serine hydrolase mechanism, as well as a conserved pentapeptide with the consensus sequence G-X-S-X-G.

[0074] Definitions

[0075] 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.

[0076] All technical and scientific terms used herein, unless otherwise defined or clearly indicated by the context, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0077] 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.

[0078] 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 was dissolved in DMSO as a substrate stock solution. The stock solution was 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 were mixed in a 96-well plate and incubated at 37°C. The released p-nitrophenol was monitored at 410 nm by a spectrophotometer.

[0079] α-L-Arabinofuranosidase: "α-L-Arabinofuranosidase" means 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. The enzyme acts on α-L-arabinofuranosides, α-L-arabans containing (1,3)- and / or (1,5)-bonds, arabinoxylans, and arabinogalactans. 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-arabinanase.

[0080] α-L-arabinofuranosidase activity: For the purposes of the present invention, α-L-arabinofuranosidase activity was determined using 5 mg of medium-viscosity wheat arabinoxylan (Megazyme International Ireland, Ltd., Bray, Co., Wicklow, Ireland) in 100 mM sodium acetate (pH 5) per ml in a total volume of 200 μl at 40 °C for 30 minutes, followed by arabinose analysis by AMINEX® HPX-87H column chromatography (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

[0081] α-xylosidase: "α-xylosidase" means an α-D-xyloside xylohydrolase (EC 3.2.1.177) that catalyzes the hydrolysis of terminal unsubstituted xylosides at the non-reducing ends of xylooligosaccharides.

[0082] 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-nitrophenol anion per minute from 1 mM p-nitrophenyl-α-D-xyloside as a substrate in 100 mM sodium citrate containing 0.01% TWEEN® 20 at 40 °C, pH 5, in a total volume of 200 μl.

[0083] β-xylosidase: "β-xylosidase" means a β-D-xyloside xylohydrolase (E.C. 3.2.1.37) that catalyzes the exohydrolysis of short β(1-4)-xylooligosaccharides to remove successive D-xylose residues from the non-reducing ends.

[0084] 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-nitrophenol anion per minute from 1 mM p-nitrophenyl-β-D-xyloside as a substrate in 100 mM sodium citrate containing 0.01% TWEEN® 20 at 40 °C, pH 5, in a total volume of 200 μl.

[0085] Carbohydrate Esterase Family 1 (CE1): Carbohydrate Esterase Family 1 is abbreviated as "CE1" herein and encompasses acetylxylan esterase (EC 3.1.1.72), cinnamoyl esterase (EC 3.1.1.-), feruloyl esterase (EC 3.1.1.73), S-formyl hydrolase (EC 3.1.2.12), diacylglycerol O-acyltransferase (EC 2.3.1.20); and trehalose 6-O-mycolytransferase (EC 2.3.1.122). The CE1 polypeptides of the present invention mainly have feruloyl esterase activity (EC 3.1.1.73) (referred to as "ferulic acid esterase activity" herein) and / or acetylxylan esterase activity (EC 3.1.1.72).

[0086] Feruloyl esterase: The term "feruloyl esterase" means 4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73), which catalyzes the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyl) group from an esterified sugar (which is usually arabinose in the "natural" substrate).

[0087] Feruloyl esterase activity: One unit of feruloyl esterase activity is equal to the amount of enzyme capable of releasing 1 micromole of p-nitrophenol per minute at pH 7 and 37 °C. 25 mM p-nitrophenyl ferulate is dissolved in DMSO and diluted to 1 mM with 100 mM PBS (pH 7) containing 1% Tween-80 as the substrate solution. 10 μl of the enzyme solution and 990 μl of the substrate solution are incubated on a thermostatic mixer at 37 °C and 600 rpm for 10 minutes. The released p-nitrophenol is monitored at 410 nm by a spectrophotometer.

[0088] 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 the 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.

[0089] 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 usually determined by an open reading frame that starts 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.

[0090] Control sequence: The term "control sequence" means a nucleic acid sequence involved in regulating the expression of a polynucleotide in a particular organism, either in vivo or in vitro. Each control sequence can be native (i.e., from the same gene) or heterologous (i.e., from a different gene) to the polynucleotide encoding the polypeptide, and can be 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.

[0091] 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.

[0092] Expression vector: An "expression vector" is a linear or circular DNA construct that contains a DNA sequence encoding a polypeptide, the coding sequence being operably linked to appropriate control sequences capable of effecting 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.

[0093] Extension: The term "extension" means the addition of one or more amino acids to the amino and / or carboxyl terminus of a polypeptide, wherein the "extended" polypeptide has feruloyl esterase and / or acetylxylan esterase activity.

[0094] Fermentation product: A "fermentation product" means a product produced by a process that includes fermentation using a fermenting organism. Fermentation products include alcohols (e.g., ethanol, methanol, butanol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, succinic acid, gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H2 and CO2); antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B 12, β-carotene); and hormones. In a preferred embodiment, the fermentation product is ethanol, e.g., fuel ethanol; drinking ethanol, i.e., potable neutral alcoholic beverages; or industrial ethanol or products 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.

[0095] Fermenting organism: "Fermenting organism" means any organism suitable for use in a fermentation process and capable of producing a desired fermentation product, including bacterial and fungal organisms, especially yeast.

[0096] Fragment: The term "fragment" means a polypeptide having a deletion of one or more amino acids at the amino and / or carboxyl terminus of the mature polypeptide, wherein the fragment has ferulic acid esterase and / or acetylxylan esterase activity.

[0097] 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. When the fusion protein is secreted, this site is cleaved, thereby releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3:568-576; Svetina et al., 2000, J. Biotechnol. 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.

[0098] 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) and catalyzes the hydrolysis of (1→4)-β-D-xylan to remove successive D-xylose residues from the non-reducing terminus.

[0099] 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 bonds in xylan.

[0100] 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.

[0101] 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.

[0102] 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 bonds in xylan.

[0103] 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 bonds 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 bonds in xylan.

[0104] 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 bonds in xylan.

[0105] 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. Endohydrolysis of (1→4)-β-D-xylosyl linkages in some glucuronyl arabinoxylans.

[0106] GH31 α-Xylosidase: "GH31 arabinofuranosidase" is an abbreviation for glycoside hydrolase family 31 α-xylosidase, which is an α-D-xylosyl xylohydrolase (EC 3.2.1.177) that catalyzes the hydrolysis of the terminal unsubstituted xyloside at the reducing end of xylooligosaccharides. Exemplary GH31 family α-xylosidases utilize a two-step double-displacement mechanism involving a covalent glycosyl-enzyme intermediate and produce products with anomeric configuration.

[0107] 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 the terminal non-reducing α-L-arabinofuranosyl residue in α-L-arabinofuranosides.

[0108] 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 the terminal non-reducing α-L-arabinofuranosyl residue in α-L-arabinofuranosides.

[0109] 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 readily determined by one skilled in the art. Thus, the initial gelatinization temperature can vary depending on the plant species, specific variety of the plant species, and 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).

[0110] 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.

[0111] 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.

[0112] 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.

[0113] 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 not found in nature. Isolated polypeptides include, but are not limited to, culture broths containing secreted polypeptides expressed in a host cell.

[0114] 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 20 to 295 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 25 to 282 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 21 to 310 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 19 to 302 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 20 to 286 of SEQ ID NO:14. In one aspect, the mature polypeptide is SEQ ID NO:15.

[0115] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" means a polynucleotide encoding a mature polypeptide having feruloyl esterase and / or acetylxylan esterase activity. In one aspect, the mature polypeptide coding sequence is nucleotides 60 to 885 of SEQ ID NO:1. In one aspect, the mature polypeptide coding sequence is nucleotides 75 to 846 of SEQ ID NO:4. In one aspect, the mature polypeptide coding sequence is nucleotides 63 to 930 of SEQ ID NO:7. In one aspect, the mature polypeptide coding sequence is nucleotides 57 to 906 of SEQ ID NO:10. In one aspect, the mature polypeptide coding sequence is nucleotides 60 to 858 of SEQ ID NO:13.

[0116] Native: The term "native" means a nucleic acid or polypeptide that occurs naturally in a host cell.

[0117] 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 indicated, nucleic acid sequences are presented in the 5' to 3' orientation.

[0118] 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 way that does not exist in nature to contain segments of nucleic acid or synthetic and containing one or more control sequences operably linked to a nucleic acid sequence.

[0119] 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.

[0120] 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 or molar percentage). In a related sense, a composition is enriched in a molecule when the concentration of the molecule is substantially increased 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 that in the starting composition.

[0121] 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.

[0122] 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 present in the cells from which the polypeptide is derived, especially other proteins and most especially other enzymes. 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 that is native or recombinantly associated with the polypeptide.

[0123] 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 present invention are preferably in substantially pure form (i.e., the formulation is substantially free of other polypeptide materials associated therewith natively or recombinantly). For example, this can be achieved by preparing the polypeptide using well-known recombinant methods or using classical purification methods.

[0124] 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".

[0125] 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 a polypeptide from a 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 filter aids or packed filter media, cloth filtration in a cassette filter, drum filtration, rotary 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 of 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 separation. Recover encompasses the separation and / or purification of the polypeptide.

[0126] 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-simplified (-nobrief) option must be specified on the command line. The output of "longest identity" marked by Needle is calculated as follows:

[0127] (Identical residues × 100) / (Alignment length - Total number of gaps in the alignment)

[0128] 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-simplified option must be specified on the command line. The output of "longest identity" marked by Needle is calculated as follows:

[0129] (Identical deoxyribonucleotides × 100) / (Alignment length – Total number of gaps in the alignment)

[0130] 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.

[0131] 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 ferulic acid esterase and / or acetylxylan esterase activity.

[0132] 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.

[0133] Distillers' spent wash: "Distillers' spent wash" refers to the centrifuged filtrate separated from whole distillers' grains, which is pumped to an evaporator to be concentrated into a slurry.

[0134] Variant: The term "variant" means a polypeptide having ferulic acid esterase and / or 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.

[0135] 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).

[0136] 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 (e.g., a protein, an amino acid or a nucleic acid sequence). In contrast, the term "non-naturally occurring" refers to any substance not found in nature (e.g., a recombinant nucleic acid and protein sequence produced in a laboratory, or a modification of a wild type sequence).

[0137] Xylanase: "Xylanase" encompasses: endo-1,4-β-xylanase (EC 3.2.1.8), which catalyzes the endohydrolysis of (1→4)-β-D-xylosidic linkages 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 linkages in some glucuronoxylans.

[0138] 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 produces 1.0 micromole of reducing sugar per minute per liter of 2 g birchwood xylan as a substrate in 50 mM sodium acetate containing 0.01% 2 at 50 °C and pH 5 (measured as glucose equivalents as described in Lever, 1972, A new reaction for colorimetric determination of carbohydrates, Anal. Biochem. 47:273 - 279). The activity of EC 3.2.1.136 xylanase can be determined at 37 °C in 0.01% X - 100 and 200 mM sodium phosphate (pH 6) using 0.2% AZCL - glucuronoxylan as a substrate. One unit of xylanase activity is defined as the amount of enzyme that produces 1.0 micromole of azurine per minute from 0.2% AZCL - glucuronoxylan as a substrate in 200 mM sodium phosphate (pH 6) at 37 °C and pH 6. Detailed Description

[0139] Carbohydrate Esterase Family 1 (CE1) Polypeptides with Ferulic Acid Esterase and / or Acetylxylan Esterase Activity

[0140] The present invention relates to Carbohydrate Esterase Family 1 (CE1) polypeptides having ferulic acid esterase and / or acetylxylan esterase activity. In one aspect, the present invention relates to polypeptides having ferulic acid esterase and / or acetylxylan esterase activity, which polypeptides are selected from the group consisting of:

[0141] (a) polypeptides having 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;

[0142] (b) polypeptides having 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;

[0143] (c) a polypeptide having 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:2;

[0144] (d) a polypeptide encoded by a polynucleotide that has 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:1;

[0145] (e) a polypeptide derived from SEQ ID NO:2, the mature polypeptide of SEQ ID NO:2, or SEQ ID NO:3 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);

[0146] (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

[0147] (g) a fragment of a polypeptide of (a), (b), (c), (d), or (e);

[0148] wherein the polypeptide has ferulic acid esterase and / or acetylxylan esterase activity.

[0149] In one aspect, the polypeptide has 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:2 or the mature polypeptide of SEQ ID NO:2.

[0150] In another aspect, the polypeptide has 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.

[0151] The polypeptide preferably comprises, consists essentially of, or consists of: the amino acid sequence of SEQ ID NO:2 or its mature polypeptide.

[0152] The polypeptide preferably comprises, consists essentially of, or consists of: amino acids 20 to 295 of SEQ ID NO:2.

[0153] The polypeptide preferably comprises, consists essentially of, or consists of: the amino acid sequence of SEQ ID NO:3.

[0154] The polypeptide may have an N-terminal and / or C-terminal extension of one or more amino acids (e.g., 1-5 amino acids).

[0155] In another aspect, the polypeptide is a fragment that contains at least 235 amino acid residues (e.g., amino acids 1 to 235 of SEQ ID NO:3), at least 248 amino acid residues (e.g., amino acids 1 to 248 of SEQ ID NO:3), or at least 262 amino acid residues (e.g., amino acids 1 to 262 of SEQ ID NO:3).

[0156] In some embodiments, the polypeptide is encoded by a polynucleotide having 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.

[0157] The polynucleotide encoding the polypeptide preferably comprises, consists essentially of, or consists of: nucleotides 60 to 885 of SEQ ID NO:1.

[0158] 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 minor properties, 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.

[0159] In one aspect, the present invention relates to a polypeptide having feruloyl esterase and / or acetylxylan esterase activity, which polypeptide is selected from the group consisting of:

[0160] (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;

[0161] (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;

[0162] (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;

[0163] (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;

[0164] (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, especially substitutions);

[0165] (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

[0166] (g) a fragment of the polypeptide of (a), (b), (c), (d), or (e);

[0167] wherein the polypeptide has feruloyl esterase activity and / or acetylxylan esterase activity.

[0168] In one aspect, the polypeptide has 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 SEQ ID NO:5 or the mature polypeptide of SEQ ID NO:5.

[0169] In another aspect, the polypeptide has 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 SEQ ID NO:6.

[0170] The polypeptide preferably comprises, consists essentially of, or consists of: the amino acid sequence of SEQ ID NO:5 or its mature polypeptide.

[0171] The polypeptide preferably comprises, consists essentially of, or consists of: amino acids 25 to 282 of SEQ ID NO:5.

[0172] The polypeptide preferably comprises, consists essentially of, or consists of: the amino acid sequence of SEQ ID NO:6.

[0173] The polypeptide may have an N-terminal and / or C-terminal extension of one or more amino acids (e.g., 1-5 amino acids).

[0174] In another aspect, the polypeptide is a fragment that contains at least 219 amino acid residues (e.g., amino acids 1 to 219 of SEQ ID NO:6), at least 232 amino acid residues (e.g., amino acids 1 to 232 of SEQ ID NO:6), or at least 245 amino acid residues (e.g., amino acids 1 to 245 of SEQ ID NO:6).

[0175] In some embodiments, the polypeptide is encoded by a polynucleotide having 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.

[0176] The polynucleotide encoding the polypeptide preferably comprises, consists essentially of, or consists of nucleotides 75 to 846 of SEQ ID NO:4.

[0177] 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 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.

[0178] In one aspect, the present invention relates to a polypeptide having feruloyl esterase and / or acetylxylan esterase activity, which polypeptide is selected from the group consisting of:

[0179] (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:8;

[0180] (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:9;

[0181] (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:8;

[0182] (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:7;

[0183] (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 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);

[0184] (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

[0185] (g) A fragment of a polypeptide of (a), (b), (c), (d), or (e);

[0186] wherein the polypeptide has feruloyl esterase activity and / or acetylxylan esterase activity.

[0187] In one aspect, the polypeptide has 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 SEQ ID NO:8 or the mature polypeptide of SEQ ID NO:8.

[0188] In another aspect, the polypeptide has 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 SEQ ID NO:9.

[0189] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:8 or its mature polypeptide.

[0190] The polypeptide preferably comprises, consists essentially of, or consists of amino acids 21 to 310 of SEQ ID NO:8.

[0191] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:9.

[0192] The polypeptide may have an N-terminal and / or C-terminal extension of one or more amino acids (e.g., 1-5 amino acids).

[0193] In another aspect, the polypeptide is a fragment that contains at least 247 amino acid residues (e.g., amino acids 1 to 247 of SEQ ID NO:9), at least 261 amino acid residues (e.g., amino acids 1 to 261 of SEQ ID NO:9), or at least 276 amino acid residues (e.g., amino acids 1 to 276 of SEQ ID NO:9).

[0194] In some embodiments, the polypeptide is encoded by a polynucleotide having 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.

[0195] The polynucleotide encoding the polypeptide preferably comprises, consists essentially of, or consists of: nucleotides 63 to 930 of SEQ ID NO:7.

[0196] 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, 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).

[0197] In one aspect, the present invention relates to polypeptides having ferulic acid esterase activity and / or acetylxylan esterase activity, the polypeptides being selected from the group consisting of:

[0198] (a) a polypeptide having 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;

[0199] (b) a polypeptide having 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;

[0200] (c) a polypeptide having 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;

[0201] (d) a polypeptide encoded by a polynucleotide having 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;

[0202] (e) A polypeptide derived from SEQ ID NO:11, the mature polypeptide of SEQ ID NO:11, or SEQ ID NO:12, 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);

[0203] (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

[0204] (g) A fragment of a polypeptide of (a), (b), (c), (d), or (e);

[0205] wherein the polypeptide has feruloyl esterase activity and / or acetylxylan esterase activity.

[0206] In one aspect, the polypeptide has 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.

[0207] In another aspect, the polypeptide has 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.

[0208] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:11 or its mature polypeptide.

[0209] The polypeptide preferably comprises, consists essentially of, or consists of amino acids 19 to 302 of SEQ ID NO:11 or its mature polypeptide.

[0210] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:12.

[0211] The polypeptide may have an N-terminal and / or C-terminal extension of one or more amino acids (e.g., 1-5 amino acids).

[0212] In another aspect, the polypeptide is a fragment that contains at least 234 amino acid residues (e.g., amino acids 1 to 234 of SEQ ID NO:12), at least 255 amino acid residues (e.g., amino acids 1 to 255 of SEQ ID NO:12), or at least 270 amino acid residues (e.g., amino acids 1 to 270 of SEQ ID NO:12).

[0213] In some embodiments, the polypeptide is encoded by a polynucleotide having 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 the mature polypeptide coding sequence of SEQ ID NO:10.

[0214] The polynucleotide encoding the polypeptide preferably comprises, consists essentially of, or consists of nucleotides 57 to 906 of SEQ ID NO:10.

[0215] 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, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. The amino acid changes can have minor properties, that is, 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 the 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).

[0216] In one aspect, the present invention relates to a polypeptide having feruloyl esterase and / or acetylxylan esterase activity, which polypeptide is selected from the group consisting of:

[0217] (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:14;

[0218] (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:15;

[0219] (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:14;

[0220] (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:13;

[0221] (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;

[0222] (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

[0223] (g) a fragment of the polypeptide of (a), (b), (c), (d), or (e);

[0224] wherein the polypeptide has ferulic acid esterase and / or acetylxylan esterase activity.

[0225] 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%, or at least 99%, or 100% sequence identity with SEQ ID NO:14 or the mature polypeptide of SEQ ID NO:14.

[0226] 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%, or at least 99%, or 100% sequence identity with SEQ ID NO:15.

[0227] The polypeptide preferably comprises, consists essentially of, or consists of: the amino acid sequence of SEQ ID NO:14 or its mature polypeptide.

[0228] The polypeptide preferably comprises, consists essentially of, or consists of: amino acids 20 to 286 of SEQ ID NO:14.

[0229] The polypeptide preferably comprises, consists essentially of, or consists of: the amino acid sequence of SEQ ID NO:15.

[0230] The polypeptide may have an N-terminal and / or C-terminal extension of one or more amino acids (e.g., 1 - 5 amino acids).

[0231] In another aspect, the polypeptide is a fragment that contains at least 227 amino acid residues (e.g., amino acids 1 to 227 of SEQ ID NO:15), at least 240 amino acid residues (e.g., amino acids 1 to 240 of SEQ ID NO:15), or at least 254 amino acid residues (e.g., amino acids 1 to 254 of SEQ ID NO:15).

[0232] In some embodiments, the polypeptide is 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%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:13.

[0233] The polynucleotide encoding the polypeptide preferably comprises, consists essentially of, or consists of nucleotides 60 to 858 of SEQ ID NO:13.

[0234] In another aspect, the polypeptide is derived from SEQ ID NO:14 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:14 by substitution, deletion, or addition of one or several 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, 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 epitope, or a binding module.

[0235] 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 feruloyl esterase and / or acetylxylan esterase activities of the resulting molecule are 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.

[0236] Using known mutagenesis, recombination, and / or shuffling methods, followed by relevant screening procedures, single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested, such as those 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).

[0237] Mutagenesis / shuffling methods can be combined with high-throughput, automated screening methods to detect the activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). Mutagenized DNA molecules encoding active polypeptides can be recovered from 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.

[0238] Carbohydrate esterase family 1 (CE1) polypeptides act on a variety of substrates using a classical serine hydrolase mechanism that involves a catalytic triad comprising a nucleophilic serine, histidine, and an acidic amino acid (such as aspartic acid and glutamic acid). The catalytic serine is located at the center of a conserved pentapeptide with the consensus sequence G-X-S-X-G. The pentapeptide segment forms a "nucleophilic elbow," which is a fingerprint for identifying CE1 family polypeptides based on the primary structure of the polypeptide. Histidine is conserved, while the acidic residue of the catalytic triad can be aspartic acid or glutamic acid. The positions of these features are shown in Table 1 below, and Figure 1 an alignment showing the conservation of these features in the mature sequences of exemplary CE1 polypeptides of the present invention is shown in.

[0239] Table 1

[0240]

[0241]

[0242] The polypeptide can be a fusion polypeptide.

[0243] In one aspect, the polypeptide is isolated.

[0244] In another aspect, the polypeptide is purified.

[0245] Source of polypeptides having feruloyl esterase and / or acetylxylan esterase activity

[0246] The polypeptides having feruloyl esterase and / or acetylxylan esterase activity of the present invention 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.

[0247] In one aspect, the polypeptide is obtained from Microsphaeropsis. In another aspect, the polypeptide is a polypeptide obtained from Microsphaeropsis amaranthi. In another aspect, the polypeptide is a polypeptide obtained from Sphaeropsis. In another aspect, the polypeptide is a polypeptide obtained from Microsphaeropsis fusca. In another aspect, the polypeptide is a polypeptide obtained from Microsphaeropsis hellebori. In another aspect, the polypeptide is a polypeptide obtained from Microsphaeropsis olivacea. In another aspect, the polypeptide is a polypeptide obtained from Microsphaeropsis ononidicola. In another aspect, the polypeptide is a polypeptide obtained from Microsphaeropsis proteae. In another aspect, the polypeptide is a polypeptide obtained from Microsphaeropsis spartii-juncei.

[0248] 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 appropriate equivalents.

[0249] The polypeptides can be identified and obtained using the above-mentioned probes from other sources, including microorganisms isolated from natural sources (e.g., soil, compost, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, compost, water, etc.). Techniques for directly isolating microorganisms and DNA from natural habitats are well known in the art. The polynucleotides encoding the polypeptides can then be obtained by similarly screening the genomic DNA or cDNA library of another microorganism or a mixed DNA sample. Once the polynucleotides encoding the polypeptides have been detected with one or more probes, the polynucleotides 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).

[0250] polynucleotide

[0251] The invention also relates to polynucleotides encoding the polypeptides of the invention, as described herein.

[0252] The polynucleotides can be genomic DNA, cDNA, synthetic DNA, synthetic RNA, mRNA, or combinations thereof. The polynucleotides can be cloned from strains of the genus Chlorella or related organisms and, thus, can be, for example, polynucleotide sequences encoding variants of the polypeptides of the invention.

[0253] In one embodiment, the polynucleotide is a subsequence encoding a fragment of the invention having feruloyl esterase and / or acetylxylan esterase activity. In one aspect, the subsequence contains at least 705 nucleotides (e.g., nucleotides 60 to 765 of SEQ ID NO:1), at least 744 nucleotides (e.g., nucleotides 60 to 804 of SEQ ID NO:1), or at least 786 nucleotides (e.g., nucleotides 60 to 846 of SEQ ID NO:1).

[0254] In one embodiment, the polynucleotide is a subsequence encoding a fragment of the invention having feruloyl esterase and / or acetylxylan esterase activity. In one aspect, the subsequence contains at least 657 nucleotides (e.g., nucleotides 75 to 732 of SEQ ID NO:4), at least 696 nucleotides (e.g., nucleotides 75 to 771 of SEQ ID NO:4), or at least 735 nucleotides (e.g., nucleotides 75 to 810 of SEQ ID NO:4).

[0255] In one embodiment, the polynucleotide is a subsequence encoding a fragment of the present invention having ferulic acid esterase and / or acetylxylan esterase activity. In one aspect, the subsequence contains at least 741 nucleotides (e.g., nucleotides 63 to 804 of SEQ ID NO:7), at least 783 nucleotides (e.g., nucleotides 63 to 846 of SEQ ID NO:7), or at least 828 nucleotides (e.g., nucleotides 63 to 891 of SEQ ID NO:7).

[0256] In one embodiment, the polynucleotide is a subsequence encoding a fragment of the present invention having ferulic acid esterase and / or acetylxylan esterase activity. In one aspect, the subsequence contains at least 702 nucleotides (e.g., nucleotides 57 to 759 of SEQ ID NO:10), at least 765 nucleotides (e.g., nucleotides 57 to 822 of SEQ ID NO:10), or at least 810 nucleotides (e.g., nucleotides 57 to 867 of SEQ ID NO:10).

[0257] In one embodiment, the polynucleotide is a subsequence encoding a fragment of the present invention having ferulic acid esterase and / or acetylxylan esterase activity. In one aspect, the subsequence contains at least 681 nucleotides (e.g., nucleotides 60 to 741 of SEQ ID NO:13), at least 720 nucleotides (e.g., nucleotides 60 to 780 of SEQ ID NO:13), or at least 762 nucleotides (e.g., nucleotides 60 to 822 of SEQ ID NO:13).

[0258] In one embodiment, the polynucleotide encoding the polypeptide of the present invention is isolated from Chlorella cells.

[0259] 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 correspond to the codon usage of the host organism intended for enzyme production, or by introducing nucleotide substitutions that may result in different amino acid sequences. For a general description of nucleotide substitutions, see, for example, Ford et al., 1991, Protein Expression and Purification 2:95-107.

[0260] In one aspect, the polynucleotide is isolated.

[0261] In another aspect, the polynucleotide is purified.

[0262] Nucleic acid construct

[0263] The present invention also relates to nucleic acid constructs comprising the polynucleotide of the present invention, wherein the polynucleotide is operably linked to one or more control sequences which direct the expression of the coding sequence in a suitable host cell under conditions compatible with these control sequences.

[0264] 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.

[0265] Promoter

[0266] The control sequence may be a promoter, i.e., a polynucleotide recognized by the host cell for expression of the polynucleotide encoding the polypeptide of the present 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.

[0267] Examples of suitable promoters for directing transcription of the polynucleotide of the present 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.

[0268] Examples of suitable promoters for directing transcription of the polynucleotide of the present invention in filamentous fungal host cells are promoters obtained from cells of the genus Aspergillus, Fusarium, Rhizomucor, and Trichoderma, 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.

[0269] 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.

[0270] Terminator

[0271] The control sequence can 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.

[0272] 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).

[0273] Preferred terminators for filamentous fungal host cells can be obtained from Aspergillus or Trichoderma species, such as the genes of Aspergillus niger glucoamylase, Trichoderma reesei β-glucosidase, Trichoderma reesei cellobiohydrolase I, and Trichoderma reesei endoglucanase I, such as the terminators described in Mukherjee et al., 2013, “Trichoderma: Biology and Applications” and Schmoll and 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology.

[0274] 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.

[0275] mRNA stabilizer

[0276] 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.

[0277] 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).

[0278] Examples of mRNA stabilizer regions of 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.

[0279] Leader sequence

[0280] The control sequence can also be a leader sequence, i.e., an mRNA untranslated region important for translation in the host cell. The leader sequence is operably linked to the 5'-end of the polynucleotide encoding the polypeptide. Any leader sequence functional in the host cell can be used.

[0281] 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.

[0282] 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.

[0283] Suitable leader sequences for yeast host cells can be obtained from the genes of the following: 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).

[0284] Polyadenylation sequence

[0285] The control sequence can also be a polyadenylation sequence, i.e., a sequence operably linked to the 3'-end of a polynucleotide, which is recognized by the host cell during transcription as a signal for adding polyadenylate residues to the transcribed mRNA. Any polyadenylation sequence functional in the host cell can be used.

[0286] Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes of the following: Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0287] Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. [Molecular Cell Biology] 15:5983-5990.

[0288] Signal peptide

[0289] The control sequence can also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a polypeptide and directs the polypeptide into the secretory pathway of the cell. The 5'-end of the coding sequence of the polynucleotide itself can contain a signal peptide coding sequence that is naturally linked to the coding sequence segment encoding the polypeptide in the translation reading frame. Alternatively, the 5'-end of the coding sequence can contain a signal peptide coding sequence that is heterologous to the coding sequence. In cases where the coding sequence does not naturally contain a signal peptide coding sequence, a heterologous signal peptide coding sequence may be required. Alternatively, the heterologous signal peptide coding sequence can 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 can be used.

[0290] Effective signal peptide coding sequences for bacterial host cells are signal peptide coding sequences obtained from the genes of the following: Bacillus NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus alpha-amylase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prsA. Additional signal peptides are described by Freudl, 2018, Microbial Cell Factories [Microbial Cell Factories] 17:52.

[0291] The effective signal peptide coding sequence of 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.

[0292] Useful signal peptides of yeast host cells are obtained from the genes of the following: Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992, ibid.

[0293] Propeptide

[0294] The control sequence may also be a propeptide coding 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 into active polypeptides by catalytic cleavage or autocatalytic cleavage of the propeptide from the pro-polypeptide. Propeptide coding sequences can be obtained from the genes of the following: Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95 / 33836), Rhizomucor miehei aspartic protease, and Saccharomyces cerevisiae α-factor.

[0295] 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 contain only a portion of the signal peptide sequence and / or only a portion of the propeptide sequence. Alternatively, the final or isolated polypeptide may contain a mixture of the mature polypeptide and a polypeptide containing a partial or full-length propeptide sequence and / or signal peptide sequence.

[0296] Regulatory sequence

[0297] It may also be desirable to add regulatory sequences that regulate the expression of polypeptides related to host cell growth. Examples of regulatory sequences are those that cause gene expression to be turned on or off in response to chemical or physical stimuli, including the presence of regulatory compounds. Regulatory sequences in prokaryotic systems include the lac, tac, and trp operon systems. In yeast, the ADH2 system or GAL1 system can be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, the Aspergillus oryzae TAKA α-amylase promoter and the Aspergillus oryzae glucoamylase promoter, the Trichoderma reesei cellobiohydrolase I promoter and the Trichoderma reesei cellobiohydrolase II promoter can be used. Other examples of regulatory sequences are those that 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.

[0298] Transcription factor

[0299] The control sequence can also be a transcription factor, i.e., a polynucleotide encoding a polypeptide that specifically binds to 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 usually attached to a specific DNA sequence adjacent to the genetic element regulated by the transcription factor. Transcription factors can regulate the expression of the protein of interest directly (i.e., by binding to its promoter to activate the transcription of the gene encoding the protein of interest) or indirectly (i.e., by binding to the promoter of another transcription factor that regulates the transcription of the gene encoding the protein of interest to activate the transcription of the other transcription factor). 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 [Subcellular Biochemistry] 52:7-23 and Balleza et al., 2009, FEMS Microbiol. Rev. [FEMS Microbiology Review] 33(1):133-151.

[0300] Expression vector

[0301] The present invention also relates to recombinant expression vectors comprising the polynucleotides, promoters, and transcriptional and translational termination signals of the present invention. Multiple nucleotides and control sequences can be ligated together to generate 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 comprising the polynucleotide into an appropriate vector for expression. When generating the expression vector, the coding sequence is positioned in the vector such that the coding sequence is operably linked to an appropriate control sequence for expression.

[0302] The recombinant expression vector can be any vector (e.g., a plasmid or a virus) that can be conveniently subjected to recombinant DNA procedures and can cause the expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear or a closed circular plasmid.

[0303] The vector can be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, 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 the 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 can be used, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon can be used.

[0304] The vector preferably contains one or more selectable markers that allow for convenient selection of 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.

[0305] 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.

[0306] For integration into the genome of the host cell, the vector can rely on the polynucleotide sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous recombination (such as homology-directed repair (HDR)) or non-homologous recombination (such as non-homologous end joining (NHEJ)).

[0307] For autonomous replication, the vector can further contain an origin of replication, which 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.

[0308] 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 can be 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 host cell genome 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.

[0309] host cell

[0310] The present invention also relates to recombinant host cells that comprise a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of the polypeptide of the present invention.

[0311] A construct or vector comprising the polynucleotide is introduced into the 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.

[0312] The host cell can be any microbial cell that can be used for the recombinant production of the polypeptide of the present invention, such as a prokaryotic cell or a fungal cell.

[0313] Prokaryotic host cells can be any Gram-positive or Gram-negative bacterium. 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.

[0314] The bacterial host cell can be any Bacillus cell, 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.

[0315] For the purposes of the present invention, Bacillus species / genus / species shall be defined as described in Patel and Gupta, 2020, Int. J. Syst. Evol. Microbiol. [International Journal of Systematic and Evolutionary Microbiology] 70: 406-438.

[0316] The bacterial host cell can also be any Streptococcus cell, including but not limited to Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus zooepidemicus cells.

[0317] 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.

[0318] 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. A person skilled in the art will be able to readily determine a suitable method for introducing DNA into a given prokaryotic cell depending, for example, 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.

[0319] The host cell can be a fungal cell. As used herein, "fungal" includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota, as well as the Oomycota and all mitosporic fungi (as defined in, for example, Hawksworth et al., Ainsworth and Bisby’s Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).

[0320] Fungal cells can be transformed by processes involving protoplast-mediated transformation, Agrobacterium-mediated transformation, electroporation, the gene gun method, 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.

[0321] 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).

[0322] 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 cells. In a preferred embodiment, the yeast host cell is a Pichia or Komagataella cell, such as Pichia pastoris cells (Komagataella phaffii).

[0323] 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.

[0324] 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.

[0325] For example, the filamentous fungal host cell may 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 avenaceum, 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.

[0326] In one aspect, the host cell is isolated.

[0327] In another aspect, the host cell is purified.

[0328] Production method

[0329] The present invention also relates to methods for producing the polypeptides of the present invention, which methods comprise (a) culturing cells that produce the polypeptide in its wild-type form under conditions conducive to the production of the polypeptide; and optionally (b) recovering the polypeptide. In one aspect, the cells are Chlorella cells. In another aspect, the cells are Chlorella amygdalina cells. In another aspect, the cells are Pycnoporus coccineus cells. In another aspect, the cells are Pycnoporus coccineus. In another aspect, the cells are Chlorella fusca cells. In another aspect, the cells are Chlorella ellipsoidea cells. In another aspect, the cells are Chlorella olivacea cells. In another aspect, the cells are Microsphaeropsis ononidicola cells. In another aspect, the cells are Chlorella variabilis cells. In another aspect, the cells are Microsphaeropsis spartii-juncei cells.

[0330] The present invention also relates to methods for producing the polypeptides of the present invention, which methods comprise (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.

[0331] 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 permit the expression and / or isolation of the polypeptide in a laboratory or industrial fermentor. Suitable media can be obtained from commercial suppliers or can be prepared according to published compositions (e.g., in the catalog 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.

[0332] The polypeptide can be detected using methods specific for the polypeptide known in the art, including but not limited to assays using specific antibodies, enzyme product formation, enzyme substrate disappearance, or determination of the relative or specific activity of the polypeptide.

[0333] The polypeptide can be recovered from the medium using methods known in the art, including but 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.

[0334] 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).

[0335] In an alternative aspect, the polypeptide is not recovered.

[0336] Particles

[0337] The invention also relates to enzyme particles / pellets comprising the polypeptide of the invention. In one embodiment, the particle comprises a core and optionally one or more coatings (outer layers) surrounding the core.

[0338] The diameter of the core (measured as the equivalent spherical diameter (volume-based average particle size)) can be 20 - 2000 μm, in particular 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 using a Malvern Mastersizer and / or the method described under ISO 13320 (2020).

[0339] In one embodiment, the core comprises a CE1 family polypeptide of the invention having ferulic acid esterase and / or acetylxylan esterase activity.

[0340] The core can include additional materials such as fillers, fibrous materials (cellulose or synthetic fibers), stabilizers, solubilizers, suspending agents, viscosity regulators, light spheres, plasticizers, salts, lubricants, and fragrances.

[0341] The core can include binders such as synthetic polymers, waxes, fats, or carbohydrates.

[0342] The core typically can include salts of polyvalent cations, reducing agents, antioxidants, peroxide decomposition catalysts, and / or acidic buffer components as a homogeneous blend.

[0343] 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.

[0344] The diameter of the core can be 20 - 2000 μm, in particular 50 - 1500 μm, 100 - 1500 μm or 250 - 1200 μm.

[0345] The core can be surrounded by at least one coating, for example 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).

[0346] 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%.

[0347] The coating is preferably at least 0.1 μm thick, in particular 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.

[0348] 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.

[0349] 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.

[0350] The salt coating may contain 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.

[0351] 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.

[0352] The salt can 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.

[0353] The salt coating may contain a single salt or a mixture of two or more salts. The salt can 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.

[0354] The salt can 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 chlorine, bromine, iodine, sulfate, sulfite, bisulfite, thiosulfate, phosphate, dihydrogen phosphate, dibasic phosphate, hypophosphite, pyrophosphate dihydrogen, 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 can be used.

[0355] The salt in the coating may have a constant humidity of more than 60%, in particular 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.

[0356] 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.

[0357] 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.

[0358] Preferably, the salt is applied as a salt solution, for example, using a fluidized bed.

[0359] 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 glycerol monoesters, and glycerol di-esters, and glycerol triesters of fatty acids. Examples of film-forming coating materials suitable for application by fluidized bed techniques are given in GB 1483591.

[0360] The particles may optionally have one or more additional coatings. Examples of suitable coating materials are polyethylene glycol (PEG), methyl hydroxypropylcellulose (MHPC), and polyvinyl alcohol (PVA). Examples of enzyme particles with multiple coatings are described in WO 93 / 07263 and WO 97 / 23606.

[0361] 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.

[0362] 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:

[0363] (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).

[0364] (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, in which 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.

[0365] (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.

[0366] (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 plate with drilled holes) sets a limit on the allowable pressure drop across the extrusion openings. In addition, when using small openings, very high extrusion pressures increase 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).

[0367] (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.

[0368] (f) Mixer granulation products, where the polypeptide-containing liquid is added to a dry powder composition of conventional granulation components. The liquid and the 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.

[0369] (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.

[0370] (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.

[0371] (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 polypeptides 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.

[0372] 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.

[0373] The particulate matter can further comprise 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.

[0374] Another example of formulating a polypeptide by using co-particulate matter is disclosed in WO 2013 / 188331.

[0375] The present invention also relates to a protected polypeptide prepared by the method disclosed in EP 238216.

[0376] Liquid formulation

[0377] The present invention also relates to a liquid composition comprising the polypeptide of the present invention. The composition can comprise 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 borates, or phenylboric acid derivatives such as 4-formylphenylboric acid).

[0378] In some embodiments, one or more fillers or one or more carrier materials are included to increase the volume of such compositions. Suitable fillers or carrier materials include, but are not limited to, various salts of sulfate, carbonate, and silicate radicals, as well as talc, clay, etc. Suitable fillers or carrier materials for liquid compositions include, but are not limited to, water or low molecular weight primary and secondary alcohols (including polyols and diols). Examples of such alcohols include, but are not limited to, methanol, ethanol, propanol, and isopropanol. In some embodiments, these compositions contain from about 5% to about 90% of such materials.

[0379] 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.

[0380] In another embodiment, the present invention relates to liquid formulations that contain:

[0381] (A) 0.001%-25% w / w of the polypeptide of the present invention having ferulic acid esterase and / or acetylxylan esterase activity;

[0382] (B) 20%-80% w / w polyol;

[0383] (C) Optionally 0.001%-2% w / w preservative; and

[0384] (D) Water.

[0385] In another embodiment, the present invention relates to liquid formulations that contain:

[0386] (A) 0.001%-25% w / w of the polypeptide of the present invention having ferulic acid esterase and / or acetylxylan esterase activity;

[0387] (B) 0.001%-2% w / w preservative;

[0388] (C) Optionally 20%-80% w / w polyol; and

[0389] (D) Water.

[0390] 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, acetates and phosphates, 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.

[0391] 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).

[0392] 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.

[0393] 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.

[0394] Composition

[0395] The present invention relates to compositions that comprise a carbohydrate esterase family 1 (CE1) polypeptide having ferulic acid esterase and / or acetylxylan esterase activity, a polypeptide having arabinofuranosidase activity towards di-substituted arabinose, a polypeptide having arabinofuranosidase activity towards mono-substituted arabinose, a polypeptide having xylanase activity, a polypeptide having β-xylosidase activity, and optionally an α-xylosidase.

[0396] The present invention contemplates the use of 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. In other words, the methods of the present invention contemplate the use of any of the compositions or exemplary polypeptides described in this section.

[0397] A. Exemplary CE1 Polypeptides Having Ferulic Acid Esterase and / or Acetylxylan Esterase Activity

[0398] Aspects of the present invention relate to compositions that comprise a combination of a CE1 polypeptide having ferulic acid esterase and / or 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 CE1 polypeptide having ferulic acid esterase and / or acetylxylan esterase activity, when used in combination with a polypeptide having arabinofuranosidase activity towards di-substituted and mono-substituted arabinose, xylanase, and β-xylosidase, increases the production of monomeric arabinose and / or xylose as compared to compositions comprising a polypeptide having arabinofuranosidase activity towards di-substituted and mono-substituted arabinose, xylanase, and β-xylosidase alone.

[0399] Exemplary CE1 polypeptides having ferulic acid esterase and / or acetylxylan esterase activity have the amino acid sequence of SEQ ID NO:3. In one embodiment, a CE1 polypeptide having ferulic acid esterase and / or acetylxylan esterase activity has an amino acid sequence of SEQ ID NO:3 with 0 to 10 conservative amino acid substitutions and has ferulic acid esterase and / or acetylxylan esterase activity. In one embodiment, the CE1 polypeptide has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:3 and has ferulic acid esterase and / or acetylxylan esterase activity. Exemplary CE1 polypeptides having ferulic acid esterase and / or acetylxylan esterase activity have the amino acid sequence of SEQ ID NO:6. In one embodiment, a CE1 polypeptide having ferulic acid esterase and / or acetylxylan esterase activity has an amino acid sequence of SEQ ID NO:6 with 0 to 10 conservative amino acid substitutions and has ferulic acid esterase and / or acetylxylan esterase activity. In one embodiment, the CE1 polypeptide has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:6 and has ferulic acid esterase and / or acetylxylan esterase activity. Exemplary CE1 polypeptides having ferulic acid esterase and / or acetylxylan esterase activity have the amino acid sequence of SEQ ID NO:9. In one embodiment, a CE1 polypeptide having ferulic acid esterase and / or acetylxylan esterase activity has an amino acid sequence of SEQ ID NO:9 with 0 to 10 conservative amino acid substitutions and has ferulic acid esterase and / or acetylxylan esterase activity. In one embodiment, the CE1 polypeptide has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:9 and has ferulic acid esterase and / or acetylxylan esterase activity. Exemplary CE1 polypeptides having ferulic acid esterase and / or acetylxylan esterase activity have the amino acid sequence of SEQ IDNO:12.In one embodiment, the CE1 polypeptide having ferulic acid esterase and / or acetylxylan esterase activity has an amino acid sequence of SEQ ID NO:12 with 0 to 10 conservative amino acid substitutions and has ferulic acid esterase and / or acetylxylan esterase activity. In one embodiment, the CE1 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 ferulic acid esterase and / or acetylxylan esterase activity. An exemplary CE1 polypeptide having ferulic acid esterase and / or acetylxylan esterase activity has the amino acid sequence of SEQ ID NO:15. In one embodiment, the CE1 polypeptide having ferulic acid esterase and / or acetylxylan esterase activity has an amino acid sequence of SEQ ID NO:15 with 0 to 10 conservative amino acid substitutions and has ferulic acid esterase and / or acetylxylan esterase activity. In one embodiment, the CE1 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 ferulic acid esterase and / or acetylxylan esterase activity.

[0400] The CE1 polypeptide having ferulic acid esterase and / or acetylxylan esterase activity can be added at a concentration between 0.0001 - 1 mg EP (enzyme protein) / g DS (for example, 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.

[0401] B. Exemplary polypeptides having arabinofuranosidase activity towards di-substituted arabinose

[0402] Aspects of the present invention relate to compositions that comprise a combination of an arabinofuranosidase active on 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 on di-substituted arabinose, when combined with a CE1 polypeptide having ferulic acid esterase and / or acetylxylan esterase activity, a polypeptide having arabinofuranosidase activity on 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 CE1 polypeptide alone, a polypeptide having arabinofuranosidase activity on mono-substituted arabinose, a xylanase, a β-xylosidase, and optionally an α-xylosidase.

[0403] In one embodiment, the polypeptide having arabinofuranosidase activity on di-substituted arabinose is a GH43 arabinofuranosidase. In one embodiment, the GH43 arabinofuranosidase is a GH43_36 arabinofuranosidase.

[0404] Exemplary GH43 arabinofuranosidases can be from the genus Humicola, Lasiodiplodia, or Poronia.

[0405] Exemplary GH43 arabinofuranosidases can be from Humicola insolens, Lasiodiplodia theobromae, or Poronia punctata.

[0406] Exemplary GH43 arabinofuranosidases have the amino acid sequence of SEQ ID NO:16. In one embodiment, the GH43 arabinofuranosidase has the amino acid sequence of SEQ ID NO:16 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:16 and has arabinofuranosidase activity. Exemplary GH43 arabinofuranosidases have the amino acid sequence of SEQ ID NO:17. In one embodiment, the GH43 arabinofuranosidase has the amino acid sequence of SEQ ID NO:17 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:17 and has arabinofuranosidase activity. Exemplary GH43 arabinofuranosidases have the amino acid sequence of SEQ ID NO:18. In one embodiment, the GH43 arabinofuranosidase has the amino acid sequence of SEQ ID NO:18 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:18 and has arabinofuranosidase activity.

[0407] 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.

[0408] C. Exemplary polypeptides having arabinofuranosidase activity against mono-substituted arabinose

[0409] Aspects of the present invention relate to compositions that comprise a combination of an arabinofuranosidase that is active on mono-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 on mono-substituted arabinose, when used in combination with a CE1 polypeptide having feruloyl esterase and / or acetylxylan esterase activity, a polypeptide having arabinofuranosidase activity on 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 CE1 polypeptide, the polypeptide having arabinofuranosidase activity on di-substituted arabinose, the xylanase, the β-xylosidase, and optionally the α-xylosidase alone.

[0410] In one embodiment, the polypeptide having arabinofuranosidase activity on mono-substituted arabinose is a GH51 arabinofuranosidase. In one embodiment, the GH51 arabinofuranosidase is a GH51_6 arabinofuranosidase.

[0411] Exemplary GH51 arabinofuranosidases can be from the genus Meripulus, the genus Lasiodiplodia, or the genus Acidiella.

[0412] Exemplary GH51 arabinofuranosidases can be from the species Meripulus giganteus, Lasiodiplodia theobromae, or Acidiella bohemica.

[0413] Exemplary GH51 arabinofuranosidases have the amino acid sequence of SEQ ID NO:19. In one embodiment, the GH51 arabinofuranosidase has the amino acid sequence of SEQ ID NO:19 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:19 and has arabinofuranosidase activity. Exemplary GH51 arabinofuranosidases have the amino acid sequence of SEQ ID NO:20. In one embodiment, the GH43 arabinofuranosidase has the amino acid sequence of SEQ ID NO:20 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:20 and has arabinofuranosidase activity. Exemplary GH51 arabinofuranosidases have the amino acid sequence of SEQ ID NO:21. In one embodiment, the GH51 arabinofuranosidase has the amino acid sequence of SEQ ID NO:21 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:21 and has arabinofuranosidase activity.

[0414] Polypeptides having arabinofuranosidase activity towards mono-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.

[0415] D. Exemplary polypeptides having xylanase activity

[0416] Aspects of the present 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 present invention contemplates that any polypeptide having xylanase activity, when used in combination with a CE1 polypeptide having ferulic acid esterase and / or 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 the CE1 polypeptide having ferulic acid esterase and / or acetylxylan esterase activity, the polypeptide having arabinofuranosidase activity towards di- and mono-substituted arabinose, the β-xylosidase, and optionally the α-xylosidase alone.

[0417] In one embodiment, the polypeptide having xylanase activity is a GH5_21 xylanase.

[0418] Exemplary GH_21 xylanases can be from the genus Bacteroides, Belliella, Chryseobacterium, or Sphingobacterium.

[0419] Exemplary GH_21 xylanases can be from the species Bacteroides cellulosilyticus CL02Y12C19, Belliella sp.-64282, Chryseobacterium sp., Chryseobacterium oncorhynchi, or Sphingobacterium sp.-64162.

[0420] Exemplary GH5_21 xylanases can be from a bioreactor metagenome, an elephant feces metagenome, a xanthan gum alkaline community O, a xanthan gum alkaline community S, or a xanthan gum alkaline community T.

[0421] Exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO:22. In one embodiment, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO:22 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:22 and has xylanase activity. Exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO:23. In one embodiment, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO:23 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:23 and has xylanase activity. Exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO:24. In one embodiment, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO:24 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:24 and has xylanase activity. Exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO:25. In one embodiment, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO:25 with 0 to 10 conservative amino acid substitutions and has xylanase activity. In 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:25 and has xylanase activity. Exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO:26.In one embodiment, the GH5_21 xylanase has an amino acid sequence of SEQ ID NO:26 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:26 and has xylanase activity. An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO:27. In one embodiment, the GH5_21 xylanase has an amino acid sequence of SEQ ID NO:27 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:27 and has xylanase activity. An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO:28. In one embodiment, the GH5_21 xylanase has an amino acid sequence of SEQ ID NO:28 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:28 and has xylanase activity. An exemplary GH5_21 xylanase has the amino acid sequence of SEQ IDNO:29. In one embodiment, the GH5_21 xylanase has an amino acid sequence of SEQ ID NO:29 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:29 and has xylanase activity. An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO:30. In one embodiment, the GH5_21 xylanase has an amino acid sequence of SEQ ID NO:30 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:30 and has xylanase activity. An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO:31. In one embodiment, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO:31 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:31 and has xylanase activity. An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO:32. In one embodiment, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO:32 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:32 and has xylanase activity. An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO:33. In one embodiment, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO:33 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:33 and has xylanase activity. An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO:34. In one embodiment, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO:34 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:34 and has xylanase activity. An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO:35. In one embodiment, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO:35 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:35 and has xylanase activity. An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO:36. In one embodiment, the GH5_21 xylanase has the amino acid sequence of SEQ ID NO:36 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:36 and has xylanase activity.

[0422] In one embodiment, the polypeptide having xylanase activity is a GH5_35 xylanase.

[0423] Exemplary GH5_35 xylanases can be from the genus Bacillus, Cohnella, or Paenibacillus.

[0424] Exemplary GH5_35 xylanases can be from the species Bacillus hemiccellulosilyticus JCM 9152, Cohnella xylanilytica, Paenibacillus chitinolyticus, or Paenibacillus sp.-62332.

[0425] Exemplary GH5_35 xylanases can be from a compost metagenome.

[0426] Exemplary GH5_35 xylanases have the amino acid sequence of SEQ ID NO:37. In one embodiment, the GH5_35 xylanase has the 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_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:37 and has xylanase activity. Exemplary GH5_35 xylanases have the amino acid sequence of SEQ ID NO:38. In one embodiment, the GH5_35 xylanase has the 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_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:38 and has xylanase activity. Exemplary GH5_35 xylanases have the amino acid sequence of SEQ ID NO:39. In one embodiment, the GH5_35 xylanase has the 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_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:39 and has xylanase activity. Exemplary GH5_35 xylanases have the amino acid sequence of SEQ ID NO:40. In one embodiment, the GH5_35 xylanase has the 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_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:40 and has xylanase activity. Exemplary GH5_35 xylanases have the amino acid sequence of SEQ ID NO:41.In one embodiment, the GH5_35 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_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:41 and has xylanase activity. In one embodiment, the polypeptide having xylanase activity is a GH30_8 xylanase.

[0427] Exemplary GH30_8 xylanases can be from the genus Bacillus.

[0428] Exemplary GH30_8 xylanases can be from the species Bacillus sp.-18423.

[0429] Exemplary GH30_8 xylanases have the amino acid sequence of SEQ ID NO:42. In one embodiment, the GH30_8 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 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:42 and has xylanase activity.

[0430] 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.

[0431] E. Exemplary polypeptides having β-xylosidase activity

[0432] 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 CE1 polypeptide having ferulic acid esterase and / or acetylxylan esterase activity, a polypeptide having arabinofuranosidase activity towards di- and mono-substituted arabinose, a polypeptide having xylanase activity, a polypeptide having β-xylosidase activity, and optionally a polypeptide having α-xylosidase activity, increases the production of monomeric arabinose and / or xylose compared to compositions comprising a CE1 polypeptide alone, a polypeptide having arabinofuranosidase activity towards di- and mono-substituted arabinose, a xylanase, and optionally an α-xylosidase.

[0433] In one embodiment, the β-xylosidase is a GH3 β-xylosidase.

[0434] Exemplary GH3 β-xylosidases can be from the genus Aspergillus or Talaromyces.

[0435] Exemplary GH3 β-xylosidases can be from the species Aspergillus fumigatus, Aspergillus nidulans, or Talaromyces emersonii.

[0436] Exemplary GH3β-xylosidases have the amino acid sequence of SEQ ID NO:43. In one embodiment, the GH3β-xylosidase has the amino acid sequence of SEQ ID NO:43 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:43 and has β-xylosidase activity. Exemplary GH3β-xylosidases have the amino acid sequence of SEQ ID NO:44. In one embodiment, the GH3β-xylosidase has the amino acid sequence of SEQ ID NO:44 with 0 to 10 conservative amino acid substitutions and has β-xylosidase activity. In 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:44 and has β-xylosidase activity. Exemplary GH3β-xylosidases have the amino acid sequence of SEQ ID NO:45. In one embodiment, the GH3β-xylosidase has the amino acid sequence of SEQ ID NO:45 with 0 to 10 conservative amino acid substitutions and has β-xylosidase activity. In 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:45 and has β-xylosidase activity.

[0437] 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.

[0438] F. Exemplary polypeptides having α-xylosidase activity

[0439] 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 CE1 polypeptide having ferulic acid esterase and / or acetylxylan esterase activity, a polypeptide having arabinofuranosidase activity towards di- and mono-substituted arabinose, a polypeptide having xylanase activity, a polypeptide having β-xylosidase activity, and optionally a polypeptide having α-xylosidase activity, increases the production of monomeric arabinose and / or xylose compared to compositions comprising a CE1 polypeptide alone, a polypeptide having arabinofuranosidase activity towards di- and mono-substituted arabinose, a xylanase, and a β-xylosidase.

[0440] In one embodiment, the α-xylosidase comprises a GH31 α-xylosidase.

[0441] Exemplary GH31 α-xylosidases can be from the genus Herbinix.

[0442] Exemplary GH31 α-xylosidases can be from the species Herbinix hemicellulosilytica.

[0443] Exemplary GH31 α-xylosidases have the amino acid sequence of SEQ ID NO:46. In one embodiment, the GH31 α-xylosidase has an amino acid sequence of SEQ ID NO:46 with 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:46 and has α-xylosidase activity.

[0444] 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.

[0445] G. Exemplary fermenting organisms

[0446] 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 (i.e., converting) sugars such as arabinose, glucose, maltose, and / or xylose 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.

[0447] 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.

[0448] 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 appreciate 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.

[0449] 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).

[0450] 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).

[0451] The fermenting organism can comprise 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).

[0452] The fermenting organisms can be in the form of a composition that comprises the fermenting organisms and naturally occurring and / or non-naturally occurring components.

[0453] The fermenting organisms can be in any viable form, including ground, dried, including active dry and instant, compressed, paste (liquid) form, etc. In one embodiment, the fermenting organisms (e.g., Saccharomyces cerevisiae strains) are dry yeasts, such as active dry yeast or instant yeast. In one embodiment, the fermenting organisms are ground yeast. In one embodiment, the fermenting organisms are compressed yeast. In one embodiment, the fermenting organisms are paste yeast.

[0454] In one embodiment, it is a composition that comprises the fermenting organisms (e.g., Saccharomyces cerevisiae strains) described herein and one or more components selected from the group consisting of: surfactants, emulsifiers, gums, swelling agents, and antioxidants and other processing aids.

[0455] The compositions described herein can comprise the fermenting organisms (e.g., Saccharomyces cerevisiae strains) described herein and any suitable surfactant. In one embodiment, one or more surfactants are anionic surfactants, cationic surfactants, and / or nonionic surfactants.

[0456] The compositions described herein can comprise the fermenting organisms (e.g., Saccharomyces cerevisiae strains) 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, fatty acid esters of propylene glycol.

[0457] In one embodiment, the composition comprises the fermenting organisms (e.g., Saccharomyces cerevisiae strains) 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.

[0458] The compositions described herein can comprise the fermenting organisms (e.g., Saccharomyces cerevisiae strains) 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.

[0459] The compositions described herein can comprise the fermenting organisms (e.g., Saccharomyces cerevisiae strains) described herein and any suitable swelling agent. In one embodiment, the swelling agent is methylcellulose or carboxymethylcellulose.

[0460] 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.

[0461] 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 a 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 and preferably 10 7 to 10 10 and especially about 5×10 7 .

[0462] Method for producing a fermentation product from an amylated starch material

[0463] One aspect of the present invention relates to a method for producing a fermentation product (e.g., fuel ethanol) from an amylated starch material, wherein during saccharification and / or fermentation, a composition comprising a CE1 polypeptide having ferulic acid esterase and / or acetylxylan esterase activity or a CE1 polypeptide having ferulic acid esterase and / or acetylxylan esterase activity is present or added.

[0464] In one embodiment, the method for producing a fermentation product from a starch-containing material comprises the following steps:

[0465] (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;

[0466] (b) saccharifying the dextrin using glucoamylase to produce fermentable sugars; and

[0467] (c) fermenting the sugars using a fermenting organism to produce a fermentation product;

[0468] wherein during the saccharification step (b) and / or the fermentation step (c), a CE1 polypeptide having ferulic acid esterase and / or acetylxylan esterase activity or a composition comprising a CE1 polypeptide having ferulic acid esterase and / or acetylxylan esterase activity is present or added.

[0469] The present invention contemplates the use of any of the CE1 polypeptides or compositions described herein 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.

[0470] The present invention contemplates the use of any of the above-exemplified CE1 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 used in a method for producing a fermentation product.

[0471] The exemplary composition used in step (b) and / or step (c) comprises a CE1 polypeptide having ferulic acid esterase and / or acetylxylan esterase activity, a polypeptide having arabinofuranosidase activity towards di-substituted arabinose, a polypeptide having arabinofuranosidase activity towards mono-substituted arabinose, a polypeptide having xylanase activity, and a polypeptide having β-xylosidase activity.

[0472] The exemplary compositions used in step (b) and / or step (c) comprise a CE1 polypeptide having ferulic acid esterase and / or 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 CE1 polypeptide having ferulic acid esterase and / or 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 CE1 polypeptide having ferulic acid esterase and / or 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 CE1 polypeptide having ferulic acid esterase and / or 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 CE1 polypeptide having ferulic acid esterase and / or 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 CE1 polypeptide having ferulic acid esterase and / or acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH30_8 xylanase, and a GH3 β-xylosidase.

[0473] The exemplary compositions used in step (b) and / or step (c) comprise a CE1 polypeptide having ferulic acid esterase and / or 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 CE1 polypeptide having ferulic acid esterase and / or 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 CE1 polypeptide having ferulic acid esterase and / or 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 CE1 polypeptide having ferulic acid esterase and / or 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 CE1 polypeptide having ferulic acid esterase and / or 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 CE1 polypeptide having ferulic acid esterase and / or acetylxylan esterase activity, a GH43_36 arabinofuranosidase, a GH51_6 arabinofuranosidase, a GH30_8 xylanase, and a GH3 β-xylosidase.

[0474] The exemplary compositions used in step (b) and / or step (c) comprise a CE1 polypeptide having ferulic acid esterase and / or 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.

[0475] The exemplary compositions used in step (b) and / or step (c) comprise a CE1 polypeptide having ferulic acid esterase and / or 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 CE1 polypeptide having ferulic acid esterase and / or 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 CE1 polypeptide having ferulic acid esterase and / or 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 CE1 polypeptide having ferulic acid esterase and / or 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 CE1 polypeptide having ferulic acid esterase and / or 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 CE1 polypeptide having ferulic acid esterase and / or 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 CE1 polypeptide having ferulic acid esterase and / or 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 CE1 polypeptide having ferulic acid esterase and / or 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 CE1 polypeptide having ferulic acid esterase and / or acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_35 xylanase, a GH3 β-xylosidase, and a GH31 α-xylosidase. The exemplary compositions used in step (b) and / or step (c) comprise a CE1 polypeptide having ferulic acid esterase and / or acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH30_8 xylanase, and a GH3 β-xylosidase, and a GH31 α-xylosidase.

[0476] 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.

[0477] 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).

[0478] 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).

[0479] 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).

[0480] Process parameters

[0481] Process parameters for producing a fermentation product (such as producing 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).

[0482] Starch - containing material

[0483] 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.

[0484] Reduction of the particle size of the starch-containing material

[0485] Before the liquefaction step (a), the particle size of the starch-containing material can be reduced, for example, by dry milling.

[0486] Slurry

[0487] Before the liquefaction step (a), a slurry can be formed that includes the starch-containing material (e.g., preferably ground) and water. Alpha-amylase and optionally protease can be added to the slurry. The slurry can be heated therebetween to a temperature above the initial gelatinization temperature of the starch-containing material to initiate gelatinization of the starch.

[0488] Jet cooking

[0489] Before adding alpha-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 at a temperature range of 100°C to 120°C for up to at least 15 minutes.

[0490] Liquefaction temperature

[0491] 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.

[0492] Liquefaction pH

[0493] 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.

[0494] Liquefaction time

[0495] 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.

[0496] Liquefying enzyme

[0497] The present invention contemplates the use of thermostable enzymes during the liquefaction step (a). It is well known in the art to use various thermostable enzymes during the liquefaction step (a), including, for example, thermostable α-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).

[0498] 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, WO94 / 02597, WO 96 / 23873, WO 96 / 23874, WO 96 / 39528, WO 97 / 41213, WO 97 / 43424, WO 99 / 19467, WO 00 / 60059, WO 2002 / 010355, WO 2002 / 092797, WO 2009 / 149130, WO 2009 / 61378, WO 2009 / 061379, WO 2009 / 061380, WO 2009 / 061381, WO 2009 / 098229, WO 2009 / 100102, WO 2010 / 115021, WO2010 / 115028, WO 2010 / 036515, WO 2011 / 082425, WO 2013 / 096305, WO 2013 / 184577, WO 2014 / 007921, WO 2014 / 164777, WO 2014 / 164800, WO 2014 / 164834, WO 2019 / 113413, WO 2019 / 113415, WO 2019 / 197318 (each of these patents is incorporated herein by reference).

[0499] Examples of suitable thermostable glucoamylases include, but are not limited to, glucoamylases described in: 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).

[0500] Examples of suitable thermostable endoglucanases include, but are not limited to, the endoglucanases described in WO 2015 / 035914 (which is incorporated herein by reference).

[0501] 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).

[0502] Examples of suitable thermostable phytases include, but are not limited to, phytases described in: WO 1996 / 28567, WO 1997 / 33976, WO 1997 / 38096, WO 1997 / 48812, WO 1998 / 05785, WO 1998 / 06856, WO 1998 / 13480, WO 1998 / 20139, WO 1998 / 028408, WO 1999 / 48330, WO 1999 / 49022, 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-containing products include BIO-FEEDPHYTASE TM 、PHYTASE NOVO TM CT or L、LIQMAX or RONOZYME TM NP、 HIPHOS、 P5000(CT)、NATUPHOS TM NG 5000。

[0503] 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, WO 2018 / 118815 A1, and WO 2018 / 169780A1 (each of these patents is incorporated herein by reference).

[0504] Examples of suitable commercially available protease-containing products include AVANTEC FORTIVA FORTIVA

[0505] 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).

[0506] 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

[0507] One or more of the enzymes described above are to be used in the methods of the present invention in an "effective amount". Guidelines for determining the effective amount of the enzymes used in the liquefaction step (a) and guidelines for performing activity assays to determine the activity of those enzymes can be found in the published patent applications cited for each of the different thermostable liquefying enzymes.

[0508] Saccharification temperature

[0509] 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.

[0510] Saccharification pH

[0511] The saccharification can be carried out within a pH range of 4 to 5. Preferably, the pH is about 4.5.

[0512] Saccharification time

[0513] The saccharification can last for about 24 hours to about 72 hours.

[0514] Fermentation time

[0515] The fermentation can last for 6 to 120 hours, 24 hours to 96 hours, or 35 hours to 60 hours.

[0516] Simultaneous saccharification and fermentation

[0517] 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 missing] °C, at 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 at a pH of 3.8 to 4.5 for 48 to 60 hours.

[0518] Saccharification and / or fermentation enzymes

[0519] 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.

[0520] 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).

[0521] 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).

[0522] 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, WO2012 / 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).

[0523] 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); AMIGASE TMand AMIGASE TM PLUS (from DSM); G-ZYME TM G900, G-ZYME TM and G990 ZR (from DuPont - Genencor).

[0524] Examples of suitable β-glucanases include, but are not limited to, the β-glucanases described in WO 2021 / 055395 (which is incorporated herein by reference).

[0525] 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).

[0526] 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).

[0527] 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).

[0528] 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.

[0529] Examples of suitable lipases include, but are not limited to, the lipases described in: WO2017 / 112533, WO2017 / 112539, and WO 2020 / 076697 (each of these patents is incorporated herein by reference).

[0530] 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).

[0531] Examples of suitable phytases include, but are not limited to, the phytases described in WO 2001 / 62947 (which is incorporated herein by reference).

[0532] Examples of suitable pectinases include, but are not limited to, the pectinases described in WO 2022 / 173694 (which is incorporated herein by reference).

[0533] Examples of suitable peroxidases include, but are not limited to, the peroxidases described in WO 2019 / 231944 (which is incorporated herein by reference).

[0534] 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).

[0535] 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).

[0536] Method for producing a fermentation product from a material containing ungelatinized starch

[0537] 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 during saccharification and / or fermentation, a composition containing a CE1 polypeptide having ferulic acid esterase and / or acetylxylan esterase activity or a CE1 polypeptide having ferulic acid esterase and / or acetylxylan esterase activity is present or added.

[0538] In one embodiment, the method for producing a fermentation product from a material containing ungelatinized starch comprises the following steps:

[0539] (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

[0540] (b) Fermenting the sugar using a fermenting organism to produce a fermentation product;

[0541] wherein during the saccharification step (a) and / or the fermentation step (b), a CE1 polypeptide having ferulic acid esterase and / or acetylxylan esterase activity or a composition containing a CE1 polypeptide having ferulic acid esterase and / or acetylxylan esterase activity is present or added.

[0542] The present invention contemplates any of the CE1 polypeptides or compositions described herein for use in methods for producing a fermentation product. 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.

[0543] The present invention contemplates the use of any of the above-exemplified CE1 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 methods for producing a fermentation product.

[0544] The exemplary composition used in step (a) and / or step (b) comprises a CE1 polypeptide having ferulic acid esterase and / or 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.

[0545] The exemplary compositions used in step (a) and / or step (b) comprise a CE1 polypeptide having ferulic acid esterase and / or 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 (a) and / or step (b) comprise a CE1 polypeptide having ferulic acid esterase and / or 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 (a) and / or step (b) comprise a CE1 polypeptide having ferulic acid esterase and / or acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5 xylanase, and a GH3 β-xylosidase. The exemplary compositions used in step (a) and / or step (b) comprise a CE1 polypeptide having ferulic acid esterase and / or acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_21 xylanase, and a GH3 β-xylosidase. The exemplary compositions used in step (a) and / or step (b) comprise a CE1 polypeptide having ferulic acid esterase and / or acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_35 xylanase, and a GH3 β-xylosidase. The exemplary compositions used in step (a) and / or step (b) comprise a CE1 polypeptide having ferulic acid esterase and / or acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH30_8 xylanase, and a GH3 β-xylosidase.

[0546] The exemplary compositions used in step (a) and / or step (b) comprise a CE1 polypeptide having ferulic acid esterase and / or 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 (a) and / or step (b) comprise a CE1 polypeptide having ferulic acid esterase and / or 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 (a) and / or step (b) comprise a CE1 polypeptide having ferulic acid esterase and / or 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 (a) and / or step (b) comprise a CE1 polypeptide having ferulic acid esterase and / or 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 (a) and / or step (b) comprise a CE1 polypeptide having ferulic acid esterase and / or 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 (a) and / or step (b) comprise a CE1 polypeptide having ferulic acid esterase and / or acetylxylan esterase activity, a GH43_36 arabinofuranosidase, a GH51_6 arabinofuranosidase, a GH30_8 xylanase, and a GH3 β-xylosidase.

[0547] The exemplary compositions used in step (a) and / or step (b) comprise a CE1 polypeptide having ferulic acid esterase and / or 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.

[0548] The exemplary compositions used in step (a) and / or step (b) comprise a CE1 polypeptide having ferulic acid esterase and / or 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 (a) and / or step (b) comprise a CE1 polypeptide having ferulic acid esterase and / or 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 (a) and / or step (b) comprise a CE1 polypeptide having ferulic acid esterase and / or 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 (a) and / or step (b) comprise a CE1 polypeptide having ferulic acid esterase and / or 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 (a) and / or step (b) comprise a CE1 polypeptide having ferulic acid esterase and / or 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 (a) and / or step (b) comprise a CE1 polypeptide having ferulic acid esterase and / or 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 (a) and / or step (b) comprise a CE1 polypeptide having ferulic acid esterase and / or 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 (a) and / or step (b) comprise a CE1 polypeptide having ferulic acid esterase and / or 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 (a) and / or step (b) comprise a CE1 polypeptide having ferulic acid esterase and / or acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH5_35 xylanase, a GH3 β-xylosidase, and a GH31 α-xylosidase. The exemplary compositions used in step (a) and / or step (b) comprise a CE1 polypeptide having ferulic acid esterase and / or acetylxylan esterase activity, a GH43 arabinofuranosidase, a GH51 arabinofuranosidase, a GH30_8 xylanase, and a GH3 β-xylosidase, and a GH31 α-xylosidase.

[0549] In one embodiment, the composition is added during saccharification step (b). In one embodiment, the composition is added during 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.

[0550] 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 particle 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).

[0551] 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).

[0552] 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).

[0553] 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).

[0554] Back - end or downstream processing

[0555] A. Recovery of fermentation products and production of stillage

[0556] 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, which 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.

[0557] 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 stillage.

[0558] 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, for example, as fuel ethanol, drinking ethanol (i.e., potable neutral alcoholic beverages), or industrial ethanol.

[0559] In some embodiments of these methods, the recovered fermentation product is substantially pure. For 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.

[0560] Suitable assays can be performed using methods known in the art to test for ethanol and contaminant production and sugar consumption. For example, ethanol products and other organic compounds can be analyzed by methods such as HPLC (high performance liquid chromatography), GC-MS (gas chromatography-mass spectrometry), and LC-MS (liquid chromatography-mass spectrometry), or other suitable analytical methods using conventional procedures well known in the art. The release of ethanol in the fermentation broth can also be tested using the culture supernatant. By using, for example, a refractive index detector for glucose and alcohols, and a UV detector for organic acids, by HPLC (Lin et al., Biotechnol. Bioeng. [Biotechnology and Bioengineering] 90:775 - 779 (2005)), or using other suitable assays and detection methods well known in the art, the by-products and residual sugars (e.g., glucose or xylose) in the fermentation medium can be quantified.

[0561] B. Processing of whole stillage

[0562] In one embodiment, the whole stillage is processed into two streams - a wet cake and a centrate. The whole stillage is separated or partitioned into a solid phase and a liquid phase by one or more methods for separating the centrate from the wet cake. The centrate is divided into two streams - stillage water, which enters the evaporator; and a countercurrent, which is recycled to the front of the plant. Any suitable separation technique can be used to separate the whole stillage into a centrate (e.g., stillage water, when pumped to the evaporator rather than the front end of the plant) and a wet cake to remove most of the liquid / water. In a preferred embodiment, separation / dehydration is performed by centrifugation. In the industry, a preferred centrifuge is a settling centrifuge, preferably a high-speed settling centrifuge. An example of a suitable centrifuge is the NX 400 steep cone series from Alfa Laval, which is a high-performance decanter. Similar settling centrifuges are also available from Flottweg. In another preferred embodiment, other conventional separation equipment such as plate / frame filter presses, belt filter presses, screw presses, gravity thickeners, and dehydrators, or similar equipment, are used for separation.

[0563] C. Processing of stillage water

[0564] Distillers' water is the term for the supernatant used in the centrifugation of whole distillers' grains. Typically, distillers' water contains 4%-8% dry solids (DS) (mainly proteins, soluble fibers, fats, fine fibers, and cell wall components), and the temperature is about 60°C - 90°C. The distillers' water stream can be condensed by evaporation to provide two process streams, including: (i) an evaporator condensate stream, which contains the condensed water removed from the distillers' water during evaporation; and (ii) a slurry stream, which contains a more concentrated stream of non-volatile dissolved and undissolved solids, such as non-fermentable sugars and oils remaining in the distillers' water due to the removal of the evaporated water.

[0565] Optionally, oil can be removed from the distillers' water, or it can be removed as an intermediate step in the evaporation process, which typically uses a series of several evaporation stages.

[0566] The slurry and / or the deoiled slurry can be introduced into a dryer together with the wet cake (from the whole distillers' grains separation step) to provide a product called dry distillers' grains with solubles, which can also be used as animal feed. In one embodiment, the slurry and / or the deoiled slurry are sprayed into one or more dryers to combine the slurry and / or the deoiled slurry with the whole distillers' grains to produce dry distillers' grains with solubles.

[0567] Distillers' water in the range of 5 - 90 vol.-%, such as between 10% - 80%, such as between 15% - 70%, such as between 20% - 60% (e.g., optionally hydrolyzed) can be recycled (as a countercurrent) to step (a). The recycled distillers' water (i.e., the countercurrent) can account for about 1 - 70 vol.-%, preferably 15 - 60 vol.-%, especially about 30 - 50 vol.-% of the slurry formed in step (a). In one embodiment, the process further includes optionally recycling at least a portion of the distillers' water stream to the slurry after oil has been extracted from the distillers' water stream.

[0568] D. Drying of the wet cake and production of dry distillers' grains and dry distillers' grains with solubles

[0569] After separating a wet cake containing about 25 - 40 wt.-%, preferably 30 - 38 wt.-% dry solids from distillers' slops water (e.g., by dehydration), it can be dried in a drum dryer, spray dryer, ring dryer, fluidized bed dryer, etc. to produce "dried distillers' grains" (DDG). DDG is a valuable feed ingredient for animals such as livestock, poultry, and fish. Preferably, DDG is provided with a moisture content of less than about 10 - 12 wt.-% to avoid mold and microbial decomposition and increase shelf life. Additionally, a high moisture content also makes transporting DDG more expensive. The wet cake is preferably dried under conditions that do not denature the proteins in the wet cake. The wet cake can be blended with a slurry separated from distillers' slops water and dried to produce DDG with solubles (DDGS). A partially dried intermediate product, such as sometimes referred to as modified wet distillers' grains, can be produced by partially drying the wet cake and optionally adding the slurry before, during, or after the drying process.

[0570] Fermentation broth formulation or cell composition

[0571] The present invention also relates to a fermentation broth formulation or cell composition comprising the polypeptide of the present invention. The fermentation broth formulation or cell composition further comprises additional components used in the fermentation process, such as cells (including host cells containing the gene encoding the polypeptide of the present invention, which are used to produce the polypeptide of interest), cell debris, biomass, fermentation medium, and / or fermentation products. In some embodiments, the composition is a cell-killed whole broth containing one or more organic acids, killed cells, and / or cell debris, and the medium.

[0572] As used herein, the term "fermentation broth" refers to a preparation produced by cell fermentation that does not undergo or undergoes minimal recovery and / or purification. For example, when a microbial culture is incubated and grown to saturation under carbon-limiting conditions that permit protein synthesis (e.g., expression of an enzyme by a host cell) and secretion of the protein into the cell culture medium, a fermentation broth is produced. The fermentation broth can contain the unfractionated or fractionated contents of the fermentation materials derived at the end of the fermentation. Typically, the fermentation broth is unfractionated and contains the spent medium and cell debris present, for example, after removing microbial cells (e.g., filamentous fungal cells) by centrifugation. In some embodiments, the fermentation broth contains the spent cell culture medium, extracellular enzymes, and viable and / or non-viable microbial cells.

[0573] In some embodiments, the fermentation broth formulation or cell composition comprises a first organic acid component (comprising at least one organic acid having 1-5 carbons and / or its salt) and a second organic acid component (comprising at least one organic acid having 6 or more carbons and / or its salt). In some embodiments, the first organic acid component is acetic acid, formic acid, propionic acid, its salt, or a mixture of two or more of the foregoing; and the second organic acid component is benzoic acid, cyclohexanecarboxylic acid, 4-methylpentanoic acid, phenylacetic acid, its salt, or a mixture of two or more of the foregoing.

[0574] In one aspect, the composition contains one or more organic acids and optionally further contains killed cells and / or cell debris. In some embodiments, these killed cells and / or cell debris are removed from the whole cell-killed culture broth to provide a composition free of these components.

[0575] The fermentation broth formulation or cell composition may further comprise preservatives and / or antimicrobial (e.g., bacteriostatic) agents, including but not limited to sorbitol, sodium chloride, potassium sorbate, and other agents known in the art.

[0576] The whole cell-killed culture broth or cell composition may contain the unfractionated contents of the fermentation material derived at the end of fermentation. Typically, the whole cell-killed culture broth or cell composition contains the spent medium and cell debris present after microbial cells (e.g., filamentous fungal cells) are incubated and grown to saturation under carbon-limiting conditions that permit protein synthesis. In some embodiments, the whole cell-killed culture broth or cell composition contains the spent cell medium, extracellular enzymes, and killed filamentous fungal cells. In some embodiments, methods known in the art can be used to permeabilize and / or lyse the microbial cells present in the whole cell-killed culture broth or composition.

[0577] The whole cell-killed culture broth or cell composition as described herein is typically a liquid, but may contain insoluble components such as killed cells, cell debris, medium components, and / or one or more insoluble enzymes. In some embodiments, the insoluble components can be removed to provide a clarified liquid composition.

[0578] The whole cell-killed culture broth formulations and cell compositions of the present invention can be produced by the methods described in WO 90 / 15861 or WO 2010 / 096673.

[0579] The present invention is further described by the following examples, which should not be construed as limiting the scope of the present invention.

[0580] Examples

[0581] Enzymes used in the examples

[0582] CE1A: An exemplary CE1 family polypeptide with ferulic acid esterase and / or acetylxylan esterase activity from Sphaeropsis sapinea, disclosed in SEQ ID NO:3.

[0583] CE1B: An exemplary CE1 family polypeptide with ferulic acid esterase and / or acetylxylan esterase activity from Sphaeropsis sapinea, disclosed in SEQ ID NO:6.

[0584] CE1C: An exemplary CE1 family polypeptide with ferulic acid esterase and / or acetylxylan esterase activity from Sphaeropsis sapinea, disclosed in SEQ ID NO:9.

[0585] CE1D: An exemplary CE1 family polypeptide with ferulic acid esterase and / or acetylxylan esterase activity from Sphaeropsis sapinea, disclosed in SEQ ID NO:12.

[0586] CE1E: A CE1 family polypeptide with ferulic acid esterase and / or acetylxylan esterase activity from Sphaeropsis sapinea, disclosed in SEQ ID NO:15.

[0587] GH43A: An exemplary GH43 arabinofuranosidase from Humicola insolens, disclosed in SEQ ID NO:16.

[0588] GH43B: An exemplary GH43 arabinofuranosidase from Lasiodiplodia theobromae, disclosed in SEQ ID NO:17.

[0589] GH43C: An exemplary GH43 arabinofuranosidase from Dothiorella gregaria, disclosed in SEQ ID NO:18.

[0590] GH51A: An exemplary GH51 arabinofuranosidase from Grifola gigantea, disclosed in SEQ ID NO:19.

[0591] GH51B: An exemplary GH51 arabinofuranosidase from Lasiodiplodia theobromae, disclosed in SEQ ID NO:20.

[0592] GH51C: An exemplary GH51 arabinofuranosidase from Acidibacter bohemicus, disclosed in SEQ ID NO:21.

[0593] GH5_21A: An exemplary GH5_21 xylanase from Bacteroides cellulosilyticus CL02T12C19, disclosed in SEQ ID NO:22.

[0594] GH5_21B: Exemplary GH5_21 xylanase from Xanthomonas campestris alkaliphilic community S, disclosed in SEQ ID NO:23.

[0595] GH5_21C: Exemplary GH5_21 xylanase from Sphingobacterium sp.-64162, disclosed in SEQ ID NO:24.

[0596] GH5_21D: Exemplary GH5_21 xylanase from Sphingobacterium sp.-64162, disclosed in SEQ ID NO:25.

[0597] GH5_21E: Exemplary GH5_21 xylanase from Xanthomonas campestris alkaliphilic community O, disclosed in SEQ ID NO:26.

[0598] GH5_21F: Exemplary GH5_21 xylanase from bioreactor metagenome, disclosed in SEQ ID NO:27.

[0599] GH5_21G: Exemplary GH5_21 xylanase from Xanthomonas campestris alkaliphilic community T, disclosed in SEQ ID NO:28.

[0600] GH5_21H: Exemplary GH5_21 xylanase from Xanthomonas campestris alkaliphilic community S, disclosed in SEQ ID NO:29.

[0601] GH5_21I: Exemplary GH5_21 xylanase from Bellilinea sp.-64282, disclosed in SEQ ID NO:30.

[0602] GH5_21J: Exemplary GH5_21 xylanase from Chryseobacterium mytili, disclosed in SEQ ID NO:31.

[0603] GH5_21K: Exemplary GH5_21 xylanase from Xanthomonas campestris alkaliphilic community T, disclosed in SEQ ID NO:32.

[0604] GH5_21L: Exemplary GH5_21 xylanase from Sphingobacterium sp., disclosed in SEQ ID NO:33.

[0605] GH5_21M: Exemplary GH5_21 xylanase from elephant feces metagenome, disclosed in SEQ ID NO:34.

[0606] GH5_21N: Exemplary GH5_21 xylanase from elephant feces metagenome, disclosed in SEQ ID NO:35.

[0607] GH5_21O: Exemplary GH5_21 xylanase from Chryseobacterium species disclosed in SEQ ID NO:36.

[0608] GH5_35A: Exemplary GH5_35 xylanase from Oceanobacillus iheyensis disclosed in SEQ ID NO:37.

[0609] GH5_35B: Exemplary GH5_35 xylanase from Paenibacillus xylanilyticus JCM 9152 disclosed in SEQ ID NO:38.

[0610] GH5_35C: Exemplary GH5_35 xylanase from Paenibacillus sp.-62332 disclosed in SEQ ID NO:39.

[0611] GH5_35D: Exemplary GH5_35 xylanase from compost metagenome disclosed in SEQ ID NO:40.

[0612] GH5_35E: Exemplary GH5_35 xylanase from Paenibacillus chitinolyticus disclosed in SEQ ID NO:41.

[0613] GH30_8: Exemplary GH30_8 xylanase from Bacillus sp.-18423 disclosed in SEQ ID NO:42.

[0614] GH3A: Exemplary GH3 β-xylosidase from Aspergillus fumigatus disclosed in SEQ ID NO:43.

[0615] GH3B: Exemplary GH3 β-xylosidase from Aspergillus nidulans disclosed in SEQ ID NO:44.

[0616] GH3C: Exemplary GH3 β-xylosidase from Talaromyces emersonii disclosed in SEQ ID NO:45.

[0617] GH31A: Exemplary GH31 α-xylosidase from Hymenobacter sp. disclosed in SEQ ID NO:46

[0618] GH8: Exemplary GH8 xylanase from Bacillus sp. KK-1 disclosed in SEQ ID NO:47.

[0619] GH10: Exemplary GH10 xylanase from Aspergillus aculeatus disclosed in SEQ ID NO:48.

[0620] GH11: Exemplary GH11 xylanase from Thermomyces lanuginosus disclosed in SEQ ID NO:49.

[0621] Liquefying enzyme blend 1: Exemplary thermostable α-amylase from Bacillus stearothermophilus disclosed in SEQ ID NO:50; Exemplary thermostable protease from Pyrococcus furiosus disclosed in SEQ ID NO:52.

[0622] Liquefying enzyme blend 2: Exemplary thermostable α-amylase from Bacillus stearothermophilus disclosed in SEQ ID NO:51; Exemplary thermostable protease from Pyrococcus furiosus disclosed in SEQ ID NO:52; Exemplary thermostable xylanase from Thermotoga maritima disclosed in SEQ ID NO:53.

[0623] Saccharifying enzyme blend: Exemplary glucoamylase from Gloeophyllum sepiarium disclosed in SEQ ID NO:54; Exemplary α-amylase from Rhizomucor pusillus disclosed in SEQ ID NO:55; Exemplary trehalase from Talaromyces funiculosus disclosed in SEQ ID NO:56; Exemplary β-glucosidase from Aspergillus fumigatus disclosed in SEQ ID NO:57; Exemplary cellobiohydrolase from Aspergillus fumigatus disclosed in SEQ ID NO:58; Exemplary endoglucanase from Trichoderma reesei disclosed in SEQ ID NO:59.

[0624] Hemicellulase blend: Polypeptide with arabinofuranosidase activity towards di-substituted arabinose (GH43A), polypeptide with arabinofuranosidase activity towards mono-substituted arabinose (GH51A), polypeptide with xylanase activity (GH5_21O), and polypeptide with β-xylosidase activity (GH3A).

[0625] Determination of Td of liquefying enzyme by differential scanning calorimetry

[0626] The thermal stability of the enzyme was determined by differential scanning calorimetry (DSC) using a VP-capillary differential scanning calorimeter (MicroCal Inc., Piscataway, NJ, USA). In the thermogram (Cp vs. T) obtained after heating an enzyme solution (approx. 0.5 mg / ml) in buffer (50 mM acetate, pH 5.0) at a constant programmed heating rate of 200 K / hour, the thermal denaturation temperature Td (°C) was taken as the top of the denaturation peak (main endothermic peak).

[0627] The sample solution and reference solution (approx. 0.2 ml) were loaded into the calorimeter from storage conditions at 10 °C (reference: buffer without enzyme), and thermally pre-equilibrated at 20 °C for 20 minutes, followed by a DSC scan from 20 °C to 120 °C. The denaturation temperature was determined with an accuracy of approximately + / -1 °C.

[0628] Strain

[0629] The fungal strain NN074640 was isolated from a soil sample collected in Brazil in 2019 by the dilution plate method using PDA medium (pH 6, 28 °C). Then it was purified by transferring single conidia onto PDA agar plates. Based on both morphological characteristics and ITS rDNA sequences, the strain NN074640 was identified as Sphaeropsis.

[0630] The yeast strain MEJI797 is MBG5012 of WO 2019 / 161227, which further expresses Pycnoporus sanguineus glucoamylase (SEQ ID NO:4 of WO 2011 / 066576) and a heterologous Rhizomucor pusillus α-amylase expression cassette (as described in WO 2013 / 006756).

[0631] Media and solutions

[0632] The PDA plate consisted of 39 g of potato dextrose agar and deionized water made up to 1 liter.

[0633] Examples

[0634] Example 1: The effect of combining xylanases from GH families 5, 8, 10, 11, and 30 with arabinofuranosidases from GH families 43 and 51 on increasing xylose and arabinose in a simultaneous saccharification and fermentation process

[0635] An industrial liquefied mash prepared with liquefying enzyme blend 1 was used for the experiment. The dry solids determined by a moisture balance were approximately 34% DS, and the pH was adjusted to pH 5.0, followed by supplementation with 3 ppm penicillin and 500 ppm urea. Simultaneous saccharification and fermentation (SSF) was carried out via small-scale fermentation. Approximately 4.0 g of industrially liquefied corn mash was added to 15 ml screw-capped bottles. To each vial was added a saccharifying enzyme blend at 0.42 AGU / g DS and an appropriate amount of the corresponding xylanase and arabinofuranosidase, and the dosing regimens are shown in Table 2. The saccharifying enzyme blend was used as a control without the addition of xylanase or arabinofuranosidase. The actual enzyme dosage was based on the exact weight of the corn slurry in each vial. After adding the enzymes, fermentation was initiated by adding 50 μL of the propagated yeast strain MEJI797. The tubes were incubated at 32 °C, and each treatment was repeated three times. After 65 h of SSF, the tubes were removed from the incubator, 50 μL of 34% H2SO4 was added, and then centrifuged at 3500 rpm for 10 min, followed by filtration through a 0.45-μm filter. Sugar concentrations were determined using an HPLC system equipped with an H column (Benson Polymeric, BP-700H, 300 × 7.8 mm).

[0636] Table 2: Arabinofuranosidase dosing regimens with or without xylanase

[0637]

[0638]

[0639] Table 3: Results

[0640] Treatment Average arabinose, ppm Arabinose increase % Control 490 0％ GH43A 770 57％ GH51A 770 57％ GH43A + GH51A 1190 142％ GH43A + GH51A + GH5_21O 2270 363％ GH43A + GH51A + GH8 1350 175％ GH43A + GH51A + GH10 1330 172％ GH43A + GH51A + GH11 1270 159％ GH43A + GH51A + GH30_8 2150 338％

[0641] % increase in arabinose = [(average arabinose experimental ppm - average arabinose control) / average arabinose control] × 100

[0642] Table 3 shows that the GH5_21 or GH30_8 xylanase in combination with GH43 and GH51 arabinofuranosidases releases the highest concentration of arabinose compared to the individual GH43 or GH51 arabinofuranosidases or their combinations without xylanase.

[0643] Example 2: Effect of the combination of a GH3 family β-xylosidase with arabinofuranosidases from GH 43 and 51 families and xylanase from GH5_21 on increasing xylose in a simultaneous saccharification and fermentation process

[0644] An industrial liquefied mash prepared with the liquefying enzyme blend 2 was used for the experiment. The dry solids determined by a moisture balance were approximately 35.9% DS, and the pH was adjusted to pH 5.0, followed by supplementation with 3 ppm penicillin and 500 ppm urea. Simultaneous saccharification and fermentation (SSF) was carried out via small-scale fermentation. Approximately 4.2 g of industrially liquefied corn mash was added to 15 ml screw-capped vials. To each vial was added a saccharifying enzyme blend at 0.42 AGU / g DS and an appropriate amount of the respective xylanase, arabinofuranosidase, and β-xylosidase (as listed in Table 4). The dosing scheme followed a fixed amount of GH5_21 xylanase, GH43 arabinofuranosidase, and GH51 arabinofuranosidase at 10 μg / g dry solids each, with or without the β-xylosidase GH3A, GH3B, or GH3C at doses of 25, 50, 100, or 200 μg / g dry solids. The saccharifying enzyme blend was used as a control without the addition of xylanase or β-xylosidase. The actual enzyme dose was based on the exact weight of the corn slurry in each vial. After adding the enzymes, fermentation was initiated by adding 50 μL of the propagated yeast strain MEJI797. The tubes were incubated at 32 °C, and each treatment was repeated three times. After 65 h of SSF, the tubes were removed from the incubator and then subjected to centrifugation at 3500 rpm for 10 min, followed by filtration through a 0.45-μm filter. Sugar concentrations were determined using an HPLC system equipped with a lead column (Benson Polymeric, BP-800Pb, 300 × 7.8 mm).

[0645] Results

[0646] Table 4 shows that β-xylosidase in combination with GH43, GH51 arabinofuranosidase, and GH5_21 xylanase significantly increased xylose release, and higher enzyme doses corresponded to higher xylose release.

[0647] Table 4

[0648]

[0649]

[0650] Example 3: Effect of combinations of GH 5 xylanase subfamilies 21 and 35 with Hi GH 43 and Mg GH51 arabinofuranosidases and AfGH3 β-xylosidase on increasing xylose and arabinose in a simultaneous saccharification and fermentation process

[0651] The industrial liquefied mash prepared with the liquefying enzyme blend 1 was used for the experiment. The dry solids determined by a moisture balance were approximately 33.7% DS, and the pH was adjusted to pH 5.0, followed by supplementation with 3 ppm penicillin and 500 ppm urea. Simultaneous saccharification and fermentation (SSF) was carried out via small-scale fermentation. Approximately 4.2 g of industrially liquefied corn mash was added to 15 ml screw-cap bottles. To each vial was added a saccharifying enzyme blend at 0.42 AGU / g DS, 10 μg / g DS of GH43A arabinofuranosidase, 10 μg / g DS of GH51A arabinofuranosidase, 25 μg / g DS of GH3A β-xylosidase, and 10 μg / g DS of the corresponding xylanase (as listed in Table 5). The saccharifying enzyme blend was used as a control without the addition of arabinofuranosidase, xylanase, or β-xylosidase. The actual enzyme dosage was based on the exact weight of the corn slurry in each vial. After adding the enzymes, fermentation was initiated by adding 50 μL of the propagated yeast strain MEJI797. The tubes were incubated at 32 °C, and each treatment was repeated three times. After 65 h of SSF, the tubes were removed from the incubator, 50 μL of 34% H2SO4 was added, and then centrifuged at 3500 rpm for 10 min, followed by filtration through a 0.45 μm filter. Sugar concentrations were determined using an HPLC system equipped with a lead column (Benson Polymeric, BP-800Pb, 300×7.8 mm).

[0652] Results

[0653] Table 5 shows that the addition of xylanases from GH5_21 and GH5_35 significantly increased the release of xylose and arabinose compared to the control or the treatment consisting of GH43, GH51 arabinofuranosidases, and GH3 β-xylosidase without xylanase.

[0654] Table 5

[0655]

[0656] Example 4: Effect of single, double, triple, or quadruple combinations of GH5_21 xylanase, GH43 and GH51 arabinofuranosidases, and GH3 β-xylosidase hemicellulases on increasing xylose and arabinose in a simultaneous saccharification and fermentation process

[0657] An industrial liquefied mash prepared with liquefaction enzyme blend 2 was used for the experiment. The dry solids determined by a moisture balance were approximately 33.4% DS, and the pH was adjusted to pH 5.0, followed by supplementation with 3 ppm penicillin and 500 ppm urea. Simultaneous saccharification and fermentation (SSF) was carried out via small-scale fermentation. Approximately 4.2 g of industrially liquefied corn mash was added to 15-ml screw-cap bottles. A saccharification enzyme blend at 0.6 AGU / g DS was added to each vial, and the dosing regimens of 10 ug / g DS GH5_21O xylanase, 10 ug / g DS of GH43A arabinofuranosidase, 10 ug / g DS of GH51A arabinofuranosidase, and / or 25 ug / g DS of GH3A β-xylosidase were followed. As a control, only the saccharification enzyme blend was added without arabinofuranosidase, xylanase, or β-xylosidase. The actual enzyme dosage was based on the exact weight of the corn slurry in each vial. After adding the enzymes, fermentation was initiated by adding 50 μL of the hydrated yeast strain MEJI797. The tubes were incubated at 32 °C, and each treatment was repeated three times. After 65 h of SSF, the tubes were removed from the incubator, 50 μL of 34% H2SO4 was added, and then centrifuged at 3500 rpm for 10 min, followed by filtration through a 0.45-μm filter. Sugar concentrations were determined using an HPLC system equipped with an H column (Benson Polymeric, BP-700H, 300 × 7.8 mm). The decantation tubes containing the wet corn mash at the end of fermentation were subjected to vacuum freeze-drying for 3 days. The dry solid weight of each tube was determined, and the residual solid was calculated as the ratio of the final solid weight to the initial solid weight.

[0658] Results

[0659] Table 6

[0660]

[0661]

[0662] Table 6 shows that the addition of GH5_21 xylanase together with GH43, GH51 arabinofuranosidase, and GH3 β-xylosidase increased xylose and arabinose release compared to the control or the treatment consisting of GH43, GH51 arabinofuranosidase, and GH3 β-xylosidase without xylanase.

[0663] Example 5: Effect of arabinofuranosidases from GH families 43 and 51 in combination with xylanase from GH family 5 subfamily 21 on increasing arabinose in a simultaneous saccharification and fermentation process

[0664] An industrial liquefied mash prepared with liquefying enzyme blend 2 was used for the experiment. The dry solids determined by a moisture balance were approximately 36% DS, and the pH was adjusted to pH 5.0, followed by supplementation with 3 ppm penicillin and 500 ppm urea. Simultaneous saccharification and fermentation (SSF) was carried out via small-scale fermentation. Approximately 4.0 g of industrially liquefied corn mash was added to 15 ml screw-cap vials. A saccharifying enzyme blend of 0.42 AGU / g DS and an appropriate combination of arabinofuranosidases from families GH43 and GH51 (as listed in Table 7) were added to each vial. The dosing scheme followed a fixed amount of GH5_21O xylanase, GH43 arabinofuranosidase, and GH51 arabinofuranosidase, each at 10 μg / g dry solids, respectively. As a control, only the saccharifying enzyme blend was used without adding xylanase or arabinofuranosidase. The actual enzyme dosage was based on the exact weight of the corn slurry in each vial. After adding the enzymes, fermentation was initiated by adding 50 μL of the propagated yeast strain MEJI797. The tubes were incubated at 32 °C, and each treatment was repeated three times. After 65 h of SSF, the tubes were removed from the incubator, 50 μL of 34% H2SO4 was added, and then centrifuged at 3500 rpm for 10 min, followed by filtration through a 0.45-μm filter. The sugar concentration was determined using an HPLC system equipped with an H column (Benson Polymeric, BP-700H, 300 × 7.8 mm).

[0665] Results

[0666] Table 7

[0667] Treatment Average arabinose, ppm Arabinose increase % Control 165 0 GH43A + GH51A 2744 1563％ GH43B + GH51A 2559 1451％ GH43A + GH51C 1842 1016％ GH43A + GH51B 2198 1232％

[0668] % Increase in arabinose = [(Average arabinose experimental ppm - Average arabinose control) / Average arabinose control] × 100

[0669] Table 7 shows that the combination of GH43 and GH51 arabinofuranosidases with GH5_21 xylanase increased arabinose compared to the control without arabinofuranosidase and xylanase.

[0670] Example 6: Effect of arabinofuranosidases from GH families 43 and 51 in combination with xylanase from GH family 5 subfamily 21 on increasing arabinose in a simultaneous saccharification and fermentation process

[0671] The industrial liquefied mash prepared with the liquefying enzyme blend 2 was used for the experiment. The dry solids determined by a moisture balance were approximately 33.8% DS, and the pH was adjusted to pH 5.0, followed by supplementation with 3 ppm penicillin and 500 ppm urea. Simultaneous saccharification and fermentation (SSF) was carried out via small-scale fermentation. Approximately 4.2 g of industrially liquefied corn mash was added to 15 ml screw-cap bottles. To each vial was added a saccharifying enzyme blend at 0.42 AGU / g DS and an appropriate combination of arabinofuranosidases from families GH43 and GH51 (as listed in Table 8). The dosing scheme followed fixed amounts of GH5_21 xylanase, GH43 arabinofuranosidase, and GH51 arabinofuranosidase, each at 10 μg / g dry solids, respectively. As a control, only the saccharifying enzyme blend was used without addition of xylanase or arabinofuranosidase. The actual enzyme dosage was based on the exact weight of the corn slurry in each vial. After adding the enzymes, fermentation was initiated by adding 50 μL of the propagated yeast strain MEJI797. The tubes were incubated at 32 °C, and each treatment was repeated three times. After 65 h of SSF, the tubes were removed from the incubator, 50 μL of 34% H2SO4 was added, and then centrifuged at 3500 rpm for 10 min, followed by filtration through a 0.45-μm filter. Sugar concentrations were determined using an HPLC system equipped with an H column (Benson Polymeric, BP-700H, 300 × 7.8 mm).

[0672] Results

[0673] Table 8

[0674] Treatment Average arabinose, ppm Arabinose increase % Control 415 0％ GH43A + GH51A 2120 410％ GH43C + GH51A 1902 358％

[0675] % Increase in arabinose = [(Average arabinose experimental ppm - Average arabinose control) / Average arabinose control] × 100

[0676] Table 8 shows that the combination of GH43 and GH1 arabinofuranosidases with GH5_21 xylanase increased arabinose release compared to the case without using xylanase and arabinofuranosidase.

[0677] Example 7: Effect of the combination of an exemplary CE1 polypeptide with ferulic acid esterase and / or acetylxylan esterase activity from Sphaeropsis sp. and exemplary polypeptides with arabinofuranosidase activity towards di-substituted arabinose, arabinofuranosidase activity towards mono-substituted arabinose, xylanase activity, and β-xylosidase activity on increasing xylose and arabinose in a simultaneous saccharification and fermentation process

[0678] An industrial liquefied mash prepared with liquefying enzyme blend 1 was used for the experiment. The dry solids determined by a moisture balance were approximately 34% DS, and the pH was adjusted to pH 5.0, followed by supplementation with 3 ppm penicillin and 500 ppm urea. Simultaneous saccharification and fermentation (SSF) was carried out via small-scale fermentation. Approximately 4.2 g of industrially liquefied corn mash was added to 10 ml screw-capped vials. To each vial was added a saccharifying enzyme blend at 0.42 AGU / g DS, along with the amounts of hemicellulase blend shown in Table 9 and the corresponding CE1 polypeptide at 10 μg / g DS shown in Table 9, after which 50 μL of the propagated yeast strain MEJI797 was added per 4.2 g of slurry. As a control, only the saccharifying enzyme blend was used without adding the hemicellulase blend or CE1 polypeptide. The actual enzyme dosage was based on the exact weight of the corn slurry in each vial. The vials were incubated at 32 °C, with each treatment repeated three times. After 65 h of SSF, the tubes were removed from the incubator and subjected to centrifugation at 3500 rpm for 10 min, followed by filtration through a 0.45 μm filter. The sugar concentration was determined using an HPLC system equipped with an H column (Benson Polymeric, BP-700H, 300×7.8 mm). The decantation tubes containing the wet corn mash at the end of fermentation were subjected to vacuum freeze-drying for 3 days. The dry solids weight of each tube was determined, and the residual solids were calculated as the ratio of the final solids weight to the initial solids weight.

[0679] Table 9: Dosage of enzymes in the hemicellulase blend

[0680] Enzyme Dose, ug / g dry solid GH21O 10 GH43A 10 GH51A 10 GH3A 25

[0681] Results

[0682] As shown in Table 10 below, the combination of CE1 polypeptide and hemicellulase blend increased xylose and arabinose release compared to the control or the hemicellulase blend without adding the CE1 polypeptide. Compared to the control, the hemicellulase blend significantly decreased the residual solids, and the addition of CE1 polypeptide further decreased the residual solids, indicating that the hemicellulase blend and its combination with CE1 polypeptide increased corn fiber degradation.

[0683] Table 10

[0684]

[0685] Example 8: The effect of increasing xylose and arabinose in a simultaneous saccharification and fermentation process by combining an exemplary CE1 polypeptide having ferulic acid esterase and / or acetylxylan esterase activity with or without an exemplary α-xylosidase, an exemplary polypeptide having arabinofuranosidase activity towards di-substituted arabinose, an exemplary polypeptide having arabinofuranosidase activity towards mono-substituted arabinose, an exemplary polypeptide having xylanase activity, and an exemplary polypeptide having β-xylosidase activity

[0686] An industrial liquefied mash prepared with liquefying enzyme blend 1 was used for the experiment. The dry solids determined by a moisture balance were approximately 35% DS, and the pH was adjusted to pH 5.0, followed by supplementation with 3 ppm penicillin and 500 ppm urea. Simultaneous saccharification and fermentation (SSF) was carried out via small-scale fermentation. Approximately 4.2 g of industrially liquefied corn mash was added to 10 ml screw-cap vials. With or without 10 μg / g DS of GH31A, a saccharifying enzyme blend at 0.42 AGU / g DS was added to each vial, along with the amounts of hemicellulase blend shown in Table 9 above and 10 μg / g DS of CE1D and CE1E polypeptides, after which 50 μL of the propagated yeast strain MEJI797 was added per 4.2 g of slurry. As a control, only the saccharifying enzyme blend was used without adding the hemicellulase blend, CE1 enzyme, or GH31A. The actual enzyme dosage was based on the exact weight of the corn slurry in each vial. The vials were incubated at 32 °C, and each treatment was repeated three times. After 65 h of SSF, the tubes were removed from the incubator and subjected to centrifugation at 3500 rpm for 10 min, followed by filtration through a 0.45-μm filter. Sugar concentrations were determined using an HPLC system equipped with a lead column (Benson Polymeric, BP-800Pb, 300 × 7.8 mm). The decantation tubes containing the wet corn mash at the end of fermentation were subjected to vacuum freeze-drying for 3 days. The dry solid weight of each tube was determined, and the residual solid was calculated as the ratio of the final solid weight to the initial solid weight.

[0687] Results

[0688] As shown in Table 11 below, the combination of CE1 polypeptides with the hemicellulase blend increased xylose and arabinose release compared to the control, a hemicellulase blend without CE1 polypeptides. When mixed with CE1 polypeptides and the hemicellulase blend, the addition of an exemplary α-xylosidase (e.g., GH31A) further increased xylose and arabinose release. Compared to the control, the hemicellulase blend significantly decreased the residual solids, and the addition of CE1 polypeptides and α-xylosidase (e.g., GH31) further decreased the residual solids, indicating that the hemicellulase blend and its combination with CE1 polypeptides and GH31 α-xylosidase increased corn fiber degradation.

[0689] Table 11

[0690]

[0691] The invention described and claimed herein is not limited to the scope of the specific aspects disclosed herein, as these aspects are intended to be illustrative of several aspects of the invention. Any equivalent aspects are intended to be within the scope of the invention. Indeed, various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In case of conflict, the present disclosure, including definitions, will control.

[0692] The invention is further defined by the following numbered paragraphs:

[0693] 1. A polypeptide having feruloyl esterase and / or acetylxylan esterase activity, said polypeptide selected from the group consisting of:

[0694] (i)

[0695] (a) a polypeptide having at least 83%, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity to SEQ ID NO:2;

[0696] (b) a polypeptide having at least 83%, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity to SEQ ID NO:3;

[0697] (c) a polypeptide having at least 83%, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO:2;

[0698] (d) A polypeptide encoded by a polynucleotide that has 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:1;

[0699] (e) A polypeptide derived from SEQ ID NO:2, the mature polypeptide of SEQ ID NO:2, or SEQ ID NO:3 and differing by 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);

[0700] (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

[0701] (g) A fragment of the polypeptide of (a), (b), (c), (d), or (e);

[0702] wherein the polypeptide has feruloyl esterase and / or acetylxylan esterase activity;

[0703] (ii)

[0704] (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;

[0705] (b) a polypeptide having at least 70% identity, at least 71% identity, at least 72% identity, at least 73% identity, at least 74% identity, at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity with SEQ ID NO:6;

[0706] (c) a polypeptide having at least 70% identity, at least 71% identity, at least 72% identity, at least 73% identity, at least 74% identity, at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO:5;

[0707] (d) a polypeptide encoded by a polynucleotide having at least 70% identity, at least 71% identity, at least 72% identity, at least 73% identity, at least 74% identity, at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity with the mature polypeptide coding sequence of SEQ ID NO:4;

[0708] (e) A polypeptide derived from SEQ ID NO:5, the mature polypeptide of SEQ ID NO:5, or SEQ ID NO:6, 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);

[0709] (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

[0710] (g) A fragment of the polypeptide of (a), (b), (c), (d), or (e);

[0711] wherein the polypeptide has feruloyl esterase activity and / or acetylxylan esterase activity;

[0712] (iii)

[0713] (a) A polypeptide having at least 70% identity, at least 71% identity, at least 72% identity, at least 73% identity, at least 74% identity, at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity with SEQ ID NO:8;

[0714] (b) a polypeptide having at least 70% identity, at least 71% identity, at least 72% identity, at least 73% identity, at least 74% identity, at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity to SEQ ID NO:9;

[0715] (c) a polypeptide having at least 70% identity, at least 71% identity, at least 72% identity, at least 73% identity, at least 74% identity, at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO:8;

[0716] (d) a polypeptide encoded by a polynucleotide having at least 70% identity, at least 71% identity, at least 72% identity, at least 73% identity, at least 74% identity, at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:7;

[0717] (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 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);

[0718] (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

[0719] (g) A fragment of the polypeptide of (a), (b), (c), (d), or (e);

[0720] wherein the polypeptide has feruloyl esterase activity and / or acetylxylan esterase activity;

[0721] (iv)

[0722] (a) A polypeptide having at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity with SEQ ID NO:11;

[0723] (b) A polypeptide having at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity with SEQ ID NO:12;

[0724] (c) a polypeptide having 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;

[0725] (d) a polypeptide encoded by a polynucleotide having 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;

[0726] (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 (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);

[0727] (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

[0728] (g) a fragment of the polypeptide of (a), (b), (c), (d), or (e);

[0729] wherein the polypeptide has ferulic acid esterase activity and / or acetylxylan esterase activity; and

[0730] (v)

[0731] A polypeptide having at least 70% identity, at least 71% identity, at least 72% identity, at least 73% identity, at least 74% identity, at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity to SEQ ID NO:14;

[0732] A polypeptide having at least 70% identity, at least 71% identity, at least 72% identity, at least 73% identity, at least 74% identity, at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity to SEQ ID NO:15;

[0733] A polypeptide having at least 70% identity, at least 71% identity, at least 72% identity, at least 73% identity, at least 74% identity, at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO:14;

[0734] (d) A polypeptide encoded by a polynucleotide that has at least 70% identity, at least 71% identity, at least 72% identity, at least 73% identity, at least 74% identity, at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:13;

[0735] (e) A polypeptide derived from SEQ ID NO:14, the mature polypeptide of SEQ ID NO:14, or SEQ ID NO:15 that differs 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);

[0736] (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

[0737] (g) A fragment of the polypeptide of (a), (b), (c), (d), or (e);

[0738] wherein the polypeptide has ferulic acid esterase activity and / or acetylxylan esterase activity.

[0739] 2. A polypeptide having ferulic acid esterase and / or acetylxylan esterase activity, the polypeptide selected from the group consisting of:

[0740] (i)

[0741] a polypeptide having 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;

[0742] a polypeptide having 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;

[0743] a polypeptide encoded by a polynucleotide having 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;

[0744] a polypeptide derived from the polypeptides of (a), (b), (c), wherein the N-terminus and / or C-terminus has been extended by the addition of one or more amino acids, and the polypeptide has ferulic acid esterase and / or acetylxylan esterase activity; and

[0745] a fragment of the polypeptide of (a), (b), (c);

[0746] wherein the polypeptide has ferulic acid esterase and / or acetylxylan esterase activity;

[0747] (ii)

[0748] a polypeptide having at least 70% identity, at least 71% identity, at least 72% identity, at least 73% identity, at least 74% identity, at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity to SEQ ID NO:5;

[0749] a polypeptide having at least 70% identity, at least 71% identity, at least 72% identity, at least 73% identity, at least 74% identity, at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity to SEQ ID NO:6;

[0750] a polypeptide encoded by a polynucleotide having at least 70% identity, at least 71% identity, at least 72% identity, at least 73% identity, at least 74% identity, at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:4;

[0751] (d) A polypeptide derived from the polypeptide of (a), (b), or (c), wherein the N-terminus and / or C-terminus has been extended by adding one or more amino acids, and the polypeptide has ferulic acid esterase and / or acetylxylan esterase activity; and

[0752] (e) A fragment of the polypeptide of (a), (b), (c);

[0753] wherein the polypeptide has ferulic acid esterase activity and / or acetylxylan esterase activity;

[0754] (iii)

[0755] (a) A polypeptide having at least 70% identity, at least 71% identity, at least 72% identity, at least 73% identity, at least 74% identity, at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity with SEQ ID NO:8;

[0756] (b) A polypeptide having at least 70% identity, at least 71% identity, at least 72% identity, at least 73% identity, at least 74% identity, at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity with SEQ ID NO:9;

[0757] (c) A polypeptide encoded by a polynucleotide that has at least 70% identity, at least 71% identity, at least 72% identity, at least 73% identity, at least 74% identity, at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:7;

[0758] (d) A polypeptide derived from the polypeptide of (a), (b), or (c), wherein the N-terminus and / or C-terminus has been extended by the addition of one or more amino acids, and the polypeptide has feruloyl esterase and / or acetylxylan esterase activity; and

[0759] (e) A fragment of the polypeptide of (a), (b), or (c);

[0760] wherein the polypeptide has feruloyl esterase activity and / or acetylxylan esterase activity;

[0761] (iv)

[0762] (a) A polypeptide that has at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity to SEQ ID NO:11;

[0763] (b) a polypeptide having at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity with SEQ ID NO:12;

[0764] (c) a polypeptide encoded by a polynucleotide having at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity with the mature polypeptide coding sequence of SEQ ID NO:10;

[0765] (d) a polypeptide derived from the polypeptide of (a), (b), or (c), wherein the N-terminus and / or C-terminus has been extended by adding one or more amino acids, and the polypeptide has ferulic acid esterase and / or acetylxylan esterase activity; and

[0766] (e) a fragment of the polypeptide of (a), (b), or (c);

[0767] wherein the polypeptide has ferulic acid esterase activity and / or acetylxylan esterase activity; and

[0768] (v)

[0769] a polypeptide having at least 70% identity, at least 71% identity, at least 72% identity, at least 73% identity, at least 74% identity, at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity to SEQ ID NO:14;

[0770] a polypeptide having at least 70% identity, at least 71% identity, at least 72% identity, at least 73% identity, at least 74% identity, at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity to SEQ ID NO:15;

[0771] a polypeptide encoded by a polynucleotide having at least 70% identity, at least 71% identity, at least 72% identity, at least 73% identity, at least 74% identity, at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:13;

[0772] (d) A polypeptide derived from the polypeptide of (a), (b), or (c), wherein the N-terminus and / or C-terminus has been extended by adding one or more amino acids, and the polypeptide has feruloyl esterase and / or acetylxylan esterase activity; and

[0773] (e) A fragment of the polypeptide of (a), (b), (c);

[0774] wherein the polypeptide has feruloyl esterase activity and / or acetylxylan esterase activity.

[0775] 3. A polypeptide having feruloyl esterase and / or acetylxylan esterase activity, the polypeptide selected from the group consisting of:

[0776] (i)

[0777] (a) A polypeptide having 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:2; or

[0778] (b) A fragment of the polypeptide of (a);

[0779] wherein the polypeptide has feruloyl esterase and / or acetylxylan esterase activity;

[0780] (ii)

[0781] (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; or

[0782] (b) A fragment of the polypeptide of (a);

[0783] ...

Claims

1. A polypeptide having ferulic acid esterase and / or acetylxylan esterase activity, said polypeptide being selected from the group consisting of: (i) (a) a polypeptide having 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:2; (b) a polypeptide having 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:3; (c) a polypeptide having 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:2; (d) a polypeptide encoded by a polynucleotide having 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:1; (e) a polypeptide derived from SEQ ID NO:2, the mature polypeptide of SEQ ID NO:2, or SEQ ID NO:3 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 adding one or more amino acids; and (g) A fragment of the polypeptide of (a), (b), (c), (d), or (e); wherein the polypeptide has ferulic acid esterase and / or acetylxylan esterase activity; (ii) (a) A polypeptide having at least 70% identity, at least 71% identity, at least 72% identity, at least 73% identity, at least 74% identity, at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity with SEQ ID NO:5; (b) A polypeptide having at least 70% identity, at least 71% identity, at least 72% identity, at least 73% identity, at least 74% identity, at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, 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 to the mature polypeptide of SEQ ID NO:5; (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: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 (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); (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 ferulic acid esterase activity and / or acetylxylan esterase activity; (iii) a polypeptide having at least 70% identity, at least 71% identity, at least 72% identity, at least 73% identity, at least 74% identity, at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity to SEQ ID NO:8; a polypeptide having at least 70% identity, at least 71% identity, at least 72% identity, at least 73% identity, at least 74% identity, at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity to SEQ ID NO:9; a polypeptide having at least 70% identity, at least 71% identity, at least 72% identity, at least 73% identity, at least 74% identity, at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity to the mature polypeptide of SEQ ID NO:8; (d) A polypeptide encoded by a polynucleotide that has at least 70% identity, at least 71% identity, at least 72% identity, at least 73% identity, at least 74% identity, at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, 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 therefrom 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 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 ferulic acid esterase activity and / or acetylxylan esterase activity; (iv) (a) A polypeptide having at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity to SEQ ID NO:11; (b) a polypeptide having at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity to SEQ ID NO:12; (c) a polypeptide having at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, 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 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, 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 (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 A fragment of the polypeptide of (g)(a), (b), (c), (d) or (e); wherein the polypeptide has feruloyl esterase activity and / or acetylxylan esterase activity; and (v) (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:14; (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:15; (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:14; (d) A polypeptide encoded by a polynucleotide that has at least 70% identity, at least 71% identity, at least 72% identity, at least 73% identity, at least 74% identity, at least 75% identity, at least 76% identity, at least 77% identity, at least 78% identity, at least 79% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, 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 and differing therefrom 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); (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 ferulic acid esterase activity and / or 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.

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.

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.

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

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

8. A composition, the composition comprising a carbohydrate esterase family 1 (CE1) polypeptide having ferulic acid esterase and / or acetylxylan esterase activity, a polypeptide having arabinofuranosidase activity towards di-substituted arabinose, a polypeptide having arabinofuranosidase activity towards mono-substituted arabinose, a xylanase, a β-xylosidase, and optionally an α-xylosidase.

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

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

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

12. The composition as claimed in any one of claims 8-11, wherein the GH5 xylanase is a GH5_21 family xylanase.

13. The composition as claimed in any one of claims 8-12, wherein the GH5 xylanase is a GH5_35 xylanase.

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

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

16. The composition as claimed in any one of claims 8-15, wherein the CE1 polypeptide having ferulic acid esterase and / or acetylxylan esterase activity is a polypeptide as described in any one of claims 1-4.

17. The composition as claimed in any one of claims 7-16, wherein the composition comprises an α-xylosidase.

18. The composition as claimed in any one of claims 7-17, wherein the α-xylosidase is a GH31 α-xylosidase.

19. A method for producing a fermentation product from a starch-containing material, the method comprising the steps of: (a) saccharifying the starch-containing material with 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 CE1 polypeptide having ferulic acid esterase activity and / or acetylxylan esterase activity or a composition comprising a CE1 polypeptide having ferulic acid esterase activity and / or acetylxylan esterase activity is present or added during the saccharification step (a) and / or the fermentation step (b).

20. The method as claimed in claim 19, wherein the CE1 polypeptide is a CE1 polypeptide as described in any one of claims 1-4 or is formulated into a particle as described in claims 5-6.

21. The method as claimed in claim 19 or 20, wherein the composition comprising the CE1 polypeptide is a 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 CE1 polypeptide having ferulic acid esterase activity and / or acetylxylan esterase activity or a composition comprising the CE1 polypeptide having ferulic acid esterase activity and / or 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 CE1 polypeptide is the CE1 polypeptide according to any one of claims 1-4, or is formulated into particles according to claims 5-6.

24. The method according to claim 22 or 23, wherein the composition comprising the CE1 polypeptide is the composition according to any one of claims 7-18.

25. A polynucleotide encoding the 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 60 to 885 of SEQ ID NO:1; (ii) SEQ ID NO:4 or nucleotides 75 to 846 of SEQ ID NO:4; (iii) SEQ ID NO:7 or nucleotides 63 to 830 of SEQ ID NO:7; (iv) SEQ ID NO:10 or nucleotides 57 to 906 of SEQ ID NO:10; and (v) SEQ ID NO:13 or nucleotides 60 to 858 of SEQ ID NO:

13.

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 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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